Thymic Epithelial Tumors Diagnostic, prognostic and physiologic implications of

heat shock protein 27 and 70

Doctoral thesis at the Medical University of Vienna for obtaining the academic degree

Doctor of Philosophy

Submitted by

Dr. med. univ. Stefan Janik

Supervisor Univ. Prof. Dr. med. univ.

Hendrik Jan Ankersmit, MBA Department of Thoracic Surgery

Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration Medical University of Vienna

Währinger Gürtel 18-20 1090 Vienna, Austria

Vienna, 06/2016

Dedicated to my father Hans and to my love Stefanie.

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Acknowledgement

I am very grateful that Hendrik Jan Ankersmit provided me the opportunity to carry

out this thesis and especially for his personal encourage and support to develop my

scientific as well as personal skills. My special thanks go out also to my colleagues

at the laboratory, who had become friends over the last years, for their personal and

technical support.

I would like to sincerely thank my Co-Supervisor and Mentor Bernhard Moser for

inspiring me scienfitically and personally and for his ability to show me how

interesting and funny science could be.

I am also thankful to Leonhard Müllauer and Ana-Iris Schiefer, colleagues of the

Clinical Institute of Pathology, for sharing their scientific knowledge with me and for

their support with important experiments within this study.

Above all, I owe particular thank to the Christian Doppler Laboratory for Cardiac and

Thoracic Diagnosis and Regeneration for funding my project and for making the

realization of this thesis possible.

Finally I want to thank particularly my parents Hans and Silvia, my sister Astrid, my

grandparents and especially my love Stefanie for their lifelong support and unlimited

love and patience.

The results of this thesis were presented at following national congresses:

Annual Meeting of the Austrian Society of Pneumology, Graz, (2015), Austria

56th Annual Meeting of the Austrian Society of Surgery, Linz, (2015), Austria

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Declaration This work was conducted at the Department of Thoracic Surgery of the Medical

University of Vienna under the supervision of Hendrik Jan Ankersmit and Bernhard

Moser in cooperation with colleagues of the Clinical Institute of Pathology.

Serum and tissues specimens were collected from patients who underwent

treatment at the Department of Thoracic Surgery, Medical University of Vienna. All

serum analysis and initial immunohistochemical stainings have been performed at

the laboratories of the Department of Thoracic Surgery. Stainings have been

reproduced from Leonhard Müllauer, Ana-Iris Schiefer and Barbara Neudert

(Clinical Institute of Pathology) by using automated staining platforms.

Doublestaining and routine work up of tissues specimens has been conducted by

the Clinical Institute of Pathology.

Data evaluation, interpretation of results and manuscript preparation was

performed by Stefan Janik (Department of Thoracic Surgery, Medical University of

Vienna) under the supervision of Bernhard Moser and Hendrik Jan Ankersmit

(Department of Thoracic Surgery, Medical University of Vienna) and with support of

Leonhard Müllauer and Ana-Iris Schiefer (Clinical Institute of Pathology).

Figures have been courtesies from Bernhard Moser (Department of Thoracic

Surgery) and Leonhard Müllauer (Clinical Institute of Pathology) or permission was

obtained from respective journals for the reuse of figures within this thesis.

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Table of Contents Acknowledgement……………………………………………...………………………. 3

Declaration………………………………………………………………………………. 4

Table of contents……………………………………………………………………….. 5

List of Figures…………………………………………………………………………… 7

List of Tables……………………………………………………………………………. 7

Abstracts in English and German…………………………………………………….. 8

Abstract………………………………………………………………………….… 8

Zusammenfassung………………………………………………………………. 9

Publication arising from this thesis…………………………………………………….11

Abbreviations……………………………………………………………………………. 11

CHAPTER I: Scientific Background…………………………………………………... 13

1. Thymus……………………………………………………………………………….. 13

1.1 Introduction……………………………………………………………………….. 13

1.1.1 Physiology………………………………………………………………….. 13

1.1.2 T-Cells………………………………………………………………………. 16

1.1.3 Histology……………………………………………………………………. 17

1.2 Thymic Epithelial Tumors……………………………………………………….. 19

1.2.1 Introduction…………………………………………………………………. 19

1.2.2 Clinical Presentation and paraneoplastic syndromes………………….. 20

1.2.3 Histologic classification of TETs…………………………………………..20

1.2.4 Masaoka-Koga staging system…………………………………………... 26

1.2.5 Diagnostic workup in TETs……………………………………………...... 30

1.2.6 Treatment of TETs - Surgery, Radiotherapy, Chemotherapy………….38

1.2.7 Targeted Therapy………………………………………………………….. 44

1.2.8 Tumor Recurrence................................................................................ 47

1.2.9 Second malignancies and risk factors ………………………………….. 48

1.2.10 Prognostic Factors………………………………………………............... 49

1.3 Thymic Hyperplasia……………………………………………………………… 51

1.3.1 Introduction…………………………………………………………………. 51

1.3.2 True Thymic Hyperplasia…………………………………………………. 52

1.3.3 Follicular Thymic Hyperplasia……………………………………………..52

1.4 Myasthenia Gravis……………………………………………………………….. 54

1.4.1 Introduction…………………………………………………………………. 54

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1.4.2 Epidemiology……………………………………………………………….. 55

1.4.3 Symptoms…………………………………………………………………... 55

1.4.4 Classification……………………………………………………………….. 56

1.4.5 MG subtypes and autoantibodies…………………………………………57

1.4.6 Intrathymic pathogenesis of MG: EOMG, TAMG………………………. 59

1.4.7 Diagnosis…………………………………………………………………… 61

1.4.8 Therapy………………………………………………………………………62

1.5 Heat Shock Proteins. ……………………………………………………………. 66

1.5.1 Introduction…………………………………………………………………. 66

1.5.2 Apoptosis …………………………………………………………………... 67

1.5.3 Regulation of Apoptosis by HSP27 and 70……………………………... 69

1.5.4 HSP27 and 70 in cancer………………………………………………….. 71

1.5.5 Heat shock proteins in our workgroup…………………………………… 72

1.6 Aims of this thesis………………………………………………………………... 72

CHAPTER II: Results…………………………………………..………………………. 73

2.1 HSP27 and 70 expression in thymic epithelial tumors and benign thymic

alterations: diagnostic, prognostic and physiologic implications……………. 73

2.1.1 Prologue……………………………………………………………………. 73

CHAPTER III: Discussion……………………………………………………………… 103

3.1 General Discussion……….……………………………………………………… 103

3.2 Conclusion and Outlook…………………………………………………………. 111

CHAPTER IV: Material and Methods………………………………………………… 115

4.1 Study Population………………………………………………………………..… 115

4.2 Immunohistochemistry…………………………………………………………… 116

4.3 Double-Staining…………………………………………………………………… 117

4.4 Evaluation of immunoreactivity………………………………………………….. 117

4.4.1 Thymic Epithelial Tumors…………………………………………………... 117

4.4.2 Non-Malignant Thymic Specimens………………………………………... 118

4.5 Enzyme-linked immunosorbent assay………………………………….....…… 118

4.6 Statistical Methods……………………………………………………………….. 118

References………………………………………………………………………………. 119

Curriculum Vitae…………………………………..……………………………………. 141

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List of Figures Figure 1: Thymic Histology and schematic illustration of T-cell maturation

(adapted from Gorgon et al.)1…………………………………………………..... 15

Figure 2: Schematic illustration of T-cell subsets (adapted from Raphael et al.

and Russ et al.) 7,11………………………………………………………………… 17 Figure 3: Thymic Involution……………………………………………………………. 19

Figure 4: WHO Classification of Thymic Epithelial Tumors………………………... 25

Figure 5: Masaoka-Koga Staging System (Detterbeck et al.)66 ..……………........ 29

Figure 6: Examples for radiological examination techniques in TETs……………. 32

Figure 7: En-bloc resection of a WHO type B2 thymoma………………………….. 39

Figure 8: Follicular Thymic Hyperplasia…………………………………………….... 53

Figure 9: Therapeutic flowchart in treatment of Myasthenia Gravis (adapted

from Gilhus et.)188………………………….………………………….…………… 64

Figure 10: Extended Thymectomy in two patients with Myasthenia Gravis……… 66

Figure 11: Extrinsic, intrinsic and execution pathway of apoptosis (adapted from

Mcllwain et al)233………................................................................................... 69

List of Tables Table 1: WHO classification of Thymic Epithelial Tumors (adapted from Rosai

et al.)41…………………………………………………………….………………... 22

Table 2: Classification of Thymic Neuroendocrine Tumors (adapted from Travis

et al. and Marx et al.)42,43…………………………………………………………..24

Table 3: Thymic Epithelial Tumors (adapted from Marx et al.)43………………….. 26

Table 4: Masaoka-Koga staging system (adapted from Detterbeck et al.)66…….. 27

Table 5: Serum Biomarkers in mediastinal tumors…………………………………. 33

Table 6: Common mediastinal pathologies – a comprehensive overview

(adapted from Marchevsky et al.)93……………………………………………… 34

Table 7: Selected Immunohistochemical Markers for the use of histologic

workup of Mediastinal Tumors (adapted from Marchevsky et al.)93………..... 38

Table 8: Recommendations for treatment of Thymic Epithelial Tumors according

to Masaoka-Koga tumor stage (adapted from Falkson et al. and Ried et

al.)126,137........................................................................................................... 44

Table 9: Definitions of tumor recurrence in Thymic Epithelial Tumors (adapted

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from Huang et al.)64…..................................................................................... 47

Table 10: Prognostic Factors in Thymic Epithelial Tumors (adapted from Kondo

et al.)162…………………………………………………………………………….. 50

Table 11: Osserman Classification of Myasthenia Gravis (adapted from

Osserman et al.)205.......................................................................................... 56

Table 12: Myasthenia Gravis Foundation of America (MGFA) – Clinical

Classification of MG (adapted from Jaretzki et al.)206………………………….. 56

Table 13: Clinical subtypes of Myasthenia Gravis (adapted from Marx et al. and

Meriggiolo et al.) 92,199……………………………………………………………... 59

Table 14: Characteristics of patients with Thymic Epithelial Tumors……………... 116

Abstracts in English and German Abstract Thymic Epithelial Tumors (TETs) represent the most common malignancies in the

anterior mediastinum in adults. According to histology the following three main

subtypes, which are associated with different clinical and oncological behavior, can

be differentiated: (1) thymomas, (2) thymic carcinomas (TCs) and (3) thymic

neuroendocrine tumors (TNETs). Interestingly, thymomas are associated with a

more benign behavior, lower rates of recurrence and better prognosis compared to

TCs and TNETs. Thymomas show also a unique association with paraneoplastic

Myasthenia Gravis (MG), whereas MG is rarely or even missing in patients with TCs

or TNETs. However, TETs represent a heterogeneous group of malignancies and

risk factors as well as pathogenesis of these malignancies still remains elusive and

tumor recurrence can occur in 10-30% of cases even decades after initial surgery.

Hence, prognostic and diagnostic biomarkers are urgently needed for tumor follow-

up and to improve outcome prognoses.

Heat shock proteins (HSPs), so-called “stress proteins” are upregulated by

different stressors and function as molecular chaperons as well as inhibitors of

apoptosis. It has been already shown that HSPs are upregulated in the vast

majority of malignancies and inhibition of HSPs was an effective strategy to target

cancer growth and metastasis. We believe that HSPs, especially the strongest

inducible HSPs (HSP27 and HSP70), might be also upregulated in TETs. Hence,

this thesis was conducted to clarify the questions whether heat shock proteins are

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involved in the pathogenesis of TETs, whether they might represent useful and

reliable prognostic and/or diagnostic markers and whether HSPs are appropriate

molecules for targeted therapy. Therefore, immunohistochemical stainings of

HSP27 and 70 were performed in 101 specimens of TETs and 24 specimens with

benigng thymic alterations. Additionally, we measured systemic HSP27 and 70

concentrations in serum of 46 patients with TETs, 33 patients with benign thymic

alterations and 49 volunteers.

HSP expression was significantly different between histologic tumor subtypes

and clinical tumor stages. Weak HSP tumor expression was associated with

significantly worse freedom from recurrence compared to TETs with more intense

HSP expression. Patients with TETs had significantly elevated HSP27 and 70

serum concentrations compared to volunteers and highest HSP serum

concentrations were detected in TETs with advanced tumor stages. Most

remarkably, serum HSP concentrations significantly decreased after complete

tumor resection, which let us assume that HSPs may represent useful biomarkers in

follow up of patients with TETs. However, although our results indicate a role of

HSPs in TETs their function as diagnostic, prognostic or even therapeutic markers

need to be further evaluated.

Zusammenfassung Epitheliale Thymustumore sind aufgrund ihrer niedrigen Inzidenz seltene

Malignome, aber machen nichtsdestotrotz den größten Anteil an mediastinalen

Tumoren aus und stellen bei Erwachsenen die häufigsten mediastinalen

Neoplasien dar. Epitheliale Thymustumore werden anhand ihrer Histomorphologie,

welche auch mit unterschiedlichem biologischem und onkologischem Verhalten der

Tumore sowie Prognose assoziiert sind, in drei Subtypen unterteilt: Thymome (1),

Thymuskarzinome (2), Neuroendorkine Thymustumore (3). Thymome,

beispielsweise, verhalten sich benigner und sind mit niedrigeren Rezidivraten und

besserer Prognose assoziiert im Vergleich zu Thymuskarzinomen und

neuroendokrinen Thymustumoren. Desweiteren treten bei PatientInnen mit

Thymomen häufig Symptome einer Myasthenia Gravis (MG) auf, während MG

selten bis nie bei PatientInnen mit Thymuskarzinom oder neuroendokrinen

Thymustumoren auftritt.

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Epitheliale Thymustumore stellen eine heterogene Gruppe von Malignome

dar, deren Pathogenese sowie Risikofaktoren sind nach wie vor unklar. Sogar

Jahre bis Jahrzehnte nach der initialen Tumoroperation können Tumorrezidive in

10-30% der Fälle auftreten und machen eine lebenslange Tumornachsorge

notwendig. Da es keine etablierten Biomarker für PatientInnen mit epithelialen

Thymustumoren gibt sind diagnostische und prognostische Marker dringend

notwendig.

“Heat shock proteins (HSPs)” oder auch “Hitze Schock Proteine” werden

durch eine Vielzahl unterschiedlicher Stressoren hochreguliert und wirken

intrazellulär als molekulare Chaperone sowie als Inhibitoren der Apoptose. Bisher

konnte eine Hochregulation von HSPs in der Mehrzahl von Malignomen gezeigt

werden und durch Hemmung von HSPs konnte das Tumorwachstum sowie die

Wahrscheinlichkeit von Metastasen verringert werden. Wir vermuten, dass HSPs,

vor allem die am stärksten induzierbaren (HSP27 und 70) auch bei Thymustumoren

hochreguliert sein könnten. Das Ziel dieser Arbeit ist es zu klären, ob HSPs an der

Pathogenese von epithelialen Thymustumoren beteiligt sind und ob HSPs als

diagnostische und/oder prognostische Marker für PatientInnen mit Thymustumoren

verwendet werde könnten. Deshalb haben wir immunohistochemische Färbungen

von HSP27 und 70 in PatientInnen mit epithelialen Thymustumoren (n=101) und

PatientInnen mit benignen Thymuspathologien (n=24) durchgeführt. Weiters haben

wir die HSP27 und 70 Konzentrationen im Serum von 46 PatientInnen mit

Thymustumoren, 33 PatientInnen mit benignen Thymuspathologien und 49

gesunden Kontrollen untersucht.

Die Mehrzahl der Thymustumore exprimierte HSP27 und 70, wobei jedoch

die Intensität vom histologischen Subtyp und Tumorstadium abhängig war und

schwache HSP Expression mit signifikant früheren Tumorrezidiven verbunden war.

Weiters konnten wir zeigen, dass die HSP27 und 70 Proteinkonzentrationen im

Serum von PatientInnen mit Thymustumoren signifikant erhöht waren im Vergleich

zu gesunden Kontrollen und dass die höchsten Serumkonzentrationen bei Tumoren

mit fortgeschrittenen Stadien auftraten. Interessanterweise waren nach kompletter

Tumorentfernung die HSP Serumkonzentrationen deutlich reduziert, was auf eine

Verbindung zwischen systemischen “Stress-Proteinen” und Thymustumoren

schließen lässt. Obwohl unsere Daten darauf hindeuten, dass HSPs in die

Pathogenese von epithelialen Thymustumoren involviert sind, muss deren Rolle als

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diagnostische, prognostische oder sogar therapeutische Marker weiter untersucht

werden.

Publication arising from the thesis Janik S, Schiefer AI, Bekos C, Hacker P, Haider T, Moser J, Klepetko W, Müllauer

L, Ankersmit HJ, Moser B. HSP27 and 70 expression in thymic epithelial tumors

and benign thymic alterations: diagnostic, prognostic and physiologic implications.

Sci Rep. 2016 Apr 21;6:24267. doi: 10.1038/srep24267.

Abbreviations AChR Acetylcholine Receptor

ADC Adenocarcinoma

AIRE Autoimmune Regulator

APC Antigen Presenting Cells

ChT Chemotherapy

CMJ Cortico-Medullary Junction

CSS Cause-Specific Survival

CT Computed Tomography

cTEC Cortical Thymic Epithelial Cells

DC Dendritic Cell

DISC Death Inducing Signaling Complex

DN Double Negative Thymocytes

DP Double Positive Thymocytes

EC Endothelial Cells

EOMG Early-Onset MG

EPP Extrapleural Pneumonectomy

ESTS European Society of Thoracic Surgeons

FADD Fas Associated Death Domain

FFR Freedom From Recurrence

FTH Follicular Thymic Hyperplasia

GC Germinal Center

GCT Germ Cell Tumor

HC Hassall’s Corpuscles

HSPs Heat Shock Proteins

ITMIG International Thymic Malignancy Interest Group

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IVIG Intravenous Immunoglobulin

LOMG Late-Onset MG

LRP-4 Lipoprotein-Related Protein 4

MEN Multiple Endocrine Neoplasia

MG Myasthenia Gravis

MGFA Myasthenia Gravis Foundation of America MHC Major Histocompatibility Complex

MNT Micronodular Thymoma

MRI Magnet Resonance Imaging

mTEC Medullary Thymic Epithelial Cells

MUSK Muscle-Specific Kinase

NET Neuroendocrine Tumors

NK-cells Natural Killer Cells

NMJ Neuromuscular Junction

OS Overall Survival

PET Positron Emission Tomography

PORT Postoperative Radiotherapy

RT Radiotherapy

SCC Squamous Cell Carcinoma

SNMG Seronegative Myasthenia Gravis

SP Single Positive Thymocytes

TAMG Thymoma Associated Myasthenia Gravis

TC Thymic Carcinoma

TCR T-cell Receptor

TEC Thymic Epithelial Cells

TGF Tumor Growth Factor

TH Thymic Hyperplasia

Th-cell T-helper Cell

TLR Toll-like Receptor

TNET Thymic Neuroendocrine Tumor

TNF Tumor Necrosis Factor

TRADD TNF Receptor Associated Death Domain

TRAIL TNF-related Apoptosis Inducing Ligand Receptor

Tregs Regulatory T-cells

TTH True Thymic Hyperplasia

VATS Video-assisted Thoracoscopy

WHO World Health Organization

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Chapter I: Scientific Background 1. Thymus 1.1 Introduction The thymus is a bilobed organ, which is located in the anterior upper mediastinum,

in front of the great vessels (e.g. superior vena cava or pulmonary trunk), on top of

the heart and behind the sternum.1 The thymus and bone marrow represent primary

lymphatic organs, where lymphocyte maturation takes place. T-cell maturation, also

known as “thymopoiesis”, takes place in the thymic gland, whereas B-cells develop

in the bone marrow. Thymopoiesis is the result of close symbiosis between

thymocytes (=early lymphocytes) and thymic epithelial cells (TECs). It is noteworthy

to mention that the terms thymocytes or early T-cells are used synonymously.

According to localization TECs can be differentiated into an outer cortical region,

which contains cortical TECs (cTECs) and into a central medullary region, which

contains medullary TECs (mTECs). The specificity as well as localization of TECs is

important to provide a highly specific three-dimensional framework that is crucial for

establishment of mature and self-tolerant T-cells.1,2 The process of T-cell maturation

is shortly summarized in the following.

1.1.1 Physiology Briefly, T-cell progenitor cells, originating from hematopoietic stem cells that are

localized in the bone marrow, reach the thymic gland via blood flow and enter the

thymus through large vessels in the corticomedullary junction (CMJ). At this point of

maturation, thymocytes do not express mature T-cell markers (either CD4 or CD8

and CD3); hence these early T-cells are termed double negative (DN).3 Within

further maturation DC cells migrate from the CMJ through the outer cortex, where

they form T-cell receptors (TCRs=CD3 pos.), which are membrane bound

heterodimer receptors that are composed of two polypeptide chains (αβ or γδ).

Constant TCR gene rearrangement enables mature T-cells to specifically recognize

diverse antigens and dysregulation of this rearrangement may result in impaired T-

cell function. Development of TCR (CD3+) further triggers proliferation and

maturation from DN T-cells into CD4 and CD8 double-positive (DP) cortical

thymocytes3, which subsequently undergo positive selection. Positive selection is a

specific process in T-cell maturation that permits survival only for those T-cells that

13

recognize major histocompatibility complexes (MHC) I or II on cTECs. According to

that positive selection warrants that only those T-cells will undergo further

maturation that are able to recognize self-antigens and therefore which will be able

to fulfill their distinct immunological functions. Additionally, depending on the affinity

of DP cells to bind class I or II MHC complexes, DP cells are going to differentiate

either into single positive (SP) CD8+ or CD4+ cells, respectively. Those cells that fail

positive selection are going to be removed.3 SP cells further migrate back to thymic

medulla, where they undergo negative selection. Negative selection is the second

specific and crucial selection process in T-cell maturation, takes place in thymic

medulla and is triggered by mTECs and dendritic cells (DCs). Both mTECs and

DCs function as antigen-presenting cells (APCs) that present the vast majority of

self-antigens in order to induce self-tolerance. Conversely to positive selection,

where thymocytes are only removed if they are not able to recognize MHC

complexes, at negative selection all thymocytes are removed which are not self

tolerant and therefore potentially autoimmune. In literature the autoimmune

regulator AIRE is considered as main transcription factor that regulates the

expression of diverse tissue-specific antigens4, which are not expressed normally in

the thymus, such as kidney or liver antigens and which therefore enables the

induction of self-tolerance against a large amount of self-antigens. Those SP T-cells

that are reactive to self-antigens are going to be removed (negative selection) and

finally only those T-cells who survived positive and negative selection will be

released as naïve T-cells into the periphery (Fig. 1).

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Figure 1: Thymic Histology and schematic illustration of T-cell maturation (adapted from Gorgon et al.)1. Hematoxylin-Eosin staining of a juvenile thymus is shown. M

indicates thymic medulla, C indicates thymic cortex and asterisk marks Hassall’s

corpuscles (A; 200xmagnification). In figure 1B (400x magnification) arrows indicate

corticomedullary junction (CMJ). Schematic illustration of T-cell maturation is shown in

figure 1C.T-cell progenitor cells (PCs) enter the thymus through vessels in the CMJ (1).

Double-negative (DN) cells proliferate and migrate to the cortex (2), where gene

rearrangement of T-cell receptor (TCR) and differentiation into double-positive (DP) cells

take place (3). DP cells undergo positive selection and differentiate into single-positive (SP)

cells (4). SP cells further migrate to the thymic medulla where they undergo negative

selection (5). Finally naïve T-cells that passed positive and negative selection are released

to the periphery via vessels in the CMJ (6).

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1.1.2 T-cells These naïve T-cells contribute to the peripheral pool of T-cells and circulate through

secondary lymphatic organs, mainly lymph nodes, where they bind antigens, get

activated and develop to effector T-cells. Detailed information regarding activation

of T- effector cells and their certain functions for the innate and adaptive immune

system are beyond the scope of this work and are only described briefly below.

Basically, T-cells are differentiated into two main subtypes including (1)

CD3+CD4+CD8- T-helper (Th) cells and (2) CD3+CD4-CD8+ cytotoxic T-cells. More

than two decades ago two subsets of Th cells, Th1 and Th2 cells were identified

based on the secretion of different cytokine profiles.5 Th1 cells are characterized by

secretion of interferon (IFN) γ and tumor necrosis factor (TNF) α, leading to

increased activation of macrophages, phagocytosis and are therefore essential for

cell-mediated immune responses, while Th2 cells secrete interleukin (IL)-4, -5 and -

13, which in turn are important for B-cell activation, immunoglobulin production and

are therefore crucial for humoral-mediated immunity.6 Since the discovery of the

first Th subsets several further subsets have been identified, all characterized by

unique cytokine profiles that play critical roles in effector response and immune cell

differentiation. Nowadays, Th cells can be differentiated into following subsets: Th1,

Th2, Th17, regulatory T-cells, Th22 and Th9.7 Th17 cells are characterized by the

secretion of proinflammatory IL-17, which leads to recruitment of neutrophils,

activation of innate immune cells and overall release of further proinflammatory

cytokines, such as TNF.8 Conversely, regulatory T-cells or Tregs are main

modulators of immune reactions in the periphery, induce tolerance by secretion of

IL-10 and tumor growth factor (TGF) β and therefore Tregs are crucial to prevent

overwhelming immune reactions.9 Finally, Th22 and Th9 are recently defined and

less known subsets compared to the better known Th subsets (Th1, 2, 17 and

Tregs). Indeed, the functions of these new subsets are widely unknown and further

investigations are necessary for a better understanding of these T-effector cells.7

Last but not least, CD8+ cytotoxic T-cells represent the second main subtype of T-

cells. Cytotoxic T-cells recognize peptides and antigens that are presented by MHC

class I receptors, which are expressed by all nucleated host cells, whereas MHC

class II receptors that are recognized by Th cells are only expressed by APCs. In

case of viral infection or other intracellular pathogens, such as cancer, foreign

antigens are processed by the affected cells and presented at the MHC Class I

16

receptors, which in turn are recognized by cytotoxic T-cells that induce apoptosis of

the affected cells.10

Figure 2: Schematic illustration of T-cell subsets (adapted from Raphael et al. and Russ et al.) 7,11. Immature precursor cells from the bone marrow enter the thymus and

develop to naïve T-cells (see also Fig.1). Depending on affinity to binding Major

Histocompatibility Complex (MHC) I or II, T-cells develop either to cytotoxic CD8+ T-cells or

to CD4+ T-helper (Th) cells. After activation of naïve Th cells by antigen presenting cells

(APCs) Th subsets develop from naïve T-cells depending on certain cytokine signals.

Depending on these cytokines, following Th subsets with different functions can be

differentiated: Th1, Th2, Th17, Th9, Th22, regulatory T-cell (Tregs).

1.1.3 Histology Interestingly, swirled epithelial cells, known as Hassall’s corpuscles (HCs), are

located in thymic medulla and were first described in human thymus more than 150

years ago.12 These characteristic and organotypical cells are commonly used as

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diagnostic criteria for identifying thymic tissue, but although HCs are known since

quite a long time, their exact function remains elusive. However, one hypothesis is

that HCs are involved in both negative selection of T-cells with removal of apoptotic

thymocytes as well as in positive selection with maturation of thymocytes. Another

hypothesis is that HCs do express thymic stromal lymphopoietin, which has been

identified as trigger for DC mediated positive selection of CD4+CD25+ regulatory T-

cells13 and therefore HCs are important for maturation of Tregs. Moreover, Wang et

al. (2012) investigated the development of mTECs and has observed that mature

mTECs express AIRE only once during a limited period of 1-2 days, followed by

decreased AIRE expression and accompanied by downregulation of MHC receptors

and finally results in loss of nuclei and formation of HCs.14 Hence, it seems most

likely that HCs represent the final stage or remnants of mTECs.

Another interesting fact of the thymus is that it undergoes characteristic

age-related changes, which are characterized by gradually replacement of thymic

parenchyma by fatty tissue and connective tissue15 and is termed thymic involution.

Indeed thymic parenchyma starts to decrease already from the first year of life15

and decreases 3% per year through middle age (35-45 years) and 1% per year

through the rest of the life.16 This characteristic involution of thymic parenchyma

with simultaneous expansion of fatty tissue and stroma, results in shrinking of

thymic parenchyma below 10% of initial thymic tissue at birth.17 The reasons for

thymic involution are largely unknown, but however reduction of thymic output of

naïve T-cells18 and peripheral T-cell pool, as consequence of thymic involution19,

has been linked to increased mortality in elderly people due to increased infections,

autoimmune diseases and certain malignancies.20 Remarkably, despite this

phenomenon of “immunosenescence”, which leads to the assumption of higher

rates of autoimmune diseases in elderly persons, the percentage of patients with

autoimmune diseases is fortunately not overwhelming and indicates that though

thymic involution is a characteristic age-related change of thymus, the impact and

influence on the immune system is limited.

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Figure 3: Thymic Involution. A well-defined thymus with regular architecture, clear-cut

lobulation, complete cortex and thin fibrous interlobular septa of a 14-year-old child is

shown (A). Thymus of a 27-year-old woman is shown (B), which shows loosening of

interlobar septa, increase of fat tissue and partial loss of thymic cortex with contact of

thymic medulla and fat tissue. Further, thymus of a 44-year-old male (C) and 57-year-old

woman (D) are shown that are characterized by more pronounced fatty involution and

nearly complete loss of cortico-medullar diversity. (100x magnification; Hematoxylin-Eosin

staining). (Courtesy from Leonhard Müllauer and created according to Ströbel et al.)2 1.2 Thymic Epithelial Tumors 1.2.1 Introduction Malignancies arising from thymic epithelial cells are classified as thymic epithelial

tumors (TETs), account for 30-50% of mediastinal tumors in adults and represent

also the most common mediastinal malignancies in adults.21,22 Nonetheless, TETs

are rare malignancies with incidences ranging from 1.3 to 3.2 per 1.000.000 person-

years.23,24 TETs generally occur in adults between the ages 50 and 60 without any

sex predominance.25,26 Interestingly, TETs show a unique and exceptional

19

association with paraneoplastic autoimmune disease Myasthenia Gravis (MG),

which is characterized by fatiquable muscle weakness and occurs in up to 30% of

patients with thymic malignancies.27 In such cases, patients are typically younger

between the age of 20 and 30 with a female predominance (see also 1.4

Myasthenia Gravis).

1.2.2 Clinical Presentation and paraneoplastic syndromes About one-third of patients with TETs are asymptomatic, while the other two-thirds

suffering from symptoms at time of diagnosis. These symptoms mainly result either

from local mediastinal tumor compression or infiltration of surrounding mediastinal

structures or from paraneoplastic syndromes.28 Local symptoms include chest pain,

cough, shortness of breath, superior vena cava syndrome, paralysis of diaphragm

(due to phrenic nerve involvement) and hoarseness (due to recurrent laryngeal

nerve infiltration). In case of pleural tumor spread pleural effusion and chest pain

are described frequently.28

As already mentioned, TETs are frequently associated with paraneoplastic

syndromes, mainly MG, which can be detected in 30-50% of patients with TETs,

whereas in turn TETs were found in 10-15% of patients with MG.27-29 Beside MG,

patients with TETs are associated with Pure Red Cell Aplasia and

Hypogammaglobulinemia (Good’s Syndrome) in up to 5% of cases, Systemic Lupus

Erythematosus (SLE) in 1.5-2% of patients with TETs and occasionally with

Syndrome of Inappropriate Antidiuretic Hormone secretion (SIADH), neuromyotonia

and polymyositis, pernicious anemia, Hashimoto’s thyroiditis or hemolytic anemia.30-

38 According to that there is an obvious association between thymic malignancies

and paraneoplastic syndromes, especially MG, which often leads often to detection

and diagnosis of thymic tumors. It’s assumed that the development of thymic

tumors is accompanied by impairment of physiologic T-cell maturation and

elimination of autoreactive T-cells, which in turn fosters the initiation of autoimmune

diseases.31

1.2.3 Histologic classification of TETs TETs represent a heterogenic group of tumors and histologically, TETs are biphasic

tumors, which are composed of epithelial and lymphocyte components.39 Based on

fundamental histomorphologic differences, reflecting also their different oncologic

20

behavior, TETs are differentiated into thymomas, thymic carcinomas (TCs) and

thymic neuroendocrine tumors (TNETs). The WHO classification is the most widely

used histologic classification system to differentiate Thymomas and TCs and is

based on the proportion of lymphocyte component and the morphological features

of the neoplastic TECs.40,41 Initially, TNETs were classified as subtype of TCs but

due to their different clinical and biological behavior as well as due to their different

cells of origin, TNETs have been separated in later modifications and now represent

a separate tumor entity.42,43 Thymomas and TCs represent the vast majority of

TETs, while TNETs comprise for less than 5% of TETs.44

Thymoma According to WHO classification thymomas are divided into two

major types: type A thymomas, which are characterized by spindle-or oval-shaped

nuclei with uniform cytology and type B thymomas, which have predominantly round

or polygonal appearances with variable lymphocyte components.26 WHO type B

thymomas are further differentiated into B1, B2 and B3 thymomas depending on the

amount of non-neoplastic lymphocytes on the one hand, and otherwise on the

morphology and degree of atypia of neoplastic TECs. Per definition, WHO type B1

thymomas show the largest amount of lymphocytes with only scant tumor cells with

less atypia, whereas B3 thymomas are characterized by minor lymphocyte

components, medium-sized tumor cells with more atypia, but still with organotypical

features.26,42 Thymomas showing type A and B features are classified as type AB

thymomas. Interestingly, although the WHO classification classifies TETs only

based on histologic and morphologic features, it reflects also their clinical and

oncologic behavior. For instance, WHO type A thymomas are characterized by a

more benign clinical behavior, almost indolent growing with less invasive courses

and 5 to 10 year overall survival rates from 80-100%, while B2 or B3 thymomas are

characterized by more aggressive behavior with pleural dissemination in up to 25%

and distant metastases in approximately 15% of cases.26,45-49 The most common

histologic subtypes are AB thymomas and B2 thymomas with 19-60% of cases,

while type A and B1 thymomas are counted in only 3.5-21% of cases.45,50-52 An

overview of the WHO classification criteria is given in table 1.

21

Table 1: WHO Classification of Thymic Epithelial Tumors (adapted from Rosai et al.)41. Type Definition Thymoma

A Tumors that are composed of neoplastic thymic epithelial cells, characterized by spindle/oval shape, lacking nuclear atypia and accompanied by few or no non-neoplastic lymphocytes

AB Tumors comprising both features of type A thymomas admixed with foci rich in lymphocytes. The segregation of these two patterns can be indistinct or sharp

B1 Tumors resemble the normal functional thymus and contain large numbers of cells that have an appearance almost indistinguishable from normal thymic cortex with areas resembling thymic medulla.

B2 Tumors in which neoplastic epithelial components appear as scattered plump cells with vesicular nuclei and distinct nucleoli among a heavy population of lymphocytes; perivascular spaces are common and resulting in a characteristic palisading effect.

B3 Tumors are composed of round or polygonal shaped neoplastic epithelial cells and exhibiting nor or mild atypia. They are admixed with minor components of lymphocytes, resulting in a sheet like growth of neoplastic epithelial cells.

Thymic Carcinoma TC Tumors exhibiting clear-cut atypia and a set of cytoarchitectural features no longer

specific to the thymus, but rather analogous to those seen in carcinomas of other organs. Thymic carcinomas lack immature lymphocytes and those that may be present are mature and usually admixed with plasma cells. Thymic Carcinomas (TCs) are, per definition, malignant epithelial tumors

with clear-cut atypia, absent organotypical (thymus-like) features and almost

invariably invasiveness.26,42 According to histologic differentiations TCs can be

further classified into 10 main subtypes, including squamous cell carcinoma (SCC),

basaloid carcinoma, mucoepidermoid carcinoma, lymphoepithelioma-like

carcinoma, clear cell carcinoma, sarcomatoid carcinoma, adenocarcinomas, NUT

(Nuclear protein in testis) carcinoma, undifferentiated carcinoma and other rare TC

subtypes.43 Among TCs, SCC is the most common subtype, which accounts for 76

to 79% of cases.53,54 Recently, our working group identified a new subtype of thymic

adenocarcinomas with mostly mucinous morphology and association with thymic

cysts. We propose that these thymic adenocarcinomas with enteric differentiation

represent a novel subtype, which might be helpful to differentiate between primary

thymic malignancies from metastatic diseases, especially from the gastrointestinal

tract.55

Initially, TCs were classified as type C thymomas to underline their thymic

epithelial origin, but subsequently this term has been eliminated again in later

classifications due to their different histologic and molecular patterns and their more

aggressive clinical and prognostic behavior of TCs compared to thymomas.42,53

22

Generally, TCs tend to represent at advanced tumor stages, more aggressive tumor

behavior, earlier tumor recurrences, shorter overall survival and worse prognosis

compared to thymomas. Two large retrospective studies, which have investigated

more than 1300 thymomas, observed infiltration of surrounding organs/tissues or

metastases in 27.1-38% of thymomas, while a comparable study of 1042

investigated cases of TCs found infiltration or metastases in up to 78% of

TCs.50,52,54 Additionally, 92.8% and 90.5% 5- and 10-year overall survival rates have

been reported for thymomas, compared to 60% and 40% 5- and 10-year overall

survival rates for patients with TCs.50,54 Once more, MG was principally missing in

patients with TCs, whereas MG is quite common in patients with thymomas.56

Thymic Neuroendocrine Tumors (TNETs) arise from thymic neuro-

endocrine cells, a minor cell population scattered within the normal thymus and

were counted priory to the group of TCs but have been later classified as own tumor

entity.44 Typically, TNETs are highly aggressive malignancies that occur

predominantly in male adults. Five – and 10-year overall survival rates of 79% and

41% have been reported, which is by comparison similar to overall survival for

patients with TCs.57,58 Histologically, TNETs are not distinguishable from

neuroendocrine tumors (NETs) origin from other organs, such as gastrointestinal or

pulmonary neuroendocrine tumors. Thus TNETs are differentiated in accordance to

the nomenclature of neuroendocrine tumors of the lung into two main subgroups:

(1) well – differentiated TNETs or Carcinoid Tumors and (2) poorly - differentiated

TNETs.42-44 Poorly - differentiated TNETs are further separated into small cell

carcinoma and large cell neuroendocrine carcinoma, whereas carcinoid tumors are

further divided into typical and atypical carcinoids. An overview of the classification

and characteristics of TNET subtypes are shown in table 2.

23

Table 2: Classification of Thymic Neuroendocrine Tumors (adapted from Travis et al.

and Marx et al.)42,43.

TNETs Subtypes Characteristics Poorly -

differentiated

Small Cell Carcinoma

- Small round, oval or spindle-shaped cells - Scant cytoplasm and prominent nuclear

molding - Well - defined cell borders - Granular nuclear chromatin - Indistinguishable from small cell carcinoma

arising from the lung Large Cell

Neuroendocrine Carcinoma

- Large cells with neuroendocrine morphology (palisading, trabeculae, nesting or rosette - like features)

- Extensive necrosis - High mitotic rates (>10 mitosis per HPF) - Positive neuroendocrine markers or

neurosecretory granules in electron microscopy

Well - differentiated or

Carcinoid Tumor

Typical Carcinoid - Polygonal cells with granular cytoplasm arranged in ribbons, festoons, solid nests and rosette-like glands

- < 2 mitosis per 10 HPF - No necrosis

Atypical Carcinoid - Necrosis present - 2-10 mitosis per HPF

TNETs, thymic neuroendocrine tumors; HPF, high power fields

It is noteworthy to mention that 30 to 50% of patients with TNETs commonly

suffer from endocrinopathies, mainly ectopic adrenocorticotropic hormone secretion

(Cushing Syndrome) and the hereditary tumor syndrome multiple endocrine

neoplasia type 1 (MEN-1), while paraneoplastic MG is typically missing.28,44 In turn,

8% of MEN-1 cases are accompanied by TNETs.59

Summarized, TETs represent a quite heterogeneous group of tumors that

can be classified into three main types (thymomas, TCs and TNETs), which are

characterized by different clinical behavior and prognosis. Although the WHO

classification is most widely used to classify TETs, there could be morphological

continuum between some thymomas and TCs, which could cause problems in

classification and causes poor interobserver reproducibility. Due to that the WHO

criteria are periodically reevaluated and redefined by specified pathologists in order

to take into account latest research results and to increase the degree of

reproducibility.40

24

Figure 4: WHO Classification of Thymic Epithelial Tumors. Hematoxylin-Eosin staining

of WHO type A (A), B1 (B), B2 (C), B3 (D), TC (E) and TNET (F) is shown. Arrows indicate

neoplastic epithelial cells within thymic medulla of a B1 thymoma. (200x magnification in A,

B, C, F and 400x magnification in D and E). TC, thymic carcinoma; TNET, thymic

neuroendocrine tumor. (Courtesy from Leonhard Müllauer).

25

Table 3: Thymic Epithelial Tumors (adapted from Marx et al.)43.

In contrast to highly aggressive, commonly infiltrative growing TCs and

TNETs and lack of paraneoplastic MG, thymomas are generally characterized by

slowly indolent growing with excellent prognosis. However, despite the mostly

indolent, non-invasive growing of thymomas, they should not be considered as

being benign due to their invasive and metastatic potential and should be treated

with the same radicalism as TCs and TNETs.

1.2.4 Masaoka-Koga staging system Initially, several different staging systems have been used for the clinical

classification of TETs. The Masaoka system and/or the Koga modification of the

Thymoma Type A thymoma (including atypical variant) Type AB thymoma Type B1 thymoma Type B2 thymoma Type B3 thymoma Other rare thymomas (micronodular thymoma, metaplastic thymoma, microscopic thymoma)

Thymic Carcinoma Squamous cell carcinoma Basaloid carcinoma Mucoepidermoid carcinoma Lymphoepithelioma-like carcinoma Clear cell carcinoma Sarcomatoid carcinoma Adenocarcinomas

Papillary adenocarcinoma Thymic carcinoma with adenoid cystic carcinoma-like features Mucinous adenocarcinoma Adenocarcinoma, NOS (not otherwise specified)

NUT carcinoma (nuclear protein in testis) Undifferentiated carcinoma Other rare thymic carcinomas

Thymic Neuroendocrine Tumors Well-differentiated TNETs (Carcinoid tumors)

Typical carcinoid Atypical carcinoid

Poorly – differentiated TNETs Small-cell carcinoma Large-cell neuroendocrine carcinoma

26

Masaoka system respectively, the French Group d’Étude des Tumeurs Thymiques

system and a TNM system were used most commonly.60-63

Nonetheless, most centers used the Koga modification of the Masaoka

system (Masaoka-Koga staging) in their reports and thus the Masaoka-Koga

staging developed to the mainly used clinical staging system of TETs and is so far

also recommended by the International Thymic Malignancy Interest Group

(ITMIG).64 The ITMIG is an organization, which has been established in 2003 by

patients suffering from thymic malignancies and their family members and has been

developed to a multispecialty organization involving more than 600 experts,

comprising thoracic surgeons, oncologists, pulmonologists, radiologists,

pathologists, neurologists and basic science researchers.65 These days research on

thymic malignancies is mainly fostered by the ITMIG and the thymic working group

of the European Society of Thoracic Surgeons (ESTS)

Nowadays TETs are staged according to the Masaoka-Koga staging system

that differentiates 4 major stages depending on the level of invasiveness (Table 4;

Fig. 5).

Table 4: Masaoka-Koga staging system (adapted from Detterbeck et al. with comments of the International Thymic Malignancy Interest Group) 66. Stage Definition

I Microscopically completely encapsulated tumor including tumors with invasion into but not through the capsule or tumors where the capsule is missing with no invasion into surrounding organs

II a Microscopic transcapsular invasion II b Macroscopic invasion into thymic or surrounding fatty tissue, or grossly

adherent but not breaking through mediastinal pleura or pericardium gross visual tumor extension into the surrounding fatty tissue and/or adherence but not invasion into neighboring organs or pleura

III Macroscopic invasion into neighboring organs including microscopic invasion into mediastinal pleura, pericardium and direct penetration of visceral pleura or into lung parenchyma; invasion into or penetration of major vascular structures and/or phrenic or vagus nerves

IV a Pleural or pericardial metastases microscopically confirmed nodules, separated from the primary tumor (not per continuitatem), including visceral, parietal, diaphragmal pleura and pericardium

IV b Lymphogenous or hematogenous metastases any nodal involvement (intrathoracic), pulmonary parenchymal nodules (not a pleural implant) and any nodal involvement outside the thorax (extrathoracic metastases)

27

In stark contrast to widely used TNM staging systems, the Masaoka-Koga

staging system primarily focuses on the local extension and invasion of tumor, while

involvement and infiltration of lymph nodes is from less importance. Recently, the

validity of TNM staging in TETs in comparison to the commonly used Masaoka-

Koga system was analyzed for 154 cases of TETs and reevaluated for prognosis

and outcome. Interestingly, 98% of WHO type A, AB, B1 thymomas (68 out of 69

cases) were restaged as stage I tumors, whereas type B2, B3 thymomas and TCs

were more frequent staged as stage III and IV tumors. Altogether, though TNM

staging systems seem to reflect the biological behavior of different WHO tumor

subtypes, it doesn’t serve as prognostic marker.67 Further studies are definitely

needed to evaluate whether the well-established and widely accepted Masaoka-

Koga staging or a new TNM staging is more appropriate for staging TETs and for

predicting outcome and tumor behavior. For 2017, new data of the ITMIG in

cooperation with IASCL (International Association for the Study of Lung Cancer) are

expected for a TNM based staging system of TETs.

However, the Masaoka-Koga staging is easy to perform, provides excellent

reproducibility, predicts outcome and represents altogether a strong independent

prognostic factor for patients with TETs (see also prognostic factors in TETs).45,68

28

Figure 5: Masaoka-Koga Staging System (Detterbeck FC et al.)66. Reprinted and

adapted with permission from Elsevier.

29

1.2.5 Diagnostic Workup in TETs A) Radiologic examination – CT, MRI, PET Principally, in two thirds of cases TETs present as asymptomatic indolent growing

tumor and symptoms, such as chest pain, hoarseness, dyspnea, just result from

tumor invasion or compression of surrounding organs, corresponding to advanced

tumor stages (Masaoka-Koga stage III-IV).28 Conversely, due to the known fact that

MG represents the most common paraneoplastic syndrome in TETs, those patients

that present with symptoms of paraneoplastic MG (approximately 30% of cases)

typically diagnosed at earlier tumor stages, which is associated with much better

prognosis.27,45 Hence, the well-known association between MG and thymic

pathologies leads to proper and fast screening of thymic tumors in all patients with

muscle weakness and enables early diagnosis of TETs at smaller tumor sizes,

lower Masaoka-Koga tumor stages and in turn correlates with higher rates of

completeness of resection, which represents a favorable prognostic factor for

survival and freedom from recurrence.45,50

Computed Tomography (CT) represents the first choice technique for

investigating mediastinal masses, regarding their distribution and invasion into

neighboring structures.69 Although chest X-ray provides several advantages, such

as low-radiation dose, low price and availability and could detect also 45-80% of

thymomas, it nonetheless lacks regarding sensitivity of smaller mediastinal tumors

and specificity to differentiate TETs from other mediastinal tumors.70,71 Due to the

great sensitivity, CT should be always performed whenever TETs or other

mediastinal tumors are clinically suspected.71

Generally, TETs have some characteristic CT appearances that help to

differentiate TETs from other tumors that can be found in the anterior mediastinum.

First, TETs are closely related to the superior pericardium. Second, TETs are

principally well defined, round or lobulated, homogenous and enhances after

application of contrast medium. Third, TETs are usually round masses with

diameters ranging from few millimeters up to 34 cm at time of diagnosis.70 Fourth,

TETs are rarely present with metastatic lymphadenopathy or with metastatic

pulmonary nodules compared to other malignancies. Nonetheless, staging of TETs

by CT remains challenging but is crucial for planning surgical procedures. A recent

review based on 129 cases of TETs could show significant correlations between

radiologic features, such as tumor size, tumor shape, tumor density, completeness

30

of capsule, involvement of surrounding tissues and pathologic Masaoka-Koga tumor

stage and confirms once more the high utility of CT in the diagnostic workup of

TETs.72 According to significant differences in CT, such as location of mediastinal

mass with respect to midline, morphology, homogeneity of attenuation, fatty

intercalation, coexisting lymphadenopathy, circumscription, mass effect and

Hounsfield Units, it is even possible to differentiate between TETs, lymphomas and

benign thymic alterations.73 Further CT could help to identify those patients who

might benefit from surgical thymectomy from those who won’t benefit.73

Magnetic resonance imaging (MRI) is rarely used in diagnostic workup of

TETs compared to CT but MRI is of course valuable in selected cases and

circumstances. Principally, the role of MRI in identification of TETs is limited due to

non-specific features of TETs in MRI and the availability of CT as excellent

diagnostic tool. Nonetheless, an Italian workgroup could recently show that the

apparent diffusion coefficient predicts both the WHO classification as well as the

Masaoka-Koga staging system and has significant prognostic impact on disease-

free survival.74 It is already known that heterogeneity of TETs is accompanied by a

more aggressive tumor behavior and tumor stage. However, heterogeneity of TETs

could not be used as characteristic feature for differentiation between invasive and

non-invasive TETs, because there exists a great overlap and spread between

different TET subtypes.75 To sum up, the use of MRI in diagnosis of TETs is limited

but there are some undeniable benefits of MRI compared to CT, such as lack of

radiation, more appropriate investigation of vessels can be done without using

contrast medium and of course its superior contrast resolution. Disadvantages of

MRI are poor resolution of lung parenchyma and relative length. Thus the main

value of MRI in TETs lies in its value for surgical planning, especially if infiltration of

surrounding organs, including great vessels or heart is suspected.71

Positron emission tomography (PET) The value of nuclear medicine in

examination of mediastinal tumors is limited. In the last decades, PET scan,

particularly in combination with CT has been widely used for staging in different

malignancies. It could be shown that the amount of tracer uptake, called

standardized uptake value (SUV), is higher in TCs compared to thymomas and in

high-grade compared to low-grade tumors. Unfortunately, SUV uptake between

low- and high-grade tumors could be overlapping and makes a general use of PET

for differentiation of TETs difficult.76-79 Clinically, PET in combination with CT (PET-

31

CT) is commonly performed for staging TETs, especially in cases of more

aggressive thymomas and TCs where metastases are possible and/or suspected.71

Altogether, CT represents the standard tool for diagnostic workup of

mediastinal tumors and MRI or PET are only used and recommended in selected

cases or for additional information. Despite radiologic examination techniques could

provide additional, useful information on the origin of tumor and its distribution,

histologic examination and workup is nonetheless necessary.

Figure 6: Examples for radiologic examination techniques in Thymic Epithelial Tumors. Chest X-rays with corresponding chest CT scans of patients with WHO type B2

thymoma (A, C) and thymic carcinoma (B, D) are shown. T indicates tumor. Coronal and

axial view of PET-CT scans of another patient with thymic carcinoma and infiltration of

sternum is shown (E, F). (Courtesy from Bernhard Moser).

32

B) Pathologic examination Preoperative pathologic examination is not necessary, whenever there is a strong

clinical suspicion for TET, such as the simultaneous presence of mediastinal mass

and paraneoplastic MG or suspect mediastinal mass with characteristic radiological

features typically related to TETs and of course small, easy to resect tumors in the

anterior mediastinum.80 However, beside TETs common mediastinal tumors, such

as germ cell tumors (GCTs), lymphomas and neuroendocrine tumors should be

always taken into account. In table 5 the use of serum biomarkers for diagnostic

workup of mediastinal tumors is illustrated.

Table 5: Serum Biomarkers in mediastinal tumors.

Tumor Serum Marker Comments Germ Cell Tumor

(GCT) AFP, βHCG 81-84 - AFP, βHCG provide prognostic

information on diagnosis, persistence of tumor after therapy or recurrence

- Predicts Overall Survival and Disease Free Survival

Lymphoma LDH 86

β2 – Microglobulin 85,86

- LDH is unspecific, reflects cellular turnover and proliferative activity

- β2 -Microglobulin reflects cellular turnover, directly related to tumor burden, associated with higher tumor stage, independent prognostic factor in lymphomas

Neuroendocrine Tumor (NET)

NSE 87,89,90

Chromogranin A 87-90

- NSE most important marker in poorly-differentiated NETs

- Chromogranin A best available biomarker for diagnosis of NET; predicts outcome, efficacy of therapy

- Chromogranin A most important marker in well-differentiated NETs

Thymic Epithelial Tumor (TET)

AChR 91,92 - no established biomarkers - AChR antibodies are not specific for

TETs, but if they are positive there is a strong clinical suspicion for TET

AFP, alpha-fetoprotein; βHCG, beta human chorionic gonadotropin; LDH, lactate dehydrogenase; NSE, neuron specific enolase; AChR, Acetylcholine Receptor.

Additionally to serum biomarkers, clinical data, such as patient age or sex,

localization of tumor, presence or absence of paraneoplastic syndromes or other

symptoms like fever, weight loss and night sweat should be always taken into

account for differential diagnosis of mediastinal tumors. Table 6 provides an

overview of common mediastinal pathologies according to localization, age and

clinical symptoms.

33

Table 6: Common mediastinal pathologies – a comprehensive overview (adapted from

Marchevsky et al.)93.

Pathology Localization Age Clinical symptoms TET Thymoma anterior adults

children 1/3 asymptomatic, 1/3 symptoms due to paraneoplastic MG; AChR antibodies are unspecific but commonly elevated in patients with TETs and paraneoplastic MG

TC anterior adults symptoms result from tumor infiltration or compression of surrounding organs (e.g. cough, dyspnea, pain,…)

TNET anterior adults NSE, Chromogranin A are serum biomarkers, which are frequently increased; accompanied by paraneoplastic endocrinopathies (Cushing Syndrome, MEN,…)

Benign Thymic

Pathology

TH anterior adults children

mainly associated with MG or incidental finding

Thymic Cyst

anterior adults children

mostly asymptomatic, incidental finding

Lymphoma anterior adults children

occasionally associated with fever, night sweat, weight loss (=B-symptoms); generalized lymph node enlargement; tumor marker: β2 – Microglobulin, LDH

GCT anterior children young adults

cough, dyspnea, chest pain due to tumor compression; large mediastinal masses AFP and βHCG are frequently increased

Neurogenic Tumors

posterior children adults

most asymptomatic; pain, paresthesia due to compression of nerves

Thyroid Tumors

superior anterior

adults symptoms related to thyroid enlargement, such as dysphagia, dyspnea, etc.; hoarseness due to recurrent laryngeal nerve involvement

Metastatic Tumors

anterior middle

adults anamnesis

TET, Thymic Epithelial Tumor; TC, Thymic Carcinoma; TNET, Thymic Neuroendocrine

Tumor; MG, Myasthenia Gravis; AChR, Acetylcholine Receptor; NSE, neuron specific

enolase; MEN, Multiple Endocrine Neoplasia; TH, Thymic Hyperplasia; LDH, lactate

dehydrogenase; GCT, Germ Cell Tumor; AFP, alpha-fetoprotein; βHCG, β-human chorionic

gonadotropin. 81-93

34

Nonetheless, anamnesis, clinical presentation and serum tumor markers are

at the best useful for the diagnostic workup of mediastinal tumors but couldn’t

replace the histologic evaluation or confirmation, respectively. Histologic evaluation,

preferential through minimal invasive techniques, is especially in those cases

recommended, whenever there is strong clinical suspicion of mediastinal tumors

other than TETs, which require specific therapies other than primary surgical tumor

resection.80 Another indication for histologic evaluation are TETs, which are too

extensive for curative surgical resection and which are thought to be initially better

treated by radio- and/or chemotherapy.80

However, there exist several ways to obtain tumor tissue for histologic

evaluation, including minimal-invasive techniques, such as ultrasound – or CT

guided biopsy, fine needle aspiration (FNA), needle core biopsy, endobronchial

ultrasound guided (EBUS) biopsy or more invasive techniques, such as

minithoracotomy, video assisted thoracoscopic surgery (VATS) and

mediastinoscopy.93 Basically, the type of used technique mainly depends on tumor

localization. While tumors that are located in the anterior mediastinum could be

easily verified by FNA, tumors in the posterior mediastinum are hardly reachable by

FNA or other minimal invasive approaches and open, surgical biopsies are more

appropriate.93 These surgical open biopsies (mediastinoscopy, VATS or

minithoracotomy), which are undeniable associated with more pain, higher levels of

invasiveness and longer hospital stays, provide pathologists larger tissue samples

than FNA or needle core biopsies and allow diagnosis with less difficulty.94

Nonetheless, several differential diagnostic problems could remain and make

further histologic workup necessary. Basically, additional immunohistochemical

stainings are predominantly performed to solve such typical problems, which are as

follows: (1) differentiation between Thymic Hyperplasia and Thymoma, especially if

only few neoplastic cells are distributed within normal thymus (e.g. B1 thymoma);

(2) WHO subtyping of thymomas; (3) differentiation between atypical thymomas

(mainly B3 thymomas) and TCs; (4) TCs vs. TNETs; (5) differentiation between

TETs, metastases and other primary tumors of the mediastinum.93 Selected

immunohistochemical markers for histologic workup of mediastinal tumors are

shown in table 7 and are briefly described in the following.

Thymomas, TCs and TNETs origin from thymic epithelial cells, therefore

TETs do express a variety of epithelial marker, such as CK5/6, CEA, CD15 and

35

calretinin.93 Chu PG and colleagues (2002) observed that even in case of malignant

degeneration and transformation, neoplastic epithelial cells still express their

organotypical keratin filaments. In case of TETs, cytokeratin 5/6 are typical keratins,

which could be used to identify thymic epithelial cells as cells of origin.95 TETs

comprise two components, a neoplastic epithelial cell component and a non-

neoplastic lymphocyte component. Lymphocytes express typical T-cell markers,

such as CD3, CD1a, CD99 and TdT (terminal deoxynucleotidyl transferase) and

could be used to differentiate between thymomas and TCs, which are per definition

characterized by loss of organotypical features, accompanied also by loss of T-cell

markers. Sometimes differentiation between B3 thymomas and TCs could be really

challenging and make additional immunohistochemical stainings, such as CD117

(c-KiT) necessary. CD117 is a transmembrane tyrosine kinase receptor that is

expressed in the majority of TCs (86%), while widely missing in thymomas.96

Especially the simultaneous expression of CD117 and CD5 are more often seen in

TCs compared to thymomas.93

Recently, the combination of several new markers has been assessed for

differentiation between B3 thymomas and thymic SCC. Interestingly, sensitivity of

GLUT-1 (Glucose Transporter-1) or MUC-1 (Mucin-1) was 100% for TCs and even

higher compared to CK5/6 (95%), CD117 (90%), CD5 (80%) and CEA (75%), while

specificity was 100% for CD5, CD117 and CEA but only 56.3% for MUC-1and 50%

for GLUT-1. Sensitivity of T-cell markers TdT and CD1a were 93.8% and 87.5% in

B3 thymomas with specificity of 100% and 95% for CD1a and TdT, respectively.97

Occasionally, the differentiation between atypical B3 thymomas, TCs and

TNETs on HE (Hematoxylin and Eosin) stainings could be also challenging. In such

cases, the positive expression of neuroendocrine markers, including synaptophysin,

chromogranin A and CD56, confirms easily the presence of tumors with

neuroendocrine differentiation.93,98

TH vs. Thymoma. The differentiation between TH and WHO type B1

thymomas, which are characterized by only few neoplastic cells diffusely distributed

within regular thymic architecture, could be another diagnostic challenge, especially

in biopsies. In HE-stainings, B1 thymomas are characterized by more pronounced

fibrous capsule, septation and strong predominance of cortical over medullary

thymic areas compared to TH.99 However, in cases that histomorphology could not

be clearly distinguished, p63 staining could be helpful to differentiate to identify

36

neoplastic epithelial cells.40

TETs vs. Lymphomas. Conversely to TETs, lymphomas originate from

hematolymphoid cells and don’t express epithelial markers, but of course do

express different lymphocyte markers, such as CD1a, CD3, CD20, CD99 or TdT.

Hence the absence of epithelial markers and the simultaneous expression of

lymphocyte markers are characteristic for lymphomas. Overexpression of either

mature T-cell marker CD3 or mature B-cell marker CD20 helps to distinguish

between B- and T-cell lymphomas.93 GCT. In children or young adults with mediastinal tumors, GCTs must be

always taken in consideration. Octamer-binding transcription factor 3/4 (OCT3/4) is

an established marker for GCTs and depending on tumor subtype further markers

could be positive, such as placental alkaline phosphatase (PLAP) in seminoma or

alpha-fetoprotein (AFP) in yolk-sac tumors.93,100

Primary tumor vs. metastases. Finally and probably the most important

diagnostic considerations are metastases. Clinical and radiological examinations

could provide essential information for differential diagnosis but at the end it is the

job of pathologists to find the right diagnosis based on the amount of clinical and

radiological information in combination with histomorphology and immuno-

histochemical staining patterns of tissue specimens. For exclusion of Non-Small

Cell Lung Cancer (NSCLC) TTF-1 (thyroid transcription factor-1), napsin and

surfactant apoprotein (S-AP) could be helpful. TTF-1 and S-AP are generally

expressed on respiratory epithelium and are used to distinguish malignancies with

pulmonary origin from non-pulmonary malignancies.101 Further, TTF-1, thyroglobulin

and PAX-8 are also expressed in thyroid cancers. Metastatic breast carcinomas

show positive expression for estrogen and progesterone receptors and

mammaglobulin. CD5 and EMA (Epithelial Membrane Antigen) are frequently

expressed by adenocarcinomas of extrathymic origin, for instance in colon cancer.93

In such cases, mediastinal metastases needs to be always excluded before the rare

diagnosis of primary adenocarcinoma of the thymus could be confirmed.

37

Table 7: Selected Immunohistochemical markers for the use of histologic workup of mediastinal tumors (adapted from Marchevsky et al.)93.

Cyto- keratin

CD3 CD45

CD99 TdT

CD1a CD20

CD117 CD5 EMA

CD56 Chromo. Synapto.

AFP PLAP Oct3/4

TTF-1 Surfact. Napsin

Thymoma + + + - - - - - TH + + + + - - - - TC + + - - + +/- - -

TNET + - - - - + - - Lymphoma - + + + - - - -

GCT +/- - - - - - + - Metastases +/- - - - +/- +/- - +

TH, Thymic Hyperplasia; TC, Thymic Carcinoma; TNET, Thymic Neuroendocrine Tumor;

GCT, Germ Cell Tumor; TdT, Terminal deoxynucleotidyl Transferase; EMA, Epithelial

Membrane Antigen; Chromo., Chromogranin A; Synapto., Synaptophysin; AFP, Alpha-

Fetoprotein; PLAP, Placental Alkaline Phosphatase; Oct3/4, Octamer-binding transcription

factor 3/4; TTF-1, Thyroid Transcription Factor-1.; Surfact., Surfactant.

1.2.6 Treatment of TETs – Surgery, Radiotherapy, Chemotherapy

I) Surgery – surgical approaches, debulking, lymph node resection For patients with TETs, radical surgical resection represents the mainstay of

therapy and it must be the aim of each surgeon to obtain radical tumor resection,

whenever possible.80 After complete tumor resection of Masaoka-Koga stage I, II, III

and IV TETs, excellent 10-year OS rates of 90%, 70%, 55% and 35% have been

reported, respectively.80 Remarkably, in case that radical tumor resection (R0) could

be achieved fro stage III TETs, long-term survival was comparable to patients with

stage I TETs.102 According to that, regardless of tumor stage, complete and radical

resection should be the primary aim whenever complete resection is thought to be

achievable. Hence, any effort should be done to achieve this aim, even if it requires

resection and reconstruction of great vessels (e.g. superior vena cava) or resection

of one phrenic nerve.103 Even in cases where multiple pleural nodules (Masaoka-

Koga stage IVa) are present and which couldn’t be treated by local resection, more

invasive pleural operations, including total, local or regional pleurectomy or maximal

invasive extrapleural pneumonectomy (EPP) should be performed to achieve

complete tumor resection. Our working group currently reevaluates on behalf of the

ESTS thymic working group the surgical therapy of TETs with pleural involvement.

38

In literature acceptable 5-year OS rates from approximately 75% have been

reported for TETs at Masaoka-Koga stage IVa.104-106

Numerous studies confirmed that completeness of surgical resection, which

of course depends on tumor stage, represents an independent and important

favorable prognostic factor in TETs.28,45,52,102,107,108 Rates of complete surgical

resection gradually decreases from 100% in stage I TETs to 88%, 49% and 29% in

stage II, III and IV tumors, respectively.80

However, whenever complete surgical tumor resection should be performed

it is essential to consider that the thymic gland, including the tumor, consists of two

main lobes and numerous microscopic thymic remnants, which are widely

distributed within the mediastinal fatty tissue.109 In fact the thymus with all

mediastinal tissue could range from the level of the thyroid gland superior to the

diaphragm inferior, bilaterally between both phrenic nerves and from the sternum

anterior to the pericardium and great vessels posteriorly.110,111 Hence, en-bloc

resections including the tumor and all mediastinal fatty tissue in total are strongly

recommended for surgical resection of all TETs. En-bloc tumor resection of a WHO

type B2 thymoma with infiltration of the right lung (Masaoka-Koga tumor stage III) is

shown in figure 7.

Figure 7: En-bloc resection of a WHO type B2 thymoma. Preoperative chest X-ray with

corresponding CT scan is shown in A and B. Surgical specimen of the WHO type B2

thymoma, Masaoka-Koga stage III is shown in C. T indicates tumor/thymoma. (Courtesy from Bernhard Moser).

Surgical approaches – initially, median sternotomy was recommended as

traditional and established surgical approach for thymectomy due to the fact that it

provides optimal overview and enables easy radical tumor resection. However, in

the last years, the use of minimal invasive surgery (VATS and robotic surgery) has

39

progressed with increasing numbers worldwide.112,113 As a result of this progress

many centers forward minimal invasive approaches, such as transcervical

approaches, VATS or robotic surgery, which represent safe alternatives with similar

results compared to open approaches.114 In addition, minimal invasive approaches

provide important advantages, such as significantly shorter hospital and insensitive

care unit lengths, less pain and lower estimated blood loss.115 However, minimal

invasive approaches should be only performed in stage I and II tumors, when no

invasion or infiltration of surrounding organs is suspected and when uncomplicated

complete tumor resection is preoperatively assumed. In case that involvement of

phrenic nerve, innominate vein, other great vessels or adherence/infiltration of

organs (e.g. lung, pericardium) is suspected, a primary open approach is strongly

recommended and conversion to open approach should be performed whenever

situation couldn’t be handled adequately by minimal invasive approach.114

Debulking - the favorable prognostic value of complete surgical tumor

resection in patients with TETs is unquestioned but the value of tumor debulking in

cases that could not be resected completely is doubtful. Recently, a meta-analysis

that included 13 studies with in total 314 patients assessed the impact of debulking

compared to diagnostic open biopsies for unresectable TETs. Interestingly,

debulking was occasionally associated with improved OS and should be principally

considered for all patients with unresectable TETs.116 Another meta-analysis

showed that although debulking surgery has affected survival for patients with

thymoma positively, these differences frequently didn’t reach statistical significance.

In patients with TCs, debulking surgery didn’t show any benefit on survival.117 So

far, the value of debulking surgery in TETs is still discussed controversial, while

some studies show beneficial effects on survival others do not. Nonetheless,

debulking is doubtless an option in patients within palliative settings to reduce tumor

volume in order to minimize tumor related symptoms (e.g. chest pain, dyspnea).

Further, patients who will need adjuvant chemo-and/or radiotherapy, tumor volume

reduction could help to minimize the need of high doses of chemo - and /or

radiotherapy and to lower damage to adjacent tissue, which of course could cause

new symptoms again.117 To sum up, so far no common recommendations exist and

the need and possibilities of debulking surgery should be considered for each

patient individually.

Lymph node resection – TNM staging systems are used for staging of the

40

majority of malignancies and is based on three basic characteristics, including

tumor size (T), lymph node involvement (N) and absence or presence of

metastases (M). Conversely in TETs, staging according to the Masaoka-Koga

staging systems is based only on the level of invasiveness (T factor), while

lymphogenous and hematogenous metastases (N and M factors) are not taken into

consideration yet and are classified only as stage IVb.61 Principally, extended

complete surgical tumor resection is recommended for all TETs whenever feasible,

but the role of lymph node sampling and resection at the time of resection is still

unclear.

TETs mainly metastasized to local lymph nodes in the anterior mediastinum

(thymoma, 90%; TC, 69%; TNET, 91%) and N status had significant impact on

survival. Interestingly, rate of lymphogenous metastases (N factor) was related to

tumor subtype and has been 1.8%, 27% and 28% for thymomas, TCs and TNETs,

respectively.118 Five year OS rates in thymomas were 96%, 62% and 20% for N0

(no metastases), N1 (metastases in anterior mediastinal lymph nodes) and N2

(metastases to intrathoracic lymph nodes other than N1). In TCs, the 5-year OS

rates for N0, N1 and N2 were 56%, 42% and 29%, respectively. According to that,

the prognosis of TETs tends to get worse with progression of N factor.118 Moreover,

5-year OS was also worse in thymomas and TCs with hematogenous metastases

(M1) compared to M0 tumors and were 57% to 95% in thymomas and 35% to 51%

in TCs, respectively. Despite that, M factor was no independent prognostic factor for

TETs.118

Altogether nodal status is an important prognostic factor in TETs and positive

nodules have adverse impact on OS.118,119 Lymphogenous metastases mostly

occurs in anterior mediastinum and the risk of lymph node metastases is increased

in TETs that infiltrate surrounding organs (Masaoka-Koga stage III) compared to

tumors without invasion (thymoma: 5.6% vs. <0.01%; TC: 28.8% vs. 14.8%; TNET:

37.5% vs. 12.5%).118 Accordingly, Weklser B et al. even recommends lymph node

dissection in all invasive TETs that are known to be associated with higher risks of

lymphogenous metastases, which in turn are associated with worse prognosis.

Moreover, selective lymph node dissection might result also in change of tumor

stage, which might be accompanied by more aggressive therapeutic regimes.119 For

2017 a new TNM classification for TETs is expected and will hopefully address the

value of lymph node dissection in TETs.

41

II) Radiotherapy Whereas surgery with complete tumor resection is recommended for all TETs,

whenever tumor resection could be achieved, indications and recommendations for

radiotherapy (RT) strongly depend on tumor stage.

In non-invasive TETs (Masaoka-Koga stage I), radical tumor resection is

usually curative and due to the negligible low risk of recurrence, postoperative RT

(PORT) is not indicated. Of course in case of incomplete resection, PORT should

be considered.

In invasive TETs (Masaoka-Koga stage II-IVa) PORT is principally

recommended to in order to improve freedom from recurrence and to improve

survival. The use of PORT in patients with stage II and III TETs after radical tumor

resection (R0) is still conversely discussed in literature and several studies have

been published that either supports or contradicts PORT.120-125 Recently, a meta-

analysis that examined survival data of more than 3000 patients, revealed that

PORT significantly improved OS in patients with complete resection of stage II and

III thymomas.120 Conversely a Japanese study, which included 1265 patients found

no beneficial effects for PORT on OS and relapse free survival (RFS) for stage II

and III thymomas, while RFS was significantly increased in TCs.125 Altogether, the

majority of studies showed beneficial effects of PORT in stage III TETs, while the

benefit of PORT in stage II TETs remains unclear. Hence, the ITMIG recommends

PORT for all stage III and IVa TETs regardless of completeness of resection,

whereas the use of PORT in stage II TETs should be discussed individually.65

Nonetheless, PORT is also recommended by ITMIG in stage IIB TETs, TETs with

close surgical margins, WHO type B thymomas and TETs with adherence to

pericardium.126 Minimal doses of 50 Gy over 5 weeks for PORT are recommend,

while doses above 60 Gy should be avoided due to the risk of radiation induced

injury .65,127

Induction Therapy - conversely to PORT the value of induction RT for

primarily (at diagnosis) not resectable TETs is unquestioned. It is the aim of

neoadjuvant (=induction) therapy to reduce the tumor volume to enable

completeness of surgical resection. Induction regimes may include RT alone,

chemotherapy (ChT) alone or combined RT and ChT.128-132 In case that RT may be

also an option for adjuvant therapy, it is crucial to carefully consider cumulative

radiation dose to avoid adverse events, such as bone marrow suppression.133

42

Definitive RT is considered for those patients with unresectable tumors or

those that are not fit enough for surgery due to medical comorbidities. If definitive

RT is considered alone for recurrent disease or for unresectable primary tumors,

radiation doses of 60-66Gy are recommended. In case of combined RT and ChT

only 54 Gy are recommended because ChT is known as sensitizer for RT and helps

to reduce radiation dose.127, 134-136

III) Chemotherapy There is no indication for ChT in early - stage TETs (stage I and II) that could be

resected primarily. Conversely, in primary unresectable TETs with advanced staged

(III and IVa) tumors, induction ChT is recommended for downstaging of TETs to

enable surgical resection as well as adjuvant ChT to improve outcome. Whether

induction ChT is necessary or not mainly depends on tumor extent in preoperative

imaging.137 Thus the main objective of induction ChT in advanced-local (III) or

non-metastatic (IVa) TETs is downstaging of tumor to enable radical tumor

resection. Data of retrospective studies clearly demonstrated that TETs, thymomas

more than TCs, are chemosensitive and partial response could be achieved in 40-

80% of cases.137,138 Most patients received combination ChT according to the PAC

schema (Cisplatin, Doxorubicin, Cyclophosphamid).139 Remarkably, induction ChT

led to increased radical tumor resection rates of 72% in advanced staged TETs,

compared to only 50% and 25% for primarily resected stage III and IVa tumors,

respectively.140 Typically, 2-4 cycles of primary ChT are administrated and contrast

medium enhanced CT will be performed 3-4 weeks after application of last cycle for

evaluation of tumor response. In case of tumor response, surgery should be

performed within 8 weeks after last ChT cycle.138 Nonetheless, the role and extent

of induction ChT is still unclear and no standardized protocols have been

established yet.137

In palliative settings the intent of ChT is completely different compared to

curative situations and main aims are reducing tumor related symptoms, improving

life quality and to achieve tumor response as far as possible. In palliative-intent

ChT, not less than 3 cycles and no more than 6 cycles are recommended.138 An

overview of treatment recommendations according to tumor stage is given in table

8.

43

Table 8: Recommendations for treatment of TETs according to Masaoka-Koga tumor stage (adapted from Falkson et al. and Ried et al.)126,137.

Masaoka-Koga Stage

Definition Therapy

Stage I completely encapsulated tumor

- complete surgical resection (R0) - RT and/or ChT are not recommended

Stage IIa microscopic transcapsular invasion

- complete surgical resection (R0) - RT and/or ChT are not recommended

Stage IIb macroscopic invasion into mediastinal fatty tissue, or grossly adherent but not

breaking through mediastinal pleura

- complete surgical resection (R0) - adjuvant RT in patients with higher risk of local recurrence (close surgical margins, R1, WHO type B thymoma, TC, tumor adherence to pericardium) - ChT is not recommended

Stage III macroscopic invasion into surrounding organs (pleura,

pericardium, lung,…)

Resectable - complete surgical resection (R0) - adjuvant RT Unresectable - Biopsy, downstaging of TET with induction ChT± RT - complete surgical resection (R0), if feasible - adjuvant RT (±ChT)

Stage IVa pleural or pericardial metastases

Resectable - complete surgical resection (R0), either initially or after induction therapy Unresectable - Biopsy, downstaging of TET with induction ChT± RT - complete surgical resection (R0), if feasible - adjuvant RT (±ChT)

Stage IVb lymphogenous or hematogenous metastases

- surgery not feasible, occasional tumor debulking in order to improve symptoms - Primarily ChT is recommended ± supportive RT - Targeted Therapy?

RT, radiotherapy; ChT, chemotherapy; TC, thymic carcinoma

1.2.7 Targeted Therapy TETs that could not be surgical resected, due to infiltration of surrounding organs

(e.g. myocardium) or presence of metastases, are candidates for primary ChT.

Although TETs are principally ChT sensitive, only 65-70% of these patients show

response to ChT.138,141 Hence, for the remaining part of patients new treatment

strategies are urgently needed.

It’s believed that targeted therapy may provide an alternative for

chemotherapy-resistant TETs. So far, many targets have been identified and

addressed in vitro, but in vivo data are still not convincing and data are based only

on single-center experiences with few cases. Despite overexpression of different

growth factors and proangiogenic molecules, including epidermal growth factor

receptors (EGFR), insulin-like growth factor 1 receptor (IGF-1R), c-KIT, vascular

44

endothelial growth factor (VEGF) receptor have been demonstrated for TETs,

results and efficacy of targeted molecule therapies are disappointing.142-145 In

literature, there exists only scarce information of targeted therapy in patients with

TETs, but nevertheless these few cases are briefly reported in in the following.

a) EGFR - The response of Cetuximab and Erlotinib, both antibodies directed

against the EGFR, has been evaluated in few cases of metastatic chemorefractory

thymomas. Partial remission was reported in three patients with metastatic,

chemorefractory thymomas after application of Cetuximab.146,147 In addition, one

case of partial remission of stage IV thymoma was reported also after application of

Erlotinib, while another report didn’t observe any response in a patient with

TC.148,149

b) KIT is a transmembrane tyrosine kinase receptor, which is encoded by the

proto-oncogene c-KIT and is overexpressed in the majority of TCs but hardly

present in thymomas.96 It could be already shown that activating c-Kit mutations

and KIT overexpression are crucial for the pathogenesis of gastrointestinal stromal

tumors (GIST).150 These mutations are mainly localized in exons 9, 11, 13 and 17

and represent also strong predictors for response to KIT inhibitors.150 Although KIT

is frequently overexpressed in TCs, mutations are only found in up to 7% of TCs,

which might be an explanation for the worse or even missing response to KIT

inhibitors in patients with TCs.26,151 Imatinib and Sorafenib are two antibodies

targeted against KIT. Giaccone and colleagues reported their single center

experience on the use of Imatinib in 7 cases of TETs, including two type B3

thymomas and 5 TCs. Two patients showed stable disease, while the other 5

patients showed even progression. Interestingly none of these cases showed KIT

mutations, which supports the thesis that KIT mutations are essential for the

efficacy of KIT inhibitors.152 Partial remission was reported for 3 cases of metastatic

TC with KIT-mutation after application of KIT-inhibitors.153-155 According to that, KIT

inhibitors, such as Imatinib and Sorafenib, are promising candidates for patients

with chemorefractory and/or recurrent TCs, if KIT mutations are present.156

c) IGF-1R - Expression of IGF-1R was found in 20% of TETs, significantly

more often in B2, B3 thymomas and TCs compared to A, AB and B1 thymomas.157

The efficacy of Cixutumumab, a monoclonal antibody that targets IGF-1R, was

investigated in a subset of 49 patients with chemorefractory TETs (37 thymomas,

12 TCs). In the thymoma cohort 14% achieved partial response, while the majority

45

showed stable disease (76%) and 10% showed even progression. In contrast,

nobody of the TC cohort showed partial response, whereas 42% showed stable

disease and 58% had progressive disease. Altogether the success of Cixutumumab

was limited but most interestingly,

9 of 37 patients with thymomas developed autoimmune conditions, most commonly

red-cell aplasia.158 Thus it might be interesting to verify, whether these autoimmune

conditions have been related to the monoclonal antibody or whether they have been

caused by inhibition of IGF-1R.

d) Multi-kinase inhibitors, such as Sunitinib and Sorafenib, target diverse

tyrosine kinase receptors, such as VEGFR, platelet-derived growth factor receptor

(PDGFR) or KIT. First clinical results of Sunitinib application in 4 patients with TETs

were promising. Partial remission was observed in 3 cases, while stable disease

with excellent metabolic response in 18F-FDG-PET could be achieved in the fourth

patient. Interestingly, withdrawal of Sunitinib resulted in prompt progression of

tumor, which could be controlled again by readministration of Sunitinib. Although it

is still not clear how Sunitinib exactly works, it is believed that Sunitinib works

independent from certain mutations based on the fact that no mutations (c-KIT,

KRAS, BRAF or EGFR) have been found in those patients who had shown

response to Sunitinib.159 In literature, a handful of reports exist that could already

demonstrate effects of Sorafenib. Administration of Sorafenib led to substantial

tumor reduction in a patient with chemoresistant TC and to 50% tumor reduction in

a case of widespread metastatic TC.160,161 Conversely one case of B3 thymoma

showed progression, whereas the other case showed stable disease.26

Summarized, diverse studies could already show promising and interesting

results concerning the application of targeted therapy in metastatic or

chemorefractory TETs. Nevertheless it should be considered that the number of

patients, which might not responded to targeted therapy, are most likely not

reported in literature and hence there is a high likelihood of selection bias. However,

further studies with larger patient cohorts are needed for a better understanding of

the pathogenesis of TETs, for a better identification of those patients that may

benefit from targeted therapy and of course to verify whether targeted therapy is a

valid treatment option in patients with metastatic TETs.

46

1.2.8 Tumor Recurrence Definition - Tumor recurrence is defined as relapse of cancer after complete tumor

eradication.64 The period between complete tumor eradication and tumor relapse or

recurrence is defined as freedom from recurrence (FFR) and strongly depends on

tumor subtype and its biologic behavior. The ITMIG recommends that already the

first time when strong clinical suspicion for tumor relapse is given, should be

defined as recurrence, even if histologic evaluation (biopsy) is missing.64

Although TETs are principally characterized by slow indolent growing,

recurrences occur in 10-30% of patients with FFR ranging from months to decades

after complete resection.162 In TCs, which are considered to be more aggressive

than thymomas, recurrences occur significantly earlier and more frequent at distant

sites compared to thymomas.64,163 As a consequence, lifelong surveillance

programs with periodic radiologic examinations are recommended and should be

performed annually at least for 10 years.64 Based on the localization of relapse,

tumor recurrences are classified into (1) local, (2) regional and (3) distant

recurrence (Table 9).163

Table 9: Definitions of tumor recurrence in Thymic Epithelial Tumors (adapted from Huang et al.)64. Local Recurrence – anterior mediastinum

• Tumor relapse occurring in bed of thymus or previously resected TET • Includes also pericardial, pleural, pulmonary tumors that are adjacent to thymus or

previously resected TET • Includes also lymph nodes that are adjacent to the thymus or previously resected

TET (includes also lymph nodes in the neck that are immediately adjacent to the upper lobes of the thymus).

• Pleural recurrences at the site of previously resected stage IVa TETs (should be specifically noted as such)

Regional Recurrence – intrathoracic recurrence non contiguous with thymus or previously resected TET

• Parietal pleural nodes • Visceral pleural nodes • Pericardial pleural nodes • Mediastinal lymph nodes not adjacent to the previously resected TET or thymus

Distant Recurrence • Each type of extrathoracic recurrence • Intra parenchymal pulmonary nodules (with lung parenchyma between nodule and

visceral pleural) Localization - Most recurrences occur in the thoracic cavity and represent

as single or multiple pleural nodules (regional recurrence). It’s supposed that pleural

spread within initial tumor dissection or tumor cells that fall down from invaded

47

capsules are responsible for this phenomenon. In a recently published study on 880

thymomas, tumor recurrences were reported in approximately 10% of cases.

Distribution of tumor recurrence was as following: 61.2% recurrences were regional,

16.5% local, 13.6% distant, 5.8% regional and distant and 2.9% of recurrences

were present local, regional and distant.164 The likelihood of tumor recurrences

strongly depend on TET subtype and range from 17% in thymomas to 28% in TCs

or even up to 38% in TNETs.44,53,165

Therapy – Similar to primary tumors, extended surgical resection represents

also the mainstay of therapy for tumor recurrences and provides an excellent

treatment option, which is safe, effective and associated with significant better

survival compared to non-surgical treatment or incomplete tumor resections.164,166

Indeed, 5-and 10-year OS rates of 73.6% and 48.3% were reported that increased

even to 82.4% and 65.4% at 5 and 10 years, respectively if radical re-resection (R0)

could be achieved.167 Application of adjuvant RT and/or ChT after primary tumor

resection didn’t significantly reduce number of recurrences or lead to prolonged

FFR or improved OS.146,167,168

1.2.9 Second malignancies and risk factors Second malignancies. TETs are associated with increased risk for 2nd

malignancies with incidences ranging from 8% to 28%.169-172 In hospital-based

studies with limited number of patients (100-200 patients in each study) 3 to 4 time-

increased risks for developing 2nd malignancies were described for patients with

TETs.169 Larger epidemiologic studies on 302, 733 and 2.171 patients with TETs

revealed only 1.5 to 2-fold higher risks for second malignancies.23,171,173 Increased

associations were found for malignancies of the digestive system (including

esophagus, stomach, colon/rectum and liver/biliary tract), soft tissue sarcoma, lung

cancer, non-melanoma skin cancer, prostate cancer, cervical cancer and Non-

Hodgkin lymphoma. 23,171,174

However, epidemiologic studies always carry the weakness of retrospective

studies in which results are based on data and information collected in the past with

the immanent possibility of wrong or incomplete data. Hence it is difficult to

differentiate between causative associations or only coincidental findings.23

48

Risk factors. Major risk factors, such as radiation, immunosuppression, viral

infection and genetic risk factors, which are involved in many malignancies have

been investigated also for TETs and are summarized in the following.

The incidence of TETs was not increased in patients with HIV (human

immunodeficiency virus) infection, AIDS (acquired immune deficiency syndrome) or

in immunosuppressed solid organ transplant recipients and indicates that

immunosuppression represent no risk factor for TETs.23

Viral infections have been identified as main risk factors and triggers in

several malignancies (e.g. human papilloma virus in cervical or oropharyngeal

cancer or hepatitis C virus in hepatocellular carcinoma) but in TETs the viral

hypothesis is controversial discussed and convincing evidence is limiting. Epstein

Barr virus (EBV) was found in a subset of TCs and was also discussed to be

involved in the initiation of paraneoplastic MG.175 However, based on the fact that

EBV infection is widely distributed worldwide compared to relatively low incidences

of TETs make a pathogenic role of EBV infection rather unlikely. Recently, human

polyomavirus 7 (HPyV7) has been identified as new promising candidate for the

etiology of TETs.92 Indeed, HPyV7 DNA was found in 62% of neoplastic TECs in

TETs.176

Radiation and/or adjuvant radiotherapy represent no risk factors for the

development of TETs.23,169,172,173

The higher incidence of TETs in Asians and Blacks compared to Whites

indicates that there might exist some kind of genetic predisposition.23 Genetic

differences might be also an explanation for the reported heterogeneity of

associated second malignancies in patients with different origin.23,169,173,174 Most

interestingly, in patients with TETs second or even third malignancies have been

described in up to 30% of case and indicate that there may exist also some kind of

genotype with “oncogenetic tendencies” that favor the initiation of TETs and further

malignancies.177

1.2.10 Prognostic Factors Completeness of surgical tumor resection, WHO classification and Masaoka-Koga

tumor stage represent the main prognostic factors in TETs. Indeed, these

prognostic factors are related to each other and represent the biologic behavior of

TET. The majority (>90%) of WHO type A and AB thymomas are indolent at time of

49

diagnosis and present at early tumor stages (stage I and II). Early tumor stages are

associated with high likelihood of complete tumor resection (nearly 100%), while

TCs are typically symptomatic at time at diagnosis, present with advanced tumor

stages (stage III or IV) and significantly lower possibilities of R0 resections.45 The

dependence of tumor subtype, Masaoka-Koga tumor stage and radicalism of tumor

resection, as well as their influence on survival are summarized in table 10.

Table 10: Prognostic Factors in Thymic Epithelial Tumors (adapted from Kondo et al.)162. Histologic subtype I II III IV Total Cases of Invasion to

neighboring organs A 4 4 0 0 8 0 0%

AB 11 5 1 0 17 1 5.9% B1 11 11 1 4 27 5 18.5% B2 3 3 2 0 8 2 25% B3 5 2 2 3 12 5 41.7% TC 2 1 11 14 28 25 89.3%

Total 36 26 17 21 100 38 38.0% Resection Recurrences OS

Tumor Stage complete % subtotal inop n % 10y a I 36 100% 0 0 1 3% 89.6% II 26 100% 0 0 2 8% 85.3% III 12 72% 2 3 7 50% 65.6% IV 10 48.5% 2 9 6 55.5% 41.6%

Total 84 84% 4 12 16 29.1% 70.5% Histologic Subtype

Resection Recurrences OS complete % subtotal inop n % 10y b

A 8 100% 0 0 0 0% 98.5% AB 17 100% 0 0 0 0% 97.5% B1 27 100% 0 0 4 15% 93% B2 8 100% 0 0 0 0% 87.5% B3 11 92% 0 1 4 36% 77% TC 13 46% 4 11 8 47% 43.5%

Total 84 84% 4 12 16 16.3% 83% TC, Thymic Carcinoma; OS, overall survival; inop, inoperable; n, number; 10y, 10 year;

mean 10ya OS was calculated of data from Okumura et al.178, Murakawa et al.179 and

Koppitz et al.180; mean 10yb OS according to histologic subtype was calculated of data from

Kondo et al.162, Falkson et al.126 and Filosso et al.52

As shown in table 10 there is a strong association between WHO tumor

subtype, clinical tumor stage and resectability. Principally, thymomas show a more

benign clinical behavior, which is characterized by lower likelihood of invasion,

50

metastases or recurrence (e.g. WHO type A, AB thymoma) and better OS

compared to other TETs. Conversely, WHO type B3 thymomas and TCs frequently

present with locally advanced tumor stages at time of diagnosis, lower rates of

complete tumor resection and significantly worse OS.52,126,162

A large European study including 2030 TETs reported resectability rates of

97%, 97%, 91%, 85%, 77%, 72% and 69% for WHO type A, AB, B1, B2, B3, TC

and TNET, respectively. Completeness of resection was achieved in 99%, 92%,

78% and 53% of stage I, II, III and IV TETs, respectively.28 Incomplete resection

(R1+R2) was associated with significantly worse OS compared to complete tumor

resection (R0) with 5-, 10- and 20-year OS rates of 83%, 47% and 34% in case of

incomplete resections and 92%, 78% and 67% in case of complete tumor

resection.45

The importance and necessity of complete surgical tumor resection for

prognosis was already discovered 1996 by Regnard et al., who observed that 10-

year OS was significantly worse for those patients who had undergone incomplete

resection compared to those who had undergone complete tumor resection (28%

vs. 76%, respectively; p<0.001). Interestingly, if complete tumor resection could be

achieved in stage III thymomas, OS has been similar to those reported for

completely resected stage I thymomas.102

Recurrence rates ranging from 10 to 30% have been published for completely

resected TETs.163,166 Thus it has been suggested that rate of tumor recurrence and

FFR may represent better measurements for TET outcomes than OS.64 Recurrence

rates gradually increases with higher tumor stages (stage I 3%, II 4%, III 22%, IV

40%) and with more malignant histologic tumor subtypes (type A 3%, AB 5%, B1

8%, B2 11%, B3 14%, TC 30%, TNET 37%).28 Even in cases of tumor recurrence,

completeness of surgical tumor resection affords good survival and should be

performed whenever feasible.107,168

1.3 Thymic Hyperplasia 1.3.1 Introduction Thymic Hyperplasia (TH) is a non-malignant enlargement of the thymic gland and

represents the most common benign thymic alteration. TH is defined as an increase

of weight and seize of the thymus compared to normal age-related thymus.

Principally, two main subtypes can be differentiated: (1) True Thymic Hyperplasia

51

(TTH) is characterized by regular microscopic morphology with regular histologic

architecture but with increased weight and size, whereas (2) Follicular Thymic

Hyperplasia (FTH), is characterized by the occurrence of germinal centers (GCs) in

thymic medulla, while weight and size could range from normal to increased.181

1.3.2 True Thymic Hyperplasia TTH is defined as increase of weight and size of thymus beyond physiologic age-

related limits. In contrast to FTH, TTH is characterized by regular histomorphology

and absence of GCs but with increased size and weight that could range from 40 g

to more than 1.000 g.182 The majority of patients with TTH are asymptomatic,

whereas the minority suffers from dyspnea, chest pain and other respiratory

symptoms caused by local bronchial compression of the enlarged thymus. The

etiology of TTH is not completely understood yet and TTH could be found in

different clinical conditions and diseases, including recovery from burn, after

chemotherapy treatment, younger patients suffering from lymphomas and in

endocrine and autoimmune diseases.182-185 The phenomenon of TTH after

chemotherapy treatment is clinically termed “rebound-hyperplasia”. However, the

majority of TTH cases are still idiopathic.

Typically, TTH represents as asymptomatic masses that are occasionally

found in the anterior mediastinum and radiologic differentiation between benign TH

and malignant TETs or other malignancies such as lymphomas and GCTs could be

rather challenging. In case that malignancy couldn’t be excluded biopsy and

histopathologic examination is necessary. If TTH is assumed, no further or specific

treatment is necessary due to the fact that TTH is typically self-limiting and

undergoes spontaneous remission. However, careful radiologic examinations (chest

CT) in short periods are recommended for surveillance. Surgical thymectomy is only

indicated if (1) malignancies are suspected and/or (2) to improve symptoms that are

caused by local compression due to the thymic enlargement.181

1.3.3 Follicular Thymic Hyperplasia FTH is characterized by normal thymic cortex and GCs that are located in thymic

medulla. Physiologically, GCs are located in secondary lymphatic organs, mainly

lymph nodes and are important for B-cell activation. Briefly, GCs consist of external

dark zones that form outer layers around the internal located light zones, which are

52

surrounded by mantle zones (Fig.8). Such dark zones contain large proliferating

preliminary staged B-cells, called centroblasts that undergo permanent somatic

mutation of their antibody variable-region genes to warrant the highest possible

ability of antigen recognition. Centroblasts further develop to non-proliferating

smaller centrocytes that are located in light zones. Finally, binding to antigen

presenting DCs and Th-cells drive centrocyte maturation to either antibody

producing plasma cells or to memory B-cells. 186

Figure 8: Follicular Thymic Hyperplasia. (A) A thymus with Follicular Thymic Hyperplasia

of a 21-year old woman is shown (100xmagnification). (B) Asterisk indicates Germinal

Center (GC) that is located in thymic medulla. Higher magnification of GC is shown in figure

8B. Lighter zone (LZ) contains non-proliferating B-cells (=centrocytes) is surrounded by a

darker zone (DZ) that contains proliferating preliminary B-cells (=centroblasts). (Courtesy from Leonhard Müllauer).

Most interestingly, FTH is mainly associated with early-onset MG (EOMG), a

subtype of MG that is characterized by an onset of myasthenic symptoms before

the age of 50 and the presence of autoantibodies directed against acetylcholine

receptor (AChR), whereas FTH is commonly missing in other MG subtypes.2

Hence, it is believed that the development/presence of GCs in EOMG patients is

related or even involved in the pathogenesis of MG. Indeed, a two-step

autoimmunization process is commonly assumed. First, autoreactive T-cells are

primed against AChR subunits, which are expressed on mTECs that trigger early

autoantibody production. Second, these autoantibodies attack myoid cells that

express AChR and which further leads to activation of APCs, immune complex

formation, chronic inflammation and promotion of GCs formation.187 However, it is

still unknown, which factors favor the development of MG and why FTH mainly

53

occurs in EOMG and not in other subtypes. Altogether there is strong evidence for

an intrathymic pathogenesis of EOMG accompanied by GCs formation. Clinical

experience based on the fact that patients with FTH benefit from thymectomy

supports the hypothesis of intrathymic pathogenesis in EOMG. Thus extended-

thymectomy is recommended for all EOMG patients, early after symptom onset in

order to improve myasthenic symptoms and reduce or even to avoid the need of

immunosuppressive drugs.188

Remarkably, FTH occurs also in other autoimmune diseases, such as Sjögren

Syndrome, Systemic Lupus Erythematosus, Rheumatoid Arthritis, Systemic

Sclerosis, Behçet disease or Grave’s disease. Additionally, FTH was also reported

in 50% of patients without autoimmune disease that died from car accidents but

interestingly only MG patients have shown benefit from thymectomy, whereas no

other autoimmune disorders have shown such benefits.188-195

1.4 Myasthenia Gravis 1.4.1 Introduction Myasthenia Gravis (MG) is an autoimmune disease that is triggered by

autoantibodies, which are directed against peptides of the neuromuscular junction

(NMJ). Most commonly antibodies are directed against the AChR in the

postsynaptic membrane, which causes muscle weakness and fatigue.196 In non-

AChR associated MG other peptides, such as muscle-specific kinase (MUSK),

lipoprotein-related protein 4 (LRP4) or Agrin are targets of autoantibodies. The

characteristic and pathognomonic symptom of MG is muscle weakness that

typically increases within repetitive or continued muscle activity and improves with

rest.

Interestingly, TETs and benign TG are frequently associated with MG and

supports the thesis that the thymus is substantially involved in the pathophysiology

of MG. However, several MG subtypes could be differentiated but not all of them

show typical thymic pathologies or do respond to thymectomy. In the following

chapter, the current knowledge concerning epidemiology, symptoms, classification,

subtypes, pathogenesis, diagnosis and treatment of MG are summarized.

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1.4.2 Epidemiology Incidences varies from 4 to 12 per million and prevalence of 40 to 180 per million

people are reported.197-199 The occurrence of MG and certain MG subtypes are

strongly influenced by sex and age. MG is three times more common in females

than males in adult MG patients younger than 50, while in patients elder than 50

men are predominately affected and incidences are nearly equal in puberty.200

Principally, MG is relatively homogenous distributed worldwide with only few

geographical variations in incidences and prevalence, except juvenile MG.188

Juvenile MG is characterized by onset of symptoms before 18 years and has a

notable higher incidence in East Asia, where 50% of cases occur even before an

age of 15 with predominantly ocular symptoms only.198-201

1.4.3 Symptoms Fatigable muscle weakness is the clinical hallmark and major symptom of all forms

of MG. Muscle weakness typically fluctuates, worsens with activity and improves

with rest. Principally, all skeletal muscles could be affected that use ACh as

transmitter at muscular endplates. Patients could suffer from following symptoms:

ocular weakness with ptosis and diplopia, bulbar weakness with dysphagia and

dysarthria, weakness of axial and limb muscles and weakness of respiratory

muscles causing dyspnea. In approximately 85% of cases ocular weakness with

ptosis and diplopia is the initial symptom and manifests almost asymmetric.188,200

Conversely, weakness of limb muscles is mainly symmetric and proximal muscles

are more affected than distal muscles.188 Bulbar weakness characterized by

painless dysphagia and dysarthria is the initial symptom in 15% of MG patients and

is sometimes misinterpreted as insult located in the brain stem.202

A minority of myasthenic patients could suffer also from weakness of

respiratory muscles, sometimes also worsened by bulbar symptoms, like dysphagia

and dysarthria, which could end up even in intubation and ventilation support in

severe cases. Such life-threatening events represent neurologic emergencies and

are called myasthenic crisis.203 Respiratory infections, trauma, microaspirations,

changes in drug regimes and surgical interventions are known predisposing factors

for myasthenic crisis.204 Management of such crises is challenging and includes

intubation with respiratory support, treatment of infections and of course monitoring

of vital parameters at intensive care units.188 Application of intravenous

55

immunoglobulin (IVIG) or plasma exchange are recommended as additional

immunosuppressive treatments with rapid effects occurring within 2-5 days.188

1.4.4 Classification MG patients are commonly classified according to symptoms and severity of

muscle weakness by the modified version of the Osserman score and the MGFA

(Myasthenia Gravis Foundation of America) score. The Osserman score was

established more than 40 years ago and gets continuously replaced by the MGFA

score within the last decade.205,206 Both classification scores distinguish four to five

main MG types with further subtypes based on clinical symptoms. Classification

criteria and description for both scores are summarized in table 11 and 12,

respectively.

Table 11: Osserman Classification of Myasthenia Gravis (adapted from Osserman et al.)205. Types Clinical Characteristics

I Ocular MG -> diplopia, ptosis IIa Mild generalized MG, slow progression, good response to drugs, no crisis IIb Moderate to severe generalized MG with skeletal and bulbar muscle weakness

less response to drugs and no myasthenic crisis III Acute fulminant MG, rapid progression of severe symptoms with poor drug

response and respiratory myasthenic crisis IV Late severe MG, same as type III but with progression of symptoms over more

than 2 years Table 12: Myasthenia Gravis Foundation of America (MGFA) – Clinical Classification of MG (adapted from Jaretzki et al.)206. Class Clinical Characteristics

I Any ocular muscle weakness -> ptosis, diplopia II Mild weakness affecting other than ocular muscles ± ocular muscle weakness of

any severity IIa Mild muscle weakness predominantly affecting limb and/or axial muscles IIb Mild muscle weakness predominantly affecting oropharyngeal and/or respiratory

muscles III Moderate weakness affecting other than ocular muscles ± ocular muscle

weakness of any severity IIIa Moderate muscle weakness predominantly affecting limb and/or axial muscles IIIb Moderate muscle weakness predominantly affecting oropharyngeal and/or

respiratory muscles IV Severe weakness affecting other than ocular muscles ± ocular muscle weakness

of any severity IVa Severe muscle weakness predominantly affecting limb and/or axial muscles IVb Severe muscle weakness predominantly affecting oropharyngeal and/or

respiratory muscles V Defined by intubation with or without mechanical ventilation. The use of feeding

tube without ventilation places the patient in class IVb

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1.4.5 MG subtypes and autoantibodies Interestingly, MG subtypes present with various specific clinical and pathological

characteristics, which are accompanied by the presence of distinct autoantibodies.

In the following, MG subtypes are briefly described depending on the presence of

autoantibodies and are summarized in table 13.

I. AChR-associated MG AChR antibodies are the most common autoantibodies in patients suffering from

MG and could be detected in the majority of myasthenic patients (80%).188

Depending on the onset of myasthenic symptoms and the absence or presence of

certain thymic pathologies, following subtypes are further differentiated: EOMG,

LOMG and TAMG.

a) Early-Onset MG (EOMG) is characterized by an onset of myasthenic

symptoms before an age of 50, is frequently associated with FTH and show good

response to thymectomy.188 EOMG are associated with HLA-DR3 and HLA-B8

subtypes with a female male ratio from 3 to 1.198,207

b) Late-Onset MG (LOMG) is defined as late onset of myasthenic symptoms

after an age of 50 years. In contrast to EOMG, LOMG shows slightly male

predisposition and no association with specific thymic pathology. Thus, these

patients might not benefit from thymectomy.188

c) Thymoma-associated MG (TAMG) is defined as presence of myasthenic

symptoms in patients with TETs. Time of diagnosis and onset of paraneoplastic

symptoms is irrelevant and AChR antibodies are detectable in most patients with

TAMG.188

II. MUSK-associated MG MUSK is a transmembrane polypeptide, located in the postsynaptic endplate and

which is involved in the maintenance of AChR clustering and thus is necessary for

normal functional integrity of the NMJ.188 MUSK antibodies are detected in only 1-

4% of myasthenic patients and rarely coexist with AChR antibodies in the same

patient.188 In contrast to AChR associated MG, MUSK associated MG is not related

to any characteristic thymic pathologies and therefore these patients principally

don’t benefit from thymectomy.188 Interestingly and in contrast to other subtypes,

patients with MUSK-associated MG mainly suffer from muscle weakness of cranial

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and bulbar muscles, resulting in facial, pharyngeal and tongue weakness which is

the first symptom in up to 40% of these patients.208

III. LRP-4 associated MG

LRP4 is an activator of MUSK and also expressed in the postsynaptic

membrane.188 LPR-4 associated MG presents most commonly with ocular or mild

generalized MG and shows a female predisposition. LRP-4 antibodies have been

detected in 2-27% of myasthenic patients without AChR and MUSK antibodies.209

The majority of patients with LRP-4 associated MG show atrophic and normal for

age thymic histology.188

IV. Seronegative MG Patients with neither detectable AChR nor MUSK or LRP-4 antibodies represent a

clinical heterogeneous group. Diagnosis could be challenging if no autoantibodies

are detectable or if symptoms are not characteristic. In such cases, other

neuromuscular, muscular or non-muscular disorders should be considered that

could cause similar myasthenic like symptoms. Interestingly, in 20-50% of patients

with seronegative MG (SNMG) with mild generalized muscle weakness low-affinity

antibodies could be detected, which were mainly IgG1 antibodies that were able to

activate complement system.199,210 Altogether, whether these low-affinity antibodies

are pathogenic or not needs to be evaluated in further studies.

V. Ocular MG If muscle weakness is restricted only to ocular muscles, causing typical symptoms

like diplopia and ptosis without generalized symptoms, patients belong to the

subgroup of ocular MG. Principally, those patients are at risk to develop generalized

symptoms but if muscle weakness is restricted to ocular muscles for more than 2

years, 90% of these patients will remain in this subgroup. AChR antibodies are

typically detectable in patients with ocular subtype of MG.188 Enhanced

susceptibility of ocular muscles results from differences in physiology and

morphology of NMJ. Ocular muscles have fever postsynaptic AChR, smaller motor

units and are permanently activated, which requires high firing of ACh and

activation or AChR.199

58

Table 13: Clinical subtypes of Myasthenia Gravis (adapted from Marx et al. and

Meriggiolo et al.) 92,199. MG

subtypes Age at onset

(years) Sex

Female:Male Antibodies Thymic Pathology

HLA associat.

EOMG ≤ 50 3:1 AChR FTH DR3-B8-A1 LOMG ≥ 50 1:2 AChR Normal or

TH DR7 and

DR15 TAMG usually 40-60

1:1 AChR TET -

MUSK variable more female MUSK Normal DR14, DR16, DQ5

LRP-4 variable variable LRP-4 Normal - SNMG variable variable unknown Normal or

TH -

Ocular MG

childhood in Asia, adult in America

and Europe

variable AChR in 50% Normal or TH

Bw 461

EOMG, early onset myasthenia gravis; LOMG, late onset myasthenia gravis, TAMG,

thymoma associated myasthenia gravis; MUSK, muscle specific kinase; LRP-4, lipoprotein-

related protein 4; SNMG, seronegative myasthenia gravis; AChR, acetylcholine receptor;

FTH, follicular thymic hyperplasia; TH, thymic hyperplasia; TET, thymic epithelial tumor. 1Bw 46 HLA association was found for Chinese patients.211

Additionally, Titin and ryanodine receptor (RyR) antibodies are also detected

in few patients with MG. Both proteins do not enter the muscle cell and thus can’t

cause characteristic myasthenic symptoms, but interestingly these antibodies are

frequently associated with TAMG or LOMG, whereas they are commonly missing in

EOMG.212 RyR antibodies were even associated with increased risk of severe MG

and should therefore be considered in treatment of MG patients.213 It is suggested

that Titin and RyR antibodies might be useful for the detection of thymoma in

EOMG patients and to identify those patients with higher risk of severe myasthenic

symptoms.213

1.4.6 Intrathymic pathogenesis of MG: EOMG, TAMG In 10 to 15% of myasthenic patients TETs are detected, while approximately 30% of

patients with TETs suffer from paraneoplastic MG.27 Interestingly those patients

with EOMG show a strong association with FTH. Thus the function of the thymic

gland in the pathogenesis of MG has been intensively elucidated in the past year

and is summarized briefly below.

59

EOMG There is strong evidence that in EOMG the autoimmunization begins

and persists in the thymus and is called intrathymic pathogenesis.92 It’s suggested

that this intrathymic pathogenesis of EOMG is a two-step process. First, intrathymic

Th-cells get primed against AChR peptides, which are expressed by mTECs and by

thymic myoid cells. This activation of T-cells leads to activation of B-cells and

generation of early autoreactive antibodies. Second, these antibodies bind to AChR

on myoid cells resulting in immune complex formation, activation of complement

system and professional APCs, GC formation and finally production of mature

polyclonal AChR antibodies that are released into the periphery.92 Physiologically,

genetic rearrangement of TCR is necessary to warrant that T-cells are able to

recognize the majority of peptides and positive and negative selection of T-cells

should eliminate autoreactive T-cells to avoid autoimmune diseases. It is assumed

that lack of intrathymic elimination of such autoreactive T-cells (negative selection),

which are directed against AChR, is responsible for the intrathymic pathogenesis of

EOMG. The presence of GCs in thymic medulla, called FTH, supports this thesis.92

TAMG In contrast to EOMG, the pathogenesis of TAMG is still unclear. It’s

assumed that deficiency of T-cell maturation and also elimination of autoreactive T-

cells cause TAMG. Following reasons are assumed to responsible for impaired T-

cell maturation. First, thymic medullary areas, where negative selection takes place,

are characteristically reduced or even absent in thymomas.92 Second, the

autoimmune regulator AIRE, which is responsible for the expression of tissue

specific autoantigens on mTECs and recognition of autoreactive T-cells is

decreased in thymomas.4 Finally, HLA-class II expression on neoplastic TECs is

reduced and leads to alteration of positive T-cell selection and together with AIRE

deficiency and reduced medullary areas to further impairment of negative T-

selection and higher likelihood of autoimmune processes.92,214 Interestingly,

although negative T-cell selection is obviously impaired in TETs and thus might

foster the development of diverse autoimmune diseases, especially MG commonly

occurs and indicates that additional active intrathymic autoimmunization processes

might be involved in TAMG. Indeed, neoplastic TECs of thymomas widely express

AChR subunits, especially epsilon-subunit, while functional AChR are missing.215,216

Additionally, Titin and RyR are expressed in the majority of thymomas and

antibodies could be detected in more than 90% of patients with TAMG but rarely in

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EOMG.217 Moreover, Ströbel et al. (2002) could show that the percentage of mature

naïve CD4 T-cells was significantly reduced in MG neg. thymomas, while patients

with TAMG showed “physiologic” levels of intratumorous naïve CD4 T-cells

compared to control thymuses.218 Hence, both the intrathymic production of naïve

CD4 T-cells, which have been linked to MG as well as the inefficient negative T-cell

selection of autoreactive T-cells might represent crucial factors in the pathogenesis

of MG.218 Thus intrathymic thymopoiesis seems to be also from fundamental

relevance for TAMG. Lack of thymopoiesis in TCs due to loss of organotypical

features could be an explanation for the missing association with MG in patients

with TC, though B3 thymomas and TC are histologically quite similar.45

To sum up, deficient negative T-cell selection favors release of autoreactive

T-cells and/or active immunization against Titin or AChR. Titin and AChR are both

expressed on neoplastic TECs and might be involved in the development of TAMG.

In the periphery, these autoreactive T-cells may get activated by interaction with

cognate antigens, which leads to B-cell stimulation, activation of antibody

production and causes myasthenic symptoms by binding and degrading antigens at

the NMJ. It’s assumed that thymomas, which produce high numbers of potentially

autoreactive T-cells, gradually replace the physiologic T-cell repertoire with

autoreactive T-cells and could be an explanation for the maintenance and self-

sustaining of myasthenic symptoms even after thymectomy.188,219

1.4.7 Diagnosis Diagnosis of MG could be challenging and laborious, especially in patients who do

not suffer from characteristic MG symptoms such as fatigable muscle weakness

that improves at rest. Unusual distribution of affected muscles, fluctuating

symptoms, bulbar symptoms or diplopia could sometimes lead to evaluation for

neurologic diseases, such as stroke, brain tumors, multiple sclerosis or psychiatric

diseases and could cause delayed diagnosis. In patients with characteristic

symptoms diagnosis could be easily confirmed by different tests, including bedside,

neurophysiological blood tests.

Bedside Test The Tensilon® test is the most common used bedside test to

verify the diagnosis of MG. The test consists of single intravenous administration of

short-acting acetylcholinesterase inhibitor Edrophonium (Tensilon®), followed by

observation of the patient if any improvement of muscle weakness occurs or not.

61

The test is classified as positive if muscle weakness improves immediately after

administration of Tensilon and decreases again after few minutes due to

degradation of the acetylcholinesterase inhibitor.

Neurophysiological tests, such as electromyography (EMG), are not

necessary in patients, which present with characteristic myasthenic symptoms and

in which serum antibodies are detectable.188 However, these tests are still important

for diagnosis in patients with SNMG and of course to exclude differential diagnosis.

Among neurophysiological tests, EMG with repetitive nerve stimulation is the most

commonly used technique for testing the NMJ. Characteristically for MG, low rates

of nerve stimulation lead to gradually decrease of muscle amplitude. Abnormal

EMG results can be found in approximately every second patient with ocular MG

and in 75% of cases with generalized MG.220

Blood Test Screening for serum autoantibodies, mainly AChR, is routinely

performed if MG is suspected. Screening for other antibodies such as MUSK, LRP-

4, Agrin, Titin or RyR are yet not established for use in daily routine.

1.4.8 Therapy It is the main therapeutic aim for MG patients to improve their muscle weakness as

far as possible or in best case even to achieve complete absence of any

myasthenic symptoms. Principally, non-invasive treatment options, including

symptomatic and immunosuppressive drug treatment, intravenous application of

immunoglobulin (IVIG) and plasma exchange could be differentiated from surgical

treatment options.188

A) Non-surgical treatment options: symptomatic, immunosuppression, IVIG,

plasma exchange

Symptomatic treatment regimes include diverse drugs that lead to increased

amounts of ACh in the NMJ. Mainly Pyridostigmine (Mestinon®) a reversible

acetylcholinesterase inhibitor is used, which inhibits the degradation of ACh, leading

to higher ACh concentrations in the NMJ and improvement of myasthenic

symptoms in all MG subgroups. Indeed the improvement of muscle weakness after

application of acetylcholinesterase inhibitor is so specific that it is even used as

diagnostic tool (=Tensilon Test) especially in patients with no detectable

autoantibodies (see also 1.4.7). Inhibition of ACh degradation is the most effective

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symptomatic treatment and even more effective than increasing the presynaptic

ACh release. Long-term use of acetylcholinesterase inhibitors is safe and

habituation has not been reported. However, optimal dose needs to be adapted for

each patient individually and represents a balance between improvement of

myasthenic symptoms and cholinergic side-effects, such as diarrhea, vomiting,

nausea, bronchoconstriction, bradycardia and increased secretion of sweat or

saliva caused by stimulation of the autonomic nerve system.

Immunosuppressive drugs are used for all MG patients who do not have

satisfying results with symptomatic treatment only. Prednisone as pro-drug or the

already activated Prednisolone are corticosteroids and represent the first-line

treatment option for all patients in which symptomatic drug treatment was not

successful. Beneficial effects typically manifest after 2-6 weeks, which is faster

compared to the majority of other immunosuppressive drugs.188 Some observational

studies suggested that Prednisone/Prednisolone treatment could even reduce the

risk to develop generalized MG.221

Azathioprine with an effective dose of 2-3mg/kg per day is recommended for

MG patients with generalized muscle weakness if immunosuppression with

Prednisone/Prednisolone is not enough.222 In contrast to corticosteroids beneficial

effects manifest delayed after a period of 6-15 months.222 Azathioprine is also

effective and safe for all MG subtypes, but regular follow-ups are necessary

especially within the first months because of the known risk of leucopenia and

hepatotoxic effects.188

Mycolphenolate mofetil in combination with corticosteroids is recommended

for mild to moderate generalized MG when initial immunosuppression failed. Little is

known about the response of different MG subtypes to Mycolphenolate mofetil but,

however, side-effects are rare and only mild headache, nausea and diarrhea have

been reported.223

Rituximab is a chimeric monoclonal antibody directed against the mature B-

cell marker CD20. The use of this antibody should be considered in cases with

moderate to severe generalized MG that do not respond sufficiently to initial

immunosuppression. Two thirds of patients with severe MG, in which initial

immunosuppression with Prednisolone and Azathioprine has failed showed

substantial improvement of symptoms under Rituximab treatment.224 Interestingly,

63

patients with MUSK antibody associated MG often show insufficient response to

initial immunosuppression but show favorable responses to Rituximab.188

Ciclosporine, Methotrexate and Tacrolimus are further immunosuppressive

drugs and are used as secondary drugs for MG. Whereas studies already

confirmed beneficial effects of Ciclosporine and Methotrexate treatment for MG, the

function und use of Tacrolimus has been conversely discussed in literature and its

value needs to be further evaluated.

Plasma exchange and IVIG are not used as standard treatment of MG but

are commonly used as short-term interventions in patients with acute worsening of

generalized MG and/or myasthenic crisis or in those myasthenic patients where

standard immunosuppressive treatment lacks.188 In figure 9 the “therapeutic ladder”

of MG treatment is illustrated.

Figure 9: Therapeutic flowchart in treatment of Myasthenia Gravis (adapted from

Gilhus)188. IVIG, intravenous immunoglobulin.

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B) Surgical treatment option: Extended Thymectomy

The surgical removal of thymus is called thymectomy. It is essential to consider that

the thymic gland is not a single encapsulated organ and rather consists of multiple

lobes that are distributed within the mediastinal fatty tissue.109 Microscopic thymic

foci can be widely distributed within the mediastinal fat ranging from the level of

thyroid gland to the diaphragm and bilaterally within both phrenic nerves.109,110

Hence, complete removal of thymus with all mediastinal tissue (=extended

thymectomy) should be the aim for every surgical approach. Diverse clinical studies

showed that incomplete surgical resection was associated with persistence of

symptoms that vanished after more extensive and complete reoperation.109,110

Principally, the more thymic tissue could be removed and the less thymus is left, the

better and higher are the remission rates. Even removal of 2g of thymic tissue has

been shown to be therapeutic.109,110

Different surgical approaches are used for extended thymectomy, including

transsternal, transcervical, VATS and robotic thymectomy. For thymectomy in

myasthenic patients with nonthymomatous MG or with only small, well-

circumscribed tumors VATS and robotic-assisted thymectomy are more frequently

used. Robotic-assisted thymectomy is performed in more than 100 centers

worldwide and tends to replace VATS thymectomy because it provides diverse

advantages, such as immense operative space, easy maneuverability and three-

dimensional impressions.112 However, both techniques are characterized by hardly

any mortality, low morbidity, short hospital stays and fast recovery. In contrast,

more invasive surgical approaches, including sternotomy or thoracotomy are used

rarely for nonthymomatous MG but are commonly used in cases where TETs with

advanced tumor stages are radiologically suspected.

Interestingly, except MG no other autoimmune diseases benefit from

thymectomy. Such improvements of symptoms occur months after thymectomy and

continue up to 2 years postoperatively.188 It is noteworthy to mention that only

patients with EOMG, which is associated with FTH and in which an intrathymic

pathogenesis is assumed, benefit from thymectomy. Thymectomy is not

recommended for patients with ocular MG, MUSK–associated or LRP4-associated

MG.188 In figure 10 surgical specimens of extended thymectomies are shown.

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Figure 10: Extended Thymectomy in two patients with Myasthenia Gravis. Tissue

specimens of a 44-year old male patient with Thymoma-associated Myasthenia Gravis

(TAMG) and WHO type B2, Masaoka-Koga stage IIb (A) and of an 18-year old female

patient with Early-onset Myasthenia Gravis (EOMG) and Follicular Thymic Hyperplasia,

FTH (B) are shown. (Courtesy from Bernhard Moser).

1.5 Heat Shock Proteins 1.5.1 Introduction Heat shock proteins (HSPs) represent a class of molecules, which are highly

conserved and detectable in nearly all eukaryotic cells.225 Physiologically, HSPs are

downregulated and hardly detectable, but get upregulated by a wide range of

different stressors, such as heat, radiation, hypoxia or reactive oxygen species. 226,227 More than 30 years ago, it could be shown that in Drosophila cells, elevated

temperatures (“heat shock”) leads to constant or even increased synthesis of a

small set of proteins, later called HSPs, while synthesis of other proteins was

decreased.228 Later two main functions of HSPs have been identified that could

explain the increased expression of HSPs within pathologic conditions. First HSPs

act as molecular chaperons that control and support intracellular protein folding,

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aggregation and transport.227 Second HSPS are potent inhibitors of the intrinsic and

extrinsic apoptotic pathways.229 Hence, upregulation of HSPs within pathologic

conditions is an attempt and rescue mechanism of cells to prevent themselves from

undergoing apoptosis, to maintain cellular homeostasis and finally to enable

survival.

According to molecular weight, HSPs can be divided into small HSPs (15-

30kDa) including HSP27 and larger HSPs such as, HSP40, HSP60, HSP70, HSP90

and HSP100.230 HSP27 and 70 are considered to be the strongest inducible HSPs

and an increase of over 200-fold was described for HSP70.225,231

1.5.2 Apoptosis Apoptosis is a form of “programmed” cell death, which occurs physiologically within

development and aging in order to maintain cellular homeostasis in cell populations

and tissues. Apoptosis is also used as defense mechanism to remove cells with

DNA damage. Apoptotic cells are characterized by cell shrinkage, convolution,

intact cell membranes, no release of cytoplasm and no inflammation. Conversely,

necrosis is not programmed, leads to cell swelling, distribution of cell membrane,

cytoplasm release and inflammation.232 Apoptosis could be activated by an intrinsic

and extrinsic pathway and is regulated by controlled activation of inactive

zymogens, known as pro-caspases, which could get either activated by active

caspases or could undergo autoactivation.233 An overview of both apoptotic

pathways is given in figure 11.

A) The intrinsic pathway The intrinsic pathway can be activated by several different stimuli, including

hypoxia, starvation, DNA damage, growth factor deprivation or radiation for

instance. Proteins of the Bcl-2 family are the main regulators of the intrinsic

pathway and these proteins coordinate mitochondrial release of cytochrome c,

which represents the main signal for initiation of apoptosis. The Bcl-2 family

comprises three classed of proteins: (1) anti-apoptotic proteins, such as Bcl-XL,

which are structurally similar to Bcl-2; (2) pro-apoptotic proteins, such as Bax and

Bak, which are also structurally similar to Bcl-2 and which antagonize the pro-

survival functions of Bcl-XL and (3) the pro-apoptotic “BH3-only” proteins (Bcl-2

homology, BH), which regulate the balance between pro- and anti-apoptotic Bcl-2

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proteins. Activation of BH3-only proteins also triggers activation of pro-apoptotic

Bax/Bak proteins, resulting once more in mitochondrial release of cytochrome c.

Cytochrome C further binds to the apoptosis protease activating factor-1 (Apaf-1) in

the cytoplasm and together they form the so-called “apoptosome” complex that

initiates the activation of procaspase-9 and finally results in activation of caspase-

3.233,234

B) The extrinsic pathway The extrinsic pathway can be activated through ligands of “death” receptor family

members, including Fas/CD95, TNF-R (tumor necrosis factor receptor), TRAIL-R1

and TRAIL-R2 (TNF-related apoptosis-inducing ligand receptor).235 Binding of

ligands (Fas, TNF, TRAIL) leads to dimerization and activation of transmembrane

death receptors and cytoplasmic recruitment of adapter molecules, such as FADD

(Fas associated death domain) or TRADD (TNF receptor associated death domain),

which results in assembly of the Death Inducing Signaling Complex (DISC).236 DISC

triggers recruitment and activation of caspase 8, which could either lead to direct

activation of procaspase 3 and downstream executioner caspases 6 and 7 or to

cleavage of BH3-only protein BID that translocates to mitochondria and triggers the

intrinsic pathway of apoptosis.233,236

C) The execution pathway The intrinsic and extrinsic pathways both result in activation of the execution

pathway, which represents the final part of apoptosis. Execution caspases 3, 6 and

7 activate cytoplasmic endonuclease that leads to degradation of nuclear,

cytoskeletal proteins and DNA fragmentation, which ends up in chromatin

condensation and formation of apoptotic bodies. Finally expression of cell surface

markers, such as phosphatidylserine or Annexin, that function as recognition protein

for phagocytes and foster early phagocytosis.232

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Figure 11: Extrinsic, intrinsic and execution pathway of apoptosis (adapted from

Mcllwain et al.)233. The extrinsic pathway is activated by binding of death ligands (FasL, Fas

ligand; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand) to

corresponding death receptors (Fas, TNF-R, TRAIL-R), which leads to recruitment of

adapter molecules (FADD, Fas associated death domain; TRADD, TNF receptor

associated death domain), formation of death-inducing signaling complex (DISC; not

shown) and activation of caspase 8. Caspase 8 could either directly initiate apoptosis by

cleaving and activating of executioner caspases 3, 6 and 7 or activates the intrinsic

apoptotic pathway by cleavage of BID, which in turn activates Bax and Bak. Proteins of the

pro-apoptotic Bcl-2 family stimulate mitochondrial release of cytochrome c. Cytochrome c

and apoptotic protease activating factor (Apaf-1) forms a complex, called apoptosomes.

Finally apoptosomes activates caspase 9 that ends up in stimulation and activation of

executioner caspases 3, 6 and 7.

1.5.3 Regulation of Apoptosis by HSP27 and 70 HSPs could modulate both the intrinsic as well as the extrinsic pathway of

apoptosis. Particularly, HSPs could influence the intrinsic pathway by inhibition and

modulation of different key proteins at distinct levels of apoptosis, which are as

follows: (1) interaction and inhibition of activating proteins of the apoptotic cascade

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(upstream of mitochondria), (2) inhibition of mitochondrial cytochrome c release

(mitochondrial level) and (3) finally by inhibition of aptosome formation (post-

mitochondrial). Extrinsic pathway is mainly modulated by interacting with activation

molecules of the extrinsic pathway and by interacting with intracellular signal

transduction. In the following the regulative role and capacity of HSP27 and 70 in

apoptosis is summarized. A review about further HSPs and their essential roles for

apoptosis are indicated elsewhere and are beyond the scope of this thesis.229

(1) Upstream of mitochondria. Extracellular factors, most commonly growth

factors, bind and trigger activation of transcellular kinases, such as Akt/PKB, which

in turn leads to intracellular activation of apotose proteins (e.g. caspase 9).237

HSP27 has been identified to initiate pro-survival signals by interaction and

activation of kinase Akt.238 Additionally, HSP27 stimulates degradation of NF-κB

inhibitor I-κBα, which leads to upregulation of anti-apoptotic proteins Bcl-2 and Bcl-

xL.239 HSP70 could also affect proteins of the Bcl-2 family, particularly pro-survival

and anti-apoptotic Bcl-2 and pro-apoptotic Bax by regulation of the tumor

suppressor protein p53. In case of DNA damage, p53 induces transcription of Bax,

while Bcl-2 transcription is repressed. In up to 50% of tumor cells mutated p53 gene

has been detected. HSP70 could bind mutated p53 proteins and could prevent their

nuclear import and their activation of apoptosis.240 Moreover HSP70 binds the

carboxyl terminus of protein kinase C (PKC) and stabilizes and prolongs lifetime of

PKC. Hence HSP70 enables rephosphorylation of proteins, resulting in prolonged

survival.241

(2) Mitochondrial Level. At mitochondrial level, HSP70 could inhibit

permeabilization of outer mitochondrial membrane and thereby prevent release of

mitochondrial molecules, such as cytochrome c or apoptosis inducing factor

(AIF).242

(3) Post-Mitochondrial Level. HSP27 inhibits the intrinsic pathway of

apoptosis by directly binding cytochrome c and thereby preventing apoptosome

formation, activation of caspase 9 and executioner caspases.243 Additionally,

HSP27 could function anti-apoptotic by binding and blocking already activated

caspase 3, could increase resistance to stressors by fostering anti-oxidant defense

of cells and stimulates decrease and neutralization of ROS (reactive oxygen

species). 244-246 Moreover, HSP70 prevents apoptosome complex formation by

inhibition of recruitment and binding of procaspase 9 to Apaf-1.247

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Extrinsic pathway of apoptosis. Binding of death ligands to death

domains/receptors stimulates the extrinsic pathway of apoptosis. HSP27 and 70

could block this pathway by inhibition of Fas- and TNF-induced apoptosis,

respectively.245 Further HSP70 could inhibit activation of Bid and thereby prevents

stimulation of the intrinsic apoptotic pathway.248

1.5.4 HSP27 and 70 in cancer Overexpression of HSPs has been described in a broad variety of malignancies,

including solitary tumors and hematological malignancies, such as colon cancer,

prostate cancer, lung cancer, oral squamous cell carcinoma, breast cancer or

hepatocellular carcinoma.249-257 It’s assumed that upregulation of HSPs in

malignancies is an adaptive response of tumor cells to promote protein

homeostasis, cell survival, to inhibit cell death and to stimulate cell proliferation.258

Remarkably, in-vitro inactivation or even knockdown of HSP27 and 70 leads to

spontaneous activation of apoptosis and confirms the pivotal role of heat shock

proteins for survival of tumor cells.247,259

HSP27 was associated with worse survival in patients with node-negative

breast cancer. In-vitro studies with human breast cancer cell lines further showed

that HSP27 expression was associated also with drug resistance of tumor

cells.249,260 Similarly, in-vitro HSP70 modulates chemoresponsiveness of pancreatic

cells, while HSP27 plays key roles in gemcitabine-resistance in pancreatic

cells.250,261 Moreover, HSP expression and association with resistance to cisplatin

has been also described for germ-cell tumor cell lines, small-cell lung carcinoma

cell lines and colon cancer cell lines for instance.262

Altogether, HSPs are principally associated with drug resistance, promotion of

tumorigenesis, increased cellular migration, poor prognosis and metastases. 259,263-

266 Recently Schweiger et al. could clearly demonstrate that in patients with

pulmonary metastasized colon cancer, stromal HSP27 expression of metastases

was associated with higher microvessel densities and worse outcome.267 Hence,

HSPs are considered as targets for novel treatment strategies for diverse

malignancies and metastases.268 First promising results have been already

published and warrant further studies to elucidate the function of HSPs for target

therapy.269,270

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1.5.5 Heat shock proteins in our workgroup Already several years ago, HSPs raised our attention when our workgroup found

out that HSP27 and 70 serum levels were increased in patients with chronic

obstructive pulmonary disease (COPD).271 Further we could show that HSP27

serum levels correlated with early radiological signs of COPD, whereas lung

functions have been still normal and indicates that HSP27 serum levels may serve

as serum marker to identify early stage COPD patients.272 Beside COPD, our

workgroup investigated also serum HSP levels in patients with lung cancer and

found out that HSP27 serum concentrations were elevated in patients with non-

small cell lung cancer (NSCLC).273 As already mentioned above Schweiger et al.

(2015) demonstrated recently that HSP27 is also expressed in stroma of pulmonary

colon cancer metastases and that expression was associated with higher

microvessel densities and worse survival.267 The angiogenic role of HSP27 has

been already described in literature. Particularly, it could be shown that extracellular

HSP27 stimulates activation of NFκB in endothelial cells, which is accompanied by

increased expression of proangiogenic factors, such as IL-8 or VEGF.274

1.6 Aims of this thesis TETs represent the most common tumor entity in the anterior mediastinum in adults

and are characterized by a unique association with paraneoplastic MG. However,

the pathogenesis of TETs is still unclear and so far neither risk factors nor

biomarkers have been established for TETs.

We strongly believe that HSPs represent promising proteins that might be

involved also in the pathobiology of TETs. Hence, the main aim of this thesis is to

evaluate the local and systemic roles of HSP27 and 70 in TETs. Therefore HSP

expression will be assessed in TETs, tumor microenvironment and will be

compared to benign thymic pathologies and physiologic thymic specimens.

Additionally serum of patients with malignant and benign thymic pathologies will be

analyzed and compared to healthy controls.

Finally the prognostic, diagnostic and physiologic implications of HSPs will

be assessed and discussed.

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Chapter II: Results 2. HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications 2.1 Prologue A broad variety of different stressors, including hypoxia, inflammation or presence

of cancer cells lead to upregulation of HSPs.230 In cancer, HSP expression is

generally related to resistance to chemotherapy, tumor progression, poor prognosis

and survival.259 HSPs are molecular chaperons and potent inhibitors of apoptosis

and it is considered that these two functions are crucial for malignancies.

In the following work we investigated the role of HSP27 and 70, which

represent also the strongest inducible HSPs, in patients with malignant and benign

thymic pathologies. Therefore we firstly analyzed HSP27 and 70 tumor expression

in patients with TETs and secondly correlated HSP expression with different

outcomes, such as overall survival, cause-specific survival and freedom from

recurrence to assess the prognostic role of HSP expression for patients with TETs.

Staining of hyperplastic and regular thymuses served as controls. Additionally, we

analyzed also HSP27 and 70 serum levels in patients of the same cohort. Finally,

we discussed the prognostic, diagnostic and physiologic role of HSPs in TETs and

paraneoplastic MG.

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HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implicationsS. Janik1,2, A. I. Schiefer3, C. Bekos1,2, P. Hacker1,2, T. Haider2,4, J. Moser5, W. Klepetko1, L. Müllauer3, H. J. Ankersmit1,2 & B. Moser1

Thymic Epithelial Tumors (TETs), the most common tumors in the anterior mediastinum in adults, show a unique association with autoimmune Myasthenia Gravis (MG) and represent a multidisciplinary diagnostic and therapeutic challenge. Neither risk factors nor established biomarkers for TETs exist. Predictive and diagnostic markers are urgently needed. Heat shock proteins (HSPs) are upregulated in several malignancies promoting tumor cell survival and metastases. We performed immunohistochemical staining of HSP27 and 70 in patients with TETs (n = 101) and patients with benign thymic alterations (n = 24). Further, serum HSP27 and 70 concentrations were determined in patients with TETs (n = 46), patients with benign thymic alterations (n = 33) and volunteers (n = 49) by using ELISA. HSPs were differentially expressed in histologic types and pathological tumor stages of TETs. Weak HSP tumor expression correlated with worse freedom from recurrence. Serum HSP concentrations were elevated in TETs and MG, correlated with clinical tumor stage and histologic subtype and decreased significantly after complete tumor resection. To conclude, we found HSP expression in the vast majority of TETs, in physiologic thymus and staining intensities in patients with TETs have been associated with prognosis. However, although interesting and promising the role of HSPs in TETs as diagnostic and prognostic or even therapeutic markers need to be further evaluated.

Thymic Epithelial Tumors (TETs) comprise thymomas, thymic carcinomas (TCs) and thymic neuroendocrine tumors (TNETs). Thymomas and TCs are the most common malignant neoplasms of the anterior mediasti-num in adults and originate from thymic epithelial cells (TECs). TETs are rare malignancies with an incidence of 0.15 to 0.32 per 100.000 person-years1,2. Thymic malignancies are histologically classified according to the World Health Organization (WHO) classification, which distinguishes six main histopathologic types based on fundamental morphological differences: types A, AB, B1, B2, B3 thymomas and TCs3. TNETs are primary neu-roendocrine tumors of the thymic gland that arise from thymic neuroendocrine cells and comprise less than 5% of TETs4,5. According to nomenclature of neuroendocrine tumors of the lung, TNETs are distinguished into well-differentiated and poorly differentiated neuroendocrine carcinomas6. For staging of TETs the Koga modifi-cation of the Masaoka Staging system (Masaoka-Koga) is recommended, which classifies TETs according to their level of invasiveness into 4 stages7.

TETs and autoimmune diseases: There is only scarce information on the pathophysiology of TETs. Neither risk factors nor biomarkers for diagnosis of TETs have been established8. TETs are often associated with a variety of paraneoplastic syndromes, most frequently Myasthenia Gravis (MG)9. TETs were detected in approximately 10

1Department of Thoracic Surgery, Division of Surgery, Medical University Vienna, Austria. 2Christian Doppler Laboratory for the Diagnosis and Regeneration of Cardiac and Thoracic Diseases, Medical University Vienna, Austria. 3Clinical Institute of Pathology, Medical University Vienna, Austria. 4University Clinic for Trauma Surgery, Medical University Vienna, Austria. 5Departments of Dermatology and Venereology and Karl Landsteiner Institute of Dermatological Research, Karl Landsteiner University of Health Sciences, St. Pölten, Austria. Correspondence and requests for materials should be addressed to B.M. (email: bernhard.moser@meduniwien.ac.at)

Received: 23 September 2015

Accepted: 21 March 2016

Published: 21 April 2016

OPEN

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to 15% of patients with MG10, whereas at least 30% of patients with TETs showed symptoms of associated parane-oplastic MG at time of diagnosis11. In contrast, up to 50% of TNETs are associated with paraneoplastic endocrine diseases, most commonly with the hereditary tumor syndrome multiple endocrine neoplasia type 1 (MEN1)-and paraneoplastic Cushing syndrome12.

MG and Thymic Abnormalities: MG is an autoimmune disease mediated by autoantibodies directed against acetylcholine receptors (AChR) or other targets at the neuromuscular junction, resulting in characteristic symp-toms, like muscle weakness13. Up to 80% of patients with MG show thymic abnormalities, comprising TETs14 and benign irregularities of the thymus, such as true thymic hyperplasia (TTH) and follicular thymic hyperplasia (FTH). FTH and TTH are clinically summarized as thymic hyperplasia (TH) characterized as macroscopic or radiological enlargement of the thymus, respectively. Microscopically FTH is characterized by the presence of lymphoid follicles with germinal centers (GCs) in the thymic medulla, whereas TTH shows a non-age related, regular lobulated thymic tissue with no or only few signs of fatty involution15.

In our previous work we have provided evidence for a role of the receptor for advanced glycation endprod-ucts (RAGE) and its ligand high mobility group box 1 (HMGB1) in MG16, TETs, TTH, FTH and regular thy-mus17. HMGB1 is an important extracellular, cytoplasmic and nuclear regulator of apoptosis and autophagy18,19. Extracellular HMGB1 stimulates autophagy via binding to RAGE, cytoplasmic HMGB1 binds to Beclin1-binding protein and nuclear HMGB1 controls autophagy by its downstream molecule heat shock protein 27 (HSP27/HSPB1)18. Vice versa heat shock protein 70 (HSP70) stimulates activation of NFκ B and c-Jun terminal kinase (JNK)/Beclin-1 resulting in a positive feedback mechanism leading to increased expression and release of HMGB120. These observations sparked our interest in the possible involvement of HSPs in thymic pathology.

Heat shock proteins (HSPs) are highly conserved proteins that are present in nearly all nucleated cells21. HSPs are classified according to their molecular weight: HSP100, HSP90, HSP70, HSP60, HSP40 and small HSPs (15–30kDa) including HSP2722. Physiologically HSPs are kept at low levels and are induced by various environmental and pathophysiological stressors, such as heat shock, radiation, hypoxia and reactive oxygen species23,24. Two main functions of HSPs are known: first HSPs act as molecular chaperones thereby playing a role in intracellular protein folding, aggregation, transport and homeostasis24,25. Second HSPs have important functions as inhibitors of caspase-dependent (e.g. HSP27, HSP70) and -independent apoptosis pathways (e.g. HSP70)26.

HSPs as targets for cancer treatment: Because of their anti-apoptotic functions HSPs are believed to be key players in the pathogenesis of malignancies and as targets for novel cancer treatment strategies27. In cell cul-ture based studies HSPs promoted tumorigenesis by increasing cellular migration28, differentiation29 and drug resistance30.

Tumor Microenvironment: Tumor cells are surrounded by a variety of cell types, such as endothelial cells (ECs), fibroblasts and macrophages. Together these cells and the extracellular matrix comprise the tumor microenvironment that contributes one of the hallmarks of cancer, which is essential for tumor promotion and progression31,32. Recently it was shown that heat shock factor 1 (HSF1), the main transcription factor for HSPs, is a potent enabler of malignancy and high stromal expression in early-stage lung and breast cancer is strongly associated with poor clinical outcome33.

In the present study we sought to investigate the role of the strongest inducible heat shock proteins (HSP27 and 70)21 in TETs, tumor microenvironment and benign thymic alterations of fetal, infantile and adult patients. Moreover we measured serum concentrations of the indicated HSPs in patients with TETs, TH and volunteers with and without MG.

ResultsHSP expression in TETs, TNETs, TH and regular thymus. Expression of HSP27 and 70 in TETs and TNETs. HSP and WHO type: Semiquantitative analysis revealed moderate to strong expression of HSP27 and 70 in all investigated types of thymomas and TCs, in contrast to low or absent expression in well-differentiated TNETs (HSP27: p < 0.001; HSP70 nuclear: p = 0.003, cytoplasmic: p = 0.061; HSP27 was detected in the cytoplas-mic but not the nuclear compartment of tumor cells, whereas HSP70 showed nuclear and cytoplasmic expression. HSP70 expression was more intense in nuclei compared to the cytoplasm (nuclear 2.39 ± 0.1 vs. cytoplasmic 2.11 ± 0.1; p = 0.023), (Table 1; Figs 1 and 2; figures with higher magnification are available as supplementary material, Suppl. Figs 1 and 2).

HSP27 expression in tumor cells correlated significantly with WHO classification (p = 0.013; r = 0.247). There was no significant correlation for HSP70 expression with WHO histology (HSP70 nuclear: p = 0.173; r = − 0.137, cytoplasmic: p = 0.437; r = − 0.078). HSP27 expression in tumor cells correlated statistically significant with nuclear and cytoplasmic HSP70 expression (p = 0.027; r = 0.221 and p = 0.019; r = 0.235).

HSP staining in TETs in comparison to immunohistochemical markers used for routine diagnosis can be found as supplementary material (Suppl. Fig. 3, Suppl. Table 1).

HSP and Masaoka-Koga stage: There was no significant difference in HSP27 staining intensities in TETs from patients diagnosed at different Masaoka-Koga stages (p = 0.770). Analysis of HSP70 staining intensities in tumor cells of different Masaoka-Koga stages revealed significant differences for nuclear (p = 0.037), but not for cyto-plasmic HSP70 expression (p = 0.261). Nuclear HSP70 expression in stage I–III was higher compared to stage IV TETs (p = 0.046), while no difference was observed for cytoplasmic HSP70 expression (p = 0.100). Nuclear HSP70 expression was significantly higher in non-invasive TETs (stage I) compared to invasive TETs (stage II–IV) (p = 0.045; Table 1). There was no statistically significant correlation between HSP27 and 70 staining inten-sities and Masaoka-Koga stage (HSP27: p = 0.082, r = 0.175; HSP70 nuclear: p = 0.106, r = − 0.162; cytoplasmic: p = 0.084, r = − 0.173).

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HSP and MG: We did not detect significant differences in staining intensities of HSP27 (p = 0.058) and HSP70 (nuclear: p = 0.656; and cytoplasmic: p = 0.558, respectively) in TETs of patients with or without MG.

HSP expression and recommended outcome measures for TETs: Overall survival (OS) and cancer specific sur-vival (CSS) in TETs with low cytoplasmic HSP27 expression were not significantly different compared to TETs with moderate to strong HSP27 expression (p = 0.867 and p = 0.352, respectively). Freedom from recurrence (FFR) was significantly worse in TETs with low HSP27 expression (p = 0.017).

Cytoplasmic HSP70 staining intensities in TETs showed no statistically significant differences in OS (p = 0.337), CSS (p = 0.291) and FFR (p = 0.082), respectively. Weak nuclear HSP70 expression in TETs was associated with significantly worse FFR compared to TETs with moderate to strong nuclear HSP70 expression (p = 0.016). There was no significant difference of OS and CSS between TETs with weak nuclear HSP70 compared to those with moderate to strong expression (p = 0.980 and p = 0.985, respectively; Table 2; Fig. 3).

HSP and tumor microenvironment. HSP27 and 70 expression in stromal cells of the tumor microenviron-ment: HSP27 and 70 were both expressed in ECs, fibroblasts, adipocytes and macrophages but were absent in lymphocytes (HSP27: p < 0.001, cytoplasmic HSP70: p < 0.001). HSP27 showed significantly stronger expres-sion in ECs compared to fibroblasts, adipocytes or macrophages (p < 0.001) and was even stronger than HSP27 expression in TETs (p = 0.035).

The strongest HSP70 expression in cells of the tumor microenvironment was found in macrophages (p < 0.001). Macrophages in the tumor microenvironment of WHO type AB thymomas showed the strongest HSP70 expression which was significantly stronger compared to macrophages of type A thymomas (p = 0.001). The weakest HSP70 expression in fibroblasts was found in WHO type B3 thymomas which was significantly lower compared to fibroblasts of the tumor microenvironment in type AB thymomas (p ≤ 0.001). It is noteworthy that cytoplasmic HSP70 expression in TETs was higher than staining intensities of stromal cells (p < 0.001).

Stromal cells showed significantly stronger expression of HSP27 2.3 ± 0.0 compared to HSP70 1.0 ± 0.0 (p < 0.001). Paraneoplastic MG had no influence on HSP expression in cells of the tumor microenvironment (Suppl. Table 2).

Benign Thymic Alterations and Regular Thymus. Role of HSPs in regular and hyperplastic thymus: In ECs we found strong expression of HSP27 in all specimens (n = 23) and strong expression of HSP70 in 95.6% of cases. Further we detected strong expression of HSP27 and 70 in medullary-(mTECs) and cortical TECs (cTECs) in all non-malignant thymic specimens (n = 24). All cTECs showed strong expression of both HSPs. All Hassall’s corpuscles in the thymic medulla expressed HSP27 and 70 (Fig. 4).

HSP expression in germinal centers of thymus of MG. Additionally, we found HSP27 and 70 expres-sion in dendritic cells (DCs) of GCs and TECs with mantle zone (MZ)-like distribution in all patients with MG

Clinicopathologic characteristics

HSP expression in TETs

Cyto HSP27 Cyto HSP70 Nucl HSP70

n mean ± SEM n mean ± SEM n mean ± SEM

WHO

A 16 2.63 ± 0.1 16 2.0 ± 0.2 16 2.44 ± 0.2

AB 14 2.54 ± 0.1 14 2.5 ± 0.2 14 2.61 ± 0.2

B1 10 2.30 ± 0.2 11 1.55 ± 0.4 11 1.86 ± 0.3

B2 21 2.79 ± 0.1 21 2.29 ± 0.2 21 2.60 ± 0.2

B3 17 2.85 ± 0.1 17 2.0 ± 0.1 17 2.38 ± 0.2

TC 15 2.93 ± 0.1 15 2.1 ± 0.2 15 2.23 ± 0.3

MNT 4 3.0 ± 0.0 4 2.5 ± 0.3 4 2.5 ± 0.3

TNET 3 0.5 ± 0.3 3 1.33 ± 0.4 3 0.33 ± 0.3

p-value < 0.001a 0.061a 0.003a

Masaoka-Koga Stage

I 23 2.52 ± 0.1 23 2.30 ± 0.1 23 2.59 ± 0.1

II 46 2.64 ± 0.1 47 2.17 ± 0.1 47 2.39 ± 0.1

III 15 2.67 ± 0.2 15 1.97 ± 0.2 15 2.37 ± 0.3

IV 16 2.63 ± 0.2 16 1.78 ± 0.3 16 1.75 ± 0.3

p-value 0.770a 0.261a 0.037a

Table 1. HSP27 and 70 expression in TETs and analysis of clinicopathologic characteristics. HSP27 and 70 were strongly expressed in all thymomas and TCs but weak or absent in TNETs. Strongest nuclear HSP70 expression was found in non-invasive TETs (Masaoka-Koga stage I), whereas the weakest expression was found in metastatic TETs (Masaoka-Koga stage IV). Paraneoplastic MG had no significant difference on HSP27 and 70 staining intensities in TETs. HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; n, number; Cyto, cytoplasmic expression; Nucl., nuclear expression; WHO, World Health Organization; TC, Thymic Carcinoma; MNT, Micronodular Thymoma; TNET, Thymic Neuroendocrine Tumor; MG, Myasthenia Gravis; SEM, standard error of the mean. aOne-way Anova.

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(FTH: n = 6). In accordance to TETs, lymphocytes were negative in all cases of non-neoplastic thymic tissues, in particular those with MG (Fig. 4).

We did not detect statistically significant difference in staining patterns between patients with or without MG, in fetal, infantile and adult thymic specimens, respectively (Suppl. Table 3).

HSPs-markers for TEC and thymic dendritic cells. To specify the type of HSP expressing thymic cells we performed sequential double-staining of fetal and adult non-malignant thymic specimens. Lu-5 staining was

Figure 1. HSP27 expression in TETs. Immunohistochemical staining of HSP27 in different histologic tumor subtypes was performed. Expression of HSP27 in thymoma WHO type A (A), AB (B), B1 (C), B2 (D), B3 (E), TC-SCC (F), MNT (G), and TNET (H), is shown (400X magnification). Hematoxylin was used for counterstaining. HSP27, Heat Shock Protein 27; TETs, Thymic Epithelial Tumors; WHO, World Health Organization; TC, Thymic Carcinoma; SCC, Squamous Cell Carcinoma; MNT, Micronodular Thymoma; TNET, Thymic Neuroendocrine Tumor.

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used as marker for TECs whereas CD68 (PGM-1) was used to identify macrophages and DCs. Double-staining of Lu-5/HSP70 and CD68/HSP70 in adult and fetal thymic specimens was performed all thymic cells that showed positive staining for HSP70 coexpressed Lu5. The majority of thymic cells showed single staining for HSP70 or CD68. Only a subset with simultaneous expression of HSP70 and CD68 was identified (Fig. 5).

Figure 2. HSP70 expression in TETs. Immunohistochemical staining of HSP70 in different histologic tumor subtypes was performed. Expression of HSP70 in thymoma WHO type A (A), AB (B), B1 (C), B2 (D), B3 (E), TC-SCC (F), MNT (G), and TNET (H), is shown (400x magnification). Hematoxylin was used for counterstaining. HSP70, Heat Shock Protein 70; TETs, Thymic Epithelial Tumors; WHO, World Health Organization; TC, Thymic Carcinoma; SCC, Squamous Cell Carcinoma; MNT, Micronodular Thymoma; TNET, Thymic Neuroendocrine Tumor.

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Analysis of HSPs in serum of patients with thymic abnormalities. HSP concentrations in TETs. Increased HSP27 and 70 serum concentrations in patients with TETs and correlation with histologic tumor subtype: There was no significant statistical difference considering age (p = 0.376) and sex (p = 0.477) in patients with TETs and healthy volunteers.

We found significantly increased serum concentrations of HSP27 and 70 in patients with TETs compared to healthy volunteers (HSP27[pg/ml]: 509.6 ± 67.2 vs. 334.6 ± 50.2; p = 0.040 and HSP70[ng/ml]: 2.0 ± 0.3 vs. 1.3 ± 0.1; p = 0.021, respectively). Further stratification of patients with TETs into patients with TCs and thymo-mas compared to volunteers revealed significant differences of HSP27 and 70 serum concentrations (HSP27[pg/ml]: 635.9 ± 118.3 vs. 412.5 ± 73.2 vs. 334.6 ± 50.2; p = 0.021 and HSP70[ng/ml]: 2.4 ± 0.5 vs. 1.7 ± 0.3 vs. 1.3 ± 0.1; p = 0.015, respectively). Post hoc comparisons revealed significant differences in HSP27 and 70 serum concentrations only in patients with TCs compared to volunteers (HSP27[pg/ml]: 635.9 ± 118.3 vs. 334.6 ± 50.2; p = 0.017 and HSP70[ng/ml]: 2.4 ± 0.5 vs. 1.3 ± 0.1; p = 0.012, respectively). Separate analysis of HSP27 and 70 serum concentrations in patients with thymomas compared to volunteers showed no significant differences

ITMIG recommended outcome measures

HSP expression in TETs

Cytoplasmic HSP27 Cytoplasmic HSP70 Nuclear HSP70

Overall Survival (OS)

weak (n = 5)

mod/str (n = 90)

weak (n = 15)

mod/str (n = 80)

weak (n = 10)

mod/str (n = 85)

1 year 100% 100% 100% 100% 100% 100%

5 year 100% 96.3% 93.3% 97.2% 100% 96.1%

10 year 66.7% 92% 81.7% 92.4% 87.5% 91.4%

p-valuea 0.867 0.377 0.980

Cancer Specific Survival (CSS) weak mod/str weak mod/str weak mod/str

1 year 100% 100% 100% 100% 100% 100%

5 year 100% 97.7% 93.3% 98.7% 100% 97.6%

10 year 66.7% 95.6% 81.7% 96.4% 87.5% 95.3%

p-valuea 0.352 0.291 0.985

Freedom From Recurrence (FFR) weak mod/str weak mod/str weak mod/str

1 year 100% 100% 100% 100% 100% 100%

5 year 60% 95.3% 72% 97.1% 60% 97.5%

10 year 60% 92% 72% 93.3% 60% 93.9%

p-valuea 0.017 0.082 0.016

Table 2. HSP expression in TETs and ITMIG recommended outcome measures. TETs with absent or weak cytoplasmic HSP27 and nuclear HSP70 staining showed significantly worse FFR. There was no significant difference for OS and CSS. ITMIG, International Thymic Malignancy Interest Group; HSP, Heat Shock Protein; TETs, Thymic Epithelial Tumors; OS, Overall Survival; CSS, Cancer Specific Survival; FFR, Freedom From Recurrence; n, number; mod/str, moderate to strong HSP expression. aLog-rank test.

Figure 3. HSP expression and freedom from recurrence in TETs. Kaplan-Meier survival plots for HSP27 (A) and HSP70 (B) and FFR are shown. The number of patients at risk for moderate to strong and weak HSP27 and 70 expression are listed for the indicated time points. HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; TETs, Thymic Epithelial Tumors; FFR, Freedom From Recurrence.

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Figure 4. HSP27 and HSP70 expression in non-malignant thymic specimens. Representative images show endothelial expression of HSP27 (A) in fetal thymus and HSP70 (B) in adult thymus with regular morphology (*asterisk). cTECs with intense subcapsular expression of HSP27 (C) in fetal thymus and HSP70 (D) in FTH is shown (arrows). mTECs and HCs express HSP27 (E) and HSP70 (F) in fetal thymus. Arrows indicate HCs. Staining of HSP27 (G) and HSP70 (H) in dendritic cells of GCs (GC) and TECs in MZ-like distribution (MZ) in FTH is shown. Arrows indicating HSP expressing dendritic cells in GCs (A-F, H: 400x magnification; G: 200x magnification). Hematoxylin was used for counterstaining. HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; cTEC, cortical Thymic Epithelial Cells; mTEC, medullary Thymic Epithelial Cells; FTH, Follicular Thymic Hyperplasia; HC, Hassall’s Corpuscles; GC, Germinal Center; MZ, TECs with marginal zone like distribution.

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(HSP27[pg/ml]: 412.5 ± 73.2 vs. 334.6 ± 50.2; p = 0.374 and HSP70[ng/ml]: 1.7 ± 0.3 vs. 1.3 ± 0.1; p = 0.161, respectively).

HSP27 and 70 serum concentrations correlated significantly with WHO tumor classification (HSP27: p = 0.040; r = 0.308 and HSP70: p = 0.021; r = 0.344). In particular, the lowest HSP serum concentrations were found in WHO type A and AB thymomas, whereas the highest concentrations were found in B3 thymomas and TCs (Table 3; Suppl. Table 4; Fig. 6).

Increased HSP serum concentrations in advanced Masaoka-Koga tumor stages. Patients with Masaoka-Koga stage III–IV TETs showed significantly higher HSP27 serum concentrations compared to stage I–II tumors (HSP27[pg/ml]: 647.8 ± 100.4 vs. 358.9 ± 78.4; p = 0.030). There was no statistically significant difference in HSP70 serum concentrations when stages I-II vs. III-IV were analyzed (HSP70[ng/ml]: 1.6 ± 0.3 vs. 2.4 ± 0.4; p = 0.111). Serum HSP concentrations of non-invasive (Masaoka-Koga I) and invasive TETs (Masaoka-Koga II–IV) were not significantly different (HSP27[pg/ml]: 446.2 ± 198.7 vs. 521.0 ± 71.9; p = 0.789 and HSP70[ng/ml]: 1.4 ± 0.4 vs. 2.1 ± 0.3; p = 0.203; Table 3).

We did not find significant correlations between Masaoka-Koga stage and HSP27 (p = 0.098; r = 0.248) or HSP70 (p = 0.084; r = 0.258), respectively.

Decreased serum concentrations of HSPs after surgical tumor resection. Longitudinal analysis revealed signifi-cantly decreased HSP27 serum concentrations after complete tumor resection (0.199 fold decrease, p = 0.015). Although we detected also a decrease of HSP70 serum concentrations after tumor resection, the difference was not statistically significant (0.118 fold decrease, p = 0.121; Table 3; Fig. 6K).

Comparison of HSP concentrations in TETs and TH. Differences in HSP serum concentrations in patients with TETs compared to TH: Analysis of HSP27 serum concentrations in patients with TETs compared to TH and volunteers showed no significant differences (HSP27[pg/ml]: TETs 509.6 ± 67.2 vs. TH 464.6 ± 80.1 vs. volunteers 334.6 ± 50.2; p = 0.115). In contrast HSP70 serum concentrations were significantly increased in patients with TETs compared to both volunteers (p = 0.028) and patients with TH (p = 0.043; HSP70[ng/ml]: TETs 2.0 ± 0.3 vs. TH 1.3 ± 0.1 vs. volunteers 1.3 ± 0.1). There was no significant difference between HSP70 serum concentrations in TH and volunteers (p = 1.000).

Figure 5. Immunophenotyping of HSP expressing cells in fetal and adult thymus with regular thymic morphology. Double-staining of Lu5-pancytokeratin (red) and HSP70 (brown) of an adult (A) and fetal thymus (C) is shown. There was complete concordance of Lu5 and HSP70 expression. Staining pattern of CD68 (PGM-1, macrophages/DCs) (red) and HSP70 (brown) double-staining is shown for an adult (B) and fetal (D) thymus. While most cells showed either distinct CD68 or HSP70 expression we found few cells that showed double staining (insert in D 630x magnification). Hematoxylin was used for counterstaining. Magnification: 400x. HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; DCs; dendritic cells.

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Separate analysis of HSP27 and 70 serum concentrations in patients with TCs, thymomas and TH revealed significantly increased HSP70 serum concentrations only in TCs compared to TH (p = 0.027), whereas there was no significant difference for HSP27 serum concentrations between those subgroups (p = 0.236; HSP27[pg/ml]: TC 635.9 ± 118.3 vs. thymoma 412.5 ± 73.2 vs. TH 464.6 ± 80.1 and HSP70[ng/ml]: TC 2.4 ± 0.5 vs. thymoma 1.7 ± 0.3 vs. TH 1.3 ± 0.1; Table 3; Fig. 6).HSP concentrations in MG. Higher HSP serum concentrations in patients with autoimmune MG: There was no significant difference in HSP27 and 70 serum concentrations in patients with TETs with or without paraneo-plastic MG (HSP27[pg/ml]: TETs MG+ 434.6 ± 120.2 vs. TETs MG− 533.2 ± 80.4; p = 0.538 and HSP70[ng/ml]: TETs MG+ 1.9 ± 0.3 vs. TETs MG− 2.0 ± 0.3; p = 0.836; Table 3).

Emphasizing on MG, we measured HSP27 and 70 serum concentrations in a subgroup of 26 patients with MG (11 TETs, 15TH) compared to 26 sex (p = 0.760) and age (p = 0.453) matched volunteers. We detected significant higher serum concentrations of HSP27 in MG patients compared to volunteers (HSP27[pg/ml]: 507.5 ± 89.5 vs. 215.5 ± 35.9; p = 0.005), whereas there was no significant difference for HSP70 (HSP70[ng/ml]: 1.5 ± 0.2 vs. 1.1 ± 0.2; p = 0.088; Table 3; Fig. 6).

DiscussionThis is the first description of HSP27 and 70 expression in human regular thymic morphology of different devel-opmental stages (fetuses, infants and adults), in non-neoplastic thymic diseases, namely TTH and FTH and neo-plasms of thymic epithelial origin.

HSPs are overexpressed in several malignancies, including colon cancer34, prostate cancer35 hepatocellular carcinoma (HCC)36, lung cancer37 and oral squamous cell carcinoma (SCC)38. HSP overexpression in cancer is related to tumor growth, resistance to chemotherapy, metastases and poor survival39,40. Overexpression of HSPs was associated with inhibition of apoptosis and favored tumor initiation and progression.These observations may also be of relevance in regard to our data on thymomas and TCs. Semiquantitative analysis of HSP 27 and 70 revealed high staining intensities in all WHO type thymomas, MNT and TCs. Nuclear and cytoplasmic HSP70

Groups n

HSP serum concentrations

HSP27 [pg/ml] p-value HSP70 [ng/ml] p-value

TETs 46 509.6(339.2) ± 456.1(67.2) 2.0(1.6) ± 1.7(0.3)

Volunteers 49 334.6(180.5) ± 351.7(50.2) 0.040b 1.3(1.1) ± 0.9(0.1) 0.021b

TC 20 635.9(419.3) ± 528.8(118.3) 2.4(2.0) ± 2.0(0.5)

Thymoma 26 412.5(279.8) ± 373.2(73.2) 1.7(1.5) ± 1.4(0.3)

Volunteers 49 334.6(180.5) ± 351.7(50.2) 0.021a 1.3(1.1) ± 0.9(0.1) 0.015a

TC 20 635.9(419.3) ± 528.8(118.3) 2.4(2.0) ± 2.0(0.5)

Volunteers 49 334.6(180.5) ± 351.7(50.2) 0.027b 1.3(1.1) ± 0.9(0.1) 0.034b

Thymoma 26 412.5(279.8) ± 373.2(73.2) 1.7(1.5) ± 1.4(0.3)

Volunteers 49 334.6(180.5) ± 351.7(50.2) 0.374b 1.3(1.1) ± 0.9(0.1) 0.161b

TETs 46 509.6(339.2) ± 456.1(67.2) 2.0(1.6) ± 1.7(0.3)

TH 33 464.6(312.8) ± 351.7(50.2) 1.3(1.1) ± 0.8(0.1)

Volunteers 49 334.6(180.5) ± 351.7(50.2) 0.115a 1.3(1.1) ± 0.9(0.1) 0.014a

TC 20 635.9(419.3) ± 528.8(118.3) 2.4(2.0) ± 2.0(0.5)

Thymoma 26 412.5(279.8) ± 373.2(73.2) 1.7(1.5) ± 1.4(0.3)

TH 33 464.6(312.8) ± 351.7(50.2) 0.236a 1.3(1.1) ± 0.8(0.1) 0.032a

TETs MG− 35 533.2(337.5) ± 475.5(80.4) 2.0(1.4) ± 1.9(0.3)

TETs MG+ 11 434.6(396.7) ± 398.8(120.2) 0.538b 1.9(1.9) ± 1.1(0.3) 0.836b

MG (TETs + TH) 26 507.5(392.5) ± 456.5(89.5) 1.5(1.4) ± 0.9(0.2)

Volunteers* 26 215.5(144.5) ± 182.8(35.9) 0.005b 1.1(0.8) ± 0.8(0.2) 0.088b

Masaoka-KogaStage

I 7 446.2(285.0) ± 525.8(198.7) 1.4(1.0) ± 1.0(0.4)

II 15 318.2(260.2) ± 280.9(72.5) 1.6(1.0) ± 1.6(0.4)

III 6 694.9(685.1) ± 452.4(184.7) 2.3(1.7) ± 2.0(0.8)

IV 18 638.3(412.0) ± 508.5(119.9) 0.153a 2.4(2.0) ± 1.9(0.4) 0.472a

Table 3. Serum concentrations of HSP27 and 70 in TETs, TH and MG. Characterization and categorization of patients with TETs according to type of TET, tumor invasiveness and presence of MG. HSP27 and 70 serum concentrations were elevated in patients with TETs, particularly in patients with TCs. There was no significant difference between TETs with or without paraneoplastic MG. In contrast separate analysis of patients with MG (70% thymomatous MG) showed significantly higher HSP27 and 70 serum concentrations compared to volunteers. n, number; HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; TETs, Thymic Epithelial Tumors; TC; Thymic Carcinoma; TH, Thymic Hyperplasia; MG, Myasthenia Gravis. aOne-way ANOVA. bIndependent-samples t-test. *Subgroup of 26 patients with MG was sex- and age-matched for separate analysis. mean (median) ± SD (SEM).

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expression gradually decreased from Masaoka-Koga tumor stage I to IV, indicating that a decrease of HSP70 expression goes along with increasing malignant tumor invasiveness and worse prognosis. Higher expression of HSPs in TETs of lower Masaoka-Koga stages is in line with SCC of the tongue where overexpression of HSP27 is associated with early tumor stages and grade 1 tumors38 and Non-Small Cell Lung Cancer (NSCLC) where over-expression of HSP27 and 70i were favorable prognostic factors for survival37. These observations are in contrast to findings of overexpression in carcinomas of other sites with worse prognosis: HSP70 overexpression in colon

Figure 6. Serum concentrations of HSP27 and 70 in patients with TETs, TH, MG and volunteers. Serum concentrations of HSP27 (A) and HSP70 (B) in patients with TETs compared to healthy volunteers are shown. Patients with TETs were further separated into patients with TC and thymoma. Serum concentrations for HSP27 (C) and HSP70 (D) are shown. Serum levels of HSP70 in patients with TETs compared to patients with TH and volunteers is shown (E). Analysis of HSP70 serum concentrations in patients with TC compared to thymomas and TH is shown (F). Serum concentrations of HSP27 in patients with MG compared to volunteers is shown (G). Spearman rho correlations for HSP27 (H) and HSP70 (I) with WHO classification is shown. Pearson correlation for HSP27 and HSP70 serum concentrations are shown (J). Paired T-Test was performed to analyze fold decrease of HSP27 and 70 serum concentrations after tumor resection (K). HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein70; TETs, Thymic Epithelial Tumors; TH, Thymic Hyperplasia; MG, Myasthenia Gravis; TC, Thymic Carcinoma; r, correlation coefficient.

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cancer with low tumor differentiation and advanced tumor stage34, strong expression of HSP27 and 60 in prostate cancer with shorter recurrence free survival35, or in HCC where high HSP70 expression correlated with higher tumor stage, greater tumor size and invasiveness36.

In our patient cohort: 1-, 5- and 10-year OS (and CSS) after primary tumor resection were 100% (100%), 96.5% (97.8%) and 90.9% (94.3%). Because of this excellent survival after complete resection with or without multimodality treatment in cases of advanced tumor stage41 and the fact that recurrences can occur even decades after primary tumor resection [Cumulative Incidence Rates (CIR) of 0.12 are reported42]-the biology of TETs is best represented by FFR43. We showed that 10-year FFR was significantly worse in TETs with weak HSP27 and 70 expression (87.5% and 60.0%, respectively) compared to TETs with stronger HSP27 and 70 expression (91.4% and 93.9%, respectively).

In contrast to gradually decreasing nuclear and cytoplasmic HSP70 expression in TETs, serum concentrations of HSP70 incrementally increased from Masaoka-Koga tumor stage I to IV. Similar observations were reported in patients with NSCLC with higher serum concentrations of HSP27 in stages IIIA-IV compared to stages I-II44.

Since there is no information on the expression of HSPs in TNETs in the peer reviewed literature we com-pare our results to HSPs in neuroendocrine tumors (NETs) of other origins. HSP90 was reported to be highly expressed for instance in pancreatic45 and pulmonary neuroendocrine tumors46. In stark contrast to the high HSP90 expression in NETs of other origins we found significantly lower expression of HSP27 and 70 in TNETs. We propose the following reasons for this discrepancy: TNETs in comparison to thymomas and NETs of other origins show a more aggressive behavior47 with 5-year CSS of 50% for well-differentiated, 20% for moderately differentiated and 0% for poorly differentiated TNETs48. The majority of TNETs present in advanced tumor stages (Masaoka-Koga Stage III-IV). TNETs showed significantly higher 5- and 10-year CIR (0.34 and 0.54, respectively) compared to thymomas12, confirming the more aggressive clinical behavior of these neoplasms.

Tumor-promoting inflammation was described as an enabling characteristic fostering multiple hallmarks of cancer: independence from growth factors, escape from programmed cell death, enhanced angiogenesis and metastasis32. Extracellular HSP27 and 70 act as danger signals providing a chronic inflammatory tumor envi-ronment that promotes tumor progression and invasion49. We found elevated serum concentrations of HSP 27 and 70 in patients with TETs and strong expression of HSP27 and 70 in macrophages of the tumor microenvi-ronment contrasting the finding of only few CD68/HSP70 double positive cells in fetal and adult non-neoplastic specimens. Longitudinal measurements showed that HSP27 serum concentrations were significantly decreased after complete surgical resection of TETs. This effect could be either related to direct release from tumor cells or indirect release from immune cells within the framework of cancer-related inflammation. Similar to our findings significantly decreased HSP27 concentrations were found after surgical resection of lung metastases in patients with metastatic colorectal cancer (CRC)50. HSP27 as a tumor marker is promising and warrants further studies.

Thymocytes that pass positive and negative selection are mature T-cells that will be released from the thy-mus. During this maturation process thymocytes migrate through the epithelial framework from thymic cortex to medulla51. Double-staining of fetal and adult regular thymic specimens showed that HSPs are constitutively expressed in TECs. The role of HSP27 and 70 in mTECs and cTECs, which form the three-dimensional frame-work in the thymus that enables T-cell maturation and development warrants future study that may improve our understanding of the basic mechanisms of the thymic microenvironment and T-cell maturation.

Involvement of HSPs in the pathogenesis of several autoimmune diseases, such as Systemic Lupus Erythematosus52, diabetes53 and multiple sclerosis54 were reported. Elevated levels of serum HSP70-antibodies could be found in patients with generalized and ocular MG55. Our finding of HSP27 and 70 expressing DCs and TECs forming marginal zone-like distributions in the GCs of thymus may add to the intrathymic pathogenesis of MG. Briefly, nicotinic AChRα epitopes are presented to autoreactive CD4+ thymic immigrants by thymic antigen presenting (possibly HSP expressing) DCs. This may lead to their activation and help in the stimulation of AChR-reactive B cells forming GCs that produce anti-AChR antibodies that is one of the hallmarks of MG development56.

Currently several HSP90 inhibitors are under investigation in preclinical and clinical phase I to III trials57. Promising results were reported while the exact mechanisms of their anti-cancer effects remain largely unknown. Especially the approach to use HSP90 inhibitors combined with other antineoplastic drugs has shown promising results58. Monotherapy with HSP inhibitors showed considerable adverse drug effects57. The observed toxicity of HSP inhibition may result from targeting the constitutive expression of HSP in rest of the body (investigated in our study the high expression in TECs, ECs, macrophages, fibroblasts and adipocytes). Combination of HSP inhibitors can reduce drug resistance and toxicity of chemotherapy57.

Limitations of the study. This study carries the weaknesses of other important studies done in orphan diseases59. The rarity of TETs and their vast array of different histological patterns, different tumor stages and association with autoimmune diseases does not allow for further significant categorization of patients. This is particularly limiting for studies on patient serum that are widely not routinely sampled. Orphan disease research on TETs and MG needs international efforts such as those of the European Society of Thoracic Surgeons (ESTS) Thymic Workgroup and the International Thymic Malignancy Interest Group (ITMIG)42,43. In order to maximize the number of available tumor tissue samples and to allow for long follow-up periods this study was carried out in a prospective and retrospective manner (median follow-up time for the analysis of tumor tissue analysis: 111.7 months). Because of excellent survival and late recurrences-up to 20 years in patients with TETs60-the reported follow-up periods are relatively short. The number of experimentally investigated TNETs is particularly small. A recent multi-institutional outcome study on TNETs (not experimental research on tumor tissue or serum) reported on 205 patients12.

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Summary. In summary, we showed for the first time that HSP27 and 70 are expressed in TETs, the thymic tumor microenvironment and regular thymus. Elevated serum concentrations were detected in patients with TETs and correlated with advanced tumor stages. Longitudinal analysis identified HSP27 serum concentration as a promising tumor marker. Altogether HSP27 and 70 may have diagnostic implications and may serve as molecular targets for therapy. Further studies are warranted for a more detailed understanding of HSPs in thymic malignancies, MG and thymic physiology.

Materials and MethodsStudy design and study population. We included 125 patients in this study who underwent thymectomy at the Department of Thoracic Surgery/Medical University Vienna between 1999 and 2014. Indications for sur-gery can be summarized as follows: 1) histologically verified TET, 2) radiological suspicion of TET (mediastinal mass) or 3) thymectomy in patients with MG.

According to histology specimens were classified as thymic malignancies or non-malignant thymic speci-mens (n = 24). We analyzed tissue specimens of 101 patients with TETs (thymoma: n = 83; TC: n = 15; TNET: n = 3) who underwent surgical resection. Mean age at surgery was 57.8 years (range 16.7–86.5). Sixty-one female (60.4%) and 40 male (39.6%) patients were included. TETs were further classified according to WHO classifica-tion system3. For the purpose of statistical analysis of this manuscript WHO mixed type thymomas were classified according to the more malignant part (e.g. B2/B3 thymoma was classified as B3 thymoma).

Non-malignant thymic specimens (n = 24) were distinguished between regular for age thymic morphol-ogy (n = 8), TTH (n = 10) and FTH (n = 6). Fetal and infantile thymic tissues for histological processing were obtained at autopsy.

Serum samples of 46 patients with TETs (11 MG pos., 35 MG neg.), 33 patients with TH (15 MG pos., 18 MG neg.) and 49 volunteers were available. For a subgroup of 16 patients with TETs pre- and postoperative serum samples were available (mean 13.94 months after surgery). For repeated measures patients had to fit to the follow-ing criteria: complete surgical resection of TET (R0), no adjuvant therapy during the last month, and no sign of recurrence or 2nd malignancy.

Ethical approval was obtained from the ethics committee of the Medical University Vienna and was conducted according to Good Scientific Practice. Written informed consent was obtained from all participating patients and volunteers and all experiments were carried out in accordance to the approved ethical guidelines.

Immunohistochemistry. Formaldehyde-fixed and paraffin embedded human benign and malignant thymic specimens were retrieved from the institutional department of pathology. Immunohistochemistry stain-ing was performed by using the automated Ventana Benchmark® platform (Ventana Medical Systems, Tucson, AZ, USA) according to standard protocol of the institutional department of pathology17. Monoclonal mouse anti-human HSP27 IgG2a (Santa Cruz, Dallas, Texas, USA), monoclonal mouse anti-human HSP70 IgG2a (Novus, Littleton, CO, USA) and anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used for staining. Omission of primary antibody served as negative control. Further information to antibody specificity of HSP27 and 70 are available as supplementary material.

Double-Staining. Sequential double-staining on formaldehyde-fixed and paraffin embedded human non-malignant thymic specimens was performed by using the automated Leica Bond III immunostainer platform (Leica Biosystems, Nussloch, Germany). The following antibodies were used: CD68 (clone PGM-1) mouse mon-oclonal antibody IgG3 (DAKO M876, Glostrup, Denmark), Lu-5 (pan cytokeratin) mouse monoclonal antibody IgG1 (Biocare Medical CM43C, Concord, CA, USA) and HSP70/HSPA1A mouse monoclonal antibody IgG2a (Novus Biological, NB600-571, Littleton, CO, USA).

Evaluation of immunoreactivity. Thymic Epithelial Tumors. For the evaluation of immunoreactivity we used a scoring system from 0 to 3 to assess staining intensity of HSP27 and 70 in TETs (0, no staining, 1, weak staining, 2, moderate staining, 3 strong staining)17. Analysis of immunoreactivity was performed by two observers blinded to Masaoka-Koga Tumor stage, presence of paraneoplastic MG and WHO type. Rating represents the overall impression/average of staining intensity of most of the tumor cells. In case that two ratings differed, the slide was reevaluated and discussed.

Non-Malignant Thymic Specimens. For the analysis of immunoreactivity in non-malignant thymic specimens a quantitative score was used (“+ ”, positive, “− ”, negative). Briefly, slides were screened at low magnification and cell types represented in non-malignant thymic tissues, such as ECs, Hassal’s corpuscles, lymphocytes, GCs, cor-tical, subcapsular and medullary TECs were evaluated.

Enzyme-linked immunosorbent assay (ELISA). HSP27 and 70 concentrations in serum samples were measured by using commercially available human HSP27 (R&D Systems, Minneapolis, MN, USA, DuoSet® Human HSP27, DY1580) and HSP70 ELISA kits (R&D Systems, Minneapolis, MN, USA, DuoSet® IC Total HSP70/HSPA1A, DYC1663). All ELISA tests were performed according to the Manufacturer’s instructions. Samples were measured in duplicates. Researchers performing the assays and data analyses were blinded to the groups associated with each sample.

Statistical methods. Statistical analysis of data was performed using SPSS software (version 21; IBM SPSS Inc., IL, USA). Mann-Whitney-U test and Kruskal-Wallis rank test were used to evaluate non-normal distribu-tions with two or more than two groups, respectively. Unpaired student’s t test and one-way ANOVA were used to compare means of two or more than two independent groups, for normal (Gaussian) distributions, respectively. Post hoc comparisons were computed with Tukey’s-B and Bonferroni correction. Paired student’s t-test was used

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for analyzing two dependent groups. The type of test used is indicated in the table and/or the result section. All data were reported as mean ± standard error of the mean (SEM) in the text of the results section and more detailed as mean (median) ± standard deviation (SEM) in tables. Chi-square test was used to compare binominal variables. The probability of making a type I error was set at α value of 0.05. The null hypothesis was rejected if the p-value was less than α . Two-tailed p-values were employed. Pearson correlation (r) was performed to analysis linear relationships between two numerical measurements. Spearman rho correlation (r) was used in case that one or both variables were ranked. Kaplan-Meier survival analysis and log-rank test were used to assess outcome measures for TETs, such as OS, CSS and FFR. GraphPad Prism6 (GraphPad Software Inc., California, USA) was used for graphical display of all box plots, Kaplan-Meier curves and correlations in this manuscript.

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AcknowledgementsWe thank Andrea Alvarez Hernandez for technical help with immunohistochemical techniques and Kevin Hubner for help with the database management. We further want to thank the Christian Doppler Laboratory for the Diagnosis and Regeneration of Cardiac and Thoracic Diseases and the research laboratories (ARGE Moser, FOLAB Chirurgie) of the department of surgery, Medical University Vienna for funding.

Author ContributionsConceived and designed the experiments: S.J., A.I.S., H.J.A. and B.M. Performed the experiments: S.J., A.I.S., C.B., P.H., T.H. and B.M. Analyzed the data: S.J., A.I.S., C.B., L.M., H.J.A. and B.M. Contributed reagents/materials/analysis tools: A.I.S., W.K., L.M., H.J.A. and B.M. Wrote the paper: S.J., A.I.S., L.M., H.J.A. and B.M.

Additional InformationSupplementary information accompanies this paper at http://www.nature.com/srepCompeting financial interests: The authors declare no competing financial interests.How to cite this article: Janik, S. et al. HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications. Sci. Rep. 6, 24267; doi: 10.1038/srep24267 (2016).

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license,

unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

87

Supplementary Material

HSP27 and 70 expression in thymic epithelial tumors and

benign thymic alterations: diagnostic, prognostic and

physiologic implications

S. Janik1,2, A. I. Schiefer3, C. Bekos1,2, P. Hacker1,2, T. Haider2,4, W. Klepetko1, L.

Müllauer3, H. J. Ankersmit1,2, B. Moser1*

1Department of Thoracic Surgery, Division of Surgery, Medical University Vienna, Austria

2Christian Doppler Laboratory for the Diagnosis and Regeneration of Cardiac and Thoracic

Diseases, Medical University Vienna, Austria

3Clinical Institute of Pathology, Medical University Vienna, Austria

4University Clinic for Trauma Surgery, Medical University Vienna, Austria

*corresponding author

Bernhard Moser, MD, PD, Assoc. Prof., FEBTS

Department of Thoracic Surgery, Division of Surgery, Medical University Vienna

Währinger Gürtel 18-20, 1090 Vienna, Austria

FAX: +43 1 40400 69770

Tel: +43 1 40400 67770

E-mail: bernhard.moser@meduniwien.ac.at

88

Supplementary Table 1

Immunohistochemical markers

WHO Cases n(F:M)

HSP27 +/-/n.p.

HSP70 +/-/n.p.

Lu 5 +/-/n.p.

CD5 +/-/n.p.

CD20 +/-/n.p.

NCAM +/-/n.p.

c-Kit +/-/n.p.

Chromo +/-/n.p.

Pax 8 +/-/n.p.

CK5/6 +/-/n.p.

A 12 (8:4) 12/0/0 11/0/0* 12/0/0 0/10/2 3/2/7 0/1/11 0/8/4 0/1/11 0/0/12 1/0/11 AB 11 (9:2) 11/0/0 11/0/0 10/0/1 1/8/2 4/7/0 0/1/10 1/9/1 0/0/11 0/1/10 0/0/11 B1 11 (7:4) 11/0/0 11/0/0 11/0/0 0/8/3 0/10/1 0/1/10 0/7/4 0/0/11 0/0/11 2/0/9 B2 17 (7:10) 17/0/0 17/0/0 14/0/3 1/12/4 3/11/3 0/1/16 2/7/8 0/0/17 1/0/16 2/0/15 B3 13 (7:6) 13/0/0 13/0/0 12/0/1 0/11/2 0/7/6 1/1/11 0/10/3 1/1/11 0/0/13 1/0/12 TC 15 (5:10) 15/0/0 15/0/0 12/0/3 10/5/0 0/7/8 1/9/5 11/2/2 0/5/10 0/1/14 8/1/6

MNT 4 (2:2) 4/0/0 4/0/0 4/0/0 0/4/0 0/4/0 0/2/2 0/4/0 0/1/3 0/0/4 0/0/4 TNET 3 (2:1) 1/2/0 2/1/0 2/0/1 0/3/0 0/1/2 2/0/1 1/0/2 3/0/0 0/0/3 0/1/2 Total

Number 86

(47:39) 84/2/0 84/1/0* 77/0/9 12/61/13 10/49/27 4/16/66 15/47/24 4/8/74 1/2/83 14/2/70

Suppl. Table 1. HSP staining and immunohistochemical markers that have been

used during routine diagnostic workup of TETs and TNETs. HSP27 and 70 were

expressed in 100% of thymomas and TCs, respectively. The epithelial marker

pancytokeratin Lu5 was expressed in 100% of TETs thus correlated with 100%

expression of HSP27 and 70 in thymomas and TCs (p<0.001; r=1.0).

Immunohistochemical markers such as CD5, CD20, NCAM, c-Kit, Chromogranin A, Pax

8 and CK5/6 were only individually performed for diagnostic purposes and were found

as follows: 17.1%, 20.8%, 11.1%, 23.0%, 11.1%, 33.3% and 93.3% of cases,

respectively.

In well-differentiated TNETs (n=3) HSP27 and 70 were expressed in 33.3% and

66.6%, respectively. Similar to Lu5 expression in thymomas and TCs, Lu5 was

expressed in 100% of TNETs. The expression of CD5, CD20, Pax8, CK5/6 c-Kit, NCAM

and Chromogranin A, in TNETs are detailed above.

WHO, World Health Organization; n(F:M), number(Female:Male);TC, Thymic

Carcinoma; MNT, Micronodular Thymoma; TNET, Thymic Neuroendocrine Tumor; HSP

27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; Lu5, Pancytokeratin;

NCAM/CD56, Neural Cell Adhesive Molecule; Chromo, Chromogranin A; Pax8, Paired

89

box protein 8; CK5/6, Cytokeratin 5/6. +/-/n.p., positive/negative/not performed. *one

case was excluded because of tissue quality

90

Supplementary Table 2

HSP expression in cells of the tumor microenvironment Clinicopathologic cyto HSP27 expression (mean±SEM)

characteristics n ECs n Fibro n Adipo n Macro

WHO A 15 2.73±0.1 16 2.16±0.2 13 1.88±0.2 14 1.93±0.2 AB 14 2.89±0.1 14 2.29±0.1 13 2.08±0.2 9 2.44±0.2 B1 9 2.89±0.1 9 2.50±0.3 9 1.61±0.3 7 2.29±0.3 B2 21 2.76±0.1 21 2.05±0.1 20 1.75±0.1 15 2.27±0.1 B3 17 2.79±0.1 17 1.97±0.2 16 1.59±0.2 15 2.23±0.1 TC 15 2.77±0.1 15 2.47±0.1 13 2.04±0.2 10 2.55±0.2

MNT 4 2.75±0.3 4 2.0±0.4 3 2.17±0.2 0 - TNET 3 3.0±0.0 3 2.83±0.2 3 1.67±0.3 2 2.0±0.0

p-value 0.879a 0.137a 0.300a 0.321a

Masaoka I 23 2.74±0.1 23 2.22±0.1 21 1.74±0.1 18 2.33±0.1 Koga II 46 2.85±0.0 46 2.16±0.1 43 1.88±0.1 31 2.27±0.1 Stage III 14 2.68±0.1 15 2.03±0.2 13 2.0±0.2 11 2.09±0.2

IV 15 2.87±0.1 15 2.57±0.2 13 1.62±0.2 12 2.21±0.2

p-value 0.343a 0.119a 0.355a 0.784a

MG Pos. 29 2.88±0.1 29 2.16±0.1 28 1.70±0.1 23 2.43±0.1 Neg. 69 2.77±0.0 70 2.24±0.1 62 1.89±0.1 49 2.16±0.1

p-value 0.126b 0.583b 0.181b 0.090b

cyto HSP70 expression (mean±SEM)

n ECs n Fibro n Adipo n Macro

WHO A 15 0.93±0.2 16 1.03±0.2 13 0.77±0.2 13 1.23±0.2 AB 14 1.32±0.2 14 1.71±0.2 12 0.79±0.2 9 2.28±0.2 B1 10 0.65±0.2 10 1.20±0.3 10 0.40±0.2 8 2.19±0.2 B2 21 1.40±0.2 21 1.12±0.2 20 0.40±0.1 15 1.53±0.2 B3 17 0.91±0.1 17 0.35±0.1 16 0.31±0.1 16 1.50±0.2 TC 15 0.87±0.2 15 1.20±0.2 13 0.69±0.2 13 1.38±0.3

MNT 4 0.63±0.1 4 1.0±0.5 3 0.67±0.4 0 - TNET 3 1.33±0.3 3 1.17±0.2 3 0.33±0.3 2 2.0±0.5

p-value 0.106a 0.001a 0.366a 0.007a

Masaoka I 23 1.11±0.2 23 1.26±0.2 20 0.55±0.2 18 1.81±0.2 Koga II 47 1.03±0.1 47 0.98±0.1 44 0.45±0.1 32 1.66±0.1 Stage III 14 1.36±0.2 14 1.33±0.2 13 0.88±0.2 14 1.43±0.2

IV 15 0.70±0.2 15 0.83±0.2 12 0.46±0.1 12 1.46±0.1 p-value 0.156a 0.188a 0.215a 0.476a

MG Pos. 29 1.16±0.2 29 0.98±0.2 27 0.44±0.1 22 1.57±0.2 Neg. 70 1.0±0.1 71 1.11±0.1 63 0.58±0.1 54 1.64±0.1

p-value 0.447b 0.522b 0.376b 0.718b

Suppl. Table 2. HSP expression in cells of the tumor microenvironment. HSP27

and 70 were expressed in ECs, fibroblasts, adipocytes and macrophages but were

missing in lymphocytes. Strongest HSP27 staining was found in ECs. Neither Masaoka-

91

Koga tumor stage nor paraneoplastic MG had influence on staining intensity of cells in

the tumor microenvironment. HSP27, Heat Shock Protein 27; HSP70, Heat Shock

Protein 70; n, number; cyto, cytoplasmic expression; WHO, World Health Organization;

TC, Thymus Carcinoma; MNT, Micronodular Thymoma; TNET, Thymic Neuroendocrine

Tumor; MG, Myasthenia Gravis; ECs, Endothelial Cells; Fibro, Fibroblasts; Adipo,

Adipocytes; Macro, Macrophages; SEM, standard error of the mean; a one-way Anova,

b independent-samples t-test.

92

Supplementary Table 3

HSP expression in cells of non-malignant thymic specimens

Characteristics HSP27 HSP70

Case Group Diagnosis Sex Age MG Lym HC mTEC cTEC GC MZ ECs Lym HC mTEC cTEC GC MZ ECs

1 Fetal Reg. Thy. F 20w - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. + 2 Fetal Reg. Thy. F 22w - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

3 Fetal Reg. Thy. M 22w - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. + 4 Infantil Reg. Thy. F 1a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

5 Infantil Reg. Thy. F 1a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

6 Infantil Reg. Thy. F 14a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

7 Adult TTH F 40a + - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

8 Adult TTH M 40a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. + 9 Adult Reg. Thy. F 35a + - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

10 Adult TTH F 27a + - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

11 Adult Reg. Thy. F 34a + - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

12 Adult TTH M 30a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

13 Adult TTH M 48a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. + 14 Adult TTH F 26a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

15 Adult TTH F 21a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

16 Adult TTH M 57a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

17 Adult TTH M 55a - - + + + n.f. n.f. + - + +/+ +/+ n.f. n.f. +

18 Adult TTH M 72a - - + + + n.f. n.f. n.f. - + +/+ +/+ n.f. n.f. n.f. 19 Adult FTH F 22a + - + + + + + + - + +/+ +/+ +/+ +/+ +

20 Adult FTH F 29a + - + + + + + + - + +/+ +/+ +/+ +/+ +

21 Adult FTH M 29a + - + + + + + + - + +/+ +/+ +/+ +/+ +

22 Adult FTH M 15a + - + + + + + + - + +/+ +/+ +/+ +/+ +

23 Adult FTH F 17a + - + + + + + + - + +/+ +/+ +/+ +/+ + 24 Adult FTH M 19a + - + + + + + + - + +/+ +/+ +/+ +/+ +

Suppl. Table 3. HSP27 and HSP70 expression in thymic physiology. HSPs were

expressed in all medullary and cortical TECs, HCs and ECs. In myasthenic patients with

FTH HSPs were expressed in DCs of GCs and in TECs with marginal zone like

distribution. HSP27, Heat Shock Protein 27; HSP70, Heat Shock Protein 70; F, Female;

M, Male,w, age in weeks of pregnancy; a, age in years; Reg. Thy.,Regular Thymus for

age; TTH, True Thymic Hyperplasia; FTH; Follicular Thymic Hyperplasia; MG,

Myasthenia Gravis; Lym, Lymphocytes; HC, Hassall’s Corpuscles; mTEC, medullary

Thymic Epithelial Cells; cTEC, cortical Thymic Epithelial Cells; GC, Germinal Center;

93

MZ, TECs with marginal zone like distribution; EC, Endothelial Cells. +, positive staining;

-, negative staining; n.f., not found; +/+, positive nuclear and cytoplasmic staining.

94

Supplementary Table 4

HSP serum concentrations

Masaoka-Koga Stage

HSP27 [pg/ml] HSP70 [ng/ml] n mean(median)±SD(SEM) p-value mean(median)±SD(SEM) p-value

I-II 22 358.9(267.4)±367.8(78.4) 1.6(1.0)±1.4(0.3) III-IV 24 647.8(429.4)±491.7(100.4) 0.028b 2.4(1.9)±1.9(0.4) 0.111b

Invasive (II-IV) 39 521.0(340.8)±449.2(71.9) 2.1(1.7)±1.8(0.3) Non-Invasive (I) 7 446.2(285.0)±525.8(198.7) 0.789b 1.4(1.0)±1.0(0.4) 0.203b

Local (I-III) 28 426.9(311.3)±407.1(76.9) 1.7(1.0)±1.6(0.3) Metastasis (IV) 18 638.3(412.0)±508.5(119.9) 0.148b 2.4(1.9)±1.9(0.4) 0.203b

Suppl. Table 4. Further stratification of HSP27 and 70 serum concentrations in

patients with TETs according to Masaoka-Koga Stage. n, number; HSP27, Heat

Shock Protein 27; HSP70, Heat Shock Protein 70; TETs, Thymic Epithelial Tumors;

b independent-samples t-test.

95

Supplementary Figure S1

Supplementary Fig.1. HSP27 expression in TETs. Immunohistochemical staining of

HSP27 in different histologic tumor subtypes was performed. Expression of HSP27 in

thymoma WHO type A (A), A-component type AB (B), B1admixed with B2 (C), B2 (D),

96

B3 (E), TC - SCC (F), MNT (G) and TNET (H) is shown (630x

magnification).Hematoxylin was used for counterstaining. HSP27, Heat Shock Protein

27; TETs, Thymic Epithelial Tumors;WHO, World Health Organization; TC, Thymic

Carcinoma; SCC, Squamous Cell Carcinoma; MNT, Micronodular Thymoma; TNET,

Thymic Neuroendocrine Tumor.

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Supplementary Figure S2

Supplementary Fig.2. HSP70 expression in TETs. Immunohistochemical staining of

HSP70 in different histologic tumor subtypes was performed. Expression of HSP70 in

thymoma WHO type A (A), A-component type AB(B), B1 (C), B2 (D), B3 (E), TC - SCC

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(F), MNT (G) and TNET (H) is shown (630x magnification).Hematoxylin was used for

counterstaining. HSP70, Heat Shock Protein 70; TETs, Thymic Epithelial Tumors;WHO,

World Health Organization; TC, Thymic Carcinoma; SCC, Squamous Cell Carcinoma;

MNT, Micronodular Thymoma; TNET, Thymic Neuroendocrine Tumor.

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Supplementary Figure S3

Epithelial cells that undergo malignant transformation retain their physiologic keratin

profiles (1). Cytokeratins are helpful to distinguish TETs from other common anterior

mediastinal tumors, such as lymphomas or germ cell tumors (1,2).

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Supplementary Fig.3. Selected immunohistochemical markers used for routine

diagnosis of TETs.Cytokeratins 5/6 are generally expressed in TECs and indeed 60-

100% of TETs express CK5/6. Lu5/pancytokeratin mouse monoclonal antibody detects

both types (acidic and basic) of cytokeratin subfamilies In our study CK5/6 and

Lu5/pancytokeratin were expressed in 87.5% and 100% of TETs, respectively. In

comparison HSP27 and 70 were expressed in 100% of TETs but either absent or weakly

expressed in TNETs. Expression of HSP27 (A), HSP70 (B), Pax 8 (C), CK5/6 (D), Lu 5

(E) and CD5 (F) in WHO type B2 thymoma is shown (400x magnification). HSP27, Heat

Shock Protein 27; HSP70, Heat Shock Protein 70; Pax 8, Paired box protein 8; CK5/6,

Cytokeratin 5/6; Lu 5, pancytokeratin.

The following antibodies were used: monoclonal mouse anti-human Lu-5

(pancytokeratin) IgG1 (Biocare Medical, Concord, CA, USA)monoclonal mouse anti-

human CD5 IgG1κ(Clone 4C7, LEICA (NovoCastra), Nussloch, Germany), monoclonal

mouse anti-human CD20 IgG2α (Clone L26, DAKO, Glostrup, Denmark), monoclonal

mouse anti-human CD56/NCAM IgG1 (Clone 1B6, LEICA (NovoCastra), Nussloch,

Germany), polyclonal rabbit anti-human CD117/c-Kit (DAKO, Glostrup, Denmark),

monoclonal mouse anti-human CK5/6 (Clone D5/16B4, EUBIO, Wien, Austria),

monoclonal mouse anti-human Chromogranin A (Clone 5H7, LEICA(NovoCastra),

Nussloch, Germany) and polyclonal rabbit anti-human PAX8 (EUBIO, Wien, Austria).

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Supplementary Information about antibody specificity to HSP27 and 70

A set of rules was used to select antibodies to HSP27 and 70 (4). The antibodies were

raised against published antigens and tested on whole cell lysates. The monoclonal

HSP70/HSPA1A Antibody (W27) was tested by western blot on transfected A549 cells

(5). The monoclonal HSP 27 (F-4) antibody was tested by western blot analysis of HSP

27 expression in mouse HSP 27 transfected 293T and ECV304 whole cell lysates (6).

Supplementary References

(1) Chu PG and Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology 40,

403-439 (2002).

(2) Chu, P. G. &Weiss, L. M. Expression of cytokeratin 5/6 in epithelial neoplasms. Mod Pathol 15,

6-10 (2002).

(3) Schröder, S., Wodzynski, A. &Padberg, B. Cytokeratin expression of benign and malignant epithelial

thyroid gland tumors. An immunohistologic study of 154 neoplasms using 8 different monoclonal

cytokeratin antibodies. Pathologe. 17(6), 425-32 (1996).

(4) Saper, C. P. A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem. 57(1),

1-5 (2009).

(5) http://www.novusbio.com/PDFs/NB600-571.pdf (11.02.2016)

(6) http://datasheets.scbt.com/sc-59562.pdf (11.02.2016)

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Chapter III: Discussion 3.1 General Discussion Malignancies represent a global burden of civilization and with approximately 14

million new cases per year and more than 8 million cancer related deaths in 2012,

cancer represents a leading cause of morbidity and mortality worldwide.275 The

WHO expects that the number of new cases will rise by about 70% over the next 20

years with increasing annual cancer cases from 14 million 2012 to 22 million per

year in the next two decades. It is a declared aim of the WHO to improve our

knowledge about risk factors and pathogenesis of malignancies, as well as about

interventions and prognostic factors in order to reduce the number of cancer related

deaths and to increase the chance of curative treatment options by early detection

and adequate treatment.276

Screening and early diagnosis are two main strategies for early cancer

detection and reduction of cancer mortality. Screening methods aim to identify

those individuals with higher risks for developing cancer and to ensure prompt

diagnosis and treatment if necessary. If there are no established screening

methods, early diagnosis is essential. Unfortunately, in absence of symptoms or in

case that malignancies are characterized by long asymptomatic periods, like it is

the case in TETs, patients are diagnosed at advanced stages when curative

treatment is often not feasible.28 Thus, it is essential to improve our knowledge

about the pathogenesis of different malignancies in order to identify possible risk

factors, to establish prognostic biomarkers and of course also to develop new

treatment options. Altogether, these findings should help clinicians to better identify

patients at risk and to predict their outcome and prognosis.

Despite considerable successes in identifying molecular changes of cancers,

there had been less progress in development and establishment of tools to identify

those patients who might benefit from certain treatment options.277 Translational

medicine with special interest in oncologic research is a relative new area of basic

research and mainly focuses on fostering the transfer of new scientific results from

bench to beside; or to say it with other words, to make new innovative findings for a

broad majority of patients feasible and useful. Especially tumor biomarkers are

considered as key strategies for personalized and individual medical therapy.278

Tumor biomarkers are defined as substances, including proteins, hormones,

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enzymes or oncogene products, which are presented in or produced by cancer cells

themselves or produced by the host microenvironment and immune system in

response to tumorigenesis or tumor progression. Such biomarkers could represent

effective tools for tumor detection, diagnosis, treatment and therapeutic monitoring

and could help to predict outcome.278

However, let’s consider the current situation of thymic malignancies, which

are the most common mediastinal tumors in adults. Nonetheless, they represent

orphan malignancies due to their relative low incidences that ranges from only 1.3

to 3.2 per 1.000.000 person-years.23,24 Complete tumor resection (R0) is the main

aim in therapy of TETs and completeness of resection, represents together with

WHO classification and Masaoka-Koga tumor stage the main prognostic factors for

patients with TETs. One-third of patients with TETs are asymptomatic at time of

diagnosis, while the majority of the remaining symptomatic two-third of patients

show symptoms related to paraneoplastic MG. Moreover, TETs are also

characterized by tumor recurrences occurring in 10-30% of cases even decades

after initial radical tumor resection (R0). So far, neither risk factors nor biomarkers

haven been identified for diagnosis and detection of TETs as well as for tumor

recurrences and to predict prognosis. Thus, lifelong surveillance with repeated

chest CT is recommended for patients with TETs and is necessary for clinicians to

detect tumor recurrences at early stages.64 Nonetheless, whenever tumor

recurrence is suspected biopsy or even surgery is necessary for confirmation or

exclusion of recurrence. Altogether the pathogenesis of TETs still remains an

enigma and sensitive and reliable biomarkers are urgently needed.

Oncoproteomics provide hope and are considered as solution to identify

novel biomarkers for malignancies that are characterized by absence or lack of

specific symptoms in early tumor stages, limited understanding and knowledge of

pathogenesis, etiology and oncogenesis.230,279 Interestingly, several oncoproteomic

studies discovered increased levels of HSPs in diverse cancer cells compared to

healthy cells.230 Indeed, HSPs have been discovered in several solid tumors, such

as lung cancer, colon cancer, prostate cancer or hepatocellular cancer and let us

assume that HSPs might be also involved in TETs.254-256,280

The following study and thesis was conducted to investigate the role HSPs in

TETs, thymic hyperplasia and paraneoplastic MG. In the first part of the study we

performed immunohistochemical stainings of HSP27 and 70 in patients with TETs,

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including thymomas, TCs and TNETs, and used a semiquantitative score to assess

staining intensities of tumor cells. Moderate to strong HSP expression was found in

all thymomas and TCs but not in TNETs. Possible reasons for this phenomenon will

be discussed below. Strong expression of HSPs in TETs is in accordance to diverse

studies that described overexpression of HSPs for colon cancer, prostate cancer,

hepatocellular cancer, lung cancer or breast cancer.254-256,280,281 It’s widely assumed

that overexpression and upregulation of HSPs in diverse malignancies might be

related to their ability to inhibit apoptosis and to promote tumor survival and

progression.225

Interestingly, cytoplasmic and nuclear HSP70 tumor expression decreased

continuously from non-invasive to metastatic TETs; or to say it in other words,

highest HSP70 expression was found in non-invasive, complete encapsulate TETs,

while the weakest expression was found in metastatic TETs. Conversely, we found

homogenous HSP27 expression within all TETs regardless of tumor stage.

Altogether we could show that HSP70 expression in neoplastic TETs is strongly

associated with tumor stage, thus indicating tumor behavior and helps to predict

outcome. Our data are in line with literature, where inverse correlation between

HSP70 tumor expression and tumor stage has been described also for other lung

cancer, melanoma or oral cancer.254,282,283

According to data of diverse in-vitro studies Sherman et al. assumes that

HSP70 is crucial for tumor initiation and at early stages of tumor development.284

For instance, in mammary epithelial cells expression of oncogene Her2 triggered

development of invasive breast cancer with 100% penetrance, while Her2

expression in HSP70 knockout mice rarely triggered cancer development.285

Interestingly it could be shown that HSP70 expression in cancer cells is important

for survival and growth, while normal cells didn’t show such dependences on

HSP70.286,287 Altogether, overexpression of HSP70 in early tumor stages indicate

an important role of HSP70 in tumor initiation, but whether this upregulation is

caused by the increased necessity of inhibition of apoptosis in early tumor stages or

by the increased necessity of chaperone activity of HSP70 needs to further

evaluated. However, we could provide strong evidence that HSP70 is involved in

tumor initiation of TETs.

Next we wanted to evaluate whether HSP expression in neoplastic TECs has

also impact on prognosis. Therefore we used a semiquantitative score to assess

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staining intensity in TETs and analyzed OS, CSS and FFR in accordance to

staining intensities. HSP27 and 70 expression was significantly weaker or even

missing in TNETs, while thymomas and TCs showed constantly moderate to strong

expression. Most remarkably, tumor recurrences occurred significantly earlier in

TETs with weak cytoplasmic HSP27 and nuclear HSP70 expression respectively, in

comparison to TETs with moderate to strong HSP expression. Ten year FFR was

87.5% and 60.0% for TETs with weak cytoplasmic HSP27 and nuclear HSP70

expression respectively, while 10-year FFR was significantly better in TETs with

strong HSP expression (HSP27: 91.4% and HSP70: 93.9%, respectively). Our data

are in line with several reports which also showed that high HSP tumor expression

represents a favorable prognostic factor that is accompanied by good survival,

including small and non small cell lung cancer, large cell neuroendocrine cancer,

osteosarcoma, oral SCC, esophageal cancer and bladder cancer.254,288-293

Nonetheless, our results are in contrast to diverse studies that reported

worse prognosis in patients with HSP overexpression, such as colon cancer,

prostate cancer, hepatocellular carcinoma or pulmonary metastases of primary

colorectal cancer.255-257,267 While the overexpression of HSPs in cancer cells can be

explained by their ability to inhibit apoptosis and by their function as chaperones in

order to maintain cellular homeostasis and to prevent cell death, the lower

expression of HSPs in advanced tumor stages and its correlation with worse

prognosis is at first surprising, but could be explained by the antitumor effects of

HSPs. Interestingly, it has been shown that HSPs could interact with the immune

system and cancer cell derived HSP70 could promote the generation of specific

cytotoxic CD8+ T-cells in both mice and humans.294-296 Due to their function as

molecular chaperones, HSPs can bind and promote tumor related antigens and

present them to APCs, similarly to major histocompatibility complexes in vivo and in

vitro resulting in stimulation of immune response.297,298 Altogether HSPs could

promote specific anti-tumor immunity, trigger production of cytotoxic CD8+ T-cells

and activation of NK-cells, which ends up in inhibition of tumor growth.299,300

Thus HSPs have dual functions in malignancies. Generally, HSP expression

is necessary for tumor initiation and tumor growth, while otherwise overexpression

could also foster increased anti-tumor immune response which in turn causes

impaired tumor growth.294 Therefore loss of HSP expression in cancer cells may

decrease anti-tumor immune response and may foster tumor promotion and

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metastasis. Consistent with that it’s not surprising that HSP70 expression in

esophageal cancer inversely correlated with depth of tumor invasion and blood

vessel invasion and positively correlated with CD4+ and CD8+ T-cells and B-cells

numbers and better OS.292 Moreover HSP70 expression in breast cancer patients

was significantly lower in those patients with lymph node metastases compared to

those without metastases at time of diagnosis. Interestingly, all patients that

suffered from tumor recurrence, metastases and/or died within 2 years had primary

tumors with low HSP70 expression.294 Torrenteguy et al. further suggests that those

selected tumor clones that managed to progress with lower amounts of HSPs,

related with lower anti-tumor immune responses, are associated with higher

malignant potential and metastases.294

As already mentioned above, TNET showed significantly weaker HSP

expression, which was in stark contrast to generally strong HSP expression in

thymomas and TCs. Hence, we next examined literature to find possible

explanations for this phenomenon of low HSP expression in TNETs. Despite the

function of HSPs in NETs has been less investigated in literature, few reports

reported high expression of HSP90 in pancreatic and pulmonary neuroendocrine

tumors, which was in contrast to our results.301,302 We consider that following

reasons may be responsible for this difference. First, TNETs are characterized by a

more aggressive behavior compared to thymomas and NETS of other origins, which

is supported and confirmed by the fact that the vast majority of TNETs present with

advanced tumor stages at time of diagnosis (Masaoka-Koga Stage III-IV).303 Worse

5-year CSS of 50%, 20% and 0% has been described for well-differentiated,

moderately-differentiated and poorly-differentiated TNETs, respectively. Further 5-

and 10-year cumulative-incidence rates (0.34 and 0.54, respectively) were also

significantly worse in TNETs compared to thymomas.165,304 Altogether the higher

aggressiveness of TNETs compared to thymomas is unquestioned but the reason

for lower HSP expression remains elusive. Second, beside strong expression of

HSPs in neoplastic TECs, we found also strong HSP27 and 70 expression in TECs

of non-malignant thymic specimens. TNETs, in contrast to other TETs, origin from

neuroendocrine thymic epithelial cells, which express neuroendocrine markers and

which are completely different compared to “normal TECs”. Thus the different origin

of tumor cells might be a further explanation for the weak or even missing

expression of HSPs in TNETs. However, whether HSP is causal related to the

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pathogenesis of TNETs or whether lower HSP expression is only an incidental

finding is unclear. However our data of TNETs indicate that decrease of HSP

expression in tumor cells seems to be related to decreased anti-tumor immune

response, which in turn fosters tumor promotion and metastases.

In stark contrast to decreasing HSP expression in advanced TETs, we found

highest HSP serum concentrations in those patients with advanced tumor stages

(Masaoka-Koga III-IV) and patients with TCs. Interestingly, the function of HSPs

mainly depends on their cellular localization: (1) intracellular HSPs are molecular

chaperones and potent inhibitors of apoptosis, which promote cellular survival, (2)

extracellular HSPs have mainly pro-inflammatory functions, while (3) membrane

bound HSPs stimulates immune response as already mentioned above. A recent

review form Juhasz K et al. (2014) about the different functions of HSP70 in

metastatic cancer revealed that the function of extracellular HSP70 mainly depends

on interaction with corresponding receptors on cells of the tumor

microenvironment.305

Briefly, HSP70 could act as danger signal by binding to Toll-like receptors

(TLR) on leukocytes, leading to activation and in turn secretion of pro-inflammatory

cytokines and NO (nitrogen oxide). Further HSP70 could also activate NK-cells or

cytotoxic CD8+ T-cells via stimulation of APC, but on the other hand HSP70 could

also suppress T-cell response by recruitment and activation of Tregs. Conversely to

HSP70, our knowledge about extracellular HSP27 is limited; however it could be

shown that extracellular HSP27 also binds to TLR2 and TLR4, which triggers

activation of immune cells and results, similar to HSP70, in pro-inflammatory

signals.306 Another important function of extracellular HSP27 is enhancing of

angiogenesis, which is of particular interest for oncologic research. It was shown

that extracellular HSP27 activates NFκB, which in turn stimulates expression of

proangiogenic factors, such as VEGF and IL-8 in endothelial cells and causes

increased vessel development.274 Schweiger et al. (2015) could already show that

HSP27 expression in tumor stroma of pulmonary metastases of colorectal cancer

was associated with microvessel density in metastases and worse outcome.267

Altogether, extracellular HSP27 and 70 are pro-inflammatory mediators that

foster chronic inflammation, which in turn increases the risk of cancer formation,

tumor progression and metastatic spread.307 Chronic inflammation is known to

permanently activate immune cells, which leads to continuous secretion of

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cytokines and chemokines that function as survival, proliferation and growth signals

for malignant cells and finally promote.307 Beside that chronic inflammation

promotes motility and invasiveness of malignant cells, enhanced angiogenesis,

escape from apoptosis or independence of growth factors and represents a

hallmark in cancer development.308-310 So far, elevated HSP27 and 70 serum levels

have been described in patients with breast cancer, ovarian cancer, colon cancer,

lung cancer or esophageal SCC compared to healthy controls.311-315 In accordance

to literature, we also detected highest HSP serum levels in TETs with higher tumors

stages and WHO type B3 thymomas and TCs, both tumor subtypes, which are

characterized by more malignant tumor behavior, higher aggressiveness and

altogether worse ass prognosis. Most remarkably, we discovered completely

inverse results of HSP70 serum levels and HSP 70 tumor expression. Indeed,

HSP70 tumor expression gradually decreased from stage I to IV TETs, while

HSP70 serum concentrations gradually increased with tumor stage. Concordant to

the thesis of cancer related chronic inflammation, we discovered strong expression

of both HSPs in macrophages of tumor microenvironment, whereas in non-

malignant thymic specimens only few macrophages showed double positive

staining of CD68/70.

Longitudinal analysis of pre -and postoperative serum of patients with TETs

revealed that HSP serum levels decreased after radical tumor resection (R0), which

were similar to serum concentrations of the control group. Similarly, Schweiger et

al. also reported significant decrease of HSP27 serum concentrations after

metastasectomy of colorectal lung metastases.267 However, the exact origin of

extracellular HSPs in TETs remains unclear and whether cancer cells directly

release HSPs or whether HSPs are produced from immune cells within the

framework of chronic inflammation is unknown. Nonetheless, we could show that

HSP serum levels are principally elevated in patients with TETs and that radical

tumor resection leads to decrease of HSP27 and 70 serum concentrations beneath

physiologic levels. Altogether our results strongly indicate also a systemic role of

HSPs in the pathobiology of thymic malignancies.

In addition to thymic malignancies, we also assessed the function of HSPs in

paraneoplastic MG and thymic physiology. Basically, the thymus is responsible for

T-cell maturation, which is controlled and coordinated by a specific arrangement of

TECs forming a three-dimensional framework. During this maturation process

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thymocytes have to migrate from thymic cortex to medulla and pass positive and

negative selection. Those thymocytes that passed this framework are released from

the thymus as mature naïve T-cells.316 Homogenous expression of HSP27 and 70

in medullary and cortical TECs of all non-malignant thymic specimens indicate a

basal function of HSPs also in normal thymus and T-cell maturation. Of course

exact function of HSPs in the complex framework of T-cell maturation remains

unclear, but however we could demonstrate that HSPs are possible markers for

TECs and further studies are definitive necessary to improve our knowledge

regarding the basic functions of HSPs in the complex framework of thymic

physiology and T-cell maturation.

MG is commonly associated with thymic pathologies, such as thymic

hyperplasia or TET that occur in the majority of myasthenic patients.27 FTH,

characterized by the presence of GCs, is the main pathologic finding in most

patients with MG and led to the assumption that GC formation is crucial for the

development of MG. Indeed, several studies could demonstrate that GC formation

is associated with production of pathognomonic AChR antibodies. Firstly,

autoreactive CD4+ T-cells recognize AChR epitopes, which are presented by TECs

or APC (e.g.DCs). Secondly, this interaction results in activation of B-cells that

fosters GC formation and finally anti-AChR antibodies production.317 We could

demonstrate that all participants of the above mentioned “intrathymic pathogenesis”

of MG, including DCs, TECs and GCs express HSP27 and 70. Hence our results

indicate once more that HSPs might be involved also in the pathogenesis of MG. In

addition to HSP expression in hyperplastic thymic specimens of myasthenic

patients, we found also elevated HSP27 and 70 serum concentrations in those

myasthenic patients compared to healthy volunteers. This is in accordance to

literature, where involvement of HSPs in diverse autoimmune diseases, including

Systemic Lupus Erythematosus, Behcet`s disease, uveitis, diabetes and multiple

sclerosis were reported.318-322 Two hypotheses concerning the role of HSPs in

autoimmune diseases are widely discussed. One thesis is related to the

extracellular pro-inflammatory functions of HSPs, which act as “danger signals” that

trigger overwhelming immune reactions, which could foster the development and

severity of autoimmune diseases. The other thesis concerns the molecular mimicry

effect of host HSPs and bacterial HSPs (50-60% homology at amino acid level) that

is important for autoantibody formation and therefore related to pathogenesis.323

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Indeed, strong similarity has been shown already between chlamydial HSP60,

human HSP60 and AChRs.324 Evidence that HSPs are relevant for the

pathogenesis of MG have accumulated in the peer reviewed literature starting in

1996, where specific antibodies to human HSP60 were described in 30.5% of

myasthenic patients in comparison to 8.5% in healthy subjects.325 Elevated HSP70

serum antibodies have been also detected in patients with ocular and generalized

MG.326 Finally, the question concerning the exact function and origin of HSPs in MG

remains at least partially unanswered but nonetheless HSPs seems to be somehow

related to MG and further studies are warranted to clarify this issue.

To sum up, we demonstrated for the first time that HSP27 and 70 are expressed in TETs and that HSP expression in neoplastic TECs predicts outcome. While we detected the lowest HSP expression in advanced tumor stages, we have found highest HSP serum concentrations in patients with advanced TETs. Interestingly, longitudinal analysis revealed that complete tumor resection led to decrease of both HSPs beneath physiologic levels. Additionally, we found also local expression of HSP in hyperplastic thymi of myasthenic patients and increased HSP serum levels in those patients. Altogether, HSP27 and 70 seem to be involved in the pathobiology of TETs and paraneoplastic MG. Thus HSPs may represent prognostic and diagnostic molecules or even candidates for targeted therapy. Nonetheless, further studies are necessary for a better understanding of HSPs in the complex system between thymic pathologies and autoimmunity.

3.2 Conclusion and Outlook The aim of this thesis was to evaluate the role of HSP27 and 70 in the pathogenesis

of TETs and to evaluate further their possible implications as (1) diagnostic and/or

prognostic biomarkers and (2) as candidates for targeted therapy.

Biomarkers can be principally differentiated into prognostic and predictive

markers; while prognostic biomarkers help to predict the natural behavior of

malignancies and indicates whether outcome tends to be good or poor, predictive

markers help to identify those patients that might respond to certain therapy

regimes.277 According to Seigneuric et al. (2010) biomarkers need to fulfill diverse

characteristics, which are as follows: (I) biomarkers should be easily measurable;

(II) markers should be elevated already at early stages of disease to enable early

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detection and therapy; (III) biomarkers should be characterized by high specificity

for the pathology of interest and finally (IV) biomarkers should also predict outcome.

Immunohistochemistry and ELISA are two basic techniques that are mainly used for

investigation of potential biomarkers.327 Despite tumor biomarkers have increasingly

gained attention as keys for individual therapy, only around 20 biomarkers are used

in clinical routine, such as prostate specific antigen (PSA) as diagnostic biomarker

in prostate cancer or thyroglobulin as early prognostic marker after total

thyroidectomy in patients with differentiated thyroid cancer.328-330 However, clinical

applications of diverse biomarkers are somewhat limited and some markers are

confined to specific malignancies, while others are present in several types of

tumors. There exists no “universal” tumor marker and the predictive, diagnostic and

prognostic value of such markers is mainly related to specific clinical settings and

questions.328 It is noteworthy to mention that HSPs are key stress proteins that are

upregulated by several different stressors and thus by definition HSPs are not

specific for cancer.327 However, numerous studies showed prognostic and

diagnostic implications of HSP expressions in diverse malignancies. For instance

HSP70 expression is useful for differentiation between low-grade hepatocellular

carcinoma and hepatocellular adenoma.331 In patients with cholangiocarcinoma

HSP27 and 70 have been identified as potential biliary markers for detection of

cholangiocarcinoma and of precursor lesions.332 Similarly, high expression of

HSP27 and 70 was associated with worse prognosis in patients with colorectal

cancer and determination of HSP tumor expression was suggested as additional

biomarker for risk stratification to better predict outcome.280 Despite HSPs are not

specific for certain malignancies, diagnostic and prognostic implications could have

been identified for diverse malignancies. We could demonstrate that HSP are

generally expressed in TETs and that HSP tumor expression predicts outcome. In

particular, 100% of thymomas and TCs expressed HSP27 and 70, which was

similar to 100% expression of pancytokeratin Lu-5 or 93.3% expression of

cytokeratin 5/6 respectively, both markers for epithelial cells. According to that

positive HSP27 and 70 expression might be useful as positive predictive marker for

the identification of thymomas and TCs and evaluation of staining intensity might be

a further marker for risk stratification to estimate prognosis and outcome. More

interestingly, although HSP27 is not specific for TETs, we found significantly

decrease of HSP27serum concentrations after complete (R0) tumor resection,

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which let us assume that HSP27 may be a promising and interesting biomarker for

longitudinal analysis in patients with TETs.

It is unquestioned that the relatively low number of included and investigated

patients with TETs represents a main limitation of our study, but represents a

common problem and weakness of studies done on orphan diseases.333 Although

our study is only basically descriptive, we are strongly convinced that HSPs

represent promising biomarkers in TETs and paraneoplastic MG and international

cooperation and support of the ITMIG and the thymic workgroup of the ESTS are

essential to further evaluate the role of HSPs in thymic malignancies and to improve

our knowledge about TETs.28,40,53,64,65,165

Targeted Therapy HSP expression in various malignancies was associated

with increased cell proliferation, tumor promotion, metastases and poor clinical

outcome.230 In fact, cancer cells depend at least somehow to the expression of

HSPs due to their anti-apoptotic as well as to their chaperonic functions. Hence

inhibition of HSPs represent promising and potential therapeutic targets in cancer

therapy.327 Indeed overexpression of HSP27 and 70 was associated with

chemoresistance in melanoma cells, colorectal carcinoma cells or pancreatic

cancer cells.334 Pocaly et al. detected even a threefold increase of HSP70 in an

Imatinib resistant lymphoma cell line simultaneous to induction of chemoresistance,

whereas HSP70 levels remained low in patients that responded to Imatinib.335

Antitumoral effects of HSP90 inhibitors have been already shown for several

malignancies in phase I to III studies, while only less is known about HSP27 and 70

inhibitors in cancer patients.269,336

However, promising results have been published concerning the use of

HSP90 inhibitors combined with other chemotherapeutic agents.270,337 Inhibition of

HSP90 induced apoptosis in colon, breast and prostate cancer cells.338,339

Unfortunately, inhibition of HSP90 leads also to compensatory upregulation of heat

shock factor 1, which in turn induces increased expression of other HSPs, mainly

HSP27 and 70 and causes treatment resistance again.340,341 Thus inhibition of

HSP90 and another HSP, such as HSP27 or 70, represents a new promising

therapeutic option. In-vitro dual targeting and inhibition of HSP70 and 90 in invasive

bladder cancer cell lines led to significantly more effective effects compared to only

single inhibition.342 Additionally, simultaneous inhibition of HSP70 improved the anti-

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growth effect of HSP90 inhibitors in hepatocellular carcinoma cells, HCT116 colon

carcinoma cells and primary human AML cells.343-345

Beside Inhibition of HSPs, immunomodulatory functions of HSPs and their

possible value for immunotherapy in cancer patients gained recently attention. In

xenograft mouse model of myeloma, HSP peptide-specific cytotoxic T-cells

effectively reduced tumor burden. Whether peptides chaperoned on HSPs or HSPs

themselves elicit a potent immune response remains unclear but clearly

demonstrates that HSPs may represent potential and novel tumor antigens for

immunotherapy of myeloma.346 Further, cancer derived HSP peptide-complex

based vaccination induced immunity against several malignancies, such as gastric,

colorectal, pancreatic or ovarian cancer, in preclinical studies and demonstrated an

excellent safety profile in phase III clinical trials for advanced melanoma and renal

cell carcinoma patients. Hence, immunotherapy based on cancer derived HSPs

emerge flexible tumor- and patient- specific therapeutic option.347

To sum up, growing evidence indicates that HSPs are useful biomarkers

that predict outcome in diverse malignancies and that HSPs represent promising

targets for cancer therapy. As already mentioned above HSPs are key stress

proteins, which are upregulated by several different stressors and thus the use of

HSPs as specific and diagnostic biomarkers in cancer is limiting. However, although

HSP27 is not specific for TETs, significantly decrease of HSP27 serum levels after

complete tumor resection make serum HSP27 an interesting marker for tumor

surveillance. While effectiveness of inhibition of HSPs has been mainly shown in

preclinical studies and in-vitro models, results are interesting and warrant further

studies. The value of HSPs for targeted therapy in patients with TETs is limiting due

to the fact that complete surgical resection either of primary tumor or tumor

recurrence represents the mainstay of therapy and is associated with excellent

survival. Nonetheless, in patients that could not undergo surgery or with

metastases, inhibition of HSP27 or 70 in combination with chemotherapeutic agents

might represent an additional therapeutic option.

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Chapter IV: Material and Methods 4.1 Study population A total of 125 patients were included in this study, which underwent surgical

resection of benign and thymic pathologies between 1999 and 2014. Histologically

verified thymic malignancies, strong clinical suspicion of TET and thymectomy in

myasthenic patients were the main indications for surgery. We included 101

patients with TETs (83 thymomas, 15 TCs, 3 TNETs) and 24 patients with benign

thymic alterations. Basic characteristics of patients with TETs are shown in table 14.

Non-malignant thymic specimens (n=24) were distinguished between regular

for age thymic morphology (n=8) and thymic hyperplasia (n=16). Patients with

thymic hyperplasia were further separated into TTH (n=10) and FTH (n=6)

depending on the absence or presence of GCs. At time of diagnosis 10 out of 24

patients (41.7%) suffered from MG. Fetal and infantile thymic tissues for histological

processing were obtained at autopsy.

Additionally, serum samples were available of 46 with TETs (11 MG pos., 35

MG neg.), 33 patients with TH (15 MG pos., 18 MG neg.) and 49 sex and age

matched volunteers, who were used as healthy control group. For longitudinal

analysis, pre- and postoperative serum samples of 16 patients with TETs with a

mean time of 13.94 months after initial surgery were available. Patients who had not

undergone complete tumor resection at initial operation (R0), those who had

adjuvant therapy within the last month of postoperative blood drawl or those who

had signs of tumor recurrence were excluded from longitudinal analysis.

The study was approved by the ethics committee of the Medical University of

Vienna and the study was conducted according to Good Scientific Practice. Written

informed consent was obtained from all participating patients and volunteers.

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Table 14: Characteristics of patients with Thymic Epithelial Tumors.

Patient Characteristics (n=101) n % Sex

Male 40 39.6 Female 61 60.4

WHO Classification MNT 4 4.0 A 16 15.8 AB 14 13.9 B1 11 10.9 B2 21 20.8 B3 17 16.8 TC 15 14.9 TNET 3 3.0

Clinical Masaoka- Koga Tumor Stage I 23 22.8 II 47 46.5 III 15 14.9 IV 16 15.8

Therapy Surgery only 54 53.4 Multimodality Treatment 57 56.4

Residual Tumor Classification R0 88 87.1 R1+2 13 12.9

Paraneoplastic Myasthenia Gravis Yes 29 28.7 No 72 71.3

Recurrence Yes 13 12.9 No 88 87.1

Multimodality Treatment includes surgery and radiotherapy ± chemotherapy in neoadjuvant

and/or adjuvant setting. n, number; MNT, Micronodular Thymoma; TC, Thymic Carcinoma;

TNET, Thymic Neuroendocrine Tumor.

4.2 Immunohistochemistry Formaldehyde-fixed and paraffin embedded human benign and malignant thymic

specimens (4μm in thickness) were retrieved from the Clinical Institute of Pathology.

First a subset of only 30 specimens was stained according to the following

protocol.348,349 Specimens were baked for 1 hour at 55°C and deparaffinized in

xylene followed by ethanols with decreasing alcohol concentration. Antigen retrieval

was performed by boiling slides at 600 watt (3x5min) in a microwave oven using

citrate buffer at pH 6.0 (Target Retrieval Solution, Dako, USA). Endogenous

peroxidase activity was blocked by application of 0.3% hydrogen peroxide. Blocking

serum of the same species as the secondary antibody was added to avoid

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unspecific protein-protein interactions. Monoclonal mouse anti-human HSP27 IgG2a

(Santa Cruz, Dallas, Texas, USA), monoclonal mouse anti-human HSP70 IgG2a

(Novus, Littleton, CO, USA) and anti-mouse IgG secondary antibodies (Vector

Laboratories, Burlingame, CA, USA) were used as antibodies. Vectastain ABC kit

(Vector Laboratories) and DAB Peroxidase kit (Vector Laboratories) were used to

amplify immunoreactivity. Counterstaining was performed by using Mayer's

Hematoxylin. Finally, sections were dehydrated and mounted by using Pertex

Mounting Media (Leica Microsystems, Germany).348,349

Immunohistochemical staining of HSP27 and 70 for all specimens was

performed/reproduced by using the automated Ventana Benchmark® platform

(Ventana Medical Systems, Tucson, AZ, USA) according to standard protocol of the

Clinical Institute of Pathology.348,349

4.3 Double-Staining Additionally, sequential double-staining on formaldehyde-fixed and paraffin

embedded human non-malignant thymic specimens was performed by using the

automated Leica Bond III immunostainer platform (Leica Biosystems, Nussloch,

Germany). We used the following antibodies: (1) HSP70/HSPA1A mouse

monoclonal antibody IgG2a (Novus Biological, NB600-571, Littleton, CO, USA). (2)

CD68 (clone PGM-1) mouse monoclonal antibody IgG3 (DAKO M876, Glostrup,

Denmark) was used for the detection of monocytes and DCs and (3) Lu-5, mouse

monoclonal antibody IgG1 (Biocare Medical CM43C, Concord, CA, USA), was used

for the identification of epithelial cells.

4.4 Evaluation of immunoreactivity Two different systems were used for evaluation of immunoreactivity in benign and

malignant thymic specimens.

4.4.1 Thymic Epithelial Tumors To assess staining intensities of HSP27 and 70 in malignant thymic specimens we

used a commonly accepted and used scoring system ranging from 0 to 3 (0, no

staining, 1, weak staining, 2, moderate staining, 3 strong staining).348 Two blinded

observers performed analysis and rating represents the overall average of staining

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intensity of the majority of tumor cells. Additionally, cells of the tumor

microenvironment, such as endothelial cells (ECs) or fibroblast were also analyzed.

In case that ratings differed, we reevaluated and discussed the slide.

4.4.2 Non-Malignant Thymic Specimens Conversely, non-malignant thymic specimens were analyzed by using a quantitative

score (“+”, positive, “-“, negative). Slides were screened at low magnification (40x)

and positive or negative staining was evaluated in ECs, GCs, lymphocytes, HCs

and TECs.

4.5 Enzyme-linked immunosorbent assay We used enzyme-linked immunosorbent assays (ELISAs) for the analysis of HSP27

and 70 serum concentrations. For this purpose commercial available human HSP27

(R&D Systems, Minneapolis, MN, USA, DuoSet® Human HSP27, DY1580) and

HSP70 ELISA kits (R&D Systems, Minneapolis, MN, USA, DuoSet®IC Total

HSP70/HSPA1A, DYC1663) were used. All ELISA tests were performed according

to the Manufacturer`s instructions and samples were always measured in

duplicates. Finally, assay plates were analyzed by using an ELISA plate reader

(Victor 3, Multilabel plate reader, PerkinElmer) at 450nm wavelength.

4.6 Statistical Methods SPSS software (version 21; IBM SPSS Inc., IL, USA) was used for statistical

analysis of our data. Non-normal distributed variables with two or more than two

groups were analyzed by using Mann-Whitney-U tests and Kruskal-Wallis rank

tests, respectively. Chi-square test was used to compare nominal variables.

Unpaired student’s t test and one-way ANOVA were used to compare means of two

or more than two independent groups with normal (Gaussian) distributions,

respectively. Tukey’s-B and Bonferroni correction were used for post hoc

comparisons. For longitudinal analysis of two dependent groups with normal

distribution paired student’s t-test was used. All data are reported as

mean±standard error of the mean (SEM) in the text and more detailed as

mean(median)±standard deviation(SEM) in tables. Two-tailed p-values were

employed for all tests. Pearson correlation (r) was performed to analyze linear

118

relationships between two metric measurements, whereas Spearman rho

correlation (r) was used in case that one or both variables were ranked. Kaplan-

Meier survival analysis and log-rank test were used to assess outcome measures

for TETs, including overall survival (OS), cause-specific survival (CSS) and freedom

from recurrence (FFR). GraphPad Prism6 (GraphPad Software Inc., California,

USA) was used for graphical display of graphs in this thesis.

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348. Moser B, Janik S, Schiefer AI, Müllauer L, Bekos C, Scharrer A, Mildner M, Rényi-Vámos F, Klepetko W, Ankersmit HJ. Expression of RAGE and HMGB1 in thymic epithelial tumors, thymic hyperplasia and regular thymic morphology. PLoS One. 2014; 9(4): e94118.

349. Janik S, Schiefer AI, Bekos C, Hacker P, Haider T, Moser J, Klepetko W, Müllauer L, Ankersmit HJ, Moser B. HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications. Sci Rep. 2016; 6: 24267.

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Curriculum Vitae

Stefan Johannes JANIK, MD Personal Background Nationality Austria Family Status Single Address Dr. Bruno Kreisky Ring 4, 2435 Ebergassing Phone Number 0664 46 25 023 Email stefan.janik@meduniwien.ac.at Date of Birth August 19th, 1990, Vienna Parents Dr. Silvia and Mag. Hans Janik Education 07/2015 Graduation Dr. med. univ. (M.D.), Medical University of

Vienna, Austria 06/2015 – present Research fellow at the Department of Ear, Nose, Throat

Diseases, Head and Neck Surgery, Medical University of Vienna

10/2014 – present MDPhD/PhD student at the Department of Thoracic Surgery, Medical University of Vienna, Austria

08/2010 – present Research fellow at the Department of Thoracic Surgery, Medical University of Vienna, Austria

10/2009 – 07/2015 Medical student at the Medical University of Vienna, Austria 07/2008 – 03/2009 Social Service as paramedic at “Grünes Kreuz” 06/2008 Graduation with distinction 2000 - 2008 Don Bosco - Gymnasium, 2442 Unterwaltersdorf,

Niederösterreich, Austria 1996 - 2000 Volksschule Judenplatz, 1010 Vienna, Austria Postgraduate Training 05/2016 – present Resident at the Department of Ear, Nose, Throat Diseases,

Head and Neck Surgery, Medical University of Vienna 01/2016 – 04/2016 Resident at the Department of Thoracic Surgery, Medical

University of Vienna 11/2015 – 01/2016 Resident at LKH Wr. Neustadt, Niederösterreich Clinical Training 03-07/2015 Department of Ear, Nose, Throat Diseases, Head and Neck

Surgery, Medical University of Vienna (16 weeks) 11/2014-03/2015 Department of Internal Medicine, Barmherzige Brüder,

Eisenstadt, Burgenland (16 weeks) 08-11/2014 Department of Thoracic Surgery, Medical University of Vienna

(16 weeks)

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09/2013 Department of Trauma Surgery, Barmherzige Brüder Eisenstadt, Burgenland (4 weeks)

08/2013 General Practitioner – Dr. Gerhard Vikydal, 2435 Ebergassing, Niederösterreich (2 weeks)

07/2013 Department of Surgery – LKH Baden, Niederöstereich (4 weeks)

09/2012 Department of Trauma Surgery – Medical University of Vienna (4weeks)

09/2011 Intensive Care Unit – Gastroenterology and Hepatology, Medical University of Vienna (4 weeks)

Congresses and Meetings 04/2016 Symposium - Management of Laryngotracheal problems II,

14th-15th April 2016, Vienna, Austria 10/2015 Annual Meeting of the Austrian Society of Pneumology, Graz,

15.-17.10. 2015, Austria 06/2015 56th Annual Meeting of the Austrian Society of Surgery, Linz,

June 3-5, 2015, Austria 01/2015 6th Wiener Head and Neck Tage, Vienna, 12.-14.1.15, Austria 12/2014 4th EACTS Meeting on Cardiac and Pulmonary Regeneration,

11-13.12.14, Berne, Switzerland 10/2014 Annual Meeting of the Austrian Society of Pneumology,

Salzburg, 2.-4.10.14, Austria 06/2014 55th Annual Meeting of the Austrian Society of Surgery, Graz,

June 25-27, 2014, Austria 01/2014 37th Seminar of the Austrian Society of Surgical Research,

Gosau, January 17. – 18. 2014, Austria 03/2011 National Symposium of the Department of Thoracic Surgery:

Focus on Thymus, Medical University of Vienna, Austria 12/2010 2nd EACTS Meeting on Cardiac and Pulmonary Regeneration,

02-04.12.10, Vienna, Austria Publications 4 articles in peer-reviewed journals (2 under submission) 14 abstracts as author or co-author at national and international meetings 7 presentations Articles

Mohammad Kasiri, Lucian Beer, Thomas Haider, Elisabeth Simader, Denise Traxler, Thomas Schweiger, Stefan Janik, Shahrokh Taghavi, Christian Gabriel, Michael Mildner, Hendrik Jan Ankersmit. Secretome of Apoptotic Peripheral Blood Mononuclear Cells Possesses Antimicrobial Activity and Induces De Novo AMPs In Vivo. (under submission) Christine Bekos, Stefan Janik, Stefanie Nickl, Jonathan Kliman, Jessica Didcock, Matthias Zimmermann, Patrick Altmann, Andreas Mitterbauer, Lukas Unger, Thomas Haider, Georg Roth, Berit Payer, Michael Koller, Robert Fritz, Christian Gäbler, Mario Kessler, Thomas Szekeres, Walter Klepetko, Hendrik Jan Ankersmit, and Bernhard Moser. Cytokine changes in non-professional marathon and half-marathon runners. (under submission)

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Janik S, Schiefer AI, Bekos C, Hacker P, Haider T, Moser J, Klepetko W, Müllauer L, Ankersmit HJ, Moser B. HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations: diagnostic, prognostic and physiologic implications. Sci Rep. 2016 Apr 21;6:24267. doi: 10.1038/srep24267. Bernhard Moser, Ana-Iris Schiefer, Stefan Janik, Alexander Marx, Helmut Prosch, Wolfgang Pohl, Barbara Neudert, Anke Scharrer, Walter Klepetko and Leonhard Müllauer. Adeoncarcinoma of the Thymus, Enteric Type. Report of 2 Cases, and Proposal for a Novel Subtype of Thymic Carcinoma. Am J Surg Pathol (2015); 39(4):541-8 Bernhard Moser, Anna Megerle, Christine Bekos, Stefan Janik, Tamás Szerafin, Peter Birner, Ana-Iris Schiefer, Michael Mildner, Irene Lang, Nika Skoro-Sajer, Shahrokh Taghavi, Walter Klepetko and Hendrik Jan Ankersmit. ocal and Systemic RAGE Axis Changes in Pulmonary Hypertension: CTEPH and iPAH. PLoS ONE, 2014 Sep 4;9(9):e106440. doi: 10.1371/journal.pone.0106440. eCollection 2014. Bernhard Moser, Stefan Janik, Ana-Iris Schiefer, Leonhard Müllauer, Christine Bekos, Anke Scharrer, Michael Mildner, Ferenc Rényi-Vámos, Walter Klepetko and Hendrik Jan Ankersmit. Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology. (PLoS ONE, 9(4): e94118. Doi: 10.1371/ journal.pone.0094118)

Abstracts

Philipp Hacker, Thomas Haider, Stefanie Nickl, Stefan Janik, Carmen Schwaiger, Thomas Raunegger, Hendrik Jan Ankersmit, Stefan Hacker. Serum HSP27 and HSP70 concentrations are increased and predict mortality in burn patients. 34th Annual Meeting of the German Society of Burn DAV2016, Bertesgaden 13.-16.01.16,Germany Philipp Hacker, Stefanie Nickl, Stefan Janik, Carmen Schwaiger, Thomas Raunegger, Hendrik Jan Ankersmit, Stefan Hacker, Thomas Haider. Systemic Lipocalin-2 concentrations are increased in burn patients. 34th Annual Meeting of the German Society of Burn DAV2016, Bertesgaden 13.-16.01.16,Germany Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Philipp Hacker, Walter Klepetko, Leonhard Müllauer, Hendrik Jan Ankersmit and Bernhard Moser. Prognostic and Clinical relevance of HSP expression in Thymic Epithelial Tumors. Annual Meeting of the Austrian Society of Pneumology, Graz 15.-17.10. 2015, Austria Bernhard Moser, Stefan Janik, Jose Ramon Matilla, Georg Lang, Clemens Aigner, Jan Ankersmit, Shahrokh Taghavi and Walter Klepetko. Prognostic Factors for Thymomas and Thymic Carcinomas: Masaoka-Koga Stage, WHO classification and Completeness of surgical resection. Annual Meeting of the Austrian Society of Pneumology, Graz 15.-17.10. 2015, Austria Bekos C, Janik S, Didcock J, Kliman J, Koller M, Fritz R, Gäbler C, Klepetko W, Ankersmit J und Moser B. Exercise-induced bronchoconstriction and cytokine changes in non-professional marathon runners. Annual Meeting of the Austrian Society of Pneumology, Graz 15.-17.10. 2015, Austria Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Phillip Hacker, Walter Klepetko, Leonhard Müllauer, Hendrik Jan Ankersmit and Bernhard Moser. Heat shock protein 27 and 70 expression in thymic epithelial tumors. 56th Annual Meeting of the Austrian Society of Surgery, Linz, June 3-5, 2015, Austria

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Stefan Janik, Ana-Iris Schiefer, Leonhard Müllauer, Christine Bekos, Anke Scharrer, Michael Mildner, Ferenc Rényi-Vámos, Walter Klepetko, Hendrik Jan Ankersmit and Bernhard Moser. The local and systemic role of RAGE in thymic epithelial tumors, thymic hyperplasia and thymic physiology. Annual Meeting of the Austrian Society of Pneumology, Salzburg 2.-4.10.14, Austria. Anna Megerle, Stefan Janik, Christine Bekos, Tamás Szerafin, Peter Birner, Ana-Iris Schiefer, Irene Lang, Nika Skoro-Sajer, Shahrokh Taghavi, Walter Klepetko, Hendrik Jan Ankersmit and Bernhard Moser. Local and systemic changes of the RAGE axis in pulmonary hypertension: CTEPH and iPAH. Annual Meeting of the Austrian Society of Pneumology, Salzburg 2.-4.10.14, Austria. Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Leonhard Müllauer, Michael Mildner, Walter Klepetko, Jan Ankersmit, Bernhard Moser. RAGE – ligand axis in the pathogenesis of thymic epithelial tumors and thymic physiology. 55th Annual Meeting of the Austrian Society of Surgery, Graz, June 25-27, 2014, Austria Christine Bekos, Stefan Janik, Stefanie Nickl, Jonathan Kliman, Jessica Didcock, Matthias Zimmermann, Patrick Altmann, Andreas Mitterbauer, Lukas Unger, Thomas Haider, Georg Roth, Berit Payer, Michael Koller, Robert Fritz, Christian Gäbler, Mario Kessler, Walter Klepetko, Hendrik Jan Ankersmit and Bernhard Moser. The RAGE axis in marathon and half-marathon runners. 55th Annual Meeting of the Austrian Society of Surgery, Graz, June 25-27, 2014, Austria Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Anke Scharrer, Michael Mildner, Ferenc Rényi-Vámos, Walter Klepetko, Jan Ankersmit, Bernhard Moser. RAGE and its ligand HMGB1 in thymic physiology, follicular hyperplasia and thymic epithelial tumors. 11th Annual Meeting of the Austrian Society of Neurology. Salzburg, March 26-29, Austria. Stefan Janik, Ana-Iris Schiefer, Leonhard Müllauer, Christine Bekos, Anke Scharrer, Michael Mildner, Ferenc Rényi-Vámos, Walter Klepetko, Jan Ankersmit, Bernhard Moser. Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology. 37th Seminar of the Austrian Society of Surgical Research, Gosau, January 17. – 18. 2014, Austria Bekos C, Zimprich F, Nickl S, Janik S, Klepetko W, Ankersmit HJ, Moser B. The Receptor of advanced Glycation Endproducts and its ligands in Myasthenia Gravis. 3rd International Conference – Advances in Clinical Neuroimmunology. Vienna, Austria. 06/2012) Bekos C, Zimprich F, Janik S, Nickl S, Klepetko W, Ankersmit HJ, Moser B. The Receptor of advanced Glycation Endproducts and its ligands in Myasthenia Gravis. 10th Annual Meeting of the Austrian Society of Neurology. Graz, Austria.

Presentations Oral presentations

Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Philipp Hacker, Walter Klepetko, Leonhard Müllauer, Hendrik Jan Ankersmit and Bernhard Moser. Prognostic and Clinical relevance of HSP expression in Thymic Epithelial Tumors. Annual Meeting of the Austrian Society of Pneumology, Graz 15.-17.10. 2015, Austria (Poster Prize)

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Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Phillip Hacker, Walter Klepetko, Leonhard Müllauer, Hendrik Jan Ankersmit and Bernhard Moser. Heat shock protein 27 and 70 expression in thymic epithelial tumors. 56th Annual Meeting of the Austrian Society of Surgery, Linz, June 3-5, 2015, Austria Stefan Janik, Ana-Iris Schiefer, Christine Bekos, Leonhard Müllauer, Michael Mildner, Walter Klepetko, Jan Ankersmit, Bernhard Moser. RAGE – ligand axis in the pathogenesis of thymic epithelial tumors and thymic physiology. 55th Annual Meeting of the Austrian Society of Surgery, Graz, June 25-27, 2014, Austria Stefan Janik, Ana-Iris Schiefer, Leonhard Müllauer, Christine Bekos, Anke Scharrer, Michael Mildner, Ferenc Rényi-Vámos, Walter Klepetko, Jan Ankersmit, Bernhard Moser. Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology. 37th Seminar of the Austrian Society of Surgical Research, Gosau, January 17. – 18. 2014, Austria

Poster Presentations

Bernhard Moser, Stefan Janik, Jose Ramon Matilla, Georg Lang, Clemens Aigner, Jan Ankersmit, Shahrokh Taghavi and Walter Klepetko. Prognostic Factors for Thymomas and Thymic Carcinomas: Masaoka-Koga Stage, WHO classification and Completeness of surgical resection. Annual Meeting of the Austrian Society of Pneumology, Graz 15.-17.10. 2015, Austria Bekos C, Janik S, Didcock J, Kliman J, Koller M, Fritz R, Gäbler C, Klepetko W, Ankersmit J und Moser B. Exercise-induced bronchoconstriction and cytokine changes in non-professional marathon runners. Annual Meeting of the Austrian Society of Pneumology, Graz 15.-17.10. 2015, Austria Stefan Janik, Ana-Iris Schiefer, Leonhard Müllauer, Christine Bekos, Anke Scharrer, Michael Mildner, Ferenc Rényi-Vámos, Walter Klepetko, Hendrik Jan Ankersmit and Bernhard Moser. The local and systemic role of RAGE in thymic epithelial tumors, thymic hyperplasia and thymic physiology. Annual Meeting of the Austrian Society of Pneumology, Salzburg 2.-4.10.14, Austria.

Scientific Skills, Current Projects Scientific Skills

Immunohistochemistry Enzyme Linked Immune Sorbent Assay (ELISA) Database management Statistical analysis Immunofluorescence-, Lightmicroscopy Cell culture, Tumor cell lines, PBMCs

Awards and Grants 10/2015 Poster Prize - Annual Meeting of the Austrian Society of

Pneumology, Graz 15.-17.10. 2015, Austria 10/2014 – 09/2015 MDPhD excellence program, Medical University of Vienna,

Austria 12/2014 Scholarship for Outstanding Academic Achievement

(Leistungsstipendium) 12/2011 Student Research Scholarship, Medical University of Vienna,

Austria (Forschungsstipendium)

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Funding All projects, studies and research at the Department of Thoracic Surgery between 10/2009 and 10/2015 were mainly supported and funded by the Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis & Regeneration. Language Skills Native German Speaker Proficient in English References Assoc. Prof. Priv. Doz. Dr. med. Bernhard Moser FEBTS, Department of Thoracic Surgery, Medical University of Vienna Univ. Prof. Dr. med. Hendrik Jan Ankersmit MBA, Department of Thoracic Surgery, Medical University of Vienna and Laboratory Head of the Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis & Regeneration.

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