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215 Problematic Cases in Pulmonary Cytopathology Stan Eilers MD Paula LaPolice CT(ASCP) Adebowale Adeniran MD, FASCP Ajay Shah MD Indra Balachandran PhD, SCT(ASCP), CFIAC Umesh Kapur MD, FASCP 2011 Annual Meeting – Las Vegas, NV AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste. 1600 Chicago, IL 60603

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Page 1: 215 Problematic Cases in Pulmonary Cytopathology Stan ...dn3g20un7godm.cloudfront.net/2011/AM11FNV/215... · 215 Problematic Cases in Pulmonary Cytopathology This course will present

215 Problematic Cases in Pulmonary Cytopathology

Stan Eilers MD Paula LaPolice CT(ASCP)

Adebowale Adeniran MD, FASCP Ajay Shah MD

Indra Balachandran PhD, SCT(ASCP), CFIAC Umesh Kapur MD, FASCP

2011 Annual Meeting – Las Vegas, NV

AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste. 1600

Chicago, IL 60603

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215 Problematic Cases in Pulmonary Cytopathology This course will present images from selected pulmonary cytology cases which proved to be challenging for participants in the ASCP NonGYN Assessment Program. The audience will participate with an interactive audience response system. The cytomorphologic criteria for the differential diagnoses will be discussed with additional examples including material from the ASCP Asset Warehouse. This session will also place special emphasis on the role of cytopathology in the personalized medicine of treating pulmonary malignancies through appropriate choice and use of ancillary and molecular technologies. Participants should expect information that is timely and germane to the practicing pathologist seeking to employ the best combination of microscopic review skills and ancillary and molecular testing methods to provide the best diagnosis and course of treatment for patients with pulmonary malignancies.

• Sharpen diagnostic skills for cytotechnologists and cytopathologists by emphasizing and illustrating diagnostic cytologic criteria in the following specific settings: the differential diagnosis of reactive atypia versus adenocarcinoma; the differential diagnosis of small cell carcinoma versus non-small cell carcinoma; and the diagnosis of pulmonary malignancies from extra pulmonary sites, specifically hepatic aspirates and pleural fluid. In each of the above mentioned scenarios, the precise diagnosis is necessary to provide the correct treatment since each entity has significantly different treatment protocols.

• Emphasize and inform the participants on the role of ancillary studies in the diagnosis and treatment of pulmonary malignancies. Specifically, the use of immunohistochemical stains in the differential diagnosis of squamous cell carcinoma, adenocarcinoma and small cell carcinoma. In addition, the role of molecular studies, primarily in adenocarcinoma, will be discussed. The specific cell type and molecular genotype is critical in developing a specific treatment plan in this era of personalized medicine. For some participants this may represent a review, but for others this represents new more detailed information.

• Increase awareness of ASCP Non-GYN Assessment Products that can be used to enhance pathologist and cytotechnologist practice-based professional work.

FACULTY: Stan Eilers MD Paula LaPolice CT(ASCP) Adebowale Adeniran MD, FASCP Ajay Shah MD Indra Balachandran PhD, SCT(ASCP), CFIAC Umesh Kapur MD, FASCP Practicing Pathologists Cytopathology Cytopathology (Non-Gynecologic) 1.0 CME/CMLE Credit Accreditation Statement: The American Society for Clinical Pathology (ASCP) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education (CME) for physicians. This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education (ACCME). Credit Designation: The ASCP designates this enduring material for a maximum of 1 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity. ASCP continuing education activities are accepted by California, Florida, and many other states for relicensure of clinical laboratory personnel. ASCP designates these activities for the indicated number of Continuing Medical Laboratory Education (CMLE) credit hours. ASCP CMLE credit hours are acceptable to meet the continuing education requirements for the ASCP Board of Registry Certification Maintenance Program. All ASCP CMLE programs are conducted at intermediate to advanced levels of learning. Continuing medical education (CME) activities offered by ASCP are acceptable for the American Board of Pathology’s Maintenance of Certification Program.

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Problematic Cases in Pulmonary Cytopathology

As Identified from the ASCP NonGYN Assessment Program

Stan Eilers, MDASCP NonGYN Assessment Committee

ChairChair

Mercy Medical Center, Cedar Rapids, IAMercy Medical Center, Clinton, IA

Weland Laboratories, Cedar Rapids, IA

ASCP CME Disclosure of Relevant Financial Relationships

In accordance with ACCME Standards, I verify that I

h l t fi i l l ti hi t di lhave no relevant financial relationships to disclose,

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

ASCP Non-GYN Committee

2010-2011 Members Pulmonary Course Faculty Italicized

•Stanley Eilers, MD, FASCP, Chair•Paula LaPolice, CT(ASCP), Vice-Chair•Perkins Mukunyadzi, MD, FASCP, Medical MemberPerkins Mukunyadzi, MD, FASCP, Medical Member•Umesh Kapur, MD, FASCP, Medical Member•Ajay Shah, MD, FASCP, Medical Member•Husain Saleh, MD, MBA, Medical Member•Adebowale Adeniran, MD, FASCP, Medical Member•Amberly Nunez, MD, Resident Member•Amy J. Wendel Spiczka, MS, SCT, MB, HTL(ASCP)CM, Cytotechnologist Member•Indra Balachandran, PhD, SCT(ASCP), CFIAC, Cytotechnologist Member•ASCP Staff: Jennifer J. Clark, SCT(ASCP)CM, Product Development Manager

ASCP Non-GYN Committee Programs

NonGYN/FNB Glass Slide Programs (10)Assessment Programs, which include detailed Educational Case Studies:

1. NonGYN Assessment – mixture of NonGYN/FNB cases2. FNB Site-Specific Assessment / Breast3. FNB Site-Specific Assessment / Thyroid4 FNB Site-Specific Assessment / Lymph Node4. FNB Site-Specific Assessment / Lymph Node5. FNB Site-Specific Assessment / Salivary Gland

Review Programs, Target Answers and Peer-comparison Statistics only

1. NonGYN Review– mixture of NonGYN/FNB cases 2. FNB Site-Specific Review / Breast3. FNB Site-Specific Review / Thyroid4. FNB Site-Specific Review / Lymph Node5. FNB Site-Specific Review / Salivary Gland

ASCP Non-GYN Committee Programs

NonGYN Digital Image ProgramA printed assessment product with digital images of 20

NonGYN/FNB cases

Cytology eLearning Modules in 6 areas:Cytology eLearning Modules in 6 areas:1. GYN2. NonGYN3. FNB4. Technical/Ancillary Studies5. Laboratory Mgt & Admin6. Safety

Importance of Pulmonary Cytology

•220, 520 new cases of lung cancer annually

•157,300 annual deaths from lung cancer

•70% of all lung cancer patients will have only cytology/small biopsy specimen

•Cell type is critical for appropriate treatment and ancillary tests

•EBUS and related procedures are increasing the number of pulmonary cytologic specimens

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What Is This Presentation About?

The cases were selected from the ASCP Non-GYN Assessment Glass Slide Program and have been reviewed by hundred of

cytologists

They represent “everyday” cases, but presented challenges to the participants

Discussion of key morphologic features and ancillary tests helpful to accurate diagnosis will be the focus of our expert faculty

Format•NonGYN Assessment Committee Members will review topic areas of interest with statistics and images from selected cases for:

- Reactive/Reparative Processes- Adenocarcinoma vs. Squamous cell carcinoma- Small cell vs NSCLC- Small cell carcinoma presenting as metastatic disease- Lung cancer in fluid cytology- Lung cancer in fluid cytology

•Discussion will include a review of cytomorphologic criteria, molecular diagnostics, and therapeutic treatments

•Additional images from the ASCP Asset Warehouse will be used as supporting material

•At program end, a brief interactive morphology quiz using the Audience Response System will be given comparing results to program participant statistics

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Problematic Cases in Pulmonary Cytopathology

As Identified from the ASCP NonGYN Assessment Program 

Paula LaPolice CT(ASCP)ASCP NonGYN Assessment Committee

Vi Ch iVice Chair

Baystate Medical CenterSpringfield, Ma

Educational Coordinator Cytology Services

ASCP CME Disclosure of Relevant Financial Relationships

In accordance with ACCME Standards, I verify that I

have no relevant financial relationships to disclose,have no relevant financial relationships to disclose,

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

Reactive Cases inPulmonary Specimens

How do we differentiate:

Reactive/Repairfrom

Neoplastic processes

Clinical Disease Features

•Cough

•Wheezing

•Increased mucus production

•Shortness of breath

•Chest pain

•Hemoptysis

Causes of a Reactive Process

•Pneumonia

•Bronchitis

•Bronchiectasis

•Bronchial asthma

•Toxin exposure

•Radiation

•Chemotherapeutic agents

•Instrumentation

Cumulative Statistics from Reference Cases Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 701 77.0%Infectious/Inflammatory Process 176 19.3%Benign Neoplasm 1 0.1%Lesion of Uncertain Biologic Potential 12 1.3%Positive for Malignancy 20 2.2%Total 910 100.0%

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pulmonary, NonGYN, Negative/Reactive

Reference Interpretation Choices Total %ATYPICAL METAPLASIA 4 0.4%BLASTOMYCOSIS 4 0.4%CARCINOID 8 0.9%NEGATIVE/REACTIVE 714 78.6%No Interpretation Selected 41 4.5%NON-SMALL CELL CARCINOMA 9 1.0%METASTATIC MELANOMA 2 0.2%MYCETOMA 119 13.1%SMALL CELL CARCINOMA 1 0.1%SQUAMOUS CELL CARCINOMA 6 0.7%Total 908 100.0%

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Reactive/Reparative Features

•Cohesive orderly flat sheets

•Abundant cytoplasm

•Nuclei-oval to round, enlarged

•Nuclear size variation

•Inflammatory background

Reactive/Reparative Features

•Chromatin bland, finely granular and evenly

distributed

•Prominent nucleoli, uniform in size and shape

•Mitosis common

•Patient history

Reactive Bronchial EpitheliumReactive Squamous Metaplasia

Reactive Case #1

A 42-year-old man presented with bilateral

pneumonia, pleural effusions, shortness of breath

and mediastinal adenopathy. A fine needle

aspiration was performed.

Reactive Case #1

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Reactive Case #1 Reactive Case #1

Reactive/Repair

Squamous Cell Ca

Reactive Case #2

A 74-year-old man presented with long standing

cough and a history of multiple myeloma for which

he had recently received chemotherapy. A bronchial

brushing was performed.

Reactive Case #2

Reactive Case #2 Reactive Case #2Reactive/Repair

Adenocarcinoma

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Common Differential Diagnoses

• Adenocarcinoma

•Squamous cell Carcinoma

Molecular/Ancillary Testing Protocols

Unfortunately there are none!

Therapeutic Options

Treat the cause

of the

reactive process to heal the injury

Key Take Away Points

•Key Cytologic Features– Patient history– Cohesive cellular sheets– Smooth nuclear membranes– Prominent nucleoli – Abundant cytoplasm– Inflammatory background

•Key Ancillary Tests– None

•Therapeutic Implications– Treat the cause of the injury

References

•DeMay RM. The Art and Science of Cytopathology. Chicago, Ill: ASCP Press 1996

•Erozan YS.,Ramzy I. Pulmonary Cytopathology. Springer 2009:29-45:103-131

•Rabb S,MD, Oweity T,MD,Hughes J,MD,Salomao D,MD, Kelly C,MD, Flynn C,MD, D”Antonio J,PhD, and Cohen M,MD. Effect of Clinical History on , , , , , y

Diagnostic Accuracy in the Cytologic Interpretation of Bronchial Brush Specimens. American Journal of Clinical Pathology 200:114:78-83

•Koss L,Melamed M. Koss’Diagnostic Cytopathology and its Histopathologic Bases. Lippincott Williams & Wilkins 2006:595-596

•Geisinger K, Stanley M, Raab S, Silverman J, Abati A. Modern Cytopathology. Churchill Livingstone; 2004:371

Thank You

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Problematic Cases in Pulmonary Cytopathology

As Identified from the ASCP NonGYN Assessment Program

Umesh Kapur, MDASCP NonGYN Assessment Committee

Medical MemberMedical Member

Assistant Professor, Department of PathologyLoyola University Medical Center

Maywood, IL

ASCP CME Disclosure of Relevant Financial Relationships

In accordance with ACCME Standards, I verify that I

have no relevant financial relationships to disclose,

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pulmonary, NonGYN, Positive for Malignancy, AdenocarcinomaCumulative Statistics from 8 Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 28 1.7%Infectious/Inflammatory Process 13 0.8%Benign Neoplasm 1 0.1%Lesion of Uncertain Biologic Potential 6 0.4%Positive for Malignancy 1590 97.1%Total 1638 100.0%ota 638 00 0%

Reference Interpretation Choices TotalADENOCARCINOMA 1382 84.4%BENIGN BRONCHIAL EPITHELIAL CELLS 15 0.9%CARCINOID 7 0.4%GOBLET CELL HYPERPLASIA 1 0.1%HISTIOCYTES 8 0.5%No Interpretation Selected 62 3.8%REACTIVE ALVEOLAR PNEUMOCYTES 7 0.4%REACTIVE BRONCHIAL EPITHELIAL CELLS 9 0.5%SMALL CELL CARCINOMA 24 1.5%SQUAMOUS CELL CARCINOMA 119 7.3%VIRAL CHANGES 4 0.2%Total 1638 100.0%

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Lung, FNA, Positive for Malignancy, AdenocarcinomaCumulative Statistics from 5 Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 31 2.3%Infectious/Inflammatory Process 38 2.9%Benign Neoplasm 74 5.6%Lesion of Uncertain Biologic Potential 22 1.7%Positive for Malignancy 1163 87.6%Total 1328 100.0%

Reference Interpretation Choices Total %ADENOCARCINOMA 822 62.0%CHONDROID HAMARTOMA 90 6.8%MESOTHELIOMA 83 6.3%METASTATIC CHONDROSARCOMA 74 5.6%METASTATIC CHORDOMA 89 6.7%METASTATIC MELANOMA 9 0.7%No Interpretation Selected 78 5.9%BENIGN/REACTIVE BRONCHIAL EPITH CELLS 24 1.7%SQUAMOUS CELL CARCINOMA 57 4.3%VIRAL CHANGES 2 0.2%

1328 100.0%

Adenocarcinoma-Cytologic Features

•Cellularity

•Flat honeycomb sheets, rosettes, acinar structures or three dimensional groups

•Medium to large size cells with abundant delicate gcytoplasm

•Intracytoplasmic mucin

•Round to oval eccentric nuclei with fine chromatin and prominent nucleoli

Adenocarcinoma-Cytologic Features

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Adenocarcinoma-Cytologic Features Look what is inside….

Targetoid mucin

Adenocarcinoma-Cytologic Features Squamous cell carcinoma-Cytologic Features

•Cellularity

•Large sheets, cohesive or dyshesive

•Polymorphic cell shapes rounded elongated (tadpole)•Polymorphic cell shapes, rounded, elongated (tadpole)

•Dense cytoplasm, keratinization, orangeophilia

•Pyknotic nucleus, nucleolus (sometimes)

•Anucleate squames

Squamous cell carcinoma-Cytologic Features

Squamous cell carcinoma - cells

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Squamous cell carcinoma-Cytologic Features What about these?

Non-small cell lung carcinomas - NSCLC

Difficulties in accurate sub-classification

•Poorly differentiated tumors

•Non-keratinizing squamous carcinoma

•Mixed /combined tumors

Immunoperoxidase stains

P63TTF-1

CK5/6TTF-1

Immunoperoxidase stains

•Several studies have evaluated immunoperoxidase stain algorithms for sub-typing non-small cell carcinoma

E ll t i di ti t b t•Excellent accuracy in predicting tumor sub-type using a panel comprising of TTF-1, P63 and CK5/6 (Rekhtman et al. & Khayyata et al.)

•Katzenstein et al. showed accurate sub-typing utilizing a novel antibody napsin A along with TTF-1, P63 and CK5/6 in small lung biopsies

Immunoperoxidase Testing Algorithm (Rekhtman et al.)

P63 (negative)

P63 (focal)

P63 (diffuse)

TTF‐1 (diffuse)

ADC ADC ADC

TTF‐1 (focal)

ADC ADC SCC

TTF‐1 (negative)

IND IND (CK5/6)

SCC

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Significance of sub-classifying

Distinction between adenocarcinoma and squamous carcinoma impacts treatment and patient outcome

– Bevacizumab therapy is contraindicated in patients with squamous cell carcinoma because of potential fatal hemorrhageg

– Adenocarcinoma histology is a strong predictor for outcome to pemetrexed therapy in advanced-stage patients

– EGFR mutation is a validated predictive marker for response and progression-free survival with EGFR-TKIs in the first-line therapy in advanced lung adenocarcinoma

– KRAS, ERCC1, RRM1, TS, EML4-Alk

Key Take Away Points• Diagnostic cytologic features present

–Squamous cell carcinoma–Adenocarcinoma

•Poorly differentiated Non-small cell carcinoma, proceed to IHC (TTF-1, P63, CK5/6 )–Non-small cell carcinoma, favor adenocarinoma–Non-small cell carcinoma favor squamous carcinoma–Non-small cell carcinoma, favor squamous carcinoma

•IHC all negative or non-contributory–Non-small cell carcinoma

•Do not use non-squamous cell carcinoma

•Most important is diagnosis, conserve tissue for IHC and molecular studies

References•Rekhtman N, Ang DC, Sima CS et al. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimen. Mod Pathol 27 May 2011. Advance online publication

•Khayyata S, Yun S, Patha T et al. Value of p63 and CK5/6 in distinguishing squamous cell carcinoma from adenocarcinoma in lung fine-needle aspiration specimens. Diagn Cytopathol 2009; 37: 178-83

•Travis WD, Brambilla E, Noguchi M et al. International association for the study of lung cancer/ American thoracic society/European respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 2011;6: 244-285

•Hasanovic A, Rekhtman N, Sigel CS. Advances in fine needle aspiration cytology for the diagnosis of pulmonary carcinoma. Patholog Res Int. 2011;2011:897292. Epub 2011 Jun 27

References•Mukhopadhyay S, Katzenstein Al. Subclassification of non-small cell lung carcinoma l

acking morphologic differentiation on biopsy specimen: Utility of an Immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol 2011;35:15-25

•DeMay RM. The Art and Science of Cytopathology. Chicago, IL: ASCP Press 1996

•Ohori NP, Santa Maria EL. Cytopathologic Diagnosis of Bronchioloalveolar CarcinomaOhori NP, Santa Maria EL. Cytopathologic Diagnosis of Bronchioloalveolar CarcinomaDoes It Correlate With the 1999 World Health Organization Definition? Am J ClinPathol 2004;122:44-50

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Problematic Cases in Pulmonary Cytopathology

As Identified from the ASCP NonGYN Assessment Program

Indra Balachandran, Ph.D.,SCT,CFIACASCP NonGYN Assessment Committee

Cytotechnologist MemberCytotechnologist Member

Associate Professor and Program Director of Cytotechnology

Albany College of Pharmacy & Health SciencesAlbany, NY

ASCP CME Disclosure of Relevant Financial Relationships

In accordance with ACCME Standards, I verify that I

have no relevant financial relationships to disclose,

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

Small cell carcinoma (SCCa) vs non small cell carcinoma (NSCCa)

Important tenets in lung cytology•Lung carcinoma in cytology is categorized into two broad histologic

categories into small cell and non-small cell carcinoma 1,2

•Important clinical, therapeutic and prognostic significance

•Clinical management of lung cancer is based mainly on stage at diagnosis and also the histologic subtype

•Most SCCa are highly disseminated with possible metastasis at the time of diagnosis

•SCCa highly aggressive tumors with high mitotic rate

•SCCa remarkable sensitivity to chemotherapeutic agents and also radiation and therefore first in line of treatment 1

•Except for a rare Stage I SCCa, surgery is not considered

Small cell carcinoma (SCCa) vs non small cell carcinoma (NSCCa)

Important tenets in lung cytology•For NSSCa surgery is the preferred modality due to the relative

chemoresistance of these tumors

•Also with the advent of targeted therapy for certain types of NSSCa, it is imperative that these are separated from SCCa and then subjected to molecular testing to detect mutation and/orsubjected to molecular testing to detect mutation and/or amplification of EGFR, KRAS or detection of Human echinoderm microtubule-associated protein, like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene to determine applicability of EGFR-TKI or other such personalized therapy 3,4,,5

Clinical Disease Features•Hemoptysis

•Dyspnea•Chest pain•Wheezing•Post-obstructive pneumonia•MalaiseDi d d k i (S i V C S d ) i h•Distended neck veins (Superior Vena Cava Syndrome) or with

recurrent laryngeal nerve paralysis•Ectopic hormone production by the tumor producing symptoms like

Cushing’s syndrome6

•Large, bulky central tumors with paratracheal, mediastinal or other central node involvement diagnosed by EBUS-TBNA•Chest X-ray often with perihilar mass or mediastinal widening•Increased propensity for metastasis to brain diagnosed in cerebrospinal

fluid cytology, and liver, pleura and other sites by FNA•Long term survival is approximately 10% at 2 years

Etiology/Pathogenesis

SCCa and other neuroendocrine tumors of the lung

•Related to cigarette smoking with a male preponderance

•All lung tumors are derived from a common entodermal stem cell•The current WHO classification based on divergent differentiation •Wherein certain group of tumors acquire specific neuroendocrine•Wherein certain group of tumors acquire specific neuroendocrine

markers such as chromogranin, synaptophysin and neural cell adhesion molecule (CD56) and are therefore classified as neuroendocrine tumors such as typical carcinoid, atypical carcinoid, small cell carcinoma and large cell neuroendocrine carcinoma

•Divergent differentiation also explains large cell neuroendocrine carcinoma and combined small cell carcinoma with non-small cell carcinoma (CSCLCs)

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Etiology/Pathogenesis

SCCa and other neuroendocrine tumors of the lung

•CSCLSs arise when a subset of non-small cell carcinoma not considered morphologically neuroendocrine express neuroendocrine differentiation with neuroendocrine markers 8 due to random acquisition of genetic or epigenetic alterations 9

• WHO classification of CSCLCs as a subset of small cell carcinoma is based on the same clonal origin with neuroendocrine differentiation concept

•This theory also explains the presence of rare cases of mixed small cell and large cell neuroendocrine carcinoma

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pulmonary, NonGYN, Small Cell Carcinoma

Cumulative Statistics from Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 29 7.6%Infectious/Inflammatory Process 184 48.0%Benign Neoplasm 4 1.0%Lesion of Uncertain Biologic Potential 7 1.8%Positive for Malignancy 159 41.5%g yTotal 383 100.0%

Reference Interpretation Choices Total %ADENOCARCINOMA 7 1.8%ASPERGILLOSIS 165 43.1%LYMPHOMA 3 0.8%NEGATIVE/REACTIVE BRONCHIAL EPITHELIAL CELLS 5 1.3%No Interpretation Selected 40 10.4%SMALL CELL CARCINOMA 136 35.5%SQUAMOUS CELL CARCINOMA 9 2.3%VIRAL CHANGES 18 4.7%Total 383 100.0%

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Lung, FNA, Small Cell Carcinoma

Cumulative Statistics from Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 0 0.0%Infectious/Inflammatory Process 1 0.2%Benign Neoplasm 1 0.2%Lesion of Uncertain Biologic Potential 4 0.9%Positive for Malignancy 421 98 6%Positive for Malignancy 421 98.6%Total 427 100.0%

Reference Interpretation Choices Total %ADENOCARCINOMA 2 0.5%No Interpretation Selected 22 5.2%NEGATIVE/REACTIVE BRONCHIAL EPITHELIAL CELLS 2 0.5%SMALL CELL CARCINOMA 397 93.6%SQUAMOUS CELL CARCINOMA 1 0.2%

424 100.0%

Cytologic Features

General characteristics of SCCa

•SCCa can present as classic oat cell carcinoma (lymphocytelike), intermediate, spindle cell type or combined type (CSCLCs)with SCCa has a squamous or adenocarcinoma component 10

• Morphology of SCCa vary also based on the type of cytology specimen under consideration and the type of cytopreparation y y

•Bronchial brush and fine needle aspiration specimens provide a direct sample of the tumor and provide a tumor with well preserved cells with all the classic SCCa criteria

•The current WHO classification does not separate the various types of SCCa based on morphology since all the three types are similar clinical presentation and response to chemotherapy

•Combines all these subcategories into one as SCCA with CSCLCs as a subset •The subcategories are a morphologic continuum and also show similar response to

neuroendocrine markers such as chromogranin, synaptophysin and CD56•All show dense core neurosecretory granules in the cytoplasm in transmission electron

microscopy

Cytologic Features

SCCa in sputum •malignant cells 1.5 times larger

than mature lymphocyte•degenerate and appear as

aggregates of oat cell variety along streaks of mucus

•anisokaryosis and cytosishi h / ti d t•high n/c ratio and scanty

cytoplasm•marked nuclear molding•areas of apoptosis and necrosis•chromatin may have the

characteristic salt and pepper appearance in better preserved malignant cells

•but may appear rather smudgy in degenerate tumor cells

•areas of crush artifact may be seen

Cytologic FeaturesIn bronchial wash/brush samples1

•better preserved cellular samples than sputum

•cells 1.5 times the size of lymphocytes

•cells exhibit high nuclear/cytoplasmic ratios•scanty delicate, basophilic cytoplasm•nuclear molding•crush artifact•apoptosis and necrosisapoptosis and necrosis•granular salt and pepper chromatin•inconscpicuous nucleoli•single file arrangements• Sturgis et al 1in their study of 76 bronchial

wash and brush specimens list three criteria i.e., nuclear molding, salt and pepper chromatin and scant cytoplasm were more than 90% sensitive and specific for small cell carcinoma

•nuclear molding most significant •increased the odds of accurate diagnosis as small

cell carcinoma by 300 fold

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Cytologic Features

SCCa in fine needle aspirations

•numerous tumor cells arranged singly and in irregular syncyial aggregates

•tumor cells may be small or intermediate in cell size and may be round to pleomorphic spindle shaped cells 2

•cytoplasm is delicate and scanty•N/C ratio of tumor cells is very high•marked apoptosis and necrosis in the

background•nuclear molding seen within aggregates•nucleoli are absent or rarely seen as

micronucleoli•chromatin is finely granular and shows salt and

pepper consistency•occasionally some of the cells may be larger

due to direct sampling of the tumor

SCCa in ThinPrep®

Courtesy: Walid Khalbuss, M.D.

Common Differential Diagnoses

• Most common differential diagnoses include basal cell or reserve cell hyperplasia, lymphoma, and small cell type of squamous carcinoma

Small cell carcinoma Reserve cell hyperplasia

Irregular aggregates of cells sheets of small uniform cellsIrregular aggregates of cells showing anisokaryosis and anisocytosis

sheets of small uniform cells and no single cells

Nuclear moldingApoptosis and necrosis

No nuclear moldingNo apoptosis and necrosis

Salt and pepper chromatin Finely granular evenly distributed chromatin

Differential diagnosis

Reserve cell or basal cell hyperplasia Small cell carcinoma

Common Differential Diagnoses

• Most common differential diagnoses include basal cell or reserve cell hyperplasia, lymphoma, and small cell type of squamous carcinoma

Small cell carcinoma Lymphoma

Irregular aggregates of cells showing anisokaryosis and

monoclonal proliferation of single malignant cells

anisokaryosis and anisocytosisNuclear moldingApoptosis and necrosis

No nuclear moldingLymphoglandular bodies may be present, nuclear debris may be seen

Salt and pepper chromatinNucleoli not prominentTTF1, Chromogranin, synaptophysin and CD56 +

Coarsely granular evenly distributed chromatin with prominent nucleoliTTF1,Chromogranin, synaptophysinand CD56- but flow cytometryillustrates immunophenotyping with various lymphoid markers

Differential diagnosis

Small cell carcinoma Large cell lymphoma

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Common Differential Diagnoses

• Most common differential diagnoses include basal cell or reserve cell hyperplasia, lymphoma, and small cell type of squamous carcinoma

Small cell carcinoma Small cell variant of squamous carcinoma 2

Irregular aggregates of cells showing anisokaryosis and anisocytosis

aggregates of uniform population of small and/or large cells with focal intercellular bridges and isolated cell keratinizationy g

Nuclear moldingMitosis and apoptosis prominent

No nuclear molding Mitosis and apoptosis not prominent

Salt and pepper chromatinNo prominent nucleoliTTF1,chromogranin, synaptophysin, CD56+CK5/6-EM shows cytoplasmic dense core granules

Finely or coarsely granular chromatin with prominent nucleoli TTF1,chromogranin, synaptophysin, CD56-CK5/6+EM shows cytoplasmic intermediate keratin filaments

Differential diagnosis

Small cell carcinoma Squamous carcinoma – small cell type

ASCP Check Sample: 2006:C-06-2(C-180)

Histologic Features

•nests, rosettes or ribbons of small cells

•peripheral palisading of the nests•necrosis•crush artifact•Azzopardi effect may be seen 11

•cells round to fusiform•scant cytoplasm•finely granular chromatin with absent

or inconspicuous nucleoli•nuclear molding difficult visualize•high mitotic rate exceeding 10/single

HPF is characteristicCourtesy: Dr. Walid Khalbuss, MD

Molecular/Ancillary Testing ProtocolsImmunohistochemistry

•TTF1 shows nuclear+

•Synaptophysin and CD56 cytoplasmic+•Chromogranin positivity depends upon the number of

neurosecretory granules and can be negative often•Apoptosis related proteins such as bcl 2 and p53 are positive12•Apoptosis related proteins such as bcl-2 and p53 are positive12

•Large cell neuroendocrine carcinoma -membrane positivity to CK7, CK18, E-cadherin and B –catenin and differentiate from SCCa

•CSCLCs with areas showing squamous differentiation CK5/6+, TTF1-

•CSCLCs with areas showing adenocarcinoma will exhibit CK7+ positivity, TTF1+ will be positive in the SCCacomponent as well as adenocarcinoma component

Molecular/Ancillary Testing Protocols

Molecular profile of SCCA 13

•Chromosomal abnormalities (mainly deletions in the short arm) on chromosome 3 very common

•N-myc and L-myc oncogenes are most commonly amplified than C mycthan C-myc

•Extent of deletions may vary between cell lines•In deletions in the short arm on chromosome 3, the minimum

common region of overlap was assigned to bands 3p23-3p24

•Loss of heterozygosity (LOH)22q13 common

Therapeutic OptionsLimited ‐ stage disease (LD) 14 Extensive stage disease (ED) 14

At the time of diagnosis, disease is confined to hemithorax of origin, the mediastinum or the supraclavicular nodes

Tumors spread beyond the supraclavicularnodes

Surgery may be considered as an option Surgery not considered

Best results with thoracic radiation therapy combined with chemotherapy

Thoracic irradiation combined with chemothrapy does not significantly improve clinical outcome. Palliative radiation in minimizing symptoms of primary tumor extensive disease especially brain, epidural or bone metastases

Chemotherapy usually a two drug combination of platinum and etoposide

Combination chemothrapy with etoposideplus cisplatin or carboplatin

Prophylactic cranial irradiation to prevent CNS recurrence

Prophylactic cranial irradiation to prevent CNS recurrence may be considered in patients with an excellent response to chemotherapy early on

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Key Take Away Points•Key Cytologic Features

–Small cells about 1.5 times the size of lymphocyte–High n/c ratio with scan delicate cytoplasm, salt and pepper chromatin

and rare to absent micronuceloli–Nuclear molding–Crush artifact, apotosis and necrosis

•Key Ancillary Testsy y–Immunohistochemistry showing characteristic synaptophysin and

CD56 positivity in the cytoplasm and TTF1 positivity in the nucleus–Electron microscopy shows cytoplasmic dense core granules–Chromosomal abnormalities in short arm of chr.3–Loss of heterozygosity (LOH)22q13

•Therapeutic Implications–Treatment for Stage 1- surgery may be considered as an option–For most SCCa chemotherapy with platinum analogues and

podophyllotoxins with or without radiation–Prophylactic brain irradiation to prevent meningeal carcinomatosis

References1. Sturgis CD, Nassar DL, D’Antonio JA et al. Cytologic features useful for

distinguishing small cell from non-small cell carcinomas in bronchial brush and was specimens. AJCP 2000; 114:197-202

2. Jing X, David O, Moroz K. Cytologic diagnosis of small cell variant of squamous cell carcinoma and its differential diagnosis. ASCP Check Sample 2006:C-06-2(C-180)

3. Kulesza P, Ramachandran K, Patel JD. Emerging concepts in the pathology and molecular biology of advanced non-small cell lung cancer. AJCP2011;136:228-238.;

4. Dacic A. Flanagan M, Cieply K et al. Significance of EGFR Protein expression and gene amplification in non-small cell carcinoma. AJCP2006;125:860-865

5. Wagner PL, Perner S, Rickman DS et al. In Situ evidence of KRAS amplification and association with increased p21 levels in non-small cell lung carcinoma. AJCP 2009; 132:500-505

6. ASCP Non-Gyn Assessment program 2007. Case FP749 and 841

7. Staymates B.MIME Non-Gyn/FNB Cytoquest Program.2004. Case No NC654.

References8. Moran C, Suster S, Coppola D et al. Neuroendocrine carcinomas of the lung.

AJCP2009;131:206-221.

9. Wagner PL, Kitabayashi N, Chen YT et al. Combined small cell lung carcinomas. Gentypic and immunophenotypic analysis of the separate morphologic components. AJCP 2009;131:376-3

10. Siddiqui MT, Fatima N. Pulmonary neuroendocrine tumors: A review of clinicopathologic and cytologic features. ASCP Check Sample 2011:C-11-5(C-443)

11 Ki i SR C l Atl f diff ti l di i i f li ti d i ti11. Kini SR. Color Atlas of differential diagnosis in exfoliative and aspiration cytopathology. 2nd edition. Philadelphia, Lippincott Williams & Wilkins, 2011.

12. Nitadori J, Ishii G, Tsuta K et al. Immunohistochemical differential diagnosis between large cell neuroendocrine carcinoma and small cell carcinoma by tissue microarray analysis with a large anitobody panel. AJCP 2006;125:682-692.

13. Ibsen JM, Walters JJ, Twentyman PR etal. Oncogene amplification and chromosomal abnormalities in small cell lung cancer. J of Cell Biochem1987; 33(4): 267-288.

14. http://www.cancer.gov/cancertopics/pdq/treatment/small-cell-lung/healthprofessional/page1

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As Identified from the ASCP NonGYN Assessment Program

Ajay Shah, MDASCP NonGYN Assessment Committee

Medical Member

Problematic Cases in Pulmonary Cytopathology

Medical DirectorFNA Clinic and Dept. of Pathology

The Toledo ClinicToledo, Ohio

In accordance with ACCME Standards, I verify that I

have no relevant financial relationships to disclose,

ASCP CME Disclosure of Relevant Financial Relationships

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

Metastatic Neuroendocrine Neoplasms from Lung

Prototype – Small Cell Carcinoma

Disease Features

• Carcinoids - Grade I – malignant because metastasizes,

rarely. More common with Grade II carcinoids

• Small Cell & Large Cell – Grade III

• Neural Type NET – rare

• Mets from lung more common than other primary site

relates to frequency

Prototype – Small Cell

• Male : Female 3-4:1 peak in 60’s

• Smokers

• Central, rarely peripheral – BUT almost always disseminated at time of diagnosis

-60 to 75% with hilar/mediastinal mets

- 2nd ry deposits often larger than 1 ry

• Products: Neuroamines & Neuropeptides

• Non specific clinical features: pl effusion, cough, hemoptysis, pop, wt. loss, abd pain, etc.

Cytology – Small Cell

• Small cell 2-4x size of lymphocytes, isolated but more commonly in synctia

• Delicate cells – traumatic effects

• High N/C ratio; scant cytoplasm

• Nuclear Molding

• Salt & Pepper to fine chromatin

• Nucleoli inconspicious

• Mitotic activity

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Metastatic to Pleural Fluid

• 83 yr old male with pleural effusion

Ref: ASCP Non-Gyn Assessment Program Case NG 763

Metastatic to Pleural Fluid

• 45 year old male with lung mass and pleural effusion

Ref: ASCP Non-Gyn Assessment Program Case NG 1270

Metastatic to Pleural Fluid

• Case 3 - 54 year old with history of invasive breast carcinoma 10 years ago now presents with 2 cm lung mass and pleural effusion.

Ref: ASCP Non-Gyn Assessment Program Case NG 1281-2

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Body Fluid, NonGYN, Metastatic Small Cell CarcinomaDiagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 55 1.7%Infectious/Inflammatory Process 0 0.0%Benign Neoplasm 0 0.0%Lesion of Uncertain Biologic Potential 18 0.6%Positive for Malignancy 3128 97.7%Total 3201 100.0%

Reference Interpretation Choices Total %BENIGN/REACTIVE MESOTHELIAL CELLS 59 1.8%BRONCHOALVEOLAR CARCINOMA 48 1.5%GERM CELL TUMOR 2 0.1%LARGE CELL UNDIFFERENTIATED CARCINOMA 14 0.4%LYMPHOMA 13 0.4%MESOTHELIOMA 9 0.3%METASTATIC ADENOCARCINOMA 134 4.2%METASTATIC MELANOMA 9 0.3%METASTATIC SMALL CELL CARCINOMA 2794 87.3%METASTATIC SQUAMOUS CELL CARCINOMA 2 0.1%No Interpretation Selected 112 3.6%RHEUMATOID EFFUSION 5 0.2%Total 3199 100.0%

Metastatic to Liver

• 73 yr old male with abnormal sputum cytology and multiple liver nodules

Ref: ASCP Non-Gyn Assessment Program Case FP 1316

SMALL CELL

Ref: ASCP Non-Gyn Assessment Program Case FP 1316

Ref: ASCP Non-Gyn Assessment Program Case FP 1320 and FP345

CARCINOID

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Ref: ASCP Non-Gyn Assessment Program FP345

Ref: ASCP Check Sample Case C11-5

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Liver/Pancreas, FNA, Metastatic Small Cell Carcinoma

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 25 2.8%Infectious/Inflammatory Process 0 0.0%Benign Neoplasm 2 0.2%Lesion of Uncertain Biologic Potential 10 1.1%Positive for Malignancy 858 95.9%Total 895 100.0%

Reference Interpretation Choices Total %CHOLANGIOCARCINOMA 22 2.5%HEPATOCELLULAR CARCINOMA 37 4.1%LYMPHOMA 7 0.8%NORMAL CELLULAR ELEMENTS 27 3.0%METASTATIC ADENOCARCINOMA 26 2.9%METASTATIC MELANOMA 5 0.6%METASTATIC SMALL CELL CARCINOMA 748 83.6%No Interpretation Selected 45 5.0%

895 100.0%

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pancreas, FNA, Metastatic Carcinoid

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 6 2.1%Infectious/Inflammatory Process 1 0.3%Benign Neoplasm 0 0.0%Lesion of Uncertain Biologic Potential 23 8.0%Positive for Malignancy 258 89.6%os t e o a g a cy 58 89 6%Total 288 100.0%

Reference Interpretation Choices Total %HEPATOCELLULAR CARCINOMA 51 17.7%NORMAL LIVER/HEPATOCYTES 4 1.4%METASTATIC ADENOCARCINOMA 8 2.8%METASTATIC CARCINOID 212 73.6%No Interpretation Selected 13 4.5%

288 100.0%

Differential Diagnosis

• Site dependent

Liver (met adenoca, HCC, Cholangioca)

Pancreas (islet cell, adenoca)

Lymph Node (met ca, lymphoma)

Pleura (met adenoca, mesothelioma)

• Age dependent

Pediatric (“neural” type more common) vs. Adult

Molecular/Ancillary Studies

• IHC :especially CD 56/NCAM, Synaptophysin, Chromogranin others include TTF-1, NSE, CD57, PAX-5, b-sCK (CAM 5.2, MNF116 )

Molecular :• Molecular : p53, Rb, Ki-67p53, k-ras-2, c-raf-1

• Morphology and Immunoprofile define NET• Molecular genetic testing for dx is limited

55 yr old dx with metastatic prostate ca 2yrs ago, rx with Lupron, then radiation 6 mo ago for low back and hip pain, now presents with supraclavicular mass – ultrasound guided FNA preformed:

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Synaptophysin Chromogranin PSASynaptophysin Chromogranin PSA

CD 56 Pan CK TTF ‐ 1control

Therapy

• Limited success over past decade in survival

• 5 year survival in Grade III NEC vs. NSCLC

(stage I resectable) 30% vs. 60%

P f NE diff ti ti i ti f th t• Presence of NE differentiation in any portion of the tumor is associated with poor prognosis –thus accurate typing particularly noting presence of any neuroendocrine morphology or differentiation is critical for Rx.

• No “targeted therapy” for NEC as yet.

Therapy

• Limited Stage – nodal mets + primary tumor covered in radiation field rad + chemo

• Higher Stage – chemo (carbo, cis-plat, VP-16)i iti ll i b t i t d linitially responsive but resistance develop

Key Take Away Points

• Key Cytology Features: salt & pepper to fine granular chromatin pattern,nuclear molding, mitosis, high N/C scant cytoplasm.

• Key Ancillary Tests:y yIHC – Synaptophysin, Chromogranin, CD56

• Therapeutic Implications:Presence of NE morphology or differentiation in any portion of tumor impacts on Rx and outcome therefore accurate tumor typing is critical!

References• Sudha R. Kini.  Color Atlas of Differential Diagnosis in Exfoliative and Aspiration 

Cytopathology,  Second Edition Lippincott Williams & Wilkins, 2011, 349‐369 

 • DeMay RM.  The Art of Science of Cytopathology 

Chicago, III:  ASCP Press 1996  

• Felix G. Fernandez, MD and Richard J. Battafarano, MD, PhD.  Large‐Cell Neutroendocrine Carcinoma of the Lung October 2006, Vol. 13, No. 4, 270‐275 

 • Cesar A. Moran, MD, Saul Suster, MD, Domenico Coppola, MD and Mark R. Wick, MD.  

Neuroendocrine Carcinomas of the Lung A Critical AnalysisNeuroendocrine Carcinomas of the Lung, A Critical Analysis Am J Clin Pathol 2009; 131, 206‐221 

 • Gabriel Sica, MD, PhD, Madeline F. Vazquez, MD, nassar Altorki, MD, Jeffrey Port, MD, 

Paul C. Lee, MD, Yifang Liu, MD, Elizabeth Hyjek, MD and Anjali Saqi, MD.   PAX‐5 Expression in Pulmonary Neuroendocrine Neoplasms, Its Usefulness in Surgical and Fine‐Needle Aspiration Biopsy Specimens Am J Clin Pathol 2008; 129, 556‐562 

 • Patrick L. Wagner, MD, Naoki Kitabayashi, Yao‐Tseng Chen, MD, PhD and Anjali Saqi, 

MD.  Combined Small Cell Lung Carcinoma, Genotypic and Immunophenotypic Analysis of the Separate Morphologic Components Am J Clin Pathol 2009; 131, 376‐382 

 • Ronald A. DeLellis, MD.  The Neuroendocrine System and Its Tumors, An Overview 

Am J Clin Pathol 2001; 115, S5‐S16  

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Problematic Cases in Pulmonary Cytopathology

As Identified from the ASCP NonGYN Assessment Program

Adebowale J. Adeniran, MDASCP NonGYN Assessment Committee

Medical MemberMedical Member

Assistant Professor of PathologyYale University School of Medicine

New Haven, CT

ASCP CME Disclosure of Relevant Financial Relationships

In accordance with ACCME Standards, I verify that I

have no relevant financial relationships to disclose,

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

ASCP CME Disclosure of Relevant Financial Relationships

In accordance with ACCME Standards, I verify that I

have no relevant financial relationships to disclose,

with proprietary entities producing health care goods

or services, that are discussed in this CME activity.

Guidelines for interpreting body cavity fluids

We need to know:

1. The significance of a positive diagnosis

2. The most reliable criteria for a diagnosis of malignancyg g y

3. The common pitfalls

4. When to be cautious

5. When to look really hard for the foreign cells

6. How to deal with the problematic case

Pleural fluid cytology

The problem

Metastatic adenocarcinoma versus benign/reactive mesothelial cells

Metastatic adenocarcinoma versus mesothelioma

Mesothelioma versus reactive mesothelial cells

Guidelines for body cavity fluids

Cells in 3-D

Large groups of cells with complex arrangement

A discrete population of cells distinct from mesothelial cells

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Case #1

An 86-year-old female with a history of pneumonia

and empyema has persistent bilateral pleural

ff i d l l thi k i Th t ieffusions and pleural thickening. Thoracocentesis

was performed and the pleural fluid was submitted

for evaluation.

Key diagnostic features

•Mostly single cell presentation with occasional clusters

•Predominantly mononuclear or binuclear cells

•Round cells with low nuclear/cytoplasmic ratiosy p

•Round, bland nuclei with small inconspicuous nucleoli

•Dense cytoplasm with clear outer rim (“lacy skirt”)

•Evidence of window-like slits between cells

MOC-31 Ber-EP4

D2-40 CK5/6

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pleural Fluid, NonGYN, Benign/Reactive Mesothelial Cells

Cumulative Statistics from Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 609 84.2%Infectious/Inflammatory Process 46 6.4%Benign Neoplasm 0 0.0%Lesion of Uncertain Biologic Potential 23 3.2%Positive for Malignancy 45 6 2%Positive for Malignancy 45 6.2%Total 723 100.0%

Reference Interpretation Choices %ADENOCARCINOMA, NOS 35 4.8%ATYPICAL LYMPHOID CELLS, R/O MALIGNANCY 16 2.2%BENIGN/REACTIVE MESOTHELIAL CELLS 609 84.2%GRANULOMATOUS INFLAMMATION 23 3.2%LYMPHOMA 2 0.3%MESOTHELIOMA 6 0.8%No Interpretation Selected 31 4.3%SMALL CELL CARCINOMA 1 0.1%Total 723 100.0%

Causes and key cytologic features of Non-neoplastic effusions

Congestive heart failureOccasional hemosiderophages

Pulmonary infarctionNon-specific mixed inflammation

PneumoniaInflammatory cells of various types depending on the nature and duration

of the pneumoniaof the pneumonia

Chemotherapy and Radiation pneumonitisNo consistent and distinctive changes

Autoimmune serositis (e.g. SLE)Characteristic LE cells and moderate amount of neutrophils

Rheumatoid pleuritisAbundant clumps of granular debris and macrophages

TuberculosisHigh proportion of lymphocytes and few mesothelial cells

Others: Hypothyroidism, Nephrotic syndrome, Cirrhosis

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Typical immunostaining patterns of Reactive mesothelial cells and Adenocarcinoma

Tumor Reactive mesothelial cells AdenocarcinomaAE1/AE3 + +

CK5/6 + -Calretinin + -HBME-1 + -/+

WT 1 +WT-1 + -D2-40 + -CEA - +

Ber-EP4 - +B72.3 - +CD15 - +

MOC-31 - +TTF-1 - +

Case #2

An 85-year-old man presented with increasing

shortness of breath. Chest film revealed a large right

l l ff i ith i t l t i f thpleural effusion with compressive atelectasis of the

right lung. Thoracocentesis yielded 800cc of

serosanguinous fluid which was centrifuged and

prepared for cytologic evaluation.

Key diagnostic features

•Cellular sample with 2-cell population

•3-D cell groups with common cell border

•Numerous large clusters

•Eccentric nuclei with marked hyperchromasia and prominent irregular nucleoliprominent irregular nucleoli

•Homogeneous delicate cytoplasm with large randomly distributed secretory vacuoles

•Lacunae (cell block sections)

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TTF-1 Ber-EP4

Calretinin D2-40

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pleural Fluid, NonGYN, Adenocarcinoma, lung primary

Cumulative Statistics from Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 21 4.7%Infectious/Inflammatory Process 7 1.6%Benign Neoplasm 0 0.0%Lesion of Uncertain Biologic Potential 2 0.4%Positive for Malignancy 421 93.3%Total 451 100.0%

Reference Interpretation Choices Total %ADENOCARCINOMA, LUNG PRIMARY 302 67.0%BENIGN/REACTIVE MESOTHELIAL CELLS 29 6.4%MESOTHELIOMA 31 7.0%METASTATIC MELANOMA 30 6.9%METASTATIC SQUAMOUS CELL CARCINOMA 37 8.2%No Interpretation Selected 22 4.5%Total 451 100.0%

Most common tumors that cause malignant pleural effusions by sex

Men WomenLung BreastLymphoma/leukemia Lungy p gGastrointestinal tract Lymphoma/leukemiaSarcoma OvaryMesothelioma Gastrointestinal tractGenitourinary (kidney, bladder, prostate) EndometriumMelanoma Mesothelioma

Cytologic differences between Malignant Mesothelioma and Adenocarcinoma

Feature Malignant Mesothelioma AdenocarcinomaCell population Monotonous mesothelial cells 2-cell population

Cellular groups 3-D, tight spheres and loose clusters with knobby bordersWindows commonly seen

Syntitial 3-D cluster or papillae with community bordersWindows unusual

Psammoma bodies Few in number when present Numerous when presentPsammoma bodies Few in number when present Numerous when present

Nucleus Usually central or paracentralMild hyperchromasiaSmall or very prominent nucleoli

Usually eccentricMarked hyperchromasiaFrequently prominent irregular nucleoli

Cytoplasm Dense center with fuzzy edges2-tone staining

Delicate, homogeneousUniform stain

Vacuoles Perinuclear and submembranous

Secretory, largeRandomly distributed

Multinucleated giant cells

Common Rare

Typical immunostaining patterns of Malignant Mesothelioma and Adenocarcinoma

Tumor Malignant mesothelioma AdenocarcinomaAE1/AE3 + +

CK5/6 + -Calretinin + -HBME-1 + -/+

WT 1 +WT-1 + -D2-40 + -CEA - +

Ber-EP4 - +B72.3 - +CD15 - +

MOC-31 - +TTF-1 - +

Case #3

A 79-year-old man was seen at the emergency room

for possible myocardial infarction. On exam a large

right pleural fluid was discovered as was thickening

of the chest wall. Thoracocentesis yielded 1000cc of

bloody fluid and was submitted for processing and

cytologic evaluation.

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Key diagnostic features

•Large clusters with scalloped (“knobby”) edges

•Giant mesothelial cells, including binucleated multinucleated forms

•Cellular clasping and “cell within cell” appearance

•Round centrally placed nucleus and prominent•Round, centrally placed nucleus and prominent nucleolus

•Dense cytoplasm with peripheral “halo”

•Normal nuclear-to-cytoplasmic ratio

•Windows

Statistical Performance of Problematic CasesCases from the ASCP NonGYN Assessment Program

Pleural Fluid, NonGYN, MesotheliomaCumulative Statistics from Reference Cases

Diagnostic Category Total %Negative/Reactive/Hyperplasia/Developmental 264 8.0%Infectious/Inflammatory Process 78 2.4%Benign Neoplasm 10 0.3%Lesion of Uncertain Biologic Potential 36 1.1%Positive for Malignancy 2925 88.3%Total 3313 100.0%

Reference Interpretation Choices %Reference Interpretation Choices %ACUTE INFLAMMATION/BACTERIAL INFECTION 38 1.1%ADENOCARCINOMA, NOS 263 7.9%BENIGN/REACTIVE MESOTHELIAL CELLS 191 5.8%CHANGES S/O PNEUMOTHORAX 27 0.8%GRANULOMATOUS INFLAMMATION 23 0.7%LYMPHOMA 26 0.8%METASTATIC GLIOBLASTOMA 8 0.2%METASTATIC MELANOMA 27 0.8%MESOTHELIOMA 2482 74.9%Negative for Malignancy 23 0.7%No Interpretation Selected 137 4.1%RADIATION THERAPY CHANGES 6 0.2%RHEUMATOID EFFUSION 20 0.6%SMALL CELL CARCINOMA 3 0.1%SQUAMOUS CELL CARCINOMA 39 1.2%Total 3313 100.0%

Common Differential Diagnoses of Mesothelioma

•Reactive mesothelial cells

•Metastatic tumor- Adenocarcinoma- Squamous cell carcinoma- Epithelioid hemangioendothelioma

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Mesothelioma versus Reactive mesothelial cells

•Mesothelioma cells are markedly larger in size

•Chromatin in mesothelioma stains variably darker and may be irregular in distribution

•Nucleoli are usually present and may be enlarged and multiplemultiple

•Macronucleoli are associated with malignancy and may be the sole criterion of malignancy

•Reactive mesothelial proliferations may show high cellularity, cytologic atypia, papillary excrescences, and entrapment

•Malignant mesotheliomas may appear bland

Mesothelioma versus Reactive mesothelial cells

• Ancillary studies are of little value in this scenario– Immunohistochemistry

• Desmin• EMA• GLUT-1

P53• P53• Ki67• Oncofetal Protein IMP3

– Molecular studies• 9p21 homozygous deletion

• Distinction remains a clinicopathologic one

• Pathologic parameter is morphologic assessment by standard H&E light microscopy

Effusion cytology caution!!!

Positive effusion = stage 4 metastatic disease

Pitfalls in body cavity fluids

• Single malignant cells

• Uniform population of tumor cells with virtually no mesothelial cells

• Reactive atypia

Clues to identifying single malignant cells

• Intracytoplasmic mucin may be a distinct, well-defined vacuole i.e. target-like, or may be multiple small, fine vacuoles

• High nuclear/cytoplasmic ratio (N/C) cells• High nuclear/cytoplasmic ratio (N/C) cells

• History-breast, gastric or other GI primaries are especially notorious for producing dispersed malignant cells

• Always look for the lurking, hiding malignant cells

Key Take Away Points

• Approach to a difficult effusion– History– Cell arrangement– Compare with obvious mesothelial cells– Cytomorphology– Prepare additional smears– Prepare cell block– Ancillary studies– Consultation– Communicate limitations

• Therapeutic Implications– Positive effusion = stage 4 metastatic disease– Mesothelioma = radical surgery + radiation & chemotherapy

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References•Cibas ES, Ducatman BS. Cytology: Diagnostic Principles and Clinical

Correlates. Sauders Elsevier, Philadelphia, PA , 2009

•Ko EC, Jhala NC, Shultz JJ, Chhieng DC. Use of a Panel of Markers in the Differential Diagnosis of Adenocarcinoma and Reactive Mesothelial Cells in Fluid Cytology. Am J Clin Pathol 2001;116:709-715

•Lee A Baloch ZW Yu G Gupta PK Mesothelial Hyperplasia with Reactive•Lee A, Baloch ZW, Yu G, Gupta PK. Mesothelial Hyperplasia with Reactive Atypia: Diagnostic Pitfalls and Role of Immunohistochemical Studies – A Case Report. Diagn Cytopathol. 2000:22:113-116

•Johnston WW. The Malignant Pleural Effusion: A Review of Cytopathologic Diagnoses of 584 specimens from 472 Consecutive Patients. Cancer. 1985;56:905-909.

•Hasteh F, Lin GY, Weidner N, Michael CW. The Use of Immunohistochemistry to Distinguish Reactive Mesothelial Cells from Malignant Mesothelioma in Cytologic Effusions. Cancer Cytopathol. 2010;118:90-96

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