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Cytology in EBUS

Myriam Remmelink MD, PhDDpt Surgical Pathology, Brussels

CONFLICT OF INTERESTS:

§ To comply with UEMS CME regulations

§ No conflict of interests

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AGENDA

§ Introduction§ Indications§ Material§ Technical considerations§ Cytologic examination§ Pitfalls§ Ancillary studies§ Conclusions05.07.18 3

EBUS - TBNA

§ Endobronchial Ultrasound-Guided Transbronchial NeedleAspiration

§ Diagnostic modality that can help provide pathologicalsampling of lung or mediastinal lesions

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Echo-endoscopeEBUS - TBNA

Echo-endoscopeEBUS - TBNA

EBUS - TBNA

EBUS - TBNA

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EBUS-TBNAEBUS - TBNA

3-5 for ech targeted lesionpasses

EBUS-TBNA

3 -5 passes / target

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• Minimally invasive• Safe• Cost effective• Real time image guidance • Provides high yield specimens

• Challenging : experience &expertise• Cannot be used to access alllymph node• May procure sample withbronchial contamination• May have non diagnostic resultsthat need additional sampling

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The IASLC lymph node map, group of lymph node stations into zones

MAIN INDICATIONS

§ (Re) Staging of lung cancer patients

§ Diagnosis of lymphadenopathy

§ Enlarged or FDG-avid mediastinal lymph node(primarly in the anterior and superior mediastinum)

§ Mass lesion within the mediastinum or centrally located inthe lung

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COMPLICATIONS

§ Low complication rate (<1%), no mortality¨ Bleeding¨ Infection¨ Pneumomediastinum¨ Pneumothorax

§ Safe

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DIAGNOSTIC PERFORMANCES

Very high specificityclose to 100%

in most studies

The sensitivity varies depending on whetherhistological or clinical follow upis used for

comparison

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DIAGNOSTIC PERFORMANCES

SAMPLING Obtaining good samples

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GOLD STANDARD : MEDIASTINOSCOPY

Specificity : 100% both

Sensibility : Best in mediastinoscopy : 91%

PRICE ! EBUS Mediastinoscopy

Morbidity <1% 0,5-2,5%

Mortality 0 0,02%

SPECIMEN COLLECTION AND PROCESSING

§ In order to optimize aspirated material, all residualmaterial in the needle and syringue, and the excessmaterial on slides or in blood clots should be collected forpotential ancillary studies

§ FNA material can be used to prepare direct smear, cellblocks

§ And submit to microbiology culture, molecular studies ifneeded05.07.18 19

ROSE OR NOT?

§ Rapid on site evaluation

§ Done by cytologist ( or telecytology ! )

§ Can provide feedback regarding adequacy of the specimen :QUANTITATIVE AND QUALITATIVE

§ Improve the diagnostic yield

§ Allow appropriate triage of the procured specimen05.07.18 20

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ROSE

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EBUS TBNA needle are long and have multiple movable parts

Thus more than one person may be needed duringslide preparation :

One can hold the needle tip and guide expelledmaterialThe other can hold the handle and plunge the syringue filled with air

SLIDE PREPARATION

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1 slide/passeAir dryed , Diff Quick staining

Formalin 10% bufferedCell block

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TRIAGE

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(Rose)Cellblock

3. Infection? Sterile container for microbiology culture

4.Lymphoproliferative disorder? Medium (RPMI) for flow cytometry

5. Molecular testing

DIFFICULTIES

§ Lack of standardized criteria

§ Wide variety of mediastinal/lung lesions to consider(always have clinical data!!!)

§ Potential contamination of the needle with normal ordysplastic bronchial elements

§ Lack of ancillary studies during ROSE05.07.18 26

DIAGNOSTIC DIFFICULTIES AND CHALLENGES

§ Non diagnostic EBUS-TBNA specimen can still be hypercellular

§ Due to numerous reactive bronchial epithelial cells, cartilage, mucoidcells

§ The high cellularity of EBUS-TBNA makes these cases difficult toscreen both at the time of Rose evaluation and final evaluation

§ Thus the focus is more on qualitative , not quantitative features

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PRACTICAL APPROACH TO EVALUATION

§ Adequate : YOU SEE SOMETHING§ Tumor cells OBVIOUS!, no rareOr§ Granulomatous inflammationOr§ Sufficent (?) number of lymphocytes and/or pigment laden

macrophages

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Sufficent?No definitive criteria but important to avoid false negative!!One slide with lymphocytes comprising 30% or more of thecellsMore than 40 lympho/HPF in most cellular area

LYMPHOCYTE COUNTS (DQ STAIN, X400)

<40 =40 >40

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ANTHRACOTIC PIGMENT LADEN MACROPHAGES

Helpful indicator of lymph node samplingDD : melanin, graphite, hemosiderin pigment

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DIAGNOSTIC DIFFICULTIES AND CHALLENGES

§ There is currently no universally accepted welldefined adequacy criteria for EBUS-TBNAspecimens

§ Pitfalls in evaluation of adequacy in EBUS-TBNA

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Bland neoplasms Mimic benign bronchial epithelial cells

Single malignant cells Difficult to identifyReactive changes withinbronchial epithelium

Mimic malignancy

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BLAND NEOPLASM

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SINGLE ATYPICAL CELLS

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REACTIVE CHANGES

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ADEQUACY OF THE SPECIMEN

§ CRITICAL

§ Non diagnostic cases >< negative case

§ Minimize the risk of false negative

§ No well-established adequacy criteria with regard to thenumber of lymphocytes needed for a negative diagnosis

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ADEQUACY

§ Major problem in EBUS : contamination!!! (bronchial cells )

§ Do I have sufficient lymphocytes to indicate that the lymph node isadequately sampled?

§ ? Germinal center fragments and clusters of anthracotic pigment-laden macrophages

§ Is there are malignant cells or granuloma among all the benign andreactive cells?

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Adequate§ Adequate specimen =

material that explains the patient’s lymphadenopathy

§ Malignant cells?§ Granulomatous

inflammation?§ Suffisant lymphoid cells

Inadequate§ Abundant bronchial

contamination§ And/or§ Blood§ Without lymphoid cells ,

granulomatous inflammation, malignant cells

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ADEQUACY OF THE SPECIMEN

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INADEQUATE

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CLINICAL MANAGEMENT

Mediastinallymphadenopathy>1 cm of FDG avid on

PET scan

EBUS TBNA

Malignant

Treatment

Indeterminate or non diagnostic

Mediastinoscopy

Benign

Médiastinoscopy Specific diagnosis

NON DIAGNOSTIC OR UNSATISFACTORY

§ Mediastinoscopy

§ Could be informative!

§ Biologic properties of the lesion!§ Lymphomas!

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NORMAL COMPONENTS

§ Recognizing normal components is CRITICAL to avoid anoverdiagnosis or misdiagnosis in these specimens

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Check adequacy

Confusing !!!!!

LYMPHOCYTES

§ Benign and reactive lymphocytes can be difficultto distinguish from§ Small cell lymphoma§ Thymic lesions§ Small blue cell tumors

§ Clinical correlation !!!§ Discussion with the clinician§ Flow cytometry, immunohistochemistry05.07.18 44

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BENIGN BRONCHIAL CONTAMINANTS

§ EBUS-TBNA needle traverses the bronchial wall§ CONTAMINATION§ Bronchial cells : presence of cilia on the apical surface arising from a

terminal bar

DD carcinoma

§ Nuclear polymorphism, hyperchromasia, nuclear enlargment05.07.18 45

CONTAMINATION , BACKGROUND MATERIAL , ARTIFACTS

§ Bronchial contamination gives rise to specimens with high baseline cellularity even in non diagnostic samples

§ Bronchial contamination such as reactive or metaplasticbronchial epithelial cell , mucous and cartilage is important to recognize because in the absence of lymphoid cells, granulomas or tumor cells, it indicates a non diagnostic sample

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CONTAMINATION , BACKGROUND MATERIAL , ARTIFACTS

§ The main type of background include blood, mucinous, necrotic,inflammatory, tigroid and lymphoid

§ Artefactual distorsion such as air drying or crush artifact

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CONTAMINATION , BACKGROUND MATERIAL , ARTIFACTS

§ Goblet cell metaplasia >< signet ring type adenocarcinoma

§ Clustering, ciliated cell, lack pleomorphism or necrosis

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CONTAMINATION , BACKGROUND MATERIAL , ARTIFACTS

Mucoid material >< mucinous adenocarcinoma

§ Dirty, entrapped bronchial cells, no cells with mucinous cytoplasmor targetoid vacuoles with central mucin droplet

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CONTAMINATION , BACKGROUND MATERIAL , ARTIFACTS

§ Lymphoid cells crushed >< small cell carcinomaI

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CONTAMINATION

§ Key features of benign or reactive cells :

§ Scant cells with atypia§ Lack of necrosis§ Lack of mitotic figures§ Spectrum of changes from benign to reactive (not two distinct

different populations)

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CONTAMINATION

§ Caution : § Poor fixation/ staining§ Obscuring inflammation§ Scant cellularity

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GRANULOMATOUS INFLAMMATION

§ First : Are you sure it is a § granuloma?

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GRANULOMATOUS INFLAMMATION

§ Second : type of granuloma?

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Correlation with• special stains• microbiology culture results• clinical findings

PULMONARY EPITHELIAL NEOPLASMS

§ Diagnosing NSCLC can be challenging given the cytomorphological overlap with

§ Granulomatous inflammation, § Reactive bronchial epithelial atypia,§ Metastatic extrapulmonary carcinomas

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PULMONARY EPITHELIAL NEOPLASMS

§ Diagnosing NSCLC can be challenging given the cytomorphological overlap with

§ Granulomatous inflammation, IHC§ Reactive bronchial epithelial atypia,§ Metastatic extrapulmonary carcinomas IHC

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PULMONARY EPITHELIAL NEOPLASMS

§ Small cell carcinomas can be difficult to differentiate from

§ Benign lymphocytes with crush artifacts, lymphoma, IHC

§ Neuroendocrine neoplasms§ NSCLC (basaloid carcinoma, LCNEC) IHC

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PULMONARY EPITHELIAL NEOPLASMS

§ Collaboration with clinicians to avoid loss of material!!!

§ Dont forget : Sparing of material for molecular studies in case of ADC, NSCLC favor ADC NSCLC-NOS

§ EBUS-TBNA is frequently used as the primary diagnostic modalities for establishing a diagnosis and for staging the patient

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ALGORITHM FOR THE WORK-UP OF NON RESECTIONLUNG CANCER SPECIMEN J Thorac Oncol, 2011

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IMMUNOHISTOCHEMICAL TYPING & CLASSIFICATION IASCL/ATS/ERS OF NSCLC

+

Moleculartesting

Moleculartesting

No test

WHO 2015

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NON PULMONARY METASTATIC CARCINOMAS

§ Metastatic non pulmonary carcinomas can overlap in cytomorphologywith lung carcinomas which can make difficult to diagnose in EBUS-TBNA

§ Especially in patients with a prior history of another tumor or in patients without a lung mass

§ It is important to obtain sufficient material for ancillary studies as IHC and potential molecular studies, in select tumor when one isconsidering a metastatic non pulmonary carcinomas

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NON PULMONARY METASTATIC CARCINOMAS

§ Careful attention devoted to clinical history and atypical morphology or the immunoprofile should raise suspicion of a non pulmonarycarcinoma

§ Site specific staining can help to determine the site of origin for a variety of non pulmonary adenocarcoma

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ADENOCARCINOMA AND IMMUNOPROFILE

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Metastatic papillary carcinoma can mimic primary pulmonary adenocarcinomawith papillary features (TTF1 positivity in both!)

METASTATIC NON PULMONARY SQUAMOUS CELLCARCINOMA

§ Paucity of site-specific markers that we can use to determine their site of origin

§ P16 ( + in situ hybridation for HPV)

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NON EPITHELIAL NEOPLASM

§ Lymphomas!!!

§ And other hematologic malignancies

§ Mesenchymal tumors

§ Germ cells tumors

§ Melanomas

§ Mesothelioma05.07.18 67

Sufficient material!Ancillary studies!

NON EPITHELIAL NEOPLASM

§ Lymphomas!!! And other hematologicmalignancies

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Flow cytometryImmuno-Cyto-chemistry

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THYMIC LESIONS AND NEOPLASMS

§ Limited reports

§ Limited to patient for whom a surgery is not feasible

§ Cytologic examination of thymoma alone cannot reliablyclassify the various subtypes of thymomas nor can theybe used to determine invasion

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TAKE HOME MESSAGE 1

§ ROSE § Quantitative and qualitative examination

§ Tumor cells OBVIOUS!, no rareOr§ Granulomatous inflammation Or§ Sufficent (?) number of lymphocytes and/or pigment

laden macrophages

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TAKE HOME MESSAGE 2

§ Contamination!

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TAKE HOME MESSAGE 3

§ Collaboration!

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LITTERATURE

Endobronchial ultrasound-transbronchial needle aspiration (EBUS/TBNA): a diagnostic challenge for mediastinal lesions.

§ Divisi D, Zaccagna G, Barone M, Gabriele F, Crisci R.

§ Ann Transl Med. 2018 Mar;6(5):92.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA)-from morphology to molecular testing.

§ Righi L, Franzi F, Montarolo F, Gatti G, Bongiovanni M, Sessa F, La Rosa S.

§ J Thorac Dis. 2017 May;9(Suppl 5):S395-S404.

Guideline for the acquisition and preparation of conventional and endobronchial ultrasound-guided transbronchial needleaspiration specimens for the diagnosis and molecular testing of patients with known or suspected lung cancer.

§ van der Heijden EH, Casal RF, Trisolini R, Steinfort DP, Hwangbo B, Nakajima T, Guldhammer-Skov B, Rossi G, Ferretti M, Herth FF, Yung R, Krasnik M; World Association for Bronchology and Interventional Pulmonology, TaskForce on Specimen Guidelines.

§ Respiration. 2014;88(6):500-17..

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