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LungLung CytologyCytologyLungLung CytologyCytologyPınar Fırat,MD,MIAC

Diagnosis of lung cancerDetection of infectionsEvaluation of interstitial diseases

LungLung CytologyCytology

Morphologic featuresApproach to small samples

LungLung CancerCancer

• Second most frequent cancer in both sexes– Most common reason of cancer deaths

• ~ 70% diagnosed at advanced stage– Small biopsies and cytology : Main diagnostic tool~40%

• Differentiating SCLC from NSCLC is importantbut not sufficient– Histologic subtype of NSCLC has a clinical relevance; – predicts efficacy and toxicity of some treatments – predicts the likelihood of molecular changes which

may lead to specific therapies.

~85%

ResponsibilityResponsibility of of pathologistpathologistin in lunglung cancercancer

• Diagnosis

• SubtypingE f li ti t l

yp g

• Molecular testsExfoliative cytologyFine needle aspirationtransthoracic/ transbronchial

Lung CytologyLung CytologyAdvantage Disadvantage

Sputum Non-invasive, non-expensive

Degenerated cells, lowsensitivity, difficulty inlocalizing the lesion

Brushing Direct sampling, wellpreserved cells

For lesions visible in bronchoscopy

Washing Sampling wide area, well Contamination with blood, Washing p g ,preserved cells

,inflammation, debris

Bronchioloalveolerlavage

Alveolar sampling

TransthoracicFNA

Direct sampling, wellpreserved cells, on site evaluation, ancillary tests

Radiology guidance, not suitable for centralmasses, pneumothorax

TransbronchialFNA (TBNA) with/without EBUS

+ diagnosis and stagingat once, both forperipheric and centralmasses

Very much dependent on technique,

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Reserve cells

Squamous metaplasia Pnömositler

Reactive changes in alveoler epitheliumInfectionsDrug toxicityInfarction

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AdenoCa

Pneumonia

ReactiveReactive• Few cells

• Morphologic continuumbetween benign and abnormal

• Cohesive groups

• Regular sheets, windows

• Many cells

• Benign-anormal arasında kontrast

• Loss of cohesion– Many single cells

• Overlapping, 3-dimention

CaCarcinomarcinoma

Regular sheets, windowsbetween cells

• Knobby contours in groups

• No membrane irregularity

• Delicate granuler chromatin– Karyopiknosis, karyoreksis,

karyolisis

• Cilia/ terminal bar

pp g,

• Smooth contoured groups

• Irregular nuclear membrane

• Course chromatin

ToTo avoidavoid falsefalse (+) (+) diagnosisdiagnosis ….….

• Compare the morphology of atypical cellswith native cells

• Do not trust poor preparation

N l f ll• Never rely on few cells

Transbronchial aspiration

Pulmonary infarction

DeMay, 1996

Transthoracic aspiration- Contaminations

Mesothelial cells

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Transbronchial aspiration-contaminations

CELLNETPATHOLOGY

For correct diagnosis• High quality preparation

University of Istanbul

ClassificationClassification of of LungLung TumorsTumors WHOWHO--20152015EpithelialEpithelial tumorstumors

• Adenocarcinoma– Lepidic– Acinar– Papillary– Micropapillary– Solid– Variants

• Invasive mucinous

• Neuroendocrine tumors– Small cell– Large cell neuroendocrine– Carcinoid tumors– Preinvasive lesion

• Large cell carcinomaas e uc ous

• Colloid• Fetal• Enteric

– Minimally invasive– Preinvasive lesions

• Atypical adenomatous hyperplasia• Adenocarcinoma in situ

• Squamous cell carcinoma– Keratinizing– Non-keratinizing– Basaloid– Preinvasive lesion

• Adenosquamous carcinoma• Pleomorphic carcinoma,

spindle and giant cellcarcinoma

• Carcinosarcoma• Pulmonary blastoma• Lymphoepithelioma like• NUT carcinoma• Salivary gland type

ClassificationClassification of of LungLung TumorsTumors WHOWHO--20152015EpithelialEpithelial tumorstumors

• Adenocarcinoma– Lepidic– Acinar– Papillary– Micropapillary– Solid– Variants

• Invasive mucinous

• Neuroendocrine tumors– Small cell– Large cell neuroendocrine– Carcinoid tumors– Preinvasive lesion

• Large cell carcinomaas e uc ous

• Colloid• Fetal• Enteric

– Minimally invasive– Preinvasive lesions

• Atypical adenomatous hyperplasia• Adenocarcinoma in situ

• Squamous cell carcinoma– Keratinizing– Non-keratinizing– Basaloid– Preinvasive lesion

• Adenosquamous carcinoma• Pleomorphic carcinoma,

spindle and giant cellcarcinoma

• Carcinosarcoma• Pulmonary blastoma• Lymphoepithelioma like• NUT carcinoma• Salivary gland type

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AdenocarcinomaAdenocarcinoma

• Glanduler differentiation– Glands, papillae, mucin

• Sheets / 3-D groups

• Nucleocytoplasmic polarity (columnar)Nucleocytoplasmic polarity (columnar)

• Fine vesicular chromatin

• Nucleoli

• Intracytoplasmic luminas

• Palisating nuclei at the periphery of the groups

Adenocarcinoma

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Tbc

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Squamous cell carcinoma

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Keratinizing SqCC Nonkeratinizing SqCC

Cohesion Single cells Groups

Cells Pleomorphic, bizaar Uniform

Cytoplasm (PAP) Orangeophilic, pink Blue-green

SquamousSquamous cellcell carcinomacarcinoma

Keratinization Keratin pearls, goast c. Dyskeratosis ±

N/C

Chromatin Pyknotic Paler

Nucleoli Insconspicious Conspicious

TTF-1

SquamousSquamous• Clusters, streaming,

whorling• Cell in cell pattern• Central, oval/

elongated nuclei

• Sheets, papillarystructures

• Acini• Exantric, round/oval

nuclei

AdenoidAdenoid

elongated nuclei• Course dense

chromatin• Small nucleoli• Dense cytoplasm• Distinct cytoplasmic

borders• Keratinized cells

• Granuler chromatin

• Large nucleoli• Pale/lacy cytoplasm• Indistinct cytoplasmic

borders• Mucin secretion

Morphologic criteria p<0.05

Squamous cellcarcinoma– Keratinized lameller

cytoplasm– Dense opaque

chromatin

Adenocarcinoma

– Monolayered sheets– Nucleocytoplasmic

polarity

73 cases

chromatin– Pseudosinsitial groups– Elongated nuclei– Pleomorphic-poligonal

cells– Streaming pattern– Cell in cell– Keratinized single cells

– Gland like structures– Papillae like structures– Intranuclear inclusions

Dr.Dilek EceDr.D.Yılmazbayhan

2010

AdenocarcinomaSquamous carcinoma

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Small Small cellcell carcinomacarcinoma

• Size (<3 lymphocyte)

• Neurosecretory granules

• Absence of nucleoli

• Hyperchromatic but powdery chromatin

• Scanty cytoplasm

• Paranuclear blue bodies, crush artifact, molding, necrosis

Small cell carcinoma

Small cell carcinoma

Sputum

Small cell carcinoma

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SCLC SCLC

Reserve cells

SCLC

Smallcell

Lymphoma

PNET

SCLC

Basaloid Ca

Basaloid Ca Basaloid Ca

Carcinoid tumorSmall cell Ca

Carcinoid tumor

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Atypical carcinoid is a resection diagnosis

Carcinoid tumor Leiomyosarcoma

AdenocarcinomaCarcinoid tumor

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• 26 carcinoid cases, 1100 interpretations• Frequently misclassified 19 cases (>20% of reponses)

were reviewed• Patterns:

– Poorly preserved pale staining cells with pale chromatinand suggestion of molding

– Numerous large spindle shaped cells– Numerous cells varying markedly in size and shape, some

are smudgy and degenerated– Hypocelluler specimen– Blood obscuring cells– Tumor cells predominantly in groups, few isolated cells

Ki-67

Small cell Ca Carcinoid Tm

Neuroendocrine features in cytologyNeuroendocrine features in cytology

• Salt&pepper (fine&coarse granular) chromatin

• Single cells, loose groups

L f t l k d l i• Loss of cytoplasm, naked nuclei

• If present cytoplasm is delicate and scanty

• Nuclear molding

• Nucleoli variable

• Cells attached to capillariesNicholson et al, Cancer Cytopathology 2000

Large cell neuroendocrine carcinomaLCNEC

Large cell neuroendocrine carcinomaLCNEC

• High grade neuroendorine neoplasm

• Morphologic and clinical features are closet ll ll i th thto small cell carcinoma rather than non-small cell

Large cell neuroendocrine carcinomaLCNEC

Large cell neuroendocrine carcinomaLCNEC

Large cell Small cell

Necrosis +++ +

Groups +++ ++

Rosettes ++ +

Molding ++ +++

Nuclear size >3L <3L

Salt&pepper chro. +++ +++

Nucleoli + -

Cytoplasm ++ -

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Large cell neuroendocrine carcinoma

OriginalOriginal cytologicalcytological diagnosisdiagnosisgivengiven toto LCNECLCNEC

• Non-small cell carcinoma

• Poorly differentiated carcinoma

• Undifferentiated carcinoma

• High grade neuroendocrine carcinomaHigh grade neuroendocrine carcinoma

• Small cell carcinoma

• Large cell neuroendocrine carcinoma

LCNEC

Basaloid Ca

Basaloid Ca Basaloid Ca

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LCNEC

Adenocarcinoma

LCNEC

Adenocarcinoma

DifferentialDifferential DxDx of LCNECof LCNEC

• LCNEC vs NSCLC:– immunhistochemical reactivity with

neuroendocrine markers accompanied bysome cytologic features suggestive of y g ggneuroendocrine morphology.

• LCNEC vs SCLC:– tight cellular clusters, nuclear size, identifiable

cytoplasm and presence of nucleoli

C/ACLCNEC

SCC

LCNEC

Cancer(Cancer Cytopathol) 2008; 114: 180-6

University of Istanbulti

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Tuberculosis AspergillosisTuberculosis Aspergillosis

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Pulmonaryhamartoma

Transthoracic aspirationsNon-diagnostic

University of Istanbul

EntitiesEntities can not be can not be diagnoseddiagnosed//subtypedsubtypedbyby smallsmall samplessamples

• In situ or minimally invasiveadenocarcinomas

• Large cell carcinomas

C i ith th• Carcinomas with more than onecomponent– Adenosquamous carcinoma

– Pleomorphic carcinoma

–

–

BX: Squamous Ca

Resection: Pleomorphic carcinoma

PSK

NSCLC NSCLC withwith spindlespindle andand giantgiant cellscells, , couldcould representrepresent pleomorphicpleomorphiccarcinomacarcinoma ((mentionmention ifif sqccsqcc oror adenoadeno componentcomponent ifif presentpresent) )

PSK

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University of Istanbul

• Subtyping is needed !

• But saving the tissue is also important !

ApproachApproach toto smallsmall biopsiesbiopsies andandcytologiccytologic samplessamples

IASLC/ATS/ERS - 2011WHO - 2015

Small cell Non-small cell

Adenoid diff

Squamous diffIHCMorpho.

IHCMorpho.

• The value of cytology and small biopsies for subtypingof NSCLCs is equivalent (directly diagnosing or favoring aof NSCLCs is equivalent (directly diagnosing or favoring a subtype)

• Concordance between cytology and histology is 93%

• If used together, at least one gives a clear-cutdiagnosis in 84% of the cases, if favoring diagnosis is also added, untyped NSCLCs reduces to 4%

101 simultaneous cytology&biopsy cases, examined seperately (blinded)

• Is it small cell? – YES Immunohistochemistry, sign out

WHO2015

ApproachApproach toto smallsmall biopsiesbiopsies andandcytologiccytologic samplessamples

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Small cell carcinoma

CD56 PSK

TTF-1LCA

• Is it small cell? – NO

• Squamous or adenocarcinoma?YES Si t

ApproachApproach toto smallsmall biopsiesbiopsies andandcytologiccytologic samplessamples

WHO2015

• YES Sign out

• I AM NOT SURE IHC

AdenoCa

Squamous Ca

AdenoCaSquamous Ca

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Kappavalues

Intercellular bridges !Single cell keratinization !Intracytoplasmic vacuoles!

Giant cells

TTF-1

p63

6.5% misdiagnosed4.2% adenocarcinoma

Immun markers for subtyping

SquamousP40

P63

CK 5/6

AdenocarcinomaTTF-1

Napsin A

CK 7

Desmocollin

.

.

.

Mucin stains

.

.

.

Immune markers for subtyping

SquamousP40

P63

CK 5/6

AdenocarcinomaTTF-1

Napsin A

SK 7

Desmocollin

.

.

.

Mucin stains

.

.

.

nuclear

cytoplasmic

• 1103 cases, TMA

Untyped NSCLC 10%

>1%

• Untyped NSCLC 10%

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Original table is modified>10%

TTF-1

Napsin A

Careful !Which cells ?

Thyroid papillary ca

Lung adenoCa

Napsin A

TG

TTF-1

2 different clones

• SPT24 Sensitive but not specific enough

• 8G7G3/1 Specific but less sensitive

Immunohistochemical Subtyping in Large Cell Lung Carcinomas

(A retrospective study with cases diagnosed between 2000‐2013)

Canan ŞAKAR , Pinar Firat

91 resection specimen 47 LCC, 25 solid/solid predominant Adeno, 20 poorly dif.

squamous

p40 SK5/6 TTF-1 Napsin-A

Sensitivity 84% 79% 60% 56%

Specificity %100 %100 %100 %100

cut-off 30% cut-off 1%

Unpublished data

p40

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p63 p40

?

Fatima N, CancerCytopathol 2011;

119: 127-33

Righi L, CancerCytopathol 2011;

117: 3416-23

TTF-1 / NapsinA

p63 / NapsinA TTF-1 / desmocollin3

How to use IHC in small samples ?

One squamous, one adenoid marker

(p40, TTF-1)– p40(+), TTF-1(-) : NSCLC, favor squamous Ca

p40(-) TTF-1(+) : NSCLC favor adeno Ca

Focal weak p40/p63 positivityNSCLC-NOS !

– p40(-), TTF-1(+) : NSCLC, favor adeno Ca

– p40(+), TTF-1(+), different cells : NSCLC, mightbe adenosquamous Ca

– p40(+), TTF-1(+), same cells: NSCLC, favoradeno Ca

– p40(-), TTF-1(-) : NSCLC, NOS

Cytokeratin / S100, CD45, CD31CDX-2, ER, clinical data

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p63TTF-

1

p63 SK5/6 TTF-1

TTF-1Do not pay attention to the

squamoid apperance in cell blocks

ApplyingApplying IHC IHC toto smallsmall samplessamples ??

• If any doubt in defining squamous or adenoiddifferentiation, then IHC is needed

• Threshold for squamous differentiationshould be high (dense eosnophilic cytoplasm, distinct intercellular borders ?? Not enough! gpseudosquamous adenoCa?)

• Use limited IHC to save the tissue formolecular tests , 1 squamous 1 adenoidmarker

• TTF-1 specific• P40 is more specific than p63

How How toto prepareprepare cellcell blocksblocks

• Clots and tissue fragments directly into formalin

• Cell suspensions– Saline, PBS, formalin, 50% ethanol, commercial fixative

solutions ….

• Centrifuge- tissue process for the cell pellet-– Commercial systems

– Ethanol, ethanol/formaline

– Plasma & thrombin/thromboplastin

• Original methodologies

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• Rinse the needle in PBS/tamponated solution

• Centrifuge

• Add few drops of pooled plasma on the

Diagn Cytopathol 2009; 37: 86-90

Reagent used forprothrombin time test

• Add few drops of pooled plasma on thesediment, mix well, than add few drops of thrombin/ thromboplastin, mix again

• Wait for 5 min

• Remove the resultant clot, wrap, cassette andstore in formalin till tissue processing

QuestionsQuestions toto answeranswer at ROSEat ROSE

• Is the specimen representing the target?– Clinical features/ radiology

• Is the amount and quality of cells sufficient for a satisfactory morphologic evaluation?y p g– What is the preliminary diagnosis ?

• Any ancillary test needed either for diagnosis orfor therapy?– How should I handle the specimen ?

GoodGood materialmaterial ……forfor diagnosisdiagnosis , , forfor therapytherapy

– Shared responsibility: • pulmonologist, • radiologist, • pathologist / cytopathologistpathologist / cytopathologist

– The most suitable sampling technique shouldbe chosen• for the patient• for the ancillary tests