cytopathologic and molecular...

COMPANION MEETING Four “Ps” of Pulmonary Cytopathology: Procedural, Predictive, Personalized and Participatory

Prof. Fernando Schmitt Medical Faculty of Porto University, Porto, Portugal

IPATIMUP General-Secretary of the International Academy of Cytology

CYTOPATHOLOGIC AND MOLECULAR DIAGNOSTIC ISSUES

IN LUNG CYTOPATHOLOGY

• The importance of cytological techniques for investigation of respiratory conditions has been recognized since the earliest days of clinical cytology.

• The last few decades have seen ample demonstration of the sensitivity and predictive value of cytodiagnosis of lung tumors and an acceptance of all cytological modalities as a basis for management.

LUNG CYTOLOGY

• Sensitivity of diagnosis of malignancy

• Predictive value of malignant diagnosis

• Tumor typing

• Select prognostic markers for targeted therapy

ACCURACY OF LUNG CYTOLOGY FOR TUMOR DIAGNOSIS AND MANAGEMENT

Methods for Cytologic Sampling of the Lung

• Sputum Spontaneously expectorated Induced

• Bronchoscopic procedures Bronchial brushing Bronchial washing BAL

• Pulmonary artery catheter sampling • Fine needle aspiration, including EBUS/EUS-FNA

SAMPLING LOCATION OF THE LESION

CENTRAL PERIPHERAL

SPUTUM 70-85% 30-60%

BRONCHIAL WASHING

70-90% 61-76%

BRONCHIAL BRUSHING

77-90% 50-70%

BAL 80-90% 70-80%

FNA 80-95% 80-95%

LUNG CYTOLOGY Sensitivity

Gray and Kocjan, 2010; Bibbo M, 2008; Layfield et al, 1996; Rosenthal DL, 1988

SENSITIVITY OF LUNG CYTOLOGY

• Multiple sampling often increases the yield.

• A negative result by any single diagnostic method cannot exclude malignancy.

• Size and depth of the lesion matters, as well as the experience of the radiologist and the pathologist

• Using a selection or combination of the different cytological diagnostic method up to 98% of central lesions and 94% of peripheral lung tumors may be diagnosed pretherapeutically.

PREDICTIVE VALUE OF MALIGNANT DIAGNOSIS

• A 100% predictive value of malignant diagnosis is the aim because cytological diagnosis is to be used for definitive management decisions.

• The positive predictive value of malignancy has been near this level for experienced cytopathologists for many years.

• But there are rare false positive cases due a non-neoplastic mimics of malignancy.

Gray and Kocjan, 2010; Bibbo M, 2008; Silverman JF, 1995; Naryshkin et at, 1993

LUNG CYTOLOGY Bronchial cell atypia

Reactive Syncytia

Ciliocytophthoria Creola bodies: hyperplastic reactive epithelium


Squamous metaplasia Vegetable cells


Basal cell hyperplasia of Bronchial epithelium Hyperplasia of type II pneumocytes

LUNG CYTOLOGY and TUMOR TYPING

Histologic Type Sputum/bronchoscopy cytological material

FNA

Squamous cell carcinoma

85%

80%

Adenocarcinoma 79% 96%

Large-cell carcinoma

30% 42%

Small-cell carcinoma

93% 95%

LUNG CYTOLOGY and TUMOR TYPING

Gray and Kocjan, 2010; Bibbo M, 2008

LUNG CYTOLOGY Morphological Aspects

• SQUAMOUS CELL CARCINOMA

– Hypercellular

– Single cells

– Keratin pearls

– Sharp cell outlines

– Dark smudgy chromatin

– karyopyknosis

LUNG CYTOLOGY Squamous cell carcinoma

• Keratinizing type

– Isolated single cells

– Spindle, caudate shapes

– Eosinophilic or

orangeophilic cytoplasm

– Necrotic background

• Non-keratinizing

– Irregular, solid sheets

– Spindle, oval nuclei

– Variable chromatin distribution

– Nucleoli may be prominent


• ADENOCARCINOMA

– Hypercellular

– Syncytial groups

– Scant or vacuolated cytoplasm

– Nuclei vesicular

– Pale chromatin

– Prominent nucleoli


• ADENOCARCINOMA (formerly BAC)

– Ball-like cell clusters

– Papillary fronds

– Nuclei are round to oval with finely granular chromatin

– Nucleoli present but inconspicuous

– Mucinous and non-mucinous

• SMALL CELL CARCINOMA

– Small cells in loose clusters with single cells

– Molding

– No or small nucleoli

– Salt and pepper chromatin

– Mitosis

– Necrosis


• LARGE CELL CARCINOMA

– Hypercellular

– Large groups

– Large cells

– Macronucleoli, sometimes multiple

– Cytoplasm outline frequently ill-defined


• CARCINOID

– Single cells

– Round cells

– Salt and pepper chromatin

– No necrosis, molding or mitosis

– Spindle-cell type


LUNG CYTOLOGY Adenoid-cystic carcinoma

LUNG CYTOLOGY Metastatic tumours

Colon carcinoma Breast carcinoma

Renal cell carcinoma Metastatic melanoma


Acute irradiation effect

LUNG CYTOLOGY Ancillary Studies

• Characterize primary tumours.

• Distinguish primary vs metastasis

• Therapeutic targets.

• Special stainings.

• Immunocytochemistry.

• Flow cytometry

• Molecular techniques.

LUNG CYTOLOGY Ancillary Studies

“The development of a huge amount of new ancillary techniques has paralleled the emergence of clinical cytology as a major diagnostic speciality. Clinical applications of these techniques have been growing in the last decade”. Acta Cytol 51, 2007

Ancillary Studies in Cytology Challenges

•To select the correct test for a limited sample quantity.

•Avoid to jump from a histological adapted technique directly to cytological material.

•Use appropriate controls for cytological material.

The histologic concept – anti-PLAP (PL8F6) in a seminoma

MGG None Acetone

96 % ethanol 10 % NBF 10 % NBF + HIER/Ci

• This survey showed wide variation in the use of ICC among different laboratories across the Europe.

• The variability in material and fixatives is a major factor preventing standardization of some procedures using the technique.

• Currently, ICC methods have been refined and high quality reagents and automation are more widely available, so it is expected that technical problems will be reduced in the near future.

• 100 articles

• Last 15 years

• 9 most used antibodies

Diagn. Cytopathol. 2011;39:245–250.

LUNG CYTOLOGY Primary tumours

• Accurate distinction between small-cell and non-small-cell carcinomas of the lung has crucial therapeutic significance. • In recent years, cytology have been increasingly used for establishing the diagnosis of lung cancer and classifying the specific tumour type.

• However, morphologic distinction between some small-cell lung carcinomas and nonkeratinizing poorly differentiated pulmonary squamous cell carcinomas can be difficult.

• Some markers can help the cytopathologist in this task.

IS TTF-1 A GOOD IMMUNOHISTOCHEMICAL MARKER TO DISTINGUISH PRIMARY FROM METASTATIC LUNG ADENOCARCINOMAS ?

Pathol Res Pract 196: 835, 2000

• A transcription factor that plays an important role in the lung specific expression of surfactant proteins. • TTF1 is expressed in up to 75% of pulmonary adenocarcinomas and is negative in metastatic adenocarcinomas (exception for thyroid carcinomas). • Positive in small cell carcinomas • Rarely positive in squamous cell carcinoma • Mucinous ADC is usually negative

LUNG Carcinoma

Small cell morphology

Yes P63

Positive

No

No

SqCC-small cell type

Chromogranin/ Synaptophysin/

CD56- Positive

Yes SCLC

P63/CK5

TTF1/Napsin A ADC

+++ -

SqCC - Or -/+

+++

EGFR Positive

Yes

Tyrosine kinase

inhibitors

Yes

No ALK

Positive Crizotinib

Cancer Cytopathology 2011

• Most small cell carcinomas are TTF-1+, P63-, CK5/6- and CRG+

• Most poorly differentiated squamous cell carcinomas are TTF-1-, P63+, CK5/6+ and CRG-

Cell-blocks Direct smears Cytospin Heat-induced retrieval Automated system

Squamous cell carcinoma Adenocarcinoma

Bevacizumab combined with chemotherapy prolongs survival for some patients with advanced NSCL cancer.

Diagnostic Cytopathology 37: 178-183, 2009

• Since patients with SCC cannot receive Bevacizumab because of a 30% mortality rate due to fatal hemoptysis, cytopathologists have been asked to specifically identify squamous differentiation in cases of NSCL, so that patients can receive appropriate treatment.

n + - nec/lost

Archival alcohol-fixed smears Heat-induced retrieval Manual technique

Modern Pathology 24: 103A, 2011

The application and diagnostic utility of immunocytochemistry on direct smears in the sub-

classification of NSCL carcinoma

Diagnosis Napsin A TTF-1 P63

ADC 9/10 9/10 2/10

SCC 0/5 0/5 5/5

• Most ADC are TTF-1+, P63-, CK5/6-

• Most SCC are TTF-1-, P63+, CK5/6+

Diagnostic Cytopathology 37: 178-183, 2009

LUNG Carcinoma

Small cell morphology

Yes P63

Positive

No

No

SqCC-small cell type

Chromogranin/ Synaptophysin/

CD56- Positive

Yes SCLC

P63/CK5

TTF1/Napsin A ADC

+++ -

SqCC - Or -/+

+++

EGFR Positive

Yes

Tyrosine kinase

inhibitors

Yes

No ALK

Positive Crizotinib


• Molecular cytopathology (MCP) can be defined as molecular studies applied on all types of cytological specimens, namely gynecology cytology, exfoliative non-gyn cytology and fine needle aspirates.

• MCP has been applied to detect specific organisms or oncologic changes at molecular levels

Equivocal respiratory cytology is a common diagnostic dilemma to both cytopathologists and clinicians. Since chromosomal alterations are a hallmark of cancer but are rare or absent in benign conditions, the recognition of them in equivocal cytological specimens might distinguish reactive from malignant cells.

7p12 – EGFR (red) 8q24 – MYC (gold)

Ch 6 – (blue) 8q24 – MYC (gold)

Multitarget FISH containing locus specific probes to chromosomes 6 and to the 5p15, 8q24 (c-myc gene) and

7p12 (EGFR gene)

Polysomy Tetrasomy

Polysomy Tetrasomy

• FISH is significantly more sensitive than conventional cytology for detecting lung cancer in bronchoscopically obtained bronchial brushing specimens. • FISH detected tumours at earlier stages and detected a higher proportion of peripheral tumours. • Additional studies are needed to determine if the higher sensitivity of FISH has the potential to improve patient survival.

• Lung metastases are frequent; and, in most instances, a previous diagnosis of malignancy already is available. • However, pulmonary metastases can be the first sign of disease and may require careful investigation to avoid an inappropriate diagnosis of primary lung carcinoma. • Adenocarcinoma, squamous cell carcinoma and small cell carcinoma are histological types that can be difficult to distinguish between primary or secondary tumours.

LUNG CYTOLOGY Primary vs. Metastasis

IS TTF-1 A GOOD IMMUNOHISTOCHEMICAL MARKER TO DISTINGUISH PRIMARY FROM METASTATIC LUNG ADENOCARCINOMAS ?

Pathol Res Pract 196: 835, 2000

• A transcription factor that plays an important role in the lung specific expression of surfactant proteins. • TTF1 is expressed in up to 75% of pulmonary adenocarcinomas and is negative in metastatic adenocarcinomas (exception for thyroid carcinomas). • Positive in small cell carcinomas • Rarely positive in squamous cell carcinoma • Mucinous ADC is usually negative

Metastasis of adenocarcinoma

• ER, PGR, AR

• CDX-2, TTF-1

• CK subtyping (CK 7, CK 20)

• CA125, PSA, PSAP

• CEA, villin

• Thyroglobulin, calcitonin

• Uroplakin

• RCC, CD10

CK subtyping

• CK7+ CK20+ urothelial, pancreas, ovarium (mucinous)

• CK7+ CK20- lung, breast, mesothel ovarium, endometrial

• CK7- CK20+ colon

• CK7- CK20- prostate, hepatoma, renal, squamous ca

TTF-1

CK7

CK20

Lung-adenocarcinoma

ADENOCARCINOMA PSA

FNA and IMMUNOCYTOCHEMISTRY Primary sites of metastasis

Schmitt et al. Acta Cytol 33: 899, 1989

ADENOCARCINOMA ER

• To date, there is no reliable immunohistochemical marker that discriminates between primary pulmonary squamous cell carcinoma (SCC) and cervical SCC metastatic to the lung. • Overexpression of p16 has been consistently observed in HPV-related cervical cancer. In lung cancer is common in small cell carcinoma, but not in SCC.

DIAGNOSIS PROGNOSIS

THERAPEUTIC TARGETS

DRIVER MUTATIONS IN NSCL CANCER

Biologic Agents and Indications in the Treatment of NSCLC

PATHOLOGY CONSIDERATIONS FOR GOOD PRACTICE

• Strategic use of small biopsy and cytology samples is important, i.e., use the minimum specimen necessary for an accurate diagnosis, to preserve as much tissue/cells as possible for potential molecular studies.

• To guide therapy for patients with advanced lung adenocarcinoma each institution should develop a multidisciplinary team that coordinates the optimal approach to obtaining and processing biopsy/cytology specimens to provide expeditious diagnostic and molecular results.

Molecular implications in Lung Cancer

• EGFR mutation is a validated predictive marker on histological and cytological samples for response with EGFR-Tkis in first line therapy in advanced lung adenocarcinomas.

• Tumors with an EGFR mutation have been associated with a more indolent course.

• EGFR and KRAS mutation are virtually mutually exclusive.

• EGFR/KRAS mutation-negative cases may have detectable fusion of EML4-ALK.

EGFR: How to detect ?

Over-expression Amplification

Mutation

Response rates to Gefitinib and Erlotinib based on Presence/absence of a Molecular Marker

EGFR, exon 21

p.Leu858Arg

EGFR mutations in NSCL cancer • The most common EGFR mutations in lung cancer are small in-frame deletions in exon 19 and a point mutation (L858R) in exon 21. • These mutations likely cause constitutive activation of the kinase and confer dramatic sensitivity to TKis gefinitib and erlotinib.

• Unfortunately, the effect of TKis is limited in time because of the emergence of drug resistance. A second mutation, a substitution T790 M in exon 20 appears in half of all patients with acquired resistance to TKis.

• Screening of EGFR mutations, both for selecting patients for treatment and for detecting the resistance mutation is extremely important.

EGFR status in cytological samples

• In NSCLC, the clinical application of TKIs targeting EGFR requires investigation of biomarkers of patient response. • Currently, the molecular detection of EGFR mutation is the most accurate method for selecting this patients.

• Because minimally invasive diagnostic procedures are most often used in a diagnostic workup of lung cancer, cytological samples are often the material available for analysis.

EGFR status in cytological samples Technical issues

• Needs to develop and validate criteria for the standardized interpretation of EGFR FISH results using cytological specimens • Minimal number of tumor cells for successful EGFR DNA sequence analysis.

CYTOLOGY HISTOLOGY

EGFR mutations in NSCL cancer Minimal number of cells

Enrichment by microdissection if necessary.

• 100 cells: ideal • 50 cells: possible • 30 cells: still possible

Savic S et al.Br J Cancer 98: 154-160, 2008

EGFR mutations in NSCL cancer Challenges

• At least one third of NSCL cancer primary diagnoses are made solely on basis of cytological specimen, where the number of tumour cells is very limited. • The low abundance of cells precludes subsequent molecular analysis. In the TRIBUTE trial only 39% of the samples contained enough tumour cells to DNA sequencing. This dropout rate needs to be reduced, especially in the context of developing personalized medicine.

• Recently, it was developed new methods to detect EGFR mutations in samples with a limited number of tumour cells and in cytologic specimens..

• This method is based on microdissection of tumor cells directly into PCR buffer, followed by amplification and determination of EGFR status by length analysis of fluorescently-labelled products or TaqMan assay. • Because there is no need for prior DNA purification the method is highly sensitive and allow detect EGFR mutations in samples containing as few as eight tumor cells.

•Whole genome amplification has allowed for high-fidelity in vitro reproduction of quality template DNA.

LUNG

Carcinoma Small cell

morphology

Yes P63

Positive

No

No

SqCC-small

cell type

Chromogranin/

Synaptophysin/

CD56-

Positive

Yes SCLC

P63/CK5

TTF1/Napsin A ADC

+++

- SqCC

- Or -/+

+++

EGFR

Positive

Yes

Tyrosine

kinase

inhibitors

Yes

No ALK

Positive Crizotinib


Reflex testing

Squamous Cell carcinoma Nonsquamous cell carcinoma

No reflex testing; only if requested by oncologists

FISH ALK

Mutation testing EGFR (exons 19 and 21; exons 18-21) Optional: KRAS (exon 2/codons 12-13)

P63, CK5, TTF1

ALK break apart FISH probe

• 3% of NSCLC

• Selected population: 13%

• Therapeutic target

• Crizotinib

• Never/light smokers

• Male > Female

• younger age (52 vs 65)

• adenocarcinoma (acinar/mucinous type)

• no EGFR/KRAS mutation (?)

• Resistance to EGFR therapy

Shaw et al., J Clin Oncol 2009;27:4247

Perner et al., Neoplasia 2008;10:298

EGFR Analysis

2011

ALK Analysis

2011

Nº. of cases: 298

17

EGFR mut: 27 (9%)

ALK + 1 (5,88%)

EGFR and ALK on Lung Cancer Our data - 2011

Tissue-cell collection/fixation

Pathologist’s review and report

Decision for EGFR mutation analysis, tumor content reviewed

microdissection, if needed

EGFR mutation testing

Final report with morphology and mutation results

1-2 Working days (except IHC)

12-24 Hours

5-7 Working days

Cytology

Pathology

Molecular testing

Final Report

If Negative FISH ALK 3-4 Working days

Workflow for Lung Carcinoma

Ancillary Studies in Lung Cytology Conclusions

• Results of ancillary tests can only be interpreted in the context of an informed, carefully considered clinical and cytological diagnosis.

• In many cases, a single test are unlikely to provide a specific diagnosis even within a limited differential diagnosis.

• Assessment of EGFR status and ALK translocations has proven to be predictive of therapeutic response and, therefore, essential to the management of patients.

Ancillary Studies in Lung Cytology Conclusions

• Various techniques using different types of cytological samples and preparations have shown promising results, with similar or higher accuracy and sensitivity when compared with surgical specimens.

• At this moment, it is important to encourage the proper collection and handling of cytological samples in new prospective clinical studies so that these novel techniques can be validated in large patient cohorts.

• Cytology claims for pressing remodeled attitudes. Small samples obtained by minimally invasive methods exploited morphological and molecular information are the model biopsies of the futures.

• Molecular and genomic techniques need to become a significant component of the training curriculum of pathology. Without our commitment, it is inevitable that these gaps will be filled by other medical specialties.

• Now is the time to engineer a core expertise in pathology that meets the future’s needs, and this opportunity cannot be missed or we run the risk of being the last generation of pathologists.

(CYTO)PATHOLOGY IS CHANGING….

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