CYTOPATHOLOGIC AND MOLECULAR DIAGNOSTIC ISSUES IN LUNG CYTOPATHOLOGY
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1 COMPANION MEETING Four Ps of Pulmonary Cytopathology: Procedural, Predictive, Personalized and Participatory CYTOPATHOLOGIC AND MOLECULAR DIAGNOSTIC ISSUES IN LUNG CYTOPATHOLOGY Prof. Fernando Schmitt Medical Faculty of Porto University, Porto, Portugal IPATIMUP General-Secretary of the International Academy of Cytology
2 LUNG CYTOLOGY 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.
3 ACCURACY OF LUNG CYTOLOGY FOR TUMOR DIAGNOSIS AND MANAGEMENT Sensitivity of diagnosis of malignancy Predictive value of malignant diagnosis Tumor typing Select prognostic markers for targeted therapy
4 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
5 LUNG CYTOLOGY Sensitivity SAMPLING LOCATION OF THE LESION CENTRAL PERIPHERAL SPUTUM 70-85% 30-60% BRONCHIAL WASHING BRONCHIAL BRUSHING 70-90% 61-76% 77-90% 50-70% BAL 80-90% 70-80% FNA 80-95% 80-95% Gray and Kocjan, 2010; Bibbo M, 2008; Layfield et al, 1996; Rosenthal DL, 1988
6 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.
7 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 nonneoplastic mimics of malignancy. Gray and Kocjan, 2010; Bibbo M, 2008; Silverman JF, 1995; Naryshkin et at, 1993
8 LUNG CYTOLOGY Bronchial cell atypia Reactive Syncytia Ciliocytophthoria Creola bodies: hyperplastic reactive epithelium
9 LUNG CYTOLOGY Bronchial cell atypia Squamous metaplasia Vegetable cells
10 LUNG CYTOLOGY Bronchial cell atypia Basal cell hyperplasia of Bronchial epithelium Hyperplasia of type II pneumocytes
11 LUNG CYTOLOGY and TUMOR TYPING
12 LUNG CYTOLOGY and TUMOR TYPING Histologic Type Squamous cell carcinoma Sputum/bronchoscopy cytological material FNA 85% 80% Adenocarcinoma 79% 96% Large-cell carcinoma Small-cell carcinoma 30% 42% 93% 95% Gray and Kocjan, 2010; Bibbo M, 2008
13 LUNG CYTOLOGY Morphological Aspects SQUAMOUS CELL CARCINOMA Hypercellular Single cells Keratin pearls Sharp cell outlines Dark smudgy chromatin karyopyknosis
14 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
15 LUNG CYTOLOGY Morphological Aspects ADENOCARCINOMA Hypercellular Syncytial groups Scant or vacuolated cytoplasm Nuclei vesicular Pale chromatin Prominent nucleoli
16 ADENOCARCINOMA (formerly BAC) Ball-like cell clusters Papillary fronds Nuclei are round to oval with finely granular chromatin Nucleoli present but inconspicuous Mucinous and nonmucinous LUNG CYTOLOGY Morphological Aspects
17 LUNG CYTOLOGY Morphological Aspects SMALL CELL CARCINOMA Small cells in loose clusters with single cells Molding No or small nucleoli Salt and pepper chromatin Mitosis Necrosis
18 LARGE CELL CARCINOMA Hypercellular Large groups Large cells Macronucleoli, sometimes multiple Cytoplasm outline frequently ill-defined LUNG CYTOLOGY Morphological Aspects
19 LUNG CYTOLOGY Morphological Aspects CARCINOID Single cells Round cells Salt and pepper chromatin No necrosis, molding or mitosis Spindle-cell type
20 LUNG CYTOLOGY Adenoid-cystic carcinoma
21 LUNG CYTOLOGY Metastatic tumours Colon carcinoma Breast carcinoma Renal cell carcinoma Metastatic melanoma
22 LUNG CYTOLOGY Morphological Aspects Acute irradiation effect
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24 LUNG CYTOLOGY Ancillary Studies Characterize primary tumours. Distinguish primary vs metastasis Therapeutic targets.
25 LUNG CYTOLOGY Ancillary Studies Special stainings. Immunocytochemistry. Flow cytometry Molecular techniques.
26 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
27 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.
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30 The histologic concept anti-plap (PL8F6) in a seminoma MGG None Acetone 96 % ethanol 10 % NBF 10 % NBF + HIER/Ci
31 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.
32 100 articles Last 15 years 9 most used antibodies Diagn. Cytopathol. 2011;39:
33 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.
34 PD
35 IS TTF-1 A GOOD IMMUNOHISTOCHEMICAL MARKER TO DISTINGUISH PRIMARY FROM METASTATIC LUNG ADENOCARCINOMAS? 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 Pathol Res Pract 196: 835, 2000
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37 Yes P63 Positive Yes SqCC-small cell type LUNG Carcinoma Small cell morphology No Chromogranin/ Synaptophysin/ CD56- Positive Yes SCLC No SqCC P63/CK5 TTF1/Napsin A - Or -/+ +++ ADC EGFR Positive Yes Tyrosine kinase inhibitors Crizotinib ALK Positive No Cancer Cytopathology 2011
38 Cell-blocks Direct smears Cytospin Heat-induced retrieval Automated system 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-
39 Squamous cell carcinoma Adenocarcinoma
40 Bevacizumab combined with chemotherapy prolongs survival for some patients with advanced NSCL cancer.
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42 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. Diagnostic Cytopathology 37: , 2009
43 n + - nec/lost
44 Archival alcohol-fixed smears Heat-induced retrieval Manual technique
45 The application and diagnostic utility of immunocytochemistry on direct smears in the subclassification of NSCL carcinoma Diagnosis Napsin A TTF-1 P63 ADC 9/10 9/10 2/10 SCC 0/5 0/5 5/5 Modern Pathology 24: 103A, 2011
46 Most ADC are TTF-1+, P63-, CK5/6- Most SCC are TTF-1-, P63+, CK5/6+ Diagnostic Cytopathology 37: , 2009
47 Yes P63 Positive Yes SqCC-small cell type LUNG Carcinoma Small cell morphology No Chromogranin/ Synaptophysin/ CD56- Positive Yes SCLC No SqCC P63/CK5 TTF1/Napsin A - Or -/+ +++ ADC EGFR Positive Yes Tyrosine kinase inhibitors Crizotinib ALK Positive No Cancer Cytopathology 2011
48 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
49 7p12 EGFR (red) 8q24 MYC (gold) 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. Ch 6 (blue) 8q24 MYC (gold)
50 Multitarget FISH containing locus specific probes to chromosomes 6 and to the 5p15, 8q24 (c-myc gene) and 7p12 (EGFR gene) Polysomy Tetrasomy
51 Polysomy Tetrasomy
52 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.
53 LUNG CYTOLOGY Primary vs. Metastasis 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.
54 IS TTF-1 A GOOD IMMUNOHISTOCHEMICAL MARKER TO DISTINGUISH PRIMARY FROM METASTATIC LUNG ADENOCARCINOMAS? 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 Pathol Res Pract 196: 835, 2000
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56 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
57 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
58 CK7 CK20 TTF-1 Lung-adenocarcinoma
59 FNA and IMMUNOCYTOCHEMISTRY Primary sites of metastasis ADENOCARCINOMA PSA ADENOCARCINOMA ER Schmitt et al. Acta Cytol 33: 899, 1989
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61 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.
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63 DIAGNOSIS PROGNOSIS THERAPEUTIC TARGETS
64 DRIVER MUTATIONS IN NSCL CANCER
65 Biologic Agents and Indications in the Treatment of NSCLC
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67 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.
68 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.
69 EGFR: How to detect? Over-expression Amplification Mutation
70 Response rates to Gefitinib and Erlotinib based on Presence/absence of a Molecular Marker EGFR, exon 21 p.leu858arg
71 EGFR mutations in NSCL cancer The most common EGFR mutations in lung cancer are small inframe 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.
72 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.
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75 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.
76 CYTOLOGY HISTOLOGY
77 EGFR mutations in NSCL cancer Minimal number of cells 100 cells: ideal 50 cells: possible 30 cells: still possible Enrichment by microdissection if necessary. Savic S et al.br J Cancer 98: , 2008
78 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..
79 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. Whole genome amplification has allowed for high-fidelity in vitro reproduction of quality template DNA. 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.
80 Yes P63 Positive Yes SqCC-small cell type LUNG Carcinoma Small cell morphology No Chromogranin/ Synaptophysin/ CD56- Positive Yes SCLC No SqCC P63/CK5 TTF1/Napsin A - Or -/+ +++ ADC EGFR Positive Yes Tyrosine kinase inhibitors Crizotinib ALK Positive No Cancer Cytopathology 2011
81 P63, CK5, TTF1 Squamous Cell carcinoma Nonsquamous cell carcinoma Reflex testing 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)
82 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 Perner et al., Neoplasia 2008;10:298 Shaw et al., J Clin Oncol 2009;27:4247
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85 EGFR and ALK on Lung Cancer Our data EGFR Analysis 2011 ALK Analysis 2011 Nº. of cases: EGFR mut: 27 (9%) ALK + 1 (5,88%)
86 Workflow for Lung Carcinoma Hours Tissue-cell collection/fixation Cytology Pathologist s review and report 1-2 Working days (except IHC) Decision for EGFR mutation analysis, tumor content reviewed Pathology microdissection, if needed 5-7 Working days EGFR mutation testing Molecular testing 3-4 Working days If Negative FISH ALK Final report with morphology and mutation results Final Report
87 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.
88 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.
89 (CYTO)PATHOLOGY IS CHANGING. 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.
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