Club de Patología Pulmonar

Transcripción

1 Club de Patología Pulmonar Mª Dolores Lozano Escario Clínica Universidad de Navarra. Pamplona. Mujer de 75 años no fumadora, que ingresa para valoración y tratamiento de una diseminación de un tumor de pulmón diagnosticado y tratado 4 años antes

2 Enero 2007 se diagnosticó de neoplasia pulmonar estadio IV. TAC y PET: - Masa pulmonar en relación con bronquio principal izquierdo - Metástasis hepáticas, suprarrenal izquierda y cerebrales Broncoscopia con PAAF y punción-biopsia de la masa pulmonar.

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6 TTF-1 CK-7

7 L858R Mutation. Exon 21

8 Adenocarcinoma de pulmón estadio IV con mutación L858R en exón 21 del gen EGFR Se inicia tratamiento con Tarceva Respuesta radiológica y estabilización de la enfermedad Revisiones periódicas

9 2011 (4 años más tarde) Progresión: PAAF de hígado y suprarrenal y adenopatías mediastínicas 10L

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13 2011

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16 1. L858R Mutation. Exon T790M Exon 20

17 Recidiva de adenocarcinoma de pulmón con mutaciónes L858R en exón 21 y T790M en exón 20 del gen EGFR. Fusión ALK.

18 Cuestiones/Comentarios: Frecuencia de la coexistencia de algunas alteraciones genéticas con implicaciones terapeúticas: EGFR y ALK/EML4 Analisis mutacional en material citológico Conveniencia de realizar estudio mutacional en las recidivas / metástasis

20 Referencia Edad Género Consumo Tabaco Koivunen et. al Casos publicados con mutaciones de EGFR y fusión ALK Dco. AP Tipo de muestra mutaciones EGFR ALK- EML4 ALK: método de detección??? AC Lobectomía Del 19 + FISH, RT-PCR Kuo et.al Tiseo et.al Zhang et.al Popat et.al Tanaka et.el Boland et.al Caso presentado 72 Mujer Nunca AC Fresco Del 19 + RT-PCR 48 Varón Nunca AC-Sq Lobectomía Del 19 + FISH? Mujer Nunca AC Lobectomía Del 19 + RT-PCR Mujer Nunca AC Lobectomía Del 19 + FISH 39 Varón Exfumador AC Biopsia transbronquial L858R + RT-PCR, FISH 72 Mujer Nunca AC Lobectomía L747-S752 + FISH Mujer Nunca AC AC w. signetring cells Citología Extensiones teñidas y ThinPrep L858R L858R + T790M? + FISH

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23 Cuestiones/Comentarios: Frecuencia de la coexistencia de algunas alteraciones genéticas con implicaciones terapeúticas: EGFR y ALK/EML4 Análisis mutacional en material citológico Conveniencia de realizar estudio mutacional en las recidivas / metástasis

24 En cáncer de pulmón, Demanda de diagnósticos precisos, rápidos y mediante métodos mínimamente invasivos Integración de test moleculares en el manejo clínico del paciente con cáncer Actualmente, el diagnóstico de cáncer de pulmón se realiza con frecuencia mediante muestras pequeñas (PAAF, biopsias transbronquiales, etc ) obtenidas mediante métodos poco invasivos

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28 Usefulness of cytological samples for the assessment of ALK gene rearrangements in NSCLC patients Tania Labiano M.D. 1, José I Echeveste M.D. 1, Luis M Seijo M.D. 2, José L Pérez-Gracia M.D. 3, Alfonso Gúrpide M.D. 3, Miguel A. Idoate M.D. 1, Mercedes Aguirre CLT 1, Isabel Zudaire M.D. 4 and María D. Lozano M.D Pathology Department, 2. Respiratory MedicineDepartment, 3. Clinical Oncology Department, at University Clinic of Navarra 4. Center for Applied Medical Research, University of Navarra. ABSTRACT Background: ALK gene rearrangement defines a new molecular subtype of NSCLC. Crizotinib, a dual MET and ALK inhibitor, shows promising efficacy in these patients. To date, determination of EML4-ALK fusions has been performed in biopsies or surgical specimens. However, advanced lung cancer is often diagnosed in cytology specimens obtained through fine needle aspiration (FNA), and frequently cytological specimens are the only tumor samples available. We evaluated the possibility of determining ALK gene rearrangements in cytological samples from NSCLC patients. Design: Between January and September 2011, 42 cytological samples from 42 NSCLC patients (25 M and 17 F) were analyzed for ALK gene rearrangements by FISH (Vysis dual colour break apart probe). Tumour samples were obtained by bronchoscopy guided FNA (21 cases-50%), EBUS-FNA (2 cases-4.8%), EUS-FNA (7 cases-16.7%), CT- FNA (2 cases-4.8%), and direct FNA (2 cases- 4.8%). We also studied 2 cavity fluids (4.8%), 4 imprints from surgical specimens (9.5%), and 2 cellblocks received for consultation. FISH analysis was performed on Papanicolau stained smears in 15 cases (35.7%), non-stained ThinPrep in 21 cases (50%), cell block in 4 cases (9.5%), and 1 in a stained ThinPrep. All cases were previously tested for EGFR and KRAS mutations. Results: Twenty-nine samples (69%) were adequate for FISH analysis. Two cases were positive (6.9%), both women and non-smokers of 36 and 73 yo having adenocarcinoma with signet ring cells. FISH analysis was done on ThinPrep unstained slides. One case had a concurrent EGFR mutation in exon 21. FISH study was unsuccessful in 13 cases. Ten of them were performed on Papanicolau stained smears (76.9%), and 3 on unstained ThinPrep (14.3%). All paraffin embedded samples, and 19 (86.4%) TrinpPrep were adequate for FISH analysis. Correlation between cytological and surgical samples has been performed so far in 4 cases, with a concordance rate of 100%. Conclusion: Determination of ALK gene rearrangements in cytological specimens is feasible. ThinPrep and cell blocks are the most suitable samples for FISH analysis, while Papanicolau stained smears provide poor results. Coexistence of ALK gene rearrangement and EGFR mutation, although rare, are not mutually exclusive. Analysis by FISH of ALK gene rearrangement on ThinPrep slides could be an option when no paraffin embedded tissue is available. RESULTS Twenty-nine out of 42 samples (69%) were adequate for FISH analysis: o 5 Cell blocks- FFPE (11,25%) o 1 Stained smear (2,25%) o 19 TrinpPrep slides (86.5%) 1/19 ( 5.26%) Stained 18/19 (94.75%) Non stained FISH analysis was unsuccessful in 13 cases ( 31%): o 10 Papanicolau stained smears (76.9%) o 3 non -stained ThinPrep slides (14.3%) (Figure 2 shows cytological sample adequacy) Two cases were positive for ALLK rearrangement (6.9%). Table 1 shows clinicopathological features of these two patients. Correlation between cytological and surgical samples has been performed so far in 4 cases, with a concordance rate of 100%. POSITIVE CASE 1 36 year -old woman. Light smoker (7.5 packs year). Presented with a 9,1 x 11 cm. mass in her left lung associated with a massive malignant effusion. At physical examination a supraclavicular enlarged lymph node was detected. Direct FNA was performed. EGFR and KRAS were wild type. Based on signet ring cell morphology, FISH analysis for ALK were performed. TTF 1 POSITIVE CASE 2 75 year - old woman. Never smoker. Diagnosed in 2007 of advanced stage of lung adenocarcinoma showing a L854R EGFR mutation in exon 21. Erlotinib (Tarceva) was administered achieving complete remission. In 2011 multiples metastases in liver, suprarenal gland and bone were detected. A EUS-guided FNA cytology from liver and left adrenal gland was performed for diagnosis and molecular studies. The tumor maintained the mutation L854R in exon 21. A new acquired mutation of TKI s resistance in exon 20 (T790M) was also detected. Based on signet ring cell morphology, FISH analysis for ALK were performed. TTF 1 BACKGROUND ALK gene rearrangement defines a new molecular subtype of NSCLC. Crizotinib, a dual MET and ALK inhibitor, shows promising efficacy in these patients 2-3. To date, determination of EML4 - ALK gene fusions has been performed in biopsies or surgical specimens 1. However, advanced lung cancer is often diagnosed in cytology specimens obtained through FNA, and frequently cytological specimens are the only tumour samples available. We evaluated the possibility of determining ALK gene rearrangements in cytological samples from NSCLC patients by FISH. PATIENTS AND METHODS Between January and September 2011, 42 cytological samples from 42 NSCLC patients (25 M and 17 F) were analyzed for ALK gene rearrangements by FISH (Vysis dual colour break apart probe). Fig. 3 Adenocarcinoma with signet cell cells (PAS positive intracytoplasmatic vacuoles). Nuclear expression of TTF1. - Figure 5. Adenocarcinoma with predominance of signet cell cells. Nuclear expression of TTF1. A B 1. L858R Mutation. Exon 21 Rapid on site evaluation (ROSE) was done in all FNA cases. Tumour samples were obtained by : Bronchoscopy guided FNA (21 cases-50%) EUS guided FNA (7 cases-16.7%) EBUS guided FNA (2 cases- 4.8%) CT guided FNA (2 cases-4.8%) Direct FNA (2 cases- 4.8%) Cavity fluids (2 cases- 4.8%) External consultation cases(2 cases- 4.8%) Surgical specimens imprints (4 cases - 9.5%) ALK FISH analysis was performed on: A C B D Non-stained ThinPrep (21 cases - 50%) Pap stained smear (15 cases %) Fig. 1 A) Transbronchial FNA of a Cytological Cell blocks (4 cases - 9.5%) subcarinal lymph node guided by Stained ThinPrep slides (1 case -2.4%) bronchoscopy. B) EUS showing the needle in a mediastnal enlarged lymph node C) CT guided FNA D) Direct FNA of All cases were previously tested for EGFR and KRAS mutations by direct sequencing a neck node. Fig. 2 Cytological sample adequacy and inadequacy for FISH analysis using different processing methods Table 1 Clinicopathological characteristics of ALK positive cases CASE 1 CASE 2 Gender Female Female Age Smoke status Light smoker (7.5 pack/year) Non smoker Cytological diagnosis Adenocarcinoma Adenocarcinoma Signet ring cell % 30 % 70% Sample procurement technique Location Type of sample for FISH analysis Direct FNA Supraclavicular lymph node Non - stained ThinPrep KRAS status WT WT EUS guided FNA Mediastinal lymph node Non - stained ThinPrep EGFR status WT L858R+T790M Fig. 4 Positive FISH test is characterized by more than 15 % of the cells with separated green and red signals (Split signals, (arrows) and clustered gene amplifications (arrow head) CONCLUSION Determination of ALK gene rearrangements in cytological specimens is feasible. ThinPrep and cell blocks are the most suitable samples for FISH analysis, while Papanicolau stained smears provide poor results. Coexistence of ALK gene rearrangement and EGFR mutation, although rare, are not mutually exclusive. Analysis by FISH of ALK gene rearrangement on ThinPrep slides could be an option when no paraffin embedded tissue is available. REFERENCES 1.Soda Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009;27(September (26)): Fig. 6 A) Positive FISH test. Split signal (arrow) B) EGFR Sanger direct sequencing showing the L858R mutation in exon 21 and the T790M mutation in exon M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448: Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363: T790M Exon 20

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30 Schematic procedure of the analyses starting from a histological (A) or cytological (B) sample. Ulivi P et al. J Clin Pathol doi: /jclinpath Copyright by the BMJ Publishing Group Ltd & Association of Clinical Pathologists. All rights reserved.

32 2007 TTF-1 CK-7 L858R Mutation. Exon 21

33 2011 TTF-1 1. L858R Mutation. Exon T790M Exon 20

34 Conclusiones y recomendaciones: Es factible realizar estudio molecular en material citológico La frecuencia de la coexistencia de algunas alteraciones genéticas (EGFR y ALK) es baja pero tiene implicaciones terapeúticas Es recomendable realizar estudio mutacional en las recidivas / metástasis

35 GRACIAS POR VUESTRA ATENCIÓN! JI Echeveste Tania Labiano Nerea Gómez Mariam Maset Miriam Montañana Merche Aguirre Mª Eugenia Echarri

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