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An Overview on Molecular Cytopathology

An Overview on Molecular Cytopathology. Manuel Salto-Tellez Professor and Chair of Molecular Pathology Queen’s University Belfast . “The Cytology sample, in the context of appropriate laboratory validations, should not be treated differently to any other sample for molecular testing".

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An Overview on Molecular Cytopathology

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  1. An Overview on Molecular Cytopathology Manuel Salto-Tellez Professor and Chair of Molecular Pathology Queen’s University Belfast
  2. “The Cytology sample, in the context of appropriate laboratory validations, should not be treated differently to any other sample for molecular testing" “The ideal sample for molecular testing is the first one available which, often, is the cytology sample" DIAGNOSTIC & CLINICAL APPLICATIONS
  3. Association for Molecular Pathology (AMP) Diagnostic Molecular Cytopathologyis the application of molecular diagnosis to those samples that are firstly analysed by cytopathologists: FNAs, effusion and exfoliative cytology Cell Blocks Diagnostic MolecularCytopathology
  4. Why Molecular Cytopathology? CELL BLOCK (X20) 1.5 mm SMEAR (X20) The right DNA/RNA protocol: Proven for the sampling method Good DNA/RNA quality checks The right (RT-)PCR protocol: Designed for FFPE materials Good internal and external controls The right materials: Enough malignant cells Enough malignant/benign ratio
  5. The increasing importance of Molecular Cytopathology
  6. DIAGNOSIS? Architecture +++ Cytology +++ IHC quality +++ Usually more challenging to obtain Tissue biopsy? Architecture + Cytology +++ IHC quality + Easier to obtain FNA cytology? M
  7. EGFR = 16.2% Han HS. ClinLung Cancer. 2011 May 20. KRAS = 5% Mariani P. Anticancer Res. 2010 Oct;30(10):4229-35. HER2 = 5.5% Houssami N. Breast Cancer Res Treat. 2011 Oct;129(3):659-74. DISCORDANCE RATE PRIMARY TUMOUR METASTATIC TUMOUR HER2 = 32.0% Mittendorf EA. Clin Cancer Res. 2009 Dec 1;15(23):7381-8. PRE- TREATMENT POST- TREATMENT
  8. Translocations in sarcomas

  9. Translocations in sarcomas

    Tumor Translocation Fusion Product Ewing’s sarcoma t(11;22) EWS-FL11 Peripheral PNET t(21;22) EWS-ERG Myxoid liposarcoma t(12;16) TLS-CHOP t(12;22) EWS-CHOP Alveolar Rhabdomyosarcoma t(2;13) PAX3-FKHR t(1;13) PAX7-FKHR Clear cell sarcoma t(12;22) EWS-ATF1 Desmoplastic small round t(11;22) EWS-WT1 cell tumor Synovial sarcoma t(X;18) SYT-SSX1/ SYT-SSX2 Myxoid chondrosarcoma t(9;22) EWS-CHN Dermatofibrosarcoma t(17;22) COL1A1-PDGFB protuberans Infantile fibrosarcoma t(12;15) ETV6-NTRK3 S T R O N G D I A G N O S T I C V A L U E
  10. Synovial Sarcoma
  11. DiagnCytopathol. 2003 Dec;29(6):341-3. Sequence confirmation of the EWS-WT1 fusion gene transcript in the peritoneal effusion of a patient with desmoplastic small round cell tumor. Chiu LL, Koay ES, Chan NH, Salto-Tellez M.
  12. We present a case of a 17 year-old male with disseminated peritoneal disease and peritoneal effusion.
  13. a b c 1 2 3 M M 1 2 3 d e f 310 bp 281/271 bp 310 bp 281/271 bp 234 bp 234 bp 194 bp 189 bp 194 bp 118 bp g h Phosphoglycerte kinase (PGK) EWS exon 7 / WT1 exon 8 EWS WT1 Reverse j WT1 EWS Forward i PAP Cam 5.2 AE1/3 d) CD99 e) Desmin f) NSE
  14. a b c 1 2 3 M M 1 2 3 d e f 310 bp 281/271 bp 310 bp 281/271 bp 234 bp 234 bp 194 bp 189 bp 194 bp 118 bp g h Phosphoglycerte kinase (PGK) EWS exon 7 / WT1 exon 8 EWS WT1 Reverse j WT1 EWS Forward i PAP Cam 5.2 AE1/3 d) CD99 e) Desmin f) NSE

  15. Translocations in sarcomas

    Tumor Translocation Fusion Product Ewing’s sarcoma t(11;22) EWS-FL11 Peripheral PNET t(21;22) EWS-ERG Myxoid liposarcoma t(12;16) TLS-CHOP t(12;22) EWS-CHOP Alveolar Rhabdomyosarcoma t(2;13) PAX3-FKHR t(1;13) PAX7-FKHR Clear cell sarcoma t(12;22) EWS-ATF1 Desmoplastic small round t(11;22) EWS-WT1 cell tumor Synovial sarcoma t(X;18) SYT-SSX1/ SYT-SSX2 Myxoid chondrosarcoma t(9;22) EWS-CHN Dermatofibrosarcoma t(17;22) COL1A1-PDGFB protuberans Infantile fibrosarcoma t(12;15) ETV6-NTRK3 S T R O N G D I A G N O S T I C V A L U E
  16. 1 2 3 M M 1 2 3 310 bp 281/271 bp 310 bp 281/271 bp 234 bp 234 bp 194 bp 189 bp 194 bp 118 bp Phosphoglycerte kinase (PGK) EWS exon 7 / WT1 exon 8 a b c PAP Cam 5.2 AE1/3 d) CD99 e) Desmin f) NSE d e f g h EWS WT1 Reverse j WT1 EWS Forward i
  17. a b c 1 2 3 M M 1 2 3 d e f 310 bp 281/271 bp 310 bp 281/271 bp 234 bp 234 bp 194 bp 189 bp 194 bp 118 bp g h Phosphoglycerte kinase (PGK) EWS exon 7 / WT1 exon 8 EWS WT1 Reverse j WT1 EWS Forward i Diagnostic Cytopathology, 2003 Dec; 29(6): 341-3. PAP Cam 5.2 AE1/3 d) CD99 e) Desmin f) NSE

  18. Haemato-oncologyLymphomas

  19. Clinical history 53 year old male 2 months of left neck and facial swelling CT: well-demarcated homogenous tumour in the left parotid gland, measuring up to 55 x 50 x 50 mm.
  20. FNAC Cytomorphology Discohesive lymphoid population Cells with irregular nuclear membrane and small nucleoli, reminiscent of centrocytes. Fewer medium to large size and show prominent nucleoli. Paucity of tingible body macrophages.
  21. Immunohistochemistry on cell block CD 20 CD 10 CD 3
  22. Molecular findings Cell block unstained sections sent for FISH for the 14:18 FL translocation Many tumour cells showed fusion signals of less than one signal diameter separation
  23. Final diagnosis: Follicular Lymphoma
  24. Follicular lymphoma and diffuse large B-cell lymphoma transformation. Diff-Quik smear (A) and cell block preparation (B) IHC CD20 (C ) IHC bcl2 (E) IHC CD3 (D) FISH IGH/BCL2 Dual Color, Dual Fusion Translocation Probe Set detected two orange/green (yellow) fusion signals in the cells, confirming the presence of t(14;18) (q32;q21) and hence the diagnosis of follicular lymphoma (F).

  25. Thyroid Pathology

  26. Nikiforov YE et al. J Clin Endocrinol Metab. 2011 Aug 31. Impact of Mutational Testing on the Diagnosis and Management of Patients with Cytologically Indeterminate Thyroid Nodules: A Prospective Analysis of 1056 FNA Samples.
  27. Molecular diagnostics and personalised / predictive medicine Gastrointestinal stromal tumour Br J Cancer 2007;96:776–82 Cytopathology 2009;20:297–303 C-kit mutations Imatinib HER2-neu FISH Mod Pathol 2003;16:79–85 Hum Pathol 2003;34:362–8 J Clin Pathol2010;63:839–42 J Clin Pathol 2011; July 14th Arch Pathol Lab Med 2011; 135(6):693-5 Trastuzumab Lapatinib Breastcancer Gastriccancer Erlotinib Gefitinib ALK inhibitor PF-02341066 Lung cancer EGFRmutations Clin Chem 2007;53:62–70 J Thorac Oncol 2011, on line EML4-ALK Colon cancer J Mol Diagn 2009;11:543–52 Pathology 2008;40:295–8 Cytopathology 2010; Oct 4th Int J Colorectal Dis 2010; Dec 3th Cetuximab K-Ras mutations GSK2118436 PLX4032 Pathology 2008;40:295–8 Cytopathology 2010; Oct 4th B-Raf mutations Malignant Melanoma Modern pathology must be a synergy of morphology, IHC and molecular Dx
  28. Gastrointestinal stromal tumor Br J Cancer 2007;96:776–82 Cytopathology. 2009; 20:297-303 C-kit mutations Imatinib c-kit Mutations
  29. Cytopathology. 2009 Oct;20(5):297-303. Comparative validation of c-kit exon 11 mutation analysis on cytology samples and corresponding surgical resections of gastrointestinal stromal tumours Pang NK, Chin SY, Nga ME, Chang AR, Ismail TM, Omar SS, Charlton A, Salto-Tellez M.
  30. Primary Extragastrointestinal Stromal Tumor of the Pleura: Report of a Unique Case With Genetic Confirmation. Long KB, et al. Am J SurgPathol. 2010 May 3 Molecular analysis of c-Kit and PDGFRA in GISTs diagnosed by EUS. Gomes AL, Bardales RH, Milanezi F, Reis RM, Schmitt F. Am J ClinPathol. 2007 Jan;127(1):89-96 Fine-needle aspiration biopsy diagnosis of gastrointestinal stromal tumors using morphology, immunocytochemistry, and mutational analysis of c-kit. Rader AE, Avery A, Wait CL, McGreeveyLS, Faigel D, Heinrich MC. Cancer. 2001 Aug 25;93(4):269-75
  31. Colon cancer B-Raf mutations J Mol Diagn.  2009; 11:543-52 Pathology 2008;40:295–8 Cytopathology 2010, Oct 4, in press Int J Colorectal Dis. 2010 Dec 3. Cetuximab K-Ras mutations KRAS mutations
  32. KRAS and BRAF mutation analysis can be reliably performed on aspirated cytological specimens of metastatic colorectal carcinoma NKB Pang, ME Nga, SY Chin, TM Ismail, GL Lim, R Soong and M Salto-Tellez
  33. Lung cancer Erlotinib Gefitinib ALK inhibitor PF-02341066 EGFRmutations Clin Chem 2007;53:62–70 JTO 2011, accepted EML4-ALK EGFR mutations
  34. Sharma et al. Nat Rev Cancer 2007
  35. Lung cancer EGFRmutations Clin Chem 2007;53:62–70 J Thorac Oncol 2011; accepted Gefitinib Erlotinib Colon cancer EGFR mutation testing – clinical materials
  36. Obtaining lung tumour samples: what are the challenges? Small Sample Revolution In samples that are getting smaller, pathologists need to generate more meaningful information Diagnostic / Therapeutic
  37. Chin et al. Clin Chem 2007
  38. RESECTION (MACRO) RESECTION (X20) 66-year-old female, non-smoker, asymptomatic, finding on routine X-ray left upper lobe lung tumour, 4×4.5 cm CELL BLOCK (X600) CELL BLOCK (X20) 1.5 mm Chin et al. Clin Chem 2007
  39. EGFR mutation testing of cytology samples: experience from NUHS Singapore A review of EGFR Mutational Analysis on Non Small Cell Lung Cancer (NSCLC) Cytology Specimens A Dhewar, B Pang, ME Nga, Q Ahmed and M Salto- Tellez Joint BSCC-NAC Annual Scientific Meeting 13-16 July 2011 Keele University
  40. EGFR - Unsatisfactory rate 2011 13.3% 9.1% Excised during surgery Guided needle biopsy (for peripheral lesions) 9.1% 5.3% Bronchoscopic biopsy (for central lesions) FNA cytology and serous effusions (for peripheral lesions)
  41. EGFR mutation testing of cytology samples: experience from NUHS Singapore A comparative study of surgical and cytological specimens for EGFR mutation testing- review of data from two major tertiary hospitals in Singapore S Gupta, JE Seet,M Salto-Tellez Joint BSCC-NAC Annual Scientific Meeting 13-16 July 2011 Keele University
  42. Diagnostic and therapeutic recommendations Tumour sampling Sampling must be as generous as possible ‘Diagnostic’ sampling Sample must be kept in the best possible conditions Not adequate for diagnosis Adequate for diagnosis Adequate for therapeutic decision making? Yes No Salto-Tellez et al. J Thorac Oncol 2011, in press In some occasions, a second biopsy may be inevitable Diagnostic opinion Therapeutic decision
  43. Pathology sequence – reflex testing Diagnostic material (bronchoscopic, needle core or cytology) NSCLC SCLC EGFR exons 18–21 mutation detection EGFR mut EGFR wt KRAS Ex 2 & 3 KRAS mut? KRAS wt? EML4-ALK EML4-ALK AMP EML4-ALK not AMP Other treatments ALK inhibitors? Other treatments KRAS-related treatment? EGFR-TKI treatment
  44. Fujii T et al. Molecular testing and Cytopathology of Pleural Effusions in NSCLC(EGFR/KRAS/AML4-ALK). O-73
  45. Molecular Diagnostics and the Specific Weight of Morphology
  46. SQUAMOUS CELL CARCINOMA ADENOCARCINOMA NON-SMALL-CELL CARCINOMA SMALL-CELL CARCINOMA
  47. Non-small cell carcinoma continuum NSCLC, FAVOUR ADENOCA NSCLC, FAVOUR SCC SCC ADENOCA NSCLC, NOS YES YES (YES) (NO) NO +++ ++(+) + (-) - Who should be tested for EGFR mutations? What are the chances of mutation detection?
  48. HER2-neu FISH Mod Pathol 2003;16:79–85 Hum Pathol 2003;34:362–8 JCP2010;63(9):839-42 JCP 2011, accepted Archives Path Lab Med 2001, in press Trastuzumab Lapatinib Breastcancer Gastriccancer Her-2/neu amplification
  49. Clinical history provided 53 year old female Hep B carrier, sAg+ve on FU Elevated AFP (37.4ug/L). History of previous gastrectomy in another hospital ?diagnosis CT abdomen showed no liver lesions
  50. Left adrenal nodule Approximately 2.0 x 2.6 cm in size Occult HCC metastasis? EUS-FNAC done
  51. Cytomorphology Many papilleroid clusters of cohesive tumour cells Branching pattern Very cellular smears
  52. Cell block Adenocarcinoma Papillary pattern Focal CK 20 + Negative for CK 7, AFP & TTF-1
  53. Morphological diagnosis: Metastatic adenocarcinoma Excerpt from conversation with the managing oncologist: “Did the patient have a gastric cancer?” “Yes, please do a Her-2 scoring on the metastasis… both FISH and IHC.” “In the cytology sample?” “Yes, the original materials a re in another hospital….., by the way, it was intestinal type adenocarcinoma of the stomach…”
  54. Her 2 IHC Heterogeneity of staining Baso lateral staining pattern No complete staining Approximately 10 % showed ++ intensity in the cell block.
  55. Chromogenic ISH Fluorescent ISH
  56. Kapila K et al. FNA in Breast Cancer: FISH, CISH and IHC Comparison P2-068 Beraki E et al. Her-2 Status by DuoSISH O-74
  57. Molecular diagnostics and personalised / predictive medicine Gastrointestinal stromal tumour Br J Cancer 2007;96:776–82 Cytopathology 2009;20:297–303 C-kit mutations Imatinib HER2-neu FISH Mod Pathol 2003;16:79–85 Hum Pathol 2003;34:362–8 J Clin Pathol2010;63:839–42 J Clin Pathol 2011; July 14th Arch Pathol Lab Med 2011; 135(6):693-5 Trastuzumab Lapatinib Breastcancer Gastriccancer Biomarker Analysis Prior to Therapeutic Treatment is Here to Stay Erlotinib Gefitinib ALK inhibitor PF-02341066 Lung cancer EGFRmutations Clin Chem 2007;53:62–70 J Thorac Oncol 2011, on line EML4-ALK Colon cancer J Mol Diagn 2009;11:543–52 Pathology 2008;40:295–8 Cytopathology 2010; Oct 4th Int J Colorectal Dis 2010; Dec 3th Cetuximab K-Ras mutations GSK2118436 PLX4032 Pathology 2008;40:295–8 Cytopathology 2010; Oct 4th B-Raf mutations Malignant Melanoma Modern pathology must be a synergy of morphology, IHC and molecular Dx
  58. “The Cytology sample, in the context of appropriate laboratory validations, should not be treated differently to any other sample for molecular testing" “The ideal sample for molecular testing is the first one available which, often, is the cytology sample" DIAGNOSTIC & CLINICAL APPLICATIONS
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