FRCPath Part 1 Self Help Session

1. FRCPath Part 1 Self Help Session Cancer Genetics Helene Schlecht

2. Discuss examples of how analysis of oncogenes may be of use currently within diagnostic laboratories.

3. Keywords • Oncogenes • KIT and PDGFRA – GIST - Imatinib • HER2 – breast cancer - Trastuzumub • EGFR – non small cell lung carcinoma - Gefitinib or Erlotinib • K-RAS – Colorectal cancer – cetuximab and pantimumab

4. Oncogenes • An oncogene is a gene that when mutated or over expressed is involved in changing a normal cell into a tumour cell. • Proto-oncogene – a normal gene that can become an oncogene. • Proto-oncogenes encode proteins that are involved in the regulation of cell growth and differentiation.

5. Oncogenes • Oncogene activation involves a gain of function. • Quantitative – an increase in unaltered product • Qualitative – production of a subtly modified product by a mutation or production of chimeric gene via chromosome rearrangement. • Usually dominant and affect only one allele.

6. Oncogenes – Activation Mechanisms • Amplification – eg HER2; MYCN • Point mutation – eg HRAS, KIT • Chromosome rearrangement – eg BCR-ABL1 • Translation to transcriptionally active region – eg MYC

7. KIT and PDGFRA in GISTs • cKIT and PDGFRA – encode transmembrane glycoproteins. Part of type III receptor tyrosine kinase family. Lasota and Miettinen 2006 Seminars in Diagnostic Pathology 23: 91

8. KIT and PDGFRA in GISTs • Gain of function mutations major driving force in pathogenesis of sporadic GISTs. • Two classes of mutations: • Regulatory domains – EC, JM • Enzymatic domains – TK1 and TK2 • Continuous receptor activation independent of ligand binding causing GISTs.

9. KIT and PDGFRA in GISTs – molecular genetics • >90% cKit-JM exon 11 (p.Trp557_Lys558del) • EC exon 9 (p.Ala502_Tyr503dup); • TK1 exon 13 (p.Lys642Glu) • TK2 exon 17 (p.Asp816Val) • PDGFRA-TK2 exon 18 (p.Asp842Val) • Also JM exon 13 (p.Val561Asp) • TK1 exon 14 (p.Asn659Lys) • Del; delins; pm; dup; complex mutations (ins and inv – rare)

10. KIT and PDGFRA in GISTs - treatment • Imatinib mesylate (Gleevec/Glivec) • Inhibits cKIT, PDGFRA and ABL tyrosine kinases. • Mutation location affects imatinib sensitivity eg. KIT exon 11 mutations better response than exon 9 and WT. • Resistant mutations: PDGFRA p.Asp842Val and KIT p.Asp816Val • Acquired resistance during imatinib-based treatment

11. KIT and PDGFRA in GISTs • Diagnosis – Pathology and DOG-1 IHC, sometimes mutational analysis. • Prognosis – mutation analysis useful – cKIT exon 11 mutations have poor prognosis. • Predictive – mutation analysis useful – cKIT exon 11 mutations responds well to imatinib. • Management • Alternative treatment – sunitinib (sutent)

12. c-RET in MEN2 • Rare hereditary complex disorder: • Medullary thyroid carcinoma, bilateral/unilateral pheochromocytoma, other hyperplasia and/or neoplasia of different endocrine tissues. • MEN2A, MEN2B, FMTC • Biochemical markers: calcitonin – MTC; catecholamine - PHEO

13. c-RET and MEN2 • Activating mutations in c-RET proto-oncogene. • RET a tyrosine receptor kinase. • Extracellular portion – cadherine-like domains, calcium-binding site and cysteine-rich domain. • Transmembrane domain. • Intracellular portion – two distinct tyrosine kinase domains.

14. c-RET and MEN2 – molecular genetics • 98% MEN2 have germline mutations in cRET exons 10,11,13,14,15,16 (cysteine rich domain and TK domains) • MEN2A/FMTC – missense mutations in cysteine rich domain eg codons 609,611, 618, 620; p.Cys634Arg • FMTC - intracellular domain eg codons 768, 790, 791. • MEN2B – mutations in intracellular tyroine kinase receptor domain eg. p.Met918Thr, p.Ala883Phe exon 15.

15. c-RET and MEN2 • Diagnosis – confirms type of MEN2 • Prognosis – MEN2B poor prognosis • Predictive – phenotype and age of onset • Management – prophylactic surgery

16. EGFRs • Epidermal growth factor receptors: EGFR, HER2, HER3, HER4. • Receptor Tyrosine kinases Ligand binding site Ectodomain Transmembrane domain Internal domain: TK1 domain RTK substrate interaction 1 2 3 4 Barros et al. 2010 Histopathology 56: 560

17. HER2 in breast cancer • HER2 has no ligand binding site. Forms heterodimers to trigger downstream pathways. • Overexpression induces mitogenic and antiapoptotic signals. • 13-23% of breast tumours show over expression HER2.

18. HER2 in breast cancer – treatment • IHC or FISH • Trastuzumub (herceptin) – increases apoptosis and cell cycle arrest. • New treatments: Antibody that inhibit HER protein dimers – Pertuzumab. • Diagnosis – pathology • Prognosis – HER2 +ve poor prognosis • Predictive – responds to trastuzumub

19. EGFR and cancer • Epidermal Growth Factor Receptor • Involved in development, growth and differentiation of several organs. • Primary signalling pathways include RAS/RAF/MEK/ERK and PI3K/AKT • Aberrant expression or activity identified in many epithelial cancers: HNSCC, NSCLC, CRC, breast cancer, pancreatic cancer and brain cancer.

20. EGFR in non small lung cancer • Activating mutations in tyrosine kinase domain: >80% point mutations in exon 21 (p.Lys858Arg) or deletions in exon 19 (codons 746-750). • Gefitinib or Erlotinib • Drug resistance eg T790M • Diagnosis – pathology • Prognosis – ? • Predictive – response to gefitinib/erlotinib

21. EGFRs Modjtahedi and Essapen 2009 Anti-Cancer Drugs 20:851

22. EGFR and K-RAS in colorectal cancer • Colorectal cancer treated with EGFR- inhibitors: pantimumab and cetuximab. • K-ras – member of Ras family of small G proteins • Activating mutations cause constitutive activation of downstream signalling pathways – resistance to EGFR inhibitors.

23. EGFR and K-RAS in colorectal cancer • Mutations in exon 2 (codons 12 and 13) compromise hydrolysis of Ras-bound GTP to GDP causing activation of Ras pathway. • Gain of function mutations in BRAF also unresponsive. • Predictive – resistance to cetuximab/ pantimumab

24. EGFRs Modjtahedi and Essapen 2009 Anti-Cancer Drugs 20:851

25. CML • BCR-ABL kinase target for tyrosine kinase inhibitor imatinib.

26. References • KIT and PDGFRA in GIST • Lasota and Miettinen 2006 Seminars in Diagnostic Pathology 23: 91 • MEN2 • Marini et al. 2006 Orphanet Journal of Rare Diseases 1:45 • EGFR and HER2 • Barros et al. 2010 Histopathology 56: 560 (HER family) • Wheeler et al. 2010 Nature Reviews 7:493 (EGFR review) • Harris and McCormick 2010 Nature Reviews 7:251 (EGFR review) • K-RAS in colorectal cancer • Fletcher and Rubin 2007 Curr Opinion Genetics and Dev. 17:3 • Siddiqui and Piperdi 2010 Ann Surg Oncol 17:1168 • Modjtahedi and Essapen 2009 Anti-Cancer Drugs 20:851