1. Jennie Bell CMGS/ACC Spring meeting 14 th April 2010

3.  Cancer cells acquire the capacity for autonomous and dysregulated proliferation Uncontrolled production of growth factors Enhanced expression of growth factor receptors ↳ Proliferation of cancer cells ↳ Induction of angiogenesis ↳ Metastasis Lung cancer cell

4. KRAS C-KIT EGFR

5.  EGFR is a transmembrane receptor belonging to a family of 4 related proteins  The majority of human epithelial cancers are marked by activation of growth factors and receptors of the epidermal growth factor receptor family

6.  KRAS is a gene in the EGFR signaling pathway  Activating mutations impair GTPase activity resulting in constitutive activation  Up to 30% of all human tumours contain a KRAS mutation  Most commonly observed in lung, colon and pancreatic cancer and haematopoietic neoplasm http://www.kras-info.com/

7.  c-KIT is a proto-oncogene and a transmembrane receptor  Ligand binding activates intracellular tyrosine kinase domain  PDGFRa is homologous to KIT and functions in a parallel pathway.  Both genes can be mutated in GIST Gastrointestinal stromal tumours.

8.  EGFR was the first growth factor receptor proposed as a target for cancer therapy.  Development of EGFR antagonists for treatment of metastatic epithelial cancers: Non-small-cell lung cancer (NSCLC) Squamous cell carcinoma of head and neck Colorectal cancer Pancreatic cancer

9. EGFR inhibitors approved for cancer treatment: Erlotinib Gefitinib Cetuximab Panitumumab

10.  Gefitinib, a small molecule tyrosine kinase inhibitor, is a targeted therapy for the treatment of patients with non-small cell lung cancer  The drug binds to the EGFR TK domain with high specificity and affinity resulting in highly effective inhibition of the aberrant signalling pathways.

11.  Imatinib is a synthetic tyrosine kinase inhibitor (Glivec, Novartis Pharmaceuticals UK) successfully used in the treatment of CML  Imatinib can block the activated receptor tyrosine kinase activity of c-kit  Targeted therapeutic approach in GIST.

12.  Not all patients respond in the same way to drug treatment  The presence or absence of a mutation can influence response  The presence of different somatic mutations within each gene can affect the action of the drug at the cellular level

13.  Clinical trials have shown that patients with certain EGFR mutations derive significant benefit from gefitinib treatment while patients without these mutations gain more benefit from standard chemotherapy  Mutations are found in four exons of the EGFR gene (exons 18 to 21).  Deletions in exon 19 and a point mutation in exon 21 (L858R) account for around 90% of all activating mutations

15.  As Gefitinib has been shown to benefit patients with particular somatic sequence changes  Essential to identify specific sequence changes in individual patients  Match sequence variants with specific patient treatment

16.  GIST patients with a somatic mutation generally have a higher response to treatment than those that do not.  Exon 11 most commonly mutated in (67% of cases) Mutations in exon 11 generally respond to treatment with Imatinib better than mutations in other exons.  Exon 9 mutations are less common (17% of cases) Exon 9 mutations have a lower response rate to Imatinib therapy vs exon 11 mutations (but a better response rate to Imatinib than c-kit "wild-type" GIST)

17.  There has been a recent paradigm shift in cancer patient treatment Broad-spectrum cytotoxic therapy molecular targeted treatment  The presence of a sequence variant within one of these genes (EGFR, c-Kit, KRAS) can determine a patient’s treatment.

18.  A patient presents with lung cancer  A biopsy is taken  A diagnosis of NSCLC should be confirmed by a Consultant Histopathologist  Subsequent molecular analysis of the tumour sample is required  Identifying patients with an EGFR mutation is a critical part of the patient care pathway  Appropriate treatment discussion with patient

19. Dr Philippe Tanière Consultant Histopathologist UHB Brendan O’Sullivan/Frances Hughes BMS, UHB Jennie Bell/Dr Fiona Macdonald Consultant Clinical Scientists, BWH http://www.egfr-info.com/EGFR-exon

20. Department of Cellular Pathology UHB West Midlands Regional Genetics BWH

21.  Partnership established in 2002  Department of Cellular Pathology, University Hospital Birmingham and West Midlands Regional Genetics Laboratory  Tumour studies in colon cancer (MSI and IHC in Lynch syndrome/HNPCC)

22.  KIT and PDGFRa analysis for GIST Sequencing exons 9, 11, 13, 17 c-kit and exons 10, 12, 14, 18 PDGFRa  KRAS analysis for lung and colon cancer Pyrosequencing exon 2 KRAS  EGFR for lung cancer Analysis of exons 18 to 21 (RQ-PCR, sequencing)

23.  All molecular work is performed within the Regional Genetics Laboratory  A basic report is issued giving details of any sequence variants identified (or not)  The results are integrated into an overall patient summary by Consultant Histopathologist

24. * Samples reported 1 st Jan to 31 st March 2010

25. Validation

26.  Service validation complete  Problem with availability of DxS kit  Currently using direct sequencing  Reports will be issued from 1 st April 2010

27.  BRAF analysis in colorectal cancer Pyrosequencing based assay  BRAF and KIT in melanoma KIT mutated in non-skin melanomas BRAF mutated in sun exposed areas  MGMT methylation in gliomas and endocrine tumours predictive to alkylant based chemotherapy  ERCC1 expression RNA based test on paraffin sections Predictive marker to response to platinum based chemotherapy (lung, pancreas, stomach and colon)

28.  Management of results for patients referred with cancer is co-ordinated by Consultant Histopathologist  Equipment and molecular expertise is provided by the Genetics Service  Molecular testing is performed to accredited standards

29.  More drug targets  Improved drug treatments  Further expansion of molecular pathology services is anticipated  Supported by strong cross-discipline collaboration

30.  Molecular Laboratory Staff  Dr Fiona Macdonald jenny.bell@bwhct.nhs.uk  Brendan O’Sullivan  Frances Hughes  Dr Philippe Tanière phillipe.taniere@uhb.nhs.uk