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Essay Plan

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  1. Discuss examples of how analysis of oncogenes may be of use currently within diagnostic laboratories (not necessarily just molecular genetics labs). E.g. Her2 in breast cancer, EGFR mutations and tyrosine kinase inhibitors in lung cancer, KIT in GIST, RAS in Colon cancer and other tumours. Include how they are used in decision making for treatment where appropriate. Fletcher JA and Rubin BP (2007) Curr Opinion Genet Dev, 17,3-7 Marini F et al (2006) Orphanet J Rare Dis, 1,,45

  2. Essay Plan • Definition of oncogene • Products and activation of oncogenes • Examples of oncogenes in disease • Breast cancer (HER2) • Lung cancer (EGFR) • GIST (KIT) • Colon cancer (RAS/BRAF) • Endocrine cancers (RET)

  3. Oncogenes Gene involved in control of cell proliferation which, when overactive, can help to transform a normal cell into a tumour cell • The products of oncogenes can be classified into 6 broad groups: • Transcription factors (e.g. MYC and JUN) • Chromatin remodelers (e.g. ALL1) • Growth factors (e.g. SIS) • Growth factor receptors (e.g. VEGFR) • Signal transducers (e.g. RAS family and ABL1) • Apoptosis regulators (e.g. BCL2) • Oncogenes can be activated by structural alterations resulting from: • Mutation • Gene fusion • Juxtaposition to enhancer elements • Amplification

  4. Breast Cancer - HER2 gene amplification HER2 gene amplification and/or protein overexpression has been identified in 10-34% of invasive breast cancers Associated with increased cell proliferation, cell motility, tumour invasiveness, progressive regional and distant metastases, accelerated angiogenesis and reduced apoptosis. HER2 positivity is associated with worse prognosis (higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy. HER2 overexpression is now accepted as a strong predictive marker for clinical benefit from trastuzumab (Herceptin)

  5. Breast Cancer (HER2) - therapy Trastuzumab (Herceptin) a monoclonal antibody that binds to the extracellular domain of HER2, improves response rates, time to progression and survival when used alone or added to chemotherapy in metastatic breast cancer Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle → reduced proliferation Trastuzumab suppresses angiogenesis by both induction of antiangiogenic factors and repression of proangiogenic factors. Trastuzumab is associated with cardiac dysfunction in 2-7% of cases. Approximately 10% of patients are unable to tolerate this drug because of pre-existing heart problems.

  6. Breast Cancer (HER2) - testing IHC - Most frequent initial test for HER2 status (2+ and 3+ = IHC positive) FISH – viewed as being the “gold standard” technique in identifying patients who would benefit from trastuzumab CISH – DNA copy number (HER2 only) Southern blotting RT-PCR (needs to be performed on intact invasive cancer) MLPA (kit available from MRCH) mRNA microarray Dimerisation status assays Circulating tumour cells

  7. Non-Small Cell Lung Cancer (NSCLC) Non-small cell lung cancer accounts for 80% of all lung cancers • any type of epithelial lung cancer other than small cell lung carcinoma • most common subtypes are squamous cell carcinoma (35%), large cell carcinoma (10%) and adenocarcinoma (27%) • - biologically aggressive and relatively insensitive to chemotherapy 31,000 new cases each year in England and Wales 30,000 deaths in England and Wales in 2007 Survival rate from diagnosis; 25% >1 year, 7% >5 years

  8. NSCLC – activating mutations in EGFR Transmembrane tyrosine kinase receptor for members of EGF family ligands (i.e EGF and TGFa) In response to ligand binding, receptors dimerise, then use ATP to autophosphorylate tyrosine residues – this elicits downstream activation and signalling cascades involved in DNA synthesis and cell proliferation Activating mutations in EGFR result in constitutive activation of EGFR

  9. In the clinic, EGFR overexpression has been associated with chemoresistance, disease progression and poor survival ~10% of patients have a rapid and often dramatic clinical response to the TKI, Gefitinib

  10. Clinical responsiveness is dependent on the mutational status of EGFR

  11. EGFR mutation spectrum Exon 19 deletions and L858R account for ~90% of patients with activating mutations Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib

  12. EGFR mutation analysis Methods: DXS kit (detection of 29 somatic mutations in the EGFR gene) Direct sequencing Fragment size analysis (exon 19 deletions) Pyrosequencing HRM dHPLC CE-SSCA Enrichment by macrodissection of tumour by Consultant Histopathologist (minimum 30% tumour cells)

  13. Gastrointestinal Stromal Tumours (GISTs) Due to gain-of-function mutations in KIT (85%) and PDGRA (5%) – receptor kinases Mutations lead to constitutive activation and phosphorylation cascade of the tyrosine residues in multiple downstream signalling molecules and activation of signal transduction pathways (e.g. Ras/MAP) involved in the regulation of cell proliferation, apoptosis, chemotaxis and adhesion. Results in uncontrolled cell growth and tumour development

  14. GISTs – therapy Treatment is with TK inhibitors – imatinib and sunitinib Imatinib is a 2-phenylaminopyrimidine derivative that functions as a specific inhibitor of a number of tyrosine kinase enzymes. It occupies the TK active site, leading to a decrease in activity. The active sites of tyrosine kinases each have a binding site for ATP. The enzymatic activity catalyzed by a tyrosine kinase is the transfer of the terminal phosphate from ATP to tyrosine residues on its substrates (tyrosine phosphorylation). Imatinib works by binding close to the ATP binding site, locking it in a closed or self-inhibited conformation, and therefore inhibiting the enzyme activity of the protein semi-competitively. Response depends on the tumour mutations status – up to 20% of patients show no response to treatment Mutations can cause resistance to imatinib by shifting its equilibrium toward the open or active conformation

  15. GISTs – mutations in KIT & PDGRA KIT Val559Ile (exon 11) and Asn822Lys (exon 17) resistant to treatment PDGRA Asp842Val and Asp846Val (exon 18) resistant to treatment During imatinib treatment, resistance often develops due to secondary KIT or PDGRA mutations (mainly secondary KIT point mutations), therefore monitoring needed. Treatment of resistant tumours with sunitinib has shown some benefit

  16. Colon cancer – K-ras KRAS is a gene that encodes a GTPase in the epidermal growth factor receptor (EGFR) signaling pathway This signaling pathway is important in the development and progression of cancer On ligand binding, the EGFR homodimerises, leading to the activation of the intracellular kinase domain. Through the small adaptor proteins Sos and Grb, the KRAS signalling cascade is activated, leading to increased proliferation Wild-type KRAS protein is active for a short period when the EGFR is stimulated The effects of the protein are tightly controlled Mutations in the Ras protein usually cause constitutive activation of Ras GTPase, which leads to overactivation of downstream signaling pathways, resulting in cell transformation and tumorigenesis Somatic mutations in the K-ras gene have been found in multiple human cancers including colorectal, lung, and pancreatic cancers

  17. Colon cancer – K-ras mutations Mutations in the K-ras gene have been found in multiple human cancers including colorectal, lung, and pancreatic cancers The development of colorectal cancer is a multistep process involving cumulative genetic mutations. Oncogenes, tumor-suppressor genes, and chromosomal deletions are required to ultimately transform colonic epithelium. Among those genes, K-ras is considered to be critical components in the development of colorectal cancer Approximately 30% to 50% of colon cancers have K-ras mutations affecting codons 12,13 and 61. KRAS mutation is predictive of a very poor response to panitumumab (Vectibix) and cetuximab (Erbitux) therapy in colorectal cancer Several commercial assays available to detect the mutations. Allele-specific and direct sequencing assays generally used.

  18. Colon cancer (K-ras) – therapy • Monoclonal antibodies against EGFR block signaling by the receptor and inhibit downstream events, including effects mediated by KRAS • When KRAS is mutated and permanently activated, blocking EGFR will not prevent events downstream of KRAS • In this case, anti-EGFR therapy will not prevent the tumor from growing and spreading • Therefore, EGFR inhibitors are only effective in KRAS wild-type tumors • In mCRC, approx. 60% of patients have KRAS wild-type tumors 18

  19. Multiple Endocrine Neoplasia - RET • Autosomal dominant endocrine cancer syndrome • 3 recognised clinical variants • MEN2A • MEN2B • Familial medullary thyroid carcinoma (FMTC) • All variants show high penetrance of medullary thyroid carcinoma (MTC) • 90% of MEN2 patients will eventually show evidence of MTC

  20. 5’ Syndrome Exon Codons 609,611,618,620 630,634 Exon 10 Exon 11 MEN2A 10 11 Cysteine Rich Domain MEN2B 15 16 883 918 Transmembrane Domain 10 11 13 14 609,611,618,620 630,634 768,790,791 804 FMTC Exon 13 Exon 14 Tyrosine Kinase 1 Tyrosine Kinase 2 Exon 15 Exon 16 3’ All 3 phenotypes due to activating germline mutations in RET, which encodes a tyrosine kinase receptor RET binds growth factors of the glial derived neurotrophic factor (GDNF) family

  21. Multiple Endocrine Neoplasia (RET) – Biochemical testing • MTC • Plasma calcitonin concentration is measured before, 2, and 5 minutes after intravenous administration of calcium • Positive test = stimulated level is > 3 times the basal level (or > 300ng/L) • Phaeochromocytoma • elevated levels of catecholamines and catecholamine metabolites in 24hr urine collection • Parathyroid adenoma • simultaneously elevated serum concentrations of calcium and parathyroid hormone (PTH)

  22. Multiple Endocrine Neoplasia (RET) – mutation analysis Timing of surgery <5 years <1 year 5-10 years Screening of exons 5, 8, 10, 11, 13, 14, 15 and 16 in the RET gene by direct sequencing Mutations in these exons are found in approximately 95% patients Used to establish or confirm the diagnosis of MEN2 in symptomatic individuals and enable predictive testing of at risk family members Mutation-positive family members – prophylactic thyroidectomy At the moment, surgical treatment offers the only chance of cure and therefore early clinical and genetic detection and prophylactic surgery are the goal in subjects at risk

  23. References • Oncogenes • Croce, 2008, NEJM, 358(5):502-511 (Review) • HER2 • Wolff et al, 2007, J. Clin. Oncol., 25 (1):118-145 • GISTs • Lasota & Miettinen, 2008, Histopathology, 53:245-266 • K-ras • Jiang et al, 2009, Cancer, Aug 15, 3609-3619 (Review)

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