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TO EVALUATE THE FREQUENCY OF KIRSTEN-RAS GENE MUTATION AND ITS ASSOCIATION WITH CLINICAL AND PATHOLOGICAL FACTORS IN COLORECTAL ADENOCARCINOMA. 6. BRIEF RESUME OF INTENDED WORK 6.1 NEED FOR THE STUDY: Colorectal Adenocarcinoma is a major cause of morbidity and mortality worldwide. Colorectal adenocarcinoma contributes to 15% of cancer related deaths. The development of colorectal adenocarcinoma is a multistep process which can arise due to accumulation of molecular alterations including chromosomal abnormalities, genetic mutations and epigenetic changes. Kirsten-RAS (K-Ras) is a gene that encodes one of the proteins in the epidermal growth factor receptor (EGFR) signaling pathway. This protein has an intrinsic GTPase activity.1 The K-ras gene may be normal (wild-type) or mutated. Mutation in K-ras gene in patients with colorectal adenocarcinoma develops early in the progression from adenoma to carcinoma. The effects of mutated K-ras leads to unregulated tumour growth and spread. Kras mutation in colorectal adenocarcinoma is associated with the advanced Dukes stage.2‚ 3 This mutation is also associated with an increased risk of relapse and death and with a significantly poorer median overall survival than patients without the mutation.4, 1 Recent studies have shown that somatic gain of function K-ras mutations, have been identified as a reliable strong negative predictor for response to anti-EGFR treatment, i.e. Cetuximab and Panitumumab in metastatic colorectal cancer.1, 5 The monoclonal antibodies bind to the EGFR and thereby inhibit cell proliferation, metastasis, and angiogenesis. Recent clinical data confirm that the efficacy of Cetuximab and Panitumumab is confined to patients bearing tumours with wild-type K-ras. K-ras mutation analysis may now be considered a new standard of care in the selection of patients for EGFR-targeted therapy.1 Hence the present study is being conducted to detect the frequency of the K-ras gene mutation by PCR sequencing technique and to correlate it with the clinical and pathological factors describing the age, sex, tumour location, Duke’s stage and differentiation in colorectal adenocarcinoma. 6.2 REVIEW OF LITERATURE: About 90% of colorectal cancers are well to moderately differentiated adenocarcinomas with variable amount of mucin production. Other microscopic types are conventional adenocarcinoma variants like mucinous carcinoma, signet ring carcinoma, adenosquamous, carcinoid etc. The classic adenoma carcinoma sequence accounts for 80% of sporadic colon tumours. K-ras encodes a small 21Kd protein in the epidermal growth factor receptor (EGFR) signaling pathway. The activation of EGFR leads to the activation of intracellular effectors involved in intracellular signaling pathways such as the G protein K-Ras.1, 3 K-Ras is a self-inactivating signal transducer, cycling from guanosine diphosphate (GDP) bound (“off” state) to guanosine triphosphate (GTP) bound (“on” state) in response to receptor activation. This response is transient because of the intrinsic GTPase activity. The K-Ras protein controls the transduction of signals required for proliferation, differentiation, and survival, mainly acting as GDP/GTP–regulated binary switches located at the inner surface of the plasma membrane.1, 6 The K-ras gene may be normal (wild-type) or mutated. Wild-type K-ras protein is active for a short period when the EGFR is stimulated. The effects of the protein are tightly controlled. When K-ras is mutated the protein is permanently turned on, even without being triggered by EGFR-mediated signaling. Activating mutations in K-ras occurring early in colorectal tumorigenesis are thought to abolish GTPase activity leading to increased and unregulated cellular proliferation and malignant transformation.1 Mutations in K-ras is a late event and is supported by the observation that mutations are present in fewer than 10% of adenomas< 1cm diameter and are found in 50% of adenomas > 1 cm diameter and 50% of invasive adenocarcinomas.6 The two monoclonal antibodies Cetuximab and Panitumumab targeting epidermal growth factor receptor (EGFR) have been approved for the palliative treatment of metastatic CRC. Somatic gain of function in K-ras mutations have been identified as a reliable strong negative predictor for the response to anti EGFR treatment in colorectal carcinoma because constitutionally activated K-ras eliminates the effect of upstream EGFR inhibition.5 Palliative Cetuximab and Panitumumab therapy of colorectal carcinoma is dependent on K-ras wild type status of tumour tissue irrespective of whether applied in combination conventional chemotherapy or as singular drugs.5 The activating oncogenic mutations in K-ras are found mostly in codons 12 and 13 of exon 1, but may also affect codon 61, in exon2.3 6.3 OBJECTIVES OF THE STUDY: 1. To detect the frequency of K-ras gene mutation (wild type/mutant) in colorectal adenocarcinoma. 2. To correlate the K-ras gene mutation status with the clinical and pathological factors in colorectal adenocarcinoma. 7. MATERIALS AND METHODS: 7.1 SOURCE OF DATA: All the formalin fixed and paraffin embedded tumour blocks of surgically resected colorectal specimens, biopsies and metastatic excisions from colorectal adenocarcinomas received in the Pathology department at M.S.Ramaiah Teaching Hospital and M.S.Ramaiah Memorial Hospital from June 2009 to June 2012. 7.2 METHOD OF COLLECTION OF DATA: 1. Clinical history will be collected from patient’s records. 2. Studies to diagnose the colorectal adenocarcinoma with tumour staging will be done as follows. a. For retrospective cases: - the clinical details and histopathological reports will be retrieved from the patient’s records. b. For prospective cases: - The clinical history will be taken. The specimens will be received in the pathology department in 10% formalin. The formalin fixed specimen will be processed in the standard manner and paraffin sections of 3-5µ thickness will be studied in detail. The stain used will be Haematoxylin and Eosin. 3. For detection of K-ras mutation statusFor retrospective and prospective casesThe paraffin embedded tissue blocks of diagnosed colorectal carcinoma will be sent and subjected to PCR followed by gene sequencing at Oncquest laboratories limited (Delhi)/Triasta laboratory (Mumbai). Procedure Four–Five 3µm thick serial sections will be cut from each paraffin block. The sections are evaluated to confirm that cancer cells are present. The tissue sample is then removed from the slide in order to perform the K-ras status test. The tumour DNA from the tissue sample is purified. The tumour DNA is checked for purity as part of quality control procedure. A highly sensitive K-ras specific test based on Polymerase chain reaction (PCR) uses labelled probes to amplify DNA in order to detect the presence of mutated K-ras. The products of amplification are measured to determine whether the tumour has wild type K-ras gene or mutant version. INCLUSION CRITERIA: Biopsies surgically resected and histologically proven colorectal adenocarcinoma patients > 18 years of age and both sexes are included. EXCLUSION CRITERIA: Tumour tissue with extensive necrosis and scanty viable tumour. SAMPLE SIZE DETERMINATION: Rationality of sample size Studies carried out on KRAS mutation in colorectal adenocarcinoma reveals that the mutation ranges from 22-40%. However majority of the studies have reported 40 % as the frequency of mutation. Considering the above mutation rate with the precision of 10% and a confidence interval of 90% estimated sample size for the present study works out to be 65. Hence it is proposed to include 65 subjects for the study. STATISTICAL ANALYSIS: The data is interpreted in terms of frequency distribution tables. The proportion of the subjects revealing mutation according to various subgroups such as clinical pathological staging, age, type of colorectal cancer will be estimated along with 95% confidence interval. Quantitative parameters such as age, duration of symptoms etc. will be summarised in terms of mean and standard deviation. The differences in the frequency of mutation between various subgroups will be tested for statistical significance by employing chi square/Fishers exact test. (p<0.05) will be considered for statistical significance. 7.3 DOES THE STUDY REQUIRE ANY INTERVENTION OR INVESTIGATIONS TO BE CONDUCTED ON HUMANS? The study will be conducted on formalin fixed paraffin embedded tumour sample from biopsies and surgically resected colorectal carcinoma. No additional financial burden will be incurred on patients. No additional intervention or investigations are done on humans in the present study. 7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION? Yes 8. LIST OF REFERENCES: 1. Ramos et al. Understanding the predictive role of K-ras for epidermal growth factor receptor – targeted therapies in colorectal cancer. Clinical colorectal cancer; 2008; 7, Suppl.2, S52-S57. 2. Naguib A, Mitrou P, Gay L, Cooke J, Luben R et al. Dietary, lifestyle and clinicopathological factors associated with BRAF and K-ras mutations arising in distinct subsets of colorectal cancers in the EPIC Norfolk study. Biomed Central Cancer 2010; 10:99. 3. Sameer et al. Mutation pattern of KRAS gene in colorectal cancer patients of Kashmir: A report. Indian journal of cancer 2009; 46: 219-25. 4. Andreyev et al. Kirsten ras mutations in patients with colorectal cancer: the `RASCAL II´ study. British Journal of cancer; 2001; 85(5): 692-696. 5. Weichert et al. KRAS Genotyping of Paraffin - Embedded Colorectal Cancer Tissue in Routine Diagnostic, Comparison of Methods and Impact of Histology. Journal of molecular diagnostics 2010; 12: 35-42. 6. Turner J. The Gastrointestinal Tract, Chapter 17 in Robbins and Cotran Pathologic Basis of Disease, 8th ed Elsevier 2010; 822-825.