Download TO EVALUATE THE FREQUENCY OF KIRSTEN

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.