Untitled Document

Globally,cancers affect 9 million people and causes 5 million deaths annually. In Malaysia, the estimated cancer incidence is 150 for every 100,000 population; that is approximately 33,000 new cases per year. Of the ten leading cancers in Malaysia, colorectal cancer ranks second after lung cancer in males while in females, it ranks sixth with breast cancer as the leading cause. The progression of colorectal cancers is a result of a series of genetic changes which involves the activation of oncogenes (cancer-causing genes) or the inactivation of tumour suppressor genes. A large number of different mutations in various genes have been reported and the analysis of these mutations at different stages of development of colorectal cancer will contribute towards a better understanding of this cancer. In addition, some of these genetic changes in oncogenes can be used as markers for diagnosis/early detection as well as prognosis of colorectal cancer. Studies to detect genetic changes in various oncogenes and tumour suppressor genes in Malaysian colorectal cancer patients are lacking, thus an objective of this study would be to demonstrate genetic alterations in selected predisposition genes in Malaysian patients.

As the development of colorectal cancer is accompanied by complex changes in gene expression patterns, there is a need to study these patterns so that novel genes involved with colorectal carcinogenesis can be identified. Such new information will serve as a basis for future studies involving the development and/or design of novel therapies or the assessment of local pharmaceutical products with anti-cancer properties. Therefore another objective of this study will be to make use of newer technologies like "microarray technology" and "real time PCR" which are powerful tools to analyse and quantify gene expression for colorectal cancer. The microarray technology is a very powerful tool which will enable us to study differential gene expression in cancer cells; it will provide information as to which genes are upregulated (over expressed) or down regulated. In addition to this information, selected genes need to be quantified to allow for a more accurate correlation to be made between gene expression levels and tumorigenesis. With real time PCR, single base changes in DNA fragments can be detected; also this technique will allow us to determine the number of copies of a particular gene that is expressed at various stages of the cancer. Such information will contribute towards our understanding of the regulation pathways that occur during progression of colorectal cancer as well as identify useful biomarkers for cancer diagnosis and prognosis.

Another important aspect that needs to be studied is the role of various biomolecules involved in mediating host protective response to cancer as this will help unravel the complex process of colorectal tumouregenesis and lead to better management of the disease. Therefore another objective of the study will be to study the various biomolecules involved so as to understand why or how tumour cells evade the host's immune system. We will need to study the type, maturation and activation of various cells of the host immune system in order to correlate the type and level of expression of various biomolecules with tumour staging and progression. The data will serve as useful prognostic markers.

Expected outputs from the study will include new information on the types of genetic mutations found in Malaysian colorectal cancer patients; expression profiles of selected predisposition genes that can help us elucidate tumour progression pathways for colorectal cancer; identification of biomarkers that can be used for early detection, diagnosis or prognosis of colorectal cancer which will lead to overall improvement in the management of colorectal cancer. In addition, specialized technologies that are acquired through the study will be made available to other interested researchers. Lastly, this project will involve researchers from at least 5 different institutions and by working together, networking and inter-institutional cooperation will be further enhanced.

Period of the project : 3 Years (2001 - 2004)

Achievements

Progress/Achievements for year one

New Process :

1- Sample collection (UKM and UPM)

2- Sample processing (UKM,IMR)
i- DNA extraction : DNA was isolated from tissue (normal and tumor) from the 9 patients by the UKM group. These DNA samples were given to IMR in November 2002
ii- RNA extraction : The IMR group used the first 2 tissue samples obtained from HKL to optimize methods for RNA extraction and cDNA synthesis. They are currently extracting RNA from the 9 samples obtained from HUKM which were recently received.

3- Mutation analysis of APC,p53,p27 and Ki ras genes (IMR and UKM)
i- Optimization of PCR : PCR conditions for all the exons of interest in the various
genes mentioned above have been optimized
ii- Mutation Analysis Studies : DNA samples extracted from the normal and tumour
tissue from 9 patients with colorectal carcinoma were received from HUKM in November 2002. The DNA content of the samples were quantified and DNA quality checked. These DNA samples will be used for mutation analysis studies of APC,p53,p27 and Ki ras genes in 2003

4- Realtime PCR studies (UKM and USM)
All major reagents have been purchased and preliminary testing of our newly acquired realtime PCR machine at USM has been completed. Currently awaiting cDNA from patients samples to be supplied by IMR.

5- Microarray studies (UNIMAS)
Their collaborators at IMR are currently processing these samples for total RNA and these will be made available to us in the very near future. In the period, while waiting for specimens, they have already tested the feasibility of performing microarray work at UNIMAS. This test has been proven successful and optimization wwork is underway. We are also working on acquiring various human array types ranging from general large human gene arrays to specific focused arrays containing genes of particular functional classes.

They have also embarked on preliminary mutation analysis work. To date, we have already optimized all parameters for investigating mutation status of 2 tumour suppressor genes (TSGs), APC and p53 on colorectal specimens. This covers both genomic (exonic) and cDNA mutations. Although our collaborators at IMR, and UPM have already embarked on the same strategy, reproducibility of mutation status by all working groups would strengthen any information pertaining to the identification of predisposition genes in local cases. Besides, the 2 TSGs mentioned they are currently optimizing experimental parameters for the p63 (a member of the p53-gene family) TSG also.

6- Characterization of role of cells (UPM)
One colour staining for phenotyping : The frequency of CD4 and CD 8+ cells infiltrating into the tumour were determined by flow cytometry. The method for determining he frequency of cells as been established.

To determine whether expresion of proapotic molecules contributed to tumour survival, fresh tumour specimens were disaggregated and stained with markers for tumour cell and bcl-2. Flow cytometry and intensity of staining of bcl-2 showed that the increased expression of bcl-2 in tumour tissue compared to normal tissue was statistically significant.

7- Immunohistochemistry
i- Various chemicals, reagents and consumables were purchased through Institute Medical Research (IMR) for tissue processing, embedding in paraffin and hematoxylin and eosin staining and immunohistochemistry.
ii- To date thirteen colorectal specimens have been collected and processed. The specimens were obtained from patients with benign and malignant lesions.
iii- The tissues were processed and embedded in paraffin. The paraffin blocks were cut into tissue sections for hematoxylin and eosin evaluation and immunohistochemical analysis.
iv- Hematoxylin and eosin (H&E) staining was completed for the specimens collected.
v- Standadization of reagents and immunostaining technique for the detection of cyclin D1 nuclearprotein is in progress.