Research Projects

Colorectal Cancer Research – Prof Rajkumar Ramesar

Colorectal Cancer Research – Prof Rajkumar Ramesar

Prof Rajkumar Ramesar

Division of Human Genetics, Faculty of Health Sciences, University of Cape Town

Title of the project

Investigation Of The Clinical, Pathological, Epidemiological And Molecular Genetic Basis Of Colorectal Cancers

Progress Made

The project has produced several outputs, including postgraduate students, publications, and direct application of the products of research in patient management.

  • Most tellingly for CANSA, the project’s overall success has recently been reflected by the University Vice-Chancellor’s Alan Pifer Award to the Principal Investigator, Professor Ramesar. The award is “in recognition of outstanding research that demonstrates relevance to the advancement and welfare of South Africa’s disadvantaged people”. Click here to read the article.
  • In addition, the project has resulted in a successful application to the Fogarty Foundation (of the NIH, USA) to fund an USA postdoctoral fellow to work in the Ramesar lab on the aspect of genetic modifiers: 2008/9
  • The project has attracted the third of its visiting scholars from Erasmus University, the Netherlands

What is the aim of the project?

Anatomical Pathology:

  • To characterise the morphology – macroscopic, microscopic pathology, the histochemical, immunohistochemical and molecular aspects of colorectal carcinomas developing in the three cohorts

Human Genetics:

  • To determine what genetic factors influence the site (specificity or non-specificity) of disease?
  • To determine what genetic factors influence the variable age of onset of disease?
  • Does the neuropsychological profile of  high risk patients influence compliance (proposed in the original project outline)

What has been achieved to date?

The project’s overall success has recently been reflected by the University Vice-Chancellor’s Alan Pifer Award to the Principal Investigator, Professor Ramesar presented at a function in April 2009. Read more…

At present, the local disease Registry in the Division, contains information from approximately 1600 individuals comprising  475 probands affected with colorectal cancer, and their at-risk relatives. DNA from  236 (out of the 475) probands was screened comprehensively for mutations in the three major mismatch repair (MMR) genes using conventional techniques for mutation detection (single stranded conformational polymorphism detection, denaturing high performance liquid chromatography, multiplex ligation dependant probe amplification and direct cycle sequencing) (Hameed et al., 2006). The selection of  patients for MMR gene mutation screening had been done on the basis of the number of first degree relatives with Lynch spectrum type cancers and the age at diagnosis.

To date, a wide range of different disease causing variations have been identified in approximately 20% of probands screened (understandably the mutation in some probands overlap). The most common disease causing variations that have been identified to date were the MLH1 1528C>T nonsense mutation, the MSH2 delCT1219 frameshift mutation and the MSH2 delGG1340 frameshift mutation.  A standard diagnostic testing procedure was developed for all of the disease-causing variations in order to offer predictive testing for at-risk individuals in extended family members.

This research has provided experience with regards to translating high tech research to families and communities that range from first world urban settings to remote rural settings. The translational work, with genetic counselling, and  clinical surveillance has provided evidence that this intervention prevents mortality and morbidity (Stupart et al, 2008).

Importantly, we have recently provided evidence that the outreach programme, with a mobile colonoscopy service, provides as good an outcome as is the norm in our tertiary setting (Anderson et al., 2007).

Our work on identifying a large founder population, with the same single hMLH1 gC1528T mutation underlying Lynch Syndrome has been extremely valuable for several reasons. To date, the mutation seems to be unique to South Africa, and is calculated to have originated approximately 500 years ago (unpublished).  The age of onset for colorectal cancer in patients with a primary mutation in one of the MMR genes ranges between 16-63 years of age.  A cohort of 200 individuals with a primary mutation in the MMR genes were selected to investigate the genetic determinants of variation in the age of onset for Lynch spectrum type cancers.  Candidate genes were selected based on their functional roles in the metabolism of xenobiotic agents (GSTT1, GSTM1, NAT2), methylation (MTHFR), cell-cycling (CCND1, ATM), cell-adhesion (E-cadherin, B-catenin) and  cell-death (Tp53).  The Kaplan Meyer survival analysis of GSTT1, GSTM1 and MTHFR yielded significant results, indicating that specific variants in these genes alter the age of onset for a cohort of colorectal cancer patients with a primary MMR gene mutation (Felix et al., 2006).

A submission to the Fogarty Foundation has resulted in funding for an American postdoctoral Fellow from Vanderbilt University, to further this exciting work on modifier genes.

Still with the modifier effects: The most significant demographic difference noted in the South African cohort of patients with a mutation in the MMR genes is the difference in the age of onset between males and females, affected with/ predisposed to colorectal cancer.  In an attempt to investigate the possible genetic determinant that may be involved in the difference in age of onset observed between males and females, X-chromosomal candidate genes involved in cell cycling, cell death and DNA methylation were selected for interrogation.  As part of a BSc(Hons) project in 2007, ATRX and TSPYL2 were investigated for their role in determining the age of onset in a cohort of male and female individuals predisposed to colorectal cancer.  From the Kaplan Meyer survival curves, allele 2 of the rs3088074 variant conferred a 40% delay in the age of onset for colorectal cancer at all ages (unpublished data).

In addition, we have described clinical heterogeneity involving the age of onset/diagnosis of disease and site of tumorigenesis in this cohort of approximately 130 affected homogeneous mutation carriers.  The families with this hMLH1 mutation have previously been managed as having a predisposition only to colorectal cancers (Goldberg et al., 2000). Now, for the first time we have provided definitive evidence that a range of gynaecological cancers, are also part of the spectrum of Lynch syndrome in these families with the c1528T mutation. Using immuno-histochemistry and microsatellite instability, we have conclusively shown that even breast cancer, which has not previously been described as part of the classic Lynch II syndrome, occurs in these families (Blokhuis et al., 2007).  This information is crucial for optimal clinical follow-up and management of females with this mutation.  As previously described our mutation-positive subjects contributed significantly to an international two year chemoprevention trial, involving complex starch and non-steroidal inflammatory drugs (REC REF: 209/2004). The study and outcomes are described in our NEJM publication (Burn et al., 2008).  Our research work looking for early markers is also progressing well (Dakshinamurthy et al., 2008), as is the work on defining the related family of cancers (Ibirogba et al., 2008).

Further qualitative work describing the various factors which influence compliance has been completed at Master’s level theses (Algar U: MSc Thesis and Kruger B: MSc Thesis); these are currently being prepared for publication. Ms Kruger’s work has already been submitted for publication (Pietersen et al.  2008). Ms Zandre Bruwer, an MSc Genetic Counselling graduate who worked on the project (her thesis is titled: Hereditary nonpolyposis colorectal cancer: comprehension of a cancer risk in conjunction with a genetic risk) is preparing a proposal to investigate compliance, socioeconomic status, ad neuropsychological profiling, for a PhD.  Ms Kusha Kalideen’s MSc thesis  (Genetic factors which lead to variable expression of cancers in different tissues in Lynch Syndrome) was successfully assessed by 2 international examiners; she will be graduating in June 2009.  Ms Jacqueline Meyer also submitted her MSc thesis (Relating genetic underpinnings of familial cancers to the phenotype, treatment outcomes, and prognosis), and will likely also be graduating in June 2009. The project has attracted a further 2 Honours students in 2009.

What do you hope to achieve in the next year?

The exciting aspect of the work will be the convergence of the research conducted by the current Masters, PhD and Postdoctoral researchers.  Comparison of the histological details of the range of tumours (using the range of antibodies mentioned in the original proposal, and microscopic details), as well as the stage of disease, the treatment modalities, and outcomes of the disease/management will provide a model for stratification of tumours towards influencing different modes of management.  Following the Blokhuis et al. (2008) study, one of the current projects (MSc:Ms Kusha Kalideen) investigates the molecular mechanism differentiating between the Lynch I (colorectal specific) and Lynch 2 phenotypes (affecting several other tissues apart from the colorectum).  This work will be important in understanding the steps leading from the primary mutation in MMR genes revealing the various steps that may ultimately result in neoplasia and disease.

Following on a review of the genes/mutations that lead to the ‘conservative (Lynch I) phenotype compared to the promiscuous (Lynch II) phenotype, the work now involves a biojnformatic interrogation of genes that may be most susceptible to mismatch errors (e.g. those with repeats in their primary sequence), and possibly involved or subject to hormonal regulation, in order to explain the range of female genitourinary malignancies.  These genes will be investigated in tumour and lymphocytic DNA from patients with extracolonic cancers.  In addition, a pilot investigation involving a gene expression array (Affymetrix U133) will be carried out on sporadic and Lynch syndrome cancer and matched lymphocytic RNA from the patients in order to gauge differential gene expression (REC REF 416/2005).  This will be valuable in identifying possible ‘susceptibility genes’.

The project is currently underway with the data already archived regarding mutation analysis in our familial cohort.  Genealogically traced family members who are already part of the clinical surveillance programme will be investigated regarding the range of cancers they’ve developed. A considerable amount of bioinformatics will be utilised in investigating gene expression in the range of tissues in which cancers have occurred, and attempt to relate this to the nature of the actual mutation.  Dr Douglas Stupart, consultant surgeon on the project, is starting his PhD, examining the role of mucins and their respective genes in the pathobiology and immunogenic characteristics displayed by the Lynch group of cancers.

In terms of expanding our study of modifier genes, Dr Sarah Owens, who is a postdoctoral researcher from Vanderbilt university in the USA, has joined the project as a Fogarty International Fellow. She will be examining a wider range of modifiers than we have previously done, using state of the art technology available in our Centre for Proteomic and Genomic Research.  Again, the major attraction to our project was the prospect of testing a wide range of candidate modifier genes in a homogeneous cohort of individuals carrying the hMLH1 C1528T mutation. She will be spending July 2007-June 2008 on this project, and contribute substantially to outputs (REC REF 211/2008).

A further aspect of current research (2008/9) investigates the appropriateness of treatment modalities for CRC.  In the first instance, we have gathered clinical records on treatment modalities and outcomes in our cohort of affected probands.  We propose to investigate the DNA from all of these individuals for genetic polymorphisms in pharmacogenes responsible for the metabolism of the most commonly used chemotherapeutic agent, 5FU.   We are particularly interested in the outcomes between those individuals who are carrying MMR gene mutations and those who are not (in the first instance), and then to correlate this to functional polymorphisms in the 5FU metabolising genes (MSc: Ms Jacqueline Meyer).  In an extension of this study, we propose to investigate a separate cohort of 147 individuals affected with late stage CRC, who took part in a clinical trial involving 5FU, and where there is clear 5 year follow-up data (Madden et al., 2005).

In terms of the clinical and translational aspects of our project, our project has been very successful in attracting visiting Fellows from Universities in the Netherlands.  Ms Mirre Rakers (a 5th year Medical Student), from Erasmus University in the Netherlands, (who followed Maria Blokhuis’ successful stay on our research project (Blokhuis et al., 2008) began to ascertain what proportion of overall colorectal cancers in the northern Cape Province are being captured during our mobile and referred colonoscopies.  The third such visiting fellow arrived recently and is currently acquiring data via the NHLS.

The access to data has been approved through the Institutional Ethics committee (REC REF 351/2008). An important aspect of the project, involving extension of our psychosocial studies and neuropsychological profiling of individuals with mutations and relating this to mechanisms to improve screening compliance will be consolidated.  Ms Zandre Bruwer, has completed an MSc in Genetic Counselling, and will be incorporating a study of psychosocial circumstance, diet/nutrition profiling in a multi-component study to better understand the interaction between professional care givers, and aspects of the subjects being managed, in order to improve our effectiveness.  Ms Bruwer will be doing this work for a PhD, and thereby be part of the projects major drive to develop capacity.  Ms Amy Northam, an MSc student will be joining the project to work on the neurpsychological aspect of the project; she will be attempting to determine if there is any correlation with aspects of temperament/personality and manifestation of disease.  This is based on previous research where ability to handle stress, and its impact on the immune system (and development of disease) was assessed.  The hMLH1 c1528 T cohort, with individuals having a wide range of age at onset (16y-78y)  is perfect for assessing this variable.

Peer-reviewed publications

  • Protocol for identifying individuals at risk for familial colorectal cancer, their genealogical tracing, and clinical management to reduce mortality and morbidity
  • DNA test kit for hMLH1; hMSH2; hMSH6 (the predominant genes involved in about 90% of familial nonpolypotic colorectal cancers)
  • An MSc (Genetic Counselling) course.  The course was instigated because of the recognition that programmes such as the current one were going to require formally trained genetic counsellors. The MSc programme which is greatly ‘oversubscribed’ reflects a growing awareness of the need for Genetic Counselling.
  • Clinical trial (CAPP) aimed at prevention of disease in mutation-positive individuals

2005-2008

  • Stupart DA, Goldberg PA, Algar U, Ramesar R. (2009).  Surveillance colonoscopy improves survival in a cohort of subjects with a single mismatch repair gene mutation. Colorectal Disease. 11(2):126-30.
  • Burn J, Bishop DT, Mecklin J-P, Macrae F, Möslein G, Olschwang S, Bisgaard ML, Ramesar R, Eccles D,  Maher ER, Bertario L, Jarvinen HR, Lindblom A, Evans DG, Lubinski J, Morrison PJ, Ho JWC, Vasen HFA, Side L, Thomas HJW, Scott R, Dunlop M, Barker G, Elliott F, Jass JR, Fodde R, Lynch HT, Mathers JC (2008). A randomised controlled trial of aspirin and resistant starch to prevent colorectal neoplasia in Lynch Syndrome (HNPCC): The CAPP2 Study, New England Journal of Medicine  11;359(24):2567-78.
  • Dakshinamurthy AG, Ramesar R, Goldberg P, Blackburn JM. (2008). Infrequent and low expression of cancer-testis antigens located on the X chromosome in colorectal cancer: implications for immunotherapy in South African populations.  Biotechnology Journal  3(11):1417-23.
  • Ibirogba SB, Algar U, Goldberg PA, Duffield M, Vorster A, Ramesar R. (2008). Clinical and pathological features of hereditary mixed polyposis syndrome: report on a South African family. South African Journal of Surgery. 46(3):90-2.
  • Blokhuis MM, Goldberg PA, Pietersen GE, Algar U, Vorster A, Govender D, Ramesar RS. (2008).  The Extracolonic Cancer Spectrum In Females With the Common ‘South African’ hMLH1 c.C1528T Mutation.  Familial Cancer 7(3):191-8.
  • Anderson DW, Goldberg PA, Algar U, Felix R, Ramesar RS. (2007).  Mobile colonoscopic surveillance provides quality care for hereditary nonpolyposis colorectal carcinoma families in South Africa. Colorectal Disease. 9(6):509-14.
  • Hameed F, Goldberg PA, Hall P, Algar U, van Wijk R, Ramesar R. (2006). Immunohistochemistry detects mismatch repair gene defects in colorectal cancer. Colorectal Disease. 8(5):411-7
  • Felix R, Bodmer WF, Fearnhead  NS, Bartlett S , Goldberg P, van der Merwe L, Ramesar RS (2006). The Influence Of Genetic Modifiers On Survival Until Diagnosis In A Cohort Of Individuals Predisposed To Colorectal Cancer With The Same Primary Mutation In The hMLH1 Gene. Mutation Research. Vol 602/1-2 pp 175 – 181
  • Madden M, Goldberg P, Geddes C, Van der Merwe L. (2005).  Double-blind randomised trial comparing 5-fluorouracil plus leucovorin to placebo for metastatic colorectal carcinoma.  Colorectal Disease. 7(5):507-12.

Conferences

2005 – 2008

  • Williams B, Pietersen E, Algar U, Craig, P, Vorster, Goldberg P, Ramesar R.  Factors influencing compliance in the CAPP2 preventative trial in South Africa. International Society for Gastrointestinal Hereditary Tumors (INSIGHT Conference Newcastle upon Tyne, UK 14-17 June 2005
  • Algar U, Goldberg PA, Ramesar R, The knowledge and attitudes of family members who have received predictive genetic test results for hereditary nonpolyposis colorectal cancer in South Africa   Poster Presentation at the First conference InSiGHT Newcastle upon Tyne, UK 14-17 June 2005.
  • Ramesar R, Algar U, Felix R, Vorster A, Pietersen E, Goldberg P. Recognition of an Inherited Basis to   Disease: A powerful tool in reducing Morbidity and Mortality in colorectal cancers. Invited presentation: South African society for Gastroenterology/Association of Surgeons of South Africa Conference,  Durban 21-24 August 2005
  • Ramesar R. Translating high technology research into a sustainable genetic service: Familial Cancer Genetics in South Africa. African Society of Human Genetics, Addis Ababa Ethiopia 3-5 June 2006
  • Ramesar R,  Vorster A, Felix R, Savanhu T, Goldberg P An Extensive Founder Mutation, MLH1g.1528C>T, in the hMLH1 Gene Underlying HNPCC is a Valuable Aide in Phenotype/Genotype Alignment and in Management in a Subpopulation in South Africa. Conference of the International Society for Gastrointestinal Hereditary Tumours (Insight) Japan, Yokohama 27-30 March 2007
  • Ramesar R, Felix R, Bodmer W, Fearnhead NS, van der Merwe L, Goldberg PGSTM1 And GSTT1 Polymorphisms As Modifiers Of Age At Diagnosis Of Hereditary Nonpolyposis   Colorectal Cancer (HNPCC) In A Homogeneous Cohort Of Individuals Carrying the Same Predisposing Mutation in hMLH1. Conference of the International Society for Gastrointestinal Hereditary Tumours (Insight) Japan, Yokohama 27-30 March 2007
  • Ramesar R: Invited Speaker. “Hereditary Colorectal cancers”. African Organisation for Research and Training in Cancer (AORTIC). 24th to 28th October 2007, Cape Town International Convention Centre, South Africa
  • Ramesar R: Invited Speaker “Genetics and Cancer” CANSA Learning Review Meeting, Mowbray,  Cape Town 13-16 November 2007
  • Ramesar R. Stratification of Cancers into Genetic Subtypes is an Aide to Clinical Management In vited Plenary Speaker:  Congress of the African Society for Human Genetics, Cairo, November 3-5, 2007
  • Ramesar R. Colorectal Cancer Genetics in Southern Africa.  Invited Speaker: American Association for the Advancement of Sciences (AAAS).  Symposium on Progress in Human Genomics Research in Africa: Science, Technology and Ethics, 14-18 February 2008, Boston International Convention Centre, Boston USA
  • Ramesar RS. GENETICS AND PUBLIC HEALTH: THE CONVINCING CASE OF LYNCH SYNDROME. InSiGHT meeting in Düsseldorf, Germany 24-28 June 2009.

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