published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

Pancreatic cancer (PC) is the fourth leading cause of cancer death in Western societies. Despite significant progress in understanding the molecular pathology of PC and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility. Affymetrix Genechipfi oligonucleotide microarrays were used to interrogate mRNA expression of PC and normal pancreas to identify molecular pathways dysregulated in PC. Analysis of these data identified altered expression of numerous components of the S100 Calcium Binding Protein Family, Retinoic Acid signalling pathway and the HOX transcriptional network in PC compared to normal pancreas. These pathways were assessed using immunohistochemistry (IHC) and in-situ hybridisation (ISH) in a cohort of patients with PC. Increased protein expression, of S100A2, S100A6 and S100P was observed in 43%, 60% and 48% of PC respectively. Expression of S100A2 was associated with a poor outcome (p = 0.009), whilst increased expression of S100A6 (p = 0.0008) and S100P (p = 0.0005) were associated with an improved outcome. Additionally, S100A2 expression was identified as an independent marker of outcome in resected tumours. Aberrant expression of retinoic acid signalling components was demonstrated in PC cell lines using semi-quantitative RT-PCR. ISH demonstrated expression of Retinoic Acid Induced 3 (RAI3), an orphan G protein coupled receptor normally expressed in the fetal lung, in 68% of PC, and this co-segregated with an improved overall survival (p = 0.026).Ectopic protein expression of HOXB2, a transcription factor normally expressed in the developing hindbrain and modulated by retinoic acid, was observed in 15% of early PanIN lesions and 38% of PC specimens. Expression of HOXB2 was associated with non-resectable tumours and was an independent predictor of poor survival in resected tumours. Suppression of HOXB2 protein expression using small interfering RNA, resulted in epithelioid trans-differentiation in the Panc-1 PC cell line, however no alteration in proliferation rates were observed compared to controls. This thesis has shown that transcript profiling and tissue validation has identified potential markers of early diagnosis and outcome in PC. Furthermore, pathways and molecules previously thought to be associated with normal human development have been implicated to play a role in the development and progression of PC. Further analyses of these markers will determine any potential role in future diagnostic and therapeutic strategies.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The work presented in this thesis deals with the association between cancers of the upper gastro-intestinal tract and the tumor suppressor gene, TP53, and can be divided into three parts: (i) the analysis of the mutational spectrum of TP53 with respect to laryngeal cancer, (ii) the analysis of the mutational spectrum of TP53 with respect to esophageal cancer and (iii) the analysis of TP53 transcriptional levels in esophageal cancer. Laryngeal cancer (LC) is the 6th most common cancer in the world and the 2nd most common respiratory cancer, with approximately 500 000 new cases per annum detected worldwide. Over the last few years, LC has become increasingly prevalent within the Coloured Community of the Western Cape. The mechanisms of tumorigenesis in LC remain unknown, although smoking and alcohol consumption are considered to be major risk factors. Mutations within the gene TP53 have been strongly implicated as playing a role in cancer development, as they are frequently found in several cancer types. We therefore screened exons 5 - 8 of TP53 for mutations in DNA from tumor biopsies (n=44) and blood samples (n=42) from Coloured LC patients, using polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Blood samples from a healthy, matched control group (n=40) were included in the study as controls. Significant correlations were found between the occurrence of LC and age and smoking, whereas daily meat consumption was a possible protective factor. In tumor-derived samples, mutations were found in 3 of the exons under investigation, representing 25% of the samples. The mutations were unique to the tumor biopsies, indicating a somatic origin for mutations. The data confirms that the region between codons 175 and 273 of TP53 is a mutational hotspot for cancers in general. This study reports 6 novel mutations within this same region. Esophageal cancer (EC) has a very high incidence in South Africa, relative to the rest of the world, and is particularly common amongst the Black Transkei population. The goal of this study was to determine whether there are differences in the TP53 mutational pattern observed in the Coloured Western Cape community as compared to that observed in the Black Transkei community. This required the analysis of the molecular structure of TP53, specifically exons 5 - 8, in a group of Coloured EC patients (n=44) treated at Tygerberg Hospital, Cape Town, South Africa. DNA obtained from tumor biopsies and blood (from patients) as well as from apparently healthy surrounding tissue was screened via PCR-SSCP and direct sequencing analysis. Only 4 nucleotide changes were observed from a total of 124 sequences obtained, of which two were novel to esophageal squamous cell carcinoma. These 4 nucleotide alterations were found only within the tumor biopsy sample set, representing 9% of the tumors investigated. This study revealed that the mutational spectrum of TP53 within the Coloured population of the Western Cape greatly differs from that of the Black community of the Transkei. This suggests that a different set of etiological factors are involved in the tumorigenic process for each of these distinct geographical communities, which is the subject of an epidemiological study undertaken by the MRC. The final part of this thesis deals with the quantification and comparison of TP53 transcription levels in esophageal cancer tumor tissue to the TP53 levels in healthy esophageal tissue obtained from patients from a unique geographical and ethnic background. The cohort used in this study consisted of Coloured patients (n=2) treated at Tygerberg Hospital. The LightCycler system was implemented in order to try to accurately quantify TP53 mRNA levels. Unfortunately, the desired results were unattainable due to unforeseen difficulties encountered during the study. These difficulties included the insufficient preservation of samples for RNA based studies. Several recommendations were made concerning future similar studies, including an improved planning strategy as well as the employment of an RNA stabilizing agent. Additionally, a few important contributions were made through this study, including the design and optimization of TP53 primers specifically intended for future RNA studies. These primers would enable the identification of the presence of TP53 RNA species as well as the absence of DNA contamination in a single PCR amplification step. Other contributions include the development of a well-optimized RNA extraction method for the extraction of RNA from tough tissues (such as the human esophageal tissue used in this study). This method makes the extraction of large quantities of RNA from small amounts of tough tissue types possible. In conclusion, this study has made a significant contribution to the field of cancer research, by shedding light on the TP53 mutational spectrum with regards to laryngeal as well as esophageal cancer in a population unique to the Western Cape. The first part of this thesis has been published in Cancer Genetics and Cytogenetics (Barnard, D., K. Lehmann, E.G. Haal, P.O. van Heiden, and l.C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarities to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145:126-132), of which a copy can be found in Appendix I. This work has also been presented (by D. Barnard) at an international conference entitled "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", held in Amsterdam, the Netherlands during the period 13 - 15 December 2002. / AFRIKAANSE OPSOMMING: Die werk wat in hierdie tesis voorgelê word handel oor die assosiasie tussen kankers van die boonste gastrointestinale weg en die tumor suppressor geen, TP53, en kan in 3 dele gedeel word, (i) die analise van die mutasiespektrum van TP53 in laringiale kanker (LK), (ii) die analise van die mutasiespektrum van TP53 in slukderm kanker (SK) en (iii) die analise van die transkripsievlakke van TP53 in SK. Laringeal kanker (LK) is die 6de algemeenste kanker in die wêreld en die 2de algemeenste respiratoriese kanker, met "n benaderde 500 000 nuwe gevalle jaarliks wêreldwyd. Oor die afgelope paar jare het LK "n toenemende probleem geraak, veral in die Kleurling gemeenskap van die Wes Kaap. Die meganismes van die tumorvorming in LK is onbekend, alhoewel rook-en alkoholgebruik vername risiko faktore is. Die voorkoms van mutasies in TP53 is verskeie kere aangetoon in verskillende kanker tipes en daar word vermoed dat dit "n rol speel in tumorvorming. In hierdie studie is dus na mutasies in eksons 5 - 8 van TP53 gesoek in tumor biopsie weefsel (n=44) en bloed isolate (n=42) van Kleurling LK pasiënte d.m.v. polimerase ketting reaksie - enkelstring konformasie polimorfisme (PKR-ESKP) analisering en direkte volgorde bepaling. Bloed monsters van "n vergelykbare groep (n=40) is ook in die studie ingesluit as "n kontrole. Betekenisvolle positiewe korrelasies is gevind tussen die voorkoms van LK en ouderdom sowel as rook. Daarmee saam is daaglikse vleisinname as potensiële beskermende faktor gevind. In tumor biopsies is mutasies in 3 van die ondersoekte eksons gevind, wat 25% van die biopsie monsters verteenwoordig. Hierdie mutasies is uniek aan die tumor biopsie weefsels en dui op "n somatiese oorsprong van mutasies. Hierdie bevindinge bevestig dat die gedeelte tussen kodons 173 - 273 van TP53 "n hipermuteerbare gebied geassosieer met kankers is. Hierdie studie bevestig 6 nuwe mutasies. Daar is 'n hoë insidensie van slukderm kanker (SK) in Suid Afrika relatief tot die res van die wêreld. Hierdie soort kanker word veral gevind by die Swart populasie van die Transkei. Die doel van hierdie studie was om verskille tussen die TP53 mutasie patroon van die Kleurling gemeenskap van die Wes Kaap en die Swart gemeenskap van die Transkei te vergelyk. Hiervoor is die molekulêre struktuur van TP53, veral eksons 5 - 8, in 'n groep Kleurling SK pasiënte (n=42) wat behandel is by Tygerberg Hospitaal, Kaapstad, Suid Afrika, geanaliseer. Analisering is gedoen deur DNS van tumor, bloed en ook oënskynlike gesonde aangrensende weefsel van dieselfde pasiënte te onderwerp aan PKR-ESKP analise en direkte volgorde bepaling. Slegs 4 nukleotied veranderings is gevind in 124 volgorde bepalings, waarvan 2 nuwe veranderings is in SK. Hierdie 4 nukleotied veranderinge verteenwoordig 9% van al die tumors wat ondersoek is in die studie. Hierdie studie bewys dat die mutasiespektrum van TP53 in die Kleurling gemeenskap van die Wes Kaap grootliks verskil van die Swart gemeenskap van die Transkei. Dit impliseer dat verskillende etiologiese faktore moontlik 'n rol mag speel op die tumorvormingsproses in die 2 afsonderlike geografiese gemeenskappe. Hierdie is die onderwerp van 'n epidemiologiese studie wat deur die MNR onderneem word. Die laaste deel van hierdie tesis handel oor die kwantifisering en vergelyking van TP53 transkripsievlakke in SK tumor weefsel teenoor TP53 vlakke in gesonde slukderm weefsel van pasiënte in 'n unieke geografiese en etniese agtergrond. Die studie populasie in hierdie projek het bestaan uit Kleurling pasiënte (n=2) wat by Tygerberg hospitaal behandel is. Die "LightCycler" sisteem is gebruik vir die akkurate kwantifisering van TP53 boodskapper RNS vlakke. Ongelukkig is die verlangde resultate nie gekry nie as gevolg van onvoorsiene probleme wat ondervind is tydens die studie. Hierdie probleme sluit in die onvoldoende preserv RNS studies. Hierdie inleiers maak dit nou moontlik om die teenwoordigheid van TP53 RNS spesies sowel as die afwesigheid van DNS kontaminasie in een PKR amplifikasie stap te kan identifiseer. 'n Ander belangrike bydrae is die ontwikkeling van 'n goed geoptimaliseerde RNS ekstraksie metode vir moeilike starre weelfsel tipes (soos menslike slukderm weefsel in hierdie studie) en maak die ekstraksie van groot hoeveelhede RNS uit klein hoeveelhede van moeilik hanteerbare weefsel tipes moontlik. Om saam te vat, hierdie studie het betekenisvolle bydraes gemaak tot die veld van kankernavorsing deur die ontrafeling van die TP53 mutasiespektrum in beide laringeale sowel as slukderm kanker, in 'n populasie uniek aan die Wes Kaap. Die eerste deel van hierdie tesis is gepubliseer in Cancer Geneties and Cytogenetics (Barnard, D., K. Lehmann, E. G. Hoal, P. D. van Heiden, and T. C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarites to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145: 126-132) en 'n afskrif van die artikel is ingesluit in Appendix I. Hierdie werk is ook voorgedra (deur D. Barnard) by 'n internasionale kongres getiteld "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", wat in Amsterdam, Nederland gehou is gedurende 13 - 15 Desember 2002

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.

published_or_final_version / Pathology / Master / Master of Medical Sciences

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.