Charles H.C.M. Buys

Deletions of the Short Arm of Chromosome 3 in Solid Tumors and the Search for Suppressor Genes

The concept that cells can become malignant upon the elimination of parts of chromosomes inhibiting cell division dates back to Boveri in 1914. Deletions occurring in tumor cells are therefore considered a first indication of possible locations of tumor suppressor gene. Approaches used to localize and identify the paradigm of tumor suppressors, RB1, have also been applied to localize tumor suppressor genes on 3p, the short arm of chromosome 3. This review discusses the methodological advantages and limitations of the various approaches. From a review of the literature on losses of 3p in different types of solid tumors it appears that some tumor types show involvement of the same region, while between others the regions involved clearly differ. Also discussed are results of functional assays of tumor suppression by transfer of part of chromosome 3 into tumor cell lines. The likelihood that a common region of deletions would contain a tumor suppressor is strongly enhanced by coincidence of that region with a chromosome fragment suppressing tumorigenicity upon introduction in tumor cells. Such a situation exists for a region in 3p21.3 as well as for one or more in 3p12-p14. The former region is considered the location of a lung cancer suppressor. The same gene or a different one in the same region may also play a role in the development of other cancers including renal cell cancer. In the latter cancer, there may be additional roles of the VHL region and/or a 3p12-p14 region. The breakpoint region of a t(3;8) originally found to be constitutively present in a family with hereditary renal cell cancer now seems to be excluded from such a role. Specific genes on 3p have been suggested to act as suppressor genes based on either their location in a common deletion region, a markedly reduced expression or presence of aberrant transcripts, their capacity to suppress tumorigenicity upon transfection in to tumor cells, the presumed function of the gene product, or a combination of several of these criteria. A number of genes are evaluated for their possible role as a tumor suppressor according to these criteria. General agreement on such a role seems to exist only for VHL. Though hMLH1 plays an obvious role in the development of specific mismatch repair-deficient cancers, it cannot revert the tumor phenotype and therefore cannot be considered a proper tumor suppressor. The involvement of VHL and MLH1 also in some specific hereditary cancers allowed to successfully apply linkage analysis for their localization. TGFBR2 might well have a tumor suppressor function. It does reduce tumorigenicity upon transfection. Other 3p genes coding for receptor proteins THRB and RARB, are unlikely candidates for tumor suppression. Present observations on a possible association of FHIT with tumor development leave a number of questions unanswered, so that provisionally it cannot be considered a tumor suppressor. Regions that have been identified as crucial in solid tumor development appear to be at the edge of synteny blocks that have been rearranged through the chromosome evolution which led to the formation of human chromosome 3. Although this may merely represent a chance occurrence, it might also reflect areas of genomic instability.

Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) (Amsterdam criteria) is often caused by mutations in mismatch repair (MMR) genes, and tumors of patients with HNPCC show microsatellite instability (MSI-high phenotype). Germline mutations of MMR genes have rarely been found in families that have HNPCC or suspected HNPCC and that do not show microsatellite instability (MSI-low phenotype). Therefore, an MSI-high phenotype is often used as an inclusion criterion for mutation testing of MMR genes. Correction of base-base mismatches is the major function of MSH6. Since mismatches present with an MSI-low phenotype, we assumed that the phenotype in patients with HNPCC-related tumors might be associated with MSH6 germline mutations. We divided 36 patients with suspected HNPCC into an MSI-low group (n=18) and an MSI-high group (n=18), on the basis of the results of MSI testing. Additionally, three unrelated patients from Amsterdam families with MSI-low tumors were investigated. All patients were screened for MSH2, MLH1, and MSH6 mutations. Four presumably causative MSH6 mutations were detected in the patients (22%) who had suspected HNPCC and MSI-low tumors. Furthermore, we detected one frameshift mutation in one of the three patients with HNPCC and MSI-low tumors. In the MSI-high group, one MSH6 missense mutation was found, but the same patient also had an MLH1 mutation, which may explain the MSI-high phenotype. These results suggest that MSH6 may be involved in a substantial proportion of patients with HNPCC or suspected HNPCC and MSI-low tumors. Our data emphasize that an MSI-low phenotype cannot be considered an exclusion criterion for mutation testing of MMR genes in general.

Molecular and Clinical Characteristics of MSH6 Variants: An Analysis of 25 Index Carriers of a Germline Variant

The MSH6 gene is one of the mismatch-repair genes involved in hereditary nonpolyposis colorectal cancer (HNPCC). Three hundred sixteen individuals who were known or suspected to have HNPCC were analyzed for MSH6 germline mutations. For 25 index patients and 8 relatives with MSH6 variants, molecular and clinical features are described. For analysis of microsatellite instability (MSI), the five consensus markers were used. Immunohistochemical analysis of the MLH1, MSH2, and MSH6 proteins was performed. Five truncating MSH6 mutations, of which one was detected seven times, were found in 12 index patients, and 10 MSH6 variants with unknown pathogenicity were found in 13 index patients. Fourteen (54%) of 26 colorectal cancers (CRCs) and endometrial cancers showed no, or only weak, MSI. Twelve of 18 tumors of truncating-mutation carriers and 3 of 17 tumors of missense-mutation carriers showed loss of MSH6 staining. Six of the families that we studied fulfilled the original Amsterdam criteria; most families with MSH6, however, were only suspected to have HNPCC. In families that did not fulfill the revised Amsterdam criteria, the prevalence of MSH6 variants is about the same as the prevalence of those in MLH1/MSH2. Endometrial cancer and/or atypical hyperplasia were diagnosed in 8 of 12 female carriers of MSH6 truncating mutations. Most CRCs were localized distally in the colon. Although, molecularly, missense variants are labeled as doubtfully pathogenic, clinical data disclose a great resemblance between missense-variant carriers and truncating-mutation carriers. We conclude that, in all patients suspected to have HNPCC, MSH6-mutation analysis should be considered. Neither MSI nor immunohistochemistry should be a definitive selection criterion for MSH6-mutation analysis.

Segregation at three loci explains familial and population risk in Hirschsprung disease

Hirschsprung disease (HSCR), the most common hereditary cause of intestinal obstruction, shows considerable variation and complex inheritance. Coding sequence mutations in RET, GDNF, EDNRB, EDN3 and SOX10 lead to long-segment (L-HSCR) and syndromic HSCR but fail to explain the transmission of the much more common short-segment form (S-HSCR). We conducted a genome scan in families with S-HSCR and identified susceptibility loci at 3p21, 10q11 and 19q12 that seem to be necessary and sufficient to explain recurrence risk and population incidence. The gene at 10q11 is probably RET, supporting its crucial role in all forms of HSCR; however, coding sequence mutations are present in only 40% of linked families, suggesting the importance of noncoding variation. Here we show oligogenic inheritance of S-HSCR, the 3p21 and 19q12 loci as RET-dependent modifiers, and a parent-of-origin effect at RET. This study demonstrates by a complete genetic dissection why the inheritance pattern of S-HSCR is nonmendelian.

A role for MLH3 in hereditary nonpolyposis colorectal cancer.

We investigated a possible role of the mismatch-repair gene MLH3 in hereditary nonpolyposis colorectal cancer by scanning for mutations in 39 HNPCC families and in 288 patients suspected of having HNPCC. We identified ten different germline MLH3 variants, one frameshift and nine missense mutations, in 12 patients suspected of HNPCC. Three of the 12 also carried a mutation in MSH6.

MSH2 and MLH1 mutations in sporadic replication error-positive colorectal carcinoma as assessed by two-dimensional DNA electrophoresis

Replication errors (RER) are frequently seen in both sporadic and hereditary forms of colorectal cancer. In hereditary nonpolyposis colorectal cancer (HNPCC), RER is associated with defects in DNA mismatch repair genes. Two of these genes, MSH2 and MLH1, account for a major share of this cancer syndrome. In order to assess the role of these genes in sporadic RER+ colorectal carcinoma, we have carried out a mutation analysis of MSH2 and MLH1 by two‐dimensional (2‐D) DNA electrophoresis, including heteroduplexing and separation in a denaturing gradient. All exons were amplified using multiplex PCR and were separated on the basis of both size and base pair composition under a single set of experimental conditions. Exons showing a spot position different from normal were sequenced. In screening 33 unselected, sporadic RER+ colorectal tumors, a germline mutation accompanied by loss of heterozygosity in tumor tissue was found in two patients. They were among the 4 patients out of the 33 screened that were diagnosed before the age of 50 years. In 8 of the remaining 31 tumors (26%), presence of somatic mutations (9 in total) could be demonstrated. While suggesting involvement of other genes in a substantial part of sporadic RER+ colorectal carcinomas, our results also demonstrate a clear role of MSH2 and MLH1 in these sporadic tumors and show that young sporadic RER+ colorectal carcinoma patients have a high probability of germline mutations. This has important implications for genetic testing and management of young colorectal cancer patients and their families. Genes Chromosom. Cancer 18:269–278, 1997.

Toward New Strategies to Select Young Endometrial Cancer Patients for Mismatch Repair Gene Mutation Analysis

PURPOSE To determine the frequency of mismatch repair (MMR) gene germline mutations in endometrial cancer patients who were diagnosed at less than 50 years of age; to relate the presence of mutations to family history, histopathologic data, presence of tumor microsatellite instability (MSI), and immunostaining; and to formulate criteria for genetic testing in these patients. PATIENTS AND METHODS Endometrial cancer patients (N = 58), who were diagnosed at less than 50 years of age, were included and questioned about their family history. Mutation analysis of the MLH1, MSH2, and MSH6 genes was performed (denaturing gradient gel electrophoresis and sequence analysis to detect small mutations and multiplex ligation-dependent probe amplification to detect large deletions or duplications). For MSI analysis, five consensus markers were used, and immunostaining of the three MMR proteins was performed. RESULTS In five of 22 patients with a positive first-degree family history for hereditary nonpolyposis colorectal cancer (HNPCC)-related cancers, pathogenic germline mutations were found (one MLH1, three MSH2, and one MSH6). Four mutation carriers belonged to families fulfilling the revised Amsterdam criteria. No mutations were found in the 35 patients without such family history (P =.006). MSI was detected in 20 of 57 cancers, among which four were from mutation carriers. In 23 of 51 cancers, one or more MMR protein was absent; in all five mutation carriers, immunostaining indicated the involved MMR gene. CONCLUSION In 23% of the young endometrial cancer patients with at least one first-degree relative with an HNPCC-related cancer, an MMR gene mutation was detected. Therefore, presence of an HNPCC-related cancer in a first-degree relative seems to be an important selection criterion for mutation analysis. Subsequent immunostaining of MMR proteins will point to the gene(s) that should be analyzed.

Involvement of multiple loci on chromosome 3 in renal cell cancer development

In renal cell carcinoma (RCC), mostly occurring as sporadic cases, the short arm of chromosome 3 is a frequent target of deletion events. Taking into account cytological classifications of RCC, the deletions appear to be characteristic of clear cell or nonpapillary RCC only. This subtype constitutes most sporadic RCC and RCC as part of the Von Hippel‐Lindau disease caused by germline mutations of VHL at 3p25. In a proportion of sporadic tumours, somatic mutations of VHL occur, again only in clear cell or nonpapillary RCC. However, in a sizable proportion of sporadic clear cell RCC, VHL mutations are absent. Therefore, a role for VHL in RCC development in general seems unlikely. Familial papillary RCC is not linked to chromosome 3. A rat model of hereditary RCC, the EKER rat, is associated with a germline mutation of the rat homologue of the tuberous sclerosis gene on human chromosome 16. Analysis of allelic losses of chromosome 3 in 143 highly informative sporadic tumours published in the literature points to a small segment of 3p21.3 as a candidate tumour‐suppressor region. A 2‐Mb fragment containing this segment suppresses tumourigenicity when present in mouse fibrosarcoma cells. A similar effect could be attributed to the region 3p12‐p14 on the basis of results from its introduction into an RCC cell line. The responsible gene should not be sought at 3p14 translocation breakpoints segregating with RCC in a few rare families because there is evidence that RCC in these cases is due to events involving VHL or another gene distal to the breakpoint. FHIT is also an unlikely candidate according to observations comparing RCC and a variety of normal tissues. Results of an analysis of sporadic patients with multiple renal tumours indicate an association of allelic losses of the VHL and 3p12‐p14 regions with adenomas and suggest that losses of the 3p21 region are necessary for malignant development to clear cell or nonpapillary RCC. Genes Chromosom. Cancer 19:59–76, 1997.

Determination of TP53 Mutation Is More Relevant Than Microsatellite Instability Status for the Prediction of Disease-Free Survival in Adjuvant-Treated Stage III Colon Cancer Patients

PURPOSE Microsatellite instability (MSI), TP53 mutation, and KRAS mutation status have been reported as prognostic factors in colon cancer. Most studies, however, have included heterogeneous groups of patients with respect to cancer stage. We determined the prognostic relevance of high-frequency MSI (MSI-H), TP53 mutations, and KRAS mutations in a well-defined group of patients with stage III colon cancer (N = 391), randomly assigned for adjuvant treatment with fluorouracil-based chemotherapy. METHODS Three hundred ninety-one tumor specimens were available. MSI was determined in 273 specimens, and mutation analyses of TP53 and KRAS were performed in 220 and 205 specimens, respectively. RESULTS In a univariate analysis, MSI-H (44 of 273; 16%) was associated with a longer disease-free survival (DFS; P = .038), but in a multivariate model adjusting for nodal involvement, histology, invasion, and grade of tumor, the association of MSI status with DFS did no longer reach statistical significance, though the risk estimate for microsatellite stability versus MSI-H tumors did not change much. Mutant TP53, found in 116 (53%) of 220 tumors, was associated with a shorter DFS, both in univariate (P = .009) and multivariate analyses (P = .018), whereas KRAS mutations (58 of 205; 28%) did not show any prognostic significance. CONCLUSION Both mutant TP53 and MSI-H seem to be prognostic indicators for disease-free survival, but only TP53 retains statistical significance after adjusting for clinical heterogeneity. Thus, in adjuvantly treated patients with stage III colon cancer, presence or absence of a TP53 mutation should be considered as a better predictor for DFS than MSI status.

Abundance of protein-bound sulfhydryl and bisulfide groups at chromosomal nucleolus organizing regions

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.