Monoallelic mutation analysis (MAMA) for identifying germline mutations.

Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC).

Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers.

Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.

Genetic mapping of a locus predisposing to human colorectal cancer.

Genetic linkage analysis was used to determine whether a specific chromosomal locus could be implicated in families with a history of early onset cancer but with no other unique features. Close linkage of disease to anonymous microsatellite markers on chromosome 2 was demonstrated in two large kindreds. The pairwise lod scores for linkage to marker D2S123 in these kindreds were 6.39 and 1.45 at zero recombination, and multipoint linkage with flanking markers resulted in lod scores of 6.47 and 6.01. These results prove the existence of a genetically determined predisposition to colorectal cancer that has important ramifications for understanding and preventing this disease.

Clues to the pathogenesis of familial colorectal cancer.

A predisposition to colorectal cancer is shown to be linked to markers on chromosome 2 in some families. Molecular features of "familial" cancers were compared with those of sporadic colon cancers. Neither the familial nor sporadic cancers showed loss of heterozygosity for chromosome 2 markers, and the incidence of mutations in KRAS, P53, and APC was similar in the two groups of tumors. Most of the familial cancers, however, had widespread alterations in short repeated DNA sequences, suggesting that numerous replication errors had occurred during tumor development. Thirteen percent of sporadic cancers had identical abnormalities and these cancers shared biologic properties with the familial cases. These data suggest a mechanism for familial tumorigenesis different from that mediated by classic tumor suppressor genes.

Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.

Endometrial carcinoma is the second most common tumor type in women with hereditary nonpolyposis colorectal carcinoma. Microsatellite instability (MI) has been observed in the inherited (hereditary nonpolyposis colorectal carcinoma-associated) form of endometrial carcinoma as well as in approximately 20% of presumably sporadic cases. Recent studies suggest that MI in many cell lines or xenografts derived from sporadic colorectal carcinomas is not attributable to mutations in four known human DNA mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, and hPMS2). Mutational analyses of these four MMR genes in endometrial carcinomas have not been previously reported. We analyzed nine sporadic MI-positive primary endometrial carcinomas for mutations in the above four MMR genes. Mutations were detected in two tumors (in hMSH2), and both of the mutations were acquired somatically. Immunohistochemical staining revealed a lack of expression of hMSH2 protein in the two tumors containing hMSH2 mutations. Our data suggest that mutations in these four known DNA MMR genes are not responsible for MI in the majority of sporadic endometrial carcinomas displaying this phenotype.

p53 Mutation and MDM2 amplification in human soft tissue sarcomas.

The p53 and MDM2 genes were analyzed in 24 human soft tissue sarcomas (11 malignant fibrous histiocytomas and 13 liposarcomas). Alterations of p53, consisting of point mutations, deletions, or overexpression, were detected in one-third (8 of 24) of the sarcomas. MDM2 gene amplification was detected in another 8 tumors, but no tumor contained an alteration of both genes. Monoclonal antibodies reactive with the human MDM2 gene product were developed, and immunohistochemical analysis revealed nuclear localization and overexpression of MDM2 in those tumors with amplified MDM2 genes. These data support the hypothesis that p53 and MDM2 genetic alterations are alternative mechanisms for inactivating the same regulatory pathway for suppressing cell growth.

Evaluation of the FHIT gene in colorectal cancers.

A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.

Amplification of cyclin genes in colorectal carcinomas.

The genetic status of cyclin genes was examined in a panel of 47 colorectal carcinoma cell lines. Cyclin D2 was found to be amplified in one tumor and cyclin E in another. In each of the two cases, the amplified cyclin gene was overexpressed at the protein or mRNA level. Cyclin D1, previously shown to be amplified in breast and other tumors, was not amplified in these cancers. These data suggest that a variety of cyclin genes can play a role in human tumorigenesis and that cyclins D2 and E are particularly important in a subset of colorectal neoplasms.

Expression of the human mismatch repair gene hMSH2 in normal and neoplastic tissues.

Hereditary nonpolyposis colorectal cancer is caused by inherited mutations of mismatch repair genes. We developed monoclonal antibodies to the prototype human mismatch repair gene hMSH2 and used them to detect an immunoreactive protein of M(r) 100,000 in mismatch-proficient cell lines. In addition, a M(r) 150,000 protein coimmunoprecipitated with the hMSH2 gene product in cell lines expressing hMSH2. Immunohistochemistry demonstrated that the hMSH2 protein was exclusively nuclear. Whereas the hMSH2 protein was expressed in a variety of tissues, the most striking pattern was observed in esophageal and intestinal epithelia, where expression was limited to the replicating compartment. Neoplastic cells within benign and malignant mismatch repair-proficient tumors expressed the protein, but no hMSH2 immunoreactivity was observed in the colorectal tumors of patients with germline hMSH2 mutation. These results have implications for tumorigenic mechanisms and, potentially, for diagnosis.

[Immunohistochemical and molecular analyis of mismatch repair genes in germ cell tumors]. / Análisis inmunohistoquimico y molecular de los genes de reparación en cáncer testicular.

UNLABELLED: Correction of misincorporated nucleotides during DNA replication (mismatch repair) distinguishes histologically similar cancers with distinct biological and clinical behavior. We investigated expression of two mismatch repair genes in testis cancer to determine the expression pattern in histologically distinct subtypes, correlate expression with genetic instability and correlate expression and genetic instability with clinical outcome. PATIENTS AND METHODS: 118 cases of testis cancer were analyzed. Immunohistochemical analysis of paraffin embedded specimens utilized monoclonal antibody for hMLH1 and hMSH2 mismatch repair proteins. Genetic instability was determined by comparing genomic DNA from microdissected matched normal and tumor cells. PCR amplification of 10 genetic markers assessed loss of heterozygosity and/or microsatellite instability. RESULTS: hMSH2 staining was associated with pathologic stage (p < 0.001) while hMLH1 staining was associated with cancer specific survival (p = 0.036). Genetic instability was detected in 94% of low hMLH1 and 92% of low hMSH2 staining tumors. Relapse and cancer specific death correlated with genetic instability (p = 0.01 and 0.04 respectively). Overall 9% of tumors exhibited reduced mismatch repair expression, microsatellite instability and an unfavorable clinical outcome. CONCLUSIONS: Mismatch repair expression and genetic instability define testis cancers with distinct molecular properties and clinical behavior. In conjunction with pathologic examination and serum tumor markers, mismatch repair expression may be an important determinant for clinical management of men with this malignancy.

Laparoscopic creation of a catheterizable cutaneous ureterovesicostomy.

Nephrectomy and creation of a cutaneous ureterovesicostomy for intermittent catheterization of the bladder traditionally requires two surgical procedures performed through separate incisions. Herein we report completion of these procedures using a transperitoneal laparoscopic approach, with the ureterovesicostomy stoma created at one of the laparoscopic working ports. The clinical course was remarkable for a shortened postoperative hospitalization (48 hours) with minimal incisional pain, and an excellent long-term result with complete bladder emptying and resolution of urinary infections. Laparoscopic application of the Mitrofanoff principle for creation of a catheterizable cutaneous ureterovesicostomy combines the advantages of both, allowing optimal preservation of ureteral vascularity, minimal morbidity, and efficient bladder evacuation.

Regulation of cytomegalovirus late-gene expression: differential use of three start sites in the transcriptional activation of ICP36 gene expression.

We have investigated the transcriptional regulation of the human cytomegalovirus gamma gene encoding the ICP36 family (p52, the major late DNA-binding protein). The ICP36 transcription unit initiates at three distinct sites which are separated by approximately 50 nucleotides and are differentially regulated during infection. At early times (8 h postinfection), only two of these start sites, the most proximal and distal site, were active whereas at late times (36 h postinfection), the middle start site was activated. Expression from this late start site was dependent upon DNA replication. Consensus TATA elements were located upstream of all three start sites, although the element upstream of the late start site was unusual in both sequence and position when compared with conventional TATA elements. Deletion analysis was used in conjunction with transient assays to define independent promoters in this region. The two early start sites and associated TATA elements functioned as separable independently regulated promoters. The region containing the late start site and TATA element but excluding either of the flanking TATA elements was inactive in transient assays. Our work establishes that the ICP36 gene is under complex early and late transcriptional regulation and that the sequences regulating transcriptional activation are temporally and spatially distinct.

Regulation of cytomegalovirus late gene expression: gamma genes are controlled by posttranscriptional events.

We investigated the control of human cytomegalovirus (CMV) late (gamma)-gene expression in human fibroblast cells. Transcriptional activity of two gamma genes, encoding ICP27, a structural component (matrix or tegument) of virions, and ICP36, a major DNA-binding protein family, was followed by analysis of steady-state RNA levels during viral infection. Synthesis of the protein products of these genes was analyzed with specific monoclonal antibodies in conjunction with sensitive immunoblot or immunoprecipitation analysis. Although accumulation of ICP27 and ICP36 was not abundant until late times, both late genes were as transcriptionally active at early times (4 h postinfection) as at late times (48 h postinfection). Reduced amounts (less than 5% of late levels) of the protein products were detected at early times, demonstrating that a small proportion of the ICP27 and ICP36 RNA made at this time was translated. These observations establish that expression of at least two CMV gamma genes is regulated through posttranscriptional events. The very early transcriptional activation of late genes and the relative importance of posttranscriptional regulation to late-gene expression distinguishes CMV from other well-studied herpesviruses and does not appear analogous to late-gene regulation in any other DNA animal virus.

The mismatch repair gene hMSH2 is mutated in the prostate cancer cell line LNCaP.

PURPOSE: Mismatch repair genes are responsible for the coordinated correction of misincorporated nucleotides formed during DNA replication. Inactivating and inherited mutations in the prototypic mismatch repair gene hMSH2 have been described in a cancer predisposition syndrome known as hereditary nonpolyposis colon cancer. Patients with hereditary nonpolyposis colon cancer are at increased risk for colon cancer and extracolonic cancers such as upper tract transitional cell carcinoma but not prostate cancer. We investigated expression of hMSH2 in prostate cancer cell lines using genetic and molecular analysis. MATERIALS AND METHODS: We used the 3 well described prostate cancer cell lines, DU145, LNCaP and PC3. Western blot analysis with monoclonal antibody to hMSH2 was used to assess expression. Southern blot and polymerase chain reaction of genomic DNA were used to identify genetic alterations in the hMSH2 gene. Single cell cloning, dinucleotide repeats and BAT-26 were used to assess the cell lines for microsatellite instability. RESULTS: The prostate cancer cell line LNCaP did not express hMSH2 and was found to have a homozygous deletion of hMSH2 exons 9 to 16, resulting in truncation of the protein. While microsatellite analysis did not reveal alterations at the BAT-26 locus, single cell cloning produced several LNCaP subclones with alteration at 1 dinucleotide repeat. CONCLUSIONS: The well described prostate cancer cell line LNCaP has a mutation in the hMSH2 gene, resulting in loss of expression and possible evidence of microsatellite instability. To our knowledge our finding is the first demonstration of a genetic alteration in hMSH2 in a prostate cancer cell line.

Expression of the human mismatch repair gene hMSH2: a potential marker for urothelial malignancy.

BACKGROUND: The human mismatch repair (MMR) gene hMSH2 (human mutS homolog-2) is a DNA repair gene that has been reported to be mutated in 40% of hereditary nonpolyposis colon cancer (HNPCC) kindreds and a small percentage of sporadic tumors. HNPCC is a cancer predisposition syndrome with an increased risk of carcinoma of the colon, endometrium, stomach, small intestine, ovary, ureter, and renal pelvis. Immunohistochemical analysis demonstrated increased hMSH2 expression in sporadic colon carcinoma and in the replicative compartment of normal epithelium. A recent immunohistochemical analysis of hMSH2 in bladder tumors correlated reduced hMSH2 expression with recurrence and higher tumor grade. In the current study, we examined hMSH2 expression in urothelial malignancy using immunohistochemical analysis and developed a molecular assay for the detection of hMSH2 expression in bladder washes. METHODS: Immunohistochemical analysis of 17 tumors from the genitourinary tract and reverse transcription coupled with polymerase chain reaction (RT-PCR) of 40 bladder washes were used to investigate hMSH2 expression in noninvasive and invasive urothelial malignancies. RESULTS: Increased expression of hMSH2 was detected in all tumors examined using immunohistochemical analysis independent of grade or stage. Reverse transcription-PCR of hMSH2 mRNA from bladder washes detected 17 of 21 patients with primary or recurrent urothelial neoplasms or tumors involving the urothelial system. Four patients with urothelial malignancies without detectable hMSH2 expression from their bladder washes had high grade lesions. Ten of 13 patients without pathologic or cystoscopic evidence of bladder tumors were negative for hMSH2 expression in bladder washes. Two patients with bladder tumors and bladder washes that were positive for hMSH2 subsequently were found to be negative for hMSH2 after treatment of their tumors and at last follow-up had remained recurrence free for at least 1 year. CONCLUSIONS: The results of the current study suggest that hMSH2 expression is increased in low and high grade urothelial neoplasms, similar to the expression pattern in sporadic colon carcinoma. However, a fraction of high grade lesions may not express hMSH2 as detected by RT-PCR from bladder washes. The ability to detect hMSH2 expression in bladder washes may allow the use of hMSH2 expression as a marker for urothelial malignancy. In addition, the ability to define hMSH2 deficient tumors using bladder washes may have prognostic significance in the treatment of patients with urothelial carcinoma.

Mismatch repair genes on chromosomes 2p and 3p account for a major share of hereditary nonpolyposis colorectal cancer families evaluable by linkage.

Two susceptibility loci for hereditary nonpolyposis colorectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.

Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.

Recent studies have shown that a locus responsible for hereditary nonpolyposis colorectal cancer (HNPCC) is on chromosome 2p and that tumors developing in these patients contain alterations in microsatellite sequences (RER+ phenotype). We have used chromosome microdissection to obtain highly polymorphic markers from chromosome 2p16. These and other markers were ordered in a panel of somatic cell hybrids and used to define a 0.8 Mb interval containing the HNPCC locus. Candidate genes were then mapped, and one was found to lie within the 0.8 Mb interval. We identified this candidate by virtue of its homology to mutS mismatch repair genes. cDNA clones were obtained and the sequence used to detect germline mutations, including those producing termination codons, in HNPCC kindreds. Somatic as well as germline mutations of the gene were identified in RER+ tumor cells. This mutS homolog is therefore likely to be responsible for HNPCC.

Análisis inmunohistoquimico y molecular de los genes de reparación en cáncer testicular / Immunohistochemical and molecular analysis of mismatch repair genes in germ cell tumors

La corrección de los nucleótidos mal incorporados durante la replicación del ADN (sistema de reparación de genes) puede determinar un potencial biológico y clínico diferente en los tumores. En este trabajo investigamos la expresión inmunohistoquímica de los genes de reparación en cáncer de testículo en los distintos tipos histológicos, correlacionando el grado de expresión con la presencia de inestabilidad microsatélite y correlacionando ambas con la evolución clínica. Pacientes y método: 118 casos de tumores testiculares fueron analizados molecularmente realizando inmunohistoquímica para hMLH1 y hMSH2. El análisis de inestabilidad microsatélite y LOH se realizó comparando ADN micro disecado de tejido tumoral y normal, el que fue amplificado mediante PCR con 10 marcadores preestablecidos. Resultados: El grado de expresión de hMSH2 se correlaciona con el estadio del tumor (p<0,001) mientras que la sobreviva cáncer específica se correlaciona con el grado de expresión de hMLH1 (p=0,036). Inestabilidad microsatélite se detectó en un 94% y 92% de los cánceres con baja expresión de hMLH1 y hMSH2 respectivamente. La recaída y muerte cáncer específica se correlacionan con el grado de inestabilidad genética (p=0,01, p=0,04). Un 9% de los tumores presentan una bajo grado de expresión de los genes de reparación, inestabilidad microsatélite y un mal pronóstico. Conclusiones: El grado de expresión de los genes de reparación así como la frecuencia de inestabilidad logran definir cánceres testiculares con diferentes propiedades moleculares y diferente pronóstico. Los genes de reparación usados en conjunto con la histología, los marcadores serológicos pueden ser determinantes en el manejo de los pacientes con tumores testiculares (AU)

Correction of misincorporated nucleotides during DNA replication (mismatch repair) distinguishes histologically similar cancers with distinct biological and clinical behavior. We investigated expression of two mismatch repair genes in testis cancer to determine the expression pattern in histologically distinct subtypes, correlate expression with genetic instability and correlate expression and genetic instability with clinical outcome. Patients and methods: 118 cases of testis cancer were analyzed. Immunohistochemical analysis of paraffin embedded specimens utilized monoclonal antibody for hMLH1 and hMSH2 mismatch repair proteins. Genetic instability was determined by comparing genomic DNA from microdissected matched normal and tumor cells. PCR amplification of 10 genetic markers assessed loss of heterozygosity and/or microsatellite instability. Results: hMSH2 staining was associated with pathologic stage (p<0.001) while hMLH1 staining was associated with cancer specific survival (p=0.036). Genetic instability was detected in 94% of low hMLH1 and 92% of low hMSH2 staining tumors. Relapse and cancer specific death correlated with genetic instability (p=0.01 and 0.04 respectively). Overall 9% of tumors exhibited reduced mismatch repair expression, microsatellite instability and an unfavorable clinical outcome. Conclusions: Mismatch repair expression and genetic instability define testis cancers with distinct molecular properties and clinical behavior. In conjunction with pathologic examination and serum tumor markers, mismatch repair expression may be an important determinant for clinical management of men with this malignancy (AU)