<Emphasis Type="Italic">K-ras,BRCA1/2</Emphasis>, and <Emphasis Type="Italic">CHEK2</Emphasis> mutations and loss of heterozygosity at 9p, 17p,and 18q in sporadic adenocarcinoma of the pancreas

The diagnostic significance of molecular markers was assessed for the most common somatic aberrations at the K-ras, TP53, CDKN2A, and MADH4 loci, as well as less common mutations of BRCA1, BRCA2, and CHEK2, arising in preinvasive stages of sporadic adenocarcinoma of the pancreas. The study was performed on paired primary pancreatic adenocarcinoma and normal pancreatic tissue specimens obtained from 37 Russian patients. Surgical adenocarcinoma specimens were subjected to manual microdissection. Mutations of K-ras codon 12 were found in 24 tumor specimens (0.65), but not in normal pancreatic tissue specimens. Mutations of BRCA1 (185delAG, 300T > G, 4153delA, 4158A > G, 5382insC), BRCA2 (695insT, 6174delT), and CHEK2 (1100delC) were not found. The informativeness of allelic losses did not differ significantly among the three tumor suppressor loci and was 60% for TP53 (GDB186817) and CDKN2A (D9S974 + D9S162) and 65.7% for MADH4 (D18S363 + D18S474) (t = 0.48). The CDKN2A locus had the highest LOH frequency of 0.95. For TP53 and MADH4 the LOH frequency was 0.62 and 0.70, respectively. In 80% of adenocarcinomas, at least one locus was characterized with LOH. The overall informativeness of the combined data on K-ras mutations and loss of heterozygosity at 9p, 17p, and 18q was 85.7%. Only 9% of the tumors were characterized with microsatellite instability.     

Mutation analysis of <Emphasis Type="Italic">K-ras</Emphasis> protooncogene in colorectal adenocarcinomas and polyps in Russian patients

To estimate diagnostic value of K-ras mutations during cancer risk group formation, they were studied in the samples of sporadic carcinomas (n = 58), benign (n = 33), and malignant (n = 13) polyps of large intestine obtained during surgery or polypectomy. Using PCR analysis, restriction analysis, SSCP analysis and automated sequencing, eight various point mutations were revealed. Six of them were located in codon 12 and two, in codon 13 of the K-ras gene. Mutation frequency in carcinomas, benign and malignant polyps was 43, 49, and 69%, respectively. In the normal tissue samples of colorectum, no changes in codons 12 and 13 in the K-ras gene were observed. Mutations in the groups of Russian patients examined partially overlapped. In patients with colorectal carcinoma the mutation frequency in the K-ras gene was not associated with disease onset age, location, and the extent of tumor differentiation while it was associated with the stage of tumor process. In polyps, the maximum mutation frequency was revealed among patients over 70 years of age as well as in the adenomas of villous histology and large size (≥1cm). No correlation between the K-ras mutation frequency and the extent of polyp dysplasia was observed.     

Human chromosome 3P regions of putative tumor-suppressor genes in renal, breast, and ovarian carcinomas

Allelic imbalances (AI) of polymorphic markers at the short arm of chromosome 3 (3p) were mapped using DNA samples of renal cell carcinoma (RCC, 80 cases), breast carcinoma (BC, 95 cases), and epithelial ovarian cancer (EOC, 50 cases) at the same dense panel of markers (up to 24 loci). Six regions with the increased AI frequency (versus the average values determined for all the analyzed 3p markers) at RCC, BC or EOC were found in 3p chromosome. Four 3p regions presumably contain tumor-suppressor genes (TSG) involved in the epithelial tumors of various types. Region between D3S2409 and D3S3667 markers in the 3p21.31 region was identified in this study for the first time. The AI peak in D3S2409-D3S3667 region was statistically significant (P < 0.001, according to Fisher) when representative sample set of 95 BC patients was analyzed. The data on increased frequency of polymorphic marker allele amplification suggest that the D3S2409-D3S3667 region contains both putative TSG and protooncogenes.     

Analysis of Mutations of the RET Proto-oncogene in Patients with Medullary Thyroid Carcinoma

The spectrum of mutations of the RET proto-oncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four RET exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six different mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four types of mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of RET mutations from three MEN 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.     

Methylation of the Promoter Region of the RASSF1A Candidate Tumor Suppressor Gene in Primary Epithelial Tumors

The methylation of the promoter CpG island of the RASSF1A tumor suppressor gene in primary tumors of 172 Muscovites with renal cell carcinoma (RCC), breast cancer (BC), or ovarian epithelial tumors (OET) was assayed by means of methylation-specific PCR (MSP) and PCR-based methylation-sensitive restriction enzyme analysis (MSRA). The MSP, MSRA, and previous bisulfite sequencing data correlated significantly with each other (P 10^–6 for Spearman's rank correlation coefficients). By MSP and MSRA, the respective methylation frequencies of the RASSF1A promoter were 86% (25/29) and 94% (50/53) in RCC, 64% (18/28) and 78% (32/41) in BC, and 59% (17/29) and 73% (33/45) in OET. Methylation-sensitive restriction enzymes (HpaII, HhaI, Bsh1236I, AciI) increased the analysis sensitivity and made it possible to establish the methylation status for 18 CpG dinucleotides of the RASSF1A promoter region. With the MSRA data, the density of methylation of the CpG island was estimated at 72% in RCC, 63% in BC, and 58% in OET (the product of the number of CpG dinucleotides and the number of specimens with RASSF1A methylation was taken as 100%). Methylation of the RASSF1A promoter region was observed in 11–35% of the DNA specimens from the histologically normal tissue adjacent to the tumor but not in the peripheral blood DNA of 15 healthy subjects. The RASSF1A methylation frequency showed no significant correlation with the stage, grade, and metastatic potential of the tumor. On the other hand, epigenetic modification of RASSF1A was considerably more frequent than hemizygous or homozygous deletions from the RASSF1A region. These results testify that methylation of the RASSF1A promoter region takes place early in carcinogenesis and is a major mechanism inactivating RASSF1A in epithelial tumors.     

Methylation of promoter region of <Emphasis Type="Italic">RAR</Emphasis>-β2 gene in renal cell,breast, and ovarian carcinomas

The protein encoded by RAR-β2 (retinoic acid receptor) gene is a member of the superfamily, of nuclear receptors of retinoids which are involved in regulation of cell differentiation and proliferation. The level of RAR-β2 mRNA is downregulated in a number of cell lines derived from human epithelial tumors. Inactivation of the RAR-β2 gene is associated with methylation of its promoter region, which is observed in various carcinomas at a frequency of 30–70%. In renal and ovarian tumors, methylation at this region is poorly studied, the data being contradictory. We report a high methylation frequency in the gene promoter region in RCC (59%, 36/61) and a somewhat lower frequency in EOC (30%, 15/50). Methylation frequency in BC (46%, 26/56) is consistent with the published data. Significant correlation of methylation frequency in promoter region of RAR-β2 gene with RCC progression (P ≤ 0.005 by Fisher’s exact test) was established.     

Identification of Hereditary Variants of Cutaneous Melanoma

The identification of hereditary variants of cutaneous melanoma and analysis of the role of hereditary factors and syndromes predisposing to cutaneous melanoma were carried out. The involvement of individual nevus phenotypes in the development of this disease was determined. Based on a survey of recent molecular biological data and our studies, the etiological and genetic heterogeneity of cutaneous melanoma is reported. In relatives of patients with cutaneous melanoma and persons with multiple pigmented nevi, the malignant tumors proved to be differentiated in the direction of the neural crist tissues and/or derivatives of cutaneous mesenchyma. Based on the evidence obtained, the approaches have been developed to formation of risk groups for the purpose of early diagnostics of cutaneous melanoma.     

Rapid detection of regionally clustered germ-line BRCA1 mutations by multiplex heteroduplex analysis. UKCCCR Familial Ovarian Cancer Study Group.

Germ-line mutations of the BRCA1 gene are responsible for a substantial proportion of families with multiple cases of early-onset breast and/or ovarian cancer. Since the isolation of BRCA1 last year, >65 distinct mutations scattered throughout the coding region have been detected, making analysis of the gene time consuming and technically challenging. We have developed a multiplex heteroduplex analysis that is designed to analyze one-quarter of the coding sequence in a single-step screening procedure and that will detect approximately 50% of all BRCA1 mutations so far reported in breast/ovarian cancer families. We have used this technique to analyze BRCA1 in 162 families with a history of breast and/or ovarian cancer and identified 12 distinct mutations in 35 families.