[Mutation analysis of K-ras 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 = 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 healthy tissue of the large intestine, 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. The maximum mutation frequency was revealed in polyps of patients over 70 years of age as well as in the adenomas of villous histology and large size ((1 cm). No correlation between the K-ras mutation frequency and the extent of polyp dysplasia was observed.

[Analysis of K-ras, BRCA1/2, CHEK2 mutations and microsatellite markers (loss of heterozygosity at 9p, 17p and 18q) in sporadic pancreas adenocarcinomas].

The purpose of this study was to investigate informativety and clinical significance of most frequent somatic alterations in K-ras, TP53, CDKN2A, MADH4 and more uncommon mutations in BRCA1, BRCA2, CHEK2 genes, which arise on preinvasive stage in sporadic pancreatic adenocarcinomas (PA), in Russian patients. We examined surgically resected and manually microdissected primary PA tissue samples and samples of normal pancreatic tissue for 37 individuals. K-ras mutations in codon 12 were found in 24 tumors (0.65) and none of normal tissue samples. No mutations were detected in BRCA1(185delAG, 300T > G, 4153delA, 4158A > G,5382insC), BRCA2 (695insT, 6174delT) and CHEK2 (1100delC) genes. Informativety for allelic loss of three tumor suppressor genes studied had not statistically significant differences: 60% - for TP53 (GDB186817) and CDKN2A (D9S974 + D9S162); and 65.7% - for MADH4 (D18S363 + D18S474) (t = 0.48). Maximal frequency of loss of heterozygosity (LOH) was observed for CDKN2A - 0.95. For TP53 and MADH4 it was 0.62 and 0.70 respectively. The tumors included 80% cases showing LOH on different chromosomal loci. The combination of K-ras mutations (c.12) and LOH at 9p, 17p and 18q resulted in a high informativety of selected molecular markers: 85.7%. Instability of microsatellites was found only in 9% of PA.

[Methylation of promoter region of RASSF1A gene and frequencies of allelic imbalances in chromosome 3 critical regions are correlated with progression of clear cell renal cell carcinoma].

The short arm of chromosome 3 (3p) contains several critical regions harboring the set of genes with tumor suppressor activities. The RASSF1A gene (LUCA region, 3p21.31) shows various functions which can be associated with tumorigenesis. Among 3p genes this gene can be most frequently methylated in epithelial tumors of various locations. Here two independent methods (methyl-specific PCR and methyl-sensitive restriction analysis) were used to show significant correlation of methylation level of promoter region of this gene with grade and clinical stage of clear cell renal cell carcinoma (RCC) for the first time. Analysis of 23 polymorphic markers of 3p using the representative set of samples (80 cases RCC), described clinically and histological, permitted to reveal significant correlation between frequency of allelic alterations in some critical regions of 3p (LUCA and AP20) and RCC progression, as distinct from the whole 3p. These data suggest that methylation of promoter region of the RASSF1A gene is associated with RCC progression, and besides, structure-functional alterations in other 3p genes can be also related with RCC progression. In addition, significant correlation between RASSF1A methylation events and allelic losses in the close polymorphic marker was shown here, pointing to the role of "two hit" model for this tumor-suppressor gene inactivation in RCC.

[Alteration of SEMA3B gene expression levels in epithelial tumors].

Gene expression decreasing in tumors permits to suggest tumor-suppressor activities for these genes. Thus, mRNA quantity decrease was found for SEMA3B gene in many cell lines of small cell (SCLC) and non-small cell lung carcinoma (NSCLC) and it is well-known that SEMA3B suppresses growth of the NCI-H1299 non-small cell lung carcinoma (NSCLC) cell line and tumor formation in nude mice. The aim of this work was to study spectrum of SEMA3B expression level in epithelial tumors of various locations. Using semi-quantitative RT-PCR it was shown for the first time decrease of SEMA3B mRNA quantity (10-250 times as much) in cell lines of renal, breast and ovarian tumors (4/11, 36%). SEMA3B expression profiles in primary tumors of five locations (kidney, lung, breast, ovary and colon) were studied for the first time. This analysis revealed decrease of mRNA quantity (5-1000 times as much) in clear cell renal cell carcinomas with significant high frequency: 25/51, 49% (cases with decrease of mRNA quantity) and 5/51, 10% (cases with increase), P < 0.0001 by Fisher exact test. In addition, the first data about comparatively frequent decrease of mRNA quantity in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal carcinomas (6/11, 54% vs. 2/11,18%) were shown. These results permitted to suggest a possible role of SEMA3B in inhibiting of growth of renal, ovarian and colorectal cancer cells.

[Two CpG-islands of SEMA3B gene: methylation in clear cell renal cell carcinoma].

Earlier in some small cell lung (SCLC) and non-small cell lung carcinoma (NSCLC) cell lines, methylation of CpG-island was found in the SEMA3B region, which belongs to the first intron according to the NCBI data base (Build 36). The aim of this work was to study methylation of two SEMA3B CpG-islands: promoter and intronic in clear cell renal cell carcinoma (RCC). Using methyl specific PCR and bisulfite sequencing, it was shown for the first time that promoter CpG-island was methylated in RCC with high frequency 56% (34/61), and intronic CpG-island - with somewhat lower frequency 35% (17/48). Significant reverse correlation was estimated between mRNA level decrease and methylation of promoter CpG-island in RCC for the first time (P < or = 0.05 by Fisher's exact test), no correlation was determined for intronic CpG-island. This result suggested that methylation of promoter CpG-island contributed into inactivation of SEMA3B gene-suppressor in RCC.

[Methylation of promoter region of RAR-beta2 gene in renal cell, breast, and ovarian carcinomas].

The protein encoded by RAR-beta (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-beta2 mRNA is downregulated in a number of cell lines derived from human epithelial tumors. Inactivation of the RAR-beta2 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-beta2 gene with RCC progression (P < or = 0.005 by Fisher's exact test) was established.

[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 suppressor genes of tumor growth (TSG) observed in the epithelial tumors of various types. Region between D3S2409 and D3S3667 markers in the 3q21.31 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 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 BRCA1/2 and CHEK2 mutations in ovarian cancer and primary multiple tumors involving the ovaries. Patients of Russian population using biochips].

Ovarian cancer (OC) is one of the leading cause of cancer death in women. Inherited BRCA1 and BRCA2 mutations strikingly increase OC risk (with lifetime risk estimates ranging at 10-60%). Mutation 1100delC in CHEK2 gene was shown to be associated with breast cancer in women carrying this mutation. Knowledge of the nature and frequency of population-specific mutations in these genes is a critical step in the development of simple and inexpensive diagnostic approaches to DNA analysis. The frequencies of 185delAG, 300T>G, 4153delA, 4158A>G, 5382insC mutations in BRCA1 gene, 695insT and 6174delT mutations in BRCA2 gene and 1100delC mutation in CHEK2 gene were analyzed using biochips in Russian OC patients. We studied 68 women who received a diagnosis of epithelial OC and 19 women with primary multiple tumors involving the ovaries. The 185delAG, 300T>G, 4153delA and 5382insC in BRCA1 gene were identified. The most prevailing mutation was 5382insC in BRCA1 gene (87.5% of all BRCA1 mutations OC patients, 50.0% in patients with primary multiple tumors involving the ovaries). No mutations in BRCA2 and CHEK2 genes were detected.

High incidence of mutations in BRCA1 and BRCA2 genes in ovarian cancer.

The incidence of mutations in the BRCA1 and BRCA2 genes in the studied sampling of 74 patients with ovarian cancer was 19%. The incidence of mutations in the Russian sampling of patients, formed without consideration for the family history, is one of the highest in European countries. Retrospective analysis showed that 9% patients carrying mutation had no family history of ovarian or breast cancer. The majority of mutations (86%) were detected in BRCA1 gene, where 5382insC mutation predominated (58%). These data suggest the possibility and advisability of screening for mutations in the BRCA1/2 genes in patients with ovarian cancer, particularly because this population includes patients without family history of ovarian and/or breast cancer.

[Activation of RHOA gene transcription in epithelial tumors may be caused by gene amplification and/or demethylation of its promotor region].

RHOA protein, a member of small GTPases family, is implicated in cell morphogenesis, adhesion, and in cell cycle regulation. RHOA gene (3p21.31) exhibits cell transformation activity, and therefore gene is considered as a potential oncogene. The aim of this study was to investigate RHOA transcription and copy number changes in three epithelial tumors (breast, renal cell and epithelial ovarian carcinomas, 45 tumor/normal pairs altogether). EII, HhaI, AciI n Bsh1236I). Hypomethylation of the RHOA promoter region in tumor DNA was observed two times more frequently than increased methylation. Moreover, all (15) cancer cases with hypomethylation of the RHOA gene showed a 2-10 fold increased expression of RHOA. It was concluded that gene copy multiplication and demethylation of the RHOA promoter region can contribute to transcription activation of this gene in epithelial tumors.

[Population genetic analysis of the association between the BRCA1 and P53 gene polymorphisms and the risk of sporadic breast cancer].

Polymorphism of two tumor-suppressor genes, BRCA1 and P53, was examined. DNA was extracted from blood leukocytes of the women affected with breast cancer (N = 151) and of the women with no clinical symptoms of tumor diseases (N = 191). Typing of the polymorphic variants was performed using PCR-RFLP method. It was demonstrated that the genetic structure of the patient group (taking into consideration BRCA1 and P53 polymorphic variants) differed from that of the control group. The group of genotypes, found exclusively among the patients, as well as the group of "resistant" genotypes revealed predominantly among the controls, was described. Detection of the genotype A1A1 B1B1 S1S1 C1C1 F1F1 J2J2, whose frequency in control group was eight times higher than in the patient group, was an additional confirmation of the existence of "resistant" genotypes. These findings point to the association between the combinations of the BRCA1 and P53 allelic variants and the risk of breast cancer.

[Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors]. / Uroven' metilirovaniia promotornoi oblasti gena RASSF1A v pervichnykh épitelial'nykh opukholikh.

Methylation of the promoter CpG-islands of the candidate tumor suppressor gene RASSF1A (3p21.31) was studied in primary tumors of kidney, breast and ovary (172 cases). Methylation-specific PCR (MSP) and methyl-sensitive restriction endonuclease digestion followed by PCR (MSRA) were applied. Statistically significant correlation (P << 10(-6)) was shown for the results of the MSP and MSRA, and the data of bisulfite sequencing reported earlier. The frequency of RASSF1A methylation according to MSP and MSRA was 86% (25/29) and 94% (50/53) in renal cell carcinoma (RCC) and 64% (18/28) and 78% (32/41)--in breast carcinoma (BC) samples, and 59% (17/29) and 73% (33/45) in ovarian epithelial tumors (OET), respectively. The use of several methyl-sensitive restriction enzymes (HpaII, HhaI, Bsh12361, AciI) enhanced the sensitivity of MSRA and allowed to analyze methylation status of 18 CpG-pairs in the RASSF1A CpG-island. Density of methylation of the RASSF1A CpG-island was 72% (644/900) in RCC, 63% (361/576) in BC, and 58% (346/594) in OET samples (18 CpG-pairs multiplied to the number of samples shown methylation were assumed as 100%). The RASSF1A gene methylation was also observed in samples of morphologically normal tissues adjacent to corresponding tumors (11-35%), but it was not detected in blood DNAs of healthy donors (0/15). The RASSF1A methylation frequency did not show significant correlation to tumor stage, grade and metastases (P = 0.3-1.0). The RASSF1A gene methylation was observed more frequently than other investigated aberrations--hemi- and homozygous deletions inside or around this gene. These observations are consistent with the hypothesis that the RASSF1A gene methylation is an early event in the carcinogenesis and one of the dominant ways of its inactivation.

[Identification of the hereditary variants of skin melanoma]. / Identifikatsiia nasledstvennykh variantov melanomy kozhi.

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.

Spectrum of mutations in BRCA1 gene in hereditary forms of breast and ovarian cancer in Russian families.

The 5382insC mutation predominated (94%) in the spectrum of detected mutations of BRCA1 gene. High incidence of this mutation in familial breast cancer detected for the first time attested to origination of 5382insC mutation from the European part of Russia. The percentage of families with mutations in BRCA1 gene and familial predisposition to ovarian cancer was significantly higher than in hereditary predisposition to breast cancer (p<0.007). These data suggest that clinical manifestation of the mutation depends on genotypical factors other than the position of this mutation in BRCA1 gene. The results prompt screening for hereditary predisposition to these diseases.

[Analysis of mutations in the RET proto-oncogene in patients with medullary thyroid tumor]. / Analiz mutatsii v protoonkogene RET u bol'nykh s medulliarnym rakom shchitovidnoi zhelezy.

The spectrum of mutations of the RET protooncogene 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 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 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 MET 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.

[Hereditary cancer: identification, genetic heterogeneity, medico-genetic consult]. / Nasledstvennyi rak: identifikatsiia, geneticheskaia geterogennost', mediko-geneticheskoe konsul'tirovanie.

The paper deals with a role of inherited factors responsible for the occurrence of malignant tumors. Inherited types of cancer are shown to occur virtually at its sites and averaged 5-15%. Formalized criteria for identifying inherited cancer diseases and their etiological and genetic heterogeneity are presented. A role of genes that genetically predispose to particular forms of cancer is shown, which allows for early (preclinical) diagnosis and prevention of cancer diseases.

[Mapping allelic deletions on the short arm of human chromosome 3 in kidney neoplasms]. / Kartirovanie allel'nykh deletsii na korotkom pleche khromosomy 3 cheloveka v opukholiakh pochki.

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL (3p26-p25), the region of homozygous deletions AP20 (3p22-p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).