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Melanoma is the most dangerous malignant disease of skin with high risk of relapsing and metastasis dissemination. The molecular biological studies implemented during last decade drastically altered our concepts about mechanisms of carcinogenesis of melanocytes. The review considers both hereditary factors of predisposition to melanoma (rare alleles of genes CDKN2A и CDK4, mutations MITF and BAP1) and somatic genetic disorders involved into carcinogenesis of melanoma. These mutations in genes causing hyper-activation of RAS-MAPK (BRAF, NRAS, MEK, NF1) и PI3K- (PTEN, AKT) of signaling pathways and also genes of tyrosine-kinase receptors KIT, ERBB4 activating transfer of signal in cell. Also, the role of сAMP and NF-κB in melanomagenesis is considered. The detection of activating mutations of key signaling pathways in oncogenes permitted to apply medications of target action many of which demonstrated a good therapeutic effect. The combined treatment of melanoma in aggregate with immune therapy is especially perspective.

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Melanoma is the most lethal malignancy of skin, which is comprised of clinically relevant molecular subsets defined by specific "driver" mutations in BRAF, NRAS, and KIT genes. Recently, the better results in melanoma treatment were obtained with the mutation-specific inhibitors that have been developed for clinical use and target only patients with particular tumor genotypes. The aim of the study was to characterize the spectrum of "driver" mutations in melanoma subtypes from 137 patients with skin melanoma and 14 patients with mucosal melanoma. In total 151 melanoma cases, the frequency of BRAF, NRAS, KIT, PDGFRA, and KRAS mutations was 55.0, 10.6, 4.0, 0.7, and 0.7%, respectively. BRAF mutations were found in 69% of cutaneous melanoma without UV exposure and in 43% of cutaneous melanoma with chronic UV exposure (p=0.045), rarely in acral and mucosal melanomas. Most of melanomas containing BRAF mutations, V600E (92%) and V600K (6.0%) were potentially sensitive to inhibitors vemurafenib and dabrafenib. NRAS mutations were more common in cutaneous melanoma with chronic UV exposure (26.0%), in acral and mucosal melanomas; the dominant mutations being Q61R/K/L (87.5%). KIT mutations were found in cutaneous melanoma with chronic UV exposure (8.7%) and mucosal one (28.6%), but not in acral melanoma. Most of KIT mutations were identified in exon 11; these tumors being sensitive to tyrosine kinase inhibitors. This is the first monitoring of BRAF, NRAS, KIT, PDGFRA, and KRAS hotspot mutations in different subtypes of melanoma for Russian population. On the base of data obtained, one can suppose that at the molecular level melanomas are heterogeneous tumors that should be tested for "driver" mutations in the each case for evaluation of the potential sensitivity to target therapy. The obtained results were used for treatment of melanoma patients.

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TNF is an inflammatory cytokine that involved in pathogenesis of different malignancies. Promoter single nucleotide polymorphism -238(G/A)TNF (rs361525) is investigated for the detection of susceptibility to the infectious, autoimmune and oncological diseases. The goal of the study was to investigate the association of-238(G/A)TNF polymorphism (rs361525) with breast cancer (BC) prognosis. -238(G/A) TNF allelic variants were detected by PCR-RFLP. We failed to reveal the genotype distributions disparity among groups with different stages of the disease, ER, PR or Her2/neu positive versus negative status. The AG genotype frequency was about 10% and there were no BC patients with AA genotype in all separated groups. However the overall survival was significantly lower for AG then for GG carriers with II stage or ER-positive BC. Our data suggest that -238(G/A)TNF polymorphism perhaps is not involved in the initiation of malignancies but it is a substantial factor of BC prognosis.

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KRAS mutations in patients with metastatic colorectal cancer (CRC) are a negative marker of the effectiveness of anti-EGFR therapy and have prognostic significance. KRAS genotyping was performed in the material from patients with metastatic CRC. KRAS mutations were determined in tumor DNA from archival biopsies of 573 patients using PCR and sequencing. Mutations in the exon 2 of the KRAS gene were detected in 36.3% of cases of CRC, while often in women (41.1%) than in men (31.2%). There were identified eight types of KRAS mutations, the most frequent--replacement of G12D (33.7%), G12V (32.7%) and G13D (12.5%). There were revealed differences in the frequency and spectrum of KRAS mutations in CRC of various locations; in tumors of the rectum dominated mutation G12V (39%). The Russian CRC patients find out a higher frequency of mutations G12V and a lower frequency of mutations G13D, than patients from Europe and it should be taken into account when assessing the response of CRC patients with different mutant KRAS status on chemotherapy and targeted therapy.

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Gastrointestinal stromal tumors (GIST) are the most common mesenchymal neoplasms of the gastrointestinal tract. Analysis of GIST morphology is necessary for selection of primary patients with the high risk of tumor progression for adjuvant treatment with gleevek following complete gross resection of KIT (CD 117)-positive GIST. In this study we've analyzed morphological parameters and survival of 120 GIST patients before target therapy. According to risk stratification of primary GIST by tumor location, size and mitotic index (mitoses per 50 visual fields) 44% of gastric GISTs, 87,5% of small bowel GISTs and 100% of rectum GISTs have been classified as high risk group. There was no significant difference between survival of patients with different type of GIST, Ki-67 proliferative index and presence of necrosis.

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Gastrointestinal stromal tumours (GIST) are the commonest mesenchymal tumours of gastrointestinal organs accounting for 1-3% of all gastrointestinal neoplasms and almost 5% of all soft tissue sarcomas. Most GIST (95%) express transmembrane receptor KIT or CD117, CD34 (70%), vimentin (80%), specific smooth muscle and neurogenic markers (with different frequency). Up to 85% of the stromal tumours have mutations in the KIT gene (exones 9, 11, 13, 17) and 3-18% in the PDGFRA (platelet-derived growth factor receptor-alpha) gene (exones 12, 14, 18). Mutations are absent in 5% of KITand PDGFRA genes. Mutational status, mitotic index, size and location of the tumour are the most informative criteria for the choice of treatment strategy and prognosis of the disease.

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Earlier we have shown high frequency of loss of heterozygosity of microsatellite marker D6S273 within HLA III class region in DNA samples from cervical intraepithelial neoplasias and cervical cancer. According to publications three genes were identified in this region. For detection of D6S273 position we used in silico analysis of mRNA sequences deposited in GenBank (NCBI) and investigated LY6G6D gene expression in tumor cell lines. LY6G6D gene exon borders were analyzed with 5'- or 3'-rapid amplification of cDNA ends. We have found that LY6G6D gene consists of 9 exons and includes two earlier identified genes G6D and G6F. Microsatellite D6S273 is located in the last 8 intron of LY6G6D gene. The third gene LY6G6E consisting of four exons is located in 6 intron of LY6G6D gene in the opposite orientation. We suggest that LY6G6D gene is coding three main mRNA transcripts in the same open reading frame but differ in exon composition: MEGT1 consists of 1-4, 8, 9 exons, G6F consists of 1-6 exons and G6D consists of 7-9 exons of LY6G6D gene. High homology with immunoglobulin superfamily within 20-120 aminoacids of MEGT1 and G6F proteins is shown by in silico translation of their mRNAs.

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Analysis of microsatellite TNFalpha marker and (-308(G/A) polymorphisms in promoter of TNFalpha gene was conducted in 167 patients with various types of sporadic breast cancer (BC) as well as in 139 healthy Russian donors. It was shown that frequency of allele 7 in TNFalpha microsatellite marker was significantly higher in BC patients than in healthy donors (17.9% versus 10.4%; P = 0.02) mainly due to the patients with invasive ductal BC (19.2% versus 10.4%; P = 0.008). The TNFalpha allele 9 was observed significantly more frequently in patients with invasive-ductal cancer (6.4% versus 1%; P= 0.01). The studies of-308(G/A)TNFalpha polymorphism in BC patients and healthy donors have shown no differences in the distribution frequency of highly secreted allele (-308A)TNFalpha. However, invasive lobular BC patients carrying (-308AG)TNFalpha genotype were observed significantly more frequently than invasive-ductal BC patients carrying the same allele (34.0 versus 17.3%; P = 0.034). Thus it has been shown for the first time that invasive-ductal and invasive-lobular BC patients differ in distribution of TNFalpha and -308(G/A)TNFalpha alleles.

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A first attempt at investigating polymorphism of microsatellite TNFa and SNP-308 (G/A) in promoter of TNFalpha was made in patients with sporadic and hereditary breast cancer. 308 (A) TNFalpha and TNFa 12 alleles frequencies were significantly higher while that of TNFa10--significantly lower in the hereditary cancer group as compared with donors as well as sporadic cancer patients. That was contributed by cases of infiltrative-lobular tumors. Conversely, because of infiltrative-ductal tumors fraction, TNFa 7 allele frequency in sporadic cancer group was significantly higher than in donors and hereditary breast cancer patients. It was suggested that polymorphism of 308 (G/A) TNFalpha and TNFa depended, first of all, on patterns of breast and, secondly, on the elevated TNFalpha expression as a factor of pathogenesis of hereditary breast cancer.

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Gastrointestinal stromal tumors (GIST) are the most common mesenchymal neoplasms of the digestive tract. Inherent overexpression of receptor tyrosine kinase KIT (CD117) and mutations in c-Kit or PDGFRA genes are highly significant prognosticators. A first Russian investigation of c-Kit and PDGFRA mutations in GIST was carried out in 60 patients. c-Kit mutations were identified in 83.3% (50/60), the most frequent being mutations in c-Kit exon 11 (73.4%, 44/60). Among them, different mutations were identified in the 5'-end of c-Kit exon 11 in 37 GISTs. Duplications in the 3'-end of c-Kit exon 11 were reported in 7 tumors. Mutations in c-Kit exon 9 (73.4%, 44/60) were found in 5 tumors (8.3 3%, 5/60) while mutations in c-Kit exon 13 (0%, 44/60) and 17 (1.7%, 1/60) were rare. PDGFRA mutations in exon 18 were identified in (8.3 3%, 5/60). Substitution D842V occurred only in one gastric epithelioid-cell GIST. The remaining PDGFRA mutations contained deletions with aminoacids 842-846. There were no c-Kit and PDGFRA mutations in five tumors. Our findings point to a significant correlation between c-Kit and PDGFRA mutations, on the one hand, and tumor site and histological pattern, on the other. Hence, c-Kit and PDGFRA mutation detection should be used as an additional prognosticator for efficacy of target therapy.

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Oncogenic human papilloma viruses (mostly HPV types 16 and 18) are the major cause of cervical intraepithelial neoplasia (CIN) that progress into cervical cancer (CC). To reveal early genetic alterations at chromosome 6 important for CC progression we have analyzed loss of heterozygosity (LOH) in DNA from 45 CIN cases, 47 microcarcinomas and 19 invasive squamous cell carcinomas stage IB. LOH analysis of DNA samples prepared with microdissection from all CIN foci as well as from CC lesions and synchronous CIN has permitted the investigation of CIN and CC heterogeneity. 79% of CC stage 1 showed LOH with 6 microsatellite markers at chromosome 6. LOH with microsatellite markers D6S276 (6p22) and TNFalpha (6p21.3) was found in 50% of CC cases. LOH frequency in CIN lesions, synchronous with CC, was higher then LOH in CIN cases without cancer, the statistical significance (p = 0.004) was shown for marker D6S291 (6p21.2). The finding suggests that high level of LOH frequency in CIN lesions may be a marker of unfavorable prognosis for CIN. Progression from microcarcinoma to invasive CC of IB stage was associated with higher LOH frequency at D6S344 (6p25) and TNFalpha (6p21.3). The early genetic alterations were found in CIN with microsatellites D6S273 and TNFalpha located at 6p21.3. Moreover the LOH frequency at D6S273 retained the same in CIN and CC cases. Based on HPV-typing, LOH analysis and X-chromosome inactivation the polyclonality of CC lesions as well as CIN was shown in a few patients.

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The results obtained in the Laboratory of Molecular Biology of Viruses, CRC carried out in the framework of the Human Genome program and devoted to the study of the activity of cell and viral genes in cervical cancer are summarized. DNA of human papillomaviruses persists in tumors both in episomal and integrative forms. Integration may occur in different regions of chromosomes. Viral transforming genes E6 and E7 are always present in tumor cells, while antibodies to these proteins are detected only in approximately 30% of patients. Loss of heterozygosity is detected on long and short arms of chromosome 6; some such cases are manifest already at the early stages of tumor progression, while others are typical of the late stages. Several genes that are potentially involved in tumorigenesis and are subject to hypermethylation in CpG islands were identified. Methylation of several genes is observed in approximately 30% of tumors. Tumor progression is associated with increased expression of p16ink4a, an inhibitor of cyclin-dependent kinases.

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To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.

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The genome of human papilloma viruses from a high-risk group (HPV types 16 and 18) has been detected in 90% of cervical tumors and, in some cases, in the adjacent normal tissues. The presence of viral DNA is the main molecular marker of this neoplasia. HPV genome may persist in the tumors as episomal and integrative forms at early and late stages of tumor progression. The status of viral DNA and the pattern of its expression are similar in all cells of this tumor cell population and seem to be a marker of tumor cell monoclonality. Antibodies to the products of viral oncogenes E6 and E7 were found only in 35% of the patients with tumor where HPV genome is present. Thus, this criteria cannot be used for diagnostic and prognostic purposes. On chromosome 6 in the cervical tumors, the specific marker of heterozygocity on loci 6p21.3 was found. The marker appears at the precancer stage and may be regarded as a marker of tumor monoclonality. Heterozygocity loss in the specific locus in the region 6q16-21 correlates with tumor progression and suggests that there are potential tumor-suppressor genes in this region of chromosome 6. A group of HPV positive tumors with a hypermethylator phenotype is described. These tumors are characterized by the simultaneous methylation and inactivation of multiple genes, including tumor suppressor genes.

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HPV16 is frequently seen in invasive cervical cancer (ICC) and cervical intraepithelial neoplasia (CIN). Its E6 gene has frequent sequence variations. Although some E6 variants have been reported to have different biochemical or biological properties, they do not show geographical identity. Moreover, the definition of 'variant' has been a source of confusion because it has been based on all departures from the 'prototype' once isolated randomly from an ICC case. We amplified the HPV16 E6 gene by PCR from fresh-frozen tissue of 104 cases of ICC and CIN from Russian patients and sequenced it in positive cases. We found that 32 of 55 (58.2%) ICC cases and 18 of 49 (36.7%) CIN cases were HPV 16-positive and we could identify 3 groups of E6 variants: group A was characterized by G at nt 350 where group B had T, and group M was a heterogeneous mixture of unique E6 variants; no significant difference existed in the distribution of the different groups between ICC and CIN; the clinically malignant (as defined by FIGO stage) order between the groups was M > A > B in ICC; in the cases with a single HPV16 E6 sequence, coexisting ICC, CIN and normal epithelium in the same patient shared the E6 variant; and 4 cases of ICC had double/multiple E6 variants. The results did not show any importance of E6 variants for ICC progression in Russian women. The results also indicated that the original HPV16 variant persisted during ICC progression, and that at a low frequency, double infections and/or mutation of variants might occur.

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Investigation on intratumoral genetic heterogeneity provides an important insight into the roles of genetic alterations in human carcinogenesis and clues to clonal origin of tumors. Intratumoral heterogeneity of genetic changes of cervical cancer has not been described so far. In this study, we analyzed the intratumoral heterogeneity of chromosome 3p deletions and X-chromosome inactivation patterns in multiple microdissected samples from each individual cervical cancer, attempting to understand the roles of 3p deletions in development of cervical cancer and its clonal origin. Totally, 120 normal and lesional samples from 14 cases of fresh cervicalcancers were analyzed. Frequency and patterns of allelic losses of 3p were assessed by polymerase chain reaction (PCR) amplification of 12 microsatellite markers flanking the frequently deleted regions of 3p, followed by Genescan analysis in an ABI 377 DNA sequencer. Loss of heterozygosity was recorded as heterogeneous pattern (LOH present in parts of samples or LOH involving different alleles among different samples) and homogeneous pattern (LOH involving identical alleles in all samples from the tumor). Allelic loss affecting at least one marker was detected in 8 of 14 cases (57%). Allelic losses, both homogeneous and heterogeneous, were frequently detected at FHIT gene region (D3S1300, 40% and 60%; D3S4103, 27.3% and 54.6%), 3p21.3-21.2 (D3S1478, 27.3% and 45.5%), and 3p24.2-22 (D3S1283, 30% and 50%). Seven of eight LOH-positive tumors exhibited homogeneous allelic loss involving at least one of these three 3p loci. Allelic losses were present in the CIN lesions synchronous with invasive lesions positive for LOH. Our findings suggest essential roles of genes on these 3p loci, particularly the FHIT gene in participating in clonal selection and early development of cervical cancer. Most interestingly, with the combination of LOH analysis and X-chromosome inactivation analysis, we provided the first clear genetic evidence of polyclonal origin of cervical invasive cancer in two of eight cases. This finding strongly suggests the importance of field defect (possible human papilloma virus) in cervical carcinogenesis.

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Comparative allelotyping of the short arm of human chromosome 3 (3p) in four types of epithelial carcinomas was performed using an identical set of polymorphic markers. In total, 117 samples of non-papillary renal cell carcinoma (RCC), non-small cell lung carcinoma (NSCLC), carcinoma of uterine cervix (CC), and breast carcinoma (BC) were screened for loss of heterozygosity (LOH) with 10 di-, tri- and tetrameric markers covering nine bands of 3p. High LOH frequencies were detected in at least one locus: RCC (36/43, 84%), BC (20/26, 77%), NSCLC (16/24, 67%), and CC (15/24, 62%). Small interstitial deletions prevailed in BC and CC whereas large continuous and discontinuous deletions were mainly found in RCC and NSCLC. Different epithelial tumors displayed unique LOH profiles with partial overlaps in 3p26.1, 3p21.31, and 3p13. The overlap around D3S2409 (3p21.31) appeared common for RCC, BC and CC.

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Chromosome 6 is frequently affected in different tumors. However, little information exists on chromosome 6 deletions in cervical cancer. We have studied loss of heterozygosity (LOH) and microsatellite instability (MIN) in 62 invasive squamous cell carcinomas of the cervix (CC) using 19 polymorphic microsatellite markers spanning both arms of chromosome 6 and one marker located at 5p15. We found that LOH at chromosome 6 is a common feature of cervical carcinomas: 90% (56/62) of CC had LOH at least at one locus and about 58% (36/62) had LOH on both arms of chromosome 6. The highest LOH incidence was shown in HLA region (6p21.3-6p21.1) with markers D6S273 and D6S276 in 52.7% and 45.2% of informative cases respectively. Frequent LOH on 6q was found at loci D6S311 (6q24-25. 1), D6S305 (6q26) and D6S281 (6q27-6qter) in 37.8%, 33.3% and 39.0% of informative cases respectively. There was no significant correlation observed between clinical parameters of cervical cancer (age, histologic grade, clinical stages and progression) and LOH frequency. Microsatellite instability was found in 3 out of 62 cases (4.8%) at three and more loci out of 20 tested. The study shows that several regions on 6p and 6q may harbour potential tumor-suppressor genes important for cervical cancer progression.

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Effects of growth factors of non-immune origin including somatotropin (ST) and platelet-derived growth factor (PDGF) on the expression of the proteins encoded by c-fos, c-myc, c-fun, and c-ets family protooncogenes were studied for the first time. The dynamics of the oncoprotein expression in activated CD(3+)-lymphocytes was investigated by immunoblotting. The accumulation of the Fos and Myc proteins was enhanced in T-lymphocytes treated with ST, PDGF, or phytohemagglutinin; the accumulation was maximum at 30-60 min and decreased in 2 h; the data indicate that the oncoproteins participate in the early lymphocyte activation by various growth factors. The Jun protein appears only in 3 h after the onset of lymphocyte activation; this suggests independent participation of Fos in the early stages of lymphocyte activation prior to the appearance of Jun, preceding the joint action of Fos and Jun within the AP-1 transcription complex. The products of the c-ets family are differentially activated by the studied growth factors. Resting lymphocytes actively accumulate the Ets-1 protein; ST and PDGF activation decreases Ets-1 expression in 2 h. The Ets-2 protein is not detected in resting cells and PDGF-activated lymphocytes, whereas lymphocyte activation by ST is associated with accumulation of Ets-2. The data suggest that the product of the c-ets-1 gene is more important in the regulation of resting cells and the product of the c-ets-2 gene is important during activation of lymphocytes by ST. The results indicate that activation of lymphocytes with growth factors of non-immune origin is mediated by several signal transduction pathways.

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