[The genetic markers and targets of target therapy of melanoma.]

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.

[ Spectrum of oncogene mutations is different in melanoma subtypes].

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.

[Analysis of the -238(G/A)TNF polymorphism in breast cancer patients].

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.

[The frequency and spectrum of KRAS mutations in metastatic colorectal cancer].

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.

[Morphological characteristics and prognostic criteria for gastrointestinal stromal tumors].

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.

[Gastrointestinal stromal tumours: clinico-morphological features, pathogenesis and modern treatment strategies].

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.

[Detection of LY6G6D gene exon-intron structure,].

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.

[Tumor necrosis factor gene polymorphisms in breast cancer patients].

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.

[c-Kit and PDGFRA mutations and clinico-morphological features of gastrointestinal stromal tumors].

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.

[Polymorphisms of tumor necrosis factor genes in sporadic and hereditary breast cancer].

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.

[Loss of heterozygosity at chromosome 6 as a marker of early genetic alterations in cervical intraepithelial neoplasias and microinvasive carcinomas].

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.

[Interaction of viral and cellular genes in cervical tumors]. / Vzaimodeistvie virusnykh i kletochnykh genov v opukholiakh sheiki matki.

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.

[Cervical carcinoma progression-associated genetic alterations on chromosome 6]. / Geneticheskie izmeneniia na khromosome 6, assotsiirovannye s progressiei raka sheiki matki.

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.

[Molecular markers of the cervical cancer]. / Molekuliarnye markery raka sheiki matki.

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.

Expression of protooncogenes during lymphocyte activation by growth factors.

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.

[Nuclear oncoprotein expression in lung precancer and cancer at various stages of tumor progression studies at the level of light- and electron-immunohistochemistry]. / Ekspressiia iadernykh onkobelkov pri predrake i rake legkovo na raznykh stadiiakh opukholevoi progressii (po dannym svetovoi i élektronnoi immunohistokhimii).

68 cases of lung carcinoma, 3 carcinoids and 15 fibrosing alveolitis with foci of adenomatosis and bronchiolo-alveolar carcinoma were studied. Oncoproteins c-fos, c-jun, c-ets-1, c-myc L and L-myc were identified in the tumour and surrounding tissue. Expression of c-fos was revealed in 79 of 138(59.4%) of proliferative and dysplastic changes of lung epithelium; c-jun in 40 of 61 (65.6%), c-ets-1 in 22 of 41 (53.7%), c-myc in 41 of 96(42.7%) and L-myc in 15 of 61 (24.6%), mainly in altered bronchial epithelium with a positive reaction to the antibodies against neuron specific enolase and S100 protein. More pronounced expression of nuclear oncoproteins, heterogeneity of their location in tissues, frequent cytoplasmic location in tumour cells were typical for lung carcinoma.

[Cell oncogene expression in normal, metaplastic, dysplastic epithelium and squamous cell carcinoma of the uterine cervix]. / [Ekspressiia kletochnykh onkogenov v normal'nom, metaplasticheskom, displasticheskom épitelii i ploskokletochnom rake sheiki matki.

Immunohistochemical analysis of the protein expression c-myc, ets 1, ets 2, TPR-met, c-fos, c-jun, c-ras-pan, p53, yes, src in 79 samples of normal, metaplastic squamous epithelium, intraepithelial and invasive squamous cell carcinoma of uterine cervix was performed using polyclonal rabbit antibodies to the synthetic peptides homologous active areas of corresponding oncoproteins. Higher content of myc, fos, ets2, p53, ras is noted in metaplasia, dysplasia and in tumours as compared to the normal tissues. Protein myc is revealed in the cytoplasm at a grave dysplasia and in the nucleus in the intraepithelial carcinoma: this may serve as a criterion at a differential diagnosis of these conditions. Expression of the oncoproteins fos, ets2, p53, src in the metaplastic squamous cell carcinoma was higher than in the true squamous cell (ectocervical) carcinoma. When compared to the advanced carcinomas, increase of ets2, p53, and at some degree that of myc, the increase is noted in the latter. Invasive carcinoma with a high level of oncoproteins showed a tendency to the synchronization of myc and ras expression. Poor prognosis was associated with a low level (before treatment) of the expression of the majority of the oncoproteins studied.

[Expression and co-expression of cellular oncogenes in the course of tumor progression in neuroendocrine lung tumors]. / Ekspressiia i koékspressiia kletochnykh onkogenov v khode opukholevoi progressii v neiroéndokrinnykh opukholiiakh legkikh.

The tumours studied were as follows: benign carcinoids, well differentiated carcinomas (atypical carcinoids) and poorly differentiated tumours (small-cell carcinoma with neuroendocrine differentiation). Oncoproteins c-myc, c-fos, c-jun, L-myc, c-sis, c-ras, c-src, c-ets-1, c-met were studied immunohistochemically in the material obtained from 25 patients during the operations. A higher expression of c-fos, c-jun, c-ets-1 and c-met is observed at early stages of progression and that of c-myc and L-myc at later stages. Enhancement of c-myc and L-myc expression correlated with the invasiveness and lymphogenic metastasizing. Co-expression and negative correlation between some oncogenes are established. The data obtained may be used for prognosis and therapy.

[In vivo identification of YB-1 protein, interacting with the enhancer of human papillomavirus (HPV) type 18, using antibodies to a synthetic peptide]. / Identifikatsiia in vivo belka YB-1, vzaimodeistvuiushchego s énkhancerom virusa papilloma cheloveka (HPV) tipa 18 s pomoshch'iu antitel k sinteticheskomu peptidu.

Enhancer sequences of human papilloma virus (HPV) type 18 were used for screening of HeLa cells cDNA library in lambda gt11 using the protein binding method. Clones with YB I gene homology sequences were isolated. This gene is coding the protein which binds the regulatory region of Y gene of main histocompatibility complex (HLA 11). The YB I transcripts were revealed in all tested samples of cervical carcinomas. To analyze the protein the rabbit antibodies were produced to synthetic peptide, which corresponds to the most hydrophilic region of the protein. This antipeptide serum allowed to identify the nuclear 42K protein in HeLa cells as well as in normal fibroblasts.

Expression of pp60c-src in human small cell and non-small cell lung carcinomas.

c-src protein was found in 60% of lung carcinomas (20 of 33 cases or primary tumours) by immunoblotting with a monoclonal antibody (Mab 327) and immunohistochemistry with serum from rabbits bearing tumours induced by Rous sarcoma virus. src protein expression was assessed in 4 small cell lung carcinomas and in an atypical carcinoid of neuroendocrine origin. However, pp60c-src was also found in non-small cell lung carcinomas: in 60-80% of adenocarcinomas and bronchiolo-alveolar cancers and in 50% of squamous cell carcinomas. In the squamous cell carcinomas, src protein was expressed more frequently in poorly differentiated than in well and moderately differentiated carcinomas. Expression of pp60c-src was not found in epithelial cells of histologically unchanged lung tissues. These results show that pp60c-src may be activated in human lung carcinomas of different histopathological types.