[Structural and functional analysis of tumor genomes and the development of test systems for early diagnosis, prognosis and cancer therapy optimization].

The article discusses results of the structural and functional analysis of molecular genetic abnormalities in various malignant tumors. Investigations have discovered more than 20 new markers for sporadic breast cancer. Several of them formed the test system, allowing the diagnosis with a specificity of 100%. Appearance of TMPRSS2/ERG4 chimeric gene is a frequent tumor-specific event, its expression is correlated with more aggressive forms of prostate cancer, may serve as a molecular marker for tumor cells and androgen assessment of tumor response to hormonal therapy. The effective systems for the early diagnosis of cervix and endometrium cancer were developed as well. Mutations in the VHL, deletions of chromosome 3 and methylation of several genes can predict the course and selection of effective therapy of clear cell kidney cancer, a number of molecular markers were identified for early diagnosis and prognosis of recurrence of bladder cancer. For diagnosis, prognosis and treatment of brain tumors we developed an effective complex system of markers. Protocol of molecular genetics investigation reveals the cause of the disease by more than 90% of patients with retinoblastoma. In order to study abnormal methylation in tumor genomes an innovative technology AFLOAT has been developed that allows to efficiently identify new markers with diagnostic value. Test systems of molecular genetic and epigenetic markers for early diagnosis and prognosis as well as for cancer therapy optimization have shown to be effective, have been approved for use in clinical practice and are being introduced into practical healthcare.

[Generation and application of dynamic standard reference intervals for analyzing results of comparative genomic hybridization].

The present work was aimed at generating the dynamic standard reference intervals (DSRI) and their application for chromosomal-aberration (CA) analysis. The evaluation of the generated DSRI was performed using the DNA samples from four patients with already known CA. High-resolution comparative genomic hybridization analysis (HR-CGH) allowed us to not only identify all of the CAs, that were not revealed by CGH, but also to detect the breakpoints and to determine the size of chromosomal imbalance.

[Correlation of p16/INK4a gene damages and protein expression in the tumor tissue of sporadic breast cancer].

A tumor emerges due to the structural and functional abnormalities occurring in the genes, which causes a change in the spectrum of protein molecules. Strong correlations between the gene damages and following changes in the protein spectrum make it possible to study different stages of carcinogenesis and to create a more complete system of molecular markers for the diagnosis of different types of tumors, which comprises protein markers and DNA markers. The present investigation has studied a correlation between the inactivation mechanisms (structural and functional) of the suppressor gene p16/INK4a, which occur at the level of DNA, and the results of its protein expression examined by immunohistochemical methods in the tumor specimens from patients with breast cancer. The investigation could indicate that p16/INK4a gene damage frequently occurred in the tumors of the above type. In the majority of study cases, molecular damages revealed in the gene diminish on its protein expression; however, there are still cases that defy generally accepted explanations.

[Novel methylation and expression markers associated with breast cancer].

We have developed a modification of methylation sensitive arbitrarily primed PCR, one of the methods of differentially methylated CpG islands in cancer cells genomes screening. Seven genes undergoing abnormal epigenetic regulation in breast cancer, SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4 and PSMF1, have been identified by this method. Methylation and loss of expression frequencies were evaluated for each of the identified genes on 100 paired (cancer/morphologically intact control) breast tissue samples. Significant frequencies of abnormal methylation were detected for SEMA6B, BIN1, and LAMC3 (38%, 18%, and 8% correspondingly). Methylation of the above genes was not characteristic for morphologically intact breast tissues. Downregulation of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4 and PSMF1 in breast cancer was as frequent as 44-94% by real-time PCR expression assay. The most pronounced functional alterations were demonstrated for SEMA6B and LAMC3 genes, which allows recommending their inclusion into the panels of carcinogenesis diagnostic panels. Fine methylation mapping was performed for the genes most frequently methylated in breast cancer (SEMA6B, BIN1, LAMC3), providing a fundamental basis for the development of effective methylation tests for these genes.

[Analysis of the correlation between genetic and immunohistochemical markers (RB1, p16 and p53 gene regultors of a cell cycle) in sporadic mammary gland carcinoma].

The results of an investigation of the RB 1, p16 and p53 genes in sporadic breast cancer through molecular and immunohistochemical studies are presented.

[Abnormal methylation of p16/CDKN2A AND p14/ARF genes GpG Islands in non-small cell lung cancer and in acute lymphoblastic leukemia]. / Sravnitel'nyi analiz anomal'nogo metilirovaniia CpG-ostrovkov, raspolozhennykh v promotornykh oblastiakh genov p16/CDKN2A i p14/ARF, pri nemelkokletochnom rake legkogo i ostrom limfoblastnom leikoze.

Multiplex methylation-sensitive PCR and methylation-specific PCR were employed in studying the methylation of CpG islands in the p16/CDKN2A and p14/ARF promoter and the first exon regions in non-small cell lung cancer (54 samples) and acute B-cell lymphoblastic leukemia (61 samples). Differences in methylation were detected between types of neoplasia as well as between CpG islands studied within the same types of tumors. High level of the p16/CDKN2A first exon CpC island methylation was revealed in non-small cell lung cancer (68%) and in acute B-cell lymphoblastic leukemia (55%) and the CpG island of p14/ARF first exon was nonmethylated in these types of tumors. The methylation of CpG-rich fragments of genes p16/CDKN2A and p14/ARF promoters was analysed. As was found out, CpG islands located in 5' areas of one and the same gene can differ in methylation frequencies. The comparison of sensitivity between methylation-specific PCR and methylation-sensitive PCR used in the methylations studies was carried out.

[Diagnostics for epigenetic pathology in hereditary and oncologic diseases]. / Diagnostika épigeneticheskoi patologii pri nasledstvennykh i onkologicheskikh zabolevaniiakh.

The review considers the epigenetic defects and their diagnostics in several hereditary disorders and tumors. Aberrant methylation of the promoter or regulatory region of a gene results in its functional inactivation, which is phenotypically similar to structural deletion. Screening tests were developed for Prader-Willi, Angelman, Wiedemann-Beckwith, and Martin-Bell syndromes and mental retardation FRAXE. The tests are based on allele methylation analysis by methylation-specific or methylation-sensitive PCR. Carcinogenesis-associated genes (RB1, CDKN2A, ARF14, HIC1, CDI, etc.) are often methylated in tumors. Tumors differ in methylation frequencies, allowing differential diagnostics. Aberrant methylation of tumor suppressor genes occurs in early carcinogenesis, and its detection may be employed in presymptomatic diagnostics of tumors.

[Abnormal methylation of several tumor suppressor genes in sporadic breast cancer]. / Anomal'noe metilirovanie nekotorykh genov-supressorov pri sporadicheskom rake molochnoi zhelezy.

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, MGMT, HIC1, and N33 promoter regions in breast carcinoma (105 tumors). Methylation was often observed for the two major suppressor genes involved in cell-cycle control through the Cdk-Rb-E2F signaling pathway, RB1 (18/105, 17%) and p16 (59/105, 56%); both genes were methylated in 13 tumors. Methylation involved p15 in two (2%) tumors; CDH1, in 83 (79%) tumors; MGMT, in eight (8%) tumors, and N33, in nine (9%) tumors. The p14 promoter was not methylated in the tumors examined.

[Analysis of the allelic polymorphism of the five X-linked (CA)n dinucleotide repeats in Russia]. / Analiz allel'nogo polimorfizma piati X-stseplennykh dinukleotidnykh povtorov (CA)n v Rossii.

A PCR-based survey of allelic polymorphism of three microsatellite markers, DXS998, DXS548, and FRAXAC1, mapped to chromosome region Xp27.3, and two microsatellite markers, DXS8091 and DXS1691 located on Xq28 was carried out using a series of DNA samples obtained from 98 unrelated individuals from Russia. The number of alleles detected on electrophregrams for each marker tested was 4, 6, 4, 5, and 3, respectively. The values of heterozygosity index for the markers examined were 0.65, 0.27, 0.38, 0.70, and 0.29, respectively. The observed distribution of the allelic frequencies for each microsatellite marker examined fitted Hardy--Weinberg expectations. The values of individualization potential determined for each marker were 0.24, 0.53, 0.43, 0.12, and 0.52, respectively. In the sample tested the genotype distribution with regard to above loci was determined. The perspectives of using the analyzed allelic polymorphisms for indirect DNA diagnostics of the monogenic diseases located in this chromosome region (X-linked mental retardations, FRAXA and FRAXE) as well as for human population genetics and personal identification is discussed.

[Profile of methylation of certain tumor growth suppressing genes in non-small cell lung cancer]. / Profil' metilirovaniia nekotorykh genov-supressorov opukholevogo rosta pri nemelkokletochnom rake legkogo.

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5' regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk-Rb-E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.

[RB1 and CDKN2A functional defects resulting in retinoblastoma]. / Funktsional'naia patologoia genov RB1 i CDKN2A, privodiashchaia k razvitiiu retinoblastomy.

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed a molecular defect in at least one allele in 51 (98%) tumors.