[Smooth muscle tumors of uncertain malignant potential]. / Gladkomyshechnye opukholi s neopredelennym zlokachestvennym potentsialom.

OBJECTIVE: To investigate microsatellite instability in smooth muscle tumors of uncertain malignant potential and to compare the results with clinical and morphological data. SUBJECT AND METHODS: Histological and immunohistochemical studies were conducted in 26 patients aged 30-63 years (mean age, 37 years) with leiomyomatosis; which revealed intravenous leiomyomatosis in 20 cases, metastasizing leiomyoma in 2, disseminated peritoneal leiomyomatosis in 3, and smooth muscle tumor of uncertain malignant potential in 1 case. Microsatellite instability was studied by fragment analysis on a genetic analyzer using a test system of six markers: D10S1146, D10S218, D10S24, D10S1213, D3S1295, and D9S942. RESULTS: Microsatellite repeat changes characteristic of leiomyosarcomas (heterozygosity loss and/or microsatellite instability in at least one locus studied) were found in 6 patients; all were clinically and morphologically diagnosed as having intravenous leiomyomatosis. In 3 of these 6 cases, leiomyomatosis was accompanied by metastases to the lungs and spread to the peritoneum; heart damage was noted in 2 cases. The data analysis did not allow identification of any significant clinical and morphological criteria for this group. CONCLUSION: Leiomyomatosis is not a transitional form from benign leiomyoma to leiomyosarcoma, as evidenced by the difference in the status of molecular markers. Analysis of molecular genetic changes in DNA from tumor tissue samples cannot categorically clarify the nature of the disease by identifying the signs of genetic instability; however, there is a need for further accumulation of experience in studying tumors of this group and in identifying the possible association with disease prognosis.

Specific Localization of Missense Mutations in the VHL Gene in Clear Cell Renal Cell Carcinoma.

Missense mutations in the VHL gene during sporadic clear cell renal cell carcinoma were studied to evaluate their localization in relation to functionally important motifs of the VHL protein. Somatic mutations were identified in 124 of 307 samples. All missense mutations in the α-domain were localized in the binding site for elongin C. Substitutions in the ß-domain (77%) were found in the HIF-binding site. Five missense mutations were absent in these sites, which illustrates their role in VHL protein formation or suppressor function of other protein cofactors. Mutation c.392A→T (p.N131I) was identified for the first time. Our results hold much promise to estimate the boundaries of functionally important sites in the VHL suppressor gene and contribute to the interpretation of a pathogenic role of mutations in direct DNA diagnostics.

[Expression of microRNA let-7a, miR-155, and miR-205 in tumor and tumor-adjacent histologically normal tissue in patients with non-small cell lung cancer]. / Ekspressiya mikroRNK let-7a, miR-155, miR-205 v opukholevoi i smezhnoi morfologicheski normal'noi tkani u patsientov nemelkokletochnym rakom legkogo.

UNLABELLED: Non-small cell lung cancer (NSCLC) is a main group of lung malignancies. Epigenetic changes are as important as genome structural changes in carcinogenesis. MicroRNA (miRNA) is a class of non-coding single-stranded RNAs that play an important role in the regulation of matrix RNA (mRNA) translation and degradation. MicroRNA expression changes occur in many cancers. According to the field cancerization theory, tumor-adjacent histologically normal tissue takes part in tumor progression by triggering cell transformation. The important clinical implication is that the fields may serve as the basis for a recurrence after surgery. Thus, the aim of our study was to determine the expression levels of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues to evaluate these changes as potential prognostic markers in NSCLC patients. METHODS: The expression of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues at 2 and 5 cm was determined by real-time PCR with subsequent quantification using a 2-ΔΔСt method. The findings were then analyzed to reveal possible associations with clinical and morphological parameters, such as age, cancer stage, and tumor grade. RESULTS: The expression of miRNA let-7a was found to be significantly lower in tumor than that in tumor-adjacent apparently normal tissue at 2 and 5 cm. In groups of patients older than 63 years with Stage III-IV NSCLC, the expressions of microRNA let-7a and miR-155 in tumor tissue were substantially lower than that in the adjacent normal tissue. Beyond that point, patients with high-grade tumors had also a significantly lower expression of miRNA let-7a in relatively adjacent apparently normal tissue. CONCLUSION: The findings suggest that miRNA let-7a and miR-155 may be used as poor prognostic markers for patients with NSCLC.

[DNA methylation in the promoter regions of the laminin family genes in normal and breast carcinoma tissues].

Extracellular glycoproteins of the laminin family are essential components of basement membranes involved in a number of biological processes, including tissue differentiation, wound healing, and tumorigenesis. We present the first comprehensive study of promoter methylation status of the genes encoding laminin chains in normal tissues (peripheral blood leucocytes, buccal epithelial cells, autopsy breast tissue samples) and in breast carcinoma samples. Based on the results of this study, we divide laminin genes into three categories. Genes, constitutively methylated in breast tissues include LAMA3A, LAMB2, LAMB3, and LAMC2. Genes prone to abnormal methylation in breast carcinoma include LAMA1, LAMA2, LAMA3B, LAMA4, LAMB1, and LAMC3. Genes that are rarely if ever methylated in breast carcinoma include LAMA5 and LAMC1. The constitutively methylated group includes all of the genes that encode subunits of laminin-5 (the historical name of laminin 332), the promoters of which were previously considered unmethylated in normal tissues and prone to abnormal methylation in breast cancer.

[High-grade prostatic intraepithelial neoplasia: state-of-the-art].

According to current views, high-grade prostatic intraepithelial neoplasia is the most likely precursor of prostate adenocarcinoma. This review gives the latest data of genetic, proteomic, and morphological analyses of this neoplasia and touches upon the probems that might arise when searching for new markers for differential diagnosis and prognosis estimation.

[Diagnostic value of estimation of ERG expression in prostate adenocarcinoma and high-grade prostatic intraepithelial neoplasia].

OBJECTIVE: to estimate the diagnostic and prognostic value of analyzing the abnormal overexpression of the chimeric protein ERG, encoded by the chimeric gene TMPRSS2/ERG, in prostatic neoplasias. MATERIAL AND METHODS: A total of 100 prostate adenocarcinoma samples were examined. The presence of tumor and high-grade prostatic intraepithelial neoplasia (hPIN) was verified by immunohistochemical tests using anti-P504S and anti-34ßE12 antibodies in serial sections; RT-PCR was employed to analyze the chimeric transcript TMPRSS2/ERG in 30 prostate adenocarcinoma samples. RESULTS: ERG expression was noted in 46% of the adenocarcinomas and in 21% of hPIN. Eight (8%) patients were observed to have heterogeneous ERG expression: the marked reaction in some tumor portions was concurrent with its complete absence in others. Furthermore, there was ERG expression in all cases of intraductal (noninvasive) carcinoma (the foci of intraductal carcinoma were assessed as atypical cribriform lesions by light microscopy). The prognostic value of ERG expression could not be determined at the current stage of the investigation. CONCLUSION: The relatively low rate of ERG-positive hPIN counts in favor of the limited role of this marker in the differential diagnosis of hPIN. ERG in combination with P504S and 34ßE12 is an informative marker for the differential diagnosis of hPIN with intraductal carcinoma.

[Intravascular leiomyomatosis].

Intravenous leiomyomatosis is a rare disease from a group of tumors with the indefinite grading potential. The paper describes two cases of intravenous leiomyomatosis with its detailed morphological pattern, molecular genetic findings, and a brief literature review. Losses of heterozygosity of microsatellite repeats thatwere located on chromosome 10 in 10q22.1 and common in uterine leiomyosarcomas were found in both cases. Investigations of the morphological and biological characteristics of leimyomatosis are important to clarify the key molecular mechanisms underlying the development of this nosological entity and to determine etiopathogenetic relationships between intravenous leiomyomatosis and other uterine smooth muscle neoplasms.

[First Russian nationwide molecular epidemiological study for melanoma: results of BRAF mutation analysis].

This report presents the initial results of the first Russian molecular epidemiological study of melanoma. The investigation included 1035 patients with stage IIIB-IV melanoma residing in various regions of Russia. Sequencing of BRAF gene revealed mutation in 627 (60.6%) tumors; c.1799T > A (p.V600E) substitution was detected in 563 cases, and other mutations in 64 melanomas. Frequency of BRAF alterations was significantly higher in patients of younger age (< 50 years: 72.9%; > or = 50 years: 57.1%; p = 0.00003). 710 melanomas included in the study were located in sun non-exposed regions of the skin; this category of tumors was characterized by the highest occurrence of BRAF mutations (63.9%). In conclusion, more than a half of Russian patients with advanced melanoma are potential candidates for the treatment of kinase inhibitors of mutated BRAF.

[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.

[Allelic imbalance in patients with non-small cell lung cancer].

Non-small cell lung cancer (NSCLC) comprises 80% of all lung cancers and is characterized by multiple genetic alterations such as loss of heterozygosity (LOH) and microsatellite instability (MSI). The aim of the study was to analyze of molecular-genetic alterations in tumor and tissue surrounding the tumor to determine genetic features of different histological types of NSCLC and its possible associations with clinicopathological parameters of patients. A microsatellite analysis of chromosomal regions 12p23.3, 2q35, 3p14.2, 3p22.2, 3p26.3, 9p22.1, 17p13.3 was performed. The frequency of genetic alterations in NSCLC was 50% in average. LOH/MSI in the tumor surrounding tissue at 2 and 5 cm. from tumor was not detected. There were statistically significant associations between LOH and/or MSI and the tumor stage, its histological type and smoking status. The found genetic alterations can be used as molecular markers of squamous cell lung cancer in difficult diagnostic cases and appraised as prognostic markers.

[Benign metastatic leiomyoma of the corpus uteri].

Benign metastasizing leiomyoma (ICD.0 8898/1) is a rare phenomenon characterized by multiple benign smooth muscle tumors (metastases) in the organs and tissues of patients with uterine leiomyoma without evidence for another tumor process. This tumor should be differentiated from leiomyosarcoma, at the same time account must be taken of the fact that its morphological criteria are not always effective. Molecular genetic testing is a more accurate method that allows valid differentiation of leiomyoma from leiomyosarcoma. Genetic testing is used to estimate losses of heterozygosity and microsatellite instability, which are characteristic of leiomyosarcoma only. The paper describes a clinical case of benign metastasizing leiomyoma in a 54-year-old patient with uterine myoma and pelvic lymph node metastasis. Molecular genetic testing was carried out using the samples obtained from primary uterine leiomyoma, morphologically altered ovarian tissue, and lymph node metastasis to determine the common origin of tumors in the uterus and lymph node and to reveal the benign or malignant nature of these neoplasms. Despite the fact that the term "benign metastasizing leiomyoma" is widely accepted in the world literature, neither these tumors nor metastases have morphological or genetic signs of malignancy so we consider the term "systemic leiomyomatosis" to better reflect the essence of this process.

[Fusion genes and transcripts in neoplasia].

Chromosomal rearrangements resulting in the formation of fusion genes are common events in carcinogenesis. There are more than 440 known fusion genes found in both malignant and benign tumors. The mechanism of transcription induced chimerism (TIC) contributes to fusion transcripts in normal human tissues. However, there is no clarity about the role of TIC in carcinogenesis. Hybrid proteins resulting from chimeric genes regarded as ideal markers which are specific for disease entities can be potential targets for the treatment due to their key roles in malignant transformation. In some tumors fusion genes may play primary role, and in the others may represent an additional mechanism during subclonal selection. The aim is to briefly review and discuss the occurrence and biologic relevance of chimeric genes in hematologic malignant diseases, sarcomas and epithelial neoplasms.

[Analysis of SYT/SSX1 and SYT/SSX2 fusion genes from synovial sarcoma].

The t(X;18)(p11;q11) translocation has been shown to be the specific alteration for synovial sarcomas. The translocation leads to production of chimeric protein SYT/SSX by fusion of SYT and SSX genes involved. The expression analysis of SYT/SSX1 and SYT/SSX2 chimeric transcripts was performed in formalin-fixed soft tissue tumour specimens and the diagnostic validity of immunohistochemistry, FISH and RT-PCR methods was compared. The chimeric transcripts were detected in 12 from 16 synovial sarcomas: 7 SYT/SSX1 and 5 SYT/SSX2 fusion variants; by fluorescence hybridization in situ (FISH) the translocation was found in 13 from 16 sarcoma samples. As synovial sarcoma represents a diagnostically challenging group, genetic analysis of translocations and chimeric transcripts is an extremely useful confirmatory diagnostic tool providing higher sensitivity than immunohistochemistry markers do.

[The loss of heterozygosity and microsatellite instability analysis in differential diagnostics of leiomyosarcoma and proliferative leiomyoma of the uterus].

Uterine leiomyosarcoma (ULMS) is rare and highly malignant smooth muscle tumor. The different diagnosis between uterine leiomyoma with high proliferative index (ULM) and ULMS is one of the basic problems in the pathology for nowadays. We had investigated the loss of heterozygosity (LOH) and microsatellite instability (MI) to find out a genetic differences between ULM and ULMS. The inicrosatellite analysis was evaluated by PCR using 6 polymorphic markers for chromosomal regions 10q22.1 (D10S1146, D010S218), 10q26.13 (D10S1213), 10p13 (D10S24), 9p21.3 (D9S942), 3p14.3 (D3S1295) in 20 patients with ULMS. 38 patients with ULM were suggested as control group. Our results have demonstrated high frequency allelic imbalance in ULMS samples (average frequency 40%). The comparative analysis between 2 studied groups of patients has been shown higher frequencies of genetic changes for ULMS. Specificity and sensitivity of the LOH and/or MI markers scores 92 and 95% accordingly.

[Molecular diagnosis of liposarcomas: identification of the chimeric genes FUS/CHOP and EWS/CHOP].

This paper presents the results of an analysis the chimeric genes FUS/CHOP and EWS/CHOP in patients diagnosed as having liposarcoma in order to make a differential diagnosis in both soft tissue tumors and various variants of liposarcoma. Liposarcomas were found in 5 of 7 cases of primary tumors: 4 chimeric transcripts of the FUS/CHOP type (5-2), a variant of alternative splicing of the FUS/CHOP type (5-2) with depletion in 14 p.n. anda rare variant of the EWS/CHOP type (7-2). Fluorescence in situ hybridization (FISH) confirmed translocations in the tumor samples with the chimeric genes being detected. Reverse transcription-polymerase chain reaction and FISH revealed no chimeric genes specific to myxoid sarcoma in a group of patients with other variants of liposarcoma. Thus, the findings support the strict specificity of the chimeric genes FUS/CHOP and EWS/CHOP for myxoid liposarcoma and the expression of these genes in most tumors of this type.

[Frequent allelic losses in tumor-associated stromal cells and tumor epitelium of prostate cancer].

It has become increasingly clear that tumor microenvironment plays a critical role in carcinogenesis. Accumulation of genetic alterations is typical not only for cancer epithelial cells but tumor-associated fibroblasts as well. Tumor epithelia, tumor-associated stroma from prostatectomy specimens of patients with prostate cancer and cells from prostatic intraepithelial neoplasia (PIN) and adjacent stroma from males with PIN were isolated by using laser capture microdissection. Microsatellite allelotyping was evaluated using 4 highly polymorphic markers for chromosomal regions 8p22, 16q23-24 and 13q14. Incidences of alterations (loss of heterozygosity or allelic imbalance) were 48% for region 8p22, 72% for 16q23 and 37% for 13q14. The LOH frequencies in tumor-associated stroma cells were very similar. Alterations at chromosome 13q were significantly associated with advanced tumor stage, whereas AI at 16q was also associated with high Gleason score and lymph node metastasis. We find some incidences of allelic imbalance in premalignant lesions in epithelial (16-27%) and stromal (7-22%) components. Our results show that the frequencies of genetic aberrations are as high in stromal cells as in tumor cells.

[Abberant methylation of p16, HIC1, N33 and GSTP1 genes in tumor epitelium and tumor-associated stromal cells of prostate cancer].

The methylation status of four genes significant in prostate carcinogenesis p16, HIC1, N33 and GSTP1, were evaluated using quantitative methylationsensitive polymerase chain reaction. Tumor epithelia, tumor-associated stroma, normal epithelia, foci of PIN and benign prostate hyperplasia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens of patients with localized prostate cancer by using laser capture microdissection. We found high levels of gene methylation in the tumor epithelium and tumor-associated stromal cells and some methylation in both hyperplastic epithelium and stromal cells in normal-appearing tissues located adjacent to tumors. Promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may play an important role in cancer development and progression. We examined the promoter methylation status of pl6, HIC1, N33 and GSTP1 in prostate biopsy fragments and prostate tissues after radical prostatectomy from patients with adenocarcinoma without laser capture microdissection. Methylation frequencies of all genes in tumor samples were considerably lower than frequencies in microdissected tumour samples (HIC1, 71 versus 89%; p16, 22 versus 78%; GSTP1, 32 versus 100%; N33, 20 versus 33%). The laser capture microdissection is required procedure in methylation studies taking into account multifocality and heterogenity of prostate cancer tissue.

[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.

[Aberrant methylation of tumor suppressor genes and allelic imbalance in cervical intraepitelial neoplasia].

We analysed 42 high-grade CIN or CIN3 samples, 42 nondysplasia tissues adjacent to CIN3. 35 smears from women without gynecological pathology were also evaluated. Methylation status of six genes (p16, MLH1, HIC1, MGMT, N33 and RB1) was determined using methylation-sensitive PCR. There is some insignificant level of methylation determined in normal smears. Methylation percentages of the genes in CIN3 were: p16, 58%; MLH1, 51%; HIC1, 84%; N33, 27%. Methylation percentages of the genes in nondysplasia adjacent tissues were also high. There is no significant difference in methylation frequencies of MGMT and RB1 determined between dysplasia and control. We identified allelic imbalance at chromosomes 5q11-q14 and 13q14 in 21% cases (9/42). The incidence of LOH was investigated in 7% (3/42) cases at region 13q14.