Oksana Kowalczuk

Expression of selected tumor suppressor and oncogenes in endometrium of women with endometriosis.

BACKGROUND It is becoming increasingly evident that the eutopic endometrium of women with endometriosis shows certain genetic alterations which are not found in the endometrium of disease-free women. The aim of the study was to compare the expression level of mammalian target of rapamycin (mTOR) tumor suppressor and oncogene-related genes in the endometrium of women with and without endometriosis as well as in ovarian endometriosis. METHODS A total of 81 regularly menstruating patients were recruited in the study. We applied the micro fluidic gene array to examine the expression of 15 human tumor suppressor and oncogenes in eutopic endometrium of 40 women with endometriosis and 41 controls without endometriosis. In 14 patients with endometriosis, gene expression was also studied in matched ovarian lesions. We studied the following genes: NF1, RHEB, mTOR, PTEN, TSC1, TSC2, KRAS, S6K1, TP53, EIF4E, LKB1, PIK3CA, BECN1, 4EBP1 and AKT1. Immunohistochemical studies were subsequently performed for selected proteins. RESULTS Of the 15 studied genes, we found significantly higher levels of oncogene AKT1 (P = 0.006) and tumor suppressor gene 4EBP1 (P = 0.01) mRNAs in the eutopic endometrium of women with endometriosis compared with control patients. Immunohistochemistry showed that 4EBP1 and AKT1 proteins were expressed in eutopic endometrium. CONCLUSIONS Our results suggest that up-regulation of AKT1 and 4EBP1 in eutopic endometrium may be associated with the pathogenesis of endometriosis, but their precise role remains to be established.

Circulating free DNA, p53 antibody and mutations of KRAS gene in endometrial cancer

This study was conducted to evaluate the significance of circulating free DNA (CFDNA), p53 antibody (p53‐Ab) and mutations of KRAS gene in the development of endometrial cancer (EC). A total of 109 patients with EC (87 patients with Type I and 22 patients with Type II) took part in this study. KRAS mutations and CFDNA were detected by means of the PCR‐RFLP and enriched by the PCR‐RFPL method. ELISA was used to analyze plasma p53‐Ab. Tissue expression of P53 protein was evaluated immunohistochemically (IHC). The frequency of KRAS mutations was especially high in Grade 2 of Type I EC. CFDNA was frequently detected in patients with early stage of Type II EC at a low level of grade. It is noteworthy that the p53‐Ab positive rate increased in the higher grade of Type I tumors. A significant difference in the number of cases with the p53‐Ab was found in the advanced stage of Type I tumors. The frequency of KRAS and p53‐Ab correlates with tumor stage only in the Type I EC. Plasma CFDNA and p53‐Ab offer a chance to develop a procedure for EC Type II diagnosis. The association between tumor cells related to CFDNA and p53‐Ab with Type II tumor suggests that it might potentially serve as a marker in predicting the prognosis and offers a possibility to individualize treatment regimen.

Circulating free DNA and p53 antibodies in plasma of patients with ovarian epithelial cancers

BACKGROUND This study was conducted in order to evaluate the significance of circulating free DNA (CFDNA), blood plasma p53 antibodies (p53-Ab) and mutations of KRAS gene in the prognosis of ovarian epithelial cancers. PATIENTS AND METHODS A total of 126 patients were included in this study. KRAS mutations and CFDNA were detected by means of the PCR-restriction fragment length polymorphism (PCR-RFLP) and enriched by the PCR-RFLP method. Enzyme-linked immunosorbent assay was used to analyze plasma p53-Ab. RESULTS KRAS mutations were detected in 27 (21.4%) of examined tumors. The frequency of KRAS mutations was especially high in mucinous cancers (P < 0.001). CFDNA and p53-Ab were frequently detected in patients with serous cancers in high grade (P < 0.001). The overall survival rate was significantly lower for patients with serous tumors and CFDNA and p53-Ab-positive than negative tumors (P = 0.022 and P < 0.001, respectively). In mucinous ovarian cancer, a worse overall survival was correlated with the KRAS mutations (P = 0.03). CONCLUSIONS The results of the present study suggested that a presence of KRAS mutations in mucinous ovarian cancer and CFDNA and p53-Ab in serous tumors was correlated with the highest risk of cancer progression.

Circulating levels of TNF-α and its soluble receptors in the plasma of patients with epithelial ovarian cancer

The significance of circulating levels of TNF-alpha and its soluble receptors (sTNF-Rs) in the plasma of patients with epithelial ovarian cancer (EOC) has not been fully elucidated. The present study was to investigate the relationship of pretreatment plasma levels of TNF-alpha, sTNFR-1 and sTNFR-2 with outcome in 126 patients with EOC. Concentrations of TNF-alpha and sTNF-Rs were determined by enzyme-linked immunosorbent assay (ELISA). Median TNF-alpha and sTNF-Rs levels were significantly higher in EOC patients than in healthy controls. High plasma levels of TNF-alpha and sTNF-Rs were correlated with tumor stage and with reduced mean survival time (MST). The results of the present study suggested that preoperative plasma TNF-alpha and sTNF-Rs levels in EOC patients correlated with the highest risk of cancer progression. Thus, the clinical value of an activated TNF system in EOC needs to be further investigated.

CXCL5 as a potential novel prognostic factor in early stage non-small cell lung cancer: results of a study of expression levels of 23 genes

As the current staging system is imprecise for estimating prognosis of early stage non-small cell lung cancer (NSCLC), it is important to identify other methods for selecting high-risk patients after failed surgical treatment. The aim of the study was to evaluate the expression of 23 genes as putative prognostic markers in early stage NSCLC. The study was performed on 109 pairs of tumor and matched unaffected lung tissue surgical specimens taken from stage I and II NSCLC patients. We evaluated the mRNA level of 23 genes using the real-time PCR method. The difference in the expression between the tumor and normal tissue for each gene was analyzed using a general linear model. The influence of gene expression on survival was analyzed by using the proportional hazards model. Eighteen out of the 23 genes showed statistically significant differences in expression between the tumor and non-tumor tissue. For 12 genes (ITGB1, ITGB3, CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCR3, CXCR4, TNF, CHKA, AGFG1, and CTC1), the expression was lower, and for six genes (ITGA5, IL8, IL6, CXCL2, CXCL3, and CXCL12), it was higher in the tumor tissue as compared to the matched normal tissue. Expression changes were more pronounced in squamous cell carcinomas than in adenocarcinomas or large cell carcinomas. Of all the analyzed genes, only CXCL5 was found to statistically significantly (p = 0.04) influence both overall and disease-free survival. Among the 23 genes previously suggested to be relevant for early staged NSCLC patients’ postoperative outcome, only CXCL5 showed a statistically significant prognostic effect.

Serum vascular endothelial growth factors C and D in patients with oesophageal cancer.

OBJECTIVE Lymph node metastasis is a characteristic of malignant cancers and is observed more frequently in oesophageal cancer than in other digestive tract cancers, making it one of the most important prognostic factors. Vascular endothelial growth factors C (VEGF-C) and D (VEGF-D) are important lymphangiogenic factors in human cancers and lymphangiogenesis is associated with lymph node metastasis. The aim of the study was to determine the correlation between pre-treatment serum levels of VEGF-C (sVEGF-C) and VEGF-D (sVEGF-D) and clinicopathologic features in patients with oesophageal cancer. METHODS Serum VEGF-C and sVEGF-D were measured by enzyme-linked immunoadsorbent assay (ELISA) on 149 patients with oesophageal cancer, 29 patients with benign oesophageal diseases and 30 healthy controls. RESULTS Serum VEGF-C and sVEGF-D levels were significantly higher in patients with oesophageal carcinoma than in the control group (p<0.001 and p=0.001, respectively) or in the benign oesophageal diseases group (p=0.04 and p=0.03, respectively). Subgroup analysis showed that lymph node metastasis (p=0.001), stage (p=0.001), tumour depth (p=0.006), resectability (p=0.002), tumour size (p=0.01), distant metastases (p=0.01) and histological grading (p=0.04) were correlated with an elevated level of sVEGF-C. Elevated levels of sVEGF-D were associated with tumour depth (p=0.002), stage (p=0.01) and lymph node metastasis (p=0.02). Among the patients (n=83) who underwent potentially curative surgery, the overall survival time (p=0.008) was shorter for patients with a high level (>8667 pg ml(-1)) of sVEGF-C than for those with a low level (<8667 pg ml(-1)), when the cut-off value was determined on the basis of the median value in oesophageal cancer patients. On univariate regression analysis, tumour size, tumour depth, stage, lymph node metastases, distant metastases, resectability and sVEGF-C were found to be significant prognostic factors. CONCLUSIONS These results suggest that pre-treatment levels of sVEGF-C and sVEGF-D reflect lymph node metastases and advanced stage of oesophageal cancer. Serum VEGF-C may be useful in predicting poor outcome for patients undergoing a potentially curative oesophagectomy.

Mutations of the KRAS oncogene in endometrial hyperplasia and carcinoma.

The aim of this study was to examine the prevalence and clinicopathological significance of KRAS point mutation in endometrial hyperplasia and carcinoma. We analysed KRAS in 11 cases of complex atypical hyperplasia and in 49 endometrial carcinomas using polymerase chain reaction associated with restriction fragment length polymorphism (PCR-RFPL). Point mutations at codon 12 of KRAS oncogene were identified in 7 of 49 (14,3%) tumor specimens and in 2 of 11 (18,2%) hyperplasias. No correlation was found between KRAS gene mutation and age at onset, histology, grade of differentiation and clinical stage. We conclude that KRAS mutation is a relatively common event in endometrial carcinogenesis, but with no prognostic value.

Serum levels of VEGF and VEGF-C in patients with endometrial cancer

Endometrial cancer (EC) is the most common type of uterine cancer. A dualistic model of endometrial tumorigenesis serves as a useful way of categorizing these cancers in terms of both etiology and clinical behavior. There are two types of EC: type I and type II. Type I is so-called estrogen-dependent, and appears mostly in pre- and perimenopausal women, it is well differentiated and therefore has a better prognosis. Type II EC is estrogen-independent, diagnosed mostly in postmenopausal women, thin and fertile women, or in women with normal menstrual cycles. It is aggressive and has a worse prognosis than type I. The aim of this study was to evaluate the relationship between the pretreatment serum levels of VEGF and VEGF-C and the outcome of EC patients. A total of 98 patients treated between 1999 and 2003 were included in this study. Circulating VEGF and VEGF-C levels were determined using ELISA kits. VEGF levels among the 76 patients with type I, and the 22 patients with type II EC were significantly higher than those found in the healthy control subjects (p < 0.001). The differences in mean values of VEGF-C were highly significant in both types of tumor examined compared to the control (p < 0.001). The results demonstrate that serum VEGF concentration correlated significantly with advanced FIGO stage in type II EC (p < 0.001). The preoperative VEGF-C level correlated with advancing tumor stages in type I EC (p < 0.05). An elevated preoperative VEGF-C was an independent risk factor for disease-specific survival in patients with type II tumors. Thus, in type II EC patients with high preoperative levels of VEGF-C, pelvic and para-aortic lymphadenectomy should be performed. However, the value of longitudinal measurements of the markers used is yet to be determined.

High prevalence of genotype 4 among hepatitis C virus-infected intravenous drug users in north-eastern Poland.

The aim of this study was to describe the distribution of hepatitis C genotypes among intravenous drug users in north‐eastern Poland. The study group included intravenous drug users recruited at a drug treatment center and a clinic for HIV‐infected patients. HCV infection was confirmed by qualitative nested RT‐PCR to test for the presence of HCV RNA. Genotypes were determined by 5′UTR sequencing and comparing the results with known genotype sequences. Among 111 HCV‐infected and HCV–RNA‐positive intravenous drug users, the most prevalent genotypes were 1 (38.7%), 3 (37.8%), and 4 (23.4%). Most infections with genotype 4 (88.5%) were found among HCV–HIV‐coinfected drug users. The study demonstrated a high prevalence of genotype 4 (23.4%) among HCV‐infected Polish drug users. J. Med. Virol. 80:615–618, 2008.

Selective gene expression profiling of mTOR-associated tumor suppressor and oncogenes in ovarian cancer

The aim of this study was to selectively profile the activation status of mammalian target of rapamycin (mTOR)-associated oncogenes and tumor suppressor genes (TSGs) in ovarian cancer specimens, healthy ovaries and benign ovarian tumors, including endometrial cysts. We used a novel type of microfluidic gene array to examine the expression of 15 human tumor suppressors and oncogenes in ovarian cancer specimens of 53 patients, benign ovarian cysts of 29 women (endometrial and simple) and 11 healthy ovaries of individuals in whom the material was obtained during total hysterectomies performed because of fibroid changes. The array was custom-designed to include the following genes: NF1, RHEB, mTOR1, AKT-1, PTEN, TSC1, TSC2, KRAS, RPS6KB1, 4EBP1, TP53, EIF4E, STK11, PIK3CA and BECN1. Confirmatory immunohistochemical detection was performed for a group of selected proteins. Particularly significant differences were observed as to the expression of PTEN (p < 0.0001), TP53 (p = 0.0003), PIK3CA (p = 0.0003) and BECN1 (p = 0.0014) which were shown to be downregulated in cancer patients when compared to healthy ovaries and benign ovarian cysts (endometrial and simple). These markers did not show association with grade or stage of the tumor. Immunohistochemistry showed that PTEN, TP53, PIK3CA and BECN1 proteins are expressed in ovarian cancer. Our results indicate that there are significant differences in the expression of some of the mTOR-related tumor suppressors and oncogenes which could be associated with the pathogenesis of ovarian cancer.