Karol Kajo

RASSF1A and CDH1 hypermethylation as potential epimarkers in breast cancer

Breast cancer is the most common cancer in women worldwide, representing 28.2% of all female malignancies. In addition to genetic changes, epigenetic events, as aberrant DNA methylation and histone modification, are responsible for cancer development. Many tumour suppressor genes are inactivated by DNA hypermethylation, which could be utilized for identification of new epigenetic biomarkers. To investigate the relation between DNA methylation level and breast cancer progression, we analysed DNA methylation in RASSF1A and CDH1 promoters using quantitative multiplex methylation-specific PCR in paraffin-embedded tumour tissues and blood samples from 92 breast cancer patients and 50 controls, respectively. The associations between RASSF1A and CDH1 methylation levels and clinico-pathological parameters were tested by Kruskal-Wallis and van der Waerden ANOVA tests. Out of 92 breast cancer patients, 76 (82.6%) manifested various levels of RASSF1A (range from 1.20 to 92.63%) and 20 (21.7%) of CDH1 (range from 1.20 to 79.62%) methylation. However, no methylation was found in 50 controls. Increasing trends in RASSF1A methylation were observed in tumour size, lymph node status and TNM stage, but only CDH1 methylation levels showed statistically significant differences between the patient subgroups in lymph node status and IHC subtype. Overall, stable relatively high RASSF1A methylation could be utilised as universal tumour marker and the less frequent but highly methylated CDH1 promoter can serve for identification of potentially metastasising tumours.

Prognostic value of programmed-death-1 receptor (PD-1) and its ligand 1 (PD-L1) in testicular germ cell tumors

BACKGROUND Testicular germ cell tumors (TGCTs) belong to the most chemosensitive solid tumors; however, a small proportion of patients fail to be cured with cisplatin-based chemotherapy. Inhibitors of PD-1/PD-L1 pathways represent a new class of promising drugs in anticancer therapy. The aim of this study was to evaluate expression and prognostic value of PD-1 and PD-L1 in TGCTs. PATIENTS AND METHODS Surgical specimens from 140 patients with TGCTs (131 with primary testicular tumor and 9 with extragonadal GCTs) were included into the translational study. PD-1 and PD-L1 expression was detected in the tumor tissue by immunohistochemistry using monoclonal antibodies, scored by the multiplicative quickscore (QS) method, compared with their expression in normal testicular tissue and correlated with clinicopathological characteristics and clinical outcome. RESULTS None of the GCTs exhibited PD-1 protein, although expression of PD-L1 was significantly higher in GCTs in comparison with normal testicular tissue (mean QS = 5.29 versus 0.32, P < 0.0001). Choriocarcinomas exhibit the highest level of PD-L1 with decreasing positivity in embryonal carcinoma, teratoma, yolk sac tumor and seminoma. PD-L1 expression was associated with poor prognostic features, including ≥3 metastatic sites, increased serum tumor markers and/or non-pulmonary visceral metastases. Patients with low PD-L1 expression had significantly better progression-free survival [hazard ratio (HR) = 0.40, 95% confidence interval (CI) 0.16-1.01, P = 0.008] and overall survival (HR = 0.43, 95% CI 0.15-1.23, P = 0.040) compared with patients with high PD-L1 expression. CONCLUSIONS In this translational study, we showed, for the first time, the prognostic value of PD-L1 expression in TGCTs and our data imply that the PD-1/PD-L1 pathway could be a novel therapeutic target in TGCTs.

Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer.

Endometrial cancer is a common gynecological malignancy with a good prognosis in early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we examined the methylation status of the GSTP1, CDH1 and RASSF1A genes in endometrioid endometrial cancer (EEC), endometrial complex hyperplasia (EHP) and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk and clinicopathological parameters. A nested two-stage methylation-specific PCR (MSP) was performed to analyze the promoter CpG methylation status of GSTP1, CDH1 and RASSF1A genes in the population studied. A total of 92 subjects were initially included in the study of which 41 EEC, 19 EHP and 20 controls were processed for final analyses. A significant difference was found between the study groups and the presence of promoter CpG hypermethylation status in the GSTP1 (p<0.05) and RASSF1A (p<0.0001) genes. RASSF1A, GSTP1 and CDH1 gene promoter methylation was present in 85.4, 68.3 and 31.4% of EEC samples when compared to that in the controls with 30.0, 35.0 and 20.0%, respectively. CpG methylation of all three investigated tumor-suppressor genes was found in 12.2% of EEC patients, in 4.2% of EHP patients and in 3.7% of the controls, respectively. Positive findings for the promoter methylation of two investigated genes were found in 48.7% of EEC patients, 26.0% of EHP patients and in 18.5% of the controls. With regard to histopathological variables and CpG methylation, we found significant correlations between the RASSF1A and GSTP1 genes and higher tumor grade, deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes. No associations were noted between promoter hypermethylation of the CDH1 gene and biological features of the endometrial cancer cases. The results indicate that aberrant CpG methylation of the promoter region in the GSTP1 and RASSF1A tumor-suppressor genes is an important event in carcinogenesis of endometrial cancer and may have an impact on tumor aggressiveness. Finally, the present study suggests that epigenetic alterations may be of diagnostic value for the better clinical management of premalignant endometrial lesions.

Melatonin potentiates the anti-tumour effect of pravastatin in rat mammary gland carcinoma model

Previous studies in the field of cancer research have suggested a possible role for statins in the reduction of risk in certain malignancies. The purpose of these studies was to examine the chemopreventive effects of pravastatin alone and in combination with pineal hormone melatonin in the N‐methyl‐N‐nitrosourea‐induced mammary carcinogenesis model. Pravastatin was given orally (1 00 mg/kg) and melatonin was added to the water (20 μg/ml). Chemoprevention began seven days prior to carcinogen administration and subsequently continued for 15 weeks until autopsy. At autopsy, mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. Parameters of experimental carcinogenesis, mechanism of action (biomarkers of apoptosis, angiogenesis and proliferation) and side effects after long‐term treatment in animals were assessed. Pravastatin alone suppressed tumour frequency by 20.5% and average tumour volume by 15% compared with controls. Combined administration of the drugs decreased tumour frequency by 69% and lengthened tumour latency by nine days compared with control animals. The ration between high and low grade carcinomas was apparently reduced in both treated groups. The analysis of carcinoma cells showed significant expression increase in caspase‐3 and caspase‐7 after pravastatin treatment; however, combined treatment even more pronounced increase in the expression of both caspases. Regarding VEGFR‐2 expression, a small effect in carcinomas of both treated groups was found. In plasma metabolism evaluation, pravastatin alone significantly decreased levels of glucose and triacylglycerols. Our results suggest a mild anti‐neoplastic effect of pravastatin in this rat mammary gland carcinoma model. Statins co‐administered with other suitable drug (e.g. melatonin) should be further evaluated for tumour‐preventive properties.

The p53 Codon 72 Exon 4 BstUI Polymorphism and Endometrial Cancer in Caucasian Women

Objectives: Studies on the association between the p53 Arg72Pro polymorphism and endometrial cancer have reported contrasting conclusions. With the exception of Asian subjects, data demonstrating the influence of this polymorphism on endometrial carcinogenesis in other races/ethnic groups, including Caucasians, are scarce. Thus, we aimed to investigate its role in the development of endometrial cancer and its association with prognostic markers. Methods: A case-control study examining the p53 codon 72 polymorphism in a total of 451 samples (121 cancer patients, 330 healthy controls) using polymerase chain reaction and sequencing techniques was conducted. Genotypes were correlated with clinico-pathological factors and age. Logistic regression analyses were used to adjust for possible confounding variables, and data were evaluated using the Pearson χ2 test. Results: We found the Pro allele and genotype frequency to be insignificantly higher in cases than controls (Pro allele: 24.8 and 22.3%, respectively; genotypes: Arg/Pro 36.36 and 34.24%, Pro/Pro 6.61 and 5.15%, respectively). Logistic regression analysis revealed an increased risk for disease in carriers of the Pro allele, with an odds ratio (OR) of 1.13 [95% confidence interval (CI) 0.73–1.76] for heterozygotes and an OR of 1.36 (95% CI 0.56–3.30) for homozygotes. Furthermore, we noted a trend for Arg/Pro + Pro/Pro towards poor tumour differentiation, angioinvasion, pelvic lymph node spread and type II carcinomas, with ORs of 1.27 (95% CI 0.60–2.66), 1.24 (95% CI 0.67–2.30), 1.21 (95% CI 0.53–2.75) and 1.69 (95% CI 0.70–4.10), respectively. Additionally, the Pro genotype was associated with a lower risk for cancer in women with early menarche (OR 1.17, 95% CI 0.60–2.28) and late menopause (OR 0.70, 95% CI 0.30–1.63). Despite the increased or decreased risk observed for some variables, none of these trends were significant. Conclusions: Our data did not demonstrate any significant difference in the prevalence of the p53 Arg72Pro genotype between patients and controls, providing evidence that this polymorphism is only weakly associated with the risk of endometrial cancer and prognostic factors in Caucasian women.

Resveratrol enhances the chemopreventive effect of celecoxib in chemically induced breast cancer in rats.

Resveratrol and celecoxib were used as chemopreventive agents in animal models of carcinogenesis, and exert antiproliferative and proapoptotic effects on cancer cells. Therefore, the aim of this study was to evaluate whether combining resveratrol with celecoxib may exert more potent anticarcinogenic effects than the single agents. Mammary carcinogenesis was initiated in 70 female Sprague–Dawley rats with N-methyl-N-nitrosourea (NMU). The chemoprevention with resveratrol, celecoxib, and their combination started 2 weeks before the first carcinogen dose and lasted until the end of the experiment. Tumor incidence and frequency, latency period, tumor volume, the expression of cyclooxygenase 2 (COX2) and growth differentiation factor 15 (GDF15), and also the formation of reactive oxygen species were analyzed using different methods. In addition, the levels of resveratrol and its metabolites in blood and selected tumor tissues were determined by high-performance liquid chromatography. Finally, the anticancer effects of the reagents were studied in the human breast cancer cell line MCF-7. Celecoxib as a single agent significantly decreased tumor frequency, prolonged tumor latency, and decreased the total number of malignant tumors compared with the NMU conditions. Tumor volume was nonsignificantly reduced (0.68±0.25 vs. 0.93±0.28 cm3). Importantly, the addition of resveratrol to celecoxib reduced tumor volume by 60% compared with celecoxib alone (from 0.68±0.25 to 0.27±0.07 cm3, P<0.05). Furthermore, the combination of resveratrol and celecoxib reduced tumor frequency by 29% compared with celecoxib alone (P=0.53). Tumor latency was not influenced by this combination compared with celecoxib alone (126.56±3.45 vs. 120.71±4.08 days). In addition, COX2 mRNA and immunoreactive protein stained on tumor sections were reduced and GDF15 protein increased significantly by the combination studied compared with the NMU conditions. In agreement with these data, a significant reduction in reactive oxygen species in blood lymphocytes of the combination was detected, which may have contributed toward the cancer-preventive effects of this application. This study showed that in NMU-induced mammary cancer in rats, the combination of resveratrol and celecoxib led to a significant reduction in all tumor parameters. In addition, in terms of tumor volume, the combination was more efficient than celecoxib as a single agent.

Pioglitazone in chemically induced mammary carcinogenesis in rats.

Data available from in-vitro and in-vivo studies suggest oncostatic properties of peroral antidiabetics, thiazolidinediones, in many types of cancer. This study is the first report on the chemopreventive effect of pioglitazone in mammary carcinogenesis in rats. Mammary carcinogenesis was induced by N-methyl-N-nitrosourea administered in two intraperitoneal doses per 50 mg/kg bodyweight on the 43rd and 50th postnatal days. Pioglitazone was administered in the diet at concentrations of 10 and 100 ppm, respectively, 12 days before the first carcinogen dose until the termination of the experiment. During the experiment, the animals were weighed weekly and palpated for the presence of mammary tumors, and the incidence, latency, tumor frequency, and tumor volume were recorded. The experiment was terminated 17 weeks after the first carcinogen dose; basic tumor growth parameters and metabolic and hormonal variables were evaluated. Pioglitazone at higher concentration decreased incidence and frequency per group from the 11th week of experiment when compared with the control group and a group receiving a lower dose. Pioglitazone at a higher dose decreased the final incidence by 38%, frequency per group by 63%, and extended latency period by 32% when compared with the control group. Our data suggest that pioglitazone and other glitazones should be further investigated for oncopreventive effects.

Human epithelial growth factor receptor 2[Ile655Val] polymorphism and risk of breast fibroadenoma.

Studies on the association between the Ile to Val polymorphism at codon 655 of the human epithelial growth factor receptor 2 (HER-2) gene and susceptibility to breast cancer have been reported for almost all ethnic populations, with both positive or negative conclusions. No study, however, has yet been focused on the possible association between this gene and its predisposition to benign breast lesions, especially on risk for fibroadenoma. We aimed to study the association of the single nucleotide polymorphism V655 HER-2 gene polymorphism with histologically verified breast fibroadenoma risk. We conducted a molecular epidemiological case–control study of 70 breast fibroadenoma cases without cellular atypia and 172 healthy female controls. We found that the Val variant allele and genotype frequency of this polymorphism is higher in cases with fibroadenoma; however, this difference was not significant (allele Val 655: 27.86 and 22.67% in fibroadenoma and controls, respectively; genotype Ile/Val: 35.71 and 38.37% and Val/Val: 10.0 and 3.49% in fibroadenoma and controls, respectively). Applying logistic regression analysis, we found an increased risk of fibroadenoma formation in carriers of the Val allele (odds ratio=1.17; 95% confidence interval=0.67–2.05), in which the highest risk was associated with homozygous genotype (odds ratio=3.07; 95% confidence interval=0.97–9.72), but this risk was not significant. Stratification by age (cut-off 45 years) revealed the highest risk of fibroadenoma among young women homozygous for the Val allele (odds ratio=3.30). The risk, however, was slightly increased (odds ratio=1.24) among older carriers of the aberrant allele in their genotype as well, but it was not significant. In spite of insignificant differences, our results indicate that HER-2 Ile655Val polymorphism, especially in a homozygous form might play some role in the etiology of breast fibroadenoma formation. The significance of this susceptibility, however, will have to be verified by larger studies.

Immunohistochemical and histomorphological analysis of rat mammary tumors after simvastatin treatment

The results of experimental studies have indicated the pleiotropic effects of statins in organism, e.g. the influence on cell cycle, apoptosis or angiogenesis. In this study, the effects of simvastatin on selected parameters of apoptosis and proliferation in chemocarcinogen-induced mammary tumorigenesis in female rats were determined. Simvastatin was administered dietary at a dose of 18 mg/kg and highly effective dose of 180 mg/kg the entire experiment (18 weeks). At autopsy mammary tumors were removed and prepared for immunohistochemical and histomorphological analysis. In treated animals (simvastatin 180 mg/kg), significant decrease by 12% in Bcl-2 protein expression and non-significant decrease by 27% of Ki67 protein expression in tumor cells compared to tumor cells in control animals were observed after semiquantitative evaluation. Morphometrical analysis has shown significant proapototic shift in Bcl-2/Bax ratio in tumor cells. In high grade control carcinoma cells, the expression of Ki67 increased by 37% (non-significantly) in comparison with control low grade carcinomas. A histomorphological analysis of malignant tumors has revealed a shift from high grade to low grade carcinomas after simvastatin treatment. The noticeable decrease of mammary tumor frequency and incidence in rats after simvastatin treatment was accompanied with antiapoptotic Blc-2 protein decrease and proapoptotic Bax protein increase in this experiment.

Myoid (Muscular) Hamartoma of the Breast: Case Report and Review of the Literature

Background: Myoid (muscular) hamartoma (MH) of the breast is a rare benign tumour-forming lesion composed of differentiated mammary glandular and stromal structures, fatty tissue, and areas of smooth muscle. It is considered to be a variant of mammary hamartoma. Case Report: We report the case of a 46-year-old woman with MH, and provide a literature review explaining the origin of smooth muscle cells. Histologically, the tumour consisted of fibrolipomatous stroma containing ductal and lobular structures of the mammary gland located mainly at the tumour borders. The glandular structures showed signs of micro- and macrocystic changes, apocrine metaplasia, and adenosis. The dominant feature was the presence of a fascicular formation of spindle cells, predominantly in central parts, with incursion between glandular structures. Immunohistochemically, foci of smooth muscle tissue were positive for desmin, smooth muscle actin, and h-caldesmon. Oestrogen and progesterone receptors (PR) showed positive expression which was markedly higher for PR. There was negative expression of CD34, S-100 protein, and CD10. Conclusions: The origin of smooth muscle cells in MH is unknown. However, it is presumed to be derived from hormonally responsive breast stromal cells by smooth muscle metaplasia, based on evidence of hormone receptor expression in the lesion.