Pawel Surowiak

Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer.

In the last decade, optimized treatment for non-small cell lung cancer had lead to improved prognosis, but the overall survival is still very short. To further understand the molecular basis of the disease we have to identify biomarkers related to survival. Here we present the development of an online tool suitable for the real-time meta-analysis of published lung cancer microarray datasets to identify biomarkers related to survival. We searched the caBIG, GEO and TCGA repositories to identify samples with published gene expression data and survival information. Univariate and multivariate Cox regression analysis, Kaplan-Meier survival plot with hazard ratio and logrank P value are calculated and plotted in R. The complete analysis tool can be accessed online at: www.kmplot.com/lung. All together 1,715 samples of ten independent datasets were integrated into the system. As a demonstration, we used the tool to validate 21 previously published survival associated biomarkers. Of these, survival was best predicted by CDK1 (p<1E-16), CD24 (p<1E-16) and CADM1 (pu200a=u200a7E-12) in adenocarcinomas and by CCNE1 (pu200a=u200a2.3E-09) and VEGF (pu200a=u200a3.3E-10) in all NSCLC patients. Additional genes significantly correlated to survival include RAD51, CDKN2A, OPN, EZH2, ANXA3, ADAM28 and ERCC1. In summary, we established an integrated database and an online tool capable of uni- and multivariate analysis for in silico validation of new biomarker candidates in non-small cell lung cancer.

Melatonin stimulates the activity of protective antioxidative enzymes in myocardial cells of rats in the course of doxorubicin intoxication.

Abstract: The study aimed at determining the effect of melatonin on the activity of protective antioxidative enzymes in the heart and of lipid peroxidation products in the course of intoxication with doxorubicin (DOX). The rats were categorized into four groups, receiving: 0.9% NaCl i.p. (NaCl control); melatonin [20u2003mg/kg body weight (b.w.)] s.c. (control Mel); DOX (2.5u2003mg/kg b.w.) i.p.; melatonin plus DOX in doses as above. All the substances were administered once in a week for four consecutive weeks. Homogenates of heart tissue were examined for activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), levels of reduced glutathione (GSH) and of lipid peroxidation indices (MDAu2003+ 4‐HDA). Administration of melatonin alone did not induce alterations in levels of MDAu2003+u20034‐HDA, GSH, or in activity of GPx, SOD or CAT, as compared to the group receiving 0.9% NaCl. GSH levels decreased following DOX but remained at normal levels following DOX and melatonin. The level of MDAu2003+u20034‐HDA increased following DOX, as compared with the control, a change prevented by the combination of DOX+melatonin. Activities of GPx, SOD and CAT were higher in groups receiving DOX and/or DOX plus melatonin than in control groups. Activity of CAT and the level of GSH in the group receiving DOX plus melatonin were significantly higher than in the group intoxicated with DOX alone. The obtained results demonstrate that, when given in parallel with DOX, melatonin protects cardiomyocytes from damaging effects of the cytostatic drug (reflected by the levels of MDAu2003+u20034‐HDA). The protective effect resulted, in part from the augmented levels of GSH and from stimulation of CAT activity by melatonin in cardiomyocytes subjected to the action of DOX.

Estrogen Receptor Alpha Expression in Ovarian Cancer Predicts Longer Overall Survival

Estrogen as a potential factor of ovarian carcinogenesis, acts via two nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), but the cellular signal pathways involved are not completely clear so far. In this study we have described the expression of ERα, detected by immunocytochemistry in 11 ovarian carcinoma cell lines and by immunohistochemistry in 43 Federation Internationale des Gyneacologistes et Obstetristes stage III ovarian carcinoma specimens prepared before and after treatment with cisplatin-based schemes. For cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma, analysis of cisplatin sensitivity in 11 ovarian carcinoma cell line was also performed. The strong nuclear ERα expression was only shown in the single A2780P cell line. Expression of ERα in tissue specimens did not reveal any correlations between histopathological parameters (histologic type and grading). We demonstrated a significant association with ERα expression in specimens from primary laparotomies (PL) and cause–specific survival. In the cases terminated by death of the patient, overall immunoreactivity score of ERα expression at PL was significantly lower than in surviving patients. In addition, Kaplan-Meier analysis revealed significantly shorter overall survival time and progression-free time in cases with lower immunoreactivity score of ERα expression at PL. Our findings support the hypothesis that aberrant hormone activity, by way of altered receptor expression, might be an important factor in the malignant transformation of ovarian cancer.

Role of exogenous melatonin in reducing the nephrotoxic effect of daunorubicin and doxorubicin in the rat

Abstract: u2002The aim of these studies was to examine the nephroprotective effect of melatonin following the anthracycline administration [daunorubicin (DNR); doxorubicin (DOX)] in rats. Application of these drugs in chemotherapy is limited because of their cardiotoxicity and nephrotoxicity. Rats of the Buffalo strain were divided into groups according to the cytostatic drug used, its dose and sequence of administration [DNR or DOX single (i.v.) dose of 10u2003mg/kg b.w., i.e. acute intoxication and 3u2003mg/kg b.w. (i.v.) weekly for 3u2003wk, subchronic intoxication]. Melatonin was administered subcutaneously before and after every injection of a cytostatic drug at a dose of 10u2003mg/kg b.w. The severity of renal alterations was examined both biochemically [levels of lipid peroxidation markers, malonyldialdehyde (MDA) and 4‐hydroxyalkenals (4‐HDA)], or histologically. A statistically significant decrease in renal damage was noted after melatonin administration to acutely or subchronically intoxicated DNR‐treated and DOX‐treated rats. Biochemical assays revealed significant decreases in MDA and 4‐HDA levels following application of melatonin during subchronic DNR or DOX intoxication. In summary, melatonin was found to exert a protective effect on the kidney, which was particularly evident after subchronic DOX and DNR intoxication, using both histological or biochemical methods.

ERM/Rho protein expression in ductal breast cancer: a 15 year follow-up.

PurposeThe aim of this study was to examine the expression of ERM (ezrin, moesin) and Rho (RhoA, RhoB and Cdc42) proteins in breast cancer (BC) patients and to investigate the relationship between the sub-cellular localisation of these proteins and clinicopathological characteristics and patient survival.MethodsThe expression and specific sub-cellular distribution of the ERM/Rho proteins was analysed by immunohistochemistry in a homogeneous group of 85 stage II ductal BC patients with a follow-up of 15xa0years.ResultsEnhanced immunoreactivity of all analysed proteins was found to be associated with the presence of lymph node metastases (ezrin, Pu2009=u20090.047, moesin, Pu2009=u20090.038, RhoA, Pu2009=u20090.024, RhoB, Pu2009=u20090.004 and Cdc42, Pu2009=u20090.047). Nuclear localisation of ezrin was found to correlate with the presence of lymph nodes metastases (Pu2009=u20090.004) and with histological de-differentiation (Pu2009=u20090.015). In contrast, we found that the nuclear topography of RhoA and Cdc42, and the perinuclear localisation of RhoB, were strongly associated with a lack of nodal metastases (Pu2009=u20090.008, Pu2009=u20090.048, Pu2009=u20090.001, respectively), whereas a decreased reactivity of RhoA in the stromal compartment of BC tumours was associated with the presence of lymph node metastases (Pu2009=u20090.011). No relationship was observed between ERM/Rho protein expression and oestrogen receptor (ER), progesterone receptor (PgR) or HER-2 reactivity in the BC cells. Also, ERM/Rho protein expression did not predict patient survival, but RhoB over-expression in the stromal compartment of the tumours was found to be associated with a better prognosis (Pu2009=u20090.0106).ConclusionsThe ERM/Rho immunoprofile and the assessment of its specific sub-cellular localisation may be instrumental for the prediction of lymph node metastases in ductal BC patients.

Role of exogenous melatonin in reducing the cardiotoxic effect of daunorubicin and doxorubicin in the rat

The aim of the studies was to examine the cardioprotective effect of melatonin during the anthracycline administration (daunorubicin, doxorubicin) in rats. Application of these drugs in chemotherapy is limited because of their cardiotoxicity. Rats of Buffalo strain were divided into groups according to the cytostatic drug used, its dose and sequence of administration (single intravenous [i.v.] dose of 10 mg/kg b.w., i.e., acute intoxication; 3 mg/kg b.w. weekly for 3 weeks, subchronic intoxication). Melatonin was administered subcutaneously before and after every injection of a cytostatic drug at a dose of 10 mg/kg b.w. The degree of cardiac muscle cell alterations was examined either histologically (Mean Total Score technique and the Billingham scale), or biochemically (levels of lipid peroxidation markers, malonyldialdehyde, and 4-hydroxyalkenals). Statistically significant decrease in cardiac muscle cell damage was noted with an aid of the Billingham scale after melatonin administration in acutely intoxicated doxorubicin-treated rats (p < 0.001). The similar phenomenon was observed using the Mean Total Score technique in case of acute daunorubicin or doxorubicin (p < 0.01 and p < 0.001, respectively) intoxications. A significant reduction in cardiac muscle cell lesions was detected either by the Billingham scale or by the Mean Total Score technique during subchronic intoxication with either of the anthracyclines when melatonin was given. Biochemical assays revealed significant decreases in malonyldialdehyde and 4-hydroxyalkenals levels following application of melatonin during either acute doxorubicin (p < 0.05) or subchronic daunorubicin (p < 0.01) intoxication. In summary, melatonin was found to exert a protective effect on the cardiac muscle cells, which was particularly evident after acute doxorubicin or subchronic daunorubicin intoxication, using either histological or biochemical methods.

Increased expression of vascular endothelial growth factor in bone marrow of multiple myeloma patients.

BACKGROUNDnAngiogenesis (neovascularization) is a multistep process in which new blood vessels grow from existing vessels. Angiogenesis is associated with the growth, dissemination, and metastasis of solid tumors. There is increasing evidence that neovascularization may be important in hematological malignancies. Several studies suggest that vascular endothelial growth factor (VEGF) is one of the most important cytokines responsible for the development, maintenance, and progression of multiple myeloma (MM) by promoting bone marrow angiogenesis. A high serum concentration of VEGF has been reported in MM patients. The aim of this study was to evaluate the expression of VEGF in the bone marrow of MM patients.nnnMETHODSnEighteen paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MM were evaluated. In addition, 10 bone marrow core biopsy specimens from adult patients without evidence of malignancy were used as controls. Bone marrow sections were stained immunohistochemically for VEGF.nnnRESULTSnOur data show that multiple myeloma is associated with an increased expression of VEGF in the bone marrow.nnnCONCLUSIONSnOur observation supports previous studies suggesting that angiogenesis may play a role in the pathophysiology of hematopoietic malignancies. The clinical significance of this phenomenon needs further investigation. However, this study provides rationale for the use of angiogenesis inhibitors in MM therapy.

Correlation between PARP-1 immunoreactivity and cytomorphological features of parthanatos, a specific cellular death in breast cancer cells.

In parthanatos, a PARP-1 (poly (ADP-ribose) polymerase 1)-mediated cell death, dissipation of mitochondrial membrane potential, large-scale DNA fragmentation and chromatin condensation were observed. In contrast to apoptosis, it does not cause apoptotic bodies formation. Although PARP-1-mediated cell death presents loss of membrane integrity similar to necrosis, it does not induce cell swelling. The purpose of the study was to correlate the immunohistochemical parameters of PARP-1 reactivity and the selected cytomorphological features of parthanatos: the lack of apoptotic bodies and the absence of necrosis in breast cancer (BC) specimens. Immunohistochemistry for PARP-1 was performed on 83 BC specimens. Correlations between parameters of PARP-1 expression and sub-cellular localisation and the presence of apoptotic bodies and necrosis were evaluated. High expression of PARP-1 (immunoreactive score ≥6) was associated with the lack of apoptotic bodies (P=0.013) and with the absence of necrosis (P=0.002). The presence of apoptotic bodies was correlated with re-distribution of PARP-1 from the nucleus to cytoplasm in BC cells (P=0.029). Additionally, a tendency was observed between necrosis and loss of nuclear PARP-1 expression (P=0.049). Our study suggests that PARP-1 may play a crucial role in induction and regulation of specific type of cellular death called parthanatos.

MRP2 (ABCC2, cMOAT) expression in nuclear envelope of primary fallopian tube cancer cells is a new unfavorable prognostic factor.

ObjectiveTo determine the prognostic value of the immunohistochemical evaluation of the multidrug resistance-associated protein 2 (MRP2) expression, together with its subcellular localization in primary fallopian tube carcinomas (PFTCs).MethodsThe immunohistochemical analysis was performed using samples originating from 70 patients with PFTCs.Results(1) We documented that MRP2 can be localized in the plasma membrane (MRP2c), as well as in the nuclear envelope (MRP2n) of the PFTC cells. (2) Patients with more advanced stage, with progression of the disease and patients who died, showed significantly higher expression of the MRP2n. (3) Univariate and multivariate analyses showed that MRP2n is an unfavorable prognostic factor in PFTCs. (4) The analysis of the classic clinicopathological data revealed that only the FIGO stage had prognostic value, both in the univariate, as well as in multivariate analysis.Conclusions(1) This study suggests that MRP2n is a new disadvantageous prognostic factor in PFTCs and (2) that expression in nuclear envelope can be associated with lower differentiation of cancer cells and their resistance to the cisplatin. (3) We have also confirmed independent prognostic value of FIGO stage in PFTCs.

Nuclear-cytoplasmic PARP-1 expression as an unfavorable prognostic marker in lymph node‑negative early breast cancer: 15-year follow-up.

PARP-1 plays an important role in DNA damage repair and maintaining genome integrity by repairing DNA single-strand breaks (SSBs) by base excision repair (BER). The aim of the present study was to examine the expression of PARP-1 in breast cancer (BC) patients and to assess the relationship between the subcellular localization of this protein and clinicopathological characteristics. The reactivity of PARP-1 was analyzed by immunohistochemistry in a homogeneous group of 83 stage II ductal BC patients with a 15-year follow-up. Immunostaining of PARP-1 was also evaluated in 4 human BC cell lines and resistance prediction profile for 11 anticancer agents was performed using 3 models of drug-resistant cell lines. Nuclear-cytoplasmic expression (NCE) was associated with shorter overall survival, which was not statistically significant during the 10-year follow-up but became statistically significant after 10 years of observation, during the 15-year follow-up (P=0.015). Analysis performed in subgroups of patients with (N+) and without (N-) nodal metastases showed that NCE was associated with poor clinical outcome in N- patients (P=0.017). Multivariate analysis confirmed a significant impact of NCE on unfavorable prognosis in N- early BC. The presence of PARP-1 NCE may be a new potential unfavorable prognostic factor in lymph node- negative early BC.