PSMB7 is associated with anthracycline resistance and is a prognostic biomarker in breast cancer.

BACKGROUND: To date individual markers have failed to correctly predict resistance against anticancer agents in breast cancer. We used gene expression patterns attributable to chemotherapy-resistant cells to detect potential new biomarkers related to anthracycline resistance. One of the genes, PSMB7, was selected for further functional studies and clinical validation. METHODS: We contrasted the expression profiles of four pairs of different human tumour cell lines and of their counterparts resistant to doxorubicin. Observed overexpression of PSMB7 in resistant cell lines was validated by immunohistochemistry. To examine its function in chemoresistance, we silenced the gene by RNA interference (RNAi) in doxorubicin-resistant MCF-7 breast cancer cells, then cell vitality was measured after doxorubicin treatment. Microarray gene expression from GEO raw microarray samples with available progression-free survival data was downloaded, and expression of PSMB7 was used for grouping samples. RESULTS: After doxorubicin treatment, 79.8+/-13.3% of resistant cells survived. Silencing of PSMB7 in resistant cells decreased survival to 31.8+/-6.4% (P>0.001). A similar effect was observed after paclitaxel treatment. In 1592 microarray samples, the patients with high PSMB7 expression had a significantly shorter survival than the patients with low expression (P<0.001). CONCLUSION: Our findings suggest that high PSMB7 expression is an unfavourable prognostic marker in breast cancer.

Nuclear maspin expression as a good prognostic factor in human epithelial ovarian carcinoma.

Maspin, a protein belonging to the serpin superfamily, seems to exert tumour-suppressive activity. Its significance in ovarian cancer prognosis is currently under investigation. In the present work, immunocytochemical maspin expression in 132 invasive epithelial ovarian carcinomas was assessed independently in the nucleus and cytoplasm, in correlation with histopathological and clinical data. Positive maspin expression was found in 117 cases: nuclear/cytoplasmatic in 71, exclusive nuclear in 29, and only cytoplasmatic in 17 cases. Cytoplasmatic maspin expression was positively correlated with tumour grade (p = 0.000), FIGO stage (p = 0.002), and distant metastases (p = 0.000) but exhibited no significant correlation with tumour type (p = 0.078). Nuclear maspin expression showed negative correlation with tumour grade (p = 0.025), FIGO stage (p = 0.05), distant metastases (p = 0.001), and cancer remission (p = 0.000) but showed no significant relationship with the patients' age (p = 0.948) or cancer subtype (p = 0.261). Kaplan-Meier survival analysis showed that strong cytoplasmatic maspin expression was correlated with shorter disease-free survival (p = 0.000) whereas strong nuclear expression was correlated with longer survival (p = 0.000). In Cox regression analysis, low nuclear maspin expression (score 2 and 3) remained a significant independent prognostic factor (p = 0.001) with a relative death risk of 5.337. The obtained results suggest that maspin expression may be a significant marker in epithelial ovarian carcinoma prognosis with its nuclear expression being a good prognostic factor.

[YB-1 as a potential target in cancer therapy]. / YB-1 als potenzielles Ziel für die Tumortherapie.

The 42-kDa multifunctional cellular protein Y-box protein 1 (YB-1) is expressed in various cancers. It is localized in the cytoplasm as well as in the nucleus. In particular, YB-1 is localized in the nuclear compartment following cellular stress, such as radiation, drug treatment, hyperthermia, or viral infection. Within the nucleus, YB-1 can act as a transcription factor, and it is involved in the regulation of important cancer-associated genes. For example, YB-1 triggers the expression of Her-2 and estrogen receptor alpha (ERalpha) in breast cancer. Thus, nuclear YB-1 appears to be a potential target for the inhibition of Her-2- and ERalpha-dependent proliferation signals, particularly with regard to resistance to Her-2-targeting drugs such as trastuzumab. In some cancers, YB-1 may be involved in regulating MDR1/P-glycoprotein, mediating classical multidrug resistance (MDR). Furthermore, YB-1 is involved in the replication of adenovirus type 5, a commonly used vector in gene therapy. Thus, YB-1 can trigger an "oncolytic" effect in YB-1 nuclear positive cancer cells treated with adenoviruses. Besides its impact as a prognostic factor, in the future the diagnostics of cellular YB-1 status may provide the basis for a virotherapy or a gene therapy incorporating adenoviruses.

Prediction of the response to chemotherapy in ovarian cancers.

Ovarian cancer represents the fifth most frequent cause of death as a result of malignant processes after cancers of the breast, large intestine, lung and stomach. Owing to the localisation of ovarian cancer, approximately 75% of cases are diagnosed at the III and IV stages of advancement according to FIGO. Because of the advanced stage of the disease surgery has to be followed by chemotherapy in most cases of ovarian cancer and therefore resistance to cytostatic drugs represents a major clinical problem. The potential to predict the response to therapy with the use of cytostatic drugs would enable the most effective drugs to be applied in individual cases, thus improving the efficiency of the treatment and restricting the development of resistance to cytostatic drugs. In the present paper the progress made so far in the prediction of the clinical course of ovarian cancer is reviewed. The significance of the expression of the ATP-binding cassette (ABC) transporters is described, including P-glycoprotein and MRP2, the principal representatives of the protein group. The importance of disturbed control of apoptosis and the overexpression of HER-2 and topoisomerase 1A are also discussed. Two sections are devoted to the most recent studies in the biology of ovarian cancer, pangenomic studies on gene expression using DNA microarrays and aberrations of DNA methylation.

Elevated metallothionein (MT) expression in invasive ductal breast cancers predicts tamoxifen resistance.

Elevated expression of the low molecular weight metallothionein (MT) proteins can be found typically in breast cancer cases with less favourable prognosis. The MT gene has been described to be potentially down-regulated by estrogen receptor alpha. The present study is aimed at examining the predictive value of MT expression for results of tamoxifen treatment in breast cancer in relation to steroid receptor status. Sixty patients with primary invasive ductal breast cancers with post-operative tamoxifen treatment were enrolled in the study. In paraffin sections of the studied tumours immmunohistochemical reactions were performed using antibodies directed against MT, estrogen receptors (ER) and progesterone receptors (PgR). Results of the immunohistochemical reactions and of clinical observations were analysed using multivariate progression analysis based on the Cox proportional hazard model. Elevated MT expression was demonstrated to be typical for cases with documented relapse of the disease (P<0.001) or terminated by death (P=0.03). Decreased ER expression was found to be typical for cases of a higher grade (P=0.02) and cases terminated by death (P=0.006). The multivariate analysis showed that elevated MT expression was characteristic for cases with shorter overall survival time (P=0.04). The data showed that MT carried an independent, and also independent from ER status, unfavourable predictive value as far as results of tamoxifen treatment were concerned.

Prognostic significance of metallothionein expression in correlation with Ki-67 expression in adenocarcinomas of large intestine.

The study aimed at determining levels of metallothionein (MT) and Ki-67 antigen expression in adenocarcinomas of large intestine and examining relation of the expression levels with various clinical and pathological variables. The studies were performed on 81 cases of large intestine adenocarcinoma. Using immunocytochemistry, expressions of MT (positive reaction in 73 cases) and of Ki-67 (positive reaction in 79 cases) antigen were examined and the obtained results were compared with, i.a., grade (G) of the tumour and depth to which intestinal wall was infiltrated by individual tumours. Patient survival analysis was also performed, as correlated to expression levels of the two antigens. The obtained results permitted to disclose that the lower was grade of histological differentiation (G2, G3), the more pronounced was expression of MT and Ki-67. Also, the deeper was neoplastic infiltration of intestinal wall, the more pronounced was MT and Ki-67 expression. Despite the relatively strong correlation between MT expression and Ki-67 expression (r=0.536; p<0.05), only Ki-67 antigen expression in large intestine adenocarcinomas was inversely correlated to survival of the patients. Ki-67 proved to be a better prognostic marker, as compared to MT, in large intestine adenocarcinomas.

Expression of metallothionein in synovial sarcoma cells.

Other authors have demonstrated earlier that cells of normal synovium contain metallothionein. The protein was also detected in several other normal cell types and in tumors derived from the cells. Metallothionein content is thought to reflect proliferative activity of neoplastic cells. Therefore, it was decided to demonstrate metallothionein expression in various types of synovial sarcoma. The present study aimed to determine metallothionein cellular expression by immunocytochemical techniques in nine cases of biphasic, six cases of monophasic (spindle cell), and five cases of poorly differentiated synovial sarcoma, and to compare the expression with those of vimentin and cytokeratin 19. Metallothionein expression was demonstrated in epithelioid cells in all cases of biphasic type sarcoma and in spindle cells in all cases of monophasic type tumors. In poorly differentiated tumors, metallothionein expression was detected in four of five cases (80%). Expression of cytokeratin 19 was typical for epithelioid cells and expression of vimentin for spindle cells of synovial sarcoma. A much less pronounced expression of the proteins was observed in poorly differentiated tumors. The results indicate that metallothionein expression may prove useful in differential diagnosis and for defining prognosis in cases of synovial sarcomas.

Correlation of histopathological and biochemical appraisal of anthracyclin-induced myocardium damage.

Treatment of various tumor types with anthracycl in cytostatic drugs (DNR--daunorubicin and DOX--doxorubicin) is limited by their high cardiotoxicity. This study was aimed at examining effectiveness of histopathological appraisal of myocardial cell injury induced by the cytostatic drugs and evaluated according to Billingham and by MTS methods as compared to results of parallel biochemical assessment of lipid peroxidation indices (MDA- malonyldialdehyde, 4-HDA-4-hydroxyalkenes). The experiments were performed on rats intoxicated with DNR or DOX in an acute manner (1 x 10 mg/kg body weight, i.v.) or a subchronic manner (3 x 3 mg/kg body weight. i.v.). Significant positive correlations were demonstrated between results of histological and biochemical appraisal in rats intoxicated in the acute manner with DNR (r=(0.91), intoxicated in the subchronic manner with DNR (r=0.90) and in rats intoxicated in the acute or subchronic manner with DOX (r=0.91, r=0.77, respectively). The obtained results have confirmed the free radical mechanism of cardiomyocyte injury induced by anthracyclines and the applied techniques of evaluating the destruction may be used independently of each other.

Suitability of tissue prints for immunocytochemical diagnosis of mammary cancer.

The present study was aimed at evaluating suitability of tissue prints for immunocytochemical evaluation of mammary cancer cells. The prints, originating from 30 cases of mammary cancer were studied using immunocytochemical reactions with monoclonal antibodies against estrogen and progesterone receptors, metallothionein (MT), P-glycoprotein and cytokeratins (clone LP34). Expression of individual antigens was assessed using the scale in which intensity of the colour reaction and percentage of positive cells were taken into account. The obtained results did not differ qualitatively or quantitatively from those obtained in paraffin sections. The studies have shown that tissue prints can be used for reliable immunocytochemical evaluation of expression of various proteins in mammary cancer cells.

Analysis of estrogen receptor (ER) and estrogen-dependent pS2 protein expression in cells of mammary ductal carcinoma.

Not every case of mammary carcinoma with expression of estrogen receptors (ER) responds by remission to anti-estrogen treatment. Possible causes of the phenomenon may involve abnormalities of the receptor or defects in mechanisms of signal transmission. One of the ways in which function of the receptor may be detected involves examination of expression of the antigens which appear as a consequence of estrogen stimulation, e.g., expression of pS2 protein. The present study was aimed at comparing ER and pS2 expression in cells of mammary carcinoma, and hence, at finding out whether the presence of ER is equivalent to the sensitivity to estrogen action. In paraffin sections of ductal mammary carcinoma samples obtained from 56 patients, immunocytochemical reactions were performed using monoclonal antibodies against ER and pS2. The results documented positive correlation between the presence of ER and the presence of pS2 in cells of mammary carcinoma (Spearman's rank correlation: r=0.43, p <0.001). Thus, the intensity of pS2 expression was directly related to the expression of ER and the latter was found to be functional.

Prognostic value of immunocytochemical estimation of estrogen receptor (ER) and of pS2 estrogen-dependent protein in cells of mammary ductal carcinoma. Analysis of five-year course of the disease.

The pS2 protein belongs to the so called estrogen-dependent proteins, which appear in cells as a result of estrogen stimulation. The present study was aimed at determining prognostic value of estrogen receptor (ER) and pS2 protein expression in mammary cancer cells. The immunocytochemical reactions were performed in paraffin sections of tissue samples from 62 patients with ductal mammary carcinoma using monoclonal antibodies against ER and pS2. The analysis included also survival time of the patients, determined during the five-year observations. The studies documented a correlation between survival time and the level of ER expression (p=0.014) and absence of correlations between survival time and pS2 expression (p=0.55). The results indicate that evaluation of ER expression in mammary cancer cells may assist in defining prognosis of the patients while parallel estimation of pS2 expression in the cells is useless in this respect.

Immunocytochemical evaluation of metallothionein (MT) expression in myoepithelial cells of ductal mammary carcinoma and its relation to survival time: analysis of 7-year course of the disease.

Myoepithelial cells participate in the development of mammary glands and have been suggested to play a role in the biology of mammary cancer. Recent studies demonstrated in the cells expression and overexpression of metallothionein (MT): a low molecular weight, cystein-rich protein which participates, i.a., in the multidrug resistance to cvtostatic drugs. The present study was aimed at examining the relation between results of immunocytochemical (ICC) detection of MT expression in myoepithelial cells, present in structures of a ductal mammary carcinoma, and survival of the patients. In sections originating from 43 patients with ductal mammary carcinoma ICC reactions were performed to detect MT and to confirm the presence of myoepithelial cells (using antibodies to smooth muscle actin). Survival of the patients was also determined in the course of 7-year observations. Statistical analysis using the Coxe's model did not detect relations between MT expression intensity, in the myoepithelium on one hand and patient survival on the other (chi2=0.003 p=0.96).

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.

[The analysis of estrogen and pregesterone receptors in leiomyomas cell in different phases of menstrual cycle]. / Analiza ekspresji receptorów estrogenowych i progesteronowych w komórkach miesniaków macicy w zaleznosci od fazy cyklu miesiecznego.

The aim of a study was immunocytochemical analysis of estrogen (ER) and progesterone (PgR) receptors in uterine leiomyomas cells derived from patients who underwent an operation in three different phases: in the follicular phase of the menstrual cycle (12 cases), in the luteal phase (20 cases) and in the postmenopausal age (18 cases). The examined receptors reflect the tissue sensibility to activity of sex steroid hormones and play an important role in the pathogenesis of uterine leiomyomas. Performed studies showed that expression of ER and PgR does not depend on the phase of the menstrual cycle and that it seems to be similar like in the postmenopausal stage. In all groups PgR expression was significantly higher then the expression of ER. In the group of patients in which the surgery was performed during the luteal phase of the menstrual cycle was demonstrated negatively correlation (-0.4846) between the expression of ER in relation to the expression of PgR.

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.

A comprehensive overview of targeted therapy in metastatic renal cell carcinoma.

Chemotherapy and immunotherapy failed to deliver decisive results in the systemic treatment of metastatic renal cell carcinoma. Agents representing the current standards operate on members of the RAS signal transduction pathway. Sunitinib (targeting vascular endothelial growth factor), temsirolimus (an inhibitor of the mammalian target of rapamycin - mTOR) and pazopanib (a multi-targeted receptor tyrosine kinase inhibitor) are used in the first line of recurrent disease. A combination of bevacizumab (inhibition of angiogenesis) plus interferon α is also first-line therapy. Second line options include everolimus (another mTOR inhibitor) as well as tyrosine kinase inhibitors for patients who previously received cytokine. We review the results of clinical investigations focusing on survival benefit for these agents. Additionally, trials focusing on new agents, including the kinase inhibitors axitinib, tivozanib, dovitinib and cediranib and monoclonal antibodies including velociximab are also discussed. In addition to published outcomes we also include follow-up and interim results of ongoing clinical trials. In summary, we give a comprehensive overview of current advances in the systemic treatment of metastatic renal cell carcinoma.

Unfavorable prognostic value of CD24 expression in sections from primary and relapsed ovarian cancer tissue.

Expression of CD24 represents a poorly recognized, unfavorable prognostic factor. Expression of the protein is supposed to facilitate extravasation of tumor cells. Our study aimed at examination of prognostic significance of CD24 estimation in samples obtained from primary surgeries (PS) and secondary cytoreductions (SCR) (after chemotherapy) in ovarian cancer patients. The analyses were performed on sections originating from 73 tumor samples. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD24. Kaplan-Meier's analysis showed that a significantly shorter overall survival time and progression-free time was demonstrated to characterize cases with cytoplasmic membranous expression of CD24 (CD24c-m) (P < 0.001). The calculations performed demonstrated also a significantly higher proportion of CD24c-m positive cases in sections from SCR as compared to that from PS (P= 0.04) and in cases of progressive disease as compared to complete response at PS and SCR (P= 0.002 and P= 0.05, respectively). Summing up, in this study, we have demonstrated a negative prognostic significance of a cytoplasmic membranous expression of CD24 in cases of ovarian cancer.

Topoisomerase 1A, HER/2neu and Ki67 expression in paired primary and relapse ovarian cancer tissue samples.

In the present study we examined prognostic value of immunohistochemical estimation of topoisomerase 1A (TOP 1A) and HER-2/neu expression in ovarian cancers treated with platinum-based drugs but not with topotecan and the relation between expression of these proteins on the one hand and intensity of proliferation (Ki67) on the other. The analyses were performed on 73 samples of ovarian carcinoma originating from 43 first-look laparotomies (FLL) and, in 30 cases, from secondary cytoreductions (SCR)(after chemotherapy) from the same patients. In paraffin sections immunohistochemical reactions were performed using antibodies directed to HER-2/neu, TOP 1A and Ki67. Kaplan-Meier's analysis disclosed a shorter overall survival time in cases with augmented expression of TOP 1A at FLL and with higher expression of Ki67 at SCR. A shorter progression-free time was detected in cases with higher proportion of Ki67 positive cells at FLL. No relationship could be disclosed between HER-2/neu expression and the studied clinicopathological parameters. The studies confirmed high value of Ki67 estimation. The augmented expression of TOP 1A was demonstrated to represent an unfavourable prognostic factor. Thus, in cases with elevated expression of TOP 1A application of topotecan-based therapeutic schemes should be considered.

Expression of aminopeptidase N/CD13 in human ovarian cancers.

Aminopeptidase N/CD13 (EC 3.4.11.2) is suggested to play a role in cancer cells invasion, and its activity can be inhibited using specific inhibitors. CD13 inhibitors evoke apoptosis of CD13-positive cancer cells. However, expression of CD13 has not been described in specimens obtained from ovarian carcinomas. Thus, in the present study, the expression of CD13 and its significance was examined in samples of ovarian cancers. The analyses were performed on sections originating from 73 tumor samples (43 from primary laparotomies [PL] and 30 from secondary cytoreductions [SCRs]). Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD13. The analysis demonstrated no relationships between the expression of CD13 on one hand and clinical variables and pathologic variables of the patients on the other hand. Expression of CD13 was demonstrated to be significantly more pronounced in samples obtained in PLs as compared to samples from SCRs (P < 0.001). Thus, the data indicate that a potential treatment of ovarian carcinoma with CD13 inhibitors should be performed before chemotherapy or in parallel to first-lapse chemotherapy.