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Background: Caspase-8 and caspase-9 (encoded by CASP8 and CASP9) are executive caspases of programmed cell death (apoptosis). Dysregulation of apoptosis plays an important role in cancer development, progression, and resistance to anticancer therapy. The goal of this work was to evaluate potential associations between polymorphisms in CASP8 and CASP9, previously linked to breast cancer risk, and the transcript levels of these genes (including their alternative anti-apoptotic variants) in tumor tissues and the clinical characteristics of the patients., Material and Methods: Sanger sequencing, high resolution melting (HRM) analysis, and allelic discrimination were used to identify polymorphisms in DNA samples isolated from tumor tissues and peripheral blood lymphocytes of 60 breast carcinoma patients. Total transcript levels of CASP8 and CASP9, and levels of alternative splicing variants CASP8L and CASP9B, were quantified by real-time PCR in tumor tissues. Clinically interesting associations were validated in DNA from lymphocytes of 615 breast carcinoma patients., Results: A haplotype in CASP9 composed of three polymorphisms rs4645978-rs2020903-rs4646034 was significantly associated with CASP9 expression in tumors, with the expression of the progesterone receptor and ERBB2, and with the TNBC subtype of breast carcinoma in the validation study. The associations between the rs3834129 polymorphism in CASP8 and stage of disease, rs6435074 with grade, expression of estrogen receptor and ERBB2, and rs6723097 with ERBB2 expression have not yet been validated. However, rs6723097 was associated with disease-free survival in patients treated with hormonal therapy., Conclusion: This study reveals a previously unknown and presumably functional (in silico) association between a haplotype in CASP9 and molecular and clinical phenotypes of breast carcinoma. The potential clinical utility of this association for prognostication of breast carcinoma should be evaluated by independent studies.Key words: breast carcinoma - caspases - polymorphisms - functional - clinical - importanceThis work was supported by grant of the CU Grant Agency No. 1444313, and grant of the Internal Grant Agency of the Czech Ministry of Health No. 15-25618A.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 3. 3. 2016Accepted: 26. 10. 2016.

Objective: The aim of the study was the analysis of the new molecular genetic immunomarkers (p53, c-erbB-2, Ki 67, bcl-2) hormonal receptors (ER, PR) and ploidy disturbances and their relation to the most important prognostic factors for endometrial cancer., Design: Prospective study., Setting: Dept. of Gynaecology and Obsterics, Laboratory of Experimental Medicine, Institute of Pathology, Institute of Molecular and Transplational Medicine, Medical Faculty and University Hospital, Olomouc., Methods: The study group consisted of 88 endometrial cancer patients. The biopsies of the tumours obtained at operations were routinely histopathologically examined. Subsequently, the immunohistochemical tumormarkers were determined. The same biopsies were examined by microdissection and flow cytometric ploidy analysis and karyotyping. The findings were compared with the most important prognostic factors for endometrial cancer, mainly with clinical stage of the disease, grade and histopathological type., Results: Aneuploidy was found in 71% in the group of poorly differentiated endometrial cancers (G3) in contrast to 47% in the group of G1 and G2 tumours. High expression of p53, Ki 67, c-erbB-2 and low rate of sex hormone receptors was found in the prognostically unfavourable group (G3)., Conclusions: Aneuploidy seems to be an important prognostical factor for endometrial cancer patients. Identification of p53, Ki 67, c-erbB-2, ER a PR is a useful tool to specify a group of prognostically unfavourable patients.

Breast cancer patients with a tumor size of < or = 1 cm are still rare but their number is increasing as a consequence of mammary screening. It is logical that the best adjuvant treatment in such cancers is often discussed with respect to the risk of relapse of the disease. The number of patients in clinical trials with very small breast cancer is quite low, and a specific clinical trial for such patients is not planned. Retrospective analysis of some clinical trials which included patients with breast cancer and with very small size shows the worse prognosis of patients with HER2 positive tumors. Indirectly, we can assume the application of adjuvant treatment with trastuzumab in such very small breast cancer. However, the decision should be individual with regards to further risk factors for the disease and risk of the treatment itself. This article is more a contemplation in order to provoke discussion on this provocative subject.

Backgrounds: Metastatic breast cancer is a disease which is not curable. Thus, prolongation of survival with preserved or improved quality of life is the aim of the treatment. Three phase III studies (E2100, AVADO and RIBBON-1) showed the benefit of adding bevacizumab to the standard 1st line chemotherapy. Higher response rate and longer progression-free survival were achieved in these studies. Bevacizumab does not increase toxicity of the chemotherapy regimens. Since 2007 bevacizumab has been registered for the treatment of patients with metastatic breast cancer., Observation: Here we present the case of a patient in which bevacizumab treatment led to excellent results. Lobular breast cancer, pT2N0M0, ER-negative, PR-negative, HER2-negative was diagnosed in a 40-year-old woman in 2003. FAC adjuvant chemotherapy was used. Six years later, in March 2009, a relapse in mediastinal lymphatic nodes, the lungs, pleura and bones was detected. A weekly regimen of paclitaxel in combination with bevacizumab started in May 2009. Paclitaxel treatment finished in November 2009, bevacizumab continued for 11 months till April 2010, when complete remission in the lungs, mediastinum and bones was confirmed. Now only bisphosphonate is being continued., Conclusion: Our experience also confirms the contribution of bevacizumab in the treatment of metastatic breast cancer.

Objective: To assess the immunohistochemical expression of p53, bcl-2, c-erb-2, Ki-67, estrogen and progesterone receptors in endometrial cancer patients., Design: Experimental study., Setting: Department of Obstetrics and Gynecology, Institute of Human Genetics, Department of Pathology, Palacky University Medical School and University Hospital, Olomouc., Methods: We studied 103 cases of primary untreated endometrial carcinoma in which the p53, bcl-2, c-erb-2, Ki-67, estrogen and progesterone receptor antigens were investigated by an immunohistochemical method. We evaluated the correlations among the immunohistochemical staining assessed by histoscore, and the age, grading, depth of invasion, stage of the neoplasia and extrauterine disease., Results: Mean age was 67 years (range 35-90). p53, bcl-2, c-erb-2, Ki-67, estrogen and progesterone receptors were positive in 49 (48%), 81 (79%). 18 (17%), 99 (96%), 73 (70%) and 87 (84%) patients respectively. There was no clear association between immunohistochemical parameters and the age of patients. p53 and Ki-67 overexpression was found to be related to poor grade of differentiation, deeper myometrial invasion, advanced stage of neoplasia and extrauterine spread of disease. Immunostaining for bcl-2 correlated inversely with FIGO stage, while c-erb-2 was overexpressed in tumors with deeper myometrial invasion. Estrogen and progesterone receptor positive tumors showed a statistically significant association with clinicopathological parameters of better clinical outcome., Conclusion: The overexpression of p53 and Ki-67 seems to indicate more malignant phenotype, while bcl-2 and c-erb-2 may have a limited role in the identification of high-risk tumors.

Introduction: Trastuzumab is a humanized monoclonal antibody directed against the HER-2 receptor. Trastuzumab-based therapy significantly improves response rate (RR), time to progression (TTP) and overall survival (OS) for women with HER-2 positive metastatic breast cancer. Despite its initial efficacy, acquired resistance to trastuzumab develops in a majority of patients with MBC, and a large subset never responds, demonstrating primary resistance. The purpose of this retrospective study was to determine prognostic factors applicable to clinical practice., Methods: We enrolled 112 women with metastatic breast cancer, who started the trastuzumab-based therapy at Masaryk Memorial Cancer Institute until January 2007. Clinical and laboratory factors, such as: patients conditions, character ofmetastatic spread, histology, estrogen, progesterone and Her-2 receptor status, Her-2/neu gene amplification, and serum tumor markers CEA, CA 15-3 and extracellular domain of Her-2 receptor (S-HER-2 ECD) were monitored. The association of all factors to response to therapy, time to progression (TTP) and overall survival (OS) was assessed., Results: In 95% patients, the trastuzumab was combined with cytostatics (83% taxanes), 88,4% of patients started the trastuzumab as the first or second-line anticancer treatment. The median TTP was 284 days (9,3 months) and the median OS was 612 days (20,1 months) for all patients, RR was 54,5%. The highest RR was associated with the first-line treatment (p<0.0001) and with HER-2 gene/Chromosome 17 ratio > 2,2 (p=0,0092). Eleven patients (9,8%) discontinued the treatment because of toxicity, 7 patients did it as a result of cardiotoxicity (6,2%). CNS metastases occurred in 31 patients (27,7%). The S-HER-2 ECD was the most frequently elevated serum marker at the time of the treatment initialization (72,5%) and at the time of the progression (55,9%). Cox regression analysis identified S-HER-2 ECD levels at the beginning and between day 90 and 130 of the trastuzumab therapy as the best predictors of TTP. On the other hand the best predictor of OS was level of CEA before the treatment started and level of S-HER-2 ECD between day 90 and 130 of the trastuzumab therapy., Conclusions: We confirmed that the only one predictive marker for response to trastuzumab therapy is a proof of HER-2 tumor positivity.The highest prevalence of S-HER-2 ECD positivity among serum tumor markers and the strong association between initial and subsequent S-HER-2 ECD serum concentrations and time to progression and overall survival make the S-HER-2 ECD the most significant prognostic marker.

Background: The latest clinical trials indicate better performance of aromatase inhibitors, compared to tamoxifen, in adjuvant hormonotherapy of breast carcinoma. The identification of molecular markers, predicting resistance to tamoxifen, could help to identify patients, which are most likely to benefit from aromatase inhibitors in up-front adjuvant hormonotherapy., Material and Methods: Tissue microarrays were constructed from archival paraffin blocks of primary tumors of 179 patients with estrogen receptor positive operable breast carcinoma in stage I-III, subsequently treated with tamoxifen for five years or until relapse, with at least 7 years follow up available. The amplifications of Her-2 and cyclin D1 genes were evaluated by fluorescence in-situ hybridization. The level of progesterone receptor (PR) and Ki67 were estimated by immunohistochemistry., Results: 54 of above patients recurred during follow up. In univariate analysis of disease free survival, the presence of more than three nodal metastases (RR=4,5 p<0,001), grade 3 (RR=2,3 p=0,035), cyclin D1 (RR=3,06 p<0,001) and Her-2 (RR=3,06 p<0,001) amplifications were identified as significant risk factors, together with the negativity of PR (RR=2,1 p=0,013). In multivariate analysis, only clinical stage III (RR=2,6 p=0,003), cyclin D1 (RR=2,7 p=0,001) and Her-2 (RR=2,1 p=0,014) amplifications proved significant. In 77 patients who received adjuvant chemotherapy no statistically significant risk factor was identified. In multivariate analysis of 102 patients without adjuvant chemotherapy only stage III (RR=6,9 p=0,001) and Her-2 amplification (RR=4,5 p=0,001) were confirmed., Conclusion: The advanced clinical stage, cyclin D1 and Her-2 gene amplifications represent factors, predicting the failure of adjuvant tamoxifen treatment, but their predictive value is much lower in patients receiving adjuvant chemotherapy. This fact indicates, they can reflect the common biological aggressiveness of tumor and need not to be tamoxifen specific.

Background: HER-2/neu protein overexpression has been shown to be an independently adverse prognostic and predictive factor in patients with breast cancer. Recently, HER-2/neu overexpression has gained therapeutic implications: It has been shown that in patients with breast cancer the use of trastuzumab/Herceptin TM, the recombinant humanized monoclonal antibody directed against extracellular domain of HER-2/neu molecule, can block the HER-2/neu protein activation and bring about a clinical remission. Following these developments, demand for pathologists to evaluating properly HER-2/neu in breast cancer specimens has been rapidly increasing., Methods and Results: In our series of 449 cases of breast cancer, HER-2/neu protein and gene were examined by means of immunohistochemistry and fluorescence in situ hybridization respectively. Results of HER-2/neu study were compared with the hormonal status, cancer grade and proliferation activity as assessed using immunohistochemistry with MIB1 antibody. All seven cases of breast cancer with strong overexpression of HER-2/neu (score 3+) manifested a gene amplification. In contrast, among 11 cases of breast cancers with mild HER-2/neu overexpression (score 2+), the gene amplification was demonstrated in 5 cases only (45%)., Conclusions: Immunohistochemical assessment and fluorescence in situ hybridization (FISH) are complementary methods for detection of HER-2/neu status in breast cancer. While immunohistochemistry is an excellent screening method, FISH should be used for the confirmation of positive results before the Herceptin treatment.

Salivary duct carcinoma (SDC) is highly malignant salivary gland tumour with aggressive clinical behaviour, characterised by its histological resemblance to invasive ductal carcinoma of the breast. Amplification of gene HER-2/neu and overexpression of its gene product have been shown to have both prognostic and treatment implications in breast cancer. The reports concerning the expression of c-erbB2/HER-2/neu in salivary gland tumours are few and controversial. Thus, eleven cases of SDC were evaluated for HER-2/neu status using immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). To the best of our knowledge, this is the first molecular genetic analysis of SDCs using FISH. HER-2/neu overexpression, identified as strong membrane staining, was observed in all but one case of SDC in majority of neoplastic cells while only four tumours, of nine cases analysed, revealed HER-2/neu gene amplification by means of FISH analysis. SDCs were associated with poor clinical outcome, 6 patients (55%) died of disseminated carcinoma within 4 to 44 months after therapy. There was no difference in outcome of patients with IHC positive-nonamplified and IHC positive-amplified tumours.

Routine assessing of HER2 status is necessary for successful treatment of carcinoma of breast by Herceptin. Our results prove comparability of well standardized semiquantitative method by Hercep Test DAKO: both negative findings (73.4% in laboratory A, and 77.4% in laboratory B) and positive findings (26.6% in laboratory A, and 22.6% in laboratory B) corresponded both with each other and with the DAKO's reference data. The study also testifies to problems with interpretation of the IHC data (primary antibody c-erbB-2-oncoprotein DAKO). The problems concern particularly the processing standards, the dilution of antibodies, the application of IHC automatic machine, and probably also the detection system. It is suggested that each laboratory develops a standardized IHC protocol, in coordination with other laboratories. Thus, standardization of both processing and evaluation of the results will be achieved. As the indication for treatment by Herceptin is connected with HER2 overexpression, it is suggested to follow the algorhythm of HER2 examination presented by Nenutil at the ROCHE Satellite symposium.

Overexpression of the ERBB-2 protein has become a target of the anti-neoplastic treatment in patients with invasive duct carcinomas of the breast with a monoclonal antibody trastuzumab (Herceptin, Genentech). From this reason the immunohistochemical (IHC) evaluation of ERBB-2 protein expression is crucial. However, there are patients in whom the IHC investigation is biased by a subjective evaluation among cases considered negative (in the range of 1+) and positive (2+), and among cases considered positive 2+ and 3+. In such cases it is advisable to complement the IHC investigation by detection of copy numbers of the ERBB2 gene with fluorescence in situ hybridization (FISH). The overexpression of the protein is caused by the gene amplification in a majority of cases. The patients, whose carcinomas show overexpression of the ERBB-2 protein and the overexpression is caused by a significant gene amplification, profit from the Herceptin therapy. In this review we focus also on the methodology of FISH in paraffin sections and tissue imprints with respect to the detection of copy numbers of chromosome 17 and the ERBB2 gene.