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Background Core needle biopsy plays a crucial role as diagnostic tool for BC. Both Ki67 and likely tumor-infiltrating lymphocytes (TILs) in the near future are determining the kind of systemic therapy. The role of TILs in BC is still an issue for clinical research, albeit preliminary results of neoadjuvant and adjuvant clinical studies already now highlight the crucial impact of TILs on therapy response and survival. Methods Evaluation of related publications (pubmed) and meeting abstracts (ASCO, SABCS). Results The monoclonal antibody Ki67 recognizing a nuclear antigene in proliferating cells is a positive marker of therapy response and superior survival. Endocrine responsive tumors of low proliferation (Ki67 < 14%/11%) respond to tamoxifen, in contrast postmenopausal tumors with higher proliferation respond better to aromatase-inhibitors. Pathological complete response (pCR)-rates increase in tumors with higher proliferation (Ki67 > 19%) vs. tumors with lower proliferation after neoadjuvant chemotherapy (NAC). pCR-rates of up to 60% can be seen in TNBC and HR-, HER2+BC, lower pCR-rates, however, in HR+, HER2- BC. Increased stromal TILs are found in 30% of TNBC and in 19% of HR-, HER2+BC. The percentage of TILs is a significant independent parameter for pCR after NAC. Lymphocyte-predominant BC (LPBC) respond with higher pCR-rates than non-LPBC or tumors without any TILs. Increased TILs in TN and HR-, HER2+ subtypes predict benefit from addition of carboplatin to NAC. TILs are also associated with improved DFS and OS among patients with TNBC and HR-, HER2+ BC. Conversly and interestingly increased TILs in patients with HR+, HER2-(luminal) BC are associated with a 10% higher risk of death per 10% increase of TILs. Interactions between immune system and cancer are complex. The cancer-immunity cycle characterizes these interactions. BC subtypes with higher number of mutations such as TNBC and HR-, HER2+BC are considered to provide a raising number of tumor-associated antigens, thereby capable to build up a higher endogenous immune response. TILs may serve as surrogate marker of both an existing endogenous immune response and the probability to respond to cancer immune therapies. As cancer co-opt immune checkpoint-pathways as a major mechanism of immune resistance, in particular, against cytotoxic T-cells, blockades of checkpoint-pathways by antibodies are one of the goals of the current cancer immunotherapy studies. Therapy studies with antigene-based strategies (vaccines) and antibodies against the immune checkpoints PD-1 and CTLA-4 and their inhibitory pathways in order to enhance cytotoxic T-cell activities against cancer cells with or without chemotherapy are underway. Conclusions It can be suggested that the use of multigene expression testing will increase in order to select more clearly primary HR+, HER2- BC patients with intermediate recurrence risk who likely may benefit from chemotherapy. Furthermore Ki67 and the multigene expression test Oncotype DX can act as dynamic markers to avoid cytostatic overtreatment and endocrine undertreatment. A data-derived optimal Ki67 cut point for pCR and DFS as well as OS is currently not feasible. The integration of stromal TILs into the immunohisto-pathological report after their evaluation has been standardized is likely helpful to determine patients who profit by additional carboplatin chemotherapy. Oncologists need an enlarged information about the tumor-microenvironment in future. The preliminary results of current BC immunotherapy studies are encouraging.

Background: Molecular breast cancer subgroups show differences with regard to prognosis and response to chemotherapy. In vitro chemotherapy sensitivity and resistance assays (CSRAs) are a means to directly evaluate tumour cell response to a given drug., Methods: Using tissue microarray (TMA) analysis, 550 invasive breast cancers were investigated for the expression of oestrogen receptor (ER), progesterone receptor (PR), vimentin, Ck5, Ck14, Ck19, HER2 and Mib-1/Ki-67. All carcinomas were subjected to in vitro CSRA analysis using three separate chemotherapy combinations. In vitro chemotherapy sensitivity was established using an adenosine triphosphate (ATP) bioluminescence assay. Results of immunohistochemical staining and in vitro response were correlated., Results: Mib-1 expression was associated with sensitivity against Paclitaxel/Epirubicin (p = 0.014) and Docetaxel/Epirubicin (p = 0.014). Mib-1 expression was positively/negatively correlated with expression of basal/luminal markers, respectively. Hierarchical clustering revealed three subtypes, resembling luminal, basal-like and HER2-like breast cancer subtypes. In vitro response for Paclitaxel/Epirubicin was most frequently observed for cases in the basal category (40.3%) compared to HER2-like (25.8%) and luminal cases (28.6%). In multivariate analysis expression of Mib-1 was not a significant independent predictor of in vitro response to chemotherapy., Conclusion: Breast cancer molecular subclasses show differences regarding in vitro chemotherapy sensitivity. These may in part explain differences observed between breast cancer molecular subtypes in vivo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Gains of chromosomes 7p and 8q are associated with poor prognosis among oestrogen receptor-positive (ER+) stage I/II breast cancer. To identify transcriptional changes associated with this breast cancer subtype, we applied suppression subtractive hybridisation method to analyse differentially expressed genes among six breast tumours with and without chromosomal 7p and 8q gains. Identified mRNAs were validated by real-time RT-PCR in tissue samples obtained from 186 patients with stage I/II breast cancer. Advanced statistical methods were applied to identify associations of mRNA expression with distant metastasis-free survival (DMFS). mRNA expression of the key enzyme of cholesterol biosynthesis, squalene epoxidase (SQLE, chromosomal location 8q24.1), was associated with ER+ 7p+/8q+ breast cancer. Distant metastasis-free survival in stage I/II breast cancer cases was significantly inversely related to SQLE mRNA in multivariate Cox analysis (P<0.001) in two independent patient cohorts of 160 patients each. The clinically favourable group associated with a low SQLE mRNA expression could be further divided by mRNA expression levels of the oestrogen-regulated zinc transporter LIV-1. The data strongly support that SQLE mRNA expression might indicate high-risk ER+ stage I/II breast cancers. Further studies on tumour tissue from standardised treated patients, for example with tamoxifen, may validate the role of SQLE as a novel diagnostic parameter for ER+ early stage breast cancers.

Targeting the oestrogen receptor, HER2 (human epidermal growth factor receptor 2) and vascular endothelial growth factor has markedly improved breast cancer therapy. New targeted therapeutic approaches to induction of apoptosis or inhibition of anti-apoptosis, cell cycle progression, signal transduction and angiogenesis are described. The molecular pathways and their inhibitory or repair mechanisms are discussed in the preclinical and clinical settings.

Breast cancer has a significant capacity to metastasize to bone. Bisphosphonates are the standard treatment for hypocalcaemia of malignancy (HCM), which is a common complication of bone metastasis. The combination of bisphosphonates with standard anticancer drugs such as paclitaxel or tamoxifen results in a synergistic apoptotic effect greater than that produced by either single agent alone. Potential antitumour effects in vitro of the two bisphosphonates zoledronic acid (Zol) and ibandronate (Ib) (each at 30 microM) combined with different anticancer drug combinations: cyclophosphamide/metotrexate/5-fluorouracil (CMF), epirubicin/cyclophosphamide (EC), epirubicin/paclitaxel (ET), and epirubicin/docetaxel (EDoc) were investigated using ATP-cell viability assay (ATP-CVA). Twenty cases of female primary, invasive breast cancer were assessed. Ibandronate and zoledronic acid alone showed an inhibitory effect on breast cancer tumour cells in vitro. The breast tumour growth inhibition effect for those two drugs amounted to 22 and 25% respectively. Inhibitory effects were clearly visible for all four combinations of anticancer drugs together with both bisphosphonates. Combinations of anticancer drugs with zoledronic acid seem to be more effective with respect to tumour growth inhibition than combinations with ibandronate.

Objective: DNA ICM allows measurement of nuclear DNA content and genotypical grading of malignancy. The aim of this study was to prove the prognostic value of DNA parameter in comparison to established prognostic factors for DFS and OS., Patients and Methods: Cytological imprints of 177 unselected primary NNBC patients were subjected to ICM. ICM parameter 2cDI, 5cEE, 9cEE, DNA mean value, proliferation fraction (SG2M) and ploidy were investigated together with established parameter like pT-stages, histology, grading, hormone receptor status and patient age regarding DFS and OS. Univariate and multivariate analysis were performed., Results: Univariate analysis revealed that except ploidy all ICM parameter and pT-stages, histology as well as grading were significant prognostic factors for DFS. However, only 2cDI and pT-stages were proved independent prognostic factors in multivariate analysis. Regarding OS 9cEE, histology and pT-stages were significant factors in univariate analysis. However, only 9cEE and pT-stages were found to be independent prognostic factors in multivariate analysis., Conclusions: DNA - ICM parameter 2cDI and 9cEE together with pT-stages were proved independent prognostic factors in NNBC patients.

Background: At present, node-negative, high-risk breast cancer patients cannot be identified with sufficient accuracy. Consequently, further strong prognostic factors are needed., Methods: Among 181 node-negative breast cancer (NNBC) patients, c-myc and HER-2/neu oncogenes were identified prospectively using double differential PCR. The possible impact of amplification of those oncogenes on disease-free survival (DFS) and overall survival was examined. Furthermore, the possible effects of adjuvant therapies on rate of recurrence and mortality in oncogene-amplified NNBC patients were investigated., Results: The prevalence rates for amplification of c-myc and HER-2/neu were 21.5% and 30.4%, respectively. On univariate analysis, c-myc-amplified NNBCs were associated with significantly shorter DFS at 36 months after the initial diagnosis (85.3% versus 97.3%). As compared with nonamplified cancers, HER-2/neu-amplified NNBCs did not exhibit any significant differences after 36 months and 95 months. Multivariate analysis indicated that c-myc amplification and tumour size, in contrast to HER-2/neu amplification, oestrogen receptor status, grading and age, were the only independent parameters for DFS. During the period of observation, we found no evidence for an impact of amplification of the oncogenes on overall survival in all cases. With respect to various adjuvant systemic therapies such as chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil; fluorouracil, epirubicin, cyclophosphamide) and endocrine therapy (tamoxifen), no significant differences were identified in oncogene-amplified NNBC patients in terms of DFS and overall survival. However, those c-myc-amplified NNBC patients who did not receive adjuvant systemic therapy exhibited significantly shorter DFS and overall survival as compared with c-myc-nonamplified patients., Conclusion: C-myc amplification appears to be a strong prognostic marker with which to predict early recurrence in NNBC patients. C-myc-amplified NNBC patients without adjuvant systemic therapy experienced shorter DFS and overall survival.

A pilot study on relationships of selected molecular factors (c-myc oncogene average gene copy numbers (AGCN); serum CEA and CA 15.3 antigen levels; tumor cells' DNA values), to the ex vivo chemosensitivity of primary female human breast cancer in a modified adenosine triphosphate cell viability chemosensitivity assay (ATP-CVA), was performed. Four drug combinations were tested. A group of 75 cases of female primary breast cancer was assessed. Numerous correlations were found among molecular factors tested but none, with the exception of tumor grading, of these reflected ex vivo chemosensitivity of tumors tested. The results suggest that the parameters tested may not be important factors related to adjuvant chemoresponsiveness of primary human breast cancer to tested drug combinations.

Objective: In order to characterise carcinoma of the breast the determination of ploidy can be used in addition to established prognostic factors such as histology and grading. The aim of the investigation was to establish the association between histology, grading and ploidy and to indicate the prognostic and predictive value of these parameters in relation to disease free survival (DFS) and overall survival (OS)., Material and Methods: 125 consecutive cases of primary breast carcinoma occurring between the years 1992-1995 were surveyed. The median follow up time lasted 45 months. Correlation analyses were carried out using the Chi-square test, Kaplan-Meier (univariate) and Cox (multivariate) methods., Results: Histology and grading showed no correlation to ploidy but seems to be of importance for DFS in node-negative breast carcinoma. Ploidy did not influence neither DFS nor OS. Ductal histology appeared to be a useful factor in predicting the response of cases treated with an anti-oestrogen (Tamoxifen) since in this group a higher relapse rate of 25% occurred., Conclusions: Ploidy appears not to be of clinical importance.

ErbB-4 protein is a recently discovered member of the ErbB family. The role of ErbB-4 protein in mammary-gland tissue has not been definitively established. To date, the expression of erbB-4 in breast tissue has been determined in only a few cases and, to the best of our knowledge, its amplification has not been examined. We therefore used the double differential polymerase chain reaction (ddPCR) for determination of the amplification profile of erbB-4 and erbB-2, another gene from the ErbB family, in human primary breast cancer specimens. We examined the amplification of the genes in 20 normal breasts and 176 invasive breast cancer samples. Amplification of erbB-2 was detected in 19% and erbB-4 in 13% of the samples studied. Co-amplification of the two oncogenes was found in only five out of 176 samples. Human breast cancer-derived cell lines in most cases overexpress both erbB-2 and erbB-4 (Beerli et al., 1995. Mol. Cell Biol. 15, 6496-6505; Han et al., 1995. Proc. Natl. Acad. Sci. USA 92, 9747-9751), but data on separate erbB-2 overexpression, without overexpression of erbB-4, were also reported (Wosikowski et al., 1997. Clin. Cancer Res. 3, 2405-2414). At the gene level, we found that co-amplification of the genes in the case of human breast cancer is rare. Moreover, an inverse association of the erbB-4 amplification with estrogen receptor activity and direct correlation with the tumor size were found. Due to these correlations, erbB-4 oncogene amplification can be assumed to be of prognostic or predictive value in the diagnosis of breast cancer.

Competitive and differential quantitative PCR methods circumvent the limiting factors of PCR which cause poor reproducibility. We describe the development and performance evaluation of another quantitative PCR method, double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. The ratio of band intensities of the PCR products in silver-stained polyacrylamide gels expresses the average gene copy number (AGCN) per cell of the erbB oncogenes. The coefficient of variability (CV) was less than 25% for an AGCN of 1. The PCR data were in correlation to the results from dot blotting. DNA image analysis did not reveal any correlation between DNA content and gene dosage deviation of the erbB oncogenes. The method was applied to normal breast tissue, benign breast diseases, breast cancer tissue and lymph node metastases. We suggest this method as being reproducible, low cost and rapid, and therefore suitable for clinical studies on erbB oncogene dosage estimation.

We have determined the average gene copy numbers (AGCN) of the erbB-1 gene, encoding the epidermal growth factor receptor (EGF-R), the erbB-2 and the erbB-3 genes in breast, ovarian, oral, and lung cancer tissue by using double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. In a retrospective study the AGCN of the erbB genes and the time up to the appearance of metastases were subjected to life-table analysis in 128 women with primary breast cancer. Patients whose breast cancer tissue showed an AGCN for erbB-1 of less than 0.4 and greater then 1.6, as expected from the literature, for erbB-2 of greater than 2.0 and for erbB-3 of less than 1.75 had decreased disease-free survival (DFS). The quotient of erbB-1 and erbB-2 AGCN was the most significant in multivariate Cox analysis followed by nodal status and progesterone receptor status. In extensive studies a similar association between erbB AGCN and metastasis was seen in ovarian cancer and oral cancer, though erbB oncogene aberrations in those entities were not as frequent as in breast cancer. The AGCN of erbB oncogenes may not be of prognostic value in untreated lung cancer patients.