The potential prognostic value of connexin 26 and 46 expression in neoadjuvant-treated breast cancer.

BACKGROUND: Several classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST) are still missing. Deregulation of gap junction channel forming connexins (Cx) has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems. METHODS: The expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA). Cx plaques of <1µm were detected with multilayer, multichannel fluorescence digital microscopy. Current classifications to assess residual tumour burden after primary systemic therapy included the EWGBSP, CPS-EG, Miller-Payne, Sataloff and NSABP systems. RESULTS: In our cohort dominated by hormone receptor (ER/PR) positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1-2 had significantly better overall survival (p=0.015) than cases with scores 3-5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (<5%) statistically correlated with better overall survival (p=0.011). Moderate or higher Cx46 expression (>20%) pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (p(pre-chemo)=0.006; Sataloff TB (p(pre-chemo)=0.005; p(post-chemo)=0.029) and in Miller-Payne G3 (p(pre-chemo)=0.002; p(post-chemo)=0.012) classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups. CONCLUSION: Our results suggest that Cx46 and Cx26 expression in breast cancer may improve the assessment of pathological response and refine intermediate prognostic subgroups of residual tumour classifications used after neoadjuvant chemotherapy.

Comparison of 5 Ki-67 antibodies regarding reproducibility and capacity to predict prognosis in breast cancer: does the antibody matter?

Although several antibodies are available for immunohistochemical detection of Ki-67, even the most commonly used MIB-1 has not been validated yet. Our aim was to compare 5 commercially available antibodies for detection of Ki-67 in terms of agreement and their ability in predicting prognosis of breast cancer. Tissue microarrays were constructed from 378 breast cancer patients' representative formalin-fixed, paraffin-embedded tumor blocks. Five antibodies were used to detect Ki-67 expression: MIB-1 using chromogenic detection and immunofluorescent-labeled MIB-1, SP-6, 30-9, poly, and B56. Semiquantitative assessment was performed by 2 pathologists independently on digitized slides. To compare the 5 antibodies, intraclass correlation and concordance correlation coefficient were used. All the antibodies but immunofluorescent-labeled MIB-1 (at 20% and 30% thresholds, P=.993 and P=.342, respectively) and B56 (at 30% threshold, P=.288) separated high- and low-risk patient groups. However, there were a significant difference (P values for all comparisons≤.005) and a moderate concordance (intraclass correlation, 0.645) between their Ki-67 labeling index scores. The highest concordance was found between MIB-1 and poly (concordance correlation coefficient=0.785) antibodies. None of the antibodies except Ki-67 labeling index as detected by poly (P=.031) at 20% threshold and lymph node status (P<.001) were significantly linked to disease-free survival in multivariate analysis. At 30% threshold, this was reduced to lymph node status (P<.001) alone. Our results showed that there are considerable differences between the different Ki-67 antibodies in their capacity to detect proliferating tumor cells and to separate low- and high-risk breast cancer patient groups.

Changes of protein expression in prostate cancer having lost its androgen sensitivity.

OBJECTIVE: The majority of prostate cancers require androgen hormones for growth, and androgen ablation is an important part of the systemic treatment of advanced prostate cancer. Nevertheless, most of these cancers eventually relapse as they become less sensitive to androgen ablation and anti-androgen treatment. Elucidating the molecular events that are responsible for the conversion of androgen-sensitive cancers to androgen-refractory tumors may reveal new therapeutic opportunities. METHODS: In the present study, we investigated nine androgen-sensitive and nine androgen-refractory prostate cancer samples to evaluate the expression levels of 10 selected proteins that have been implicated in oncogenesis and cancer progression. RESULTS: Our immunohistochemical data show that three of the investigated proteins (i.e., minichromosome maintenance-2, methylguanine-DNA methyltransferase, and androgen receptor) are expressed at significantly different levels in the androgen-refractory cancer samples than in the androgen-sensitive tumors, whereas the expression levels of the seven other studied proteins (i.e., ß-catenin, p27, p21, p16, Ki67, hypoxia-inducible factor 1 alpha, and geminin) are not significantly different regarding the two groups. CONCLUSIONS: Our data suggest that the increased expression of minichromosome maintenance-2 and decreased expression of methylguanine-DNA methyltransferase related to androgen receptor are indicative of the androgen-refractory stage in prostate cancer. Further studies are required to determine whether these expression changes play a causative role in the transition of androgen-sensitive to androgen-refractory prostate cancer.

Connexin 43 communication channels in follicular dendritic cell development and in follicular lymphomas.

Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to "ex vivo" germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinal centers and in malformed follicles of follicular lymphomas and showed strong upregulation in newly generated and/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and "ex vivo" germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression.

Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia.

In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals.

Correlations of differentially expressed gap junction connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with breast cancer progression and prognosis.

BACKGROUND AND AIMS: Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers. MATERIALS AND METHODS: Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models. RESULTS: The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other's prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively. CONCLUSION: Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

Cell cycle analysis can differentiate thin melanomas from dysplastic nevi and reveals accelerated replication in thick melanomas.

Cell replication integrates aberrations of cell cycle regulation and diverse upstream pathways which all can contribute to melanoma development and progression. In this study, cell cycle regulatory proteins were detected in situ in benign and malignant melanocytic tumors to allow correlation of major cell cycle fractions (G1, S-G2, and G2-M) with melanoma evolution. Dysplastic nevi expressed early cell cycle markers (cyclin D1 and cyclin-dependent kinase 2; Cdk2) significantly more (p < 0.05) than common nevi. Post-G1 phase markers such as cyclin A, geminin, topoisomerase IIα (peaking at S-G2) and aurora kinase B (peaking at G2-M) were expressed in thin (≤1 mm) melanomas but not in dysplastic nevi, suggesting that dysplastic melanocytes engaged in the cell cycle do not complete replication and remain arrested in G1 phase. In malignant melanomas, the expression of general and post-G1 phase markers correlated well with each other implying negligible cell cycle arrest. Post-G1 phase markers and Ki67 but none of the early markers cyclin D1, Cdk2 or minichromosome maintenance protein 6 (Mcm6) were expressed significantly more often in thick (>1 mm) than in thin melanomas. Marker expression did not differ between metastatic melanomas and thick melanomas, with the exception of aurora kinase A of which the expression was higher in metastatic melanomas. Combined detection of cyclin A (post-G1 phase) with Mcm6 (replication licensing) and Ki67 correctly classified thin melanomas and dysplastic nevi in 95.9 % of the original samples and in 93.2 % of cross-validated grouped cases at 89.5 % sensitivity and 92.6 % specificity. Therefore, cell cycle phase marker detection can indicate malignancy in early melanocytic lesions and accelerated cell cycle progression during vertical melanoma growth.

Stability and prognostic value of Slug, Sox9 and Sox10 expression in breast cancers treated with neoadjuvant chemotherapy.

BACKGROUND: Expression of transcription-factors as Slug and Sox9 was recently described to determine mammary stem-cell state. Sox10 was previously shown to be present also in breast cancer. Protein overexpression of Slug, Sox9 and Sox10 were associated with poor overall survival and with triple-negative phenotype in breast cancer. In this study we tested the stability of Slug, Sox9 and Sox10 expression during chemotherapy and addressed their prognostic role of in neoadjuvant treated primary breast-cancer and their correlation to pathological-response and overall survival. METHODS: We analyzed immunohistochemical expression of Slug, Sox9 and Sox10 in tissue microarrays of 96 breast cancers prior to and after neoadjuvant chemotherapy. Expression was evaluated in invasive tumor cells and in tumor stroma and scored as 0, 1+, 2+ 3+. Expression-profile prior to and after chemotherapy was correlated to overall survival (Kaplan Meier) and with established clinico-pathological parameter. RESULTS: Sox9, Sox10 and Slug were expressed in 82-96% of the tumor cells prior to chemotherapy. Slug was expressed in 97% of the cases in tumor stroma before therapy. Change in expression-profile after chemotherapy occurred only in Slug expression in tumor-cells (decreased from 82 to 51%, p = 0.0001, Fisher's exact test). The other markers showed no significant change after chemotherapy. Stromal Sox9 expression (0 to 2+) correlated to better overall survival after chemotherapy (p = 0.004) and reached almost statistical significance prior to chemotherapy (p = 0.065). There was no correlation between Sox9 and hormone-receptor expression. In multivariate-analysis, the stromal Sox9 expression after chemotherapy proved to be an independent and better prognostic marker than hormone-receptor status. Other clinico-pathological parameter (as HER2-status or pathological-stage) showed no correlation to the analyzed markers. CONCLUSION: Strong stromal Sox9 expression in breast cancer after chemotherapy was found to bear negative prognostic information and was associated with shortened overall survival. Slug expression was significantly changed (reduced) in samples after neoadjuvant chemotherapy.