Jan C. Brase

Circulating miRNAs are correlated with tumor progression in prostate cancer

Circulating miRNAs have recently been indicated as practicable and promising biomarkers for noninvasive diagnosis in various tumor entities. However, cell‐free miRNAs have not been found to correlate with clinicopathological variables in epithelial carcinomas. To learn more about the potential clinical relevance of circulating miRNAs in prostate cancer, we screened 667 miRNAs in serum samples from patients with metastatic (n = 7) and localized prostate cancer (n = 14). Various miRNAs were highly abundant in the sera of patients with metastatic disease, and five upregulated miRNAs (miRNA‐375, miRNA‐9*, miRNA‐141, miRNA‐200b and miRNA‐516a‐3p) were selected for further validation. In the first validation study (n = 45), selected miRNAs were analyzed in a prospectively collected serum set taken from different prostate cancer risk groups. Most of the selected miRNAs were significantly correlated with adverse risk factors when different clinicopathological variables were analyzed. Circulating miRNA‐375 and miRNA‐141 turned out to be the most pronounced markers for high‐risk tumors. Their levels also correlated with high Gleason score or lymph‐node positive status in a second independent validation study (n = 71). In addition, the expression levels of miRNA‐375 and miRNA‐141 were monitored in 72 prostate tissue samples (36 tumor vs. 36 benign). Both miRNAs were highly expressed in all samples and significantly upregulated in the tumors compared to normal tissues. Overall, our observations suggest that miRNA‐375 and miRNA‐141 expression is enhanced in prostate cancer specimens and their release into the blood is further associated with advanced cancer disease.

Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy With or Without Carboplatin in Human Epidermal Growth Factor Receptor 2–Positive and Triple-Negative Primary Breast Cancers

PURPOSE Modulation of immunologic interactions in cancer tissue is a promising therapeutic strategy. To investigate the immunogenicity of human epidermal growth factor receptor 2 (HER2) -positive and triple-negative (TN) breast cancers (BCs), we evaluated tumor-infiltrating lymphocytes (TILs) and immunologically relevant genes in the neoadjuvant GeparSixto trial. PATIENTS AND METHODS GeparSixto investigated the effect of adding carboplatin (Cb) to an anthracycline-plus-taxane combination (PM) on pathologic complete response (pCR). A total of 580 tumors were evaluated before random assignment for stromal TILs and lymphocyte-predominant BC (LPBC). mRNA expression of immune-activating (CXCL9, CCL5, CD8A, CD80, CXCL13, IGKC, CD21) as well as immunosuppressive factors (IDO1, PD-1, PD-L1, CTLA4, FOXP3) was measured in 481 tumors. RESULTS Increased levels of stromal TILs predicted pCR in univariable (P < .001) and multivariable analyses (P < .001). pCR rate was 59.9% in LPBC and 33.8% for non-LPBC (P < .001). pCR rates ≥ 75% were observed in patients with LPBC tumors treated with PMCb, with a significant test for interaction with therapy in the complete (P = .002) and HER2-positive (P = .006), but not the TNBC, cohorts. Hierarchic clustering of mRNA markers revealed three immune subtypes with different pCR rates (P < .001). All 12 immune mRNA markers were predictive for increased pCR. The highest odds ratios (ORs) were observed for PD-L1 (OR, 1.57; 95% CI, 1.34 to 1.86; P < .001) and CCL5 (OR, 1.41; 95% CI, 1.23 to 1.62; P < .001). CONCLUSION Immunologic factors were highly significant predictors of therapy response in the GeparSixto trial, particularly in patients treated with Cb. After further standardization, they could be included in histopathologic assessment of BC.

Serum microRNAs as non-invasive biomarkers for cancer

Human serum and other body fluids are rich resources for the identification of novel biomarkers, which can be measured in routine clinical diagnosis. microRNAs are small non-coding RNA molecules, which have an important function in regulating RNA stability and gene expression. The deregulation of microRNAs has been linked to cancer development and tumor progression. Recently, it has been reported that serum and other body fluids contain sufficiently stable microRNA signatures. Thus, the profiles of circulating microRNAs have been explored in a variety of studies aiming at the identification of novel non-invasive biomarkers.In this review, we discuss recent findings indicating that circulating microRNAs are useful as non-invasive biomarkers for different tumor types. Additionally, we summarize the knowledge about the mechanism of microRNA release and the putative functional roles of circulating microRNAs. Although several challenges remain to be addressed, circulating microRNAs have the potential to be useful for the diagnosis and prognosis of cancer diseases.

ERG Status Is Unrelated to PSA Recurrence in Radically Operated Prostate Cancer in the Absence of Antihormonal Therapy

Purpose: About 50% of prostate cancers have TMPRSS2–ERG fusions with concurrent ERG overexpression. The aim of this study was to determine whether clinical differences exist between ERG-positive and ERG-negative cancers in surgically treated patients not exposed to antihormonal therapy. A secondary aim was to search for differences between these tumor classes. Experimental Design: A tissue microarray containing samples from more than 2,800 prostate cancers with clinical data was analyzed for ERG alterations by immunohistochemistry and FISH. Results were compared with tumor phenotype, biochemical recurrence, and molecular features considered important for prostate cancer. The effect of ERG on androgen receptor (AR)-dependent transcription was analyzed in cell lines. Results: ERG expression was found in 52.4% of 2,805 cancers with a 95% concordance between ERG expression and ERG gene rearrangement detected by FISH. ERG expression was unrelated to clinical outcome and tumor phenotype. Differences in AMACR, Annexin A3, Bcl2, CD10, ALCAM, chromogranin A, epidermal growth factor receptor, HER2, mTOR, p53, and synaptophysin status were significant but minimal in absolute numbers. The most striking difference was found for AR expression, which was markedly higher in ERG-positive cancers. In vitro studies showed ERG-dependent impairment of AR-mediated transcriptional activity. Conclusions: The striking similarities between these two types of prostate cancers rules out a major impact of ERG on tumor aggressiveness in early, not hormonally treated cancer. The marked difference in AR levels between ERG-positive and -negative cancers supports a systematic difference in potential response to hormonal therapy as previously observed in clinical trials. Clin Cancer Res; 17(18); 5878–88. ©2011 AACR.

The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2- breast cancer patients.

Background:ER+/HER2− breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy.Methods:A total of 1702 postmenopausal ER+/HER2− breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan–Meier method and Cox regression analysis were used in an early (0–5 years) and late time interval (>5 years post diagnosis).Results:EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up.Conclusion:The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.

EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer

Background In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed. Patients and methods We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan–Meier survival analysis. Results After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%–61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years. Conclusion The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.

TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-β signaling

BackgroundTMPRSS2-ERG gene fusions occur in about 50% of all prostate cancer cases and represent promising markers for molecular subtyping. Although TMPRSS2-ERG fusion seems to be a critical event in prostate cancer, the precise functional role in cancer development and progression is still unclear.MethodsWe studied large-scale gene expression profiles in 47 prostate tumor tissue samples and in 48 normal prostate tissue samples taken from the non-suspect area of clinical low-risk tumors using Affymetrix GeneChip Exon 1.0 ST microarrays.ResultsComparison of gene expression levels among TMPRSS2-ERG fusion-positive and negative tumors as well as benign samples demonstrated a distinct transcriptional program induced by the gene fusion event. Well-known biomarkers for prostate cancer detection like CRISP3 were found to be associated with the gene fusion status. WNT and TGF-β/BMP signaling pathways were significantly associated with genes upregulated in TMPRSS2-ERG fusion-positive tumors.ConclusionsThe TMPRSS2-ERG gene fusion results in the modulation of transcriptional patterns and cellular pathways with potential consequences for prostate cancer progression. Well-known biomarkers for prostate cancer detection were found to be associated with the gene fusion. Our results suggest that the fusion status should be considered in retrospective and future studies to assess biomarkers for prostate cancer detection, progression and targeted therapy.

Genome-wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with miR-26a Hypermethylation

UNLABELLED Prostate cancer is the second most common cancer among men worldwide. Alterations in the DNA methylation pattern can be one of the leading causes for prostate cancer formation. This study is the first high-throughput sequencing study investigating genome-wide DNA methylation patterns in a large cohort of 51 tumor and 53 benign prostate samples using methylated DNA immunoprecipitation sequencing. Comparative analyses identified more than 147,000 cancer-associated epigenetic alterations. In addition, global methylation patterns show significant differences based on the TMPRSS2-ERG rearrangement status. We propose the hypermethylation of miR-26a as an alternative pathway of ERG rearrangement-independent EZH2 activation. The observed increase in differential methylation events in fusion-negative tumors can explain the tumorigenic process in the absence of genomic rearrangements. SIGNIFICANCE In contrast to TMPRSS2-ERG -rearranged tumors, the pathomechanism for gene fusion-negative tumors is completely unclear. Using a sequencing-based approach, our work uncovers significant global epigenetic alterations in TMPRSS2-ERG gene fusion-negative tumors and provides a mechanistic explanation for the tumor formation process.

ERBB2 and TOP2A in Breast Cancer: A Comprehensive Analysis of Gene Amplification, RNA Levels, and Protein Expression and Their Influence on Prognosis and Prediction

Purpose: The prognostic and predictive relevance of epidermal growth factor receptor 2 (ERBB2) and topoisomerase II α (TOP2A) have long been a matter of debate. However, the correlation of DNA amplification, RNA levels, and protein expression and their prognostic role and association with anthracycline responses in node-negative breast cancer have not yet been evaluated. Experimental Design: We first analyzed TOP2A and ERBB2 at the levels of gene amplification, and RNA and protein expression, and studied their correlations. Additionally, TOP2A and ERBB2 were analyzed in 782 node-negative breast carcinomas in patients who did not receive systemic therapy and in 80 patients treated with epirubicin and cyclophosphamide (EC) prior to surgery. Results: TOP2A gene amplification did not correlate with protein expression (P = 0.283) and showed an association with gene expression with only borderline significance (P = 0.047). By contrast, TOP2A RNA levels correlated with protein expression (P < 0.001). TOP2A gene expression was significantly associated with the metastasis-free interval (MFI; P < 0.001) and was associated with complete remission in patients treated with EC (P = 0.002). In contrast to TOP2A, ERBB2 gene amplification correlated with RNA level (P < 0.001) and protein expression (P < 0.001). ERBB2 gene expression was associated with the MFI only in estrogen receptor–positive carcinomas, whereas ERBB2 protein expression (P = 0.032) was associated with MFI in the entire cohort. Conclusions: Overall, our study indicates that the TOP2A RNA level is a good prognostic marker and is also associated with a favorable response to anthracyclin-based therapy. By contrast, ESR1 was associated with poorer responses to anthracyclin-based therapy, whereas the association with ERBB2 RNA was not significant. Clin Cancer Res; 16(8); 2391–401. ©2010 AACR.

Clinical validation of the EndoPredict test in node-positive, chemotherapy-treated ER+/HER2− breast cancer patients: results from the GEICAM 9906 trial

IntroductionEndoPredict (EP) is an RNA-based multigene test that predicts the likelihood of distant recurrence in patients with estrogen receptor-positive (ER+), human epidermal growth factor receptor 2–negative (HER2−) breast cancer (BC) who are being treated with adjuvant endocrine therapy. Herein we report the prospective-retrospective clinical validation of EP in the node-positive, chemotherapy-treated, ER+/HER2− BC patients in the GEICAM 9906 trial.MethodsThe patients (N = 1,246) were treated either with six cycles of fluorouracil, epirubicin and cyclophosphamide (FEC) or with four cycles of FEC followed by eight weekly courses of paclitaxel (FEC-P), as well as with endocrine therapy if they had hormone receptor–positive disease. The patients were assigned to EP risk categories (low or high) according to prespecified cutoff levels. The primary endpoint in the clinical validation of EP was distant metastasis-free survival (MFS). Metastasis rates were estimated using the Kaplan-Meier method, and multivariate analysis was performed using Cox regression.ResultsThe molecular EP score and the combined molecular and clinical EPclin score were successfully determined in 555 ER+/HER2− tumors from the 800 available samples in the GEICAM 9906 trial. On the basis of the EP, 25% of patients (n = 141) were classified as low risk. MFS was 93% in the low-risk group and 70% in the high-risk group (absolute risk reduction = 23%, hazard ratio (HR) = 4.8, 95% confidence interval (CI) = 2.5 to 9.5; P < 0.0001). Multivariate analysis showed that, in this ER+/HER2− cohort, EP results are an independent prognostic parameter after adjustment for age, grade, lymph node status, tumor size, treatment arm, ER and progesterone receptor (PR) status and proliferation index (Ki67). Using the predefined EPclin score, 13% of patients (n = 74) were assigned to the low-risk group, who had excellent outcomes and no distant recurrence events (absolute risk reduction vs high-risk group = 28%; P < 0.0001). Furthermore, EP was prognostic in premenopausal patients (HR = 6.7, 95% CI = 2.4 to 18.3; P = 0.0002) and postmenopausal patients (HR = 3.3, 95% CI = 1.3 to 8.5; P = 0.0109). There were no statistically significant differences in MFS between treatment arms (FEC vs FEC-P) in either the high- or low-risk groups. The interaction test results between the chemotherapy arm and the EP score were not significant.ConclusionsEP is an independent prognostic parameter in node-positive, ER+/HER2− BC patients treated with adjuvant chemotherapy followed by hormone therapy. EP did not predict a greater efficacy of FEC-P compared to FEC alone.