Bruno V. Sinn

Cutoff Finder: A Comprehensive and Straightforward Web Application Enabling Rapid Biomarker Cutoff Optimization

Gene or protein expression data are usually represented by metric or at least ordinal variables. In order to translate a continuous variable into a clinical decision, it is necessary to determine a cutoff point and to stratify patients into two groups each requiring a different kind of treatment. Currently, there is no standard method or standard software for biomarker cutoff determination. Therefore, we developed Cutoff Finder, a bundle of optimization and visualization methods for cutoff determination that is accessible online. While one of the methods for cutoff optimization is based solely on the distribution of the marker under investigation, other methods optimize the correlation of the dichotomization with respect to an outcome or survival variable. We illustrate the functionality of Cutoff Finder by the analysis of the gene expression of estrogen receptor (ER) and progesterone receptor (PgR) in breast cancer tissues. This distribution of these important markers is analyzed and correlated with immunohistologically determined ER status and distant metastasis free survival. Cutoff Finder is expected to fill a relevant gap in the available biometric software repertoire and will enable faster optimization of new diagnostic biomarkers. The tool can be accessed at http://molpath.charite.de/cutoff.

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.

Neoadjuvant carboplatin in patients with triple-negative and HER2-positive early breast cancer (GeparSixto; GBG 66): a randomised phase 2 trial

BACKGROUND Preclinical data suggest that triple-negative breast cancers are sensitive to interstrand crosslinking agents, and that synergy may exist for the combination of a taxane, trastuzumab, and a platinum salt for HER2-positive breast cancer. We therefore aimed to assess the efficacy of the addition of carboplatin to neoadjuvant therapy for triple-negative and HER2-positive breast cancer. METHODS Patients with previously untreated, non-metastatic, stage II-III, triple-negative breast cancer and HER2-positive breast cancer were enrolled. Patients were treated for 18 weeks with paclitaxel (80 mg/m(2) once a week) and non-pegylated liposomal doxorubicin (20 mg/m(2) once a week). Patients with triple-negative breast cancer received simultaneous bevacizumab (15 mg/kg intravenously every 3 weeks). Patients with HER2-positive disease received simultaneous trastuzumab (8 mg/kg initial dose with subsequent doses of 6 mg/kg intravenously every 3 weeks) and lapatinib (750 mg daily). Patients were randomly assigned in a 1:1 ratio with dynamic allocation and minimisation, stratified by biological subtype and Ki-67 level to receive, at the same time as the backbone regimens, either carboplatin (AUC 1·5 [2·0 for the first 329 patients] once a week) or no carboplatin. The primary endpoint the proportion of patients who achieved a pathological complete response (defined as ypT0 ypN0), analysed for all patients who started treatment; a p value of less than 0·2 was deemed significant for the primary endpoint. This trial is registered with ClinicalTrials.gov, number NCT01426880. FINDINGS 296 patients were randomly assigned to receive carboplatin and 299 to no additional carboplatin, of whom 295 and 293 started treatment, respectively. In this final analysis, 129 patients (43·7%, 95% CI 38·1-49·4) in the carboplatin group achieved a pathological complete response, compared with 108 patients (36·9%, 31·3-42·4) without carboplatin (odds ratio 1·33, 95% CI 0·96-1·85; p=0·107). Of the patients with triple-negative breast cancer, 84 (53·2%, 54·4-60·9) of 158 patients achieved a pathological complete response with carboplatin, compared with 58 (36·9%, 29·4-44·5) of 157 without (p=0·005). Of the patients with HER2-positive tumours, 45 (32·8%, 25·0-40·7) of 137 patients achieved a pathological complete response with carboplatin compared with 50 (36·8%, 28·7-44·9) of 136 without (p=0·581; test for interaction p=0·015). Haematological and non-haematological toxic effects that were significantly more common in the carboplatin group than in the no-carboplatin group included grade 3 or 4 neutropenia (192 [65%] vs 79 [27%]), grade 3 or 4 anaemia (45 [15%] vs one [<1%]), grade 3 or 4 thrombocytopenia (42 [14%] vs one [<1%]), and grade 3 or 4 diarrhoea (51 [17%] vs 32 [11%]); carboplatin was more often associated with dose discontinuations (141 [48%] with carboplatin and 114 [39%] without carboplatin; p=0·031). The frequency of grade 3 or 4 haematological events decreased from 82% (n=135) to 70% (n=92) and grade 3 or 4 non-haematological events from 78% (n=128) to 59% (n=77) in the carboplatin arm when the dose of carboplatin was reduced from AUC 2·0 to 1·5. INTERPRETATION The addition of neoadjuvant carboplatin to a regimen of a taxane, an anthracycline, and targeted therapy significantly increases the proportion of patients achieving a pathological complete response. This regimen seems to increase responses in patients with triple-negative breast cancer, but not in those with HER2-positive breast cancer. FUNDING GlaxoSmithKline, Roche, and Teva.

Prospective Validation of Immunological Infiltrate for Prediction of Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer - A Substudy of the Neoadjuvant GeparQuinto Trial

Introduction We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT. Patients and Methods The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher. Results Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients (14.3%, p<0.001). LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis (LPBC: OR 2.7, p = 0.003, strLy: OR 1.2, p = 0.01). The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate (OR 1.2, p = 0.01) but not in multivariate logistic regression analysis (OR 1.2, p = 0.11). Conclusion Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies.

Prognostic significance of Dicer expression in ovarian cancer - link to global microRNA changes and oestrogen receptor expression

MicroRNA (miRNA) deregulation is a hallmark of human cancer. However, the mechanisms underlying miRNA alteration and the specific role of proteins involved in miRNA processing remains to be elucidated. Dicer is a key enzyme in the miRNA processing pathway that is essential for the production of mature miRNAs from their precursors. We tested the hypothesis that Dicer has biological and clinical relevance in ovarian cancer, using a range of methods including in vitro manipulation of Dicer expression. We observed down‐regulation of Dicer in a subgroup of ovarian carcinomas, and found that decreased Dicer expression correlates significantly with reduced patient survival in serous cancers and advanced disease stages. Moreover, microarray and functional analysis suggest that reduced Dicer expression is connected with a global down‐regulation of the microRNAome and with gene expression changes, particularly reduced expression of oestrogen receptor (ER) mRNA and protein in tumour tissue and in cell culture. Our data suggest a common mechanism for miRNAs changes by alterations in the basic machinery controlling miRNA biogenesis, of which Dicer is a central enzyme. These alterations of miRNA processing are of prognostic value and may play a role in the molecular pathogenesis of ovarian carcinoma and, possibly, other tumours. Knowledge of these molecular pathways may help toward new targeted therapeutic approaches for ovarian cancer. Copyright

Ki67 measured after neoadjuvant chemotherapy for primary breast cancer

Purpose: The value of Ki67 measured on residual disease after neoadjuvant chemotherapy is not sufficiently described. Experimental Design: Participants of the GeparTrio study with primary breast cancer randomly received neoadjuvant response-guided [8 cycles TAC (docetaxel/doxorubicin/cyclophosphamide) in responding and TAC-NX (vinorelbine/capecitabine) in nonresponding patients] or conventional (6 cycles TAC) chemotherapy according to interim response assessment. Ki-67 levels were centrally measured immunohistochemically after neoadjuvant treatment if tumor tissue was available. Here, we analyze 1,151 patients having a pathologic complete response (pCR; n, 484), or residual disease with low (0–15%), intermediate (15.1–35%), or high (35.1–100%) posttreatment Ki67 levels in 488, 77, and 102 patients, respectively. Results: Patients with high posttreatment Ki67 levels showed higher risk for disease relapse (P < 0.0001) and death (P < 0.0001) compared with patients with low or intermediate Ki67 levels. Patients with low Ki67 levels showed a comparable outcome to patients with a pCR (P = 0.211 for disease-free and P = 0.779 for overall survival). Posttreatment Ki67 levels provided more prognostic information than pretreatment Ki67 levels or changes of Ki67 from pre- to posttreatment. Information on pCR plus posttreatment Ki67 levels surmount the prognostic information of pCR alone in hormone–receptor-positive disease [hazard ratios (HR), 1.82–5.88] but not in hormone–receptor-negative disease (HR: 0.61–1.73). Patients with conventional and response-guided treatment did not show a different distribution of posttreatment Ki67 (P = 0.965). Conclusions: Posttreatment Ki67 levels provide prognostic information for patients with hormone–receptor-positive breast cancer and residual disease after neoadjuvant chemotherapy. Levels were not prognostic for outcome after response-guided chemotherapy. High posttreatment Ki67 indicates the need for innovative postneoadjuvant treatments. Clin Cancer Res; 19(16); 4521–31. ©2013 AACR.

Decentral gene expression analysis for ER+/Her2− breast cancer: results of a proficiency testing program for the EndoPredict assay

Gene expression profiles provide important information about the biology of breast tumors and can be used to develop prognostic tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone–receptor-positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside, it is essential to evaluate the test–performance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict (EP) assay in seven different molecular pathology laboratories in Germany, Austria, and Switzerland. A set of ten formalin-fixed paraffin-embedded tumors was tested in the different labs, and the variance and accuracy of the EndoPredict assays were determined using predefined reference values. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of gene expression in luminal breast cancer.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell–autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Standardized evaluation of tumor-infiltrating lymphocytes in breast cancer : results of the ring studies of the international immuno-oncology biomarker working group

Multiple independent studies have shown that tumor-infiltrating lymphocytes (TIL) are prognostic in breast cancer with potential relevance for response to immune-checkpoint inhibitor therapy. Although many groups are currently evaluating TIL, there is no standardized system for diagnostic applications. This study reports the results of two ring studies investigating TIL conducted by the International Working Group on Immuno-oncology Biomarkers. The study aim was to determine the intraclass correlation coefficient (ICC) for evaluation of TIL by different pathologists. A total of 120 slides were evaluated by a large group of pathologists with a web-based system in ring study 1 and a more advanced software-system in ring study 2 that included an integrated feedback with standardized reference images. The predefined aim for successful ring studies 1 and 2 was an ICC above 0.7 (lower limit of 95% confidence interval (CI)). In ring study 1 the prespecified endpoint was not reached (ICC: 0.70; 95% CI: 0.62–0.78). On the basis of an analysis of sources of variation, we developed a more advanced digital image evaluation system for ring study 2, which improved the ICC to 0.89 (95% CI: 0.85–0.92). The Fleiss’ kappa value for <60 vs ≥60% TIL improved from 0.45 (ring study 1) to 0.63 in RS2 and the mean concordance improved from 88 to 92%. This large international standardization project shows that reproducible evaluation of TIL is feasible in breast cancer. This opens the way for standardized reporting of tumor immunological parameters in clinical studies and diagnostic practice. The software-guided image evaluation approach used in ring study 2 may be of value as a tool for evaluation of TIL in clinical trials and diagnostic practice. The experience gained from this approach might be applicable to the standardization of other diagnostic parameters in histopathology.

SPARC expression in resected pancreatic cancer patients treated with gemcitabine: results from the CONKO-001 study

BACKGROUND Previous investigations in pancreatic cancer suggested a prognostic role for secreted protein acidic and rich in cysteine (SPARC) expression in the peritumoral stroma but not for cytoplasmic SPARC expression. The aim of this study was to evaluate the impact of SPARC expression in pancreatic cancer patients treated with gemcitabine compared with untreated patients. PATIENTS AND METHODS CONKO-001 was a prospective randomized phase III study investigating the role of adjuvant gemcitabine when compared with observation. Tissue samples of 160 patients were available for SPARC immunohistochemistry on tissue microarrays to evaluate its impact on patient outcome. RESULTS Strong stromal SPARC expression was associated with worse disease-free survival (DFS) and overall survival (OS) in the overall study population (DFS: P = 0.005, OS: P = 0.033). Its negative prognostic impact was restricted to patients treated with gemcitabine (DFS: P = 0.007, OS: P = 0.006). High cytoplasmic SPARC expression also was associated with worse patient outcome (DFS: P = 0.041, OS: P = 0.011). Again the effect was restricted to patients treated with gemcitabine (DFS: P = 0.002, OS: P = 0.003). In multivariable analysis, SPARC expression was independently predictive of patient outcome. CONCLUSIONS Our data confirm the prognostic significance of SPARC expression after curatively intended resection. The negative prognostic impact was restricted to patients who received adjuvant treatment with gemcitabine, suggesting SPARC as a predictive marker for response to gemcitabine.