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Purpose Sphingosine-kinase-1 (SPHK1) is a key enzyme of sphingolipid metabolism which is involved in ovarian cancer pathogenesis, progression and mechanisms of drug resistance. It is overexpressed in a variety of cancer subtypes. We investigated SPHK1 expression as a prognostic factor in epithelial ovarian cancer patients. Methods Expression analysis of SPHK1 was performed on formalin-fixed paraffin-embedded tissue from 1005 ovarian cancer patients with different histological subtypes using immunohistochemistry. Staining intensity of positive tumor cells was assessed semi-quantitatively, and results were correlated with clinicopathological characteristics and survival. Results In our ovarian cancer collective, high levels of SPHK1 expression correlated significantly with complete surgical tumor resection (p = 0.002) and lower FIGO stage (p = 0.04). Progression-free and overall survival were further significantly longer in patients with high-grade serous ovarian cancer and overexpression of SPHK1 (p = 0.002 and p = 0.006, respectively). Conclusion Our data identify high levels of SPHK1 expression as a potential favorable prognostic marker in ovarian cancer patients.

Purpose: We evaluated mRNA signatures to predict response to neoadjuvant PD-L1 inhibition in combination with chemotherapy in early triple-negative breast cancer. Experimental Design: Targeted mRNA sequencing of 2,559 transcripts was performed in formalin-fixed, paraffin-embedded samples from 162 patients of the GeparNuevo trial. We focused on validation of four predefined gene signatures and differential gene expression analyses for new predictive markers. Results: Two signatures [GeparSixto signature (G6-Sig) and IFN signature (IFN-Sig)] were predictive for treatment response in a multivariate model including treatment arm [G6-Sig: OR, 1.558; 95% confidence interval (CI), 1.130–2.182; P = 0.008 and IFN-Sig: OR, 1.695; 95% CI, 1.234–2.376; P = 0.002), while the CYT metric predicted pathologic complete response (pCR) in the durvalumab arm, and the proliferation-associated gene signature in the placebo arm. Expression of PD-L1 mRNA was associated with better response in both arms, indicating that increased levels of PD-L1 are a general predictor of neoadjuvant therapy response. In an exploratory analysis, we identified seven genes that were higher expressed in responders in the durvalumab arm, but not the placebo arm: HLA-A, HLA-B, TAP1, GBP1, CXCL10, STAT1, and CD38. These genes were associated with cellular antigen processing and presentation and IFN signaling. Conclusions: Immune-associated signatures are associated with pCR after chemotherapy, but might be of limited use for the prediction of response to additional immune checkpoint blockade. Gene expressions related to antigen presentation and IFN signaling might be interesting candidates for further evaluation.

Background: The RxPONDER and TAILORx trials demonstrated benefit from adjuvant chemotherapy in patients < 50 years with node-positive breast cancer and Recurrence Score (RS) 0-25, and with node-negative disease and RS 16-25, respectively. Neither trial showed benefit in older women with RS < 26. It is unclear what explains the interaction between age and adjuvant chemotherapy benefit. Methods: We analyzed transcriptomic and genomic data from n=4,507 ER+/HER2- breast cancers to compare differences in estrogen receptor (ER), proliferation, and immune-related gene expressions, and somatic mutation patterns and mutation burden between younger (< 50 years of age) and older (>55 years) patients. We restricted our analysis to patients in the lower 80% range of in silico RS distribution to mimic the RxPONDER and TAILORx populations. Results: Five data sets were analyzed independently to assess consistency of results (TCGA n=530; microarray cohort A n=865; Cohort B n=609, METABRIC n=867, SCAN-B n=1636). Older patients had significantly higher somatic mutation burden and more frequent copy number gain in ESR1, LATS1, ARID1B, SGK1, and MYB genes (odds ratio [OR] > 8.5, FDR< 0.05), but lower frequency of GATA3 mutations (12% versus 26%, P< 0.0001). Younger patients had higher rate of ESR1 copy number loss (OR: 0.45, FDR: 0.03). There was no difference in proliferation-related gene expression. ESR1 mRNA expression was significantly lower in younger women in all cohorts (P < 0.001). A regression model of ESR1 mRNA expression using age and ER IHC positivity indicated that lower ER expression in younger patients is primarily driven by lower ESR1 mRNA per cancer cell and not by fewer ER positive cells. We also assessed four gene signatures associated with endocrine therapy sensitivity including a 4-gene ERS, a 7-gene ERS-Lum, a 106-gene ERS-Pos signature, and a 59-gene ERS-Neg signature associated with endocrine resistance. In the TCGA and METABRIC cohorts, the ERS, ERS-Lum, and ERS-Pos signatures were all lower (FDR< 0.03) while the ERS-Neg signature was higher (FDR< 0.001) in younger patients. Similarly, in both microarray cohorts, and in the SCAN-B-cohort, the ERS-Pos signature was lower and the ERS-Neg signature was higher in younger patients (FDR< 0.002). Next, we assessed 4 different immune cell signatures that have been associated with response to chemotherapy. In the TCGA, B-cell, T-cell, Mast-cell, and TIS signatures were significantly higher (FDR Citation Format: Tao Qing, Thomas Karn, Mariya Rozenblit, Julia Foldi, Michal Marczyk, Naing Lin Shan, Kim Blenman, uwe Holtrich, Kevin Kalinsky, Funda Meric-Bernstam, Lajos Pusztai. Molecular differences between younger versus older estrogen receptor positive/human epidermal growth factor receptor-2 negative breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-09.

Background: The implementation of immune checkpoint inhibitors in the therapy of different cancer types has provided promising results, but only a limited number of patients respond. Therefore, biomarkers to identify these responding patients are urgently needed. Methods: The GeparNuevo was a randomized, double-blind phase II trial in which triple-negative breast cancer (TNBC) patients were treated with neoadjuvant chemotherapy (NACT) consisting of nanoparticle albumin-bound paclitaxel in an initial phase followed by treatment with epirubicin and cyclophosphamide. Placebo or durvalumab were given throughout the neo-adjuvant treatment and in the “window” sub-cohort also prior to chemotherapy. Primary objective of this report was to evaluate changes in the blood immune cell repertoires of TNBC patients receiving durvalumab (anti-PD-L1) versus placebo in combination with NACT. At up to 4 different time points during therapy, blood samples were taken and underwent immunomonitoring using multicolor flow cytometry. The absolute counts of the major immune cell subtypes in the blood as well as the frequencies of different immune cell subpopulations and their functional phenotypes along treatment were determined and correlated to clinico-pathologic characteristics of the patients and to treatment response. Results: 120 out of 174 patients included in the GeparNuevo trial underwent blood immunomonitoring; 63 patients belonged to the “window” sub-cohort. Durvalumab administration almost completely blocked the detection of the inhibitory ligand PD-L1 and induced changes in the composition of the immune cell subpopulations. Evaluation of the “window” sub-cohort, in which an enhanced, but not significant pathological clinical response was observed within the immunomonitored patients, identified different markers correlating with clinical response to durvalumab. Higher frequencies of CD4+ T cells at recruitment as well as increased frequencies of T cells bearing the gamma delta TCR along treatment were some of the characteristics of patients responding to durvalumab treatment. Conclusions: The flow cytometry-based immunomonitoring of the clinical trial identified different immune-relevant biomarkers at recruitment as well as during treatment that predict clinical response to durvalumab. After validation of this data in an independent patient cohort, these markers could be implemented for an improved patient stratification to immunotherapy. Citation Format: Chiara Massa, Thomas Karn, Karsten Weber, Andreas Schneeweiss, Claus Hanusch, Jens-Uwe Blohmer, Dirk-Michael Zahm, Christian Jackisch, Marion van Mackelenbergh, Jörg Thomalla, Frederik Marmé, Jens Huober, Volkmar Müller, Christian Schem, Anja Müller, Elmar Stickeler, Katharina Biehl, Peter A. Fasching, Michael Untch, Sibylle Loibl, Carsten Denkert, Barbara Seliger. Immunological and clinical consequences of durvalumab treatment in combination to neoadjuvant chemotherapy in triple-negative breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-04.

Background: It is well known that immunological pathways are relevant for response to classical neoadjuvant chemotherapy as well as combined chemo-immunotherapy. In addition, it has been shown that combined chemo-immunotherapy significantly improves survival, even in the context of only moderate effects on pCR. Due to the window therapy with durvalumab-alone and the option to analyze multiple consecutive biopsies, the GeparNuevo trial offers the opportunity to 1) determine gene expression patterns for pCR and DDFS endpoints 2) identify pathways most relevant for pCR and DDFS 3) identify genes specifically regulated by immunotherapy (comparison of samples pre-and post-window) 4) identify genes specifically regulated by chemotherapy (comparison of samples pre-Tx and after 4 cycles of chemotherapy 5) identify longitudinal patterns of gene expression by comparison of up to four time points and 6) identify changes in the tumor microenvironment by spatial sequencing of tumor cell and stroma areas. Methods: 292 tumor samples were evaluated by gene expression analysis: 162 pretherapeutic core biopsies, 79 post-window biopsies, 32 biopsies during chemotherapy and 19 biopsies of the residual tumor after therapy. These samples were analyzed by HTG OBP panel targeting 2549 genes which are assigned to 25 different biological mechanisms or cellular pathways. In addition, spatial profiling was compared in a subset of pre-and post-window samples using Nanostring GeoMx spatial profiling system. Endpoints were pCR and DDFS. Results: A total of more than 600 genes were significantly associated with either the pCR or the DDFS endpoint in either the complete GeparNuevo cohort or one of the two therapy arms. Interestingly, there was a large number of predictive or prognostic genes (n=247 for pCR and n=179 for DDFS) in the durvalumab arm, while the number of genes in the placebo arm was considerably lower (n=113 for pCR and n=61 for DDFS). We used existing pathway information for HTG OBP panel to analyze the contribution of different cellular processes to pCR and DDFS signatures in different therapy arms. Immune pathways were particularly relevant for durvalumab signatures (pCR and DDFS), while cell cycle related gene expression patterns were particularly involved in signatures predictive of pCR in both therapy arms. To further assign genes to the cellular response to durvalumab-alone or chemotherapy-alone, we compared gene expression patterns in durvalumab arm before and after the window phase (gene expression patterns induced by one dose of durvalumab) with gene expression patterns in placebo arm before and after 4 cycles of chemotherapy. Further longitudinal alterations were analyzed by comparison of longitudinal samples for 4 different time-points (a: before NACT, n=162; b: after window phase, n=79; c: after 4 cycles, n=31 and d: at surgery, n=19). Using the Nanostring GeoMx spatial RNA profiling system guided by cytokeratine immunofluorescence, we compared areas with high tumor cell content with stromal areas with or without TILs. In combination with the HTG gene expression data, we were able allocate the changes induced by durvalumab vs chemotherapy to the stromal cell and tumor cell compartment, indicating a re-organization of the tumor-microenvironment. Conclusions: In our analysis, we show that immune gene signatures are particularly relevant for neoadjuvant response to durvalumab as well as prognosis after durvalumab treatment, while proliferation signatures are involved in pCR-signatures after durvalumab as well as chemotherapy. The spatial analysis showed that relevant changes occur in the stromal compartment, indicating a re-organization of the tumor microenvironment. The parallel targeting of immune- and proliferation pathways might explain why a combined immunotherapy-chemotherapy approach is more successful than each single therapy strategy alone. Citation Format: Carsten Denkert, Andreas Schneeweiss, Julia Rey, Akira Hattesohl, Thomas Karn, Michael Braun, Paul Jank, Jens Huober, Hans-Peter Sinn, Dirk-Michael Zahm, Claus Hanusch, Frederik Marmé, Jenny Furlanetto, Jörg Thomalla, Jens-Uwe Blohmer, Marion van Mackelenbergh, Thorsten Stiewe, Peter Staib, Christian Jackisch, Julia Teply-Szymanski, Peter A. Fasching, Bruno V. Sinn, Michael Untch, Karsten Weber, Sibylle Loibl. PD4-02 Spatial and temporal heterogeneity of predictive and prognostic signatures in triple-negative breast cancer treated with neoadjuvant combination immune-chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-02.

Background: Biomarkers are needed to prognosticate and predict efficacy of therapy for patients undergoing neoadjuvant chemotherapy for HER2-negative breast cancer (BC). Caveolin genes (CAV1, CAV2) are responsible for formation of caveolae, which are 50-100 nM membrane invaginations implicated in endocytosis and transcytosis of nutrients and substances, including albumin. Overexpression of caveolin family genes has been implicated in driving BC progression, but also in predicting response to nab-paclitaxel. We hypothesized that high CAV1/2 expression would correlate with poor clinical outcomes, but that patients with high CAV1/2 expression would have better outcomes with nab-paclitaxel based chemotherapy. Methods: We correlated tumor CAV1/2 RNA expression from available RNA-Seq data with pathologic complete response (pCR) and clinical outcomes (disease-free survival, DFS, and overall survival, OS) in the GeparSepto (G7) clinical trial, which randomized patients with early stage BC to preoperative paclitaxel versus nab-paclitaxel-based chemotherapy regimens. CAV1/2 log-transformed values were analyzed as a continuous variable and dichotomized about the mean for each gene. Multivariable logistic regression models were generated, and included age, T-stage, N-stage, tumor grade (G3 vs G1-2), Ki67 (continuous), and histology (non-ductal versus ductal). Results: There was RNA-Seq expression data available for 279 out of 810 HER2-negative patients, of whom 26.5% were hormone receptor (HR)-negative (triple negative). CAV1 and CAV2 expression values were directly correlated with each other (Pearson coefficient 0.452). There was no difference in CAV1 expression between HR-negative and HR-positive patients, but there was significant up-regulation of CAV2 expression (mean) in HR-negative patients (p=0.003). With regards to pCR, high CAV1/2 expression was associated with a strong benefit from nab-paclitaxel compared to paclitaxel (OR(CAV1)=4.92 (1.70-14.22) and OR(CAV2)=5.39 (1.76-16.47)) in contrast to low CAV1/2 expression (OR(CAV1)=0.94 (0.38-2.34) and OR(CAV2)=0.95 (0.39-2.29)) in multivariate modeling (tests for interaction p=0.023 and p=0.019). For CAV1 this effect continued beyond pCR with regards to survival: high CAV1 expression was associated with superior survival in the nab-paclitaxel arm (HR(DFS)=0.53 (0.27-1.05), HR(OS)=0.29 (0.11-0.77)) but not low CAV1 expression (HR(DFS)=1.37 (0.62-3.04), HR(OS)=2.47 (0.78-7.80)) in multivariate modeling (test for interaction p=0.077 (DFS) and p=0.005 (OS)). No significant interactions in hazard ratios for DFS and OS were detected for CAV2. With regard to prognostic effects, CAV2 expression was significantly associated with worse DFS and OS for all patients. In particular, high CAV2 expression was associated with poor prognosis for HR-negative patients in multivariate modeling (OR(DFS)=4.40 (1.44-13.46), OR(OS)=9.38 (1.13-77.75)), but not for HR-positive patients (OR(DFS)=1.60 (0.84-3.05), OR(OS)=2.05 (0.92-4.58)) (test for interaction p=0.125 and p=0.186). Conclusions: Higher CAV2 expression is associated with worse DFS and OS, in particular for HR-negative patients, confirming potential prognostic roles for CAV1/2. No significant differences in DFS and OS based on CAV1/2 expression were noted for patients who received nab-paclitaxel based treatment, but the odds of obtaining pCR were improved for patients with high CAV1/2 expression. Taken together, these findings suggest that CAV1/2 expression may offset the negative prognostic factor associated with higher CAV1/2 expression in patients treated with nab-paclitaxel regimens by enhancing the efficacy of treatment, perhaps through increased nab-paclitaxel endocytosis/transcytosis. Citation Format: Terence Williams, Andreas Schneeweiss, Christian Jackisch, Changxian Shen, Karsten Weber, Peter Fasching, Carsten Denkert, Jenny Furlanetto, Ernst Heinmoller, Sabine Schmatloch, Thomas Karn, Christopher Szeto, Marion van Mackelenbergh, Valentina Nekljudova, Elmar Stickeler, Patrick Soon-Shiong, Christian Schem, Barbara Fleige, Volkmar Muller, Frederik Marme, Michael Untch, Sibylle Loibl. Caveolin gene expression predicts for response and clinical outcomes of patients treated with preoperative paclitaxel-based chemotherapy regimens in early stage breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-01.

Background: Tumor immune markers such as tumor infiltrating lymphocytes (TILs) or expression-based profiles have been correlated with both response to neoadjuvant chemotherapy and prognosis in early breast cancer (BC) patients. Some chemotherapies, such as paclitaxel, lead to the development of TILs and in some cases, suppression of regulatory T-cells. Therefore, assessment of the tumor microenvironment (TME) could provide important information for clinical decision-making. The aim of this study was to test if RNAseq-based TME classification of BC tumors is predictive of pathological complete Response (pCR) and prognosis in the neoadjuvant GeparSepto (G7) trial (NCT01583426). Methods: We performed a retrospective-prospective analysis of a subset of 810 subjects of the total of 1207 patients of the G7 trial. In G7 HER2-negative early high-risk BC patients were studied to determine if nab-paclitaxel is superior to solvent-based paclitaxel. In addition to the taxane paclitaxel, both treatment arms received epirubicin plus cyclophosphamide before surgery. For this analysis, a subset of 279 HER2 negative patients with sufficient quality of pretherapeutic core biopsies to perform whole-transcriptome RNAseq (~200x106 reads per tumor) was used. Based on RNAseq data, immune activity classification was provided by ImmunityBio (Culver City, CA) by comparison of expression of 23 immune-cell-specific gene signatures as described by Bindea et al. (Immunity, 2013) to those from a reference population of 1467 similarly-profiled unselected tumor samples from a large tumor database (NantOmics, Culver City, CA). Unsupervised hierarchical clustering of inferred immune activities revealed 3 distinct groups termed “hot”, “warm”, and “cold” clusters. Logistic regression analysis based on age, trial arm, tumor size, nodal status, Ki-67, hormone-receptor (HR) status and immune activity cluster (hot/warm vs. cold) as independent variables was performed to predict pCR (ypT0/ypN0). Cox regression analysis with the same covariates was also performed to predict disease-free survival (DFS) and overall survival (OS). Results: Of the 279 patients, 67 had a pCR (24%). The analyzed subset was similar to the main HER2 negative population (pCR-rate: 22%). Patients with a “hot/warm” or “cold” immune activity assessment had a pCR in 30% and 13% of the cases, respectively. The odds-ratio of the multivariate logistic regression analysis was 2.17 (95% CI: 1.00-4.71, p=0.0512). With regard to DFS and OS, T follicular helper cell B-cell, and T-cell signatures seemed to play a prominent role, and the hazard ratios (also “hot/warm” vs. “cold”) for the multivariate analyses were 0.38 (95% CI: 0.21-0.66; p=0.0007) and 0.34 (95%CI: 0.16-0.72, p= 0.0045), respectively. Within the 23 individual immune-cell-specific gene signatures, CD56dimNatural Killer (NK), type 1 helper T-cells, and CD8+ T-cell signatures seemed to be closely associated with achievement of a pCR. RNAseq-based deconvolution of immune-cell activity was corroborated by IHC-based TIL scoring. Immune-hot/warm patients had more intratumoral lymphocytes compared to cold tumors (mean: 11.6% vs. 4.9%, p Citation Format: Peter A Fasching, Carsten Denkert, Stephen Benz, Karsten E Weber, Christopher Szeto, Jan Budczies, Andreas Schneeweiss, Elmar Stickeler, Sabine Schmatloch, Christian Jackisch, Thomas Karn, Hans Peter Sinn, Mathias Warm, Marion van Mackelenbergh, Sharooz Rabizadeh, Christian Schem, Ernst Heinmöller, Volkmar Müller, Frederik Marmé, Patrick Soon-Shiong, Valentina Nekljudova, Michael Untch, Sibylle Loibl. Tumor immune-cell activity assessed by RNAseq is an independent predictor of therapy response and prognosis after neoadjuvant chemotherapy in HER2 negative breast cancer patients - An analysis of the GeparSepto trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-08.

Acid ceramidase (ASAH1) is a key player in sphingolipid metabolism and signaling. It has prognostic value for several cancers, but histotype-specific analyses of ovarian cancer are not yet available. We used three retrospective TMA cohorts encompassing a total of 1106 ovarian cancers with follow-up data for immunohistochemical analysis of acid ceramidase (ASAH1) expression. Patients with sub-optimal debulking and persistent residual tumor after surgery introduced bias in the prognostic analysis and were excluded from further studies. Overall, we detected an association of ASAH1 expression with better prognosis in ovarian cancer patients. ASAH1 expression differed between histological ovarian cancer histotypes with most frequent expression in endometrioid and clear cell ovarian cancer, which are both associated with good prognosis. Stratified subgroup analyses within these histotypes did not reveal significant survival differences, but the power of the analysis may be limited by smaller sample sizes. In contrast to breast cancer, we found only a modest concordance between estrogen receptor status and ASAH1 expression within the endometrioid ovarian cancer histotype. In an exploratory analysis of estrogen receptor negative endometrioid ovarian cancer, ASAH1 expression was associated with significantly better overall survival (P = 0.007). Acid ceramidase is most frequently expressed in endometrioid and clear cell histotypes and could add independent prognostic value to estrogen receptor in endometrioid ovarian cancer. Modulating sphingolipid metabolism may lead to novel therapeutic intervention strategies for this disease.

BMC cancer 22(1), 1040 (2022). doi:10.1186/s12885-022-10109-1, Published by BioMed Central, London

British journal of cancer : BJC 121(12), 1009-1015 (2019). doi:10.1038/s41416-019-0630-3, Published by Nature Publ. Group, Edinburgh

Purpose:Next-generation sequencing (NGS) can be used for comprehensive investigation of molecular events in breast cancer. We evaluated the relevance of genomic alterations for response to neoadjuvant chemotherapy (NACT) in the GeparSepto trial.Experimental Design:Eight hundred fifty-one pretherapeutic formalin-fixed paraffin-embedded (FFPE) core biopsies from GeparSepto study were sequenced. The panel included 16 genes for mutational (AKT1, BRAF, CDH1, EGFR, ERBB2, ESR1, FBXW7, FGFR2, HRAS, KRAS, NRAS, SF3B1, TP53, HNF1A, PIK3CA, and PTEN) and 8 genes for copy-number alteration analysis (CCND1, ERBB2, FGFR1, PAK1, PIK3CA, TOP2A, TP53, and ZNF703).Results:The most common genomic alterations were mutations of TP53 (38.4%) and PIK3CA (21.5%), and 8 different amplifications (TOP2A 34.9%; ERBB2 30.6%; ZNF703 30.1%; TP53 21.9%; PIK3CA 24.1%; CCND1 17.7%; PAK1 14.9%; FGFR 12.6%). All other alterations had a prevalence of less than 5%. The genetic heterogeneity in different breast cancer subtypes [lum/HER2neg vs. HER2pos vs. triple-negative breast cancer (TNBC)] was significantly linked to differences in NACT response. A significantly reduced pathologic complete response rate was observed in PIK3CA-mutated breast cancer [PIK3CAmut: 23.0% vs. wild-type (wt) 38.8%, P < 0.0001] in particular in the HER2pos subcohort [multivariate OR = 0.43 (95% CI, 0.24–0.79), P = 0.006]. An increased response to nab-paclitaxel was observed only in PIK3CAwt breast cancer, with univariate significance for the complete cohort (P = 0.009) and the TNBC (P = 0.013) and multivariate significance in the HER2pos subcohort (test for interaction P = 0.0074).Conclusions:High genetic heterogeneity was observed in different breast cancer subtypes. Our study shows that FFPE-based NGS can be used to identify markers of therapy resistance in clinical study cohorts. PIK3CA mutations could be a major mediator of therapy resistance in breast cancer.

We analyzed the predictive potential of pretreatment soluble carbonic anhydrase IX levels (sCAIX) for the efficacy of bevacizumab in the phase III neoadjuvant GeparQuinto trial. sCAIX was determined by enzyme-linked immunosorbent assay (ELISA). Correlations between sCAIX and pathological complete response (pCR), disease-free and overall survival (DFS, OS) were assessed with logistic and Cox proportional hazard regression models using bootstrapping for robust estimates and internal validation. 1,160 HER2-negative patient sera were analyzed, of whom 577 received bevacizumab. Patients with low pretreatment sCAIX had decreased pCR rates (12.1 vs. 20.1%, p = 0.012) and poorer DFS (adjusted 5-year DFS 71.4 vs. 80.5 months, p = 0.010) compared to patients with high sCAIX when treated with neoadjuvant chemotherapy (NCT). For patients with low sCAIX, pCR rates significantly improved upon addition of bevacizumab to NCT (12.1 vs. 20.4%; p = 0.017), which was not the case in patients with high sCAIX (20.1% for NCT vs. 17.0% for NCT-B, p = 0.913). When analyzing DFS we found that bevacizumab improved 5-year DFS for patients with low sCAIX numerically but not significantly (71.4 vs. 78.5 months; log rank 0.234). In contrast, addition of bevacizumab worsened 5-year DFS for patients with high sCAIX (81 vs. 73.6 months, log-rank 0.025). By assessing sCAIX levels we identified a patient cohort in breast cancer that is potentially undertreated with NCT alone. Bevacizumab improved pCR rates in this group, suggesting sCAIX is a predictive biomarker for bevacizumab with regards to treatment response. Our data also show that bevacizumab is not beneficial in patients with high sCAIX.

583 Background: Immunotherapy is entering clinical practice as a promising new neoadjuvant therapeutic approach in triple-negative breast cancer, and it is important to identify biomarkers to focus this therapy on those patients that have the highest benefit. Interestingly, an improved survival outcome is observed in pCR and non-pCR patients, which raises the hypothesis that biomarkers might also be different for pCR prediction as well as prognosis. In this study, we investigated this hypothesis in the neoadjuvant GeparNuevo trial. Methods: A total of 174 patients were randomized to receive neoadjuvant chemotherapy with durvalumab vs. placebo. HTG EdgeSeq mRNA analysis was performed for a total of 2549 genes in 162 pretherapeutic core biopsies. In addition, tumor-infiltrating lymphocytes (stromal and intratumoural) as well as PD-L1 protein expression was evaluated by IHC. We systematically compared the distant disease-free survival (DDFS) of 5 predefined gene signatures (including the GeparSixto immune signature) as well as 12 single mRNA markers identified in previous projects between treatment arms using univariate Cox proportional-hazard regression analyses. In addition, exploratory biomarker analyses were performed. Results: The PSIP1 gene expression (per 1 unit hazard ratio [HR]: 0.58 95%CI 0.41-0.83; p=0.002), TAP1 (per 1 unit HR: 0.68 95%CI 0.48-0.95; p=0.025) as well as stromal TILs (sTILs) (per 10% HR: 0.73 95%CI 0.56-0.95; p=0.019) were significant for improved DDFS in the complete cohort. In the placebo arm PSIP1 (HR 0.50 95%CI 0.29-0.87; p=0.014) as well as sTILs (HR 0.73 95%CI 0.53-0.99; p=0.044) were significant for improved DDFS. In the durvalumab arm, the gene expression of PSIP1 (HR 0.54 95%CI 0.31-0.94; p=0.029), PD-L1/CD274 (per 1 unit HR: 0.41 95%CI 0.21-0.77; p=0.006), CD38 (per 1 unit: HR 0.52 95%CI 0.29-0.92; p=0.026) as well as the GeparSixto immune signature (per 1 unit HR: 0.51 95%CI 0.27-0.97; p=0.041) were significant for improved DDFS, with a positive test for interaction with treatment arm for PD-L1/CD274 (interaction p=0.020). Additional analyses, including multivariate Cox regressions for DDFS as well as systematic comparisons for biomarkers for DDFS and for pCR, will be presented. Conclusions: Our analysis suggests that biomarkers for immune response are linked to improved survival with neoadjuvant durvalumab therapy and that in this setting, survival biomarkers are not identical to pCR biomarkers. The results are a basis for a further dissection of the contribution of pCR to survival effects of immunotherapy.

581 Background: It is commonly anticipated that a high tumor mutational burden (TMB) is a predictor of response to immune checkpoint blockade (ICB). We previously showed that triple-negative breast cancer (TNBC) from the GeparNuevo study with high TMB displayed increased response both to neoadjuvant chemo-ICB with durvalumab but also to chemotherapy alone, with no significant interaction with treatment arm (Karn et al. Ann Oncol 2020). In contrast, we also observed that cases with very low TMB more often displayed a pCR after treatment with chemo-ICB than with chemotherapy alone. This may in fact suggest a benefit of ICB to those TNBC with rather low TMB. Methods: We have analyzed the distant disease-free survival (DDFS) of GeparNuevo patients according to TMB and treatment arm (neoadjuvant chemotherapy plus durvalumab or chemotherapy plus placebo). For TMB (mut/Mb) we applied the identical cutoff of the upper tertile as in our previous analysis. Results: The median follow-up of the time-to-event data was 43.7 months. Data of TMB was available in 149 of 174 patients. We found that within the high-TMB tumors (durvalumab: n=27; placebo: n=23), DDFS was similar between both arms of the trial (durvalumab vs. placebo: HR (hazard ratio) 0.95 [95%CI 0.19-4.69], p=0.95). Strikingly however, within the low-TMB group (durvalumab: n=47; placebo: n=52) we observed a significantly better DDFS in the durvalumab-chemotherapy combination arm, in contrast to the arm treated only with chemotherapy (durvalumab vs. placebo: HR 0.23 [95%CI 0.06-0.79], p=0.02; interaction test for TMB and treatment arm p=0.17). The observation was also robust to alternative TMB cutoffs. Similar results were obtained for invasive disease-free survival. Conclusions: Our results show, in contrast to other published data, that patients with early TNBC and low TMB/neoantigen counts may benefit from short-term neoadjuvant durvalumab treatment, while for those with high TMB, durvalumab plus chemotherapy does not improve efficacy over chemotherapy alone.

Background Current genetic and genomic tests measuring homologous recombination deficiency (HRD) show limited predictive value. This study compares the performance of an immunohistology-based RAD51 test with genetic/genomic tests to identify patients with HRD primary triple-negative breast cancer (TNBC) and evaluates its accuracy to select patients sensitive to platinum-based neoadjuvant chemotherapy (NACT). Patients and methods This is a retrospective, blinded, biomarker analysis from the GeparSixto randomized clinical trial. TNBC patients received neoadjuvant paclitaxel plus Myocet®-nonpegylated liposomal doxorubicin (PM) or PM plus carboplatin (PMCb), both arms including bevacizumab. Formalin-fixed paraffin-embedded (FFPE) tumor samples were laid on tissue microarrays. RAD51, BRCA1 and γH2AX were quantified using an immunofluorescence assay. The predictive value of RAD51 was assessed by regression models. Concordance analyses were carried out between RAD51 score and tumor BRCA (tBRCA) status or genomic HRD score (Myriad myChoice®). Associations with pathological complete response (pCR) and survival were studied. Functional HRD was predefined as a RAD51 score ≤10% (RAD51-low). Results Functional HRD by RAD51-low was evidenced in 81/133 tumors (61%). RAD51 identified 93% tBRCA-mutated tumors and 45% non-tBRCA mutant cases as functional HRD. The concordance between RAD51 and genomic HRD was 87% [95% confidence interval (CI) 79% to 93%]. In patients with RAD51-high tumors, pCR was similar between treatment arms [PMCb 31% versus PM 39%, odds ratio (OR) 0.71, 0.23-2.24, P = 0.56]. Patients with RAD51-low tumors benefited from PMCb (pCR 66% versus 33%, OR 3.96, 1.56-10.05, P = 0.004; interaction test P = 0.02). This benefit maintained statistical significance in the multivariate analysis. Carboplatin addition showed similar disease-free survival in the RAD51-high [hazard ratio (HR) 0.40, log-rank P = 0.11] and RAD51-low (0.45, P = 0.11) groups. Conclusions The RAD51 test identifies tumors with functional HRD and is highly concordant with tBRCA mutation and genomic HRD. RAD51 independently predicts clinical benefit from adding Cb to NACT in TNBC. Our results support further development to incorporate RAD51 testing in clinical decision-making.

Young women receiving chemotherapy for early breast cancer (EBC) have a high probability for ovarian failure, defined by chemotherapy-induced amenorrhea (CIA) as a surrogate. CIA is insufficiently reliable and reproducible. We analysed chemotherapy-induced ovarian failure (CIOF) by assessing hormone parameters, CIA, and antral follicle count (AFC).Blood samples of women aged ≤45 years treated with anthracycline/taxane-based chemotherapy for EBC from four neoadjuvant/adjuvant trials were collected at baseline, at the end of treatment (EOT), and at 6, 12, 18, and 24 months after EOT. Centrally assessed oestradiol (cutoff52.2 ng/L) and follicle-stimulating hormone (cutoff12.4IU/L) were used to define CIOF for patients with baseline premenopausal hormone levels, anti-Müllerian hormone (AMH), and AFC to assess ovarian reserve. Further analyses included CIA, regain of premenopausal hormone levels, and disease-free survival (DFS) also in subgroups.Six hundred ninety-six patients aged ≤45 years had premenopausal hormone levels at baseline. Overall, 85.1% (592/696) experienced CIOF at EOT, and 147 of 592 had further hormone measurements after EOT. Of those, 32.7% (48/147) regained premenopausal hormone levels after 6 months, 57.9% (66/114) regained premenopausal hormone levels after 12 months, 83.0% (73/88) regained premenopausal hormone levels after 18 months, and 89.2% (74/83) regained premenopausal hormone levels after 24 months. After 24 months, 72.4% (21/29) of patients without CIOF and 100% (14/14) with CIOF had low AMH levels. Four-year DFS without CIOF versus CIOF was 65.9% versus 84.6% (hazard ratio [HR] = 2.09, 95% confidence interval [CI]: 1.37-3.19; P 0.001); in hormone receptor positive 61.8% versus 87.5% (HR = 2.69, 95% CI: 1.57-4.60; P 0.001); in30 years 68.3% versus 92.6% (HR = 4.87, 95% CI: 1.05-22.63; P = 0.026).Most premenopausal women experienced CIOF after chemotherapy for EBC. After 2 years, nearly all regain premenopausal hormone levels. CIOF was associated with better DFS, especially in patients with hormone receptor-positive EBC or aged30 years.

[This corrects the article DOI: 10.1371/journal.pone.0238021.].

Clinical epigenetics 13(1), 184 (2021). doi:10.1186/s13148-021-01176-5, Published by BioMed Central, [S.l.]

BMC cancer 21(1), 920 (2021). doi:10.1186/s12885-021-08656-0, Published by Springer, Berlin ; Heidelberg

Background: Breast cancer is one of the most common malignancies during pregnancy. Although breast cancer in pregnancy (BCP) is still a rare event (1 in 3000 to 10000 pregnancies), the incidence is likely to increase as more women tend to delay childbearing into later life and the overall lifetime cancer risk increases with age. Pregnancy presents a complex and unique immunological condition. Pregnant women are widely considered to be in an immunosuppressed state, making them more susceptible to infectious diseases. Recent studies have shown similarities between malignancies and the semi-allogenic fetus in terms of immune evasion strategies, for example upregulation of non-classical human leukocyte antigen G (HLA-G). The loss or downregulation of HLA class I is also a way to escape anti-tumor immunity. The aim of this study was to investigate the tumor biology and immunology of pregnant breast cancer patients and the impact of pregnancy on the immunological characteristics of the breast cancer. Methods: 196 of 2831 patient enrolled in the BCP registry (GBG 29) had available tumor material. After identifying representative tumor regions, tissue microarrays (TMAs) of formalin-fixed paraffin embedded core biopsies or surgical specimens from 126 pregnant breast cancer patients treated with neo-(adjuvant) chemotherapy were constructed. TMAs were stained via immunohistochemistry to assess estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), Ki-67 (5%), HLA-G (≤5% vs >5%), TIGIT and Nectin4 as well as hematoxylin-eosin for the prevalence of tumor-infiltrating lymphocytes (TILs, ≤30% vs 31-60% vs >60%). Results: Median age of the patients was 34 (range 26 - 47) years. At the time of diagnosis 50.8% of patients had cT2 tumor and 36.8% nodal involvement. The pre-dominant histological tumor type was ductal or ductal-lobular-invasive carcinoma (89.5%) with poor differentiation (G3: 68.3%). 78.5% of patients were centrally HER2 negative, 42.1% ER-positive and 53.2% PgR-positive; 53.3% had a high expression of Ki-67 (≥20%). With regards to breast cancer subtypes, most patients had either TNBC (34.7%) or HER2-/HR+ breast cancer (43.8%). HLA class I expression (≤5%) was downregulated in 21.3% of patients while 46.3% showed upregulation of HLA-G expression (>5%). Analyzing HLA-G as continuous variable demonstrated an increased but not significant median expression of HLA-G in T3-4 (N=27) compared to T1-2 (N=99) tumor stage (12.4% vs 3.4%, p=0.226). TILs were detected in 62 out of 126 analyzed patients, of whom 93.5% (N=58) had a low expression of TILs (≤30%). Analysis of TIGIT and Nectin4 is ongoing. Conclusions: The high prevalence of low TILs expression in breast cancer during pregnancy might be a sign of reduced maternal immunity associated with pregnancy. HLA class I downregulation detected in our cohort suggests that it is an independent factor of anti-tumor immunity. Additional immune markers and data from a non-pregnant cohort are currently evaluated to make valid conclusions regarding gestational effects on classical and immunological tumor features. Those data will be presented at the meeting. Citation Format: Kristin Galas, Moritz Gleitsmann, Julia Rey, Christine Solbach, Isabell Witzel, Thomas Karn, Andreas Schneeweiss, Bruno Sinn, Tanja Fehm, Carsten Denkert, Volkmar Müller, Anne-Sophie Litmeyer, Christian Schem, Paul Jank, Frederik Marmé, Jenny Furlanetto, Peter A. Fasching, Elmar Stickeler, Olaf Ortmann, Marion van Mackelenbergh, Valentina Nekljudova, Sibylle Loibl. Immunological markers in patients with breast cancer occurring during pregnancy - Results from GBG BCP study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-14.

Different endogenous and exogenous mutational processes act over the evolutionary history of a malignant tumor, driven by abnormal DNA editing, mutagens or age-related DNA alterations, among others, to generate the specific mutational landscape of each individual tumor. The signatures of these mutational processes can be identified in large genomic datasets. We investigated the hypothesis that genomic patterns of mutational signatures are associated with the clinical behavior of breast cancer, in particular chemotherapy response and survival, with a particular focus on therapy-resistant disease.Whole exome sequencing was carried out in 405 pretherapeutic samples from the prospective neoadjuvant multicenter GeparSepto study. We analyzed 11 mutational signatures including biological processes such as APOBEC-mutagenesis, homologous recombination deficiency (HRD), mismatch repair deficiency and also age-related or tobacco-induced alterations.Different subgroups of breast carcinomas were defined mainly by differences in HRD-related and APOBEC-related mutational signatures and significant differences between hormone-receptor (HR)-negative and HR-positive tumors as well as correlations with age, Ki-67 and immunological parameters were observed. We could identify mutational processes that were linked to increased pathological complete response rates to neoadjuvant chemotherapy with high significance. In univariate analyses for HR-positive tumors signatures, S3 (HRD, P0.001) and S13 (APOBEC, P = 0.001) as well as exonic mutation rate (P = 0.002) were significantly correlated with increased pathological complete response rates. The signatures S3 (HRD, P = 0.006) and S4 (tobacco, P = 0.011) were prognostic for reduced disease-free survival of patients with chemotherapy-resistant tumors.The results of this investigation suggest that the clinical behavior of a tumor, in particular, response to neoadjuvant chemotherapy and disease-free survival of therapy-resistant tumors, could be predicted by the composition of mutational signatures as an indicator of the individual genomic history of a tumor. After additional validations, mutational signatures might be used to identify tumors with an increased response rate to neoadjuvant chemotherapy and to define therapy-resistant subgroups for future therapeutic interventions.

Synthetic lethal combination induces ferroptosis., Identification of targeted therapies for TNBC is an urgent medical need. Using a drug combination screen reliant on synthetic lethal interactions, we identified clinically relevant combination therapies for different TNBC subtypes. Two drug combinations targeting the BET family were further explored. The first, targeting BET and CXCR2, is specific for mesenchymal TNBC and induces apoptosis, whereas the second, targeting BET and the proteasome, is effective for major TNBC subtypes and triggers ferroptosis. Ferroptosis was induced at low drug doses and was associated with increased cellular iron and decreased glutathione levels, concomitant with reduced levels of GPX4 and key glutathione biosynthesis genes. Further functional studies, analysis of clinical datasets and breast cancer specimens revealed a unique vulnerability of TNBC to ferroptosis inducers, enrichment of ferroptosis gene signature, and differential expression of key proteins that increase labile iron and decrease glutathione levels. This study identified potent combination therapies for TNBC and unveiled ferroptosis as a promising therapeutic strategy.

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive form of breast cancer (BC). Due to the absence of targets such as HER2 or hormone receptors, early TNBC is treated with surgery and chemotherapy. Since TNBC is also considered the most immunogenic type of BC with tumor infiltrating lymphocytes that are predictive for chemotherapy response and prognostic for patients′ survival, many different immunotherapeutic strategies are currently explored in clinical trials for the treatment of this disease. In order to efficiently combine chemotherapy with immunotherapy, it is important to evaluate the effect of chemotherapy on immune cells in vivo.MethodsPeripheral blood was taken from 56 patients with TNBC undergoing neoadjuvant chemotherapy with nanoparticle albumin-bound paclitaxel (Nab-Pac) followed by epirubicin and cyclophosphamide (EC) at three different time points. Multicolor flow cytometry was used to characterize the immune cell composition and functional properties along neoadjuvant chemotherapy.ResultsWhereas the first phase of the neoadjuvant chemotherapy did not significantly alter the patients′ immune cell composition, after the second phase of chemotherapeutic administration most B cells (>90%) were lost and the frequency of natural killer (NK) cells and CD4+ T lymphocytes decreased approximately to 50%. In contrast, the frequency of CD8+ T cells were less affected.ConclusionsDespite late consequences of Nab-Pac cannot be ruled out, these data suggest that different chemotherapeutics might have distinct effects on the immune cell repertoire and that different immune cell populations exhibit a specific susceptibility to these chemotherapies with B and NK cells being more affected than T cells. This might also have an impact on the combination of chemotherapies with immunotherapies.Trial registration numberNCT02685059.

Background The predictive value of tumor mutational burden (TMB), alone or in combination with an immune gene expression profile (GEP), for response to neoadjuvant therapy in early triple negative breast cancer (TNBC) is currently not known, either for immune checkpoint blockade (ICB) or conventional chemotherapy. Patients and methods We obtained both whole exome sequencing and RNA-Seq data from pretreatment samples of 149 TNBC of the recent neoadjuvant ICB trial, GeparNuevo. In a predefined analysis, we assessed the predictive value of TMB and a previously developed immune GEP for pathological complete remission (pCR). Results Median TMB was 1.52 mut/Mb (range 0.02–7.65) and was significantly higher in patients with pCR (median 1.87 versus 1.39; P = 0.005). In multivariate analysis, odds ratios for pCR per mut/Mb were 2.06 [95% confidence intervals (CI) 1.33–3.20, P = 0.001] among all patients, 1.77 (95% CI 1.00–3.13, P = 0.049) in the durvalumab treatment arm, and 2.82 (95% CI 1.21–6.54, P = 0.016) in the placebo treatment arm, respectively. We also found that both continuous TMB and immune GEP (or tumor infiltrating lymphocytes) independently predicted pCR. When we stratified patients in groups based on the upper tertile of TMB and median GEP, we observed a pCR rate of 82% (95% CI 60% to 95%) in the group with both high TMB and GEP in contrast to only 28% (95% CI 16% to 43%) in the group with both low TMB and GEP. Conclusions TMB and immune GEP add independent value for pCR prediction. Our results recommend further analysis of TMB in combination with immune parameters to individually tailor therapies in breast cancer.

hTFM in primary vulvar cancer is an important prognostic factor. Ideally, a diameter of 8 mm should be achieved after primary surgery. The role of VIN III persistence after primary surgery in vulvar cancer is still unclear. The main objective of the current study was to study the role of residual VIN III re-excision and compare differences in disease-free survival among patients with different hTFM and in primary vulvar cancer.Forty-two patients with residual adjacent VIN III after primary surgery for vulvar cancer which were operated between 2000 and 2016 in our clinic were enrolled in this retrospective study. Re-excision rates for residual adjacent VIN III were calculated. According to the histological margin patients were divided into three group: 3, 3-8 and 8 mm. Univariate and multivariate analyses were conducted using the Kaplan-Meier method and Cox proportional hazards models, respectively.The vast majority of patients had pT1b stage (57.1%), grading G2 (71.4%) and lymph node-negative (45.3%) disease at first diagnosis. The re-excision rate was 57.1%. The 5-year disease-free survival (DFS) rates in patients with 3, 3-8 and 8 mm hTFM were 50.0, 50.0 and 81.0%, respectively (p = 0.032). The 5-year DFS rates in patients with re-excision and without re-excision for VIN III were 77.3 and 52.9%, respectively (p = 0.060). In univariate analysis was solely hTFM 8 mm a prognostic factor for DFS (p = 0.017).hTFM may be a potential prognostic indicator for DFS in vulvar cancer patients. Re-excision for residual adjacent VIN III could not be established as a prognostic factor for DFS after primary surgery in squamous cell cancer of vulva.

Summary Background Tumour-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) and HER2-positive breast cancer, but their role in luminal breast cancer and the effect of TILs on prognosis in all subtypes is less clear. Here, we assessed the relevance of TILs for chemotherapy response and prognosis in patients with TNBC, HER2-positive breast cancer, and luminal–HER2-negative breast cancer. Methods Patients with primary breast cancer who were treated with neoadjuvant combination chemotherapy were included from six randomised trials done by the German Breast Cancer Group. Pretherapeutic core biopsies from 3771 patients included in these studies were assessed for the number of stromal TILs by standardised methods according to the guidelines of the International TIL working group. TILs were analysed both as a continuous parameter and in three predefined groups of low (0–10% immune cells in stromal tissue within the tumour), intermediate (11–59%), and high TILs (≥60%). We used these data in univariable and multivariable statistical models to assess the association between TIL concentration and pathological complete response in all patients, and between the amount of TILs and disease-free survival and overall survival in 2560 patients from five of the six clinical trial cohorts. Findings In the luminal–HER2-negative breast cancer subtype, a pathological complete response (pCR) was achieved in 45 (6%) of 759 patients with low TILs, 48 (11%) of 435 with intermediate TILs, and 49 (28%) of 172 with high TILs. In the HER2-positive subtype, pCR was observed in 194 (32%) of 605 patients with low TILs, 198 (39%) of 512 with intermediate TILs, and 127 (48%) of 262 with high TILs. Finally, in the TNBC subtype, pCR was achieved in 80 (31%) of 260 patients with low TILs, 117 (31%) of 373 with intermediate TILs, and 136 (50%) of 273 with high TILs (p 2 test for trend). In the univariable analysis, a 10% increase in TILs was associated with longer disease-free survival in TNBC (hazard ratio [HR] 0·93 [95% CI 0·87–0·98], p=0·011) and HER2-positive breast cancer (0·94 [0·89–0·99], p=0·017), but not in luminal–HER2-negative tumours (1·02 [0·96–1·09], p=0·46). The increase in TILs was also associated with longer overall survival in TNBC (0·92 [0·86–0·99], p=0·032), but had no association in HER2-positive breast cancer (0·94 [0·86–1·02], p=0·11), and was associated with shorter overall survival in luminal–HER2-negative tumours (1·10 [1·02–1·19], p=0·011). Interpretation Increased TIL concentration predicted response to neoadjuvant chemotherapy in all molecular subtypes assessed, and was also associated with a survival benefit in HER2-positive breast cancer and TNBC. By contrast, increased TILs were an adverse prognostic factor for survival in luminal–HER2-negative breast cancer, suggesting a different biology of the immunological infiltrate in this subtype. Our data support the hypothesis that breast cancer is immunogenic and might be targetable by immune-modulating therapies. In light of the results in luminal breast cancer, further research investigating the interaction of the immune system with different types of endocrine therapy is warranted. Funding Deutsche Krebshilfe and European Commission.

Background/Aim: Totally implanted venous access devices (TIVAD) are increasingly used in the treatment of cancer patients. The aim of this study was to assess the incidence of early and late complications resulting from subcutaneous TIVADs in patients with breast cancer. Materials and Methods: Between 2004 and 2009, we reviewed patients with breast cancer who had a TIVAD placed. Early and late complications, as well as risk factors for TIVAD-associated thrombosis were retrospectively assessed. Results: A total of 281 patients were included. Complications occurred in 26% of patients, the majority of which were late complications (21.4%.) The development of TIVAD associated thrombosis was the most frequent late complication (16.4%). In the univariate analysis followed by a multivariate model, risk factors for TIVAD associated thrombosis were not identified. Only within the subgroup of metastatic breast cancer patients an increased risk of TIVAD-associated thrombosis of left compared to right venous access was detected (p=0.015). Conclusion: TIVAD implantation done in a gynecological outpatient setting is feasible and safe.

BMC cancer 21(1), 1024 (2021). doi:10.1186/s12885-021-08754-z, Published by BioMed Central, London

Claudin-1 is a tight junction protein that has been demonstrated to be involved in tumorigenesis and tumor progression in various types of solid tumors. In the present study, the protein expression of claudin-1 in squamous cervical cancer tissues obtained from 106 patients was analyzed by immunohistochemistry. In addition, the grade of claudin-1 expression was analyzed for associations with certain clinicopathological parameters. A significant overexpression of claudin-1 was detected in the tumor cells, when compared with that in the peritumoral stroma. There was no significant association between claudin-1 expression and FIGO stage, tumor size, grading or the appearance of distant metastases. Cervical cancer patients scoring positive for claudin-1 protein expression tended to exhibit more lymph node metastasis (28.3%), compared with claudin-1-negative patients (7.1%). Regarding overall survival, the results of the present study suggest a better prognosis for claudin-1-negative patients. In order to elucidate whether claudin-1 overexpression has a significant prognostic impact on squamous cervical cancer, further studies are required.

Purpose: The BRCA1-like profile identifies tumors with a defect in homologous recombination due to inactivation of BRCA1. This profile has been shown to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break-inducing chemotherapy. We tested the predictive potential of the BRCA1-like profile for adjuvant non-myeloablative, intensified dose-dense chemotherapy in the GAIN trial. Methods: Lymph node positive breast cancer patients were randomized to 3 × 3 dose-dense cycles of intensified epirubicin, paclitaxel, and cyclophosphamide (ETC) or 4 cycles concurrent epirubicin and cyclophosphamide followed by 10 cycles of weekly paclitaxel combined with 4 cycles capecitabine (EC-TX). Only triple negative breast cancer patients (TNBC) for whom tissue was available were included in these planned analyses. BRCA1-like or non-BRCA1-like copy number profiles were derived from low coverage sequencing data. Results: 119 out of 163 TNBC patients (73%) had a BRCA1-like profile. After median follow-up of 83 months, disease free survival (DFS) was not significantly different between BRCA1-like and non-BRCA1-like patients [adjusted hazard ratio (adj.HR) 1.02; 95% confidence interval (CI) 0.55–1.86], neither was overall survival (OS; adj.HR 1.26; 95% CI 0.58–2.71). When split by BRCA1-like status, DFS and OS were not significantly different between treatments. However, EC-TX seemed to result in a trend to an improvement in DFS in patients with a BRCA1-like tumor, while the reverse accounted for ETC treatment in patients with a non-BRCA1-like tumor (p for interaction = 0.094). Conclusions: The BRCA1-like profile is not associated with survival benefit for a non-myeloablative, intensified regimen in this study population. Considering the limited cohort size, capecitabine might have additional benefit for TNBC patients.

The presence of tumor-infiltrating lymphocytes (TIL) is a favorable prognostic factor in breast cancer, but what drives immune infiltration remains unknown. Here we examine if clonal heterogeneity, total mutation load, neoantigen load, copy number variations (CNV), gene- or pathway-level somatic mutations, or germline polymorphisms (SNP) are associated with immune metagene expression in breast cancer subtypes. Thirteen published immune metagenes correlated separately with genomic metrics in the three major breast cancer subtypes. We analyzed RNA-Seq, DNA copy number, mutation and germline SNP data of 627 ER+, 207 HER2+, and 191 triple-negative (TNBC) cancers from The Cancer Genome Atlas. P-values were adjusted for multiple comparisons, and permutation testing was used to assess false discovery rates. Increased immune metagene expression associated significantly with lower clonal heterogeneity estimated by MATH score in all subtypes and with a trend for lower overall mutation, neoantigen, and CNV loads in TNBC and HER2+ cancers. In ER+ cancers, mutation load, neoantigen load, and CNV load weakly but positively associated with immune infiltration, which reached significance for overall mutation load only. No highly recurrent single gene or pathway level mutations associated with immune infiltration. High immune gene expression and lower clonal heterogeneity in TNBC and HER2+ cancers suggest an immune pruning effect and equilibrium between immune surveillance and clonal expansion. Thus, immune checkpoint inhibitors may tip the balance in favor of immune surveillance in these cancers. Cancer Res; 77(12); 3317–24. ©2017 AACR.

Background: Tumors with infiltrating lymphocytes (TIL) demonstrate a better prognosis particularly in TNBC and HER2 positive breast cancer. Two competing hypothesis predict contrasting relationships of TILs and genomic heterogeneity. On one hand, a strong immune response may lead to “pruning” of intratumor heterogeneity by eliminating immunogenic clones resulting in a near equilibrium, hence better prognosis, while cancers that escape the surveillance may evolve towards greater clonal heterogeneity and genomic complexity. In some cancers, the predicted neoantigens are less frequent than expected by chance also suggesting immune mediated elimination of neoplastic clones (Rooney et al. 2015). Studies also showed an inverse association between immune cell infiltration and intratumor clonal heterogeneity (Morris et al. 2016). On the other hand, cancers with greater genomic instability and mutational burden will have larger clonal heterogeneity and therefore more neoantigens and greater immune infiltration. Indeed, a positive correlation between overall mutation load and immune activity in the tumor microenvironment was observed in pooled data across a broad range of cancer types (Brown et al. 2014, Rooney et al. 2015, Schumacher and Schreiber 2015). Methods: We assessed these two competing hypothesis and examined the relationship between genomic complexity and immune gene expression in different breast cancer subtypes. We used previously described immune metagene expression (DNA microarray n=655) as measures of immune infiltration in the TCGA data set (RNA-Seq n=1215). We compared somatic mutations, mutation count, neoantigen load, clonal heterogeneity metrics and the distribution of mutations in 119 canonical cancer genes and 12 cancer pathways between good and poor prognosis TNBC (n=208) corresponding to high and low immune infiltration. Results:A positive but weak correlation between mutation count and immune metagene expression was observed when all breast cancer subtypes were analyzed together (P=0.08). This was driven by the generally higher mutation count and immune infiltration in TNBC. When TNBC was analyzed separately, good prognosis TNBC with high immune infiltration had lower total mutation count (P=0.021) and predicted neo-antigen count (P=0.035). Clonal heterogeneity was also lower in good prognosis TNBC (P=0.001). There was a strong inverse relationship of dispersion in mutation variant allele frequencies and immune metagene expression. CASP8 was the top enriched mutation in TNBC with high immune infiltration (P=0.007 with no adjustment for multiple testing). Conclusions:High immune infiltration is associated with reduced intratumor heterogeneity in TNBC suggesting immune sculpting of the tumor and a near equilibrium between the cancer and immune surveillance. Surgical resection of the primary tumor may tilt the balance towards the immune system resulting in the better prognosis of high-TIL TNBC. TNBC with low immune infiltration has greater clonal heterogeneity and mutation load and may represent the consequence of escape from immune surveillance. Mutation of CASP8 may be one way to evade tumor cell killing in high-TIL TNBC as previously noted. Citation Format: Karn T, Jiang T, Hatzis C, Sänger N, El-Balat A, Holtrich U, Becker S, Bianchini G, Pusztai L. Immune sculpting of the triple negative breast cancer genome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-07.

BACKGROUND/AIM Borderline ovarian tumors (BOTs) have a less aggressive behavior than invasive epithelial ovarian tumors. Still some patients relapse or succumb to disease. Molecular markers that reliably predict prognosis are lacking. Insulin-like growth factor II mRNA-binding protein (IMP3) has been suggested as a prognostic marker in colorectal, hepatocellular, and ovarian clear-cell carcinomas. MATERIALS AND METHODS We analyzed the expression of IMP3 by immunohistochemistry in a cohort of 140 BOT and its association with histopathological features. RESULTS We found no association of IMP3 expression with patients' age, FIGO stage, microinvasion, and presence of implants. In contrast, IMP3 expression correlated to mucinous subtype of BOTs (42.2% vs. 9.5% among other subtypes) (p

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous disease with poor prognosis and no effective targeted therapies. EGFR is highly expressed in basal-like TNBC and is considered as a potential therapeutic target. However, EGFR targeting exerts only marginal clinical benefits, possibly due to activation of compensatory signaling pathways, which are frequently associated with HER3 upregulation. Here we show that concomitant targeting of EGFR and the nonreceptor tyrosine kinases PYK2/FAK synergistically inhibits the proliferation of basal-like TNBC cells in vitro and attenuates tumor growth in a mouse xenograft model. Dual targeting of EGFR and PYK2/FAK inhibited complementary key growth and survival pathways mediated by AKT, S6K, STAT3, and ERK1/2 activation. PYK2 inhibition also abrogated HER3 upregulation in response to EGFR antagonists, thereby circumventing HER3-associated drug resistance. Mechanistically, PYK2 inhibition facilitated the proteasomal degradation of HER3 while inducing upregulation of NDRG1 (N-myc downstream regulated 1 gene). NDRG1 enhanced the interaction of HER3 with the ubiquitin ligase NEDD4, while PYK2, which interacts with NEDD4 and HER3, interfered with NEDD4–HER3 binding, suggesting that the PYK2–NDRG1–NEDD4 circuit has a critical role in receptor degradation, drug response, and resistance mechanism. Our studies offer a preclinical proof of concept for a strategy of cotargeting the EGFR and PYK2/FAK kinases to improve TNBC therapy. Cancer Res; 77(1); 86–99. ©2016 AACR.

Background. High-TMB (HTMB) is an emerging promising agnostic biomarker for predicting benefit from immune-checkpoint inhibitors, independently of tumor type. At ASCO 2019, the TAPUR trial reported an interesting 21% ORR in heavily pretreated metastatic BC patients with very HTMB [vHTMB, ≥9 mutations/megabase (Muts/Mb)]. We aimed to define the differential gene expression and methylation landscape between low and high TMB in each BC subtype. Methods. In TCGA, we identified 848 patients with WES data available for TMB estimation. [ER+/HER2- (LumA by PAM50)) n = 364; ER+/HER2- (LumB by PAM50), n = 147; HER2+, n = 158; and TN, n = 179]. High TMB was defined according to two different cut-offs: ≥9 (vHTMB) and≥5 Muts/Mb (HTMB). The second arbitrary cut-off was used to define a larger group allowing to better characterize the different molecular landscapes associated with high and low TMB in each BC subtype. The HTMB group was compared with an equal number of tumors with low TMB. We assessed the differential RNA expression and methylation of single genes and pathways (defined using Gene Ontology - GO). “Common” genes and pathways were defined as recurrently associated with TMB (p Citation Format: Luca Licata, Barbara Galbardi, Balázs Győrffy, Thomas Karn, Lorenzo Sica, Alessia Rognone, Patrizia Zucchinelli, Daniela Aldrighetti, Stefania Zambelli, Luca Gianni, Giampaolo Bianchini. Molecular differences between high and low tumor mutational burden (TMB) across breast cancer (BC) subtypes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-09.

The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and in vitro siRNA/CRISPR and in vivo functional assays. We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell (815–53,714) and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5–100 μg/ml) or de novo generated Fabs (20 μg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development.

Background: Remodeling of extracellular matrix through collagen degradation is a crucial step in the metastatic cascade. The aim of this study was to evaluate the potential clinical relevance of the serum collagen degradation markers (CDM) C3M and C4M during neoadjuvant chemotherapy for breast cancer. Methods: Patients from the GeparQuinto phase 3 trial with untreated HER2-positive operable or locally advanced breast cancer were enrolled between 7 November 2007, and 9 July 2010, and randomly assigned to receive neoadjuvant treatment with EC/docetaxel with either trastuzumab or lapatinib. Blood samples were collected at baseline, after four cycles of chemotherapy and at surgery. Cutoff values were determined using validated cutoff finder software (C3M: Low &le, 9.00 ng/mL, high &gt, 9.00 ng/mL, C4M: Low &le, 40.91 ng/mL, high &gt, 40.91 ng/mL). Results: 157 patients were included in this analysis. At baseline, 11.7% and 14.8% of patients had high C3M and C4M serum levels, respectively. No correlation was observed between CDM and classical clinical-pathological factors. Patients with high levels of CDM were significantly more likely to achieve a pathological complete response (pCR, defined as ypT0 ypN0) than patients with low levels (C3M: 66.7% vs. 25.7%, p = 0.002, C4M: 52.7% vs. 26.6%, p = 0.031). Median levels of both markers were lower at the time of surgery than at baseline. In the multivariate analysis including clinical-pathological factors and C3M levels at baseline and changes in C3M levels between baseline and after four cycles of therapy, only C3M levels at baseline (p = 0.035, OR 4.469, 95%-CI 1.115&ndash, 17.919) independently predicted pCR. In a similar model including clinical-pathological factors and C4M, only C4M levels at baseline (p = 0.028, OR 6.203, 95%-CI 1.220&ndash, 31.546) and tumor size (p = 0.035, OR 4.900, 95%-CI 1.122&ndash, 21.393) were independent predictors of pCR. High C3M levels at baseline did not correlate with survival in the entire cohort but were associated with worse disease-free survival (DFS, p = 0.029, 5-year DFS 40.0% vs. 74.9%) and overall survival (OS, p = 0.020, 5-year OS 60.0% vs. 88.3%) in the subgroup of patients randomized to lapatinib. In the trastuzumab arm, C3M did not correlate with survival. In the entire patient cohort, high levels of C4M at baseline were significantly associated with shorter DFS (p = 0.001, 5-year DFS 53.1% vs. 81.6%) but not with OS. When treatment arms were considered separately, the association with DFS was still significant (p = 0.014, 5-year DFS 44.4% vs. 77.0% in the lapatinib arm, p = 0.023, 5-year DFS 62.5% vs. 86.2% in the trastuzumab arm). Conclusions: Collagen degradation markers are associated with response to neoadjuvant therapy and seem to play a role in breast cancer.

Purpose: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug. Experimental Design: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays. Results: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21–2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00–4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41–2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28–4.51; P = 0.006) with a significant interaction with bevacizumab treatment (P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected (P = 0.025). Conclusions: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition.

Background Recent studies revealed that maternal and embryonic contributions impact on HLA-G protein expression and might contribute to pregnancy success or failure. The main objective of this study was to examine the paternal levels of the immunoregulatory soluble human leukocyte antigen-G (sHLA-G) protein in seminal plasma and testicular biopsy samples during artificial reproductive technique (ART) treatment and to investigate possible correlations with other semen parameters, age, and pregnancy outcome of the female partner. Methods Soluble HLA-G levels of 106 seminal plasma samples and eight testicular biopsy samples were determined using a commercial sHLA-G Enzyme-linked immunosorbent assay (ELISA) kit. Results We observed a significant negative correlation of male age with total sHLA-G amount (P 0.023, R -0.221) and semen volume (P = 0.047, R -0.193). Testicular biopsy samples were analyzed and tested positively with sHLA-G ELISA. Levels of sHLA-G in seminal plasma samples from men with normozoospermia did not deviate significantly from those with reduced semen quality. No significant difference of sHLA-G levels in seminal plasma and pregnancy outcome of the female partner was detected. Our data showed that age of men with normozoospermia was significantly lower when the female partner conceived after ART treatment (P = 0.016, Mann-Whitney U test). Conclusion High sHLA-G levels in seminal plasma of the male partner appear not to be required for pregnancy but might contribute among other factors to the success of establishing and maintaining pregnancy through long-term priming of the female uterine milieu.

Journal of Cell Science was made aware of duplicated α-tubulin control blots in [Fig. 3][1]C and 3G of J. Cell Sci. (2014) 127, [4740-4749][2] ([doi:10.1242/jcs.155721][3]). The journal approached the Weizmann Institute of Science and asked them to review the original data supplied by the

Immune cell infiltration in breast cancer is important for the patient's prognosis and response to systemic therapies including immunotherapy. We sought to investigate the prevalence of tumour-infiltrating lymphocytes (TILs) and their association with immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) in high-risk, node-positive breast cancer of the adjuvant German Adjuvant Intergroup Node-positive (GAIN-1) trial.We evaluated TILs by haematoxylin and eosin staining and PD-1 and PD-L1 (SP263 assay) expression by immunohistochemistry in 1318 formalin-fixed, paraffin-embedded breast carcinomas. The association of TILs with PD-1, PD-L1, molecular intrinsic subtypes, outcome and therapy regimens (dose-dense [dd] epirubicin, paclitaxel and cyclophosphamide [EPC] and dd epirubicin, cyclophosphamide, paclitaxel and capecitabine [EC-PwX]) was statistically tested.Overall TILs density was significantly associated with the expression of PD-1 and PD-L1 in immune cells (each p 0.0001) and PD-L1 in tumour cells (p = 0.0051). TILs were more common in triple-negative breast cancer (TNBC) and human epidermal growth factor receptor 2 (HER2)-positive tumours (each p 0.0001). On multivariate Cox regression analyses, patients with breast cancer without TILs had an unfavourable disease-free survival (DFS) in the EPC arm compared with the EC-PwX arm (hazard ratio [HR] = 0.69 [0.44-1.06], p = 0.0915); but no differences were seen in tumours with TILs (HR = 1.24 [0.92-1.67], p = 0.1566, interaction p = 0.0336). PD-1-positive immune cells in TNBC were associated with a significantly better DFS (HR = 0.50 [0.25-0.99], p = 0.0457). PD-L1 expression had no impact on patient outcome.TILs predict the benefit of intensified ddEPC compared with ddEC-PwX therapy in node-positive, high-risk breast cancer. TILs, PD-1 and PD-L1 are linked to each other indicating tumour immunogenicity. Moreover, PD-1-positive immune cells have a positive prognostic impact in TNBC.NCT00196872.

More than 70% of breast cancers contain lymphocytic infiltration in the stroma, and preclinical studies suggest that immunoediting and partial control of cancer progression by the local immune microenvironment operate in most breast cancers. Consistent with this hypothesis, a large number of studies demonstrated a favorable prognostic and chemotherapy response predictive role for immune infiltration in breast cancer. The evidence is particularly strong for triple-negative and HER2-positive cancers. The development of clinically effective immune checkpoint inhibitors now provides an opportunity to test the therapeutic potential of augmenting the local antitumor immune response. Several phase I clinical trials using single-agent anti–PD-1 and anti–PD-L1 antibodies demonstrated objective tumor response rates, with remarkably durable responses, in heavily pretreated, metastatic, triple-negative cancers and somewhat lower responses in estrogen receptor–positive cancers. Currently, close to 50 ongoing, or soon to open, clinical trials evaluate the role of this new treatment modality in breast cancer. Clin Cancer Res; 22(9); 2105–10. ©2016 AACR.

Purpose: In spite of improvements of average benefit from adjuvant/neoadjuvant treatments, there are still individual patients with early breast cancer at high risk of relapse. We explored the association with outcome of robust gene cluster–based metagenes linked to proliferation, ER-related genes, and immune response to identify those high-risk patients. Experimental Design: A total of 3,847 publicly available gene-expression profiles were analyzed (untreated, N = 826; tamoxifen-treated, N = 685; chemotherapy-treated, N = 1,150). Genes poorly performing in formalin-fixed samples were removed. Outcomes of interest were pathologic-complete response (pCR) and distant metastasis-free survival (DMFS). In ER+HER2−, the proliferation and ER-related metagenes were combined to define three risk groups. In HER2+ and ER−HER2− risk groups were defined by tertiles of an immune-related metagene. Results: The high-proliferation/low-ER group of ER+HER2− breast cancer had significantly higher pCR rate [OR, 5.01 (1.76–17.99), P = 0.005], but poorer outcome [HR = 3.73 (1.63–8.51), P = 0.0018] than the low-proliferation/high-ER. A similar association with outcome applied to patients with residual disease (RD) after neoadjuvant chemotherapy (P = 0.01). In ER−HER2− and HER2+ breast cancer, immune metagene in the high tertile was linked to higher pCR [33.7% vs. 11.6% in high and low tertile, respectively; OR, 3.87 (1.79–8.95); P = 0.0009]. In ER−HER2−, after adjuvant/neoadjuvant chemotherapy, 5-year DMFS was 85.4% for high-tertile immune metagene, and 43.9% for low tertile. The outcome association was similar in patients with RD (P = 0.0055). In HER2+ breast cancer treated with chemotherapy the association with risk of relapse was not significant. Conclusions: We developed metagene-based predictors able to define low and high risk of relapse after adjuvant/neoadjuvant therapy. High-risk patients so defined should be preferably considered for trials with investigational agents. Clin Cancer Res; 22(2); 337–45. ©2015 AACR.

e18061 Background: Risk factors for invasive recurrence of borderline tumours (BOT) of the ovary are needed. The actin-bundling protein Fascin-1 (FSCN1) is involved in alteration of adhesion and cell motility and has been associated with invasion and metastasis in different cancers. Methods: We studied a retrospective cohort of 124 bordeline tumors of the ovary (BOT) with validated diagnosis by an independent pathologist for expression of FSCN1 as potential prognostic biomarker. Results: We found strong expression of the actin-bundling protein FSCN1 in 62 of these tumors. FSCN1 positive patients were slightly younger (46.9±2.2 vs. 53.3±2.3 years, P = 0.049). Expression of FSCN1 was more frequently detected in BOT with serous compared to mucinous histotype (58/78 [74.4%] vs. 3/41 [7.3%], p < 0.001), and in cases with micropapillary pattern (25/28 [89.3%] vs. 38/98 [38.8%], P < 0.001). In addition, a non significant trend was observed for more frequent expression of FSCN1 among cases with implants (8/10 [80.0%] vs. 55/116 [47.4%], P = 0.095). When we compared progression free survival of the patients we found numerical lower but not statistically significant PFS rates of 90.5±6.4% vs. 94.4±5.4% for 10 years, and 81.4±10.3% vs. 94.4±5.4% for 15 years, in BOT with FSCN1 expression compared to those tumors negative for FSCN1, respectively. Conclusions: Our analysis suggests an association of FSCN1 expression in BOT with micropapillary pattern and implants, which have been linked to inferior prognosis. Cases with FSCN1 expression in our cohort displayed numerical lower PFS rates prompting further studies of this marker.

Background: Breast cancer is one of the most common malignancies during pregnancy. Guidelines for breast cancer treatment during pregnancy demonstrating that breast cancer during pregnancy can be treated similarly to non-pregnancy-associated breast cancer, except for hormonal and anti-HER2 therapies. Nonetheless, a decreased birth weight is often observed in newborns. Therefore, this project aims to analyze the effects of chemotherapy and the impact of cancer progression on the placenta. Methods: Placentas from breast cancer patients (n=63) and non-cancer participants (n=20) enrolled in Breast Cancer in Pregnancy (BCP) study were collected after delivery and embedded in paraffin. Sections were stained with Hematoxylin-Eosin (HE) and IHC (Ki-67, cPARP, p27Kip1); trophoblast morphology was evaluated by an established scoring system and distribution of immunohistochemical markers was assessed. Additionally, epigenetic analyses (cytosine methylation) for the following genes were performed: LINE-1 (general methylation), IGF2-H19 (birth weight), EPO (hypoxia), BDNF (neurobiological development), glucocorticoid receptor, HSD11B2 (inactivation of glucocorticoids), estrogen receptor, P-glycoprotein, CYP-3A4 (drug metabolism). Results: HE staining revealed significant damage of trophoblast nuclei and membranes in placentas from breast cancer patients compared to controls (mean score of damage 1.9/1.8 vs 0.8/0.7, p Citation Format: Karolin Fröhlich, Torsten Plösch, Fenja Seither, Volkmar Müller, Thomas Karn, Elmar Stickeler, Christian Schem, Christine Solbach, Amelie Lupp, Rikst N Verkaik-Schakel, Gitta Turowski, Peter Mallmann, Marion van Mackelenbergh, Bruno Sinn, Valentina Nekljudova, Carsten Denkert, Pamela Schittler, Udo Markert, Sibylle Loibl. Histological and epigenetic analyses of placenta tissue from breast cancer patients and healthy participants [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-08.