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Introduction: HER2-positive breast cancer is considered aggressive, but due to the development of targeted drugs in the past 20 years, a substantial improvement of therapy results can be postulated. Proven evidence is provided by data of randomized clinical trials. The present study aimed at demonstrating the improvement of therapy results over time using single-center real world data. Material and Methods: In our center, we prospectively built a consecutive tumor and data base from 2000 to 2020 and identified patients with early HER2-positive breast cancer (n=396). The cohort was divided in four groups by year of diagnosis according to the changing therapy concepts: A) 2000-2004 (no HER2-directed therapy, n=83), B) 2005-2011 (trastuzumab, n=96), C) 2012-2017 (trastuzumab/pertuzumab, n=135), D) 2018-2020 (T-DM1 in patients with non-pCR), n=55). HER2 was measured by IHC and ISH corresponding to the ASCO-CAP guidelines. HER2-directed therapy was indicated according to the respective national guidelines (AGO). The median follow-up was 58 months in the entire cohort (1-266), group A 84 months (10-266), group B 94 months (2-201), group C 56 months (1-118), group D 32 months (8-53). The primary endpoint was overall survival (OS), and secondary endpoint was iDFS. Kaplan-Meier estimates were calculated and multivariate analyses were performed using SPSS 28 (IBM, Armonk, NY, USA). Results: Overall, 57% of the 396 patients had an age of 50 years or more, 85% had a NST histology, 54% of the tumors were larger than 2cm, 45% were node-positive; 34% were steroid hormone receptor negative. HER2-directed therapy was delivered to 7 of 83 pts in group A (8.4%), to 62 of 96 pts in group B (64.6%), to 119 of 135 in group C (88.15%), and to 50 of 55 pts in group D (90.91%), resp.. Overall, in the first 3 years we observed 19 deaths and the probability for OS was 89.7 % for group A, 92.2 % for group B, 96.4 % for group C and 100% for group D, resp.; iDFS was 77.4% in A, 86.8% in B, 94.9% in C and 95.2% in D, resp.. After adjustment for nodal status, grading and steroid hormone receptor status, we calculated the effect size of the incremental improvement of treatment for OS and iDFS as HRs referred to group A (Table 1). Conclusion: With this prospectively established single center cohort, we are able to confirm a significant improvement of the treatment results in patients with HER2-positive early breast cancer over the last 20 years applying individualized HER2-directed therapies. Table 1. Multivariate analyses of OS and iDFS adjusted for nodal status, grading and hormone receptor status. Citation Format: Christoph Thomssen, Julia Engel, Kristin Reinhardt, Hans-Georg Strauß, Regina Große, Susanne Barrot, Marcus Bauer, Lisa van Uden, Sandy Kaufhold, Kathleen Schüler, Vanessa Wieder, Eva J. Kantelhardt, Martina Vetter. HER2-directed therapy in early breast cancer – improvement over 20 years [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 P1-11-11.

Purpose Phosphatidylinositide-3-kinase (PI3K) regulates proliferation and apoptosis; somatic PIK3CA-mutations may activate these processes. Aim of this study was to determine the prevalence of PIK3CA-mutations in a cohort of early stage breast cancer patients and the association to the course of disease. Patients and methods From an unselected cohort of 1270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT 01592825) 1123 tumours were tested for the three PIK3CA hotspot-mutations H1047R, E545K, and E542K by qPCR. Primary objectives were the prevalence of somatic PIK3CA-mutations and their association to tumour characteristics. Secondary objective was the association of PIK3CA-mutations to recurrence-free interval (RFI) and overall survival. Results PIK3CA-mutation rate was 26.7% (300 of 1123). PIK3CA-mutations were significantly more frequent in steroid hormone-receptor (SHR)-positive HER2-negative (31.4%), and G1 and G2 tumours (32.8%). Overall, we did not observe a significant association of PIK3CA-mutations to RFI. In SHR-positive BCs with PIK3CA-mutations, a strong trend for impaired RFI was observed (adjusted HR 1.64, 95% CI 0.958–2.807), whilst in SHR-negative BCs PIK3CA-mutations were insignificantly associated with improved RFI (adjusted HR 0.49; 95% CI 0.152–1.597). Of note, we observed a significantly detrimental prognostic impact of PIK3CA-mutations on RFI in SHR-positive, HER2-negative BCs if only aromatase inhibitors were administered as adjuvant therapy (adjusted HR 4.44, 95% CI 1.385–13.920), whilst no impact was observed in tamoxifen treated patients. Conclusion This cohort study speficies the overall mutation rate of PIK3CA in early breast cancer. The impact of PIK3CA-mutations on RFI and OS was heterogeneous. Our results suggest that estrogen deprivation failes to be active in case of PIK3CA-mutation.

The low overall survival rates of breast cancer (BC) patients in sub-Saharan Africa (SSA) are driven by regionally differing tumor biology, advanced tumor stages at diagnosis and limited access to therapy. However, it is not known whether regional differences in the composition of the tumor microenvironment (TME) exist and affect patients’ prognosis. In this international, multicentre cohort study, 1,237 formalin-fixed, paraffin-embedded BC samples, including samples of the ‘African Breast Cancer-Disparities in Outcomes (ABC-DO) Study’, were analyzed. The immune cell phenotypes, their spatial distribution in the TME and immune escape mechanisms of BC samples from SSA and Germany (n=117) were investigated using histomorphology, conventional and multiplex immunohistochemistry (IHC), and RNA expression analysis. The data revealed no regional differences in the number of tumor-infiltrating lymphocytes (TILs) in the 1,237 SSA BC samples, while the distribution of TILs in different BC IHC subtypes showed regional diversity, particularly when compared to German samples. Higher TIL densities were associated with better survival in the SSA cohort (n=400), but regional differences concerning the predictive value of TILs existed. High numbers of CD163+ macrophages and CD3+CD8+ T cells accompanied by reduced cytotoxicity, altered IL10 and IFNγ levels and downregulation of MHC class I components were predominantly detected in BC samples from Western SSA. Features of non-immunogenic BC phenotypes were associated with reduced patient survival (n=131). We therefore conclude that regional diversity in the distribution of BC subtypes, TME composition and immune escape mechanisms should be considered for therapy decisions in SSA and the design of personalized therapies.

Rho GDP dissociation inhibitor β (Rho-GDIβ), an inhibitor of Rho GTPases, is primarily expressed by hematopoietic cells but is also found in epithelial cancer cells. Recently, we have identified Rho-GDIβ as a target of the transcription factor Ets1. Here, we show that, in breast cancer cells, Ets1 regulates Rho-GDIβ expression and binds to the upstream region of the Rho-GDIβ gene. Furthermore, in primary breast cancer, Rho-GDIβ is coexpressed with Ets1. Studying the function of Rho-GDIβ in breast cancer, we found that a Rho-GDIβ–specific small interfering RNA increased cellular migration but also decreased the expression of cyclooxygenase-2 (Cox-2) oncogene as shown by microarray, quantitative reverse transcription-PCR, and Western blot analyses. Further studies revealed that Rho-GDIβ regulates Cox-2 gene at least partly on the transcriptional level, most likely by activating nuclear factor of activated T cells 1 (NFAT-1). Vav-1, an interaction partner of Rho-GDIβ, was also found to interfere with Cox-2 expression and NFAT-1 cellular distribution, suggesting a cooperative action of Rho-GDIβ and Vav-1 on Cox-2 expression. To explore the importance of Rho-GDIβ for the survival of breast cancer patients, two cohorts, including 263 and 117 patients, were analyzed for clinical outcome in relation to Rho-GDIβ RNA and protein levels, respectively. Expression of Rho-GDIβ was not associated with either disease-free or overall survival in the two patient population. Our data suggest that the expression of Rho-GDIβ in breast cancer is neither beneficial nor disadvantageous to the patient. This may be the net effect of two opposing activities of Rho-GDIβ, one that suppresses tumor progression by inhibiting migration and the other that stimulates it by enhancing Cox-2 expression. [Cancer Res 2007;67(22):10694–702]

Supplementary Tables 1-5 from Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

Supplementary Figure 2 from Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

Supplementary Figure 1 from Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

Introduction. Neoadjuvant chemotherapy (NACT) is the standard of care in aggressive breast cancer, including triple negative breast cancer (TNBC). Cytotoxic drugs specifically target proliferating cells, however, patient outcome is variable. Tumor physiology and response to therapy are orchestrated by an intricate interplay between cancer, stromal and immune cells collectively forming the tumor microenvironment. We have recently uncovered a feedback mechanism of tumor cells and fibroblasts, involving IFNB1 signaling, that supports tumor cells in the recovery from chemotherapy-induced stress. Here, we wanted to assess whether targets of IFNB1 signaling in fibroblasts and tumor cells would qualify as predictive markers of pathological complete response (pCR) after neoadjuvant therapy (NACT) as prognostic markers for the course of the disease. Methods and Patients. RNA-sequencing data from in vitro experiments found the GO-term GO:0051607 ‘defense response to virus’ significantly enriched. Twentyfour genes intersected between differentially expressed genes and the genes of this GO-term. We selected three of the encoded proteins a) interferon induced with helicase C domain 1 IFIH1, b) interferon alpha-inducible protein ISG15, c) 2'-5'-oligoadenylate synthetase OAS1 to test their expression in human specimens of TNBCs after NACT by immunohistochemistry (IHC). None of the respective genes correlated with recurrence free survival when tested in treatment-naïve tumor biopsies (KM Plotter). A prospective consecutively enrolled cohort (2000 - 2021) was available with an overall pCR rate of 46%. pCR was defined by no invasive cancer cell in breast or axilla (ypT0 N0). The median follow-up for iDFS was 36.2 months (6-154) and for OS 39.3 months (6-214).Primary objective was the correlation between IFIH1, ISG15 and OAS1 protein expression in the residual tumor by non-pCR or in the tumor bed by pCR. Second objective was the association of IFIH1, ISG15 and OAS1 protein expression to invasive disease-free survival (iDFS) and overall survival (OS). Results. To date, IHC staining has been established for IFIH1, ISG15 and OAS1. In representative stainings of FFPE tissue samples with pCR we did not detect any positive signal for IFIH1 and ISG15 in stromal cells like fibroblast or lymphocytes. Slight to strong protein expression was detected by non-pCR in cancer cells, stromal cells and tumour infiltrating lymphocytes. In contrast OAS1 was expressed especially strong in lymphocytes by pCR or non-pCR. Cancer cell showed moderate OAS1 expression. IHC analysis and of the entire cohort is in progress including the analyses of the association of these markers to pCR, non-pCR and iDFS/OS. Conclusion. Using samples from our consecutive, multicentre enrolled cohort, an association between the expression of markers of an IFNB1-triggered antiviral response and pCR and survival was demonstrated in patients of the TNBC subgroup. Analysis of the entire cohort is necessary to potentially demonstrate applicability of an interferon-response as predictor of survival. Citation Format: Marcus Bauer, Martina Vetter, Ana Maia, Efstathios Vlachavas, Brigitta Michels, Mireia Berdiel-Acer, Kathleen Schüler, Alessandra Morselli, Manio Skarlatou, Christoph Thomssen, Stefan Wiemann. Communication between tumor cells and fibroblasts as a prognostic factor of NACT in TNBC [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 P1-08-15.

Background. Guidelines recommend broad use of carboplatin (CBDCA) for neoadjuvant therapy (NACT) of patients with triple negative breast cancer (TNBC). However, current evidence is contradictory. The aim of this prospective observational study was to assess efficacy and feasibility of CBDCA-containing neoadjuvant therapy in clinical routine. Patients and Methods. Patients who received NACT for early TNBC (n=150) were consecutively enrolled between 2000 and 2021. The following regimens were used: dose-dense epirubicin-cyclophosphamide (ddEC) followed by taxanes (n=62); CBDCA-taxane combination followed by ddEC (n=49), CBDCA-taxane combination, no anthracyclines (n=25), other chemotherapy regimen (n=14). Some CBDCA therapies were given every 3 weeks (n=18), the majority was given in a weekly fashion (n=56). The efficacy and feasibility including toxicities of these carboplatin regimen were analyzed. Median follow-up iDFS: 36.2 months (6-154), median Follow-up OS 39.3 months (6-214).Primary objective was the pCR rate (ypT0 N0) in patients who were treated with CBDCA in comparison to those who received standard non-platinum containing NACT. Secondary objectives were toxicity, therapy adherence, cancer associated recurrences and association of pCR rate with invasive disease-free survival (iDFS) and overall survival (OS). Results. Half of the patients (n=74) were treated with CBDCA, two third (69%) of them received the complete intended cycles if necessary with primary GCSF-support. Overall, the addition of CBDCA slightly increased the pCR rate from 43.4% to 48.6% with a 1.8 times reduced risk for iDFS events. The highest pCR rate was observed in those patients (n=50) who had received all CBDCA-courses as planned (50% vs 45%). By 48 months, pCR with CBDCA treatment was associated with an improved course of the disease (86.3 % iDFS probability), followed by non-pCR and CBDCA-therapy (81.1% iDFS probability), pCR and no platinum (79.8%) and non-pCR and no platinum (70.7%). Considering OS, patients without CBDCA and no pCR had the highest mortality (19.5%), all other groups had over 90% OS-probability. Dose-modification of CBDCA was associated to pCR (4 of 5 patients), whilst with discontinuation of CBDCA, 12 of 20 patients had residual disease. Although neutropenia, thrombocytopenia or anemia occurred in 36 patients, CBDCA discontinuation due to hematological effects was only necessary in 10 cases. Conclusion. In our prospective cohort, the addition of CBDCA to neoadjuvant chemotherapy for patients with TNBC was highly effective. Even after dose-modification of CBDCA (e.g. for toxicity) it seems to be effective compared to discontinuation or no inclusion of CBDCA. Our data support the current recommendations to include CBDCA in neoadjuvant therapy for TNBC. Citation Format: Christoph Thomssen, Kathleen Schüler, Marcus Bauer, Kristin Reinhardt, Hans-Gerorg Strauß, Martina Vetter. Efficacy of neoadjuvant systemic carboplatin therapy in triple-negative breast cancer [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 P2-12-05.

Introduction. Tumor cells are influenced by their microenvironment, including immune infiltration. These tumor infiltrating lymphocytes (TILs) are considered to have prognostic and predictive value for patients with early breast cancer. The aim of this study was to evaluate the distribution of TILs and the association with survival in unselected sample of breast cancers. Patients and Methods. From a prospective, multicenter cohort of 1,270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT 01592825), 1,136 samples were evaluated for TIL infiltration inside the borders of the invasive tumor, following the recommendations of the international TILs working group. TILs were assigned to one of these three TILs groups: low TILs (60%) and additionally scored as a continuous parameter per 10% increment. Primary objective was the distribution of TILs in different groups dependent on hormone receptor and HER2 status of the tumor (IHC group). Second objective was the association of TILs with recurrence free interval (RFI) and overall survival (OS) in univariate and multivariate analyses. The median observation time was 62 months (1-123). Results. Dependent on the IHC types a specific distribution of TILs was found: more than 60% TILs were detected in 1.5% (12 of 828) of hormone receptor (HR) positive and HER2 negative tumors, 9.7% (17 of 175) of HER2 positive tumors with any HR status and 18.8% (25 of 133) of triple negative breast cancer (TNBC). Patients with HR positive and HER2 negative tumors showed no impact of TILs with regard to RFI and OS. In the HER2 positive group with more than 60% TILs, no RFI event was detected, and with less than 60% TILs, 15% of the patients had an RFI event. The probability of OS was 94% (TILs >60%) and 81% (TILS ≤60%). For TNBC and the same TIL cut off, 88% had no RFI and OS event, compared to 70% with no event for RFI and 74% for OS. Applying 10% increments of TILs to the entire cohort, the multivariate analysis revealed a hazard ratio of 0.895 (95% CI 0.796-1.007) for RFI and a significant hazard ratio of 0.890 (95% CI 0.794-0.997) for OS. Considering the IHC groups, a 10% increment of TILs in HER2 positive tumors led to an increase of RFI (0.792, 95% CI 0.601-1.043) and OS (0.713, 95% CI 0.533-0.955, p Citation Format: Kathleen Schüler, Daniel Bethmann, Tilmann Lantzsch, Christoph Uleer, Volker Hanf, Susanne Peschel, Jutta John, Marleen Pöhler, Joerg Buchmann, Karl-Friedrich Buerrig, Edith Weigert, Eva Johanna Kantelhardt, Christoph Thomssen, Martina Vetter. Tumor infiltrating lymphocytes as a prognostic factor [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 P4-07-15.

Contains fulltext : 290914.pdf (Publisher’s version ) (Open Access) BACKGROUND: In node-negative breast cancer (NNBC), a high risk of recurrence is determined by clinico-pathological or tumor-biological assessment. Taxanes may improve adjuvant chemotherapy. METHODS: NNBC 3-Europe, the first randomized phase-3 trial in node-negative breast cancer (BC) with tumor-biological risk assessment, recruited 4146 node-negative breast cancer patients from 2002 to 2009 in 153 centers. Risk assessment was performed by clinico-pathological factors (43%) or biomarkers (uPA/PAI-1, urokinase-type plasminogen activator/its inhibitor PAI-1). High-risk patients received six courses 5-fluorouracil (500 mg/m(2)), epirubicin (100 mg/m(2)), cyclophosphamide (500 mg/m(2)) (FEC), or three courses FEC followed by three courses docetaxel 100 mg/m(2) (FEC-Doc). Primary endpoint was disease-free survival (DFS). RESULTS: In the intent-to-treat population, 1286 patients had received FEC-Doc, and 1255 received FEC. Median follow-up was 45 months. Tumor characteristics were equally distributed; 90.6% of tested tumors had high uPA/PAI-1-concentrations. Planned courses were given in 84.4% (FEC-Doc) and 91.5% (FEC). Five-year-DFS was 93.2% (95% C.I. 91.1-94.8) with FEC-Doc and 93.7% (91.7-95.3) with FEC. Five-year-overall survival was 97.0% (95.4-98.0) for FEC-Doc and 96.6% % (94.9-97.8) for FEC. CONCLUSIONS: With adequate adjuvant chemotherapy, even high-risk node-negative breast cancer patients have an excellent prognosis. Docetaxel did not further reduce the rate of early recurrences and led to significantly more treatment discontinuations.

Purpose The recent development of multi-gene assays for gene expression profiling has contributed significantly to the understanding of the clinically and biologically heterogeneous breast cancer (BC) disease. PAM50 is one of these assays used to stratify BC patients and individualize treatment. The present study was conducted to characterize PAM50-based intrinsic subtypes among Ethiopian BC patients. Patients and methods Formalin-fixed paraffin-embedded tissues were collected from 334 BC patients who attended five different Ethiopian health facilities. All samples were assessed using the PAM50 algorithm for intrinsic subtyping. Results The tumor samples were classified into PAM50 intrinsic subtypes as follows: 104 samples (31.1%) were luminal A, 91 samples (27.2%) were luminal B, 62 samples (18.6%) were HER2-enriched and 77 samples (23.1%) were basal-like. The intrinsic subtypes were found to be associated with clinical and histopathological parameters such as steroid hormone receptor status, HER2 status, Ki-67 proliferation index and tumor differentiation, but not with age, tumor size or histological type. An immunohistochemistry-based classification of tumors (IHC groups) was found to correlate with intrinsic subtypes. Conclusion The distribution of the intrinsic subtypes confirms previous immunohistochemistry-based studies from Ethiopia showing potentially endocrine-sensitive tumors in more than half of the patients. Health workers in primary or secondary level health care facilities can be trained to offer endocrine therapy to improve breast cancer care. Additionally, the findings indicate that PAM50-based classification offers a robust method for the molecular classification of tumors in the Ethiopian context.

HER3 is highly expressed in luminal breast cancer subtypes. Its activation by NRG1 promotes activation of AKT and ERK1/2, contributing to tumour progression and therapy resistance. HER3-targeting agents that block this activation, are currently under phase 1/2 clinical studies, and although they have shown favorable tolerability, their activity as a single agent has proven to be limited. Here we show that phosphorylation and activation of HER3 in luminal breast cancer cells occurs in a paracrine manner and is mediated by NRG1 expressed by cancer-associated fibroblasts (CAFs). Moreover, we uncover an autocrine role of NRG1 in CAFs. This occurs independently of HER3 and results in the induction of a strong migratory and pro-fibrotic phenotype, describing a subset of CAFs with elevated expression of NRG1 and an associated transcriptomic profile that determines their functional properties. Finally, we identified Hyaluronan Synthase 2(HAS2), a targetable molecule strongly correlated withNRG1, as an attractive player supporting NRG1 - autocrine signaling in CAFs.

Introduction: Triple-negative breast cancer (TNBC) is considered the most aggressive type of breast cancer (BC) with limited options for therapy. TNBC is a heterogeneous disease and tumors have been classified into TNBC subtypes using gene expression profiling to distinguish basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor (LAR), and one nonclassifiable group (called unstable). Objectives: The aim of this study was to verify the clinical relevance of molecular subtyping of TNBCs to improve the individual indication of systemic therapy. Patients and Methods: Molecular subtyping was performed in 124 (82%) of 152 TNBC tumors that were obtained from a prospective, multicenter cohort including 1,270 histopathologically confirmed invasive, nonmetastatic BCs (NCT 01592825). Treatment was guideline-based. TNBC subtypes were correlated with recurrence-free interval (RFI) and overall survival (OS) after 5 years of observation. Results: Using PAM50 analysis, 87% of the tumors were typed as basal with an inferior clinical outcome compared to patients with nonbasal tumors. Using the TNBCtype-6 classifier, we identified 23 (15%) of TNBCs as LAR subtype. After standard adjuvant or neoadjuvant chemotherapy, patients with LAR subtype showed the most events for 5-year RFI (66.7 vs. 80.6%) and the poorest probability of 5-year OS (60.0 vs. 84.4%) compared to patients with non-LAR disease (RFI: adjusted hazard ratio [aHR] = 1.87, 95% confidence interval [CI] 0.69–5.05, p = 0.211; OS: aHR = 2.74, 95% CI 1.06–7.10, p = 0.037). Conclusion: Molecular analysis and subtyping of TNBC may be relevant to identify patients with LAR subtype. These cancers seem to be less sensitive to conventional chemotherapy, and new treatment options, including androgen receptor-blocking agents and immune checkpoint inhibitors, have to be explored.

Introduction: Endocrine therapy for breast cancer (BC) patients is highly underutilized in rural Ethiopia and other African countries. Objective: This study aims to assess the feasibility of and adherence to tamoxifen therapy in rural Ethiopia. Methods: We ascertained the hormone receptor (HR) status in 101 women diagnosed with BC from January 2010 to December 2015 and who had surgery in Aira Hospital, in rural Ethiopia. From 2013, tamoxifen was offered to patients with HR-positive (HR+) tumors. Prescription refill records and a structured questionnaire were used to assess receipt of and adherence to tamoxifen. Results: Of the 101 BC patients tested for HR status during the study period, 66 (65%) patients were HR+ and were eligible for tamoxifen treatment. However, 15 of the HR+ patients died before tamoxifen became available in 2013. Of the remaining 51 HR+ patients, 26 (51%) initiated tamoxifen but only 9 of them (35%) adhered to therapy (medication possession rate ≥80%, median observation 16.2 months). After 1 year, 52% of the patients were still adherent, and 9 patients had discontinued therapy. The reasons for non-initiation of tamoxifen included patient factors (n = 5), including financial hardship or lack of transportation, and health care provider factors (n = 12). Conclusions: Endocrine therapy for BC patients seems feasible in rural Western Ethiopia, although non-adherence due to financial hardship and a less developed health care infrastructure remains a major challenge. We postulate that the implementation of breast nurses could reduce patient and health system barriers and improve initiation of and adherence to endocrine treatment.

In breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical Phase II–III trials. The objective of this study was to explore the clinical relevance of farnesyltransferase β-subunit (FNTB) single nucleotide promoter polymorphisms (FNTB-173 6G > 5G (rs3215788), -609 G > C (rs11623866) and -179 T > A (rs192403314)) in early breast cancer. FNTB genotyping was performed by pyrosequencing in 797 patients from a prospective multicentre observational PiA trial (NCT 01592825). In the total cohort, the FNTB-173 6G > 5G polymorphism was an independent predictor of RFI (HR = 0.568; 95% CI = 0.339–0.949, p = 0.031), OS (HR = 0.629; 95% CI = 0.403–0.980, p = 0.040) and BCSS (HR = 0.433; 95% CI = 0.213–0.882; p = 0.021), whereas the FNTB-609 G > C polymorphism was an independent predictor of RFI (HR = 0.453; 95% CI = 0.226–0.910, p = 0.026) and BCSS (HR = 0.227; 95% CI = 0.075–0.687, p = 0.009). Subtype analysis revealed the independent prognostic relevance of FNTB promoter polymorphisms, particularly in TNBC but not in luminal or HER2-positive intrinsic subtypes. Finally, we used electrophoretic mobility shift assays (EMSAs) to confirm in vitro that the polymorphism FNTB-173 6G > 5G resulted in the differential binding of nuclear proteins from five different breast cancer cell lines. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms (i) are independent prognostic biomarkers, particularly in patients with early TNBC, and (ii) could modulate FNTB’s transcriptional activity.

Introduction: Triple negative breast cancer (TNBC) is, in general, associated with unfavorable prognosis. However, TNBC it is a heterogenous disease. Thus, we analyzed the prognosis (RFI, OS) of TNBC in association to the molecular subtypes (according to Lehmann et al. 2011) and the prognostic markers uPA (urokinase-type plasminogen activator) and its inhibitor (uPA/PAI-1) within a prospective cohort of breast cancer patients. Material & Methods: The PiA-study was designed as a multicenter cohort of early BC patients (n=1270, 2009 – 2011), treated according to national guidelines from six centers in Germany (Prognostic assessment in routine Application, NCT 01592825). All 153 patients with TNBC (12%) received chemotherapy (neoadjuvant, n=99, or adjuvant, n=54). Tumor characteristics were based on local pathology. IN 81 TNBC patients, fresh frozen tissue was obtained for central uPA/PAI-1 testing by ELISA (FEMTELLE BioMedica Diagnostic) was available. RNA of formalin fixed and paraffin embedded (FFPE) TNBC tumor material (n=125) was analyzed using the GeneChip® U133 Plus 2.0 (Affymetrix). The online Vanderbilt predictor tool was used to determine molecular subtypes (http://cbc.mc.vanderbilt.edu/tnbc). Results: In the total cohort, 89.5% (CI 87.7-91.3) were free of RFI events after 5 years, the TNBC patients showed the poorest outcome with 72.8% (CI 65.5-80.1). In TNBC patients with a low uPA/PAI-1 status significantly more were free of RFI events than in patients with high uPA/PAI-1 status: 93% (CI 79.4-106.4) and 70.5% (CI 58.7-82.3), respectively. By the type 6-Vanderbilt tool, molecular subtypes were assigned as follows: BL1 18%, BL2 12%, IM 22%, M 12% MSL 6%, LAR: 14%, and UNS 16%. Using the type 4 tool we found for BL1 30%, BL2 18%, M 15%, LAR 18%, and UNS 19%. In all survival analysis, the patients with LAR subtype (n=23) had the lowest survival probability, with BL2 subtype N=22) the best: for 5 year RFI 58.7% (CI 29.4-71.8) versus 81.6% (CI 65.3-97.9). In those 54 TNBC patients who were treated with neoadjuvant chemotherapy, 46% of the patients had a pathologically confirmed complete response (ypT0/is ypN0). Counclusion: TNBC is a heterogeneous disease. Patients with TNBC and low uPA/PAI-1 expression have a very low rate of disease recurrence. Particular attention has to be drawn to patients with the LAR subtype, since it seems to have the worst prognosis and may require androgen receptor directed treatment. Outcome results of different endpointsn=125BL1 (n=38)BL2 (n=22)M (n=18)LAR (n=23)UNS (n=24)OS (95% CI)80.3% (67.2-93.4)95.2% (86.2-104.2)71.8% (50.8-92.8)65.2% (44.4-84.4)78.4% (61.5-95.3)RFS (95% CI)78.1% (64.6-91.6)81.6% (65.3-97.9)66.7% (44.9-88.5)50.6% (29.4-71.8)78.6% (62-95.2)RFI (95% CI)78.1% (64.6-91.6)81.6% (65.3-97.9)66.7% (44.9-88.5)58.7% (37.3-80.1)91.3% (79.7-102.9)BCSS (95% CI)80.3% (67.2-93.4)95.2% (86.2-104.2)71.8% (50.8-92.8)82.4% (66.7-98.1)90.9% (78.9-102.9) Citation Format: Martina Vetter, Carolin Hartung, Tilmann Lantzsch, Volker Hanf, Christoph Uleer, Susanne Peschel, Jutta John, Joerg Buchmann, Edith Weigert, Karl-Friedrich Buerrig, Eva-Johanna Kantelhardt, Christoph Thomssen. Prospective analysis of molecular subtypes in triple negative 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 P4-06-13.

// Marcus Bauer 1 , Eva Johanna Kantelhardt 2, 3 , Thorsten Stiewe 4, 5, 6 , Andrea Nist 4 , Marco Mernberger 4 , Katharina Politt 4 , Volker Hanf 7 , Tilmann Lantzsch 8 , Christoph Uleer 9 , Susanne Peschel 10 , Jutta John 11 , Jorg Buchmann 12 , Edith Weigert 13 , Karl-Friedrich Burrig 14 , Claudia Wickenhauser 1 , Christoph Thomssen 2 , Frank Bartel 1, * and Martina Vetter 2, * 1 Institute of Pathology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany 2 Department of Gynaecology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany 3 Institute of Medical Epidemiology, Biostatistics and Informatics, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany 4 Institute of Molecular Oncology, Universities of Giessen and Marburg Lung Center, German Center for Lung Research (DZL), Marburg, Germany 5 Genomics Core Facility, Philipps-University, Universities of Giessen and Marburg Lung Center, German Center for Lung Research (DZL), Marburg, Germany 6 Universities of Giessen and Marburg Lung Center, German Center for Lung Research (DZL), Marburg, Germany 7 Department of Gynaecology, Hospital Fuerth, Fuerth, Germany 8 Department of Gynaecology, Hospital St. Elisabeth and St. Barbara, Halle (Saale), Germany 9 Onkologische Praxis Uleer, Hildesheim, Germany 10 Department of Gynaecology, St. Bernward Hospital, Hildesheim, Germany 11 Department of Gynaecology, Helios Hospital Hildesheim, Hildesheim, Germany 12 Institute of Pathology, Hospital Martha-Maria, Halle (Saale), Germany 13 Institute of Pathology, Hospital Fuerth, Fuerth, Germany 14 Institute of Pathology Hildesheim, Hildesheim, Germany * Co-senior authors and contributed equally to this work Correspondence to: Frank Bartel, email: frank.bartel@medizin.uni-halle.de Keywords: p53; mutation; tumor suppression; breast cancer; single nucleotide polymorphism Received: September 18, 2018 Accepted: February 15, 2019 Published: March 08, 2019 ABSTRACT Background: Genetic factors play a substantial role in breast cancer etiology. Genes encoding proteins that have key functions in the DNA damage response, such as p53 and its inhibitors MDM2 and MDMX, are most likely candidates to harbor allelic variants that influence breast cancer susceptibility. The aim of our study was to comprehensively analyze the impact of SNPs in the TP53 , MDM2 , and MDMX genes in conjunction with TP53 mutational status regarding the onset and progression of breast cancer. Methods: In specimen from 815 breast cancer patients, five SNPs within the selected genes were analyzed: TP53 – Arg72Pro (rs1042522), MDM2 – SNP285 (rs2279744), SNP309 (rs117039649); MDMX – SNP31826 (rs1563828), and SNP34091 (rs4245739). Classification of the tumors was evaluated by histomorphology. Subtyping according hormone receptor status, HER2-status and proliferation rate enabled provision of the clinico-pathological surrogate of intrinsic subtypes. Results: The homozygous C-allele of MDM2 SNP285 was significantly associated with a younger age-at-diagnosis of 44.2 years, in contrast to G/G- and G/C-patients (62.4, 62.7 yrs., respectively; p = 0.0007; log-Rank-test). In contrast, there was no difference regarding the age-at-diagnosis for patients with the respective genotypes of MDM2 SNP309 ( p = 0.799; log-Rank-test). In patients with estrogen receptor (ER)-positive and TP53 -mutated tumors, however, the T/T-genotype of the MDM2 SNP309 was significantly associated with an earlier average age-at-diagnosis compared with T/G+G/G-patients (53.5 vs. 68.2 yrs; p = 0.002; log-Rank-test). In the triple-negative subgroup, the G/G-patients had an average age-at-diagnosis of 51 years compared with 63 years for SNP309T carriers ( p = 0.004; log-Rank-test) indicating a susceptibility of the G/G genotype for the development of triple negative breast cancer. Patients with the A/A-genotype of MDMX SNP31826 with ER-negative tumors were diagnosed 11 years earlier compared with patients and ER-positive tumors (53.2 vs. 64.4 yrs; p = 0.025, log-Rank-test). Furthermore, in luminal B-like patients (HER2-independent) the C/C-genotype of MDMX SNP34091 was significantly correlated with a decreased event-free survival compared with the A/A-genotype ( p < 0.001; log-Rank-test). Conclusions: We showed that SNPs in the MDM2 and MDMX genes affect at least in part the onset and progression of breast cancer dependent on the ER-status. Our findings provide further evidence for the distinct etiological pathways in ER-negative and ER-positive breast cancers.

BACKGROUND: The minor allele of two caspase-8 polymorphisms, namely CASP8 -652 6N InsDel and CASP8 Asp302His, were shown to promote survival of T-lymphocytes and were repeatedly associated with reduced breast cancer susceptibility. However, besides some preliminary findings, clinical relevance of these polymorphisms in patients with already existing primary breast cancer has not yet been established. Considering an immunomodulatory and potentially tumor-protective role of these caspase-8 variants, we genotyped 785 primary breast cancer patients and correlated caspase-8 variants with i) disease-free survival (DFS) and ii) the presence of tumor infiltrating lymphocytes (TILs). METHODS: Primary breast cancer samples were collected at the Martin-Luther University, Halle-Wittenberg between 2009 and 2011 as part of the multicenter prospective PiA trial (NCT 01592825). The majority of patients had luminal-like tumors (75.9%), followed by triple negative (10.1%), luminal-Her2-like (9.6%) and Her2-enriched tumors (4.5%). Genotyping was performed by pyrosequencing, TILs status was assessed by hematoxylin and eosin staining. RESULTS: The CASP8 -652 deletion was significantly associated with improved DFS in an allele-dose dependent manner (p=0.027). Homozygosity for the -652 6N Del variant was an independent predictor for improved DFS (p=0.005). In patients with a 302His/His genotype, there was no event of recurrence during the entire observation time. Combined analysis of diplotypes revealed that both polymorphisms had an influence on DFS (p=0.029). Interestingly, patients with the 302His/His variant among the unstratified patient cohort and among the luminal-like subtype alone had tumors with very low lymphocyte infiltration (0-10% TILs in 65% of cases compared to 31% of cases for other genotypes, p=0.025). CONCLUSION: In line with previous epidemiological findings, we propose a prognostically favorable role of the CASP8 -652 6N Del and the Asp302His variant in primary breast cancer patients. Moreover, we suggest for the first time a role of the Asp302His variant in immunosurveillance and lymphocyte infiltration of breast cancer. Our findings strongly encourage further analyze of these genetic variants as a biomarker for prognostic and immunotherapeutic considerations. Citation Format: Wimberger P, Bachmann HS, Kuhlmann JD, Link T, Kröber E, Thomssen C, Malle B, Vetter M, Kantelhardt EJ. Association of Caspase 8 polymorphisms with TILs and disease-free survival in primary breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-03.

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HG-SOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here we show, that the IGF2 mRNA binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HG-SOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in a RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signaling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rational for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HG-SOC.Graphical Abstract

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.

Breast cancer is the most common cancer in women worldwide. The tumor microenvironment contributes to tumor progression by inducing cell dissemination from the primary tumor and metastasis. TGFβ signaling is involved in breast cancer progression and is specifically elevated during metastatic transformation in aggressive breast cancer. In this study, we performed genomewide correlation analysis of TGFBR2 expression in a panel of 51 breast cancer cell lines and identified that MET is coregulated with TGFBR2. This correlation was confirmed at the protein level in breast cancer cell lines and human tumor tissues. Flow cytometric analysis of luminal and basal-like breast cancer cell lines and examination of 801 tumor specimens from a prospective cohort of breast cancer patients using reverse phase protein arrays revealed that expression of TGFBR2 and MET is increased in basal-like breast cancer cell lines, as well as in triple-negative breast cancer tumor tissues, compared to other subtypes. Using real-time cell analysis technology, we demonstrated that TGFβ1 triggered hepatocyte growth factor (HGF)-induced and MET-dependent migration in vitro. Bioinformatic analysis predicted that TGFβ1 induces expression of C-ets-1 as a candidate transcription factor regulating MET expression. Indeed, TGFβ1-induced expression of ETS1 and breast cancer cell migration was blocked by knockdown of ETS1. Further, we identified that MET is a direct target of miR-128-3p and that this miRNA is negatively regulated by TGFβ1. Overexpression of miR-128-3p reduced MET expression and abrogated HGF-induced cell migration of invasive breast cancer cells. In conclusion, we have identified that TGFβ1 regulates HGF-induced and MET-mediated cell migration, through positive regulation of C-ets-1 and negative regulation of miR-128-3p expression in basal-like breast cancer cell lines and in triple-negative breast cancer tissue.

Background: Breast cancer patients at high risk for recurrence are treated with anthracycline-based chemotherapy, but not all patients do equally benefit from such a regimen. To further improve therapy decision-making, biomarkers predicting outcome are of high unmet medical need. Methods: The percent DNA methylation ratio (PMR) of the promoter gene coding for the Paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time polymerase chain reaction (PCR) test. The multicenter study was conducted in routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue from 205 lymph node-positive breast cancer patients treated with adjuvant anthracycline-based chemotherapy. Results: The cut-off for the PITX2 methylation status (PMR = 12) was confirmed in a randomly selected cohort (n = 60) and validated (n = 145) prospectively with disease-free survival (DFS) at the 10-year follow-up. DFS was significantly different between the PMR ≤ 12 versus the PMR > 12 group with a hazard ratio (HR) of 2.74 (p < 0.001) in the validation cohort and also for the patient subgroup treated additionally with endocrine therapy (HR 2.47; p = 0.001). Conclusions: Early-stage lymph node-positive breast cancer patients with low PITX2 methylation do benefit from adjuvant anthracycline-based chemotherapy. Patients with a high PITX2 DNA methylation ratio, approximately 30%, show poor outcome and should thus be considered for alternative chemotherapy regimens.

Introduction The PiA-study (Prognostic assessment in routine Application, NCT 01592825) was designed as a representative cohort of breast cancer patients to estimate the proportions of traditional and modern prognostic factors. The ASCO-recommended biomarkers uPA (urokinase-type plasminogen activator) and its inhibitor PAI-1 were used for biological risk assessment particularly for intermediate risk breast cancer patients and disease-free survival of the patients after 5 yrs of follow-up (F/U) was calculated. Material & Methods Between 2009 and 2011, 1,074 non-metastasized, primarily operated breast cancer patients from six centers in Germany were included. From 815 patients, fresh frozen tissue was obtained and processed for central testing uPA/PAI-1 by ELISA (FEMTELLE®, Sekisui Diagnostics GmbH). Low uPA/PAI-1 status is defined by uPA and PAI-1 concentrations below the published cut-offs, high status means one or both were higher than the corresponding cut-offs. Tumor characteristics were based on local pathology. The centers had to follow the national guidelines. In low-risk patients, adjuvant chemotherapy was spared. The median F/U is 56 months (range 0-78). Results In the total cohort of 1,074 patients, 166 had G1- and 237 had G3-tumors. Of the 671 patients with a G2-tumor, the following were allocated to the high-risk group: node-positive (n=371), younger than 35 yrs (n=17), and triple-negative (TN) or HER2-positive (n=118). For 253 tumors of the remaining 355 patients with an intermediate risk of recurrence (pN0, G2, HR positive, HER2-negative, ≥35 yrs), uPA/PAI-1 status was available. 126 (49.8%) were allocated to the low-risk group, one patient had a recurrence. At 5 yrs, in the total cohort 90.6% (95% CI, 89.5-91.7) of the patients were free of invasive disease. Of 114 HER2-positive tumors, 94 (82.4 %) had a high uPA/PAI-1 status, only one of the 38 HR negative/HER2 positive tumors had a low uPA/PAI-1 status. In the TN group, the majority of tumors had a high uPA/PAI-1 status (66 of 81; 81.5 %). In 30 patients lymph nodes were involved, 18.5% (n=15) had a low uPA/PAI-1 status, one event was detected. In N pos. patients with an high uPA/PAI-1 6 events were observed. Conclusion Testing for uPA/PAI-1 in the daily routine is feasible, fresh frozen tissue has been prepared from 76% of the tumors of the recruited patients, 37% of them had a low risk status. Using uPA/PAI-1, about half of the node-negative patients with an intermediate risk of recurrence were allocated to a group with an extremely low risk of recurrence and thus chemotherapy could be spared. Also in node-positive disease, uPA/PAI-1 has a prognostic impact. Tab 1: Proportion of the subgroups according to IHC, grading and uPA/PAI-1-statusTumor typetotallow uPA/PAI-1 statushigh uPA/PAI-1 statusn=815 (100%)n=304 (37%)n=511 (63%)Luminal A-like tumors:HRpos., HER2neg., G1, G2515 (63.2%)240 (78.9%)275 (53.9%)Luminal B/HER2-negative-like tumors:HRpos., HER2neg., G3104 (12.8%)29 (9.5%)75 (14.7%)Luminal B/HER2-positive-like tumors:HR pos., HER2 pos., all grades77 (9.4%)19 (6.2%)58 (11.4%)HER2-positive (nonluminal) - like tumors:HRneg., HER2pos., all grades38 (4.7%)1 (0.3%)37 (7.3%)TN tumors:HRneg., HER2neg., all grades81 (9.9%)15 (4.9%)66 (12.9%) Citation Format: Vetter M, Hartung C, Hanf V, Lantzsch T, Uleer C, Peschel S, John J, Buchmann J, Bürrig K-F, Weigert E, Thomssen C, Kantelhardt EJ. The ASCO-recommended prognostic factors uPA/PAI-1 in a multicenter cohort study (PiA) [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 P2-05-12.

Purpose The objective of this study was to compare tumor characteristics, biomarkers, and surrogate subtypes of breast cancer between Sudanese and German women. Methods Tumor characteristics and immunohistochemistry markers (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) were collected from the routine assessment of consecutive patients with invasive breast cancer diagnosed from 2010 to 2015 (Gezira University Pathology Laboratory, Gezira, Sudan) and from 1999 to 2013 (Breast Centre, Martin-Luther-University, Halle, Germany). Results A total of 2,492 patients (German [n = 1,932] and Sudanese [n = 560]) were included. Age at diagnosis ranged from 20 to 94 years. Sudanese women were, on average, 10 years younger than German women, with a mean (± standard deviation) age of 48.8 (13.5) and 58.6 (12.4) years, respectively. The Sudanese women had a higher grade, larger tumor, and more lymph node positivity compared with German women. ER-, PR-, and HER2-negative proportions were 55%, 61.8%, and 71.3%, respectively, for Sudanese women versus 22.7%, 32.3%, and 82.5%, respectively, for German women. The triple-negative subtype was more prevalent in Sudanese women (34.5%) than in German women (14.2%). The strongest factor associated with ER-negative disease was grade III (odds ratio, 19.6; 95% CI 11.6 to 33.4; P < .001). Sudanese patients were at higher risk for ER-negative breast cancer, with an odds ratio of 2.01 ( P = .001; adjusted for age, size, nodal status, histologic type, and grade). Stratified by grade, the influence of origin was observed in grade I and grade II tumors, but not in grade III tumors. Conclusion Sudanese women had more aggressive tumor characteristics and unfavorable prognostic biomarkers. After adjustment, Sudanese origin was still associated with hormone receptor–negative disease, especially in grade I and II tumors. These findings suggest differences in tumor biology among these ethnic groups.

The caspase 8 variants CASP8 -652 6N InsDel and Asp302His have previously been identified to promote survival of T-lymphocytes and to indicate reduced breast cancer susceptibility. Besides some preliminary findings, prognostic relevance of these polymorphisms in patients with existing breast cancer has not been investigated. Considering an immunomodulatory role of these polymorphisms, we genotyped 785 early breast cancer patients and correlated caspase 8 variants with disease-free survival (DFS) and the presence of tumor infiltrating lymphocytes (TILs). Early breast cancer specimens were collected as part of the multicenter prospective PiA study. Genotyping was performed by pyrosequencing, TILs status was assessed using hematoxylin & eosin staining. The CASP8 -652Del variant was significantly associated with improved DFS in an allele-dose dependent manner (p = 0.027). Homozygosity for the -652Del variant was an independent predictor for improved DFS (HR = 0.36; 95% CI = 0.174–0.726; p = 0.005). In patients with the 302HisHis genotype, there was no event of recurrence during observation time. Combined analysis of diplotypes revealed an influence of both polymorphisms on DFS (p = 0.029). Interestingly, patients with the 302HisHis variant among the unstratified patient cohort (and among the luminal-like subtype, by trend) had tumors with lower lymphocyte infiltration (p = 0.025). We propose a prognostically favorable role of the -652Del and the 302His variant in primary breast cancer and suggest for the first time an association between polymorphisms in apoptosis-related genes and the immunophenotype in breast cancer. Our findings encourage further investigation of caspase 8 polymorphisms as biomarkers for prognostic and immunotherapeutic considerations.

e24258Background: The minor allele of two caspase-8 polymorphisms, namely CASP8 -652 6N InsDel and CASP8 Asp302His, were shown to promote survival of T-lymphocytes and were repeatedly associated wi...

The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.

Background: ASCO Tumor Marker Guidelines 2007 recommended clinical routine use of the invasion markers uPA and PAI-1 for risk assessment in node-negative breast cancer patients (Harris et al. JCO 2007; 25:5287), and in some countries, e.g. Germany and France, risk assessment using these markers is broadly used. We wanted to evaluate the impact of uPA/PAI-1 on identifying patients with low risk of recurrence also in the daily routine in order to demonstrate that >25 years since their first description, these markers are still valuable. Material and Methods: We identified a cohort of 227 patients who were tested for uPA and PAI-1 in the clinical routine before 2012. Fresh frozen tissue of the primary tumor was obtained at biopsy or operation and processed for testing by a commercially available ELISA (FEMTELLE®, Sekisui Diagnostics GmbH) as previously described (Thomssen et al. JNCI 2009;101:1028). Tumor and patient characteristics were documented and all patients were regularly followed. Tumor concentrations below 3 ng/mg protein for uPA and below 14 ng/mg protein for PAI-1 were considered indicating low risk of recurrence (Harbeck et al. EJC 2013; 49:1825). Disease-free survival was defined as survival free from metastasis and loco-regional recurrence. Results: In our cohort, 86 patients had low tumor levels of uPA/PAI-1 (37.9 %). The median follow-up was 38.9 months (0.4 – 113.5 months). Adjuvant chemotherapy was delivered to 25 of 86 patients (29.1%) in the low risk group and to 85 of 141 patients (60.3%) in the high risk group; if steroid hormone receptor status was positive, generally adjuvant endocrine therapy for five years was advised. Using immunohistochemical subtyping, 73 of 86 patients with low uPA/PAI-1 values were luminal-like, 9 patients had a HER2-positive tumor and 3 patients had a triple negative breast cancer (TNBC). In patients with uPA or PAI-1 or both elevated, 105 of 141 patients had a luminal-like cancer, 12 patients were HER2 positive and 22 had TNBC; 1 case unknown. At 60 months of follow-up, patients with low uPA and/or PAI-1 tumor values had not experienced any recurrence, while in the high risk group 7 recurrences were observed although adequate adjuvant therapy was delivered (log-rank p=0.07). In node-negative pts with low uPA/PAI-1 values (n=72; pN0 70, cN0 2), no recurrences were observed, in 104 high risk patients 5 recurrences were observed (p=0.057). Conclusions: This observation confirms that also in daily routine, patients with a very low risk of recurrence can be identified by testing for uPA/PAI1. This group of pts comprises nearly 40% of pts and in these patients further evaluation with expensive predictive tests can be avoided and - above all - potentially toxic adjuvant chemotherapy can be spared. Citation Format: Thomssen C, Kantelhardt E-J, Grosse R, Papendick N, Steer S, Buchmann J, Wickenhauser C, Vetter M. The ASCO-recommended biomarkers urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 define a subgroup of patients with very low risk of recurrence under routine conditions. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-22.

Introduction: The burden of breast cancer is escalating across the globe and becoming a major public health problem in Ethiopia. Considering the fact that breast cancer is a heterogeneous disease in its nature, it demands a coordinated multimeric approach for diagnosis, treatment and management. Therefore, NanoString nCounter-based gene expression analysis might be employed for breast cancer intrinsic molecular subtyping, since it is a robust, highly reproducible tool and allows to simultaneously explore the expression of hundreds of genes in a single reaction tube. The aim of the study was to determine the intrinsic molecular subtypes of breast cancer samples from Ethiopian patients using a tailored NanoString nCounter gene expression assay for formalin-fixed, paraffin-embedded (FFPE) material. Methods: The present study was carried out to determine the molecular subtypes of breast cancer in Ethiopian patients. Archived FFPE tumor samples from public hospitals in Ethiopia were retrieved and blocks with clinical and pathologic data were used. From each sample total RNA was extracted by miRNeasy kit, then total RNA concentration and quality were determined and high-quality RNA was subjected to NanoString-based nCounter gene expression analysis using a tailored assay consisting of molecular and immunologic markers. For each gene, gene-fold change expression is given when compared to the reference cut-off value. The study focused on the analyses of estrogen receptor 1(ESR1), progestrone receptor (PGR), erythroblastic oncogene B (ERBB2) genes expression and the intrinsic molecular subtypes done using PAM 50 assay. Statistical analysis was carried out using SPSS software. Results: A total of 164 FFPE breast cancer blocks were analyzed for protein (ER, PR and Her2) expression status and mRNA expression levels. 86/164 (52.4%) breast cancer patients were tumor grade 3. More than 50% of tumor samples were ER and PR positive. In contrast to hormone receptors, only 20 (12.2%) breast tumor samples were found to be positive for HER-2. With regard to the mRNA expression levels of the respective genes, 106 (64.6%), 43 (26.2%) and 143 (87.2%) of breast cancer lesions analyzed showed an upregulation of ESR-1, PGR and ERBB2 genes, respectively. The samples with ER and PR positive results demonstrated by immunohistochemistry (IHC) showed nCounter levels from -60 to 124 (median=7) and -379 to 9 (median= -2), respectively. Samples with HER-2 positive results by IHC showed nCounter levels from -1 to 60 (median=17.5). Using PAM50 algorithm, we found 32 (19.5%) luminal A, 33 (20.1%) luminal B, 24 (14.6%) HER-2 enriched, 29 (17.7%) basal and 17 (10.4%) normal type breast cancer. Conclusion: According to the RNA expression assay, the majority of breast cancer subtypes were luminal A and luminal B and the lowest was normal type. In addition, the large majority of the cases analyzed were hormone receptor positive. Thus, hormonal therapy is of high importance for breast cancer care in Ethiopia. Citation Format: Zelalem Desalegn Woldesonbet, Martina Vetter, Meron Yohannes Nigussie, Tamrat Abebe Zeleke, Yonas Bekuretsion, Mahlet Arayselassie, Mathewos Assefa, Abebe Bekele, Endale Anberber, Claudia Wickenhauser, Eva J. Kantelhardt, Jürgen Bukur, Barbara Seliger. NanoString nCounter-based gene expression assay for evaluation of breast cancer molecular subtypes in Ethiopian patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C031.

Background: Breast cancer is the most common cancer overall, with an estimated 2.4 million incident cases in 2015. Current treatment of breast cancer is mainly surgery, chemotherapy, radiotherapy, and hormonal therapy. Although these approaches appear to be effective, they have failed to result in long-term cure, especially for aggressive HER2-positive and triple-negative breast cancer. This particularly holds for sub-Saharan Africa with a dominance of aggressive breast cancer subtypes with very limited treatment options and almost no research activities in place. As a result, continuous exploration of alternative treatment strategies is urgently required. Consequently, a collaborative research program has been established between Addis Ababa University, Ethiopia, and Martin-Luther-University, Germany, to enhance breast cancer research in Ethiopia. As a pilot study in Germany, we have assessed the in vitro potency of the three inhibitors romidepsin, trametinib, and lapatinib targeting HDAC, MEK and HER2/EGFR, respectively, in different breast cancer cell lines. Methods: Seven distinct breast cancer cell lines (MCF7, SKBR3, T47D, BT474, BT20, HCC1806 and HCC1937) of known HER-2 and hormone status were used to evaluate the antiproliferative effect of romidepsin, lapatinib, and trametinib. Cells cultivated in complete RPMI media were seeded in to 96 well plates (3,000 cells/well) and incubated at 370C and 5% CO2 for 24hrs. The cells were then treated for 24, 48, and 72 hours with different concentrations of the drugs. Cells exposed to solvent alone served as controls. XTT cell viability assay was used to determine proliferation of cells following manufacturers' instruction. IC50 value was calculated (Compusyn software) for each drug against the different cell lines to determine the antiproliferative effect. Results: Romidepsin exhibited a strong antiproliferative effect against all breast cancer cell lines tested, resulting in a marked decrease in cell viability with increasing concentrations, which highly varied between the breast cancer cell lines analyzed. The calculated IC50 values 48 hrs after the onset of treatment ranged between 2.2 and 52 nM; T47D was the most sensitive. Lapatinib treatment of the EGFR/HER-2 expressing BT20, SKBR3 and BT474 caused dose-dependent suppression of cell viability against SKBR3 and BT474 with an IC50 value of 450 and 77 nM, respectively. In contrast, BT20 cells were resistant to this treatment and were the only breast cancer cell line with an enhanced sensitivity to increasing concentrations of trametinib (IC50 =2.79 μM). Conclusion: In sum, romidepsin might improve the treatment efficacy of breast cancer though it needs to be further explored in vitro as well as in vivo alone and in combination with other therapies. Although trametinib has been suggested to be insufficient for monotherapy of breast cancer in general, it might be still a candidate for the treatment of basal-like breast cancers due to the sensitivity of BT20 cells. Citation Format: Meron Yohannes Nigussie, Jürgen Bukur, Zelalem Desalegn Woldesonbet, Tamrat Abebe Zeleke, Yonas Bekuretsion, Mahlet Arayselassie, Mathewos Assefa, Abebe Bekele, Endale Anberber, Martina Vetter, Claudia Wickenhauser, Eva J Kantelhardt, Barbara Seliger. Antiproliferative efficacy of different targeted drugs on breast cancer cell lines of distinct subtypes [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B020.

Motivation Several gene expression-based risk scores and subtype classifiers for breast cancer were developed to distinguish high- and low-risk patients. Evaluating the performance of these classifiers helps to decide which classifiers should be used in clinical practice for personal therapeutic recommendations. So far, studies that compared multiple classifiers in large independent patient cohorts mostly used microarray measurements. qPCR-based classifiers were not included in the comparison or had to be adapted to the different experimental platforms. Results We used a prospective study of 726 early breast cancer patients from seven certified German breast cancer centers. Patients were treated according to national guidelines and the expressions of 94 selected genes were measured by the mid-throughput qPCR platform Fluidigm. Clinical and pathological data including outcome over five years is available. Using these data, we could compare the performance of six classifiers (scmgene and research versions of PAM50, ROR-S, recurrence score, EndoPredict and GGI). Similar to other studies, we found a similar or even higher concordance between most of the classifiers and most were also able to differentiate high- and low-risk patients. The classifiers that were originally developed for microarray data still performed similarly using the Fluidigm data. Therefore, Fluidigm can be used to measure the gene expressions needed by several classifiers for a large cohort with little effort. In addition, we provide an interactive report of the results, which enables a transparent, in-depth comparison of classifiers and their prediction of individual patients. Availability and implementation https://services.bio.ifi.lmu.de/pia/. Supplementary information Supplementary data are available at Bioinformatics online.

Mixed-Materials parts have great light-weight potential for the automotive application to reduce the carbon footprint. But the joining of fibre composite plastic sheets to metal sheets is in practical application limited to adhesive bonding or mechanical joining with additional fastener elements due to the large differences in physical properties. A new process chain based on plastic joining without fastener elements is proposed and first results on the mechanism and on the achievable strength of the new joints are shown. The process chain consists of three steps: First joining pins are added to the sheet metal by an additive manufacturing process. In a second step these pins are pierced through the fibre composite sheet with a local heating of the thermoplastic in an overlap setup. In the third and last step the joint is created by forming the pins with the upsetting process to create a shape lock. The shear strength of the joined specimens was tested in a tensile testing machine. The paper shows that even with a non-optimized initial setup joints can be realised and that the new process chain is a possible alternative to adhesive bonding.

Human epidermal growth factor receptor 2 (ErbB2) is overexpressed in a variety of human malignancies. Moreover, ErbB2 has been reported to influence cancer patient survival and progression of different tumor entities. However, information regarding the prognostic impact of ErbB2 in soft tissue sarcoma (STS) patients is limited and conflicting.ErbB2 mRNA and protein levels were defined by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), and the prognostic impact of ErbB2 mRNA and protein levels in tumor tissue of 124 soft tissue sarcoma patients were investigated.The median ErbB2 mRNA expression level in tumor tissue was decreased 3.9-fold compared to non-neoplastic surrounding tissue (p = 0.001). Furthermore, an increased ErbB2 mRNA expression level was associated with an improved tumor-specific survival (p = 0.01, log rank test). Multivariate Cox's proportional hazard regression analyses revealed an increased ErbB2 mRNA expression level as an independent favorable prognostic factor for tumor-specific survival of STS patients (n = 124; RR = 3.0; 95 % CI = 1.6-5.7; p 0.001). In addition, multivariate Cox's proportional hazard regression analyses showed that an increased ErbB2 protein expression level correlated with poorer recurrence-free survival of STS patients (n = 47; RR = 9.9; 95 % CI = 1.7-59.7; p = 0.012), in particular for STS patients who received postoperative radiotherapy (n = 27; RR = 17.9; 95 % CI = 1.3-247.7; p = 0.031).This study suggests an inverse prognostic value of ErbB2 mRNA and protein expression level.