Supplementary Figure 1 from Coordinates in the Universe of Node-Negative Breast Cancer Revisited

Supplementary Tables 1, 3, 5-6, Figures 1-6 from The Humoral Immune System Has a Key Prognostic Impact in Node-Negative Breast Cancer

8 Supplementary Figures and 2 Supplementary Tables

Supplementary Data from ERBB2 Induces an Antiapoptotic Expression Pattern of Bcl-2 Family Members in Node-Negative Breast Cancer

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

Published classifiers for prediction of pCR (a), study centers that contributed to the EXPRESSION trial (b), baseline characteristics of patients in the EXPRESSION trial (c), publicly available breast cancer datasets (d), comparison of patients who achieved versus not achieved a pCR (e), association of clinicopathologic parameters with pCR (f)

Supplementary Table 4 from The Humoral Immune System Has a Key Prognostic Impact in Node-Negative Breast Cancer

Supplementary Table 2 from The Humoral Immune System Has a Key Prognostic Impact in Node-Negative Breast Cancer

Classifier performance in the subgroup of ER+/HER2-patients

Background: Recently, novel antibody-drug conjugates (ADCs) showed clinical activity in a subset of advanced HER2-negative patients. We investigated the prognostic significance of HER2-low and HER2-zero tumors.Patients and Methods: The retrospective cohort study included 410 consecutive node-negative breast cancer patients without adjuvant systemic therapy treated between 1985 and 2000 (median follow-up 16.73 [IQR 8.58-23.45] years). 351 (85.6%) were HER-2 negative and subdivided into HER2-zero and HER2-low. HER2 gene expression was available in 170 (48.4%) patients. Differences in HER2 status for immunohistochemistry, gene expression and clinico-pathologic parameters were assessed using Fisher's exact test, Pearson’s correlation and Mann-Whitney test. Prognosis was investigated using the Kaplan-Meier method and Cox regression analyses.Results: 198 (56.4%) of the 351 HER2-negative patients had HER2-low tumors and 153 (43.6%) were HER2-zero. Significant differences between HER2-zero and HER2-low tumors were found in histologic grade (P=0.001), Ki-67 (P=0.013), and HER2 gene expression (P=0.002). HER2-low patients had significantly longer disease-free survival (DFS) (15-year rate: 67.5% [95% CI 61.0-74.7] vs 47.3% [95% CI 39.9-56.1], P<0.001) and overall survival (OS) (15-year rate: 75.4% [95% CI 69.4-81.9] vs 66.8% [95% CI 59.5-74.9], P=0.009). The results of multivariable analyses confirmed the independent prognostic significance of HER2 status (DFS: HR 0.556, 95% CI 0.409-0.755, P<0.001; OS: HR 0.664, 95% CI 0.467-0.945, P=0.023).Conclusion: Our results argue for additional clinically relevant subtypes of HER2-negative breast cancer with different biology and prognosis. In particular, advanced HER2-low patients may be future suitable candidates for novel ADCs.

Background: Breast carcinoma is the most common and one of the most malignant tumors in women. Multimodal standard therapy currently increases the 5-year survival rate to over 80%. For some years now, complementary medicine has also been playing an increasingly role in the field of cancer therapy. In particular, mistletoe extract is used in breast cancer therapy to reduce the side effects of tumor therapy and to improve patients' quality of life. Different approaches exist to stimulate the immune system. In this in vitro study, the effects of exosomes from mistletoe extract treated tumor cells on the cytotoxic behavior of immune cells were investigated. Material and Methods: Two human breast cancer cell lines MCF-7 and SK-BR3 were used. Mistletoe extract preparation Viscum album M at a concentration of 10 μg/ml was used to treat tumor cells. After 48 h incubation, exosomes were isolated from the cell culture supernatant of tumor cells. These were then used for stimulation of T cell line MOLT4 and NK cell line KHYG-1. Fluorescence-activated cell sorting (FACS) and a cytotoxicity assay (LDH assay) were used to examine the immune cells. The focus was laid on the analysis of surface markers and cytotoxicity of immune cells against tumor cells. Results: The LDH assays showed significant differences with respect to the cytotoxicity of the immune cells. The NK cell line KHYG-1 showed a significant increase in cytotoxicity after incubation with the exosomes derived from mistletoe extract-treated tumor cells MCF7 compared to the NK cells that remained untreated (57,9% ± 22,6% vs. 86,8% ± 12,8%). FACS analysis shows significant differences in surface markers CD3 and CD45 on the T cell line after incubation with the exosomes isolated from the tumor cells treated with mistletoe extract. Conclusion: Based on the results, we conclude that mistletoe extract has a positive effect on the cytotoxicity of immune cells. Thus, there is an assumption of a stimulation effect of the patient's immune system. It is already proved by clinical studies that mistletoe extract does not exert negative influence on standard therapy procedures nor does it lead to unexpected side effects. Therefore, the use of mistletoe extract could be an integral part of standard therapy in breast cancer patients in the future. No conflict of interest.

AbstractPurpose: Expression-based classifiers to predict pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) are not routinely used in the clinic. We aimed to build and validate a classifier for pCR after NACT. Patients and Methods: We performed a prospective multicenter study (EXPRESSION) including 114 patients treated with anthracycline/taxane-based NACT. Pretreatment core needle biopsies from 91 patients were used for gene expression analysis and classifier construction, followed by validation in five external cohorts (n = 619). Results: A 20-gene classifier established in the EXPRESSION cohort using a Youden index–based cut-off point predicted pCR in the validation cohorts with an accuracy, AUC, negative predictive value (NPV), positive predictive value, sensitivity, and specificity of 0.811, 0.768, 0.829, 0.587, 0.216, and 0.962, respectively. Alternatively, aiming for a high NPV by defining the cut-off point for classification based on the complete responder with the lowest predicted probability of pCR in the EXPRESSION cohort led to an NPV of 0.960 upon external validation. With this extreme-low cut-off point, a recommendation to not treat with anthracycline/taxane-based NACT would be possible for 121 of 619 unselected patients (19.5%) and 112 of 322 patients with luminal breast cancer (34.8%). The analysis of the molecular subtypes showed that the identification of patients who do not achieve a pCR by the 20-gene classifier was particularly relevant in luminal breast cancer. Conclusions: The novel 20-gene classifier reliably identifies patients who do not achieve a pCR in about one third of luminal breast cancers in both the EXPRESSION and combined validation cohorts.

We studied the prognostic impact of tumor immunoglobulin kappa C (IGKC) mRNA expression as a marker of the humoral immune system in the FinHer trial patient population, where 1010 patients with early breast cancer were randomly allocated to either docetaxel-containing or vinorelbine-containing adjuvant chemotherapy. HER2-positive patients were additionally allocated to either trastuzumab or no trastuzumab. Hormone receptor-positive patients received tamoxifen. IGKC was evaluated in 909 tumors using quantitative real-time polymerase chain reaction, and the influence on distant disease-free survival (DDFS) was examined using univariable and multivariable Cox regression and Kaplan–Meier estimates. Interactions were analyzed using Cox regression. IGKC expression, included as continuous variable, was independently associated with DDFS in a multivariable analysis also including age, molecular subtype, grade, and pT and pN stage (HR 0.930, 95% CI 0.870–0.995, p = 0.034). An independent association with DDFS was also found in a subset analysis of triple-negative breast cancers (TNBC) (HR 0.843, 95% CI 0.724–0.983, p = 0.029), but not in luminal (HR 0.957, 95% CI 0.867–1.056, p = 0.383) or HER2-positive (HR 0.933, 95% CI 0.826–1.055, p = 0.271) cancers. No significant interaction between IGKC and chemotherapy or trastuzumab administration was detected (Pinteraction = 0.855 and 0.684, respectively). These results show that humoral immunity beneficially influences the DDFS of patients with early TNBC.

Interferons are crucial for adaptive immunity and play an important role in the immune landscape of breast cancer. Using microarray-based gene expression analysis, we examined the subtype-specific prognostic significance of interferon-γ (IFN-γ) as a single gene as well as an IFN-γ signature covering the signaling pathway in 461 breast cancer patients. Prognostic significance of IFN-γ, as well as the IFN-γ signature for metastasis-free survival (MFS), were examined using Kaplan–Meier as well as univariate and multivariate Cox regression analyses in the whole cohort and in different molecular subtypes. The independent prognostic significance of IFN-γ as a single gene was limited to basal-like breast cancer (hazard ratio (HR) 2.779, 95% confidence interval (95% CI) 1.117–6.919, p = 0.028). In contrast, the IFN-γ-associated gene signature was an independent prognostic factor in the whole cohort (HR 2.287, 95% CI 1.410–3.633, p < 0.001) as well as in the basal-like (HR 3.458, 95% CI 1.154–10.359, p = 0.027) and luminal B (HR 2.690, 95% CI 1.416–5.112, p = 0.003) molecular subtypes. These results underline the subtype-dependent prognostic influence of the immune system in early breast cancer.

Introduction/ Purpose of study Targeting M2 macrophages as an immunosuppressive cell population within the tumor-microenvironment has become an important effort within preclinical research to achieve and enhance anti-tumor efficacy. In contrast to reprogramming approaches, we focused on effective and specific depletion of M2 macrophages (Mphs) by a new antibody-drug-conjugate (ADC) comprising a CD206-binding antibody conjugated to a nicotinamide phosphoribosyl transferase (NAMPT) toxophore which was proved to be very potent in cell killing. Description of procedures Human (hu) and mouse (ms) cross-reactive CD206 antibodies were identified by phage display using recombinant hu/ms CD206-extracellular domains. Selected clones showing strong CD206 binding by ELISA were reformatted into huIgG1 format and cellular targeting confirmed by FACS binding and internalization using CD206-transfected HEK cells. Antibodies were conjugated to a NAMPT toxophore by Cys-conjugation using two different non-cleavable linkers. Murine peritoneal Mphs collected after thioglycolate stimulation were polarized to M1 (LPS + IFNγ) or M2 (IL4 + IL13 + PGE2) within 24h and characterized for cytokines and typical markers via FACS (CD206, CD163, CD80/MHCII) and qPCR (iNOS, TGFβ, ARG1). After 96h treatment with either CD206- or isotype-NAMPT-ADCs, viability of Mphs was determined. In vivo pharmacokinetics (PK) of unconjugated antibodies were analyzed in plasma of female BALB/c mice for up to 72h. Summary of data Of the resulting 57 hu/ms cross-reactive CD206-binding antibodies, TPP-17829 and TPP-17836 were identified with single-digit nanomolar binding affinity and strong internalization in CD206-transfected cells as a prerequisite for an ADC approach. Conjugation of the antibodies to NAMPT toxophores yielded drug-antibody-ratios of 4-7 as determined by SEC-UV without significant aggregation. These two CD206-NAMPT-ADCs successfully depleted polarized M2-Mphs characterized by high CD206 expression, whereas isotype-NAMPT-ADC controls had no effect. While the unconjugated NAMPT toxophore was potent in depleting primary low-proliferating M1/M2 Mphs, by comparison, the anti-proliferative toxophore KSP (kinesin-spindle-protein inhibitor) was completely ineffective. PK analysis of TPP-17829 and TPP-17836, however, revealed a rapid plasma clearance within the first hours, most likely target-mediated via the liver, rendering them unacceptable for in vivo efficacy testing as NAMPT-ADCs. Conclusions CD206-NAMPT-ADCs demonstrated proof of concept for specific depletion of M2-Mphs in vitro. However, specific in vivo depletion of M2 Mphs from tumors will be challenging with respect to fast plasma clearance observed for anti-CD206 antibodies. Citation Format: Sandra Berndt, Katharina Filarsky, Patrick Smith, Niels Boehnke, Markus Berger, Fionnuala McAleese Eser, Hans-Georg Lerchen, Phillip Ellinger, Mathias Gehrmann, Jim Wu, Dominic Hildebrand, Bertolt Kreft, Uwe Gritzan. Effective depletion of M2 macrophages by CD206-NAMPT-ADCs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 677.

Hsp70-1A—the major stress-inducible member of the HSP70 chaperone family—is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.

A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.