BackgroundTriple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC.MethodsCath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs).ResultsHigh CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFβ decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy.ConclusionCath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0498-z) contains supplementary material, which is available to authorized users.

Background: In the triple-negative breast cancer (TNBC) group, the luminal androgen receptor subtype is characterized by expression of androgen receptor (AR) and lack of estrogen receptor and cytokeratin 5/6 expression. Cathepsin D (Cath-D) is overproduced and hypersecreted by breast cancer (BC) cells and is a poor prognostic marker. We recently showed that in TNBC, Cath-D is a potential target for antibody-based therapy. This study evaluated the frequency of AR/Cath-D co-expression and its prognostic value in a large series of patients with non-metastatic TNBC. Methods: AR and Cath-D expression was evaluated by immunohistochemistry in 147 non-metastatic TNBC. The threshold for AR positivity (AR+) was set at ≥1% of stained cells, and the threshold for Cath-D positivity (Cath-D+) was moderate/strong staining intensity. Lymphocyte density, macrophage infiltration, PD-L1 and programmed cell death (PD-1) expression were assessed. Results: Scarff-Bloom-Richardson grade 1–2 and lymph node invasion were more frequent, while macrophage infiltration was less frequent in AR+/Cath-D+ tumors (62.7%). In multivariate analyses, higher tumor size, no adjuvant chemotherapy and AR/Cath-D co-expression were independent prognostic factors of worse overall survival. Conclusions: AR/Cath-D co-expression independently predicted overall survival. Patients with TNBC in which AR and Cath-D are co-expressed could be eligible for combinatory therapy with androgen antagonists and anti-Cath-D human antibodies.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background: Microarrays studies identified the subtype of luminal androgen receptor (LAR) among triple-negative breast cancer (TNBC). This subgroup is distinct of basal-like tumors and is characterized by the lack of ER, CK5/6 expression but the expression of genes that are usually expressed by ER+ luminal tumors like androgen receptor (AR). Using immunohistochemistry (IHC) AR is expressed in 8-58% of TNBC and its prognostic value is controverted. The aspartic protease cathepsin D (cath-D) is overproduced and hypersecreted by breast cancer (BC) cells and is often described as a marker of poor prognosis. We have already shown that cath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. We aimed at evaluating co-expression AR/cath-D-associated profiles and its prognostic value in a large retrospective series of patients with non-metastatic TNBC with a long follow-up. Patients and methods: AR and cath-D expression were evaluated by IHC in tissue microarrays of 147 patients with non-metastatic TNBC treated in our center between 2002 and 2012. Positivity threshold was set at ≥1% nuclear staining for AR. In tumor epithelial BC with vesicular and peripheral membrane labeling, cath-D signal was scored as absent (0%), low (<20%), moderate (20-50%) or high (>50%). Tumors were defined as cath-D+ for staining ≥ 20%. Basal-like phenotype (CK5/6 and/or EGFR+), lymphocytic infiltration, PD-L1 expression and macrophages infiltration were also assessed. Results: Median age was 61.6 years (range 30.2-98.6). 53.1% of tumors were classified pT1 and 61.2% pN0. We found 86.2% of ductal carcinomas, 6.9% of lobular carcinomas and 6.9% of other histological types. SBR grade 1-2 represented 11% of tumors. A basal-like phenotype was observed in 61.6% of cases. Adjuvant chemotherapy (ACT) was delivered in 68% of patients. 72.8% of patients had AR+ tumors. Among the 142 patients with available AR/cath-D co-expression 62.7% had AR+ and cath-D+ tumors. AR+/cath-D+ tumors exhibited more frequently: lymph node invasion (p=0.04), less frequently macrophages infiltration (p=0.04) and a trend of lower nuclear grade (1/2) (p=0.06) than others TNBC. There was no significant difference regarding basal-like phenotype, lymphocytic infiltration or PD-L1 expression. With a median follow-up of 5.4 years, there was a trend for a lower relapse-free survival (RFS) for patients with AR+/cath-D+ tumors (p=0.09): 3-years RFS were 67.4% (CI 95% [54.1-77.6]) and 81.9% (CI 95% [68.0-90.1]) for AR+/cath-D+ and the others TNBC, respectively. 5-years RFS were 57.6% (CI 95% [43.0-69.7]) and 71.4% (CI 95% [55.4-82.5]) for AR+/cath-D+ and the others TNBC, respectively. Tumor size, nodal status and ACT were also statistically correlated to RFS. In univariate analysis, age (p=0.01), tumor size (p=0.002), nodal status (p=0.004), ACT (p=0.004) were significantly associated with overall survival (OS). There was a trend for AR/cath-D co-expression (p=0.086). In multivariate analyses, tumor size (p=0.002), ACT (p<0.001) and AR/cath-D co-expression (p<0.001) were independent prognostic factors. Conclusions: In this series, almost 63% of TNBC had an AR/cath-D co-expression with distinct clinicopathological characteristics. AR+/cath-D+ co-expression independently predicted OS. Patients with AR+/cath-D+ tumors tended to have higher risk of late recurrences than patients with others TNBC. These biomarkers could be useful to identify a specific subgroup of TNBC with worse prognosis and could have therapeutic implications: anti-androgens are under investigation; pre-clinical studies are ongoing with anti-cath-D antibodies. Citation Format: Hanane Mansouri, Lindsay Alcaraz, Caroline Mollevi, Aude Mallavialle, William Jacot, Florence Boissière-Michot, Joelle Simony-Lafontaine, Valérie Laurent-Matha, Pascal Roger, Emmanuelle Liaudet-Coopman, Séverine Guiu. Prognostic value of androgen receptor and cathepsin D co-expression in non-metastatic triple-negative breast cancer and correlation with other biomarkers [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 P6-05-11.

PurposeTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutics. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited.Experimental DesignThis study analyzed the prognostic value of tumor and stromal cell SPARC expression in a large series of 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). Fibrosis, tumor-associated macrophage (TAM) infiltration, tumor-infiltrating lymphocyte (TIL) density, PD-L1 and PD-1 expression were assessed. Tumor and stromal cell SPARC expression was studied by immunofluorescence, western blotting, and meta-analysis of published single-cell mRNA sequencing data. The biological role of fibroblast-secreted SPARC was analyzed using cell adhesion, wound healing, Transwell-based motility and invasion, and tumor spheroid assays.ResultsSPARC expression was detected in cancer cells (42.4%), cancer-associated fibroblasts (CAFs) (88.1%), TAMs (77.1%), endothelial cells (75.2%), and TILs (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. SPARC expression in CAFs was an independent prognostic factor in multivariate analysis. Tumor and stromal cell SPARC expression was observed in TNBC cytosols, patient-derived xenografts, and cell lines. SPARC was expressed by different CAF subsets, including myofibroblasts and inflammatory CAFs. Fibroblast-secreted SPARC inhibited TNBC cell adhesion and stimulated their migration and invasion.ConclusionsSPARC expression in CAFs is an independent predictor of recurrence-free survival in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC-targeted therapy.Statement of translational relevanceHere, we identified a subgroup of patients with triple-negative breast cancer (TNBC) with worse prognosis and eligible for therapies that target extracellular matrix proteins in the tumor stroma. Specifically, we found that expression of the matricellular protein SPARC in cancer-associated fibroblasts (CAFs) is an independent prognostic marker of poor outcome in TNBC. Furthermore, we showed that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved/associated with many tumor-related processes. We then found that SPARC secreted by fibroblasts has a pro-tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our results support the need to consider SPARC expressed/secreted by CAFs as a novel therapeutic target in TNBC in the context of treatments to modulate the tumor stroma.

No abstract