Breast cancer (BC) is the leading cause of cancer deaths in women. Patients with estrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative or low (HER2-; HER2low) BC exhibit a late recurrence pattern and resistance to endocrine therapy. Antibody-drug conjugates (ADCs) are new anticancer agents that currently show a remarkable acceleration in preclinical and clinical development. The aspartic protease cathepsin D (cath-D), a poor prognosis marker in BC, is a tumor cell-associated protein with pro-tumor activity that is highly and specifically secreted by BC cells. We recently showed using the human anti-cath-D antibody F1M1 that cath-D is a potent target for antibody-based therapy in BC. Here, we asked whether cath-D is a suitable target for ADC-based therapy in non-amplified ER+/HER2low BC. To this aim, we bioconjugated F1M1 to the cytotoxic payload monomethyl auristatin F (MMAF) via a non-cleavable linker (for the release of a non-cell permeable MMAF metabolite) and to its cell permeable derivative monomethyl auristatin E (MMAE) via a protease-sensitive cleavable linker, to form the ADCs F1M1-MMAF and F1M1-MMAE respectively, with a drug-to-antibody ratio close to four. These F1M1-based ADCs were specifically internalized and showed cytotoxic activity in the nanomolar range in the ER+/HER2low MCF-7 and endocrine therapy-resistant ER+/HER2low MCF-7/LCC2 BC cell lines. In vivo, three injections of F1M1-based ADCs led to long-lasting tumor growth inhibition in mice harboring MCF-7/LCC2 cell xenografts. In conclusion, we showed for the first time that cath-D is a potent target for ADC in BC.

Cathepsins, the most abundant lysosomal proteases, have key functions in cell maintenance and homeostasis. They are overexpressed and hypersecreted in cancer and associated with poor prognosis. Secreted cathepsins display pro-tumour activities in the tumour microenvironment and thus represent interesting molecular targets in oncology. Recently, several antibody-based cancer therapies have targeted the pro-tumour activity of the extracellular cathepsin pool, altering several cancer hallmarks, but not the intracellular cathepsin levels that are often crucial for cell homeostasis. In this mini-review, we describe advances in antibodies against extracellular cathepsins in cancer, and their effect on the proteolytic cascade, matrix remodelling, proliferation, and modulation of the anti-cancer immune response. We also discuss the add-on value of combination strategies (anti-cathepsin antibodies with chemotherapy and/or biologics) that make anti-cathepsin antibodies a new opportunity for disease management.

Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype characterized by aggressive clinical behavior and poor prognosis. The immune landscape associated with TNBC often reveals high immunogenicity. Therefore, immunotherapy, which has demonstrated its efficacy in different cancer types, could be a promising strategy for TNBC, given the limited therapeutic options currently available besides conventional chemotherapy. The aspartic protease cathepsin D (cath-D) is a tumor cell-associated extracellular protein with protumor activity, a marker of poor prognosis, and a target for antibody-based therapy in TNBC. This commentary provides a synopsis/narrative summary of the development of anti-cath-D antibodies in different formats, their key roles in restoring the antitumor immunity, particularly via activation of tumor-infiltrating natural killer cells, and their dual antitumor effects on cancer cells and stromal cancer-associated fibroblasts, suggesting their interest for clinical use in the light of the current clinical knowledge on TNBC.

BACKGROUND AND PURPOSE:Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2+ BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts). EXPERIMENTAL APPROACH:CathD expression was evaluated by western blotting and immunofluorescence, and antibody binding to CathD by ELISA. Antibody anti-tumour efficacy was investigated in mouse models. Immune cell recruitment and activation were assessed by immunohistochemistry, immunophenotyping, and RT-qPCR. KEY RESULTS:F1 and F1M1 antibodies remodelled the tumour immune landscape. Both antibodies promoted innate antitumour immunity by preventing the recruitment of immunosuppressive M2-polarized tumour-associated macrophages (TAMs) and by activating natural killer cells in the tumour microenvironment of both models. This translated into a reduction of T-cell exhaustion markers in the tumour microenvironment that could be locally supported by enhanced activation of anti-tumour antigen-presenting cell (M1-polarized TAMs and cDC1 cells) functions. Both antibodies inhibited tumour growth in the highly-immunogenic E0771 model, but only marginally in the immune-excluded TUBO model, indicating that anti-CathD immunotherapy is more relevant for BC with a high immune cell infiltrate, as often observed in TNBC. CONCLUSION AND IMPLICATION:Anti-CathD antibody-based therapy triggers the anti-tumour innate and adaptive immunity in preclinical models of BC and is a promising immunotherapy for immunogenic TNBC. LINKED ARTICLES:This article is part of a themed issue Immunotherapy in Cancer. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v183.6/issuetoc.

Introduction Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC.Methods Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide.Results Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity.Conclusions F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

BackgroundIn triple-negative breast cancer (TNBC), the most immunogenic breast cancer type, tumor-infiltrating lymphocytes (TILs) are an independent prognostic factor. Tertiary lymphoid structures (TLS) are an important TILs source, but they are not integrated in the current prognostic criteria.MethodsIn this retrospective study, TLS were assessed in hematein-eosin-saffron-stained (HES) histological sections from 397 early, chemotherapy-naive TNBC samples after primary surgical resection. Their association with i) classical clinicopathological features, ii) TILs and CD3+, CD8+, CD20+ lymphoid populations, iii) CD68+, CD163+, CD11b+, CD66b+ myeloid populations, and iv) expression of the PD1/PD-L1 and PVR/TIGIT axis immune checkpoint components and their prognostic significance were evaluated.ResultsTLS were observed in 88.2% of samples, mainly in peritumoral areas (86.1%). Increased amount of peritumoral TLS (PT-TLS) was significantly associated with younger age (p<0.001), smaller tumor size and higher tumor grade (both, p<0.001), HER2null tumors (versus HER2low tumors, p<0.002), and non-lobular histological type (p<0.016). TNBC with higher PT-TLS abundance displayed more often a basal-like (p<0.001) and not molecular-apocrine phenotype (p<0.001). TLS abundance was associated with TILs and hot tumor inflammatory pattern (both, p<0.001). Remarkably, PT-TLS abundance was positively associated with the density of the analyzed lymphoid (CD3+, CD8+, CD20+) and myeloid (CD68+, CD163+, CD11b+) cell populations (all p<0.001), with the exception of CD66b+ cells, as well as with expression of the PD1/PD-L1 and TIGIT/PVR immune checkpoint markers. In univariate analysis, beside the classical clinicopathological factors (tumor size, node involvement and adjuvant chemotherapy), TILs, hot tumors and PT-TLS were significantly associated with clinical outcome. Moreover, the risk of relapse was inversely correlated with PT-TLS abundance (Kaplan-Meier analysis). In multivariate analysis, pathological stage, adjuvant chemotherapy and PT-TLS remained correlated with relapse-free survival.ConclusionOur results suggest that TLS are a frequent feature in early TNBC and that their presence, particularly at the tumor periphery, recapitulates the tumor immune microenvironment. In our series, their prognostic value outperformed that of TILs. Therefore, their easy quantification on routine HES sections and their integration into the factors classically analyzed by pathologists could improve the clinical management of TNBC, a breast cancer type whose prognosis remains too poor.

Journal of the European Academy of Dermatology and VenereologyEarly View LETTER TO THE EDITOR A deep-learning algorithm to localize basal cell carcinoma foci on Mohs surgery frozen sections Hugo Bonnefille, Hugo Bonnefille Department of Dermatology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorMarwan Abbas, Marwan Abbas LIRMM, Montpellier University, CNRS, Montpellier, FranceSearch for more papers by this authorPascal Roger, Pascal Roger Department of Pathology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorFrançois Habib, François Habib Department of Dermatology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorFarzaneh Masset, Farzaneh Masset Department of Pathology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorMarc Chaumont, Marc Chaumont LIRMM, Montpellier University, CNRS, Montpellier, France Université de Nîmes, Nîmes, FranceSearch for more papers by this authorGerard Subsol, Gerard Subsol LIRMM, Montpellier University, CNRS, Montpellier, FranceSearch for more papers by this authorPierre E. Stoebner, Corresponding Author Pierre E. Stoebner pierre.stoebner@chu-nimes.fr orcid.org/0000-0001-8928-8933 Department of Dermatology, CHU Carémeau, Montpellier University, Nîmes, France IRCM, INSERM U1194, Montpellier, France Correspondence Pierre E. Stoebner, Department of Dermatology, CHU Carémeau, Montpellier University, 30000 Nîmes, France. Email: pierre.stoebner@chu-nimes.frSearch for more papers by this author Hugo Bonnefille, Hugo Bonnefille Department of Dermatology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorMarwan Abbas, Marwan Abbas LIRMM, Montpellier University, CNRS, Montpellier, FranceSearch for more papers by this authorPascal Roger, Pascal Roger Department of Pathology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorFrançois Habib, François Habib Department of Dermatology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorFarzaneh Masset, Farzaneh Masset Department of Pathology, CHU Carémeau, Montpellier University, Nîmes, FranceSearch for more papers by this authorMarc Chaumont, Marc Chaumont LIRMM, Montpellier University, CNRS, Montpellier, France Université de Nîmes, Nîmes, FranceSearch for more papers by this authorGerard Subsol, Gerard Subsol LIRMM, Montpellier University, CNRS, Montpellier, FranceSearch for more papers by this authorPierre E. Stoebner, Corresponding Author Pierre E. Stoebner pierre.stoebner@chu-nimes.fr orcid.org/0000-0001-8928-8933 Department of Dermatology, CHU Carémeau, Montpellier University, Nîmes, France IRCM, INSERM U1194, Montpellier, France Correspondence Pierre E. Stoebner, Department of Dermatology, CHU Carémeau, Montpellier University, 30000 Nîmes, France. Email: pierre.stoebner@chu-nimes.frSearch for more papers by this author First published: 16 January 2023 https://doi.org/10.1111/jdv.18859 Hugo Bonnefille and Marwan Abbas are co-first authors. Gerard Subsol and Pierre E. Stoebner are co-senior authors. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Early ViewOnline Version of Record before inclusion in an issue RelatedInformation

Purpose Triple-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 Design This 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. Results SPARC 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. Conclusions SPARC 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 relevance Here, 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.

Background: Therapeutic strategies targeting neovessels responsible for musculoskeletal chronic pain have emerged, including neovessels embolization. Our study aimed to develop a large animal model of patellar tendinopathy with neovascularization. Methods: Nine 3-month-old male piglets (18 patellar tendons) received percutaneous injections of increasing doses of collagenase (0 to 50 mg) at day 0 (D0). Tendinopathy was evaluated by ultrasound (D7 and D14). Neovascularization was evaluated visually and on angiographies. Bonar score was used for histological analysis (D14). Correlations were evaluated using Spearman’s rank (Rs) test. Results: Research protocol was well tolerated. All tendons were enlarged with a median increase of 31.58% [25–40.28] at D7 (p = 0.244) at D7 and 57.52% [48.41–91.45] at D14 (p = 0.065). Tendons with collagenase injection had more hypoechoic changes, with one tendon rupture (p = 0.012). Neovascularization was reported above 5 mg collagenase (p < 0.01) at D7 and D14 with dose-related neovessels induction (Rs = 0.8, p < 0.001). The Bonar score increased above 5 mg collagenase, correlated with the dose (Rs = 0.666, p = 0.003). Conclusions: The study shows the feasibility, safety and reproducibility of this new large animal model of patellar tendinopathy with neovascularization after collagenase injection. It will allow studying new treatments on direct embolization of neovessels by endovascular approach.

Background: Ex vivo confocal microscopy using fusion mode and digital staining (EVCM) scans unfixed fresh tissue and produces rapidly digitally stained images of very similar quality to classical pathology. We investigated whether EVCM could represent an alternative to the standard histological examination of the pretherapeutic basal cell carcinoma (BCC) punch biopsies. Objectives: The objective of the study was to assess diagnostic accuracy of EVCM versus traditional histopathological examination for diagnosing and subtyping clinically suspicious lesions of BCC in 3-mm fresh and nonfixed punch biopsies. Methods: In this prospective monocentric observational study, patients with clinically suspected BCC were consecutively enrolled. Punch biopsies were imaged using EVCM and subsequently processed for standard histologic examination (gold standard). EVCM images were examined by a dermatopathologist blinded to clinical aspect of the lesion and histopathological results. Concordance between the EVCM and histology analysis was calculated with Cohen’s kappa (κ) statistic. Results: Sixty-six patients were recruited, and 106 biopsies were analyzed. EVCM correctly diagnosed 70/73 BCCs and 31/33 non-BCC lesions, corresponding to a sensitivity of 96% and a specificity of 94% (positive predictive value = 97%, negative predictive value = 91%). The EVCM assessment led to over-staging and under-staging of BCC subtypes in 5% and 11% of cases, respectively. It led to over-staging and under-staging of BCC depths in 5% and 15%, respectively. The kappa coefficient for concordance was 0.78 (95% confidence interval [CI]: 0.69–0.88) when considering BCC subtypes and 0.81 (95% CI: 0.72–0.90) when considering BCC depths. Conclusions: These results render EVCM as a promising option for “real-time” pretreatment evaluation of clinically suspected BCC lesions. Further larger randomized studies are needed to assess the efficiency of EVCM versus standard care in patients with clinically suspected BCC.

La chirurgie micrographique de Mohs (CMM) « classique » permet l'exérèse des cancers cutanés avec contrôle anatomopathologique extemporané de 100 % des berges tumorales. Elle est peu pratiquée en France en raison de difficultés logistiques qui pourraient être résolues en partie par une semi-automatisation de la lecture anatomopathologique. L'objectif de notre travail était d'élaborer un algorithme d'intelligence artificielle (IA) performant capable de localiser les foyers de carcinome basocellulaire (CBC) au sein de lames histologiques de CMM via un réseau neuronal convolutif (RNC). Les lames issues des procédures CMM réalisées dans notre centre entre 2016 et 2021 étaient incluses puis numérisées (grossissement x20). Les foyers de CBC étaient délimités manuellement via le logiciel Qupath. Les limites étaient systématiquement vérifiées par un anatomopathologiste compétent en CMM. Les images tissulaires de 71 lames issues de 49 patients étaient découpées en patchs de 256 × 256 pixels (200 × 200 μm). Le nombre de patchs était démultiplié (rotations, color augmentation). 106 567 patchs étaient obtenus; 7972 contenant du CBC formaient la classe « pathologique », 98595 formaient la classe « sain ». Ces patchs constituaient la base d'entraînement du RNC (Resnet-34). 10 lames issues de 10 patients différents formaient la base de test. 13 602 patchs tissulaires étaient automatiquement extraits puis classifiés par le RNC pré-entraîné. Premièrement, on déterminait un seuil de classification sain/pathologique pour que notre modèle soit cliniquement performant. Puis, la qualité de superposition des foyers de CBC prédits par le RNC et la vérité terrain était évaluée. Le principe de la métrique était que chaque foyer était considéré localisé si l'algorithme trouvait au moins un patch pathologique en son sein. L'AUC-ROC de notre algorithme était de 0,9787. Avec le seuil optimal cliniquement pertinent déterminé à 0,21, le RNC classait correctement 12945/13602 patchs (VP= 1171, FN = 293, FP = 364, VN = 11774) soit une Se de 0,80, une Sp de 0,97, une VPP de 0,76, une VPN de 0,98. Mais les patchs classés comme FN contenaient peu de CBC (7,4 % en moyenne), ils correspondaient majoritairement à des patchs en bordure de foyer tumoral. Pour les 10 lames de test, l'approche métrique permettait de localiser correctement 212/214 foyers, (FN = 2, FP = 67). Soit une Se de 0,99 et une VPP de 0,75. Les foyers FP avaient une taille moyenne de 1,4 patchs vs. 4,3 patchs pour les VP. Par sa bonne sensibilité, notre algorithme ouvre la voie de la CMM assistée par IA. Ses performances pourront être améliorées en augmentant la base d'apprentissage par un recrutement multicentrique, en intégrant plus de sous-types histologiques rares (sclérodermiformes) et avec les améliorations technologiques dont bénéficieront les RNC.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/-knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

Background: The aim of this study was to assess the impact of the initial therapeutic strategy on oncologic outcomes in patients with HPV-positive OPSCC. Methods: All p16-positive OPSCCs treated from 2009 to 2014 in 7 centers were retrospectively included and classified according to the therapeutic strategy: surgical strategy (surgery +/- adjuvant radiotherapy and chemotherapy) vs. non-surgical strategy (definitive radiotherapy +/- chemotherapy). Univariate, multivariate propensity score matching analyses were performed to compare overall (OS), diseasespecific (DSS) and recurrence-free survival (RFS). Results: 382 patients were included (surgical group: 144; non-surgical group: 238). Five-year OS, DSS and RFS were 89.2, 96.8 and 83.9% in the surgical group and 84.2, 87.1 and 70.4% in the non-surgical group, respectively. These differences were statistically significant for DSS and RFS after multivariate analysis, but only for RFS after propensity score matching analysis.

The last international guidelines on HER2 determination in breast cancer have been updated in 2018 by the American Society of Clinical Oncology and College of American Pathologists, on the basis of a twenty-year practice and results of numerous clinical trials. Moreover, the emerging HER2-low concept for 1+ and 2+ non amplified breast cancers lead to refine French practices for HER2 status assessment. The GEFPICS group, composed of expert pathologists, herein presents the latest French recommendations for HER2 status evaluation in breast cancer, taking into account the ASCO/CAP guidelines and introducing the HER2-low concept. In the era of personalized medicine, HER2 status assessment remains one of the most important biomarkers in breast cancer and its quality guaranties the optimal patients' care. French pathologists' commitment in theranostic biomarker quality is more than ever required to provide the most efficient cares in oncology. (C) 2021 Elsevier Masson SAS. All rights reserved.

INTRODUCTION:Therapeutic management of oropharyngeal squamous cell carcinomas (OPSCC) is still debated. Since the role of HPV was demonstrated, few studies have focused on HPV-negative OPSCC. The aim of our study was to assess the impact of therapeutic strategy (surgical vs. non-surgical) on oncologic outcomes in patients with HPV-negative OPSCC. MATERIAL AND METHOD:All p16-negative OPSCCs treated from 2009 to 2014 in 7 tertiary-care centers were included in this retrospective study and were classified according to the therapeutic strategy: surgical strategy (surgery ± adjuvant radiotherapy and chemotherapy) vs. non-surgical strategy (definitive radiotherapy ± chemotherapy). Patients not eligible for surgery (unresectable tumor, poor general-health status) were excluded. Univariate, multivariate and propensity score matching analyses were performed to compare overall (OS), disease-specific (DSS) and recurrence-free survival (RFS). RESULTS:Four hundred seventy-four (474) patients were included in the study (surgical group: 196; non-surgical group: 278). Five-year OS, DSS and RFS were 76.5, 81.3 and 61.3%, respectively, in the surgical group and 49.9, 61.8 and 43.4%, respectively, in the non-surgical group. The favorable impact of primary surgical treatment on oncologic outcomes was statistically significant after multivariate analysis. This effect was more marked for locally-advanced than for early-stage tumors. Propensity score matching analysis confirmed the prognostic impact of primary surgical treatment for RFS. CONCLUSION:Therapeutic strategy is an independent prognostic factor in patients with p16-negative OPSCC and primary surgical treatment is associated with improved OS, DSS and RFS. These results suggest that surgical strategy is a reliable option for advanced stage OPSCC.

INTRODUCTION:Although Human Papilloma Virus (HPV)-driven oropharyngeal cancer (OPC) prognosis is significantly better than that of other head and neck cancers, up to 25% of cases will recur within 5 years. Data on the pattern of disease recurrence and efficiency of salvage treatment are still sparse. MATERIAL AND METHOD:Observational study of all recurrent OPCs diagnosed, following a curative intent treatment, in seven French centers from 2009 to 2014. p16 Immunohistochemistry was used to determine HPV status. Clinical characteristics, distribution of recurrence site, and treatment modalities were compared by HPV tumor status. Overall survival was examined using Kaplan-Meier and multivariate Cox regression modeling. RESULTS:350 recurrent OPC patients (246 p16-negative and 104 p16-positive patients). The site of recurrence was more frequently locoregional for p16-negative patients (65.4% versus 52.9% in p16-positive patients) and metastatic for p16-positive patients (47.1% versus 34.6% in p16-patients, p = 0.03). Time from diagnosis to recurrence did not differ between p16-positive and p16-negative patients (12 and 9.6 months, respectively, p-value = 0.2), as the main site of distant metastasis (all p-values ≥0.10). Overall and relapse-free survival following the first recurrence did not differ according to p16 status (p-values from log-rank 0.30 and 0.40, respectively). In multivariate analysis, prognosis factors for overall survival in p16-negative patients were distant metastasis (HR 2.11, 95% CI 1.30-3.43) and concurrent local and regional recurrences (HR 2.20, 95% CI 1.24-3.88). CONCLUSION:With the exception of the initial site of recurrence, the pattern of disease relapse and the efficiency of salvage treatment are not different between p16-positive and negative OPCs.

Abstract 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.

Background: The current pathological tumour-node-metastasis (pTNM) classification for upper tract urothelial carcinoma (UTUC) does not include any risk stratification of pT3 renal pelvicalyceal tumours. Objective: To assess the prognostic impact of pT3 subclassification in a multicentre cohort of patients with UTUC of the renal pelvicalyceal system undergoing radical nephroureterectomy (RNU). Design, setting, and participants: Data from all consecutive patients treated with RNU for pT3 renal pelvicalyceal UTUC at 14 French centres from 1995 to 2013 were reviewed retrospectively. Intervention: A central pathology review (CPR) was used to stratify pT3 patients into those with infiltration of the renal parenchyma on a microscopic level (pT3a) versus those with infiltration of the renal parenchyma visible on gross inspection of the resection specimen and/or invasion of peripelvic fat (pT3b). Outcome measurements and statistical analysis: Inverse probability weighting (IPW)-adjusted Cox regression analyses were used to compare recurrence-free survival (RFS) and cancer-specific survival (CSS) between pT3a and pT3b patients. Results and limitations: Overall, 202 patients were included and further stratified into pT3a (n = 98; 48.5%) and pT3b (n = 104; 51.5%) subgroups. Median time to follow-up in the weighted population was 68 (interquartile range, 50-95) mo. In IPW-adjusted Cox regression analyses, pT3b versus pT3a substage was associated with a significant adverse effect on RFS (hazard ratio [HR] = 2.02; 95% confidence interval [CI] = [1.36-3.01]; p < 0.001) and CSS (HR = 1.84; 95% CI = [1.20-2.82]; p = 0.005). The study is limited by its retrospective design. Conclusions: Using IPW-adjusted analyses after the CPR, we observed that RNU patients with pT3b renal pelvicalyceal UTUC had adverse prognosis as compared with those with pT3a disease. As such, this subclassification could help refine the current pTNM system for UTUC. Patient summary: In this report, we looked at the prognostic interest of stratifying patients with pT3 renal pelvicalyceal upper tract urothelial carcinoma based on the extent of local invasion. We found that those with extensive infiltration (pT3b) had adverse prognosis as compared with those with limited infiltration (pT3a). This information could be provided on pathology reports to further guide clinical decision making. (c) 2020 European Association of Urology. Published by Elsevier B.V. All rights reserved.

INTRODUCTION:Patients with oropharyngeal squamous cell carcinoma (OPSCC) display a significant risk of synchronous primary neoplasia (SPN) which could impact their management. The aims of this study were to evaluate the risk and distribution of SPN in OPSCC patients according to their HPV (p16) status, the predictive factors of SPN and the impact of SPN on therapeutic strategy and oncologic outcomes. MATERIAL AND METHODS:All OPSCC patients treated from 2009 to 2014 were included in this multicentric retrospective study. Univariate analyses were conducted using Chi-2 and Fisher exact tests. For multivariate analyses, all variables associated with a p ≤ 0.10 in univariate analysis were included in logistic regression models. RESULTS:Among the 1291 patients included in this study, 75 (5.8%) displayed a SPN which was preferentially located in the upper aerodigestive tract, lung and esophagus. Comorbidity level (p = 0.03), alcohol (p = 0.005) and tobacco (p = 0.01) consumptions, and p16 tumor status (p < 0.0001) were significant predictors of SPN. In multivariate analysis, p16+ status was significantly associated with a lower risk of SPN (OR = 0.251, IC95% [0.133;0.474]). Patients with a SPN were more frequently referred for non-curative treatment (p = 0.02). In patients treated with curative intent, there was no impact of SPN on the therapeutic strategy (surgical vs. non-surgical treatment). We observed no overall survival differences between patients with or without SPN. CONCLUSION:P16 tumor status is the main predictive factor of SPN in OPSCC patients. This study provides crucial results which should help adapt the initial work-up and the global management of OPSCC patients.

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.