A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

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.

Anti-cathepsin D immunotherapy triggers both innate and adaptive anti-tumour immunity in breast cancer.

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.

SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. 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. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.

Diagnostic Accuracy of Digital Staining ex vivo Confocal Microscopy for Diagnosing and Subtyping Basal Cell Carcinoma in Fresh Pretherapeutic Punch Biopsies: A Monocentric Prospective Study.

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.

Metachronous second primary neoplasia in oropharyngeal cancer patients: Impact of tumor HPV status. A GETTEC multicentric study.

INTRODUCTION: Patients with oropharyngeal squamous cell carcinoma (OPSCC) display a significant risk to develop a metachronous second primary neoplasia (MSPN). HPV and non-HPV-related OPSCC are 2 distinct entities with biological, clinical and prognostic differences. The aims of our study were to analyze the impact of tumor HPV status and other relevant clinical factors, such as tobacco and/or alcohol (T/A) consumption, on the risk and distribution of MSPN in OPSCC patients and to assess the impact of MSPN on patient survival. MATERIAL AND METHODS: All OPSCC patients treated from 2009 to 2014 were included in this multicentric retrospective study. P16 immunohistochemical expression was used as a surrogate maker of tumor HPV status. The impact of tumor p16 status on the risk of MSPN was assessed in uni- and multivariate analyses. Overall survival (OS) was determined by Kaplan-Meier analysis. RESULTS: Among the 1291 patients included in this study, 138 (10.7%) displayed a MSPN which was preferentially located in the head and neck area (H&N), lung and esophagus. Multivariate analyses showed that p16- tumor status (p = 0.003), T/A consumption (p = 0.005) and soft palate tumor site (p = 0.009) were significantly associated with a higher risk of MSPN. We found no impact of p16 tumor status on the median time between index OPSCC diagnosis and MSPN development, but a higher proportion of MSPN arising outside the H&N, lung and esophagus was found in p16 + than in p16- patients. MSPN development had an unfavorable impact (p = 0.04) on OS only in the p16 + patient group. CONCLUSION: P16 tumor status and T/A consumption were the main predictive factors of MSPN in OPSCC patients. This study provides crucial results with a view to tailoring global management and follow-up of OPSCC patients.

[2021 update of the GEFPICS' recommendations for HER2 status assessment in invasive breast cancer in France]. / Mise à jour 2021 des recommandations du GEFPICS pour l'évaluation du statut HER2 dans les cancers infiltrants du sein en France.

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.

A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment.

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.

Prognostic Impact of pT3 Subclassification in a Multicentre Cohort of Patients with Urothelial Carcinoma of the Renal Pelvicalyceal System Undergoing Radical Nephroureterectomy: A Propensity Score-weighted Analysis After Central Pathology Review.

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.

Upfront surgery or definitive radiotherapy for p16+ oropharyngeal cancer. A GETTEC multicentric study.

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), disease-specific (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. CONCLUSION: In p16+ OPSCC patients, upfront surgery results in higher RFS than definitive radiotherapy ± chemotherapy but does not impact OS.

Oropharyngeal cancer: First relapse description and prognostic factor of salvage treatment according to p16 status, a GETTEC multicentric study.

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.

Collagenase-Induced Patellar Tendinopathy with Neovascularization: First Results towards a Piglet Model of Musculoskeletal Embolization.

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.

Upfront surgery or definitive radiotherapy for patients with p16-negative oropharyngeal squamous cell carcinoma. A GETTEC multicentric study.

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.

Synchronous primary neoplasia in patients with oropharyngeal cancer: Impact of tumor HPV status. A GETTEC multicentric study.

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.

Co-Expression of Androgen Receptor and Cathepsin D Defines a Triple-Negative Breast Cancer Subgroup with Poorer Overall Survival.

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.

Characterisation of tumour microenvironment and immune checkpoints in primary central nervous system diffuse large B cell lymphomas.

Primary central nervous system diffuse large B cell lymphoma (PCNS-DLBCL) is a rare and aggressive entity of diffuse large B cell lymphoma (DLBCL). Elements of the tumour microenvironment (TME) including tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs) have been associated with survival in DLBCL but their composition and prognostic impact in PCNS-DLBCL are unknown. Programmed cell death-1 (PD1)/programmed death-ligand 1 (PD-L1) immune checkpoint may represent a therapeutic option. Here, we aimed to characterise PD1/PDL1 immune checkpoints and the composition of the TME in PCNS-DLBCL. We collected tumour tissue and clinical data from 57 PCNS-DLBCL and used immunohistochemistry to examine TAMs (CD68, CD163), TILs (CD3, CD4, CD8, PD1) and tumour B cells (PAX5/PDL1 double stains, PDL1). The PDL1 gene was evaluated by fluorescence in situ hybridization (FISH). PAX5/PDL1 identified PDL1 expression by tumour B cells in 10/57 cases (17.5%). PDL1 gene translocation was a recurrent cytogenetic alteration in PNCS-DLBCL (8/47.17%) and was correlated with PDL1 positive expression in tumour B cells. The TME consisted predominantly of CD163 (+) M2 TAMs and CD8 (+) TILs. Most TAMs expressed PDL1 and most TILs expressed PD1. The density of TAMs and TILs did not associate with outcome. We showed that expression of PD1 on TILs and PDL1 on TAMs, but not the expression of PDL1 on tumour B cells was correlated with better prognosis. These findings support a significant role of TME composition and PD1/PDL1 crosstalk in PCNS-DLBCL pathogenesis and bring new insights to the targeted therapy of this aggressive lymphoma.

[GEFPICS' guidelines for management of breast cancer tissue samples in the neoadjuvant setting]. / Recommandations du GEFPICS pour la prise en charge des prélèvements dans le cadre du traitement néoadjuvant du cancer du sein.

Neoadjuvant therapy is an increasing treatment option in the management of breast cancer. The tumor response to neoadjuvant therapy, especially the pathological complete response, is a validated endpoint frequently used in clinical trials. However, there is still a lack of standardization for the surgical specimen management in the neoadjuvant setting. This leads to heterogeneity in the specimen handling and might lead to significant bias for the prognostic assessment of patients or in clinical trials. The GEFPICS group, composed of expert breast cancer pathologists, herein presents guidelines for the management of breast and axillary specimen before treatment (management of biopsy, items of the pathological report) and after neoadjuvant therapy (specimen handling, histological assessment of response, items of the pathological report and response grading systems).

Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies.

BACKGROUND: Triple-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. METHODS: Cath-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). RESULTS: High 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 TGFß decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy. CONCLUSION: Cath-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.

Phenotypes of Atopic Dermatitis Depending on the Timing of Onset and Progression in Childhood.

Importance: Atopic dermatitis is an inflammatory, pruritic skin disease that often occurs in early infancy with a chronic course. However, a specific description of subtypes of atopic dermatitis depending on the timing of onset and progression of the disease in childhood is lacking. Objective: To identify different phenotypes of atopic dermatitis using a definition based on symptoms before age 6 years and to determine whether some subtypes are more at risk for developing other allergic diseases. Design, Setting, and Participants: The Protection Against Allergy Study in Rural Environments (PASTURE) is a European birth cohort where pregnant women were recruited between August 2002 and March 2005 and divided in 2 groups dependent on whether they lived on a farm. Children from this cohort with data on atopic dermatitis from birth to 6 years of age were included. Exposures: Atopic dermatitis, defined as an itchy rash on typical locations from birth to 6 years. Main Outcomes and Measures: The latent class analysis was used to identify subtypes of atopic dermatitis in childhood based on the course of symptoms. Multivariable logistic regressions were used to analyze the association between atopic dermatitis phenotypes and other allergic diseases. Results: We included 1038 children; of these, 506 were girls. The latent class analysis model with the best fit to PASTURE data separated 4 phenotypes of atopic dermatitis in childhood: 2 early phenotypes with onset before age 2 years (early transient [n = 96; 9.2%] and early persistent [n = 67; 6.5%]), the late phenotype with onset at age 2 years or older (n = 50; 4.8%), and the never/infrequent phenotype (n = 825; 79.5%), defined as children with no atopic dermatitis. Children with both parents with history of allergies were 5 times more at risk to develop atopic dermatitis with an early-persistent phenotype compared with children with parents with no history of allergies. Both early phenotypes were strongly associated with food allergy. The risk of developing asthma was significantly increased among the early-persistent phenotype (adjusted odds ratio, 2.87; 95% CI, 1.31-6.31). The late phenotype was only positively associated with allergic rhinitis. Conclusions and Relevance: Using latent class analysis, 4 phenotypes of atopic dermatitis were identified depending on the onset and course of the disease. The prevalence of asthma and food allergy by 6 years of age was strongly increased among children with early phenotypes (within age 2 years), especially with persistent symptoms. These findings are important for the development of strategies in allergy prevention.

Antifibrotic, Antioxidant, and Immunomodulatory Effects of Mesenchymal Stem Cells in HOCl-Induced Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a rare intractable disease with unmet medical need and fibrosis-related mortality. Absence of efficient treatments has prompted the development of novel therapeutic strategies, among which mesenchymal stem cells/stromal cells (MSCs) or progenitor stromal cells appear to be one of the most attractive options. The purpose of this study was to use the murine model of hypochlorite-induced SSc to investigate the systemic effects of MSCs on the main features of the diffuse form of the disease: skin and lung fibrosis, autoimmunity, and oxidative status. METHODS: We compared the effects of different doses of MSCs (2.5 × 10(5) , 5 × 10(5) , and 10(6) ) infused at different time points. Skin thickness was assessed during the experiment. At the time of euthanasia, biologic parameters were quantified in blood and tissues (by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, assessment of collagen content). Assessments of histology and immunostaining were also performed. RESULTS: A lower expression of markers of fibrosis (Col1, Col3, Tgfb1, and aSma) was observed in both skin and lung following MSC infusion, which was consistent with histologic improvement and was inversely proportional to the injected dose. Importantly, sera from treated mice exhibited lower levels of anti-Scl-70 autoantibodies and enhanced antioxidant capacity, confirming the systemic effect of MSCs. Of interest, MSC administration was efficient in both the preventive and the curative approach. We further provide evidence that MSCs exerted an antifibrotic role by normalizing extracellular matrix remodeling parameters as well as reducing proinflammatory cytokine levels and increasing antioxidant defenses. CONCLUSION: The results of this study demonstrate the beneficial and systemic effects of MSC administration in the HOCl murine model of diffuse SSc, which is a promising finding from a clinical perspective.