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Correlation of mRNA for NK cell markers with prognosis in prostate cancer (public datasets).

The field of immunotherapy for solid tumors, such as prostate cancer, has been recently focusing on therapies that can counter tumor-mediated immunosuppression. Precise quantification and characterization of the immune infiltrates in tumors is crucial to improve treatment efficacy. Natural killer (NK) cells, major components of the antitumor immune system, have never been isolated from prostate tumors, despite their suspected role in disease progression. Here, we examined the frequency, phenotype, and functions of NK cells infiltrating control and tumor prostate tissues. NK cell infiltrates in prostate tissues were mainly CD56 (NCAM1)-positive and displayed an unexpected immature, but activated, phenotype with low or no cytotoxic potential. Furthermore, we show that TGFβ1 (TGFB1) is highly secreted into the prostate environment and partly mediates the immunosuppressive effects on NK cells. In addition to this basal level of immunotolerance to NK cells, the prostate environment became further resistant to NK cell–mediated immunity upon cancer cell infiltration. Coculture experiments revealed that prostate cancer cells induced the expression of inhibitory receptor (ILT2/LILRB1) and downregulated the expression of activating receptors NKp46 (NCR1), NKG2D (KLRK1), and CD16 (FCGR3) by NK cells, thus preventing their recognition of tumor cells. Notably, blood levels of NKp46 were also decreased in prostate cancer patients and were inversely correlated with levels of prostate-specific antigen, the main prognostic factor in prostate cancer. Our study shows that a strong immunosuppressive environment impairs NK cell function at multiple levels in prostate cancer and provides a rationale for the design of therapies that restore NK cell efficiency in the prostate tumor microenvironment. Cancer Res; 76(8); 2153–65. ©2016 AACR.

List of the antibodies (Name, Clone, Provider) and isotype controls used for NK cell phenotype and functional experiments.

Legend for Supplementary Tables S1-S2 and Supplementary Figures S1-S4.

Simple Summary Vδ2+ γδ T cells have potent antitumor properties both in vitro and in murine preclinical models of breast cancers. However, in the context of human breast cancer, there is a lack of information for potential phenotypic alterations of this crucial immune cell subset. This is partly due to Vδ2+ γδ T cells scarcity in surgical specimens. To break this deadlock, we assessed Vδ2+ γδ T cell phenotypes using untreated breast cancer patients’ peripheral blood, so-called minimally invasive “liquid biopsy”. We show that circulating Vδ2+ γδ T cell phenotypic alterations are already established at diagnosis and related to tumor progression. Notably, terminally differentiated Vδ2+ γδ T cells expressing canonical markers of replicative senescence and exhaustion were significantly associated with tumor-draining lymph node invasion. Our results shed light on the interest of using liquid biopsy to monitor rare events and support Vδ2+ γδ T cell involvement in breast cancer pathogenesis and progression. Abstract The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αβ T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αβ T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients’ peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients’ peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.

Resume Les lesions mammaires a risque ou lesions frontieres representent un panel de lesions heterogenes dont la prise en charge n’est pas a l’heure actuelle standardisee. Les difficultes d’interpretation histopathologique de ces entites lesionnelles, le manque d’homogeneite des resultats de la litterature ainsi que les changements recents de la classification de l’Organisation mondiale de la sante contribuent a favoriser la prise en charge de ces lesions au cas par cas. Ainsi, la confrontation multidisciplinaire et particulierement radioanatomopathologique prend toute sa valeur afin de ne pas surtraiter les patientes ou au contraire de ne pas sous-estimer le potentiel d’agressivite des lesions mammaires.

Background Genomic-based approaches in advanced breast cancer (ABC) were recently demonstrated as feasible in the clinical practice, but only a limited number of patients were actually treated with targeted therapies matching genomic alterations, with low antitumor activity. We conducted a pilot study to evaluate whether precision medicine using NGS and aCGH could be implemented prospectively at a single center in ABC patients. In addition, we examined whether PDX could be derived from ABC and thus could help inform therapeutic decision. Methods ABC patients accessible to tumor biopsy were prospectively enrolled at the Institut Paoli-Calmettes in the BC-BIO study (ClinicalTrials.gov, NCT01521676). Tumor tissue from locally recurrent or metastatic disease was immediately frozen after dedicated biopsy. Genomic profiling included high-resolution 4x180K aCGH (Agilent Technologies, Massy, France) and DNA sequencing, using a library of 365 cancer candidate genes (HaloPlex target enrichment kit, Agilent technologies, Santa Clara, CA, USA) and MiSeq analyzer (Illumina, San Diego, CA, USA) with 2x150-bp, paired-end at about 300x coverage. In a subset of patients, fresh tumor was implanted orthotopically in humanized cleared fat pads of NSG mice for establishing xenotransplants. Results A total of 34 ABC patients were included, with the following characteristics: median age 54 years (35-77); molecular subtypes: 11 triple-negative (32%), 12 luminal non-HER2 (35%), 4 luminal HER2 (12%), 3 HER2 non-luminal (9%), and 4 unknown (12%); 33 with previous chemotherapy (97%); 22 with previous endocrine treatment (35%); 7 with previous anti-HER2 (21%). Tumor biopsies were obtained from liver (15), skin (6), peritoneum (4), breast (3), node (3), lung (1), pleura (1), and ascitis (1), with a median tumor cellularity of 70% (range 10-90%). aCGH and NGS were available from 34 and 33 patients, respectively. An actionable target was found in 28 patients (82%), corresponding to 66 targets, including 37 mutations (8 in PIK3CA, 7 TP53, 4 ESR1, 2 AKT1, 2 BRCA2, 2 HER2), 22 amplifications (7 for CCND1, 2 CCNE1, 2 FGFR1, 2 IGF1R) and 7 homozygous deletions (3 for PTEN, 2 CDKN2A/B,1 BRCA2, 1 STK11). A targeted therapeutic proposal was possible, either in a clinical trial (N=18, 52%) or using already registered drugs (N=17, 50%). Ten patients actually received a targeted treatment, 1 of them experienced objective response and 1 showed stable disease for more than 6 months. Of 26 patients subjected to mouse implantation, 10 had successful xenografting (6 triple-negative, 2 HER2, 1 luminal non-HER2, 1 subtype non-attributed), with a median time to reach 10 mm of 148 days. These PDX will be used as models to understand the patient's therapeutic response. Conclusion Precision medicine using high-throughput DNA sequencing and aCGH can be implemented at a single center in the context of clinical practice and may allow direct therapeutic proposal in 1/3 of patients, but antitumor activity was minimal. PDX may be obtained in a significant fraction of patients, especially in triple-negative and HER2 subtypes, and could phenotypically complement genomic data. Citation Format: Gonçalves A, Bertucci F, Chaffanet M, Guille A, Garnier S, Adelaide J, Carbuccia N, Brunelle S, Piana G, Cabaud O, Thomassin-Piana J, Paciencia-Gros M, Chereau-Ewald E, Lambaudie E, Sabatier R, Tarpin C, Provansal M, Jalaguier-Coudray A, Extra J-M, Sarran A, Pakradouni J, Viens P, Lopez M, Ginestier C, Charafe-Jauffret E, Birnbaum D. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH) and patient-derived tumor xenograft (PDX) for precision medicine in advanced breast cancer: A single-center prospective study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-23.

Resume Le depistage systematique du cancer du sein a engendre une augmentation significative des prelevements biopsiques mammaires. La validation d’un resultat histologique est une etape majeure dans la prise en charge des patientes ; la correlation des images radiologiques et histologiques est essentielle a une prise en charge adequate. De plus, il s’agit d’une obligation de la Societe francaise de radiologie (SFR) 2013 1 . Lors de la reception du resultat, le radiologue doit s’assurer qu’il est concordant avec l’anomalie senologique biopsiee. Il n’est pas toujours possible, pour des raisons d’organisation, de partager ces resultats lors d’une reunion pluridisciplinaire ; il est donc important d’utiliser des outils permettant de « parler le meme langage » entre les acteurs des differentes specialites ; la classification europeenne des biopsies mammaires repond parfaitement a cette necessite.

The field of immunotherapy for solid tumors, such as prostate cancer, has been recently focusing on therapies that can counter tumor-mediated immunosuppression. Precise quantification and characterization of the immune infiltrates in tumors is crucial to improve treatment efficacy. Natural killer (NK) cells, major components of the antitumor immune system, have never been isolated from prostate tumors, despite their suspected role in disease progression. Here, we examined the frequency, phenotype, and functions of NK cells infiltrating control and tumor prostate tissues. NK cell infiltrates in prostate tissues were mainly CD56 (NCAM1)-positive and displayed an unexpected immature, but activated, phenotype with low or no cytotoxic potential. Furthermore, we show that TGFβ1 (TGFB1) is highly secreted into the prostate environment and partly mediates the immunosuppressive effects on NK cells. In addition to this basal level of immunotolerance to NK cells, the prostate environment became further resistant to NK cell–mediated immunity upon cancer cell infiltration. Coculture experiments revealed that prostate cancer cells induced the expression of inhibitory receptor (ILT2/LILRB1) and downregulated the expression of activating receptors NKp46 (NCR1), NKG2D (KLRK1), and CD16 (FCGR3) by NK cells, thus preventing their recognition of tumor cells. Notably, blood levels of NKp46 were also decreased in prostate cancer patients and were inversely correlated with levels of prostate-specific antigen, the main prognostic factor in prostate cancer. Our study shows that a strong immunosuppressive environment impairs NK cell function at multiple levels in prostate cancer and provides a rationale for the design of therapies that restore NK cell efficiency in the prostate tumor microenvironment. Cancer Res; 76(8); 2153–65. ©2016 AACR.