Regulatory T cells infiltrate the tumor-induced tertiary lymphoïd structures and are associated with poor clinical outcome in NSCLC.

On one hand, regulatory T cells (Tregs) play an immunosuppressive activity in most solid tumors but not all. On the other hand, the organization of tumor-infiltrating immune cells into tertiary lymphoid structures (TLS) is associated with long-term survival in most cancers. Here, we investigated the role of Tregs in the context of Non-Small Cell Lung Cancer (NSCLC)-associated TLS. We observed that Tregs show a similar immune profile in TLS and non-TLS areas. Autologous tumor-infiltrating Tregs inhibit the proliferation and cytokine secretion of CD4+ conventional T cells, a capacity which is recovered by antibodies against Cytotoxic T-Lymphocyte-Associated protein-4 (CTLA-4) and Glucocorticoid-Induced TNFR-Related protein (GITR) but not against other immune checkpoint (ICP) molecules. Tregs in the whole tumor, including in TLS, are associated with a poor outcome of NSCLC patients, and combination with TLS-dendritic cells (DCs) and CD8+ T cells allows higher overall survival discrimination. Thus, Targeting Tregs especially in TLS may represent a major challenge in order to boost anti-tumor immune responses initiated in TLS.

Intracellular Factor H Drives Tumor Progression Independently of the Complement Cascade.

The complement system is a powerful and druggable innate immune component of the tumor microenvironment. Nevertheless, it is challenging to elucidate the exact mechanisms by which complement affects tumor growth. In this study, we examined the processes by which the master complement regulator factor H (FH) affects clear cell renal cell carcinoma (ccRCC) and lung cancer, two cancers in which complement overactivation predicts poor prognosis. FH was present in two distinct cellular compartments: the membranous (mb-FH) and intracellular (int-FH) compartments. Int-FH resided in lysosomes and colocalized with C3. In ccRCC and lung adenocarcinoma, FH exerted protumoral action through an intracellular, noncanonical mechanism. FH silencing in ccRCC cell lines resulted in decreased proliferation, due to cell-cycle arrest and increased mortality, and this was associated with increased p53 phosphorylation and NFκB translocation to the nucleus. Moreover, the migration of the FH-silenced cells was reduced, likely due to altered morphology. These effects were cell type-specific because no modifications occurred upon CFH silencing in other FH-expressing cells tested: tubular cells (from which ccRCC originates), endothelial cells (human umbilical vein endothelial cells), and squamous cell lung cancer cells. Consistent with this, in ccRCC and lung adenocarcinoma, but not in lung squamous cell carcinoma, int-FH conferred poor prognosis in patient cohorts. Mb-FH performed its canonical function of complement regulation but had no impact on tumor cell phenotype or patient survival. The discovery of intracellular functions for FH redefines the role of the protein in tumor progression and its use as a prognostic biomarker or potential therapeutic target.See article by Daugan et al., p. 891 (36).

Complement C1s and C4d as Prognostic Biomarkers in Renal Cancer: Emergence of Noncanonical Functions of C1s.

The complement system plays a complex role in cancer. In clear cell renal cell carcinoma (ccRCC), local production of complement proteins drives tumor progression, but the mechanisms by which they do this are poorly understood. We found that complement activation, as reflected by high plasma C4d or as C4d deposits at the tumor site, was associated with poor prognosis in two cohorts of patients with ccRCC. High expression of the C4-activating enzyme C1s by tumor cells was associated with poor prognosis in three cohorts. Multivariate Cox analysis revealed that the prognostic value of C1s was independent from complement deposits, suggesting the possibility of complement cascade-unrelated, protumoral functions for C1s. Silencing of C1s in cancer cell lines resulted in decreased proliferation and viability of the cells and in increased activation of T cells in in vitro cocultures. Tumors expressing high levels of C1s showed high infiltration of macrophages and T cells. Modification of the tumor cell phenotype and T-cell activation were independent of extracellular C1s levels, suggesting that C1s was acting in an intracellular, noncanonical manner. In conclusion, our data point to C1s playing a dual role in promoting ccRCC progression by triggering complement activation and by modulating the tumor cell phenotype and tumor microenvironment in a complement cascade-independent, noncanonical manner. Overexpression of C1s by tumor cells could be a new escape mechanism to promote tumor progression.See related Spotlight by Magrini and Garlanda, p. 855. See article by Daugan et al., p. 909 (40).

Tertiary Lymphoid Structure-B Cells Narrow Regulatory T Cells Impact in Lung Cancer Patients.

The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4+ T cell clonality. Here, we investigated the relationship between the presence of TLS-B cells and CD4+ T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4+ T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4+ T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the TLS-Bhigh tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-Bhigh Treglow patients had the best clinical outcomes. Overall, the correlation between the density of TLS-Bhigh tumors with early differentiated, activated and non-regulatory CD4+ T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.

CD103+CD8+ TRM Cells Accumulate in Tumors of Anti-PD-1-Responder Lung Cancer Patients and Are Tumor-Reactive Lymphocytes Enriched with Tc17.

Accumulation of CD103+CD8+ resident memory T (TRM) cells in human lung tumors has been associated with a favorable prognosis. However, the contribution of TRM to anti-tumor immunity and to the response to immune checkpoint blockade has not been clearly established. Using quantitative multiplex immunofluorescence on cohorts of non-small cell lung cancer patients treated with anti-PD-(L)1, we show that an increased density of CD103+CD8+ lymphocytes in immunotherapy-naive tumors is associated with greatly improved outcomes. The density of CD103+CD8+ cells increases during immunotherapy in most responder, but not in non-responder, patients. CD103+CD8+ cells co-express CD49a and CD69 and display a molecular profile characterized by the expression of PD-1 and CD39. CD103+CD8+ tumor TRM, but not CD103-CD8+ tumor-infiltrating counterparts, express Aiolos, phosphorylated STAT-3, and IL-17; demonstrate enhanced proliferation and cytotoxicity toward autologous cancer cells; and frequently display oligoclonal expansion of TCR-ß clonotypes. These results explain why CD103+CD8+ TRM are associated with better outcomes in anti-PD-(L)1-treated patients.

Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions.

BACKGROUND: Natural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor immune response. Emerging data reveal NK cell dysfunction within the tumor microenvironment (TME) through checkpoint inhibitory molecules associated with a regulatory phenotype. OBJECTIVE: We aimed at analyzing the gene expression profile of intratumoral NK cells compared with non-tumorous NK cells, and to characterize their inhibitory function in the TME. METHODS: NK cells were sorted from human lung tumor tissue and compared with non- tumoral distant lungs. RESULTS: In the current study, we identify a unique gene signature of NK cell dysfunction in human non-small cell lung carcinoma (NSCLC). First, transcriptomic analysis reveals significant changes related to migratory pattern with a downregulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CX3C chemokine receptor 1 (CX3CR1) and overexpression of C-X-C chemokine receptor type 5 (CXCR5) and C-X-C chemokine receptor type 6 (CXCR6). Second, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and killer cell lectin like receptor (KLRC1) inhibitory molecules were increased in intratumoral NK cells, and CTLA-4 blockade could partially restore MHC class II level on dendritic cell (DC) that was impaired during the DCs/NK cell cross talk. Finally, NK cell density impacts the positive prognostic value of CD8+ T cells in NSCLC. CONCLUSIONS: These findings demonstrate novel molecular cues associated with NK cell inhibitory functions in NSCLC.

Risk factors associated with myasthenia gravis in thymoma patients: The potential role of thymic germinal centers.

Thymomas are associated with a very high risk of developing Myasthenia Gravis (MG). Our objectives were to identify histological and biological parameters to allow early diagnosis of thymoma patients susceptible to developing MG. We conducted a detailed retrospective analysis from a patient database, searching for differences between patients with thymoma-associated MG (MGT, n = 409) and thymoma without MG (TOMA, n = 111) in comparison with nonthymomatous MG patients (MG, n = 1246). We also performed multiplex and single molecule arrays to measure the serum levels of cytokines in these groups of patients and controls (n = 14-22). We identified a set of parameters associated with MG development in thymoma patients: 1) detection of anti-acetylcholine receptor (AChR) antibodies, 2) development of B1 or B2 thymoma subtypes, 3) presence of ectopic thymic germinal centers (GCs), 4) local invasiveness of thymoma, and 5) being a woman under 50 years old. Among these parameters, 58.8% of MGT patients displayed GCs with a positive correlation between the number of GCs and anti-AChR titers. By immunohistochemistry, we found thymic GCs in the adjacent tissues of thymomas encircled by high endothelial venules (HEVs) that could favor peripheral cell recruitment. We also clearly associated MG symptoms with higher IFN-γ, IL-1ß and sCD40L serum levels, specifically in MGT patients compared to TOMA patients. Altogether, these analyses allowed the clear identification of histological, in particular the presence of GCs, and biological parameters that would facilitate the evaluation of the probability of the MG outcome postoperatively in thymoma patients.

High IGF1R protein expression correlates with disease-free survival of patients with stage III colon cancer.

PURPOSE: The aim of this study was to investigate the association between expression of insulin-like growth factor-1 receptor (IGF1R) and its ligand, IGF-II, and disease-free survival (DFS) in patients with stage III colon cancer (CC). METHODS: In this retrospective study we included consecutive patients who underwent curative surgery for stage III CC. IGF1R and IGF-II/IGF2 status were evaluated in tumour samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Associations of markers with DFS were analysed using Cox proportional hazards models. RESULTS: Hundred and fifty-one CC patients were included (median age, 66.6 years; female, 54.3%). Low levels of IGF1R and IGF-II protein expression were observed in 16.1% and 10.7% of the cases, respectively. No significant differences in clinicopathological characteristics between patients with tumours expressing low IGF1R or IGF-II protein levels and those with high levels were observed. A low IGF1R protein expression was found to be significantly associated with a shorter DFS (HR 3.32; 95% CI, 1.7-6.31; p = 0.0003), while no association was observed between IGF-II protein expression and DFS (HR 0.91; 95% CI, 0.28-2.96; p = 0.87). In a multivariate analysis, IGF1R protein status remained an independent prognostic factor for DFS (HR 2.73; 95% CI, 1.40-5.31; p = 0.003). Furthermore, we found that neither IGF1R nor IGF2 mRNA expression levels as measured by qRT-PCR correlated with the respective protein expression levels as assessed by immunohistochemistry. Neither of the mRNA expression levels was significantly associated with DFS. CONCLUSIONS: From our data we conclude that low IGF1R protein expression represents a poor prognostic biomarker in stage III colon cancer.

Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1.

Neuropilin-1 (Nrp-1) is a marker for murine CD4+FoxP3+ regulatory T (Treg) cells, a subset of human CD4+ Treg cells, and a population of CD8+ T cells infiltrating certain solid tumours. However, whether Nrp-1 regulates tumour-specific CD8 T-cell responses is still unclear. Here we show that Nrp-1 defines a subset of CD8+ T cells displaying PD-1hi status and infiltrating human lung cancer. Interaction of Nrp-1 with its ligand semaphorin-3A inhibits migration and tumour-specific lytic function of cytotoxic T lymphocytes. In vivo, Nrp-1+PD-1hi CD8+ tumour-infiltrating lymphocytes (TIL) in B16F10 melanoma are enriched for tumour-reactive T cells exhibiting an exhausted state, expressing Tim-3, LAG-3 and CTLA-4 inhibitory receptors. Anti-Nrp-1 neutralising antibodies enhance the migration and cytotoxicity of Nrp-1+PD-1hi CD8+ TIL ex vivo, while in vivo immunotherapeutic blockade of Nrp-1 synergises with anti-PD-1 to enhance CD8+ T-cell proliferation, cytotoxicity and tumour control. Thus, Nrp-1 could be a target for developing combined immunotherapies.

Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth.

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

Aggressiveness of Localized Prostate Cancer: the Key Value of Testosterone Deficiency Evaluated by Both Total and Bioavailable Testosterone: AndroCan Study Results.

Failure rates after first-line treatment of localized prostate cancer (PCa) treatment remain high. Improvements to patient selection and identification of at-risk patients are central to reducing mortality. We aimed to determine if cancer aggressiveness correlates with androgen levels in patients undergoing radical prostatectomy for localized PCa. We performed a prospective, multicenter cohort study between June 2013 and June 2016, involving men with localized PCa scheduled to undergo radical prostatectomy. Clinical and hormonal patient data (testosterone deficiency, defined by total testosterone (TT) levels < 300 ng/dL and/or bioavailable testosterone (BT) levels < 80 ng/dL) were prospectively collected, along with pathological assessment of preoperative biopsy and subsequent radical prostatectomy specimens, using predominant Gleason pattern (prdGP) 3/4 grading. Of 1343 patients analyzed, 912 (68%) had prdGP3 PCa and 431 (32%) had high-grade (prdGP4, i.e., ISUP ≥ 3) disease on prostatectomy specimens. Only moderate concordance in prdGP scores between prostate biopsies and prostatectomy specimens was found. Compared with patients with prdGP3 tumors (i.e., ISUP ≤ 2), significantly more patients with prdGP4 cancers had demonstrable hypogonadism, characterized either by BT levels (17.4% vs. 10.7%, p < 0.001) or TT levels (14.2% vs. 9.7%, p = 0.020). BT levels were also lower in patients with prdGP4 tumors compared to those with prdGP3 disease. Testosterone deficiency (defined by TT and/or BT levels) was independently associated with higher PCa aggressiveness. BT is a predictive factor for prdGP4 disease, and evaluating both TT and BT to define hypogonadism is valuable in preoperative assessment of PCa (AndroCan Trial: NCT02235142).

Intraoperative confocal laser endomicroscopy for real-time in vivo tissue characterization during surgical procedures.

INTRODUCTION: Probe-based confocal laser endomicroscopy (pCLE) is an innovative technique providing real-time, in vivo optical biopsies. A previous ex vivo phase of the study (PERSEE) allowed identifying accurate pCLE criteria for the diagnosis of hepatic and peritoneal surgical specimens. This study aimed at evaluating the pCLE role for in vivo intra-abdominal tissue characterization during digestive cancer surgical procedures. METHODS: Between October 2014 and July 2015, consecutive patients diagnosed with digestive cancers and scheduled for a surgical resection or an exploratory laparoscopy were prospectively enrolled. Endomicroscopic images were acquired using a motorized Confocal Miniprobe™ with a bending distal tip providing easy access to abdominal organs. It was connected to an endomicroscopy system that allowed near-infrared illumination (at a wavelength of 785 nm) in conjunction with indocyanine green for contrast agent. A live audiovisual transmission was established between the surgeon and the pathologist for real-time interpretation of optical biopsies. Intraoperative pCLE performance for the diagnosis of suspicious nodules was assessed using corresponding surgical histopathology as reference standard. RESULTS: 21 consecutive patients were successfully enrolled. Live audiovisual transmission between the surgeon and the pathologist was successfully established in all cases. 62 pCLE sequences were acquired from different tissues [peritoneum (n = 27), liver (n = 21), lymph node (n = 4), diaphragm (n = 3), colon (n = 3), stomach (n = 2), and adrenal gland (n = 2)]. Malignant tissues were identified by fluorescently enhanced irregular cancerous tubes contrasting with dark glandular lumen and extracellular matrix. pCLE sensitivities and specificities were 67% and 100%, and 38% and 100% for peritoneal and hepatic carcinogenesis, respectively. One benign incident was reported during the trial with no patient consequence. CONCLUSIONS: Real-time intraoperative pCLE with near-infrared illumination is feasible and safe, provides additional information in terms of tissue characterization, and, in combination with telepathology, allows interactive collaboration between the surgeon and the pathologist during surgical procedures. Trial registration clinicaltrials.gov Identifier: NCT02312167.

The clinical role of the TME in solid cancer.

The highly complex and heterogenous ecosystem of a tumour not only contains malignant cells, but also interacting cells from the host such as endothelial cells, stromal fibroblasts, and a variety of immune cells that control tumour growth and invasion. It is well established that anti-tumour immunity is a critical hurdle that must be overcome for tumours to initiate, grow and spread and that anti-tumour immunity can be modulated using current immunotherapies to achieve meaningful anti-tumour clinical responses. Pioneering studies in melanoma, ovarian and colorectal cancer have demonstrated that certain features of the tumour immune microenvironment (TME)-in particular, the degree of tumour infiltration by cytotoxic T cells-can predict a patient's clinical outcome. More recently, studies in renal cell cancer have highlighted the importance of assessing the phenotype of the infiltrating T cells to predict early relapse. Furthermore, intricate interactions with non-immune cellular players such as endothelial cells and fibroblasts modulate the clinical impact of immune cells in the TME. Here, we review the critical components of the TME in solid tumours and how they shape the immune cell contexture, and we summarise numerous studies evaluating its clinical significance from a prognostic and theranostic perspective.

Human preprocalcitonin self-antigen generates TAP-dependent and -independent epitopes triggering optimised T-cell responses toward immune-escaped tumours.

Tumours often evade CD8 T-cell immunity by downregulating TAP. T-cell epitopes associated with impaired peptide processing are immunogenic non-mutated neoantigens that emerge during tumour immune evasion. The preprocalcitonin (ppCT)16-25 neoepitope belongs to this category of antigens. Here we show that most human lung tumours display altered expression of TAP and frequently express ppCT self-antigen. We also show that ppCT includes HLA-A2-restricted epitopes that are processed by TAP-independent and -dependent pathways. Processing occurs in either the endoplasmic reticulum, by signal peptidase and signal peptide peptidase, or in the cytosol after release of a signal peptide precursor or retrotranslocation of a procalcitonin substrate by endoplasmic-reticulum-associated degradation. Remarkably, ppCT peptide-based immunotherapy induces efficient T-cell responses toward antigen processing and presenting machinery-impaired tumours transplanted into HLA-A*0201-transgenic mice and in NOD-scid-Il2rγnull mice adoptively transferred with human PBMC. Thus, ppCT-specific T lymphocytes are promising effectors for treatment of tumours that have escaped immune recognition.

TNFR2/BIRC3-TRAF1 signaling pathway as a novel NK cell immune checkpoint in cancer.

Natural Killer (NK) cells control metastatic dissemination of murine tumors and are an important prognostic factor in several human malignancies. However, tumor cells hijack many of the NK cell functional features compromising their tumoricidal activity. Here, we show a deleterious role of the TNFα/TNFR2/BIRC3/TRAF1 signaling cascade in NK cells from the tumor microenvironment (TME). TNFα induces BIRC3/cIAP2 transcripts and reduces NKp46/NCR1 transcription and surface expression on NK cells, promoting metastases dissemination in mice and poor prognosis in GIST patients. NKp30 engagement, by promoting the release of TNFα, also contributes to BIRC3 upregulation, and more so in patients expressing predominantly NKp30C isoforms. These findings reveal that in the absence of IL-12 or a Th1-geared TME, TNFα can be considered as a negative regulatory cytokine for innate effectors.

Expression of LLT1 and its receptor CD161 in lung cancer is associated with better clinical outcome.

Co-stimulatory and inhibitory receptors expressed by immune cells in the tumor microenvironment modulate the immune response and cancer progression. Their expression and regulation are still not fully characterized and a better understanding of these mechanisms is needed to improve current immunotherapies. Our previous work has identified a novel ligand/receptor pair, LLT1/CD161, that modulates immune responses. Here, we extensively characterize its expression in non-small cell lung cancer (NSCLC). We show that LLT1 expression is restricted to germinal center (GC) B cells within tertiary lymphoid structures (TLS), representing a new hallmark of the presence of active TLS in the tumor microenvironment. CD161-expressing immune cells are found at the vicinity of these structures, with a global enrichment of NSCLC tumors in CD161+ CD4+ and CD8+ T cells as compared to normal distant lung and peripheral blood. CD161+ CD4+ T cells are more activated and produce Th1-cytokines at a higher frequency than their matched CD161-negative counterparts. Interestingly, CD161+ CD4+ T cells highly express OX40 co-stimulatory receptor, less frequently 4-1BB, and display an activated but not completely exhausted PD-1-positive Tim-3-negative phenotype. Finally, a meta-analysis revealed a positive association of CLEC2D (coding for LLT1) and KLRB1 (coding for CD161) gene expression with favorable outcome in NSCLC, independently of the size of T and B cell infiltrates. These data are consistent with a positive impact of LLT1/CD161 on NSCLC patient survival, and make CD161-expressing CD4+ T cells ideal candidates for efficient anti-tumor recall responses.

mTOR pathway activation drives lung cell senescence and emphysema.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung cell senescence may be a major pathogenic factor. Here, we investigated the potential role for mTOR signaling in lung cell senescence and alterations in COPD using lung tissue and derived cultured cells from patients with COPD and from age- and sex-matched control smokers. Cell senescence in COPD was linked to mTOR activation, and mTOR inhibition by low-dose rapamycin prevented cell senescence and inhibited the proinflammatory senescence-associated secretory phenotype. To explore whether mTOR activation was a causal pathogenic factor, we developed transgenic mice exhibiting mTOR overactivity in lung vascular cells or alveolar epithelial cells. In this model, mTOR activation was sufficient to induce lung cell senescence and to mimic COPD lung alterations, with the rapid development of lung emphysema, pulmonary hypertension, and inflammation. These findings support a causal relationship between mTOR activation, lung cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a potentially new therapeutic target in COPD.

Preclinical ex vivo evaluation of the diagnostic performance of a new device for in situ label-free fluorescence spectral analysis of breast masses.

OBJECTIVES: To assess the diagnostic performance of a new device for in situ label-free fluorescence spectral analysis of breast masses in freshly removed surgical specimens, in preparation for its clinical development. METHODS: Sixty-four breast masses from consenting patients who had undergone either a lumpectomy or a mastectomy were included. Label-free fluorescence spectral acquisitions were obtained with a 25G fibre-containing needle inserted into the mass. Data from benign and malignant masses were compared to establish the most discriminating thresholds and measurement algorithms. Accuracy was verified using the bootstrap method. RESULTS: The final histological examination revealed 44 invasive carcinomas and 20 benign lesions. The maximum intensity of fluorescence signal was discriminant between benign and malignant masses (p < .0001) whatever their sizes. Statistical analysis indicated that choosing five random measurements per mass was the best compromise to obtain high sensitivity and high negative predictive value with the fewest measurements. Thus, malignant tumours were identified with a mean sensitivity, specificity, negative and positive predictive value of 98.8%, 85.4%, 97.2% and 93.5%, respectively. CONCLUSION: This new in situ tissue autofluorescence evaluation device allows accurate discrimination between benign and malignant breast masses and deserves clinical development. KEY POINTS: • A new device allows in situ label-free fluorescence analysis of ex vivo breast masses • Maximum fluorescence intensity discriminates benign from malignant masses (p < .0001) • Five random measurements allow a high negative predictive value (97.2%).

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.

Paxillin Binding to the Cytoplasmic Domain of CD103 Promotes Cell Adhesion and Effector Functions for CD8+ Resident Memory T Cells in Tumors.

CD8+/CD103+ tissue-resident memory T cells (TRM cells) accumulate in several human solid tumors, where they have been associated with a favorable prognosis. However, the role of CD103, the α subunit of the integrin αEß7 (also known as CD103), in the retention and functions of these TRM is undefined. In this report, we investigated the role of CD103 cytoplasmic domain and the focal adhesion-associated protein paxillin (Pxn) in downstream signaling and functional activities triggered through αE/CD103 chain. Binding to immobilized recombinant (r)E-cadherin-Fc of CD103 integrin expressed on tumor-specific CTL clones promotes phosphorylation of Pxn and Pyk2 and binding of Pxn to the αE/CD103 subunit tail. Inhibition of Pxn phosphorylation by the Src inhibitor saracatinib or its knockdown via shRNA dramatically altered adhesion and spreading of freshly isolated CD8+/CD103+ lung tumor-infiltrating lymphocytes and CD103+ tumor-specific CTL clones. Inhibition of Pxn phosphorylation with saracatinib in these CTL clones also severely compromised their functional activities toward autologous tumor cells. Using Jurkat T cells as a model to study CD103 integrin activation, we demonstrated a key role of serine residue S1163 of the αE chain intracellular domain in polarization of CD103 and recruitment of lysosomes and Pxn at the contact zone of T lymphocytes with rE-cadherin-Fc-coated beads. Overall, our results show how Pxn binding to the CD103 cytoplasmic tail triggers αEß7 integrin outside-in signaling that promotes CD8+ T-cell migratory behavior and effector functions. These results also explain the more favorable prognosis associated with retention of TRM cells in the tumor microenvironment. Cancer Res; 77(24); 7072-82. ©2017 AACR.