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The purpose of this study was to assess the heat strain experienced by children during unstructured physical activity outdoors in a temperate continental summer climate. Eighteen children (7 girls, 12.1 ± 1.7 years) performed up to 4 h of outdoor free-play (duration: 218 ± 33 min; air temperature of 24.5 ± 3.9°C and relative humidity of 66.2 ± 9.2%). Urine specific gravity (USG) was measured pre- and post-free-play, while body core temperature (Tco, ingestible pill) and heart rate (HR) were measured continuously. Physiological strain index (PSI) was calculated from Tco and HR (scale: 0 (none) to 10 (very high)). Activity levels were categorized as rest, light, moderate, and vigorous based on the metabolic equivalent of task, estimated from video analysis. Most children were euhydrated pre (78%, USG ≤ 1.020), but not post-free-play (28%, USG ≤ 1.020). Mean and peak Tco, HR, and PSI responses were 37.8 ± 0.3°C and 38.4 ± 0.3°C, 133 ± 14 bpm and 180 ± 12 bpm, and 4.7 ± 1.1 (low) and 7.4 ± 1.0 (high), respectively. All children reached peak Tco≥38.0°C, with seven ≥38.5°C, and the highest at 38.9°C. The children spent 58 ± 15% of free-play engaged in moderate-to-vigorous intensity physical activity. During free-play, all of the children performed moderate-to-vigorous intensity physical activity, which was associated with pronounced elevations in heat strain.

In the current study, in vitro neuroprotective effect of carob (Ceratonia siliqua L.) pulp (wild and cultivated varieties) at the unripe and ripe stage was investigated through testing against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), linked to Alzheimer’s disease (AD). As neurodegenerative disorders are strongly related to oxidative damage and metal accumulation, the antioxidant activity of carob extracts was determined by DPPH, ABTS, ferric reducing power (FRP), CUPRAC and ferrous iron chelating (FIC) assays at both ripening stages. Total phenolic (TPC), total flavonoids (TFC) and total condensed tannin contents (CTC) were also assessed. At the unripe stage, the samples of both varieties exhibited an interesting inhibitory effect against AChE and BChE with a strong antioxidant activity by scavenging free radicals and by acting as reducing agents. The result showed that the concentrations of TPC, TFC, and CTC were decreased significantly (P

Purpose:We report efficacy and safety with extended follow-up, and exploratory biomarker analyses from the phase II CheckMate 275 trial to identify biomarkers of response to nivolumab in platinum-resistant metastatic or unresectable urothelial carcinoma (mUC).Patients and Methods:Patients received nivolumab 3 mg/kg once every 2 weeks until disease progression, unacceptable toxicity, or other protocol-defined reasons. The primary endpoint was objective response rate (ORR) per blinded independent review committee (BIRC; using RECIST v1.1) in all treated patients and by tumor PD-L1 expression. Key secondary endpoints were progression-free survival (PFS) per BIRC using RECIST v1.1 and overall survival (OS) in all patients and by PD-L1 expression. Exploratory endpoints included safety and biomarker analyses of tumor mutational burden (TMB), PD-L1, and previously identified mutational signatures.Results:Of 270 treated patients, 139 had evaluable TMB. With 33.7 months' minimum follow-up, ORR per BIRC, median PFS, and median OS [95% confidence interval (CI)] in all treated patients were 20.7% (16.1–26.1), 1.9 months (1.9–2.3), and 8.6 months (6.1–11.3), respectively. No new safety signals were identified. Higher TMB was associated (P < 0.05) with improved ORR [OR (95% CI): 2.13 (1.26–3.60)], PFS [HR: 0.75 (0.61–0.92)], and OS [HR: 0.73 (0.58–0.91)]. TMB combined with PD-L1 better predicted ORR, PFS, and OS than PD-L1 alone. Higher mutational signature 2 score was associated with better OS but did not improve the predictive value of TMB.Conclusions:These results support the durable antitumor activity of nivolumab and suggest that TMB may enrich for better response in mUC. Future studies of TMB/PD-L1 as biomarkers for response to nivolumab in randomized trials are warranted.See related commentary by Swami et al., p. 5059

NEW FINDINGS What is the central question of this study? Black-African descendants are thought, by some, to possess genotypic adaptations conducive to survival in hot climates. We therefore assessed whether Canadian residents of black-African descent display enhanced whole-body total heat loss (evaporative plus dry heat exchange) in comparison to Caucasian Canadians during exercise eliciting matched heat-loss requirements in dry heat. What is the main finding and its importance? Neither whole-body total heat loss nor body heat storage differed significantly between groups, irrespective of the exercise intensity. Our findings indicate that genotypic adaptations associated with ethnicity do not appreciably modify whole-body heat exchange during exercise-heat stress. ABSTRACT Ethnicity has long been thought to modulate thermoregulatory function; however, an evaluation of whole-body heat exchange in men of black-African descent and Caucasian men (white-European descendants), born and raised in the same climate, during exercise eliciting matched heat-loss requirements remained unavailable. We therefore used direct calorimetry to assess whole-body total heat loss (evaporative plus dry heat exchange) in young (18-30 years of age), second-generation (or higher) black-African (n = 11) and Caucasian (n = 11) men. Participants performed three 30 min bouts of semi-recumbent cycling at fixed metabolic heat productions (and therefore matched heat-loss requirements between groups) of 200 (light), 250 (moderate) and 300 W m-2 (vigorous), each followed by 15 min recovery, in dry heat (40°C, ∼13% relative humidity). Across all exercise bouts, dry (P = 0.435) and evaporative (P = 0.600) heat exchange did not differ significantly between groups. As such, total heat loss during light, moderate and vigorous exercise was similar between groups (P = 0.777), averaging [mean (SD)] 177 (10), 217 (13) and 244 (20) W m-2 in black-African men and 172 (13), 212 (17) and 244 (17) W m-2 in Caucasian men. Accordingly, body heat storage across all exercise bouts (summation of metabolic heat production and total heat loss) was also similar between the black-African [568 (142) kJ] and Caucasian groups [623 (124) kJ; P = 0.356]. We demonstrated that, when assessed in young, second-generation (or higher) black-African and Caucasian men during exercise eliciting matched heat-loss requirements in dry heat, ethnicity did not significantly modulate whole-body dry and evaporative heat exchange or the resulting changes in total heat loss and body heat storage.

PURPOSE CheckMate 032 is an open-label, multicohort study that includes patients with unresectable locally advanced or metastatic urothelial carcinoma (mUC) treated with nivolumab 3 mg/kg monotherapy every 2 weeks (NIVO3), nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks (NIVO3+IPI1), or nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks for four doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks (NIVO1+IPI3). We report on the expanded NIVO1+IPI3 cohort and extended follow-up for the NIVO3 and NIVO3+IPI1 cohorts. METHODS Patients with platinum-pretreated mUC were enrolled in this phase I/II multicenter study to receive NIVO3, NIVO3+IPI1, or NIVO1+IPI3 until disease progression or unacceptable toxicity. Primary end point was investigator-assessed objective response rate per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, including duration of response. RESULTS Seventy-eight patients were treated with NIVO3 (minimum follow-up, 37.7 months), 104 with NIVO3+IPI1 (minimum follow-up, 38.8 months), and 92 with NIVO1+IPI3 (minimum follow-up, 7.9 months). Objective response rate was 25.6%, 26.9%, and 38.0% in the NIVO3, NIVO3+IPI1, and NIVO1+IPI3 arms, respectively. Median duration of response was more than 22 months in all arms. Grade 3 or 4 treatment-related adverse events occurred in 21 (26.9%), 32 (30.8%), and 36 (39.1%) patients treated with NIVO3, NIVO3+IPI1, and NIVO1+IPI3, respectively. Grade 5 treatment-related pneumonitis occurred in one patient each in the NIVO3 and NIVO3+IPI1 arms. CONCLUSION With longer follow-up, NIVO3 demonstrated sustained antitumor activity alone and in combination with ipilimumab. NIVO1+IPI3 provided the greatest antitumor activity of all regimens, with a manageable safety profile. This result not only supports additional study of NIVO1+IPI3 in mUC, but demonstrates the potential benefit of immunotherapy combinations in this disease.

Purpose To examine the extent of regional variations in cutaneous vasodilatation during rapid and gradual local thermal hyperaemia (LTH) in young adults. Methods In thirty young adults (21 ± 3 years, 15 females), cutaneous vascular conductance, normalized to maximum local skin heating at 44 °C (%CVCmax), was assessed at the upper chest, abdomen, dorsal arm, dorsal forearm, thigh, and medial calf during rapid (33–42 °C at 1 °C·20 s−1) and gradual (33–42 °C at 1 °C·5 min−1) LTH on separate days. For both protocols, local temperatures were held at 42 °C for up to 35 min, followed by 20–30 min at 44 °C. During rapid LTH, between-region responses were evaluated at baseline, the initial vasodilator peak, and 42 °C plateau. During gradual LTH, responses were assessed at baseline and the 42 °C plateau. Results There were significant main effects of body region on %CVCmax for the initial peak and plateau during rapid LTH and for the plateau during gradual LTH (all P 0.05). The magnitudes of between-region differences varied across the body (~1–17% range). The greatest effects were observed for the abdomen, wherein responses were consistently lower compared to other regions. Further, responses were consistent between males and females across all body regions and heating phases. Conclusion Regional variations in the cutaneous vasodilator response to local heating are evident for rapid and gradual LTH in young adults, with the largest effects observed for the abdomen, albeit regional differences were similar between sexes.

International audience; Genomic copy number variants (CNVs) are routinely identified and reported back to patients with neuropsychiatric disorders, but their quantitative effects on essential traits such as cognitive ability are poorly documented. We have recently shown that the effect size of deletions on cognitive ability can be statistically predicted using measures of intolerance to haploinsufficiency. However, the effect sizes of duplications remain unknown. It is also unknown if the effect of multigenic CNVs are driven by a few genes intolerant to haploinsufficiency or distributed across tolerant genes as well. Here, we identified all CNVs > 50 kilobases in 24,092 individuals from unselected and autism cohorts with assessments of general intelligence. Statistical models used measures of intolerance to haploinsufficiency of genes included in CNVs to predict their effect size on intelligence. Intolerant genes decrease general intelligence by 0.8 and 2.6 points of intelligence quotient when duplicated or deleted, respectively. Effect sizes showed no heterogeneity across cohorts. Validation analyses demonstrated that models could predict CNV effect sizes with 78% accuracy. Data on the inheritance of 27,766 CNVs showed that deletions and duplications with the same effect size on intelligence occur de novo at the same frequency. We estimated that around 10,000 intolerant and tolerant genes negatively affect intelligence when deleted, and less than 2% have large effect sizes. Genes encompassed in CNVs were not enriched in any GOterms but gene regulation and brain expression were GOterms overrepresented in the intolerant subgroup. Such pervasive effects on cognition may be related to emergent properties of the genome not restricted to a limited number of biological pathways.

International audience; Objective:Deleterious copy number variants (CNVs) are identified in up to 20% of individuals with autism. However, levels of autism risk conferred by most rare CNVs remain unknown. The authors recently developed statistical models to estimate the effect size on IQ of all CNVs, including undocumented ones. In this study, the authors extended this model to autism susceptibility.Methods:The authors identified CNVs in two autism populations (Simons Simplex Collection and MSSNG) and two unselected populations (IMAGEN and Saguenay Youth Study). Statistical models were used to test nine quantitative variables associated with genes encompassed in CNVs to explain their effects on IQ, autism susceptibility, and behavioral domains.Results:The “probability of being loss-of-function intolerant” (pLI) best explains the effect of CNVs on IQ and autism risk. Deleting 1 point of pLI decreases IQ by 2.6 points in autism and unselected populations. The effect of duplications on IQ is threefold smaller. Autism susceptibility increases when deleting or duplicating any point of pLI. This is true for individuals with high or low IQ and after removing de novo and known recurrent neuropsychiatric CNVs. When CNV effects on IQ are accounted for, autism susceptibility remains mostly unchanged for duplications but decreases for deletions. Model estimates for autism risk overlap with previously published observations. Deletions and duplications differentially affect social communication, behavior, and phonological memory, whereas both equally affect motor skills.Conclusions:Autism risk conferred by duplications is less influenced by IQ compared with deletions. The model applied in this study, trained on CNVs encompassing >4,500 genes, suggests highly polygenic properties of gene dosage with respect to autism risk and IQ loss. These models will help to interpret CNVs identified in the clinic.

Outcomes for patients with melanoma have improved over the past decade as a result of the development and FDA approval of immunotherapies targeting cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death ligand 1 (PD-L1). However, these therapies do not benefit all patients, and an area of intensive research investigation is identifying biomarkers that can predict which patients are most likely to benefit from them. Here, we report exploratory analyses of the associations of tumor mutational burden (TMB), a 4-gene inflammatory gene expression signature, and BRAF mutation status with tumor response, progression-free survival, and overall survival in patients with advanced melanoma treated as part of the CheckMate 066 and 067 phase III clinical trials evaluating immuno-oncology therapies. In patients enrolled in CheckMate 067 receiving the anti–PD-1 inhibitor nivolumab (NIVO) alone or in combination with the anti–CTLA-4 inhibitor ipilimumab (IPI) or IPI alone, longer survival appeared to associate with high (>median) versus low (≤median) TMB and with high versus low inflammatory signature scores. For NIVO-treated patients, the results regarding TMB association were confirmed in CheckMate 066. In addition, improved survival was observed with high TMB and absence of BRAF mutation. Weak correlations were observed between PD-L1, TMB, and the inflammatory signature. Combined assessment of TMB, inflammatory gene expression signature, and BRAF mutation status may be predictive for response to immune checkpoint blockade in advanced melanoma.

Summary Background Patients with metastatic urothelial carcinoma have a dismal prognosis and few treatment options after first-line chemotherapy. Responses to second-line treatment are uncommon. We assessed nivolumab, a fully human IgG4 PD-1 immune checkpoint inhibitor antibody, for safety and activity in patients with metastatic or surgically unresectable urothelial carcinoma whose disease progressed or recurred despite previous treatment with at least one platinum-based chemotherapy regimen. Methods In this multicentre, phase 2, single-arm study, patients aged 18 years or older with metastatic or surgically unresectable locally advanced urothelial carcinoma, measurable disease (according to Response Evaluation Criteria In Solid Tumors v1.1), Eastern Cooperative Oncology Group performance statuses of 0 or 1, and available tumour samples for biomarker analysis received nivolumab 3 mg/kg intravenously every 2 weeks until disease progression and clinical deterioration, unacceptable toxicity, or other protocol-defined reasons. The primary endpoint was overall objective response confirmed by blinded independent review committee in all treated patients and by tumour PD-L1 expression (≥5% and ≥1%). This trial is registered with ClinicalTrials.gov, number NCT02387996, and is completed. Follow-up is still ongoing. Findings Between March 9, 2015, and Oct 16, 2015, 270 patients from 63 sites in 11 countries received nivolumab, and 265 were evaluated for activity. Median follow-up for overall survival was 7·00 months (IQR 2·96–8·77). Confirmed objective response was achieved in 52 (19·6%, 95% CI 15·0–24·9) of 265 patients. Confirmed objective response was achieved in 23 (28·4%, 95% CI 18·9–39·5) of the 81 patients with PD-L1 expression of 5% or greater, 29 (23·8%, 95% CI 16·5–32·3) of the 122 patients with PD-L1 expression of 1% or greater, and 23 (16·1%, 95% CI 10·5–23·1) of the 143 patients with PD-L1 expression of less than 1%. Grade 3–4 treatment-related adverse events occurred in 48 (18%) of 270 patients—most commonly grade 3 fatigue and diarrhoea, which each occurred in five patients. Three deaths were attributed to treatment (pneumonitis, acute respiratory failure, and cardiovascular failure). Interpretation Nivolumab monotherapy provided meaningful clinical benefit, irrespective of PD-L1 expression, and was associated with an acceptable safety profile in previously treated patients with metastatic or surgically unresectable urothelial carcinoma. Funding Bristol-Myers Squibb.

Prior studies have demonstrated that fibroblast receptor 3 (FGFR3)-mutant urothelial cancers (UCs) are associated with decreased T-cell infiltration. As FGFR3 mutations are enriched in luminal-like UC and luminal-like UC has been shown to be relatively less responsive to PD-1/PD-L1 inhibition (checkpoint inhibition [CPI]), these data have led to the speculation that FGFR3 mutations may be causally related to poor T-cell infiltration and that UC patients harboring FGFR3 mutations may be suboptimal candidates for CPI. Using data derived from two clinical trials exploring CPI in metastatic UC, we demonstrate no statistically significant difference in response rates in patients with FGFR3-mutant versus wild-type UC. We present hypothesis-generating data, suggesting that similar response rates may be explained by a "balancing out" of previously identified independent positive and negative predictors of CPI sensitivity; that is, compared with FGFR3 wild-type UC, FGFR3-mutant UC is associated with a similar tumor mutational burden, lower T-cell infiltration, but also lower stromal/transforming growth factor beta (TGF-β) signals. Based on our findings, FGFR3 mutation status is not a biomarker of resistance to CPI. Indeed, the single-agent activity of both FGFR3 inhibitors and CPI in FGFR3-mutant UC, and potential non-cross resistance provide a strong pragmatic rationale for combination approaches. PATIENT SUMMARY: In this report, we examined the impact of a mutated gene found in a subset of urothelial cancers on response to treatment with immunotherapy. We found that patients with tumors harboring mutations in the gene FGFR3 respond to immunotherapy similarly to patients without such mutations.

International audience; Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.

Immune checkpoint therapy (ICT) can produce durable antitumor responses in metastatic urothelial carcinoma (mUCC); however, the responses are not universal. Despite multiple approvals of ICT in mUCC, we lack predictive biomarkers to guide patient selection. The identification of biomarkers may require interrogation of both the tumor mutational status and the immune microenvironment. Through multi-platform immuno-genomic analyses of baseline tumor tissues, we identified the mutation of AT-rich interactive domain-containing protein 1A (ARID1A) in tumor cells and expression of immune cytokine CXCL13 in the baseline tumor tissues as two predictors of clinical responses in a discovery cohort (n = 31). Further, reverse translational studies revealed that CXCL13-/- tumor-bearing mice were resistant to ICT, whereas ARID1A knockdown enhanced sensitivity to ICT in a murine model of bladder cancer. Next, we tested the clinical relevance of ARID1A mutation and baseline CXCL13 expression in two independent confirmatory cohorts (CheckMate275 and IMvigor210). We found that ARID1A mutation and expression of CXCL13 in the baseline tumor tissues correlated with improved overall survival (OS) in both confirmatory cohorts (CheckMate275, CXCL13 data, n = 217; ARID1A data, n = 139, and IMvigor210, CXCL13 data, n = 348; ARID1A data, n = 275). We then interrogated CXCL13 expression plus ARID1A mutation as a combination biomarker in predicting response to ICT in CheckMate275 and IMvigor210. Combination of the two biomarkers in baseline tumor tissues suggested improved OS compared to either single biomarker. Cumulatively, this study revealed that the combination of CXCL13 plus ARID1A may improve prediction capability for patients receiving ICT.

Summary Metastatic castration-resistant prostate cancer (mCRPC) is immunologically "cold" and predominantly resistant to immune checkpoint therapy due to few tumor-infiltrating T cells. Ipilimumab (anti-CTLA-4) or anti-PD-1/PD-L1 monotherapy failed to show a significant benefit. Although the PD-1/PD-L1 pathway is minimally expressed in prostate tumors, we previously demonstrated that PD-1/PD-L1 expression increases as a compensatory inhibitory pathway in parallel with an ipilimumab-induced increase in tumor-infiltrating T cells. Here, we report the largest trial to date in mCRPC with anti-CTLA-4 plus anti-PD-1 (nivolumab 1 mg/kg plus ipilimumab 3 mg/kg; CheckMate 650, NCT02985957). With median follow-ups of 11.9 and 13.5 months in cohorts 1 (pre-chemotherapy; n = 45) and 2 (post-chemotherapy; n = 45), objective response rate was 25% and 10%, and median overall survival was 19.0 and 15.2 months, respectively. Four patients, two in each cohort, had complete responses. Exploratory studies identify potential biomarkers of response. Grade 3–4 treatment-related adverse events have occurred in ∼42%–53% of patients, with four treatment-related deaths. Therefore, dose/schedule modifications have been implemented.

Genomic Copy Number Variants (CNVs) are routinely identified and reported back to patients with neuropsychiatric disorders, but their quantitative effects on essential traits such as cognitive ability are poorly documented. We have recently shown that the effect-size of deletions on cognitive ability can be statistically predicted using measures of intolerance to haploinsufficiency. However, the effect-sizes of duplications remain unknown. It is also unknown if the effect of multigenic CNVs are driven by a few genes intolerant to haploinsufficiency or distributed across tolerant genes as well.Here, we identified all CNVs >50 kilobases in 24,092 individuals from unselected and autism cohorts with assessments of general intelligence. Statistical models used measures of intolerance to haploinsufficiency of genes included in CNVs to predict their effect-size on intelligence. Intolerant genes decrease general intelligence by 0.8 and 2.6 points of IQ when duplicated or deleted, respectively. Effect-sizes showed no heterogeneity across cohorts. Validation analyses demonstrated that models could predict CNV effect-sizes with 78% accuracy. Data on the inheritance of 27,766 CNVs showed that deletions and duplications with the same effect-size on intelligence occur de novo at the same frequency.We estimated that around 10,000 intolerant and tolerant genes negatively affect intelligence when deleted, and less than 2% have large effect-sizes. Genes encompassed in CNVs were not enriched in any GOterms but gene regulation and brain expression were GOterms overrepresented in the intolerant subgroup. Such pervasive effects on cognition may be related to emergent properties of the genome not restricted to a limited number of biological pathways.

ObjectiveDeleterious copy number variants (CNVs) are identified in up to 20% of individuals with autism. However, only 13 genomic loci have been formally associated with autism because the majority of CNVs are too rare to perform individual association studies. To investigate the implication of undocumented CNVs in neurodevelopmental disorders, we recently developed a new framework to estimate their effect-size on intelligence quotient (IQ) and sought to extend this approach to autism susceptibility and multiple cognitive domains.MethodsWe identified CNVs in two autism samples (Simons Simplex Collection and MSSNG) and two unselected populations (IMAGEN and Saguenay Youth Study). Statistical models integrating scores of genes encompassed in CNVs were used to explain their effect on autism susceptibility and multiple cognitive domains.ResultsAmong 9 scores of genes, the “probability-of-being loss-of-function intolerant” (pLI) best explains the effect of CNVs on IQ and autism risk. Deletions decrease IQ by a mean of 2.6 points per point of pLI. The effect of duplications on IQ is three-fold smaller. The odd ratios for autism increases when deleting or duplicating any point of pLI. This increased autism risk is similar in subgroups of individuals below or above median IQ. Once CNV effects on IQ are accounted for, autism susceptibility remains mostly unchanged for duplications but decreases for deletions. Model estimates for autism risk overlap with previously published observations. Deletions and duplications differentially affect social communication, behaviour, and phonological memory, whereas both equally affect motor skills.ConclusionsAutism risk conferred by duplications is less influenced by IQ compared to deletions. CNVs increase autism risk similarly in individuals with high and low IQ. Our model, trained on CNVs encompassing >4,500 genes, suggests highly polygenic properties of gene dosage with respect to autism risk. These models will help interpreting CNVs identified in the clinic.

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all tested cell lines. The soft cell confiner is thus a valuable tool to decipher the effect of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.Graphical abstractA unique tool to analyze the role of long-term effect of mechanical confinement in normal and pathological conditions

Snake venoms contain various molecules that can be used as tools in the diagnosis and in the treatment of hemostatic disorders. This study reports the isolation and functional characterization of a new thrombin-like enzyme and its role in the modulation of platelet aggregation and coagulation. The molecule was purified by gel filtration, anion exchange chromatography and reverse-phase-HPLC on C8 column; its molecular weight was determined. Natural and synthetic substrates were used to evaluate its enzymatic activities. The fibrinogenolytic activity was tested electrophoretically and by reverse-phase-HPLC on C18 column. Otherwise, the effect on blood coagulation and deficient plasma factors were also evaluated. The mechanism by which a thrombin-like enzyme VLCV (thrombin-like enzyme)-induced platelet aggregation was explored in presence of ticlopidin, clopidogrel and aspirin. VLCV (45 kDa) isolated from Vipera lebetina as a thrombin-like enzyme seems to be able to modulate platelet function. This enzyme showed an amidolytic activity by hydrolyzing the chromogenic-specific substrate of thrombin and the α-chain of fibrinogen. It is also able to clot human plasma and the deficient human plasma in factor X, suggesting that it is involved in the intrinsic and common pathways. The aggregating effect of VLCV is more sensitive to ticlopidine than to the clopidogrel suggesting the involvement of ADP/P2Y12/PI3K pathway. VLCV seems to be able to promote human platelet aggregation suggesting an interaction between P2Y12 and PAR1. Due to its ability to replace the missing factor X and its proaggregating activity, VLCV could be used as molecular tool to better understand the hemostasis mechanism.

International audience; Estrogens are major regulators of the mammary gland development, notably during puberty, via estrogen receptor (ER) activation, leading to the proliferation and differentiation of mammary cells. In addition to estrogens, the bone morphogenetic proteins (BMPs) family is involved in breast stem cell/progenitor commitment. However, these two pathways that synergistically contribute to the biology of the normal mammary gland have also been described to initiate and/or promote breast cancer development. In addition to intrinsic events, lifestyle habits and exposure to environmental cues are key risk factors for cancer in general, and especially for breast cancer. In the latter case, bisphenol A (BPA), an estrogen-mimetic compound, is a critical pollutant both in terms of the quantities released in our environment and of its known and speculated effects on mammary gland biology. In this review, we summarize the current knowledge on the actions of BMPs and estrogens in both normal mammary gland development and breast cancer initiation, dissemination, and resistance to treatment, focusing on the dysregulations of these processes by BPA but also by other bisphenols, including BPS and BPF, initially considered as safer alternatives to BPA.

Lower clearance of immune checkpoint inhibitors is a predictor of improved overall survival (OS) in patients with advanced cancer. We investigated a novel approach using machine learning to identify a baseline composite cytokine signature via clearance, which, in turn, could be associated with OS in advanced melanoma. Peripheral nivolumab clearance and cytokine data from patients treated with nivolumab in two phase III studies (n = 468 (pooled)) and another phase III study (n = 158) were used for machine-learning model development and validation, respectively. Random forest (Boruta) algorithm was used for feature selection and classification of nivolumab clearance. The 16 top-ranking baseline inflammatory cytokines reflecting immune-cell modulation were selected as a composite signature to predict nivolumab clearance (area under the curve (AUC) = 0.75; accuracy = 0.7). Predicted clearance (high vs. low) via the cytokine signature was significantly associated with OS across all three studies (P < 0.01), regardless of treatment (nivolumab vs. chemotherapy).

The broad use of RNA-sequencing technologies held a promise of improved diagnostic tools based on comprehensive transcript sets. However, mining human transcriptome data for disease biomarkers in clinical specimens is restricted by the limited power of conventional reference-based protocols relying on uniquely mapped reads and transcript annotations. Here, we implemented a blind reference-free computational protocol, DE-kupl, to directly infer RNA variations of any origin, including yet unreferenced RNAs, from high coverage total stranded RNA-sequencing datasets of tissue origin. As a bench test, this protocol was powered for detection of RNA subsequences embedded into unannotated putative long noncoding (lnc)RNAs expressed in prostate cancer tissues. Through filtering and visual inspection of 1,179 candidates, we defined 21 lncRNA probes that were further validated for robust tumor-specific expression by NanoString single molecule-based RNA measurements in 144 tissue specimens. Predictive modeling yielded a restricted probe panel enabling over 90% of true positive detection of cancer in an independent dataset from The Cancer Genome Atlas. Remarkably, this clinical signature made of only 9 unannotated lncRNAs largely outperformed PCA3, the only RNA biomarker approved by the Food and Drug Administration agency, specifically, in detection of high-risk prostate tumors. The proposed reference-free computational workflow is modular, highly sensitive and robust and can be applied to any pathology and any clinical application.

PURPOSE Nivolumab 1 mg/kg plus ipilimumab 3 mg/kg (NIVO1+IPI3) is approved for first-line treatment of patients with advanced melanoma in several countries. We conducted a phase IIIb/IV study (CheckMate 511) to determine if nivolumab 3 mg/kg plus ipilimumab 1 mg/kg (NIVO3+IPI1) improves the safety profile of the combination. PATIENTS AND METHODS Patients (N = 360) age 18 years or older with previously untreated, unresectable stage III or IV melanoma were randomly assigned 1:1 to NIVO3+IPI1 or NIVO1+IPI3 once every 3 weeks for four doses. After 6 weeks, all patients received NIVO 480 mg once every 4 weeks until disease progression or unacceptable toxicity. The primary end point was a comparison of the incidence of treatment-related grade 3 to 5 adverse events (AEs) between groups. Secondary end points included descriptive analyses of objective response rate, progression-free survival, and overall survival. The study was not designed to formally demonstrate noninferiority of NIVO3+IPI1 to NIVO1+IPI3 for efficacy end points. RESULTS At a minimum follow-up of 12 months, incidence of treatment-related grade 3 to 5 AEs was 34% with NIVO3+IPI1 versus 48% with NIVO1+IPI3 ( P = .006). In descriptive analyses, objective response rate was 45.6% in the NIVO3+IPI1 group and 50.6% in the NIVO1+IPI3 group, with complete responses in 15.0% and 13.5% of patients, respectively. Median progression-free survival was 9.9 months in the NIVO3+IPI1 group and 8.9 months in the NIVO1+IPI3 group. Median overall survival was not reached in either group. CONCLUSION The CheckMate 511 study met its primary end point, demonstrating a significantly lower incidence of treatment-related grade 3-5 AEs with NIVO3+IPI1 versus NIVO1+IPI3. Descriptive analyses showed that there were no meaningful differences between the groups for any efficacy end point, although longer follow up may help to better characterize efficacy outcomes.

Introduction Key-enzyme inhibition is a recent approach for the treatment of some health disorders, including diabetes, skin hyperpigmentation, Alzheimer's disease, and some microbial infections. Looking for potential natural multi-target inhibitors, flavonoid-rich extracts from the peel of Ficus carica fruits were simultaneously tested against five enzymes and various methods were used to evaluate their antioxidant activity. Methods Extracts from peels from F. carica cultivars (Bakor Noir, Bouankik, Azenjer, and Tazegaght) were prepared using methanol acidified by 5% citric acid. Inhibitory activities of extracts at 10 mg/mL were measured in vitro against α-glucosidase, tyrosinase, urease, acetylcholinesterase, and butyrylcholinesterase. Also, the antioxidant activity was evaluated using free radical scavenging, iron and copper chelating, and reducing assays. Results Extracts were efficient in inhibiting tyrosinase and α-glucosidase (IC50 values from 0.09 to 0.45, and 1.18 to 3.38 mg/mL, respectively). However, a weak inhibition was observed against urease and cholinesterases. Among the antioxidant activities studied, extracts were more efficient for copper chelation and radical scavenging (IC50 values from 0.05 to 0.13, and 0.30 to 1.33 mg/mL, respectively). Phytochemical analysis revealed the extraction of large quantities of flavonoids (up to 50.4 mg/g of peels), mainly flavonols (up to 81% of total flavonoids). Given the specific extract composition, including two fractions (flavonoids and citric acid), a strong synergy was observed for enzyme inhibition and metal chelation. Conclusion Flavonoid-rich extracts from F. carica could be considered as multifunctional bioactive ingredients to be used in anti-hyperglycemic and skin-whitening pharmaceutical formulations.

488 Background: Immune checkpoint therapy can produce durable anti-tumor responses in metastatic urothelial carcinoma (mUCC); however, the responses are not universal. Despite multiple approvals of immune checkpoint therapy in mUCC, we lack predictive biomarkers to guide patient selection. Therefore, there is a critical need to develop clinically useful biomarkers to refine patient selection. The single biomarker studies either focused on tumor mutations or immune response biomarkers, which may limit predictive power due to lack of integration between cancer cell biology and immune cell responses. The identification of biomarkers may require interrogation of both the tumor mutational status and the immune microenvironment. Methods: We performed retrospective multi-platform immuno-genomic analyses of pre-treatment tumor tissues in a discovery cohort (n = 31). Next, we tested the clinical relevance of ARID1A mutation and pre-treatment CXCL13 expression in two independent confirmatory cohorts (CheckMate275 and IMvigor210). Additionally, we performed reverse translational studies using murine model of bladder cancer to demonstrate direct association of the biomarkers in anti-PD-(L)-1 mediated anti-tumor immunity. Results: We identified genomic mutation of AT-rich interactive domain-containing protein 1A ( ARID1A) in tumor cells and expression of immune cytokine CXCL13 in the pre-treatment tumor tissues as two predictors of clinical responses. We found that ARID1A mutation and expression of CXCL13 in the baseline tumor tissues correlated with improved overall survival (OS) in both confirmatory cohorts (CheckMate275, CXCL13 data, n = 217; ARID1A data, n = 139, and IMvigor210, CXCL13 data, n = 348; ARID1A data, n = 275). Further, reverse translational studies revealed that CXCL13−/− tumor-bearing mice were resistant to immune checkpoint therapy whereas ARID1A knockdown enhanced sensitivity to immune checkpoint therapy in a murine model of bladder cancer. We then interrogated CXCL13 expression plus ARID1A mutation as a combination biomarker in predicting response to immune checkpoint therapy in CheckMate275 and IMvigor210. Combination of the 2 biomarkers in baseline tumor tissues showed improved OS compared to either single biomarker. Conclusions: Cumulatively, this study revealed that the combination of CXCL13 plus ARID1A mutation may improve patient selection in mUCC for immune checkpoint therapy.

Objective To examine the contributions of calcium-activated K+ (KCa) channels and nitric oxide synthase (NOS) to adenosine triphosphate (ATP)-induced cutaneous vasodilation in healthy older adults. Methods In eleven older adults (69 ± 2 years, 5 females), cutaneous vascular conductance, normalized to maximum vasodilation (%CVCmax) was assessed at four dorsal forearm skin sites that were continuously perfused with either 1) lactated Ringer solution (Control), 2) 50 mM tetraethylammonium (TEA, KCa channel blocker), 3) 10 mM Nω-nitro-L-arginine (L-NNA, NOS inhibitor), or 4) combined 50 mM TEA +10 mM L-NNA, via microdialysis. Local skin temperature was fixed at 33 °C at all sites with local heaters throughout the protocol while the cutaneous vasodilator response was assessed during coadministration of ATP (0.03, 0.3, 3, 30, 300 mM; 20 min per dose), followed by 50 mM sodium nitroprusside and local skin heating to 43 °C to achieve maximum vasodilation (20–30 min). Results Blockade of KCa channels blunted %CVCmax relative to Control from 0.3 to 300 mM ATP (All P 0.05). Conclusion In healthy older adults, KCa channels play an important role in modulating ATP-induced cutaneous vasodilation, while the NOS contribution to this response is negligible.

Background Hyoscyamus albus L. ( Solanaceae ) an old medicinal plant is a rich source of tropane and nortropane alkaloids which confers to this plant a number of very interesting and beneficial therapeutic effects. Purpose Calystegines that are polyhydroxylated alkaloids and imino-sugars poccess significant glycosidases inhibitory activities and are therefore good candidats for the treatment of diabetes mellitus. Study design Calystegines extracted from Hyoscyamys albus seeds were tested for teir acute oral toxicity and investigated for their in-vivo antidiabetic effect on Streptozotocine induced diabetes in mice. Methodes Calystegines were extracted from the seeds plant using an Ion exchange column; the remaining extract was then administrated orally to mice at several single doses for acute toxicity assay. A dose of 130 mg/kg streptozotocine was injected to mice to induce diabetes mellitus, and diabetic mice were treated orally during 20 days with 10 mg/kg and 20 mg/kg calystegines and 20 mg/kg glibenclamide as the reference drug. Results Acute oral toxicity showed that calystegines are not toxic up to a dose of 2000 mg/kg with absence of any signs of intoxication and damages in Liver and kidney tissues. The nortropane alkaloids markedly reduced blood glucose levels and lipid parameters of diabetic mice to normal concentrations after 20 days of treatment at 10 mg/kg and 20 mg/ kg ( p 0.05). Histopathological study of diabetic mice pancreas indicated that calystegines of Hyoscyamus albus have minimized streptozotocine damages on β-cells of islets of langerhans, stimulated β-cells regeneration and improved with this insulin secretion. Conclusion The findings of this study suggest that calystegines are potent antidiabetic agents with antihyperglicemic and hypolipidemic effects, and a protective fonction on pancreas in streptozotocin induced diabetes in mice.

This study aimed to assess Lawsonia inermis L. and honey mixture effects on burn wounds and hair growth in rabbits. Nine male Oryctolagus cuniculus local rabbits were allocated randomly in 3 groups. After antisepsis and local anesthesia, four circular burns were created on the animal’s backs. Immediately after burning, the wounds were covered with honey in three rabbits (HON group), another group was treated first with honey and then L. inermis powder was added on honey (LI_HON group). Three untreated animals were used as control (CRL group). The treatment of rabbits was applied once daily (6/7 days) until 24th day post burns. The animals were observed for their general state, aspects of wounds and the healing times were also recorded. At 35th day post burns, the hair growth was investigated in the different groups. LI_HON group has recorded the best results compared to other groups; a short significant necrosis duration (LI_HON Vs CRL, p

The study aimed to evaluate Pistacia Lentiscus Virgin Fatty Oil (PLVFO) effects on lipidemic profile and carcass characteristics in rabbits. 15 male adult rabbits were allocated in three groups of 5 each. The first was not treated and served as control (CRL), the second was gavaged by Egg Yolk (EY) at a dose of 7 mL kg-1 BW (6/7 days), the third was treated as EY and then PLVFO was applied at a dose of 1 mL kg-1 BW (6/7 days). At 28th day of experiment, the biochemical profiles were determined and then animals were sacrified for anatomo-pathological studies and carcass characteristics investigation. PLVFO has resulted in non significant decrease of total cholesterol, a significant decrease of LDL-C (PLVFO Vs EY, ANOVA, p

Cancers infiltrated with T-cells are associated with a higher likelihood of response to PD-1/PD-L1 blockade. Counterintuitively, a correlation between epithelial–mesenchymal transition (EMT)-related gene expression and T-cell infiltration has been observed across tumor types. Here we demonstrate, using The Cancer Genome Atlas (TCGA) urothelial cancer dataset, that although a gene expression-based measure of infiltrating T-cell abundance and EMT-related gene expression are positively correlated, these signatures convey disparate prognostic information. We further demonstrate that non-hematopoietic stromal cells are a major source of EMT-related gene expression in bulk urothelial cancer transcriptomes. Finally, using a cohort of patients with metastatic urothelial cancer treated with a PD-1 inhibitor, nivolumab, we demonstrate that in patients with T-cell infiltrated tumors, higher EMT/stroma-related gene expression is associated with lower response rates and shorter progression-free and overall survival. Together, our findings suggest a stroma-mediated source of immune resistance in urothelial cancer and provide rationale for co-targeting PD-1 and stromal elements., Although T-cell infiltration is correlated with EMT-related gene expression in urothelial cancer specimens, here, the authors report EMT-related signatures in urothelial cancer arise mainly from stromal cells. Increased EMT-related gene expression in T-cell infiltrated tumors is associated with an attenuated response to immune checkpoint blockade, providing a rationale for therapeutic co-targeting PD-1 and stromal elements.

Background The antitumor activity of programmed death-1 (PD-1)/PD-1 ligand 1 (PD-L1) inhibition alone is limited in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). Combining immunotherapy with standard-of-care chemotherapy could improve outcomes in mCRPC. We report interim analysis results for the nivolumab + docetaxel (NIVO+DOCE) treatment arm from CheckMate 9KD. Methods CheckMate 9KD is a phase II study of NIVO in combination with DOCE, rucaparib, or enzalutamide. Eligible pts had confirmed metastatic adenocarcinoma of the prostate, ongoing androgen deprivation therapy, and evaluable tumor biopsy. Pts assigned to NIVO+DOCE (chemotherapy-naive and received ≤2 prior second-generation hormonal therapies) received NIVO 360mg every 3 weeks + DOCE 75mg/m2 every 3 weeks + prednisone 5mg twice daily for ≤10 cycles followed by NIVO 480mg every 4 weeks alone until disease progression or unacceptable toxicity (up to 2 years). Coprimary endpoints were objective response rate (ORR) and prostate-specific antigen response rate (PSA-RR; defined as≥50% PSA reduction from baseline). Secondary endpoints included radiographic progression-free survival (rPFS), and safety/tolerability in all treated pts. Association of biomarkers with efficacy was an exploratory endpoint. Results This interim analysis included 41 pts in the NIVO+DOCE arm with a minimum follow-up of 28 weeks, of whom 19 (46.3%) had measurable disease. At the time of the analysis, 24 (58.5%) had discontinued study treatment. ORR in pts with measurable disease was 36.8% (95% confidence interval [CI], 16.3–61.6) with 1 complete response and 6 partial responses. Confirmed PSA-RR was 46.3% (95% CI, 30.7–62.6). Median rPFS was 8.2 months (95% CI, 6.6–not estimable). The 6-month rPFS rate was 71.5%. Any-grade and grade 3/4 treatment-related adverse events occurred in 92.7% and 48.8% of pts, respectively. Associations of biomarkers are also evaluated. Conclusions The combination of NIVO+DOCE showed encouraging clinical activity in pts with mCRPC, with a safety profile consistent with those of the individual agents. A phase III trial is warranted to further evaluate NIVO+DOCE in mCRPC. Clinical trial identification NCT03338790. Editorial acknowledgement Professional medical writing assistance was provided by Nicolette Belletier, PhD, of Parexel, and funded by Bristol-Myers Squibb. Legal entity responsible for the study Bristol-Myers Squibb. Funding Bristol-Myers Squibb and Ono Pharmaceutical Company Limited. Disclosure K. Fizazi: Advisory / Consultancy: Janssen; Advisory / Consultancy: Astellas; Advisory / Consultancy: Bayer; Advisory / Consultancy: Orion. A. Rezazadeh Kalebasty: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Genentech; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Exelexis; Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Janssen; Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: AZ; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bayer; Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution): BMS; Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Eisai; Research grant / Funding (institution): Macrogenics; Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Astellas; Research grant / Funding (institution): Beyond Spring; Research grant / Funding (institution): Bioclin/Rainier Therapeutics; Research grant / Funding (institution): Clovis; Research grant / Funding (institution): Bavarian; Research grant / Funding (institution): Nordic; Research grant / Funding (institution): Seattle Genetics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pfizer; Shareholder / Stockholder / Stock options: ECOM; Honoraria (self), Speaker Bureau / Expert testimony: Sanofi; Honoraria (self), Speaker Bureau / Expert testimony: Amgen; Travel / Accommodation / Expenses: Prometheus Labs. J.C. Vazquez Limon: Non-remunerated activity/ies, Personal Fees and non-financial support: AstraZeneca; Non-remunerated activity/ies, Non-financial support: Merck; Non-remunerated activity/ies, Personal fees: Roche; Non-remunerated activity/ies, Personal fees: Bristol-Myers Squibb. A.J. Armstrong: Research grant / Funding (institution): BMS; Advisory / Consultancy, Research grant / Funding (institution): Janssen; Advisory / Consultancy, Research grant / Funding (institution): Pfizer/Astellas; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Dendreon; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Byer; Research grant / Funding (institution): Constellation; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Genentech/Roche; Advisory / Consultancy: Clovis. B. Sharkey: Full / Part-time employment: Bristol-Myers Squibb. A. Saci: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. J. Li: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. X. Wang: Full / Part-time employment: Bristol-Myers Squibb. M. Ciprotti: Full / Part-time employment: Bristol-Myers Squibb. P. Sathyanarayana: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. F. Saad: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Astellas; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Janssen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Bayer; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Sanofi; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): BMS; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Pfizer. D.P. Petrylak: Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Bellicum; Research grant / Funding (institution): Clovis; Advisory / Consultancy, Research grant / Funding (institution): Dendreon; Research grant / Funding (institution): Eli Lilly; Research grant / Funding (institution): Endocyte ; Advisory / Consultancy: Exelixis; Advisory / Consultancy: Ferring; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Innocrin; Advisory / Consultancy, Research grant / Funding (institution): Johnson and Johnson; Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): Millineum; Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Progenics; Research grant / Funding (institution): Roche Labatories; Advisory / Consultancy, Research grant / Funding (institution): Sanofi Aventis; Research grant / Funding (institution): Sotio; Speaker Bureau / Expert testimony, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: Tyme Pharmaceuticals. R.K. Pachynski: Advisory / Consultancy, Research grant / Funding (institution): BMS; Speaker Bureau / Expert testimony: Dendreon; Advisory / Consultancy: EMD Serono/Pfizer; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Genentech/Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Genomic Health; Research grant / Funding (institution): Janssen; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony: Merck; Advisory / Consultancy: Jounce Therapeutics. C. Drake: Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: Compugen; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy, Research grant / Funding (institution): Janssen Oncology; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Astellas Medivation; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Tizona Therapeutics; Advisory / Consultancy: Merck; Advisory / Consultancy: Pierre Fabre; Advisory / Consultancy: F-Star; Advisory / Consultancy, Shareholder / Stockholder / Stock options: KLEO; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Harpoon; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Shattuck Labs; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Werewolf; Advisory / Consultancy: Genocea; Advisory / Consultancy: Sanofi Aventis; Advisory / Consultancy, Licensing / Royalties: Amplimmune; Advisory / Consultancy, Licensing / Royalties: BMS; Advisory / Consultancy: EMD. All other authors have declared no conflicts of interest.

The widely-used herbicide atrazine (ATZ) is detected in ground and surface water in many countries. Several studies in animals have demonstrated that ATZ has endocrine-disrupting effects on male and female reproduction in many vertebrate species. In this study, we investigated the effects of ATZ exposure on meiosis, a key step in gametogenesis in mammals. The treatment was initiated before oocyte entry into meiosis, which occurs during the embryonic period in females. We found that embryonic exposure to ATZ increases the level of 8-oxo-guanine in the nucleus of meiotic cells, reflecting oxidative stress and affecting meiotic double-strand break repair, chromosome synapsis and crossover numbers. Finally, embryonic exposure to ATZ reduces the number of primordial follicles and increases the incidence of multi-oocyte follicles in adult mice. Our data demonstrate that embryonic exposure to ATZ disrupts prophase I of meiosis and affects normal follicle formation in female mice.

Background: Data suggest that peripheral factors such as circulating cytokines could function as predictive indicators of clinical response to specific therapies or as general prognostic indicators of outcome in patients with cancer. Previous studies have focused on evaluating the association between individual cytokines and clinical outcome. There is no clear consensus on how or which of these cytokines should be selected for prediction of treatment outcome. Clearance of immunotherapeutic agents such as the anti-PD-1 nivolumab (NIVO) has been shown to be a predictor of best overall response and overall survival (OS) across multiple indications. Determination of clearance requires post-treatment pharmacokinetic (PK) samples which negates its utility as a baseline prognostic factor. We investigated a novel machine learning approach to identify a baseline composite cytokine signature that correlates with the established efficacy response relationship for NIVO clearance in advanced melanoma. This approach, which is capable of integrating multivariate factors into a composite feature, may provide a more accurate reflection of underlying biologic factors that contribute to outcome following treatment. Methods: Peripheral serum PK (NIVO clearance) and pharmacodynamic (Myriad Rules Based Medicine-customized inflammatory cytokine panel) data from 2 phase 3 studies (NCT01721772; NCT01844505) of NIVO monotherapy in patients with melanoma (n = 471) were used for machine learning model development (training data set). A third phase 3 study of NIVO in advanced melanoma (NCT01721746) was included in model application (test data set; n = 158). Random forest, a tree-based ensemble learning method, was used for feature selection and classification (low vs high) of NIVO clearance. Kaplan-Meier analyses and log-rank p values were used to assess statistical difference in long-term OS between predicted high- vs low-clearance groups. Results: A panel of ~20 baseline inflammatory cytokines related to immune cell modulation were selected by the machine learning model to predict clearance of NIVO monotherapy in patients with advanced melanoma (area under the curve of receiver operating characteristic: 0.79). The predicted clearance value from the cytokine signature was significantly associated with OS across all 3 studies, including patients treated with NIVO or dacarbazine (control) (p Conclusions: We developed a machine learning approach to identify a serum prognostic cytokine signature based on the relationship between NIVO clearance and response. The strong association of the defined cytokine signature with OS from NIVO and dacarbazine suggests a prognostic role for drug clearance. This cytokine signature has the potential to be a prognostic biomarker in melanoma and may be used for balancing and randomization of patients in clinical studies. Citation Format: Rui Wang, Xiao Shao, Junying Zheng, Abdel Saci, Max Qian, Irene Pak, Amit Roy, Akintunde Bello, Jasmine Rizzo, Fareeda Hosein, Rebecca A. Moss, Megan Wind-Rotolo, Yan Feng. A machine learning approach to identify a peripheral prognostic cytokine signature via nivolumab clearance in patients with advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2273.

Background: Metastatic or surgically unresectable urothelial cancer (UC) is a disease with high unmet need. Nivolumab monotherapy demonstrated clinical benefit in patients with platinum-resistant UC (CheckMate 275; NCT02387996). Because not all patients respond to nivolumab therapy, identifying biomarkers for response is of critical importance. In the CheckMate 275 cohort, higher tumor mutational burden (TMB) was associated with longer overall survival (OS) (1). Previous studies have shown that certain mutational signatures, combinations of mutation types arising from specific mutagenesis processes, were associated with mutational burden and clinical outcome in UC (2,3). We hypothesized that specific mutational signatures may be associated with response to nivolumab in CheckMate 275. Methods: Whole exome sequencing data and clinical annotations for 139 archival, tumor-normal paired samples from patients enrolled in the CheckMate 275 cohort were collected and analyzed. Sequencing reads were processed and somatic variants called as described previously (4). Percentage of mutations across 30 mutational signatures were generated using deconstructSigs, which infers signature activity with given known signatures (5). Association of the most prevalent mutational signatures with TMB, previously known clinical biomarkers, and clinical efficacy (OS, progression free survival [PFS], and objective response [OR]) were examined. Results: We identified age-related (signature 1), endogenous mutagenesis-related APOBEC (signatures 2 and 13) and UV-induced (signature 7) as the most prevalent mutational signatures in UC. When examining their correlations with TMB, signature 1 showed negative correlation, signatures 2 and 13 showed high positive correlation, and signature 7 showed low correlation. Higher signature 2 scores were associated with better OS, but were poor predictors for PFS and OR. Overall, while signature 2 added predictive value over previously known clinical biomarkers such as PD-L1 expression, serum hemoglobin level, and presence of liver metastasis, it did not improve on the predictive performance of TMB. Conclusions: An APOBEC-related mutational signature (signature 2) was associated with OS in patients with advanced UC treated with nivolumab. This signature was correlated with TMB and did not improve upon TMB as a predictive biomarker. Our work demonstrates the importance of including TMB as a covariate when analyzing mutational signatures. References: 1. Galsky MD et al. Ann Oncol 2017;28(suppl 5). Abstract 848PD 2. Glaser AP et al. Oncotarget 2018;9:4537-48 3. Roberts SA et al. Nat Genet 2013;45:970-6 4. Carbone DP et al. NEJM 2017;376:2415-26 5. Forbes SA et al. Nucleic Acids Res 2017;45:D777-83 Citation Format: G. Celine Han, Peter M. Szabo, Abdel Saci, Alice M. Walsh, Natallia Kalinava, Ariella Sasson, Bruce Fischer, Matthew D. Galsky, Padmanee Sharma. Mutational signatures as biomarkers of response to nivolumab in metastatic bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4890.

Background: Increased tumor mutational burden (TMB) and inflammation are associated with improved clinical outcomes to immuno-oncology (I-O) therapy in many tumor types. Genomic correlates of response to nivolumab (N) vs dacarbazine (D) (CheckMate [CM] 066; NCT01721772) and N+ipilimumab (I) combination therapy or N vs I (CM 067; NCT01844505) were evaluated for association of TMB and an inflammatory gene signature with clinical outcomes. Methods: In pretreatment tumor samples from eligible patients (pts), TMB was analyzed by whole-exome sequencing using median number of total missense mutations to define high vs low TMB (TMB-H/TMB-L). Associations of TMB with progression-free survival (PFS) and overall survival (OS) were evaluated per protocol-defined exploratory analyses using 4-year follow-up data for both trials. PFS and OS associations with an inflammatory signature were assessed by RNAseq in CM 067 samples only and grouped into tertiles for analysis. HR and 95% CI were calculated using Cox modeling and P values were calculated using the Wald test. Results: In CM 066, TMB was evaluable in 122 of 411 pts. PFS and OS were significantly longer in pts treated with N, but not with D, for TMB-H vs TMB-L tumors. The benefit of PFS and OS for N vs D was greater in pts with TMB-H than TMB-L (Table). In CM 067, 583 of 937 pts were evaluable for TMB and 299 were evaluable using an inflammatory signature. PFS and OS were longer in each treatment (tx) arm in pts with TMB-H vs TMB-L (Table). Across tx arms, a numerical benefit was observed for PFS and OS in pts with high vs low inflammatory signature (Table). TMB-H and a high inflammatory signature score were independently associated with clinical outcomes to I-O. Conclusions: High TMB or high inflammatory signature was associated with benefit to I-O. TMB status did not differentiate between N+I and N, and higher inflammatory status increased the likelihood of benefit for N+I and N, suggesting further evaluation of these biomarkers to characterize the response to I-O regimens in melanoma. Association of TMB (CM 066) or TMB and inflammatory signature (CM 067) with PFS and OSCategorical comparisonClinical measureHR (95% CI; P value)CM 066NDPatients with evaluable TMB, n (high, low)53 (23, 30)69 (38, 31)TMB-H vs TMB-LPFSOS0.33 (0.16-0.69; 0.0031)0.43 (0.2-0.91; 0.027)0.67 (0.39-1.10; 0.13)0.66 (0.39-1.10; 0.13)N vs D in TMB-HPFSOS0.28 (0.14-0.59; 0.00083)0.35 (0.17-0.73; 0.0046)N vs D in TMB-LPFSOS0.57 (0.33-0.99; 0.047)0.55 (0.31-0.98; 0.044)Categorical comparisonClinical measureHR (95% CI; P value)CM 067N+INIPatients with evaluable TMB, n (high, low)197 (94, 103)192 (95, 97)194 (101, 93)TMB-H vs TMB-LPFSOS0.56 (0.39-0.81; 0.002)0.53 (0.34-0.81; 0.0032)0.45 (0.31-0.65; 0.00002)0.48 (0.31-0.72; 0.00046)0.64 (0.47-0.87; 0.005)0.59 (0.42-0.83; 0.0022)N+I vs N in TMB-HPFSOS0.95 (0.63-1.40; 0.81)0.97 (0.60-1.60; 0.88)-N+I vs N in TMB-LPFSOS0.77 (0.56-1.10; 0.12)0.87 (0.61-1.30; 0.46)-Patients with evaluable inflammatory signature, n (low, med, high)93 (33, 32, 28)106 (39, 31, 36)100 (28, 36, 36)Inflammatory signature, high vs lowPFSOS0.38 (0.18-0.80; 0.01)0.53 (0.24-1.18; 0.12)0.60 (0.34-1.00; 0.069)0.35 (0.18-0.69; 0.0025)0.48 (0.27-0.85; 0.012)0.52 (0.27-1.00; 0.049) Citation Format: F. Stephen Hodi, Jedd D. Wolchok, Dirk Schadendorf, James Larkin, Max Qian, Abdel Saci, Tina C. Young, Sujaya Srinivasan, Han Chang, Megan Wind-Rotolo, Jasmine I. Rizzo, Donald G. Jackson, Paolo A. Ascierto. Genomic analyses and immunotherapy in advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT037.

142 Background: Immune checkpoint inhibitor monotherapy has shown limited clinical benefit in patients (pts) with prostate cancer, likely due to an immunologically “cold” tumor microenvironment. We report preplanned interim efficacy/safety for NIVO + IPI in pts with mCRPC from CheckMate 650. Methods: Asymptomatic/minimally symptomatic pts who progressed after 2nd-generation hormone therapy and have not received chemotherapy for mCRPC (cohort 1) and pts who progressed after taxane-based chemotherapy (cohort 2) were included. Treatment was NIVO 1 mg/kg + IPI 3 mg/kg Q3W for 4 doses, then NIVO 480 mg every 4 weeks. Coprimary endpoints: objective response rate (ORR) and radiographic PFS per PCWG2. Safety is a secondary endpoint. Exploratory endpoints include correlation of biomarkers with efficacy. Results: 78 pts had a minimum follow-up of 6 months; among pts with baseline measurable disease, ORR was 26% and 10% in cohorts 1 and 2 (Table). In both cohorts, ORR was higher in pts with PD-L1 ≥1%, DNA damage repair (DDR), homologous recombination deficiency (HRD), or above-median tumor mutational burden (TMB). Of all PSA responding pts (Table), 4 had PSA

This analysis provides long-term follow-up in patients with BRAF wild-type advanced melanoma receiving first-line therapy based on anti-programmed cell death 1 receptor inhibitors.To compare the 3-year survival with nivolumab vs that with dacarbazine in patients with previously untreated BRAF wild-type advanced melanoma.This follow-up of a randomized phase 3 trial analyzed 3-year overall survival data from the randomized, controlled, double-blind CheckMate 066 phase 3 clinical trial. For this ongoing, multicenter academic institution trial, patients were enrolled from January 2013 through February 2014. Eligible patients were 18 years or older with confirmed unresectable previously untreated stage III or IV melanoma and an Eastern Cooperative Oncology Group performance status of 0 or 1 but without a BRAF mutation.Patients were treated until progression or unacceptable toxic events with nivolumab (3 mg/kg every 2 weeks plus dacarbazine-matched placebo every 3 weeks) or dacarbazine (1000 mg/m2 every 3 weeks plus nivolumab-matched placebo every 2 weeks).Overall survival.At minimum follow-ups of 38.4 months among 210 participants in the nivolumab group (median age, 64 years [range, 18-86 years]; 57.6% male) and 38.5 months among 208 participants in the dacarbazine group (median age, 66 years [range, 25-87 years]; 60.1% male), 3-year overall survival rates were 51.2% (95% CI, 44.1%-57.9%) and 21.6% (95% CI, 16.1%-27.6%), respectively. The median overall survival was 37.5 months (95% CI, 25.5 months-not reached) in the nivolumab group and 11.2 months (95% CI, 9.6-13.0 months) in the dacarbazine group (hazard ratio, 0.46; 95% CI, 0.36-0.59; P .001). Complete and partial responses, respectively, were reported for 19.0% (40 of 210) and 23.8% (50 of 210) of patients in the nivolumab group compared with 1.4% (3 of 208) and 13.0% (27 of 208) of patients in the dacarbazine group. Additional analyses were performed on outcomes with subsequent therapies. Treatment-related grade 3/4 adverse events occurred in 15.0% (31 of 206) of nivolumab-treated patients and in 17.6% (36 of 205) of dacarbazine-treated patients. There were no deaths due to study drug toxic effects.Nivolumab led to improved 3-year overall survival vs dacarbazine in patients with previously untreated BRAF wild-type advanced melanoma.ClinicalTrials.gov identifier: NCT01721772.

Introduction: Nivolumab monotherapy has already shown efficacy and safety in the open-label, single-arm, phase II CheckMate 275 trial (objective response rate [ORR], 19.6%). Here, we report updated results with a minimum follow-up of 21.3 months together with additional exploratory biomarker analyses. Methods: Patients received nivolumab 3 mg/kg IV every 2 weeks until disease progression or unacceptable toxicity. The primary endpoint was confirmed ORR by blinded independent review committee (per Response Evaluation Criteria in Solid Tumors v.1.1). Secondary endpoints included progression-free survival (PFS) and overall survival (OS). ORR was further examined by the duration of response (DoR) and time to response. Outcomes were evaluated in all patients and by tumor cell programmed death-1 ligand 1 (PD-L1) expression (cutoff ≥1% by Dako immunohistochemical staining). Myeloid-derived suppressor cells (MDSCs), defined as Lin− CD14+ HLA-DR-low/negative monocytic myeloid cells, were measured by flow cytometry in the circulation at baseline, and archival pretreatment biopsies were assessed for interferon gamma (IFN-γ) gene expression signature to assess associations with OS. Results: There were 270 treated patients. Median follow-up was 24.5 months. ORR was 20.4% for all treated patients. There were nine additional complete responses (CRs) since results with median follow-up of 11.5 months were reported (17; 6.3%). Of the 55 responders, 72.7% had DoR ≥6 months, 54.5% had DoR ≥12 months, and 30.9% had an ongoing response. Median (95% confidence interval [CI]) DoR for all treated patients was 17.7 (11.5-22.0) months. Median (95% CI) PFS and OS were 1.9 (1.9-2.3) and 8.6 (6.1-11.3) months, respectively. OS benefit was observed across tumor PD-L1 expression levels. The 1-year and 2-year OS rates (95% CI) were 40.3% (34.4-46.2) and 29.4% (23.9-35.1), respectively. The most common any-grade treatment-related adverse events were fatigue (18.1%), diarrhea (12.2%), pruritus (11.5%), and decreased appetite (9.3%). Lower vs higher baseline MDSCs were associated with longer OS in all patients and independent of tumor PD-L1 expression ( Conclusions: In this updated analysis of CheckMate 275, durable efficacy was observed with additional CRs since the previous report. No new safety signals were observed. A combination of immune-associated tumor and circulating biomarkers (as defined in this study) may identify patients with particularly favorable outcomes with nivolumab. Citation Format: Padmanee Sharma, Ari Baron, Andrea Necchi, Elizabeth R. Plimack, Sumanta K. Pal, Jens Bedke, Jose A. Arranz, Daniel Vaena, Marc-Oliver Grimm, Sergio Bracarda, Margitta Retz, Arlene Siefker-Radtke, Chikara Ohyama, Gary Grossfeld, Haolan Lu, Abdel Saci, Hao Tang, Matthew D. Galsky. Nivolumab monotherapy in patients with advanced platinum-resistant urothelial carcinoma: Efficacy and safety update and association between biomarkers and overall survival in CheckMate 275 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT178.

511 Background: Prior studies have correlated FGFR3 mutations (mut) and increased PPARg expression with decreased T-cell infiltration raising the possibility that these might represent mediators of PD-1/PD-L1 blockade resistance in luminal UC and targets for combination strategies. The causality of these associations remains unclear and the clinical relevance in PD-1/PD-L1 blockade treated patients has been underexplored. Methods: Archival tumor specimens from patients enrolled on CheckMate 275, a phase 2 trial of nivo in patients with platinum-resistant metastatic UC, underwent whole exome sequencing (WES) and gene expression profiling (HTG EdgeSeq). CD8 expression was determined by immunohistochemistry. Results: WES and gene expression data was available for 139 and 217 patients, respectively; the baseline characteristics and outcomes of both cohorts were similar to the full CheckMate 275 cohort. FGFR3mut were detected in 15 patients (11%) and correlated with higher FGFR3 gene expression. Neither FGFR3mut nor FGFR gene expression correlated with CD8 expression (Pearson's r -0.13; p=0.16) or with response rate (RR), progression-free survival (PFS), or overall survival (OS); RR in FGFRmut vs. FGFRwt = 20% vs 20%. PPARg gene expression was inversely correlated with CD8 (Pearson’s r -0.44; P

There is to date no effective way of preventing or curing neurodegenerative diseases such as Alzheimer disease or transmissible spongiform encephalopathies. The idea of treating those conditions by immunological approaches has progressively emerged over the last ten years. Encouraging results have been reported in Alzheimer disease and in peripheral forms of mouse prion diseases following passive injection of Abs or active immunization against the peptides or proteins presumably at the origin of those disorders. Still, major difficulties persist due to some characteristics of those conditions such as slow evolution, brain location, uncertainties regarding precise pathogenic pathways, and, above all, the fact that the target Ag is self, meaning that it is poorly immunogenic and potentially harmful if tolerance was transgressed. To analyze some of those difficulties, we are developing adoptive cell transfer approaches. In this study, lymphocytes sensitized against the prion protein in nontolerant Prnp−/− mice were transferred into histocompatible wild-type recipients which were partly or totally devoid of their own lymphocytes. Under such conditions, we found that the engrafted T lymphocytes resisted peripheral tolerance, remained reactive for several months against epitopes of the prion protein, and significantly attenuated the progression of prions in secondary lymphoid organs with subsequent delay in the evolution of the neurological disease. Interestingly, those protective T lymphocytes secreted lymphokines and migrated more readily into the host CNS but did not appear to be engaged in cooperation with host B cells for Ab production.

We address here the issue of prioritizing non-coding mutations in the tumoral genome. To this aim, we created two independent computational models. The first (germline) model estimates purifying selection based on population SNP data. The second (somatic) model estimates tumor mutation density based on whole genome tumor sequencing. We show that each model reflects a different set of constraints acting either on the normal or tumor genome, and we identify the specific genome features that most contribute to these constraints. Importantly, we show that the somatic mutation model carries independent functional information that can be used to narrow down the non-coding regions that may be relevant to cancer progression. On this basis, we identify positions in non-coding RNAs and the non-coding parts of mRNAs that are both under purifying selection in the germline and protected from mutation in tumors, thus introducing a new strategy for future detection of cancer driver elements in the expressed non-coding genome., Author Summary Cancer cells undergo a mutation/selection process that resembles that of any living cell. Most mutations in cancer cell DNA occur in the so-called "non-coding" regions that represent 98.5% of the genome length. Pinning down which of these mutations contribute to the fitness of cancer cells would be important for identifying new "cancer drivers", which may in turn lead to future treatments. Unfortunately, predicting the impact of a non-coding DNA alteration remains extremely difficult. In this study, we analyze millions of non-coding cancer mutations and show cancer-specific mutational patterns can be used to predict non-coding regions that are preserved from mutations and may thus be important for cancer cell survival. Combining this information with population data, we propose a new scoring system that should help prioritize important non-coding mutations in future studies.

Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.