Variant-to-function mapping of late-onset Alzheimer's disease GWAS signals in human microglial cell models implicates RTFDC1 at the CASS4 locus.

Late-onset Alzheimer's disease (LOAD) research has principally focused on neurons over the years due to their known role in the production of amyloid beta plaques and neurofibrillary tangles. In contrast, recent genomic studies of LOAD have implicated microglia as culprits of the prolonged inflammation exacerbating the neurodegeneration observed in patient brains. Indeed, recent LOAD genome-wide association studies (GWAS) have reported multiple loci near genes related to microglial function, including TREM2, ABI3, and CR1. However, GWAS alone cannot pinpoint underlying causal variants or effector genes at such loci, as most signals reside in non-coding regions of the genome and could presumably confer their influence frequently via long-range regulatory interactions. We elected to carry out a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models (iPSC-derived microglia and HMC3) in order to physically map interactions between LOAD GWAS-implicated candidate causal variants and their corresponding putative effector genes. Notably, we observed consistent evidence that rs6024870 at the GWAS CASS4 locus contacted the promoter of nearby gene, RTFDC1. We subsequently observed a directionallly consistent decrease in RTFDC1 expression with the the protective minor A allele of rs6024870 via both luciferase assays in HMC3 cells and expression studies in primary human microglia. Through CRISPR-Cas9-mediated deletion of the putative regulatory region harboring rs6024870 in HMC3 cells, we observed increased pro-inflammatory cytokine secretion and decreased DNA double strand break repair related, at least in part, to RTFDC1 expression levels. Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD 'CASS4' GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene.

Vaginal lacerations during laparoscopic hysterectomy for endometrial cancer and local recurrence risk.

Objective: Laparoscopic surgery (MIS) offers equivalent oncologic outcomes as compared to open surgery, while causing lesser morbidity and resulting in a faster recovery. Vaginal extraction of specimens may cause vaginal or perineal lacerations (VL). The objective of this retrospective study was to assess local recurrence rates compared between cases with vaginal laceration (VL) or without vaginal lacerations (NL). Methods: We identified patients with endometrial cancer who underwent MIS between 2014 and 2018. We assessed the rate of local recurrence between patients in VL and NL cohorts. The study included all histologic subtypes and stages while benign final pathology, synchronous primaries or cases that required laparotomy for extraction were excluded. Results: 338 MIS cases were evaluable of which 40 cases had a vaginal laceration during specimen extraction. There was no significant difference in age, race, presence of LVSI, stage, grade, histology or use of vaginal brachytherapy between cohorts. Cases with vaginal lacerations were significantly associated with a higher median BMI and larger uterine size. The VL cohort was more likely to have received adjuvant treatment. In early stage disease, more cases had non-endometrioid histology in the VL group and had increased incidence of chemotherapy and radiation use as well. There were no cases of isolated vaginal recurrence (0/40) in the VL group as compared to an incidence of 2 % (7/298) in the NL group with a relative risk of 0.48 (CI: 0.03-8.36, p = 0.62). There were 4 cases of pelvic recurrence (4/40) in the VL group and 2 cases in the NL group (2/298) with a relative risk of 2.13 (CI: 0.46-9.89, p = 0.34). Conclusions: In endometrial cancer cases, we did not observe a significantly increased risk of vaginal or pelvic recurrence after a vaginal laceration at the time of specimen removal.

Randomized Placebo-Controlled, Biomarker-Stratified Phase Ib Microbiome Modulation in Melanoma: Impact of Antibiotic Preconditioning on Microbiome and Immunity.

Gut-microbiota modulation shows promise in improving immune-checkpoint blockade (ICB) response; however, precision biomarker-driven, placebo-controlled trials are lacking. We performed a multicenter, randomized placebo-controlled, biomarker-stratified phase I trial in patients with ICB-naïve metastatic melanoma using SER-401, an orally delivered Firmicutesenriched spore formulation. Fecal microbiota signatures were characterized at baseline; patients were stratified by high versus low Ruminococcaceae abundance prior to randomization to the SER-401 arm (oral vancomycin-preconditioning/SER-401 alone/nivolumab + SER-401), versus the placebo arm [placebo antibiotic/placebo microbiome modulation (PMM)/nivolumab + PMM (NCT03817125)]. Analysis of 14 accrued patients demonstrated that treatment with SER-401 + nivolumab was safe, with an overall response rate of 25% in the SER-401 arm and 67% in the placebo arm (though the study was underpowered related to poor accrual during the COVID-19 pandemic). Translational analyses demonstrated that vancomycin preconditioning was associated with the disruption of the gut microbiota and impaired immunity, with incomplete recovery at ICB administration (particularly in patients with high baseline Ruminococcaceae). These results have important implications for future microbiome modulation trials. Significance: This first-of-its-kind, placebo-controlled, randomized biomarker-driven microbiome modulation trial demonstrated that vancomycin + SER-401 and anti-PD-1 are safe in melanoma patients. Although limited by poor accrual during the pandemic, important insights were gained via translational analyses, suggesting that antibiotic preconditioning and interventional drug dosing regimens should be carefully considered when designing such trials.

A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Purpose: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, CLPP activators which reduce OXPHOS indirectly and have demonstrated safety in patients. Experimental Design: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201, ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic effects in vivo in UM liver metastasis models. Results: CLPP expression was confirmed in primary and mUM patient samples. ONC201/212 treatment of UM cell lines in vitro decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis. ONC212 increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Conclusion: Imipridones are a promising strategy for further testing and development in mUM.

Gut Microbiome in Patients With Early-Stage and Late-Stage Melanoma.

Importance: The gut microbiome modulates the immune system and responses to immunotherapy in patients with late-stage melanoma. It is unknown whether fecal microbiota profiles differ between healthy individuals and patients with melanoma or if microbiota profiles differ among patients with different stages of melanoma. Defining gut microbiota profiles in individuals without melanoma and those with early-stage and late-stage melanoma may reveal features associated with disease progression. Objective: To characterize and compare gut microbiota profiles between healthy volunteers and patients with melanoma and between patients with early-stage and late-stage melanoma. Design, Setting, and Participants: This single-site case-control study took place at an academic comprehensive cancer center. Fecal samples were collected from systemic treatment-naive patients with stage I to IV melanoma from June 1, 2015, to January 31, 2019, and from healthy volunteers from June 1, 2021, to January 31, 2022. Patients were followed up for disease recurrence until November 30, 2021. Main Outcomes and Measures: Fecal microbiota was profiled by 16S ribosomal RNA sequencing. Clinical and pathologic characteristics, treatment, and disease recurrence were extracted from electronic medical records. Fecal microbiome diversity, taxonomic profiles and inferred functional profiles were compared between groups. Results: A total of 228 participants were enrolled (126 men [55.3%]; median age, 59 [range, 21-90] years), including 49 volunteers without melanoma, 38 patients with early-stage melanoma (29 with stage I or melanoma in situ and 9 with stage II), and 141 with late-stage melanoma (66 with stage III and 75 with stage IV). Community differences were observed between patients with melanoma and volunteers. Patients with melanoma had a higher relative abundance of Fusobacterium compared with controls on univariate analysis (0.19% vs 0.003%; P < .001), but this association was attenuated when adjusted for covariates (log2 fold change of 5.18 vs controls; P = .09). Microbiomes were distinct between patients with early-stage and late-stage melanoma. Early-stage melanoma had a higher alpha diversity (Inverse Simpson Index 14.6 [IQR, 9.8-23.0] vs 10.8 [IQR, 7.2-16.8]; P = .003), and a higher abundance of the genus Roseburia on univariate analysis (2.4% vs 1.2%; P < .001) though statistical significance was lost with covariate adjustment (log2 fold change of 0.86 vs controls; P = .13). Multiple functional pathways were differentially enriched between groups. No associations were observed between the microbial taxa and disease recurrence in patients with stage III melanoma treated with adjuvant immunotherapy. Conclusions and Relevance: The findings of this case-control study suggest that fecal microbiota profiles were significantly different among patients with melanoma and controls and between patients with early-stage and late-stage melanoma. Prospective investigations of the gut microbiome and changes that occur with disease progression may identify future microbial targets for intervention.

Concurrent intrathecal and intravenous nivolumab in leptomeningeal disease: phase 1 trial interim results.

There is a critical need for effective treatments for leptomeningeal disease (LMD). Here, we report the interim analysis results of an ongoing single-arm, first-in-human phase 1/1b study of concurrent intrathecal (IT) and intravenous (IV) nivolumab in patients with melanoma and LMD. The primary endpoints are determination of safety and the recommended IT nivolumab dose. The secondary endpoint is overall survival (OS). Patients are treated with IT nivolumab alone in cycle 1 and IV nivolumab is included in subsequent cycles. We treated 25 patients with metastatic melanoma using 5, 10, 20 and 50 mg of IT nivolumab. There were no dose-limiting toxicities at any dose level. The recommended IT dose of nivolumab is 50 mg (with IV nivolumab 240 mg) every 2 weeks. Median OS was 4.9 months, with 44% and 26% OS rates at 26 and 52 weeks, respectively. These initial results suggest that concurrent IT and IV nivolumab is safe and feasible with potential efficacy in patients with melanoma LMD, including in patients who had previously received anti-PD1 therapy. Accrual to the study continues, including in patients with lung cancer. ClinicalTrials.gov registration: NCT03025256 .

Uterine leiomyosarcomas harboring MAP2K4 gene amplification are sensitive in vivo to PLX8725, a novel MAP2K4 inhibitor.

INTRODUCTION: Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification. METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed. RESULTS: Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot. CONCLUSION: PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

Obesity Is Associated with Altered Tumor Metabolism in Metastatic Melanoma.

PURPOSE: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI). EXPERIMENTAL DESIGN: Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371). RESULTS: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI. CONCLUSIONS: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies. See related commentary by Smalley, p. 5.

Changes in outcomes and factors associated with survival in melanoma patients with brain metastases.

BACKGROUND: Treatment options for patients with melanoma brain metastasis (MBM) have changed significantly in the last decade. Few studies have evaluated changes in outcomes and factors associated with survival in MBM patients over time. The aim of this study is to evaluate changes in clinical features and overall survival (OS) for MBM patients. METHODS: Patients diagnosed with MBMs from 1/1/2009 to 12/31/2013 (Prior Era; PE) and 1/1/2014 to 12/31/2018 (Current Era; CE) at The University of Texas MD Anderson Cancer Center were included in this retrospective analysis. The primary outcome measure was OS. Log-rank test assessed differences between groups; multivariable analyses were performed with Cox proportional hazards models and recursive partitioning analysis (RPA). RESULTS: A total of 791 MBM patients (PE, n = 332; CE, n = 459) were included in analysis. Median OS from MBM diagnosis was 10.3 months (95% CI, 8.9-12.4) and improved in the CE vs PE (14.4 vs 10.3 months, P < .001). Elevated serum lactate dehydrogenase (LDH) was the only factor associated with worse OS in both PE and CE patients. Factors associated with survival in CE MBM patients included patient age, primary tumor Breslow thickness, prior immunotherapy, leptomeningeal disease, symptomatic MBMs, and whole brain radiation therapy. Several factors associated with OS in the PE were not significant in the CE. RPA demonstrated that elevated serum LDH and prior immunotherapy treatment are the most important determinants of survival in CE MBM patients. CONCLUSIONS: OS and factors associated with OS have changed for MBM patients. This information can inform contemporary patient management and clinical investigations.

A clinical bulbar assessment scale (CBAS) for amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Comprehensive and valid bulbar assessment scales for use within amyotrophic lateral sclerosis (ALS) clinics are critically needed. The aims of this study are to develop the Clinical Bulbar Assessment Scale (CBAS) and complete preliminary validation. METHODS: The authors selected CBAS items from among the literature and expert opinion, and content validity ratio (CVR) was calculated. Following consent, the CBAS was administered to a pilot sample of English-speaking adults with El Escorial defined ALS (N = 54) from a multidisciplinary clinic, characterizing speech, swallowing, and extrabulbar features. Criterion validity was assessed by correlating CBAS scores with commonly used ALS scales, and internal consistency reliability was obtained. RESULTS: Expert raters reported strong agreement for the CBAS items (CVR = 1.00; 100% agreement). CBAS scores yielded a moderate, significant, negative correlation with ALS Functional Rating Scale-Revised (ALSFRS-R) total scores (r = -0.652, p < .001), and a strong, significant, negative correlation with ALSFRS-R bulbar subscale scores (r = -0.795, p < .001). There was a strong, significant, positive correlation with Center for Neurologic Studies Bulbar Function Scale (CNS-BFS) scores (r = 0.819, p < .001). CBAS scores were significantly higher for bulbar onset (mean = 38.9% of total possible points, SD = 22.6) than spinal onset (mean = 18.7%, SD = 15.8; p = .004). Internal consistency reliability (Cronbach's alpha) values were: (a) total CBAS, α = 0.889; (b) Speech subscale, α = 0.903; and (c) Swallowing subscale, α = 0.801. DISCUSSION: The CBAS represents a novel means of standardized bulbar data collection using measures of speech, swallowing, respiratory, and cognitive-linguistic skills. Preliminary evidence suggests the CBAS is a valid, reliable scale for clinical assessment of bulbar dysfunction.

Neoadjuvant relatlimab and nivolumab in resectable melanoma.

Relatlimab and nivolumab combination immunotherapy improves progression-free survival over nivolumab monotherapy in patients with unresectable advanced melanoma1. We investigated this regimen in patients with resectable clinical stage III or oligometastatic stage IV melanoma (NCT02519322). Patients received two neoadjuvant doses (nivolumab 480 mg and relatlimab 160 mg intravenously every 4 weeks) followed by surgery, and then ten doses of adjuvant combination therapy. The primary end point was pathologic complete response (pCR) rate2. The combination resulted in 57% pCR rate and 70% overall pathologic response rate among 30 patients treated. The radiographic response rate using Response Evaluation Criteria in Solid Tumors 1.1 was 57%. No grade 3-4 immune-related adverse events were observed in the neoadjuvant setting. The 1- and 2-year recurrence-free survival rate was 100% and 92% for patients with any pathologic response, compared to 88% and 55% for patients who did not have a pathologic response (P = 0.005). Increased immune cell infiltration at baseline, and decrease in M2 macrophages during treatment, were associated with pathologic response. Our results indicate that neoadjuvant relatlimab and nivolumab induces a high pCR rate. Safety during neoadjuvant therapy is favourable compared to other combination immunotherapy regimens. These data, in combination with the results of the RELATIVITY-047 trial1, provide further confirmation of the efficacy and safety of this new immunotherapy regimen.

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

Neoadjuvant immunotherapy across cancers: meeting report from the Immunotherapy Bridge-December 1st-2nd, 2021.

After the success of immunotherapy in the treatment of advanced metastatic cancer, further evaluation in earlier settings, including high-risk, surgically-resectable disease is underway. Potential benefits of a neoadjuvant immunotherapeutic approach include presurgical tumor shrinkage, reduced surgical morbidity, early eradication of micrometastases and prevention of distant disease, and greater antigen-specific T cell response. For some cancers, pathologic response has been established as a surrogate measure for long-term outcomes, therefore offering the ability for early and objective assessment of treatment efficacy and the potential to inform and personalize adjuvant treatment clinical decision-making. Leveraging the neoadjuvant treatment setting offers the ability to deeply interrogate longitudinal tissue in order to gain translatable, pan-malignancy insights into response and mechanisms of resistance to immunotherapy. Neoadjuvant immunotherapy across cancers was a focus of discussion at the virtual Immunotherapy Bridge meeting (December 1-2, 2021). Clinical, biomarker, and pathologic insights from prostate, breast, colon, and non-small-cell lung cancers, melanoma and non-melanoma skin cancers were discussed and are summarized in this report.

Androgen receptor blockade promotes response to BRAF/MEK-targeted therapy.

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed1,2. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.

Hallmarks of Resistance to Immune-Checkpoint Inhibitors.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.