A single relapse induces worsening of disability and health-related quality of life in patients with neuromyelitis optica spectrum disorder.

Background: Cumulative damage from multiple relapses in neuromyelitis optica spectrum disorder (NMOSD) is associated with poor health-related quality of life (HRQoL) and long-term disability in patients positive for anti-aquaporin 4 antibodies (AQP4+). This study assessed the effect of an individual relapse on HRQoL and disability outcomes in AQP4+ NMOSD. Methods: Post hoc analyses of data pooled from the PREVENT study and its open-label extension, which evaluated the efficacy and safety of eculizumab in AQP4+ NMOSD, examined the effect of a single relapse on 3 disability and 4 HRQoL outcome measures. Assuming the effect of 1 relapse extends to multiple relapses, an extrapolation was done to assess the effect of 2 relapses on these outcomes. Results: In 27 patients (placebo: n = 20; eculizumab: n = 7) experiencing an independently adjudicated relapse, 1 relapse led to significantly worse disability (modified Rankin Scale and Expanded Disability Status Scale [EDSS]) and HRQoL (36-item Short-Form Health Survey mental and physical component summaries; European Quality of Life 5-Dimension questionnaire 3-Level visual analogue scale and utility index) scores. In 4 of 7 outcomes, clinically meaningful worsening was more likely for relapsing versus non-relapsing patients (n = 116). Extrapolating the effect of 2 relapses predicted that clinically meaningful worsening was more likely in 6 out of 7 outcomes, including EDSS, for patients experiencing multiple relapses versus patients experiencing no relapses. Conclusion: Findings from these clinical trial data demonstrate that a single NMOSD relapse can worsen disability and HRQoL, underscoring the role of relapse prevention in improving long-term outcomes in patients with AQP4+ NMOSD.

Eculizumab monotherapy for NMOSD: Data from PREVENT and its open-label extension.

During PREVENT (a phase 3, randomized, double-blind, placebo-controlled, time-to-event study) and its open-label extension (interim analysis), 33 adults with aquaporin-4 immunoglobulin G-positive neuromyelitis optica spectrum disorder (AQP4-IgG + NMOSD) received eculizumab monotherapy for a median of 2.8 years (range, 14 weeks-5.2 years). At 192 weeks (~4 years), 96% of these patients were free from adjudicated relapses (Kaplan-Meier analysis; 95% confidence interval, 75.7-99.4). During PREVENT, 95% (20/21) of patients receiving eculizumab monotherapy had no disability worsening. Eculizumab monotherapy provides effective long-term relapse prevention, relieving the chronic immunosuppression burden in patients with AQP4-IgG + NMOSD. ClinicalTrials.gov; PREVENT: NCT01892345; open-label extension: NCT02003144.

Eculizumab in Asian patients with anti-aquaporin-IgG-positive neuromyelitis optica spectrum disorder: A subgroup analysis from the randomized phase 3 PREVENT trial and its open-label extension.

Background Eculizumab, a terminal complement inhibitor, significantly reduced the risk of relapse compared with placebo in patients with anti-aquaporin-4 immunoglobulin G-positive (AQP4+) neuromyelitis optica spectrum disorder (NMOSD) in the PREVENT trial. We report efficacy and safety analyses in Asian patients in PREVENT and its open-label extension (OLE). Methods PREVENT was a double-blind, randomized, phase 3 trial. Patients with AQP4+ NMOSD were randomly assigned (2:1) to receive intravenous eculizumab (maintenance dose, 1200 mg/2 weeks) or placebo. Patients who completed PREVENT could receive eculizumab in an OLE. Analyses were performed in a prespecified subgroup of Asian patients. Results Of 143 patients enrolled, 52 (36.4%) were included in the Asian subgroup (eculizumab, n = 37; placebo, n = 15); 45 Asian patients received eculizumab in the OLE. Most Asian patients (86.5%) received concomitant immunosuppressive therapy. During PREVENT, one adjudicated relapse occurred in patients receiving eculizumab and six occurred in patients receiving placebo in the Asian subgroup (hazard ratio, 0.05; 95% confidence interval: 0.01-0.35; p = 0.0002). An estimated 95.2% of Asian patients remained relapse-free after 144 weeks of eculizumab treatment. Upper respiratory tract infections, headache, and nasopharyngitis were the most common adverse events with eculizumab in the Asian subgroup. Conclusion Eculizumab reduces the risk of relapse in Asian patients with AQP4+ NMOSD, with a benefit-risk profile similar to the overall PREVENT population. The benefits of eculizumab were maintained during long-term therapy. Clinical trial registration ClinicalTrials.gov identifiers: NCT01892345 (PREVENT); NCT02003144 (open-label extension).

Long-Term Safety and Efficacy of Eculizumab in Aquaporin-4 IgG-Positive NMOSD.

OBJECTIVE: During PREVENT (NCT01892345), eculizumab significantly reduced relapse risk versus placebo in patients with aquaporin-4 immunoglobulin G-positive neuromyelitis optica spectrum disorder (AQP4-IgG+ NMOSD). We report an interim analysis of PREVENT's ongoing open-label extension (OLE; NCT02003144) evaluating eculizumab's long-term safety and efficacy. METHODS: Patients who completed PREVENT could enroll in the OLE to receive eculizumab (maintenance dose = 1,200 mg/2 weeks, after a blinded induction phase). Safety and efficacy data from PREVENT and its OLE (interim data cut, July 31, 2019) were combined for this analysis. RESULTS: Across PREVENT and the OLE, 137 patients received eculizumab and were monitored for a median (range) of 133.3 weeks (5.1-276.9 weeks), for a combined total of 362.3 patient-years (PY). Treatment-related adverse event (AE) and serious adverse event (SAE) rates were 183.5 in 100 PY and 8.6 in 100 PY, respectively. Serious infection rates were 10.2 in 100 PY in eculizumab-treated patients versus 15.1 in 100 PY in the PREVENT placebo group. No patient developed a meningococcal infection. At 192 weeks (3.7 years), 94.4% (95% confidence interval [CI], 88.6-97.3) of patients remained adjudicated relapse-free. The adjudicated annualized relapse rate was 0.025 (95% CI = 0.013-0.048) in all eculizumab-treated patients versus 0.350 (95% CI = 0.199-0.616) in the PREVENT placebo group. During the OLE, 37% of patients (44 of 119 patients) stopped or decreased background immunosuppressive therapy use. INTERPRETATION: This analysis demonstrates that eculizumab's long-term safety profile in NMOSD is consistent with its established profile across other indications. This analysis also demonstrated the sustained ability of long-term eculizumab treatment to reduce relapse risk in patients with AQP4-IgG+ NMOSD. ANN NEUROL 2021;89:1088-1098.

Long-term outcomes of peginterferon beta-1a in multiple sclerosis: results from the ADVANCE extension study, ATTAIN.

BACKGROUND: ADVANCE was a phase III trial of the efficacy and safety of subcutaneous peginterferon beta-1a 125 µg every 2 or 4 weeks in patients with relapsing-remitting multiple sclerosis (RRMS). ATTAIN was a 2-year extension study of ADVANCE. The aim was to evaluate the long-term safety, tolerability, and efficacy of peginterferon beta-1a 125 µg every 2 or 4 weeks in ATTAIN. METHODS: ADVANCE dosing schedules were maintained in ATTAIN, except that every-4-weeks dosing patients were switched to every-2-weeks dosing after conversion of the study to an open-label protocol. ATTAIN was considered complete when the last patient completed the 96-week extension study. Primary endpoints included adverse event (AE) and serious AE (SAE) incidence. Secondary endpoints included relapse, magnetic resonance imaging, and disability outcomes. RESULTS: Of the 1512 patients randomized in ADVANCE, 1076 (71%) continued treatment in ATTAIN; of these, 842 (78%) completed the open-label extension study. During ATTAIN, 478 patients (87%) in the every-2-weeks group and 471 patients (89%) in the every-4-weeks group experienced an AE; SAEs were reported in 90 patients (16%) in the every-2-weeks group and 113 patients (21%) in the every-4-weeks group. The most frequent AEs reported were injection site reactions and flu-like symptoms, both of which numerically decreased over time. Peginterferon beta-1a every 2 weeks versus every 4 weeks significantly reduced the adjusted annualized relapse rate over 6 years (0.188 versus 0.263, p = 0.0052) and the risk of relapse over 5 years (36% versus 49%, p = 0.0018). Fewer new T1, new/newly enlarging T2, and gadolinium-enhancing magnetic resonance imaging lesions were observed with every-2-weeks dosing than every-4-weeks dosing over 4 years. CONCLUSIONS: Results from the ADVANCE extension study, ATTAIN, confirm the favorable long-term safety and tolerability profile of peginterferon beta-1a in patients with RRMS and provide additional evidence for the clinical and radiological benefits associated with this therapy.

Peginterferon ß-1a every 2 weeks increased achievement of no evidence of disease activity over 4 years in the ADVANCE and ATTAIN studies in patients with relapsing-remitting multiple sclerosis.

BACKGROUND: No evidence of disease activity (NEDA) is a composite measurement, incorporating clinical and magnetic resonance imaging (MRI) elements of disease activity to sensitively evaluate the therapeutic efficacy of treatments for relapsing-remitting multiple sclerosis (RRMS). OBJECTIVE: To assess the NEDA status of patients treated with peginterferon ß-1a in the ADVANCE and ATTAIN studies and explore its predictive value on longer-term clinical outcomes. METHODS: ATTAIN was a 2-year extension of the pivotal 2-year ADVANCE study of peginterferon ß-1a for RRMS. Achievement of clinical NEDA, MRI NEDA, or overall NEDA was calculated cumulatively and by year over 4 years. Clinical outcomes during ATTAIN were analyzed based on NEDA status at the end of ADVANCE. RESULTS: Significantly more patients treated with peginterferon ß-1a every 2 weeks than every 4 weeks achieved clinical NEDA (60.6% versus 50.6%, p = 0.0063) and MRI NEDA (28.3% versus 15.8%, p = 0.0005) through year 4 and overall NEDA through year 3 (20.9% versus 13.9%, p = 0.0160). Over 4 years, 15.8% of patients in the every 2 weeks group and 10.7% of patients in the every 4 weeks group maintained overall NEDA (p = 0.0584). Achievement of clinical NEDA, MRI NEDA, or overall NEDA in ADVANCE was predictive of annualized relapse rate in ATTAIN; achievement of clinical NEDA in ADVANCE was also predictive of NEDA achievement and confirmed disability worsening in ATTAIN. CONCLUSIONS: Peginterferon ß-1a every 2 weeks is associated with higher levels of NEDA compared with placebo in year 1 or peginterferon ß-1a every 4 weeks in years 2-4. Overall NEDA within the first 2 years of treatment may be prognostic of long-term clinical outcomes.Clinicaltrials.gov: NCT01332019.

Matching-adjusted comparisons demonstrate better clinical outcomes with SC peginterferon beta-1a every two weeks than with SC interferon beta-1a three times per week.

BACKGROUND: Subcutaneous (SC) peginterferon beta-1a and SC interferon beta-1a (IFN beta-1a) have demonstrated efficacy in treating relapsing-remitting multiple sclerosis (RRMS) but have never been compared in direct head-to-head clinical trials, the gold-standard comparison. A well-balanced matching-adjusted comparison of weighted individual patient data on SC peginterferon beta-1a, and aggregate data from published phase 3 clinical trials of SC IFN beta-1a, was conducted to provide additional information on the comparative efficacy of these two agents. METHODS: Individual patient data from a study of SC peginterferon beta-1a 125 mcg every two weeks (ADVANCE) and pooled summary data from four published studies of SC IFN beta-1a 44 mcg three times per week (OPERA I and II, CARE-MS I and II) with similar populations were utilized. A comparison was conducted by weighting individual peginterferon beta-1a-treated patients, using estimated propensity of enrolling in SC IFN beta-1a treatment to match multiple key aggregate baseline characteristics of SC IFN beta-1a-treated patients. After matching, weighted annualized relapse rate (ARR), 24-week confirmed disability worsening (CDW), and clinical no evidence of disease activity (clinical-NEDA) were calculated and compared for peginterferon beta-1a and SC IFN beta-1a. RESULTS: After matching, baseline characteristics were well balanced across treatment groups. At 2 years, ARR after matching was 0.256 for patients receiving peginterferon beta-1a (effective n = 376) and 0.335 for those receiving SC IFN beta-1a (n = 1218) (P = 0.0901). The percentage of patients who were relapse free over 2 years was significantly higher with peginterferon beta-1a than with SC IFN beta-1a (75.1% vs. 57.4% [after matching], P < 0.0001). The peginterferon beta-1a treatment group had a significantly lower proportion of patients with 24-week CDW compared with SC IFN beta-1a (after matching 6.5% vs. 13.2%; P = 0.0007). Clinical-NEDA occurred in a significantly higher proportion of patients treated with SC peginterferon beta-1a versus SC IFN beta-1a (74.1% vs. 48.1%; P < 0.0001). CONCLUSIONS: This matching-adjusted comparison using data from four phase 3 trials with SC IFN beta-1a formulations demonstrated that patients with RRMS treated with SC peginterferon beta-1a 125 mcg every two weeks achieved better clinical outcomes than patients who received SC IFN beta-1a 44 mcg three times per week.

The EDSS-Plus, an improved endpoint for disability progression in secondary progressive multiple sclerosis.

BACKGROUND: The Expanded Disability Status Scale (EDSS) has wide scientific and regulatory precedent but limited ability to detect clinically relevant disability progression in secondary progressive multiple sclerosis (SPMS) patients, partly due to a lack of meaningful measurement of short-distance ambulatory and upper-extremity function. OBJECTIVE: To present a rationale for a composite endpoint adding the timed 25-foot walk (T25FW) and 9-Hole Peg Test (9HPT) to EDSS for SPMS disability progression assessment. METHODS: Using the International Multiple Sclerosis Secondary Progressive Avonex Clinical Trial (IMPACT) placebo arm ( n = 215) data, we analyzed disability progression using a novel progression endpoint, "EDSS-Plus," defined as progression on ⩾1 of 3 components (EDSS, T25FW, and/or 9HPT) confirmed ⩾24 weeks apart and with a ⩾20% minimum threshold change for T25FW and 9HPT. RESULTS: Over 2 years, subjects classified as T25FW, 9HPT (dominant hand), or 9HPT (non-dominant hand) progressors worsened on average by 103.4%, 69.0%, and 59.2%, respectively, while non-progressors' times remained largely unchanged. Using EDSS-Plus, 59.5% of the patients had 24-week confirmed disability progression versus 24.7% (EDSS), 41.9% (T25FW), and 34.4% (9HPT (either hand)) on each component alone. CONCLUSION: The 24-week confirmed minimum worsening of ⩾20% for T25FW and 9HPT clearly separates SPMS progressors from non-progressors. We propose that EDSS-Plus may represent an improved endpoint to identify SPMS disability progression.

Subgroup and sensitivity analyses of annualized relapse rate over 2 years in the ADVANCE trial of peginterferon beta-1a in patients with relapsing-remitting multiple sclerosis.

ADVANCE was a 2-year, double-blind, placebo-controlled, Phase 3 study in 1512 patients aged 18-65 years with relapsing-remitting multiple sclerosis, which demonstrated that peginterferon beta-1a 125 mcg administered subcutaneously every 2 or 4 weeks led to significant reductions in annualized relapse rate (ARR) compared with placebo. This analysis examined ARR over 2 years in ADVANCE across subgroups. Patients were treated with peginterferon beta-1a every 2 weeks or every 4 weeks, or placebo during Year 1. Thereafter, patients on placebo were re-randomized to peginterferon beta-1a every 2 weeks or every 4 weeks (delayed treatment). Subgroup analyses were conducted by demographics and baseline disease characteristics. The following results compared ARR in these subgroups for patients in continuous 2-week treatment versus continuous 4-week treatment. ARR was similar in most demographic and baseline disease characteristic subgroups evaluated within the peginterferon beta-1a every-2-week arm or every-4-week arm over 2 years. Although for both doses some differences in the point estimates for ARR were noted among the subgroups, considerable overlap in the confidence intervals suggested that the efficacy of peginterferon beta-1a is similar in all patients irrespective of gender, age, body weight, geographical region, and disease activity at initiation of treatment. Within each peginterferon beta-1a dosing group, ARR was generally similar across most subgroups.

COMPARE: Pharmacokinetic profiles of subcutaneous peginterferon beta-1a and subcutaneous interferon beta-1a over 2 weeks in healthy subjects.

AIM: Subcutaneous (s.c.) peginterferon beta-1a injected once every 2 weeks and s.c. interferon beta-1a injected three times per week (Rebif®) have demonstrated efficacy in relapsing-remitting multiple sclerosis, but direct comparisons of pharmacological activity and tolerability between the two products are lacking. COMPARE was an open label, crossover, pharmacokinetic (PK) study evaluating drug exposure and the safety and tolerability of s.c. peginterferon beta-1a and s.c. interferon beta-1a, over 2 weeks in healthy subjects. METHODS: Thirty healthy subjects received one dose of peginterferon beta-1a (125 µg s.c.) or six doses of interferon beta-1a (44 µg s.c.) over 2 weeks, followed by the alternate treatment after a 2 week washout period. Drug concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) and PK parameters including cumulative area under the concentration-time curve (AUC0-336h ) over 2 weeks and maximum observed serum concentrations (Cmax ) were estimated using a non-compartmental analysis. RESULTS: The PK analysis population comprised 26 subjects for each treatment. Drug exposure (AUC0-336h ) was 60% higher with s.c. peginterferon than with s.c. interferon beta-1a (117.4 ng ml(-1) h, 95% confidence interval 95.6, 144.3 vs. 73.1 ng ml(-1) h, 95% confidence interval 61.2, 87.3, respectively; P < 0.0001). Injection-site reactions (ISRs) were the most common adverse events (AEs) observed with both treatments. Numerically lower frequencies and incidence rates of ISRs, headache, myalgia and chills were observed with s.c. peginterferon beta-1a. CONCLUSIONS: One dose of s.c. peginterferon delivered significantly greater drug exposure than s.c. interferon beta-1a three times a week over 2 weeks, and a lower frequency of AEs.

A miRNA-Based Signature Detected in Primary Melanoma Tissue Predicts Development of Brain Metastasis.

PURPOSE: Brain metastasis is the major cause of mortality among patients with melanoma. A molecular prognostic test that can reliably stratify patients at initial melanoma diagnosis by risk of developing brain metastasis may inform the clinical management of these patients. EXPERIMENTAL DESIGN: We performed a retrospective, cohort-based study analyzing genome-wide and targeted microRNA expression profiling of primary melanoma tumors of three patient cohorts (n = 92, 119, and 45) with extensive clinical follow-up. We used Cox regression analysis to establish a microRNA-based signature that improves the ability of the current clinicopathologic staging system to predict the development of brain metastasis. RESULTS: Our analyses identified a 4-microRNA (miR-150-5p, miR-15b-5p, miR-16-5p, and miR-374b-3p) prognostic signature that, in combination with stage, distinguished primary melanomas that metastasized to the brain from nonrecurrent and non-brain metastatic primary tumors (training cohort: C-index = 81.4%, validation cohort: C-index = 67.4%, independent cohort: C-index = 76.9%). Corresponding Kaplan-Meier curves of high- versus low-risk patients displayed a clear separation in brain metastasis-free and overall survival (training: P < 0.001; P < 0.001, validation: P = 0.033; P = 0.007, independent: P = 0.021; P = 0.022, respectively). Finally, of the microRNA in the prognostic model, we found that the expression of a key lymphocyte miRNA, miR-150-5p, which is less abundant in primary melanomas metastatic to brain, correlated with presence of CD45(+) tumor-infiltrating lymphocytes. CONCLUSIONS: A prognostic assay based on the described miRNA expression signature combined with the currently used staging criteria may improve accuracy of primary melanoma patient prognoses and aid clinical management of patients, including selection for adjuvant treatment or clinical trials of adjuvant therapies.

Melanoma risk loci as determinants of melanoma recurrence and survival.

BACKGROUND: Steadily high melanoma mortality rates urge for the availability of novel biomarkers with a more personalized ability to predict melanoma clinical outcomes. Germline risk variants are promising candidates for this purpose; however, their prognostic potential in melanoma has never been systematically tested. METHODS: We examined the effect of 108 melanoma susceptibility single nucleotide polymorphisms (SNPs), associated in recent GWAS with melanoma and melanoma-related phenotypes, on recurrence-free survival (RFS) and overall survival (OS), in 891 prospectively accrued melanoma patients. Cox proportional hazards models (Cox PH) were used to test the associations between 108 melanoma risk SNPs and RFS and OS adjusted by age at diagnosis, gender, tumor stage, histological subtype and other primary tumor characteristics. RESULTS: We identified significant associations for rs7538876 (RCC2) with RFS (HR=1.48, 95% CI=1.20-1.83, p=0.0005) and rs9960018 (DLGAP1) with both RFS and OS (HR=1.43, 95% CI=1.07-1.91, p=0.01, HR=1.52, 95% CI=1.09-2.12, p=0.01, respectively) using multivariable Cox PH models. In addition, we developed a logistic regression model that incorporates rs7538876, rs9960018, primary tumor histological type and stage at diagnosis that has an improved discriminatory ability to classify 3-year recurrence (AUC=82%) compared to histological type and stage alone (AUC=78%). CONCLUSIONS: We identified associations between melanoma risk variants and melanoma outcomes. The significant associations observed for rs7538876 and rs9960018 suggest a biological implication of these loci in melanoma progression. The observed predictive patterns of associated variants with clinical end-points suggest for the first time the potential for utilization of genetic risk markers in melanoma prognostication.

Partially Linear Single Index Cox Regression Model in Nested Case-Control Studies.

The nested case-control (NCC) design is widely used in epidemiologic studies as a cost-effective subcohort sampling method to study the association between a disease and its potential risk factors. NCC data are commonly analyzed using Thomas' partial likelihood approach under the Cox proportional hazards model assumption. However, the linear modeling form in the Cox model may be insufficient for practical applications, especially when there are a large number of risk factors under investigation. In this paper, we consider a partially linear single index proportional hazard model, which includes a linear component for covariates of interest to yield easily interpretable results and a nonparametric single index component to adjust for multiple confounders effectively. We propose to approximate the nonparametric single index function by polynomial splines and estimate the parameters of interest using an iterative algorithm based on the partial likelihood. Asymptotic properties of the resulting estimators are established. The proposed methods are evaluated using simulations and applied to an NCC study of ovarian cancer.

Application of the asthma phenotype algorithm from the Severe Asthma Research Program to an urban population.

RATIONALE: Identification and characterization of asthma phenotypes are challenging due to disease complexity and heterogeneity. The Severe Asthma Research Program (SARP) used unsupervised cluster analysis to define 5 phenotypically distinct asthma clusters that they replicated using 3 variables in a simplified algorithm. We evaluated whether this simplified SARP algorithm could be used in a separate and diverse urban asthma population to recreate these 5 phenotypic clusters. METHODS: The SARP simplified algorithm was applied to adults with asthma recruited to the New York University/Bellevue Asthma Registry (NYUBAR) to classify patients into five groups. The clinical phenotypes were summarized and compared. RESULTS: Asthma subjects in NYUBAR (n = 471) were predominantly women (70%) and Hispanic (57%), which were demographically different from the SARP population. The clinical phenotypes of the five groups generated by the simplified SARP algorithm were distinct across groups and distributed similarly to those described for the SARP population. Groups 1 and 2 (6 and 63%, respectively) had predominantly childhood onset atopic asthma. Groups 4 and 5 (20%) were older, with the longest duration of asthma, increased symptoms and exacerbations. Group 4 subjects were the most atopic and had the highest peripheral eosinophils. Group 3 (10%) had the least atopy, but included older obese women with adult-onset asthma, and increased exacerbations. CONCLUSIONS: Application of the simplified SARP algorithm to the NYUBAR yielded groups that were phenotypically distinct and useful to characterize disease heterogeneity. Differences across NYUBAR groups support phenotypic variation and support the use of the simplified SARP algorithm for classification of asthma phenotypes in future prospective studies to investigate treatment and outcome differences between these distinct groups. TRIAL REGISTRATION: Clinicaltrials.gov NCT00212537.

Serum microRNAs as biomarkers for recurrence in melanoma.

BACKGROUND: Identification of melanoma patients at high risk for recurrence and monitoring for recurrence are critical for informed management decisions. We hypothesized that serum microRNAs (miRNAs) could provide prognostic information at the time of diagnosis unaccounted for by the current staging system and could be useful in detecting recurrence after resection. METHODS: We screened 355 miRNAs in sera from 80 melanoma patients at primary diagnosis (discovery cohort) using a unique quantitative reverse transcription-PCR (qRT-PCR) panel. Cox proportional hazard models and Kaplan-Meier recurrence-free survival (RFS) curves were used to identify a miRNA signature with prognostic potential adjusting for stage. We then tested the miRNA signature in an independent cohort of 50 primary melanoma patients (validation cohort). Logistic regression analysis was performed to determine if the miRNA signature can determine risk of recurrence in both cohorts. Selected miRNAs were measured longitudinally in subsets of patients pre-/post-operatively and pre-/post-recurrence. RESULTS: A signature of 5 miRNAs successfully classified melanoma patients into high and low recurrence risk groups with significant separation of RFS in both discovery and validation cohorts (p = 0.0036, p = 0.0093, respectively). Significant separation of RFS was maintained when a logistic model containing the same signature set was used to predict recurrence risk in both discovery and validation cohorts (p < 0.0001, p = 0.033, respectively). Longitudinal expression of 4 miRNAs in a subset of patients was dynamic, suggesting miRNAs can be associated with tumor burden. CONCLUSION: Our data demonstrate that serum miRNAs can improve accuracy in identifying primary melanoma patients with high recurrence risk and in monitoring melanoma tumor burden over time.

The novel gamma secretase inhibitor RO4929097 reduces the tumor initiating potential of melanoma.

Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma.

miR-30b/30d regulation of GalNAc transferases enhances invasion and immunosuppression during metastasis.

To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.

Quantitative evaluation of all hexamers as exonic splicing elements.

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 3' splice sites tend to be single stranded, and effective 5' splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms.

Outcomes of the RESTOR-MV Trial (Randomized Evaluation of a Surgical Treatment for Off-Pump Repair of the Mitral Valve).

OBJECTIVES: we sought to determine whether patients with functional mitral regurgitation (FMR) would benefit from ventricular reshaping by the Coapsys device (Myocor, Inc., Maple Grove, Minnesota). BACKGROUND: FMR occurs when ventricular remodeling impairs valve function. Coapsys is a ventricular shape change device placed without cardiopulmonary bypass to reduce FMR. It compresses the mitral annulus and reshapes the ventricle. We hypothesized that Coapsys for FMR would improve clinical outcomes compared with standard therapies. METHODS: RESTOR-MV (Randomized Evaluation of a Surgical Treatment for Off-Pump Repair of the Mitral Valve) was a randomized, prospective, multicenter study of patients with FMR and coronary disease with core laboratory analysis. After enrollment, patients were stratified to the standard indicated surgery: either coronary artery bypass graft alone or coronary artery bypass graft with mitral valve repair. In each stratum, randomization was to either control (indicated surgery) or treatment (coronary artery bypass graft with Coapsys ventricular reshaping). RESULTS: the study was terminated when the sponsor failed to secure ongoing funding; 165 patients were randomized. Control and Coapsys both produced decreases in left ventricular (LV) end-diastolic dimension and MR at 2 years (p < 0.001); Coapsys provided a greater decrease in LV end-diastolic dimension (p = 0.021). Control had lower MR grades during follow-up (p = 0.01). Coapsys showed a survival advantage compared with control at 2 years (87% vs. 77%) (hazard ratio: 0.421; 95% confidence interval: 0.200 to 0.886; stratified log-rank test; p = 0.038). Complication-free survival (including death, stroke, myocardial infarction, and valve reoperation) was significantly greater with Coapsys at 2 years (85% vs. 71%) (hazard ratio: 0.372; 95% confidence interval: 0.185 to 0.749; adjusted log-rank test; p = 0.019). CONCLUSIONS: analysis of RESTOR-MV indicates that patients with FMR requiring revascularization treated with ventricular reshaping rather than standard surgery had improved survival and a significant decrease in major adverse outcomes. This trial validates the concept of the ventricular reshaping strategy in this subset of patients with heart failure. (Randomized Evaluation of a Surgical Treatment for Off-Pump Repair of the Mitral Valve [RESTOR-MV]; NCT00120276).