A seamless phase II/III design with dose optimization for oncology drug development.

The US FDA's Project Optimus initiative that emphasizes dose optimization prior to marketing approval represents a pivotal shift in oncology drug development. It has a ripple effect for rethinking what changes may be made to conventional pivotal trial designs to incorporate a dose optimization component. Aligned with this initiative, we propose a novel seamless phase II/III design with dose optimization (SDDO framework). The proposed design starts with dose optimization in a randomized setting, leading to an interim analysis focused on optimal dose selection, trial continuation decisions, and sample size re-estimation (SSR). Based on the decision at interim analysis, patient enrollment continues for both the selected dose arm and control arm, and the significance of treatment effects will be determined at final analysis. The SDDO framework offers increased flexibility and cost-efficiency through sample size adjustment, while stringently controlling the Type I error. This proposed design also facilitates both accelerated approval (AA) and regular approval in a "one-trial" approach. Extensive simulation studies confirm that our design reliably identifies the optimal dosage and makes preferable decisions with a reduced sample size while retaining statistical power.

Ramucirumab plus pembrolizumab in patients with previously treated advanced non-small-cell lung cancer, gastro-oesophageal cancer, or urothelial carcinomas (JVDF): a multicohort, non-randomised, open-label, phase 1a/b trial.

BACKGROUND: Pre-clinical and clinical evidence suggests that simultaneous blockade of VEGF receptor-2 (VEGFR-2) and PD-1 or PD-L1 enhances antigen-specific T-cell migration, antitumour activity, and has favourable toxicity. In this study, we aimed to assess the safety and preliminary antitumour activity of ramucirumab (an IgG1 VEGFR-2 antagonist) combined with pembrolizumab (an IgG4 PD-1 antagonist) in patients with previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma, non-small-cell lung cancer, or urothelial carcinoma. METHODS: We did a multicohort, non-randomised, open-label, phase 1a/b trial at 16 academic medical centres, hospitals, and clinics in the USA, France, Germany, Spain, and the UK. We enrolled adult patients aged 18 years or older with histologically confirmed gastric or gastro-oesophageal junction adenocarcinoma (cohorts A and B), non-small-cell lung cancer (cohort C), or urothelial carcinoma (cohort D), whose disease had progressed on one or two lines of previous therapy (for those with gastric or gastro-oesophageal junction adenocarcinoma) or one to three lines of previous therapy (for those with non-small-cell lung cancer and urothelial carcinoma) that included platinum (for all tumour types) or fluoropyrimidine or both (for gastric or gastro-oesophageal junction adenocarcinoma). Eligibility criteria included presence of measurable disease and an Eastern Cooperative Oncology Group performance status of 0-1. Patients with previously untreated gastric or gastro-oesophageal junction adenocarcinoma and non-small-cell lung cancer were also enrolled (in two additional separate cohorts); the results for these cohorts will be reported separately. The first 21-day treatment cycle was a dose-limiting toxicity observation period (phase 1a; safety run-in), followed by a phase 1b cohort expansion stage. Pembrolizumab 200 mg was administered intravenously on day 1, and intravenous ramucirumab was administered at 8 mg/kg on days 1 and 8 for cohort A or at 10 mg/kg on day 1 for cohorts B, C, and D, every 3 weeks, until disease progression or other discontinuation criteria were met. The primary endpoint was the safety and tolerability of ramucirumab in combination with pembrolizumab assessed by the incidence of adverse events in both phase 1a and 1b and as dose-limiting toxicities during phase 1a. The safety and activity analysis set included all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02443324, and is no longer enrolling patients. FINDINGS: Between July 30, 2015 and June 24, 2016, we enrolled and treated 92 patients (41 with gastric or gastro-oesophageal junction adenocarcinoma, 27 with non-small-cell lung cancer, and 24 with urothelial carcinoma). Median follow-up was 32·8 months (IQR 28·1-33·6). During the first cycle of treatment (phase 1a safety run-in; n=11), one patient with gastro-oesophageal junction adenocarcinoma who received the 8 mg/kg dose of ramucirumab had grade 3 abdominal pain, colitis, hepatitis, interstitial lung disease, and jaundice, and grade 4 cholestasis, and died on treatment on day 40; the death was deemed related to progressive disease. No additional dose-limiting toxicities occurred and the decision was made to maintain the full planned doses of ramucirumab and pembrolizumab in phase 1b (n=81). Treatment-related adverse events occurred in 75 (82%) of 92 patients, the most common of which was fatigue (in 33 patients [36%]), predominantly of grade 1 or 2 severity. 22 patients (24%) had one or more treatment-related adverse events of grade 3 or worse, most commonly hypertension (six patients; 7%) and colitis (five patients; 5%). Serious adverse events occurred in 53 (58%) of 92 patients, and were deemed related to treatment in 22 (24%) patients. The most common treatment-related serious adverse events were abdominal pain in patients with gastric or gastro-oesophageal junction adenocarcinoma (in three [7%] of 41 patients); asthenia and myocardial infarction in patients with non-small-cell lung cancer (two [7%] of 27 patients), and colitis in patients with urothelial carcinoma (two [8%] of 24 patients). Six (7%) of 92 patients discontinued treatment because of treatment-related adverse events, and one death (from pulmonary sepsis in a patient with gastric or gastro-oesophageal junction adenocarcinoma) was deemed related to treatment. The number of patients achieving an objective response was three (7%; 95% CI 1·5-19·9) of 41 in the gastric or gastro-oesophageal junction adenocarcinoma cohort, eight (30%; 13·8-50·2) of 27 in the non-small-cell lung cancer cohort, and three (13%, 2·7-32·4) in the urothelial carcinoma cohort. INTERPRETATION: Ramucirumab in combination with pembrolizumab showed a manageable safety profile with favourable antitumour activity in patients with previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma, non-small-cell lung cancer, and urothelial carcinoma. Our results contribute to the growing evidence that supports dual inhibition of the VEGF-VEGFR2 and PD-1-PD-L1 pathways. This combination could be further explored with or without chemotherapy, especially for patients with tumours for which single-agent checkpoint inhibitors have shown no additional benefit over chemotherapy. FUNDING: Eli Lilly and Company, and Merck and Co.

Galactosylated chitosan triptolide nanoparticles for overcoming hepatocellular carcinoma: Enhanced therapeutic efficacy, low toxicity, and validated network regulatory mechanisms.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Current therapies present significant limitations. Triptolide (TP) is highly effective against multiple cancers including HCC. However, high toxicity, low water solubility, and unknown therapeutic targets limit its clinical application. Herein, we designed galactosylated-chitosan-TP-nanoparticles (GC-TP-NPs) with high drug loading capacities for targeted delivery to HCC. In addition to a sustained release pattern, an efficient asialoglycoprotein receptor mediated cellular uptake in vitro, and high liver tumor accumulation in vivo, GC-TP-NPs showed lower systemic and male reproductive toxicities than free TP. Importantly, GC-TP-NPs retained the anti-cancer activities of the free TP, exerting the same pro-apoptotic and anti-proliferative effects on HCC cells in vitro, and displayed higher efficacies in reducing tumor sizes in vivo. Further investigation revealed that GC-TP-NPs induced cancer cell apoptosis via blocking TNF/NF-κB/BCL2 signaling. Collectively, GC-TP-NP represents a promising candidate in halting liver cancer progression while minimizing systemic toxicity.

Ramucirumab Plus Pembrolizumab in Patients with Previously Treated Advanced or Metastatic Biliary Tract Cancer: Nonrandomized, Open-Label, Phase I Trial (JVDF).

LESSONS LEARNED: Ramucirumab plus pembrolizumab revealed no unexpected safety findings in patients with advanced or metastatic biliary tract cancer, which is consistent with reports of other tumor cohorts within this phase Ia/b trial.Ramucirumab plus pembrolizumab did not demonstrate an improvement in overall survival when compared with historical controls in biomarker unselected, heavily pretreated patients with advanced or metastatic biliary tract cancer.Patients with programmed death-ligand 1 (PD-L1)-positive tumors had improved overall survival compared with patients with PD-L1-negative disease. BACKGROUND: Few treatment options exist for patients with advanced biliary tract cancer (BTC) following progression on gemcitabine-cisplatin. Preclinical evidence suggests that simultaneous blockade of vascular endothelial growth factor receptor 2 (VEGFR-2) and programmed death 1 (PD-1) or programmed death-ligand 1 (PD-L1) enhances antitumor effects. We assessed the safety and efficacy of ramucirumab, an IgG1 VEGFR-2 antagonist, with pembrolizumab, an IgG4 PD-1 antagonist, in biomarker-unselected patients with previously treated advanced or metastatic BTC. METHODS: Patients had previously treated advanced or metastatic adenocarcinoma of the gallbladder, intrahepatic and extrahepatic bile ducts, or ampulla of Vater. Ramucirumab 8 mg/kg was administered intravenously on days 1 and 8 with intravenous pembrolizumab 200 mg on day 1 every 3 weeks. The primary endpoint was safety and tolerability of the combination. Secondary endpoints included objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: Twenty-six patients were treated at 12 centers in five countries. Hypertension was the most common grade 3 treatment-related adverse event (TRAE), occurring in five patients. One patient experienced a grade 4 TRAE (neutropenia), and no treatment-related deaths occurred. Objective response rate was 4%. Median progression-free survival and overall survival were 1.6 months and 6.4 months, respectively. CONCLUSION: Ramucirumab-pembrolizumab showed limited clinical activity with infrequent grade 3-4 TRAEs in patients with biomarker-unselected progressive BTC.

Enhancement of the adaptive signature design for learning and confirming in a single pivotal trial.

Because of the complexity of cancer biology, often the target pathway is not well understood at the time that phase III trials are initiated. A 2-stage trial design was previously proposed for identifying a subgroup of interest in a learn stage, on the basis of 1 or more baseline biomarkers, and then subsequently confirming it in a confirmation stage. In this article, we discuss some practical aspects of this type of design and describe an enhancement to this approach that can be built into the study randomization to increase the robustness of the evaluation. Furthermore, we show via simulation studies how the proportion of patients allocated to the learn stage versus the confirm stage impacts the power and provide recommendations.

A Multi-Arm Two-Stage (MATS) design for proof-of-concept and dose optimization in early-phase oncology trials.

The Project Optimus initiative by the FDA's Oncology Center of Excellence is widely viewed as a groundbreaking effort to change the status quo of conventional dose-finding strategies in oncology. Unlike in other therapeutic areas where multiple doses are evaluated thoroughly in dose ranging studies, early-phase oncology dose-finding studies are characterized by the practice of identifying a single dose, such as the maximum tolerated dose (MTD) or the recommended phase 2 dose (RP2D). Following the spirit of Project Optimus, we propose an Multi-Arm Two-Stage (MATS) design for proof-of-concept (PoC) and dose optimization that allows the evaluation of two selected doses from a dose-escalation trial. The design assesses the higher dose first across multiple indications in the first stage, and adaptively enters the second stage for an indication if the higher dose exhibits promising anti-tumor activities. In the second stage, a randomized comparison between the higher and lower doses is conducted to achieve PoC and dose optimization. A Bayesian hierarchical model governs the statistical inference and decision making by borrowing information across doses, indications, and stages. Our simulation studies show that the proposed MATS design yield desirable performance. An R Shiny application has been developed and made available at https://matsdesign.shinyapps.io/mats/.

SPA: Single patient acceleration in oncology dose-escalation trials.

Efficient identification of the optimal dose and dosing scheme is one of the most critical and challenging tasks in early-phase oncology trials. The results are far-reaching because advancing a sub-optimal dose to late-stage development may not only jeopardize patients' safety or fail to deliver desired efficacy, but also be costly to sponsors as refined doses must be evaluated further before seeking regulatory approval. A good dose-escalation design is anticipated to yield high accuracy of selecting the correct dose while using fewer patients and keeping the trial duration short. Recently, treating a single patient at each lower dose level until certain events are triggered to switch to larger cohorts has gained much popularity. We name this approach "Single Patient Acceleration" (SPA), which is essentially a variant of the Accelerated Titration Design (ATD) by Simon et al. [25]. Although literature on novel dose-escalation methods is abundant in the past decade, there is a surprisingly lack of research on evaluating the ATD/SPA framework. In this article, we conduct comprehensive simulations to evaluate the performance of dose-escalation designs with or without SPA, and show that SPA improves design efficiency with similar or better accuracy to those without the "single patient" component under certain circumstances (e.g., slow initial enrollment, or the true maximum tolerated dose is at higher candidate dose levels). Potential safety concerns as a cost of efficiency improvement are also investigated in a quantitative manner to illustrate a comprehensive benefit-risk profile of SPA. Practical considerations and recommendations in using SPA are also discussed.

Phase 1 Expansion Cohort of Ramucirumab Plus Pembrolizumab in Advanced Treatment-Naive NSCLC.

INTRODUCTION: Data of first-line ramucirumab plus pembrolizumab treatment of programmed death-ligand 1 (PD-L1)-positive NSCLC (cohort E) are reported (NCT02443324). METHODS: In this multicenter, open-label phase 1a/b trial, patients received ramucirumab 10 mg/kg and pembrolizumab 200 mg every 21 days for up to 35 cycles. PD-L1 positivity was defined as tumor proportion score (TPS) greater than or equal to 1%. Exploratory NanoString biomarker analyses included three T-cell signatures (T-cell-inflamed, Gajewski, and effector T cells) and CD274 gene expression. RESULTS: Cohort E included 26 patients. Treatment-related adverse events of any grade occurred in 22 patients (84.6%). Treatment-related adverse events of grade greater than or equal to 3 were reported in 11 patients (42.3%); the most frequent was hypertension (n = 4, 15.4%). Objective response rate was 42.3% in the treated population and 56.3% and 22.2% for patients with high (TPS ≥ 50%) and lower levels (TPS 1%-49%) of PD-L1 expression, respectively. Median progression-free survival (PFS) in the treated population was 9.3 months, and 12-month and 18-month PFS rates were 45% each. Median PFS was not reached in patients with PD-L1 TPS greater than or equal to 50% and was 4.2 months in patients with PD-L1 TPS 1% to 49%. Median overall survival was not reached in the treated population, and 12-month and 18-month overall survival rates were 73% and 64%, respectively. Biomarker data suggested a positive association among clinical response, three T-cell signatures, CD274 gene expression, and PD-L1 immunohistochemistry. CONCLUSIONS: First-line therapy with ramucirumab plus pembrolizumab has a manageable safety profile in patients with NSCLC, and the efficacy signal seems to be strongest in tumors with high PD-L1 expression.

Ramucirumab and durvalumab for previously treated, advanced non-small-cell lung cancer, gastric/gastro-oesophageal junction adenocarcinoma, or hepatocellular carcinoma: An open-label, phase Ia/b study (JVDJ).

BACKGROUND: Emerging evidence supports combining immune checkpoint inhibitors (ICIs) with conventional or targeted therapies to enhance ICI antitumour activity and broaden the spectrum of patients who respond to ICIs. Here, we present the safety and preliminary efficacy of ramucirumab, an anti-VEGFR2 IgG1, plus durvalumab, an anti-PD-L1 IgG1, in previously treated patients with advanced non-small-cell lung cancer (NSCLC), gastric/gastro-oesophageal junction adenocarcinoma (gastric/GEJ), or hepatocellular carcinoma (HCC). PATIENTS AND METHODS: A 25-centre, phase Ia/b single-arm, non-randomised, multi-cohort study was undertaken in patients with advanced/metastatic disease, Eastern Cooperative Oncology Group performance status, 0-1, progression on prior therapy, no prior ramucirumab or immunotherapy and any PD-L1 status. Patients received ramucirumab (10 mg/kg) plus durvalumab (1125 mg) intravenously Q3W (NSCLC), or ramucirumab (8 mg/kg) plus durvalumab (750 mg) Q2W (gastric/GEJ, HCC). RESULTS: Phase Ia treatment was found safe for phase Ib expansion; final enrolment was NSCLC (n = 28), gastric/GEJ (n = 29), HCC (n = 28). Grade ≥3 treatment-related adverse events occurred in 32.1%, 37.9% and 42.9% of patients, respectively. The most common were fatigue (35.7%), hypertension (34.5%) and diarrhoea (28.6%), respectively. Two patients died owing to an adverse event; one was treatment-related (hepatitis acute, HCC cohort). Objective response rate was 11% for NSCLC and HCC and 21% for gastric/GEJ. Median progression-free survival and overall survival were, respectively, 2.7 and 11 months in NSCLC; 2.6 and 12.4 months in gastric/GEJ; 4.4 and 10.7 months in HCC, with more prolonged survival in patients with high PD-L1 expression. CONCLUSION: Ramucirumab/durvalumab exhibited manageable safety. The combination showed antitumour activity in all cohorts, particularly in patients with high PD-L1 expression.

Ramucirumab in Combination with Pembrolizumab in Treatment-Naïve Advanced Gastric or GEJ Adenocarcinoma: Safety and Antitumor Activity from the Phase 1a/b JVDF Trial.

Ramucirumab (anti-VEGFR2) plus pembrolizumab (anti-PD1) demonstrated promising antitumor activity and tolerability among patients with previously treated advanced cancers, supporting growing evidence that combination therapies modulating the tumor microenvironment may expand the spectrum of patients who respond to checkpoint inhibitors. Here we present the results of this combination in first-line patients with metastatic G/GEJ cancer. Twenty-eight patients (≥18 years) with no prior systemic chemotherapy in the advanced/metastatic setting received ramucirumab (8 mg/kg days 1 and 8) plus pembrolizumab (200 mg day 1) every 3 weeks as part of JVDF phase 1a/b study. The primary endpoint was safety. Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), and overall survival (OS). Tumors were PD-L1-positive (combined positive score ≥ 1) in 19 and -negative in 6 patients. Eighteen patients experienced grade 3 treatment-related adverse events, most commonly hypertension (14%) and elevated alanine/aspartate aminotransferase (11% each), with no grade 4 or 5 reported. The ORR was 25% (PD-L1-positive, 32%; PD-L1-negative, 17%) with duration of response not reached. PFS was 5.6 months (PD-L1-positive, 8.6 months; PD-L1-negative, 4.3 months), and OS 14.6 months (PD-L1-positive, 17.3 months; PD-L1-negative, 11.3 months). Acknowledging study design limitations, ramucirumab plus pembrolizumab had encouraging durable clinical activity with no unexpected toxicities in treatment-naïve biomarker-unselected metastatic G/GEJ cancer, and improved outcomes in patients with PD-L1-positive tumors.

EGFR Gene Copy Number by FISH May Predict Outcome of Necitumumab in Squamous Lung Carcinomas: Analysis from the SQUIRE Study.

INTRODUCTION: Necitumumab is a monoclonal antibody targeting EGFR. In the SQUIRE trial, the addition of necitumumab to chemotherapy for squamous cell lung cancer significantly improved overall survival (OS) (hazard ratio [HR] = 0.84); in a post hoc analysis, EGFR copy number gain determined by fluorescence in situ hybridization (FISH) showed a trend toward improved OS (HR = 0.70) and progression-free survival (PFS) (HR = 0.71) with the addition of necitumumab. We present the analysis of granular EGFR FISH data from SQUIRE to examine the potential predictive role of high polysomy and gene amplification, as both were included in the FISH-positive category. METHODS: Available specimens from SQUIRE underwent FISH analysis in a central laboratory, and each sample was evaluated by using the Colorado EGFR scoring criteria. The correlation of granular FISH parameters with clinical outcomes was assessed. RESULTS: Samples were available for 557 of 1093 patients; 208 patients (37.3%) were FISH-positive, including 167 (30.0%) with high polysomy and 41 (7.4%) with gene amplification. In patients with high polysomy, the addition of necitumumab resulted in a statistically significant increase in PFS (6.08 versus 5.13 months [p = 0.044]) and nonstatistically significant increase in OS (12.6 versus 9.5 months [p = 0.133]); among patients with gene amplification, the addition of necitumumab did not significantly improve PFS (7.4 versus 5.6 months; [p = 0.334]) but did improve OS (14.8 versus 7.6 months; [p = 0.033]). CONCLUSIONS: EGFR copy number gain by FISH might have a role as a predictive biomarker for necitumumab in squamous cell lung cancer. In our opinion, these data encourage further studies to prospectively evaluate this potential biomarker.

Ramucirumab Safety in East Asian Patients: A Meta-Analysis of Six Global, Randomized, Double-Blind, Placebo-Controlled, Phase III Clinical Trials.

Purpose Several ramucirumab trials have reported a higher incidence of selected adverse events (AEs) in East Asian (EA) patients with cancer versus non-EA patients. A meta-analysis was conducted across six completed phase III trials to establish the safety parameters of ramucirumab in EA compared with non-EA patients. Materials and Methods Six global, randomized, double-blind, placebo-controlled, phase III registration trials investigating ramucirumab were assessed. Relative risks (RRs) and 95% CIs were calculated for selected all-grade and grade ≥ 3 AEs using fixed-effects and mixed-effects models. Ratio of RR and number needed to harm were calculated for AEs (all grade and grade ≥ 3) between EA and non-EA patients. Results Of 4,996 randomly assigned patients receiving ramucirumab or placebo, 802 (16.1%) were EA (ramucirumab, n = 411; placebo, n = 391) and 4,194 were non-EA (ramucirumab, n = 2,337; placebo, n = 1,857). Patient baseline characteristics were generally balanced between treatment arms in EA and non-EA patients, excluding sex and body weight. Grade ≥ 3 AEs possibly associated with ramucirumab, which were increased in EA versus non-EA patients, included neutropenia (42.1% v 25.5%, respectively) and proteinuria (3.9% v 0.6%, respectively). There was an increase in the RR of several grade ≥ 3 AEs, including hypertension and proteinuria, in ramucirumab-treated EA and non-EA patients compared with placebo. The ratio of RR revealed no significant differences between EA and non-EA patients for all-grade and grade ≥ 3 AEs. Conclusion Despite the enhanced propensity of selected AEs in EA patients relative to non-EA patients, there were no substantial differences in the RR for AEs possibly associated with ramucirumab in these phase III trials.

Peroxiredoxin 6 Overexpression Induces Anxiolytic and Depression-Like Behaviors by Regulating the Serotonergic Pathway in Mice

Peroxiredoxin 6 (PRDX6) is a bifunctional protein with both glutathione peroxidase and calcium-independent phospholipase activity. Recently, we reported that PRDX6 plays an important role in dopaminergic neurodegeneration in Parkinson’s disease.However, the relationship between PRDX6 function and emotional behavior remains elusive. In the present study, we examined depression- and anxiety-like behaviors in PRDX6-overexpressing transgenic (PRDX6-Tg) mice using the forced swim test, tail suspension test, open field paradigm, and elevated plus-maze. PRDX6-Tg mice exhibited depression-like behaviors and low anxiety. In particular, female PRDX6-Tg mice exhibited anxiolytic behavior in the open field test. Furthermore, the serotonin content in the cortex and 5-hydroxytryptophan-induced head twitch response were both reduced in PRDX6-Tg mice. Interestingly, levels of dopa decarboxylase expression in the cortex were decreased in male PRDX6-Tg mice but not in female mice. Our findings provide novel insights into the role of PRDX6 in 5-HT synthesis and suggest that PRDX6 overexpression can induce depression-like behaviors via downregulation of the serotonergic neuronal system.

The level of residual dispersion variation and the power of differential expression tests for RNA-Seq data.

RNA-Sequencing (RNA-Seq) has been widely adopted for quantifying gene expression changes in comparative transcriptome analysis. For detecting differentially expressed genes, a variety of statistical methods based on the negative binomial (NB) distribution have been proposed. These methods differ in the ways they handle the NB nuisance parameters (i.e., the dispersion parameters associated with each gene) to save power, such as by using a dispersion model to exploit an apparent relationship between the dispersion parameter and the NB mean. Presumably, dispersion models with fewer parameters will result in greater power if the models are correct, but will produce misleading conclusions if not. This paper investigates this power and robustness trade-off by assessing rates of identifying true differential expression using the various methods under realistic assumptions about NB dispersion parameters. Our results indicate that the relative performances of the different methods are closely related to the level of dispersion variation unexplained by the dispersion model. We propose a simple statistic to quantify the level of residual dispersion variation from a fitted dispersion model and show that the magnitude of this statistic gives hints about whether and how much we can gain statistical power by a dispersion-modeling approach.

Goodness-of-fit tests and model diagnostics for negative binomial regression of RNA sequencing data.

This work is about assessing model adequacy for negative binomial (NB) regression, particularly (1) assessing the adequacy of the NB assumption, and (2) assessing the appropriateness of models for NB dispersion parameters. Tools for the first are appropriate for NB regression generally; those for the second are primarily intended for RNA sequencing (RNA-Seq) data analysis. The typically small number of biological samples and large number of genes in RNA-Seq analysis motivate us to address the trade-offs between robustness and statistical power using NB regression models. One widely-used power-saving strategy, for example, is to assume some commonalities of NB dispersion parameters across genes via simple models relating them to mean expression rates, and many such models have been proposed. As RNA-Seq analysis is becoming ever more popular, it is appropriate to make more thorough investigations into power and robustness of the resulting methods, and into practical tools for model assessment. In this article, we propose simulation-based statistical tests and diagnostic graphics to address model adequacy. We provide simulated and real data examples to illustrate that our proposed methods are effective for detecting the misspecification of the NB mean-variance relationship as well as judging the adequacy of fit of several NB dispersion models.

Limonene Inhibits Methamphetamine-Induced Sensitizations via the Regulation of Dopamine Receptor Supersensitivity

Limonene is a cyclic terpene found in citrus essential oils and inhibits methamphetamine-induced locomotor activity. Drug dependence is a severe neuropsychiatric condition that depends in part on changes in neurotransmission and neuroadaptation, induced by exposure to recreational drugs such as morphine and methamphetamine. In this study, we investigated the effects of limonene on the psychological dependence induced by drug abuse. The development of sensitization, dopamine receptor supersensitivity, and conditioned place preferences in rats was measured following administration of limonene (10 or 20 mg/kg) and methamphetamine (1 mg/kg) for 4 days. Limonene inhibits methamphetamine-induced sensitization to locomotor activity. Expression of dopamine receptor supersensitivity induced by apomorphine, a dopamine receptor agonist, was significantly reduced in limonene-pretreated rats. However, there was no significant difference in methamphetamine-induced conditioned place preferences between the limonene and control groups. These results suggest that limonene may ameliorate drug addiction-related behaviors by regulating postsynaptic dopamine receptor supersensitivity.

Length bias correction in gene ontology enrichment analysis using logistic regression.

When assessing differential gene expression from RNA sequencing data, commonly used statistical tests tend to have greater power to detect differential expression of genes encoding longer transcripts. This phenomenon, called "length bias", will influence subsequent analyses such as Gene Ontology enrichment analysis. In the presence of length bias, Gene Ontology categories that include longer genes are more likely to be identified as enriched. These categories, however, are not necessarily biologically more relevant. We show that one can effectively adjust for length bias in Gene Ontology analysis by including transcript length as a covariate in a logistic regression model. The logistic regression model makes the statistical issue underlying length bias more transparent: transcript length becomes a confounding factor when it correlates with both the Gene Ontology membership and the significance of the differential expression test. The inclusion of the transcript length as a covariate allows one to investigate the direct correlation between the Gene Ontology membership and the significance of testing differential expression, conditional on the transcript length. We present both real and simulated data examples to show that the logistic regression approach is simple, effective, and flexible.

Synthetic Cannabinoid-Induced Immunosuppression Augments Cerebellar Dysfunction in Tetanus-Toxin Treated Mice

Synthetic cannabinoids are one of most abused new psychoactive substances. The recreational use of abused drug has aroused serious concerns about the consequences of these drugs on infection. However, the effects of synthetic cannabinoid on resistance to tetanus toxin are not fully understood yet. In the present study, we aimed to determine if the administration of synthetic cannabinoids increase the susceptibility to tetanus toxin-induced motor behavioral deficit and functional changes in cerebellar neurons in mice. Furthermore, we measured T lymphocytes marker levels, such as CD8 and CD4 which against tetanus toxin. JWH-210 administration decreased expression levels of T cell activators including cluster of differentiation (CD) 3ε, CD3γ, CD74p31, and CD74p41. In addition, we demonstrated that JWH-210 induced motor impairment and decrement of vesicle-associated membrane proteins 2 levels in the cerebellum of mice treated with tetanus toxin. Furthermore, cerebellar glutamatergic neuronal homeostasis was hampered by JWH-210 administration, as evidenced by increased glutamate concentration levels in the cerebellum. These results suggest that JWH-210 may increase the vulnerability to tetanus toxin via the regulation of immune function.

Power of association tests in the presence of multiple causal variants.

We show that the statistical power of a single single-nucleotide polymorphism (SNP) score test for genetic association reflects the cumulative effect of all causal SNPs that are correlated with the test SNP. Statistical significance of a score test can sometimes be explained by the collective effect of weak correlations between the test SNP and multiple causal SNPs. In a finite population, weak but significant correlations between the test SNP and the causal SNPs can arise by chance alone. As a consequence, when a single-SNP score test shows significance, the causal SNPs contributing to the power of the test are not necessarily located near the test SNP, nor do they have to be in linkage disequilibrium with the test SNP. These findings are confirmed with the Genetic Analysis Workshop 17 mini-exome data. The findings of this study highlight the often overlooked importance of long-range and weak linkage disequilibrium in genetic association studies.

Altered Brain Activation in Ventral Frontal-Striatal Regions Following a 16-week Pharmacotherapy in Unmedicated Obsessive-Compulsive Disorder

Recent studies have reported that cognitive inflexibility associated with impairments in a frontal-striatal circuit and parietal region is a core cognitive deficit of obsessive-compulsive disorder (OCD). However, few studies have examined progressive changes in these regions following clinical improvement in obsessive-compulsive symptoms. To determine if treatment changes the aberrant activation pattern associated with task switching in OCD, we examined the activation patterns in brain areas after treatment. The study was conducted on 10 unmedicated OCD patients and 20 matched controls using event-related functional magnetic resonance imaging. Treatment improved the clinical symptoms measured by the Yale-Brown Obsessive Compulsive Scale and behavioral flexibility indicated by the switching cost. At baseline, OCD showed significantly less activation in the dorsal and ventral frontal-striatal circuit and parietal regions under the task-switch minus task-repeat condition compared with controls. After treatment, the neural responses in the ventral frontal-striatal circuit in OCD were partially normalized, whereas the activation deficit in dorsal frontoparietal regions that mediate shifting attention or behavioral flexibility persisted. It is suggested that altered brain activation in ventral frontal-striatal regions in OCD patients is associated with their cognitive flexibility and changes in these regions may underlie the pathophysiology of OCD.