Winnie S. Liang

Multiple myeloma is a treatable, but currently incurable, hematological malignancy of plasma cells characterized by diverse and complex tumor genetics for which precision medicine approaches to treatment are lacking. The Multiple Myeloma Research Foundation’s Relating Clinical Outcomes in Multiple Myeloma to Personal Assessment of Genetic Profile study (NCT01454297) is a longitudinal, observational clinical study of newly diagnosed patients with multiple myeloma (n = 1,143) where tumor samples are characterized using whole-genome sequencing, whole-exome sequencing and RNA sequencing at diagnosis and progression, and clinical data are collected every 3 months. Analyses of the baseline cohort identified genes that are the target of recurrent gain-of-function and loss-of-function events. Consensus clustering identified 8 and 12 unique copy number and expression subtypes of myeloma, respectively, identifying high-risk genetic subtypes and elucidating many of the molecular underpinnings of these unique biological groups. Analysis of serial samples showed that 25.5% of patients transition to a high-risk expression subtype at progression. We observed robust expression of immunotherapy targets in this subtype, suggesting a potential therapeutic option.

The posterior cingulate cortex (PCC) is a key hub of the default mode network underlying autobiographical memory retrieval, which falters early in the progression of Alzheimer’s disease (AD). We recently performed RNA sequencing of post-mortem PCC tissue samples from 26 elderly Rush Religious Orders Study participants who came to autopsy with an ante-mortem diagnosis of no cognitive impairment but who collectively displayed a range of Braak I–IV neurofibrillary tangle stages. Notably, cognitively unimpaired subjects displaying high Braak stages may represent cognitive resilience to AD pathology. Transcriptomic data revealed elevated synaptic and ATP-related gene expression in Braak Stages III/IV compared with Stages I/II, suggesting these pathways may be related to PCC resilience. We also mined expression profiles for small non-coding micro-RNAs (miRNAs), which regulate mRNA stability and may represent an underexplored potential mechanism of resilience through the fine-tuning of gene expression within complex cellular networks. Twelve miRNAs were identified as differentially expressed between Braak Stages I/II and III/IV. However, the extent to which the levels of all identified miRNAs were associated with subject demographics, neuropsychological test performance and/or neuropathological diagnostic criteria within this cohort was not explored. Here, we report that a total of 667 miRNAs are significantly associated (rho > 0.38, P < 0.05) with subject variables. There were significant positive correlations between miRNA expression levels and age, perceptual orientation and perceptual speed. By contrast, higher miRNA levels correlated negatively with semantic and episodic memory. Higher expression of 15 miRNAs associated with lower Braak Stages I–II and 47 miRNAs were associated with higher Braak Stages III–IV, suggesting additional mechanistic influences of PCC miRNA expression with resilience. Pathway analysis showed enrichment for miRNAs operating in pathways related to lysine degradation and fatty acid synthesis and metabolism. Finally, we demonstrated that the 12 resilience-related miRNAs differentially expressed in Braak Stages I/II versus Braak Stages III/IV were predicted to regulate mRNAs related to amyloid processing, tau and inflammation. In summary, we demonstrate a dynamic state wherein differential PCC miRNA levels are associated with cognitive performance and post-mortem neuropathological AD diagnostic criteria in cognitively intact elders. We posit these relationships may inform miRNA transcriptional alterations within the PCC relevant to potential early protective (resilience) or pathogenic (pre-clinical or prodromal) responses to disease pathogenesis and thus may be therapeutic targets.

The posterior cingulate cortex, a key hub of the default mode network, underlies autobiographical memory retrieval and displays hypometabolic changes early in Alzheimer disease. To obtain an unbiased understanding of the molecular pathobiology of the aged posterior cingulate cortex, we performed RNA sequencing (RNA-seq) on tissue obtained from 26 participants of the Rush Religious Orders Study (11 males/15 females; aged 76–96 years) with a pre-mortem clinical diagnosis of no cognitive impairment and post-mortem neurofibrillary tangle Braak Stages I/II, III, and IV. Transcriptomic data were gathered using next-generation sequencing of RNA extracted from posterior cingulate cortex generating an average of 60 million paired reads per subject. Normalized expression of RNA-seq data was calculated using a global gene annotation and a microRNA profile. Differential expression (DESeq2, edgeR) using Braak staging as the comparison structure isolated genes for dimensional scaling, associative network building and functional clustering. Curated genes were correlated with the Mini-Mental State Examination and semantic, working and episodic memory, visuospatial ability, and a composite Global Cognitive Score. Regulatory mechanisms were determined by co-expression networks with microRNAs and an overlap of transcription factor binding sites. Analysis revealed 750 genes and 12 microRNAs significantly differentially expressed between Braak Stages I/II and III/IV and an associated six groups of transcription factor binding sites. Inputting significantly different gene/network data into a functional annotation clustering model revealed elevated presynaptic, postsynaptic and ATP-related expression in Braak Stages III and IV compared with Stages I/II, suggesting these pathways are integral for cognitive resilience seen in unimpaired elderly subjects. Principal component analysis and Kruskal–Wallis testing did not associate Braak stage with cognitive function. However, Spearman correlations between genes and cognitive test scores followed by network analysis revealed upregulation of classes of synaptic genes positively associated with performance on the visuospatial perceptual orientation domain. Upregulation of key synaptic genes suggests a role for these transcripts and associated synaptic pathways in cognitive resilience seen in elders despite Alzheimer disease pathology and dementia.

PURPOSE: Hyperthermic intraperitoneal chemotherapy (HIPEC) confers a survival benefit in epithelial ovarian cancer (EOC) and in preclinical models. However, the molecular changes induced by HIPEC have not been corroborated in humans. PATIENTS AND METHODS: A feasibility trial evaluated clinical and safety outcomes of HIPEC with cisplatin during optimal cytoreductive surgery (CRS) in patients with EOC diagnosed with stage III, IV, or recurrent EOC. Pre- and post-HIPEC biopsies were comprehensively profiled with genomic and transcriptomic sequencing to identify mutational and RNAseq signatures correlating with response; the tumor microenvironment was profiled to identify potential immune biomarkers; and transcriptional signatures of tumors and normal samples before and after HIPEC were compared to investigate HIPEC-induced acute transcriptional changes. RESULTS: Thirty-five patients had HIPEC at the time of optimal CRS; all patients had optimal CRS. The median progression-free survival (PFS) was 24.7 months for primary patients and 22.4 for recurrent patients. There were no grade 4 or 5 adverse events. Anemia was the most common grade 3 adverse event (43%). Hierarchical cluster analyses identified distinct transcriptomic signatures of good versus poor responders to HIPEC correlating with a PFS of 29.9 versus 7.3 months, respectively. Among good responders, significant HIPEC-induced molecular changes included immune pathway upregulation and DNA repair pathway downregulation. Within cancer islands, % programmed cell death protein 1 expression in CD8+ T cells significantly increased after HIPEC. An exceptional responder (PFS 58 months) demonstrated the highest programmed cell death protein 1 increase. Heat shock proteins comprised the top differentially upregulated genes in HIPEC-treated tumors. CONCLUSION: Distinct transcriptomic signatures identify responders to HIPEC, and preclinical model findings are confirmed for the first time in a human cohort.

The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor–normal genomic DNA (gDNA) samples derived from a breast cancer cell line—which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations—and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking ‘tumor-only’ or ‘matched tumor–normal’ analyses.

Multiple myeloma is a treatable, but currently incurable, hematological malignancy of plasma cells characterized by diverse and complex tumor genetics for which precision medicine approaches to treatment are lacking. The MMRF CoMMpass study is a longitudinal, observational clinical study of newly diagnosed multiple myeloma patients where tumor samples are characterized using whole genome, exome, and RNA sequencing at diagnosis and progression, and clinical data is collected every three months. Analyses of the baseline cohort identified genes that are the target of recurrent gain- and loss-of-function events. Consensus clustering identified 8 and 12 unique copy number and expression subtypes of myeloma, respectively, identifying high- risk genetic subtypes and elucidating many of the molecular underpinnings of these unique biological groups. Analysis of serial samples showed 25.5% of patients transition to a high-risk expression subtype at progression. We observed robust expression of immunotherapy targets in this subtype, suggesting a potential therapeutic option.

Here we present a novel statistical approach to splicing outlier and differential splicing detection, implemented in a software package called Bisbee. We leverage Bisbee’s prediction of protein level effects to benchmark using matched RNAseq and mass spectrometry data from normal tissues. Bisbee exhibits improved sensitivity and specificity over existing approaches. We applied Bisbee to confirm a pathogenic splicing event in a rare disease and to identify tumor-specific splice isoforms associated with an oncogenic splice factor mutation. We also identified common tumor associated splice isoforms replicated in an independent dataset, demonstrating the utility of Bisbee in discovering disease relevant splice variants.

Circular RNAs (circRNAs) are evolutionarily conserved RNA species that are formed when exons “back-splice” to each other. Current computational algorithms to detect these back-splicing junctions produce divergent results, and hence there is a need for a method to distinguish true-positive circRNAs. To this end, we developed Assembly based CircRNA Validator (ACValidator) for in silico verification of circRNAs. ACValidator extracts reads from a user-defined window on either side of a circRNA junction and assembles them to generate contigs. These contigs are aligned against the circRNA sequence to find contigs spanning the back-spliced junction. When evaluated on simulated datasets, ACValidator achieved over ∼80% sensitivity on datasets with an average of 10 circRNA-supporting reads and with read lengths of at least 100 bp. In experimental datasets, ACValidator produced higher verification percentages for samples treated with ribonuclease R compared to nontreated samples. Our workflow is applicable to non-polyA-selected RNAseq datasets and can also be used as a candidate selection strategy for prioritizing experimental validations. All workflow scripts are freely accessible on our GitHub page https://github.com/tgen/ACValidator along with detailed instructions to set up and run ACValidator.