Copy number changes, including loss of tumor suppressors and gain/amplification of oncogenes, and copy neutral-loss of heterozygosity (CN-LOH) are important genomic aberrations in oncology for diagnosis, prognosis and therapy selection. Without a publicly available, curated database for annotation/interpretation of these types of events across multiple neoplasms, labs that perform copy number and CN-LOH assessments must build their own interpretative databases either proactively or as they encounter new events, and likely a combination of both of these methods. We describe an internally curated database of over 2300 annotations across multiple cancers that speeds interpretation of cytogenomic array cases by importing BioDiscovery's Nexus Copy NumberTM software-analyzed copy number and CN-LOH events into a Genospace FullViewTM interface which instantly matches the events with related diagnostic, prognostic and therapeutic annotations, including FDA biomarker and NCCN guideline associations, as well as clinical trials for enhanced interpretation and instantaneously adding annotations to reports along with references, trials, sites, table highlights, etc. The process of creating and annually updating the database as well as examples of each step of analysis/annotating/interpreting/reporting will demonstrate the effectiveness of such an approach in a high volume oncology laboratory.

Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.

Familial aggregation of Waldenström macroglobulinemia (WM) cases, and the clustering of B-cell lymphoproliferative disorders among first-degree relatives of WM patients, has been reported. Nevertheless, the possible contribution of inherited susceptibility to familial WM remains unrevealed. We performed whole exome sequencing on germ line DNA obtained from 4 family members in which coinheritance for WM was documented in 3 of them, and screened additional independent 246 cases by using gene-specific mutation sequencing. Among the shared germ line variants, LAPTM5(c403t) and HCLS1(g496a) were the most recurrent, being present in 3/3 affected members of the index family, detected in 8% of the unrelated familial cases, and present in 0.5% of the nonfamilial cases and in <0.05 of a control population. LAPTM5 and HCLS1 appeared as relevant WM candidate genes that characterized familial WM individuals and were also functionally relevant to the tumor clone. These findings highlight potentially novel contributors for the genetic predisposition to familial WM and indicate that LAPTM5(c403t) and HCLS1(g496a) may represent predisposition alleles in patients with familial WM.

The development of targeted therapies based on specific genomic alterations has altered the treatment and management of lung and colorectal cancers. Chromosomal microarray (CMA) has allowed identification of copy number variations (CNVs) in lung and colorectal cancers in great detail, and next-generation sequencing (NGS) is used extensively to analyze the genome of cancers for molecular subtyping and use of molecularly guided therapies. The main objective of this study was to evaluate the utility of combining CMA and NGS for a comprehensive genomic assessment of lung and colorectal adenocarcinomas, especially for detecting drug targets. We compared the results from NGS and CMA data from 60 lung and 51 colorectal tumors. From CMA analysis, 33% were amplified, 89% showed gains, 75% showed losses and 41% demonstrated loss of heterozygosity; pathogenic variants were identified in 81% of colon and 67% lung specimens through NGS. KRAS mutations commonly occurred with loss in TP53 and there was significant loss of BRCA1 and NF1 among male patients with lung cancer. For clinically actionable targets, 23% had targetable CNVs when no pathogenic variants were detected by NGS. The data thus indicate that combining the two approaches provides significant benefit in a routine clinical setting not available by NGS alone.

We hypothesize that bronchial epithelium microRNAs (miRNAs) regulate airway gene expression and may be used to better understand the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). We propose a novel approach to identify miRNAs functionally associated with disease by computing the differential connectivity (DC) of miRNA/mRNA association networks between disease and normal states. By measuring the change in the structure of the miRNA/mRNA disease specific networks we may identify microRNAs which regulate genes that contribute to the disease. We integrate miRNA expression via small RNA sequencing and gene expression data from 182 subjects (129 COPD cases/53 controls). In the differential connectivity (DC) method, each miRNA is assigned a DC score, which captures the overall difference in the pairwise microRNA-gene correlation strengths between case and control …

We hypothesize that bronchial epithelium microRNAs (miRNAs) regulate airway gene expression and may be used to better understand the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). We propose a novel approach to identify miRNAs functionally associated with disease by computing the differential connectivity (DC) of miRNA/mRNA association networks between disease and normal states. By measuring the change in the structure of the miRNA/mRNA disease specific networks we may identify microRNAs which regulate genes that contribute to the disease. We integrate miRNA expression via small RNA sequencing and gene expression data from 182 subjects (129 COPD cases/53 controls). In the differential connectivity (DC) method, each miRNA is assigned a DC score, which captures the overall difference in the pairwise microRNA-gene correlation strengths between case and control …

Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log(2) number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

Methods:COPD (n= 150) or ILD (n= 149) and from normal lung tissue (n= 65). Reads mapping to a microRNA locus were grouped into those with the canonical seed and those with an alternative seed due to variation in the 5’start position (isomiRs). 253 of these samples were also profiled with gene expression and SNP microarrays.

Purpose Genetic predisposition plays a major role in the etiology of melanoma, but known genetic markers only account for a limited fraction of family-history-associated melanoma cases. Expression microarrays have offered the opportunity to identify further genomic profiles correlated with family history of melanoma. We aimed to distinguish mRNA expression signatures between melanoma cases with and without a family history of melanoma. Methods Based on the Nurses’ Health Study, family history was defined as having one or more first-degree family members diagnosed with melanoma. Melanoma diagnosis was confirmed by reviewing pathology reports, and tumor blocks were collected by mail from across the USA. Genomic interrogation was accomplished through evaluating expression profiling of formalin-fixed paraffin-embedded tissues from 78 primary cutaneous invasive melanoma cases, on either a 6K or whole-genome (24K) Illumina gene chip. Gene set enrichment analysis was performed for each batch to determine the differentially enriched pathways and key contributing genes. Results The CXC chemokine receptor 4 (CXCR4) pathway was consistently up-regulated within cases of familial melanoma in both platforms. Leading edge analysis showed four genes from the CXCR4 pathway, including MAPK1 , PLCG1 , CRK , and PTK2 , were among the core members that contributed to the enrichment of this pathway. There was no association between the enrichment of CXCR4 pathway and NRAS , BRAF mutation, or Breslow thickness of the primary melanoma cases. Conclusions We found that the CXCR4 pathway might constitute a novel susceptibility pathway associated with family history of melanoma in first-degree relatives.

Rationale: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome. Objectives: To identify methylation marks that modify gene expression in IPF lung. Methods: We assessed DNA methylation (comprehensive highthroughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation-gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis. Measurements and Main Results: We identified 2,130 differentially methylated regions (DMRs; <5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P < 2.2 X 10(-16)). We validated 13/15 DMRs by targeted analysis of methylation. Methylation-expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P < 2.2 X 10(-16)). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL. Conclusions: These results suggest that DNA methylation may be involved in the pathogenesis of IPF.

Methods: As part of the Lung Genomics Research Consortium, we sequenced the small RNA in lung tissue samples from subjects with COPD (n= 150) or ILD (n= 149) and from normal lung tissue (n= 65). Reads mapping to a microRNA locus were grouped into those with the canonical seed and those with an alternative seed due to variation in the 5'start position (isomiRs). 253 of these samples were also profiled with gene expression and SNP microarrays.Results: The expression levels of 309 canonical microRNAs and 242 isomiRs were significantly different between patients with disease and controls (FDR< 0.05). Unsupervised clustering of these microRNAs revealed four distinct groups of subjects with distinct clinical and pathological characteristics including groups enriched for COPD subjects, ILD subjects, or an admixture of both ILD and COPD subjects. Separate causality networks linking SNPs with …

METHODS: Methylation and expression profiles on lung tissue from 94 IPF and 67 control subjects were collected on the Comprehensive High-throughput Arrays for Relative Methylation (CHARM) and Agilent SurePrint G3 platforms, respectively. Methylation arrays were scaled and normalized using the CHARM package and 50% of probes with most variance across all samples were used in further analysis. Expression arrays were normalized using the Robust Multi-array Average (RMA). Limma package was used for expression and Matrix eQTL was used for QTL analysis.RESULTS: After controlling for age, gender, smoking status, and technical variables, we identified 1315 genes differentially expressed at 5% false discovery rate (FDR) and with> 2 fold change in IPF compared to control. QTL analysis identified methylation marks that control expression of these genes both in cis and trans, with an enrichment for …

Background Rhesus macaques (RMs) inoculated with live-attenuated Rev-Independent Nef¯ simian immunodeficiency virus (Rev-Ind Nef¯SIV) as adults or neonates controlled viremia to undetectable levels and showed no signs of immunodeficiency over 6-8 years of follow-up. We tested the capacity of this live-attenuated virus to protect RMs against pathogenic, heterologous SIVsmE660 challenges. Methodology/Principal Findings Three groups of four RM were inoculated with Rev-Ind Nef¯SIV and compared. Group 1 was inoculated 8 years prior and again 15 months before low dose intrarectal challenges with SIVsmE660. Group 2 animals were inoculated with Rev-Ind Nef¯SIV at 15 months and Group 3 at 2 weeks prior to the SIVsmE660 challenges, respectively. Group 4 served as unvaccinated controls. All RMs underwent repeated weekly low-dose intrarectal challenges with SIVsmE660. Surprisingly, all RMs with acute live-attenuated virus infection (Group 3) became superinfected with the challenge virus, in contrast to the two other vaccine groups (Groups 1 and 2) (P=0.006 for each) and controls (Group 4) (P=0.022). Gene expression analysis showed significant upregulation of innate immune response-related chemokines and their receptors, most notably CCR5 in Group 3 animals during acute infection with Rev-Ind Nef¯SIV. Conclusions/Significance We conclude that although Rev-Ind Nef¯SIV remained apathogenic, acute replication of the vaccine strain was not protective but associated with increased acquisition of heterologous mucosal SIVsmE660 challenges.

(COPD). Cigarette smoking is the primary risk factor for COPD and smoking has been associated with changes in DNA methylation. Assessing the contribution of epigenetic mechanisms to COPD pathogenesis is critical and methylation changes associated with smoking must be carefully considered.Using samples from the Lung Tissue Research Consortium (LTRC) lung tissue DNA methylation profiles were collected from 61 Methods: severe/very severe COPD patients and 43 controls using Comprehensive High-throughput Arrays for Relative Methylation (CHARM). COPD cases were selected based on spirometric classification of GOLD 3 or 4 (Global Initiative for Chronic Obstructive Lung Disease). Percent methylation estimates from the normalized dataset were fit to disease status, age, gender, pack-years of smoking, and years since stopping smoking in a linear regression model. DNA methylation changes …