Analysis of the genomic landscapes of Barbadian and Nigerian women with triple negative breast cancer.

PURPOSE: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that disproportionately affects women of African ancestry (WAA) and is often associated with poor survival. Although there is a high prevalence of TNBC across West Africa and in women of the African diaspora, there has been no comprehensive genomics study to investigate the mutational profile of ancestrally related women across the Caribbean and West Africa. METHODS: This multisite cross-sectional study used 31 formalin-fixed paraffin-embedded (FFPE) samples from Barbadian and Nigerian TNBC participants. High-resolution whole exome sequencing (WES) was performed on the Barbadian and Nigerian TNBC samples to identify their mutational profiles and comparisons were made to African American, European American and Asian American sequencing data obtained from The Cancer Genome Atlas (TCGA). Whole exome sequencing was conducted on tumors with an average of 382 × coverage and 4335 × coverage for pooled germline non-tumor samples. RESULTS: Variants detected at high frequency in our WAA cohorts were found in the following genes NBPF12, PLIN4, TP53 and BRCA1. In the TCGA TNBC cases, these genes had a lower mutation rate, except for TP53 (32% in our cohort; 63% in TCGA-African American; 67% in TCGA-European American; 63% in TCGA-Asian). For all altered genes, there were no differences in frequency of mutations between WAA TNBC groups including the TCGA-African American cohort. For copy number variants, high frequency alterations were observed in PIK3CA, TP53, FGFR2 and HIF1AN genes. CONCLUSION: This study provides novel insights into the underlying genomic alterations in WAA TNBC samples and shines light on the importance of inclusion of under-represented populations in cancer genomics and biomarker studies.

High prevalence of deleterious mutations in concomitant nonsyndromic cleft and outflow tract heart defects.

Our previous work demonstrating enrichment of outflow tract (OFT) congenital heart disease (CHD) in children with cleft lip and/or palate (CL/P) suggests derangements in common underlying developmental pathways. The current pilot study examines the underlying genetics of concomitant nonsyndromic CL/P and OFT CHD phenotype. Of 575 patients who underwent CL/P surgery at Children's Hospital Los Angeles, seven with OFT CHD, negative chromosomal microarray analysis, and no recognizable syndromic association were recruited with their parents (as available). Whole genome sequencing of blood samples paired with whole-blood-based RNA sequencing for probands was performed. A pathogenic or potentially pathogenic variant was identified in 6/7 (85.7%) probands. A total of seven candidate genes were mutated (CHD7, SMARCA4, MED12, APOB, RNF213, SETX, and JAG1). Gene ontology analysis of variants predicted involvement in binding (100%), regulation of transcription (42.9%), and helicase activity (42.9%). Four patients (57.1%) expressed gene variants (CHD7, SMARCA4, MED12, and RNF213) previously involved in the Wnt signaling pathway. Our pilot analysis of a small cohort of patients with combined CL/P and OFT CHD phenotype suggests a potentially significant prevalence of deleterious mutations. In our cohort, an overrepresentation of mutations in molecules associated with Wnt-signaling was found. These variants may represent an expanded phenotypic heterogeneity within known monogenic disease genes or provide novel evidence of shared developmental pathways. The mechanistic implications of these mutations and subsequent developmental derangements resulting in the CL/P and OFT CHD phenotype require further analysis in a larger cohort of patients.

Cancer transcriptomic profiling from rapidly enriched circulating tumor cells.

Transcriptomic profiling of metastatic cancer can illuminate mechanisms of progression and lead to new therapies, but standard biopsy is invasive and reflects only a single metastatic site. In contrast, circulating tumor cell (CTC) profiling is noninvasive and repeatable, reflecting the dynamic and systemic nature of advanced disease. To date, transcriptomic profiling of CTCs has not delivered on its full potential, because white blood cells (WBCs) vastly outnumber CTCs. Current profiling strategies either lack cancer sensitivity and specificity or require specialized CTC capture protocols that are not readily scalable to large patient cohorts. Here, we describe a new strategy for rapid CTC enrichment and transcriptomic profiling using commercially available WBC depletion, microfluidic enrichment and RNA sequencing. When applied to blood samples from patients with advanced prostate cancer (PC), transcriptomes from enriched samples cluster with cancer positive controls and previously undetectable prostate-specific transcripts become readily measurable. Gene set enrichment analysis reveals multiple significantly enriched signaling pathways associated with PC, as well as novel pathways that merit further study. This accessible and scalable approach yields cancer-specific transcriptomic data and can be applied repeatedly and noninvasively in large cancer patient cohorts to discover new therapeutic targets in advanced disease.

Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma.

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.

Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases.

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations.

Histological Validation of 11Carbon-Acetate Positron Emission Tomography/Computerized Tomography in Detecting Lymph Node Metastases in Prostate Cancer.

PURPOSE: Conventional imaging cannot definitively detect nodal metastases of prostate cancer. We histologically validated C-acetate positron emission tomography/computerized tomography to identify nodal metastases, examining prostate cancer factors that influence detection rates. MATERIALS AND METHODS: Patients with C-acetate avid positron emission tomography/computerized tomography imaged pelvic/retroperitoneal lymph nodes underwent high extended robotic lymphadenectomy. A standardized mapping template comprising 8 predetermined anatomical regions was dissected during lymphadenectomy, allowing for matched, region based analysis and comparison of imaging and histological data. RESULTS: In 25 patients a total of 2,149 lymph nodes were excised (mean 86 per patient, range 27 to 136) and 528 (22%) harbored metastases (mean 21 positive nodes per patient, range 0 to 109). A total of 174 anatomical regions had matching imaging histological data. C-acetate positron emission tomography/computerized tomography accurately identified 48 node-positive regions and accurately ruled out 88 regions as metastasis-free. C-acetate sensitivity, specificity, and positive and negative predictive values were 67%, 84%, 74% and 79%, respectively. An increasing, histologically measured metastatic lesion size in long axis diameter of 5 or less, 6 to 10, 11 to 15, 16 to 20 and 21 mm or greater correlated with improved C-acetate detection rates of 45%, 62%, 81%, 89% and 100%, respectively. Each standard uptake value unit increase correlated with a 1.9 mm increase in nodal long axis diameter (p <0.001) and a 1.2 mm increase in short axis diameter (p <0.001). Positive C-acetate positron emission tomography/computerized tomography findings correlated with histological lymph node size (long axis diameter 12 mm and short axis diameter 6 mm), metastatic lesion size (long axis diameter 11 mm and short axis diameter 6 mm) and extranodal extension (positive 88% vs false-negative 58%, p = 0.005). CONCLUSIONS: C-acetate positron emission tomography/computerized tomography can identify prostate cancer metastatic nodal disease. However, it underestimates the true cephalad extent of nodal involvement, performing better in the pelvis than in the retroperitoneum. Standard uptake value, histological nodal size, intranodal metastasis size and extranodal extension correlate with cancer bearing nodes.

Lack of APC somatic mutation is associated with early-onset colorectal cancer in African Americans.

African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared with other US populations. They present with more right-sided, microsatellite stable disease and are diagnosed at earlier ages compared with non-Hispanic Whites (NHWs). To gain insight into these trends, we conducted exome sequencing (n = 45), copy number (n = 33) and methylation analysis (n = 11) of microsatellite stable AA CRCs. Results were compared with data from The Cancer Genome Atlas (TCGA). Two of the 45 tumors contained POLE mutations. In the remaining 43 tumors, only 27 (63%) contained loss-of-function mutations in APC compared with 80% of TCGA NHW CRCs. APC-mutation-negative CRCs were associated with an earlier onset of CRC (P = 0.01). They were also associated with lower overall mutation burden, fewer copy number variants and a DNA methylation signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease. Three of the APC-mutation-negative CRCs had loss-of-function mutations in BCL9L. Mutations in driver genes identified by TCGA exome analysis were less frequent in AA CRC cases than TCGA NHWs. Genes that regulate the WNT signaling pathway, including SOX9, GATA6, TET1, GLIS1 and FAT1, were differentially hypermethylated in APC-mutation-negative CRCs, suggesting a novel mechanism for cancer development in these tumors. In summary, we have identified a subtype of CRC that is associated with younger age of diagnosis, lack of APC mutation, microsatellite and chromosome stability, lower mutation burden and distinctive methylation changes.

A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs.

Type I collagen is composed of two α1(I) polypeptides and one α2(I) polypeptide and is the most abundant protein in the human body. Expression of type I collagen is primarily controlled at the level of mRNA stability and translation. Coordinated translation of α(I) and α2(I) mRNAs is necessary for efficient folding of the corresponding peptides into the collagen heterotrimer. In the 5' untranslated region (5' UTR), collagen mRNAs have a unique 5' stem-loop structure (5' SL). La ribonucleoprotein domain family member 6 (LARP6) is the protein that binds 5' SL with high affinity and specificity and coordinates their translation. Here we show that RNA helicase A (RHA) is tethered to the 5' SL of collagen mRNAs by interaction with the C-terminal domain of LARP6. In vivo, collagen mRNAs immunoprecipitate with RHA in an LARP6-dependent manner. Knockdown of RHA prevents formation of polysomes on collagen mRNAs and dramatically reduces synthesis of collagen protein, without affecting the level of the mRNAs. A reporter mRNA with collagen 5' SL is translated three times more efficiently in the presence of RHA than the same reporter without the 5' SL, indicating that the 5' SL is the cis-acting element conferring the regulation. During activation of quiescent cells into collagen-producing cells, expression of RHA is highly up-regulated. We postulate that RHA is recruited to the 5' UTR of collagen mRNAs by LARP6 to facilitate their translation. Thus, RHA has been discovered as a critical factor for synthesis of the most abundant protein in the human body.

Correction: Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis.

[This corrects the article DOI: 10.1371/journal.pone.0065897.].

Common mitochondrial deletions in RNA-Seq: evaluation of bulk, single-cell, and spatial transcriptomic datasets.

Common mitochondrial DNA (mtDNA) deletions are large structural variants in the mitochondrial genome that accumulate in metabolically active tissues with age and have been investigated in various diseases. We applied the Splice-Break2 pipeline (designed for high-throughput quantification of mtDNA deletions) to human RNA-Seq datasets and describe the methodological considerations for evaluating common deletions in bulk, single-cell, and spatial transcriptomics datasets. A robust evaluation of 1570 samples from 14 RNA-Seq studies showed: (i) the abundance of some common deletions detected in PCR-amplified mtDNA correlates with levels observed in RNA-Seq data; (ii) RNA-Seq library preparation method has a strong effect on deletion detection; (iii) deletions had a significant, positive correlation with age in brain and muscle; (iv) deletions were enriched in cortical grey matter, specifically in layers 3 and 5; and (v) brain regions with dopaminergic neurons (i.e., substantia nigra, ventral tegmental area, and caudate nucleus) had remarkable enrichment of common mtDNA deletions.

Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification.

OBJECTIVE: Comprehensive and reliable genome-wide variant analysis of a small number of cells has been challenging due to genome coverage bias, PCR over-cycling, and the requirement of expensive technologies. To comprehensively identify genome alterations in single colon crypts that reflect genome heterogeneity of stem cells, we developed a method to construct whole-genome sequencing libraries from single colon crypts without DNA extraction, whole-genome amplification, or increased PCR enrichment cycles. RESULTS: We present post-alignment statistics of 81 single-crypts (each contains four- to eight-fold less DNA than the requirement of conventional methods) and 16 bulk-tissue libraries to demonstrate the consistent success in obtaining reliable coverage, both in depth (≥ 30X) and breadth (≥ 92% of the genome covered at ≥ 10X depth), of the human genome. These single-crypt libraries are of comparable quality as libraries generated with the conventional method using high quality and quantities of purified DNA. Conceivably, our method can be applied to small biopsy samples from many tissues and can be combined with single cell targeted sequencing to comprehensively profile cancer genomes and their evolution. The broad potential application of this method offers expanded possibilities in cost-effectively examining genome heterogeneity in small numbers of cells at high resolution.

Impact of Genomic and Clinical Factors on Outcome of Children ≥18 Months of Age with Stage 3 Neuroblastoma with Unfavorable Histology and without MYCN Amplification: A Children's Oncology Group (COG) Report.

PURPOSE: Patients ≥18 months of age with International Neuroblastoma Staging System (INSS) stage 3 unfavorable histology (UH), MYCN-nonamplified (MYCN-NA) tumors have favorable survival rates compared with other high-risk neuroblastoma populations. The impact of select clinical and biological factors on overall survival (OS) and event-free survival (EFS) were evaluated. EXPERIMENTAL DESIGN: Patients enrolled on Children's Oncology Group (COG) A3973 (n = 34), ANBL0532 (n = 27), and/or biology protocol ANBL00B1 (n = 72) were analyzed. Tumors with available DNA (n = 65) and RNA (n = 42) were subjected to whole-exome sequencing (WES) and RNA sequencing. WES analyses and gene expression profiling were evaluated for their impact on survival. Multivariate analyses of EFS/OS using significant factors from univariate analyses were performed. RESULTS: 5-year EFS/OS for patients treated with high-risk therapy on A3973 and ANBL0532 were 73.0% ± 8.1%/87.9% ± 5.9% and 61.4% ± 10.2%/73.0% ± 9.2%, respectively (P = 0.1286 and P = 0.2180). In the A3973/ANBL0532 cohort, patients with less than partial response (PR; n = 5) at end-induction had poor outcomes (5-year EFS/OS: 0%/20.0% ± 17.9%. Univariate analyses of WES data revealed that subjects whose tumors had chromosome 1p or 11q loss/LOH and chromosome 5 or 9 segmental chromosomal aberrations had inferior EFS compared with those with tumors without these aberrations. Multivariate analysis revealed that 11q loss/LOH was an independent predictor of inferior OS [HR, 3.116 (95% confidence interval, 1.034-9.389), P = 0.0435]. CONCLUSIONS: Patients ≥18 months of age at diagnosis who had tumors with UH and MYCN-NA INSS stage 3 neuroblastoma assigned to high-risk therapy had an 81.6% ± 5.3% 5-year OS. Less than PR to induction therapy and chromosome 11q loss/LOH are independent predictors of inferior outcome and identify patients who should be eligible for future high-risk clinical trials.

Genome Analysis Using Whole-Exome Sequencing of Non-Syndromic Cleft Lip and/or Palate from Malagasy Trios Identifies Variants Associated with Cilium-Related Pathways and Asian Genetic Ancestry.

BACKGROUND: Orofacial clefts (OFCs) are common congenital disabilities that can occur as isolated non-syndromic events or as part of Mendelian syndromes. OFC risk factors vary due to differences in regional environmental exposures, genetic variants, and ethnicities. In recent years, significant progress has been made in understanding OFCs, due to advances in sequencing and genotyping technologies. Despite these advances, very little is known about the genetic interplay in the Malagasy population. METHODS: Here, we performed high-resolution whole-exome sequencing (WES) on non-syndromic cleft lip with or without palate (nCL/P) trios in the Malagasy population (78 individuals from 26 families (trios)). To integrate the impact of genetic ancestry admixture, we computed both global and local ancestries. RESULTS: Participants demonstrated a high percentage of both African and Asian admixture. We identified damaging variants in primary cilium-mediated pathway genes WNT5B (one family), GPC4 (one family), co-occurrence in MSX1 (five families), WDR11 (one family), and tubulin stabilizer SEPTIN9 (one family). Furthermore, we identified an autosomal homozygous damaging variant in PHGDH (one family) gene that may impact metabiotic activity. Lastly, all variants were predicted to reside on local Asian genetic ancestry admixed alleles. CONCLUSION: Our results from examining the Malagasy genome provide limited support for the hypothesis that germline variants in primary cilia may be risk factors for nCL/P, and outline the importance of integrating local ancestry components better to understand the multi-ethnic impact on nCL/P.

Evolutionary conservation of the fidelity of transcription.

Accurate transcription is required for the faithful expression of genetic information. However, relatively little is known about the molecular mechanisms that control the fidelity of transcription, or the conservation of these mechanisms across the tree of life. To address these issues, we measured the error rate of transcription in five organisms of increasing complexity and found that the error rate of RNA polymerase II ranges from 2.9 × 10-6 ± 1.9 × 10-7/bp in yeast to 4.0 × 10-6 ± 5.2 × 10-7/bp in worms, 5.69 × 10-6 ± 8.2 × 10-7/bp in flies, 4.9 × 10-6 ± 3.6 × 10-7/bp in mouse cells and 4.7 × 10-6 ± 9.9 × 10-8/bp in human cells. These error rates were modified by various factors including aging, mutagen treatment and gene modifications. For example, the deletion or modification of several related genes increased the error rate substantially in both yeast and human cells. This research highlights the evolutionary conservation of factors that control the fidelity of transcription. Additionally, these experiments provide a reasonable estimate of the error rate of transcription in human cells and identify disease alleles in a subunit of RNA polymerase II that display error-prone transcription. Finally, we provide evidence suggesting that the error rate and spectrum of transcription co-evolved with our genetic code.

Extracellular signal-regulated kinase-2 within the ventral tegmental area regulates responses to stress.

Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors.

Imaging of type I procollagen biosynthesis in cells reveals biogenesis in highly organized bodies; Collagenosomes.

Mechanistic aspects of type I procollagen biosynthesis in cells are poorly understood. To provide more insight into this process we designed a system to directly image type I procollagen biogenesis by co-expression of fluorescently labeled full size procollagen α1(I) and one α2(I) polypeptides. High resolution images show that collagen α1(I) and α2(I) polypeptides are produced in coordination in discrete structures on the ER membrane, which we termed the collagenosomes. Collagenosomes are disk shaped bodies, 0.5-1 µM in diameter and 200-400 nm thick, in the core of which folding of procollagen takes place. Collagenosomes are intimately associated with the ER membrane and their formation requires intact translational machinery, suggesting that they are the sites of nascent procollagen biogenesis. Collagenosomes show little co-localization with the COPII transport vesicles, which export type I procollagen from the ER, suggesting that these two structures are distinct. LARP6 is the protein which regulates translation of type I collagen mRNAs. The characteristic organization of collagenosomes depends on binding of LARP6 to collagen mRNAs. Without LARP6 regulation, collagenosomes are poorly organized and the folding of α1(I) and α2(I) polypeptides into procollagen in their cores is diminished. This indicates that formation of collagenosomes is dependent on regulated translation of collagen mRNAs. In live cells the size, number and shape of collagenosomes show little change within several hours, suggesting that they are stable structures of type I procollagen biogenesis. This is the first report of structural organization of type I collagen biogenesis in collagenosomes, while the fluorescent reporter system based on simultaneous imaging of both type I collagen polypeptides will enable the detailed elucidation of their structure and function.

Role of Ki-67, MRE11, and PD-L1 as Predictive Biomarkers for Recurrence Pattern in Muscle-invasive Bladder Cancer.

BACKGROUND/AIM: Muscle invasive bladder cancer (MIBC) is an aggressive disease with high rates of local recurrence following radical cystectomy (RC). Currently, there are no clinically validated biomarkers to predict local only recurrence (LOR) and guide adjuvant treatment decisions. This pilot study evaluated the role of Ki-67, MRE11 and PD-L1 as predictive biomarkers for recurrence patterns in patients undergoing RC for MIBC. PATIENTS AND METHODS: Our institutional cystectomy database containing cases from 1992-2014 was queried for patients with local only recurrence (LOR), and case-matched to patients with distant recurrence (DR) and no recurrence (NR). Clinicopathological data were collected and a tissue microarray was analyzed for presence of Ki-67, MRE11, and PD-L1 using immunofluorescence and immunohistochemistry. RESULTS: Pathologic specimens from 42 patients (18 NR, 16 LOR, and 8 DR) were reviewed. Compared to normal bladder tissue, tumors had increased expression of Ki-67 (p<0.01) and PD-L1 (p<0.05). High Ki-67 was associated with recurrence pattern (local vs. distant) on univariate analysis (p<0.05). Ki-67 cell density varied by recurrence type: LOR (1354 cells/mm2), DR (557 cells/mm2) and NR (1111 cells/mm2) (p=0.034). CONCLUSION: Our selected biomarkers could distinguish MIBC from normal bladder tissue but could not classify samples by recurrence pattern.

Wild-Type KRAS Allele Effects on Druggable Targets in KRAS Mutant Lung Adenocarcinomas.

KRAS mutations are one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC) and in lung adenocarcinomas in particular. Development of therapeutics targeting KRAS has been incredibly challenging, prompting indirect inhibition of downstream targets such as MEK and ERK. Such inhibitors, unfortunately, come with limited clinical efficacy, and therefore the demand for developing novel therapeutic strategies remains an urgent need for these patients. Exploring the influence of wild-type (WT) KRAS on druggable targets can uncover new vulnerabilities for the treatment of KRAS mutant lung adenocarcinomas. Using commercially available KRAS mutant lung adenocarcinoma cell lines, we explored the influence of WT KRAS on signaling networks and druggable targets. Expression and/or activation of 183 signaling proteins, most of which are targets of FDA-approved drugs, were captured by reverse-phase protein microarray (RPPA). Selected findings were validated on a cohort of 23 surgical biospecimens using the RPPA. Kinase-driven signatures associated with the presence of the KRAS WT allele were detected along the MAPK and AKT/mTOR signaling pathway and alterations of cell cycle regulators. FoxM1 emerged as a potential vulnerability of tumors retaining the KRAS WT allele both in cell lines and in the clinical samples. Our findings suggest that loss of WT KRAS impacts on signaling events and druggable targets in KRAS mutant lung adenocarcinomas.

Comprehensive molecular profiling of UV-induced metastatic melanoma in Nme1/Nme2-deficient mice reveals novel markers of survival in human patients.

Hepatocyte growth factor-overexpressing mice that harbor a deletion of the Ink4a/p16 locus (HP mice) form melanomas with low metastatic potential in response to UV irradiation. Here we report that these tumors become highly metastatic following hemizygous deletion of the Nme1 and Nme2 metastasis suppressor genes (HPN mice). Whole-genome sequencing of melanomas from HPN mice revealed a striking increase in lung metastatic activity that is associated with missense mutations in eight signature genes (Arhgap35, Atp8b4, Brca1, Ift172, Kif21b, Nckap5, Pcdha2, and Zfp869). RNA-seq analysis of transcriptomes from HP and HPN primary melanomas identified a 32-gene signature (HPN lung metastasis signature) for which decreased expression is strongly associated with lung metastatic potential. Analysis of transcriptome data from The Cancer Genome Atlas revealed expression profiles of these genes that predict improved survival of patients with cutaneous or uveal melanoma. Silencing of three representative HPN lung metastasis signature genes (ARRDC3, NYNRIN, RND3) in human melanoma cells resulted in increased invasive activity, consistent with roles for these genes as mediators of the metastasis suppressor function of NME1 and NME2. In conclusion, our studies have identified a family of genes that mediate suppression of melanoma lung metastasis, and which may serve as prognostic markers and/or therapeutic targets for clinical management of metastatic melanoma.

What Genes Can Tell: A Closer Look at Vestibular Schwannoma.

OBJECTIVE: Comprehensive molecular profiling of radioresistant and cystic vestibular schwannoma (VS) subtypes. STUDY DESIGN: Our study utilized whole-exome sequencing (WES), RNA-sequencing (RNAseq), and correlated clinical data from 12 samples (2 samples of solid sporadic subtype, 8 with cystic changes, and 2 previously irradiated). SETTING: Academic medical center. PATIENTS: Patients diagnosed with VS who required surgical treatment. Inclusion: Cystic and radioresistant tumors matched to age and tumor volume, with solid sporadic VS samples as control; Exclusion: NF-2 patients. INTERVENTION(S): WES using custom probes for copy number analysis. A modified version of the Agilent Human Whole Exome sequencing hybrid capture system was used to process samples. Recurrent variants were identified and compared between groups. Leukocyte-derived DNA was utilized as internal control to reduce false-positives. MAIN OUTCOME MEASURE(S): Analysis of genetic landscape of VS subtypes (naive solid VS, cystic VS, and previously irradiated VS) by performing deep next-generation sequencing. RESULTS: WES data achieved a mean coverage of 202X and RNAseq generated an average of 74 million total reads. As a group, 25% of samples had 22q loss. Somatic analysis identified previously reported genes and multiple novel mutations across samples. Differential expression analysis of RNAseq data found significantly mutated genes such as COL6A3, CLMP, ART4, Lumican that were shared by both cystic VS and irradiated VS, but not seen in sporadic VS. CONCLUSIONS: Using WES we were able to demonstrate that cystic and irradiated samples are subtypes of VS with an increased mutation burden and a unique genetic fingerprint. We identified differences between the genomic and molecular profile of cystic VS and radioresistant VS. Our results help advance the understanding of the pathophysiology of these tumor subtypes and suggest possible molecular targets for novel treatment strategies.