Comprehensive Genomic Study Research Articles

Lack of Significant Differences in Somatic Alterations between MGUS, SMM and Symptomatic Multiple Myeloma: A Result from Comprehensive Genomic Profiling Study

Somatic alterations including single nucleotide variants (SNVs), copy number alterations (CNAs) and structural variants (SVs) are an important component and the hallmark of Multiple Myeloma (MM) with certain alterations having clinical implications. Recently, the chronological order of such events was explained in detailed studies however previous study has compared the frequency and clonality of such events in early stage plasma cell disorder (MGUS or SMM) and their comparison with MM.Here we performed whole genome (WGS) and RNA sequencing (RNAseq) on 67 samples (8 Unique paired samples from patients with MGUS and when progressed to MM, 10 paired samples at SMM stage and then progressed to MM, as well as non-progressor SMM (NP-SMM) patients who have not progressed after at least 5 years of follow-up. In addition, we profiled CNAs and their clonality level in 164 MGUS, 147 SMM and 336 newly diagnosed MM patients to construct the clonal structure and frequency of somatic alterations.We did not observe significant difference in overall frequency of mutations at MGUS (median=5662 SNVs, IQR= [5252 - 7864]) and SMM stage (median=5308 SNVs, IQR=[4236 - 6125] ) and at progression to MM (median= 5983 SNVs, IQR=[5097 - 8916]). Overall mutational load increased from precursor stage to MM by only 5% with no recurrent change and the difference was not statistically significant (p > 0.05). Similar trend was also observed for SVs [median=36, IQR= [25.25 -40.75] at MGUS, median=34, IQR= [12-52] at SMM, median=27, IQR= [27-41] at MM]. In fact, some of the samples had lower SNV and SV load in MM compared to precursor stages even though that had increased cancer cell fraction. Copy number analysis on paired samples also showed >90% similarity only with few non-recurrent focal CNAs. We have compared WGS data from non-progressor SMM with progressor SMM and newly-diagnosed MM. The non-progressor SMM patients showed identical CNAs and not significantly different Ig translocation frequencies to both other groups. Except for NRAS mutations, mutation frequency in NP-SMM was similar to others. Finally, by using data from 647 patients at three stages (MGUS, SMM and MM), we compared the frequency of CNAs at each time point. Similar to MM, we observed almost equal frequency of hyperdiploid and non-hyperdiploid SMM cases. In hyperdiploid SMM, clonal copy number gain was observed in 95% of patients involving two of the three chromosomes 9, 15 and 19. Moreover, a similar pattern was observed in other hyperdiploid chromosomes including chromosomes 3,5,7,11 and 21. The observed event frequency and clonality levels in SMM were identical to one observed in MM as well as MGUS. Non-hyperdiploid SMM group had early and clonal events targeting chromosome 13 deletion with gain of 1q, 11 and/or 9 at the same frequency level as in MM. The only significant difference was observed in subclonal deletions targeting some of the infrequent deletion events observed in HMM and NHMM. In conclusion, our data from longitudinal analysis of paired precursor and symptomatic MM samples as well as cross sectional data highlight lack of significant differences in genomic changes between precursor conditions and symptomatic myeloma. This lack of progressive and unique genomic change suggests the need for additional transcriptomic, epigenomic or tumor micro environment changes to accelerate the tumor development. Disclosures Bolli: Celgene: Honoraria; Gilead: Other: travel expenses; Novartis: Honoraria. Moreau:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Munshi:Celgene: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy.

Ductal and acinar carcinomas of the prostate: A comparative comprehensive genomic profiling study.

271 Background: Prostatic ductal carcinoma (PDC) is an uncommon centrally located prostate with a characteristic tubulopapillary histology. PDC shares some biomarker features with the more common prostatic acinar carcinoma (PAC) including PSA expression. Methods: In this comparative comprehensive genomic profiling (CGP) study, FFPE samples from 61 clinically advanced PDC and 4,132 PAC were profiled for all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Results: The age, frequency of genomic alterations (GA) per tumor and TP53 GA of PDA and PAC were similar (Table). TMPRSS2:ERG fusions and AR GA were more often identified in PAC than PDC whereas PTEN GA were more common in PDC than PAC. Targetable GA were identified at similar frequencies in both groups focused on BRCA2 (PARP inhibitors), BRAF (BRAF/MEK inhibitors) and PIK3CA (MTOR inhibitors). ATM GA (PARP inhibitors) were more common in PAC than PDC. CDK12 GA potentially associated with immunotherapy (IO) benefit were similar in PDA and PAC as were the frequencies of MSI-High status, median TMB and high TMB levels. Conclusions: The pathologic features of PDC and PAC have been classically maintained as representative of 2 different tumor types with potentially contrasting histogenesis. In the current CGP-based study, PDA and PAC did not display significant differences in genomic signatures. CGP may reveal biomarkers that could direct patients to targeted (PARP, MTOR and BRAF/MEK inhibitors) or immunotherapies ( CDK12 GA, MSI-High or high TMB status) in both PDA and PAC. [Table: see text]

Comparative genomics revealed the gene evolution and functional divergence of magnesium transporter families in Saccharum

BackgroundSugarcane served as the model plant for discovery of the C4 photosynthetic pathway. Magnesium is the central atom of chlorophyll, and thus is considered as a critical nutrient for plant development and photosynthesis. In plants, the magnesium transporter (MGT) family is composed of a number of membrane proteins, which play crucial roles in maintaining Mg homeostasis. However, to date there is no information available on the genomics of MGTs in sugarcane due to the complexity of the Saccharum genome.ResultsHere, we identified 10 MGTs from the Saccharum spontaneum genome. Phylogenetic analysis of MGTs suggested that the MGTs contained at least 5 last common ancestors before the origin of angiosperms. Gene structure analysis suggested that MGTs family of dicotyledon may be accompanied by intron loss and pseudoexon phenomena during evolution. The pairwise synonymous substitution rates corresponding to a divergence time ranged from 142.3 to 236.6 Mya, demonstrating that the MGTs are an ancient gene family in plants. Both the phylogeny and Ks analyses indicated that SsMGT1/SsMGT2 originated from the recent ρWGD, and SsMGT7/SsMGT8 originated from the recent σ WGD. These 4 recently duplicated genes were shown low expression levels and assumed to be functionally redundant. MGT6, MGT9 and MGT10 weredominant genes in the MGT family and werepredicted to be located inthe chloroplast. Of the 3 dominant MGTs, SsMGT6 expression levels were found to be induced in the light period, while SsMGT9 and SsMTG10 displayed high expression levels in the dark period. These results suggested that SsMGT6 may have a function complementary to SsMGT9 and SsMTG10 that follows thecircadian clock for MGT in the leaf tissues of S. spontaneum. MGT3, MGT7 and MGT10 had higher expression levels Insaccharum officinarum than in S. spontaneum, suggesting their functional divergence after the split of S. spontaneum and S. officinarum.ConclusionsThis study of gene evolution and expression of MGTs in S. spontaneum provided basis for the comprehensive genomic study of the entire MGT genes family in Saccharum. The results are valuable for further functional analyses of MGT genes and utilization of the MGTs for Saccharum genetic improvement.

Comparative Genome-Wide Survey of Single Nucleotide Variation Uncovers the Genetic Diversity and Potential Biomedical Applications among Six Macaca Species

Macaca is of great importance in evolutionary and biomedical research. Aiming at elucidating genetic diversity patterns and potential biomedical applications of macaques, we characterized single nucleotide variations (SNVs) of six Macaca species based on the reference genome of Macaca mulatta. Using eight whole-genome sequences, representing the most comprehensive genomic SNV study in Macaca to date, we focused on discovery and comparison of nonsynonymous SNVs (nsSNVs) with bioinformatic tools. We observed that SNV distribution patterns were generally congruent among the eight individuals. Outlier tests of nsSNV distribution patterns detected 319 bins with significantly distinct genetic divergence among macaques, including differences in genes associated with taste transduction, homologous recombination, and fat and protein digestion. Genes with specific nsSNVs in various macaques were differentially enriched for metabolism pathways, such as glycolysis, protein digestion and absorption. On average, 24.95% and 11.67% specific nsSNVs were putatively deleterious according to PolyPhen2 and SIFT4G, respectively, among which the shared deleterious SNVs were located in 564–1981 genes. These genes displayed enrichment signals in the ‘obesity-related traits’ disease category for all surveyed macaques, confirming that they were suitable models for obesity related studies. Additional enriched disease categories were observed in some macaques, exhibiting promising potential for biomedical application. Positively selected genes identified by PAML in most tested Macaca species played roles in immune and nervous system, growth and development, and fat metabolism. We propose that metabolism and body size play important roles in the evolutionary adaptation of macaques.

Genetic landscape of hepatitis B virus–associated diffuse large B-cell lymphoma

AbstractHepatitis B virus (HBV) infection is endemic in some parts of Asia, Africa, and South America and remains to be a significant public health problem in these areas. It is known as a leading risk factor for the development of hepatocellular carcinoma, but epidemiological studies have also shown that the infection may increase the incidence of several types of B-cell lymphoma. Here, by characterizing altogether 275 Chinese diffuse large B-cell lymphoma (DLBCL) patients, we showed that patients with concomitant HBV infection (surface antigen positive [HBsAg+]) are characterized by a younger age, a more advanced disease stage at diagnosis, and reduced overall survival. Furthermore, by whole-genome/exome sequencing of 96 tumors and the respective peripheral blood samples and targeted sequencing of 179 tumors from these patients, we observed an enhanced rate of mutagenesis and a distinct set of mutation targets in HBsAg+ DLBCL genomes, which could be partially explained by the activities of APOBEC and activation-induced cytidine deaminase. By transcriptome analysis, we further showed that the HBV-associated gene expression signature is contributed by the enrichment of genes regulated by BCL6, FOXO1, and ZFP36L1. Finally, by analysis of immunoglobulin heavy chain gene sequences, we showed that an antigen-independent mechanism, rather than a chronic antigenic simulation model, is favored in HBV-related lymphomagenesis. Taken together, we present the first comprehensive genomic and transcriptomic study that suggests a link between HBV infection and B-cell malignancy. The genetic alterations identified in this study may also provide opportunities for development of novel therapeutic strategies.

A comparative bioinformatic analysis of C9orf72.

C9orf72 is associated with frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS), both of which are devastating neurodegenerative diseases. Findings suggest that an expanded hexanucleotide repeat in the non-coding region of the C9orf72 gene is the most common cause of familial FTD and ALS. Despite considerable efforts being made towards discerning the possible disease-causing mechanism/s of this repeat expansion mutation, the biological function of C9orf72 remains unclear. Here, we present the first comprehensive genomic study on C9orf72 gene. Analysis of the genomic level organization of C9orf72 across select species revealed architectural similarity of syntenic regions between human and mouse but a lack of conservation of the repeat-harboring intron 1 sequence. Information generated in this study provides a broad genomic perspective of C9orf72 which would form a basis for subsequent experimental approaches and facilitate future mechanistic and functional studies on this gene.

General paucity of genomic alteration and low tumor mutation burden in refractory and metastatic hepatoblastoma: comprehensive genomic profiling study

Hepatoblastoma (HBL) is a hepatic malignancy of infants and young children, which is often cured by combinations of surgery and chemotherapy. Management of refractory and metastatic HBL is challenging. Comprehensive genomic profiling was performed on 31 refractory and metastatic HBL using a hybrid-capture, adaptor ligation-based next-generation sequencing assay. Tumor mutation burden (TMB) was calculated from a minimum of 1.11 Mb of sequenced DNA and reported as mutations/Mb. The results were analyzed for all classes of genomic alterations (GA). Activating CTNNB1 mutation was the most frequent GA seen in 19 (61%) of cases. All 3 (100%) mixed epithelial and mesenchymal HBL harbored CTNNB1 mutation. The small cell undifferentiated subtype showed SMARCB1 loss in both cases. There was no significant further correlation of GA with histologic subtype. In addition to the potential targeting of CTNNB1, other rarely identified possible targetable GA included ERBB4 (6%) and FBXW7, SRC and BRCA2 (each at 3%). The mean TMB was 3.5 mut/Mb, the median was 1.7 mut/Mb. There were 2 HBL with ≥10 mut/Mb. No alterations in TP53 were identified, and alterations in the DNA repair pathways were rare. Refractory and metastatic HBL is characterized by a general paucity of GA and is dominated by frequent CTNNB1 mutation and overall low TMB. Although potentially targetable GA are seen on occasion in HBL and a small number of cases have high TMB with potential to respond to immune checkpoint inhibitors, advanced HBL will remain a treatment challenge.

Genome-Wide Analysis Identifies MEN1 and MAX Mutations and a Neuroendocrine-Like Molecular Heterogeneity in Quadruple WT GIST

Quadruple wild-type (WT) gastrointestinal stromal tumor (GIST) is a genomic subgroup lacking KIT/PDGFRA/RAS pathway mutations, with an intact succinate dehydrogenase (SDH) complex. The aim of this work is to perform a wide comprehensive genomic study on quadruple WT GIST to improve the characterization of these patients. We selected 14 clinical cases of quadruple WT GIST, of which nine cases showed sufficient DNA quality for whole exome sequencing (WES). NF1 alterations were identified directly by WES. Gene expression from whole transcriptome sequencing (WTS) and miRNA profiling were performed using fresh-frozen, quadruple WT GIST tissue specimens and compared with SDH and KIT/PDGFRA-mutant GIST. WES identified an average of 18 somatic mutations per sample. The most relevant somatic oncogenic mutations identified were in TP53, MEN1, MAX, FGF1R, CHD4, and CTDNN2. No somatic alterations in NF1 were identified in the analyzed cohort. A total of 247 mRNA transcripts and 66 miRNAs were differentially expressed specifically in quadruple WT GIST. Overexpression of specific molecular markers (COL22A1 and CALCRL) and genes involved in neural and neuroendocrine lineage (ASCL1, Family B GPCRs) were detected and further supported by predicted miRNA target analysis. Quadruple WT GIST show a specific genetic signature that deviates significantly from that of KIT/PDGFRA-mutant and SDH-mutant GIST. Mutations in MEN1 and MAX genes, a neural-committed phenotype and upregulation of the master neuroendocrine regulator ASCL1, support a genetic similarity with neuroendocrine tumors, with whom they also share the great variability in oncogenic driver genes.Implications: This study provides novel insights into the biology of quadruple WT GIST that potentially resembles neuroendocrine tumors and should promote the development of specific therapeutic approaches. Mol Cancer Res; 15(5); 553-62. ©2017 AACR.

Evolution and expression of the fructokinase gene family in Saccharum

BackgroundSugarcane is an important sugar crop contributing up to about 80% of the world sugar production. Efforts to characterize the genes involved in sugar metabolism at the molecular level are growing since increasing sugar content is a major goal in the breeding of new sugarcane varieties. Fructokinases (FRK) are the main fructose phosphorylating enzymes with high substrate specificity and affinity.ResultsIn this study, by combining comparative genomics approaches with BAC resources, seven fructokinase genes were identified in S. spontaneum. Phylogenetic analysis based on representative monocotyledon and dicotyledon plant species suggested that the FRK gene family is ancient and its evolutionary history can be traced in duplicated descending order: SsFRK4, SsFRK6/SsFRK7,SsFRK5, SsFRK3 and SsFRK1/SsFRK2. Among the close orthologs, the number and position of exons in FRKs were conserved; in contrast, the size of introns varied among the paralogous FRKs in Saccharum. Genomic constraints were analyzed within the gene alleles and between S. spontaneum and Sorghum bicolor, and gene expression analysis was performed under drought stress and with exogenous applications of plant hormones. FRK1, which was under strong functional constraint selection, was conserved among the gene allelic haplotypes, and displayed dominant expression levels among the gene families in the control conditions, suggesting that FRK1 plays a major role in the phosphorylation of fructose. FRK3 and FRK5 were dramatically induced under drought stress, and FRK5 was also found to increase its expression levels in the mature stage of Saccharum. Similarly, FRK3 and FRK5 were induced in response to drought stress in Saccharum. FRK2 and FRK7 displayed lower expression levels than the other FRK family members; FRK2 was under strong genomic selection constraints whereas FRK7 was under neutral selection. FRK7 may have become functionally redundant in Saccharum through pseudogenization. FRK4 and FRK6 shared the most similar expression pattern: FRK4 was revealed to have higher expression levels in mature tissues than in premature tissues of Saccharum, and FRK6 presented a slight increase under drought stress.ConclusionsOur study presents a comprehensive genomic study of the entire FRK gene family in Saccharum, providing the foundations for approaches to characterize the molecular mechanism regulated by the SsFRK family in sugarcane.

First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation

BackgroundNeurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined.Case presentationHere, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansionConclusionsThis is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.

Comparative Mitogenomic Analyses of Praying Mantises (Dictyoptera, Mantodea): Origin and Evolution of Unusual Intergenic Gaps.

Praying mantises are a diverse group of predatory insects. Although some Mantodea mitogenomes have been reported, a comprehensive comparative and evolutionary genomic study is lacking for this group. In the present study, four new mitogenomes were sequenced, annotated, and compared to the previously published mitogenomes of other Mantodea species. Most Mantodea mitogenomes share a typical set of mitochondrial genes and a putative control region (CR). Additionally, and most intriguingly, another large non-coding region (LNC) was detected between trnM and ND2 in all six Paramantini mitogenomes examined. The main section in this common region of Paramantini may have initially originated from the corresponding control region for each species, whereas sequence differences between the LNCs and CRs and phylogenetic analyses indicate that LNC and CR are largely independently evolving. Namely, the LNC (the duplicated CR) may have subsequently degenerated during evolution. Furthermore, evidence suggests that special intergenic gaps have been introduced in some species through gene rearrangement and duplication. These gaps are actually the original abutting sequences of migrated or duplicated genes. Some gaps (G5 and G6) are homologous to the 5' and 3' surrounding regions of the duplicated gene in the original gene order, and another specific gap (G7) has tandem repeats. We analysed the phylogenetic relationships of fifteen Mantodea species using 37 concatenated mitochondrial genes and detected several synapomorphies unique to species in some clades.

Structure, phylogeny, allelic haplotypes and expression of sucrose transporter gene families in Saccharum.

BackgroundSugarcane is an economically important crop contributing to about 80 % of the world sugar production. Increasing efforts in molecular biological studies have been performed for improving the sugar yield and other relevant important agronomic traits. However, due to sugarcane’s complicated genomes, it is still challenging to study the genetic basis of traits, such as sucrose accumulation. Sucrose transporters (SUTs) are critical for both phloem loading in source tissue and sucrose uptaking in sink tissue, and are considered to be the control points for regulating sucrose storage. However, no genomic study for sugarcane sucrose transporter (SsSUT) families has been reported up to date.ResultsBy using comparative genomics and bacterial artificial chromosomes (BACs), six SUT genes were identified and characterized in S. spontaenum. Phylogenetic analyses revealed that the two pairs SsSUTs (SsSUT1/SsSUT3 and SsSUT5/SsSUT6) could be clustered together into two separate monocot specific SUT groups, while SsSUT2 and SsSUT4 were separated into the other two groups, with members from both dicot and monocot species. Gene structure comparison demonstrated that the number and position of exons/introns in SUTs were highly conserved among the close orthologs; in contrast, there were variations among the paralogous SUTs in Sacchuarm. Though with the high polyploidy level, gene allelic haplotype comparative analysis showed that the examined four SsSUT members exhibited conservations of gene structures and amino acid sequences among the allelic haplotypes accompanied by variations of intron sizes. Gene expression analyses were performed for tissues from seedlings under drought stress and mature plants of three Saccharum species (S.officinarnum, S.spotaneum and S.robustum). Both SUT1 and SUT4 expressed abundantly at different conditions. SUT2 had similar expression level in all of the examined tissues, but SUT3 was undetectable. Both of SUT5 and SUT6 had lower expression level than other gene member, and expressed stronger in source leaves and are likely to play roles in phloem loading. In the seeding plant leave under water stress, four genes SUT1, SUT2, SUT4 and SUT5 were detectable. In these detectable genes, SUT1 and SUT4 were down regulated, while, SUT2 and SUT5 were up regulated.ConclusionsIn this study, we presented the first comprehensive genomic study for a whole gene family, the SUT family, in Saccharum. We speculated that there were six SUT members in the S. spotaneum genome. Out of the six members, SsSUTs, SsSUT5 and SsSUT6 were recent duplication genes accompanied by rapid evolution, while, SsSUT2 and SsSUT4 were the ancient members in the families. Despite the high polypoidy genome, functional redundancy may not exist among the SUTs allelic haplotypes supported by the evidence of strong purifying selection of the gene allele. SUT3 could be a low active member in the family because it is undetectable in our study, but it might not be a pseudogene because it harbored integrated gene structure. SUT1 and SUT4 were the main members for the sucrose transporter, while, these SUTs had sub-functional divergence in response to sucrose accumulation and plant development in Saccharum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2419-6) contains supplementary material, which is available to authorized users.

Clinically advanced and metastatic pure mucinous carcinoma of the breast: a comprehensive genomic profiling study.

Pure mucinous breast carcinoma (pmucBC) is a distinctive variant of breast cancer (BC) featuring an excellent overall prognosis. However, on rare occasions, pmucBC pursues an aggressive clinical course. We queried whether comprehensive genomic profiling (CGP) would uncover clinically relevant genomic alterations (CRGA) that could lead to targeted therapy treatment for patients with an advanced and metastatic form of pmucBC. From a series of 51,238 total cancer samples, which included 5605 cases of clinically advanced BC and 22 cases of stage IV pmucBC, DNA was extracted from 40 microns of FFPE sections. Comprehensive genomic profiling was performed using a hybrid-capture, adaptor ligation-based next generation sequencing assay to a mean coverage depth of 564X. The results were analyzed for all classes of genomic alterations (GA) including base substitutions, insertions and deletions, select rearrangements, and copy number changes. Clinically relevant genomic alterations were defined as those indicating possible treatment with anti-cancer drugs on the market or in registered clinical trials. Samples were obtained from breast (11), lymph nodes (3), chest wall (2), liver (2), soft tissue (2), bone (1), and pleura (1). The median age of the 22 pmucBC patients was 57 years (range 32-79 years). Three pmucBCs were grade 1, 17 were grade 2, and 2 were grade 3. Twenty-one (95 %) pmucBC were ER+, 18 (82 %) were PR+, and 3 (14 %) were HER2+ by IHC and/or FISH. A total of 132 GA were identified (6.0 GA per tumor), including 53 CRGA, for a mean of 2.4 GA per tumor. Amplification of FGFR1 or ZNF703, located within the same amplicon, was found in 8 of 22 cases (36 %). This enrichment of FGFR1 amplification in 36 % of pmucBC versus 11 % of non-mucinous ER+ BC (601 cases) was significant (p < 0.005). Other frequently altered genes of interest in pmucBC were CCND1 and the FGF3/FGF4/FGF19 amplicon (27 %), often co-amplified together. ERBB2/HER2 alterations were identified in 5 pmucBC (23 %): ERBB2 amplification was found in 3 of 3 cases (100 %) that were HER2+ by IHC and/or FISH; 1 pmucBC was negative for HER2 overexpression by IHC, but positive for amplification by CGP; and 2 pmucBC harbored the ERBB2 substitutions D769Y and V777L (one sample also featured ERBB2 amplification). The enrichment of ERBB2 GA in metastatic pmucBC versus non-metastatic primary pmucBC was significant (p = 0.03). CRGA were also found in 20 additional genes including PIK3CA (5), BRCA1 (1), TSC2 (1), STK11 (1), AKT3 (1), and ESR1 (1). Metastatic pmucBC is a distinct form of breast cancer that features a relatively high frequency of CRGA, including a significant enrichment of FGFR1 alterations and a high frequency of ERBB2 alterations when compared with non-metastatic pmucBC. These findings suggest that CGP can identify a variety of known and emerging therapy targets that have the potential to improve outcomes for patients with clinically advanced and metastatic forms of this disease.

Genome-Wide Analysis Uncovers Novel Recurrent Alterations in Primary Central Nervous System Lymphomas.

Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma confined to the central nervous system. Whether there is a PCNSL-specific genomic signature and, if so, how it differs from systemic diffuse large B-cell lymphoma (DLBCL) is uncertain. We performed a comprehensive genomic study of tumor samples from 19 immunocompetent PCNSL patients. Testing comprised array-comparative genomic hybridization and whole exome sequencing. Biallelic inactivation of TOX and PRKCD was recurrently found in PCNSL but not in systemic DLBCL, suggesting a specific role in PCNSL pathogenesis. In addition, we found a high prevalence of MYD88 mutations (79%) and CDKN2A biallelic loss (60%). Several genes recurrently affected in PCNSL were common with systemic DLBCL, including loss of TNFAIP3, PRDM1, GNA13, TMEM30A, TBL1XR1, B2M, CD58, activating mutations of CD79B, CARD11, and translocations IgH-BCL6. Overall, B-cell receptor/Toll-like receptor/NF-κB pathways were altered in >90% of PNCSL, highlighting its value for targeted therapeutic approaches. Furthermore, integrated analysis showed enrichment of pathways associated with immune response, proliferation, apoptosis, and lymphocyte differentiation. In summary, genome-wide analysis uncovered novel recurrent alterations, including TOX and PRKCD, helping to differentiate PCNSL from systemic DLBCL and related lymphomas.

Dichotomy of Genetic Abnormalities in PEComas With Therapeutic Implications.

Perivascular epithelioid cell neoplasms (PEComa) are a family of rare mesenchymal tumors with hybrid myo-melanocytic differentiation. Although most PEComas harbor loss-of-function TSC1/TSC2 mutations, a small subset were reported to carry TFE3 gene rearrangements. As no comprehensive genomic study has addressed the molecular classification of PEComa, we sought to investigate by multiple methodologies the incidence and spectrum of genetic abnormalities and their potential genotype-phenotype correlations in a large group of 38 PEComas. The tumors were located in soft tissue (11 cases) and visceral sites (27) including uterus, kidney, liver, lung, and urinary bladder. Combined RNA sequencing and fluorescence in situ hybridization analysis identified 9 (23%) TFE3 gene-rearranged tumors, with 3 cases showing an SFPQ/PSF-TFE3 fusion and 1 case showing a novel DVL2-TFE3 gene fusion. The TFE3-positive lesions showed a distinctive nested/alveolar morphology and were equally distributed between soft tissue and visceral sites. In addition, novel RAD51B gene rearrangements were identified in 3 (8%) uterine PEComas, which showed a complex fusion pattern and were fused to RRAGB/OPHN1 genes in 2 cases. Other nonrecurrent gene fusions, HTR4-ST3GAL1 and RASSF1-PDZRN3, were identified in 2 cases. Targeted exome sequencing using the IMPACT assay was used to address whether the presence of gene fusions is mutually exclusive from TSC gene abnormalities. TSC2 mutations were identified in 80% of the TFE3 fusion-negative cases tested. Coexistent TP53 mutations were identified in 63% of the TSC2-mutated PEComas. Our results showed that TFE3-rearranged PEComas lacked coexisting TSC2 mutations, indicating alternative pathways of tumorigenesis. In summary, this comprehensive genetic analysis significantly expands our understanding of molecular alterations in PEComas and brings forth the genetic heterogeneity of these tumors.

The chimerical genome of Isla del Coco feral pigs (Costa Rica), an isolated population since 1793 but with remarkable levels of diversity.

The history of domestic species and of their wild ancestors is not a simple one, and feral processes can clarify key aspects of this history, including the adaptive processes triggered by new environments. Here, we provide a comprehensive genomic study of Isla del Coco (Costa Rica) feral pigs, a unique population that was allegedly founded by two individuals and has remained isolated since 1793. Using SNP arrays and genome sequencing, we show that Cocos pigs are hybrids between Asian and European pigs, as are modern international pig breeds. This conclusively shows that, as early as the 18th century, British vessels were loading crossbred pigs in Great Britain and transporting them overseas. We find that the Y chromosome has Asian origin, which has not been reported in any international pig breed. Chinese haplotypes seem to have been transmitted independently between Cocos and other pig breeds, suggesting independent introgression events and a complex pattern of admixing. Although data are compatible with a founder population of N = 2, variability levels are as high in Cocos pigs as in international pig breeds (~1.9 SNPs/kb) and higher than in European wild boars or local breeds (~1.7 SNPs/kb). Nevertheless, we also report a 10-Mb region with a marked decrease in variability across all samples that contains four genes (CPE, H3F3C, SC4MOL and KHL2) previously identified as highly differentiated between wild and domestic pigs. This work therefore illustrates how feral population genomic studies can help to resolve the history of domestic species and associated admixture events.

GENOME-WIDE ANALYSIS UNCOVERS RECURRENT ALTERATIONS IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMAS (PCNSL)

BACKGROUND: Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma (NHL) confined to the CNS. Whether there is a PCNSL-specific genomic signature and, if so, how it differs from systemic DLBCL is uncertain. To address this gap in critical knowledge we performed a comprehensive genomic study in a cohort of 19 immunocompetent PCNSL patients by combining aCGH, whole exome sequencing and mate-pair whole genome sequencing. METHODS: 1) Tumor samples from 19 immunocompetent (HIV-, EBV-) PCNSL. 2) DNA from frozen tissue or from formalin-fixed paraffin embedded (FFPE) tissues. 3) All samples analyzed using a combination of aCGH, whole exome sequencing, mate-pair whole genome sequencing, targeted sequencing and FISH. 4) Overall survival estimated using Kaplan Meier method. RESULTS: We found a complex karyotype with a median of 21 copy-number abnormalities, 6 structural abnormalities and 3.3 non-synonymous mutations per Mb. We found a remarkably high prevalence of MYD88 activating mutations (79%) and CDKN2A biallelic loss (60%). Biallelic losses of TOX (a regulator of T-cell development), PRKCD (protein kinase C delta) and chromosomal breakpoints in DLGAP1 (discs large-associated protein 1) were recurrently found in PCNSL but not in systemic DLBCL, suggesting a specific role in PCNSL pathogenesis. Several other genes recurrently affected in PCNSL are common with systemic DLBCL of the ABC-type, such as loss of TNFAIP3, PRDM1, GNA13, TMEM30A, TBL1XR1, B2M and CD58, activating mutations of CD79B, CARD11, BRAF3 and translocations of IgH-BCL6. Overall, components of the NF-□B signaling pathway were altered in >90% of PNCSL, highlighting the potential value of this pathway for targeted therapeutic approaches. Furthermore, integrated analysis also showed an enrichment of networks associated with immune response, proliferation, regulation of apoptosis, and lymphocyte differentiation. CONCLUSIONS: We report a PCNSL genomic landscape closely related to ABC-DLBCL but with critical elements that support the existence of a subset of PCNSL-specific abnormalities, helping to genetically differentiate PCNSL from systemic DLBCL and related lymphomas. SECONDARY CATEGORY: Immunobiology & Immunotherapy.

The Genomic Landscape Of Primary Central Nervous System Lymphomas

Primary central nervous system lymphoma (PCNSL) is an aggressive and incurable variant of non-Hodgkin lymphoma (NHL) that is confined to the central nervous system. Most PCNSL (90%) are part of the immune-privileged site-associated diffuse large B-cell lymphomas (DLBCL). Unlike nodal DLBCL, only a limited number of genetic studies have been performed in PCNSL, partly due to lack of available tissue specimens. Because of the fragmented knowledge of the genomic basis, it is still a matter of debate whether they differ from systemic DLBCL with respect to their molecular features and pathogenesis and also if there is a CNS specific signature. We performed a comprehensive genomic study in a cohort of 22 immunocompetent (HIV- and EBV-) PCNSL. DNA was extracted from FFPE tissues (N=15) and frozen tissue (N=7). Samples were analyzed using a combination of aCGH (N=18), exome sequencing (N=10), mate-pair genome sequencing (N=2) and targeted sequencing (N=8). We found a complex karyotype with a median of 21 copy-number abnormalities (range 10–47), 6 structural abnormalities, 6 frameshift indels and 67 nonsynonymous mutations. By integrating mutation and copy number data we found a group of genes recurrently mutated and/or deleted in PNCSL. Remarkable findings were the high prevalence of MYD88 activating mutations (L265P/M232T/V217F), found in 69% of cases and the biallelic loss of CDKN2A (60%). A subset of recurrent abnormalities was exclusively found in PCNSL, and not being previously identified in systemic DLBCL. Thus, 11% of PCNSL have biallelic inactivation of TOX (a regulator of T-cell development) and PRKCD (protein kinase C delta), while another 17% of cases show focal monoallelic deletions/mutations in these genes. Finally, recurrent mutations have been identified in ATM, which have not been found in nodal DLBCL. Several other genes affected have been previously identified in nodal DLBCL, such as biallelic loss of TNFAIP3 (16%), PRDM1 (16%), GNA13, TMEM30A, B2M and CD58 (11% each), activating mutations of CD79B (28%) and CARD11 (19%) and translocations of BCL6 (22%). Components of the NF-kB pathways were altered in >90% of PNCSL. Pathway analysis also showed an enrichment of networks associated with immune response, proliferation, regulation of apoptosis and lymphocyte differentiation and activation. Finally, we searched for associations between genetic alterations and clinical outcome. We showed that deletions of 6q21 (PRDM1) and 6q23 (TNFAIP3) were both associated with shorter overall survival (p=0.007 and p=0.03, respectively). In summary, we report a genomic background in PCNSL similar to post-GC DLBCL but reinforcing the existence of a subset of abnormalities specific to PCNSL, suggesting their potential relevance in the disease pathogenesis. Additionally, the results obtained from FFPE samples are encouraging and larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the disease. Disclosures:Stewart:Onyx: Consultancy, Research Funding; Millenium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Patents & Royalties.

Detection and genomic interrogation of invasive circulating tumor cells (iCTCs) derived from men with metastatic castration resistant prostate cancer (mCRPC).

11048 Background: Isolation, enumeration, and genomic profiling of CRPC CTCs offers the potential to discover genetic changes that occur in advanced disease. The Vitatex VitaCap platform captures CTCs based on their ability to invade a collagenous matrix (CAM), allows for capture of invasive CTCs (iCTCs) independent of EpCAM status, and yields viable cells suitable for comprehensive genomic study. Here we sought to compare CTC yields between the CAM and CellSearch platforms, to determine the utility of prostate-specific membrane antigen (PSMA) as an iCTC biomarker, to identify iCTC clusters and iCTCs expressing stem-like markers, and to explore the feasibility of iCTC epigenomic analysis. Methods: CTCs were isolated and enumerated simultaneously using the CellSearch and CAM platforms in 23 men with mCRPC. CAM-isolated iCTCs were defined as EpCAM+PSMA+ and were enumerated immunocytochemically (ICC) and by flow cytometry. iCTC clusters were enumerated by ICC. The Illumina Infinium HumanMethylation27 BeadChip was used to determine whole genome methylation status for CAM isolated cells. Results: 35 samples were collected for CAM analysis. A median of 27 (range 0-800) and 23 (range 2-390) iCTCs/ml were detected by ICC and flow respectively. In a subset of 20 samples, a median of 7 CTCs/ml (range 0-85) were detected by the CellSearch platform. CTCs were detectable by either CAM or CellSearch in &gt;95% of samples. iCTC clusters were observed in 23% of samples with a median 7 clusters/ml (range 1-200). iCTCs expressing stem-like markers CD44 and Seprase were detected in 70% and 97% of samples by ICC and flow respectively, with a median of 9/ml (range 1-264) by flow. The iCTC methylation profile highly resembled mCRPC. Conclusions: The CAM and CellSearch platforms yield comparable CTC counts. iCTC clusters and iCTCs expressing stem-like markers are detectable using the CAM platform, and the iCTC methylome closely resembles that of mCRPC. Correlation with clinical data may yield further insight into the functional significance of these findings.

Genomic analysis of Meckel–Gruber syndrome in Arabs reveals marked genetic heterogeneity and novel candidate genes

Meckel-Gruber syndrome (MKS, OMIM #249000) is a multiple congenital malformation syndrome that represents the severe end of the ciliopathy phenotypic spectrum. Despite the relatively common occurrence of this syndrome among Arabs, little is known about its genetic architecture in this population. This is a series of 18 Arab families with MKS, who were evaluated clinically and studied using autozygome-guided mutation analysis and exome sequencing. We show that autozygome-guided candidate gene analysis identified the underlying mutation in the majority (n=12, 71%). Exome sequencing revealed a likely pathogenic mutation in three novel candidate MKS disease genes. These include C5orf42, Ellis-van-Creveld disease gene EVC2 and SEC8 (also known as EXOC4), which encodes an exocyst protein with an established role in ciliogenesis. This is the largest and most comprehensive genomic study on MKS in Arabs and the results, in addition to revealing genetic and allelic heterogeneity, suggest that previously reported disease genes and the novel candidates uncovered by this study account for the overwhelming majority of MKS patients in our population.