Copy Neutral Loss Of Heterozygosity Events Research Articles

Mutation rates and fitness consequences of mosaic chromosomal alterations in blood

Mosaic chromosomal alterations (mCAs) are common in cancers and can arise decades before diagnosis. A quantitative understanding of the rate at which these events occur, and their functional consequences, could improve cancer risk prediction and our understanding of somatic evolution. Using mCA clone size estimates from the blood of approximately 500,000 UK Biobank participants, we estimate mutation rates and fitness consequences of acquired gain, loss and copy-neutral loss of heterozygosity events. Most mCAs have moderate to high fitness effects but occur at a low rate, being more than tenfold less common than equivalently fit single-nucleotide variants. Notable exceptions are mosaic loss of X and Y, which we estimate have roughly 1,000-fold higher mutation rates than autosomal mCAs. Although the way in which most mCAs increase in prevalence with age is consistent with constant growth rates, some mCAs exhibit different behavior, suggesting that their fitness may depend on inherited variants, extrinsic factors or distributions of fitness effects.

Copy-Neutral Loss-of-Heterozygosity Adds Diagnostic and Prognostic Information to the Molecular Profiling of Hematological Malignancies

Background: Copy-neutral loss-of-heterozygosity (CN-LOH) - not detectable by chromosome banding analysis - is gaining importance as a prognostic factor and can either cause the duplication of an activating mutation in an oncogene, the deletion of a tumor suppressor gene or the gain/loss of specific methylated regions. However, examination for possible CN-LOH in hematological diagnostics is at present not routinely performed and, hence, data regarding the occurrence of CN-LOH across different entities as well as the association of relevant genes is limited.Aim: (1) Frequency assessment of CN-LOH by target enrichment sequencing (TES) in a diagnostic setting, (2) evaluation of whole genome sequencing (WGS) data to estimate the prevalence of CN-LOH in a larger cohort, to pinpoint relevant genes for CN-LOHs with so far unknown associations, and to determine cross-entity variability.Patients and Methods: 1196 patients (507 female, 689 male, median age: 66 years), sent between 04/2021-07/2021 for diagnostic work-up, were analyzed by TES with a median coverage of 1765x for the gene panel and 52x for the CNV spike-in panel (IDT, Coralville, IA). Amplification-free WGS libraries of 3851 different patients were sequenced with a median coverage of 102x. Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner). Cnvkit (v 0.9.9) was used to call copy number variations (CNVs) and CN-LOH for TES and HadoopCNV (Yang et al. 2017) was used to call CN-LOH for WGS.Results: 1196 patients were analyzed by TES. For 10% of the patients at least one CN-LOH event was detected without any association to age or gender but a slightly higher incidence in myeloid compared to lymphoid neoplasms (10% vs 6%). In 14 patients, CN-LOH affected more than one chromosome arm. CN-LOH occurred most frequently in 4q (n = 15), 7q (n = 16), 9p (n = 25) and 11q (n = 10). As expected, 4q CN-LOH co-occurred with high variant allele frequencies (VAF) of TET2. Based on WGS data, 4q CN-LOH occurred predominately in AML (35%), CMML (22%), and MDS (20%). In rare cases, 4q CN-LOH was associated with FBXW7 variants in T-ALL. 7q CN-LOH occurred nearly exclusively in myeloid neoplasms (95%) and was associated with high VAFs in EZH2 in 69% of TES and 82% of WGS cases. CUX1 variants with high VAFs were detected in 80% (TES) and 45% (WGS) of the remaining cases, respectively. The well-known 9p CN-LOH led to JAK2V617F homozygosity in all myeloid neoplasms and occurred most often in MPNs. In T-ALL, regions of 9p CN-LOH harbored CDKN2A/B deletions. 11q CN-LOH occurred more often in myeloid than lymphoid neoplasms (79% vs 21%) and was associated with CBL variants in 61% and KMT2A-PTD in 19% of the cases. In contrast, ATM was the relevant gene in all lymphoid cases with 11q CN-LOH. CN-LOH in 11p was detected less frequently and only in 25% of cases an association with WT1 variants could be identified.Our WGS data confirmed the known associations between 1p CN-LOH and high allele burden in MPL, CSF3R and NRAS, 2p CN-LOH and DNMT3A variants, 13q CN-LOH and FLT3-ITD, the near exclusive occurrence of 16p CN-LOH in follicular lymphoma (FL, 98%) with high CREBBP-mutant allele burden , 17p CN-LOH and TP53 homozygosity, and the exclusive occurrence of 21q CN-LOH in AML and its association with RUNX1 mutations. Besides, 12q CN-LOH was associated with KMT2D in FL, with SH2B3 in MDS/MPN overlaps and in rare cases with KDM2B. For 17q CN-LOH the relevant gene was not unequivocally identifiable with high mutant allele variants in SRSF2, STAT5B, and NF1. 18q CN-LOH was a very rare event but consistently associated with a high VAF of MBD2, which presumably influences cell proliferation (Cheng et al. 2018). 19q CN-LOH was mostly (63%) associated with a high VAF of CEBPA variants, except for patients with hairy cell leukemia: in these cases nonsense mutations in CIC (VAF > 90%) were detected. CN-LOH in 22q was more common in myeloid malignancies (65% vs 35%) and associated with PRR14L mutations in the majority of myeloid cases (62%). Of note, this association occurred neither in AML samples nor in lymphoid neoplasms. No recurrent mutations were found for 6p and 14q CN-LOHs. For all other chromosomes, CN-LOH events were very rare.Conclusions: By using a CNV spike-in panel, TES adds additional diagnostic and prognostic information by enabling simultaneous detection of selected gene mutations and genome-wide CNVs, as well as CN-LOH, without increase in sequencing costs and turn-around times. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

A genome-wide analysis of the molecular alterations occurring in the adenomatous and carcinomatous components of the same tumor based on the adenoma-carcinoma sequence.

Identification of molecular alterations occurring in the adenomatous and carcinomatous components within the same tumor would greatly enhance understanding of the neoplastic progression of colorectal cancer. We examined somatic copy number alterations (SCNAs) and mRNA expression at the corresponding loci involved in the adenoma–carcinoma sequence in the isolated adenomatous and cancer glands of the same tumor in 15 cases of microsatellite‐stable “carcinoma in adenoma,” using genome‐wide SNP and global gene expression arrays. Multiple copy‐neutral loss of heterozygosity events were detected at 4q13.2, 15q15.1, and 14q24.3 in the adenomatous component and at 4q13.2, 15q15.1, and 14q24.3 in the carcinomatous component. There were significant differences in the copy number (CN) gain frequencies at 20q11.21–q13.33, 8q13.3, 8p23.1, and 8q21.2–q22.2 between the adenomatous and carcinomatous components. Finally, we found a high frequency of five genotypes involving CN gain with upregulated expression of the corresponding gene (RPS21, MIR3654, RSP20, SNORD54, or ASPH) in the carcinomatous component, whereas none of these genotypes were detected in the adenomatous component. This finding is interesting in that CN gain with upregulated gene expression may enhance gene function and play a crucial role in the progression of an adenoma into a carcinomatous lesion.

Recurrent Copy Number Variants Are Highly Prevalent in Acute Myeloid Leukemia

Introduction: Large chromosomal copy number variants (CNVs) detected by conventional karyotyping are common in AML; however, small segmental CNVs or copy-neutral loss of heterozygosity (CN-LOH) are not detected by conventional techniques and require sequence-based approaches. Specifically, acquired CN-LOH occurs through duplication of genetic material and subsequent loss of the reciprocal chromosome resulting in a hemizygous state. CN-LOH has been demonstrated in AML, mostly occurring in regions of known recurrent mutations (ex: FLT3 , JAK2 , TET2 ), but its overall prevalence and involvement in AML pathogenesis has yet to be determined. Initial studies regarding the significance of CNVs suggest possible prognostic impact. We aimed to explore the prevalence of segmental CNVs and CN-LOH in a cohort of pediatric and adult AML specimens that underwent clinical comprehensive genomic profiling (CGP) (FoundationOne Heme Sequencing).Methods: CGP for targeted DNA and RNA sequencing was performed on 932 patients with AML (n=179 pediatric age ≤ 18 years, n=753 adult age ≥ 19 years). Specimen sites included bone marrow, peripheral blood, and biopsy of extramedullary disease. Following strict quality control (QC) measures to exclude any samples with transplant history, contamination, low purity, and lack of aneuploidy, all level CNV and LOH events were reported using a proprietary algorithm. A total of 323 samples met QC standards for LOH events, with all level copy number analyses available for 236 (73%) patients. We analyzed the loci of 91 genes to determine the type and prevalence of CNV events for amplifications (CNA; CN>2), deletions (CND; CN<2), CN-LOH, copy-loss LOH (CL-LOH) events at the genomic or chromosome level excluding sex chromosomes.Results: Among the 323 samples (n=59 pediatric, n=264 adult), the prevalence of LOH events was 91% (294). Identified LOH events included single gene LOH or chromosomal LOH, including arm or whole chromosome. Chromosomal events occurred in 172 (53%) of patients, and most frequently involved the following arms: 7q (n=59), 7p (n=57), 13q (n=25), 18q (n=23), 18p (n=22), 5q (n=19), and 17p (n=19). Among patients with chromosomal LOH events, 131 (76%) had events in the 7 chromosomal arms with the highest frequency (Fig 1A). Genomic LOH events were detected in 287 (89%) patients, and were detected in all 91 of the genes analyzed, with a frequency of 1-58. Further, CN-LOH events were detected in 162 (50%) patients occurring in 89 (98%) of genes analyzed (Fig 1B), with a frequency of 1-17. Among the 20 genes with highest frequency of LOH, 11 of them occurred at the 7q and 13q regions, which is consistent with previous findings that these are areas of frequent genomic losses. LOH, including CN-LOH, events overall commonly occurred in genes located in the 5q, 7p, 13q, 16q, and 17p regions. Recurrent LOH events in 3 of the most commonly affect genes occurred in approximately 31% of patients (EZH2 , n=103, BRAF , n=102, and KMT2C , n=99), all located on 7q. CN-LOH events were detected in oncogenes such as CDK6 , MET , FLT3 , JAK2 , ABL1 .Among the 236 samples evaluable for CNA assessment, 217 (92%) had at least one CNA or CND detected. CNAs were detected in 163 patients (69%) and occurred in all 91 of the genes analyzed (Fig 1C). Among samples with CNAs detected, the median number of somatically acquired CNAs was 5 (range 1-91). Recurring CNAs were detected across all chromosomes, but were most common in genes located on chromosome 1q, 8, 11q, 13q, 17q, 19, and 21q. CNDs were detected in 165 patients (70%) and occurred in 83 of genes (91%) analyzed. Among samples with CNDs detected, the median number of CNDs was 6 (range 1-58).Conclusion: CNVs and LOH events were commonly detected in this sample of 323 AML specimens. We show that LOH is highly prevalent, with genomic events detected in 89% of cases analyzed and CN-LOH events specifically detected in 50%. We found highly recurrent genes located at a few distinct chromosomal loci, suggesting some regions may be more prone to chromosomal instability. Future research in AML is needed to understand the mechanisms behind genomic and large-scale chromosomal disruptions, including CN-LOH. Identification of segmental CNVs and CN-LOH may identify cryptic variants that might aid in more appropriate diagnosis, management, disease monitoring, as well aid in the interpretation of mutation data as these events might alter the variant allele fraction of sequence variants. [Display omitted] DisclosuresHe:Foundation Medicine: Employment, Other: stock. Sun:Foundation Medicine: Employment, Other: Shareholder. Severson:Foundation Medicine: Employment, Other: Stock. Bailey:Foundation Medicine: Employment, Other: stock. Morley:Foundation Medicine: Employment, Other: Stock. Balasubramanian:Foundation Medicine: Employment, Other: stock. Erlich:Foundation Medicine: Employment, Other: stock. Lipson:Foundation Medicine: Employment, Other: stock. Otto:Foundation Medicine: Employment, Other: stock. Vergillo:Foundation Medicine: Employment, Other: stock. Ross:Foundation Medicine Inc: Employment, Other: stockholder. Mughal:Foundation Medicine Inc: Other: stockholder. Stephens:Foundation Medicine: Employment, Other: stock. Miller:Foundation Medicine Inc: Employment, Other: stockholder. He:Foundation Medicine Inc: Employment, Other: stockholder.

A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms.

Simple SummaryChromosomal abnormalities and somatic mutations are found in patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in around 50–80% of cases. The identification of these alterations is important for the accurate diagnosis and prognostic classification of these patients. Often, an apparently normal or failed karyotype might lead to an inadequate estimation of the prognostic risk, and several strategies should be combined to solve these cases. The aim of this study was to introduce a novel next-generation sequencing (NGS)-based strategy for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this approach on a large cohort of patients by comparing our findings with those obtained with standard-of-care methods (i.e., karyotype and SNP-arrays). We show that our platform represents a significant improvement on current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

Abstract 1327: Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells

Abstract Background: Genome-wide analysis of Loss-of-heterozygosity (LoH) has been shown to be important in several contexts, including the assessment of BRCAness signature, a marker of homologous repair deficiency in several cancer types associated with efficacy of platinum therapy and PARP inhibitors. Methods to infer genome-wide LoH status require high-coverage whole-genome sequencing or low-pass sequencing of a large number of single cells. We present a method to infer genome-wide LoH status of as low as one single-cell from low-pass sequencing of libraries produced by Ampli1™ LowPass kit, at a fraction of the cost of current NGS-based methods. Methods: We used low-pass sequencing data generated from 294 single Circulating Tumor Cells (CTCs) from prostate, multiple myeloma and lung cancer patients, and from 24 single cells from 8 cell lines. DNA amplified with Ampli1™ WGA kit (based on ligation-mediated PCR) combined with Ampli1 LowPass kit for Illumina generates a reduced genome representation, whereby a significant fraction of fragments is expected to be covered by more than one read. We reasoned that, even at a mean coverage insufficient for variant calling (≤0.05x), a statistically significant decrease in loci showing 2 alleles should be expected in LoH regions. A pileup of mapped sequences at dbSNP loci was obtained (minor allele frequency ≥5%; read depth ≥2). Mono- and bi-allelic loci (pileup containing 1 or 2 different alleles, respectively), were counted in each uniform copy-number (CN) genome segment within a single chromosome arm. Regions with CN equal to cell ploidy were used as internal reference. A one-sided Fisher test on biallelic sites depletion (multiplicity corrected p-value threshold of 0.05) was used to call LoH on each region. Results: Compared with libraries obtained using random shearing, Ampli1 LowPass single-cell libraries showed an increase in mean coverage of covered genome from 1.1 to 1.9 starting from the same number of reads (1M) thus increasing the number of loci for LoH calling. Analysis of 10 patients' single cells with known LoH events, corresponding to regions at copy number 1, detected 1006 events out of 1142. Several (492) copy neutral LoH events or LoHs in copy gained regions were also detected. Representatives of such events were verified successfully by high pass sequencing of the same library. Performance was confirmed by analysis of 24 single cells from cell lines whose LoH status was inferred from CCLE database. Conclusions: This innovative method leverages reduced genome representation inherent in Ampli1 LowPass libraries, to compute genome-wide LoH along with copy number (CN) status, meeting the need for LoH/CN analysis at single-cell level on rare-cells such as CTCs. Citation Format: Alberto Ferrarini, Claudio Forcato, Marianna Garonzi, Genny Buson, Massimiliano Pellicano, Francesca Fontana, Nicolò Manaresi. Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1327.

Mapping the Evolution of the Mutational Landscape of Acute Myeloid Leukemia from Diagnosis to Post-Transplantation Relapse and Its Interplay with Immune Evasion Mechanisms

Background Over the last decade, the advent of next-generation sequencing (NGS) has dramatically improved our understanding of the genomic landscape that characterize acute myeloid leukemia at time of diagnosis, leading to the identification of new prognostic subsets and therapeutic targets. Still, few studies have addressed how this landscape changes upon treatment, and in particular after allogeneic hematopoietic cell transplantation (allo-HCT). Moreover, the links between the mutational profile of AML and mechanisms of post-transplantation immune evasion remain to date largely unknown. Methods Taking advantage of the HaloPlex High Sensitivity (HS) technology, which allows a more precise definition of mutations and clonal architecture through the use of unique molecular identifiers, we designed and tested a NGS panel covering genes and miRNAs known to be involved in the pathogenesis of myeloid malignancies (n=112), in the DNA damage response (n=50) or in immune-related processes (n=30). By using an Illumina HiSeq2500n instrument,we sequenced a total of 200 AML samples collected from 131 patients, including 84 diagnoses, 45 relapses after chemotherapy (CT) and 71 relapses after allo-HCT, 31 of which characterized by genomic loss of HLA (Vago et al, N Engl J Med, 2009). Variant calling was performed using a pipeline based on the FreeBayes algorithm. Results Sequencing coverage was uniform across all target amplicons, with a 855x average depth-of-sequencing, and a mean of 122 unique barcodes for each region. Among the 84 diagnosis samples we identified 253 oncogenic mutations, with a median of 3 mutations per patient (range 0-8). Both the frequency and the prognostic value of mutations detected at diagnosis were in line with the largest published dataset on AML genomics (Papaemmanuil et al, N Engl J Med, 2016; R2= 0.88, p&amp;lt;0.0001). The overall burden of oncogenic mutations did not change significantly neither at relapses after CT nor after allo-HCT. Analyzing the profile of known oncogenic drivers of diagnoses and of the two relapse cohorts we documented high similarity between the three time-points, with significant differences limited to FLT3-ITD (present in 29% patients at diagnosis vs 49% and 48% at relapses after CT and allo-HSCT, p=0.02 and p=0.01 respectively) and WT1 mutations (5% vs 20% and 28%, p=0.006 and p&amp;lt;0.0001 respectively; Figure 1A). Also when comparing post-transplantation HLA loss relapses with their "classical" counterparts we could find no significant difference in the frequency or co-occurrence pattern of known AML driver mutations. Still, in HLA loss relapses we documented a significantly higher frequency of variants of unknown oncogenic potential befalling in genes implicated in the DNA damage response, including those belonging to the Fanconi Anemia pathway (35% of mutations in HLA loss vs 10% in classical relapses, p&amp;lt;0.0001; Figure 1B). This intriguing observation suggests that, in these cases, double strand breaks might be more frequently repaired through homologous recombination, and that this in turn might result in a higher likelihood of copy-neutral loss of heterozygosity events, the molecular mechanism at the basis of HLA loss. Finally, thanks to our panel, we were able to track possible routes of leukemic clonal evolution between diagnosis and post-transplantation relapse: interestingly, in several cases emergence of an HLA loss subclone out-competed clones carrying known oncogenic drivers, suggesting that after allo-HCT the immune privilege might outperform the proliferative advantage (Figure 1C). Discussion Taken together, our data highlight a complex interplay between the genomic landscape of AML and the environmental selective pressure represented by the graft-versus-leukemia effect. Whereas in fact some genomic alterations that carried little consequence before transplant become able in this setting to promote specific patterns of immune escape, others, that had driven AML biology before, turn out to be dispensable after allo-HCT. Understanding and tracking these evolutionary dynamics may provide new tools and targets to treat, or even prevent, AML post-transplantation relapses. Figure 1 Disclosures Sala: Gilead Company: Honoraria. Gentner:Genenta Science: Consultancy, Equity Ownership, Research Funding. Vago:GenDx: Research Funding; Moderna Therapeutics: Research Funding.

Recurrent genetic HLA loss in AML relapsed after matched unrelated allogeneic hematopoietic cell transplantation

AbstractImmune evasion is a hallmark of cancer and a central mechanism underlying acquired resistance to immune therapy. In allogeneic hematopoietic cell transplantation (alloHCT), late relapses can arise after prolonged alloreactive T-cell control, but the molecular mechanisms of immune escape remain unclear. To identify mechanisms of immune evasion, we performed a genetic analysis of serial samples from 25 patients with myeloid malignancies who relapsed ≥1 year after alloHCT. Using targeted sequencing and microarray analysis to determine HLA allele-specific copy number, we identified copy-neutral loss of heterozygosity events and focal deletions spanning class 1 HLA genes in 2 of 12 recipients of matched unrelated-donor HCT and in 1 of 4 recipients of mismatched unrelated-donor HCT. Relapsed clones, although highly related to their antecedent pretransplantation malignancies, frequently acquired additional mutations in transcription factors and mitogenic signaling genes. Previously, the study of relapse after haploidentical HCT established the paradigm of immune evasion via loss of mismatched HLA. Here, in the context of matched unrelated-donor HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention.

The Genomic and Transcriptomic Landscape of Systemic Mastocytosis

Background and AimSystemic mastocytosis (SM) is characterized by a heterogeneous spectrum of disease variants with different clinical features and prognosis. In indolent SM (ISM), the clinical course is usually stable and patients (pts) have a normal life expectancy. In contrast, aggressive SM (ASM) and mast cell leukemia (MCL) are characterized by severe organ damage and are fatal forms with short survival times. All forms of SM share the presence of activating mutations in the KIT tyrosine kinase (most frequently, the D816V mutation), which suggests that cooperating events might be responsible for the profoundly different clinical presentation and outcome of advanced SM (ASM and MCL) as compared to ISM. However, no robust studies using genome-wide approaches to scan for additional molecular aberrations in ASM and MCL have been published to date. This prompted us to use exome and RNA sequencing and single nucleotide polymorphism (SNP)-arrays to search for novel (and potentially druggable) molecular lesions.MethodsSeven pts with MCL, 6 pts with ASM and 2 pts with ISM were studied using whole exome sequencing (WES) and single nucleotide polymorphism (SNP)-arrays. WES (80x) was performed on a Hiseq 2500 (Illumina). SNP-arrays were done using Cytoscan HD Arrays (Affymetrix). Matched normal/mast cell DNA was analyzed in all but 2 archival MCL cases for whom no germline DNA source was available. RNA sequencing was also performed in 3/7 MCL and 1/6 ASM cases for whom high quality RNA was available. Data analysis and integration was performed according to in-house optimized pipelines.ResultsSNP-arrays found arm-level copy number (CN) or copy-neutral loss-of-heterozygosity (CN-LOH) events in 5/7 MCL pts (loss of 7p, 8p, 9q, 12p, 13q and CN-LOH at 4q, 18q and 21q). In contrast, ASM and ISM showed only focal CN and CN-LOH events. Overall, a median of 28 (range, 20-78) and 77 (range, 55-132) submicroscopic CN and CN-LOH events, respectively, were detected. The number of events did not significant differ between ISM, ASM and MCL. A total of 54, 102 and 1835 genes were significantly found to be recurrently involved in loss, gain and CN-LOH events, respectively. The average number of nonsynonymous mutations identified by WES was 15 in ISM and 35 in ASM and MCL. Signature analysis showed that the majority of sequence changes were C>T transitions and C>A transversions. Among genes recurrently mutated in ≥2 patients or concurrently identified by WES and SNP-arrays or known to be associated with cancer, some had already been reported to be mutated in SM: ASXL1, TET2, CBL, RUNX1, NRAS, IDH1, SRSF2, SF3B1. Alterations were also found in genes not previously implicated in SM, including RUNX3, MLL2, MLL3, CDC27, TP53BP1, CCND3, NCOR2, BCORL1, ATM, WRN, ARID1B, ARID3B, KDM1B, ARID4A, SETD1A, SETD1B, PRDM1. Pathway analysis showed enrichment of genetic lesions affecting the following cellular processes: PI3K/Akt and MAPK pathways, calcium signaling, chromatin modification, DNA methylation and DNA damage repair. RNA-seq results, filtered against those of a set of normal bone marrow samples, revealed a high number of RNA chimeras involving two adjacent genes in the same transcription orientation and invariably joining the pre-last exon of the upstream gene to the second exon of the downstream gene - a fusion pattern suggestive of intergenic splicing following transcription read-through events. Deletions at the intergenic region was excluded by SNP-arrays for all the predicted transcription-induced chimeras. In addition, in all the 4 pts analyzed, retention of some or all introns in mature transcripts was observed for some genes, including known tumor suppressors (TS) and other key genes like TP53, BAX, ERCC2, WRN, FANCD2, ATR; U2AF2, U2AF1, SRSF6.Conclusions•SNP-array and WES analysis of SM revealed a heterogeneous landscape of molecular alterations, although some key cellular pathways were found to be recurrently altered.•More interestingly, RNA-seq uncovered perturbation of transcript elongation and splicing in all the advanced SM pts analyzed. Intron retention, in particular, seems to be a novel recurrent mechanism of TS gene inactivation at the posttranslational level in advanced SM. The mechanisms leading to intron retention and their cellular consequences deserve further investigation.Supported by AIRC (project code 16996), progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresSoverini:Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Cavo:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Martinelli:MSD: Consultancy; Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau.

Genomic-Wide Analysis By High Resolution SNP Array Identifies Novel Genomic Alteration in Acute Myeloid Leukemia

Introduction: Novel array-based technique-single-nucleotide polymorphism (SNP) microarray can detect cytogenetic lesions mostly involving structural alterations with losses or gains of chromosomic material. These abnormalities are predictive of response and can help define therapeutic strategies. SNP microarray can also detect copy-neutral loss of heterozygosity (CN-LOH), which has a described role in Acute Myeloid Leukemia (AML) by inducing oncogene duplication, tumor suppressor inhibition and epigenetic reprogramming.Aim: To improve conventional cytogenetic analysis and identify new genes relevant to leukemogenesis by SNP array-based genotyping.Materials and Methods: We analyzed 279 AML patients (pts) at diagnosis by SNP Array 6.0 or Cytoscan HD Array (Affymetrix). Thirty-four samples were also analyzed by Whole Exome Sequencing WES (HiSeq 2000,Illumina). SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team, while WES data were analyzed by GATK and MuTect.Results: Copy Number Alterations (CNAs) were scattered across all chromosomes (chrs). All pts showed CNA events: 44.4% of CN gain, 21% of CN loss and 34.6% of CN-LOH.Single copy gains mainly affected chrs X, 1, 2, 4, 9 and 8. Duplications occurred at chrs 2, 3, 5 and 14. Heterozygous loss events were detected in chrs 3, 5 and 14, while regions of deletion were located in chrs 6, 7 and 22. The CN-LOH event was the most common event and involved chrs 1, 2, 3, 4, 5, 6 and 8. We studied the deletome profile in our cohort (Fig. 1) pf pts in order to define the minimal common deletion region.SNP array analysis showed that several genes were preferentially deleted, including ADAM5 (12,9%), PHF6 (12,2%), AGPS (10%), SOX6 (7,9%), WT1 (6,5%), CRLF2 (5,4%) and LRRK1 (4,3%); while the genes preferentially amplified were GPC3 (70,25%), FLT3 (44,8%), FGF13 (36,2%), KIT (31,54%), AFF2 (31,5%), ETS1 (26,52%), MITF (22,2%), CASK (16,5%), CDY1 (14,33%), MECP2 (14,33%), FOXP2 (14%) and SMAD4 (12,9%).Single-copy losses and deletions were enriched (p<0,001) for genes mapping into the following pathways: cancer-related (87 genes), metabolism (154 genes), ErbB signaling (34 genes), MAPK signaling (55 genes), ubiquitin mediated proteolysis (33 genes). Thirty-eight loss and 3 deletion events were shared by at least 14 pts (5%). Ten genes were deleted in at least 10 patients (3,6%).Concerning single copy gain and amplified genes, the functional pathways significantly represented in our cohort were: cancer-related (103 genes), regulation of actin cytoskeleton (60 genes), MAPK signaling (64 genes), metabolic (171 genes) and cell adhesion molecules pathways (CAMs, 5 genes). Regarding gain events, 24 genes were shared by at least 42 pts (15%), 184 genes were shared by at least 14 pts (5%) and 58 genes by at least 28 pts (10%); whereas 15 genes have duplication events (in homozygosis) shared by at least 14 pts (5%).Significant CN-LOH events included genes mapping to metabolic pathways (249 genes), pathways in cancer (103 genes), regulation of actin cytoskeleton (67 genes), calcium signaling pathways (57 genes), WNT signaling pathways (51 genes) and protein digestion and absorption (34 genes). CN-LOH events in 119 genes were shared by at least 14 patients (5%).Finally, we could distinguish 3 clusters of pts, each one characterized by a peculiar pattern of amplified or deleted genes. In order to define relevant pathogenic mechanisms in our cohort, we combined the deletome profile with WES data obtained from 34 pts. Interestingly, we found deletion of genes which are also targeted by mutations (BRCA2, LRRK1). Moreover, some deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations (CASK deletion and MPP6 mutation, CDK6 deletion and PPM1B mutation, MAPT deletion and SPAG5 mutation).Conclusion: By SNP array we have identified CNAs involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on the prognosis of AML pts. Minimal deleted regions deserve further investigation in order to identify new candidate oncogenes which could be relevant AML biomarkers.Acknowledgment: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12(L. Bolondi), FP7 NGS-PTL project.MCF and VG equally contributed to this work. [Display omitted] DisclosuresSoverini:Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Abstract B1-56: Distinct roles of copy number and loss-of-heterozygosity in predicting prognosis for breast cancer patients

Abstract Purpose: Novel molecular stratification of the breast cancer population based on genomic aberrations has been recently reported.[1] The main focus has been on deriving prognostic classifiers by integrating gene expression and copy number aberrations (CNAs) data, but the role of other events, such as loss-of-heterozygosity (LOH), has not been extensively characterized in this integrated landscape. Here we consider the respective role of CNAs and LOH in breast cancer, in the context of an integrated genomic analysis, and their impact on clinical outcome. Methods: Currently, retrospective data with gene expression, CNA and LOH were used from 219 patients with early primary breast cancer who were resected in the period 1989-2003.[2] CNA and LOH calls were made using OncoSNP v1.4, a Bayesian model driven genomic analysis tool to call genomic aberrations and characterize LOH status, and statistical analyses were performed in R v3.0.1. The evaluated clinical outcome was distant-recurrence free survival (DRFS). Two gene sets were tested: A) general cancer drivers from the COSMIC cancer gene census and B) all genes across the genome. Significantly amplified (increased DNA copy number with respect to normal tissue), copy neutral (no change in copy number) and deleted cancer drivers were identified by deriving null distributions from permutation analysis. Survival analysis was done using both LogRank and Cox Proportional Hazards methods. The effect of LOH on the gene expression was tested using Mann-Whitney. Benjamini and Hochberg method was used to adjust for multiple testing. Results: A higher than expected LOH occurrence was detected in 168 out of 523 COSMIC defined cancer drivers. Of these, 10 were significantly amplified, 94 were copy neutral and 64 deleted. Within these three copy number groups, patients with LOH vs no LOH were compared for DRFS. LOH was a prognostic event in 14 out of 94 copy neutral group of genes, while 4 and 2 genes were found in the amplified and deleted genes respectively. Furthermore, tumors showing copy neutral LOH events had worse outcome than tumors where these genes are amplified (no LOH) or deleted (LOH). A whole genome analysis showed that a higher global LOH frequency, across the entire genome, is associated with poor prognosis as measured by a decreased DRFS, irrespective of the global level of CNAs in the tumour. Furthermore, 386 of the 5184 genes that showed a higher than expected LOH occurrence, were significantly associated with DRFS with respect to LOH status, and 51 of these were so independently of CNAs and global LOH. Conclusions: LOH in copy neutral patients has an effect on clinical outcome in a subset of cancer drivers and overall genes, irrespective of the global genomic alteration status. This finding supports an approach of measuring and integrating multiple genomic data information, which expectantly improves the accuracy of identifying subgroups of patients, resulting in improved treatment selection. Ongoing work, which will be presented at the meeting, is focused on characterizing these events, and confirming these analyses in the large TCGA dataset[3] with multiple cancer types.

Genomic Architecture and Treatment Response In Pediatric Acute Myeloid Leukemia: A Report From The Children's Oncology Group

BackgroundChildhood acute myeloid leukemia (AML) is characterized by chromosomal instability and requires intensive therapy for cure. Here we present a large-scale study from 3 Children's Oncology Group (COG) trials to define the genomic architectural profiles of pediatric AML and to describe whole-arm gains and losses and focal rearrangements using SNP microarrays, accounting for mutation status, non-neoplastic cell infiltration and aneuploidy. We also investigate the association between somatic copy number aberrations (CNAs) and event free survival (EFS). Patients and MethodsA total of 505 matched tumor-remission samples from 459 children with de novo AML were obtained from the COG studies AAML-0531, AAML-03P1, and CCG-2961. 254 paired tumor-remission samples were genotyped on the Affymetrix SNP 6.0 chip at the University of Washington, Seattle, WA, and 251 on the Illumina 2.5M OmniQuad at the Children's Hospital of Philadelphia, PA. All genotyping output was converted to log R ratio and B-allele frequency values and annotations updated to hg19, GRCh37. Illumina intensities were additionally corrected for patterns of genomic wave.A matched allele-specific copy number analysis of tumors (ASCAT) was performed using ASCAT 2.2. A total of 246 (98%) Illumina and 247 (97%) Affymetrix samples passed quality control criteria. ASCAT profiles of patients genotyped on both platforms were manually reviewed and those with highest false CNA calls excluded. Samples with a low signal-to-noise ratio were either eliminated or visually annotated. All CNA segments were manually inspected and false positives removed. The resulting 452 ASCAT profiles were stratified into the risk categories 1) favorable, e.g. inv(16), t(16;16), t(8;21), NPM1, and CEBPα, 2) standard, e.g. normal karyotype, +8, +21, +22, del(7q), del(9q), abnormal 11q23, or other structural changes, and 3) poor, e.g. -5, -7, del(5q), or FLT3/ITD+. GISTIC 2.0 was used to identify genomic areas with significant recurrent aberrations, e.g. amplifications, deletions, and copy-neutral loss-of-heterozygosity (CN-LOH) events. Finally, we investigated whether profiles of genomic instability in the AML genome are predictive of 3 year EFS by using the total number of CNAs as a measure for allelic imbalance. ResultsThe inter-platform concordance for samples genotyped on the two platforms was very high. On average leukemic samples acquire 1.14 somatic CNAs, with a mean of 1.1, 1.3, and 0.8 in the favorable, standard, and poor risk groups respectively (Fig 1). CN-LOH events are observed in 14% (n = 64) of the patients, with 28% involving chromosome 13, and others involving the arms of 11p (23%), 1p (11%), 9p (8%), 7q (6%), 19q (6%), and 3q (5%). Known mutations in AML were enriched in recurrent focal CNA regions, as shown by amplifications on 17q24 (*TK1), 1q32, 3q28, 11q23 (*MLL), 6q27 (*DLL1), 2q32.1, and 4q35.2 (Fig 2). Focal CN-LOH regions were confined to 11p15.5 (*NUP98, *PICALM, *WT1), 1p36.3 (*RUNX3, *NRAS), 9p24.3 (*MLLT3), 3q25.3, 6p23 and 7q35 (*MLL3). Deletions include 7q36.1 (*MLL3, *EZH2), 16p13.11 (*MYH11), 9q21.32, 11p13 (*WT1), 2q37.1 (*IDH1, *DNMT3A), 10p12.31 (*MLLT10), 11q23.3 (*MLL), 16q22.1 (*CBFB), and 1p36.3 (*RUNX3). The association between CNA status and 3 year EFS approached statistical significance for all patients (Table 1), and EFS was significantly lower in standard risk patients with CNAs (51% no CNA vs 34% with CNAs, P 0.03). CNA status did not alter the event risk in the other risk groups. [Display omitted] [Display omitted] Table 1Prognostic Properties of CNAsRisk categoryCNATotal3yr EFSLogrank POverall021554% ± 7%0.0871+23547% ± 7%Favorable08066% ± 11%0.8091+8765% ± 10%Standard08051% ± 11%0.0301+10034% ± 10%Poor04842% ± 15%0.3761+4137% ± 15% ConclusionsThe number of CNAs occurring in this cohort is lower than previously reported. However, the presence of somatic CNAs in standard risk patients is significantly associated with a worse treatment outcome that is similar to the poor risk group. This study confirms the established regions of CNA enrichment in pediatric AML and identifies novel regions that may involve driving mediators of tumor fitness and/or acquired resistance to targeted therapies. Disclosures:No relevant conflicts of interest to declare.

Genome‐wide analysis of recurrent copy‐number alterations and copy‐neutral loss of heterozygosity in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the eighth most commonly diagnosed malignancy worldwide, and it is generally characterized by a poor prognosis. The aim of our study has been to identify possible recurring genomic abnormalities in this malignancy, likely to have a key role in pathogenesis. The single-nucleotide polymorphism (SNP)-array data relative to 19 HNSCC samples (submitted by Poage et al., PloS ONE 2010; 5: e9651), accessible at NCBI GEO database (GSE20939), were analyzed using criteria that take into account both genotyping and intensity data. By this method, we determined the number and localization of recurrent copy-neutral loss of heterozygosity (CN-LOH) regions and compared them with recurrent somatic copy-number alterations (CNAs). Single-nucleotide polymorphism-array data analysis allowed us to detect, for the first time in HNSCC, chromosomal segment of CN-LOHs in addition to CNAs. Chromosomal alterations have been detected in 14 (73.7%) of 19 samples, and the 12.1% of all alterations observed (LOHs, gains, and CN-LOHs) were CN-LOHs. The most recurrent gain events, occurring in 78.5% of cases (11 samples), were harbored within 8q21.11-q21.13 and 8q23.1-q24.22 loci, while the most recurrent loss and CN-LOH events were present at 3p21.31-p21.1 (57.1%; 8 samples) and 17p13.3-p13.1 (28.6%; 4 samples) loci, respectively. The investigated chromosomal regions, in particular those with CN-LOH, harbored some interesting genes, such as HIC1, DOCK8, KANK1, and NOTCH1 whose role, mutations and epigenetic modifications in HNSCC deserve to be investigated, in order to understand the significance of CN-LOH events in HNSCC pathogenesis.

Copy neutral loss of heterozygosity is more frequent in older ovarian cancer patients

Loss of heterozygosity (LOH) is a common type of genomic alterations in ovarian cancer. Analyzing 74,415 copy neutral LOH events in 513 serous ovarian adenocarcinomas samples from the Cancer Genome Atlas, we report that the frequency of LOH events increases with age. Similar trend is observed for LOH involving chromosome 17, which is frequently implicated in ovarian cancer. The results are consistent when we analyze data from the Boston high-grade serous cancer cohort. We further show that germ line and somatic mutations in BRCA1 (in chromosome 17) and BRCA2 (in chromosome 13) loci are not necessary to establish the pattern. We also report significant age-related changes in expression patterns for several genes in the homologous recombination (HR) pathway, such as BRCA1, RAD50, RAD52, XRCC2, XRCC3, and MRE11A in these patient samples. Furthermore, we develop a metric for pathway-level imbalance, and show that increased imbalance in the HR pathway, i.e., increase in expression of some HR genes and decrease in expression of others, is common and correlates significantly with the frequency of LOH events in the patient samples. Taken together, it is highly likely that aging and deregulation of HR pathway contribute to the increased incidence of copy-neutral LOH in ovarian cancer patients.

Integrated analysis of copy number and loss of heterozygosity in primary breast carcinomas using high-density SNP array

Breast cancer is a heterogeneous disease, marked by extensive chromosomal aberrations. In this study, we aimed to explicate the underlying chromosomal copy number (CN) alterations and loss of heterozygosity (LOH) implicated in a cohort of Malaysian hospital-based primary breast carcinoma samples using a single nucleotide polymorphism (SNP) array platform. The analysis was conducted by hybridizing the extracted DNA of 70 primary breast carcinomas and 37 normal peripheral blood samples to the Affymetrix 250K Sty SNP arrays. Locus-specific CN aberrations and LOH were statistically summarized using the binary segmentation algorithm and hidden Markov model. Selected genes from the SNP array analysis were also validated using quantitative real-time PCR. The merging of CN and LOH data fabricated distinctive integrated alteration profiles, which were comprised of finely demarcated minimal sites of aberrations. The most prevalent gains (≥ 30%) were detected at the 8q arm: 8q23.1, 8q23.3, 8q24.11, 8q24.13, 8q24.21, 8q24.22, 8q24.23 and 8q24.3, whilst the most ubiquitous losses (≥ 20%) were noted at the 8p12, 8p21.1, 8p21.2, 8p21.1-p21.2, 8p21.3, 8p22, 8p23.1, 8p23.1‑p23.2, 8p23.3, 17p11.2, 17p12, 17p11.2-p12, 17p13.1 and 17p13.2 regions. Copy-neutral LOH was characterized as the most prevailing LOH event, in which the most frequent distributions (≥ 30%) were revealed at 3p21.31, 5q33.2, 12q24.12, 12q24.12‑q24.13 and 14q23.1. These findings offer compre-hensive genome-wide views on breast cancer genomic changes, where the most recurrent gain, loss and copy-neutral LOH events were harboured within the 8q24.21, 8p21.1 and 14q23.1 loci, respectively. This will facilitate the uncovering of true driver genes pertinent to breast cancer biology and the develop-ment of prospective therapeutics.