Detection Of Copy Number Alterations Research Articles

PB1662 INTEGRATION OF GENOME‐WIDE ANALYSIS INTO CLINICAL DIAGNOSIS OF ACUTE LYMPHOBLASTIC LEUKEMIA: HOW MUCH CAN WE IMPROVE CYTOGENETIC TESTING?

Background:Acute lymphoblastic leukemia (ALL) is a heterogeneous, aggressive disease, and most common in children. It is a leading cause of cancer death in young and adult, too. However, about 30% of pediatric and 50% of adult ALL patients still lack defined genetic hallmarks of biological and clinical significance. In addition, complex karyotype‐ALL (CX‐ALL) promoting a wide spectrum of diverse genetic alterations with striking genomic instability, are overall often neglected by research.Aims:The overall goal of this study was to characterize karyotypes and genetic content of 25 ALL cases and to rule out if one single molecular approach may be sufficient to detect genomic aberrations in these patients.Methods:ALL cases were retrospectively studied by multitude multicolor banding (mMCB), fluorescence in situ hybridization (FISH) using selected locus‐specific probes and array‐comparative genomic hybridization (aCGH).Results:Most strikingly, 13 of the studied 25 ALLs (∼55%) revealed abnormal karyotypes, applying mMCB: structural abnormalities were present in 12 and even numerical ones were identified in 2 cases. Overall, 123 copy number alterations (CNAs) were identified as loss (∼80%) or as gains (∼20%). Furthermore, highly complex karyotypes were detected by aCGH and mMCB in 4 out of the 13 aberrant cases. The latter four patients had either deletions or amplifications in genes involved in lymphoid development, cell cycle, Hematopoietic stem cells development and/or DNA repair (IKZF1, CDKN2A/B, PAX5, BIRC3, ATM, RB1, RUNX1), as shown by FISH, and also presented acquired marker chromosomes.Summary/Conclusion:Molecular approaches are suited to identify cryptic aberrations and potential target genes involved in leukemogenesis and progression of the disease. The present work demonstrated that aCGH is a highly efficient tool for detection of CNAs in ALL. However, while aCGH provide data on imbalanced genomic alterations, molecular cytogenetics additionally detects different leukemic subclones within one sample, as well as balanced translocations leading to tumor‐specific fusion genes. Thus, still no single approach is sufficient to comprehensively characterize all acquired aberrations in ALL.

PS1367 COMPREHENSIVE GENETIC ANALYSIS OF MULTIPLE MYELOMA IN JAPAN

Background:Next generation sequencing (NGS) technology has enabled comprehensive analyses of genomic abnormalities occurring in cancer. Previous genetic studies using large cohorts of patients with multiple myeloma (MM) have revealed the genomic landscape of MM. Although some of these genomic abnormalities are believed to be associated with the molecular pathogenesis of MM and/or clinical outcome, these relationships are not fully understood. In addition, most previous studies have been performed in European and American cohorts. Thus, the genomic profiles of Asian or Japanese MM patients remains unclear.Aims:We aimed to clarify the genomic profiles of Japanese patients with MM and to explore genomic alterations correlated to the clinical or pathological features of MM.Methods:The cohort in this study included 138 Japanese patients, newly diagnosed with MM between Dec. 2013 and Jan. 2018. The median age at diagnosis was 69 years (36–87), and the male to female ratio was 1:1. Tumor DNA was isolated from CD138‐positive bone marrow plasma cells, and germline control DNA was derived from peripheral blood mononuclear cells. A total of 340 genes related to MM and immunoglobulin (Ig) gene rearrangement regions were analyzed for somatic mutations and clonal Ig rearrangements by targeted capture sequencing. Single nucleotide polymorphism‐baits were also included to detect copy number alterations (CNA) by sequencing. IgH‐CCND1 translocations were detected, and expression levels of CCND1 mRNA were measured using FISH and RQ/RT‐PCR, respectively.Results:Clonal Ig gene rearrangements in myeloma‐derived cells were identified in all patients. In total, 136/138 patients (98.6%) harbored at least one single‐nucleotide variant or indel, with a median of 4 (0–12). KRAS was the most commonly mutated gene (48/138, 34.8%), and abnormalities in RAS/MAPK pathway genes were present in 85/138 patients (61.6%). In copy number analysis, 130 /138 patients (94.2%) had some kind of CNA and 52/138 (37.7%) were considered to be hyperdiploid subtypes according to WHO classification. Analysis of relationships between somatic mutations and CNA revealed MAX gene mutations in 9 patients (6.5%). All of these were accompanied by loss of heterozygosity (LOH) at chr14q23. An expression analysis was performed in 92 (66.7%) patients. Overexpression of CCND1 was observed in all 24 patients with t(11;14) as previously reported. Moreover, patients with a gain of CCND1 and uniparental disomy (UPD) of chr11 showed CCND1 overexpression. Del(17p) was observed in 13/138 patients (9.4%) and was predictive of shorter overall survival (OS) (p < 0.001).In cases with t(11;14), the presence of a KRAS mutation also tended to predict shorter OS (p = 0.0379).Figure:Frequency of mutated genes in this cohort.Summary/Conclusion:To the best of our knowledge, this is the first report of a comprehensive genetic analysis of Japanese MM patients. Although most of our results were compatible with those of the previous reports, hyperdiploid cases were less frequent, and KRAS mutations were more frequent than in other cohorts. These characteristics, although based on small numbers, may represent racial differences. In addition, all of the patients who had MAX mutations displayed LOH in this cohort. Because the function of MAX is not fully understood in MM, this correlation may add to our understanding of the molecular pathogenesis of myeloma.image

S135 NGS‐BASED COPY‐NUMBER ANALYSIS IN MORE THAN 2,000 PATIENTS WITH MYELOID NEOPLASMS

Background:Copy‐number alteration (CNA) is a hallmark of cancer genomes and has been implicated in the development of human cancers, including myeloid neoplasms. We developed a novel, next‐generation sequencing‐based platform for highly sensitive detection of CNAs, which was applied to sequencing data from more than 2,000 patients.Aims:To delineate a comprehensive landscape of CNAs in myeloid neoplasms and differential impacts of CNAs on clinical and biological phenotype.Methods:Whole‐exome sequencing (WES) was performed on samples from 260 patients with de novo acute myeloid leukemia (de novo AML, n = 136), myelodysplastic syndrome (MDS, n = 75), myelodysplastic/myeloproliferative neoplasms (MDS/MPN, n = 32), or AML‐MDS (n = 17). Additionally, almost 2,000 cases with de novo AML, MDS, MDS/MPN, AML‐MDS, or myeloproliferative neoplasms (MPN) were examined by targeted deep sequencing, 763 of which were pre‐transplantation peripheral blood provided by Japan Marrow Donor Program.Total copy numbers and allele‐specific copy numbers (ASCNs) were quantified based on sequencing depths and allelic ratios on genome‐wide probes. To enhance the precision of analyses, copy‐number signals were corrected for multiple known biases (e.g. those due to GC content, ASCN, and PCR duplication). We also corrected residual biases by comparing signals in a tumor sample with those in normal samples. Copy‐number polymorphisms reported in Database of Genomic Variants were excluded.Results:Analyses on 260 cases using WES identified a total of 821 CNAs (497 deletions, 246 amplifications, 78 UPD), where 57% of the patients harbored at least one alteration. Using GISTIC 2.0 algorism, we identified significantly altered regions involving myeloid driver genes: losses of 7q22.1 (CUX1), 12p13.2 (ETV6), 17p13.1 (TP53), and 17q11.2 (NF1), and gains of 3q26–27 (EVI1), 8q24.21 (MYC), 11q23.3 (MLL), 11q24–25 (ETS1), 13q12.2 (FLT3), 21q22.2 (ERG). Comparing between myelodysplasia (MDS, MDS/MPN, AML‐MDS) and de novo AML, deletions of 13q14 were significantly enriched in myelodysplasia (Odds ratio [OR]: 5.07, P = 0.040) while amplifications of 11q24–25 (OR: 5.54, P = 0.028), and 21q22.2 (OR: 6.10, P = 0.020) in de novo AML, suggesting a specific role of these events in each disease entity.Expanding our findings, we combined cases examined by WES with those examined by targeted deep sequencing. In the whole cohort, the landscape of CNAs is largely consistent with that in WES data, and we additionally highlighted less frequent but recurrent alterations, resulting in a comprehensive landscape of CNAs in myeloid neoplasms. This allowed us to assess the associations of CNAs with disease characteristics in details. A set of CNAs correlated with specific disease subtypes. For example, del(5q), del(7q), del(12p), and loss of heterozygosity (LOH) of 17p were enriched in AML‐MDS as compared with MDS, while LOH of 4q and 11q were enriched in Chronic myelomonocytic leukemia. In prognostic analyses, del(16q), amp(11q), amp(19q), amp(22q) were most strongly associated with poor survivals among all CNAs (Hazard ratio: >2.0, P < 0.001), independently of complex karyotypes or TP53 mutations.Summary/Conclusion:We obtained the comprehensive landscape of CNAs in myeloid neoplasms based on the sequencing data from more than 2,000 patients. We found that CNAs targeted specific myeloid driver genes and were differentially correlated with disease subtypes and prognosis. Collectively, our analysis uncovered the clinical and biological implications of CNAs in myeloid neoplasms.

Chromosomal microarray analysis is superior in identifying cryptic aberrations in patients with acute lymphoblastic leukemia at diagnosis/relapse as a single assay

Conventional cytogenetics (CC) is important in diagnosis, therapy, monitoring of post-transplant bone marrow, and prognosis assessment of acute lymphoblastic leukemia (ALL). However, due to the nature of ALL, CC often encounters difficulties of complex karyotype, poor chromosome morphology, low mitotic index, or normal cells dividing only. In contrast, chromosomal microarray analysis (CMA) showed a specificity >99% and a sensitivity of 100% in chronic lymphocytic leukemia (CLL) patients. Here, we report our experience with CMA on adult ALL patients. Thirty-three bone marrow/blood samples from ALL patients (aged 18-79years, median 44) at diagnosis/relapse, analyzed by CC and/or fluorescence in situ hybridization (FISH), were recruited. Chromosomal microarray analysis results were compared with CC. Fluorescence in situ hybridization analysis, if available, was applied when there was a discrepancy. Copy-neutral loss-of-heterozygosity (CN-LOH) was found in 8 cases (24.2%). Only CN-LOH at 9p was recurrent (3 cases, 9.1%). Copy number alterations (CNAs) were detected in 6 of 9 cases (66.7%) with normal karyotypes, in 3 of 5 cases (60.0%) with sole "balanced" translocations, and in 18 of 19 cases (94.7%) with complex karyotypes. Common CNAs involved CDKN2A/2B (30.3%), IKZF1 (27.3%), PAX5 (9.1%), RB1 (9.1%), BTG1 (6.7%), and ETV6 (6.7%), which regulate cell cycle, B lymphopoiesis, or act as tumor suppressors in ALL. Copy number alteration detection rate by CMA was 81.8% (27 of 33 cases) as compared to 57.6% (19 of 33 cases) by CC. Incorporation of CMA as a routine clinical test at the time of diagnosis/relapse, in conjunction with CC and/or FISH, is highly recommended.

A novel subpopulation of circulating tumor cells in patients with cholangiocarcinoma.

e15637 Background: In cholangiocarcinoma (CCA), molecular analysis during disease progression is rarely feasible due to low access to tissue. Circulating tumor cells (CTCs) could overcome this issue. Data on CTCs in CCA are presently insufficient to promote their use in clinical management due to their low detection rates. Improved methods to capture all CTC subpopulation are therefore needed. Methods: Peripheral blood (PB) was longitudinally collected from patients with metastatic/unresectable CCA (intrahepatic, extrahepatic and gallbladder). CTCs were enriched from PB (10mL) with Parsortix, labeled for epithelial and leukocyte markers, and analyzed in the DEPArray to collect i) single CTCs positive for epithelial markers (eCTC) and ii) single double negative cells (DNC) lacking epithelial and leukocyte markers. DNCs underwent WGA and low-pass WGS to detect copy number alterations. DNCs with aberrant genotype were defined as non-conventional CTCs (ncCTCs). Results: Forty-one blood samples were collected from 21 patients receiving gemcitabine and/or platinum-based regimens. We detected 18 eCTCs and 73 ncCTCs, respectively in 8/41 (19%, median number = 1.5, range 1-5) and 31/41 (76%, median number = 2, range 1-7) samples. By considering also ncCTCs, CTC-positivity increased by 4.4-fold (83% of CTC+ samples). All untreated patients were CTC+, and CTC- samples (n = 7) were collected only on-treatment. In 18 patients, the detection of eCTCs ( ≥1eCTC/10mL blood) at baseline was associated with a shorter overall survival than in eCTC- patients (7.0 vs 19 months, p = 0.03). CTC count fluctuations during treatment mirrored patient response. Indeed, in responding patients CTCs were not detectable. In some patients, CTC counts were more informative on treatment outcome than imaging and Ca19.9 levels. Conclusions: We developed a new single-cell protocol to detect two distinct CTC subpopulations (eCTC and ncCTC) in patients with CCA. Baseline eCTCs detected by our method inform prognosis whereas ncCTCs provide hints on treatment response. The possibility to collect and molecularly characterize CTC from all patients with our protocol, opens the way to future treatment planning based on CTC molecular asset.

Tumor Heterogeneity Index to Detect Human Epidermal Growth Factor Receptor 2 Amplification by Next-Generation Sequencing: A Direct Comparison Study with Immunohistochemistry

Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) is common in gastric cancer (GC). However, a direct comparison between the results of HER2 immunohistochemistry (IHC) and next-generation sequencing (NGS)-based cancer panel tests has not been explored in GC. We aimed to determine optimal thresholds of HER2 overexpression to be detected by NGS with the data of 168 metastatic GC cases with known expression levels of HER2 by IHC and the copy number alteration of ERBB2 obtained by NGS test by applying tumor heterogeneity index (THI) and receiver operating characteristic curve. GC tissues were obtained via biopsy (N = 103) and gastrectomy (N = 65). HER2 3+by IHC was observed in 27 cases (16%), and 14 of 27 HER2 IHC 3+ cases (52%) showed intratumoral heterogeneity (<90% of tumor cells are HER2 3+). Of 27 HER2 3+ cases, 19 (70%) were detected by NGS. All eight cases with discrepant IHC and NGS results harbored intratumoral heterogeneity. The receiver operating characteristic curve analysis showed that the optimal value of THI to be detected by NGS was a score of 72 for biopsy specimens and 120 for resection specimens. In conclusion, intratumoral heterogeneity of HER2 expression is observed in 52% of metastatic GC cases. NGS laboratories should be aware that accuracy of ERBB2 copy number alteration detection by NGS is influenced by the THI.

An accessible GenePattern notebook for the copy number variation analysis of Illumina Infinium DNA methylation arrays

Illumina Infinium DNA methylation arrays are a cost-effective technology to measure DNA methylation at CpG sites genome-wide and across cohorts of normal and cancer samples. While copy number alterations are commonly inferred from array-CGH, SNP arrays, or whole-genome DNA sequencing, Illumina Infinium DNA methylation arrays have been shown to detect copy number alterations at comparable sensitivity. Here we present an accessible, interactive GenePattern notebook for the analysis of copy number variation using Illumina Infinium DNA methylation arrays. The notebook provides a graphical user interface to a workflow using the R/Bioconductor packages minfi and conumee. The environment allows analysis to be performed without the installation of the R software environment, the packages and dependencies, and without the need to write or manipulate code.

Nano-GLADIATOR: real-time detection of copy number alterations from nanopore sequencing data

The past few years have seen the emergence of nanopore-based sequencing technologies which interrogate single molecule of DNA and generate reads sequentially. In this paper, we demonstrate that, thanks to the sequentiality of the nanopore process, the data generated in the first tens of minutes of a typical MinION/GridION run can be exploited to resolve the alterations of a human genome at a karyotype level with a resolution in the order of tens of Mb, while the data produced in the first 6-12h allow to obtain a resolution comparable to currently available array-based technologies, and thanks to a novel probabilistic approach are capable to predict the allelic fraction of genomic alteration with high accuracy. To exploit the unique characteristics of nanopore sequencing data we developed a novel software tool, Nano-GLADIATOR, that is capable to perform copy number variants/alterations detection and allelic fraction prediction during the sequencing run ('On-line' mode) and after experiment completion ('Off-line' mode). We tested Nano-GLADIATOR on publicly available ('Off-line' mode) and on novel whole genome sequencing dataset generated with MinION device ('On-line' mode) showing that our tool is capable to perform real-time copy number alterations detection obtaining good results with respect to other state-of-the-art tools. Nano-GLADIATOR is freely available at https://sourceforge.net/projects/nanogladiator/. Supplementary data are available at Bioinformatics online.

Highly Multiplexed Fluorescence in Situ Hybridization for in Situ Genomics

The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. We demonstrate the reliability of this multiplex approach on normal human lymphocytes, metaphase spreads of transformed cell lines, and cultured circulating tumor cells. It also opens the door to the development of gene panels for more comprehensive analysis of copy number changes in tissue, including the study of heterogeneity and of high-throughput clinical assays that could provide rapid quantification of gene copy numbers in samples with limited cellularity, such as circulating tumor cells.

An accessible GenePattern notebook for the copy number variation analysis of Illumina Infinium DNA methylation arrays.

Illumina Infinium DNA methylation arrays are a cost-effective technology to measure DNA methylation at CpG sites genome-wide and across cohorts of normal and cancer samples. While copy number alterations are commonly inferred from array-CGH, SNP arrays, or whole-genome DNA sequencing, Illumina Infinium DNA methylation arrays have been shown to detect copy number alterations at comparable sensitivity. Here we present an accessible, interactive GenePattern notebook for the analysis of copy number variation using Illumina Infinium DNA methylation arrays. The notebook provides a graphical user interface to a workflow using the R/Bioconductor packages minfi and conumee. The environment allows analysis to be performed without the installation of the R software environment, the packages and dependencies, and without the need to write or manipulate code.

Detection of mutations in SF3B1, EIF1AX and GNAQ in primary orbital melanoma by candidate gene analysis

BackgroundOcular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest primary site), conjunctiva or the orbit. Primary orbital melanoma (POM) is exceedingly rare, with approximately 60 cases reported to date. Despite recent advances in our understanding of the genetics of primary uveal and conjunctival melanomas, this information is lacking for POM.MethodsDNA was extracted from 12 POM tissues, with matched germline DNA (where available). MLPA was conducted to detect chromosomal alterations and Sanger sequencing used to identify point mutations in candidate melanoma driver genes (BRAF, NRAS, KRAS, GNA11, GNAQ), and other genes implicated in melanoma prognosis (EIF1AX, SF3B1). Immunohistochemistry was performed to analyse BAP1 nuclear expression.ResultsMLPA detected copy number alterations in chromosomes 1p, 3, 6 and 8. Sequencing of melanoma driver genes revealed GNAQ (p.Q209L) mutations in two samples; although it is possible that these samples represent extraocular spread of an occult uveal melanoma. A recurrent mutation in SF3B1 (p.R625H) was observed in indolent, but not aggressive, tumours; a mutation in EIF1AX (p.N4S) was detected in one patient with non-aggressive disease.ConclusionsEIF1AX and SF3B1 mutations appear have a role in determining the clinical course of POM and detection of these changes could have clinical significance. Further in depth analysis of this rare group using differing ‘omic technologies will provide novel insights into tumour pathogenesis.

Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project

PurposeFresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS. MethodsWe conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples. ResultsWe found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80°C or 65°C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs). ConclusionWe present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.

Abstract 1033: Patient-derived xenografts for prognostication and personalized treatment for head and neck squamous cell carcinoma

Abstract Overall outcomes for HPV-negative head and neck squamous cell carcinoma (HNSCC) remain poor with 5-year overall survival rates of 50-60%. Oral squamous cell carcinoma (OSCC), the most common subtype of HPV-negative HNSCC, is typically treated with surgery, and clinico-pathologic features are used to identify patients in need of adjuvant therapies such as radiation therapy (RT), or radiation plus concurrent chemotherapy (CRT). It is clear from the rate of loco-regional or distant failures that more accurate methods of risk stratification would greatly improve outcomes for OSCC patients. This requires biomarkers to identify patients that will benefit from adjuvant RT or CRT but currently there are no validated molecular biomarkers that have been clinically implemented for the personalized treatment of OSCC. In addition to biomarkers for better risk stratification, there is also a need for novel therapeutic strategies leading to improved outcomes. Recently patient-derived xenografts (PDXs) have been shown to faithfully recapitulate human tumor biology and predict drug responses, supporting their relevance as preclinical models for new drug development. Upon subcutaneous implantation of HNSCC specimens into NOD/SCID/IL2Rγ-/- mice, 161 of 243 samples (66%) successfully formed patient-derived xenografts (PDX). Using univariable and multivariable analyses, the ability to form a PDX correlated significantly with adverse clinical outcomes, and specifically, patients with palpable PDX-formation within 8 weeks experienced particularly poor outcomes (hazard ratio for overall survival = 3.0). A cohort of engrafting and non-engrafting samples were sequenced using a targeted sequencing panel designed for both mutational and copy number alteration detection. The overall frequency of somatic genomic alterations detected was similar to The Cancer Genome Atlas cohort and interestingly, successful engraftment correlated to amplification of the CCND1 gene. Twelve PDX models were treated with the CDK4/6 inhibitor, abemaciclib; 5 of 6 models with CCND1 amplifications and/or CDKN2A mutations responded to abemaciclib treatment, whereas only 1 of 6 models lacking these alterations responded. These results demonstrate the potential of using PDX models to identify novel targeted therapies for HNSCC patients who have the poorest outcomes. In the future, PDX avatars could also be exploited to individualize treatment for patients at high risk of relapse following definitive treatment. Citation Format: Christina Karamboulas, Jeffrey P. Bruce, Andrew J. Hope, Jalna Meens, Shao Hui Huang, Jie Su, Fei-Fei Liu, Trevor J. Pugh, Scott V. Bratman, Wei Xu, Laurie E. Ailles. Patient-derived xenografts for prognostication and personalized treatment for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1033.

Abstract 5346: Recurrent genomic copy alterations deregulate the transcriptome and key signaling networks in head and neck cancer cell lines and tumors with worse prognosis

Abstract Human cancer cell lines have been important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations and subtypes found in tumors could further accelerate functional and therapeutic discoveries. We conducted an integrated analysis of the genomic and transcriptomic profiles of 15 human papillomavirus negative (HPV−) and 11 HPV(+) head and neck squamous cell carcinoma (HNSCC) lines, to identify models of important molecular subtypes uncovered among 279 HNSCC tumors from The Cancer Genome Atlas (TCGA). We developed analytic approaches to detect copy number alterations (CNAs) driving altered expression and predict deleterious mutations in HNSCC lines for comparison with TCGA. Strikingly, HNSCC lines displayed recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of a cluster of ~100 mRNAs encoding multiple known and understudied candidate oncogenes. These CNAs, together with loss and under-expression or mutations of putative tumor suppressor genes, recapitulate genomic alterations found in more aggressive HNSCC tumor subtypes. Among these, concurrent 3q26.3 amplification and TP53 mutation in HPV(−) HNSCCs is associated with worse overall survival. Common and distinctive CNAs, transcriptome, and mutations converge on biologically and therapeutically important signal pathways and functions in HNSCCs differing in HPV status, including PI3K, Hippo, TGF-β, Wnt/β-catenin, TP63, TP53/death, cell cycle/mitosis, stemness/differentiation, chromatin remodelling/DNA replication, post-transcriptional regulation, and mitochondrial biosynthesis. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with historic and recently derived HNSCC lines, and provide valuable models for future preclinical and therapeutic studies in subsets of HNSCCs with worse prognosis. Supported by NIDCD/NIH intramural projects ZIA-DC-000073, ZIA-DC-000074. This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov) Citation Format: Hui Cheng, Xinping Yang, Han Si, Anthony Saleh, Wenming Xiao, Jamie Coupar, Susanne M. Gollin, Robert L. Ferris, Natalia Issaeva, Wendell G. Yarbrough, Mark E. Prince, Thomas E. Carey, Carter Van Waes, Zhong Chen. Recurrent genomic copy alterations deregulate the transcriptome and key signaling networks in head and neck cancer cell lines and tumors with worse prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5346.

Abstract 1183: Targeted sequencing of cell-free DNA data enables comprehensive profiling of tumor copy number landscape from blood

Abstract Background: The evolution of cancer genomes within a single tumor creates mixed cell populations with divergent somatic mutational architectures. Inference of tumor subpopulations has been disproportionately focused on the assessment of somatic point mutations, whereas computational methods targeting copy number alterations (CNA) in targeted sequencing data remain underdeveloped. Methods: Using a large database of clinical cell-free DNA (cfDNA) sequencing data (Guardant Health, CA), we developed a coverage-based probabilistic model to simultaneously normalize molecular coverage, segment the genome, predict copy number alterations, and estimate the tumor content in cfDNA samples, while accounting for mixtures of cell populations. This model was technically validated using tissue fluorescence in situ hybridization (FISH) data and then applied to a unique set of cfDNA sequencing data from &amp;gt;5,000 normal and clinical samples spanning multiple cancer types, where model predictions were compared to the observed allelic frequencies of somatic driver mutations and heterozygous germline SNPs. Results: Technical validation against FISH-derived tissue copy number estimates demonstrated high concordance with model estimates. Analysis of clinical samples demonstrated a wide range of copy number architectures, including prevalent copy number neutral loss-of-heterozygosity, large chromosomal deletions, and high focal amplifications, all of which are not easily detected and/or differentiated with standard sequencing analysis approaches on highly-fragmented cfDNA. Conclusion: Our results show that probabilistic modeling of coverage data generated from targeted cfDNA sequencing can detect and differentiate heterogeneous tumor populations with diverse somatic variations, CNA, and LOH landscape. This method may enable improvements in CNA detection accuracy, sensitivity, and specificity and provides more precise interrogation of tumor fraction and clonal architecture. Citation Format: Catalin Barbacioru, Eric Collisson, Darya Chudova, Justin Odegaard, Richard Lanman, AmirAli Talasaz. Targeted sequencing of cell-free DNA data enables comprehensive profiling of tumor copy number landscape from blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1183.

Abstract 422: Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications

Abstract Next Generation Sequencing is increasingly used in translational cancer research and as a diagnostic test. Most tumor specimens available for testing are formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE-derived DNA is typically damaged, causing difficulties for standard library preparation methods. Frayed ends prevent blunt-ended double-stranded adapter ligation, used for whole genome sequencing (WGS) and targeted bait hybridization library preparation. Fragmented DNA and modified bases interfere with PCR, which underlies amplification based targeted sequencing. We present a single-stranded library preparation method especially forgiving for low quality FFPE-derived DNA. By excising damaged bases and using single-stranded adapter ligation, we omit repair aimed at generating blunt-ended double-stranded DNA, and eliminate the need for whole genome PCR. After ligation of the first adapter, libraries can either undergo second adapter ligation to generate WGS libraries, or be used as input to oligo-selective sequencing (OS-Seq) target capture. We show that our FFPE-derived PCR-free WGS libraries exhibit coverage depth and uniformity highly similar to double-stranded PCR-free libraries of matched cell line (non-FFPE) materials. Additionally, we present performance of our WGS library preparation on clinical FFEP samples. We further demonstrate reproducible high and uniform coverage for reference standards and clinical FFPE samples in targeted capture. We show a lack of correlation between DNA quality and sequencing metrics such as on-target rate (R2=0.005), uniformity (R2=0.0002), and coverage (R2=0.06), with successful removal of C&amp;gt;T FFPE artifacts comparable to standard repair methods. We demonstrate sensitive copy number alteration detection through analysis of coverage depth, feasible due to the absence of pre-capture PCR. In conclusion, our single-stranded library preparation method is forgiving for sample quality and provides uniform and high coverage as the basis for somatic variant calling. Citation Format: Anna Vilborg. Single-stranded library preparation enables unbiased sequencing of damaged, FFPE-derived DNA for both targeted gene capture and WGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 422.

Clinical-grade validation of whole genome sequencing reveals robust detection of low-frequency variants and copy number alterations in CLL.

The 100000 Genome Project aims to develop a diagnostics platform by introducing whole genome sequencing (WGS) into clinical practice. Samples from patients with chronic lymphocytic leukaemia were subjected to WGS. WGS detection of single nucleotide variants and insertion/deletions were validated by targeted next generation sequencing showing high concordance (96·3%), also for detection of sub-clonal variants and low-frequency TP53 variants. Copy number alteration detection was verified by fluorescent insitu hybridisation and genome-wide single nucleotide polymorphism array (concordances of 86·7% and 92·9%, respectively), confirming adequate sensitivity by WGS. Our results confirm that WGS can provide comprehensive genomic characterisation for clinical trials, drug discovery and, ultimately, precision medicine.

Multigene sequencing, copy number alteration detection, and survival with first-line PD-1 based therapy in patients with mucosal melanoma.

e21579Background: Mucosal melanomas (McM) have lower tumor mutational burden (TMB) and higher fraction of genome with alterations (FGA) than cutaneous melanomas. Sequencing efforts to date suggest ...

Identification of copy number alterations in colon cancer from analysis of amplicon-based next generation sequencing data.

The objective of this study was to determine the feasibility to detect copy number alterations in colon cancer samples using Next Generation Sequencing data and to elucidate the association between copy number alterations in specific genes and the development of cancer in different colon segments. We report the successful detection of somatic changes in gene copy number in 37 colon cancer patients by analysis of sequencing data through Amplicon CNA Algorithm. Overall, we have found a total of 748 significant copy number alterations in 230 significant genes, of which 143 showed CN losses and 87 showed CN gains. Validation of results was performed on 20 representative genes by quantitative qPCR and/or immunostaining. By this analysis, we have identified 4 genes that were subjected to copy number alterations in tumors arising in all colon segments (defined “common genes”) and the presence of copy number alterations in 14 genes that were significantly associated to one specific site (defined “site-associated genes”). Finally, copy number alterations in ASXL1, TSC1 and IL7R turned out to be clinically relevant since the loss of TSC1 and IL7R was associated with advanced stages and/or reduced survival whereas copy number gain of ASXL1 was associated with good prognosis.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.