Somatic Copy Number Alterations Profiles Research Articles

LabelSeg: segment annotation for tumor copy number alteration profiles.

Somatic copy number alterations (SCNAs) are a predominant type of oncogenomic alterations that affect a large proportion of the genome in the majority of cancer samples. Current technologies allow high-throughput measurement of such copy number aberrations, generating results consisting of frequently large sets of SCNA segments. However, the automated annotation and integration of such data are particularly challenging because the measured signals reflect biased, relative copy number ratios. In this study, we introduce labelSeg, an algorithm designed for rapid and accurate annotation of CNA segments, with the aim of enhancing the interpretation of tumor SCNA profiles. Leveraging density-based clustering and exploiting the length-amplitude relationships of SCNA, our algorithm proficiently identifies distinct relative copy number states from individual segment profiles. Its compatibility with most CNA measurement platforms makes it suitable for large-scale integrative data analysis. We confirmed its performance on both simulated and sample-derived data from The Cancer Genome Atlas reference dataset, and we demonstrated its utility in integrating heterogeneous segment profiles from different data sources and measurement platforms. Our comparative and integrative analysis revealed common SCNA patterns in cancer and protein-coding genes with a strong correlation between SCNA and messenger RNA expression, promoting the investigation into the role of SCNA in cancer development.

Novel insights into the pathogenesis of follicular lymphoma by molecular profiling of localized and systemic disease forms

Knowledge on the pathogenesis of FL is mainly based on data derived from advanced/systemic stages of FL (sFL) and only small cohorts of localized FL (lFL) have been characterized intensively so far. Comprehensive analysis with profiling of somatic copy number alterations (SCNA) and whole exome sequencing (WES) was performed in 147 lFL and 122 sFL. Putative targets were analyzed for gene and protein expression. Overall, lFL and sFL, as well as BCL2 translocation-positive (BCL2+) and –negative (BCL2−) FL showed overlapping features in SCNA and mutational profiles. Significant differences between lFL and sFL, however, were detected for SCNA frequencies, e.g., in 18q-gains (14% lFL vs. 36% sFL; p = 0.0003). Although rare in lFL, gains in 18q21 were associated with inferior progression-free survival (PFS). The mutational landscape of lFL and sFL included typical genetic lesions. However, ARID1A mutations were significantly more often detected in sFL (29%) compared to lFL (6%, p = 0.0001). In BCL2 + FL mutations in KMT2D, BCL2, ABL2, IGLL5 and ARID1A were enriched, while STAT6 mutations more frequently occurred in BCL2- FL. Although the landscape of lFL and sFL showed overlapping features, molecular profiling revealed novel insights and identified gains in 18q21 as prognostic marker in lFL.

Abstract 3553: Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes

Abstract Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high-yield liquid biopsy enabling in vivo detection of tumor-derived cell-free DNA (cfDNA) thus overcoming the contraindication of biopsy. Somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on RB1 gene, have been able to be identified in the same clinical samples but with two separate sequencing runs. In this study, we first developed a single targeted sequencing method to identify both SCNAs and RB1 SNVs. With this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye. Materials and Methods: 11 paired AH and Rb tumor samples were included in the study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated with QIAgen commercial kits. DNAs were constructed into whole genome libraries followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MycN genes, the all-exon regions of BCOR and CREBBP genes, and a whole genome CNV backbone. Libraries were submitted to Illumina HiSeq 4000 platform for fastq data generation with 2 × 150bp mode. The bioinformatics pipeline was optimized to generate SCNA profiles from targeted sequencing reads, along with SNV calling. Results: For SCNA profiles, 11/11 AH samples (100%) and 8/11 tumor samples (72.72%) have positive RB-SCNA signatures. Strong concordances were observed between AH and tumor SCNA profiles (median = 90.1%) with targeted sequencing reads. In total, 9 disease-driving RB1 SNVs were identified in 6 patients (54.5%). 7/9 (77.8%) of the variants were shared between AH and tumor samples, while the AH and tumor each contained one unique SNV. In all SNVs, the AH displayed a higher allele frequency. Notably, 4/11 samples have focal RB1 gene deletion detected with SCNA profiling, which may explain the difficulties of RB1 SNV detection in some samples. Further, one case was driven by a MYCN gene amplification with no RB1 alterations. Conclusions: This study presented highlights the utility of a single method to call both SVNs and SCNAs in a single clinical sample, with enriched tumor information detected in AH compared with tumor. This study further strengthens the utility of AH as a liquid biopsy platform for Rb eyes. Citation Format: Liya Xu, Mike J. Schmidt, Rishvanth K. Prakabar, Peter Kuhn, James Hicks, Jesse Berry. Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3553.

Low-cost and clinically applicable copy number profiling using repeat DNA

BackgroundSomatic copy number alterations (SCNAs) are an important class of genomic alteration in cancer. They are frequently observed in cancer samples, with studies showing that, on average, SCNAs affect 34% of a cancer cell’s genome. Furthermore, SCNAs have been shown to be major drivers of tumour development and have been associated with response to therapy and prognosis. Large-scale cancer genome studies suggest that tumours are driven by somatic copy number alterations (SCNAs) or single-nucleotide variants (SNVs). Despite the frequency of SCNAs and their clinical relevance, the use of genomics assays in the clinic is biased towards targeted gene panels, which identify SNVs but provide limited scope to detect SCNAs throughout the genome. There is a need for a comparably low-cost and simple method for high-resolution SCNA profiling.ResultsWe present conliga, a fully probabilistic method that infers SCNA profiles from a low-cost, simple, and clinically-relevant assay (FAST-SeqS). When applied to 11 high-purity oesophageal adenocarcinoma samples, we obtain good agreement (Spearman’s rank correlation coefficient, rs=0.94) between conliga’s inferred SCNA profiles using FAST-SeqS data (approximately £14 per sample) and those inferred by ASCAT using high-coverage WGS (gold-standard). We find that conliga outperforms CNVkit (rs=0.89), also applied to FAST-SeqS data, and is comparable to QDNAseq (rs=0.96) applied to low-coverage WGS, which is approximately four-fold more expensive, more laborious and less clinically-relevant. By performing an in silico dilution series experiment, we find that conliga is particularly suited to detecting SCNAs in low tumour purity samples. At two million reads per sample, conliga is able to detect SCNAs in all nine samples at 3% tumour purity and as low as 0.5% purity in one sample. Crucially, we show that conliga’s hidden state information can be used to decide when a sample is abnormal or normal, whereas CNVkit and QDNAseq cannot provide this critical information.ConclusionsWe show that conliga provides high-resolution SCNA profiles using a convenient, low-cost assay. We believe conliga makes FAST-SeqS a more clinically valuable assay as well as a useful research tool, enabling inexpensive and fast copy number profiling of pre-malignant and cancer samples.

Abstract 1967: Potential of aqueous humor as a liquid biopsy for uveal melanoma

Abstract Introduction: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and arises from the uvea (iris, ciliary body and choroid). Although there are prognostic biomarkers available, identifying them requires intraocular tumor biopsy. Blood biopsy has emerged as a significantly less invasive alternative that also offers the ability to longitudinally track disease. However, blood biopsy has been unsuccessful for clinical use in identifying prognostic biomarkers in UM due to low detection rates. Thus, eye-specific aqueous humor (AH) biopsy may be a better alternative. Methods: This case series includes 20 primary UM patients with 13 (65%) choroid and 7 (35%) ciliary body (CB) tumors. AH samples were collected prior to brachytherapy plaque placement (n = 17), after brachytherapy (n = 19) and at enucleation (n = 1). A tumor wash sample (n = 1) was collected after routine fine needle aspiration biopsy. Nucleic acids ([NA], dsDNA, RNA, ssDNA and miRNA) were quantitated by Qubit Assay Kits. All AH samples and cells from tumor washing underwent DNA isolation and was followed by shallow whole-genome sequencing for copy-number profiling. Results: Comparison of pre- and post-radiation samples revealed significantly higher concentration of ssDNA (n = 10 pairs, P = 0.039) and miRNA (n = 10 pairs, P = 0.008) in choroid post- samples and significantly higher concentration of dsDNA (n = 7 pairs, P = 0.031) and miRNA (n = 7 pairs, P = 0.016) in CB post- samples. Between choroid (n = 12) and CB (n = 7) post- samples, there was a significantly higher concentration of dsDNA (P = 0.022) and RNA (P = 0.009) in post- CB samples, suggesting NA content is released from the disturbance of tumor cells by radiation and due to the proximity of the tumor to the AH in CB. Highly recurrent UM somatic copy number alterations (SCNAs) of monosomy 3, 6p gain, 6q loss and 8q gain were identified in post- AH samples. There was a significant difference between altered SCNA profiles in CB and choroid (P= 0.038). Altered SCNA profiles were found in 1/12 (8.3%) and 4/7 (57.1%) post-radiation AH samples of choroid and CB, respectively. No SCNAs were found in the same eyes’ pre-radiation sample. A high concordance of DNA profiles between a tumor wash and post- AH sample was identified (r = 0.978) and UM mutations GNAQ and BAP1 were detected in 3/6 (50%) post- AH with 2/5 (40%) matching tumor samples, demonstrating AH dsDNA’s tumor-origin. Conclusions: The AH is a source of cfDNA, with a higher yield of NAs after radiation in UM. Given the significant increase in NA concentration after radiation and in CB compared to choroid tumors, we hypothesize that all detected NAs post-radiation are tumor-derived due to apoptosis of tumor cells and subsequent release of cfDNA and due to the proximity of the CB tumor to AH. These results represent the first time that UM SCNAs and mutations were identified in cfDNA isolated from the AH, and this suggests that the AH can serve as a liquid biopsy for UM, especially in CB tumors. Citation Format: Deborah H. Im, Chen-Ching Peng, Liya Xu, Mary E. Kim, Dejerianne Ostrow, Venkata Yellapantula, Moiz Bootwalla, Jaclyn A. Biegel, Xiaowu Gai, Peter Kuhn, James Hicks, Jesse L. Berry. Potential of aqueous humor as a liquid biopsy for uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1967.

Comparison of somatic mutation landscapes in Chinese versus European breast cancer patients.

SummaryRecent genomic studies suggest that Asian breast cancer (BC) may have distinct somatic features; however, most comparisons of BC genomic features across populations did not account for differences in age, subtype, and sequencing methods. In this study, we analyzed whole-exome sequencing (WES) data to characterize somatic copy number alterations (SCNAs) and mutation profiles in 98 Hong Kong BC (HKBC) patients and compared with those from The Cancer Genome Atlas of European ancestry (TCGA-EA, N = 686), which had similar distributions of age at diagnosis and PAM50 subtypes as in HKBC. We developed a two-sample Poisson model to compare driver gene selection pressure, which reflects the effect sizes of cancer driver genes, while accounting for differences in sample size, sequencing platforms, depths, and mutation calling methods. We found that somatic mutation and SCNA profiles were overall very similar between HKBC and TCGA-EA. The selection pressure for small insertions and deletions (indels) in GATA3 (false discovery rate (FDR) corrected p < 0.01) and single-nucleotide variants (SNVs) in TP53 (nominal p = 0.02, FDR corrected p = 0.28) was lower in HKBC than in TCGA-EA. Among the 13 signatures of single-base substitutions (SBS) that are common in BC, we found a suggestively higher contribution of SBS18 and a lower contribution of SBS1 in HKBC than in TCGA-EA, while the two APOBEC-induced signatures showed similar prevalence. Our results suggest that the genomic landscape of BC was largely very similar between HKBC and TCGA-EA, despite suggestive differences in some driver genes and mutational signatures that warrant future investigations in large and diverse Asian populations.

Somatic Copy Number Alterations in Human Cancers: An Analysis of Publicly Available Data From The Cancer Genome Atlas.

Somatic copy number alterations (SCNAs) are a pervasive trait of human cancers that contributes to tumorigenesis by affecting the dosage of multiple genes at the same time. In the past decade, The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) initiatives have generated and made publicly available SCNA genomic profiles from thousands of tumor samples across multiple cancer types. Here, we present a comprehensive analysis of 853,218 SCNAs across 10,729 tumor samples belonging to 32 cancer types using TCGA data. We then discuss current models for how SCNAs likely arise during carcinogenesis and how genomic SCNA profiles can inform clinical practice. Lastly, we highlight open questions in the field of cancer-associated SCNAs.

Clinical Impact of Somatic Copy Number Alterations in Circulating Tumor DNA from Diverse Lymphoma Subtypes

Introduction:Somatic copy number alterations (SCNAs) constitute a major class of genomic aberrations known to have functional significance across cancers, including prognostic value in lymphoma. For example, diffuse large B-cell lymphoma (DLBCL) patients with tumors harboring SCNAs in MYC, BCL2, or TP53 tend to have inferior outcomes (Quesada, ASH 2016). However, detection of copy number alterations without invasive tissue biopsies remains difficult. We applied Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq), a targeted high-throughput sequencing platform, to noninvasively detect and monitor genome-wide SCNAs in patients with DLBCL, primary mediastinal B-cell lymphoma (PMBCL), and classical Hodgkin lymphoma (cHL).Methods: We profiled pre-treatment blood samples from 209 patients with diverse lymphomas including 168 DLBCL, 20 cHL, and 21 PMBCL, using a custom 314kb gene panel targeting recurrently altered regions in these cancers. Additionally, we sequenced 139 tumor biopsies to use as matched tissue gold standards for SCNA confirmation. In each sample, we interrogated the sequencing read distribution to summarize focal and broad SCNAs directly from plasma. All thresholds were tuned for 95% specificity, which were empirically verified in independent withheld healthy controls. Results were orthogonally validated through tissue karyotyping, fluorescence in situ hybridization (FISH), and whole genome sequencing (WGS) (Fig 1A and Fig 1B).Results: We determined our limit of detection (LOD) for detecting SCNAs in plasma at ~1% circulating tumor fraction with 77% sensitivity; we observed higher sensitivity above 5% circulating tumor fraction (sensitivity = 95%). In our cohort, 70% of patients had over 1% circulating tumor burden with representation from all clinical stages. In DLBCL patients, we detected gains in MYC (12%), BCL2 (24%), BCL6 (19%), and PD-L1 (23%), as well as losses in TP53 (27%) and CDKN2A (19%). SCNAs detected in plasma were 85% concordant with those detected in matched tumor biopsies. Additionally, PD-L1 copy number gains were more frequently detected in both PMBCL (63%, p= 0.026) and cHL (71%, p= 0.012) than in DLBCL (23%), consistent with prior reports (Chapuy B, Blood 2016; Roemer MG, J. Clin. Oncol. 2016).We also demonstrate that noninvasive detection of copy number gains in MYC and BCL2, as well as loss of TP53, are negatively prognostic for survival (p < 0.0001, p= 0.0013, p < 0.0001, respectively) (Fig 1C). Furthermore, we identified associations between DLBCL cell-of-origin classification and specific SCNAs, with non-GCB DLBCL enriched for BCL2, BCL6, and PD-L1 copy number gains (39% vs 17%; 31% vs 15%, and 39% vs 25%, respectively). Circulating tumor burden was not statistically different between GCB and non-GCB patients. TP53 losses were more frequently observed in relapsed/refractory patients compared to treatment naïve patients (22% vs 45%, p= 0.023).We performed a preliminary analysis monitoring disease over time in three patients with clear SCNAs detected in plasma at diagnosis. Across longitudinal blood samples (n= 42), SCNA-based disease detection demonstrated 95% concordance with mutation-based disease detection, with a LOD down to ~0.1% circulating tumor burden. Our results suggest noninvasive SCNA profiling could provide an independent method for detecting residual disease, particularly in tumors characterized by structural alterations. Further validation and expansion of this monitoring method will be explored and presented.Conclusions: Noninvasive identification of genome-wide SCNAs is feasible via targeted next generation sequencing. Our approach allows for both genotyping and monitoring of copy number gains and losses from plasma, without requiring matched germline or tissue specimens. This strategy not only allows for the noninvasive genotyping of tumor SCNAs, but also provides an orthogonal method for monitoring residual disease from liquid biopsies using next generation sequencing.Figure 1: A) Matched tissue FISH staining confirms arm-level PD-L1 amplification (red) detected in plasma. D9Z1 (green) was used as an assay control. B) SCNAs detected in plasma were concordant with karyotype analysis of a matched tumor biopsy. C) Copy number alterations in MYC, BCL2, and TP53 are adversely prognostic for survival in R-CHOP treated DLBCL patients. [Display omitted] DisclosuresHüttmann:Bristol-Myers Squibb, Takeda, Celgene, Roche: Honoraria; Gilead, Amgen: Other: Travel cost. Gaidano:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Westin:Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Apotex: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Advani:Bayer Healthcare Pharmaceuticals: Research Funding; Gilead: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Nanostring: Consultancy; Spectrum: Consultancy; Millennium: Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Agensys: Research Funding; Infinity: Research Funding; Juno Therapeutics: Consultancy; Kura: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy; Cell Medica: Research Funding; Sutro: Consultancy. Diehn:Varian Medical Systems: Research Funding; Roche: Consultancy; Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy. Alizadeh:Genentech: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; CiberMed: Consultancy.

Highlights from the literature

<h3>Abstract</h3> Cell-free DNA (cfDNA) measured via liquid biopsies provides a way for minimally-invasive monitoring of tumour evolutionary dynamics during therapy. Here we present liquidCNA, a method to track subclonal evolution...

Abstract 452: Simultaneous identification of clinically relevant RB1 Mutations and somatic copy number alterations in the Aqueous Humor of retinoblastoma eyes

Abstract Purpose: Retinoblastoma (RB) tumorigenesis is associated with mutations in the RB1 tumour suppressor gene and somatic copy number alterations (SCNAs). Both alleles of the RB1 gene must be mutated for tumour development. The initial RB1 mutation may be germline or somatic. However, the somatic RB1 mutation was only detectable in enucleated RB eyes, when the tumour samples were available. The aqueous humor (AH) can serve as a surrogate to direct tumour biopsy and can be done in vivo. In this study, we demonstrate that both whole genome SCNAs and RB1 mutations can be simultaneously assayed in the AH liquid biopsy. Methods: AH from 7 eyes of 6 RB patients and 4 matched tumor samples were included. AH was extracted via clear corneal paracentesis. Cell-free DNA (cfDNA) was isolated and constructed into whole genome libraries. Low pass whole genome sequencing was performed for copy number alteration analysis. The same libraries were further used for capture-based targeted NGS for mutation detection. Custom hybridization capture panel manufactured from Agilent includes the whole gene region of RB1 gene, MCYN gene, and all exons of BCOR and CREBBP genes (Agilent SureSelect Tier1). Captured libraries were sequenced on Illumina platform with paired-end 150 cycles mode. Bioinformatics analysis was performed for single-nucleotide variant (SNV) and loss of heterozygosity (LOH) detection using an in-house pipeline (information available on request). Comparison was also made to clinically available peripheral blood RB1 mutation screening (CHLA, CPM). Results: Somatic whole genome SCNAs were detected in all 4 enucleated tumours. Among 7 evaluated AH samples, 5 contained SCNAs and the remaining 2 were flat without alterations under current analysis setting. Mutational analysis of tumour DNA from 4 enucleated eyes identified 6 RB1 SNVs. Mutation results from AH cfDNA were concordant with results obtained from corresponding tumour tissue and peripheral blood screening, when available. In the 3 AH samples without paired tumours, both RB1 hits were identified with high variant allele frequency (VAF), indicating high tumour fraction in AH of RB eyes. Conclusions: Use of a customized hybridization capture approach facilitated identification of RB1 mutation(s), together with low pass whole genome analysis for SCNA profiling in the same AH liquid biopsy provides the opportunity to identify the genomic and genetic alterations simultaneously for one diseased eye. This refined approach provides the potential to further investigate the spectrum of RB1 mutations and SCNA subtypes in both enucleated and salvaged RB eyes. It is crucial to distinguish between these alternative mechanisms, for the wider implications for management of patient and family members. Citation Format: Liya Xu, Lishuang Shen, Ashley Polski, Rishvanth K. Prabakar, Mark Reid, Peter Kuhn, David E. Cobrinik, James Hicks, Xiaowu Gai, Jesse Berry. Simultaneous identification of clinically relevant RB1 Mutations and somatic copy number alterations in the Aqueous Humor of retinoblastoma eyes [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 452.

Somatic copy number profiling from hepatocellular carcinoma circulating tumor cells

Somatic copy number alterations (SCNAs) are important genetic drivers of many cancers. We investigated the feasibility of obtaining SCNA profiles from circulating tumor cells (CTCs) as a molecular liquid biopsy for hepatocellular carcinoma (HCC). CTCs from ten HCC patients underwent SCNA profiling. The Cancer Genome Atlas (TCGA) SCNA data were used to develop a cancer origin classification model, which was then evaluated for classifying 44 CTCs from multiple cancer types. Sequencing of 18 CTC samples (median: 4 CTCs/sample) from 10 HCC patients using a low-resolution whole-genome sequencing strategy (median: 0.88 million reads/sample) revealed frequent SCNAs in previously reported HCC regions such as 8q amplifications and 17p deletions. SCNA profiling revealed that CTCs share a median of 80% concordance with the primary tumor. CTCs had SCNAs not seen in the primary tumor, some with prognostic implications. Using a SCNA profiling model, the tissue of origin was correctly identified for 32/44 (73%) CTCs from 12/16 (75%) patients with different cancer types.

Integrative analysis reveals driver long non-coding RNAs in osteosarcoma.

Transcriptome profiling of osteosarcoma (OS) by next generation sequencing technology (NGS) has been broadly performed by previous researches, which uncovers a large number protein-coding driver genes, facilitates our understanding of the molecular mechanisms of OS formation, progression and metastasis. Recently, more and more researchers realize the importance of long non-coding RNAs (lncRNAs) on the development of OS. However, few studies focus on discovering driver lncRNAs.Here we collected somatic copy number alterations (SCNAs) and gene expression profiles of 84 samples from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project. The RNA sequencing data detected 13,903 expressed lncRNAs, 157 of which were previously reported to be associated with cancer based on the annotations from Lnc2Cancer database.By analyzing the SNP array data, several significant SCNAs were detected, such as the amplifications on chromosomes 1q, 4q, 17p, 17q, and 19q, and deletions on 1q, 3q, 9p, 10q, and 15q. With the SCNA and gene expression profiles, we identified 167 driver genes by integrative analysis, including 162 novel driver lncRNAs, 2 lncRNAs reported to be associated with OS, and another 3 associated with other cancers. Furthermore, functional characterization and survival analysis revealed that RP11-241F15.10 may function as a tumor suppressor in OS, and loss of function may contribute to activation of Wnt signaling pathway.This study not only facilitates our understanding of the oncogenic or tumor-suppressor role of lncRNAs in OS, but also provides potential therapies for the patients with OS with metastasis or relapse.

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44− tumor cells in colon cancer by single-cell sequencing

ABSTRACTCancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAMhighCD44+ tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAMhighCD44− population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAMhighCD44+) and DTC (EpCAMhighCD44−) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45−EpCAMhighCD44+ and CD45−EpCAMhighCD44− populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer

BackgroundColorectal cancer is a heterogeneous group of malignancies with complex molecular subtypes. While colon cancer has been widely investigated, studies on rectal cancer are very limited. Here, we performed multi-region whole-exome sequencing and single-cell whole-genome sequencing to examine the genomic intratumor heterogeneity (ITH) of rectal tumors.MethodsWe sequenced nine tumor regions and 88 single cells from two rectal cancer patients with tumors of the same molecular classification and characterized their mutation profiles and somatic copy number alterations (SCNAs) at the multi-region and the single-cell levels.ResultsA variable extent of genomic heterogeneity was observed between the two patients, and the degree of ITH increased when analyzed on the single-cell level. We found that major SCNAs were early events in cancer development and inherited steadily. Single-cell sequencing revealed mutations and SCNAs which were hidden in bulk sequencing. In summary, we studied the ITH of rectal cancer at regional and single-cell resolution and demonstrated that variable heterogeneity existed in two patients. The mutational scenarios and SCNA profiles of two patients with treatment naïve from the same molecular subtype are quite different.ConclusionsOur results suggest each tumor possesses its own architecture, which may result in different diagnosis, prognosis, and drug responses. Remarkable ITH exists in the two patients we have studied, providing a preliminary impression of ITH in rectal cancer.

Abstract 1749: Stability of copy-number profiles defines two molecular subtypes during urothelial carcinoma’s evolution

Abstract Background: Somatic copy-number alterations (SCNAs) are distinct molecular events occurring in muscle-invasive urothelial carcinoma (UC). Defining the biologic role and the timing of SCNAs in UC’s evolution is critical for understanding disease biology and for precision therapy. Methods: To examine whether distinct SCNAs define specific subtypes of UC and characterize the evolutionary dynamics of SCNA acquisition, we conducted whole-exome sequencing (WES) of a discovery cohort of 44 advanced UC tumors and matching germline samples, including 13 pairs of matched primary and metastatic tumors prospectively collected before and after chemotherapy. We queried WES data from 285 UCs from The Cancer Genome Atlas (TCGA) as a validation cohort. We performed a novel in-depth allele-specific copy-number analysis in 2503 cancer genes using the CLONET computational framework, which accounts for tumor ploidy and cellularity to infer the clonal hierarchy of SCNAs. Results: Analysis of SCNAs from tumor pairs within each patient revealed punctuated evolution of SCNAs occurring early in UC’s development followed by minor changes (clonal stasis) throughout the tumor’s lifetime. Two distinct clusters of UCs with strikingly different SCNA profiles in the discovery cohort emerged. Cluster A was defined by diploid copy number background and focal deletions in the 9p21 region (CDKN2A, CDKN2B and MTAP). Cluster B was defined by genome-wide duplications and several enriched amplifications including 1q21.1 (SETDB1 and MLLT11), (P = 0.0002) and 6p22.3 (E2F3), (P = 0.001). TP53 mutations (P = 0.0001) were enriched in the cluster B, consistent with the role of functional p53 as a “guardian of the genome” preventing chromosomal instability. SCNAs clusters A and B were confirmed in the TCGA validation cohort. Cluster A was enriched with tumors belonging to TCGA bladder cancer cluster III (“basal/squamous-like”) (P = 0.02). Conclusions: Using novel allele-specific SCNAs analysis, we define two distinct clusters of UC presenting a framework for understanding this aspect of UC’s biology. In contrast to somatic mutations, SCNAs are clonally static during each tumor’s evolution. This data has important implications for precision medicine for UC. Citation Format: Bishoy M. Faltas, Davide Prandi, Scott T. Tagawa, David Nanus, Ana M. Molina, Himisha Beltran, Francesca Demichelis, Mark A. Rubin. Stability of copy-number profiles defines two molecular subtypes during urothelial carcinoma’s evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1749. doi:10.1158/1538-7445.AM2017-1749

Punctuated evolution of copy-number alterations to define two molecular subtypes of muscle-invasive urothelial carcinoma.

299 Background: Somatic copy-number alterations (SCNAs) are distinct molecular events occurring in muscle-invasive urothelial carcinoma (UC). Defining the biologic role and the timing of SCNAs in UC’s evolution is critical for precision therapy. Methods: To examine whether distinct SCNAs define specific subtypes of UC and characterize the evolutionary dynamics of SCNA acquisition, we conducted whole-exome sequencing (WES) of a discovery cohort of 44 advanced UC tumors and matching germline samples, including 13 pairs of matched primary and metastatic tumors prospectively collected before and after chemotherapy. We queried WES data from 285 UCs from The Cancer Genome Atlas (TCGA) as a validation cohort. We performed a novel in-depth allele-specific copy-number analysis in 2503 cancer genes using the CLONET computational framework, which accounts for tumor ploidy and cellularity to infer the clonal hierarchy of SCNAs. Results: Analysis of SCNAs from tumor pairs within each patient revealed punctuated evolution of SCNAs occurring early in UC’s development followed by minor changes (clonal stasis) throughout the tumor’s lifetime. Two distinct clusters of UCs with strikingly different SCNA profiles in the discovery cohort emerged. Cluster A was defined by diploid copy number background and focal deletions in the 9p21 region (CDKN2A, CDKN2B and MTAP). Cluster B was defined by genome-wide duplications and several enriched amplifications including 1q21.1 (SETDB1 and MLLT11), (P = 0.0002) and 6p22.3 (E2F3), (P = 0.001). TP53 mutations (P = 0.0001) were enriched in the cluster B, consistent with the role of functional p53 as a “guardian of the genome” preventing chromosomal instability. SCNAs clusters A and B were confirmed in the TCGA validation cohort. Cluster A was enriched with tumors belonging to TCGA bladder cancer cluster III (“basal/squamous-like”) (P = 0.02). Conclusions: Using novel allele-specific SCNAs analysis, we define two distinct clusters of UC presenting a framework for understanding this aspect of UC’s biology. In contrast to somatic mutations, SCNAs are clonally static during each tumor’s evolution. This data has important implications for precision medicine for UC.

A Meta-Analysis of Retinoblastoma Copy Numbers Refines the List of Possible Driver Genes Involved in Tumor Progression.

BackgroundWhile RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes.MethodsWe performed SCNA profiling of 45 primary retinoblastoma samples and eight retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N = 310).ResultsComprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N = 34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades.InterpretationSince focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.

Genome-wide profiling of copy number alterations in cancer: focus on melanoma

Thanks to a never-before detailed view of the human genome, the last decade has brought to light the notion of DNA copy number variation (CNV) as the pivotal force contributing to population genomic diversity and evolution. It is as well clear now that cancer typically results in loosened control over genomic integrity and that the acquisition of somatic copy number alterations (SCNAs), whether confined to specific genes or affecting entire chromosome arms, is likely to be a fundamental prerequisite to the adaptive pressure that drives oncogenesis. This review gives a brief overview of key developments in genome-wide SCNA profiling, with specific emphasis on array-based techniques and deep-sequencing, which indeed enabled us to identify the large majority of genomic regions undergoing frequent alteration in human cancers and defining recognizable clinical phenotype. Alongside with the prospective to take advantage for future personalized precision medicine, high-throughput SCNA analysis have already proven diagnostic and prognostic potential, particularly for those clinically unpredictable and therapy-refractory tumors, such us cutaneous melanoma. Biomedical Reviews 2013; 24: 11-24.

GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers

We describe methods with enhanced power and specificity to identify genes targeted by somatic copy-number alterations (SCNAs) that drive cancer growth. By separating SCNA profiles into underlying arm-level and focal alterations, we improve the estimation of background rates for each category. We additionally describe a probabilistic method for defining the boundaries of selected-for SCNA regions with user-defined confidence. Here we detail this revised computational approach, GISTIC2.0, and validate its performance in real and simulated datasets.