Analysis Of Somatic Copy Number Alterations Research Articles

Not Only RET but NF1 and Chromosomal Instability Are Seen in Young Patients with Sporadic Medullary Thyroid Carcinoma.

Genetic analysis of sporadic medullary thyroid carcinoma (MTC) has revealed somatic variants in RET, RAS, and occasionally other genes. However, around 20% of patients with sporadic MTC lack a known genetic driver. To uncover potential new somatic or germline drivers, we analyze a distinct cohort of patients with sporadic, very early-onset, and aggressive MTC. Germline and somatic DNA exome sequencing was performed in 19 patients, previously tested negative for germline RET variants. Exome sequencing of 19 germline samples confirmed the absence of RET and identified an NF1 pathogenic variant in 1 patient. Somatic sequencing was successful in 15 tumors revealing RET variants in 80%, predominantly p.Met918Thr, which was associated with disease aggressiveness. In RET-negative tumors, pathogenic variants were found in HRAS and NF1. The NF1 germline and somatic variants were observed in a patient without a prior clinical diagnosis of neurofibromatosis type 1, demonstrating that the loss of heterozygosity of NF1 functions as a potential MTC driver. Somatic copy number alterations analysis revealed chromosomal alterations in 53.3% of tumors, predominantly in RET-positive cases, with losses in chromosomes 9 and 22 being the most prevalent. This study reveals that within a cohort of early-onset nonhereditary MTC, RET remains the major driver gene. In RET-negative tumors, NF1 and RAS are drivers of sporadic MTC. In addition, in young patients without a RET germline mutation, a careful clinical evaluation with a consideration of germline NF1 gene analysis is ideal to exclude Neurofibromatosis type 1 (NF1).

Harnessing Cell-Free DNA Biological Features for Early Treatment Response Prediction in High Grade B-Cell Lymphoma

Introduction The application of circulating cell-free DNA (cfDNA) has attracted recent interest for cancer detection and monitoring. The presence of somatic copy number alterations (SCNAs) can be used to differentiate between cancer cases and healthy controls (PMID: 29109393). This information can be retrieved from low-coverage whole genome sequencing (lcWGS) and used to determine the proportion of tumor derived cfDNA (ctDNA) in a tube of blood at a reasonable cost. Previous studies observed that cancer patients tend to have a higher fraction of shorter cfDNA fragments compared to healthy controls, irrespective of the presence of genomic events (PMID: 30404863). Here, we evaluate if a combination of cfDNA genomic and epigenomic features that can be retrieved from a single run of lcWGS, has potential for the early prediction of end of induction (EoI) response, in patients with high-grade lymphoma with MYC and BCL2 and/or BLC6 rearrangements (HGBL-DH/TH). Methods In the HOVON-152 phase II trial (NCT03620578) HGBL-DH/TH patients were treated with induction immunochemotherapy with one cycle of R-CHOP followed by five cycles of DA-EPOCH-R. Patients in complete metabolic response (CMR, defined as Deauville score 1-3) at the end of induction receive 1-year nivolumab consolidation. Blood was collected from 40 patients using PAXgene ccfDNA tubes after one cycle (timepoint 1, T1), three (T2) and six (T3) cycles of treatment. At T3, response to induction treatment was determined using 18F-FDG-PET scan. Patients with CMR at EoI were considered responders. In this study, we included 40 out of 97 patients from the HOVON-152 phase II trial, and enriched for patients with no CMR. Plasma was separated using dual centrifugation protocol, and cfDNA was isolated using the QIASymphony kit (QIAGEN). Sequencing libraries were prepared using the ThruPLEX Plasma-seq kit (Takara). lcWGS was performed on a Novaseq 6000 (Illumina). Somatic copy number aberrations (SCNAs) were retrieved with ichorCNA, insert size profiles were generated using Picard tools. SCNAs were classified as detected when the tumor fraction quantified with ichorCNA exceeded 3%. Results We assessed the detection of SCNAs and the SCNA-derived tumor fraction using the ichorCNA software. The ctDNA tumor fraction was found to be significantly increased in non-responders (NR, n=24) compared to responders (R, n=16), irrespective of sampling timepoint (Wilcoxon, p<0.001). At T1, the tumor fraction was significantly elevated in non-responders (Wilcoxon, p=0.014), and SCNAs were detected in 6/16 (38%) of the responders versus 19/24 (79%) of the non-responders, with negative predictive value (NPV): 71%, positive predictive value (PPV): 74%. Therefore, non-responders tend to have a higher fraction of tumor-derived signal which is already apparent at earlier timepoints. To evaluate the potential of using cfDNA fragmentation for the early detection of response, we retrieved the fragment size profiles. The proportion of short fragments (P20-150 bp), was found to be increased in non-responders compared to responders at T1 (Wilcoxon, p<0.001) and T2 (Wilcoxon, p=0.033), which suggests that the plasma of non-responders contains a higher fraction of tumor-signal (Figure 1A). Previous reports leveraged the size properties of ctDNA in order to increase the sensitivity of SCNA based methods (PMID: 30404863). In order to utilize both epigenomic and genomic properties to increase the detection rate of tumor-signal, we performed in silico size selection to computationally filter out fragments in the 20-150bp size range. Upon repeating the SCNA analysis, we observed that 20/24 (83%) non-responders and 1/15 (19%) responders had detectable ctDNA at T1 (Figure 1B). This yields a NPV and PPV of 80% and 86% respectively for EoI response detection at T1. By leveraging the fragmentomic properties of cfDNA, the sensitivity of SCNA-based methods can be improved, which leads to an increased detection of response at earlier timepoints. Discussion In patients with HGBL-DH/TH, after one cycle of R-CHOP, the presence of SCNAs enhanced by fragment size analysis in plasma, is significantly associated with an unfavourable EoI response, with a NPV and PPV of 80% and 86% respectively. This novel approach can be performed without prior knowledge of genetic alterations, and may have high potential to guide early risk-adapted treatment strategies in HGBL-DH/TH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic landscape of indolent and aggressive Kaposi sarcomas.

Kaposi sarcoma (KS) is a rare skin tumour caused by herpesvirus 8 infection and characterized by either indolence or an aggressive course necessitating systemic therapies. The genetic basis of this difference remains unknown. To explore the tumour mutational burden in indolent and aggressive KS. We performed whole-exome sequencing on a cohort of 21 KS patients. We compared genetic landscape including tumor mutational burden between the two forms of indolent and agressive KS. Aggressive KS tumours had a significantly higher TMB and a larger cumulative number of deleterious mutations than indolent KS tumours. In addition, all aggressive tumours had at least three deleterious mutations, whereas most indolent tumours harboured only one or no predicted deleterious mutations. Deleterious mutations listed in the Cancer Gene Census were detected exclusively in patients with aggressive disease. An analysis of somatic copy-number alterations (SCNA) revealed a tendency towards higher number of alterations in aggressive KS. These data suggest that SCNA alterations and an increase in mutational burden promote aggressive KS and that it might be more appropriate to consider indolent KS as an opportunistic skin disease rather than a cancer.

Longitudinal circulating tumor DNA (ctDNA) whole-exome sequencing (WES) in the phase Ib/II trial of palbociclib and bazedoxifene reveals genomic dynamics and clonal evolution with the acquisition of treatment resistance in hormone receptor-positive, HER2-negative (HR+ HER2-), advanced breast cancer (ABC).

1058 Background: Patients (pts) with HR+ HER2- ABC ultimately develop endocrine resistance. To gain insights into the genetic mechanisms of resistance we performed WES on serial plasma samples from endocrine resistant pts treated on a clinical trial (NCT02448771). Methods: Plasma samples were collected at baseline (n=36), day 1 of cycle 2 (n=33), and at the end of treatment (EOT, n=33). Samples were subjected to ultra-low passage (ULP, 0.19-0.57X) WGS to determine ctDNA tumor fraction (TF) for the selection of samples (TF&gt;0.03) for subsequent WES (193X). Somatic single nucleotide variations, somatic copy number alteration (SCNA), phylogeny, tumor mutational burden, mutational signatures, and germline analyses were performed. Results: All 102 samples underwent successful ULP and 68 WES. Overall, most frequent pathogenic mutations were in ESR1 and PIK3CA. At baseline, 32% of pts had ESR1 mutation and 21% PIK3CA mutation. There was no association between ESR1 mutations and PFS. In contrast, baseline PIK3CA mutations were detected only in pts who did not have a clinical benefit, and were associated with worse PFS compared to pts with wild-type PIK3CA (1.8 vs. 3.9 months, respectively, HR=0.2, 95% CI 0.06-0.6, P=0.0019, log-rank test). Additionally, pts with a baseline truncating mutation, mostly in tumor suppressor genes ( TP53, MEN1, RB1, CDKN1B, NF1, TP53BP1, TP63, SMAD2/4, ARID1A, KMT2C), also had a significantly worse PFS (1.7 vs 3.8 months, HR=0.3, 95% CI 0.1-0.7, P=0.006, log-rank test). At EOT, 20% (4/20) of pts with matched baseline samples had newly acquired mutations that are suggestive of mechanisms of acquired resistance and offer potential therapeutic targets (e.g. ERBB2, PIK3CA). SCNA analysis showed that in all pts there were at least 2 SCNAs in cancer-related driver genes, most common in CCND1 and ELF3. Moreover, in all samples we identified at least 1 SCNA related to a potential mechanism of resistance. To better understand tumor heterogeneity and sub-clonal architecture we performed an evolutionary analysis (sufficient TF≥0.15, available in n=7). Phylogenetic analysis revealed sub-clonal dynamics that could explain the acquisition of resistance in at least three pts (3/7), and identified novel genes which might have role in endocrine resistance (e.g. DCAF13, ZFHX3). Conclusions: Our results demonstrate the feasibility and utility of serial WES in a clinical trial. Serial ctDNA WES and evolutionary studies enabled us to discover novel potential genomic mechanisms of tumor progression, and identified PIK3CA mutations as a candidate biomarker of resistance to the combination of palbociclib and bazedoxifene, which may apply to other next generation endocrine treatments. Clinical trial information: NCT02448771.

Comprehensive Somatic Copy Number Analysis Using Aqueous Humor Liquid Biopsy for Retinoblastoma

Simple SummaryAqueous humor (AH) liquid biopsy is an enriched source of cell-free circulating tumor-derived DNA for retinoblastoma (RB). The use of this AH liquid biopsy allows for genomic analysis of eyes in the absence of tumor tissue. Development of this platform was critical because direct tumor biopsy is prohibited in RB due to risk of extraocular tumor spread. In this retrospective study, we provide comprehensive, whole-genome analysis of the somatic copy number alterations (SCNAs) in 68 eyes of 64 RB patients. We show that the prevalence of specific SCNAs differ between eyes that required immediate enucleation (surgical removal) and eyes that were attempted to be saved but subsequently failed treatment, requiring secondary enucleation. Increases in chromosomal instability, or higher number of broad genomic alterations, predict higher risk clinical and biomarker features in these eyes. Prospective analyses are needed to further determine the clinical relevance and application of these findings.Aqueous humor (AH) liquid biopsy has been established as a surrogate tumor biopsy for retinoblastoma (RB). Previous AH studies have focused on highly recurrent RB somatic copy number alterations (SCNAs) including gain of 1q, 2p, 6p, and loss of 13q and 16q. In this retrospective study, we provide a comprehensive, whole-genome analysis of RB SCNAs and evaluate associated clinical features for 68 eyes of 64 RB patients from whom AH was obtained between December 2014 and October 2020. Shallow whole-genome sequencing of AH cell-free DNA was performed to assess for SCNAs. The prevalence of specific non-highly recurrent SCNAs, such as 20q gain and 8p loss, differed between primarily and secondarily enucleated eyes. Increases in chromosomal instability predict more advanced seeding morphology (p = 0.015); later age of diagnosis (p < 0.0001); greater odds of an endophytic tumor growth pattern (without retinal detachment; p = 0.047); tumor heights >10 mm (p = 0.09); and containing 6p gain, a biomarker of poor ocular prognosis (p = 0.004). The AH liquid biopsy platform is a high-yield method of whole-genome RB SCNA analysis, and SCNAs are associated with numerous clinical findings in RB eyes. Prospective analyses are encouraged to further elucidate the clinical relevance of specific SCNAs in RB.

Whole-exome sequencing of alpha-fetoprotein producing gastric carcinoma reveals genomic profile and therapeutic targets

Alpha-fetoprotein producing gastric carcinoma (AFPGC) is a rare and aggressive subtype of gastric cancer. However, little is known about the genomic features of this disease. We perform whole-exome sequencing analysis of AFPGC, and identify 34 significantly mutated genes. Somatic copy number alterations analysis reveals several significant focal amplifications (e.g. 19q12, 17q12) and focal deletions (e.g. 1p36.11, 9p21.3), and some of these negatively affect the patient prognosis. Comparative analyses reveal that AFPGC has distinct genomic features from gastric cancer of The Cancer Genome Atlas as well as four molecular subtypes. Several frequently altered genes with potential as therapeutic targets are identified in AFPGC. Further analysis reveals that AFPGC with amplification of CCNE1 at 19q12 and/or ERBB2 at 17q12 show poorer survival and more aggressive. Subsequently, based on our established patient-derived xenograft models for AFPGC, translational research is performed and the therapeutic value of targeting CCNE1 and ERBB2 is validated. In this work, we provide an understanding of genomic characteristics of AFPGC and propose a platform to explore and validate the genome-guided personalized treatment for this disease.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9,MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent lossesprobably occurred before whole-genome doubling, that was foundas a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9,MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cellcarcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Genome-wide somatic copy number alteration analysis and database construction for cervical cancer.

Cervical cancer is a common gynecological malignancy with high incidence and mortality. Somatic copy number alterations (CNAs) play an important role in identifying tumor suppressor genes and oncogenes and are a useful diagnostic indicator for many cancer types. However, the genomic landscape of CNAs in cervical cancer has not yet been comprehensively characterized. In the present study, we collected 974 cervical cancer samples from different data sources. All samples were analyzed by genomic arrays to obtain high-resolution CNAs. Focal genomic regions with CNA events and potential cancer driver genes were identified by GISTIC2.0. Meanwhile, we constructed a comprehensive cervical cancer database by PHP and self-written Perl and R scripts. In total, 54 recurrent regions of amplification and deletion were detected. Frequently altered tumor suppressor genes were found in these regions, including PIK3CA, ERBB2, EP300 and FBXW7. CNA hotspots and related enriched functional categories were also identified. The incidence of chromothripsis in cervical cancer was estimated to be 6.06%, and the chromosome pulverization hotspot regions were detected. Based on the curated data, we developed CNAdbCC (http://cailab.labshare.cn/CNAdbCC/), a comprehensive database for copy number alterations in cervical cancer. We provide a user-friendly Web interface for data mining and visualization. It is the most comprehensive public database devoted exclusively to genomic alterations in cervical cancer. These results extend our molecular understanding of cervical cancer. The database will enable researchers to explore specific CNA patterns in this lethal cancer and facilitate the discovery of therapeutic candidates.

Identification of candidate cancer drivers by integrative Epi-DNA and Gene Expression (iEDGE) data analysis

The emergence of large-scale multi-omics data warrants method development for data integration. Genomic studies from cancer patients have identified epigenetic and genetic regulators – such as methylation marks, somatic mutations, and somatic copy number alterations (SCNAs), among others – as predictive features of cancer outcome. However, identification of “driver genes” associated with a given alteration remains a challenge. To this end, we developed a computational tool, iEDGE, to model cis and trans effects of (epi-)DNA alterations and identify potential cis driver genes, where cis and trans genes denote those genes falling within and outside the genomic boundaries of a given (epi-)genetic alteration, respectively. iEDGE first identifies the cis and trans gene expression signatures associated with the presence/absence of a particular epi-DNA alteration across samples. It then applies tests of statistical mediation to determine the cis genes predictive of the trans gene expression. Finally, cis and trans effects are annotated by pathway enrichment analysis to gain insights into the underlying regulatory networks. We used iEDGE to perform integrative analysis of SCNAs and gene expression data from breast cancer and 18 additional cancer types included in The Cancer Genome Atlas (TCGA). Notably, cis gene drivers identified by iEDGE were found to be significantly enriched for known driver genes from multiple compendia of validated oncogenes and tumor suppressors, suggesting that the remainder are of equal importance. Furthermore, predicted drivers were enriched for functionally relevant cancer genes with amplification-driven dependencies, which are of potential prognostic and therapeutic value. All the analyses results are accessible at https://montilab.bu.edu/iEDGE. In summary, integrative analysis of SCNAs and gene expression using iEDGE successfully identified known cancer driver genes and putative cancer therapeutic targets across 19 cancer types in the TCGA. The proposed method can easily be applied to the integration of gene expression profiles with other epi-DNA assays in a variety of disease contexts.

Comprehensive Genomic Review of TCGA Head and Neck Squamous Cell Carcinomas (HNSCC).

The aim of this present study was to comprehensively describe somatic DNA alterations and transcriptional alterations in the last extension of the HNSCC subsets in TCGA, encompassing a total of 528 tumours. In order to achieve this goal, transcriptional analysis, functional enrichment assays, survival analysis, somatic copy number alteration analysis and somatic alteration analysis were carried out. A total of 3491 deregulated genes were found in HNSCC patients, and the functional analysis carried out determined that tissue development and cell differentiation were the most relevant signalling pathways in upregulated and downregulated genes, respectively. Somatic copy number alteration analysis showed a “top five” altered HNSCC genes: CDKN2A (deleted in 32.03% of patients), CDKN2B (deleted in 28.34% of patients), PPFIA1 (amplified in 26.02% of patients), FADD (amplified in 25.63% of patients) and ANO1 (amplified in 25.44% of patients). Somatic mutations analysis revealed TP53 mutation in 72% of the tumour samples followed by TTN (39%), FAT1 (23%) and MUC16 (19%). Another interesting result is the mutual exclusivity pattern that was discovered between the TP53 and PIK3CA mutations, and the co-occurrence of CDKN2A with the TP53 and FAT1 alterations. On analysis to relate differential expression genes and somatic copy number alterations, some genes were overexpressed and amplified, for example, FOXL2, but other deleted genes also showed overexpression, such as CDKN2A. Survival analysis revealed that overexpression of some oncogenes, such as EGFR, CDK6 or CDK4 were associated with poorer prognosis tumours. These new findings help us to develop new therapies and programs for the prevention of HNSCC.

Abstract 2908: Mesothelioma phylogeny reveal MTAP as a solitary clonal deletion, exposing vulnerability to the PRMT5 perturbagen, quinacrine

Abstract Background: Malignant Pleural Mesothelioma (MPM) remains an incurable cancer that is caused by asbestos, and for which there is a paucity of effective therapy. Stratified medicine for MPM is in its infancy. We hypothesized that deciphering the phylogenetic architecture of mesothelioma would yield a census of recurrent clonal homozygous copy number losses as potential therapeutic vulnerabilities. Methods and Results: We prospectively enrolled 125 patients with MPM undergoing radical pleurectomy decortication, into the MEDUSA (Mesothelioma Evolution: DrUgging Somatic Alterations) study. Multi-region whole exome sequencing was conducted on 106 tumours from 20 patients (Medusa20 cohort). Up to 5 consistent regions were sampled: apex, pericardium, anterior/ posterior costophrenic angles, and the oblique fissure. For each patient, matching whole blood DNA was also whole exome sequenced to allow identification of tumour- specific somatic variations. Somatic copy number alterations (SCNAs) in each tumour region were called using SEQUENZA. We inferred phylogeny for each patient’s tumour using the SCNA calls by maximum parsimony (TUMULT), which revealed branched evolution in all MPMs. The total number of SCNAs ranged from 78 to 380 across the cohort with biphasic MPMs exhibiting a significantly larger total and clonal SCNA burden compared to epithelioid MPMs (p=0.024) . Only 9p21 which harbours CDKNA and methylthioadenosine phosphorylase (MTAP), exhibited clonal homozygous loss in 3 patients (15%). Clonal heterozygous loss was seen in 2 patients (10%). A further 5 patients showed with evidence of parallel evolution involving MTAP loss in distant MPM regions (25%), with one patient's MPM having late homozygous deletion in a single branch (5%). MTAP loss was validated by array based SCNA analysis and was found to be negatively prognostic in an independent cohort. Protein arginine methyltransferase 5 (PRMT5) has been recently identified as a vulnerability in MTAP deleted cancer. We found that siRNA silencing of PRMT5, caused MTAP selective loss of clonogenicity with proliferative arrest. Utilizing the connectivity map, quinacrine was validated as a PRMT5 perturbagen, which suppressed c-jun-dependent PRMT5 expression without inhibiting its methyltransferase activity. Quinacrine phenocopied PRMT5 siRNA, reducing global symmetrical arginine dimethylation of histone H4 (H4R3me2S). Finally, exogenous wild-type PRMT5 rescued quinacrine-mediated cell arrest in MTAP-negative cells, an effect not seen using the PMRT5 E444Q methyltransferase dead mutant. Conclusion: MTAP deletion is a clonal homozygous event in mesothelioma, with potential as a therapeutically tractable Achilles heel, via PRMT5 silencing using a repurposed small molecule, quinacrine. Citation Format: Sara Busacca, Lee Brannan, Apostolos Nakas, Annabel Sharkey, Chiara Riganti, David Waller, Cathy Richards, Iris Salaroglio, Vladan Milosevic, Peter Wells-Jordan, Alan Dawson, Michael Sheaff, John LeQuesne, Aarti Gaba, Robert Hastings, Luke Martinson, Jin-Li Lo, Amrita Bajaj, Paul Boutros, Tom John, Bibhusal Thapa, Gareth Wilson, Jacqui Shaw, Charles Swanton, Frank Dudbridge, Edward Hollox, Dean A. Fennell. Mesothelioma phylogeny reveal MTAP as a solitary clonal deletion, exposing vulnerability to the PRMT5 perturbagen, quinacrine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2908.

Abstract P2-03-09: Autocrine motility factor signaling pathway promotes aggressive behavior and migration in breast cancer

Abstract Background: Autocrine motility factor (AMF) is secreted by cancer cells and acts in an autocrine or paracrine fashion to bind to its receptor, AMFR, at the cell surface of tumor cells. The AMF-AMFR pathway has been shown to promote proliferation, anti-apoptosis, motility and migration, invasion, and metastasis pathways in various cancers. However, our understanding of the AMF-AMFR pathway in breast cancer is limited. We propose that the AMF signaling pathway is an unexplored mechanism for breast cancer tumor aggression and its receptor, AMFR, may be a potential therapeutic target. Methods: Tumor tissue obtained from consented female (n = 31) and male (n = 1) patients were analyzed by the Affymetrix OncoscanTM genome-wide microarray platform and examined for somatic copy-number alterations (SCNAs) of AMFR. cBioPortal was used to investigate SCNA of AMFR and gene expression of AMF on primary breast cancer tumors from METABRIC (n = 1,784) and TCGA Pan-Cancer Atlas (n = 981) datasets. In vitro, AMF and AMFR gene expression in luminal A (MCF-7) and triple-negative breast cancer (MDA-MB-231) cell lines were assessed by qPCR. Cell migration assays were performed on MDA-MB-231 cells to investigate their migration towards AMF with AMFR present or knocked down by siRNA. Results: Microarray analysis of 32 tumors revealed that a single-copy loss of AMFR occurred 79% of the time in luminal A tumors (n = 19); 67% of the time in luminal B tumors (n = 6); 33% of the time in ER+, PR+, HER2+ tumors (n = 3); and 0% of the time in triple-negative breast cancer (TNBC) tumors (n = 4), suggesting that the loss of AMFR results in less aggressive tumors that have good overall prognosis. To extend our findings to a larger patient cohort, SCNA analysis of the METABRIC and TCGA Pan-Cancer Atlas datasets revealed that single-copy loss of AMFR occurred in 64.53% and 68.14% of luminal A tumors, 58.51% and 59.90% of luminal B tumors, 23.62% and 48.72% of HER2 overexpression tumors, and 29.80% and 39.77% of TNBC tumors, respectively. Therefore, AMFR appears most frequently deleted in the tumor genomes of good prognosis breast cancer molecular subtypes (luminal tumors). Gene expression analysis of AMF in the METABRIC (using z-scores) and TCGA Pan-Cancer Atlas (using batch-normalized numbers) datasets revealed median mRNA expressions of -0.3351 and 4862 in luminal A tumors, -0.05415 and 5841 in luminal B tumors, 0.5758 and 9390 in HER2 overexpression tumors, and 0.754 and 8798 in TNBC tumors, respectively, suggesting that the AMF-AMFR pathway is more active in aggressive breast cancers. Similarly, we observed that AMF and AMFR are transcriptionally overexpressed by 7-fold and 16-fold, respectively, in the TNBC cell line MDA-MB-231 compared to the luminal A breast cancer cell line MCF-7. When AMFR is knocked down in MDA-MB-231 cells, focused migration towards AMF is abolished. Conclusion: AMF-AMFR pathway activity correlates with aggressive cancer cell behavior and enhanced migration in breast cancer. Single-copy loss of AMFR in tumor genomes is associated with less aggressive tumors with better overall prognosis. AMFR may be an attractive therapeutic target. Citation Format: Liu C, Price JD, Maroni D, Stevens JM, VanDyke AZ, Althof PA, Mondal P, Sanmann JN, Thayer SP. Autocrine motility factor signaling pathway promotes aggressive behavior and migration in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-09.

Using SAAS-CNV to Detect and Characterize Somatic Copy Number Alterations in Cancer Genomes from Next Generation Sequencing and SNP Array Data.

Somatic copy number alterations (SCNAs) are profound in cancer genomes at different stages: oncogenesis, progression, and metastasis. Accurate detection and characterization of SCNA landscape at genome-wide scale are of great importance. Next-generation sequencing and SNP array are current technology of choice for SCNA analysis. They are able to quantify SCNA with high resolution and meanwhile raise great challenges in data analysis. To this end, we have developed an R package saasCNV for SCNA analysis using (1) whole-genome sequencing (WGS), (2) whole-exome sequencing (WES) or (3) whole-genome SNP array data. In this chapter, we provide the features of the package and step-by-step instructions in detail.

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

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

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.

Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking.

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.

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.

Abstract 3992: Modes of metastasis evolution in human and murine cancer revealed by whole exome sequencing

Abstract Despite that fact that 90% of cancer deaths are due to metastasis, relatively little is known about how metastases evolve from primary cancers. We subjected 101 trios consisting of primary tumor, brain metastasis, and normal reference tissue to whole exome sequencing (WES). To analyze the data, we developed novel computational tools to derive high quality allelic copy-number profiles directly from the WES data. These were used to perform an integrative analysis of somatic copy-number alterations (SCNAs) and somatic single nucleotide variants (SSNVs). This analysis allowed us to estimate the clonal architecture of the primary and metastatic samples from each patient, and to reconstruct a single phylogenetic tree relating all of the subclones detected in both samples. We determined that all of the matched metastases analyzed likely formed from a single clone. In all cases, we observed a sibling relationship between the metastasis and primary tumor samples, indicating that they share a common ancestor, but ongoing evolution in the primary tumor resulted in fully clonal mutations in the primary biopsy that were not present in the metastasis sample. An important corollary of this is that in no case was a metastasis-founding subclone detected in a primary tumor sample. Using mutational spectra associated with tobacco exposure in lung cancers, we determined that most of the evolution leading up to the brain metastasis like occurred within the lung. In contrast with these findings, analysis of related primary tumor and metastasis families in a mouse model of small cell lung cancer using identical methodology revealed evidence of more variable patterns of metastatic spread. Analysis of draining lymph nodes revealed that multiple subclones of the primary contributed to these metastases. In addition, we uncovered evidence for the sequential spread of metastases, consistent with multiregion sequencing of primary and metastatic human pancreatic cancer. The extension of this finding to a mouse cancer model suggests this may be a more frequent feature of cancer metastasis, and raises the possibility of genetically dissecting these processes in this experimental system. Citation Format: Scott L. Carter, Priscilla K. Brastianos, David G. McFadden, Thales Papagiannakopoulos, Amaro Taylor-Weiner, Kristian Cibulskis, Tyler Jacks, William Hahn, Gad Getz. Modes of metastasis evolution in human and murine cancer revealed by whole exome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3992. doi:10.1158/1538-7445.AM2014-3992

Abstract 3306: Aberrant expression of ribosomal protein p1 and its pseudogene in breast tumors

Abstract Introduction: Ribosome biogenesis and translation control are essential cellular processes in growing cancer cells. Defects in ribosomal components can alter the pattern of gene expression, cell cycle progression and cell growth. Human ribosomes consist of four RNA species and 79 ribosomal proteins with 2090 processed and 16 duplicated pseudogenes. Specifically, 60S Acidic Ribosomal Protein P1 (RPLP1) is expressed in a tissue specific manner with multiple processed pseudogenes. We accidentally identified the expressed pseudogene 4 of RPLP1 (RPLP1P4) and determined genetic and molecular mechanism of RPLP1 and RPLP1P4 expression in breast cancer cells. Methods: Expression levels of RPLP1 and RPLP1P4 were quantified using qRT-PCR with total RNAs isolated from 7 normal tissues and 20 breast tumor tissues of 5 different subtypes. Cell cycle progression was analyzed cytofluorimetrically with the Beckman Coulter Gallios Flow Cytometer. Gene expression profiles were performed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Array and analyzed using Partek Genomic Suite. Somatic copy number alterations (SCNA) of RPLP1/RPLP1P4 were detected with the custom designed TaqMan assays using ABI 7900HT real time PCR system. CopyCaller™ Software was used for the SCNA analysis with RNase P as a reference. Results: Because breast cancer subtypes have distinct origins and molecular characteristics, we compared expression of RPLP1 and RPLP1P4 in 5 subtypes of breast tumors. Although RPLP1/RPLP1P4 is ubiquitously expressed in normal and cancer tissues, their expression levels are significantly increased in the basal-like subtype of breast cancer compared to other subtypes with a 2.23 ± 1.06 fold increase in basal-like subtype compared to non-basal subtypes. Interfering RPLP1 expression with siRNA dramatically decreased cell proliferation up to the level of complete inhibition of cell proliferation. FACS data revealed reduced S phase cells in RPLP1 silencing-cells compared to control, suggesting that RPLP1-mediated regulation of cell proliferation is partly mediated through regulation of cell cycle progression. Analysis of gene expression profiles displayed down-regulation of genes involved in cellular metabolism and DNA replication. When we assessed 44 formalin fixed paraffin embedded breast tissues for SCNA of RPLP1/RPLP1P4, we observed RPLP1 amplifications in 44% of tumor DNAs with no amplification observed in normal tissues. The clear difference between tumors and normal tissues demonstrates frequent RPLP1 amplification events during tumorigenesis, which possibly drives an increase in RPLP1 expression and cell proliferation in cancer. Conclusion: Our data suggest a subtype-specific increased expression of a ribosomal protein and its pseudogene, potentially driven by copy number alterations in breast tumors. Future studies to understand the contributions of ribosomal protein genes and their pseudogenes to human neoplasia are warranted. Citation Format: Yoo J. Han, Olufunmilayo I. Olopade. Aberrant expression of ribosomal protein p1 and its pseudogene in breast tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2014-3306