Genome-wide Copy Number Aberrations Research Articles

A molecular study of pediatric pilomyxoid and pilocytic astrocytomas: Genome-wide copy number screening, retrospective analysis of clinicopathological features and long-term clinical outcome.

Pilocytic Astrocytoma (PA) is the most common pediatric brain tumors. PAs are slow-growing tumors with high survival rates. However, a distinct subgroup of tumors defined as pilomyxoid astrocytoma (PMA) presents unique histological characteristics and have more aggressive clinical course. The studies on genetics of PMA are scarce. In this study, we report one of the largest cohort of pediatric patients with pilomyxoid (PMA) and pilocytic astrocytomas (PA) in Saudi population providing a comprehensive clinical picture, retrospective analysis with long-term follow-up, genome-wide copy number changes, and clinical outcome of these pediatric tumors. We examined and compared genome-wide copy number aberrations (CNAs) and the clinical outcome of the patients with PA and PMA. The median progression free survival for the whole cohort was 156 months and it was 111 months for the PMA, however, not statistically significantly different between the groups (log-rank test, P = 0.726). We have identified 41 CNAs (34 gains and 7 losses) in all tested patients. Our study yielded the previously reported KIAA1549-BRAF Fusion gene in over 88% of the tested patients (89% and 80% in PMA and PA, respectively). Besides the fusion gene, twelve patients had additional genomic CNAs. Furthermore, pathway and gene network analyses of genes in the fusion region revealed alterations in retinoic acid mediated apoptosis and MAPK signaling pathways and key hub genes that may potentially be involved in tumor growth and progression, including BRAF, LUC7L2, MKRN1, RICTOR, TP53, HIPK2, HNF4A, POU5F, and SOX4. Our study is the first report of a large cohort of patients with PMA and PA in the Saudi population that provides detailed clinical features, genomic copy number changes, and outcome of these pediatric tumors and may help better diagnosis and characterization of PMA.

Candidate targets of copy number deletion events across 17 cancer types.

Genome variation is the direct cause of cancer and driver of its clonal evolution. While the impact of many point mutations can be evaluated through their modification of individual genomic elements, even a single copy number aberration (CNA) may encompass hundreds of genes and therefore pose challenges to untangle potentially complex functional effects. However, consistent, recurring and disease-specific patterns in the genome-wide CNA landscape imply that particular CNA may promote cancer-type-specific characteristics. Discerning essential cancer-promoting alterations from the inherent co-dependency in CNA would improve the understanding of mechanisms of CNA and provide new insights into cancer biology and potential therapeutic targets. Here we implement a model using segmental breakpoints to discover non-random gene coverage by copy number deletion (CND). With a diverse set of cancer types from multiple resources, this model identified common and cancer-type-specific oncogenes and tumor suppressor genes as well as cancer-promoting functional pathways. Confirmed by differential expression analysis of data from corresponding cancer types, the results show that for most cancer types, despite dissimilarity of their CND landscapes, similar canonical pathways are affected. In 25 analyses of 17 cancer types, we have identified 19 to 169 significant genes by copy deletion, including RB1, PTEN and CDKN2A as the most significantly deleted genes among all cancer types. We have also shown a shared dependence on core pathways for cancer progression in different cancers as well as cancer type separation by genome-wide significance scores. While this work provides a reference for gene specific significance in many cancers, it chiefly contributes a general framework to derive genome-wide significance and molecular insights in CND profiles with a potential for the analysis of rare cancer types as well as non-coding regions.

Low-pass whole-genome and targeted sequencing of cell-free DNA from cerebrospinal fluid in pediatric patients with central nervous system tumors.

Central nervous system tumors are the most common pediatric solid tumors and the most frequent cause of cancer-related morbidity in childhood. Significant advances in understanding the molecular features of these tumors have facilitated the development of liquid biopsy assays that may aid in diagnosis and monitoring response to therapy. In this report, we describe our comprehensive liquid biopsy platform for detection of genome-wide copy number aberrations, sequence variants, and gene fusions using cerebrospinal fluid (CSF) from pediatric patients with brain, spinal cord, and peripheral nervous system tumors. Cell-free DNA was isolated from the CSF from 55 patients, including 47 patients with tumors and 8 controls. Abnormalities in cell-free DNA were detected in 24 (51%) patients including 11 with copy number alterations, 9 with sequence variants, and 7 with KIAA1549::BRAF fusions. Positive findings were obtained in patients spanning histologic subtypes, tumor grades, and anatomic locations. This study demonstrates the feasibility of employing this platform in routine clinical care in upfront diagnostic and monitoring settings. Future studies are required to determine the utility of this approach for assessing response to therapy and long-term surveillance.

Abstract 2784: A robust copy number based clonality classification for multiple pulmonary tumors in routine pathology practice by shallow next generation sequencing of formalin fixed clinical specimens

Abstract Multiple synchronous or metachronous independent tumors in one patient, particularly those with thoracic involvement, can be as high as 20%. Defining whether a patient has multiple primary cancers (MPC) or metastatic disease is an urgent clinical need due to differences in treatment strategies, with curative intend for MPCs. Discerning the clonal and non-clonal relationship of two lesions for metastases and MPCs, respectively, has been done since the 1970s by analyzing histological characteristics, which nowadays are complemented with genomic features. However, one or few mutation features are insufficient to unequivocally diagnose tumor clonality, due to intratumoral heterogeneity and common mutations. We and others have demonstrated convincing clonality classification by genome-wide copy number aberrations (CNAs) analysis, which is the recommendation from the International Association for the Study of Lung Cancer (IASLC), independent of histological differences or similarities. Notwithstanding, a gold standard clonality assessment is currently lacking such that quality evaluation and calibration of the diagnostic tests are not possible. Gold-standard data: Genetic data from non-clonal cancer samples has become virtually endless by means of Whole Exome and Whole Genome sequencing (WES and WGS) since the mutational discrepancies by WES between two MPCs from a single lung cancer patient is the same as those of two primary cancers from different patients. Therefore, publicly available WES or WGS data from tumors of different lung cancer patients can serve as a gold standard reference for non-clonality. A gold standard reference for clonal samples can be derived from WES data from multiple biopsies of one patient, i.e. from the TRACERx initiative. These datasets now allow for the first time to calibrate CNA clonality testing against a gold standard and calculate the sensitivity and specificity of multiple lung tumors in one patient. Diagnostic test: Shallow Whole Genome Sequencing (sWGS) based genome-wide CNA analysis on Formalin-Fixed Paraffin-Embedded (FFPE) specimens has been used as the diagnostic routine at the Cancer Center Amsterdam (CCA), Amsterdam UMC over the past 5 years. The pipeline was calibrated against gold standard clonal and non-clonal WES data. We present sensitivity and specificity for an adequate, fast, and more economic clonality test based on genome-wide CNAs. Citation Format: Bárbara Andrade Barbosa, Teodora Radonic, Yongsoo Kim, Bauke Ylstra. A robust copy number based clonality classification for multiple pulmonary tumors in routine pathology practice by shallow next generation sequencing of formalin fixed clinical specimens [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 2784.

Abstract 5263: Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome

Abstract Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Although conventional cytogenetic analysis (CCA) has been mandatory in clinical routine to detect recurrent genomic abnormalities in AML/MDS, it presents a few obvious shortcomings, such as time-consuming in vitro cell-culturing procedure, low resolution (no smaller than 10 Mbp), and subjective bias introduced from the visual interpretation by cytogeneticists. With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations not only those well-characterized clinically actionable mutations but also cryptic alternations yet to be defined and annotated. In this study a shallow whole-genome sequencing (sWGS) based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow cells of 107 newly diagnosed patients with AML (n=63, 58.9%) or MDS (n=44, 41.1%). Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth. Genome-wide CNAs was analyzed for each sample using an automated pipeline. European Leukemia Net (ELN) or Revised International Prognostic Scoring System (IPSS-R)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. It demonstrated 98.1% concordance of CNA profiles at individual patient level with cytogenetics and/or FISH. It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients (15%) by presenting new information. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. In summary, LeukoPrint provided an automated, convenient and cost-effective approach to describe genomic CNA profiles in AML/MDS. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the standard ELN/IPSS-R guidelines. Citation Format: Xiaodong Lyu, Tao Li, Dandan Zhu, Mengna Zhang, Yuexin Cheng, Yan Chen, Zhenling Li, Shiyong Li, Wei Wu, Shuaipeng Geng, Jingshuai Li, Xiangxiang He, Yangwei Li, Yinyin Chang, Zunmin Zhu, Mao Mao, Yongping Song. Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome [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 5263.

Abstract 6084: Recurrent genomic patterns of MPNST evolution correlate with clinical outcome

Abstract Neurofibromatosis type 1 (NF1) is the most common tumor predisposition syndrome, and is associated with an aggressive soft-tissue sarcoma, malignant peripheral nerve sheath tumours (MPNSTs), the greatest cause of mortality in people with NF1. The only potentially curative therapy involves en bloc resection with negative margins, which is not always appropriate. Even therapy with curative intent is associated with poor overall survival for both sporadic and NF1-related MPNSTs. The development of novel therapies has been largely hindered by a poor understanding of the molecular events underpinning MPNST pathogenesis. We report a comprehensive multi-omic study of MPNST evolution based on whole genome sequencing, transcriptomic and methylation profiling data on 95 tumors (64 NF1-related; 31 sporadic). In all cases, the early events in MPNST evolution involve biallelic inactivation of NF1 followed by inactivation of CDKN2A, as well as mutations in TP53 or PRC2 complex genes in a subset of cases. Analysis of the genomic architecture revealed distinct pathways of tumor evolution that can be identified through H3K27 trimethylation (H3K27me3) status. Integration of these data allows us to propose several mechanistic tumor evolution models. Tumors with H3K27me3 loss evolve through extensive copy number aberrations (CNAs) including haploidization followed by whole genome doubling and chromosome 8 amplifications, whereas tumors with H3K27me3 retention evolve through extensive chromosome instability and chromothripsis. Taken together, these genome-wide CNA profiles act as a surrogate for the loss of H3K27me3 status and correlate with prognosis, suggesting that CNA profiling of cell-free DNA could be incorporated in clinical decision-making. Citation Format: Isidro Cortes Ciriano, Chris D. Steele, Katherine Piculell, Alyaa Al-Ibraheemi, Vanessa Eulo, Marilyn M. Bui, Aikaterini Chatzipli, Brendan C. Dickson, Dana C. Borcherding, Alon Galor, Jesse Hart, Andrew Feber, Kevin B. Jones, Justin T. Jordan, Raymond H. Kim, Daniel Lindsay, Colin Miller, Yoshihiro Nishida, Jonathan Serrano, Nicole J. Ullrich, David Viskochil, Xia Wang, Matija Snuderl, Paula Proszek, Peter J. Park, Adrienne M. Flanagan, Angela C. Hirbe, Nischalan Pillay, David T. Miller, The Genomics of MPNST (GeM) Consortium. Recurrent genomic patterns of MPNST evolution correlate with clinical outcome [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 6084.

Whole-genome sequencing as an alternative to analyze copy number abnormalities in AML, MDS and MM.

e19023 Background: Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and multiple myeloma (MM). With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations. Methods: 178 patients who met the clinical manifestations of blood malignancies were included in the study, including 89 patients with AML, 48 with MDS and 41 with MM. Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth (shallow whole-genome sequencing, sWGS). Genome-wide CNAs greater than 5 Mbp was analyzed for each sample. European Leukemia Net (ELN), Revised International Prognostic Scoring System (IPSS-R) or Revised International Staging System (R-ISS)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. Results: In this study, CNA events were identified in 109 (73.6%) patients via sWGS. By calculating frequencies of recurrent gains and losses at chromosome arm level, some recurrent events were detected like gains of chr 8 (observed in 12 AML and 9 MDS patients), chr 1 (observed in 18 MM patients), losses of chr 7 (observed in 9 AML and 3 MDS patients) and chr 13 (observed in 16 MM patients), which were consistent with previously reported cytogenetic events. Besides, amplifications of odd-numbered chromosomes were detected in 15(36.6%) of patients with multiple myeloma, including chromosomes 3, 5, 7, 9, 11, 15, 19 and/or 21. And the trisomy of chr 5, 7 or 9 are mainly observed with a frequency of 66.7%. Among 145 patients with CCA results, a high concordance of 97.7% (98.5%, 97.6% and 97.1% in AML, MDS and MM respectively) was achieved in CNA profiles at individual patient level with cytogenetics and/or FISH. Noteworthily, CNA events were detected in 20 of 28 (71.4%) MM patients with negative karyotyping results. Moreover, sWGS improved the overall diagnostic yield by reassign 18 patients (9 with AML and 9 with MDS) to risk categories with poorer prognosis. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. Conclusions: In summary, a streamlined sWGS provides an accurate, convenient and cost-effective approach to describe genomic profiles of CNAs in patients with AML, MDS and MM. Clinical trial information: ChiCTR2100052276.

Improved Uveal Melanoma Copy Number Subtypes Including an Ultra-High-Risk Group.

PurposeTo evaluate the clinical relevance of low-frequency copy number aberrations (CNAs) in uveal melanoma (UM) and to discern residual genomic and clinical heterogeneity within established molecular subtypes based on genome-wide CNA profiling of 921 primary tumors.DesignRetrospective single-center case series.ParticipantsPatients with primary UM referred for genetic testing between 2008 and 2016 (n = 921). The Cancer Genome Atlas cohort with clinical outcome data available (n = 70) was used to validate findings.MethodsGenome-wide CNAs were generated for primary tumors from 921 patients and for 19 metastatic UM (mUM) in the liver. Of the 921 patients, metastatic outcome was known for 678 patients with a median time to metastasis of 4.5 years. The primary tumors were processed on the Affymetrix arrays SNP-5.0 (n = 140), SNP-6.0 (n = 359), or CytoScanHD (n = 422), and the metastatic tumors on the CytoScanHD array (n = 19). Recurrent CNAs were identified, and the prognostic effect of individual CNAs and multiple CNA clustering strategies, including more specific molecular subgroups with rare CNAs, were evaluated.Main Outcome MeasuresCNA recurrence, and effect of CNAs and derived molecular subtypes on metastatic-free survival.ResultsGenomic profiling revealed CNAs associated with risk of metastasis and demonstrated a strong association between chromosomal instability and patient prognosis. Using standard prognostic CNAs, 6 clusters were detected, and inclusion of chromosome 16q deletion revealed an additional cluster. Of these 7 genomic clusters, 5 patient groups showed distinct rates of metastasis, indicating that different genomic patterns can have similar patient outcomes. A small group of patients with a significantly higher rate of metastasis was characterized by monosomy 3, 8q amplification, and deletion of 1p or 16q. Although this ultra–high-risk group accounts for only 7% of this cohort, 88% demonstrated metastasis within 4 years, compared with 45% in the second-highest risk group.ConclusionsThese results suggest that 1p and 16q deletion should be incorporated in clinical assays to assess prognosis at diagnosis and to guide enrollment in clinical trials for adjuvant therapies.

Molecular Profiling of Relapsed and Refractory Myeloma Identifies Frequent Bi-Allelic Driver Events — Interim Results of the ‘Myeloma UK Seven’ Biomarker Trial

IntroductionPomalidomide and low-dose dexamethasone (PomDex) are approved for relapsed and refractory myeloma (RRMM) with Overall Response Rates of about 30%, including durable remissions. Combining PomDex with biologic agents can improve response, but there are currently no markers to predict which patients benefit from the addition of drugs to PomDex alone. Myeloma UK (MUK) seven is a multi-center UK phase II trial that is designed to generate evidence for improved outcomes and treatment stratification in RRMM by: 1) building a bio-repository for molecular characterization of RRMM and discovery of predictive markers for response 2) patient randomisation to Cyclophosphamide-PomDex (CyPomDex) or PomDex to investigate if the cost-effective CyPomDex combination increases progression free survival compared to PomDex 3) a flexible design that allows addition of trial arms with other promising PomDex combinations. We report high patient and investigator bio-sampling adherence and first comprehensive genetic profiling results in RRMM using a novel molecular assay platform.MethodsMUKseven (NCT02406222) will in total recruit 250 RRMM patients who are currently randomised 1:1 to CyPomDex or PomDex therapy. Registered patients undergo bone marrow sampling at screening, Cycle 1 Day 14, Cycle 4 Day 14 and at disease progression, and peripheral blood sampling on Cycle 1 Day 1. Patient consent includes mandatory bio-sampling at screening. Material is shipped to central laboratories for processing and storage of CD138-purified (>95%) MM tumor and non-tumor bone marrow microenvironment cells. Participants are currently recruited from over 30 NHS Hospitals in the MUK Early Phase Clinical Network.Digital multiplex ligation-dependent probe amplification (dMLPA®; MRC Holland) is a novel assay for high-throughput genome-wide copy number aberration (CNA) profiling. The MM specific D006 assay contains 160 probes that interrogate regions frequently affected by CNA in MM, including hotspots CDKN2C, CKS1B, MYC and regions encoding for therapeutic targets such as MCL1 or SLAMF7. In addition, 219 probes are distributed over all chromosomal arms and inform a virtual karyogram. The targeted design allows for analysis of over 80 patient samples in a single MiSeq (Illumina) run, using 10-20 ng DNA input material.ResultsBetween March 2016 and July 2017 a total of 83 patients were randomised to MUKseven with a median follow up of 6 cycles (1-13). Bone marrow material was received by central laboratories for all patients (100%) at screening, 69/79 (87.3%) at Cycle 1 day 14, 47/54 (87.0%) at Cycle 4 day 14 and 13/29 (44.8%) at disease progression. Peripheral blood was received for 73/79 (92.4%) on Cycle 1 Day 1. These return rates are higher than those reported for recent commercial trials. Of importance, for 52% of patients, matched bone marrow samples from earlier disease stages collected as part of UK academic research studies were available.We profiled the first 81 RRMM samples from trial entry with dMLPA and compared CNA frequency to a recently published newly diagnosed MM (NDMM) cohort which was assessed with MLPA (Shah V, Leukemia 2017). We found significantly more focal bi-allelic CNA of driver lesions in RRMM than in NDMM (P = 0.002). These included homozygous deletions of CDKN2C (6.2 vs 1.9%) and RB1 (3.7 vs 0.8%), amplifications of MYC (8.6 vs 2.3%), and heterozygous losses of TP53 (16.0 vs 9.6%). There were significantly more RRMM patients with one or more of the following bi-allelic changes: CDKN2C, RB1 or CYLD homozygous loss, or 1q amplification (18.5 vs 8.2%; P = 0.007), and 3.7% of patients in the RRMM cohort showed more than one bi-allelic change. We identified frequent homozygous deletions in NFκB pathway genes such as CYLD (4.9%), and recurrent amplifications of therapeutic targets like MCL1 and SLAMF7 on chromosome 1 (8.6% amplifications). The trial is recruiting and profiling of additional cases as well as longitudinal profiling of cases with material from earlier disease stages is ongoing.ConclusionWe demonstrate highly successful conduct of a multi-center biomarker discovery trial by the Myeloma UK Clinical Trial Network and the UK Myeloma Research Alliance. Our results demonstrate that bi-allelic CNA increase significantly in RRMM and some of these may help guide therapy. Recruitment and analyses of additional cases enrolled into MUKseven are ongoing and updated data will be presented at the meeting. DisclosuresPawlyn:Takeda: Honoraria, Other: Travel support; Janssen: Other: Travel support; Celgene: Honoraria, Other: Travel support. Atanesyan:MRC-Holland: Employment. Savola:MRC-Holland: Employment. Thakurta:Celgene Corporation: Employment, Equity Ownership. Garg:Janssen: Other: travel support, Research Funding, Speakers Bureau; Takeda: Other: travel support; Novartis: Other: travel support, Research Funding. Cook:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetcs: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau. Boyd:Janssen: Honoraria, Other: Travel support; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Kaiser:Takeda: Consultancy; BMS: Consultancy, Other: Travel expenses; Janssen: Honoraria; Chugai: Consultancy; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Chromosome 20 loss is characteristic of breast implant–associated anaplastic large cell lymphoma

AbstractBreast implant–associated anaplastic large cell lymphoma (BIA-ALCL) is a very rare type of T-cell lymphoma that is uniquely caused by a single environmental stimulus. Here, we present a comprehensive genetic analysis of a relatively large series of BIA-ALCL (n = 29), for which genome-wide chromosomal copy number aberrations (CNAs) and mutational profiles for a subset (n = 7) were determined. For comparison, CNAs for anaplastic lymphoma kinase (ALK)− nodal anaplastic large cell lymphomas (ALCLs; n = 24) were obtained. CNAs were detected in 94% of BIA-ALCLs, with losses at chromosome 20q13.13 in 66% of the samples. Loss of 20q13.13 is characteristic of BIA-ALCL compared with other classes of ALCL, such as primary cutaneous ALCL and systemic type ALK+ and ALK− ALCL. Mutational patterns confirm that the interleukin-6–JAK1–STAT3 pathway is deregulated. Although this is commonly observed across various types of T-cell lymphomas, the extent of deregulation is significantly higher in BIA-ALCL, as indicated by phosphorylated STAT3 immunohistochemistry. The characteristic loss of chromosome 20 in BIA-ALCL provides further justification to recognize BIA-ALCL as a separate disease entity. Moreover, CNA analysis may serve as a parameter for future diagnostic assays for women with breast implants to distinguish seroma caused by BIA-ALCL from other causes of seroma accumulation, such as infection or trauma.

MBCL-08. INTEGRATIVE MOLECULAR ANALYSIS OF PATIENT-MATCHED DIAGNOSTIC AND RELAPSED MEDULLOBLASTOMAS

INTRODUCTIONThe next generation of clinical trials for relapsed medulloblastoma demands a thorough understanding of the clinical behavior of relapsed tumors as well as the molecular relationship to their diagnostic counterparts.METHODSA multi-institutional molecular cohort of patient-matched (n=126 patients) diagnostic MBs and relapses/subsequent malignancies was profiled by DNA methylation array. Entity, subgroup classification, and genome-wide copy-number aberrations were assigned while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations.RESULTSComprised of WNT (2%), SHH (41%), Group 3 (18%), Group 4 (39%), primary tumors retained subgroup affiliation at relapse with the notable exception of 10% of cases. The majority (8/13) of discrepant classifications were determined to be secondary glioblastomas. Additionally, rare (n=3) subgroup-switching events of Group 4 primary tumors to Group 3 relapses were identified coincident with MYC/MYCN pathway alterations. Amongst truly relapsing MBs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with Group 3 (55% of alterations shared) and Group 4 tumors (63% alterations shared) sharing a larger proportion of cytogenetic alterations compared to SHH tumors (42% alterations shared; Chi-square p-value < 0.001). Subgroup- and gene-specific patterns of conservation and divergence amongst putative driver genes were also observed.CONCLUSIONIntegrated molecular analysis of relapsed MB discloses potential mechanisms underlying treatment failure and disease recurrence while motivating rational implementation of relapse-specific therapies. The degree of genetic divergence between primary and relapsed MBs varied by subgroup but suggested considerably higher conservation than prior estimates.

Single B-Cell Genomic Analyses Differentiate Vitreoretinal Lymphoma from Chronic Inflammation

To test whether analyzing DEPArray (Menarini Silicon Biosystems) isolated single B cells from the vitreous fluid can reveal crucial genomic and clinicopathological features to distinguish patients with vitreoretinal lymphoma (VRL) from those with chronic inflammation using immunoglobulin heavy chain (IGH), disease biomarker myeloid differentiation primary response 88 (MYD88)L265P mutation, and copy number profiling. A single-center, retrospective study. Remnant vitreous biopsies from 7 patients with VRL and 4 patients with chronic inflammation were acquired for molecular analysis. Vitreous fluid samples were prefixed in PreservCyt (Hologic) and underwent cytologic analysis and immunohistochemistry examination. Single cells were isolated using the DEPArray NxT system, followed by downstream genomic analysis. The frequencies of the dominant IGH and MYD88L265P mutation and the genome-wide copy number aberration (CNA) profiles of individual vitreous-isolated B cells were characterized. An average of 10 to 13 vitreous B cells were used in the single-cell IGH and MYD88 analyses. Higher frequencies of dominant IGH (88.8% ± 13.2%) and MYD88L265P mutations (35.0% ± 31.3%) were detected in patients with VRL than in patients with chronic inflammation (65.9% ± 13.4% and 1.5% ± 2.6% for IGH and MYD88L265P, respectively). In a cytology-proven VRL case, all 15 vitreous isolated B cells were derived from the same clone with 100% paired IGH: immunoglobulin light chain (IGK) sequences. Genome-wide copy number profiling revealed a high degree of similarity between B cells from the same patient with VRL, with extensive gains and losses at the same areas across the whole genome. In addition, 14 of 15 B cells showed a BCL2/JH t(14;18) translocation, confirming cellular malignancy with a clonal origin. Clustering analysis of the copy number profiles revealed that malignant B cells derived from different patients with VRL had no common genome-wide signatures. Single B-cell genomic characterization of the IGH, MYD88L265P mutation, and copy number profile enables VRL diagnosis. Because our study involved only a small cohort, these meaningful proof-of-concept data now warrant further investigation in a larger patient cohort.

Clinical Validation of Somatic Mutation Detection by the OncoScan CNV Plus Assay

The OncoScan CNV Plus Assay (OS+) is a single-nucleotide polymorphism microarray platform that can detect 74 hotspot somatic mutations (SMs) in nine genes via molecular inversion probes. We report validation of the SM component of OS+using a cohort of pediatric high-grade brain tumor specimens. SM calls were generated from 46 brain tumor cases, most tested orthogonally via bidirectional Sanger sequencing. The initial calling algorithm result showed that 31 tumors were positive and 15 were negative for SM, with a total of 71 OS+SM calls [28 high-confidence (HC) and 43 low-confidence (LC)]. Sanger sequencing was performed for 54 of the 71 calls (27 HC and 27 LC), as well as for 21 randomly selected hotspots across the 15 OS+negative cases. HC calls (except EGFR) Sanger sequencing confirmed positive, negative calls confirmed negative, but none of the LC calls were Sanger-confirmed positive. An update of the OS+algorithm resolved the LC calls, but of the 11 HC SM EGFR calls, Sanger sequencing confirmed only one. Two PTEN SM calls by OS+ in two separate cases were also negative per Sanger sequencing. We conclude that a majority of HC OS+SM calls were accurate, except calls identified in EGFR and PTEN. Clinically, we report SMs identified by OS+only after Sanger sequencing verification.

Clinical, Pathology, Genetic, and Molecular Features of Colorectal Tumors in Adolescents and Adults 25 Years or Younger

Colorectal cancers (CRCs) are rare in adolescents and adults ages 25 years or younger. We analyzed clinical, pathology, and molecular features of colorectal tumors from adolescents and young adults in an effort to improve genetic counseling, surveillance, and, ultimately, treatment and outcomes. We analyzed clinical data and molecular and genetic features of colorectal tumor tissues from 139 adolescents or young adults (age, ≤25 y; median age, 23 y; 58% male), collected from 2000 through 2017; tumor tissues and clinical data were obtained from the nationwide network and registry of histopathology and cytopathology and The Netherlands Cancer Registry, respectively. DNA samples from tumors were analyzed for microsatellite instability, mutations in 56 genes, and genome-wide somatic copy number aberrations. Mucinous and/or signet ring cell components were observed in 33% of tumor samples. A genetic tumor risk syndrome was confirmed for 39% of cases. Factors associated with shorter survival time included younger age at diagnosis, signet ring cell carcinoma, the absence of a genetic tumor risk syndrome, and diagnosis at an advanced stage of disease. Compared with colorectal tumors from patients ages 60 years or older in the Cancer Genome Atlas, higher proportions of tumors from adolescents or young adults were microsatellite stable with nearly diploid genomes, or contained somatic mutations in TP53 and POLE, whereas lower proportions contained mutations in APC. We found clinical, molecular, and genetic features of CRCs in adolescents or young adults to differ from those of patients older than age 60 years. In 39% of patients a genetic tumor risk syndrome was identified. These findings provide insight into the pathogenesis of CRC in young patients and suggest new strategies for clinical management. Performing genetic and molecular analyses for every individual diagnosed with CRC at age 25 years or younger would aid in this optimization.

PATH-54. MULTI-DIMENSIONAL MOLECULAR CHARACTERIZATION OF PATIENT-MATCHED MEDULLOBLASTOMA AT DIAGNOSIS AND RELAPSE

Abstract INTRODUCTION Recurrent medulloblastoma (MB) confers an abysmal prognosis with ~10% 5-year overall survival. Optimal treatment paradigms for relapsed disease are largely unknown. Conservation of molecular subgroup at relapse has been described and divergent clonal evolution implicated, yet multi-dimensional molecular characterization of larger cohorts are warranted to substantiate these findings and to disclose potential mechanisms underlying treatment failure and disease recurrence. METHODS A multi-institutional series of 85 patient-matched, primary MBs and their relapses was profiled by DNA methylation array (Illumina 450K/850K). Entity and molecular subgroup classifications were assigned using random forest tumor classifiers (Molecular Neuropathology v.11b4). Genome-wide copy-number aberrations were also inferred from these data while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (1%), SHH (41%), Group 3 (22%), Group 4 (35%), primary tumors largely retained subgroup affiliation at relapse with the notable exception of 14% of cases. The majority (8/12) of discrepant classifications were determined to be secondary glioblastomas, while three Group 4 primary tumors relapsed as Group 3. Amongst conserved pairs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with an average of 0.8 (range 0–5) primary-specific and 2.0 (range 0–11) relapse-specific cytogenetic alterations. Despite high global methylation correlations between primary/relapse pairs (median 0.95), epigenetic evolution at relapse is suggested by the lower degree of conservation amongst promoter-associated probes (median 0.83, p &lt; 2.2e-16). CONCLUSION Secondary malignancy, particularly glioblastoma, may masquerade as relapsed MB, highlighting the utility of molecular characterization for relapsed disease and necessity for vigilant clinical surveillance. By deciphering the evolution of MB from diagnosis to relapse, a fundamental understanding of disease pathogenesis may be garnered, motivating rational and targeted clinical interventions in early therapy and at the time of relapse.

Profiling of Circulating Free DNA Using Targeted and Genome-wide Sequencing in Patients with SCLC

IntroductionSCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient stratification and response monitoring.MethodsWe applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA [the percentage of genomic regions amplified]), Z-score (a measure of standard deviation), and Moran’s I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample.ResultsGenome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival.ConclusionsWe demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.

Loss of 1p36.33 Frequent in Low-Grade Serous Ovarian Cancer

BACKGROUND: Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian carcinoma. Limited data regarding the molecular-genetic background exist beyond mutations in the RAS signaling pathway. There is a growing need to better characterize these tumors due to chemoresistance and limited therapeutic options in advanced or recurrent disease. METHODS: We performed genome-wide copy number aberration (CNA) profiles and mutation hotspot screening (KRAS, BRAF, NRAS, ERBB2, PIK3CA, TP53) in 38 LGSOC tumor samples. RESULTS: We detected mutations in the RAS-signaling pathway in 36.8% of cases, including seven KRAS, four BRAF, and three NRAS mutations. We identified two mutations in PIK3CA and one mutation in MAP3K1, EGFR, and TP53. CNAs were detected in 86.5% of cases. None of the focal aberrations was correlated with specific clinical characteristics. The most frequently detected CNA was loss of 1p36.33 in 54.1% of cases, with a trend towards lower progression-free survival and overall survival in patients with 1p36.33 loss. CONCLUSIONS: Activating RAS mutations were dominant in our series, with supplementary detection of two PIK3CA mutations which may lead to therapeutic options. Furthermore, we detected 1p36.33 deletions in half of the cases, indicating a role in tumorigenesis, and these deletions may serve as a prognostic marker.

Prognostic effect of whole chromosomal aberration signatures in standard-risk, non-WNT/non-SHH medulloblastoma: a retrospective, molecular analysis of the HIT-SIOP PNET 4 trial.

SummaryBackgroundMost children with medulloblastoma fall within the standard-risk clinical disease group defined by absence of high-risk features (metastatic disease, large-cell/anaplastic histology, and MYC amplification), which includes 50–60% of patients and has a 5-year event-free survival of 75–85%. Within standard-risk medulloblastoma, patients in the WNT subgroup are established as having a favourable prognosis; however, outcome prediction for the remaining majority of patients is imprecise. We sought to identify novel prognostic biomarkers to enable improved risk-adapted therapies.MethodsThe HIT-SIOP PNET 4 trial recruited 338 patients aged 4–21 years with medulloblastoma between Jan 1, 2001, and Dec 31, 2006, in 120 treatment institutions in seven European countries to investigate hyperfractionated radiotherapy versus standard radiotherapy. In this retrospective analysis, we assessed the remaining tumour samples from patients in the HIT-SIOP PNET 4 trial (n=136). We assessed the clinical behaviour of the molecularly defined WNT and SHH subgroups, and identified novel independent prognostic markers and models for standard-risk patients with non-WNT/non-SHH disease. Because of the scarcity and low quality of available genomic material, we used a mass spectrometry-minimal methylation classifier assay (MS-MIMIC) to assess methylation subgroup and a molecular inversion probe array to detect genome-wide copy number aberrations. Prognostic biomarkers and models identified were validated in an independent, demographically matched cohort (n=70) of medulloblastoma patients with non-WNT/non-SHH standard-risk disease treated with conventional therapies (maximal surgical resection followed by adjuvant craniospinal irradiation [all patients] and chemotherapy [65 of 70 patients], at UK Children's Cancer and Leukaemia Group and European Society for Paediatric Oncology (SIOPE) associated treatment centres between 1990 and 2014. These samples were analysed by Illumina 450k DNA methylation microarray. HIT-SIOP PNET 4 is registered with ClinicalTrials.gov, number NCT01351870.FindingsWe analysed methylation subgroup, genome-wide copy number aberrations, and mutational features in 136 assessable tumour samples from the HIT-SIOP PNET 4 cohort, representing 40% of the 338 patients in the trial cohort. This cohort of 136 samples consisted of 28 (21%) classified as WNT, 17 (13%) as SHH, and 91 (67%) as non-WNT/non-SHH (we considered Group3 and Group4 medulloblastoma together in our analysis because of their similar molecular and clinical features). Favourable outcomes for WNT tumours were confirmed in patients younger than 16 years, and all relapse events in SHH (four [24%] of 17) occurred in patients with TP53 mutation (TP53mut) or chromosome 17p loss. A novel whole chromosomal aberration signature associated with increased ploidy and multiple non-random whole chromosomal aberrations was identified in 38 (42%) of the 91 samples from patients with non-WNT/non-SHH medulloblastoma in the HIT-SIOP PNET 4 cohort. Biomarkers associated with this whole chromosomal aberration signature (at least two of chromosome 7 gain, chromosome 8 loss, and chromosome 11 loss) predicted favourable prognosis. Patients with non-WNT/non-SHH medulloblastoma could be reclassified by these markers as having favourable-risk or high-risk disease. In patients in the HIT-SIOP PNET4 cohort with non-WNT/non-SHH medulloblastoma, with a median follow-up of 6·7 years (IQR 5·8–8·2), 5-year event-free survival was 100% in the favourable-risk group and 68% (95% CI 57·5–82·7; p=0·00014) in the high-risk group. In the validation cohort, with a median follow-up of 5·6 years (IQR 3·1–8·1), 5-year event-free survival was 94·7% (95% CI 85·2–100) in the favourable-risk group and 58·6% (95% CI 45·1–76·1) in the high-risk group (hazard ratio 9·41, 95% CI 1·25–70·57; p=0·029). Our comprehensive molecular investigation identified subgroup-specific risk models which allowed 69 (51%) of 134 accessible patients from the standard-risk medulloblastoma HIT-SIOP PNET 4 cohort to be assigned to a favourable-risk group.InterpretationWe define a whole chromosomal signature that allows the assignment of non-WNT/non-SHH medulloblastoma patients normally classified as standard-risk into favourable-risk and high-risk categories. In addition to patients younger than 16 years with WNT tumours, patients with non-WNT/non-SHH tumours with our defined whole chromosomal aberration signature and patients with SHH-TP53wild-type tumours should be considered for therapy de-escalation in future biomarker-driven, risk-adapted clinical trials. The remaining subgroups of patients with high-risk medulloblastoma might benefit from more intensive therapies.FundingCancer Research UK, Swedish Childhood Cancer Foundation, French Ministry of Health/French National Cancer Institute, and the German Children's Cancer Foundation.

9 Liquid biopsy in small cell lung cancer (SCLC)

IntroductionAnalysis of circulating cell-free DNA (cfDNA) and circulating tumour cells (CTCs) obtained from the peripheral blood of patients with cancer is a powerful and minimally invasive approach that allows the reconstruction of tumour genomes, with potential as a companion diagnostic for both patient stratification and monitoring. Here, we use cfDNA next-generation sequencing (NGS) analysis to assess and compare circulating tumour DNA (ctDNA) profiles in patients with SCLC.Material and methodsNGS libraries were prepared from cfDNA isolated from the pre-treatment peripheral blood samples from 69 patients and 32 cancer-free controls. Libraries were subjected to wholegenome sequencing (WGS) to establish genome wide copy number aberrations (CNA) as well as targeted mutation analysis of 110 genes frequently altered in SCLC. In addition CellSearch, an epitope dependent enrichment platform was used to enumerate CTCs from a parallel blood sample.Results and discussionsCNA patterns revealed SCLC associated changes including losses on chromosomes 3 p, 5q and 17 p and gains on chromosome 3q and 5 p as well as amplification of MYC in 21/69 (30%) and SOX2 in 36/69 (52%) as well losses on FHIT in 40/69 (58%), RASSFI in 38/69 (55%) and RB1 in 24/69 (35%) patients. CNA metrics enabled detection of tumour associated changes in 58/64 (84%) patients, Targeted NGS detected tumour associated mutations in 59/63 (94%) with TP53 mutations detected in 50 patients (85% of patients with any detectable ctDNA). CNA and somatic mutations were detected in both limited stage (LS-SCLC, confined to 1 hemithorax) and extensive stage (ES-SCLC, with distant metastases) with statistically significant differences seen for CNA metrics and variant allele frequencies of mutations consistent with higher levels of ctDNA in ES-SCLC. However, no significant differences between ES and LS were observed in the mutation patterns with respect to DNA damage repair, RAS and PI3K and transcriptional regulation pathways between the two stages of SCLC. There was considerable overlap between the detection of ctDNA and CTC counts, with cfDNA readouts detecting tumour related changes in 93% and CTCs detected only in 78% of patients.ConclusionWe have established sensitive methods for detecting ctDNA in SCLC and combined with CTC enumeration we have an effective liquid biopsy applicable to 98% of patients in this cohort. Future work will involve utilising this optimised NGS approach in an independent cohort of patients to correlate cfDNA metrics with clinical outcome.

Abstract 5602: Detection of circulating cell-free tumor DNA (ctDNA) in patients with small cell lung cancer (SCLC)

Abstract Introduction: Tumor genomes can be reconstructed from the molecular information obtained from circulating cell-free DNA (cfDNA) and circulating tumor cells (CTCs) obtained from the peripheral blood of patients with cancer. The analysis of cfDNA and CTCs is a minimally invasive approach and represents a powerful research tool, with potential as a companion diagnostic for both patient stratification and monitoring. Here, we use cfDNA next-generation sequencing (NGS) analysis to assess and compare ctDNA profiles in patients with SCLC. Methods: Whole genome sequencing (WGS) libraries were prepared from cfDNA isolated from the pre-treatment peripheral blood samples from 69 patients and 32 cancer-free controls. Libraries were subjected to WGS to establish genome wide copy number aberrations (CNA) as well as targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated from CNA such as Percent Genome Altered (PGA; percentage of genomic regions altered), Z-score (measure of standard deviation) and Moran's I (measure of spatial autocorrelation). In addition CellSearch®, an epitope dependent enrichment platform was used to enumerate CTCs from a parallel blood sample. Results: An examination of CNA patterns revealed SCLC associated changes such as losses on chromosomes 3p, 5q and 17p and gains on chromosome 3q and 5p as well as amplification of MYC in 21/69 (30%) and SOX2 in 36/69 (52%) as well losses on FHIT in 40/69 (58%), RASSFI in 38/69(55%) and RB1 in 24/69 (35%) patients. A combination of three CNA metrics enabled detection of tumor associated changes in 58/64 (84%) patients, with Moran's I emerging as the most sensitive CNA metric for detecting ctDNA. Targeted NGS detected tumor associated mutations in 60/64 (94%) with TP53 mutations detected in 50 patients (83% of patients with any detectable ctDNA). Somatic mutations and CNA were detected in both limited stage SCLC (LS-SCLC, confined to 1 hemithorax) and extensive stage (ES-SCLC, with distant metastases) with statistically significant differences seen for CNA metrics and variant allele frequencies of mutations consistent with higher levels of ctDNA in ES-SCLC. However, no significant differences between ES and LS were observed in the mutation patterns with respect to DNA damage repair, RAS and PI3K and transcriptional regulation pathways between the two stages of SCLC. There was considerable overlap between the detection of ctDNA and CTC counts, with cfDNA NGS readouts detecting tumor related changes in 96% of patients and CTCs were detected only in 76% of patients. Conclusion: We have established sensitive methods for detecting ctDNA in cfDNA and combined with CTC enumeration we have an effective liquid biopsy for 98% of patients (96% in LS and 100% in ES) in this cohort. Future work will involve utilising this optimised NGS approach in an independent cohort of patients to correlate cfDNA metrics with clinical outcome. Citation Format: Sumitra Mohan, Victoria Foy, Hui Sun Leong, Pietà G. Schofield, Mahmood Ayub, Nigel K. Smith, Sudhakar Sahoo, Chang Sik-Kim, Lynsey Priest, Mathew Carter, Hedley T. Carr, Crispin Miller, Corinne Finn-Faivre, Fiona Blackhall, Dominic G. Rothwell, Caroline Dive, Gerard Brady. Detection of circulating cell-free tumor DNA (ctDNA) in patients with small cell lung cancer (SCLC) [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 5602.