Recurrent Copy Number Alterations Research Articles

The genomic landscape of Vk*MYC myeloma highlights shared pathways of transformation between mice and humans

Multiple myeloma (MM) is a heterogeneous disease characterized by frequent MYC translocations. Sporadic MYC activation in the germinal center of genetically engineered Vk*MYC mice is sufficient to induce plasma cell tumors in which a variety of secondary mutations are spontaneously acquired and selected over time. Analysis of 119 Vk*MYC myeloma reveals recurrent copy number alterations, structural variations, chromothripsis, driver mutations, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational activity, and a progressive decrease in immunoglobulin transcription that inversely correlates with proliferation. Moreover, we identify frequent insertional mutagenesis by endogenous retro-elements as a murine specific mechanism to activate NF-kB and IL6 signaling pathways shared with human MM. Despite the increased genomic complexity associated with progression, advanced tumors remain dependent on MYC. In summary, here we credential the Vk*MYC mouse as a unique resource to explore MM genomic evolution and describe a fully annotated collection of diverse and immortalized murine MM tumors.

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-06.

Single nucleotide polymorphism (SNP) chromosomal microarray as a diagnostic tool for mucinous tubular and spindle cell carcinoma: A validation study

Mucinous tubular and spindle cell carcinoma (MTSCC) shows significant overlap with papillary renal cell carcinoma (PRCC), and harbor recurrent copy-number alterations (CNA).We evaluated 16 RCC with features suggestive of MTSCC using chromosomal microarrays.The cohort was comprised of 8 females and males, each, with an age range of 33–79 years (median, 59), and a tumor size range of 3.4–15.5 cm (median, 5.0). Half the tumors were high-grade (8/16, 50%) with features such as necrosis, marked cytologic atypia, and sarcomatoid differentiation, and 5/16 (31%) were high stage (≥pT3a). Three (of 16, 19%) cases had a predominant (>95%) spindle cell component, whereas 5/16 (31%) were composed of a predominant (>95%) epithelial component. Most cases (12/16, 75%) exhibited a myxoid background and/or extravasated mucin, at least focally.Twelve (of 16, 75%) cases demonstrated CNA diagnostic of MTSCC (losses of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22). In addition, 2 high-grade tumors showed loss of CDKN2A/B, and gain of 1q, respectively, both of which are associated with aggressive behavior. Three (of 16, 19%) cases, demonstrated nonspecific CNA, and did not meet diagnostic criteria for established RCC subtypes. One (of 16, 6%) low-grade epithelial predominant tumor (biopsy) demonstrated characteristic gains of 7, 17, and loss of Y, diagnostic of PRCC.MTSCC can be a morphologically heterogenous tumor. Our study validates the detection of characteristic chromosomal CNA for diagnostic use that may be useful in challenging cases with unusual spindle cell or epithelial predominant features, as well as in high-grade tumors.

Abstract 6941: Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA)

Abstract Retinoblastoma is a pediatric retinal cancer that initiates in response to biallelic RB1 loss or MYCN amplification in a cone precursor cell of origin. After initial emergence, RB1-/- retinoblastomas progress to more aggressive forms by accumulation of single nucleotide variants (SNVs) and highly recurrent somatic copy number alterations including 1q+, 2p+, 6p+ and 16q- (RB SCNAs), resulting in heterogeneous mixtures of subclonal cell populations. SCNAs are enriched in highly evolved and aggressive tumor subtypes and show prognostic value in retinoblastoma liquid biopsies. However, the transcriptomic effects of retinoblastoma SCNAs are poorly understood. Single cell methods may enable comparison of tumor subclones within emerging retinoblastoma tumors and thereby provide insight into tumor progression. We retrieved single cell RNA sequencing data for 25 retinoblastoma tumors and inferred SCNAs and subclone phylogenies from scRNAseq information using a recently published tool, Numbat. We identified tumors with clonal variation in RB SCNAs and excluded samples with poor transcriptome quality or excessive additional SCNAs. We proceeded with analysis of 11 tumors with subclonal heterogeneity for an SCNA of interest, including 10 with 1q+, eight with 2p+, four with 6p+, and six with 16q-. After removal of nontumor cells, we defined cell clusters, cluster marker genes, and cell cycle phase gene signatures, and we compared cell state distributions for subclones with an acquired SCNA relative to their immediate antecedents. These analyses revealed recurrently discriminated transcriptomic clusters corresponding to six discrete cell cycle states (post-mitotic G1, G1, G1/S, S, S/G2, G2/M) and variable cell stress states, including a cluster with hypoxia and heat shock related marker gene expression in all samples. In each tumor, SCNA-defined tumor subclones had a distinct cell state distribution. Tumor subclones with acquired 1q+ were enriched in G2 states and depleted in G1 states. Subclones with 2p+ were enriched in G2 states. Subclones with 6p+ were enriched in S phase states. Subclones with 16q- were diminished in post-mitotic cell states. Tumor clones with 1q+ and 16q- were most strongly enriched for G2 phase cells. Such 1q+/16q- tumor cells were significantly differentially distributed in three tumor replicates. Tumor clones with other RB SCNAs were enriched over diploid clones in proliferating G2 cells without SCNA specific effects. To identify candidate drivers of SCNA-specific transcriptomic effects we measured RB SCNA clonal variation in gene expression within and across transcriptomic states. We identified recurrently differentially expressed in-segment genes in 1q+, 2p+, 6p+, and 16q-, as well as out-of-segment genes. Finally, we propose molecular mechanisms underlying SCNA recurrence that may enable tumor evolution in retinoblastoma. Citation Format: Kevin Stachelek, David E. Cobrinik. Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6941.

Abstract 869: Single-cell DNA replication dynamics in genomically unstable cancers

Abstract Background: DNA replication and cell cycle regulation are frequently disrupted as part of a cancer’s progression toward uncontrolled proliferation, generating somatic copy number alterations (CNAs) and producing intratumoral heterogeneity that drives subsequent evolution. Structural variation and CNAs have been shown to impact epigenetic and chromatin states, but our ability to assess their impact on DNA replication timing (RT) and cell proliferation rates remains limited. Single-cell whole genome sequencing (scWGS) is a powerful method for studying clonal heterogeneity and CNAs, and has the potential to provide greater insight into DNA replication dynamics in aneuploid populations. However, computational identification of S-phase cells and distinguishing inherited somatic CNAs from transient DNA replication changes remain challenging. Methods: We present a new method, PERT, which uses a Bayesian framework to model read depth as a combination of somatic copy number, replication, and sequencing bias, enabling estimation of DNA replication profiles and cell cycle phase from scWGS data. Unlike previous approaches, PERT provides unbiased estimates of RT and cell cycle phase which allows for analysis of previously uncharacterized cell types using any scWGS platform. These unique properties enable PERT to perform novel analysis such as estimating clone-specific proliferation rates and studying the interplay between RT and somatic CNAs during tumor evolution. We applied PERT to a cohort of >50,000 scWGS cells obtained from a collection of genomically unstable breast and ovarian cell lines, xenografts and primary cancer tissues. Results: Clone RT profiles correlated with future copy number changes in serially passaged cell lines. Cell type was the strongest determinant of RT heterogeneity, while whole genome doubling (WGD) and mutational signature had weaker RT associations but were associated with accumulation of late S-phase cells. Recurrent CNAs affecting chromosome X had striking impact on RT, with loss of the inactive X allele shifting replication earlier, and loss of inactive Xq resulting in reactivation of Xp. Analysis of time series xenografts illustrated that cell cycle distributions approximate clone proliferation. This relationship enabled us to observe that highly proliferative clones were the most chemosensitive in cisplatin-treated xenografts and, separately, present novel evidence that WGD leads to slower proliferation. Conclusions: Our analysis implicates cell type as the strongest determinant of RT with chrX being the locus of highest RT variation due to X-inactivation. Separately, quantification of S-phase cells enables interrogation of the on- and off-treatment fitness of genetically distinct subclones. This work leads to better understanding of how DNA replication dynamics drive and are further modulated by genomic instability. Citation Format: Adam C. Weiner, Marc Williams, Hongyu Shi, Ignacio Vazquez-Garcia, Sohrab Salehi, Nicole Rusk, Sohrab P. Shah, Andrew McPherson. Single-cell DNA replication dynamics in genomically unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 869.

TCGAnalyzeR: An Online Pan-Cancer Tool for Integrative Visualization of Molecular and Clinical Data of Cancer Patients for Cohort and Associated Gene Discovery.

For humans, the parallel processing capability of visual recognition allows for faster comprehension of complex scenes and patterns. This is essential, especially for clinicians interpreting big data for whom the visualization tools play an even more vital role in transforming raw big data into clinical decision making by managing the inherent complexity and monitoring patterns interactively in real time. The Cancer Genome Atlas (TCGA) database's size and data variety challenge the effective utilization of this valuable resource by clinicians and biologists. We re-analyzed the five molecular data types, i.e., mutation, transcriptome profile, copy number variation, miRNA, and methylation data, of ~11,000 cancer patients with all 33 cancer types and integrated the existing TCGA patient cohorts from the literature into a free and efficient web application: TCGAnalyzeR. TCGAnalyzeR provides an integrative visualization of pre-analyzed TCGA data with several novel modules: (i) simple nucleotide variations with driver prediction; (ii) recurrent copy number alterations; (iii) differential expression in tumor versus normal, with pathway and the survival analysis; (iv) TCGA clinical data including metastasis and survival analysis; (v) external subcohorts from the literature, curatedTCGAData, and BiocOncoTK R packages; (vi) internal patient clusters determined using an iClusterPlus R package or signature-based expression analysis of five molecular data types. TCGAnalyzeR integrated the multi-omics, pan-cancer TCGA with ~120 subcohorts from the literature along with clipboard panels, thus allowing users to create their own subcohorts, compare against existing external subcohorts (MSI, Immune, PAM50, Triple Negative, IDH1, miRNA, metastasis, etc.) along with our internal patient clusters, and visualize cohort-centric or gene-centric results interactively using TCGAnalyzeR.

Multiplex Detection of Copy Number Alterations in Multiple Myeloma Including Emerging Therapeutic Targets By Digitalmlpa

Recent advances in immunotherapy have highlighted that in multiple myeloma (MM), besides risk-determining copy number alterations (CNAs) there is a need for investigation of CNAs in CAR-T cell therapy-related targets (e.g. BCMA, GPRC5D, FcRH5). DigitalMLPA technology has been successfully used for characterization of recurrent CNAs on chromosome arms 1p, 1q, 13q and 17p in newly diagnosed and relapsed MM cases, as well as in cancer cell lines of MM origin. Adding probes aimed at CAR-T cell therapy-related target genes complements the already included predictive target probes ( IKZF1/3, IRF4, MYC, RPL5, SLAMF7 and BRAF V600E point mutation) in SALSA® digitalMLPA™ Probemix D006 Multiple Myeloma. In addition, due to a large overlap of affected genomic regions between hematologic neoplasms, we aimed to demonstrate usefulness of this multiplex assay not only on cell lines of MM origin, but also of myeloid and lymphoid lineages. Methods. D006 Multiple Myeloma digitalMLPA probemix contains (i) probes targeting chromosomal arms with recurrent CNAs, including 1p (33 probes), 1q (29 probes), 13q (23 probes), 17p (20 probes); (ii) probes for promising predictive targets such as BCMA, CRBN, GPRC5D, FcRH5, IKZF1/3, IRF4, MYC, RPL5, SLAMF7 - 2-4 probes per gene, and BRAF V600E mutation-specific probe; (iii) 278 probes for subtelomeric, pericentromeric and middle regions of chromosomal arms for all chromosomes; and (iv) control probes for quality control and troubleshooting. DigitalMLPA reactions were performed with 20-40 ng DNA from 29 cancer cell lines of lymphoid and myeloid lineage, 16 of which were of MM origin. Commercial blood-derived genomic DNA samples from healthy individuals were used as references for data normalization using data analysis software Coffalyser digitalMLPA™. Results. Losses of 1p were among the most frequent CNAs in MM cell lines (n=13) and were mainly local (1p21.1-p22.1), including homozygous deletion of CDKN2C in MM cell lines co-occurring with TENT5C subclonal loss in 2 of those. 13q loss (encompassing predominantly the whole arm) was detected in 13 MM cell lines. 1q and 17p losses were present in 12 MM cell lines. One MM cell line showed loss of BCMA (LOPRA-1) and one of CRBN (EJM), 6 had loss of GPRC5D, 9 had MYC gain. None of the MM cell lines carried the BRAF V600E mutation. In the EOL-1 myeloid lineage cell line, trisomy of chromosomes 4, 6, 8 and 19 were correctly identified by increased ratios for all probes covering these chromosomes: 24, 27, 20 and 11 probes respectively; and in the HG-3 lymphoid lineage cell line 11q25 gain and 13q loss was detected. Examples of CNAs detected by D006 Multiple Myeloma probemix are described in Table 1. Copy number ratios of predictive target genes in MM cell lines are shown in Table 2. Ratios between 0.7-1.3 (green cells) indicate normal copy number, ratios below 0.7-1.3 range indicate copy number loss (red cells) and higher ratios indicate gains (blue cells). CNAs detected by digitalMLPA were highly concordant with those reported in public databases. Conclusions. digitalMLPA technology is well suited for multiplex CNA detection of routinely analyzed genomic regions in MM and provides a unique opportunity to research molecular genetic markers of emerging significance in MM in the same reaction. In addition, this MM assay also gives the possibility to analyze gross CNAs in other hematopoietic neoplasms with overlapping affected regions. Input DNA requirements that are a fraction of current WGS, simple protocol and possibility of combining of digitalMLPA libraries with other NGS libraries make D006 Multiple Myeloma digitalMLPA probemix a reliable, cost-effective and robust method to detect well-established and emerging CNAs.

Whole Genome and Transcriptome Sequencing of 21 Paired Chronic and Blast Phase CML Cases: Acquisition of Genomic Alterations, Changes in the Transcriptomic Profiles and Occurrence of B-Cell Receptor Rearrangements

Background: Since the introduction of tyrosine kinase inhibitor therapy survival has substantially improved in CML. However, a subset of patients become resistant and progress to blast phase. Still the underlying genomic changes are not completely understood. Cohort & Methods: We studied 21 CML patients with paired sample material available from diagnosis in chronic phase (CP) and from blast phase (BP). All patients received a tyrosine kinase inhibitor starting at diagnosis of CML CP. Samples were analyzed by cytomorphology, chromosome banding analysis, WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp). B-cell receptor (BCR) rearrangements were analyzed with MiXCR software. The transcriptomic phenotype (TP) of each CP and BP sample was estimated by mapping their profiles to representative cases diagnosed with B-ALL, T-ALL, MDS, CMML, AML and CML CP. Results: The median time between diagnosis of CP and BP was 14.7 months (range: 4.7 - 137 months). While 18/21 CML in CP showed a TP corresponding to CML CP, one case each showed a TP corresponding to CMML, AML and B-ALL, respectively, although bone marrow blast counts were 6.5%, 5.5% and 5.5%, respectively. Of note, the case with the CMML-TP harbored a p190 BCR:: ABL1 isoform and also resembled CMML in bone marrow morphology. These three cases progressed to BP after 5.8, 5.3, and 8.9 months. The TP in BP corresponded to B-ALL in 13 cases, while 4, 3, and 1 case showed an AML-, CML CP- and MDS-TP, respectively (figure). In CP recurrent genomic alterations in addition to the BCR:: ABL1 fusion were mutations in ASXL1 (n=5), DNMT3A, TET2, RUNX1, and MGA (2 cases each). In BP the genomic landscape was more complex. Additional genetic alterations compared to CP were detected in all cases. Recurrently gained mutations in BP were mutations in WT1, BCOR, and SETD2 (2 cases each). The gain of copy number alterations (CNA) was more frequent. Recurrent CNA were: loss 9p (n=6), gain Ph-chromosome (n=4), loss 7q (n=3), gain 21q (n=3), gain 1q, loss 3q, gain 8q, loss 12p, and loss 13q in 2 patients each. Only three balanced structural variants were gained: a MECOM-, a NUP98:: HHEX- and a KMT2A:: MLLT3-rearrangement. Further, in 11/21 BP resistance mutations within the BCR:: ABL1 kinase domain were detected. The variant allele frequency (VAF) of RUNX1 mutations increased in BP in both cases with RUNX1 mutation present in CP (5% to 42%, time to BP: 4.7 months; 8% to 59%, time to BP: 11.7 months). DNMT3A and MGA mutations were about stable (VAF CP/BP: DNMT3A: 48%/37%, 33%/46%; MGA: 52%/36%, 40%/52%), while TET2 mutations were stable in one case (46%/49%) and decreased in the second (57%/27%). ASXL1 mutations were stable in 2 cases with an AML-TP in BP and lost in 3/5 cases with a B-ALL-TP in BP. BP with a lymphoid TP differed substantially with respect to the genomic profile from BP with myeloid TP. WTS data revealed a clonal BCR rearrangement in 8/13 BP with B-ALL-TP, of which 4 rearrangements are predicted to be productive. Further, CDKN2A/B deletions, IKZF1 deletions, and SETD2 mutations were only found in BP with B-ALL-TP. Thus, CML in BP with a B-ALL-TP showed a genomic landscape similar to B-ALL with BCR:: ABL1-rearrangement. In contrast CML BP cases with an AML-TP (n=4) had gained either rearrangements typically found in AML such as a KMT2A:: MLLT3 rearrangement and a MECOM rearrangement or acquired mutations in RUNX1, BCOR and WT1, or a CN-LOH 11p in combination with a WT1 VAF increase from 51% to 94%. Cases showing still a CML CP-TP (n=3) in BP acquired rather few additional genetic abnormalities (mutations in BCOR, WT1, CTCF; NUP98:: HHEX-rearrangement, gain of Ph-chromosome, loss of 11p, 17q), which were more myeloid in appearance. Conclusions: 1) Extensive genetic profiling indicated a substantial clonal evolution in the progression from CP to BP CML including loss of ASXL1 mutations, expansion of RUNX1 mutated clones, multiple CNA, and the frequent acquisition of a BCR rearrangement in BP with a transcriptomic phenotype resembling B-ALL. 2) A subset of CML cases in CP already showed a transcriptomic phenotype resembling acute leukemia indicating a rapid progression to BP. 3) The presence of a RUNX1 mutated subclone or a clonal BCR rearrangement seem to represent a warning signal in CML CP. 4) The prognostic validity of an extended genetic profiling including ASXL1, RUNX1, BCR rearrangement, and transcriptional signature in CML CP needs to be assessed in forthcoming prospective studies.

Pediatric Sertoli-Leydig Cell Tumors of the Ovary: An Integrated Study of Clinicopathological Features, Pan-cancer-Targeted Next-generation Sequencing and Chromosomal Microarray Analysis From a Single Institution.

Sertoli-Leydig cell tumors (SLCTs) are currently classified into 3 molecular subtypes: DICER1 -mutant (younger patient age), FOXL2 -mutant, and DICER1/FOXL2 -wildtype. However, it is not clear whether all pediatric SLCTs are DICER1 -mutant molecular subtypes and whether other molecular genetic aberrations besides DICER1 are involved in the pathogenesis and prognosis of these tumors. We studied comprehensive data for 8 cases of pediatric SLCTs, including clinicopathological features, pan-cancer-targeted next-generation sequencing/OncoKids panel, and chromosomal microarray analysis, to further analyze the correlation among clinicopathological features, molecular genetic aberrations, and prognosis. The ages of the patients ranged from 4 to 16 years (median, 14y). Seven cases were moderately differentiated, and one was poorly differentiated with heterologous mesenchymal elements. Two cases had heterologous epithelium or retiform elements. Follow-up was available for all 8 patients (median, 49.5mo). Seven patients were alive without evidence of recurrence or metastasis, and only case 5 developed metastases (synchronous bilateral pulmonary tumors with rhabdomyosarcomatous differentiation). All 8 tumors were found to harbor somatic hotspot DICER1 mutations, and 5 patients carried germline DICER1 mutations (2 of them had the phenotype of DICER1 syndrome). Together with recent studies, the DICER1 mutation frequency is 100% in pediatric SLCTs (n=27, age≤16y). Copy number alterations were detected in 3 tumors; the only recurrent copy number alterations was the gain of whole chromosome 6 in case 5 and case 8. This is the first report describing clinicopathological features and molecular alterations in pediatric SLCTs. Our results demonstrate that all pediatric SLCTs belong to the DICER1 -mutant molecular subtype, highlighting that somatic hotspot DICER1 mutation detection has high sensitivity (100%) for the auxiliary diagnosis of pediatric SLCTs (age ≤16y). Some pediatric SLCTs harbor molecular genetic aberrations other than DICER1 mutation, and their significance needs further study.

Recurrent Somatic Copy Number Alterations and Their Association with Oncogene Expression Levels in High-Grade Ovarian Serous Carcinoma.

Somatic copy number alterations (SCNAs) are frequently observed in high-grade ovarian serous carcinoma (HGOSC). However, their impact on gene expression levels has not been systematically assessed. In this study, we explored the relationship between recurrent SCNA and gene expression using The Cancer Genome Atlas Pan Cancer dataset (OSC, TCGA, PanCancer Atlas) to identify cancer-related genes in HGOSC. We then investigated any association between highly correlated cancer genes and clinicopathological parameters, including age of diagnosis, disease stage, overall survival (OS), and progression-free survival (PFS). A total of 772 genes with recurrent SCNAs were observed. SCNA and mRNA expression levels were highly correlated for 274 genes; 24 genes were classified as a Tier 1 gene in the Cancer Gene Census in the Catalogue of Somatic Mutations in Cancer (CGC-COSMIC). Of these, 11 Tier 1 genes had highly correlated SCNA and mRNA expression levels: TBL1XR1, PIK3CA, UBR5, EIF3E, RAD21, EXT1, RECQL4, KRAS, PRKACA, BRD4, and TPM4. There was no association between gene amplification and disease stage or PFS. EIF3E, RAD21, and EXT1 were more frequently amplified in younger patients, specifically those under the age of 55 years. Patients with tumors carrying PRKACA, BRD4, or TPM4 amplification were associated with a significantly shorter OS. RECQL4 amplification was more frequent in younger patients, and tumors with this amplification were associated with a significantly better OS.

Impact of Rare Structural Variant Events in Newly Diagnosed Multiple Myeloma.

Whole-genome sequencing (WGS) of patients with newly diagnosed multiple myeloma (NDMM) has shown recurrent structural variant (SV) involvement in distinct regions of the genome (i.e., hotspots) and causing recurrent copy-number alterations. Together with canonical immunoglobulin translocations, these SVs are recognized as "recurrent SVs." More than half of SVs were not involved in recurrent events. The significance of these "rare SVs" has not been previously examined. In this study, we utilize 752 WGS and 591 RNA sequencing data from patients with NDMM to determine the role of rare SVs in myeloma pathogenesis. Ninety-four percent of patients harbored at least one rare SV event. Rare SVs showed an SV class-specific enrichment within genes and superenhancers associated with outlier gene expression. Furthermore, known myeloma driver genes recurrently impacted by point mutations were dysregulated by rare SVs. Overall, we demonstrate the association of rare SVs with aberrant gene expression supporting a potential driver role in myeloma pathogenesis.

DNA cytosine deamination is associated with recurrent Somatic Copy Number Alterations in stomach adenocarcinoma.

Stomach Adenocarcinoma (STAD) is a leading cause of death worldwide. Somatic Copy Number Alterations (SCNAs), which result in Homologous recombination (HR) deficiency in double-strand break repair, are associated with the progression of STAD. However, the landscape of frequent breakpoints of SCNAs (hotspots) and their functional impacts remain poorly understood. In this study, we aimed to explore the frequency and impact of these hotspots in 332 STAD patients and 1,043 cancer cells using data from the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE). We studied the rates of DSB (Double-Strand Breaks) loci in STAD patients by employing the Non-Homogeneous Poisson Distribution (λ), based on which we identified 145 DSB-hotspots with genes affected. We further verified DNA cytosine deamination as a critical process underlying the burden of DSB in STAD. Finally, we illustrated the clinical impact of the significant biological processes. Our findings highlighted the relationship between DNA cytosine deamination and SCNA in cancer was associated with recurrent Somatic Copy Number Alterations in STAD.

OR01-03 DNA Copy Number Profiles In Recurrent And Stable Non-functioning Pituitary Adenomas

Abstract Disclosure: G. Johannsson: None. M. Persson: None. S. Schlaffer: None. A. Fehr: None. C. Örndal: Employee; Self; Unilabs AB. W. Seager: None. M. Buchfelder: None. G. Stenman: None. D.S. Olsson: Consulting Fee; Self; Ipsen, Novo Nordisk, Sandoz. Grant Recipient; Self; Pfizer Global R&D. Employee; Self; AstraZeneca. Background: Residual tumors are common after primary surgery in patients with non-functioning pituitary adenoma (NFPA), 30-50% of these will experience tumor progression. Tumor control is of vital importance for patient outcome since tumor progression is strongly associated with excess morbidity and mortality. The aim of the study was to explore new potential biomarkers of tumor progression by studying the DNA copy number profiles of NFPAs with either a stable residual tumor behavior or marked tumor progression. Methods: In this case-controlled study, 72 patients operated for NFPA were selected. The progression group (n = 39) included patients [age at diagnosis (mean ± SD) 41.3±14.0 years] with marked tumor progression (30 patients required ≥3 surgical procedures). The stable group (n = 33; 61.1±10.2 years) had either a stable residual tumor without evidence of tumor progression during follow-up (n = 25) or no recurrence during follow-up (n = 8). The mean follow-up time in the stable group was 6.2 years (range 3.6-15 years). ArrayCGH analysis was performed on DNAs isolated from fresh frozen tumor tissue using the Human Genome CGH Microarray 244K and 180K oligonucleotide arrays. Copy number alterations (CNAs) were validated using FISH on formalin-fixed paraffin-embedded sections. Results: CNAs were identified in 29 tumors (40%). A total of 183 CNAs were detected in the 72 tumors, including 75 segmental alterations and 108 gains and losses of whole chromosomes and chromosome arms. The average number of CNAs per tumor was 2.5 (SD 5.9). CNAs were more common in the progression group compared to the stable group (Mean no. of CNAs 3.7 vs 1.2; P = 0.060). Twenty-four tumors (72%) in the stable group had normal profiles whereas only 19 (49%) in the progression group lacked CNAs. Eighteen CNAs were recurrent, defined as minimal common regions of deletion or gain in ≥4 tumors. The most frequently lost region was 11q21-q23.3 (n = 10). Deletions involving 10q23.21-q23.3 were seen in both the progression group (n = 5) and the stable group (n = 2) and included an approximately 2.8 Mb minimal common region harboring the tumor suppressor gene PTEN. The most frequent gains involved whole chromosomes 5 (n = 8), 7 (n = 10) and 12 (n = 7). Recurrent CNAs involving 10q (PTEN probe), 11q (MAML2 probe), and chromosomes 5 and 7 were confirmed using FISH. Conclusion: In this explorative study, we found a higher number of CNAs in NFPAs with marked tumor progression compared to stable adenomas without the need for reintervention during the long-term follow-up. Interestingly, segmental losses of chromosome 10q23.21-q23.3, including the tumor suppressor gene PTEN, were overrepresented in patients with marked tumor progression. Presentation: Thursday, June 15, 2023

Abstract A008: Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis

Abstract Wilms Tumor (WT), or nephroblastoma, is the most common pediatric renal cancer. The most prevalent epigenetic alterations in WT occur on chromosome 11p15, accounting for at least two-thirds of WT. These same changes on chromosome 11p15 also cause Beckwith-Wiedemann Syndrome (BWS). BWS is a pediatric overgrowth disorder affecting numerous tissues, including the kidney, and up to 28% of patients with BWS develop cancer. As patients with BWS have the same 11p15 epigenetic and structural changes that arise in some sporadic WT, BWS-WT provides a unique opportunity to investigate the specific molecular drivers by which 11p15 alterations induce oncogenesis. Using clinical data and patient samples from the International BWS Registry and Biorepository, we used whole exome sequencing, methylation array, and RNA-sequencing technologies to examine the genomic and epigenomic landscape of BWS-WT compared to control kidney samples. We found several large-scale recurrent copy number alterations (CNAs) in the BWS-WT cohort across cancer-associated chr1p36, WT-associated chr16q, and at a novel chr15q site. Two BWS-WT presented with an above average number of genomic alterations; one had mutations in telomere maintenance genes, and the other had mutations in mismatch repair genes. Recurrent mutations in genes associated with sporadic WT, such as TP53, CTNNB1, and WT1 were not present in BWS samples. We found other single nucleotide variants (SNVs) in BCORL1, ASXL1, ATM and AXL genes in the BWS cohort. We further compared the BWS data to publicly available non-syndromic/nonBWS data, stratified based on 11p15 status (normal or altered), and the molecular signaling differences were investigated. Differential methylation studies showed enrichment of biological processes related to the Wnt signaling, integrin signaling, insulin receptor signaling, and BMP signaling pathways in BWS-WT. Altered-11p15 nonBWS-WT showed enrichment of cell cycle and DNA damage checkpoint processes, as well as TORC1/TOR signaling and Wnt signaling pathways, as assayed by differential methylation. RNA sequencing data showed differential gene expression of Wnt signaling along with other distinct pathways including Notch, BMP, PPAR and, NIK/NF-kappaB signaling pathways in BWS-WT. Based on an interactome study, CTNNB1 exhibited the broadest range of interactions, and its overexpression was observed in BWS-WT, although not specifically mutated in the BWS samples. Weighted gene coexpression network analysis (WGCNA) showed that cell cycle-related processes and DNA repair mechanisms are shared features of WT with alterations in chromosome 11p15. While BWS-WT predisposition and 11p15 alterations in nonBWS-WT are well-established, this study is the first to focus on stratifying tumors by this characteristic. Data presented herein suggest a mechanism in which 11p15-altered genomes predispose cells to oncogenic transitions. Further investigation of our findings may serve to identify novel diagnostic or therapeutic targets in WT oncogenesis. Citation Format: Snehal Dinkar Nirgude, Natali Sobel Naveh, Sanam Kavai, Jennifer Kalish. Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A008.

Abstract A017: Molecular analysis of matched PIN, invasive prostate cancer, and adjacent normal prostate tissue samples reveal distinct transcriptional signatures and clonal relationships

Abstract Background: Prostate cancer (PCa) is highly heterogeneous, and while some tumors remain indolent throughout a patient's lifetime, others are highly aggressive, leading to significant morbidity and death. The current methods for PCa diagnosis and prognosis are highly suboptimal, resulting in over-diagnosis and overtreatment of many clinically insignificant tumors. To understand the nature of PCa heterogeneity, we conducted comprehensive genomic analyses on matched samples of adjacent normal prostate tissue, prostate intraepithelial neoplasia (PIN), and invasive PCa. Methods: To investigate the changes cells undergo during PCa development and progression, we analyzed genomic and transcriptomic alterations in 92 adjacent normal, 80 PIN, and 99 PCa samples from 34 patients. For each sample type, both epithelial and stromal components were analyzed separately using laser capture microdissection of formalin fixed paraffin embedded tissue. Transcriptomic analysis was performed using smart3-SEQ and copy number analysis was performed by low-pass whole genome sequencing (WGS). The samples were analyzed as three separate data sets. Results: We studied the spectrum of molecular changes present in both epithelial and stromal samples. A negative-binomial regression model was used to identify differentially expressed genes between adjacent normal prostate tissue and invasive PCa in epithelium and stroma separately using the discovery data set. The resulting gene lists were used to perform non-negative matrix factorization clustering of all samples in the three datasets separately. This analysis identified two epithelial and two stromal clusters with distinct RNA expression profiles in all three data sets. For invasive PCa samples, all except three epithelial samples and five stromal samples clustered in the ‘invasive-like’ clusters. Similarly for adjacent normal samples, all except three of the epithelial and six of the stromal samples clustered as ‘normal-like’ across the three datasets. For PIN samples, 54% of epithelial and 62% of stromal samples classified as ‘normal-like’. We performed phylogenetic analysis of WGS data and identified PIN lesions with and without clear clonal relation to invasive PCa lesions. Recurrent copy number variations were identified in both PIN and PCa lesions. Conclusion. We identified two distinct epithelial and two distinct stromal expression clusters with ‘invasive-like’ and ‘normal-like’ signatures. WGS analysis revealed recurrent copy number alterations in PIN and PCa lesions, and evidence of clonal relationships between PIN and invasive PCa. These studies provide new insight into PIN biology and the relationship between PIN and invasive PCa. Citation Format: Siri H. Strand, Okyaz Eminaga, Sujay Vennam, Chunfang Zhu, Jason Wang, Sushama Varma, Rosalie Nolley, Christian Kunder, Jonathan Pollack, Andreas Roeder, Karina D. Sorensen, James D. Brooks, Robert B. West. Molecular analysis of matched PIN, invasive prostate cancer, and adjacent normal prostate tissue samples reveal distinct transcriptional signatures and clonal relationships [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A017.

Mitogen-induced defective mitosis transforms neural progenitor cells.

Chromosome instability (CIN) with recurrent copy number alterations is a feature of many solid tumors, including glioblastoma (GBM), yet the genes that regulate cell division are rarely mutated in cancers. Here, we show that the brain-abundant mitogen, platelet-derived growth factor-A (PDGFA) fails to induce the expression of kinetochore and spindle assembly checkpoint genes leading to defective mitosis in neural progenitor cells (NPCs). Using a recently reported in vitro model of the initiation of high-grade gliomas from murine NPCs, we investigated the immediate effects of PDGFA exposure on the nuclear and mitotic phenotypes and patterns of gene and protein expression in NPCs, a putative GBM cell of origin. NPCs divided abnormally in defined media containing PDGFA with P53-dependent effects. In wild-type cells, defective mitosis was associated with P53 activation and cell death, but in some null cells, defective mitosis was tolerated. Surviving cells had unstable genomes and proliferated in the presence of PDGFA accumulating random and clonal chromosomal rearrangements. The outcome of this process was a population of tumorigenic NPCs with recurrent gains and losses of chromosomal regions that were syntenic to those recurrently gained and lost in human GBM. By stimulating proliferation without setting the stage for successful mitosis, PDGFA-transformed NPCs lacking P53 function. Our work describes a mechanism of transformation of NPCs by a brain-associated mitogen, raising the possibility that the unique genomic architecture of GBM is an adaptation to defective mitosis that ensures the survival of affected cells.

SOX11 regulates SWI/SNF complex components as member of the adrenergic neuroblastoma core regulatory circuitry

The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.

Clinicopathologic and Molecular Characteristics of Epstein-Barr Virus–Associated Smooth Muscle Tumor Compared With Those of Leiomyoma and Leiomyosarcoma

Epstein-Barr virus (EBV)–associated smooth muscle tumors (EBV-SMTs) are rare smooth muscle neoplasms exclusively associated with immunosuppression, such as in patients with HIV/AIDS, posttransplant, and congenital immunodeficiency. However, the genomic landscape of EBV-SMTs is poorly understood. Leiomyosarcomas harbor genomic instability and multiple recurrent DNA copy number alterations, whereas leiomyomas lack such changes. Thus, this study aimed to fill this knowledge gap by characterizing copy number alterations in EBV-SMTs and correlating this information with clinicopathologic characteristics. Our study investigated and compared the pathologic characteristics and copy number profiles of 9 EBV-SMTs (from 7 post-transplant and AIDS patients), 6 leiomyomas, and 7 leiomyosarcomas, using chromosomal microarray platforms. Our results showed a lower copy number alteration burden in EBV-SMTs and leiomyoma than in leiomyosarcoma. This contrast in the molecular profile between EBV-SMTs and leiomyosarcoma is concordant with the different clinical behaviors and pathologic characteristics exhibited by these tumors. Despite having an overall copy number alteration profile closer to leiomyoma, recurrent copy number gain of oncogenes, such as RUNX1, CCND2, and ETS2, was found in EBV-SMTs. Epigenetic alterations may play an important role in tumorigenesis as recurrent copy number gains were found in histone deacetylases. A gene enrichment analysis also demonstrated enrichment of genes involved in the host response to viral infection, suggesting that the tumor immune microenvironment may play an important role in EBV-SMT tumorigenesis.

Molecular profiling of EBV associated diffuse large B-cell lymphoma

Epstein-Barr virus (EBV) associated diffuse large B-cell lymphoma (DLBCL) represents a rare aggressive B-cell lymphoma subtype characterized by an adverse clinical outcome. EBV infection of lymphoma cells has been associated with different lymphoma subtypes while the precise role of EBV in lymphomagenesis and specific molecular characteristics of these lymphomas remain elusive. To further unravel the biology of EBV associated DLBCL, we present a comprehensive molecular analysis of overall 60 primary EBV positive (EBV+) DLBCLs using targeted sequencing of cancer candidate genes (CCGs) and genome-wide determination of recurrent somatic copy number alterations (SCNAs) in 46 cases, respectively. Applying the LymphGen classifier 2.0, we found that less than 20% of primary EBV + DLBCLs correspond to one of the established molecular DLBCL subtypes underscoring the unique biology of this entity. We have identified recurrent mutations activating the oncogenic JAK-STAT and NOTCH pathways as well as frequent amplifications of 9p24.1 contributing to immune escape by PD-L1 overexpression. Our findings enable further functional preclinical and clinical studies exploring the therapeutic potential of targeting these aberrations in patients with EBV + DLBCL to improve outcome.

Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.