Frequent Copy Number Alterations Research Articles

CDKN2A copy number alteration in bladder cancer: Integrative analysis in patient-derived xenografts and cancer patients

Bladder cancer (BlCa) is an extensively heterogeneous disease that leads to great variability in tumor evolution scenarios and lifelong patients’ surveillance, emphasizing the need for modern minimally-invasive precision medicine. Herein, we have explored the clinical significance of copy-number alterations (CNAs) in BlCa. CNAs profiling was performed in 15 patient-derived xenografts (PDXs) and validated in TCGA-BLCA (n=408) and Lindgren et al. (n=143) cohorts. CDKN2A copy-number loss was identified as the most frequent CNA in bladder tumors, associated with reduced CDKN2A expression, tumors of papillary phenotype and prolonged PDX survival. Study’s screening cohort consisted of 243 BlCa patients and CDKN2A copy-number was assessed in genomic DNA and cell-free DNA (cfDNA) from 217 tumors and 189 pre-treatment serum samples, respectively. CDKN2A copy-number loss was correlated with superior disease-free and progression-free survival of non-muscle-invasive bladder cancer (NMIBC) patients. Moreover, higher CDKN2A index (CDKN2A/LEP ratio) in pre-treatment cfDNA was associated with advanced tumor stage, grade and short-term NMIBC progression to invasive disease, while multivariate models fitted for CDKN2A index in pre-treatment cfDNA offered superior risk-stratification of T1/high grade and EORTC high-risk patients, enhancing prediction of treatment outcome. CDKN2A copy-number status could serve as minimally-invasive tool in improving risk-stratification and supporting personalized prognosis in BlCa.

Abstract PO4-14-07: Genomic and immune profiling of breast cancer brain metastases

Abstract BACKGROUND: Brain metastases (BrM) arising from breast cancer (BC) are an increasing consequence of advanced disease, with up to half of patients (pts) with metastatic HER2+ or triple negative breast cancer experiencing central nervous system (CNS) recurrence. The genomic alterations driving CNS recurrence, along with contribution of the immune microenvironment, particularly by BC subtype remains unclear. METHODS: We characterized BrM from a cohort of n=42 BC pts by sequencing whole-exome DNA and total RNA libraries from frozen (n=31) and FFPE (n=34) BCBrM, FFPE extracranial (ECT, n=12) and blood DNA (n=26) tissues from the Duke Brain Tumor Biorepository. Analyses conducted and planned include inference of PAM50 intrinsic subtypes, somatic mutations, copy number alterations, immune cell type decompositions by CIBERSORTx, differential RNA expression, driver analysis, and associations with clinical outcomes. RESULTS: PAM50 subtypes across 31 frozen BrM were 23% Luminal (Lum) A, 13% LumB, 33% HER2-enriched, 27% Basal-like, 3% Normal-like. For 34 FFPE BrM, 15% were LumA, 24% LumB, 33% HER2-enriched, 24% Basal-like, 3% Normal-like. Across 26 paired FFPE and frozen BrM, subtype discrepancy was seen in 23% (6) cases of which frozen to FFPE classification differed: 3 LumA to LumB, 1 LumB to LumA, 1 Normal to LumB, and 1 Basal to LumB. Among 10 paired FFPE BrM and ECT, subtype concordance was observed in 70%. Discordance was seen in 1 case each for ECT to BrM: LumB to HER2, LumB to LumA, Basal to Normal-like. WES demonstrated frequent copy number alterations (CNA) in clinically-relevant genes including TGFB1, NOTCH1, CDK4, and ERBB3, in both BrM and ECT. For pts with matched blood, ERBB3 CNA (gain and loss) were more commonly found in FFPE BrM (7/19, 37%) compared to ECT (1/8, 13%). Among BrM, the most frequently altered clinically-relevant genes included TP53 (~75%), PTEN, EGFR, RB1, PIK3CA, NF1 and ESR1 (all ≤15%); in ECT, TP53 (67%), BRCA2, FBXW7, and ATM (all ≤33%). PI3K pathway genes (e.g. PTEN, PIK3CA) alterations were exclusive to BrM. One pt’s ECT showed an ATM mutation that was not observed in the paired BrM. Conversely, BRAF and CCND2 mutations were observed in BrM, but not ECT for 1 pt each. Inferring the relative abundance of immune populations in frozen BrM illustrated that 23% were CD4+ resting memory T cells, 25% M2 tumor-permissive macrophages, 13% M0 macrophages; M1 tumor-inhibiting macrophages were only 2%. When comparing immune cell populations between FFPE BrM and ECT, ECT had more M1 macrophages (Chi-sq 4.23, P = 0.04), while other immune cell populations were of similar relative abundance. Immune cell fractions did not vary by subtype in BrM or ECT with one exception: M2 macrophages were lower in Basal compared to LumB tissues (frozen BrM: Chi-sq = 9.28, P = 0.05; FFPE BrM: Chi-sq = 4.61, P = 0.33; FFPE EC: Chi-sq = 9.78, P = 0.04). In n=8 pt-matched FFPE BrM/ECT, hallmark pathways upregulated in BrM included MYC and E2F targets and oxidative phosphorylation, while those with lower expression in BrM included epithelial-mesenchymal transition, interferon gamma response, and JAK-STAT signaling. CONCLUSION: This analysis showed moderate discrepancy in subtype call of BrM by tissue preparation (frozen vs. FFPE), with LumB classification showing the highest discrepancy, and more commonly called in FFPE tissues. Subtype concordance between ECT and BrM was relatively high. Analysis of CNA illustrated deletions and amplifications in targetable genes, notably ERBB3 preferentially in BrM compared to ECT. Mutational analysis identified targetable alterations exclusive to BrM; this knowledge could lead to BrM-targeted treatments. Inferred immune cell populations illustrated a tumor permissive microenvironment in BrM. Therapeutic strategies repolarizing macrophages toward a tumor-inhibiting phenotype in BrM are warranted. Analyses of associations between genomic data and clinical outcomes, as well as driver analyses, are ongoing. Citation Format: Amanda Van Swearingen, Marissa Lee, Layne Rogers, Alexander Sibley, Pixu Shi, Xiaodi Qin, Michael Goodin, Kouros Owzar, Carey Anders. Genomic and immune profiling of breast cancer brain metastases [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 PO4-14-07.

Mutational landscape of inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples.MethodsWe retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes.ResultsThe number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1–2, ESCAT LOE I–II), similarly to patients with non-IBC.ConclusionsWe provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.

Haploinsufficiency of phosphodiesterase 10A activates PI3K/AKT signaling independent of PTEN to induce an aggressive glioma phenotype.

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

Abstract 4358: Genomic profiling of residual kidney structures in dialysis patients

Abstract Background: Chronic kidney disease is frequently associated with persistent inflammation, which results in fibrosis in the stroma, a reduced number of renal tubules, the formation of multiple cystic lesions, ultimately terminating in renal failure and hemodialysis. The high incidence of renal cell carcinoma (RCC) in hemodialysis patients suggests a relationship between tissue remodeling by chronic inflammation and carcinogenesis. However, little is known about the genetic background of cancer development from remaining tubules and cystic lesions in hemodialysis patients. Methods: We enrolled 5 patients under hemodialysis who were accompanied by acquired cystic kidney disease and who underwent total nephrectomy for renal cancer. Surgical specimens were fixed with alcohol-based solution and paraffin-embedded, and after H&E staining, subjected to laser capture microdissection (LCM) to collect remaining tubules and cysts containing approximately 200 cells. DNA was extracted and analyzed for somatic mutations and copy number alteration by whole-exome sequencing. Results: In total, we collected 161 LCM samples, including 118 from proximal tubules, 17 from collecting ducts, and 26 from cysts. The median variant allele frequencies of detected mutations were 0.237 in proximal tubules, 0.133 in collecting duct, and 0.381 in cysts, indicating larger clonal expansion in proximal tubules and cysts than in collecting ducts. Proximal tubules and cysts contained recurrent mutations in FAT1 (11% and 8%, respectively), STAG2 (5% and 8%), and PTEN (3% and 4%), whereas no recurrent driver mutations were identified in collecting ducts. In copy number analysis, 26% of proximal tubules, 35% of collecting ducts, and 80% of cysts had copy number alterations. Of note, samples from cysts had more copy number alterations compared to remaining tubules (on average, 2.3 vs. 1.1). Proximal tubules had recurrent copy number gains of chromosomes 3, 7, 10, 18, and 20, and loss of chromosome 22 and samples from collecting ducts showed gains of chromosome 7 and loss of chromosome 18. In cysts, gains of chromosomes 2, 3, 7, 10, 12, and 16 and loss of chromosomes 15, 16, 21, and 22 were frequently observed. Copy number profile in cysts was similar to that of papillary RCC and acquired cystic disease-associated RCC. Conclusion: In the end-stage cystic kidney, both proximal tubules and cysts exhibited an enrichment of driver mutations commonly found in papillary RCC and RCC with acquired cystic disease, reflecting the fact that the incidence of these two types of RCC increases as the duration of dialysis becomes longer. In addition, clear cell RCC drivers, such as VHL mutation and loss of chromosome 3, were not observed, possibly explaining the rare occurrence of clear cell RCC in hemodialysis patients. Since cysts had more frequent copy number alterations than remaining tubules, cysts in the end-stage kidney might be precursor lesions of RCC. Citation Format: Kosuke Ieiri, Nobuyuki Kakiuchi, Tomonori Hirano, Koichi Watanabe, Hirona Maeda, Hiroko Tanaka, Satoru Miyano, Dai Takamatsu, Takashi Matsumoto, Keisuke Monji, Masaki Shiota, Junichi Inokuchi, Hideki Makishima, Masatoshi Eto, Seishi Ogawa. Genomic profiling of residual kidney structures in dialysis patients [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 4358.

Abstract 3480: Alterations in the tumor microenvironment and key signaling pathways in naturally-occurring canine gliomas mirror those seen in human patients

Abstract Background: Malignant gliomas are the most common type of primary brain tumors, with patient outcomes that are generally dismal. Recently, pet dogs have been assessed as a valuable model in which to study gliomas. Gliomas in pet dogs occur at similar rates to those seen in humans, and a recent study comparing canine and human gliomas identified high similarity in terms of mutational rates, aneuploidy, the timing of mutations, and methylation patterns. Here, we further compare naturally-occurring canine and human gliomas, focusing on the tumor microenvironment (TME) and key pathways dysregulated in human gliomas. Methods: Spontaneously occurring gliomas from adult and pediatric human, and canine patients were utilized for this study. RNA, whole genome/whole exome, and bisulfite sequencing data from adult and canine samples were downloaded from the Genomic Data Commons and the National Cancer Institute’s Integrated Canine Data Commons, respectively. Pediatric tumor RNA-seq and genomic data were obtained from the Gabriella Miller Kids First database. Normal adult human and canine brain tissue served as control tissue. Only tumors with a clear diagnosis of astrocytoma, oligodendroglioma, or oligoastrocytoma were included in this study. Results: Overall, we found that key canonical pathways that are altered in human gliomas are also altered in canine gliomas. A comparison of high-grade gliomas in pet dogs to normal canine brain tissues showed significant abnormal gene expression in 3 canonical oncogenic pathways of human GBM: the TP53, RB1, and RTK/RAS/PI3K pathways. Similarities between species included increased expression of PDGF, and receptors PDGFRA and PDGFRB. Frequent copy number alterations and mutations were also observed in PDGFRA and EGFR. Differences between species include the mutation rate of TP53, which we found to be mutated frequently in adults but not in pediatric or canine gliomas, however, expression of TP53 as assessed by RNA-seq was significantly increased in both adult and canine gliomas. IDH1, which drives methylation, was significantly overexpressed in adults but underexpressed in canine. Correspondingly, each of the promotor regions of TERT, PD1 and MGMT was hypermethylated in adults, but hypomethylated in canine. Although an analysis of the TME from RNA-seq data revealed an influx of immune cells in adult, pediatric and canine gliomas, there was a concurrent influx of immunosuppressive cells. Gene expression profiles of astrocytomas and oligodendrogliomas show differences in alterations of immune-related and TME-specific pathways. Conclusion: We found that similar TME and pathway alterations were observed between human and canine gliomas, and that TME alterations were different between canine astrocytomas and oligodendrogliomas. The canine TME, like that in humans, appears to be immunosuppressive. Citation Format: Nadia A. Lanman, Sagar Utturkar, Harish Kothandaraman, Deepika Dhawan, Danni Liu, Min Zhang, Matthew Beyers, Deborah W. Knapp, R. Timothy Bentley. Alterations in the tumor microenvironment and key signaling pathways in naturally-occurring canine gliomas mirror those seen in human patients [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 3480.

Abstract 2517: The amplification of TPX2 is a potential biomarker of oxaliplatin-sensitivity of colorectal cancers (CRCs) with CIN phenotype

Abstract The amplification of Chr20q11.21 harboring TPX2 gene is the most frequent copy number alteration among the cases of colorectal cancers (CRCs), especially left-sided ones. We retrospectively examined 2828 cases of CRC patients who took cancer-genome panel tests in Japan between June 2019 and November 2022. The time to first-line treatment failure was longer with regimen with oxaliplatin than with those with irinotecan in CRC patients with Chr20q11,21 amplification (12.2 versus 9.2 months, p=0.0481). Oxaliplatin induces double-strand breaks (DSBs) and then cellular apoptosis. A key event of the DNA damage response to DSBs for cellular survival is the extensive phosphorylation of Ser139 of Histone H2AX adjacent to the chromosomal break. TPX2 whose gene resides in Chr20q11,21 has been reported to repress the phosphorylation of H2AX upon ionizing radiation. The purpose of this study is to identify the relationship between the amplification of TPX2 and oxaliplatin sensitivity in human CRCs. The knockdown of TPX2 decreased the oxaliplatin-susceptibility of COLO320 and Caco-2, CIN phenotype CRC cell lines which highly expressed TPX2. However, TPX2 knockdown did not reduce the oxaliplatin sensitivity of HCT-116, an MSI phenotype CRC cell line with low-level of TPX2 expression. The knockdown of TPX2 in CIN phenotype CRC cells, not in the MSI phenotype CRC cells, enhanced the phosphorylation of H2AX upon the DNA-damage induced by oxaliplatin. These findings suggest that amplification of TPX2 contributes to the oxaliplatin-susceptibility of CRCs with CIN phenotype through DNA repair machinery. Citation Format: Shohei Ueno, Taichi Isobe, Ryosuke Taguchi, Kenji Tsuchihashi, Koichi Akashi, Eishi Baba. The amplification of TPX2 is a potential biomarker of oxaliplatin-sensitivity of colorectal cancers (CRCs) with CIN phenotype [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 2517.

Abstract 3928: Genomic landscape and estimation of immune infiltration of soft tissue sarcoma histology subtypes from the ORIEN network

Abstract Sarcomas encompass a group of malignant diseases arising from mesenchymal origins. Given their rarity and diversity, a fundamental understanding of the genomic underpinnings for many sarcoma subtypes is still lacking. Studies are often limited to one or several of the more common subtypes or a narrow evaluation of a broader sampling. We therefore report on one of the largest comprehensive omics evaluation in sarcomas to date, including whole exome sequencing (WES; n = 1170) and RNA-sequencing (n = 983) of tissues from 29 different sarcoma histologic subtypes collected at 13 institutions in the US as part of the Oncology Research Information Exchange Network (ORIEN). We identified recurrent somatic mutations previously identified in sarcomas (e.g. TP53, KIT) as well as other cancer types (e.g. BRCA1). The burden of putatively pathogenic driver point mutations was higher in metastatic samples (median = 3) as compared to primary tumor samples (median = 2; p < 0.001). We observed frequent copy number alterations including whole genome doubling more commonly in metastatic compared to primary tumors (23.4% vs. 16.9%; p = 0.0.25). Inspection of gene expression dimensionality reduction (UMAP) showed separation of gastrointestinal stromal tumors (GISTs), leiomyosarcomas, myxoid liposarcomas, and well/de-differentiated liposarcomas from the other histologies. Differential expression analysis for these four histologies with gene set enrichment analysis highlights the diversity of disease-specific pathways and need for sarcoma subtype-specific translational focus. Estimation of immune cell abundances based on RNA-seq followed by hierarchical clustering identified five immune subtypes. The subtypes ranged from low (clusters A, B) to high (clusters D, E) immune infiltration with higher abundance of T, B, Natural Killer (NK), and myeloid cells (FDR < 0.01). Intermediate immune group C was predominantly composed by GISTs and marked by an enrichment for NK cells (FDR < 0.01) compared to all groups except the immune “hot” group E; however, this immune group exhibited modest infiltration by other immune cell types. Notably, we observed significant differences in the overall survival of patients with sarcomas in immune enriched (C, D, E) compared to immune depleted clusters (A, B; p = 0.002). In summary, we report the genomic and expressional landscape of over 1000 sarcomas, representing one of the largest comprehensive profiling efforts in this disease. We identify the mutational and copy number variation landscape and observe differences between primary and metastatic samples. We highlight expression pathways that are enriched in histologic subtypes that cluster most distinctly from others, providing a subtype-specific roadmap for further translational efforts. Finally, we define immune enriched or depleted sarcoma subgroupings that carry a prognostic impact. Citation Format: Alex C. Soupir, Oscar E. Ospina, Dale Hedges, Jamie K. Teer, Michael D. Radmacher, David M. McKean, Nathan Seligson, Martin McCarter, Breelyn Wilkey, Greg Riedlinger, John Groundland, Benjamin J. Miller, Bryan Schneider, Reema Patel, Abdul Rafeh-Naqash, Stephen Edge, Bodour Salhia, Chris Moskaluk, Maggy Johns, Michelle L. Churchman, Oliver Hampton, David Liebner, Brooke L. Fridley, Andrew S. Brohl. Genomic landscape and estimation of immune infiltration of soft tissue sarcoma histology subtypes from the ORIEN network [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 3928.

Identification of prognostic models for glycosylation-related subtypes and tumor microenvironment infiltration characteristics in clear cell renal cell cancer

One of the most fatal forms of cancer of the urinary system, renal cell carcinoma (RCC), significantly negatively impacts human health. Recent research reveals that abnormal glycosylation contributes to the growth and spread of tumors. However, there is no information on the function of genes related to glycosylation in RCC. In this study, we created a technique that can be used to guide the choice of immunotherapy and chemotherapy regimens for RCC patients while predicting their survival prognosis. The Cancer Genome Atlas (TCGA) provided us with patient information, while the GeneCards database allowed us to collect genes involved in glycosylation. GSE29609 was used as external validation to assess the accuracy of prognostic models. The "ConsensusClusterPlus" program created molecular subtypes based on genes relevant to glycosylation discovered using differential expression analysis and univariate Cox analysis. We examined immune cell infiltration as measured by estimate, CIBERSORT, TIMER, and ssGSEA algorithms, Tumor Immune Dysfunction and Exclusion (TIDE) and exclusion of tumour stemness indices (TSIs) based on glycosylation-related molecular subtypes and risk profiles. Stratification, somatic mutation, nomogram creation, and chemotherapy response prediction were carried out based on risk factors. We built and verified 16 gene signatures associated with the prognosis of ccRCC patients, which are independent prognostic variables, and identified glycosylation-related genes by bioinformatics research. Cluster 2 is associated with lower human leukocyte antigen expression, worse overall survival, higher immunological checkpoints, and higher immune escape scores. In addition, cluster 2 had significantly better angiogenic activity, mesenchymal EMT, and stem ability scores. Higher immune checkpoint genes and human leukocyte antigens are associated with lower overall survival and a higher risk score. Higher estimated and immune scores, lesser tumor purity, lower mesenchymal EMT, and higher stem scores were all characteristics of the high-risk group. High amounts of tumor-infiltrating lymphocytes, a high mutation load, and a high copy number alteration frequency were present in the high-risk group.Discussion.According to our research, the 16-gene prognostic signature may be helpful in predicting prognosis and developing individualized treatments for patients with renal clear cell carcinoma, which may result in new personalized management options for these patients.

Comparison of genomic alterations in Epstein-Barr virus-positive and Epstein-Barr virus-negative diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV-posDLBCL) is an aggressive B-cell lymphoma that often presents similar morphological and immune phenotype features to that of EBV-negative DLBCL (EBV-negDLBCL). To better understand their difference in genomic landscape, we performed whole-exome sequencing (WES) of EBV-posDLBCL and EBV-negDLBCL. This analysis revealed a new mutational signature 17 (unknown) and signature 29 (smoking) in EBV-posDLBCL as well as a specific mutational signature 24 (associated with aflatoxin) in EBV-negDLBCL. Compared with EBV-negDLBCL, more somatic copy number alterations (CNAs) and deletions were detected in EBV-posDLBCL (p = 0.01). The most frequent CNAs specifically detected in EBV-posDLBCL were gains at 9p24.1 (PDL1 and JAK2), 8q22.2-q24.23 (DEPTOR and MYC), and 7q31.31-q32.2 (MET), which were validated in additional EBV-posDLBCL cases. Overall, 53.7% (22/41) and 62.9% (22/35) of the cases expressed PD-L1 and c-MET, respectively, in neoplastic cells, whereas only 15.4% (4/26) expressed c-MYC. Neoplastic c-MET expression was positively correlated with PD-L1 (p < 0.001) and MYC expression (p = 0.016). However, EBV-posDLBCL cases did not show any differences in overall survival between PD-L1-, c-MET-, or c-MYC-positive and -negative cases or between age-related groups. Analysis of the association between somatic mutation load and EBV status showed no difference in the distribution of tumor mutant burden between the two lymphomas (p = 0.41). Recurrent mutations in EBV-posDLBCL implicated several genes, including DCAF8L1, KLF2, and NOL9, while in EBV-negDLBCL, ANK2, BPTF, and CNIH3 were more frequently mutated. Additionally, PIM1 is the most altered gene in all the WES-detected cases. Our results confirm that genomic alteration differs significantly between EBV-posDLBCL and EBV-negDLBCL, and reveal new genetic alterations in EBV-posDLBCL. The positive correlation of c-MET and PD-L1/c-Myc expression may be involved in the pathogenesis of EBV-posDLBCL, which is should be explored prospectively in trials involving MET-directed therapies.

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.

Ubiquitin-Mediated Degradation of MORF4L1 By FBXO11 Suppresses Cholesterol Biosynthesis in T Cell Acute Lymphoblastic Leukemia

The substrate receptor of SKP1-Cullin1-FBOX complex, FBXO11, is frequently mutated in both diffuse large B cell lymphoma and acute myeloid leukemia, and typically functions as a tumor suppressor by targeting substrates for ubiquitin-mediated degradation. Although FBXO11 is also mutated in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines, the underlying mechanism in T-ALL development remains to be established. Here, we identified that both endogenous activation and exogenous overexpression of FBXO11 could suppress the growth of T-ALL cell in vitro and in vivo. While conversely, loss of FBXO11 accelerates the T-ALL cell proliferation. Transcriptomic and total quantitative proteomic analyses in FBXO11-depleted T-ALL cells reveal activation of the cholesterol biosynthesis pathway with accumulation of intracellular cholesterol levels. Importantly, we identified a chromatin regulator MORF4L1, mortality factor 4 like protein 1, as a functional substrate of FBXO11 in T-ALL cells. We first observed that FBXO11 exhibited frequent copy number alterations in T-ALL cell lines and in patient cohorts from public datasets. Moreover, by CRISPR SAM (Synergistic Activation Mediator) endogenous activation and lentiviral mediated gene overexpression, we validated that FBXO11 played a role in inhibiting tumor cell proliferation in T-ALL cells both in vitro and in vivo. Conversely, loss of FBXO11 accelerated T-ALL cell proliferation. Taken together, our results suggest that FBXO11 functions as a tumor suppressor in T-ALL. Mechanistically, we uncovered that the absence of FBXO11 could activate the cholesterol synthesis signaling pathway from both the RNA-seq and the TMT proteomic analysis in FBXO11-depleted MOLT4 cells compared to control cells. Concordantly, loss of FBO11 resulted in the accumulation of intracellular cholesterol level, suggesting that FBXO11 may suppress cholesterol biosynthesis in T-ALL cells. To understand how FBXO11 controls cholesterol biosynthesis, we further identified the potential substrates from the combined quantitative transcriptome and proteome analysis. MORF4L1, a chromatin modifier involved in histone acetylation, was identified as a key substrate of FBXO11 based on its upregulation at the protein level in FBXO11-depleted cells. Moreover, our data suggest MORF4L1 is a regulator of cholesterol biosynthesis in T-ALL. The inhibition of MORF4L1 suppressed the cellular growth of T-ALL cell lines, and led to the downregulation of the expression of cholesterol-related genes, such as gene HMGCR and HMGCS1. Of note, suppression of MORF4L1 and its cholesterol biosynthesis target genes such as HMGCR, HMGCS1 could rescue the accelerated growth effect after loss of FBXO11, indicating a functional role for MORF4L1 in T-ALL development. In summary, our data support a model wherein the degradation of MORF4L1 by FBXO11 suppresses leukemia cell growth by controlling cholesterol biosynthesis. The identification of this regulatory mechanism is required for T-ALL and provides valuable insights into the metabolic vulnerabilities of leukemia.

Molecular Characteristics and Clinical Significance of Germ Cell Tumors

Germ cell tumors(GCT), which predominantly emerge in the early to middle teenage years among males, affect the pineal gland, followed by the neurohypophysis, often presenting with site-specific symptoms. Diagnosis hinges on imaging, tumor markers(HCG and AFP), and pathological evaluation. The radiation dose/coverage and chemotherapy intensity are tailored to the distinction between the germinoma and non-germinoma types. Surgical resection is reserved for residual non-germinomas. Biological investigations have revealed frequent mutations in the RAS, MAPK, and PI3K pathways, with no obvious structural variations. These mutations are more prevalent in germinomas than in non-germinomas. Germinomas exhibit a strikingly low methylation status across the genome, mirroring the state of primordial germ cells(PGC), deemed as the cells of origin. Mitosis/meiosis-related genes are highly expressed in germinoma, which is another supporting evidence of PGCs as cells of origin. In contrast, non-germinomas display transcriptomic features that differentiate them into tissue formation and organogenesis. Frequent copy number alterations are another hallmark of GCTs. Among these, 12p gain has been identified as a negative prognostic factor in non-germinomas. Pathologically confirmed tumor cell content serves as a poor prognostic indicator in germinomas and requires external validation as a reliable marker. Given the significant long-term sequelae stemming from treatment burdens in vulnerable young patients, a need for targeted therapy has arisen. Ongoing genomic studies are exploring the pathogenesis and uncovering potential leads for the establishment of precision medicine.

Genomic analysis of spinal meningiomas: correlation with histopathological grade.

Spinal meningiomas are one of the most common primary intradural tumors of the adult spine. Spinal meningiomas typically have a benign course with low rates of recurrence. Recent advances in genomic profiling have provided valuable information on meningioma biology and natural history, but these studies have focused primarily on cranial meningiomas. Chromosomal copy number analysis of meningiomas has been shown to be a valuable molecular profiling technique for distinguishing benign from aggressive tumors. The Integrated Grade for Meningioma is a novel grading scheme that uses mitotic index and copy-number profile to identify aggressive tumors at high risk for recurrence. The integrated grade has been shown to be a better predictor of tumor behavior than WHO grade alone. The objective of this study was to evaluate the chromosomal copy-number profile of spinal meningiomas, and to correlate these findings with the assigned WHO grades. The authors evaluated 94 spinal meningiomas treated surgically at their institution between 2002 and 2022. The histopathological results including WHO grade, mitotic index, presence of atypical features, and MIB-1 index were recorded. Chromosomal copy number as determined by institutional whole-genome DNA copy-number profiling was available for 57 tumors. The WHO grades of the cohort consisted of 81 (86%) WHO grade 1 tumors and 13 (14%) WHO grade 2 tumors. In tumors for which copy-number profiling was available, 44 (77%) of 57 demonstrated loss of 22q/NF2. Notably absent were frequent high-risk copy number alterations including loss of 1p, 3p, 4p/q, 6p/q, 10p/q, 14q, 18p/q, 19p/q, and focal loss of CDKN2A on 9p. Of the 9 WHO grade 2 tumors for which copy-number profiling was available, 6 tumors were reclassified to a lower risk profile (integrated grade 1). This analysis suggests that spinal meningiomas exhibit overwhelmingly indolent biology, as supported by their benign integrated grade. These findings have implications in the surgical management of these patients in relation to the need for complete Simpson grade I resection, as well as the potential avoidance of adjuvant therapy following surgery in the setting of otherwise frequently benign pathology.

Analysis of the Mutational Landscape of Osteosarcomas Identifies Genes Related to Metastasis and Prognosis and Disrupted Biological Pathways of Immune Response and Bone Development.

Osteosarcoma (OS) is the most prevalent type of bone tumor, but slow progress has been achieved in disentangling the full set of genomic events involved in its initiation and progression. We assessed by NGS the mutational spectrum of 28 primary OSs from Brazilian patients, and identified 445 potentially deleterious SNVs/indels and 1176 copy number alterations (CNAs). TP53 was the most recurrently mutated gene, with an overall rate of ~60%, considering SNVs/indels and CNAs. The most frequent CNAs (~60%) were gains at 1q21.2q21.3, 6p21.1, and 8q13.3q24.22, and losses at 10q26 and 13q14.3q21.1. Seven cases presented CNA patterns reminiscent of complex events (chromothripsis and chromoanasynthesis). Putative RB1 and TP53 germline variants were found in five samples associated with metastasis at diagnosis along with complex genomic patterns of CNAs. PTPRQ, KNL1, ZFHX4, and DMD alterations were prevalent in metastatic or deceased patients, being potentially indicative of poor prognosis. TNFRSF11B, involved in skeletal system development and maintenance, emerged as a candidate for osteosarcomagenesis due to its biological function and a high frequency of copy number gains. A protein-protein network enrichment highlighted biological pathways involved in immunity and bone development. Our findings reinforced the high genomic OS instability and heterogeneity, and led to the identification of novel disrupted genes deserving further evaluation as biomarkers due to their association with poor outcomes.

Evolution of genomic profiles in primary and recurrent brain gliomas.

e14042 Background: Diffuse gliomas are highly heterogenous tumors with variable biological behaviors, including high intratumor and extratumor heterogeneity, and with occurrence of recurrent lesions in majority of patients. During disease progression, gliomas undergo cellular and genomic evolution with newly acquired genetic properties. However, the mechanism of this complicated process associated with treatment failure is poorly understood. In this retrospective study, we performed cytogenomic analyses of primary and recurrent tumors in five patients with diffuse glioma who underwent surgical resections or biopsies of multiple recurrences. Methods: One primary and at least two recurrent freshly resected tumor tissues from each patient were analyzed using combination of cytogenomic methods. To assess specific and random copy number alterations (CNAs), I-FISH (Abbott Molecular, MetaSystems), array CGH/SNP (Agilent) and MLPA (MRC Holland) were performed. Methylation of MGMT promoter was investigated by methylation-specific MLPA (MRC Holland) and mutations of 67 genes associated with solid tumors were assessed by target NGS (Archer VariantPlex Solid Tumor, Invitae). Results: All five patients (three men and two women) experienced recurrence with newly acquired genetic or epigenetic changes. We observed a higher frequency of CNAs in recurrent gliomas than in primary tumors. Moreover, several aberrations were not detected in recurrence despite being found in earlier samples. As a primary event we proved mutation R132H of IDH1 gene. In addition, we detected methylation of the MGMT promoter, CDKN2A/B homozygous deletion, and RB1 deletion as later events that were probably associated with higher tumor grades. Besides the typical genomic changes, we detected many aberrations with unknown or unclear prognostic relevance (e.g. inframe deletion in TP53, p.Met243_Asn247del, etc.). The progression to a higher grade of glioma occurred in four patients. Conclusions: The evolutional patterns in glioma depend on clonal selection caused by CNAs, mutations, genetic drift, intratumor heterogeneity and/or the patient’s treatment. Recurrence may arise from one major tumor clone or from one or more subclones within the primary tumor through. Integrated cytogenomic analyses of genetic/epigenetic profiles of primary and all recurrent tissues can contribute to a better understanding of mechanisms responsible for these processes and to identification of new alterations related to gliomas progression and/or resistance to treatment. These biomarkers could subsequently serve as new therapeutic targets for personalized treatment.

IDENTIFICATION OF GENOMIC BIOMARKERS OF DISEASE PROGRESSION AND SURVIVAL IN NEWLY‐DIAGNOSED PRIMARY CNS LYMPHOMA

Introduction: There is a significant unmet need for robust genomic biomarkers that identify patients with newly-diagnosed primary CNS lymphoma (PCNSL) at high risk of progression, both during standard methotrexate-based induction as well as after dose-intensive consolidation. Methods: We performed targeted next-generation sequencing of 529 cancer genes of formalin-fixed, paraffin-embedded tumors from 74 patients with newly diagnosed primary CNS diffuse large B-cell lymphoma using the UCSF500 Cancer Panel and the Institute for Human Genetics CLIA Laboratory. The assay detects structural variants including single nucleotide variants, small insertions and deletions, and copy-number alterations (CNAs). Sixty-four EBV-negative PCNSL patients treated with a standard methotrexate, temozolomide, rituximab (MTR) induction were available for complete analysis. Results: We identified frequent mutations involving MYD88 (77%), CD79B (41.9%), ETV6 (27%) and BTG1 (25.7%) as well as frequent CNA involving 6q loss (62.5%), 9p loss (34.4%) and 12 gain (31.3%). Three genomic alterations were most significantly associated with early disease progression in PCNSL: (1) Alterations at 6p21.3, predominantly copy neutral loss of heterozygosity (CN-LOH) at the HLA Class I (HLA-A, B, C) and Class II loci (HLA-DR, DP, DQ, TAP1) as well as homozygous deletion of HLA Class II; (2) Mutations of tumor suppressor genes BTG1 and ETV6. Overall, these genomic alterations were detected in 28 out of 31 progression events in the cohort. The subgroup with either CN-LOH or homozygous deletion at 6p had the earliest progression, followed by the subgroup with mutations at either BTG1/ETV6 and no 6p CN-LOH or homozygous deletion, compared to no alterations at these 3 loci. 10 of 18 tumors with these alterations at 6p also contained BTG1 and/or ETV6 mutations. Survival analysis demonstrates that the subgroup with 6p CN-LOH or homozygous deletion had the earliest progression, followed by the subgroup with BTG1/ETV6 mutations compared to no alterations at these 3 loci (PFS p = 3.7e-5, OS p = 0.0017, logrank test, Figure 1). Multivariate Cox proportional hazard analysis demonstrated a significant 3.24-fold increased risk of progression with 6p CN-LOH/homozygous deletion or BTG1/ETV6 mutations (p = 3.5e-5, adjusted by Age, KPS, IELSG, MSKCC, IOL, Maintenance, and Consolidation). The research was funded by: National Cancer Institute NIH R01CA139-83-01A1 Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, genomics, epigenomics, and other-omics No conflicts of interests pertinent to the abstract.

Loss of BLK expression as a potential predictor of poor prognosis and immune checkpoint blockade response in NSCLC and contribute to tumor progression

BackgroundImmune checkpoint blockade (ICB) has been proved to have significant anti-tumor effect in the clinical treatment of non-small cell lung cancer (NSCLC). Therefore, biomarkers predicting ICB response can provide better treatment for patients with NSCLC. MethodsDifferential expression genes (DEGs) were identified by ImmuCellAI database. Copy number alteration (CNA) was analyzed by cBioPortal. The predicted efficiency of 4 genes on cancer immunotherapy was assessed by ROC analysis. The survival value of BLK was analyzed by Kaplan-Meier plotter and Prognoscan analysis. Clinical significance of BLK IHC-TMA score in NSCLC was also explored. The CCK-8 assay, wound healing assay, western blot assay in vitro and subcutaneous xenograft experiments in vivo were used for investigating the functions of BLK. The RNA-sequencing were performed to screen BLK regulated genes and conducted for GO/KEGG enrichment analysis. The transcriptional regulatory factor of BLK promoter region was predicted by ChIP-seq analysis. Results39 common DEGs between ICB Response (R) group and No Response (NR) group with NSCLC were identified, in which the CNA frequency of BLK deletion (> 6%) was found. The predicted efficiency of BLK on immunotherapy was performed best in NSCLC (AUC>0.7). Low expression of BLK was related to NSCLC with significantly poor prognosis. BLK overexpression can inhibit growth of NSCLC via activating apoptosis pathway, inhibiting the G2M checkpoint and Glycolysis pathway. The enrichment analysis indicated that BLK regulated genes related to oncogenic potential in NSCLC. Besides, BLK expression was inhibited via H3K27me3 modification in A549 and H1299 cells. BLK mRNA level was negatively correlated with methylation and positively correlated with the tumor purity in NSCLC. ConclusionOur study provides strong evidence that low expression of BLK may serve as a biomarker for poor prognosis in NSCLC, while response to ICB therapy and contributes to NSCLC tumor progression.

Abstract 6066: Genomic analysis of end-stage renal disease

Abstract Background: Chronic kidney disease is frequently associated with persistent inflammation, which results in fibrosis in the stroma, a reduced number of renal tubules, the formation of multiple cystic lesions, ultimately terminating in renal failure and hemodialysis. Given the high incidence of renal cell carcinoma (RCC) in hemodialysis patients, suggesting a relationship between tissue remodeling by chronic inflammation and carcinogenesis. However, little is known about the genetic background of cancer development from remaining tubules and cystic lesions in hemodialysis patients. Method: We enrolled 5 patients under hemodialysis who were accompanied by acquired cystic kidney disease and underwent radical nephrectomy for RCC. Surgical specimens were fixed with alcohol-based solution and paraffin-embedded, and after H&amp;E staining, subjected to laser capture microdissection (LCM) to collect remaining tubules and cysts containing approximately 200 cells. DNA was extracted and analyzed for somatic mutations and copy number alteration by whole-exome sequencing. Result: In total, we collected 161 LCM samples, including 118 from proximal tubules, 17 from collecting ducts, and 26 from cysts. Median variant allele frequencies of detected mutations were 0.237 in proximal tubules, 0.133 in collecting duct, and 0.381 in cysts, indicating larger clonal expansion in proximal tubules and cysts than in collecting ducts. Proximal tubules and cysts contained recurrent mutations in FAT1 (11% and 8%, respectively), STAG2 (5% and 8%), and PTEN (3% and 4%), whereas no recurrent driver mutations were identified in collecting ducts. In copy number analysis, 26% of proximal tubules, 35% of collecting ducts, and 80% of cysts had copy number alterations. Of note, samples from cysts had more copy number alterations compared to remaining tubules (on average, 2.3 vs. 1.1). Proximal tubules had recurrent copy number gains of chromosomes 3, 7, 10, 18, and 20, and loss of chromosome 22 and samples from collecting ducts showed gains of chromosome 7 and loss of chromosome 18. In cysts, gains of chromosomes 2, 3, 7, 10, 12, and 16 and loss of chromosomes 15, 16, 21, and 22 were frequently observed. Copy number profile in cysts was similar to that of papillary RCC and acquired cystic disease-associated RCC. Conclusion: In the end-stage cystic kidney, proximal tubules and cysts showed an enrichment of driver mutations commonly found in papillary RCC and RCC with acquired cystic disease, reflecting the fact that the incidence of these two types of RCC increases as the duration of dialysis becomes longer. In addition, clear cell RCC drivers, such as VHL mutation and loss of chromosome 3, were not observed, possibly explaining the rare occurrence of clear cell RCC in hemodialysis patients. Since cysts had more frequent copy number alterations than remaining tubules, cysts in the end-stage kidney might be precursor lesions of RCC. Citation Format: Kosuke Ieiri, Nobuyuki Kakiuchi, Tomonori Hirano, Tomomi Nishimura, Koichi Watanabe, Hiroko Tanaka, Satoru Miyano, Dai Takamatsu, Keisuke Monji, Eiji Kashiwagi, Masaki Shiota, Junichi Inokuchi, Masatoshi Eto, Seishi Ogawa. Genomic analysis of end-stage renal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6066.

Somatic mutation landscape in a cohort of meningiomas that have undergone grade progression

BackgroundA subset of meningiomas progress in histopathological grade but drivers of progression are poorly understood. We aimed to identify somatic mutations and copy number alterations (CNAs) associated with grade progression in a unique matched tumour dataset.MethodsUtilising a prospective database, we identified 10 patients with meningiomas that had undergone grade progression and for whom matched pre- and post-progression tissue (n = 50 samples) was available for targeted next-generation sequencing.ResultsMutations in NF2 were identified in 4/10 patients, of these 94% were non-skull base tumours. In one patient, three different NF2 mutations were identified in four tumours. NF2 mutated tumours showed large-scale CNAs, with highly recurrent losses in 1p, 10, 22q, and frequent CNAs on chromosomes 2, 3 and 4. There was a correlation between grade and CNAs in two patients. Two patients with tumours without detected NF2 mutations showed a combination of loss and high gain on chromosome 17q. Mutations in SETD2, TP53, TERT promoter and NF2 were not uniform across recurrent tumours, however did not correspond with the onset of grade progression.ConclusionMeningiomas that progress in grade generally have a mutational profile already detectable in the pre-progressed tumour, suggesting an aggressive phenotype. CNA profiling shows frequent alterations in NF2 mutated tumours compared to non NF2 mutated tumours. The pattern of CNAs may be associated with grade progression in a subset of cases.