Patterns Of Copy Number Alterations Research Articles

Abstract PO1-07-02: Sensitive tumor detection, accurate quantification, and ER subtyping using low-pass methylome of liquid biopsy samples from patients (pts) with metastatic breast cancer (mBC)

Abstract Background The presence and amount of blood circulating tumor DNA (Tumor Content, TC) is emerging as a clinically relevant factor in many cancers, including BC. In the metastatic setting, a commonly used tumor tissue agnostic approach involves analyzing Copy Number Alterations (CNA) from low-pass whole-genome sequencing (lpWGS) by ichorCNA. However, with a lower limit of detection of 3%, it is unsuitable for low TC applications, such as monitoring disease in response to treatment. Here we report on the use of low-pass whole genome bisulfite sequencing (lpWGBS) coupled with a novel tool, METER (METhylation analyzER), to overcome current limitations of lpWGS, enabling sensitive TC detection, accurate quantification, and ER subtyping from mBC liquid biopsies. Methods METER is a computational tool to analyse TC exploiting BC-specific Differentially Methylated Sites (DMS) and Regions (DMR) in lpWGBS data. To define BC DMSs and DMRs, Rocker-meth was applied to the WGBS profiles of 30 BC tissue (BASIS dataset) and 23 healthy donor cell free DNA (cfDNA) (Fox-Fisher 2021). METER consists of three modules: 1) METER-quant, a DMS-based quantifier of TC; 2) METER-detect, a DMR-based z-score method to classify samples as TC+ (METER+) or TC- (METER-); 3) METER-subtype, to infer ER status via Robust Partial Correlation applied to ER+ vs. ER- BC DMR. We generated lpWGBS data (coverage 0.5-1X) of 135 cfDNA samples from 58 pts (45 ER+, 13 ER-, 55 (95%) up to 3 previous lines of treatment; before starting treatment (T0, n=58), after the first cycle (T1, n=25), and at progression (T2, n=22)) and 30 from healthy individuals as controls. ichorCNA using sensitive parameters (allowing normal fraction up to 0.99) was used to estimate reference TC. A leave-one-out strategy applied to the control samples was used to estimate the false discovery rate (FDR) of METER-detect classification. METER+/- pts were tested for association with OS and PFS using the log-rank test and compared with ichorCNA detection (TC by ichorCNA above or below 3%, ichorCNA+/-). Results TC of 105 cfDNA BC samples by METER-quant was concordant with estimates from the state-of-the-art tool ichorCNA (R >0.90, p< 1e-10). In 22 pts with complete longitudinal data, METER-quant median TC of 0.05 at T0 (IQR 0.02-0.12), 0.02 at T1 (0.01-0.07), and 0.06 at T2 (0.03-0.11) were obtained, with significant difference observed for T0 vs T1 and T2 vs T1 (paired Wilcoxon p< 0.05). Across all time points, METER-detect classified 43% of the n=42 ichorCNA- samples as METER+. The reliability and prognostic performance of METER and ichorCNA detection classification were then compared using clinical outcome data. Based on T0 data, METER+ pts had significantly worse OS than METER- (HR=4.2 CI=2.0-8.9, p< 0.001). This effect was stronger than using ichorCNA (HR=2.3 CI=1.3-4.0, p=0.006). Worse PFS at T0 was observed for METER+ compared with METER- pts (HR=3.7 CI=1.8-7.9, p< 0.001), while no significant association was observed for ichorCNA+ pts (HR=1.4 CI=0.8-2.5, p=0.2). Of note, ichorCNA-/METER+ pts (N=11) at T0 had worse PFS compared with ichorCNA-/METER- (N=11) (HR=3.7 CI 1.4-9.7, p=0.005). Considering the 73 METER+ and 53 samples with METER-quant >5%, METER-subtype showed an accuracy of 0.84 (CI=0.73-0.92) and 0.94 (CI=0.83-0.99) respectively in classifying ER status based on IHC. Discussion lpWGBS enables tumor tissue agnostic analysis and concurrent investigation of complementary molecular features, including CNA and DNA methylation patterns. In a small and heterogeneous cohort, METER showed comparable performance to state-of-the-art tools in terms of TC quantification (ichorCNA) and ER subtyping (IHC), while offering enhanced sensitivity and ensuring a low FDR. METER showed promising prognostic stratification capabilities, particularly for patients with low TC. Validation in an extended prospective cohort is currently ongoing. Citation Format: Marta Paoli, Agostina Nardone, Francesca Galardi, Chiara Biagioni, Dario Romagnoli, Francesca De Luca, Gian Marco Franceschini, Ilenia Migliaccio, Marta Pestrin, Giuseppina Sanna, Emanuela Risi, Luca Livraghi, Erica Moretti, Laura Biganzoli, Luca Malorni, Francesca Demichelis, Matteo Benelli. Sensitive tumor detection, accurate quantification, and ER subtyping using low-pass methylome of liquid biopsy samples from patients (pts) with metastatic breast cancer (mBC) [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 PO1-07-02.

Abstract 7419: Seq2Karyotype (S2K): A method for deconvoluting heterogeneity of copy number alterations using single-sample whole-genome sequencing data

Abstract Broad copy number alterations (CNAs) at chromosomal band resolution have been used as cancer diagnostic and prognostic biomarkers for decades. These events have been characterized by cytogenetic imaging, an approach which is powerful for assessing heterogeneity but limited for locus fine mapping. While CNA detection by next-generation sequencing has become a standard analysis, existing methods rarely model CNA heterogeneity in bulk tumor samples and often require a paired normal sample for control. To overcome these limitations, we developed Seq2Karyotype (S2K, https://github.com/chenlab-sj/Seq2Karyotype), a new algorithm for in-silico karyotyping using single-sample whole-genome sequencing (WGS) data. S2K performs joint modeling of read-depth and allelic imbalance (AI) of high-quality heterozygous SNPs to identify reference diploid regions, followed by modeling and segmenting the deviation from the reference. Empirical coverage and AI of segmented regions are fitted to models of single and admixed CNAs to estimate clonality. The final karyotyping considers both the model fitness and minimization of evolution steps.To evaluate S2K’s performance, we analyzed two cell lines commonly used for benchmark test: COLO829 (melanoma) and HCT1395 (breast cancer); both had single-cell (sc) WGS for validation. For COLO829, S2K replicated the four populations detected by scWGS but derived a different clonality estimate. The predominant clone, defined by loss of 1p, 10p, and chr18, was estimated to have 67% cellular fraction (CF) by bulk sample analysis of S2K in contrast to the 10% CF by scWGS analysis. In HCT1395, S2K identified three new CNAs present in >50% of the cells of the matching germline sample which were subsequently validated by FISH, karyotyping and SKY mapping.To demonstrate S2K’s utility, we analyzed three diverse data sets: 17 neuroblastoma cell lines, 24 pediatric AML samples with karyotyping data, and two blood samples from children with myelodysplastic syndromes (MDS) known to harbor mosaic uniparental disomy (UPD). CNA-based intra-tumor heterogeneity was detected in 88% (15/17) of the neuroblastoma cell lines, comprised of 2-4 distinct populations with diverse ranges of CF (~10%-90%) and varying CNA patterns (e.g. admixture of tetraploid and diploid cells), which were validated by cytogenetics or scWGS. In the patient AML samples, S2K detected >95% of the previously reported cytogenetic events and 30% of additional copy-neutral loss-of-heterozygosity events. The mosaic UPD events in MDS patients were detected with projected clonality of 60% and 25%, respectively. These results not only demonstrate the accuracy of in-silico karyotyping performed by S2K but also reveal the dynamic intra-tumor heterogeneity in cancer cell lines, which may impact the design and interpretation of future experiments using these cell lines. Citation Format: Limeng Pu, Karol Szlachta, Virginia Valentine, Xiaolong Chen, Jian Wang, Dennis Kennetz, Daniel Putnam, Sivaraman Natarajan, Li Dong, Thomas Look, Marcin Wlodarski, Lu Wang, Steven Burden, John Easton, Xiang Chen, Jinghui Zhang. Seq2Karyotype (S2K): A method for deconvoluting heterogeneity of copy number alterations using single-sample whole-genome sequencing data [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 7419.

Genetic Profile of Myeloid Neoplasms Developed with Fanconi Anemia and Aldehyde Degradation Deficiency Syndrome

Background Fanconi anemia (FA) is an inherited bone marrow failure syndrome which develops on the basis of defective cellular responses to DNA damage. Aldehyde degradation deficiency (ADD) syndrome has been recently found as a disease caused by the defect of degradation of endogenous formaldehyde, which shares similar clinical features with FA. Although genomic abnormalities in these diseases have been associated with the high propensity of myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), the mechanism of the early development of MDS/AML remains unclear. Therefore, in this study, we aimed to understand the mechanism of the development of hematological malignancies by analyzing the genomic abnormalities seen in these diseases. Method We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) for 2 AML and 6 MDS developed in Japanese patients with FA (n = 6) and ADD syndrome (n = 2). Skin or bone marrow fibroblasts derived from the same patients were also studied as germline controls. We investigated somatic mutations, structural variants (SVs), and copy number alterations (CNAs) by WES and WGS. Mutational signature was analyzed to understand the mutational processes in these diseases. Result The number of somatic mutations and CNAs in AML identified by WES were 24 and 5 per patient on average, respectively, which were higher than those in MDS (6.8 mutations and 1.8 CNAs per patient) (P=0.07 and 0.06, respectively). However, no significant difference was observed for the number of SVs identified by WGS between MDS (35 counts per patient) and AML (52 counts per patient). Somatic mutations affected known driver genes in myeloid malignancies, such as RUNX1 (n = 1), ASXL1 (n = 1), SRSF2 (n = 1) and NRAS (n = 1) (Figure1). SVs also involved known driver genes in myeloid malignancies, such as CBL (n = 1), KDM6A (n = 1), PHF6 (n = 1), and RUNX1 (n = 1). In total, RUNX1 was most frequently altered (2/6, 33%). CNAs were more frequently accumulated in recurrent regions, including +1q (n = 5), +3q (n = 4), and -7q (n = 2) (Figure1). As previously reported, MDM4 and EVI1 were contained in +1q and +3q, respectively. Higher cancer cell fraction implied that most of these copy number changes were clonal, which suggested the early acquisition of copy number changes and their driver role in leukemic transformation. For mutational signatures, three known signatures were identified, which included clock-like SBS1 and SBS5 caused by endogenous mutational processes, and SBS3 related to defective homologous recombination. In addition, a novel mutational signature (Sig-A) was also detected. SBS3 and Sig-A were mainly detected in patients with FA and ADD syndrome, respectively, indicating that the different mutational processes are operating in the two diseases. Conclusion WES and WGS revealed the genetic landscape of FA and ADD syndrome mutation accumulation. Myeloid neoplasms related with FA and ADD syndrome were characterized by a unique pattern of driver mutations and CNAs, such as RUNX1 mutations/SVs, +1q and +3q. Although the two diseases share some clinical features, our study has revealed the different mutational processes in these diseases. Further investigation is warranted to reveal the underling mechanisms of mutation accumulation and the role of driver mutations in these diseases.

Single Cell Multi Omics and Spatial Analysis Reveals Plasmablast Signature Malignant Cells As a Key of Intratumor Heterogeneity in Primary Central Nervous System Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) constitutes approximately 5% of extranodal lymphomas. More than 90% of PCNSL are diagnosed as diffuse large B-cell lymphomas (DLBCL), mainly classified as the activated B cell (ABC) subtype. Recent genetic analyses have identified key molecular drivers of PCNSL, including MYD88, CARD11 and CD79B. However, molecular analyses using bulk samples have provided limited insights into the heterogeneity in lymphoma cells of PCNSL. To improve our understanding in the biology of PCNSL, we conducted a multimodal single-cell analyses and integrated single-cell and spatial analyses to characterize the molecular properties of PCNSL tumors. Methods:Our study included seven PCNSL patients and two patients with reactive lymph nodes (RLN). We performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) combined with B-cell and T-cell receptor (BCR and TCR) analysis on samples from 3 PCNSL and 2 RLN patients, while the remaining PCNSL patients' samples were subjected to single-cell transcriptomic sequencing. Additionally, six formalin-fixed and paraffin-embedded (FFPE) tissues from the patients analyzed in the single-cell sequencing (except for one patient) were performed with GeoMx digital spatial profiling (DSP). Result: To ensure data quality, we filtered out low-quality cells and doublets, resulting in a dataset of 41,556 single cells. B cells represented the most abundant cluster, followed by T cells and monocytes. Copy number alterations (CNAs) data derived from InferCNV analysis showed distinct CNAs patterns in lymphoma cells of each patient. UMAP plot projection analysis revealed the presence of a distinct subtype of malignant cells within the B-cell population, which was characterized as high expression of plasmablast (PBL)-associated surface proteins, including SLAMF7, CD39, and CD38. Enrichment analysis based on germinal center (GC) gene expression signatures (Holmes AB et al. J Exp Med. 2020) revealed a broader spectrum of B-cell differentiation states in malignant B cells. Single-cell BCR sequencing further demonstrated that different GC signature state cells harbored the same BCR repertoire within each patient, suggesting a common origin of malignant cells. Non-negative matrix factorization (NMF) analysis confirmed the enrichment of PBL signature scores in the distinct malignant cell population observed in the UMAP plot, consistent with DPE analysis (Figure A). Interestingly, the proportion of PBL signature cells varied among patients. Signaling interaction analysis highlighted CD70-CD27 and SPP1-CD44 pathways as enriched in tumor cells. PBL signature cells received TNFSF13B and TNFSF13 signaling through TNFRSF17 (BCMA), whereas other tumor cells expressed TNFRSF13C (BAFF-R) and received TNFSF13B signaling alone. We further analyzed two public bulk RNA-seq data (Bruno A et al. Neuro Oncol 2018, n = 6; Radke J et al. Nat Commun 2022, n=38) in PCNSL to decompose tumor components. Tumor PBL components were identified in most PCNSL cases, although several cases exhibited a low presence of tumor PBL population. Of note, analysis including these public datasets with our single-cell cohort showed that PCNSL samples with high tumor PBL proportion had unfavorable prognosis . Additionally, we performed DSP to integrate the single-cell and spatial multi-omics analyses. We deconvoluted the cell composition by using transcriptomic data derived from each region of interest (ROI) and found that the cell proportions in ROI were consistent with the corresponding single-cell results. However, there were differential profiles of cell composition according to patients rather than ROIs, suggesting that PCNSL tumors may display spatial homogeneity (Figure B). Conclusion: Through the application of single-cell and spatial transcriptomic analysis, we identified a distinct subtype expressing the PBL signature gene. These findings have important therapeutic implications and provide new insights into the mechanisms of relapse in PCNSL.

Cracking the pattern of tumor evolution based on single-cell copy number alterations.

Copy number alterations (CNAs) are a key characteristic of tumor development and progression. The accumulation of various CNAs during tumor development plays a critical role in driving tumor evolution. Heterogeneous clones driven by distinct CNAs have different selective advantages, leading to differential patterns of tumor evolution that are essential for developing effective cancer therapies. Recent advances in single-cell sequencing technology have enabled genome-wide copy number profiling of tumor cell populations at single-cell resolution. This has made it possible to explore the evolutionary patterns of CNAs and accurately discover the mechanisms of intra-tumor heterogeneity. Here, we propose a two-step statistical approach that distinguishes neutral, linear, branching and punctuated evolutionary patterns for a tumor cell population based on single-cell copy number profiles. We assessed our approach using a variety of simulated and real single-cell genomic and transcriptomic datasets, demonstrating its high accuracy and robustness in predicting tumor evolutionary patterns. We applied our approach to single-cell DNA sequencing data from 20 breast cancer patients and observed that punctuated evolution is the dominant evolutionary pattern in breast cancer. Similar conclusions were drawn when applying the approach to single-cell RNA sequencing data obtained from 132 various cancer patients. Moreover, we found that differential immune cell infiltration is associated with specific evolutionary patterns. The source code of our study is available at https://github.com/FangWang-SYSU/PTEM.

Distinct patterns of copy number alterations may predict poor outcome in central nervous system germ cell tumors

We have previously reported that 12p gain may predict the presence of malignant components and poor prognosis for CNS germ cell tumor (GCT). Recently, 3p25.3 gain was identified as an independent predictor of poor prognosis for testicular GCT. Eighty-one CNS GCTs were analyzed. Copy number was calculated using methylation arrays. Five cases (6.2%) showed 3p25.3 gain, but only among the 40 non-germinomatous GCTs (NGGCTs) (5/40, 12.5%; p = 0.03). Among NGGCTs, those with a yolk sac tumor component showed a significantly higher frequency of 3p25.3 gain (18.2%) than those without (1.5%; p = 0.048). NGGCTs with gain showed significantly shorter progression-free survival (PFS) than those without (p = 0.047). The 3p25.3 gain and 12p gain were independent from each other. The combination of 3p25.3 gain and/or 12p gain was more frequent among NGGCTs with malignant components (69%) than among those without (29%; p = 0.02). Germinomas containing a higher number of copy number alterations showed shorter PFS than those with fewer (p = 0.03). Taken together, a finding of 3p25.3 gain may be a copy number alteration specific to NGGCTs and in combination with 12p gain could serve as a marker of negative prognosis or treatment resistance. Germinoma with frequent chromosomal instability may constitute an unfavorable subgroup.

A clinically applicable molecular classification of oncocytic cell thyroid nodules.

Whole-chromosome instability with near-whole genome haploidization (GH) and subsequent endoreduplication is considered a main genomic driver in the tumorigenesis of oncocytic cell thyroid neoplasms (OCN). These copy number alterations (CNA) occur less frequently in oncocytic thyroid adenoma (OA) than in oncocytic carcinoma (OCA), suggesting a continuous process. The current study described the CNA patterns in a cohort of 30 benign and malignant OCN, observed using a next generation sequencing (NGS) panel that assesses genome-wide loss of heterozygosity (LOH) and chromosomal imbalances using 1,500 single nucleotide polymorphisms (SNP) across all autosomes and the X chromosome in DNA derived from cytological and histological samples. Observed CNA patterns were verified using multiparameter DNA flow cytometry with or without whole-genome SNP array analysis and lesser-allele intensity-ratio (LAIR) analysis. On CNA-LOH analysis using the NGS panel, GH type CNA were observed 4 of 11 (36%) OA and 14 of 16 OCA (88%). Endoreduplication was suspected in 8 of 16 (50%) OCA, all with more extensive GH type CNA (p<0.001). Reciprocal chromosomal imbalance type CNA, characterized by (imbalanced) chromosomal copy number gains and associated with benign disease, were observed in 6 of 11 (55%) OA and one equivocal case of OCA. CNA patterns were different between the histopathological subgroups (p<0.001). By applying the structured interpretation and considerations provided by the current study, CNA-LOH analysis using an NGS panel that is feasible for daily practice may be of great added value to the widespread application of molecular diagnostics in the diagnosis and risk stratification of OCN.

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.

Cancer genomes tolerate deleterious coding mutations through somatic copy number amplifications of wild-type regions

Cancers evolve under the accumulation of thousands of somatic mutations and chromosomal aberrations. While most coding mutations are deleterious, almost all protein-coding genes lack detectable signals of negative selection. This raises the question of how tumors tolerate such large amounts of deleterious mutations. Using 8,690 tumor samples from The Cancer Genome Atlas, we demonstrate that copy number amplifications frequently cover haploinsufficient genes in mutation-prone regions. This could increase tolerance towards the deleterious impact of mutations by creating safe copies of wild-type regions and, hence, protecting the genes therein. Our findings demonstrate that these potential buffering events are highly influenced by gene functions, essentiality, and mutation impact and that they occur early during tumor evolution. We show how cancer type-specific mutation landscapes drive copy number alteration patterns across cancer types. Ultimately, our work paves the way for the detection of novel cancer vulnerabilities by revealing genes that fall within amplifications likely selected during evolution to mitigate the effect of mutations.

The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

AbstractIntrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21–ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

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.

A contemporary guide to chromosomal copy number profiling in the diagnosis of renal cell carcinoma.

The routine clinical implementation of molecular methods other than fluorescence in situ hybridization in the evaluation of renal neoplasia is currently limited, as the current standard of care primarily involves a combination of morphologic and immunophenotypic analysis of such tumors. Amongst various molecular techniques, global copy number profiling using single nucleotide polymorphism-based microarrays, colloquially referred to as SNP-arrays, is being increasingly utilized to profile renal tumors, as several subtypes have characteristic recurrent patterns of copy number alterations. Recurrent copy number alterations in common tumor types include loss of chromosome 3p in clear cell renal cell carcinoma (RCC), gain of chromosomes 7 and 17 in papillary RCC and multiple losses in chromosomes 1, 2, 6, 10, 13, 17, and 21 in chromophobe RCC. Such assays are being increasingly utilized in the clinical setting. Herein, we discuss some common clinical applications of such testing that includes high yield diagnostic and prognostic applications. Diagnostic utility includes evaluation of tumor types that are primarily defined by underlying copy number alterations, establishing the underlying subtype in high grade dedifferentiated (unclassified) renal tumors, as well as assessment of loss of heterozygosity, which is an important component in the workup for germline alterations in tumor suppressor genes. Universal adoption of these techniques across clinical laboratories will likely be significantly affected by variables such as cost, reimbursement, and turnaround time.

The kinetics of blast clearance are associated with copy number alterations in childhood B-cell acute lymphoblastic leukemia

We analyzed the pattern of whole-genome copy number alterations (CNAs) and their association with the kinetics of blast clearance during the induction treatment among 195 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) who displayed intermediate or high levels of minimal residual disease (MRD). Using unsupervised hierarchical clustering of CNAs > 5 Mbp, we dissected three clusters of leukemic samples with distinct kinetics of blast clearance [A – early slow responders (n=105), B – patients with persistent leukemia (n=24), C – fast responders with the low but detectable disease at the end of induction (n=66)] that corresponded with the patients’ clinical features, the microdeletion profile,the presence of gene fusions and patients survival. Low incidence of large CNAs and chromosomal numerical aberrations occurred in cluster A which included ALL samples showing recurrent microdeletions within the genes encoding transcription factors (i.e., IKZF1, PAX5, ETV6, and ERG), DNA repair genes (XRCC3 and TOX), or harboring chromothriptic pattern of CNAs. Low hyperdiploid karyotype with trisomy 8 or hypodiploidy was predominantly observed in cluster B. Whereas cluster C included almost exclusively high-hyperdiploid ALL samples with concomitant mutations in RAS pathway genes. The pattern of CNAs influences the kinetics of leukemic cell clearance and selected aberrations affecting DNA repair genes may contribute to BCP-ALL chemoresistance.

Genomic copy number alterations as biomarkers for triple negative pregnancy-associated breast cancer

BackgroundPABC, commonly defined as breast cancer diagnosed during or ≤ 1 year after pregnancy, accounts for 7% of all breast cancers in women ≤ 45 years. Compared to age-matched non-PABC patients, PABC is characterized by a particularly aggressive histopathologic profile with poorly differentiated and estrogen- and progesterone receptor negative tumors and associated high mortality rates. This study assessed the genomic background of triple-negative PABC tumors by detection of copy number alterations (CNAs).MethodsMLPA was used to compare CNAs in breast cancer-associated chromosomal loci between triple-negative PABC- and subtype-matched non-PABC patients. Both CNA patterns were evaluated by cluster analysis; associations between individual gene CNAs, pathological characteristics and survival were explored.ResultsTriple-negative PABC tumors exhibited unique CNAs compared to non-PABC tumors, including enrichment for TOP2A copy number loss, an independent predictor of worse overall survival (HR 8.96, p = 0.020). Cluster analysis based on CNA profiles identified a triple-negative PABC-subgroup with a particularly poor prognosis, characterized by chromosome 8p copy number loss. Individual gene CNAs analysis revealed that FGFR1 copy number loss on chromosome 8p11.23 was an independent predictor of poor outcome in multivariate analysis (HR 3.59, p = 0.053) and predicted the development of distant metastases (p = 0.048).ConclusionThis study provides novel insights into the biology of triple-negative PABC tumors suggesting that CNAs, particularly 8p loss and TOP2A loss, are involved in the development of breast cancer during pregnancy. FGFR1 loss and TOP2A loss seem to be promising new biomarkers that independently identify subgroups of PABC patients with poor prognosis. These genomic biomarkers may provide clues for personalized therapy.

Signatures of copy number alterations in human cancer

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage–fusion–bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3–5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Abstract 49: Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets

Abstract Purpose: Previous studies implicated viral oncoproteins E6 and E7, of which targets include but are not limited to tumor suppressors p53 and pRb, in human papillomavirus (HPV) associated tumorigenesis. However, HPV-associated tumors must accumulate additional genomic alterations as HPV oncoproteins alone are insufficient for tumor formation in vivo. Experimental Design: Targeted sequencing was conducted with a panel of over 800 cancer related genomic regions and genes in a novel cohort of 525 head and neck squamous cell carcinoma (HNSCC) patients, including 252 HPV(+) and 273 HPV(-) tumors. Variant calling and filtering, followed by analysis with computational tools such as MutSig for somatic mutations and DiNAMIC.Duo for recurrent copy number alterations (CNAs), defined the catalog of driver alterations that are specific to HPV(+) and HPV(-) tumors. Pathway analysis was then used to elucidate the functional role of somatic alterations in the context of previously described HNSCC perturbations and HPV pathophysiology. Results: Integrative genomic analysis in one of the largest HPV(+) HNSCC cohorts to date revealed differential patterns of somatic mutations and CNAs in E6/E7 targets between HPV(+) and HPV(-) tumors. Specifically, EP300, an activator of p53 and target of E6, and retinoblastoma (RB) family members RB1 (pRb) and RBL2 (p130), which are known targets of E7 mediated degradation, were selected as significantly mutated by MutSig in HPV(+) samples only (EP300 &amp; RB1 P&amp;lt;0.001, RBL2 P=0.01), while RBL1 (p107) was significant for both HPV(+) and HPV(-) samples (P&amp;lt;0.001, P=0.02 respectively). Higher fractions of mutations in HPV(+) vs. HPV(-) tumors were observed for EP300 (13% vs 6%, Fisher’s exact test P=0.006), RB1 (7% vs 3%, P=0.07), RBL2 (3% vs 1%, P=0.09), and FOXM1 (4% vs 1%, P=0.02), another cell cycle pathway target of E7, but not for RBL1 (4% vs. 3%, P=1). Lower copy numbers were detected in HPV(+) tumors compared to HPV(-) for chromosome regions containing RB1 (P=0.03), RBL1 (P=0.08), RBL2 (P&amp;lt;0.001), and for another inhibitory target of E6, FADD (P&amp;lt;0.001), which is an activator of the proapoptotic protein caspase-8. Additionally, significant copy number gains were detected in HPV(+) vs. HPV(-) tumors within the genomic region containing E2F1 (P&amp;lt;0.001), the transcription factor whose inhibition by RB family proteins is abrogated in the presence of E7, thus leading to abnormal cell cycle progression and proliferation. Conclusions: Previous studies suggest HPV-associated tumors lack significant levels of somatic alterations in canonical tumor suppressor genes that are known targets of viral oncoproteins. Here, we show additional selective pressure exists for somatic alteration of E6/E7 target genes in at least a subset of HPV(+) HNSCCs, which further deregulate cell cycle and apoptosis pathways in these tumors. Citation Format: Jeremiah Holt, Heejoon Jo, Xiaobei Zhao, Hyo Young Choi, Vonn Walter, Paul Little, Benjamin Wahle, Angela Mazul, Jose P. Zevallos, David Neil Hayes. Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 49.

Abstract 54: Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies

Abstract The use of sequencing-based assays for clinical management of pediatric cancer patients has become increasingly common. However, for many pediatric patients, gene panel based sequencing tests yield few actionable results. Given the complex genomic alterations present in many pediatric cancers, especially high-risk solid tumors, we hypothesized that an unbiased approach might reveal more actionable findings and lead to a more comprehensive understanding of these diseases. To accomplish this, we integrated whole-genome sequencing (WGS) with RNAseq in the analysis of a pediatric oncology cohort, with a focus on longitudinal cases to capture potential tumor evolution in metastatic or treated cases. Our cohort consists of 269 high-risk pediatric oncology patients, including patients with relapsed/refractory disease, metastatic disease at diagnosis, prior cancer history, a rare diagnosis, or an estimated overall survival &amp;lt;50%. Solid tumors, CNS tumors, and leukemia/lymphomas are all represented. In total, 391 samples were characterized using WGS (tumor ~60X; germline ~30X) and/or RNAseq (tumor, polyA selected, ≥20 million reads). For 85 of these patients, multiple samples were collected at different time points (diagnosis, resection, relapse, etc.) to identify changes in the cancer over time. If panel testing was performed as part of their clinical care, a comparison to the integrated WGS/RNA analysis was made. WGS was used to identify variants (SNVs), structural rearrangements (SVs), mutational signatures, and copy-number alterations (CNAs). RNAseq was used to identify gene expression outliers, gene fusions, and confirm the expression of variants identified using WGS. The combination of WGS and RNAseq was then used to identify and prioritize potentially actionable variants for each patient. Our results show that the integration of WGS and RNAseq can provide more and higher-quality actionable information than either modality alone, whilst also capturing the majority of actionable variants detected by panel sequencing. RNAseq identified not only druggable fusions and expression outliers, but also many rare and novel fusions. WGS provided fusion validation but highlighted the limitations of WGS alone in identifying fusions resulting from complex SVs. Conversely, WGS was adept at capturing genome-wide patterns of CNAs and loss of heterozygosity that are missed by gene-centric panels. Further RNAseq integration enabled prioritization of expressed SNVs as well as CNAs and SVs that significantly alter gene expression. We also used WGS to extract mutational signatures and tracked their evolution across longitudinal samples. We found potentially biologically significant differences in therapy-induced mutations caused by platinum and alkylating agents. Our unbiased approach has enabled further discovery that advances our understanding of these rare and highly aggressive malignancies. Citation Format: Henry J. Martell, Avanthi Tayi Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Maria V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, E. Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 54.

Copy number alteration signatures as biomarkers in cancer: a review.

Within certain cancers, extensive copy number alterations (CNAs) contribute to a complex and heterogenic genomic profile. This makes it difficult to understand and unravel the distinct molecular dynamics shaping the disease while preventing clinically effective patient stratification. CNA signature analysis represents a novel genomic stratification tool for probing this complexity, offering an intricate framework for deriving CNA patterns at the molecular level. This allows the underlying genomic mechanisms of specific cancers to be revealed, leading to the potential identification of therapeutic targets and prognostic associations. This review outlines the molecular and methodological basis of CNA signatures and focuses on recent advances highlighting their clinical utility, limitations and prospective future as novel diagnostic and prognostic cancer biomarkers.

Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients

Metastatic progression is the main cause of death in cancer patients, whereas the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we assembled MSK-MET, a pan-cancer cohort of over 25,000 patients with metastatic diseases. By analyzing genomic and clinical data from this cohort, we identified associations between genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma, and HR+/HER2+ breast ductal carcinoma, but not in others, including colorectal cancer and high-grade serous ovarian cancer, where copy-number alteration patterns may be established early in tumor development. We also identified somatic alterations associated with metastatic burden and specific target organs. Our data offer a valuable resource for the investigation of the biological basis for metastatic spread and highlight the complex role of chromosomal instability in cancer progression.

Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis.

Oral leukoplakia is the most common type of oral potentially malignant disorders and considered a precursor lesion to oral squamous cell carcinoma. However, a predictor of oral leukoplakia prognosis has not yet been identified. We investigated whether copy number alteration patterns may effectively predict the prognostic outcomes of oral leukoplakia using routinely processed paraffin sections. Comparison of copy number alteration patterns between oral leukoplakia with hyperplasia (HOL, n=22) and dysplasia (DOL, n=21) showed that oral leukoplakia with dysplasia had a higher copy number alteration rate (86%) than oral leukoplakia with hyperplasia (46%). Oral leukoplakia with dysplasia exhibited a wider range of genomic variations across all chromosomes compared with oral leukoplakia with hyperplasia. We also examined a retrospective cohort of 477 patients with oral leukoplakia with hyperplasia with detailed follow-up information. The malignant transformation (MT, n=19) and leukoplakia recurrence (LR, n=253) groups had higher frequencies of aneuploidy events and copy number loss rate than the free of disease (FD, n=205) group. Together, our results revealed the association between the degree of copy number alterations and the histological grade of oral leukoplakia and demonstrated that copy number alteration may be effective for prognosis prediction in oral leukoplakia patients with hyperplasia.