B-allele Frequency Research Articles

PATH-56. LOSS OF HETEROZYGOSITY OF CDKN2A/B DEFINES AN AGGRESSIVE SUBSET OF RECURRENT, HIGH-GRADE MENINGIOMAS

Abstract INTRODUCTION Loss of heterozygosity (LOH) occurs when one gene allele is lost, leaving the cell with a single functional copy. This genetic alteration drives tumor progression by disabling critical regulatory pathways. Currently, CDKN2A/B homozygous loss is a criterion for WHO Grade 3 meningiomas. However, the impact of CDKN2A/B LOH and other copy-number alterations remains unclear. This study investigates the effects of LOH in the CDKN2A/B tumor suppressor gene on progression-free survival (PFS) and overall survival (OS) in meningiomas. METHODS The study included adult patients with sporadic primary or recurrent meningiomas who underwent resection and whole exome sequencing. LOH and copy-number alterations were confirmed on visual inspection of b-allele frequency and mean ratio graphs. Samples of unsuitable quality for evaluation were excluded. CDKN2A/B copy number and LOH were associated with PFS and OS. RESULTS The sample included 333 meningiomas (64.6% female [n=215], 18.8% recurrent meningiomas [n=61], and 50 cases of postoperative recurrence [15%]). CDKN2A/B was intact in 88.5% of samples (n=295), while 38 samples (11.4%) exhibited CDKN2A/B LOH. This included homozygous deletions (n=2, 5.2%), heterozygous deletions (n=11, 28.9%), amplifications (n=12, 31.6%), and copy-neutral LOH (n=13, 34.2%). All copy-number alterations were associated with an increased risk of recurrence compared to CDKN2A/B intact samples, so CDKN2A/B LOH was used for subsequent analysis. CDKN2A/B LOH was more frequently associated with prior recurrence (22.9% vs. 8.8%; p=0.004), prior radiation (46% vs. 15.6%; p<0.0001), and WHO Grade 2 and 3 tumors (63.2% vs. 42.7%; p=0.02). CDKN2A/B LOH was associated with worse PFS (HR 3.97 [1.3-11.9]; p=0.01) and worse OS (HR 2.9 [1.03-8.2]; p=0.0429) only in recurrent, high-grade meningiomas, independent of the fraction of the genome altered, mutation count, and Simpson grade. Postoperative radiation therapy did not improve PFS (HR 3.24 [1.06-9.85]; p=0.038). CONCLUSION CDKN2A/B LOH is enriched in high-grade, recurrent meningiomas and confers worse PFS and OS despite postoperative radiotherapy. CDKN2A/B LOH is a high-risk biomarker that predicts tumor behavior and patient outcome.

Incidence of haploidy and triploidy in trophectoderm biopsies of blastocysts derived from normally and abnormally fertilized oocytes.

We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos. A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters. Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35years and for morphologically high-grade embryos. Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.

Aligning genotyping and copy number data in single trophectoderm biopsies for aneuploidy prediction: uncovering incomplete concordance.

To what extent can genotype analysis aid in the classification of (mosaic) aneuploid embryos diagnosed through copy number analysis of a trophectoderm (TE) biopsy? In a small portion of embryos, genotype analysis revealed signatures of meiotic or uniform aneuploidy in those diagnosed with intermediate copy number changes, and signatures of presumed mitotic or putative mosaic aneuploidy in those diagnosed with full copy number changes. Comprehensive chromosome screening (CCS) for preimplantation genetic testing has provided valuable insights into the prevalence of (mosaic) chromosomal aneuploidy at the blastocyst stage. However, diagnosis of (mosaic) aneuploidy often relies solely on (intermediate) copy number analysis of a single TE biopsy. Integrating genotype information allows for independent assessment of the origin and degree of aneuploidy. Yet, studies aligning both datasets to predict (putative mosaic) aneuploidy in embryos remain scarce. A single TE biopsy was collected from 1560 embryos derived from 221 couples tested for a monogenic disorder (n = 218) or microdeletion-/microduplication syndrome (n = 3). TE samples were subjected to both copy number and genotyping analysis. Copy number and SNP genotyping analysis were conducted using GENType. Unbalanced chromosomal anomalies ≥10 Mb (or ≥20 Mb for copy number calls <50%) were classified by degree, based on low-range intermediate (LR, 30-50%), high-range intermediate (HR, 50-70%) or full (>70%) copy number changes. These categories were further subjected to genotyping analysis to ascertain the origin (and/or degree) of aneuploidy. For chromosomal gains, the meiotic division of origin (meiotic I/II versus non-meiotic or presumed mitotic) was established by studying the haplotypes. The level of monosomy (uniform versus putative mosaic) in the biopsy could be ascertained from the B-allele frequencies. For segmental aneuploidies, genotyping was restricted to deletions. Of 1479 analysed embryos, 24% (n = 356) exhibited a whole-chromosome aneuploidy, with 19% (n = 280) showing full copy number changes suggestive of uniform aneuploidy. Among 258 embryos further investigated by genotyping, 95% of trisomies with full copy number changes were identified to be of meiotic origin. For monosomies, a complete loss of heterozygosity (LOH) in the biopsy was observed in 97% of cases, yielding a 96% concordance rate at the embryo level (n = 248/258). Interestingly, 4% of embryos (n = 10/258) showed SNP signatures of non-meiotic gain or putative mosaic loss instead. Meanwhile, 5% of embryos (n = 76/1479) solely displayed HR (2.5%; n = 37) or LR (2.6%; n = 39) intermediate copy number changes, with an additional 2% showing both intermediate and full copy number changes. Among embryos with HR intermediate copy number changes where genotyping was feasible (n = 25/37), 92% (n = 23/25) showed SNP signatures consistent with putative mosaic aneuploidy. However, 8% (n = 2/25) exhibited evidence of meiotic trisomy (9%) or complete LOH in the biopsy (7%). In the LR intermediate group, 1 of 33 (3%) genotyped embryos displayed complete LOH. Furthermore, segmental aneuploidy was detected in 7% of embryos (n = 108/1479) (or 9% (n = 139) with added whole-chromosome aneuploidy). These errors were often (52%) characterized by intermediate copy number values, which closely aligned with genotyping data when examined (94-100%). N/A. The findings were based on single TE biopsies and the true extent of mosaicism was not validated through embryo dissection. Moreover, evidence of absence of a meiotic origin for a trisomy should not be construed as definitive proof of a mitotic error. Additionally, a genotyping diagnosis was not always attainable due to the absence of a recombination event necessary to discern between meiotic II and non-meiotic trisomy, or the unavailability of DNA from both parents. Interpreting (intermediate) copy number changes of a single TE biopsy alone as evidence for (mosaic) aneuploidy in the embryo remains suboptimal. Integrating genotype information alongside the copy number status could provide a more comprehensive assessment of the embryo's genetic makeup, within and beyond the single TE biopsy. By identifying meiotic aberrations, especially in presumed mosaic embryos, we underscore the potential value of genotyping analysis as a deselection tool, ultimately striving to reduce adverse clinical outcomes. L.D.W. was supported by the Research Foundation Flanders (FWO; 1S74621N). M.B., K.T., F.V.M., S.J., A.V.T., V.S., D.S., A.D., and S.S. are supported by Ghent University Hospital. B.M. was funded by Ghent University. The authors have no conflicts of interest.

Genome-wide analysis and visualization of copy number with CNVpytor in igv.js.

Copy number variation (CNV) and alteration (CNA) analysis is a crucial component in many genomic studies and its applications span from basic research to clinic diagnostics and personalized medicine. CNVpytor is a tool featuring a read depth-based caller and combined read depth and B-allele frequency (BAF) based 2D caller to find CNVs and CNAs. The tool stores processed intermediate data and CNV/CNA calls in a compact HDF5 file-pytor file. Here, we describe a new track in igv.js that utilizes pytor and whole genome variant files as input for on-the-fly read depth and BAF visualization, CNV/CNA calling and analysis. Embedding into HTML pages and Jupiter Notebooks enables convenient remote data access and visualization simplifying interpretation and analysis of omics data. The CNVpytor track is integrated with igv.js and available at https://github.com/igvteam/igv.js. The documentation is available at https://github.com/igvteam/igv.js/wiki/cnvpytor. Usage can be tested in the IGV-Web app at https://igv.org/app and also on https://github.com/abyzovlab/CNVpytor.

O-155 Limited utility of mosaicism reporting in PGT-A: independent confirmation from a European multisite, double-blinded analysis including 4293 embryo transfers following the first US experience

Abstract Study question Are results showing limited clinical utility of mosaicism reporting in PGT from a blinded mosaic embryo transfer American study, confirmed in a European setting? Summary answer Even between different patient populations and clinical settings, putative mosaicism reporting has limited clinical utility for embryo selection when co-evaluated with other clinical/embryological factors. What is known already Intermediate chromosomal copy number (ICN) results in NGS-based PGT-A are commonly interpreted as embryo mosaicism. A recent prospective, non-selection study in America showed that reporting mosaicism would not significantly improve embryo selection. At odds with this, results suggesting that embryo selection based on putative mosaicism can improve clinical outcomes continue to be reported, possibly reflecting limitations in accurate mosaicism identification or retrospective analysis biases. This study replicates the design of previously reported American study, using the same PGT-A assay, examining the predictive value of whole-chromosome ICN (wICN) and segmental ICN (sICN) following concurrent copy-number and genotyping-PGT in a European setting. Study design, size, duration A multisite double-blinded study involving 14 European IVF clinics was conducted from April 2022 to July 2023, including 3365 patients and 4293 single embryo transfers. Embryos, reported as negative for uniform aneuploidies, including those with wICN or sICN, were chosen for transfer based solely on standard developmental and morphological evaluation. Primary outcome measure was live birth rate defined as any pregnancy reaching the 24th week of gestation (LBR). Participants/materials, setting, methods Trophectoderm biopsies were analysed using a validated targeted-NGS assay with approximately 5000 loci across the genome, providing both quantitative and genotyping information to support aneuploidy classification. Putative mosaicism was inferred from ICN deviations from the expected two copies for autosomes and partly supported by corresponding SNP B-allele frequency patterns. Related clinical and embryological variables were analysed in a multivariate analysis and a Random Forrest regression used to model the clinical utility of putative mosaicism. Main results and the role of chance Of the embryos transferred, 6.8% (293/4293) were wICN only; 6.2% (269/4293) were sICN only and 0.5% (23/4293) were a combination of wICN and sICN. The detected ICN ranged from 23%-84% with no overrepresentation of any individual chromosomes. The LBR of embryos in the control group (without ICN), sICN and wICN were 46.8% (1736/3708; 95%CI:45.2-48.4%), 36.4% (98/269; 95%CI:30.9-42.3%) and 39.2% (115/293; 95%CI:33.8-44.9%) respectively. The miscarriage rate for control, sICN and wICN were 12.9% (257/1993; 95%CI:11.5-14-4%), 19.7% (24/122; 95%CI:13.6-27-6%) and 16.7% (23/138; 95%CI:11.4-23.8%), respectively. Logistic regression (P = 0.05) showed that the presence of sICN (OR: 0.7; 95%CI: 0.54-0.9) and wICN (OR: 0.62; 95%CI: 0.36-1.07) were modestly but significantly associated with LBR along with other embryological and clinical factors including embryo morphology (OR: 0.89; 95%CI: 0.84-0.96), day of biopsy (OR: 0.65;95%CI: 0.56-0.75), previous ET failures (OR: 0.79; 95%CI: 0.71-0.88) that were more strongly associated. No significant effect was observed of any specific intermediate CN cut-off on the association with LBR. Random Forrest regression, considering all correlating variables, showed a LBR prediction (AUC) equal to 0.578 without putative mosaicism and AUC of 0.583 with putative mosaicism included, a negligible increase considering the low incidence of mosaicism in our clinical setting along with its modest predictive value. Limitations, reasons for caution Follow-up prenatal and post-natal data at this time was not available, hence conclusions about these outcomes wasn’t possible. Despite the overall large sample size, the analysis of specific effects at different ICN values lacked sufficient power. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings Although putative mosaicism was associated with modestly impaired pregnancy rate, two independent studies now confirm that reporting mosaicism has limited impact when prospectively selecting embryos for transfer based on standard embryological parameters. Considering the established downsides, including potential embryo disposal, the practice of mosaicism reporting in PGT is questionable. Trial registration number Not applicable

HATCHet2: clone- and haplotype-specific copy number inference from bulk tumor sequencing data.

Bulk DNA sequencing of multiple samples from the same tumor is becoming common, yet most methods to infer copy-number aberrations (CNAs) from this data analyze individual samples independently. We introduce HATCHet2, an algorithm to identify haplotype- and clone-specific CNAs simultaneously from multiple bulk samples. HATCHet2 extends the earlier HATCHet method by improving identification of focal CNAs and introducing a novel statistic, the minor haplotype B-allele frequency (mhBAF), that enables identification of mirrored-subclonal CNAs. We demonstrate HATCHet2's improved accuracy using simulations and a single-cell sequencing dataset. HATCHet2 analysis of 10 prostate cancer patients reveals previously unreported mirrored-subclonal CNAs affecting cancer genes.

Abstract 3954: Increased mosaic chromosomal alterations among survivors of childhood acute myeloid leukemia

Abstract Background: We previously characterized point mutations of clonal hematopoiesis (CH) including single nucleotide variants or small insertion/deletions residing in a set of cancer-related genes (e.g., DNMT3A and TET2), and found that CH point mutations occurred at a much higher prevalence in childhood cancer survivors (CCS) than in age-sex-race matched community controls. However, mosaic chromosomal alterations (mCAs), another form of CH with megabase-scale structural alterations (copy gain, copy loss and copy-neutral loss of heterozygosity), have not been examined. Methods: Detection of mCAs was performed on whole-genome sequencing (WGS, 30X) data from 5-year CCS with the Mosaic Chromosomal Alterations (MoChA) caller which utilizes phased genotypes, coverage, and B allele frequency (BAF) at heterozygous sites (HETS). We implemented the pipeline established by the TOPMed consortium to detect low mutant cell fractions (CF ≥1%). Variant filtration criteria: minor allele frequency &amp;lt;1%, the read depth of either allele &amp;lt;5, and only one variant retained in each 1Kb window. Called mCA filtration criteria: &amp;lt;100 HETS; lod score &amp;lt;5; relative coverage &amp;gt;2.9 or BAF deviation &amp;gt;0.16 and relative coverage &amp;gt;2.5. Autosomal mCAs (CF ≥1%, HETS &amp;gt;100 and length &amp;gt;1Mb) retained. Multivariable Cox models evaluated the association between mCAs and specific late-effects including all-cause mortality, obesity, myocardial infarction (MI) or cardiomyopathy (CMP); multivariable logistic models evaluated the association between mCAs and childhood cancer diagnoses. All models were adjusted for age, sex and therapeutic exposures. Results: In our evaluation of 4,330 CCS (median age at blood draw: 26.8 years) and 433 controls (33.6 years) with WGS data from the St. Jude Lifetime Cohort, the prevalence of mCAs was slightly higher among CCS than community controls (4.83% vs. 4.16%). Among CCS, mCAs were associated with therapeutic exposures (alkylating agents: odds ratio [OR]=1.48, 95% CI=1.09-2.00; antimetabolites: 1.53, 1.02-2.31; corticosteroids: 0.60, 0.40-0.91). In contrast to recent mCA studies in the general population, we observed no co-occurrence with CH point mutations, and no associations with late-effects including all-cause mortality, obesity, MI or CMP. Interestingly, mCAs were significantly associated with the primary diagnosis of acute myeloid leukemia (AML, OR=2.86, 95% CI=1.63-5.01). Furthermore, this finding was replicated in 2898 5-year survivors from the Childhood Cancer Survivor Study (OR=3.77, 95% CI=1.94-7.33). Conclusion: mCAs were not associated with age-related morbidities and mortality among CCS. However, we identified a striking association between mCAs and previous diagnoses of AML independent of treatment exposures. If validated among AML patients at the time of diagnosis, mCAs, which reflect the underlying genome instability, may serve as a diagnostic biomarker for AML. Citation Format: Xijun Zhang, Na Qin, Qian Dong, Cheng Chen, John Easton, Heather Mulder, Xiang Chen, Kyla Shelton, Cindy Im, Yutaka Yasui, Greg Armstrong, Kirsten K. Ness, Melissa M. Hudson, Paul Auer, Jinghui Zhang, Zhaoming Wang. Increased mosaic chromosomal alterations among survivors of childhood acute myeloid leukemia [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 3954.

Abstract 3492: Comprehensive analysis of hematological malignancies with optical genome mapping and Bionano VIA™Software

Abstract The current standard for analysis of samples with suspected hematological cancer is karyotyping, fluorescence in situ hybridization (FISH), and/or chromosomal microarray (CMA). However, these methods have several limitations for the detection of structural variants (SVs) or copy number variants (CNV) that are smaller than 5 Mbp (karyotyping). Additionally, they are limited in their ability to detect low allele fraction (LAF) events and cannot detect the absence of heterozygosity (AOH) or the loss of heterozygosity (AOH/LOH - FISH and karyotyping). Optical genome mapping (OGM) from Bionano can enable accurate detection of SV and CNV events of sizes down to 5 kbp, with high sensitivity to detect LAF events down to 5% variant allele fraction. Bionano’s VIA™ software provides visualization and analysis support for OGM SV events including an improved Circos plot and ideogram for whole genome review. VIA incorporates CNV calling from OGM data using the SNP-FASST3 algorithm which also allows for accurate copy number calling at allele fractions as low as 10%. CNV detection performance was evaluated using 280 CNV events ranging in size from 175 kbp to 8 Mbp on the 22 autosomal chromosomes. Sensitivity and PPV were calculated for each size range separately. Using SNP-FASST3, VIA also adds the ability to detect AOH/LOH events from OGM data. By analyzing locations where known SNPs disrupt the specific nucleotide sequence motif used by the DLE-1 labeling enzyme, a B-allele frequency can be calculated and enable AOH/LOH detection at low allelic fractions. AOH/LOH performance using SNP-FASST3 was evaluated using a cohort of constitutional and cancer samples analyzed with orthogonal methods as well as with simulated events. Sensitivity for 20-25 Mbp AOH events was 92% with PPV greater than 90% at 25% aberrant cell fraction. The VIA 7.0 release also includes the ability to create a customizable decision tree using supplied reference annotation files for CNV, AOH and SV event pre-classification. VIA resources include a panel of target variants derived from various medical societies for clinical analysis guidelines specific to Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) and an aggregation of all hematological malignancy guidelines (pan-heme). These methods, combined with tools for expert review and reclassification of variants, allow users to conduct whole genome interpretation and report results. These new features in VIA provide a streamlined workflow for analyzing heterogeneous hematological cancer samples and demonstrate the analytical performance of updated approaches for detection of CNV and AOH/LOH regions at low allele fractions using optical genome mapping alone. Citation Format: Jacob Wisotsky, Aliz Raski, Westley Sherman, Megan Roytman, Samer Al-Saffar, Jen Hauenstein, Andy Wing Chun Pang, Viren Wasnikar, Neil Miller. Comprehensive analysis of hematological malignancies with optical genome mapping and Bionano VIA™Software [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 3492.

Abstract 2820: The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation

Abstract Background and Aim: We have reported genetically-engineered mouse models for colonic neoplasia by using our novel CDX2P-Cre mice, in which Cre is restrictedly expressed in colonic epithelial cells. Although colonic tumors are generated in CDX2P-Cre;Apcflox/+ (CPC;Apc) mice with highly penetrant rate, greater number and deeper invasion of colonic tumors were observed in CDX2P-Cre;Apcflox/+;Ptenflox/+ (CPC;Apc+Pten), possibly resulting from haploinsufficiency of Pten gene. We have also confirmed shorter lifespan in CPC;Apc+Pten than that in CPC;Apc (AACR Annual Meeting 2018). Here, we test if these phenotypes come from one copy loss of Pten itself or secondary genetic and/or transcriptomic alterations induced by Pten loss by using whole genome sequence (WGS) and RNA sequence from macroscopically-dissected colonic tumors (bulk RNA-seq). Methods: WGS for five tumors and five livers as counterpart controls in each mouse model was performed by DNBSEQ platforms. Bulk RNA-seq was performed for three tumors in each model. Results: In the analysis using B-allele frequency, copy-neutral LOH occurred in chromosome 18 including Apc loci specifically in colonic neoplasia in both CPC;Apc and CPC;Apc+Pten mice. In contrast, LOH was not observed in chromosome 19 including Pten loci in CPC;Apc+Pten mice. We also confirmed remarkably decreased coverage of reads in reference mapping in Apc locus, and there was a slight decrease of coverage in Pten locus. There were no additional driver gene mutations in CPC;Apc or CPC;Apc+Pten in WGS. All these results suggested just one copy loss of Pten resulting in aggressive tumor phenotype in CPC;Apc+Pten mice. In RNA-seq and subsequent GSEA comparing colonic tumors in CPC;Apc+Pten with those in CPC;Apc revealed upregulation of some signal pathways including mTORC1. Supporting this observation, number of colon tumors, especially adenocarcinomas, were decreased by rapamycin administration to CPC;Apc+Pten. Similarly, rapamycin was more effective in tumorids from CPC;Apc+Pten those from CPC;Apc. Conclusion: WGS reveals one copy loss of Pten locus is the only genetic alteration in CPC;Apc+Pten when compared with CPC;Apc. One copy loss of Pten is sufficient to upregulate mTORC1 pathway in a CPC;Apc+Pten mouse model, resulting in promotion of tumor formation and invasion. Citation Format: Haruki Sada, Hiroaki Niitsu, Hikaru Nakahara, Naoya Sakamoto, Hirotaka Tashiro, Hideki Ohdan, Takao Hinoi. The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation [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 2820.

Abstract 4895: Optical genome mapping analysis across multiple solid tumor types in Bionano VIATM software

Abstract Solid tumors can show highly complex somatic structural and copy number variants (CNVs) of multiple classes. These variants can reveal mechanisms behind carcinogenesis, including the amplification of oncogenes, deletion or inactivation of tumor suppressor genes, and fusions to create new oncogenes. High degrees of heterogeneity within solid tumors can complicate analysis. Optical Genome Mapping (OGM) is a comprehensive technology which combines the resolution of molecular-level data with the scale of cytogenomic analysis. ≈5-15mg sections of freshly frozen tumor are homogenized in buffer to release ultra-high molecular weight DNA. Long fragments are then labeled at a regular 6mer, then linearized and imaged with native long-range structure (≥150kbp) preserved. In silico molecules are assembled into maps and aligned to a reference genome to detect structural variants with sensitivity to ≥5% allele fraction. Bionano VIATM software provides tools for visualization and interpretation of structural variants detected with OGM. VIA also provides CNV calling and detection of Absence of Heterozygosity (AOH) and allelic imbalance using SNP-FASST3, a segmentation algorithm for detecting mosaic events in OGM and other types of data. VIA calculates a B-Allele frequency (BAF) distribution from OGM data and adds a genome-wide BAF track to the applications suite, enabling calling of allelic imbalance events such as AOH. Visualized together, the overlay of structural variant information on copy number data provides context for chromosome fusions underlying copy number change. Additionally, balanced fusions are revealed consistently in cases where the impact is copy neutral. To demonstrate the utility of integrative analysis of structural, copy number, and allelic imbalance variants in solid tumor datasets, we processed a pilot set of six diverse solid tumors through Bionano software. Brain, breast, kidney, lung, ovarian, and prostate tumor data which had undergone previous analysis in Bionano Solve 3.7 were freshly analyzed in Solve 3.8 and VIA 7.0. In the lung tumor dataset there are clear allelic imbalance segments spanning most of chromosome 4 with no concomitant copy number change. In VIA, these segments can be merged on examination, converted to AOH calls, and further specified to reflect mosaic copy neutral loss of heterozygosity (CN-LOH). In the complex kidney tumor dataset, it is difficult to resolve ploidy state based on copy number data alone. This ambiguity is resolvable with VIA, by leveraging the copy number and BAF-based allelic imbalance distributions and recentering probes to a manually defined diploid region. The comprehensive OGM data approach facilitated by VIA can streamline analysis in challenging solid tumor datasets. Structural variant visualization, CNV detection and AOH detection are available in VIA 7.0. Citation Format: Samer I. Al-Saffar, Benjamin Clifford, Aliz Raksi, Alex Hastie, Neil Miller, Alka Chaubey. Optical genome mapping analysis across multiple solid tumor types in Bionano VIATM software [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 4895.

Abstract 4343: Revealing ovarian cancer copy number variation in single cells

Abstract The mortality rate associated with ovarian cancer (OvCa) is disproportionately high in comparison to its incidence rate. This is partly due to the heterogeneous nature of the disease, which reduces treatment efficacy and contributes to high rates of relapse and chemotherapy resistance. Most OvCa are epithelial in origin and can be classified into four main subtypes: serous, mucinous, endometrioid, and clear cell. Of these, high grade serous ovarian cancer (HGSOC) is the deadliest. Epithelial ovarian carcinomas (EOC) typically exhibit widespread chromosomal and arm-level copy number abnormalities across most of the genome; in HGSOC, focal amplifications and microdeletions are especially prevalent and indicative of high genomic instability. To understand the heterogeneity of aneuploidy in EOC and HGSOC, we performed shallow single-cell whole genome sequencing on four EOC samples: two HGSOC, one clear cell, and one mixed clear cell and endometrioid. All samples were late stage and treatment naïve, and one sample had a known BRCA2 mutation. Sequencing data was processed by two complementary methods to call copy number alterations. First, we used the Cell Ranger DNA pipeline (10x Genomics) to align cell-identified sequencing reads to human reference genome GRCh38 for coverage-based copy number estimation. Resulting copy number calls were cleaned up for mappability, quality, and noisiness. Each sample was then subject to clustering and subclustering analysis using maximum likelihood genetic clustering algorithms. All samples exhibited a high level of aneuploidy, including characteristic alterations known to be associated with EOC. Two tumors contained readily distinguishable clonal populations, and all samples contained main tumor clones that could be further divided by unique subclonal characteristics. Evidence of polyploidy was also seen in all four specimens, with some tumor clusters exhibiting triploid and tetraploid baselines. In parallel, sequencing data was analyzed by the Copy-number Haplotype Inference in Single-cell by Evolutionary Links (CHISEL) algorithm. CHISEL utilizes both binned read depth ratio and B-allele frequency data to determine allele- and haplotype-specific copy numbers in single cells. Results from CHISEL confirmed the copy number calls from Cell Ranger DNA, and revealed widespread loss of heterozygosity in all samples. These findings were corroborated with allele-specific copy number data derived from matched tumor-normal whole exome sequencing. Furthermore, CHISEL detected polyploidy in one-third of the tumor cells with no preference for the A or B alleles. Overall, our findings highlight that the known heterogeneity of ovarian cancer extends to the level of aneuploidy and CNAs, shedding light on factors which pose significant barriers to effective personalized medicine implementation. Citation Format: Yuxin Jin, Rania Bassiouni, Lee D. Gibbs, Jing Qian, Solomon Rotimi, Michelle G. Webb, Seeta Rajpara, David W. Craig, Lynda D. Roman, John D. Carpten. Revealing ovarian cancer copy number variation in single cells [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 4343.

O-290 Clinical utility of putative mosaicism detected using concurrent copy-number and genotyping PGT method: outcomes from multisite, prospective, non-selection study including 9828 single embryo transfer cycles

Abstract Study question What is the clinical utility and associated outcomes of mosaic whole chromosome or segmental aneuploidies detected using concurrent copy-number and genotyping analysis in PGT-A cycles? Summary answer Although high-level whole-chromosome mosaicism is linked to reduced sustained implantation, it has limited clinical significance in PGT-A cycles when co-evaluated with other clinical/ embryological factors What is known already NGS-based PGT-A can detect intermediate chromosomal copy number (CN), commonly interpreted as mosaic chromosomal aneuploidies in embryos. A prospective non-selection approach is the most effective way to assess the clinical utility of reporting putative mosaicism findings in PGT-A, wherein the presence of mosaicism is not disclosed and does not influence embryo selection. Conflicting results have been reported previously, possibly due to technological limitations in mosaicism assessment or retrospective analysis methods. This study reports the results of the largest multisite prospective non-selection clinical study examining the predictive value of whole-chromosome and segmental mosaicism, assessed through combined CN and genotyping data analysis. Study design, size, duration A multisite study involving seven IVF clinics was conducted from Feb 2020 to Oct 2022, including 6951 patients and 9828 single embryo transfers. The study involved a prospective non-selection approach, where embryos suspected of having whole chromosomal or segmental mosaicism were reported as negative for non-mosaic aneuploidies. Embryos were chosen for transfer based solely on standard morphological features. The primary outcome was sustained implantation rate (SIR) defined as pregnancy continuing beyond 8 weeks of gestation. Participants/materials, setting, methods In this study, the trophectoderm biopsies were analyzed using a custom, targeted NGS assay that examined approximately 5000 loci across the genome, providing genotyping information to support aneuploidy classification. Mosaicism was identified by any copy number deviation from the expected two copies (LogR plots) and confirmed by corresponding SNP B-allele frequency (BAF) patterns. Confounding factors, such as clinical and embryological variables, were controlled for in the multivariate analysis. Main results and the role of chance The average female age in this cohort was 34.9 years (SD = 4.1), with aneuploidy rate of 30% in embryos and SIR of 61.2%. Of the embryos transferred, 6.5% (636/9828) were whole chromosomal mosaic (WCM) only; 9.6% (947/9828) were segmental mosaic (SM) only and 1% (83/9829) were a combination of WCM and SM. The rate of putative mosaicism ranged from 15%-89%. The SIR of embryos in the control group (non-mosaic), SM and WCM were 62% (5190/8328; 95%CI), 58% (549/947;95%CI) and 50.3% (320/636; 95%CI P &amp;lt; 0.01) respectively. A logistic model found that the level of WCM was associated to SIR, along with other embryological and clinical factors. In particular, WCM with a CN difference &amp;gt;50% as well as poor embryo morphology were associated with lower SIR (OR = 0.5; 95% CI:0.32-0.76), but low-level (&amp;lt;50%) mosaicism was not significant (NS). Notably, female age (OR = 0.98; 95% CI:0.97-0.99 per year), BMI (0.98; 95% CI:0.98-0.99) and previous ET failures (OR = 0.58; 95% CI:0.5-0.68) were strongly associated with SIR. A predictive model, taking into account all relevant variables, yielded a significant stratification of SIR, from 43% to 68%. Given the low incidence of WCM in our clinical setting, the multivariate SIR prediction (AUC) was 0.580 without WCM and 0.585 with mosaicism included. Limitations, reasons for caution This study did not have prenatal and post-natal data available at the time of the abstract's writing, hence conclusions about these outcomes wasn’t possible. Despite the large sample size, chromosome specific analysis was not feasible. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings In this non-selection study, WCM of &amp;gt; 50% variation was associated with lower SIR. However, high-level WCM has a minimal overall impact on SIR when co-evaluating with other clinical/ embryological parameters. Decisions on reporting criteria for these findings must weigh the risk of discarding potentially viable embryos with substantial reproductive potential. Trial registration number Not applicable

MOSAIC CHROMOSOMAL ALTERATIONS AND LONGEVITY

Abstract Mosaic chromosomal alterations (mCAs) are structural alterations that are associated with mortality, age, cancer, cardiovascular disease, and diverse infections. The distribution of mCAs in long-lived subjects and individuals with familial longevity is not well described. We applied MOsaic CHromosomal Alteration (MoChA) caller on genome-wide genotype samples of 2025 centenarians, their siblings, and offspring and 273 unrelated controls from the New England Centenarian Study (NECS) and 3642 subjects with familial longevity and 920 controls from the Long-Life Family Study (LLFS). MoChA utilizes a Hidden Markov Model to detect mCA-induced deviations in allelic balance at heterozygous sites with Log R Ratio and B-allele frequency (BAF) with phased genotype information. We analyzed somatic mCAs in samples with genome-wide BAF phase concordance less than 0.51, LOD score greater than 10, and estimated cell fraction less than 50%. The results in the two studies showed that autosomal mCAs spanning over 100 kbase pairs increase with older age until approximately 102 years. However, the prevalence of the subjects with mCAs tends to plateau after that age, suggesting that the accumulation of mCAs is less prevalent in long-lived subjects. We also found that offspring and siblings of centenarians accumulate less autosomal mCAs (fixed-effect meta-analysis for NECS and LLFS: RR=0.78, p=0.033) compared to unrelated controls. In addition, consistent with results from other studies, mCAs are associated with increased risk for mortality (HR=1.08, p=0.02) and sex (Male RR=1.37, p=4.15e-05), and impact incident events of cancer, dementia, diabetes, and cardiovascular diseases even at extreme old ages.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

P-552 Analysis of the genomewide BAF profiles of selected SNPs allows reliable aneuploidy detection in preimplantation embryos, independent of haplotyping

Abstract Study question Can we reliably detect aneuploidy in trophectoderm samples using SNP array data, independent of haplotyping? Summary answer We identified all chromosomes with meiotic copy number gains and non-mosaic copy number losses larger than 5Mb that had been detected with Karyomapping. What is known already Currently, generic methods such as Karyomapping (Vitrolife) and OnePGT (Agilent), are commonly used for preimplantation genetic testing of monogenic disorders (PGT-M). The genome-wide approach has the advantage that also aneuploidy can be detected by visualizing the haplotypes, the raw B-allele frequency (BAF) and the copynumber (CN) or Log2 ratio (Log2R) values. However, these haplotyping methods have considerable shortcomings for PGT-A including the need of reference DNA samples from both sides of the family and the long time required to access all haplotype information. We aimed to circumvent these shortcomings with our own proprietary method. Study design, size, duration We retrospectively re-analyzed the available raw SNP array data from 359 embryos with trophectoderm biopsy between September 1 2015 and December 31 2017 that were diagnosed with Karyomapping (Vitrolife) as being not genetically transferable. The local ethical committee approved this study under number B.U.N. 143201731745. Written informed consent was available. We intended to identify anomalies &amp;gt;5Mb that are detectable by haplotyping. Participants/materials, setting, methods SNPs were categorized based on the parental SNP genotypes. Next, the BAF values for each category of SNPs (cBAF) were extracted from the raw SNP array data and visualized on genomewide plots. The obtained cBAF plots were analyzed for the presence of aneuploidy together with raw BAF and Log2R SNP data. The results from the interpretation of these profiles were compared with the findings previously obtained with Karyomapping (Log2R, raw BAF and haplotype profiles). Main results and the role of chance A low level maternal contamination was observed in 5 embryos by analysis of cBAF profiles that had not been detected using Karyomapping. In five other embryos, an abnormal ploidy was observed with both methods. In the remaining 8027 chromosomes from 349 embryos, all 70 chromosomes with both parental homolog (BPH) anomalies larger than 5Mb detected with Karyomapping were identified by analysis of cBAF profiles. In 68 chromosomes the type (segmental or whole chromosome) and the copy number (+1 or + 2 copies) of the detected BPH anomaly was identical with both methods. All 93 chromosomes with a copy number loss larger than 5Mb detected with Karyomapping were identified by analysis of cBAF profiles. For 92 out of 93 chromosomes the type of non-mosaic copy number loss was identical (segmental or whole chromosome). Out of 17 single parental homolog (SPH) copy number gains interpreted as non-mosaic by Karyomapping, 16 were interpreted as non-mosaic based on cBAF profiles. In one instance, the SPH copy number detected with Karyomapping was interpreted as a mosaic SPH trisomy using cBAF. Two chromosomes with mosaic anomalies and one chromosome without diagnosis with Karyomapping were interpreted as non-mosaic anomalies using cBAF. Limitations, reasons for caution In case of consanguinity aneuploidy may be more difficult to detect. The limit of detection for the size of segmental anomalies remains to be determined. Wider implications of the findings By analyzing the cBAF profiles we were able to detect all types of chromosome anomaly that are detectable by haplotyping. We were able to identify meiotic BPH copy number gains and determine the parent of origin for all anomalies without haplotyping and hence without the need for DNA from familymembers. Trial registration number not applicable

GENType: all-in-one preimplantation genetic testing by pedigree haplotyping and copy number profiling suitable for third-party reproduction.

Is it possible to develop a comprehensive pipeline for all-in-one preimplantation genetic testing (PGT), also suitable for parents-only haplotyping and, for the first time, third-party reproduction? Optimized reduced representation sequencing (RRS) by GENType, along with a novel analysis platform (Hopla), enables cheap, accurate and comprehensive PGT of blastocysts, even without the inclusion of additional family members or both biological parents for genome-wide embryo haplotyping. Several haplotyping strategies have proven to be effective for comprehensive PGT. However, these methods often rely on microarray technology, whole-genome sequencing (WGS) or a combination of strategies, hindering sample throughput and cost-efficiency. Moreover, existing tools (including other RRS-based strategies) require both prospective biological parents for embryo haplotyping, impeding application in a third-party reproduction setting. This study included a total of 257 samples. Preliminary technical validation was performed on 81 samples handpicked from commercially available cell lines. Subsequently, a clinical validation was performed on a total of 72 trophectoderm biopsies from 24 blastocysts, tested for a monogenic disorder (PGT-M) (n = 15) and/or (sub)chromosomal aneuploidy (PGT-SR/PGT-A) (n = 9). Once validated, our pipeline was implemented in a diagnostic setting on 104 blastocysts for comprehensive PGT. Samples were whole-genome amplified (WGA) and processed by GENType. Quality metrics, genome-wide haplotypes, b-allele frequencies (BAFs) and copy number profiles were generated by Hopla. PGT-M results were deduced from relative haplotypes, while PGT-SR/PGT-A results were inferred from read-count analysis and BAF profiles. Parents-only haplotyping was assessed by excluding additional family members from analysis and using an independently diagnosed embryo as phasing reference. Suitability for third-party reproduction through single-parent haplotyping was evaluated by excluding one biological parent from analysis. Results were validated against reference PGT methods. Genome-wide haplotypes of single cells were highly accurate (mean > 99%) compared to bulk DNA. Unbalanced chromosomal abnormalities (>5 Mb) were detected by GENType. For both PGT-M as well as PGT-SR/PGT-A, our technology demonstrated 100% concordance with reference PGT methods for diverse WGA methods. Equally, for parents-only haplotyping and single-parent haplotyping (of autosomal dominant disorders and X-linked disorders), PGT-M results were fully concordant. Furthermore, the origin of trisomies in PGT-M embryos was correctly deciphered by Hopla. Intrinsic to linkage-analysis strategies, de novo single-nucleotide variants remain elusive. Moreover, parents-only haplotyping is not a stand-alone approach and requires prior diagnosis of at least one reference embryo by an independent technology (i.e. direct mutation analysis) for haplotype phasing. Using a haplotyping approach, the presence of a homologous recombination site across the chromosome is biologically required to distinguish meiotic II errors from mitotic errors during trisomy origin investigation. We offer a generic, fully automatable and accurate pipeline for PGT-M, PGT-A and PGT-SR as well as trisomy origin investigation without the need for personalized assays, microarray technology or WGS. The unique ability to perform single-parent assisted haplotyping of embryos paves the way for cost-effective PGT in a third-party reproduction setting. L.D.W. is supported by the Research Foundation Flanders (FWO; 1S74619N). L.R. and B.M. are funded by Ghent University and M.B., S.S., K.T., F.V.M. and A.D. are supported by Ghent University Hospital. Research in the N.C. lab was funded by Ghent University, VIB and Kom op Tegen Kanker. A.D.K and N.C. are co-inventors of patent WO2017162754A1. The other authors have no conflicts of interest. N/A.

Patients with polyclonal hepatocellular carcinoma are at a high risk of early recurrence and have a poor recurrence-free survival period

Background/purpose of the studyTumor heterogeneity based on copy number variations is associated with the evolution of cancer and its clinical grade. Clonal composition (CC) represents the number of clones based on the distribution of B-allele frequency (BAF) obtained from a genome-wide single nucleotide polymorphism (SNP) array. A higher CC number represents a high degree of heterogeneity. We hypothesized and evaluated that the CC number in hepatocellular carcinoma (HCC) tissues might be associated with the clinical outcomes of patients.MethodsSomatic mutation, whole transcriptome, and CC number based on copy number variations of 36 frozen tissue samples of operably resected HCC tissues were analyzed by targeted deep sequencing, transcriptome analysis, and SNP array.ResultsThe samples were classified into the heterogeneous tumors as poly-CC (n = 26) and the homogeneous tumors as mono-CC (n = 8). The patients with poly-CC had a higher rate of early recurrence and a significantly shorter recurrence-free survival period than the mono-CC patients (7.0 months vs. not reached, p = 0.0084). No differences in pathogenic non-synonymous mutations, such as TP53, were observed between the two groups when targeted deep sequencing was applied. A transcriptome analysis showed that cell cycle-related pathways were enriched in the poly-CC tumors, compared to the mono-CC tumors. Poly-CC HCC is highly proliferative and has a high risk of early recurrence.ConclusionCC is a possible candidate biomarker for predicting the risk of early postoperative recurrence and warrants further investigation.

Hematopoietic mosaic chromosomal alterations in the New England Centenarian Study.

Mosaic chromosomal alterations (mCAs) are structural alterations that include deletions, duplications, or copy-neutral loss of heterozygosity. mCAs are reported to be associated with survival, age, cancer, and cardiovascular disease. Previous studies of mCAs in large population-based cohorts (UK Biobank, MGBB, BioBank Japan, and FinnGen) have demonstrated a steady increase of mCAs as people age. The distribution of mCAs in centenarians and their offspring is not well characterized. We applied MOsaic CHromosomal Alteration (MoChA) caller on 2298 genome-wide genotype samples of 1582 centenarians, 443 centenarians’ offspring, and 273 unrelated controls from the New England Centenarian Study (NECS). Integrating Log R ratio and B-allele frequency (BAF) intensities with genotype phase information, MoChA employs a Hidden Markov Model to detect mCA-induced deviations in allelic balance at heterozygous sites consistent with genotype phase in the DNA microarray data. We analyzed mCAs spanning over 100 k base pairs, with an estimated cell fraction less than 50%, within samples with genome-wide BAF phase concordance across phased heterozygous sites less than 0.51, and with LOD score of more than 10 for the model based on BAF and genotype phase. Our analysis showed that somatic mCAs increase with older age up to approximately 102 years, but the prevalence of the subjects with mCAs tend to decrease after that age, thus suggesting that accumulation of mCAs is less prevalent in long-lived individuals. We also used Poisson regression to show that centenarians and their offspring tend to accumulate less mCA (RR = 0.63, p=0.045) compared to the controls.

CNVpytor: a tool for copy number variation detection and analysis from read depth and allele imbalance in whole-genome sequencing.

BackgroundDetecting copy number variations (CNVs) and copy number alterations (CNAs) based on whole-genome sequencing data is important for personalized genomics and treatment. CNVnator is one of the most popular tools for CNV/CNA discovery and analysis based on read depth.FindingsHerein, we present an extension of CNVnator developed in Python—CNVpytor. CNVpytor inherits the reimplemented core engine of its predecessor and extends visualization, modularization, performance, and functionality. Additionally, CNVpytor uses B-allele frequency likelihood information from single-nucleotide polymorphisms and small indels data as additional evidence for CNVs/CNAs and as primary information for copy number–neutral losses of heterozygosity.ConclusionsCNVpytor is significantly faster than CNVnator—particularly for parsing alignment files (2–20 times faster)—and has (20–50 times) smaller intermediate files. CNV calls can be filtered using several criteria, annotated, and merged over multiple samples. Modular architecture allows it to be used in shared and cloud environments such as Google Colab and Jupyter notebook. Data can be exported into JBrowse, while a lightweight plugin version of CNVpytor for JBrowse enables nearly instant and GUI-assisted analysis of CNVs by any user. CNVpytor release and the source code are available on GitHub at https://github.com/abyzovlab/CNVpytor under the MIT license.

LiquidCNA: Tracking subclonal evolution from longitudinal liquid biopsies using somatic copy number alterations

SummaryCell-free DNA (cfDNA) measured via liquid biopsies provides a way for minimally invasive monitoring of tumor evolutionary dynamics during therapy. Here we present liquidCNA, a method to track subclonal evolution from longitudinally collected cfDNA samples sequenced through cost-effective low-pass whole-genome sequencing. LiquidCNA utilizes somatic copy number alteration (SCNA) to simultaneously genotype and quantify the size of the dominant subclone without requiring B-allele frequency information, matched-normal samples, or prior knowledge on the genetic identity of the emerging clone. We demonstrate the accuracy of liquidCNA in synthetically generated sample sets and in vitro mixtures of cancer cell lines. In vivo application in patients with metastatic lung cancer reveals the progressive emergence of a novel tumor subpopulation. LiquidCNA is straightforward to use, is computationally inexpensive, and enables continuous monitoring of subclonal evolution to understand and control-therapy-induced resistance.