Loss Of Heterozygosity Events Research Articles

O-095 Assessment of genome editing outcomes in human preimplantation embryos subjected to CRISPR-Cas9 – most loss of heterozygosity (LOH) events are caused by DNA repair deficiency

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable

Loss of heterozygosity impacts MHC expression on the immune microenvironment in CDK12-mutated prostate cancer

BackgroundIn prostate cancer (PCa), well-established biomarkers such as MSI status, TMB high, and PDL1 expression serve as reliable indicators for favorable responses to immunotherapy. Recent studies have suggested a potential association between CDK12 mutations and immunotherapy response; however, the precise mechanisms through which CDK12 mutation may influence immune response remain unclear. A plausible explanation for immune evasion in this subset of CDK12-mutated PCa may be reduced MHC expression.ResultsUsing genomic data of CDK12-mutated PCa from 48 primary and 10 metastatic public domain samples and a retrospective cohort of 53 low-intermediate risk primary PCa, we investigated how variation in the expression of the MHC genes affected associated downstream pathways. We classified the patients based on gene expression quartiles of MHC-related genes and categorized the tumors into “High” and “Low” expression levels. CDK12-mutated tumors with higher MHC-expressed pathways were associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. Consistent with an inflamed tumor microenvironment (TME) phenotype, digital cytometric analyses identified increased CD8 + T cells, B cells, γδ T cells, and M1 Macrophages in this group. In contrast, CDK12-mutated tumors with lower MHC expression exhibited features consistent with an immune cold TME phenotype and immunoediting. Significantly, low MHC expression was also associated with chromosome 6 loss of heterozygosity (LOH) affecting the entire HLA gene cluster. These LOH events were observed in both major clonal and minor subclonal populations of tumor cells. In our retrospective study of 53 primary PCa cases from this Institute, we found a 4% (2/53) prevalence of CDK12 mutations, with the confirmation of this defect in one tumor through Sanger sequencing. In keeping with our analysis of public domain data this tumor exhibited low MHC expression at the RNA level. More extensive studies will be required to determine whether reduced HLA expression is generally associated with primary tumors or is a specific feature of CDK12 mutated PCa.ConclusionsThese data show that analysis of CDK12 alteration, in the context of MHC expression levels, and LOH status may offer improved predictive value for outcomes in this potentially actionable genomic subgroup of PCa. In addition, these findings highlight the need to explore novel therapeutic strategies to enhance MHC expression in CDK12-defective PCa to improve immunotherapy responses.

Abstract 6235: Clinical and phenotypic consequences of HLA mediated antigen presentation deficiency in pancreatic adenocarcinoma at bulk and single-cell resolution

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal neoplasm of the pancreas characterized by a low survival rate and limited treatment options. Despite the success of immunotherapy in various cancer types, its efficacy in PDAC remains low. The underlying reasons for this discrepancy are not fully understood, despite PDAC exhibiting a moderate mutation burden. One possible determinant could be the loss of heterozygosity (LOH) at the human leukocyte antigen (HLA) loci, resulting in compromised antigen presentation. In this work, we aim to identify and validate HLA LOH events in two independent cohorts to gain insights into both their prevalence and clinical and phenotypic impact on PDAC. Methods: We applied LOHHLA, a specialized pipeline to identify HLA LOH events from paired whole genome sequencing to two independent cohorts comprising over 650 patients. We contextualized these results with the paired transcriptome-wide gene expression data and immunohistochemistry stains. In addition, we developed a machine learning classifier to predict HLA LOH from transcriptomic data. We transfer this classifier to single cell RNA sequencing data to identify the impact of (subclonal) HLA LOH on the tumor microenvironment. Results: HLA LOH events occur in approximately 30% of PDAC patients, with about 50% of these deletions being focal deletions. We find evidence that focal but not non-focal LOH is a driver of immune escape. Overall, we find that HLA LOH events are early genetic alterations and observe a significant association between HLA LOH and the Basal PDAC expression subtype. In addition, we find that HLA expression is the most important determinant of lymphocyte infiltration followed by an intact HLA locus. Moreover, our transcriptomic classifier allowed us to accurately identify HLA LOH events from RNA sequencing data, which we successfully validated in an independent cohort. Finally, we applied this classifier to single cell sequencing data and identified phenotypic differences in multiple compartments of the tumor microenvironment between samples with intact HLA and HLA LOH. Conclusions: In conclusion, our study provides the most in depth characterization of the consequences of altered antigen presentation in PDAC to date. We have demonstrated that HLA LOH occurs in a substantial proportion of patients. Overall, these findings contribute to a better understanding of the immune landscape in PDAC and may have implications for the development of immunotherapeutic strategies tailored to this challenging cancer type. Citation Format: Michael J. Geuenich, Barbara Gruenwald, Grainne O'Kane, Chengxin Yu, Tan Tiak Ju, Amy Zhang, Oumaima Hamza, Gun Ho Jang, Faiyaz Notta, Steven Gallinger, Kieran R. Campbell. Clinical and phenotypic consequences of HLA mediated antigen presentation deficiency in pancreatic adenocarcinoma at bulk and single-cell resolution [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 6235.

Abstract A041: Characterizing antigen presentation associated immune escape mechanisms in pancreatic adenocarcinoma using an integrative computational approach

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal neoplasm of the pancreas characterized by a low survival rate and limited treatment options. Despite the success of immunotherapy in various cancer types, its efficacy in PDAC remains low. The underlying reasons for this discrepancy are not fully understood, despite PDAC exhibiting a moderate mutation burden. One possible determinant could be the loss of heterozygosity (LOH) at the human leukocyte antigen (HLA) loci, resulting in compromised antigen presentation. In this work, we aim to identify and validate HLA LOH events in two independent cohorts to gain insights into both their prevalence and clinical and phenotypic impact on PDAC. Methods: We applied LOHHLA, a specialized pipeline to identify HLA LOH events from paired whole genome sequencing to two independent cohorts comprising over 650 patients. We contextualized these results with the paired transcriptome-wide gene expression data and immunohistochemistry stains. In addition, we developed a machine learning classifier to predict HLA LOH from transcriptomic data. We transfer this classifier to single cell RNA sequencing data to identify the impact of (subclonal) HLA LOH on the tumor microenvironment. Results: HLA LOH events occur in approximately 30% of PDAC patients, with about 50% of these deletions being focal deletions. These events are early genetic alterations and lead to a dosage compensation of the retained HLA genes. We observed a significant association between HLA LOH and the Basal PDAC expression subtype. In addition, we find that HLA expression is the most important determinant of lymphocyte infiltration followed by non-LOH at the HLA locus. Moreover, our transcriptomic classifier allowed us to accurately identify HLA LOH events from RNA sequencing data, which we successfully validated in an independent cohort. Finally, we applied this classifier to single cell sequencing data and identified phenotypic differences in multiple compartments of the tumor microenvironment between samples with intact HLA and HLA LOH. Conclusions: In conclusion, our study provides the most in depth characterization of the consequences of altered antigen presentation in PDAC to date. We have demonstrated that HLA LOH occurs in a substantial proportion of patients. Overall, these findings contribute to a better understanding of the immune landscape in PDAC and may have implications for the development of immunotherapeutic strategies tailored to this challenging cancer type. Citation Format: Michael J. Geuenich, Barbara Gruenwald, Amy Zhang, Oumaima Hamza, Gun Ho Jang, Grainne O’Kane, Faiyaz Notta, Steven Gallinger, Kieran R. Campbell. Characterizing antigen presentation associated immune escape mechanisms in pancreatic adenocarcinoma using an integrative computational approach [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A041.

Inherited BRCA1 and RNF43 pathogenic variants in a familial colorectal cancer type X family

AbstractGenetic susceptibility to familial colorectal cancer (CRC), including for individuals classified as Familial Colorectal Cancer Type X (FCCTX), remains poorly understood. We describe a multi-generation CRC-affected family segregating pathogenic variants in both BRCA1, a gene associated with breast and ovarian cancer and RNF43, a gene associated with Serrated Polyposis Syndrome (SPS). A single family out of 105 families meeting the criteria for FCCTX (Amsterdam I family history criteria with mismatch repair (MMR)-proficient CRCs) recruited to the Australasian Colorectal Cancer Family Registry (ACCFR; 1998–2008) that underwent whole exome sequencing (WES), was selected for further testing. CRC and polyp tissue from four carriers were molecularly characterized including a single CRC that underwent WES to determine tumor mutational signatures and loss of heterozygosity (LOH) events. Ten carriers of a germline pathogenic variant BRCA1:c.2681_2682delAA p.Lys894ThrfsTer8 and eight carriers of a germline pathogenic variant RNF43:c.988 C > T p.Arg330Ter were identified in this family. Seven members carried both variants, four of which developed CRC. A single carrier of the RNF43 variant met the 2019 World Health Organization (WHO2019) criteria for SPS, developing a BRAF p.V600 wildtype CRC. Loss of the wildtype allele for both BRCA1 and RNF43 variants was observed in three CRC tumors while a LOH event across chromosome 17q encompassing both genes was observed in a CRC. Tumor mutational signature analysis identified the homologous recombination deficiency (HRD)-associated COSMIC signatures SBS3 and ID6 in a CRC for a carrier of both variants. Our findings show digenic inheritance of pathogenic variants in BRCA1 and RNF43 segregating with CRC in a FCCTX family. LOH and evidence of BRCA1-associated HRD supports the importance of both these tumor suppressor genes in CRC tumorigenesis.

Molecular underpinnings and environmental drivers of loss of heterozygosity in Drosophila intestinal stem cells

During development and aging, genome mutation leading to loss of heterozygosity (LOH) can uncover recessive phenotypes within tissue compartments. This phenomenon occurs in normal human tissues and is prevalent in pathological genetic conditions and cancers. While studies in yeast have defined DNA repair mechanisms that can promote LOH, the predominant pathways and environmental triggers in somatic tissues of multicellular organisms are not well understood. Here, we investigate mechanisms underlying LOH in intestinal stem cells in Drosophila. Infection with the pathogenic bacteria, Erwinia carotovora carotovora 15, but not Pseudomonas entomophila, increases LOH frequency. Using whole genome sequencing of somatic LOH events, we demonstrate that they arise primarily via mitotic recombination. Molecular features and genetic evidence argue against a break-induced replication mechanism and instead support cross-over via double Holliday junction-based repair. This study provides a mechanistic understanding of mitotic recombination, an important mediator of LOH, and its effects on stem cells invivo.

Bomedemstat (IMG-7289), an LSD1 Inhibitor, Manages the Signs and Symptoms of Essential Thrombocythemia (ET) While Reducing the Burden of Cells Homozygous for Driver Mutations

The ultimate goal in the treatment of ET is to eliminate mutant cells with the capacity to both proliferate and self-renew. Lysine-specific demethylase-1, LSD1, is an enzyme critical for regulating the proliferation of hematopoietic stem cells and the maturation of progenitors (Sprussel et al. 2012). Bomedemstat is an orally active LSD1 inhibitor that reduces platelets, inflammatory cytokines, and the mutant cell burden in mouse models of myeloproliferative neoplasms (Jutzi et. al., 2019). All current treatments - hydroxyurea, interferon, or anagrelide - have their limitations and do not substantially affect the natural history of ET. IMG-7289-CTP-201 was a global, open-label, Phase 2b study of bomedemstat (IMG-7289, MK-3543) taken once daily for 24+ weeks in patients with ET resistant to or intolerant of at least one standard treatment (NCT04254978). The primary endpoint was reduction of platelets to <400 x 10 9/L in the absence of new thromboembolic events. Exploratory endpoints included changes in frequencies of mutant alleles (VAF) assessed by deep sequencing (median exonic depth =1784 reads) of 261 genes of germline and somatic DNA. Homozygosity of mutant alleles was imputed when VAF >50% and/or loss-of-heterozygosity (LOH) was evident based on the difference between the minor allele frequency of flanking single nucleotide polymorphisms (“MAF method”) in germline versus granulocyteDNA. Single cell genotypes (scDNA) (Tapestri ®) were determined in stem/progenitor (CD34+) and monocytes (CD14+) cells in patients with LOH (using the MAF method) showing a decrease in LOH in whom follow-up samples were available. For patients treated ≥24 wks, 95% (61/64) had reduced platelet counts to ≤400 x 10 9/L without new thromboembolic events, in a median time of 10 wks. In the first 48 weeks, four patients experienced a thromboembolic event ( per PI) (1 PE, 2 TIAs, and 1 transient global amnesia). Of the 14/73 (19%) patients with Day 1 WBC count >10 x 10 9/L, 100% treated for ≥24 wks had reduced the WBC count to ≤10 x 10 9/L; mean Hb remained stable. In patients with baseline MPN-SAF TSS >20 (28/73), at Wk 24, 78% (18/23) experienced an improvement with 52% (12/23) improving ≥10 points. The median baseline score for worst fatigue was 4.0 (N=73); by Wk 24, the score had improved to 3.0 (N=62). Among all patients (N=73), the most common AEs regardless of causality were dysgeusia (55%), constipation (38%), thrombocytopenia (34%), and arthralgia (27%). Twenty-four patients reported serious AEs (SAE), with related events ( per PI) in 4 patients. Twenty-one patients discontinued treatment, 11 due to AEs (3 for dysgeusia), 7 subject's decisions, 1 PI decision, 1 disease progression, and 1 unrelated death due to aspiration pneumonia. There have been no safety signals or deaths related to drug in patients treated for up to 979 days. At Wk 24, 86% patients remained on study and 75% at Wk 48. At ~Wk 24, 39 of 46 (85%) of evaluable patients had a decrease in driver VAF (Fig. 1). The greatest changes in VAF occurred in patients with cells homozygous for a driver mutation (Fig. 1). Nine of ten patients with homozygous driver clones had follow-up data: by bulk sequencing of germline+granulocyte DNA, 7 showed a reduction in the proportion of homozygous cells. In all cases tested, scDNA of CD14+ (monocytes) and CD34+ (stem/progenitor) cells were qualitatively similar to results obtained with bulk granulocyte sequencing (Fig.2). By bulk sequencing, the proportion of homozygous cells dropped by a mean of 66% (-4% to -100% (-100% = homozygosity not detectable)). Decreases in LOH by bulk and sc sequencing during treatment with bomedemstat were identified in patients with homozygosity at JAK2, CALR, and MPL. This study demonstrates that treatment with bomedemstat results in a consistent hematologic response in the majority of patients with ET. Further, deep bulk sequencing of germline+granulocyte DNA reveals LOH ( i.e., homozygosity) events with high accuracy that enables determination of the distribution of homozygote, heterozygote and wild-type genotypes. Furthermore, this study shows that sequencing germline+granulocyte DNA provides a reliable qualitative view of the impact of treatment on the genotypic distribution among CD34+ cells as revealed by scDNA genotyping. Finally, bomedemstat showed activity reducing stem/progenitor cells homozygous for mutations in JAK2, CALR, and MPL.

CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via non-homologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Nonlethal Furfural Exposure Causes Genomic Alterations and Adaptability Evolution in Saccharomyces cerevisiae.

Furfural is a major inhibitor found in lignocellulosic hydrolysate, a promising feedstock for the biofermentation industry. In this study, we aimed to investigate the potential impact of this furan-derived chemical on yeast genome integrity and phenotypic evolution by using genetic screening systems and high-throughput analyses. Our results showed that the rates of aneuploidy, chromosomal rearrangements (including large deletions and duplications), and loss of heterozygosity (LOH) increased by 50-fold, 23-fold, and 4-fold, respectively, when yeast cells were cultured in medium containing a nonlethal dose of furfural (0.6 g/L). We observed significantly different ratios of genetic events between untreated and furfural-exposed cells, indicating that furfural exposure induced a unique pattern of genomic instability. Furfural exposure also increased the proportion of CG-to-TA and CG-to-AT base substitutions among point mutations, which was correlated with DNA oxidative damage. Interestingly, although monosomy of chromosomes often results in the slower growth of yeast under spontaneous conditions, we found that monosomic chromosome IX contributed to the enhanced furfural tolerance. Additionally, terminal LOH events on the right arm of chromosome IV, which led to homozygosity of the SSD1 allele, were associated with furfural resistance. This study sheds light on the mechanisms underlying the influence of furfural on yeast genome integrity and adaptability evolution. IMPORTANCE Industrial microorganisms are often exposed to multiple environmental stressors and inhibitors during their application. This study demonstrates that nonlethal concentrations of furfural in the culture medium can significantly induce genome instability in the yeast Saccharomyces cerevisiae. Notably, furfural-exposed yeast cells displayed frequent chromosome aberrations, indicating the potent teratogenicity of this inhibitor. We identified specific genomic alterations, including monosomic chromosome IX and loss of heterozygosity of the right arm of chromosome IV, that confer furfural tolerance to a diploid S. cerevisiae strain. These findings enhance our understanding of how microorganisms evolve and adapt to stressful environments and offer insights for developing strategies to improve their performance in industrial applications.

Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targetedGENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens

BackgroundHybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events.ResultsWe found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids.ConclusionsOur results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events—as expected by mitotic repair of rare chromosomal breaks—but rather of acute episodes involving many LOH events in a short period of time.

Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays.

Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.

Abstract 240: Comprehensive analytical solution to measure HRD genomic scars from SNP arrays, pan-cancer NGS panels, and optical genome mapping (OGM)

Abstract Homologous recombination deficiency (HRD) status has been shown to be an effective pharmacogenetic biomarker for determining the efficacy of PARP inhibitor therapy across various tumor types. A functional assessment of the genomic instability associated with HRD is critical as more than half of HRD positive tumors do not have a known causative mutation. Genomic instability is commonly achieved through assessment of three characteristic genomic scars: Loss of heterozygosity (LOH), Telomeric Allelic Imbalance (TAI), & Large-Scale State Transitions (LST) but is challenging due to the need for robust detection of copy number and allelic events genome-wide. As the technology commonly used to analyze tumors varies, we sought to compare the concordance of commonly used platforms to measure genomic instability. Bionano VIA™ software provides a standardized analytical measurement of the genomic scars across technologies, including SNP arrays, pan-cancer gene panels, and optical genome mapping (OGM). The software is uniquely capable to profile solid tumors from pan-cancer sequencing panels, such as the Illumina TruSight Oncology 500 (TSO500) panel, by employing a dynamic binning approach of sequencing data to detect copy number variant (CNV) and LOH events. OGM can detect additional classes of structural rearrangements missed by other technologies to provide new insights into genomic instability. In this study, tumor samples were run on multiple technologies and analyzed in VIA to provide a measurement of the genomic scars. A comparative analysis of each technology’s performance to detect genomic instability through a standardized analytical approach will be described to provide insight into the most effective technologies to assess HRD. Citation Format: Daniel Saul, Megan Roytman, Viren Wasnikar, Rachel Burnside, Alex Hastie, Alka Chaubey. Comprehensive analytical solution to measure HRD genomic scars from SNP arrays, pan-cancer NGS panels, and optical genome mapping (OGM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 240.

Loss of Heterozygosity Spectrum Depends on Ploidy Level in Natural Yeast Populations.

The appearance of genomic variations such as loss of heterozygosity (LOH) has a significant impact on phenotypic diversity observed in a population. Recent large-scale yeast population genomic surveys have shown a high frequency of these events in natural isolates and more particularly in polyploids. However, the frequency, extent, and spectrum of LOH in polyploid organisms have never been explored and are poorly characterized to date. Here, we accumulated 5,163 LOH events over 1,875 generations in 76 mutation accumulation (MA) lines comprising nine natural heterozygous diploid, triploid, and tetraploid natural S. cerevisiae isolates from different ecological and geographical origins. We found that the rate and spectrum of LOH are variable across ploidy levels. Of the total accumulated LOH events, 8.5%, 21%, and 70.5% of them were found in diploid, triploid, and tetraploid MA lines, respectively. Our results clearly show that the frequency of generated LOH events increases with ploidy level. In fact, the cumulative LOH rates were estimated to be 9.3 × 10-3, 2.2 × 10-2, and 8.4 × 10-2 events per division for diploids, triploids, and tetraploids, respectively. In addition, a clear bias toward the accumulation of interstitial and short LOH tracts is observed in triploids and tetraploids compared with diploids. The variation of the frequency and spectrum of LOH events across ploidy level could be related to the genomic instability, characterizing higher ploidy isolates.

Reduced sister chromatid cohesion acts as a tumor penetrance modifier.

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.

P-802 Distinct genetic impact of CRISPR/Cas9 gene correction in human embryos compared to induced pluripotent stem cells

Abstract Study question Does CRISPR/Cas9 gene editing have a distinct genetic impact in human embryos compared to induced pluripotent stem cells (iPSCs)? Summary answer IPSCs display a difference in repair events, such as template usage, compared to the germline which indicates a distinct genetic impact by CRISPR/Cas9 gene editing. What is known already CRISPR/Cas9 is utilized to induce targeted DNA editing. To introduce specific changes, such as mutational correction, a DNA template is generally administered, stimulating homology-directed repair. Recent human germline editing studies aiming for mutational repair created a moderate amount of embryos which solely carried wild-type alleles. Remarkably, no template use was observed, but instead loss-of-heterozygosity (LOH) was demonstrated. This is the presence of only one (the wild-type) allele, either caused by gene conversion events using the wild type allele or chromosome loss. Up to date, no iPSCs studies have evaluated LOH events when attempting to correct a heterozygous mutation. Study design, size, duration A guide RNA targeting the mutant allele, and a repair template containing the wild-type allele and a synonymous variant to track template usage were designed. For iPSCs targeting, the components were nucleofected after which DNA was extracted from the whole well (n = 3) or from individual colonies (n = 33). For human embryo targeting, the components were injected simultaneously with sperm into donated spare oocytes (n = 32). DNA was extracted from embryos after 3-6 days of in vitro culture. Participants/materials, setting, methods Sperm and renal cells, from which the iPSCs were derived, were donated by a patient with a heterozygous base pair substitution in PLCZ1 (c.136-1G>C) causing fertilization failure. To overcome fertilization failure, assisted oocyte activation was employed during ICSI. For the embryos, next-generation sequencing (NGS), short tandem repeat (STR) analysis and a whole genome single nucleotide polymorphism (SNP) assay were performed. For the iPSCs, NGS and a targeted SNP assay were carried out. Main results and the role of chance The genetic events in embryos originating from mutant sperm (n = 32) displayed following distribution: 19% showed the untargeted mutant allele, 56% showed additional mutagenesis and 25% showed only the wild-type allele. In the latter group, the template was never utilized, pointing to LOH. STR analysis indeed revealed LOH events of different lengths in these embryos. SNP analysis of one embryo, originating from mutant sperm but displaying the wild-type allele, did not demonstrate LOH (with a detection limit of 500bp). These findings suggest the occurrence of gene conversion, which rejects the formerly stated hypothesis that the observed LOH in human embryos can mostly be attributed to chromosome loss. In the iPSCs, whole-well (n = 3) and single colony (n = 33) data demonstrated a similar trend in genetic event distribution compared to the embryos, with the main difference that in 30% of the corrected reads/colonies, template use was observed. LOH was further analysed in nineteen single colonies. In all the colonies (3/3) corrected with the template, no LOH was present. When the colonies displayed additional mutagenesis, 7% (1/13) contained LOH events. In 33% (1/3) of the colonies showing only the wild-type allele (without signs of template use), LOH was observed. Limitations, reasons for caution SNP assays have a higher resolution compared to STR analysis, and enable distinction of LOH events between ‘gene conversion’ and ‘chromosome loss’. Therefore, more embryos and iPSC colonies will be analysed with SNP assays. However, informative SNPs define the resolution of our assay (currently 500 bp from Cas9 cut site). Wider implications of the findings Our results show that template usage seems to differ between human embryos and iPSCs, but that iPSCs do not solely rely on template use as well. This demonstrates that embryo experiments to study the genetic impact of CRISPR/Cas9 cannot fully be replaced by iPSCs experiments. Trial registration number Not Applicable

Multiple Stochastic Parameters Influence Genome Dynamics in a Heterozygous Diploid Eukaryotic Model.

The heterozygous diploid genome of Candida albicans displays frequent genomic rearrangements, in particular loss-of-heterozygosity (LOH) events, which can be seen on all eight chromosomes and affect both laboratory and clinical strains. LOHs, which are often the consequence of DNA damage repair, can be observed upon stresses reminiscent of the host environment, and result in homozygous regions of various sizes depending on the molecular mechanisms at their origins. Recent studies have shed light on the biological importance of these frequent and ubiquitous LOH events in C. albicans. In diploid Saccharomyces cerevisiae, LOH facilitates the passage of recessive beneficial mutations through Haldane’s sieve, allowing rapid evolutionary adaptation. This also appears to be true in C. albicans, where the full potential of an adaptive mutation is often only observed upon LOH, as illustrated in the case of antifungal resistance and niche adaptation. To understand the genome-wide dynamics of LOH events in C. albicans, we constructed a collection of 15 strains, each one carrying a LOH reporter system on a different chromosome arm. This system involves the insertion of two fluorescent marker genes in a neutral genomic region on both homologs, allowing spontaneous LOH events to be detected by monitoring the loss of one of the fluorescent markers using flow cytometry. Using this collection, we observed significant LOH frequency differences between genomic loci in standard laboratory growth conditions; however, we further demonstrated that comparable heterogeneity was also observed for a given genomic locus between independent strains. Additionally, upon exposure to stress, three outcomes could be observed in C. albicans, where individual strains displayed increases, decreases, or no effect of stress in terms of LOH frequency. Our results argue against a general stress response triggering overall genome instability. Indeed, we showed that the heterogeneity of LOH frequency in C. albicans is present at various levels, inter-strain, intra-strain, and inter-chromosomes, suggesting that LOH events may occur stochastically within a cell, though the genetic background potentially impacts genome stability in terms of LOH throughout the genome in both basal and stress conditions. This heterogeneity in terms of genome stability may serve as an important adaptive strategy for the predominantly clonal human opportunistic pathogen C. albicans, by quickly generating a wide spectrum of genetic variation combinations potentially permitting subsistence in a rapidly evolving environment.

Abstract 2590: Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma

Abstract Objective: Outcomes for patients with metastatic or relapsed osteosarcoma (OS) remain poor. Many OS samples have demonstrated a high proportion of mutational signature 3 (ms3), suggestive of homologous recombination DNA repair deficiency (HRD) or a BRCAness molecular phenotype. The HRD score, which quantifies chromosomal structural rearrangements by combining loss of heterozygosity (LOH), large state transitions (LST), and telomere allelic imbalances (TAI), has been used to assess HRD status in patients with breast and ovarian cancer, with high scores found to be predictive of PARP inhibitor sensitivity. While high HRD scores have been described in OS, little is known about how HRD scores vary by stage, site, pre vs post-therapy, presence of homologous recombination pathway or TP53 alterations, and their correlation to ms3 contribution. Methods: To address this issue, we evaluated 48 OS samples with tumor purity ≥ 20% from 46 patients who had 80x whole genome sequencing (WGS) performed through our pediatric expanded genomics platform. HRD scores were then estimated using scarHRD, a computational tool, which quantifies LOH, LST, and TAI events from WGS data and has shown a good correlation to SNP-array based methods. Results: Of the 48 samples analyzed, 25 samples were from primary sites and 23 from metastatic sites of disease. The median HRD score was 45 with a minimum score of 6 and a maximum score of 79. The 25th, 75th, and 90th percentiles of values were 35.7, 57, and 66.3 respectively. There was a significant difference in HRD score in patients with localized disease (n=22) vs metastatic disease (n=26) at time of diagnosis with median HRD (mHRD) scores of 41 and 51.5 (p = 0.027). The presence of a TP53 structural variant or single nucleotide variant (mHRD=47) was associated with higher HRD scores than TP53 wild-type samples (mHRD=29.5, p=0.04). There was no statistically significant difference in HRD scores in pre vs post-treated samples or primary site vs metastatic site samples. Samples with homologous recombination pathway alterations (n=10) were not associated with increased HRD score. There was a positive correlation (Spearman’s rho=0.425, p=0.001) between the number of mutations attributed to ms3 and HRD score. Conclusions: The range and distribution of HRD scores in our OS cohort closely resemble BRCA1/2 deficient breast cancer. Patients with metastatic disease at diagnosis were found to have higher HRD scores, representing a higher degree of genomic instability and increasing potential to accumulate resistance mutations. Higher HRD scores in TP53 mutated samples may be due to chromosomal instability caused by mechanisms other than HRD. The BRCAness phenotype in OS is now being targeted in a phase II clinical trial, combining the PARP inhibitor olaparib with the ATR inhibitor ceralasertib (NCT0441706), which includes integrated correlative biology to prospectively assess biomarkers of response such as HRD score. Citation Format: Michael David Kinnaman, Julia Pena, Max Levine, Nancy Bouvier, Elli Papaemmanuil, Suzanne Forrest, Katherine Janeway, Paul Meyers, Julia Glade Bender. Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma [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 2590.

Abstract 754: Pan cancer analysis of germline variants in the CPTAC dataset highlights their significance and functional importance in cancer development

Abstract Germline variants play important roles in cancer susceptibility and development, but identification and interpretation of germline variation impacting cancer risk remains a challenge. There has been little research on the impact of germline variants on somatic global/phosphoproteomics landscapes, and even less so for their impact on other tumor characteristics. Here we use cross-cancer genomic, transcriptomic, and global/phosphoproteomics datasets from CPTAC (Clinical Proteomic Tumor Analysis Consortium) to study the global impact of germline variants (common and rare) and the differential impact of germline vs somatic mutations on oncogenic processes, the tumor microenvironment (TME), and treatment responses. We analyze data from 1,093 cases across 10 cancer types (BR, CO, OV, EC, HNSCC, PDAC, LSCC, LUAD, GBM, and ccRCC). We investigate the impact of rare pathogenic variants across cancer types, discerning cancer-gene associations enriched with predisposing variants. We find novel instances of cancer-gene associations not found using TCGA data alone. Loss of heterozygosity (LOH) analyses of germline variants show that BRCA2 may be involved in predisposition not only in BRCA and OV, but also in LUAD. We also see ATM LOH events in ccRCC, CO, and LSCC. Analyses of the effects of germline vs somatic mutations on gene, protein, and phosphoprotein expression revealed significantly different protein expression levels in carriers of pathogenic germline variants vs somatic mutations, notably within core MMR-genes (MSH2, MSH6, PMS2). We examine trans-effects of germline variants on interacting proteins using proteomic/phosphoproteomic data, finding multiple trans-effects of BRCA1 pathogenic variants on the phosphoprotein expression of genes known to interact with BRCA1. Finally, we explore associations of germline and somatic causal events, noting proteomic and mutational signatures indicative of mutational processes taking place in the cell. We see direct relationships between mutations in core gene members of key DNA-damage response pathways (mismatch repair, homologous recombination repair) and the respective signatures associated with those pathways’ deficiencies. Additional analyses include: quantitative trait locus (QTL) analyses to assess the impact of common germline variants on gene (eQTL), protein (pQTL), and phosphoprotein levels (phQTL); analyses of the impact of germline variants on protein signaling during oncogenesis by mapping germline variants to post-translational modification sites); impact of germline variants on the immune landscape and tumor microenvironment (TME); and analyses of paired tumor and Normal Adjacent Tissue (NAT) samples for the discovery of immunogenic germline variants. This study illustrates the broad influence of germline variants on mutational landscapes and their functional impact on cancer. Citation Format: Fernanda Martins Rodrigues, Nadezhda V. Terekhanova, Oncogenic Drivers, Pathways Group, Clinical Proteomic Tumor Analysis Consortium, Gad Getz, Li Ding. Pan cancer analysis of germline variants in the CPTAC dataset highlights their significance and functional importance in cancer development [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 754.

Loss-of-heterozygosity facilitates a fitness valley crossing in experimentally evolved multicellular yeast.

Determining how adaptive possibilities do or do not become evolutionary realities is central to understanding the tempo and mode of evolutionary change. Some of the simplest evolutionary landscapes arise from underdominance at a single locus where the fitness valley consists of only one less-fit genotype. Despite their potential for rapid evolutionary change, few such examples have been investigated. We capitalized on an experimental system in which a significant evolutionary shift, the transition from uni-to-multicellularity, was observed in asexual diploid populations of Saccharomyces cerevisiae experimentally selected for increased settling rates. The multicellular phenotype results from recessive single-locus mutations that undergo loss-of-heterozygosity (LOH) events. By reconstructing the necessary heterozygous intermediate steps, we found that the evolution of multicellularity involves a decrease in size during the first steps. Heterozygous genotypes are 20% smaller in size than genotypes with functional alleles. Nevertheless, populations of heterozygotes give rise to multicellular genotypes more readily than unicellular genotypes with two functional alleles, by rapid LOH events. LOH drives adaptation that may enable rapid evolution in diploid yeast. Together these results show discordance between the phenotypic and genotypic multicellular transition. The evolutionary path to multicellularity, and the adaptive benefits of increased size, requires initial size reductions.