Allelic Imbalance Research Articles

Homologous Recombination Deficiency (HRD) in Hematological Malignancies

Introduction: The inability of a cell to repair the double-stranded breaks because of a defective homologous recombination repair (HRR) pathway results in Homologous Recombination Deficiency (HRD). The defective DNA repair leads to genomic instability and tumorigenesis, which has a characteristic scar predictive of the HRD phenotype. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi). However, there has been limited investigation of HRD phenotype in hematological malignancies, and herein, we aim to evaluate HRD phenotype using Optical genome mapping (OGM) technology in comparison to large gene next-generation sequencing (NGS) panel. Methods: This retrospective study included the analysis of 60 hematological malignancy samples that were evaluated with a 523-gene next-generation sequencing (NGS) panel and OGM. The HRD scores were calculated using a combination of three HRD signatures that included loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST). Results: In the 60 hematological malignancy cases analyzed with OGM and 523-gene NGS panel, the HRD scores were 11 (±9.8) with OGM compared to 2.4 (±3.8) with 523-gene NGS panel. OGM detected 67% additional structural variants that resulted from HRD in these cases. Conclusion: This study highlights the HRD scars that are missed by current technologies used for accessing HRD phenotype and presents OGM as an alternative tool with high resolution and sensitivity to accurately assess the HRD phenotype. We recommend the use of OGM for HRD phenotype evaluation as the most sensitive method, that could potentially lead to the differentiation of tumors with or without HRD phenotype in the ongoing clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Homologous Recombination DNA Repair Deficiency Score Is Independent Prognostic Variable in Myelodysplastic Syndrome/ Acute Myeloid Leukemia with Complex Karyotype

Introduction Homologous Recombination Deficiency (HRD) score is a measure of genomic complexity caused by disruption/suppression of the homologous recombination repair (HRR) pathway due to specific gene mutations (BRCA1/2, PALB2, RAD51, RAD51C, RAD51D), or epigenetic events. HRD score, which reflects underlying deficiency/mutations in the genes of this pathway, predicts adverse prognosis in ovarian/ breast cancer with favorable responses to PARP inhibitors. In hematological malignancies, although complex karyotype is a known poor prognostic marker, the clinical significance of HRD has not been explored. The goal of this study was to evaluate the clinical utility of HRD score in patients with AML/MDS, specifically those associated with complex karyotype. Methods We identified consecutive 101 treatment naïve MDS/AML [28 MDS; 73 AML) patients who underwent both clinical grade chromosomal microarray analysis (CMA) testing (Agilent aCGH+SNP 180K) and targeted 81-gene myeloid next-general sequencing mutation analysis (Illumina) in CLIA-certified lab at diagnosis. Following evaluation of CNA(s) and CN-LOH, composite HRD scores [combination of loss-of-heterozygosity (LOH >15Mb but <whole chromosome), telomeric allelic imbalance (TAI) >10Mb with telomere involvement and not crossing centromere, and large-scale state transitions (LST) between CNA(s) and CN-LOH >10Mb, excluding aneuploidy] were generated using algorithms from Nx Clinical v6.2 (BioDiscovery, Bionano Genomics). HRD calculation on the platform was previously validated on 34 hematological malignancies using Illumina SNP 850K. Clinico-pathological information was collected from the medical records. Overall survival was calculated from the time of diagnosis to death or last follow-up. Results The study cohort included 101 AML/MDS [28 MDS, 73 AML] with a median age of 67 years (range, 1-89). The median bone marrow blast percentage was 38% (0-95). Baseline characteristics are shown. The median HRD score for the entire cohort was 6 (range, 0-39). Of 11 patients with normal karyotype, 8 (73%) patients had HRD score of 0, while rest had a positive score due to CN-LOH. Sixteen had 1-2 abnormalities [median HRD score: 3, range, 0-10]. The remaining 74 patients had complex karyotype (CK, defined as 3 or more abnormalities) which was the focus of the rest of the analysis. The median HRD score was 8 (range, 0-39). Forty-three (58%) CK-AML/MDS cases had TP53 mutations. As expected, CK-AML/MDS cases had a higher median HRD score compared to non-complex cases [median HRD: 3; p=0.005]. TP53mut CK-AML/MDS had a significantly higher number of chromosomal aberrations [16 (1-39)] compared to TP53wt CK-AML/MDS [16 (1-39) vs. 5.5 (2-11); p<0.0001], but 11 (15%) CK-AML/MDS patients had HRD of 0 due to whole chromosome gains/losses (aneuploidy). TP53mut AML/MDS cases had a significantly higher median HRD [15 (0-39)] compared to TP53wt AML/MDS cases [3 (range, 0-11); p=<0.0001]. Over the follow-up time period, the median overall survival (OS) was 7.6 months. A higher HRD score correlated with worse outcome (p=0.001). An HRD score of 12 was the best cut-off based on outcomes (9.4 vs. 2.6 months; p=0.0002; HR 2.46; CI: 1.51-4]. By univariate analysis, age, CK, TP53 mutations and HRD score (both as continuous and categorical variable based on a cut-off of 12) associated with survival, but not gender or diagnosis (MDS vs. AML). By multivariate analysis, only age and HRD score (HR 2.3 95% CI: 1.387-3.666; p=0.001) retained independent prognostic significance. Conclusions This is the first study to explore a potential role for HRD as a prognostic marker in HM. CK-AML/MDS cases with TP53 mutations have a significantly higher HRD score compared to those with wild-type TP53. HRD score is an independent prognostic biomarker with a potential therapeutic opportunity for treatment using DNA damaging drugs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Homologous Recombination Deficiency and Tumor Mutational Burden as Clinical Biomarkers for Melanoma

Abstract Introduction/Objective Homologous recombination deficiency (HRD) cancers have high levels of genetic instability, potentially being targets for platinum agents or poly-ADP ribose polymerase inhibitors. Tumor mutational burden (TMB), which is used to guide immunotherapy, is thought to be associated with HRD. This study aimed to evaluate the frequency of HRD and its correlation with other genomic parameters in melanoma. Methods/Case Report A total of 14 cases with the diagnosis of melanoma were found in the genomic database between March 1, 2021 to July 30, 2021. One case had an EWSR1-ATF1 translocation and was excluded. Copy number variations and absence of heterozygosity detection were analyzed by the NxClinical software (BioDiscovery, El Segundo, CA). HRD was scored by three DNA-based measures of genomic instability (loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST)). HRD-LOH is the number of LOH regions &amp;gt; 15 Mb and do not cross the centromere. HRD-TAI is the number of regions &amp;gt; 10 Mb with allelic imbalance that extend to one of the subtelomeres and do not cross the centromere. HRD-LST is the number of break points between regions &amp;gt; 10 Mb. A high HRD score and LOH percentage were defined as ≥ 42 and ≥ 16%, respectively. Results (if a Case Study enter NA) Of the 13 cases, 10 had a high TMB and 3 had a low TMB. A high HRD score was found in 3 cases; high LOH percentage was found in 4 cases. PDL-1 was expressed in 8 of the 11 cases tested. A high HRD score correlated with high LOH percentage by Pearson’s chi-squared test (p &amp;lt; 0.01). No significant association was found between TMB and HRD score or LOH percentage. Common alterations among cases with high TMB included mutations in NRAS (3/13; 23.1%), CDKN2A (2/13; 15.4%), BRAF (2/13; 15.4%), BRCA2 (2/13; 15.4%), RAF1 (2/13; 15.4%), RET (1/13; 7.7%), IDH1 (1/13; 7.7%), PALB2 (1/13; 7.7%), BRCA1 (1/13; 7.7%), ERBB2 (1/13; 7.7%), NF1 (1/13; 7.7%), MLH1 (1/13; 7.7%), ATM (1/13; 7.7%) and MET (1/13; 7.7%). One case had amplifications in CCND1, FGF 3/4/19 and MET. Among cases with low TMB, alterations included MYC amplification (1/3; 33.3%) and BRAF mutations (2/3; 66.6%). Conclusion In our study, HRD score is not associated with TMB status. There is a strong correlation between HRD score and LOH percentage, suggesting that LOH is a major component in the genomic scarring seen in HRD. HRD may be an independent biomarker of immunogenicity to guide immunotherapy.

Association between the rs3812316 Single Nucleotide Variant of the MLXIPL Gene and Alpha-Linolenic Acid Intake with Triglycerides in Mexican Mestizo Women.

The carbohydrate response element binding protein (ChREBP) is a key transcription factor to understand the gene-diet-nutrient relationship that leads to metabolic diseases. We aimed to analyze the association between the rs17145750 and rs3812316 SNVs (single nucleotide variants) of the MLXIPL gene with dietary, anthropometric, and biochemical variables in Mexican Mestizo subjects. This is a cross-sectional study of 587 individuals. Genotyping was performed by allelic discrimination. In addition, liver and adipose tissue biopsies were obtained from a subgroup of 24 subjects to analyze the expression of the MLXIPL gene. An in silico test of the protein stability and allelic imbalance showed that rs17145750 and rs3812316 showed a high rate of joint heritability in a highly conserved area. The G allele of rs3812316 was associated with lower triglyceride levels (OR = -0.070 ± 0.027, p &lt; 0.011, 95% CI = -0.124 to -0.016), the production of an unstable protein (ΔΔG -0.83 kcal/mol), and probably lower tissue mRNA levels. In addition, we found independent factors that also influence triglyceride levels, such as insulin resistance, HDL-c, and dietary protein intake in women. Our data showed that the association of rs3812316 on triglycerides was only observed in patients with an inadequate alpha-linolenic acid intake (1.97 ± 0.03 vs. 2.11 ± 0.01 log mg/dL, p &lt; 0.001).

114. Comparison of optical genome mapping, CMA, and 523-gene NGS panel for Homologous Recombination Deficiency calculation

Homologous Recombination Deficiency (HRD) is characterized by the inability of a cell to repair the double-stranded breaks using the homologous recombination repair (HRR) pathway. The deficiency of the HRR pathway results in defective DNA repair, leading to genomic instability and tumorigenesis. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]?ribose) polymerase (PARP) inhibitors (PARPi). However, there are no standardized methods to define, measure, and report HRD in diagnostic laboratories. Herein, we compare optical genome mapping (OGM), chromosomal microarray (CMA), and 523-gene NGS panel for HRD scar calculations. Traditionally, CMA has been used to calculate either loss of heterozygosity (LOH), or a combination of LOH, telomeric allelic imbalance (TAI), and large scale transitions (LST), where a combination is more accurate than LOH score alone for HRD calculation. We demonstrate the use of a 523-gene NGS panel to detect HRD using NxClinical software with automated HRD calculation using LOH, TAI, and LST. More interestingly, we compare OGM with CMA and NGS panel and highlight the potential advantages of using OGM, which reveals the genomic instability with a much higher resolution compared to other methodologies. For example, in a case of glioblastoma, the HRD score with CMA was 7 (TAI:1, HRD-LOH: 2, LST: 4) compared to 29 with OGM (TAI:1, HRD-LOH:2, LST:25). The HRD definition, measurement, and reporting criteria need to be standardized for each platform, while OGM might be the most sensitive platform for HRD calculation.

Integrative Genomic Tests in Clinical Oncology.

Many clinical decisions in oncology practice rely on the presence or absence of an alteration in a single genetic locus, be it a pathogenic variant in a hereditary cancer gene or activating mutation in a drug target. In addition, there are integrative tests that produce continuous variables and evaluate complex characteristics of the entire tumor genome. Microsatellite instability (MSI) analysis identifies tumors with the accumulation of mutations in short repetitive nucleotide sequences. This procedure is utilized in Lynch syndrome diagnostic pipelines and for the selection of patients for immunotherapy. MSI analysis is well-established for colorectal malignancies, but its applications in other cancer types lack standardization and require additional research. Homologous repair deficiency (HRD) indicates tumor sensitivity to PARP inhibitors and some cytotoxic drugs. HRD-related "genomic scars" are manifested by a characteristic pattern of allelic imbalances, accumulation of deletions with flanking homology, and specific mutation signatures. The detection of the genetic consequences of HRD is particularly sophisticated and expensive, as it involves either whole genome sequencing (WGS) or the utilization of large next-generation sequencing (NGS) panels. Tumor mutation burden (TMB) can be determined by whole exome sequencing (WES) or middle-throughput NGS multigene testing. Although TMB is regarded as an agnostic indicator of tumor sensitivity to immunotherapy, the clinical utility of this test is proven only for a few cancer types.

CSMD1 Shows Complex Patterns of Somatic Copy Number Alterations and Expressions of mRNAs and Target Micro RNAs in Esophageal Squamous Cell Carcinoma.

Simple SummaryHuman Cub and Sushi Multiple Domains 1 (CSMD1) is a novel candidate tumor-suppressor gene. We investigated CSMD1 in esophageal squamous cell carcinoma (ESCC) by performing an integrated analysis of somatic DNA alterations (i.e., copy number alteration, allelic imbalance, and loss of heterozygosity) with RNA expressions (mRNA and target miRNAs) on specimens from the same ESCC patients, using data from SNP, miRNA, and RT-PCR arrays. Our results indicate that the CSMD1 gene may play a role in the development of ESCC through complex patterns involving somatic alterations and mRNA expression. Furthermore, somatic copy number alterations in SNPs located in non-coding regions of CSMD1 appear to influence expression of both this gene and its target miRNAs.Background: Human Cub and Sushi Multiple Domains 1 (CSMD1) is a novel candidate tumor-suppressor gene that codes for multiple domains, including complement regulatory and adhesion proteins, and has recently been shown to have alterations in multiple cancers. We investigated CSMD1 in esophageal squamous cell carcinoma (ESCC) by performing an integrated analysis on somatic copy number alterations (CNAs), including copy-number gain or loss, allelic imbalance (AI), loss of heterozygosity (LOH), and the expressions of mRNA and its target miRNAs on specimens from the same patients with ESCC. Results: (i) Two-thirds of ESCC patients had all three types of alterations studied—somatic DNA alterations in 70%, and abnormal expressions of CSMD1 RNA in 69% and in target miRNAs in 66%; patterns among these alterations were complex. (ii) In total, 97% of 888 CSMD1 SNPs studied showed somatic DNA alterations, with most located near exons 4–11, 24–25, 39–40, 55–56, and 69–70. (iii) In total, 68% of SNPs with a CNA were correlated with expression of CSMD1. (iv) A total of 33 correlations between non-coding SNPs and expression of CSMD1 target miRs were found. Conclusions: Our results indicate that the CSMD1 gene may play a role in ESCC through complex patterns of DNA alterations and RNA and miRNA expressions. Alterations in some somatic SNPs in non-coding regions of CSMD1 appear to influence expression of this gene and its target miRNAs.

Genetic risk of osteoarthritis operates during human skeletogenesis.

Osteoarthritis (OA) is a polygenic disease of older people resulting in the breakdown of cartilage within articular joints. Although it is a leading cause of disability, there are no disease-modifying therapies. Evidence is emerging to support the origins of OA in skeletogenesis. Whereas methylation quantitative trait loci (mQTLs) co-localizing with OA genome-wide association study signals have been identified in aged human cartilage and used to identify effector genes and variants, such analyses have never been conducted during human development. Here, for the first time, we have investigated the developmental origins of OA genetic risk at seven well-characterized OA risk loci, comprising 39 OA-mQTL CpGs, in human fetal limb (FL) and cartilage (FC) tissues using a range of molecular genetic techniques. We identified significant OA-mQTLs at 14 and 29 CpGs in FL and FC tissues, respectively, and compared our results with aged cartilage samples (AC). Differential methylation was observed at 26 sites between FC and AC, with the majority becoming actively hypermethylated in old age. Notably, 6/9 OA effector genes showed allelic expression imbalances during fetal development. Finally, we conducted ATAC-sequencing in cartilage from the developing and aged hip and knee to identify accessible chromatin regions and found enrichment for transcription factor binding motifs including SOX9 and FOS/JUN. For the first time, we have demonstrated the activity of OA-mQTLs and expression imbalance of OA effector genes during human skeletogenesis. We show striking differences in the spatiotemporal function of these loci, contributing to our understanding of OA aetiology, with implications for the timing and strategy of pharmacological interventions.

ABCL-371 STR Loci Allele Imbalance at 9p24.1 in Primary Mediastinal B-Cell Lymphoma

Molecular factors that determine the prognosis of immunotherapy response in primary mediastinal B-cell lymphoma (PMBCL) are not yet described. PD-L1/L2 expression levels in tumor samples have demonstrated clinical utility for many solid tumors. PD-L1/L2 expression levels could be affected by chromosomal lesions on 9p24.1 (losses or gains). Short tandem repeat (STR) analysis of appropriate markers is the simplest way to detect these lesions. To analyze possible associations between STR loci allele imbalance (AI) on 9p24.1 and PD-L1 expression levels in tumor cells from PMBCL patients. Two STR Loci (gt) n and (ttat)m on 9p24.1 in tumor cells were analyzed for a cohort of 36 PMBCL patients admitted to the National Medical Research Center for Hematology (Moscow, Russia) from 2013 to 2022. For six patients, STR data were compared with PD-L1 expression assessed by immunohistochemistry. DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR analysis for AI was performed by PCR with original primers. The fragment analysis was performed on an ABI3130 Genetic Analyzer. Inclusion criteria: de novo diagnosed PMBCL patients. pretreatment. The heterozygosity level for the (gt)n locus was 31 of 36 patients (86%), and AI was found in 16 of 31 (51.6%). The heterozygosity level for the (ttat)m locus was 20 of 36 patients (56%), and AI was found in 9 of 20 (45%). One patient was homozygous for both loci. No patients were found with AI at one locus and a normal heterozygous variant at the second locus. PD-L1 expression in tumor cells was evaluated for six patients. In two patients with aberrant STR loci, PD-L1 expression was found in 75%-100% of tumor cells. None of the four patients with normal 9p24.1 STR markers had PD-L1-positive cells. The high incidence of AI in the 9p24.1 locus indicates its possible involvement in the pathogenesis of PMBCL. The association between AI and PD-L1 expression in tumor cells requires confirmation in an extended cohort of patients. This study was supported by Rakfond grant 2/2020.

Poster: ABCL-371 STR Loci Allele Imbalance at 9p24.1 in Primary Mediastinal B-Cell Lymphoma

Poster: ABCL-371 STR Loci Allele Imbalance at 9p24.1 in Primary Mediastinal B-Cell Lymphoma

Breaking the reproductive barrier of divergent species to explore the genomic landscape.

Background: Climate change will have significant consequences for species. Species range shifts induce the emergence of new hybrid zones or the spatial displacement of pre-existing ones. These hybrid zones may become more porous as alleles are passed from one species to another. Currently, hybridization between highly divergent species living in sympatry seems extremely limited. Indeed, this phenomenon involves breaking two barriers. The first is the pre-mating barrier, related to the reproductive phenology of the two species. The second is the post-zygotic barrier, related to the genetic divergence between these species. Here, we were interested in identifying new hybridization patterns and potential implications, especially in the context of environmental modifications. Methods: We sampled Telestes souffia and Parachondrostoma toxostoma wild specimens from different locations across France and genotyped them for SNP markers. We identified discriminant loci using F1-hybrid specimens and parental species and performed principal component analysis and Bayesian model-based clustering to analyze phylogenetic information. Furthermore, we assessed deviation in allele frequency from F1 to F2 and for Hardy–Weinberg equilibrium for F2 and assessed gene function associated with two F2 cohorts. Results: We demonstrate that by breaking the ecological barrier, massive introgressive hybridization is possible between two endemic lineages of Cyprinidae belonging to two distinct genera. For both cohorts studied (=2 cm and >2 cm), a large majority of loci (>88%) presented no deviation in allele frequency and no departure from the Hardy–Weinberg equilibrium. For individuals beyond the 2 cm stage, two phenomena were observed. The first was an allelic imbalance in favor of P. toxostoma, for some genomic regions, with genes involved in developmental regulatory processes, cytoskeletal organization, and chromosome organization. The second was an excess of heterozygous loci coupled with an equilibrium of allelic frequencies for genes involved in immune response and kidney/liver development. Moreover, the 2 cm-sized specimens with high mortality yielded a particular genomic signature. Conclusion: Our study displayed important results for understanding the early stages of hybridization between divergent lineages and predicting the emergence of future hybrid zones in the wild. Moreover, this hybridization generates a wide spectrum of hybrids that are a potential source of important evolutionary novelties.

Precise identification of cancer cells from allelic imbalances in single cell transcriptomes

A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data.

In-house testing for homologous recombination repair deficiency (HRD) testing in ovarian carcinoma: a feasibility study comparing AmoyDx HRD Focus panel with Myriad myChoiceCDx assay.

Homologous recombination repair (HRR) is the main mechanism of repair of DNA double-strand breaks. Its deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs). BRCA1/2 mutations and/or other aberrations in genes of HRR are well known causes of HRD and genomic instability. Poly ADP-ribose polymerase inhibitors (PARPi) have revolutionized the management of BRCA mutant EOCs and demonstrated activity in HRD tumor cells. Determining HRD status can provide informations on the magnitude of benefit for PARPi therapy. Myriad MyChoice CDx is a next generation sequencing- based in vitro diagnostic test that assesses the Genomic Instability Score (GIS) which is an algorithmic measurement of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions using DNA isolated from formalin-fixed paraffin embedded tumor tissue specimens. However Myriad MyChoice CDx, is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy. In this report, we described our experience in performing the HRD Focus AmoyDx (Amoy Diagnostics Ltd, Xiamen, Fujian, China) on the same samples of EOCs evaluated with Myriad MyChoiceCDx assay. The overall percent agreement between AmoyDx and Myriad was 87.8% (65 of 74 tumors tested). All the 36 AmoyDx negative cases were confirmed to be negative by Myriad (negative predictive value, 100%). The concordance of the results with the gold standard Myriad MyChoice CDx assay suggest the feasibility and reliability of HRD testing in diagnostic laboratories with high-throughput NGS platforms and qualified personnel.

576P Genome instability index used for the optimization of molecular classification of endometrial carcinoma

The molecular classification of endometrial carcinoma (EC) has divided patients into four distinct prognostic subtypes based on the genomic abnormalities. However, in clinical application, the genomic copy number index lacks a simple and accurate detection method, and the method of using p53 immunohistochemistry or TP53 mutation as a surrogate for copy number status needs to be optimized. Here, we explored the application of genome instability (GI) index in molecular classification of EC. We assessed a retrospective cohort of EC from Beijing Cancer Hospital between 2011 and 2020. Genomic profiling of tumor tissues from EC patients was sequenced by OncoScreen Plus Panel of 520 cancer-related genes, and tumor immune microenvironment was evaluated using multiplex immunofluorescence staining. GI index was calculated as the sum of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions scores. 143 patients were enrolled in this study, with the median follow-up time of 38.77 months. Molecular analysis firstly identified four different prognostic subgroups: POLE mutations (mut), MSI-H, TP53 wildtype, and TP53 mut. We also divided patients into two groups based on GI index: GI-high and GI-low, and patients with GI-high were found in three subgroups except for POLE mut. Further analysis showed that GI status had a more important effect on prognosis than TP53 mutation. Therefore, we proposed a three-group molecular classification based on POLE mutation and GI index: POLE mut (n=9, 6.5%), GI-low (n=116, 82.9%), and GI-high (n=15, 10.7%). Patients with GI-high had much poorer survival than those with GI-low [disease-free survival: hazard ratio (HR), 17.87, 95% CI, 6.67-50.00, P < 0.001; overall survival: HR, 16.75, 95% CI, 4.76-50.00, P < 0.001]. The application in TCGA-UCEC cohort containing 481 EC patients confirmed that this new method worked well for risk stratification of EC. Further analysis showed that patients in GI-high group had higher tumor grade, aneuploidy and lower T cell infiltrations. The one-stop method of GI index combined with POLE mutation offers more accurate and stable disease risk stratification and demonstrates the potential for guiding therapy for EC patients.

Allelic expression imbalance in articular cartilage and subchondral bone refined genome-wide association signals in osteoarthritis.

To present an unbiased approach to identify positional transcript single nucleotide polymorphisms (SNPs) of osteoarthritis (OA) risk loci by allelic expression imbalance (AEI) analyses using RNA sequencing of articular cartilage and subchondral bone from OA patients. RNA sequencing from 65 articular cartilage and 24 subchondral bone from OA patients was used for AEI analysis. AEI was determined for all genes present in the 100 regions reported by the genome-wide association studies (GWAS) catalog that were also expressed in cartilage or bone. The count fraction of the alternative allele (φ) was calculated for each heterozygous individual with the risk SNP or with the SNP in linkage disequilibrium (LD) with it (r2 > 0.6). Furthermore, a meta-analysis was performed to generate a meta-φ (null hypothesis median φ = 0.49) and P-value for each SNP. We identified 30 transcript SNPs (28 in cartilage and two in subchondral bone) subject to AEI in 29 genes. Notably, 10 transcript SNPs were located in genes not previously reported in the GWAS catalog, including two long intergenic non-coding RNAs (lincRNAs), MALAT1 (meta-φ = 0.54, FDR = 1.7×10-4) and ILF3-DT (meta-φ = 0.6, FDR = 1.75×10-5). Moreover, 12 drugs were interacting with seven genes displaying AEI, of which seven drugs have been already approved. By prioritizing proxy transcript SNPs that mark AEI in cartilage and/or subchondral bone at loci harbouring GWAS signals, we present an unbiased approach to identify the most likely functional OA risk-SNP and gene. We identified 10 new potential OA risk genes ready for further translation towards underlying biological mechanisms.

In Vivo Clonal Analysis Reveals Random Monoallelic Expression in Lymphocytes That Traces Back to Hematopoietic Stem Cells.

Evaluating the epigenetic landscape in the stem cell compartment at the single-cell level is essential to assess the cells’ heterogeneity and predict their fate. Here, using a genome-wide transcriptomics approach in vivo, we evaluated the allelic expression imbalance in the progeny of single hematopoietic cells (HSCs) as a read-out of epigenetic marking. After 4 months of extensive proliferation and differentiation, we found that X-chromosome inactivation (XCI) is tightly maintained in all single-HSC derived hematopoietic cells. In contrast, the vast majority of the autosomal genes did not show clonal patterns of random monoallelic expression (RME). However, a persistent allele-specific autosomal transcription in HSCs and their progeny was found in a rare number of cases, none of which has been previously reported. These data show that: 1) XCI and RME in the autosomal chromosomes are driven by different mechanisms; 2) the previously reported high frequency of genes under RME in clones expanded in vitro (up to 15%) is not found in clones undergoing multiple differentiation steps in vivo; 3) prior to differentiation, HSCs have stable patterns of autosomal RME. We propose that most RME patterns in autosomal chromosomes are erased and established de novo during cell lineage differentiation.

Detection of DNA copy number alterations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of single nucleotide polymorphisms.

Copy number alterations (CNAs) are frequently found in malignant tissues. Different approaches have been used for CNA detection. However, it is not easy to detect a large panel of CNA targets in heterogenous tumors. We have developed a CNAs detection approach through quantitatively analyzed allelic imbalance by allelotyping single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the copy number changes were quantified by real-competitive PCR (rcPCR) to distinguish loss of heterozygosity (LOH) and genomic amplification. The approach was used to validate the CNA regions detected by next generation sequencing (NGS) in early-stage lung carcinoma. CNAs were detected in heterogeneous DNA samples where tumor DNA is present at only 10% through the SNP based allelotyping. In addition, two different types of CNAs (loss of heterozygosity and chromosome amplification) were able to be distinguished quantitatively by rcPCR. Validation on a total of 41 SNPs from the selected CNA regions showed that copy number changes did occur, and the tissues from early-stage lung carcinoma were distinguished from normal. CNA detection by MALDI-TOF MS can be used for validating potentially interesting genomic regions identified from next generation sequencing, and for detecting CNAs in tumor tissues consisting of a mixture of neoplastic and normal cells.

Homologous recombination deficiency (HRD) score predicts response to platinum-based chemotherapies in Chinese high grade serous ovarian cancer patients (188)

<b>Objectives:</b> Homologous recombination repair deficiency (HRD), as a major feature of high-grade serous ovarian, is associated with the efficacy of PARP inhibitor (PARPi) therapy. Tumors present with HRD are characterized by a defect in DNA double-strand break repair, which is thought to render particularly sensitive to inter-strand cross-linking agents, including platinum analogs. However, whether HRD status confers sensitivity to platinum-based chemotherapies in high-grade serous ovarian patients remains poorly defined. <b>Methods:</b> In this study, we used the machine learning method to build a genomic scar (GS) model to predict HRD, which takes different types of chromosomal copy numbers into consideration for the calculation of genomic instability. GS score ≥50 was defined as HRD positive, accounting for 94.5% of <i>BRCA</i>-deficient events. HRD score, consisting of heterozygous (LOH), telomeric allelic imbalance (TAI), and Large-scale transition (LST), was also determined (HRD-positive [score ≥42] versus HRD-negative [score <42]), and both GS score and HRD score impacts on clinical efficacy were explored. <b>Results:</b> A total of 94 high-grade serous ovarian patients who received first-line platinum-based chemotherapy but no further PARPi maintenance therapy was picked out and analyzed for the predictive value of GS score and HRD score in platinum-based chemotherapy efficacy. GSS-positive was observed in 78/94 (82.9%) patients, and HRD-positive was observed in 74/94 (78.7%) patients, concordance between GS score and HRD score was 91%. Progression-free survival (PFS) (GSS: HR: 0.604; p=0.00082; HRD: HR: 0.64; p=0.00134), overall survival (OS) (GSS: HR: 0.482; p=0.00155; HRD: HR: 0.496; p=0.00231), and platinum-free interval (PFI) (GSS: HR: 0.621; p=0.00204; HRD: HR: 0.664; p=0.00482) were significantly longer in both GS-positive and HRD-positive groups (Figure 1). In <i>BRCA</i>-wildtype patients, survival differences were also seen in GSS-positive versus GSS-negative (p=0.007674) and HRD-positive versus HRD-negative (p=0.017588) populations (Figure 2). Platinum-resistant patients with a PFI of less than six months show a significantly lower GSS-positive rate and HRD-positive rate than platinum-sensitive patients (PFI>6 months) (Figure 2).Fig. 1 <b>Conclusions:</b> HRD status identifies high-grade serous ovarian patients and remains a significant predictor of response to platinum-based chemotherapy. GSS showed high concordance with the HRD score, which implied the high robustness of the GS model.

Detection of molecular signatures of homologous recombination deficiency in endometrial cancer (180)

<b>Objectives:</b> Homologous recombination repair deficiency (HRD) is observed in endometrial cancer (EC). Tumors harboring HRD are candidates for poly-ADP-ribose polymerase inhibitor (PARP inhibitor), and several PARP inhibitor trials in EC are underway. However, the prevalence and mechanism of HRD in endometrial cancer are less characterized. <b>Methods:</b> Ninety-five patients with endometrial cancer with a median follow-up of 55 months were included in this study. The homologous repair status was determined using genomics scar scores (including loss-of-heterozygosity, large-scale transitions, number of telomeric allelic imbalances, and ploidy). In addition, microsatellite instability (MSI), tumor mutation burden (TMB), and genomic mutation landscape were obtained using a panel-based next-generation sequencing assay. <b>Results:</b> Among 95 patients, we detected 15/95 (15.8%) patients with HRD (HRD score > 0). Homologous repair pathway and DNA damage pathway alterations did not contribute to HRD. Patients with HRD were associated with increased <i>TP53</i> mutation (HRD vs HRP, 92.3% vs 15.4%, p < 0.0001) and lower TMB (HRD vs HRP; 2.8 vs 26.3, p=0.02). Although patients with HRD presented significantly higher stages (p = 0.03), only marginal p values were found related to recurrence rate (p = 0.08). Median PFS was numerically higher in HRD (20 months, 95% CI: 14.1 - 25.9) versus HRP (40 months, 95% CI: 7.1 -72.9). Additionally, the HRD score was not related to the clinical outcomes of. platinum-based chemotherapy (<i>n</i> = 45) or immunotherapy (<i>n</i> = 20) for the treatment of recurrence. <b>Conclusions:</b> A subset of endometrial tumors has genomic features suggestive of HRD and has a tendency towards a worse prognosis. However, EC patients with HRD may be more likely to benefit from therapies, such as PARP inhibitors.

Noninvasive fetal genotyping of single nucleotide variants and linkage analysis for prenatal diagnosis of monogenic disorders

BackgroundHigh-cost, time-consuming and complex processes of several current approaches limit the use of noninvasive prenatal diagnosis (NIPD) for monogenic disorders in clinical application. Thus, a more cost-effective and easily implementable approach is required.MethodsWe established a low-cost and convenient test to noninvasively deduce fetal genotypes of the mutation and single nucleotide polymorphisms (SNPs) loci by means of targeted amplification combined with deep sequencing of maternal genomic and plasma DNA. The sequential probability ratio test was performed to detect the allelic imbalance in maternal plasma. This method can be employed to directly examine familial pathogenic mutations in the fetal genome, as well as infer the inheritance of parental haplotypes through a group of selected SNPs linked to the pathogenic mutation.ResultsThe fetal mutations in 17 families with different types of monogenic disorders including hemophilia A, von Willebrand disease type 3, Duchenne muscular dystrophy, hyper-IgM type 1, glutaric acidemia type I, Nagashima-type palmoplantar keratosis, and familial exudative vitreoretinopathy were identified in the study. The mutations included various forms: point mutations, gene inversion, deletions/insertions and duplication. The results of 12 families were verified by sequencing of amniotic fluid samples, the accuracy of the approach in fetal genotyping at the mutation and SNPs loci was 98.85% (172/174 loci), and the no-call rate was 28.98% (71/245 loci). The overall accuracy was 12/12 (100%). Moreover, the approach was successfully applied in plasma samples with a fetal fraction as low as 2.3%.ConclusionsWe have shown in this study that the approach is a cost-effective, less time consuming and accurate method for NIPD of monogenic disorders.