Clinical Genomics Research Articles

P-340 Reconciling diversity in health and genomic data collection with the regulation of AI in clinical genomics

Abstract Study question How far the existing and emerging EU regulations address concerns about lack of diversity in genomic databases which used to train AI in genomics? Summary answer The main regulations in the European Union provide no clear answers for reconciling the need for more diverse genetic data with regulatory standards. What is known already AI researchers need high quality genomic data that reflect the underlying diversity of human populations to develop and validate safe, effective, and equitable tools for genomic medicine. The problem is that most of the genomic data that are available to develop such tools are of insufficient quality, due to inappropriate use of population descriptors, and over representative of individuals of European ancestry. We used the example of developing algorithms to calculate polygenic risk scores ( PRS) and concerns about their accuracy for various sub-populations to illustrate our discussion. Study design, size, duration We have analysed the relevant EU regulations including, the upcoming AI Act, Medical Devices Regulation ( MDR), In Vitro Diagnostic Medical Devices ( IVDR) and the GDPR to assess how the concerns related to lack of diversity in genetic/genomic databases have been addressed. Participants/materials, setting, methods NA. Main results and the role of chance The main regulations for clinical AI in the European Union, namely those regulating medical devices, provide no clear answers for reconciling the need for more diverse genetic data with regulatory standards. On the other hand, some of the data protection regulation requirements arising from the EU General Data Protection Regulation (GDPR) may further challenge collection of sensitive attributes such as race/ancestry which might be needed for brining diversity to databases. On the other hand, several interesting impending new regulations addressing AI and algorithms such as the EU’s AI Act may help in some ways, but still fail to ultimately grapple with the fundamental obstacles in producing the diverse genomic data needed to achieve regulatory objectives. In particular, the proposed requirements related to data quality and transparency need to be accompanied by further guidance on how to define representativeness and how to address long-term impact on fairness concerns in the context of medical AI, when the developed tools are not of optimal use for populations not descending from European ancestry. Limitations, reasons for caution NA. Wider implications of the findings The legal analysis provided in this paper will provide insights for broader types of AI tools which may be implemented in the context of Assisted Reproductive Technologies and beyond. Trial registration number NA

P-272 Cannabis negatively impacts female fertility: an in vitro and clinical investigation

Abstract Study question How does tetrahydrocannabinol (THC) impacts human oocyte maturation and female fertility? Summary answer THC alters the oocyte’s RNA profile and affects meiotic spindle formation resulting in an increased rate of aneuploidy in the oocyte and in the blastocyst. What is known already In the past decades, cannabis consumption and legalization has been increasing globally. It is the third most used substance by reproductive aged people and our group demonstrated that tetrahydrocannabinol (THC) reaches the ovarian follicle. THC is known to impact male fertility, but due to the challenges associated with investigating the female gamete in humans, there is no study about the human oocyte. Animal studies have shown that THC impacts oocyte maturation in a concentration dependent manner, but between models, the results are conflicting. Study design, size, duration For the in vitro exposure assessment, GV oocytes naïve to THC were collected from consenting patients undergoing IVF-ICSI from July 2022 to June 2024 (IRB: Veritas #16518). Per patient, oocytes were split into three groups: Control, THC1 (physiological concentration of THC, based on previous human studies) and THC2 (supra-physiological concentration, based on animal studies), and cultured for 24h in our standard clinical in vitro maturation media. Participants/materials, setting, methods Single-cell RNAseq and pathway analysis were conducted to identify key pathways altered by THC treatments (n = 86 from 24 patients). Spindle morphology was assessed using confocal microscopy. Polar body biopsy was performed and ploidy assessed using our clinical low pass whole genome sequencing pipeline. Also, we conducted a multi-year retrospective study (n = 1059 patients) by measuring the concentration of THC in their follicular fluid by mass spectrometry and collecting and comparing IVF outcomes between cases and controls. Main results and the role of chance In vitro, THC treatment marginally increased the maturation rate (46% for Ctrl (n = 103), 51% for THC1 (n = 103) and 58% for THC2 (n = 102)). Using time-lapse imaging, no difference was observed between key developmental stages of oocyte maturation including GVBD and polar body extrusion. Single-cell RNAseq revealed significant alterations to the transcriptome profile of THC treated oocytes. When treated with THC1 (physiological concentration), 89 upregulated and 227 downregulated genes were identified, this was exaggerated when treated with THC2 (supraphysiologic concentration) 402 upregulated and 62 downregulated genes were identified. Pathway analysis showed that differentially expressed genes in THC1 group were associated with immune system and synaptic transmission, while THC2 group showed genes enriched in meiotic spindle formation and apoptosis. Moreover, preliminary results demonstrated abnormal spindle configuration and misaligned chromosomes in THC-treated groups and a concurrent decrease in the number of euploid oocytes. Furthermore, retrospective analysis of clinical IVF outcomes revealed that patients whose FF was positive for THC (62 patients, 6%) also had a slight increased maturation rate (77% vs 73%, p=ns), but a significant reduction of their embryo euploid rate when compared to control patients (59% vs 70%, p < 0.01). Limitations, reasons for caution The in vitro exposure to THC utilized GV oocytes that did not mature after ovarian stimulation, so they might have a reduced capacity of becoming MII. The THC measurement was assessed only on the day of retrieval and we do not know the route, dose, or duration of THC consumption. Wider implications of the findings This is the first study investigating the impact of cannabis on human oocytes. Our data suggests that cannabis consumption may reduce the number of euploid embryos available for transfer. With cannabis legalization increasing worldwide, further investigation into its consequences is critical for clinical consultation and legalization guidelines. Trial registration number Not applicable

Molecular characteristics of patients with colorectal signet-ring cell carcinoma with different ABO blood groups

BackgroundColorectal signet-ring cell carcinoma (SRCC) is a rare subtype of malignant adenocarcinoma, accounting for approximately 1 % of colorectal cancer (CRC) cases. Its biomarkers and molecular characteristics remain controversial, and there are no specific therapeutic targets or strategies for its clinical treatment. MethodsA retrospective study was conducted between January 2010 and December 2021. 1058 colorectal cancer cases from the Sun Yat-sen University Cancer Center and 489 cases from the Tumor Genome Atlas Project were included in the analysis, of which 64 were SRCC. Data extraction included patient demographics, blood types and risk factors, including clinical variables and genomics (either a 19-gene panel NGS or 1021-gene panel NGS). Univariate analyses were performed to identify factors significantly associated with overall survival. ResultsThe blood groups of 27 (42.2 %), 18 (28.1 %), 12 (18.8 %), and seven (10.9 %) patients were classified as O, A, B, and AB, respectively. We found that O was a unique blood group characterized by a low frequency of KRAS mutations, a high frequency of heterozygosity at each HLA class I locus, and a high tumor mutational burden (TMB). Patients in blood group A with high-frequency KRAS mutations and those in blood group B with anemia and metabolic abnormalities required targeted treatment. Furthermore, genetic alterations in SRCC differed from those in adenocarcinoma and mucinous adenocarcinoma. ConclusionsOur study revealed genomic changes in SRCC patients across different blood groups, which could advance the understanding and precise treatment of colorectal SRCC.

The CRISPR-Cas System and Clinical Applications of CRISPR-Based Gene Editing in Hematology with a Focus on Inherited Germline Predisposition to Hematologic Malignancies.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single-guide RNA (sgRNA)-based gene editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, highly instructive, and inspiring for future advances in medicine. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent β-thalassemia has renewed hope for treating other hematologic diseases, including patients with a germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-inspired gene therapies. The purpose of this paper is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy, preceded by a brief description of the current tools being used in clinical genome editing; and third, a presentation of the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with a germline predisposition to hematologic malignancies.

Established the prediction model of early-stage non-small cell lung cancer spread through air spaces (STAS) by radiomics and genomics features.

This study was aimed to establish a prediction model for spread through air spaces (STAS) in early-stage non-small cell lung cancer based on imaging and genomic features. We retrospectively collected 204 patients (47 STAS+ and 157 STAS-) with non-small cell lung cancer who underwent surgical treatment in the Jinling Hospital from January 2021 to December 2021. Their preoperative CT images, genetic testing data (including next-generation sequencing data from other hospitals), and clinical data were collected. Patients were randomly divided into training and testing cohorts (7:3). The study included a total of 204 eligible patients. STAS were found in 47 (23.0%) patients, and no STAS were found in 157 (77.0%) patients. The receiver operating characteristic curve showed that radiomics model, clinical genomics model, and mixed model had good predictive performance (area under the curve [AUC]=0.85; AUC=0.70; AUC=0.85). The prediction model based on radiomics and genomics features has a good prediction performance for STAS.

Deep learning-based pathological prediction of lymph node metastasis for patient with renal cell carcinoma from primary whole slide images

BackgroundMetastasis renal cell carcinoma (RCC) patients have extremely high mortality rate. A predictive model for RCC micrometastasis based on pathomics could be beneficial for clinicians to make treatment decisions.MethodsA total of 895 formalin-fixed and paraffin-embedded whole slide images (WSIs) derived from three cohorts, including Shanghai General Hospital (SGH), Clinical Proteomic Tumor Analysis Consortium (CPTAC) and Cancer Genome Atlas (TCGA) cohorts, and another 588 frozen section WSIs from TCGA dataset were involved in the study. The deep learning-based strategy for predicting lymphatic metastasis was developed based on WSIs through clustering-constrained-attention multiple-instance learning method and verified among the three cohorts. The performance of the model was further verified in frozen-pathological sections. In addition, the model was also tested the prognosis prediction of patients with RCC in multi-source patient cohorts.ResultsThe AUC of the lymphatic metastasis prediction performance was 0.836, 0.865 and 0.812 in TCGA, SGH and CPTAC cohorts, respectively. The performance on frozen section WSIs was with the AUC of 0.801. Patients with high deep learning-based prediction of lymph node metastasis values showed worse prognosis.ConclusionsIn this study, we developed and verified a deep learning-based strategy for predicting lymphatic metastasis from primary RCC WSIs, which could be applied in frozen-pathological sections and act as a prognostic factor for RCC to distinguished patients with worse survival outcomes.

Genetic variation near GRB10 associated with bone growth and osteosarcoma risk in canine and human populations

BackgroundCanine and human osteosarcoma are similar in clinical presentation and tumor genomics. Giant breed dogs experience elevated osteosarcoma incidence, and taller stature remains a consistent risk factor for human osteosarcoma. Whether evolutionarily conserved genes contribute to both human and canine osteosarcoma predisposition merits evaluation. MethodsA multi-center sample of childhood osteosarcoma patients and controls underwent genome-wide genotyping and imputation. Ancestry-adjusted SNP associations were calculated within each dataset using logistic regression, then meta-analyzed across the three datasets, totaling 1091 patients and 3026 controls. Ten regions previously associated with canine osteosarcoma risk were mapped to the human genome, spanning ∼6 Mb. We prioritized association testing of 5985 human SNPs mapping to candidate osteosarcoma risk regions detected in Irish wolfhounds, the largest dog breed studied. Secondary analyses explored 6289 additional human SNPs mapping to candidate osteosarcoma risk regions identified in Rottweilers and greyhounds. ResultsFourteen SNPs were associated with human osteosarcoma risk after adjustment for multiple comparisons, all within a 42 kb region of human Chromosome 7p12.1. The lead variant was rs17454681 (OR=1.25, 95 %CI: 1.12–1.39; P=4.1×10−5), and independent risk variants were not observed in conditional analyses. While the associated region spanned 2.1 Mb and contained eight genes in Irish wolfhounds, associations were localized to a 50-fold smaller region of the human genome and strongly implicate GRB10 (growth factor receptor-bound protein 10) in canine and human osteosarcoma predisposition. PheWAS analysis in UK Biobank data identified noteworthy associations of the rs17454681 risk allele with varied measures of height and pubertal timing. ConclusionsOur comparative oncology analysis identified a novel human osteosarcoma risk allele near GRB10, a growth inhibitor that suppresses activated receptor tyrosine kinases including IGF1R, PDGFRB, and EGFR. Epidemiologists may benefit from leveraging cross-species comparisons to identify haplotypes in highly susceptible but genetically homogenous populations of domesticated animals, then fine-mapping these associations in diverse human populations.

The Immunopeptidomics Ontology (ImPO).

The adaptive immune response plays a vital role in eliminating infected and aberrant cells from the body. This process hinges on the presentation of short peptides by major histocompatibility complex Class I molecules on the cell surface. Immunopeptidomics, the study of peptides displayed on cells, delves into the wide variety of these peptides. Understanding the mechanisms behind antigen processing and presentation is crucial for effectively evaluating cancer immunotherapies. As an emerging domain, immunopeptidomics currently lacks standardization-there is neither an established terminology nor formally defined semantics-a critical concern considering the complexity, heterogeneity, and growing volume of data involved in immunopeptidomics studies. Additionally, there is a disconnection between how the proteomics community delivers the information about antigen presentation and its uptake by the clinical genomics community. Considering the significant relevance of immunopeptidomics in cancer, this shortcoming must be addressed to bridge the gap between research and clinical practice. In this work, we detail the development of the ImmunoPeptidomics Ontology, ImPO, the first effort at standardizing the terminology and semantics in the domain. ImPO aims to encapsulate and systematize data generated by immunopeptidomics experimental processes and bioinformatics analysis. ImPO establishes cross-references to 24 relevant ontologies, including the National Cancer Institute Thesaurus, Mondo Disease Ontology, Logical Observation Identifier Names and Codes and Experimental Factor Ontology. Although ImPO was developed using expert knowledge to characterize a large and representative data collection, it may be readily used to encode other datasets within the domain. Ultimately, ImPO facilitates data integration and analysis, enabling querying, inference and knowledge generation and importantly bridging the gap between the clinical proteomics and genomics communities. As the field of immunogenomics uses protein-level immunopeptidomics data, we expect ImPO to play a key role in supporting a rich and standardized description of the large-scale data that emerging high-throughput technologies are expected to bring in the near future. Ontology URL: https://zenodo.org/record/10237571 Project GitHub: https://github.com/liseda-lab/ImPO/blob/main/ImPO.owl.

The pathway alteration load as a pan-cancer determinant of outcome of targeted therapies: Results from the Drug Rediscovery Protocol (DRUP).

3138 Background: Although targeted treatment (TT) has improved outcomes for many patients with cancer, the emergence of primary or secondary resistance remains a common challenge. A frequently suggested contributing mechanism to this resistance is the existence of parallel activated oncogenic signaling pathways that remain untargeted and support tumor survival. Therefore, we hypothesized that an increased number of altered oncogenic signaling pathways (Pathway Alteration Load, PAL) predicts reduced benefit of TT which targets only one altered pathway. Methods: The Drug Rediscovery Protocol (DRUP) is a prospective, pan-cancer, non-randomized clinical trial (NCT02925234) in which patients with treatment-refractory metastatic cancer with an actionable molecular profile are treated with matched approved targeted- and immunotherapies outside their registered indication. All patients treated with TT for whom clinical outcome and whole genome sequencing results of a pre-treatment biopsy were available were included. The PAL was determined by counting the number of proliferation pathways impacted by somatic driver alterations, using an established pathway classification scheme (Sanchez-Vega et al., Cell, 2018). Influence of PAL on clinical benefit rate (CBR, defined as objective response or stable disease ≥ 16 weeks according to RECIST v1.1 or RANO), progression-free survival (PFS) and overall survival (OS) was evaluated. Cox proportional-hazards models, stratified by tumor type, were used to adjust outcomes for drug, performance status, previous treatment lines, tumor mutational burden and cancer cell ploidy. Results were validated using the independent Hartwig database of metastatic cancers. Results: In all 154 patients treated with single pathway-directed TT, PAL was 0 in 3, 1 in 20, 2 in 37, 3 in 56, 4 in 31, 5 in 6 and 6 in 1 patient(s). Patients with a PAL below the median of 3 ( N= 60) demonstrated a higher CBR (41.7% vs 25.5%, odds ratio [OR] 0.48, P = 0.051), longer PFS (median 4.7 vs 2.9 months, adjusted hazard ratio [aHR] 1.70, P = 0.020) and OS (median 13.7 vs 5.6 months, aHR 3.80, P< 0.001), compared to those with PAL ≥3. Results were consistent across different PAL cut-offs and when applying PAL continuously (aHR for PFS 1.41, P = <0.001; aHR for OS 1.77, P = <0.001). In the independent Hartwig cohort, 258 patients treated with single pathway-directed TT were identified and similar results were found for CBR (54.2% vs 36.7%, OR 2.04, P = 0.009) and PFS (7.0 vs 4.2 months, aHR 1.55, P = 0.009), but not for OS (14.7 vs 8.2 months, aHR 1.37, P = 0.111). Conclusions: In our population, PAL emerged as a pan-cancer predictor of resistance to TT. Our findings provide support for more refined patient selection for TT and highlight the rationale for combinatorial treatment strategies, especially in patients with multiple affected pathways. Clinical trial information: NCT02925234 .

Inherited Germline Variants in Urinary Tract Cancer: A Multicenter Whole-Exome Sequencing Analysis and Correlation With Clinical Features and Tumor Genomics.

This study investigates a real-world multicenter cohort of patients with urinary tract cancer (UTC), with primary disease sites including the bladder, urethra, and upper tract, who enrolled for research molecular testing of their germline and tumor. The purpose of this study was to evaluate factors that could affect the likelihood of identifying a clinically actionable germline pathogenic variant (PV). Patients with UTC were identified from 10 cancer institutes of the Oncology Research Information Exchange Network consortium. The data set comprised abstracted clinical data with germline and tumor genomic data, and comparative analyses were conducted. Clinically actionable germline PVs in cancer predisposition genes were identified in 16 (4.5%) of 354 patients. A higher proportion of patients with the urethra and the upper tract as the primary sites of disease had PVs with a prevalence of 11% (5/45), compared with only 3.6% (11/308) in those with the bladder as the primary site of disease (P = .04). There were no significant differences in markers of genomic instability (such as tumor mutational burden, microsatellite instability [MSI], and loss of heterozygosity, copy number, and chromosomal instability) between those with PVs and those without (P > .05). Of the PVs identified, 10 (62%) were in homologous recombination repair (HRR) genes, three (19%) in mismatch repair (MMR) genes, and three (19%) in genes associated with other pathways. Tissue-based assessment of genomic instability, such as MSI, does not reliably indicate germline PV. A comprehensive clinical germline testing approach that includes HRR genes in addition to MMR genes is likely to yield PVs in approximately one of 10 patients with nonbladder primary disease sites such as the upper tract and the urethra.

Variant Detection in 3′ Exons of PMS2 Using Exome Sequencing Data

PMS2 is one of the mismatch repair genes included in routine genetic testing for Lynch syndrome, colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics (ACMG) secondary findings gene list in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read based next generation sequencing (NGS) technologies is complicated by the presence of the pseudogene PMS2CL and often supplemented by long-range based approaches such as long-range polymerase chain reaction (LR-PCR) or long-read based next generation sequencing, which increases the complexity and cost. Here, we described a bioinformatics homology triage workflow that can eliminate the need for long-read based testing for PMS2 for the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost.

The impact of clinical genome sequencing in a global population with suspected rare genetic disease

There is mounting evidence of the value of clinical genome sequencing (cGS) in individuals with suspected rare genetic disease (RGD), but cGS performance and impact on clinical care in a diverse population drawn from both high-income countries (HICs) and low- and middle-income countries (LMICs) has not been investigated. The iHope program, a philanthropic cGS initiative, established a network of 24 clinical sites in eight countries through which it provided cGS to individuals with signs or symptoms of an RGD and constrained access to molecular testing. A total of 1,004 individuals (median age, 6.5 years; 53.5% male) with diverse ancestral backgrounds (51.8% non-majority European) were assessed from June 2016 to September 2021. The diagnostic yield of cGS was 41.4% (416/1,004), with individuals from LMIC sites 1.7 times more likely to receive a positive test result compared to HIC sites (LMIC 56.5% [195/345] vs. HIC 33.5% [221/659], OR 2.6, 95% CI 1.9-3.4, p<0.0001). A change in diagnostic evaluation occurred in 76.9% (514/668) of individuals. Change of management, inclusive of specialty referrals, imaging and testing, therapeutic interventions, and palliative care, was reported in 41.4% (285/694) of individuals, which increased to 69.2% (480/694) when genetic counseling and avoidance of additional testing were also included. Individuals from LMIC sites were as likely as their HIC counterparts to experience a change in diagnostic evaluation (OR 6.1, 95% CI 1.1-∞, p= 0.05) and change of management (OR 0.9, 95% CI 0.5-1.3, p= 0.49). Increased access to genomic testing may support diagnostic equity and the reduction of global health care disparities.

Genomic Newborn Screening for Pediatric Cancer Predisposition Syndromes: A Holistic Approach.

As next-generation sequencing (NGS) has become more widely used, germline and rare genetic variations responsible for inherited illnesses, including cancer predisposition syndromes (CPSs) that account for up to 10% of childhood malignancies, have been found. The CPSs are a group of germline genetic disorders that have been identified as risk factors for pediatric cancer development. Excluding a few "classic" CPSs, there is no agreement regarding when and how to conduct germline genetic diagnostic studies in children with cancer due to the constant evolution of knowledge in NGS technologies. Various clinical screening tools have been suggested to aid in the identification of individuals who are at greater risk, using diverse strategies and with varied outcomes. We present here an overview of the primary clinical and molecular characteristics of various CPSs and summarize the existing clinical genomics data on the prevalence of CPSs in pediatric cancer patients. Additionally, we discuss several ethical issues, challenges, limitations, cost-effectiveness, and integration of genomic newborn screening for CPSs into a healthcare system. Furthermore, we assess the effectiveness of commonly utilized decision-support tools in identifying patients who may benefit from genetic counseling and/or direct genetic testing. This investigation highlights a tailored and systematic approach utilizing medical newborn screening tools such as the genome sequencing of high-risk newborns for CPSs, which could be a practical and cost-effective strategy in pediatric cancer care.

Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) andCEP135(NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

The updates of the ACMG variant interpretation guidelines affect the pathogenicity determination of OTOF gene variations in patients with auditory neuropathy

Objective: To compare the differences between the variation interpretation standards and guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 (The 2015ACMG/AMP guideline) and the Deafness Specialist Group of the Clinical Genome Resource (ClinGen) in 2018 for hereditary hearing loss (Healing loss, HL) issued the expert specification of the variation interpretation guide (The 2018 HL-EP guideline) in evaluating the pathogenicity of OTOF gene variation in patients with auditory neuropathy. Methods: Thirty-eight auditory neuropathy patients with OTOF gene variant were selected as the study subjects (23 males and 15 females, aged 0.3-25.9 years). Using whole-genome sequencing, whole exome sequencing or target region sequencing (Panel) combined with Sanger sequencing, 38 cases were found to carry more than two OTOF mutation sites. A total of 59 candidate variants were independently interpreted based on the 2015 ACMG/AMP guideline and 2018 HL-EP guideline. Compared with the judgment results in 2015 ACMG/AMP guideline, the variants interpreted as lower pathogenic classifications in the 2018 HL-EP guideline were defined as downgraded variants, and the variants regarded as higher pathogenic classifications were defined as upgraded variants. Statistical analysis was conducted using SPSS 20.0. Results: The concordance rate of variant classification between the guidelines was 72.9%(43/59). The 13.6%(8/59) of variants were upgraded and 13.6% (8/59) of variants downgraded in the classifications of the 2018 HL-EP guideline. A couple of rules saw significant differences between the guidelines (PVS1, PM3, PP2, PP3 and PP5). The distribution of pathogenicity of splicing mutation was statistically different (P=0.013). Conclusions: The 2018 HL-EP guideline is inconsistent with the 2015 ACMG/AMP guideline, when judging the pathogenicity of OTOF gene variants in patients with auditory neuropathy. Through the deletion and refinement of evidence and the breaking of solidification thinking, the 2018 HL-EP guideline makes the pathogenicity grading more traceable and improves the credibility.

Development and validation of a stakeholder-driven, self-contained electronic informed consent platform for trio-based genomic research studies.

Increasingly long and complex informed consents have yielded studies demonstrating comparatively low participant comprehension and satisfaction with traditional face-to-face approaches. In parallel, interest in electronic consents for clinical and research genomics has steadily increased, yet limited data are available for trio-based genomic discovery studies. We describe the design, development, implementation, and validation of an electronic iConsent application for trio-based genomic research deployed to support genomic studies of cerebral palsy. iConsent development incorporated stakeholder perspectives including researchers, patient advocates, institutional review board members, and genomic data-sharing considerations. The iConsent platform integrated principles derived from prior electronic consenting research and elements of multimedia learning theory. Participant comprehension was assessed in an interactive teachback format. The iConsent application achieved nine of ten proposed desiderata for effective patient-focused electronic consenting for genomic research. Overall, participants demonstrated high comprehension and retention of key human subjects' considerations. Enrollees reported high levels of satisfaction with the iConsent, and we found that participant comprehension, iConsent clarity, privacy protections, and study goal explanations were associated with overall satisfaction. Although opportunities exist to optimize iConsent, we show that such an approach is feasible, can satisfy multiple stakeholder requirements, and can realize high participant satisfaction and comprehension while increasing study reach.

Novel protein-truncating variants of a chromatin-modifying gene MSL2 in syndromic neurodevelopmental disorders

Numerous large scale genomic studies have uncovered rare but recurrent pathogenetic variants in a significant number of genes encoding epigenetic machinery in cases with neurodevelopmental disorders (NDD) especially autism spectrum disorder (ASD). These findings provide strong support for the functional importance of epigenetic regulators in neurodevelopment. After the clinical genomics evaluation of the patients using exome sequencing, we have identified, three novel protein-truncating variants (PTVs) in the MSL2 gene (OMIM: 614802) which encodes a chromatin modifying enzyme. MSL2 modifies chromatin through both mono-ubiquitination of histone 2B on lysine 34 (K34) and acetylation of histone H4 on lysine 16 (K16). We reported first time the detailed clinical features associated with 3 MSL2 PTVs. There are 15 PTVs (13 de novo) reported from the large genomics studies (12 cases) or ClinVar (3 cases) of NDD, ASD, and developmental disorders (DD) but the specific clinical features for these cases are not described. Taken together, our descriptions of dysmorphic face and other features support the causal role of MSL2 in a likely syndromic neurodevelopmental disorder and add MSL2 to a growing list of epigenetic genes implicated in ASD.

Abstract PO1-14-08: Real-world clinical genomics study of HR+/HER2- metastatic breast cancers treated by CDK4/6i plus endocrine therapies revealed a drug resistant tumor segment characterized by ER independence

Abstract Background CDK4/6 inhibitors (CDK4/6i) plus endocrine therapies (ET) are the standard-of-care for hormone receptor–positive/human epidermal receptor 2–negative metastatic breast cancer (HR+/HER2− mBC). However, drug resistance remains a major unmet need. Investigations of drug resistance mechanisms has been hampered by a dearth of tumor molecular profiling data from the post-treatment setting. To address this challenge, we have conducted a real-world clinical genomics study to better understand the molecular mechanism of CDK4/6i resistance as well as to stratify patients based on integrated multi-omics profiles. Methods We retrospectively analyzed a multi-omics dataset of 400 HR+/HER2- mBC patients who had received CDK4/6i plus ET and developed progressive disease (PD) from the de-identified Tempus database. Pre-treatment and post-progression biopsies were taken  1 year prior to starting the CDK4/6i treatment or following PD respectively. Tempus xT next-generation sequencing (DNA-seq of 648 genes) and RNA sequencing assays were performed on 427 tumor FFPE samples, including 200 pre-treatment, 227 post-progression and 26 longitudinal pairs. Results The median age of the patients was 57 (54.9-57.4) and median progression free survival (PFS) is 379 (341-433) days. Two genes were found to harbor a significant increase in genomic alteration frequencies (GAF) after adjusting for FDR at post-progression vs. pre-treatment – ESR1 (41.9% vs. 15%, p=5.4e-10), RB1 (13.2% vs. 3%, p=8.5e-05). ESR1 and RB1 also harbored high frequencies of acquired genomic alterations among 26 paired samples at 34.6% and 11.5% respectively. TP53 mutation at baseline was significantly associated with shorter PFS at baseline (p=4.23e-05, HR=2.081) and TP53 GAF significantly increased after PD (37% vs. 28.5%, p=0.039). BRCA1/2 pathogenic mutations (p=1.63e-04, HR=3.066), APOBEC mutation signature S13 (p=0.0125, HR=1.55) and CCNE1 gene expression (p=0.024, HR=1.46) were significantly associated with shorter PFS. APOBEC signature (p=0.0035) and CCNE1 expression (p=1.33e-06) also significantly increased post-progression. Among the top molecular features associated with longer PFS were markers of estrogen signaling such as PGR gene expression (p=6.76e-04, HR=0.565) and the Hallmark estrogen response signature (p=0.021, HR=0.679). Applying a multi-omics pattern recognition algorithm, we identified a molecularly distinct cluster (IC1) characterized by down-regulation of estrogen signaling. IC1 is significantly associated with shorter PFS (p=3.72e-05, HR=0.22) and increased from 4% pre-treatment to 23% post-progression (p=7.3e-08). Further, IC1 is strongly enriched in markers previously implicated in CDK4/6i resistance including CCNE1 expression, RB1 mutation and MYC/E2F activation. We then developed machine learning models to predict gene-level dependency trained on cancer cell line expression and CRISPR-KO screen data. These models predicted decreased dependency on ESR1 and CDK4 and increased dependency on CDK2 in IC1, strengthening the association between ER independence and CDK4/6i resistance. Conclusions Our real-world clinical genomics study identified a comprehensive list of biomarkers associated with resistance to CDK4/6i plus ET and estimated patient prevalence for these markers in the post-treatment setting. Integrated and machine-learning analyses identified a subset of aggressive tumors with estrogen independence characteristics that are implicated in CDK4/6i resistance and suggested new therapeutic strategies. Citation Format: Zhengyan Kan, Ji Wen, Jennifer Webster, Vinicius Bonato, Whijae Roh, Xinmeng Jasmine Mu, Paul Rejto, Jadwiga Bienkowska. Real-world clinical genomics study of HR+/HER2- metastatic breast cancers treated by CDK4/6i plus endocrine therapies revealed a drug resistant tumor segment characterized by ER independence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-08.

Abstract PO4-19-02: Genetic risk estimation in breast cancer and assessing health disparities

Abstract Background: Breast Cancer (BC) incidence and the distribution of BC risk factors vary between racial and ethnic groups within the United States. It has been demonstrated that providing education on individualized breast cancer risk will improve patient uptake to recommended BC screening and prevention strategies. Most clinical risk assessment tools, including the validated IBIS model were derived from data using non-Hispanic white women. Consortia have also now identified over 300 common genetic susceptibility loci for BC, summarized by the polygenic risk score (PRS). When combined with clinical risk assessment tools, such as the IBIS or BCRAT models, the PRS can further refine risk estimation for patients. To date, most studies on the use of PRS have been done in non-Hispanic white women. While these PRS still perform well in racial minorities, there has been a recognized need to improve racial and ethnic diversity in genomic research cohorts. Herein, we aim to combine clinical risk assessment models that are already used in routine clinical practice with information derived from PRS testing in women of racial minorities to determine if this can improve risk estimation, patient understanding, and uptake to recommended breast cancer screening and prevention strategies. Design: This is a minimal risk prospective study with a single arm incorporating the PRS into a standard breast cancer risk reduction consultation, followed by annual surveys over 10 years to determine if and how the information provided by the PRS influenced patient decisions regarding recommended BC screening and prevention. Patients will have IBIS and BCRAT risks calculated at baseline visit. A survey of patient perceptions/understanding of risk, intention to undergo screening, and use of preventive medicine is completed after baseline visit prior to receiving the PRS results. Blood sample is obtained at baseline and DNA samples are analyzed for approximately 300 SNPs on a custom-designed, targeted SNP panel from ThermoFisher Scientific by the Mayo Clinic Genomics Laboratory. This PRS result is then combined with the clinical risk assessments (IBIS and BCRAT scores) using the R package Individualized Coherent Absolute Risk Estimators (iCare) tool to provide a new estimate of breast cancer risk for 5year, 10year, and lifetime risk. Patients are seen for follow-up to review results and then complete a second survey to assess their understanding of the results, BC risk, and how the PRS impacted their decision to undergo screening/prevention strategies. Eligibility Criteria: Women who self-identify as African American/Black or Hispanic/Latinx between 30-75 years old with any of the following: 1.)IBIS score of ≥5% for the 10 year risk OR BCRAT score of ≥ 3 % for the 5 year risk. 2.) History of biopsy proven atypical hyperplasia 3.) History of biopsy proven lobular carcinoma in situ. Specific Aims: Aim 1: The primary aim of this study is to explore if the addition of PRS to the BCRAT and IBIS score will improve intentions to undergo recommended breast cancer screening strategies such as mammography, MRI, or molecular breast imaging in women of underserved racial minorities Aim 2: To explore if the addition of the PRS to the BCRAT and IBIS risk score will aid women of racial minorities in deciding whether to take preventative endocrine therapy. Aim 3: To understand how individualized risk assessment and information on PRS may alter perceived risk of breast cancer. Statistical Methods Analysis will be mostly descriptive. Continuous variables will be summarized as mean (standard deviation) or median (range) and categorical variables will be reported as frequency (percentage). Kaplan-Meier method will be used to estimate the long-term cumulative risk of cancer. Current Accrual: 3. Target Accrual: 50 Citation Format: Lauren Cornell, Sandhya Pruthi, Linda Hasadsri, Sarah McLaughlin. Genetic risk estimation in breast cancer and assessing health disparities [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-02.

CAVaLRi: An Algorithm for Rapid Identification of Diagnostic Germline Variation

Clinical exome and genome sequencing (ES/GS) have become indispensable diagnostic tools for rare genetic diseases (RGD). However, the interpretation of ES/GS presents a substantial operational challenge in clinical settings. Test interpretation requires the review of hundreds of genetic variants, a task that has become increasingly challenging given the rising use of ES/GS. In response, we present Clinical Assessment of Variants by Likelihood Ratios (CAVaLRi), which employ a modified likelihood ratio (LR) framework to assign diagnostic probabilities to candidate germline disease genes. CAVaLRi models aspects of the clinical variant assessment process, taking into consideration the predicted impact of the variant, the proband and parental genotypes, and the proband’s clinical characteristics. It also factors in computational phenotype noise and weighs the relative significance of genotype, phenotype, and variant segregation information. We trained and tested CAVaLRi on variant and phenotype data from an internal cohort of 655 clinical ES cases. For validation, CAVaLRi’s performance was benchmarked against four leading gene prioritization algorithms (Exomiser’s hiPHIVE and PhenIX prioritizers, LIRICAL, and XRare) using a distinct cohort of 12,832 ES cases. Our findings reveal that CAVaLRi significantly outperforms its counterparts when clinician-curated phenotype sets are used, as evidenced by its superior precision-recall curve (PR AUC: 0.701) and average diagnostic gene rank (1.59). Notably, even when substituting highly focused clinician-curated phenotype sets with large and potentially nonspecific computationally derived phenotypes, CAVaLRi retains its precision (PR AUC: 0.658; diagnostic gene average rank: 1.68) and markedly outperforms other tools. In a large, heterogeneous validation cohort, CAVaLRi stood out as the most precise prioritization algorithm (PR AUC: 0.335; average diagnostic rank: 1.91). In conclusion, CAVaLRi presents a robust solution for prioritizing diagnostic genes, surpassing current methods. It demonstrates resilience to noisy, computationally-derived phenotypes, providing a scalable strategy to help labs focus on the most diagnostically relevant variants, thus addressing the growing demand for ES/GS interpretation.