Whole-exome Sequencing Of DNA Research Articles

Sudden cardiac death in young athletes: exome sequencing results

BACKGROUND: Ranking risks and prevention of sudden cardiac death in professional young athletes is a crucial unresolved problem in sports medicine. Intense physical exertion can lead to adaptive changes in the cardiovascular system, which can mask inherited diseases with predominant myocardial involvement, such as hypertrophic cardiomyopathy. Undiagnosed cases of such diseases can be a risk factor for fatal outcomes associated with training and competitive activities, making this problem extremely relevant.
 AIM: To conduct a diagnostic search for possible molecular causes of cardiovascular diseases associated with a high risk of sudden death in young athletes.
 MATERIALS AND METHODS: Whole-exome sequencing of DNA extracted from two groups of biomaterials was performed: nine autopsy samples of heart tissues from young athletes who died during intense physical exertion and three venous blood samples from active athletes of the Russian national team. Obtained data were subjected to bioinformatic analysis and clinical interpretation.
 RESULTS: In total, 12 DNA samples were analyzed using a panel of 277 genes associated with the development of cardiovascular diseases with a high risk of sudden death. In 4 of 12 samples (33.3%), variants possibly related to the phenotype were found upon clinical interpretation of the sequencing results. A pathogenic variant was found in the MYBPC3 gene, leading to the development of hypertrophic cardiomyopathy (OMIM: 115197), and a potential pathogenic variant was found in the TRPM4 gene, associated with progressive familial heart block type IB (OMIM: 618531). Moreover, two variants with uncertain clinical significance were found in CAV3 and SCN1B.
 CONCLUSION: Currently, data from high-throughput sequencing are collected to identify the molecular basis of cardiovascular diseases with a high risk of sudden cardiac death. Identifying young athletes who are carriers of pathogenic and possibly pathogenic gene variants is beneficial for preventing fatal outcomes and personalizing medical support for professional athletes.

Abstract 6466: Multi-omics analysis of molecular characteristics and transformation mechanisms of stage I-III micropapillary lung adenocarcinoma

Abstract Background: The micropapillary pattern (MIP) is recognized as a high-grade histological subtype in lung adenocarcinoma (LUAD), with its presence often indicative of a poorer prognosis. Despite distinctive molecular characteristics of MIP, there is a research gap regarding the molecular mechanisms underlying micropapillary transformation and prognosis factors for micropapillary LUAD. Methods: Surgically resected tumor samples from 101 patients with stage I-III LUAD and MIP components exceeding 30% were collected. The tumor samples were microdissected to separate MIP components from non-MIP components, and both fractions underwent RNA and DNA whole-exome sequencing (WES). The molecular profiles between MIP and non-MIP components, along with MIP-naïve LUAD tissues from an external cohort, were compared. Associations between molecular characteristics and recurrence-free survival were also assessed. Results: The genomic landscapes of MIP and non-MIP components within tumor tissues featuring MIP patterns exhibited remarkable similarity. At the transcriptomic level, MIP components displayed elevated PRB4 expression (FDR < 0.001), an up-regulated cell cycle pathway (P = 0.004), and reduced natural killer cell-mediated cytotoxicity (P = 0.066). Nevertheless, in comparison to MIP-naïve LUAD tissues, the MIP components showed higher chromosomal instability (P < 0.001) and revealed eighteen enriched somatic alterations, encompassing EGFR mutations, EGFR amplifications, and CDKN2A/CDKN2B deletions, all linked to up-regulations in cell proliferation pathways and down-regulated immune pathways. In the context of MIP transformation, shared mutations were observed in 97.8% (91/93) of patients between MIP and non-MIP components within the same tissues, suggesting a common origin. The subclonal fractions were comparable between paired MIP and non-MIP components, while a significant co-occurrence of EGFR amplification, CDKN2A deletion, and CDKN2B deletion was identified in MIP components. A comprehensive analysis combining differential gene expression and Cox analysis, employing a both-direction stepwise Akaike Information Criteria selection, identified the high expressions of five genes (GALNT4, MIAT, FOSL1, LAT2, SMN2) as adverse factors associated with the recurrence of micropapillary LUAD. Conclusions: We conducted an in-depth analysis of the molecular characteristics and transformation mechanisms of micropapillary lung adenocarcinoma, employing microdissection techniques to investigate at both genomic and transcriptomic levels within a substantial cohort, providing insights for the precision medicine of this aggressive cancer subtype. Citation Format: Kang Shao, Shugeng Gao, Fengwei Tan, Anqi Shao, Xiaoli Feng, Qi Xue, Yang Qu, Bo Zheng, Wei Zheng, Hanlin Chen, Qiuxiang Ou, Haimeng Tang. Multi-omics analysis of molecular characteristics and transformation mechanisms of stage I-III micropapillary lung adenocarcinoma [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 6466.

Variants in the ZNF469 gene in families with Brittle cornea syndrome and keratoconus

BackgroundBrittle cornea syndrome 1 (BCS1) is a rare autosomal recessive disorder characterized by corneal and sclera thinning and fragility that is caused by zinc finger protein 469 (ZNF469) gene mutation. Keratoconus is another disease related to corneal thinning. Several reports have linked ZNF469 variants and keratoconus. We recruited a four-generation BCS1 family and two keratoconus families to explore pathogenic ZNF469 variants. MethodsThis study included 11 members from a family with BCS1, 2 families with keratoconus, 368 sporadic keratoconus patients and 325 unrelated healthy controls. Whole exome sequencing of DNA from peripheral blood and cross species conservation analysis was used to investigate and verify ZNF469 variants. ResultsA new homozygous frameshift mutation c. 6727del (p.Asp2243Thr fs*8) in ZNF469 was detected in the BSC1 family. Two ZNF469 heterozygous variants g.88494671G > A (c.793G > A, p.G265S, rs754776767) were detected in keratoconus family 1 and a heterozygous missense variant g.88498262G > A (c.4384G > A, p.D1462 N, rs577890057) was found in keratoconus family 2. Based on the American College of Medical Genetics and Genomics guidelines, rs577890057 and rs754776767 were predicted to be variants of uncertain significance. c. 6727del (p. Asp2243Thr fs*8) in ZNF469 was identified to be pathogenic. ConclusionsWe identified a new homozygous frameshift mutation and two heterozygous missense variations in ZNF469 in the three families. Our findings extend the spectrum of ZNF469 variants associated with keratoconus.

SEC31A may be associated with pituitary hormone deficiency and gonadal dysgenesis.

Disorders/differences of sex development (DSD) result from variants in many different human genes but, frequently, have no detectable molecular cause. Detailed clinical and genetic phenotyping was conducted on a family with three children. A Sec31a animal model and functional studies were used to investigate the significance of the findings. By trio whole-exome DNA sequencing we detected a heterozygous de novo nonsense SEC31A variant, in three children of healthy non-consanguineous parents. The children had different combinations of disorders that included complete gonadal dysgenesis and multiple pituitary hormone deficiency. SEC31A encodes a component of the COPII coat protein complex, necessary for intracellular anterograde vesicle-mediated transport between the endoplasmic reticulum (ER) and Golgi. CRISPR-Cas9 targeted knockout of the orthologous Sec31a gene region resulted in early embryonic lethality in homozygous mice. mRNA expression of ER-stress genes ATF4 and CHOP was increased in the children, suggesting defective protein transport. The pLI score of the gene, from gnomAD data, is 0.02. SEC31A might underlie a previously unrecognised clinical syndrome comprising gonadal dysgenesis, multiple pituitary hormone deficiencies, dysmorphic features and developmental delay. However, a variant that remains undetected, in a different gene, may alternatively be causal in this family.

Multimodal integration of neuroimaging and genetic data for the diagnosis of mood disorders based on computer vision models

Mood disorders, particularly major depressive disorder (MDD) and bipolar disorder (BD), are often underdiagnosed, leading to substantial morbidity. Harnessing the potential of emerging methodologies, we propose a novel multimodal fusion approach that integrates patient-oriented brain structural magnetic resonance imaging (sMRI) scans with DNA whole-exome sequencing (WES) data. Multimodal data fusion aims to improve the detection of mood disorders by employing established deep-learning architectures for computer vision and machine-learning strategies. We analyzed brain imaging genetic data of 321 East Asian individuals, including 147 patients with MDD, 78 patients with BD, and 96 healthy controls. We developed and evaluated six fusion models by leveraging common computer vision models in image classification: Vision Transformer (ViT), Inception-V3, and ResNet50, in conjunction with advanced machine-learning techniques (XGBoost and LightGBM) known for high-dimensional data analysis. Model validation was performed using a 10-fold cross-validation. Our ViT ⊕ XGBoost fusion model with MRI scans, genomic Single Nucleotide polymorphism (SNP) data, and unweighted polygenic risk score (PRS) outperformed baseline models, achieving an incremental area under the curve (AUC) of 0.2162 (32.03% increase) and 0.0675 (+8.19%) and incremental accuracy of 0.1455 (+25.14%) and 0.0849 (+13.28%) compared to SNP-only and image-only baseline models, respectively. Our findings highlight the opportunity to refine mood disorder diagnostics by demonstrating the transformative potential of integrating diverse, yet complementary, data modalities and methodologies.

Oncogenic fusions in renal cell carcinoma.

475 Background: Detecting actionable or diagnostic fusions in solid tumors may significantly alter clinical decisions, including therapy selection. Some fusions have matched drugs approved for use in the patient’s cancer, or in another cancer. Others have published evidence of benefit with particular drugs but are not included in guidelines, and some may represent an inclusion criterion for clinical trials. While diagnostic fusions may not have associated therapies, they are cancer-defining and can lead to re-classification of tumors. Here we describe actionable and diagnostic fusions we have observed in renal cell carcinoma (RCC). Methods: Tumor samples from patients diagnosed with RCC were analyzed using the OncoExTra assay, which identifies somatic mutations and gene fusions through tumor-normal, whole-exome DNA and whole-transcriptome RNA sequencing. All actionable fusions, defined as those with associated FDA-approved targeted therapies in any cancer type, those that made patients eligible for an active clinical trial, or those with evidence in guidelines or the literature for possible matched therapies in any cancer, are detectable using this assay. We examined samples from patients with RCC who had the OncoExtra assay performed between May 2018 and Mar 2023 and identified both actionable and diagnostic fusions. Results: Among 389 tumor samples from patients diagnosed with RCC, there were 11 (2.8%) actionable and 1 (0.3%) diagnostic fusion identified. The actionable fusions included 6 (1.5%) TFE3, 1 (0.3%) ALK, 1 (0.3%) EGFR, 1 (0.3%) ERBB4, 1 (0.3%) YAP and 1 (0.3%) FGFR2, and the diagnostic fusion was NAB2/STAT6. The 6 TFE3 oncogene fusions involved 4 different partner genes ( PRCC was the partner in 3 fusions). ALK, EGFR, ERBB and FGFR fusions are found across a variety of cancers, and all have matched FDA-approved drug therapies in at least one cancer type. For example, ALK fusions, including the observed EML4/ALK fusion, which is common in non-small cell lung cancer, have several matched ALK-inhibitor therapies. TFE3 (transcription factor binding to IGHM enhancer 3) gene fusions are a diagnostic genomic alteration in microphthalmia-associated transcription factor (MiT) translocation RCC, which is a rare but aggressive subtype of RCC. While TFE3 fusions do not have FDA-approved matched therapies in any cancer, they are known to result in PIK3K/AKT/mTOR and MET activation, suggesting inhibitors of these pathways may be considered in these patients. The NAB2/STAT6 diagnostic fusion is characteristic of solitary fibrous tumors, suggesting that the patient likely had this kind of tumor. Conclusions: Whole-transcriptome sequencing allowed the identification of actionable fusions with the potential to affect clinical decisions regarding therapy in 2.8% of RCC patient tumors. In addition, one patient was found to have a fusion that is characteristic of a solitary fibrous tumor, demonstrating the power of genomic profiling to aid diagnosis.

Association of the occurrence of single-nucleotide genomic variants in the genes of brain morphogenesis with a predisposition to endogenous depression in the Russian population

Recent research indicates that some types of mental illnesses (schizophrenia, autism, depressive disorders) may be associated with impaired functioning of a number of genes, including those involved in brain morphogenesis. To assess the possible contribution of brain morphogenesis genes in the formation of predisposition to depressive disorders in Russian population, we performed whole-exome sequencing of genomic DNA of such patients. We identified 166 missense genomic variants in 66 genes (out of 140 studied) involved in the formation of brain tissue. The prevalence of some of them was estimated by allele-specific PCR. For the first time, a significantly higher frequency of occurrence of genomic variants rs17445840‑T (CDH2 gene), rs12923655‑C (CDH3 gene), rs1227051‑G/A (CDH23 gene), and rs12500437‑G/T (DCHS2 gene) was shown in a group of patients suffering from endogenous depressive disorder, and an association of some of the identified genomic variants with gender was established. The data obtained confirm the previously stated assumption that genes of brain tissue morphogenesis may be associated with a predisposition to the development of mental and cognitive disorders. The functional significance of the identified genetic variants remains to be established. The identification of pathogenic genomic variants with the confirmation of their functional significance allows better understanding of the pathogenesis of mental disorders and opens prospects for the development of approaches to objective diagnosis of such diseases, their early prevention, and pathogenetic therapy.

Genomic characterization of vulvar squamous cell carcinoma reveals differential gene expression based on clinical outcome

ObjectiveThe greatest challenge in the management of vulvar squamous cell carcinoma (VSCC) is treatment of recurrent disease where options for surgery and radiation have been exhausted, or treatment of disease where distant metastasis is present. Identification of mutations differentially expressed between tumor from patients who died of aggressive disease and tumor from patients with an indolent course could reveal novel prognostic indicators and guide development of therapeutic drugs. MethodsFrom 202 consecutive patients with VSCC, patients who recurred and died of disease (group A) were identified and matched by age, tumor size, depth of invasion and nodal status with those whose disease did not recur (group B). Tumors from 21 patients were subjected to whole exome sequencing of DNA and RNA, immunohistochemistry (IHC) antibodies of PD-L1 and P16, and in-situ hybridization (ISH) for high-risk HPV. ResultsAnalysis of DNA and RNA revealed six genes that were strongly differentially expressed between group A and B: TGM3, ACVR2A, ROS1, NFEL2, CCND1 and BCL6. Clinically relevant DNA mutations were significantly greater in group A versus B: 7 vs 2.3 mutations per patient. The most common genomic alterations were mutations in TP53 and the promoter region of TERT. Other common genomic events include alterations of FAT1, CDKN2A, PIK3CA, CCND1, and LRP1B. All samples were MSI stable and tumor mutational burden (TMB) was similar in groups A and B. Most VSCC specimens (81%) were positive for PD-L1. ConclusionsACVR2A and TGM3 are significantly under-expressed in tumors with poor outcome, suggesting they may play a role in tumor suppression. Clinical outcome of VSCC appears independent of MSI, TMB, or PD-L1 status.

Deep histopathology genotype–phenotype analysis of focal cortical dysplasia type II differentiates between the GATOR1-altered autophagocytic subtype IIa and MTOR-altered migration deficient subtype IIb

Focal cortical dysplasia type II (FCDII) is the most common cause of drug-resistant focal epilepsy in children. Herein, we performed a deep histopathology-based genotype–phenotype analysis to further elucidate the clinico-pathological and genetic presentation of FCDIIa compared to FCDIIb. Seventeen individuals with histopathologically confirmed diagnosis of FCD ILAE Type II and a pathogenic variant detected in brain derived DNA whole-exome sequencing or mTOR gene panel sequencing were included in this study. Clinical data were directly available from each contributing centre. Histopathological analyses were performed from formalin-fixed, paraffin-embedded tissue samples using haematoxylin–eosin and immunohistochemistry for NF-SMI32, NeuN, pS6, p62, and vimentin. Ten individuals carried loss-of-function variants in the GATOR1 complex encoding genes DEPDC5 (n = 7) and NPRL3 (n = 3), or gain-of-function variants in MTOR (n = 7). Whereas individuals with GATOR1 variants only presented with FCDIIa, i.e., lack of balloon cells, individuals with MTOR variants presented with both histopathology subtypes, FCDIIa and FCDIIb. Interestingly, 50% of GATOR1-positive cases showed a unique and predominantly vacuolizing phenotype with p62 immunofluorescent aggregates in autophagosomes. All cases with GATOR1 alterations had neurosurgery in the frontal lobe and the majority was confined to the cortical ribbon not affecting the white matter. This pattern was reflected by subtle or negative MRI findings in seven individuals with GATOR1 variants. Nonetheless, all individuals were seizure-free after surgery except four individuals carrying a DEPDC5 variant. We describe a yet underrecognized genotype–phenotype correlation of GATOR1 variants with FCDIIa in the frontal lobe. These lesions were histopathologically characterized by abnormally vacuolizing cells suggestive of an autophagy-altered phenotype. In contrast, individuals with FCDIIb and brain somatic MTOR variants showed larger lesions on MRI including the white matter, suggesting compromised neural cell migration.

Characterization of gene expression and biological pathways in Wilms tumor

BackgroundAlthough mortality for low stage (I/II) Wilms tumor (WT) has greatly improved with dose-intense multimodality and multi-agent interventions, advanced stage (III/IV) tumors remain associated with relapse, mortality and increased treatment-related morbidity. We aim to identify targets unique to the microenvironment of advanced stage WT to develop novel treatments. MethodsRetrospective review of patients treated for WT at a single institution from 2000 to 2021. Following review by a pediatric pathologist, samples underwent bulk RNA-sequencing with differential gene expression and pathway enrichment analysis. Groups analyzed included low vs advanced stage and neoadjuvant chemotherapy-treated vs treatment-naïve tumors. In neoadjuvant chemotherapy-treated patients with subsequent recurrence, genes predictive of recurrence were identified using Boruta Random Forest Feature Selection and whole-exome DNA-Sequencing. ResultsInitial analysis of 15 samples showed enrichment for cell cycle progression E2F genes with downregulation of interferon alpha and KRAS signaling genes in advanced compared to low stage tumors. Subsequent analysis of 51 tumors with stratification of RNA-sequencing profiles by treatment status showed notable differences among neoadjuvant chemotherapy-treated tumors. A subcluster of advanced stage tumors lacked enrichment of immune-related genes and pathways observed within a subcluster of immune-enriched low stage tumors, including an activating variant of Catenin beta-1. Among patients with recurrence, transmembrane protein 31 was downregulated and flavin containing dimethylaniline monoxygenase 3 upregulated. ConclusionsGenetic and cellular composition differ between low and advanced stage WT, most notably a significant reduction in immune-related genes/pathways in neoadjuvant chemotherapy-treated tumors. Significant variability exists between same stage tumors, requiring further investigation for reliable treatment targets.

Determination of Carrier Frequency of Actionable Pathogenic Variants in Autosomal Recessive Genetic Diseases in the Turkish Cypriot Population.

Whole-exome DNA sequencing is a rich source of clinically useful information for specialists, patients, and their families, as well as elucidating the genetic basis of monogenic and complex diseases in clinical diagnosis. However, interpreting and reporting variants encompassing exome and genome sequence analysis outcome data are one of the greatest challenges of the genomic era. In this study, we aimed to investigate the frequency and allele frequency spectrum of single nucleotide variants accepted as recessive disease carrier status in Turkish Cypriot exomes. The same sequencing platform and data processing line were used for the analysis of data from 100 Turkish Cypriot whole-exome sequence analysis. Identified variants were classified according to ACMG guidelines, and pathogenic variants were confirmed in other databases such as ClinVar, HGMD, Varsome, etc. Pathogenic variants were detected in 68 genes out of 100 whole-exome sequence data. The carriage rate was the highest in the CYP21A2 gene, causing 21-hydroxylase deficiency (14.70%), 11.76% in the HBB gene causing β-thalassemia, 10.29% in the BTD gene causing biotinidase deficiency, 8.82% in the CFTR gene causing cystic fibrosis, 8.82% in the RBM8A gene causing thrombocytopenia-absent radius syndrome, which is an ultra-rare disease, and 5.88% in the GAA gene causing glycogen storage disease II. The carriage of pathogenic variants in other genes causing the disease (GJB2, PAH, GALC, CYP11B2, COL4A3, HBA1, etc.) was determined as less than 5.00%. Also, the identified variations in the mentioned gene within the examined population were reported. The most prevalent mutation in North Cyprus was a missense variant (c.1360 C>T, p.Pro454Ser) detected in the CYP21A2 gene (rs6445), and the most frequently seen variant in the HBB gene was c.93-21G>A (rs35004220). We investigated reported pathogenic variants by estimating the lower and upper limits of carrier and population frequencies for autosomal recessive diseases, for which exome sequencing may reveal additional medically relevant information. Determining the lower and upper limits of these frequencies will shed light on preventive medicine practices and governmental actions.

Genomic and transcriptomic analyses of thyroid cancers identify DICER1 somatic mutations in adult follicular-patterned RAS-like tumors.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (TC). Several genomic and transcriptomic studies explored the molecular landscape of follicular cell-derived TCs, and BRAFV600E, RAS mutations, and gene fusions are well-established drivers. DICER1 mutations were described in specific sets of TC patients but represent a rare event in adult TC patients. Here, we report the molecular characterization of 30 retrospective follicular cell-derived thyroid tumors, comprising PTCs (90%) and poorly differentiated TCs (10%), collected at our Institute. We performed DNA whole-exome sequencing using patient-matched control for somatic mutation calling, and targeted RNA-seq for gene fusion detection. Transcriptional profiles established in the same cohort by microarray were investigated using three signaling-related gene signatures derived from The Cancer Genome Atlas (TCGA). The occurrence of BRAFV600E (44%), RAS mutations (13%), and gene fusions (13%) was confirmed in our cohort. In addition, in two patients lacking known drivers, mutations of the DICER1 gene (p.D1709N and p.D1810V) were identified. DICER1 mutations occur in two adult patients with follicular-pattern lesions, and in one of them a second concurrent DICER1 mutation (p.R459*) is also observed. Additional putative drivers include ROS1 gene (p.P2130A mutation), identified in a patient with a rare solid-trabecular subtype of PTC. Transcriptomics indicates that DICER1 tumors are RAS-like, whereas the ROS1-mutated tumor displays a borderline RAS-/BRAF-like subtype. We also provide an overview of DICER1 and ROS1 mutations in thyroid lesions by investigating the COSMIC database. Even though small, our series recapitulates the genetic background of PTC. Furthermore, we identified DICER1 mutations, one of which is previously unreported in thyroid lesions. For these less common alterations and for patients with unknown drivers, we provide signaling information applying TCGA-derived classification.

Primary complex motor stereotypies are associated with de novo damaging DNA coding mutations that identify KDM5B as a risk gene.

Motor stereotypies are common in children with autism spectrum disorder (ASD), intellectual disability, or sensory deprivation, as well as in typically developing children ("primary" stereotypies, pCMS). The precise pathophysiological mechanism for motor stereotypies is unknown, although genetic etiologies have been suggested. In this study, we perform whole-exome DNA sequencing in 129 parent-child trios with pCMS and 853 control trios (118 cases and 750 controls after quality control). We report an increased rate of de novo predicted-damaging DNA coding variants in pCMS versus controls, identifying KDM5B as a high-confidence risk gene and estimating 184 genes conferring risk. Genes harboring de novo damaging variants in pCMS probands show significant overlap with those in Tourette syndrome, ASD, and those in ASD probands with high versus low stereotypy scores. An exploratory analysis of these pCMS gene expression patterns finds clustering within the cortex and striatum during early mid-fetal development. Exploratory gene ontology and network analyses highlight functional convergence in calcium ion transport, demethylation, cell signaling, cell cycle and development. Continued sequencing of pCMS trios will identify additional risk genes and provide greater insights into biological mechanisms of stereotypies across diagnostic boundaries.

Genomic alterations related to HPV infection status in a cohort of Chinese prostate cancer patients

BackgroundHuman papillomavirus (HPV) has been proposed as a potential pathogenetic organism involved in prostate cancer (PCa), but the association between HPV infection and relevant genomic changes in PCa is poorly understood.MethodsTo evaluate the relationship between HPV genotypes and genomic alterations in PCa, HPV capture sequencing of DNA isolated from 59 Han Chinese PCa patients was performed using an Illumina HiSeq2500. Additionally, whole-exome sequencing of DNA from these 59 PCa tissue samples and matched normal tissues was carried out using the BGI DNBSEQ platform. HPV infection status and genotyping were determined, and the genetic disparities between HPV-positive and HPV-negative PCa were evaluated.ResultsThe presence of the high-risk HPV genome was identified in 16.9% of our cohort, and HPV16 was the most frequent genotype detected. The overall mutational burden in HPV-positive and HPV-negative PCa was similar, with an average of 2.68/Mb versus 2.58/Mb, respectively, in the targeted whole-exome region. HPV-negative tumors showed a mutational spectrum concordant with published PCa analyses with enrichment for mutations in SPOP, FOXA1, and MED12. HPV-positive tumors showed more mutations in KMT2C, KMT2D and ERCC2. Copy number alterations per sample were comparable between the two groups. However, the significantly amplified or deleted regions of the two groups only partially overlapped. We identified amplifications in oncogenes, including FCGR2B and CCND1, and deletions of tumor suppressors, such as CCNC and RB1, only in HPV-negative tumors. HPV-positive tumors showed unique deletions of tumor suppressors such as NTRK1 and JAK1.ConclusionsThe genomic mutational landscape of PCa differs based on HPV infection status. This work adds evidence for the direct involvement of HPV in PCa etiology. Different genomic features render HPV-positive PCa a unique subpopulation that might benefit from virus-targeted therapy.

BG06 Beyond keratin: when simplex is not so simple

Abstract Epidermolysis bullosa simplex (EBS) is a heterogeneous, congenital, mucocutaneous fragility syndrome, characterized ultrastructurally by cleavage within the basal keratinocyte. Seventy-five per cent of cases are attributable to mutations in KRT5 and KRT14, the vast majority of which are dominant negative mutations, resulting in impaired keratin intermediate filament dynamics within the basal layer of the epidermis. The presenting phenotype, including extracutaneous manifestations and overall severity, is often closely linked to the underlying genetic mutation; however, genotype–­phenotype correlation is poorly resolved in a minority of cases, and there is an approximately additional 15% of patients in whom genetic abnormalities are not identified. However, recent advances in next-generation sequencing technologies have expanded the mutational database. We describe two cases of EBS with rare underlying mutations and distinct clinical phenotypes. A 46-year-old man presented with widespread blistering primarily affecting his hands, feet, elbows and knees since childhood. He had unaffected, consanguineous parents and one of his three siblings was affected. Other presenting features included dystrophic nails, focal plantar keratoderma, truncal mottled pigmentation and large areas of hypopigmentation on the back. We considered a diagnosis of ‘EBS with mottled pigmentation’, but the autosomal recessive mode of inheritance and dystrophic nails were atypical. DNA whole-exome sequencing (WES) and bidirectional Sanger sequencing revealed a novel homozygous PLEC mutation [c.94dupG p.(Ala32GlyfsTer3)]. Pathogenic PLEC variants are known to cause autosomal dominant EBS, the Ogna type (MIM131950), autosomal recessive EBS with muscular dystrophy (MIM226670) and autosomal recessive EBS with pyloric atresia (MIM612138). To date, our patient has not manifested any signs of progressive muscle weakness, but the presenting age can vary. The second patient was a 30-year-old woman who presented with blistering on her flexures, feet, hands and friction sites since the age of 4 months. She had unaffected, consanguineous parents and eight unaffected siblings. WES and confirmatory bidirectional Sanger sequencing identified a homozygous nonsense mutation in DST [c.3370C>T p.(Gln1124Ter)]. DST encodes the epithelial isoform of bullous pemphigoid antigen-1, present in the skin, muscle and neuronal tissues. Only 11 patients with DST mutation-induced EBS have been reported to date. The cutaneous phenotype is reported to be blisters, postinflammatory dyspigmentation, scarring, nail dystrophy, keratoderma and hyperhidrosis. If the affected exon is present in neuronal isoforms and neuronal tissue, neurological symptoms are expected. Unlike the patient reported elsewhere who harbours exactly the same mutation, our patient has reported no neurological issues to date. These cases highlight the importance of unravelling genotype–phenotype correlation in rare autosomal recessive forms of EBS, with possible implications for future prognosis and management.

Combined immunodeficiency and impaired PI3K signaling in a patient with biallelic LCP2 variants

Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T-and B-cell defects. We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.

The clonal heterogeneity of colon cancer with liver metastases

Background Colon cancer with liver metastases (CCLM) characterized by genetic heterogeneity is an evolutionary process leading to variations in response to selective pressure, but the underlying evolutionary models still remains unclear.Methods Total of 30 samples, including primary tumor and two to four matched liver metastases from 8 treatment-naïve patients with CCLM were collected, and subjected to whole-exome DNA sequencing. PyClone was used to calculate intra and inter-tumor heterogeneity, LICHeE was used to reconstruct the cancer phylogeny trees and investigate the subclonal composition.ResultsThe genetic differences were observed between primary and metastatic lesions, as well as among multiple metastases in all patients. The natural history models of colorectal cancer in each case were identified, including parallel, linear, and branching evolution. Liver metastases could originate from primary lesions or other metastases. Pathway and process enrichment analysis also showed obvious heterogeneity and enhancement of several molecular functions.ConclusionsOur data reveal the genetic and heterogeneity between primary and metastatic lesions, as well as among multiple metastases and provide genomic evidence for clonal heterogeneity for CCLM.

Abstract 6762: Tumor immune microenvironment &genomic features of non-small cell lung carcinomas in patients with HIV (PWH)

Abstract Introduction: Lung cancer is the leading cause of cancer-related death among PWH with a relative risk that is over 3-fold higher compared to the uninfected population. In addition, HIV-infected patients with non-small cell lung cancer (NSCLC) have a significantly worse prognosis. The determinants for these associations remain uncertain. Methods: We investigated the immune contexture of 18 primary NSCLCs from PWH and 19 clinicopathologically matched NSCLCs from HIV-negative cases represented in tissue microarrays using 37-plex Imaging Mass Cytometry (IMC) panels including cell phenotype markers (CK,CD4,CD8,CD20,CD45RO,CD25,etc), immunomodulatory targets (PD-L1,PD-L2,B7-H3,B7-H4,B2M,etc) and DNA damage response/repair (DDR) indicators (γH2AX, RAD51,BRCA1,53BP1,etc). The samples were analyzed using single-cell tissue segmentation and spatial analysis. A subset of cases was also studied using whole exome DNA sequencing (WES) and mRNA sequencing (RNAseq). Results: NSCLCs from PWH showed comparable levels of tumor infiltrating lymphocytes (TILs) relative to HIV-negative cases with a trend towards a lower CD4+/CD8+ cell ratio. However, CD4+ and CD8+ TILs in PWH showed higher levels of markers associated with T-cell activation and dysfunction including GZB, CD25, Ki-67, PD-1, LAG-3, and TIM-3 than non-HIV tumors. The epithelial tumor cells in HIV-associated cases showed higher levels of the immunomodulatory ligands PD-L1, CD47, B7-H4 and VISTA; and altered levels of the DDR markers γH2AX, BRCA1 and 53BP1 relative to the non-HIV controls. Differences were also seen in tumor-associated macrophages consistent with an immune regulatory environment. The spatial analysis of tumor and immune cells revealed a distinct topographic distribution of these cells in HIV-associated malignancies. WES analysis showed a higher nonsynonymous tumor mutational burden in HIV-associated tumors (400 vs 202 mean mutations/exome) with increased predicted HLA class-I neoantigens and a numerically higher immunoediting score than the non-HIV group. Exploratory analysis identified enrichment in RYR2 and KIAA1671 gene mutations in HIV-associated malignancies. RNAseq revealed distinct mRNA expression profiles of HIV-positive and negative NSCLCs with a fraction of transcripts showing differential expression associated with tumor suppressor functions, interleukin signaling and DDR. Conclusion: NSCLCs in PWH display profound immunological and genomic alterations suggesting defective immunosurveillance. Our study reveals novel characteristics of HIV-associated NSCLC with possible clinical implications for this growing and understudied population. Citation Format: Shruti Desai, Kishu Ranjan, Syim Salahuddin, Ramsey Yusuf, Jianlei Gu, Daiwei Tang, Yong Kong, Barani Kumar Rajendran, Hongyu Zhao, Yuval Kluger, Sarah Goldberg, Brinda Emu, Kurt Alex Schalper. Tumor immune microenvironment &genomic features of non-small cell lung carcinomas in patients with HIV (PWH) [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 6762.

Abstract 2150: CLDN18/ARHGAP26 fusions in gastric cancers

Abstract Background Previous work has shown that young females, who present with genomically-stable, poorly-differentiated, diffuse gastric cancer harbor CLDN18/ARHGAP26 fusions more often than older individuals with intestinal type or non-diffuse gastric cancer histology. This fusion usually occurs between exon 5 of CLDN18 and exon 12 of ARHGAP26, is associated with a poor prognosis, and confers a propensity for metastasis. We investigated the prevalence of this fusion and characterized the fusion-positive patient population among gastric cancer patients whose tumor samples underwent OncoExtraTM assay testing. Methods Tumor and paired-normal samples from 171 gastric cancer patients aged 18-90 years were sequenced with the OncoExtraTM assay. This assay uses whole-exome DNA sequencing to detect somatic single base substitutions, indels, copy number alterations, and whole transcriptome RNA sequencing to detect gene fusions and alternative transcripts. The RNA sequencing data were used to identify CLDN18/ARHGAP26 fusions. Results The 171 patients were well balanced between sexes: 91 (53.2%) men, 80 (46.8%) women. Most patients were older: 44 (25.7%) ≤50 years old, 127 (74.3%) >50 years old. There were 6 (3.5%) patient samples with a CLDN18/ARHGAP26 fusion. The fusion was found more often in females (5 of 80, 6.3%) than males (1 of 91, 1.1%) and younger patients (3 of 44, 6.8%, ≤50 years; 3 of 127, 2.3%, >50 years). All 6 had diffuse histology by pathology, variably described as poorly differentiated, diffuse, or signet ring subtype. Notably, the 2 patients with mixed histology did not have metastases reported on the pathology report, while the 4 whose histology was described as diffuse or signet ring cell type were metastatic. In all cases the fusion involved exon 5 of CLDN18 and exon 12 of ARHGAP26. Further analysis revealed that five of the fusion-containing tumors had alterations in genes/pathways known to be oncogenic drivers of cell proliferation, specifically 1 ERBB2 (2 mutations present), 1 SWI/SNF pathway (ARID1A and PBRM1 both altered), 2 MET (both amplifications), and 1 PI3K pathway (PI3KCA altered). The sixth tumor sample possessed no obvious driver alterations: only TP53 and WRN were altered. Five of the 6 tumors had low tumor mutational burden (TMB) and were microsatellite stable, the sixth had intermediate TMB, and none had evidence for genomic instability. No CLDN18 fusions were found in any other solid tumors (7050 samples). Conclusions The OncoExtraTM assay detected gastric cancer CLDN18/ARHGAP26 fusions at a frequency (3.5%) similar to previous reports. The majority were female and of younger age compared to patients lacking this fusion. Five of the 6 tumors had additional oncogenic alterations associated with cell proliferation. Given its frequency in poorly differentiated gastric cancer with metastatic potential, as well as its prevalence in a relatively young cohort, this fusion is an attractive target for ongoing drug development. Citation Format: Deborah Josefson, David W. Hall, Jess Hoag, Ariane Kemkes, Janine LoBello, Snehal G. Thakkar, Gargi Basu. CLDN18/ARHGAP26 fusions in gastric cancers [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 2150.

Abstract P5-14-12: ESR1-alterations in HR+HER2- breast cancer patients

Abstract Introduction/Background Endocrine therapy remains the fundamental treatment for advanced HR+ breast cancer (BC). For those patients who become refractory to endocrine therapy, resistance may be associated with mutations, amplifications, and fusions in the ESR1-encoded estrogen receptor. Here we examine the frequency of ESR1 alterations and the associated genomic landscape. Methods HR+/HER2- BC samples were sequenced with the OncomapTM ExTra assay, which uses whole-exome DNA sequencing with germline subtraction to detect somatic single nucleotide substitutions, indels, and copy number alterations (CNAs), and uses RNA sequencing to detect gene fusions. Tumor mutational burden (TMB) and microsatellite instability (MSI) are also reported. For analysis, BC samples were grouped by site: local (primary breast or regional lymph node) versus metastatic. Prior treatment history was unknown. Testing for a possible association between ESR1 and other biomarkers (genes, MSI, and TMB) was done using Fisher’s Exact Test (p≤0.05). Results A total of 988 HR+HER2- breast cancer patient samples were included in the analysis. Of these, 821 (83.1%) were local samples and the remaining 167 were metastatic samples, with liver (63, 37.7%), bone (20, 12.0%), skin (16, 9.6%) and chest wall (15, 9.0%) being the most common locations. ESR1 alterations were present in 84 tumor samples, 37 local and 47 metastatic, representing 4.5% and 28.1% of samples, respectively. ESR1 alterations included missense mutations (54 samples), fusions (29 samples), and amplifications (8 samples). The most common missense mutations were Y537S (20 samples, 37.0%), D538Q (20 samples, 37.0%), and E380Q (6 samples, 11.1%), which were located in the ligand binding domain and included both clonal and subclonal events. There were 30 ESR1 fusions identified, 17 (2.1%) in local and 13 in metastatic (7.7%) samples. Most fusions (24 samples, 82.8%) involved the same partner, CCDC170, while the other 6 fusions had unique partners. Examination of the 143 other biomarkers altered among ESR1-altered samples revealed 15 genes and MSI-high status that appeared to be over-represented (Table 1). However, none of the associations were statistically significant after correcting for multiple comparisons. Further, there was no evidence that the prevalence of ERBB2, TP53, AKT1 and PIK3CA alterations differed by ESR1 status (Table 1). Conclusions ESR1 alterations were significantly more common in HR+/HER2- metastatic breast cancer samples compared to local samples. Comprehensive genomic profiling with RNA sequencing identified both common and rare ESR1 fusions, which were most frequent in the metastatic samples. No significant difference in the molecular profile of ESR1 altered vs ESR1 wildtype samples was found in this cohort. Clinical trials with novel selective ER degraders (SERDs) to target these ESR1 alterations are ongoing. Table 1. Biomarkers that showed the highest association with ESR1 and other notable breast cancer biomarkers. Citation Format: Gargi Basu, Sameer S. Udhane, Susan Dombrowski, Lenny Hong, Fadel Alyaqoub, Michelle Barbi de Moura, Thiruppavai Chandrasekaran, Turgut Dogruluk, David Driscoll, Aimee Jalkanen, Pawan Noel, Szabolcs Szelinger, Min Wang, David Hall, Jess Hoag, Janine Lobello, Frederick Baehner, Snehal Thakkar, Joyce O’Shaughnessy. ESR1-alterations in HR+HER2- breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-12.