Multiple Gene Mutations Research Articles

Targeted DNA Sequencing of Cutaneous Melanoma Identifies Prognostic and Predictive Alterations.

Cutaneous melanoma (CM) can be molecularly classified into four groups: BRAF mutant, NRAS mutant, NF1 mutant and triple wild-type (TWT) tumors lacking any of these three alterations. In the era of immune checkpoint inhibition (ICI) and targeted molecular therapy, the clinical significance of these groups remains unclear. Here, we integrate targeted DNA sequencing with comprehensive clinical follow-up in CM patients. This was a retrospective cohort study that assessed clinical and molecular features from patients with localized or metastatic CM who underwent targeted next-generation sequencing as part of routine clinical care. A total of 254 patients with CM who had a CLIA-certified targeted sequencing assay performed on their tumor tissue were included. Of the 254 patients with cutaneous melanoma, 77 were BRAF mutant (30.3%), 77 were NRAS mutant (30.3%), 47 were NF1 mutant (18.5%), 33 were TWT (13.0%) and the remaining 20 (7.9%) carried mutations in multiple driver genes (BRAF/NRAS/NF1 co-mutated). The majority of this co-mutation group carried mutations in NF1 (n = 19 or 90%) with co-occurring mutations in BRAF or NRAS, often with a weaker oncogenic variant. Consistently, NF1 mutant tumors harbored numerous significantly co-altered genes compared to BRAF or NRAS mutant tumors. The majority of TWT tumors (n = 29, 87.9%) harbor a pathogenic mutation within a known Ras/MAPK signaling pathway component. Of the 154 cases with available TMB data, the median TMB was 20 (range 0.7-266 mutations/Mb). A total of 14 cases (9.1%) were classified as having a low TMB (≤5 mutations/Mb), 64 of 154 (41.6%) had an intermediate TMB (>5 and ≤20 mutations/Mb), 40 of 154 (26.0%) had a high TMB (>20 and ≤50 mutations/Mb) and 36 of 154 (23.4%) were classified as having a very high TMB (>50 mutations/Mb). NRAS mutant melanoma demonstrated significantly decreased overall survival on multivariable analysis (HR for death 2.95, 95% CI 1.13-7.69, p = 0.027, log-rank test) compared with other TCGA molecular subgroups. Of the 116 patients in our cohort with available treatment data, 36 received a combination of dual ICI with anti-CTLA4 and anti-PD1 inhibition as first-line therapy. Elevated TMB was associated with significantly longer progression-free survival following dual-agent ICI (HR 0.26, 95% CI 0.07-0.90, p = 0.033, log-rank test). NRAS mutation in CMs correlated with significantly worse overall survival. Elevated TMB was associated with increased progression-free survival for patients treated with a combination of dual ICI, supporting the potential utility of TMB as a predictive biomarker for ICI response in melanoma.

Abstract 7356: 3 UTR variations in esophageal squamous cell carcinoma in African Americans

Abstract Esophageal squamous cell carcinoma (ESCC) predominantly affects African Americans (AA). Esophageal Cancer (EC) consists of two major histological subtypes: esophageal adenocarcinoma and squamous cell carcinoma. ESCC ranks among the most lethal cancers and is particularly aggressive among AA, comprising about 75% of total ESCC patients. The 5-year survival rate for localized EC in AA patients is 25%; in contrast, the survival rate for EC patients of Caucasian origin is 48%. However, for metastatic disease, the survival rate decreases dramatically to 5% regardless of race. To conduct an extensive mutation screening, we performed whole exome sequencing (WES) in 10 paired AA ESCC and control tissues. Mutations included nonsynonymous mutations in multiple genes and copy number variations. Here, we report on further analysis of the WES data to identify 3 UTR noncoding region alterations, comprising the highest proportion of noncoding variations. Whole exome sequencing was performed using Agilent SureSelect XT Human All Exon V6 + UTR kit on matched normal-tumor samples from 10 AA patients (nine males and one female) with advanced-stage ESCC. The ages of patients ranged from 53 to 80 years. All patients, except for one, reported tobacco use and alcohol consumption. Our analysis yielded 5378 variants in noncoding regions in our cohort. The most variations were found in the 3UTR location, with 46% of all noncoding variations. In our dataset, variations in the 3UTR region of 48 genes were predicted by CScape to assert an oncogenic effect. The highest ten oncogenic predictive scores are for ETV1, FOXO3, MSI2, TCF7L2, KAT6A, FOXO1, IRF4, TRIM24, ELK4, and FGFR1 genes. Approximately 2500 genes having 3’UTR variations are over-presented in intracellular signaling and cell-cell communications, transcriptional regulation of pluripotent stem cells by OCT4, SOX2, NANOG, generic RNA polymerase II transcription, RHO GTPase cycle, and regulation of cytoskeletal remodeling. The combined effects of noncoding and coding region mutations may largely impact poor prognosis in AA with ESCC and yield targets for developing candidate therapeutics. Citation Format: Ceyda Erkizan, Robert Wadleigh. 3 UTR variations in esophageal squamous cell carcinoma in African Americans [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 7356.

Whole-genome sequencing of SARS-CoV-2 from the initial cases of domestic cat infections in Canada.

Two cat nasal swabs from Canada's earliest confirmed SARS-CoV-2 positive domestic cats were sequenced to over 99% SARS-CoV-2 genome coverage. One cat had lineage A.23.1 SARS-CoV-2 not reported before in animals. Both sequences have multiple spike gene mutations and clustered closely with human-derived sequences in the global SARS-CoV-2 phylogenetic tree.

Prevalence and changing antimicrobial resistance profiles of Shigella spp. isolated from diarrheal patients in Kolkata during 2011-2019.

The primary aim of this study was to investigate the occurrence, characteristics, and antimicrobial resistance patterns of various Shigella serogroups isolated from patients with acute diarrhea of the Infectious Diseases Hospital in Kolkata from 2011-2019. During the study period, Shigella isolates were tested for their serogroups, antibiotic resistance pattern and virulence gene profiles. A total of 5.8% of Shigella spp. were isolated, among which S. flexneri (76.1%) was the highest, followed by S. sonnei (18.7%), S. boydii (3.4%), and S. dysenteriae (1.8%). Antimicrobial resistance against nalidixic acid was higher in almost all the Shigella isolates, while the resistance to β-lactamases, fluoroquinolones, tetracycline, and chloramphenicol diverged. The occurrence of multidrug resistance was found to be linked with various genes encoding drug-resistance, multiple mutations in the topoisomerase genes, and mobile genetic elements. All the isolates were positive for the invasion plasmid antigen H gene (ipaH). Dendrogram analysis of the plasmid and pulsed-field electrophoresis (PFGE) profiles revealed 70-80% clonal similarity among each Shigella serotype. This comprehensive long-term surveillance report highlights the clonal diversity of clinical Shigella strains circulating in Kolkata, India, and shows alarming resistance trends towards recommended antibiotics. The elucidation of this study's outcome is helpful not only in identifying emerging antimicrobial resistance patterns of Shigella spp. but also in developing treatment guidelines appropriate for this region.

A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic pathogen of increasing clinical importance due to its poor clinical outcomes and limited treatment options. Drug discovery and development in this highly antibiotic-resistant species will require further understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods for facile genetic engineering are relatively inefficient. This study reports on the first application of CRISPR/Cas9 for gene editing in M. abscessus using a dual-plasmid workflow. We establish that our method is easily programmable, efficient, and versatile for genetic disruptions in M. abscessus. This is a critical advancement to facilitating targeted gene function studies in this emerging pathogen.

Autosomal Dominant Tubulointerstitial Kidney Disease Case with Sever Acute Kidney Injury in Activities Beyond the Usual: An Unusual Presentation of the Disease

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD) is associated with multiple gene mutations and is recognized under various names, including medullary cystic kidney disease type 2, familial juvenile hyperuricemic nephropathy, uromodulin-associated kidney disease, and medullary cystic kidney disease type 2. A comprehensive understanding of these rare disorders can shed light on the pattern of tubulointerstitial fibrosis observed in various forms of chronic kidney disease. Case Presentation: Here, a case of a patient is presented who experienced acute kidney injury (AKI) following severe physical activity and an atypical manifestation of this genetic disorder. Typically, ADTKD leads to a gradual loss of kidney function, but the patient presented with AKI. The patient's renal function returned to normal following appropriate therapy. The patient's brother had a similar history of renal disease following physical activities, prompting suspicion of a shared genetic disorder. Conclusions: A genome-wide association study (GWAS) was conducted, and the results revealed the presence of the mucin 1 (MUC1) mutation, confirming the diagnosis of ADTKD. This case highlights the importance of recognizing atypical presentations of genetic disorders and utilizing advanced genetic studies for accurate diagnosis and management.

Gemtuzumab Ozogamicin in Acute Myeloid Leukemia: Efficacy, Toxicity, and Resistance Mechanisms-A Systematic Review.

Acute myeloid leukemia (AML) is a diverse group of leukemias characterized by the uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells with chromosomal rearrangements and multiple gene mutations and the impairment of normal hematopoiesis. Current efforts to improve AML outcomes have focused on developing targeted therapies that may allow for improved antileukemic effects while reducing toxicity significantly. Gemtuzumab ozogamicin (GO) is one of the most thoroughly studied molecularly targeted therapies in adults. GO is a monoclonal antibody against CD33 IgG4 linked to the cytotoxic drug calicheamicin DMH. The use of GO as a chemotherapeutic agent is not generalized for all patients who suffer from AML, particularly for those whose health prevents them from using intensive conventional chemotherapy, in which case it can be used on its own, and those who have suffered a first relapse, where its combination with other chemotherapeutic agents is possible. This systematic review aimed to comprehensively evaluate GO, focusing on its molecular structure, mode of action, pharmacokinetics, recommended dosage, resistance mechanisms, and associated toxicities to provide valuable information on the potential benefits and risks associated with its clinical use. A systematic review of eight scientific articles from 2018 to 2023 was conducted using PRISMA analysis. The results showed that GO treatment activates proapoptotic pathways and induces double-strand breaks, initiating DNA repair mechanisms. Cells defective in DNA repair pathways are susceptible to GO cytotoxicity. GO has recommended doses for newly diagnosed CD33+ AML in combination or as a single agent. Depending on the treatment regimen and patient status, GO doses vary for induction, consolidation, and continuation cycles. Multidrug resistance (MDR) involving P-glycoprotein (P-gp) is associated with GO resistance. The overexpression of P-gp reduces GO cytotoxicity; inhibitors of P-gp can restore sensitivity. Mitochondrial pathway activation and survival signaling pathways are linked to GO resistance. Other resistance mechanisms include altered pharmacokinetics, reduced binding ability, and anti-apoptotic mechanisms. GO has limited extramedullary toxicity compared to other AML treatments and may cause hepatic veno-occlusive disease (HVOD). The incidence of hepatic HVOD after GO therapy is higher in patients with high tumor burden. Hematological side effects and hepatotoxicity are prominent, with thrombocytopenia and neutropenia observed. In conclusion, GO's reintroduction in 2017 followed a thorough FDA review considering its altered dose, dosing schedule, and target population. The drug's mechanism involves CD33 targeting and calicheamicin-induced DNA damage, leading to apoptosis and resistance mechanisms, including MDR and survival signaling, which impact treatment outcomes. Despite limited extramedullary toxicity, GO is associated with hematological side effects and hepatotoxicity.

Towards A Cell Biology of Alzheimer’s Disease

In many neurodegenerative disorders, including Alzheimer's Disease (AD), the dysfunction of multiple cell types causes neuronal cell death, but the precise cellular processes involved remain incompletely understood for any neurodegenerative disease. Strikingly, synapses are often affected early during pathogenesis, probably because presynaptic terminals are distant outstations of neurons that are the most vulnerable part of a neuron. In AD, multiple mutations in APP and presenilin genes cause rare familial cases, while the ApoE4 variant of the ApoE gene represents the strongest genetic risk factor for sporadic AD in the general population. APP and presenilin gene mutations are thought to induce AD pathogenesis by overproducing pathogenic Abeta variants, and ApoE4 is thought to influence Abeta clearance, but how Abeta might incite AD pathogenesis and how ApoE4 might predispose to AD pathogenesis remains incompletely understood. Moreover, compelling evidence implicates microglial and possibly astrocytic dysfunction in AD pathogenesis. In my lab, we have taken a cell-biological approach to these questions with a focus on synapses because of their prominent role in AD, recognizing that synapse impairments in AD may also be secondary to microglial dysfunction. We have examined how pathogenic APP mutations, chronic impairments of presenilin function, or ApoE4 may act on synapses, using human neurons trans-differentiated from ES and iPS cells as a model. In my presentation, I will discuss the current status of this project which is far from providing a definitive account of AD pathogenesis but may offer a basis for understanding the most basic processes underlying such pathogenesis.

Omics analysis of Alzheimer’s disease stratified by the microglial polygenic effect

AbstractBackgroundRecent single‐cell omics analyses have revealed that microglia change into reactive microglia when Aβ accumulates in the brain and exhibit Aβ phagocytosis. However, reactive microglia are less likely to be induced in TREM2 mutation carriers. This microglia‐centred pathological mechanism may be considered one of the pathologies of Alzheimer’s disease (AD). However, a single gene mutation, such as a TREM2 mutation, can rarely explain the pathology of AD. Therefore, polygenic effects involving multiple gene mutations have been proposed. In this study, we propose a polygenic risk score (PRS) to quantify the microglial polygenic effect. We stratified patient groups by this PRS and examined differences in pathogenesis based on omics analysis using postmortem brains.MethodWhole‐genome sequencing and bulk brain tissue RNA‐seq of the frontal cortex were performed using postmortem brain samples from 100 subjects with Braak senile plaque (SP) and neurofibrillary tangle (NFT) staging information. In addition, single‐nuclei RNA‐seq (snRNA‐seq) was performed on 15 of these subjects (healthy subjects: 8 subjects, AD patients: 7 subjects).ResultWe divided AD patients into low and high groups by microglial PRS and performed bulk brain tissue RNA‐seq analysis. We identified 112 differentially expressed genes and found that those genes were related to autophagy and inflammation. To further validate these findings at the single‐cell level, we identified cell clusters containing microglia based on snRNA‐seq. Gene expression analysis of microglia in 3 AD patients with low PRS and 4 AD patients with high PRS suggested altered transcriptional profiles of immune cells and phagocytosis. The expression of APOE and TYROBP, signature genes of reactive microglia, was significantly upregulated in the AD patients with low PRS compared to the AD patients with high PRS and healthy subjects. In contrast, CX3CR1 and P2RY12, signature genes of homeostatic microglia, were downregulated.ConclusionThe stratification analysis of AD by PRS suggests that microglial reactivity differs among AD patient groups. Future detailed microglial observations in postmortem brains from AD patients with low PRS and those with high PRS will further elaborate our results.

Strengthening the Diagnosis of Drug-Resistant Tuberculosis Using NGS-Based Approaches and Bioinformatics Pipelines for Data Analysis in India.

In India, drug-resistant tuberculosis (DR-TB) is a major public health issue and a significant challenge to stop TB program. An estimated 27% of new TB cases and 44% of previously treated TB cases are resistant to at least one anti-TB drug. The conventional methods for DR-TB diagnosis are time-consuming and have limitations, leading to delays in treatment initiation and the spread of the disease. Next-generation sequencing (NGS) based approaches have emerged as a promising tool for diagnosing DR-TB, simultaneously offering rapid and accurate detection of resistance mutations in multiple genes. NGS-based approaches generate a large amount of data, which requires efficient and reliable bioinformatics pipelines for data analysis. TBProfiler and Mykrobe are the bioinformatics pipelines that have been created to analyze NGS data for the diagnosis of DR-TB. These pipelines use reference-based and machine-learning approaches to detect resistance mutations and predict drug susceptibility, enabling clinicians to make informed treatment decisions. Implementing NGS-based approaches and bioinformatics pipelines for DR-TB diagnosis can potentially improve patient outcomes by facilitating early detection of drug resistance and guiding personalized treatment regimens. However, the widespread adoption of these approaches in India faces several challenges, including high costs, limited infrastructure, and a lack of trained personnel. Addressing these challenges requires concerted effort to ensure equitable access to and effective implementation of these innovative technologies.

NGS Profile and the Mathematical Prediction Model for Venetoclax Combination Therapy in HM-Screen-Japan 02 Study

[Introduction] Azacitidine and venetoclax combination therapy (Aza/Ven) is a novel strategy for acute myeloid leukemia (AML). Although this regimen is widely used as a first line treatment not only for unfit/elderly patients but also for fit/young patients in the real-world situation, appropriate patient selection is an important issue. HM-SCREEN-Japan 02 (UMIN-CTR UMIN000046371) is a Japanese multicenter prospective observational study to evaluate the clinical utility of targeted sequencing. In this study, we performed the sub-analysis of newly diagnosed AML patients who received Aza/Ven as a 1 st line treatment to elucidate the genetic background of AML. Furthermore, we proposed a novel mathematical model to predict the response of Aza/Ven. [Methods] AmoyDx® Myeloid Blood Cancer Panel for acute myeloid leukemia was used for the targeted sequencing. We performed linear discriminant analysis to predict complete response patients by Aza/Ven based on clinical information before treatment. We utilized three clinical quantities for the analysis: Wilms Tumor-1 (WT1) mRNA expression level, the number of “favorable mutations”, and that of “unfavorable mutations”. Favorable and unfavorable mutations were defined as IDH1, IDH2, NPM1, and CEBPA mutations, and ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations, respectively, based on the previous reports (NEJM 2019, Blood 2022). The data were standardized and utilized for the linear discriminant analysis. Statistical analyses were performed using R version 4.2.3 software (R Foundation for Statistical Computation, Vienna, Austria). [Results] Total 158 patients were enrolled in our study. Among 48 patients who received Aza/Ven as a 1 st line therapy, NGS analyses were performed prior to treatment in 38 patients. Their median age was 70.5 (39-92) years old. AML with myelodysplasia-related changes was the major subtype (71%) and 37 percent of the cases had high risk cytogenetic abnormalities at diagnosis. After 1 st line Aza/Ven treatment, complete remission (CR)/ CR with incomplete blood count recovery (CRi) was achieved in 63% of the cases while the remaining 37% were non-CR. There was a trend that patients with NPM1 and IDH1/2 mutations achieved CR/CRi, while patients with RUNX1 mutation were not tended to achieve CR (Figure 1). Next, we performed linear discriminant analysis of treatment response using three pretreatment clinical factors. Linear discriminant analysis found a linear combination of prognostic factors that best separates two classes in the group of patients. Thirty-seven patients were employed to determine the discriminant function D, given by D = 0.638 × x − 0.76 × y + 0.68 × z + 0.145. Here x, y, and z represented the WT1 quantity in peripheral blood, the number of “favorable mutations”, and that of “unfavorable mutations” after standardization, respectively. Using this model, 81.1% of cases were correctly classified, and so were 75.7% of cases with leave-one-out cross validation method (Table 1). [Discussions] As previously reported (NEJM 2019, Blood adv 2020), IDH1/2 and NPM1 mutations tended to be sensitive to Aza/Ven in our cohort. However, it would be hard to predict the response of Aza/Ven by a single gene mutation because multiple gene mutations were co-existed in most of the cases (Figure 1) and clinical factors including tumor markers might also affect the outcome. Our mathematical model, involving gene mutations and WT1, could efficiently predict the response of Aza/Ven, which may support the selection of 1 st line treatment. In conclusion, our study revealed the genetic landscape of real-world Aza/Ven therapy and provided a potential prognostic model.

Integrated Molecular Characterization of HER2-Low Breast Cancer Using Next Generation Sequencing (NGS).

Based on immunohistochemistry (IHC) and in situ hybridization (ISH), HER2-low breast cancers (BC) subtype-defined as IHC1+ or IHC2+/ISH- tumors-emerged and represent more than half of all BC. We evaluated the performance of NGS for integrated molecular characterization of HER2-low BC, including identification of actionable molecular targets, copy number variation (CNV), and microsatellite instability (MSI) analysis. Thirty-one BC specimens (11 HER2+, 10 HER2-, and 10 HER2-low) were routinely analyzed using IHC and ISH, and were selected and analyzed using NGS for gene mutations including ESR1, PIK3CA, AKT1, ERBB2, TP53, BRCA1, and BRCA2, CNV, and MSI. CNV values for the ERBB2 gene were significantly (p < 0.001) different between HER2+, and either HER2-low or HER2- tumors with mean values of 7.8 (SD = 6.8), 1.9 (SD = 0.3), and 2.0 (SD = 0.3), respectively. Using 3.25 as the cutoff value, 96.8% overall concordance of HER2 status was achieved between IHC and NGS compared to IHC and ISH. Using NGS, gene mutations and amplifications were detected in 68% (21/31) and 19% (6/31) of the cases, respectively. One case of MSI was detected in a HER2-negative and ISH unamplified case. Beside IHC, NGS allows the identification of HER2-low subtype simultaneously, with the detection of multiple actionable gene mutations being helpful for molecular board treatment selection.

Investigation of Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G promoter polymorphism and other thrombophilia genes in patients with acute pulmonary embolism

Abstract Background Venous thromboembolism (VTE) is a serious threat to life and health, commonly known as the silent killer. Recent study has found that VTE is a polygenetic disease and more than 60% of deep vein thrombosis risk is influenced by genetic factors [1]. PAI-1 is a serine protease with negative feedback to fibrinolysis, activating and inhibiting coagulation [2]. The aim of this study was to elucidate the clinical impact of the PAI-1 4G/5G polymorphism and other thrombophilia in patients with acute PE. Methods A total of 58 subjects with acute PE and 60 healthy controls, were recruited in Institute of Cardiology. The accuracy of the RT-PCR method for detecting F5 G1691A (FVL) and PAI-1 4G/5G polymorphisms was evaluated by using sequencing method as the gold standard. Moreover, the association of the PAI-1 4G/5G polymorphism with susceptibility, treatment efficacy and recurrence status were explored. Eventually, we evaluated the association with inherited thrombophilia genes, such as the coagulation Factor V Leiden, prothrombin G20210A, FGB-455 G/A and methylenetetrahydrofolate reductase C677T and 1298 A/C. Results It was noted that the vast majority of patients with acute PE (87.9%) present polymorphism in the PAI-1 gene (29.3% homozygous (4G/4G) and 58.6% heterozygous (5G/4G)). Eleven patients out of 58 (19% of patients) showed factor V Leiden mutation and 19 patients out of 58 (32,7% of patients) showed mutation in FGB-455 G/A (2 homozygotes); MTHFR C677T gene mutation was found in 55,2%, while prothrombin 20210A gene mutation was presented in 6 patients (10.3% of patients). Nine patients (15,5% of patients) presented with chronic complication of VTE and 38.7% of patients showed recurrent pulmonary embolism. More than a half of patients (51.9%) had multiple gene mutation and only one presented with mutations in three studied genes. It showed a significant difference in the occurrence of other thromboses in the body and significant increase in the recurrence of pulmonary embolism and residual complications in these patients. It was also observed that individuals with the 4G/5G genotype had lower neutrophil counts and neutrophil-to-lymphocyte ratio (NLR) than the 5G/5G genotype. Furthermore, it was underlined that the patients with the 5G/5G genotype were more likely to achieve complete recanalization compared to the 4G/4G genotype. Individuals carrying the 5G/5G genotype were more likely to develop a recurrence-free status as compared to those with the 4G/4G or 4G/5G genotypes. PAI-1 antigen levels in the VTE group were significantly higher than those in the HC group. Conclusion The PAI-1 4G/5G polymorphism has potential value in assessing the prognosis of patients with VTE. This study has laid the foundation for the application of PAI-1 4G/5G polymorphism in the personalized management and monitoring of patients with VTE. According to this study results PAI-1 4G/5G polymorphism could be included in laboratory testing panel of thrombophilia.

Hermansky-Pudlak Syndrome: A Comprehensive Review of Clinical Presentation, Pathogenesis, and Therapeutic Strategies

Hermansky-Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder characterized by a complex clinical phenotype, including oculocutaneous albinism, bleeding diathesis, and a predisposition to pulmonary fibrosis. This article provides a comprehensive examination of HPS, encompassing its genetic underpinnings, molecular mechanisms, clinical manifestations, and emerging therapeutic interventions. HPS is primarily attributed to mutations in multiple genes, causing defects in the biogenesis of lysosome-related organelles, leading to platelet and granule abnormalities, which contribute to the bleeding diathesis. In addition, the oculocutaneous albinism associated with HPS is a result of impaired melanin synthesis. The article explores recent advancements in understanding HPS pathogenesis, such as aberrant platelet function and the dysregulation of various cellular pathways. Additionally, we discuss diagnostic challenges and the need for a multidisciplinary approach in managing patients with HPS. Furthermore, this review highlights the evolving landscape of therapeutic strategies, including experimental therapies targeting the underlying genetic defects, advances in supportive care for bleeding disorders, and novel approaches for managing pulmonary fibrosis. In conclusion, this article underscores the importance of ongoing research in HPS, emphasizing the potential for improved diagnosis and treatment outcomes in patients with this intriguing and complex syndrome.

Abstract 18931: A PCSK9 and APOB Double Heterozygote Presenting Phenotypically Similar to Homozygous Familial Hypercholesterolemia

Introduction: Familial hypercholesterolemia (FH) is an inherited disorder associated with mutations in APOB, PCSK9 and LDLR genes. Heterozygous FH (HeFH) generally has one gene mutation. Homozygous FH (HoFH) has two mutations in the same gene and is characterized by vastly elevated plasma LDL-C, accelerated ASCVD and resistance to standard lipid-lowering therapy. Combinations of mutations can present with a phenotype similar to HoFH. We present a double heterozygous patient with unreported mutations in PCSK9 and APOB genes, exhibiting clinical features of HoFH. Hypothesis: Patients with multiple gene mutations for FH can present clinically similar to HoFH and would benefit from novel lipid lowering therapies Methods: 73-year-old female with a history of diabetes and hyperlipidemia presented in 2018 with dyspnea on exertion. Cholesterol (TC) and triglycerides (TG) were 430 and 715 mg/dl respectively, on rosuvastatin 40 mg and omega 3 fatty acids. Physical exam showed arcus senilis. A nuclear stress test showed ST depressions in inferolateral leads but normal scintigraphy. Genetic testing revealed APOB and PCSK9 mutations. She was then treated with rosuvastatin, icosapentyl ethyl, ezetimibe and fenofibrate. TC, LDL, TG and ApoB levels were 463, 236, 747 and &gt;240 mg/dl respectively. Evolocumab was added, with reduction of TC, TG and ApoB levels to 326, 432 and 182mg/dl, despite maximal available therapy. Results: Genetic testing revealed APOB variant NP_000375.2:p.Gln3378His and PCSK9 variant NP_777596.2:pAla544Thr, which were unreported mutations with unknown clinical significance. Genetically, this patient is a double heterozygote but expresses a phenotype similar to HoFH due to a poor response to maximum therapy. Conclusions: Although usually genetically defined, the phenotype of HoFH can present with different genetic variations. Chaudhry et al established genetic diagnosis of HoFH in 0.9% of patients initially diagnosed as HeFH, of which 3 were true homozygotes and 3 were compound heterozygotes. These findings support the idea that various heterozygous combinations of mutations in the genes for LDLR, APOB and PCSK9, may present clinically similar to HoFH. Patients with these mutations could benefit from novel agents such as evinacumab.

A Prospective Multicenter Phase II Study of Orelabrutinib-Lenalidomide-Rituximab (OLR) in Patients with Untreated Mantle Cell Lymphoma (MCL) in China (POLARIS Study): Preliminary Analysis on Efficacy, Safety, Mutation Spectrum and Impact of Mutation Profiling on Treatment Responses

Background: Recently, the first-line treatment of MCL has been continuously evolving with the introduction of targeted agents and chemo-free regimens. It is well-known that bruton's tyrosine kinase (BTK) inhibitors were effective with MCL patients (pts). Orelabrutinib (O), a novel highly selective BTK inhibitor, has shown high response rates and good tolerability in R/R MCL (Song et al. Blood. 2020). Besides, O combined with rituximab (R) could preserve NK cell mediated ADCC induced by R and enhance the apoptosis of tumor (Yu et al. Mol Ther Oncolytics. 2021). Thus, we hypothesized that OLR would exhibit synergistic antitumor activity in MCL and improve the depth and durability of responses. Here, we reported the preliminary data on efficacy, safety, and mutation spectrum of OLR regimen in untreated MCL from the POLARIS study (NCT05076097). Methods: Pts ≥18 years with untreated MCL were enrolled in this prospective, multicenter, single-arm phase 2 study. All pts received OLR-induction therapy on a 28-day cycle for 6 cycles (O, 150 mg/d; L, 15 mg on days 1-21, then 20 mg of cycles 2-6 if tolerated or 10 mg if not tolerated; R, 375 mg/m 2 on days 1, 8, 15, 22, then day 1 of cycles 3, 5), followed by OLR-maintenance therapy for up to 18 cycles. Primary endpoint was complete response rate (CRR) after 6 cycles of induction treatment. Secondary endpoints included overall response rate (ORR), time to response (TTR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Exploratory endpoint was to explore the prognostic value of detecting peripheral blood (PB) ctDNA by NGS. Results: As of July 10, 2023,a total of 28 pts (male, 89.3%; median age, 60.0 y [range, 53.0-64.5]) were enrolled. Baseline characteristics included 96.4% Eastern Cooperative Oncology Group performance status of 0-1, 82.1% stage III-IV disease, 57.1% Ki67 index (≥30%), 75.0% low-risk MIPI scores, 42.9% bone marrow involvement, and 60.7% maximum lesion diameter of ≥5 cm. Twenty-one pts (75.0%) who have completed 6 cycles of induction therapy were evaluable for response, 16 (76.2%) had a complete response (CR) and 5 (23.8%) had a partial response (PR), with an ORR of 100% ( Figure 1). In addition, 18 of 21 pts were available for minimal residual disease (MRD) analysis, and PB-MRD and bone marrow MRD (BM-MRD) of the 18 pts were negative. The results of CRR were reported across different subgroups stratified by Ki67 index (&amp;lt;30% vs. ≥30%, 66.7% and 83.3%), MIPI scores (low vs. intermediate, 81.3% and 60.0%), maximum lesion diameter (&amp;lt;5 cm vs. ≥5 cm, 87.5% and 69.2%), and bone marrow involvement (yes vs. no, 83.3% and 73.3%). With a median follow-up of 11.0 months, the median TTR was 3.0 months (range, 2.8-3.4). Median DOR and median PFS were not reached, with the estimated 12-month DOR rate and PFS rate of 90.9% and 92.3%, respectively. Mutationalspectrum (475 panels) was determined in 23 pts. The spectrum of gene mutations is depicted by PB samples and tissue samples at baseline in Figure 2. The most common mutated genes in tissues samples were: ATM (48%), KMT2D (43%), CCND1 (17%), SMARCA4(17%), ARID2 (13%), BCOR (13%), BIRC3 (13%), NFKBIE (13%), NOTCH1 (13%), ACTB (9%), CDK12 (9%), FANCC(9%),FAT1(9%),KLF2(9%),MEF2B(9%),MET(9%),SETD2(9%), TP53 (13%),TERT(9%). The most common mutated genes in PB samples were: ATM (48%), KMT2D (39%), DNMT3A (17%), BIRC3 (13%), CCND1 (13%), TP53 (13%), BCOR (9%), CARD11 (9%), CBLB (9%), CRYBG1 (9%), FAT1(9%), NOTCH1 (9%), GRIN2A (9%), KDM5A (9%), KLHL6(9%), KRAS (9%), MAP3K14 (9%). For all gene mutations, 75.4% (43/57) detected in tissue were also found in PB samples. Further, more additional mutated genes can be detected in plasma. Meanwhile, 4 of 23 pts had a PR, 1 of 4 had multiple gene mutations in PB samples, including BTK and TP53. This suggests that ctDNA detection may potentially predict that these gene mutations are associated with depth of remission. Twenty-six (92.9%) pts experienced adverse events (AEs). The most common AEs (any grades, ≥3 grades) were neutropenia (64.3%, 39.3%), leukopenia (60.7%, 14.3%), thrombocytopenia (35.7%, 7.1%), lymphopenia (35.7%, 3.6%), and COVID-19 infection (28.6%, 7.1%). At the time of data cut-off, 26 pts remained on study. No deaths were observed. Conclusions: The preliminary findings show that the OLR exerted synergistic antitumor activity, with manageable toxicity in untreated MCL. Detection of PB ctDNA by NGS method may help to predict prognosis.

Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

The PitA protein contributes to colistin susceptibility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of problematic infections in individuals with predisposing conditions. Infections can be treated with colistin but some isolates are resistant to this antibiotic. To better understand the genetic basis of resistance, we experimentally evolved 19 independent resistant mutants from the susceptible laboratory strain PAO1. Whole genome sequencing identified mutations in multiple genes including phoQ and pmrB that have previously been associated with resistance, pitA that encodes a phosphate transporter, and carB and eno that encode enzymes of metabolism. Individual mutations were engineered into the genome of strain PAO1. Mutations in pitA, pmrB and phoQ increased the minimum inhibitory concentration (MIC) for colistin 8-fold, making the bacteria resistant. Engineered pitA/phoQ and pitA/pmrB double mutants had higher MICs than single mutants, demonstrating additive effects on colistin susceptibility. Single carB and eno mutations did not increase the MIC suggesting that their effect is dependent on the presence of other mutations. Many of the resistant mutants had increased susceptibility to β-lactams and lower growth rates than the parental strain demonstrating that colistin resistance can impose a fitness cost. Two hundred and fourteen P. aeruginosa isolates from a range of sources were tested and 18 (7.8%) were colistin resistant. Sequence variants in genes identified by experimental evolution were present in the 18 resistant isolates and may contribute to resistance. Overall our results identify pitA mutations as novel contributors to colistin resistance and demonstrate that resistance can reduce fitness of the bacteria.

Data-adaptive and pathway-based tests for association studies between somatic mutations and germline variations in human cancers.

Cancer is a disease driven by a combination of inherited genetic variants and somatic mutations. Recently available large-scale sequencing data of cancer genomes have provided an unprecedented opportunity to study the interactions between them. However, previous studies on this topic have been limited by simple, low statistical power tests such as Fisher's exact test. In this paper, we design data-adaptive and pathway-based tests based on the score statistic for association studies between somatic mutations and germline variations. Previous research has shown that two single-nucleotide polymorphism(SNP)-set-based association tests, adaptive sum of powered score (aSPU) and data-adaptive pathway-based(aSPUpath) tests, increase the power in genome-wide association studies (GWASs) with a single disease trait in a case-control study. We extend aSPU and aSPUpath to multi-traits, that is, somatic mutations of multiple genes in a cohort study, allowing extensive information aggregation at both SNP and gene levels. -values from different parameters assuming varying genetic architecture are combined to yield data-adaptive tests for somatic mutations and germline variations. Extensive simulations show that, in comparison with some commonly used methods, our data-adaptive somatic mutations/germline variations tests can be applied to multiple germline SNPs/genes/pathways, and generally have much higher statistical powers while maintaining the appropriate type I error. The proposed tests are applied to a large-scale real-world International Cancer Genome Consortiumwhole genome sequencing data set of 2583 subjects, detecting more significant and biologically relevant associations compared with the other existing methods on both gene and pathway levels. Our study has systematically identified the associations between various germline variations and somatic mutations across different cancer types, which potentially provides valuable utility for cancer risk prediction, prognosis, and therapeutics.

Ischemic cerebrovascular disease caused by genetic mutation and patent foramen ovale

Abstract Objectives The search for genetic mutations is very important in younger patients and other age groups with a history of recurrent cerebrovascular diseases (CVD) and a family history of other causes to be excluded. The aim of this study is to define the characteristics of genetic mutations in the etiology of ischemic stroke. Methods Twenty-three patients with acute CVD in the last 1 year and only genetic mutations acknowledged in the etiology were retrospectively analyzed. We determined the frequency of the genetic mutations that are observed in cerebral arterial events (CAE) and cerebral venous thrombosis (CVT). Results All patients had at least one genetic mutation and 19 of them had arterial events and 4 had venous thrombosis. MTHFR mutation was the most common mutation and PAI-1 mutation was the second in line for the arterial events. PAI 4G/5G, MTHFR A1298 and FV mutations were most frequently observed in venous events. Patent foramen ovale (PFO) was detected in 14 patients (%74) with CAE. Conclusions We concluded that multiple gene mutations may significantly increase the development of CVD. CVD is most commonly associated with MTHFR, PAI-1 or FV gene mutations and is most commonly seen in CAE. MTHFR mutations showed moderate linear correlation in the development of arterial events and FXII and FXIII mutations in venous events. The association of thrombophilia and PFO is high in patients who have undergone CAE, especially responsible for recurrent events. This study will need to be confirmed by prospective studies with larger sample and control group.