Re-analysis of Next-generation Sequencing Data in Patients with Hypertrophic Cardiomyopathy: Contribution of Spliceogenic MYBPC3 Variants in an Italian Cohort.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac muscle disease characterized by clinical and genetic heterogeneity. Genetic testing can reveal the presence of disease-causing variants in genes encoding sarcomere proteins. However, it yields inconclusive or negative results in 40-60% of HCM cases, owing to, among other causes, technical limitations such as the inability to detect pathogenic intronic variants. Therefore, we aimed to increase the diagnostic yield of molecular analysis for HCM by improving the in-silico detection of intronic variants in MYBPC3 that may escape detection by algorithms normally used with tagged diagnostic panels. We included 142 HCM probands with negative results in Illumina TruSight Cardio panel analysis, including exonic regions of 174 cardiomyopathy genes. Raw data were re-analyzed using existing bioinformatics tools. The spliceogenic variant c.1224-80G>A was detected in three patients (2.1%), leading us to reconsider their molecular diagnosis. These patients showed late onset and mild symptoms, although no peculiar phenotypic characteristics were shared. Collectively, rare spliceogenic MYBPC3 variants may play a role in causing HCM, and their systematic detection should be performed to provide more comprehensive solutions in genetic testing using multigenic panels.

A pilot study of precision treatment for patients with lung cancer pain by Longteng Tongluo recipe using serum genomics.

OBJECTIVE: To investigate the efficacy of Longteng Tongluo recipe (, LTTL) combined with three-step analgesia for the treatment of lung cancer pain, and the changes in serum miRNA expressions before- and after treatment with LTTL and its correlation with lung cancer pain. The possible mechanism underlying LTTL effects on the treatment of lung cancer pain was conducted. METHODS: The pilot study was conducted at the oncology ward of the Yueyang Hospital and the Longhua Hospital between March 2018 and October 2019. A prospective, single-blind, placebo controlled, randomized clinical trial of LTTL or placebo combined with three-step analgesia treatments were administered to 24 cancer pain patients diagnosed with lung cancer. Analgesic efficacy was investigated as the primary outcome. Equivalent morphine consumption and numerical rating scale (NRS) scores were used as the secondary outcome. In the present study, we utilized deep sequencing techniques to compare the differential miRNA expressions in serum samples obtained from two groups: the lung cancer pain treatment group (LTTL + three-step analgesia) and the control group (placebo + three-step analgesia). Next, we employed the target prediction database to investigate the target genes for differential miRNA expressions and Gene Ontology (GO) analysis along with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to examine the roles and the major biochemical and signaling pathways related to the differentially expressed target genes, respectively. RESULTS: LTTL treatment significantly reduces the NRS score (P = 0.021) as compared to those before treatment, along with significant reductions in the total morphine equivalent consumption (P = 0.007) and the average daily equivalent morphine consumption (P = 0.003) as opposed to the control group. The expressions of 31 miRNAs differed considerably between the two groups of patients (≥ 2 times up-modulated or down-regulated between these groups, P<0.05). For instance, the miRNAs expression levels for patients before treatment (has-miR-2110 and has-miR-7d-3p) were significantly enhanced as compared to the healthy people, after LTTL treatment, the expressions of miR-2110 and miR-7d-3p in patients with lung cancer pain reduced significantly. Studies show that the above two miRNAs were significantly associated with lung cancer pain, which could mediate lung cancer pain. Furthermore, we identified 355 genes as potential targets of the 31 differentially expressed miRNAs. Pathway enrichment analyses using KEGG and GO analysis indicated that these target genes may play a crucial role in the development and modulation of lung cancer pain. CONCLUSION: LTTL demonstrated a discernible impact on alleviating lung cancer pain and its mechanism of action may be related to the downregulation of has-miR-2110 and has-miR-7d-3p expressions. This pilot study provides support for further exploration of LTTL in patients with lung cancer pain.

Colorectal cancer with a germline BRCA1 variant inherited paternally: a case report.

BRCA genes have well-known associations with breast and ovarian cancers. However, variations in the BRCA gene, especially germline variations, have also been reported in colorectal cancer (CRC). We present the case of a rectal cancer with a germline BRCA1 variation inherited from the paternal side. A 39-year-old male was admitted with rectal cancer. The patient underwent surgical resection and the pathologic diagnosis was adenocarcinoma. Next-generation sequencing was performed and a BRCA1 variant was detected. Reviewing the public database and considering the young age of the patient, the variant was suggested to be germline. The patient's father had had prostate cancer and next-generation sequencing testing revealed an identical BRCA1 variant. In the BRCA cancer group, there is relatively little attention paid to male cancers. The accumulation of male CRC cases linked to BRCA variations may help clarify the potential pathological relationship between the two.

Comprehensive Analysis of Microsatellite Instability in Canine Cancers: Implications for Comparative Oncology and Personalized Veterinary Medicine.

Microsatellite instability (MSI) is a crucial feature in cancer biology, yet its prevalence and significance in canine cancers remain largely unexplored. This study conducted a comprehensive analysis of MSI across 10 distinct canine cancer histotypes using whole-exome sequencing data from 692 tumor-normal sample pairs. MSI was detected in 64% of tumors, with prevalence varying significantly among cancer types. B-cell lymphomas exhibited the highest MSI burden, contrasting with human studies. A novel "MSI-burden" score was developed, correlating significantly with tumor mutational burden. MSI-high (MSI-H) tumors showed elevated somatic mutation counts compared to MSI-low and microsatellite stable tumors. The study identified 3632 recurrent MSI-affected genomic regions across cancer types. Notably, seven of the ten cancer types exhibited MSI-H tumors, with prevalence ranging from 1.5% in melanomas to 37% in B-cell lymphomas. These findings highlight the potential importance of MSI in canine cancer biology and suggest opportunities for targeted therapies, particularly immunotherapies. The high prevalence of MSI in canine cancers, especially in B-cell lymphomas, warrants further investigation into its mechanistic role and potential as a biomarker for prognosis and treatment response.

Genetic profiling and diagnostic strategies for patients with ectodermal dysplasias in Korea.

BACKGROUND: Ectodermal dysplasia (ED) is a rare genetic disorder that affects structures derived from the ectodermal germ layer. RESULTS: In this study, we analyzed the genetic profiles of 27 Korean patients with ED. Whole exome sequencing (WES) was performed on 23 patients, and targeted panel sequencing was conducted on the remaining 4 patients. Among the patients in the cohort, 74.1% (20/27) tested positive for ED. Of these positive cases, EDA and EDAR mutations were found in 80% (16/20). Notably, 23.1% (3/13) of EDA-positive cases exhibited copy number variations. Among the 23 patients who underwent WES, we conducted a virtual panel analysis of eight well-known genes, resulting in diagnoses for 56.5% (13/23) of the cases. Additionally, further analysis of approximately 5,000 OMIM genes identified four more cases, increasing the overall positivity rate by approximately 17%. These findings underscore the potential of WES for improving the diagnostic yield of ED. Remarkably, 94.1% of the patients manifesting the complete triad of ED symptoms (hair/skin/dental) displayed detectable EDA/EDAR mutations. In contrast, none of the 7 patients without these three symptoms exhibited EDA/EDAR mutations. CONCLUSIONS: When conducting molecular diagnostics for ED, opting for targeted sequencing of EDA/EDAR mutations is advisable for cases with classical symptoms, while WES is deemed an effective strategy for cases in which these symptoms are absent.

Gut microbial assessment among Hylobatidae at the National Wildlife Rescue Centre, Peninsular Malaysia.

IMPORTANCE: Recent developments in genetic analytical techniques have enabled the comprehensive analysis of gastrointestinal symbiotic bacteria as a screening tool for animal health conditions, especially the endangered gibbons at the National Wildlife Rescue Centre (NWRC). OBJECTIVE: High-throughput sequencing based on 16S ribosomal RNA genes was used to determine the baseline gut bacterial composition and identify potential pathogenic bacteria among three endangered gibbons housed in the NWRC. METHODS: Feces were collected from 14 individuals (Hylobates lar, n = 9; Hylobates agilis, n = 4; and Symphalangus syndactylus, n = 1) from March to November 2022. Amplicon sequencing were conducted by targeting V3-V4 region. RESULTS: The fecal microbial community of the study gibbons was dominated by Bacteroidetes and Firmicutes (phylum level), Prevotellaceae and Lachnospiraceae/Muribaculaceae (family level), and Prevotella (and its subgroups) (genera level). This trend suggests that the microbial community composition of the study gibbons differed insignificantly from previously reported conspecific or closely related gibbon species. CONCLUSIONS AND RELEVANCE: This study showed no serious health problems that require immediate attention. However, relatively low alpha diversity and few potential bacteria related to gastrointestinal diseases and streptococcal infections were detected. Information on microbial composition is essential as a guideline to sustain a healthy gut condition of captive gibbons in NWRC, especially before releasing this primate back into the wild or semi-wild environment. Further enhanced husbandry environments in the NWRC are expected through continuous health monitoring and increase diversity of the gut microbiota through diet diversification.

Genetic testing and new variants in diagnosis of congenital ichthyoses.

BACKGROUND: The aim of this study was to evaluate how diagnostic practice in congenital ichthyoses has evolved during the years 2000-2020 and what kind of gene variants of congenital ichthyosis have been found. METHODS: The study cohort of this register-based research consisted of a total of 88 patients, whose diagnostic testing was conducted, and ichthyosis diagnoses set at the Department of Dermatology and the Department of Clinical Genetics at Tampere University Hospital during the years 2000-2020. RESULTS: Diagnosis of ichthyosis was confirmed with genetic testing in 33 cases, and with conventional diagnostic methods, such as clinical findings, skin biopsy and family history of ichthyoses, in 55 cases. We observed four novel variants in patients with the clinical diagnoses of congenital ichthyoses. CONCLUSION: When genetic testing became available, it was offered primarily to patients with severe forms of ichthyosis. During the study period next-generation sequencing became the genetic testing method of choice providing new opportunities in diagnostics.

Phenotype-genotype spectrum of a cohort of congenital muscular dystrophies: a single-centre experience from India.

Congenital Muscular Dystrophies (CMD) are phenotypically and genotypically heterogenous disorders with a prevalence of 0.68 to 2.5/100,000, contributing to significant morbidity and mortality. We aimed to study the phenotype-genotype spectrum of genetically confirmed cases of CMD. This was retrospective & descriptive study done at a quaternary care referral centre in south India. Genetically confirmed cases of CMDs seen between 2010 to 2020 were recruited. Detailed clinical history, including pedigree, MRI brain/muscle, next generation sequencing results of 61 CMD cases were collected. Collagen VI-related dystrophy (COL6-RD) (36%) was the most common subtype with variants frequently seen in COL6A1 gene. Other CMDs identified were LAMA2-RD (26%), alpha-dystroglycan-RD (19%), LMNA-RD (8%), CHKB-RD (7%) and SEPN1-RD (3%). Similar to previous cohorts, overall, missense variants were common in COL-6 RD. Variants in triple helical domain (THD) of COL6-RD were seen in 11/22 patients, 5 of whom were ambulatory contrary to previous literature citing severe disease with these variants. However, our follow-up period was shorter. In the LAMA2-RD, 2/16 patients were ambulatory & all 16 carried truncating variants. Among dystroglycanopathies, FKRP-RD was the commonest. Milder phenotype of FKRP- RD was observed with variant c.1343C > T, which was also a recurrent variant in our cohort. p.Arg249Trp variant in LMNA-CMD associated with early loss of ambulation was also identified in 1/5 of our patients who expired at age 2.8 years. The current retrospective series provides detailed clinical features and mutation patterns of genetically confirmed cases of CMD from a single center in India.

Mismatch Repair (MMR) Gene Mutation Carriers Have Favorable Outcome in Colorectal and Endometrial Cancer: A Prospective Cohort Study.

Germline (Lynch syndrome, LS) and somatic deficiencies of mismatch repair proteins (MMRd) are linked to colorectal and endometrial cancer; however, their prognostic impact in Asian populations remains unclear. This prospective cohort study aimed to determine the prevalence and outcome of germline and somatic MMRd in cancer patients suspected of LS. Patients with colorectal or endometrial cancer suspected of LS were enrolled and underwent gene sequencing for germline MMRd (gMMRd) and immunohistochemistry staining of MMR proteins in a subset of the pathological samples (pMMRd). Among the 451 enrolled patients, 36 patients were gMMRd (+). Compared with gMMRd (-) patients, the 10-year relapse-free survival in gMMRd (+) patients was significantly higher (100% vs. 77.9%; p = 0.006), whereas the 10-year overall survival was similar (100% vs. 90.9%; p = 0.12). Among the 102 gMMRd (-) patients with available pMMR status, 13.7% were pMMRd (+). The 5-year relapse-free survival was 62.9% in gMMRd (-) pMMRd (+) patients and 35.0% in gMMRd (-) pMMRd (-) patients, both lower than gMMRd (+) patients (100%; p < 0.001). This study showed that having LS confers a favorable outcome in colorectal and endometrial cancer patients and highlights the importance of germline genetic testing following the detection of somatic MMRd.

High-throughput sequencing analysis of differential microRNA expression in the process of blocking the progression of chronic atrophic gastritis to gastric cancer by Xianglian Huazhuo formula.

OBJECTIVE: To explore the mechanism of Xianglian Huazhuo formula (, XLHZ) blocking the development of chronic atrophic gastritis (CAG) to gastric cancer (GC) through bioinformatics analysis and in vitro. METHODS: Pathological morphology of gastric mucosa of rats were observed. High-throughput sequencing was used to analyze the miRNA expression profile of gastric mucosa. The miRanda, miRDB and miRWalk databases were used to predict the differential target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for differential target genes. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs and target genes. Western blot, EdU, wound healing and flow cytometry were used to observe the effect of XLHZ on epithelial-mesenchymal transition (EMT) markers, proliferation, migration, apoptosis and cell cycle of CAG cells in vitro. RESULTS: A total of five differentially expressed miRNAs and four differential target genes were screened in this study. GO analysis showed that the target genes were enriched in regulation of neuron development, regulation of transcription factor activity and regulation of RNA polymerase. KEGG pathways database differences in gene enrichment of target genes in the Wnt signaling pathway, Phospholipase D signaling pathway and mitogen-activated protein kinase signaling pathway. qRT-PCR confirmed that miRNAs and its target genes were consistent with the screening results. In vitro, our study revealed that XLHZ could increase the expression of E-cadherin, decrease the expression of transforming growth factor ß1, vimentin and ß-catenin, inhibite the proliferation and migration of CAG cells, cause cell cycle arrest at G0/G1 and G2/M phase, induce the apoptosis of CAG cells, and prevent the progression of CAG to GC. CONCLUSION: This study provided a new idea for the mechanism of blocking the progression of CAG to GC by XLHZ, which may be related to the expression of miR-20a-3p, miR-320-3p, miR-34b-5p, miR-483-3p and miR-883-3p and their target genes transferrin receptor, nuclear receptor subfamily 4 member 2, delta like canonical Notch ligand 1 and a kinase anchor protein 12 in CAG. In the future, we will continue to investigate the linkage between the active ingredients of XLHZ and the relevant miRNAs and their target genes, so as to provide more sufficient experimental basis for clinically effective prevention of CAG to GC.

The impact of the European Society of Cardiology guidelines and whole exome sequencing on genetic testing in hereditary cardiac diseases.

In the last decade, an incredible improvement has been made in elucidating the genetic bases of cardiomyopathies. Here we report the impact of either the European Society of Cardiology (ESC) guidelines or the use of whole exome sequencing (WES) in terms of a number of variants of uncertain significance (VUS) and missed diagnoses in a series of 260 patients affected by inherited cardiac disorders. Samples were analyzed using a targeted gene panel of 128 cardiac-related genes and/or WES in a subset of patients, with a three-tier approach. Analyzing (i) only a subset of genes related to the clinical presentation, strictly following the ESC guidelines, 20.77% positive test were assessed. The incremental diagnostic rate for (ii) the whole gene panel, and (iii) the WES was 4.71% and 11.67%, respectively. The diverse analytical approaches increased the number of VUSs and incidental findings. Indeed, the use of WES highlights that there is a small percentage of syndromic conditions that standard analysis would not have detected. Moreover, the use of targeted sequencing coupled with "narrow" analytical approach prevents the detection of variants in actionable genes that could allow for preventive treatment. Our data suggest that genetic testing might aid clinicians in the diagnosis of inheritable cardiac disorders.

Prevalence and molecular characterization of hepatitis E virus (HEV) from wild rodents in Hubei Province, China.

Hepatitis E, caused by the hepatitis E virus (HEV), is a global public health issue. Low similarity between the gene sequences of mouse and human HEV led to the belief that the risk of human infection was low. Recent reports of chronic and acute hepatitis E caused by murine HEV infection in humans in Hong Kong have raised global concerns. Therefore, it is crucial to investigate the epidemiology and prevalence of HEV in China. We comprehensively analyzed different rodent HEV strains to understand rocahepevirus occurrence in Hubei Province, China. The HEV positivity rate for was 6.43% (73/1136). We identified seven near-full-length rocahepevirus strains and detected rat HEV antigens in tissues from different mouse species. HEV has extensive tissue tropism and a high viral load in the liver. We highlight the genetic diversity of HEVs in rodents and underscore the importance of paying attention to their variation and evolution.

Uncommon pathogen misidentification of Herbaspirillum huttiense as Burkholderia cepacia in bacteremia: a case report.

Herbaspirillum huttiense is an opportunistic pathogen associated with rare cases of bacteremia. In this case report, H huttiense was isolated from blood samples collected from an intravenous catheter (incubated for 20.8 hours) and a peripheral vein (incubated for 14.16 hours) of a lung adenocarcinoma patient. Positive blood culture bottles were subjected to smear preparation, and Gram staining and microscopic examination revealed the presence of gram-negative rods in both aerobic bottles. We used the VITEK MS automatic microbial mass spectrometry system, VITEK 2 Compact automatic microbial analysis system, and high-throughput nucleic acid sequencing for accurate identification of the isolate. It is noteworthy that although the VITEK 2 Compact identified the isolate as Burkholderia cepacia, confirmation through VITEK MS mass spectrometry and 16S ribosomal DNA (rDNA) sequencing identified it as H huttiense. Subsequently, antimicrobial susceptibility testing was performed using the broth microdilution method, following the guidelines for nonfermenting gram-negative bacilli provided by the Clinical and Laboratory Standards Institute. This case highlights the possibility of misidentification of H huttiense as B cepacia by VITEK 2 Compact in certain situations, emphasizing the importance of considering uncommon pathogens, such as H huttiense, in the context of bacteremia in cancer patients.

KCNJ3 is a novel candidate gene for autosomal dominant pure hereditary spastic paraplegia identified using whole genome sequencing.

Hereditary spastic paraplegia (HSP) is a group of familial diseases characterized by progressive corticospinal tract degeneration. Clinically, patients present with lower-limb spasticity and weakness. To date, more than 80 genetic HSP types have been identified. Despite advances in molecular genetics, novel HSP gene discoveries are ongoing, with a low genetic diagnostic yield. In this study, we aimed to determine pathogenic variants in a family with HSP, which was not diagnosed through conventional genetic testing. We clinically characterized a large family and conducted whole genome sequencing (WGS) analysis of four affected and three unaffected individuals in the family to identify the genetic cause of HSP. This family had autosomal dominant pure (uncomplicated) late childhood-onset HSP. The patients' symptoms accelerated between the ages of 20 and 30. Brain magnetic resonance images typically showed white matter changes, a thin corpus callosum, and cerebellar atrophy. We identified a heterozygous missense variant, KCNJ3 c.1297T>G (p.Leu433Val), through WGS and family genetic analysis, confirmed by Sanger sequencing. We suggest that the identification of KCNJ3 c.1297T>G (p.Leu433Val) constitutes the discovery of a potential novel gene responsible for HSP in this family. This is the first study to report the possible role of a KCNJ3 variant in HSP pathogenesis. Our findings further expand the phenotypic and genotypic spectrum of HSP.

Microbial composition and diversity in intraradicular biofilm formed in situ: New concepts based on next-generation sequencing.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMBs) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n = 10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the MicrobiomeAnalyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.

Microbial composition and diversity in intraradicular biofilm formed in situ: New concepts based on next-generation sequencing.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMB) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n = 10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the Microbiome Analyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.

Multiple Primary Cancers With Hematologic Malignancies and Germline Predisposition: A Case Series.

The term "multiple primary (MP) cancers" refers to the existence of more than one cancer in the same patient. The combination of MP cancers with hematological malignancies is relatively uncommon. In this study, we present five patients diagnosed with MP cancers concomitant with hematological malignancies. We comprehensively analyzed their clinical characteristics, cytogenetic profiles, and germline and somatic variants. As first primaries, two patients had solid cancer not followed by cytotoxic therapy and three had hematologic cancer, followed by cytotoxic therapy. The second primaries were all hematologic malignancies that did not meet the criteria for therapy-related myeloid neoplasm. Notably, two (40%) out of the five patients harbored pathogenic potential/presumed germline variants in cancer predisposition genes. Therefore, germline variant testing should be considered when MP cancers with hematological malignancies require consideration for related donor stem cell transplantation.

Complete DPYD genotyping combined with dihydropyrimidine dehydrogenase phenotyping to prevent fluoropyrimidine toxicity: A retrospective study.

INTRODUCTION: In April 2019, French authorities mandated dihydropyrimidine dehydrogenase (DPD) screening, specifically testing uracilemia, to mitigate the risk of toxicity associated with fluoropyrimidine-based chemotherapy. However, this subject is still of debate as there is no consensus on a standardized DPD deficiency screening test. We conducted a real-life retrospective study with the aim of assessing the impact of DPD screening on the occurrence of severe toxicity and exploring the potential benefits of complete genotyping using next-generation sequencing. METHODS: All adult patients consecutively treated with 5-fluorouracil (5-FU) or its oral prodrug at six cancer centers between March 2018 and February 2019 were considered for inclusion. Dihydropyrimidine dehydrogenase deficiency screening included gene encoding DPD (DPYD) genotyping using complete genome sequencing and DPD phenotyping (uracilemia or dihydrouracilemia/uracilemia ratio) or both tests. Associations between each DPD screening method and (i) severe (grade ≥3) early toxicity and (ii) fluoropyrimidine dose reduction in the second chemotherapy cycle were evaluated using multivariable logistic regression analysis. Furthermore, we assessed the concordance between DPD genotype and phenotype using Cohen's kappa. RESULTS: A total of 551 patients were included. Most patients were tested for DPD deficiency (86%) including DPYD genotyping only (6%), DPD phenotyping only (8%), or both (72%). Complete DPD deficiency was not detected in the study population. Severe early toxicity events were observed in 73 patients (13%), with two patients (0.30%) presenting grade 5 toxicity. Despite the numerically higher toxicity rate in untested patients, the occurrence of severe toxicity was not significantly associated with the DPD screening method (p = 0.69). Concordance between the DPD genotype and phenotype was weak (Cohen's kappa of 0.14). CONCLUSION: Due to insufficient numbers, our study was not able to demonstrate any added value of DPYD genotyping using complete genome sequencing to prevent 5-FU toxicity. The optimal strategy for DPD screening before fluoropyrimidine-based chemotherapy requires further clinical evaluation.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Exploring Molecular Genetic Alterations and RAF Fusions in Melanoma: A Belvarafenib Expanded Access Program in Patients with RAS/RAF-Mutant Melanoma.

BACKGROUND: Melanoma incidence is on the rise in East Asia, yet studies of the molecular landscape are lacking in this population. We examined patients with melanoma who underwent next-generation sequencing (NGS) at a single tertiary center in South Korea, focusing on patients harboring NRAS or RAF alterations who received belvarafenib, a pan-RAF dimer inhibitor, through the Expanded Access Program (EAP). PATIENTS AND METHODS: Data were collected from 192 patients with melanoma who underwent NGS between November 2017 and May 2023. Variant call format data were obtained and annotated. Patients in the EAP received 450 mg twice daily doses of belvarafenib. RESULTS: Alterations in the RAS/RTK pathway were the most prevalent, with BRAF and NRAS alteration rates of 22.4% and 17.7%, respectively. NGS enabled additional detection of fusion mutations, including 6 BRAF and 1 RAF1 fusion. Sixteen patients with NRAS or RAF alterations received belvarafenib through the EAP, and disease control was observed in 50%, with 2 patients demonstrating remarkable responses. CONCLUSIONS: Our study highlights the value of NGS in detecting BRAF, NRAS mutations and RAF fusions, expanding possibilities for targeted therapies in malignant melanoma. Belvarafenib showed clinical benefit in patients harboring these alterations. Ongoing trials will provide further insights into the safety and efficacy of belvarafenib.