Next-generation Sequencing Analysis Research Articles

A phase 2, multicenter, clinical trial of CPX-351 in older patients with secondary or high-risk acute myeloid leukemia: PETHEMA-LAMVYX.

LAMVYX was a multicenter, single-arm, phase 2 trial designed to validate the safety and efficacy of CPX-351 in patients aged 60-75 years with newly diagnosed, secondary acute myeloid leukemia and to generate evidence on key issues not addressed in the preceding regulatory pivotal trial. The primary end point of the study was the complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate after induction. Eligible patients were recommended to undergo allogeneic hematopoietic stem cell transplantation after the first consolidation cycle. Alternatively, patients could undergo up to six maintenance cycles with CPX-351. Twenty-nine patients (49%; 95% exact confidence interval [CI], 37%-62%) patients achieved a CR/CRi after one or two cycles of induction, with a measurable residual disease negativity rate of 67% as assessed by centralized, multiparameter flow cytometry. Among patients who had serial next-generation sequencing analyses available, clearance of somatic mutations that were present at diagnosis was achieved in 7 (35%). The median follow-up among survivors was 16.8 months (range, 8.7-24.3 months). The median event-free survival was 3.0 months (95% CI, 1.4-7.3 months), and the median overall survival was 7.4 months (95% CI, 3.7-12.7 months). In landmark analyses at day +100 from diagnosis, the 1-year overall and event-free survival rate among patients who underwent allogeneic hematopoietic stem cell transplantation was 70% (95% CI, 47%-100%) and 70% (95% CI, 47%-100%), respectively. The corresponding values were 89% (95% CI, 71%-100%) and 44% (95% CI, 21%-92%), respectively, for patients who entered the maintenance phase. No significant longitudinal changes were observed in severity index or quality-of-life visual analog scale scores. The current data provide novel insights that might inform the clinical positioning and optimal use of CPX-351, complementing previous results (ClinicalTrials.gov identifier NCT04230239).

Clinicopathologic and Genomic Features of Invasive Stratified Mucin-producing Carcinoma of the Uterine Cervix Coexisting With High-grade Squamous Intraepithelial Lesion.

Invasive stratified mucin-producing carcinoma (ISMC) is a specific type of adenocarcinoma of the cervix, which is associated with human papillomavirus infection and often coexists with other types of carcinomas. However, given its rarity, understanding of this disease remains insufficient. We present a unique case of ISMC of the cervix coexisting with a high-grade squamous intraepithelial lesion (HSIL). In addition to histologic and immunohistochemical feature observation, genomic profiling of the 2 lesions was performed. Histologically, the ISMC and HSIL lesions were independent of each other. Aside from the typical morphology, various architectural features of ISMC were observed. Immunohistochemically, the ISMC and HSIL lesions were strongly and diffusely positive for p16 and exhibited high Ki-67 expression. The ISMC lesion was also positive for CK7, MUC5AC, and MUC6, while it was negative for PAX-8. The HSIL lesion was positive for CK5/6 and p40. The combined positive score of PD-L1 was 55. The other markers were all negative in both lesions, and the p53 was wild-type. Next-generation sequencing analysis revealed multiple gene mutations in the ISMC and HSIL lesions. A total of 88 gene mutations were identified in the ISMC lesion, while 20 gene mutations were identified in the HSIL lesion. Three mutations (ERBB2, histidine decarboxylase gene [HDC], and BSN) were detected in the ISMC and HSIL lesions. Both lesions had a low tumor mutation burden and microsatellite-stable status. No copy number-associated variants or structural variations were identified in either lesion. These results suggest that patients with ISMC may benefit from PD-L1 immunotherapy and targeted therapy.

Vaccine-induced human monoclonal antibodies to PfRH5 show broadly neutralizing activity against P. falciparum clinical isolates

Vaccines to the Plasmodium falciparum reticulocyte binding-like protein homologue 5 (PfRH5) target the blood-stage of the parasite life cycle. PfRH5 has the potential to trigger the production of strain-transcendent antibodies and has proven its efficacy both in pre-clinical and early clinical studies. Vaccine-induced monoclonal antibodies (mAbs) to PfRH5 showed promising outcomes against cultured P. falciparum laboratory strains from distinct geographic areas. Here, we assessed the functional impact of vaccine-induced anti-PfRH5 mAbs on more genetically diverse P. falciparum clinical isolates. We used mAbs previously isolated from single B cells of UK adult PfRH5 vaccinees and used ex-vivo growth inhibition activity (GIA) assays to assess their efficacy against P. falciparum clinical isolates. Next-generation sequencing (NGS) was used to assess the breadth of genetic diversity in P. falciparum clinical isolates and to infer the genotype/phenotype relationship involved in antibody susceptibility. We showed a dose-dependent inhibition of clinical isolates with three main GIA groups: high, medium and low. Except for one isolate, our data show no significant differences in the mAb GIA profile between P. falciparum clinical isolates and the 3D7 reference strain, which harbors the vaccine allele. We also observed an additive relationship for mAb combinations, whereby the combination of GIA-low and GIA-medium antibodies resulted in increased GIA, having important implications for the contribution of specific clones within polyclonal IgG responses. While our NGS analysis showed the occurrence of novel mutations in the pfrh5 gene, these mutations were predicted to have little or no functional impact on the antigen structure or recognition by known mAbs. Our present findings complement earlier reports on the strain transcendent potential of anti-PfRH5 mAbs and constitute, to our knowledge, the first report on the susceptibility of P. falciparum clinical isolates from natural infections to vaccine-induced human mAbs to PfRH5.

Case report: A rare case of meningoencephalitis caused by Mycobacterium gordonae

Meningoencephalitis, an infectious disease affecting the nervous system, is primarily caused by a variety of pathogens. Non-tuberculous mycobacteria (NTM) have emerged as the leading causative agent of infections worldwide, but central nervous system infections resulting from NTM are infrequent in individuals with functioning immune systems. This case report highlights the diagnosis and treatment of a 26-year-old female patient who developed headaches 2 months post double eyelid surgery and was subsequently diagnosed with NTM meningoencephalitis through metagenomic next-generation sequencing (mNGS) analysis of cerebrospinal fluid. The patient underwent a comprehensive diagnostic and therapeutic protocol, resulting in a positive clinical outcome.

Distribution and classifications of PKHD1 gene variants in a Turkish population using the next generation sequencing method.

Autosomal recessive polycystic kidney disease is an inherited kidney disease. This study aims to detect rare and common DNA variants of the PKHD1 gene using next-generation sequencing (NGS) and to classify them in terms of being pathogenic according to The American College of Medical Genetics and Genomics. NGS analysis was performed on the DNA of 304 patients who were referred to Ege University Molecular Medicine Laboratory with suspected polycystic kidney disease. As a result, a total of 82 different DNA variants, 16 of which were novel, were detected. The breakdown of the variants found is as follows: 73 (89.02%) were missense variants, six (7.32%) nonsense variants, two (2.44%) frameshift deletions, and one (1.22%) nonframeshift deletion. According to The American College of Medical Genetics and Genomics classification of these variants, 26 were benign (Class 5), two were likely benign (Class 4), 36 were of uncertain significance (Class 3), and nine were likely pathogenic (Class 2), nine of which are pathogenic variants (Class 1). Heterozygosity was found in 39 (63.9%) patients, homozygosity in six (9.8%) patients, compound heterozygosity in 12 (19.7%) patients, and complex genotype in four (6.6%) patients in which variants in Class 1, Class 2 and Class 3 were determined according to ACMG classification. When the exon distributions of the DNA variants detected in the PKHD1 gene were analyzed, the most common exons of the DNA variant are exon 32 (n = 9), exon 58 (n = 8), exon 67 (n = 6), exon 61 (n = 5), 30 exons (n = 4). This fast and economical molecular diagnostic approach will provide a reliable prenatal diagnostic option, enabling definitive disease diagnosis and the identification of carriers.

Myeloid neoplasm with concurrent BCR::ABL1 translocation, and JAK2 and SF3B1 co-mutations with severe fibrosis: a case report

Abstract Introduction/Objective Myeloproliferative neoplasms (MPNs) arise from hematopoietic stem cells with somatically altered tyrosine kinase signaling. Classification of MPNs is based on hematologic, histopathologic and molecular characteristics including the presence of BCR::ABL1 and JAK2 V617F. Methods/Case Report We present a 72-year-old male with a myeloid neoplasm and severe fibrosis, with concurrent BCR::ABL1 translocation, and co-mutations in JAK2 and SF3B1. Initially, the patient was diagnosed with anemia, thrombocytosis and folic acid deficiency and was prescribed supplemental folic acid. At follow up, normocytic anemia and thrombocytosis were seen. Bone marrow biopsy showed overtly fibrotic marrow with maturing trilineage hematopoiesis, markedly increased megakaryopoiesis and ring sideroblasts. Chromosome analysis and sequential GTG-fluorescence in situ hybridization (FISH) of metaphases using the dual-fusion three color probe BCR/ABL1- ASS1 revealed abnormal results. A t(9;22)(q34;q11.2) was detected along with an atypical FISH signal pattern with a single BCR::ABL1 fusion on the derivative chromosome 22 instead of the typical two fusions, resulting in loss of one BCR::ABL1 fusion signal including loss of the ASS1 on the derivative chromosome 9. Next-generation sequencing (NGS) analysis detected double driver mutations, JAK2 p. Val617Phe and SF3B1 p. Lys700Glu in the patient. JAK2 V617F mutation was further confirmed by real-time quantitative PCR (qPCR) with variant allele frequencies of 39.7%. Additionally, the patient was positive for BCR::ABL1 p190 fusion transcript (10%). The presence of JAK2 and SF3B1 co-mutations along with ring sideroblasts and the patient’s clinical presentation suggests the diagnosis of myelodysplastic/myeloproliferative neoplasm with thrombocytosis and SF3B1 mutation. Results (if a Case Study enter NA) NA Conclusion Although, the detection of t(9;22) by chromosome analysis is a diagnostic feature of chronic myeloid leukemia (CML), our case adds to the limited information on co-existence of BCR::ABL1 and JAK2 V617F with SF3B1 mutations in severe fibrosis. The frequency of co-occurrence, the temporal order of acquisition and clonal relationship of these alterations is poorly understood, and it is also unclear for this case since there is no prior bone marrow biopsy or diagnostic work up. Literature search of these rare cases show that some patients with similar findings progress to myelofibrosis as in this patient. It is possible that the presence of two mutated tyrosine kinase genes accelerates disease progression.

Multigene Panel Next-Generation Sequencing Techniques in the Management of Patients with Metastatic Colorectal Carcinoma: The Way Forward for Personalized Treatment? A Single-Center Experience

The efficacy and cost-effectiveness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remain uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), and BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in five patients (2.8%) and SMO in four patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in three patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on number of mutations. In total, 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

AML typical mutations (CEBPA, FLT3, NPM1) identify a high-risk chronic myelomonocytic leukemia independent of CPSS molecular

AbstractMutations commonly associated with acute myeloid leukemia (AML), such as CEBPA, FLT3, IDH1/2, and NPM1, are rarely found in chronic myelomonocytic leukemia (CMML), and their prognostic significance in CMML has not been clearly identified. In 127 patients with CMML, we have retrospectively analyzed next-generation sequencing and polymerase chain reaction data from bone marrow samples collected at the time of CMML diagnosis. Seven patients harbored CEBPA mutations, 8 FLT3 mutations, 12 IDH1 mutations, 26 IDH2 mutations, and 11 NPM1 mutations. Patients with CMML harboring CEBPA, FLT3, and/or NPM1 mutations (mutCFN) more frequently had the myeloproliferative subtype, a high prevalence of severe cytopenia, and elevated blast counts. Regardless of their CMML Prognostic Scoring System molecular classification, mutCFN patients with CMML had a poor prognosis, and the multivariate analysis identified mutCFN as an independent marker of overall survival. The genetic profile of these mutCFN patients with CMML closely resembled that of patients with AML, with higher-risk clinical characteristics. Our findings lead us to suggest including the assessment of these mutations in CMML prognostic models and treating these patients with AML-type therapies, including intensive chemotherapy and allogeneic stem cell transplantation, whenever feasible. Furthermore, certain targeted therapies approved for use in AML should be considered.

Chinese Herbal Medicines as Natural Alternative Products to Antibiotics in Weaned Piglets through Intestinal Microbiota Regulation.

During the growth process of weaned piglets, digestive problems such as gastrointestinal disorders and diarrhea are common. Farmers usually use antibiotics to help piglets grow smoothly. However, the overuse of antibiotics can lead to antibiotic resistance issues. Therefore, this study chose to use plant extracts as feed additives to explore their potential as alternatives to antibiotics. Additionally, Tilmicosin was used as the antibiotic because it is widely used in treating respiratory infections in piglets. Since traditional Chinese medicine often uses natural products, we selected Guizhi Li-Zhong (GLZ) extract as an alternative to antibiotics. The experiment involved 126 piglets, each 4 weeks old, which were randomly assigned to one of four groups: the sham group (basal diet without supplements, 10.3 ± 0.4 kg, n = 31), the low-dose GLZ group (basal diet with 0.05% GLZ, 10.9 ± 0.4 kg, n = 32), the regular-dose GLZ group (basal diet with 0.2% GLZ, 10.6 ± 0.4 kg, n = 32), and the regular-dose Tilmicosin antibiotic group (basal diet with 0.2% Tilmicosin, 10.2 ± 0.3 kg, n = 31). We recorded and compared the survival rate, growth rate, feed conversion ratio, and diarrhea incidence among four groups of weaned piglets from the 4th to the 10th weeks of age. Then, we examined the oxidative stress, inflammation, and apoptosis in small intestine tissue (jejunum and ileum) through immunohistochemistry and Western blot and compared the gut microbiota in large intestine tissue (colon and rectum) through a next-generation sequencing (NGS) analysis. Our results showed that weaned piglets supplemented with 0.05% and 0.2% GLZ had better survival rates, growth rates (p < 0.01), and feed conversion ratios (p < 0.01) compared to those receiving sham treatment. Even weaned piglets supplemented with 0.2% GLZ performed better than those supplemented with 0.2% Tilmicosin antibiotics (p < 0.05). Furthermore, the incidence of diarrhea and small intestine injury (indicated by oxidative stress-, inflammation-, and apoptosis-related proteins) in piglets supplemented with 0.05% and 0.2% GLZ was lower than in piglets receiving sham treatment (p < 0.05). Even piglets supplemented with 0.2% GLZ had less injury than those supplemented with 0.2% Tilmicosin antibiotics (p < 0.05). The NGS results further showed that GLZ treatment significantly improved beneficial bacteria in weaned piglets (p < 0.05), while antibiotic treatment reduced beneficial bacteria (p < 0.05). In summary, we recommend adding GLZ to the feed as an alternative to antibiotics. This not only effectively reduces intestinal damage but also improves the gut microbiota, thereby promoting the growth of weaning piglets.

Metagenomic Analysis of Bacterial Communities in Bee Bread in Türkiye

This study aims to investigate the bacterial community structure in bee bread samples collected from 10 provinces of Türkiye using next-generation sequencing (NGS) and metagenomic analysis. Bacterial genomic DNA was extracted and sequenced using Illumina MiSeq. Bioinformatic analysis involved quality assessment, OTU classification, principal coordinate analysis (PCoA), and diversity index calculations. Heatmap and PCoA were utilized to explore the impact of locality and ecological zones on microbial diversity. Metagenomic analysis of 12 bee bread samples revealed 276,583 high-quality sequencing reads. The dominant bacterial phyla identified were Proteobacteria, Actinobacteria, Cyanobacteria, and Firmicutes. At the genus level, Streptomyces, Streptococcus, Bacillus, and Synechococcus were the most abundant, with Streptomyces and Bacillus playing key roles in the fermentation process of bee bread. The Shannon diversity index (ranging from 2.92 to 4.26) and Simpson's index (0.83 to 0.95) indicated high species diversity and relative abundance in bee bread. The study underscores the need for locality-specific approaches in beekeeping management and highlights the potential significance of beneficial bacterial taxa, particularly those involved in fermentation, in contributing to the nutritional and health properties of bee bread. These findings provide a foundation for further research on the microbial dynamics that support bee colony health.

Clear Cell Adenocarcinoma of the Urinary Tract Primary to the Renal Pelvis: A Multi-institutional Clinicopathologic and Molecular Study of Five Patients.

Clear cell adenocarcinoma (CCA) of the urinary tract is a rare malignancy and tumors involving the renal pelvis are notably sparse in the literature, with only 5 other patients reported. We present 5 patients, 4 women, and 1 man, with CCA of the renal pelvis. The age at presentation ranged from 29 to 81 years. The tumor size ranged from 4.5 to 8.0 cm. Tumors exhibited shared morphologic and immunohistochemical features with CCA of the female genital tract and those originating in the bladder and urethra, including cells with large nuclei, prominent nucleoli, nuclear hobnailing, and scant clear cytoplasm. Common immunohistochemical findings included reactivity for PAX8, CK7, HNF1β, and Napsin-A. One of the tumors arose in the background of a mixed epithelial and stromal tumor. Another tumor occurred in a renal allograft and tumor cells were positive for the BK virus, demonstrated by SV40 immunohistochemistry. All tumors were negative for TFE3 and TFEB rearrangement and lacked TERT alterations. Follow-up was limited with no recurrence in 4 patients at a maximum of 20 months follow-up and 1 patient died of an unrelated cause at 25 months of follow-up. Next-generation sequencing analysis of all 5 CCAs revealed mutations within genes implicated in DNA damage repair and chromatin remodeling pathways, including ATM, BRCA1, BRCA2, ARID1A, DICER1, SMAD4, NOTCH1, and MYC amplification. These molecular findings underscore the dysregulation of fundamental cellular processes essential for genomic integrity maintenance.

Analyzing the Mutational Landscape of Prostate Cancer Susceptibility Genes through Next-Generation Sequencing (NGS) Analysis

Prostate cancer, a significant public health concern, exhibits a complex genetic landscape influenced by a variety of susceptibility genes. This study deciphers the intricate genetic makeup of prostate cancer by examining mutations in 14 prostate cancer susceptibility genes. The Next-Generation Sequencing (NGS) approach was employed on a cohort of 45 prostate cancer patients to detect mutations. The gnomAD database was used to analyze mutational frequencies in the Asian population suffering from prostate cancer. The cBioPortal database was employed to check the presence of observed mutations in The Cancer Genome Atlas (TCGA) dataset. In addition, this study employed RT-qPCR and Immunohistochemistry (IHC) techniques were utilized to check the functional consequences of the observed pathogenic mutations. Finally, Metascape and DrugBank resources were used for gene enrichment and drug prediction analyses. Results of the NGS analysis revealed a total of 29 mutations (pathogenic and benign) within the examined prostate cancer cohort, distributed across four key genes (BRCA1/2, TP53, and PMS2) out of the total analyzed 14 genes. Pathogenic mutations in BRCA1/2 and TP53 genes are of particular interest due to their fundamental roles in DNA repair, cell cycle regulation, and tumor suppression. Functional consequence analyses (RT-qPCR and IHC) demonstrated the down-regulation of BRCA1/2 and TP53 genes in prostate cancer samples with pathogenic mutations, reinforcing the disruption of their tumor suppressor roles. Lastly, drug prediction analysis uncovered promising therapeutic options by identifying drugs capable of enhancing the mRNA expression of these genes. This opens new avenues for tailored treatment strategies aimed at restoring normal cellular functions of the BRCA1/2 and TP53 genes. In conclusion, this study provides a comprehensive view of genetic mutations in prostate cancer susceptibility genes, ranging from benign to pathogenic. It emphasizes the genetic complexity of prostate cancer and offers insights into potential mechanisms driving this malignancy. These findings lay the groundwork for further research, personalized treatment approaches, and enhanced clinical management of prostate cancer.

Anticancer potential of isoalantolactone in testicular cancer: an analysis of cytotoxicity, apoptosis, and signaling pathways.

Testicular cancer, a highly prevalent malignancy among young adults, has witnessed an alarming rise in recent decades. This study delves into the therapeutic potential of isoalantolactone (IATL), a natural product extracted from Inula helenium and Inula racemosa, against testicular cancer. Employing MTT assays and flow cytometry, we observed a dose-dependent reduction in cell viability and induction of cell cycle arrest at sub-G1 phase with increasing IATL concentrations. Furthermore, Annexin V/PI dual staining revealed IATL-induced apoptosis. Human Apoptosis Array analysis demonstrated IATL's influence on HIF-1α and TNF R1 expression, implicating its role in cancer cell growth and death regulation. Next-generation sequencing (NGS) and pathway analysis highlighted the involvement of ferroptosis and HIF-1 signaling in IATL-mediated effects. Western blotting validated the downregulation of key proteins associated with apoptosis inhibition and activation, confirming IATL's potential as an anticancer agent. Moreover, IATL induced ferroptosis by modulating expression levels of GPX4, xCT, NRF2, and HO-1. Our findings shed light on IATL's multifaceted anticancer mechanisms, emphasizing its potential as a therapeutic candidate for testicular cancer.

Pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma: additional evidence to support they are a single disease with variation in the histologic spectrum.

Pediatric-type follicular lymphoma (PTFL) and pediatric nodal marginal zone lymphoma (PNMZL) are two rare indolent B-cell lymphomas with overlapping features. Recently, cases showing hybridizing features of PTFL and PNMZL have been reported. Herein, we retrospectively analyzed the clinicopathologic features of 59 patients, including 39 with PTFL, 5 with PNMZL, and 15 with mixed-type tumors (MTT). And next-generation sequencing analysis was performed on 3 PTFL, 2 PNMZL, and 2 MTT cases. In addition, previously published mutational data of 96 PTFLs, 25 PNMZLs, and 46 MTTs were also analyzed. There were 52 male and 7 female patients, with a median age of 17years. Most patients (96.6%) had lymph node involvement in the head and neck region and were diagnosed with stage I disease. Among the 50 patients (85%) with telephone follow-up, 44 (88%) adopted a watch-and-wait strategy after surgical resection of the lesions. Only one PTFL patient experienced a relapse 6months after diagnosis. Microscopically, not only the MTT cases showed a composite form of enlarged follicles and interfollicular lymphocytic proliferation producing a progressively transformed germinal center (PTGC) pattern, but also focal follicles with a PTGC-like pattern were observed in PTFL cases. Genetically, the most frequently mutated genes were TNFRSF14 (in 3 PTFLs and 2 MTTs), MAP2K1 (in 2 PTFLs, 1 PNMZL and 1 MTT), and IRF8 (in 2 MTTs and 1 PNMZL). Based on the similar or overlapping clinical, pathologic, and genetic features, PTFL and PNMZL are likely to represent two different histologic patterns of the same disease.

Analysis of the life cycle of Helicobacter pylori bacteriophage KHP40 belonging to the genus Schmidvirus.

Bacteriophage (phage) KHP40 was previously isolated from the supernatant of a culture of Helicobacter pylori KMT83 cells. In this study, we analyzed the infection characteristics of KHP40, phage release pattern from KMT83 cells, and state of KHP40 DNA in KMT83 cells. The findings revealed that KHP40 phage showed varied adsorption efficiencies for different strains, long latent periods, and small burst sizes. Additionally, KHP40 activity was maintained at pH 2.5-12. KHP40 phages were released during the vegetative growth phase of the KMT83 cells. PCR analysis demonstrated that KHP40 DNA was stably maintained in KMT83 clones. Next-generation sequencing analysis revealed the presence of two distinct types of circular double-stranded DNA in H. pylori KMT83 cells. One was an H. pylori-specific DNA consisting of 1,578,403 bp, and the other was a 26,412 bp sequence that represented the episomal form of phage KHP40 DNA. Furthermore, defective KHP40-lysogenic DNA was detected in the H. pylori-specific DNA, the deleted portion of which appeared to have been transferred to another location in the bacterial genome. These findings indicate that KHP40 DNA exists in both episomal and defectively lysogenized states in KMT83 cells, and active phages are produced from KHP40-episomal DNA.

Nodule-specific NRF2-targeted upregulation in patients with KEAP1 mutations and familial nontoxic multinodular goiter.

Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2 related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter. This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations. Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole transcript expression array and immunohistochemistry. We found five probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H), which had no meaningful concomitance with mutations of other goiter-associated genes in germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating < 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations. KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Deciphering microbiome and fungi-bacteria interactions in chronic wound infections using metagenomic sequencing.

Chronic wounds caused by infections impose a considerable global healthcare burden. The microbial features of these infections and possible correlations between bacteria and fungi may influence wound healing. However, metagenomic next-generation sequencing (mNGS) analyses of these features remain sparse. Therefore, we performed mNGS on chronic wound infection samples to investigate features and correlations between the bacteriome and mycobiome in 66 patients (28: chronic wounds; 38: non-chronic wounds). Microbial community characteristics in patients with wound infections, microbiome-systemic inflammation associations, and bacteria-fungi correlations were analyzed. Infections constituted the primary cause of wounds in this study. Nontuberculous mycobacteria (23%) and Mycobacterium tuberculosis (13%) were the most common pathogens associated with chronic wounds, whereas Staphylococcus aureus (15%) was the most prevalent in non-chronic wound infections. Patients with chronic wound infections had a higher abundance of Pseudomonas aeruginosa than those without chronic wounds. Microbes with a high relative abundance in chronic wound infections were less significantly associated with plasma inflammatory factors than those in non-chronic wound infections. Additionally, a positive correlation between Candida glabrata and P. aeruginosa and an association between Malassezia restricta and anaerobic species were detected in patients with chronic wound infections. Our results further support the hypothesis that P. aeruginosa is a microbial biomarker of chronic wound infection regardless of the causative pathogens. Moreover, we propose a positive correlation between C. glabrata and P. aeruginosa in chronic wound infections, which advances the current understanding of fungi-bacteria correlations in patients with chronic wound infections.

12427 MKRN3 Methylation Abnormalities In Patients With Pubertal Disorders

Abstract Disclosure: N.C. Grosek: None. T. Silva: None. J.C. Magnotto: None. A. Latronico: None. A.P. Canton: None. Y. Chan: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Central Precocious Puberty (CPP) is caused by early reactivation of pulsatile GnRH secretion before age 8 years in girls and 9 years in boys. Delayed puberty (DP) is defined by the absence or incomplete development of sexual characteristics by age 13 years in girls and 14 years in boys. Environmental cues affect the HPG axis and timing of puberty via epigenetic mechanisms such as DNA methylation (DNAm). Inactivating mutations in the maternally imprinted Makorin Ring Finger 3 (MKRN3) gene are the most common genetic defect associated with CPP, representing ∼9% of patients with sporadic and familial CPP. Genomic imprinting occurs through differential methylation of CpG islands at a gene promoter or enhancer region. Differentially methylated regions (DMRs) regulate genetic imprinting. We hypothesized that transcription factor binding sites (TFBS) for ZFP57 (CpG57) andNRF1 (CpGs54+55) within the islet 3.7kb upstream of MKRN3 (MRKN3 islet) may impact expression. This study aimed to analyze if aberrant MKRN3 islet DNAm changes are associated with disorders of pubertal timing. Methods and Results: Germline DNA from 2 control patients was used to analyze the MKRN3 promoter region and islet DNAm profile. Bisulfite-treated DNA was amplified by PCR with primers targeting MKRN3 promoter and islet and submitted to Sanger sequencing. All CpG islands in the promoter region were 100% methylated, but those in the MKRN3 islet were differentially methylated with ∼50-60% methylated. DNA from our CPP cohort (n=71) underwent Next-Generation Sequencing analysis of bisulfite-treated DNA of these regions to identify the DMR potentially associated with MKRN3 imprinting. The MKRN3 islet covered CpG59-CpG49. Next, samples from our CPP cohort and patients with DP (n=44) and normal pubertal timing (n=33; 45% prepubertal, 55% pubertal) underwent pyrosequencing analyses of MKRN3islet. To compare DNAm levels between controls and our cohorts at each CpG island, T-tests were used: reported as mean±SEM. In the CPP cohort, 4/11 CpGs were significantly hypomethylated compared to controls (CpGs 54, 53, 51, 50). Specifically, the NRF1 TFBS was hypomethylated compared to controls (37.8 vs 41.6 ± 1.1, p&amp;lt;0.001) and DP (41.4 vs 43.8 ± 0.83, p=0.044). In the DP group, we found no significant difference in DNAm compared to controls. But they had hypermethylation of 4/11 CpGs compared to the CPP group, including the NRF1 TFBS (CpGs 55, 53, 51,50). Neither the CPP nor DP cohorts had significant DNAm differences at the ZFP57 TFBS compared to controls. Conclusion: Our study showed hypomethylation of 4 out of 11 CpGs in MKRN3 islet, including the NRF1 TFBS, in CPP compared to control and DP groups. On the other hand, NRF1 TFBS was hypermethylated in DPP compared to CPP. Further studies are needed to determine if these alternations in methylation are associated with MKRN3 expression levels and pubertal initiation. Presentation: 6/1/2024

12672 MKRN3 Methylation Abnormalities In Patients With Pubertal Disorders

Abstract Disclosure: N.C. Grosek: None. T. Silva: None. J.C. Magnotto: None. A. Latronico: None. A.P. Canton: None. Y. Chan: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Central Precocious Puberty (CPP) is caused by early reactivation of pulsatile GnRH secretion before age 8 years in girls and 9 years in boys. Delayed puberty (DP) is defined by the absence or incomplete development of sexual characteristics by age 13 years in girls and 14 years in boys. Environmental cues affect the HPG axis and timing of puberty via epigenetic mechanisms such as DNA methylation (DNAm). Inactivating mutations in the maternally imprinted Makorin Ring Finger 3 (MKRN3) gene are the most common genetic defect associated with CPP, representing ∼9% of patients with sporadic and familial CPP. Genomic imprinting occurs through differential methylation of CpG islands at a gene promoter or enhancer region. Differentially methylated regions (DMRs) regulate genetic imprinting. We hypothesized that transcription factor binding sites (TFBS) for ZFP57 (CpG57) andNRF1 (CpGs54+55) within the islet 3.7kb upstream of MKRN3 (MRKN3 islet) may impact expression. This study aimed to analyze if aberrant MKRN3 islet DNAm changes are associated with disorders of pubertal timing. Methods and Results: Germline DNA from 2 control patients was used to analyze the MKRN3 promoter region and islet DNAm profile. Bisulfite-treated DNA was amplified by PCR with primers targeting MKRN3 promoter and islet and submitted to Sanger sequencing. All CpG islands in the promoter region were 100% methylated, but those in the MKRN3 islet were differentially methylated with ∼50-60% methylated. DNA from our CPP cohort (n=71) underwent Next-Generation Sequencing analysis of bisulfite-treated DNA of these regions to identify the DMR potentially associated with MKRN3 imprinting. The MKRN3 islet covered CpG59-CpG49. Next, samples from our CPP cohort and patients with DP (n=44) and normal pubertal timing (n=33; 45% prepubertal, 55% pubertal) underwent pyrosequencing analyses of MKRN3islet. To compare DNAm levels between controls and our cohorts at each CpG island, T-tests were used: reported as mean±SEM. In the CPP cohort, 4/11 CpGs were significantly hypomethylated compared to controls (CpGs 54, 53, 51, 50). Specifically, the NRF1 TFBS was hypomethylated compared to controls (37.8 vs 41.6 ± 1.1, p&amp;lt;0.001) and DP (41.4 vs 43.8 ± 0.83, p=0.044). In the DP group, we found no significant difference in DNAm compared to controls. But they had hypermethylation of 4/11 CpGs compared to the CPP group, including the NRF1 TFBS (CpGs 55, 53, 51,50). Neither the CPP nor DP cohorts had significant DNAm differences at the ZFP57 TFBS compared to controls. Conclusion: Our study showed hypomethylation of 4 out of 11 CpGs in MKRN3 islet, including the NRF1 TFBS, in CPP compared to control and DP groups. On the other hand, NRF1 TFBS was hypermethylated in DPP compared to CPP. Further studies are needed to determine if these alternations in methylation are associated with MKRN3 expression levels and pubertal initiation. Presentation: 6/1/2024

Spitz melanoma with MAP3K8::ABLIM1 rearrangement: a case report with review of the literature

BackgroundSpitz tumors are relatively uncommon melanocytic lesions, typically affecting a relatively younger population but can be encountered at any age. They are characterized by a proliferation of melanocytes with epithelioid and/or spindled cytomorphology features, and interpretation is often challenging. The majority of these tumors are driven by kinase fusions or HRAS mutations. MAP3K8 fusions, although rare, are characteristic genomic events in Spitz tumors, especially in more atypical or malignant lesions.Case presentationHere, we present the case of a 43-year-old woman with a clinically cystic mass in her right groin, histologically characterized as a spindle and epithelioid cell malignant tumor. Immunohistochemistry revealed diffuse expression of S100 protein, tyrosinase and SOX10, patchy weak PRAME, HMB45 and Melan-A reactivity, and negative staining for BRAF V600E. Next-generation sequencing analysis revealed the presence of a MAP3K8::ABLIM1 fusion gene, as well as GRIN2A and TERT promoter mutations. The morphology, immunohistochemistry and molecular analysis confirmed Spitz melanoma with molecular features suggesting a worse prognosis.ConclusionThis case introduces a novel fusion partner of MAP3K8 in the context of Spitz melanoma and expands the morphologic and molecular spectrum of Spitz melanoma.