High Prevalence Of Mutations Research Articles

Association Study Between Kynurenine 3-Monooxygenase (KMO) Gene and Parkinson's Disease Patients.

The influence of various risk factors such as aging, intricate cellular molecular processes, and lifestyle factors like smoking, alcohol consumption, caffeine intake, and occupational factors has received increased focus in relation to the risk and development of Parkinson's disease (PD). Limited research has been conducted on the assessment of lifestyle impact on kynurenine 3-monooxygenase (KMO) gene in PD. A total of 164 subjects, including 82 PD cases and 82 healthy individuals, were recruited based on specific inclusion and exclusion criteria. The severity of PD and clinical assessment were evaluated using the Unified Parkinson's Disease Rating Scale (UPDRS) and Hoehn and Yahr (HY) scaling. Sanger sequencing was performed to analyse the KMO gene in the recruited subjects, and case-control studies were conducted. The UPDRS assessment revealed significant impairments in smell, tremors, walking, and posture instability in the late-onset PD cohorts. The HY scaling indicated a higher proportion of late-onset cohorts in stage 2. Moreover, both alcoholic and non-alcoholic groups showed significantly increased levels of 3-HK in late-onset PD. Gene analysis identified missense variants at position g.241593373T > A (rs752312199) and intronic variants at positions g.241592623A > G (rs640718), g.241592800C > A (rs990388262), g.241592802A > C (rs1350160268), g.241592808T > C (rs1478255936), and g.241592812G > T (rs948928931). The alterations in the KMO gene were found to influence the levels of kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK). Genomic analysis revealed a high prevalence of missense mutations in the late-onset PD groups, leading to a decline in 3-HK levels in patients. This leads to the reduction of the progression of disease in late-onset groups which shows that this mutation may lead to the protective effect on the PD subjects. This study suggests the use of KYNA and 3-HK as potential biomarkers in analysing the progression of disease. This study is limited by its small sample size. To overcome this limitation, a larger study involving in greater number of participants is needed to thoroughly investigate the KMO gene and KP metabolites, to enhance our understanding of Parkinson's disease progression, and to enhance diagnostic capabilities.

Familial FTD in China:progress and prospects

AbstractBackgroundFrontotemporal dementia (FTD) is a set of clinical syndromes characterized by progressive abnormalities in behavior, executive function, language, with a strong genetic background. However, the familiar FTD studies were rare in China. Here we aim to review the previous cross‐sectional reports of Chinese FTD patients with gene mutations, as well as the update status of the longitudinal familiar FTD study by the Chinese Familial Frontotemporal Lobar Degeneration Consortium (CFFC).MethodTo review current status of Chinese FTD patients with gene mutations, we performed a comprehensive electronic searches of Medline, PubMed, and Embase databases were performed by using the combination of a number of medical subject headings, Emtree subject headings, and free‐text terms (“frontotemporal lobar degeneration” or “frontotemporal dementia” for clinical categories; “Chinese” or “China” for ethnics). To start a multi‐center longitudinal cohort of familiar FTD, we established the CFFC in October 2022 by involving 7 top teaching hospitals across China. Patients with MAPT, GRN and C9orf72 mutations were enrolled and followed up according the designed framework.ResultBased on the previous reports, genetic mutations accounted for 5.13%‐27.9% as reported in a few cross‐sectional cohort of Chinese FTD patients. Mostly reported pathogenic variants were MAPT, followed by GRN, TBK1, CHCHD10, C9orf72 repeat expansions, VCP, and SQSTM1. Up till now, there were 26 pedigrees with MAPT mutations, 11 pedigrees with GRN mutations and 9 pedigrees with C9orf72 repeat expansions mutations have been enrolled in the longitudinal multi‐center cohort study by CFFC.ConclusionThe high prevalence of MAPT mutations in Chinese patients with FTD, may implies a genetic heterogeneity between Chinese and other ethnics. Longitudinal studies with larger sample size to modeling the trajectory of biomarkers in genetic mutation carriers for earlier diagnosis and intervention are needed in the future.

Abstract LB_B26: Efficacy and tolerability of a novel pan-RAS inhibitor with a unique mechanism of selectivity in mouse models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the U.S. This is frequently due to advanced disease at time of diagnosis as well as the high prevalence of KRAS driver mutations. Currently available targeted therapeutics are designed to covalently inactivate only KRAS-G12C mutants, whereas the most common mutations in PDAC are KRAS-G12D, KRAS-G12V, and KRAS-G12R. Thus, the need for novel RAS-targeted therapeutics with efficacy in tumors with these more common mutations is of utmost urgency to reduce PDAC disease burden. Our novel class of pan-RAS inhibitors have unique biological and attractive drug-like properties. We synthesized an orally bioavailable prodrug, ADT-1004, which generates the active species, ADT-007, that inhibits growth of PDAC tumors. ADT-007 inhibits PDAC cell growth in vitro with IC50 as low as 2 nM regardless of RAS mutant allele, inhibits RAS activation, and downstream MAPK/AKT signaling. Differentiated normal epithelial cells, hepatic cells, and tumor cells with wild type RAS are insensitive to ADT-007 due to enzymatic detoxification providing unique selectivity for a pan-RAS inhibitor. ADT-1004 is well tolerated in vivo, producing sustained ADT-007 concentrations well above IC50 values in plasma. Daily oral ADT-1004 administration results in significant inhibiteion of tumor growth in both mouse orthotopic and PDX models of PDAC tumors accompanied by a reduction in RAS signaling. Citation Format: Adam B Keeton, Yulia Y Maxuitenko, Jeremy B Foote, Chung-Hui Huang, Kristy L Berry, Khalda Fadlalla, Ganji P Nagaraju, Bandi DS Reddy, Xi Chen, Bassel F El-Rayes, Donald J Buchsbaum, Gary A Piazza. Efficacy and tolerability of a novel pan-RAS inhibitor with a unique mechanism of selectivity in mouse models of pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B26.

Mutational Analysis of the Genome Stability Factor BOD1L1 in Myeloproliferative Neoplasm

The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations. To investigate the contribution that mutations might make to the pathogenesis or clinical course of MPNs, 261 genes recurrently mutated in myeloid malignancies were sequenced in DNA from germline and blood samples from patients with AML, ET and MF enrolled in studies of the LSD1 inhibitor bomedemstat. Earlier work identified somatic mutations inthegene BOD1L1 in isolated megakaryocytes in 6 of 12 patients with MPN with mutations suggestive of loss-of-function (Guo et al., 2017). BOD1L1 plays a key role in multiple pathways of cellular DNA repair in conjunction with the histone methyltransferase SETD1A, which regulates transcription and DNA repair (Higgs et al. 2018; Hoshii et al. 2018. We previously demonstrated that BOD1L1 prevents uncontrolled resection of damaged DNA forks under conditions of replication stress, and that its loss leads to severe genome instability (Higgs et al., 2015, 2018). In addition, BOD1L1 promotes repair of DNA double-strand breaks after ionizing radiation through its interaction with the DNA repair protein RIF1 (Bayley et al., 2022). This sequencing-based analysis identified specific germline mutations in BOD1L1 that were 3-6 times more prevalent in MPN patients compared to a control population (gnomAD). This enrichment of BOD1L1 mutations was confirmed in whole-exome sequencing from an independent data set (EGA) of 183 patients with MPNs. We predicted these highly deleterious mutations, or at least a subset, may compromise the DNA repair functions of BOD1L1 and potentially influence the development and/or progression of MPN. To investigate this prediction, we disrupted BOD1L1 using a doxycycline-inducible CRISPR-Cas9 system in HeLa cells and complemented them with gRNA-resistant wild-type BOD1L1,or one of six mutants distributed throughout the protein. None of the mutants affected BOD1L1 protein expression; however, two of the six variants were defective for RIF1 binding by co-immunoprecipitation/proximity ligation, failed to recruit this key DNA repair factor to radiation-induced double-strand breaks, and compromised repair of these breaks. Moreover, single molecule DNA fiber analyses revealed that these same two mutants were unable to protect stalled replication forks against resection, leading to elevated levels of genomic damage. To explore the functional consequences of BOD1L1 loss-of-function on megakaryocytes, the gene was silenced using shRNA in human megakaryoblastic JAK2 V617F SET-2 cells, differentiated with thrombopoietin, and analyzed by flow cytometry. These cells were CD41-positive, consistent with megakaryocytic differentiation, and showed increased DNA damage (γH2AX) compared to controls. Depletion of BOD1L1 also lead to a greater number of cells with 2n nuclear content, and fewer with ≥8n, compared with controls, suggesting that the loss of BOD1L1 led to impaired polyploidization likely due to unrepaired DNA damage and subsequent cell death. In summary, we have shown that MPN patients have a high prevalence of mutations in the key genome instability factor BOD1L1 and that a subset of these mutations compromised its DNA repair functions and increased genome instability. The presence of BOD1L1 somatic mutations in megakaryocytes in MF patients may deregulate polyploidization, altering megakaryocyte function that may contribute to the development of bone marrow fibrosis. That no patients with BOD1L1 germline mutations progressed to AML in these clinical studies, coupled with the fact that these variants were not enriched in patients with AML, invites the possibility that BOD1L1 mutations may be synthetically lethal with other somatic mutations that result in genome instability and, hence, protect against progression to AML.

Whole Exome Sequencing of Patients with Adult-Onset Evans Syndrome: A Cohort of 80 Patients

Introduction Loss of tolerance toward several self-antigens suggests an underlying genetic predisposition. A previous study (1) conducted in children with Evans syndrome revealed a high prevalence of pathogenic mutations in genes involved in primary immunodeficiencies. Whether these findings are restricted to children is unknown. In this study, we assessed the prevalence of constitutional genetic variants in a cohort of patients with adult onset Evans syndrome. Patients and methods We consecutively included from November 2021 to June 2023 patients that had been diagnosed with adult onset (≥18 years) Evans syndrome (association of immune thrombocytopenia (ITP), warm autoimmune hemolytic anemia (wAIHA) and/or autoimmune neutropenia (AIN) according to international guidelines (2,3)), and identified in France throughout the network of the national reference center for adult' autoimmune cytopenia. We excluded patients with a systemic lupus (according to Systemic Lupus International Collaborating Clinics criteria (4)), and patients with indolent B cell malignancies at Evans syndrome onset. Clinical and biological data were retrospectively collected using a standardized form. Patients underwent whole-exome sequencing after written and informed consent was obtained. Gene variants already published and/or with in vitro confirmed consequences were considered as genetic diagnoses. Clinical interpretation of variants was performed according to the ACMG guidelines (5). A patient was classified as having a genetic diagnosis if carryinghemizygous/heterozygous pathogenic or likely pathogenic variant (for X-linked or autosomal dominant conditions), or 2 pathogenic or likely pathogenic variants in the same gene (for autosomal recessive conditions). Results We identified 80 patients (40% female, median age 31 years at Evans syndrome diagnosis [range 18-76]) with adult-onset Evans syndrome from 19 centers. Median age at first cytopenia was 28 years [range 1-73], and 13 patients (16%) were under 18 years at first cytopenia. 78 patients (96%) had ITP (chronic in 87%), 59 (73%) had wAIHA (with at least one relapse in 58%), and 38 (47%) had AIN (chronic in 74%). Median delay between first and second cytopenia was 18 months (range 0 - 329) and 28 patients (35%) had synchronous onset of cytopenia. Twenty-four patients (30%) had associated immune manifestation (involving endocrine system in 9%, lung in 9%, gastrointestinal tract in 6%, skin in 5%, central nervous system in 4%) and 28% had antinuclear antibody titers ≥ 1/160. Lymphoproliferation was found in 56%, including lymphadenopathy in 41% and splenomegaly in 35%. Nine patients (11%) had lymphoma or cancer history. Twenty patients (25%) had humoral immune defect, including 13 (16%) with a common variable immunodeficiency (CVID)-like phenotype. In the whole cohort, 9 patients (11.25%) had a genetic diagnosis that could be linked to Evans syndrome, with heterozygous variants in CTLA4, STAT3, NFKB1, CYBB, TNFAIP3, DDX41, MYH9 and homozygous variant in PIK3CG. Twenty (25%) patients had genetic testing prior to this study (targeted Sanger sequencing [N=3], or NGS panel [N=17]), resulting in 2 genetic diagnoses ( CYBB and TNFAIP3). A family history of immune manifestations in first-degree relatives was found in 56% of the patients with genetic diagnosis versus 20% of patients without (P=0.0308). However, the proportion of patients with CVID-like phenotype, associated immune disorder, lymphoproliferation, cancer/lymphoma, positive antinuclear antibodies, or pediatric onset of first cytopenia was not significantly increased in patients with genetic diagnosis. Discussion As expected, the proportion of genetic diagnoses in patients with adult onset Evans syndrome was lower that what was previously described in children, but led to a significant impact on clinical management in some patients. We did not find clinical or biological features associated with a genetic diagnosis, suggesting that genetic testing should be considered in every adult patient diagnosed with Evans syndrome in the absence of any obvious underlying cause or condition. References (1) Hadjadj J et al., Blood 2019; 134: 9-21. (2) Provan D et al., Blood Adv 2019; 3: 3780-817. (3) Jäger U et al., Blood Rev 2020; 41: 100648. (4) Petri M et al., Arthritis Rheum 2012; 64: 2677-86. (5) Richards S et al., Genet Med 2015; 17: 405-24.

1-Year Data Analysis from a Cancer Center Molecular Tumor Board Dedicated to Clonal Hematopoiesis

Background Nearly 25% of cancer patients harbor clonal hematopoiesis of indeterminate potential (CHIP). Detection of clonal hematopoiesis (CH) in routine clinical practice is increasing due to incidental findings during sequencing analysis of circulating cell-free DNA (cfDNA) or exploration of unexplained cytopenia. To improve clinical management of these patients in our institution, we set up a multidisciplinary molecular tumor board (MTB) dedicated to patient cases with CH, CHIP, and clonal cytopenia of undetermined significance (CCUS) in 2022. Methods Between June 2022 and June 2023, our MTB reviewed a total of 345 cases, of which 212 referred by the solid tumor MTB and 133 referred by hematologists. The former 212 cases with CH were identified through cfDNA sequencing (FoundationOne Liquid CDx) within the Gustave Roussy Cancer Profiling study (NCT04932525), which includes some genes recurrently mutated in hematologic malignancies ( ASXL1, DNMT3A, IDH1, IDH2, JAK2, MPL, MYD88, SF3B1, TET2, U2AF1). A variant allele frequency (VAF) of at least 10% for the CH mutation was required to refer cases to the MTB, except for JAK2, MPL, and MYD88 mutations. The latter 133 cases were screened for CH mutations on peripheral blood leukocytes using a next-generation sequencing (NGS) assay targeting 174 genes recurrently mutated in hematologic malignancies. In addition to molecular data, standard clinical and biological parameters were recorded for each individual case. Results Patients referred by the solid tumor MTB In this cohort of 212 patients, median age was 72 years old (range, 48-91), sex ratio (M/F) was 1.4, median prior anticancer treatment lines was 2 (0-9), and time between MTB and cancer diagnosis was 2.3 years (0.2-34). The most frequent tumor types were lung (23%), colorectal (20%), prostate (19%), gynecological (12%) and breast (9%) cancers. The most frequently mutated genes were DNMT3A (59%), TET2 (30%), ASXL1 (24.5%), JAK2 (23%), SF3B1 (7.5%), and MYD88 (6%), with a median VAF of 4.2%, 3.0%, 8.5%, 1.0%, 5.0%, and 0.20%, respectively. Fifty percent of patients harbored more than 1 mutation. Six patients had a known history of myeloproliferative neoplasm (MPN). The proportion of cases with CH, CHIP, and CCUS was 15%, 59%, and 17%, respectively. The CH risk score of myeloid malignancy (CHRS, Weeks et al.) could be calculated in 126 cases but may underestimate risk due to the limited gene list considered, and revealed 69 (55%) low-risk, 52 (41%) intermediate-risk, and 5 (4%) high-risk patients. Our MTB recommended hematologic consultation for additional investigations in 58 patients, of which 22 were actually referred (main reason for not referring patients was advanced cancer disease). Four patients were diagnosed with hematologic malignancy (1 MPN, 1 chronic myeloid leukemia, 2 myelodysplastic syndromes [MDS]). Patients referred by a hematologist In this cohort of 133 patients, median age was 69 years old (53-89), and sex ratio (M/F) was 0.6. Eighty-six percent of patients had a cancer history, median prior anticancer treatment lines was 2 (0-7), time between MTB and cancer diagnosis was 3.5 years (0.2-34). The most frequent tumor types were lymphoid disorders (39%), breast cancer (15%), and gynecological cancer (15%). The proportion of CH, CHIP, and CCUS was 11%, 33%, and 41%, respectively. The most frequently mutated genes were DNMT3A (48,5%), PPM1D (24%), TET2 (22%), TP53 (17%), ASXL1 (6%), JAK2 (4.5%) and CHEK2 (4.5%), with a VAF of 2.5%, 2,3%, 4.0%, 2.0%, 8.0%, 1.0%, and 2.5%, respectively. Sixty percent of patients harbored more than 1 mutation. The CHRS could be calculated in 98 cases and revealed 34% low-risk, 50% intermediate-risk, and 16% high-risk CH patients. No need for a specific follow-up was recommended by the MTB in 33 patients. Among the 85 patients with hematologic follow-up, 3 (for which the CHRS was intermediate in 2 cases and high in 1 at the time of MTB evaluation) rapidly progressed to a myeloid malignancy ( Figure 1) (2 MDS and 1 MDS/MPN). Conclusions Our study highlights the utility of a MTB dedicated to CH, CHIP and CCUS for the identification of high-risk patients, early detection of hematologic malignancies, and appropriate therapeutic management. Long-term follow-up is needed to validate the CHRS but the design of a specific score for patients with cancer may be helpful due to the specific features of this population (high prevalence of PPM1D mutations, post-treatment cytopenia).

High Prevalence of IDH Mutation in Myeloid Neoplasm with Concomitant Autoimmune Rheumatic Disorders

BACKGROUND: The association between myeloid neoplasm (MN) and autoimmune rheumatological diseases (AIRD) is increasingly recognized. Furthermore, prolonged cytotoxic disease modifying anti-rheumatological drugs (DMARD) for AIRD may potentially induce therapy-related MN (t-MN), a lethal haematological malignancy with dismal outcomes. However, the genetic association and mechanism underpinning immune dysfunction in patients with between MN and AIRD is not fully elucidated. METHODS: In this collaborative study across the two disciplines and continents (Australia and USA), we assessed the burden of well-defined AIRD in a large cohort of MN (n=1702) that included myelodysplastic syndrome (MDS; n=861), acute myeloid leukaemia (AML; n=640), myeloproliferative neoplasm (MPN; n=89) and MDS/MPN overlap (n=112). We characterized the cytogenetic and mutational landscape of MN with AIRD (MN-AIRD) and without AIRD. In order to assess the mechanistic link between AIRD and MN, we investigated the bone marrow microenvironment in MN-AIRD vs. well-curated age matched controls. RESULTS: 132 (7.75%) of MN (n=1702) had concomitant AIRD. Although association of AIRD was observed across all subtypes of MN, enrichment of AIRD was observed in patients with MDS, MDS/MPN and MPN compared to AML (9.5% vs. 9.8% vs 10.1% vs. 4.67%; P <0.001). Inflammatory arthritis (n=65; 49.24%) followed by inflammatory connective tissue diseases (CTD, n=23; 17.42%), polymyalgia rheumatica (PMR, n=18; 13.63%) and vasculitis (n=16; 12.12%) were the most prevalent AIRD in the MN cohort. Notably, 87.7% of inflammatory arthritis were rheumatoid arthritis (RA; n=57). Striking survival differences were observed between t-MN without AIRD and MN-AIRD (14.0 vs. 33.7 months; P <0.0001) and t-MDS no-AIRD and MDS-AIRD (16.1 vs. 33.4 months, P =0.001). Chromosomal abnormalities including complex karyotype, monosomal karyotype, del 5q, del 7q, marker chromosome and TP53 mutations were enriched in t-MN following independent primary cancer (t-MN without AIRD). However striking enrichment of IDH1 and IDH2 (henceforth referred as IDH) mutations in MN with AIRD compared to MN without AIRD (20% vs. 11%, P =0.02), particularly in MDS-AIRD compared to MDS without AIRD (21% vs. 7%; P =0.001). The enrichment of IDH mutations were particularly prevalent in in MN cases with RA compared to other AIRD (34% vs. 11%; P =0.005). While IDH mutations were observed in only 8% and 6% of PMR and inflammatory CTD patients, respectively. Surprisingly higher frequency of IDH mutations was observed in evaluable seronegative compared to seropositive RA patients (62% vs. 0%; P =0.006). We also observed that majority of IDH mutations in seronegative RA were IDH1 (6/8; 75%) while IDH2 mutations were common other AIRD (6/6; 100%) ( P =0.009). We also assessed the impact of DMARDs on genomics of myeloid neoplasm. The majority of MN-AIRD patients treated with methotrexate did not harbor poor risk cytogenetic and mutation features such as complex karyotype, monosomal karyotype, del 5q, del 7q and/or TP53 mutation. In contrast, the majority of patients who had cyclophosphamide harbored high risk cytogenetic and molecular features. To assess pathogenic mechanisms linking MN and AIRD, we evaluated bone marrow microenvironment in myeloid neoplasm patients with and without AIRD. Bone marrow mesenchymal stromal cells (MSC) isolated from MN-AIRD were highly senescent compared to age matched healthy controls, MN without AIRD, with defective proliferating and differentiation capacity of MN-AIRD and secretion of highly proinflammatory cytokines and chemokines. CONCLUSIONS: In summary, our key findings include: 7.8% of MN were associated with AIRD with stronger association of AIRD with MDS and MDS/MPN compared to AML and striking enrichment of IDH mutations in MN with inflammatory arthritis compared to MN with other AIRD (29% vs 11%; P =0.02), especially in seronegative RA compared to seropositive RA (62% vs. 0%; P =0.006). In contrast to cyclophosphamide and azathioprine, majority of cases treated with methotrexate did not harbor poor risk cytogenetic and/or TP53 mutation. As methotrexate is widely used as an anchor drug in many rheumatology treatment regimens, this is a highly significant finding of clinical relevance.

High Prevalence of MYD88 and CD79B Mutations and PD-L1 Alterations in Bone Marrow Large B Cell Lymphoma Associated with Hemophagocytic Lymphohistiocytosis

Introduction: Bone marrow large B cell lymphoma associated with hemophagocytic lymphohistiocytosis (HLH) is a rare disease with a highly aggressive clinical course. Until now, the molecular genomics of bone marrow large B cell lymphoma associated with HLH remains undetermined. Methods: Cases of bone marrow large B cell lymphoma with HLH were included. The inclusion criteria were as follows: (1) diagnosis of large B cell lymphoma presenting initially in the bone marrow; (2) no overt lymph node involvement or tumor formation; (3) fulfillment of five or more of the eight HLH-2004 diagnostic criteria. (4) cases with available samples. Genomic DNA was extracted from bone marrow cells or the plasma. A panel comprising 475 hematological malignancy-related genes were used for targeted gene sequencing. The prepared library was sequenced using Illumina HiSeq 4000. Molecular classification was predicted by using the LymphGen algorithm. Results: A total of 23 cases were included. The median age was 63. 71.4% of patients were the non-GCB subtype (10/14) based on the Hans algorithm and 50% (9/18) of cases showed CD5 expression. Somatic mutations were identified in all the cases. The most common disrupted genes were PIM1 (12/23, 52.2%), PD-L1 (9/23, 39.1%), MYD88 (8/23, 34.8%), BCL6 (6/23, 26.1%), CD79B (6/23, 26.1%), IRF4 (6/23, 26.1%) and TET2 (6/23, 26.1%) (Figure 1). For nine cases with PD-L1 aberrations, seven cases had PD-L1 structural variants (SVs) and one case showed PD-L1 amplification (Figure 2). All the six cases with BCL6 aberrations showed BCL6 SVs (Figure 2). For six cases with BCL6 SVs, four cases showed immunoglobulin heavy chain ( IGH)- BCL6 translocations and two cases harbored IKZF1- BCL6 translocations. Regarding molecular classification, eight cases (34.8%) were classified as the MCD subtype, two cases the BN2 subtype, two cases the ST2 subtype, and two cases the composite subtype. Conclusion: To the best of our knowledge, this is the first study exploring the molecular genetics of bone marrow large B cell lymphoma with HLH. Our study suggesting bone marrow large B cell lymphoma with HLH showed frequent MYD88/CD79B and PD-L1 alterations. Our study provided clues for the pathogenesis of bone marrow large B cell lymphoma with HLH and identified potential druggable targets for future clinical trials.

Characterizing Hereditary Hemolytic Anemias in a Hispanic Cohort Using Next Generation Sequencing

Background: Hereditary hemolytic anemias (HHA) are a heterogeneous group of nonimmune disorders characterized by increased red blood cell (RBC) destruction due to abnormalities of hemoglobin stability, defects of RBC metabolism, and disorders of RBC hydration. Although uncommon, they comprise important causes of anemia in children and adults by causing chronic or episodic hemolysis. Clinical, laboratory, and genetic heterogeneity characterize this group of disorders and make recognition challenging. The advent of next generation sequencing (NGS) has made accurate genetic diagnoses attainable, with an increasing number of studies reporting clinical utility in HHA. For patients with suspected HHA, multigene testing is available incorporating an NGS panel associated genes. However, there is limited data on the use of multigene NGS to characterize HHA in the Hispanic population. Herein, we describe results from NGS testing of a pilot cohort referred for HHA investigation to hematology clinics along the US/Mexico border. Methods: Pediatric and adult hematology clinics at Texas Tech University in El Paso, TX collaborated for testing on HHA referral and clinical case review. The diagnostic panel (AnemiaID/PerkinElmer/Revvity®) included 51 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions were covered. Targeted gene capture and library construction for NGS were performed using a Whole Blood and Saliva kit and 150 base pair paired-end sequencing was done on Illumina® NGS systems at target average coverage of 80×. NGS output data were summarized descriptively for all patients and clinically correlated for diagnosis. Results: In this pilot cohort, 11 patients were tested during September 2022 to June 2023, age ranging 7-75 years, with females accounting for 64% of the group. Findings are reported in the Table below; Pathogenic variants leading to a definitive diagnosis were identified in 8 of 11 cases (73%). The most frequently mutated genes were SPTA1 followed by PIEZO1, SPTB, RHAG, GSR, SLC4A1 and TPI1. Approximately 58% of the mutations were reported as novel. Diagnoses included, most commonly, Hereditary Spherocytosis followed by Hereditary Pyropoikilocytosis and Congenital Dyserythropoietic Anemia. Compound heterozygosity leading to complex interactions was noted in 4 cases (36%), involving SPTA1 low expression alleles in trans (alpha-LELY and alpha-LEPRA) in 2 cases. Conclusion: Hereditary hemolytic anemia in the Hispanic population is characterized by complex molecular interactions and a high prevalence of novel mutations in RBC cytoskeleton/enzyme genes. NGS has clinical utility and should be considered for accurate diagnosis. Further research into the molecular basis and genetic spectrum of HHA in the Hispanic population is needed.

PATH-36. CLASSIFICATION OF DIFFUSE GLIOMA SUBTYPES USING A NEXT-GENERATION SEQUENCING PANEL IN ACCORDANCE WITH THE 2021 WORLD HEALTH ORGANIZATION CRITERIA

Abstract Diffuse gliomas, which are the most common primary brain tumors, exhibit significant variations in their characteristics such as morphology, location, genetic alterations, and response to treatment. In 2021, the World Health Organization (WHO) introduced updated guidelines for glioma classification based on molecular alterations. This study aimed to classify 476 primary diffuse glioma samples utilizing a next-generation sequencing OncoPanel AMC, targeted DNA sequencing, in accordance with the 2021 WHO criteria. The samples were divided into 3 subtypes: oligodendroglioma (ODG; n=70), diffuse astrocytoma (DA; n=65), and glioblastoma (GBM; n=341). The median age of patients diagnosed with GBM (62 years) was significantly higher compared to those with ODG (50 years) and DA (42 years) (Page=0.056 [ODG vs.DA], 3.06×10-11 [ODG vs.GBM], and 3.06×10-11 [DA vs.GBM]). Gender and tumor mutational burden did not exhibit significant associations with the subtypes. Consistent with the 2021 WHO criteria, ODG samples exhibited frequent mutations in IDH1 (91%), NOTCH1 (27%), FUBP1 (23%), CIC (20%), and IDH2 (9%). DA samples had a high prevalence of mutations in IDH1 (98%), TP53 (91%), ATRX (78%), and IDH2 (2%). In contrast, GBM samples showed mutations in IDH-wildtype and EGFR (18%). Cox multivariate regression analysis of 341 GBM patients (median survival=15.9 months) revealed that older age, lack of MGMT promoter methylation, and impaired performance status (Karnofsky performance scale< 80) were significantly associated with survival outcome (HRage=1.40, Page=7.64×10-3, HRMGMT=1.43, PMGMT=6.98×10-3, and HRKPS=1.79, PKPS=3.05×10-6). In summary, by utilizing the AMC OncoPanel and the 2021 WHO criteria, we successfully classified diffuse glioma into three distinct.

BIOM-12. ULTRA-DEEP WHOLE GENOME SEQUENCING UNCOVERS A HIGH PREVALENCE OF GLIOBLASTOMA-DERIVED CELL-FREE DNA IN PLASMA

Abstract The detection of glioblastoma-derived circulating cell-free DNA (ccfDNA) has proven formidable because tumor-specific mutations are hidden among the abundance of ccfDNA from healthy cells. While capture-enrichment panels provide the necessary read depth to detect low-frequency variants, searches are constrained to a narrow range of targets and largely limited to single nucleotide variants (SNVs). Here, we use ultra-deep whole genome sequencing (WGS) to broaden the search for somatic mutations across the entire genome. In addition, a kmer-based reference-free variant caller, which mitigates the bias and errors that accrue during alignment, is used to extend the mutation search beyond SNVs to include insertions/deletions (INDELS) and structural variants (SVs) such as large INDELS ( >20 bp) and tandem duplications. In tumor DNA and ccfDNA, we achieve a WGS (GRCh38) mean read depth of 263X and 225X, respectively. WGS of matched buffy coat DNA and sequencing reads from the 1000 Genome Database were used to eliminate germline polymorphisms, sequencing artifacts, and common variants. We detected 20,488 somatic mutations in tumor DNA of which 17.3% affected a coding sequence, 31.9% were INDELS, 91 were large INDELs, and 18 were tandem duplications. A tumor-informed search identified 489 of these somatic mutations in ccfDNA which included a large INDEL and a similar portion of mutations affecting a coding sequence. Using an unguided search for glioblastoma-derived ccfDNA uncovered an additional 8,794 somatic mutations of which 16.6% involved a coding sequence, 60.7% were INDELS, 30 were large INDELS, and 3 were tandem duplications. The high prevalence of somatic mutations discovered in ccfDNA using an unbiased approach highlights the low yield of tumor-informed searches in spatially genetically heterogeneous cancers like glioblastoma. Our findings demonstrate that ultra-deep WGS detects glioblastoma-derived ccfDNA by broadening the search for variants across the entire genome and through the inclusion of INDELS and SVs.

Exploration of the Tumour Biological Significance of PCLO in Gastric Cancer: Results from a Large Central European Cohort

Introduction: A recent multiregional whole-exome sequencing of 48 tumour samples from 9 gastric adenocarcinomas discovered PCLO mutations in 23 (47.9%) tumour samples. Based on that unexpected high prevalence of PCLO mutations, we hypothesized a tumour biological significance of PCLO in gastric cancer (GC). Methods: Tumour samples (whole tissue sections) obtained from 466 patients resected for therapy-naive GC were stained with an anti-PCLO antibody. The histoscore for tumour cells and the presence of immunostaining of stromal cells and tumour vessels was documented for each case. An algorithm for PCLO immunopositivity was formed and correlated with clinicopathological patient characteristics. Results: 175 GCs were classified as PCLO positive within tumour cells, and 291 as negative. Stromal cells were positive for PCLO in 106 cases and tumour vessels in 84. PCLO-positive GCs more often showed an intestinal phenotype, a lower T category and were more commonly associated with Helicobacter pylori infection. A separate analysis of PCLO expression in intestinal and diffuse type GCs, respectively, showed no significant correlations. Patients with PCLO negative/low tumour cells showed a shortened overall (14.0 ± 1.4 vs. 16.0 ± 1.8 months) and tumour-specific survival (15.0 ± 1.6 months vs. 17.9 ± 3.6). Comparison of PCLOs genotype with its phenotype in 48 tumour samples obtained from nine cases showed no direct correlations with missense mutations. Conclusion: Our data provide evidence that PCLO is differentially expressed in GC and might delay tumour progression.

The Presence of Ring Chromosomes in Patients with Myeloid Neoplasms Is Predictive of a Poor Outcome

Cytogenomic analysis is an important tool in the risk stratification of patients with hematologic malignancies. Complex chromosomal alterations are associated with a poor prognosis (Döhner et al., Blood 2022). Ring chromosomes (RC) are circular chromosomal structures associated with loss or gain/amplification of genetic material and are associated with poor prognosis in acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) (Rosenbaum et al., Human Path 2017). Due to their rarity, the impact of RC on response to therapy has not yet been well characterized. We further examined the significance of RC in AML, MDS and myeloproliferative neoplasms (MPN) by retrospectively analyzing records of patients identified to have RC and myeloid neoplasms at our institution between 2017 and 2023 and collected demographic, diagnostic, treatment, molecular, cytogenomic, and outcomes data. We identified 27 cases with RC with clinical data available for 25. Demographic and treatment details are described in Table 1. The most common chromosomes involved were r(11) and r(7), 6 and 4 cases respectively, followed by r(6) and r(19) with 2 cases each. The most frequently observed diagnosis was AML in 12 patients (48%) followed by MDS with 9 cases (36%) and MPN in 4 cases (16%). In 17 cases (68%) the RC was cytogenetically detected at diagnosis and was not observed in the context of prior therapy. Remarkably, mutations in TP53 were present in 14 cases (56%) and accompanied by loss of 17p (bi-allelic) in 7 cases (28%). The next most commonly observed gene mutations were in DNMT3A (7 cases), TET2 (4 cases) and ASXL1 (3 cases). In 11 cases (44%) the alterations in TP53 were not associated with prior therapy for other malignancies. The median time from prior chemotherapy to detection of the RC was 5.1 years (range 0.6 to 13.5 years). Chromothripsis (a single DNA catastrophic event resulting in extensive genomic rearrangements of one or a few chromosomes) as detected by CGH+SNP array and homogenous staining regions (autonomously replicating extra-chromosomal elements that are frequently associated with gene amplification) were observed in 3 patients each. RC were the sole genetic abnormality in 5 patients. A Kaplan-Meier analysis of median overall survival (mOS) in patients with AML and MDS with RC at diagnosis revealed that patients who received supportive care had significantly shorter mOS than patients who received hypomethylating agents (HMA), HMA/Venetoclax(VEN), or anthracycline-based chemotherapy (log rank test, p = 0.001). A significant difference in mOS between patients who received HMA alone versus HMA/VEN versus chemotherapy was not observed (11.4 vs 3.1 vs 7.3 months, p = 0.8) (Figure 1). Patients who received any of the therapies outlined above likewise demonstrated a significantly longer progression free survival (log rank test, p = 0.001). There was no effect on mOS for any of the other tested prognostic parameters, including the presence of a complex karyotype, TP53 allele status, or therapy-related neoplasm, nor for pair-wise interactions between those categories and first-line therapies (p > 0.05 for all tested associations). These data represent a large single-institution cohort of patients with RC and is the first to investigate the impact of therapy and molecular profiles in this population. We observed a high prevalence of TP53 mutations and loss of 17p, however we lacked longitudinal data to make causal inferences regarding the relationship between these abnormalities and ring formation. Of note, 68% of patients did not have prior exposure to therapy, and 36% of all patients lacked TP53 mutations or chromosomal p53 loss, indicating a high rate of de novo ring formation. This is a novel finding perhaps suggesting that inherent chromosomal instability characterizes these patients. Comparing response to type of therapy and outcomes of patients, we conclude that the mOS in patients with RC without therapy is significantly shortened compared with any of the therapeutic options described above, and that the addition of VEN to HMA did not positively influence mOS or PFS compared to HMA alone or chemotherapy in our cohort. Furthermore, the presence of a complex karyotype or a mutation and/or deletion of TP53 with a RC was not independently associated with inferior mOS, suggesting that the presence of a ring chromosome alone may be an important adverse risk factor in myeloid malignancies.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine–pyrimethamine in central Africa: a cross-sectional study

Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. Toulouse Institute for Infectious and Inflammatory Diseases.

Next-generation sequencing in dermatology.

Over the past decade, Next-Generation Sequencing (NGS) has advanced our understanding, diagnosis, and management of several areas within dermatology. NGS has emerged as a powerful tool for diagnosing genetic diseases of the skin, improving upon traditional PCR-based techniques limited by significant genetic heterogeneity associated with these disorders. Epidermolysis bullosa and ichthyosis are two of the most extensively studied genetic diseases of the skin, with a well-characterized spectrum of genetic changes occurring in these conditions. NGS has also played a critical role in expanding the mutational landscape of cutaneous squamous cell carcinoma, enhancing our understanding of its molecular pathogenesis. Similarly, genetic testing has greatly benefited melanoma diagnosis and treatment, primarily due to the high prevalence of BRAF hot spot mutations and other well-characterized genetic alterations. Additionally, NGS provides a valuable tool for measuring tumor mutational burden, which can aid in management of melanoma. Lastly, NGS demonstrates promise in improving the sensitivity of diagnosing cutaneous T-cell lymphoma. This article provides a comprehensive summary of NGS applications in the diagnosis and management of genodermatoses, cutaneous squamous cell carcinoma, melanoma, and cutaneous T-cell lymphoma, highlighting the impact of NGS on the field of dermatology.

Rapidly progressive multiple system atrophy in a patient carrying LRRK2 G2019S mutation.

Multiple system atrophy (MSA) is considered a primarily sporadic neurodegenerative disease, but the role of genetic is poorly understood. We present a female patient of Moroccan origin who developed a rapidly progressive non-levodopa responsive parkinsonism, gait and balance problems, and dysautonomia including severe bulbar symptoms. She was diagnosed with MSA Parkinsonian-type (MSA-P) and suddenly died at night at 58 years of age. Reduced striatal DAT-SPECT, putaminal hyperintensity on T2-MRI, and hypometabolism with FDG-PET were present. Genetic testing documented a G2019S mutation in the LRRK2 gene. A skin biopsy was obtained and used to perform alpha-synuclein RT-QuIC, which was negative, and immunohistochemical analysis, which demonstrated abnormal alpha-synuclein deposits in cutaneous nerves. Elevated blood neurofilament light chain levels were also documented. LRRK2 mutations are the most common cause of monogenic Parkinson's disease (PD) and G2019S is the most frequent variant. Our patient presented with biological, clinical, and radiological features of MSA, but genetic testing revealed a G2019S LRRK2 mutation, which has been previously reported only in one other case of pathologically proven MSA but with mild progression. In our patient, post-mortem confirmation could not be performed, but RT-QuIC and immunohistochemical findings on skin biopsy support the diagnosis of MSA. G2019S LRRK2 may be linked to an increased risk of MSA. Cases of atypical parkinsonism with rapid disease course should be screened for PD-related genes especially in populations with a high prevalence of mutations in known genes.

HIV Drug Resistance Mutations and Subtype Profiles among Pregnant Women of Ho Chi Minh City, South Vietnam.

According to the latest data released by UNAIDS, the global number of people living with HIV (PLHIV) in 2021 was 38.4 million, with 1.5 million new HIV infections. In different countries, a significant proportion of these cases occur in the adult fertile population aged 15-49 years. According to UNAIDS, Vietnam had a national HIV prevalence of 0.3% of the total population at the end of 2019, with approximately 230,000 PLHIV. The most effective way to prevent mother-to-child transmission of HIV is ART to reduce maternal viral load. HIV-infected pregnant women should undergo monthly monitoring, especially before the expected date of delivery. The aim of our work was to analyze subtypic structure and drug-resistant variants of HIV in pregnant women in Ho Chi Minh City. The study material was blood plasma samples from HIV-infected pregnant women: 31 women showed virological failure of ART, and 30 women had not previously received therapy. HIV-1 genotyping and mutation detection were performed based on analysis of the nucleotide sequences of the pol gene region. More than 98% of sequences genotyped as HIV-1 sub-subtype CRF01_AE. When assessing the occurrence of drug resistance mutations, genetic resistance to any drug was detected in 74.41% (95% CI: 62.71-85.54%) of patients. These included resistance mutations to protease inhibitors in 60.66% (95% CI: 47.31-72.93%) of patients, to NRTIs in 8.20% (95% CI: 2.72-18.10%), and to NNRTIs in 44.26% (95% CI: 31.55-57.52%). Mutations associated with NRTI (2) and NNRTI (8) resistance as well as PI mutations (12), including minor ones, were identified. The high prevalence of drug resistance mutations found in this study among pregnant women, both in therapeutically naive individuals and in patients with virological failure of ART, indicates that currently used regimens in Vietnam are insufficient to prevent vertical HIV infection.

Next-Generation Sequencing-Based Analysis of Clinical and Pathological Features of PIK3CA-Mutated Breast Cancer.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is a well-known oncogene with a high prevalence of mutation in breast cancer patients. The effect of the mutation is a deregulation in phosphatidylinositol 3-kinase-related pathways, and, consequently, in unrestricted cell growth and differentiation. With the advent of precision oncology, PIK3CA has emerged as a pivotal treatment target, culminating in the recent approval of alpelisib. Despite years of research on this genetic alteration, certain aspects of its influence on the prognosis of breast cancer remain ambiguous. The purpose of this analysis is to characterize the clinical picture of breast cancer patients with PIK3CA mutation in comparison to the PIK3CA-wild-type group. We examined 103 tumor samples from 100 breast cancer patients using a next-generation sequencing panel. Presence of the mutation was linked to an older age at diagnosis, a lower expression of Ki67 protein, a greater percentage of tumors expressing progesterone receptors, and a notably higher incidence of metastatic disease at presentation. No significant differences were identified in overall and progression-free survival between the two groups. Our findings enhance the understanding of how PIK3CA mutations shape the clinical and prognostic landscape for breast cancer patients.

Prognostic relevance of clonal hematopoiesis in myeloid neoplastic transformation in patients with follicular lymphoma treated with radioimmunotherapy.

While novel radioisotope therapies continue to advance cancer care, reports of therapy-related myeloid neoplasms (t-MN) have generated concern. The prevalence and role of clonal hematopoiesis (CH) in this process remain to be defined. We hypothesized that: (i) CH is prevalent in relapsed follicular lymphoma and is associated with t-MN transformation, and (ii) radiation in the form of radioimmunotherapy (RIT) plays a role in clonal progression. In this retrospective cohort study, we evaluated the prevalence and prognostic impact of CH on clinical outcomes in 58 heavily pre-treated follicular lymphoma patients who received RIT. Patients had been given a median of four lines of therapy before RIT. The prevalence of CH prior to RIT was 46%, while it was 67% (P=0.15) during the course of RIT and subsequent therapies in the paired samples. Fourteen (24%) patients developed t-MN. Patients with t-MN had a higher variant allele fraction (38% vs. 15%; P=0.02) and clonal complexity (P=0.03) than those without. The spectrum of CH differed from that in age-related CH, with a high prevalence of DNA damage repair and response pathway mutations, absence of spliceosome mutations, and a paucity of signaling mutations. While there were no clear clinical associations between RIT and t-MN, or overall survival, patients with t-MN had a higher mutant clonal burden, along with extensive chromosomal abnormalities (median survival, afer t-MN diagnosis, 0.9 months). The baseline prevalence of CH was high, with an increase in prevalence on exposure to RIT and subsequent therapies. The high rates of t-MN with marked clonal complexities and extensive chromosomal damage underscore the importance of better identifying and studying genotoxic stressors accentuated by therapeutic modalities.

Association of MTHFR polymorphism, folic acid and vitamin B12 with serum homocysteine levels in pregnant women.

Homocysteine is known to be associated with adverse vascular and metabolic effects, as well as pregnancy complications. Its serum levels are influenced by the function of the enzyme methylenetetrahydrofolate reductase (MTHFR) and the dietary intake of folic acid, vitamin B12, and methionine. In this cross-sectional study, we investigated the association of genetic polymorphisms of the MTHFR gene with vitamin status in pregnant women during mandatory folic acid supplementation. The study included 102 pregnant women between 24 and 28 weeks of gestation who were attending regular outpatient examinations at the maternity clinic. Homocysteine, folic acid, vitamin B12 levels, and MTHFR gene polymorphisms (C677T and A1298C) were analyzed. Significant associations were found between vitamin B12 and folic acid levels with homocysteine (P < 0.001), with lower serum levels of these vitamins being associated with higher homocysteine levels. Surprisingly, there was no significant association between MTHFR genetic polymorphisms and serum homocysteine levels, likely attributed to the supplementation of folic acid and vitamin B12 in vitamin supplements for pregnant women, which counteracts the effect of the mutation. Remarkably, a high prevalence of MTHFR gene mutations was found, with the C677T polymorphism present in 56.9% and the A1298C polymorphism in 87.2% of pregnant women. These findings emphasize the importance of adequate folic acid and vitamin B12 intake during pregnancy to regulate homocysteine levels. Although the MTHFR gene mutations were highly prevalent, their influence on homocysteine levels in this population appears to be mitigated by vitamin supplementation. Further research is warranted to explore the impact of these mutations on other aspects of pregnancy outcomes. The trial is registrated at Clinicaltrail.gov (NCT04952324).