Age-Related Clonal Hematopoiesis Is a Precursor for Adult Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) represents 20% of adult leukemias. Recent technologic advances have enabled detailed characterization of the genetic basis of leukemogenesis in ALL, including somatic structural DNA rearrangements and sequence mutations that disrupt lymphoid development, signaling, tumor suppression, and epigenetic modification. These studies also showed differences in the molecular profiles of pediatric vs adult ALL. However, adults with ALL, especially older adults (≥40 years), were underrepresented in these large series. Clinical outcomes of older adults with ALL are inferior to younger patients (<40 years) and the molecular basis for these differences is not completely understood. Hematopoietic stem cells accumulate DNA mutations with aging, and age-related clonal hematopoiesis (ARCH) has been linked to increased incidence of myeloid malignancies. The prevalence of ARCH increases logarithmically as the population ages, but its role in lymphoid leukemogenesis has not been fully established. We hypothesize that ARCH is a common precursor lesion for the development of ALL in older adults, and patients with ARCH-associated ALL have different clinical outcomes compared to patients whose disease do not harbor these mutations. We retrospectively studied adults with ALL treated at the University of Chicago and Moffitt Cancer Center between July 2014 and April 2021. Genetic profiling of tumor samples was performed by using Miseq Illumina next-generation sequencing (NGS) platform with a comprehensive sequencing panel covering commonly mutated myeloid and lymphoid genes. We classified pathogenicity using American College of Medical Genetics and Genomics guidelines. In total, 345 patients were studied: 286 (83%) had B-ALL, 49 (14%) had T-ALL and 10 (3%) had early T-precursor (ETP)-ALL. Overall, median age at diagnosis was 47 years (range, 18-88 years), and 211 (61%) were ≥40 years at diagnosis; 154 (45%) were women. Cytogenetic groups were as follows: 24% had Ph+ ALL, 13% had Ph-like ALL, and 3% had ALL with KMT2A rearrangement. The most frequent mutation in our adult ALL cohort was the loss of CDKN2A gene (32%), followed by mutations in TP53 (17%), IKZF1 (16%), NOTCH1 (9%), NRAS (9%), and JAK2 (6%) genes. Mutations involving the recurrently mutated genes in ARCH were seen in 110 of 345 patients (32%) with the following order of frequency: TP53 (17%), DNMT3A (5%), TET2 (4%), RUNX1 (3.5%), ASXL1 (3%), IDH1/2 (2%), BCORL1 (2%), EZH2 (1%), CUX1 (1%), and U2AF1 (1%) (Figure 1A). ARCH-associated mutations were more common in older adults (≥40 years) compared to young adults (41% vs 17%, p< 0.0001). Variant allelic frequencies (VAFs) for the ARCH-associated mutations were higher than the mutations involving signaling pathways, which suggests the ancestral nature of the former and secondary nature of the latter (Figure 1B). We further observed clonal dynamics in patients with serial diagnosis, remission and relapse samples available for sequencing. Founder ARCH clones re-emerged at the time of relapse (patient 92 and 100), and were also detectable at the time of complete remission with persistent measurable residual disease (patient 100) (Figure 1C). The overall survival (OS) for patients with ARCH-associated ALL was shorter than patients without ARCH, but the difference did not reach statistical significance (median OS, 39 months vs 84 months, p= 0.16) (Figure 1D). Our results indicate that ARCH is commonly identified as an ancestral event in older adults with ALL, with TP53 mutations being the most prevalent. Unlike patients with AML and TP53 mutations, patients with ALL and ARCH-associated mutations had comparable clinical outcomes to patients without ARCH. This may reflect the frequent use of antibody-based therapies (i.e. blinatumomab and inotuzumab) at diagnosis (on a clinical trial basis) or relapse in the two centers where these patients were treated. Collectively, these data suggest that ARCH may constitute a fertile soil for acute lymphoblastic leukemogenesis and further studies are warranted to interrogate the dynamic interplay between myeloid and lymphoid compartments of these patients. Figure 1 Figure 1. Disclosures Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Shah: BeiGene: Consultancy, Honoraria; Incyte: Research Funding; Acrotech/Spectrum: Honoraria; Novartis: Consultancy, Other: Expenses; Pfizer: Consultancy, Other: Expenses; Amgen: Consultancy; Servier Genetics: Other; Jazz Pharmaceuticals: Research Funding; Precision Biosciences: Consultancy; Pharmacyclics/Janssen: Honoraria, Other: Expenses; Kite, a Gilead Company: Consultancy, Honoraria, Other: Expenses, Research Funding; Adaptive Biotechnologies: Consultancy; Bristol-Myers Squibb/Celgene: Consultancy, Other: Expenses.

Molecular Characterization of Drug-Resistant Mycobacterium tuberculosis Isolated from HIV-Positive Patients in Rivers State, Nigeria

Aim: Drug resistant tuberculosis is a major challenge in the global bid to control the disease burden and improve treatment outcomes of DRTB infected individuals. Methods: The Line Probe Assay (LPA) was used to assess the drug resistance pattern and gene mutations in 260 sputum specimens collected consecutively from 260 adult, HIV sero-positive subjects presenting with symptoms suggestive of TB in clinical settings across Rivers State, Nigeria. Results: The results showed a 61.2% (n = 159) prevalence of TB among all study subjects. LPA analysis showed that 16 (10.1%) were multidrug resistant strains, 17 (10.7%) Rifampicin (RIF) monoresistant strains, 24 (15.1%) Isonaizid (INH) monoresistant strains and 102 (64.2%) drug susceptible strains. Among all 32 RIF-resistant strains, 24 (75%) had a mutation in rpoB S531L at the MUT3 band. Another mutation was observed at rpoB H526D (1/17) in RIF-monoresistant strains but not in MDR-TB strains. In INH resistant strains, mutations were observed at the katG gene [32/39 (82.1%)] which constituted 13/15 (86.7%) in MDR-TB strains and 19/24 (79.2%) in INH-monoresistant strains (p =0.002). Overall frequency of inhA mutation was 6/39 (15.4%); MDR-TB strains [2/15 (13.3%)] and INH-monoresistant strains [4/24 (16.7%), p = 0.2]. Combined KatG and inhA mutation was found in 1/39 (2.6%) of INH-monoresistant strains and in none of MDR-TB strains. Of the single inhA mutation bands, only 1/39 (4.2%) MUT 3B (T8A mutation) was observed in INH-monoresistant strains. Conclusion: The results showed that drug-resistant TB is prevalent in Rivers State with various gene mutations observed in the different TB strains isolated, making LPA analysis a necessity in the bid to improving prevention and control efforts in the region.

SSB facilitates fork substrate discrimination by PriA

PriA is a member of the SuperFamily 2 helicase family. Its role in vivo is to reload the primosome onto stalled replication forks resulting in the restart of the previously stalled DNA replication process. SSB is known to play key roles in mediating activities at replication forks and it is known to bind to PriA. To gain mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriA in vitro in the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by DNA-bound SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for the E.coli protein. SSB, while enhancing the activity of PriA on its preferred fork, both decreases the affinity of the helicase for other forks and decreases catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3’-OH placed at the end of the nascent leading strand at the fork. When both the 3’-OH and SSB are present, the maximum effect is observed. This ensures that PriA will only load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.

GENE-32. SYNTHETIC LETHAL INTERACTIONS WITH IDH1R132H IN GLIOMA STEM-LIKE CELLS

Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.

Detection of fimC gene as fingerprinting for Salmonella typhimurium isolates by using Polymerase Chain Reaction

A total of 480 fecal samples were collected from children (less than 3 years old) , ofboth sexes suffering from diarrhea who admitted to The Teaching Hospital of Maternity andPediatrics in Al- Diwaniya governorate, Salmonella spp. were isolated and identified usingbacterial culturing on selective media, in addition to, biochemical and Mini API 20E andserotyping by monovalent antisera. Polymerase chain reaction (PCR) was used to detect fimCgene encoding for biosynthesis of fimC of Salmonella typhimurium. The results revealed thatthe rate of Salmonella isolates in fecal samples of patients were (38/480) 7.9% using culturaland Mini API20 E, The results of serotyping revealed that isolates there were 34 belong toSalmonella spp. Of these isolates 30 belong to S . typhimurium, while the remainingbelong to S. enteritidis ( 2 isolates) and S. meunchen (2 isolates), when the PCR techniquewas used to detect the presence of fimC gene, 32 Salmonella isolates were belong to S.typhimurium appeared to contain this gene . The results of this study revealed that thePCR technique had a high specifity (100%) in detection of S. typhimurium in comparison toserotyping.