Single Cell Sequencing of Pediatric Acute Myeloid Leukemia Reveals Clonal Evolution to Relapse on Combination Chemotherapy with Sorafenib

Introduction: Relapse of pediatric acute myeloid leukemia (AML) remains a leading cause of childhood cancer mortality, and leukemias with activation of the Fms-like tyrosine kinase 3 (FLT3) are particularly susceptible to relapsed disease. Risk-directed therapy to prevent relapse is based both on genetic changes known to drive drug resistance, and measurable residual disease (MRD) at the end of induction therapy (EOI). In adult AML, resistance to type II FLT3-inhibitors, like sorafenib, is primarily driven by on-target FLT3 kinase domain (KD) mutations. However, the resistance mechanisms for pediatric leukemias, which are treated on combination therapies, have not been fully elucidated. MRD is considered the among the most predictive markers of future relapsed disease. It has been assumed that the major clone at the time of MRD assessment will predict the majority clone at relapse. However, this assumption has not been proven. The definition of the most specific genetic and MRD markers of relapse are essential to prognosticate and personalize therapy to prevent relapsed disease. Methods: We performed single cell sequencing (SCS) with a high-throughput DNA sequencing platform, Mission Bio Tapestri, on bone marrow or peripheral blood samples from 24 samples from 8 pediatric patients treated on COG AAML1031 with serial samples from diagnosis, EOI, and relapse. Results: We analyzed a total of 94,833 cells from 8 pediatric patients (median cells per patient 12,428) all treated on AAML1031. SCS revealed a sensitive and specific description of clonal evolution on the combination of sorafenib with cytotoxic chemotherapy. The FLT3 internal tandem duplication (ITD) was controlled by the therapy in only half of the patients. In five of the patients, the FLT3-ITD was present in multiple clones. The FLT3-ITD co-mutated with additional mutations (NRAS, SH2B3, WT1, TET2, or NPM1) in half of the patients. However, the presence of a co-mutation did not necessarily correlate with whether or not the ITD-containing clone persisted at the time of relapse. Of the leukemias whose relapse was not driven by FLT3, the most likely mutational driver of resistance was NRAS. Notably, however, despite the fact that FLT3 KD mutations make up the bulk of mutational resistance to type II FLT3i such as sorafenib in adult patients, there were no on-target FLT3 mutations found in any of these pediatric patients. Further, SCS allows for an unprecedented depth of analysis of the genetic complexity of pediatric AML. Phylogenic analysis revealed that the same mutations may arise independently in different cells (NPM1 W288fs, NRAS G60E). Additionally, the same gene may be mutated twice within the same cell (WT1, TET2). These data, consistent with our prior work, suggest that some leukemias may have a predilection to mutations within specific loci. Finally, although there is a standing assumption that the dominant MRD population will proliferate into relapsed disease, in 3/8 patients, the dominant MRD clone did not predict the dominant relapse clone. Conclusions: SCS allows for direct measurement of clonal hierarchy and evolution, phylogeny, co-mutational status, and zygosity, which can only be inferred through traditional bulk NGS. The mutational mechanisms of resistance seen in adult leukemias treated with sorafenib monotherapy are not necessarily relevant to the pediatric population; rather than on-target FLT3 mutations, off target mutations including NRAS are found. This corroborates prior findings that off-target RAS pathway mutations may drive resistance to FLT3i. Non-RAS off-target mutations found in this cohort do not necessarily predict sorafenib resistance, so may be passenger mutations. The lack of consistent resistance mutations suggests that other mechanisms of resistance such as epigenetic modifications may also drive resistance to combination chemotherapy with FLT3i in pediatric leukemia. Further, SCS exposes more genetic complexity in pediatric AML than has previously been appreciated: the same mutation may independently arise in more than one cell or the same cell may have multiple mutations within the same gene. Finally, the sensitivity of SCS reveals that the major clone at the time of MRD assessment is not necessarily the major clone at relapse. This suggests a benefit of more frequent MRD monitoring to track clonal evolution in real time. Disclosures Smith: Daiichi Sankyo: Consultancy; Revolutions Medicine: Research Funding; AbbVie: Research Funding; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Molecular characteristic of rifampin-sensitive and resistant isolates and characteristics of rpoB gene mutations in MRSA

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) infections have become a leading cause of bacterial infections in both healthcare and community settings. Mutations in the rpoB gene cause resistance to rifampin (RIF R ), a critical antibiotic for treatment of multidrug-resistant S. aureus . The aim of this study was to detect the molecular characteristics of RIF R MRSA and analysis the rpoB gene mutations involved in RIF resistance. MethodsA total of 49 RIF R MRSA and 38 RIF S MRSA isolates collected from seven cities in China were analyzed by multilocus sequence typing, staphylococcus chromosomal cassette mec (SCC mec ) typing, spa typing, and rpoB gene mutations. ResultsST239-III-t030 (35/49, 71.4%), the major clone in RIF R MRSA isolates; ST45-IV-t116 (16/38, 42.1%), the major clone in RIF S MRSA isolates with rpoB mutations. RIF R MRSA isolates were resistance to erythromycin, ciprofloxacin, tetracycline, gentamicin, and clindamycin. By contrast, RIF S MRSA isolates with rpoB mutation were more susceptible to ciprofloxacin, tetracycline, and gentamicin. Forty-three (87.8%) isolates present the mutational change H481N and L466S, conferring 128-512 μg/ml RIF resistance. The four isolates with RIF MIC > 1024 μg/ml had additional amino acid substitution: H481N, L466S, A473T (n=2); H481Y (n=2), associated with a high-level RIF resistance. Of 38 RIF S MRSA isolates, two mutations were observed, including H481N (n=37) and A477D (n=1). ConclusionThe predominant RIF R MRSA clones in China were ST239III-t030. Molecular character, antibiotic resistant profiles, and rpoB mutations between RIF R MRSA and RIFS MRSA were diverse. Antibiotics for treating patients with MRSA infections can be selected based on molecular characteristics.

A single Proteus mirabilis lineage from human and animal sources: a hidden reservoir of OXA-23 or OXA-58 carbapenemases in Enterobacterales

In Enterobacterales, the most common carbapenemases are Ambler’s class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.

Ighv Mutational Status By Deep Next Generation Sequencing Refines Ighv Sanger Sequencing Classification in Patients with Chronic Lymphocytic Leukaemia

Introduction: Determination of the mutational status of rearranged immunoglobulin heavy chain variable (IgHV) genes in patients with Chronic Lymphocytic Leukaemia (CLL), is considered one of the most important prognostic factors: patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; ≤98%). Mutational status, is currently determined by Sanger sequencing (Sseq) that allows the analysis of the major clone, however, international guidelines recommend caution in assigning mutational status in cases with "Borderline" IgHV identity (97-97.9%), and cases with double rearrangements with discordant mutational status. Objective: Analyze and determine the mutational status of the IgHV locus by High-throughput sequencing (HTS), in a cohort of CLL patients (n=51) with unclassifiable Sseq results: borderline status (n=22); double rearrangements (n=27) with discordant mutational status (n=2). Methods: We included 51 DNA samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2019 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and analysis of IgHV rearrangements were performed on DNA conforming to the updated ERIC recommendations. In all the cases we were able to determinate the IGVH identity. To switch high-throughput sequencing to the clinical practice, we assessed the reliability of different library preparation methods to sequence IGH locus in patients with CLL. Amplification was performed using the Sequencing Multiplex Kit based on IGH FR (forward primers) and consensus JH (reverse primer) multiplex. PCR products were purified using Magsi-NGS Prep magnetic beads (Magnamedics Diagnostics), normalized and pooled to create a library for sequencing using a MiSeq equipment. To simplify and make automatic the analysis of the same we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Results: This approach led to the identification of a dominant clone IgHV in all cases (n=51). Instead, the percentage of identity calculated by HTS analysis varies in: - 15/22 borderline cases whose mutational status could be recalculated into 10 MM and 5 UM. The rest 7 remaining in borderline group. - We could identify both clones in 29 double rearrangements cases, with concordant mutational status except 2/29 undetermined cases, included in UM group regarding HTS results. Our tool led to the identification of a dominant clonotypic IgHV in all cases, and when compared the HTS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, 15 out of 22 (68,18%), could be reclassified. This case showed a major clone with productive rearrangement mutated by Sseq but unmutated by HTS. Conclusions: Analyze and determine the mutational status of the IgHV locus by HTS, would potentially reveal multiple rearrangements and increase the prognostic precision of IgHV mutation analysis. IgHV-HTS classification is able to precisely classify patients with borderline status or/and multiple IgHV rearrangements for which Sseq is inconclusive. In this case, it has been possible to improved prognostication for 17 out of 24 patients. This is helping us to discover the advantages of the data obtained by HTS compared with current Sseq standard technique. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Terol: Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Research Funding; Roche: Consultancy; Astra Zeneca: Consultancy.

Spread of a Major Clone of Salmonella enterica Serotype Enteritidis in Poultry and in Salmonellosis Outbreaks in Southern Brazil

ABSTRACT Salmonella spp. are among the most important agents of foodborne diseases all over the world. Human Salmonella outbreaks are often associated with the consumption of poultry products (meat and eggs), and one of the most prevalent serotypes associated with these products is Salmonella Enteritidis. Brazil is one of the most important poultry exporters in the world. In southern Brazil, three closely related clones of Salmonella Enteritidis have been responsible for the majority of foodborne Salmonella outbreaks over the past decade. However, until now, there has been little information regarding the clonal relationship among the Brazilian Salmonella strains of avian origin and those involved in foodborne outbreaks. Therefore, the aim of the present study was to complete the molecular characterization of Salmonella Enteritidis strains isolated from poultry and food sources involved in Salmonella outbreaks. PCR ribotyping was performed to discriminate the strains into different ribotype profiles according to the banding pattern amplification. This technique was able to differentiate the Salmonella Enteritidis strains into two banding patterns: R2 and R4. R2 accounted for 98.7% of the strains. DNA sequencing of the 600-bp fragment, present in all ribotypes, was applied to confirm this result. The sequences generated showed high levels of similarity, ranging from 99.7 to 100%, and were grouped into a single cluster. These results suggest that there is a clonal relationship among the Salmonella Enteritidis strains responsible for several salmonellosis outbreaks and the strains collected from poultry sources.

Genomic Architecture and Clonal Dynamics of Early Relapsed BCP-ALL

Relapse represents the most common cause of therapy failure in B-cell precursor ALL acute lymphoblastic leukemia (BCP-ALL), and is caused by selective outgrowth of therapy-resistant leukemic cells. Two-third of BCP-ALL relapses present after treatment, i.e. after two years. These relapses may originate from leukemic (sub)clones that remained in a quiescent state during treatment or that could not be reached by the chemotherapeutics. Relapses that occur during treatment are different in that they display clonal outgrowth in the presence of chemotherapeutics, and these patients have poorer outcomes. The aim of this study is to explore the genomic abnormalities in leukemia with early relapse, and investigate the clonal dynamics of relapses that arise during treatment. We included 17 BCP-ALL cases which relapse during treatment (<2yrs) according to DCOG protocols ALL9, ALL10 or ALL11. Median remission time was 1.08 yrs (range 0.48-1.95). Whole exome sequencing was performed on DNA isolated at time of first diagnosis, complete remission and relapse from bone marrow or peripheral blood, with an average read depth on target of 108x. After mapping of the reads, variants were called using HaplotypeCaller. In total, we identified 1771 somatic mutations in 1562 genes. Per case, a median of 21 mutations were detected at diagnosis (range 10-630) and 31 at relapse (range 10-652). A hypermutation profile was observed in one diagnosis-relapse pair, and two additional relapses. All cases harbored mutations shared between diagnosis and relapse, which were mostly part of the major clone at both time points. However, the fraction of shared mutations varied considerably between cases, ranging from <10% in 3 cases to >80% in 4 cases. Based on the clonal dynamics, 3 distinct groups were recognized. Group I includes two cases, with relapses within 6 months, in which the (sub)clonal mutation spectrum between diagnosis and relapse was identical. Group II (n=10) presented with a relapse closely resembling the major clone at diagnosis. Mostly, these relapses acquired new mutations and they often branched off from the major clone already before the time of diagnosis. Finally, Group III (n=5) consists of cases in which the relapse originates from a minor subclone at diagnosis that hardly resembled the major clone, suggesting a clonal switch during treatment. Next, we analyzed the genes with mutations that were predicted to be damaging (truncating and non-synonymous conserved missense variants). We performed pathway analysis for these genes and identified RAS pathway genes to be frequently mutated among shared mutations, while mutations in genes involved in epigenetic regulation, chromatin condensation and regulation of transcription were acquired. In total, 7 of the genes with (predicted) pathogenic mutations in relapse were affected in at least two cases, including known genes like KRAS, CREBBP, and WHSC1 (NSD2). CREBBP mutations were never part of the major clone at diagnosis and were present in cases with numerical chromosomal aberrations. Both cases with hotspot E1099K mutation in WHSC1 were t(1;19) translocation-positive. Recent studies showed that somatic mutational signatures, composed of the six substitution subtypes in a 3-nucleotide context, expose specific biological processes underlying tumor development, including defects in genomic maintenance and repair. Currently 30 mutational signatures have been described [http://cancer.sanger.ac.uk/cosmic/signatures]. Despite the low number of mutations in most samples, we identified at least 5 of these signatures, including the most common signature 1, a signature associated with aberrant AID/APOBEC activity (signature 2), and three signatures associated with mismatch repair deficiency (6, 15, 26). Most cases carried multiple signatures, but signature 2 was very prominent is one relapse and one diagnosis-relapse pair. Most signatures were preserved from diagnosis to relapse suggesting that the same mutational processes remained active. Taken together, our results show considerable heterogeneity in the group of children with early relapse of BCP-ALL. Two cases with the shortest remission times relapsed without notifiable somatic changes, whereas most other early relapses appeared to arise from minor or newly appearing subclones. These findings demonstrate the strong clonal selection that occurs during treatment in cases with very early relapse. Disclosures No relevant conflicts of interest to declare.

Using Whole Exome Sequencing in Pediatric Acute Lymphoblastic Leukemia Germline, Diagnosis, and Relapse Trios to Discover Novel Relapse Enriched Mutations for Clonal Backtracking By Ddpcr

While the outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically over the last four decades, the prognosis for those who relapse remains dismal, especially for those who relapse while on therapy. In fact, relapsed disease remains a leading cause of cancer related mortality in children. To date, various studies have discovered a number of somatic alterations that contribute to driving relapse and have provided profound insight into the selective forces that lead to clonal outgrowth of drug resistant populations, however these lists are not yet comprehensive. We analyzed 13 pediatric ALL patients treated according to Nordic NOPHO ALL protocols and explored a comprehensive collection of germline, diagnosis, relapse, and maintenance samples. Whole exome sequencing (WES) was performed on all available germline, diagnosis, and relapse samples to find somatic missense mutations enriched in the relapse samples versus the diagnosis and/or germline samples. Sequencing reads were aligned to the human genome (build hg19/GRCh37) using the Burrows-Wheeler Aligner (BWA) and single-nucleotide somatic variants were generated with MuTect. ANNOVAR was used to annotate variants with functional consequences and identify if the variant was contained in dbSNP, ExAC, 1000 Genomes project, and COSMIC databases. Nine of the NOPHO patients were analyzed as trios (WES of germline, diagnosis, and relapse), three of the patients as Diagnosis-Relapse duos and one as a Germline-Relapse duo. Candidate relapse driving mutations were identified as present at high levels in the relapse sample, but were undetectable in germline or low to absent in the diagnosis sample. Missense mutations had to be enriched by ≥5% in the relapse sample versus diagnosis/germline to be included for further consideration. Relapse specific candidates were further prioritized based on tumor percentage (≥ 20%), bioinformatic tools predicting a missense change being deleterious or damaging to protein function, and literature reviews for insight into the biological pathway potentially affected.Eight of the thirteen patients contained mutations in genes previously reported to be enriched and are involved in nucleoside metabolism/synthesis, histone acetylation, transcription regulation, or cell signaling/growth through the Ras pathway. Interestingly, a majority of the patients contained novel relapse specific genes in a major clone that met the criteria for drivers (Table 1). These novel candidates are involved in a wide array of cellular processes such as cell adhesion/migration, RNA polymerase II/transcription, circadian rhythm, the unfolded protein response, RNA transport, epigenetic regulation, DNA methylation, and kinases. Knowing the exact relapse specific mutations for each patient allows use of droplet digital PCR (ddPCR) to track the emergence of specific candidate mutations from peripheral blood samples (range of 2-68 per patient, Table 1) collected from these patients prior to relapse. Thus far, we have successfully backtracked the emergence of the NT5C2 p.R367Q mutation (.2% Minor Allele Frequency (MAF)) just over a month before frank relapse in patient 8142, using ddPCR. Tracking these mutations offers insight into which mutations drive relapse and the speed at which the relapse clones emerge. Probes for ddPCR to detect our top candidates have been developed and are currently being applied. Ultimately, candidate mutations emerging with the major clone will undergo functional testing to understand the mechanism by which the mutation drives relapse. Through these approaches, we will be able to pinpoint what mutation(s) and combinations thereof drive relapse through clonal survival during maintenance therapy. Disclosures No relevant conflicts of interest to declare.

Mutational and Clonal Dynamics in Patient-Derived Xenografts of Acute Myeloid Leukemia

Patient-derived xenografts have emerged as an attractive model to faithfully recapitulate acute myeloid leukemia (AML) in vivo and to test the therapeutic efficacy of new treatment regiments. The most efficient host for AML cells appears to be the NSGS mouse strain engineered to express the human cytokines SCF, GM-CSF and FLT3L. However, little is known about how the underlying genomics affect engraftment in this model and how the genomics and clonality of the patient samples are affected by the successional passaging in vivo. To address these questions, we transplanted cells from 27 AML patients to NSGS mice. First, we correlated engraftment to leukemia-associated mutations determined by whole-exome sequencing. The frequency of engraftment in primary recipients for the most common mutations in AML were as follows; FLT3-ITD: 29% (2/7), NPM1: 75% (9/12), DNMT3A: 55% (6/11), IDH1/2: 54% (7/13), TET2: 100% (5/5), all samples: 54% (13/24). Although samples carrying the FLT3-ITD mutation engrafted with relatively low frequency, they generated more prolific disease, with cell numbers several fold higher than for any other patient. Samples transplanted to multiple mice showed strikingly similar characteristics. Next, we determined changes in mutation patterns and clonal composition by whole-exome sequencing of human myeloid cells sorted from primary and secondary recipient mice. For all the 11 patients analyzed, the variant allele frequencies (VAF) of the mutations found in the patient sample were increased to or maintained at around 50% by the first passage in vivo. This corresponds to a heterozygous mutation being present in the whole cell population and indicates that the xenotransplantation model enriches the leukemic cells. Importantly, no novel mutations in known AML-associated genes were detected after either the first or second passage in mice, demonstrating that the genotype of the patient sample is preserved during expansion in vivo. We then studied the clonal evolution in the 3 patients who presented with multiple clones. Notably, all 3 cases displayed drastic changes in the allele frequencies of specific mutations. One of the patients had what appeared to be 2 clones at diagnosis, a major clone with 8 AML-associated mutations (VAF 30-55%) and a minor clone with an additional NRAS mutation (VAF 5%). After one passage in vivo, the BCOR mutation in the major clone had disappeared (VAF 52% to 0%), while the other mutations in the major clone were maintained or slightly increased and the NRAS mutation had increased drastically (VAF 5% to 46%). This shows that the minor clone containing the NRAS mutation must have branched from a cell with all the presumed major clone mutations except that in BCOR and that this subclone completely outcompeted the major clone in vivo. This clonal composition also remained upon secondary transplantation. The second patient presented with a major clone carrying 9 mutations (VAF 35-55%) and a small subclone containing an SMC3 mutation (VAF 2%). Also in this case, the small subclone vastly outgrew the presumed founding clone in multiple mice to a VAF of 25-35%. The third patient carried mutations in 11 genes at diagnosis. Upon in vivo passaging, the lowest-frequency mutation, in IDH2, had markedly increased (VAF 10% to 36%), whereas the 3 second-lowest frequency mutations had completely disappeared (VAF 20-23% to 0%), while the high-frequency mutations remained at close to 50%. This development reveals 3 subclones at diagnosis; a founding clone that had given rise to 2 independent subclones, the largest of which was lost upon transplantation and the smallest of which vastly expanded. Hence, for all 3 patients with multiple clones, the smallest subclone drastically expanded in vivo at the expense of the others. This may reflect the biology in the patient, where subclones can only reach detectable levels by expanding much more rapidly than the founding clone. We show that this process continues in the xenografts and may thus model the evolution from diagnosis to relapse. In conclusion, our results suggest that AML patient cells generally maintain their genotype during passaging in vivo but that clonal competition drastically alters the mutational landscape, emphasizing the need for genetic characterization of patient-derived xenografts. Disclosures Fioretos: Cantargia: Equity Ownership.