Remission Samples Research Articles

Genomic Determinants of Outcome in Acute Lymphoblastic Leukemia.

Although cure rates for childhood acute lymphoblastic leukemia (ALL) exceed 90%, ALL remains a leading cause of cancer death in children. Half of relapses arise in children initially classified with standard-risk (SR) disease. To identify genomic determinants of relapse in children with SR ALL, we performed genome and transcriptome sequencing of diagnostic and remission samples of children with SR (n = 1,381) or high-risk B-ALL with favorable cytogenetic features (n = 115) enrolled on Children's Oncology Group trials. We used a case-control study design analyzing 439 patients who relapsed and 1,057 who remained in complete remission for at least 5 years. Genomic subtype was associated with relapse, which occurred in approximately 50% of cases of PAX5-altered ALL (odds ratio [OR], 3.31 [95% CI, 2.17 to 5.03]; P = 3.18 × 10-8). Within high-hyperdiploid ALL, gain of chromosome 10 with disomy of chromosome 7 was associated with favorable outcome (OR, 0.27 [95% CI, 0.17 to 0.42]; P = 8.02 × 10-10; St Jude Children's Research Hospital validation cohort: OR, 0.22 [95% CI, 0.05 to 0.80]; P = .009), and disomy of chromosomes 10 and 17 with gain of chromosome 6 was associated with relapse (OR, 7.16 [95% CI, 2.63 to 21.51]; P = 2.19 × 10-5; validation cohort: OR, 21.32 [95% CI, 3.62 to 119.30]; P = .0004). Genomic alterations were associated with relapse in a subtype-dependent manner, including alterations of INO80 in ETV6::RUNX1 ALL, IKZF1, and CREBBP in high-hyperdiploid ALL and FHIT in BCR::ABL1-like ALL. Genomic alterations were also associated with the presence of minimal residual disease, including NRAS and CREBBP in high-hyperdiploid ALL. Genetic subtype, patterns of aneuploidy, and secondary genomic alterations determine risk of relapse in childhood ALL. Comprehensive genomic analysis is required for optimal risk stratification.

New ABL1 Kinase Domain Mutations in BCR::ABL1-Positive Acute Lymphoblastic Leukemia.

Since the development of the first-generation Tyrosine Kinase Inhibitor (TKI), it has played a crucial role in the treatment of BCR::ABL1-positive acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). However, ABL1 kinase domain (ABL1 KD) mutations confer resistance to several TKIs. These mutations have been extensively studied in chronic myeloid leukemia (CML) but less so in BCR::ABL1-positive acute lymphoblastic leukemia (ALL). Our study aimed to analyze the the ABL1 KD mutations in 97 consecutive newly-diagnosed adults with BCR::ABL1-positive ALL before therapy, in cytogenetic complete remission and at relapse with next generation sequencing (NGS). The relationship between ABL1 KD mutations and TKI selection was also analyzed. Previously unreported ABL1 KD mutations R239G, F401V/L, R516L and K262T were the most prevalent in pre-therapy and cytogenetic remission samples, whereas T315I/P and P-loop mutations were most prevalent in relapse samples. R239G, F401V/L, R516L and K262T are related to the BCR::ABL1 structure, whereas T315I/P and P-loop mutations directly alter kinase activity. BaF3 cells transfected with ABL1 KD F401V, K262T, R239G, or R516L mutations were resistant to imatinib but strongly inhibited by olverembatinib with IC50 values of 0.73 to 1.52nM. Meanwhile, olverembatinib had advantages in increasing complete molecular response (CMR) and good prognosis. Overall, our findings indicate the prevalence and impact of new ABL1 KD mutations in BCR::ABL1-positive ALL patients, highlighting the necessity for effective therapies targetingthese mutations.

The genomic basis of childhood T-lineage acute lymphoblastic leukaemia.

T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease.

SJPedPanel: A Pan-Cancer Gene Panel for Childhood Malignancies to Enhance Cancer Monitoring and Early Detection.

The purpose of the study was to design a pan-cancer gene panel for childhood malignancies and validate it using clinically characterized patient samples. In addition to 5,275 coding exons, SJPedPanel also covers 297 introns for fusions/structural variations and 7,590 polymorphic sites for copy-number alterations. Capture uniformity and limit of detection are determined by targeted sequencing of cell lines using dilution experiment. We validate its coverage by in silico analysis of an established real-time clinical genomics (RTCG) cohort of 253 patients. We further validate its performance by targeted resequencing of 113 patient samples from the RTCG cohort. We demonstrate its power in analyzing low tumor burden specimens using morphologic remission and monitoring samples. Among the 485 pathogenic variants reported in RTCG cohort, SJPedPanel covered 86% of variants, including 82% of 90 rearrangements responsible for fusion oncoproteins. In our targeted resequencing cohort, 91% of 389 pathogenic variants are detected. The gene panel enabled us to detect ∼95% of variants at allele fraction (AF) 0.5%, whereas the detection rate is ∼80% at AF 0.2%. The panel detected low-frequency driver alterations from morphologic leukemia remission samples and relapse-enriched alterations from monitoring samples, demonstrating its power for cancer monitoring and early detection. SJPedPanel enables the cost-effective detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions by targeted sequencing of ∼0.15% of human genome for childhood malignancies. It will enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection.

A comparative study of anti–ADAMTS-13 antibody dynamics in immune-mediated thrombotic thrombocytopenic purpura

BackgroundThrombotic thrombocytopenic purpura, particularly its immune-mediated variant (iTTP), necessitates accurate diagnostic approaches for effective management. ObjectivesTo compare a chemiluminescence immunoassay (CLIA) and an enzyme-linked immunosorbent assay (ELISA) for testing ADAMTS-13 activity and detecting anti–ADAMTS-13 autoantibodies (AAbs) in patients with iTTP. MethodsThis study involved 31 paired samples from 12 iTTP patients. ADAMTS-13 activity was measured using the HemosIL AcuStar (Instrumentation Laboratory, CLIA) and Technozym (Technoclone) activity assay (ELISA). The presence of AAbs was assessed using Technozym ADAMTS-13-INH assay (ELISA) and HemosIL AcuStar activity (CLIA) within a Bethesda assay following mixing with normal pool plasma. von Willebrand factor (VWF) multimers were analyzed using the HYDRASYS-2 SCAN system and the HYDRAGEL 5- or 11-VW Multimer kits (Sebia). VWF activity levels were measured with the HemosIL AcuStar VWF:GPIbR on the ACL AcuStar Analyzer (IL). ResultsFor ADAMTS-13 activity, a strong linear relationship and no bias between CLIA and ELISA were confirmed (slope = 1.01 [0.91, 1.11], intercept = 0.00 [−0.47, 0]). However, significant discrepancies were found in AAb detection during remission phases with ADAMTS-13 activity between 10% and 50%, with CLIA and ELISA showing significant divergence (P < .001, Cohen’s g = 0.34). Consistently, VWF multimers and activity levels exhibited significantly different values between remission samples with ADAMTS-13 activity below 50% and above 50%. In longitudinal analysis of patients with multiple iTTP relapses, positivity to CLIA appears to precede ELISA in predicting exacerbations. ConclusionWhile CLIA and ELISA might be interchangeable for assessing ADAMTS-13 activity, they are not equivalent for detecting AAbs, particularly in patients in clinical remission with ADAMTS-13 activity between 10% and 50%.

Abstract 1763: Long-read sequencing of pediatric leukemia identifies clinically relevant genomic rearrangements

Abstract Clinical detection of genomic rearrangements currently depends on a range of complementary and overlapping methods including fluorescence in situ hybridization (FISH), karyotyping, and DNA and/or RNA sequencing. This requirement for multiple tests results in higher total costs, extended analysis time, and increased usage of sometimes limited tumor material. In our laboratory, we apply long high-fidelity reads to human samples using PacBio circular consensus sequencing (HiFi-GS) of 12-18kb DNA fragments, which offers better structural variant detection relative to short-read sequencing. Here, we assess the ability of HiFi-GS to detect clinically relevant genomic rearrangements (CRGRs) in pediatric leukemia. We selected 11 pediatric leukemia cases with corresponding normal (remission) samples, including 7 cases of B-cell acute lymphoblastic leukemia (B-ALL) and 4 cases of acute myeloid leukemia (AML); 5 of these 11 leukemia cases had known CRGRs as below. The remaining 6 leukemia cases had also undergone clinical genetic profiling via karyotyping, FISH, microarray, and sequencing yet a clinical grade genomic driver was not identified. DNA from each sample was sequenced on a Revio instrument (PacBio, Menlo Park, CA) to a target depth of 30x. The PacBio Human Whole Genome Sequencing (WGS) workflow was used to process HiFi reads for haplotagged alignment and phasing, followed by somatic structural variant calling using Severus. Subsequent analysis focused on the presence of break ends in known cancer-relevant genes. We found that HiFi-GS detected the known CRGRs in all 5 cases with prior findings, providing precise break ends and clarifying sometimes unclear cytogenetic observations (such as exact partner genes), in these cases. These included detection of an ETV6-RUNX1 fusion, NUP98-NSD1 fusion, KMT2A-AFF1 fusion, KMT2A-AFDN fusion, and RUNX1-RUNX1T1 fusion. HiFi sequencing also detected CRGRs in 2 of the 6 “undiagnosed cases”; namely, one case of ZNF384-TCF3 fusion and one case of KMT2A-MLLT10 fusion. The former can be cytogenetically cryptic, while the latter can often be missed due to its potentially complex nature. Our results demonstrate that HiFi-GS can reliably detect CRGRs, bringing us closer to the promise of a single comprehensive genetic test for cancer characterization. Additional work examining other somatic structural and copy number variants (i.e., inversions, deletions, and duplications) in these cases, as well as oncogenic sequence variants, is ongoing. Citation Format: Byunggil Yoo, Ayse Keskus, Chengpeng Bi, Lisa Lansdon, Tanveer Ahmad, Irina Pushel, Adam Walter, Margaret Gibson, Erin Guest, Tomi Pastinen, Mikhail Kolmogorov, Midhat S. Farooqi. Long-read sequencing of pediatric leukemia identifies clinically relevant genomic rearrangements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1763.

Differentiated pattern of complement system activation between MOG-IgG-associated disease and AQP4-IgG-positive neuromyelitis optica spectrum disorder.

Myelin oligodendrocyte glycoprotein antibody (MOG) immunoglobulin G (IgG)-associated disease (MOGAD) has clinical and pathophysiological features that are similar to but distinct from those of aquaporin-4 antibody (AQP4-IgG)-positive neuromyelitis optica spectrum disorders (AQP4-NMOSD). MOG-IgG and AQP4-IgG, mostly of the IgG1 subtype, can both activate the complement system. Therefore, we investigated whether the levels of serum complement components, regulators, and activation products differ between MOGAD and AQP4-NMOSD, and if complement analytes can be utilized to differentiate between these diseases. The sera of patients with MOGAD (from during an attack and remission; N=19 and N=9, respectively) and AQP4-NMOSD (N=35 and N=17), and healthy controls (N=38) were analyzed for C1q-binding circulating immune complex (CIC-C1q), C1 inhibitor (C1-INH), factor H (FH), C3, iC3b, and soluble terminal complement complex (sC5b-9). In attack samples, the levels of C1-INH, FH, and iC3b were higher in the MOGAD group than in the NMOSD group (all, p<0.001), while the level of sC5b-9 was increased only in the NMOSD group. In MOGAD, there were no differences in the concentrations of complement analytes based on disease status. However, within AQP4-NMOSD, remission samples indicated a higher C1-INH level than attack samples (p=0.003). Notably, AQP4-NMOSD patients on medications during attack showed lower levels of iC3b (p<0.001) and higher levels of C3 (p=0.008), C1-INH (p=0.004), and sC5b-9 (p<0.001) compared to those not on medication. Among patients not on medication at the time of attack sampling, serum MOG-IgG cell-based assay (CBA) score had a positive correlation with iC3b and C1-INH levels (rho=0.764 and p=0.010, and rho=0.629 and p=0.049, respectively), and AQP4-IgG CBA score had a positive correlation with C1-INH level (rho=0.836, p=0.003). This study indicates a higher prominence of complement pathway activation and subsequent C3 degradation in MOGAD compared to AQP4-NMOSD. On the other hand, the production of terminal complement complexes (TCC) was found to be more substantial in AQP4-NMOSD than in MOGAD. These findings suggest a strong regulation of the complement system, implying its potential involvement in the pathogenesis of MOGAD through mechanisms that extend beyond TCC formation.

Peak ADAMTS13 activity to assess ADAMTS13 conformation and risk of relapse in immune-mediated thrombotic thrombocytopenic purpura

AbstractPrevious studies have demonstrated that >38% of patients with immune-mediated thrombotic thrombocytopenic purpura in remission with activity >50% had an open ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) conformation. We assessed ADAMTS13 conformation in remission (ADAMTS13 activity >60%), focusing on peak ADAMTS13 activity levels and longitudinal assessment in 420 samples across 157 patients. Fewer cases had an open conformation at peak ADAMTS13 activity than unselected remission samples with ADAMTS13 activity >60% (23% vs 43%). Patients with a closed ADAMTS13 conformation at peak ADAMTS13 activity had an eightfold lower relapse rate in the subsequent year (9% vs 46%) and a fivefold lower relapse rate within 2 years (23% vs 62%) compared with cases with an open conformation. Patients with an open conformation at peak ADAMTS13 activity required preemptive anti-CD20 treatment earlier than those with a closed conformation (median, 10 vs 25 months). Longitudinally, an open conformation was evident at, and often preceded relapse. When the conformation was already open before relapse, an increase in the conformation index at relapse was seen despite the undetectable anti-ADAMTS13 immunoglobulin G (IgG) antibody. In cases with detectable anti-ADAMTS13 IgG antibody, these became undetectable before achieving a closed conformation, highlighting the relapse risk even with undetectable anti-ADAMTS13 IgG antibody and the clinical utility of open/closed during monitoring. To our knowledge, this is the first study to show an association between relapse risk and ADAMTS13 conformation when activity levels are at a peak. The open conformation identifies antibody-mediated subclinical disease that is not detectable by the current ADAMTS13 testing.

Short-Chain Fatty Acid Production by Gut Microbiota Predicts Treatment Response in Multiple Myeloma.

The gut microbiota plays important roles in health and disease. We questioned whether the gut microbiota and related metabolites are altered in monoclonal gammopathies and evaluated their potential role in multiple myeloma and its response to treatment. We used 16S rRNA sequencing to characterize and compare the gut microbiota of patients with monoclonal gammopathy of undetermined significance (n = 11), smoldering multiple myeloma (n = 9), newly diagnosed multiple myeloma (n = 11), relapsed/refractory multiple myeloma (n = 6), or with complete remission (n = 9). Short-chain fatty acids (SCFA) were quantified in serum and tested in cell lines. Relevant metabolites were validated in a second cohort of 62 patients. Significant differences in alpha- and beta diversity were present across the groups and both were lower in patients with relapse/refractory disease and higher in patients with complete remission after treatment. Differences were found in the abundance of several microbiota taxa across disease progression and in response to treatment. Bacteria involved in SCFA production, including Prevotella, Blautia, Weissella, and Agathobacter, were more represented in the premalignant or complete remission samples, and patients with higher levels of Agathobacter showed better overall survival. Serum levels of butyrate and propionate decreased across disease progression and butyrate was positively associated with a better response. Both metabolites had antiproliferative effects in multiple myeloma cell lines. We demonstrate that SCFAs metabolites and the gut microbiota associated with their production might have beneficial effects in disease evolution and response to treatment, underscoring its therapeutic potential and value as a predictor.

Allele-Specific Expression of Leukemia Genes Is Associated with Pathogenicity in Poor Risk AML

Introduction: An imbalance in gene expression of the different alleles of a gene (allele-specific expression or ASE) is an intriguing genome-wide and dynamic phenomenon that could have an important role in tumorigenesis. Little is known about the causes and consequences of the differential cis- and trans-regulation on resultant ASE, aside from the impact of copy number alterations (CNAs) or imprinting. Acute myeloid leukemia (AML) is an aggressive clonal hematopoietic stem cell malignancy, with a low mutational burden in comparison with other cancers, where the impact of ASE may constitute a novel mechanism of pathogenesis. Aim: Application of Whole Genome Sequencing (WGS), RNA-seq and Oxford Nanopore methylation sequencing to study the ASE landscape in a cohort of poor risk AML patients and shed light on the mechanisms triggering this phenomenon as well as its temporal dynamics. Methods: ASE landscape was explored using paired germline and tumour WGS and RNA-seq on diagnostic bone marrow or peripheral blood samples from 33 cytogenetically poor risk AML patients, and additional RNA-seq from 18 samples corresponding to follow-up timepoints (including remission, relapse and refractory samples) from eight of the patients.Heterozygous SNPs were identified in the genomic DNA, and allele frequencies in RNA-seq reads were used to quantify ASE, after filtering for CNAs in the tumor DNA. Cas9-enriched Oxford Nanopore sequencing was then performed on available patient samples at diagnosis and subsequent time points. Allele-specific methylation was determined on the Nanopore sequencing using Guppy v6.3. Results: In the diagnostic cohort, we identified 4,802 protein-coding genes exhibiting ASE in at least one sample, with a median of 169 genes per sample (range 119-468, with one outlier sample showing ASE in 1,705 genes). Overall, 283 protein-coding genes showed ASE in ≥ 20% patients across the 33 samples. These genes included known imprinted genes and the transcription factor GATA2, where ASE was previously associated with AML, validating our ASE pipeline and filtering criteria. We also identified for the first time recurrent ASE in other myeloid genes such as KMT2A (20%), the homeobox genes PBX2 (41%), PBX3 (20%), HOXA6 (20%) and HOXB cluster ( HOXB2 (31%), HOXB3 (27%), HOXB5 (40%), HOXB6 (30%)); and the myeloid differentiation genes CDKN2A (40%), PDGFRB (25%) and PRTN3 (57%). Eighteen primary AML samples from 8/33 patients had paired diagnostic, remission and relapse or refractory material, allowing us to track the dynamic ASE pattern. RNA-seq and Sanger sequencing of cDNA in these samples showed that the ASE observed at diagnosis in HOXB2, HOXB3, HOXB6, PBX3 and GATA2 was lost in the corresponding remission sample (bi-allelic expression), with ASE returning at relapse and maintained in the refractory samples. In order to explore the epigenetic mechanisms underlying these allele-specific changes in expression, Cas9-enriched Oxford Nanopore sequencing was performed in 2 patients with available DNA at diagnosis and sequential time points, to determine the pattern of allele-specific methylation of the promoter region of GATA2 (patient 1) and HOXB2 (patient 2). In patient 1 at diagnosis, 90% of CpG sites were methylated at the GATA2 promoter region on one allele versus 61% in the other allele, with the hypomethylated allele corresponding to the allele that is overexpressed in the ASE sample. However, in the remission sample, the proportion of methylated CpG sites on the two alleles was similar (83% and 73%, respectively), in agreement with the expression of both GATA2 alleles at this stage. Likewise, in the diagnostic sample of patient 2, 6% of CpG sites were methylated in the HOXB2 promoter region of one allele, corresponding to the overexpressed allele in the ASE sample, while the other promoter allele had 17% of CpG sites methylated; with no differences in CpG methylation between alleles in the remission samples (9% versus 9%), again correlating with the bi-allelic expression of HOXB2 at remission. Conclusions: Combining WGS and RNA-seq in AML primary samples, we have identified key leukemia genes showing recurrent dynamic ASE associated with disease evolution: since it is found at diagnosis, lost in remission, but recovered in relapse and persisting in refractory stages. Our results also suggest allele-specific methylation as the potential mechanism orchestrating this phenomenon.

High Dimensional Clustering and Identification of Immune Cell Hybrid Populations in B-ALL across the Disease Continuum

Introduction: Nearly half of adults with acute lymphoblastic leukemia (ALL) will experience relapse of their disease, and those with relapsed ALL carry a worse prognosis than those who remain in remission. The influence of the tumor immune microenvironment (TME) on relapse and survival outcomes in ALL is not well understood. Mass cytometry (CyTOF) enables immune profiling based on single cell protein expression and can identify phenotypic diversity of cells in the TME that cannot reliably be detected by standard flow cytometry. Recently, T-cell/monocyte hybrid cells have been discovered in patients with uveal melanoma and latent tuberculosis infections. However, cells expressing CD19, a surface antigen expressed on B-ALL cells, and markers of other cells in the TME including T cells and tumor-associated macrophages (TAMs), have not been described in ALL or other hematologic malignancies. The presence of CD3/CD163 hybrid cells in the TME may have predictive and prognostic implications for adults with ALL, as TAMs play a role in suppressing antitumor immunity which can lead to T-cell exhaustion. In this study, we identify the presence of multilineage hybrid cells in peripheral blood (PB) and bone marrow (BM) samples of patients with B-ALL alongside accompanying evolution of the TME at each disease stage. Methods: PB (N=16) and BM aspirate (N=16) isolates were collected from patients with B-ALL at diagnosis, relapse, or remission, as well as from healthy donors (HDs, N=7) for comparison of cellular profiles. A 28-marker CYTOF panel was designed to evaluate cell surface and intracellular markers of interest in the myeloid compartment. Antibodies were purchased pre-conjugated or conjugated in house based on manufacturer guidelines (Standard BioTools). Samples were barcoded with palladium isotopes prior to staining to reduce the impact of batch effect, and were then analyzed on a first generation CyTOF system (Standard BioTools). Following background attenuation and singlet gating in FlowJo (BD), the CATALYST R package enabled deconvolution and visualization of single cell data, including dimensional reduction techniques (tSNE) and clustering of all cell populations. An iterative approach was taken to determine a stable configuration of clusters across sample populations to protect from over-fragmentation of data, further informed by K-means clustering using the elbow method. Heatmaps were then generated based on median scaled expression of markers contained in each cluster. Results: The CyTOF panel identified several different myeloid cell populations in newly diagnosed, relapsed, and remission samples in both PB and BM compared to HDs. Most interestingly, quantifiable levels of a cluster of cells co-expressing CD19, CD3 and CD163 as well as a second cluster of cells expressing CD19 and CD163 were detected in newly diagnosed and relapsed patients, but not in HDs. A single patient with the largest proportion of these cell populations in their PB at the time of relapse presented with an aggressive leukemia variant and relapsed multiple times prior to succumbing to illness. On investigation of BM aspirates, the triple positive hybrid population was found in a similar distribution amongst the newly diagnosed and relapsed patients, as well as affecting one patient who achieved remission. This patient also had a highly aggressive leukemia that was refractory to chemotherapy and immunotherapy, and only achieved this remission following CAR-T cell therapy. She subsequently underwent stem cell transplant and is in remission. Conclusions: Our findings add to the literature highlighting the utility of high parameter single cell capture techniques in detecting rare cell populations in the leukemic TME. Further, we identified two unique hybrid cell populations that are reliably detected across different disease stages in patients with B-ALL. Importantly, these populations persisted following stringent gating protocols to rule out doublets and debris. Although our sample size was too small to demonstrate a prognostic impact of these hybrid cell populations in patients with B-ALL, the identification of these populations alone is a significant finding. Further investigation and patient sample accrual are warranted to better understand the role of these cell types in tumor biology, which may pave the way towards new prognostic models that can leverage well-established immune monitoring techniques.

Genomic Characterization of Acute Myeloid Leukemia with Aberrations of Chromosome 7: A Multinational Cohort of 523 Patients

Introduction: Aberrations of chromosome 7 [abn(7)] are found in ≈ 10% of newly diagnosed acute myeloid leukemia (AML) and associate with a dismal prognosis. A large-scale comprehensive exploration of the underlying genetic heterogeneity in AML with abn(7) has yet to be performed and could add essential insights into the outcome of this poorly understood patient group. Methods: We collected diagnostic samples from 523 adult AML patients (median age 59 years) with abn(7). Whole-exome sequencing (WES) was performed to discover potentially underestimated genetic lesions in 61 paired diagnostic / remission samples. Subsequently, a gene panel including 66 genes and a SNP backbone for copy-number aberration (CNA) detection was designed and applied to the remaining 471 samples. The majority of patients (78%) were diagnosed with de novo (dn) AML, whereas 22% had secondary (s) or therapy-related (t) AML. Intensive induction treatment was administered to 80% of the patients, while 36 % underwent allogenic stem cell transplantation. Apart from 43% of patients with concomitant complex karyotype (abn(7)/CK+), 24% had -7 as a sole abnormality (-7 sole) and 13% del(7q) sole. Results: A mean of 15.3 single-nucleotide variants (SNVs) and 7.5 CNAs per patient were found by WES. Here, the most frequent recurrent SNVs were identified in TP53 (29.5%), followed by mutations (mut) in genes involved in epigenetic regulation ( DNMT3A, TET2, ASXL1, IDH2), transcription factors ( RUNX1), and genes affecting RAS-signaling ( NF1, KRAS), Figure 1. Targeted sequencing revealed 1829 SNVs with a VAF≥ 2% in 64 genes (mean: 3.8 SNVs / patient). 30% of patients harbored at least one mutation in genes located within the commonly deleted region of 7q, most frequently KMT2C, EZH2CUX1, SAMD9L, SAMD9, LUC7L2 and BRAF. The number of driver gene mutations was higher in CK- than in CK+ patients (4.5 vs. 3.3 SNVs). We found KRAS (OR 3.76, CI 1.17-16.87, P= .044) and RUNX1 (OR 3.61, CI 1.62-8.78, P= .003) mutations to be enriched in -7 sole patients, and FLT3 mutations to be associated with del(7q) sole status (OR 0.33, CI 0.13-0.82, P= .019). With respect to previously unknown lesions, a high amount of KMT2C mutations (16.6%) and recurrent alterations in FAT1 and TACC2 were discovered (6.4% each; Fig. 1). For KMT2C - located on chr7q36.1 - a total of 98 SNVs with mainly missense (73.5%) and truncating (20.4%) mutations at known cancer hotspots were noted. KMT2Cmut was associated with dnAML and AML with maturation morphology. In the entire cohort, the most common co-occurring chromosomal alterations discovered by high-resolution CNA analysis were deletions in chromosomes 5, 17, and 12. Our approach enabled the identification of small fragment deletions (≤10Mb) affecting the TP53, NF1, and ETV6 loci in 5-9% of all cases, which were missed by conventional G-banding. Cancer Cell Fraction and Bradley-Terry models were used to simulate the sequential order of genomic aberrations. While mutations in TP53 and epigenetic-related genes were early events, -7 and del(7q) were often subclonal and SNVs in NRAS, KMT2C very late events in leukemogenesis. Survival analyses in intensively-treated patients (n= 414) revealed that 61% reached complete remission, 67% relapsed, and median overall survival (OS) was 11.9 months. Abn TP53 and high WBC count were independently associated with shorter relapse-free survival (RFS). For OS, besides older age and high WBC count, we identified abn TP53, PTPN11mut, TET2mut,-5, and -18 as poor prognostic factors in multivariate analysis (Table 1). In contrast, IDH2mutconferred an independent favorable prognostic effect for RFS and OS. Notably, abn TP53 outcompeted the prognostic impact of CK+ (Table 1). Compared to TP53wt, patients with abn TP53/CK+ and abn TP53/CK- had a similar poor outcome with median RFS of 6 and 4 months (CK+/CK-, P&amp;lt;.001) and OS of 6.8 and 8.6 months (CK+/CK-, P&amp;lt;.001). In contrast to other genomic studies in myelodysplastic syndrome, we found abn TP53 to be associated with poor outcome irrespective of the single- or multihit mutation status following definitions of the latest ICC classification (Blood, 2022). Conclusion: Our results offer novel insights into the genomic landscape and clonal trajectory of AML with abn(7). This work unravels formerly underestimated genetic lesions ( KMT2Cmut) and alterations with high prognostic impact (abn TP53 and IDH2mut) for better future risk stratification.

Mitochondrial dsRNA from B-Precursor Acute Lymphoblastic Leukemia Cells Induce Formation of Cancer Associated Fibroblast in the Bone Marrow Leading to Formation of a Chemoprotective Bone Marrow Niche

Precursor B-cell Acute Lymphoblastic Leukemia (B-ALL) is typically a highly chemosensitive malignancy with the majority of children being cured by chemotherapy alone. Adult ALL is often chemosensitive at initial treatment, but long term disease free survival is less common. It is widely accepted that outcome in ALL closely relates to underlying genetic lesion (Moorman, Hematologica 2016) but no specific mechanisms by which ALL genetics influence outcome have been identified. We have previously demonstrated that an ‘activated mesenchymal stromal cell (MSC) niche’ develops in response to reactive oxygen species (ROS) -inducing chemotherapy in which mitochondria are actively transferred along tunnelling nanotubes to ‘rescue’ B-ALL cells from chemotherapy (Burt, et al Blood 2019). According to recent evidence that chemotherapy resistant ALL sub-clones may exist at diagnosis (Dobson et al Cancer Discovery 2020), we hypothesised that B-ALL cells may themselves directly activate bone marrow stromal cells to generate cancer associated fibroblasts (CAF) from MSC. To test this hypothesis, we first used our previously described in vitro niche model wherein either the HS27a MSC cell line or healthy donor (HD) MSCs are co-cultured with either B-ALL cell lines or primary patient B-ALL cells. Three of five B-ALL cell lines and 3 of 4 primary B-ALL samples tested, induced activation of the MSCs, as characterised by cytomorphological changes identified by confocal microscopy, up-regulation of CAF-relevant gene expression and increased IL6, IL8 and CCL2 protein secretion. The ability of the B-ALL cell lines to induce CAF correlated directly with the intrinsic ROS levels of the cell as determined by CellROX assay. In vivo, the CAF-inducing ALL cell lines also activated murine MSCs when established as xenografts in NOD-SCID gamma (NSG) mice. Immunohistochemistry of NSG murine femora revealed a very significant expansion in nestin+ cells after exposure to CAF-induing ALL cells compared to non-CAF-inducing controls. We established that the ALL cells activated MSC without contact, both from exposure of MSC to ALL cells in transwell and from exposure to B-ALL cell conditioned media (CM). RNA sequencing of HS27a stromal cells exposed to CAF-inducing B-ALL cell CM compared with non-CAF inducing controls unexpectedly showed that the most highly upregulated pathways were those involved in RNA sensing. Using a smaller gene panel selected from among the RNA-sensing pathway genes, we confirmed that this was also the case for primary, HD MSCs upon exposure to primary ALL patient samples. As a result, we sought evidence for the role of mitochondrial dsRNA, which has been shown to activate the anti-viral signalling pathway (Dhir et al, Nature 2018). Using the J2 anti-dsRNA monoclonal antibody in both confocal and flow cytometric assays, we observed very significantly higher levels of dsRNA in MSC which had been exposed to CAF inducing B-ALL cell CM than in non-CAF inducing B-ALL or MSC alone controls. We confirmed by RT-PCR for mitochondrial genes on dsRNA immunoprecipitated from CAF inducing B-ALL cell CM that the dsRNA was of mitochondrial origin. Using ELISA, we quantified dsRNA from total cell RNA extracted from paired primary B-ALL patient samples at diagnosis and in remission. We found significantly higher levels of dsRNA at diagnosis than in remission, with it being undetectable in most remission samples. Moreover, we confirmed by flow cytometry the presence of dsRNA in 6 B-ALL patient derived xenografts which was absent in the control NSG mice without leukemia. We used DMSO to degrade dsRNA from CAF inducing B-ALL cell CM, as is often done in Sanger sequencing and confirmed the degradation by ELISA. We then quantified gene expression of a selection of stromal cell activation marker genes, as well as genes in the RNA sensing pathway after exposure to either DMSO-treated B-ALL cell CM or non-treated control. There was a 2 to 4-fold reduction in expression of IL6, IL8 as well as of RIG-I, MX2, IFIH1, MAVS and OAS1 expression in DMSO-treated condition compared to control. We confirmed that IL6, IL8 and CCL2 protein secretion was also significantly abrogated in the test but not the control condition. We propose a model in which ROS-induced mitochondrial dysfunction in ALL cells leads to release of dsRNA which in turn activates bone marrow stromal cells leading to protective niche formation, despite lack of prior exposure to cytotoxic agents.

BAX Mutated Clonal Hematopoiesis Arises Following Treatment with the BCL2 Inhibitor Class of Therapeutics across a Range of Hematological and Non-Hematological Neoplasms

Clonal hematopoiesis (CH) is an age-related phenomenon characterized by the expansion of genomically mutated hematopoietic clones with a selective fitness advantage. Venetoclax, a selective BCL2 inhibitor, has established efficacy in hematological malignancies such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Our previous studies described the emergence of deleterious BAX variants within the myeloid compartment of patients (pts) receiving venetoclax for CLL (Blombery, Blood2022). Whether this phenomenon is specific to venetoclax and the CLL disease context is currently unknown. We therefore aimed to investigate the prevalence and pattern of BAX-mutatedCH across different hematological and non-hematological neoplasms in response to BCL2 inhibitor therapy (both venetoclax and BGB-11417). We first examined pts with AML in complete morphologic remission after venetoclax exposure in both the newly diagnosed (CAVEAT; Chua, JCO 2020) and early relapse (VALDAC; Tiong, ASH 2022) settings using unique molecular index-based error-corrected next generation sequencing with a sensitivity of 0.5% (QIAseq targeted DNA panel). BAX variants were detected in remission samples in 11/20 (55%) and 9/26 (35%) pts in these cohorts, respectively, after a median of 9- and 4-months post venetoclax. This represented a significant higher incidence compared to remission samples from pts with newly diagnosed AML post conventional cytotoxic chemotherapy without venetoclax (6/87 [7%]; adjusted p&amp;lt;0.01 for both pairwise comparisons). Having observed the increased frequency of BAX-mutated CH in remission in myeloid (AML) and lymphoid (CLL) neoplasms, we then investigated whether BAX variants may be acquired in response to BCL2 inhibitor therapy outside the context of hematological neoplasm. We assessed a cohort of pts with metastatic breast cancer who received venetoclax in combination with tamoxifen (mBEP; Lok, Cancer Discover 2019), and detected BAX variants from peripheral blood samples in 5/16 (31%) pts (median age 66) after a median duration of 20 (range 13-44) months on venetoclax. In contrast, no BAX variants were detected in a similarly aged control cohort of 50 pts (median age 62) with breast cancer without exposure to venetoclax (p=0.01). The rates of any CH-related mutation were 75% and 50% pts, respectively (p=0.09). No pt developed MDS or AML during follow up in the mBEP cohort. Finally, to explore whether emergence of BAX-mutated CH is specific to venetoclax as an agent, we studied a cohort of 9 pts with CLL who had received BGB-11417, a more potent and selective BCL2 inhibitor; median treatment duration 13 months. Notably, we observed the emergence of BAX variants in 2/9 (22%) pts in this cohort. The characteristics of the BAX variants (n=88) in 28 pts (AML, CLL or breast cancer) are summarized in Figure 1; 3 variants (in 3 pts) were detectable in pre-treatment samples. Over half of the missense (n=22) or in-frame variants (n=1) were clustered in the C-terminus of the BAX protein, while truncating variants (n=65) were observed throughout the gene. The median number of BAX variants per pt was 2, with 32% of pts having ≥3 variants (max=13). The median VAF was 1.4%, and only 16% of variants had a VAF &amp;gt;5%. The number and VAF of BAX variants did not significantly correlate with the duration of therapy. Among 12 pts with AML who had serial testing (median inter-test interval 10 months), BAX variants (n=24) did not significantly change in VAF (median Δ 0.7%) with time after initial acquisition (Pearson's coefficient 0.17). To clarify the compartments affected by BAX variants we performed single-cell combined DNA/protein sequencing (Tapestri) of a sample with multiple BAX variants and demonstrated their presence in multiple independent clones with myeloid-biased lineage but also within the natural killer cell compartment ( Figure 2). In summary, the emergence of BAX-mutated CH appears to be a generalized phenomenon of adaptive hematopoiesis that occurs across both hematological and non-hematological malignancy settings as well being observed with other BCL2 inhibitors consistent with a therapeutic class effect. Further research is needed to understand the contribution of this phenomenon to the subsequent development of clinically relevant hematological abnormalities and its implications for future therapeutic resistance.

A Novel and Cost-Efficient Measurable Residual Disease (MRD) Detection Assay Using a Single Molecule Molecular Inversion Probe-Based Next Generation Sequencing (NGS) Assay Predicts Overall Survival in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Introduction Relapse remains the main reason for poor outcomes of patients (pts) with acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the preferred post-remission treatment in pts at high risk of relapse. The presence of measurable residual disease (MRD) after intensive frontline treatment or before or after HSCT has emerged as an important predictor of relapse. Various methods are used for MRD detection, including flow cytometry and molecular assays detecting individual gene mutations (mut) such as NPM1mut. Next generation sequencing (NGS)-based assays covering multiple genes commonly mutated in AML may allow sensitive MRD detection in a broad range of pts with diverse genetic characteristics. Methods We established a targeted, error corrected NGS MRD assay utilizing single-molecule molecular inversion probes (smMIPs). The smMIP protocol uses a hybridization-capture approach to selectively enrich and amplify both DNA strands of 92 regions in 32 genes in a single reaction. Unique molecular identifiers (UMIs) facilitate computational correction of sequencing errors to enable reliable detection of variants ≥0.5% variant allele frequency (VAF), with the additional advantage of relatively low reagent and sequencing costs of &amp;lt; $100 per sample. The smMIP assay was used to evaluate MRD status on pre-HSCT bone marrow samples from 98 AML pts who received an HSCT in first (n=83) or second (n=15) complete remission with or without count recovery. Median age at HSCT was 59 (range 20-75) years, 42% were female; 70% had de novo AML, 19% had an antecedent myeloid neoplasm and 11% had AML post cytotoxic therapy. European LeukemiaNet (ELN)-2022 risk groups at diagnosis were favorable in 11%, intermediate in 33%, adverse in 20% and unknown in 22%. All pts had received intensive induction chemotherapy. Twenty-nine percent received a myeloablative conditioning regimen (MAC), while 23% received reduced intensity conditioning (RIC) and 48% a non-myeloablative (NMA) conditioning regimen. Donors were matched related (18%), haploidentical (6%), or matched (55%) or mismatched (21%) unrelated. The median follow-up for pts alive was 34.8 months. Results Overall, we detected 190 variants in 71 (72%) of 98 pre-HSCT remission samples. The most commonly affected genes were DNMT3A (in 37% of pts), TET2 (21%), PPM1D (12%), IDH2 (10%) and TP53 (5%). Twenty-nine percent of pts had only variants affecting the clonal hematopoiesis-related genes DNMT3A, TET2 or ASXL1 (‘DTA‘), while 44% of patients had ≥1 variant involving a non-DTA gene. MRD-positive pts, defined as those with ≥1 non-DTA variant, had similar baseline and HSCT-related characteristics (includingage, sex, ELN risk group, de novo vs. secondary AML) compared to pts without non-DTA variants. Overall survival (OS) was significantly inferior in NGS MRD-positive pts compared to those with no non-DTA variant (Figure A, 5-year OS, 49% vs. 85%, P=.011). MRD-positive pts also had a non-significant higher cumulative incidence of relapse at 5 years (46% vs. 24%, P=.14), while non-relapse mortality was similar in both groups (P=.72). We next performed multivariable analyses, considering variables associated with OS at an univariable P&amp;lt;.10 (MRD status, patient sex, therapy-related AML, conditioning intensity), and using stepwise backward variable selection to identify factors significantly associated with OS (Figure B). In the final multivariable model, pre-HSCT MRD detection remained associated with inferior OS (hazard ratio 2.51; 95% confidence interval, 1.07 - 5.91; P=.035). Female sex associated with favorable OS, while myeloablative conditioning (MAC) showed a borderline significant association with superior OS. Of note, we did not detect an interaction between conditioning intensity and detection of MRD with regard to OS, i.e. the prognostic association of MRD detection was not different between pts receiving myeloablative or less-intensive conditioning. However, this analysis was limited by the small patient number. Conclusion Detection of MRD by targeted NGS using the smMIPs approach provides prognostic information in pts undergoing allogeneic HSCT, including those without established molecular markers such as NPM1mut. This method is applicable to a large proportion of AML pts and is cost-efficient for broad clinical use. S.M.K. and T.H., and M.J. and K.H.M. contributed equally to this abstract.

Personalized Residual Disease Detection in Acute Myeloid Leukemia with a Multiplexed Genomic Fingerprint Predicts Patient Outcomes

Background: Acute myeloid leukemia (AML) is the most common leukemia in adults. While therapeutic options for AML have continued to improve, nearly half of all patients who achieve clinical remission will relapse within several years after diagnosis. Risk factors for relapse include age, cytogenetics, specific gene mutations, and the detection of residual disease after treatment. Current molecular approaches for measuring residual disease are designed primarily around a single gene. This limits both the sensitivity and the applicability of the assay to all patients, as AML is a highly heterogenous genetic disease. In this study, we demonstrate the utility of a comprehensive, personalized, multiplexed genomic assay that exhibits high sensitivity, excellent quantitation, and promising performance across tissue types. This approach enables the serial, non-invasive monitoring of patients with AML and the prediction of patient outcome. Methods: Serial samples of bone marrow, peripheral blood, and urine were collected at multiple time points from 12 patients with AML who were treated with standard-of-care therapy. Time points range from presentation to relapse, with a median of 4 time points per patient (range of 2-10). A median of 7 samples were collected per patient (range of 2 to 16). DNA was extracted from the peripheral blood mononuclear cells (PBMCs), the cell-free fraction of peripheral blood, bone marrow mononuclear cells, and the cell-free fraction of urine. Deep whole genome sequencing of paired presentation and remission samples identified hundreds of candidate leukemia-associated somatic single-nucleotide variants comprising the personalized genomic fingerprint of each patient's disease. A representative set of variants (n=30-50) was selected for each patient, and a personalized set of primers was designed for the Multiplex Accurate Sensitive Quantification (MASQ) next-generation sequencing protocol. The sensitivity of this assay allows for single variants to be quantified down to at least 1 in 100,000, resulting in an overall sensitivity below 1 in 1 million for residual disease detection. Quantification of the individual variants from the fingerprint was evaluated in samples of all tissue types collected (bone marrow, blood cells, blood cell-free DNA, and urine cell-free DNA) and all time points collected (presentation, post-induction, post-consolidation, and follow-up visits). Results: Serial monitoring of the leukemia genomic fingerprint enables risk assessment after each stage of treatment. Using Multiplex Accurate Sensitive Quantification (MASQ), we detected leukemia-associated variants in the blood and bone marrow of all patients during remission, at levels ranging from less than 10^-6 to 10^-2. We find consistent low signal, less than 10^-4, in both bone marrow and blood in patients with good prognosis. In contrast, we find high signal in remission, greater than 10^-3, in patients with poor prognosis. In the analysis of serial samples, frequencies drop over time with additional treatment and increase preceding clinical identification of relapse. Figure 1A shows the trajectory of 50 variants over 5 time points in PBMCs, preceding relapse. The overall signal increases at 7 months prior to clinical relapse and again at 5 months prior to clinical relapse. Figure 1B shows a comparison of the aggregate estimates of the leukemia genomic fingerprint in the cellular and cell-free components of the blood. While total DNA yields from the cell-free fraction of blood are lower than from PBMCs, we find that the frequency of the leukemia genomic fingerprint in the two sources of DNA is highly similar. Bone marrow and peripheral blood also show highly similar measurements of residual disease, suggesting that non-invasive monitoring with only a blood draw is a promising approach. Signal of the leukemia fingerprint is also detectable in urine, but at reduced levels and much more variable across patients. Conclusion: Our study shows the capability and utility of a personalized genomic residual disease detection method using MASQ to monitor patients and predict patient outcome. The high sensitivity and accurate quantitation of MASQ are demonstrated in a case study where relapse could be detected molecularly 7 months prior to clinical identification. Collection and analysis of a larger case series is ongoing.

Epigenetic Classification of Acute Myeloid Leukemia Revealed By Genome-Wide Chromatin Profiling

Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease and is predominantly defined by genetic abnormalities and morphology. However, many lines of evidence suggest the role of epigenetic alterations in the pathogenesis and the classification of AML, whereas no large-scale comprehensive analyses of both genetic and epigenetic alterations in AML have been reported. Methods We enrolled a total of 1,564 primary AML cases (524 from Japanese and 1,040 from Swedish cohorts), whose diagnostic samples were subjected to ATAC sequencing together with targeted capture sequencing using an in-house gene panel including 331 known AML driver genes and an additional 1,158-1,317 probes for copy number detection. We also performed RNA (N=1,398) and H3K27ac-targeted ChIP sequencing (N=80). Normal bone marrow samples from 25 donors as well as 15 remission samples were analyzed by ATAC-seq as a control. Results ATAC-seq revealed approximately 300,000 reproducible peaks in primary AML. Most peaks were found in gene-distal regions, and explained a large variance across samples. Among all peaks, 72% were not detected in control samples and thus, specific to AML. Analysis of the correlations between ATAC-seq accessibility and gene expression revealed a subset of regulatory elements that regulate nearby gene expression; each peak was linked to a median of 1 gene (0-31), while each gene was associated with a median of 11 peaks (0-110). We next estimated the cellular composition of the bulk AML samples by deconvolution analysis using ATAC-seq data. The predominance of hematopoietic stem cell (HSC), monocyte, and erythroid signatures was associated with French-American-British (FAB) subtypes of minimal differentiation/without maturation subtypes (M0/M1), myelomonocytic/monoblastic/monocytic subtypes (M4/M5), and erythroid subtype (M6), respectively, suggesting bulk ATAC-seq of AML samples reflected the differentiation potential of leukemic cells. The ATAC-based clustering identified 11 distinct epigenetic subgroups, including three well-known genetic classes defined by gene fusions, such as PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11. By contrast, some genetic classes defined by a single mutation, such as NPM1 or CEBPA mutation, were further classified into multiple distinct epigenetic subgroups. For example, the NPM1-mutated subtype was separated into four subgroups based on the varying degrees of ATAC signatures associated with HSC and monocytes. Each subgroup of NPM1-mutated AML showed different clinical characteristics such as white blood cell counts, age, and prognosis, and co-mutation pattern in FLT3, DNMT3A, and IDH1/ IDH2 mutations. Gene ontology analysis revealed that the subgroup of NPM1-mutated AML with high HSC signature showed upregulated TGF-β signaling, while one with monocytic signature showed upregulated TNF-α signaling and interferon-γ response. Next, we evaluated transcription factor (TF) binding sites in a genome-wide manner, using TF motifs and foot printing in upregulated ATAC-peaks in each subgroup and revealed distinct profiles of activated TFs in each epigenetic subgroup of NPM1-mutated AML. In the subgroup characterized by high HSC signature, HSC-related TFs that belong to MECOM, HOX, and RUNX families, were more active. By contrast, other TFs, such as IRF, CEBP, and JUN/FOS families, were more enriched in the subgroup showing high monocyte signature. By developing a prediction model for these subgroups of NPM1-mutated AML based on the gene expression profile, we reproduced these subgroups and validated clinical and molecular features in an external Beat AML cohort. Utilizing ex vivo drug sensitivity dataset in the Beat AML, we identified candidate drugs showing subgroup-specific effectiveness such as panobinostat for NPM1-mutated AML with monocytic differentiation. Finally, ChIP-seq analysis identified a number of super-enhancers in the vicinity of leukemia-related TFs and oncogenes. Conclusion Through comprehensive profiling of chromatin accessibility and gene mutations/expression in a large cohort of AML patients, we demonstrate the epigenetic heterogeneity and identify novel AML subgroups that had unique epigenetic and clinical features. Our findings highlight the role of epigenetic alterations in AML pathogenesis and impact on molecular classification of AML.

Measurable Residual IDH2 before Allogeneic Transplant for Acute Myeloid Leukemia

Introduction: Detection of measurable residual disease (MRD) in AML patients during remission is associated with adverse clinical outcomes after allogeneic hematopoietic cell transplant (alloHCT), including recent strong evidence from our group regarding residual NPM1 and/or FLT3-ITD variants (PMID: 36881031). Large studies examining the significance of other potential MRD targets, including isocitrate dehydrogenase isoform 2 ( IDH2), are needed. IDH2, which is mutated in approximately 12% of AML patients, is of particular interest due to the possibility of targeted clinical intervention. Methods: Patients over the age of 18 in first complete remission (CR1) with IDH2 mutated AML undergoing alloHCT transplant at a CIBMTR site between 2013-2019 with remission blood samples within 100 days of alloHCT and available clinical data were included in this study. Residual variants in IDH2, NPM1, and FLT3genes were detected by ultrasensitive error-corrected next-generation sequencing (NGS-MRD) on genomic DNA from pre-conditioning blood and validated by digital droplet PCR (ddPCR). The primary outcomes were overall survival (OS) and cumulative incidence of relapse with non-relapse mortality as a competing risk. The day of transplant was considered as time 0, and the median follow-up time was calculated for censored patients. Kaplan-Meier estimation (log-rank tests), Cox proportional hazards models, and Fine-Gray regression models were fitted to examine the clinical outcomes. Results: Among the 257 IDH2-mutated AML patients in this study, 57 (23%) experienced relapse, with 41 events occurring within 12 months after alloHCT. The median follow-up time was 25 months. Among 247 patients with pre-transplant remission flow cytometry results reported, 25% were positive, but no difference in rates of relapse or OS was observed between positive and negative patients. NGS MRD analysis detected residual IDH2 variants in remission samples of 130 patients (51%) with variant allele fraction (VAF) ranging from 0.05-56% (median: 3%), and 100% validation by ddPCR. NGS MRD IDH2 positive patients had increased rates of relapse (p = 0.03; 3yr: +11%, 95% CI: 0.2% to 22%) and decreased OS (p &amp;lt; 0.001; 3yr: -25%, 95% CI: -13% to -38%) compared to those who tested negative (Figure 1A). These OS differences were still observed between NGS MRD IDH2 positive and negative patients, regardless of age stratification (under 60yr p &amp;lt; 0.001, 3yr: -28%, 95% CI: -12% to -43%; over 60yr p = 0.04, 3yr: -19%, 95% CI: -1% to -40%); however, only patients over 60yrs had increased rates of relapse (p = 0.007; 3yr: +21, 95% CI: 6% to 36%). The impact of co-mutation status was examined by subdividing the cohort based on the presence of NPM1 or FLT3-ITD mutations at baseline. Residual IDH2 variants in patients without baseline NPM1 or FLT3-ITD co-mutations (n = 151) were associated with decreased OS and increased relapse ( IDH2+ vs IDH2-; OS p = 0.005, 3yr: -23%, 95% CI: -6% to -40%; Relapse p = 0.01, 3yr: +19, 95% CI: 6% to 32%; Figure 1B). In contrast, residual IDH2 variants were not predictive of relapse in co-mutated patients (n=106, p = 0.5). In these patients, the only prognostic marker for relapse was NPM1 and/or FLT3-ITD MRD, with similar relapse risk between IDH2 only positive and MRD negative groups (p &amp;lt; 0.001; 3yr: 83% vs. 11% vs. 15%; Figure 1B). In multivariable analysis, IDH2 MRD positivity was associated with decreased OS (HR 2.5, 95% CI: 1.5-4.2, p &amp;lt; 0.001) and increased rates of relapse (HR 2.1, 95% CI: 1.4-5.7, p = 0.003) after adjusting for other characteristics. Conclusion: In this study, we demonstrate that adult patients with IDH2-mutated AML who have IDH2 variants detected in CR1 prior to first alloHCT experienced increased relapse rates and decreased overall survival. However, this association is significant only in patients without baseline NPM1 and FLT3-ITD mutations. In co-mutated patients, NPM1 and/or FLT3-ITD MRD serve as superior prognostic markers. Subgroup analyses show the potential influence of factors such as conditioning intensity and age on clinical outcomes. Nevertheless, larger studies with balanced subgroups and more comprehensive baseline mutational information, including IDH2 mutation subtype, are needed to better define the clinical relevance of IDH2 MRD in this context.

Modeling complement activation on human glomerular microvascular endothelial cells.

Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.

Effect of clonal hematopoiesis in remission on hematopoiesis recovery in patients with NPM1 mutated acute myeloid leukemia after chemotherapy

Objective: To investigate the effect of clonal hematopoiesis (CH) in remission on hematopoiesis recovery in patients with NPM1 mutated acute myeloid leukemia (AML) after chemotherapy. Methods: Retrospective analysis was performed on 86 patients with NPM1(mut) AML newly diagnosed and treated in the First Affiliated Hospital of Soochow University between July 2016 and June 2019. Their clinical data and NGS test results at diagnosis were analyzed. Moreover, bone marrow samples in remission were tested using Sanger sequencing. The log-rank test was used to analyze the difference in hematopoietic recovery, and Cox proportional hazard models were used to analyze the prognostic factors affecting hematopoietic recovery. Results: The median age of the 86 NPM1(mut) AML patients was 50 years (15-69 years). There were 39 males and 47 females. Forty-one patients were induced with intensity chemotherapy ("7 + 3"), whereas 45 patients were treated with low-dose cytarabine-based induction chemotherapy. At diagnosis, The most common mutations in the patients were FLT3, DNMT3A, TET2, and IDH1/IDH2 mutations. CH-associated mutations persisted in 21 patients during remission, and the mutations were DNMT3A, TET2, ASXL1, and IDH1/IDH2. The recovery time of neutrophils in patients with CH-associated mutations in remission was consistent with that in patients without CH in remission (P=0.282) but the recovery time of platelets in patients with CH in remission was significantly longer[26 (95% CI 21-32) days vs 25 (95% CI 23-26) days, P=0.032]. Furthermore, univariate analysis indicated that age, induced chemotherapy program, and CH in remission were risk factors for platelet recovery, whereas multivariate analysis indicated that induced chemotherapy program and CH in remission were independent risk factors for platelet recovery (HR=0.454, P=0.001 and HR=0.520, P=0.027, respectively) . Conclusion: CH in remission delays the hematopoietic recovery of patients with NPM1(mut) AML after chemotherapy.