Myeloid Disease Research Articles

The Easix Score Predicts Overall Survival and Late Relapse in Patients Undergoing Reduced Intensity Conditioned Allogeneic Stem Cell Transplantation for Myeloid Disease

Background Allogeneic hematopoietic stem cell transplantation (alloSCT) is an effective treatment for myeloid malignancies, however outcomes post-transplant are variable and predictive scores limited. The Endothelial Activation and Stress Index (EASIX) measured at key timepoints pre- and post-transplant has been shown to predict outcomes in alloSCT, including overall survival, non-relapse mortality and incidence of sinusoidal obstructive syndrome (SOS). It can be readily calculated using standard biomarkers (LDH (U/L) x creatinine (mg/dL)/platelets (x109/L). We sought to examine its predictive role in a homogenous population of patients. Methods We conducted a retrospective national review of 117 patients undergoing reduced-intensity alloSCT for myeloid disease including AML (n=56), MDS (n=35) and MPN (n=26). All included received fludarabine, busulfan and rabbit anti-thymocyte globulin (Flu/Bu/ATG) conditioning from 2015-2020. We calculated the EASIX score on the day prior to conditioning, on day 0, at onset of acute graft versus host disease and 1 year post transplant. Univariate analysis was conducted in the STATA statistical package. Results At the pre-conditioning time-point the mean EASIX was 3.11 (0.3-17.54). Increased scores correlated with and were reliably predictive of reduced overall survival (p=0.034) (fig. 1). Higher scores at this time-point did not predict chronic GVHD or relapse, however an increased rate of acute GVHD was seen in those with lower EASIX (p=0.012). At day 0 the mean EASIX was 15.38 (1.07-179.02), however we could not demonstrate a predictive effect on SOS as no events occurred in this cohort. Nor did we identify a predictive value at this point for GVHD risk or relapse. At onset of acute GVHD (n=67, mean 3.52, 0.44-51.43), the EASIX score was predictive of overall survival (p=0.027) but did not predict risk of chronic GVHD or relapse. At 1 year post-transplant (n=88, mean 2.09, 0.48-13.59) a higher score showed a trend towards decreased overall survival (p=0.1) but was associated with an increased risk of subsequent relapse (p=0.01)(fig. 2). Conclusions The EASIX score is an easily applicable clinical tool using real world biomarkers readily available at serial time-points in transplant recipients. When applied to our homogenously treated cohort it retains its value in predicting overall mortality at the key time points of pre-conditioning and at onset of GVHD. Adoption of this simple score may be of benefit in risk stratifying the transplant process for reduced intensity conditioned allograft recipients, particularly when combined with established prediction tools for non-relapse mortality such as the HCT-CI. In addition to this, we have shown that the EASIX score may be a useful predictive tool for late relapse in those patients who are alive at 1 year post transplant. We suspect that this effect is primarily reflective of those patients with persistent thrombocytopenia and raised LDH, both markers of potential disease activity. Importantly, the use of this score in the reduced intensity population may be particularly relevant due to the increasing age of transplant eligibility and its use as a surrogate of endothelial dysregulation and potential for end organ complications through the transplant process. To assess the underlying T cell mediated and cytokine secretome effects on endothelial dysfunction, an observational study is ongoing at our institution, examining the interplay of immunomodulatory therapy, graft T cell content, early T and NK cell recovery and the cytokine secretion patterns at various time points before, during and after transplant in reduced intensity conditioned patients with myeloid disease. We plan to correlate these data with serial EASIX assessments to identify the determinants of the underlying biological process and identify potential time-points and biological targets for future study.

Targeting IDH1-Mutated Pre-Leukemic Hematopoietic Stem Cells in Myeloid Disease, Including CCUS and AML

Introduction: Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been reported in clonal hematopoiesis and are recurrent, early events in the development of acute myeloid leukemia (AML). A subsequent stepwise acquisition of additional genetic aberrations leads to the development of frank leukemia. At the time of AML diagnosis, rare pre-leukemic hematopoietic stem cells (pHSC) can be detected containing some, but not all, of the mutations in the corresponding leukemia, although these pHSCs are phenotypically indistinguishable from residual wildtype hematopoietic stem cells (HSC). The frequency of pHSCs at the time of diagnosis is known to correlate to overall survival in AML patients (Corces et al, Nat Genet 2016), indicating the potential relevance of therapeutically targeting these cells to prevent relapse. Methods: To model these rare pre-leukemic cells, we introduced IDH1 R132H and IDH2 R140Q mutants or their wildtype counterparts into human CD34+ hematopoietic stem and progenitor cells (HSPC) using CRISPR/Cas9 and AAV6-mediated homology directed repair (HDR). Successfully edited cells were then sorted by FACS based on GFP expression for subsequent analyses including 2HG quantification, colony formation, differentiation capacity, RNA sequencing, and metabolic assays. Additionally, primary pHSCs from diagnostic bone marrow aspirates from AML patients were also isolated by FACS and subjected to treatment, colony formation assays, and genotyping by ddPCR. Results: Human CD34+ HSPCs engineered to express either IDH1 R132H or IDH2 R140Q produced 2HG and showed a striking reduction in colony formation and differentiation capacity, recapitulating the cytopenic phenotype observed in IDH-mutated clonal cytopenia of undetermined significance (CCUS). RNA-seq of these cells revealed large transcriptomic changes shared between IDH1 R132H and IDH2 R140Q mutations, but gene set enrichment analyses revealed the specific and significant downregulation of oxidative phosphorylation-related genes in R132H-mutated cells. These R132H cells were further confirmed to have lower basal metabolic activity coupled to a higher mitochondrial dependency as determined by SCENITH (Argüello et al, Cell Metab 2021). These metabolic alterations suggested that it might be possible to selectively therapeutically target R132H mutated cells. To evaluate this, we treated HSPCs engineered to express GFP, IDH1 wildtype, or IDH1 R132H, with the known mutant-specific inhibitor ivosidenib, the OXPHOS-inhibitor IACS-010759, or DMSO as a control. Strikingly, ivosidenib restored the defect in colony formation conferred by R132H and increased the total number of colonies whereas IACS-010759 effectively inhibited R132H colony formation (Figure 1A). To further investigate this vulnerability, we sorted pHSCs from diagnostic bone marrow samples from primary IDH1 and IDH2-mutated AML samples according to the phenotype CD45dimCD3-CD19-CD20-CD34+CD38-CD99-TIM3-. These cells were subjected to treatment with DMSO, ivosidenib, enasidenib, or IACS-010759 and subsequently plated for colony formation. Because the sorted cell population contained a mixture of mutant pHSCs and residual wildtype HSCs, all colonies were picked and genotyped for IDH mutations using ddPCR, revealing that only IDH1 wildtype colonies grew out after IACS-010759 treatment whereas all other conditions resulted in a mixture of wildtype and mutant colonies (Figure 1B). Conclusions: The OXPHOS-inhibitor IACS-010759 can eradicate IDH1-mutated pHSCs whereas ivosidenib cannot. This effect is specific for IDH1 mutations and the same effect is not seen in IDH2-mutated pHSCs. These results have potential clinical implications when considering therapeutic options in AML as well as IDH1-mutated pre-leukemic conditions such as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Feasibility of Real-World Assessment of Patient-Reported Outcomes in Acute Myeloid Leukemia By Cytogenetic Risk and Treatment Status

Background Prior research has demonstrated the benefit of gathering patient-reported-outcomes (PRO's) in oncology practice for clinical outcomes, health resource utilization and quality of life. The FDA guidance on Core Patient Reported Outcomes in Cancer Clinical Trials further highlights the importance of PRO's for capturing disease, treatment, and functional impacts of oncology therapy. For patients with Acute Myeloid Leukemia (AML), disease mortality and survey compliance pose particular challenges to gathering PRO data. Methods The study cohort was derived from users of Patient Reported Outcomes mobile platform (PROmpt®), an application for remote patient reporting and provider symptom management that is employed in routine care in 30+ integrated cancer treatment centers in the United States. Patients age 18+, with confirmed AML diagnosis from March, 2021 to May, 2022 were enrolled in the platform at treatment initiation and provided with weekly surveys. Weekly assessments included symptom burden (PRO-CTCAE), function impact (PROMIS 4A), and overall quality of life (EORTC QLQ C29, C30). Clinical, biomarker, and treatment data were obtained from electronic medical record structured data. Patients were stratified by cytogenetic risk (Poor vs Favorable/Intermediate/Unknown) and treatment type (Curative Therapy vs Maintenance Therapy). Baseline demographics and characteristics were assessed. Symptom burden, functional impact and quality-of-life impact were described longitudinally Results The overall cohort (n=65) had a median age of 69 (range 20-87). Among the persons with reported frailty status (n=42), there were 11 (26%) Fit, 21 (50%) (Intermediate Fit) and 10 (24%) Frail. Across all unique patient-weeks, the most frequent symptoms reported were fatigue (19%), decreased appetite (15%), general pain (14%) and nausea (13%). From Weeks 1 to 12, the average number of symptoms reported per patient per week ranged from 1.3 to 2.7, the averages collectively decreasing over time. Patients receiving Maintenance Therapy were directionally older and less fit than patients receiving Curative Therapy, mean age 72 vs 67 years and Intermediate-Fit/Frail 72 vs 50%, respectively. Over 12 weeks of treatment, PROMIS 4A Composite Function Scores were also consistently higher among patients receiving Maintenance Therapy compared to patients receiving Curative Therapy (Figure 1). In contrast, patients with Poor Cytogenetic Risk showed similar age and fitness status compared to patients with Favorable, Intermediate or Unknown Cytogenetic Risk. However, patients with Poor Cytogenetic Risk consistently reported higher PROMIS 4A Composite Function Scores over the 12 weeks of treatment compared to those with Favorable, Intermediate, or Unknown Cytogenetic Risk (Figure 2). Conclusion This study demonstrates the feasibility of gathering PRO's with real-world data among a cohort of patients with AML using a remote symptom monitoring platform, across baseline prognostic and therapeutic factors. Although results are limited due to small sample sizes, higher levels in overall function for patients on Maintenance Therapy and those with Poor Cytogenetic Risk underscore the value of routine follow-up, which should include PRO assessment throughout the entire therapeutic process. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Early Transcriptional Activity of Vtr-297 - Results from an Ongoing Phase I Clinical Trial

VTR-297 (Trichostatin A) is a pan-inhibitor of histone deacetylases types 1, 2, 4, 6, and 10. Histone deacetylase inhibitors (HDACi) are well-known small molecules that increase the acetylation of lysine residues by inhibiting the activity of histone deacetylase enzymes. These anticancer agents affect epigenetic and non-epigenetic transcriptional regulation and are already part of effective clinical practice and hematology regimens. As HDACi are known for their relatively wide transcriptional modulation, we have conducted a high throughput screen to elucidate early transcriptional effects related to exposure to Trichostatin A (TSA) on patient-derived, clinical time-series samples. NCT03838926 is an ongoing, open-label, dose-escalation, phase I clinical trial for subjects with relapsed or refractory hematologic malignancies. Enrolled patients were assigned to receive one currently tested dose (1.25 / 2.50 / 5.00 mg; IV infusion) in 28-day cycles, 3-times a week for three weeks with one week off (IV administration days 1,3,5,8,10,12,15,17 and 19) until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint was safety/tolerability and maximum tolerated dose (MTD) of VTR-297 (TSA). Currently, twelve patients enrolled in the study, including Multiple Myeloma (MM, n=5), Non-Hodgkin's Lymphoma (NHL, n=5), and Hodgkin's Lymphoma (HL, n=2) across the first three dose-escalation cohorts that completed (1.25 / 2.50 / 5.00mg). No significant drug-related toxicities were reported, and MTD was not reached (recruitment is currently ongoing in the fourth dose cohort of 10mg). As a part of the exploratory analysis of whole blood (PBMC), patient samples were collected on cycle1 day1 (C1D1; pre-infusion, 15min-post, 30min-post, 1h-post, 2h-post, and 6h-post infusion) to capture any transcriptional changes (RNAseq) related to VTR-297 treatment. In the current analysis, samples were collected and analyzed in respective batches (15min / 30min / 1h / 2h / 6h) and compared with combined baseline samples (pre-infusion). Using Deseq2 to ascertain transcriptionally affected genes (pval<0.05, up/down relative fold-change 1.5x), we detected 136 differentially expressed genes across at least one of the time point analyzed. Among the detected genes, we found novel and previously described genes related to the core pathophysiology of myeloid and lymphoid disease (e.g., iron metabolism, vascular metastasis, and tyrosine kinase activity). In conclusion, these preliminary signatures offer early biomarker information related to a specific transcriptional activity elicited by intravenously delivered VTR-297 in the clinical setting. In an ongoing clinical study, we will further monitor, validate, and expand on these findings. Further observations at higher, safe, and clinically meaningful doses will complement our current, early picture of VTR-297 activity and how it can be used to optimize TSA-based treatments.

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dosage, Safety and Efficacy of Posaconazole As Primary Antifungal Prophylaxis in High-Risk Recipients of Allogeneic Stem Cell Transplantation

Introduction: The use of anti-mold prophylaxis with anti-aspergillus activity is recommended for patients (pts) undergoing allogeneic hematopoietic stem cell transplantation (Allo-HSCT) with high risk of fungal infection (Maertens, J Antimicrob Chemother 2018). Yet, few data are available regarding the use of posaconazole. Methods: This retrospective study included all Allo-HSCT adult recipients having benefited from posaconazole therapy in our center as a primary anti-fungal prophylaxis for high-risk of fungal infection. The latter was defined as use of i)an alternative donor, ii)sequential conditioning regimen, iii)a regimen associated with severe mucositis, iv) post-transplant cyclophosphamide (PT-Cy) or v) previous allo-HSCT. Posaconazole was programmed to be initiated at day (D) 0 or D5/6 (in case of PT-Cy) and administered up to day 100. We report here on the residual concentration of posaconazole during the neutropenic phase then on the tolerance and efficacy of such prophylaxis in this cohort. Results: Seventy high-risk pts (males 63.3%, median age 54 years old, range 20-73) received Allo-HSCT between April 2020 and December 2021 for a myeloid (n=51), lymphoid (n=17) or non-malignant hematological (n=2) disease and received posaconazole as a primary prophylaxis. The Sorror score at transplant time was ≥ 3 for 24 pts (33.8%). Twenty-two patients had already received posaconazole as a primary prophylaxis before Allo-SCT including 26 during the neutropenic phase of their induction for AML or MDS, 3 for prolonged neutropenia, 2 during treatment with alemtuzumab for T-cell leukemia/lymphoma, and 1 during a previous AlloHCST. Four patients had also received other antifungal agentsbefore Allo-SCT (caspofongine (n=1), voriconazole (n=1) or liposomal amphotericin B (n=2)) because of a suspected mold infection which has been finally excluded. All pts had at least one high-risk criterion of fungal infection. These included the use of an alternative donor in 42 pts (haplo-identical donor n= 41, umbilical cord blood n=1), sequential conditioning regimen in 9, with PT-Cy in 57, second or third allo-HSCT in 8 and conditioning regimen associated with severe mucositis in 11 (TBI 8-12gy n=3, thiotepa-based n=8). All but one pts received posaconazole using the gastroresistant tablet form at a dose of 300mg/day. One patient received the drinkable suspension form at a dose of 200mg 3 times a day. Following prophylaxis initiation, serum posaconazole concentrations were available in 51 and 16 patients at medians of 9 and 39 days, respectively. A protective residual level of posaconazole (> 0.5mg/L) was achieved in 57% of the patients (n=29) at first assessment and 87.5% (n=12) at second evaluation. Posaconazole was stopped early due to related-toxicity in 9 cases (12.6%), including liver toxicity in 7 pts, QT prolongation in 1 and skin hyperesthesia in 1. Treatment was restarted without recurrence of toxicity in 2 cases. Posaconazole was stopped early in 2 patients due to non-related toxicity, including one veno-occlusive disease and one hepatic acute GVHD. Posaconazole was temporary discontinued in 4 patients due to a suspected fungal infection before being restarted. Most patients (n=59/70, 84.2%) did not present fungal infections while being under posaconazole therapy. Only 11 cases were documented with this complication (Table 1), including 8 invasive pulmonary aspergillosis (IPA; possible n=3; probable n=5), one possible invasive fungal infection and 2 mucormycoses (1 with possible IPA). Fungal infections occurred despite documentation of a protective residual level of posaconazole (> 0.5mg/L) in 5 of 6 evaluable patients. Of note, 9/11 patients had received PTCY as GVHD prophylaxis. Posaconazole was stopped (before D7 in 3) and other antifungal drugs started (voriconazole or liposomal amphotericin B) allowing to fungal infection cure in 5 pts. However, 7 pts died including 2 of an uncontrolled fungal infection. Conclusion: Posaconazole is a well-tolerated and efficient primary prophylaxis in recipients of Allo-HSCT at high-risk of fungal infection. As some fungal infections occured very early after D0 while prophylaxis started only a D5/6 after PTCY, our results suggests to propose posaconazole as early as the beginning of the conditionning together with dose monitoring. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Diagnostic Utility of Whole Genome Sequencing in Adults with B-Other Acute Lymphoblastic Leukaemia

Background B-other ALL represents a working definition for patients with B cell precursor (BCP) ALL without a known primary chromosomal abnormality at diagnosis. In this study we use whole genome sequencing (WGS) to characterize the molecular pathogenesis of adult B-other cases (age ≥ 25yrs) from the UKALL14 trial (NCT01085617). Methods Of 652 patients aged 25-65yrs enrolled onto UKALL14, 333 (51%) had B-other ALL. Sufficient material was available to screen 156/333 B-other cases for recurrent Ph-like fusion events (CRLF2, JAK2, ABL1, ABL2 & PDGFRB) using FISH and MLPA (kit P335). This identified 28 (18%) "Ph-like" fusion events (21 CRLF2, 5 ABL-class and 2 JAK2). Of the remaining 128 B-other cases 57 had available samples for tumor normal paired WGS (read depth 60x and 30x respectively). Diagnostic RNA was available for 33 of the 57 cases Bioinformatic analysis was performed to determine small somatic mutations; single nucleotide variants (SNVs) and insertion/deletions (INDELs) as well as copy number aberrations (CNA) and structural variants (SV). We developed WGS approaches to identify IGH enhancer hijack and DUX4 rearrangements, utilizing gGnome and GRIDSS respectively. For 33 cases RNA-seq data was analyzed to validate fusion genes and genetic classification into one of 18 ALL subtypes using the ALLSorts v2 classifier. Results 52/57 cases passed QC. In 5 (10%) cases an established WHO2016 genetic subtype was identified that was missed by standard of care including three high-risk (2 low hypodiploid and a near haploid) and 2 standard risk (high hyperdiploid and TCF3::PBX1) cases. Cytogenetic analysis for these 5 cases either failed (n=2) or produced a normal karyotype (n=3). The WGS inferred tumor purity for these 5 cases was variable with a median of 46% and a range of 33% to 96% compared to 89% (range 13%-99%) for the remaining cases (n=47) in the dataset. Among the remaining 47 B-other cases, WGS analysis identified abnormalities enabling 41 (87%) cases to be classified into a genetic ALL subtype: DUX4 rearrangements (DUX4r, n=8), PAX5alt (n=7), PAX5 P80R (n=4), ZNF384 rearrangements (ZNF384r, n=5), ZEB2/CEBP (n=4), MEF2D rearrangements (MEF2Dr, n=3), UBTF::ATXN7L3 fusions (n=2), IDH1/2 mutations (n=2), JAK-STAT abnormalities (n=2), along with single cases of IGK::BCL2, IGH::CEBPA, IGH::ID4 and IGH::MIR125B1. WGS resolves 8 cases with complex karyotype into one of 6 different genomic subtypes: 2xDUX4r, 2xMEF2Dr, ZEB2/CEBP, UBTF::ATXN7L3, IGK::BCL2 & IGH::ID4. The MEF2Dr subtype (n=3), is enriched in chromothripsis (2/3), and complex SVs targeting the MEF2D gene. Lastly, in all cases with PAX5 mutations (PAX5 P80R (n=4) and PAX5alt (n=4)) we observe bi-allelic targeting of locus by mutations, deletions or LOH. The remaining 6 (13%) B-other cases could not be classified into a recognized B-other subtype. A MYO18A::FGFR1 fusion (t(8;17)(p11;q23)), which most likely maps to the WHO2016 "Lymphoid neoplasms with FGFR1 rearrangement" classification. Two cases had clonal frameshifts in genes associated with T-ALL (PTPN2 p.A108fs*5 and WT1 p.V371fs*14 respectively). A case with a somatic ETV6 p.R399C previously reported in germline risk with a ZNF292::KDM6A fusion. Lastly a case with a clonal mutation typically associated with myeloid disease ASXL1 p.R417*. Among the 52 cases studied, WGS only failed to identify a driver or putative driver in one case, which had the lowest burden of SNVs (861) and an estimated tumor purity, 28%. For the 31 cases with both DNA and RNA, WGS and RNA analysis were concordant for 100% of cases, with RNA identifying an additional KDM6A::CGA fusion. In 3 cases WGS provides evidence that would have challenged diagnosis and/or treatment; IGK::BCL2 with BCL2 and MYC overexpression, IGH::DUX4 with hypermutation (MSH6 loss) and IGH::ID4 with homozygous loss of CD58 and concurrent LOH of HLA-B. Conclusions WGS assigned 88% (46/52) of cases called B-other to an established genetic subtype of ALL. This includes 5 cases with WHO2016 defined subtypes, 3 were high-risk ploidy subtypes that would have changed their UKALL14 risk group and post-induction treatment in the absence of other risk factors. Among the true B-other cases, 87% (41/47) were assigned to one of the newly described genetic subtypes of ALL. Taken together our findings demonstrate the clinical utility of WGS as a diagnostic assay to inform and improve the management of adult B-Other ALL patients in the future.

Systematic Identification of Autosomal and Y-Encoded Minor Histocompatibility Antigens Reveals Predictors of Chronic Gvhd and Candidate GVL Targets

At the core of the therapeutic effect of HLA-matched allogeneic hematopoietic cell transplantation (allo-HCT) is T cell alloreactivity against minor histocompatibility antigens (mHAgs), polymorphic peptides resulting from donor-recipient disparity at sites of single nucleotide gene polymorphisms (SNPs). Despite their crucial role in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD), only few mHAgs have been characterized to date. To systematically identify autosomal and Y-chromosome encoded HLA-I restricted mHAgs, we devised a computational pipeline based on: (i) comparison of whole exomes (WES) from paired donor-recipient DNA to define recipient-restricted exonic non-synonymous polymorphisms; and (ii) generation of expression filters by incorporating published single cell expression profiles from normal and malignant hematopoietic cells and GvHD target organs. For each GvHD tissue (skin, liver, GI, lung, oral mucosa and lacrimal gland), a large single cell database combining ≥1 independent datasets was created to cluster and annotate tissue-resident cell types. Cell-type specific genes were defined based on their total number of reads (CPM>5 in any cluster), thus creating an expression atlas capturing less frequent albeit biologically relevant cell types underestimated in bulk expression profiles. For GvL predictions, we built an AML-specific filter by defining AML-expressed genes from single cell and bulk data, and by subtracting genes with expression in non-hemopoietic tissues, per transcriptome and proteome GTEx databases. Candidate GvL/GvHD mHAgs prediction was performed by applying the HLAthena binding prediction tool on recipient-restricted SNPs expressed in GvL or GvHD tissues. To validate the pipeline predictive power, we used a panel of 95 HLA class I monoallelic lines generated from B721.221 cells, which we previously profiled by WES, RNASeq and immunopeptidomics. By assigning the B721.221 genome as surrogate 'HCT recipient’ and the reference hg19 genome as surrogate 'donor', we identified recipient-restricted SNPs, whose class I-restricted presentation we confirmed by mass spectrometry for a median of 5 (range 1-16) predicted epitopes/ allele. For 2 HLA-A0201 epitopes encompassing SNPs with an allelic frequency <0.001, we validated immunogenicity through stimulation of PBMCs from HLA-A0201 healthy donors (HDs) with corresponding synthetic peptides. For Y-encoded mHAgs, we predicted a median of 62 (range 24-107) epitopes/HLA-I allele from 9 genes expressed (>1 TPM) in ≥1 non germline tissue. Analysis of 12 male immunopeptidomes confirmed presentation of 3 known Y mHAgs and 26 novel predicted epitopes across 18 alleles. To estimate the fraction of immunogenic predicted binders, we tested all Y epitopes predicted across 3 common alleles (A0201, B1801, C0501). Of 215 Y peptides tested, 53 (~25%) elicited an antigen-specific T cell response in PBMCs from female HDs, as per dextramer-based detection of ≥0.2% CD8 cells. To test the prognostic potential of the mHAg load on cGvHD risk, we performed WES on 220 donor-recipient pairs treated with matched-related-donor allo-HCT for myeloid disease at DFCI between 2013-2020. Our logistic regression analysis used organ-specific NIH moderate/severe cGvHD as outcome; as covariates, we included tissue-specific mHAg load and standard HCT variables (disease type and prognostic risk, conditioning intensity, graft type, GvHD prophylaxis, female into male [F→M] transplant, relapse). The burden of lung-expressed mHAgs was correlated with lung GvHD occurrence (P=0.023). Liver GvHD was associated with F→M transplants (P=0.023). Subgroup analysis further showed liver GvHD to be correlated with the number of epitopes predicted from the RPS14Y gene (P=0.037). For identification of AML-specific mHAgs, a median of 31 (range 13-50) mHAgs/patient were predicted in our cohort from 269 genes preferentially expressed in AML, with a trend for a protective effect of their load against relapse (P=0.058). Of note, 39 SNPs generated putative mHAgs shared by >10 patients and covering 8 common HLA-I alleles in Caucasians, thereby representing potential targets for post-HCT immunotherapy. Overall, we report the generation of a robust analytic pipeline which enables the broadening of the repertoire of mHAgs available for donor selection and for predicting, monitoring or manipulating GvL and GvHD after allo-HCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Patterns and Outcomes of Patients with Primary or Secondary Acute Myeloid Leukemia By Type of Site (Academic or Community/Government): A CONNECT® Myeloid Registry Study

Introduction Up to 30% of acute myeloid leukemias (AML) evolve from prior hematologic disorders and are classified as secondary AML. Secondary AML is traditionally associated with worse prognosis than de novo, or primary, AML, resulting in inferior response rates and poorer survival. The treatment landscape for AML has evolved recently with the introduction of venetoclax (VEN) and the availability of targeted agents. To better understand evolution of the management of secondary AML, we evaluated treatment patterns and outcomes in patients (pts) with primary AML vs secondary AML treated in real-world clinical practice and enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011) by type of site (academic or community/government). Methods Pts were classified as having either primary or secondary AML based on history of myelodysplastic syndrome (MDS) or progression from MDS to AML. Overall survival (OS) from the date of AML diagnosis, estimated by the Kaplan-Meier method, was evaluated in each group according to type of site (academic or community/government), utilization of allogeneic hematopoietic stem cell transplantation (alloHSCT), and tumor protein p53 (TP53) mutational status. Time to adoption of a VEN-based therapy, defined as first use of VEN-based therapy following Food and Drug Administration (FDA) approval in this setting, was evaluated using a Cox model with factors for age (< 75 and ≥ 75 y), type of site, and geographic region. Results A total of 891 pts in the Registry with a diagnosis of AML were assessed (primary AML, n = 676; secondary AML, n = 215). Median (range) ages were 70 (55-97) y in the primary AML group and 74 (19-93) y in the secondary AML group. Most pts were male (primary AML, 61.6%; secondary AML, 61.5%) and most were treated at community or government centers (primary AML, 60.2%; secondary AML, 66.0%). Pts with primary AML had significantly longer survival than pts with secondary AML; median OS (95% confidence interval [CI]) was 15.6 (13.6-17.4) mo and 5.1 (3.6-6.7) mo, respectively (P < 0.001). Median OS for pts with primary AML and secondary AML treated at academic centers was 17.0 mo and 5.6 mo, respectively (hazard ratio [HR] [95% CI], 0.41 [0.30-0.54], P < 0.001) and in community/government centers was 14.1 mo and 5.2 mo, respectively (HR [95% CI], 0.53 [0.43-0.66], P < 0.001). There was no significant difference in median OS in pts treated at academic centers compared with pts treated at community/government centers for both primary AML (academic vs community/government centers: 17.0 mo [95% CI, 14.7-20.7] vs 14.1 mo [11.0-16.9]; Figure 1A) and secondary AML (5.0 mo [95% CI, 3.0-7.2] vs 5.1 mo [3.7-7.3]; Figure 1B). Significantly longer median OS in pts with primary AML vs secondary AML was observed regardless of alloHSCT status (no alloHSCT: 11.7 mo vs 4.5 mo; HR [95% CI], 0.53 [0.44-0.64], P < 0.001; with alloHSCT: 64.3 mo [35.8-NC] vs 24.9 mo [6.8-48.9], P = 0.002]. Longer median OS was observed among pts who received alloHSCT compared with those without alloHSCT: primary AML, 41.1 mo vs 12.8 mo; secondary AML, 24.9 mo vs 4.4 mo; additional analyses are warranted to account for immortal time bias. Regardless of TP53 mutation status, pts with primary AML had significantly longer median OS than pts with secondary AML (no mutation: 22.0 mo [17.6-26.1] vs 6.2 mo [2.5-12.6], P < 0.001; with mutation: 8.8 mo [3.9-14.2] vs 3.8 mo [1.4-6.8], P = 0.014). VEN-based therapies were adopted more quickly by pts aged ≥ 75 y vs < 75 y (P < 0.001) and those treated at community or government centers vs academic centers (P = 0.045); no association between geographic region and time to adoption of VEN-based therapies was observed. Conclusions Overall, outcomes of pts with AML in the CONNECT® Myeloid Disease Registry are consistent with previously reported outcomes. Median OS is longer in primary AML vs secondary AML regardless of whether pts receive alloHSCT or have mutations in TP53. This analysis suggests that contrary to commonly held perceptions, site of care does not affect overall pt outcome and that VEN adoption occurs sooner at community centers. The clinically meaningful increase in OS in pts receiving alloHSCT, when compared with pts without alloHSCT, regardless of primary or secondary AML further emphasizes the importance of providing pts with the opportunity for transplant. These observations merit further study into potential differences in treatment patterns and outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FLT3-ITD Does Not Predict Inferior Prognosis Compared with FLT3-wild Type in Acute Myeloid Leukemia (AML) Patients Age ≥ 60 Years: A Retrospective Cohort Study

IntroductionFLT3 mutations are found in up to 30% of AML patients (pts), with 80% being internal tandem duplications (ITD). Based largely on data in younger pts from an era prior to the use of FLT3 inhibitors or hypomethylating agents (HMA) and venetoclax (Ven), FLT3-ITD mutated (FLT3-ITD+) AML is associated with higher relapse rates and decreased survival compared to FLT3 wild-type (WT) disease. Post hoc analysis from the VIALE-A trial demonstrated comparable outcomes between pts with FLT3-mutated vsFLT3-WT AML treated with HMA+Ven. Further, the AMLSG16-10 trial demonstrated improved survival among pts aged 60-70 with FLT-ITD+ AML treated with "7+3” and midostaurin compared to historical controls. To determine the clinical significance of FLT3-ITD+ in older adults with AML, we assessed the prevalence of FLT3-ITD mutations in pts with AML age ≥ 60 years treated at Dana Farber Cancer Institute (DFCI), evaluated whether FLT3-ITD+ is associated with an inferior prognosis in this age group and identified other specific characteristics affecting survival. Methods We included older (age ≥ 60) pts diagnosed with AML at DFCI between August 2015 and September 2021 and treated with: daunorubicin and cytarabine ("7+3") with or without a third drug; liposomal daunorubicin and cytarabine (CPX351); or HMA-based therapy (HMA+/-Ven). Groups were defined by presence (FLT3-ITD+) or absence (FLT3-ITD-) of FLT3-ITD mutation. We excluded pts in whom diagnostic molecular analysis was not performed, those who had acute promyelocytic leukemia or who received other treatments. Overall survival (OS) was evaluated by the Kaplan-Meier method. Cox regression models were fitted to assess the effect of FLT3-ITD mutations and additional covariates (age, prior myeloid disease, cytogenetics, molecular groups, treatment, and transplant as time-dependent covariate) on OS. Results Of the 380 pts, 53 (14%) were FLT3-ITD+ and 327 (85%) FLT3-ITD-. The median age was 69 years (range 60-89) and was similar between groups (68 [range 60-87] vs 70 [range (60-89], respectively, p=0.18). Intensive chemotherapy (IC) was used in 179 pts: 111 (62%) with "7+3", 20 (11%) with "7+3"+midostaurin (15 [47%] in FLT-ITD+ and 5 [3%] in FLT-ITD- in pts with FLT-TKD mutation), 16 (9%) with "7+3"+other non-FLT3 inhibitor investigational therapy and 32 (18%) with CPX351. HMA+Ven or HMA was used in 111 (29%) and 90 (24%) of pts, respectively. Baseline characteristics in FLT3-ITD+ and FLT3-ITD- pts differed by rates of normal karyotype, monosomal and/or complex karyotype (both p<0.001), and specific molecular co-mutations: (TP53 [p=0.032], secondary type mutations [0.045] and NPM1 [p=0.001]. Table). The median OS of the entire cohort was 13.6 months (CI 95% 11-16) and did not differ between pts with FLT3-ITD+vsFLT3-ITD- (median OS 15.4 [CI 95% 11-28] vs 13.1 [CI 95% 11-16], p=0.33). The median OS was similar between FLT3-ITD+vsFLT3-ITD- pts among those who received IC: 24.8 months (CI 95% 15-NA) vs 19.1 (CI 95% 13-30; p=0.43) and HMA-based-therapy: 9.33 months (CI 95% 6-NA) vs 9.9 (CI 95% 8-14; p=0.95), respectively (Figure). The addition of midostaurin could potentially ameliorate an adverse impact of a FLT3-ITD mutation. Thus, we conducted a sensitivity analysis in IC-treated pts who did not receive midostaurin, which demonstrated similar outcomes in FLT3-ITD+ ( n=17) vsFLT-ITD- (n=142): Median OS 15.4 months (CI 95% 4-NR) vs 17.5 (CI 95% 13-30), respectively, p=0.97. In a multivariable analysis ( MVA), FLT3-ITD status was not associated with OS (HR 1.1 [CI 95% 0.7-1.6] p=0.66). Factors associated with OS in the MVA were: treatment with HMA+Ven vs HMA (HR 0.62 [CI 95% 0.4-0.9], p=0.01), age ≥ 70 years (HR 1.03 [CI 95% 1.003-1.1], p=0.03), monosomal and/or complex karyotype vs normal karyotype (HR 1.67 [CI 95% 1.2-2.4], p=0.009) and TP53 mutations (HR 1.63 [CI 95% 1.1-2.3], p=0.005). Of note, treatment with intensive therapy vs HMA was not associated with OS (0.98 [CI 95% 0.7-1.5], p=0.93). Conclusion In older pts with AML, FLT3-ITD mutations were less common than reported in younger pts and were not associated with an adverse prognosis. Age ≥ 70 years, complex/ monosomal karyotype and presence of TP53 were associated with worse survival. Compared to HMA, therapy with HMA+Ven was associated with improved OS, while treatment with IC was not. The prognostic significance of a FLT3-ITD mutation should be re-evaluated in older pts with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Patterns and Outcomes of Patients with NPM1+ Acute Myeloid Leukemia (AML) Enrolled in the CONNECT® Myeloid Disease Registry

Background Mutations in NPM1 (NPM1+) occur in ∼30% of patients with AML. NPM1+ has been associated with favorable outcomes following intensive chemotherapy (IC) and demonstrated positive prognostic value in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) mutations. Recently, several studies have reported differences in outcomes in patients with NPM1 mutations based on age and treatment. In this analysis we evaluated treatment patterns and outcomes in AML based on NPM1 mutational status and primary induction therapy in patients who were enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011). Methods Patients aged 55 years or older with AML were grouped by known NPM1 status (NPM1+ vs NPM1‒) and induction therapy (non-IC vs IC). Overall survival (OS) and event-free survival (EFS) were evaluated using the Cox model adjusted for age. For the EFS analyses, an event was defined as death, disease progression, or refractory to therapy. Results A total of 483 patients with known NPM1 status were assessed (NPM1+, n = 123; NPM1‒, n = 360). Most patients were male (NPM1+, 61.5%; NPM1‒, 60.7%) and white (NPM1+, 78.0%; NPM1‒, 85.6%; Table). Median age, Eastern Cooperative Oncology Group performance status (ECOG PS), and frailty group were similar in the NPM1+ and NPM1‒ groups. Favorable and adverse European LeukemiaNet (ELN) risk scores at baseline were different in the NPM1+ and NPM1‒ groups. The majority of patients received non-IC regimens as induction therapy (NPM1+, 69.1%; NPM1-, 75.0%). Overall, patients with NPM1+ AML had significantly longer median OS (26 mo) compared with patients with NPM1- AML (15 mo) (HR [95% CI]: NPM1+ vs NPM1- 0.58 [0.44-0.76]; P < 0.01). When analyzed by type of induction regimen (non-IC vs IC), although OS remained numerically longer for patients in the NPM1+ groups, the differences were not statistically significant. Among patients who received non-IC regimens, median OS was 19 and 14 mo in the NPM1+ and NPM1- groups, respectively (Figure). Among patients who received IC regimens, median OS was 24 and 16 mo in the NPM1+ and NPM1- groups, respectively. There was a significant improvement in EFS for patients in the NPM1+ group vs the NPM1- group (20 mo vs 11 mo; hazard ratio [HR] [95% CI], NPM1+ vs NPM1- 0.61 [0.47-0.80]; P < 0.01). This was also true for patients treated with non-IC regimens (15 vs 11 mo in the NPM1+ and NPM1- groups, respectively; HR [95% CI], NPM1+ vs NPM1- 0.67 [0.46-1.00]; P = 0.048). In contrast with the non-IC treated regimens, in patients treated with IC regimens, NPM1+ status was not associated with significantly longer EFS compared to NPM1- status. Patients with known NPM1 status were further evaluated for additional mutations and the following differences were noted: the frequency of mutations in tumor protein p53 (TP53) (1.6% vs 18.6%), RUNX1 (0.8% vs 14.8%), ASXL1 (2.4% vs 17.6%) IDH2 (8.9% vs 15.7%), and NRAS/KRAS (8.9% vs 14.3%) was lower in patients with NPM1+ vs NPM1- AML, respectively, whereas the frequency of mutations in FLT3-ITD (35.8% vs 17.1%), FLT3- tyrosine kinase domain (TKD) (14.6% vs 6.2%), DNMT3A (23.6% vs 15.7%), and IDH1 (14.6% vs 8.6%) was higher in patients with NPM1+ vs NPM1- AML, respectively. Conclusions Overall, patients with NPM1+ AML had longer OS and EFS than patients with NPM1‒ AML. This was observed among patients with NPM1+ AML for both non-IC and IC induction regimens. Patterns of co-mutations differed between the NPM1+ and NPM1- groups and is a likely contributing factor to differences between outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discordant Cellular and Humoral Responses to a 3-Dose COVID-19 mRNA Vaccination Schedule in Patients with Hematologic Malignancies

Introduction Studies in 2-dose COVID-19 vaccinated hematology patients demonstrated reduced humoral immune responses compared to healthy individuals. It has become common practice to offer these patients a 3rd COVID-19 vaccination but data substantiating this are scarce. We quantitatively and qualitatively evaluated humoral as well as cellular responses to a 3-dose vaccination schedule, followed by a 4th vaccination, in a prospectively designed, large and diverse cohort of patients with hematologic malignancies. Methods We prospectively quantified SARS-CoV-2-specific spike glycoprotein 1 immunoglobulin G (S1 IgG) concentration against the first serum standard for COVID-19 (20/136; provided by the National Institute for Biological Standards Control), antibody neutralization capacity of wild-type virus and variants of concern, and SARS-CoV-2-specific CD4+ and CD8+ T cell responses by activation marker expression, cytokine production and peptide-HLA tetramer staining in patients with hematologic malignancies prior to, and 4 weeks after each COVID-19 vaccination. Antibody concentrations were compared to those obtained by age-matched healthy individuals, extracted from a population-based study conducted by the Dutch National Institute for Public Health and the Environment. Previously infected participants, identified as having nucleocapsid (N) IgG >14.3 BAU/ml, were excluded from further analysis. Results In total, 692 patients with a hematologic malignancy were included in the study. In 673 evaluable patients S1 IgG concentrations were significantly lower after the primary 2-dose mRNA-1273 schedule, compared to concentrations in age-matched healthy individuals (Haggenburg et al., Blood Adv 2022). A 3rd mRNA-1273 vaccination led to S1 IgG concentrations comparable to those obtained by age-matched healthy individuals after the primary 2-dose mRNA-1273 schedule (Figure 1). Moreover, a 3rd vaccination significantly improved antibody neutralization capacity against SARS-CoV-2 wild type and variants of concern (not shown). The rise in S1 IgG concentration after the 3rd vaccination was most pronounced in patients with a recovering immune system, but potent responses were also observed in patients with persistent immunodeficiencies. S1 IgG antibody concentrations remained low in patients receiving, or shortly after, anti-CD20 therapy, CD19-directed chimeric antigen receptor T cell therapy, and in chronic lymphocytic leukemia patients receiving ibrutinib (Figure 1; Haggenburg et al., medRxiv 2022). In most B cell depleted patients, spike-specific CD4+ and, most prominently, CD8+ T cell responses were generated. A dichotomy between humoral and cellular immune responses was also observed in other patient groups: patients with myeloproliferative disease receiving ruxolitinib, multiple myeloma patients receiving immunomodulatory drugs or daratumumab, and patients with chronic graft versus host disease obtained adequate S1 IgG concentrations, but less pronounced SARS-CoV-2-specific, cytokine-producing CD4+ and/or CD8+ T cell responses after each vaccination. This is in contrast to multiple myeloma patients receiving 1st line remission-induction therapy followed by high-dose melphalan and autologous HCT, who generated antibody and CD4+ T cell responses comparable to healthy individuals. In patients with myeloid disease receiving hypomethylating agents S1 IgG antibody and T cell responses were both impaired. A 4th vaccination further improved S1 IgG concentrations in the majority of patients; its effect on cellular immunity is currently being evaluated. Conclusion After a 3-dose mRNA vaccination schedule, the majority of patients with hematologic malignancies obtained adequate antibody concentrations and robust CD4+ T cell responses. However, T and B cell responses were often discordant: the presence of adequate SARS-CoV-2 specific antibody concentrations was not necessarily accompanied by robust T cell immunity and vice versa. The impact of discordant T and B cell responses on vaccine effectiveness requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clofarabine and Busulfan Myeloablative Conditioning in Allogeneic Hematopoietic Cell Transplantation for Patients With Active Myeloid Malignancies

Patients with refractory or relapsed and refractory myeloid malignancies have a poor prognosis. Allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning (MAC) in patients with active, chemotherapy-refractory myeloid disease is historically associated with high rates of relapse and nonrelapse mortality (NRM). A MAC regimen combining clofarabine with busulfan (Clo/Bu4) has been reported to exhibit antileukemic activity with acceptable toxicity in patients age ≤70 years. Here we describe the clinical outcomes of a real-world population of patients with active myeloid malignancies undergoing allogeneic HCT with Clo/Bu4 MAC. In a single-center retrospective descriptive analysis, we identified patients who underwent HCT for myeloid malignancies not in remission using Clo/Bu4 MAC between 2012 and 2020. We report event-free survival (EFS) and overall survival (OS), cumulative incidences of relapse and NRM, and the incidence and severity of acute and chronic graft-versus-host disease (GVHD). We identified 69 patients with a median age of 60 years (range, 22 to 70 years). Most patients had relapsed/refractory or primary refractory acute myelogenous leukemia (AML; n=55) or refractory myelodysplastic syndrome (MDS; n=12); 1 patient had chronic myelogenous leukemia, and 1 patient had a blastic plasmacytoid dendritic cell neoplasm. Fifty patients (72.5%) had complete remission at day 100 post-transplantation. Two-year EFS and OS were 30% (95% confidence interval [CI], 20% to 44%) and 40% (95% CI, 29% to 54%), respectively. Patients with AML had a 2-year EFS and OS of 28% (95% CI, 18% to 44%) and 38% (95% CI, 27% to 54%), respectively; those with MDS had a 2-year EFS and OS of 47% (95% CI, 25% to 88%) and 56% (95% CI, 33% to 94%), respectively. The cumulative incidence of relapse at 2 years was 39% (95% CI, 27% to 51%) for all patients, including 45% (95% CI, 31% to 58%) in the patients with AML and 18% (95% CI, 2% to 45%) in those with MDS. NRM at 2 years was 31% (95% CI, 20% to 42%), including 27% (95% CI, 15% to 39%) in patients with AML and 35% (95% CI, 10% to 63%) in those with MDS. The total incidence of acute GVHD (aGVHD) of any severity was 80%, and the incidence of grade III-IV aGVHD was 22%. In patients who achieved remission, those who required systemic immunosuppression for aGVHD (58%) had poorer 2-year EFS (29% versus 54%; P=.05) and 2-year OS (39% versus 70%; P=.04) compared to those who did not. The 2-year cumulative incidence of chronic GVHD was 44% (95% CI, 28% to 58%). Clo/Bu4 MAC followed by allogeneic HCT for patients with active myeloid malignancies is an effective transplantation strategy for patients up to age 70, particularly those with advanced MDS. The high incidence of and poor outcomes associated with aGVHD highlight the importance of optimizing preventative strategies.

Rare Germline ATM Variants Influence the Development of Chronic Lymphocytic Leukemia.

Germline missense variants of unknown significance in cancer-related genes are increasingly being identified with the expanding use of next-generation sequencing. The ataxia telangiectasia-mutated (ATM) gene on chromosome 11 has more than 1,000 germline missense variants of unknown significance and is a tumor suppressor. We aimed to determine if rare germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared with other hematologic malignancies and if they influence the clinical characteristics of CLL. We identified 3,128 patients (including 825 patients with CLL) in our hematologic malignancy clinic who had received clinical-grade sequencing of the entire coding region of ATM. We ascertained the comparative frequencies of germline ATM variants in categories of hematologic neoplasms, and, in patients with CLL, we determined whether these variants affected CLL-associated characteristics such as somatic 11q deletion. Rare germline ATM variants are present in 24% of patients with CLL, significantly greater than that in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). Patients with CLL with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of patients with CLL, is associated with a three-fold increase in rates of somatic 11q deletion, and is a hypomorph in cell-based assays. Germline ATM variants cluster within CLL and affect the phenotype of CLL that develops, implying that some of these variants (such as ATM p.L2307F) have functional significance and should not be ignored. Further studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL.

Hoxa9/meis1-transgenic zebrafish develops acute myeloid leukaemia-like disease with rapid onset and high penetrance.

HOXA9 and MEIS1 are co-expressed in over 50% of acute myeloid leukaemia (AML) and play essential roles in leukaemogenesis, but the mechanisms involved are poorly understood. Diverse animal models offer valuable tools to recapitulate different aspects of AML and link in vitro studies to clinical trials. We generated a double transgenic zebrafish that enables hoxa9 overexpression in blood cells under the draculin (drl) regulatory element and an inducible expression of meis1 through a heat shock promoter. After induction, Tg(drl:hoxa9;hsp70:meis1) embryos developed a preleukaemic state with reduced myeloid and erythroid differentiation coupled with the poor production of haematopoietic stem cells and myeloid progenitors. Importantly, most adult Tg(drl:hoxa9;hsp70:meis1) fish at 3 months old showed abundant accumulations of immature myeloid precursors, interrupted differentiation and anaemia in the kidney marrow, and infiltration of myeloid precursors in peripheral blood, resembling human AML. Genome-wide transcriptional analysis also confirmed AML transformation by the transgene. Moreover, the dihydroorotate dehydrogenase (DHODH) inhibitor that reduces leukaemogenesis in mammals effectively restored haematopoiesis in Tg(drl:hoxa9;hsp70:meis1) embryos and improved their late survival. Thus, Tg(drl:hoxa9;hsp70:meis1) zebrafish is a rapid-onset high-penetrance AML-like disease model, which provides a novel tool to harness the unique advantages of zebrafish for mechanistic studies and drug screening against HOXA9/MEIS1 overexpressed high-risk AML.

AML-251 Overall Survival (OS) With Intensive Chemotherapy (IC) vs Non-IC in Patients With Newly Diagnosed (ND) AML from the Connect® Myeloid Disease Registry Ineligible for Randomized Clinical Trials (RCT)

Patients with acute myeloid leukemia (AML) in RCTs may not reflect the population seen in clinical practice due to strict eligibility criteria. To evaluate outcomes in clinical practice with IC vs venetoclax-containing regimens based on the VIALE-A trial eligibility criteria in patients with AML from the Connect® Myeloid Disease Registry (NCT01688011). Patients were stratified into 3 groups: 1) eligible: met all VIALE-A inclusion criteria; 2) unfit: ineligible for VIALE-A; 3) fit: ineligible for a venetoclax-based regimen in VIALE-A (would have qualified for IC). Baseline characteristics were summarized by group. Induction regimens included IC or venetoclax-based therapies. OS was estimated by Kaplan-Meier method. Hazard ratios (HR) for induction regimens were estimated using Cox models adjusted for age, ELN risk, ECOG, frailty score, and comorbidity index. Of 734 patients (Dec 2021), 61% were male; 84% were white; median age 71 years. Only 26% of patients (n=192) were eligible for VIALE-A; 45% (n=327) and 29% (n=215) were ineligible due to unfitness and overall fitness, respectively. The main reason for VIALE-A ineligibility was high overall comorbidity grade (n=265 [36%]). At baseline, fit patients intended to undergo transplant more often than unfit patients. Median OS (mOS) for eligible, unfit, and fit patients was 14, 10, and 22 months, respectively (HR [95%CI], eligible vs unfit, 1.02 [0.83-1.25], P-value, NS; eligible vs fit, 1.77 [1.38-2.25], P<0.0001; unfit vs fit, 1.74 [1.40-2.16], P<0.0001). Unfit patients receiving IC had significantly longer mOS (14 vs 6 months, respectively; HR, 0.51 [95%CI, 0.27-0.98]; P=0.042) and were more likely to receive a transplant (16% [n=17] vs 1% [n=1], respectively) vs those receiving venetoclax-based regimens. Eligible patients receiving IC (n=31) tended to have shorter mOS (13 months) vs patients receiving venetoclax-based therapies (n=27; 23 months; HR, 1.45 [95%CI, 0.66-3.17]; P-value, NS). Most patients with ND AML in the Connect® Myeloid Disease Registry would have been ineligible for VIALE-A due to being too fit or unfit. Among patients ineligible for an RCT due to unfitness, there was an association with increased OS in patients receiving IC vs those receiving venetoclax-based therapies.

The yin-yang of immunity: Immune dysregulation in myelodysplastic syndrome with different risk stratification.

Myelodysplastic syndrome (MDS) is a heterogeneous group of myeloid clonal diseases with diverse clinical courses, and immune dysregulation plays an important role in the pathogenesis of MDS. However, immune dysregulation is complex and heterogeneous in the development of MDS. Lower-risk MDS (LR-MDS) is mainly characterized by immune hyperfunction and increased apoptosis, and the immunosuppressive therapy shows a good response. Instead, higher-risk MDS (HR-MDS) is characterized by immune suppression and immune escape, and the immune activation therapy may improve the survival of HR-MDS. Furthermore, the immune dysregulation of some MDS changes dynamically which is characterized by the coexistence and mutual transformation of immune hyperfunction and immune suppression. Taken together, the authors think that the immune dysregulation in MDS with different risk stratification can be summarized by an advanced philosophical thought “Yin-Yang theory” in ancient China, meaning that the opposing forces may actually be interdependent and interconvertible. Clarifying the mechanism of immune dysregulation in MDS with different risk stratification can provide the new basis for diagnosis and clinical treatment. This review focuses on the manifestations and roles of immune dysregulation in the different risk MDS, and summarizes the latest progress of immunotherapy in MDS.

CircNFIX knockdown inhibited AML tumorigenicity by the miR-876-3p/TRIM31 axis

ABSTRACT Background Acute myeloid leukemia (AML) is one of the most common malignant myeloid diseases in adults with a dismal prognosis. We aimed to explore the effects of circNFIX on the proliferation and apoptosis of AML cells. Methods The expressions of circNFIX, miR-876-3p and tripartite motif (TRIM) 31 in the bone marrow specimens of AML patients and AML cell lines were detected by qRT-PCR or western blot. Cell proliferation was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-ethynyl-29-deoxyuridine (EdU) assays. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was used to detect protein expression. The relationship between miR-876-3p and circNFIX or TRIM31 was identified by dual-luciferase reporter assay or RNA pull-down assay. Results The expression level of circNFIX was significantly increased in the bone marrow samples of AML patients and AML cells when compared with normal controls. CircNFIX silencing inhibited AML cell proliferation and promoted apoptosis. Inhibition of miR-876-3p reversed the effect of circNFIX knockdown on AML cell progression. In addition, circNFIX indirectly regulated TRIM31 through miR-876-3p. Further, TRIM31 overexpression counteracted the effect of circNFIX silencing on AML cell proliferation and apoptosis. Conclusion CircNFIX knockdown could suppress the proliferation and induce the apoptosis of AML cells by targeting the miR-876-3p/TRIM31 axis.

An early glimpse at azacitidine plus venetoclax for myelodysplastic syndromes

Myelodysplastic syndromes, chronic myelomonocytic leukaemia, and acute myeloid leukaemia comprise a spectrum of heterogenous myeloid diseases characterised by abnormal haematopoiesis, a block in differentiation, and haematopoietic insufficiency resulting in cytopenias. These multiclonal processes show considerable biologic, phenotypic, genetic, and clinical overlap. In recognition of this continuum, WHO has recently renamed the former category of myelodysplastic syndromes with excess blasts-2 (defined as having ≥10% and <20% bone marrow blasts) to “myelodysplastic syndromes/acute myeloid leukaemia”.

A Synopsis Clonal Hematopoiesis of Indeterminate Potential in Hematology.

Simple SummaryMutations are not the norm, yet they exist. Having some mutations can infer information about a precancerous state. Clonal hematopoiesis of indeterminate potential is a condition of recurrent somatic mutations in the blood of otherwise healthy adults. In this review, we unravel the role of these mutations in multiple myeloma.Clonal hematopoiesis of indeterminate potential can be defined as genetic mutations that correlate in hematologic neoplasia such as myelodysplastic syndrome. Patients with cytopenia increasingly undergo molecular genetic tests of peripheral blood or bone marrow for diagnostic purposes. Recently, a new entity has been demarcated to lessen the risk of incorrect diagnoses of hematologic malignancies. This new entity is a potential precursor of myeloid diseases, analogous to monoclonal gammopathy of undetermined significance as a potential precursor of multiple myeloma.