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Abstract Background Therapy-related acute myeloid leukemia (t-AML) is a secondary, generally incurable, malignancy attributable to chemotherapy exposure. Although there is a genetic component to t-AML susceptibility in mice, the relevant loci and the mechanism(s) by which they contribute to t-AML are largely unknown. An improved understanding of susceptibility factors and the biological processes in which they act may lead to the development of t-AML prevention strategies. Results In this work we applied an integrated genomics strategy in inbred strains of mice to find novel factors that might contribute to susceptibility. We found that the pre-exposure transcriptional state of hematopoietic stem/progenitor cells predicts susceptibility status. More than 900 genes were differentially expressed between susceptible and resistant strains and were highly enriched in the apoptotic program, but it remained unclear which genes, if any, contribute directly to t-AML susceptibility. To address this issue, we integrated gene expression data with genetic information, including single nucleotide polymorphisms (SNPs) and DNA copy number variants (CNVs), to identify genetic networks underlying t-AML susceptibility. The 30 t-AML susceptibility networks we found are robust: they were validated in independent, previously published expression data, and different analytical methods converge on them. Further, the networks are enriched in genes involved in cell cycle and DNA repair (pathways not discovered in traditional differential expression analysis), suggesting that these processes contribute to t-AML susceptibility. Within these networks, the putative regulators (e.g., Parp2, Casp9, Polr1b) are the most likely to have a non-redundant role in the pathogenesis of t-AML. While identifying these networks, we found that current CNVR and SNP-based haplotype maps in mice represented distinct sources of genetic variation contributing to expression variation, implying that mapping studies utilizing either source alone will have reduced sensitivity. Conclusion The identification and prioritization of genes and networks not previously implicated in t-AML generates novel hypotheses on the biology and treatment of this disease that will be the focus of future research.

Background: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease.Methods: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation.Results: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes ( HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro.Conclusions: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.). [ABSTRACT FROM AUTHOR]

Background: Precision oncology relies on molecular diagnostics, and the value-proposition of modern healthcare networks promises a higher standard of care across partner sites. We present the results of a clinical pilot to standardize precision oncology workflows., Methods: Workflows are defined as the development, roll-out, and updating of disease-specific molecular order sets. We tracked the timeline, composition, and effort of consensus meetings to define the combination of molecular tests. To assess clinical impact, we examined order set adoption over a two-year period (before and after roll-out) across all gastrointestinal and hepatopancreatobiliary (GI) malignancies, and by provider location within the network., Results: Development of 12 disease center-specific order sets took ~9 months, and the average number of tests per indication changed from 2.9 to 2.8 (P = .74). After roll-out, we identified significant increases in requests for GI patients (17%; P < .001), compliance with testing recommendations (9%; P < .001), and the fraction of "abnormal" results (6%; P < .001). Of 1088 GI patients, only 3 received targeted agents based on findings derived from non-recommended orders (1 before and 2 after roll-out); indicating that our practice did not negatively affect patient treatments. Preliminary analysis showed 99% compliance by providers in network sites, confirming the adoption of the order sets across the network., Conclusion: Our study details the effort of establishing precision oncology workflows, the adoption pattern, and the absence of harm from the reduction of non-recommended orders. Establishing a modifiable communication tool for molecular testing is an essential component to optimize patient care via precision oncology., (© The Author(s) 2022. Published by Oxford University Press.)

Chemotherapies approved for defined subgroups promise personalized oncologic care, but their off-label impact is unclear. Lenalidomide is approved for lower-risk, transfusion-dependent (TD) myelodysplastic syndromes (MDS) with del(5q), but frequently used in MDS outside this indication. We characterized lenalidomide use and outcomes among non-TD patients with MDS. Patients 65 or older diagnosed with MDS between 2007 and 2013 were identified using SEER; linked Medicare claims were evaluated for transfusions, lenalidomide use, and incident toxicities. TD was ≥2 transfusion episodes within an 8-week period; responses were transfusion independence (TI) and ≥50% transfusion reduction (minor response). We compared overall survival for non-TD patients receiving lenalidomide versus those not receiving lenalidomide, matched on disease and patient characteristics. We identified 676 patients who had received lenalidomide, including 275 (40.7%) TD and 401 (59.3%) non-TD; 18.5% (125/676) had zero claims for RBC transfusion prior to receiving lenalidomide. Incident toxicities among patients prescribed lenalidomide were similar in TD and non-TD groups, except incident thromboembolic events were higher among non-TD patients (10.8% vs. 6.0%, P = .04). Comparing 191 non-TD patients receiving lenalidomide within 6 months of MDS diagnosis to risk-matched MDS controls, lenalidomide was not associated with improved OS (P = .78). Among TD patients (n = 275), 31% achieved TI, and 30% achieved minor hematologic response, with a median time to TI of 4.1 weeks. In conclusion, we confirmed the benefit of lenalidomide among TD patients with MDS; however, many non-TD patients also received lenalidomide. These patients experienced accompanying toxicity without evidence of benefit in terms of transfusion needs or overall survival., (© 2018 Wiley Periodicals, Inc.)

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by frequent resistance to available chemotherapeutic agents. The basic therapy for patients with AML has changed little over the past 30 years. Improvements in outcome in recent decades in younger adult cohorts have generally been ascribed to better supportive care (ie, transfusion and antimicrobial therapy); older adults with AML continue to fare poorly. The explosion of new knowledge regarding the AML genome has yet to be translated into therapeutic benefit, but analysis of specific molecular features in AML samples has enabled the field to more accurately parse out prognosis and assign appropriate therapies (eg, chemotherapy vs allogeneic stem cell transplantation) for groups of patients. Cytogenetic analysis, whether by metaphase or interphase analysis, has been the main tool used to divide patients into varying prognostic subsets, but it has been modified in recent years to include assessment of mutations in a small number of genes. In the past several years, new technologies have provided strategies to interrogate individual cancer genomes in a broad and in-depth fashion. The present article discusses the potential of these new technologies, particularly gene panel and whole-exome or whole-genome sequencing, to improve diagnosis, prognosis, and therapeutic outcome in AML., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Myelodysplastic syndromes (MDS) are a heterogenous group of hematologic malignancies characterized by clonal expansion of BM myeloid cells with impaired differentiation. The identification of recurrent mutations in MDS samples has led to new insights into the pathophysiology of these disorders. Of particular interest is the recent recognition that genes involved in the regulation of histone function (EZH2, ASXL1, and UTX) and DNA methylation (DNMT3A, IDH1/IDH2, and TET2) are recurrently mutated in MDS, providing an important link between genetic and epigenetic alterations in this disease. The mechanism by which these mutated genes contribute to disease pathogenesis is an active area of research, with a current focus on which downstream target genes may be affected. Recent advances from sequencing studies suggest that multiple mutations are required for MDS initiation and progression to acute myeloid leukemia (AML). The past several years have yielded many new insights, but the complete genetic landscape of MDS is not yet known. Moreover, few (if any) of the findings are sufficiently robust to be incorporated into routine clinical practice at this time. Additional studies will be required to understand the prognostic implications of these mutations for treatment response, progression to AML, and survival.

In this issue of Blood, Watanabe-Okochi and colleagues use a mouse bone marrow transplantation model to demonstrate that mutant alleles of AML1 (RUNX1) can initiate a myelodysplastic syndrome (MDS) that progresses to acute myelogenous leukemia (AML) in association with overexpression of Evi1.

Hydroxychloroquine (HCQ) is an immunosuppressive lysosomotropic amine that has activity against graft-versus-host disease (GVHD). In a single-institution phase III trial, 95 recipients of allogeneic peripheral blood stem cell (PBSC) transplantation were randomized to receive, in a double-blind fashion, and in addition to prophylactic cyclosporine A (CSA), HCQ, or placebo starting 21 days pretransplant and continued until day +365. HCQ was very well tolerated and not associated with side effects. Overall, the incidence of acute GVHD (aGVHD) was 59% in both arms, and severe aGVHD occurred in 11% (HCQ) and 14% (placebo) (P = .76). Sixty percent and 78% of patients developed chronic GVHD (cGVHD) in the HCQ and the placebo arms, respectively (P = .15). With a median follow-up of 18 months, relapse-free and overall survivals (OS) were comparable in both groups. In summary, in this randomized trial, the addition of HCQ to single-agent CSA had no effects on aGVHD or cGVHD or survival.

Satellite cells are tissue-specific stem cells critical for skeletal muscle growth and regeneration. Upon exposure to appropriate stimuli, satellite cells produce progeny myoblasts. Heterogeneity within a population of myoblasts ensures that a subset of myoblasts readily differentiate to form myotubes, whereas other myoblasts remain undifferentiated and thus available for future muscle growth. The mechanisms that contribute to this heterogeneity in myoblasts are largely unknown. We show that satellite cells are Sca-1(neg) but give rise to myoblasts that are heterogeneous for sca-1 expression. The majority of myoblasts are sca-1(neg), rapidly divide, and are capable of undergoing myogenic differentiation to form myotubes. In contrast, a minority population is sca-1(pos), divides slower, and does not readily form myotubes. Sca-1 expression is not static but rather dynamically modulated by the microenvironment. Gain-of-function and loss-of-function experiments demonstrate that sca-1 has a functional role in regulating proliferation and differentiation of myoblasts. Myofiber size of sca-1 null muscles is altered in an age-dependent manner, with increased size observed in younger mice and decreased size in older mice. These studies reveal a novel system that reversibly modulates the myogenic behavior of myoblasts. These studies provide evidence that, rather than being a fixed property, myoblast heterogeneity can be modulated by the microenvironment.

Calcineurin inhibitor-induced central nervous system toxicities are uncommon and often resolve after discontinuation of the offending drug. The long-term outcome of these patients is, however, unknown. Resolution of symptoms occurred in 70% of 30 allografted recipients who developed calcineurin inhibitor-induced neurotoxicity. When patients were rechallenged with the same or a different calcineurin inhibitor, symptoms recurred in 41%, leading to permanent discontinuation of the drug. De novo or progressive acute graft-versus-host disease (GVHD) was observed in 54% of patients at a median of 7 d (range 1-70 d) after initial onset of neurotoxicity. The prognosis was grim, with 24 (80%) of these patients dying a median 33 d after the onset of neurotoxicity (range 2-594 d). GVHD and/or infection occurred in 54% and were the most common primary causes of death. We conclude that calcineurin inhibitor-induced neurotoxicity is frequently reversible but associated with a poor prognosis.

The Ly-6 family includes a number of highly homologous, low molecular weight glycophosphatidylinositol-linked proteins expressed on hematopoietic and lymphoid cells. The best characterized family member is Sca-1 (Ly-6A/E), an antigen commonly used for purification of murine pluripotent hematopoietic cells. We sought to characterize the genomic locus surrounding the Sca-1 gene. We identified several overlapping P1 artificial chromosomes containing the Sca-1 gene and mapped one of these to mouse chromosome 15D3.1-3.3, the region previously shown to contain members of the murine Ly-6 gene family. We then mapped this clone and found that the Sca-2 gene lies 35.4 kilobase (kb) downstream of Sca-1 in the opposite transcriptional orientation. This is the first direct demonstration of physical linkage of Ly-6 genes. A novel gene, highly homologous to Sca-1 was identified and localized 13.4 kb downstream of Sca-1. This gene, which we designated Ly-6M, shares several structural features conserved among members of the Ly-6 family. Ly-6M messenger RNA (mRNA) is easily detectable in hematopoietic tissue (bone marrow, spleen, thymus, peritoneal macrophages) as well as kidney and lung. No mRNA expression was detected in heart, stomach, liver, small intestine, brain, or skin. Ly-6M protein is detectable on 10% to 15% of peripheral blood leukocytes, including monocytes and a subpopulation of B220(+) cells. Ly-6M is broadly distributed in the bone marrow, with prominent expression on monocytes and myeloid precursors. The identification and characterization of Ly-6M adds a new member to a complex family of homologous, tightly linked genes that have proven extremely useful reagents for defining populations within the hematopoietic system.

Granzyme (gzm) B-deficient cytotoxic lymphocytes (CTL) have a severe defect in the rapid induction of target cell apoptosis that is almost completely corrected by prolonged incubation of the CTL effectors and their targets. We show in this report that perforin-dependent, gzmB-independent cytotoxicity is caused by gzmA (or tightly linked genes). CTL deficient for gzmA and gzmB retain normal perforin function, but these CTL have a cytotoxic defect in vivo that is as severe as perforin-deficient CTL. Collectively, these results suggest that perforin provides target cell access and/or trafficking signals for the gzms, and that the gzms themselves deliver the lethal hits. The gzmA pathway appears to function independently from gzmB and may therefore provide a critical "back-up" system when gzmB is inhibited in the target cell.

CD8+ cytotoxic lymphocytes, natural killer cells and lymphokine-activated killer cells depend primarily on the perforin/granzyme system to kill their targets, while CD4+ T cells utilize Fas and other mechanisms to induce cell death. The molecular mechanisms used by these pathways to induce target cell apoptosis may converge on common death substrates.

A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro experiments (Heusel et al, Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8+ CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class I-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class II-dependent GVHD was not altered using gzm B -/- CD4+ effectors. In a hybrid resistance model, gzm B -/- hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.