Hemophagocytic Lymphohistiocytosis Gene Variants in Severe Aplastic Anemia and Their Impact on Hematopoietic Cell Transplantation Outcomes.

Germline genetic testing for patients with severe aplastic anemia (SAA) is recommended to guide treatment, including the use of immunosuppressive therapy and/or adjustment of hematopoietic cell transplantation (HCT) modalities. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition often associated with cytopenias with autosomal recessive (AR) or X-linked recessive (XLR) inheritance. HLH is part of the SAA differential diagnosis, and genetic testing may identify variants in HLH genes in patients with SAA. The impact of pathogenic/likely pathogenic (P/LP) variants in HLH genes on HCT outcomes in SAA is unclear. In this study, we aimed to determine the frequency of HLH gene variants in a large cohort of patients with acquired SAA and to evaluate their association(s) with HCT outcomes. The Transplant Outcomes in Aplastic Anemia project, a collaboration between the National Cancer Institute and the Center for International Blood and Marrow Transplant Research, collected genomic and clinical data from 824 patients who underwent HCT for SAA between 1989 and 2015. We excluded 140 patients with inherited bone marrow failure syndromes and used exome sequencing data from the remaining 684 patients with acquired SAA to identify P/LP variants in 14 HLH-associated genes (11 AR, 3 XLR) curated using American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) criteria. Deleterious variants of uncertain significance (del-VUS) were defined as those not meeting the ACMG/AMP P/LP criteria but with damaging predictions in ≥3 of 5 meta-predictors (BayesDel, REVEL, CADD, MetaSVM, and/or EIGEN). The Kaplan-Meier estimator was used to calculate the probability of overall survival (OS) after HCT, and the cumulative incidence calculator was used for other HCT outcomes, accounting for relevant competing risks. There were 46 HLH variants in 49 of the 684 patients (7.2%). Seventeen variants in 19 patients (2.8%) were P/LP; 8 of these were loss-of-function variants. Among the 19 patients with P/LP HLH variants, 16 (84%) had monoallelic variants in genes with AR inheritance, and 3 had variants in XLR genes. PRF1 was the most frequently affected gene (in 8 of the 19 patients). We found no statistically significant differences in transplantation-related factors between patients with and those without P/LP HLH variants. The 5-year survival probability was 89% (95% confidence interval [CI], 72% to 99%) in patients with P/LP HLH variants and 70% (95% CI, 53% to 85%) in those with del-VUS HLH variants, compared to 66% (95% CI, 62% to 70%) in those without variants (P = .16, log-rank test). The median time to neutrophil engraftment was 16 days for patients with P/LP HLH variants and 18 days in those with del-VUS HLH variants or without variants combined (P = .01, Gray's test). No statistically significant associations between P/LP HLH variants and the risk of acute or chronic graft-versus-host disease were noted. In this large cohort of patients with acquired SAA, we found that 2.8% of patients harbored a P/LP variant in an HLH gene. No negative effects of HLH gene variants on post-HCT survival were noted. The small number of patients with P/LP HLH variants limits the study's ability to provide conclusive evidence; nonetheless, our data suggest that there is no need for special transplantation considerations for patients with SAA carrying P/LP variants.

Clinical Correlates of Venetoclax-Based Combination Sensitivities to Augment Acute Myeloid Leukemia Therapy.

The BCL2 inhibitor venetoclax combined with the hypomethylating agent azacytidine shows significant clinical benefit in a subset of patients with acute myeloid leukemia (AML); however, resistance limits response and durability. We prospectively profiled the ex vivo activity of 25 venetoclax-inclusive combinations on primary AML patient samples to identify those with improved potency and synergy compared with venetoclax + azacytidine (Ven + azacytidine). Combination sensitivities correlated with tumor cell state to discern three patterns: primitive selectivity resembling Ven + azacytidine, monocytic selectivity, and broad efficacy independent of cell state. Incorporation of immunophenotype, mutation, and cytogenetic features further stratified combination sensitivity for distinct patient subtypes. We dissect the biology underlying the broad, cell state-independent efficacy for the combination of venetoclax plus the JAK1/2 inhibitor ruxolitinib. Together, these findings support opportunities for expanding the impact of venetoclax-based drug combinations in AML by leveraging clinical and molecular biomarkers associated with ex vivo responses. SIGNIFICANCE: By mapping drug sensitivity data to clinical features and tumor cell state, we identify novel venetoclax combinations targeting patient subtypes who lack sensitivity to Ven + azacytidine. This provides a framework for a taxonomy of AML informed by readily available sets of clinical and genetic features obtained as part of standard care. See related commentary by Becker, p. 437 . This article is featured in Selected Articles from This Issue, p. 419.

Chasing leukemia differentiation through induction therapy, relapse and transplantation.

Despite rapid advances in our understanding of acute myeloid leukemia (AML), the disease remains challenging to treat with 5-year survival for adult patients 20 years or older estimated to be 26% (Cancer 2021). The use of new targeted therapies including BCL2, IDH1/IDH2, and FLT3 inhibitors has revolutionized treatment approaches but also changed the disease trajectory with unique modes of resistance. Recent studies have shown that stem cell maturation state drives expression level and/or dependence on various pathways, critical to determining drug response. Instead of anticipating these changes, we remain behind the curve chasing the next expanded clone. This review will focus on current approaches to treatment in AML, including defining the significance of blast differentiation state on chemotherapeutic response, signaling pathway dependence, metabolism, immune response, and phenotypic changes. We conclude that multimodal treatment approaches are necessary to target both the immature and mature clones, thereby, sustaining drug response.

Optimizing the cryopreservation and post-thaw recovery of natural killer cells is critical for the success of off-the-shelf platforms.

Natural killer (NK) cells are a promising allogeneic, off-the-shelf, cellular immunotherapy product. These cells can be manipulated ex vivo, genetically edited to enhance tumor targeting and expanded to produce large cell banks for multiple patient infusions. Therapeutic efficacy of these products depends on the recovery of viable and functional cells post-thaw. Post-thaw loss of viability and cytolytic activity results in large, and often variable, discrepancies between the intended cell dose (based on counts at cryopreservation) and the actual dose administered. Compared to their highly activated state in fresh culture, post-thaw NK cells demonstrate critical changes in cytokine production, cytotoxic activity, in vivo proliferation and migration. When these NK cells are introduced into the highly immunosuppressive tumor microenvironment, the functional changes induced by cryopreservation further limits the clinical potential of these products. This report will review the impact of cryopreservation on ex vivo expanded NK cells and outlines strategies described in published studies to recover post-thaw function.

Reversible suppression of T cell function in the bone marrow microenvironment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.

MicroRNA regulation of natural killer cell development and function in leukemia.

MicroRNAs (miRNAs) are now recognized as important regulators of all cellular processes, including immune function and cancer survival. These evolutionary preserved, single-stranded, non-coding RNA molecules mediate important functional effects primarily through post-transcriptional regulation of protein expression. MiRNAs are known to mediate multiple oncogenic pathways in tumor cells, both tumor promoting and tumor suppressing. In addition to a direct tumor cell effect, miRNAs have also been shown to play a critical role in immune cell development, function and survival. Here we expand on previous reports to evaluate miRNA regulation in natural killer (NK) cells primarily in humans and focus on their influence on NK cell development and function in the setting of hematologic malignancies. In addition, we highlight the most recent miRNA discoveries in hematologic malignancies and discuss areas of future exploration relevant to the translational field of innate immunology and miRNA-based therapeutic intervention.

Human AML activates the aryl hydrocarbon receptor pathway to impair NK cell development and function.

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.

Acute Myeloid Leukemia: A Concise Review.

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder characterized by immature myeloid cell proliferation and bone marrow failure. Cytogenetics and mutation testing remain a critical prognostic tool for post induction treatment. Despite rapid advances in the field including new drug targets and increased understanding of the biology, AML treatment remains unchanged for the past three decades with the majority of patients eventually relapsing and dying of the disease. Allogenic transplant remains the best chance for cure for patients with intermediate or high risk disease. In this review, we discuss the landmark genetic studies that have improved outcome prediction and novel therapies.

Headache prevalence following recovery from TTP and aHUS.

Thrombotic thrombocytopenia purpura (TTP) and atypical hemolytic uremic syndromes (aHUS) are distinct clinical disorders characterized by hemolytic anemia, thrombocytopenia, microthrombi, and end organ damage. TTP is characterized by a low ADAMTS13 activity level at diagnosis of <10 % ADAMTS13 activity, while aHUS is characterized as having >10 % ADAMTS13 activity. Despite clinical remission, survivors of thrombotic microangiopathy suffer significant comorbidity and decreased quality of life (QOL) than their healthy counterparts. The reason for this is unclear. Is it a lingering effect from their initial acute episode or ongoing subclinical injury/inflammation despite clinical remission? Common clinical complaints validated in practice include increased depression, deficits in memory, concentration, mood, and mental endurance. We suspect headaches may be an important clinical tool toward understanding patient morbidity and decreased QOL. To date, no studies report headache frequency or severity in this population. To answer this question, adult patients >3 months since their last acute episode of TTP or aHUS were approached to take a Headache Impact Test (HIT-6) survey. Between June 1, 2013 and May 30, 2014, 31 patients in remission (21 patients with prior TTP and 10 patients with prior aHUS) completed the HIT-6 survey. The survey scores were then compared to the HIT-6 normative population data established by Qualitymetric incorporated. Overall, TTP patients had a significantly higher average HIT-6 score of 59.9 compared to an average HIT-6 score of 51 seen in sex-matched controls (SD 9.6, p value 0.002). No significant difference was seen in the HIT-6 scores of aHUS patients. Of TTP patients studied, approximately 57 % (12/21) had three or more episodes and were >24 months since the last episode. The average time since last acute episode in TTP patients was 37.5 months. There was no significant correlation between TTP survivor HIT-6 scores and the number of prior episodes (1, 2, or >3) or timing from the last episode (3-6, 7-12, 13-24, or >24 months). About 19/21 (90 %) patients, who are TTP survivors, also had a normal ADAMTS13 activity level (>10 %) on the day of the survey. Our study suggests that headaches have significant impact on TTP survivors and should be followed in the clinical setting to prevent undue patient morbidity. Larger studies are needed to understand how headaches impact long-term survival and risk of relapse.