Meghali Goswami

A multigene array for measurable residual disease detection in AML patients undergoing SCT

AML is a diagnosis encompassing a diverse group of myeloid malignancies. Heterogeneous genetic etiology, together with the potential for oligoclonality within the individual patient, have made the identification of a single high-sensitivity marker of disease burden challenging. We developed a multiple gene measurable residual disease (MG-MRD) RQ–PCR array for the high-sensitivity detection of AML, retrospectively tested on 74 patients who underwent allo-SCT at the NHLBI in the period 1994–2012. MG-MRD testing on peripheral blood samples prior to transplantation demonstrated excellent concordance with traditional BM-based evaluation and improved risk stratification for post-transplant relapse and OS outcomes. Pre-SCT assessment by MG-MRD predicted all clinical relapses occurring in the first 100 days after allo-SCT compared with 57% sensitivity using WT1 RQ–PCR alone. Nine patients who were negative for WT1 prior to transplantation were correctly reclassified into a high-risk MG-MRD-positive group, associated with 100% post-transplant mortality. This study provides proof of principle that a multiple gene approach may be superior to the use of WT1 expression alone for AML residual disease detection.

Expression of putative targets of immunotherapy in acute myeloid leukemia and healthy tissues

The ability to target myeloid malignancies using immunotherapy through means other than allogeneic transplantation depends on the capability to target leukemic clones while sparing normal tissues. It is now possible to generate clinical grade ex-vivo expanded T cells specific for leukemia-associated antigens (LAAs) for use in adoptive cell therapy.1 Although a variety of putative LAAs in acute myeloid leukemia (AML) have been identified for use as potential targets for immunotherapy2, 3, 4, 5, 6, 7, 8 and consensus panels have attempted to prioritize generic cancer antigens,9 a comprehensive evidence-based list of AML antigen targets has not yet been established. As a first step toward this goal, we therefore analyzed, using quantitative real-time PCR, the gene expression of 65 potential LAAs (Supplementary Table S1) in de-identified, clinically annotated samples from 48 newly diagnosed untreated AML patients that were collected under institutional review board-approved protocols from three NCCN cancer centers.

Immunological effects of hypomethylating agents

ABSTRACT Introduction: Epigenetic changes resulting from aberrant methylation patterns are a recurrent observation in hematologic malignancies. Hypomethylating agents have a well-established role in the management of patients with high-risk myelodysplastic syndrome or acute myeloid leukemia. In addition to the direct effects of hypomethylating agents on cancer cells, there are several lines of evidence indicating a role for immune-mediated anti-tumor benefits from hypomethylating therapy. Areas covered: We reviewed the clinical and basic science literature for the effects of hypomethylating agents, including the most commonly utilized therapeutics azacitidine and decitabine, on immune cell subsets. We summarized the effects of hypomethylating agents on the frequency and function of natural killer cells, T cells, and dendritic cells. In particular, we highlight the effects of hypomethylating agents on expression of immune checkpoint inhibitors, leukemia-associated antigens, and endogenous retroviral elements. Expert commentary: In vitro and ex vivo studies indicate mixed effects on the function of natural killer, dendritic cells and T cells following treatment with hypomethylating agents. Clinical correlates of immune function have suggested that hypomethylating agents have immunomodulatory functions with the potential to synergize with immune checkpoint therapy for the treatment of hematologic malignancy, and has become an active area of clinical research.

Impaired B cell immunity in acute myeloid leukemia patients after chemotherapy

BackgroundChanges in adaptive immune cells after chemotherapy in adult acute myeloid leukemia (AML) may have implications for the success of immunotherapy. This study was designed to determine the functional capacity of the immune system in adult patients with AML who have completed chemotherapy and are potential candidates for immunotherapy.MethodsWe used the response to seasonal influenza vaccination as a surrogate for the robustness of the immune system in 10 AML patients in a complete remission post-chemotherapy and performed genetic, phenotypic, and functional characterization of adaptive immune cell subsets.ResultsOnly 2 patients generated protective titers in response to vaccination, and a majority of patients had abnormal frequencies of transitional and memory B-cells. B-cell receptor sequencing showed a B-cell repertoire with little evidence of somatic hypermutation in most patients. Conversely, frequencies of T-cell populations were similar to those seen in healthy controls, and cytotoxic T-cells demonstrated antigen-specific activity after vaccination. Effector T-cells had increased PD-1 expression in AML patients least removed from chemotherapy.ConclusionOur results suggest that while some aspects of cellular immunity recover quickly, humoral immunity is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. The observed B-cell abnormalities may explain the poor response to vaccination often seen in AML patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immunity and increased PD-1 expression shortly after chemotherapy might have implications for the success of several modalities of immunotherapy.

Novel Antigen Targets for Immunotherapy of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) was the first malignancy for which immunotherapy, in the form of allogeneic hematopoietic stem cell transplantation (allo-HSCT), was integrated into the standard of care. Allo-HSCT however is an imperfect therapy associated with significant morbidity and mortality while offering only incomplete prevention of AML clinical relapse. These limitations have motivated the search for AML-related antigens that might be used as more specific and effective targets of immunotherapy. While historically such investigations have focused on protein targets expressed uniquely in AML or at significantly higher levels than in normal tissues, this article will review recent discoveries which have identified a novel selection of potential antigen targets for AML immunotherapy, such as non-protein targets including lipids and carbohydrates, neo-antigens created from genetic somatic mutations or altered splicing and post-translational modification of protein targets, together with innovative ways to target overexpressed protein targets presented by cell surface peptide-MHC complexes. These novel antigens represent promising candidates for further development as targets of AML immunotherapy.

Molecular measurable residual disease testing of blood during AML cytotoxic therapy for early prediction of clinical response

PURPOSE Measurable residual disease (MRD) testing after initial chemotherapy treatment can predict relapse and survival in AML. However, it has not been established if repeat molecular or genetic testing during chemotherapy can offer information regarding the chemotherapy sensitivity of the leukemic clone. PATIENTS AND METHODS Blood from 45 adult AML patients at day 1 and 4 of induction (n = 35) or salvage (n = 10) cytotoxic chemotherapy was collected for both quantitative real-time PCR (qPCR) assessment (WT1) and next generation sequencing (NGS, >500x depth) of 49 gene regions recurrently mutated in MDS/AML. RESULTS The median age was 62 (23-78); 42% achieved a complete response. WT1 was overexpressed in most patients tested but was uninformative for very early MRD assessment. A median of 4 non-synonymous variants (range 0-7) were detected by DNA sequencing of blood on day 1 of therapy (median VAF: 29%). Only two patients had no variants detectable. All mutations remained detectable in blood on day 4 of intensive chemotherapy and remarkably the ratio of mutated to wild-type sequence was often maintained. This phenomenon was not limited to variants in DNMT3A, TET2 and ASXL1. The kinetics of NPM1 and TP53 variant burden early during chemotherapy appeared to be exceptions and exhibited consistent trends in this cohort. CONCLUSIONS Molecular testing of blood on day 4 of chemotherapy is not predictive of clinical response in AML. The observed stability in variant allele frequency suggests that cytotoxic therapy may have a limited therapeutic index for clones circulating in blood containing these mutations. Further validation is required to confirm the utility of monitoring NPM1 and TP53 kinetics in blood during cytotoxic therapy.

Human Bone Marrow Assessment by Single Cell RNA Sequencing, Mass Cytometry and Flow Cytometry

New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from twenty healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated good correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference dataset and highlights opportunities for further refinement.