Durable responses in acute lymphoblastic leukaemia with the use of FLT3 and IDH inhibitors.

Data regarding the use of FMS-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1/2 (IDH1/2) inhibitors in acute lymphoblastic leukaemia (ALL) are lacking. We identified 14 patients with FLT3- or IDH1/2-mutated ALL. Three early T-cell precursor-ALL patients received FLT3 or IDH2 inhibitors. Patient 1 maintains a complete remission (CR) with enasidenib after intolerance to chemotherapy. Patient 2 maintained a CR for 27 months after treatment with enasidenib for relapsed disease. Patient 3 was treated with venetoclax and gilteritinib at the time of relapse and maintained a CR with gilteritinib for 8 months. These cases suggest that FLT3 and IDH inhibitors could represent a viable therapeutic option for ALL patients with these mutations.

Sequential tumor molecular profiling identifies likely germline variants.

PURPOSE: To identify likely germline DNA variants from sequential tumor profiling data from hematopoietic malignancies (HMs). METHODS: The coefficient of variance was calculated from variant allele frequency of next-generation sequencing assays. Variants' likelihood of being germline was ranked on a 1 to 5 scale. Outcomes were examined in patients with such variants. RESULTS: In a pilot set of 33 genes, 89% of grade 1, 77% of grade 2, 62% of grade 3, 52% of grade 4, and 21% of grade 5 variants were confirmed to be germline. Among those, 22% were pathogenic or likely pathogenic in genes recognized as conferring hereditary HM risk, including BRCA1/2, CHEK2, CSF3R, and DDX41. To determine if this approach identified genes with known autosomal dominant inheritance, we analyzed sequential data from 1336 genes in 1135 HM patients. Among unique variants, 16% occurred in hereditary HM genes, and 15% were deleterious. Patients with grade 1/2 alleles had decreased survival 2 years after initial molecular testing (78% versus 88%, P = .0037) and increased all-cause mortality compared with those without (hazard ratio 2.02, 95% CI 1.18-3.46, P = .019). CONCLUSION: Variant germline status may be predicted using sequential tumor profiling and patients with likely germline variants experience inferior outcomes compared with those without.

Socioeconomic determinants of the biology and outcomes of acute lymphoblastic leukemia in adults.

ABSTRACT: Various socioeconomic and biologic factors affect cancer health disparities and differences in health outcomes. To better characterize the socioeconomic vs biologic determinants of acute lymphoblastic leukemia (ALL) outcomes, we conducted a single-institution, retrospective analysis of adult patients with ALL treated at the University of Chicago (UChicago) from 2010 to 2022 and compared our outcomes with the US national data (the Surveillance, Epidemiology, and End Results [SEER] database). Among 221 adult patients with ALL treated at UChicago, BCR::ABL1 was more frequent in patients with higher body mass index (BMI; odds ratio [OR], 7.64; 95% confidence interval [CI], 1.17-49.9) and non-Hispanic Black (NHB) ancestry (59% vs 24% in non-Hispanic White (NHW) and 20% in Hispanic patients; P = .001). In a multivariable analysis, age (hazard ratio [HR], 6.93; 95% CI, 2.27-21.1) and higher BMI at diagnosis (HR, 10.3; 95% CI, 2.56-41.5) were independent predictors of poor overall survival (OS). In contrast, race or income were not predictors of OS in the UChicago cohort. Analysis of the national SEER database (2010-2020) demonstrated worse survival outcomes in Hispanic and NHB patients than in NHW patients among adolescent and young adults (AYAs) but not in older adults (aged >40 years). Both AYA and older adult patients with higher median household income had better OS than those with lower income. Therefore, multidisciplinary medical care coupled with essential supportive care services offered at centers experienced in ALL care may alleviate the socioeconomic disparities in ALL outcomes in the United States.

Loss of thymocyte competition underlies the tumor suppressive functions of the E2a transcription factor in T-ALL.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is frequently associated with increased expression of the E protein transcription factor inhibitors TAL1 and LYL1. In mouse models, ectopic expression of TAL1 or LYL1 in T cell progenitors, or inactivation of E2A, is sufficient to predispose mice to develop T-ALL. How E2A suppresses thymocyte transformation is currently unknown. Here, we show that early deletion of E2a, prior to the DN3 stage, was required for robust leukemogenesis and was associated with alterations in thymus cellularity, T cell differentiation, and gene expression in immature CD4+CD8+ thymocytes. Introduction of wild-type thymocytes into mice with early deletion of E2a prevented leukemogenesis, or delayed disease onset, and impacted the expression of multiple genes associated with transformation and genome instability. Our data indicate that E2A suppresses leukemogenesis by promoting T cell development and enforcing inter-thymocyte competition, a mechanism that is emerging as a safeguard against thymocyte transformation. These studies have implications for understanding how multiple essential regulators of T cell development suppress T-ALL and support the hypothesis that thymocyte competition suppresses leukemogenesis.

Acute Lymphoblastic Leukemia with Myeloid Mutations Is a High-Risk Disease Associated with Clonal Hematopoiesis.

Myeloid neoplasms arise from preexisting clonal hematopoiesis (CH); however, the role of CH in the pathogenesis of acute lymphoblastic leukemia (ALL) is unknown. We found that 18% of adult ALL cases harbored TP53, and 16% had myeloid CH-associated gene mutations. ALL with myeloid mutations (MyM) had distinct genetic and clinical characteristics, associated with inferior survival. By using single-cell proteogenomic analysis, we demonstrated that myeloid mutations were present years before the diagnosis of ALL, and a subset of these clones expanded over time to manifest as dominant clones in ALL. Single-cell RNA sequencing revealed upregulation of genes associated with cell survival and resistance to apoptosis in B-ALL with MyM, which responds better to newer immunotherapeutic approaches. These findings define ALL with MyM as a high-risk disease that can arise from antecedent CH and offer new mechanistic insights to develop better therapeutic and preventative strategies. SIGNIFICANCE: CH is a precursor lesion for lymphoblastic leukemogenesis. ALL with MyM has distinct genetic and clinical characteristics, associated with adverse survival outcomes after chemotherapy. CH can precede ALL years before diagnosis, and ALL with MyM is enriched with activated T cells that respond to immunotherapies such as blinatumomab. See related commentary by Iacobucci, p. 142.

Dual Targeting of Apoptotic and Signaling Pathways in T-Lineage Acute Lymphoblastic Leukemia.

PURPOSE: Relapsed T-acute lymphoblastic leukemia (T-ALL) has limited treatment options. We investigated mechanisms of resistance to BH3 mimetics in T-ALL to develop rational combination strategies. We also looked at the preclinical efficacy of NWP-0476, a novel BCL-2/BCL-xL inhibitor, as single agent and combination therapy in T-ALL. EXPERIMENTAL DESIGN: We used BH3 profiling as a predictive tool for BH3 mimetic response in T-ALL. Using isogenic control, venetoclax-resistant (ven-R) and NWP-0476-resistant (NWP-R) cells, phosphokinase array was performed to identify differentially regulated signaling pathways. RESULTS: Typical T-ALL cells had increased dependence on BCL-xL, whereas early T-precursor (ETP)-ALL cells had higher BCL-2 dependence for survival. BCL-2/BCL-xL dual inhibitors were effective against both subtypes of T-lineage ALL. A 71-protein human phosphokinase array showed increased LCK activity in ven-R cells, and increased ACK1 activity in ven-R and NWP-R cells. We hypothesized that pre-TCR and ACK1 signaling pathways are drivers of resistance to BCL-2 and BCL-xL inhibition, respectively. First, we silenced LCK gene in T-ALL cell lines, which resulted in increased sensitivity to BCL-2 inhibition. Mechanistically, LCK activated NF-κB pathway and the expression of BCL-xL. Silencing ACK1 gene resulted in increased sensitivity to both BCL-2 and BCL-xL inhibitors. ACK1 signaling upregulated AKT pathway, which inhibited the pro-apoptotic function of BAD. In a T-ALL patient-derived xenograft model, combination of NWP-0476 and dasatinib demonstrated synergy without major organ toxicity. CONCLUSIONS: LCK and ACK1 signaling pathways are critical regulators of BH3 mimetic resistance in T-ALL. Combination of BH3 mimetics with tyrosine kinase inhibitors might be effective against relapsed T-ALL.

Loss of thymocyte competition underlies the tumor suppressive functions of the E2a transcription factor in T lymphocyte acute lymphoblastic leukemia.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is frequently associated with increased expression of the E protein transcription factor inhibitors TAL1 and LYL1. In mouse models, ectopic expression of Tal1 or Lyl1 in T cell progenitors or inactivation of E2a, is sufficient to predispose mice to develop T-ALL. How E2a suppresses thymocyte transformation is currently unknown. Here, we show that early deletion of E2a , prior to the DN3 stage, was required for robust leukemogenesis and was associated with alterations in thymus cellularity, T cell differentiation, and gene expression in immature CD4+CD8+ thymocytes. Introduction of wild-type thymocytes into mice with early deletion of E2a prevented leukemogenesis, or delayed disease onset, and impacted the expression of multiple genes associated with transformation and genome instability. Our data indicate that E2a suppresses leukemogenesis by promoting T cell development and enforcing inter-thymocyte competition, a mechanism that is emerging as a safeguard against thymocyte transformation. These studies have implications for understanding how multiple essential regulators of T cell development suppress T-ALL and support the hypothesis that thymus cellularity is a determinant of leukemogenesis.

Paired bone marrow and peripheral blood samples demonstrate lack of widespread dissemination of some CH clones.

Clonal hematopoiesis (CH) represents clonal expansion of mutated hematopoietic stem cells detectable in the peripheral blood or bone marrow through next generation sequencing. The current prevailing model posits that CH mutations detected in the peripheral blood mirror bone marrow mutations with clones widely disseminated across hematopoietic compartments. We sought to test the hypothesis that all clones are disseminated throughout hematopoietic tissues by comparing CH in hip vs peripheral blood specimens collected at the time of hip replacement surgery. Here, we show that patients with osteoarthritis have a high prevalence of CH, which involve genes encoding epigenetic modifiers and DNA damage repair pathway proteins. Importantly, we illustrate that CH, including clones with variant allele frequencies >10%, can be confined to specific bone marrow spaces and may be eliminated through surgical excision. Future work will define whether clones with somatic mutations in particular genes or clonal fractions of certain sizes are either more likely to be localized or are slower to disseminate into the peripheral blood and other bony sites.

Allogeneic hematopoietic stem cell transplant outcomes in adults with inherited myeloid malignancies.

There is increasing recognition that pathogenic germ line variants drive the development of hematopoietic cancers in many individuals. Currently, patients with hereditary hematologic malignancies (HHMs) receive similar standard therapies and hematopoietic stem cell transplant (HSCT) approaches as those with sporadic disease. We hypothesize that patients with myeloid malignancies and deleterious germ line predisposition variants have different posttransplant outcomes than those without such alleles. We studied 472 patients with myeloid neoplasms, of whom 26% had deleterious germ line variants and 34% underwent HSCT. Deleterious germ line variants in CHEK2 and DDX41 were most commonly seen in American and Australian cohorts, respectively. Patients with deleterious germ line DDX41 variants had a higher incidence of severe (stage 3-4) acute graft-versus-host disease (GVHD) (38%) than recipients with deleterious CHEK2 variants (0%), other HHM variants (12%), or patients without such germ line variants (9%) (P = .002). Importantly, the use of posttransplant cyclophosphamide reduced the risk of severe acute GVHD in patients receiving HSCT for deleterious germ line DDX41-associated myeloid neoplasms (0% vs 53%, P = .03). Based on these results, we advocate the use of posttransplant cyclophosphamide when individuals with deleterious germ line DDX41 variants undergo allogeneic HSCT for myeloid malignancies, even when transplantation has been performed using wild-type donors.

Measurable residual disease in acute lymphoblastic leukemia: methods and clinical context in adult patients.

Measurable residual disease (MRD) is the most powerful independent predictor of risk of relapse and long-term survival in adults and children with acute lymphoblastic leukemia (ALL). For almost all patients with ALL there is a reliable method to evaluate MRD, which can be done using multi-color flow cytometry, quantitative polymerase chain reaction to detect specific fusion transcripts or immunoglobulin/T-cell receptor gene rearrangements, and high-throughput next-generation sequencing. While next-generation sequencing-based MRD detection has been increasingly utilized in clinical practice due to its high sensitivity, the clinical significance of very low MRD levels (<10-4) is not fully characterized. Several new immunotherapy approaches including blinatumomab, inotuzumab ozogamicin, and chimeric antigen receptor T-cell therapies have demonstrated efficacy in eradicating MRD in patients with B-ALL. However, new approaches to target MRD in patients with T-ALL remain an unmet need. As our MRD detection assays become more sensitive and expanding novel therapeutics enter clinical development, the future of ALL therapy will increasingly utilize MRD as a criterion to either intensify or modify therapy to prevent relapse or de-escalate therapy to reduce treatment-related morbidity and mortality.

B-cell lymphoma-2 downregulation is a useful feature supporting a neoplastic phenotype in mature T-cell lymphomas.

Normal T cells express high levels of B-cell lymphoma-2 (BCL2) protein, and data regarding BCL2 expression status and its diagnostic utility in T-cell lymphoma are scarce. We evaluated BCL2 expression in a series of mature T-cell lymphoproliferations (TCLs) including indolent and more recently recognized entities (follicular helper T-cell [TFH] lymphomas). Sixty-six neoplastic biopsies (60 patients) representing mature nodal, extranodal, and leukemia T-cell neoplasms were collected from three institutes (2 US and 1 Japan) and were compared with reactive T cells in 8 benign tissues/blood and 9 T cell-rich B-cell proliferations. BCL2 immunostaining was performed and scored based on intensity-weighted H-score (0-300). Next-generation sequencing (NGS; 5 cases), BCL2 gene sequencing, and real-time polymerase chain reaction (PCR; 3 cases) were conducted. Association of H-score with overall survival (using proportional hazards modeling) was assessed in nonleukemic TCLs. Most TCLs showed significantly downregulated median BCL2 H-score (125, range: 18-300) with the exception of T-cell prolymphocytic leukemia and hepatosplenic T-cell lymphoma, both of which showed uniform strong retention of BCL2 as did the 8 reactive tissues (median H-score: 280; p = 0.000). Notably all TFH lymphoma CD4 neoplastic T cells, subcutaneous panniculitis-like T-cell lymphoma, CD8 adipocyte-rimming T cells, and T-cell large lymphocyte leukemia with pathogenic STAT5B and TP53 mutation showed BCL2 downregulation. No BCL2 mutations were observed by NGS or sequencing with decreased BCL2 mRNA transcripts by real-time PCR. BCL2 downregulation is pervasive among many TCLs and unrelated to any mutations. There is utility for BCL2 immunostaining in some challenging situations as discussed in this article.

Cytopenias After CD19 Chimeric Antigen Receptor T-Cells (CAR-T) Therapy for Diffuse Large B-Cell Lymphomas or Transformed Follicular Lymphoma: A Single Institution Experience.

INTRODUCTION: Patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) have poor outcomes. Treatment with CD19 chimeric antigen receptor (CAR-T) cells, tisagenlecleucel and axicabtagene ciloleucel, has been associated with improved outcomes. Cytopenias were observed in clinical trials with both products; however, little is known regarding the patterns and outcomes of these cytopenias. SUBJECTS AND METHODS: We reviewed DLBCL patients (n=32) receiving either product between January and September 2018 at our institution. RESULTS: Median duration of leukopenia, neutropenia, lymphopenia, anemia, and thrombocytopenia was 49, 9, 117.5, 125, and 95.5 days after CAR-T infusion, respectively. Filgrastim was used in 63% of patients, and 50% of patients received red cell or platelet transfusions. With the exception of neutropenia, increase in the duration of cytopenia of any lineage was associated with improvement in progression-free survival, and in overall survival in case of anemia. There was no association between the duration of cytopenias with either cytokine release syndrome or neurotoxicity. DISCUSSION: Our data suggest a correlation between cytopenias and survival outcomes after CD19 CAR-T therapy. If validated, cytopenia may be proven useful as a biomarker of response and survival after CAR-T therapy.

Genetics of Myelodysplastic Syndromes.

Myelodysplastic syndrome (MDS) describes a heterogeneous group of bone marrow diseases, now understood to reflect numerous germline and somatic drivers, characterized by recurrent cytogenetic abnormalities and gene mutations. Precursor conditions including clonal hematopoiesis of indeterminate potential and clonal cytopenia of undetermined significance confer risk for MDS as well as other hematopoietic malignancies and cardiovascular complications. The future is likely to bring an understanding of those individuals who are at the highest risk of progression to MDS and preventive strategies to prevent malignant transformation.

Genomic analysis of cellular hierarchy in acute myeloid leukemia using ultrasensitive LC-FACSeq.

Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

Cedazuridine/decitabine: from preclinical to clinical development in myeloid malignancies.

Since the US Food and Drug Administration (FDA) approvals of parenteral decitabine and azacitidine, DNA methyltransferase inhibitors, otherwise referred to as DNA hypomethylating agents (HMAs), have been a mainstay in the treatment of higher-risk myelodysplastic syndromes. The development of oral HMAs has been an area of active interest; however, oral bioavailability has been quite poor due to rapid metabolism by cytidine deaminase (CDA). This led to the development of the novel CDA inhibitor cedazuridine, which was combined with an oral formulation of decitabine. Preclinical work demonstrated a pharmacokinetic and pharmacodynamic profile approximate to parenteral decitabine, leading to early-phase clinical trials of oral cedazuridine-decitabine (C-DEC) in myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML). A combination of oral decitabine 35 mg with oral cedazuridine 100 mg was established as the recommended phase 2 dose. Phase 2 data confirmed bioequivalence of C-DEC when compared with parenteral decitabine, and a larger phase 3 trial has demonstrated similar results, leading to the FDA approval of C-DEC for use in intermediate/high-risk myelodysplastic syndrome (MDS) and CMML. This review will focus upon the current role of HMA therapy in MDS/CMML, preclinical and clinical development of C-DEC, and potential roles of oral HMA therapy in myeloid malignancies moving forward.

Pretreatment with LCK inhibitors chemosensitizes cisplatin-resistant endometrioid ovarian tumors.

BACKGROUND: Ovarian cancer is the most fatal gynecologic malignancy in the United States. While chemotherapy is effective in the vast majority of ovarian cancer patients, recurrence and resistance to standard systemic therapy is nearly inevitable. We discovered that activation of the non-receptor tyrosine kinase Lymphocyte Cell-Specific Protein-Tyrosine Kinase (LCK) promoted cisplatin resistance. Here, we hypothesized that treating high grade, platinum resistant endometrioid cancer cells with an LCK inhibitor (LCKi) followed by co-treatment with cisplatin would lead to increased cisplatin efficacy. Our objective was to assess clinical outcomes associated with increased LCK expression, test our hypothesis of utilizing LCKi as pre-treatment followed by co-treatment with cisplatin in platinum resistant ovarian cancer in vitro, and evaluate our findings in vivo to assess LCKi applicability as a therapeutic agent. RESULTS: Kaplan-Meier (KM) plotter data indicated LCK expression is associated with significantly worse median progression-free survival (HR 3.19, p = 0.02), and a trend toward decreased overall survival in endometrioid ovarian tumors with elevated LCK expression (HR 2.45, p = 0.41). In vitro, cisplatin resistant ovarian endometrioid cells treated first with LCKi followed by combination LCKi-cisplatin treatment showed decreased cell viability and increased apoptosis. Immunoblot studies revealed LCKi led to increased expression of phosphorylated H2A histone family X ([Formula: see text]-H2AX), a marker for DNA damage. In vivo results demonstrate treatment with LCKi followed by LCKi-cisplatin led to significantly slowed tumor growth. CONCLUSIONS: We identified a strategy to therapeutically target cisplatin resistant endometrioid ovarian cancer leading to chemosensitization to platinum chemotherapy via treatment with LCKi followed by co-treatment with LCKi-cisplatin.