Inflammatory signals from fatty bone marrow support DNMT3A driven clonal hematopoiesis.

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3AMut-HSCs when exposed to FBM. DNMT3AMut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3AMut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3AMut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3AMut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Aspergillus galactomannan detection in exhaled breath condensate compared to bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in immunocompromised patients.

OBJECTIVES: Exhaled breath condensate (EBC) is a noninvasive means of sampling the airways that has shown significant promise in the diagnosis of many disorders. There have been no reports of its usefulness in the detection of galactomannan (GM), a component of the cell wall of Aspergillus. The suitability of EBC for the detection of GM for the diagnosis of invasive aspergillosis (IA) using the Platelia Aspergillus enzyme-linked immunosorbent assay was investigated. METHODS: Prospective, cross-sectional study of lung transplant recipient and haemotologic malignancy patients at a university centre. EBC samples were compared to concomitant bronchoalveolar lavage (BAL) samples among lung transplant recipients and healthy controls. EBC was collected over 10 minutes using a refrigerated condenser according to the European Respiratory Society/American Thoracic Society recommendations, with the BAL performed immediately thereafter. RESULTS: A total of 476 EBC specimens with 444 matched BAL specimens collected from lung transplant recipients (n = 197) or haemotologic malignancy patients (n = 133) were examined. Both diluted and untreated EBC optical density (OD) values (0.0830, interquartile range (IQR) 0.0680-0.1040; and 0.1130, IQR 0.0940-0.1383), respectively, from all patients regardless of clinical syndrome were significantly higher than OD values in healthy control EBCs (0.0508, IQR 0.0597-0.0652; p < 0.0001). However, the OD index values did not correlate with the diagnosis of IA (44 samples were associated with IA). Furthermore, no significant correlation was found between EBC GM and the matched BAL specimen. CONCLUSIONS: GM is detectable in EBC; however, no correlation between OD index values and IA was noted in lung transplant recipients.

CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 in combination with NKs from AML patients in remission.

Despite the heterogeneity of acute myeloid leukemia (AML), overexpression of the interleukin-3 receptor-α (CD123) on both the more differentiated leukemic blast and leukemic stem cells (LSCs) provides a therapeutic target for antibody treatment. Here we present data on the potential clinical activity of the monoclonal antibody CSL362, which binds to CD123 with high affinity. We first validated the expression of CD123 by 100% (52/52) of patient samples and the correlation of NPM1 and FLT3-ITD mutations with the high frequency of CD123 in AML. In vitro studies demonstrated that CSL362 potently induced antibody-dependent cell cytotoxicity (ADCC) of AML blasts including CD34+CD38-CD123+ LSCs by natural killer cells (NKs). Importantly, compared with healthy donor (HD) NKs, NKs drawn from AML patients in remission had a comparable ADCC activity against leukemic cells; of note, during remission, immature NKs were five times higher in AML patients than that in HDs. Significantly, we report a case where leukemic cells were resistant to autologous ADCC; however, the blasts were effectively lysed by CSL362 together with donor-derived NKs after allogeneic hematopoietic stem cell transplantation. These studies highlight CSL362 as a promising therapeutic option following chemotherapy and transplant so as to improve the outcome of AML patients.

Expression of CD4 is correlated with an unfavorable prognosis in wild-type NPM1, FLT3-ITD-negative cytogenetically normal adult acute myeloid leukemia.

INTRODUCTION: In the cytogenetically normal population of AML (CN-AML), FLT3-ITD-positive and wild-type NPM1 is correlated with a worse outcome, and FLT3-ITD-negative with NPM1-mut is correlated with a better outcome. This leaves a large subpopulation of CN-AML patients without NPM1 or FLT3-ITD mutations with heterogeneous outcomes with overall survivals (OS) ranging from several weeks to years. Therefore, new prognostic markers are needed to better risk stratify this subset of patients. METHODS: The retrospective study included 60 de novo adult AML patients diagnosed at our institution with normal karyotype, no FLT3-ITD or NPM1 mutations, and who did not receive allogeneic hematopoietic stem cell transplantations. We investigated the prognostic significance of immunophenotypic markers and clinical laboratory features in this double-negative population. RESULTS: Older age (>60) and CD4 expression (14%) were significantly correlated with shorter event-free survival (EFS) (P < 0.001, P = 0.016, respectively). Expression of CD56 (12%), as well as lack of CD34 expression (19% of the cases), was also associated with a worse EFS (P = 0.048, P = 0.028, respectively). On multivariable analysis, CD4 expression and old age (>60) were identified as independent predictors for worse EFS (P = 0.016; P = 0.001, respectively) and OS (P = 0.048; P = 0.028, respectively). CONCLUSIONS: Our results indicate that CD4 expression and older age are adverse prognostic factors in wild-type NPM1, FLT3-ITD-negative CN-AML.

Successful treatment of gastrointestinal mucormycosis in an adult with acute leukemia: case report and literature review.

Mucormycosis has emerged as an important cause of invasive fungal infection in patients with hematologic malignancies. Gastrointestinal mucormycosis is an unusual presentation of this invasive fungal infection, and it causes considerable morbidity and mortality. Such outcomes are due in part to a nonspecific presentation that results in delays in diagnosis and treatment. Successful treatment of gastrointestinal mucormycosis involves surgical debridement and appropriate antifungal therapy.

Biological and clinical consequences of NPM1 mutations in AML.

Acute myeloid leukemia (AML) is characterized by accumulation of myeloid cells in the bone marrow because of impaired differentiation and proliferation, resulting in hematopoietic insufficiency. NPM1 is one of the most commonly mutated genes in AML, present in 20-30% of cases. Mutations in NPM1 represent a distinct entity in the World Health Organization (WHO) classification and commonly indicate a better risk prognosis. In this review, we discuss the many functions of NPM1, the consequence of mutations in NPM1 and possible mechanisms through which mutations lead to leukemogenesis. We also discuss clinical consequences of mutations, associated gene expression patterns and the role of NPM1 mutations in informing prognosis and therapeutic decisions and predicting relapse in AML.

Venous thromboembolism prevention during asparaginase-based therapy for acute lymphoblastic leukemia.

BACKGROUND: Venous thromboembolism (vte) is a recognized complication in patients treated with asparaginase-containing chemotherapy regimens; the optimal preventive strategy is unclear. We assessed the safety and efficacy of prophylaxis using low-dose low molecular weight heparin in adult patients with acute lymphoblastic leukemia in complete remission treated with an asparaginase-based post-remission chemotherapy regimen. METHODS: As part of the intensification phase of the Dana-Farber Cancer Institute 91-01 regimen, asparaginase was administered weekly to 41 consecutive patients for 21-30 weeks; these patients also received prophylaxis with enoxaparin 40 mg daily (60 mg for patients ≥80 kg). Outcomes were assessed against outcomes in a comparable cohort of 99 patients who received the same chemotherapy regimen without anticoagulation prophylaxis. RESULTS: The overall rate of symptomatic venous thrombosis was not significantly different in the prophylaxis and non-prophylaxis cohorts (18.92% and 21.74% respectively). Among patients receiving prophylaxis, vte occurred in higher proportion in those who weighed at least 80 kg (42.86% vs. 4.35%, p = 0.0070). No major bleeding complications occurred in the prophylaxis group (minor bleeding: 8.1%). CONCLUSIONS: Prophylaxis with low-dose enoxaparin during the intensification phase was safe, but was not associated with a lower overall proportion of vte.

INPP4B overexpression is associated with poor clinical outcome and therapy resistance in acute myeloid leukemia.

In this study, we investigated the role of inositol polyphosphate-4-phosphatase, type-II (INPP4B) in acute myeloid leukemia (AML). We observed that AML patients with high levels of INPP4B (INPP4B(high)) had poor response to induction therapy, shorter event-free survival and shorter overall survival. Multivariate analyses demonstrated that INPP4B(high) was an independent predictor of poor prognosis, significantly improving current predictive models, where it outperformed conventional biomarkers including FLT3-ITD and NPM1. Furthermore, INPP4B(high) effectively segregated relative risk in AML patients with normal cytogenetics. The role of INPP4B on the biology of leukemic cells was assessed in vitro. Overexpression of INPP4B in AML cell lines enhanced colony formation potential, recapitulated the chemotherapy resistance observed in AML patients and promoted proliferation in a phosphatase-dependent, and Akt-independent manner. These findings reveal that INPP4B(high) has an unexpected role consistent with oncogenesis in AML, in contrast to its previously reported tumor-suppressive role in epithelial cancers. Overall, we propose that INPP4B is a novel prognostic biomarker in AML that has potential to be translated into clinical practice both as a disease marker and therapeutic target.

Dominant-negative Ikaros cooperates with BCR-ABL1 to induce human acute myeloid leukemia in xenografts.

Historically, our understanding of mechanisms underlying human leukemogenesis are inferred from genetically engineered mouse models. Relatively, few models that use primary human cells recapitulate the full leukemic transformation as assayed in xenografts and myeloid transformation is infrequent. We report a humanized experimental leukemia model where xenografts develop aggressive acute myeloid leukemia (AML) with disseminated myeloid sarcomas within 4 weeks following transplantation of cord blood transduced with vectors expressing BCR-ABL1 and a dominant-negative isoform of IKAROS, Ik6. Ik6 induced transcriptional programs in BCR-ABL1-transduced progenitors that contained repressed B-cell progenitor programs, along with strong stemness, proliferation and granulocyte-monocytic progenitor (GMP) signatures-a novel combination not induced in control groups. Thus, wild-type IKAROS restrains stemness properties and has tumor suppressor activity in BCR-ABL1-initiated leukemia. Although IKAROS mutations/deletions are common in lymphoid transformation, they are found also at low frequency in AML that progress from a prior myeloproliferative neoplasm (MPN) state. Our experimental system provides an excellent model to gain insight into these rare cases of AML transformation and the properties conferred by IKAROS loss of function as a secondary mutation. More generally, our data points to the importance of deregulated stemness/lineage commitment programs in human myeloid leukemogenesis.

Evidence-based guidelines for the use of tyrosine kinase inhibitors in adults with Philadelphia chromosome-positive or BCR-ABL-positive acute lymphoblastic leukemia: a Canadian consensus.

Adult Philadelphia chromosome-positive (Ph+) or BCR-ABL-positive (BCR-ABL+) acute lymphoblastic leukemia (all) is an acute leukemia previously associated with a high relapse rate, short disease-free survival, and poor overall survival. In adults, allogeneic hematopoietic cell transplant in first remission remains the only proven curative strategy for transplant-eligible patients. The introduction of tyrosine kinase inhibitors (tkis) in the treatment of patients with Ph+ or BCR-ABL+ all has significantly improved the depth and duration of complete remission, allowing more patients to proceed to transplantation. Although tkis are now considered a standard of care in this setting, few randomized trials have examined the optimal use of tkis in patients with Ph+ all. Questions of major importance remain, including the best way to administer these medications, the choice of tki to administer, and the schedule and the duration to use. We present the results of a systematic review of the literature with consensus recommendations based on the available evidence.

Do quality of life or physical function at diagnosis predict short-term outcomes during intensive chemotherapy in AML?

BACKGROUND: Intensive chemotherapy (IC) used to treat acute myeloid leukemia (AML) is associated with toxicity, particularly in older adults. Emerging data suggest that baseline quality of life (QOL) and physical function may predict outcomes in oncology, although data in AML are limited. We investigated the association between baseline QOL and physical function with short-term treatment outcomes in adults and elderly AML patients. MATERIALS AND METHODS: We conducted a prospective, longitudinal study of adults (age 18+) AML patients undergoing IC. Before starting IC, patients completed the European Organisation for the Research and Treatment of Cancer (EORTC) 30-item questionnaire (QLQ-C30) and Functional Assessment of Cancer Therapy Fatigue subscale (FACT-Fatigue) in addition to physical function tests (grip strength, timed chair stands, 2-min walk test). Outcomes included 60-day mortality, intensive care unit (ICU) admission and achievement of complete remission (CR). Logistic regression was carried out to evaluate each outcome. RESULTS: Of the 239 patients (median age 57.5 years), 56.7% were male and median Charlson comorbidity score was 0. Sixty-day mortality, ICU admission and CR occurred in 9 (3.7%), 15 (6.3%) and 167 (69.9%) patients, respectively. Using univariate regression, neither QOL nor physical function at presentation was predictive of 60-day mortality (all P > 0.05), whereas ICU admission (P < 0.001) and remission status at 30 days (P = 0.007) were. Fatigue (P = 0.004) and role functioning (P = 0.003) were predictors of ICU admission; QOL and physical function were not. A higher Charlson score predicted ICU admission (P = 0.01) and remission status (P = 0.002). The cytogenetic risk group was associated with achievement of CR (P = 0.02); QOL and physical function were not (all P > 0.05). Findings were similar when patients age 60+ were examined. Relationships between fatigue and role functioning with ICU admission deserve further exploration. CONCLUSIONS: Baseline QOL and physical function tests in this prospective study were not associated with short-term mortality, ICU admission or achievement of CR after the first cycle of chemotherapy.

The prognostic impact of FLT3-ITD and NPM1 mutations in patients with relapsed acute myeloid leukemia and intermediate-risk cytogenetics.

Internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) and nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis, but less is known about their utility to predict outcomes at relapse. We retrospectively analysed outcomes of 70 patients with relapsed, intermediate-risk karyotype AML who received a uniform reinduction regimen, with respect to FLT3-ITD and NPM1 mutation status and first complete remission (CR1) duration. CR1 duration, but not molecular status, was significantly correlated with CR2 rate. On univariate analysis, patients with mutated FLT3-ITD (FLT3+) had significantly worse overall survival (OS) compared with those with neither an NPM1 nor FLT3-ITD mutation (NPM1-/FLT3-). On multivariate analysis, shorter CR1 duration was significantly correlated with inferior OS at relapse (P<0.0001), while FLT3 and NPM1 mutation status and age were not significantly correlated with OS. Patients who subsequently underwent allogeneic stem cell transplant (alloSCT) had a superior OS regardless of CR1 duration, but outcomes were better in patients with CR1 duration>12 months. In intermediate-risk karyotype AML patients receiving reinduction, CR1 duration remains the most important predictor of OS at relapse; FLT3-ITD and NPM1 status are not independent predictors of survival.

Gads (Grb2-related adaptor downstream of Shc) is required for BCR-ABL-mediated lymphoid leukemia.

Philadelphia chromosome-positive leukemias, including chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (B-ALL), are driven by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice receiving whole bone marrow transduced with BCR-ABL. Y177 of BCR-ABL is critical to the development of myeloid disease, and phosphorylation of Y177 has been shown to induce GRB2 binding to BCR-ABL, followed by activation of the Ras and phosphoinositide 3 kinase signaling pathways. We show that the GRB2-related adapter protein, GADS, also associates with BCR-ABL, specifically through Y177 and demonstrate that BCR-ABL-driven lymphoid disease requires Gads. BCR-ABL transduction of Gads(-/-) bone marrow results in short latency myeloid disease within 3-4 weeks of transplant, while wild-type mice succumb to both a longer latency lymphoid and myeloid diseases. We report that GADS mediates a unique BCR-ABL complex with SLP-76 in BCR-ABL-positive cell lines and B-ALL patient samples. These data suggest that GADS mediates lymphoid disease downstream of BCR-ABL through the recruitment of specific signaling intermediates.

Elevated calpain activity in acute myelogenous leukemia correlates with decreased calpastatin expression.

Calpains are intracellular cysteine proteases that have crucial roles in many physiological and pathological processes. Elevated calpain activity has been associated with many pathological states. Calpain inhibition can be protective or lethal depending on the context. Previous work has shown that c-myc transformation regulates calpain activity by suppressing calpastatin, the endogenous negative regulator of calpain. Here, we have investigated calpain activity in primary acute myelogenous leukemia (AML) blast cells. Calpain activity was heterogeneous and greatly elevated over a wide range in AML blast cells, with no correlation to FAB classification. Activity was particularly elevated in the CD34+CD38- enriched fraction compared with the CD34+CD38+ fraction. Treatment of the cells with the specific calpain inhibitor, PD150606, induced significant apoptosis in AML blast cells but not in normal equivalent cells. Sensitivity to calpain inhibition correlated with calpain activity and preferentially targeted CD34+CD38- cells. There was no correlation between calpain activity and p-ERK levels, suggesting the ras pathway may not be a major contributor to calpain activity in AML. A significant negative correlation existed between calpain activity and calpastatin, suggesting calpastatin is the major regulator of activity in these cells. Analysis of previously published microarray data from a variety of AML patients demonstrated a significant negative correlation between calpastatin and c-myc expression. Patients who achieved a complete remission had significantly lower calpain activity than those who had no response to treatment. Taken together, these results demonstrate elevated calpain activity in AML, anti-leukemic activity of calpain inhibition and prognostic potential of calpain activity measurement.

Older age is associated with similar quality of life and physical function compared to younger age during intensive chemotherapy for acute myeloid leukemia.

We examined the quality of life (QOL) and physical function over the first three cycles of intensive chemotherapy in 103 newly diagnosed younger (18-59 years, n=64) and older adults (age 60 or older, n=39) with acute myeloid leukemia. Both QOL and physical function were worse than normative data. QOL was fairly stable over time and similar in both age groups, whereas physical function generally improved over time, although the improvement was somewhat greater in younger than older adults. Compared to younger adults, older adults tolerate intensive chemotherapy quite well from QOL and physical function perspectives.

Phase I/II multicenter study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of AZD4877 in patients with refractory acute myeloid leukemia.

Eg5 (kinesin spindle protein) is a microtubule motor protein, essential for centrosome separation during mitosis. This Phase I/II, open-label, multicenter, two-part study investigated AZD4877, a potent Eg5 inhibitor, in patients with acute myeloid leukemia. Primary objectives were to determine the maximum tolerated dose (MTD) (part A), assess efficacy (part B) and determine the pharmacokinetic profile (parts A and B). Secondary objectives included assessment of safety and tolerability. AZD4877 was administered at a range of doses (2, 4, 7, 10, 13, 16 and 18 mg/day) as a 1-hour intravenous infusion on three consecutive days of a continuous 2-week schedule. The MTD in part A was defined as 16 mg/day based on dose-limiting stomatitis at 16 and 18 mg/day, hyperbilirubinemia at 16 mg/day and palmar-plantar erythrodysesthesia syndrome at 18 mg/day. Systemic exposure to AZD4877 generally increased with increasing dose whereas half-life was not dose dependent. No evaluable patients experienced a complete remission (CR) or CR with incomplete blood count recovery (CRi), demonstrating no evidence of AZD4877 efficacy in this population. Evidence of monoasters in all but the 4 mg/day dose group provided proof of mechanism for AZD4877. This study was terminated due to lack of efficacy. (ClinicalTrials.gov identifier NCT00486265).

Anti-leukemia effect of ex vivo expanded DNT cells from AML patients: a potential novel autologous T-cell adoptive immunotherapy.

CD3(+)CD56(-), CD4 and CD8 double negative T (DNT) cells comprise 1-3% of peripheral blood (PB) mononuclear cells. Their role in tumor immunity remains largely unknown due to their limited numbers and lack of effective methods to expand them. Here we developed a novel protocol by which DNT cells can be expanded ex vivo to therapeutic levels in 2 weeks from 13 of 16 acute myeloid leukemia (AML) patients during chemotherapy-induced complete remission. The expanded DNT cells expressed similar or higher levels of interferon-γ and tumor necrosis factor-α and Granzyme B as that seen in bulk activated CD8T cells from the same patient but significantly higher levels of perforin. The expanded DNT cells could effectively kill both allogeneic and autologous primary CD34(+) leukemic blasts isolated from PB of AML patients in a perforin-dependant manner. These results demonstrate, for the first time, that DNT cells from AML patients can be expanded ex vivo even after intensive chemotherapy, and are effective at killing both allogeneic and autologous primary leukemic blasts. These findings warrant studies further exploring the potential of DNT cells as a novel adjuvant immunotherapy to decrease the risk of relapse in patients with AML and, perhaps, other cancers.

A phase I/II study of imatinib plus reinduction therapy for c-kit-positive relapsed/refractory acute myeloid leukemia: inhibition of Akt activation correlates with complete response.

This phase I/II study evaluated imatinib as a c-kit inhibitor combined with mitoxantrone, etoposide and cytarabine therapy for patients with primary refractory or relapsed c-kit+ acute myeloid leukemia (AML). Imatinib was escalated through three dose levels in successive six patient cohorts. The combination was well tolerated up to 400 mg/day imatinib. Of 21 patients treated at this dose, 13 (62%) achieved complete response (CR), 7 (33%) were non-responders and one died during induction. The CR rate was 80% in patients with standard-risk karyotype versus 33% in patients with adverse karyotype. The CR rate for primary non-responders was 6/14 (43%) versus 7/7 (100%) for relapsed patients. AML blasts from peripheral blood were assayed for phosphorylated Akt (pAkt) and phosphorylated ERK (pERK) by flow cytometry before to and after imatinib dosing. Of eight patients achieving CR with reinduction, seven demonstrated marked (≥60%) pAkt inhibition with imatinib therapy. In contrast, all the six non-responders to reinduction demonstrated <60% pAkt inhibition (P=0.005). There was no correlation between pERK inhibition and response to therapy. These results indicate that lack of pAkt inhibition in vivo is associated with resistance to reinduction therapy using this regimen. Further studies using agents that are able to inhibit Akt more effectively are warranted.