Veena Fauble

Indication and management of allogeneic stem cell transplantation in primary myelofibrosis: A consensus process by an EBMT/ELN international working group

The aim of this work is to produce recommendations on the management of allogeneic stem cell transplantation (allo-SCT) in primary myelofibrosis (PMF). A comprehensive systematic review of articles released from 1999 to 2015 (January) was used as a source of scientific evidence. Recommendations were produced using a Delphi process involving a panel of 23 experts appointed by the European LeukemiaNet and European Blood and Marrow Transplantation Group. Key questions included patient selection, donor selection, pre-transplant management, conditioning regimen, post-transplant management, prevention and management of relapse after transplant. Patients with intermediate-2- or high-risk disease and age <70 years should be considered as candidates for allo-SCT. Patients with intermediate-1-risk disease and age <65 years should be considered as candidates if they present with either refractory, transfusion-dependent anemia, or a percentage of blasts in peripheral blood (PB) >2%, or adverse cytogenetics. Pre-transplant splenectomy should be decided on a case by case basis. Patients with intermediate-2- or high-risk disease lacking an human leukocyte antigen (HLA)-matched sibling or unrelated donor, should be enrolled in a protocol using HLA non-identical donors. PB was considered the most appropriate source of hematopoietic stem cells for HLA-matched sibling and unrelated donor transplants. The optimal intensity of the conditioning regimen still needs to be defined. Strategies such as discontinuation of immune-suppressive drugs, donor lymphocyte infusion or both were deemed appropriate to avoid clinical relapse. In conclusion, we provided consensus-based recommendations aimed to optimize allo-SCT in PMF. Unmet clinical needs were highlighted.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Outcomes of Allogeneic Hematopoietic Cell Transplantation in Patients with Myelofibrosis with Prior Exposure to Janus Kinase 1/2 Inhibitors

The impact of Janus kinase (JAK) 1/2 inhibitor therapy before allogeneic hematopoietic cell transplantation (HCT) has not been studied in a large cohort in myelofibrosis (MF). In this retrospective multicenter study, we analyzed outcomes of patients who underwent HCT for MF with prior exposure to JAK1/2 inhibitors. One hundred consecutive patients from participating centers were analyzed, and based on clinical status and response to JAK1/2 inhibitors at the time of HCT, patients were stratified into 5 groups: (1) clinical improvement (n = 23), (2) stable disease (n = 31), (3) new cytopenia/increasing blasts/intolerance (n = 15), (4) progressive disease: splenomegaly (n = 18), and (5) progressive disease: leukemic transformation (LT) (n = 13). Overall survival (OS) at 2 years was 61% (95% confidence interval [CI], 49% to 71%). OS was 91% (95% CI, 69% to 98%) for those who experienced clinical improvement and 32% (95% CI, 8% to 59%) for those who developed LT on JAK1/2 inhibitors. In multivariable analysis, response to JAK1/2 inhibitors (P = .03), dynamic international prognostic scoring system score (P = .003), and donor type (P = .006) were independent predictors of survival. Among the 66 patients who remained on JAK1/2 inhibitors until stopped for HCT, 2 patients developed serious adverse events necessitating delay of HCT and another 8 patients had symptoms with lesser severity. Adverse events were more common in patients who started tapering or abruptly stopped their regular dose ≥6 days before conditioning therapy. We conclude that prior exposure to JAK1/2 inhibitors did not adversely affect post-transplantation outcomes. Our data suggest that JAK1/2 inhibitors should be continued near to the start of conditioning therapy. The favorable outcomes of patients who experienced clinical improvement with JAK1/2 inhibitor therapy before HCT were particularly encouraging, and need further prospective validation.

Allogeneic stem cell transplantation for myeloproliferative neoplasm in blast phase

The prognosis for patients with Philadelphia-negative myeloproliferative neoplasms (MPN) who evolve into acute myeloid leukemia (AML) or blast phase (MPN-BP) is extremely poor. Although allogeneic stem cell transplantation (allo-SCT) is considered potentially curative, very few patients have been reported who have undergone allo-SCT for MPN-BP; therefore the success rate remains unknown. In a retrospective review, we identified 13 patients with an MPN transformation to blast phase after a median 9 years (range 5 months to 30 years); 8 (median age 55) continued to allo-SCT within 6 months. Induction chemotherapy cleared blood/marrow blasts in 60% (6/10) (2 declined therapy, 1 had early death). At the time of allo-SCT, 5/8 patients were in complete remission (CR) of their leukemia or had returned to MPN chronic phase (CP), 2 had residual blood blasts and 1 was refractory with >5% marrow blasts. At follow-up (median 20.3 months), 6 patients are alive in CR of both their leukemia/MPN. All 5 patients in CR/CP at pre-allo-SCT remain alive in remission, while 2/3 with persistent blood/marrow blasts relapsed and expired. We conclude that MPN-BP can be cured by allo-SCT in a significant percentage of patients, but that adequate leukemic clearance prior to allo-SCT offers an optimal outcome.

Ex vivo activity of BCL-2 family inhibitors ABT-199 and ABT-737 combined with 5-azacytidine in myeloid malignancies

Novel targeted therapies for the treatment of acute myeloid leukemia (AML) and other advanced myeloid malignancies are urgently needed. Recently we reported results from in vitro studies comparing the BCL-2, BCL-XL and BCL-w inhibitor ABT-737 (preclinical compound with similar inhibitory profile to ABT-263/navitoclax) with the selective BCL-2 inhibitor ABT-199, as single-agent and in combination with 5-azacytidine (5-Aza) in AML-derived cell lines [1]. This study demonstrated greater in vitro potency of ABT-737 in most AML-derived cell lines, as compared to ABT-199, including greater sensitization to 5-Aza. Additionally, we demonstrated that the combination of ABT-737 and 5-Aza exhibits strong synergy in short-term ex vivo cultures of myeloid malignancies, including de novo and secondary AML, myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPNs). Tsao et al. reported synergistic activity between 5-Aza and ABT-737 in AML as well [2]. Recently, Pan et al. published a comparison between single-agent ABT-737 or ABT-263 and ABT-199 in vitro and in animal models, demonstrating potent ABT-199 activity in AML, although ABT-263 was more potent in some primary samples [3]. However, none of these studies directly compared the potential of ABT-737 versus ABT-199 to sensitize the anti-leukemic activity of 5-Aza. Therefore, we directly compared the ability of ABT-737 versus ABT-199 to synergize with 5-Aza in myeloid malignancies using short-term ex vivo cultures of primary AML and MDS/chronic myelomonocytic leukemia (CMML) samples. In primary AML and MDS/CMML samples tested in ex vivo drug dose combination response assays (n = 5), all samples showed synergy, and the synergistic effect with 5-Aza was similar for ABT-199 and ABT-737 [Figures 1(A), 1(C) and 1(D)], making this the first published evidence, to our knowledge, that ABT-199 also synergizes with 5-Aza in a limited number of AML and MDS/CMML samples ex vivo. The level of ex vivo synergy for ABT-737 and 5-Aza was comparable to that previously published [1]. Synergy occurred at low nM doses of ABT-737 and ABT-199, mostly in the range of 40–160 nM for both compounds, doses which most frequently corresponded to maximal synergy. The respective 5-Aza concentrations shown in Figure 1 are 10–15% maximal effective concentration (EC10–15) doses for AML samples and EC10–20 doses for MDS/CMML samples, corresponding to 2.5 µM 5-Aza. Antagonism with 5-Aza was observed at high nanomolar doses (> 640 nM) of both ABT compounds, or with exceedingly high (> 200 µM), clinically irrelevant doses of 5-Aza (data not shown). Figure 1 5-Azacytidine synergy in combination with ABT-199 or ABT-737. 5-Azacytidine was applied to ex vivo cultures as a single dose upon experiment initiation and the readout for synergy analyses with CalcuSyn was relative cell number as measured with Cell Titer-Glo ... We also compared the single-agent activity of ABT-199 versus ABT-737 ex vivo. As highlighted above, we previously showed that, as single agent, ABT-737 was more potent than ABT-199 in vitro. However, consistent with recently published data [3], we find that ex vivo ABT-199 and ABT-737 were more similarly potent in AML samples, with EC50 values < 20 nM for both agents [Figure 1(B) and Tables I and ​andII].II]. Thus, the AML-derived cell line panel we previously evaluated had a greater dependency on BCL-XL and/or BCL-w in vitro than the primary samples we analyzed ex vivo. This is also consistent with our recent observations from BH3 profiling assays. BH3 profiling is a functional assay that uses BH3 peptides to indirectly assess induction of mitochondrial outer membrane permeabilization as a surrogate measurement of apoptosis. The BH3 peptides used are referred to as BH3 metrics when associated with BH3 profiling outputs. Previously, we showed that HRK (specific BH3 binding partner for BCL-XL) was important as a BH3 metric in vitro [4,5]; however, NOXA (MCL-1 specific binding partner) as a BH3 metric more strongly correlated with clinical 5-Aza response [1]. While single-agent EC50 values of ABT-737 and ABT-199 were more similar ex vivo than in vitro ABT-737 was still significantly more potent in some ex vivo samples [Figure 1(B) and Table 1]. Our observations are consistent with a recent report [3] showing that although ABT-199 and ABT-737/263 are often equipotent in ex vivo AML samples, some samples are more sensitive to ABT-737/263, while fewer samples are more sensitive to ABT-199. Th us, BCL-XL and BCL-w may play a more important role in modulating apoptosis in a number of cases of AML. Conversely, BCL-2 and hence ABT-199 may have a greater role in other cases of AML. Collectively, these data also support our previous suggestion, that dual BCL-2 family targeting drugs (i.e. BCL-XL and BCL-2, or BCL-XL and MCL-1) could hold more potential for the treatment of some myeloid neoplasms. However, whether more selective or broader BCL-2 family targeting compounds will have greater activity in myeloid malignancies remains an open question, since oncogenic dependency and compound selectivity influence therapeutic index. For example, the on-target side-effect of thrombocytopenia arising from BCL-XL inhibition by navitoclax has prevented further development in leukemias for now, while ABT-199 is currently being investigated as single-agent in an ongoing trial in patients with AML (ClinicalTrials.gov; NCT01994837). Which agent may be more advantageous, and for which patients, can ultimately only be determined clinically. Table I p-Values associated with single-agent ABT-737 and ABT-199 comparisons in AML samples with diff erential activity*. Table II Characteristics of primary samples testedex vivo. The pre-clinical data presented by our and Dr. Konopleva’s groups is promising with regard to the activity of ABT-737 in combination with 5-Aza. Herein we present the first evidence that ABT-199 also potently synergizes with 5-Aza in primary AML and MDS/CMML samples ex vivo. Based on our preclinical data, it can be suggested that such a combination may also be an effective strategy for patients refractory/resistant to monotherapy with 5-Aza or BCL-2 family inhibitors. Navitoclax may still have a role in some cases of AML where additional anti-apoptotic BCL-2 family members such as BCL-XL or BCL-w are operating, such as in erythroid-differentiated disease with high BCL-XL expression [6,7]. Additionally, MCL-1 is thought to be a critical anti-apoptotic gene in AML [8,9]. Evidence suggesting that 5-Aza can reduce MCL-1 protein levels in primary samples [2] provides further impetus to support 5-Aza in combination with ABT-199 or navitoclax, because neither of these compounds inhibits MCL-1. Several mechanistic and clinical questions remain unanswered regarding monotherapy with BCL-2 family inhibitors, as well as their potential to be combined with 5-Aza. A large body of evidence underlies the theory that a therapeutic index for cytotoxic chemotherapy is, at least in part, a function of apoptotic differences between normal and malignant cells/tissues, termed “apoptotic primedness,” not merely differences in proliferation rate as conventionally thought [10,11]. Thus, it is conceivable that BCL-2 family targeting agents may be effective as single-agent therapy, especially in diseases dependent on BCL-2, such as chronic lymphocytic leukemia, where clinical responses have already been observed [12]. In AML, the contribution of anti-apoptotic proteins BCL-2, BCL-XL and MCL-1 in maintaining leukemogenesis alone or combined is not completely resolved. Furthermore, it is not known whether 5-Aza induces additional pro-apoptotic signals that increase the “apoptotic primedness,” resulting in increased therapeutic benefit in combination with BCL-2 family targeting compounds. It is possible that dual or multiple targeting of BCL-2 family proteins may be therapeutically impractical due to side-effects; however, it is likely that a therapeutic index may even be greater for broader BCL-2 family inhibition given that neoplastic myeloid cells persist at a higher apoptotic threshold, likely mediated by redundant expression of BCL-2 and other anti-apoptotic family members. Evidence that BCL-2 and MCL-1 may be selectively up-regulated in leukemic stem cells compared to normal hematopoietic stem cells also supports this concept [8,9,13]. In summary, we show that ABT-199 compared to ABT-737 can result in similarly potent 5-Aza sensitization in AML and MDS ex vivo. Therefore, clinical trials combining 5-Aza with navitoclax, as we previously proposed, or with ABT-199 for the treatment of advanced myeloid malignancies are warranted.

Ruxolitinib in clinical practice for therapy of myelofibrosis: Single USA center experience following Food and Drug Administration approval

Myelofi brosis is a myeloproliferative neoplasm (MPN) characterized by increased bone marrow fi brosis, abnormal blood counts with peripheral cytopenias, extramedullary hematopoiesis, splenomegaly and profound constitutional symptoms [1,2]. Presenting as a primary disease or arising from polycythemia vera or essential thrombocythemia, myelofi brosis incurs a signifi cant symptom burden for patients, and results in a reduced survival of 2 – 11 years [3 – 7]. Amongst MPNs, myelofi brosis has represented a unique challenge to eff orts aimed at palliating symptoms or impacting disease course. Th e 2005 discovery of the gain-of-function JAK2V617F mutation present in a signifi cant proportion of patients with polycythemia vera, essential thrombocythemia and myelofi brosis opened a new avenue of investigation into potential novel therapies [8,9]. Among the various JAK2 inhibitor agents that have been developed, ruxolitinib was the fi rst to receive Food and Drug Administration (FDA) approval in the USA for the treatment of intermediate- and high-risk myelofi brosis. Th e Controlled Myelofi brosis Study with Oral JAK Inhibitor Treatment Trials compared ruxolitinib therapy in patients with intermediate-2 or high-risk myelofi brosis with placebo (COMFORT-I) and with best available therapy (COMFORT-II) [10,11]. In both trials, patients in the ruxolitinib group experienced signifi cant clinical benefi ts, including reduction in spleen size and amelioration of debilitating symptoms coupled with a tolerable side-eff ect profi le. In this study, we report the initial clinical experience with ruxolitinib outside of the clinical trial setting at our high-volume MPN program. Approval for the analysis was obtained from the Mayo Clinic Institutional Review Board. Patients with myelofi brosis including primary myelofi brosis (PMF), post-polycythemia vera (post-PV MF) and post-essential thrombocythemia (post-ET MF) were included. All patients had a Dynamic International Prognostic Scoring System (DIPSS) score of intermediate-1/2 risk or high risk at the start of therapy [12]. Clinical and hematopathological data, including bone marrow biopsy, were reviewed in all cases, and the diagnosis was made in accordance with the 2008 World Health Organization criteria. Baseline patient information, physical examination results, laboratory data, hematopathology reports and interim follow-up information were acquired via chart review, with baseline obtained within 1 month of initiating therapy. All patients were prescribed ruxolitinib within 6 months of FDA approval. Ruxolitinib was started at 20 mg twice daily for patients with platelet counts of 200 10 9 /L or hemoglobin concentrations 10 g/dL. Patients with values below these were started on lower doses at the discretion of the providing physician. Doses were adjusted for lack of effi cacy or excess toxicity. Complete blood counts were checked on a weekly basis for the fi rst 2 months, then monthly thereafter. Drug effi cacy and toxicity were determined at follow-up visits and through telephone conversations with patients. Symptoms were assessed through a verbal response during review of systems questioning. Spleen size was determined by physical examination. Descriptive statistics were determined using means, medians, ranges and standard deviations for continuous variables. Categorical variables were evaluated using frequencies and relative frequencies.

Impact of race and ethnicity on outcomes and health care utilization after allogeneic hematopoietic cell transplantation

Abstract Disparities in outcomes after hematopoietic cell transplant (HCT) are reported mostly by registry studies. We examined the association of self-reported race and ethnicity with outcomes and health care utilization after allogeneic HCT in a single center study. Clinical and socioeconomic data of 296 adult patients who underwent allogeneic HCT from November 2003 to October 2012 were analyzed. Survival was compared between non-Hispanic Whites (NHW) and minority patients using Cox proportional hazards regression. Some 73% of patients were NHW and 27% were racial/ethnic minority patients. More minority patients were younger and had lower socioeconomic status. Both unadjusted and adjusted overall and progression-free survival were comparable between the two groups. High risk disease, poor performance score and Medicare/Tricare were significant predictors of mortality. Health care utilization was comparable between the two groups. Homogeneity of medical care for allogeneic HCT may help overcome racial/ethnic disparities, but not those due to patients’ primary insurance.

Allo-SCT for myelofibrosis: reversing the chronic phase in the JAK inhibitor era?

At present, allo-SCT is the only curative treatment for patients with myelofibrosis (MF). Unfortunately, a significant proportion of candidate patients are considered transplant ineligible due to their poor general condition and advanced age at the time of diagnosis. The approval of the first JAK inhibitor, ruxolitinib, for patients with advanced MF in 2011 has had a qualified impact on the treatment algorithm. The drug affords substantial improvement in MF-associated symptoms and splenomegaly but no major effect on the natural history. There has, therefore, been considerable support for assessing the drug’s candidacy in the peritransplant period. The drug’s precise impact on clinical outcome following allo-SCT is currently not known; nor are the drug’s long-term efficacy and safety known. Considering the rarity of MF and the small proportion of patients who undergo allo-SCT, well designed collaborative efforts are required. In order to address some of the principal challenges, an expert panel of laboratory and clinical experts in this field was established, and an independent workshop held during the 54th American Society of Hematology Annual Meeting in New Orleans, USA on 6 December 2013, and the European Hematology Associations Annual Meeting in Milan, Italy on 13 June 2014. This document summarizes the results of these efforts.

Myeloid neoplasm with eosinophilia associated with isolated extramedullary FIP1L1/PDGFRA rearrangement

Myeloid neoplasms with eosinophilia associated with PDGFRA rearrangement are very responsive to tyrosine kinase inhibitors (TKIs). Herein, we report a case of a 53-year-old man with eosinophilia and a well-differentiated extramedullary myeloid tumor with evidence of FIP1L1/PDGFRA rearrangement by fluorescent in situ hybridization in the extramedullary tissue. His bone marrow evaluation revealed a hypercellular marrow with eosinophilia but without evidence of a FIP1L1/PDGFRA rearrangement. The patient was treated with imatinib at a dose of 100 mg daily and responded with normalization of his peripheral eosinophil count. The case raises the possibility that an extramedullary myeloid tumor may represent a primary site for PDGFRA rearrangement, and highlights the importance of performing cytogenetic testing on extramedullary tissue. Detection of the chromosomal rearrangement is critical for initiation of effective targeted therapy that can improve patient outcomes.

Comparison of reduced intensity conditioning regimens used in patients undergoing hematopoietic stem cell transplantation for myelofibrosis

The aim of this study is to compare clinical outcomes of patients who underwent allogeneic stem cell transplantation (HCT) for myelofibrosis with reduced intensity conditioning (RIC) using either Busulfan Fludarabine (BuFlu), Fludarabine Bis-chlorethyl-nitroso-urea/ carmustine Melphalan (FBM) or Fludarabine Melphalan (FluMel) regimens. Sixty-one patients were identified who underwent HCT with one of these RIC regimens. Overall survival (OS) was not different in the 3 groups. However, 100% donor chimerism was seen in more frequently at day +30 and day +100 in patients who received FBM or FluMel than BuFlu, in both CD3 and CD33 fractions. For instance, 100% donor chimerism in CD33 fraction was present in 100% patients in FBM cohort, 90% in FluMel cohort while 44% in BuFlu cohort at day +100. Acute graft-versus host disease, grade 2–4 and grade 3–4, was not statistically different in the 3 groups (BuFlu 47 and 35%, FBM 68 and 27%, FluMel 68 and 46%; p = 0.31 and 0.45). Relapses and non-relapse mortality was also not statistically significantly different. Our study shows similar OS with these 3 RIC regimens in myelofibrosis; although donor chimerism at day +30 and day +100 was better in patients who received FBM and FluMel.