2009

ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n and children, and revealed the potentiation of cytarabine cytotoxicity ex vivo. Given the promising results of schedule A, we designed and conducted a phase II study of triapine 105 mg/m 2 daily followed by fludarabine 30 mg/m 2 daily for 5 days in 37 patients with aggressive MPN and secondary, transformed AML. We confirm our previous findings that this combination of agents is clinically active in accelerated MPN and secondary AML derived from MPNs, and further identify the JAK2 V617F mutation as a potential predictor of response to sequential ribonucleotide reductase inhibition. Methods Patients Between August 2006 and November 2010, 37 adults with pathological confirmation of accelerated MPN (>5% blasts in bone marrow, new onset/worsening myelofibrosis, new onset or >25% increase in hepatomegaly/splenomegaly, or new onset/worsening constitutional symptoms), CML-BC, CMML (>5% blasts), or secondary AML, arising from a preexisting MPN or CMML, defined by standard criteria, Treatment plan Triapine was administered as a 4-h intravenous infusion daily for 5 consecutive days at a dose of 105 mg/m 2 /day. Fludarabine 30 mg/m 2 /day was administered as a 30-min intravenous infusion daily, beginning within 1 h of completion of the triapine infusion, on each of the 5 consecutive days. Each cycle was 21 days in duration. Patients were eligible to receive additional cycles of treatment until disease progression or toxicity. Measurement of toxicities Non-hematologic toxicities were graded according to National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. Treatment of triapine was interrupted for grade 3 non-hematologic toxicities for up to 48 h. All subsequent doses were withheld until toxicity resolved to grade ≤1 (or baseline). Measurement of response Bone marrow aspiration and biopsies were performed prior to treatment, during week 3 of the first cycle, at the time of hematologic recovery from all cycles of therapy (defined as neutrophil count >500/mm 3 and platelets >20,000/mm 3 independently of transfusion), or at any time that leukemia regrowth was suspected. The overall response rate was defined as complete remission, partial remission, or hematologic improvement, lasting for ≥30 days. Given the different subsets of diseases, standardized response criteria were used for CMML (the Myelodysplastic Syndrome International Working Group criteria), 33 CMML transforming to acute myeloid leukemia (standard AML response criteria) 35 , and transformation of MPN to secondary AML (Mascarenhas et al.). 36 Statistical considerations The primary objectives of this study were to determine the efficacy and toxicity of triapine followed by fludarabine in patients with aggressive MPN. The study was designed to detect an improvement in overall response rate from 10-30% with a twosided type 1 error rate of 5% and 90% power using a two-stage approach. A stopping requirement was placed such that the study would terminate if fewer than three out of 18 patients had a response. An additional 24 patients were planned but the study was completed after a total of 37 patients had been enrolled because of lack of drug availability. Overall survival was calculated from the first day of treatment to death or last known follow-up and estimated using the KaplanMeier method. A landmark analysis 37 was performed to compare response groups using day 42 of treatment as the landmark time. Overall survival was compared between groups of patients using Cox proportional hazard models, adjusting for age and gender. The database was locked on June 1, 2013. Analyses were completed using statistical freeware R version 3.0.1. 38 Results Patients' characteristics The pretreatment clinical demographics of the 37 patients enrolled in this trial are outlined in Toxicity A total of 100 cycles of triapine and fludarabine were administered to 37 patients, with a median of three cycles per patient (range, 1-6). Nine out of 37 (24%) patients received four or more cycles of therapy. Early death (prior to day 30) occurred in 4/37 (11%) patients (secondary AML: n=2, CML: n=2) due to sepsis (n=2), multi-organ failure (n=1), or cardiogenic shock (n=1). As presented in Clinical outcomes The overall response rate to triapine and fludarabine in this population was 49% (18/37). In the first stage, nine of 18 patients achieved a response, with nine of the remaining 19 patients achieving a response in the second stage. Overall, nine patients achieved a complete remission (24%) (including one patient with complete remission with incomplete platelet recovery), four patients achieved a partial remission (11%), and five patients achieved hematologic improvement (14%). An additional five patients had stable disease for at least one cycle. The overall response rate for each disease subset was: CMML 57% (4/7), MPN 60% (3/5), CML-BC 25% (1/4), and secondary AML 48% (10/21) ( All six patients with a JAK2 V617F mutation had a response, including three (50%) who achieved a complete J.F. Zeidner et al. 674 haematologica | 2014; 99(4) © F e r r a t a S t o r t i F o u n d a t i o n remission. In comparison, the overall response rate in patients with wild-type JAK2 was 29% (4/14 patients, P=0.01), with an overall complete remission rate of 14% (2/14 patients, P=0.13). Each of the three patients with JAK2 V617F mutations achieving a complete remission had secondary AML arising from MPN (myelofibrosis: n=2, essential thrombocythemia: n=1). The two patients who achieved a complete remission with a known wildtype JAK2 had secondary AML (from antecedent CMML: n=1) and CML-BC (n=1). The median overall survival of the study population was 6.9 months (95% CI: 4.3-10.7 months), with the 6-month and 12-month overall survival rates being 54% and 24%, respectively. The median overall survival of responders (patients achieving complete or partial remission or having a hematologic improvement) and complete responders was 10.6 months (95% CI: 8.7-15.8 2 months) and 10.6 months (95% CI: 8.7-21.5 months), respectively. Patients with a known JAK2 V617F mutation had a median overall survival of 10.5 months (95% CI: 5.0-41.5 months) compared with 5.6 months (95% CI: 2.7-30.4 months) in patients without a JAK2 V617F mutation (adjusted P=0.17). The median duration of first response to relapse or death was 121 days (range, 52-422 days). Five patients received an allogeneic SCT after therapy with triapine and fludarabine (CMML: n=2, CML: n=2, secondary AML: n=1). The median age of patients receiving an allogeneic SCT was 60 years (range, 43-68). Three patients (CML: n=2, CMML: n=1) had myeloablative conditioning and two patients (secondary AML: n=1, CML: n=1) had non-myeloablative conditioning. Two of the five patients (CMML: n=1, CML: n=1) who underwent an allogeneic SCT had a complete response to triapine and fludarabine, two patients (CMML: n=1, CML-BC: n=1) had refractory disease on study and then received additional treatment prior to allogeneic SCT, and one patient with secondary AML had stable disease prior to allogeneic SCT. Three patients (CML: n=2, CMML: n=1) died of complications of the allogeneic SCT (infection: n=2, hepatic sinusoidal obstructive syndrome: n=1), including the two patients who achieved a complete remission, and two patients relapsed after the transplant. One patient with secondary AML (arising from CMML) achieved stable disease on study and then had a non-myeloablative haploidentical allogeneic SCT (5 months after being enrolling into the study), with a subsequent relapse 17 months after Discussion MPN continue to be a major therapeutic challenge, especially once they undergo aggressive transformation. This phase II study involving triapine, a novel ribonucleotide reductase M2 inhibitor, combined with fludarabine (an M1 ribonucleotide reductase inhibitor), extends our initial observations that this regimen has clinical activity in aggressive/accelerated phase MPN including transformations into AML. This study met its primary end-point of an overall response rate >30% (49%). The complete remission rate of 24% in this heavily pretreated population of patients is promising. Patients with secondary AML had an overall response rate and complete remission rate of 48% and 33%, respectively, which is particularly encouraging in these patients who have a dismal prognosis. There did not seem to be an upper age limit of responders, as patients responded to therapy up to the age of 75 years. The encouraging response rates (n=6/6) in patients with JAK2 V617F mutations are of particular interest, and reached statistical significance (P=0.01) when compared to those in JAK2 wild-type patients (overall response rate: 100% versus 29%). The three patients who achieved a complete remission with JAK2 V617F mutations had secondary AML derived from a JAK2 V617F MPN clone. The results of triapine and fludarabine in MPN (including transformations to AML) with JAK2 V617F mutations are intriguing; however, given the small number of patients in these analyses, larger scale trials will need to be performed to confirm these suggestions. Landmark analyses comparing responders to nonresponders by day 42 of treatment did not reveal significant differences in overall survival. This landmark analysis does, however, have its limitations. The time point of 42 days was chosen prior to study accrual as the time at which an initial response rate should be noted for each patient in the study. However, seven of nine complete responders achieved a complete remission after day 42 of treatment (range, 21-96 days) and these seven patients were thus not listed among those with a complete remission for statistical comparisons using the landmark analysis. Additionally, if a patient who had a partial remission or hematologic improvement before day 42 went on to achieve a complete remission after day 42, this response was not included in the specific comparison of complete remission versus no complete remission for the landmark statistical comparisons. Nonetheless, the clinical responses were short-lived, with the median duration of response being 4 months, emphasizing the high degree of chemoresistance seen in accelerated MPN. Moreover, the median overall survival of 6.9 months underscores the extremely poor prognosis of the patients in this study. Because of the short duration of response, this combination of agents may work best as a bridge to further therapy, such as allogeneic SCT. However, only five patients were able to receive an allogeneic SCT after treatment with these ribonucleotide reductase inhibitors (including 4/5 who died), which may reflect the older age of patients in the study (median age: 64 years). Three of the five patients who were able to undergo allogeneic SCT were given myeloablative preparative regimens. As allogeneic SCT techniques become more refined, it may be possible to select patients who could benefit from a non-myeloablative preparative regimen compared to a myeloablative one. The combination of triapine and fludarabine led to unique toxicities, most notably acute pulmonary toxicities, such as methemoglobinemia and metabolic acidosis. These toxicities are distinctively related to the triapine infusion. Although these toxicities were not inconsequential, they often resolved promptly without intervention and without subsequent organ dysfunction. Upon dose reduction of triapine in ten patients, there were no additional grade ≥3 toxicities. Contemporaneous inhibition of the M1 and M2 subunits of ribonucleotide reductase is a concept worthy of further exploration in patients with accelerated MPN. Although triapine is 100-1000 fold more potent than hydroxyurea, the therapeutic efficacy of triapine and hydroxyurea has never been compared. It would be rational to further explore hydroxyurea in combination with other cytotoxic agents and M1 ribonucleotide reductase inhibitors (such as fludarabine, gemcitabine and clofarabine) for synergistic ribonucleotide reductase inhibition. This approach may be better tolerated than the combination of triapine and fludarabine. Given the epigenetic dysregulation seen in patients with accelerated MPN (with or without transformation to AML), additional treatment approaches for these patients include hypomethylating agents, such as azacitidine. Since this study was conceived and executed, novel JAK2 inhibitors, such as ruxolitinib, have emerged as integral parts of the therapeutic armamentarium for MPN. 42 Given the encouraging results of this study, simultaneous inhibition of the M1 and M2 subunits of ribonucleotide reductase should be investigated with JAK2 inhibitors, either in combination or in sequence. Hydroxyurea was shown in one study to increase STAT-5 phosphorylation in a dose-dependent manner, thus leading to activation of the STAT-JAK pathway. J.F. Zeidner et al. 676 haematologica | 2014; 99(4) © F e r r a t a S t o r t i F o u n d a t i o n V617F mutations, it would be worth exploring whether M2 ribonucleotide reductase inhibitors or combination ribonucleotide reductase inhibition has any significant impact on JAK2 allele burden. JAK2 inhibitors, such as ruxolitinib, seem to be clinically effective in myelofibrosis patients with or without JAK2 V617F mutations and it is unclear whether these agents work by JAK1 or JAK2 inhibition, with or without modulation of inflammatory cytokines. In summary, sequential ribonucleotide reductase inhibition with an M2 inhibitor (triapine) followed by an M1 inhibitor (fludarabine) led to an overall response rate of 49% in patients with accelerated MPN, CML, CMML and secondary AML. Responses were seen in all disease states, including secondary AML, and were particularly notable in patients with JAK2 V617F mutations. The responses were, however, temporary, which underscores the need for further investigative trials in this population of patients. Future studies combining ribonucleotide reductase inhibitors, with or without the addition of JAK2 inhibitors, should be explored in patients with secondary AML and high-risk accelerated MPN. © F e r r a t a S t o r t i F o u n d a t i o n Acknowledgment