Minimally-Myelosuppressive Asparaginase-Containing Induction Regimen for Treatment of a Jehovah’s Witness with mutant IDH1/NPM1/NRAS Acute Myeloid Leukemia

Treatment of patients with acute myeloid leukemia (AML) who do not wish to accept blood product transfusion, including Jehovah’s Witnesses, is extremely challenging. The use of conventional chemotherapy for induction of complete remission (CR) results in profound anemia and thrombocytopenia requiring frequent transfusions of blood products, without which such treatment will be life-threatening. Finding a well tolerable, minimally myelosuppressive induction regimen for such patients with AML is a clear example of area of unmet medical need. Here, we report a successful treatment of a 52-year-old Jehovah’s Witness with newly diagnosed AML with peg-asparaginase, vincristine and methylprednisolone. The AML was characterized with normal karyotype, and mutations in isocitrate dehydrogenase 1 (IDH1-Arg132Ser), nucleophosmin 1 (NPM1-Trp289Cysfs*12) and neuroblastoma RAS viral oncogene homolog (NRAS-G1y12Va1). After one 28-day cycle of treatment, the patient achieved complete remission with incomplete count recovery (CRi) and after the second cycle, he achieved CR with full blood count recovery. The patient has never received any blood products. Notwithstanding that myeloperoxidase-induced oxidative degradation of vincristine results in its lack of activity as monotherapy in AML, its combination with corticosteroid and asparaginase has resulted in a robust remission in this patient. Diminished steroid clearance by asparaginase activity as well as reduction in serum glutamine level induced by glutaminase enzymatic activity of asparaginase may have contributed to effective killing of the myeloblasts that carry IDH1/NPM1/NRAS mutations. In conclusion, asparaginase-containing regimens, which are approved for treatment of acute lymphoblastic leukemia (ALL) but not AML, can be used to treat patients with AML who do not accept blood transfusion.


Introduction
Treatment of individuals who do not accept transfusion of blood products, including Jehovah's Witnesses, with acute myeloid leukemia (AML) is exceptionally challenging [1][2][3]. Conventional remission induction regimens containing cytotoxic agents such as cytarabine and anthracyclines often are not offered to these patients, because chemotherapy results in profound myelosuppression requiring frequent red blood cell and platelet transfusions without which severe anemia and thrombocytopenia are life-threatening [4][5][6]. On the other hand, without induction treatment, these patients rapidly will succumb to their leukemia.
Asparaginase products, which are approved for treatment of acute lymphoblastic leukemia (ALL) [7][8][9], but not AML, do not suppress the bone marrow and can provide a treatment option for patients with AML who do not wish to receive blood transfusion [10,11]. In this article, we report and discuss a successful treatment of a Jehovah's Witness with newly diagnosed AML with an asparaginase-containing regimen.
He tolerated the chemotherapy regimen well without developing any major adverse events. Even though, his hemoglobin and platelet counts remained low (Figure 2), he did not experience any symptoms indicating cardio-respiratory compromise or any bleeding, and did not receive any blood product transfusion.  Twenty eight days after initiation of the chemotherapeutic agents, a bone marrow aspiration and biopsy was repeated which showed cellular marrow with no morphological or immunophenotypic evidence of AML ( Figure 1C,D) with a decrease in blasts from 55% by flow cytometry to <1%. On the same day, complete blood count (CBC) showed WBC 1.4ˆ10 9 /L with 53% granulocyte, no blasts, hemoglobin 4.5 g/dL, and platelet 50ˆ10 9 /L (Figure 2), confirming the achievement of complete remission with incomplete count recovery (CRi). Due to logistical issues including insurance coverage, serum glutamine and 2-hydroxyglutarate (2-HG) levels could not be measured.
One week later, the patient developed edema in the right upper extremity where had a peripherally inserted central catheter (PICC) as well as left lower extremity edema. The duplex ultrasound revealed acute thrombus in the right distal subclavian vein, right axillary vein and right brachial vein as well as acute, occlusive deep venous thrombosis (DVT) of the paired left peroneal veins (isolated, axial calf DVT) without extension proximally into the left popliteal vein or the more-proximal deep veins of the left leg. Since at this point patient had adequate platelet recovery to 142ˆ10 9 /L, he was treated with full dose low molecular weight heparin. His symptoms had resolved in a few days.
Forty nine days after diagnosis and initiation of the first cycle of induction treatment, he began the second cycle of the same chemotherapy regimen with pegasparaginase, vincristine, and prednisone (50% dose reduced) as an outpatient. Patient remained on anticoagulation and tolerated the chemotherapy well except for development of Grade 2 vincristine-induced peripheral neuropathy. The CBC upon completion of the second cycle showed WBC 9.2ˆ10 9 /L with 54% granulocytes, no blasts, hemoglobin 10.4 g/dL, and platelets 255ˆ10 9 /L ( Figure 2). His bone marrow aspiration and biopsy revealed normocellular bone marrow for age (cellularity 50%-60%) with full-spectrum trilineage hematopoiesis, and no morphological or immunophenotypic evidence of involvement by AML, confirming the achievement of CR ( Figure 1E,F). The re-analysis of the bone marrow cells for the myeloid mutation panel revealed no somatic mutations or disease specific alterations, including the original IDH1, NPM1, and NRAS indicating achievement of molecular remission. His performance status and organ functions have remained normal.

Discussion
Both E. coli and Erwinia-derived asparaginase products including long acting pegasparaginase (polyethylene glycol L-asparaginase) possess a dual asparaginase and glutaminase enzymatic activity that diminishes both serum asparagine and glutamine levels by deamidating them to aspartate and glutamate, respectively [12]. It is believed that the cytotoxicity of asparaginase against lymphoblasts is due to auxotrophic nature of these cells for asparagine; however, the therapeutic value of asparaginases against myeloblasts is reported to be due to glutamine depletion resulting in disruption of protein synthesis downstream of mammalian target of rapamycin (mTOR) causing strong apoptotic and autophagic responses [13][14][15][16].
Mutations in IDH1 and IDH2 have been reported in approximately 20% of de novo AML [17][18][19][20][21], and their presence is an unfavorable prognostic factor according to most studies [22][23][24][25][26]. It has been suggested that IDH mutations confer adverse prognostic effect in patients with AML whose myeloblasts lack NPM1 mutation [26,27]. Wild-type IDHs are NADP-dependent enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), with production of NADPH [28]. Altered amino acids in mutant IDH are located in the catalytic pocket of the enzymes resulting in converting α-KG to 2-hydroxyglutarate (2-HG) with the consumption of NADPH. Heterozygous mutations resulting in a single amino acid change at arginine 132 (R132H, R132C, R132G, R132S) of IDH1 and arginine 140 (R140Q, R140W) or arginine 172 (R172K, R172G) of IDH2 have been reported [18,20]. By 13 C and isotope-labeling experiments, it has been shown that the primary source for α-KG in AML cells with IDH mutation is glutamine [18]. Subsequently, it has been demonstrated that in vitro interruption of glutamine metabolism preferentially slows the growth of primary AML cells with mutant IDH versus wild type IDH [29,30]. Clinical studies aiming at the evaluation of safety and efficacy of asparaginase products in patients with AML with or without IDH mutations are ongoing (NCT02283190 and NCT01810705).
The major adverse events of asparaginase products, reported from clinical trials involving patients with ALL, include anaphylaxis and serious allergic reactions, thrombosis, pancreatitis, glucose intolerance, coagulopathy, and hepatotoxicity. Interestingly, myelosuppression does not occur frequently and seriously after asparaginase administration. Also the myelosuppressive effect of vincristine compared to other conventional chemotherapeutic agents is minimal.
The fact that asparaginase and vincristine are minimally myelosuppressive is important for patients such as Jehovah's Witnesses that do not accept blood product transfusion; as it is well known that the use of conventional chemotherapy for remission induction in AML results in life-threatening anemia and thrombocytopenia, requiring blood product transfusions with an average of 10.8 and 8.5 units of red blood cell and platelet transfusions, respectively [31]. We combined asparaginase and steroid with vincristine to increase their anti-leukemic effect, while maintaining the minimal myelosuppressive property of the regimen. Vincristine, a cell cycle-specific vinca alkaloid which induces metaphase arrest, as a single agent is not active against myeloblasts in AML [32]. Overexpression of multi-drug resistance proteins and myeloperoxidase-induced oxidation and degradation of vincristine are the main causes of inherent resistance of AML cells to vincristine as monotherapy [33,34]. While a synergistic or additive anti-leukemic activity between vincristine and asparaginase is not well known; decreased clearance of steroid with sustained asparaginase activity combined with prolonged asparaginase half-life due to immunomodulatory effect of steroid on anti-asparaginase antibody suggest synergism between asparaginase and steroid products [35,36].

Conclusion
A minimally myelosuppressive regimen including pegasparaginase, vincristine and steroid was used to induce durable complete remission in a patient with mutant IDH1/NPM1/NRAS AML who did not want to receive blood product transfusions. Moving forward, we plan to continue using non-myelosuppressive mono-or combination-therapies as consolidation regimens for this patient. We also plan to prospectively test the effect of this combination for similar patients with prospective measurement of serum biomarkers.