Evidence for Clinical Differentiation and Differentiation Syndrome in Patients With Acute Myeloid Leukemia and IDH1 Mutations Treated With the Targeted Mutant IDH1 Inhibitor, AG-120

Abstract
We describe 3 patients with relapsed/refractory acute myeloid leukemia who developed clinically-apparent differentiation concurrent with clinical response during monotherapy with AG-120, a novel oral inhibitor of mutant isocitrate dehydrogenase 1. Symptoms included marked leukocytosis and exuberant neutrophil re- covery among other clinically apparent constitutional manifestations. Awareness of the potential for differ- entiation syndrome with such inhibitors, and prompt identification and intervention, are essential to facilitate clinical resolution.Background: Cancer-associated isocitrate dehydrogenase (IDH) mutations block normal cellular differentiation via production of the oncometabolite, R-2-hydroxyglutarate. In patients with acute myeloid leukemia (AML) receiving targeted mutant IDH inhibitor therapy, neutrophil recovery within the setting of clinical differentiation syndrome (DS) has been anecdotally described. Patients and Methods: We describe 3 patients who developed clinically apparent DS during monotherapy with the mutant IDH1 inhibitor, AG-120, for relapsed/refractory AML. Results: AGe120-induced differentiation commenced within the first 60 days of treatment, notably in the same time frame as clinical response, strengthening the purported mechanism of targeted mutant IDH inhibitor therapy via successful myeloid maturation. Symptoms of DS were nonspecific and included culture-negative fever, edema, hypotension, malaise, and pleural and/or pericardial effusions, in addition to marked neutrophil-predominant leukocytosis. Conclusion: DS can occur during treatment with targeted mutant IDH1 inhibitor therapy. Patients might present with nonspecific clinical manifestations often in the setting of leukocytosis related to exuberant neutrophil recovery. Prompt identification and initiation of treatment interventions, including hydroxyurea, corticosteroids, and/or consideration of temporary treatment discontinuation, are important to facilitate prompt resolution.

Introduction
Differentiation syndrome (DS) is a potentially fatal complication of effective leukemia treatment first described in patients with acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA).1 The reported incidence in APL ranges from 2% to 27%,2 likely because of the heterogeneity and range of clinical symptoms,as well as imprecise diagnostic criteria. In APL, signs and symptoms of DS have been described 2 to 47 days after treatment initiation, and include increasing white blood cell count (WBC) and absolute neutrophil count (ANC), culture-negative fever, weight gain, edema, dyspnea, interstitial infiltrates, pleural effusion, pericardial effusion, hypotension, and renal failure.1,3,4 The underlying path- ophysiology remains poorly understood, but is thought to be related to release of inflammatory vasoactive cytokines and tissue infiltra- tion by briskly maturing cells.2,5 Myeloid differentiation and clinical DS have also been described in patients with acute myeloid leu- kemia (AML) receiving therapy with fms-related tyrosine kinase 3 inhibitors and hypomethylating agents, including neutrophilic skin infiltrates retaining the aberrant FLT3-internal tandem duplication mutation in some instances. Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations are noted in approximately 20% of patients with AML.8 Cancer- associated IDH1/2 mutations block normal cellular differentiation and drive tumorigenesis by promoting abnormal reduction of a- ketoglutarate (a-KG) to the oncometabolite, R-2-hydroxyglutarate (2-HG).9,10 2-HG accumulation inhibits multiple a-KG-depen- dent dioxygenases, including histone and DNA demethylases, which regulate the cellular epigenetic state.11 The first-in-human phase I clinical study of the novel, oral mutant IDH1 inhibitor, AG-120, is ongoing (ClinicalTrials.gov NCT02074839), and early results in 66 patients indicate that monotherapy is well tolerated, with an overall response rate of 36% according to International Working Group (IWG) criteria in a primarily relapsed/refractory AML population.12 Responses occur without a period of bone marrow aplasia, unlike standard cytoreductive therapy. Neutrophil recovery in the setting of a clinical DS in patients receiving mutant IDH inhibitor therapy has been anecdotally described, but the clinical patterns of differentiation have not been previously reported. Herein, we describe 3 patients treated at our institution who developed clinically apparent differentiation and DS during AG-120 monotherapy for relapsed/refractory AML.

Results
A 53-year-old man was found to have incidental anemia during a routine physical examination. Results of complete blood count (CBC), bone marrow aspirate and biopsy, and cytogenetic and molecular studies at presentation are shown in Table 1. He received intensive cytarabine-based induction AML chemotherapy, which was associated with complications (Table 1) and end of cycle 1 staging bone marrow showed primary refractory disease with 77% myeloblasts.Because of the presence of an IDH1-R132C mutation, the pa- tient was transitioned to AG-120 500 mg/d orally on 28-day continuous cycles. His CBC on day 1 of therapy showed a WBC of 21.7 × 109/L, with 1% neutrophils and 95% circulating blasts(Table 1; Figure 1A). A day 14 bone marrow aspiration showedpersistent AML, with 37% myeloblasts and at completion of cycle 1, blasts had reduced to 18% and he started cycle 2 without dose adjustments or treatment interruption (Table 1). On cycle 2, day 1, his WBC was 29 × 109/L with 24% neutrophils and 13% circu-lating blasts. On cycle 2, day 5, he reported a mild backache, anddenied having fever, chills, cough, shortness of breath, nausea, vomiting, diarrhea, or urinary symptoms. His physical examination was normal except for mild 1+ nontender bilateral pedal edema. The CBC showed an increase in WBC to 42.5 × 109/L, with 64% neutrophils and 6% circulating blasts (Table 1; Figure 1A). Therewere no signs or symptoms of infection and he was maintained with empiric antimicrobial prophylaxis and hydroxyurea at 2 g/d was started. On cycle 2, day 8, he described symptoms of mild vertigo, poor appetite, and anorexia, and was found to be tachycardic and mildly hypotensive (100/59 mm Hg) with an otherwise unre- markable physical examination. An electrocardiogram (EKG) confirmed sinus tachycardia with no ST/T wave changes. An in-fectious evaluation including urine, sputum, and blood cultures wasnegative at 7 days. Hydroxyurea was reduced to 1 g/d. On cycle 2, day 12, the WBC was 12.4 × 109/L (76% neutrophils and 0% circulating blasts; Table 1).

Hydroxyurea was discontinued 8 daysafter initiation. In the absence of any infectious etiology, the rise in leukocyte count with a predominance of mature neutrophils, concomitant reduction in circulating myeloblasts, platelet recovery, and mild clinical symptoms of hypotension, tachycardia, malaise, vertigo, and lower back pain that spontaneously resolved, were suggestive of DS.The patient achieved a complete response after 3 cycles of AG- 120 and transitioned to a matched related donor allogeneic stem cell transplant, and remains in an ongoing complete remission 2 months after transplantation with successful engraftment. Laboratory parameters for a 45-year-old man who presented with fatigue are shown in Table 1. He was treated with front-line AML induction therapy (Table 1). Because of primary refractory disease after cycle 1, he was treated with AG-221 (mutant IDH2 inhibitor) 100 mg daily for 4 cycles without evidence of response. He then received azacitidine (75 mg/m2 intravenously on days 2-8) and nivolumab (3 mg/kg on days 1 and 14) as part of a clinical trial for 4 cycles without response.Because of the persistence of his IDH1-R132C mutation, he started AG-120, 500 mg daily in the phase I trial. Laboratory pa-rameters on cycle 1, day 1 are shown in Table 1 and included a WBC of 3.1 × 109/L with 6% neutrophils and 65% circulating blasts. Over the first 2 weeks of AG-120 treatment, peripheralmyeloblasts steadily declined from 65% to 23% (Figure 1B). On cycle 1, day 19, he was hospitalized for acute onset chest pain and shortness of breath with exertion, without cough or upper respira- tory complaints (vital signs and CBC are shown in Table 1, and included a WBC of 25.4 × 109/L with 39% neutrophils and 6%circulating blasts). An EKG showed sinus tachycardia and diffuse ST segment elevation concerning for pericarditis.

A 2-view chest x- ray identified left lower lung opacities representing possible atelec- tasis or early pneumonia, with a small left pleural effusion and enlarged cardiac silhouette suggestive of pericardial effusion. The patient was admitted to the intensive care unit and empiric broad- spectrum antibiotics were started for possible pneumonia, predni- sone 1 mg/kg for 3 days for pericarditis, and furosemide diuresis. An EKG on day 2 showed a sustained left ventricular ejection fraction of 59%, with a moderate to large pericardial effusion with no right ventricular chamber collapse. A repeat EKG 2 days later showed significant improvement in pericardial effusion with medical man- agement. His clinical status improved and he was discharged after 5 days (cycle 1, day 23) with a further 1-week tapering course of steroids. AG-120 was continued during hospitalization and upon hospital discharge. All cultures during hospitalization were negative.By cycle 2, day 1 of AG-120, the WBC had increased to 87.2 ×109/L with 40% neutrophils and 6% circulating blasts (Table 1, Figure 1B) and bone marrow blast count was stable (18%). Review of systems and physical examination were unremarkable. He received a 5-day course of hydroxyurea (2 g twice daily) to control leukocytosis. His WBC decreased over the next 2 weeks, normalizing by cycle 3, day 1 (Table 1). At that time, repeat assessment identified a completeremission with incomplete platelet recovery, with 3% myeloblasts in ahypercellular marrow with no morphologic evidence of residual leukemia, and full neutrophil and hemoglobin (Hb) recovery. In the absence of infection, the pericardial effusion followed by a dramatic rise in leukocyte count with predominantly mature neutrophils was highly suggestive of a clinically-significant DS.A 51-year-old man with fever and fatigue was diagnosed with AML. A CBC on admission showed a WBC of 7.7 × 109/L (0% neutrophils and 73% circulating blasts; Table 1). After induction therapy, which was associated with multiple complications (Table 1), he had primary refractory disease.The patient was then treated with AG-120 1200 mg daily in the phase I trial.In cycle 1, his WBC improved from 0.5 × 109/L on day 1 with 5% neutrophils and 76% circulating blasts, to 25 × 109/L on day 12, with 22% neutrophils and 50% circulating blasts (Table 1).Treatment with hydroxyurea 2 g/d orally was started. On day 15 the bone marrow blast count had decreased from 69% to 37% (Table 1; Figure 1C). The rapid rise in WBC count was associated with an increase in ANC and decrease of peripheral blast and bone marrow blast percentages, coinciding with treatment response and laboratory evidence of differentiation. Full count recovery with absence of leukemic blasts in the bone marrow and peripheral blood, leading to an IWG-defined complete response, was obtained after 2 cycles of AG-120. Leukocytosis was managed with 2 weeks of tapering hydroxyurea, which was discontinued at the start of cycle 3. HisWBC was 2.9 × 109/L and ANC 2250 at that time. After 3 cycles ofAG-120, he underwent a matched unrelated donor stem cell transplant.

Discussion
The discovery of recurrent pathogenic IDH mutations and the advent of mutant IDH inhibitors has been one of the most sig- nificant translational advances in the management of AML in the past decade. AG-120 inhibits the mutant IDH1 enzyme and re- duces aberrant serum 2-HG levels, which induces differentiation of leukemia cells.13The pathophysiology of DS in AML remains incompletely un- derstood. In APL, ATRA and arsenic trioxide are thought to induce release of cytokines from differentiating myeloid cells, leading to excessive inflammatory response,2,5 and might increase expression of cell surface integrins, which could increase adhesion of myeloid cells to vascular endothelium, thereby facilitating extravasation.2,5 The DS observed in patients treated with mutant IDH inhibitor therapy might be due to similar phenomena when treatment removes the differentiation block in the malignant myeloid clone, leading to a rapid increase in differentiated neutrophils.In all 3 patients, manifestations of AGe120-induced DS occurred concurrently with early evidence of clinical response, suggesting that DS is related to treatment response and successful myeloid maturation. Further analyses are needed to confirm this association. In case 1, laboratory and clinical symptoms were first noted on cycle 2, day 5, including leukocytosis, mild pedal edema, malaise, asymptomatic hypotension, and tachycardia, without fever or other constitutional manifestations, and hydroxyurea alone was used with gradual resolution of symptoms.

In case 2, signs and symptoms began on cycle 1, day 19 with low-grade fever, leuko- cytosis, and pericardial and pleural effusions. Tapering prednisone resulted in complete resolution of pericarditis, pericardial effusion, and clinical symptoms. A second discrete episode of differentiation seemed to begin at the start of cycle 2, with extreme leukocytosis that stabilized with hydroxyurea alone. In case 3, laboratory pa- rameters consistent with myeloblast differentiation were first noted on cycle 1, day 12 without apparent clinical manifestations. Hy- droxyurea was initiated for a short tapering course because of the rapid leukocytosis, and AG-120 was not held.Signs and symptoms of DS are not specific. They can includefever, edema, weight gain, leukocytosis, rash, hypotension, renal dysfunction, and pleural and pericardial effusions. A rising leukocyte count, comprising increasing neutrophils with a parallel decrease in leukemic blasts, was the most consistent finding among our patients experiencing AGe120-related DS. We did not observe significant weight gain or renal failure. In relapsed/refractory AML, many of these nonspecific clinical findings can also be related to systemicinfections or progressive leukemia, and care must be taken to accurately identify and appropriately manage mutant IDH inhibitor-induced DS. We recommend prompt initiation of hy- droxyurea (suggested dose 2-4 g/d orally, titrated daily as needed to control leukocytosis), corticosteroids (suggested dose, 10 mg dexa- methasone every 12 hours for 3 days or until improvement), furosemide (40-80 mg/d) in patients with clinically apparent effusions, and consideration of temporary hold of mutant IDH inhib- itor therapy until clinical improvement.

Conclusion
Differentiation syndrome can occur during treatment with the mutant IDH1 inhibitor, AG-120. The timing of onset and the signs and symptoms can vary, and might present with dramatic clinical manifestations in addition to marked leukocytosis and exuberant neutrophil recovery. DS should be considered in all patients treated with AG-120 experiencing rapid neutrophil-predominant leukocytosis, culture-negative fever, constitutional symptoms, and pleural or pericardial effusions. Steroid therapy appears successful in clinically significant DS, and oral hydroxyurea can mitigate rapid leukocy- tosis. Ongoing research will improve the identification of risk factors and better define this syndrome in patients treated with targeted “differentiating” AG-120 therapy.