A phase I study of lenalidomide plus chemotherapy with idarubicin and cytarabine in patients with relapsed or refractory acute myeloid leukemia and high‐risk myelodysplastic syndrome

Abstract Patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) have poor outcomes and hematopoietic cell transplantation (HCT) is the only curative treatment. New targeted therapies improved survival in select patients with specific mutations, however management of patients without these molecular alterations is an unmet need. We conducted a phase one study of lenalidomide in combination with cytarabine/idarubicin salvage chemotherapy in patients with R/R AML and high‐risk myelodysplastic syndromes. A total of 33 patients were enrolled in the study (30 AML, 3 MDS), and treated at three dose levels with 3 + 3 design. Dose‐limiting toxicity (DLT) was seen in eight patients, including four hematologic DLTs. The most commonly observed non‐hematologic serious adverse events were febrile neutropenia, rash, sepsis and renal injury. Dose level −1, consisting of 25 mg/d lenalidomide D1‐21, 1 g/m2 cytarabine D5‐8, and 8 mg/m2 idarubicin D5‐7 was determined to be the maximum tolerated dose. Note, 15/33 (45%) of patients were able to receive pre‐planned 21 days of lenalidomide. Overall, 18 patients achieved complete remission (CR) (n = 14) or CR with incomplete count recovery (CRi) (n = 4) with total CR/CRi rate of 56%. The 1‐year and 2‐year overall survival (OS) were 24% and 10%, respectively. Among responders, 10/18 underwent allogeneic HCT and had a 1‐year OS of 40%. There was no molecular pattern associated with response. These data demonstrate that the combination had clinical activity in R/R AML. This regimen should be further investigated for patients who relapsed after HCT, and as a bridge therapy to HCT. (ClinicalTrials.gov identifier: NCT01132586).


| INTRODUCTION
The initial treatment for most fit patients with newly diagnosed acute myeloid leukemia (AML) consists of the combination of an anthracycline (daunorubicin or idarubicin) and cytarabine, which has led to remission rates approaching 60%-80%. 1 However, patients who do not respond to initial chemotherapy or experience relapse after initial complete remission (CR) have dismal outcomes. 2 Hematopoietic cell transplantation (HCT) has curative potential in a minority of fit relapsed/refractory (R/R) AML patients who can achieve CR after salvage therapy or those with a low Duval score. 3 Response rates with commonly used cytarabine-based reinduction regimens (eg, mitoxantrone, etoposide, cytarabine [MEC] or fludarabine, cytarabine, filgrastim [FLAG]) can reach 40%-60% if CR duration was 1 year or longer. But, this rate drops to 10%-15% in cases with shorter CR duration and multiple lines of prior therapy. 4 Therefore, new therapeutic strategies that are tolerable and more efficacious are needed to improve outcomes in R/R AML patients.
Lenalidomide is an immunomodulatory agent, approved by the US Food and Drug Administration for the management of multiple myeloma, myelodysplastic syndrome (MDS) with del(5q) and certain subtypes of lymphoma. 5 The mechanism of action of lenalidomide is incompletely understood, but previous investigations proposed activation of innate and adaptive immune responses, altered cell cycling, disruption of marrow microenvironment, ubiquitination of critical targets and induction of expression of the tumor suppressor genes. 6 Its unique clinical activity in MDS with del(5q) was shown to be associated with the degradation of casein kinase 1A1, resulting in death of malignant progenitor cells. 7 In AML mouse models, lenalidomide treatment was shown to exhibit antitumorigenic activity through increased expression of mirR-181a, which resulted in increased sensitivity to cytotoxic chemotherapy. 8 We have previously demonstrated single agent activity of lenalidomide in patients with R/R AML. 9 The dose was safely escalated to 50 mg daily for 21 days of a 28-day cycle with a CR rate of 16% (5/31). Fatigue was the dose limiting toxicity (DLT) but remissions were durable, lasting 5 to 14 months. Remissions with full donor chimerism were also observed in post-transplant patients, suggesting a possible immunomodulatory effect. Similarly, a recent study investigating the combination of lenalidomide with azacitidine in AML patients who relapsed after allogeneic HCT, reported a 47% (7/15) response rate after three cycles of treatment. 10 The maximum tolerated dose (MTD) was determined as 25 mg daily, which did not reverse biologic features of Tcell exhaustion. When lenalidomide was combined with intermediate-dose cytarabine in a phase one study of R/R AML patients, MTD for lenalidomide was 10 mg daily, overall response rate was 41% (13/32) with 16% (5/32) CR and 16% (5/32) CR with incomplete count recovery (CRi). 11 Finally, lenalidomide in combination with MEC re-induction therapy in R/R AML resulted in 34% (12/35) CR rate with MTD of 50 mg daily. 12 The drug was also tested in the first-line setting as a single agent or in combination with other chemotherapy regimens for previously untreated AML. [13][14][15][16][17][18][19][20][21] Given the clinical activity of single agent lenalidomide in AML and preclinical studies suggestive of synergy with chemotherapy, we conducted a phase one study of lenalidomide in combination with cytarabine and idarubicin re-induction chemotherapy, to evaluate the safety and tolerability of this combination in patients with R/R AML and high-risk MDS.

| Eligibility criteria and study design
The study (NCT01132586) was approved by the Institutional Review Board of The Ohio State University, conducted in accordance with the Declaration of Helsinki, and all patients provided written informed consent. Eligible patients were adults aged 18 to 65 with a confirmed pathologic diagnosis of AML or high-risk MDS which had recurred or was refractory to at least one prior line of therapy. Patients who could not attain CR after one round of induction therapy containing anthracycline plus cytarabine (100-200 mg/m 2 every 24 hours for 7 days) were considered to have refractory disease and were eligible for this study. High-risk MDS was defined as International Prognostic Scoring System (IPSS) score of 1.5 or higher. 22 Cytogenetic risk for AML patients was defined per European LeukemiaNet 2017 criteria. 23 Patients with secondary AML and therapy-related myeloid neoplasms were eligible. Patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status of zero to two, and adequate organ function including a total bilirubin <2 mg/dL, ALT and AST <2.5× upper limit of normal, left ventricular ejection fraction ≥40%, creatinine <2 mg/dL and calculated creatinine clearance ≥50 mL/min. Patients who had previously received lenalidomide, cytarabine and/or idarubicin were eligible provided that the combination of the three agents had never been administered, and that no lenalidomide had been administered for at least 6 months. Patients were excluded if they had active central nervous system disease, uncontrolled systemic infections, and if any chemotherapy or radiotherapy within 2 weeks prior to enrollment. All participants had to agree to practice appropriate contraception. Hydroxyurea was permitted until day 5 if necessary to maintain white blood cell count (WBC) <40 000/μL. Patients were treated with 21-day course of oral lenalidomide in addition to 96-hour continuous intravenous (IV) cytarabine infusion administered on days 5-8 and idarubicin administered IV as 1-hour infusion on days 5, 6 and 7 ( Figure 1A). Lenalidomide dose was escalated in separate cohorts using a classic 3 + 3 phase one design to determine the MTD with this combination. No intrapatient dose escalation was allowed. After establishment of the MTD, a planned 12-patient expansion group was included to better estimate the tolerability of this dose and to provide a preliminary efficacy assessment.
All patients underwent bone marrow biopsy at baseline, on day 5 after single-agent lenalidomide therapy, and on day 28 after the completion of induction cycle. Response was assessed according to the International Working Group criteria for AML and MDS. 24,25 Patients who achieved CR were evaluated for allogeneic HCT or a single cycle of consolidation therapy on this trial, which consisted of oral lenalidomide dosed as during the induction, cytarabine on days 5-7, and idarubicin on days 5 and 6.
F I G U R E 1 A, Study schema for 21-day re-induction cycle in relapsed/refractory acute myeloid leukemia or high-risk myelodysplastic syndrome cohort. Patients underwent bone marrow (BM) biopsy prior to study entry and on day 5 after single-agent lenalidomide therapy. Cytarabine was started on day 5 and administered as 96-hour continuous infusion. Idarubicin (Ida) was given intravenously once a day on days 5, 6, 7 and each day the infusion lasts 1 hour. The table shows dosing schema for the study drugs at dose levels −1, 1 and 2. B, Best response to trial therapy according to dose level. CR, complete response; CRi, CR with incomplete count recovery. C, Kaplan-Meier overall survival and relapse-free survival curves of all patients treated on this study, as well as comparison between patients who achieved remission on this study (responders) and patients who were refractory to study regimen (non-responders) [Color figure can be viewed at wileyonlinelibrary.com]

| Safety assessment
Adverse events were graded per the Common Terminology Criteria for Adverse Events (version 4.0). The DLT was defined during cycle one of therapy. It is recognized that drug-related toxicity in this population may be difficult to ascertain given the aggressive nature of the disease. Nonhematologic grade four toxicities were defined as DLTs if they were drug-related. Toxicity attributed to any of the three agents was considered dose-limiting. Hematologic DLT was defined as failure to recover absolute neutrophil count (ANC <500/μL) or platelet count (<25 000/μL) by day 42 in the absence of persistent disease. If the DLT occurred in two or more patients at a single dose level, then that dose was deemed intolerable and the next lower dose level was expanded. Given the high prevalence of infectious complications associated with cytopenias, infection-related complications were not considered DLT unless severity or duration was longer than expected with conventional treatment. Alopecia, nausea and vomiting controllable with antiemetic therapy, line-associated venous thrombosis, transient and correctable electrolyte abnormalities were also not considered DLT.

| Mutational analysis
Viably preserved patient bone marrow and blood samples were sequenced using targeted amplicon sequencing on the HiSeq (Illumina) platforms to detect common myeloid mutations as described before. 26

| Outcomes and statistical analysis
The primary objective was to determine the MTD of lenalidomide in combination with conventional chemotherapy in patients with R/R

| RESULTS
A total of 33 patients were enrolled in the study (30 AML, three MDS) and their clinical characteristics are summarized in Table 1. None of the patients had prior lenalidomide exposure.

| Toxicity profile and MTD assessment
Toxicity data were collected for 33 patients and ≥grade three nonhematologic adverse events are shown in Table 2. A total of eight DLTs (24%) were observed with four being hematologic DLTs. At dose level one, three patients were initially enrolled, of whom one of them experienced hematologic DLT (neutropenia). Three more patients were enrolled at this dose level, one had grade four confusion (requiring intensive-care unit stay) which was likely multifactorial and not drug-related. We subsequently enrolled three patients at dose level 2, one had prolonged grade four neutropenia and one patient died on day 5 of the study due to pulmonary hemorrhage after bronchoscopic procedure. Three more patients were enrolled at this dose level, one developed heart failure symptoms due to new onset reduced left ventricular ejection fraction, thought possibly due to anthracycline toxicity. Therefore, the dose was reduced back to dose level 1 with three more patients enrolled, two of whom had prolonged grade four neutropenia and one patient died from intracranial hemorrhage. This led to further de-escalation to dose level −1, which delivered the same lenalidomide dose (25 mg daily) as the previous dose level, but both idarubicin and cytarabine were dose-reduced (see Figure 1). The initial six patients enrolled at this level did not experience DLT, therefore this dose level was determined to be the MTD and recommended to pursue for the phase II dose level. Twelve subsequent patients were enrolled for pre-planned dose expansion to further assess safety and tolerability.
Only one patient experienced serious adverse event (SAE) with acute renal failure, septic shock and respiratory failure.

| Clinical responses
Twenty-nine out of 33 enrolled patients were evaluable for efficacy ( Figure 1B) The median OS for the entire cohort was 6 months, and the median RFS for patients who achieved CR/CRi was 4.4 months ( Figure 1C). The

| Molecular profile and response
Next-generation sequencing for recurrent somatic mutations was performed at study entry in 24 patients (Figure 2A). There was no clear association between mutation patterns and response to therapy.
Twelve patients also had serial pre-treatment and post-treatment samples available for analysis ( Figure 2B). Among seven patients with

| DISCUSSION
Effective therapeutic options for patients with R/R AML are limited.
Intensive chemotherapy is often less effective than in the front-line setting and is associated with substantial toxicity. Several novel targeted therapies demonstrated significant clinical activity in R/R AML, but their use is restricted to specific biologic subtypes of AML such as patients with mutated FLT3 or IDH genes. Therefore, the most common approach for fit patients with R/R disease is to achieve remission followed by allogeneic HCT. Historically, remission rates with salvage chemotherapy have been reported at 16%-25%. 4 In this phase one study, we found that lenalidomide given for 21 days in combination with  12 In this study, we could safely administer lenalidomide 25 mg/d with a dose-adjusted chemotherapy regimen. However, administration of this dose beyond 10 days was associated with increased toxicity, which supports the 10-day schedule of the previous study.
Preclinical data suggest that lenalidomide may sensitize leukemic blasts to cytotoxic chemotherapy. 8 This was also observed in comparative analysis of paired marrow samples obtained before and after lenalidomide treatment of patients participated on phase one lenalidomide plus MEC study. 28 Lenalidomide enhanced apoptotic priming in vivo and dynamic BH3 profiling assay was suggested as a biomarker to predict response to combination. Embarking on this encouraging mechanistic data, a recent phase two study of 222 newly diagnosed older AML patients compared standard 7 + 3 induction chemotherapy with or without lenalidomide at a dose of 20 mg daily on days 1-21. 21 Although the combination was well tolerated, addition of lenalidomide did not improve the CR/CRi rate, event-free survival or OS. Given the importance of sequencing these drugs, future studies would be warranted to investigate the impact of pretreatment with higher doses of lenalidomide. It should also be noted that therapeutic activity of lenalidomide was more pronounced with prolonged treatment in other diseases (eg, myeloma). Therefore, a maintenance treatment strategy should also be considered in AML.
Lenalidomide has several immunomodulatory effects, including stimulation of innate, cellular and humoral responses. 6 Initial reports of lenalidomide in AML relapsed after HCT showed promising antileukemic activity at a cost of severe GVHD. 9 However, a recent phase one study in this setting demonstrated that a salvage regimen with 7 days of azacitidine followed by 25 mg daily lenalidomide on days 10-30 (of a 30-day cycle) was tolerable with acceptable rates of GVHD and 47% clinical response. 10 It should be noted that patients in these series received either alemtuzumab or anti-thymocyte globulin for GVHD prophylaxis as opposed to T-replete allografts used in other lenalidomide maintenance studies. Additional studies are needed to optimize the lenalidomide containing salvage and maintenance regimens for post-HCT AML patients.
In addition to its incompletely understood mechanism of action, the molecular subgroup of AML patients who may derive the most benefit from lenalidomide is not clearly defined. Given its efficacy in MDS with del(5q), prior studies focused on testing this drug in AML patients with del(5q) and showed only modest benefit. 13 We reported single agent activity of lenalidomide in AML patients with isolated trisomy 13 9,29 and another brief report demonstrated clinical activity of lenalidomide in combination with hypomethylating agents in AML patients with inv(3). 30 Due to the low frequency of these abnormalities in AML, lenalidomide clinical trials including the present study lack the statistical power to analyze individual karyotypic abnormalities.
Similarly, our study and others could not identify a myeloid mutation signature predicting response to lenalidomide. 12 This study has several limitations. Given the phase I nature of the study, data on efficacy of the combination regimen should be inter-