Fludarabine, Cytarabine and Gentuzumab Ozogamicin (FLA-GO) as Salvage Therapy and Bridge to Transplant in Adult Relapsed Acute Myeloid Leukemia (AML) Patients

Special Article - Acute and Chronic Myeloid Leukemia

Ann Hematol Oncol. 2017; 4(4): 1145.

Fludarabine, Cytarabine and Gentuzumab Ozogamicin (FLA-GO) as Salvage Therapy and Bridge to Transplant in Adult Relapsed Acute Myeloid Leukemia (AML) Patients

Cefalo M¹, Del Principe MI¹*, Buccisano F¹, Di Piazza F¹, Ottaviani L¹, Maurillo L¹, Sarlo C², De Santis G¹, Consalvo MI¹, De Bellis E¹, De Angelis G¹, Zizzari A¹, Paterno G¹, Divona M¹, Postorino M¹, Del Poeta G¹, Sconocchia G³, Arcese W¹, Amadori S¹ and Venditti A¹

¹Cattedra di Ematologia, Dipartimento di Biomedicina e Prevenzione, Università Degli Studi di Roma “Tor Vergata”, Italy

²Ematologia, Policlinico Universitario-Campus Biomedico, Italy

³Istituto di Farmacologia Translazionale, Dipartimento di Medicina, CNR, Italy

*Corresponding author: Maria Ilaria Del Principe, Cattedra di Ematologia, Dipartimento di Biomedicina e Prevenzione, Università Degli Studi di Roma “Tor Vergata”, Roma, Italy

Received: January 25, 2017; Accepted: March 16, 2017; Published: March 31, 2017


Twenty-seven patients affected by relapsed/refractory acute myeloid leukemia (AML) were treated with a combination of fludarabine, cytarabine; (FLA) and gentuzumabozogamicin(GO). Complete remission (CR) rate was 37% (10/27 patients), with one patient (4%) dying early because of treatment related infection. Six out of 10 patients (60%) who achieved a second CR were able to receive allogeneic SCT. Although the difference was not statistically significant, likely due to the small sample size, we observed that CR rate was higher in patients< 60 years and with a first CR duration >12 months. Two-years cumulative incidence of relapse and overall survival were 20±13% and 20±8%, respectively. In conclusion, although based on a small sample size, our study suggests that the association of FLA plus GO has a favorable toxicity profile and might be used as a bridge to transplant in young patients, especially when the duration of first CR is = 12 months.

Keywords: Acute myeloid leukemia, Gentuzumabozogamicin, CD33 antigen, Salvage therapy


Prognosis of adult patients with relapsed/refractory acute myeloid leukemia (AML) is generally unsatisfactory with a 5-year overall survival (OS) of 10-30% [1-4]. Such a dismal outcome is well explained with a lack in availability of effective salvage options and with the problematic access to allogeneic stem cell transplantation (ASCT), which remains the only potential curative approach.

Salvage regimens generally rely on the delivery of high-dose cytarabine (ARA-C), alone or in combination, followed by ASCT [5]. Among combinatorial approaches, the association of fludarabine and ARA-C (FLA) was found to be superior to ARA-C single agent. In fact, fludarabine modulates the pharmacologic activation of ARA-C [6-8] therefore increasing the rate of accumulation of its active metabolite ARA-C 5’-triphosphate [6].

Besides attempts to ameliorate the efficacy of conventional chemotherapy, developing and implementing programs of “targeted therapy” has represented a further expedient to approach difficultto- treat diseases such as relapsed/refractory AML. Among agents able to target specific leukemic antigens or pathways, Gentuzumab Ozogamicin (GO) is one of the most extensively investigated. GO is a humanized anti-CD33 monoclonal antibody conjugated to a semisynthetic derivative of calicheamicin, a cytotoxic drug that is released inside the leukemic cells after the internalization of the immunoconjugate. CD33 is a 67-kD transmembrane cell surface glycoprotein receptor, which represents an attractive target for immunotherapy since it is expressed on the external surface of leukemic blasts in more than 80% of patients with AML, but not on normal precursor hematopoietic cells [4]. Either as a single agent or in combination with chemotherapy, GO was reported to improve overall responses rate in adults with relapsed/refractory AMLs [8-10].

Based on these premises, we conducted a retrospective analysis of 27 adult patients with relapsed/refractory AML, to evaluate the efficacy and the safety of the association FLA plus GO (FLA-GO) and to assess the feasibility of ASCT after FLA-GO.

Patients and Methods


Twenty-seven adult patients with refractory/relapsed AML treated with FLA-GO regimen between 2006 and 2010 at the Policlinico Tor Vergata of Rome, were analyzed for the purposes of the present study. Approval for this study was obtained from the institutional review board. All patients received written informed consent in accordance with the declaration of Helsinki. Inclusion criteria were relapsed/ refractory AML, age >18 years, ECOG performance status= 2 [11], normal renal and liver function (creatinine = 2 mg/dl, total bilirubin = 2 mg/dl), adequate cardiac function (LVFE =50%). Patients with Acute Promyelocitic Leukemia were excluded.

Routine molecular, karyotypic and immunophenotypic analysis

Total RNA was extracted from Ficoll-Hypaque isolated bone marrow mononuclear cells collected both at diagnosis and at disease relapse using standard procedures [12] and reverse-transcribed with random hexamers as primers [13]. Diagnostic molecular studies for recurrent translocations were performed according to previously published methods [13]. DNA was also extracted using a columnbased Qiagen kit protocol. Fms-like tyrosine kinase- internal tandem duplication (FLT3 ITD) and nucleophosmin1 (NPM1) gene mutations were investigated using protocols reported elsewhere [14- 17].

Conventional karyotyping was performed on bone marrow aspirates after short-term culture and analysed after G-banding. The description of the karyotype was according to the International System for Human Cytogenetic Nomenclature [18].

Bone marrow samples were also collected for immunophenotyping with a standard panel of antibodies [19]. Briefly, each antibody was incubated with 1-2x106 cells in a 100μl of volume, and isotypematched antibodies were used as negative controls. After incubation, cells were re-suspended in 0.5 ml PBS and analyzed with a flow cytometer (Facs Canto; Becton Dickinson). Percentage of CD33 positive blasts was determined drawing a proper gate on the leukemic population in the FSC/SSC plot.

Treatment schedule

The chemotherapy regimen consisted in a 30-minute infusion of intravenous fludarabine, 30 mg/m2/day on days 1-5 and, on the same days, in 2-hour infusion of intravenous ARA-C, 2 g/m2/day. In order to generate the best pharmacologic interaction between fludarabine and ARA-C, infusion of ARA-C began 4 hours after the end of that of fludarabine. On day 6, intravenous GO (Mylotarg, Pfizer, New York, NY, and Ben Venue Laboratories, Bedford, OH, USA) was infused at the dose of 6 mg/m2 Thirty minutes before GO administration, all patients received a pre-medication with methylprednisolone (40 mg, flat dose ). Duration of GO infusion was two hours. All patients had a central venous catheter (CVC) inserted, all received fluoroquinolonebased and anti-fungal prophylaxis from the start of chemotherapy until resolution of neutropenia or introduction of empirical antibiotic therapy for febrile neutropenia. When transfusions were required, blood components were irradiated before infusion [20].

Response definition and safety monitoring

Bone marrow aspiration for determination of response was performed on day + 28±2 from the start of chemotherapy. Response criteria were those of the International Working Group [21] based on whichCR was defined as a bone marrow blasts count <5%, absolute neutrophil count >1x109/l, platelets count >100x109/l and transfusions independence. Partial Remission (PR) was defined as a bone marrow blasts percentage of 5% to 25%. Toxicity was evaluated according to Common Terminology Criteria for Adverse Events [22]. Early death (ED) was defined as death within 31 days following day 1 of chemotherapy administration.

Outcome definition and statistical analysis

Descriptive statistics are presented including median and range for continuous variables, absolute and relative frequencies for categorical variables. The relapse rate after FLA-GO was assessed by the cumulative incidence (CI) function, using the competing risks method and considering death without relapse like competing event. The Kaplan-Meier product-limit method was used to estimate and plot the OS. Differences between groups were determined using the Gray’s test and the log-rank test for the CI measures and for OS, respectively. A p value of <0.05 was considered statistically significant. All the analysis were conducted using software R version 2.15.0.


The clinical and biological characteristics of the 27 patients are summarized in Table 1. Twenty patients were males, 7 females. Median age was 58 years (range 22-71 years). Five patients had an AML primarily refractory to a first induction course and 22 were in first relapse after a frontline induction regimen combining anthracycline, etoposide and ARA-C. Among patients in first relapse, 2 relapsed after ASCT and 1 after autologous SCT. In 14 of 22 (64%) patients who relapsed, duration of first CR (CR1) was =12 months. Overall, median duration of CR1 was 9 months (range 2-110). At the time of resistance or relapse, cytogenetic data were available in 25 of 27 (93%) patients: 2 (8%), 15 (55%) and 8 (29%) had a favorable-, intermediate- and unfavorable-risk karyotype, respectively [23]. From a molecular point of view, 8 of the 27 (30%) patients had FLT3 ITD mutation and 3 (11%) had both NPM1 and FLT3-ITD mutation. Median percentage of CD33 expression was 88% (range 4-100%) with 19 patients (70%) showing positivity in more than 80% of the leukemic blasts.