Robert E. Gallagher

Prognostic relevance of integrated genetic profiling in acute myeloid leukemia

BACKGROUND Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML. METHODS We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients. RESULTS We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67). CONCLUSIONS We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).

Arsenic trioxide improves event-free and overall survival for adults with acute promyelocytic leukemia: North American Leukemia Intergroup Study C9710

Arsenic trioxide (As(2)O(3)) is a highly effective treatment for patients with relapsed acute promyelocytic leukemia (APL); its role as consolidation treatment for patients in first remission has not been defined. We randomized 481 patients (age ≥ 15 years) with untreated APL to either a standard induction regimen of tretinoin, cytarabine, and daunorubicin, followed by 2 courses of consolidation therapy with tretinoin plus daunorubicin, or to the same induction and consolidation regimen plus two 25-day courses of As(2)O(3) consolidation immediately after induction. After consolidation, patients were randomly assigned to one year of maintenance therapy with either tretinoin alone or in combination with methotrexate and mercaptopurine. Ninety percent of patients on each arm achieved remission and were eligible to receive their assigned consolidation therapy. Event-free survival, the primary end point, was significantly better for patients assigned to receive As(2)O(3) consolidation, 80% compared with 63% at 3 years (stratified log-rank test, P < .0001). Survival, a secondary end point, was better in the As(2)O(3) arm, 86% compared with 81% at 3 years (P = .059). Disease-free survival, a secondary end point, was significantly better in the As(2)O(3) arm, 90% compared with 70% at 3 years (P < .0001). The addition of As(2)O(3) consolidation to standard induction and consolidation therapy significantly improves event-free and disease-free survival in adults with newly diagnosed APL. This trial was registered at clinicaltrials.gov (NCT00003934).

Single Cycle of Arsenic Trioxide–Based Consolidation Chemotherapy Spares Anthracycline Exposure in the Primary Management of Acute Promyelocytic Leukemia

PURPOSE Event-free survival following all-trans-retinoic acid (ATRA) -based therapy for acute promyelocytic leukemia (APL) averages 70% at 5 years. While arsenic trioxide (ATO) can induce remissions in 95% of relapsed patients, few studies have addressed the integration of ATO into the primary management of APL. This study examines the efficacy of a single cycle of ATO-based consolidation therapy in a treatment regimen designed to decrease exposure to other cytotoxic agents. PATIENTS AND METHODS After induction with ATRA and daunorubicin (DRN), untreated patients with APL received 3 days of cytarabine and DRN followed by 30 doses of ATO beginning on day 8. Molecular remitters received 2 years of risk-based maintenance therapy. Results Forty-one of 45 patients receiving induction therapy achieved remission; four patients died (one before treatment was initiated). Thirty-seven patients received consolidation and maintenance; of these one patient relapsed (CNS) and one died in remission during maintenance therapy (hepatic sickle cell crisis). With a median follow-up of 2.7 years, estimated disease-free survival was 90%; overall survival for all patients was 88%. Despite a total anthracycline dose of only 360 mg/m(2), cardiac ejection fraction decreased by > or = 20% in 20% of patients. CONCLUSION These data, combined with other recent studies using ATO in the primary management of APL, demonstrate the important role that ATO can play in the primary management of this curable disease. Future studies should continue to focus on reducing the toxicity of treatment without increasing the relapse rate.

Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases

The primary objective of this study was to determine whether caspases are involved in arsenic trioxide(ATO)-induced apoptosis of human myeloid leukemia cells. A secondary objective was to determine whether apoptosis induced by ATO compared with VP-16 is differentially affected by an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), which has been reported to inhibit apoptosis induced by some chemotherapeutic agents. NB4 and HL60 cells were incubated with ATO in the presence and absence of the caspase protease inhibitors Z-VAD.fmk or Y-VAD. cho. Apoptosis was assessed by morphology, DNA laddering and flow cytometry. Poly (ADP-ribose) polymerase (PARP) cleavage was used as a marker for the activation of caspases. PARP cleavage occurred during ATO-induced apoptosis in both NB4 and HL60 cells. Z-VAD.fmk, a broad-spectrum inhibtor, could block ATO-induced apoptosis and PARP cleavage, whilst Y-VAD. cho, a selective inhibitor of caspase 1, had no such effect. PMA pre-incubation for up to 8 hours under conditions known to activate PKC had no effect on either ATO- or VP-16-induced apoptosis. We conclude that in cultured myeloid leukemia cells ATO-induced apoptosis is executed by caspases from the distal, PARP-cleaving part of the activation cascade and that PKC activation has no effect on apoptosis induced by either ATO or VP-16 in these cells.

Inhibition of all-trans-retinoic acid metabolism by fluconazole in vitro and in patients with acute promyelocytic leukemia

All-trans-retinoic acid induces acute promyelocytic leukemia cell differentiation in vitro, and it produces greater than 90% complete remissions in patients with acute promyelocytic leukemia. Despite the high response rate, the majority of patients relapse with continued trans-retinoic acid therapy, and disease progression has been observed to be accompanied by an increase in the metabolism of trans-retinoic acid in the patients. In this study, the pharmacokinetic disposition of trans-retinoic acid was determined by HPLC in patients with acute promyelocytic leukemia before and after concurrent therapy with the triazole antimycotic agent fluconazole. Treatment with trans-retinoic acid for 1 week reduced the area under the plasma trans-retinoic acid concentration vs time curve in one patient by 67%, from 277 to 91 ng/mL/hr. Trans-retinoic acid pharmacokinetics were repeated after the second dose of fluconazole, administered 1 hour prior to the retinoid, and the AUC was found to be 401 ng/mL/hr, a greater than 4-fold increase from the pre-fluconazole level. A similar, though more modest, effect of fluconazole was seen in a second acute promyelocytic leukemia patient. The effect of fluconazole on trans-retinoic acid metabolism was examined in vitro using isolated human hepatic microsomes. Fluconazole inhibited the NADPH-dependent cytochrome P450-mediated catabolism of trans-retinoic acid in a concentration-dependent manner. Although fluconazole was approximately one-half as potent an inhibitor when compared with ketoconazole, a related antifungal drug, 60-90% inhibition was observed at the concentrations of fluconazole measured in the acute promyelocytic leukemia patients. Neither fluconazole nor ketoconazole inhibited lipid hydroperoxide-mediated metabolism of trans-retinoic acid. Since fluconazole is a well-tolerated agent frequently administered to leukemia patients, its use in combination with trans-retinoic acid merits further consideration.

A surrogate marker profile for PML/RARα expressing acute promyelocytic leukemia and the association of immunophenotypic markers with morphologic and molecular subtypes

The availability of genotype‐specific therapy for PML/RARαpos acute promyelocytic leukemia (APL) requires that this disease be precisely diagnosed. Immunophenotypic characteristics heretofore proclaimed as reliably characterizing APL (HLA‐DRlow, CD34low, P‐glycoproteinlow myeloid phenotype) do not differentiate from APL‐like immune profiles unassociated with the PML/RARα fusion transcript.

Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Cell cycle-dependent reactivity with the monoclonal antibody Ki-67 during myeloid cell differentiation☆

The specificity and sensitivity of the monoclonal antibody Ki-67 in identifying proliferating cell compartments was tested with the human promyelocytic leukemia cell line HL-60 using multi-parameter flow cytometry. While correlated measurements of DNA content and Ki-67 immunofluorescence indicated that the antigen was present in all phases of the cell cycle, reactivity with the antibody was highest in proliferating S and G2+M cells. The analysis of the BrdU content of cells sorted on the basis of reactivity with Ki-67 showed a correlation between Ki-67 reactivity and BrdU uptake. In HL-60 cells induced to differentiate with dimethyl sulfoxide (DMSO), the loss of reactivity with Ki-67 paralleled the exit of cells from the cell cycle. This was not observed in DMSO-resistant HL-60 cells. These results validate the usefulness of the Ki-67 antibody for determining the proliferative stage of mammalian cells in culture.

Improving acute promyelocytic leukemia (APL) outcome in developing countries through networking, results of the International Consortium on APL.

Thanks to modern treatment with all-trans retinoic acid and chemotherapy, acute promyelocytic leukemia (APL) is now the most curable type of leukemia. However, this progress has not yielded equivalent benefit in developing countries. The International Consortium on Acute Promyelocytic Leukemia (IC-APL) was established to create a network of institutions in developing countries that would exchange experience and data and receive support from well-established US and European cooperative groups. The IC-APL formulated expeditious diagnostic, treatment, and supportive guidelines that were adapted to local circumstances. APL was chosen as a model disease because of the potential impact on improved diagnosis and treatment. The project included 4 national coordinators and reference laboratories, common clinical record forms, 5 subcommittees, and laboratory and data management training programs. In addition, participating institutions held regular virtual and face-to-face meetings. Complete hematological remission was achieved in 153/180 (85%) patients and 27 (15%) died during induction. After a median follow-up of 28 months, the 2-year cumulative incidence of relapse, overall survival (OS), and disease-free survival (DFS) were 4.5%, 80%, and 91%, respectively. The establishment of the IC-APL network resulted in a decrease of almost 50% in early mortality and an improvement in OS of almost 30% compared with historical controls, resulting in OS and DFS similar to those reported in developed countries.

Treatment-influenced associations of PML-RARα mutations, FLT3 mutations, and additional chromosome abnormalities in relapsed acute promyelocytic leukemia

Mutations in the all-trans retinoic acid (ATRA)-targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse. Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD+ were also associated with reduced postrelapse survival.