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Blood, 1 August 2007, Vol. 110, No. 3, pp. 1025-1028. Prepublished online as a Blood First Edition Paper on April 10, 2007; DOI 10.1182/blood-2006-12-061283. This Article Abstract Full Text (PDF) All Versions of this Article: blood-2006-12-061283v1 110/3/1025    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Tamburini, J. Articles by Bouscary, D. Search for Related Content PubMed PubMed Citation Articles by Tamburini, J. Articles by Bouscary, D. Related Collections Neoplasia Signal Transduction Brief Reports Clinical Trials and Observations Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  NEOPLASIA Brief Report Constitutive phosphoinositide 3-kinase/Akt activation represents a favorable prognostic factor in de novo acute myelogenous leukemia patients Jerome Tamburini1,4,6, Caroline Elie4,7, Valérie Bardet1,5, Nicolas Chapuis1,4, Sophie Park1,4,6, Philippe Broët8, Pascale Cornillet-Lefebvre9, Bruno Lioure10, Valérie Ugo11, Odile Blanchet12, Norbert Ifrah13, Francis Witz14, François Dreyfus1,4,6, Patrick Mayeux1,4, Catherine Lacombe1,5, and Didier Bouscary1,4,6 1 Institut Cochin, Département d'Hématologie, Paris; 2 Institut National de la Santé et de la Recherche Médicale (INSERM), U567, Paris; 3 Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, Paris; 4 Université Paris Descartes, Faculté de Médecine René Descartes, Unité Mixte de Recherche (UMR)-S 8104, Paris; 5 Laboratoire d'Hématologie, Assistance Publique–Hôpitaux de Paris, Hôpital Cochin, Paris; 6 Service de Médecine Interne–Hématologie, Assistance Publique–Hôpitaux de Paris, Hôpital Cochin, Paris; 7 Département de Biostatistique, Hôpital Cochin, Paris; 8 JE 2492, Faculté de Médecine Paris Sud; 9 Laboratoire d'Hématologie, Centre Hospitalo–Universitaire (CHU) Reims; 10 Département d'Hématologie et d'Oncologie, Hôpitaux Universitaires, Strasbourg; 11 Laboratoire d'Hématologie, CHU Brest; 12 Laboratoire d'Hématologie, CHU Angers; 13 Service des Maladies du Sang, CHU Angers; 14 Service d'Hématologie, Hôpital Brabois, Nancy, France    Abstract Top Abstract Introduction Patients, materials, and methods Results and discussion Authorship References   The phosphoinositide 3-kinase (PI3K/Akt) pathway is activated in acute myelogenous leukemia (AML) and is promising for targeted inhibition. Ninety-two patients with primary AML were analyzed for PI3K/Akt constitutive activation. Fifty percent of the patients presented with constitutive PI3K activation (PI3K +). No difference was observed between PI3K + and PI3K – groups concerning age, sex, white blood cell count, lactate dehydrogenase (LDH) level, bone marrow blast cells, French-American-British (FAB) classification, cytogenetics, RAS or nucleophosmin (NPM) mutations. Slightly more FLT3-ITD was detected in the PI3K – group (P = .048). The complete remission rate was similar between the 2 groups. With a median follow-up of 26 months, we observed for PI3K + and PI3K – patients, respectively, 56% and 33% overall survival (P = .001) and 72% and 41% relapse-free survival (P = .001). Constitutive PI3K/Akt activity is a favorable prognosis factor in AML, even after adjustment for FLT3-ITD, and may confer a particular sensitivity to chemotherapy. A better understanding of the downstream effectors of the PI3K/Akt pathway is needed before targeting in AML.    Introduction Top Abstract Introduction Patients, materials, and methods Results and discussion Authorship References   Several recent articles have highlighted that the class IA phosphoinositide 3-kinase/serine-threonine kinase (PI3K)/Akt pathway is activated in acute myelogenous leukemia (AML), both in blast cells and in the immature CD34 +, CD38Low, and CD123 + population.1–4 Most PI3K activity in AML is caused by the p110 isoform of PI3K, the activity of which can be specifically inhibited by IC87114.5,6 The mechanisms leading to PI3K activation are unclear and no activating mutation in the PI3KCD gene coding this isoform has been identified.7 Two studies have suggested that the prognosis of AML patients with an upregulated PI3K/Akt pathway is worst, suggesting that this pathway could constitute a therapeutic target.2,8 To ascertain this hypothesis, we prospectively studied constitutive PI3K/Akt/FOXO3A activation in 92 de novo AML patients.    Patients, materials, and methods Top Abstract Introduction Patients, materials, and methods Results and discussion Authorship References   Patients Ninety-two patients with de novo AML treated in the AML-2001 trial from the Groupe Ouest-Est des Leucémies et Autres Maladies du Sang (GOELAMS) were included, after approval by the GOELAMS Institutional Review Board and receipt of signed informed consent in accordance with the Declaration of Helsinki. The patients received cytosine-arabinoside (200 mg/m2, days 1-7) and daunorubicin (60 mg/m2, days 1-3) or idarubicin (8 mg/m2, days 1-5), and a second course (daunorubicin 35 mg/m2, days 17-18 or idarubicin 8 mg/m2, days 17-18, and cytosine-arabinoside 1 g/m2 every 12 hours days 17-20) if bone marrow blasts were more than 5% at day 15. All patients in complete remission received low-dose consolidation with daunorubicin (60 mg/m2, days 1-2) or idarubicin (12 mg/m2, days 1-2), and cytosine-arabinoside (100 mg/m2, days 1-7). Patients with HLA-identical siblings underwent allogenic stem cell transplant (up front until 50 years old; after a consolidation and with a reduced intensity conditioning after 51 years old). Other patients received one consolidation (idarubicin 12 mg/m2 or daunorubicin 60 mg/m2, days 1-2 and cytosine-arabinoside 3g/m2 every 12 hours days 1-4), followed by autologous stem cell transplantation or high-dose chemotherapy in favorable-risk karyotypes. Cell processing and PI3K/Akt activity detection Fresh blast cells from the bone marrow were studied at diagnosis for PI3K/Akt activation and FOXO3A (a Forkhead transcription factor) phosphorylation. Highly infiltrated samples (75 [81%] of 92) were studied by Western blot, and others (17 [19%] of 92) by flow cytometry. We previously reported that both methods gave identical results when tested on the same samples.4 Blast cells were starved for 4 hours in cytokine and serum-free medium before testing for constitutive or FLT3-ligand (a growth factor for hematopoietic progenitors)–stimulated Akt and FOXO3A phosphorylation by Western blot analysis. Antibodies used were anti-p-Akt (Ser 473 and Thr 308, Cell Signaling Technologies, Danvers, MA), anti-pFOXO3A (Thr 32; Upstate Biotechnology, Charlottesville, VA), anti-beta-actin (Sigma, St Louis, MO), and anti-Akt1/2 (Cell Signaling Technologies). Dual color flow cytometry of Akt Ser 473 phosphorylation in the CD45low leukemic population was performed as described.4 FLT3, N-RAS, K-RAS, and NPM gene mutational status These analyses were performed as described.7,9,10,11 Statistical analysis Statistical analysis is described in Figure 1B and 1C. View larger version (9K): [in this window] [in a new window]   Figure 1. Patients with AML can be separated into 2 groups: with constitutive activation of PI3K (PI3K + group) and without PI3K activation (PI3K – group). (A) Blast cells from patients 1 and 2 were analyzed for PI3K/Akt activation at the indicated times, after starvation in serum- and cytokine-free medium, to determine constitutive and sustained activation of this signaling pathway. Proteins from 106 cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot using antibodies directed against p-Akt (Ser 473) and pFOXO3A (Thr 32). To ensure equal loading, blots were reprobed with anti-actin or anti-Akt antibodies. LY294002 was used at 25 µM for 30 minutes to inhibit PI3K/Akt. The functionality of the PI3K/Akt pathway was tested in patient 2, after 15-minute stimulation with FLT3 ligand at 50 ng/mL. Vertical lines were drawn to indicate cuts made to put together nonadjacent regions from the same gel. (B) Kaplan-Meier curves for overall survival according to the PI3K status. Classical log-rank test was used to compare survival distribution between subgroups. We tested the proportional hazard hypothesis using a test proposed by Grambsch et al.14 To investigate for a mixed effect (early and late effect) of PI3K/Akt activation, we tested the null hypothesis of no short-term and no long-term effect, using the model proposed by Broët et al.15 This latter test allows for testing 3 null hypotheses: (1) no long-term and short-term effect; (2) no short-term effect; and (3) no long-term effect (whatever a short-term effect is). Here, the long-term effect represents the effect on the proportion of patients alive after 30 months. (C) Cumulative incidence curves for relapse according to the PI3K status. Relapse probabilities were obtained from crude cumulative incidence estimates, treating death before relapse and relapse as competing risk events. Crude cumulative incidence curves for relapse were compared between subgroups using the Gray test.16      Results and discussion Top Abstract Introduction Patients, materials, and methods Results and discussion Authorship References   Constitutive activation of PI3K is detected in 50% of patients We first determined that a 4-hour starvation clearly divided AML patients into 2 groups: one with PI3K constitutive activation (PI3K +) and the other without PI3K activation (PI3K –; Figure 1A). In patient 1 (PI3K + ), Akt (Ser 473) and FOXO3A (Thr 32) phosphorylations were detected with the same intensity during 8 hours of starvation. LY294002 inhibited both phosphorylations. Patient 2 is representative of the PI3K – group and had no detectable Akt or FOXO3a phosphorylation. The functionality of the PI3K/Akt pathway was detected by stimulating the cells with FLT3-ligand as shown in patient 2. All PI3K – samples presented an activation of the pathway after cytokine stimulation. In 15 samples tested, a perfect correlation between Akt phosphorylation on Ser 473 and on Thr 308 residues was found (data not shown). Foxo3A phosphorylation was only detected in PI3K + patients. The PI3K + group represented 46 (50%) of 92 patients. Association between PI3K status and clinical and biologic data Patient characteristics according to their PI3K status are reported in Table 1. There was no difference between the 2 groups for age, sex, FAB subtypes, cytogenetic subgroups, myelodysplastic syndrome (MDS) features, white blood cell counts, LDH levels, bone marrow blast percentage, or tumor burden. The rates of N-RAS/K-RAS and NPM mutations were similar between PI3K + and PI3K – patients. FLT3-ITD were slightly more frequent in the PI3K – group (P = .048; Table 1). View this table: [in this window] [in a new window]   Table 1. Characteristics of the 92 patients according to their PI3K/Akt status   Overall survival and relapse-free survival are better in patients with constitutive PI3K Complete remission was obtained in 74 (80%) of 92 patients with no difference between the 2 groups (P = .60). With a median follow-up of 26 months (range, 3 days to 37 months), 48 patients died. Overall survival was 45% (95% confidence interval [CI], 34%–58%) for the entire population. Figure 1B shows a clear nonproportional effect between the 2 groups, confirmed by the test by Grambsch et al14 (P = .002). A 2-phase effect is present (Figure 1B), with 2 hazard rates crossing each other at approximately 1 year. When testing for short-term and long-term effects, the test is highly significant (P = .014), with PI3K activation being associated with a better long-term survival. For an actually unknown reason, slightly more patients in the PI3K + group died from infection before 12 months without AML relapse (short-term effect, P = .027). Nevertheless, the overall survival rate at 30 months was 56% (95% CI, 41%-76%) in the PI3K + group and 33% (95% CI, 21%-52%) in the PI3K – group. Thirty patients relapsed. Crude cumulative incidence curves for relapse are presented in Figure 1C. Relapse free survival was 72% (95% CI, 54%-89%) in the PI3K + group and 41% (95% CI, 24%-58%) in the PI3K – group (P = .001). Because there was more FLT3-ITD in the PI3K – patients, and a nonsignificant imbalance in term of unfavorable cytogenetics (PI3K + 16% and PI3K – 30%), explorative multivariate models were performed. The PI3K activation still played a significant prognostic role on overall survival and relapse-free survival after adjustment for FLT3-ITD (P = .01 and P = .007, respectively) and cytogenetics (P = .014 and .008, respectively). Two studies have correlated Akt phosphorylation to an adverse overall survival in AML.2,8 In their study, Kornblau et al8 did not starve blast cells before testing for Akt phosphorylation, and they did not test for constitutive PI3 activation. Accordingly, they observed that 91% of blast samples presented Akt phosphorylation, whereas we observed constitutive activation in 50% of AML samples only. Moreover, the populations studied were not similar, because all patients in our study had de novo AML, whereas 32% in Kornblau's study had AML secondary to MDS or chemotherapy.8 In their study, Min et al2 observed that Akt was undetectable in pAkt-negative patients. Clearly, their samples are different from those tested in our study in which pAkt could be induced in all PI3K – samples stimulated by FLT3-ligand. One hypothesis for the lower relapse rate of PI3K + patients may be that PI3K drives immature leukemic populations in the S phase, increasing their susceptibility to cell cycle-dependant chemotherapies. Overall, these results suggest that precise identification of Akt targets is needed before targeting this pathway. As recently reported in cancer models, inhibiting the Akt pathway may lead to unexpected dark side effects.12,13    Authorship Top Abstract Introduction Patients, materials, and methods Results and discussion Authorship References   Contribution: J.T., V.B., and N.C. performed research. C.E. and P.B. performed the statistical analysis. S.P., B.L., N.I., F.W., and F.D. provided patient samples. P.C.L., V.H., and O.B. performed molecular analysis. P.M., C.L., and D.B. designed research and wrote the paper. J.T. and C.E. contributed equally to this work. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Didier Bouscary, Département d'Hématologie, Institut Cochin, 27 rue du Faubourg Saint-Jacques, F-75014 Paris, France; e-mail: bouscary{at}cochin.inserm.fr.    Acknowledgments   The authors thank all participating investigators from the GOELAMS (Groupe Ouest-Est d'Etude des Leucémies et Autres Maladies du Sang). This work was supported by l'Association Laurette Fugain and by grants from the Association pour la Recherche contre le Cancer (ARC).    Footnotes   Submitted December 4, 2006; accepted March 26, 2007. Prepublished online as Blood First Edition Paper, April 10, 2007 DOI: 10.1182/blood-2006-12-061283 The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.    References Top Abstract Introduction Patients, materials, and methods Results and discussion Authorship References   Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood 2003; 102:972–980.[Abstract/Free Full Text] Min YH, Eom JI, Cheong JW, et al. Constitu-tive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable. Leukemia 2003; 17:995–997.[CrossRef][Medline] [Order article via Infotrieve] Grandage VL, Gale RE, Linch DC, Khwaja A. PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF-kappaB, Mapkinase and p53 pathways. Leukemia 2005; 19:586–594.[Medline] [Order article via Infotrieve] Bardet V, Tamburini J, Ifrah N, et al. Single cell analysis of phosphoinositide 3-kinase/Akt and ERK activation in acute myeloid leukemia by flow cytometry. Haematologica 2006; 91:757–764.[Abstract/Free Full Text] Sujobert P, Bardet V, Cornillet-Lefebvre P, et al. Essential role for the p110delta isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia. Blood 2005; 106:1063–1066.[Abstract/Free Full Text] Billottet C, Grandage VL, Gale RE, et al. A selective inhibitor of the p110delta isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16. Oncogene 2006; 25:6648–6659.[CrossRef][Medline] [Order article via Infotrieve] Cornillet-Lefebvre P, Cuccuini W, Bardet V, et al. Constitutive phosphoinositide 3-kinase activation in acute myeloid leukemia is not due to p110delta mutations. Leukemia 2006; 20:374–376.[CrossRef][Medline] [Order article via Infotrieve] Kornblau SM, Womble M, Qiu YH, et al. Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia. Blood 2006; 108:2358–2365.[Abstract/Free Full Text] Nakao M, Yokota S, Iwai T, et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996; 10:1911–1918.[Medline] [Order article via Infotrieve] Bezieau S, Devilder MC, Avet-Loiseau H, et al. High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis. Hum Mutat 2001; 18:212–224.[CrossRef][Medline] [Order article via Infotrieve] Bardet V, Costa LD, Elie C, et al. Nucleophosmin status may influence the therapeutic decision in de novo acute myeloid leukemia with normal karyotype. Leukemia 2006; 20:1644–1646.[CrossRef][Medline] [Order article via Infotrieve] Sawyers CL. Will kinase inhibitors have a dark side? N Engl J Med 2006; 355:313–315.[Free Full Text] Yoeli-Lerner M, Yiu GK, Rabinovitz I, Erhardt P, Jauliac S, Toker A. Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT. Mol Cell 2005; 20:539–550.[CrossRef][Medline] [Order article via Infotrieve] Grambsch PM and Therneau TM. Proportional hazards tests and diagnostics based on weighted residuals. Biometrika 1994; 81:515–526.[Abstract/Free Full Text] Broet P, De Rycke Y, Tubert-Bitter P, Lellouch J, Asselain B, Moreau T. A semiparametric approach for the two-sample comparison of survival times with long-term survivors. Biometrics 2001; 57:844–852.[CrossRef][Medline] [Order article via Infotrieve] Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a competing risk. Ann Stat 1988; 16:1141–1154.[CrossRef] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J. Tamburini, A. S. Green, V. Bardet, N. Chapuis, S. Park, L. Willems, M. Uzunov, N. Ifrah, F. Dreyfus, C. Lacombe, et al. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: blood-2006-12-061283v1 110/3/1025    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Tamburini, J. Articles by Bouscary, D. Search for Related Content PubMed PubMed Citation Articles by Tamburini, J. Articles by Bouscary, D. Related Collections Neoplasia Signal Transduction Brief Reports Clinical Trials and Observations Social Bookmarking                   What's this?

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