Evidence of Myeloid Differentiation in Non-M3 Acute ...

[Cancer Biology & Therapy 6:1, 18-21, January 2007]; ©2007 Landes Bioscience

Clinical Case Report

Evidence of Myeloid Differentiation in Non-M3 Acute Myeloid Leukemia Treated with the Retinoid X Receptor Agonist Bexarotene Donald E. Tsai1 Selina M. Luger1 Allison Kemner1 Cezary Swider1 Ami Goradia2 Ewa Tomczak2 Doris DiPatri2 Adam Bagg2 Peter Nowell2 Alison W. Loren1 Alexander Perl1 Stephen Schuster1 James E. Thompson1 David Porter1 Charlambos Andreadis1 Edward A. Stadtmauer1 Steven Goldstein1 Richard Ghalie3 Martin Carroll1

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All‑trans‑retinoic acid has dramatically changed the treatment paradigm for acute promyelocytic leukemia, however, it has no significant activity in non-M3 acute myeloid leukemia (AML). In vitro, bexarotene, a retinoid X receptor agonist inhibits the prolifera‑ tion of non-M3 AML cell lines and induces differentiation of leukemic blasts from patients. We hypothesized that there may be similar activity in patients with AML. We report on two patients with relapsed or refractory non-M3 AML treated with bexarotene monotherapy. After initiating treatment, both patients showed leukemic differentiation in their peripheral blood and reduction in bone marrow blasts to less than 5%. One patient had a significant improvement in her platelet count with loss of platelet transfusion needs. Differentiation syndrome occurred in one patient and was successfully treated with steroids and discon‑ tinuation of bexarotene. These data suggest that bexarotene has clinical activity in non-M3 AML and may be able to induce myeloid differentiation in vivo.

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Retinoids play a critical role in the maturation and proliferation of a wide variety of cell types. These actions make retinoids potential therapeutic agents in the treatment of cancer.1 All‑trans‑retinoic acid (ATRA) has dramatically changed the treatment and prognosis of patients with acute promyelocytic leukemia (APML).2 APML is characterized by the t(15; 17) translocation, which results in the production of aberrant retinoic acid receptors and subsequent interruption of myeloid differentiation.3 ATRA, a retinoid receptor agonist, is able to overcome this differentiation arrest and allow for normal maturation of the leukemic blasts.2,3 With the introduction of ATRA, the outcome of APML has improved significantly so that 80% of patients may now expect long term relapse free survival.4 While ATRA has significantly improved the overall survival in patients with APML, it has no significant clinical activity in non-M3 AML. Bexarotene is a novel retinoid X receptor agonist that is used for the treatment of cutaneous T cell lymphoma (CTCL) and has been tested for activity in the treatment of other malignancies.5‑8 In vitro, bexarotene has long been known to inhibit clonal growth and induce differentiation of the HL‑60 leukemic cell line as well as leukemic cells from patients with AML.9 Recent reports have suggested that bexarotene may have enhanced differentiation effects in primary AML cells when subordination by RAR protein is released with a cAMP agonist.10 We hypothesized that bexarotene would have activity in patients with non-M3 AML. Here we describe 2 individuals with non-M3 AML who were treated with bexarotene on our institutional review board approved phase I clinical trial.

Division, Department of Medicine and 2Department of Pathology and Laboratory Medicine; University of Pennsylvania Medical Center, Philadelphia, Pennsylvania USA

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3Ligand Pharmaceuticals; San Diego, California USA

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*Correspondence to: Donald E. Tsai; 16 Penn Tower, 3400 Spruce Street; Philadelphia, Pennsylvania 19104 USA; Tel.: 215.614.0037; Fax: 215.662.4064; Email: [email protected]

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Original manuscript submitted: 11/21/06 Manuscript accepted: 11/26/06

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Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3619

Materials and Methods

acute myeloid leukemia, bexarotene, retinoic acid, retinoic X receptor, leukemia, myeloid differentiation, retinoic acid syndrome

Patient summary. Patient 1. The patient is a 69‑year‑old white female with a history of myelodysplastic syndrome (MDS) that transformed into AML‑M2 with chromosome studies showing 46, XX, del (1)(p13), del (5)(q31q35), ‑11, +mar [9]. She was initially treated with 2 courses of induction chemotherapy consisting of daunorubicin and cytarabine on a clinical study with either placebo or zosuquidar. At recovery, her bone marrow biopsy showed recurrent AML (15% cellularity & 14% blasts) with persistent transfusion dependent pancytopenia. She received 1 cycle of azacitidine with no response. At the initiation of bexarotene (150 mg/m2 daily), she was neutropenic (WBC 1.7 x 103/ mL, ANC 806/mL) and transfusion dependent, requiring three transfusions of platelets per week and red blood cells weekly. Her pretreatment bone marrow biopsy showed 15%

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Key words

Acknowledgements See page e4.

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Bexarotene Induced Myeloid Differentiation

cellularity with 10% blasts (Fig. 1A and B). Over the next four months, her platelet and neutrophil count increased and she became independent of platelet transfusions. Bone marrow biopsy on D+143 showed MDS with 50% cellularity and no evidence of residual AML (Fig. 1C and D). On D+220, peripheral blood neutrophils were purified by flow cytometry and analyzed for morphology (Fig. 1E) and tested by FISH for the markers associated with the original AML diagnostic samples. Purified neutrophils were 92% positive for the 5q‑deletion defined with the 5q31 VYSIS EGR1 probe (Fig. 1F). While taking bexarotene, the patient remained RBC transfusion dependent and pancytopenic with a median WBC 1.8 x 103/ mL (range 1.1‑3.9), ANC 540/mL (range 20–1782) and platelet 35 x 103/mL (21–60). The patient ultimately developed progression of her AML and expired from pneumonia on D+425. Patient 2. The patient is a 57 year‑old white male with AML M2 with t(8; 21)(q22q22). His induction chemotherapy consisted of daunorubicin and cytarabine. He achieved a CR after one course of induction chemotherapy. He then underwent two cycle of consolidative high dose cytarabine followed by an autologous stem cell transplant conditioned with busulfan and cyclophosphamide. Six months after transplant, he relapsed and underwent reinduction with mitoxantrone, etoposide and cytarabine with no response. Twenty‑nine days after his last chemotherapy, he was started on bexarotene (150 mg/m2 daily). On D+1 of therapy, WBC was Figure 1. Patient # 1 Studies. (A) Bone marrow biopsy pre-bexarotene (5x magnification), 1.7 x 103/mL and contained 18% blasts consistent (B) Bone marrow aspirate prebexarotene (100x magnification), (C) Bone marrow biopsy with persistent AML. His bone marrow had 40% bexarotene D+143 (5x magnification), (D) Bone marrow aspirate bexarotene D+143 (40x cellularity with 20% blasts (Fig. 2A). On D+34, he magnification), (E) Flow cytometry purified peripheral blood neutrophils using antibodies to was hospitalized with a progressive cough, shortness CD16 and CD45, (F) FISH on flow cytometry purified peripheral blood neutrophils using probe of breath and paroxysmal nocturnal dyspnea. In CEP 5/5q. the prior 18 days, his WBC and ANC had gone from 3.7 x 103/mL and 1,480 /mL, respectively, to 27.9 x 103/mL 3 Stain Set (Fisher Diagnostic, Middeltown VA). FISH analysis was and 14,076 /mL (Fig. 2B). Chest CT showed a new pericardial performed in the clinical cytogenetics laboratory of the University effusion. Bexarotene was discontinued on D+34 and he was started of Pennsylvania Cancer Center. Cells were incubated for 24 hours on dexamethasone 10 mg BID. His peripheral blood blasts decreased in cell culture medium, treated with EGTA/KCl hypotonic, fixed significantly by D+38 (WBC 23.9 x 103/mL, ANC 21,280 /mL and with methanol‑acetic acid solution and dropped onto glass slides. blasts 3%). Peripheral blood smear demonstrated atypical neutrophils VYSIS LSI 5q31EGR1/5p15.2D5S23,D5S21 dual color, and t(8; (Fig. 2C). Flow sorted peripheral blood neutrophils tested by FISH 21) AML1/ETO, dual color dual fusion probes were used for the demonstrated that 100% of these neutrophils contained t(8,21) in‑situ hybridization. (Fig. 2D). His symptoms resolved within 48 hours of initiating steroids. Follow‑up echocardiogram on D+50 showed resolution of Results and Discussion the pericardial effusion. Bone marrow biopsy on D+41 showed 80% We report on two patients with relapsed or refractory non-M3 cellularity and 3% blasts with significant myeloid differentiation AML who were treated with bexarotene monotherapy. Bexarotene (Fig. 2E). Off bexarotene, he ultimately developed recurrent AML. is an oral synthetic retinoid analog that is typically used for the He elected to receive no further therapy and expired on D+99. Flow separation and FISH analysis. Peripheral blood cells treatment of cutaneous T cell lymphoma (CTCL). In a phase I trial were collected in heparinized tubes and processed with hypotonic in non-M3 AML being performed at our institution, we have seen lysis to remove red blood cells. Cells were washed and stained evidence of activity in a cohort of patients treated at a relatively low with antibodies to CD16, a granulocytic marker, and CD45, a dose level (150 mg/m2) compared to drug doses (300 mg/m2) that are pan leukocyte marker (Becton Dickinson). Cells were sorted on typically used in CTCL. In these patients, evidence for induction of a Becton Dickson FACS Aria based on side scatter and forward myeloid differentiation similar to that seen with ATRA in APML was scatter properties into granulocytic cells and mononuclear cells. seen. Both patients had improvements in their ANC with evidence Granulocytic purity was confirmed by cytospin stained with HEMA of leukemic blast maturation. Evidence that this is true leukemic www.landesbioscience.com


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Figure 2. Patient #2 studies. (A) Bone marrow aspirate prebexarotene (100x magnification), (B) Graph of WBC, ANC and blast count while on bexarotene. Time is measured in days from the start of bexarotene. (C) Peripheral blood neutrophils on D+38 of bexarotene (100x magnification), (D) FISH on flow cytometry purified peripheral blood neutrophils using probe to t(8; 21) AML1/ETO, (E) Bone marrow aspirate bexarotene D+41 (40x magnification).

maturation and not recovery of normal blood cell production is shown by the high percentage of peripheral blood neutrophils containing their respective leukemic cytogenetic abnormality. Neutrophils from a control patient with AML who had not received bexarotene did not show evidence of the leukemic cytogenetic abnormality (data not shown). There were significant reductions in leukemic blasts in both patient’s bone marrow to less than 5%. Patient #1, who remained on bexarotene for over a year, had a sustained improvement in her platelet counts, allowing her to be free from platelet transfusions for a number of months. These initial signs of activity are encouraging especially given the low dose of bexarotene being used. Importantly, these results are consistent with the hypothesis that activation of the RXR through agonists such as bexarotene will stimulate differentiation in non-M3 AML. 20

In addition to the hematologic effects of bexarotene, we have seen evidence of differentiation syndrome induced by bexarotene therapy. This includes patient #2 described above and one other patient not included in this report who had similar findings. The common toxicity of bexarotene is nonhematologic, mild and reversible, consisting of hypertriglyceridemia, hypothyroidism, rash, and headache. No pericardial effusions or syndromes such as described above have ever been reported in non-AML patients treated with bexarotene. Patient #2 developed respiratory distress and a pericardial effusion in the setting of a rapidly rising ANC and evidence of leukemic differentiation in the bone marrow. Consistent with differentiation syndrome due to bexarotene, the symptoms and findings resolved rapidly with the cessation of bexarotene and initiation of steroids. Differentiation syndrome has been described as a side effect of


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leukemic differentiation seen in APML that is responding to arsenic trioxide or ATRA. It has not been described in any other setting. This is the first report of differentiation syndrome in non-M3 AML and may reflect myeloid differentiating activity due to bexarotene. Retinoic acid receptors are involved in many biologic processes and fall into separate subclasses including retinoic acid receptor (RAR) and retinoid X receptor (RXR). While ATRA selectively binds RAR’s, bexarotene selectively binds and activates the RXR subclass.1 The specific role of RXR receptors in myeloid differentiation and leukemogenesis is unknown but a number of lines of evidence suggest that activation of RXR receptors may induce myeloid differentiation and/or apoptosis of leukemic cells.9 RAR and RXR receptors are able to interact to form active dimer and heterodimer complexes. It is possible that the myeloid differentiation effects we have seen with bexarotene in non-M3 AML reflect the RXR initiation of a downstream RAR/RXR complex normally triggered through an alternate RAR pathway by ATRA in APML. The proper AML subtypes that might respond and benefit from bexarotene remain to be determined. Bexarotene appears to have activity in non-M3 AML as manifested by decreased bone marrow blasts, leukemic blast differentiation, improvement in transfusion needs and the occurrence of differentiation syndrome. The optimal use of bexarotene in non-M3 AML remains to be determined. Clinical trials of bexarotene in AML are ongoing at our institution. Acknowledgements

Acknowledgements We would like to thank the patients who enrolled and the nurses who assisted with performance of the ongoing clinical trial. We thank Patty Sanchez for critical reading of the manuscript and members of the Carroll laboratory for helpful discussion. References 1. Garattini E, Terao M. Atypical retinoids: An expanding series of anti‑leukemia and anti‑cancer agents endowed with selective apoptotic activity. J Chemother 2004; 16(Suppl 4):70‑3. 2. Warrell RP Jr, Frankel SR, Miller WH Jr, Scheinberg DA, Itri LM, Hittelman WN, Vyas R, Andreeff M, Tafuri A, Jakubowski A, et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all‑trans‑retinoic acid). N Engl J Med 1991; 324:1385‑93. 3. Melnick A, Licht JD. Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999; 93:3167‑215. 4. Lengfelder E, Saussele S, Weisser A, Buchner T, Hehlmann R. Treatment concepts of acute promyelocytic leukemia. Crit Rev Oncol Hematol 2005; 56:261‑74. 5. Boehm MF, Zhang L, Zhi L, McClurg MR, Berger E, Wagoner M, Mais DE, Suto CM, Davies JA, Heyman RA, et al. Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J Med Chem 1995; 38:3146‑55. 6. Duvic M, Hymes K, Heald P, Breneman D, Martin AG, Myskowski P, Crowley C, Yocum RC; Bexarotene Worldwide Study Group. Bexarotene is effective and safe for treatment of refractory advanced‑stage cutaneous T‑cell lymphoma: Multinational phase II‑III trial results. J Clin Oncol 2001; 19:2456‑71. 7. Dragnev KH, Petty WJ, Shah S, Biddle A, Desai NB, Memoli V, Rigas JR, Dmitrovsky E. Bexarotene and erlotinib for aerodigestive tract cancer. J Clin Oncol 2005; 23:8757‑64. 8. Tsai DE, Aqui NA, Vogl DT, Bloom RD, Schuster SJ, Nasta SD, Wasik MA. Successful treatment of T‑cell post‑transplant lymphoproliferative disorder with the retinoid analog bexarotene. Am J Transplant 2005; 5:2070‑3. 9. Kizaki M, Dawson MI, Heyman R, Elster E, Morosetti R, Pakkala S, Chen DL, Ueno H, Chao W, Morikawa M, Ikeda Y, Heber D, Pfahl M, Koeffler HP. Effects of novel retinoid X receptor‑selective ligands on myeloid leukemia differentiation and proliferation in vitro. Blood 1996; 87:1977‑84. 10. Altucci L, Rossin A, Hirsch O, Nebbioso A, Vitoux D, Wilhelm E, Guidez F, De Simone M, Schiavone EM, Grimwade D, Zelent A, de The H, Gronemeyer H. Rexinoid‑triggered differentiation and tumor‑selective apoptosis of acute myeloid leukemia by protein kinase A‑mediated desubordination of retinoid X receptor. Cancer Res 2005; 65:8754‑65.

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