ORIGINAL ARTICLE. Bone Marrow Transplantation (2015) 50, 40 44; doi: /bmt ; published online 22 September 2014

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1 Bone Marrow Transplantation (215) 5, Macmillan Pulishers Limited All rights reserved /15 ORIGINAL ARTICLE Delayed hematopoietic recovery after auto-sct in patients receiving arsenic trioxide-ased therapy for acute promyelocytic leukemia: a multi-center analysis GN Mannis 1, AC Logan 1, AD Leavitt 2, M Yanada 3, J Hwang 4, RL Olin 1, LE Damon 1, C Andreadis 1,WZAi 1, KM Gaensler 1, CC Greene 1, NK Gupta 1, LD Kaplan 1, A Mahindra 1, Y Miyazaki 5, T Naoe 6, S Ohtake 7, PH Sayre 1, CC Smith 1, JM Venstrom 1, JL Wolf 1, L Caallero 2, N Emi 3 and TG Martin 1 A potential link etween arsenic (ATO)-ased therapy and delayed hematopoietic recovery after autologous hematopoietic SCT (HSCT) for acute promyelocytic leukemia (APL) has previously een reported. We retrospectively reviewed the clinical histories of 58 patients undergoing autologous HSCT for APL at 21 institutions in the United States and Japan. Thirty-three (56%) of the patients received ATO-ased therapy prior to stem cell collection. Delayed neutrophil engraftment occurred in 1 patients (17%): 9 of the 1 patients (9%) received prior ATO (representing 27% of all ATO-treated patients), compared with 1 of the 1 patients (1%) not previously treated with ATO (representing 4% of all ATO-naïve patients; Po.1). Compared with ATO-naïve patients, ATO-treated patients experienced significantly longer times to ANC recovery (median 12 days vs 9 days, Po.1). In multivariate analysis, the only significant independent predictor of delayed neutrophil engraftment was prior treatment with ATO (hazard ratio 4.87; Po.1). Of the availale stem cell aliquots from APL patients, the median viale post-thaw CD34+ cell recovery was significantly lower than that of cryopreserved autologous stem cell products from patients with non-apl AML. Our findings suggest that ATO exposure prior to CD34+ cell harvest has deleterious effects on hematopoietic recovery after autologous HSCT. Bone Marrow Transplantation (215) 5, 4 44; doi:1.138/mt ; pulished online 22 Septemer 214 INTRODUCTION Acute promyelocytic leukemia (APL) accounts for ~ 5 8% of adult AMLs, representing 6 8 new cases annually in the United States. 1 3 Although CR rates may exceed 9% with upfront therapy for APL, as many as 15 3% of patients will relapse after initially receiving ATRA- and cytotoxic chemotherapy-ased therapies. 4 9 Arsenic trioxide (ATO) is a more recent addition to the APL therapeutic arsenal. Initially proven effective in achieving remissions in relapsed APL, ATO plus ATRA was recently demonstrated to e not-inferior to ATRA plus chemotherapy in the front-line setting for low- and intermediate-risk patients. 1,11 For APL patients who relapse and susequently attain a CR2, and for some APL patients who are deemed high-risk for relapse in CR1, autologous hematopoietic SCT (HSCT) continues to have an important therapeutic role Little is known aout the impact of ATO on susequent autologous HSCT, ut two cases of delayed hematopoietic recovery in patients treated with ATO have een reported. 16 To etter understand the impact of ATO therapy on hematopoietic recovery, we retrospectively reviewed the histories of all patients who underwent autologous HSCT for APL at our institution since We additionally included data from all APL patients who underwent autologous HSCT in the only pulished prospective trial of ATO therapy prior to autologous HSCT y the Japan Adult Leukemia Study Group (JALSG). 17 We identify an association etween ATO-ased APL therapy and delayed hematopoietic recovery following autologous HSCT. PATIENTS AND METHODS We retrospectively analyzed the medical records of 35 consecutive adult APL patients (age 18 years) who underwent autologous HSCT at the University of California, San Francisco (UCSF) Medical Center etween January 1994 and July 212. A contemporaneous control cohort of patients who underwent autologous HSCT for non-apl AML in CR1 or CR2 was included for analysis of neutrophil and plt engraftment. The study was approved y the UCSF Committee on Human Research. Patients in the JALSG cohort were enrolled at 2 centers etween Decemer 25 and June 29, with data collected prospectively upon study entry, as previously reported. 17 Autologous HSCT Patients at UCSF underwent consolidation with i.v. cytaraine 16 g/m 2 and etoposide 4 mg/kg over 4 days, followed y PBSC moilization with G-CSF 1 μg/kg per day and PBSC collection upon neutrophil recovery. The target 1 Division of Hematologic Malignancies and Blood and Marrow Transplantation, Department of Medicine, University of California, San Francisco, Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, USA; 2 Departments of Laoratory Medicine, University of California, San Francisco, San Francisco, CA, USA; 3 Department of Hematology, Fujita Health University School of Medicine, Toyoake, Japan; 4 Department of Biostatistics, University of California, San Francisco, San Francisco, CA, USA; 5 Department of Hematology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; 6 Department of Hematology, Nagoya University Graduate School of Medicine, Nagoya, Japan and 7 Department of Clinical Laoratory Science, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan. Correspondence: Professor TG Martin, Division of Hematologic Malignancies and Blood and Marrow Transplantation, Department of Medicine, University of California, San Francisco, 4 Parnassus Avenue, Room A522, San Francisco, CA 94143, USA. tmartin@medicine.ucsf.edu Received 11 March 214; revised 28 July 214; accepted 7 August 214; pulished online 22 Septemer 214

2 CD34+ cell dose was per kg, with a minimum dose of per kg. Prior to transplant, patients received preparative therapy consisting of BU 12.8/16 mg/kg (i.v./po) divided into 16 doses, and a single dose of i.v. etoposide 6 mg/kg 3 days prior to HSCT. On the day of HSCT, the moilized PBSC product was thawed and immediately infused without cell washing or other manipulations. Patients in the JALSG cohort underwent consolidation with i.v. cytaraine 16 g/m 2 over 4 days followed y PBSC moilization with G-CSF eginning at day 6, as descried previously. 17 A minimum CD34+ cell collection of 2x1 6 per kg was required. The conditioning regimen consisted of BU 12 mg/kg PO divided into 12 doses and melphalan 14 mg/m 2 divided into 2 doses. Definition of outcomes Neutrophil engraftment was defined as the first of 3 consecutive days with an ANC45 per μl. To facilitate merging the UCSF and JALSG data sets, plt engraftment was defined as the first of 3 consecutive days during hospitalization with an unsupported plt count 43 per μl. If patients did not achieve plt engraftment y the end of their transplant hospitalization, the date of engraftment was defined as the first unsupported post-hospitalization plt count 43 per μl. For the purposes of statistical comparison, delayed hematopoietic recovery was defined as requiring 414 days after stem cell infusion to achieve an ANC45 per μl, which was 2 s.d. aove the mean numer of days to achieve neutrophil engraftment in patients not receiving ATO therapy. Similarly, delayed plt recovery was defined as requiring 49 days after HSCT to achieve an unsupported plt count 43 per μl. CD34 viaility assays Cryopreserved cell samples were thawed at 37 o C and immediately resuspended in a 1:1 dextran 4/alumin solution. Cell suspensions were equilirated at room temperature for 1 min and an aliquot was then stained for viaility using Beckman Coulter antiodies (Beckman Coulter, Brea, CA, USA) to CD34 (phycoerythrin-conjugated) and 7-aminoactinomycin D. Cells were interrogated using a Beckman Coulter FC5 (Beckman Coulter) with 1 events collected for each sample. Analysis was performed using CXP cytometer software (Beckman Coulter). Statistical analysis Normally distriuted continuous data are reported as mean (± s.d.), and time data are reported as median (range). Categorical data are reported as frequency (%). Multivariate analyses were performed using SAS software (SAS Institute Inc., Cory, NC, USA). A P-value of o.5 was considered significant. Kaplan Meier and cumulative incidence curves were generated using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) and statistical differences were assessed via Fisher s exact, Log-Rank and Wilcoxon analyses. RESULTS Patient characteristics Thirty-five APL patients (15 men and 2 women) underwent a total of 36 autologous HSCTs in the UCSF cohort (Tale 1). One patient relapsed 43 years after his first autologous HSCT and received a second autologous HSCT. One ATO-treated patient was excluded from analysis due to contamination of the initial CD34+ cell collection with Candida krusei. In the JALSG cohort, 23 APL patients (13 men and 1 women) underwent autologous HSCT. Twenty-four transplants (41%) were performed for patients in CR1 and 35 (59%) for patients in CR2. All transplants in the JALSG cohort were performed for patients in CR2 vs 12 of 36 transplants (33%) in the UCSF cohort. Thirty-three of the 59 transplants (56%) were performed for patients who received ATO-ased therapy prior to autologous stem cell collection. A significantly higher proportion of patients who received ATO were transplanted in CR2 (94 vs 15%, Po.1). There were otherwise no significant differences in clinical characteristics or risk features etween the ATO-treated and the ATO-naïve transplant recipients (Tale 1). Patients in the UCSF cohort were Tale 1. Characteristics Patient characteristics No ATO (n = 26) ATO (n = 33) P-value Total transplants, n (%) JALSG () 23 (69.7) UCSF 26 (1) 1 (3.3) Age at diagnosis, years (range) 44 (19 6) 45 (18 63).84 Age at HSCT, years (range) 45 (2 61) 46 (2 64).35 Male gender, n (%) 11 (42.3) 17 (51.5).48 Remission status at time of HSCT, n (%) CR1 22 (84.6) 2 (6.1) o.1 CR2 4 (15.4) 31 (93.9) Total CD34+ cells collected x1 6 per kg, mean (s.d.) (63.3) (45.2).14 Areviations: ATO = arsenic trioxide; HSCT = hematopoietic SCT; JALSG = Japan Adult Leukemia Study Group; UCSF = University of California, San Francisco. infused a mean CD34+ cell dose of per kg (range ; data unavailale for the JALSG cohort). Delayed hematopoietic recovery Compared with ATO-naïve patients, ATO-treated patients experienced significantly longer time to ANC recovery in univariate analysis (median 12 vs 9 days, Po.1) (Figure 1a). Plt recovery was not significantly delayed in univariate analysis ut showed a trend toward longer time-to-engraftment in the ATO-treated group (median 18 vs 13 days, P =.26) (Figure 1). In the UCSF cohort, ATO-treated patients required significantly longer transplant hospitalizations (median 3 vs 25 days, Po.1; data unavailale for the JALSG cohort) (Figure 1c). Ten cases (17%) of delayed neutrophil engraftment were identified, five (14%) in the UCSF cohort and five (22%) in the JALSG cohort. Nine of the 33 patients (27%) who received prior ATO experienced delayed neutrophil engraftment, compared with 1 of the 26 patients (3.8%) not previously treated with ATO (P =.32). In a multivariate analysis including remission status (CR1 vs CR2) and conditioning regimen (UCSF vs JALSG), the only significant independent predictor of delayed neutrophil engraftment was prior treatment with ATO (hazard ratio 4.86, confidence interval , Po.1). Delayed plt recovery was independently predicted y either prior ATO-ased therapy (hazard ratio 4.13, confidence interval , P =.4) or y undergoing transplant in Japan (hazard ratio 2.71, confidence interval , P =.28). In multivariate analysis (Tale 2), remission status was not a predictor of time to hematopoietic recovery. Similarly, neither the ATO dose per kilogram, nor the CD34+ cell dose infused (data availale for UCSF cohort only) significantly impacted hematopoietic recovery in univariate analysis. To further explore whether delayed hematopoietic recovery may have een attriutale to a higher proportion of ATO-treated patients undergoing transplant in CR2, we evaluated the time to hematopoietic recovery of 65 consecutive non-apl AML patients who underwent autologous stem cell transplant in CR1 (n = 51) or CR2 (n = 14) at UCSF. There was no significant difference in the median time to neutrophil engraftment (median 11 days for oth; P =.95) (Supplementary Figure 1A) or plt engraftment (median 19 vs 16 days, P =.1) (Supplementary Figure 1B). Post-thaw CD34+ cell viaility Cryopreserved aliquots from the initial stem cell collection were availale for seven patients in the UCSF cohort (four ATO-treated Macmillan Pulishers Limited Bone Marrow Transplantation (215) 4 44

3 42 a ANC >5/µL (%) Plts >3,/µL (%) c hospital discharge (%) P= P=.255 Tale 2. Multivariate analysis of predictors of neutrophil and plt recovery Outcome and predictors RR (95% CI) P-value ANC45 ATO 4.87 ( ) o.1 JALSG conditioning.97 ( ).94 CR1.64 ( ).29 Plts43 ATO 4.13 ( ).4 JALSG conditioning 2.71 ( ).28 CR1.62 ( ).24 Areviations: ATO = Arsenic trioxide; CI = confidence interval; JALSG = Japan Adult Leukemia Study Group; RR = recovery rate. and three ATO-naïve) to test post-thaw CD34+ cell viaility. In the seven APL patient samples, the median post-thaw CD34+ cell viaility was 65% (range %). The median CD34+ cell viaility in the four ATO-treated patients was not significantly different 4 P< Figure 1. Patients exposed to ATO prior to hematopoietic stem and progenitor cell collection experience (a) delayed recovery of ANC (median 12 vs 9 days, Po.1), () a non-significant trend toward delayed plts recovery (median 18 vs 13 days, P =.26), and (c) require longer hospitalizations in the UCSF cohort (3 vs 25 days, Po.1) following autologous SCT. a Viaility (%) Side Scatter Total Events CD34-PE APL P =.5 CD34+ cells 7-AAD Non-APL Figure 2. Post-thaw CD34+ cell viaility. (a) Representative FACS plot of CD34+ cell post-thaw viaility of cryopreserved autograft aliquots using 7-amino-actinomycin D staining. () Viale CD34+ hematopoietic progenitor cell recovery is lower in APL patients (median 65%, range %) in comparison with non-apl AML patients (median 81%, range 49 92%; P =.5). Bars on graph represent median and interquartile range. than the three ATO-naïve patient samples. By comparison, however, median CD34+ cell viaility was significantly higher (81%, range 49 92%; P =.5) in a random selection of 15 cryopreserved autologous stem cell products from patients with non-apl AML (Figure 2). CD34+ cell yield prior to cryopreservation was not different etween ATO-treated and ATO-naïve patients. Clinical outcomes With median follow-up of 4.5 years (range.2 18), 7 of the 58 patients have died (12%; 5 from relapsed disease). Nine of the 1 patients (9%) who experienced delayed neutrophil engraftment remain alive and 28 of 3 patients (93%) with follow-up longer than 5 years after autologous HSCT remain alive. Median relapse-free (RFS) and OS have not een reached, although statistically the RFS and OS were oth shorter in patients receiving ATO prior to stem cell collection (Figure 3), likely reflecting the higher proportion of ATO-treated patients eing transplanted in CR2. In the UCSF cohort, delayed neutrophil engraftment was clinically significant in all cases. Four patients all of whom were treated with ATO required additional stem cell infusions, and all required significantly more RBC (median 6 vs 2 units, P =.1) and plt transfusions (median 5 vs 3 units, P =.22), as well as longer hospitalizations (median 3 vs 25 days, Po.1). DISCUSSION ATO is increasingly used as oth front-line and as salvage therapy for APL. In this study, we analyzed the impact of pre-transplant ATO-ased therapy on susequent hematopoietic recovery after autologous HSCT. We found a significant association etween ATO-ased therapy and delayed hematopoietic recovery following Bone Marrow Transplantation (215) Macmillan Pulishers Limited

4 a Relapse-free survival (%) Overall survival (%) Years post-sct Years post-sct Figure 3. Kaplan Meier curves. (a) Relapse-free and () OS was decreased in vs non- patients, ut is likely confounded y a higher proportion of patients transplanted in CR2 in the group. Kaplan Meier curves are truncated at 6 years for improved visualization. autologous HSCT. This was associated with an increased need for transfusion support and longer hospitalizations. We hypothesize that impaired post-thaw CD34+ cell viaility in patients with APL may interact with prior ATO exposure, susequently leading to delayed post-transplant hematopoietic recovery. APL cells are uniquely sensitive to the aility of ATO to promote differentiation, inhiit proliferation and induce apoptosis via its effect on the PML-RARα transcript. 18 These effects are dosedependent in vitro, with induction of apoptosis via oth PML- RARα-dependent and -independent mechanisms at high concentrations. 19 PML-RARα-independent mechanisms have the potential for off-target effects that may underlie the delay in normal hematopoietic recovery seen in our study. For example, in APL NB4 cells in vitro, this includes a H 2 O 2 -dependent pathway that results from sensitivity of the intracellular glutathione redox system to ATO. 2 In myeloma cell lines, anti-proliferative effects of ATO may e mediated through inhiition of the transcription factor nuclear-factor-kappa B and interference with IL-6 activity. 21 In vitro exposure of non-apl leukemic cell lines to ATO has antiangiogenic properties, inhiiting vascular endothelial growth factor production and preventing capillary growth. 22 The iological mechanism of delayed hematopoietic recovery in our study warrants further investigation. The present study has several limitations. As a retrospective study, the aility to further define the iologic mechanism of delayed hematopoietic recovery was limited y the availaility of stored stem cell aliquots. In addition, as a merged data set from multiple centers, institutional protocols for stem cell collection, storage and transplantation differed slightly. Nonetheless, in multivariate analysis, delayed neutrophil engraftment was not significantly associated with institution. In summary, this study confirms an association etween ATO exposure prior to autologous stem cell harvest and delayed hematopoietic recovery after autologous HSCT for APL. Our study identified impaired CD34+ cell viaility in patients with APL relative to non-apl AML, suggesting that APL autografts may e vulnerale to detrimental functional impacts of prior ATO exposure, although neither a clear causal relationship nor a specific iologic mechanism is yet estalished. Because of increasing usage of ATO in front-line APL therapy, larger studies are warranted to validate these findings and to determine the potential mechanism of delayed hematopoietic recovery after autologous HSCT and whether it can e mitigated. CONFLICT OF INTEREST The authors declare no conflict of interest. ACKNOWLEDGEMENTS We would like to thank Jennifer Rasmussen and Katrina De La Cruz of the UCSF Blood and Marrow Transplant Laoratory for their expert technical assistance with the CD34+ cell viaility assays, and Nicole Foley and Sara Halvestine of the UCSF Division of Blood and Marrow Transplantation for their assistance with data management. This work was supported in part y an American Society of Hematology Research Training Award for Fellows (GNM), a Grant-in-Aid for Cancer Research from the Ministry of Health, Laour and Welfare of Japan (Clinical Cancer Research 23-4); and the National Cancer Center Research and Development Fund (26-A-24). REFERENCES 1 Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ et al. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 1998; 339: Yamamoto JF, Goodman MT. 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