RESEARCH ARTICLE. Introduction Wiley Periodicals, Inc.

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1 De novo acute myeloid leukemia with 20 29% blasts is less aggressive than acute myeloid leukemia with 30% blasts in older adults: a Bone Marrow Pathology Group study AJH Robert Paul Hasserjian, 1 * Federico Campigotto, 2 Veronica Klepeis, 1 Bin Fu, 3 Sa A. Wang, 3 Carlos Bueso-Ramos, 3 Michael Joseph Cascio, 4 Heesun Joyce Rogers, 5 Eric Darryl Hsi, 5 Craig Soderquist, 6 Adam Bagg, 6 Jiong Yan, 7 Rachel Ochs, 7 Attilio Orazi, 7 Frank Moore, 8 Amer Mahmoud, 8 Tracy Irene George, 8 Kathryn Foucar, 8 Jamie Odem, 9 Cassie Booth, 9 William Morice, 9 Daniel J. DeAngelo, 10 David Steensma, 10 Richard Maury Stone, 10 Donna Neuberg, 2 and Daniel Alan Arber 4 It is controversial whether acute myeloid leukemia (AML) patients with 20 29% bone marrow (BM) blasts, formerly referred to as refractory anemia with excess blasts in transformation (RAEBT), should be considered AML or myelodysplastic syndrome (MDS) for the purposes of treatment and prognostication. We retrospectively studied 571 de novo AML in patients aged >50 years, including 142 RAEBT and 429 with 30% blasts (AML30), as well as 151 patients with 10 19% BM blasts (RAEB2). RAEBT patients were older and had lower white blood count, but higher hemoglobin, platelet count, and karyotype risk scores compared to AML30, while these features were similar to RAEB2. FLT3 and NPM1 mutations and monocytic morphology occurred more commonly in AML30 than in RAEBT. RAEBT patients were treated less often with induction therapy than AML30, whereas allogeneic stem cell transplant frequency was similar. The median and 4-year OS of RAEBT patients were longer than those of AML30 patients (20.5 vs 12.0 months and 28.6% vs 20.4%, respectively, P ); this difference in OS was manifested in patients in the intermediate UKMRC karyotype risk group, whereas OS of RAEBT patients and AML30 patients in the adverse karyotype risk group were not significantly different. Multivariable analysis showed that RAEBT (P < ), hemoglobin (P ), UKMRC karyotype risk group (P ), normal BM karyotype (P ), treatment with induction therapy (P < ), and stem cell transplant (P < ) were associated with longer OS. Our findings favor considering de novo RAEBT as a favorable prognostic subgroup of AML. Am. J. Hematol. 89:E193 E199, VC 2014 Wiley Periodicals, Inc. Introduction The original French American British (FAB) classification of myelodysplastic syndromes (MDS) included a category in which myeloblasts comprise 20 29% of bone marrow (BM) cells, termed refractory anemia with excess blasts in transformation (RAEBT) [1]. In the 2001 WHO Classification of myeloid neoplasms, the myeloblast count used to define acute myeloid leukemia (AML) was lowered from 30% to 20% of the BM cells or peripheral blood (PB) leukocytes, thus eliminating the MDS category of RAEBT and subsuming it into AML. This was based on the prevalent practice of treating patients with 20 29% BM blasts with induction chemotherapy and direct evidence from some studies showing that outcomes in RAEBT patients following induction therapy were similar to AML patients with 30% BM blasts [2]. In contrast, both the original [3] and revised [4] International Prognostic Scoring System of MDS (IPSS and IPSS-R, respectively) found similar outcomes in patients with 10 19% and 20 29% blasts. In the IPSS-R, patients with 20 29% BM blasts, who would be diagnosed as AML according to the WHO Classification, are considered within the same risk stratum as patients with 10 19% blasts, most of whom would be diagnosed as high-grade MDS (refractory anemia with excess blasts-2, RAEB2) in the WHO Classification. Unlike the Estey et al. study and other studies that provided the basis for reducing the blast count threshold for AML [5], the IPSS and IPSS-R studies only included patients who had not received disease-modifying therapy. The hypomethylating agents azacitidine and decitabine are important therapies for MDS and have been shown to prolong the survival of highrisk MDS patients [6]. Since 2001, hypomethylating agents have also been used increasingly to treat older AML patients or patients ineligible for induction chemotherapy. In one recent study of AML patients 65 years, the outcome with induction chemotherapy was similar to that with Additional Supporting Information may be found in the online version of this article. 1 Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; 2 Department of Biostatistics and Computational Biology, Dana Farber Cancer Institute, Boston, Massachusetts; 3 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4 Department of Pathology, Stanford University, Stanford, California; 5 Department of Pathology, Cleveland Clinic, Cleveland, Ohio; 6 Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania; 7 Department of Pathology, Weill Cornell Medical College, New York, New Yort; 8 Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; 9 Department of Pathology, Mayo Clinic, Rochester, Minnesota; 10 Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts. Conflict of interest: Nothing to report. *Correspondence to: Robert P. Hasserjian, Department of Pathology, WRN244, Massachusetts General Hospital, 55 Fruit Street, Boston, MA rhasserjian@partners.org Received for publication: 2 June 2014; Revised: 11 July 2014; Accepted: 15 July 2014 Am. J. Hematol. 89:E193 E199, Published online: 16 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: /ajh VC 2014 Wiley Periodicals, Inc. doi: /ajh American Journal of Hematology, Vol. 89, No. 11, November

2 Hasserjian et al. RESEARCH ARTICLE hypomethylating agent therapy [7] and in that study, a lower BM blast count was an independent favorable prognostic factor for survival. Another recent study found a median overall survival of 24.5 months in older adult RAEBT patients treated with azacitidine [8], which is longer than the reported 7 12 month median survival of unselected older adult AML patients treated with various therapies [8 11]. These data suggest that in the current era, RAEBT patients may have a more favorable prognosis compared to AML patients with higher BM blast counts. We conducted this multi-institutional retrospective study to compare the clinical features, pathology, and genetics of de novo RAEBT patients to de novo AML patients with 30% blasts and to de novo high-grade MDS patients with 10 19% blasts. Cases of favorableprognosis karyotype AML were excluded, as well as cases diagnosed in patients <50 years of age, which include a high proportion of favorable karyotype cases. We also compared the treatment approaches and outcome of RAEBT patients to higher blast-count AML patients. Our aim was to determine if de novo RAEBT has clinicopathologic features more akin to high-grade MDS or to AML with- 30% blasts, and if low blast count represents an independent prognostic factor in older adult AML patients. We restricted our study to patients diagnosed since 2004, when hypomethylating agents were used more widely to treat AML and by which time cases with 20% blasts were being diagnosed as AML following the publication of the 2001 WHO Classification. Methods Patients. We searched the pathology files of seven institutions for newly diagnosed AML cases in patients aged 50 years after 1/1/2004. Patients with history of prior cytotoxic chemotherapy, radiation therapy, or any myeloid neoplasm were excluded, as were cases with t(15;17), inv (16)/t(16;16), or t(8;21) cytogenetic abnormalities. All cases had 20% BM blasts, based on count of aspirate smears or touch preparations; cases with inadequate smears were included if examination of the BM biopsy confirmed 20% blasts and if distinction between 20 30% and >30% blasts could be made and validated by at least two observers. Cases were divided into two groups, with 20 29% BM blasts (RAEBT group) and 30% BM blasts (AML30 group). In addition, we retrieved cases of newly diagnosed de novo MDS or AML with 10 19% BM blasts (RAEB2 group) from the same institutions over the same time period. Clinical information at presentation and follow-up information, including overall survival (OS) from the date of diagnosis, were retrieved from the electronic medical record at each institution. As this study was retrospective, treatment decisions were based on the practice at each respective institution where the cases were diagnosed. Treatments administered were obtained from the electronic medical records and were categorized as supportive care only (including growth factors, steroids, or hydroxyurea), low-intensity therapies (lenalidomide, low-dose cytarabine, and other cytotoxic investigational agents), hypomethylating agents, induction therapy (standard induction therapy employing anthracycline and cytarabine), and allogeneic stem cell transplant (SCT). This study was approved by the Institutional Review Boards of all participating institutions and was conducted by members of the Bone Marrow Pathology Group. Pathology. Pathologic features recorded for each case included BM cellularity, FAB Classification subtype (for RAEBT and AML30), 2008 WHO Classification subtype, BM blast count (based on the aspirate smear or estimated on the trephine biopsy if the aspirate smear was inadequate) and dysplasia in the nonblast cells. Dysplasia was scored in each lineage as <10%, 10 50%, or >50% dysplastic cells based on examination of both BM aspirate smears and trephine biopsy; criteria for dysplasia were according to the 2008 WHO Classification [12]. Cytogenetics and molecular genetic studies. All cases with available karyotype were stratified according to the Comprehensive Cytogenetic Scoring System of the IPSS-R [13] and the United Kingdom Medical Research Council criteria for AML (UKMRC) [14]. RAEBT and AML cases were categorized as AML-MRC if specific myelodysplasia-related cytogenetic abnormalities were identified or if >50% dysplastic cells were identified in 2 hematopoietic lineages [15]. Information on FLT3 and NPM1 mutation status was recorded when available. A validation group of 53 additional de novo AML cases, meeting the same inclusion criteria as the study patients and with known FLT3 and NPM1 mutation status, was obtained from the pathology files of Mayo Clinic. Statistical analysis. Fisher s exact test and Wilcoxon test were used to compare categorical and continuous variables between groups, respectively. OS from diagnosis was estimated using the method of Kaplan and Meier and the log-rank test was used to compare OS between groups. Multivariable subsequent to univariate Cox proportional hazards regression models were used to assess the impact of the patient groups along with other risk factors on OS. The proportional hazard assumption was checked in each of the univariate Cox regression analyses. A 2- sided P-value less than 0.05 was considered statistically significant. Results Clinical and morphologic features A total of 571 AML cases were retrieved, including 142 with 20 29% BM blasts (RAEBT) and 429 with 30% BM blasts (AML30). Cases represented all consecutive diagnoses meeting inclusion criteria at each institution, or were a subset of cases based on either random selection of all cases diagnosed over the entire study period or all consecutive cases diagnosed over a shorter time period. The breakdown of cases by institution and selection process is shown in Supporting Information Table I. An additional 151 RAEB2 group cases with 10 19% BM blasts were retrieved. Following the WHO Classification, these comprised mostly RAEB2 (135 cases), but also included acute erythroid leukemia (with 10 19% BM blasts, but with blasts comprising 20% of the nonerythroid cells, 14 cases), and AML with 10 19% BM blasts but 20% PB blasts (two cases). Blast count was based on the biopsy because of insufficient smears in eight (6%) RAEBT, 19 (4%) AML30, and 13 (9%) RAEB2 cases. Clinical features of the RAEBT patients in comparison to the AML30 and RAEB2 patients are shown in Table I. RAEBT patients were older (P < ) and had lower median WBC (P < ) and PB blast percentage (P < ), but higher hemoglobin (P ) and platelet count (P ) compared to AML30 patients. There were no significant differences in age or blood counts (with the exception of higher PB blast percentage in RAEBT) between RAEBT and RAEB2 patients. The morphologic and genetic features of the patient groups are shown in Table I. RAEBT differed from AML30 with respect to BM cellularity (P < ), morphologic dysplasia (P < for all three lineages), and monocytic differentiation (FAB M4 or M5) (P < ). In contrast, the morphologic features of RAEBT and RAEB2 were similar, with the exception of a higher proportion of RAEB2 cases showing megakaryocytic dysplasia (P ). Cytogenetics and FLT3/NPM1 mutation status The karyotype profile of RAEBT generally differed from AML30, in that a higher proportion of RAEBT cases had adverse UKMRC karyotype (P ), abnormal karyotype (P ), and cytogenetic changes defining AML-MRC [15] (P ), while IPSS-R karyotype risk grouping was not significantly different (P ). The karyotype risk profile of RAEBT was similar to RAEB2 (Table I). Although FLT3 and NPM1 mutation analyses were only available in a limited subset of patients, FLT3 ITD mutations were less frequent in RAEBT (3/94, 4%) compared to AML30 (59/246, 24%) (P < ) and NPM1 mutations were also less frequent in RAEBT (1/55, 2%) compared to AML30 (46/150, 31%) (P < ). In a separate validation cohort of de novo AML patients aged 50 years from Mayo Clinic, 0/12 RAEBT compared to 13/40 AML30 had a FLT3 mutation (P ) and 1/12 RAEBT compared to 18/35 AML30 had NPM1 mutation (P ). Treatment and survival The treatments administered to the AML30 and RAEBT patients are shown in Table II. The use of hypomethylating agents was more frequent and use of induction therapy was less frequent (both as the first treatment and at any time during the disease course) in RAEBT compared with AML30. When analyzed by patient age decades, this difference was driven by strikingly different treatment proportions in the patients aged years (Table II). Treatment and outcome 194 American Journal of Hematology, Vol. 89, No. 11, November 2014 doi: /ajh.23808

3 RAEBT is less aggressive than high blast-count AML TABLE I. Comparison of RAEBT, AML30, and RAEB2 Clinical Features at Presentation RAEBT (n 5 142) AML30 (n 5 429) P value (RAEBT versus AML30 RAEB2 (n 5 151) P value (RAEBT versus RAEB2) Gender, M:F 89:53 252: : Median age, y < Age 60 y 111/142 (78%) 271/429 (63%) /151 (80%) 0.77 Median white blood count (310 9 /L) a < Median hemoglobin (g/dl) a Median platelet count (310 9 /L) a Median peripheral blood blasts, % a < Median bone marrow cellularity a 60% 90% < % 0.40 Median [range] bone marrow blasts 24% [20 29%] 67% [30 100%] NA 13% [10 19%] NA Erythroid lineage dysplasia (>10%) 75/134 (56%) 133/414 (32%) < /142 (65%) 0.11 Myeloid lineage dysplasia (>10%) 75/130 (58%) 106/416 (25%) < /144 (52%) 0.40 Megakaryocytic dysplasia (>10%) 66/127(52%) 116/411 (28%) < /131(68%) 0.01 FAB M4 or M5 23/115 (20%) 137/342 (39%) < NA NA WHO diagnosis of AML-MRC b 68/126 (54%) 157/403 (39%) NA NA Adverse UKMRC karyotype 49/135 (36%) 110/408 (27%) /142 (39%) 0.62 Normal karyotype 60/135 (44%) 222/408 (54%) /142 (39%) 0.39 IPSS-R karyotype groups NA NA 0.77 Very low/low 64 (45%) 61 (40%) Intermediate 26 (18%) 26 (17%) High/very high 45 (32%) 54 (36%) Unknown 7 (5%) 10 (7%) a White blood count data missing in 6 AML30 and 5 RAEB2; hemoglobin data missing in 10 AML30 and 4 RAEB2; platelet count data missing in 1 RAEBT, 7 AML30, and 3 RAEB2; peripheral blood blast data missing in 4 RAEBT, 28 AML 30, and 14 RAEB2; bone marrow cellularity data missing in 6 RAEBT, 31 AML30, and 9 RAEB2. b By either morphology or karyotype criteria. NA, not applicable. TABLE II. Treatments Administered to RAEBT and AML30 Patients RAEBT (n 5 142) AML30 (n 5 429) P value Supportive care only 11 (8%) 36 (8%) Hypomethylating agents as 36 (25%) 36 (8%) < first therapy Age /31 (5%) 1/158 (1%) 0.07 Age /46 (26%) 7/138 (5%) < Age 70 22/65 (34%) 28/132 (21%) 0.08 Hypomethylating agents at 49 (35%) 78 (18%) any time Induction therapy as first 73 (51%) 310 (72%) < therapy Age /31 (90%) 147/158 (93%) 0.70 Age /46 (61%) 118/138 (86%) < Age 70 17/65 (26%) 45/132 (34%) 0.33 Induction therapy at any time 82 (58%) 317 (74%) Allogeneic SCT at any time 41 (29%) 118 (27%) 0.75 data for RAEB2 patients were not analyzed, since the vast majority of these patients were not diagnosed with AML and thus would not be eligible for induction therapy at most institutions. A similar proportion of RAEBT (29%) and AML30 patients (27%) received SCT, between 2 and 42 months (for RAEBT patients) and between 1 and 47 months (for AML30 patients) after diagnosis. A competing risk analysis of time to SCT where death was considered the competing risk in RAEBT and AML30 patients revealed no significant difference in the cumulative incidence of SCT in these two groups (Gray s test: P ). The median follow-up time of all patients was 55.5 months. The median OS of RAEBT patients was 20.5 (90% CI: ) months, which was longer than the median OS of AML30 patients of 12.0 (90% CI: ) months (P , log rank test) (Fig. 1A). The 1-year and 4-year survival probabilities were 60.2% (90% CI: %) and 28.6% (90% CI: %), respectively, for RAEBT patients and 50.7% (90% CI: %) and 20.4% (90% CI: %), respectively, for AML30 patients. After excluding the eight RAEBT and 19 AML30 patients whose diagnoses were made on BM biopsy blast count estimate, median OS of RAEBT patients was still significantly longer than AML30 patients (P ). Considering patients 60 years old, OS of RAEBT patients (n 5 111) was longer than AML30 patients (n 5 271) (P ), while considering patients years old, OS of RAEBT patients (n 5 31) was also longer than AML30 patients (n 5 158)(P ). Univariate analysis of factors influencing OS was performed on the AML30 and RAEBT combined group and is shown in Table III. An abnormal karyotype of any kind, as well as a higher-risk karyotype score (assessed by either UKMRC or WHO AML-MRC-defining karyotype) were associated with shorter OS. Within the UKMRC intermediaterisk karyotype group, RAEBT patients had longer OS compared to AML30 patients (Fig. 1B, P ), while there was no significant difference between the two groups within the adverse-risk karyotype group (Fig. 1C, P ). Analysis of the subset of patients with available FLT3 mutation data showed that among the UKMRC intermediate-risk patients with wild-type FLT3, RAEBT patients (N 5 57) had longer OS than AML30 patients, (N 5 121, Fig. 1D, P ). Longer OS was also seen in the RAEBT group compared to AML30 among UKMRC intermediate-risk patients lacking NPM1 mutation (P ) and lacking both NPM1 and FLT3 mutations (P ) (data not shown). Comparison of OS between NPM1- mutated RAEBT and AML30 was not possible, because only one RAEBT patient had NPM1 mutation. Stepwise multivariable Cox regression analysis (including all variables with P value <0.2 in the univariate analysis) identified AML group type (RAEBT or AML30), hemoglobin level, UKMRC risk group, any karyotypic abnormality, treatment with induction therapy at any time, and allogeneic SCT, as independent predictors of OS in the combined AML30 and RAEBT group (Table IV). FLT3 and NPM1 mutation status were not included in the multivariable analysis because of a high proportion of missing values. Univariate analysis of factors influencing OS within the RAEBT group alone revealed doi: /ajh American Journal of Hematology, Vol. 89, No. 11, November

4 Hasserjian et al. RESEARCH ARTICLE Figure 1. (A) Kaplan Meier analysis of overall survival of RAEBT (n 5 142) and AML30 (n 5 429) patients. Overall survival RAEBT patients is longer than AML30 patients (P ). (B) Among patients with UKMRC intermediate-risk karyotype, overall survival is longer in RAEBT patients (n 5 86) than in AML30 patients (n 5 296, P ). (C) Among patients with UKMRC adverse-risk karyotype, there is no significant difference in overall survival between RAEBT (n 5 49) and AML30 patients (n 5 110, P ). (D) Kaplan Meier analysis of overall survival of intermediate-risk karyotype RAEBT (n 5 57) and AML30 patients (n 5 121) with wild-type FLT3. Within this subgroup, overall survival of RAEBT patients is longer than AML30 patients (P ). similar findings to the combined RAEBT and AML30 group; in addition, platelet count, BM cellularity, and erythroid lineage dysplasia were associated with OS (Supporting Information Table II). The final multivariable model shown in Table IV was expanded to test whether the effects of intensive therapies on OS were similar in the RAEBT and AML30 groups. Two separate expanded models with an interaction term between disease group (RAEBT or AML30) and induction chemotherapy first, and then between disease group and SCT, were considered. The inclusion of the interaction terms neither added significantly to the model nor altered the coefficients of factors already in the model. Hence the odds ratios estimations and the P- values were similar with or without the interaction terms. Both these aspects suggest that the effects of intensive therapy (induction chemotherapy or SCT) on OS were similar in the two groups. We also separately analyzed patients who were treated with any disease-modifying therapies (hypomethylating agents or intensive therapy, representing 119 RAEBT and 269 AML30 patients) versus those who received only low-intensity therapies or supportive care (representing 23 RAEBT and 61 AML30 patients). OS of RAEBT patients was longer than the AML30 patients both in the disease-modifying therapy subgroup (P ) and in the low-intensity therapy/supportive care subgroup (P ) (log rank test). Discussion In this retrospective study, we analyzed the clinicopathologic features and outcome of older adult de novo RAEBT patients treated at multiple institutions and compared them to AML patients presenting with 30% blasts. We found that the clinical features (age and blood counts) of RAEBT patients at presentation resembled de novo RAEB2 and differed from AML30 patients. Despite less frequent treatment with induction therapy, RAEBT patients survived longer than AML patients with higher blast counts. Our study included patients aged 50 years, but we found that the favorable prognostic impact of RAEBT was maintained in the subgroup of patients 60 years old (P by log-rank test). The median survival of RAEBT patients in our study was 20.5 (90% CI: ) months, which is longer than the median survivals of 3 16 months reported in several prior studies [2,16,17]. However, many of these studies included a high proportion of patients with secondary AML (who have inferior survival to de novo AML patients) [18], or examined older patients than our series; the median survival of de novo RAEBT patients aged >50 years treated with azacitidine reported by Fenaux et al. [8] was 24 months, comparable to our study. The outcome of AML patients is influenced by a number of factors reflecting both the disease biology and patient characteristics [19]. AML-MRC is defined in the WHO Classification as a subtype of AML characterized by a prior history of MDS, significant background morphologic dysplasia, and/or high-risk karyotypic abnormalities similar to those found in MDS, and has an inferior prognosis to AML, not otherwise specified (AML-NOS) [20]. The relatively high incidence of dysplasia and the karyotype profile of RAEBT in our study resulted in a higher proportion of RAEBT cases (54%) being classified as AML-MRC than that of AML30 (39%). However, the RAEBT patients survived longer than AML30 patients, because of a significantly longer survival of RAEBT compared to AML30 in the 196 American Journal of Hematology, Vol. 89, No. 11, November 2014 doi: /ajh.23808

5 TABLE III. Univariate Analysis of Factors Influencing OS in Combined RAEBT and AML30 Patients (n 5 571) Risk factor at diagnosis P value HR [95% CI] RAEBT [ ] Female gender [ ] Age group, y (reference 50 59) [ ] < [ ] 80 < [ ] WBC, /L (reference <2.1) a [ ] [ ] [ ] Hemoglobin, g/dl (reference <8.3) a [ ] [ ] [ ] Platelets, /L (reference <34) a [ ] [ ] [ ] PB blasts, % (reference <2) a [ ] [ ] [ ] BM cellularity, % (reference <60) a [ ] [ ] [ ] Erythroid dysplasia, % (reference <10) [ ] > [ ] Myeloid dysplasia, % (reference <10) [ ] > [ ] Megakaryocytic dysplasia, % (reference <10) [ ] > [ ] Myelodysplasia-related morphology [ ] present (>50% dysplastic cells in 2 lineages) UKMRC adverse risk karyotype < [ ] Abnormal karyotype < [ ] AML-MRC karyotype < [ ] WHO diagnosis of AML-MRC b < [ ] Treatment (at any time) Induction therapy [ ] Allogeneic SCT < [ ] a Cutpoints for these variables were chosen based on the method of quartiles. b By either morphology or karyotype. subgroup of patients with UKMRC intermediate-risk karyotype (P value ); the outcome of patients with adverse-risk karyotype was not significantly different between RAEBT and AML30 patients (P value ). These findings indicate that low blast count (20 29%) is a favorable prognostic factor in older AML patients with intermediate-risk karyotype. On multivariable analysis, the favorable impact of RAEBT was independent of other prognostic factors and treatment modality. In the limited subgroup of patients with available FLT3 mutation analysis, we found that the favorable outcome of RAEBT patients relative to AML30 was maintained in the FLT3 wildtype intermediate UKMRC karyotype-risk patients (P ). Thus, the longer survival of the RAEBT patients does not appear to be explained only by the relatively low incidence of FLT3 mutation in this group. High white blood cell count and high numbers of circulating PB blasts are associated with the FLT3 mutation and are negative prognostic factors in AML [21]. However, in our study, which included low BM blast count (20 29%) as a variable, neither white RAEBT is less aggressive than high blast-count AML TABLE IV. Multivariable Analysis of Factors Influencing OS in RAEBT and AML30 Patients (N 5 533) Risk factor at diagnosis P value HR [95% CI] RAEBT < [ ] Hemoglobin 10.3 g/dl a [ ] Intermediate UKMRC karyotype risk [ ] Normal karyotype [ ] Induction therapy at any time < [ ] Allogeneic SCT < [ ] a Cut-point for hemoglobin based on method of quartiles from univariate analysis. Additional Supporting Information may be found in the online version of this article. Supplementary Information blood cell count nor PB blast percentage were independent prognostic factors for OS in all AML patients. PB blast percentage (11% vs. 0%, OR % CI: ) within the RAEBT group was a prognostic factor, as has been shown in prior studies [16,22]. The results of our study differ from those of Estey et al. [2], which found that RAEBT and AML30 patients treated with induction chemotherapy had similar outcomes, and formed part of the rationale for inclusion of RAEBT within AML in the 2001 WHO Classification. One possible explanation for this difference is the advent of hypomethylating agent therapy for AML. These agents have been shown to prolong the survival of RAEBT patients compared to those treated with supportive care [8,17] and may be more effective in RAEBT than in AML30 [7]. Also, in our series, 29% of RAEBT and 27% of AML30 patients received allogeneic SCT, compared to <2% of patients in the Estey et al. study. In addition, we excluded patients with secondary AML, who comprised a substantial subset of the patients in the Estey et al. study and whose outcome appears to be influenced by prognostic parameters that are different from de novo AML [23]. Another study by Arber et al. found no difference in survival between RAEBT (N 5 24) and AML30 (N 5 276) patients, but this was in a much younger patient population (median age 41) than our series and also included patients who had progressed from prior MDS or had therapy-related disease [24].Since the favorable outcome of RAEBT was manifested only in the intermediate-karyotype risk patients in our study, cohorts that include a high proportion of patients with adverse-risk karyotype (such as therapy-related or secondary AML) may not reveal a difference in outcome between RAEBT and higher blast-count AML. We did not compare outcome in RAEBT to RAEB2 in our retrospective study, since the vast majority of the latter patients would not be eligible for induction therapy at most institutions. In two studies restricted to patients who did not receive intensive therapy, OS of RAEBT and RAEB2 patients were similar [4,25]. One possible explanation for the more favorable outcome in RAEBT compared to AML30 is that RAEBT represents an earlier stage of AML. However, the 8.5 month-longer median OS of RAEBT patients is not likely explained by lead time bias alone. More likely is the possibility that RAEBT lacking adverse-risk karyotype exhibits an intrinsic disease biology that is different from higher blast-count AML. For example, the fact that RAEBT manifests not only by lower BM blast count but also lower BM cellularity, peripheral blood leukocyte count, and PB blast percentage raises the possibility that the leukemic clone in RAEBT may have lower proliferative capacity compared to AML30, which may reflect a different mutational profile. We did find a lower incidence of FLT3 and NPM1 mutations in RAEBT compared to AML30 in our series (and confirmed this in a separate validation cohort of patients from Mayo Clinic), but many patients were diagnosed and treated before testing for FLT3 and doi: /ajh American Journal of Hematology, Vol. 89, No. 11, November

6 Hasserjian et al. NPM1 mutations were mandated in the 2008 WHO Classification and these data were missing on a significant subset of patients in the main cohort. We did not examine the incidence of other AMLassociated gene mutations in RAEBT and AML30, and thus we could not assess any association of the overall gene mutation profile with blast count. In the NCCN guidelines for MDS, RAEBT is considered to be part of the MDS spectrum if it arises in patients with a history of MDS or if patients manifest slowly progressive disease ( store/login/login.aspx?returnurl5http:// physician_gls/pdf/mds.pdf). Although we did not have sufficient information on longitudinal blood counts of the patients prior to diagnosis to analyze the tempo of their cytopenias, the less marked cytopenias compared to AML30 patients suggest a more slowly progressive disease in RAEBT patients at the time of clinical presentation. The BM morphology of RAEBT patients resembled RAEB2 more closely than AML30 in terms of lower cellularity and more prominent dysplasia in all hematopoietic lineages; one caveat is that RAEBT and RAEB2 (by definition) have a higher percentage of nonblast cells than AML30, which may facilitate the identification of dysplasia. The subsets of RAEBT patients with adverse-risk and MDSrelated karyotypes displayed similar outcome to comparable AML30 patients, suggesting that adverse-risk karyotype AMLs are biologically similar irrespective of blast count and generally have uniformly poor prognosis. This is consistent with the aggressive behavior of genetically complex myeloid neoplasms, including therapy-related AML and MDS, in which outcome is not prominently influenced by BM blast percentage [26]. In our series, morphologic dysplasia was not associated with outcome and did not add to cytogenetic risk in identifying patients with poor outcome either within the entire AML cohort or within the RAEBT group. Prior studies have shown conflicting results as to the prognostic value of multilineage dysplasia in AML outcome [20,27]. In this retrospective analysis, we also found that the treatment approach of RAEBT patients differed from AML30 patients. RAEBT patients were more likely to be treated with hypomethylating agents, while AML30 patients aged were more likely to be treated initially with induction therapy compared with RAEBT patients. This may reflect the perception that AML patients with low blast count benefit more from hypomethylating agents than patients with higher blast count, as been shown in some studies [7], but not others [28,29], and the results of the MDS-001 study, in which patients with 20 30% blasts treated with azacitidine fared better than those randomized to receive conventional care (doctor s predetermined choice of low dose cytarabine, induction chemotherapy, or supportive care) [10]. In addition, induction therapy may be used more frequently in patients with high circulating blast counts, who may have symptoms that demand rapid cytoreduction. Because our study was retrospective and involved multiple institutions, the types of therapies the patients received were variable and thus we could not compare the effectiveness of specific types of therapies used in defined patient subpopulations, nor could we determine the factors that led to the use of particular therapies in patient subgroups. However, based on analysis of intensive treatments in our multivariable analysis, the benefits of intensive therapies (both induction chemotherapy and SCT) appeared to be similar in RAEBT and AML30 patients in our study. In summary, we found that among older adult de novo AML patients who received various treatment modalities, a low BM blast count (20 29%) was associated with a favorable prognosis. This favorable outcome is manifested in the subgroup of RAEBT patients with UKMRC intermediate-risk karyotype, who comprise the majority of de novo RAEBT patients. Conversely, survival of patients with 20 29% blasts and adverse karyotype was similar to that of patients with 30% blasts and adverse karyotype. Our findings favor considering AML with 20 29% BM blasts and lacking high-risk karyotype as a favorable prognostic subgroup of AML, NOS. These patients appear to manifest a disease that could be considered as a less aggressive, hypoproliferative form of AML with some similarities to RAEB2. Further study is needed to determine the optimal treatment for these patients and to examine the interaction of gene mutation risk profiles with low blast count in influencing AML outcome. Future AML trials should include BM blast count as a variable, in order to determine if RAEBT patients may respond differently to specific therapeutic regimens as compared to AML patients with higher blast counts. Our data also suggest that RAEBT patients with adverse-risk karyotype are appropriately included along with higher blast-count AML patients within the WHO category of AML with myelodysplasiarelated changes. Acknowledgments RESEARCH ARTICLE The authors thank Hildreth Curran for assistance in preparing the manuscript. The members of the Bone Marrow Pathology Group are Adam Bagg, Carlos Bueso-Ramos, Kathryn Foucar, Robert Hasserjian, Eric Hsi, Sa Wang, Daniel Arber, Bill Morice, Attilio Orazi, and John Anastasi. References 1. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982;51: Estey E, Thall P, Beran M, et al. Effect of diagnosis (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, or acute myeloid leukemia [AML]) on outcome of AML-type chemotherapy. Blood 1997;90: Greenberg P, Cox C, LeBeau MM, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997;89: Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012;120: Estey E, Pierce S, Kantarjian H, et al. Treatment of myelodysplastic syndromes with AML-type chemotherapy. Leuk Lymphoma. 1993;11(Suppl 2): Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al. Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: A randomised, open-label, phase III study. Lancet Oncol 2009;10: Quintas-Cardama A, Ravandi F, Liu-Dumlao T, et al. Epigenetic therapy is associated with similar survival compared with intensive chemotherapy in older patients with newly diagnosed acute myeloid leukemia. Blood 2012;120: Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al. Azacitidine prolongs overall survival compared with conventional care regimens in elderly patients with low bone marrow blast count acute myeloid leukemia. J Clin Oncol 2010;28: Anderson JE, Kopecky KJ, Willman CL, et al. Outcome after induction chemotherapy for older patients with acute myeloid leukemia is not improved with mitoxantrone and etoposide compared to cytarabine and daunorubicin: A Southwest Oncology Group study. Blood 2002; 100: Dombret H, Raffoux E, Gardin C. Acute myeloid leukemia in the elderly. Semin Oncol 2008; 35: Kantarjian H, O Brien S, Cortes J, et al. Results of intensive chemotherapy in 998 patients age 65 years or older with acute myeloid leukemia or high-risk myelodysplastic syndrome: predictive prognostic models for outcome. Cancer 2006;106: Brunning RD, Orazi A, Germing U, et al. Myelodysplastic syndromes/neoplasms, overview. In: Swerdlow SH, Campo E, Harris NL, et al., editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: International Agency for Research on Cancer (IARC); pp Schanz J, Tuchler H, Sole F, et al. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. J Clin Oncol 20120;30: Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: Determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116: Arber DA, Brunning RD, Orazi A, et al. Acute myeloid leukaemia with myelodysplasia-related changes. In: Swerdlow SH, Campo E, Harris NL, 198 American Journal of Hematology, Vol. 89, No. 11, November 2014 doi: /ajh.23808

7 RAEBT is less aggressive than high blast-count AML et al., editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: International Agency for Research on Cancer (IARC); pp Breccia M, Latagliata R, Carmosino I, et al. Refractory anaemia with excess of blasts in transformation re-evaluated with the WHO criteria: Identification of subgroups with different survival. Acta Haematol 2007;117: Kantarjian HM, Thomas XG, Dmoszynska A, et al. Multicenter, randomized, open-label, phase III trial of decitabine versus patient choice, with physician advice, of either supportive care or low-dose cytarabine for the treatment of older patients with newly diagnosed acute myeloid leukemia. J Clin Oncol 2012;30: Wheatley K, Brookes CL, Howman AJ, et al. Prognostic factor analysis of the survival of elderly patients with AML in the MRC AML11 and LRF AML14 trials. Br J Haematol 2009;145: Dohner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: Recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010;115: Weinberg OK, Seetharam M, Ren L, et al. Clinical characterization of acute myeloid leukemia with myelodysplasia-related changes as defined by the 2008 WHO classification system. Blood 2009;113: Whitman SP, Maharry K, Radmacher MD, et al. FLT3 internal tandem duplication associates with adverse outcome and gene- and microrna-expression signatures in patients 60 years of age or older with primary cytogenetically normal acute myeloid leukemia: A Cancer and Leukemia Group B study. Blood 2010;116: Strupp C, Gattermann N, Giagounidis A, et al. Refractory anemia with excess of blasts in transformation: Analysis of reclassification according to the WHO proposals. Leuk Res 2003;27: Okuyama N, Sperr WR, Kadar K, et al. Prognosis of acute myeloid leukemia transformed from myelodysplastic syndromes: A multicenter retrospective study. Leuk Res 2013;37: Arber DA, Stein AS, Carter NH, Ikle D, Forman SJ, Slovak ML. Prognostic impact of acute myeloid leukemia classification. Importance of detection of recurring cytogenetic abnormalities and multilineage dysplasia on survival. Am J Clin Pathol 2003;119: Germing U, Strupp C, Kuendgen A, et al. Prospective validation of the WHO proposals for the classification of myelodysplastic syndromes. Haematologica 2006;91: Singh ZN, Huo D, Anastasi J, et al. Therapyrelated myelodysplastic syndrome: Morphologic subclassification may not be clinically relevant. Am J Clin Pathol 2007;127: Haferlach T, Schoch C, Loffler H, et al. Morphologic dysplasia in de novo acute myeloid leukemia (AML) is related to unfavorable cytogenetics but has no independent prognostic relevance under the conditions of intensive induction therapy: Results of a multiparameter analysis from the German AML Cooperative Group studies. J Clin Oncol 2003;21: Pleyer L, Stauder R, Burgstaller S, et al. Azacitidine in patients with WHO-defined AML results of 155 patients from the Austrian Azacitidine Registry of the AGMT-Study Group. J Hematol Oncol 2013;6: van der Helm LH, Veeger NJ, Kooy M, et al. Azacitidine results in comparable outcome in newly diagnosed AML patients with more or less than 30% bone marrow blasts. Leuk Res 2013;37: doi: /ajh American Journal of Hematology, Vol. 89, No. 11, November