Mayo Prognostic Model for WHO-Defined Chronic

ORIGINAL ARTICLE Mayo prognostic model for WHO-deﬁned chronic myelomonocytic leukemia: ASXL1 and spliceosome component mutations and outcomes

MM Patnaik1, E Padron2, RR LaBorde1, TL Lasho1, CM Finke1, CA Hanson3, JM Hodneﬁeld3, RA Knudson4, RP Ketterling4, A Al-kali1, A Pardanani1, NA Ali2, RS Komroji2 and A Tefferi1

We evaluated the prognostic relevance of several clinical and laboratory parameters in 226 Mayo Clinic patients with chronic myelomonocytic leukemia (CMML): 152 (67%) males and median age 71 years. At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. In univariate analysis, significant risk factors for survival included anemia, thrombocytopenia, increased levels of white blood cells, absolute neutrophils, absolute monocyte count (AMC), absolute lymphocytes, peripheral blood and bone marrow blasts, and presence of circulating immature myeloid cells (IMCs). Spliceosome component (P ¼ 0.4) and ASXL1 mutations (P ¼ 0.37) had no impact survival. On multivariable analysis, increased AMC (410 Â 109/l, relative risk (RR) 2.5, 95% confidence interval (CI) 1.7–3.8), presence of circulating IMC (RR 2.0, 95% CI 1.4–2.7), decreased hemoglobin (o10 g/dl, RR 1.6, 99% CI 1.2–2.2) and decreased platelet count (o100 Â 109/l, RR 1.4, 99% CI 1.0–1.9) remained significant. Using these four risk factors, a new prognostic model for overall (high risk, RR 4.4, 95% CI 2.9–6.7; intermediate risk, RR 2.0, 95% CI 1.4–2.9) and leukemia-free survival (high risk, RR 4.9, 95% CI 1.9–12.8; intermediate risk, RR 2.6, 95% CI 1.1–5.9) performed better than other conventional risk models and was validated in an independent cohort of 268 CMML patients.

Leukemia (2013) 27, 1504–1510; doi:10.1038/leu.2013.88 Keywords: survival; prognosis; CMML; ASXL1

INTRODUCTION subsequently applied to 212 CMML patients in the Dusseldorf 7 Chronic myelomonocytic leukemia (CMML) is a clonal, hemato- registry; in a univariate analysis, circulating IMC had no poietic stem cell disorder, with overlapping features of myelodys- prognostic impact, whereas on multivariable analysis, elevated plastic syndromes (MDSs) and myeloproliferative neoplasms. lactate dehydrogenase, BM blast count 410%, male gender, 9 CMML is characterized by persistent peripheral blood (PB) hemoglobin o12 g/dl and ALC 42.5 Â 10 /l were independently 7 monocytosis 41 Â 109/l, absence of the BCR-ABL1 fusion, absence prognostic. The Dusseldorf score classified patients into three risk of rearrangements of the PDGFRA or PDGFRB genes, absence of categories, with median survivals of 93 (low), 26 (intermediate) 7 X20% myeloblasts or promonocytes in the blood and bone and 11 (high) months, respectively. The Spanish cytogenetic risk- marrow (BM) and presence of dysplasia in one or more myeloid stratification system analyzed the role of karyotype in patients 8 lineages.1 Based on the 2008 World Health Organization (WHO) with CMML. Based on their analysis, three cytogenetic risk classification system, CMML is further subcategorized into CMML-1 categories were identified: low risk (diploid karyotype or loss of (o5% circulating blasts and o10% BM blasts) and CMML-2 chromosome Y as a single anomaly), intermediate risk (all other (5–19% circulating blasts, 10–19% BM blasts or when Auer rods abnormalities except those mentioned in low- and high-risk are present irrespective of the blast count), with median overall categories) and high risk (trisomy 8, abnormalities of chromosome survivals (OSs) of approximately 20 and 15 months, respectively.1,2 7 or complex karyotype); with 5-year OS for the three groups 8 Numerous prognostic models have attempted to risk stratify being 35%, 26% and 4%, respectively (Po0.001). This 8 patients with CMML. In this regard, the value of Bournemouth, stratification system did not predict leukemic transformation (LT). Lille and the International Prognostic Scoring Systems (IPSS) is In 2008, the Global MDAPS (G-MDAPS) was developed limited, as they were designed primarily for patients with MDS, for patients with de novo MDS, secondary MDS and CMML 9 excluding CMML patients with a proliferative phenotype.3–5 The (n ¼ 1915). On a multivariable analysis, independent prognostic MD Anderson Prognostic Scoring System (MDAPS) was developed factors included older age, poor performance status, in a cohort of 213 CMML patients and identified a hemoglobin thrombocytopenia, anemia, increased BM blasts, leukocytosis 9 level 12 g/dl, presence of circulating immature myeloid cells (420 Â 10 /l), chromosome 7 or complex cytogenetic o 9 (IMCs), absolute lymphocyte count (ALC) 42.5 Â 109/l andX10% abnormalities and a prior history of red blood cell transfusions. BM blasts as independent predictors for inferior survival.6 This model identified four prognostic groups with median This model identified four subgroups of patients with median survivals of 54 (low), 25 (intermediate 1), 14 (intermediate 2) 9 survivals of 24, 15, 8 and 5 months respectively.6 The MDAPS was and 6 months (high), respectively. The G-MDAPS was validated in

1Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA; 2Malignant Hematology and Immunology Program, H. Lee Moffitt Cancer Center, Tampa, FL, USA; 3Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA and 4Division of Cytogenetics, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA. Correspondence: Professor A Tefferi, Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA. E-mail: [email protected] Received 13 March 2013; accepted 20 March 2013; accepted article preview online 27 March 2013; advance online publication, 19 April 2013 Prognosis in chronic myelomonocytic leukemia MM Patnaik et al 1505 176 patients with CMML and leukocytosis (412 Â 109/l), with Kaplan–Meier method and compared by the log-rank test. Cox propor- median survivals in the four risk categories being 33, 19, 12 and tional hazard regression model was used for multivariable analysis. 8 months, respectively (Po0.01);9 limitations for G-MDAPS P-values o0.05 were considered significant. The Stat View (SAS Institute, included the inclusion of patients with upto 30% BM blasts Cary, NC, USA) statistical package was used for all calculations. (oligoblastic AML). Molecular abnormalities are detected in B90% of patients with CMML.10,11 These are broadly categorized into mutations involving RESULTS epigenetic regulators (EZH2, ASXL1, TET2, DNMT3A, IDH1 and Two-hundred and twenty-six patients with WHO-defined CMML IDH2),10,12,13 spliceosome components (SF3B1, SRSF2, U2AF35 and and who were seen at the Mayo Clinic from 1997 to 2007 were ZRSR2),1,14,15 DNA-damage response genes (TP53)16 and included in the current study. The median age of the cohort was transcription regulators and signaling molecules (JAK2, KRAS, 71 years (range 20–90 years), with 67% of the patients being NRAS, NPM1 and RUNX1).11,17 Thus far, in CMML, loss-of-function males. Table 1 outlines the presenting clinical and laboratory gene mutations involving ASXL1 and EZH2 has been associated features and subsequent events in the 226 study patients with with poor outcome.12,15 Given the inconsistency among currently CMML, stratified by PB and BM blasts. One-hundred and ninety- available CMML prognostic models, their lack of strict adherence one patients (84%) had CMML-1 with a median OS of 21 months, to the WHO classification system when selecting study patients whereas 35 patients had CMML-2 with a median OS of 15 months. and the discovery of molecular abnormalities potentially having Patients with CMML-2 were more likely to have lower an impact on survival, we undertook the current study with 226 hemoglobin levels (P ¼ 0.04), higher AMC (P ¼ 0.03), lower platelet single-center study patients strictly defined by the WHO, fully counts (P ¼ 0.0007), higher likelihood of circulating blasts and IMC annotated for karyotype and also analyzed for reportedly (Po0.0001), and higher risk of karyotypic abnormalities prognostic somatic mutations. We then validated the results in (Po0.0001). They were also more likely to be assigned higher an independent cohort of 268 patients from the H. Lee Moffitt risk scores based on the MDAPS and the G-MDAPS. There, Cancer Center in Tampa, Florida. however, was no difference in the distribution of ASXL1 and spliceosome component mutations (Table 1). One-hundred and fifty (66%) patients had a diploid karyotype, MATERIALS AND METHODS whereas 11 (5%) had no Y chromosome. Seven (3%) patients had an abnormality of chromosome 7 (monosomy 7-6 and del7q-1), The current study was approved by the Mayo Clinic institutional review board. Study eligibility criteria included availability of PB smear, BM 16 (7%) had þ 8 (9 as a single abnormality and 7 with an histology and cytogenetic information at the time of referral to the Mayo additional structural abnormality), 8 (4%) had a monosomal Clinic. The diagnoses of CMML, including subclassification into CMML-1 or karyotype and 2 (1%) patients had a complex karyotype without CMML-2, and documentation regarding the presence or absence of ring monosomies. Other cytogenetic abnormalities included del20q-5 sideroblasts and LT were according to the 2008 WHO criteria.1 All complete (2%), þ 21-3 (1%), þ 14-2 (1%), þ 11-2 (1%) and isochromosome blood count differentials and PB smears were evaluated for presence of 17q-2(1%). Eighteen patients (8%) had chromosomal transloca- circulating IMCs, defined by the presence of any of the following cells in tions that did not fit into any specific category. Fluorescence in situ 6 circulation: myeloblasts, myelocytes, metamyelocytes and promyelocytes. hybridization testing for BCR-ABL1 and PDGFRA/B was negative Karyotype risk designation and CMML risk stratification were according to in all cases. The Spanish cytogenetic risk-stratification system the Spanish cytogenetic risk-stratification system,8 the MDAPS6 and the G-MDAPS, respectively.9 identified 163 (72%) patients in the low-risk category, 31 (14%) in In the analysis of prognostic factors, variables included were age, sex, the intermediate-risk category and 32 (14%) in the high-risk hemoglobin, white blood cell count (WBC), absolute neutrophil count, category, respectively. There was no difference in outcomes in a absolute monocyte count (AMC), ALC, platelet count, PB and BM blasts, comparison between CMML patients with a monosomal karyotype circulating IMC, BM cellularity, percentage of BM ring sideroblasts, and those with a complex karyotype without monosomies. WHO morphological subcategories (CMML-1 vs CMML-2), karyotype ASXL1 mutations were detected in 87 (49%) out of 179 analyzed groups based on the Spanish cytogenetic risk-stratification system, MDAPS patients, 72% (63) being male, with a median age of 70 years and G-MDAPS prognostic risk categories, ASXL1 mutations and spliceo- (range 27–86 years). The most common ASXL1 mutation was some component mutations, involving SF3B1, SRSF2 and U2AF35 c.1934dupG;p.Gly646TrpfsX12 (n ¼ 47, 54%), followed by the (also referred to as U2AF1). At the time of CMML diagnosis, DNA from BM or PB was extracted using 1900_1922_ del seen in 10 (11%) patients. Table 2 describes conventional methods. ASXL1, SF3B1 and SRSF2 mutation analysis was the ASXL1 mutational prevalence in 179 patients with CMML. performed according to previously published methods.19–20 For detection ASXL1-mutated patients were found to have a higher WBC of U2AF35 mutations, we applied the standard PCR techniques and (P ¼ 0.009), higher AMC (P ¼ 0.008), higher prevalence of circulat- bidirectional sequencing. For U2AF35, we amplified two areas with known ing IMC (P ¼ 0.03) and were more likely to have concomitant mutations that included residues S34 and Q157. Briefly, two separate PCR mutations of the U2AF35 gene (P ¼ 0.03). There was no difference reactions were performed and the primers used were as follows: (for S34) in age (P ¼ 0.6), gender (P ¼ 0.23), hemoglobin values (P ¼ 0.07), Forward: 50-GGTGCTTAATACCACGGAAAA-30, Reverse: 50-AGTCGATCACCTG 0 0 0 platelet count (P ¼ 0.09), ALC (P ¼ 0.79), PB (P ¼ 0.5) and BM (0.27) CCTCACT-3 ; (for Q157) Forward: 5 -GCCTCGTGTGCATTCTCTG-3 , Reverse: blasts, WHO histological categories (P ¼ 0.3) and the incidence of 50-CTTTTCAGTTTCGCCGTGAG-30. PCR amplification conditions were the same for both U2AF35 reactions and included a primary denaturation of SRSF2 (P ¼ 0.57) and SF3B1 (P ¼ 0.43) mutations. Prognostic risk 95 1C for 2 min, followed by 35 cycles of: 95 1C for 30 s, 57 1C for 45 s and stratification using the Spanish cytogenetic risk-stratification 72 1C for 45 s, and ended with a final extension of 72 1C for 5 min. Products system (P ¼ 0.17), MDAPS (P ¼ 0.2) and the G-MDAPS (0.32) were purified before sequencing. demonstrated no difference between mutated and unmutated All statistical analyses considered clinical and laboratory parameters patients. Thirty-seven (42%), 13 (14%) and 2 (2%) patients had obtained at the time of referral to the Mayo Clinic, which in most instances concomitant ASXL1 and SRSF2, U2AF35 and SF3B1 mutations, coincided with time of BM biopsy at the Mayo Clinic and study sample respectively. Because of the question on whether c.1934dupG;p.- collection. Differences in the distribution of continuous variables between Gly646TrpfsX12 is truly an ASXL1 gene mutation vs a somatic categories were analyzed by either Mann–Whitney (for comparison of two alteration,12 we reanalyzed the entire cohort, excluding the 47 groups) or Kruskal–Wallis (comparison of three or more groups) test. Patient groups with nominal variables were compared by w2-test. OS was patients with this mutation and found no difference in our results calculated from the date of first referral to date of death (uncensored) or (Supplementary figure one). Sixteen ASXL1 mutations were last contact (censored). Leukemia-free survival was calculated from the novel and have not been reported to date (Table 2). date of first referral to the date of LT (uncensored) or death/last contact ASXL1 mutations had no effect on the OS (P ¼ 0.37) or leukemia (censored). Overall and leukemia-free survival curves were prepared by the free survival (0.09) (Table 3 and Figure 1).

& 2013 Macmillan Publishers Limited Leukemia (2013) 1504 – 1510 Prognosis in chronic myelomonocytic leukemia MM Patnaik et al 1506 Table 1. Clinical and laboratory features and subsequent events in 226 patients with World Health Organization deﬁned chronic myelomonocytic leukemia

Variable All patients with Patients with Patients with P-value CMML-1 CMML (n ¼ 226) CMML-1 (n ¼ 191) CMML-2 (n ¼ 35) vs CMML-2

Age in years; median (range) 71 (20–90) 71 (20–88) 70 (20–90) 0.57 Males; n (%) 152 (67) 135 (71) 17 (49) 0.01 Hemoglobin g/dl; median (range) 10.4 (6.4–15.6) 10.5 (6.4–15.6) 10 (6.4–14.4) 0.04 WBC Â 109/l; median (range) 12.4 (1.3–302) 12 (1.3–302) 19.5 (2.1–133.5) 0.15 ANC Â 109/l; median (range) 5.8 (0.1–143) 5.7 (0.1–143) 6.3 (0.2–114) 0.2 AMC Â 109/l; median (range) 2.7 (1–40) 2.5 (1–38) 5.1 (1–40) 0.03 ALC Â 109/l; median (range) 1.6 (0–22) 1.6 (0–22) 2.2 (0.7–10) 0.14 Platelets Â 109/l; median (range) 92.5 (8–1100) 98 (8–1100) 48 (17–325) 0.0007 Presence of circulating immature myeloid cells; n (%) 110 (49) 80 (42) 30 (86) o0.0001 PB blast %; median (range) 0 (0–19) 0 (0–4) 1 (0–19) o0.0001 BM blast %; median (range) 4 (0–19) 3 (0–9) 13 (10–19) o0.0001 BM cellularity %; median (range) 70 (30–100) 70 (30–95) 70 (30–100) 0.56

Spliceosome mutational analysis; n (%) SF3B1 13 (6) 10 (5) 3 (9) 0.44 SRSF2 90 (40) 77 (40) 13 (37) 0.69 U2AF35 20 (9) 19 (10) 1 (3) 0.17

*ASXL1 mutational status; n (%) 87 (49) 70 (47) 17 (58) 0.31

Spanish cytogenetic risk stratiﬁcation; n (%) Low 163 (72) 150 (79) 13 (37) o0.0001 Intermediate 31 (14) 22 (12) 9 (26) High 32 (14) 19 (10) 13 (37)

MD Anderson prognostic risk categories; n (%) Low 104 (46) 104 (54) 0 (0) o0.0001 Intermediate 1 65 (29) 57 (30) 8 (23) Intermediate 2 47 (21) 30 (16) 17 (49) High 10 (4) 0 (0) 10 (29)

Global MD Anderson prognostic risk categories; n (%) Low 43 (19) 38 (88) 5 (12) 0.0008 Intermediate 1 84 (37) 78 (93) 6 (7) Intermediate 2 51 (23) 43 (84) 8 (16) High 48 (21) 32 (67) 16 (33)

Leukemic transformations; n (%) 32 (14) 19 (10) 12 (34) 0.0001 Deaths; n (%) 176 (78) 137 (72) 31 (89) 0.39 Abbreviations: ALC, absolute lymphocyte count; AMC, absolute monocyte count; ANC, absolute neutrophil count; ASXL1, additional sex combs 1 gene; BM, bone marrow; CMML, chronic myelomonocytic leukemia; PB, peripheral blood; SF3B1, splicing factor 3B subunit 1; SRSF2, serine/arginine-rich splicing factor 2; U2AF35, U2 small-nuclear RNA auxiliary factor 1; WBC, white blood cell count. *ASXL1 gene sequencing was limited to 179 patients, 150 with CMML-1 and 29 with CMML-2, because of limited availability of DNA.

Ninety patients (40%) had SRSF2 mutations, 13 (6%) had SF3B1 (P ¼ 0.01), WHO morphological categories (P ¼ 0.02), MDAPS mutations and 20 (9%) had U2AF35 mutations. One-hundred and (Po0.0001), G-MDAPS (P ¼ 0.01) and the Spanish cytogenetic twenty-three (54%) patients had at least one of the three risk-stratification system (P ¼ 0.04) (Table 3). As mentioned above, spliceosome mutations. Notably, all three spliceosome mutations ASXL1 and spliceosome component mutations had no impact on were mutually exclusive. There was no statistically significant OS. On multivariable analysis that included the aforementioned difference, among the three mutation groups (SRSF2 vs SF3B1 vs variables, only increased AMC (410 Â 109/l, relative risk (RR) 2.5, U2AF35), in prognostically relevant parameters.1 The only notable 95% confidence interval (CI) 1.7–3.8), presence of circulating IMC difference was that patients with the SF3B1 mutations had a higher (RR 2.0, 95% CI 1.4–2.7), decreased hemoglobin (o10 g/dl, RR percentage of BM ring sideroblast (Po0.0001), lower median WBC 1.6, 99% CI 1.2–2.2) and decreased platelet count (o100 Â 109/l, (P ¼ 0.04) and a lower ALC (P ¼ 0.045). In univariate analysis, the RR 1.4, 99% CI 1.0–1.9) retained significance. Using these four presence of SRSF2, SF3B1 or U2AF35 mutations had no prognostic independent risk factors, we prepared a new prognostic risk impact on OS or leukemia free survival (Table 3 and Figure 1).1 model consisting of three risk categories: low risk (0 risk factors), At a median follow-up of 15 months, considering all 226 intermediate risk (one risk factor) and high risk (two or more risk patients, 176 (78%) deaths and 32 (14%) LTs were documented. factors), with median survivals of 32, 18.5 and 10 months, Median survivals were 22 months for CMML-1 and 14 months for respectively (Figure 2). The Mayo prognostic model performed CMML-2. On a univariate analysis, factors adversely influencing OS better than the MDAPS, G-MDAPS and the Spanish cytogenetic included high WBC (Po0.0001), high absolute neutrophil count risk models (Figure 2). This model was also predictive of LT: high (P ¼ 0.0005), high ALC (P ¼ 0.005), high AMC (Po0.0001), low risk, RR 4.9 (95% CI 1.9–12.8) and intermediate risk, RR 2.6 (1.1–5.9). platelet count (P ¼ 0.012), presence of circulating IMC (Po0.0001), Individual parameters of independent significance for LT included PB blasts (P ¼ 0.002), BM blasts (P ¼ 0.005), BM cellularity PB blast count and AMC 410 Â 109/l. The Mayo prognostic model

Leukemia (2013) 1504 – 1510 & 2013 Macmillan Publishers Limited Prognosis in chronic myelomonocytic leukemia MM Patnaik et al 1507 Table 2. Spectrum of ASXL1 mutations in 179 patients with WHO-deﬁned chronic myelomonocytic leukemia

ASXL1 gene ASXL1-mutation base-pair Number of Previously Mutation region affected substitution followed by patients (%) described ID (COSM) (exon number) amino-acid change

1 1782C4A;C594X 1 (1%) Yes 110708 1 1900-1922_del 10 (11%) Yes 219102 1 1900 A4T;R634X 1 (1%) Yes 1012900 1 1934_insG 47 (54%) Yes 34210 1 1972 G4T;G658X 1 (1%) No 1 2059_delG 1 (1%) No 1 2077C4T;R693X 2 (4%) Yes 51388 1 2161_delC 1 (1%) No 2 2209_insCA 1 (1%) No 2 2291_insC 1 (1%) No 2 2310-2311_delAG 1 (1%) No 2 2355_delT 1 (1%) No 2 2433_delT 1 (1%) No 2 2512 A4T;K838X COSM145115 1 (1%) Yes 145115 2 2577_delC 1 (1%) No 2 2580-2581_delAG 1 (1%) No 2 2601_delG 1 (1%) No 3 2893C4T;R965X COSM267971 1 (1%) Yes 267971 3 2989_insT 1 (1%) No 3 3202C4T;R1068X COSM41715 2 (4%) Yes 41715 4 3306 G4T;E1102D rs139115934 COSM36205 MAF:0.01 2 (4%) Yes 36205 5 3935C4T;A1312V 1 (1%) Yes 97046 5 3947 G4A; R1316H 1 (1%) No 5 3965C4G;P1322G COSM36204 rs141930107 1 (1%) Yes 36204 5 4128_insG 1 (1%) Yes 53215 6 4189 G4A; G1397S rs146464648 COSM133033 1 (1%) Yes 133033 6 4285 T4G;S1429A 1 (1%) No 6 4304 G4C;S1435I 1 (1%) No 6 4382C4T;P1461L 1 (1%) No

Table 3. Univariate and multivariable overall and leukemia-free survival analysis for 226 patients with chronic myelomonocytic leukemia

Variables Overall survival Leukemia-free survival

Univariate Multivariable Univariate Multivariable analysis P-value analysis P-value analysis P-value analysis P-value

Age 0.89 0.07 Sex 0.92 0.10 Hemoglobin level 0.003 0.0002 0.15 White blood cell count o0.0001 NS 0.008 NS Absolute neutrophil count 0.0005 NS 0.02 NS Absolute lymphocyte count 0.005 NS 0.0008 NS Absolute monocyte count o0.0001 0.0003 0.01 0.01 Platelet count 0.012 0.0065 0.03 NS Circulating immature myeloid cells o0.0001 0.0002 0.003 NS Peripheral blood blast % 0.002 NS 0.0001 0.003 Bone marrow blast % 0.005 NS 0.0002 NS Bone marrow cellularity % 0.01 NS 0.50 Bone marrow rings % 0.36 NS 0.40 SRSF2 mutational status 0.67 0.55 SF3B1 mutation status 0.80 0.91 U2AF35 mutation status 0.49 0.37 ASXL1-mutation status 0.37 0.73 MD Anderson prognostic scoring system o0.0001 NS 0.0004 0.0094 Global MD Anderson prognostic scoring system 0.01 NS 0.34 Spanish cytogenetic risk categories 0.04 NS 0.03 NS WHO morphological categories (CMML-1 vs CMML-2) 0.02 NS o0.0001 NS Abbreviations: ASXL1, additional sex combs 1 gene; CMML, chronic myelomonocytic leukemia; SF3B1, splicing factor 3B subunit 1; SRSF2, serine/arginine-rich splicing factor 2; U2AF35, U2 small-nuclear RNA auxiliary factor 1; WHO, World Health Organization. was then validated in an independent cohort of 268 patients with (69%) were male and 227 (85%) patients met the 2008 WHO WHO-defined CMML at the H. Lee Moffitt Cancer Center in Tampa, criteria for CMML-1. The three risk groups had median survivals of Florida (Supplementary Table 1 and Supplementary figure two). 66 (low risk), 36 (intermediate risk) and 18 (high risk) months, The median age of this cohort was 70 years (range 30–90); 185 respectively (Po0.0005).

& 2013 Macmillan Publishers Limited Leukemia (2013) 1504 – 1510 Prognosis in chronic myelomonocytic leukemia MM Patnaik et al 1508 Over-all survival of 224 CMML patients stratified Over-all survival of 196 CMML patients stratified by SRSF2 mutational status. by ASXL1 mutational status. 1 CMML patients with SRSF2 mutations, CMML patients without ASXL1 mutations, 1 n=90, median survival 24 months n=92, median survival 22 months 0.8 CMML patients without SRSF2 mutations, CMML patients with ASXL1 mutations, 0.8 n=134, median survival 16 months n=87, median survival 20 months 0.6 0.6

Survival 0.4 P=0.33

Survival 0.4 P=0.6 0.2 0.2

0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Months Months

Over-all survival of 212 CMML patients stratified Over-all survival of 222 CMML patients stratified by SF3B1 mutational status. by U2AF35 mutational status.

CMML patients with SF3B1 mutations, CMML patients with U2AF35 mutations, 1 n=13 , median survival 17 months 1 n=20 , median survival 12 months CMML patients without SF3B1 mutations, CMML patients without U2AF35 mutations, 0.8 n=199, median survival 20 months 0.8 n=202 , median survival 20 months

0.6 0.6

0.4 0.4 P=0.48 Survival Survival P=0.8

0.2 0.2

0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Months Months

Figure 1. Impact on OS of SRSF2, ASXL1, SF3B1 and V2AK35 mutations in patients with CMML.

Mayo Clinic Risk Stratification Model for CMML Global MD Anderson Prognostic System for CMML

1 Low risk, n= 43; median survival 33 months. 1 Low risk , n = 115; median survival P<0.0001 0.8 Intermediate-1 risk, n= 84; median 32 months. survival 19 months. 0.8 Intermediate risk , n= 58; median Intermediate-2 risk, n= 51; survival 18.5 months. 0.6 median survival 17 months. High risk, n=54; median survival 10 High risk, n= 48; median survival 0.6 months. 11 months. 0.4

Survival 0.4 P=0.02

0.2 0.2

0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Months

MD Anderson Prognostic System for CMML Spanish stratification based on karyotypic abnormalities

1 Low risk, n= 105, median survival P<0.0001 33 months. Intermediate-1 risk, n= 65, median 1 0.8 survival 17.5 months Low risk, n= 159 , median survival 22 months. Intermediate-2 risk, n= 47, median 0.8 0.6 survival 11 months Intermediate risk, n= 31, median survival 20 months High risk, n = 10; median survival 0.6 10 months High risk, n = 32; median survival 0.4

Survival 8 months

Survival 0.4 P=0.04 0.2 0.2

0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Months Months Figure 2. Comparison of the Mayo prognostic model for CMML, with the MD Anderson model, Global MD Anderson model and the Spanish cytogenetic risk-stratiﬁcation system.

DISCUSSION French–American–British classification of CMML (Bournemouth, The presence of overlapping features between MDS and Modified Bournemouth and Lille) or are not primarily designed 3–5,21 myeloproliferative neoplasm has given rise to ambiguity with for CMML (IPSS, revised IPSS and G-MDAPS). The commonly regard to diagnosis and prognostication of patients with CMML. used MDAPS was developed on a cohort of 213 CMML patients A number of prognostic systems are either based on the old and identified four risk groups based on the hemoglobin level,

Leukemia (2013) 1504 – 1510 & 2013 Macmillan Publishers Limited Prognosis in chronic myelomonocytic leukemia MM Patnaik et al 1509 presence of IMC, ALC and BM blasts.6 The adverse prognostic consisting of three risk categories: low risk (0 risk factors), value conferred by these factors was validated by the Dusseldorf intermediate risk (one risk factor) and high risk (two or more risk model, with the exception of circulating IMC.7 In addition, factors), with median survivals of 32, 18.5 and 10 months, the Dusseldorf scoring system identified elevated lactate respectively (Figure 2). This model was validated in an indepen- dehydrogenase and male gender as negative prognosticators dent cohort of 268 CMML patients at the H. Lee Moffitt Cancer for survival.7 In general, high ALC have been associated Center, in Tampa, Florida. In our cohort of CMML patients, the with favorable outcomes in hematological malignancies, and its Mayo prognostic model outperformed the MDAPS, G-MDAPS and inclusion as an adverse prognosticator in the MDAPS is the Spanish cytogenetic risk models. perplexing.10,22 In our study, high ALC, although prognostic in a univariate analysis, lost its prognostic significance on a multi variable analysis. In addition, contrary to the aforementioned models, we found that a high AMC was independently associated CONFLICT OF INTEREST with a shortened OS. The G-MDAPS was developed for patients The authors declare no conflict of interest. with de novo MDS, secondary MDS and CMML and identified older age, poor performance status, thrombocytopenia, anemia, increased BM blasts, leukocytosis, chromosome 7 or complex ACKNOWLEDGEMENTS cytogenetic abnormalities and transfusion dependence as 9 Current study is supported in part by grants from the ‘Myeloproliferative Disorders independent factors associated with a shorter OS. In our study, Foundation, Chicago, IL, USA’ and ‘The Henry J. Predolin Foundation for Research in although the G-MDAPS stratified patients into four risk categories, Leukemia, Mayo Clinic, Rochester, MN, USA’. the Mayo prognostic model was more effective and was able to risk stratify within each of the G-MDAPS risk groups. 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