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TO THE EDITOR: The discovery of prognostically informative mutations in patients with primary myelofibrosis (PMF)[1][1] prompted the development of mutation-enhanced risk scores.[2][2][⇓][3][⇓][4][⇓][5]-[6][6] Among these mutations, those in ASXL1 are invariably associated with adverse

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.

The discovery of mutations in calreticulin (CALR) in patients with primary myelofibrosis (PMF)1, 2 prompted a reappraisal of the clinical correlates and prognostic impact of the so-called driver mutations that include JAK2V617F, MPLW515L/K/A and CALR in ~60%, 5–10% and 20–25% of patients, respectively. As compared with their JAK2V617F counterpart, PMF patients harboring CALR mutations showed younger age, higher platelet and lower hemoglobin and leukocyte counts. The cumulative incidence of anemia, leukocytosis and thrombocytopenia was significantly lower in CALR-mutated patients who were also less likely to be red cell transfusion-dependent;3, 4 in addition, they had significantly longer large splenomegaly-free survival compared with the other genotypes as well as patients lacking the three driver mutations (triple-negative (TN) patients).3, 4 Interestingly, spliceosome mutations were significantly less represented in CALR-mutated patients; however, no additional molecular or cytogenetic correlate was highlighted.3 These data suggested a milder disease in patients harboring the CALR mutation, and conceivably the presence of CALR mutation was associated with better overall survival (OS) when compared with JAK2V617F- and MPLW515-mutated patients, and particularly TN patients.3, 4 In multivariable analysis, CALR mutations had a favorable impact on survival that was independent of International Prognostic Scoring System (IPSS), Dinamic IPSS (DIPSS)4 or DIPSS-plus risk stratification,3 and also of ASXL1 mutation, known for its dismal impact on survival in PMF.5 At this regard, we found that DIPSS-plus-independent OS was significantly longer in CALR-mutated/ASXL1-unmutated compared with CALR-unmutated/ASXL1-mutated patients.6

Background: We reported that mutations in ASXL1, EZH2, IDH1/2 and SRSF2 are negative prognostic variables for survival in primary myelofibrosis (PMF). Patients harboring any one of these mutations comprise IPSS and DIPSS-plus independent high molecular risk (HMR) category. Conversely, prognostic variables in secondary myelofibrosis (sMF), including post-polycythemia vera (PPV) and post-essential thrombocythemia (PET) MF are not defined. Aims: The aim was to evaluate the correlations of HMR mutational status with hematologic characteristics and clinical presentation, and the role for outcome predition, in PPV- and PET-MF. Methods: PPV- and PET-MF were diagnosed by IWG-MRT criteria; all pts provided informed consent. Previously published methods were used to screen mutations involving JAK2,MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results: A series of 200 sMF pts from 3 Italian centres was collected: 108 were PPV-MF (54%), 92 PET-MF (46%). PPV-MF cohort: Median age was 65y. Median follow up from PPV-MF diagnosis 4.7y (0.2-25.9y) and median time from PV to PPV-MF 10y (1.08-30.7y). Death occurred in 34 pts (31.8%), 7 pts (6.5%) developed leukemia. Median OS from PPV-MF diagnosis was 9.5y (7.1-11.9y). Frequency of mutations was: JAK2V617F 100% (median allele burden 77%, range 23-100), ASXL1 17.8%, EZH2 3.7%, IDH 5.6%, SRSF2 1%; 29 patients (26.9%) were classified as HMR cases. Hematologic characteristics: median leucocytes 13.0x109/L, hemoglobin 13.3g/dL, platelets 328x109/L, blasts ≥1% 23.4%. Constitutional symptoms in 45.8%, splenomegaly 94.1% (43.3% >10cm from LCM), pruritus 49%; median percentage of BM cellularity was 90% (25-100%) and grade 3 fibrosis in 16.8%. Overall 73 patients were evaluable for karyotype and abnormalities were detected in 42.5%. We did not find significant correlations between individual mutations or HMR category and hematologic and clinical characteristics. PET-MF cohort: Median age was 63.6y. Median follow up from the PET diagnosis 3.3y (0.2-14.5y), median time from ET to PET-MF 11.8y (0.9-30.6y). Death occurred in 30 pts (32.6%), 11 pts (12.0%) developed leukemia. Median OS from PET-MF diagnosis was 9.2y (4.9-13.6y). Frequency of mutations was: JAK2V617F 48.9% (median allele burden 49%, range 0-100), CALR 40.2%, MPL 4.3% and triple negativity (TN) 6.5%; ASXL1 29.3%, EZH2 6.5%, IDH 1.1%, SRSF2 3.3%; 33 pts (35.9%) were HMR. Hematologic characteristics: median leucokytes 8.0x109/L, hemoglobin 10.9 g/dL, platelets 375x109/L, blasts ≥1% 26.4%. Constitutional symptoms 35.4% of pts, splenomegaly 82% (18.2% >10cm from LCM), pruritus i31%; median percentage of BM cellularity was 70% (15-90%) and grade 3 fibrosis was found in 31.7%. Overall 56 patients were evaluable for karyotype and abnormalities were detected in 25.0%. By correlating hematologic and clinical characteristics with unique mutations and HMR category, we found that HMR mutated pts presented greater leukocytosis (P=0.04) and higher JAK2V617F allele burden (P=0.017) than pts in the low-molecular risk –LMR- category (ie, wild-type for ASXL1, EZH2, SRSF2, IDH1/2). Comparison of PPV-MF and PET-MF: a comparison of hematologic and clinical characteristics according to the diagnosis sMF disclosed that PPV-MF pts presented more frequently splenomegaly (P=0.008; for >10cm from LCM P Univariate analysis disclosed significant correlations between shortened survival and mutated ASXL1 (P=0.02, HR 2.2 95% CI, 1.02-4.8) or EZH2 (P=0.05, HR 5.0 95% CI, 1.0-40.7) in PPV-MF. A HMR category was associated with reduced survival in PPV-MF: median survival 6.1y versus 9.5yr LMR (HR 1.07, 95%CI 1.0-4.4; P=0.04). In PET-MF survival was 4.8y in HMR versus 10.9y in LMR (HR1.6, 95%CI 0.8-3.4; P=0.2). Conclusion: We conclude that a HMR status is associated with shorter survival in sMF, but the overal impact is narrower than in primary MF, even though the rate of mutations is similar (Table1). Disclosures No relevant conflicts of interest to declare.

Background: Mutations in the gene calreticulin (CALR) were recently discovered in 60-80% of patients (pts) with primary myelofibrosis (PMF) and essential thrombocythemia (ET) who were un-mutated for JAK2V617F and MPLW515. CALR mutated PMF pts had better overall survival (OS) compared with JAK2V617F or MPLW515 mutated while in ET CALR mutations were associated with lower incidence of thrombosis although the effect on survival was not significant. Conversely, there is no information concerning the impact of CALRmutation on disease phenotype and prognosis in post-essential thrombocythemia myelofibrosis (PET-MF). Aims: The aim of the study was to assess whether CALR mutational status and/or allele burden had clinical and/or prognostic relevance in PET-MF compared with JAK2, MPLmutated or triple-negative (TN) pts. Methods: ET and PET-MF were diagnosed by 2008 WHO and IWG-MRT criteria respectively; all pts provided an informed consent. Genotyping for CALR, JAK2V617F and MPLW515 was performed in granulocytes using allele specific RTQ-PCR (JAK2, MPL), capillary electrophoresis and direct sequencing (CALR, MPL). The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results: A series of 147 PET-MF pts from 4 Italian centres was collected. Pts median age was 63y. Median follow up from PET diagnosis was 3.2y (0.07-18.8y) and the median time from ET to PET diagnosis was 11.6y (0.9-30.6y). Death occurred in 38 pts (26%) and 14 pts (9.5%) developed acute leukemia (AML). The median OS in the entire series calculated from PET-MF diagnosis was 10.9y (7.1-14.7y). Frequency of mutations was: CALR 16%, JAK2V617F 77%; MPLW515 4.3%; TN 2.8%. The frequency of CALR mutations in PET-MF patients was superimposable to that observed in a control group of 576 ET patients from our Institution (15.5%) and slightly lower compared with other series (20-25%). Type of CALRmutations was: 59.6% type 1, 23.1%, type 2, 17.3% others, significantly different (P=0.023) from ET: 46% type 1, 38% type 2, 16% others. Median CALR allele burden in PET-MF was 56% (20%-100%) with no significant differences in the CALR mutation subtypes (57.5% in type 1, 47.5% in type 2 and 45.0% in others); however, the median mutant allele burden of CALR-mutated PET-MF patients was significantly higher than in ET patients (33%, range 2%-52%; n=100) (P10 cm) splenomegaly (28.6% vs 14% in CALR+, 7.1 in MPL+ and 25% in TN pts; P=0.02) and constitutional symptoms (50% vs 18.8% in CALR+, 45% in MPL+ and 12.5% in TN pts; P=0.002). The interval from ET to PET-MF was significantly longer in CALR+ pts (14.5y) compared with JAK2+ (10.2y) and TN patients (11.0y; P=0.04 for both) and similar to MPL+ (14y). There was a reduced rate of death (13.5%) in CALR+ compared with JAK2+ (30.6%), MPL+ (21.4%) and TN (66.7%) pts (P=0.005), although Kaplan Meier estimates did not reach a statistically significant difference. Finally, there were less AML transformation in CALR+ pts (1.9%) compared with JAK2+ (13.9%), MPL+(7.1%) and TN (22.2%) (P=0.04). Conclusion: These results show that CALR mutation is associated with delayed transformation of ET to PET-MF, a milder disease in terms of splenomegaly and symptom burden and a reduced risk of death compared with JAK2V617F PET-MF pts and more in general with MPL mutated and TN pts.In addition, similar to findings in primary MF and unlike in ET, PET-MF is characterized by prevalence of type 1 CALR mutations. Disclosures No relevant conflicts of interest to declare.