Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis.

Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.

Extramedullary blastic transformation of primary myelofibrosis in the form of disseminated myeloid sarcoma: a case report and review of the literature.

Splenomegaly is a key clinical manifestation of myelofibrosis, and splenectomy is currently indicated in patients with drug refractory, symptomatic splenomegaly or with the aim of improving refractory cytopenias. Transformation to acute myeloid leukemia occurs in up to 20% of patients with myelofibrosis, while cases of myeloid sarcoma have been reported very unfrequently. In this manuscript, we report the case of a 60-year-old man with a history of primary myelofibrosis who underwent splenectomy because of drug-refractory massive splenomegaly, systemic symptoms and anemia. At the opening of the peritoneal cavity, the spleen resulted massively enlarged and tenaciously entrapped by a pervasive neoplastic-like tissue. The extensive involvement of the abdomen fatally complicated the surgical procedure. At postmortem examination, the spleen showed a diffuse infiltration of immature cells that were also found in the peritoneum, bowel, liver, lungs and myocardium. After immunohistochemical, cytogenetic, flow cytometric and molecular characterization of neoplastic population, a diagnosis of disseminated myeloid sarcoma of the spleen was made. This case report highlights a very unusual case of myeloid sarcoma originating from the spleen in a patient with myelofibrosis who had no evidence of bone marrow or peripheral blood involvement by leukemic cells. Molecular characterization showed that leukemic cells originated from the founding clone of the chronic phase. The sarcoma could not be suspected based on clinical findings and was diagnosed only at the time of surgical procedure and autopsy. This case suggests that leukemic transformation of myelofibrosis can originate outside the bone marrow and, presumably rarely, present as a granulocytic sarcoma.

Mutation-enhanced international prognostic systems for essential thrombocythaemia and polycythaemia vera.

Survival prediction in essential thrombocythaemia (ET) and polycythaemia vera (PV) is currently based on clinically-derived variables; we examined the possibility of integrating genetic information for predicting survival. To this end, 906 molecularly-annotated patients (416 Mayo Clinic; 490 University of Florence, Italy), including 502 ET and 404 PV, were recruited. Multivariable analysis identified spliceosome mutations to adversely affect overall (SF3B1, SRSF2 in ET and SRSF2 in PV) and myelofibrosis-free (U2AF1, SF3B1 in ET) survival; TP53 mutations predicted leukaemic transformation in ET; "adverse" mutations occurred in 51 (10%) ET and 8 (2%) PV patients. We confirmed the independent survival effect of adverse mutations [hazard ratio (HR) 2·4, 95% CI 1·6-3·5], age >60 years (6·6, 4·6-9·7), male sex (1·8, 1·3-2·4) and leukocytosis ≥11 × 109 /l (1·6, 1·1-2·2), in ET, and adverse mutations (7·8, 3·1-17·0), age >67 years (5·4, 3·6-8·1), leukocytosis ≥15 × 109 /l (2·8, 1·8-4·2) and thrombosis history (2·0, 1·4-2·9), in PV. HR-based risk point allocation allowed development of three-tiered mutation-enhanced international prognostic systems (MIPSS) which were validated in both cohorts and performance was shown to be superior to conventional scoring systems. Spliceosome mutations enhance survival prediction in ET and PV and identify patients at risk for fibrotic progression. TP53 mutations predict leukaemic transformation in ET.

Myelodysplasia as assessed by multiparameter flow cytometry refines prognostic stratification provided by genotypic risk in systemic mastocytosis.

Systemic mastocytosis (SM) is characterized by extreme heterogeneity of manifestations and prognosis. Several disease-related biomarkers, including clinical, hematological and molecular variables, have been correlated with prognosis. Although relevant, the mutation profile closely reflects the WHO classification that has per se prognostic value. High-risk mutations (HRM) are largely confined to advanced forms, and thus fail in providing information regarding progression and outcome in the not-advanced variants. In this work, we studied hematopoietic cells by multi-parameter flow cytometry (MFC) in order to highlight dysplastic traits that might provide insights into outcome. A score previously validated for myelodysplastic syndromes, with high reproducibility in standard diagnostics, was used. The application of an MFC score to a cohort of 71 SM cases, concurrently genotyped for configuring a HRM category, resulted in the identification of two separate patients' categories (MFC+ and MFC-) characterized by significantly different clinical and laboratory features at presentation. The extent of dysplasia by MFC tended to parallel WHO-category and genotype-related stratification. MFC+ patients had shorter survival compared to MFC- ones, for whom the incidence of progression and/or death was virtually null. Of note, MFC score remained prognostically informative in unadvanced subsets. Furthermore, the integration of MFC and HRM was an independent predictor for outcome, also overcoming WHO-categories in multivariate analysis for EFS. Our results support the use of MFC analysis in the evaluation of patients with SM, alone and in combination with HRM, for refinement of prognosis assessment.

A case report of systemic mastocytosis associated with multiple hematologic non-mast cell lineage diseases.

Systemic mastocytosis (SM) is a hematological malignancy characterized by extracutaneous infiltration by atypical mast cells. Together with indolent SM, aggressive SM, and mast cell leukemia, the World Health Organization (WHO) recognizes another major disease subgroup: SM with an associated hematological neoplasm, which is characterized by the presence of a concurrent neoplasm, more commonly, a chronic myelomonocytic leukemia. While KIT D816V is commonly regarded as the driver mutation, the clinical presentation of SM is extremely varied. Treatment of SM might not be simple, but now more specific therapies tailored toward prognostic subgroups of patients have been developed. Here, we report a detailed description of clinical management and biological features of a systemic mastocytocis case associated with multiple hematologic non-mast cell lineage diseases.

Mutation landscape in patients with myelofibrosis receiving ruxolitinib or hydroxyurea.

Refractoriness to ruxolitinib in patients with myelofibrosis (MF) was associated with clonal evolution; however, whether genetic instability is promoted by ruxolitinib remains unsettled. We evaluated the mutation landscape in 71 MF patients receiving ruxolitinib (n = 46) and hydroxyurea (n = 25) and correlated with response. A spleen volume response (SVR) was obtained in 57% and 12%, respectively. Highly heterogenous patterns of mutation acquisition/loss and/or changes of variant allele frequency (VAF) were observed in the 2 patient groups without remarkable differences. In patients receiving ruxolitinib, driver mutation type and high-molecular risk profile (HMR) at baseline did not impact on response rate, while HMR and sole ASXL1 mutations predicted for SVR loss at 3 years. In patients with SVR, a decrease of ≥ 20% of JAK2V617F VAF predicted for SVR duration. VAF increase of non-driver mutations and clonal progression at follow-up correlated with SVR loss and treatment discontinuation, and clonal progression also predicted for shorter survival. These data indicate that (i) ruxolitinib does not appreciably promote clonal evolution compared with hydroxyurea, (ii) VAF increase of pre-existing and/or (ii) acquisition of new mutations while on treatment correlated with higher rate of discontinuation and/or death, and (iv) reduction of JAK2V617F VAF associated with SVR duration.

Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

MIPSS70: Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients With Primary Myelofibrosis.

Purpose To develop a prognostic system for transplantation-age patients with primary myelofibrosis (PMF) that integrates clinical, cytogenetic, and mutation data. Patients and Methods The study included 805 patients with PMF age ≤ 70 years recruited from multiple Italian centers and the Mayo Clinic (Rochester, MN), forming two independent learning and validation cohorts. A Cox multivariable model was used to select from among a list of 22 variables those that were predictive of overall survival (OS). Integrated clinical and genetic prognostic models with (MIPSS70-plus) or without (MIPSS70) cytogenetic information were developed. Results Multivariable analysis identified the following as significant risk factors for OS: hemoglobin < 100 g/L, leukocytes > 25 × 109/L, platelets < 100 × 109/L, circulating blasts ≥ 2%, bone marrow fibrosis grade ≥ 2, constitutional symptoms, absence of CALR type-1 mutation, presence of high-molecular risk mutation (ie, ASXL1, EZH2, SRSF2, IDH1/ 2), and presence of two or more high-molecular risk mutations. By assigning hazard ratio (HR)-weighted points to these variables, three risk categories were delineated for the MIPSS70 model; 5-year OS was 95% in low-risk, 70% in intermediate-risk, and 29% in high-risk categories, corresponding to median OS of 27.7 years (95% CI, 22 to 34 years), 7.1 years (95% CI, 6.2 to 8.1 years), and 2.3 years (95% CI, 1.9 to 2.7 years), respectively. In the MIPSS70-plus model, which included cytogenetic information, four risk categories were delineated, with 5-year OS of 91% in low-risk, 66% in intermediate-risk (HR, 3.2; 95% CI, 1.9 to 5.2), 42% in high-risk (HR, 6.4; 95% CI, 4.1 to 10.0), and 7% very high-risk categories (HR, 17.0; 95% CI, 9.8 to 29.2). Both models remained effective after inclusion of older patients in the analysis. Conclusion MIPSS70 and MIPSS70-plus provide complementary systems of risk stratification for transplantation-age patients with PMF and integrate prognostically relevant clinical, cytogenetic, and mutation data.

Presentation and outcome of patients with 2016 WHO diagnosis of prefibrotic and overt primary myelofibrosis.

The 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1, SRSF2, IDH1/2, EZH2) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this "real-life" study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum.

Prognostic impact of bone marrow fibrosis in primary myelofibrosis. A study of the AGIMM group on 490 patients.

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow-up information. We found that fibrosis grade 2 and greater on a 0-3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918-922, 2016. © 2016 Wiley Periodicals, Inc.

Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group.

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 incidence and morphology of Monckeberg's medial calcification in banked vascular segments from a monocentric donor population.

Little is known about the real incidence and the clinical relevance of the enigmatic Monckeberg's medial calcification in the patency of the femoral artery allograft. Here we present a retrospective study on 143 multiorgan donors (mean age 38 years, range 14-59 years), to describe the incidence and the morphological features of vascular calcifications in banked femoral arteries suitable for clinical use. In the present series, focal vascular calcifications were present in 36 (25 %) cases, 23 cases localized in the intima, 7 in the media, and 6 were mixed. No correlation was found between the incidence of calcifications and the classical cardiovascular clinical risk factors (n = 9); only hypertension correlated with the medial localization, but not with the incidence, of the calcification (P = 0.017). While the macroscopic exclusion criteria of vascular grafts include atheromatous and not-atheromatous lesions, we ignore the actual impact of Monckeberg's medial calcification on vessel transplantation and allograft life. In our opinion this is a very important topic, since when the histological criteria for Monckeberg's calcification diagnosis are used, 25 % of our young donors population was affected. Whether Monckeberg's medial calcification is a stable arterial condition, apparently underestimated in the general population, or a dynamic process evolving with age and atherosclerosis, or a banking-related vascular alteration, still remain an open issue deserving further studies with subjects of different ages.

Targeted deep sequencing in polycythemia vera and essential thrombocythemia.

Polycythemia vera (PV) is characterized by JAK2 and essential thrombocythemia (ET) by JAK2, calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) mutations; we describe the occurrence and prognostic relevance of DNA sequence variants/mutations other than JAK2/CALR/MPL. A myeloid neoplasm-relevant 27-gene panel was used for next-generation sequencing of bone marrow or whole blood DNA and conventional tools were used for analysis. "Adverse variants/mutations" were identified by age-adjusted multivariable analysis of impact on overall, leukemia-free, or myelofibrosis-free survival. Fifty-three percent of 133 Mayo Clinic patients with PV and 53% of 183 with ET harbored 1 or more sequence variants/mutations other than JAK2/CALR/MPL; the most frequent were TET2 and ASXL1. "Adverse variants/mutations" in PV included ASXL1, SRSF2, and IDH2 and in ET SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2; combined prevalence was 15% and 15%, respectively. Adverse variants/mutations were associated with inferior survival in both PV (median, 7.7 vs 16.9 years) and ET (median, 9 vs 22 years) and the effect was independent of conventional prognostic models with respective hazard ratio (95% confidence interval) of 2.8 (1.5-5.1) and 2.6 (1.4-4.8); these observations were validated in 215 Italian patients with PV and 174 with ET. In both Mayo Clinic and Italian cohorts, leukemic or fibrotic progression was also predicted by adverse variants/mutations. Number of mutations did not provide additional prognostic information. We conclude that targeted deep sequencing in PV and ET allows for genetic risk stratification that is independent of clinically derived prognostic models.