Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients.

The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.

Quantitative assessment of JAK2 V617F and CALR mutations in Philadelphia negative myeloproliferative neoplasms.

BACKGROUND: Philadelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. PATIENTS AND METHODS: Quantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. RESULTS: Twelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). CONCLUSION: Our study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.

Assessment of rotation thromboelastometry parameters in patients with essential thrombocythemia at diagnosis and after hydroxyurea therapy.

Patients with essential thrombocythemia suffer from thrombotic complications that are the main source of mortality. Due to its complex pathogenesis, no existing single laboratory method is able to identify the patients at highest risk for developing thrombosis. Twenty patients with essential thrombocythemia at diagnosis, 15 healthy volunteers and 20 patients treated with hydroxyurea were compared with regard to certain rotation thromboelastometry parameters. Clotting time (CT), clot formation time (CFT), α-angle, and maximum clot firmness (MCF) were assessed by using the INTEM, EXTEM, FIBTEM, and NATEM tests. Patients with essential thrombocythemia at diagnosis demonstrated significantly higher mean platelet count and markedly lower mean red blood count than controls. CT and CFT readings were found to be markedly lower in essential thrombocythemia patients at diagnosis than in the control group according to the EXTEM test. Patients at diagnosis had markedly lower CT values (EXTEM, FIBTEM) than patients on hydroxyurea therapy. Alpha angle values were markedly higher in essential thrombocythemia patients at diagnosis than in controls, according to the EXTEM, FIBTEM and NATEM tests. MCF readings were significantly higher in essential thrombocythemia patients at diagnosis than in controls according to EXTEM, INTEM, FIBTEM, and NATEM tests. Patients on hydroxyurea therapy had markedly lower MCF values according to EXTEM test than patients at diagnosis. Patients with essential thrombocythemia demonstrate a prothrombotic state at the time of diagnosis, which is reflected in changes by certain rotation thromboelastometry parameters. The hydroxyurea therapy induces downregulation of the prothrombotic features seen in essential thrombocythemia patients at diagnosis.

It is time to change thrombosis risk assessment for PV and ET?

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms to be diagnosed according to the WHO classification. Molecular profiling must include the analysis of JAK2 (looking for the V617F point-mutation in PV and ET, screening exon 12 for mutations only in V617F-negative PV), CALR and MPL mutations (both in V617F-negative ET). The current risk stratification to predict thrombosis requires two parameters: age over 60 years and prior history of thrombosis. On the basis of these two risk factors patients can be stratified in low-risk and high-risk and receive a proper treatment. However, a modern stratification of thrombotic risk might consider "new" low-risk patients: conventional low-risk plus absence of leukocytosis from diagnosis onwards and a hematocrit level below 45% during the course of disease for PV; conventional low-risk plus absence of leukocytosis from diagnosis onwards, JAK2 negativity, CALR positivity, and absence of cardiovascular risk factors for ET.

Clinical significance of clonality assessment in JAK2V617F-negative essential thrombocythemia.

JAK2V617F-negative essential thrombocythemia (ET) is a heterogeneous disease including clonal cases and others without evidence of clonality. However, it is unknown if the detection of myeloid clonality in JAK2V617F-negative ET patients confers a different clinical outcome than those in whom clonal hematopoiesis cannot be demonstrated. The objective of the present study was to evaluate the clinical significance of clonality assessment in patients with JAK2V617F-negative ET. Clonality investigation including mutational status of MPL, TET2, and ASXL1 genes and human androgen receptor (HUMARA) assay was performed in 73 JAK2V617F-negative cases out of 186 subjects consecutively diagnosed with ET in a single institution, at diagnosis or during follow-up. Mutations in MPL, TET2, and ASXL1 were observed in 7, 4, and 2 cases, respectively, whereas clonality by HUMARA assay was demonstrated in 21 out of 46 (46 %) female patients. With a median follow-up of 8 years, death, thrombosis, bleeding, and disease transformation were registered in 7, 10, 8, and 6 patients, respectively. No differences in thrombosis, bleeding or survival were observed according to clonality assessment. The probability of disease transformation at 10 years was higher in patients showing clonal hematopoiesis by presenting mutations in either MPL, TET2, or ASXL1 (64 versus 2 % in patients without mutations, p < 0.001) and in those with HUMARA clonality (35 versus 0 % in patients with polyclonal hematopoiesis, p < 0.004). In conclusion, disease transformation is associated with evidence of clonality in JAK2V617F-negative ET.

Assessment of spleen size using gamma camera scintigraphy in newly diagnosed patients with essential thrombocythaemia and polycythaemia vera.

By using gamma camera imaging the spleen size was assessed in 18 consecutive patients with essential thrombocythaemia (ET) and in 18 consecutive patients with polycythaemia vera (PV). All ET and PV patients were newly diagnosed and had not received any myelosuppressive therapy prior to study. The spleen areas in both posterior and left lateral projections were determined. Eighteen consecutive patients with idiopathic thrombocytopenic purpura (ITP) served as a control group since by definition they do not present with splenic enlargement; in these latter subjects the mean posterior and left lateral splenic areas were almost identical (48 +/- 15 and 47 +/- 17 cm2, respectively). In comparison with this control group patients with ET and PC had significantly larger spleens. In both ET and in PV patients the left lateral spleen scan area exceeded the posterior one. Patients with PV had larger splenic areas in both projections than did patients with ET, but the differences were not statistically significant. Compared to the ITP patients it was found that at least 50% of the ET patients and at least 61% of the PV patients at diagnosis presented with splenomegaly.