Alberto Alvarez-Larrán

Red cell mass measurement in patients with clinically suspected diagnosis of polycythemia vera or essential thrombocythemia

The cut off for hemoglobin or hematocrit that indicates the need for an isotopic red cell mass study was investigated in 179 patients with a presumptive diagnosis of polycythemia vera or essential thrombocythemia. Hematocrit showed better diagnostic accuracy than hemoglobin. Hemoglobin over 18.5 g/dL in males or over 16.5 g/dL in females showed a high specificity indicating that red cell mass study could be avoided in such cases, but it showed low sensitivity leading to 46% false negatives. The best value of hematocrit to indicate a red cell mass study was 0.50 L/L in males (specificity 75%, sensitivity 87.5%) and 0.48 L/L in females (specificity 73%, sensitivity 94%). Lowering the hematocrit threshold to 0.48 L/L in males increased sensitivity up to 95%. A red cell mass study should be performed in patients with suspected diagnosis of essential thrombocythemia or polycythemia vera and with hematocrit between 0.48 L/L and 0.52 L/L.

WHO-histological criteria for myeloproliferative neoplasms: reproducibility, diagnostic accuracy and correlation with gene mutations and clinical outcomes

Bone marrow histology is included in the diagnostic criteria of myeloproliferative neoplasms (MPNs). However, some concerns have emerged about its reproducibility. To evaluate the diagnostic accuracy of histology and to assess its correlation with presence of mutations and clinical outcomes, two pathologists reviewed the bone marrow biopsies corresponding to 211 patients with MPN. Despite the low agreement in the evaluation of individual histopathological characteristics, the concordance among pathologists when establishing the diagnosis was good (Kappa index 0·67). The specificity of histology was 100%, 98·5% and 98% in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), respectively, whereas the sensitivity of histological diagnosis was low in PV and ET (32·5% and 54% respectively) and acceptable in PMF (75%). Thirteen out of 146 (9%) patients with clinical ET were diagnosed as prefibrotic PMF. No histological agreement or MPN otherwise unspecified was more frequently observed in JAK2 V617F‐positive ET than in CALR‐mutated cases, whereas megakaryocytic abnormalities and prefibrotic PMF were more frequently observed in CALR‐mutated ET. In conclusion, histological criteria of MPN have a limited diagnostic accuracy due to low sensitivity. Patients with JAK2 V617F‐positive MPN have a heterogeneous histology while CALR‐positive ET is associated with megakaryocyte abnormalities and prefibrotic PMF.

JAK2V617F monitoring in polycythemia vera and essential thrombocythemia: clinical usefulness for predicting myelofibrotic transformation and thrombotic events.

The JAK2V617F allele burden has been identified as a risk factor for vascular events and myelofibrotic transformation in polycythemia vera (PV) and essential thrombocythemia (ET). However, all previous studies have evaluated a single time point JAK2V617F measurement. Therefore, the frequency and the clinical significance of changes in the JAK2V617F mutant load occurring during the disease evolution remain unknown. In the present study, JAK2V617F monitoring was performed during the follow‐up of 347 patients (PV = 163, ET = 184). According to their JAK2V617F evolutionary patterns, patients were stratified as stable < 50% (n = 261), stable ≥50% (n = 52), progressive increase (n = 24) and unexplained decrease (n = 10). After a 2,453 person‐years follow‐up, a total of 59 thrombotic events, 16 major hemorrhages, and 27 cases of myelofibrotic transformations were registered. At multivariate analyses, patients with a persistently high (≥50%) or unsteady JAK2V617F load during follow‐up had an increased risk of myelofibrotic transformation (Incidence rate ratio [IRR]: 20.7, 95% CI: 6.5–65.4; P < 0.001) and a trend for a higher incidence of thrombosis (IRR: 1.7, 1–3.3; P = 0.05) than patients with a stable allele burden below 50%. In conclusion, JAK2V617F monitoring could be useful in patients with PV and ET for predicting diseases complications, especially myelofibrotic transformation. Am. J. Hematol. 89:517–523, 2014.

Discrimination between tumour epithelium and stroma via perception-based features

In this work we propose the use of image features based on visual perception for discriminating epithelium and stroma in histological images. In particular, we assess the capability of the following five visual features to correctly discriminate epithelium from stroma in digitised tissue micro-arrays of colorectal cancer: coarseness, contrast, directionality, line-likeliness and roughness. The use of features directly related to human perception makes it possible to evaluate the tissue?s appearance on the basis of a set of meaningful parameters; moreover, the number of features used to discriminate epithelium from stroma is very small. In the experiments we used histologically-verified, well-defined images of epithelium and stroma to train three classifiers based on Support Vector Machines (SVM), Nearest Neighbour rule (1-NN) and Naive Bayes rule (NB). We optimised SVM?s parameters on a validation set, and estimated the accuracy of the three classifiers on a independent test set. The experiments demonstrate that the proposed features can correctly discriminate epithelium from stroma with state-of-the-art accuracy. HighlightsTumour-stroma ratio (TSR) is an independent prognostic factor for a number of oncologic diseases.Manual assessment of TSR is challenging and suffers from poor intra- and interobserver agreement.We propose a computer-assisted approach based on perceptual features for discriminating between epithelium and stroma.The method can correctly differentiate epithelium from stroma with overall accuracy of ? 97 % .

Influence of JAK2 46/1 haplotype in the natural evolution of JAK2V617F allele burden in patients with myeloproliferative neoplasms

JAK2V617F allele burden was prospectively measured in untreated patients with polycythaemia vera (PV, n=26) or essential thrombocythaemia (ET, n=36) and compared according to JAK2 46/1 haplotype status. The mean increase in JAK2V617F allele burden per year was 1%, 0.8% and 6% for PV patients with the JAK2 46/1 haplotype in negative, heterozygous and homozygous status, respectively (p<0.001). The JAK2 46/1 haplotype had no influence in JAK2V617 allele burden in ET. In conclusion, untreated PV patients homozygous for the JAK2 46/1 haplotype show a progressive increase in the JAK2V617F allele burden during the evolution of the disease.

Impact of genotype on leukaemic transformation in polycythaemia vera and essential thrombocythaemia

The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow‐up of 7·2 years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2–0·9, P = 0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9–4·6, P = 0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR‐mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1–5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1–3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F‐positive. JAK2 V617F‐mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F‐negative leukaemia. Survival was longer in JAK2 V617F‐unmutated leukaemia (343 days vs. 95 days, P = 0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F‐negative clone is TP53 independent and shows better survival.

Masked polycythaemia vera: presenting features, response to treatment and clinical outcomes

Masked polycythaemia vera (PV) has been proposed as a new entity with poorer outcome than overt PV. In this study, the initial clinical and laboratory characteristics, response to treatment and outcome of masked and overt PV were compared using red cell mass and haemoglobin or haematocrit levels for the distinction between both entities. Sixty‐eight of 151 PV patients (45%) were classified as masked PV according to World Health Organisation diagnostic criteria, whereas 16 (11%) were classified as masked PV using the British Committee for Standards in Haematology (BCSH). In comparison with overt PV, a higher platelet count and a lower JAK2V617F allele burden at diagnosis were observed in masked PV. Patients with masked PV needed lower phlebotomies and responded faster to hydroxcarbamide than those with overt PV. Complete haematological response was more frequently achieved in masked than in overt PV (79% vs. 58%, P = 0.001). There were no significant differences in the duration of haematological response, the rate of resistance or intolerance to hydroxycarbamide and the probability of molecular response according to type of PV (masked vs. overt). Overall survival, rate of thrombosis and major bleeding, and probability of transformation was superimposable among patients with masked and overt PV.

The role of serum erythropoietin level and JAK2 V617F allele burden in the diagnosis of polycythaemia vera.

Low serum erythropoietin (EPO) is a minor criterion of Polycythaemia Vera (PV) but its diagnostic usefulness relies on studies performed before the discovery of JAK2 V617F mutation. The objective of the present study was to evaluate the diagnostic accuracy of serum EPO and JAK2 V617F allele burden as markers of PV as well as the combination of different diagnostic criteria in 287 patients (99 with PV, 137 with Essential Thrombocythaemia and 51 with non‐clonal erythrocytosis). Low EPO showed good diagnostic accuracy as a marker for PV, with the area under the curve (AUC) of the chemiluminescent‐enhanced enzyme immunoassay (CEIA) being better than that of radioimmunoassay (RIA) (0·87 and 0·76 for CEIA and RIA, respectively). JAK2 V617F quantification displayed an excellent diagnostic accuracy, with an AUC of 0·95. A haematocrit >52% (males) or >48% (females) plus the presence of the JAK2 V617F mutation had a sensitivity and specificity of 79% and 97%, respectively. Adding low EPO or the JAK2 V617F allele burden did not improve the diagnostic accuracy for PV whereas the inclusion of both improved the sensitivity up to 83% and maintaining 96% specificity. Haematocrit and qualitative JAK2 V617F mutation allow a reliable diagnosis of PV. Incorporation of EPO and/or JAK2 V617F mutant load does not improve the diagnostic accuracy.

Dynamics of JAK2 V617F allele burden of CD34+ haematopoietic progenitor cells in patients treated with ruxolitinib

Ruxolitinib has demonstrated splenomegaly reduction and symptom relief in patients with polycythaemia vera (PV) and myelofibrosis (MF) (Harrison et al, 2012; Verstovsek et al, 2012, 2014; Vannucchi et al, 2015) with limited effect on granulocytic JAK2 V617F mutant load (Harrison et al, 2012; Vannucchi et al, 2015). However, the effect of ruxolitinib on the JAK2 V617F allelle burden at the progenitor level is still unknown. We have assessed the effect of ruxolitinib on haematopoietic stem cells (HSCs, CD34 CD38 ), haematopoietic progenitor cells (HPCs, CD34 CD38) and granulocytes from seven patients with JAK2 V617F myeloproliferative neoplasms (PV, n = 4; secondary MF, n = 3). HPCs and HSCs were isolated from peripheral blood in all patients. Isolation of cell subpopulations and quantification of JAK2 V617F allele burden was performed in circulating HSCs, HPCs and granulocytes before starting ruxolitinib and after 6 and 12 months of therapy as previously described (Angona et al, 2015). Additional mutations in TET2 (whole coding sequence), DNMT3A (exon 23), TP53 (exons 2–10), ASXL1 (exon 12), SF3B1 (exons 14 and 15), SRSF2 (exon 1) and U2AF1 (exons 2 and 6–7) genes were studied in granulocytes by Sanger sequencing or pirosequencing using a Next Generation Sequencing (NGS) 454 GS Junior platform (Roche Applied Science, Mannhion, Germany). Mutations detected were also analysed by NGS in DNA extracted from granulocytes, HSCs and HPCs after 6 and 12 months of therapy. The study was approved by the Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Ruxolitinib was indicated as second-line treatment in all seven patients. Palpable splenomegaly was present in six patients before starting ruxolitinib with a reduction during the treatment in all of them. Most patients had marked improvement in symptoms. Two patients discontinued ruxolitinib after 6 months of treatment, due to severe anaemia (n = 1) and transformation to acute leukaemia (n = 1). The quantification of JAK2 V617F allele burden in HSCs, HPCs and granulocytes during ruxolitinib therapy is shown in Fig 1. In HSCs, JAK2 V617F allele burden remained stable in five patients (>50% and <50% in three and two patients, respectively), decreased from 54% to 27% and duplicated from 22% to 47% in one patient each (Fig 1A). A reduction of JAK2 V617F allele burden in HPCs was observed after 6 months of therapy in three patients but this reduction was not confirmed at 1 year (Fig 1B). The granulocytic JAK2 V617F allele burden remained stable in all but one patient (Patient 2) in whom a progressive increase, from 37% at baseline to 87% at 12 months, was observed (Fig 1C). One patient (Patient 4) showed additional mutations in TET2 (p.R1179 fs and p.E1483X). The mutational burden of both TET2 mutations remained stable in HSCs and granulocytes during ruxolitinib treatment (Fig 2). The allele burden of TET2 p.R1179 fs remained stable in HPCs whereas TET2 p.E1483X presented a mild progressive increase from 13% to 33 9%. To the best of our knowledge, this is the first study evaluating the effect of ruxolitinib on circulating HSCs and HPCs. Our results showed that ruxolitinib had a minimal impact on the mutant JAK2 V617F allele burden in PV and secondary MF patients, both in granulocytes and CD34 cell subpopulations, suggesting a minimal inhibition of the bone marrow JAK2 V617F-mutated cells. It is noteworthy that the six patients with palpable splenomegaly prior to therapy experienced a reduction of the spleen size during treatment in contrast with the absence of JAK2 V617F allele burden variation in bone marrow progenitor cells in all patients. This could be explained by differences in the microenvironment of the bone marrow and the spleen, with bone marrow progenitors being less sensitive to this treatment in contrast to those stem cells present in the spleen. In this regard, Wang et al (2014) recently showed that AZD1480, a JAK1/2/3 inhibitor, may induce apoptosis in myeloproliferative neoplasm progenitor cells in the spleens of MF patients with a subsequent reduction in splenomegaly. It has been reported that the presence of mutations in additional genes other than JAK2 V617F may play a role in the transformation of chronic phase PV and MF to acute leukaemia (Swierczek et al, 2011; Brecqueville et al, 2012). In our series, one PV patient harboured two mutations in the TET2 gene, without significant mutational burden variation in any of the cell populations during ruxolitinib treatment. In this sense, if ruxolitinib has an effect on HSCs, its role might be different depend on whether JAK2 V617F and TET2 mutations coexist in the same clone or are present in two independent clones. In the first situation, we would expect that ruxolitinib, by inhibiting JAK2, also had some effect on TET2 mutational load. By contrast, if the two mutations are present in different clones, JAK2 inhibition

Performance of the myelofibrosis secondary to PV and ET-prognostic model (MYSEC-PM) in a series of 262 patients from the Spanish registry of myelofibrosis

Performance of the myelofibrosis secondary to PV and ET-prognostic model (MYSEC-PM) in a series of 262 patients from the Spanish registry of myelofibrosis