Hematopoietic clonal dominance, stem cell mutations, and evolutionary pattern of JAK2V617F allele burden in polycythemia vera.

OBJECTIVES: Clonal dominance is characteristic of patients with post-polycythemia vera myelofibrosis (post-PV MF), whereas patients in chronic phase usually display polyclonal hematopoiesis. The aim of this work was to study the mutational burden of JAK2V617F at the progenitor level in patients with PV and correlate it with the evolutive phase of the disease and the presence of mutations in genes different to JAK2V617F. METHODS: JAK2V617F was measured in stem cells, progenitor cells, and granulocytes of 45 patients with PV (early chronic phase n = 26, late chronic phase n = 10, post-PV MF n = 9). In addition, screening of TET2, DNMT3A, ASXL1, SF3B1, SRSF2, U2AF1, and TP53 was performed with quantification of the mutation in CD34+ cells in positive cases. Moreover, we assessed whether JAK2V617F allele burden in granulocytes (at a single time point or monitoring) could be used as a surrogate of clonal dominance. RESULTS: Ten patients presented clonal dominance at progenitor level (PV at diagnosis n = 2, late chronic phase n = 1, post-PV MF n = 7). Additional mutations were identified in four patients at diagnosis, three in TET2, and one in DNMT3A gene, with clonal dominance present in three of them. At PV diagnosis, clonal dominance was demonstrated only in patients with additional mutations. JAK2V617F monitoring showed better diagnostic accuracy than single time point measurement as a marker of clonal dominance. CONCLUSIONS: Clonal dominance may be present at diagnosis, especially in those cases carrying other mutations. JAK2V617F monitoring during follow-up could help in the identification of patients with clonal dominance.

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.

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.

Busulfan in patients with polycythemia vera or essential thrombocythemia refractory or intolerant to hydroxyurea.

Therapeutic options for patients with polycythemia vera (PV) and essential thrombocythemia (ET) resistant or intolerant to hydroxyurea are limited. Busulfan is effective as first-line therapy, but there is scarce information on this drug as second-line treatment. The efficacy of busulfan in patients with advanced PV or ET refractory or intolerant to hydroxyurea was assessed in 36 patients (PV n = 15, ET n = 21) treated for a median of 256 days. Complete hematological response (CHR) was achieved in 83 % of patients, after a median time of 203 days (range 92-313). The probability of sustained CHR at 1 and 2 years was 87 and 62 %, respectively. Time to CHR was shorter in patients treated with ≥14 mg of busulfan per week than with lower doses (141 versus 336 days, p = 0.01). Partial molecular response was achieved in three out of nine (33 %) patients. Busulfan was stopped in 27 patients (75 %) due to CHR achievement in 18 cases (67 %), hematological toxicity in 8 cases (30 %), and disease transformation in 1 case. With a median follow-up of 721 days, six patients have died, with the probability of survival at 2 years being 85 %. The probability of thrombosis at 2 years was 11 %. Transformation into acute leukemia or myelodysplastic syndrome was observed in three cases, all of them in a JAK2V617F-negative clone carrying additional mutations. Busulfan, at a dose of 2 mg/day, is an effective option for elderly patients with PV or ET who fail to hydroxyurea, but a significant rate of transformation was observed.

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 disease's complications, especially myelofibrotic transformation.

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.

TET2, ASXL1, IDH1, IDH2, and c-CBL genes in JAK2- and MPL-negative myeloproliferative neoplasms.

Mutations in the TET2 and ASXL1 genes have been described in approximately 14% and 8% of patients, respectively, with classic myeloproliferative neoplasms (MPN), but their role as possible new diagnostic molecular markers is still inconclusive. In addition, other genes such as IDH1, IDH2, and c-CBL have also been reported in several myeloid neoplasms. We have studied the mutational status of TET2 (complete coding region), ASXL1 (exon12), IDH1 (R132), IDH2 (R140 and R172), and c-CBL (exons 8 and 9) in 62 MPN patients (52 essential thrombocythemia (ET), five polycythemia vera (PV), and five primary myelofibrosis (PMF)) negative for both JAK2 (V617F and exon 12) and MPL (exon 10) mutations. Pathogenic alterations in the TET2 gene were detected in three out 52 ET cases (4.8%). ASXL1 gene pathogenic mutations were also detected in three cases (two ET and one PMF). One ET patient harbored, simultaneously, one TET2 and one ASXL1 mutations. Mutations in the TET2 and ASXL1 genes showed no association with the JAK2 46/1 haplotype. Analysis of a JAK2V617F-positive cohort of 50 ET patients showed no mutations in either the TET2 or ASXL1 genes. Regarding IDH1, IDH2, and c-CBL genes, no mutations were found in any patient. In conclusion, TET2 and ASXL1 pathogenic mutations are found in 8% of MPN lacking JAK2 and MPL mutations, whereas IDH1, IDH2, and c-CBL mutations are not detected in this subset of patients.

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.

Modulation of JAK2 V617F allele burden dynamics by hydroxycarbamide in polycythaemia vera and essential thrombocythaemia patients.

The modulation of JAK2 V617F allele burden dynamics was prospectively analysed in 47 patients (26 polycythaemia vera [PV] and 21 essential thrombocythaemia [ET]) treated with first-line hydroxyurea (HU) and compared with the JAK2 V617F dynamics of a control group of 45 PV and ET patients. A partial molecular response (PMR), according to European Leukaemia Net criteria, was observed in 27/47 (57%) patients. Median time to PMR was 14 months (3-66) with a probability of PMR at 3 years of 57%. A significant decrease in JAK2 V617F allele load was observed at 36 months both in PV and ET patients, being the reduction in PV higher than in ET patients (P = 0·01). A haematocrit ≥0·45 L/L was associated with a higher probability of attaining a PMR (HR:3·4; 95%CI:1·02-11·6, P = 0·04). Control group showed a slight increase of JAK2 V617F allele burden over time. The reduction in the mutated allele load comparing treated patients versus controls was highly significant both in PV and ET, demonstrating a clear effect of HU on the JAK2 V617F allele burden. In conclusion, first-line HU can attain PMR in more than 50% of newly diagnosed PV and ET patients, with a continuous decrease of the JAK2 V617F allele burden in PV patients during treatment.

Clinical evaluation of the European LeukaemiaNet criteria for clinicohaematological response and resistance/intolerance to hydroxycarbamide in essential thrombocythaemia.

Standardized criteria of response to treatment and a unified definition of resistance/intolerance to hydroxycarbamide (HC) in essential thrombocythaemia (ET) have been proposed by the European LeukaemiaNet (ELN). We have retrospectively evaluated such criteria in 166 ET patients treated with HC for a median of 4·5 years. Overall, 134 patients achieved a complete clinicohaematological response (CR) and 25 a partial response. Thirty-three patients met at least one of the ELN criteria defining resistance (n = 15) or intolerance (n = 21) to HC. Fifteen cases developed anaemia with thrombocytosis, which was associated with a high incidence of myelofibrosis and death from any cause. Other definitions of resistance were less useful. Factors determining the thrombotic risk were a history of prior thrombosis and a baseline leucocyte count >10 × 109/ l. Of note, patients achieving a CR, even if sustained during the entire follow-up, did not benefit from a lower incidence of thrombosis or an improved survival. In conclusion, most ET patients respond to HC, but the achievement of response, as defined by the ELN, does not correlate with the patients' outcome. The best discriminating ELN criterion of resistance to HC was the detection of anaemia, which also identified a subgroup of patients with poor prognosis.

Postpolycythaemic myelofibrosis: frequency and risk factors for this complication in 116 patients.

Postpolycythaemic myelofibrosis (PPMF) is a known complication of polycythaemia vera (PV) but information regarding its incidence and predisposing factors is not well defined. In 116 subjects consecutively diagnosed with PV in a single institution (median age 62 years, range: 20-88), the probability of PPMF was analysed by the Kaplan-Meier method, followed by the log-rank test. With a mean follow-up of 8 years (95% confidence interval: 6.6-9), 17 patients had evolved into PPMF (15%). The probability of evolution to PPMF was 16% at 10 years and 34% at 15 years. Age, gender, spleen size, leucocytosis, thrombocytosis or cytoreductive treatment were not associated with an increased risk of PPMF. The actuarial probability of PPMF at 15 years was higher in those patients presenting at diagnosis with endogenous megakaryocytic colony formation (59% when present versus 10% when absent, P = 0.03), an elevated serum lactate dehydrogenase (LDH) level (69% vs. 23% in patients with normal LDH, P = 0.04), and in those who were heterozygous for the JAK2 V617F mutation (55% vs. 17% in heterozygotes, P = 0.04). In conclusion, PPMF is a frequent complication in PV patients at 15 years with the risk being higher in patients with increased LDH, endogenous megakaryocytic colony formation or a high JAK2 V617F allele burden.

JAK2 exon 12 mutations in polycythemia vera or idiopathic erythrocytosis.

JAK2 exon 12 mutations were detected in 4 out of 20 polycythemia vera and idiopathic erythrocytosis V617F-negative patients and were only present in the myeloid lineage. Initial hematologic data of these patients differ from those of V617F-positive patients, but there is no difference in thrombotic development and myelofibrotic transformation.

[Essential thrombocythemia: baseline characteristics and risk factors for survival and thrombosis in a series of 214 patients]. / Trombocitemia esencial: características iniciales y factores de riesgo de supervivencia y trombosis en una serie de 214 pacientes.

BACKGROUND AND OBJECTIVE: Two prognostic models to predict overall survival and thrombosis-free survival have been proposed: International Prognostic Score for Essential Thrombocythemia (IPSET) and IPSET-Thrombosis, respectively, based on age, leukocytes count, history of previous thrombosis, the presence of cardiovascular risk factors and the JAK2 mutational status. The aim of the present study was to assess the clinical and biological characteristics at diagnosis and during evolution in essential thrombocythemia (ET) patients as well as the factors associated with survival and thrombosis and the usefulness of these new prognostic models. PATIENTS AND METHODS: We have evaluated the clinical data and the mutation status of JAK2, MPL and calreticulin of 214 ET patients diagnosed in a single center between 1985 and 2012, classified according to classical risk stratification, IPSET and IPSET-Thrombosis. RESULTS: With a median follow-up of 6.9 years, overall survival was not associated with any variable by multivariate analysis. Thrombotic history and leukocytes>10×10(9)/l were associated with thrombosis-free survival (TFS). In our series, IPSET prognostic systems of survival and thrombosis did not provide more clinically relevant information regarding the classic risk of thrombosis stratification. CONCLUSION: Thrombotic history and leukocytosis>10×10(9)/l were significantly associated with lower TFS, while the prognostic IPSET-Thrombosis system did not provide more information than classical thrombotic risk assessment.

[Molecular characterization of atypical chronic myeloid leukemia and chronic neutrophilic leukemia]. / Caracterización molecular de la leucemia mieloide crónica atípica y la leucemia neutrofílica crónica.

BACKGROUND AND OBJECTIVE: Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) display similar clinical and hematological characteristics. The objective of the present study was to determine the mutational status of SETBP1 and CSF3R in these diseases. PATIENTS AND METHOD: The mutational status of SETBP1 and CSF3R was studied in 7 patients with aCML (n = 3), CNL (n = 1) and unclassifiable myeloproliferative neoplasms (MPN-u) (n = 3). Additionally, mutations in ASXL1, SRSF2, IDH1/2, DNMT3A, and RUNX1 were also analyzed. RESULTS: SETBP1 mutations (G870S and G872R) were detected in 2 patients with MPN-u, and one of them also presented mutations in SRSF2 (P95H) and ASXL1 (E635fs). The CNL case showed mutations in CSFR3 (T618I), SETBP1 (G870S) and SRSF2 (P95H). No patient classified as aCML had mutations in SETBP1 or CSF3R. One of the patients with mutations evolved to acute myeloid leukemia, while the other 2 had disease progression without transformation to overt leukemia. CONCLUSION: The knowledge of the molecular alterations involved in these rare diseases is useful in the diagnosis and may have an impact on both prognosis and therapy.

Genetic predisposition to molecular response in patients with myeloproliferative neoplasms treated with hydroxycarbamide.

JAK2V617F allele burden was prospectively measured in polycythemia vera (PV, n=52) and essential thrombocythemia (ET, n=39) patients receiving hydroxycarbamide (HC) and analyzed according to JAK2 46/1 haplotype and genotype of SLC14A1, SLC14A2 and ARG2 urea transporters. Molecular response (MR) was obtained in 68.7% and 38.9% of PV patients with GG and AA or GA genotype in SLC14A2, respectively (p=0.07). No significant differences were observed neither in PV nor in ET according to JAK2 46/1 haplotype, SLC14A1 and ARG2. In conclusion, JAK2 46/1 haplotype does not influence MR in HC treated patients and urea transporters polymorphisms display a minimal effect.

Leukemic transformation driven by an ASXL1 mutation after a JAK2V617F-positive primary myelofibrosis: clonal evolution and hierarchy revealed by next-generation sequencing.

We have characterized the molecular changes underlying the transformation of a JAK2V617F+-myelofibrosis with trisomy 8, into a JAK2V617F-negative leukemia. Leukemic clone did not carry JAK2V617F mutation, but showed ASXL1 mutation (R693X). This mutation was identified in a low percentage at diagnosis by next-generation sequencing. Using this technology in serial specimens during the follow-up, we observed a progressive expansion of the ASXL1-mutated minor clone, whereas the JAK2V617F+-clone carrying trisomy 8 decreased. Hematologic progression occurred simultaneously with an ASXL1-R693X-negative lung-cancer. This is the first report showing a clear association between the expansion of an ASXL1-mutated clone and the leukemic transformation of myelofibrosis.

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.

A sporadic multiple gastrointestinal stromal tumor with unique clinical and molecular features.

Most sporadic gastrointestinal stromal tumors occur as solitary lesions, whereas a multicentric appearance involving the stomach, the small intestine, or both sites is suspicious for lesions developed in the setting of hereditary or idiopathic tumor syndromes or metastatic disease. The rare occurrence of multiple sporadic gastrointestinal stromal tumors has been recently reported in the literature. Here, we report a case of multiple sporadic gastrointestinal stromal tumors affecting the small intestine in a 61-year-old man, unique with regard to the number of lesions (>30) and the molecular profile. Four different mutations of KIT involving exons 11, 13, and 17 were present among 4 of 10 excised tumors. In addition, BRAF p.V600E mutation was detected in 5 tumors and was mutually exclusive with KIT mutations. To our knowledge, this is the first time a case of a synchronic multisporadic gastrointestinal stromal tumor outstanding for the high number of lesions, which are of independent origin, is reported.

Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer.

INTRODUCTION: Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. METHODS: ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. RESULTS: One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. CONCLUSION: The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.