Florinda Gilsanz

High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations: A Diagnostic Tool for Myeloproliferative Neoplasms

JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations.

Breast Cancer–Specific mRNA Transcripts Presence in Peripheral Blood After Adjuvant Chemotherapy Predicts Poor Survival Among High-Risk Breast Cancer Patients Treated With High-Dose Chemotherapy With Peripheral Blood Stem Cell Support

PURPOSE To study the prognostic significance of the presence of breast cancer-specific mRNA transcripts in peripheral blood (PB), defined by serial analysis of gene expression, in high-risk breast cancer (HRBC) patients undergoing high-dose chemotherapy after receiving adjuvant chemotherapy. METHODS From 1994 to 2000, 84 HRBC patients (median age, 44 years; > 10 nodes; 74%) received adjuvant chemotherapy (fluorouracil, epirubicin, and cyclophosphamide for six cycles [83%] or doxorubicin and cyclophosphamide followed by paclitaxel) before undergoing one course of cyclophosphamide plus thiotepa plus carboplatin (STAMP V). Radiotherapy or hormone therapy was administered whenever indicated. Aliquots of apheresis-mononuclear blood cells were frozen from each patient. mRNA was isolated using an automatic nucleic acid extractor based on the magnetic beads technology; reverse transcription was performed using random hexamers. Cytokeratin 19, HER-2, P1B, PS2, and EGP2 transcripts were quantified to B-glucuronidase by real-time polymerase chain reaction (RT-PCR) using a linear DNA probe marked with a quencher and reporter fluorophores used in RT-PCR. Presence of PB micrometastases, estrogen receptor and progesterone receptor status, tumor size, age, tumor grade, number of nodes affected, and treatment with paclitaxel were included in the statistical analysis. RESULTS Median follow-up was 68.3 months (range, 6 months to 103 months). Forty-seven relapses (56%) and 35 deaths (41.7%) were registered. Both tumor size and presence of micrometastases reached statistical significance according to the Cox multivariate model. Relapse hazard ratio (HR) for those patients with PB micrometastases was 269% (P = .006); death HR, 300% (P = .011). Time relapse was 53 months longer for patients without micrometastases: 31.3 v 84.2 months (P = .021). CONCLUSION PB micrometastases presence after adjuvant chemotherapy predicts both relapse and death more powerful than classical factors in HRBC patients undergoing high-dose chemotherapy. Micrometastases search using a gene panel appears to be a more accurate procedure than classical approaches involving only one or two genes.

Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases.

Several sensitive methods for the detection of JAK2 V617F mutation have been published recently, most of them based on Real Time polymerase chain reaction (PCR). However, only some of them have performed studies of diagnostic validity. This study compares three methods based on Real Time PCR to detect JAK2 V617F mutation: two based on hybridization probes (HP) and peptide nucleic acid probe (PNA) and a third employing allele specific oligonucleotide primers for JAK2 V617F quantification. One hundred forty-nine healthy subjects, 61 essential thrombocythemia (ET), 32 polycythemia vera (PV), 38 secondary thrombocytoses, and 35 secondary erythrocytoses were included. Validity test study for JAK2 617 HP PCR in PV Sensitivity (Se) was 88% and in Specificity (Sp), 100%. In ET, Se was 57% and Sp, 100%. For JAK2 617 PNA PCR in PV, Se was 94% and Sp, 97.8%. In ET, Se was 70% and Sp, 95.7%. In JAK2 V671F allelo-specific-oligonucleotide (ASO) quantitative PCR (qPCR), cutoff point of 1% was established by receiving operating characteristic (ROC) curves. In PV, Se was 93.8% and Sp, 98.5%. In ET, Se was 80% and Sp, 95.9%. Two percent of the healthy subjects were positive by JAK2 617 PNA PCR and 2% by JAK2 617 ASO qPCR. JAK2 V617F mutation was detected in healthy subjects by cloning and sequencing. JAK2 617 HP is an adequate test in differential diagnosis for both erythrocytosis and thrombocytosis. When JAK2 V617F allele burden is low, JAK2 617 ASO qPCR should be performed. Simultaneous determination of JAK2 V617F and PRV-1 overexpression does not improve the diagnostic value of JAK2 V617F tests in MPD.

Clinical significance of Gata-1, Gata-2, EKLF, and c-MPL expression in acute myeloid leukemia†

The aim of this study was to evaluate the biological correlation and prognostic impact of Gata‐1, Gata‐2, EKLF, and c‐MPL transcript level in a group of 41 acute mieloid leukemia (AML) patients. Gata‐1 overexpression was related to advanced age and a low percentage of bone marrow blasts and was associated with the expression of CD34 antigen and lymphoid T markers. The negative impact of Gata‐1 expression on the probability of achieving complete remission has been confirmed. Gata‐2 overexpression was associated with a low percentage of blasts in BM and males. Expression of c‐MPL was associated with CD34+ AML and M2 FAB AML subtype. A higher expression of EKLF was found in secondary AML versus primary AML. Nevertheless, patients expressing EKLF had a longer overall survival and event free survival than those patients that did not express EKLF. Our study has identified expression of EKLF as a factor with a favorable impact on prognosis in AML. Am. J. Hematol, 2009.

Epigenomic profiling in polycythaemia vera and essential thrombocythaemia shows low levels of aberrant DNA methylation

Aims The purpose of this study was to compare the DNA-methylation signature in classic chronic Philadelphia negative myeloproliferative neoplasms (MPN), polycythaemia vera (PV) and essential thrombocythaemia (ET), in order to obtain a global insight into DNA-methylation changes associated with these malignancies. Methods Thirty-five MPN samples from 11 ET JAK2 V617F, 12 ET JAK2 wild type (WT) and 12 PV JAK2 V617F patients as well as 12 from healthy donors were analysed. DNA samples extracted from whole peripheral blood were hybridised to the ‘HumanMethylation27 DNA Analysis BeadChip.’ Results All groups showed a very homogeneous methylation pattern. Only the ZNF577 gene showed a differential methylation profile between PV JAK2 V617F positive and controls. This aberrant methylation was correlated with a differential gene expression of ZNF577. No aberrant hypermethylation was found in the SOCS-1 and SOCS-3 genes. Conclusions According to our results, an aberrant methylation pattern does not seem to play a crucial role in MPN pathogenesis; nor does it justify phenotypical differences between PV and ET.

Inhibition of related JAK/STAT pathways with molecular targeted drugs shows strong synergy with ruxolitinib in chronic myeloproliferative neoplasm

This study aimed to assess the antitumour effects, molecular mechanisms of action, and potential synergy of ruxolitinib with sorafenib, KNK437, dasatinib, and perifosine, in Philadelphia‐negative chronic myeloproliferative neoplasms (MPN). Cytotoxic and cytostatic effects of the different compounds were determined in the JAK2 V617F‐positive cell lines, HEL and Ba/F3 JAK2V617F EPOR, and in primary mononuclear and bone marrow CD34‐positive cells from 19 MPN patients. Ruxolitinib [50% inhibitory concentration (IC50)PV = 15 nmol/l], as well as sorafenib ( IC50 PV=8μmol/l ), KNK437 ( IC50 PV=100μmol/l ), and perifosine ( IC50 PV=15μmol/l ), were able to inhibit proliferation in cell line models and in primary cells from MPN patients. Dasatinib, KNK437, and sorafenib showed a strong synergistic effect in combination with ruxolitinib [combination index (CI)PV < 0·3]. Western blot confirmed that ruxolitinib blocked ERK, and consequently STAT5 activation, sorafenib inhibited ERK, P38 and STAT5, dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the JAK2 protein, reducing its expression. Inhibiting JAK2‐related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of ruxolitinib with inhibitors that target these pathways has a strong synergistic effect, which may be due to decreased activation of the common effector, STAT5.

Long-Term Follow-Up of Donor Chimerism and Tolerance After Human Liver Transplantation

We aimed to quantify peripheral donor chimerism (DC) and to analyze its association with graft and recipient outcome. Forty‐two liver transplant recipients and their respective donors were studied, providing a total of 148 posttransplantation serum samples. DC was assessed with real‐time quantitative polymerase chain reaction (qPCR) to detect polymorphic markers. DC did not decrease with time post‐transplantation and was higher in child recipients versus adults and in recipients of deceased donor liver transplants versus recipients of live donor liver transplants. Higher levels of DC were detected in Rh‐positive blood group donors, in O blood group recipients versus A blood group recipients, and in recipients with hepatitis C virus versus recipients with alcoholic cirrhosis. High DC was associated with patients with organ damage due to recurrent disease and rejection. Stable, high levels of DC, in the absence of other major clinical events, may thus be a marker of transplantation tolerance, and this knowledge may help to tailor immunosuppressive treatment. In conclusion, qPCR is a useful technique for DC follow‐up in liver transplantation, although the evolution of DC levels should be analyzed in accordance with the clinical outcome of the patient. Liver Transpl 15:581–591, 2009.

Comparative intracellular cytokine production by in vitro stimulated T lymphocytes from human umbilical cord blood (HUCB) and adult peripheral blood (APB)

To date over 400 HUCB transplants have been reported from different centers. It has been suggested that there is a reduced graft‐versus‐host‐disease (GVHD) with HUCB compared to bone marrow transplantation. Since cytokine production by a cell is an indication of the cells function it is important to determinate the differences between APB and HUCB with respect to production of these soluble factors. Our aim was to analyse the intracellular cytokine production by HUCB and APB T lymphocytes with and emphasize on their possible role in GVHD. Heparinized HUCB samples from 8 normal full‐term deliveries and 10 normal blood donors were stimulated 4 hours at 37∘C and 5% CO2 with phorbol 12‐myristate 13‐acetate (PMA) and Ionomycin in the presence of brefeldine. Afterwards cells were stained with CD3, CD4 or CD8 in different combinations. Finally, after cell permeabilization, cells were stained with Il‐2, Il‐4 or IFN‐γ. Data acquisition was performed on a FACScan flow cytometer. Compared to APB, HUCB T lymphocytes produced less Il‐2, Il‐4 and IFN‐γ. In HUCB, Il‐2, Il‐4 and IFN‐γ were produced predominantly by CD4+ T cells. In APB, Il‐2 and Il‐4 were also produced predominantly by CD4+ cells compared with CD8+ T lymphocytes, however, IFN‐γ was produced by both CD4+ and CD8+ T cells. These results indicate that there are clear differences in the cytokine profile between T cells in APB and HUCB.

Chronic ethanol abuse and membrane fluidity changes in liver disease

The long-term effect of ethanol on human red cell membrane fluidity was studied, by fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene as a probe, in 11 healthy subjects, 9 chronic alcoholics without evidence of liver disease, 12 chronic alcoholics with biopsy-proven alcoholic liver disease and 9 abstemious patients with chronic active liver disease, most of them cirrhosis of the liver. Fluorescence polarization values were not significantly different in the two groups without liver disease. Patients with alcoholic and non-alcoholic liver disease showed higher fluorescence polarization values than patients without liver disease. These changes correlated with the severity of liver dysfunction and were not related to alcohol consumption. In conclusion, the decrease in fluidity of the erythrocyte membrane in alcoholic patients with chronic liver disease, is related to liver dysfunction but not to chronic ethanol ingestion. Changes in membrane fluidity in chronic alcoholics are found only in the presence of liver disease.

Treatment of leg ulcers in beta-thalassaemia intermedia: use of platelet-derived wound healing factors from the patient's own platelets.

The interesting report by Josifova et al (2001), describescomplete healing of an intractable ankle ulcer of nearly20 years duration after 141 d of treatment with acombination of platelet-derived wound-healing factors(PDWHFs) in a patient with b-thalassaemia intermedia.We have used the same procedure except that thepatient’s own platelets were used as a source of PDWHFsin a b-thalassaemia intermedia patient with an ankle ulcerof .4 years duration who was unresponsive to otherprocedures, including free skin grafting. Use of the patient’sown platelets is potentially safer, and because thalassaemiaintermedia patients usually have thrombocytosis it isparticularly attractive.Platelets were obtained by apheresis from the patient,a 26-year-old, transfusion-independent splenectomizedman with haemoglobin levels around 9 g/dl. Morethan 900 910 /l platelets, were aliquoted in single-dose 2 ml vials under sterile conditions and frozen at2808C. The preparation was thawed at room tempera-ture before application. We applied a sterile gauze swabmoistened with the thawed PDWHFs and firmlybandaged the area, every day from d 1 to d 10 andwhen granulation tissue developed, on alternative days(d 11 to d 32).The ulcer surface area was 13 cm