miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk.

Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a-/- mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling.

Treatment-free remission following frontline nilotinib in patients with chronic myeloid leukemia in chronic phase: results from the ENESTfreedom study.

The single-arm, phase 2 ENESTfreedom trial assessed the potential for treatment-free remission (TFR; i.e., the ability to maintain a molecular response after stopping therapy) following frontline nilotinib treatment. Patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase with MR4.5 (BCR-ABL1⩽0.0032% on the International Scale (BCR-ABL1IS)) and ⩾2 years of frontline nilotinib therapy were enrolled. Patients with sustained deep molecular response during the 1-year nilotinib consolidation phase were eligible to stop treatment and enter the TFR phase. Patients with loss of major molecular response (MMR; BCR-ABL1IS⩽0.1%) during the TFR phase reinitiated nilotinib. In total, 215 patients entered the consolidation phase, of whom 190 entered the TFR phase. The median duration of nilotinib before stopping treatment was 43.5 months. At 48 weeks after stopping nilotinib, 98 patients (51.6%; 95% confidence interval, 44.2-58.9%) remained in MMR or better (primary end point). Of the 86 patients who restarted nilotinib in the treatment reinitiation phase after loss of MMR, 98.8% and 88.4%, respectively, regained MMR and MR4.5 by the data cutoff date. Consistent with prior reports of imatinib-treated patients, musculoskeletal pain-related events were reported in 24.7% of patients in the TFR phase (consolidation phase, 16.3%).

MYC antagonizes the differentiation induced by imatinib in chronic myeloid leukemia cells through downregulation of p27(KIP1.).

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and dasatinib. In the CML-derived K562 cell line, low concentrations of imatinib induce proliferative arrest and erythroid differentiation. We found that imatinib upregulated the cell cycle inhibitor p27(KIP1) (p27) in a time- and -concentration dependent manner, and that the extent of imatinib-mediated differentiation was severely decreased in cells with depleted p27. MYC (c-Myc) is a transcription factor frequently deregulated in human cancer. MYC is overexpressed in untreated CML and is associated to poor response to imatinib. Using K562 sublines with conditional MYC expression (induced by Zn(2+) or activated by 4-hydroxy-tamoxifen) we show that MYC prevented the erythroid differentiation induced by imatinib and dasatinib. The differentiation inhibition is not due to increased proliferation of MYC-expressing clones or enhanced apoptosis of differentiated cells. As p27 overexpression is reported to induce erythroid differentiation in K562, we explored the effect of MYC on imatinib-dependent induction of p27. We show that MYC abrogated the imatinib-induced upregulation of p27 concomitantly with the differentiation inhibition, suggesting that MYC inhibits differentiation by antagonizing the imatinib-mediated upregulation of p27. This effect occurs mainly by p27 protein destabilization. This was in part due to MYC-dependent induction of SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation.

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia.

There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.

Comparative study of BCR-ABL1 quantification: Xpert assay, a feasible solution to standardization concerns.

The level of BCR-ABL1 reached after treatment with tyrosine kinase inhibitors is an effective marker of the therapeutic response and a good survival predictor in chronic myeloid leukemia (CML) patients. However, no agreement has yet been achieved about either the standardization of the technique to determine BCR-ABL1 or the interpretation of the results. The aim of this study was to compare the method currently recommended by the European Leukemia Net, which includes the application of a conversion factor to express the results in international scale, with an automated method (Xpert BCR-ABL™, Cepheid). BCR-ABL1 transcript quantification was performed in 117 samples from CML patients in two different laboratories by both methods, and the results were compared by statistical procedures. A high linear correlation was obtained in the results between the two methods. The concordance at logarithmic intervals reached 62 %. When the major molecular response (MMR) was analyzed, 85 % agreement was achieved. The automated method provides reproducible results and does not show significant differences compared with the traditional method. As a clinical tool, Xpert correctly classified the patients in MMR and can be considered a useful alternative for the molecular follow-up of CML patients.

Acute promyelocytic leukemia developing after radiotherapy for prostate cancer in a patient with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is rarely associated with secondary acute myelogenous leukemia (AML) usually due to chemotherapy or radiotherapy. No cases of concomitant CLL and acute promyelocytic leukemia (APL) have been found in the literature. Nevertheless, up to 12% of therapy-related AML cases are classified as APL. Of these latter, most are related to topoisomerase treatment, with a few acute cases occurring after radiotherapy. We report here a patient with an untreated CLL who developed APL 2 years after radiotherapy for prostate carcinoma.

Pretreatment characteristics and clinical outcome of acute promyelocytic leukaemia patients according to the PML-RAR alpha isoforms: a study of the PETHEMA group.

Of 167 newly diagnosed acute promyelocytic leukaemia patients, 83 patients were long (L)-form (50%), eight variable (V)-form (5%) and 76 short (S)-form (45%). The V-form and S-form groups presented a significantly higher percentage of patients with white blood cell counts > 10 x 10(9)/l (P < 0.05). The S-form cases displayed a significantly higher number of cases with M3v microgranular features (P = 0.005) and CD34 expression (P < 0.0001). There were no differences between the three isoforms in complete remission (CR) rate (overall CR 90%), but the 3-year disease-free survival was lower for V-form cases than it was for L- and S-form cases (62% vs. 94% and 89%, P = 0.056). We conclude that the V-form and S-form types are associated with some negative prognostic features at diagnosis. However, our data were only able to demonstrate an association with adverse prognosis in the V-form type and, moreover, as the number of cases was limited, needs to be confirmed in large, uniformly treated series.

p190 BCR-ABL rearrangement in chronic myeloid leukemia and acute lymphoblastic leukemia.

A minority of chronic myeloid leukemia (CML) cases have breakpoint in the minor cluster region (m-bcr) of the BCR-ABL fusion gene. We report a patient with Ph-positive acute lymphoblastic leukemia and m-bcr breakpoint at diagnosis. The patient was treated with chemotherapy followed by an autologous peripheral blood stem cell transplantation, achieving a clinical and hematological complete remission but with persistence of the Philadelphia chromosome. One year later, she developed leukocytosis with a blood picture consistent with CML. She was treated with hydroxyurea and interferon alpha with no response. This is the second case of m-bcr CML reported presenting with features of lymphoid blast crisis or acute lymphoblastic leukemia.

Detection of a t(8;21)(q22;q22) in a case of M5 acute monoblastic leukemia.

Although the translocation (8;21) is the single most common structural rearrangement reported in acute myeloblastic leukemia (AML), it is rarely seen in AML FAB type M5. We describe a case of a 51-year-old male with a diagnosis of acute monoblastic leukemia (AML M5b with hemophagocytic component) whose karyotype showed at (8;21)(q22;22). To our knowledge, this is the first report of this translocation in an AML M5b. The t(8;21) has been associated with a good prognosis, but our patient suffered a fast and fatal evolution.

Differential regulation of Max and role of c-Myc during erythroid and myelomonocytic differentiation of K562 cells.

The protein Max binds to c-Myc and the heterodimer c-Myc/Max seems to be the active form in vivo. While the expression of c-myc is extensively regulated, no major changes in max expression have been reported so far with respect to differentiation. We have studied the expression of c-Myc and Max during in vitro differentiation of the bipotent human myeloid leukemia K562 cell line. This cell model system allowed us to compare c-Myc and Max expression during differentiation along erythroid (induced by 1-beta-D-arabinofuranosyl-cytosine) and myelomonocytic lineages (induced by 12-0-tetradecanoylphorbol-13-acetate). We found that c-myc expression decreased as a result of both differentiating treatments. The expression level of max remained unchanged during myelomonocytic differentiation. In contrast, max mRNA and protein were dramatically down-regulated during erythroid differentiation of K562 cells, thus demonstrating that max gene is subjected to regulation during differentiation. We also studied the expression of the other two described members of the c-Myc network: mxi1 and mad. mxi1 expression increased during erythroid differentiation but was strongly down-regulated during myelomonocytic differentiation of K562. mad was constitutively expressed during K562 erythroid differentiation and slightly increased during induction of the myelomonocytic pathway. We have obtained K562 sublines stably transfected with a zinc-inducible c-myc gene. In these clones the overexpression of c-Myc did not interfere with TPA-induced myelomonocytic differentiation. In contrast, erythroid differentiation was significantly inhibited upon c-myc induction despite the down-regulation of endogenous max expression. These results suggest a differential role for c-Myc in the human myeloid cell differentiation depending on the cell lineage.

[Bone marrow aplasia and gold salts. Review of the literature apropos of 2 cases]. / Aplasia medular y sales de oro. Actualización de la literatura a propósito de dos casos.

Bone marrow aplasia remains the most serious adverse effect with gold therapy. Its prevalence is still a controversial issue and at present it is not possible to give accurate figures on its frequency. Two patients diagnosed of rheumatoid arthritis are reported. They underwent chrysotherapy and developed bone marrow aplasia within a 2-month period of therapy. The pathogenic mechanism of blood dyscrasias secondary to gold salts is still unknown. The best available method in the prevention of this serious condition is the periodical obtention of complete blood counts.

[Chronic eosinophilic pneumonia in a patient treated with allogenic bone marrow transplantation]. / Neumonía eosinófila crónica en un paciente tratado con trasplante de médula ósea alogénico.

A 39 year old patient diagnosed of severe aplastic anemia and treated with allogenic bone marrow transplantation and who presented chronic eosinophilic pneumonia eight months after the transplant is presented. The patient had no previous history of asthma or atopy. Conditioning was performed with cyclophosphamide and total body irradiation. Prophylaxis of the graft versus host disease was carried out with cyclosporin and short course of methotrexate. At day 30 mild graft versus host disease appeared which spontaneously resolved. A progressive increase in the number of eosinophils was observed from day +40 reaching 1.05 x 10(9)/l at day +180 coinciding with suspension of the cyclosporine. The patient remained asymptomatic with no evidence of chronic graft versus host disease. At 8 months following allogenic transplantation the patient developed three episodes of fever, cough, moderate dyspnea and pulmonary infiltrates. Respiratory tests showed a restrictive pattern. Bronchoalveolar lavage contained 20% of eosinophils. Upon lung biopsy alveolar infiltration by eosinophils, lymphocytes and mononuclear cells was observed. Diagnosis of chronic eosinophilic pneumonia was made with initiation of steroid treatment. A drastic response was observed. The patient remained asymptomatic without recurrence and without evidence of chronic graft versus host disease. This picture may have been caused by the donor eosinophils given that retrospective evaluation demonstrated a persistent moderate eosinophilia in the donor.

Down-regulation of c-myc gene is not obligatory for growth inhibition and differentiation of human myeloid leukemia cells.

Suppression of c-myc expression is observed during induced differentiation of several myeloid cell lines and it has been attributed to the cell growth arrest that accompanies terminal differentiation. To dissect the role of c-Myc in the proliferation-differentiation switch we have studied c-myc expression in K562 cells exposed to several chemical agents. This model system allowed us to discriminate between the growth arrest and differentiation phenomena as well as the induction of differentiation along two different lineages (erythroid and myelomonocytic). Our results showed that c-myc expression did not significantly decrease when growth inhibition is reversible, either by treatment with a differentiating agent such as hydroxyurea (which induced erythroid differentiation) or by a non-differentiating agent such as interferon-alpha. In contrast, c-myc expression decreased when cells underwent terminal differentiation, either along the myelomonocytic (by 12-O-tetradecanoylphorbol-13-acetate) or erythroid (by 1-beta-D-arabinofuranosylcytosine) lineages. These results indicated that c-myc down-regulation is not obligatory for growth arrest and non-terminal differentiation of human myeloid cells. In contrast, c-myc down-regulation occurred in terminal differentiation, but induction of myelomonocytic differentiation resulted in a greater loss of c-myc mRNA than induction of erythroid differentiation.

Induction of apolipoprotein E expression during erythroid differentiation of human K562 leukemia cells.

Apolipoprotein E (ApoE) is the only apolipoprotein that is expressed in extrahepatic tissues. ApoE expression was studied in leukemia K562 cells differentiated towards erythroid or myelomonocytic lineages. When K562 cells were differentiated into the erythroid lineage by addition either of 1-beta-D-arabinofuranosylcytosine or hydroxyurea, an increase in ApoE mRNA and protein was detected. A weaker ApoE induction was also observed during phorbol ester-induced myelomonocytic differentiation. Previous work has associated ApoE expression to monocytic differentiation. The findings reported here indicate that ApoE overexpression is not associated with a specific lineage in myeloid differentiation and that may play a role in erythroid differentiation.