Low-burden TP53 mutations in chronic phase of myeloproliferative neoplasms: association with age, hydroxyurea administration, disease type and JAK2 mutational status.

The multistep process of TP53 mutation expansion during myeloproliferative neoplasm (MPN) transformation into acute myeloid leukemia (AML) has been documented retrospectively. It is currently unknown how common TP53 mutations with low variant allele frequency (VAF) are, whether they are linked to hydroxyurea (HU) cytoreduction, and what disease progression risk they carry. Using ultra-deep next-generation sequencing, we examined 254 MPN patients treated with HU, interferon alpha-2a or anagrelide and 85 untreated patients. We found TP53 mutations in 50 cases (0.2-16.3% VAF), regardless of disease subtype, driver gene status and cytoreduction. Both therapy and TP53 mutations were strongly associated with older age. Over-time analysis showed that the mutations may be undetectable at diagnosis and slowly increase during disease course. Although three patients with TP53 mutations progressed to TP53-mutated or TP53-wild-type AML, we did not observe a significant age-independent impact on overall survival during the follow-up. Further, we showed that complete p53 inactivation alone led to neither blast transformation nor HU resistance. Altogether, we revealed patient's age as the strongest factor affecting low-burden TP53 mutation incidence in MPN and found no significant age-independent association between TP53 mutations and hydroxyurea. Mutations may persist at low levels for years without an immediate risk of progression.

Clinical impact of bone marrow morphology for the diagnosis of essential thrombocythemia: comparison between the BCSH and the WHO criteria.

Essential thrombocythemia (ET) is currently diagnosed either by the British Committee of Standards in Haematology (BCSH) criteria that are predominantly based on exclusion and not necessarily on bone marrow (BM) morphology, or the World Health Organization (WHO) criteria that require BM examination as essential criterion. We studied the morphological and clinical features in patients diagnosed according either to the BCSH (n=238) or the WHO guidelines (n=232). The BCSH-defined ET cohort was re-evaluated by applying the WHO classification. At presentation, patients of the BCSH group showed significantly higher values of serum lactate dehydrogenase and had palpable splenomegaly more frequently. Following the WHO criteria, the re-evaluation of the BCSH-diagnosed ET cohort displayed a heterogeneous population with 141 (59.2%) ET, 77 (32.4%) prefibrotic primary myelofibrosis (prePMF), 16 (6.7%) polycythemia vera and 4 (1.7%) primary myelofibrosis. Contrasting WHO-confirmed ET, the BCSH cohort revealed a significant worsening of fibrosis-free survival and prognosis. As demonstrated by the clinical data and different outcomes between WHO-diagnosed ET and prePMF, these adverse features were generated by the inadvertent inclusion of prePMF to the BCSH group. Taken together, the diagnosis of ET without a scrutinized examination of BM biopsy specimens will generate a heterogeneous cohort of patients impairing an appropriate clinical management.

Survival and prognosis among 1545 patients with contemporary polycythemia vera: an international study.

Under the auspices of an International Working Group, seven centers submitted diagnostic and follow-up information on 1545 patients with World Health Organization-defined polycythemia vera (PV). At diagnosis, median age was 61 years (51% females); thrombocytosis and venous thrombosis were more frequent in women and arterial thrombosis and abnormal karyotype in men. Considering patients from the center with the most mature follow-up information (n=337 with 44% of patients followed to death), median survival (14.1 years) was significantly worse than that of the age- and sex-matched US population (P<0.001). In multivariable analysis, survival for the entire study cohort (n=1545) was adversely affected by older age, leukocytosis, venous thrombosis and abnormal karyotype; a prognostic model that included the first three parameters delineated risk groups with median survivals of 10.9-27.8 years (hazard ratio (HR), 10.7; 95% confidence interval (CI): 7.7-15.0). Pruritus was identified as a favorable risk factor for survival. Cumulative hazard of leukemic transformation, with death as a competing risk, was 2.3% at 10 years and 5.5% at 15 years; risk factors included older age, abnormal karyotype and leukocytes ≥15 × 10(9)/l. Leukemic transformation was associated with treatment exposure to pipobroman or P32/chlorambucil. We found no association between leukemic transformation and hydroxyurea or busulfan use.

Deletions of the transcription factor Ikaros in myeloproliferative neoplasms.

Transformation to acute leukemia is a major complication of myeloproliferative neoplasms (MPNs), however, the genetic changes leading to transformation remain largely unknown. We screened nine patients with post-MPN leukemia for chromosomal aberrations using microarray karyotyping. Deletions on the short arm of chromosome 7 (del7p) emerged as a recurrent defect. We mapped the common deleted region to the IKZF1 gene, which encodes the transcription factor Ikaros. We further examined the frequency of IKZF1 deletions in a total of 29 post-MPN leukemia and 526 MPN patients without transformation and observed a strong association of IKZF1 deletions with post-MPN leukemia in two independent cohorts. Patients with IKZF1 loss showed complex karyotypes, and del7p was a late event in the genetic evolution of the MPN clone. IKZF1 deletions were observed in both undifferentiated and differentiated myeloid cell types, indicating that IKZF1 loss does not cause differentiation arrest but rather renders progenitors susceptible to transformation, most likely through chromosomal instability. Induced Ikzf1 haploinsufficiency in primary murine progenitors resulted in elevated Stat5 phosphorylation and increased cytokine-dependent growth, suggesting that reduced expression of IKZF1 is sufficient to perturb growth regulation. Thus, IKZF1 loss is an important step in the leukemic transformation of a subpopulation of MPN patients.

Autocrine cell suicide in a Burkitt lymphoma cell line (Daudi) induced by interferon alpha: involvement of tumor necrosis factor as ligand for the CD95 receptor.

The CD95 receptor, a member of the tumor necrosis factor (TNF) receptor superfamily, mediates signals for cell death on specific ligand or antibody engagement. It was hypothesized that interferon alpha (IFN-alpha) induces apoptosis through activation of the CD95-mediated pathway and that CD95 and ligands of the death domain may belong to the group of IFN-stimulated genes. Therefore, the effect of IFN-alpha on CD95-CD95L expression, on the release of TNF-alpha, and on TNF receptor 1 expression in an IFN-sensitive human Burkitt lymphoma cell line (Daudi) was investigated. After 5 days' incubation, apoptosis in 81% of IFN-alpha-treated Daudi cells was preceded by a release of TNF-alpha and an induction of CD95 receptor expression. Although supernatants of IFN-treated Daudi cells induced apoptosis of CD95-sensitive Jurkat cells, CD95L was undetectable on protein or on messenger RNA levels, and the weak initial expression of TNF receptor 1 increased only slightly during IFN treatment. Surprisingly, binding of TNF-alpha to CD95 was observed and confirmed by 3 different techniques-enzyme-linked immunosorbent assay using immobilized CD95:Fc-immunoglobulin G, immunoprecipitation assay using CD95 receptor precipitates of Daudi cells, and binding of sodium iodide 125-TNF-alpha to Daudi cells, which was strongly stimulated by IFN-alpha and inhibited by CD95L, CD95:Fc, unlabeled TNF-alpha, and anti-TNF-alpha antibody. Preincubation of Daudi cells with antagonists of the CD95-mediated pathway resulted in an inhibition of IFN-alpha-mediated cell death. The present investigation shows that IFN-alpha induces autocrine cell suicide of Daudi cells by a cross-talk between the CD95 receptor and TNF-alpha. The CD95 receptor can be considered a third TNF receptor, in addition to p55 and p75.

Arsenic trioxide and ascorbic acid: synergy with potential implications for the treatment of acute myeloid leukaemia?

Arsenic trioxide (As2O3) induces remission in a high proportion of patients with acute promyelocytic leukaemia (APL) via induction of apoptosis. Preliminary reports suggest that the apoptotic effect of As2O3 is not specific for APL but can also be observed in non-APL acute myeloid leukaemia (AML) cells, although these are less sensitive than APL cells. Ascorbic acid has recently been demonstrated to enhance the apoptotic effect of As2O3. We have therefore evaluated combined As2O3/ascorbic acid treatment in various clinical samples of AML. Our results indicate a significant synergistic effect of As2O3 and ascorbic acid, suggesting a possible future role of As2O3/ascorbic acid combination therapy in patients with AML.

Macrocytic anemia and thrombocytosis associated with thymoma: a case report.

Thymomas are often associated with autoimmune disorders. We report on a 45-year-old female patient with thymoma and hypogammaglobulinemia (Good's syndrome) who developed symptomatic macrocytic anemia (Hb 4.4 g/dl, MCV 112 fl) and thrombocytosis (Plt 442 G/l). Besides hypogammaglobulinemia (IgG 589 mg/dl), an inverted ratio of CD4(+)/CD8(+) cells was seen. The bone marrow biopsy showed a slightly hypercellular bone marrow with normal granulopoiesis, normal megakaryopoiesis and a mild dyserythropoiesis without any ring-sideroblasts. The in-vitro stem cell culture from the bone marrow revealed an atypical growth of macroclusters, reduced BFU-E and CFU-GEMM colony growth, whereas the CFU-GM colony growth was within the normal range. The chromosomal analysis showed a normal karyotype. The plasma vitamin B(12) and folate levels were within normal ranges, and we could not detect any autoantibodies. These findings excluded the differential diagnoses pure red cell aplasia (PRCA) and pernicious anemia. After resection of the thymoma of mixed cell type, the macrocytic anemia and thrombocytosis disappeared. The clinical course was complicated by a cerebral palsy and a life-threatening fungal septicemia after surgery. In the third year after thymectomy, hyporegenerative macrocytic anemia and thrombocytosis reappeared and an immunosuppressive treatment with prednisolone (1 mg/kg BW) was started. After initiation of the prednisolone therapy, reticulocyte counts increased and macrocytic anemia as well as thrombocytosis disappeared. The normalization of these laboratory parameters during glucocorticoid therapy suggests that in rare cases the constellation of macrocytic anemia, thrombocytosis and hypogammaglobulinemia may be due to an underlying immunologic mechanism.

The effect of interferon alpha on myeloproliferation and vascular complications in polycythemia vera.

The effect of interferon alpha (IFN) on myeloproliferation and vascular complications was studied in 32 patients (17 female, 15 male; median age 60.5 yr) with polycythemia vera (PV). IFN therapy was initiated at a median time of 19 months after diagnosis. Ten patients were pretreated with chemotherapy in addition to phlebotomy. IFN dose was 12 megaU/wk during the first year, 9 megaU/wk during the second year and 12 megaU/wk thereafter. During IFN alpha treatment hematocrit level was 45.7% and remained at this level after the second year of treatment, compared to 46.5% before IFN. The frequency of phlebotomy before IFN was 0.49/month and dropped to 0.19/month (p <0.0005) during the first year of IFN treatment. IFN normalized high platelet and leukocyte counts in a majority of patients. The incidence of deep venous thromboses was 3.6%/yr before IFN alpha and 1.8%/yr during the first year of treatment. IFN-induced side-effects were mainly flu-like symptoms, fever, fatigue and arthralgia. In conclusion, IFN allowed the reduction of the dose of chemotherapy and decreased the need of phlebotomy. Despite improvement of hematological parameters, it is still uncertain whether IFN alpha can improve clinical symptoms in PV.

MDR1 gene expression in chronic lymphocytic leukemia.

In order to assess the clinical role of the MDR1 gene in chronic lymphocytic leukemia (CLL), we determined its expression in the leukemic cells of 39 patients with CLL and compared this with other clinical and laboratory parameters. MDR1 RNA expression was detected in 29 patients. MDR1 RNA transcripts were independent of age, treatment status of the patients and the clinical stage of CLL, but correlated with the white blood cell count and MDR2 RNA transcripts. Expression of the tumor suppressor gene p53 was found in 30 out of 37 patients and was associated with MDR1 RNA expression (P < 0.001). Immunocytochemistry using the monoclonal antibody C219 was performed in 38 patients, and in 28 cases, more than 5% of the leukemic cells were found to express cell surface P-glycoprotein. P-glycoprotein expression correlated with the expression of MDR1 RNA (P = 0.048).