Svetlana Krichevsky

Age related microsatellite instability in T cells from healthy individuals.

Many immune functions decline with age and may jeopardize the elderly, as illustrated, for example by the significantly higher mortality rate from influenza in old age. Although innate and humoral immunity are affected by aging, it is the T cell compartment, which manifests most alterations. The mechanisms behind these alterations are still unclear, and several explanations have been offered including thymic involution and Telomere attrition leading to cell senescence. Age related accumulation of mutations has been documented and could serve as an additional mechanism of T cell dysfunction. One effective repair mechanism capable of rectifying errors in DNA replications is the mismatch repair (MMR) system. We previously reported a comparative examination of individual DNA samples from blood cells obtained at 10 year intervals from young and old subjects. We showed significantly higher rates of microsatellite instability (MSI), an indicator of MMR dysfunction in older subjects, compared to young. In the present study we confirm this result, using direct automated sequencing and in addition, we demonstrate that as CD8 lymphocytes from aged individuals, undergo repeated population doublings (PDs) in culture, they develop MSI. CD4 clones that also undergo repeated PDs in culture develop significant MSI as well. Elucidation of this previously unexplored facet of lymphocyte dynamics in relation to aging may help identify novel mechanisms of immunosenescence and pathways that could serve as targets for interventions to restore immune function.

Treatment of Chronic Myeloid Leukemia Cells with Imatinib (STI571) Impairs p53 Accumulation in Response to DNA Damage

Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.

Reversed BCR/ABL rearrangement detected by FISH in Philadelphia negative chronic myelocytic leukemia

We investigated leukemic cells from a patient with chronic myelocytic leukemia (CML) and a normal 46,XX karyotype. Molecular studies revealed rearrangement of the M-bcr region and formation of BCR/ABL fusion mRNA with b3a2 configuration. Fluorescence in situ hybridization (FISH) using the abl probe showed signal on both chromosomes 9 band q34, while the bcr probe hybridized to one chromosome 22 and to one chromosome 9. In this case, as in three other cases recently described (Hagemeijer et al. and Nachava et al.), the bcr/abl rearrangement is shown to be on 9q34, instead of the usual location on 22q11.

Janus Kinase V617F mutation in cigarette smokers

The JAK2 V617F mutation is responsible for the constitutive activation of the erythropoietin receptor signaling pathway in most cases of polycythemia vera (PV). The mutation has also been described in healthy people. As smoking may result in secondary polycythemia, the goal of this trial was to examine the effect of smoking on the prevalence of the JAK2 mutation and its correlation to erythrocytosis. The study was case–control. Hospitalized smokers (n = 81) and nonsmokers (n = 61) were recruited. Serum was drawn for complete blood count, erythropoietin, ferritin and venous blood gases. JAK2 mutation was analyzed by highly sensitive allele‐specific Quantitative Real Time PCR. The JAK2 mutation was found in 29/81 (35.8%) of smokers in comparison to only 9/61 (14.8%) of the control group (P = 0.007). The frequency of the mutation among smokers who were positive for the JAK2 mutation had a mean of 6.78 × 10−4 ± 1.08 × 10−3 vs. 1.51 × 10−4 ± 2.04 × 10−4 among nonsmokers (P = 0.027). Both frequencies are much lower than those found in PV. There was a medium correlation between older age and mutation frequency in nonsmokers (r= 0.67, P = 0.043). Hematocrit was higher in smokers (47.8 ± 6 vs. 41.7 ± 4.7, P < 0.0001), but no correlation was found to JAK2 mutation. In a cohort of hospitalized smokers and nonsmokers, JAK2 mutation was more prevalent and found in higher frequencies among smokers than nonsmokers. We suggest that accelerated erythropoiesis renders the cells susceptible to JAK2 mutation. Am. J. Hematol., 2011.

Factors related to the development of acquired von Willebrand syndrome in patients with essential thrombocythemia and polycythemia vera

OBJECTIVE We characterized acquired von Willebrand syndrome (AVWS) among essential thrombocythemia (ET) and polycythemia vera (PV) patients. METHODS A review of patients with ET or PV evaluated for AVWS. RESULTS Of 116 patients with ET, 64 (55%) developed AVWS; of 57 with PV, 28 (49%) developed AVWS. Median platelet counts of ET and PV patients who developed AVWS were 920×109/L and 679×109/L, respectively (P=0.01). Of patients who developed AVWS, 69.5% had platelet counts below 1000×109/L. Bleeding was more common in patients with AVWS, among both ET and PV patients (P<0.001). VWF:RCo levels and VWF:RCo/VWF:Ag ratio were lower among JAK2 V617F positive- vs. JAK2 V617F negative- ET patients (P=0.02 and P=0.002, respectively); whereas VWF:Ag levels were comparable (P=0.96). ET patients harboring the JAK2 V617F mutation were more likely to develop AVWS than were calreticulin-positive patients (70.3% vs. 45.7%, P=0.02), despite lower platelet counts (median 773 vs. 920×109/L, P=0.05). In multivariable analysis, younger age (P=0.002), platelet count (P<0.001), hemoglobin level (P=0.01) and JAK2 V617F mutation (P=0.01) independently predicted the development of AVWS among ET patients; whereas only platelet count predicted its development among PV patients (P<0.001). CONCLUSION Among ET and PV patients, AVWS was common and associated with higher bleeding rates and higher platelet count; nonetheless, most AVWS patients had platelet counts under 1000×109/L. Thus, AVWS screening should be included in routine assessment of ET and PV patients. Among ET patients, JAK2 V617F was a main driver for the development of AVWS.

The prognostic value of bone marrow involvement at the molecular level in aggressive lymphoma

Abstract We retrospectively studied the prognostic role of molecular (gene rearrangement, GRR) bone marrow (BM) involvement in diffuse large B-cell lymphoma (DLBCL, 424 patients) and in peripheral T-cell lymphoma (PTCL, 67 patients). When correlating BM GRR to histological findings at diagnosis, the GRR test was more sensitive (p = 0.036) but less specific (p < 0.0001) in PTCL than in DLBCL. For DLBCL (but not PTCL), a positive BM GRR correlated with advanced stage (p = 0.0001) and high IPI (p = 0.002), and worsened the progression free survival (PFS) (p = 0.05) and overall survival (OS) (p = 0.01), irrespective of rituximab treatment. Histologic negative/GRR positive cases had worse PFS/OS (p < 0.0001) than histologic/GRR double negative cases, however BM GRR was not an independent prognostic survival factor. End-of-treatment BM GRR did not predict survival. We conclude that BM GRR is unjustified as a prognostic tool for PTCL and should be reserved for a subset of DLBCL patients with negative histology of the BM.

The JAK2V617F mutation in normal individuals takes place in differentiating cells

The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 17.3% (17/98) of normal donors; the mutant allele burden was, however, very low (<0.01% compared to >1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34+ cells. Erythropoietin-stimulated differentiation of normal CD34+ cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F.