Miroslaw Majewski

Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3‐kinase (PI‐3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI‐3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI‐3k activation failed to stimulate Akt kinase, a recently identified PI‐3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI‐3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI‐3k specific inhibitor at low concentrations, suppressed BCR/ABL‐dependent colony formation of murine marrow cells, and that a kinase‐deficient Akt mutant with dominant‐negative activity inhibited BCR/ABL‐dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation‐defective SH2 domain BCR/ABL mutants to induce growth factor‐independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c‐Myc and Bcl‐2; in contrast, expression of a constitutively active Akt mutant induced Bcl‐2 and c‐Myc expression, and stimulated the transcription activation function of c‐Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI‐3k activation and document the essential role of the PI‐3k/Akt pathway in BCR/ABL leukemogenesis.

Multilevel Dysregulation of STAT3 Activation in Anaplastic Lymphoma Kinase-Positive T/Null-Cell Lymphoma

Accumulating evidence indicates that expression of anaplastic lymphoma kinase (ALK), typically due to t(2;5) translocation, defines a distinct type of T/null-cell lymphoma (TCL). The resulting nucleophosmin (NPM) /ALK chimeric kinase is constitutively active and oncogenic. Downstream effector molecules triggered by NPM/ALK remain, however, largely unidentified. Here we report that NPM/ALK induces continuous activation of STAT3. STAT3 displayed tyrosine phosphorylation and DNA binding in all (four of four) ALK+ TCL cell lines tested. The activation of STAT3 was selective because none of the other known STATs was consistently tyrosine phosphorylated in these cell lines. In addition, malignant cells in tissue sections from all (10 of 10) ALK+ TCL patients expressed tyrosine-phosphorylated STAT3. Transfection of BaF3 cells with NPM/ALK resulted in tyrosine phosphorylation of STAT3. Furthermore, STAT3 was constitutively associated with NPM/ALK in the ALK+ TCL cell lines. Additional studies into the mechanisms of STAT3 activation revealed that the ALK+ TCL cells expressed a positive regulator of STAT3 activation, protein phosphatase 2A (PP2A), which was constitutively associated with STAT3. Treatment with the PP2A inhibitor calyculin A abrogated tyrosine phosphorylation of STAT3. Finally, ALK+ T cells failed to express a negative regulator of activated STAT3, protein inhibitor of activated STAT3. These data indicate that NPM/ALK activates STAT3 and that PP2A and lack of protein inhibitor of activated STAT3 may be important in maintaining STAT3 in the activated state in the ALK+ TCL cells. These results also suggest that activated STAT3, which is known to display oncogenic properties, as well as its regulatory molecules may represent attractive targets for novel therapies in ALK+ TCL.

Immunosuppressive tor kinase inhibitor everolimus (RAD) suppresses growth of cells derived from posttransplant lymphoproliferative disorder at allograft-protecting doses

Background. Posttransplant lymphoproliferative disorders (PTLDs) represent a life-threatening complication of standard immunosuppressive therapy. The impact of novel, rapamycin-related immunosuppressive drugs on the pathogenesis of PTLDs remains undefined. Methods. We tested the effect of everolimus (RAD, Novartis Pharma AG, Basel, Switzerland) on human PTLD-derived cells using in vitro assays and an in vivo severe combined immunodeficiency disease mouse xenotransplant model. Results. Everolimus profoundly inhibited the proliferation, cell-cycle progression, and survival of the PTLD-1 cell line established from a pulmonary PTLD. Equally profound inhibition of PTLD-1 growth was achieved in vivo at well-tolerated everolimus doses of 0.5 to 5 mg/kg per day. Five mg/kg per day of everolimus, given once per day, inhibited PTLD-1 tumor volume gain by more than 10-fold in treated mice compared with untreated mice. Because the subsequent pharmacokinetic analysis indicated rapid everolimus absorption, distribution, and clearance in mice (with a half-life of 3 to 6 hr and maximum drug blood concentration reached after 0.5 to 1 hr), treatment was changed to a twice-daily regimen. Everolimus given twice daily at 0.5 mg/kg per dose inhibited tumor-volume gain by more than 60-fold and at 0.25 mg/kg per dose by more than 10-fold. Similar everolimus doses were required to prevent graft rejection in a mouse heart allotransplantation model; the highest dose tested (1.5 mg/kg twice daily) resulted in long-term graft survival in all mice that underwent transplantation. Conclusions. Everolimus displays a potent inhibitory effect on PTLD-derived cells in vitro and in vivo in a dose range leading to prevention of allograft rejection and may prove effective in both the prevention and treatment of PTLDs in transplant patients.

A single weekly dose of imatinib is sufficient to induce and maintain remission of chronic eosinophilic leukaemia in FIP1L1-PDGFRA-expressing patients

Hypereosinophilic syndrome (HES) is defined as chronic, unexplained hypereosinophilia with organ involvement. A subset of HES patients presents an interstitial deletion in chromosome 4q12, which leads to the expression of an imatinib‐responsive fusion gene, FIP1L1‐PDGFRA. These patients are diagnosed as chronic eosinophilic leukaemia (CEL). We treated seven CEL and HES patients, six of which expressed FIP1L1‐PDGFRA, with imatinib using initial daily doses ranging from 100 to 400 mg. In a remission maintenance phase, the patients were treated with imatinib once weekly. All imatinib‐treated patients achieved a complete haematological remission (CHR), and five of the six patients with FIP1L1‐PDGFRA expression exhibited molecular remission. The decreased imatinib doses were as follows: 200 mg/week in three patients, 100 mg/week in two patients and 100 mg/d in the remaining two patients. For remission maintenance, imatinib doses were set at 100 mg/week in five patients and 200 mg/week in two patients. At a median follow‐up of 30 months all patients remained in CHR and FIP1L1‐PDGFRA expression was undetectable in five of the six FIP1L1‐PDGFRA‐expressing patients. These data suggest that a single weekly dose of imatinib is sufficient to maintain remission in FIP1L1‐PDGFRA‐ positive CEL patients.

T-cell abnormalities are present at high frequencies in patients with hypereosinophilic syndrome

A T-cell clone, identified by clonal rearrangement of the T-cell receptor and by the presence of aberrant T-cell immunophenotype in peripheral blood, defines lymphocytic variant of hypereosinophilic syndrome. This study shows that T-cell abnormalities are present at high frequencies in patients with hypereosinophilic syndrome. See related perspective article on page 1188. Background A T-cell clone, thought to be the source of eosinophilopoietic cytokines, identified by clonal rearrangement of the T-cell receptor and by the presence of aberrant T-cell immunophenotype in peripheral blood defines lymphocytic variant of hypereosinophilic syndrome (L-HES). Design and Methods Peripheral blood samples from 42 patients who satisfied the diagnostic criteria for HES were studied for T-cell receptor clonal rearrangement by polymerase chain reaction according to BIOMED-2. The T-cell immunophenotype population was assessed in peripheral blood by flow cytometry. The FIP1L1-PDGFRA fusion gene was detected by nested polymerase chain reaction. Results Forty-two HES patients (18 males and 24 females) with a median age at diagnosis of 56 years (range 17–84) were examined in this study. Their median white blood cell count was 12.9×109/L (range 5.3–121), with an absolute eosinophil count of 4.5×109/L (range 1.5–99) and a median eosinophilic bone marrow infiltration of 30% (range 11–64). Among the 42 patients, clonal T-cell receptor rearrangements were detected in 18 patients (42.8%). Patients with T-cell receptor clonality included: T-cell receptor β in 15 patients (35%), T-cell receptor γ in 9 (21%) and T-cell receptor δ in 9 (21%) patients, respectively. Clonality was detected in all three T-cell receptor loci in 4 cases, in two loci in 7 patients and in one T-cell receptor locus in the remaining 7 patients. The FIP1L1-PDGFRA fusion transcript was absent in all but 2 patients with T-cell receptor clonality. Three patients out of 42 revealed an aberrant T-cell immunophenotype. In some patients, an abnormal CD4:CD8 ratio was demonstrated. Conclusions T-cell abnormalities are present at high frequencies in patients with HES.

Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy

IntroductionThe purpose of this study was to analyze the data of patients with T-cell large granular lymphocyte (T-LGL) lymphocytosis associated with inflammatory arthropathy or with no arthritis symptoms.MethodsClinical, serological as well as histopathological, immuhistochemical, and flow cytometric evaluations of blood/bone marrow of 21 patients with T-LGL lymphocytosis were performed. The bone marrow samples were also investigated for T-cell receptor (TCR) and immunoglobulin (IG) gene rearrangements by polymerase chain reaction with heteroduplex analysis.ResultsNeutropenia was observed in 21 patients, splenomegaly in 10, autoimmune diseases such as rheumatoid arthritis (RA) in 9, unclassified arthritis resembling RA in 2, and autoimmune thyroiditis in 5 patients. T-LGL leukemia was recognized in 19 cases. Features of Felty syndrome were observed in all RA patients, representing a spectrum of T-LGL proliferations from reactive polyclonal through transitional between reactive and monoclonal to T-LGL leukemia. Bone marrow trephines from T-LGL leukemia patients showed interstitial clusters and intrasinusoidal linear infiltrations of CD3+/CD8+/CD57+/granzyme B+ lymphocytes, reactive lymphoid nodules, and decreased or normal granulocyte precursor count with left-shifted maturation. In three-color flow cytometry (FCM), T-LGL leukemia cells demonstrated CD2, CD3, and CD8 expression as well as a combination of CD16, CD56, or CD57. Abnormalities of other T-cell antigen expressions (especially CD5, CD7, and CD43) were also detected. In patients with polyclonal T-LGL lymphocytosis, T cells were dispersed in the bone marrow and the expression of pan-T-cell antigens in FCM was normal. Molecular studies revealed TCRB and TCRG gene rearrangements in 13 patients and TCRB, TCRG, and TCRD in 4 patients. The most frequently rearranged regions of variable genes were Vβ-Jβ1, Jβ2 and Vγ If Vγ10-Jγ. Moreover, in 4 patients, additional rearrangements of IG kappa and lambda variable genes of B cells were also observed.ConclusionRA and neutropenia patients represented a continuous spectrum of T-LGL proliferations, although monoclonal expansions were most frequently observed. The histopathological pattern and immunophenotype of bone marrow infiltration as well as molecular characteristics were similar in T-LGL leukemia patients with and without arthritis.

Expression of SHP-1 phosphatase indicates post-germinal center cell derivation of B-cell posttransplant lymphoproliferative disorders

SHP-1 tyrosine phosphatase acts as a negative regulator of signaling by receptors for growth factors, cytokines, and chemokines and by receptors involved in immune response. Our recent study showed that SHP-1 is tightly regulated at various stages of B-cell differentiation and is expressed in the mantle and marginal zones, interfollicular B cells, and plasma cells, whereas it is nondetectable in germinal center cells. In this study we evaluated expression of SHP-1 in vitro and in vivo in nine cell lines representing three different types of EBV+ B-cell populations closely resembling or derived from posttransplant lymphoproliferative disorders (PTLDs). Furthermore, we examined tissue samples from 58 patients with B-cell PTLDs, both EBV+ (85% of the cases analyzed) and EBV− (15%). SHP-1 protein was strongly expressed in all cell lines and PTLD cases. In addition, the PTLD cases were essentially negative for germinal center B-cell markers: none expressed CD10 and only one expressed BCL-6. More than 40% expressed a late post-germinal B-cell marker, CD138. The universal expression of SHP-1, lack of expression of CD10 and BCL-6, and frequent expression of CD138 suggest that PTLDs are derived from post-germinal center B cells regardless of the EBV cell infection status. Based on the immunophenotype, B-cell PTLDs could be divided into two broad categories corresponding to the early (CD10−/BCL-6−/SHP-1+/CD138−) and late (CD10−/BCL-6−/SHP-1+/CD138+) post-germinal center cells. By being expressed earlier, SHP-1 is a more sensitive marker of post-germinal center B cells than CD138, which is seen on the terminally differentiated immunoblasts and plasma cells.

High incidence of ancestral HLA haplotype 8.1 and monoclonal incomplete DH–JH immunoglobulin heavy chain gene rearrangement in persistent polyclonal B-cell lymphocytosis

Dear Editor, Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare, benign, and usually asymptomatic entity, characterized by sustained B-cell CD5 lymphocytosis with normal surface ./1 chains ratio, with bilobulated and/or binuclear cells and elevated serum polyclonal IgM level. The increased frequency of HLA-DR7 haplotype and chromosomal clonal aberrations in particular concerning the chromosome 3 suggest a genetic background of this entity [1, 4]. We studied four women with PPBL, aged 48 to 51 years, for the clonality of rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in peripheral blood lymphocytes by 14 multiplex PCR reactions with different primers: three VH–JH (IGH), two DH–JH (IGH), two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), and one TCR delta (TCRD) [5]. Peripheral lymphocytes were also studied with classical cytogenetic and FISH analyses, using 3q/3pspecific probes. All patients and 200 healthy ethnically matched controls were HLA typed for A, B, Cw, and DRB1 genes by PCR-based sequence-specific primer amplification using SSP kits (Olerup, Sweden). Three out of four patients studied were asymptomatic, while one presented recurrent bacterial infections and decreased serum IgA and IgG levels. In all patients studied, peripheral lymphocytosis ranged from 6.1 to 10.5×10/l for a minimum of 5 years, with predominance of CD19/CD5 cells. Binucleated/ bilobulated lymphocytes were present on the blood smear, surface kappa/lambda chains ratios were normal, and serum levels of polyclonal IgM were increased. Cytogenetic studies revealed abnormalities in one patient (+i(3)(q10), +18, i(18)(q10), rob(13;14)(10;q10), with the presence of i(3)(q10) in 10% of interphase nuclei). No patients revealed the monoclonality in IGH VH–JH, IGK, IGL, nor TCR genes, but we found incomplete monoclonal DH7–JH IGH rearrangements in two patients. The observed DH–JH genes rearrangements were not related to other upstream DH or downstream JH genes. HLA typing showed DRB1*07 allele in 2/4 patients and in 58/200 controls (p=0.38). We found significantly higher frequency of Cw*07–B*08– Ann Hematol (2008) 87:597–598 DOI 10.1007/s00277-007-0434-z

Biclonal T-cell receptor gammadelta+ large granular lymphocyte leukemia associated with rheumatoid arthritis.

T-cell large granular lymphocyte (T-LGL) leukemia is a rare disorder of mature activated cytotoxic T lymphocytes with monoclonal T-cell receptor (TCR) genes rearrangements. T-LGL leukemias usually express CD3þ/CD47/CD8þ/CD57þ/TCRabþ although other variants like: CD3þ/CD4þ/CD87/ TCRabþ or CD3þ/CD4þ/CD8þ/TCRabþ have also been observed [1]. TCRgdþ LGL leukemias represent a rare (about 5%) subset of CD3þ T-LGL leukemias with a clinical presentation similar to TCRabþ LGL leukemia. The clinical course is indolent, associated with cytopenias (mainly neutropenia), autoimmune disorders and less often, splenomegaly. The most common phenotype of leukemic cells is CD2þ/CD3þ/CD47/CD5þ/CD7þ/CD8þ/ CD57þ and a variable expression of CD16, CD56, CD11b, CD11c [2,3]. Two large series of patients with gdþ T-LGL leukemia have recently been published [2,3], but to our knowledge only one case of a well documented biclonal T-LGL leukemia was reported [4]. We present a case of T-LGL leukaemia associated with long-lasting rheumatoid arthritis and proliferation of two immunophenotypically slightly different populations of T-cells (CD3þ/ CD47/CD8þ and CD3þ/CD47/CD87) detected by flow cytometry (FCM) as well as the presence of distinct TCRG and TCRD genes rearrangements. A 58-year-old female was referred to the Department of Hematology, Institute of Hematology and Transfusion Medicine for investigation of mild leucocytosis and lymphocytosis persisting for 1 year. The patient had a 32-year history of rheumatoid arthritis with severe changes in numerous joints and positive rheumatoid factor, anticyclic citrullinated peptide antibodies and antinuclear antibodies. The medical history included bilateral hip and right knee replacement surgery and autoimmune thyroiditis. The patient has been treated with Meloxicam 15 mg and Methylprednisolone 6 mg daily as well as 7.5 mg Methotrexate once a week. Blood counts were as follows: white blood cells 9.26 10/L, lymphocytes 6.66 10/L, neutrophils 1.76 10/L, hemoglobin 13.4 g/dL and platelets 2306 10/L. In peripheral blood smear majority of lymphocytes had abundant cytoplasm and azurophilic granules consistent with LGLs. There was no hepatosplenomegaly or lymphadenopathy on physical examination and ultrasonography. Biochemical markers were normal except for polyclonal hypergammaglobulinemia. The patient was seronegative for EBV, CMV, HBV, HCV, HIV and HTLV-I. Bone marrow was normocellular in histopathological evaluation of trephine biopsy. Immunohistochemical stainings revealed subtle interstitial clusters and intrasinusoidal linear infiltrations of small