Andrea Camera

Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab

In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59(-)). The proportion of C3(+) RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom (51)Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess (51)Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.

Significant reduction of the hybrid BCR/ABL transcripts after induction and consolidation therapy is a powerful predictor of treatment response in adult Philadelphia-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has a dismal prognosis. We prospectively evaluated minimal residual disease (MRD) by measuring BCR/ABL levels with a quantitative real-time PCR procedure after induction and after consolidation in 45 adults with Ph+ ALL who obtained complete hematological remission after a high-dose daunorubicin induction schedule. At diagnosis, the mean BCR-ABL/GUS ratio was 1.55±1.78. A total of 42 patients evaluable for outcome analysis were operationally divided into two MRD groups: good molecular responders (GMRs; n=28) with >2 log reduction of residual disease after induction and >3 log reduction after consolidation therapy, and poor molecular responders (PMRs; n=14) who, despite complete hematological remission, had a higher MRD at both time points. In GMR, the actuarial probability of relapse-free, disease-free and overall survival at two years was 38, 27 and 48%, respectively, as compared to 0, 0 and 0% in PMR (P=0.0035, 0.0076 and 0.0026, respectively). Salvage therapy induced a second sustained complete hematological remission in three GMR patients, but in no PMR patient. Our data indicate that, as already shown in children, adult Ph+ ALL patients have a heterogeneous sensitivity to treatment, and that early quantification of residual disease is a prognostic parameter in this disease.

Acute lymphoblastic leukaemia occurring as second malignancy : report of the GIMEMA Archive of Adult Acute Leukaemia

Between July 1992 and June 1996, 901 new cases of adult acute lymphoblastic leukaemia were recorded in the GIMEMA Archive of Adult Acute Leukaemia; 21 of them (2.3%) had a previous primary malignancy (PM). We found that secondary acute lymphoblastic leukaemia cases (sALL) presented with older age, a high incidence of pre‐pre‐B immunophenotype and a significantly higher prevalence of cancer among relatives compared to de novo ALL.

Treatment of adults with acute lymphoblastic leukaemia in first bone marrow relapse: results of the ALL R-87 protocol

Sixty‐one adults aged <55 years with acute lymphoblastic leukaemia (ALL) in first bone marrow relapse were enrolled in an Italian cooperative study (ALL R‐87 protocol) from 12 GIMEMA Institutions. The treatment programme consisted of: (1) an induction phase with intermediate‐dose cytarabine (IDARA‐C 1 g/m2, 6 h daily infusion ×6 d) plus idarubicin (IDA; 5 mg/m2/d × 6 d) and prednisone (40 mg/m2/d × 21 d), (2) a consolidation phase followed by (3) bone marrow transplant (BMT). Median first complete remission (CR) duration was 8.5 months (range 1–54 months). 34/61 patients achieved CR (56%); 24 (39%) failed to respond and three (5%) died during induction. Most responders (24 patients) could not enter the BMT programme; 15 relapsed early (median time to relapse 2 months); nine were withdrawn due to toxicity and one died in CR of infection. Nine of the 34 CRs underwent BMT (five autologous and four allogeneic). Three of the four allotransplanted patients are alive in continuous CR at 22, 43 and 63 months; only one of the five who underwent an autologous BMT is alive in CR at 46 months.

CD200/OX2, a cell surface molecule with immuno-regulatory function, is consistently expressed on hairy cell leukaemia neoplastic cells

CD200 (formerly called OX2) is a transmembrane glycoprotein with immunosuppressive functions. It is expressed on normal B-lymphocytes, T-lymphocytes, dendritic cells and several solid tissues (Kawasaki & Farrar, 2008). CD200 receptor expression is limited to myeloid leucocytes and a subset of Tlymphocytes (Kawasaki & Farrar, 2008). In mouse systems, the binding of CD200 to its receptor (i) decreases the production of T-helper cell type 1 (Th1)-like cytokines, such as interleukin (IL)-2 and interferon c, (ii) increases the release of Th2-like cytokines, such as IL-10 and IL-4 (Gorczynski, 2001) and (iii) promotes the in vitro differentiation of T cells toward CD4CD25Foxp3 Treg lymphocytes (Gorczynski et al, 2008). CD200 is constantly overexpressed on chronic lymphocytic leukaemia (CLL) cells (McWhirter et al, 2006). The addition of CLL cells to mixed lymphocyte reactions causes an immunological shift from a Th1-like response to a Th2-like response, confirming that CD200 plays an important role in controlling T-cytotoxic immune response (McWhirter et al, 2006). Starting from these data, we extended the investigation of CD200 expression to another B-chronic lymphoproliferative disorder i.e. hairy cell leukaemia (HCL). Hairy cell leukaemia is a distinct disease entity in the World Health Organization (WHO) classification, displaying unique clinico-pathological and biological features (Tiacci et al, 2006). As hairy cells display a specific immunophenotype, multicolour flow cytometry is currently the best tool for HCL diagnosis. A total of 10 specimens (six peripheral blood samples and four bone marrow aspirates), collected from 10 patients with newly diagnosed HCL, were studied. As normal controls we analysed 10 peripheral blood specimens and two bone marrow aspirates from 12 healthy donors. An aliquot (50 ll) of each sample was incubated at 4 C for 30 min in the presence of appropriate amounts of monoclonal antibodies. The mixtures were then diluted 1:20 in ammonium chloride lysing solution, incubated at room temperature for 10 min and finally washed prior to flow cytometric analysis with the FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA). The following antigens were analysed: CD200, SmIg-kappa, SmIg-lambda, CD45, CD19, CD5, CD23, CD20, CD22, CD103, CD11c, CD25, CD43, CD10, CD3, CD56 and CD81. Hairy cells were gated as CD45CD19 ‘monocytoid cells’ (i.e. cells with light scatter features typical of monocytes). In addition, in the majority of cases, we also were able to perform a full immunological gate on CD45 CD19CD103CD11c cells. With regard to the normal controls included in our study, B-lymphocytes were simply gated as CD45CD19 cells. In all specimens cell doublets and debris were excluded from our analysis by forward-scatter versus side-scatter dotplot examination. To set the cut-off point to distinguish between CD200 negative and positive cells, we used the ‘Fluorescence Minus One’ technique as described by Perfetto et al (2004). A single case was arbitrarily judged CD200 positive when the percentage of positive cells (PPC) was higher than 30%. All HCL samples were CD200 positive with PPC and median fluorescence intensity (MFI) median values of 99 (25th–75th percentile 92–99) and 3016 (25th–75th percentile 1382–5430), respectively. Although CD200 was positive in 12 out of 12 normal controls, the PPC and MFI median values were of 71 (25th–75th percentile 64–83) and 582 (25th–75th percentile 406–725), respectively. Differences in PPCs and MFIs between HCLs and normal controls were statistically significant (Mann–Whitney U, two-tailed testing, P < 0Æ0001). Data regarding MFI analysis are shown in Fig 1. Whereas normal controls showed weak CD200 fluorescence intensity with a bimodal distribution, HCL samples showed bright CD200 expression in a homogeneous pattern (Fig 2). This is the first documented direct evidence of CD200 overexpression in HCL. As described above, CD200 promotes Th2-like cytokines synthesis. IL-4 and IL-10 are reported to reduce anti-tumour cytotoxic T cell response (McWhirter

Characterization of two novel cell lines, DERL-2 (CD56 + /CD3 + /TCRγδ + ) and DERL-7 (CD56 + /CD3 − /TCRγδ − ), derived from a single patient with CD56 + non-Hodgkin's lymphoma

Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic γδ T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRγδ+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRγδ+ while DERL-7 was CD56+/CD3−/TcRγδ−. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes β, γ and δ, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.

Immunophenotype of Acute Lymphoblastic Leukemia Cells: The Experience of the Italian Cooperative Group (Gimema)

The immunophenotype of 304 adult lymphoblastic leukemias (> 18 years) diagnosed on the basis of the FAB criteria was determined at the time of diagnosis using a panel of monoclonal antibodies. The series comprised cases diagnosed and immunophenotyped in 43 Italian centers (GIMEMA Cooperative Group) between April 1988 and June 1991. The immunophenotypic characterization consisted of two consecutive steps. The initial screening was based on the reactivity for TdT, HLA-Dr, CD7, CD10, CD13, CD19, CD24, CD33 and CD41. According to the results obtained, the second level of investigation assessed the positivity for intra cytoplasmic (Cy) Ig, CD1a, CD2, CD3, CD4, CD5, CD8 and CD20. Based on the hierarchical expression of the different B- and T-cell related antigens, each case was assigned to a given differentiation stage. B-lineage ALL were classified in five subgroups (B0-B4) and T-lineage ALL in four subgroups (T0-T3). Cases in which the blasts were lymphoid according to the FAB criteria, but expressed myeloid antigens in association with B- and T-lymphoid markers were defined as hybrid leukemias. As expected, CD10+ cases (B2-B3) were the most frequent within the B-lineage ALL (83.2% of cases). CyIg+ (B3) accounted for about 20% of CD10+ ALL. Twenty eight cases (13.4%) were at a pre-cALL stage (B0-B1) and of these, 8 (3.8% of the total series) were positive only for TdT and HLA-Dr (B0). Intermediate and mature thymic phenotypes (T2-T3) were predominant within the T-ALL (67.2%) groups. Five cases, were positive only for TdT and CD7 (CD5+), and classified as T0. 9.2% of cases fulfilled the definition of hybrid leukemia, largely in view of the co-expression of B-lymphoid and myeloid markers.(ABSTRACT TRUNCATED AT 250 WORDS)

M4 acute myeloid leukemia: the role of eosinophilia and cytogenetics in treatment response and survival. The GIMEMA experience.

This analysis on a large GIMEMA population of patients with M4-acute myeloid leukemia confirmed the favorable prognostic role of inv(16), demonstrated the good prognostic role of eosinophilia and revealed an enhancement of the effect when the two factors were both present. See related perspective on page 976. Background Myelomonocytic acute myeloid leukemia (M4-AML) is frequently associated with the cytogenetic marker inv(16) and/or the presence of eosinophilia. The aim of this study was to analyze the incidence and prognostic role of these factors in a large series of patients. Design and Methods Adult patients with acute myeloid leukemia consecutively enrolled in the GIMEMA trials AML10 and LAM99p were retrospectively analyzed. Results Among 1686 patients, 400 cases of M4-AML were identified; of these, 78% had neither eosinophilia nor inv(16), 6% had eosinophilia only, 8% had inv(16) only and 8% had both. Univariate analysis showed that both eosinophilia and inv(16) were correlated with a higher probability of complete remission, lower resistance to chemotherapy and increased overall survival. Multivariate analysis showed that the simultaneous presence of the two factors significantly increased the probabilities of both complete remission and overall survival. The presence of only one of the two factors also increased the probabilities of complete remission and overall survival, but not to a statistically significant extent. The relapse-free survival of the responding patients was not influenced by the two factors. Conclusions In a large series of patients with M4-AML we confirmed the favorable role of inv(16), but the weight of this factor among the whole M4 population was of limited relevance. Eosinophilia, which affects a small proportion of cases, also emerged as a favorable prognostic factor. Based on the results of this large case population, overall and relapse-free survival rates of patients with M4-AML are not significantly better than those of patients with non-M4 AML, while the concomitant presence of both inv(16) and eosinophilia was associated with a significantly improved prognosis.

Secondary haematological neoplasm after treatment of adult acute lymphoblastic leukaemia: analysis of 1170 adult ALL patients enrolled in the GIMEMA trials

Between 1983 and 1994 the incidence of secondary haematological neoplasms (SHM) was evaluated in 1170 new cases of ALL enrolled in the GIMEMA trials. Of the 942 patients who achieved complete remission (CR); seven developed a SHM: four AMLs and three NHLs.