Gianpietro Semenzato

BRAF Mutations in Hairy-Cell Leukemia

BACKGROUND Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Aberrant Wnt/β-Catenin Pathway Activation in Idiopathic Pulmonary Fibrosis

To investigate the molecular events that may underpin dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we analyzed the expression patterns of β-catenin on 20 IPF/UIP lung samples, together with two downstream target genes of Wnt signaling, cyclin-D1, and matrilysin. In 18 of 20 cases of IPF/UIP investigated on serial sections, nuclear β-catenin immunoreactivity and abnormal levels of cyclin-D1 and matrilysin were demonstrated in proliferative bronchiolar lesions (basal-cell hyperplasia, squamous metaplasia, bronchiolization, honeycombing). The nature of these lesions was precisely defined using specific markers (ΔN-p63, surfactant-protein-A, cytokeratin-5). Interestingly, nuclear β-catenin accumulation was also demonstrated in fibroblast foci in most (16 of 20) IPF/UIP samples, often associated with bronchiolar lesions. Similar features were not observed in normal lung and other fibrosing pulmonary diseases (diffuse alveolar damage, organizing pneumonia, nonspecific interstitial pneumonia, desquamative interstitial pneumonia). Sequence analysis performed on DNA extracted from three samples of IPF/UIP did not reveal abnormalities affecting the β-catenin gene. On the basis of these findings new models for IPF/UIP pathogenesis can be hypothesized, centered on the aberrant activation of Wnt/β-catenin signaling, with eventual triggering of divergent epithelial regeneration at bronchiolo-alveolar junctions and epithelial-mesenchymal-transitions, leading to severe and irreversible remodeling of the pulmonary tissue.

Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis

B cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Analysis of B cells freshly isolated from 40 patients with chronic lymphocytic leukemia demonstrated that the Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, displays anomalous properties. Lyn is remarkably overexpressed at the protein level in leukemic cells as compared with normal B lymphocytes, with a substantial aliquot of the kinase anomalously present in the cytosol. Whereas in normal B lymphocytes Lyn activation is dependent on B cell-receptor stimulation, in resting malignant cells, the constitutive activity of the kinase accounts for high basal protein tyrosine phosphorylation and low responsiveness to IgM ligation. Addition of the Lyn inhibitors PP2 and SU6656 to leukemic cell cultures restores cell apoptosis, and treatment of malignant cells with drugs that induce cell apoptosis decreases both activity and amount of the tyrosine kinase. These findings suggest a direct correlation between high basal Lyn activity and defects in the induction of apoptosis in leukemic cells. They also support a critical role for Lyn in B-CLL pathogenesis and identify this tyrosine kinase as a potential therapeutic target.

CXCR3 and Its Ligand CXCL10 Are Expressed by Inflammatory Cells Infiltrating Lung Allografts and Mediate Chemotaxis of T Cells at Sites of Rejection

The attraction of T lymphocytes into the pulmonary parenchyma represents an essential step in mechanisms ultimately leading to lung allograft rejection. In this study we evaluated whether IP-10 (CXCL10), a chemokine that is induced by interferon-gamma and stimulates the directional migration of activated T cells, plays a role in regulating the trafficking of effector T cells during lung allograft rejection episodes. Immunohistochemical examination showed that areas characterized by acute cellular rejection (grades 1 to 4) and active obliterative bronchiolitis (chronic rejection, Ca) were infiltrated by T cells expressing CXCR3, i.e., the specific receptor for CXCL10. In parallel, T cells accumulating in the bronchoalveolar lavage of lung transplant recipients with rejection episodes were CXCR3+ and exhibited a strong in vitro migratory capability in response to CXCL10. In lung biopsies, CXCL10 was abundantly expressed by graft-infiltrating macrophages and occasionally by epithelial cells. Alveolar macrophages expressed and secreted definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3+ T-cell line 300-19; the secretory capability of alveolar macrophages was up-regulated by preincubation with interferon-gamma. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the bronchoalveolar lavage in individuals with rejection episodes. These data indicate the role of the CXCR3/CXCL10 interactions in the recruitment of lymphocytes at sites of lung rejection and provide a rationale for the use of agents that block the CXCR3/CXCL10 axis in the treatment of lung allograft rejection.

The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis

B- and T-cell recirculation is crucial for the function of the immune system, with the control of cell migration being mainly mediated by several chemokines and their receptors. In this study, we investigated the expression and function of CXCR3 on normal and malignant B cells from 65 patients with chronic lymphoproliferative disorders (CLDs). Although CXCR3 is lacking on CD5(+) and CD5(-) B cells from healthy subjects, it is expressed on leukemic B lymphocytes from all (31/31) patients with chronic lymphocytic leukemia (CLL). The presence of CXCR3 was heterogeneous in other B-cell disorders, being expressed in 2 of 7 patients with mantle cell lymphoma (MCL), 4 of 12 patients with hairy cell leukemia (HCL), and 11 of 15 patients with other subtypes of non-Hodgkins lymphomas (NHLs). Chemotaxis assay shows that normal B cells from healthy subjects do not migrate in response to IFN-inducible protein 10 (IP-10) and IFN-gamma-induced monokine (Mig). In contrast, a definite migration in response to IP-10 and Mig has been observed in all malignant B cells from patients with CLL, but not in patients with HCL or MCL (1/7 cases tested). Neoplastic B cells from other NHLs showed a heterogenous pattern. The migration elicited by IP-10 and Mig was inhibited by blocking CXCR3. No effect of IP-10 and Mig chemokines was observed on the cytosolic calcium concentration in malignant B cells. The data reported here demonstrate that CXCR3 is expressed on malignant B cells from CLDs, particularly in patients with CLL, and represents a fully functional receptor involved in chemotaxis of malignant B lymphocytes.

Identification of NKp80, a novel triggering molecule expressed by human NK cells

The ability of NK cells to kill a wide range of tumor or virally infected target cells as well as normal allogeneic T cell blasts appears to depend upon the concerted action of multiple triggering NK receptors. In this study, using two specific monoclonal antibodies [(mAb) MA152 and LAP171], we identified a triggering NK receptor expressed at the cell surface as a dimer of approximately 80 kDa (NKp80). NKp80 is expressed by virtually all fresh or activated NK cells and by a minor subset of T cells characterized by the CD56 surface antigen. NKp80 surface expression was also detected in all CD3– and in 6 / 10 CD3+ large granular lymphocyte expansions derived from patients with lymphoproliferative disease of granular lymphocytes. In polyclonal NK cells, mAb‐mediated cross‐linking of NKp80 resulted in induction of cytolytic activity and Ca2 + mobilization. A marked heterogeneity existed in the magnitude of the cytolytic responses of different NK cell clones to anti‐NKp80 mAb. This heterogeneity correlated with the surface density of NKp46 molecules expressed by different NK clones. The mAb‐mediated masking of NKp80 led to a partial inhibition of the NK‐mediated lysis of appropriate allogeneic phytohemagglutinin‐induced T cell blasts, while it had no effect on the lysis of different tumor target cells, including T cell leukemia cells. These data suggest that NKp80 recognizes a ligand on normal T cells that may be down‐regulated during tumor transformation. Molecular cloning of the cDNA coding for NKp80 revealed a type II transmembrane molecule of 231 amino acids identical to the putative protein encoded by a recently identified cDNA termed KLRF1.

Sarcoidosis is a Th1/Th17 multisystem disorder

Background and aims Sarcoidosis is characterised by a compartmentalisation of CD4+ T helper 1 (Th1) lymphocytes and activated macrophages in involved organs, including the lung. Recently, Th17 effector CD4+ T cells have been claimed to be involved in the pathogenesis of granuloma formation. The objective of this study was to investigate the involvement of Th17 cells in the pathogenesis of sarcoidosis. Methods Peripheral and pulmonary Th17 cells were evaluated by flow cytometry, real-time PCR, immunohistochemistry analyses and functional assays in patients with sarcoidosis in different phases of the disease and in control subjects. Results Th17 cells were detected both in the peripheral blood (4.72±2.27% of CD4+ T cells) and in the bronchoalveolar lavage (BAL) (8.81±2.25% of CD4+ T lymphocytes) of patients with sarcoidosis and T cell alveolitis. Immunohistochemical analysis of lung and lymph node specimens showed that interleukin 17 (IL-17)+/CD4+ T cells infiltrate sarcoid tissues surrounding the central core of the granuloma. IL-17 was expressed by macrophages infiltrating sarcoid tissue and/or forming the granuloma core (7.88±2.40% of alveolar macrophages). Analysis of some lung specimens highlighted the persistence of IL-17+/CD4+ T cells in relapsed patients and their absence in the recovered cases. Migratory assays demonstrated the ability of the Th17 cell to respond to the chemotactic stimulus CCL20—that is, the CCR6 ligand (74.8±8.5 vs 7.6±2.8 migrating BAL lymphocytes/high-powered field, with and without CCL20, respectively). Conclusions Th17 cells participate in the alveolitic/granuloma phase and also to the progression towards the fibrotic phase of the disease. The recruitment of this cell subset may be driven by CCL20 chemokine.

High serum level of the soluble form of CD30 molecule in the early phase of HIV-1 infection as an independent predictor of progression to AIDS.

ObjectiveTo determine the serum levels of the soluble form of the CD30 (sCD30) activation molecule in the early phase of HIV-1 infection, and to investigate the possible correlation with evolution to AIDS. MethodssCD30 values were determined by an enzyme-linked immunosorbent assay (ELISA) on serum samples collected at the time of the first evidence of HIV-1 infection in 110 individuals with a median follow-up of 56 months (range, 12–88 months), at the A1 (74 cases) or A2 (36 cases) stages of the 1993 revised Centers for Disease Control and Prevention classification. The data were evaluated using established clinical and immunological parameters, including circulating CD4+ T-cell count. The controls were 110 blood donors and 51 HIV-1 -negative subjects belonging to groups at risk for HIV-1 infection. ResultsElevated sCD30 levels ( 20 U/ml) were found in 83.6% of HIV-1 -infected cases and in 47% of at-risk seronegatives. Data analysis revealed that HIV-1-infected patients with higher sCD30 levels (>35 U/ml) experienced faster disease progression (P = 0.0002). This was also the case in patients at the earliest stage (A1) of HIV infection (P = 0.0027). In these latter cases the predictive value of sCD30 was independent of the initial absolute number of circulating CD4+lymphocytes. ConclusionsSerum levels of sCD30 are increased in the large majority of patients in the early phase of HIV-1 infection and represent an indicator of progression to AIDS independent of other prognostic parameters.

New pathogenetic insights into the sarcoid granuloma.

Although the most important enigma of sarcoidosis, ie, its etiology, remains an unsolved problem, the past few years have seen remarkable advances in understanding general immunologic and molecular aspects of the mechanisms leading to granuloma formation in sarcoidosis. It is now clear that during the sarcoid inflammatory process several cytokines are secreted at sites of disease activity; in addition, high affinity receptors for cytokines participating in the granuloma development have been recently identified. This article provides a detailed description of recent data that have clarified cellular interactions governing the dynamics of granuloma formation in sarcoidosis. Together, these new results provide important insights that can refocus efforts at developing immunotherapeutic methods of inhibiting cytokine production at sites of granuloma formation.

Combination of Rituximab, Bendamustine, and Cytarabine for Patients With Mantle-Cell Non-Hodgkin Lymphoma Ineligible for Intensive Regimens or Autologous Transplantation

PURPOSE The combination of bendamustine (B) and rituximab (R) is efficacious, with favorable toxicity in mantle-cell lymphoma (MCL). In this phase II study, we combined cytarabine with R and B (R-BAC) in patients with MCL age ≥ 65 years who were previously untreated or relapsed or refractory (R/R) after one prior immunochemotherapy treatment. PATIENTS AND METHODS In stage one, we established the maximum-tolerated dose (MTD) of cytarabine in R-BAC. In stage two, patients received R (375 mg/m(2) intravenously [IV] on day 1), B (70 mg/m(2) IV on days 2 and 3), and cytarabine (MTD IV on days 2 to 4) every 28 days for four to six cycles. The primary end point (overall response rate [ORR]) was evaluated by positron emission tomography. Secondary end points included safety, progression-free survival (PFS), response duration, and overall survival. RESULTS Forty patients (median age, 70 years; 20 previously untreated patients) were enrolled; 93% had Ann Arbor stage III/IV disease; 49% had high Mantle Cell International Prognostic Index scores, with 15% blastoid histology. All R/R patients (35% refractory) had previously received R-containing regimens. The cytarabine MTD used in stage two was 800 mg/m(2), and R-BAC was well tolerated, with an 85% treatment completion rate. The major toxicity was transient grades 3 to 4 thrombocytopenia (87% of patients); febrile neutropenia occurred in 12%. The ORR was 100% (95% complete response [CR]) for previously untreated and 80% (70% CR) for R/R patients. The 2-year PFS rate (± standard deviation) was 95% ± 5% for untreated and 70% ± 10% for R/R patients. CONCLUSION R-BAC is well tolerated and active against MCL.