Livio Trentin

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.).

Early Interim 2-[18F]Fluoro-2-Deoxy-D-Glucose Positron Emission Tomography Is Prognostically Superior to International Prognostic Score in Advanced-Stage Hodgkin's Lymphoma: A Report From a Joint Italian-Danish Study

PURPOSE Starting from November 2001, 260 newly diagnosed patients with Hodgkins lymphoma (HL) were consecutively enrolled in parallel Italian and Danish prospective trials to evaluate the prognostic role of an early interim 2-[(18)F]fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) scan and the International Prognostic Score (IPS) in advanced HL, treated with conventional ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) therapy. PATIENTS AND METHODS Most patients (n = 190) presented with advanced disease (stages IIB through IVB), whereas 70 presented in stage IIA with adverse prognostic factors. All but 11 patients were treated with standard ABVD therapy followed by consolidation radiotherapy in case of bulky presentation or residual tumor mass. Conventional radiologic staging was performed at baseline. FDG-PET scan was performed at baseline and after two courses of ABVD (PET-2). No treatment change was allowed on the basis of the PET-2 results. RESULTS After a median follow-up of 2.19 years (range, 0.32 to 5.18 years), 205 patients were in continued complete remission and two patients were in partial remission. Forty-three patients progressed during therapy or immediately after, whereas 10 patients relapsed. The 2-year progression-free survival for patients with positive PET-2 results was 12.8% and for patients with negative PET-2 results was 95.0% (P < .0001). In univariate analysis, the treatment outcome was significantly associated with PET-2 (P < .0001), stage IV (P < .0001), WBC more than 15,000 (P < .0001), lymphopenia (P < .001), IPS as a continuous variable (P < .0001), extranodal involvement (P < .0001), and bulky disease (P = .012). In multivariate analyses, only PET-2 turned out to be significant (P < .0001). CONCLUSION PET-2 overshadows the prognostic value of IPS and emerges as the single most important tool for planning of risk-adapted treatment in advanced HL.

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 lymphoproliferative disease of granular lymphocytes. A heterogeneous disorder ranging from indolent to aggressive conditions

A multiparameter analysis, which included the evaluation of clinical features, cell morphology, karyo‐type, phenotypic and functional immunologic findings, and T‐cell receptor beta‐chain configuration was performed on 34 patients with lymphoproliferative disease of granular lymphocytes (LDGL). The two‐fold aim of the study was to identify the most useful tools that would more accurately characterize these patients and to deal with the problem of classifying these lymphoproliferative disorders. The data presented in this article suggest that a single parameter may not be sufficient to define the nature of the proliferating cells or to predict the clinical course of the disease and prognosis for the patient. The use of a multiparameter approach, however, may reach this goal, thus providing important prognostic and therapeutic information. Our study supports the concept that lymphoproliferative disease of granular lymphocytes is a heterogeneous disorder that ranges from indolent and possibly reactive conditions to the manifestation of aggressive malignancies.

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.

Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.

The predictive role of interim positron emission tomography for Hodgkin lymphoma treatment outcome is confirmed using the interpretation criteria of the Deauville five-point scale

A retrospective, international, multicenter study was undertaken to assess: (i) the prognostic role of ‘interim’ positron emission tomography performed during treatment with doxorubicin, bleomycin, vinblastine and dacarbazine in patients with Hodgkin lymphoma; and (ii) the reproducibility of the Deauville five-point scale for the interpretation of interim positron emission tomography scan. Two hundred and sixty patients with newly diagnosed Hodgkin lymphoma were enrolled. Fifty-three patients with early unfavorable and 207 with advanced-stage disease were treated with doxorubicin, bleomycin, vinblastine and dacarbazine ± involved-field or consolidation radiotherapy. Positron emission tomography scan was performed at baseline and after two cycles of chemotherapy. Treatment was not changed according to the results of the interim scan. An international panel of six expert reviewers independently reported the scans using the Deauville five-point scale, blinded to treatment outcome. Forty-five scans were scored as positive (17.3%) and 215 (82.7%) as negative. After a median follow up of 37.0 (2–110) months, 252 patients are alive and eight have died. The 3-year progression-free survival rate was 83% for the whole study population, 28% for patients with interim positive scans and 95% for patients with interim negative scans (P<0.0001). The sensitivity, specificity, and negative and positive predictive values of interim positron emission tomography scans for predicting treatment outcome were 0.73, 0.94, 0.94 and 0.73, respectively. Binary concordance amongst reviewers was good (Cohen’s kappa 0.69–0.84). In conclusion, the prognostic role and validity of the Deauville five-point scale for interpretation of interim positron emission tomography scans have been confirmed by the present study.

Chlorambucil plus rituximab with or without maintenance rituximab as first‐line treatment for elderly chronic lymphocytic leukemia patients

In a phase II trial, we evaluated chlorambucil and rituximab (CLB‐R) as first‐line induction treatment with or without R as maintenance for elderly chronic lymphocytic leukemia (CLL) patients. Treatment consisted of eight 28‐day cycles of CLB (8 mg/m2/day, days 1–7) and R (day 1 of cycle 3, 375 mg/m2; cycles 4–8, 500 mg/m2). Responders were randomized to 12 8‐week doses of R (375 mg/m2) or observation. As per intention‐to‐treat analysis, 82.4% (95% CI, 74.25–90.46%) of 85 patients achieved an overall response (OR), 16.5% a complete response (CR), 2.4% a CR with incomplete bone marrow recovery. The OR was similar across Binet stages (A 86.4%, B 81.6%, and C 78.6%) and age categories (60–64 years, 92.3%; 65–69, 85.2%; 70–74, 75.0%; ≥75, 81.0%). CLB‐R was well tolerated. After a median follow‐up of 34.2 months, the median progression‐free survival (PFS) was 34.7 months (95% CI, 33.1–39.5). TP53 abnormalities, complex karyotype, and low CD20 gene expression predicted lack of response; SF3B1 mutation and BIRC3 disruption low CR rates. IGHV mutations significantly predicted PFS. R maintenance tended towards a better PFS than observation and was safe and most beneficial for patients in partial response and for unmutated IGHV cases. CLB‐R represents a promising option for elderly CLL patients. Am. J. Hematol. 89:480–486, 2014.

Increased Serum Levels of Soluble Interleukin-2 Receptor in Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis

In this study we investigated the serum levels of a released soluble form of the interleukin-2 receptor (sIL-2R) in 42 patients with rheumatoid arthritis and in 12 cases of systemic lupus erythematosus. Data were evaluated in relationship to the clinical phase and compared with those observed in normal controls (N=56) and in osteoarthritis (N = 7). Increased levels were observed in both rheumatoid arthritis (mean ± SE, 604±49 U/ml) and systemic lupus erythematosus (1438±481 U/ml). These values were significantly higher than in control (256±15 U/ml;P<0.001) and in osteoarthritis (298±33 U/ml;P<0.001) groups. In addition, the highest values were associated with the active phases of both rheumatoid arthritis (active vs inactive, 771±78 vs 451±39 U/ml;P<0.001) and systemic lupus erythematosus (active vs inactive, 2108±489 vs 499±75 U/ml;P<0.001). Our findings suggest that the detection of sIL-2R in rheumatoid arthritis and in systemic lupus erythematosus may represent a good marker of disease activity, which indirectly indicates the ongoing activation and/or proliferation of immunoreactive cells which are involved in the pathogenetic events of these autoimmune conditions.