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B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced H 2 O 2 is thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous H 2 O 2 in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous H 2 O 2 in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low catalase expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous H 2 O 2 in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of catalase were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL., (© 2018 by The American Society of Hematology.)

It is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n = 64) and a validation cohort (VC; n = 84) of patients with chronic lymphocytic leukemia with "normal" FISH. An abnormal karyotype was found in 21.5% and 35.7% of cases in the LC and VC, respectively, and was associated with a lower immunophenotypic score (P = .030 in the LC, P = .035 in the VC), advanced stage (P = .040 in the VC), and need for treatment (P = .002 in the LC, P = < .0001 in the VC). The abnormal karyotype correlated with shorter time to first treatment and shorter survival in both the LC and the VC, representing the strongest prognostic parameter. In patients with chronic lymphocytic leukemia with normal FISH, karyotypic aberrations by conventional cytogenetics with novel mitogens identify a subset of cases with adverse prognostic features.

Although many literature data are available on the role of Notch signaling in T-cell acute lymphoblastic leukemia (ALL) biology, the importance of this molecular pathway in the development of B-lineage ALL (B-ALL) cells in the BM microenvironment is unknown so far. In this study, we used anti-Notch molecules neutralizing Abs and γ-secretase inhibitor (GSI) XII to investigate the role of the Notch signaling pathway in the promotion of human B-ALL cell survival in presence of stromal cell support. The treatment with combinations of anti-Notch molecule neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of stromal cells from normal donors and B-ALL patients. Interestingly, the inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. Our data suggest that the stromal cell-mediated antiapoptotic effect on B- ALL cells is mediated by Notch-3 and -4 or Jagged-1/-2 and DLL-1 in a synergistic manner.

All-trans-retinoic acid (ATRA) has greatly modified the prognosis of acute promyelocytic leukemia; however, the role of maintenance in patients in molecular complete remission after consolidation treatment is still debated. From July 1993 to May 2000, 807 genetically proven newly diagnosed acute promyelocytic leukemia patients received ATRA plus idarubicin as induction, followed by 3 intensive consolidation courses. Thereafter, patients reverse-transcribed polymerase chain reaction-negative for the PML-RARA fusion gene were randomized into 4 arms: oral 6-mercaptopurine and intramuscular methotrexate (arm 1); ATRA alone (arm 2); 3 months of arm1 alternating to 15 days of arm 2 (arm 3); and no further therapy (arm 4). Starting from February 1997, randomization was limited to ATRA-containing arms only (arms 2 and 3). Complete remission was achieved in 761 of 807 (94.3%) patients, and 681 completed the consolidation program. Of these, 664 (97.5%) were evaluated for the PML-RARA fusion gene, and 586 of 646 (90.7%) who tested reverse-transcribed polymerase chain reaction-negative were randomized to maintenance. The event-free survival estimate at 12 years was 68.9% (95% confidence interval, 66.4%-71.4%), and no differences in disease-free survival at 12 years were observed among the maintenance arms.

The role of neutrophils as key players in the regulation of innate and adaptive immune responses is increasingly being recognized. We report that human neutrophils establish a network with both natural killer (NK) cells and 6-sulfo LacNAc(+) dendritic cells (slanDCs), which ultimately serves to up-regulate NK-derived interferonγ (IFNγ). This network involves direct reciprocal interactions and positive amplification loops mediated by cell-derived cytokines. Accordingly, we show that after lipopolysaccharide + interleukin-2 (IL-2) or IL-15/IL-18 stimulation, neutrophils directly interact with and potentiate the activity of both slanDCs and NK cells. On the one hand, neutrophils augment the release of IL-12p70 by slanDCs via a CD18/ intercellular adhesion molecule-1 (ICAM-1) interaction that stimulates activated NK cells to produce IFNγ. IFNγ further potentiates the interaction between neutrophils and slanDCs and the release of slanDC-derived IL-12p70, thus creating a positive feedback loop. On the other hand, neutrophils directly co-stimulate NK cells via CD18/ICAM-3, leading to the production of IFNγ. Colocalization of neutrophils, NK cells, and slanDCs, as well as of IL-12p70 and IFNγ, in inflamed tissues of Crohn disease and psoriasis provides strong evidence for a novel cellular and cytokine cooperation within the innate immune system in which neutrophils act as amplifiers of NK cell/slanDC-mediated responses.

The prevalence, clinical characteristics, and prognostic significance of immune thrombocytopenia (IT) in patients with chronic lymphocytic leukemia (CLL) have not been clearly determined. To clarify this, we retrospectively analyzed 1278 consecutive newly diagnosed patients with CLL. Criteria for IT diagnosis included the following: rapid (< 2 weeks) and severe fall (half of the initial level and below 100 x 10(9)/L) in platelet count; normal or augmented megakaryocytes in bone marrow; no or limited (not palpable) splenomegaly; no cytotoxic treatment in the preceding month. Sixty-four patients (5%) were diagnosed with IT. The median time to IT from CLL diagnosis was 13 months (range, 0-81 months), and median platelet count at IT diagnosis was 14 x 10(9)/L (range, 1-71 x 10(9)/L). Fifty-six of the 64 patients (87%) received treatment for IT. The probability of responding to treatment for IT was significantly higher for patients receiving chemotherapy with or without steroids than for patients treated with intravenous immunoglobulins with or without steroids (P = .01). The development of IT was significantly associated with unmutated IgVh, a positive direct antiglobulin test, and the occurrence of autoimmune hemolytic anemia. Patients with CLL and IT had poorer survival than other patients with CLL (5-year overall survival 64% vs 82%, P < .001), and this effect was independent from common clinical prognostic variables.

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21).

Epidermal growth factor receptor-1 (EGFR-1/HER-1/ErbB-1) regulates proliferation and cell fate during epidermal development. HER-1 is activated by several EGF-family ligands including heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogenic and chemotactic molecule that participates in tissue repair, tumor growth, and other tissue-modeling phenomena, such as angiogenesis and fibrogenesis. We found that mesenchymal stem cells (MSCs), the precursors of different mesenchymal tissues with a role in processes in which HB-EGF is often involved, normally express HER-1, but not HB-EGF itself. Under the effect of HB-EGF, MSCs proliferate more rapidly and persistently, without undergoing spontaneous differentiation. This effect occurs in a dose-dependent fashion, and is specific, direct, and HER-1 mediated, as it is inhibited by anti-HER-1 and anti-HB-EGF blocking antibodies. Moreover, HB-EGF reversibly prevents adipogenic, osteogenic, and chondrogenic differentiation induced with specific media. These data show that HB-EGF/HER-1 signaling is relevant to MSC biology, by regulating both proliferation and differentiation.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily exerting cytotoxic activities toward tumor cells. Herein, we demonstrate that therapeutic concentrations of interferon alpha (IFNalpha) stimulate the expression of high levels of TRAIL mRNA and the release of elevated amounts of a soluble bioactive form of TRAIL (sTRAIL) in both human neutrophils and monocytes. Supernatants harvested from IFNalpha-treated neutrophils/monocytes elicited, on TRAIL-sensitive leukemic cell lines, proapoptotic activities that were significantly reduced by either a combination of TRAIL-R1/Fc and TRAIL-R2/Fc chimeras or neutralizing anti-TRAIL, anti-TRAIL-R1, and anti-TRAIL-R2 antibodies, suggesting that they were mediated by released sTRAIL acting on both TRAIL receptors. Since diseases such as chronic myeloid leukemia (CML) and melanoma are effectively treated with IFNalpha,we also demonstrate that CML neutrophils and peripheral blood mononuclear cells (PBMCs) cultured with IFNalpha at therapeutic concentrations retain the capacity of releasing sTRAIL, suggesting that CML leukocytes, in vivo, might represent an important source of sTRAIL. In this regard, we show that sTRAIL serum levels as well as leukocyte-associated TRAIL significantly increase in melanoma patients following IFNalpha administration. Collectively, these findings indicate that sTRAIL released by IFNalpha-activated neutrophils and monocytes contributes not only to the immunoregulatory actions but also to the therapeutic activities of IFNalpha.

To determine the clinical significance of BCL-2 expression in Hodgkin-Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD), we correlated its expression with presenting clinical and laboratory features and failure-free survival (FFS). Eligible patients were untreated and negative for HIV-1; they had biopsy-proven cHD. BCL-2 expression was determined immunohistochemically in available pretreatment tissue biopsy specimens without knowledge of clinical outcome. Tumors were considered positive if any HRS cells expressed BCL-2. We identified 707 patients with cHD, whose median age was 30 years; 54% were men. HRS cells expressed BCL-2 in 359 (65%) of 551 nodular sclerosis, 67 (47%) of 143 mixed cellularity, and all 5 lymphocyte depletion. For all patients, the 5-year FFS was 74% versus 84% for tumors with versus without BCL-2 expression (P =.0016, by log-rank test). For the 412 patients treated with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens, the 5-year FFS for tumors with versus without BCL-2 expression was 74% versus 88% (P =.001, by log-rank test); for the 233 patients with Ann Arbor stage I or II, FFS was 84% versus 92% (P =.04, by log-rank test); and for the 179 patients with Ann Arbor stage III or IV, FFS was 62% versus 81% (P =.006, by log-rank test). Multivariate analysis confirmed that BCL-2 expression is independently associated with inferior FFS along with age 45 or older, Ann Arbor stage IV, low serum albumin and high serum lactate dehydrogenase levels. We conclude that BCL-2 is frequently expressed by HRS cells in cHD and is associated with inferior FFS in patients treated with ABVD or equivalent regimens.

Little is known about the prognostic role of multidrug resistance (MDR) in adults with newly diagnosed acute lymphoblastic leukemia (ALL). In the context of the GIMEMA ALL0496 protocol, we evaluated the impact of MDR1 (protein expression and function) on the achievement of complete remission (CR) and clinical outcome. Flow cytometric analysis of MDR1 expression (D) and function (rhodamine-123 efflux) was obtained in 203 and 158 patients, respectively. MDR1 expression was detected in 44 (21.7%) of 203 patients, and function was found in 23 (14.6%) of 158 (14.6%) patients. Expression of the multidrug resistance-associated protein 1 (MRP1) and lung-resistance protein (LRP) evaluated in 43 samples was found in 13 and 26 patients, respectively. Among the 200 patients evaluable for the clinical correlation study, 125 (79.6%) of 157 without MDR1 expression achieved CR compared with 23 (53.5%) of 43 with MDR1 expression (P =.001). At univariate analysis, MDR1 expression was significantly associated with CR when considered as a dichotomized (P =.001) or continuous (P =.01) variable. At multivariate analysis, dichotomized evaluation of MDR1 expression independently predicted CR (P =.004) with age (P =.03) and CD34 (P =.03); as a continuous variable, MDR1 expression (P =.03) was the only significant factor other than CD34 (P =.01). MDR1 function failed to predict achievement of CR or of MRP1 and LRP expression. MDR1 expression did not correlate with CR duration, nor did it predict for survival duration. These results demonstrate that MDR1 expression in de novo adult ALL is an independent predictor of CR achievement.

The tumor necrosis factor receptor family molecule CD30 is expressed by activated and memory T cells, depending on IL-4 stimulation preferentially in association with Th0- and Th2-type responses. It mediates pleiotropic effects primarily of the inhibitory type. Arguing that CD30(+) cells have a peculiar redistribution in disease, it is demonstrated here, in the Hodgkin-derived L540 cell line (an established model for studying CD30 signaling), that CD30 regulates the prototypic lymphoid chemokine receptor CXCR4 (CD184), which plays an important role in many organ systems and is a coreceptor for human immunodeficiency virus-1 entry. CD30 stimulation with agonistic antibodies in L540 cells led to the accumulation of CXCR4 mRNA, which reached a plateau after 4 hours and did not require protein synthesis. It has been reported recently that CD30 up-regulates the transcription of CCR7 mRNA in YT lymphoma cells. After mRNA transcription, membrane expression of CXCR4 in L540 cells increased as early as 12 hours, reached a plateau after 24 hours (MFI +/- SD, 839 +/- 122 vs basal 168 +/- 28; P <.01) and was still increased after 5 days, permitting enhanced sensitivity to the chemotactic activity of CXCR4-ligand CXCL12 (CI +/- SD, 10 +/- 1 vs basal 5 +/- 2; P <.01). CD30 cross-linking also induced the release of CCL5 and CCL3 and the up-regulation of membrane binding capacity for CCL3 and CCL4 and decreased proliferative activity. This new regulatory role of CD30 may be relevant for T-cell maturation and effector responses and for promoting cancer biology.

Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized by the deregulated expression of cyclin D1 by t(11;14). The molecular mechanisms responsible for MCL's clinical behavior remain unclear. The authors have investigated the expression of p53, E2F-1, and the CDK inhibitors p27 and p21 in 110 MCLs, relating their expression to proliferative activity (Ki-67). For comparison, they have similarly analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL) lymphomas. p53 was detected more frequently in large-cell MCL (l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of 19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high, correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs (12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27 positive. Reverse transcription-polymerase chain reaction and in vitro protein degradation assays demonstrated that MCLs have normal p27 mRNA expression but increased p27 protein degradation activity via the proteasome pathway. Correlation of MCL p53 and p27 expression with clinical data showed an association between reduced overall survival rates and the overexpression of p53 (P =.001), the loss of p27 (P =. 002), or both. Loss of p27 identified patients with a worse clinical outcome among p53 negative cases (P =.002). These findings demonstrated that MCL has a distinct cell cycle protein expression similar to that of high-grade lymphoma. The loss of p27 and the overexpression of p53 in MCL are prognostic markers that identify patients at high risk. The demonstration that low levels of p27 in MCL result from enhanced proteasome-mediated degradation should encourage additional clinical trials. (Blood. 2000;95:619-626) (Blood. 2000;95:619-626)

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a widely expressed EGF superfamily member that induces mitogenic and/or chemotactic activities toward different cell types through binding to EGF receptors 1 or 4. Membrane-bound HB-EGF exerts growth activity and adhesion capabilities and possesses the unique property of being the receptor for diphtheria toxin (DT). Using molecular and functional techniques, we show that human polymorphonuclear granulocytes (PMN), which did not express HB-EGF in resting conditions, expressed it at mRNA and protein level, following incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Other classic agonists for PMN (including lipopolysaccharide, phagocytable particles, tumor necrosis factor-alpha, or G-CSF) failed to induce HB-EGF. The effects of GM-CSF on HB-EGF mRNA levels were concentration-dependent, reached a plateau after 1 to 2 hours of stimulation, and did not require protein synthesis. After GM-CSF treatment, membrane-bound HB-EGF was detected by flow cytometry. At the same time, PMN acquired sensitivity to the apoptosis-promoting effect of DT, which, moreover, specifically suppressed the GM-CSF-induced priming of formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion release. Finally, soluble HB-EGF was detected in the PMN culture medium by a specific enzyme-linked immunosorbent assay. Thus, we provide evidence that HB-EGF is specifically inducible by GM-CSF in PMN and represents a novel peptide to be included in the repertoire of PMN-derived cytokines.

Preliminary reports suggested a prognostic significance for serum levels of soluble CD30 (sCD30) in patients with Hodgkin's disease (HD). In this study, we investigated the prognostic impact of sCD30 concentration at diagnosis in relation to the other recognized prognostic parameters in 303 patients with HD observed in three different institutions between 1984 and 1996. sCD30 levels were correlated with stage, presence of B symptoms, and tumor burden. High sCD30 levels entailed a higher risk of poor outcome, and the event-free survival (EFS) probability at 5 years for patients with sCD30 levels >/=100 and less than 100 U/mL was 59.9% (95% confidence interval [CI], 40.6% to 65.9%) and 87.5% (95% CI, 81.5% to 91.6%), respectively (P < .001). On the basis of the results of univariate analysis of 14 pretreatment characteristics, we included five prognostic factors (high sCD30 serum level, stage III-IV, B symptoms, low hemoglobin level, and age >/=50 years) into a multivariate model. High sCD30 and advanced stage were independently associated with an unfavorable prognosis. Their combined evaluation identified patients at high risk (stages III and IV and sCD30 >/=100 U/mL: EFS, 46.9%) and low risk (stages I and II with sCD30 <100 U/mL: EFS, 88. 7%) of treatment failure (P < .001). We conclude that the combined evaluation of sCD30 serum level and stage at presentation identifies patients with HD at high risk of an unfavorable outcome.