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Clinical or biological parameters useful to predict progression during treatment in real-life setting with ibrutinib, idelalisib and venetoclax in relapsed/refractory chronic lymphocytic leukemia (CLL) are still debated. We conducted a multi-center retrospective study on CLL patients treated with ibrutinib and/or idelalisib who were switched to venetoclax for progression or due to adverse events to identify any clinical and/or biological parameters useful to predict progression during treatment with venetoclax. Of all the 128 evaluable patients, 81 had received ibrutinib prior to switching to venetoclax, 35 had received idelalisib and 12 both. When comparing the three subgroups, we did not notice any statistical difference in terms of clinical or biological features. No variable at baseline and at different time points during the follow-up (at 6, 12, 18 and 24 months) was found to predict progression nor to have significance for Progression Free Survival (PFS) in the ibrutinib group and in the idelalisib group and in subgroups according to the line of treatment. Analyzing the data of the venetoclax treatment, after a median follow up of 14.3 months, median PFS was not reached and estimated 3-year PFS was 54%. Of the 128 patients treated with venetoclax, 28 (22%) experienced progressive disease. At multivariate analysis for predictive factors for progression, lymph node diameter >56.5 mm before starting treatment emerged as an independent risk factor for progression. The lymph node predictive role for progression during venetoclax treatment could be a new parameter that deserves to be investigate in future studies., (© 2023 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

Chronic lymphocytic leukaemia (CLL) is characterised by the expansion of a neoplastic mature B cell clone. CLL clinical outcome is very heterogeneous, with some subjects never requiring therapy and some showing an aggressive disease. Genetic and epigenetic alterations and pro-inflammatory microenvironment influence CLL progression and prognosis. The involvement of immune-mediated mechanisms in CLL control needs to be investigated. We analyse the activation profile of innate and adaptive cytotoxic immune effectors in a cohort of 26 CLL patients with stable disease, as key elements for immune-mediated control of cancer progression. We observed an increase in CD54 expression and interferon (IFN)-γ production by cytotoxic T cells (CTL). CTL ability to recognise tumour-targets depends on human leukocyte antigens (HLA)-class I expression. We observed a decreased expression of HLA-A and HLA-BC on B cells of CLL subjects, associated with a significant reduction in intracellular calnexin that is relevant for HLA surface expression. Natural killer (NK) cells and CTL from CLL subjects show an increased expression of the activating receptor KIR2DS2 and a reduction of 3DL1 and NKG2A inhibiting molecules. Therefore, an activation profile characterises CTL and NK cells of CLL subjects with stable disease. This profile is conceivable with the functional involvement of cytotoxic effectors in CLL control.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, whose presentation and clinical course are highly variable. Identification of novel prognostic factors may contribute to improving the CLL classification and providing indications for treatment options. The zinc finger protein ZNF224 plays a key role in cell transformation, through the control of apoptotic and survival pathways. In this study, we evaluated the potential application of ZNF224 as a novel marker of CLL progression and therapy responsiveness. To this aim, we analyzed ZNF224 expression levels in B lymphocytes from CLL patients at different stages of the disease and in patients showing different treatment outcomes. The expression of ZNF224 was significantly increased in disease progression and dramatically decreased in patients in complete remission after chemotherapy. Gene expression correlation analysis performed on datasets of CLL patients revealed that ZNF224 expression was well correlated with that of some prognostic and predictive markers. Moreover, bioinformatic analysis coupled ZNF224 to NF-κB pathway, and experimental data demonstrated that RNA interference of ZNF224 reduced the activity of the NF-κB survival pathway in CLL cells. Consistently with a pro-survival role, ZNF224 knockdown raised spontaneous and drug-induced apoptosis and inhibited the proliferation of peripheral blood mononuclear cells from CLL patients. Our findings provide evidence for the involvement of ZNF224 in the survival of CLL cells via NF-κB pathway modulation, and also suggest ZNF224 as a prognostic and predictive molecular marker of CLL disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Catapano, Sepe, Toscano, Paolella, Chiurazzi, Barbato, Bruzzese, Arianna, Grosso, Romano, Romano, Costanzo and Cesaro.)

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL., Competing Interests: The authors have declared that no competing interests exist.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by variable clinical courses among different patients. This notion was supported by the possible coexistence of two or more independent CLL clones within the same patients, identified by the characterization of the B cell receptor immunoglobulin (BcR IG) idiotypic sequence. By using the antigen-binding site of the BcR IG as bait, the identification and isolation of aggressive and drug-resistance leukemic B-cell clones could allow a deeper biological and molecular investigation. Indeed, by the screening of phage display libraries, we previously selected a peptide binder of the idiotypic region of CLL BCR IGs expressing the unmutated rearrangement IGHV1-69 and used it as a probe to perform a peptide-based cell sorting by flow cytometry in peripheral blood samples from patients with CLL. Since the IGHV1-69 clones persisted during the follow-up time in both patients, we explored the possibility of these clones having acquired an evolutive advantage compared to the other coexisting clones in terms of a higher expression of genes involved in the survival and apoptosis escape processes. To this end, we studied the expression patterns of a panel of genes involved in apoptosis regulation and in NF-kB-dependent pro-survival signals by comparative qRT-PCR assays. According to the results, IGHV1-69 clones showed a higher expression of pro-survival and anti-apoptotic genes as compared to the other CLL clones with different immunogenetic characteristics. Moreover, these IGHV1-69 clones did not carry any characteristic genetic lesions, indicating the relevance of our approach in performing a comprehensive molecular characterization of single tumor clones, as well as for designing new personalized therapeutic approaches for the most aggressive and persistent tumor clones.

Competing Interests: None declared.

Introduction: In patients with primary immune thrombocytopenia (ITP), a short course of steroids is routinely given as first-line therapy. However, the response is often transient and additional therapy is usually needed. Thrombopoietin receptor agonists (TPO-RAs) are frequently used as second-line therapy, although there is little clinical guidance on the timing of their administration and on tapering/discontinuation of the drug. To provide clinical recommendations, we used the Delphi technique to obtain consensus for statements regarding administration and on tapering/discontinuation of second-line TPO-RAs among a group of Italian clinicians with expertise in management of ITP., Methods: The Delphi process was used to obtain agreement on five statements regarding initiation and on tapering/discontinuation of second-line TPO-RAs. Agreement was considered when 75% of participants approved the statement. Eleven experts participated in the voting., Results: Full consensus was reached for three of the five statements. The experts held that an early switch from corticosteroids to a TPO-RA has the dual advantage of sparing patients from corticosteroid abuse and improve long-term clinical outcomes. All felt that dose reduction of TPO-RAs can be considered in patients with a stable response and platelet count >100 × 10 9 /L that is maintained for at least 6 months in the absence of concomitant treatments, although there was less agreement in patients with a platelet count >50 × 10 9 /L. Near consensus was reached regarding the statement that early treatment with a TPO-RA is associated with an increase in clinically significant partial or complete response. The experts also agreed that optimization of tapering and discontinuation of TPO-RA therapy in selected patients can improve the quality of life., Conclusion: The present consensus can help to provide guidance on use of TPO-RAs in daily practice in patients with ITP., Plain Language Summary: Second-line administration of thrombopoietin receptor agonists in immune thrombocytopenia There is little guidance on the timing of administration and tapering/discontinuation of thrombopoietin receptor agonists (TPO-RAs) in patients with primary immune thrombocytopenia (ITP).The Delphi technique was used to obtain consensus for five statements.The present consensus among Italian clinicians aims to provide guidance on second-line use of TPO-RAs for patients with ITP in daily practice., Competing Interests: Conflict of interest statement: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: CM received honoraria from Amgen and Novartis for serving on advisory boards; SS received honoraria from CSL, AMGEN, Novartis, Novo Nordisk, SOBI, and Bayer; ZF received honoraria and funding from Novartis, Amgen, and Grifols; PF received honoraria from Novartis; FB received consultation honoraria from Amgen, Novartis, and Momenta; BC received honoraria from Novartis and Amgen; BA has received honoraria from Amgen, Novartis, Novo Nordisk, Bayer, and Takeda; GG, SPR, BE, CF, CG, RE, VA have no conflict of interest to declare., (© The Author(s), 2021.)

The efficacy and safety of thrombopoietin receptor agonists (TRAs) in older patients with primary immune thrombocytopenia (ITP) are unknown. We investigated TRA response and switch, thrombotic/hemorrhagic risk, and sustained responses off-treatment (SROTs) in 384 patients with ITP aged ≥60 years. After 3 months, 82.5% and 74.3% of eltrombopag- and romiplostim-treated patients, respectively, achieved a response; 66.7% maintained the response (median follow-up, 2.7 years). Eighty-five (22.2%) patients switched to the alternative TRA; although no cross-toxicity was observed, 83.3% of resistant patients had a response after the switch. Thirty-four major thromboses (3 fatal) and 14 major hemorrhages (none fatal) occurred in 18 and 10 patients, respectively, while on TRAs and were associated with thrombosis history (subdistribution hazard ratio, 2.04, P = .05) and platelet count <20 × 109/L (subdistribution hazard ratio, 1.69; P = .04), respectively, at TRA start. A recurrent event occurred in 15.6% of patients surviving thrombosis, in all cases but 1 during persisting TRA treatment (incidence rate, 7.7 per 100 patient-years). All recurrences occurred in the absence of adequate antithrombotic secondary prophylaxis. Sixty-two (16.5%) responding patients discontinued TRAs; 53 (13.8%) patients maintained SROTs, which were associated with TRA discontinuation in complete response (P < .001). Very old age (≥75 years; 41.1%) was associated with the more frequent start of TRAs in the persistent/acute phase but not with response or thrombotic/hemorrhagic risk. TRAs are effective in older patients with ITP, with no fatal hemorrhages and with SROTs in a significant portion of patients. Caution is warranted in patients with a history of thrombosis, and a careful risk/benefit balance should be considered., (© 2021 by The American Society of Hematology.)

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maisano, Iaccino, D’Ambrosio, Chiurazzi, Dattilo, Scalise, Gentile, Vecchio, Nisticò, Aloisio, De Sensi, Fiume, Quinto and Mimmi.)

The current COVID-19 pandemic requires revisiting our current approach to major blood disorders, including ITP (Immune Thrombocytopenia), stirring up the production of several disease-specific practical guidelines. This report describes an updated version of consensus-based practical guidelines on the management of ITP, adapted to the Italian health system and social context. It highlights the role of the hematologist in offering guidance for choosing differentiated approaches in relation to specific circumstances and is intended to provide them with a useful tool for sharing the decision-making process with their patients. Probably, the greatest risk to avoid for a patient with suspected, ongoing or relapsed ITP - that is not severe enough to place him or her at risk for major bleeding - is to be infected in non-hospital and hospital healthcare settings. This risk must be carefully considered when adapting the diagnostic and therapeutic approach. More in detail, the document first addresses the appropriate management for COVID-19 negative patients with newly diagnosed ITP or who experience a relapse of previous ITP, according to first and second lines of treatment and then the management of COVID-19 positive patients according to their severity, from paucisymptomatic to those requiring admission to Intensive Cure Units (ICU). The pros and cons of the different treatments required to correct platelet count are discussed, as are some specific situations, including chronic ITP, splenectomy, thromboembolic complication and anti COVID-19 vaccination., Competing Interests: Competing interests: The authors declare no conflict of Interest.

Thrombopoietin receptor agonists (TPO-RAs) are currently indicated for continuous treatment of chronic primary immune thrombocytopenia (ITP). However, there is growing evidence that TPO-RAs can also trigger sustained response in 10-30% of cases after treatment tapering and discontinuation. Therefore, at least for selected responding patients, it might be rational to plan TPO-RA interruption to exploit off-treatment response. Intriguingly, complete or partial responses with TPO-RAs are frequently observed when treatments are initiated early, suggesting that unknown immune-related mechanisms may be involved in this phenomenon. The sustained responses observed after interruption of TPO-RAs may be interpreted as a recovery of immunological tolerance; thus, the re-establishment of immunological equilibrium might be primarily responsible for the observed off-treatment effect. Importantly, these findings may indicate that anticipated TPO-RA usage can lead to improved responses, and that optimized tapering and interruption in selected patients can furthermore improve prognoses. On the base of this rationale, a series of real-life considerations have been generated by a panel of Experts to elucidate possible novel criteria and modalities to identify subgroups of patients who can benefit from tapering and/or discontinuation of TPO-RAs. Towards this aim, the results of a survey of ITP experts are herein reported, reflecting a snapshot of current real-life experience on early discontinuation of TPO-RA-based therapy. The present manuscript also highlights the importance of future translational studies on novel prognostic and predictive biomarkers that can stratify patients and facilitate the clinical choice for second-line treatment of ITP., Competing Interests: Declaration of Competing Interest The authors declare potential conflict of interest with Novartis Corp., (Copyright © 2019. Published by Elsevier Ltd.)

Background: The incidence of peripherally inserted central catheter (PICC)-related adverse events has been uncertain in the setting of acute myeloid leukemia (AML) compared with the incidence of centrally inserted central catheter (CICC) adverse events., Patients and Methods: We conducted a monocentric, randomized trial of patients with previously untreated AML. Of the 93 patients, 46 had received a PICC and 47 had received a CICC as frontline intravascular device. Thereafter, all patients underwent intensive chemotherapy for hematologic remission induction. The primary endpoint was catheter-related (CR)-bloodstream infection (BSI) and venous thrombosis (VT) rate. The secondary endpoints catheter malfunction, catheter removal, and patient overall survival., Results: The CR-BSI and CR-VT rate in the PICC and CICC groups was 13% and 49%, respectively, with a difference of 36 percentage points (relative risk for CR-BSI or CR-VT, 0.266; P = .0003). The CR-BSI incidence was 1.4 and 7.8 per 1000 catheters daily in the PICC and CICC groups, respectively. Among the CR thromboses, the symptomatic VT rate was 2.1% in the PICC group and 10.6% in the CICC group. In the CICC group, 16 of the 47 patients (34%) had the catheter removed for BSI (n = 5), septic thrombophlebitis (n = 4), VT (n = 2), or malfunction (n = 5) a median of 7 days after insertion. In the PICC group, only 6 of the 46 patients (13%) required catheter removal for VT (n = 2) or malfunction (n = 4). At a median follow-up of 30 days, 6 patients in the CICC group died of CR complications versus none of the patients in the PICC group (P = .012). Using PICCs, the reduction in BSI and symptomatic VT decreased mortality from CR infection and venous thromboembolism. In contrast, the CICC approach led to early catheter removal mostly for difficult-to-treat infectious pathogens., Conclusion: Our data have confirmed that BSI and symptomatic VT are the major complications affecting frontline central intravascular device-related morbidity in the leukemia setting. The use of a PICC is safer than that of a CICC and maintains the effectiveness for patients with AML undergoing chemotherapy, with an approximate fourfold lower combined risk of infection or thrombosis at 30 days., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy with a variable clinical outcome. Biomarkers of CLL progression are required for optimising prognosis and therapy. The Inhibitor of Bruton's tyrosine kinase-isoform α (IBTKα) gene encodes a substrate receptor of Cullin 3-dependent E3 ubiquitin ligase, and promotes cell survival in response to the reticulum stress. Searching for novel markers of CLL progression, we analysed the expression of IBTKα in the peripheral blood B-cells of CLL patients, before and after first line therapy causing remission. The expression of IBTKα was significantly increased in disease progression, and decreased in remission after chemotherapy. Consistently with a pro-survival action, RNA interference of IBTKα increased the spontaneous and Fludarabine-induced apoptosis of MEC-1 CLL cells, and impaired the cell cycle of DeFew B-lymphoma cells by promoting the arrest in G0/G1 phase and apoptosis. Consistently, RNA interference of IBTKα up regulated the expression of pro-apoptotic genes, including TNF, CRADD, CASP7, BNIP3 and BIRC3. Our results indicate that IBTKα is a novel marker of CLL progression promoting cell growth and resistance to apoptosis. In this view, IBTKα may represent an attractive cancer drug target for counteracting the therapy-resistance of tumour cells.

Neutropenic enterocolitis (NEC) is an abdominal infection reported primarily in patients with acute myeloid leukemia (AML) following chemotherapy, especially cytarabine, a notable efficacious cytotoxic agent for AML remission. Specific data regarding the impact of different cytarabine schedules and/or antibacterial regimens for NEC are sparse. The aim of the study was to identify the predictors of outcome within 30 days of NEC onset. NEC episodes were retrospectively pinpointed among 440 patients with newly diagnosed AML hospitalized in our Institution, over a 10-year period, for receiving chemotherapy protocols with 100-6000 mg/m 2 daily of cytarabine. Two subgroups, survivors versus nonsurvivors, were compared by using logistic regression analysis. NEC was documented in 100 of 420 (23.8%) analyzed patients: 42.5% had received high-dose cytarabine, whereas 19% and 15% intermediate-dose and standard-dose cytarabine, respectively (P < 0.001). The 30-day NEC attributable mortality rate was 23%. In univariate analysis, antileukemic protocols containing robust dosages of cytarabine were significantly associated with high mortality (P < 0.001); whereas, standard-dose cytarabine and prompt initiation (at the ultrasonographic appearance of intestinal mural thickening) of NEC therapy with antibiotic combinations including tigecycline were significantly associated with low mortality. In multivariate analysis, high-dose cytarabine-containing chemotherapy was the independent predictor of poor outcome (odds ratio [OR]: 0.109; 95% confidence interval [CI]: 0.032-0.364; P < 0.001), whereas ultrasonography-driven NEC therapy with antibiotic regimens including tigecycline was associated with a favorable outcome (OR: 13.161; 95% CI: 1.587-109.17; P = 0.017). Chemotherapy schedules with robust dosages of cytarabine for AML remission are associated with a high rate of NEC incidence and attributable. Vigorous antibacterial therapy, triggered off pathologic ultrasonographic findings, with drug combinations which have broad antimicrobial coverage and good gut penetration, specifically those also including tigecycline, may be effective in improving 30-day survival rate after NEC onset., (© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Two noninferiority, randomized, controlled trials were conducted in parallel comparing the safety and efficacy of platelets treated with Intercept or Mirasol pathogen-reduction technologies versus standard platelets., Study Design and Methods: The primary endpoint was the percentage of hematology patients who developed World Health Organization Grade 2 or greater bleeding. A noninferiority margin of 11% was chosen based on expected Grade 2 or greater bleeding in 20% of controls. The study was closed for financial restrictions before reaching the planned sample size of 828 patients, and an intention-to-treat analysis was conducted on 424 evaluable patients., Results: In the Intercept trial (113 treated vs. 115 control patients), the absolute risk difference in Grade 2 or greater bleeding was 6.1%, with an upper one-sided 97.5% confidence limit of 19.2%. The absolute risk difference in the Mirasol trial (99 treated vs. 97 control patients) was 4.1%, and the upper one-sided 97.5% confidence limit was 18.4%. Neither absolute risk difference was statistically significant. In both trials, posttransfusion platelet count increments were significantly lower in treated versus control patients. Mean blood component use in treated patients versus controls was 54% higher (95% confidence interval, 36%-74%; Intercept) and 34% higher (95% confidence interval, 16%-54%; Mirasol) for platelets and 23% higher (95% confidence interval, 8%-39%; Intercept) and 32% higher (95% confidence interval, 10%-57%; Mirasol) for red blood cells. Unexpected reactions and adverse events were not reported. Mortality did not differ significantly between treated and control patients., Conclusion: Although conclusions on noninferiority could not be drawn due to low statistical power, the study provides additional information on the safety and efficacy of pathogen-reduced platelets treated with two commercial pathogen-reduction technologies., (© 2017 AABB.)

Chronic lymphocytic leukaemia (CLL) is associated with apoptosis resistance and defective control of cell growth. Our study describes for the first time a critical role in CLL for the KRAB-zinc finger protein ZNF224. High ZNF224 transcript levels were detected in CLL patients with respect to control cells. Moreover, ZNF224 expression was significantly lowered after conventional chemotherapy treatment in a subset of CLL patients. By in vitro experiments we confirmed that ZNF224 expression is suppressed by fludarabine and demonstrated that ZNF224 is involved in apoptosis resistance in CLL cells. Moreover, we showed that ZNF224 positively modulates cyclin D3 gene expression. Consistently, we observed that alteration of ZNF224 expression leads to defects in cell cycle control. All together, our results strongly suggest that in CLL cells high expression level of ZNF224 can lead to inappropriate cell growth and apoptosis resistance, thus contributing to CLL progression. Targeting ZNF224 could thus improve CLL response to therapy., (© The Author 2016. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.)

Chronic lymphocytic leukemia (CLL) B cells are phenotypically identified by surface expression of CD5 and CD23 antigens. Infrequently, patients with a monoclonal B cell lymphocytosis clinically resembling classic B-CLL have been found to harbor leukemic B cells lacking expression of the CD5 antigen. Little information is available concerning such CLL-like lymphoproliferative syndromes. Here, we provide phenotypic and clinical characteristics of 13 patients with CD5-negative chronic lymphoproliferative disorders selected from among 400 B-CLL patients followed up at a single academic center. Phenotypic analysis was carried out by flow cytometry using a broad panel of monoclonal antibodies including activation, costimulatory, adhesion, and growth factor receptor molecules. Moreover, intracellular staining and stimulation experiments were performed to investigate whether CD5 antigen was either retained in the cytoplasm of clonal B cells or not expressed due to defective cellular activation, respectively. Overall, CD5-negative leukemic cells were found to express significantly different levels of several membrane molecules, including CD95, CD69, CD23, CD25, CD80, and CD20, compared to "classic" CLL B cells. CD5 antigen was not detected in the cytoplasm of CD5-negative clonal B cells, nor could it be induced following in vitro activation. CD3+ T cell proportions were found to be less affected in CD5-negative patients than in classic B-CLL. Although these data suggest that CD5-negative clonal B cells are phenotypically different from classic B-CLL, clinical outcomes were similar to those shown by B-CLL patients, with most of the patients experiencing a long-lasting disease requiring chemotherapeutic intervention at some time during the disease course.

About 10% of the human immunodeficiency virus (HIV) patients show thrombocytopenia. We describe the case of an HIV/HCV-positive patient whose autoimmune thrombocytopenia resolved with the addition of raltegravir to previous highly active antiretroviral therapy (HAART). It is noteworthy that the effect on platelet count appeared to be independent of viral load suppression, which was achieved with previous antiretroviral regimens. In fact, it has been suggested that the positive effect exerted by raltegravir on autoimmune diseases is due to its inhibition on herpes viruses, and hence on activation of endogenous human retroviruses. This consideration, if confirmed, could open new avenues in the treatment of autoimmune thrombocytopenia in the HIV setting.

Background: Septic thrombophlebitis increases patient morbidity and mortality following metastatic infections, pulmonary emboli, and/or septic shock. Central venous catheter (CVC) removal for occult septic thrombophlebitis challenges current strategy in neutropenic patients., Patients and Methods: We prospectively evaluated infection-related mortality in 100 acute leukemia patients, with CVC-related bloodstream infection (CRBSI) after chemotherapy, who systematically underwent ultrasonography to identify the need for catheter removal. Their infection-related mortality was compared with that of a historical cohort of 100 acute leukemia patients, with CRBSI after chemotherapy, managed with a clinically driven strategy. Appropriate antimicrobial therapy was administered in all patients analyzed., Results: In the prospective series, 30/100 patients required catheter removal for ultrasonography-detected septic thrombophlebitis after 1 median day from BSI onset; 70/100 patients without septic thrombophlebitis retained their CVC. In the historical cohort, 60/100 patients removed the catheter (persistent fever, 40 patients; persistent BSI, 10 patients; or clinically manifest septic thrombophlebitis, 10 patients) after 8 median days from BSI onset; 40/100 patients retained the CVC because they had not clinical findings of complicated infection. At 30 days median follow-up, one patient died for infection in the ultrasonography-assisted group versus 17 patients in the historical cohort (P<0.01). With the ultrasonography-driven strategy, early septic thrombophlebitis detection and prompt CVC removal decrease infection-related mortality, whereas clinically driven strategy leads to inappropriate number, reasons, and timeliness of CVC removal., Conclusion: Ultrasonography is an easy imaging diagnostic tool enabling effective and safe management of patients with acute leukemia and CRBSI.

Background: Loss of response to transforming growth factor-beta (TGF-beta ) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-beta signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-beta signal in chronic lymphocytic leukemia., Design and Methods: We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-beta signal was investigated by western blotting and flow cytometry. The levels of Bcl2-family members and death-associated-protein kinase were also investigated by western blotting, whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was obtained by gene silencing with short interfering RNA., Results: Twenty-two out of 62 chronic lymphocytic leukemia samples were sensitive to TGF-beta-induced apoptosis. All but two of the responsive samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples that were not sensitive to TGF-beta were sensitive to FK506. Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2 phosphorylation and nuclear translocation. Accordingly, death-associated-protein kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2 and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. FK506 removed FKBP12 from its binding to the TGF-beta-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12., Conclusions: Our study shows that most chronic lymphocytic leukemia cells escape the homeostatic control of TGF-beta and that FK506 restores the TGF-beta signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-beta receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway.

B-cell chronic lymphocytic leukaemia (B-CLL) cells fail to undergo apoptosis. The mechanism underlying this resistance to cell death is still largely unknown. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) effectively kills tumour cells but not normal cells, and thus represents an attractive tool for the treatment of cancer. Unfortunately, lymphocytes from B-CLL patients are resistant to TRAIL-mediated apoptosis. Thus, we aimed to study the involvement of PED, a DED-family member with a broad antiapoptotic action, in this resistance. We demonstrate that B lymphocytes obtained from patients with B-CLL express high levels of PED. Treatment of B-CLL cells with specific PED antisense oligonucleotides, a protein synthesis inhibitor or HDAC inhibitors, induced a significant downregulation of PED and sensitized these cells to TRAIL-induced cell death. These findings suggest a direct involvement of PED in resistance to TRAIL-induced apoptosis in B-CLL. It also identifies this DED-family member as a potential therapeutic target for this form of leukaemia., ((c) 2006 Wiley-Liss, Inc.)

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of long-lived and well-differentiated clonal B-lymphocytes in peripheral blood, lymphoid tissue and bone marrow. Although B-CLL pathogenesis is not entirely understood, the progressive increase in lymphocyte counts coupled with the very low proportion of proliferating cells suggests that B-CLL may be primarily determined by defective apoptosis. Consistently, freshly analyzed CLL B-cells express very low levels of membrane CD95, one of the best-known receptors involved in triggering apoptosis. In this study, CD95 upregulation on CLL B-cells was induced by culturing clonal B-cells in the presence of supernatants from preactivated autologous T-lymphocytes. Intracellular cytokine staining of preactivated autologous T-lymphocytes using monoclonal antibodies (moAbs) specific for Th1 or Th2 cytokines, namely interleukin (IL)-2, IL-4, IL-5, IL-10 and interferon (IFN)-gamma, showed these cells to be positive for IL-2 and IFN-gamma. Blocking experiments using moAbs specific for IL-2 and/or IFN-gamma revealed that CD95 upregulation on CLL B-cells was mainly driven by IFN-gamma. However, CD95-expressing CLL B-cells were demonstrated to be resistant to CD95-mediated apoptosis, thus arguing against strategies aimed at exploiting CD95-mediated apoptosis for immunotherapy of B-CLL.

Background and Objectives: The outcome of adult patients with acute lymphoblastic leukemia (ALL) is discouraging, only about 30% of them becoming long-term survivors. A small fraction of patients are resistant to the first line treatment, while most patients relapse within two years of achieving complete remission (CR). No standard treatment exists for refractory or relapsed patients. The GIMEMA group designed a phase II trial for adult ALL patients with refractory or relapsed disease., Design and Methods: Patients aged >15 years with primary refractory or relapsed ALL were eligible for this study. The salvage strategy included a single high dose of idarubicin combined with high dose cytarabine, followed by consolidation therapy leading to a stem cell transplant procedure according to donor availability., Results: From 1998 to 2002, 135 patients were enrolled. Seventy-five patients (55%) achieved CR, including 12 Philadelphia-positive cases; 44 patients had persistent leukemia and 16 died during reinduction. Fifty patients received a stem cell transplant: 19 from an HL-A identical sibling, 16 from an unrelated donor, 7 from a haploidentical relative, 2 received cord blood, and 6 had an autotransplant. The median disease-free and overall survival were both short (5.0 and 6.4 months, respectively); however, after a median follow-up of 40 months, 13 patients are alive, 10 of whom are free of disease (9 transplanted), while 3 are alive with leukemia., Interpretation and Conclusions: The treatment induced CR in a high percentage of poor prognosis patients, thus rendering a transplant procedure feasible in most of them. However, significant rates of transplant-related mortality and post-transplant relapse encourage the search for more effective and less toxic conditioning regimens.

Profound immune dysfunction is a constant feature in B-cell chronic lymphocytic leukemia (B-CLL) patients. Immunological abnormalities include hypogammaglobulinemia, impaired immunoglobulin class switching and diminished germinal center formation. This state of immune suppression renders B-CLL patients highly susceptible to infections, which contribute greatly to morbidity and mortality in this disease. Impaired T cell function in B-CLL is well-documented and has been suggested to result from inhibitory effects exerted by malignant B lymphocytes. Because the presence of leukemic cells may represent a major obstacle to efficient T cell activation, T lymphocytes were separated from CLL B cells, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4h, and then cocultured with autologous leukemic B cells both at a 1:1 ratio or at the same ratio as in vivo for 24-40 h. CLL B cell expression of CD86 and CD95 was markedly upregulated using this approach, whereas CD80 expression was augmented only in a minority of patients; these effects were partially preserved even when preactivated T cells were rechallenged with CLL B cells at the same low T/B cell ratio as that observed in vivo. Finally, CD80 upregulation on CLL B cells appeared to be mainly dependent on CD40L-mediated stimulation, whereas CD86 and CD95 expression was efficiently augmented by soluble factors released by preactivated T lymphocytes. In conclusion, efficient activation of T lymphocytes in B-CLL may be achieved which, in turn, may result in enhanced antigen-presenting capacity and susceptibility to apoptosis of leukemic cells via CD86 and CD95 upregulation, respectively.

Pseudothrombocytopenia (PTCP) is the consequence of an EDTA-activated platelet agglutination, resulting in a spuriously low platelet count. We report the case of a 54-year-old man with EDTA-dependent PTCP associated with liver cirrhosis. He couldn't undergo endoscopic examination and dental care for two years because of a previous diagnosis of severe thrombocytopenia secondary to liver cirrhosis. Lack of PTCP recognition may lead the physician to misdiagnosis and mismanagement of the patient.

We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean +/- SE: 28.5 +/- 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% +/- 7.8% on average (P = .0077). Because the CD40 antigen activates NF-kappaB/Rel transcription factors in B cells, and NF-kappaB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-kappaB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-kappaB/Rel activity; p50, RelA, and c-Rel components of the NF-kappaB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-kappaB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-kappaB/Rel levels. To determine the involvement of NF-kappaB/Rel activity in the G28-5-mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-kappaB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-kappaB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-kappaB/Rel inhibitors, could improve the therapeutic effect of fludarabine., (Copyright 1998 by The American Society of Hematology.)

Starting from the observation that a number of consecutive patients with non-Hodgkin's lymphoma (NHL) resulted positive for hepatitis C virus (HCV) antibodies on routine testing, we set up a survey for HCV contact prevalence in all patients with lymphoproliferative disorders (LPD) followed in our institution. We searched for HCV antibodies by a third-generation ELISA technique, followed by a confirmation test (RIBA III); serum viral RNA and HCV genotype were investigated by a RT-PCR technique. We screened a total of 315 patients suffering from B-NHL (91), multiple myeloma (56), MGUS (48), chronic lymphocytic leukemia (57), Waldentrom's macroglobulinemia (13), Hodgkin's disease (HD)(43), and T-NHL (9). While only 1 of 52 patients with a non-B-LPD (HD or T-NHL) had signs of HCV contact (i.e., 1.9%, which is in the range of the normal population in the South of Italy), 59 of 263 patients with a B-LPD (22.4%) had HCV antibodies or RNA, or both, with no major differences among the various types of disorders, except for WM, in which the rate was higher (61.5%). The same prevalence was found for patients tested at diagnosis or during the follow-up, and in transfused or never-transfused patients. Only a few patients were aware of having a liver disease; one-half of HCV-positive patients never had transaminase increase. A review of data from Central and Northern Italy is included, showing similar findings; a report from Japan has confirmed such an association, while limited surveys in England have not revealed any correlation. These findings may have important biological and clinical implications.

BACKGROUND. A number of side effects have been observed in patients treated for hematological diseases with alpha-interferon (IFN). In several cases side effects consisted of immunological disorders. Autoimmune hemolytic anemia (AIHA) is the most typical example of an IFN-induced immune-mediated complication. CASE SERIES. In 10 years we observed 9 patients with various hematological disorders who developed AIHA during IFN treatment. The interval between the start of IFN treatment and the onset of acute hemolysis suggests a dual pattern of occurrence: (1) early onset (interval 1 to 21 days), seen in patients who had anti-RBC antibodies before IFN treatment; (2) late onset (interval 3-38 months), in patients with no history of anti-RBC antibodies at the start of treatment. Discontinuation of IFN, often associated with prednisone treatment, caused prompt hematological recovery in all cases; anti-erythrocyte antibodies persisted in the first group of patients and disappeared in the second. CONCLUSIONS. In rare cases IFN may cause AIHA. The immunological derangement caused by IFN seems to act at two different levels: enhanced destruction of antibody-coated RBCs and induction of autoreactive B-cells. As for the possibility of other preexisting immunological disorders, AIHA (even latent) is a contraindication to IFN treatment. Patients treated with IFN need accurate monitoring to guard against for the development of autoimmune disorders.

Background: In a recently reported study, low doses of intravenous immunoglobulins (IVIG) were shown to be as effective as high doses in protecting chronic lymphocytic leukemia (CLL) patients against infections, although a control group was not included. With this background we started a crossover study of low-dose IVIG prophylaxis aimed at investigating its superiority over empirical treatment of infections., Materials and Methods: Forty-two CLL patients with hypogammaglobulinemia (IgG < 600 mg/dL) and/or a history of at least one episode of severe infection in the 6 months preceding inclusion in the study were randomly allocated to receive either an infusion of 300 mg/kg IVIG every 4 weeks for 6 months or no treatment. Then they were switched to observation or IVIG for another 12 months; finally, they received IVIG or no therapy for 6 more months., Results: A significantly lower incidence of infectious episodes was observed during IVIG prophylaxis in 30 patients who completed the 6-month period of either observation or IVIG therapy. The same applied to the 17 patients who completed 12 months of either observation or IVIG prophylaxis. Interestingly, the restoration of serum IgG levels obtained in 17 out of 25 patients (mean percent value of IgG increase, 41.8%) did not parallel a decrease in the number of infectious episodes., Conclusions: A protective effect against infections is demonstrated for low-dose IVIG in the present study. A benefit was shown in patients who completed either 12 or 6 months of IVIG prophylaxis; however, even this low-dose treatment is not a cost effective way to prevent infection in CLL patients.