Search

The combination of elotuzumab, lenalidomide, and dexamethasone (EloRd) enhanced the clinical benefit over Rd with a manageable toxicity profile in the ELOQUENT-2 trial, leading to its approval in relapsed/refractory multiple myeloma (RRMM). The present study is a 3-year follow-up update of a previously published Italian real-life RRMM cohort of patients treated with EloRd. This revised analysis entered 319 RRMM patients accrued in 41 Italian centers. After a median follow-up of 36 months (range 6-55), 236 patients experienced disease progression or died. Median progression-free survival (PFS) and overall survival (OS) were 18.4 and 34 months, respectively. The updated multivariate analyses showed a significant reduction of PFS and OS benefit magnitude only in cases with International Staging System stage III. Major adverse events included grade 3/4 neutropenia (18.5%), anemia (15.4%), lymphocytopenia (12.5%), and thrombocytopenia (10.7%), while infection rates and pneumonia were 33.9% and 18.9%, respectively. No new safety signals with longer follow-up have been observed. Of 319 patients, 245 (76.7%) reached at least a partial remission. A significantly lower response rate was found in patients previously exposed to lenalidomide. In conclusion, our study confirms that EloRd is a safe and effective regimen for RRMM patients, maintaining benefits across multiple unfavorable subgroups., (© 2022 John Wiley & Sons Ltd.)

Subcutaneous (SC) rituximab may be beneficial in terms of convenience and tolerability, with potentially fewer and less severe administration-related reactions (ARRs) compared to the intravenous (IV) form. This report presents the results of a phase IIIb study conducted in Italy. The study included adult patients with CD20+ DLBCL or FL having received at least one full dose of IV RTX 375 mg/m 2 during induction or maintenance. Patients on induction received ≥4 cycles of RTX SC 1400 mg plus standard chemotherapy and FL patients on maintenance received ≥6 cycles of RTX SC. Overall, 159 patients (73 DLBCL, 86 FL) were enrolled: 103 (54 DLBCL, 49 FL) completed induction and 42 patients with FL completed 12 maintenance cycles. ARRs were reported in 10 patients (6.3%), 3 (4.2%) with DLBCL and 7 (8.1%) with FL, all of mild severity, and resolved without dose delay/discontinuation. Treatment-emergent adverse events (TEAEs) and serious adverse events occurred in 41 (25.9%) and 14 patients (8.9%), respectively. Two patients with DLBCL had fatal events: Klebsiella infection (related to rituximab) and septic shock (related to chemotherapy). Neutropenia (14 patients, 8.9%) was the most common treatment-related TEAE. Two patients with DLBCL (2.8%) and 6 with FL (7.0%) discontinued rituximab due to TEAEs. 65.2% and 69.7% of patients with DLBCL and 67.9% and 73.6% of patients with FL had complete response (CR) and CR unconfirmed, respectively. The median time to events (EFS, PFS, and OS) was not estimable due to the low rate of events. At a median follow-up of 29.5 and 47.8 months in patients with DLBCL and FL, respectively, EFS, PFS, and OS were 70.8%, 70.8%, and 80.6% in patients with DLBCL and 77.9%, 77.9%, and 95.3% in patients with FL, respectively. The switch from IV to SC rituximab in patients with DLBCL and FL was associated with low risk of ARRs and satisfactory response in both groups. This trial was registered with NCT01987505., Competing Interests: Gianluca Gaidano discloses roles in advisory boards or speakers' bureaus of AbbVie, Astra-Zeneca, Janssen, Roche, and Sunesys. Marco Ladetto declares in the last five years relationships in terms of consultancy, participation in advisory boards, invitation to scientific meetings, institutional research support and contracts with AbbVie, Acerta, Amgen, Archigen, ADC Therapeutics, BeiGene, Celgene, Gilead, J&J, Jazz, Roche, Sandoz, and Takeda. Luca Arcaini received advisory honoraria or speaker's bureau honoraria from Roche, Celgene, Janssen-Cilag, Verastem, EUSA Pharma, Incyte, and Sanofi and research support from Gilead. Ferreri Andres J. M. declares the following: speaker fee from Adienne; research grants from BMS, BeiGene, Pharmacyclics, Hutchison MediPharma, Amgen, Genmab, ADC Therapeutics, Gilead, Novartis, and Pfizer; advisory boards from Gilead, Novartis, Juno, and PletixaPharm; inventor of patents on NGR-hTNF/RCHOP in relapsed or refractory PCNSL and SNGR-hTNF in brain tumours. Maurizio Martelli declares the following: consultancy for Roche, Celgene, Janssen, Sandoz, Novartis, and Gilead; Membership on an Entity's Board or Advisory Committees for Roche, Celgene, Janssen, Sandoz, Novartis, Gilead, and Servier. No conflicts of interest are declared by the other authors regarding the publication of this article., (Copyright © 2022 Mario Petrini et al.)

We investigated the feasibility and activity of an intensified dose-dense ABVD (dd-ABVD) regimen in patients with early-stage unfavorable Hodgkin lymphoma (HL). This prospective, multicenter, phase II study enrolled 96 patients with newly diagnosed, unfavorable stage I or II classical HL. The patients received four cycles of dd-ABVD followed by radiotherapy. Interim PET (PET-2) was mandatory after two courses. Primary endpoints were the evaluation of dd-ABVD feasibility and activity (incidence of PET-2 negativity). The feasibility endpoint was achieved with 48/52 (92.3%) patients receiving > 85% of the programmed dose. The mean dose intensity in the overall patient population (n = 96) was 93.7%, and the median duration of dd-ABVD was 85 days (range, 14-115) versus an expected duration of 84 days. PET-2 was available for 92/96 (95.8%) patients, of whom 79 were PET-2 negative (85.9%). In total, 90 (93.8%) patients showed complete response at the end of treatment. With a follow-up of 80.9 months (3.3-103.2), the median progression-free survival (PFS) and overall survival (OS) were not reached. At 84 months, PFS and OS rates were 88.4% and 95.7%, respectively. No evidence for a difference in PFS or OS was observed for PET-2-negative and PET-2-positive patients. Infections were documented in 8.3% and febrile neutropenia in 6.2% of cases. Four patients died: one had alveolitis at cycle 3, one death was unrelated to treatment, and two died from a secondary cancer. dd-ABVD is feasible and demonstrates activity in early-stage unfavorable HL. The predictive role of PET-2 positivity in early-stage unfavorable HL remains controversial. The study was registered in the EudraCT (reference number, 2011-003,191-36) and the ClinicalTrials.gov (reference number, NCT02247869) databases., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Objectives: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only., Methods: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study., Results: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders., Conclusions: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Up to 30% immune thrombocytopenia (ITP) patients achieve a sustained remission off-treatment (SROT) after discontinuation of thrombopoietin receptor agonists (TPO-RAs). Factors predictive of response are lacking. Patients aged ≥18 years with newly diagnosed or persistent ITP were treated with eltrombopag for 24 weeks. Primary end-point was SROT: the proportion of responders that were able to taper and discontinue eltrombopag maintaining the response during a period of observation (PO) of six months. Secondary end-points included the association between some immunological parameters (TPO serum levels, cytokines and lymphocyte subsets) and response. Fifty-one patients were evaluable. Primary end-point was achieved in 13/51 (25%) treated patients and 13/34 (38%) patients who started the tapering. Baseline TPO levels were not associated with response at week 24 nor with SROT. Higher baseline levels of IL-10, IL-4, TNF-α and osteopontin were negative factors predictive of response (P = 0·001, 0·008, 0·02 and 0·03 respectively). This study confirms that SROT is feasible for a proportion of ITP patients treated with eltrombopag. Some biological parameters were predictive of response., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

The primary aim of this study was to describe the use of primary anti-infective prophylaxis (AP) in common clinical practice in patients affected by immune thrombocytopenia (ITP) and treated with RTX. Population studied consisted of patients affected by ITP (age ≥ 18 years) who had received at least one dose of RTX from January 2008 to June 2018. Five Italian haematology centres participated in the current study. Data were retrospectively collected: demographic data (age, gender), concomitant comorbidities and previous therapies for ITP, characteristics of AP, the occurrence of infections and their management. The ITP cohort consisted of 67 patients sub-grouped into two categories according to the administration of AP: (1) treated with AP (N= 34; 51%) and (2) not treated with AP (N=33, 49%). AP consisted of combined trimethoprim/sulfamethoxazole (TMP/SMX) and acyclovir (AC) in half of patients. TPM/SMX as a single agent was adopted in 32% patients and one patient received only AC. Overall, infections were experienced in 15% of patients during follow-up with a similar proportion in the 2 groups (treated and not treated) of patients (14.7% vs 15%). Clinical course of infections was however, less severe in patients treated with AP, where all infections were grade 2 and did not require hospitalization. In neither group of patients was reported Pneumocystis pneumonia. In conclusion, despite the absence of clear evidence, our analysis shows that AP in patients with ITP receiving RTX is frequently adopted, even if in the absence of well-defined criteria. Prophylaxis administration is quite consistent within the same haematological Center; thus, it seems related to clinicians' experience.

Objective and Methods: In order to assess the efficacy of brentuximab vedotin (Bv) in combination with bendamustine (B) in multiple relapsed or refractory (RR) classic Hodgkin lymphoma (cHL), medical records of 47 patients treated with BvB in second relapse or beyond were reviewed., Results: The median number of previous treatments was 2 (1-4). Bv was given at 1.8 mg/kg on day 1 and bendamustine at 90 mg/m 2 on days 1 and 2 of a 21-day cycle. The median number of BvB cycles was 4 (2-7), and all patients were evaluable for efficacy. The CR and OR rates were 49% and 79%, respectively; 67% of responding patients and 2 in stable disease proceeded to a SCT procedure. After a median follow-up of 19 months (5-47), median PFS was 18 months (95%CI: 23-29), and the 2-year OS was 72%. Significantly longer PFS and OS were observed in patients attaining a major clinical response to treatment and in those who received consolidation with SCT. Fifteen (32%) patients experienced severe (G > 2) toxicity. The main toxicities were neutropenia (23%), gastrointestinal (10%), peripheral sensory neuropathy (11%), and infection (4%)., Conclusion: Our real-world results suggest that BvB is an effective third-line rescue and bridge-to-transplant regimen for RR-cHL patients., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Despite widespread use of decitabine to treat acute myeloid leukaemia (AML), data on its effectiveness and safety in the real-world setting are scanty. Thus, to analyze the performance of decitabine in clinical practice, we pooled together patient-level data of three multicentric observational studies conducted since 2013 throughout Italy, including 306 elderly AML patients (median age 75 years), unfit for intensive chemotherapy, treated with first-line decitabine therapy at the registered schedule of 20 mg/m 2 /iv daily for 5 days every 4 weeks. Overall response rate (ORR), overall survival (OS) curves, and multivariate hazard ratios (HRs) of all-cause mortality were computed. Overall, 1940 cycles of therapy were administered (median, 5 cycles/patient). A total of 148 subjects were responders and, therefore, ORR was 48.4%. Seventy-one patients (23.2%) had complete remission, 32 (10.5%) had partial remission, and 45 (14.7%) had haematologic improvement. Median OS was 11.6 months for patients with favourable-intermediate cytogenetic risk and 7.9 months for those with adverse cytogenetic risk. Median relapse-free survival after CR was 10.9 months (95% confidence interval [CI]: 8.7-16.0). In multivariate analysis, mortality was higher in patients with adverse cytogenetic risk (HR=1.58; 95% CI: 1.13-2.21) and increased continuously with white blood cell (WBC) count (HR=1.12; 95% CI: 1.06-1.18). A total of 183 infectious adverse events occurred in 136 patients mainly (>90%) within the first five cycles of therapy. This pooled analysis of clinical care studies confirmed, outside of clinical trials, the effectiveness of decitabine as first-line therapy for AML in elderly patients unfit for intensive chemotherapy. An adverse cytogenetic profile and a higher WBC count at diagnosis were, in this real life setting, unfavourable predictors of survival., (© 2019 John Wiley & Sons, Ltd.)

Sequential use of the TPO-RAs romiplostim and eltrombopag in ITP patients failing either agent was retrospectively evaluated to assess efficacy and impact of clinical characteristics on outcome. Patients were grouped into 5 categories: efficacy issues: 1 st TPO-RA failure; loss of response; non-efficacy issues: platelet fluctuations; patient's preference; adverse event development. Either one TPO-RA sequence was analyzed at 3 month and at last follow-up. 106/546 patients on TPO-RA underwent switch and 65% achieved, regained or maintained a short- term response independent of switch sequence, gender or age; lower response rates were associated with lines of previous therapy; disease duration lowers probability to respond. Clinically, patients switched for efficacy issue did not differ from those switched for non-efficacy issues. Response was achieved/regained in 57.8% of patients switched for efficacy issues, the lowest response rates were observed in non-responders to 1 st TPO-RA; 80% of patients switched for non-efficacy issues maintained a response. Platelet fluctuation resolved in 44.4%. Of the 49 patients evaluable for long-term outcome, 27 were in response on therapy; 16 discontinued the TPO-RA for reasons other than efficacy, while only 6 were non responders. We confirm the efficacy of TPO-RA switch; once achieved, response to the 2 nd TPO-RA seems durable., (© 2017 Wiley Periodicals, Inc.)

Purpose: Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL., Experimental Design: First, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N = 40) and validation (N = 78) sets., Results: s-Arg-1 was higher in patients with advanced stage (p = 0.045), B-symptoms (p = 0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p = 0.012). Baseline levels of s-Arg-1 > 200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2.Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p = 0.0004 and p = 0.012).Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N = 63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N = 46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N = 9) with a 3-Y PFS of 37% (p = 0.0004)., Conclusions: We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a novel potential biomarker for HL prognosis.

Objective: We performed an external and multicentric validation of the predictive value of abdominal computed tomography (aCT) on time to first treatment (TTFT) in early stage chronic lymphocytic leukemia (CLL) patients., Methods: aCT was performed at diagnosis in 181 Rai 0 patients enrolled in the O-CLL1-GISL trial (clinicaltrial.gov ID:NCT00917549)., Results: Fifty-five patients showed an abnormal aCT. Patients with an abnormal aCT showed a significantly shorter TTFT than those with normal aCT (P < 0.0001). At multivariate analysis, aCT (P = 0.011), β-2 microglobulin (P = 0.019), and CD38 expression (P = 0.047) correlated with TTFT. Following IWCLL 2008 criteria, 112 (61.9%) cases remained at Rai 0, while 69 (38.1%) satisfied the criteria of clinical monoclonal B-cell lymphocytosis (cMBL). Reclassified Rai 0 patients with an abnormal aCT showed a significantly shorter TTFT than those with a normal aCT (P < 0.0001). At multivariate analysis, only aCT (P = 0.011) correlated with TTFT. Eleven cMBL cases (15.9%) showed an abnormal aCT and were reclassified as small lymphocytic lymphomas (SLL); nonetheless, TTFT was similar for cMBLs and SLLs., Conclusion: Our results confirm the ability of the abnormal aCT to predict progression in early stage cases., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

ZAP-70 is a marker of clinical outcome in chronic lymphocytic leukemia (CLL), however its assessment suffers from a lack of standardization consensus. To identify novel markers able to surrogate IGHV mutational status, CD19(+)CD5(+)-B-lymphocytes from 216 patients enrolled in a prospective study (ClinicalTrial.gov Identifier:NCT00917540), underwent gene expression profiling. Samples were split into CLL-Training (n=102) and CLL-Validation (n=114) sets, and an independent supervised analysis for IGHV mutational status was performed considering all genes with gene expression equal or above that of ZAP-70. Thirty-one genes (23 up- and 8 down-regulated) and 23 genes (18 up- and 5 down-regulated) satisfied these criteria in the CLL-Training and CLL-Validation sets, respectively, and 20 common genes (15 up and 5 down) were found to be differentially regulated in both sets. Two (SNORA70F, NRIP1) of the down-regulated and 6 (SEPT10, ZNF667, TGFBR3, MBOAT1, LPL, CRY1) of the up-regulated genes were significantly associated with a reduced risk of disease progression in both sets. Forcing the afore-mentioned genes in a Cox multivariate model together with IGHV mutational status, only CRY1 (HR=2.3, 95% CI: 1.1-4.9, P=.027) and MBOAT1 (HR=2.1, 95% CI: 1.1-3.7, P=.018) retained their independent prognostic impact, supporting the hypothesis that these genes may potentially act as surrogates for predicting IGHV mutational status., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the attempt to find a peripheral blood biological marker that could mirror the dysregulated microenvironment of Hodgkin Lymphoma (HL), we analysed the amount of myeloid-derived suppressor cells (MDSC), including the three main sub-types (monocytic, granulocytic and CD34 + fraction). The absolute MDSC count was investigated in 60 consecutive newly diagnosed HL patients and correlated with clinical variables at diagnosis and outcome. Patients received standard-of-care chemotherapy with the exception of interim fluorodeoxyglucose positron emission tomography (PET-2)-positive patients, who were switched early to a salvage regimen. All MDSC subsets were increased in HL patients compared to normal subjects (P < 0·0001) and were higher in non-responders. However, a strong prognostic significance was limited to immature (CD34(+) ) MDSC. A cut-off level of 0·0045 × 10(9) /l for CD34(+) MDSC resulted in 89% (95% confidence interval [CI] 52-99%) sensitivity and 92% (95% CI 81-98%) specificity. The positive predictive value to predict progression-free survival was 0·90 for PET-2 and 0·98 for CD34(+) MDSC count; the negative predictive value was 0·57 for PET-2 and 0·73 for CD34(+) MDSC. PFS was significantly shorter in patients with more than 0·0045 × 10(9) CD34(+) MDSC cells/l at diagnosis and/or PET-2 positivity (P < 0·0001). In conclusion, all circulating MDSC subsets are increased in HL; CD34(+) MDSC predict short PFS, similarly to PET-2 but with the advantage of being available at diagnosis., (© 2014 John Wiley & Sons Ltd.)

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B-cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10(-4) ) and CD38 (P < 1 × 10(-4) ) and ZAP-70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B-cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion., (Copyright © 2012 Wiley Periodicals, Inc.)

Several trials comparing the efficacy of standard melphalan and prednisone (MP) therapy with MP plus thalidomide (MPT) in elderly patients with multiple myeloma (MM) have been reported, with inconsistent results. The primary goal of our study was to evaluate the efficacy and toxicity of MP versus MPT in newly diagnosed patients with MM who were transplant-ineligible or over age 65. A total of 135 patients were enrolled. Either minimal response or better or partial response or better were more frequent with MPT treatment (p = 0.001). After a median follow-up of 30 months, median progression-free survival (PFS) and overall survival (OS) were 33 and 52 months for MPT versus 22 and 32 months for MP, respectively. The comparison showed a significant advantage for MPT versus MP in PFS (p = 0.02) and only a trend for OS (p = 0.07). Severe adverse events were observed more frequently with MPT. In conclusion, our results show an improved activity of MPT at a cost of increased toxicity. We believe that MPT can be considered one of the new standard of care for elderly or transplant-ineligible patients with MM.

DNA aneuploidy has been used as a genetic marker of malignancy in multiple myeloma (MM). CD38 and CD138 expression and absence of CD22 and CD19 may define plasmacells (PC). Several authors support evidences of circulating plasmacells, and their role in relapse after autologous stem cell transplantation has been hypothesised. The existence of B-lymphocytes belonging to the myeloma clone is still controversial. If CD19 or CD22 positive B-lymphocytes are part of the myeloma clone, there should be evidence of myeloma-specific genetic markers in this population. Using DNA content measurement in combination with CD19 or CD38 detection in a multiparametric flow cytometry analysis, we studied bone marrow and peripheral blood of 10 aneuploid MM patients. In the bone marrows of all these 10 aneuploid patients (100%), we detected CD38(++) aneuploid plasmacells ( 27 +/- 17%, mean +/- S.D.) and a small number of CD19(+) aneuploid lymphocytes ( 0.11 +/- 0.074%). In 100% of these patients, we also detected CD38(++) aneuploid circulating plasmacells ( 0.6 +/- 0.9 %) and a small number of CD19(+) aneuploid lymphocytes (0.03 +/- 0.04%). In this study, we detected aneuploid CD19(+) lymphocytes and CD38(++) plasmacells in bone marrow and peripheral blood of all MM patients. A crucial role for the detection of aneuploid CD19(+) cells was played by the acquisition of a sufficient number of CD19(+) lymphocytes by using a "live gate" acquisition and "continuous gating" analysis. With the techniques used in this study, it was possible to detect aneuploid B lymphoid cells among normal diploid B cells. The significance of this finding is controversial and opened to different interpretations.

Expression of two MDR genes, BCRP and MDR1, was evaluated by real-time PCR technique in 51 AML patients. Fifty-six percent expressed the BCRP gene, with the 48.2% showing intermediate levels. Eighty-eight percent expressed the MDR1, with 23.8% of cases at high expression. A significant correlation between BCRP and MDR1 values was found by regression analysis. Either levels of BCRP or MDR1 did not correlate with clinical characteristics of patients at diagnosis.

Acute promyelocytic leukemia (APL) is characterized by onset at a young age and a life-threatening hemorrhagic diathesis, which is attributed to a disseminated intravascular coagulation (DIC)-like coagulopathy. The discovery of all-trans-retinoic acid has changed the course of APL treatment by reducing the onset of DIC and inducing a complete and durable remission in more than 90% of patients. The occurrence of APL during pregnancy is not a frequent event, but the management of these patients raises many therapeutic and ethical dilemmas and requires a careful clinical case evaluation of fetal and maternal risk, coagulation status, the parents' wishes, and therapeutic options. Here we describe 3 patients with APL diagnosed during pregnancy. Clinical data and the therapeutic approaches are presented. In the discussion, we analyze clinical decisions and therapeutic options and compare our cases with those found in the literature.

We evaluated the presence of P-glycoprotein (P-gp)-170, multidrug resistance protein (MRP), lung resistance protein (LRP)-56 and Bcl-2 in CD19-positive cells from 100 cases of chronic lymphocytic leukaemia (CLL). P-gp-170 was found in 73% of the CLL cases with no significant difference regarding stage or previous treatment. LRP-56 protein was homogeneously distributed with no differences for stage or treatment. MRP protein was detected at a low level of expression in 49.4% of CLL patients with no differences for stage or treatment. Bcl-2 protein was expressed at a high level in all CLL patients and higher levels were found in the advanced stage. This leads us to conclude that P-gp, MRP, LRP-56 and Bcl-2 are frequently expressed in CLL. P-gp, MRP and LRP are not correlated to stage or previous treatment. Bcl-2 is higher in advanced-stage patients. The clinical and biological significance of these zMDR mechanisms in CLL remains to be fully explained.

Background: Programmed cell death (PCD) is characterized by a sequence of tightly regulated events that result in the activation of caspases and in internucleosomal DNA cleavage. Late apoptotic events such as DNA-strand breaks can be assayed by in situ end labeling (ISEL) and DNA measurement (sub G1) using flow cytometry. Phosphatidylserine (PS) redistribution from the inner plasma membrane leaflet to the outer leaflet, an early event in PCD, can be detected by annexin V (AxV) binding to PS. AxV-fluorescein isothiocyanate (FITC) fluorescence intensity is variable and characterizes different cell populations, denoted here as AxV-negative (AxV(neg)), AxV-low-positive (AxV(lo)), and AxV-high-positive (AxV(hi))., Methods: We investigate the correlation of three methods (ISEL, sub G1 DNA content, and AxV assay) for detecting apoptosis with focus on differences between populations with different levels of PS. We also examined the expression of PCD-regulating Bcl-2 family members in these cell populations by reverse transcription-polymerase chain reaction (RT-PCR). Chronic lymphocytic leukemia (CLL) cells exposed to fludarabine (FAMP) were used as an in vitro model. Cells with different PS/AxV levels were separated using fluorescence-activated cell sorting (FACS)., Results: Only purified AxV(hi) cells had high positivity in the ISEL and sub G1 assays (94 +/- 0.6%, 88.6 +/- 6.6%, and 98.6 +/- 0.6%, respectively), indicating that late apoptotic cells are detected equally by all three methods. In the AxV(lo) population, ISEL was positive in 21% +/- 13% and DNA sub G1 in 20% +/- 6.6% of cells, suggesting that AxV identifies early apoptotic cells better than the other assays. Anti-apoptotic Bcl-2 and Bcl-X(L) were upregulated by FAMP when cells entered apoptosis (AxV(lo)), as was pro-apo- ptotic Bcl-X(S), which was undetectable in nonapoptotic AxV(neg) cells. Pro-apoptotic Bax was only expressed in AxV(neg) and AxV(lo) cells. Late apoptotic AxV(hi) cells did not express Bcl-X(S) or Bax., Results: (1) AxV staining is more sensitive than sub G1 or ISEL in detecting early apoptotic cells; (2) only late apoptotic cells are equally detected by all assays; (3) AxV is a valuable tool in the detection and isolation of apoptotic cells at different stages of PCD; and (4) pro-apoptotic Bcl-X(S) and Bax are expressed at early, not late, stages of apoptosis., (Copyright 2000 Wiley-Liss, Inc.)

We have investigated the effect of nerve growth factor (NGF) in the androgen-dependent, prostate adenocarcinoma LNCaP cell line. Exposure of LNCaP cells to NGF resulted in a significant increase of cell proliferation. The effect was concentration dependent and equally present in serum- or charcoal-stripped serum-supplemented and serum-deprived conditions. The mitogenic action of NGF was accompanied by an enhanced expression of prostate-specific antigen (PSA) and resulted additive to the proliferative effect of dihydrotestosterone. The proliferative effect of NGF appeared to be mediated by the high-affinity NGF receptor, p140trka. Only p140trka, but not the low-affinity NGF receptor, p75LNGFR, was expressed in LNCaP cells; both the proliferative response and the phosphorylation of p140trka upon NGF treatment were prevented by the tyrosine kinase inhibitor K252a. LNCaP cells transiently transfected with the cDNA encoding for p75LNGFR appeared more sensitive to NGF, as demonstrated by the increased number of p75LNGFR-transfected LNCaP cells exposed for 72 h to NGF compared with wild LNCaP cultures. However, p75LNGFR-transfected LNCaP cells rapidly underwent apoptotic death when deprived of NGF. Our study demonstrates the physiological relevance of NGF in the regulation of prostate cell proliferation and the relative contribution of the high- and low-affinity NGF receptors in this control.

Chronic myeloid leukemia (CML) is a hematological malignancy resulting from clonal expansion and massive accumulation of leukemic myeloid cells that retain differentiation and maturation capacity. Since CML cell accumulation has been related to apoptosis inhibition by the product of the BCR-ABL gene, attempts to eradicate leukemic cells would require therapeutic drugs able to overcome this inherent resistance. Here, we investigated in vitro the apoptotic effect of all-trans retinoic acid (ATRA) and cytosine arabinoside (ARA-C), employed alone, in combination or in sequence, on freshly isolated cells from 10 patients with chronic-phase CML. Our cell cultures showed that both ATRA and ARA-C were able to induce apoptosis in CML cells, even if ARA-C resulted more effective than ATRA. The combined use of ATRA and ARA-C seemed to have only an additive effect while the sequential use did not show any advantage. These in vitro observations indicate that ATRA and ARA-C may be effective in reducing CML cells through apoptosis induction, suggesting that it could be worthwhile to examine ATRA and ARA-C combinations in the therapy of CML.

Reduced or absent neutrophil alkaline phosphatase (NAP) activity is a common feature of neutrophilic granulocytes from patients with chronic myeloid leukemia (CML). In this study we examined whether NAP activity could be restored in vitro by stimulating CML cells with different promoters such as all-trans-retinoic acid (ATRA), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). The results obtained indicated that ATRA and G-CSF, either alone or in combination, were effective in inducing NAP activity in CML cells, whereas GM-CSF was not. Further, NAP restoration in ATRA- and G-CSF-treated cultures was accompanied by increased morphologic differentiation of the CML clone. It might be concluded that the CML clone could be driven in vitro by ATRA and G-CSF both to achieve granulocytic maturation and to correct functional NAP-related defects.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

Cyclin B1 plays a critical role in regulating cell-cycle progression from G2 through M phase (including exit from M phase). In this study, we investigated the relationship between taxol-induced M-phase arrest, disruption of the cyclin B1-regulation pathway and apoptosis in KB cells. Continuous exposure of KB cells to 0.5 microg/ml taxol caused mitotic arrest and >90% cell death at 48 hr. Mitotic blockade peaked at 24 hr, with 68% of cells in mitosis at that time compared with 3% at baseline, and decreased thereafter. Apoptosis assessed by morphological changes and DNA ladder fragmentation was a later event, peaking at 48 hr (later time points were not studied). Taxol also caused an increase in cyclin B1 accumulation, as assessed by Western blot analysis, and stimulated cyclin B1-dependent kinase. Cyclin B1 accumulation and kinase stimulation peaked at 12 and 24 hr, respectively, at which times they were 5-fold and 90-fold higher than in control untreated cells. These effects decreased thereafter. All taxol-induced cellular effects were abrogated by the protein and RNA synthesis inhibitors cycloheximide and actinomycin D. In contrast, the endonuclease inhibitors aurintricarboxilic acid and zinc markedly inhibited taxol-induced DNA ladder fragmentation without altering taxol-induced cell-cycle arrest, cyclin B1 accumulation, activation of cyclin B1 kinase activity and cytotoxicity. We conclude that taxol-induced stimulation of cyclin B1-dependent kinase activity parallels mitotic arrest, is more pronounced than mitotic arrest and precedes the induction of programmed cell death.

Fludarabine (F-ara-A), an adenine nucleoside analog with efficacy in B-cell chronic lymphocytic leukemia (B-CLL), has also been shown to have a long-lasting suppressive effect on T lymphocytes. In heterogeneous clinical samples, apoptosis cannot be detected by standard methods in small cellular subsets. We developed, therefore, a combined assay of in situ end-labeling of nicked DNA by terminal deoxynucleotide transferase, with measurements of cellular DNA content and surface antigens (CD3, CD4, CD8, and CD19) by multiparametric flow cytometry. This assay was used to determine F-ara-A-induced apoptosis in different lymphocyte subsets from CLL patients and normal controls treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2 protein levels. We observed a direct effect of F-ara-A on both B-CLL and T lymphocytes. The response to F-ara-A in B-CLL lymphocytes in vitro was Rai stage-dependent, the early-stages being more responsive (P = .01). Higher levels of spontaneous apoptosis were observed in B-CLL lymphocytes from early stage patients (P = .02). No difference was observed in spontaneous apoptosis of normal T cells in B-CLL, although T lymphocytes in late-stage disease were more sensitive to F-ara-A-induced apoptosis. Incubation with cyclosporin A did not affect B-CLL and T-lymphocyte survival compared with control cultures. Results suggested a direct apoptotic effect of F-ara-A on B-CLL lymphocytes that decreases with increasing clinical stage. No correlation was found between bcl-2 and spontaneous or F-ara-A-induced apoptosis. Apoptosis occurred at all cell-cycle stages and was not restricted to cells in S phase. The mechanisms of this stage-dependent apoptosis in CLL remain to be elucidated.

Monoclonal antibodies to very late antigen 4 (VLA-4) recognize the alpha4beta1 integrin receptor. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34-selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34-selected cells was shown to induce apoptosis of CD34-selected cells in these CD34-selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34-selected cells. Given that there is no difference between the alpha4beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+-selected precursor cells proliferate at a higher rate when these cells are plated on recombinant vascular cell adhesion molecule 1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function that results from the anchorage-dependent growth of the CD34-selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy.

Chronic lymphocytic leukaemia (CLL) is an indolent lymphoproliferative disorder manifested by low growth fraction and prolonged survival of the malignant cells. The mechanisms that enable CLL cells to live longer and to resist apoptosis remain unclear. Because the malignant CLL cells express CD40 and Fas receptors, which can transduce cell-survival and cell-death signals, we examined the role of CD40 in the growth regulation of CLL cells and its interaction with Fas-mediated and fludarabine-induced apoptosis in vitro. Primary CLL cells underwent spontaneous apoptosis in culture, which was enhanced by exogenous human Fas ligand (FasL) or fludarabine. Exogenous CD40L rescued CLL cells from spontaneous apoptosis in a dose-dependent manner, and caused CLL cells to resist apoptosis induced by FasL or fludarabine. Patients' autologous plasma rescued CLL cells from spontaneous apoptosis, an effect that could be reversed with anti-CD40 ligand (CD40L) antibodies. The levels of soluble CD40 ligand in the sera of 51 CLL patients and 55 healthy donors were determined by enzyme-linked immunosorbent assay. The mean soluble CD40L level in normal donors was 0.29 ng/ml compared to a mean value of 0.80 ng/ml in CLL patients (P < 0.001). CD40L up-regulated bcl-X(L) mRNA but not bcl-2 in CLL cells within 3-6 h in culture. Our results demonstrated that serum of patients with CLL contained elevated levels of biologically active soluble CD40L, and that CD40L can prolong survival of CLL cells and mediate their resistance to FasL and fludarabine in vitro.

Incorporation of fludarabine, 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A), into replicating DNA inhibits further chain elongation and is the critical event in F-ara-A-mediated cytotoxicity. We have used the normal cellular process of nucleotide excision repair to create an opportunity for F-ara-A incorporation into the DNA of noncycling cells. Irradiation of quiescent lymphocytes with UV light (254 nm, 0. 5-30 J/m2) in the presence of [3H]F-ara-A produced a dose-dependent increase in F-ara-A monophosphate incorporation into DNA that reflected a 60-70% inhibition of DNA repair at 2 h. Lymphocytes pretreated with 3 micrometer F-ara-A for 2 h before irradiation with 0.5 or 2.0 J/m2 were incubated for 24 h in the presence or absence of F-ara-A. Morphological features of apoptosis and DNA cleavage into high molecular weight fragments were increased in cells treated with UV plus F-ara-A compared to those treated with UV or F-ara-A alone. FACScan analysis confirmed the morphological and biochemical results. A 2-h pulse of F-ara-A produced intracellular F-ara-ATP levels of 40 micrometer, and removal of F-ara-A from the media resulted in a monophasic elimination (r2 = 0.88) in F-ara-ATP levels with a half-life of 5.6 h. Lymphocytes undergoing apoptosis demonstrated a G0 DNA content, indicating that entry into the cell cycle was not required. This study demonstrates that F-ara-AMP is incorporated into DNA during UV-induced repair in quiescent lymphocytes and that this is associated with the inhibition of ongoing DNA repair and an increased incidence of apoptosis. Combinational therapies involving fludarabine with agents and modalities that initiate DNA repair may have clinical relevance in the treatment of human malignancies.

CD30, a member of the tumour necrosis receptor superfamily, is physiologically expressed on a subpopulation of T helper cells in normal individuals but is also expressed on several malignant and virally transformed cells. Its ligand (CD30L) is a pleiotropic cellular transmembrane protein that can induce cell death in several CD30+ cell lines. CD30L expression has been reported on activated human peripheral blood T lymphocytes and macrophages but not on B cells. Here we show that the CD30L is expressed on resting normal and on malignant B cells in addition to both CD4+ and CD8+ subsets of activated T cells, making it the second tumour necrosis family member, in addition to the CD27 ligand, that can be expressed on both T and B cells. These findings raise the possibility that the CD30L has a role in B-cell/T-cell communication and that B and T cells are likely to be involved in the growth regulation of CD30+ tumours.

The growth and tumorigenicity of murine lung cancer cells transfected with an antisense cyclin D1 construct were evaluated in studies pertaining to mouse lung carcinogenesis. This antisense construct inhibited the expression of cyclin D in these cells, significantly reducing both their in vitro proliferation and tumorigenicity in nude mice relative to control cells. These data may have implications regarding the treatment of human neoplasms of aerodigestive tract origin that either overexpress the cyclin D oncogene or exhibit mutations that influence cell cycle progression via cyclin D-dependent mechanisms.

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 (p21) inhibits cellular proliferation and has been implicated in p53-dependent apoptosis. In this study, we examined the regulation of p21 in the K562 erythroleukemia cell line using a human temperature-sensitive mutant of p53, 143Ala. We showed that 143Ala at the permissive temperature (32 degrees C) activated the expression of p21. Stable cell lines expressing 143Ala or other p53 mutants were established. We then examined whether elevation of p21 promotes apoptosis and sensitized cells to radiation-induced apoptosis. Our results showed that p21 elevation did not promote or sensitize K562 cells to apoptosis.

A unique mRNA produced by the t(15;17) (q22-24;q11-21) translocation in the leukemic cells of acute promyelocytic leukemia patients encodes a chimeric protein, PML/RARalpha, which is formed by the fusion of the retinoic acid receptor alpha (RARalpha) and the promyelocytic locus gene (PML). This translocation is often the only visible karyotypic aberration present which is detected in almost 100% of acute promyelocytic leukemia patients. As an initial step to study the role of PML/RARalpha in leukemogenesis, we attempted to express the fusion protein in hematopoietic cells through retrovirus-mediated gene transfer of the retroviral vector, pGPRCHT, which contains the PML/RARalpha cDNA. Transduction of the PML/RARalpha cDNA fragment used in this vector, which extends from the position 31 bp to the position 2638 bp in a transcription unit driven by the Moloney murine sarcoma virus LTR, was found to abrogate the growth factor dependence of TF-1 cells. In addition, introduction of PML/RARalpha into TF-1 cells can protect these cells from the apoptosis usually induced in TF-1 cells by growth factor withdrawal, as measured by three assays for apoptosis: morphology, DNA ladder formation, and end labeling of nicked DNA with fluorescent-conjugated nucleotide precursors followed by a fluorescence-activated cell sorting assay. These data suggest that the PML/RARalpha fusion protein may inhibit programmed cell death in myeloid cells.