Search

To verify the clinical usefulness of extracellular cyclic nucleotide determination as a tumor marker, plasma cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured in 70 normal subjects and 173 acute leukemia patients studied in different stages of their disease. Mean plasma cAMP levels were similar in leukemic and normal subjects, although in 48 patients in the active stage of the disease, first diagnosis, or relapse, the cAMP values were below the normal range, and most of these patients failed to respond to chemotherapy. Plasma cGMP levels were markedly elevated in untreated patients, normalized in all patients who attained complete remission, and increased promptly to pretreatment values in patients who relapsed, suggesting that their determination may be useful to monitor the patients' response to treatment.

To verify the clinical usefulness of extracellular cyclic nucleotide determination as a tumor marker, plasma cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured in 70 normal subjects and 173 acute leukemia patients studied in different stages of their disease. Mean plasma cAMP levels were similar in leukemic and normal subjects, although in 48 patients in the active stage of the disease, first diagnosis, or relapse, the cAMP values were below the normal range, and most of these patients failed to respond to chemotherapy. Plasma cGMP levels were markedly elevated in untreated patients, normalized in all patients who attained complete remission, and increased promptly to pretreatment values in patients who relapsed, suggesting that their determination may be useful to monitor the patients’ response to treatment.© 1987 byGrune & Stratton, Inc.0006-4971/87/6906-00I0$3.00/0

The presenting clinico-hematologic features of 386 patients with nonmyelomatous monoclonal gammopathy (MG) were correlated with the frequency of malignant transformation to evaluate the most important variables conditioning its evolution into multiple myeloma (MM) or Waldenstrom macroglobulinemia (WM). Most of the patients (335) had monoclonal gammopathy of undetermined significance (MGUS: 39 IgA, 242 IgG, 54 IgM): the remaining 51 patients (12 IgA, 39 IgG) fulfilled all of the MGUS diagnostic criteria (according to Durie) except that bone marrow plasma cell (BMPC) content was 10% to 30%, and so they were defined as having monoclonal gammopathy of borderline significance (MGBS). There were no significant differences between the MGUS and MGBS groups in terms of age, sex, or median follow-up. After a median follow- up of 70 and 53 months, respectively, 23 of 335 MGUS and 19 of 51 MGBS patients had undergone a malignant evolution. Univariate analysis of the IgA and IgG patients showed that the cumulative probability of the disease evolving into MM correlated with diagnostic definition (MGBS v MGUS), BMPC content (> or = 10% v < 5% and < or = 5% v > 5%) and reduced serum polyclonal Ig. In the IgG cases, there was also a significant correlation with detectable Bence Jones proteinuria, serum monoclonal component (MC) levels and age at diagnosis (> 70 v < = or 55 years). In the IgG cases as a whole, the same variables remained in the Cox model where the BMPC percentage was considered after natural logarithmic transformation and the monoclonal component as g/dL value. The relative risks of developing MM are the following: 2.4 for each 1 g/dL increase of IgG, serum MC, 3.5 for detectable light chain proteinuria, 4.4 for the increase of 1 unit in log. BMPC percentage, 6.1 for age > 70, 3.6 and 13.1 for a reduction in one or two polyclonal Ig. In conclusion, our study allows the identification of a particular subset of MGUS patients (MC < = or 1.5 g/dL, BMPC < 5%, no reduction in polyclonal Ig and no detectable light chain proteinuria) at very low- risk of evolution, who can be considered as having benign monoclonal gammopathies. We also describe a previously undefined group of MG patients (with monoclonal gammopathy of borderline significance) who are at high-risk of malignant evolution. These findings could have a considerable impact on the cost/benefit ratio of monitoring programs in these patients.

Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Colombi:MolMed: Employment. Lambiase:MolMed: Employment. Bordignon:MolMed SpA: Employment.

Background:Haploidentical HSCT is a readily available platform for most patients lacking an HLA-matched donor, but the T-cell depleted (TCD) approach without any post-transplant donor cell therapy is impaired by high non-relapse mortality (NRM) due to slow IR, while the T-cell replete (TCR) approach followed by cyclophosphamide and immunosuppressive therapy is hampered by high chronic GvHD and relapse incidence (RI)

Bonini: MolMed S.p.A.: Consultancy. Colombi:MolMed: Employment. Lambiase:MolMed S.p.A: Employment. Bordignon:MolMed: Employment.

Niederwieser: Novartis, Gentium, Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Colombi:MolMed: Employment. Antonio:MolMed: Employment. Bordignon:MolMed: Employment.

Background: Haploidentical family donors represent the ideal solution to offer a potential cure for every high-risk leukemia patient undergoing HSCT. Widespread use of haploidentical HSCT has been historically limited by high rates of late non-relapse mortality (NRM) and relapse, which are associated with the delayed immune reconstitution (IR) due to either ex vivo T-cell depletion or in vivo post-HSCT cyclophosphamide given as graft-vs-host disease (GvHD) prevention. We have previously shown in a phase 2 trial (TK007, Lancet Oncol 2009;10:489) that TK cells (donor T cells genetically modified to express the HSV-TK suicide gene) can safely induce an early IR when given after T-cell depleted haploidentical HSCT

Introduction

Castagnetti: Bristol Myers Squibb: Honoraria; Novartis: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Baccarani:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria.

β-herpesviruses, such as CMV and HHV6, are important pathogen in transplanted patients. The morbidity because of CMV reactivation after allogeneic stem cell transplantation (SCT) has led to the monitoring of this virus and to introduction of preemptive therapy. However, CMV infection is still one of the most challenging complications, because CMV disease may occur as life-threatening pneumonitis, and may increase the risk of opportunistic infections. HHV6 reactivation has been demonstrated after SCT and this virus is recognized as important pathogen, either by direct infection or via interaction with CMV. Innate and adaptive immune response against these viruses involves the activation of Toll-like receptors (TLRs). TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs). Viral nucleic acids and viral structural proteins, such as glycoproteins, are considered as PAMPs. Endosomal TLRs (TLR3, 7, 8 and 9) recognize viral nucleic acids and some surface TLRs may be involved in the detection of structural proteins. Some clinical and experimental evidences indicate that CMV and HHV-6 can modulate the immune system and influence the immune reconstitution after SCT. However, the role of TLRs in this complex interplay remains unclear, especially in the setting of allogeneic SCT.The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to CMV and HHV6 reactivation in the early period after allogeneic SCT.CMV and HHV6 reactivation was monitored weekly by quantitative real-time PCR until the second month after SCT. The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and in any case before CMV or HHV6 reactivation. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls.CMV reactivation within 2 months after transplantation was observed in 13 out of 33 patients. CMV pneumonitis was observed in 1 patient. HHV-6 reactivation was detected in 1 patient. Median age was 45 years (range, 22–64) and 21 patients were male. TLRs expression and function did not significantly differ in controls and patients without CMV. Lymphocytes of patients with CMV reactivation showed an increased expression of TLR5 (4,1±2,4 vs 2,0±1,7 p=0,008). TLR8 expression was lower on monocytes with CMV reactivation (0,8±0,9 vs 2,0±1,7 p=0,03). MCP-1 relative induction post-stimulation of TLR1 and 8 was significantly decreased in patients with CMV reactivation (p<0,04).Surface TLR2 and intracellular TLR3 and 9 are reported to recognize CMV by some authors. In our study, surface TLR5 and intracellular TLR8 seem to be involved in the interaction between CMV and the immune system of transplanted patients. In particular, TLR8 could play a protective role. MCP-1 production upon TLR1 and 8 activation negatively correlates with CMV reactivation. The defective immune system after SCT could explain these results, which could be confirmed by the assessment of a larger number of patients and the analysis of other possible interfering factors.No relevant conflicts of interest to declare.

The introduction of Imatinib has significantly improved the outcome for patients with Ph+ CML. The complete cytogenetic response (CCgR) is a strong and confirmed predictor of improved long-term outcome. According to current recommendations, Imatinib (IM) should be continued indefinitely. However, optimal responders can be eligible for investigational trials of treatment discontinuation.This study (ClinicalTrials.gov NCT 00858806) describes the effects of a policy of intermittent Imatinib (INTERIM) treatment (one month on/one month off) on cytogenetic and molecular responses in a selected population of patients ≥ 65 years old who were receiving treatment with Imatinib for > 2 years and were in stable complete cytogenetic response (CCgR). The primary endpoint of the study was the proportion of patients who maintained CCgR after 1 year of INTERIM. The secondary endpoint was the level of BCR-ABL transcripts during INTERIMCytogenetic and molecular responses were monitored by FISH and RT-Q-PCR every 3 months. The definition of CCgR, and of CCgR loss was based on CBA of marrow metaphases which was performed at baseline and in all the patients who became FISH positive (BCR-ABL–positive nuclei > 1%). Major molecular response (MMR), corresponding to a 3-log reduction in BCR-ABL transcript level from the standardized baseline, was defined as BCR-ABL ≤0.1%IS and was indicated as MR3.0. For the purposes of this study, complete molecular response (CMR) was defined as a 4-log reduction in BCR-ABL transcript level (<0.01%IS), with a sensitivity of at least 10,000 ABL copies, and is indicated as MR4.0. In case of loss of CCgR or MMR, Ph+ additional cytogenetic abnormalities (ACA) and BCR-ABL kinase domain (KD) point mutation analysis were also performed.Seventy-six patients have been enrolled. Six patients (8%) lost CCgR (CBA positive), and 3 other patients became FISH positive while remaining CBA negative. At 12 months, the probability of maintaining CBA negativity was 92% (95% CI 86–98%), while the probability of maintaining FISH negativity was 87% (95% CI 79–94%). None of the factors that were examined by univariate and multivariate analysis were found to be associated with an higher probability of either loosing the CCgR (CBA) or showing a FISH positivity (> 1%), with the exception of the duration of imatinib therapy (HR = 0.23, 95% CI 0.008–0.73, P =.01). Among patients with prior Imatinib treatment longer or shorter than 48 months, the probability of maintaining FISH negativity was 94% (95% CI 88–100%) vs 71% (95% CI 53–89%), respectively, HR = 0.23 (P =.007). All the 6 patients who lost CCgR regained the CCgR with daily Imatinib, at the same dose, defined by FISH negativity after 3 to 9 months (4/6 also CBA negative, 2 patients refused bone marrow aspiration). At baseline, all but one patient (99%) were in MMR (MR3.0, BCR-ABL ≤0.1%IS), and 63 patients (83%) were in MR4.0 (<0.01%IS). After one year of intermittent Imatinib, 18/76 patients (24%) lost MR3.0 and 25/63 (39%) patients lost MR4.0. No patient lost complete hematologic response, progressed to accelerated or blastic phase, developed ACA in Ph+ cells, or developed BCR-ABL mutations. After one year, the remaining 70 patients were allowed to go back to continuous treatment, or to continue the intermittent treatment. During the first six months of follow-up (month 13 to 18), 1 of 70 discontinued Imatinib for atrial fibrillation and 9 of 70 went back to continuous treatment because FISH negativity loss (1 patient) or MMR (MR3.0) loss (8 patients). All the patients continue to be regularly observed and monitored and the first twelve months of follow-up will be presented.The intermittent use of imatinib in older patients in stable CCgR after continuous imatinib treatment results in the transient loss of the CCgR in a minority (8%) of the patients. However, the disease burden at the molecular level significantly increased. A policy of intermittent treatment may be an alternative both to chronic continuous treatment and to treatment discontinuation, particularly in the elderly. However, a longer follow up is required before drawing final conclusions.This work was supported in part by EuropeanLeukemiaNet through the European Treatment and Outcome Study (EUTOS) and by Cofin 2009.Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Di Raimondo:celgene: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, and in the pathogenesis of autoimmune and graft-versus-host (GVHD) diseases. Pattern recognition receptors such as Toll-like receptors (TLRs) play a key role in the cross-talk between innate and adaptive immune system. TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs), such as common protein, carbohydrate or DNA/RNA pattern motifs. TLRs are also receptors for endogenous ligands and damaged tissue, suggesting that both pathogen-derived molecules and products of damaged tissue can trigger signals which are responsible for the regulation of innate and adaptive immune responses. Extracellular ligands are recognized by surface TLRs (TLR1,TLR2,TLR4,TLR5, and TLR6). Intracellular TLRs (TLR3,TLR7,TLR8 and TLR9) bind mainly to foreign nucleic acids and sometimes detect self DNA/RNA.Very little is known about expression and function of TLRs in vivo in patients who underwent allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to the onset of acute GVHD.The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and at the onset of GVHD. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls.We analyzed 17 healthy donors and 34 patients. Median age was 46 years (range, 22–64) and 22 patients were male. Acute GVHD developed in 19 patients (12 with grade >=2). Clinical and transplant characteristics did not differ in patients with and without GVHD. Lymphocytes and monocytes of patients with acute GVHD showed higher levels of TLR5 (3,5±2,3 vs1,9±1,6 p=0,03; 25,8±25,9 vs 9,0±5,0 p=0,02) and a decreased expression of TLR1 (2,5±2,8 vs 4,3±2,8 p=0,02; 21,4±21,9 vs 54,9±37,4 p=0,005) and TLR9 (63,8±30,4 vs 111,1±62,9 p=0,03; 85,3±73,9 vs 164,2±90,6 p=0,01). IFN-gamma relative induction post-stimulation of TLR2,3,4 and 9 was significantly decreased in patients with acute GVHD (p< 0,04).TLRs show a different profile of expression in patients with acute GVHD in comparison with patients without it. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. In particular, a decreased expression of TLR-9 (receptor of hypomethylated DNA) on lymphocytes and monocytes can promote TLR-7 activation, inducing type I interferons and other pro-inflammatory cytokines. TLR-1 and −5, which are ligands for bacterial cell wall, could also be involved in the pathogenesis of GVHD. Moreover, acute GVHD negatively correlates with IFN-gamma production upon TLR2,3,4 and 9 activation. The assessment of a larger number of patients could be useful to understand the complex interplay among pathogens, self or non-self DNA and RNA, and the immune system.No relevant conflicts of interest to declare.

Abstract 1682

Abstract 4924

Abstract 4071

Abstract 786

This work was supported in part by CML-Leukemia Net and Progetto Regione Lombardia.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Azacitidine (AZA) at a dose of 75 mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates and prolongation of survival in high-risk MDS patients (pts) (Fenaux, 2009). However few data are hitherto available concerning the efficacy and safety of Aza in lower risk MDS. A lower dose regimen, AZA 5 (75 mg/mq daily, subcutaneously, for 5 consecutive days every 4 weeks) have shown to induce response rates consistent with the currently approved schedule (Lyons, 2009), however in this study pts were not classified according to IPSS risk.The use of AZA in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial, AZA 5 regimen was administered to IPSS low-or-intermediate-1 risk pts, for a total of 8 courses, in order to evaluate its efficacy and safety. Furthermore pharmacogenomic studies (GEP, SNP) cytokine network and PI-PLC-beta1 methylation and gene expression, before and at the end of 4th and 8th course of Aza treatment, were planned to identify new biological markers to predict the response.From September 2008 to February 2010, 34 patients (24 males, 10 females), with a median age of 71 (56-84) yrs, with symptomatic transfusion-dependent anemia, previously unresponsive to erythropoietin (EPO) or not expected to respond to EPO, or with severe neutropenia or thrombocytopenia, were enrolled into the study. According to WHO classification, 15 pts had RA, 6 RARS, 7 RCMD and 6 RAEB-1.At present time 30/34 pts are evaluable: 23/30 pts (77%) completed the treatment plan (8 courses), 3/30 pts (10%) are ongoing and 4/30 (13%) died during the treatment period. According to the 2006 International Working Group criteria, overall response rate (ORR) was 60,9 % (14/23 pts): 5 pts (21,7%) achieved complete remission (CR), while 9 pts (39,1%) showed an hematologic improvement (HI) (7 erythroid responses, 1 erythroid/platelet response and 1 neutrophil/platelet response). 9/23 pts (39%) maintained a stable disease (SD). Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%). 4 pts (11,7%) died during treatment (2 pts after the 1th cycle and 2 pts after the 4th course) because of septic shock, gastrointestinal hemorrage, pneumonia, and respiratory distress, respectively. The median duration of response was 3,5 months (range 1–14 months). Surprisingly, 3/14 patients (2 CR and 1 HI erythroid) showed a long duration of response (11, 13 and 14 months, respectively), still ongoing, after discontinuation of AZA. Preliminary data on the lipid signalling pathways suggested a direct correlation between the demethylating effect on PI-PLC-beta1 and responsiveness to treatment.Our study shows that AZA low-dose schedule may be a feasible and effective treatment for low-risk MDS pts and may induce durable responses. Despite AZA safety, extreme caution is needed in pts with age-related comorbidities and/or with severe neutropenia or thrombocytopenia, especially in low-risk MDS. Furthermore, PI-PLC–β1 demethylation and gene expression could represent a new biological marker to predict the clinical response to AZAOff Label Use: In Italy the use of Azicitidine for Low-Risk Myelodysplastic patients is off-label. The use of azacitidine in our study is part of a Phase II clinical trial. Finelli:Celgene: Consultancy.

Abstract 4029

Abstract 233

Abstract 3412

Elderly CML patients treated with Imatinib (IM) in early chronic phase (CP) have similar cytogenetic response and survival compared with younger patients, but they show a lower compliance to standard IM therapy (400 mg/day).The aim of the study is to investigate if CCgR that has been achieved with standard (daily administration) IM therapy can be maintained with the same dose of IM given intermittently (INTERIM).The study population is represented by elderly patients (≥ 65 years old) with Ph+ CML and with stable CCgR after at least 2 years of standard IM therapy (daily administration). IM is given at the same dose that was given at the time of enrollment by the following intermittent schedule: 1 week on / 1 week off for the 1st month; 2 weeks on / 2 weeks off for the 2nd and 3rd month; 1 month on / 1 month off from the 4th month thereafter. In cases of loss of CCgR INTERIM was stopped and standard therapy (daily administration) was resumed. After 12 months, the patients who are in continuous CCgR are advised to continue the intermittent study schedule and to be followed indefinitely. The CgR status was evaluated at baseline (by conventional cytogenetics on bone marrow and FISH on peripheral-blood) and every 3 months during the study (only by FISH on peripheral-blood). If FISH (% of Ph+ cells) increased more than 1% in two consecutive examinations, evaluation of marrow cells metaphases was performed to confirm the loss of CCgR and to check for additional cytogenetic abnormalities. Quantitative molecular assessment of BCR-ABL transcript by RQ-PCR on peripheral blood was due at baseline and every 3 months during the study and mutational analysis of ABL was performed in case of loss of CCgR.One-hundred and fourteen patients have been considered eligible, but 17 (15%) refused to enter into the protocol. Out of 97 enrolled patients, 87 started INTERIM, 5 patients (5%) went off the study for major protocol violation before the 3rd month and, at present, 82 patients are ongoing. Of these 82 patients, 52, 30 and 11 completed the 3rd, 6th and 9th month, respectively. The preliminary results of the first 6 months are here reported. The distribution of patients according to FISH results is shown in Fig. 1. Only 1/68 pts (at 6th month) showed an increased >1% in Ph+ cells by FISH but he maintained a CCgR when checked by conventional cytogenetic. As showed in Fig. 2, 96 to 87% of patients maintained a major molecular response MMR (≤0,1) according to International Scale (IS).This study is trying to test the minimum effective dose of Imatinib to maintain the CCgR in elderly CML patients with stable CCgR. The preliminary results at 6 months do not show negative trends both for cytogenetic and molecular response. Therefore, the study is ongoing and all patients are expected to complete the trial time (12 months).No relevant conflicts of interest to declare.

Nucleoside 5-Azacitidine (5-Aza) administered at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates, reduces progression to acute myeloid leukaemia (AML) and increases overall survival in the high risk MDS patients.The use of 5-Aza in the earlier phases of MDS could reduce the proliferative advantage of MDS clone and favours the regrowth of normal hematopoiesis. In the setting of low-risk MDS patients lower doses of 5-Aza could be enough to induce hematologic responses. We attempted to use an alternative schedule, 75 mg/mq subcutaneous daily for 5 consecutive days every 28 days, for a total of 8 courses, to evaluate its efficacy and tolerability in low risk MDS patients. Pharmacogenomic studies (gene expression profile and single nucleotide polymorphism analysis), cytokine network and phosphatidylinositol-phospholipase C – β1 (PI-PLC - β1) expression, before and after 5-Aza treatment, were planned to identify new biological markers to predict the response.From September 2008 to August 2009, 25 patients were enrolled into the study. According to WHO criteria 12 patients had refractory anemia (RA), 5 patients refractory cytopenia with multilineage dysplasia (RCMD), 7 patients refractory anemia with excess blasts-1 (RAEB-1) and 1 patient refractory anemia with ringed sideroblasts (RARS). All patients were classified as Low Risk (IPSS score 0-1). Age at diagnosis ranged between 56 and 84 years. All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006.9/25 (36%) patients completed at least 4 courses of therapy and only 6/25 (24%) patients finished the treatment plan (8 courses). Five patients obtained an hematologic improvement (3 erythroid response and 2 neutrophil response) whereas 4 patients maintained a stable disease. The others 16 patients are ongoing. The drug was very well tolerated. WHO grade III hematologic toxicity consisted in neutropenia (1 case) and thrombocytopenia (1 case) was observed in only two patients, but it was transitory and no delay of treatment was necessary.Our preliminary results show that the 5-Aza five days/monthly schedule is very well tolerated and it appears to induce hematologic improvements as well as the seven days/monthly schedule, at least in the low-risk MDS setting. Biological studies will be useful to elucidate the genetic bases of sensititvity or resistance to 5-AZA and to optimise the therapy.Acknowledgments: this work was supported by MIUR 2008.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 4851

Abstract 4186

Abstract 860

Standard therapy with Imatinib (i.e. daily administration) significantly prolongs the survival of Ph+ CML patients who obtain a complete cytogenetic response (CCgR). Elderly patients (i.e. > 65 yrs) have similar cytogenetic responses and survival, but they usually show a low compliance. This prospective multicenter trial addresses the issue of intermittent administration of Imatinib (INTERIM) in Ph+ CML patients older than 65 years with stable complete cytogenetic response (CCgR) after at least 2 years of standard therapy with IM. Lower doses of IM could be sufficient to maintain the CCgR, to improve the tolerance and to reduce the costs of therapy. For this purpose, IM will be given at the same dose that was given at the time of enrollment by the following intermittent schedule: 1 week on/1 week off for the 1stmonth (weeks 1–4); 2 weeks on/2 weeks off for the 2ndand 3rdmonth (weeks 5–12); 1 month on/1 month off from the 4thmonth thereafter (weeks 13 on). The primary objective of the study is to evaluate the proportion of patients who remain in CCgR with INTERIM. The cytogenetic response (CgR) status will be evaluated at baseline (by conventional cytogenetics on bone marrow and FISH on peripheral-blood) and every 3 months during the study (only by FISH on peripheral-blood). If FISH documents a variation of the baseline value of more than 1% in two consecutive examinations, evaluation of marrow cells metaphases will be performed to confirm the loss of CCgR and to check for additional cytogenetic abnormalities (ACA). In case of loss of CCgR INTERIM will be stopped and standard therapy (daily administration) will be resumed. Quantitative molecular assessment of BCR-ABL transcript by RQ-PCR on peripheral blood is due at baseline and every 3 months during the study and mutational analysis of ABL will be performed in case of loss of CCgR. After 12 months, the patients who are in continuous CCgR are advised to continue the intermittent study schedule and to be followed indefinitely. The study started in May 2008 and, at present, 18 patients have been enrolled. Preliminary data on the first six months will be presented.

Background. Haploidentical family donors represent the ideal solution to offer to every patient with high risk leukemia the potential cure of hematopoietic stem cell transplantation. Extensive application of haploidentical transplantation (haplo-SCT) has been limited by high rate of late transplant related mortality (TRM) and relapse associated with the delayed immune reconstitution (IR) secondary to the procedures of profound T-cell depletion required for severe graft-vs-host-disease (GvHD) prevention. Methods. In a haplo-SCT phase I-II multicenter, open, non-randomized trial sponsored by MolMed SpA, we infused donor lymphocytes genetically engineered to express the suicide gene herpes simplex thymidine kinase (TK-DLI) to induce early IR, while selectively controlling GvHD. We enrolled 54 patients (pts) -median age 51- with high-risk hematologic malignancies. Thirty-two patients were in remission at transplantation. Results. After myeloablative conditioning regimen, 50 pts received a median 11×10^6/ kg CD34+ and 1.1×10^4/kg CD3+ after Clinimacs CD34+ selection. Median time to engraftment of neutrophils > 1.0 ×10^9/L and platelets > 50 ×10^9/L was 14 days. TK cells could always be prepared in the appropriate timeframe, and no drop out secondary to inadequate cell manipulation occurred. Twenty-eight pts received TK-DLI at a dose of CD3+ cells of 0.9–40 ×10^6/kg: 22 pts obtained prompt IR with CD3+>100/mcl at day +75 (median) from haplo-SCT and day +23 from TK-DLI. Eleven pts developed GvHD (10 acute GvHD grade I-IV and 1 chronic GvHD) that was always abrogated by the suicide gene induction; no progression from acute GvHD to chronic GvHD and no GvHD-related death or long-term complication occurred. No acute or chronic adverse event related to the gene transfer procedure was observed during extended follow-up. With a median follow-up of 178 days (range 2–1821), the 3-year TRM in intention to-treat (ITT) analysis was 40% with last mortality event at d+166. Significantly, the cumulative infectious mortality was 10% in TK-treated immune-reconstituted patients. Immune reconstitution obtained with TK-cells infusion correlated with rapid development of a wide T-cell repertoire and detection of high frequencies of T-cells specific for opportunistic pathogens. Initially, TK-cells represented the predominant T cell component in reconstituting patients and showed a effector memory phenotype, an oligoclonal repertoire and a persistent ganciclovir sensitivity. At later times, untransduced cells progressively expanded and became the prevalent circulating lymphocyte population. This reconstitution kinetic was observed only in patients with TK-cell engraftment, suggesting a critical homeostatic contribution of TK-cells early after transplant. One year after TK cells infusion the repertoire and distribution of T cell subsets completely normalized. In ITT, the median leukemia-free survival (LFS) for patients transplanted with advanced chemoresistant disease was at d+169, while patients in remission at the time of transplantation showed an overall survival in ITT of 51% at 1 year. Conclusions. This multicenter trial confirm the safety and potential benefit in improving survival of the gene transfer technology integrated with a standard therapeutic procedure such as allogeneic SCT. The conditional benefit for patients treated with TK cells was remarkable as indicated by a complete abrogation of late infectious mortality provided by the fast and effective immune reconstitution. In the future, this technology based on genetic engineering of activated donor lymphocytes with higher allo-reactive potentialwill potentially impact also the transplant outcome of patients with refractory disease.

Standard therapy with Imatinib (i.e. daily administration) significantly prolongs the survival of Ph+ CML patients who obtain a complete cytogenetic response (CCgR). Elderly patients (i.e. > 65 yrs) have similar cytogenetic responses and survival, but they usually show a low compliance. This prospective multicenter trial addresses the issue of intermittent administration of Imatinib (INTERIM) in Ph+ CML patients older than 65 years with stable complete cytogenetic response (CCgR) after at least 2 years of standard therapy with IM. Lower doses of IM could be sufficient to maintain the CCgR, to improve the tolerance and to reduce the costs of therapy. For this purpose, IM will be given at the same dose that was given at the time of enrollment by the following intermittent schedule: 1 week on/1 week off for the 1st month (weeks 1–4); 2 weeks on/2 weeks off for the 2nd and 3rd month (weeks 5–12); 1 month on/1 month off from the 4th month thereafter (weeks 13 on). The primary objective of the study is to evaluate the proportion of patients who remain in CCgR with INTERIM. The cytogenetic response (CgR) status will be evaluated at baseline (by conventional cytogenetics on bone marrow and FISH on peripheral-blood) and every 3 months during the study (only by FISH on peripheral-blood). If FISH documents a variation of the baseline value of more than 1% in two consecutive examinations, evaluation of marrow cells metaphases will be performed to confirm the loss of CCgR and to check for additional cytogenetic abnormalities (ACA). In case of loss of CCgR INTERIM will be stopped and standard therapy (daily administration) will be resumed. Quantitative molecular assessment of BCR-ABL transcript by RQ-PCR on peripheral blood is due at baseline and every 3 months during the study and mutational analysis of ABL will be performed in case of loss of CCgR. After 12 months, the patients who are in continuous CCgR are advised to continue the intermittent study schedule and to be followed indefinitely. The study started in May 2008 and, at present, 18 patients have been enrolled. Preliminary data on the first six months will be presented.

Background.Haploidentical family donors represent the ideal solution to offer to every patient with high risk leukemia the potential cure of hematopoietic stem cell transplantation. Extensive application of haploidentical transplantation (haplo-SCT) has been limited by high rate of late transplant related mortality (TRM) and relapse associated with the delayed immune reconstitution (IR) secondary to the procedures of profound T-cell depletion required for severe graft-vs-host-disease (GvHD) prevention.

T-cell depletion (TCD) of allogeneic stem cells transplantation (HSCT) is the most powerful approach to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. However, the prolonged immune deficiency resulting from TCD significantly impairs the outcome of HSCT from a haploidentical family donor (haplo-HSCT). In a phase I-II clinical trial (protocol MMTK007), we showed that the add-back of suicide-gene transduced donor lymphocytes (TK+ cells) provides early immune-reconstitution (IR), crucial to avoid opportunistic infections and disease relapse. While the achievement of an early and sustained IR significantly reduced the incidence of infectious complications and mortality, the kinetic of abatement of viral reactivations differed in different patients, suggesting variability in the repertoire, function and persistence of viral-specific T cells. To gain insights on the activity of the post-transplant immune system, we focused on immune responses to cytomegalovirus (CMV) and Ebstein Barr virus (EBV) as clinical paradigm of an effective immune system. In our trial 29 patients (median age 52y) were transplanted for high-risk leukemia. Seventeen pts received TK-DLI starting at day +42, 14 pts obtained prompt immune reconstitution with CD3+ >100/mcl at day +86 (median) from SCT and day +21 from TK-DLI. Twelve/14 experienced CMV reactivation, 3 reactivation/patient (median), each reactivation required 14 days (median) of pre-emptive treatment with foscarnet to achieve a complete clearance; in 2/38 events, a CMV antigenemia persistent during foscarnet treatment was cleared by ganciclovir administration. In this series, the last CMV reactivation requiring pre-emptive treatment happened at day +84 from SCT and day +19 from TK-DLI. Nine/14 experienced EBV reactivation, 1 reactivation/patient (median), 6 patients required treatment with rituximab (375 mg/mq weekly) for 3 cycles (median). In 3 cases we documented EBV lymphoproliferative disease, that were completely controlled with the treatment. The proportion of lymphocytes committed to produce IFN-γ upon CMV or EBV stimulation normalized within the first 3 months post transplant. The analysis of CMV and EBV T-cell response showed a prevalent host-restriction pattern, suggesting active modulation of the T-cell repertoire by the host environment. The TCR-Vβ repertoire progressively changed from oligoclonal into polyclonal and normalized by 1 year after transplant. The progressive acquirement of a protective, host-restricted, anti-viral response, highly correlated with a decline in the occurrence of any infectious event, that were nearly abrogated by 6th month post transplant. The overall cumulative infectious mortality beyond day +100 post transplant was 12,5% in TK treated patients demonstrating the efficacy of this strategy in building a protective immune system.

The somatic mutation JAK2 V617F has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMF). In typical forms of myelodysplastic syndromes (MDS), JAK2 V617F mutation is rarely present (2–5%); on the contrary, it has been found with higher prevalence in patients with RARS-T (i.e. MDS/MPD-U with platelet count >600×109/L and ringed sideroblasts more than 15%) and in a subgroup of MDS patients with isolated 5q deletion and a proliferative bone marrow. In this study we analysed the JAK2 V617F mutational status in 53 MDS patients (26 males, 27 females; median age at the time of the study 76 years, range 45–91). Patients were classified as follows: 4 cases 5q- syndrome, 3 RCMD, 5 MDS/MPD, 1 MDS-U, 23 RA, 12 RARS, 5 RAEB. DNA was extracted from purified granulocytes; all samples were analyzed by allele-specific polymerase chain reaction (PCR), according to Baxter el al (2005). DNA samples were further subjected to direct sequencing for confirmatory testing. The JAK2 V617F mutation was present in 3 cases, with an overall frequency of 5%. With respect to MDS subtype, 1 patient had RA and 2 RARS. Among the 12 RARS patients, the two V617F postive displayed thrombocytosis (680×109/L and 649×109/L), whereas none of the 10 RARS V617F negative patients showed high platelet counts (median Plt 157×109/L, range 5–422×109/L). In one JAK2 mutant case, thrombocytosis required treatment with hydroxyurea. Moreover, the two V617F positive RARS patients displayed higher WBC count (6.2×109/L and 8.5×109/L) than the V617F negatives (median WBC 4.05×109/L); no difference was observed in Hb levels. The JAK2 positive RA patient had 10% of sideroblasts in bone marrow, normal platelet and WBC count and no proliferative characteristics; since the occurrence of the mutation may be an early event and preceed the classical manifestations of MDS/MPD, a longer follow-up is necessary to determine its possible prognostic significance. Considering the V617F negative MDS cases, only one patient, diagnosed as MDS/MPD, showed a platelet count >600×109/L. In conclusion, we confirmed recent reports showing that JAK2 V617F is present with low prevalence (about 5%) in MDS; in particular, the JAK2 mutation identifies a subset of MDS patients with and “overlap” syndrome, characterised by proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis, who may benefit from JAK2 specifically targeted therapies.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to aryl hydrocarbon receptor (AhR), a member of the erb-A family, allowing, after DNA binding, gene expression regulation. TCDD has a large number of biological effects, as skin and cardiovascular disease, diabetes and cancer. An increase in myeloid leukemia, Hodgkin’s and non-Hodgkin’s lymphomas, was observed after 15 years in the population exposed to TCDD after the 1976 accident in Seveso, Italy. In the present study, we analyzed the in vitro effect of TCDD exposure on human CD34+ progenitor cells from G-CSF stimulated leukapheresis of 4 normal donors. Gene expression modulation induced by TCDD was analysed on highly purified (>96%) CD34+ cells, after exposure to 10 nM of TCDD for 12 hrs. Gene expression profiles have been generated by high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed with a supervised approach (DNA-Chip Analyzer, dChip 2006). The differential expression of 257 transcripts (150 up regulated and 106 down-regulated) distinguished the 4 TCDD treated from the 4 untreated control samples. Interestingly, a number of the differentially expressed genes were involved in skin and cardiovascular diseases, diabetes, and different cancers, all of which have been associated with TCDD exposure. Among skin diseases, defects in laminin beta 3, ALOX12B and keratin 2a, all downregulated in TCDD exposed CD34+ cells, are associated with development of epidermolysis bullosa, erythroderma ichthyosiform, and Siemens ichthyosis bullosa, respectively. As far as diabetes and cardiovascular disease pathogenesis are concerned, defects of SLC2A4 gene, dowregulated by TCDD, is associated with the development of non insulin dependent diabetes, while epoxide hydrolase 2 and EGR2 are associated with cardiovascular disease. Among cancer related genes, ARHGAP26 and ABL2 are associated with juvenile myelomonocytic leukemia and acute myeloid leukemia with eosinophilia, respectively. Nevertheless, the expression of numerous other genes potentially involved in hemopoiesis/leukemogenesis, was modulated by TCDD exposure. Among these examples are c-kit ligand, LIF receptor, pre B lymphocyte gene 1, piwi-like 2, FLT3 ligand, chemokine ligands 14 and 15, and cdk2, that were all up regulated, while MLL4, wnt inhibitory factor 1, chemokine ligand 7, and lymphoid blast crisis oncogene, were down regulated. In conclusion, the gene expression pattern induced by TCDD exposure on the CD34+ normal progenitor cells is consistent with the spectrum of TCDD induced toxicities/diseases. In particular, it provides the basis for a possible role of TCDD in the neoplastic transformation of hemopoietic stem cells and support the epidemiologic data of increased hematologic cancer risk in the population exposed accidentally to the substance.

The somatic mutation JAK2 V617F has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMF). In typical forms of myelodysplastic syndromes (MDS), JAK2 V617F mutation is rarely present (2–5%); on the contrary, it has been found with higher prevalence in patients with RARS-T (i.e. MDS/MPD-U with platelet count >600×109/L and ringed sideroblasts more than 15%) and in a subgroup of MDS patients with isolated 5q deletion and a proliferative bone marrow. In this study we analysed the JAK2 V617F mutational status in 53 MDS patients (26 males, 27 females; median age at the time of the study 76 years, range 45–91). Patients were classified as follows: 4 cases 5q- syndrome, 3 RCMD, 5 MDS/MPD, 1 MDS-U, 23 RA, 12 RARS, 5 RAEB. DNA was extracted from purified granulocytes; all samples were analyzed by allele-specific polymerase chain reaction (PCR), according to Baxter el al (2005). DNA samples were further subjected to direct sequencing for confirmatory testing. The JAK2 V617F mutation was present in 3 cases, with an overall frequency of 5%. With respect to MDS subtype, 1 patient had RA and 2 RARS. Among the 12 RARS patients, the two V617F postive displayed thrombocytosis (680×109/L and 649×109/L), whereas none of the 10 RARS V617F negative patients showed high platelet counts (median Plt 157×109/L, range 5–422×109/L). In one JAK2 mutant case, thrombocytosis required treatment with hydroxyurea. Moreover, the two V617F positive RARS patients displayed higher WBC count (6.2×109/L and 8.5×109/L) than the V617F negatives (median WBC 4.05×109/L); no difference was observed in Hb levels. The JAK2 positive RA patient had 10% of sideroblasts in bone marrow, normal platelet and WBC count and no proliferative characteristics; since the occurrence of the mutation may be an early event and preceed the classical manifestations of MDS/MPD, a longer follow-up is necessary to determine its possible prognostic significance. Considering the V617F negative MDS cases, only one patient, diagnosed as MDS/MPD, showed a platelet count >600×109/L. In conclusion, we confirmed recent reports showing that JAK2 V617F is present with low prevalence (about 5%) in MDS; in particular, the JAK2 mutation identifies a subset of MDS patients with and “overlap” syndrome, characterised by proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis, who may benefit from JAK2 specifically targeted therapies.

T-cell depletion (TCD) of allogeneic stem cells transplantation (HSCT) is the most powerful approach to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. However, the prolonged immune deficiency resulting from TCD significantly impairs the outcome of HSCT from a haploidentical family donor (haplo-HSCT). In a phase I-II clinical trial (protocol MMTK007), we showed that the add-back of suicide-gene transduced donor lymphocytes (TK+ cells) provides early immune-reconstitution (IR), crucial to avoid opportunistic infections and disease relapse. While the achievement of an early and sustained IR significantly reduced the incidence of infectious complications and mortality, the kinetic of abatement of viral reactivations differed in different patients, suggesting variability in the repertoire, function and persistence of viral-specific T cells. To gain insights on the activity of the post-transplant immune system, we focused on immune responses to cytomegalovirus (CMV) and Ebstein Barr virus (EBV) as clinical paradigm of an effective immune system. In our trial 29 patients (median age 52y) were transplanted for high-risk leukemia. Seventeen pts received TK-DLI starting at day +42, 14 pts obtained prompt immune reconstitution with CD3+>100/mcl at day +86 (median) from SCT and day +21 from TK-DLI. Twelve/14 experienced CMV reactivation, 3 reactivation/patient (median), each reactivation required 14 days (median) of pre-emptive treatment with foscarnet to achieve a complete clearance; in 2/38 events, a CMV antigenemia persistent during foscarnet treatment was cleared by ganciclovir administration. In this series, the last CMV reactivation requiring pre-emptive treatment happened at day +84 from SCT and day +19 from TK-DLI. Nine/14 experienced EBV reactivation, 1 reactivation/patient (median), 6 patients required treatment with rituximab (375 mg/mq weekly) for 3 cycles (median). In 3 cases we documented EBV lymphoproliferative disease, that were completely controlled with the treatment. The proportion of lymphocytes committed to produce IFN-γ upon CMV or EBV stimulation normalized within the first 3 months post transplant. The analysis of CMV and EBV T-cell response showed a prevalent host-restriction pattern, suggesting active modulation of the T-cell repertoire by the host environment. The TCR-Vβ repertoire progressively changed from oligoclonal into polyclonal and normalized by 1 year after transplant. The progressive acquirement of a protective, host-restricted, anti-viral response, highly correlated with a decline in the occurrence of any infectious event, that were nearly abrogated by 6th month post transplant. The overall cumulative infectious mortality beyond day +100 post transplant was 12,5% in TK treated patients demonstrating the efficacy of this strategy in building a protective immune system.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to aryl hydrocarbon receptor (AhR), a member of the erb-A family, allowing, after DNA binding, gene expression regulation. TCDD has a large number of biological effects, as skin and cardiovascular disease, diabetes and cancer. An increase in myeloid leukemia, Hodgkin's and non-Hodgkin's lymphomas, was observed after 15 years in the population exposed to TCDD after the 1976 accident in Seveso, Italy. In the present study, we analyzed the in vitro effect of TCDD exposure on human CD34+progenitor cells from G-CSF stimulated leukapheresis of 4 normal donors. Gene expression modulation induced by TCDD was analysed on highly purified (>96%) CD34+cells, after exposure to 10 nM of TCDD for 12 hrs. Gene expression profiles have been generated by high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed with a supervised approach (DNA-Chip Analyzer, dChip 2006). The differential expression of 257 transcripts (150 up regulated and 106 down-regulated) distinguished the 4 TCDD treated from the 4 untreated control samples. Interestingly, a number of the differentially expressed genes were involved in skin and cardiovascular diseases, diabetes, and different cancers, all of which have been associated with TCDD exposure. Among skin diseases, defects in laminin beta 3, ALOX12B and keratin 2a, all downregulated in TCDD exposed CD34+ cells, are associated with development of epidermolysis bullosa, erythroderma ichthyosiform, and Siemens ichthyosis bullosa, respectively. As far as diabetes and cardiovascular disease pathogenesis are concerned, defects of SLC2A4 gene, dowregulated by TCDD, is associated with the development of non insulin dependent diabetes, while epoxide hydrolase 2 and EGR2 are associated with cardiovascular disease. Among cancer related genes, ARHGAP26 and ABL2 are associated with juvenile myelomonocytic leukemia and acute myeloid leukemia with eosinophilia, respectively. Nevertheless, the expression of numerous other genes potentially involved in hemopoiesis/leukemogenesis, was modulated by TCDD exposure. Among these examples are c-kit ligand, LIF receptor, pre B lymphocyte gene 1, piwi-like 2, FLT3 ligand, chemokine ligands 14 and 15, and cdk2, that were all up regulated, while MLL4, wnt inhibitory factor 1, chemokine ligand 7, and lymphoid blast crisis oncogene, were down regulated. In conclusion, the gene expression pattern induced by TCDD exposure on the CD34+normal progenitor cells is consistent with the spectrum of TCDD induced toxicities/diseases. In particular, it provides the basis for a possible role of TCDD in the neoplastic transformation of hemopoietic stem cells and support the epidemiologic data of increased hematologic cancer risk in the population exposed accidentally to the substance.

T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months. High numbers of T cell precursors specific for CMV and EBV were detected at immune reconstitution (median of 86 and 69 gIFN specific spots/10^5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0.002). Six pts developed acute (GvHD), (grade I to IV) that was always completely abrogated by the suicide system. Overall survival of TK cells treated patients is 50% at three years. These results indicate that TK-DLI abolish late mortality after CD34+ haplo-SCT in adults. A phase III multicentric study will start in 2007 to validate prospectively the advantage of TK-DLI in haplo-SCT.

T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months.

The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ >100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT. We believe that these results are due to the rapid development of a wide T cell repertoire obtained by TK cell infusions. Immunological follow-up showed Th1/Tc1 effector memory T cells, with a wide TCR repertoire in the first 3 months after SCT in all patients. High frequencies of T cells specific for CMV (median: 35 and 93 spots/105 cells with CMV-infected donor and host fibroblasts) and EBV (median: 58 and 41 spots/105 cells with donor and host EBV-LCL) were detected by gIFN ELISpot at time of immunereconstitution, and correlated with complete control of viral infections. Normalization of the T cell repertoire was documented by spectratype, immune-phenotype for naïve and memory T cell subsets and gIFN ELIspot 6 months after treatment. A phase III randomized multicentric study will start in 2006.

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by peripheral thrombocytosis and abnormal proliferation of megakariocytes in the bone marrow. Even thought thrombosis is frequently associated to ET, the risk factors of this clinical complication are still controversial. The aim of this retrospective, single institution study was to investigate clinical and laboratory characteristics associated with the occurrence of thrombotic events, with the purpose of identifying subgroups of patients who could benefit from antiaggregant and/or cytostatic treatment. 306 consecutive ET patients (109 men and 197 females, median age 58 yr) diagnosed between January 1979 and December 2002 were included in the study. At the time of analysis, 196 patients were still alive with a median follow up of 96 months. The following variables were investigated for the association with thrombotic complications: age, platelet count, previous history of thrombotic events, time from diagnosis, treatment with antiaggregant/cytostatic drugs, and cardiovascular risk factors such as arterial hypertension, obesity, hypercolesterolemia, diabetes, cigarette smoking. At the time of last follow up, 46 patients (15%) experienced at least one thrombotic event. The occurrence of thrombotic events was observed in 26/64 (40.6%) patients with previous history of thrombosis and in 20/242 (8.3%) patients with no previous history of thrombosis (p<0.0001 Fisher’s exact test, odd ratio 7.6). A significant difference between the two groups of patients was also confirmed when Kaplan Meier estimates of thrombosis-free survival were compared by log-rank test (p<0.0001). By logistic regression, platelet number at diagnosis did not associate with occurrence of thrombosis in the whole patient population. When patients without previous history of thrombosis were stratified according to the number of cardiovascular risk factors (none vs one vs more than one), a significant correlation with occurrence of thrombotic events was observed (Mantel-Haenszel Chi-square 5.47, p<0.05). This study confirms that history of thrombosis is strongly related with risk of further thrombotic events in patients with ET, whereas platelet number at diagnosis does not seem to represent a prognostic factor. In patients with no previous history of thrombosis, the presence of other cardiovascular risk factors has to be taken into account when establishing the therapeutic approach.

The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ >100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT.

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by peripheral thrombocytosis and abnormal proliferation of megakariocytes in the bone marrow. Even thought thrombosis is frequently associated to ET, the risk factors of this clinical complication are still controversial. The aim of this retrospective, single institution study was to investigate clinical and laboratory characteristics associated with the occurrence of thrombotic events, with the purpose of identifying subgroups of patients who could benefit from antiaggregant and/or cytostatic treatment. 306 consecutive ET patients (109 men and 197 females, median age 58 yr) diagnosed between January 1979 and December 2002 were included in the study. At the time of analysis, 196 patients were still alive with a median follow up of 96 months. The following variables were investigated for the association with thrombotic complications: age, platelet count, previous history of thrombotic events, time from diagnosis, treatment with antiaggregant/cytostatic drugs, and cardiovascular risk factors such as arterial hypertension, obesity, hypercolesterolemia, diabetes, cigarette smoking. At the time of last follow up, 46 patients (15%) experienced at least one thrombotic event. The occurrence of thrombotic events was observed in 26/64 (40.6%) patients with previous history of thrombosis and in 20/242 (8.3%) patients with no previous history of thrombosis (p<0.0001 Fisher's exact test, odd ratio 7.6). A significant difference between the two groups of patients was also confirmed when Kaplan Meier estimates of thrombosis-free survival were compared by log-rank test (p<0.0001). By logistic regression, platelet number at diagnosis did not associate with occurrence of thrombosis in the whole patient population. When patients without previous history of thrombosis were stratified according to the number of cardiovascular risk factors (none vs one vs more than one), a significant correlation with occurrence of thrombotic events was observed (Mantel-Haenszel Chi-square 5.47, p<0.05). This study confirms that history of thrombosis is strongly related with risk of further thrombotic events in patients with ET, whereas platelet number at diagnosis does not seem to represent a prognostic factor. In patients with no previous history of thrombosis, the presence of other cardiovascular risk factors has to be taken into account when establishing the therapeutic approach.

Haplo-identical stem cell transplantation (haplo-SCT) is a therapeutic option for pts with high risk hematologic malignancies lacking an HLA matched donor. The full exploitation of haplo-SCT is limited by delayed immune reconstitution and prolonged risk of life-threatening infections. We previously identified in an academic haplo-SCT trial, effective doses of donor lymphocytes genetically engineered to express a suicide gene (the herpes simplex thymidine kinase (TK-DLI) as an effective tool for preventing disease relapse and promoting immune reconstitution. In case of GvHD, TK-DLI could be selectively eliminated by ganciclovir. A phase II multicenter trial (MM TK007), aimed at verifying the therapeutic activity of early add-backs of TK+ cells after haplo-SCT in inducing a stable immune reconstitution, started in 2002. 20 pts were recruited and transplanted for AML (16, 8 of whom post MDS), ALL (2), advanced NHL and HD (2); disease status at SCT was CR1(5), CR2(7), refractory(8). Median time from diagnosis to SCT was 394 d (95-3752), median age was 52 (17–62). A median of 12.3x106/kg (7.3–15.5) CD34+ selected cells (Clinimacs) and 1.05x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning regimen. GvHD prophylaxis was never administered post-SCT. 18/20 pts engrafted with a median time of 13 d (8–21), for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-DLI. The statistical end-point of the study was to achieve immune reconstitution in 7/18 treated pts. So far, 10 pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42. Although this study will continue up to 18 treated pts, the primary end-point has been already achieved since 7/9 evaluable pts obtained CD3+ >100/μl at a median time of 76 d (61–99) from SCT and 21 d (14–31) from TK-DLI. Transduced cells were documented ex vivo in all pts; median counts at peak of immune reconstitution were 483 CD3+/μl, 304 CD8+/μl, 130 CD4+/μl. Prompt immune reconstitution resulted in complete control of viral infections and virtually eliminated late infections frequently reported after haplo-SCT. In 1 pt a grade II biopsy-proven acute GvHD, involving skin and liver was observed and completely abrogated by ganciclovir (10 mg/kg/day) in the absence of immunosuppressive drugs. One pt relapsed at d 300; overall, 6 pts are alive in CR at d +423, +363, +317, +255, +109, +104. The preliminary results of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from infectious mortality after haplo-SCT while providing an effective and selective treatment for GvHD. The potential of this strategy in preventing disease relapse is promising and will remain the main focus of the long-term follow-up.

Haplo-identical stem cell transplantation (haplo-SCT) is a therapeutic option for pts with high risk hematologic malignancies lacking an HLA matched donor. The full exploitation of haplo-SCT is limited by delayed immune reconstitution and prolonged risk of life-threatening infections. We previously identified in an academic haplo-SCT trial, effective doses of donor lymphocytes genetically engineered to express a suicide gene (the herpes simplex thymidine kinase (TK-DLI) as an effective tool for preventing disease relapse and promoting immune reconstitution. In case of GvHD, TK-DLI could be selectively eliminated by ganciclovir. A phase II multicenter trial (MM TK007), aimed at verifying the therapeutic activity of early add-backs of TK+ cells after haplo-SCT in inducing a stable immune reconstitution, started in 2002. 20 pts were recruited and transplanted for AML (16, 8 of whom post MDS), ALL (2), advanced NHL and HD (2); disease status at SCT was CR1(5), CR2(7), refractory(8). Median time from diagnosis to SCT was 394 d (95-3752), median age was 52 (17–62). A median of 12.3x106/kg (7.3–15.5) CD34+ selected cells (Clinimacs) and 1.05x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning regimen. GvHD prophylaxis was never administered post-SCT. 18/20 pts engrafted with a median time of 13 d (8–21), for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-DLI. The statistical end-point of the study was to achieve immune reconstitution in 7/18 treated pts. So far, 10 pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42. Although this study will continue up to 18 treated pts, the primary end-point has been already achieved since 7/9 evaluable pts obtained CD3+ >100/μl at a median time of 76 d (61–99) from SCT and 21 d (14–31) from TK-DLI. Transduced cells were documented ex vivo in all pts; median counts at peak of immune reconstitution were 483 CD3+/μl, 304 CD8+/μl, 130 CD4+/μl. Prompt immune reconstitution resulted in complete control of viral infections and virtually eliminated late infections frequently reported after haplo-SCT. In 1 pt a grade II biopsy-proven acute GvHD, involving skin and liver was observed and completely abrogated by ganciclovir (10 mg/kg/day) in the absence of immunosuppressive drugs. One pt relapsed at d 300; overall, 6 pts are alive in CR at d +423, +363, +317, +255, +109, +104. The preliminary results of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from infectious mortality after haplo-SCT while providing an effective and selective treatment for GvHD. The potential of this strategy in preventing disease relapse is promising and will remain the main focus of the long-term follow-up.