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Different studies concerning gender differences in motor performance of handwriting movements have been carried out in an age range up to 17 years. In this preliminary study, we investigated the influence of gender on handwriting in a sample of young subjects (21-28 years old) in order to examine the possible differences in handwriting process due to gender in this specific age range. The relationship between gender and some kinematic aspects of handwriting was studied by analyzing the script of 84 (42 females and 42 males) university students, randomly recruited at University of Trieste that performed a copying task acquired by a digital tablet and analyzed by a suitable proprietary software. Significant differences between genders were observed in the number of strokes/s and strokes/cm, in the stroke length and duration as well as in the horizontal peak velocity of strokes and pen lift duration. The differences support the hypothesis that females, in this specific age range, write in a less fragmented way, with higher velocity and shorter motor planning time that male peers. In conclusion, although the limited number of candidates, the study confirms that many kinematic parameters can be profitably used to quantify objectively differences between genders in handwriting process

The role of (18)FDG-PET/CT during follow-up of patients affected by Hodgkin's lymphoma (HL) in complete remission after treatment is not fully elucidated, since a wide use of F-18 fluorodeoxyglucose positron emission tomography/computed tomography ((18)FDG-PET/CT) in this setting could be limited by a relative high rate of false-positive results. Herein, we summarize a retrospective analysis of 27 patients with Hodgkin's lymphoma in complete remission after the first-line (n = 20) or salvage (n = 7) therapy receiving serial (18)FDG-PET/CT scans during follow-up. Out of 165 scans, 13 were suspected for relapse, which was confirmed in seven patients. All relapses were correctly identified by (18)FDG-PET/CT positivity, with a 100% sensitivity; false-positive rate was 46% and negative predictive value was 100%. True-positive findings were mostly associated with multiple sites, subdiaphragmatic involvement, and/or previous sites of disease. According to our results, we conclude that performing routine PET/CT scan during follow-up of those patients who are at high risk of relapse would be advisable, although caution must be adopted when interpreting PET/CT results due to the relatively high rate of false-positive findings. If FDG abnormal uptake is present at multiple nodal sites, subdiaphragmatic lymph nodes, or previous sites of disease, histological verification of PET abnormal findings is warranted.

The extensive exploitation of the antitumor effect of donor lymphocytes infused after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is limited by the risk of graft-versus-host disease (GvHD). To overcome this limitation, we investigated the therapeutic potential of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes simplex virus (TK) in 23 patients experiencing recurrence of hematologic malignancies after allo-HSCT. Long-term follow-up of infused patients included analysis of engraftment of genetically engineered lymphocytes, in vivo assessment of antitumor effect, and control of GvHD by ganciclovir. All 17 patients evaluable for engraftment and graft-versus-leukemia (GvL) had circulating TK+ cells detectable beginning at a median time of 18 days. Eleven patients (65%) experienced a substantial clinical benefit resulting in 6 (35%) complete remissions and 5 (29%) partial responses. The antitumor effect tightly correlated with the in vivo received ganciclovir, resulting in elimination of TK+ cells and effective and selective treatment of GvHD. Immunization against HSV-TK was observed in 7 patients but did not preclude an effective GvL. These data validate the feasibility, safety, and efficacy of TK+ cells in the context of allografting and represent the basis for a broader application of this technology. The extensive exploitation of the antitumor effect of donor lymphocytes infused after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is limited by the risk of graft-versus-host disease (GvHD). To overcome this limitation, we investigated the therapeutic potential of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes simplex virus (TK) in 23 patients experiencing recurrence of hematologic malignancies after allo-HSCT. Long-term follow-up of infused patients included analysis of engraftment of genetically engineered lymphocytes, in vivo assessment of antitumor effect, and control of GvHD by ganciclovir. All 17 patients evaluable for engraftment and graft-versus-leukemia (GvL) had circulating TK+ cells detectable beginning at a median time of 18 days. Eleven patients (65%) experienced a substantial clinical benefit resulting in 6 (35%) complete remissions and 5 (29%) partial responses. The antitumor effect tightly correlated with the in vivo received ganciclovir, resulting in elimination of TK+ cells and effective and selective treatment of GvHD. Immunization against HSV-TK was observed in 7 patients but did not preclude an effective GvL. These data validate the feasibility, safety, and efficacy of TK+ cells in the context of allografting and represent the basis for a broader application of this technology.

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo , with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo . Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

The feasibility and toxicity of allogeneic stem cell transplantation after nonmyeloablative conditioning including thiotepa, fludarabine, and cyclophosphamide have been investigated in 6 patients with breast cancer and 7 patients with renal cell cancer. The program included the use of escalating doses of donor lymphocyte infusions (DLI) and/or interferon alpha (IFNα) for patients showing no tumor response and no graft-versus-host disease (GVHD). Patients were at high risk of transplant-related mortality (TRM) because of age, advanced stage, and previous treatments. We observed a partial remission in 4 renal cancer and in 2 breast cancer patients (one at the molecular level in the bone marrow), occurring after cyclosporine withdrawal or after DLI and/or IFNα. All the responses were accompanied by the occurrence of acute GVHD. We conclude that reduced-intensity allogeneic stem cell transplantation is a feasible procedure in renal and breast cancer, and that the exploitation of graft-versus-tumor effect after DLI is a promising finding.

7037 Background: Stem cell transplantation from a HLA-compatible sibling donor is an adoptive immunotherapy for cytokine-refractory, metastatic clear-cell renal cell cancer (RCC). However, the recent introduction of targeted therapy compounds has reduced the interest in this therapeutic strategy. We have reanalyzed our series with the aim to assess long-term benefit from allografting. Methods: Twenty-five RCC patients (Table) received a reduced-intensity allograft from an HLA-identical sibling donor after a thiotepa, fludarabine, and cyclophosphamide conditioning regimen, and a cyclosporine-based GVHD prophylaxis. Allogeneic peripheral blood hematopoietic cells were collected by apheresis after filgrastim treatment of the donor. Results: Best response to allograft was evaluable in 24 patients: 1 complete remission, 4 partial remissions, 12 minor response or stable disease, 7 progressive disease. Six patients died because of transplant related mortality (TRM) at day +12, +71, +86, +151, +259, +478. Cause of death was infection in four cases, GVHD in one case, and acute renal failure in one case. Fourteen patients died for progressive disease at median 415 (36–958) days from transplant. One-year survival was 48%, and 5-yr survival was 20%. At a median observation time of 65 months, 5 patients are alive, one in CR, one in PR, and three with stable disease. At multivariate analysis, CRP value before transplant, number of CD34+ infused cells and disease status at +90 significantly correlated with survival. Survival of patients at favourable/intermediate-risk according to the MSKCC score that underwent allografting was better in comparison to the survival predicted by historical controls. Conclusions: Transplantation is able to induce long-term disease control in a fraction (20%) of relapsed RCC patients. Identifying patients who could benefit from allografting, and incorporating molecularly targeted drugs in the transplant regimen, could further decrease progression and prolong overall survival. [Table: see text] No significant financial relationships to disclose.

Background Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. Methods In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per mu L or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. Findings From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. Interpretation Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. Background Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. Methods In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per mu L or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. Findings From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. Interpretation Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. Background Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality.Methods In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per mu L or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124.Findings From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure.Interpretation Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation.

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.

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.

Simultaneous isobutene etherification/dimerization reactions have been studied in the liquid phase using macroporous sulfonic acid resins. It has been found that, in the presence of methanol, oligomerization to species heavier than dimers may be greatly reduced with respect to data reported in the literature for isobutene dimerization. Products of the described synthesis are MTBE and a mixture of branched C8 hydrocarbons having excellent properties for gasoline blending. These two products may be obtained in a wide range of relative ratios. The influence of methanol (i.e., methanol/isobutene molar ratio), feedstock type, and reactor design on product composition and byproducts formation is discussed.

Autologous bone marrow transplantation (ABMT) for hematologic malignancies is associated with a high relapse rate. Interleukin-2 (IL-2) administration is a therapy that may prevent relapse if used when the tumor burden is minimal. In this study we administered recombinant IL-2 (rIL-2) therapy to 12 patients affected by hematologic malignancies either before or after autologous stem cell transplantation (ASCT). rIL-2 was given by a 6 day continuous intravenous infusion with escalating doses, up to 18 x 10(6)/m2/day, depending on patient tolerance. The functional immune responses of the patients were assessed as natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activities and in vitro interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) synthesis. During rIL-2 treatment, the expected side effects occurred; only 3 patients, who showed severe cardiovascular toxicity, required suspension of the treatment. All toxicities reversed after the end of the therapy. Immunologic monitoring was carried out the day before starting rIL-2 infusion and then repeated on days 3, 7, and 14 after rIL-2 was discontinued. Following every rIL-2 course, a pronounced increase in CD3+, CD8+, CD56+ cells was found, with a peak value on day 3. The NK and LAK activities showed a significant increase on day 3 (p < 0.001) over pretherapy values; the increase lasted until day 14, although the difference at later time points was not significant. Before transplant the synthesis of both IFN-gamma and TNF-alpha decreased following rIL-2 therapy, whereas higher levels of these lymphokines were found after posttransplant rIL-2 courses.(ABSTRACT TRUNCATED AT 250 WORDS)