Elisabetta Todisco

Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I–II study

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 muL 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. FUNDING MolMed SpA, Italian Association for Cancer Research.

Predictive value of early 18F‐fluorodeoxyglucose positron emission tomography (FDG‐PET) during salvage chemotherapy in relapsing/refractory Hodgkin lymphoma (HL) treated with high‐dose chemotherapy

This retrospective study evaluated whether early 2‐[fluorine‐18]fluoro‐2‐deoxy‐D‐glucose positron emission tomography (FDG‐PET) after two cycles of salvage chemotherapy (PET2) could predict survival after high‐dose chemotherapy (HDC). Twenty‐four Hodgkin lymphoma (HL) patients were included. PET2 was negative in 58% and positive in 42% of patients. Ninety per cent of patients (9/10) with positive PET2 relapsed after HDC while all but one patient with negative PET2 maintained a complete remission. The 2‐year progression‐free survival was 93% vs. 10% for patients with negative and positive PET2, respectively (P < 0.001). This study shows that interim PET can predict the outcome after high‐dose chemotherapy in HL patients.

Factors affecting successful mobilization with plerixafor: an Italian prospective survey in 215 patients with multiple myeloma and lymphoma

Although the efficacy of plerixafor in peripheral blood stem cell (PBSC) mobilization has been explored in several studies, factors associated with successful plerixafor mobilization after administration of granulocyte–colony‐stimulating factor (G‐CSF), with or without chemotherapy, have not been investigated. We analyzed data on PBSC mobilization from a large Italian database of lymphoma and myeloma plerixafor‐treated patients.

CD40 ligand-stimulated B cell precursor leukemic cells elicit interferon-γ production by autologous bone marrow T cells in childhood acute lymphoblastic leukemia

Childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, collected from bone marrow (BM) at diagnosis, were cultured, after thawing, on allogeneic human bone marrow stroma (HBMS) for 48 h in the presence of a soluble trimeric CD40 ligand (stCD40L) molecule. HBMS maintained leukemic cells viability in all tested cases (mean viability 85%). Under these culture conditions we noticed upregulation or de novo expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) in 22/22, 15/23 and 21/23 cases, respectively. Upregulation, in terms of fluorescence intensity, was also observed in the expression of MHC I, MHC II, CD54 (ICAM 1) and CD58 (LFA 3) molecules. HBMS alone, although to a lesser extent, was able to induce modulation of these molecules, but not CD80, in a similar proportion of cases. Neither stCD40L nor HBMS induced modulation of CD10 and CD34 molecules. Moreover, in 4/4 tested cases, stCD40L-stimulated ALL cells were able to induce allogeneic T cells proliferation. To evaluate whether leukemia-reactive T cells were detectable in the BM of ALL patients at diagnosis, stCD40L-stimulated ALL cells were co-cultured with autologous T cells (ratio 1:1), isolated from BM at diagnosis, for 4 days and a 24 h ELISPOT assay was applied to detect the presence of interferon-gamma (IFN-γ)-producing cells. In four of seven cases IFN-γ-producing cells were detected with frequencies of 1/900, 1/1560, 1/2150 and 1/1575 autologous T cells. These data confirm that stCD40L exposure can activate the antigen-presenting cell (APC) capacity of BCP-ALL cells cultured on HBMS and that ELISPOT assay can be used to measure the frequency of leukemia-reactive autologous T cells in the BM of ALL patients even after short-term culture with stCD40L-stimulated ALL cells.

Early discharge after high-dose melphalan and peripheral blood stem cell reinfusion in patients with hematological and non-hematological disease

The purpose of this study was to analyse our experience of early discharge 2 days after high-dose melphalan (HDM) (Day-1) followed by peripheral blood stem cell re-infusion (Day-0) and re-admission on Day +5 in patients with hematological diseases or solid tumors. From 2000 to November 2005, seven patients received tandem Melphalan 200 mg/m2 HDM with peripheral blood stem cells transplantation (PBSC-T), 130 a single HDM, for a total of 144 procedures. In 123 of them, patients were discharged on Day +1 for re-admission on Day +5 or earlier in the event of complications. Antibiotic prophylaxis was not used. Patients were hospitalised in positive-pressure reverse isolation room during the neutropenic period. Of the 123 procedures eligible for our mixed inpatient–outpatient management regimen, six (5%) required early re-admission for complications. Full engraftment was achieved in all cases. Median time to neutrophil count >0.5 × 109/µL and >1 × 109/µL were 12 and 14 days, respectively. Median time to platelet recovery (>20 × 109/µL) was 13 days. Severe extra-hematological toxicities occurred in 78 (63%) patients: all had oral mucositis and five had associated diarrhoea. During hospitalisation, 94/123 (76%) experienced febrile neutropenia, 20/94 (21%) had documented infection and 74/94 (79%) were considered fever of unknown origin. Median fever duration was 1 day (range 0–11). Median duration of antibiotic treatment was 6 days (range 3–26). Median time to discharge (from Day 0) was 16 days (range 11–57). There was no mortality by on Day +100. Our experience of early discharge after HDM and PBSC-T with re-admission on Day +5 is safe and feasible with acceptable frequency of hematological and extra-hematological toxicities. The regimen allows reduced hospital stay and hence cost savings.

Decision analysis of allogeneic hematopoietic stem cell transplantation for patients with myelodysplastic syndrome stratified according to the revised International Prognostic Scoring System.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Crucial questions in clinical decision-making include the definition of optimal timing of the procedure and the benefit of cytoreduction before transplant in high-risk patients. We carried out a decision analysis on 1728 MDS who received supportive care, transplantation or hypomethylating agents (HMAs). Risk assessment was based on the revised International Prognostic Scoring System (IPSS-R). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of different treatment policies on survival. Life expectancy increased when transplantation was delayed from the initial stages to intermediate IPSS-R risk (gain-of-life expectancy 5.3, 4.7 and 2.8 years for patients aged ⩽55, 60 and 65 years, respectively), and then decreased for higher risks. Modeling decision analysis on IPSS-R versus original IPSS changed transplantation policy in 29% of patients, resulting in a 2-year gain in life expectancy. In advanced stages, HMAs given before transplant is associated with a 2-year gain-of-life expectancy, especially in older patients. These results provide a preliminary evidence to maximize the effectiveness of allo-SCT in MDS.

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.

Inhibition of chronic graft-vs-host disease with retention of anti-myeloma effects by the proteasome inhibitor bortezomib.

Graft-vs-host-disease (GVHD) is a major complication of allogeneic stem cell transplantation (ASCT) [1]. Bortezomib is a proteasome inhibitor that has recently been approved for myeloma treatment. It has been shown to have numerous biological effects, including inhibition of NF-kB activation [2]. NF-kB is a transcription factor that has been detected in most cell types and that controls the expression of a number of genes involved in mediating immune and inflammatory responses [3]. A recent study on murine acute graft-vs-host disease (aGVHD) models has shown that bortezomib induces a selective depletion of alloreactive T lymphocytes by decreasing the production of Th1 cytokines [4]; it inhibits aGVHD and preserves graft-versus-tumor (GVT) effects [5]; and also that NF-kB is the target of bortezomib activity for aGVHD prevention [6]. However, there are only sparse data available on the effect of bortezomib on human GVHD [7]. We report a case of extensive chronic GVHD (cGVHD) and post-ASCT extramedullary myeloma progression, which were both responsive to bortezomib. A 49-year-old Caucasian patient with IgGk Stage III A multiple myeloma (Durie and Salmon) upon diagnosis, in partial remission because of a persistent positive immunofixation [8] after conventional chemotherapy and high-dose melphalan, received lenograstim-stimulated CD34þ cells from an HLAidentical sibling (on Day 0) after reduced-intensity conditioning with fludarabine 30 mg/m/die 73, 72, 71 i.v. and cyclophosphamide 300 mg/m/die 73, 72, 71 i.v. GVHD prophylaxis consisted of cyclosporine A and short-course methotrexate. Neutropenia and thrombocytopenia were not observed. Disease evaluation after ASCT showed stable disease. Full donor chimerism (revealed by PCR analysis) on total marrow and peripheral cells was reached six months after transplant and coincided with cyclosporine withdrawal. Eight months after ASCT the patient developed extensive cGvHD with involvement of the liver, skin, and bilateral conjunctiva and mouth mucosa, which was treated with cyclosporine and steroids until complete remission was reached. Sixteen months after ASCT the patient experienced extramedullary disease progression as revealed by a huge mass (12.56 9 cm) extending to the upper left chest wall, reappearance of monoclonal immunoglobulin (M component) by electrophoresis, and absence of bone marrow infiltration by plasma cells. The chest mass biopsy confirmed the presence of myelomatous cells. Cyclosporine was tapered and stopped, and loco-regional radiotherapy (total 20 Gy) was administered without any disease response. Then the patient received three monthly escalating doses of donor lymphocytes (DLIs, 16 10 kg; 56 10 kg; 16 10 kg) from his HLA-identical donor without any perceivable reduction of the extramedullary mass. Therefore, in June 2005, a few days after the third DLI, he was started on standard-dose bortezomib (1.3 mg/m i.v. for four times on Days þ1, þ4, þ8, þ11) to be

In Vitro Killing of Colorectal Carcinoma Cells by Autologous Activated NK Cells is Boosted by Anti-Epidermal Growth Factor Receptor-induced ADCC Regardless of RAS Mutation Status

Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients’ NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.

Clinical management of peripherally inserted central catheters compared to conventional central venous catheters in patients with hematological malignancies: A large multicenter study of the REL GROUP (Rete Ematologica Lombarda - Lombardy Hematologic Network, Italy)

Tao Cai , Xiang Chen, Jinchen Li, Bingwu Xiang, Liu Yang, Yidian Liu, Qiuli Chen, Zhouwen He, Kevin Sun, P. Paul Liu Institute of Genomic Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, China National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland Physical Medicine and Rehabilitation Center, Second Affiliated Hospital of Wenzhou Medical University and Yuying Children’s Hospital, Wenzhou, Zhejiang, China Genetic Metabolic CentraLand of Laboratory, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, China Department of Neurology, First Affiliated Hospital of Nanhua University, Hengyang, Hunan, China Oncogenesis and Development Section, National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, Maryland