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.

Donor lymphocyte infusion after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) is considered the cornerstone in the treatment of several malignant and not malignant hematological diseases. However, relapse of hematological disease after allo-SCT is considered the most challenging point in the field. The risk can be reduced through optimal patients, donor and disease selection before allo-SCT, but harnessing donor immune system is an appealing way to treat or avoid disease relapse. Donor lymphocyte infusion (DLI) is a simple and effective therapy after allo-SCT. In this paper, the efficacy of DLI will be analyzed in different hematological diseases, focusing also on their therapeutic or pre-emptive use.

Erythrocyte exchange and leukapheresis in pregnancy.

Hematological diseases in pregnancy should be carefully managed with a multidisciplinary approach, which should include obstetrics, hematology and, in selected patients, apheresis professionals. Hematological malignancies in pregnant women are rare, but the attending physicians should be aware that the use of cytotoxic drugs, tyrosine-kinase inhibitors or differentiating agents such as all-trans retinoic acid (ATRA) during the first trimester of pregnancy might be teratogenic and, in turn, induce fetal abnormalities or abortion. Thus, in pregnant patients with either acute or chronic leukemia presenting with symptomatic hyperleukocytosis, leukocytapheresis (LA) could be considered as a bridge therapeutic option. Furthermore, sickle cell disease (SCD) in pregnant women is usually managed only with supportive care, i.e. packed red blood cell (RBC) transfusion to prevent excessive hemoglobin decrease, hydration and prevention of acute sickling crisis. Nevertheless, selected patients at high risk for placental detachment due to vasoocclusive acute crisis or with multiple pregnancies may benefit from prophylactic erythrocyte exchange (EEX). Both LA and EEX must be carried out by well trained personnel and the patients (and the fetus) must be under close clinical and instrumental monitoring. In the present paper, recent indications for performing either LA or EEX in pregnant patients are reviewed.

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.

Kinetics of the use of cryopreserved autologous stem cell grafts: a GITMO-SIDEM survey.

BACKGROUND AIMS: Hematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient. METHODS: We conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU. RESULTS: In our cohort, 79% of patients collected >5 × 106 CD34+ cells/kg, and 3.4% collected <2 × 106 CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 × 106 CD34/kg and lower age groups. CONCLUSIONS: These data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.

A policy for the disposal of autologous hematopoietic progenitor cells: report from an Italian consensus panel.

BACKGROUND: Autologous stem cell transplantation (ASCT) requires collection and cryopreservation of hematopoietic progenitor cells (HPCs), which in turn may be partially or never reinfused. Thus, HPC storage has become a logistic, ethical, and economic issue. SIDEM, GITMO, and CNT/ISS endorsed a project aimed to define national criteria for HPC disposal aimed to guarantee appropriateness and equity. STUDY DESIGN AND METHODS: A multidisciplinary panel was convened including HPC harvest and manipulation experts from apheresis units, hematologists with clinical expertise in ASCT, a representative of the national health authority, and a bioethicist. An analytic hierarchy process (AHP) was carried out to select disposal criteria. RESULTS: The AHP selected two criteria for prompt disposal of freshly collected HPCs: an abnormal freezing procedure causing highly reduced viability or major microbiology contamination. Moreover, AHP selected six major criteria, each one of them allowing for the disposal of stored HPC units: patient death, withdrawal of consent to ASCT, contraindications or loss of indications to ASCT, a damaged label that prevents correct identification of the unit, and time elapsed since harvest longer than 10 years. Three minor criteria were additionally identified that allowed to anticipate disposal only provided that viability levels are below the limit of acceptance: a documented cold chain interruption, loss of bag integrity, and total amount of stored CD34+ cells lower than 1 × 10(6) /kg or lower than 2 × 10(6)/kg in patients with a successfully completed stem cell transplantation program. CONCLUSIONS: A formal consensus process allowed SIDEM and GITMO to propose a policy for autologous HPC disposal that fulfills clinical, ethical, and economic criteria.

Central venous catheter insertion in peripheral blood hematopoietic stem cell sibling donors: the SIdEM (Italian Society of Hemapheresis and Cell Manipulation) point of view.

Collection of peripheral blood hematopoietic stem cells (PBSC) is the practice of choice for graft procurement in both autologous and allogeneic setting. The success of this procedure depends on the use of adequate vascular accesses. Well-sized peripheral veins are the first option in autologous and allogeneic donations. In autologous setting, in case of lack of adequate veins, central venous catheters (CVC) may be used for collection. In the allogeneic setting, although available data have shown the safety of the use of CVC, there are still some controversies about the possible insertion of a CVC in donors. A specific policy from competent registries is usually applied in the different countries to regulate the use of CVC in unrelated donors. In siblings, the question is still undefined due both to the lack of shared guidelines and to the specific characteristics of this donation. In fact, in not so rare cases, larger stem cell doses for specific cell manipulations (e.g., T/B cell depletion in the haploidentical setting) are needed. The lack of international rules or standard that forbid the use of a CVC in siblings and published data that document the safety of this procedure, allowed the Società Italiana di Emaferesi e Manipolazione Cellulare (SIdEM) national Board to identify a possible, shared, operational approach to address this issue by a case-specific risk-benefit assessment.

Extracorporeal photopheresis for the treatment of acute and chronic graft-versus-host disease in adults and children: best practice recommendations from an Italian Society of Hemapheresis and Cell Manipulation (SIdEM) and Italian Group for Bone Marrow Transplantation (GITMO) consensus process.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective treatment for both acute and chronic graft-versus-host disease (GVHD) in children and adults. Despite the large use of this treatment, a large heterogeneity in current application of ECP has been reported so far and recent evidence brought novel issues into some specific topics. Consensus-based recommendations ameliorate the appropriateness in daily clinical practice and, in turn, optimize the use of health care resources. STUDY DESIGN AND METHODS: Two Italian scientific societies, the Italian Society of Hemapheresis and Cell Manipulation (SIdEM) and the Italian Group for Bone Marrow Transplantation (GITMO), joined to develop and disseminate recommendations on appropriate application of ECP treatment in patients with GVHD. Accordingly, SIdEM and GITMO named an expert panel that first selected 16 questions that were considered relevant for clinical practice: the questions were subsequently addressed through a revision of the available literature and in consensus meetings. The whole group discussed the proposed recommendations according to the nominal group technique. RESULTS: The above-described approach in turn allowed the panel to agree on 47 practice recommendations. SIdEM and GITMO will disseminate such recommendations to the national transplant centers. CONCLUSION: In conclusion, SIdEM and GITMO have made a scientific effort to provide a useful tool to physicians involved in the field, thus supporting daily clinical practice, as well as strategic decisions in the setting of ECP treatment of GVHD.

Erythrocyte-exchange with the OPTIA™ cell separator in patients with sickle-cell disease.

Erythrocyte-exchange (EEX) has proven to be a very useful tool in sickle-cell disease (SCD) patients either during acute painful crisis unresponsive to hydration and/or analgesia or as a prophylactic treatment in high risk patients in those who do not tolerate hydroxyurea (HU), with the aim of lowering HbS levels. EEX may be performed either by using continuous- or discontinuous flow devices, the former being of choice in children or in low-weight patients. Thus, a low extracorporeal blood volume (EBV) could allow for a better and safer procedure management. In this study we compared EEX procedure performed with the recently released OPTIA device with EEX procedures performed using the COBE Spectra device (EBV 185 vs 270 mL, respectively). Twenty-one EEX (4 as emergency treatment) were performed in 12 patients with the Spectra device and 25 (9 as emergency treatment) in 15 patients with the OPTIA device. All the procedures were well tolerated and uneventful. We did not observe significant differences between the two devices as to pre- and post-EEX parameters, namely in target hematocrit and in HbS reduction. Noteworthy, due to the lowest EBV allowed by the OPTIA device, an EEX procedure performed in a 13 Kg- child did not require a preliminary priming of the circuit. In conclusion, the OPTIA device proved to be as effective as the Spectra device in treating SCD patients either during sickling crisis or as prophylactic therapy. The OPTIA device can be safely used in the pediatric setting since it allows a lower EBV.

Best practice for peripheral blood progenitor cell mobilization and collection in adults and children: results of a Società Italiana Di Emaferesi e Manipolazione Cellulare (SIDEM) and Gruppo Italiano Trapianto Midollo Osseo (GITMO) consensus process.

BACKGROUND: A large heterogeneity in current mobilization and collection practices is perceived. Moreover, recent evidence introduced novel issues into some specific topics. Optimization of the clinical practice, through the adoption of clinical practice guidelines, previously proved to reduce health care resource use. STUDY DESIGN AND METHODS: Two Italian scientific societies, Società Italiana Di Emaferesi e Manipolazione Cellulare (SIDEM) and Gruppo Italiano Trapianto Midollo Osseo (GITMO), perceived the need of hematologists and transfusionists to share a common paradigm in the setting of hematopoietic stem cell transplantation (SCT). The aim of the current position paper is to provide common definitions and criteria for mobilization and collection of peripheral blood stem cells both in autologous and in the allogeneic setting. Current international and national standards (i.e., International Society of Hematotherapy and Graft Engineering) and recommendations (i.e., European Group for Blood and Marrow Transplantation) were harmonized with the Panel recommendations. RESULTS: The Expert Panel consisted of nine members (five transfusionists and four hematologists with both clinical and scientific experience of SCT in both pediatric and adult setting) and one methodologist and first convened on April 19, 2010: they in turn agreed on the questions to be answered by the project. Available literature was reviewed by one expert and the methodologist and presented to the other members. Statements were then formulated. SIDEM and GITMO planned an informal meeting of the Panel every 2 years to discuss relevant updates and possible changes to the recommendations. CONCLUSION: The efforts of the expert panel members allowed to set up and share a common approach to the mobilization, enumeration, and collection issues in the field of both autologous and allogeneic peripheral blood SCT.

High concentration plasma-reduced plateletapheresis concentrates.

Single-donor hyperconcentrated plateletapheresis (dry-platelets) collection has been introduced in the 90's as a part of the newly developed multi-component collection strategy. This approach allowed to safely collect multiple components from a single apheresis donation, i.e. RBC, FFP and/or plateletpheresis units. Dry-platelets are usually resuspended in additive solution to maintain an adequate pH during the storage period until use. Some concern existed about possible higher degrees of platelet activation in dry-platelets units when compared to standard concentration (1.0-1.6 × 10(6)/µL platelets) units and its possible correlation with lower in vivo efficiency and/or survival of the former units. Several authors investigated this specific issue, and dry-platelets units proved to be equally effective than standard concentration plateletpheresis units in recipients. The use of dry-platelets units may reduce (i) the risk of passive infusion of naturally occurring ABO-related hemolytic antibodies when donor O platelets are given to group A, B, or AB recipient, (ii) the risk of TRALI when multiparous donors undergo plateletpheresis. Furthermore, dry-platelet collection may allow for an increased amount of FFP sent to industry. Finally, hyperconcentrated platelet units may be used for "niche" indications, such as intrauterine platelet transfusion or, in case of autologous dry-platelet collection, for further freezing for long term storage in selected patients within onco-hematological settings.

GMP-based CD133(+) cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population.

Despite advances in graft-versus-host-disease (GVHD) treatment, it is estimated that overall survival (OS) at 2 years for hematopoietic cell transplantation (HCT) recipients who experience steroid-resistant GVHD is 10%. Among recent therapeutic approaches for GVHD treatment, mesenchymal stromal cells (MSCs) hold a key position. We describe a multicenter experience of 11 pediatric patients diagnosed with acute or chronic GVHD (aGVHD, cGVHD) treated for compassionate use with GMP-grade unrelated HLA-disparate donors' bone marrow-derived MSCs, expanded in platelet-lysate (PL)-containing medium. Eleven patients (aged 4-15 years) received intravenous (i.v.) MSCs for aGVHD or cGVHD, which was resistant to multiple lines of immunosuppression. The median dose was 1.2 x 10(6)/kg (range: 0.7-3.7 x 10(6)/kg). No acute side effects were observed, and no late side effects were reported at a median follow-up of 8 months (range: 4-18 months). Overall response was obtained in 71.4% of patients, with complete response in 23.8% of cases. None of our patients presented GVHD progression upon MSC administration, but 4 patients presented GVHD recurrence 2 to 5 months after infusion. Two patients developed chronic limited GVHD. This study underlines the safety of PL-expanded MSC use in children. MSC efficacy seems to be greater in aGVHD than in cGVHD, even after failure of multiple lines of immunosuppression.

Mononuclear cell collection in patients treated with extracorporeal photochemotherapy by using the off-line method: a comparison between COBE Spectra AutoPbsc version 6.1 and Amicus cell separators.

BACKGROUND: The first step (MNC collection) during extracorporeal photochemotherapy by using the off-line method is of paramount importance, since the product should be highly MNC-enriched with low RBC count to avoid UV-A intercepting by erythrocytes. MATERIAL AND METHODS: 10 patients (nine with chronic GvHD and one with bullous pemphigoid) underwent MNC collection on subsequent days either with the COBE Spectra AutoPbsc version 6.1 or the Amicus Crescendo cell separator. Total and differential WBC count were performed on peripheral blood samples before procedure and on the yields. The procedure parameters were recorded for comparison between the two devices. Analysis included descriptive statistics and paired t-test. RESULTS: No clinically relevant side effect with the exception of mild paresthesias was observed during the 20 MNC collections. Furthermore, no difference were observed as to pre-collection hematological parameters. The mean total nucleated cell count in the yield was 6.99 ± 2.39 × 10(9) with a MNC content of 5.9 ± 2.19 × 10(9) in the Spectra group and 6.15 ± 2.02 × 10(9) with 5.29 ± 2.39 × 10(9) in the Amicus group (P = 0.407 and P = 0.540, respectively). PLT content was higher in the yield from the Spectra group when compared to the Amicus group (1.54 × 10(11) ± 0.74 vs. 0.53 × 10(11) ± 0.34, P = 0.001). Moreover, no difference were observed in the RBC content in the product : 3.38 mL ± 1.91 vs. 4.59 mL ± 2.09, P = 0.191, when COBE Spectra and Amicus were compared, respectively. CONCLUSIONS: Both devices provide a satisfactory MNC collection for further UV-A treatment.

Rituximab as pre-emptive treatment in patients with thrombotic thrombocytopenic purpura and evidence of anti-ADAMTS13 autoantibodies.

Thrombotic thrombocytopenic purpura (TTP) is a rare and severe disease characterized by thrombocytopenia, microangiopathic haemolytic anemia, neurological and renal involvement associated with deficiency of the von Willebrand factor-cleaving protease, ADAMTS13. Persistence of high titers of anti-ADAMTS13 autoantibodies predisposes to relapsing TTP. Since relapses are associated with high morbidity and mortality rates, the optimal therapeutic option should be a pre-emptive treatment able to deplete anti-ADAMTS13 autoantibodies and avoid relapses. Five patients who presented with persistence of undetectable ADAMTS13 activity and high titers of autoantibodies, were treated with rituximab as pre-emptive therapy during remission. Four of them were affected by relapsing TTP and one was treated after the first episode. ADAMTS13 activity ranging from 15% to 75% with disappearance of inhibitors was achieved after three months in all patients, and persisted >20% without inhibitors at six months. In three patients disease-free status is still ongoing after 29, 24 and six months, respectively. Relapses were documented in two patients during follow-up: in one patient remission lasted 51 months; while in the other patient relapse occurred after 13 months. Results demonstrated that rituximab used as pre-emptive treatment may be effective in maintaining a sustained remission in patients with anti-ADAMTS13 antibodies in whom other treatments failed to limit the production of inhibitors, and suggests that re-treatment with rituximab should be considered when ADAMTS13 activity decreases and inhibitors reappear into the circulation, to avoid a new relapse.