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Genetic subgroups; Immune reconstitution; Pretransplantation infections Subgrupos genéticos; Reconstitución inmune; Infecciones previas al trasplante Subgrups genètics; Reconstitució immune; Infeccions prèvies al trasplantament Background Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for patients with severe combined immunodeficiency (SCID), a group of monogenic immune disorders with an otherwise fatal outcome. Objective We performed a comprehensive multicenter analysis of genotype-specific HSCT outcome, including detailed analysis of immune reconstitution (IR) and the predictive value for clinical outcome. Methods HSCT outcome was studied in 338 patients with genetically confirmed SCID who underwent transplantation in 2006-2014 and who were registered in the SCETIDE registry. In a representative subgroup of 152 patients, data on IR and long-term clinical outcome were analyzed. Results Two-year OS was similar with matched family and unrelated donors and better than mismatched donor HSCT (P < .001). The 2-year event-free survival (EFS) was similar in matched and mismatched unrelated donor and less favorable in mismatched related donor (MMRD) HSCT (P < .001). Genetic subgroups did not differ in 2-year OS (P = .1) and EFS (P = .073). In multivariate analysis, pretransplantation infections and use of MMRDs were associated with less favorable OS and EFS. With a median follow-up of 6.2 years (range, 2.0-11.8 years), 73 of 152 patients in the IR cohort were alive and well without Ig dependency. IL-2 receptor gamma chain/Janus kinase 3/IL-7 receptor–deficient SCID, myeloablative conditioning, matched donor HSCT, and naive CD4 T lymphocytes >0.5 × 10e3/μL at +1 year were identified as independent predictors of favorable clinical and immunologic outcome. Conclusion Recent advances in HSCT in SCID patients have resulted in improved OS and EFS in all genotypes and donor types. To achieve a favorable long-term outcome, treatment strategies should aim for optimal naive CD4 T lymphocyte regeneration.

Leukocyte Adhesion Deficiency type 1 (LAD-1) is a rare primary immunodeficiency due to mutations in the gene encoding for the common β-chain of the β2 integrin family (CD18). Herein, we describe clinical manifestations and long-term complications of eight LAD-1 patients. Four LAD-1 patients were treated with hematopoietic stem cell transplantation (HSCT), while the remaining four, including two with moderate LAD-1 deficiency, received continuous antibiotic prophylaxis. Untreated patients presented numerous infections and autoimmune manifestations. In particular, two of them developed renal and intestinal autoimmune diseases, despite the expression of Beta-2 integrin was partially conserved. Other two LAD-1 patients developed type 1 diabetes and autoimmune cytopenia after HSCT, suggesting that HSCT is effective for preventing infections in LAD-1, but does not prevent the risk of the autoimmune complications.

PURPOSE: Complete signal transducer and activator of transcription 1 (STAT1) deficiency is a rare autosomal recessive condition characterized by impairment of intracellular signaling from both type I and type II interferons (IFN). Affected patients are prone to early severe mycobacterial and viral infections, which usually result in death before 18 months of age. We previously reported a patient affected by complete STAT1 deficiency who underwent hematopoietic stem cell transplantation (HSCT). Here, we describe the transplantation procedures and long-term outcomes. METHODS: The patient, who had suffered multiple life-threatening mycobacterial and viral infections in the first years of life, underwent HSCT at 4 years of age from a partially matched (HLA compatibility 8/10) unrelated donor after a myeloablative conditioning regimen consisting of busulfan, cyclophosphamide, and anti-thymocyte globulin. RESULTS: Hematological reconstitution was detected at d+15, with full donor engraftment demonstrated by molecular analysis of leukocytes. Several complications occurred in the post-transplantation phase, including acute graft versus host disease, posterior reversible encephalopathy, thrombotic thrombocytopenic purpura, bilateral keratoconjunctivitis with complete loss of vision, and chronic lower limb lymphedema. Analysis of STAT1 in CD3+ cells at 90 and 120 days after HSCT by flow cytometry showed normal STAT1 phosphorylation levels in response to IFN-α. CONCLUSIONS: Notably, no severe infections occurred after discharge (day + 90) during a 9-year follow-up, suggesting that normal response to IFNs in hematopoietic cells is sufficient to provide protection in humans.

We describe the case of a child affected by severe combined immunodeficiency (SCID) with adenosine deaminase (ADA) deficiency showing a maternal T-cell engraftment, a finding that has never been reported before. The presence of engrafted maternal T cells was misleading. Although ADA enzymatic levels were suggestive of ADA-SCID, the child did not present the classical signs of ADA deficiency; therefore, the initial diagnosis was of a conventional SCID. However, ADA toxic metabolites and molecular characterization confirmed this diagnosis. Polyethylene glycol-modified bovine (PEG) ADA therapy progressively decreased the number of maternal engrafted T cells. The child was grafted with full bone marrow from a matched unrelated donor, after a reduced conditioning regimen, and the result was the complete immunological reconstitution., Highlights • Maternal engrafted T-cell in ADA-SCID • Engrafted T cells can be misleading for diagnosis • Diagnostic testing is critical

Less than 25% of children who require hematopoietic stem cell transplantation (HSCT) for primary immunodeficiencies (PIDs) or genetic hematological diseases have an HLA-identical sibling. For them, a matched unrelated donor (MUD), although baring a greater risk of graft failure, delayed engraftment and immune reconstitution, and severe graft-versus-host disease (GvHD), represents a valid alternative. The stem cell source is also important, as unprocessed peripheral blood stem cells (PBSCs) contain 5 to 10 times more T cells than bone marrow (BM)-derived grafts, a major risk especially for small children with PID. A CD34+ positive selection can mitigate HLA compatibility issues, but the resulting CD3+ T cell depletion hampers engraftment and facilitates infections. To mitigate those problems, we decided to add back a certain number of T cells (30 × 106 cells/kg body weight [BW]) to the positive CD34+ selection derived from MUD BM or PBSCs and report the results in terms of time to engraftment and immune reconstitution, GvHD incidence, infections, and survival. Our aim was to show not only the feasibility and clinical efficacy of this addback but also that PBSC-derived CD34+ selected grafts with calibrated T cell addback would be equivalent to BM-derived grafts. We analyzed retrospectively our single-center cohort of 76 children (median age, 1.9 years) affected by PID (61) and hematological diseases (15) who received a total of 79 MUD HSCTs with CD34+ selection and addback of 30 × 106 CD3+ cells/kg BW between 2001 and 2019. We used descriptive and analytic statistics (chi-square, Student's t-test, Mann–Whitney U test, as appropriate) and constructed Kaplan–Meier curves using the log-rank test to compare patients grafted with BM or PBSC-derived inocula. The two groups showed no statistically significant differences in terms of age, sex, HLA-mismatch, or amount of CD3+ cells/kg BW added back to the CD34+ selection. However, the latter being higher in the PBSC group (P = .0001). Overall engraftment rate was 96% (73/76) and occurred faster in the PBSC group than in BM recipients: polymorphonuclear cells, 16 versus 21 days (P = .006); platelets, 15 versus 22 days (P = .001). GvHD incidence was low. No acute GvHD was diagnosed in 24 children, whereas grades I, II, III, and IV occurred in 19, 28, five, and three children, respectively (P not significant). Chronic GvHD was seen in only two children. The CD4+ count at six months after HSCT was higher in PBSC recipients as compared to those receiving BM (184 versus 88 CD4+ cells; P = .003). Overall survival for the whole cohort was 80% at 10 years, with no significant difference between the two stem cell sources (P not significant). Viral infections occurred among five of the PBSC grafted children and 14 in the BM group (P not significant), and no patient suffered from post-transplant lymphoproliferative disorder (PTLD). The results we present show that an addback of 30 × 106 donor CD3+ cells/kg recipient BW to a MUD BM or PBSC-derived CD34+ selection gives promising results in infants and young children undergoing HSCT for PID or hematological diseases. Furthermore, with this manipulation the inherent limits of PBSC-derived grafts can be overcome, allowing both swift engraftment and immune reconstitution without an increase in GvHD, infections, or PTLD.

We carried out a retrospective analysis of 27 patients with Adenosine Deaminase (ADA) deficiency diagnosed in a single center from 1997 to the 2013, for evaluating whether data regarding types of disease-inducing mutations, biochemical and immunological features as well as clinical outcomes of patients treated with enzyme replacement or transplantation, were comparable to those obtained in multicenter studies. The ADA deficiency diagnosis was performed with biochemical, immunological and molecular techniques. Ten patients treated with hematopoietic stem cell transplantation and three in treatment with enzyme replacement were followed up in our center. Twenty-four different mutations were identified and five were not previously reported. Identical mutations were found among patients from the same Romani ethnic group or from the same geographical region. A more rapid recovery was observed in enzyme replacement treated patients in comparison with those transplanted that, however, showed a continuous and long-lasting improvement both in terms of immune and metabolic recovery. The data obtained in our single center are comparable with those that have been reported in multicenter surveys.

We describe a previously healthy 14-year-old girl with acute onset autoimmune hemolytic anemia, associated with severe but transient lymphopenia during corticosteroid therapy, without infectious episodes during follow-up. After detailed investigations to rule out an underlying immunodeficiency, we detected a heterozygous ADA gene mutation. This was associated with slightly increased blood levels of adenosine and deoxyadenosine nucleotides and with reduced ADA activity in red blood cells, but within the normal range. This observation suggests that heterozygous ADA mutation might be a predisposing factor for lymphopenia in patients receiving corticosteroid therapy.

Chronic Granulomatous Disease (CGD) is caused by the failure of the phagocytes to kill pathogens. We carried out a retrospective analysis of cellular, molecular and clinical features of 14 young patients (mean age at the onset of symptoms and diagnosis: 10 and 25 months, respectively), 7 with autosomal recessive and 7 X-linked form, referred to the Children's Hospital of Brescia between 1999 and 2016. Two new mutations were found, one localized in the CYBB and one in the NCF1 genes. Twelve patients were followed in our institution; the average length of their follow-up after diagnosis was 66 months in X-linked patients and 126 months in autosomal recessive inheritance. The overall survival was 67%, 40% in X-linked and 86% in autosomal recessive form. Eight patients were treated with HSCT. We did not find a clear correlation between the clinical symptoms and the type of mutation, but the fine characterization of the patients was mandatory for therapeutic option, genetic counseling and prenatal diagnosis.

Partial depletion of TCR alpha/beta + / CD19 + cells in matched unrelated transplantation of three patients with osteopetrosis

Switched and IgM memory B cells execute different and noninterchangeable functions. We studied memory B cells in children of different ages, in peripheral blood and spleen and compared them with those of children born asplenic or unable to build germinal centers. We show that, whereas switched memory B cells are mostly generated in the germinal centers at all ages, IgM memory B cells can be distinct in three types with different developmental history. Innate IgM memory B cells, the largest pool in infants, are generated in the spleen by a germinal center-independent mechanism. With age, if the spleen is present and germinal centers are functional, innate IgM memory B cells are remodelled and accumulate somatic mutations. The third type of IgM memory B cell is a by-product of the germinal center reaction. Our data suggest that the B-cell memory developmental program is implemented during the first 5-6 years of life.

A major problem in the field of stem cell transplantation is the difficulty to monitor the efficacy of immune reconstitution. By modifying the widely used method of measuring T-cell receptor excision circles (TRECs) and the recently proposed kappa-deleting recombination excision circles (KRECs) assay, we set up a duplex Real-Time PCR that allowed the simultaneous quantification of newly produced T and B cells in children with primary immunodeficiency undergone to transplantation. We found that lymphocyte recovery involves the mobilization of both new T and B cells from production and maturation sites, and that the increase of TRECs and KRECs can be or strictly associated or independent one from the other. Some patients showed a "lymphocyte rebound" which is followed by a progressive decrease of newly produced T and B lymphocytes starting about 2years after transplantation. In other patients, TRECs and KRECs number remained very low for the entire period of study.

BMT is curative in almost 75% of children affected by severe primary immunodeficiencies (PIDs). Recently, the chance of cure has increased thanks to the availability of matched unrelated donors (MUDs). Nevertheless, besides the conventional indications to BMT (profound or absent T-cell function, profound or absent natural killer function, known syndromes with T-cell deficiencies), indications to BMT for PIDs affecting the quality of life or having an expectation of life that does not exceed the third-fourth decade remain unclear. Infact, if it is evident that the survival rate in an infant grafted for a PID with a MUD is expected to be more than 80%, alternative treatments such as gene therapy are now available.

We retrospectively analyzed the outcome of hematopoietic stem cell transplantations (HSCT) performed at our Center between 1991 and 2002 in 11 unselected patients with Omenn syndrome, a variant of severe combined immunodeficiency. The patients' mean age at the time of the first HSCT was 8.4 months. Two patients received two, and one patient three, HSCT procedures. The resulting 15 HSCT derived in seven cases from HLA-haploidentical parents, in four patients from matched unrelated donors, in three cases from an HLA phenotypically identical related donor, and in one case from an HLA genotypically identical family donor. Nine out of 11 patients are alive and immunoreconstituted 30-146 months after transplantation. At the time of the most recent evaluation, all of the nine survivors had normal T-cell function, and eight of them had developed normal antibody production. This study demonstrates an overall mortality of 18.2%, which is substantially lower than previously reported. Early recognition of OS, rapid initiation of adequate supportive treatment and HSCT lead to improved outcome for this otherwise fatal disease, regardless of the origin and matching of hematopoietic stem cells.

Purpose: High-dose therapy (HDT) and peripheral-blood stem-cell transplantation (PBSCT) in HIV-associated lymphoma (HIV-Ly) has been recently reported in selected patients. We describe the results of a multi-institutional program of HDT and PBSCT as salvage therapy in HIV-Ly responsive to highly active antiretroviral therapy (HAART) in unselected patients. Patients and Methods: Patients with resistant or relapsed HIV-Ly after first-line chemotherapy (CT) underwent PBSC collection after a course of second-line CT or cyclophosphamide and granulocyte colony-stimulating factor. Patients with chemotherapy-sensitive disease received carmustine, etoposide, cytarabine, and melphalan (BEAM regimen) and PBSC reinfusion. Effective HAART was maintained during the entire program. Results: Sixteen consecutive patients entered the program. Adequate collection of PBSC was obtained in 80% of patients (median CD34+ cells 6.8 × 106/kg). Three patients had early progression. Ten patients (62%) received PBSCT with prompt engraftment in all patients (neutrophils and platelet engraftment after a median of 10 days [range, 8 to 10 days] and 13 days [range, 8 to 18 days], respectively). No patients died as a result of opportunistic or other infections or treatment-related complications. Eight of nine assessable patients achieved complete remission (one patient after radiotherapy for residual disease) and one patient achieved partial remission. Two patients experienced relapse and died at +10 and +14 months. Six patients are alive and disease free at a median of 8 months after transplantation. Conclusion: Our data confirm that HDT plus PBSCT is feasible and active as salvage therapy in HIV-Ly on a multi-institutional basis and in unselected HAART-responding patients. HIV infection should no longer preclude the opportunity of HDT in patients with lymphoma.

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Summary Background X-linked severe combined immunodeficiency (SCIDX1) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common γ chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDX1 can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. Methods A male fetus was diagnosed as having SCIDX1 by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E resetting. Chimerism analysis was by HLA-DQα typing and γ-chain staining on cord blood. Findings A healthy 3·6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3·5 months of age the infant is well and his T-cell counts and function are normal. Interpretation In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

The presence of myxovirus resistance protein A (MxA) RNA was studied in 55 febrile children with primary immunodeficiency, 27 of whom underwent hematopoietic cell transplantation, and in 28 age-matched controls. The level of MxA RNA was above the cutoff, established as the 95th percentile found in controls, with primary immunodeficiency either undergoing transplantation or not in febrile patients, and with a documented diagnosis of infection by adenovirus, cytomegalovirus, Epstein-Barr virus, respiratory syncytial virus, and rotavirus. The presence of rare viral infections, unrecognized among those that more frequently occur in patients with primary immunodeficiency and in patients undergoing transplantation, may explain the high MxA RNA levels observed in some patients with fever but undetectable genomes or antibodies for the more common viruses. The level of MxA in febrile patients with acute graft versus host disease was below the cutoff, with a median level comparable with that observed in patients with primary immunodeficiency, who did not undergo transplantation and were without fever and infections, but significantly lower compared with controls. The level of MxA was well correlated with viral infections in follow-up samples. These data indicate that the measurement of MxA RNA is simple and useful to detect viral infections and in distinguishing them from acute graft versus host disease after allogeneic cell transplantation.

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle+ lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA–treated patients, it was accompanied by a significant and progressive decrease in circulating CD19+ lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle+ cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.

The aim of the most recent studies on regenerative medicine was to focus on capability of stem cells deriving not only from haematopoietic system, but also from other organ and tissues, to regenerate damaged tissues. Stem cells derived from foetal annexes such as cord blood, placenta and amniotic fluid can be currently used in the effort to treat prenatally diagnosed genetic diseases. Cells derived from cord blood have been used since 1988 as an alternative source to realize stem cell transplantation. Compared with bone marrow, cord blood has shown the advantages of quick availability, less risk of GHVD, together with higher compatibility rates, and less risk of infections. Mesenchymal stem cells (MSCs) are multi-potent stem cells able to differentiate into different lineages, including osteocytes, chondrocytes, and adipocytes. Because of their trafficking capacity to injured tissues, clinical trials have been started evaluating the use of MSCs in the treatment of metabolic diseases like Hurler syndrome and metachromatic leukodystrophy, or Osteogenesis Imperfecta. MSCs were initially identified in adult bone marrow (BM-MSC), but cells resembling BM-MSCs have also been found in other tissues, both adult (peripheral blood, synovial membrane) and foetal (peripheral blood, liver, spleen, placenta, umbilical cord, and amniotic membrane). BM-MSCs have been widely used in clinical applications, as for cell-based therapy of Osteogenesis Imperfecta and metabolic diseases. In addition, human multi-potent MSCs present in second-trimester amniotic fluids may be a good target for prenatal gene therapy because of their expandability, their ability to differentiate into multiple lineages and their high transduction efficiency.

A case of Kaposi's sarcoma in a child with no serologic evidence of human immunodeficiency virus (HIV) infection is reported. A 7-year-old boy with Stage IV non-Hodgkin's lymphoma, after conventional chemotherapy, underwent autologous bone marrow transplantation (ABMT). Five months later he presented with supraclavicular mass and mediastinal enlargement. A bone marrow biopsy showed hypoplasia with no signs of the underlying disease, whereas the excised mass revealed a typical histologic pattern of Kaposi's sarcoma. The child is currently being treated with recombinant alpha-interferon (alpha-IFN) and regression of the disease has been achieved.

In an attempt to define a biological marker of monocyte hyperactivation in the course of infantile histiocytosis, the spontaneous nitroblue tetrazolium (NBT) reduction assay was applied to monocytes from 13 children with Langerhans cell histiocytosis (LCH), familial haemophagocytic lymphohistiocytosis (FHL), juvenile xanthogranuloma or malignant histiocytosis. Significant increase in NBT reduction was observed in the patients with both active LCH and FHL in comparison with control subjects, who were either healthy or affected by different conditions. A close relationship between spontaneous reduction rate and clinical condition of the patients was evident in patients tested at diagnosis, during remission and during the course of disease reactivation. Interleukin-1 (IL-1) production by monocytes was also evaluated: the patients with LCH and FHL displayed a significant increase in in vitro IL-1 production by lipopolysaccharide-stimulated monocytes. In our experience the spontaneous NBT reduction assay was a sensitive, quite specific, low-cost and reproducible test for the evaluation of children with histiocytosis. Useful information may be obtained at diagnosis but also during the clinical course of disease by using this marker of monocyte spontaneous activation.

Background Currently, hematopoietic stem cell transplantation allows long-term survival in a high proportion of infants with congenital severe T-cell immunodeficiency. However, relatively little is known of their long-term quality of life. Objective We sought to assess the long-term immune reconstitution and clinical status in children treated with stem cell transplantation for severe T-cell immunodeficiency. Methods Immune function and clinical status have been analyzed in a cohort of 40 patients with severe T-cell immunodeficiency who are alive at a follow-up of at least 5 years after transplantation. Results Most patients have attained normal T- and B-cell function. Weight and height were normal at last follow-up in most patients. Endocrine and severe neurologic abnormalities have been observed in 17.5% and 10% of the patients, respectively. Conclusions These data indicate that with current management strategies, stem cell transplantation can lead to long-term survival and good quality of life in the majority of patients with severe T-cell immunodeficiency. Clinical implications Prompt recognition of congenital severe T-cell immunodeficiency, followed by stem cell transplantation, allows excellent perspectives of long-term survival and good quality of life for these otherwise fatal disorders.

The treatment of Wiskott-Aldrich syndrome (WAS), a once uniformly fatal disorder, has evolved considerably as the use of hematopoietic stem cell transplant has become more widespread. For the majority of patients who lack an human leukocyte antigen-identical sibling, closely matched unrelated donor bone marrow transplant (MUD BMT) at an early age is an excellent option that nevertheless is not uniformly chosen. We retrospectively analyzed our experience with transplantation in 23 patients with WAS from 1990 to 2005 at the University of Brescia, Italy, of whom 16 received MUD BMT. Myeloablative chemotherapy was well tolerated with median neutrophil engraftment at day 18, and no cases of grade III or IV graft-vs-host disease. Overall survival was very good with 78.2% (18/23) of the whole cohort and 81.2% (13/16) of MUD BMT recipients surviving. Among 18 survivors, full donor engraftment was detected in 12 patients, and stable mixed chimerism in all blood lineages in four patients. Deaths were limited to patients who had received mismatched related BMT or who had severe clinical symptomatology at the time of transplantation, further emphasizing the safety and efficacy of MUD BMT when performed early in the clinical course of WAS.

Aims There is increasing evidence that stem cell (SC) mobilization to the heart and their differentiation into cardiac cells is a naturally occurring process. We sought to assess the safety and feasibility of granulocyte-colony stimulating factor (G-CSF) administration in humans to enhance SC mobilization and left ventricle (LV) injury repair during myocardial infarction (MI). Methods and results Twenty patients with STEMI (mean age, 61±10 years), of whom 14 were submitted to primary percutaneous coronary intervention, were randomized to G-CSF (5 µg/kg/day s.c. for 4 consecutive days) or placebo. At entry and then at months 3 and 6, 99mTc-sestamibi gated-SPECT was performed to estimate extension of perfusion defect (PD) and LV function. The study drug was well tolerated and induced a significant increase of white blood count, CD34+ cells, and CD34+ cells coexpressing AC133 and VEGFR-2. At follow-up, treated and placebo groups did not differ for the angiographic coronary late loss and showed a similar pattern of PD recovery, whereas in the former at 6 months LVEF and especially LVEDV tended to be relatively higher ( P =0.068) and lower ( P =0.054), respectively. Conclusion G-CSF administration in acute MI patients was feasible and did not lead to any clinical or angiographic adverse events and resulted in CD34+ and CD34+AC133+VEGFR2+ cell mobilization.

In utero transplantation of hematopoietic stem cells allows immune reconstitution of fetuses with severe combined immunodeficiency. The objective of this work was to study the quality of T-cell reconstitution following this procedure.We evaluated the kinetics and extent of T-cell reconstitution in five infants with severe combined immune deficiency (SCID), three with a B+ and two with a B- phenotype, who received haploidentical stem cell transplantation before birth. To this end, we measured the frequency of T-cell receptor excision circles (TREC) and the diversity of the T-cell repertoire.In utero transplantation led to engraftment of donor-derived T lymphocytes which attained normal numbers in four infants, who are in good health. In the three patients with a B+ phenotype, generation of a heterogeneous T-cell repertoire was associated with development of TREC levels comparable to those of SCID patients treated by post-natal transplantation and of healthy babies. Of the two patients with a B- phenotype, one developed mixed T-cell chimerism and a substantial number of circulating T cells, associated with a variable heterogeneity of the T-cell repertoire; TREC levels were normal soon after birth, but declined thereafter. The remaining B- patient remained lymphopenic with a skewed T-cell repertoire and very low TREC levels. This patient eventually required transplantation from a matched unrelated donor at 5 years of age, but died of EBV-related lymphoproliferative disease.These data indicate that in utero transplantation of fetuses with B+ SCID allows generation of newly diversified T lymphocytes and ensures long-term reconstitution of cell-mediated immunity.

Uncontrolled-rate freezing (URF) techniques, which are fast and easy, could represent an attractive alternative to controlled-rate cryopreservation procedures which are time consuming and require high-level technical abilities. It was the aim of the present study to evaluate, on a routine basis, whether URF might spare primitive hematopoietic progenitors and maintain engrafting capacity.One-hundred and nineteen peripheral blood progenitor cells (PBPC) collections from 104 patients with hematologic malignancies were cryopreserved in bags, with an URF procedure, in a cryoprotectant solution consisting of PBS, HSA and 10% DMSO and stored in liquid nitrogen. PBPC bags were tested before cryopreservation and at thawing for primitive (LTC-IC) and committed hematopoietic progenitors (CFU-Mix, BFU-E, CFU-GM) by means of long- and short-term culture assays, respectively. In addition, PBPC bags were evaluated for CD34+ cell numbers.Although thawing was associated with a statistically significant reduction of the absolute number of nucleated cells, recovery of LTC-IC, CFU-Mix, BFU-E, CFU-GM and CD34+ cells was not affected by the freezing/thawing procedures. No adverse effects were reported at thawing and only mild transient reactions were recorded in 22 patients during reinfusion of cryopreserved PBPC. All the patients underwent myeloablative therapy followed by reinfusion of PBPC, and prompt and rapid hematopoietic recovery was obtained in all patients.Our freezing procedure is fast and easy, and allows rapid hematopoietic recovery after myeloablative therapy by sparing primitive and committed hematopoietic progenitors. Our study strongly supports technical improvements aimed at cost reduction and feasibility of routine freezing procedures.

Introduction Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia (CML), effectively prolonging overall survival. Because discontinuation of treatment is associated with molecular relapse, IM is required indefinitely to maintain operational cure. To evaluate the degree of response to therapy and to highlight the persistence of the disease after treatment, patients should be monitored routinely. The gold standard for diagnosing CML is the cytogenetic analysis, a direct not-sensitive method to detect Ph-positive cells. Quantitative real-time RT-PCR (qRT-PCR) provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells and cannot detect transcriptionally silent leukemic stem cells. Methods Here we will propose a new sensitive approach to directly detect the number of leukemic cells using a DNA-based biomarker specific for each patient. We applied targeted next-generation sequencing for the identification of genomic BCR-ABL1 fusion junctions, and we developed a sensitive new approach to detect the number of leukemic cells by a DNA Q-PCR assay based on the genomic break-point, with a formula to calculate the number of Ph+ cells. The percentage of the leukemic cells (LC) was calculated using the following formula: %LC= (2/(2Δct+1))*100, where ΔCt is the difference between the amplification cycles of the BCR-ABL1 and BCR reactions. The number of LC was calculated by multiplying the total number of cells analyzed in each sample by the percentage of LC calculated by the ΔCt formula. We then defined a limit of quantization and a limit of sensitivity in the evaluation of minimal residual disease (MRD), as described by guidelines for the detection of MRD by genomic Q-PCR in acute lymphoblastic leukemia (ALL). We defined a “quantitative range” of detection, the portion of the standard curve in which the MRD levels can be quantified reproducibly and accurately, and we defined the “limit of sensitivity”, the lowest MRD level that still can be detected, although not in all replicates. We thus calculated the exact number of leukemic cells only when the MRD fell within the range of quantization. The detection of MRD at the limit of sensitivity was indicated as positive but not quantified. Results We monitored eight CML patients treated with Imatinib for 8 years. We tested the same samples by patient specific Q-PCR, and in parallel by cytogenetic analysis and by standard qRT-PCR. In all samples positive for chimeric transcripts we measured corresponding chimeric genomic DNA (gDNA) by Q-PCR, confirming the sensitivity of the Q-PCR method. According to conventional criteria, undetectable levels of BCR-ABL1 mRNA assessed by qRT-PCR are indicative of complete molecular response (CMR), but in 33.3% (45/135) samples with undetectable levels of mRNA, we detected the persistence of transcriptionally-silent leukemic cells. However, we never found samples negative by gDNA Q-PCR and positive by RNA-based qRT-PCR (Figure 1). Thirty-six of 135 samples were also analyzed cytogenetically until the achievement of CCyR. As expected, Ph+cells were detected only in 25% (9/36) and 22,2% (8/36) of samples by CBA and I-FISH, respectively, whereas BCR-ABL1 mRNA was detected by qRT-PCR in 83.3% (30/36) of samples and Ph+ cells were detected by genomic Q-PCR in 91.7% (33/36) of samples (Figure 1). Finally, the separation of different cell populations from blood and bone marrow revealed the presence of a population of transcriptionally silent cancer stem cells. The gDNA based Q-PCR analysis performed on highly purified (immunomagnetically selected ) CD34+and CD3+ cells confirmed the presence of a population of transcriptionally silent cancer stem cells. Conclusions The demonstration of positive gDNA Q-PCR in 33.3% of samples negative for the RNA qRT-PCR could partially explain why some patients lose MMR and CMR and others do not, when IM is discontinued for brief periods. The gDNA based Q-PCR could be used to supplement conventional techniques, providing clinicians with additional information about disease status and response in determining whether to stop or alter therapy. Acknowledgments to AIRC and AIL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1687 Background: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder resulting from the t(9;22)(q34;q11) balanced reciprocal translocation within a pluripotent stem cell (SC). The resulting Philadelphia (Ph) chromosome produces BCR-ABL1 fusion gene coding for a deregulated Abl tyrosine-kinase with constitutive and tumorigenic activity. The first line therapy of CML is imatinib mesylate, which targets Bcr-Abl protein, inhibiting proliferation pathways. Complete cytogenetic response can be achieved in 95% of patients treated in the early chronic phase (CP)1. Molecular monitoring of minimal residual disease is crucial to detect poor responses to imatinib and optimizing treatment with second generation tyrosine-kinase inhibitors or allogeneic stem cell transplantation. Residual leukemia is assessed by a quantitative manner evaluating levels of BCR-ABL1 transcripts by real-time reverse transcriptase PCR (qRT-PCR). Although qRT-PCR detects mRNA levels in a very sensitive manner, the negative result is difficult to interpret, because undetectable levels of chimeric transcript can reflect either an effective elimination of leukemia cells, or the presence of a quiescent leukemia SC transcriptionally silent. Methods: We developed a novel highly sensitive method to identify quiescent leukemic cells through quantitative real-time PCR (Q-PCR) targeting the genomic breakpoint sequence1. In CML each patient shows a unique genomic fusion sequence1, that need to be characterized in order to design a specific genomic assay. We selected 14 patients with CML diagnosed in the early CP. We identified junctions sequences by long-range PCR2. We carried out Q-PCR assay using common primer forward and probe in BCR, and 2 different primers reverse, in ABL or BCR, used as control1. The percentage of leukemic cells (LCs) was calculated using the derivation of the δCt formula1: LC= [100*(2/2δCt+1)]/n], where δCt is the difference between amplification cycles of BCR-ABL1 and BCR reactions, and n is the number of experimental replicates. We tested the sensitivity and the efficiency of the method on K562 cell line. According to the human C value, K562 were diluted in normal commercial genomic DNA until 10−4 dilutions. Eight CML patients in early CP were the object of this study. A patient specific Q-PCR assay was performed on DNA obtained at diagnosis and subsequently applied to monitor minimal residual disease during Imatinib treatment for up to 8 years, for a total of 61 samples. In parallel the same peripheral blood samples were tested by standard qRT-PCR, and the percentage of residual disease (international scale) measured by mRNA was compared with DNA analysis. Results: Positive levels of mRNA were obtained in 79% of samples analyzed by qRT-PCR,while we detected Ph-positive cells in 92% of samples. In all positive samples for chimeric transcript we measured positive levels of corresponding genomic DNA, confirming the sensitivity of the Q-PCR method. In 13% of samples, with undetectable levels of mRNA, we observed the persistence of quiescent leukemic cells, transcriptionally silent like shown by patient 2 in figure 1. This could probably indicate the presence of pluripotent LSCs or progenitor cells, that does not respond to imatinib treatment. Finally undetectable levels of mRNA were confirmed by a correspondent DNA negativity in 8,2% of the samples. This datum should be investigated further in order to establish if the disease was been eradicated. Patients negative by mRNA detection in several consecutive follow-ups could be candidates to stop imatinib therapy. The development of a DNA base technique could be a powerful tool to evaluate the effective presence/absence of leukemic cells. Patient 8 resulted negative at 70 months monitored by RNA and DNA technique could be a candidate to stop the therapy (figure 2). Conclusion: Although the initial characterization of genomic breakpoint sequence is still time consuming, it may provide a patient-specific DNA biomarker that can be used to detect the presence of quiescent leukemic cells otherwise undetectable using a conventional qRT-PCR. The DNA genomic Q-PCR could be a very sensitive and direct technique to detect minimal residual disease in CML patients treated with tyrosine-kinase inhibitors and allogeneic transplantation. We thank AIL Varese and Bergamo. Disclosures: No relevant conflicts of interest to declare.

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked, recessive disorder, with a high mortality rate at early age due to hemorrhages, infections, and lymphoid malignancies. The molecular pathogenesis of the disease is unknown. Carrier females of WAS are clinically and immunologically normal, thus precluding carrier detection by simple laboratory tests. Major advances in molecular genetics have allowed mapping of the WAS gene to the pericentromeric short arm of the X chromosome, and have made carrier detection and prenatal diagnosis feasible by segregation analysis with closely linked polymorphic DNA markers. Furthermore, the observation that carriers of WAS exhibit a unilateral inactivation of the X chromosome in hematopoietic cells has provided a new tool for carrier detection. However, critical interpretation of molecular analysis data is essential to provide accurate genetic counseling to WAS families.

Bone marrow transplantation (BMT) using HLA-partially matched family donors has produced disappointing results (25–30% of long-term survivors) in patients with severe aplastic anemia. We describe two children affected by severe aplastic anemia, not responsive to immunosuppressive therapy, who underwent allogeneic bone marrow transplantation using a HLA-partially matched family donor. Both cases presented 2 first class HLA-antigens (A and B) disparity between donor and recipient. The pretransplant conditioning regimen consisted of cyclophosphamide, thoracoabdominal irradiation, cytosine-arabinoside, and antilymphocyte globulin. As graft versus host disease (GVHD) prophylaxis, Cyclosporine-A was administered at usual dosages for 6 months. A full marrow engraftment was observed in both cases. Only grade I acute GVHD, promptly responsive to corticosteroid therapy, developed with no chronic GVHD. Five months after transplant, both children progressively developed hypertension, renal function impairment, thrombocytopenia, and severe normochromic anemia, with erythropoietin serum levels lower than expected for the haematocrit. After antihypertension treatment and supportive therapy, the clinical picture progressively improved, while treatment with recombinant human erythropoietin completely corrected the long-lasting anemia. The two children are alive and well 28 months after the transplant, with a Karnofsky score of 100% and a normal peripheral blood count. The authors suggest that, once immunosuppressive therapy has failed, BMT from donors other than HLA-identical sibling is a feasible approach in children affected by severe aplastic anemia, not having an HLA-identical donor. © 1993 Wiley-Liss, Inc.

Neuramide, a tissue extract having antiviral action against influenza A virus and immunostimulant action, was analyzed by preparative size-exclusion high-performance liquid chromatography (HPLC) and a low-molecular-weight fraction responsible for the antiviral action was isolated after reversed-phase HPLC. Four fractions having immunostimulant activity were also isolated, as evidenced by their potentiating action in the human lymphocyte proliferation induced by phytohaemagglutinin.

X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis.A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood.A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal.In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

The recent introduction of highly active antiretroviral therapy (HAART) has allowed an aggressive treatment approach in the management of HIV-associated lymphoma (HIV-Ly). Within the Italian Cooperative Group on AIDS and Tumors (GICAT) in 2000 we started a multiinstitutional program of high dose therapy (HDT) and autologous peripheral blood stem cell transplantation (PBSCT) for HIV + patients (pts) with refractory or relapsed lymphoma. CNS lymphoma were excluded. So far, 26 pts entered the study: 10 HD (five 1st relapse, two 2nd relapse, three refractory) and 16 NHL (eight 1st relapse, one 2nd relapse, two partial remission (PR), five refractory). Median age was 38 (28–56); CD4 count 207/cmm (17–506); detectable HIV viremia: 6 pts (23%). Performance status (PS): 0–1, 7 pts (27%); 2, 19 pts (73%). Ann Arbor stage of lymphoma: II, 4 pts (15%); III, 8 pts (31%), IV, 14 pts (54%); B symptoms: 9 pts (35%); bone marrow involvement: 7 pts (27%). Median duration of last remission was 6 months (mo) (1–53). Pts received 2nd line standard dose chemotherapy as debulking treatment. Six pts were refractory and 5 died for disease progression (one is on treatment); 17 of 20 pts with chemosensitive disease successfully mobilized an adequate number of CD34+ after Cyclophosphamide + G-CSF or G-CSF-supported CT. Two pts had early disease progression, and 15 pts received the BEAM regimen and PBSCT, according to the protocol. Engraftment was prompt in all. Pts received HAART during the entire program. Neither treatment-related death nor HIV related death were registered. Fourteen pts achieved complete remission and 1 partial remission. Relapse occurred in 3 (mo +5, +8, +12); 13 are alive and 11 disease-free at 12 mo (2–28) after transplant. The median survival of the entire series (26 pts) from the study entry was 17 mo with 51% of the pts alive after a median f-up of 17 mo. From the analysis of prognostic factor, PS, Ann Arbor stage IV, B symptoms and CD4 count at study entry showed a correlation with survival by univariate, but stage IV disease was the only factor proved to correlate independently with survival on multivariate analysis. The projected overall survival for pts entering the study with stage IV disease (12 pts) or less than stage IV (14 pts) was respectively 90% vs 12% at 2 years (P < 0.001). Our data showed encouraging overall results with an intensive treatment strategy as salvage therapy for HIV-Ly. Ann Arbor stage was the most important predictor of survival. Pts with stage IV disease had poor prognosis, mainly because of chemoresistent disease, suggesting that these pts might benefit from the use of dose intensification earlier in the course of their disease.

Cell-mediated cytotoxicity is considered to play a major role in immune defense and in particular in the killing of virus-infected and neoplastic cells. It appears to have some interesting implications when considering the infectious risk of acute lymphoblastic leukemia (ALL) children during immunosuppressive chemotherapy and the role of self-defense against minimal residual disease. We have studied natural killer (NK) activity and lymphokine-activated killer (LAK) activity in children during and after treatment for ALL. We observed that peripheral blood mononuclear cells in 22 children undergoing maintenance chemotherapy displayed significantly depressed NK activity compared with normal controls even when the proportion of NK cells was normal. LAK activity was also considered in 43 ALL children during and after maintenance chemotherapy. We observed that LAK activity was persistently comparable with that of normal controls. It seems definite that NK activity impairment is transient and is completely restored in ALL children a few months after chemotherapy has been successfully completed. The evidence that LAK activity is not impaired in ALL children may have some implications in view of a possible immunomodulatory approach in the presence of refractory disease.

We studied interleukin 1 (IL-1) and interleukin 2 (IL-2) production in unstimulated and stimulated cultures from 27 young diabetic patients and 21 age-matched healthy subjects. In unstimulated cultures monocytes from newly diagnosed patients produced significantly higher levels of IL-1 than controls. In lipopolysaccharide (LPS)-stimulated cultures, IL-1 production in patients with fresh and long-standing diabetes was no different from that of controls. IL-2 production was low or absent in unstimulated cultures from insulin-dependent diabetes mellitus (IDDM) patients and controls. In phytohaemagglutinin (PHA)-stimulated cultures both patient groups produced significantly less IL-2 than controls. No correlation was observed between IL-1, IL-2 production and HbA1 levels or the presence of HLA-DR3 or DR4. Our data on “spontaneous” IL-1 production support the hypothesis that monocytes from some newly diagnosed IDDM patients may circulate in a “preactivated” state. The low levels of IL-2 might be explained by an abnormal consumption or by the presence of increased soluble IL-2 receptor levels or by a serum factor which interferes with IL-2 production. Alternatively, it may be a genetically determined trait.

Expression of CD45R0 antigen has been evaluated on the surface of T lymphocyte subsets obtained from cord blood and on peripheral blood lymphocytes (PBL) from both term and preterm neonates. In some experiments, expression of the same antigen has also been evaluated after in vitro activation of cord blood lymphocytes (CBL) with phytohemagglutinin (PHA) or in mixed lymphocyte culture. The CD45R0 molecule was found to be present on a significantly lower percentage of CD3+, CD4+ and CD8+ CBL as compared with the same subsets evaluated in adult individuals. Moreover, at variance with adult PBL, the great majority of CBL displayed a low level of fluorescence when stained with UCHL1 (CD45R0-specific monoclonal antibody), and both PHA and allogeneic stimuli were able to strongly increase the expression of the CD45R0 molecule on CBL. The percentage of CD45R0+ PBL obtained from both term and preterm neonates on the 1st day of life was comparable to that of CBL, however the expression of this molecule increases remarkably within a few days in the majority of term neonates, while it seems to take more time for the antigen to be fully expressed in preterm infants.

We report on a thrombocytopenic female belonging to a pedigree with the Wiskott-Aldrich syndrome (WAS). Restriction fragment length polymorphism (RFLP) analysis with probe M27 beta, closely linked to the WAS gene, demonstrated that she is a carrier of WAS. Both small-sized and normal-sized platelets were present, suggesting that, unlike the vast majority of WAS carriers, she does not manifest nonrandom X-chromosome inactivation in the thrombopoietic cell lineage. Study of X-chromosome inactivation by means of RFLP and methylation analysis demonstrated that the pattern of X-chromosome inactivation was nonrandom in T lymphocytes, but random in granulocytes. While this is the first complete report on the occurrence of thrombocytopenia in a carrier female of WAS as the result of atypical lyonization, it also suggests that expression of the WAS gene occurs at (or extends up to) a later stage than the multipotent stem cell along the hematopoietic differentiation pathway.