Bone marrow CD34+ molecular chimerism as an early predictor of relapse after allogeneic stem cell transplantation in patients with acute myeloid leukemia.

Background: Minimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients. Methods: We retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation. Results: The cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1st month (p=0.008 and p<0.001) and at 3rd month (p<0.001 for both). By combining chimerism and WT1 at 3rd month, 13 patients with chimerism < 97.5% or WT1 > 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213. Conclusions: Our results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1st and 3rd month) are useful MRD markers. When considered together at 3rd month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1. Further studies are necessary to confirm this preliminary observation.

CD34+ Stem Cell Selection and CD3+ T Cell Add-Back from Matched Unrelated Adult Donors in Children with Primary Immunodeficiencies and Hematological Diseases.

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.

Maternal T-cell engraftment impedes with diagnosis of a SCID-ADA patient.

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.

Diagnosis, Treatment and Long-Term Follow Up of Patients with ADA Deficiency: a Single-Center Experience.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The data obtained in our single center are comparable with those that have been reported in multicenter surveys.

Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia.

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.

Long-lasting production of new T and B cells and T-cell repertoire diversity in patients with primary immunodeficiency who had undergone stem cell transplantation: a single-centre experience.

Levels of Kappa-deleting recombination excision circles (KRECs), T-cell receptor excision circles (TRECs), and T-cell repertoire diversity were evaluated in 1038 samples of 124 children with primary immunodeficiency, of whom 102 (54 with severe combined immunodeficiency and 48 with other types of immunodeficiency) underwent hematopoietic stem cell transplantation. Twenty-two not transplanted patients with primary immunodeficiency were used as controls. Only data of patients from whom at least five samples were sent to the clinical laboratory for routine monitoring of lymphocyte reconstitutions were included in the analysis. The mean time of the follow-up was 8 years. The long-lasting posttransplantation kinetics of KREC and TREC production occurred similarly in patients with severe combined immunodeficiency and with other types of immunodeficiency and, in both groups, the T-cell reconstitution was more efficient than in nontransplanted children. Although thymic output decreased in older transplanted patients, the degree of T-cell repertoire diversity, after an initial increase, remained stable during the observation period. However, the presence of graft-versus-host disease and ablative conditioning seemed to play a role in the time-related shaping of T-cell repertoire. Overall, our data suggest that long-term B- and T-cell reconstitution was equally achieved in children with severe combined immunodeficiency and with other types of primary immunodeficiency.