Francesca Scuderi

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children.

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 105 PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 105 PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached ⩾20 000 copies per 105 PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as ⩾20 000 geq per 105 PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.

Unrelated HSCT in an adolescent affected by congenital erythropoietic porphyria

Abstract: CEP is a rare inborn error of porphyrin–heme synthesis. Clinical manifestations can range from mild to severe and include erythrodontia, reddish‐colored urine, and hemolytic anemia that can be mild or severe and may result in splenomegaly. Completely avoiding exposure to the sun is crucial. Attempts to reduce erythropoiesis and to lower circulating porphyrin levels by means of erythrocyte transfusions have been successful in reducing the expression of the disease. However, the complications of a chronic transfusion regimen are potentially severe. Successful bone marrow transplantation has been reported in CEP. We report a case of successful bone marrow transplantation and prolonged follow‐up in an adolescent CEP patient.

Three consecutive related bone marrow transplants for juvenile myelomonocytic leukaemia

Abstract:  Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is the only cure for juvenile myelomonocytic leukaemia (JMML), but relapse remains the major cause of failure. A second transplant may be considered a way to induce the graft vs. leukaemia effect in patients who relapse after their first HSCT. We describe a 7‐month‐old girl with JMML who relapsed after a first, related allo‐HSCT, and who again relapsed 8 months after the second transplant, despite discontinuation of immusuppressive therapy. She underwent a third allogeneic transplant from another related donor. At the time of this report the patient is in complete remission 26 months after the third transplant. We suggest that a third allo‐HSCT may be taken into consideration for JMML patients who experience relapse, even after two previous transplants.

Monosomal complex karyotype in pediatric mixed phenotype acute leukemia

We report on a pediatric case of mixed phenotype acute leukemia with myeloid and T-lymphoid differentiation, a single myeloblastic cell population, and a monosomal complex karyotype. The patient, a 5-year-old girl, responded to acute myeloid leukemia-oriented therapy that was decided based on the morphological appearance of blast cells. In this study, we analyzed the patients peculiar chromosomal abnormalities, as evaluated by array comparative genomic hybridization in combination with multicolor fluorescence in situ hybridization and cytogenetic analyses.

Clinical and morphological practices in the diagnosis of transplant-associated microangiopathy: a study on behalf of Transplant Complications Working Party of the EBMT

Transplant-associated thrombotic microangiopathy (TA-TMA) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). This study evaluated clinical and morphological practices of TA-TMA diagnosis in EBMT centers. Two questionnaires, one for transplant physician and one for morphologist, and also a set of electronic blood slides from 10 patients with TA-TMA and 10 control patients with various erythrocyte abnormalities, were implemented for evaluation. Seventeen EBMT centers participated in the study. Regarding criteria used for TA-TMA diagnosis, centers reported as follows: 41% of centers used the International Working Group (IWG) criteria, 41% used “overall TA-TMA” criteria and 18% used physician’s decision. The threshold of schistocytes to establish TA-TMA diagnosis in the participating centers was significantly associated with morphological results of test cases evaluations (p = 0.002). The mean number of schistocytes reported from blood slide analyses were 4.3 ± 4.5% for TA-TMA cases (range 0–19.6%, coefficient of variation (CV) 0.7) and 1.3 ± 1.6% for control cases (range 0–8.3%, CV 0.8). Half of the centers reported schistocyte levels below 4% for 7/10 TA-TMA cases. The intracenter variability was low, indicating differences in the institutional practices of morphological evaluation. In conclusion, the survey identified the need for the standardization of TA-TMA morphological diagnosis.

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages’ maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment.

EFFICIENCY OF RETROVIRAL TRANSDUCTION OF CD 34+ CELLS FROM UMBILICAL CORD BLOOD DEPENDS ON GROWTH FACTOR STIMULATION. 134

The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of stable transduction. The great potentiality of umbilical cord blood CD34+ cells prompted us to evaluate which factor(s) play a major role(s) in influencing the transduction efficiency of retroviral vectors. CD34+ cells, isolated with an immunomagnetic device (MiniMacs, Miltenyi Biotech, Germany), 90-95% pure as assessed by cytofluorimetric analysis, were transduced with the LXSN retroviral vector carrying the neomicine resistance gene. Infection was made with virus-containing supernatant of Am12 env+ packaging cell line, having a titer of 2×106 PFU/ml. All the infections were performed using a multiplicity of infection of 10 PFU/cell. Multiple or single infections were performed with the following schedules: i) up to 6 infection lasting one hour over 3 days; ii) a 72 hr infections substituting fresh viral supernatant and cytokines every 24 hrs; iii) a single 72 hr infection. In most experiments, CD34+ cells were preincubated with different cytokine cocktails for 24 or 48 hrs before infection. Efficiency of transduction was evaluated both by clonogenic assays and semiquantitative PCR analysis performed either immediately or after 7 day expansion in the presence of cytokines and G-418. G-418-resistant CFU-E, BFU-E and CFU-GM colonies were scored after 7 and 14 days. We provided evidence that the efficiency of transduction was mainly dependent on cell preincubation with growth factors rather than on the number of infections. Precisely, infection performed on freshly isolated CD34+ was almost ineffective (0.4%) while 48 hr preincubation in the presence of SCF*+IL-6+IL-3 allowed to reach a level of transduction up to 40% regardeless of the number of infections. In this respect it is noteworthy that a single infection, lasting as long as a multiple infection course gave identical results. In conclusion the results obtained suggest that high efficiency of transduction of umbilical cord blood CD34+ cells can be achieved in liquid, stroma free, cultures.

HUMAN UMBILICAL CORD BLOOD CD34+ CELL EXPANSION IN THE PRESENCE OF FLT-LIGAND(FL). 133

Human umbilical cord blood (HUCB) is an important source of progenitor cells which are able to reconstitute hematopoiesis. The in vitro expansion of isolated CD34 positive cells should provide a sufficient number of hematopoietic cells for transplants in large reciplents and would increase vector-mediated gene transfer efficiency. In vitro expansion of progenitor cells requires the identification of the cytokine combinations which result in the greatest increase of committed cells, without exhausting the early progenitor pool. In this study we tested the effect of three colony stimulating-factor (CSF) combinations [SCF*+IL-6(CSF2); SCF*+IL-6+FL (CSF3); SCF*+IL-6+FL+IL-3 (CSF4)] on CD34+ cells isolated(MiniMACS, Miltenyi Biotec, Germany) from HUCB. CD34+ cells, ≥97% pure in all samples, were ≥97% CD33- (range 97-99%) and ≥97% CD38+ (range 97-99.8%). The absolute number of total cells and cell subsets, evaluated after 7 and 11 days of liquid cultures, showed the following median fold increase:Table

233 PREFERENTIAL USAGE OF INCOMPLETE REARRANGEMENTS IN PRECURSOR B-ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL)

Southern blot analysis of T-cell receptor TCR genes rearrangements is usefull for diagnostic studies on the clonality of lymphoproliferative diseases and for a better assigment of the cell lineage of leukemic cells.We analyzed 26 cases of precursor B-ALL in aim to detect the presence of TCR-δ gene rearrangements. In fact lineage crossover of gene rearrangements have been osserved quite frequently in neoplastic cells. We found 6/26 patients in germline configuration, 5/26 deleted and 15/26 with at least one allele reananged. Taking into account previous studies (Breit et al. 1993) we were able to define exactly in 8/26 cases the gene segments involved in the rearrangement; DNA extracted from bone marrow samples was digested with Eco RI, Hind III and Bgl II restriction enzymes. The hybridization with TCRJδ 1 probe showed that the majority of the rearrangements were Vδ2Dδ3 and Dδ2Dδ3.It is possible that the crossover rearrangements reflect the earliest steps of recombinational event in rearranging antigen-receptor gene loci. Besides the Vδ2Dδ3 rearrangement, the most frequently observed in precursor B-ALL, may be the earliest recombinational event in TCR-δ gene during normal T-cell ontogeny. Amplification by PCR technique, using specific primers revealed the presence of a specific band of leukemic clone and in one case we found the sequence of junctional region between Vδ2 and Dδ3 gene segments. In this particular case, the insertion of 6 nucleotides creates a unique clonal marker of leukemic cells that can be used for the detection of minimal residual disease (MRD).