B-NHL Cases in a Tertiary Pediatric Hematology-Oncology Department: A 20-Year Retrospective Cohort Study.

Non-Hodgkin lymphoma (NHL) is among the five most common pediatric cancer diagnoses in children and adolescents and consists of a heterogeneous group of lymphoid tissue malignancies -with B-cell-derived NHL accounting for nearly 80% of cases. Novel and high-throughput diagnostic tools have significantly increased our understanding of B-NHL biology and molecular pathogenesis, leading to new NHL classifications and treatment options. This retrospective cohort study investigated 17 cases of both mature B-cell NHL (Burkitt lymphoma or BL; Diffuse large B-cell lymphoma or DLBCL; Primary mediastinal large B-cell lymphoma or PMBCL; Follicular lymphoma or FL) and immature B-cell progenitor NHL (B-lymphoblastic lymphoma or BLL) that were treated in a tertiary Pediatric Hematology-Oncology Department during the last 20 years. Modern NHL protocols for children, adolescents, and young adults, along with the addition of rituximab, are safe and efficient (100% overall survival; one relapse). Elevated ESR was more prevalent than elevated LDH. Analyses have focused on immune reconstitution (grade ≥3 infections, lymphocyte and immunoglobulin levels recovery) and body-mass-index changes post-treatment, late effects (in 53% of patients), and the presence of histology markers BCL2, BCL6, CD30, cMYC, and Ki-67%. One patient was diagnosed with a second malignant neoplasm (papillary thyroid cancer).

The Diverse Genomic Landscape of Diamond-Blackfan Anemia: Two Novel Variants and a Mini-Review.

Diamond-Blackfan anemia (DBA) is a ribosomopathy characterized by bone marrow erythroid hypoplasia, which typically presents with severe anemia within the first months of life. DBA is typically attributed to a heterozygous mutation in a ribosomal protein (RP) gene along with a defect in the ribosomal RNA (rRNA) maturation or levels. Besides classic DBA, DBA-like disease has been described with variations in 16 genes (primarily in GATA1, followed by ADA2 alias CECR1, HEATR3, and TSR2). To date, more than a thousand variants have been reported in RP genes. Splice variants represent 6% of identifiable genetic defects in DBA, while their prevalence is 14.3% when focusing on pathogenic and likely pathogenic (P/LP) variants, thus highlighting the impact of such alterations in RP translation and, subsequently, in ribosome levels. We hereby present two cases with novel pathogenic splice variants in RPS17 and RPS26. Associations of DBA-related variants with specific phenotypic features and malignancies and the molecular consequences of pathogenic variations for each DBA-related gene are discussed. The determinants of the spontaneous remission, cancer development, variable expression of the same variants between families, and selectivity of RP defects towards the erythroid lineage remain to be elucidated.

Biological Features of Bone Marrow Mesenchymal Stromal Cells in Childhood Acute Lymphoblastic Leukemia.

OBJECTIVE: Mesenchymal stromal cells (MSCs) have a supportive role in hematopoiesis and as components of the bone marrow (BM) microenvironment may present alterations during acute lymphoblastic leukemia (ALL) and be affected by chemotherapeutic agents. We examined the biological and functional characteristics of MSCs in ALL diagnosis and treatment and their effect on MSC qualitative properties. MATERIALS AND METHODS: Immunophenotypic characterization, evaluation of clonogenicity, and proliferative capacity were measured. Apoptotic features, cell-cycle analysis, and stromal cell-derived factor 1α and angiopoietin-1 levels in MSC supernatant at diagnosis and in different phases of treatment were assessed. Chemotherapy was administered according to the Berlin-Frankfurt-Munster-2000 protocol. BM samples from children with solid tumors without BM involvement were used as the control group. RESULTS: The morphology, the immunophenotypic profile, and the apoptotic characteristics of the MSCs were not affected by leukemia. The secretion of factors involved in the trafficking of hematopoietic cells in the BM seems to be upregulated at diagnosis in comparison to the treatment phases. MSCs are influenced by the disease in terms of their functional characteristics such as clonogenicity and proliferation rate. These effects cease as soon as treatment is initiated. Chemotherapy does not seem to exert any effect on any of the MSC features examined. CONCLUSION: MSCs from children with ALL are affected by their interaction with the leukemic environment, but this phenomenon ceases upon treatment initiation, while no effect is observed by chemotherapy itself.

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells.

The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood.

BACKGROUND AIMS: Umbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols. METHODS: We assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133(+) subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control. RESULTS: The mononuclear cell fraction expressed 0.53±0.06% CD133. The corresponding value for CD34 was 1.64±0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93±1.25% CD34 while, after the corresponding process, CD133(+) expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133(+) yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC). CONCLUSIONS: CD34 isolation by the immunomagnetic method results in highly pure CD34(+) population, while the efficient and reproducible yield of a pure CD133(+) population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133(+) cells.

Ciprofloxacin decreases survival in HT-29 cells via the induction of TGF-beta1 secretion and enhances the anti-proliferative effect of 5-fluorouracil.

BACKGROUND AND PURPOSE: Fluoroquinolones are potent anti-microbial agents with multiple effects on host cells and tissues. Previous studies have highlighted their pro-apoptotic effect on human cancer cells and an immunoregulatory role in animal models of inflammatory bowel disease. We examined the effect of ciprofloxacin on proliferation, cell cycle and apoptosis of HT-29 cells, a human colonic epithelial cell line sensitive to transforming growth factor (TGF)-beta1-mediated growth inhibition and its role in TGF-beta1 production. We also examined the effect of ciprofloxacin on proliferation of HT-29 cells in combination with 5-fluorouracil (5-FU), a well-established pro-apoptotic agent. EXPERIMENTAL APPROACH: Using subconfluent cultures of HT-29 and Caco-2 cells, we studied the effect of ciprofloxacin, TGF-beta1 and 5-FU on proliferation, apoptosis, necrosis and cell cycle. The effect of ciprofloxacin on TGF-beta1 mRNA expression and production was studied in RNA extracts and cell culture supernatants respectively, using confluent cultures. KEY RESULTS: Ciprofloxacin decreased proliferation of HT-29 cells in a concentration- and time-dependent manner. This was mediated by accumulation of HT-29 cells into the S-phase but without any effect on apoptosis or necrosis. Additionally, ciprofloxacin enhanced the antiproliferative effect of 5-FU. Interestingly, ciprofloxacin was found to up-regulate TGF-beta1 production by HT-29 cells and its anti-proliferative effect was abolished when TGF-beta1 was blocked. Confirming this mechanism further, ciprofloxacin had no effect on Caco-2, a human colonic epithelial cell line that lacks functional TGF-beta1 receptors. CONCLUSIONS AND IMPLICATIONS: We demonstrate a novel anti-proliferative and immunoregulatory effect of ciprofloxacin on human intestinal epithelial cells mediated via TGF-beta1.

Biologic characteristics of mesenchymal stromal cells and their clinical applications in pediatric patients.

In the past few years, intensive research in the understanding of the biologic characteristics of the mesenchymal stromal cells has already led to some early clinical applications. The aim of this review is to summarize the latest information from basic science advances and the outcome of their use in clinical practice with a particular focus in pediatric patients. The minimum criteria required to identify mesenchymal stromal cells, their immunosuppressive-nonimmunogenic properties and their attribution in the treatment of graft-versus-host disease, in the acceleration of hematopoietic recovery, in tissue repair/tissue engineering and in the treatment of selected inherited disorders are discussed. Appropriate preclinical models, completion of ongoing and development of new clinical trials will establish the role of these cells in the treatment of both adult and pediatric patients.