Assessment of genomic instability and proliferation index in cultured lymphocytes of patients with Down syndrome, congenital anomalies and aplastic anaemia.

One hundred and fifteen cases [Down Syndrome (DS) n = 75, Multiple Congenital Anomalies (MCA) n = 15 and Aplastic Anaemia (AA) n = 25], with respect to their nature of predisposition to cancer, were selected for clinical, cytogenetic and cyto-molecular studies to understand the severity of genomic instability according to the nature of the different diseases. Cytogenetic studies included chromosomal aberration (CA) assays and cytokinesis block micronucleus cytome (CBMN-Cyt) assays. In DS, MCA and AA, average frequencies of nuclear anomalies (NA) were 0.015 ±â€¯0.0006, 0.021 ±â€¯0.00123, 0.031 ±â€¯0.00098, respectively and CA were 0.107 ±â€¯0.003, 0.105 ±â€¯0.008, 0.158 ±â€¯0.006, respectively per metaphase. The extent of genomic instability in patients analysed by CBMN-Cyt assays and CA assays was statistically significant in all groups. Comparatively decreased cytokinesis block proliferation index (CBPI) observed in AA patients of 1.59 ±â€¯0.05, support the assumption that decreased levels of CBPI indicate increased genomic damage. Furthermore, we performed peptide nucleic acid fluorescence in situ hybridisation (PNA FISH) analysis to understand the mechanisms behind genomic instability and telomere dysfunction. PNA FISH showed increased frequencies of telomere signal free ends (0.98 ±â€¯0.13) in individuals with higher genomic instability. Therefore, the results demonstrate that increased chromosomal instability along with higher telomere attrition or loss may initiate gross DNA damage and leads to chromosomal instability, which is an important mechanism for triggering genomic instability - an important hallmark of cancer cells.

Manejo infectológico de la aplasia medular severa en pacientes pediátricos / Prevention and management of infections in children with severe aplastic anemia

La aplasia medular es una enfermedad poco frecuente en pediatría, siendo el tratamiento de elección en las formas severas el trasplante de células progenitoras hematopoyéticas (TCPH). Gracias a los avances en TCPH, los nuevos tratamientos inmunosupresores y al adecuado tratamiento de sostén, se ha logrado en las últimas décadas una franca disminución de la mortalidad asociada a esta patología. Es por ello que uno de los principales desafíos consiste en prevenir la aparición de infecciones asociadas a la neutropenia severa y prolongada que padecen estos pacientes, siendo actualmente las infecciones bacterianas y fúngicas una de las principales causas de morbimortalidad. Por otra parte, la mayoría de las guías de manejo y tratamiento de sostén se basan en recomendaciones de expertos, siendo la evidencia escasa, más aún en pediatría. Gran parte de las recomendaciones de tratamiento empírico se basan en guías de neutropenia febril de pacientes hemato-oncológicos. A su vez, existe gran variabilidad, de acuerdo al centro de atención, en cuanto al uso de antimicrobianos para profilaxis primaria, debiéndose tener en cuenta la mayor propensión a presentar infecciones invasivas por hongos filamentosos y, en el caso de pacientes con linfopenia marcada, de enfermedad por P jirovecii a la hora de valorar la indicación de profilaxis de estos pacientes.Se detallarán a continuación las principales recomendaciones sobre manejo de prevención de infecciones y tratamiento precoz de pacientes pediátricos con aplasia medular severa.

Aplastic anemia (AA) is a rare condition in children. Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for severe idiopatic AA. Survival in severe AA has markedly improved in the past decades due to advances in HSCT, immunosuppressive and biologic drugs, as well as supportive care. Since bacterial and fungal infections are one of the principal causes of morbidity and mortality in AA, one of the main challenges is to prevent the appearance of infections associated with severe and prolonged neutropenia. Most guidelines of treatment and prophylaxis are based on expert recommendations. Given the lack of controlled studies in children with AA, most recommendations of empiric treatment rely on guidelines for febrile-neutropenia management in hemato-oncologycal patients. A great variability exists in the use of antimicrobials for primary prophylaxis among different institutions. Due to the fact that patients with severe and prolonged AA present high incidence of filamentous fungal infections, an adecuate antifungal prophylaxis is recommended. In the case of severe lymphopenia, prophylaxis against P jirovecii should also be considered. Recommendations in prophylaxis and early treatment of infections in severe pediatric AA are detailed

High-dose cyclophosphamide compared with antithymocyte globulin for treatment of acquired severe aplastic anemia.

A modified regimen of high-dose cyclophosphamide (CTX) plus cyclosporine (CsA) was adopted for patients with severe or very severe aplastic anemia, and the effectiveness was compared with a regimen of antithymocyte globulin (ATG) plus CsA. A total of 121 patients enrolled in this study received either CTX plus CsA (CTX group, 48 cases) or ATG plus CsA (ATG group, 73 cases). The early death rate was 4.2% in the CTX group and 8.2% in the ATG group, showing no significant difference (p = 0.312). The total response rate in the CTX and ATG groups was 54.2% and 57.5% at 3 months, 64.6% and 72.6% at 6 months, and 72.9% and 78.1% at 12 months, respectively (p > 0.05). The overall 5-year survival rate was 81.2% and 80.7%, and the event-free survival rate was 68.2% and 67.3% in the CTX and ATG groups, respectively (p > 0.05). The total medical cost of the CTX group was 54.8% less than that of ATG regimen (p = 0.000). In summary, treatment of severe or very severe aplastic anemia with CTX plus CsA has effectiveness that is comparable to a conventional regimen and less costly.

[Comparative assessment of myelocariocytes cell cycle in patient with hemodepressions]. / Sravnitel'naia otsenka kletochnogo tsikla mielokariotsitov bol'nykh gemodepressiiami.

The proliferative activity of bone marrow cells was studied by flow cytofluorometry in 8 patients with aplastic anemia (AA), 6 with myelodyspastic syndrome (MDS), and 26 normal subjects aged 21-40 years. The myelokaryocyte proliferation was inhibited at the G0-1 stages of cell cycle, the number of cells in the S phase was decreased, and the coefficient of variations cV of DNA cytofluorometry values were increased in AA patients in comparison with the norm. By contrast, in MDS patients the number of cells in the G0-1 phase was decreased and that of cells in the S phase increased in comparison with the norm and with AA patients; cV was increased. These differences in the cell cycle characteristics are suggested to be used as a differential diagnostic criterion in AA and MDS.

The assessment of the hematopoietic reservoir after immunosuppressive therapy or bone marrow transplantation in severe aplastic anemia.

We investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P < .00001) both for IS and BMT patients. Their number did not improve with time and was not affected by transplant or treatment-related variables. When IS patients were compared with BMT we found comparable numbers of CFU-GEMM (P = .8) and LTC-IC (P = .9), but lower numbers of BFU-E and CFU-GM (P = .05 and P = .004, respectively), suggestive of a persistent suppressive mechanism. These data indicate that LTC-IC numbers are severely reduced in BMT and IS patients, contradicting the common belief that the former are fully reconstituted as compared with the latter. In addition, the number of mature cells and committed progenitors does not seem to reflect the real size of the hematopoietic reservoir and few stem cells may be sufficient to guarantee normal hematopoiesis long term.

Aplastic anemia: assessment of myeloid progenitor cells in the bone marrow and blood provides prognostic information.

15 patients with aplastic anemia were prospectively followed after having measurements of myeloid progenitor cells in bone marrow and blood. Treatment included androgens, low or high dose steroids and standardized supportive care. The median length of survival was 5.8 months. When patients were grouped according to the numbers of myeloid progenitor cells present in their blood and bone marrow, we found that the survival length of aplastic patients with higher progenitor cell numbers was prolonged when compared to that of patients with lower numbers of these cells. The prognostic information obtained from such in vitro cultures was particularly indicative when the patients were grouped according to 'growth' and 'no growth': the absence of colony-forming myeloid stem cells was associated with survival times significantly shorter than those of patients whose cells maintained their colony-forming capacity. Besides initial numbers of myeloid progenitor cells, only initial numbers of granulocytes were related to survival length. Thus, the measurement of myeloid progenitor cells in bone marrow and blood can be of prognostic value in patients with aplastic anemia.