Mutational analysis of the cell cycle inhibitor Kip1/p27 in childhood leukemia.

BACKGROUND: Cyclin-dependent kinases (CDKs) and cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. Kip1/p27 is the main cyclin-dependent kinase inhibitor, which arrests cell division inhibiting G1-S transition. Kip1/p27 seems to play a critical role in the pathogenesis of several human malignancies and its lower expression has been shown to correlate with a poor prognosis in adult solid tumors. METHODS: Bone marrow blasts from 49 children with leukemia, 37 acute lymphoblastic leukemia (ALL), and 12 acute myeloid leukemia (AML) were studied. Exon 3 of Kip1/p27 was amplified using the polymerase chain reaction technique (PCR). Single strand conformational polymorphism and heterodouplex analysis were performed to detect DNA sequence with altered conformations and were subsequently sequenced to document mutations. RESULTS: Mutations in Kip1/p27 gene were detected in 2 out of 3 T-ALL, 6 out of 12 AML patients, and only 1 out of 34 B lineage ALL cases. Although the patient groups are small, a highly significant relation of the mutation status with the type of leukemia (P = 0.0037) and the risk group according to treatment protocols (P = 0.00021) was estimated. A statistically significant difference in the white blood count was observed (P = 0.019) between the mutated and non-mutated patient groups although no statistically significant association of the mutation status with the hemoglobin and platelets values, karyotype, age, sex, disease progression, and outcome was determined. CONCLUSIONS: Based upon these results, the Kip1/p27 mutations should be considered for further prospective testing as an additional parameter for risk stratification and treatment of childhood leukemia.

Adhesion molecules, endogenous granulocyte colony-stimulating factor levels and replating capacity of progenitors in autoimmune neutropenia of childhood.

AIM: To investigate the role of granulocyte colony-stimulating factor (G-CSF) and adhesion molecules and the response of bone marrow to peripheral cytopenia in autoimmune neutropenia of childhood (AIN). METHODS: Thirty-five children with AIN, 25 with acute leukaemia in remission, 10 of whom developed chemotherapy-associated neutropenia, and 28 non-neutropenic age-matched children were studied. The methods included haemopoietic progenitor cells' colony growth, replating of colony-forming unit-granulocyte macrophage (CFU-GM) of the 7th day and ELISA for the detection of serum levels of cytokines and adhesion molecules. RESULTS: In cases of severe autoimmune neutropenia, haemopoietic progenitors showed increased proliferative capacity compared to the control group (p = 0.03). Intercellular adhesion molecule-1 (ICAM-1), E-selectin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels inversely correlated with neutrophil counts (r = -0.8, p < 0.001 for ICAM-1, r = -0.5, p = 0.04 for E-selectin, r = -0.58, p = 0.01 for TNF-alpha, r = -0.62, p = 0.04 for IL-1beta). Serum ICAM-1, TNF-alpha and IL-1beta levels correlated positively with G-CSF levels (r = 0.47, p = 0.03 for ICAM-1, r = 0.65, p = 0.01 for TNF-alpha, r = 0.67, p = 0.04 for IL-1beta). Serum G-CSF levels were widely distributed and did not correlate with neutrophil counts (r = -0.44, p = 0.09). In secondary neutropenias the respective levels were lower than those in autoimmune neutropenia. CONCLUSIONS: Haemopoietic progenitors show increased proliferative capacity in cases of severe autoimmune neutropenia. G-CSF seems to act as an inducer of endothelium activation. The degree of neutropenia correlates with serum ICAM-1, E-selectin, TNF-alpha and IL-1beta levels, indicating the existence of an activated endothelium and presumably of a latent, low-grade, inflammatory process in severe autoimmune neutropenia.

Mutations of retinoblastoma gene (Rb-1) as a prognostic factor in children with acute leukemia and neuroblastoma.

Rb-1 is a tumor suppressor gene encoding for a nuclear phosphoprotein acting as a cell cycle regulator, normally expressed in hematopoietic cells and more often inactivated by point mutations with predominance for exons 20-24. The aim of this study is to correlate the retinoblastoma-1 (Rb-1) gene mutations with the prognosis and progression of childhood acute leukemia and neuroblastoma. Bone marrow slides from 26 children with leukemia (18 acute lymphoblastic leukemia [ALL] and 8 acute myeloid leukemia [AML]) and 4 children with neuroblastoma were studied. Exons 20, 21, and 22 were amplified using the polymerase chain reaction technique. Single strand conformational polymorphism (SSCP) and heterodoublex analysis were performed to detect mutations. In ALL cases, two samples in exon 20 (11.11%), one in exon 21 (5.56%), and four in exon 22 (22.22%) had altered conformation. All but one of these cases were classified as high-risk leukemia patients who either relapsed or never achieved remission. Two of the AML cases who did not achieve remission and one of the neuroblastoma cases with concomitant bone marrow infiltration had altered conformation as well. The SSCP and heterodoublex analysis showed that all but one who did not belong to the high-risk group had the same altered conformation. These data suggest that Rb-1 gene could possibly be used as an independent prognostic factor for the acute leukemia of childhood and result in the intensification of chemotherapy. In solid tumors with bone marrow involvement it could play a role as a marker of aggressive disease.

Soluble transferrin receptor levels and soluble transferrin receptor/log ferritin index in the evaluation of erythropoietic status in childhood infections and malignancy.

UNLABELLED: Soluble transferrin receptor (sTfR) is a new diagnostic tool for determining iron status and erythropoietic activity. The increased concentrations of sTfR in patients with iron deficiency reflect the hyperplasia of erythroid precursors. The objective of this study was to evaluate sTfR and sTfR/log ferritin index (sTfR-F) values in healthy children (n = 64), full-term neonates (n = 18), children with iron deficiency (n = 16), hemolytic anemia (n = 7), beta-thalassemia traits (n = 18), respiratory infections (n = 41) and malignancies (n = 13), and to compare these parameters for the different subgroups with those of healthy children. The sTfR levels were increased in children with iron deficiency in the same way as in adults (p < 0.0001) and in cases of increased erythropoietic activity, such as during the neonatal period (p < 0.0001), and of hemolytic anemias (p = 0.006). The index was significantly increased in iron deficiency (p < 0.0001) and decreased in neonates (p = 0.011). Children carriers of beta-thalassemia were found to have increased sTfR values (p = 0.015), but not sTfR/log ferritin index (p = 0.491), a finding suggesting that use of both parameters is necessary for distinguishing between those with and those without iron deficiency. In children with upper respiratory infection, the sTfR levels were close to normal, while the index was found to be low. In order to evaluate the iron status in infections, we further subdivided the children into two groups according to the value of ferritin, with the cut-off point at 35 microg/L. Children with ferritin level above 35 microg/L experienced normal sTfR levels but very low index, a finding which could enable the use of these two parameters for distinguishing patients with infection without concomitant iron deficiency. In the group of malignancies under chemotherapy both indices were low (p = 0.005, p < 0.0001) mainly due to myelosuppression. CONCLUSION: The interpretation of both sTfR and sTfR/log ferritin index is useful in the evaluation of iron status and erythropoietic activity, especially in children with heterozygous beta-thalassemia, infection and malignancies.