DNA Methylation Adds Prognostic Value to Minimal Residual Disease Status in Pediatric T-Cell Acute Lymphoblastic Leukemia.

BACKGROUND: Despite increased knowledge about genetic aberrations in pediatric T-cell acute lymphoblastic leukemia (T-ALL), no clinically feasible treatment-stratifying marker exists at diagnosis. Instead patients are enrolled in intensive induction therapies with substantial side effects. In modern protocols, therapy response is monitored by minimal residual disease (MRD) analysis and used for postinduction risk group stratification. DNA methylation profiling is a candidate for subtype discrimination at diagnosis and we investigated its role as a prognostic marker in pediatric T-ALL. PROCEDURE: Sixty-five diagnostic T-ALL samples from Nordic pediatric patients treated according to the Nordic Society of Pediatric Hematology and Oncology ALL 2008 (NOPHO ALL 2008) protocol were analyzed by HumMeth450K genome wide DNA methylation arrays. Methylation status was analyzed in relation to clinical data and early T-cell precursor (ETP) phenotype. RESULTS: Two distinct CpG island methylator phenotype (CIMP) groups were identified. Patients with a CIMP-negative profile had an inferior response to treatment compared to CIMP-positive patients (3-year cumulative incidence of relapse (CIR3y ) rate: 29% vs. 6%, P = 0.01). Most importantly, CIMP classification at diagnosis allowed subgrouping of high-risk T-ALL patients (MRD ≥0.1% at day 29) into two groups with significant differences in outcome (CIR3y rates: CIMP negative 50% vs. CIMP positive 12%; P = 0.02). These groups did not differ regarding ETP phenotype, but the CIMP-negative group was younger (P = 0.02) and had higher white blood cell count at diagnosis (P = 0.004) compared with the CIMP-positive group. CONCLUSIONS: CIMP classification at diagnosis in combination with MRD during induction therapy is a strong candidate for further risk classification and could confer important information in treatment decision making.

Lower hemoglobin with lower ferritin: It is not just a question of anemia.

AIM: To study the association between blood hemoglobin concentration (b-hemoglobin) and serum ferritin concentration (s-ferritin) in an ambulant patient population without inflammation and with normal kidney function. METHODS: In ambulant, adult patients with normal values of s-CRP and s-creatinine, median b-hemoglobin and the fraction with anemia was compared in groups with lower s-ferritin from a level of 100 µg/L. The 10, 50 and 90 percentiles of b-hemoglobin were modelled as functions of s-ferritin using quantile regression. RESULTS: Among 3206 women the entire b-hemoglobin distribution was shifted downwards in patients with s-ferritin less than 20 µg/L. Accordingly, the median b-hemoglobin was statistically significantly lower and the fraction with anemia was higher. In 1246 men the findings were similar, except that the turning point toward lower b-hemoglobin was at a s-ferritin level of 30 µg/L. CONCLUSIONS: Low s-ferritin is associated with decreased b-hemoglobin in many more subjects than those labelled anemic.

The diagnostic accuracy of unbound iron binding capacity (UIBC) as a test for empty iron stores.

BACKGROUND: Unbound iron binding capacity (UIBC) in serum, which is s-total iron binding capacity (2 times s- transferrin) minus s-iron, may be a more accurate marker of empty iron stores than serum transferrin saturation. Previously we have shown this for healthy females of childbearing age. METHODS: Now we used receiver operating characteristic (ROC) curve analysis to compare the diagnostic accuracy of s-iron, s-transferrin, s-transferrin saturation and s-UIBC in diagnosing empty iron stores in 29,251 female and 19,652 male outpatients. Empty iron stores were defined as s-ferritin less than 10, 15 or 20 µg/L. RESULTS: At all definitions of empty iron stores s-UIBC had a better diagnostic accuracy than the other tests in both male and female outpatients, with an area under the ROC curve of 0.85-0.97. Also in subpopulations with elevated s-CRP or low b-hemoglobin s-UIBC was more accurate than the other tests. All tests performed better in males than in females, and generally they were more accurate in adults than in children. CONCLUSION: When diagnosing empty iron stores calculation of s-UIBC is a better way to utilize the information in s-iron and s-transferrin than the calculation of s-transferrin saturation.

Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions.

One of the MLL fusion partners in leukemia is the SEPT6 gene, which belongs to the evolutionarily conserved family of genes of septins. In this work we aimed to characterize at both the RNA and DNA levels three acute myeloid leukemias with cytogenetic evidence of a rearrangement between 11q23 and Xq24. Molecular analysis led to the identification of several MLL-SEPT6 fusion transcripts in all cases, including a novel MLL-SEPT6 rearrangement (MLL exon 6 fused with SEPT6 exon 2). Genomic DNA breakpoints were found inside or near Alu or LINE repeats in the MLL breakpoint cluster region, whereas the breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II consensus cleavage site. These data suggest that a non-homologous end-joining repair mechanism may be involved in the generation of MLL-SEPT6 rearrangements in acute myeloid leukemia.

Neutrophil dysfunction after biomaterial contact in an in vitro model of cardiopulmonary bypass.

OBJECTIVE: Cardiopulmonary bypass (CPB) induces neutrophil degranulation and superoxide anion production in vivo. We hypothesized that CPB-associated neutrophil dysregulation alters neutrophil adhesion to vascular endothelial cells and the extracellular matrix. METHODS: We, therefore, recirculated neutrophils in polyvinyl chloride (PVC) tubing using a roller pump model and thereafter measured adhesion to cultured microvascular endothelial cells and gelatin-coated surfaces. Recirculation-induced neutrophil priming or exhaustion was tested by boosting with phorbol myristate-acetate (PMA) or N-formyl-methiolyl-leucyl-phenylalanine (FMLP) before quantification of adhesion. RESULTS AND CONCLUSION: After recirculation, neutrophils retained their adhesive capability to vascular endothelial cells, whereas adhesion to gelatin increased. This increase was not seen when the neutrophils were recirculated with a rotator instead of a roller pump, indicating that not only the pump mode but also foreign surface contact was of significance. The neutrophil PMA response after recirculation was not altered compared to resting neutrophils prestimulated with PMA. Recirculated neutrophils adhered less to cultured vascular endothelial cells after FMLP activation and more to gelatin compared to resting neutrophils prestimulated with FMLP. It is conceivable that dysregulation of neutrophil adhesive capability may play a part in the development of tissue damage after CPB.

Concentration-dependent effect of abciximab on platelets and neutrophils in a model of cardiopulmonary bypass.

Abciximab is a GPIIb/IIIa antagonist used in percutaneous coronary interventions to avoid platelet activation, thrombosis and inflammation. We investigated whether abciximab influenced platelet activation and platelet interaction with neutrophils and polyvinyl chloride (PVC) in a cardiopulmonary bypass (CPB) model. Isolated platelets, preincubated with and without 0.1-20 microg/mL of abciximab, were resuspended with neutrophils in plasma and recirculated by a roller pump. Platelet, but not neutrophil adhesion to PVC was inhibited by abciximab. Only high doses of abciximab reduced platelet aggregation size, but simultaneously increased platelet-neutrophil aggregation. Abciximab had no effect on platelet CD62P expression or degranulation, but platelet activation on platelet-neutrophil aggregates increased with high doses. Only low doses inhibited neutrophil degranulation. The concentration-dependent effect of abciximab on platelet-neutrophil interaction reduces its usefulness and stresses the dependency on experimental design in the evaluation of abciximab. Our study does not support the use of abciximab alone in CPB. However, incorporation of surface-coating the biomaterial with abciximab may be an interesting option.