High-resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia.

BACKGROUND: Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML). The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and a partial tandem duplication within the mixed-lineage leukemia (MLL-PTD) genes in childhood AML. PROCEDURE: Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluorescent dye SYTO-82 based high-resolution melting (HRM) curve analysis to detect FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR. RESULTS: The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event-free survival (EFS). Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring. CONCLUSION: High-resolution melting was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost-effective screening of the FLT3 and NPM1 mutations in AML patients.

Clinical significance of tumor-associated inflammatory cells in metastatic neuroblastoma.

PURPOSE: Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature. METHODS: Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts. RESULTS: Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively. CONCLUSION: These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.

TrkA expression in peripheral neuroblastic tumors: prognostic significance and biological relevance.

BACKGROUND: This study was conducted to investigate the prognostic significance and biologic relevance of trkA expression levels in peripheral neuroblastic tumors (pNTs) (i.e., neuroblastoma, ganglioneuroblastoma, and ganglioneuroma). METHODS: Levels of trkA expression from a total of 265 pNTs were determined by quantitative polymerase chain reaction analysis with Genescan software. The results were analyzed according to histopathology (favorable histology [FH] vs. unfavorable histology [UH] according to the International Neuroblastoma Pathology Classification) and MYCN tumor status (amplified vs. nonamplified) along with clinical stage and outcomes of the patients. RESULTS: The levels of trkA expression differed significantly between the group of patients who were alive and well (n = 170 patients) and the group that had progressed or died (n = 95 patients) and between the group that was alive (n = 188 patients) and the group that died (n = 77 patients). However, the trkA expression levels were not independent predictors of clinical outcome when the proportional hazards model contained the known prognostic variables of clinical stage, histopathology, and MYCN status (all tests were done in 196 patients). In the neuroblastoma category (n = 173 tumors), tumors in the FH/nonamplified MYCN subset (n = 112 tumors) expressed higher levels of trkA and showed an age-dependent neuroblastic differentiation: They were classified into either a poorly differentiated subtype (n = 91 tumors; all patients age < 1.5 years at diagnosis) or a differentiating subtype (n = 21 tumors; 57% of patients ages 1.5-5.0 years). Tumors in the UH/amplified MYCN subset (n = 30 tumors) expressed significantly lower levels of trkA and showed very limited neuroblastic differentiation. Tumors in the FH/amplified MYCN subset were very rare (n = 3 tumors) and expressed higher levels of trkA. Tumors in the UH/nonamplified MYCN subset (n = 28 tumors) had trkA levels in a wide range and showed limited neuroblastic differentiation. CONCLUSIONS: For patients with pNTs, levels of trkA expression did not add significant information to prognostic grouping, as defined by the combination of clinical stage, histopathology, and MYCN status. There was a biologically relevant correlation between molecular properties (trkA expression and MYCN status) and histopathologic features of the tumors in the neuroblastoma category.