Prognosis prediction with immunohistochemical features and MYCN amplification in neuroblastoma

다국어 초록 (Multilingual Abstract)

Introduction: Neuroblastoma is a childhood malignancy which showed highly heterogeneous clinical course. Many genetic and genomic features as well as clinical characteristics have been studied and incorporated in the modern prognostic classification of neuroblastoma to divide the risk groups. In this study, MYCN amplification, anaplastic lymphoma kinase (ALK) mutation and amplification, ALK protein expression, loss of the nuclear ATRX protein, and TERT protein expression were studied to investigate if there are any correlations between these molecular characteristics and clinical features or outcomes.
Materials and methods: Among the 104 patients who were histologically diagnosed with neuroblastoma and treated at Seoul National University Children’s Hospital between January 2002 to July 2012, 72 patients whose initial tumor samples were evaluable were enrolled in this study. Formalin-fixed, paraffin-embedded tumor tissues obtained during surgery or biopsy were used. Mutation analysis for exons 23, 24, and 25 of the ALK gene was performed with PCR amplification and direct sequencing. Immunohistochemical staining was performed on 4-μm thick tissue microarray sections using an ALK monoclonal antibody, a polyclonal antibody against ATRX and a monoclonal anti-TERT antibody. Fluorescence in situ hybridization (FISH) was performed to investigate MYCN and ALK amplification.
Results: Seven (10.0%) patients had MYCN amplification at initial diagnosis. Patients who had MYCN amplification were statistically younger than the other patients (1.6±0.8 years vs 3.1±3.7 years, P=0.010), and relapse rate was significantly higher in patients with MYCN amplification compared to the other patients (77.8% vs 19.4%, P<0.001). Forty (55.6%) patients showed ALK expression, and incidence ALK expression increased according to the increasing tumor stage (P=0.001). Relapse rate was significantly higher in ALK+ patients compared to the ALK- patients (47.5% vs 11.3%, P=0.007). ALK mutation was found only in 2 (4.1%) patients among 49 patients whose tumor samples were sufficient for DNA extraction, and ALK amplification was not found in any of the 65 patients whose tumor samples were evaluable for ALK FISH study. Nine (13.0%) patients showed loss of nuclear ATRX protein, and loss of nuclear ATRX protein was found in 2 different populations. One is the older, stage IV patients showing indolent disease course with ALK expression, and the other is young children with lower stage ALK- neuroblastomas having better prognosis. Patients without TERT expression seemed to be associated with high relapse rate, but there was no statistical significance.
Conclusion: Both MYCN amplification and ALK expression had strong prognostic significance in this study. Although ALK mutation was rare and no amplification was observed, ALK protein expression was found in significant number of patients and was correlated with advanced stage neuroblastoma and poor outcome. With these results, ALK targeted therapy could be considered as a valid therapeutic strategy for relapsed/refractory neuroblastoma patients having ALK expression. Regarding the meaning of ATRX and TERT expression in neuroblastoma, further study is required.

목차 (Table of Contents)

• Abstract i
• Contents iv
• List of Tables vi
• List of Figures vii
• List of abbreviations and symbols viii
• Introduction １
• Materials and methods ５
• Patients and specimens ５
• Immunohistochemical staining ６
• Fluorescence in situ hybridization (FISH) 7
• Mutation analysis ７
• Statistical analysis ９
• Results 10
• Clinical characteristics 10
• MYCN amplification 12
• ALK protein expression 14
• ALK amplification and mutation 16
• Loss of nuclear ATRX protein 18
• TERT protein expression 20
• Combination of IHC features and MYCN amplification 22
• Discussion 24
• References 34
• 국문 초록 42
• 감사의 글 45