High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma

Abstract

Purpose: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14(ARF) pathway in 9 of 17 (53%) neuroblastoma cell lines established at relapse.

Hypothesis: Inactivation of the p53/MDM2/p14(ARF) pathway develops during treatment and contributes to neuroblastoma relapse.

Methods: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14(ARF) methylation and deletion by methylation-specific PCR and duplex PCR, respectively, and MDM2 amplification by fluorescent in situ hybridization.

Results: Abnormalities in the p53 pathway were identified in 20 of 41 (49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6 of 41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, postchemotherapy, and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio, 3.4; 95% confidence interval, 1.2-9.9; P = 0.02). Upstream defects were present in 35% of cases: MDM2 amplification in 3 cases, all at diagnosis and relapse and p14(ARF) inactivation in 12 of 41 (29%) cases: 3 had p14(ARF) methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse.

Conclusions: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects in which p53-independent therapies are indicated.

Figure 1. p53 status and function in neuroblastoma at different stages of treatment

(a) Case 14, (ai) codon 203 mutation (aii) DHPLC chromatogram. (b-d) Case 1 (bi) codon 270 mutation present post-chemotherapy and relapse but not at diagnosis. (bii) DHPLC chromatogram (c) p53 and p21 immunohistochemistry showing lack of p21 expression at relapse (scale bar = 50μm) (d) Codon 270 mutation-specific PCR.

Figure 1. p53 status and function in neuroblastoma at different stages of treatment

(a) Case 14, (ai) codon 203 mutation (aii) DHPLC chromatogram. (b-d) Case 1 (bi) codon 270 mutation present post-chemotherapy and relapse but not at diagnosis. (bii) DHPLC chromatogram (c) p53 and p21 immunohistochemistry showing lack of p21 expression at relapse (scale bar = 50μm) (d) Codon 270 mutation-specific PCR.

Figure 2. MDM2 amplification in neuroblastoma

(a & b) Case 4 (ai) MDM2 amplification by FISH on a relapsed tumour imprint (×100), (aii) Non-MDM2 amplified tumour. (b) 50K SNP arrays log2ratio from relapsed tumour DNA showing (bi) MDM2 amplification (12q15) (bii) no MYCN amplification (2p24). (c) Case 19, MDM2 amplification by FISH on paraffin sections from (ci) diagnostic tumour and (cii) relapse.

Figure 3. p14^ARF abnormalities in neuroblastoma

(a & b) Case 9 (a) MSP showing unmethylated p14^ARF (diagnosis) and partial methylation (progression) (b) p14^ARF mRNA expression showing decreased expression at progression. (c) Duplex PCR showing (ci) homozygous deletion at diagnosis and relapse (case 20) and other relapsed tumours, (cii) the presence of p14^ARF in constitutional DNA from cases homozygously deleted for p14^ARF.