Comprehensive analysis of tumoral DNA content reveals clonal ploidy heterogeneity as a marker with prognostic significance in locoregional neuroblastoma

The clinical management of locoregional neuroblastoma (LR NB) is controversial. In a previous study we showed that diploidy was a strong prognostic predictor of outcome and detected the existence of clonal ploidy heterogeneity in a select group of cases. The aims of this study were (1) to assess the frequency of ploidy heterogeneity in LR NB, (2) to ascertain the best method to detect heteterogeneity, and (3) to correlate ploidy populations with clinical outcome. We undertook a comprehensive analysis of tumoral DNA content in 38 LR NBs comparing (1) flow cytometry (FCM), (2) karyotyping, (3) interphase fluorescence in situ hybridization, and (4) laser-scanning cytometry (LSC). Tumor ploidy heterogeneity was found by all methodologies. By FCM, all tumors with aneuploid peaks had detectable diploid DNA peaks. By LSC, all tumors with diploid and hyperploid peaks were GD2-positive in both, consistent with their tumoral origin. A predominant near-triploid clonal population (ratio diploid vs. triploid, <2.5) was observed in most nonprogressing LR NB tumors, and a predominant diploid clone (ratio di- vs. triploid, >2.5) in most progressing LR NB cases. Multivariate analysis was performed to evaluate the prognostic value of tumor ploidy assayed by different methods versus age, INSS (International Neuroblastoma Staging System) stage, and MYCN status. FCM was the most powerful prognostic factor related to poor prognosis (overall survival, P = 0.02; progression-free survival, P = 0.01). These results provide strong evidence for clonal ploidy heterogeneity in LR NB and clonal evolution toward diploidy in progressing LR NB.