Molecular classification of hepatoblastoma and prognostic value of the HB 16‐gene signature
Authors: Marie‐Annick Buendia, Carolina Armengol, Stefano Cairo
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. TO THE EDITOR: We read with interest the article recently published by Sumazin et al.1 regarding the identification of novel molecular and prognostic subgroups in hepatoblastoma (HB), the most frequent form of liver cancer during infancy. The authors combined genetic and transcriptomic approaches to characterize a set of treatment‐naïve tumors and identify several prognostic markers and signatures that they validate in an independent cohort. Interestingly, among markers associated with poor prognosis, they observe high expression of LIN28B, and its correlation with low expression of let‐7 microRNAs (miRNAs), but not with enhanced expression of c‐Myc. We broached this question in a study of miRNA expression profiles in 65 HBs and showed that poor prognostic tumors are characterized by activated Myc signaling associated with enhanced expression of LIN28B and down‐regulation of let‐7 miRNAs.2 Although the levels of MYC transcripts are not markedly different between HB and normal liver, MYCN gene expression is strongly up‐regulated in HBs, particularly in aggressive tumors.3 Moreover, the interplay of β‐catenin mutation and Myc signaling in HB pathogenesis has been demonstrated in a recent landmark paper.4 Sumazin et al.1 describe three risk‐stratifying molecular subtypes of HB that differ mainly by activation of hepatic progenitor cell markers and metabolic pathways, and found significant correlation with the C1/C2 molecular classification that we developed earlier.3 However, the authors state that "subtype prediction using the 16‐gene signature of Cairo et al. was not prognostically predictive (P > 0.1)" and that gene set enrichment analysis (GSEA) "identified multiple sets of differentially expressed genes identified by Cairo et al. but not their 16‐gene signature." With regard to the first statement, Supporting Figure S131 shows that the authors clustered the samples with the 16‐gene signature, but relative gene expression levels were not normalized, and probe set redundancy was not taken into account. Therefore, they used 28 probe sets instead of 16, which distorts the relative weight of each marker. As shown in our Figure 1, when hierarchical clustering is performed using the data of Sumazin et al. and by attributing an equal weight to each gene, the 16‐gene signature nicely stratifies samples into 2 HB groups clearly associated with the HB1, HB2, and HB3 clusters identified by the authors and with patient outcome.Figure 1: Top: Hierarchical clustering of mRNA profiles in 50 untreated HBs and five normal liver samples (labeled in cyan) from Sumazin et al.1 using the 16‐gene signature.3 Samples in yellow identify a subcluster associated with poor outcome (A = alive; D = dead of disease; R = relapse). Event‐free survival status: 8/18 versus 2/31 (P = 0.0026 [Fisher's exact test]). Bottom: GSEA using HBs from Sumazin et al. and two gene sets of eight genes down‐regulated (left) and eight up‐regulated (right) in C2 from the 16‐gene signature.Second, Supporting Figure S141 shows the GSEA that was run using the CAIRO_HEPATOBLASTOMA_ POOR_SURVIVAL signature from the MsigDB database containing all 16 genes. As mentioned by the authors, this signature is composed of two sets of eight genes with opposite trends of expression. Therefore, to be used appropriately for GSEA, the signature should have been split into two lists. As shown in Fig. 1, by doing so (two lists of eight genes up‐regulated or down‐regulated in C2) and by using the same HB samples used by the authors to perform the analysis, each gene set is significantly enriched in the expected prognosis group. Taken together, these data confirm the association of the 16‐gene signature with the patient outcome in an independent cohort of samples obtained before treatment. High‐throughput studies of extensive cohorts of patients such as the one by Sumazin et al. are of high interest to increase our knowledge of this rare pediatric liver cancer. The Paediatric Hepatic International Tumour Trial (NCT03017326) that will begin in 2017 provides an ideal opportunity to validate biological markers that will improve the clinical management of HB.