• hepatocellular carcinoma

Thomas Longerich and collaborators1 recently published an intriguing observation in hepatocellular adenoma (HCA) showing an activation of the Wnt/ß-catenin signalling pathway without CTNNB1 or APC mutations. The authors identified a recurrent deletion leading to a fusion between a short interspersed nuclear element (SINE) sequence and RSPO2 gene in three HCAs and three hepatocellular carcinomas (HCCs) all activated for ß-catenin, including one tumour with CTNNB1 mutations and five tumours without APC or CTNNB1 mutations. The authors proposed RSPO2fusion as a recurrent mechanism of ß-catenin activation in liver tumourigenesis.

Following this original observation, we analysed the expression and rearrangement of RSPO2 in a series of 10 HCAs and 163 HCCs analysed with RNAseq. We identified a correlation of RSPO2 mRNA expression with several genes known to be positively regulated by ß-catenin, including GLUL (R²=0.51, p=3.39×10⁻²⁸) and TBX3 (R²=0.46, p=5.38×10⁻²⁵) (figure 1A). These results suggest that RSPO2 is a target of the ß-catenin activation, and accordingly an overexpression of RSPO2 was observed in CTNNB1-mutated HCA and HCC. Then, we identified seven HCC samples with abnormal RSPO2 transcripts. All these seven tumours showed a strong activation of Wnt/ß-catenin pathway related to classical activating mutations in the exon 3 of CTNNB1 in six cases and AXIN1 mutation in the remaining. All abnormal transcripts fused RSPO2 exon 2 or 3 to an adjacent RNA transcribed sequence located 58 813–58 922 nucleotides upstream (chr8:108,141,620–108,141,729, hg38) (figure 1B). RSPO2 exon 2 boundaries were similar to that described by Longerich and collaborators,1 whereas the upstream sequence was variable. In two of the tumours with abnormal RSPO2 transcript, we performed whole-genome sequencing and we observed no focal deletions at the DNA level. Overall, this phenomenon is well known as transcriptional read-through events occurring in two transcribed neighbouring sequences, without DNA deletion.2 Gene read-throughs are frequently the consequence of the high transcription level but not the cause of an overexpression that is due in all our cases to classical activating mutations of CTNNB1.

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Figure 1

Expression and read-through events of RSPO2 in HCA and HCC. (A) Correlation between RSPO2 expression and GLUL and TBX3, two target genes of Wnt/ß-catenin pathway in 163 HCCs and 10 HCAs analysed by RNA sequencing (Spearman test). Results were represented in FPKM. CTNNB1-mutated tumours were represented by large circles and tumours with RSPO2 read-throughs in dark red. (B) RNAseq analyses showed read-throughs involving RSPO2 in three HCCs (#197T, #996T, #2102) with activation of the Wnt/ß-catenin pathway due to CTNNB1 activating mutations; #603N is a control normal liver. No focal RSPO2 deletion was observed in the whole-genome analysis of #197T HCC. HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; M, mutated; NM, non-mutated; SINE, short interspersed nuclear element.

Misinterpretation of read-throughs and deletion can occur frequently in targeted resequencing of poor-quality nucleic acid samples. Indeed, Longerich and colleagues1 performed genomic analyses in DNA and RNA extracted from formalin-fixed paraffin-embedded (FFPE) that can produced an artefact of sequencing or sequences difficult to interpret.3 Also, high sensitivity of the targeted resequencing kit method using chimeric primers could induce false-positive identification of fusion genes corresponding to read-throughs. Moreover, CTNNB1 primers used in this study were not able to identify exon 3 deletion that has been recurrently identified in HCA, leading to an underestimation of the CTNNB1 activating alterations.4 5

In conclusion, RSPO2 mRNA fusion described by Longerich et al 1 in liver tumours activated for ß-catenin is generated by read-throughs in the absence of chromosome deletion. These read-throughs are more likely the consequence of a high level of gene expression in liver tumours with high ß-catenin activation than a cause of ß-catenin activation.