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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.
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.
Contributors J-CN, QB and JZ-R: generation and analysis of data, and writing and approval of the manuscript.
Competing interests None declared.
Provenance and peer review Not commissioned; internally peer reviewed.
Patient consent for publication Not required.
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