Detection of Low Levels of DNAJB1::PRKACA Fusion KinaseRequires the Utilization of Sensitive Assays and CarefulMethodological Planning. Reply to Palaz et al. Independent Multi-Cohort RNA-Seq Analysis Does Not Support Recurrent DNAJB1::PRKACA Fusion i

In 2024, we published a paper which described the identification of the fusion DNAJB1::PRKACA kinase in young patients with aggressive hepatoblastoma (HBL) and with biliary atresia (BA). In our study, we used sensitive molecular and cellular techniques and found that about 70% of our analyzed HBL samples showed varying levels of DNAJB1::PRKACA expression. In total, 15–20% of those fusion-positive HBL samples had DNAJB1::PRKACA levels comparable to those observed in FLC, whereas the remaining samples exhibited lower kinase levels. Palaz et al. analyzed five HBL datasets and one BA RNA-Seq dataset with Arriba and found no DNAJB1::PRKACA fusion transcript. Based on their analysis, the authors concluded that DNAJB1::PRKACA is not expressed in HBL patients. They further stated that the identification of DNAJB1::PRKACA fusion in either HBL or BA requires orthogonal molecular validation to confirm the fusion event at the DNA or RNA level. Therefore, we conducted Sanger sequencing on RT-PCR products from several fusion-positive HBL samples and three BA samples. Both HBL and BA cases showed the presence of the DNAJB1::PRKACA fusion transcript, identical to that detected in FLC. These results verify that DNAJB1::PRKACA is present in some HBL and BA patients, as has already been demonstrated using sensitive molecular and cellular techniques. Arriba analysis of current RNA-Seq data may not be sensitive enough for detecting low DNAJB1::PRKACA levels in HBL cases, especially if they are mixed with fusion-negative HBL specimens. Thus, we found that several independent methods, such as immunoanalysis and RT-PCR sequencing, effectively detect DNAJB1::PRKACA in HBL and BA cases. Our study also highlights that detecting low levels of DNAJB1::PRKACA requires individual analysis of HBL and BA patients within the same study, using FLC as the control.