{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T04:22:14Z","timestamp":1787458934923,"version":"build-2736575974"},"reference-count":19,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["R01 CA278834"],"award-info":[{"award-number":["R01 CA278834"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>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\u201320% 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.<\/jats:p>","DOI":"10.3390\/cancers18162706","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T08:14:01Z","timestamp":1787300041000},"page":"2706","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["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 in Hepatoblastoma or Biliary Atresia. Comment on \u201cFleifil et al. DNAJB1-PKAc Kinase Is Expressed in Young Patients with Pediatric Liver Cancers and Enhances Carcinogenic Pathways. Cancers\u00a02025, 17, 83\u201d"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2313-6213","authenticated-orcid":false,"given":"Yasmeen","family":"Fleifil","sequence":"first","affiliation":[{"name":"Division of General and Thoracic Surgery, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6194-9805","authenticated-orcid":false,"given":"Ruhi","family":"Gulati","sequence":"additional","affiliation":[{"name":"Division of General and Thoracic Surgery, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7450-4248","authenticated-orcid":false,"given":"Katherine","family":"Jennings","sequence":"additional","affiliation":[{"name":"Department of Neurology, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1395-9475","authenticated-orcid":false,"given":"Alexander","family":"Miethke","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Hepatology & Nutrition, Cincinnati Children Hospital Medical Center, Cincinnati, OH 45229, USA"},{"name":"Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9802-5948","authenticated-orcid":false,"given":"Alexander","family":"Bondoc","sequence":"additional","affiliation":[{"name":"Division of General and Thoracic Surgery, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA"},{"name":"Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregory","family":"Tiao","sequence":"additional","affiliation":[{"name":"Division of Abdominal Transplantation, Stanford University, Palo Alto, CA 94304, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-0259-4550","authenticated-orcid":false,"given":"Rebekah","family":"Karns","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Hepatology & Nutrition, Cincinnati Children Hospital Medical Center, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lubov","family":"Timchenko","sequence":"additional","affiliation":[{"name":"Department of Neurology, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikolai","family":"Timchenko","sequence":"additional","affiliation":[{"name":"Division of General and Thoracic Surgery, Cincinnati Children\u2019s Hospital Medical Center, Cincinnati, OH 45229, USA"},{"name":"Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1163","DOI":"10.1002\/pbc.25970","article-title":"Fibrolamellar Hepatocellular Carcinoma: Mechanistic Distinction from Adult Hepatocellular Carcinoma","volume":"63","author":"Riggle","year":"2016","journal-title":"Pediatr. 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