Despite being the most common liver cancer in children, hepatoblastoma (HB) is a rare neoplasm. Consequently, few pretreatment tumors have been molecularly profiled, and there are no validated prognostic or therapeutic biomarkers for HB patients. We report on the first large-scale effort to profile pretreatment HBs at diagnosis. Our analysis of 88 clinically annotated HBs revealed three risk-stratifying molecular subtypes that are characterized by differential activation of hepatic progenitor cell markers and metabolic pathways: high-risk tumors were characterized by up-regulated nuclear factor, erythroid 2-like 2 activity; high lin-28 homolog B, high mobility group AT-hook 2, spalt-like transcription factor 4, and alpha-fetoprotein expression; and high coordinated expression of oncofetal proteins and stem-cell markers, while low-risk tumors had low lin-28 homolog B and lethal-7 expression and high hepatic nuclear factor 1 alpha activity. Conclusion: Analysis of immunohistochemical assays using antibodies targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis; our results pave the way for clinical collaborative studies to validate candidate biomarkers and test their potential to improve outcome for HB patients. (HEPATOLOGY 2017;65:104-121).H epatoblastoma (HB) is the most common pediatric liver tumor. It has an annual incidence rate of approximately 1.8 diagnosed cases per million in the United States, and this rate is increasing at more than 4.3% annually.(1) HBs are embryonal neoplasms that are most commonly diagnosed during the first 3 years of life. They are believed to arise from hepatic cell precursors and are characterized by heterogeneous histological patterns reminiscent of liver developmental stages.(2) Therapeutic strategies combining surgical resection and chemotherapy have improved outcomes for children with HB, but the prognosis for patients with advanced or chemotherapy-refractory disease remains poor.(1) In addition, the most effective platinum-based agents for treatment of HB often lead to serious long-term adverse effects, including ototoxicity and nephrotoxicity.(1)We describe the results of a comprehensive genomic analysis of the largest set of clinically annotated HBs reported to date. Such efforts have previously identified

Background: Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes. Patients and methods: Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation. Results: RET G810C/S mutations at the solvent front and RET Y806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RET M918T/V804M/L , who initially responded to selpercatinib and developed resistance. RET G810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RETselpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft. Conclusions: RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

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Myxoid/round cell liposarcoma is characterized by the recurrent translocations t(12;16)(q13;p11) and, less commonly, t(12;22)(q13;q12), which fuse FUS or EWSR1, respectively, to DDIT3 on chromosome 12. Although a number of different variant breakpoints have been described, greater than 90% of all cases have one of the three different FUS-DDIT3 fusions, which may have clinical significance. To identify the individual breakpoints, a sequence-specific assay such as reverse transcription-PCR (RT-PCR) is needed. In this study, we optimized primer design to develop an RT-PCR assay for the detection of the most common translocations in formalinfixed paraffin-embedded tissue specimens. We compared our assay with primers previously published for testing formalin-fixed paraffin-embedded specimens and achieved the most consistent results with our primers. We obtained RNA from 32 MLS cases, of which 27 carried one of the three common FUS-DDIT3 chimeric transcript types. Four of the negative cases were from very small biopsies with very low RNA concentration. One case was consistently negative by RT-PCR, but showed a FUS rearrangement by fluorescent in situ hybridization, suggesting that it may harbor one of the rarer FUS-DDIT3 chimeric types. In addition to the common fusions, our assay also identified a novel FUS-DDIT3 fusion between exon 9 of FUS and exon 3 of DDIT3 in one of the cases.