Simon, Sanford M

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug–response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.

Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary liver cancer that affects primarily adolescents and young adults. It is associated with a poor overall prognosis. There is a need to better define risk factors, but small sample size has limited such studies. An FLC patient registry now provides data sufficient for statistically robust inferences. We leveraged a unique patient community–based FLC registry to analyze the prognostic impact of demographic and clinical characteristics evident at diagnosis. Variables were analyzed using Cox proportional hazards regression to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). In multivariable models of 149 patients (88 females and 61 males), female gender was associated with statistically significant improved survival with HR of 0.52 (95% CI 0.29–0.93). Factors evident at diagnosis that are associated with worse survival included the presence of 10 or more tumors within the liver (HR 7.1; 95% CI 2.4–21.04), and metastases at diagnosis (HR 2.17; 95% CI 1.19–3.94). Positive lymph nodes at diagnosis, despite being found significantly associated with worse survival in a univariate analysis, did not remain significant when adjusted for covariates in a multivariable analysis. We found no statistically significant effect of age at diagnosis nor tumor size at diagnosis on survival. Female gender may confer a favorable prognosis in FLC. Established high‐risk prognostic factors that we confirmed in this Registry included the diagnostic presence of numerous intrahepatic tumors, and metastases. This is the first study derived from a FLC patient community–based registry, and highlights how registries of rare tumors can empower patients to meaningfully advance clinical and scientific discoveries.

Abstract To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. Significance: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor. This article is highlighted in the In This Issue feature, p. 2355

Myxoid hepatic adenomas are a rare subtype of hepatic adenomas with distinctive deposition of extracellular myxoid material between the hepatic plates. A total of 9 cases were identified in 6 women and 3 men with an average of 59±12 years. The myxoid adenomas were single tumors in 5 cases and multiple in 4 cases. In 1 case with multiple adenomas, the myxoid adenoma arose in the background of GNAS-mutated hepatic adenomatosis. Myxoid hepatic adenomas had a high frequency of malignant transformation (N=5 cases). They were characterized at the molecular level by HNF1A inactivating mutations, leading to loss of LFABP protein expression. In addition, myxoid adenomas had recurrent mutations in genes within the protein kinase A (PKA) pathway or in genes that regulate the PKA pathway: GNAS, CDKN1B (encodes p27), and RNF123. In sum, myxoid adenomas are rare, occur in older-aged persons, have a high risk of malignant transformation, and are characterized by the combined inactivation of HNF1A and additional mutations that appear to cluster in the PKA pathway.

AbstractFibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver malignancy in adolescents and young adults. Surgery is the mainstay of therapy for primary and metastatic disease. Most patients relapse, with development of both local and distant metastases. Brain metastases from solid tumors are rare in the pediatric and young adult population. Here, we document three patients with brain metastases from FLHCC, confirmed by histology and molecular characterization of the chimeric fusion DNAJB1–PRKACA, each necessitating neurosurgical intervention. These observations highlight the ability of FLHCC to metastasize to the brain and suggest the need for surveillance neuroimaging for patients with advanced‐stage disease.

Fibrolamellar carcinomas are characterized by activation of protein kinase A, a kinase composed of catalytic and regulatory subunits. PRKACA encodes a catalytic subunit of protein kinase A, and almost all fibrolamellar carcinomas have a heterozygous 400‐kb deletion that leads to the fusion of DNAJB1 and PRKACA . The resulting DNAJB1‐PRKACA fusion transcript is believed to activate protein kinase A by dysregulation of the catalytic portion of the protein. In contrast, PRKAR1A encodes one of the regulatory subunits of protein kinase A. We hypothesized that loss of function of this regulatory unit could also lead to protein kinase A activation and thus to fibrolamellar carcinoma. Because PRKAR1A mutations underlie the Carney complex, we searched for liver tumors in individuals with the Carney complex. We identified 3 individuals with fibrolamellar carcinomas and a personal history of the Carney complex. All three tumors displayed the typical morphology of fibrolamellar carcinoma and were positive for arginase, cytokeratin 7, and cluster of differentiation 68. Fluorescence in situ hybridization was negative for PRKACA rearrangements. However, PRKAR1A sequencing identified pathogenic mutations in two of two cases with successful sequencing. In addition, all three cases were negative for PRKAR1A protein expression, consistent with inactivation of this key regulatory unit of protein kinase A. We also identified one additional fibrolamellar carcinoma in an individual without a documented history of the Carney complex who was negative for PRKACA rearrangements but had loss of PRKAR1A protein expression as well as PRKAR1A mutations. Conclusion : Fibrolamellar carcinoma can be part of the Carney complex; in this setting, fibrolamellar carcinomas have inactivating PRKAR1A mutations instead of the DNAJB1‐PRKACA fusion gene found in sporadic fibrolamellar carcinomas, providing an alternate means for activation of protein kinase A. (H epatology 2017).