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.

Fibrolamellar Hepatocellular Carcinoma (FLC) is a rare liver cancer affecting adolescents and young adults, with no gender or ethnicity predilection and without history of underlying viral hepatitis, cirrhosis, or other known risk factors. Almost all FLC patients present a somatic heterozygous deletion in chromosome 19p13.12, DNAJB1::PRKACA, which is sufficient to drive FLC in mice. A few studies comparing FLC tumors with adjacent non-transformed liver (normal) samples revealed many transcriptional differences. However, there were done in very small datasets and analyzed using different bioinformatic methods, resulting in just 18-47% agreement between them. This study aims to comprehensively characterize the transcriptome of FLC at bulk and spatial single-cell resolution. The whole transcriptome of 109 FLC frozen patient samples, the largest RNA-seq dataset of FLC to date, was sequenced using different library preparation and ribosomal depletion methods. Only paired tumor and normal tissue samples resected from the same patient were used and divided into two groups: exploration (3 datasets, 67 samples) and testing (2 datasets, 17 samples). Additionally, as external validation datasets, RNA-seq samples from previously published studies were collected, including Sorenson et al. (FLC: 26, normal: 9, paired: 8), the TCGA-LIHC study (FLC: 6, normal: 1, paired: 1), Hirsch et al. (FLC: 15, normal: 3, paired: 0) and Francisco et al. (FLC: 27, normal: 10, paired: 9). All were reanalyzed using state-of-the-art bioinformatic methods: mapped to the Human Genome GRCh38.103 and transcripts quantified using Salmon 1.6.0, unsupervised clustering exploration using PCA, tSNE and UMAP, differential expression calculated using DESeq2 1.28.1, and checking for detectability and consistency among datasets. We found 857 up- and 988 down-regulated genes presenting the same dysregulation in the exploration datasets and confirmed in the testing and external datasets. We call these genes the transcriptional FLC signature. The FLC signature was further characterized by comparing it with the genes differentially expressed in other liver cancers: hepatocellular carcinoma (41 paired samples), hepatoblastoma (22 paired samples), and cholangiocarcinoma (27 paired samples). We found 276 up- and 352 down-regulated genes altered in other liver cancers as well as FLC, but 156 up- and 68 down-regulated only in FLC. The 112 genes with the strongest dysregulation (56 up and 56 down) were used for a MERFISH screening, providing for the first time a single-cell spatial transcriptomic characterization of FLC. This showed clear differential expression patterns in tumor, normal, stromal, and infiltrating immune cells, allowing the identification of how different cell types contribute to the transcriptional FLC signature. Citation Format: David Requena, Aldhair Medico, Luis F. Soto, Mahsa Shirani, James A. Saltsman, Gadi Lalazar, Michael P. LaQuaglia, Sanford M. Simon. Bulk and spatial single-cell transcriptomic characterization of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1516.

In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurrence and occasionally causes death. Using mass spectrometry, we quantitatively analyzed the proteomes of FLC patient’s tumor and adjacent normal, to find pathways that are changed in FLC. These data identified multiple proteins that were altered in the proteome, among these, enzymes involved in metabolism of ammonia. These results were confirmed with immunofluorescence demonstrating that these alterations occur in all tumor cells. These results suggest that FLC cells have defects in the two primary ammonia detoxification pathways in the liver, which are responsible for detoxification of 70% of the ammonia in the body: 1) consumption of ammonia by glutamine synthetase (GLUL), and 2) addition of ammonia by ornithine carbamoyltransferase (OTC) to the urea cycle. Additionally, they also generate extra ammonia because of upregulation of glutaminase (GLS). This was tested with a targeted metabolomics of the reactants and products of these enzymes. The results were consistent with both a loss of the two pathways for consumption of ammonia activation of a pathway for generating ammonia. This production of ammonia is consistent with the observation that surgical resection of fibrolamellar reduces the ammonia in patients. All FLC patients with hyperammonemic encephalopathy and documented urine test results showed increased urinary orotic acid, evidence of blockage of the OTC pathway. This study implies that hyperammonemic encephalopathy in FLC may require alternatives to commonly used therapies. Citation Format: Mahsa Shirani, Solomon Levin, Michael D. Tomasini, James Knox, Bassem Shebl, David Requena, Jackson Clark, Søren Heissel, Hanan Alwaseem, Rodrigo Surjan, Ron Lahasky, Henrik Molina, Barbara Lyons, Rachael D. Migler, Philip Coffino, Sanford M. Simon. Urea cycle metabolism is disturbed in Fibrolamellar carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6729.