Abstract A33: Discovery of chimeric transcripts involving APC and TERT in pediatric HCC by RNA sequencing
Authors: Haines Katherine , Roy Angshumoy , Wang Linghua , Sumazin Pavel , Covington Kyle R. , Muzny Donna M. , Kumar Vijetha , Doddapaneni Harsha , Chao Hsu , Wheeler David A. , Tomlinson Gail , Parsons D. Williams , Plon Sharon E. , Lopez-Terrada Dolores 
Published: May 2016

Background: Hepatocellular carcinoma (HCC) is a rare pediatric liver tumor with a poor prognosis. A characteristic DNAJB1-PRKACA gene fusion has been identified in a specific subtype, fibrolamelar HCC (FL-HCC). In the majority of pediatric cases of non-FL-HCC, a genetic cause has not been identified. We hypothesize that gene fusions could play a role in the tumorigenesis of pediatric HCC. The goal of this project is to utilize RNA sequencing to identify chimeric transcripts in pediatric HCC that could improve molecular characterization and identify potential oncogenic drivers of this disease.

Materials and Methods: We have used RNA sequencing (RNA-seq) to survey a cohort of 8 FL-HCCs, 4 pediatric HCCs, and 6 normal liver samples for chimeric transcripts. High quality RNA (RIN: 6.6-9.7) was extracted from fresh-frozen tissue and strand-specific, poly-A+ RNA-seq libraries were prepared for Illumina sequencing. Approximately 85 million paired-end reads (42.5 million fragments) of 2 x 100 bp length were generated per sample. Fusion transcripts in the tumor samples were detected using deFuse (v.0.6.1) on FASTQ files followed by subtraction of fusions also called in the normal liver dataset. The remaining calls in the HCC dataset were then filtered for the COSMIC Cancer Gene Census list to identify fusions involving known oncogenes or tumor suppressor genes. Candidate fusions were verified using BLAST and validated by RT-PCR.

Results: On average, 150 fusion transcripts were predicted per tumor sample using the deFuse algorithm. Further filtering by the normal liver dataset reduced the number of calls in the tumor datasets by ~60%. As expected, DNAJB1-PRKACA fusions were identified in the FL-HCC cohort with no additional fusions detected. Filtering by the COSMIC Cancer Gene Census list resulted in five additional fusion calls in HCC (three unique events in two tumors) two of which were confirmed by RT-PCR. The first event is an inversion within the APC and AP3B1 genes that results in the two in-frame fusion transcripts, APC-AP3B1 and APC3B1-APC, but no full-length APC transcript. The second event is a deletion encompassing the TERT promoter that results in the in-frame fusion LPCAT1-TERT. An increase in TERT expression is seen in this tumor as compared to both normal liver and other pediatric HCC tumors. Filtering for in-frame fusions involving non-COSMIC genes did not reveal any fusions in the two remaining HCCs and further analysis of these cases is ongoing.

Conclusion: The detection of chimeric transcripts by RNA-seq has allowed us to identify two unique structural events involving known cancer genes in two pediatric non-fibrolamellar HCCs. The chimeric transcripts found in these tumors provide further insight into the tumorigenic events of pediatric HCC.

Supported by research grants from Cancer Prevention and Research Institute of Texas RP101195 and RP120715 and National Institute of General Medical Sciences T32GM008307.

Citation Format: Katherine Haines, Angshumoy Roy, Linghua Wang, Pavel Sumazin, Kyle R. Covington, Donna M. Muzny, Vijetha Kumar, Harsha Doddapaneni, Hsu Chao, David A. Wheeler, Gail Tomlinson, D. Williams Parsons, Sharon E. Plon, Dolores Lopez-Terrada. Discovery of chimeric transcripts involving APC and TERT in pediatric HCC by RNA sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A33.