Exome Sequencing of Fresh-frozen or Formalin-fixed Paraffin-embedded B6C3F1/N Mouse Hepatocellular Carcinomas Arising Either Spontaneously or due to Chronic Chemical Exposure.

Scott S Auerbach,Robert C Sills,Arun R Pandiri,Ruchir Shah,Ramesh C Kovi,Miaofei Xu,Dhiral Phadke,Thai-Vu Ton,Mark J Hoenerhoff,B Alex Merrick,Hue-Hua L Hong,Debra J Taxman

doi:10.1177/0192623318789398

Abstract

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide; however, the mutational properties of HCC-associated carcinogens remain largely uncharacterized. We hypothesized that mechanisms underlying chemical-induced HCC can be characterized by evaluating the mutational spectra of these tumors. To test this hypothesis, we performed exome sequencing of B6C3F1/N HCCs that arose either spontaneously in vehicle controls ( n = 3) or due to chronic exposure to gingko biloba extract (GBE; n = 4) or methyleugenol (MEG; n = 3). Most archived tumor samples are available as formalin-fixed paraffin-embedded (FFPE) blocks, rather than fresh-frozen (FF) samples; hence, exome sequencing from paired FF and FFPE samples was compared. FF and FFPE samples showed 63% to 70% mutation concordance. Multiple known (e.g., Ctnnb1T41A, BrafV637E) and novel (e.g., Erbb4C559S, Card10A700V, and Klf11P358L) mutations in cancer-related genes were identified. The overall mutational burden was greater for MEG than for GBE or spontaneous HCC samples. To characterize the mutagenic mechanisms, we analyzed the mutational spectra in the HCCs according to their trinucleotide motifs. The MEG tumors clustered closest to Catalogue of Somatic Mutations in Cancer signatures 4 and 24, which are, respectively, associated with benzo(a)pyrene- and aflatoxin-induced HCCs in humans. These results establish a novel approach for classifying liver carcinogens and understanding the mechanisms of hepatocellular carcinogenesis.

Highlights

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide; the mutational properties of HCC-associated carcinogens remain largely uncharacterized
Analysis of the number of total and aligned reads from each of the tumor and normal tissue samples verified that a high percentage (> 90%) of both the FF and formalin-fixed paraffinembedded (FFPE) samples aligned to the C3H/HeN genome (Online Supplementary Table S1)
We found that the low number of supporting reads and noisy signal in the FFPE normal samples may be the cause of the discordance between the FF and FFPE samples for the Ctnnb1 gene

Summary

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide; the mutational properties of HCC-associated carcinogens remain largely uncharacterized. We hypothesized that mechanisms underlying chemical-induced HCC can be characterized by evaluating the mutational spectra of these tumors To test this hypothesis, we performed exome sequencing of B6C3F1/N HCCs that arose either spontaneously in vehicle controls (n 1⁄4 3) or due to chronic exposure to gingko biloba extract (GBE; n 1⁄4 4) or methyleugenol (MEG; n 1⁄4 3). The MEG tumors clustered closest to Catalogue of Somatic Mutations in Cancer signatures 4 and 24, which are, respectively, associated with benzo(a)pyrene- and aflatoxin-induced HCCs in humans These results establish a novel approach for classifying liver carcinogens and understanding the mechanisms of hepatocellular carcinogenesis. To determine the feasibility of using NTP FFPE samples, we prepared paired FF and FFPE samples from each HCC and subjected them to exome sequencing in parallel

Methods

Results

Conclusion