1. Ultra-Deep Sequencing of Multiplex-PCR Enriched Hotspot and
Disease-Related Targets in Hepatocellular Carcinoma
Wafa Amer1, Alexander Quaas1, Uta Drebber1, Hannah Eischeid1, Ulrike Koitzsch1, Anne Arens2, Claudia Vollbrecht1, Reinhard Büttner1, Margarete Odenthal1
1Institute for Pathology, University Hospital of Cologne, Germany
2QIAGEN, Inc, Hilden, Germany
BACKGROUND
RESULTS
Hepatocellular carcinoma (HCC) is the most common liver malignancy and ranks as one of the most critical worldwide health threats with regards to mortality.
The key to significantly altering the prognosis and minimizing tumour-related deaths is early detection of HCC followed by appropriate treatment.
Next generation sequencing (NGS) technologies provide the simultaneous analysis of genomic mutations in numerous target regions with high sensitivity.
Point mutations and small insertions/deletions (indels) in tumor relevant genes have been identified and proposed as new indicators in hepatocellular carcinoma prognosis.
The HCC Gene Panel for Mutation Analysis
Genomic Mutation Pattern in Hotspot Loci of HCC Nodules
We obtained 394 amplicons from two hotspot cancer gene panels with median coverage rate of 96%.
OBJECTIVES
Point mutations and small insertions/deletions (indels) in tumor relevant genes have been identified, with frequencies of nearly one third of mutations in the N-terminus of the β-catenin (CTNNB1) and 20 % of the mutations in the DNA binding domain of TP53.
Different nodules of one liver biopsy showed mostly but not always similar variant pattern, but to a different extend compared to what we expected from histological classification of the hepatocellular dedifferentiation.
Genomic Mutation Pattern in Hotspot Loci of HCC Nodules
Based on data by whole exome analysis, a hotspot targeted multiplex PCR approach was combined with ultra-deep sequencing and applied to hepatocellular carcinoma representing the genetic landscape in HCC and different grade of progression.
MATERIAL & METHOD
Workflow for Ultra-Deep Sequencing of HCC Related Cancer Genes
CONCLUSIONS
We established a fast pipeline of mutation analysis of a tumor specific, hotspot gene panel covering diagnostic relevant loci of HCC by reads.
Further tumor nodules are going to get sequenced to find a representative mutation status, for tackling the shortcomings of conventional anti-cancer therapy and providing a better understanding of molecular events leading to hepatocarcino-genesis.
72 HCC nodules of different etiology and grades were macrodissected and DNA extracted.
Two panels of hotspot cancer genes were selected based on the COSMIC database ( and recent cancer research studies. Followed by primer design using the algorithm of ABI Life technologies (Darmstadt, GER).
At least 3000 genomic copies were amplified by multiplex PCR using our designed primer sets, and 394 amplicons of each sample were generated and sequenced by means of the MiSeq platform (Illumina).
Conventional Sanger sequencing served as a reference technology.
Each macrodissected HCC nodule was sequenced by more than one million reads covering each targeted hotspot locus between approximately 400 and 2,000 reads.
Data analysis was performed using an in house developed bioinformatics pipeline optimized for the diagnostic workflow.
Variants were detected in exons of APC, ARID1A, AXIN1, BRAF, CDKN2A, KRAS, MMP9, HNF1, and PTEN.
Variants of beta catenin and TP53 gene were detected with high frequency .
CONTACT INFORMATION
Wafa Amer
Team of Dr. Margarete Odenthal
Institute for Pathology, University Hospital of Cologne
Kerpener Str. 62
50937 Cologne, Germany