Amanda J. Craig

A pilot study of ultra-deep targeted sequencing of plasma DNA identifies driver mutations in hepatocellular carcinoma

Cellular components of solid tumors including DNA are released into the bloodstream, but data on circulating-free DNA (cfDNA) in hepatocellular carcinoma (HCC) are still scarce. This study aimed at analyzing mutations in cfDNA and their correlation with tissue mutations in patients with HCC. We included 8 HCC patients treated with surgical resection for whom we collected paired tissue and plasma/serum samples. We analyzed 45 specimens, including multiregional tumor tissue sampling (n = 24), peripheral blood mononuclear cells (PMBC, n = 8), plasma (n = 8) and serum (n = 5). Ultra-deep sequencing (5500× coverage) of all exons was performed in a targeted panel of 58 genes, including frequent HCC driver genes and druggable mutations. Mutations detected in plasma included known HCC oncogenes and tumor suppressors (e.g., TERT promoter, TP53, and NTRK3) as well as a candidate druggable mutation (JAK1). This approach increased the detection rates previously reported for mutations in plasma of HCC patients. A thorough characterization of cis mutations found in plasma confirmed their tumoral origin, which provides definitive evidence of the release of HCC-derived DNA fragments into the bloodstream. This study demonstrates that ultra-deep sequencing of cfDNA is feasible and can confidently detect somatic mutations found in tissue; these data reinforce the role of plasma DNA as a promising minimally invasive tool to interrogate HCC genetics.

High-density single cell mRNA sequencing to characterize circulating tumor cells in hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) release tumor cells to the bloodstream, which can be detected using cell surface markers. Despite numerous reports suggest a direct correlation between the number of circulating tumor cells (CTCs) and poor clinical outcomes, few studies have provided a thorough molecular characterization of CTCs. Due to the limited access to tissue samples in patients at advanced stages of HCC, it is crucial to develop new technologies to identify HCC cancer drivers in routine clinical conditions. Here, we describe a method that sequentially combines image flow cytometry and high density single-cell mRNA sequencing to identify CTCs in HCC patients. Genome wide expression profiling of CTCs using this approach demonstrates CTC heterogeneity and helps detect known oncogenic drivers in HCC such as IGF2. This integrated approach provides a novel tool for biomarker development in HCC using liquid biopsy.

Diagnostic and Prognostic Performance of Liquid Biopsy in Hepatocellular Carcinoma

Liver cancer is currently the second leading cause of cancer-related deaths worldwide. A majority of patients are diagnosed at intermediate or advanced stage and are no longer candidates for curative treatment. Molecular characterization of the tumor would presumably provide valuable insights for developing novel treatments, but tissue samples are difficult to obtain, especially for nonsurgical patients. Therefore, liquid biopsy represents a noninvasive means to access the genomic information of the tumor and can easily be repeated to monitor tumor evolution. Recent technological progress and preliminary results suggest that liquid biopsy could play a central role in the future management of liver cancer.

Tumor Heterogeneity and Resistance to Targeted Therapies in Hepatocellular Carcinoma

Hepatocellular carcinoma is a heterogeneous disease, both phenotypically and genetically. This heterogeneity can be observed over a spectrum ranging among tumors of different patients, multiple nodules of one patient and even within a single tumor nodule. As demonstrated in other tumor types, this diversity may contribute to the development of drug resistance. The mechanisms behind intra-tumoral heterogeneity are not yet fully understood, and potential theories include the clonal evolution or cancer stem cells differentiation hierarchy. In recent years, many technologies have helped assess clonal content of tumors, including multi-regional and single cell next generation sequencing. Changes in clonal content over time and during treatment can now be easily tracked, which will provide novel insights into cancer evolution and mechanisms of treatment resistance. This will be critical to provide novel therapeutic targets for second and third line therapies in hepatocellular carcinoma.

Divergent evolutionary trajectories in transplanted tumor models

Human-derived tumor models are becoming popular in the context of personalized medicine, but a new study shows that these models could be less representative of primary tumors than previously thought, particularly when using late passages.

Liver capsule: Molecular-based signatures in hepatocellular carcinoma.

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Abstract 2383: Multiregional RNA sequencing identifies intratumor transcriptomic heterogeneity in a subset of early-stage hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is known to have histological intra-tumor heterogeneity. Little is known about the underlying molecular contributions to this observation, or its potential impact on resistance to therapies. AIMS: 1) To evaluate intra-tumoral molecular heterogeneity of primary HCCs using multi-regional RNA-seq; 2) to correlate molecular alterations with histological features at distinct areas. METHODS: We analyzed 55 fresh-frozen tissues from 10 patients with BCLC-A HCC treated with surgical resection. Multi-regional sampling included 38 HCCs and 16 adjacent non-tumoral tissues (average 5.4 samples per patient). We performed HE 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2383.