Genomic characterization of esophageal squamous cell carcinoma for tumor heterogeneity and biomarkers associated with treatment outcomes

Abstract

Patients with esophageal squamous cell carcinoma (ESCC) show an overall 5-year survival rate ranging from 15% to 25%. Both the intra-tumor heterogeneity and inter-patient heterogeneity impede effective therapy in ESCC. Multi-regional sampling has confirmed the existence of tumor heterogeneity in many cancers. We are interested in the inter- and intra- tumoral heterogeneity and evolution of tumor. Besides, we hypothesize that circulating tumor cells (CTCs) would reflect the tumor heterogeneity of primary sites and that tumor heterogeneity may contribute to chemoresistance in ESCC. In this study, both treatment-naive ESCC and ESCC-related synchronous cancers, along with their CTCs, were studied. Tissues with more than 30% cancer cells were selected for whole-exome sequencing (WES). In the meantime, CTCs were isolated using microfluidic chips using 20 ml of fresh blood. The WES data were processed following the Genome Analysis Toolkit (GATK) best practice and in-house pipeline for single nucleotide variants (SNVs), insertion and deletions (INDELs) and copy number variations (CNVs). Various degrees of intra-tumor heterogeneity were observed in ESCC. Trunk and branch mutations were identified for each case. In contrast, the five synchronous cancer cases show completely distinct mutation landscapes and CNVs between the primary sites except for selected genes. Common mutated genes (TP53, ZFHX4), etc and chromosome 3q amplification were observed in both tumors. Mutation signatures relating to aging, Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) and microsatellite instability (MSI) were seen among ESCC patients. Surprisingly, a mutation signature related to alcohol consumption was prevalent in ESCC-related dual cancers. Only 40% ESCC patients showed this signature. This signature may offer hints in further studies in multiple synchronous tumors in the esophagus and head and neck. Comparisons between the primary tumor and circulating-tumor DNA (ctDNA) detected shared clonal mutations in TP53, PIK3CA and EYS. Hong Kong clinicians demonstrated that complete pathological response in the primary tumor after the cisplatin therapy was a prognostic marker for better survival, while the traditional Tumor node metastasis (TNM) staging of the patients before treatment failed to reflect this. However, little is known about the mutation profiles between good responders (patients with no viable tumor cells after therapy) and poor responders (more than 50% viable tumor cells remaining after therapy). Archived formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks of 37 good responders and 42 poor responders were screened for somatic SNVs and CNVs targeting 130 cancer druggable genes. I found that some pathogenic TP53 mutations occur more frequently in poor responders, although this did not reach statistical significance. Copy number amplification in CDK5, DPYD, EGFR, ARAF and MCL1 and deletion in AXL and KIT may be associated with good response to chemotherapy. These retrospective Next-generation sequencing (NGS) studies with Formalin-fixed, paraffin-embedded (FFPE) discovered potential biomarkers associated with cisplatin responses and offer hints for future studies. In summary, my projects integrate both prospective studies with fresh tissues and retrospective studies using FFPE tissue blocks from ESCC patients. Identification of potentially useful biomarkers from tissues and CTCs may aid diagnosis of patients to facilitate effective treatments.