Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer

Abstract

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

Keywords: EC; NGS; SETBP1; cell-free DNA; cfDNA; circulating tumor DNA; ctDNA; sequencing.

Figure 1

Consort diagram of EC patient samples and workflow.

Figure 2

Oncoplot of detected somatic mutations and copy number changes in 20 EC tumors. The patients were sorted to group them by mutated genes.

Figure 3

Oncoplot of affected pathways in 20 EC tumors, based on mutated genes. The patients were sorted to group them by affected cancer pathways.

Figure 4

Amounts of cfDNA in baseline plasma (time of diagnosis, i.e., before neoadjuvant therapy or tumor resection) available for 19 of 21 EC patients. Patients with ESCC (FR13 and FR27) are marked with a red triangle. The patients with EAC are marked with a round blue dot.

Figure 5

Mutant allele frequency in baseline plasma samples (time of diagnosis) detected in 13 of 19 EC patients. The figure shows the maximal variant allele frequency (VAF) of the most prominent somatic mutation detected in cfDNA. The maximal VAF was 25.4% (FR43). For FR11, FR24, FR29, FR37, and FR59 no confident somatic single nucleotide substitutions were detected in cfDNA, possibly reflecting low VAFs in the TT (7%, 2%, 1%, 7%, and 4%). Patients with ESCC (FR13 and FR27) are marked with a red triangle. The patients with EAC are marked with a round blue dot.

Figure 6

Longitudinal blood sampling of EC patients, their staging, and their clinical course. On the left side, the patient IDs, affected gene(s), and type(s) of displayed mutation are given. In the middle, the VAF of tumor mutations is given at the sampling times (M: month; M0, M4, etc.). The straight lines between sampling time points are drawn to facilitate comparison and do not represent the VAF between sampling time points. Only the most prominent mutation is depicted, except for patient FR52 with two similarly high VAFs in TET2 and NOTCH1. The red bar (Neo) shows the length of neoadjuvant therapy. On the right side, the postoperative UICC classification and response and/or metastasis manifestation (met) are given.