Patient and Tumor-Specific Multiplexed Circulating Tumor DNA Response to Multi-Modality Therapy for Esophageal and Rectal Cancer: Preliminary Results of a Pilot Study

Abstract

As non-operative management (NOM) of locally-advanced esophageal (EC) and rectal cancer (RC) is becoming more prevalent, highly sensitive and specific serum biomarkers of treatment response are needed. In this pilot study, we hypothesized that serially monitored patient and tumor-specific mutations in circulating tumor DNA (ctDNA) throughout multi-modality treatment using a novel, highly sensitive quantification method would closely associate with clinical outcomes. Plasma specimens were collected at baseline, through neoadjuvant therapy (NAT), immediately prior to surgery and in follow up. In each patient (n=4 for EC, n=2 for RC), we identified 30-50 tumor-specific mutations from whole exome sequencing using tumor tissue to longitudinally monitor ctDNA in plasma. Using a patient-specific hybrid capture technique (DIDAseq, dual-index degenerate adaptor sequencing - a custom library sequencing approach that significantly reduces polymerase errors), we were able to identify mutant alleles at a frequency of 1 in 10-50k site observations. This variant allele frequency was used to estimate mutant genomes/mL plasma. All patients had detectable ctDNA at baseline. For all but one patient, tumor-specific ctDNA levels showed a marked decrease during NAT. Three of four EC patients underwent esophagectomy, including one whose ctDNA levels became undetectable prior to surgery, with surgical pathology revealing a pathologic complete response (pCR). Another EC patient had low persistent ctDNA levels following CRT with final pathology revealing minimal residual disease. One patient with oligometastatic EC controlled with dual-checkpoint inhibition received CRT prior to esophagectomy as this was the only site of active disease. Throughout CRT, ctDNA levels persistently increased, correlating with radiographic/endoscopic progression and resistance to CRT, with ctDNA levels precipitously dropping following esophagectomy. Shortly after esophagectomy, distant metastatic disease was confirmed with biopsy. Post-hoc analysis of plasma revealed presence of metastasis-specific ctDNA at time-points prior to esophagectomy, representing sub-clinical distant metastatic progression. Two RC patients were treated with NOM consisting of NAC and CRT. Both patients exhibited a clinical complete response and were closely observed rather than proceeding with surgery. CtDNA levels were persistently detectable for both patients; ultimately with local recurrence confirmed on endoscopy 6 and 10 months following NAT. In this pilot study, patient and tumor-specific ctDNA analysis throughout multi-modality therapy for EC and RC was shown to be of practical use in assessment of treatment response, confirming pCR and predicting local recurrences with significant clinical lead times. However, the identification of metastasis-only ctDNA may confound efforts for early detection. Further studies with additional patients are on-going.