The use of personalized biomarkers and liquid biopsies to monitor treatment response and disease recurrence in locally advanced rectal cancer after neoadjuvant chemoradiation

Abstract

Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the mainstay treatment for locally advanced rectal cancer. Variable degrees of tumor regression are observed after nCRT and alternative treatment strategies, including close surveillance without immediate surgery, have been investigated to spare patients with complete tumor regression from potentially adverse outcomes of radical surgery. However, clinical and radiological assessment of response does not allow accurate identification of patients with complete response. In addition, surveillance for recurrence is similarly important for these patients, as early detection of recurrence allows salvage resections and adjuvant interventions. We report the use of liquid biopsies and personalized biomarkers for monitoring treatment response to nCRT and detecting residual disease and recurrence in patients with rectal cancer. We sequenced the whole-genome of four rectal tumors to identify patient-specific chromosomal rearrangements that were used to monitor circulating tumor DNA (ctDNA) in liquid biopsies collected at diagnosis and during nCRT and follow-up. We compared ctDNA levels to clinical, radiological and pathological response to nCRT. Our results indicate that personalized biomarkers and liquid biopsies may not be sensitive for the detection of microscopic residual disease. However, it can be efficiently used to monitor treatment response to nCRT and detect disease recurrence, preceding increases in CEA levels and radiological diagnosis. Similar good results were observed when assessing tumor response to systemic therapy and disease progression. Our study supports the use of personalized biomarkers and liquid biopsies to tailor the management of rectal cancer patients, however, replication in a larger cohort is necessary to introduce this strategy into clinical practice.

Keywords: ctDNA; liquid biopsies; neoadjuvant therapy; personalized biomarkers; rectal cancer.

Figure 1. Patient-specific chromosomal rearrangements represented as Circos-plots

Chromosome representations are show around the outer cycle and are in a clockwise orientation starting from chromosome 1. Interchromosomal translocations are represented by colored lines linking two chromosomes. Intrachromosomal deletions and inversions are represented by gray lines. * Indicates patient-specific chromosomal rearrangements selected for ctDNA monitoring.

Figure 2. Detection of tumor-specific chromosomal rearrangements in liquid biopsies from Patient #1

Serial blood samples were collected prospectively at diagnosis (biopsy), during the resting interval (weeks 3 and 9), at the time of clinical evaluation of response (week 13), and during follow-up (weeks 40, 46 and 84). The initial treatment for Patient #1 included nCRT and radical surgery for removal of residual tumor. Subsequently, the patient was submitted to a hepatectomy after the diagnosis of liver metastasis and offered palliative treatment after detection of bone metastasis. TaqMan assays amplifying patient specific chromosomal rearrangements (T01 and T02) and a single copy non-rearranged genomic region (RNAse P) were designed to measure ctDNA and total cell-free DNA levels, respectively. ctDNA levels are plotted as relative amplifiable copies/ml of plasma. The horizontal dashed line indicates ctDNA detection limit. CEA levels in μg/ml are plotted as solid bars.

Figure 3. Detection of tumor-specific chromosomal rearrangements in liquid biopsies from Patient #2

Serial blood samples were collected prospectively at diagnosis (biopsy), during the resting interval (weeks 1, 6 and 8), at the time of clinical evaluation of response (week 13), and during follow-up (weeks 52, 80 and 124). The initial treatment for Patient #2 included nCRT and radical surgery for removal of residual tumor. TaqMan assays amplifying patient specific chromosomal rearrangements (D01 and D02) and a single copy non-rearranged genomic region (RNAse P) were designed to measure ctDNA and total cell-free DNA levels, respectively. ctDNA levels are plotted as relative amplifiable copies/ml of plasma. The horizontal dashed line indicates ctDNA detection limit. CEA levels in μg/ml are plotted as solid bars.

Figure 4. Detection of tumor-specific chromosomal rearrangements in liquid biopsies from Patient #3

Serial blood samples were collected prospectively at diagnosis (biopsy), at the end of nCRT (week 0) during the resting interval (week 6), at the time of clinical evaluation of response (week 13), and during follow-up (weeks 70 and 118). The initial treatment for Patient #1 included nCRT and radical surgery for removal of residual tumor. TaqMan assays amplifying patient specific chromosomal rearrangements (T01 and T02) and a single copy non-rearranged genomic region (RNAse P) were designed to measure ctDNA and total cell-free DNA levels, respectively. ctDNA levels are plotted as relative amplifiable copies/ml of plasma. The horizontal dashed line indicates ctDNA detection limit. CEA levels in μg/ml are plotted as solid bars.

Figure 5. Detection of tumor-specific chromosomal rearrangements in liquid biopsies from Patient #4

Serial blood samples were collected prospectively at diagnosis (biopsy), during the resting interval (weeks 3, 6 and 9), at the time of clinical evaluation of response (week 13), and during follow-up (weeks 46, 62, 72, 84, 158, 171 and 177). The initial treatment for Patient #4 included nCRT and the Watch and Wait Approach. Subsequently, the patient was submitted to different lines of chemotherapy (CT). TaqMan assays amplifying patient specific chromosomal rearrangements (I01 and D02) and a single copy non-rearranged genomic region (RNAse P) were designed to measure ctDNA and total cell-free DNA levels, respectively. ctDNA levels are plotted as relative amplifiable copies/ml of plasma. The horizontal dashed line indicates ctDNA detection limit. CEA levels in μg/ml are plotted as solid bars.