Tumor genomic profiling and personalized tracking of circulating tumor DNA in Vietnamese colorectal cancer patients

Abstract

Background: Colorectal cancer (CRC) is the fifth most common cancer with rising prevalence in Vietnam. However, there is no data about the mutational landscape and actionable alterations in the Vietnamese patients. During post-operative surveillance, clinical tools are limited to stratify risk of recurrence and detect residual disease.

Method: In this prospective multi-center study, 103 CRC patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissue and paired white blood cells were sequenced to profile all tumor-derived somatic mutations in 95 cancer-associated genes. Our bioinformatic algorithm identified top mutations unique for individual patient, which were then used to monitor the presence of circulating tumor DNA (ctDNA) in serial plasma samples.

Results: The top mutated genes in our cohort were APC, TP53 and KRAS. 41.7% of the patients harbored KRAS and NRAS mutations predictive of resistance to Cetuximab and Panitumumab respectively; 41.7% had mutations targeted by either approved or experimental drugs. Using a personalized subset of top ranked mutations, we detected ctDNA in 90.5% of the pre-operative plasma samples, whereas carcinoembryonic antigen (CEA) was elevated in only 41.3% of them. Interim analysis after 16-month follow-up revealed post-operative detection of ctDNA in two patients that had recurrence, with the lead time of 4-10.5 months ahead of clinical diagnosis. CEA failed to predict recurrence in both cases.

Conclusion: Our assay showed promising dual clinical utilities in residual cancer surveillance and actionable mutation profiling for targeted therapies in CRC patients. This could lay foundation to empower precision cancer medicine in Vietnam and other developing countries.

Keywords: circulating tumor (ctDNA); minimal residual disease (MRD); mutational landscape; next-generation sequencing (NGS); somatic mutation.

Conflict of interest statement

V-AH, T-AT, TH, DQN, Y-TL, H-NN, HG and LT are current employees of Gene Solutions, Vietnam. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Nguyen, Nguyen, Nguyen Hoang, Tran, Le Trinh, Le, Nguyen Tran, Pham, Dinh, Nguyen, Nguyen The, Mai, Chu, Pham, Nguyen, Ngo Ha, Nguyen, Nguyen, Lu, Do Thi, Truong, Nguyen, Nguyen, Giang and Tu.

Figures

Figure 1

Schematic of study design and K-Track® assay. (A) 103 patients with primary colorectal cancer stage I-IV, eligible for curative-intent surgery were enrolled. Serial plasma samples were collected before surgery and at scheduled visits after surgery. FFPE samples of surgically removed tumors were also collected. Clinical outcomes were recorded at each visit. (B) Genomic DNA of paired FFPE and WBC were sequenced to profile all tumor-specific somatic alterations in 95 cancer-associated genes. Top 5 mutations were selected by our K-Track® scoring algorithm and then used to monitor ctDNA presence in plasma samples by a bespoke multiplex PCR assay and ultra-deep sequencing at an average of 100,000X.

Figure 2

Mutational spectrum of 95 genes in the Vietnamese colorectal cancer patients. (A) The average number of tumor-derived mutations was 7 mutations per patient and not different by stage. (B) The mutation burden was not different by the tumor site. (C) Pie chart showing the distribution of mutation classes identified in 95 genes. (D) The top 25 significantly mutated genes in our cohort. (E) Mutation frequency of top mutated genes in our cohort was compared with published datasets of Caucasian and Asian cohorts. (F) Mutually exclusive and co-occurring mutated genes in our dataset. *P < 0.05; Kruskal-Wallis and post hoc Dunn’s test for (A, B).

Figure 3

Oncogenic signaling pathways and actionable alterations in the Vietnamese colorectal cancer patients. (A) The top three signaling pathways with frequent oncogenic alterations in our cohort were Wnt/β-catenin signaling, genome integrity, and MAPK signaling. (B) Proportions of patients harboring mutations in KRAS and NRAS predictive of resistance to Cetuximab and Panitumumab respectively. Frequency of the specific resistance mutations was also illustrated. (C) Proportions of patients carrying mutations that are candidate biomarkers for response to drugs with compelling clinical evidence (level 3) or laboratory evidence (level 4) as classified by the OncoKB database.

Figure 4

Detection of ctDNA in plasma samples. (A) The average number of mutations selected to track was 5 mutations per patient regardless of cancer stage. (B) Detection rate of ctDNA in pre-operative plasma samples was 90.5%. (C) Pre-operative ctDNA detection rate was associated with TNM stage, as the rate in stage I was significantly lower than in stage II and III. Nodal involvement, histological grade and CEA level status did not affect the detection rate. (D) Pre-operative CEA level was found elevated (≥5 ng/mL) in only 41.3% patients. (E) Total levels of cfDNA were not different between pre-operative and post-operative plasma samples while ctDNA and CEA levels significantly reduced after surgery. *P < 0.05; Kruskal-Wallis and post hoc Dunn’s test for (A); Chi-squared test and Fisher’s exact test for (C); Mann-Whitney U test for (E).

Figure 5

Longitudinal monitoring of ctDNA and clinical outcomes of patients. (A) Swimmer plot depicting ctDNA results over time and incidence of relapse in 19 patients that had been followed up for at least 16 months. This was an interim analysis as the study is on-going. (B, C) Longitudinal plot showing the mean VAF of ctDNA, CEA level, treatment and clinical status over time of patients ZMC002 and ZMC006. Molecular relapse detection was 4 months earlier than clinically diagnosed relapse in patient ZMC006. CEA level was still normal at the time point when ctDNA was found positive. Op, operation, CRT, chemoradiotherapy.