Plasma mutation profile of precursor lesions and colorectal cancer using the Oncomine Colon cfDNA Assay

doi:10.21203/rs.3.rs-4694299/v1

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Research Article

Plasma mutation profile of precursor lesions and colorectal cancer using the Oncomine Colon cfDNA Assay

https://doi.org/10.21203/rs.3.rs-4694299/v1

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Background

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Early detection of precursor lesions or early-stage cancer could hamper cancer development or improve survival rates. Liquid biopsy, which detects tumor biomarkers, such as mutations, in blood, is a promising avenue for cancer screening.

Aim

To assess the presence of genetic variants in plasma cell-free tumor DNA from patients with precursor lesions and colorectal cancer using the commercial Oncomine Colon cfDNA Assay.

Material and Methods

Cell-free DNA (cfDNA) samples from the blood plasma of 52 Brazilian patients were analyzed. Eight patients did not have any significant lesions (five normal colonoscopies and three hyperplastic polyps), 24 exhibited precursor lesions (13 nonadvanced adenomas, ten advanced adenomas, and one sessile serrated lesion), and 20 patients with cancer (CRC). The mutation profile of 14 CRC-associated genes were determined by next-generation sequencing (NGS) using the Oncomine Colon cfDNA Assay in the Ion Torrent PGM/S5 sequencer.

Results

Thirty-three variants were detected in eight genes (TP53, PIK3CA, FBXW7, APC, BRAF, GNAS, KRAS, and SMAD4). No variants were detected in the AKT1, CTNNB1, EGFR, ERBB2, MAP2K1 and NRAS genes. All variants were considered pathogenic and classified as missense or truncating. The TP53 gene harbored the most variants (48.48%), followed by the KRAS gene (15.15%) and the APC gene (9.09%). It was possible to detect the presence of at least one variant in cfDNA in 60% of CRC patients (12/20) and 25% of precursor lesions (6/24), which included variants in three patients with nonadvanced adenoma (3/13–23.08%) and three with advanced adenomas (3/10–30%). No variants were detected in the eight patients with normal findings during colonoscopy. The detection of mutations showed a sensitivity of 60% and a specificity of 100% for detecting CRC and a sensitivity of 50% and a specificity of 100% for detecting advanced lesions.

Conclusion

The detection of plasma NGS-identified mutations could assist in early screening and diagnostic of CRC in a noninvasive manner.

Colorectal cancer

precursor lesion

liquid biopsy

screening

NGS

plasma

Colorectal cancer (CRC) is the second leading cause of cancer death for both men and women worldwide, accounting for more than 9% of all cancer deaths [1]. The estimated incidence for Brazil, according to the Brazilian National Cancer Institute, is over 45,000 CRC cases for 2023, presenting an elevated cancer mortality rate [2, 3]. In this scenario, the early diagnosis enables a timely treatment at the initial stage of the disease and is recognized as an effective way to improve the survival rate of patients [4, 5].

Currently, colonoscopy and noninvasive methods, such as the fecal occult blood test, have been used in CRC screening as strategies for reducing mortality caused by this cancer [5, 6].

Although colonoscopy-based screening has proven to be highly effective in detecting precursor lesions and CRC, it is a costly and invasive procedure with a risk for complications; therefore, large-scale use of this approach is challenging [7]. Stool-based tests also have limitations, associated with false-positive results leading to unnecessary endoscopic examinations (mainly guaiac-based tests) and a false-negative rate of approximately 35% [8]. Moreover, the sensitivity of the fecal immunochemical test (FIT) for advanced adenomas can be as low as 40% [9]. Our group recently published the initial findings from a colorectal cancer screening initiative in a specific region of Brazil. The program utilizes the fecal immunochemical test (FIT) as the initial screening method, followed by a diagnostic colonoscopy for positive cases. Our results highlighted impressive metrics, including high test completion rates, overall participation, successful colonoscopy procedures, and early cancer detection at its initial stages. Nevertheless, we did observe a notable occurrence of positive FIT results not aligning with positive findings during subsequent colonoscopies [10]. Therefore, new molecular screening strategies are emerging to improve early CRC detection [11, 12] and screening outcome results [13].

The analysis of circulating cell-free DNA (cfDNA) in blood is a promising noninvasive approach for CRC detection and management [14, 15]. Recently, knowledge of the molecular profile of lesions has led to the development of new, noninvasive tests based on detecting these molecular alterations. Over the last few years, the rapid development of next-generation sequencing (NGS) technology has made it feasible for clinical application. Recently, a muti-test was developed (CancerSeek) for detecting multiple types of cancer, including CRC, combining the detection of mutations in ctDNA using NGS and determining the levels of circulating proteins [16].

Our group recently reported the mutational profile of precursor lesions and CRC in lesions of a Brazilian population [17, 18]. Herein, we intend to evaluate the mutation profile in liquid biopsy, blood plasma from patients with precursor lesions, and colorectal cancer samples from the Barretos Cancer Hospital CRC screening program using the NGS Oncomine Colon cfDNA Assay, which contains 14 colorectal cancer-related genes.

Study population and sample collection

We evaluated 52 patients who underwent colonoscopy for diagnosis or after a positive FIT test at Barretos Cancer Hospital from 2015 to 2019 [19][10]. The Research Institutional Board of the Barretos Cancer Hospital approved the study (753/2013 and 1060/2015), and all patients signed informed consent before enrollment.

The detected lesions were classified according to the WHO criteria [20] as previously reported [10][21]. Subjects with a personal history of familial adenomatous polyposis (FAP) or Lynch syndrome and a prior history of CRC or other types of cancer were excluded. Tumors were staged according to the Joint Committee on Cancer (AJCC) 7th edition TNM staging system [22]. When more than one lesion was detected per patient, the classification was based on the worst lesion detected following the order of severity: adenocarcinoma (cancer), advanced adenoma (AA), nonadvanced adenoma (NAA), sessile serrated lesion (SSL), and hyperplastic polyp (HP).

Patient peripheral blood was collected in EDTA tubes (BD vacuntainer®), and after centrifugation at 1500 rpm for 10 min at 4°C, the plasma was separated, stored in aliquots, and frozen at -80°C until processing [23].

Cell-free DNA (cfDNA) isolation and quality control

After a second centrifugation step of 16000 × g for 10 min at 4°C, cell-free DNA was isolated from 1 to 4mL of plasma by using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. The isolated cell-free DNA was quantified using the Qubit™ dsDNA High Sensitivity Assay Kit with a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA). cfDNA purity and size distribution were assessed using TapeStation 2200 (Agilent®) with a High Sensitivity D1000 ScreenTape system (Agilent®).

Mutation profile by next-generation sequencing

cfDNA was used for variant detection of 14 CRC-associated genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, and APC) using the commercial Oncomine Colon cfDNA Assay (Thermo Fisher Scientific, Austin, TX). In brief, 1 to 27ng of cell-free DNA was used to amplify 49 amplicons covering 236 hotspots and indels of the 14 genes according to the manufacturer’s instructions. Libraries were quantified using Ion Library TaqMan® Quantitation Kit (Thermo Fisher Scientific®). The quantified libraries were pooled and subjected to automated preparation and enrichment using 50pM of purified libraries in Ion Chef System (Life Technologies, Carlsbad, CA) and Ion 540 Kit-Chef (Thermo Fisher Scientific®). Sequencing was performed using Ion PGM Hi-Q View Sequencing Kit (Life Technologies, Carlsbad, CA) and Ion 540 Kit-Chef (Thermo Fisher Scientific®) on Ion Torrent PGM/S5 sequencer, respectively.

Sequencing data was processed in the Torrent Server, and the Human genome build 19 (hg19 or GRCh37) was used as the reference genome for aligning the sequenced reads using the Torrent Mapping Alignment Program (TMAP) in Torrent Suite™ Software (Thermo Fisher). Variant calling and annotation were performed using the workflow for Oncomine Colon Liquid Biopsy—w1.8 – DNA in the Ion Reporter™ Software (version 5.18) with default parameters.

Statistical analysis

All the statistical analyses were performed using SPSS software (v.21) and R software (v. 3.6.1). Categorical variables were compared using Fisher’s exact test and the chi-square test. The nonparametric data were analyzed using the Kruskal–Wallis and Dunn–Bonferroni post hoc correction. A p value less than 0.05 was considered to indicate statistical significance.

Clinical characterization of the population

The clinicopathological features of the 52 patients are summarized in Table 1. The average age was 61.14 years, and 53.8% of patients were female. The study population comprised eight patients with normal colonoscopy (n = 5) or hyperplastic polyps (n = 3) (N), 13 with nonadvanced adenomas (NAA), 10 with advanced adenomas (AA), 1 with a sessile serrated lesion (SSL) and 20 with colorectal cancer (CRC) at different stages, with stage III being the most frequent (45%) (Table 1).

cfDNA and NGS quality control and metrics

Among the 52 cases, a median of 17.94 ng (range of 6.64 to 27.06 ng) of cfDNA was used as input for sequencing analysis, with 14.9ng (range 8.94- 20ng) for normal samples, 20ng (range 18.98-20ng) for hyperplastic polyps, 16.77ng (range 7.09-20ng) for non-advanced adenomas, 19.16ng (range 10.27-20 ng) for advanced adenomas, 12.3ng for the only SSA sample analyzed, and 13.30ng (range 6.64 -27.07ng) for cancer.

The median value of all samples for median read coverage was 9658 (range 2021–28617), and the median molecular coverage was 1354 (range 320–3221). The distribution of median read coverage and the median molecular coverage according to the type of lesion, the input of cfDNA, and the chip used for sequencing are represented in Supplementary Fig. 1. The average number of reads per functional molecule with a unique molecular index was 7.96 (± 6.6). The average coverage per amplicon, number of mapped reads, on target percent and mean depth per sample are shown in Supplementary Fig. 2 and Supplementary Table 1.

Genetic variants detected in ctDNA of precursor lesions and colorectal cancer

We observed genetic variants in 34.62% of the participants (18 of 52). No variants were detected in participants with normal colonoscopy or hyperplastic polyps or in SSL participants. It was possible to detect the presence of at least one variant in the ctDNA of 6 adenoma participants (6/24–25%), including variants in 3 patients with nonadvanced adenoma (3/13–23.08%), 3 with advanced adenomas (3/10–30%) and twelve with CRC (12/20–60%).

The presence and number of plasma mutations per case were greater in cancer patients than in the other participants, and they were significantly greater in CRC patients when compared with participants with normal colonoscopy or hyperplastic polyps (p = 0.044 and 0.031, respectively) (Table 2 and Fig. 1A).

The number of mutations observed per case varied from none (the majority) to 4 (one cancer patient) (Fig. 1A). The presence and number of cfDNA mutations were not associated with cancer stage (Table 2). However, differences were observed in cancer grade, with more variants detected in poorly differentiated cancer than in differentiated cancer (adjusted p = 0.043) (Fig. 1B). The molecular frequency (mutant allele frequency) tended to be greater in plasma from cancer patients when compared to non-advanced and advanced adenoma (Fig. 1C) or more advanced stages of disease (stage III and stage IV) (Fig. 1D), yet not statistically significant (p = 0.63 and p = 0.49, respectively).

Thirty-three variants were detected in eight genes (Table 3). The variants were classified as missense (90.91%) or nonsense (9.09%) and considered pathogenic. The TP53 gene had the greatest number of variants, accounting for 48.48% of all variants, followed by KRAS (15.15%) and APC (9.09%) (Fig. 2).

The sensitivity, specificity and positive and negative predictive values were calculated for each lesion group (cancer, advanced adenoma, nonadvanced adenoma, and cancer + advanced adenoma). Plasma NGS showed a sensitivity of 60% for cancer patients and 50% for cancer + advanced adenoma group, while it had a sensitivity of only 30% and 23.1% for advanced adenoma patients and nonadvanced adenoma patients, respectively (Table 4, Fig. 3).

In the present study, we investigated, for the first time, the mutation profile in plasma cfDNA of Brazilian patients diagnosed with precursor lesions and colorectal cancer using the commercial Oncomine Colon cfDNA Assay.

The colorectal screening process comprises the use of colonoscopy, a gold standard method that allows the detection and removal of precursor lesions and colorectal tumors, with a reduction in morbidity and mortality [24]. A simple, noninvasive test with high sensitivity for detecting CRC and its precursor lesions and with high compliance of the population could help reduce the incidence and mortality of this cancer. Therefore, blood tests could improve compliance and outcomes in CRC screening programs [25]. Limitations in the use of blood molecular tests for the screening of asymptomatic, average-risk individuals and early detection rely on the suboptimal limit of detection for early stages CRC and precursor lesions. The release of circulating tumor DNA (ctDNA) and its detection are associated with initial tumor burden, suggesting that tumor volume and the presence of metastasis are significantly related to the detection of ctDNA in plasma [26, 27]. In addition, the small proportion of ctDNA in the total circulating cell-free DNA (the fraction of ctDNA that can span less than 0.01%) constitutes another issue to overcome in detection methodology and the feasible implementation for clinical application [28, 29].

Studies evaluating the presence of mutations in plasma as a potential utility of liquid biopsy in detecting early stages of CRC have been reported [16, 30]. However, mutation analysis via liquid biopsy of patients with precursor lesions is scarce. Analysis of single-gene ctDNA mutations shows low accuracy and is unlikely to be suitable for screening methods [31, 32].

In our work, the feasibility of a noninvasive mutation-based biomarker was tested using plasma samples and NGS in CRC screening program participants and colorectal cancer patients. A total of 35 mutations were identified in 53 samples, with a greater percentage of mutated samples (63.63%) and average number of mutations (1.27 mutations per sample) in CRC samples, compared to precursor lesions (adenoma) (19.23% of samples analyzed harbored mutations and 0.27 mutations per sample). Notably, using the same sequencing panel, Eikenboom et al. [33] observed a mutation rate of 0.6 mutations per sample in CRC, and mutations were not detected in AA. Low or no mutation rates were detected in colorectal precursor lesions using other sensitive techniques, such as QClamp XNA-based PCR and droplet digital PCR [34, 35]. Although cfDNA is a stable molecule with potential for CRC screening analysis, the proportion of ctDNA and the amount released by tumors in precursor stages and early stages of cancer can be a challenge in the analysis of mutations in the plasma of screening candidates [36]. This fact is reflected in the low number of mutations detected in precursor lesions in this study, even in advanced adenomas. In addition, the allele frequency of detected mutations was greater in cancer samples, corroborating the data showing that more advanced lesions release more DNA molecules into the bloodstream.

Studies have demonstrated the potential for the detection of precursor lesions using markers based on gene expression [37, 38] or DNA methylation [39], in addition to DNA mutation [40, 41]. Our group reported that the combination of SEPT9 and BMP3 methylation in ctDNA and age > 60 years achieved 74.0% sensitivity and 54.0% specificity for detecting AA + CRC compared to normal samples study [42], indicating that despite good sensitivity, cfDNA methylation presented poorer specificity when compared to mutation biomarker in the current work. Enthusiastic results were recently reported, by simultaneous analysis of cfDNA genomic alterations, aberrant methylation status, and fragmentomic patterns using Shield ^TM test [43]. The authors showed a sensitivity of 83.1% for CRC and 13.2% for advanced adenomas and sessile serrated lesions, with a specificity of 89.6% for the test [43].

In the present study, we obtained a ROC curve (AUC 0.8) value, indicating the good performance of ctDNA mutation in terms of sensitivity and specificity in detecting CRC. However, for adenoma detection, the sensitivity decreases to 30% due to the low concentration of ctDNA released from precursor lesions. Similar results were observed using a sequencing cfDNA panel, with a higher sensitivity (53.8%) and low specificity (92.3%) [44]. Another justification for the low sensitivity of adenoma detection relies on the performance of the Oncomine Colon cfDNA Assay. This platform requires 20ng of the input of cfDNA used for sequencing to achieve a limit of detection of 0.1%, which may decrease the sensitivity for detecting variants in precursor lesions.

Despite the low sensitivity for detecting nonadvanced lesions, in our study, no mutated ctDNA was detected in participants with normal colons. This high specificity demonstrated for the mutation-based biomarker could render this test a good candidate to complement stool-based tests in clinical practice and thus avoid unnecessary invasive diagnostic procedures. Due to the small number of samples analyzed, stratification by cancer stage for mutation analysis was not performed.

In conclusion, we showed that the commercial Oncomine Colon cfDNA Assay represents a feasible approach for mutation detection in a minimally invasive way. Concerning the power of this tool for colorectal cancer screening of ctDNA from plasma samples, improvements should be made to increase the accuracy of precursor lesions detection.

Ethics approval and consent to participate

The Research Institutional Board of the Barretos Cancer Hospital approved the study (753/2013 and 1060/2015), and all patients signed informed consent before enrollment.

Availability of data and materials

The data are available and will be shared upon reasonable request to corresponding author.

Conflict of interest

The authors have declared no competing interests.

Funding

The Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer, Brazil) supported this study. MBR received a postdoc fellowship from the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES, Brazil). WS was a recipient of a fellowship in the postgraduate program of Barretos Cancer Hospital. RMR is a recipient of the National Council for Scientific and Technological Development (CNPq, Brazil) Productivity Fellowship.

Author Contributions

M.B.R. and W.S. designed and performed the experiments, analyzed the data and wrote the manuscript. A.B.L performed the experiments. M.B.R., W.S. A.BL., A.C.C, M.T.R, F.S., R.M.R. and D.P.G. performed data curation. A.C.C. contributed to the interpretation of the results. R.M.R. and D.P.G. conceived the original idea and supervised the project. R.M.R. and D.P.G. edited and revised the manuscript. All authors critically reviewed and approved the ﬁnal manuscript.

Acknowledgments

The authors thank the Barretos Cancer Hospital and Biobank for processing blood samples and the Colorectal Cancer Group.

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Tables 1 to 4 are available in the Supplementary Files section.

No competing interests reported.

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Plasma mutation profile of precursor lesions and colorectal cancer using the Oncomine Colon cfDNA Assay

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Abstract

Background

Aim

Material and Methods

Results

Conclusion

Figures

Introduction

Material and Methods

Study population and sample collection

Cell-free DNA (cfDNA) isolation and quality control

Mutation profile by next-generation sequencing

Statistical analysis

Results

Clinical characterization of the population

cfDNA and NGS quality control and metrics

Genetic variants detected in ctDNA of precursor lesions and colorectal cancer

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1