Discovering methylation markers and development of a sense-antisense and dual-MGB probe PCR assay in plasma for colorectal cancer early detection

doi:10.21203/rs.3.rs-4838443/v1

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Discovering methylation markers and development of a sense-antisense and dual-MGB probe PCR assay in plasma for colorectal cancer early detection

https://doi.org/10.21203/rs.3.rs-4838443/v1

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Background: Screening for colorectal cancer (CRC) using plasma cell-free DNA (cfDNA) methylation is more challenging than stool testing due to the low abundance of cfDNA. Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity.

Methods: A total of 17 existing 450K microarray datasets including tissue, healthy white blood cell (WBC) and plasma cfDNA data from public databases were used to identify differentially methylated CpGs (DMCs) common to CRC and adenoma. The methylation status of candidate DMCs was confirmed by Sanger sequencing with CRC and normal tissues. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples using the SADMP method.

Results: A total of 2237 DMCs showed overlap between the cancer vs. normal and adenoma vs. normal groups. Of these, 75 were hypomethylated in 30 other non-CRC cancers. After LASSO regression, this number was reduced to eight. Two of these, NTMT1 and MAP3K14-AS1, were identified as promising candidate markers following WBC validation and primer/probe design evaluation. The SADMP technology demonstrated the ability to amplify the detection signal to approximately twice the original level. Overall, the dual-target SADMP assay demonstrated a sensitivity of 84.8% for CRC (stage I: 75.0%), a sensitivity of 32.0% for advanced adenomas (AA), and a specificity of 91.5% in controls.

Conclusions: The dual-target assay demonstrated high performance for CRC and AA detection in plasma-based tests, suggesting that it may serve as a promising noninvasive tool for CRC detection.

Cancer Biology

colorectal cancer

methylation

NTMT1

MAP3K14-AS1

The majority of early-stage advanced colorectal neoplasms, including colorectal cancer (CRC) and advanced adenoma (AA) are curable, particularly AA, which can be removed at the time of diagnosis by colonoscopy. Consequently, the early detection of CRC and AA represents a highly valuable endeavor. A number of tests based on cell-free DNA (cfDNA) methylation have been developed and have shown good performance for CRC detection. Epi proColon [1] is the first blood-based test utilized for the early detection of CRC and was developed based on methylated cfDNA of Septin9. The updated version of Epi proColon® 2.0 CE demonstrated an improved sensitivity of 74.8%-81% and specificity of 96.3%-99% in a prospective cohort study [2]. The Chinese version of Epi proColon simplified sample processing with a single reaction system in a larger volume (60 ul) instead of a 2/3 algorithm (20 ul for three runs), resulting a sensitivity of 73% and a specificity of 94.5% [3]. However, its sensitivity for detecting polyps or AA was relatively low in most studies, ranging from 8-40% [4-6]. Due to this relatively poor sensitivity, particularly for precancerous lesions, the US Preventive Services Task Force and the American Cancer Society currently do not include the test in their CRC screening guidelines. Moreover, plasma methylated Septin9 is not a CRC specific marker that it showed an ability to detect multiple cancer types, including hepatocellular carcinoma [7], gastric cancer [8],cervical cancer [9], and breast cancer [10]. Therefore, developing a sensitive and specific blood assay for CRC screening is meaningful.

Due to the disparate mechanisms by which cancer signals enter the blood and stool, it is probable that the markers are not universal, and the accuracy of CRC markers in the blood is typically low in comparison to those in stool. Consequently, an ab initio search for blood methylation markers for early CRC and AA is imperative. Furthermore, the detection of the methylation signal of cell-free tumor DNA (ctDNA) in plasma is challenging due to the presence of ctDNA fragments released by multiple organs or tissues, as well as DNA damage caused by bisulfite conversion [11]. Therefore, the identification of suitable markers and the development of effective detection techniques are essential to ensure optimal performance [12]. Classical PCR-based methylation detection techniques are often developed based on single-strand bisulfite converted DNA (BS-DNA), leaving the information of the other strand unused [13]. Furthermore, only one Taqman MGB probe is usually designed to provide fluorescent signals. Theoretically, designing primers for both sense and antisense strand DNA simultaneously or using multiple MGB probes will improve the sensitivity of a marker to detect methylation signals by enhancing fluorescent signals. Sarah Ø. Jensen et al [14] made the first attempt to design a pair of primers for both sense and antisense strands, thereby enhancing the performance of three targets for CRC detection. Additionally, the dual-strand technique was also successfully applied to methylated HOXA9 in ovarian cancer to improve the detection sensitivity [15].

In this study, we utilized all publicly available methylation data for CRC and adenomas to identify methylation markers, with a particular focus on those that are hypermethylated in adenomas. Furthermore, in order to enhance the ability of candidate markers to detect low-abundance cfDNA methylation signals in plasma, we attempted to apply dual-strand and dual-MGB probe techniques simultaneously, which we called the sense-antisense and dual-MGB probe (SADMP) technique, to develop a novel CRC plasma test. Finally, the test performance was comprehensively assessed in our recruited training and validation cohorts.

Data preparation

Thirteen methylation datasets, which were GSE77954, GSE101764, GSE131013, GSE199057, GSE164811, GSE193535, GSE129364, GSE139404, GSE107352, GSE75546, GSE77965, GSE68060 and EMTAB6450 from public databases were selected based on three criteria: 1) were generated by Illumina HumanMethylation 450k BeadChip and had the raw IDAT files, 2) the sample size was greater than 10, and 3) consisted of CRC or adenoma or normal adjacent tissue (NAT). All the IDAT files were then processed using the minfi tool [16] to obtain methylation β values. They were integrated as a single dataset (n= 1165), which we defined as Phase I discovery set for candidate marker identification.

Level 3 methylation data for 31 cancer types were retrieved from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Data for 8258 primary tumor tissues corresponding to 31 cancer types and their 710 NATs were retained. The 31 cancer types included ACC (n=79), BLCA (n=412), BRCA (n=778), CESC (n=306), CHOL (n=36), CRC (n=379), DLBC (n=48), ESCA (n=183), GBM (n=137), HNSC (n=523), KICH (n=65), KIRC (n=312), KIRP (n=271), LGG (n=513), LIHC (n=374), LUAD (n=456), LUSC (n=364), MESO (n=87), OV (n=10), PAAD (n=183), PCPG (n=178), PRAD (n=495), SARC (n=257), SKCM (n=104), STAD (n=393), TGCT (n=133), THCA (n=503), THYM (n=124), UCEC (n=418), UCS (n=57), UVM (n=80). The TCGA dataset was defined as Phase II discovery dataset. DMCs were deemed eligible if they exhibited methylation levels <0.2 on other cancer types (non-CRC), >=0.55 on CRC, and <0.15 on 710 NATs.

The other three datasets, GSE48684 [17],GSE40279 [18], and GSE122126 [19], generated by the same platform of Illumina HumanMethylation BeadChip in Gene Expression Omnibus (GEO) were used as validation sets to verify the methylation status of candidate differentially methylated CpGs (DMCs). The GSE48684 cohort consisted of 105 qualified samples, of which 41 were NAT and 64 were CRC. The GSE40279 cohort comprised whole blood cell (WBC) samples collected from 656 healthy individuals. The GSE122126 cohort included three CRC and 12 normal plasma samples.

Sample collection

This case-control study enrolled 772 cases, including 60 tissue samples (30 CRC and 30 NATs) and 712 plasma samples from the First Affiliated Hospital of Zhengzhou University between April 2022 and June 2022. Tissue samples were obtained from the preserved formalin-fixed paraffin-embedded (FFPE) sections collected from surgical patients. The plasma cohort was randomly divided into a training set and a validation set in a 2:1 ratio. The training set comprised 474 participants, including 115 healthy blood donors, 123 non-digestive disease patients (NDD), 65 intestinal disease patients (ID), 14 polyps patients, 10 non-advanced adenoma patients (Non-AA), 16 AA, 125 CRC patients, and 6 patients with other cancers. In the validation set, there were 235 participants, including 57 healthy blood donors, 60 NDD, 30 intestinal ID, 6 polyps patients, 5 Non-AA, 9 AA, 66 CRC patients, and 2 patients with other cancers (Supplemental Table 1). This study was approved by the ethics committee. Each participant signed an informed consent form. Patients with polyps, adenomas, CRC and other cancers were further confirmed by histopathological examinations. The included CRC patients were required to meet the following criteria: 1) did not receive any radiotherapy, chemotherapy or surgery, 2) ages older than 18. All CRC patients were classified as I, II, III, and IV stages according to the American Joint Cancer Committee (AJCC) staging system.

Differential methylation analysis

We performed differential methylation analysis using the rank-sum test on three groups in the discovery set: cancer vs. normal (NAT), adenoma vs. normal, and cancer vs. adenoma. DMCs were defined as significant if they meet two criteria: an FDR < 0.05 and a fold change ranking in the top 1% of all probes. The DMCs were then classified as hyper- or hypo- DMCs based on whether they exhibited high or low methylation levels in the tumor or adenoma samples compared to the normal samples. LASSO regression was implemented in the R package 'glmnet' to reduce the number of features. LASSO regression was repeated 100 times, and the frequencies of probes with non-zero coefficients in the regressions were counted.

Tissue and plasma DNA extraction

Genomic DNA from tissue samples and plasma cfDNA was isolated using the FFPE DNA rapid extraction kit (TianGen, Beijing) and the plasma/serum cfDNA extraction kit (Ammunition Life-tech, Wuhan), respectively, according to the protocols. Briefly, cfDNA extraction was divided into two steps. First, approximately 10 ml of blood was drawn from participants at room temperature and then centrifuged twice with 1300g and 14000g for 10 min at 4°C within 2 hours. Then, about 2 ml of plasma was retained for cfDNA purification. Purified cfDNA was eluted in 45 ul TE buffer and then treated with bisulfite using a DNA bisulfite modification kit (Ammunition Life-tech, Wuhan) in accordance with the instructions described in the aforementioned study [20], or stored at -80 °C until required.

Sanger sequencing

Sanger sequencing (ABI 3730XL, GENEWIZ, Suzhou) was performed for the target regions of NTMT1 and MAP3K14-AS1 after bisulfite treatment to confirm their methylation status on 30 CRC tissue samples and 30 NATs. The sequencing primers are shown in Supplemental Table 2. Following bisulfite treatment, cytosine in methylated CpG sites remained cytosine, while unmethylated cytosine was converted to thymine. Sanger sequencing results thus allowed for the direct determination of the methylation status of target regions.

Developing the SADMP and methylation-specific PCR (MSP) technique on plasma

The location and sequences of MSP primers/ probes are displayed in Supplemental Table 3. For NTMT1, two pairs of methylation primers targeted the sense and antisense strands, and their corresponding MGB probes were designed. For MAP3K14-AS1, the MGB-probe 1 and MGB-probe 2 were designed according to the antisense strand sequence and the reverse complementary sequence of the antisense strand.

Gradient dilutions of synthetic plasmids or plasmid mixtures are employed as templates for the determination of test parameters. Specifically, standard curve experiments were carried out to assess the amplification efficiency of each pair of primers. The experiments consisted of five ten-fold dilutions of fully methylated plasmid DNA templates with five replicates at each dilution (1, 10, 100, 1000, and 10000 copies per run). Primer tolerance experiments were designed to assess the specific amplification ability of methylated primers against methylated templates. Eight concentration dilutions of fully methylated DNA templates (0, 1, 5, 10, 50, 100, 200, and 400 copies/μL) mixed with a high concentration of unmethylated plasmid (10⁷ copies/μL) were prepared to evaluate the ability of methylated primers selectively amplifying methylated templates under the background of unmethylated templates. Each concentration experiment was repeated five times.

The amplification system of MSP was shown in Supplemental Table S4. The test employed a triplex PCR with a FAM channel for the internal reference gene ACTB, a ROX channel for NTMT1 (dual-strands), and VIC channel for MAP3K14-AS1 (dual-MGB probes). The PCR procedure was pre-denaturation at 95°C for 5 min (step 1), denaturation at 95°C for 15s (step 2), annealing at 60°C for 30s (step 3), repeating step 2 and 3 forty-five times in plasmid samples and fifity times in plasma samples on the 7500 Fast Real-Time PCR System (Applied Biosystems, USA), the cycle threshold (Ct) was assigned to 50 for the target without amplification.

Statistical analysis

The data processing and analysis in this study were conducted using R software (version 4.1.0). The ‘glm’ function was employed to fit the logistic regression model with a parameter of ‘family=binomial’. Receiver operating characteristic (ROC) curve analysis was performed using the ‘pROC’ package, and the area under the ROC curve (AUC) was subsequently calculated to assess the test classification performance. The optimal sensitivity and specificity of the test were estimated when Youden’s index reached its maximal value. Sensitivity and specificity were calculated using the following formulas:

And the Youden index = sensitivity+specificity-1.

The rank-sum test and Kruskal test were employed for the comparisons between two groups and the comparisons between multiple groups, respectively. The Chi-square test was utilized for the comparisons between categorical variables. The Other statistical methods used in this study were described in the corresponding results.

Study design and participant characteristics

The study is divided into four steps, as illustrated in Fig. 1. In the initial step, candidate markers were identified in a set of 1165 samples from 13 datasets. Of these, 452 were normal, 168 were adenomas, and 545 were CRC. These markers were then narrowed down using data from 31 cancer types in the TCGA dataset, DMCs that were hypermethylated in cancer other than CRC were filtered out. The available DMCs were further validated in three independent datesets. In the second step, the methylation status of candidate CpGs was confirmed by Sanger sequencing using 30 CRC tissues and 30 NATs. In the third step, the SADMP technique and MSP system were established. This involved designing appropriate primers, optimizing the qPCR amplification system, and evaluating the technical parameters of the assay. In the last step, the developed assay was tested on training and validation sets of plasma. Its performance was assessed by estimating indicators such as sensitivity, specificity, and AUC.

Landscape of the methylation patterns of the 13 discovery datasets

The methylation levels of adenoma and cancer samples were found to be lower than normal, as shown in Fig. 2A. The methylation density curves for the three groups displayed bimodal distributions with hyper- and hypomethylation peaks, respectively (Fig. 2B). The hyper-methylation peaks of adenoma and tumor samples were lower than those of normal samples, indicating higher methylation levels in normal samples.

We employed t-SEN to analyze and visualize the structure of the discovery set and identified significant differences between normal and cancer samples. However, adenomas exhibited overlap with both normal and cancer samples (Fig. 2C). We then selected the top 1% of most variable probes to cluster the discovery set using the K-means algorithm. The results showed that both normal and cancer samples clustered together, while adenomas were separated into two subgroups with a remarkable difference. They showed both high (Methy-H) and low (Methy-L) methylation status and were closer to cancer and normal samples, respectively (Fig. 2D).

Further analysis revealed that tubular adenomas constituted the largest proportion of Methy-L adenomas (23/57 or 40.35%), while villous adenomas constituted the largest proportion of Methy-H adenomas (47/73 or 64.38%). To eliminate any potential bias between the datasets, we conducted Fisher’s test separately for the Methy-H and Methy-L adenomas, with the datasets serving as the stratification factor. However, we did not identify any significant differences (P > 0.05).

Identification of candidate DMCs

First, we conducted a comparative analysis of the DMCs between cancer, adenoma, and normal samples using the 13 discovery datasets. The three comparisons (cancer vs. normal, adenoma vs. normal, and cancer vs. adenoma) yielded 3000, 3051, and 1545 DMCs, respectively. Notably, these DMCs were predominantly hyper-DMCs (Fig. 3A). Further investigations revealed that the majority of DMCs were distributed in upstream regions of genes (5'UTR, TSS1500, TSS500, and 1st Exon), except for cancer vs. adenoma where DMCs were mainly located in gene bodies (Fig. 3B). We observed an extremely high proportion of overlapping DMCs between cancer vs. normal and adenoma vs. normal, which was significantly higher than cancer vs. adenoma (Fig. 3C). To identify the most appropriate DMCs, we focused on those that were overlapped between cancer vs. normal and adenoma vs. normal (2237 probes) and evaluated their methylation levels on 31 cancer types of TCGA. This yielded 75 accessible probes (Fig. 3D). LASSO regression was employed to reduce the number of DMCs, with eight presented in 100 replicates with non-zero coefficients (Fig. 3E).

The methylation levels of NTMT1 and MAP3K14-AS1 in validation sets

The methylation levels of the eight probes were then analyzed in 656 WBC samples, and seven DMCs exhibited relatively low methylation levels (less than 0.1) with the exception of cg17892556 (Fig. 4A). Therefore, the seven DMCs are recognized as the most promising markers.

Two specific DNA methylation sites, cg14015706 and cg08247376, were selected for further analysis as they were suitable for primer/probe design. These sites are located in the first exon of NTMT1 and 200 bp upstream of the MAP3K14-AS1 transcription start site, respectively. The GSE48684 dataset revealed significantly higher methylation levels for both probes in cancer samples than in normal samples (Fig. 4B). Furthermore, the two probes demonstrated hypermethylation in CRC plasma and hypomethylation in healthy plasma (Fig. 4C), indicating a high consistency of the methylation status between tissue and plasma.

The SADMP technique

The Sanger sequencing of the target regions revealed that the CpGs sites among the amplicons were more frequently methylated in CRC than NATs, which provided the foundation for the development of SADMP technique. For NTMT1, two pairs of methylation primers targeted the sense and antisense strands, and their corresponding MGB probes were designed (Fig. 5A). For MAP3K14-AS1, the MGB-probe1 and MGB-porbe2 were designed according to the antisense strand sequence and the reverse complementary sequence of antisense strand (Fig. 5F). The estimated amplification efficiencies of MTNT1 and MAP3K14-AS1 were 98.14% and 110.08%, respectively (Fig. 5B and 5G). The sense and anti-sense strands were transformed into completely different DNA sequences after bisulfite treatment and both could be used as PCR templates. It can be postulated that the copy numbers detected by any single pair of primers or single MGB-probe assay should be half of the diluted copy numbers (including sense and anti-sense strands), and that the SADMP assay should be equal to the diluted copy numbers. For NTMT1, the detected copy numbers by sense and antisense strand assays were slightly less than half of the theoretical copy numbers with slopes of 0.39 and 0.31, respectively. The detected copy numbers by dual-strand assay were approximately 0.73 times of theoretical copy numbers, which was approximately two-fold compared to any single-strand assay (Fig. 5C), as expected. Meanwhile, Ct value of dual-strand assay was almost one cycle earlier than that of any single-strand assay (average △Ct = 1.21, Fig. 5D), which was consistent with the theoretical △Ct value (should be one). The MAP3K14-AS1 SADMP showed similar results, with the number of copies detected was doubled compared to the single probe assay (Fig. 5H), and the Ct values were shifted forward by 0.99 cycles (Fig. 5I).

Additionally, in the serially diluted experiment, no amplification cures for both targets were observed when a high proportion of unmethylated DNA was used as templates (the methylated DNA copies were 0) (Fig. 5E and 5G). These results suggested that the developed assays were explicitly targeted for methylated DNAs even at the high background of unmethylated DNAs. We also found that the NTMT1 SADMP assay was able to robustly detect methylated DNAs at a concentration of 10 copies/µL (Fig. 5E), while it was 5 copies/µL for the MAP3K14-AS1 SADMP ( Fig. 5G).

Validation and evaluation of biomarkers using MSP in plasma samples

In the plasma sample set, the Ct values of ACTB exhibited a slight decrease in trend from the healthy control groups to the cancer group (p < 0.0001) (Fig. 6A). Ct values of NTMT1 and MAP3K14-AS1 were much lower in CRC samples and AA samples than other control groups (Fig. 6B and 6C).

The Ct values could discriminate the CRC and AA samples from healthy samples very well. Furthermore, the Ct values-based result determination exhibited favorable outcomes in a previous study [21, 22]. Consequently, the Ct values were utilized as the result analysis indicators. In order to determine the algorithm and cutoff, ROC curve analysis was conducted in the training set. In the CRC vs control groups, the AUC values for NTMT1 and MAP3K14-AS1 were 0.882 (95% CI: 0.838–0.925) and 0.785 (95% CI: 0.729–0.840), respectively (Fig. 6D). In the CRC + AA vs control groups, the AUC values for NTMT1 and MAP3K14-AS1 were 0.852 (95% CI: 0.807–0.898) and 0.761 (95% CI: 0.707–0.815), respectively (Fig. 6E).

As AA was precancerous lesions, it was also benefit patients from detecting it. So we use the CRC + AA vs control groups of training set for further analysis. The performance of the combination of the two biomarker were evaluated by two strategies (Table 1). Strategy 1 involved the construction of a logistic regression model, with an estimated AUC value was 0.881 (95% CI: 0.840–0.922), the optimal sensitivity for CRC and specificity of 86.4% and 91.0%, respectively (Table 1). Strategy 2 was the 1/2 algorithm, which determined a positive measurement when the Ct of any single marker was less than its corresponding threshold. The cutoff was determined as Ct value corresponding to the maximum Youden index in the training dataset using CRC + AA vs control data. The cutoff values were 48.2 and 48.4 for NTMT1 and MAP3K14-AS1, respectively. At these cutoff, the two target sensitivities were 80.0% and 59.2%, with specificities of 92.8% and 97.0%, respectively (Fig. 6F). The sensitivity and specificity of the combination of the two biomarkers were 86.4% and 91.0% respectively (Fig. 6F). Notably, strategy 2 yielded an identical sensitivity and specificity to strategy 1. Given that strategy 2 is considerably more straightforward for physicians to interpret test results in clinical practice, we have adopted the 1/2 algorithm as the combination algorithm for the dual-target test.

Table 1

Diagnostic performance of single biomarker and marker combination in training set
Biomarker & combination	AUC (95% CI)	Sensitivity (CRC)	Sensitivity (CRC + AA)	Specificity	Cutoff	Combination method
NTMT1	0.852 (0.807–0.898)	80.0%	74.5%	92.8%	48.2	/
MAP3K14-AS1	0.761 (0.707–0.815)	59.2%	54.6%	97.0%	48.4	/
NTMT1 & MAP3K14-AS1	0.881 (0.840–0.922)	86.4%	80.8%	91.0%	0.1491	Logistic regression
NTMT1 & MAP3K14-AS1	/	86.4%	80.8%	91.0%	48.2, 48.4	1/2 algorithm

After the algorithm and cutoff was set, the validation set and all sample set were analyzed. The validation set had a 81.8% sensitivity and 92.5% specificity (Fig. 6G) and the all sample set had a 84.8% sensitivity and 91.5% specificity (Fig. 6H). The dual-target test had a higher sensitivity than that of any single biomarker and a slightly lower specificity in the three data sets (Fig. 6F-H). It indicated that the combination of the two biomarkers were better than single biomarker.

Performance in subgroups of plasma samples

The performance of the dual-target test was evaluated in subgroups of all plasma samples. The test demonstrated a detection rate of 26.7% in non-AA samples and a detection rate of 32.0% in AA samples (Fig. 7A). It exhibited sensitivities of 75.0%, 81.2% and 90.3% in stages I, II and III-IV of CRC samples, respectively (Fig. 7B). The test exhibited specificities of 95.9%, 92.3%, 84.2% and 90.9% in healthy, NDD, ID and polyps samples, respectively (Fig. 7C). The positive predictive value (PPV), negative predictive value (NPV) and accuracy for the dual-target test were 79.4%, 94.0% and 89.6%, respectively (Fig. 7D).

With regard to the detection rates in adenoma and different stages of CRC, the dual-target test demonstrated superior performance compared to that of a single biomarker (Fig. 7A-B). With respect to specificities in subgroups, the dual-target test exhibited a marginal decline in value compared to that of a single biomarker (Fig. 7C). However, with regard to NPV and accuracy, the dual-target test demonstrated superior performance compared to that of a single biomarker (Fig. 7D). With regard to PPV, the dual-target test exhibited a slight decline in performance relative to that of a single biomarker (Fig. 7D).

It is commonly accepted that patients diagnosed with CRC at an early stage can be treated more effectively and have a better prognosis. Several stool DNA-based tests have been developed that demonstrate excellent performance in detecting CRCs at their early stages [23–26]. Blood sampling is more acceptable than stool sampling, but blood-based tests are less reported and usually exhibit lower sensitivities than stool-DNA tests, ranging from 47–87% [27]. This study presented a systematic pipeline for the discovery of methylation markers from scratch, test development, and evaluation in training and validation plasma sets. The SADMP technique enhanced the ability to detect methylation signals. Following a comprehensive evaluation, the test obtained an overall sensitivity and specificity of 84.8% and 91.5%, respectively, for the detection of CRC at a volume of 2 ml plasma.

Adenoma and CRC displayed lower methylation levels overall, with the exception of regulatory regions (Fig. 2B and 3B), a finding that has been reported in previous study [17]. Previous studies have focused on the CpG Island Methylator Phenotype (CIMP) found in CRC. This study found that tubular adenomas are common in the methy-L subclass, while villous adenomas are more often in methy-H subclass. Previous studies have indicated that CIMP is rarely found in tubular adenomas, but frequently in tubulovillous and villous adenomas [28], which is in accordance with this study. The large proportion of overlapping DMCs between cancer vs normal and adenoma vs normal indicated that many CpGs have undergone aberrant methylation events at the precancerous stage, which provides robust evidence for discovering the methylation markers for CRC early detection. It should be noted that the CRC markers are not necessarily applicable to adenomas, as a significant proportion of the DMCs in the Cancer vs Normal group were not present in the Adenoma vs Normal group, as illustrated in Fig. 3C. This emphasises the importance of this study's inclusion of adenoma samples in the marker discovery set and the selection of DMCs in which cancers and adenomas overlap in a way that many other studies of CRC markers have not done.

Blood-based tests are more susceptible to interfering diseases and may result in a high false-positive rate. Therefore, specificity is a critical indicator. In the marker discovery step, 31 cancer types in the TCGA database and WBC were used to control the low methylation levels of candidate markers in other tissues and blood background, effectively attenuating the interference of unintended cfDNAs. In the assay development phase, we designed highly selective MSP primers that did not show normal amplification curves even when unmethylated DNAs were used as templates at 10⁷ copies (Fig. 5E and 5J). These aforementioned measures guarantee a high specificity in plasma samples. Two combination algorithms were utilized to assess the performance of the dual-target test in the training set, and both algorithms indicated that the combined markers had better AUC values and higher sensitivities than any single marker (Fig. 6D and 6E). However, the dual-target test showed a decreased specificity compared to both single markers, from 92.8% and 97.0–91.0% (Fig. 6H), which was also observed in other studies [29, 30]. When healthy individuals were selected as controls, the specificity improved to 95.9% (Fig. 7C), which is comparable to the SEPT9 test [2]. These data demonstrate the excellent performance of the dual-target test in detecting CRC.

A number of studies [31–35] have demonstrated that the methylation levels of NTMT1 (whose antisense chain counterpart is C9orf50) and MAP3K14-AS1 can be employed for the screening and diagnosis of CRC. In particular, the study conducted by Sarah Ø Jensen et al [34]. indicated that the C9orf50 methylation assay exhibited a plasma sensitivity of 76% and specificity of 91% for CRC. Ludovic Barault and colleagues [35] demonstrated that the MAP3K14-AS1 methylation assay in plasma exhibited a sensitivity of 69.8% and a specificity of 100% for CRC. The dual-strand technique has been proven to enhance the performance of markers in previous studies [14, 15]. This technique was also observed to be effective in our study. By detecting the methylation signals of NTMT1 sense- and antisense-strand simultaneously, the Ct value of the dual-strand assay was able to shift forward by one compared to the single-strand assay (Fig. 5D). In contrast to previous studies, the current study also included two MGB probes located downstream of forward and reverse primers of MAP3K14-AS1. During PCR strand extension, the polymerase enzymes cleaved the 5-primer sequence of probes and released two fluorescent groups. The dual-MGB probe technique theoretically doubled the fluorescent signals when both probes shared the same channel, leading to an earlier Ct value similar to that of the dual-strand technique (Fig. 5I). Serial dilution experiments confirmed the superiority of dual-MGB probes over one MGB probe (Fig. 5H-J). These results suggest that applying the SADMP technique can be a feasible strategy to enhance the detection sensitivity of candidate markers.

Early diagnosis or screening techniques are essential to improve patient survival time, particularly when curable treatments are available. Studies have shown that the 5-year survival rate of early detected CRC is almost 90%, while it was only 20% for advanced CRC [36]. The dual-target test showed a sensitivity of 75.0% and 81.2% for stage I and stage II CRC detection, notably, the dual-target test obtained a positive detection rate of 32.00% (8/25) for AA (Fig. 7B and 7C), implying its ability to detect early CRC and precancerous lesions.

The current study has some limitations that may hamper the interpretation of these results. 1) Participants in this study were enrolled from a single center, which may bias these results. 2) the SADMP techniques may not applicable for all candidate markers. The dual-strand technique may be attempted when both sense and antisense strands are suitable for designing MSP primers, while the multiple MGB probe technique is limited by the amplicon length, which is usually less than 100 bp.

In this study, we employed several public databases of adenomas and CRC for marker discovering, and ultimately identified two promising markers, NTMT1 and MAP3K14-AS1. We then constructed the SADMP technology based on these two markers, which enhanced the sensitivity of the detection. The dual-target assay has a high sensitivity for AA and early stage CRC, and its clinical application value merits further investigation.

Advanced adenoma

CRC

Colorectal cancer

cfDNA

Cell-free DNA

SADMP

Sense-antisense and dual-MGB probe

NAT

Normal adjacent tissue

TCGA

The Cancer Genome Atlas

GEO

Gene Expression Omnibus

DMC

Differentially methylated CpGs

WBC

Whole blood cell (WBC)

FFPE

Formalin-fixed paraffin-embedded

NDD

Non-digestive disease patients

Intestinal disease

Non-AA

Non-advanced adenoma

MSP

Methylation-specific PCR

ROC

Receiver operating characteristic

AUC

Area under the ROC curve

PPV

Positive predictive value

NPV

Negative predictive value

Ethics approval and consent to participate

This study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University (approval number: 2022-KY-0631-002). Each participant signed an informed consent form.

Clinical Trial Registration

This study is a sub-project of the Clinical Study of Pan-cancer DNA Methylation Test in plasma (Clinical Trials ID: NCT05685524)

Availability of data and materials

The datasets (GSE77954, GSE101764, GSE131013, GSE164811, GSE193535, GSE129364, GSE139404, GSE107352, GSE75546, GSE77965, GSE199057, GSE68060, GSE48684, GSE40279 and GSE122126) supporting the conclusions of this article are available in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). The E-MTAB-6450 data is available in EMBL biostudies (https://www.ebi.ac.uk/biostudies/arrayexpress). The source code and intermediate data can be accessed from git-hub (https://github.com/amsinfor/CRC-methylation).

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by science and technology project of Henan Province (232102310028).

Authors' contributions

QK Y and YT Z conceptualized and designed the study. YT Z，ZJ W and DH Z developed the SADMP technique. ZJ W QN Y, and X L provided clinical samples and patient information. X L and SL D conducted cfDNA extraction and bisulfite converted. YT Z, ZJ W and T Z performed data analysis and prepared the manuscript. XP L validated the study results. All the authors reviewed the manuscript.

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The authors declare no competing interests.

SupplementalTablesRSpreprint.xlsx
Supplementary materials Supplemental Table 1. The clinical features of training and validation cohorts used in this study Supplemental Table 2. The information of primers for Sanger sequencing in this study Supplemental Table 3. The information of primers and MGB probes used in this study Supplemental Table 4. The amplification system of the MSP

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Discovering methylation markers and development of a sense-antisense and dual-MGB probe PCR assay in plasma for colorectal cancer early detection

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Abstract

Figures

Background

Methods

Data preparation

Sample collection

Differential methylation analysis

Tissue and plasma DNA extraction

Sanger sequencing

Developing the SADMP and methylation-specific PCR (MSP) technique on plasma

Statistical analysis

Results

Study design and participant characteristics

Landscape of the methylation patterns of the 13 discovery datasets

Identification of candidate DMCs

The methylation levels of NTMT1 and MAP3K14-AS1 in validation sets

The SADMP technique

Validation and evaluation of biomarkers using MSP in plasma samples

Performance in subgroups of plasma samples

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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