MAPRE1 as a Plasma Biomarker for Early-Stage Colorectal Cancer and Adenomas

Colorectal cancer is the third most common cancer and the third leading cause of cancer death in both men and women in the United States (1). Most sporadic colorectal cancers develop slowly over many years and often progress from early to advanced adenoma and then to invasive colorectal cancer (2). Colorectal cancer is potentially curable if detected at an early stage; the 5-year survival rates for colorectal cancer are approximately 91% for localized disease but only about 13% if distant metastasis has occurred. Therefore, detecting adenoma and early-stage colorectal cancer is an attractive approach to reducing colorectal cancer mortality rates.

Colonoscopy is considered the gold standard for colorectal cancer screening owing to its ability to visualize the complete colon and to remove neoplastic lesions (3), but stool- or blood-based tests for colorectal cancer are more convenient, more cost effective, and less invasive than colonoscopy. Several clinical trials have reported that colorectal cancer screening with the fecal occult blood test reduced colorectal cancer–related mortality by approximately 16% (4). Although fecal occult blood tests have limited ability to detect adenomas, Imperiale and colleagues have recently reported that a stool DNA test combined with a fecal immunohistochemistry test provided higher sensitivity for detecting colorectal cancer and, to a lesser extent, advanced precancerous lesions (5).

Several potential blood-based biomarkers for early detection of colorectal cancer or for colorectal cancer risk assessment have been described (6–9). Carcinoembryonic antigen (CEA) is a circulating biomarker for colorectal cancer that is used in the clinical setting for monitoring therapy outcomes in patients with advanced disease and for predicting prognosis (10–12). However, CEA alone lacks the sensitivity and specificity to be used for early detection of colorectal cancer (12, 13). Additional biomarkers that complement CEA are needed for reliable and noninvasive detection of early-stage colorectal cancer.

We have previously undertaken a discovery study of potential circulating colorectal cancer biomarkers using mass spectrometry applied to prediagnostic samples from the Women's Health Initiative cohort, which resulted in the identification of several biomarker candidates (14). Prominent among the candidates was MAPRE1, which is known to bind APC (15, 16), a commonly mutated protein in colorectal cancer (17) and which plays a role in microtubule stabilization (18). The association between increased levels of circulating MAPRE1 and colorectal cancer was validated in independent plasma sample sets that consisted of newly diagnosed and prediagnostic colorectal cancer cases.

In the current study, we sought to determine the performance of MAPRE1 together with other candidate biomarkers for detecting disease in blood samples from patients with various stages of colorectal cancer or with adenoma collected under the auspices of the National Cancer Institute Early Detection Research Network.

All human plasma samples were obtained following Institutional Review Board approval and informed consent. Plasma samples were collected through a consortium led by a Clinical Validation Center of the National Cancer Institute Early Detection Research Network at the University of Michigan. The samples were collected before any treatment at the time of diagnosis with adenoma (N = 60), early-stage colorectal cancer [N = 30; stage I (N = 11) and stage II (N = 19)], or late-stage colorectal cancer [N = 30; stage III (N = 21) and stage IV (N = 9)]. Plasma samples from healthy controls were collected at the time of colonoscopy (N = 60; Supplementary Table S1).

Mass spectrometry analysis of human plasma samples was done as previously described (14, 19).

All animal experiments were conducted in accordance with institutional and national guidelines and regulations with approval by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center. Details on mouse models, plasma sample preparation, and mass spectrometry analysis of those samples are provided in Supplementary Methods.

A mass spectrometry analysis of colorectal cancer cell lines was performed as previously described (20). Eight colorectal cancer cell lines (HCT116, SW480, LoVo, SW620, HT29, SW48, Colo205, and Caco-2) were purchased from the ATCC and grown in RPMI 1640 medium (Pierce) that contained 10% dialyzed FBS (Invitrogen), 1% penicillin and streptomycin cocktail, and ¹³C-lysine instead of regular lysine for seven passages, according to the standard SILAC protocol (21). Protein expression was estimated using normalized spectral counts (20).

In this study, we have expanded the validation of blood-based biomarkers for colorectal cancer and adenoma to include 22 candidates for which antibodies were available for assay using antibody microarrays (Supplementary Tables S2 and S3). High-density antibody array analysis was performed as previously described (22, 23). Briefly, albumin and IgG were depleted from plasma samples, 200 μg of the remaining plasma proteins from either cases or controls were labeled with Cy5, and a reference sample (a pool of plasma from seven healthy individuals) was labeled with Cy3. Array images were obtained using a GenePix 4000B microarray scanner (Molecular Devices), and the scanned array images were analyzed using GenePix Pro 6.0 image analysis software. The raw GenePix Array List file was aligned and resized to fit the individual spot features.

Plasma levels of CEA were measured with an ELISA kit (R&D Systems) according to the manufacturer's protocol.

Tissue microarrays used in this study comprised 20 normal colonic tissues, 10 colon adenomas, and 66 colorectal cancers (Folio Biosciences). The slides were deparaffinized, rehydrated, and antigen retrieval was performed using a pressure cooker with 0.01 mol/L citrate buffer at pH 6.0. Intrinsic peroxidase activity was blocked using 1% hydrogen peroxide, and 10% normal goat serum (KPL) was used for 30 minutes to block nonspecific antibody binding. The slides were then incubated overnight at 4°C with MAPRE1 antibody (1:200 dilution; Thermo Fisher Scientific). After incubation with the horseradish peroxidase–conjugated secondary antibody (Millipore) for 1 hour at room temperature, antigen signals were detected using the 2-Solution Diaminobenzidine Kit (Cell Signaling) and counterstained with hematoxylin. The intensity of staining (0: negative, 1+: weakly positive, 2+: moderately positive, and 3+: strongly positive) and percentage of staining distribution in the tumor cells (from 0% to 100% of the cells) were evaluated for each tissue microarray core. Two independent scoring was performed in a blind manner. Then the scoring was reviewed together again when discordant. In this study, samples in which 10% or more of the cells with 1+ staining intensity were considered as positive.

For the high-density antibody array analysis, the change in the signal compared with the reference was calculated as log₂. Technical sources of variation were normalized using Loess procedures, including within-array print-tip Loess and between-array quantile normalization. Following normalization, triplicate features were summarized using their median values. Individual biomarker performance was assessed using P values calculated by a one-sided Mann–Whitney U test, sensitivity, specificity, and the area under the curve (AUC) determined by a receiver operating characteristic analysis. The P values for significance of the biomarker panel combining MAPRE1 and CEA were subjected to a likelihood ratio test.

To develop an “OR” rule combination model, MAPRE1 and CEA were used as anchor biomarkers to develop a combination rule in which a patient was considered a case if any of the biomarkers exceeded a designated threshold or was ruled out as a case if all biomarkers were below those thresholds (24). The sensitivities at 90% specificity and at 95% specificity of the MAPRE1, CEA, and AK1 (“OR” rule combination model) were compared with those of MAPRE1 alone, CEA alone, and MAPRE1 plus CEA (linear combination). The P values for the differences in sensitivity were calculated by the McNemar test (25), and a bootstrap analysis repeated 1,000 times. The differences were considered significant when P values were less than 0.05.

Plasma CEA levels were significantly higher in each colorectal cancer group than in healthy controls (P = 0.0246 for early colorectal cancer, P < 0.0001 for advanced colorectal cancer, and P = 0.0002 for total colorectal cancer, Mann–Whitney U test), but not significantly different between the adenoma and healthy control groups (P = 0.4886, Mann–Whitney U test; Table 1). In contrast, the plasma levels of MAPRE1 were significantly elevated in both the adenoma and the colorectal cancer groups compared with healthy controls (P < 0.0001 for adenoma, P = 0.0003 for early colorectal cancer, P = 0.0025 for advanced colorectal cancer, and P < 0.0001 for total colorectal cancer, Mann–Whitney U test).

Protein expression of MAPRE1 was examined in colon adenoma and colorectal cancer tissues. Among 20 normal colonic tissues, 10 colon adenomas, and 66 colorectal cancers in the tissue microarray, 9 (90.0%) colon adenomas and 45 (66.2%) colorectal cancers were MAPRE1 positive while MAPRE1 expression was observed in only 5 (25.0%) normal colonic tissues (Supplementary Fig. S1), suggesting that plasma MAPRE1 would be derived from colorectal tumors.

The combination of MAPRE1 and CEA yielded AUCs of 0.793 for early colorectal cancer and 0.731 for adenoma (Table 1 and Fig. 1). The AUC of the two biomarkers together was significantly better than that of CEA alone (P < 0.0001 for adenoma, early colorectal cancer, and total colorectal cancer, and P = 0.0014 for advanced colorectal cancer, likelihood ratio test) or of MAPRE1 alone (P = 0.0007 for adenoma, P = 0.0004 for early colorectal cancer, P = 0.0003 for advanced colorectal cancer, and P < 0.0001 for total colorectal cancer, likelihood ratio test). The combination of MAPRE1 and CEA yielded a sensitivity of 0.400 at 95% specificity in early colorectal cancers compared with healthy controls, whereas the sensitivities of CEA alone and MAPRE1 alone at 95% specificity were noticeably lower for detecting early colorectal cancer, indicating an additive effect of the combination of MAPRE1 and CEA for detecting early colorectal cancer.

We determined the individual performances of biomarker candidates for which antibodies were printed on the microarrays (Table 2). Three proteins (AK1, CLIC1, and SOD1) were significantly elevated in adenoma and in each colorectal cancer group compared with healthy controls (P < 0.05, Mann–Whitney U test). Five additional proteins (AZGP1, CALR, LGALS3BP, SPARC, and ZYX) exhibited significantly elevated levels in at least one disease group compared with healthy controls. AK1, CLIC1, and SOD1 had been previously identified in a proteomic analysis of prediagnostic plasma samples from the Women's Health Initiative cohort (Supplementary Table S2). Mass spectrometry analysis yielded substantial peptide coverage for AK1, CLIC1, and SOD1 (Fig. 2), indicating the occurrence of full-length proteins for these three biomarkers in human plasma. To determine whether these proteins originated from tumor cells, we performed proteomic analysis of three different compartments (media, surface, and whole-cell extract) of eight colorectal cancer cell lines. CLIC1 and SOD1 were broadly expressed in the media of colorectal cancer cell lines (Supplementary Table S4), whereas AK1 was identified in the media of three colorectal cancer cell lines.

To further investigate the association of increased circulating levels of these biomarker candidates with early colorectal cancer development, we used mass spectrometry to profile plasma samples collected at different timepoints during tumor development from two genetically engineered colorectal cancer mouse models. Levels of AK1 and SOD1 were elevated in plasma samples from Apc- and Msh2-deficient mice (26) with low-grade and high-grade adenomas compared with controls (Fig. 3). CLIC1 levels were elevated at preclinical timepoints (at 2 weeks and 4 weeks after induction of Kras^G12D) in a mouse with oncogenic Kras-induced colorectal cancer compared with controls (unpublished mouse model; Fig. 3). These findings further support a biologic basis for the association between increased plasma levels of these three biomarker candidates and early development of colorectal cancer.

Because CEA was not significantly elevated in adenoma but was significantly elevated in colorectal cancers at all stages, we investigated whether an “OR” rule (24), rather than a linear combination rule, better discriminates between cases (adenomas or colorectal cancers) and controls. To rule in an asymptomatic person for subsequent work-up (i.e., colonoscopy) who otherwise does not plan to take part in screening, a biomarker test with sensitivity at 90% or with 95% specificity would have potential clinical utility. As reported in Table 1, the combination of MAPRE1 and CEA using logistic regression yielded a sensitivity of 0.400 at 95% specificity in a comparison of early colorectal cancer and healthy controls and a sensitivity of only 0.167 at 95% specificity in a comparison of adenoma and healthy controls. Therefore, we assessed the potential of an “OR” rule strategy in which any biomarker in the panel exceeding a certain threshold identifies a patient as a case and in which all biomarkers in the panel not exceeding their designated thresholds would rule out a patient as a case. The “OR” rule combination of MAPRE1, CEA, and AK1 yielded significantly higher sensitivity at 95% specificity than the combination of MAPRE1 and CEA in a comparison of adenoma with controls (0.167 sensitivity for MAPRE1 and CEA and 0.350 sensitivity for the “OR” rule combination of MAPRE1, CEA, and AK1; P = 0.015, McNemar test; P = 0.099, bootstrap) and in a comparison of advanced colorectal cancer with controls (0.233 sensitivity for MAPRE1 and CEA and 0.500 sensitivity for the “OR” rule combination of MAPRE1, CEA, and AK1; P = 0.013, McNemar test; P = 0.071, bootstrap; Table 3). For early colorectal cancer compared with controls, the sensitivity at 95% specificity of the “OR” rule combination of MAPRE1, CEA, and AK1 (0.467) was higher than that of MAPRE1 and CEA (0.400); however, the difference between these sensitivities did not reach statistical significance (Table 3). The sensitivities of the “OR” rule combination of MAPRE1, CEA, and AK1 at 90% specificity were 0.483 for adenoma, 0.533 for early colorectal cancer, and 0.633 for advanced colorectal cancer, and the sensitivities of MAPRE1 and CEA at 90% specificity were 0.400 for adenoma, 0.433 for early colorectal cancer, and 0.400 for advanced colorectal cancer. These findings suggest an incremental increase in diagnostic performance due to integrating AK1 as a biomarker with MAPRE1 and CEA in screening for adenoma.

Our study focused on testing the merits of MAPRE1 as a circulating biomarker for colorectal cancer and adenomas, both alone and in combination with CEA. We also explored the potential contributions of additional candidate biomarkers. Our prior finding of increased levels of MAPRE1 in prediagnostic colorectal cancer plasmas (14), which led to the present study, is of particular interest, considering the biologic significance of this protein in the context of colorectal cancer (15–17). Concordant with the results of immunohistochemical staining of MAPRE1 in this study, recent studies have demonstrated that MAPRE1 is upregulated in premalignant colon mucosa of a rat model of chemically induced colorectal cancer and an APC-mutant rat model (27), suggesting that MAPRE1 expression is associated with early field carcinogenesis of colorectal cancer. Overexpression of MAPRE1 also has been associated with poor prognosis in colorectal cancer (28). In this study, we validated that plasma MAPRE1 levels are significantly associated with colorectal cancer and adenoma using an independent sample set from the University of Michigan. MAPRE1 with and without CEA was significantly associated with early colorectal cancer and adenoma.

Currently, the repertoire of biomarkers with demonstrated ability to detect adenoma is quite limited. In a recent validation study of a multitarget stool DNA test for colorectal cancer screening, the sensitivity for advanced precancerous lesions was 42.4% with DNA testing and 23.8% with a fecal immunochemical test. The rate of detection of polyps with high-grade dysplasia was 69.2% with DNA testing and 46.2% with the fecal immunochemical test (5). Plasma methylated SEPT9 has been shown to be a biomarker for colorectal cancer in a screening setting, with a sensitivity of 48.2% and a specificity of 91.5%, but its sensitivity for advanced adenoma was only 11.2% (6). For MAPRE1 and CEA, the sensitivity at 90% specificity was 40% for adenoma and 43% for early colorectal cancer (Table 3), suggesting that MAPRE1 combined with CEA may improve current blood-based screening for both adenoma and colorectal cancer. Further studies will be warranted to validate the performance of MAPRE1 together with CEA, particularly in high-risk adenoma (villous histology, high-grade dysplasia, size larger than 1 cm, or 3 or more adenomas).

We also determined the individual performances of additional biomarker candidates. Eight candidates were significantly associated with disease in one or more groups, of which three candidates (AK1, CLIC1, and SOD1) were each significantly associated with adenoma, early-stage colorectal cancer, advanced-stage colorectal cancer, and total colorectal cancer. These three candidates were previously identified in a proteomic analysis of prediagnostic plasma samples from the Women's Health Initiative cohort (Supplementary Table S2).

Evidence also suggests that a systemic increase in circulating AK1 may occur very early in the development of colorectal neoplasia. We found that plasma levels of AK1 were significantly higher in mice with low-grade as well as those with high-grade adenoma compared with controls and that AK1 was elevated in plasma samples from human adenoma and colorectal cancer cases compared with controls. Recent studies have indicated a role of AK1 in the bloodstream in regulating metabolism of extracellular AMP, ADP, and ATP (29), which are associated with immunosuppression and tumor progression (30). Expression of AK1 increases in response to obesity and metabolic syndrome, which are known risk factors of colorectal cancer (31–33).

We also found increases in plasma levels of CLIC1 in preclinical colorectal cancer and SOD1 in adenomas compared with controls in mouse experiments, and those biomarkers were also elevated in plasma samples from human adenoma and colorectal cancer cases compared with controls. CLIC1 is a previously characterized plasma biomarker for nasopharyngeal carcinoma and has been found to promote cell migration and invasion in colorectal cancer (34–37). SOD1 protein expression in colorectal cancer tumors and precancerous colon tissues from chemically induced colorectal cancer mouse or rat models has been investigated, with variable results (34, 38–41).

Although the “OR” rule combination of MAPRE1, CEA, and AK1 yielded the highest sensitivity in this study compared with the Lasso logistic regression model and two other machine learning methods (optimal linear combination by maximizing the partial area under the receiver operating characteristic curve and combination of biomarkers using fractional polynomials; data not shown), the merits of the “OR” rule will need to be assessed through further testing in additional, larger sample sets. The bootstrap test used the decision rule applied to subjects not included in the bootstrap sample; thus, the test does not have the bias associated with estimating performance using the same sample set used for training.

Our previous discovery studies and our validation studies presented here stem from an in-depth quantitative proteome analysis of plasma that addresses the intended application of blood-based screening for adenoma and early colorectal cancer in combination with other screening modalities. Plasma contains a rich assortment of circulating molecules and cellular materials that can inform us about tumor development, including tumor-derived DNA (6, 42), noncoding RNAs (43, 44), autoantibodies (45, 46), and metabolites (47). There remains a pressing need to determine the relevance and relative contributions of the various potential biomarkers in detecting adenoma and early colorectal cancer. Our validation of MAPRE1 as a circulating biomarker for adenoma and colorectal cancer provides a rationale for further studies into the development of a blood-based biomarker approach using MAPRE1 that complements current screening modalities for colorectal cancer.

No potential conflicts of interest were disclosed.

Conception and design: A. Taguchi, R. Kucherlapati, A.T. Boutin, Y.A. Wang, Z. Feng, P.D. Lampe, S.M. Hanash

Development of methodology: A. Taguchi, J.-h. Rho, S.C. Tripathi, Y.A. Wang, P.D. Lampe

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Taguchi, J.-h. Rho, H. Xu, S.C. Tripathi, H. Wang, D.E. Brenner, M. Kucherlapati, A.T. Boutin, R.A. DePinho

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Taguchi, Q. Yan, Y. Zhang, Y. Zhao, H. Xu, R. Kucherlapati, Z. Feng

Writing, review, and/or revision of the manuscript: A. Taguchi, Q. Yan, H. Xu, S.C. Tripathi, D.E. Brenner, M. Kucherlapati, R. Kucherlapati, Z. Feng, P.D. Lampe, S.M. Hanash

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Taguchi, J.-h. Rho, S.C. Tripathi

Study supervision: A. Taguchi, P.D. Lampe, S.M. Hanash

This work was supported by U01 CA152746 (P.D. Lampe and S.M. Hanash) from the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.