Jing W, Qin L, Zhang XH, Pan F, Ren RR, Li Y, Zhang Y, Peng LH, Wang ZK, Zhang XL. Diagnostic efficacy of fecal calprotectin combined with a high-sensitivity quantitative fecal immunochemical test for colorectal tumors. World J Gastroenterol 2026; 32(28): 121267 [DOI: 10.3748/wjg.121267]
Corresponding Author of This Article
Xiu-Li Zhang, MD, Chief, Microbiota Division, Department of Gastroenterology and Hepatology, The First Medical Center of Chinese People’s Liberation Army (PLA) General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China. zhangxl70@126.com
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Jing W, Qin L, Zhang XH, Pan F, Ren RR, Li Y, Zhang Y, Peng LH, Wang ZK, Zhang XL. Diagnostic efficacy of fecal calprotectin combined with a high-sensitivity quantitative fecal immunochemical test for colorectal tumors. World J Gastroenterol 2026; 32(28): 121267 [DOI: 10.3748/wjg.121267]
Wei Jing, Lei Qin, Xiao-Han Zhang, Fei Pan, Rong-Rong Ren, Yan Li, Yan Zhang, Li-Hua Peng, Zi-Kai Wang, Xiu-Li Zhang, Microbiota Division, Department of Gastroenterology and Hepatology, The First Medical Center of Chinese People’s Liberation Army (PLA) General Hospital, Beijing 100853, China
Wei Jing, Xiao-Han Zhang, Fei Pan, Li-Hua Peng, Zi-Kai Wang, Xiu-Li Zhang, School of Medicine, Nankai University, Tianjin 300071, China
Author contributions: Jing W and Qin L contributed equally to this work, as co-first authors; Jing W, Qin L, and Zhang XL were responsible for the study design; Jing W was responsible for data analysis and writing of the original draft; Qin L was responsible for data collection and curation, and writing of the original draft; Zhang XH, Pan F, Ren RR, Li Y, and Zhang Y carried out the study implementation; Peng LH and Wang ZK provided reagents; all authors have read and approved the final manuscript.
AI contribution statement: The authors take full responsibility and accountability for all content of this manuscript, including any portions for which AI tools were used as assistive technologies. All AI-assisted outputs were carefully reviewed, validated, and approved by the authors. AI tools were not used to generate original scientific data, perform independent scientific analyses, or draw scientific conclusions.
Institutional review board statement: This study was approved by the Ethics Committee of the First Medical Center of Chinese People’s Liberation Army General Hospital, No. S2025-886-01.
Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: The 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.
STROBE statement: The authors have read the STROBE Statement—a checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-a checklist of items.
Data sharing statement: No additional data are available.
Corresponding author: Xiu-Li Zhang, MD, Chief, Microbiota Division, Department of Gastroenterology and Hepatology, The First Medical Center of Chinese People’s Liberation Army (PLA) General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China. zhangxl70@126.com
Received: March 20, 2026 Revised: June 2, 2026 Accepted: June 22, 2026 Published online: July 28, 2026 Processing time: 116 Days and 19 Hours
Abstract
BACKGROUND
Fecal calprotectin (FC) has demonstrated good efficacy in assessing disease activity in inflammatory bowel disease; however, its value in detecting colorectal cancer (CRC) remains unclear. Although most symptomatic patients actually present with normal or minor pathological findings, they often still undergo colonoscopy in many clinical scenarios, resulting in unnecessary utilization of healthcare resources.
AIM
To investigate the diagnostic value of FC in colorectal tumors, and to determine whether the combination of FC with high-sensitivity quantitative fecal immunochemical test (hs-qFIT) can improve diagnostic efficacy for colorectal tumors compared to either test alone.
METHODS
We enrolled medium- and high-risk individuals for CRC who underwent colonoscopy in the Department of Gastroenterology and Hepatology or received surgical treatment in the Department of General Surgery at the First Medical Center of Chinese People’s Liberation Army General Hospital between March 2024 and January 2026. FC and hs-qFIT were performed prior to colonoscopy or gastrointestinal surgery. The diagnostic performance of FC, hs-qFIT, and their combination for CRC and adenomas was evaluated by calculating sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC).
RESULTS
A total of 120 subjects were included in this study. The sensitivity of FC for diagnosing CRC was 77.5% [95% confidence interval (CI): 61.7%-88.6%], the specificity was 65% (95%CI: 48.5%-78.8%), the positive predictive value (PPV) was 68.9% (95%CI: 53.5%-81.2%), the negative predictive value was 74.3% (95%CI: 57.1%-86.8%), and the AUC was 0.797 (95%CI: 0.697-0.897). The combination of FC and hs-qFIT increased the sensitivity for diagnosing CRC to 87.5% (95%CI: 73%-95.8%), increased the negative predictive value to 82.8% (95%CI: 64.7%-93.2%), and achieved an AUC of 0.866 (0.784-0.949), indicating that the combined strategy improves diagnostic performance for CRC. However, the combined test missed 12.5% of CRC cases. This strategy may help determine the urgency of further evaluation, but double-negative results cannot yet completely rule out a diagnosis of CRC. The combination of FC and hs-qFIT increased the sensitivity for diagnosing any colorectal lesion to 76.3% (95%CI: 65.5%-84.7%) and the PPV to 79.2% (95%CI: 68.5%-87.2%). Although the performance was poorer than that for CRC, the combined test may provide some guidance for referral of patients with colorectal lesions.
CONCLUSION
FC serves as a reliable biomarker for diagnosing CRC, and its combination with hs-qFIT further enhances both the sensitivity and negative predictive value for CRC detection, thereby improving the overall diagnostic efficacy for colorectal tumors.
Core Tip: Fecal calprotectin is a calcium-containing antimicrobial protein complex. Currently, it is primarily used in clinical practice for the diagnosis and monitoring of inflammatory bowel disease; however, its diagnostic value in colorectal tumors remains to be established. This paper draws a preliminary conclusion that fecal calprotectin can serve as a reliable biomarker for colorectal cancer. When combined with the fecal high-sensitivity quantitative immunochemical test, calprotectin increases the sensitivity for diagnosing colorectal cancer to 87.5%, raises the negative predictive value to 82.8%, and achieves an area under the receiver operating characteristic curve of 0.866. The combination of two biomarkers enhances the diagnostic efficacy for colorectal tumors.
Citation: Jing W, Qin L, Zhang XH, Pan F, Ren RR, Li Y, Zhang Y, Peng LH, Wang ZK, Zhang XL. Diagnostic efficacy of fecal calprotectin combined with a high-sensitivity quantitative fecal immunochemical test for colorectal tumors. World J Gastroenterol 2026; 32(28): 121267
Colorectal cancer (CRC) is currently the third most common malignancy and the second leading cause of cancer death[1]. The global incidence of CRC is projected to increase to 2.5 million by 2035[2]. The overall survival rate for advanced CRC is low, with a 5-year survival rate of only 50%[3]. The majority of patients are already in the intermediate or advanced stages at the time of diagnosis[4]. Due to their rising incidence and mortality rates year by year, colorectal tumors have become a public health issue requiring urgent solutions. The prognosis of CRC depends mainly on the tumor stage at diagnosis[5]; therefore, to achieve early diagnosis and treatment, it is crucial to find effective non-invasive biomarkers for CRC. Mutation of the APC gene is considered a key initial step in the formation of benign adenomas. Healthy intestinal epithelial cells can develop into adenomas within 5 years to 10 years, and subsequently transform into malignant cells[6]. The development of most CRCs follows the “adenoma to low-grade intraepithelial neoplasia to high-grade intraepithelial neoplasia to carcinoma” sequence[7], a relatively slow and linear progression that provides an important window of opportunity for the prevention, early diagnosis, and early treatment of colorectal tumors. Digestive symptoms (such as abdominal pain, change in bowel habits, and rectal bleeding) are common reasons for visits to primary healthcare facilities[8]. While the majority of symptomatic patients actually have normal findings or only minor lesions[9], many still undergo colonoscopy in many situations, resulting in unnecessary waste of resources and exposing patients to non-negligible risks of endoscopy-related complications[10]. Total colonoscopy and pathological results are the gold standard for diagnosing colorectal tumors; however, given the specialized nature of the procedure, its invasiveness, and the associated risks of complications which cannot be ignored[11], colonoscopy is not suitable for large-scale population-based screening for colorectal tumors. Screening for CRC can reduce both its incidence and mortality[4]. Organized screening programs are currently being implemented globally, aiming to increase early detection rates and reduce the incidence and mortality of CRC.
Fecal immunochemical test (FIT) detects human hemoglobin antigens in stool using specific antibodies, enabling quantitative measurement of fecal hemoglobin concentrations to assess intestinal bleeding. Compared with the traditional guaiac-based fecal occult blood test (FOBT), this technique is not affected by dietary or drug interference and offers higher sensitivity and stability[12]. Currently, the “two-step” strategy whereby individuals with positive FIT results subsequently undergo colonoscopy remains the most widely adopted approach for CRC screening worldwide. This study employs a high-sensitivity quantitative FIT (hs-qFIT), which lowers the detection limit of fecal hemoglobin to 10 ng/mL. A meta-analysis by Imperiale et al[13], encompassing 31 studies, reported that at a threshold of 10 μg/g, the sensitivity of a single FIT for detecting CRC was 91%, and for advanced adenoma (AA), 40%. At a threshold greater than 20 μg/g, the sensitivity for CRC decreased to 71%, and for AA to 27%. These findings indicate that reducing the FIT threshold enhances the detection of both CRC and AA, thereby improving diagnostic sensitivity.
Fecal calprotectin (FC) is a calcium-containing antibacterial protein complex belonging to the S-100 protein family, composed of three polypeptide chains. It accounts for approximately 60% of the cytosolic proteins in neutrophils and is released during acute and chronic inflammation of the gastrointestinal tract wall[14]. As a sensitive marker of intestinal inflammation, FC detection is currently primarily used clinically for the diagnosis and monitoring of inflammatory bowel disease (IBD)[15]; however, FC levels can also be elevated in various gastrointestinal disorders, including colitis and malignant tumors[16]. Previous studies have shown that FC levels are elevated in patients with CRC and return to normal levels after surgical resection[17]. CRC is often accompanied by varying degrees of local acute inflammatory response[18]. Research has found that intestinal inflammation can drive carcinogenesis, and the presence of an inflammatory microenvironment is a key hallmark of cancer[19]. Because FC reflects the inflammatory status of the intestines, it may hold potential value in the screening for colorectal tumors. FC is resistant to enzymatic degradation, remaining stable at room temperature for up to 7 days[20] and is easy to detect. In a study published in 2016[21], researchers analyzed FC levels in 654 patients referred due to alarm symptoms and suspected CRC (according to United Kingdom National Institute for Health and Care Excellence criteria). The results showed that at a cut-off value of 50 μg/g, the test had a negative predictive value (NPV) of 98.6% for CRC; when polyps larger than 10 mm were included in the analysis, the NPV was 97.2%. In a recent systematic review, we synthesized the progress of research in this field and evaluated the diagnostic value of FC for colorectal tumors[22]. Our findings demonstrated that the utility of FC is characterized by high sensitivity and moderate specificity, with its diagnostic performance correlating with tumor stage and location. More importantly, the review identified critical knowledge gaps in the current literature, notably that “the diagnostic efficacy of combining FC with other biomarkers has yet to be fully validated”. Therefore, the present study was designed to assess the diagnostic value of FC for colorectal tumors and to investigate whether the combination of FC and hs-qFIT offers superior diagnostic performance compared to either test alone, with the goal of identifying a more effective non-invasive screening strategy.
MATERIALS AND METHODS
Materials
Subjects undergoing colonoscopy in the Department of Gastroenterology or surgical treatment in the Department of General Gastrointestinal Surgery at the First Medical Center of Chinese People’s Liberation Army General Hospital from March 2024 to January 2026 were enrolled, and basic demographic information was recorded for each subject. Inclusion criteria were: (1) Age: 18-75 years, regardless of sex; (2) Assessed as being at intermediate or high risk for CRC according to the Asia Pacific Colorectal Screening scoring system; and (3) Voluntarily participating in this study and signing the informed consent form, and able to cooperate with sampling and follow-up. Exclusion criteria were: (1) History of CRC or surgical history, IBD, ischemic colitis, or other intestinal diseases that may cause colorectal bleeding; (2) Recent bloody stools, hematuria, severe diarrhea, or watery stools; (3) Women who are menstruating, pregnant, or breastfeeding; (4) Severe underlying diseases making tolerance of a complete colonoscopy difficult; and (5) Conditions such as intellectual disability, dementia, or poor performance status making provision of a stool sample difficult. This study was approved by the Ethics Committee of the First Medical Center of Chinese People’s Liberation Army General Hospital, No. S2025-886-01.
Fecal sample collection and testing/analysis
All participants received one-on-one instruction to ensure full understanding of the sample collection requirements. There were no restrictions on medication or diet for any subjects, who independently collected stool samples using the provided collection tubes. Within one week prior to colonoscopy, all participants used the sampling probes of the specialized collection tubes for hs-qFIT and FC testing to quantitatively collect approximately 2 mg of stool by broadly and evenly scraping the surface of the fecal sample (to comprehensively capture trace hemoglobin on the stool surface and minimize missed diagnoses). The sampling probe was then inserted into the collection tube to ensure the stool dissolved in 2 mL of hemoglobin lysate within the tube. After completing stool sample collection, participants returned the tubes to the study investigators. Laboratory personnel, who were blinded to patient information and colonoscopy results, performed hs-qFIT and FC testing using the AC-SCREEN® detection system (Dalian Zhongfu Bioengineering Co., Ltd.), which can quantitatively measure hemoglobin at concentrations ≥ 10 ng/mL and FC at concentrations ≥ 30 μg/g. In this study, the conversion ratio between ng/mL, the unit used for hs-qFIT, and μg/g, the unit mentioned in the literature is 1:1.
Colonoscopy and pathological examination
All patients underwent colonoscopy following standard bowel preparation, with a Boston Bowel Preparation Score of ≥ 6 considered indicative of adequate preparation. A complete colonoscopy was defined as one that reached the cecum or was halted due to luminal obstruction by malignant lesions. Biopsies or resections were performed on all lesions detected during colonoscopy, and their histopathological characteristics were evaluated. Based on the histological characteristics of the most severe lesion, colonoscopy findings were classified as CRC, adenoma, or no abnormality. AA was defined as adenomas with a maximum diameter of ≥ 10 mm, tubulovillous or villous adenomas, or those exhibiting high-grade intraepithelial neoplasia. All pathological diagnoses were independently conducted by two chief pathologists; in cases of diagnostic discrepancy, a third chief pathologist’s conclusion was adopted as definitive. Both endoscopists and pathologists were blinded to the fecal test results.
Statistical analysis
Statistical analysis of the data was performed using SPSS 27.0. Normally distributed measurement data are expressed as mean ± SD, while non-normally distributed measurement data are presented as median (lower quartile, upper quartile). Categorical data are reported as counts and percentages. Normally distributed data were analyzed using one-way analysis of variance, non-normally distributed or unknown distribution data were assessed using the Kruskal-Wallis H test for k independent samples, and categorical variables were evaluated using the χ2 test. A binary logistic regression analysis was performed with CRC diagnosis as the dependent variable, and FC, hs-qFIT, age, and alcohol consumption were simultaneously entered into the model to adjust for the effects of confounding factors. Sensitivity, specificity, positive predictive value (PPV), and NPV of each fecal test (both individually and in combination) were calculated for relevant pathologies and various types of lesions. Additionally, for colorectal-related pathologies, the area under the receiver operating characteristic curve (AUC) was calculated for FC, hs-qFIT, and their combination. The Shapiro-Wilk test was used to assess data normality, and the Kruskal-Wallis H test was employed to compare differences between groups. A P < 0.05 was considered statistically significant.
RESULTS
Baseline characteristics of the study participants
Study participants: As of now, a total of 120 patients aged 18-75 years at intermediate or high risk for CRC have been enrolled in this study, including 40 cases (33.3%) of CRC, 40 cases (33.3%) of adenoma, and 40 cases (33.3%) in the control group. Fecal FC and hs-qFIT tests were completed for all patients prior to undergoing colonoscopy or gastrointestinal surgery. Analysis of baseline clinical data from the 120 enrolled subjects included 55 males (45.8%) and 65 females (54.2%). Among them, 26 cases (21.7%) had a history of smoking, 38 cases (31.7%) had a history of alcohol consumption, and 23 cases (19.2%) had a family history of digestive system diseases. The mean ages of the control group, adenoma group, and CRC group were 49.68 ± 11.67 years, 50.15 ± 10.49 years, and 54.93 ± 8.45 years, respectively. Details regarding height, weight, body mass index, and medical history are shown in Table 1. Table 1 shows highly significant differences among the three groups in age (P = 0.022) and alcohol consumption (P = 0.003). To control for the confounding effects of age and alcohol consumption, we performed a binary logistic regression analysis. With CRC diagnosis as the dependent variable, FC, hs-qFIT, age, and alcohol consumption were simultaneously entered into the model. The results showed that after adjusting for age and alcohol consumption, FC [odds ratio (OR) = 1.002, 95% confidence interval (CI): 1.000-1.004, P = 0.043] and hs-qFIT (OR = 1.008, 95%CI: 1.002-1.014, P = 0.008) remained significantly associated with CRC, indicating that both are independent predictors of CRC.
Table 1 Baseline characteristics of the study participants, mean ± SD/n (%).
Reasons for undergoing colonoscopy among the enrolled participants: Health check-up (40%), abdominal pain or discomfort (25%), bloody stool (11.7%), change in bowel habits (9.2%), diarrhea (4.2%), constipation (3.3%), and other reasons (6.7%).
Reasons for undergoing colonoscopy among patients with CRC: Bloody stool (27.5%), health check-up (22.5%), abdominal pain or discomfort (17.5%), change in bowel habits (20%), constipation (2.5%), and other reasons (10%).
Statistical analysis results
Fecal test results: In this study, the overall sample was divided into three groups for statistical analysis: The control group, the adenoma group, and the CRC group. Among the 120 enrolled subjects, 55 (45.8%) were FC negative and 65 (54.2%) were FC positive; 70 (58.3%) were hs-qFIT negative and 50 (41.7%) were hs-qFIT positive. The median FC levels in the control group, adenoma group, and CRC group were 21.21 μg/g (21.21-53.08 μg/g), 26.91 μg/g (21.21-86.43 μg/g), and 177.24 μg/g (43.82-906.39 μg/g), respectively. The median hs-qFIT levels in the control group, adenoma group, and CRC group were 7.07 ng/mL (7.07-7.07 ng/mL), 7.07 ng/mL (7.07-32.45 ng/mL), and 125.67 ng/mL (19.27-184.02 ng/mL), respectively (normality tests for the two markers in the three groups using the Shapiro-Wilk test showed P < 0.001, indicating that the data did not follow a normal distribution).
Comparison between groups: The Kruskal-Wallis H test was performed on FC and hs-qFIT levels across the three groups, and the results indicated that the differences were statistically significant (P < 0.001). Subsequent pairwise comparisons showed that the difference in FC between the control group and the adenoma group was not significant (adjusted P = 0.547 > 0.05 after Bonferroni correction). However, significant differences in FC were found between the control group and the CRC group, as well as between the adenoma group and the CRC group (P < 0.001), with FC levels in the CRC group being significantly higher than those in the control group and the adenoma group. For hs-qFIT, significant differences were observed between the control group and the adenoma group (adjusted P = 0.039 < 0.05 after Bonferroni correction), as well as between the control group and the CRC group, and between the adenoma group and the CRC group (P < 0.001). Hs-qFIT levels in the CRC group were significantly higher than those in the control group and the adenoma group. These results indicate that although there was a difference in FC levels between the adenoma group and the normal control group, it did not reach statistical significance, whereas both biomarkers in the CRC group were significantly elevated compared to the control and adenoma groups. The distribution differences of FC and hs-qFIT among the different groups are illustrated in Figure 1, respectively.
Figure 1 The distribution of fecal calprotectin and high-sensitivity quantitative fecal immunochemical test in the control, adenoma, and colorectal cancer groups.
A: The difference in fecal calprotectin (FC) between the control group and the adenoma group was not significant (adjusted P = 0.547 > 0.05 after Bonferroni correction). Significant differences in FC were found between the control group and the colorectal cancer (CRC) group, as well as between the adenoma group and the CRC group (P < 0.001), with FC levels in the CRC group being significantly higher than those in the control group and the adenoma group; B: Significant differences were observed between the control group and the adenoma group (adjusted P = 0.039 < 0.05 after Bonferroni correction), as well as between the control group and the CRC group, and between the adenoma group and the CRC group (P < 0.001). High-sensitivity quantitative fecal immunochemical test levels in the CRC group were significantly higher than those in the control group and the adenoma group. FC: Fecal calprotectin; CRC: Colorectal cancer; hs-qFIT: High-sensitivity quantitative fecal immunochemical test.
Diagnostic performance of FC and hs-qFIT for colorectal tumors
Diagnostic performance of FC across different disease groups: In the adenoma group, FC detection showed a sensitivity of 50% (95%CI: 33.9%-66.1%), specificity of 65% (95%CI: 48.5%-78.8%), PPV of 58.8% (95%CI: 41.4%-74.5%), NPV of 56.5% (95%CI: 41.6%-70.6%), and an AUC of 0.591 (95%CI: 0.466-0.716). In the CRC group, FC detection demonstrated a sensitivity of 77.5% (95%CI: 61.7%-88.6%), specificity of 65% (95%CI: 48.5%-78.8%), PPV of 68.9% (95%CI: 53.5%-81.2%), NPV of 74.3% (95%CI: 57.1%-86.8%), and an AUC of 0.797 (95%CI: 0.697-0.897). In patients with intestinal lesions, FC detection exhibited a sensitivity of 63.8% (95%CI: 52.2%-74.1%), specificity of 65% (95%CI: 48.5%-78.8%), PPV of 78.5% (95%CI: 66.8%-87.2%), NPV of 47.3% (95%CI: 34%-61%), and an AUC of 0.694 (95%CI: 0.6-0.787), as shown in Table 2.
Table 2 Diagnostic performance of fecal calprotectin across different disease groups.
Diagnostic performance of hs-qFIT across different disease groups: As shown in Table 3, in the adenoma group, hs-qFIT detection demonstrated a sensitivity of 37.5% (95%CI: 22.9%-54.3%), specificity of 92.5% (95%CI: 79.7%-98.1%), PPV of 83.3% (95%CI: 60.5%-95.4%), NPV of 59.7% (95%CI: 46.6%-71.6%), and an AUC value of 0.653 (95%CI: 0.532-0.774). In the CRC group, hs-qFIT detection showed a sensitivity of 80% (95%CI: 64.4%-90.5%), specificity of 92.5% (95%CI: 79.7%-98.1%), PPV of 91.4% (95%CI: 77.2%-97.8%), NPV of 82.2% (95%CI: 67.9%-91.5%), and an AUC of 0.872 (95%CI: 0.786-0.958). In patients with intestinal lesions, hs-qFIT detection exhibited a sensitivity of 58.8% (95%CI: 47.1%-69.7%), specificity of 92.5% (95%CI: 79.7%-98.1%), PPV of 94% (95%CI: 83%-98.6%), NPV of 52.9% (95%CI: 40.6%-64.9%), and an AUC of 0.762 (95%CI: 0.677-0.848), as detailed in Table 3. The diagnostic performance of hs-qFIT progressively improved with increasing severity of colorectal tumors.
Table 3 Diagnostic performance of high-sensitivity quantitative fecal immunochemical test across different disease groups.
Diagnostic performance of combined FC and hs-qFIT: As shown in Table 4, the sensitivity of the combined FC and hs-qFIT test (parallel testing) for diagnosing adenomas was 65% (95%CI: 48.5%-78.8%), specificity was 60% (95%CI: 43.5%-75%), PPV was 61.9% (95%CI: 46.2%-76%), NPV was 63.2% (95%CI: 46.6%-77.8%), positive likelihood ratio (LR+) was 1.63 (1.04-2.53), negative likelihood ratio (LR-) was 0.58 (0.36-0.95), and the AUC value was 0.662 (95%CI: 0.543-0.782), with the receiver operating characteristic curve shown in Figure 2A. These results indicate that although the sensitivity of the combined test was higher than that of either test alone, the combined test is not reliable for diagnosing adenomas and is not recommended as a routine diagnostic method for adenomas. The sensitivity of the combined FC and hs-qFIT test for diagnosing CRC was 87.5% (95%CI: 73%-95.8%), specificity was 60% (95%CI: 43.5%-75%), PPV was 68.6% (95%CI: 54.9%-80.2%), NPV was 82.8% (95%CI: 64.7%-93.2%), LR+ was 2.19 (1.47-3.26), LR- was 0.21 (0.09-0.49), and the AUC value was 0.866 (95%CI: 0.784-0.949), with the receiver operating characteristic curve shown in Figure 2B. These results demonstrate that the diagnostic performance of the combined test for CRC is superior to that of either test alone. It cannot be ignored that 5 out of 40 CRC patients were missed. The characteristics of these false-negative CRC cases (double-negative on both fecal tests) are described in Table 5, which details for each patient the tumor size, stage, location, histology, clinical presentation, fecal test values, whether the specimen was fresh or frozen, and the presence of anemia or overt blood loss.
Figure 2 Receiver operating characteristic curves of fecal calprotectin, high-sensitivity quantitative fecal immunochemical test, and their combination.
A: Diagnosing adenomas. The area under the receiver operating characteristic curve (AUC) value of fecal calprotectin (FC) for diagnosing adenoma was 0.591 [95% confidence interval (CI): 0.466-0.716], the AUC value of high-sensitivity quantitative fecal immunochemical test (hs-qFIT) for diagnosing adenoma was 0.653 (95%CI: 0.532-0.774), and the AUC value of the combination of the two for diagnosing adenoma was 0.662 (95%CI: 0.543-0.782); B: Diagnosing colorectal cancer (CRC). The AUC value of FC for diagnosing CRC was 0.797 (95%CI: 0.697-0.897), the AUC value of hs-qFIT for diagnosing CRC was 0.872 (95%CI: 0.786-0.958), and the AUC value of the combination of the two for diagnosing CRC was 0.866 (95%CI: 0.784-0.949). FC: Fecal calprotectin; hs-qFIT: High-sensitivity quantitative fecal immunochemical test.
Table 4 Diagnostic performance of the combined test across different disease groups.
Differences between left-sided colon, right-sided colon, and rectum
In this study, the CRC disease group was divided into left-sided colon, right-sided colon, and rectal cancer groups. The Shapiro-Wilk test showed P < 0.001, indicating that the data did not follow a normal distribution. The Kruskal-Wallis H test was used to compare FC and hs-qFIT levels among the three groups, and the results showed no significant differences between the groups (P = 0.396, P = 0.91). As shown in Figure 3, FC and hs-qFIT levels in the left colon, right colon, and rectum were compared. The results showed that hs-qFIT levels were 122.2 (9-185.2) ng/mL in the left colon, 127.2 (24.3-165.1) ng/mL in the right colon, and 129.5 (25.4-181) ng/mL in the rectum, indicating that hs-qFIT levels were higher in rectal cancer patients than in colon cancer patients, which is consistent with the detection principle of hs-qFIT. FC levels were 132.4 μg/g (21.2-326.9 μg/g) in left-sided colon cancer, 807.1 μg/g (21.2-1002.7 μg/g) in right-sided colon cancer, and 238.6 μg/g (76.4-1217 μg/g) in rectal cancer, showing that FC levels were significantly higher in right-sided colon cancer than in left-sided colon cancer.
Figure 3 Distribution differences in left-sided colon, right-sided colon, and rectum.
A: High-sensitivity quantitative fecal immunochemical test (hs-qFIT). Hs-qFIT levels were 1222 ng/mL (9-185.2 ng/mL) in left-sided colon cancer, 127.2 ng/mL (24.3-165.1 ng/mL) in right-sided colon cancer, and 129.5 ng/mL (25.4-181 ng/mL) in rectal cancer; B: Fecal calprotectin (FC). FC levels were 1324 μg/g (21.2-326.9 μg/g) in left-sided colon cancer, 807.1 μg/g (21.2-1002.7 μg/g) in right-sided colon cancer, and 238.6 μg/g (76.4-1217 μg/g) in rectal cancer. FC: Fecal calprotectin; hs-qFIT: High-sensitivity quantitative fecal immunochemical test.
DISCUSSION
The mortality rate of CRC ranks second among all tumors[1]. CRC screening can effectively reduce the morbidity and mortality of CRC[23]. To date, there is a lack of ideal differential diagnostic methods for lower gastrointestinal diseases in clinical practice. In actual clinical work, preliminary diagnosis is primarily based on medical history and clinical manifestations, but the misdiagnosis rate is relatively high[24,25]. Moreover, by the time clinical manifestations such as abdominal pain and hematochezia appear, the tumor has generally progressed to the middle or late stage. An analysis of the reasons for undergoing colonoscopy among the overall participants included in this study showed that the majority (40%) underwent colonoscopy for health check-ups. Among CRC patients, however, the proportion undergoing colonoscopy due to hematochezia, changes in bowel habits, abdominal pain, or abdominal discomfort was significantly increased, while the proportion undergoing colonoscopy for health check-ups was significantly reduced (22.5%). The results indicate that early-stage colorectal tumors often lack specific clinical manifestations. When warning symptoms such as hematochezia, abdominal pain, or changes in bowel habits appear, they often signify that the intestinal lesions have already reached the middle or late stage. Therefore, identifying biomarkers with high accuracy is crucial for achieving early diagnosis and treatment. Currently, colonoscopy and pathological results remain the gold standard for the diagnosis of colorectal tumors. However, this examination carries risks of complications such as bleeding and perforation. Due to its invasive nature and high medical costs, patient compliance is reduced. Hence, finding a novel non-invasive screening marker for colorectal tumors to optimize the stratification of patients requiring colonoscopy is particularly important and has become a research focus of common concern for both clinical practitioners and the patient population.
FIT, which detects fecal hemoglobin concentration using specific antibodies, has been established as an effective non-invasive screening method for colorectal tumors[26]. FC assesses inflammatory activity by quantifying the degree of intestinal inflammatory cell infiltration, whereas FIT reflects the extent of the lesion by detecting the amount of intestinal mucosal bleeding[27]. Current clinical practice guidelines recommend using FIT for fecal occult blood screening in symptomatic patients, with a cutoff value of 10 μg/g[28,29]. Multiple studies support this recommendation, reporting its high NPV for CRC and AAs[30,31]. Some researchers recommend using a threshold of 20 μg/g[32], as it maintains similar diagnostic performance while potentially reducing the need for colonoscopy[33]. The FC test is a non-invasive diagnostic method for assessing intestinal inflammation by measuring calprotectin levels in feces and can accurately differentiate between IBD and IBS (irritable bowel syndrome)[34]. International guidelines recommend FC testing for IBD patients to assess disease activity and guide treatment[35]. However, research on whether FC has a diagnostic role in intestinal tumors is currently insufficient, and its diagnostic value for CRC remains inconclusive. FC is released during neutrophil death or damage[36] and is believed to have a regulatory effect on components of the inflammatory process[37]. Besides playing a role in coordinating the acute inflammatory environment, FC also controls cell proliferation, differentiation, and apoptosis. For example, the S100A8/S100A9 complex regulates the tumor microenvironment in various tumors, activating and triggering tumorigenesis[38]; mice lacking S100A9 are protected from intestinal tumor development and inflammation[39]. The fact that inflammation promotes the occurrence and development of CRC provides the theoretical basis for using FC, an intestinal inflammation marker, to diagnose CRC[19]. Intestinal inflammation can drive carcinogenesis, promoting cancer formation and progression, and the presence of an inflammatory microenvironment is a key hallmark of cancer[19]. Since FC reflects the inflammatory state of the intestine, it may have some value in screening for colorectal tumors. Regarding the diagnostic performance of FC for colorectal tumors, relevant studies exist, but a consensus has not yet been reached. In a 2022 retrospective study by Blad et al[40], which included 124 CRC patients, 98 CRC patients (79%) had FC ≥ 50 μg/g, indicating that elevated FC is common in CRC patients and that FC has high sensitivity for CRC diagnosis. This is similar to the finding in this study where 31/40 (77.5%) CRC patients had FC ≥ 30 μg/g. In a 2022 study by Ross et al[41], which included 352 FOBT-positive patients, the sensitivity for diagnosing CRC at an FC cutoff of 50 μg/g was 92.8%, with a specificity of 41.7%. FC sensitivity increased sequentially as tumors progressed from non-advanced to malignant, with the highest sensitivity observed for malignant tumors. In the study by Lué et al[42], which included 404 patients (including 16 with CRC and 39 with AA), it was concluded that the combination of FOBT and FC had better diagnostic accuracy and lower cost compared to using either test alone; it could not only directly avoid unnecessary colonoscopies and prioritize high-risk patients but also reduce the additional costs associated with these interventions and their potential complications. In a 2023 study by Lanas et al[43], which included 1224 patients (69 diagnosed with CRC, 5.6%), when the fecal hemoglobin cutoff was 10 μg/g, the NPV for CRC was 98.6% and sensitivity was 79.7%. When combined with an FC cutoff of 150 μg/g, the sensitivity of the fecal test was further improved. Fecal hemoglobin and FC could serve as effective triage tools to help identify symptomatic patients at high risk for CRC. The results of this study show that using FC alone for CRC diagnosis had a sensitivity of 77.5% (95%CI: 61.7%-88.6%) and specificity of 65% (95%CI: 48.5%-78.8%). Using hs-qFIT alone for CRC diagnosis had a sensitivity of 80% (95%CI: 64.4%-90.5%) and specificity of 92.5% (95%CI: 79.7%-98.1%). The combination of FC and hs-qFIT increased the sensitivity for CRC to 87.5% (95%CI: 73%-95.8%), increased the NPV to 82.8% (95%CI: 64.7%-93.2%), with an LR+ of 2.19 (1.47-3.26), an LR- of 0.21 (0.09-0.49), and an AUC value of 0.866 (95%CI: 0.784-0.949). The results indicate that the sensitivity of the combined test for diagnosing CRC is significantly higher than that of either test alone; however, the specificity is lower, resulting in a higher false positive rate. Positive results still require further confirmation. The relatively high NPV suggests that double-negative results have high reliability. However, it cannot be ignored that the combined test missed 12.5% of CRC cases. Given that this study measured diagnostic accuracy at a single time point during the same diagnostic encounter, without prospective follow-up and subsequent testing of the double-negative results, we cannot yet conclude that double-negative results are absolutely safe. In clinical practice, the decision to use parallel testing should be based on patient risk, clinical symptoms, and clinical goals. For high-risk CRC individuals or symptomatic patients, parallel testing may help determine urgency but cannot completely rule out a diagnosis of CRC. A meta-analysis showed that FC has a sensitivity and specificity of 83% and 61% for CRC diagnosis, respectively, which is similar to the findings of this study[18]. Combining FC and hs-qFIT testing increased the sensitivity for diagnosing intestinal lesions to 76.3% (95%CI: 65.5%-84.7%) and the PPV to 79.2% (95%CI: 68.5%-87.2%), with an AUC value of 0.764 (95%CI: 0.68-0.848). Although the performance was inferior to that for CRC, the combined test offers some guidance for referring patients with intestinal lesions. The combined FC and hs-qFIT test for adenoma detection showed a sensitivity of 65% (95%CI: 48.5%-78.8%), a specificity of 60% (95%CI: 43.5%-75%), an AUC value of 0.662 (95%CI: 0.543-0.782), an LR+ of 1.63 (1.04-2.53), and an LR- of 0.58 (0.36-0.95). The results indicate that although the sensitivity of the combined test is higher than that of either test alone, the combined test is not reliable for diagnosing adenomas and is not recommended as a routine diagnostic method for adenomas.
This study compared FC levels among left-sided colon cancer, right-sided colon cancer, and rectal cancer. The results showed that the median FC level was 132.4 μg/g (21.2-326.9 μg/g) for left-sided colon cancer, 807.1 μg/g (21.2-1002.7 μg/g) for right-sided colon cancer, and 238.6 μg/g (76.4-1217 μg/g) for rectal cancer. In a review by Baran et al[44] examining the differences between left-sided and right-sided CRC, the authors concluded that right-sided and left-sided CRCs differ in terms of embryonic origin and tumor histology. Compared to left-sided CRC, which exhibit higher chromosomal instability, right-sided CRC is primarily characterized by high microsatellite instability and KRAS or BRAF mutations. Colorectal tumors located in the right colon may possess more immunogenic tumor characteristics than those in the left colon, theoretically leading to higher FC levels. The current findings, indicating that FC levels are higher in right-sided colon cancer than in left-sided colon cancer, align with the theory proposed in previous literature that the inflammatory burden is greater in the right colon compared to the left colon. However, the sample sizes within each disease group (left-sided colon, right-sided colon, and rectal cancer) in this study were small, necessitating further expansion of the sample size to enhance inter-group comparability and the reliability of the research.
FC and hs-qFIT are both non-invasive detection methods based on fecal samples, offering simple operation and high patient acceptance, making them particularly suitable for clinical scenarios such as hematochezia and changes in bowel habits. Hs-qFIT can serve as a reliable biomarker for ruling out CRC, while combining FC with hs-qFIT can further enhance the sensitivity and NPV for CRC diagnosis. A single combined test aids in objectively assessing the necessity and urgency of subsequent examinations, thereby enabling effective patient triage in clinical practice: For symptomatic patients or those undergoing outpatient screening, if both test results are positive, colonoscopy should be prioritized. If both results are negative, prospective follow-up of the patient is recommended to assess for interval cancer or delayed diagnosis. If only one of the two tests is positive, elective repeat fecal marker testing is advised. This strategy can not only improve the detection rate of severe colorectal lesions by accurately identifying high-risk patients requiring priority management but also significantly reduce unnecessary colonoscopies, lessen patients’ medical burden and colonoscopy-related risks, alleviate the workload on gastrointestinal endoscopy units, promote the rational allocation of medical resources, and enhance social benefits. Large-scale clinical trials are still needed in the future to further validate its diagnostic performance and cost-effectiveness.
This study has several limitations. First, as a single-center study conducted in an urban tertiary hospital, its results may not be generalizable to primary care institutions in rural areas, and future multi-center geographically diverse studies are needed. Second, the sample size included in this study was relatively small, necessitating future multi-center, large-sample clinical research to further validate its diagnostic performance. Third, the cohort in this study compared 40 controls, 40 adenoma patients, and 40 CRC patients, artificially creating a relatively high prevalence. The reported predictive values are derived from an enriched study sample, which may lead to an artificial elevation of diagnostic performance. Additionally, some fecal specimens were tested after freezing (storage temperature: -80 °C, storage duration: < 1 week, without undergoing freeze-thaw cycles), which could potentially lead to degradation of hemoglobin or calprotectin; however, studies have shown that FIT and FC values remain reliable after long-term freezing[45,46]. Future research should also expand the CRC sample size to clarify the relationship between combined FC and hs-qFIT testing and colorectal tumor staging and location, and to explore its association with tumor molecular characteristics (such as KRAS mutations, BRAF mutations, etc.).
CONCLUSION
FC is a biological marker reflecting intestinal inflammatory activity. The combined detection of FC and hs-qFIT can improve the diagnostic efficacy for colorectal tumors. In this study, the combination of FC and hs-qFIT further increased the sensitivity and NPV for CRC diagnosis; however, false-negative patients still exist. Therefore, double-negative results should not be used alone to rule out CRC, especially in symptomatic or high-risk patients. This combined test may serve as an auxiliary triage tool for prioritizing colonoscopy rather than as an exclusionary test. The diagnostic performance of the combined test for adenomas is poor, and it is not recommended for routine adenoma screening. The combination of FC and hs-qFIT can be used as a pre-endoscopy triage aid, and a negative combined test result may also alleviate patient anxiety. Incorporating this strategy into clinical practice may help reduce the excessive burden on healthcare systems. The sample size of this study is relatively small, and large-scale prospective studies are needed in the future to analyze its clinical cost-effectiveness. In summary, the combination of FC and hs-qFIT fecal testing should be incorporated into the non-invasive screening system for individuals at high risk of CRC to enable prioritized triage for colonoscopy. Those without warning symptoms and with negative results on both fecal tests can be managed conservatively; conversely, those with warning symptoms and positive results on both tests require prioritized and prompt colonoscopy. This combined testing strategy may serve as an auxiliary pre-endoscopy triage tool.
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Footnotes
Peer review: Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific quality: Grade A, Grade B, Grade B, Grade B, Grade B
Novelty: Grade A, Grade B, Grade B, Grade B, Grade C
Creativity or innovation: Grade B, Grade B, Grade B, Grade B, Grade C
P-Reviewer: Chen H, MD, China; Despalatovic BR, Assistant Professor, Croatia; Shelat VG, Associate Professor, Singapore S-Editor: Fan M L-Editor: A P-Editor: Yang YQ
