Diagnostic value of serum fibrin degradation complex DR-70 combined with conventional tumor biomarkers in colorectal cancer

Abstract

Objective

Most patients with colorectal cancer (CRC) show no early symptoms, and tumor markers have low sensitivity and specificity. We therefore investigated the ability of serum fibrin degradation complex DR-70 plus traditional tumor markers to diagnose CRC.

Methods

We retrospectively screened patients with CRC or non-malignant colorectal diseases, as well as healthy individuals, for inclusion in this study. The individuals’ clinical characteristics were recorded, and serum samples were collected. Expression levels of DR-70 and conventional tumor markers were measured by enzyme-linked immunosorbent assay and electrochemiluminescence.

Results

DR-70 levels differed significantly among patients with CRC, patients with benign colorectal diseases, and healthy individuals. Receiver operating characteristic curve analysis identified DR-70 as a conventional tumor marker with the highest sensitivity and the second-highest specificity after carcinoembryonic antigen.

Conclusions

This study identified DR-70 as a reliable marker for the detection, differentiation, and progression of CRC, with good sensitivity and specificity. DR-70 measurement could greatly improve the efficacy of CRC diagnosis when used together with other tumor markers.

Introduction

Colorectal cancer (CRC) is a malignant neoplasm affecting the digestive tract. It ranks as the third most frequent malignant tumor, following breast cancer and lung cancer. Relatively favorable subsets of cancers of undefined primary origin (CUP) have recently emerged, including colorectal CUP. This new clinical entity is treated as CRC, and contributes to the increased incidence of CRC. ¹ CRC is also the second leading cause of cancer-related mortality. ² Although older patients are more prone to severe postoperative complications, there is currently no consensus regarding the effect of age on survival outcomes, and the prognosis of older patients may be confounded by differences in stage at presentation, tumor site, preexisting comorbidities, and the type of treatment received. ³ The prevalence and mortality rates of CRC in China are increasing as a result of population aging, rapid advancements in the social economy, and a change in lifestyle patterns.^4,5

CRC occurs as a sequential progression involving histological, morphological, and genetic alterations that accrue gradually over time. ⁶ Most patients have no evident clinical manifestations during the initial phases of the disease, but subsequently develop progressive alterations in bowel habits, accompanied by symptoms such as abdominal pain, diarrhea, and bloody stools, leading to severe manifestations including intestinal obstruction, weight loss, and anemia in the advanced stages. ⁷ The lack of effective early screening methods means that many individuals are diagnosed late, thus missing the optimal window for diagnosis and treatment and resulting in poor prognoses. It is therefore imperative to enhance the screening methods for CRC. Immune checkpoint molecules, such as programmed cell death protein 1 and cytotoxic T-lymphocyte associated protein 4 act as negative regulators of the immune response and are expressed on the surface of immune cells. Immune cell programmed death-ligand 1 expression was significantly higher in DNA mismatch repair (MMR)-deficient (microsatellite instability-high, MSI-H) compared with MMR-proficient (microsatellite instability-low, MSI-L) tumors, with no differences among different MSI-H molecular subtypes. ⁸ The recommended screening for defective MMR includes immunohistochemistry and/or MSI testing; however, distilling the biological and technical heterogeneity of MSI testing into usable data is challenging, and immunohistochemistry testing for the MMR machinery has been reported to produce different results for a given germline mutation, possibly due to somatic mutations. ⁹ Serum tumor markers have been widely used to detect malignant tumors, primarily because of their inherent benefits, including simplicity of use, rapidity, and high levels of safety. Nevertheless, the sensitivity and specificity of tumor markers frequently employed in the clinical diagnosis of CRC are suboptimal.^10–12

The serum fibrinogen degradation complex DR-70 is generated through the action of plasmin on fibrinogen, resulting in degradation products with different molecular weights. It is normally phagocytosed by the reticuloendothelial system, metabolized in the liver, and excreted by the kidneys. Notably however, DR-70 is also a protein group that has been linked to malignant tumors, ¹³ and it has recently been shown to detect 13 types of malignant tumors, including gastric, colorectal, liver, breast, ovarian, and uterine cancers.^14–16

In this study, we examined serum DR-70 expression levels in individuals with CRC or benign colorectal diseases and in healthy individuals, using enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence, and clinical data collection. We also aimed to explore the potential association between DR-70 expression and clinical characteristics, and to evaluate the diagnostic value of DR-70 combined with conventional tumor markers for CRC, as a fresh approach for diagnosing CRC.

Materials and methods

Research participants

This investigation involved patients with primary CRC or benign colorectal diseases (mostly polyps, benign colorectal tumors, or ulcerative colitis) who were admitted to Zhejiang Provincial People’s Hospital between October 2020 and December 2021. Simultaneously, a control group of healthy individuals was chosen at random over the same period. All participants or their families signed informed consent forms for participation in this study. The inclusion criteria for the three respective groups were: 1) individuals who underwent medical intervention at Zhejiang Provincial People’s Hospital between October 2020 and December 2021, and were diagnosed with primary CRC based on endoscopic examinations, pathological diagnoses, or surgical procedures, and who received no radiotherapy or chemotherapy prior to surgery; 2) individuals diagnosed with benign colorectal diseases, including colorectal polyps, benign colorectal tumors, ulcerative colitis, or other diseases, who received no radiotherapy or chemotherapy prior to the surgery; and 3) healthy individuals with no infectious diseases, such as hepatitis A, B, or C, AIDS, or syphilis, no colorectal lesions on endoscopy, and normal liver, kidney, and lung functions. The exclusion criteria were: 1) individuals with other primary malignant tumors, including non-CRC gastrointestinal malignancies; 2) individuals with severe target organ damage manifesting as liver failure, kidney failure, congestive heart failure, uncontrolled infections, or recent severe trauma, burns, and similar conditions; and 3) individuals with conditions that might affect the results of DR-70 testing, such as cellulitis, bronchiectasis, rheumatic disease, hemolysis, hyperlipidemia, pulmonary embolism, or the use of anticoagulants.

Clinical information gathered for the participants included age, sex, clinical diagnosis, lesion location, and clinical staging. Clinical staging of CRC was carried out in accordance with the eighth edition of the TNM staging principles created by the American Joint Committee on Cancer (AJCC).

ELISA

Fasting peripheral blood samples (5 mL) were collected from all participants 2 to 3 days before endoscopy or surgery and stored at 25°C for 30 minutes. After coagulation and contraction, blood clots were centrifuged for 10 minutes at 500 × g. The serum was aspirated into a test tube and refrigerated at −20°C until testing. Levels of carcinoembryonic antigen (CEA), cancer antigen (CA) 12-5, CA19-9, CA15-3, and CA72-4 in the supernatant were quantified by ELISA, according to the manufacturer’s protocol (Roche Diagnostics GmbH, Basel, Switzerland). DR-70 levels were also detected by ELISA (Suzhou MicroDiag Biomedicine Co., Ltd., Suzhou, Jiangsu, China).

Electrochemiluminescence assay

Expression levels of tumor markers were detected in all samples using a fully automated Roche Cobas 8000 chemiluminescence immunoassay analyzer, in accordance with the manufacturer’s guidelines.

Statistical analysis

The data were analyzed statistically using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA) and the experimental data were analyzed and plotted using GraphPad Prism8 (Domatics, Boston, MA USA). Discrete variables were characterized as frequency and frequency distributions, and continuous variables were described and analyzed as mean ± standard deviation. Continuous variables with a normal distribution were compared between two or more groups using independent-sample t-tests and one-way ANOVA. For parameters that did not follow a normal distribution, Mann–Whitney and Kruskal–Wallis tests were used to verify paired differences and compare multiple independent data groups. A P value < 0.05 indicated a statistically significant difference between the case and control groups. We determined the sensitivity, specificity, positive predictive value, and negative predictive value of the tumor markers by receiver operating characteristic curve (ROC) analysis using GraphPad Prism8 with the Youden index (sensitivity +specificity − 1) as the optimal cut-off value. The maximum sensitivity and specificity of the optimal cut-off value were calculated using the abscissa and ordinate of the ROC curve, and the diagnostic performance was then described based on the sensitivity, specificity, and area under the curve (AUC).

Results

Demographics and clinical parameters

This study included 116 patients with primary CRC and 40 patients with benign colorectal diseases (mostly polyps, benign colorectal tumors, or ulcerative colitis) who met the inclusion criteria, and 100 healthy control individuals. The demographics of the participants are shown in Table 1. Patients with CRC had significantly different lesion locations compared with patients with benign colorectal diseases (P < 0.05). There was no significant difference in sex ratios among the three groups, but patients with CRC were significantly older than those with benign colorectal diseases and healthy controls (P < 0.001).

Table 1.

Clinical parameters and demographics of individuals with colorectal cancer or benign colorectal diseases, and healthy controls.

	Control group (n = 100)	Case group		Total	P
		Benign colorectal diseases (n = 40)	Colorectal cancer (n = 116)
Frequency
Sex
Male	52 (52%)	22 (55%)	67 (57.8%)	141 (55.1%)	0.700
Female	48 (48%)	18 (45%)	49 (42.2%)	115 (44.9%)
Location of lesion
Colon	/	29 (72.5%)	61(52.6%)	90 (57.7%)	0.028*
Rectum	/	11 (27.5%)	55 (47.4%)	66 (42.3%)
Mean ± SD
Age (years)	46.87 ± 12.77	54.35 ± 12.68	62.12 ± 12.73	54.95 ± 14.49	<0.001*
CEA (μg/L)	1.78 ± 1.10	2.15 ± 1.76	167.00 ± 1316.13	76.70 ± 887.68	<0.001*
CA125 (U/mL)	12.17 ± 7.70	10.25 ± 6.73	19.59 ± 25.64	15.23 ± 18.51	<0.001*
CA19-9 (U/mL)	10.92 ± 8.17	8.91 ± 6.46	88.78 ± 271.10	45.89 ± 186.30	<0.001*
CA15-3 (U/mL)	9.88 ± 5.32	11.57 ± 6.35	14.82 ± 8.38	12.38 ± 7.35	<0.001*
CA72-4 (U/mL)	3.79 ± 5.85	3.66 ± 4.24	13.74 ± 58.78	8.28 ± 39.99	<0.001*
DR-70 (μg/mL)	7.81 ± 4.59	9.57 ± 4.59	22.33 ± 19.64	14.66 ± 15.31	<0.001*

*P < 0.05, statistically significant.

We investigated tumor marker expression in the three groups. Patients with CRC had significantly higher expression levels of CEA, CA125, CA19-9, CA15-3, CA72-4, and DR-70 relative to the benign colorectal disease and control groups (P < 0.001) (Table 1). Pairwise analysis of variance showed significant differences in DR-70 levels between patients with CRC and the other groups (P < 0.001). There was no significant difference in DR-70 expression levels between the benign colorectal disease and control groups (Figure 1; P = 0.7711). DR-70 may thus be a promising biomarker for CRC screening and differentiation.

Figure 1.

DR-70 expression levels in patients with colorectal cancer or benign colorectal disease and in healthy individuals. DR-70 expression levels detected by enzyme-linked immunosorbent assay. Data presented as mean ± standard deviation of at least 40 independent experiments. ***P < 0.001

ns, not significant.

Associations between clinical characteristics and DR-70 expression

We measured DR-70 levels using a DR-70 assay kit and defined a level of ≤5.0 μg/mL as negative and >15.0 μg/mL as positive. We compared categorical variables of clinical parameters between the case and control groups using χ² or Fisher’s exact tests (Table 2). There was no difference in DR-70 expression between the sexes, but DR-70 expression levels differed significantly across age groups (P < 0.001). Expression levels of DR-70 in patients with benign colorectal diseases or CRC differed depending on the lesion location (P = 0.031), with colon lesions associated with a higher DR-70 positivity rate then rectal lesions (52.2% vs. 34.8%). We also investigated the association between DR-70 expression and distant metastases of CRC, and showed that patients with distant metastases had a higher DR-70 positivity rate (77.3%) than patients without distant metastases (51.1%) (P = 0.026). The AJCC 8th version of the TNM staging system divides CRC into stages I, II, III, and IV, and the DR-70 positivity rate increased with increasing tumor stage (P = 0.005). The above results show that DR-70 expression depended on age, lesion location, clinical stage, and the presence of distant metastasis, but not on sex.

Table 2.

Associations between DR-70 and clinical features

	DR-70		Total	χ2	P
	Negative	Positive
Sex
Male	100 (70.9%)	41 (29.1%)	141	0.007	0.932
Female	81 (70.4%)	34 (29.6%)	115
Total	181	75	256
Age (years)
≤55	110 (86.6%)	17 (13.4%)	127	30.80	<0.001*
>55	71 (55.0%)	58 (45.0%)	129
Total	181	75	256
Location of lesion
Colon	43 (47.8%)	47 (52.2%)	90	4.646	0.031*
Rectum	43 (65.2%)	23 (34.8%)	66
Total	86	70	156
Distant metastasis
Yes	5 (22.7%)	17 (77.3%)	22	4.971	0.026*
No	46 (48.9%)	48 (51.1%)	94
Total	51	65	116
TNM stage
I	20 (66.7%)	10 (33.3%)	30	12.88	0.005*
II	16 (50.0%)	16 (50.0%)	32
III	10 (31.3%)	22 (68.8%)	32
IV	5 (22.7%)	17 (77.3%)	22
Total	51	65	116

*P < 0.05, statistically significant.

Diagnostic values of different tumor markers

We compared the diagnostic efficacies of tumor biomarkers in CRC using the ROC curve AUC, optimal cut-off value, and sensitivity and specificity. DR-70 had the highest AUC (0.8655) for CRC, followed by CEA (AUC = 0.8386), suggesting that DR-70 was a more accurate tumor marker (Table 3, Figure 2). The sensitivity and specificity of the tumor markers were determined by analyzing ROC curves with horizontal and vertical coordinates, and the optimal cut-off value was calculated by the Youden index (Youden index = sensitivity + specificity − 1). DR-70 had the highest sensitivity (87.1%) and second-highest specificity (70.7%), with an optimal cut-off value of 11.34 μg/mL. ROC curve analyses also revealed significant differences in the diagnostic efficacy of DR-70 compared with CA12-5 (P = 0.024), CA19-9 (P = 0.047), CA15-3 (P = 0.048), and CA72-4 (P = 0.031) (Table S1).

Table 3.

Receiver operating characteristic curve analysis of tumor markers.

	AUC	Cut-off	Sensitivity (%)	Specificity (%)
CEA	0.8386	2.45 μg/L	76.4	76.7
CA125	0.6320	10.85 U/mL	62.9	60.3
CA19-9	0.6681	15.05 U/mL	84.3	51.7
CA15-3	0.6698	9.90 U/mL	58.6	69.8
CA72-4	0.6420	2.15 U/mL	62.9	60.3
DR-70	0.8655	11.34 μg/mL	87.1	70.7

AUC, area under the curve; CEA, carcinoembryonic antigen; CA, cancer antigen.

Figure 2.

Receiver operating characteristic (ROC) curves of various tumor markers. Diagnostic accuracy, sensitivity, and specificity of carcinoembryonic antigen (CEA), cancer antigen (CA) 125, CA19-9, CA15-3, CA72-4, and DR-70 for colorectal cancer based on ROC curves. AUC, area under the curve.

Diagnostic value of combined tumor marker detection

Considering the limited sensitivity and specificity of traditional single-item tests, we investigated the detection capabilities of multiple tumor markers, using the cut-off values in Table 3 to distinguish between positive and negative expression. Using all five conventional tumor markers (CEA, CA125, CA19-9, CA15-3, and CA72-4) had a sensitivity of 55.2%, specificity of 92.9%, positive predictive value of 86.5%, and negative predictive value of 71.4% (Table 4, Figure 3). The low sensitivity and negative predictive value thus limited the usefulness of these five indicators.

Table 4.

Diagnostic efficacies of tumor markers.

Detection marker	CRC (n = 116)	Non-CRC (n = 140)	AUC	Sensitivity (%)	Specificity (%)	Positive predictive value (%)	Negative predictive value (%)
Combined detection
DR-70+CEA				74.1	92.9	89.6	81.3
(+)	86	10	0.8771
(−)	30	130
DR-70+CEA+CA125				74.1	90.7	86.9	80.9
(+)	86	13	0.8713
(−)	30	127
DR-70+CEA+CA19-9				77.6	90.7	87.4	83.0
(+)	90	13	0.8825
(−)	26	127
DR-70+CEA+CA15-3				75.0	92.9	89.7	81.8
(+)	87	10	0.8779
(−)	29	130
DR-70+CEA+CA72-4				77.6	91.4	88.2	83.1
(+)	90	12	0.8619
(−)	26	128
CEA+CA125+CA19-9+CA15-3+CA72-4				55.2	92.9	86.5	71.4
(+)	64	10	0.7125
(−)	52	130
DR-70+CEA+CA125+CA19-9+CA15-3+CA72-4				81.0	87.1	83.9	84.7
(+)	94	18	0.8917
(−)	22	122

CRC, colorectal cancer; AUC, area under the curve; CEA, carcinoembryonic antigen; CA, cancer antigen.

Figure 3.

Sensitivity, specificity, negative predictive value, and positive predictive value of combined detection. Sensitivity, specificity, negative predictive value, and positive predictive value of tumor markers detected by enzyme-linked immunosorbent assay. CEA, carcinoembryonic antigen; CA, cancer antigen.

After combining DR-70 with the above conventional indicators, the combinations of DR-70 + CEA + CA19-9 and DR-70 + CEA + CA72-4 showed the highest sensitivity rates of 77.6%, while DR-70 + CEA, DR-70 + CEA + CA15-3, and CEA + CA125 + CA19-9 + CA15-3 + CA72-4 all had specificities of 92.9%. Compared with DR-70 alone, the addition of CEA reduced its sensitivity (77.6% vs 87.1%) and increased its specificity (92.9% vs 70.7%) for diagnosis. These results suggest that sensitivity and specificity for CRC diagnosis were largely determined by DR-70 and CEA.

The combination of DR-70, CEA, CA125, CA19-9, CA15-3, and CA72-4 for detection yielded the highest sensitivity of 81.0% and a negative predictive value of 84.7%, with an AUC of 0.8917, indicating the highest diagnostic efficiency compared with the other combinations.

Discussion

Most cases of CRC are sporadic and progress slowly over a few years, developing from normal mucosa, to adenoma, to cancer. ¹⁷ The prevention and cure of CRC through its early diagnosis and the removal of precancerous lesions or early tumors would greatly improve the quality of life and prognosis of patients with CRC. The gold standard for diagnosing CRC is colonoscopy in conjunction with pathological biopsy, and biomarker testing is also recommended as a part of the standard investigation in patients with suspected CRC. Among several major genetic mutations in CRC, RAS mutation is correlated with oncological aggressiveness and pathologic response to chemotherapy. There is also growing evidence that inflammation drives the development of the disease. Numerous studies have accordingly investigated the predictive and prognostic roles of various blood-based inflammatory markers, including neutrophil–lymphocyte ratio, lymphocyte–monocyte ratio, and platelet–lymphocyte ratio. In addition, the roles of micro RNAs (miRNAs) as tumor suppressor genes and oncogenes, and their diagnostic, prognostic, and predictive implications are also being explored. Specifically, upregulation of miR-126 was correlated with bevacizumab resistance, whereas overexpression of miR-31, miR-100, miR-125b, and downregulation of miR-7, were associated with resistance to cetuximab. ¹⁸

Large-scale colonoscopy is not a clinically practical approach for testing for CRC, and less-intrusive and more-affordable techniques are required. We therefore collected data from 256 participants with or without CRC and compared their demographic characteristics and clinical factors. In addition to differences in standard serum tumor markers, patients with CRC had higher expression levels of DR-70 compared with healthy individuals and patients with benign colorectal diseases.

Malignant tumors enhance thrombin and fibrin production, increasing blood hypercoagulability and thrombosis risk, and facilitating the generation of fibrin degradation products through the catalytic activities of plasmin and protease. ¹⁹ The local synthesis of thrombin and the subsequent formation and degradation of fibrin may play a significant role in the proliferation and dissemination of neoplastic cells.^20,21 The assessment of DR-70 thus has the potential to function as a clinical diagnostic tool for cancer.^22,23 In this study, we investigated the relationship between DR-70 expression and clinical features, and showed that DR-70 expression was related to age, lesion location, clinical staging, and the occurrence of distant metastasis in CRC. In terms of staging, DR-70 positivity was highest among patients with clinical stage IV disease, while the positivity rate among patients with distant metastases was 77.3%. These results suggest that DR-70 may be a useful tumor marker for monitoring disease progression in patients with CRC.

An optimal tumor marker should exhibit both high sensitivity and specificity; however, the present repertoire of tumor markers does not meet these ideal criteria. CEA, CA125, CA19-9, CA15-3, and CA72-4 are typically used as tumor markers for assessing individuals with CRC. Of these, CEA has demonstrated the ability to diagnose a range of cancers in clinical practice; however, certain inflammatory conditions, including inflammatory bowel disease, bronchitis, and pancreatitis, also elicit elevated serum CEA levels, ²⁴ and CEA can also be influenced by circumstances such as pregnancy and prolonged smoking.^25,26 Similarly, CA125 has low sensitivity for predicting CRC recurrence but can be used as a prognostic indicator for gastric, endometrial, pancreatic, and other cancers. ²⁷ DR-70 can be used as a screening tool for serological malignancy in clinical practice, but elevated levels cannot identify specific malignancies. The current results showed that DR-70 had the highest sensitivity (87.1%) and second-highest specificity (second to CEA, 70.7%) compared with traditional tumor markers for CRC. Single-agent detection, however, still cannot meet the needs of clinical diagnosis. We therefore examined the use of combined markers, and showed that DR-70 and CEA played major roles in determining the sensitivity and specificity for diagnosing CRC, while the combined use of CEA, CA125, CA19-9, CA15-3, CA72-4, and DR-70 increased the sensitivity and negative predictive value to 81.0% and 84.7%, respectively. The potential challenges for clinical promotion of our present study may include rising costs and increased financial burden on patients.

In summary, this study showed that DR-70 may be a potentially reliable marker for the early detection and differentiation of CRC, as well as serving as an indicator of disease progression. The combination of DR-70 with other tumor markers can increase their diagnostic efficiency for CRC.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This retrospective study was approved by the Ethics Committee of Zhejiang Provincial People’s Hospital (approval no. 2021QT150).

Supplementary material

Supplemental material for this article is available online.

ORCID iD

Lili Yu https://orcid.org/0000-0003-2064-7576