Evaluation of platelet indices as diagnostic biomarkers for colorectal cancer

Altered platelet indices, including platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT), have been found in various cancer types. This study aimed to evaluate the role of platelet indices as potential biomarkers for the diagnosis of colorectal cancer (CRC), and to assess the association between platelet indices and CRC clinicopathological characteristics. The study included 783 subjects with CRC, 463 subjects with colorectal adenomas (CA), and 689 control subjects from June 2015 to October 2017. All participants’ clinicopathological characteristics were collected and analyzed. Here, we found that PC, MPV and PCT levels in CRC patients were significantly higher than those in CA patients and healthy participants (p < 0.001); however, PDW level in CRC patients was significantly higher than that in healthy participants while lower than that in CA patients. Receiver-operating characteristic (ROC) analysis indicated that combined detection of PCT and CEA appears to be a more effective marker to distinguish CRC patients from CA patients, with 70% sensitivity and 83% specificity. Among CRC patients, PC and PCT levels were associated with TNM stages and tumor size; MPV and PCT levels were associated with vascular invasion. Our findings suggest that altered PC, MPV and PCT levels might serve as potential biomarkers for the diagnosis and prognosis of CRC.

cancer is significantly lower than that in healthy individuals 37 . Among the limitations of current research on the clinical significance of platelet indices in CRC are the following: First, the patient sample sizes of these studies are all less than 250, which might lead to a weaker power for the statistical significance. Second, there is no study for the clinical significance of PCT in CRC. Third, no paper to date clarified the diagnostic role of platelet indices in conjunction with other markers in patients with CRC, although it was widely recognized that biomarker combinations might have better diagnostic value than individual markers. Taken together, the clinical significance of PC, MPV, PDW and PCT in CRC still remains elusive.
In this study, we measured the levels of PC, MPV, PDW and PCT in a relatively large sample size, including patients with CRC, patients with colorectal adenomas (CA), and healthy controls. The diagnostic and prognostic value of platelet indices in CRC were examined in detail.

Results
The levels of PC, MPV, PDW and PCT in CRC patients. A total of 783 patients with CRC, 463 patients with colorectal adenomas (CA), and a control group of 689 healthy participants were enrolled in this study. PC, MPV, PDW and PCT levels in CRC patients, CA patients and healthy participants are shown in Fig. 1. The levels of PC, MPV and PCT were significantly higher in CRC patients than in CA patients and healthy participants; the level of PDW in CRC patients was significantly higher compared to healthy participants while lower than that in CA patients (Fig. 1). Our data demonstrated that altered PC, MPV, PDW and PCT levels are found in CRC patients compare to CA patients and healthy participants.
Evaluation of PC, MPV and PCT as potential diagnostic biomarker for CRC. We assessed the role of PC, MPV and PCT as biomarkers for clinical diagnosis of CRC in comparison to CEA and CA19-9. Consistent with previous reports 38 , CEA and CA19-9 levels were significantly higher in CRC patients than in CA patients and healthy controls (Fig. 2a). Further analysis showed CRC patients with CEA level at least 5 ng/mL (the standard cut-off value) had elevated the levels of PC and PCT, but this phenomenon was not observed with MPV (Fig. 2b); In addition, PC, MPV and PCT levels showed no significant correlation with CA19-9 level of at least 37 U/mL (the standard cut-off value) (Fig. 2b).
Next, we used a Receiver-operating characteristic (ROC) analysis to assess the availability of PC, MPV and PCT in the differential diagnosis of CRC patients and CA patients. The analysis showed the area under the ROC curve (AUC) for PC, MPV, PCT, CEA and CA19-9 were 0.706, 0.663, 0.765, 0.740 and 0.612, respectively (Table 1, Fig. 3a), indicating that PCT was the best one among these markers. When a PCT cut-off value of 0.230 was applied, we distinguished CRC patients from CA patients with a sensitivity of 64% and a specificity of 80% (Table 1). Using a CEA cutoff of 5 ng/mL led to a sensitivity of 41% and a specificity of 90%, and using a CEA cutoff of 37 U/mL yielded a sensitivity of 16% and a specificity of 94% in distinguishing CRC patients from CA patients.
Then, PCT in conjunction with CEA or CA19-9 were further analyzed for combined detection. As shown in Table 1 and Fig. 3b, the AUC of the combined detection of PCT and CEA was significantly higher than the AUC of CEA alone, the combined detection of CEA and CA19-9, and the combined detection of PCT and CA19-9. Importantly, when PCT and CEA were combined, sensitivity and specificity are 70% and 83%, respectively.
Taken together, these results suggested that combined detection of PCT and CEA performs better than the detection of CEA alone, combined detection of CA19-9 and PCT, and combined detection of CEA and CA19-9 in discriminating CRC patients from CA patients, with higher sensitivity and specificity.
The relationship between platelet indices and clinicopathological characteristics in CRC patients. As shown in Table 2, the level of PC was significantly greater in patients with more advanced TNM stages (p < 0.0001). Further analysis showed that the level of PC was significantly associated with age, location, primary tumor stages (pT stages) and tumor size. However, no significant correlation was observed between PC level and gender, lymph node stages (pN stages), distant metastasis stages (pM stages), vascular invasion and differentiation ( Table 2). As for MPV, there was significant correlation between MPV and vascular invasion ( Table 2). PCT was associated with the tumor location, TNM stages, pT stages, pN stages, vascular invasion, and tumor size. However, none of the clinicopathological features was significantly associated with the PDW. Taken together, these data indicated that PC and MPV and PCT correlate with clinicopathological characteristics in patients with CRC.

Discussion
In this study, we are the first to find that PCT plays an important role in the diagnosis of CRC, and the combined detection of PCT and CEA can achieve better diagnostic performance than combined detection of CEA and CA19-9 in discriminating CRC patients from CA patients, with higher sensitivity and specificity. Moreover, among CRC patients, PC and PCT levels are associated with TNM stages and tumor size; MPV and PCT levels are associated with vascular invasion. These results indicated that PC, MPV and PCT might function as potential diagnostic and prognostic markers for CRC.
Previous studies have suggested that platelets play a significant role in cancer progression and metastasis [11][12][13] . The relatively low cost, high reproducibility and high applicability of laboratory measurement for PC, MPV, PDW and PCT make them suitable for clinical usability. Growing numbers of evidence suggested that PC, MPV, PDW and PCT can be potentially developed as markers for the diagnosis and prognosis of various cancers [20][21][22][23] .
The colorectal adenoma (CA), a precursor lesion of CRC, is a benign glandular tumor of the colon and the rectum. The majority of CRC arise from transformation of CA, but only 5% of CA progress to cancer. It is difficult to discriminate CRC patients from CA patients, unless the colonoscopy screening is adopted 39 . Colonoscopy screening is not a reliable screening method due to their invasiveness of the procedure, high cost, inappropriate perception of risk, dietary restrictions. Previous studies have suggested that PC, MPV, PDW and PCT have a     [20][21][22][23] , whether PC, MPV, PDW and PCT can serve as markers for discriminating CRC patients from CA patients remains unknown. First, we found that PC, MPV, PCT and PDW levels were significantly higher in CRC patients compared to CA patients and healthy participants. The level of PDW in CRC patients was significantly higher compared to healthy participants while lower than that in CA patients. Then, we evaluated the performance of PC, MPV and PCT as the diagnostic marker of CRC by ROC analysis, and the results showed that PCT can discriminate CRC patients from CA patients with better performance than CEA and CA19-9, which are widely used as markers for the diagnosis of CRC 40 . It was widely recognized that marker combinations have better diagnostic performance than individual markers. PCT in conjunction with CEA or CA19-9 were further analyzed. We found that combined detection of PCT and CEA discriminates CRC patients from CA patients with higher sensitivity and specificity.
Next, we assessed the association between platelet indices and clinicopathological characteristics of CRC patients. Previous studies found that increased PC is correlated with poorer prognosis in various types of cancer [41][42][43][44] . Our results showed that increased PC in CRC patients was not only associated with the TNM stages but also showed strong correlation with tumor size. MPV shows the average platelet volume in the blood, to our knowledge, we report here for the first time that MPV was associated with vascular invasion, but our results failed to demonstrate the association between MPV and the tumor differentiation of CRC 45 . PCT, a factor that is calculated by PC and MPV, is now generally recognized to reflect platelet activity and the percentage of platelets in blood 19 . To date, PCT is only found to be higher in epithelial ovarian cancer, papillary thyroid carcinoma 14 , and pancreatic adenocarcinoma 46 . Our results firstly showed that PCT was increased in CRC patients. Moreover, increased PCT level was associated with TNM stages, vascular invasion, and tumor size. PDW is a coefficient of variation of the platelet volume average, which was only described by one study reporting that elevated PDW is significantly associated with TNM stage and predicted worse prognosis in non-metastatic CRC patients 36 . However, our results showed that none of the clinicopathological features was associated with the PDW in CRC patients. Taken together, these findings showed that PC and PCT levels are associated with TNM stages and tumor size, and MPV and PCT levels are associated with vascular invasion in CRC patients. Considering that TNM stage, tumor size and vascular invasion are poor prognostic factors for CRC patients 47,48 , PC, MPV and PCT should be employed as potential markers for the prognosis of CRC.
There are several possible explanations for our findings. On the one hand, tumor cells can produce factors leading to the proliferation and differentiation of megakaryocyte, thereby promoting platelet production and activation 11 . On the other hand, activated platelets not only can protect tumor cells against cytolysis, but also release some growth factors contributing to cancer cell growth, metastases and invasion 11,13,[49][50][51][52] . These processes initiate a cross-talk between platelets and tumor, which promotes tumor growth and invasion, and platelet production and activity.
This study is limited in the following aspects. First, the short follow-up duration of the study with single-centered retrospective design might raise bias towards sample selection and analysis. Second, we failed to collect data on overall survival, thus we didn't evaluate the relationship of platelet indices with overall survival in CRC patients. Third, racial diversity can be a confounding factor since only Chinese ethnicity was included in the study. Therefore, more follow up studies on populations with higher racial, geological and national diversity in separate laboratories should be carried out to confirm the results herein.
In summary, despite these limitations, our study is the first study that systematically evaluated the role of PC, MPV, PDW and PCT in CRC in a relatively large sample size. Our results indicated that combined detection of PCT and CEA should be considered as potential effective markers for the clinical diagnosis of CRC. Also, PC, MPV and PCT levels can be potentially utilized as biomarkers for the prognosis of CRC. However, our data is still preliminary and the clinical application of platelet indices requires further investigation by additional clinical studies in a larger and diversified population.

Materials and Methods
Patients' characteristics. Samples  Analysis of serum CEA and CA19-9 levels. Peripheral venous blood was obtained from newly diagnosed without receiving any treatment CRC patients. The serum levels of CEA and CA19-9 were determined by a Cobas 6000 Analyzer (Roche Diagnostics, Mannheim, Germany). According to the manufacturer's instructions, the cut-off value for normal CEA is less than 5 ng/mL, and that for normal CA19-9 is less than 37 U/mL. Scientific REPoRTS | (2018) 8:11814 | DOI:10.1038/s41598-018-29293-x Statistical analysis. Statistical analysis was performed using SPSS version 21.0 software or GraphPad Prism version 5.0. All data were expressed as means ± standard error of mean (SEM). The levels of PC, MPV, PDW and PCT of CRC, CA and healthy controls were analyzed using unpaired Student's t test. Receiver-operating characteristic (ROC) curves were used to evaluate the diagnostic role of PC, MPV and PCT in distinguishing CRC patients from CA patients. The diagnostic accuracy of the ROC curve was determined by area under the curve (AUC), and the AUC value closer to 1 indicates that the diagnostic test is perfect. The Youden index was used to determine the optimal cut-off value for PC, MPV and PCT to differentiate between CRC patients and CA patients 53 . Differences in PC, MPV, PDW and PCT as grouped by clinicopathological characteristics were compared by an unpaired Student's t test or ANOVA with Student-Newman-Keuls tests. All tests were two-tailed and a threshold of P < 0.05 was defined as statistically significant.