Coagulation markers as independent predictors of prostate cancer aggressiveness: a retrospective cohort study

Coagulation system activation is commonly observed in tumor patients, including prostate cancer (PCa), with coagulation markers proposed as potential prognostic indicators for cancer severity. However, the correlation between these markers and clinicopathological features in PCa remains unclear. Thus, this study investigates the association between comprehensive coagulation markers and clinicopathological characteristics in PCa patients. A retrospective evaluation of 162 PCa patients diagnosed and categorized into low-intermediate-risk or high-risk groups based on clinical and pathological features was conducted. Coagulation markers, including fibrinogen (FIB), d-dimer (DD), activated partial thromboplastin time (APTT), prothrombin time (PT), prothrombin activity (PTA), thrombin time (TT), platelet count (PLT), and international normalized ratio (INR), were assessed. Univariate and multivariate logistic regression analyses were performed to determine associations with clinicopathological features. FIB and DD were confirmed as independent factors associated with high-risk PCa. Furthermore, FIB and DD levels showed significant positive correlations with clinical parameters, including PSA levels, ISUP grade, T stage, N stage, and M stage. Our findings suggest that FIB and DD hold promise as independent prognostic biomarkers for risk stratification in PCa. These coagulation markers may aid in assessing PCa severity and guiding personalized treatment strategies.

Prostate cancer (PCa) represents a critical global health challenge.It ranks as the fourth most common malignancy worldwide and exhibits the second-highest frequency among male neoplasms.The morbidity and mortality of PCa have exhibited a concerning upward trend globally in recent years 1 .Currently, the pathological classification, Gleason grading system, and clinicopathological staging are widely employed in clinical settings to assess the severity and prognostic implications of PCa.However, the majority of the aforementioned parameters necessitate acquisition via prostate biopsy.Regrettably, the positivity rate of prostate biopsy is often suboptimal and may be accompanied by hemorrhage, infectious complications, and other hazards 2 .Thus, how to accurately identify the severity of PCa before prostate biopsy is an essential direction of current research, which would be beneficial in PCa risk stratification.
A multitude of investigations has established a close relationship between abnormal coagulation function and the advancement of cancer 3 .In the presence of tumors, coagulation-related mechanisms within the tumor stroma and surrounding microenvironment can become activated.The degree of activation has been associated with tumor cell proliferation and metastasis [4][5][6][7] .Abnormal activation of the coagulation system can expedite malignant proliferation, invasion, and metastasis through the augmentation of tumor vascular adhesion, promotion of tumor growth and angiogenesis, and facilitation of the tumor's immune system evasion [8][9][10] .The intricate interplay between malignant tumors and the coagulation system fosters a hypercoagulable state in tumor patients, and varying levels of coagulation status may delineate the underlying biological attributes of tumors.Consequently, peripheral blood coagulation parameters, which mirror the systemic coagulation state, hold promise as potential indicators for tumor risk stratification.The levels of coagulation markers have been found to be significantly correlated with the clinicopathological features of malignancies, such as gastric cancer, breast cancer, and lung cancer [11][12][13] .However, a paucity of research has investigated the correlation between coagulation markers and the clinicopathological features of PCa.Moreover, current studies have concentrated on individual coagulation markers, such as activated partial thromboplastin time (APTT) or prothrombin time (PT), while disregarding

Data collection
We retrospectively gathered the clinicopathological data of 162 patients diagnosed with PCa.The dataset encompassed pertinent clinical characteristics such as the preoperative laboratory test, imaging results, and postoperative pathological results.Furthermore, baseline characteristics such as age, smoking history, diabetes, and hypertension were included in the analysis (Table 1).All relevant clinicopathological data were obtained from medical records, and all prostate biopsy specimens were sent to the pathology department for diagnostic evaluation.The prostate cancer pathology grade was performed based on the 2014 International Society of Urological  Pathology (ISUP) classification system 14 .The clinical stage of PCa was determined by magnetic resonance imaging (MRI) and adhered to the 2017 edition of the American Joint Committee on Cancer (AJCC) TNM staging system for malignant tumors 15 .Following the ethical standards outlined in the Declaration of Helsinki, the Ethics Committee of the Second Affiliated Hospital of University of South China approved this study.Informed consent was obtained from all participants prior to their inclusion in the study.

Statistical analysis
The normality of the distribution of quantitative variables was assessed using the Shapiro-Wilk test.For variables with a normal distribution, an independent-sample T-test or one-way ANOVA was applied, and descriptive statistics were reported as mean ± standard deviation (SD).For variables with a non-normal distribution, the Mann-Whitney U test or Kruskal-Wallis H test was applied and presented as medians (Q1-Q3).Categorical variables were presented as frequencies and percentages and analyzed using the chi-square test.All PCa participants were previously categorized into low-intermediate-risk and high-risk groups, as previously described 16 .

Comparison of clinicopathological characteristics and coagulation markers between low-intermediate-risk and high-risk PCa patients
In the present study, patients were stratified into two distinct categories of risk: the low-intermediate-risk group (n = 56, 34.57%), and the high-risk group (n = 106, 65.43%).Our study compared the clinicopathological characteristics and coagulation markers between these two groups.The findings of the present investigation demonstrated significant statistical dissimilarities in PSA levels, ISUP grade, T stage, N stage, and M stage between the two study cohorts (p < 0.001 for all), while no noteworthy distinctions in age, smoking history, hypertension, or diabetes were detected between the groups (p > 0.05 for all) (Table 2).Notably, the levels of FIB and DD were significantly elevated in the high-risk cohort in contrast to the low-risk group (p < 0.001 for both), while PLT, PT, PTA, APTT, INR, and TT were found to be similar between the two groups (p > 0.05 for all) (Fig. 2, Table 2).

Plasma FIB levels correlate with PCa severity
We investigated the association between plasma FIB levels and a range of clinical and pathological characteristics of PCa patients, using Spearman's rank correlation test.Our findings demonstrated a significant positive association between plasma FIB levels and various indicators of PCa severity, including PSA levels (r = 0.583, p < 0.001), ISUP grade (r = 0.515, p < 0.001), T stage (r = 0.682, p < 0.001), N stage (r = 0.463, p < 0.001), and M stage (r = 0.507, p < 0.001) (Table 3).
Then, patients with PCa were categorized into three groups according to tertiles of plasma FIB levels, as previously described.Statistical analysis revealed significant differences in PSA levels, ISUP grade, T stage, regional lymph node metastasis, and distant metastasis among the three groups (p < 0.001 for all), while no significant differences were found in age, smoking history, hypertension, or diabetes among the three groups (p > 0.05 for all) (Table 4).Specifically, there was a statistically significant trend of increasing PSA levels, late-stage (stage T3 + T4) percentages, and high-grade (ISUP grade 4 + 5) percentages with increasing FIB levels.Moreover, a significant increase in the proportion of patients with regional lymph node metastasis and distant metastasis was observed with increasing FIB levels (Fig. 3).In relation to coagulation markers, significant differences in DD, www.nature.com/scientificreports/PTA, TT, and INR were observed among the three FIB groups (p < 0.001, p = 0.001, p = 0.006, p = 0.002).However, no significant differences were found in PLT, PT, and APTT among the three groups (p > 0.05 for all) (Table 4).

Plasma DD levels correlate with PCa severity
Based on our analysis using Spearman's rank correlation test, a significant positive correlation was observed between plasma DD levels and various clinical and pathological characteristics of PCa patients, including PSA levels (r = 0.457, p < 0.001), ISUP grade (r = 0.393, p < 0.001), T stage (r = 0.461, p < 0.001), N stage (r = 0.356, p < 0.001), and M stage (r = 0.409, p < 0.001) (Table 5).Subsequently, patients diagnosed with PCa were classified into three groups according to the tertiles of DD levels, as described previously.Statistical analysis revealed significant differences in PSA levels, ISUP grade, T stage, regional lymph node metastasis, and distant metastasis among the three groups (p < 0.001 for all), while there were no significant differences observed among the three groups in terms of age, smoking history, hypertension, or diabetes (p > 0.05 for all) (Table 6).Notably, there was a significant trend of increasing PSA levels, late-stage (stage T3 + T4) percentages, and high-grade (ISUP grade 4 + 5) percentages with increasing DD levels.Furthermore, our analysis demonstrated a significant rise in the proportion of patients with regional lymph node metastasis and distant metastasis in correlation with increasing DD levels (Fig. 4).In terms of coagulation markers, our findings suggest significant differences in FIB across the three DD groups (p < 0.001).Nonetheless, no significant differences were noted among the three groups in terms of PLT, PT, PTA, APTT, TT, and INR (p > 0.05 for all) (Table 6).

Discussion
As molecular biology research on tumors continues to deepen, an increasing amount of evidence suggests that the coagulation system may be involved in mediating the initiation and progression of malignant tumors [17][18][19] .
Our study is the first to comprehensively investigate the association between comprehensive coagulation markers and low-or high-risk PCa, and we have confirmed that the coagulation markers FIB and DD are closely associated with the invasiveness of PCa.We observed a positive correlation between plasma levels of FIB and DD in patients with PCa and more advanced tumor stages, including T, N, and M stages.Meanwhile, levels of PSA and ISUP grades also increased with higher levels of FIB and DD.Moreover, our study demonstrated that FIB and DD are independent risk factors for high-risk PCa.Elevated plasma levels of FIB and DD may indicate unfavorable clinicopathological features and a poorer prognosis.Therefore, the integration of plasma levels of FIB and DD into the existing PCa risk stratification system may assist clinicians in formulating more reasonable treatment strategies and appropriate follow-up plans.
FIB is a high-molecular-weight soluble glycoprotein consisting of three peptide chains connected by 29 disulfide bonds and having a molecular weight of 340 kDa.It is a critical protein in the body's coagulation process.FIB may promote the progression of PCa through multiple mechanisms.Firstly, FIB can create a protective fibrin shield surrounding tumor cells, enhancing their resistance to endogenous immune mechanisms and providing a stable structural framework for the extracellular matrix of tumor cells.This supports the action of cell factors such as vascular endothelial growth factor and fibroblast growth factor on tumor cells, ultimately promoting tumor proliferation and angiogenesis 20 .Secondly, FIB receptors on tumor cells can facilitate the connection of   FIB molecules to the tumor cells, which enhances tumor cell endothelial adhesion in the vascular system of the target organ and promotes tumor metastasis 21 .Additionally, FIB can promote tumor cell adhesion to platelets through β3-integrin-mediated pathways, leading to the formation of PLT-tumor cell aggregates, which may confer protection against immune system recognition and promote tumor cell metastasis 22 .It has been reported that the absence of fibrinogen can considerably diminish the spontaneous metastatic potential of invasive tumor cell lines, including those that disseminate through the blood and lymphatic systems 23 .Prior investigations have demonstrated that plasma FIB levels are independent prognostic factors for overall survival, cancer-specific survival, and progression-free survival among patients diagnosed with PCa 24 .Song et al. recently reported similar findings, demonstrating a significant association between elevated plasma FIB levels and reduced survival rates in PCa patients 25 .These findings collectively indicate a correlation between plasma FIB levels and the prognosis of patients with PCa.Nevertheless, the association between plasma FIB levels and clinicopathological features of PCa remains poorly understood, as only a limited number of studies have investigated this relationship.Previous studies have documented a positive association between plasma FIB levels and PSA levels, T stage, and Gleason score in patients with PCa 26 , consistent with our results.Furthermore, Xie et al. suggested that plasma FIB levels before treatment in PCa patients were positively correlated with bone metastasis burden and that fibrinogen may be a potential predictive factor for high bone metastasis burden in PCa patients 27 .Our findings also support a positive correlation between high plasma FIB levels and distant metastases, which is in line with previous studies.The aforementioned research findings unequivocally establish a substantial correlation between plasma FIB levels and the severity of PCa.However, previous studies examining the relationship between plasma FIB levels and PCa failed to take other coagulation markers into account.In contrast, our study comprehensively investigated the correlation between comprehensive coagulation markers and the clinicopathological features of PCa.DD is a stable end product resulting from the degradation of cross-linked fibrin and a specific marker of fibrinolysis.Its level is known to increase with hyperactivity of the fibrinolytic system.Prior investigations have demonstrated that DD plasma levels are markedly elevated in individuals with PCa relative to those with benign prostatic hyperplasia 28 .The elevated plasma level of DD is strongly associated with an elevated risk of mortality in patients with PCa, and it may act as an independent risk factor for an unfavorable prognosis in such individuals 29 .Additionally, in another study, it was reported that the plasma level of DD was markedly higher in patients with late-stage PCa compared to those with localized disease 30 .In our investigation, we stratified PCa patients into  www.nature.com/scientificreports/three groups based on their DD levels and found the percentage of patients with late-stage PCa significantly increased with rising DD levels (from G1 to G3), which is consistent with the aforementioned research findings.The mechanism underlying the elevation of plasma FIB and DD levels in PCa patients exhibiting unfavorable clinicopathological features remains incompletely elucidated.PCa cells can increase plasma FIB levels by secreting related cytokines or through endogenous synthesis.Furthermore, PCa cells can cause dysfunction in the body's fibrinolysis system, leading to elevated plasma FIB and DD levels 31 .We hypothesize that high-risk PCa cells may have a stronger ability to secrete cytokines and synthesize FIB than cells corresponding to lowintermediate risk PCa.This hypothesis requires further investigation.
In our study, we did not observe any significant association between PLT, APTT, PT, PTA, TT, INR, and the severity of PCa in patients.Moreover, PLT, APTT, PT, PTA, TT, and INR demonstrated no elevation or reduction with escalating FIB and DD levels (Tables 4 and 6).
The activation of PLT is an indispensable prerequisite for enhanced coagulation function.Malignant neoplasms have been shown to induce dysregulation of coagulation function in the body, leading to a rapid increase in the circulating PLT count 32,33 .PLT in the circulation can shield tumor cells from immune system attacks and other pro-apoptotic stimuli and promote tumor growth and metastasis by regulating the tumor microenvironment, releasing growth factors, promoting angiogenesis, and inducing epithelial-mesenchymal transition [34][35][36] .

Figure 1 .
Figure 1.Flowchart of the patient selection process.

Figure 2 .
Figure 2. Comparison of coagulation markers in low-intermediate-risk and high-risk groups of patients.(A) High-risk PCa is related to fibrinogen level.(B) High-risk PCa is related to d-dimer level.***p < 0.001.

Figure 3 .
Figure 3.Comparison of clinicopathological characteristics in patients stratified into tertiles by plasma fibrinogen levels.(A) PSA levels in PCa patients are associated with fibrinogen levels.(B) The tumor T stage of patients with PCa is associated with fibrinogen levels.(C) The ISUP grade of patients with PCa is associated with fibrinogen levels.(D) The tumor N stage of patients with PCa is associated with fibrinogen levels.(E) The tumor M stage of patients with PCa is associated with fibrinogen levels.*0.01 ≤ p < 0.05, **0.001 ≤ P < 0.01, ***p < 0.001.

Table 2 .
A descriptive analysis of the baseline characteristics of study participants stratified by the risk of PCa.Significant values are in bold.

Table 3 .
Correlation between FIB and clinicopathological characteristics of PCa.

Table 4 .
Clinical characteristics of subjects categorized by tertiles of plasma FIB levels.Significant values are in bold.

Table 5 .
Correlation between DD and clinicopathological characteristics of PCa.

Table 6 .
Clinical characteristics of subjects categorized by tertiles of plasma DD levels.Significant values are in bold.