The potential of folic acid and homocysteine as novel serum markers for venous thromboembolism in cancer patients

Abstract

Venous thromboembolism (VTE) is a common coagulative dysfunctional complication of cancer patients. The present study aimed to determine the association and diagnostic values of serum homocysteine (Hcy) and folic acid levels with VTE in cancer patients. We enrolled 700 cancer patients and 100 healthy subjects in our study. All cancer patients, with or without VTE, underwent measurement of serum Hcy and folic acid levels and coagulative markers including D-Dimer, factor VIII, fibrinogen and tissue plasminogen activator. The diagnostic values of Hcy and folic acid were assessed by receiver operative characteristic (ROC) analysis. Correlations between Hcy and folic acid and coagulative factors were determined. Among the 700 patients with malignant tumors recruited in our study, a total of 89 patients had VTE combined within three months, and 611 patients did not have VTE. Cancer patients with VTE had significantly higher levels of Hcy and significantly lower levels of folic acid in serum. Both Hcy and folic acid in serum demonstrated high sensitivity and specificity in diagnosing VTE in cancer patients and showed strong correlations to coagulative markers. Hcy and folic acid, which have strong correlations to coagulative markers, are potential novel serum markers for stratifying VTE risk in cancer patients.

Keywords

Venous thromboembolism cancer homocysteine folic acid coagulation

1 Introduction

Pulmonary embolism (PE) and deep venous thrombosis (DVT) are together described as venous thromboembolism (VTE), which is a common complication in cancer inpatients [1], COVID-19 [2, 3], pulmonary embolism [4], obesity [5], or nephrotic syndrome [6]. VTE can occur at all cancer stages and during therapy, and VTE is an important cause of death of cancer patients [7]. A preventative strategy to lower the risk of VTE is the prophylactic anticoagulant therapy, in which low molecular weight heparin sodium is infused into cancer patients [8]. However, clinical adoption of prophylactic anticoagulant therapy is limited because of bleeding complications, which may outweigh its benefits, particularly for those with low risk of VTE [9, 10]. Therefore, identifying cancer patients at high risk for VTE is a critical task [7].

Serum biomarkers are desirable tools for cost-effectively and non-invasively assessing VTE risk, including Homocysteine (Hcy) and D-Dimer [11, 12]. Hcy is a toxic sulfur-containing amino acid found in the interconversion pathway of methionine and cysteine [13]. It has been shown that high levels of Hcy and its derivatives in blood can alter platelet aggregation, increase platelet thromboxane and coagulative factors production, thereby promoting coagulation [14]. Together, these constitute the factors that promote thrombosis. Another key player of the coagulation regulation is folic acid, which exists in animal foods and leafy green plants. Cancer patients are known to possess low plasma folate level as tumor cells must utilize folate for de novo purine synthesis [15, 16]. Low plasma folate levels are also linked to cancer via DNA methylation, an epigenetic modification critical for regulation on the genomic level [17]. Despite these prior findings on significant roles of Hcy and folic acid, clear evidence on the performance of Hcy and folic acid as biomarkers of VTE in cancer patients is still in lack and measurement of serum Hcy and folic acid levels has not been incorporated in the clinical management of cancer patients.

Herein, we aim to address the need for a robust serum biomarker for VTE in patients by investigating the correlations of Hcy and folic acid to VTE development in cancer patients. Using clinical samples, our data showed that cancer patients with VTE had significantly higher levels of Hcy and significantly lower levels of folic acid in serum. Both Hcy and folic acid in serum demonstrated high sensitivity and specificity in diagnosing VTE in cancer patients and showed strong correlations to coagulative markers.

2 Materials and methods

2.1 Subjects

Seven hundred patients with malignant tumors who came to our hospital during recent three years (2019–2021) were selected. All patients were newly diagnosed malignant tumor patients and their diagnosis was confirmed by pathological analysis. All patients were staged according to the criteria established by the 7th edition of the American Joint Committee on Cancer and the 2009 International Union Against Cancer. The study was approved by Cangzhou Central Hospital.

The following inclusion and exclusion criteria were used: inclusion criteria: (i) all patients were newly diagnosed malignant tumor patients in our hospital, and all were confirmed by pathological examination; (ii) prognosis > 3 months; (iii) patients had complete clinical data, informed consent for this study, and all cooperated with the follow-up. Exclusion criteria: (i) vascular embolism in the last 3 months or continuous application of heparin, vitamin K antagonist and other drugs affecting coagulation function; (ii) chemotherapy in the last 3 months, surgery and radiotherapy in the last 1 month; (iii) serious systemic infection in the last 1 week; (iv) combined cardiac, hepatic, renal and other organ insufficiency or familial prone to thrombosis.

2.2 Diagnosis of VTE and follow-study

VTE was diagnosed according to the Chinese Expert Guidelines for the Prevention and Treatment of Tumor-Related Venous Thromboembolism (2015 edition). All patients were instructed to have regular outpatient follow-up for 3 months, with VTE as the endpoint of follow-up, which was confirmed by ultrasound, computed tomography (CT) and angiography, etc. All patients were followed up and no case was missed. Based on the follow-up results, all patients were divided into VTE and non-VTE groups.

2.3 Measurement of Hcy and folate levels

Fasting venous blood specimens were collected from patients with malignant tumors before treatment. Centrifuged serum was used to detect Hcy and folate levels. The Hcy assay was performed by the circulating enzyme method, and the kit was produced by Beijing Jiuqiang Company, and the serum folate level was determined by electrochemical immunoassay. Fasting venous blood was also drawn in anticoagulation tubes with sodium citrate, centrifuged and then measured by sysmexCA-550 automatic coagulometer and supporting reagents for D-dimer (D-D), factor VIII (FVIII), fibrinogen (FIB), tissue plasminogen activator (FIB) and tissue plasminogen activator (t-PA).

2.4 Statistical analysis

Categorical data presented are number of cases (percentage). The comparisons of categorical data between the two groups were done by Fisher’s exact test or Chi-square test. One-way ANOVA followed by a Dunn’s multiple comparisons test were used to determine the statistical significance of Hcy and folic acid levels among different groups. Pearson correlation analysis was used to determine correlation between Hcy and folic acid levels. P < 0.05 was considered significant. The receiver operating characteristics (ROC) curves were used to determine the specificity and sensitivity of Hcy and Folic acid in diagnosing VTE in cancer patients.

3 Results

3.1 Study design and patient characteristics

Among the 700 patients with malignant tumors recruited in our study, a total of 89 patients had VTE combined within three months, and 611 patients did not have VTE. One hundred patients who performed physical examinations in our hospital were selected as healthy controls in our study.

Table 1 shows a comparison of the underlying conditions between cancer patients who developed VTE and those who did not. It was revealed that those who are > 60 years old (p < 0.001), had high-grade tumors (p = 0.011), and undergone surgery (p = 0.006) and chemotherapy (p = 0.021) had a higher risk of developing VTE. There was no significant difference between gender, comorbidity and tumor type in determining risk of VTE. In addition, none of the 100 healthy controls had any of the above complications.

Table 1
Baseline characteristics of malignant tumor patients with and without venous thromboembolism (VTE)

Non-VTE (n = 611) VTE (n = 89) p value

Age (years)

≤60 259 (42.4%) 17 (19.1%) < 0.001

> 60 352 (57.6%) 72 (80.9%)

Gender

Male 347 (56.8%) 48 (53.9%) 0.648

Female 264 (43.2%) 41 (46.1%)

Accompanying disease

Diabetes 71 (11.6%) 13 (14.6%) 0.387

Hypertension 197 (32.2%) 35 (39.3%) 0.187

Hyperlipidemia 236 (38.6%) 40 (44.9%) 0.296

Smoking history 259 (42.4%) 32 (35.9%) 0.301

Tumor type

Lung cancer 128 (20.9%) 15 (16.9%) 0.155

Pancreatic cancer 74 (12.1%) 15 (16.9%)

Gastric cancer 52 (8.5%) 10 (11.2%)

Neuroglioma 27 (4.4%) 8 (8.9%)

Breast cancer 68 (11.1%) 7 (7.9%)

Lymph cancer 59 (9.7%) 12 (13.5%)

Colorectal cancer 65 (10.6%) 6 (6.7%)

Prostatic cancer 49 (8.0%) 9 (10.1%)

Others 89 (14.6%) 7 (7.9%)

Pathological stages

I 233 (38.1%) 21 (23.6%) 0.011

II 196 (32.1%) 27 (30.3%)

III 104 (17.0%) 23 (25.9%)

IV 78 (12.8%) 18 (20.2%)

Surgery 287 (46.9%) 56 (62.9%) 0.006

Radiotherapy 235 (38.5%) 38 (42.7%) 0.486

Chemotherapy 435 (71.2%) 74 (83.1%) 0.021

	Non-VTE (n = 611)	VTE (n = 89)	p value
Age (years)
≤60	259 (42.4%)	17 (19.1%)	< 0.001
> 60	352 (57.6%)	72 (80.9%)
Gender
Male	347 (56.8%)	48 (53.9%)	0.648
Female	264 (43.2%)	41 (46.1%)
Accompanying disease
Diabetes	71 (11.6%)	13 (14.6%)	0.387
Hypertension	197 (32.2%)	35 (39.3%)	0.187
Hyperlipidemia	236 (38.6%)	40 (44.9%)	0.296
Smoking history	259 (42.4%)	32 (35.9%)	0.301
Tumor type
Lung cancer	128 (20.9%)	15 (16.9%)	0.155
Pancreatic cancer	74 (12.1%)	15 (16.9%)
Gastric cancer	52 (8.5%)	10 (11.2%)
Neuroglioma	27 (4.4%)	8 (8.9%)
Breast cancer	68 (11.1%)	7 (7.9%)
Lymph cancer	59 (9.7%)	12 (13.5%)
Colorectal cancer	65 (10.6%)	6 (6.7%)
Prostatic cancer	49 (8.0%)	9 (10.1%)
Others	89 (14.6%)	7 (7.9%)
Pathological stages
I	233 (38.1%)	21 (23.6%)	0.011
II	196 (32.1%)	27 (30.3%)
III	104 (17.0%)	23 (25.9%)
IV	78 (12.8%)	18 (20.2%)
Surgery	287 (46.9%)	56 (62.9%)	0.006
Radiotherapy	235 (38.5%)	38 (42.7%)	0.486
Chemotherapy	435 (71.2%)	74 (83.1%)	0.021

The data presented are n (percentage). The comparisons of data between the two groups were done by Fisher’s exact test or Chi-square test.

3.2 Homocysteine upregulation and folic acid downregulation are characteristics of cancer patient with VTE

Figure 1 shows the analysis of serum levels of Hcy and folate in healthy controls, cancer patients with VTE and cancer patients without VTE. It can be seen that cancer patients with VTE had significantly higher concentrations of Hcy (Fig. 1a) and significantly lower levels of folic acid (Fig. 1b) in serum, demonstrating a significant negative correlation between the Hcy and folic acid (Fig. 1c, r = –0.36, p < 0.001).

Fig. 1

Changes of homocysteine (a) and folic acid (b) levels in serum among malignant tumor patients with venous thromboembolism and their correlation (c). n = 100 for HC, 611 for Non-VTE and 89 for VTE. Box plot showing all the data.

To analyze the diagnostic values of Hcy and folic acid in cancer patients with VTE, we performed ROC analysis to calculate specificity, sensitivity and AUC of Hcy and folic acid. The cut-off values, sensitivity, specificity, and area under the curve (AUC) for Hcy and folic acid are shown in Fig. 2a and b, respectively. Using a cut off value of 15.99 nmol/mL for Hcy and 12.28 nmmol/L for folic acid, the two serum markers demonstrated high diagnostic values (sensitivity = 64.04%, specificity = 88.22%, AUC = 0.82, for Hcy; sensitivity = 78.65%, specificity = 78.892%, AUC = 0.85, for Hcy).

Fig. 2

ROC analysis of serum homocysteine (a) and folic acid (b) levels on malignant tumor patients complicated with venous thromboembolism from normal malignant tumor patients.

3.3 Serum Hcy and folic acid levels are significantly correlated with upregulated serum coagulative markers

We then measured serum levels of D-Dimer (D-D, Fig. 3a), factor VIII (FVIII, Fig. 3b), Fibrinogen (FIB, Fig. 3c), and t-PA (Fig. 3d) in healthy controls, non-VTE and VTE cancer patients. Our data indicated that for all measurements, VTE group demonstrated marked higher level of coagulative markers compared to non-VTE and HC groups (p < 0.001). Non-VTE group also demonstrated higher D-D and FIB levels than HC group (p < 0.001), but no statistically significances were seen for FVII and t-PA levels between non-VTE and HC group.

Fig. 3

Changes of coagulation function indexes including D-Dimer (D-D, a), factor VIII (FVIII, b), Fibrinogen (FIB, c) and tissue plasminogen activator (t-PA, d) in serum among malignant tumor patients with venous thromboembolism. n = 100 for HC, 611 for Non-VTE and 89 for VTE. Box plot showing all the data.

By analyzing how folic acid and Hcy levels correlated with patient D-D, FVII, FIB and t-PA levels, we found that significant negative correlations could be found between folic acid and all coagulative markers, and significant positive correlations could be found between Hcy and all coagulative markers (Fig. 4a–h). This piece of evidence further supports the important diagnostic roles of folic acid and Hcy in VTE of cancer patients.

Fig. 4

Pearson correlation analysis of serum folic acid levels with D-Dimer (D-D, a), factor VIII (FVIII, b), Fibrinogen (FIB, c) and tissue plasminogen activator (t-PA, d); serum homocysteine levels with D-D (e), FVIII (f), FIB (g) and t-PA (h) in malignant tumor patients with venous thromboembolism. n = 89.

4 Discussions

Cancer patients are characterized by disturbed Hcy and folic acid metabolisms, which have long been considered to culminate in VTE development. Previous studies showed that hyperhomocysteinemia is associated with VTE occurrence [18] but conflicting results exist [19] partly because Hcy is also thought to drive oncogenesis through a number of processes such as endothelial disturbances caused by Hcy-mediated free-radicals generation [20]. Our study strived to clarify the role of Hcy as a biomarker for VTE using pathologically confirmed VTE cases, which could facilitate translation of Hcy in the clinical setting. In our study, we showed that cancer patients without VTE indeed had an elevated level of Hcy, but the increase was not statistically significant. In contrast, cancer patients with VTE demonstrated a markedly increased Hcy level. This data clarified that Hcy upregulation is presumably a specific biomarker of VTE rather than a biomarker for cancer. Similarly, we found that folic acid downregulation is a specific indicator of VTE rather than cancer. We for the first time established that both Hcy and folic acid levels are robust diagnostic biomarkers of VTE in cancer patients and the AUC values in the ROC analysis showed a high sensitivity and specificity for both Hcy and folic acid.

In addition, we found a strong negative correlation between Hcy and folic acid, which supports the notion that direct regulation of Hcy levels by folic acid exists [21]. In response to a low cellular methionine level, methionine is synthesized by remethylating Hcy in a betaine- or folate-dependent pathway. In the folate-dependent pathway, a methyl group is added to methionine by methionine synthase using tetrahydrofolate as the substrate, the generation of which requires exogenous folate. Low folate levels consequently limit the activity of methionine synthase and affect the remethylation pathway [22]. As a result, low folate levels lead to a high plasma Hcy level and vice versa.

Our data showed that Hcy and folic acid levels had strong correlations to coagulation biomarkers including D-Dimer, factor VIII, fibrinogen and tissue plasminogen activator, which have been used as VTE predictors in multiple clinical settings [23]. This finding further verified that Hcy and folic acid are associated with coagulation to mediate VTE. Compared with these coagulation biomarkers, which thus far has to be used in a particular setting, e.g., prior to chemotherapy [24], or combined with other biomarkers or patient characteristics [25, 26], we showed that Hcy and folic acid levels can serve as independent predictors of VTE, thereby providing powerful tools to improve monitoring of VTE risks.

It should be noted that the mechanisms of Hcy and folic acid in promoting VTE are complex and unclarified. Further mechanistic studies are warranted to advance Hcy and folic acid as new VTE biomarkers. Moreover, we did not elucidate how the use of clinical chemotherapeutic agents, which could alter Hcy and folic acid concentrations [27], could affect the diagnostic values of Hcy and folic acid in VTE. Also, studies are needed to stratify the applicable cut-off value fo Hcy and folic acid to confer the highest sensitivity and specificity and characteristic of patients, e.g., age [28], could affect the diagnostic accuracies.

5 Conclusion

In summary, here we conducted a clinical study to explore the values of serum Hcy and folic acid levels in determining VTE risk in cancer patients. Our data indicated that high Hcy and low folic acid levels are robust predictors of VTE risk in cancer patients and they strongly correlate with coagulation markers.

Competing interests

The authors declare that they have no competing interests.

Funding

None.

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