Thrombogenesis-associated genetic determinants as predictors of thromboembolism and prognosis in cervical cancer

Venous thromboembolism (VTE) is a leading cause of death among cancer patients. Khorana score (KS) is the most studied tool to predict cancer-related VTE, however, it exerts poor sensitivity. Several single-nucleotide polymorphisms (SNPs) have been associated with VTE risk in the general population, but whether they are predictors of cancer-related VTE is a matter of discussion. Compared to other solid tumours, little is known about VTE in the setting of cervical cancer (CC) and whether thrombogenesis-related polymorphisms could be valuable biomarkers in patients with this neoplasia. This study aims to analyse the effect of VTE occurrence on the prognosis of CC patients, explore the predictive capability of KS and the impact of thrombogenesis-related polymorphisms on CC-related VTE incidence and patients’ prognosis regardless of VTE. A profile of eight SNPs was evaluated. A retrospective hospital-based cohort study was conducted with 400 CC patients under chemoradiotherapy. SNP genotyping was carried on by using TaqMan® Allelic Discrimination methodology. Time to VTE occurrence and overall survival were the two measures of clinical outcome evaluated. The results indicated that VTE occurrence (8.5%) had a significant impact on the patient’s survival (log-rank test, P < 0.001). KS showed poor performance (KS ≥ 3, χ2, P = 0.191). PROCR rs10747514 and RGS7 rs2502448 were significantly associated with the risk of CC-related VTE development (P = 0.021 and P = 0.006, respectively) and represented valuable prognostic biomarkers regardless of VTE (P = 0.004 and P = 0.010, respectively). Thus, thrombogenesis-related genetic polymorphisms may constitute valuable biomarkers among CC patients allowing a more personalized clinical intervention.


Materials and methods
Study cohort description. A retrospective hospital-based cohort study was conducted with cytological and histologically diagnosed CC patients with European ancestry, admitted to the Clinic of Gynaecology of the Portuguese Institute of Oncology of Porto (IPOP), from February 2002 to October 2009 and from May 2017 to October 2021, for the first-line treatment, from whom biological samples were available in our biobank. Patients were included if they had disease staged as IB2 to IVA and were submitted to CCRT as the first line treatment, with weekly administration of 40 mg/m 2 of cisplatin during external radiotherapy, and regarding on response followed or not by brachytherapy. Patients were excluded if they were under 18 years old, had done surgery (a known VTE risk factor) at least 6 months before CCRT or were only admitted for a second opinion. A total of 400 CC patients from the north region of Portugal were enrolled. Tumour staging was made according to the International Federation of Gynaecology and Obstetrics (FIGO) staging system 31 .
Patients were classified as having a VTE history in the setting of cancer based on their medical files. No active screening for VTE was made since this measure is not included in the clinical routine procedures at IPOP. As such, there was no accountability for clinically asymptomatic events, which represent the majority of cancer-related VTE events and are also considered to negatively impact a patient's prognosis, although to a lesser extent 32,33 . Data concerning demographic and clinicopathological factors, as well as the follow-up of the patients, were also obtained from their medical files. The characterization of the study cohort (n = 400) according to the VTE status (with VTE vs. without clinical evidence of VTE) is given in Table 1. The mean follow-up was 155.3 months (standard deviation (SD) = 7.6 months).
This study was approved by the ethics committee at IPOP (CES IPO: 287A/014). A written consent according to the principles of the Helsinki Declaration was obtained from each patient before their enrolment in this study.
Sample collection and genomic DNA extraction. Peripheral venous blood samples of each patient were obtained using a standard technique and collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes.
Polymorphism genotyping. Polymorphism genotyping was conducted using the TaqMan® Allelic Discrimination methodology in a StepOne Plus Real-time Polymerase Chain Reaction (Real-time PCR) system (Applied Biosystems). Each reaction was performed using 2.5 µL of TaqPath™ ProAmp™ Master Mix (1×), 2.375 µL of sterile water, 0.125 µL of TaqMan® Genotyping Assay Mix (assays ID provided in Table 2) and 1.0 µL of genomic DNA, making a total volume of 6 µL. The thermal cycling conditions for DNA amplification were the following: (1)  Ethics approval. This study was approved by the ethics committee at IPOP (CES IPO:287A/014). A written consent according to the principles of the Helsinki Declaration was obtained from each patient before their enrolment in this study.

Results
Genotype distribution of the thrombogenesis-related genetic variants. The genotype distribution of each selected SNP and the respective TaqMan assay are represented in Table 2 (Table 1). VTE incidence was 10% and 30% among the former and the latter, respectively (χ 2 , P = 0.191). Considering the new recommended cut-off scores (KS ≥ 2, high risk; 0-1, low risk), 55 out of the 90 patients were considered as having low risk and 35 were high risk ( Table 1). The disease frequency was 14% and 22% among the former and the latter, respectively (χ 2 , P = 0.883). Considering only the original cut-offs, which had better performance, KS was found to be only marginally associated with time to VTE occurrence (P = 0.078). Stratified analyses according to FIGO stage (III/IV vs. I/II) and patient's age (< 50 vs. ≥ 50 years) also did not reveal a significant predictive impact on the score. Nevertheless, KS showed a prognostic value among the 90 patients (log-rank test, P = 0.046). Namely, patients with a score ≥ 3 had a lower 10-year OS compared to the ones with a score < 3 (mean 10-year OS of 33.4 months (SD = 5.9 months) and 48.2 months (SD = 2.0 months), respectively). As it was only possible to determine the score for 90 patients and given its poor performance, further analyses integrating the score were not conducted.

Thrombogenesis-related genetic variants: impact on VTE occurrence and clinical outcome.
Considering the entire cohort of CC patients, for ZFPM2 rs4734879, OTUD7A rs7164569, ITGB3 rs5918, GSR rs3779647, F11 rs4253417 and CNTN6 rs6764623, no statistically significant association with the time to VTE occurrence and patient's prognosis was observed (P > 0.05). In opposition, PROCR rs10747514 and RGS7 rs2502448 polymorphisms were shown to have a significant impact.
The polymorphism PROCR rs10747514 presented a marginal association with VTE development (AA vs. AG/GG; χ 2 , P = 0.101). When analysing the time to a VTE event, the patients with the AA genotype (A being the minor allele; Table 2) exhibited a mean time of 100.8 months (SD = 8.8 months), whereas the G allele carriers presented a mean time of 113.7 months (SD = 1.7 months) under a recessive genetic model (AA vs. AG/GG; log-rank test, P = 0.021; Fig. 2a). The prothrombotic effect of the AA genotype was confirmed by univariate Cox regression analysis (AA vs. AG/GG; HR 3.463; 95% CI 1.128-10.369; P = 0.030). Furthermore, the multivariate analysis also corroborated the predictive impact of rs10747514 (Table 3). Specifically, carriers of the AA genotype had a fourfold increase in the 10-year risk of VTE occurrence compared to G allele genotypes adjusted for FIGO stage (AA vs. AG/GG; adjusted HR (aHR) 4.273; 95% CI 1.342-13.601; P = 0.014; Table 3). The variant PROCR rs10747514 was also shown to have a potential prognostic value among CC patients (AA vs. AG/GG, log-rank test, P = 0.040; Fig. 2b). Specifically, patients with the AA genotype presented a mean 10-year OS of 73.7 months (SD = 10.8 months), whereas carriers of G allele genotypes exhibited 95.3 months (SD = 2.8 months), suggesting a detrimental impact of the AA genotype. By stratifying the analysis according to the patients' VTE status (with vs. without clinical evidence of VTE), a significant impact of PROCR rs10747514 (Fig. 2c) on patients' 10-year OS was observed among the group of patients without clinical evidence of VTE (log-rank test, P = 0.004; Fig. 2c) but not among those with VTE (AA vs. AG/GG; log-rank test, P = 0.087). In the group without clinical evidence of VTE, the carriers of the AA genotype had a lower 10-year OS compared to the G allele genotypes carriers,   Table 4). The polymorphism RGS7 rs2502448 presented a significant association with VTE development (CT/CC vs. TT; χ 2 , P = 0.009). The patients with the C allele (minor allele; Table 2) presented a mean time to VTE of 108.5 months (SD = 2.9 months), contrasting with the 118.8 months (SD = 1.2 months) exhibited by TT genotype carriers under a dominant genetic model (CT/CC vs. TT; log-rank test, P = 0.006; Fig. 3a). This result suggested a prothrombotic effect of the C allele, which was corroborated by univariate Cox regression analysis (CT/CC vs. TT; HR 10.062; 95% CI 1.329-76.179; P = 0.025). Likewise, multivariate analysis showed that the patients with C allele genotypes had a tenfold increase in the 10-year risk of VTE occurrence compared to the TT genotype adjusted for FIGO stage (CT/CC vs. TT; aHR 10.672; 95% CI 1.409-80.813; P = 0.022; Table 3). The variant RGS7 rs2502448 was also shown to have a potential prognostic value among CC patients given the marginal association (CT/CC vs. TT; log-rank test, P = 0.086; Fig. 3b). Specifically, C allele carriers presented a mean 10-year OS of 97.1 months (SD = 3.5 months), while the ones with the TT genotype exhibited a mean OS of 86.0 months (SD = 4.9 months), indicating a protective effect of the C allele. By stratifying the analysis according to the patients' VTE status, a significant impact of the variant (Fig. 3c) on patients' 10-year OS was observed only among those without clinical evidence of VTE (log-rank test, P = 0.010; Fig. 3c). In this group, carriers of the C allele had a higher 10-year OS compared to patients with the TT genotype, confirming the protective effect of the C allele (mean OS of 102.2 months (SD = 3.4 months) and 85.7 months (SD = 4.9 months), respectively; CT/ CC vs. TT; log-rank test, P = 0.010; Fig. 3c).
Considering solely those without clinical evidence of VTE, stratified analyses according to age (< 50 vs. ≥ 50 years) and FIGO stage (III/IV vs. I/II) were performed (Fig. 3d,e, respectively). The polymorphism rs2502448 was found to have a statistically significant impact on the 10-year OS of only young patients (CT/CC vs. TT; log-rank test, P = 0.019; Fig. 3d). Additionally, regarding the FIGO stage, a significant impact of rs2502448 on the 10-year OS was only observed for patients at FIGO I/II stages (CT/CC vs. TT; log-rank test, P = 0.005; Fig. 3e). In line with the previous results, univariate Cox regression analysis confirmed the prognostic value of RGS7 rs2502448 among the patients without clinical evidence of VTE. Specifically, C allele carriers had a 50% reduction in the 10-year risk of death compared to the ones with the TT genotype (CT/CC vs. TT; HR 0.513; 95% CI 0.305-0.862; P = 0.012). Likewise, according to multivariate analysis, the C allele was also associated with a 50% reduction in the 10-year risk of death compared to the TT genotype when adjusted for FIGO stage (CT/ CC vs. TT; aHR 0.490; 95% CI 0.288-0.835; P = 0.009; Table 4).
Given the observed predictive and prognostic value of PROCR rs10747514 and RGS7 rs2502448, multivariate analyses considering both polymorphisms were conducted adjusted for FIGO stage (III/IV vs. I/II) and age (< 50 vs. ≥ 50 years) ( Table 5). The two polymorphisms had an addictive impact on CC-related VTE and patient survival.

Discussion
Cancer is a well-known major risk factor for VTE. A close interplay between tumour cells and the haemostatic components has been well-recognized, with thrombogenesis paralleling tumour progression and aggressive behaviour 1,2,25,40 . In concordance, beyond being highly incident, VTE imposes a negative impact on the prognosis of oncological patients 24 . As previously mentioned, data concerning VTE incidence and effect in CC patients, as well as the underlying mechanisms, is not as solidified [25][26][27] . Based on the somewhat scarce evidence, the frequency of venous thrombogenesis in these patients can reach up to 30% depending on multiple factors, including the type of therapeutic intervention 25 . Nevertheless, even in VTE absence, cancer patients often present a blood hypercoagulation state, which reflects the deregulated activity of certain haemostatic components involved in coagulation activation. As such, regardless of VTE status, haemostatic components present in the tumour microenvironment might favour cervical tumorigenesis, consequently deteriorating the patients' prognosis 25,27,35 .
In this cohort study, 34 patients (8.5% of 400) presented clinical evidence of VTE in the setting of malignancy. However, as patients were not actively assessed for VTE presence, events among the ones without clinical evidence of the disease cannot be excluded. Nevertheless, the VTE incidence reported in the present study is comparable to the ones stated in the literature for CC patients under chemoradiotherapy (3% 41 , 4% 42 , 6% 43 , 8% 44 and 15% 30 ). According to the results, VTE patients had a lower 10-year OS compared to their counterparts (log-rank test, P < 0.001). More specifically, those with VTE presented almost a threefold increase in the risk of death compared to the ones without clinical evidence of the disease (HR 2.522; 95% CI 1.530-4.159; P < 0.001), suggesting that VTE significantly deteriorates CC patients' prognosis (log-rank test, P < 0.001). Indeed, as previously stated, thromboembolism is known to negatively affect the prognosis of cancer patients. Furthermore, beyond the disease itself, tumour cells are known to hijack haemostatic components present in the surrounding microenvironment to fuel their progression towards metastasis, meanwhile disturbing the haemostatic imbalance www.nature.com/scientificreports/ towards thrombogenesis. Given the negative impact of VTE on the prognosis of CC patients, better thromboprophylaxis measures need to be explored, which also demands an improvement of VTE risk prediction models. The most studied tool for the prediction of cancer-related VTE is KS 18,19 . In this study, the score had poor performance. While KS ≥ 2 was not found to be associated with increased VTE risk (χ 2 , P = 0.883), KS ≥ 3 was only marginally associated with the disease (χ 2 , P = 0.191) and time to VTE occurrence (≥ 3 vs. < 3, log-rank test, P = 0.078). The patients with KS ≥ 3 (high-risk) had a VTE incidence of 30%, while the ones with KS = 1-2 (intermediate risk) had a disease incidence of 10%, which is somewhat comparable to the results of a previous study among patients with gynaecological tumours 45 . Also following the literature, the original cut-off score (≥ 3) seems to be more appropriate to determine VTE risk among these patients 46 . Although out of the scope of this study, KS ≥ 3 was found to predict a worse prognosis (≥ 3 vs. < 3, log-rank test, P = 0.046). Overall, data on the discriminatory performance of KS among cancer patients is inconsistent 20,47,48 . Further investigation is, however, required to validate these findings among CC patients, particularly in larger cohorts and considering other treatments.
As for the polymorphisms, starting with rs10747514, this intronic variant located in PROCR leads to the substitution of a guanine (G) for an adenine (A) 49 . The gene PROCR located in chromosome 20 encodes for the human endothelial protein C receptor (EPCR), a transmembrane glycoprotein predominantly present in the membrane of endothelial cells, being engaged in haemostasis [50][51][52][53] . Specifically, this protein is implicated in the protein C (PC) anticoagulant pathway by acting as the cellular receptor of PC, a serine protease with anticoagulant activity [52][53][54] . During haemostasis triggering, circulating PC binds to EPCR, leading to the generation of APC. Once dissociated from EPCR, APC together with its cofactor protein S (PS) inhibits the activated coagulation factor V (FVa) and the activated coagulation factor VIII (FVIIIa), consequently downregulating thrombin generation and preventing venous thrombogenesis 53,55 . As for PROCR rs10747514, its functional consequence is unclear. Indeed, intronic variants may affect either gene expression, for instance by disrupting a transcription factor binding site or changing the protein function [196,197]. Thus, it is plausible that PROCR rs10747514 might   56,57 . Beyond EPCR's implication in the haemostatic pathway, this protein has also been suggested to interfere with carcinogenesis 58,59 . Indeed, EPCR through coagulation-independent mechanisms plays an important role in several tumorigenic-related processes, including apoptosis, angiogenesis, inflammation, cell migration and proliferation 52,55,60 . For instance, the interaction of APC with EPCR and protease-activated receptors (PAR) allows the upregulation of the antiapoptotic protein B-cell lymphoma 2 (BCL-2), as well as the downregulation of p53, Bax and caspases 3, 8 and 9, therefore promoting cell survival 55 . Altogether, growing evidence strongly suggests a tumour-promoting effect of EPCR 52,55,58,[60][61][62] . Concordantly, in this study, PROCR rs10747514 was found to have a prognostic value among CC patients, with the AA genotype exhibiting a detrimental impact on the 10-year OS of the patients (AA vs. AG/GG; log-rank test, P = 0.040). When stratifying the analysis according to VTE status, a significant prognostic impact of the PROCR rs10747514 was only observed among the group of patients without clinical evidence of VTE occurrence (AA vs. AG/GG; log-rank test, P = 0.004). The most likely explanation for the absence of a prognostic value in the context of VTE is the reduced number of VTE patients with available information concerning the polymorphism genotypes (n = 17), which may have prevented the detection of a significant association (AA vs. AG/GG; log-rank test, P = 0.087). Nevertheless, considering solely the patients without clinical evidence of VTE, the PROCR rs10747514 AA genotype was associated with a lower 10-year OS compared to the G allele genotypes in the group of younger (AA vs. AG/GG; log-rank test, P = 0.005) and early disease stage CC patients (AA vs. AG/GG; log-rank test, P = 0.003), confirming the negative impact of the AA genotype. Indeed, the patient's age and cancer stage are well-known factors related to thrombogenesis 1 . Specifically, age combined with sex hormonal changes is reported to influence the expression levels of proteins involved in the coagulation system 63 . Namely, activation of the PC pathway is suppressed with ageing, which is thought to be one of the mechanisms underlying age-associated thrombosis 64 . Therefore, assuming that younger patients have a higher PROCR expression, the AA genotype might impose an additive effect by increasing EPCR activity, consequently favouring cancer pathways. As for the cancer stage, most genetic polymorphisms associated with thrombogenesis seem to be only relevant before metastasis. These variants might contribute to metastatic dissemination, but once it happens other biological mechanisms might be more preponderant in cancer progression 4 . Univariate (AA vs. AG/GG; HR 2.578; 95% CI 1.308-5.083; P = 0.006) and multivariate Cox regression analysis adjusted for FIGO stage (AA vs. AG/GG; aHR 2.612; 95% CI 1.322-5.161; P = 0.006) confirmed the prognostic value of PROCR rs10747514 in the cohort of CC patients without clinical evidence of VTE. Namely, patients carrying the AA genotype had a threefold increase in 10-year risk of death compared to their counterparts in both analyses. Altogether, it was hypothesized that the AA genotype might be associated with higher EPCR levels and/or activity, thereby promoting tumorigenesis and deteriorating the CC patients' clinical outcome. However, the prothrombotic effect of the AA genotype observed in this study (AA vs. AG/GG; aHR 4.273; 95% CI 1.342-13.601; P = 0.014) seems to oppose this hypothesis. Although future functional studies are required to elucidate the underlying biological processes, one possible explanation could be an increased EPCR expression and/or activity paralleling with PC trapping by EPCR, thus impairing the anticoagulation pathway while promoting cancer progression through coagulation-independent mechanisms in individuals without symptomatic VTE. Overall, PROCR rs10747514 may be a useful biomarker to predict VTE among CC patients and assess their prognosis even in the absence of venous thrombogenesis.
The intronic variant rs2502448 located in RGS7 results in a substitution of a thymine (T) to a cytosine (C). The gene RGS7 is located in chromosome 1 and encodes for the Regulator of G-protein signalling 7 (RGS7) 49 . This protein belongs to the multifunctional superfamily of regulators of G-protein signalling (RGS) responsible for controlling the signalling cascades of G-protein, an enzyme engaged in cell signalling transduction. The G-protein activity is actively controlled by RGSs (including RGS7) and G protein-coupled receptors (GPCRs). While the former inhibits G-protein signalling through the hydrolysis of guanosine triphosphate (GTP), the latter stimulates GTP-binding leading to G-protein activation [65][66][67][68] . Among its several functions, RGS7 has been previously described to play a role in platelet function, with some genetic polymorphisms within RGS7 demonstrated to be associated with a risk for venous thrombosis and ischemic stroke 66,67,[69][70][71] . As GPCRs are crucial for platelet activity, RGS7, an antagonist of GPCR signalling transduction, might prevent platelet aggregation, consequently reducing the risk of venous thrombogenesis 65,68,72 . Although its functional consequence is unclear, as an intronic variant, RGS7 rs2502448 might alter RGS7 expression and/or protein functionality, favouring VTE development. Thus, considering that the RGS7 rs2502448 C allele was previously associated with higher platelet reactivity under acetylsalicylic acid (ASA; also known as aspirin) treatment (odds ratio (OR) 3.45; CI 1.82-6.53), and given the potential RGS7 negative regulation of platelet activation and aggregation, the C allele might be linked to lower protein expression and/or activity, favouring venous thrombogenesis 36,71 . Concordantly, in this study, the RGS7 rs2502448 C allele was associated with a lower time to VTE, indicating a prothrombotic effect of this allele among CC patients (log-rank test, P = 0.006). This observation was further confirmed by both univariate (CT/CC vs. TT; HR 10.062; 95% CI 1.329-76.179; P = 0.025) and multivariate Cox regression analysis adjusted for FIGO stage (CT/CC vs. TT; aHR 10.672; 95% CI 1.409-80.813; P = 0.022). Specifically, patients carrying the C allele genotypes had a tenfold increase in the 10-year risk of VTE compared to their counterparts.

Scientific Reports
| (2023) 13:9519 | https://doi.org/10.1038/s41598-023-36161-w www.nature.com/scientificreports/ Beyond haemostasis, RGS play several roles, many of those involved in malignancy. RGS expression levels have been reported to be deregulated in a tissue-specific manner, particularly RGS1, mediating cellular processes such as survival, proliferation, and migration [73][74][75][76] . Inclusively, genetic variants in RGS genes have been found to have a prognostic value among oncological patients 76,77 . Little is known, however, about the role of RGS7 in tumorigenesis, particularly in the context of CC since RGS most likely acts in a tissue-specific manner 76 . In the present study, RGS7 rs2502448 was marginally associated with the 10-year OS of the CC patients, with the C allele exhibiting a benefit impact (CT/CC vs. TT; log-rank test, P = 0.086). Based on the previously mentioned hypothesis that the C allele might be linked to lower RGS7 expression and/or activity, thereby favouring venous thrombogenesis, RGS7 may exert oncogenic roles, which would explain the beneficial impact associated with the RGS7 rs2502448 C allele. Nevertheless, additional studies are required to explore the role of RGS7 in cervical tumorigenesis. Like PROCR rs10747514, a significant impact of RGS7 rs2502448 on patients' 10-year OS was only observed among the group of patients without clinical evidence of VTE (CT/CC vs. TT; log-rank test, P = 0.010). Among these patients, the RGS7 rs2502448 C allele was associated with a higher 10-year OS compared to the TT genotypes in the group of younger (CT/CC vs. TT; log-rank test, P = 0.019) and early disease stage CC patients (CT/CC vs. TT; log-rank test, P = 0.005), corroborating the beneficial impact of C allele. Indeed, age-associated changes in the expression level of RGS proteins have been reported 78,79 . Thus, it was hypothesized that younger patients may have a higher expression of RGS7, meaning that in these patients the polymorphism rs2502448 may have an additive impact. As for the influence of the cancer stage, as previously mentioned, genetic polymorphisms associated with thrombogenesis are thought to be more impacting in a premetastatic context, favouring events towards metastasis. Concordantly, RGS7 rs2502448 was previously found to be associated with the 5-year OS of epithelial ovarian cancer (EOC) patients at early disease stages (log-rank test, P = 0.035) but not at advanced stages, suggesting a potential role of this variant in metastasis regardless of tumour type 36 . The prognostic value of RGS7 rs2502448 in patients without clinical evidence of VTE was corroborated by both univariate (CT/CC vs. TT; HR 0.513; 95% CI 0.305-0.862; P = 0.012) and multivariate Cox regression analysis adjusted for FIGO stage (CT/CC vs. TT; aHR 0.490; 95% CI 0.288-0.835; P = 0.009). Namely, patients carrying the C allele had a 50% reduction in the 10-year risk of death compared to their counterparts in both analyses. Altogether, it was hypothesized that the C allele may lead to lower expression levels of RGS7, thus having a beneficial effect on CC patients' clinical outcomes, assuming that the protein has oncogenic roles. Our research group previously found that, among EOC patients, the rs2502448 C allele was also associated with a higher 5-year OS compared to the TT genotype (log-rank test, P = 0.035), also leading to the hypothesis of lower protein levels associated with the C allele 36 . Overall, like PROCR rs10747514, RGS7 rs2502448 seems to be a valuable tool to predict VTE among CC patients and assess their prognosis even in the absence of the disease.
The remaining evaluated polymorphisms were not associated with VTE development or the patient's prognosis (P > 0.05). Nevertheless, as this is a preliminary study, additional investigation with a larger cohort is required to validate this study's findings. Briefly, the intronic variant rs4734879 located within ZFPM2 is thought to be associated with the expression levels of Vascular endothelial growth factor A (VEGFA) and other haemostatic components implicated in thrombogenesis 49,[80][81][82][83] . Overall, in the context of cancer-related VTE, this variant has not been validated yet, at least to the best of our knowledge 4 . In a previous study conducted by our research group, ZFPM2 rs4734879 was found to be a prognostic factor among EOC patients, which was attributed to its potential influence in tumour neo-angiogenesis 35 . The synonymous variant rs7164569 located in OTUD7A might be associated with the downregulation of Nuclear Factor Kappa B (NF-kB), which positively regulates platelet activity 49,84 . Since OTUD7A acts towards NF-kB inhibition, this deubiquitinase might impair platelet responses decreasing the risk of thrombogenesis [85][86][87][88][89] . However, no study has yet validated the role of rs7164569 in cancer-related VTE 4 . As the NF-kB signalling pathway is implicated in several processes associated with tumorigenesis, OTUD7A by inhibiting it may suppress these cellular processes, suggesting that this deubiquitinase may impose a protective effect in cancer progression 87,88 . To the best of our knowledge, however, the variant rs7164569 has not yet been linked to the prognosis of oncological patients 35 . The missense variant ITGB3 rs5918 has been extensively studied given its putative impact on platelet responses and venous thrombogenesis in the general population 37,49,90 . Until now, however, no study has yet demonstrated the role of this polymorphism Figure 3. (a) Time to VTE occurrence by Kaplan-Meier and Log-rank test for CC patients, according to RGS7 rs2502448 genotypes (n = 254). Patients with the C allele had a lower time to VTE occurrence compared to patients with the TT genotype (mean time to VTE of 118.8 months (SD = 2.9 months) and 108.5 months (SD = 1.2 months), respectively; log-rank test, P = 0.006). (b) 10-year overall survival (OS) by Kaplan-Meier and Log-rank test for CC patients, according to RGS7 rs2502448 (n = 255). Patients with the C allele had a higher 10-year OS compared to patients with the TT genotype (mean 10-year OS of 97.1 months (SD = 3.5 months) and 86.0 months (SD = 4.9 months), respectively; log-rank test, P = 0.086). (c) 10-year overall survival (OS) by Kaplan-Meier and Log-rank test for CC patients without clinical evidence of VTE, according to RGS7 rs2502448 genotypes (n = 238). Patients with the C allele genotypes had a higher 10-year OS compared to patients with the TT genotype (mean 10-year OS of 102.2 months (SD = 3.4 months) and 85.7 months (SD = 4. 9 months), respectively; log-rank test, P = 0.010). (d) 10-year overall survival (OS) by Kaplan-Meier and Logrank test for young CC patients (< 50 years) and without clinical evidence of VTE, according to RGS7 rs2502448 genotypes (n = 128). Patients with the C allele had a higher 10-year OS compared to TT genotype carriers (mean OS of 105.2 months (SD = 4.4 months) and 84.4 months (SD = 6.7 months), respectively; log-rank test, P = 0.019). (e) 10-year overall survival (OS) by Kaplan-Meier and Log-rank test for CC patients at FIGO I/II disease stages and without clinical evidence of VTE, according to RGS7 rs2502448 genotypes (n = 182). Patients with the C allele genotypes had a higher 10-year OS compared to patients with the TT genotype (mean 10-year OS of 104.7 months (SD = 3.6 months) and 85.9 months (SD = 5.4 months), respectively; log-rank test, P = 0.005).  4 . Beyond platelet activation, which itself promotes cancer progression since platelets are engaged in many processes towards tumour dissemination, GP IIb/IIIa is implicated in several signalling pathways involved in tumour progression. Concordantly, the variant showed a prognostic value among EOC patients 36 . The intronic variant GSR rs3779647 was previously associated with platelet activation, however, to the best of our knowledge, this genetic variant has not been linked to cancer-related VTE 4,49,71,91 . The polymorphism was previously associated with EOC patients' survival, which is thought to be due to the contribution of GSR to resistance to platinum-based chemotherapy through its role in glutathione metabolism 36,92 . The intronic variant rs4253417 locates within the F11 gene that encodes for the coagulation factor XI (FXI), which is implicated in the intrinsic coagulation pathway culminating with thrombin generation and consequently fibrin deposition 49 www.nature.com/scientificreports/ The variant rs4253417 was found to be associated with FXI circulating levels but has not been validated in the context of malignancy 4,95 . No direct role of FXI in tumorigenesis is known, however, it may indirectly influence cancer pathways by modulating the activity and/or levels of downstream coagulation proteins [96][97][98][99][100][101][102][103][104][105] . The intergenic variant rs6764623 locates close to CNTN6, which encodes for a protein that seems to trigger the Notch pathway, which mediates inflammatory responses implicated in cardiovascular disorders 49,106 . Although unclear, the polymorphism is thought to regulate gene expression through cis and trans effects, potentially influencing CNTN6 levels 35 . The variant has been associated with a thrombotic risk in the general population, however, the same has not yet been demonstrated among oncological patients 4,85 . Also, rs6764623 was shown to have a prognostic value among EOC patients, which could be due to the multiple roles of the Notch signalling pathway in carcinogenesis 35 .
The most notable limitation of the study was its retrospective nature, which prevented the collection of relevant data. Specifically, as it is well known, several factors modulate the risk for VTE development, including, but not limited to, other comorbidities (for instance, haematological diseases), pregnancy, breastfeeding, use of oral contraception and a history of VTE, which were not accounted for in this study due to lack of information for most patients 1 . Furthermore, data concerning the use of anticoagulants or antiaggregating agents prior to cancer diagnosis were also unavailable and given that no active screening of VTE is included in the clinical routine procedures, asymptomatic VTE events are not accounted for, which could partially explain the poor performance of KS. Inclusively, as VTE was not actively screened, the distribution of patients with and without VTE could be different, which represents an intrinsic limitation in this study. Another important data missing was the specific cause of death among these patients, which prevented the assessment of cancer specific survival, as well as a possible VTE-related mortality. Likewise, information on the discontinuation of antineoplastic treatment due to VTE, which may impact the patients' survival, is also missing. Lastly, the temporal gap regarding the patients' recruitment (February 2002 to October 2009 and May 2017 to October 2021) due to the requirement of biological samples may have led to selection bias.

Conclusion and future perspectives
Even when there is a successful antithrombotic treatment, VTE is associated with a spectrum of complications that deteriorate cancer patients' prognosis 107 . Thus, the primary effort of investigation on cancer-related VTE should be focusing on the disease prevention. Altogether, the results of the present study corroborate the detrimental effect VTE has on the survival of CC patients and suggest that the genetic variants PROCR rs10747514 and RGS7 rs2502448 could be useful predictive biomarkers of thrombogenesis in these patients, in addition to serving as potential tools for prognosis assessment regardless of VTE status. In opposition, the KS was only marginally associated with CC-related VTE, showcasing a poor performance. Overall, the present study highlights the need for a more tailored thromboprophylaxis among oncological patients and better and more personalized cancer management considering the role of haemostatic components in tumorigenesis. In this sense, thrombogenesisassociated genetic polymorphisms might be the bridge to connect both worlds. However, as this is a preliminary study with limitations, additional investigation in larger cohorts and considering other relevant factors are required to validate the results. Prospective studies with active screening of VTE are also a must to better explore the role of PROCR rs10747514 and RGS7 rs2502448 and assess the performance of KS among CC patients. Likewise, it would be relevant to compare the predictive ability of the thrombogenesis-related polymorphisms with the KS and investigate potential drug targets that could be used for both thromboprophylaxis and CC treatment.