Plasma Fibrinogen Acts as a Predictive Factor for Pathologic Complete Response to Neoadjuvant Chemotherapy in Breast Cancer: A Retrospective Study of 1035 Chinese Breast Cancer Patients

Yihua Wang Chongqing Medical University First A liated Hospital Yu Wang Chongqing Medical University First A liated Hospital Rui Chen Chongqing Medical University First A liated Hospital Zhenrong Tang Chongqing Medical University First A liated Hospital Yang Peng Chongqing Medical University First A liated Hospital Shengchun Liu (  liushengchun1968@163.com ) Chongqing Medical University First A liated Hospital https://orcid.org/0000-0002-9933-3643


Background
Breast cancer is currently the most common cancer (25% of all cancer cases) and is one of the leading causes of cancer-related death (15% of cancer deaths) among females worldwide [1]. Neoadjuvant chemotherapy (NAC) has become an integral part of the systematic treatment of breast cancer; NAC is used to convert unresectable to resectable cancers, to increase the rate of success for breast-conserving surgery and to evaluate the response to chemotherapy regimens before surgery [2]. It has been recognized that patients with a pathological complete response (pCR) after NAC have a signi cant survival advantage over those with residual invasive disease [3,4].
Novel predictive biomarkers that can predict the pCR prior to NAC are valuable for making individualized treatment decisions and for maximizing e cacy in breast cancer patients. A series of studies have suggested an association between the haemostatic system and tumour biology [5,6]. Different molecular mechanisms can cause the onset of a hypercoagulable state, and hypercoagulability in cancer patients has been implicated in angiogenesis, tumour cell invasion, tumour progression, and metastatic spread [6].
Subclinical hypercoagulable states have also been demonstrated in breast cancer patients [7,8]. Fibrinogen (Fib), a key coagulation factor mainly synthesized by the liver, could be converted to brin by activated thrombin [9]. Previous studies have demonstrated that increased plasma Fib levels are frequently observed in cancer patients, and Fib has been shown to play a vital role in tumorigenesis and to contribute to angiogenesis, stroma formation, and hematogenous metastasis of tumour cells [10,11].
Recent studies have shown that elevated pre-treatment plasma Fib levels are associated with poor prognosis in breast cancer [12][13][14]. However, there have been very few studies on the correlation between the pre-treatment plasma Fib level and the pCR to NAC in breast cancer patients. The aim of the present study was to evaluate the relationship between the pre-treatment plasma Fib level and the pCR to NAC in breast cancer patients and assess the role of Fib as a predictive factor.

Patients and treatments
This retrospective study includes 1312 invasive breast cancer patients whose diagnoses were con rmed by histology and who received NAC from April 2012 to March 2019. The exclusion criteria for all participants were as follows: (1) patients with distant metastasis, bilateral breast cancer or male breast cancer, (2) patients with concurrent liver diseases, autoimmune diseases, haematological diseases or continuous anticoagulant treatment, (3) patients who were pregnant or had previous cancer or concomitant cancer, (4) patients who had radiotherapy, chemotherapy or surgery in the previous 3 months, (5) patients who received 2 cycles of NAC or no surgery, or (6) patients with incomplete data.
Ultimately, 1035 patients were eligible for analysis ( Figure 1). Medical records were reviewed to collect pre-treatment clinical data, such as age, menopausal status, tumour size, lymph node involvement, clinical stage (the 7th edition of the American Joint Committee on Cancer TNM Staging System) [15], histological subtype, oestrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, Ki67 index, and cycles of NAC. All patients received at least 2 but not more than 8 cycles of preoperative treatment with the TEC regimen: cyclophosphamide (500 mg/m 2 ), epirubicin (75 mg/m 2 ), and docetaxel (75 mg/m 2 ) every 3 weeks. In our study, trastuzumab treatment was not used in patients with HER2-positive status in neoadjuvant set, due to the limitations of the medical security system.

Ethical statements
This research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of our hospital (No. 2020-59), who deemed that written informed consent was not necessary due to the retrospective nature of the research.
Blood sample analysis Samples of 5 mL venous blood were collected in tubes with sodium citrate before NAC for the plasma coagulation test. The obtained blood samples were processed within 24 h to detect the plasma coagulation parameters, including Fib, prothrombin time (PT), prothrombin time ratio (PTR), international normalized ratio (INR), activated partial thromboplastin time (APTT), prothrombin activity (PTA), thrombin time (TT), brinogen degradation product (FDP), and D-dimer (DD) with a Sysmex CS5100 automatic coagulation analyser (Sysmex Corporation, Kobe, Japan) in centre laboratory of our hospital.

Immunohistochemical staining and scoring
All breast cancer specimens were confirmed by core needle biopsy and tested using immunohistochemistry to determine the tumour subtype. According to the 2011 St. Gallen consensus, [16] the ER and PR status were considered positive if > 1% tumour cells were stained and the HER2 status was considered positive if > 10% of the tumour cells exhibited a 3+ score by IHC or a > 2.2-fold change compared to the expression of CEP17 in tumour cells via uorescence in situ hybridization. Regarding Ki67, between 400 and 500 cells were counted to calculate the percentage of positive tumour cell nuclei, including hot spots, and 14% was de ned as the optimal cut-off value. Hormone receptor (HR) positive was de ned as ER and/or PR positive, while HR negative is de ned as ER and PR negative. Based on HR status and HER2 status, the patients were classi ed according to the following subtypes: HR (+) HER2 (-), HR (+) HER2 (+), HR (-) HER2 (+), and HR (-) HER2 (-).

Evaluation of chemotherapy response
A pCR was de ned as the absence of residual invasive tumour lesions in any breast tissue and lymph node (ypT0ypN0 or ypT0/is ypN0) [17]. Participants were classi ed into the pCR group and the non-pCR group.

Statistical methods
Data were analysed using SPSS (version 25.0) software (SPSS Inc., Chicago, IL, USA). The levels of plasma coagulation parameters were expressed as the median and interquartile range (IQR) because they are nonnormally distributed continuous variables, and the levels in the pCR group and the non-pCR group were compared by the Mann-Whitney U test and Wilcoxon signed-rank test. Receiver operating characteristic (ROC) curve analysis and the Youden index were used to calculate the optimal cut-off value for pre-treatment plasma Fib levels. Categorical variables were represented as numbers and percentages and compared via Chi-square and Fisher's tests. Signi cant factors for pCR in univariate analyses were included in the multivariate logistic regression model with a forward LR method. Odds ratios (ORs) and 95% con dence intervals (CIs) with two-sided P values were used. Statistical signi cance was de ned as a two-sided P value <0.05.

Association between coagulation factors and pCR
The plasma coagulation parameters were compared between the pCR group and the non-pCR group ( Table 2). The median value of Fib was signi cantly increased in non-pCR patients compared with pCR patients (3.05 (2.63-3.48) g/L vs 2.90 (2.53-3.28) g/L, P = 0.008) (Figure 2A). However, no signi cant differences in PT, PTR, INR, APTT, PTA, TT, FDP, and DD were noted between the pCR group and the non-pCR group (all P > 0.05). Relationship between Fib status and clinicopathological characteristics ROC curve analysis and the Youden index were used to calculate the optimal cut-off value for Fib. Our results indicated that the optimal cut-off value for Fib was 3.145 g/L (P = 0.008) ( Figure 2B). Then, based on their low or high Fib status (Fib < 3.145 g/L and ≥ 3.145 g/L), the patients were divided into groups as shown in Table 3. The relationships between Fib status and clinicopathological characteristics in our study were assessed by the chi-square test. Our results indicated that the low Fib status was signi cantly associated with premenopausal or perimenopausal status (P < 0.001), ≤ 5cm tumour size (P = 0.001), positive HR status (P = 0.003) and > 14% Ki67 index (P = 0.028). However, we failed to detect relationships between Fib status and other clinical characteristics, including age (P = 0.299), clinical lymph node involvement status (P = 0.699), histological subtype (P = 0.503), and HER2 status (P = 0.210).
Considering clinical practice and statistical power, TT, Fib status, tumour size, lymph node involvement, HR status, HER2 status, Ki67 index, and chemotherapy cycles were included in the multivariate logistic regression analysis. The results indicated that Fib status (P = 0.002), tumour size (P = 0.001), lymph node involvement (P < 0.001), HR status (P = 0.001), and Ki67 index (P < 0.001) were independently correlated with the pCR to NAC. After adjusting for other factors in the logistic regression model, Fib was an independent predictive factor for pCR to NAC, and low Fib status (Fib < 3.145 g/L) was strongly associated with a better pCR rate (OR = 2.365, 95% CI = 1.354-4.133, P = 0.002) ( Table 5).

Discussion
Nowadays, NAC has become an integral part of the systematic treatment of breast cancer [1]. A major advantage of this strategy is the ability to observe the tumour response to chemotherapy regimens before surgery [2]. It is well known that the prognosis of patients with locally advanced breast cancer is closely related to whether pCR is achieved after NAC. Patients with pCR after NAC could have a better prognosis than those without [3,4]. In this study, we examined 1035 consecutive breast cancer patients who received NAC to provide evidence for the predictive value of Fib in pCR. The main nding of our analysis is that a lower pre-treatment Fib level is a signi cant independent predictor of pCR (OR = 2.365, 95% CI = 1.354-4.133, P = 0.002) and was associated with premenopausal or perimenopausal status (P < 0.001), ≤ 5cm tumour size (P = 0.001), positive HR status (P = 0.003) and > 14% Ki67 index (P = 0.028) in breast cancer patients.
Fib is a 340 kDa glycoprotein that is mainly synthesized in the liver upon in ammatory stimulation by IL-6 and IL-1β [9]. It is generally regarded as a key factor of coagulation and brinolytic activation. Recently, A lot of research shows that an elevated pre-treatment plasma Fib level is closely associated with poor prognosis in patients with various malignant tumours [18][19][20][21], including breast cancer [12][13][14]. In a large retrospective study of 2073 consecutive breast cancer patients, Wen et al. [12] reported that an elevated preoperative plasma Fib level was an independent prognostic factor for overall survival in breast cancer patients who underwent surgical treatment (p=0.001). Additionally, a retrospective analysis including 520 consecutive breast cancer patients found that an increased pre-treatment plasma Fib level was closely associated with shorter disease-speci c survival (p=0.042) [13].
Previous studies have demonstrated that Fib can also be endogenously synthesized by breast cancer cells themselves [6,22]. Several mechanisms can explain the contribution of Fib to tumour cell in ltration and expansion. First, due to the strong procoagulant effect of tumour cells, a large amount of Fib is aggregated around tumour cells and converted into brin, which is involved in metastasis and new vessel formation and promotes the formation of tumour stromal tissue [22,23]. Second, Shu et al. demonstrated that high concentrations of Fib can induce epithelial-mesenchymal transition [24], which confers migration, invasion and metastasis capacities to tumour cells and renders tumour cells resistant to multiple drugs [25]. Moreover, Fib can promote tumour proliferation and stimulate angiogenesis through interactions with broblast growth factor 2 and vascular endothelial growth factor [9,26]. In addition, Fib can act as a bridge between the tumour and the host cell and support the adhesion of tumour cells to the vascular endothelium of target organs [26,27].
Few studies have reported the relationship between the pre-treatment Fib level and pCR to NAC in breast cancer. In a study including 67 breast cancer patients receiving NAC, Mei et al revealed that patients who had decreased Fib levels after NAC had a better clinical response than patients who had stable or increased Fib levels [28]. In our centre, implantable venous access port systems or peripherally inserted central venous catheters are routinely provided to breast cancer patients before chemotherapy, which may promote the hypercoagulable state of the blood. The comparison of plasma Fib levels before and after chemotherapy may be unreliable. Therefore, we focused on the relationship between the pretreatment plasm Fib status and the pCR of NAC in breast cancer patients.
In our study, the median value of Fib was signi cantly increased in pCR patients compared with non-pCR patients (3.05 (2.63-3.48) g/L vs 2.90 (2.53-3.28) g/L, P = 0.008), while no signi cant differences in PT, PTR, INR, APTT, PTA, FDP, and DD were noted between the pCR group and the non-pCR group. Some studies suggest that DD may be related to the clinical stage and prognosis of breast cancer [29,30]. But we failed to observe the relationship between DD and pCR to NAC in breast patients. According to ROC curve analysis and the Youden index, the optimal cut-off value was 3.145 g/L (P = 0.008). This value is discrepant with those previously reported [12][13][14] due to the differences in sample sources and predicted targets. A higher pCR rate was noted in the low Fib group (a value less than 3.145 g/L) (P = 0.003). Adjusted for other clinicopathological factors in the logistic regression model, low Fib status was still strongly associated with a better pCR rate (OR = 2.365, 95% CI = 1.354-4.133, P = 0.002). However, in 84 oesophageal cancer patients, higher pre-treatment plasma Fib levels were found to be signi cantly associated with a better histological response to neoadjuvant treatment [31]. This nding seems to contradict our results. This may be due to differences in sample size and tumour nature. Moreover, our results showed that lower plasma Fib levels were signi cantly associated with premenopausal or perimenopausal status (P < 0.001), ≤ 5cm tumour size (P = 0.001), positive HR status (P = 0.003) and > 14% Ki67 index (P = 0.028). These are similar to published studies about Fib and clinicopathological factors in breast cancer [12][13][14].
There were some limitations to this study. First, due to the retrospective nature of the current study, a selection bias is unavoidable. To limit interference factors, some exclusion criteria were set, but the relationship between these factors and breast cancer was not evaluated. Second, most patients received NAC for 4 cycles in our centre, so the pCR rates of this cohort were lower than those reported in other literature [32]. Third, long-term follow-up has not been completed of our cohort, so we cannot provide survival data for these patients to further study the relationship between the pre-treatment Fib level and the prognosis. However, this study may offer more evidence and recognition for the relationship between Fib and breast cancer and facilitate the administration of NAC therapy to achieve a better pCR rate. Further prospective trials are needed to confirm the predictive significance of Fib in breast cancer.

Conclusions
This study demonstrates that Fib was signi cantly associated with menopausal status, tumour size, ER status, PR status, and molecular subtype. More importantly, preoperative plasma low Fib status (Fib < 3.145 g/L) is an independent predictive factor for pCR to NAC in breast cancer patients. Breast cancer patients with low pre-treatment Fib levels should be recommended to receive NAC in order to achieve pCR and obtain clinical bene ts.

Declarations
Ethics approval and consent to participate This research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of our hospital (No. 2020-59), who deemed that written informed consent was not necessary due to the retrospective nature of the research.

Consent to publication
Not applicable.
Availability of data and materials 29. Lu Y, Zhang L, Zhang Q, Zhang Y, Chen D, Lou J, Jiang J, Ren C: The association of D-dimer with clinicopathological features of breast cancer and its usefulness in differential diagnosis: A systematic review and meta-analysis. PloS one 2019, 14 (9).  Figure 1