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Hemostatic alterations are frequently seen in patients with cancer, the degree of which has been found to correlate with the clinical progression of the disease [1]. Tumour cells produce tissue factor, cancer procoagulant, plasminogen activators and other factors that interact with the coagulation system, the fibrinolytic system and vascular or blood cells such that they disrupt the normal homeostasis and balance between activation and inhibition of the coagulation and fibrinolytic systems [2-4]. Rather than serving as a mere trigger for increased thromboembolic events, cancer-induced hemostatic activity has also been shown to promote tumour growth and cancer cell dissemination [5,6]. Indeed, both coagulation assays and high levels of circulating biomarkers indicative of coagulation and fibrinolysis activation have been associated with decreased survival in several tumour types [7-12]. One of the biomarkers for this up-regulation is plasma fibrinogen. Several studies have associated elevated plasma fibrinogen levels with tumour progression, invasiveness, metastasis, and poor prognosis, suggesting that plasma fibrinogen concentration could be used as a risk indicator for some tumors [13-19]. Moreover, other studies have also associated plasma fibrinogen levels with tumour response to therapy indicating that plasma fibrinogen levels could be used as a predictor for tumour response to therapy for some malignancies [20-22]. However, the relationship between plasma fibrinogen levels and prognosis in oral cancer remains uncertain [11]. Furthermore, there are hardly any similar studies in the Kenyan population. The purpose of this study was to evaluate the relationship between pre-treatment plasma fibrinogen levels and common prognostic clinico- pathologic parameters in patients presenting with OSCC at two tertiary teaching hospitals in Nairobi.

In this study 31 participants (15 men and 16 women) were recruited consecutively from among oral cancer patients presenting at the Kenyatta National and University of Nairobi Dental Hospitals over a one-year period. A detailed medical history, thorough clinical examination, chest X-ray, Computed Tomography (CT) of the head and neck and histopathological examination of OSCC biopsies were conducted for all patients. The following details of clinico-pathological parameters were also recorded: age, sex, history of smoking, tumour site and size, nodal status, distant metastasis, tumour stage and differentiation. Patients suffering from acute infectious diseases were excluded from the study due to the acute phase response characteristics of fibrinogen.

Plasma fibrinogen measurement

Five milliliters of peripheral venous blood for pre-operative fibrinogen analysis were collected in Vacutainer tubes (Becton Dickson, Rutherford, NJ, USA) containing 3.8% sodium citrate. The plasma fibrinogen concentration was determined by the prothrombintime- derived fibrinogen (FIBPT) assay using an automated photometric coagulation analyzer; ACL Elite-Pro^® and thromboplastin reagent; HemosILRecombiPlasTin (Instrumentation Laboratories), according to the manufacturer’s instruction [23,24]. HemosILRecombiPlasTin contains thromboplastin that is a liposomal preparation of recombinant human tissue factor in a synthetic phospholipid blend, calcium chloride buffer and a preservative. The addition of this reagent to the test plasma initiates the activation of the extrinsic coagulation pathway which ultimately results in the conversion of fibrinogen to fibrin.

This method is not a direct determination of plasma fibrinogen but an estimation of the fibrinogen concentration from the absorbance changes at 450 nm (delta OD) during the clotting process of the PT on an automated photo optical coagulometers [23-26]. To perform the FIBPT assay, the analyzer measures the total change in sample absorbance at 671 nm from the endpoint of the prothrombin reaction. The total change in optical density is directly proportional to the fibrinogen content of the sample [26]. The reference range was set between 242 mg/dl and 362 mg/dl. Plasma fibrinogen levels higher than 362 mg/dl indicated hyperfibrinogenemia.

Statistical analysis

The continuous data were expressed as the mean ± standard deviation and the categorical data were expressed as percentages (%). Continuous variables were compared using unpaired t-test and one way analysis of variance. Categorical variables were compared using chi-squared test, Mann-Whitney U test, Fishers exact test or Kruskal- Wallis test where appropriate. A p-value<0.005 was considered as having been statistically significant in all statistical analyses. SPSS for Windows version 9.0 computer program (SPSS Inc., Chicago, Illinois, USA) was used for statistical analysis.

Baseline patient characteristics

The socio-demographic and clinico-pathologic parameters evaluated, including sex, age, tumour site, tumour size, histological grade, tumour stage and the pretreatment plasma fibrinogen level are shown in Table 1. There were 31 patients among whom 16 were female and 15 were male with a median age of 56.7 years (range: 28-77 years, mean: 56.5 years). The most frequently involved anatomical sites were the tongue (excluding the base) 10 (32%) and gingivae 8 (26%). The other sites accounted for the remaining 13 cases (48%) which were evenly distributed. Out of the 31 patients 24 (77.4%) had stage 4 disease, 1 (3.2%) had stage 3 disease and 6 (19.4%) had stage 2 disease. Twenty-two (75.9%) patients had well differentiated (grade 1) tumors, seven (24.1%) had moderately differentiated (grade 2) tumors and two did not have histological grading available.

Table 1: Relationship between Pre-intervention plasma fibrinogen levels and socio-demographic/Clinico-pathologic parameters.

The mean plasma fibrinogen was 543.7 mg/dl (+/- 145.5), with a range from 299 mg/dl to 911 mg/dl. Out of 31 patients 30 (96.8%) had elevated plasma fibrinogen levels above the reference range (>362 mg/dl) and only one (3.2%) patient had a plasma fibrinogen level of 299 mg/dl. The relation between plasma fibrinogen concentration and clinic-pathologic variables is summarized in the table below. Except for gender, there was no significant association between plasma fibrinogen concentration and clinico-pathologic characteristics including age, sites, sizes, stage, or grade.

Fibrinogen, a soluble 340-kDa glycoprotein synthesized in the liver is transformed through the activity of activated thrombin into insoluble fibrin which is a key coagulation factor in platelet aggregation, clot formation, wound healing and coagulation [27-29]. Several studies have shown that plasma fibrinogen levels are up-regulated in various cancer types including head and neck cancer [30,31] and may indicate cancer progression, metastasis, and recurrence [32-36].

The pathogenesis of this coagulation impairment is complex and involves general risk factors which are common to all categories of patients such as advanced age, prolonged immobility, a prior history of thrombosis, high leukocyte and platelet counts, obesity, immobility, and co- morbid conditions; and other cancer-specific factors such as tumour type, grade, and disease stage. It is the relationship between pretreatment plasma fibrinogen levels and common prognostic clinic-pathological parameters in patients presenting with OSCC cell carcinoma that was the subject of this investigation.

The baseline socio-demographic and clinico-pathologic characteristics of our patients were typical of this population [37] with most of the patients aged over 40 years and an almost 1:1 male to female ratio. The plasma fibrinogen levels were up regulated in 97% of the patients. This differs markedly from a similar study [33] in which 61% of patients presented with low serum fibrinogen concentration and 31% presented with high serum fibrinogen concentration. Our study also confirmed the effect of age and gender on serum fibrinogen levels [38]. Younger patients tended to have lower serum fibrinogen levels compared to older patients and mean plasma fibrinogen levels between males and females clearly demonstrated a gender bias. A few studies have shown that the female gender, age, body mass index (BMI) and alcohol abstinence are directly associated with fibrinogen levels [38]. Most importantly, our results support the findings in other studies that have shown a positive correlation between serum fibrinogen concentration and cancer prognostic factors [11,39-41]. Although statistical significance could not be demonstrated the mean serum fibrinogen levels were higher in the larger tumors, the higher grades, and the more advanced disease.

Consistent with other studies, this study has shown that plasma fibrinogen levels are up regulated in OSCC. Furthermore, the levels of plasma fibrinogen were higher in the more severe diseases indicating that plasma fibrinogen levels could serve as an independent biomarker for oral cancer prognosis. However, further studies with larger samples are required to confirm this.

Our sincere thanks go to the administrative organs of the Kenyatta National Hospital and School of Dental Sciences, University of Nairobi, for the support accorded in the conduct of this work. This study was approved by The Kenyatta National Hospital and The University of Nairobi, Ethics and Research Committee (KNH/UoNERC: P70/02/2015).

Authors have no financial or any other interest in the publication of this work.

Dr. SK Bere: Designed and carried out clinical and laboratory research.

Prof. JF Onyango: Supervised the research and edited the final report.

Dr. AW Njiru: Assisted in the supervision of the clinical research.

Dr. EO Dimba: Assisted in the supervision of the laboratory research.