Immunohistochemical analysis of tissue factor expression in gastric carcinoma: correlations with prognosis and survival

Toneto Análise da expressão imuno-histoquímica do fator tecidual no carcinoma gástrico: correlações com prognóstico e sobrevida. 8 Rev Col Bras Cir 45(6):e2030 REFERÊNCIAS 1. World Health Organization, I.A.f.R.o.C [Internet]. Estimated number of deaths, both sexes, worldwide (top 10 cancer sites) in 2012. Available from: https:// gco.iarc.fr/today/online=-analysis-multi-barsmmodecancer&mode_population=continents&population=900&sex=0&cancer=29&type=1&statistic=0&prevalence=0&color_palette=default 2. Park JY, von Karsa L, Herrero R. Prevention strategies for gastric cancer: a global perspective. Clin Endosc. 2014;47(6):478-89. 3. The Society for Surgery of the Alimentary Tract [Internet]. Patient Care Guidelines. Surgical Treatment of Gastric Cancer. Beverly (MA): SSAT. c2004 [cited 2017]. Available from: http://www.ssat.com/cgi-bin/ guidelines_SurgicalTreatmentGastricCancer_EN.cgi 4. Anand M, Brat DJ. Oncogenic regulation of tissue factor and thrombosis in cancer. 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The effectiveness of the new (7th) UICC N classification in the prognosis evaluation of gastric cancer patients: a comparative study between the 5th/6th and 7th UICC N classification. Gastric Cancer. 2011;14(2):166-71. 22. Howson CP, Hiyama T, Wynder EL. The decline of gastric cancer: epidemiology of an unplanned triumph. Epidemiol Rev. 1986;8:1-27. Toneto Análise da expressão imuno-histoquímica do fator tecidual no carcinoma gástrico: correlações com prognóstico e sobrevida. 9 Rev Col Bras Cir 45(6):e2030 23. Abib AR, Oliveira IM, Koifman S. Histopatologia do câncer de estômago (classificação de Lauren) em amostra de pacientes hospitalares no Rio de Janeiro, 1980-1995. Cad Saúde Pública. 1997;13(Supl 1):S99-S104. 24. Marigo C, Okuyama MH, Santo GC. Tipos histológicos e mortalidade por câncer gástrico em São Paulo. Cad Saúde Pública.1997;13(Supl1):S93-7. 25. Kakkar AK, Chinswangwatanakul V, Tebbutt S, Lemoine NR, Williamson RC. A characterization of the coagulant and fibrinolitic profile of human pancreatic carcinoma cells. Haemostasis. 1998;28(1):1-6. 26. Shoji M, Hancock WW, Abe K, Micko C, Casper KA, Baine RM, et al. Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer. Am J Pathol. 1998;152(2):399-411. Recebido em: 09/10/2018 Aceito para publicação em: 11/10/2018 Conflito de interesse: nenhum. Fonte de financiamento: nenhuma. Endereço para correspondência: Gustavo Franco Carvalhal E-mail: gcarvalhal@terra.com.br


INTRODUCTION
G astric cancer is the most frequent malignant neoplasm of the gastrointestinal tract.It is the third leading cause of death among all cancers 1 .
Although its incidence has been recently reduced in developed countries, it is still a heavy burden for underdeveloped countries in Latin America, and a major issue in some countries in Asia, such as Japan 2 .The mainstay of treatment is still an aggressive surgical approach, which is not devoid of risks and complications.In spite of controversies, adjuvant treatments with chemotherapy and radiation have been increasingly employed 3 .The discovery of novel biomarkers in this disease may lead to developments in therapeutic strategies for these tumors.
In 1865, Armand Trousseau described for the first time the association between cancer, coagulation and thrombosis, in a cohort of patients with gastrointestinal tumors 4 .Apparently, one of the most important agents responsible for these associations is the Tissue Factor (TF) 5 , TF is a transmembrane glycoprotein that plays a pivotal role in the extrinsic coagulation pathway by interacting with Factor VII.TF is expressed in fibroblasts in the adventicea of blood vessels, solid organ capsules, epithelial cells in the skin and mucosas, cells of the endometrial stroma, and astrocytes from the central nervous system 6 , supposedly acting as a natural haemostatic barrier 7 .When exposed, TF initiates the coagulation cascade, resulting in the production of fibrin 8,9 .An increased expression of TF was also described in a variety of solid malignant neoplasms, including melanoma, breast, prostate and lungs.In some studies, an increased TF expression was also correlated with a worse clinical prognosis [10][11][12][13] .
Two distinct groups reported the immunohistochemical expression of TF in gastric carcinoma.A Japanese study with 207 patients has shown that an increased expression of TF was associated with a worse prognosis, whereas an Australian study with 160 patients did not reproduce such findings 14,15

Immunohistochemistry
Formalin fixed, paraffin embedded tumor blocks were cut in 3µm sections.Antigen retrieval was performed with Tris/EDTA (20mM Tris/0.65mMEDTA) at a pH of 9.0 and with tap water at 99°C.Endogenous peroxidase blockage was performed with a 3% peridrol solution (H 2 O 2 in methylic acid) for 30 minutes, and slides were washed with 5% saline in PBS to reduce background.Slides were then incubated with anti-Tissue Factor mice anti-human type 1 antibody in 1:30 dilution and with anti-CD34 mice monoclonal antibody in a dilution of 1:400.We used the detection system Kit Dako LSAB + peroxidase.Slides were treated with the chromogen 3,3'-diaminoazobenzidine (DAB) and with PBS buffer in a solution of 0.002% hydrogen peroxidase, counterstained with hematoxylin, dehydrated, clarified and mounted.

Tissue Factor Expression
The quantification of TF expression was performed according to the reactivity to the antibody, with an objective lens with 100X magnification.We classified TF expression into four categories: 1 (0% to 25% of cancer cells stained), 2 (26% to 50% of cancer cells stained), 3 (51% to 75% of cancer cells stained) or 4 (76% to 100% of cancer cells stained) (Figure 1).

Statistical Analysis and Ethics
We described quantitative data through means and standard deviations and qualitative data, as percentages.We performed comparisons between quantitative variables using the Student's t test.For qualitative data, we used the Chi-square (x 2 ) test or Fisher's exact test for the comparisons.
We carried out the survival analyses with the Kaplan-Meier method, and applied the log-rank test for the comparison between the curves.We created a multivariable model using variables that were clinically relevant (Cox regression model).The study protocol was revised and approved by the Ethics and Research Committee of our Institution (protocol 345/06).

RESULTS
In three years, we performed 101 gastric cancer resections at the Department of Surgery of our university hospital.Of these, we included 50 (49.5%) in the study.The main causes for exclusion were <15 lymph nodes identified (in 32 patients) and residual disease (in 16 patients).In three patients, tissue blocks were unavailable.Of the 50 remaining patients, we excluded four of the followup analyses due to postoperative deaths during the first admission (early postoperative mortality), being included only in the analyses of prevalence.
Overall, 38 patients were male, and 12 were female.
Tumors were located in the middle third of the stomach in 18 cases, in the proximal third in 14 cases, and in the distal third in 13 cases.Mean tumor size was 6.52cm (±4.14), the smallest measuring 0.7cm and the largest measuring 20cm in its greatest diameters.Macroscopically, the most common form of presentation was the ulcerative-infiltrative (Borrmann classification).Concerning tumor differentiation, the most common tumors were moderately differentiated (G2), representing 46% of our sample.2).Due to the small number of cases with a lesser intensity of expression, we joined groups 1, 2 and 3 into a new category (0-75% of positively stained cancer cells).Therefore, for the purpose of statistical analyses, we categorized patients into two groups: low expression (scores 1 to 3) and high expression (score 4).

Figure 1. A) Gastric adenocarcinoma with less than 25% of cells stained, category 1; B) Gastric adenocarcinoma with practically all cells stained, category 4; (50X magnification).
We compared TF expression with known prognostic factors in gastric cancer such as tumor size, Borrmann and Lauren classifications, invasion of the gastric wall, the presence of positive lymph nodes, pathologic staging and tumor differentiation.
There was no statistical correlation between TF expression and any of these prognostic factors (Table 3).Mean follow-up was 28.9 months (range Patients with an increased expression of TF had a mean survival of 33.6 months (95%CI=25.3-41.9;SE=4.2), whereas those with low expression had a mean survival of 9.74 months (95%CI=4.51-14.97;SE=2.6).This result was statistically significant (p=0.0017).Figure 3 depicts the survival curves of patients with low and increased TF expression.

Figure 3. Survival curves for patients with high intensity (>75% of cancer cells) and low intensity (<75% of cancer cells) TF immunohistochemical expression.
As TF expression was a statistically significant factor influencing overall survival, we performed a multivariate regression analysis adjusted for the most well known prognostic factors in gastric cancer.In this analysis, TF expression was not an independent prognostic factor when adjusting for age, tumor stage, grade, and Lauren and Borrmann classifications (HR 0.58; 95%CI=0.21-1.56;p=0.28).

DISCUSSION
Surgical resection is the most effective treatment for gastric adenocarcinoma.However, the aggressiveness of this neoplasm and its propensity for local, nodal and systemic progression makes early diagnosis difficult, and a potentially curative surgery is not an option for roughly half of the patients at presenation 3 .The poor prognosis of the disease and the elevated levels of recurrence and deaths with conventional treatment justify the search for novel therapeutic targets.
Most of our patients were male and older than 60 years, which is accordance with the literature.In spite of the recent decline in incidence of gastric cancers in developed countries, it remains one of the main causes of morbidity and mortality in Brazil, according to data published by the Brazilian National Cancer Institute (INCA) 16 .
Differently from American data, in which most tumors were proximal 17 , in our study most tumors occurred in the distal third of the organ.
The localization of the lesion is important, since it will define the type and extension of the resection, the extent of the lymphadenectomy and the type of reconstruction, all factors that may influence the procedure's morbidity and the mortality 18 .
The most important prognostic factor at the time of the diagnosis in gastric cancer is tumor stage 3 .Unfortunately, most gastric cancers in our series were advanced at the time of diagnosis.In only two patients the tumors were classified as early.
In the Brazilian literature, in more than half of the cases there were distant metastases at diagnosis 19 .
The percentage of metastatic cases at diagnosis is also high in the international literature 17 .In our series, most patients were classified as stage III or IV, which may explain the increased postoperative mortality (8%) observed.In the published literature, the main determinants of postoperative mortality are the presence of concomitant disease, the presence of lymph node metastases, tumor size, surgical experience and patient's age.Patients with stages III or IV tumors have a five times greater mortality than patients with stages I or II tumors 20 .
The extension of the tumors across the gastric wall and the involvement of lymph nodes are the main factors that will define TNM staging in gastric carcinomas 21 .In figure 2, we confirm that survival is directly related to staging in our sample.
Cure is often possible for patients diagnosed with tumors at early stages.

R E S U M O
Most patients (96%) presented with tumors infiltrating the muscle layer, characterized as advanced, and in 39 cases (78%) the tumors invaded the serous layer or adjacent organs.In 33 cases (66%) there was at least one metastatic lymph node.Two patients (4%) had distant metastases at the time of surgery, one in the left lobe of the liver and the other a single implant in the small bowel; both lesions were resected along with the gastric lesions.The patients' main clinical and pathological characteristics are shown in table 1.Most tumor cells demonstrated intense reactivity for Tissue Factor, and 100% of tumors expressed the protein.In the quantification of TF expression, 39 cases (78%) showed an intense expression (76-100% of cancer cells stained) (Table one 1 to 60 months).Only 19 (38%) patients were alive at the time of analysis.Of the 31 deaths, four (8%) were due to postoperative complications, and the remaining due to disease progression.In 22 patients, it was possible to determine the main recurrence site: diffuse peritoneal carcinomatosis in 12, isolated peritoneal recurrence in three, liver in three, anastomotic site in two, lung in one patient and spleen in one.Disease stage influenced overall survival, as represented in figure 2.

Table 2 .
Distribution of Tissue Factor expression in our sample.

Table 3 .
Correlation between TF expression and prognostic factors.
This finding may explain the results of the survival analysis shown in figure3, in which we describe a significantly worse survival for patients with a low TF expression compared with that of patients with an increased TF expression.In multivariate analyses, however, adjusting for other prognostic factors, the degree of TF expression did not independently predict prognosis.TF was present in tissues from all gastric cancers in our cohort.Although the degree of expression did not correlate with other prognostic factors or with survival, we believe that it may be a useful target for anticancer therapies in the future.estudar a expressão do fator tecidual (FT) e sua correlação com o prognóstico e sobrevida em pacientes com carcinoma gástrico.Métodos: verificamos a expressão imuno-histoquímica do FT em 50 espécimes de adenocarcinomas gástricos de pacientes submetidos a tratamento cirúrgico com intenção curativa.A intensidade da sua expressão foi comparada com dados clínicos e patológicos, estadiamento TNM, fatores prognósticos e sobrevida.Resultados: houve expressão do FT em todos os tumores; a intensidade de expressão do FT não foi associada com estágio TNM, variáveis clínicas ou patológicas ou sobrevida geral.Conclusão: este estudo mostra que o FT tem uma expressão elevada em carcinoma gástrico, mas que este não é útil como marcador de prognóstico.