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

ABSTRACT

Objective:

to study the expression of the tissue factor (TF) and its correlation with prognosis and survival in patients with gastric carcinoma.

Methods:

we measured the immunohistochemical expression of TF in 50 specimens of gastric adenocarcinomas from patients submitted to curative surgery. We then compared the intensity of its expression with clinical and pathological data, TNM staging, prognostic factors and survival.

Results:

all tumors displayed TF expression; the intensity of TF expression was not associated with TNM stage, clinical or pathological variables or general survival.

Conclusion:

TF has a high expression in gastric carcinoma, but that it is not useful as a prognostic marker.

Keywords:
Thromboplastin; Immunohistochemistry; Stomach Neoplasms; Prognosis

RESUMO

Objetivo:

estudar a expressão do fator tecidual (FT) e sua correlação com o prognostico 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.

Descritores:
Tromboplastina; Imuno-Histoquímica; Neoplasias Gástricas; Prognóstico

INTRODUCTION

Gastric cancer is the most frequent malignant neoplasm of the gastrointestinal tract. It is the third leading cause of death among all cancers¹. 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 Japan². 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 employed³. 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 tumors⁴. Apparently, one of the most important agents responsible for these associations is the Tissue Factor (TF)⁵, TF is a trans-membrane 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 system⁶, supposedly acting as a natural haemostatic barrier⁷. 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^10-13.

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.

Our aim was to describe the immunohistochemical expression of TF in neoplastic tissues obtained from patients with gastric adenocarcinomas treated with curative intent in South America. We also wanted to correlate TF expression with clinical and pathologic variables, and with overall survival.

METHODS

Our patients sample is a prospective cohort of patients with gastric adenocarcinoma treated at a university hospital in Southern Brazil who underwent gastric resections at our Institution from 2000 to 2003. Patients were followed until death of for a minimum period of 24 months after surgery.

Exclusion criteria were incomplete resection of the tumor, less than 15 lymph nodes identified at the pathologic report, inadequate pathology specimen, history of previous radiation therapy or chemotherapy, and history of previous diagnosis of cancer.

We compared TF expression with the variables age, gender, location, size and aspect of the tumor in the stomach, tumor stage, tumor grade, Lauren's classification, and follow-up time (defined as the time interval between the date of surgery and the last follow-up visit).

Immunohistochemistry

Formalin fixed, paraffin embedded tumor blocks were cut in 3µm sections. Antigen retrieval was performed with Tris/EDTA (20mM Tris/0.65mM EDTA) at a pH of 9.0 and with tap water at 99°C. Endogenous peroxidase blockage was performed with a 3% peridrol solution (H₂O₂ 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).

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).

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 (x²) 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 follow-up analyses due to postoperative deaths during the first admission (early postoperative mortality), being included only in the analyses of prevalence.

Patients mean age was 62.9 years (SD=11.3). 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.

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.

Table 1
Patients characteristics (N=50).

Age at admission in years: mean [±SD]	62.9 [±11.3]
Gender [male:female]	3.1:1
Tumor size in cm: mean [±SD]	6.5 [±4.1]
Location in stomach
Upper	14 (28%)
Middle	18 (36%)
Lower	13 (26%)
Entire	3 (6%)
Gastric stump	2 (4%)
Borrmann type
0	2 (4%)
1	0 (0%)
2	15 (30%)
3	19 (38%)
4	14 (28%)
Lauren subtype
Intestinal	20 (40%)
Diffuse	21 (42%)
Mixed	9 (18%)
Tumor differentiation
G1	1 (2%)
G2	23 (46%)
G3	17 (34%)
G4	9 (18%)
TNM staging
I	10 (20%)
II	5 (10%)
III	17 (34%)
IV	18 (36%)

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 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).

Table 2
Distribution of Tissue Factor expression in our sample.

TF expression.	Specimens
1 (0-25%)	2 (4)
2 (26-50%)	5 (10)
3 (51-75%)	4 (8)
4 (76-100%)	39 (78)

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).

Table 3
Correlation between TF expression and prognostic factors.

Variable	Low TF expression	High TF expression	p
	(N=11)	(N=39)
Age, years	64.8±8.5	62.4±12	0.54
Tumor size (cm)	6.8±3.2	6.4±4.5	0.78
Borrmann IV	3 (27.3)	11 (29.7)	0.99
Diffuse Lauren	8 (72.7)	13 (33.3)	0.47
T3-T4	10 (90.9)	29 (74.4)	0.45
Positive lymph nodes	10 (90.9)	23 (59.0)	0.11
G3-G4	8 (72.7)	18 (46.2)	0.22
Stage III-IV	10 (90.9)	25 (64.1)	0.18

Mean follow-up was 28.9 months (range 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.

Figure 2
Overall survival by stage

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.

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).

Figure 3
Survival curves for patients with high intensity (>75% of cancer cells) and low intensity (<75% of cancer cells) TF immunohistochemical expression

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 presenation³. 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 proximal¹⁷, 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 mortality¹⁸.

The most important prognostic factor at the time of the diagnosis in gastric cancer is tumor stage³. 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 diagnosis¹⁹. The percentage of metastatic cases at diagnosis is also high in the international literature¹⁷. 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 tumors²⁰.

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 carcinomas²¹. 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. As the incidence of gastric carcinoma in Brazil does not justify screening the whole population through radiographic or endoscopic exams, health policies that help identify individuals at increased risk for these neoplasms are necessary.

Other important factor of poor prognosis in gastric cancer is the Borrmann classification, which evaluates the penetration across the gastric wall. The fact that 66% of our patients were classified as having tumors Borrmann III or IV may explain their poor survival rates. Lauren's classification stratifying tumors into intestinal and diffuse types is often used clinically, since it is simple and reproducible. Epidemiologically, the intestinal type is more common in areas where the risk of developing gastric carcinoma is higher, whereas the diffuse type has a uniform distribution across areas of high and low risk of gastric cancers. It is speculated that the intestinal and diffuse types of gastric cancers are really two distinct diseases, with peculiar characteristics and specific risk factors²². In Brazil, a study by the National Cancer Institute (INCA) suggested a decrease in the incidence of intestinal type tumors in Rio²³, whereas another study with more than 600 patients in São Paulo has shown that the intestinal type is present in more than 60% of cases²⁴. In our study, the small percentage of patients with intestinal type tumors (40%) contrasts with the national literature from Brazil, in which the intestinal type is usually the predominant cancer.

The expression of TF and its value as a prognostic factor in cancer has been studied in a variety of tumors, but not in gastric cancer. In other tumor types, an increased TF expression is usually associated with greater tumor aggressiveness and with worse prognosis²⁵. The relationship between cancer and coagulation is complex and implies that the TF produced by malignant neoplasms may be involved with a system of intracellular signalization that acts independently from the coagulation cascade in promoting tumor development²⁶. In our study, there was immunohistochemical expression of TF in the gastric cancer cells of all 50 patients. This degree of expression suggests that it may be a useful therapeutic target in the management of this disease. Although in most cases TF expression was high, for the purpose of analyses we have divided patients into groups of high and low TF expression to try to correlate the findings with known prognostic factors and with survival.

The analyses of our results were surprising in many ways. As shown in table 3, although statistical significance was not reached, most known factors of poor prognosis in gastric cancer were more associated with a low expression of TF, except Borrmann's classification. This finding may explain the results of the survival analysis shown in figure 3, 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.

• Source of funding: none.

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Publication Dates

• Publication in this collection
  29 Nov 2018
• Date of issue
  2018

History

• Received
  09 Oct 2018
• Accepted
  11 Oct 2018