Yonago Acta medica 2009;52:21–25

Clinical Findings on Fibroblast Activation Protein in
Patients with Gastric Cancer
Youji Fukumoto, Yoshinori Yamada, Kenji Fukuda, Hiroaki Saito, Shigeru Tatebe,Shunichi Tsujitani and Masahide Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504 Japan

Human fibroblast activation protein (FAP) is a 97-kDa surface glycoprotein expressed intumor-associated fibroblasts. In this study, we immunohistochemically examined FAP
levels in surgically resected gastric carcinomas and explored their association with clinicopathological findings and prognosis. Sections of paraffin-embedded specimens wereobtained from 100 patients with advanced gastric cancer between 1989 and 2001 at ourinstitution, and they were stained with an anti-FAP antibody. Expression of FAP wasdetected in 64 patients (64%). Lymphatic vessel invasion was observed in 90% of FAPpositive patients (P = 0.015). Blood vessel invasion was observed in 98% of FAP-positivepatients (P < 0.001). The disease-specific 5-year survival rate of in the 64 patients withFAP-positive tumors (22%) was significantly lower than in the 36 patients with FAPnegative tumors (34%, P = 0.036). This indicates that vessel invasion is connected withthe expression of FAP and that a positive finding of FAP confer a worse prognosis in thepatients with gastric cancer.
Key words: fibroblast activation protein; gastric cancer; immunohistochemistry; prognosis

Gastric cancer is one of the most malignant tumor
types having a dismal prognosis. Lymph nodemetastasis, hematogenic metastasis, and peritoneal metastasis have all been reported to have astrong impact on prognosis for gastric cancer patients. Tumor cell invasion and subsequent distantspread via blood and lymph vessels are criticalsteps in tumor progression. In the process of tissue invasion and metastasis, cancer cells produce
enzymes that degrade basement membranes andextracellular matrix (Liotta et al., 1991). During
disease progression, interactions between cancer
cells and mesenchyme-derived stromal cells, suchas fibroblasts, myofibroblasts, endothelial cells,smooth muscle and hematopoietic cells occur.
However, the relationship between cancer cells

and mesenchyme-derived stromal cells has notbeen well described (Mueller and Fusenig, 2004;
Huber et al., 2005; Joyce, 2005; Orimo et al., 2005;
Kalluri and Zeisberg, 2006; Elinborg et al., 2008).
Human fibroblast activation protein (FAP)
is a 97-kDa cell surface glycoprotein with gelatinase and depeptidyl peptidase activity (PineiroSanchez et al., 1997; Park et al., 1999). This
protein is expressed in stromal fibroblasts locatedin and around the tumor. The distribution ofFAP is unique. FAP is not expressed by normalfibroblasts, but is expressed transiently in healing wound tissues (Ghersi et al., 2002), chronic
inflammatory conditions such as cirrhosis (Levyet al., 2002) and some fetal mesenchymal tissues.
Recently, FAP produced by stromal fibroblasts

Abbreviations: FAP, fibroblast activation protein; PBS, phosphate buffered saline21

Y. Fukumoto et al.

Specimens were dewaxed in xylene, rehydrated
in ethanol, treated with 0.3% hydrogen peroxide
in methanol for 15 min, and then heated in anautoclave oven (120˚C) for 20 min. They werethen washed with phosphate-buffered saline(PBS) and preblocked with 10% gout saline for 10min. After washing with PBS, the samples wereincubated overnight at 4˚C with an anti-FAP polyclonal anitibody (ab53066; Abcam, Tokyo) diluted
at 1:50. Specimens were then washed in PBS andincubated with anti-rabbit immunoglobulin conjugated to biotin for 10 min, followed by incubation with a streptavidin peroxidase complex for afurther 5 min. Following another wash with PBS,
immunohistochemical labeling was visualizedusing freshly prepared diaminobenzidine tetrahydrochloride. Sections were counterstained withhematoxylin.

was reported to play an important role in tumor
cell progression (Allinen et al., 2004; Narra et al.,2007). Cheng et al. (2002) reported that forced
FAP overexpression by tumor cells in an animalmodel results in a significant enhancement of tumor growth.
In the present study, we investigated the clinical significance of stromal fibroblast FAP expression in gastric cancer by the immunohistochemical examination. We examined the relationshipbetween FAP expression and clinicopathologicalfindings in patients with gastric cancer. We alsocomment on the clinical importance of FAP expression in stromal fibroblasts.

Materials and Methods
Patients

Scoring system

The medical records of corresponding patientswere retrospectively reviewed. The 100 primary
gastric cancer patients were enrolled in this study.
The stages of their tumors were III and IV. They
underwent gastrectomy at our institution between
1989 and 2001. The age of patients ranged from33 to 93 years (average 66.3 years); 72 were maleand 28 were female. The clinicopathologicalfindings were described according to the JapaneseClassification of Gastric Cancer by our hospital
pathologists (Japan Research Society for GastricCancer, 1995). All patients had undergone distal,proximal or total gastrectomy with dissection oflevel 1 and 2 regional lymph nodes (D2). Curative operations were performed in all patients.
The follow-up periods for survivors ranged from 1
to 180 months (median, 39 months) after surgery.
Informed consent was obtained from all subjects
and/or their guardians.

Stromal fibroblasts with FAP immunoreactivity
were quantified by 2 independent observers whoevaluated at least 1,000 fibroblasts in consecutive sections of neoplastic tissues. Stromal fibroblasts were classified into two categories basedon FAP expression: i) negative staining (negativeor equivocal staining or when less than 10% cellswere detected) and ii) positive staining (when 10%or more cells were detected).
Statistical analysis
The chi-square test was used to compare thedifferences between the two groups. The significance of differences among means was determined by the Mann-Whitney U test. The overall
and disease-free survival periods were estimatedusing the Kaplan-Meier method and compared using a two-sided log rank test. Cox’s proportionalhazards regression model was used to estimate thepredictive power of FAP expression on clinicaloutcome. Two-sided tests were computed, and P< 0.05 was considered statistically significant.

Immunohistochemistry
A streptavidin-biotin kit (Histofine SAB-PO kit;Nichirei, Tokyo, Japan) was used for staining offormalin-fixed and paraffin-embedded specimens.
22

FAP expression on gastric cancer

Results
FAP expression and clinicopathologicalfindings
FAP expression was detected in stromal fibroblasts around or within cancer tissue (Fig. 1). It
was not detected in cancer cells or stromal cells
located in normal gastric tissues in the resected
specimens. FAP-positive stromal fibroblasts weredetected in 64 patients (64%). The clinicopathological characteristics of the 64 patients withFAP-positive stromal fibroblasts were comparedwith those of the 36 patients who were FAP negative (Table 1). Lymphatic vessel invasion was observed in 90% of FAP-positive patients comparedwith 70% of FAP-negative patients (P = 0.015).
Blood vessel invasion was observed in 98% of theformer group compared with 19% of the lattergroup (P < 0.001). There was no significant difference with FAP expression level and histologicaltype of gastric cancer.

Fig. 1. Immunohistochemical staining pattern for FAP
in a gastric carcinoma. Expression of FAP is detected instromal fibroblasts. Bar = 50 µm.

Prognosis of patients
The overall 5-year survival rate among the 100
patients with stage III and IV gastric cancer was
29%. The disease-specific 5-year survival rate
among the 64 patients with FAP-positive tumors(22%) was significantly lower than that of the 36
patients with FAP-negative tumors (34%, P = 0.036,Table 1. Expression of FAP and clinicopathological parameters

Age (year)		
GenderMaleFemale
Tumor size (cm)		
Cancer stroma relationshipMed, IntSci
Histology*DifferentiatedUndifferentiatedLymph node metastasis
AbsentPresent
Lymphatic vessel invasion
AbsentPresent
Blood vessel invasion
AbsentPresent
StageIIIIVFAP expression
Positive
Negative

P value

67.2 ± 11.747
17
8.6 ± 3.7
3131
24
40
361
658
163
43
21

0.26
0.816964.6 ± 9.825
117.6 ± 3.9
1720
1224
630
1125
29
729
70.23
0.07600.82850.35360.0151< 0.0001
0.172

FAP, fibroblast activation protein; Int, intermediate type; Med, medullary type; Sci, scirrhous type.
* Differentiated, papillary or tubular adenocarcinoma; undifferenciated, poorly differenciated or undifferenciated adenocarcinoma or signet ring cell carcinoma.

23

Y. Fukumoto et al.

cancer. Our data showed that FAP expression iscorrelated with lymphatic and blood vessel invasion and caused a worse prognosis in patientswith advanced gastric cancer. FAP expression
has been described to be present predominantlyin the tumor stroma of epithelial malignancies(Garin-Chese et al., 1990; Ariga et al., 2001), andits presence has been associated with increased
microvessel density (Huang et al., 2004). FAP
activity has also been suggested to participate in
the growth of human colorectal tumor xenografts,
because inhibition of FAP enzymatic activity inthese tumors resulted in tumor growth attenuation(Cheng et al., 2002; Cheng et al., 2005). We havefound strong relationship between FAP expression in tumor-associated fibroblast and invasion ofcancer cells into microvessels of the gastric wall.
These reports suggest that FAP-dependent pathways may play an important role in epithelial cancer invasion, tumor angiogenesis, and subsequent
growth and metastasis.
We have found a statistically significant association between the expression of FAP andshortened survival time in patients. We have also
found that the expression of FAP is an independent prognostic factor as well as the histological stage. It has been proposed that scirrhous
gastric cancer has a worse prognosis, but in ourstudy there was no siginificant difference in FAP
expression between patients with scirrhous-type
cancer and other types of cancer (P = 0.076). FAP
pathways may predominate early in the course ofsmaller tumors to facilitate tumor invasion andtumor motility, which are required for metastases
to occur, thus leading to the enhanced inductionof FAP expression in early-stage disease. In advanced tumors, a relatively persistent elevation of
FAP levels may signify a more aggressive tumor
and a continued additive contribution of FAP-dependent pathways to tumor invasion and growth.
Thus high FAP levels may confer a worse prognosis even in those with advanced disease (Leonardet al., 2007).
In conclusion, we found that stromal FAP
levels are related to stage III and IV gastric can-Survival rate

1.0

P = 0.036

0.8

0.6

0.4

FAP negative (n = 36)

0.2

FAP positive (n = 64)
025

50

75

100125150

175200(month)

Time to deathFig. 2. Survival is estimated using the Kaplan-Meiermethods, which produce the disease-specific survival
curves of patients with stage III and IV gastric carcinoma according to the expression of FAP. FAP-positive
cases have lower survival rates.

Fig. 2). Multivariate analysis was performed witha Cox proportional hazards model using the covariates of tumor histology, histological stage andexpression of FAP (Table 2). FAP expression was
not detected as a prognostic factor independent of
the histological stage (P = 0.072).

Discussion
In the present study, we have immunohistochemically demonstrated the frequent occurrence of
FAP expression in surgically resected gastric
Table 2. Multivariate survival analysis by theCox proportional hazards model
Variable

Hazardratio		
Tumor histology*Differentiated/
undifferentiatedHistological stage
Stage III/IVFAP expression
Positive/negative95%
Confidence
intervalP
value

0.861

0.542–1.368

0.527

3.39

2.044–5.622< 0.001

1.557

0.956–2.535

0.072

FAP, fibroblast activation protein.
* Differentiated, papillary or tubular adenocarcinoma;
undifferenciated, poorly differenciated or undifferenciated adenocarcinoma or signet ring cell carcinoma.

24

FAP expression on gastric cancer

cer, and that the expression of FAP is associatedwith decreased survival time. If successful, stroma-directed therapy may provide clinical benefits
in a broad spectrum of indications, based on theconsistent presence and remarkable abundance ofstromal compartments in human carcinomas.

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Corresponding author: Youji Fukumoto25