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Molecular markers (PECAM-1, ICAM-3, HLA-DR) determine prognosis in primary non-Hodgkin's gastric lymphoma patients.

AIM
To investigate the prognostic significance of PECAM-1, ICAM-3 and HLA-DR antigens in patients with primary non-Hodgkin's gastric lymphoma.


METHODS
We immunohistochemically studied PECAM-1, ICAM-3 and HLA-DR antigen expression in 36 B-cell MALT-type primary gastric lymphoma patients. Ten non-malignant and ten healthy gastric tissue specimens were used as controls. Clinicopathological and survival data were correlated with the staining results.


RESULTS
HLA-DR antigen expression was detected in 33 gastric lymphoma patients (91.7%) and 6 non-malignant patients (54.5%). PECAM-1 stained tumor cells of 10 patients (27.8%), endothelial cells of 9 patients (25%) and inflammatory infiltrate of 4 patients (40%) with benign gastric disease. ICAM-3 expression was observed on the tumor cells of 17 patients (47.2%), while 5 non-malignant patients (50%) were stained positive as well. None of the healthy controls was stained for any of the genes studied. In the multivariate analysis, HLA-DR antigen and PECAM-1 were proved to be statistically significant independent prognostic factors associated with a favourable and an unfavourable prognosis respectively (P=0.009 and P=0.003). In the univariate analysis, PECAM-1(+)/ICAM-3(-) and HLA-DR(-)/ICAM-3(-) patients exhibited a significantly decreased overall survival compared to those with the exactly opposite gene expression patterns (P=0.0041 and P=0.0091, respectively). Those patients who were HLA-DR(+)/ICAM-3(+)/PECAM-1(-) (n=8) had a significantly higher survival rate compared to the rest of the group (n=24) (P=0.0289).


CONCLUSION
PECAM-1, ICAM-3 and HLA-DR are representative markers of tumor expansion potential and host immune surveillance respectively. Their combined use may help us to identify high-risk patients who could benefit from more aggressive therapeutic protocols.


INTRODUCTION
Primary gastric B-cell non-Hodgkin's lymphomas are uncommon tumors, constituting less than 2% of all primary gastric malignancies [1] . Numerous clinicopathologic studies have identified stage and grade as the most important prognostic factors [1,2] . Still there are a number of patients with favourable stage and grade that exhibit an aggressive phenotype. A second line of molecular prognostic markers has been introduced lately to better describe this clinical entity.
Several different molecules (bcl-2, p53, PCNA, c-fos, c-myc and Ki67) and biologic pathways have been implicated in the initiation and progress of primary non-Hodgkin's gastric lymphoma [3] . Immunohistochemical tracing of these molecules in gastric lymphoma patients has already been employed not only for the diagnosis but for the determination of prognosis as well [4] .
HLA-DR antigen is a class II MHC membrane-bound glycoprotein, which plays an important role in the regulation of the immune response [5] . Although it is normally expressed exclusively by antigen presenting cells of the immune system [6] , it shows variable expression in malignancies [7,8] . During oncogenesis, it is modified to affect tumor cell behavior by decreasing or enhancing specific antitumor immune mechanisms [9] .
Cell adhesion molecules are membrane glycoproteins that play a major role in neoplastic disease by interfering with cell-matrix and cell-cell interactions. They are also believed to participate in host immune surveillance by providing antigen non-specific recognition between Thcells, APCs, Tc-cells, NK-cells and their potential targets. ICAM-3 and PECAM-1 are considered to be representative members of the immunoglobulin Ig superfamily of cell adhesion molecules (CAMs). ICAM-3 is constitutively highly expressed by leukocytes especially in lymphomas and myelomas [10] . It is postulated that it may constitute the critical ligand for the initiation of lymphocyte immune responses [11] with possible antitumoral properties. Tumor expansion is angiogenesis dependent [12,13] , a function which is orchestrated by a constant interaction between tumor cells and host cells. This "cross talk" can lead either to cell cycle arrest and tumor regression or to tumor progression [14,15] . PECAM-1 (platelet/endothelial cell adhesion molecule) is considered to be an accurate measure for the assessment of vascular proliferation on tumor sections [12] determining prognosis in a variety of tumors [16,17] . Moreover, PECAM' s-1 expression by immunocompetent cells [11] as well as its regulatory role in the extravasation of leukocytes [18] , suggests a potential pathway through which lymphoid tumors Although the above molecules appear to represent interacting pathways responsible for the antitumoral response and tumor progression, up to day very little information is available on their role in the pathogenesis and prognosis of primary gastric lymphoma [19][20][21] . The aim of this study was to analyze the immunohistochemical expression of ICAM-3 (CD50), PECAM-1 (CD31) and HLA-DR antigens in a group of patients operated for primary non-Hodgkin's gastric lymphoma with respect to their clinicopathological characteristics and clinical outcome.

MATERIALS AND METHODS
We retrospectively recruited 36 B-cell MALT-type non-Hodgkin's primary gastric lymphoma patients (76.6%). All of them were diagnosed and treated during the period from 1991 to 1997 at the First Department of Propaedeutic Surgery of the Hippokration Hospital, Athens Medical School. Follow-up time measurements were specifically interrupted by death of the affected individuals from any cause. In this regard, follow-up time ranged between 15 d and 135.5 mo, with a mean of 60.5 ± 38.8 mo and a median of 68.5 mo. Favorable treatment outcome was defined as undetectable disease at the most recent followup. Unfavorable treatment outcome was defined as tumor recurrence, either locally or distantly, or death due to the tumor. Four patients had inadequate 5-year survival data and were excluded from the survival analysis. All reported deaths were attributed to the underlying disease, thus the overall survival corresponded to tumor-associated survival. Our study also included 10 gastric tissue specimens of non-malignant origin (Analytically this group consisted of 4 patients with gastritis and 6 patients with non malignant ulcer of the stomach, none of whom was submitted to any kind of surgery) and 10 healthy control tissue specimens. The characteristics of gastric lymphoma patients as well as information regarding adjuvant therapy are summarized in Table 1.
All patients were diagnosed by endoscopic biopsy and preoperatively evaluated by bone marrow biopsy, chest radiography, abdominal CT and indirect laryngoscopy. The mean age of disease presentation was 58.39 ± 15.39 years (median: 59 years).
Staging was performed according to the Japanese Classification of Gastric Carcinoma by the Japanese Gastric Cancer Association [22] . Seventeen patients (47.2%) were stage I, 7 (19.4%) stage II, 6 (16.7%) stage III and 6 (16.7%) stage IV. Histopathologic examination was undertaken according to the working formulation [23] . Fifteen (41.7%) were found to be low grade lymphomas (grade I), 7 (19.4%) intermediate grade lymphomas (grade II) and 14 (38.9%) high grade lymphomas (grade III). The presence of H pylori-associated MALT-type lymphoma was not determined in the majority of cases and this information was not included in our statistical analysis. Ten patients (27.7%) received a total gastrectomy and 20 (55.5%) a subtotal gastrectomy, while there was no information regarding the type of surgery for 6 (16.8%) patients. All patients had macroscopically clear margins, while microscopically www.wjgnet.com involved resection margins were detected in 5 patients (13.9%). Intraoperative staging consisted of biopsies of the liver and any enlarged abdominal lymph nodes. Splenectomy was performed only if the spleen was directly invaded. Adjuvant chemotherapy was administered to 25 patients (69.4%), 3 of whom (8.3%) received a combination of adjuvant chemo/radiotherapy. Eight patients (22.3%) did not receive any kind of supplemental therapy, while no data were retrieved for three more (Table 1).
Microvessels identified with PECAM-1 staining were counted on three 400 × fields (A=0.302 mm 2 ) within areas of maximum vascularity and the mean microvessel count was calculated for each area.

Statistical analysis
Data were expressed as mean ±SD. Survival analysis was performed using the Kaplan-Meier method with the log rank test. To determine independent prognostic factors we used the Cox proportional hazards model, which estimates the odds ratio in 95% confidence intervals. Univariate analyses comparing subgroups of patients were performed with the chi-square test (Pearson, Mantel-Haenzel test for linear association). The results of the morphometric examination were studied in various levels of the variables examined by the Student's t-test or the one-way ANOVA. The Levene test for homogeneity of variance was performed before the application of t-test or ANOVA. Non-parametric tests such as the Mann-Whitney test and the Kruskal -Wallis one-way ANOVA were applied in every case where the requirements of the t-test or the oneway ANOVA were not met. For all statistical tests, P < 0.05 was considered statistically significant.
HLA-DR antigen expression on tumor and peritumoral stromal cells was observed in 91.7%(n = 33) of gastric lymphoma patients ( Figure 2C).The proportion of HLA-DR positive tumor and peritumoral stromal cells was equally distributed in gastric lymphoma patients with different stage, grade, gender and type of surgery. HLA-DR was also expressed on stromal cells in 2 (50%) gastritis and 4 (66.6%) non malignant gastric ulcer control cases.
Several patients co-expressed two or more of the markers studied. The patterns of different protein coexpression are demonstrated in Table 4. None of the 10 healthy controls expressed any of the molecules studied.

Survival analysis
Univariate analysis showed that ICAM-3 expression was associated with improved 5-year survival rate (78.6% vs 55.6%, P = 0.1701). PECAM-1 expression on gastric lymphoma tumor cells was associated with significantly decreased 5-year survival rate (28.6% vs 76%, P = 0.0078) ( Figure 3A). Although the presence of PECAM-1 (+) vessels within the tumor was associated with decreased overall    survival rate (55.6% vs 69.6%), this difference was not proved to be statistically significant (P = 0.4067). Nevertheless, patients who showed PECAM-1 expression both on their endothelial cells (n = 4), presented a decreased overall survival rate compared to the rest of the group (25% vs 71.4%, P = 0.0403).

A B
Different levels of proportional HLA-DR antigen expression were sequentially evaluated for their prognostic value. HLA-DR antigen expression in more than 15% of the tumor cells (n = 26, 72.2%) was associated with an increased 5-year survival rate (75% vs 37.5%, P = 0.0469) ( Figure 3B). A corresponding though non-significant increase in the overall survival rate was also observed in those gastric lymphoma patients expressing HLA-DR antigen in more than 15% of their peritumoral stromal cells (n = 19, 52.8%) (66.67 % vs 64.29 %, P = 0.8593).
Univariate analysis revealed that patients´ gender, tumor stage, histologic grade and marginal status were not associated with their overall survival rate as demonstrated in Table 5.
To identify the independent prognostic factors that would predict survival, multivariate analysis was performed. The analysis included HLA-DR antigen and PECAM-1 upregulation (which was proved to be statistically significant prognostic markers in univariate analysis), age of the patients, tumor stage and tumor grade. Four patients were excluded from the process due to inadequate 5-year survival data. Multivariate Cox regression analysis revealed that PECAM and HLA-DR antigen expressions were the only statistically significant independent prognostic variables in the group of gastric lymphoma patients (P = 0.005 and P = 0.016, respectively). Similar results were obtained when PECAM-1 and HLA-DR antigen expressions were the only covariates submitted to multivariate analysis (P = 0.003 and P = 0.009, respectively). The results of multivariate analysis are depicted in Table 6 and Table 7.

DISCUSSION
Primary non-Hodgkin's gastric lymphoma represents a rare malignant tumor comprising 2-5% of all cases of malignant gastric tumor [27,28] . The 5-year survival rate ranges between 57% [29] and 96% for IE and IIE patients [30] , while it has been considered as low as 25% when all stages are grouped together. Several molecular markers like P27, cyclin E [31] and bcl-6 [32] have been recently assessed for their prognostic value. Still, most of them have no independent prognostic value.
In malignant lymphoma patients, clinical outcome and prognosis appear to depend largely on host immune response and vascular invasion. The current study attempted to clarify the prognostic value of HLA-DR antigen, ICAM-3 and PECAM-1 since they represent specific markers of regional immune reactions, cell-cell interactions and transendothelial migration.
HLA-DR antigen expression is a marker of host immune response in human malignant neoplasms. Its expression in tumor cells has been reported to be related with a favorable prognosis in patients with different types of cancer such as breast cancer [33] and squamous cell laryngeal carcinoma [34] . In our multivariate analysis, HLA-DR antigen expression was found to be a statistically significant independent prognostic factor associated    Table 4 represents the 15% cut-off level used in our survival analysis. with a favorable clinical outcome (P = 0.0199). This is in agreement with previous reports [33,34] . The expression of HLA-DR antigen in tumor cells has a modulating effect on the host immune response, possibly by helping in the presentation of tumor associated antigens to T-lymphocytes [35] , thus enabling the immune system to inhibit cancer growth. Regarding peritumoral stroma cells, the presence of tumor infiltrating leukocytes (TIL) invading the site of malignancy, suggests that the immune system is capable of reacting with tumors. The presence of such immune infiltrates has been documented in numerous reports and is often associated with improved prognosis [36,37] . In our primary gastric lymphoma group of patients, HLA-DR antigen expression in peritumoral stromal cells was associated with an increased (though non-significant) survival rate. Cell adhesion molecules are membrane glycoproteins that play a major role in neoplastic disease. Four primary groups of cell adhesion molecules have been described including the integrin receptor family, the immunoglobin superfamily, selectins and cadherins [38] . PECAM-1 constitutes a significant representative of the immunoglobulin superfamily and has been applied with considerable accuracy as an angiogenesis marker in several kinds of human neoplasia [16,17,39,40] .

Characteristics
A significant number of gastric lymphoma patients expressed PECAM-1 in their tumor cells (n = 10, 27.8%). Since PECAM-1 is constitutively expressed not only on the surface of endothelial cells, but on platelets, leukocytes, monocytes, neutrophils and selected T cell subsets as well [11] , our findings can be attributed to the lymphoid origin of the malignancy. PECAM-1 positive staining on gastric lymphoma tumor cells was found to be a statistically significant independent prognostic factor, associated with unfavorable prognosis (P = 0.0029). Although there are several reports [17,39] correlating increased angiogenesis with an unfavorable prognosis, this is the first report   [18] , constituting a possible regulator of the metastatic process in lymphoid tumors. Its presence on the surface of tumor cells can be therefore associated with altered cellular adhesivity, enabling malignant cells to dissociate from their primary sites, leading to tumor growth and metastasis [41][42][43] .
The detection of PECAM-1 (+) vessels within the tumor was related to an unfavorable, though nonsignificant clinical outcome. This difference reached statistical significance only for gastric lymphoma patients (n = 4) who showed PECAM-1 expression both in their tumor cells and in their endothelial cells (P = 0.0403). Although the fact that PECAM-1 expression in tumor endothelial cells has been associated with unfavorable prognosis in several tumors [2,16,17,40,[44][45][46] , tumors with PECAM-1 (+) endothelia exhibit an extended [47] overall survival rate.
Vascular density at the tumor margins is significantly lower than that within tumor stroma [12] . Additionally, gastric lymphoma patients with lymph node involvement present a significantly higher number of PECAM-1 (+) stromal vessel counts (7.285 vs 3.753, P = 0.002). It appears that the density of PECAM-1 (+) stromal microvessels/mm 2 correlates with the lymphatic metastatic pathway. Several studies have reported that PECAM-1 is a non-specific angiogenesis marker, which stains both lymphatic (weaker staining) and blood vessel endothelial cells [48,49] . New proliferating capillaries in the tumor stroma have fragmented basement membranes [50] , while endothelial cells at the tips of growing capillaries secrete collagenases and plasminogen activators [51] . These properties facilitate the metastatic process and may explain the association between tumor stromal vessel counts and lymph node metastasis as observed in our study.
Antigen-dependent and/or independent interactions between target cells and lymphocytes are required for the initiation of a specific immune response. In addition to antigen-specific interactions, "accessory" cell-surface molecules, ICAMs [52,53] , mediate an antigenindependent, non-specific adhesion between the reactant cells, which is considered the primary step in activation of lymphocytes [54] . These events often take place in a host against malignant cells. Recent work has indicated that resting leukocytes express a third ligand, ICAM-3, which appears to be the major ligand for LFA-1 in initiating phases of immune response [10,55] . Although it is poorly investigated, ICAM-3 upregulation has been well documented in lymphoid tumors [56,57] . In our gastric lymphoma group of patients, ICAM-3 upregulation was associated with a non-significantly improved 5-year survival rate. The fact that ICAM-3 expression level is associated with increased tumor surface and thickness provides evidence that its regulation is directly proportional to the tumor burden.  [58] , who observed that the co-expression of HLA class I molecules and ICAM-1 is a significant predictor of increased disease-free survival in patients with primary esophageal carcinomas. It appears that the lack of expression for both HLA-DR and ICAM-3 [HLA-DR(-)/ ICAM-3(-)] in gastric lymphoma patients is associated with a compromised host immune response against the tumor as well as with an unfavourable prognosis compared to those who were HLA-DR(+)/ICAM-3(+) (n = 10, P = 0.0091). On the contrary, gastric lymphomas expressing both HLA-DR and ICAM-3 but not PECAM-1 have not only a more potent host immune surveillance, but a negative expansion potential and a growth disadvantage as well.
In our study, PECAM-1 and HLA-DR antigen expressions were proved to be statistically significant independent prognostic factors in a group of patients with primary B-cell MALT-type non-Hodgkin's gastric lymphoma, suggesting that upregulation of both PECAM-1 and HLA-DR antigen is closely related to the clinical phenotype exhibited by the affected cases. Furthermore, it appears that ICAM-3, PECAM-1 and HLA-DR represent complementary biologic pathways associating host immune surveillance with non-specific intercellular interactions and endothelial transmigration. Their combined study in gastric lymphoma patients may amplify their prognostic accuracy and provide a better description of the biologic behaviour of these tumors.

ACKNOWLEDGMENTS
The authors are indebted to the Athens University and the Greek Ministry of Health and Welfare for sponsoring this study.