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Clinical Significance of CLDN18.2 Expression in Metastatic Diffuse-Type Gastric Cancer

Purpose Isoform 2 of tight junction protein claudin-18 (CLDN18.2) is a potential target for gastric cancer treatment. A treatment targeting CLDN18.2 has shown promising results in gastric cancer. We investigated the clinical significance of CLDN18.2 and other cell-adherens junction molecules (Rho GTPase-activating protein [RhoGAP] and E-cadherin) in metastatic diffuse-type gastric cancer (mDGC). Materials and Methods We evaluated CLDN18.2, RhoGAP, and E-cadherin expression using two-plex immunofluorescence and quantitative data analysis of H-scores of 77 consecutive mDGC patients who received first-line platinum-based chemotherapy between March 2015 and February 2017. Results CLDN18.2 and E-cadherin expression was significantly lower in patients with peritoneal metastasis (PM) than those without PM at the time of diagnosis (P=0.010 and 0.013, respectively), whereas it was significantly higher in patients who never developed PM from diagnosis to death than in those who did (P=0.001 and 0.003, respectively). Meanwhile, CLDN18.2 and E-cadherin expression levels were significantly higher in patients with bone metastasis than in those without bone metastasis (P=0.010 and 0.001, respectively). Moreover, we identified a positive correlation between the expression of CLDN18.2 and E-cadherin (P<0.001), RhoGAP and CLDN18.2 (P=0.004), and RhoGAP and E-cadherin (P=0.001). Conversely, CLDN18.2, RhoGAP, and E-cadherin expression was not associated with chemotherapy response and survival. Conclusions CLDN18.2 expression was reduced in patients with PM but significantly intact in those with bone metastasis. Furthermore, CLDN18.2 expression was positively correlated with other adherens junction molecules, which is clinically associated with mDGC and PM pathogenesis.


INTRODUCTION
Gastric cancer (GC) is the fifth most common cancer worldwide and the third leading cause of cancer-related death [1]. In South Korea, GC is the most common cancer in men and the fourth most frequently diagnosed cancer in women [2,3]. It has been categorized into two main histological subtypes based on Lauren's criteria: intestinal and diffuse [2,4]. Diffuse-type GC (DGC) is associated with a worse prognosis, occurrence at an earlier age, and the highest recurrence frequency [5]. DGCs are poorly differentiated histologically; they are characterized by a lack of intercellular adhesion molecules and disrupted tight junction molecules, and often have poorly cohesive and scattered signet-ring cell morphology, which predisposes to invasion and growth patterns [6]. DGC is more frequently associated with peritoneal metastasis (PM) than intestinal-type GC [7]. PM is the most frequent metastatic pattern of GC, although Paget's 'seed and soil' hypothesis has been considered the fundamental theory of metastasis; however, the mechanisms underlying peritoneal dissemination are yet to be elucidated [8]. According to this hypothesis, detachment of cancer cells from the primary tumor is the first step in peritoneal dissemination; thus, cancer cell detachment is considered important in PM [9]. The loss of cell-cell adhesion systems may result in the dissemination of single carcinoma cells from the primary tumor sites and trigger remodeling of the actin cytoskeleton, leading to the development of a mesenchymal phenotype and the dispersal of carcinoma cells [10]. The importance of the loss of cell-cell adhesion molecules such as E-cadherin in the context of DGC initiation and PM formation has been evaluated in several studies [11,12].
Claudin-18 (CLDN18), a member of the claudin family, is a component of tight junctions that regulates paracellular barrier functions. DGC is associated with an inter-chromosomal translocation between CLDN18 and ARHGAP (the gene encoding Rho GTPase-activating protein [RhoGAP], which contributes to the organization of actin and microtubule cytoskeletons), resulting in the generation of a RhoGAP domain-containing fusion protein with impaired function of CLDN18 and RhoGAP [13][14][15][16][17]. A previous in vitro study reported that CLDN18-ARHGAP26 fusion-positive cell lines showed impaired barrier properties, reduced cell-cell adhesion, and augmented invasiveness [18]. In younger patients with DGCs, this fusion status can act as a predictor of metastasis, such as peritoneal dissemination [15]. Moreover, gastric CLDN18 expression is related to not only size and invasiveness but also to potential metastatic ability and patient outcome [19]. Additionally, CLDN18-ARHGAP26/6 or the RhoGAP domain-containing fusion protein is associated with poor prognosis of DGC [14,16]. Yang et al. [14] performed western blot analysis and migration assay to confirm the functional effects of the CLDN18-ARHGAP fusion gene and revealed its poor prognostic role; its migration capacity was more enhanced compared to that of the control vector.
The expression of isoform 2 of CLDN18 (CLDN18.2) has been limited to differentiated epithelial cells in the gastric mucosa and primary gastric malignancies, emphasizing its potential as a candidate for targeted therapy [20]. Recently, a treatment strategy targeting CLDN18.2 has shown promising results in inoperable or recurrent GC patients [21,22].
Hereby, in this study, we aimed to investigate the clinical significance of CLDN18.2 and other adherens junction molecules (RhoGAP and E-cadherin) in metastatic DGC (mDGC).

Study population and design
This study included a total of 77 patients with mDGC who were treated with first-line platinum-based chemotherapy between March 2015 and February 2017 at Seoul St. Mary's Hospital, the Catholic University of Korea. We performed surgical biopsy and gastric endoscopic biopsy of the primary gastric tumor lesion. Computed tomography (CT) and bone scans were performed for staging. If needed, additional imaging scans, such as magnetic resonance imaging and positron emission tomography/CT, were performed. Patients were subsequently evaluated for their response to chemotherapy after 6±2 weeks based on their radiological imaging results. Radiological changes were evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1 [23]. The tumor location was classified as upper/middle/lower third. An immunohistochemical score of 3+ or 2+ was considered to determine human epidermal growth factor receptor 2 positivity by fluorescence in situ hybridization analysis. For patients with unclear diagnosis of PM using radiological examination, diagnostic laparoscopy was performed. If the patient showed definite PM on CT or other imaging modalities, diagnostic laparoscopy was not performed.

Image acquisition and quantitative data analysis
Slides were scanned using the PerkinElmer Vectra 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer), and images were analyzed using the inForm software and TIBCO Spotfire (PerkinElmer). To acquire reliable unmixed images, representative slides exposed to each emission spectrum and unstained tissue slides were used. Each of the individually stained sections (E-cadherin, RhoGAP, and CLDN18.2; Opal690) were used to establish the spectral library of fluorophores required for multispectral analysis. This spectral library served as a reference for target quantitation; the intensity of each fluorescent target was extracted from the multispectral data by linear unmixing. Each cell was identified using DAPI. Our two pathologists selected areas of interest in the primary tumor section. The total number of CLDN18.2-, RhoGAP-, and E-cadherin-positive cells was considered the total number of cell infiltrations in the tissue. The fluorescence intensity of CLDN18.2, RhoGAP, and E-cadherin in the tissue samples was determined based on the H-score: strong (3+), intermediate (2+), weak (1+), or no (0) membranous staining. The H-score of each tissue sample was calculated using the following formula: H-score=[(3×the percentage of strongly stained cells)+(2×the percentage of moderately stained cells)+(the percentage of weakly stained cells)]. H-scores ranged from 0 to 300 [24].

Statistical analysis
The correlations between the H-scores of CLDN18.2, RhoGAP, and E-cadherin, and the clinicopathological factors and radiological responses were analyzed using the independentsample t-test. The correlation between H-scores among each marker was assessed by Pearson's correlation analysis. Overall survival (OS) and progression-free survival (PFS) were calculated from the start date of first-line palliative chemotherapy until the date of death and disease progression or death, respectively. For survival analyses, data from living patients or those with no disease progression were censored from the last follow-up date. Cox proportional hazards regression analysis was performed to identify the risk factors for overall mortality. Survival curves were generated using the Kaplan-Meier method and compared using the log-rank test. All analyses were performed using the SPSS software (version 24; IBM Corp., Armonk, NY, USA), and a two-sided P-value of <0.05 was considered significant.

Correlation between clinicopathological factors and H-scores of CLDN18.2, RhoGAP, and E-cadherin
We investigated the correlation between cell-adherens junction protein expression and clinicopathological factors (

Survival and chemotherapy response based on CLDN18.2, RhoGAP, and E-cadherin expression
We investigated the tumor response after first-line chemotherapy treatment based on CLDN18.2, RhoGAP, and E-cadherin expression (  RhoGAP, and E-cadherin positivity was determined based on the median value (Fig. 5).
CLDN18.2, RhoGAP, and E-cadherin positivity were not associated with OS and PFS as evidenced by multivariate analyses (Supplementary Table 1).

DISCUSSION
The aim of the present study was to evaluate the clinical significance of CLDN18.2 expression in mDGC. To the best of our knowledge, this is the first study to investigate the clinical significance of CLDN18.2 expression by immunofluorescence in mDGCs.
We investigated the association between the expression of adherens junction molecules and PM as several previous studies have suggested that the loss of adherens junction structure stability is associated with PM [12,25]. Yonemura et al. [12] demonstrated that reduced expression of E-cadherin and high expression of S100A4 promote PM, serosal involvement, and infiltrative tumor growth. Togano et al. [25] suggested that loss of E-cadherin expression is a critical step for PM of GC with sub-serosal invasion. To date, little is known about the clinical impact of CLDN18.2 expression in metastatic GC. Loss of CLDN18.2 expression is associated with an increased proliferative and invasive potential of GC [26,27]. We found that CLDN18.2 and E-cadherin expression was lower in patients with PM than in those without PM. Notably, CLDN18.2 and E-cadherin expression was significantly higher in patients who never developed PM until death than in those who developed PM. Our results suggest that adherens junction instability may be involved in the progression and formation of PM during the course of the disease.   Remarkably, CLDN18.2 and E-cadherin expression was significantly higher in patients with bone metastases in the present study. In fact, bone metastasis and PM showed a nearly exclusive pattern (PM rates: 17% [2/12] and 74% [48/65] in patients with and without bone metastasis, respectively). Furthermore, among the five patients who never developed PM until death, three patients had initial bone metastasis. These results are in line with those reported in previous studies, which showed that bone metastasis is not frequently synchronous with PM [28], indicating that the mechanisms of organotropic metastasis are clearly different. Furthermore, our results reveal that loss of adherens junction integrity, which is generally accepted as the first step in PM formation, is more important in PM than in metastases to other sites such as bone, which occurs mostly through hematogenous spreading [29].

Significance of CLDN18.2 Expression in mDGC
Furthermore, we found a positive correlation between CLDN18.2, RhoGAP, and E-cadherin expression. Several studies have proposed expression-related interactions between different adherens junction proteins. Lu et al. [30] investigated the function of claudin-7 with respect to the regulation of cell proliferation and maintenance of epithelial cell attachment via the engagement of integrin β1. Wu et al. [31] reported that EpCAM modulates adhesion and tight junction function by regulating the intracellular localization and degradation of selected claudins. To date, the precise mechanism of interaction between adherens junction proteins is not well understood. A previous study suggested that intercellular junctions not only come apart but also undergo regulatory changes, including a phenomenon known as a cadherin switch, in which epithelial cells lose E-cadherin expression and start expressing N-cadherin during epithelial-mesenchymal transition [32]. However, our findings cannot explain the mechanism of interaction between CLDN18. correlation between the expression of these adherens junction molecules may result in their ability to disrupt cellular cohesiveness synergistically. Further studies investigating these mechanisms are warranted.
We found no difference in chemotherapy response and survival with respect to the expression of adherens junction molecules. Cell-adherens junction molecules play an important role in the epithelial-mesenchymal transition and influence chemosensitivity of cells [33]. Skalova et al. [34] proposed that claudin-1 and claudin-3 play a role in the response to chemotherapy in breast cancer. Yang et al. [14] demonstrated that patients with in-frame fusion genes containing the RhoGAP domain have aggressive DGCs. Moreover, Wang et al. [35] revealed that loss of E-cadherin is associated with poor prognosis. However, there is no clear consensus regarding the chemosensitivity and prognosis of cell-adherens junction molecules in mDGCs. In addition, none of the patients had a measurable lesion, a characteristic feature of metastatic GC, causing a small subset size (54/77, 70%) and incomplete investigation of the chemotherapy response. Furthermore, the present study is retrospective in nature and is therefore not appropriate for investigating survival. Further studies are needed to confirm this.
Our study has several limitations. First, the association between cell-adherens junction molecules was not confirmed by other experimental methods. Recent studies have investigated these markers using several methods, such as whole genome sequencing and sequencing [13,14,16]. A previous study found that the fusion of CLDN18.2 and ARHGAP26, which includes the RhoGAP domain, was frequently observed in mDGC [15]. However, immunostaining is a practical method for expression analysis. In fact, a recent clinical trial investigating an anti-CLDN18.2 antibody for treating GC used immunohistochemistry as a predictive marker; thus, we believe our study has clinical implications. Furthermore, digital pathology has several advantages with respect to accuracy. As there is currently no optimal cut-off or standard for evaluating these markers, our results can provide valuable clinical information to clinicians evaluating specimens by immunostaining. Additionally, we evaluated the positivity of CLDN18.2, RhoGAP, and E-cadherin in only tumor cells. However, previous studies have shown that CLDN18.2, RhoGAP, and E-cadherin are not only expressed in tumor cells but also in normal gastric mucosa [20,[36][37][38]. A previous study showed that CLDN18.2 expression level is significantly lower in GC than in the surrounding normal gastric mucosa and that downregulation of CLDN18.2 is associated with the proliferative potential of GC, suggesting its role in GC progression [26]. In our study, the expression of cell-adherens junction molecules in normal gastric mucosa was not assessed owing to tumor heterogeneity in slide imaging and the absence of normal control tissue in every patient. Our study did not show the clinical significance of CLDN18.2 and other molecules in terms of survival and prognosis; therefore, analysis of correlation of these proteins between cancer and normal tissues, such as the ratio of CLDN18.2 expression in tumor to normal mucosa, may provide meaningful results. Further studies are required to evaluate the patterns of cell-adherens molecules between tumor and normal mucosa. Second, our cohort included a limited number of patients. Therefore, the results should be interpreted with caution. An extensive study with a larger sample size is needed to confirm our findings. Finally, our study is retrospective, and a prospective study is also required.
Collectively, we evaluated the clinical significance of cell-adherens junction molecules, including CLDN18.2, which are being investigated as targets in GC treatment. We found that CLDN18.2 expression was significantly lower in patients with PM but intact in those with bone metastasis, which may be associated with the first step of the "seed and soil"