In the present study, RORα nuclear expression was detected in the gastric epithelium of all of the chronic gastritis, adjacent non-tumor, and GC patients. The majority of the chronic gastritis patients had a high RORα H-score, while the majority of the GC cases had a low RORα H-score. RORα was significantly decreased in GC cases compared to adjacent non-tumor and chronic gastritis cases and it was also significantly downregulated in adjacent non-tumor cells compared to chronic gastritis. These findings were consistent with others who found that RORα expression in GC tissues was significantly lower than in normal mucosa and adjacent non-tumor tissue [31, 32] In addition, lower RORα expression was documented in other types of tumors compared to normal tissues and precancerous lesions [33, 34, 35]. These findings suggested that RORα may play a role in multistep carcinogenesis as a tumor suppressor gene.
Low RORα H-score was significantly associated with unfavorable prognostic parameters in the studied GC such as diffuse type, high grade, positive lymph nodes, and high tumor budding. These findings were in agreement with Wang et al. (2017) and Su et al. (2019) who found that low RORα expression in GC was associated with dedifferentiation, tumor size, TNM stage, and lymph node metastasis. These data suggest that RORα could be used as a biomarker for GC tumor progression, aggressiveness, and poor prognosis [31, 32]
The present study detected the relation between RORα and tumor budding in GC as low RORα H-score was associated with high tumor budding. This could be explained by the fact that inhibiting RORα increased tumor cell proliferation, EMT, migration, and invasion.
In the current study, LAPTM4B was expressed in 49.7% of the chronic gastritis cases in contrast 100% of expression in GC cases. LAPTM4B expression showed significant difference between chronic gastritis cases and adjacent non tumor, between chronic gastritis and GC cases and between adjacent non-tumor and GC cases with the highest expression in GC cases followed by adjacent non-tumor tissue and the lowest expression in chronic gastritis cases. These findings were consistent with Zhou et al. (2019) who found that LAPTM4B expression in GC was higher than in normal surrounding tissue [36]. These findings suggested that LAPTM4B may play a role in the early stages of gastric carcinogenesis, and that greater expression of LAPTM4B could predict the progression of gastric lesions to precancerous lesions and subsequently GC [37].
The current study showed that high LAPTM4B significantly associated with poor prognostic pathological parameters of GC such as large tumor size, high tumor budding and lymph node positivity. These findings were in line with Xiao et al. (2013) who found that increased LAPTM4B expression in breast carcinoma was associated with TNM stage, lymph node metastases, and recurrence [38]. These findings may indicate that LAPTM4B is involved in GC proliferation, invasion, and metastasis.
The positive association between LAPTM4B expression in GC cells and TILs that was shown in our study was not explained in previous studies up our search. However, it could be explained by the findings that presence of H.pylori infection is associated with both high inflammatory infiltrate and LAPTM4B expression [39, 40].
The present study detected different E-Cadherin membranous expression patterns. In chronic gastritis cases 55.7% showed homogeneous membranous expression, while in GC only 8.2% showed homogeneous membranous expression.
As regard E-Cadherin expression pattern, there was a statistically significant difference between chronic gastritis and GC cases with the lowest percentage of homogenous expression in GC cases. These findings were consistent with others who suggest the role of E-Cadherin in multistep carcinogenesis [41, 42].
This study demonstrate the correlation between poor prognostic features such diffuse type GC, positive lymph nodes, advanced AJCC stage groups (III&IV), and increased tumor budding and heterogenous E-Cadherin expression in our GC patients. These findings were consistent with Di Bartolomeo et al. (2016) who found that abnormal E-Cadherin expression was related to advanced disease stage and poor outcome in GC patients [43].Furthermore, high-grade tumor budding was significantly associated with lower E-Cadherin expression in other epithelial tumors as esophageal cancer, colorectal cancer and tongue cancer [44, 45, 46].
Correlation between the studied immunohistochemical markers in this research showed significant negative correlation between LAPTM4B expression and RORα H -score in chronic gastritis group. This may be due to the fact that RORα is a tumor suppressor gene with high expression in normal tissues, whereas LAPTM4B is a proto-oncogene with low expression in normal tissues [36, 47]. While in GC cases, RORα H-score and the percent of E-Cadherin positivity in tumor cells had a significant co-parallel positive correlation. This may be explained by Su et al. (2019) who found that RORα enhanced E-Cadherin protein levels. Also in GC cells, it was found that knocking down of RORα reduced its inhibitory effects on the Wnt/b-catenin pathway, resulting in increased cell proliferation, EMT, migration, and invasion [32]. These findings may suggest that RORα inhibits EMT and GC cell invasion. In contrast, present work showed significant inverse association between LAPTM4B expression and E-Cadherin expression pattern in GC. According to Zhou et al. (2019) LAPTM4B overexpression lowered E-Cadherin expression [36, 47]. These results indicate that LAPTM4B may enhance EMT, increases cell migration and invasion. The present study failed to find any significant correlation between RORα and LAPTM4B in GC, and no previous studies had investigated the correlation between the two immunohistochemical markers.
When the immunoprofiles for the examined cases were collated, the immunohistochemical profile (Low RORα, high LAPTM4B and negative or heterogeneous E-Cadherin) was more predominant in GC cases than chronic gastritis. Most of GC cases with this immunoprofile showed large tumor size, high grade, positive LVI, perineural invasion, positive lymph node invasion, advanced T stage, advanced AJCC stage group and high tumor budding. These findings suggested that the immunohistochemical profile (Low RORα, high LAPTM4B and negative or heterogeneous E-Cadherin) could be used to detect GC transition and predict tumor aggressiveness.
Univariate analysis of overall survival showed that positive lymph nodes, absence of tumor necrosis, N3 stage group, presence of distant metastasis, AJCC stage IV, positive surgical margin, moderate and high grade of tumor budding and low RORα H-score all had unfavorable prognostic impact. On the other hand, multivariate COX-regression analysis failed to reveal the independent prognostic factor affecting patients ‘overall survival. While, using the recurrence free survival younger age, positive surgical margin, diffuse histological type, absence of necrosis and high grade of tumor budding were the most independent prognostic factors. According to Roberto et al. (2018) depth of invasion, lymph nodes involvement, and residual tumor were significant prognostic factors in GC [48]. In addition, Isobe et al. (2013) reported that young patients with GC have significantly higher incidences of diffuse-type histologic findings and both locally advanced and metastatic disease at presentation [49, 50].On the other hand Lee et al. (2019) found that the prognosis of GC varied with age, and young patients suffered a higher survival rate after surgery compared to elderly patients [51, 52].
The association between necrosis and good prognosis could be explained by the fact that necrosis, which is characterized by the breakdown of cytoplasmic membrane integrity, leads to the release of intracellular danger signals into the environment and the activation of immune responses against tumor cells. This induces tumor cell death and thus increases response to chemotherapeutic agents [53]. However, multiple studies found that the presence of necrosis is associated with a poor prognosis in many solid tumors. This has been proposed that the presence of necrotic cells promotes angiogenesis, endothelial cell proliferation, induces vasculature, and increases migration and invasion, all of which contribute to cancer progression [45, 55].
In agreement with the present study, Wang et al. (2021) reported that GC patients with high RORα expression levels had the best prognosis [56]. Furthermore, low RORα expression in HCC has been associated with shorter overall and disease-free survival [34]. In another study, reduced RORα mRNA expression was associated with poor prognosis in breast cancer [57].
The current study failed to find any prognostic significance for LAPTM4B. On the other hand, elevated LAPTM4B levels have been linked to a poor prognosis in a variety of malignancies, including GC, nasopharyngeal carcinoma, ovarian carcinoma, neck squamous cell carcinoma, breast cancer, and lung adenocarcinoma [37, 58, 12, 59, 60]. The present study found no prognostic significance of E-Cadherin. On the other hand, several studies found that abnormal E-Cadherin expression is associated with invasiveness, advanced tumor stage and poor prognosis in a variety of malignancies, including GC, hepatocellular carcinoma, ovarian, CRC, and breast cancers [61, 62, 63].