Comparative Studies On The Immunophenotype And P14^ARF, P16(INK4a) Changes Between Cardiac And Distal Gastric Carcinoma

During the past decades, the incidence of gastric cancer has been decreased with the change of the life-style. But the subsites of gastric carcinoma underwent significant changes. The incidence of adenocarcinoma at antrum or distal stomach has been decreased, while that at esophagogastric junction (EGJ, the proximal stomach and distal esophagus) was increased in most developed countries. In 1996, Siewert definited the adenocarcinomas located in EGJ as adenocarcinoma of esophagogastric junction (AEG) and classified them into three types (Type I, II and III) according to the tumor center location. In the past several years, the incidence of adenocarcinoma of EGJ region was also found to be increased in the high incidence area of esohageal and gastric cancer in China, but the reason was still unclear. In our country, the absolute majority of the adenocarcinomas of esophagogastric junction belong to Siewert II and III subtype, so we generally refered the adenocarcinomas in these subsites as gastric cardiac adenocarcinoma (GCA).Recent study showed that GCA was different from other gastric cancers in many ways, such as biological behavior and etiological factors. Accordingly, it might be a special subtype or independent entity of gastric carcinoma. Maeda et al reported that GCA has a higher rate of lymph node metastasis and unfavourable prognosis than noncardia tumors. Meanwhile, Hp infection was proven to be a key factor in gastric distal adenocarcinomas (GDA), while the role of it involed in GCA is controversial now. Up to now, little study about the incidence and putative differences between GCA and GDA was conducted in China.As we all know, abnormality of cell cycle is an important mechanism in carcinogenesis. P16^INK4a and p14^ARF, the members of INK4a/ARF family, could regulate the cell cycle distribution by participating in the pathway of Rb (p16^INK4a-Rb) and p53 (p14^ARF-p53), and play the critical role in tumorigeness and progression. Inactivation of p16^INK4a and p14^ARF could be detected in many human tumors including skin cancer, pancreatic cancer, pancreatic cancer, esophageal carcinoma, lung cancer. Moreover, inactivation of p16^INK4a and p14^ARF was also found in gastroenteric tumor, and the expression of p16^INK4a and p14^ARF was associated with histological differentiation and histotype in gastric carcinomas. By now, there is no study on the expression of p16^INK4a and p14^ARF between GCA and GDA. In addition, Cytokeratins (CK) is used as diagnostic marker in distinguishing different types of carcinomas, as its composition is highly heterogeneous depending on epithelial cell type. Mucins (MUC) are high molecular weight glycoproteins at surface epithelia that play important roles in carcinogenesis, Especially, MUC2 is a marker of intestinal metaplasia regarding of its staining in intestinal goblet cells, was related with histotype in gastric carcinomas.So in this study, we studied the possible difference between GCA and GDA in tumorigeness and progression in human primary gastric carcinomas.The contents of this study included the following four parts: 1. Comparison of the expression of immunophenotypic markers including CKs and MUCs between GCA and GDA. 2. Differences in the expression of p14^ARF and p16^INK4a between GCA and GDA. 3. The similarity and differences in the deletions of p16^INK4a and p14^ARF genes between GCA and GDA. 4. Difference of p14^ARF and p16^INK4a mythelations between GCA and GDA. The aim of this study is to explore the possible differences in the afore-mentioned four aspects between gastric cardiac and distal adenocarcinomas, to reveal the possible reason of the increased incidence of GCA in our country.This study includes four parts:PartⅠComparison of the expressions of immunophenotypic markers of CKs and MUCs between GCA and GDAObjective: To explore the possible difference of immunophenotype between GCA and GDA by detecting the expression of CKs and MUCs. Methods: Ninety-nine patients with gastric cardiac and distal adenocarcinomas consecutively operated at the second hospital of Hebei Medical Universty and Cixian, Zanhuang County Hospital in Hebei Province between January 2006 and December 2009 were included in the study. All patients were underwent a preoperative upper gastrointestinal endoscopy (with biopsy specimens routinely performed). Fifty patients with cardiac carcinoma attended in this study were chosen according to the tumor center within 1 cm above and 2 cm below the anatomic esophagogastric junction (EGJ). Forty-nine cases with the tumor at antrum were operated with different surgical approaches. To compare the different immunophenotype between GCA and GDA, the expression of CKs (CK7, CK14, CK19 and CK20) and MUCs (MUC1, MUC2 and MUC5AC) was detected by immunohistochemistry method.Results:1.1 The expression of CKs and MUCs in GCA in comparison with GDAImmunohistochemistry results showed that the expression of CK14 and MUC2 displayed significantly difference between GCA and GDA. The expression of CK14 was significantly higher in GCA than that in GDA (30.0% vs 10.2%, P<0.05). Inversely, MUC2 expression was lower in GCA than that in GDA (52.0% vs 71.4%, P<0.05). Furthermore, CK14 expression was associated with histological differentiation and lymph node metastasis in GCA. Expression of CK14 was significantly lower in GCA with moderate/poor differentiation than that in well differentiation (22.0% vs 66.7%, P<0.05), and it was also decreased in GCA with lymph node metastasis compared to that without lymph node metastasis (17.9% vs 45.5%, P<0.05). But CK14 expression was not related with the extent of differentiation in GDA (P>0.05). As for the other molecules including CK7, CK19, CK20, MUC1 and MUC5AC, no significant differences could be found in their immunoreactivity between GCA and GDA (P>0.05).1.2 Comparative Studies of CK7, CK20 and combined expression of CK7/CK20 between GCA and GDA The expression of CK7 and CK20 showed no significant differences between GCA and GDA (CK7: 86.0% vs 79.6%, P>0.05; CK20: 56.0% vs 40.8%, P>0.05). In GDA, CK20 expression was associated with the differentiation of tumors. The expression of CK20 was lower in GDA with moderate/poor differentiation than that in well differentiation (33.3% vs 70.0%, P<0.05). However, expression of CK20 was not related with the differentiation and lymph node metastasis in GCA (P>0.05).The coexpression of CK7/CK20 results showed that the expression rate of CK7+/CK20-, CK7+/CK20+, CK7-/CK20+ and CK7-/CK20- in GCA was 40.0%, 46.0%, 10.0% and 4.0%, respectively. And the corresponding value of CK7/CK20 expression in GDA was 46.9%, 32.7%, 8.2% and 12.2%, respectively. These data showed no statistically significant difference within the two groups (P>0.05) and no distinct association with histological differentiation (P>0.05).PartⅡThe difference of p16^INK4a and p14^ARF protein expression between GCA and GDAObjective: To explore the possible difference of p16^INK4a and p14^ARF expression between GCA and GDA.Methods: Forty-four samples from cardia and 40 tumors from distal schomach were chosen by the procedure above mentioned. Meanwhile, twenty-five samples with normal gastric mucosa were used as control group, obtained from gastric stump resected at the corresponding time period. Expressions of p16^INK4a and p14^ARF were detected in two subtypic tumors by immunohistochemistry.Results:2.1 Expression of p14^ARF in gastric tumors2.1.1 The expression of p14^ARF in GCA and GDAP14^ARF immunoreaction was located in cell nucleus and/or cytoplasm. The values of p14^ARF expression were 84.0% and 45.2% in normal gastric mucosa and tumors, respectivly, showing significantly statistics (P<0.05). Moreover, expression of p14^ARF was lower in GCA than that in GDA (34.1% vs 57.5%, P<0.05).2.1.2 Relationship between p14^ARF and clinical pathological characters in GCA and GDAThere was no relationship between p14^ARF protein expression and the sex, age, histological differentiation and lymph node metastasis of the patients with GDA (P>0.05). Wherease, it appeared that p14^ARF protein is related with the differentiation and lymph node metastasis in GCA. Only 18.5% of GCA with poor differetiation showed positve reaction, which was significantly lower than that in well differented tumors (58.8%, P<0.05). And also the p14^ARF expression was lower in tumors with lymph node metastasis than that without metastasis (17.4% vs 52.4%, P<0.05). No relationship between p14^ARF and sex, age in cardiac tumors was found (P>0.05).Moreover, p14^ARF expression displayed markedly difference in the GCA and GDA subgroup with poor differentiation. GDA with poor differentiation showed higher reaction than cardiac ones (55.2% vs 18.5%, P<0.05). And the expression of p14^ARF protein was statisticly downregulated in GCA with lymph node metastasis when compared with the GDA with lymph node metastasis (17.4% vs 57.1%, P<0.05).2.2 Expression of p16^INK4a in gastric tumors2.1.1 The expression of p16^INK4a in GCA and GDAImmunohistochemisty results showed that only 58.3% gastric carcinomas displayed positive reaction by p16^INK4a antigen, which was markedly lower than normal gastric mucosa (96.0%, P<0.05). Furthermore, expression of p16^INK4a was significantly lower in GCA than that in GDA (43.2% vs 75.0%, P<0.05).2.2.2 Relationship between p16^INK4a and clinical pathological characters in GCA and GDAThere were no relationships between p16^INK4a expression and the age, sex, hitological differentiation and lymph node metastasis of the patients with GCA and GDA (P>0.05). However, the different expression of p16^INK4a in two subtypic tumors with poor differentiation was detected, and the values were 37.0% and 72.4% in GCA and GDA with poor differentiation, respectively. Meanwhile, p16^INK4a expression was lower in GCA with lymph node metastasis than that in GDA with lymph node metastasis (39.1% vs 75.0%, P<0.05).PartⅢThe difference of p16^INK4a and p14^ARF gene deletion between GCA and GDAObjective: To explore the possible differences of p16^INK4a and p14^ARF gene deletions between GCA and GDA.Methods: Forty-four samples from cardia and 40 tumors from distal schomach were chosen by the procedure above mentioned. Meanwhile, fifteen samples with normal gastric mucosa were used as control group, obtained from gastric stump resected at the corresponding time period. Deletions of p16^INK4a (E1αand E2) and p14^ARF (E1βand E2) were detected in two subtypic tumors by PCR assay.Results:3.1 Comparative study of p16^INK4a gene deletion in E1αand E2 exon between GCA and GDA.PCR results showed that there was no deletion of E1αand E2 exon in normal gastric mucosa. While in 84 cases of gastric carcinomas, E1α, E2 exon deletion was seen in 9.5% (8/84) and 8.3% (7/84) cases, respectively, and the total deletion proportion of p16^INK4a gene in E1αand E2 exon was 17.9% (15/84) in gastric carcinomas.In 44 cases of GCA, deletion of E1α, E2 and the E1αcombined with E2 was 4 cases (9.1%), 5 cases (11.4%) and 9 cases (20.5%), respectively. In 40 cases of GDA, deletion of E1α, E2 was 10.0% (4/40) and 5.0% (2/40), respectively, and p16^INK4a (E1αand E2) deletion was 15.0% (6/40). There was no significant differences on E1α, E2 and p16^INK4a (E1αand E2) between GCA and GDA (P>0.05).3.2 Comparative study of p14^ARF gene deletion in E1βand E2 exon between GCA and GDA.PCR results showed that there is no deletion of E1βand E2 exon in normal gastric mucosa. In 84 cases of gastric carcinomas, deletion of E1β, E2 could be seen in 6.0% (5/84) and 8.3% (7/84) cases, respectively, and otal deletion proportion of p14^ARF (E1βand E2) was 14.3% (12/84).In 44 cases of GCA, deletion of E1β, E2 and E1βcombined with E2 was 3 cases (6.8%), 5 cases (11.4%) and 18.2% cases (8/44), respectively. Besides, in 40 cases of GDA, deletion of E1β, E2 was 5.0% (2/40) and 5.0% (2/40), respectively, and p16^INK4a (E1αand E2) deletion was 10.0% (4/40). There was no significant differences on E1α, E2 and p14^ARF (E1βand E2) between GCA and GDA (P>0.05).3.3 Relationship of p14^ARF and p16^INK4a expressions and gene homozygous deletionIn 84 cases of gastric carcinomas, 35 out of 84 cases displayed negative expression of p16^INK4a, and 15 out of 84 cases showed p16^INK4a gene deletion in E1αand E2 exon. Simultaneously, the p16^INK4a protein expression was negative among all the 15 cases with p16^INK4a gene deletion. And in all cases, negative expression of p14^ARF was shown in 46 cases. The p14^ARF gene deletion (E1βand E2) was only seen in 12 cases, accompanying by negative immunoreaction of p14^ARF antigen. The results may suggest that deletion of p14^ARF and p16^INK4a was one of the mechanisms leading to protein inactivation.PartⅣCompasion of p16^INK4a and p14^ARF methylation between GCA and GDAObjective: To explore the possible difference of p16^INK4a and p14^ARF methylation between gastric cardiac and distal adenocarcinomas, and study the mechanism of p16^INK4a and p14^ARF inactivation in gastric tumors.Methods: Fifty-two cardiac adenocarcinomas, and 39 distal ones and 15 cases of normal gastric mucosa were detected by Methylmion Specific PCR (MSP) for p16^INK4a and p14^ARF methylation. Meanwhile, 10 cases of tumors from cardia and 8 cases from distal stomach were used to total RNA obtaining to detect expressions of p16^INK4a and p14^ARF at mRNA level by Real-time PCR. Results:4.1 p14^ARF methylation by MSP4.1.1 Comparative study of p14^ARF gene methylation between GCA and GDAMSP result showed that the frequency of p14^ARF methylation in gastric tumors was markedly higher than that in normal gastric mucosa (56.0% vs 13.3%, P<0.05). Hypemethylation of p14^ARF was 61.5% and 43.6% in GCA and GDA, respectively, which was higher than that in normal gastric mucosa (P<0.05). Moreover, p14^ARF hypemethylation in cardiac carcinomas was higher than in distal ones (P<0.05).4.1.2 Relationship of p14^ARF hypermethylation with clinical pathological characters between GCA and GDAP14^ARF methylation was not associated with the age, sex, histological differentiation and lymph node metastasis of the patients with GDA (P>0.05). P14^ARF methylation in GCA with poor differentiation was higher than that with well differentiation (78.1% vs 45.5%, P<0.05), but there was no relationship between p14^ARF methylation and sex, age and lymph node metastasis in GCA (P>0.05). Furthermore, different frequency of p14^ARF methylation between GCA and GDA with poor differentiation was detected (78.1% vs 48.0%, P<0.05).4.2 Methylation of p16^INK4a by MSP4.2.1 Comparative study of p16^INK4a methylation between GCA and GDAPromoter methylation was detected in 63.7% of gastric tumors, higher than that of normal mucosa (20.0%, P<0.05). Methylation of p16^INK4a was 73.1% and 51.3% in gastric cardiac and distal adenocarcinomas, respectively, and promoter methylation was more frenquency in GCA than distal ones (P<0.05).4.2.2 Relationship of p14^ARF hyermethylation with clinical pathological characters between GCA and GDANo correlation was found between promoter hyermethylation and clinical pathological parameters in both tumors from two sites (P>0.05). However, promoter hypermethylation of p14^ARF was detected in 81.3% of cardiac tumors and in 56.0% of distal ones with poor differentiation, and the former was higher than the latter (P<0.05).4.3 Effection of promoter hypermethylation on mRNA expression of p14^ARF and p16^INK4a in gastric tumorsEighteen fresh samples of gastric carcinomas were selected, including 10 of cardiac carcinomas and 8 of distal ones. And they were classified two groups according to methylation status. Among 18 cases, 10 cases were positive regarding of p14^ARF methylation, 8 cases were negative. According to p16^INK4a methylation, half of cases were positive. Expression of p14^ARF and p16^INK4a at mRNA level were detected by Real-time PCR further, and the result showed that expression of p14^ARF and p16^INK4a in hypermethylated group was significantly lower when compared with that in unmethylated group (P<0.05). It may indicate that methylation of p14^ARF and p16^INK4a genes could inhibit the expression of p14^ARF and p16^INK4a at mRNA level.Conclusion:1 Higher expression of CK14 and lower expression of MUC2 in gastric cardiac carcinomas than in distal ones may indicate that GCA is different from GDA in histotype and histological origin.2 The expression of p14^ARF and p16^INK4a protein was significant lower in GCA that in GDA, and p14^ARF expression was related with differentiation and lymph node metastasis in GCA. However, the expression of both p14^ARF and p16^INK4a protein was not associated with differentiation and lymph node metastasis in GDA. Therefore, there were differences in expression of p14^ARF and p16^INK4a and its clinical pathlogical significance between GCA and GDA.3 Expressions of p16^INK4a and p14^ARF protein were negative in the cases with p16^INK4a and p14^ARF gene deletion. However, there was no significant difference in the occurance of deletions of p16^INK4a and p14^ARF gene between GCA and GDA, suggesting that there might be no correlation between deletion of p16^INK4a and p14^ARF gene and subsites of gastric carcinomas.4 Methylation levels of both p14^ARF and p16^INK4a were higher in GCA than that in GDA. The differences in the methylation level may be the important contributing factor to the differences in the mRNA and protein between cardiac adenocarcinoma and distal gastric adenocarcinoma.