Methylation And Expression Of P16,APC And RARβ In Gastric Cardia Precancerous And Cancerous Lesions From The Same Patient: Comparison With Esophageal Precancerous And Cancerous Lesions From The Same High Incidence Area

1 BACKGROUNDIn epidemiology, gastric cardia adenocarcinoma (GCA) in China is characterized by its striking geographic distraction and together occurrence with esophageal cancer (EC); GCA is quite different in pathogenesis and prevalence with distal gastric cancer: the incidence for distal gastric cancer is decreasing worldwide; in contrast, the prevalence for GCA remains in a high level. In western countries, the incidence for adenocarcinoma at gastro-esophageal junction, including GCA is dramatically increasing, one of the fastest increasing malignant diseases. These phenomenon indicate that there may be similar risk factors and molecular changes involved in GCA and EC. Obviously, to characterize the molecular changes in multistage carcinogenesis of GCA and EC from the same high incidence area is much desirable in elucidating the molecular mechanisms of GCA and EC, identifying molecular biomarkers for high-risk subject screening and early detection for GCA and EC.Linzhou (formerly Linxian) in Henan Province has been well documented as the highest incidence area for both GCA and EC. However, because of the similar symptom (dysphagia), historically, GCA was included into EC; even until 2002, UICC remains not to classify GCA as a single entity. Accumulated data for molecular changes are focused on EC, it is largely unknown for molecular alteration in GCA. Recent studies by us and other laboratories indicate that alterations of tumor suppressor genes p16, APC, RARβare common events in esophageal squamous cell carcinoma (SCC) (p16), and adenocarcinomas at gastro-esophageal junction (p16, APC, RARβ); p16 methylation may play an important role in carcinogenesis of SCC and adenocarcinoma at gastro-esophageal junction. However, the information for methylation and expression changes of p16, APC and RARβin gastric cardia carcinogenesis is very limited, not alone the comparison for these molecular changes between GCA and SCC from the same high-incidence area, which have not been reported. Thus, the present studies were undertaken to highlight the mechanisms of GCA and to identify the molecular biomarkers for high-risk subject screening and early detection for GCA by comparison the methylation and expression of p16, APC and RARβbetween the GCA and SCC from the same high incidence area in Henan, and to characterize these changes with clinical pathology (differentiation, metastasis, invasion and TNM staging).2. MATERIALS AND METHODS2.1 SubjectsGastric cardia: A total of 38 surgically resected human GCA specimens were recruited from Linzhou City Center Hospital and Yaocun Esophageal Cancer Hospital in Linzhou, Henan, the highest incidence area for both GCA and EC, including 28 males with an average age of 59±8 years old and 10 females with an average age of 56±13 years old.Esophagus: All the 45 surgically resected human EC specimens were collected from the same hospitals as above. Of the 45 cases, 31 were males with an average age of 57±6 years old and 14 females with an average age of 54±9 years old.None of the patients had received chemotherapy and/or radiotherapy before operation. All the cases were confirmed as GCA or SCC respectively by histopathology. TNM staging was undertaken based on the criteria by AJCC (2002, AJCC).2.2 Tissue collection and processingWithin 2 hours after surgical excision, the specimens were cut into two-halves longitudinally. Half of the specimen was fixed with 85% ethanol, paraffin-embedded, and sectioned at 5um for histopathological evaluation and immunohistochemical analysis. The other part of the specimens was stored in the liquid nitrogen first and then transferred in a -80℃freezer for RNA, DNA and protein extraction.2.3 Criteria for histopathological examinationWith the criteria established by our research group, the gastric cardia epithelia was classified into normal (NOR), dysplasia (DYS) and GCA; and the esophageal epithelia was classified into NOR, basal cell hyperplasia (BCH), DYS, carcinoma in situ (CIS), SCC based on the cell morphological changes and tissue architecture.2.4 Immunohistochemical analysisAvidin-biotin-peroxidase complex (ABC) method was used to analyze the expression of P16, APC and RARβ. The classification of immunoreactivity was based on the immunostaining intensity (score as 0, 1, 2, 3) and cell proportion of positive immunostaining (score as 0, 1, 2, 3, 4).2.5 Western blotWestern blot method was used for p16 protein detection in gastric cardia precancerous and cancerous lesions.2.6 MSPPhol-chloroform method was used to isolate DNA from tissues. The value of A₂₆₀/A₂₈₀ was used to evaluate the purity and integrity of DNA samples. All of the DNA samples generated the ratios was＞1.8. Methylation-specific PCR (MSP) was undertaken to determine the expression and methylation of P16, APC and RARβin GCA and SCC.2.7 RT-PCRTrizol reagent (GibcoBRL, Life technologies, USA) was used to isolate total RNA from frozen tissue samples according to protocol provided by supplier. The value of A₂₆₀/A₂₈₀ was used to evaluate the purity and integrity of RNA samples. All of the RNA samples generated the ratios between 1.6-1.8. Reverse transcription was performed using 3mg of extracted total RNA mixed with reaction mixture in a final concentration of 0.5mM dNTPs, 0.025mg/mL oligo dT, 5mM MgCl₂, 0.01M DTT, 2U/M1 RNaseOUT^TM inhibitor, and 2.5U/mL SSâ…¡RT. Following RT, PCR was carried out in a reaction volume of 30mL with final concentration of 1.5mM MgCl₂, 0.1mM dNTPs, 0.05Taq DNA polymerase, and primer pair for SCCA1. Beta-actin was used for internal normalization. The reaction was initiated at 94℃for 10min followed by 35cycles at 94℃C for 30s, 55℃for lmin, and 72℃for lmin, and final extension at 72℃for 10min.2.8 StatisticsChi-squared test and Kappa test were performed to compare "the difference between groups.3 RESULTS3.1 Histopathological observationThirty-eight cases of GCA were confirmed as GCA, including 5 cases with well-differentiation, 18 with moderately-differentiation and 15 with poorly-differentiation. Eighty-four foci of gastric cardia cancerous and precancerous lesions were detected, including 33 foci of NOR and 13 foci of DYS. TNM staging: there were 11 cases in stageâ…¡(29%), 21 cases in stageâ…¢(55%), and 6 cases with stageâ…£(16%).Forty-five cases of EC were confirmed as SCC, including 6 cases with welldifferentiation, 32 with moderately-differentiation and 7 with poorly-differentiation. A hundred thirty-five foci of esophageal cancerous and precancerous lesions were detected, including 45 foci of NOR and 45 foci of DYS. TNM staging: there were 15 cases in stageâ… (33%), 16 in stageâ…¡(37%), and 14 in stageâ…¢(30%).3.2 p16 protein expressionGastric cardia: Positive expression rate of p16 in normal gastric cardia epithelial tissue was apparently higher than in GCA tissues (73%, 24/33 vs. 37%, 14/38), and was similar with DYS tissue(62%, 8/13). The difference was significant between NOR and GCA, DYS and GCA (P＜0.05).The rate of p16 in normal esophageal epithelial tissue was apparently higher than in SCC and DYS tissues (74%, 20/27 vs. 44%, 14/32). There was significant difference between NOR and SCC, NOR and DYS, and DYS and SCC(P＜0.05).Comparison for p16 expression in gastric cardia and esophagus: Although decreasing tendency for p16 positive immunostaining rate was observed with the lesions progressed both in GCA and SCC. The decreasing extent for p16 immunostaining rate in esophagus was apparently higher than in gastric cardia; p16 positive rates in GCA and gastric DYS were 4.5 and 5 folds higher than in SCC and esophageal DYS, the difference was significance (P＜0.05). However, P16 positive rate in gastric cardia epithelium was lower than in normal esophageal epithelium tissues (P＜0.05).3.3 p16 MethylationGastric cardia: Methylation rate of p16 gene in GCA (84%, 32/38) was obviously higher than in DYS (69%, 9/13) (P＜0.05). However methylation rate of p16 gene was similar in normal and DYS tissue.Esophagus: Methylation rate of p16 gene in GCA was similar in SCC (73%, 33/45) and DYS tissue (71%, 32/45), which was higher than in normal esophageal epithelium tissue (33%, 15/45). The methylation rates between these lesions was significant (P＜0.05)..Comparison for p16 methylation in gastric cardia and esophagus: In contrast with p16 protein expression, with lesions progressed, an increasing tendency for p16 methylation was observed both in gastric cardia and esophagus, p16 methylation rate in gastric cardia DYS was lower than in esophageal DYS, but the difference was not significant. P16 methylation rate in normal gastric cardia epithelium was higher than in normal esophageal tissue(P＜0.05).3.4 Comparison of p16 promoter methylation and p16 protein expression in different severities of lesions from the same patientGastric cardia: The concordance rate of p16 promoter methylation and p16 negative protein expression in GCA, normal cardia epithelia and DYS was 64% (24/38), 36% (12/33), and 38%(5/13), respectively.Esophagus: The concordance rate of p16 promoter methylation and p16 negative protein expression in SCC, normal esophageal epithelia and DYS was 82% (37/45), 62% (28/45), and 73% (33/45), respectively.3.5 Comparison of p16 promoter methylation and p16 protein and mRNA expressions in different severities of lesions from the same patientPositive expression rate of p16 mRNA in normal cardia epithelia, DYS and GCA was 55% (18/33), 53% (8/15) and 50% (19/38), respectively. No significant difference was observed among them. The concordance rate of p16 promoter methylation, p16 mRNA and p16 protein expression in Western blot and immunohistochemical analysis was only 20% (2/8).3.6 Correlation of p16 protein expression and clinicopahtological parameters in SCC and GCA3.6.1 P16 protein expression and clinicopahtological parameters in GCAP16 positive immunostaining rate increased apparently from poorly-(27%, 4/15) to moderate-(28%, 5/18) and to well-differentiated (100%, 5/5) GCA (P＜0.05). However, no difference was observed for p16 protein expression in lymph node metastasis and tumor invasive depth.3.6.2 Comparison of p16 protein expression and clinicopahtological parameters in SCC.Absent or low p16 expression was correlated with lymph node metastasis (P＜0.05). However, no correlation for p16 protein expression and SCC differentiation and tumor invasive depth was observed.3.7 Correlation of p16 promoter methylation and clinicopahtological parameters in SCC and GCA 3.7.1 Correlation of p16 promoter methylation and clinicopahtological parameters in GCANo correlation was observed between p16 methylation and differentiation lymph node metastasis and tumor invasive depth.3.7.2 Correlation of p16 promoter methylation and clinicopahtological parameters in SCC.P16 promoter methylation rate decreased from moderate-to well-differentiated SCC(P＜0.05). However, no significant difference was observed for lymph node metastasis and tumor invasive depth in p16 promoter methylation.3.8 Promoter methylation of APC and RARβon gastric cardia precancerous and cancerous lesionsPositive expression rate of APC protein in normal cardia epithelia, DYS and GCA was 9% (3/33), 8% (1/13) and 13% (5/38), respectively. Positive expression rate of APC mRNA in normal cardia epithelia, DYS and GCA was 9% (3/33), 8% (1/13) and 5% (2/38), respectively. APC promoter methylation rate in GCA (53%, 17/32) was slightly higher than DYS(36%, 4/11)and normal gastric cardia epithelia (44%, 12/27) without statistical significance(P＞0.05).Positive expression rate of RARβprotein in normal cardia epithelia, DYS and GCAwas 9% (3/33), 8% (1/13) and 16% (6/38), respectively. Positive expression rate of RARβmRNA in normal cardia epithelia, DYS and GCA was 15% (5/33), 15%(2/13) and 13% (5/38), respectively. RARβpromoter methylation rate in GCA (13%,4/32) was slightly higher than DYS (9%, 1/11) and normal gastric cardia epithelia (11%, 3/27) without statistical significance(P＞0.05).4. CONCLUSIONS4.1 With the gastric cardia epithelial lesion progressed from normal to DYS to GCA, an decreasing tendency for p16 protein expression and increasing tendency for p16 methylation were observed, and similar results were observed in esophageal carcinogenesis, which suggest thatâ‘ aberrant p16 methylation and protein expression are early molecular event in gastric cardia and esophageal carcinogenesis; â‘¡p16 promoter methylation may be one of mechanisms for aberrant expression of p16 protein;â‘¢Analogous changes in molecular events indicate the possibility of similar risk factors involved in GCA and SCC.4.2 High consistent alterations of p16 promoter methylation in GCA and DYS, similarly in SCC and esophageal precancerous lesions indicate that p16 promoter methylation may be one of important candidate biomarkers for high-risk subject screening and early detection of GCA and SCC.4.3 Distinct p16 promoter methylation in morphologically similar DYS in esophagus and cardia may be molecular basis for reversibility of precancerous lesions. Promoter methylation and loss of protein expression of p16 may be one of key factors underlying progression of mild or moderate to severe lesions.4.4 The increased p16 protein expression and decreased p16 methylation may be the molecule markers for poor prognosis in GCA and SCC.4.5 The p16 methylation rate of GCA in Chinese people is apparently lower than in adenocarcinoma at gastro-esophageal junction from western countries, suggesting that there are different molecular mechanisms involved in GCA from China and western countries, and GCA in China should be considered as an independent disease.