INK4/ARF and gastric carcinogenesis

Gastric cancer (GC): general aspects

GC remains an aggressive disease that has a huge impact on global health. In the last few decades, the overall incidence of GC has been decreasing, but it still remains the fourth most common type of cancer and is the second leading cause of cancer-related death worldwide. The decline in its incidence is due to the use of antibiotics, changes in the consumption of chilled foods, increased use of preventive examinations and the eradication of H. pylori (1,2).

The incidence rates can vary by up to ten times around the world. Although Japan and Korea have the highest GC rates in the world, nearly two-thirds of stomach cancers occur in developing countries. High incidence areas include East Asia, Eastern Europe and South and Central America. Low incidence areas are found in South Asia, North America, East Africa, Australia and New Zealand (3).

One reason for this variation is the complex etiology dependent on a combination of environmental, host and genetic factors (4,5). One should emphasize the production of reactive oxygen species that leads to oxidative damage to DNA, mutations and aberrant DNA methylation (mainly hypermethylation of CpG islands in the promoter regions of certain genes), which results in the silencing of the tumor suppressor gene. In general, the prognosis for patients with gastric adenocarcinoma remains poor, and little progress has been made in improving the long-term survival rate (6,7).

Lauren (8) proposed the most used and widespread GC classification. He distinguishes between two main types of gastric carcinoma based on histological features: intestinal (well differentiated) and diffuse (poorly differentiated). Depending on the classification criteria and population studied, the proportion of intestinal and diffuse cancer varies (9-12). This classification is widely and routinely used by pathologists, epidemiologists and clinicians for the evaluation of gastric adenocarcinoma, particularly with respect to their incidence and etiology, although this is with limited value in relation to therapeutic decisions (13).

The intestinal form is composed of malignant cells that are bound to form structures similar to functional glands of the gastrointestinal tract. Its development has been characterized by a series of sequential steps that begin with gastritis, which ultimately leads to mucosal atrophy (atrophic gastritis) followed by intestinal metaplasia, dysplasia, carcinoma, and subsequent metastatic spread (14). This pattern reflects a more differentiated cancer, and is the most common type in high-risk populations (15,16).

The diffuse subtype of GC is considered more aggressive than the intestinal type, is originated from native cells of the gastric mucosa and tends to be poorly differentiated (17). Despite being associated with an H. pylori infection, it is more associated with the loss of E-cadherin expression and, unlike the intestinal type, is not associated with any pre-neoplastic lesions. Due to its aggressive characteristics, the diffuse type can invade surrounding tissues and organs, for example, the duodenum and esophagus (2).

Genetic abnormalities involved in the pathogenesis and progression of gastric carcinoma were identified, and several alterations were correlated with the prognosis. Molecular mechanisms, such as changes in oncogenes, tumor suppressor genes, and cell adhesion molecules, are involved in gastric carcinogenesis. Moreover, genetic instability and changes in cytokines and growth factors contribute to the complexity of pathways involved in gastric carcinogenesis (14,18).

Among these genetic abnormalities, the loss of the functions of genes encoded by the INK4-ARF locus occurs frequently in cancers, which raises questions about the alterations in biochemical pathways of these proteins when transformed into cancer cells (19,20).

INK4/ARF locus

The INK4/ARF locus is located on the human chromosome 9p21, spans approximately 35 kilobases and encodes five types of tumor suppressor genes: p16^INK4A, p15^INK4B, p14^ARF (P19^ARF in the mouse), p16^INK4Aγ and P12 (21-25): the physiological functions of some of these remain unknown (26). Additionally, it is known that this locus has the CDKN2BAS gene (also known as CDKN2b anti-sense), which is responsible for producing a noncoding antisense RNA named ANRIL, which plays a regulatory function on CDKN2A and CDKN2B expression (27,28).

The most studied proteins produced by this locus (p16^INK4A, p15^INK4B and p14^ARF) interact with other proteins that activate two critical anti-proliferative pathways (Rb and p53 pathways) which play important roles in cell cycle inhibition, senescence, and stress-induced apoptosis (29). Functionally, both p16^INK4A and p15^INK4B bind specifically to CDK4 and CDK6, inhibiting the activity of cyclin D-dependent kinases, consequently blocking the cell proliferation by preventing phosphorylation of Rb, resulting in a G1 arrest (30,31). p14^ARF, encoded in part by an alternative reading frame within the second of three exons that comprise the INK4A gene, acts primarily by inhibiting murine double minute 2 (MDM 2)-mediated ubiquitination and degradation of p53, triggering p53-dependent cell cycle arrest and apoptosis (30).

Given the central role in tumor suppression of INK4-ARF in modulating activities of Rb and p53 pathways, it is not surprising that genetic and epigenetic changes in this locus are frequently detected in the majority of tumor types (29,32).

Some studies have shown that the INK4/ARF locus is tightly controlled, and polycomb group complexes are required to initiate and maintain the silenced state (29,33,34). The polycomb repressive complex 1 (PRC1) proteins (BMI1, PCGF1, PCGF2/MEL18, CBX2, CBX7, CBX8, and RING1B), and the PRC2 proteins (EED, SUZ12, and EZH2) have been shown to directly bind to and repress the locus (33,35-37).

Genetic alterations in INK4/ARF in gastric carcinoma

Genomic instability

Genomic instability is considered a hallmark of human cancers (38), which results in several genetic aberrations, from gene (changes in a single nucleotide) to structural levels (structural changes, losses and gains of entire chromosomes) (38,39). Therefore, these aberrations can affect the expression of tumor suppressor genes, oncogenes, DNA repair genes (genome stability genes), growth regulators, and cell cycle checkpoint control genes (40). Currently, the genomic instability can be classified into two types: chromosomal instability (CIN) and microsatellite instability (MSI) (39).

CIN and MSI

CIN is characteristic of various tumors, including GCs, which commonly are associated with chromosomal aberrations responsible for major modifications of DNA content, i.e., changes in chromosome copy numbers, high-level loss of heterozygosity (LOH), and gene deletions and/or amplifications (41). Most GCs exhibit significant CIN at several chromosome arms, including 9p (41-44); these instabilities have been associated with a shorter survival in GC patients (45).

Array-based comparative genomic hybridization (aCGH) is a powerful method used to identify alterations of DNA copy number changes on a genome-wide scale (46). This method has been applied to a number of solid tumors, including GCs (47,48), revealing several regions of consensus change in DNA copy numbers, indicating the possible location of candidate oncogenes or tumor suppressor genes involved in gastric tumorigenesis (49). In particular, the chromosome 9p21.3, which has the tumor suppressor genes p16^INK4A and p15^INK4B, is known to be deleted frequently in GCs, particularly in undifferentiated GC (50-52).

Fan et al. (52) explored aCGH profiles of 64 human GC samples and eight GC cell lines using bacterial artificial chromosomes (BAC). They found out that the most frequent homozygous deleted region was 9p21 (8/72), and that homozygous deleted regions were higher in the cell lines than in the primary GC tumor samples. Weiss et al. (48) similarly observed that the loss of 9p21.3 was present in 29% of gastric adenocarcinoma.

Although many studies have investigated mutations or deletions of genes in 9p21, there has been no precise characterization of the break-points. Lee et al. (50) analyzed the entire set of large homozygous deletions in six human GC cell lines (SNU-1, SNU-5, SNU-16, SNU-520, SNU-638, and SNU-668) through genome and transcriptome approaches, and defined 9p21.3 homozygous deletions precisely. Moreover, they investigated the effect of the 9p21.3 deletions on gene expression by transcriptome sequencing, finding that no genes within the 9p21.3 deletion region were expressed in SNU-16 and SNU-668 compared to other GC cell lines analyzed.

Some studies recognize that the loss of chromosome 9p has been reported to occur more frequently in cases of GCs that are relapsed or histologically malignant (53,54).

In the literature, there are few studies reporting alterations in the p14^ARF gene. Tang et al. (55) showed that homozygous deletion of p16^INK4A and p14^ARF was present in 35.4% of the GC cases analyzed, and the loss rate of p14^ARF mRNA was 45.8% (22/48) in GC. When analyzing 11 cell lines for mRNA expression, homozygous deletion, mutation, and promoter methylation, Iida et al. (56) did not find any mutation in the whole coding region, but demonstrated that the p14^ARF gene was more frequently inactivated by homozygous deletion or methylation in diffuse-type GC cell lines (5/7, 71.4%) than in intestinal ones; thus suggesting that p14^ARF may be involved in the tumorigenic process in diffuse-type GCs.

Another type of genomic instability, commonly recognized in GC, is MSI. In GC tissues, MSI was a frequent event, with an average frequency varying from 25% to 33.9% (57-59). Zhang et al. (60) showed that the LOH frequencies of D9S171 and D9S1604 microsatellite loci in GC, located upstream of the p16^INK4A gene, were 15% and 50%, respectively. Moreover, the LOH frequency in well-differentiated GC tissue was lower than in the moderately and poorly differentiated GC tissue without any significant difference (P>0.05).

Mutations and polymorphisms

The number of studies linking genetic polymorphisms and GC has increased exponentially over the past decades, in parallel with major advances in sequencing and genotyping, resulting in the identification of polymorphisms that may be useful indicators for assessing the risk of GC (39,61). However, it is worth noting that the results derived from polymorphism studies still need to be carefully interpreted, as these biomarkers are generally population dependent, with a strong ethnic influence (39).

Li et al. (62), studying 9p21 single nucleotide polymorphisms (SNPs) from eight genome-wide association studies (GWAS), including studies of esophageal squamous cell carcinoma (ESCC), GC, pancreatic cancer, renal cell carcinoma (RCC), lung cancer (LC), breast cancer (BrC), bladder cancer (BC) and prostate cancer (PrC), identified that the SNP on rs3731239 (p16^INK4A intronic) was significantly associated with an increased risk of GC, and that it overlapped with weak but potential enhancers, leading them to suggest that SNPs in the CDKN2A/2B-AS1/2B cluster may modulate disease susceptibility, primarily through regulating expression levels of genes in the cluster.

Kim et al. (63) investigated the importance of p16^INK4A and p15^INK4B mutations in four stomach cancer cell lines and 14 stomach adenocarcinomas. They detected mutations in both genes only in stomach cancer cell lines; however, in the SNU5 cell line, they discovered a nonsense mutation (CGA→TGA/Arg→Stop) at codon 72 of the CDKN2 gene. The absence of mutations in the p16^INK4A and p15^INK4B genes in stomach cancer tissues suggests that mutations in both genes may not be a critical genetic change in GC pathways.

INK4/ARF methylation and GC

Methylation is an important characteristic of DNA and is responsible for events such as gene regulation, X chromosome inactivation, aging and cancer. Several studies have identified methylation contribution to both oncogene activation and silencing of tumor suppressor genes in the development of cancers (64).

The inactivation of the p16^INK4A gene by hypermethylation has been regarded as an important mechanism for initiation and development of GC (65). It is widely accepted that gene promoter methylation can repress gene expression at the transcriptional level, promoting the inactivation of tumor suppressor genes, leading to the development of malignant tumors (66).

The hypermethylation of p16^INK4A may also be an important biomarker for identifying the possible malignant potential of precancerous lesions. In a study by Sun et al. (65), for the first time, a positive correlation between the aberrant methylation of the p16^INK4A gene with the malignant transformation of gastric dysplasia was observed. This methylation pattern was also present in all of the GC samples that progressed from methylated gastric dysplasia lesions.

Iida et al. (56), using cell lines of the Human Science Research bank (Osaka, Japan), 7 (21.7%) of 32 samples of diffuse type, and 6 (21.4%) of 28 samples of intestinal type GC showed methylation of the p16INK4A gene, showing the lack of correlation between the methylation of this gene and the histological type.

In their experiment, Song et al. (64) analyzed the methylation profile of the p16^INK4A gene in 322 samples from patients diagnosed with GC in Huizhou Municipal Central Hospital of Guangdong (China) and reported that 75% (242/322) were hypermethylated. Unfortunately, no comparison with the histological type was performed; however, it is important to highlight the association between DNA hypermethylation with MTHRF polymorphisms C677T and C1298A evidenced in this study.

Mino et al. (67) studied 35 patients with GC at Osaka City University Hospital, and observed the hypermethylation of the p16^INK4A gene in 12/35 (34.3%) cancerous tissues: a frequency significantly higher than that of non-cancerous tissues. Although no relationship was observed between p16^INK4A promoter hypermethylation and clinicopathologic characteristics, it seems that this event must be considered a biomarker of gastric carcinogenesis. This was also observed by do Nascimento Borges et al. (18) in a Brazilian population.

Finally, in a meta-analysis performed by Peng et al. (68), nine clinical trials with 487 GC patients and 271 healthy controls showed that the methylation rate of the p16^INK4A gene in GC was significantly higher than in the healthy control, showing that the p16^INK4A gene promoter array is a useful method for the diagnosis of GC.

The loss of function of p16 protein results in increasing activity in the corresponding kinase, leading to the phosphorylation of the pRb protein and resulting in the loss of cell cycle control. The transcriptional inactivation due to methylation of the promoter may also be linked to tumor cell proliferation and neovascularization (69).

Some evidence points to a close relationship between MSI and the aberrant promoter methylation of the p16^INK4A gene (70,71). Shim et al. (70) found that 13/21 (61.9%) tumors that were MSI positive had the p16^INK4A promoter methylation, as opposed to 24/67 (35.8%) MSI negative tumors. These results suggest that MSI was associated not with a p16 protein loss, but rather with the methylation status of the gene.

There are few reports in the literature regarding the p15^INK4B gene methylation profile in GC. do Nascimento Borges et al. (18) analyzed 35 tumoral and 37 non-tumoral samples from a Brazilian population and found a high methylation frequency in the promoter region (25.9% and 27.3%, respectively). Interestingly, a correlation was noticed between patients under 60 years old and p15^INK4B hypermethylation, suggesting a relationship between p15^INK4B hypermethylation and the aging process.

Lee et al. (72) analyzed the p15 methylation pattern in 54 patients with gastric adenocarcinoma and observed 68.5% with promoter methylation, which is a frequency similar to that obtained from a patient’s serum (55.65%).

Promoter methylation of the p14^ARF gene has been found in a great number of cancers including GCs (56,73-75).

Tsujimoto et al. (76) found a positive correlation between p14^ARF promoter methylation and GC. Regarding the difference between histological types, p14^ARF methylation was more frequently found in the early stages of intestinal type cancers and in the advanced stages of cancers of the diffuse type. These results are in agreement with those found by Iida et al. (56), who investigated the methylation status of p14^ARF in 11 GC cell lines and 62 primary GCs, and found hypermethylation patterns in 9% and 35%, respectively, leading to the hypothesis that p14^ARF methylation might also occur as an early event in a fraction of intestinal type cancers.

Concluding remarks

INK4/ARF, especially 9p21 deletion and p16^INK4A promoter hypermethylation, may be used as a marker of gastric carcinogenesis, whereas mutations and polymorphisms should be carefully used, as they have a strong ethnic influence.

Acknowledgements

None.

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

References

1. Yan WF, Sun PC, Nie CF, et al. Cyclooxygenase-2 polymorphisms were associated with the risk of gastric cancer: evidence from a meta-analysis based on case-control studies. Tumour Biol 2013;34:3323-30. [PubMed]
2. Carcas LP. Gastric cancer review. J Carcinog 2014;13:14. [PubMed]
3. Crew KD, Neugut AI. Epidemiology of gastric cancer. World J Gastroenterol 2006;12:354-62. [PubMed]
4. Saukkonen K, Rintahaka J, Sivula A, et al. Cyclooxygenase-2 and gastric carcinogenesis. APMIS 2003;111:915-25. [PubMed]
5. Lochhead P, El-Omar EM. Gastric cancer. Br Med Bull 2008;85:87-100. [PubMed]
6. Piazuelo MB, Correa P. Gastric cáncer: Overview. Colomb Med (Cali) 2013;44:192-201. [PubMed]
7. Zheng L, Wang L, Ajani J, et al. Molecular basis of gastric cancer development and progression. Gastric Cancer 2004;7:61-77. [PubMed]
8. Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. an attempt at a histo-clinical classification. Acta Pathol Microbiol Scand 1965;64:31-49. [PubMed]
9. Stemmermann GN, Nomura AM, Kolonel LN, et al. Gastric carcinoma: pathology findings in a multiethnic population. Cancer 2002;95:744-50. [PubMed]
10. Vauhkonen M, Vauhkonen H, Sipponen P. Pathology and molecular biology of gastric cancer. Best Pract Res Clin Gastroenterol 2006;20:651-74. [PubMed]
11. iazuelo MB, Epplein M, Correa P. Gastric cancer: an infectious disease. Infect Dis Clin North Am 2010;24:853-69, vii. [PubMed]
12. Borges Bdo N, Santos Eda S, Bastos CE, et al. Promoter polymorphisms and methylation of E-cadherin (CDH1) and KIT in gastric cancer patients from northern Brazil. Anticancer Res 2010;30:2225-33. [PubMed]
13. Yakirevich E, Resnick MB. Pathology of gastric cancer and its precursor lesions. Gastroenterol Clin North Am 2013;42:261-84. [PubMed]
14. Smith MG, Hold GL, Tahara E, et al. Cellular and molecular aspects of gastric cancer. World J Gastroenterol 2006;12:2979-90. [PubMed]
15. Shibata N, Watari J, Fujiya M, et al. Cell kinetics and genetic instabilities in differentiated type early gastric cancers with different mucin phenotype. Hum Pathol 2003;34:32-40. [PubMed]
16. Grabsch HI, Tan P. Gastric cancer pathology and underlying molecular mechanisms. Dig Surg 2013;30:150-8. [PubMed]
17. Dicken BJ, Bigam DL, Cass C, et al. Gastric adenocarcinoma: review and considerations for future directions. Ann Surg 2005;241:27-39. [PubMed]
18. do Nascimento Borges B, Burbano RM, Harada ML. Analysis of the methylation patterns of the p16 INK4A, p15 INK4B, and APC genes in gastric adenocarcinoma patients from a Brazilian population. Tumour Biol 2013;34:2127-33. [PubMed]
19. Lowe SW, Sherr CJ. Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 2003;13:77-83. [PubMed]
20. Simboeck E, Ribeiro JD, Teichmann S, et al. Epigenetics and senescence: learning from the INK4-ARF locus. Biochem Pharmacol 2011;82:1361-70. [PubMed]
21. Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436-40. [PubMed]
22. Hannon GJ, Beach D. p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. Nature 1994;371:257-61. [PubMed]
23. Mao L, Merlo A, Bedi G, et al. A novel p16INK4A transcript. Cancer Res 1995;55:2995-7. [PubMed]
24. Lin YC, Diccianni MB, Kim Y, et al. Human p16gamma, a novel transcriptional variant of p16(INK4A), coexpresses with p16(INK4A) in cancer cells and inhibits cell-cycle progression. Oncogene 2007;26:7017-27. [PubMed]
25. Robertson KD, Jones PA. Tissue-specific alternative splicing in the human INK4a/ARF cell cycle regulatory locus. Oncogene 1999;18:3810-20. [PubMed]
26. Li J, Poi MJ, Tsai MD. Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer. Biochemistry 2011;50:5566-82. [PubMed]
27. Cunnington MS, Keavney B. Genetic mechanisms mediating atherosclerosis susceptibility at the chromosome 9p21 locus. Curr Atheroscler Rep 2011;13:193-201. [PubMed]
28. Pasmant E, Laurendeau I, Héron D, et al. Characterization of a germ-line deletion, including the entire INK4/ARF locus, in a melanoma-neural system tumor family: identification of ANRIL, an antisense noncoding RNA whose expression coclusters with ARF. Cancer Res 2007;67:3963-9. [PubMed]
29. Gil J, Peters G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 2006;7:667-77. [PubMed]
30. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13:1501-12. [PubMed]
31. Sherr CJ. Parsing Ink4a/Arf: "pure" p16-null mice. Cell 2001;106:531-4. [PubMed]
32. Sherr CJ. Ink4-Arf locus in cancer and aging. Wiley Interdiscip Rev Dev Biol 2012;1:731-41. [PubMed]
33. Aguilo F, Zhou MM, Walsh MJ. Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression. Cancer Res 2011;71:5365-9. [PubMed]
34. Popov N, Gil J. Epigenetic regulation of the INK4b-ARF-INK4a locus: in sickness and in health. Epigenetics 2010;5:685-90. [PubMed]
35. Bernard D, Martinez-Leal JF, Rizzo S, et al. CBX7 controls the growth of normal and tumor-derived prostate cells by repressing the Ink4a/Arf locus. Oncogene 2005;24:5543-51. [PubMed]
36. Gil J, Bernard D, Martínez D, et al. Polycomb CBX7 has a unifying role in cellular lifespan. Nat Cell Biol 2004;6:67-72. [PubMed]
37. Maertens GN, El Messaoudi-Aubert S, Racek T, et al. Several distinct polycomb complexes regulate and co-localize on the INK4a tumor suppressor locus. PLoS One 2009;4:e6380. [PubMed]
38. Negrini S, Gorgoulis VG, Halazonetis TD. Genomic instability--an evolving hallmark of cancer. Nat Rev Mol Cell Biol 2010;11:220-8. [PubMed]
39. Pinheiro Ddo R, Ferreira WA, Barros MB, et al. Perspectives on new biomarkers in gastric cancer: diagnostic and prognostic applications. World J Gastroenterol 2014;20:11574-85. [PubMed]
40. Albertson DG, Pinkel D. Genomic microarrays in human genetic disease and cancer. Hum Mol Genet 2003;12:R145-52. [PubMed]
41. Nobili S, Bruno L, Landini I, et al. Genomic and genetic alterations influence the progression of gastric cancer. World J Gastroenterol 2011;17:290-9. [PubMed]
42. Buffart TE, van Grieken NC, Tijssen M, et al. High resolution analysis of DNA copy-number aberrations of chromosomes 8, 13, and 20 in gastric cancers. Virchows Arch 2009;455:213-23. [PubMed]
43. Kimura Y, Noguchi T, Kawahara K, et al. Genetic alterations in 102 primary gastric cancers by comparative genomic hybridization: gain of 20q and loss of 18q are associated with tumor progression. Mod Pathol 2004;17:1328-37. [PubMed]
44. Panani AD. Cytogenetic and molecular aspects of gastric cancer: clinical implications. Cancer Lett 2008;266:99-115. [PubMed]
45. Suzuki K, Ohnami S, Tanabe C, et al. The genomic damage estimated by arbitrarily primed PCR DNA fingerprinting is useful for the prognosis of gastric cancer. Gastroenterology 2003;125:1330-40. [PubMed]
46. Kallioniemi A. CGH microarrays and cancer. Curr Opin Biotechnol 2008;19:36-40. [PubMed]
47. Gorringe KL, Boussioutas A, Bowtell DD. Novel regions of chromosomal amplification at 6p21, 5p13, and 12q14 in gastric cancer identified by array comparative genomic hybridization. Genes Chromosomes Cancer 2005;42:247-59. [PubMed]
48. Weiss MM, Kuipers EJ, Postma C, et al. Genomic alterations in primary gastric adenocarcinomas correlate with clinicopathological characteristics and survival. Cell Oncol 2004;26:307-17. [PubMed]
49. Uppal DS, Powell SM. Genetics/genomics/proteomics of gastric adenocarcinoma. Gastroenterol Clin North Am 2013;42:241-60. [PubMed]
50. Lee B, Yoon K, Lee S, et al. Homozygous deletions at 3p22, 5p14, 6q15, and 9p21 result in aberrant expression of tumor suppressor genes in gastric cancer. Genes Chromosomes Cancer 2015;54:142-55. [PubMed]
51. Takada H, Imoto I, Tsuda H, et al. Screening of DNA copy-number aberrations in gastric cancer cell lines by array-based comparative genomic hybridization. Cancer Sci 2005;96:100-10. [PubMed]
52. Fan B, Dachrut S, Coral H, et al. Integration of DNA copy number alterations and transcriptional expression analysis in human gastric cancer. PLoS One 2012;7:e29824. [PubMed]
53. Choi SW, Choi JR, Chung YJ, et al. Prognostic implications of microsatellite genotypes in gastric carcinoma. Int J Cancer 2000;89:378-83. [PubMed]
54. Kang JU, Kang JJ, Kwon KC, et al. Genetic alterations in primary gastric carcinomas correlated with clinicopathological variables by array comparative genomic hybridization. J Korean Med Sci 2006;21:656-65. [PubMed]
55. Tang S, Luo H, Yu J, et al. Relationship between alterations of p16(INK4a) and p14(ARF) genes of CDKN2A locus and gastric carcinogenesis. Chin Med J (Engl) 2003;116:1083-7. [PubMed]
56. Iida S, Akiyama Y, Nakajima T, et al. Alterations and hypermethylation of the p14(ARF) gene in gastric cancer. Int J Cancer 2000;87:654-8. [PubMed]
57. Wang Y, Ke Y, Ning T, et al. Studies of microsatellite instability in Chinese gastric cancer tissues. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 1998;15:155-7. [PubMed]
58. Fang DC, Zhou XD, Luo YH, et al. Microsatellite instability and the loss of heterologous of tumor suppressor gene in gastric cancer. Shijie Huaren Xiaohua Zazhi 1999;7:479-81.
59. Fang DC, Luo YH, Yang SM, et al. Mutation analysis of APC gene in gastric cancer with microsatellite instability. World J Gastroenterol 2002;8:787-91. [PubMed]
60. Zhang QX, Ding Y, Le XP, et al. Studies on microsatellite instability in p16 gene and expression of hMSH2 mRNA in human gastric cancer tissues. World J Gastroenterol 2003;9:437-41. [PubMed]
61. Loh M, Koh KX, Yeo BH, et al. Meta-analysis of genetic polymorphisms and gastric cancer risk: variability in associations according to race. Eur J Cancer 2009;45:2562-8. [PubMed]
62. Li WQ, Pfeiffer RM, Hyland PL, et al. Genetic polymorphisms in the 9p21 region associated with risk of multiple cancers. Carcinogenesis 2014;35:2698-705. [PubMed]
63. Kim JR, Kim SY, Lee BH, et al. Mutations of CDKN2 (MTS1/p16(INK4A)) and MTS2/p15(INK4B) genes in human stomach, hepatocellular, and cholangio-carcinomas. Experimental & Molecular Medicine 1997;29:151-6.
64. Song B, Ai J, Kong X, et al. Aberrant DNA Methylation of P16, MGMT, and hMLH1 Genes in Combination with MTHFR C677T Genetic Polymorphism in gastric cancer. Pak J Med Sci 2013;29:1338-43. [PubMed]
65. Sun Y, Deng D, You WC, et al. Methylation of p16 CpG islands associated with malignant transformation of gastric dysplasia in a population-based study. Clin Cancer Res 2004;10:5087-93. [PubMed]
66. Jeong YJ, Jeong HY, Lee SM, et al. Promoter methylation status of the FHIT gene and Fhit expression: association with HER2/neu status in breast cancer patients. Oncol Rep 2013;30:2270-8. [PubMed]
67. Mino A, Onoda N, Yashiro M, et al. Frequent p16 CpG island hypermethylation in primary remnant gastric cancer suggesting an independent carcinogenic pathway. Oncol Rep 2006;15:615-20. [PubMed]
68. Peng D, Zhang H, Sun G. The relationship between P16 gene promoter methylation and gastric cancer: a meta-analysis based on Chinese patients. J Cancer Res Ther 2014;10 Suppl:292-5. [PubMed]
69. Wang HL, Zhou PY, Liu P, et al. Role of p16 gene promoter methylation in gastric carcinogenesis: a meta-analysis. Mol Biol Rep 2014;41:4481-92. [PubMed]
70. Shim YH, Kang GH, Ro JY. Correlation of p16 hypermethylation with p16 protein loss in sporadic gastric carcinomas. Lab Invest 2000;80:689-95. [PubMed]
71. Suzuki H, Itoh F, Toyota M, et al. Distinct methylation pattern and microsatellite instability in sporadic gastric cancer. Int J Cancer 1999;83:309-13. [PubMed]
72. Lee TL, Leung WK, Chan MW, et al. Detection of gene promoter hypermethylation in the tumor and serum of patients with gastric carcinoma. Clin Cancer Res 2002;8:1761-6. [PubMed]
73. Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 1995;55:4525-30. [PubMed]
74. Esteller M, Tortola S, Toyota M, et al. Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status. Cancer Res 2000;60:129-33. [PubMed]
75. Song SH, Jong HS, Choi HH, et al. Methylation of specific CpG sites in the promoter region could significantly down-regulate p16(INK4a) expression in gastric adenocarcinoma. Int J Cancer 2000;87:236-40. [PubMed]
76. Tsujimoto H, Hagiwara A, Sugihara H, et al. Promoter methylations of p16INK4a and p14ARF genes in early and advanced gastric cancer. Correlations of the modes of their occurrence with histologic type. Pathol Res Pract 2002;198:785-94. [PubMed]