Wif-1Methylation And β-catenin Expression In Colorectal Serrated Lessions

[Background and purpose]Serrated lesions are a group of colorectal adenoma/polyps characterizedmorphologically by a serrated structure of the crypt epithelium. Due to the adjustmentmechanism of apoptosis is inhibited, epithelial cells continue to accumulate and convexto the glandular, then formate micro-papillary within fold structure, which constitutesthe serrated form. Serrated lesions were divided into hyperplastic polyps, sessileserrated adenoma/polyps and traditional serrated adenoma in WHO classification oftumors of the digestive system(2010). Depending on the type of mucus HPs weredivided into microvesicula HP, goblet cellric HP and mucin poor HP, while according todysplasia SSA/P can be divided into with and without dysplasia. Recent studies hadpointed out that there were two serrated pathways. One called sessile serrated pathway,mainly because of BRAF mutation and developed to MVHP and SSA/P. MLH1gene was silenced by methylation of CpG island promoter region, then cellular atypia ofSSA/P transformated rapidly to malignant lession. Another way was not related toBRAF mutation but K-ras mutation, called traditional serrated pathway, including theGCHP and TSA. Some research considered that MSI-L cancer was caused bymethylation of MGMT gene in TSA. Currently, there are still lack of researches aboutmethylation of other related genes in serrated lesions.Wnt/β-catenin signaling pathway made an important role in regulating cell growth andproliferation, its abnormal activation had been found closely to associate with a varietyof human tumors. Wnt inhibitory factor-1is antagonistic gene in this pathway.Hypermethylation of Wnt inhibitory factor-1in a variety of tumors had been proved toexist, and hypermethylation of the promoter were involved in development of tumors.Methylation rate of Wif-1promoter region was higher in colorectal cancer in literatures,while there are no reports about methylation status of Wif-1gene in colorectal serratedlesions.Methylation status of Wif-1and expression of β-catenin were observed for the first timein each serrated group, to investigate the relationship among methylation of Wif-1,expression of β-catenin and cancerous pathway of serrated lessions through comparingeach serrated group with CRC and normal colon tissue. And to explore the role ofassistant diagnosis of serrated lesions with detecting methylation of Wif-1gene.[Method]A total of4417cases of colorectal polyps from The Military General Hospital of BeijingPLA during a five-year period were retrospectively reviewed. The serrated lesions wereclassified based on WHO(2010) standards. Amongst4417colorectal polyps studied,586cases of serrated lesions were found. From these serrated lesions, we selected52cases of HP,41cases of SSA and23cases of TSA, while selected24cases of colorectalcancer and24cases of normal mucosa as controls for immunohistochemical staining ofβ-catenin. SYBR Green PCR was used to examine the Wif-1promoter methylation in29cases of HP,29cases of SSA,19cases of TSA,24cases of colorectal cancer and24cases of nomal mucosa from each group. [Results]The abnormal positive rate of β-catenin was gradually increased in normal colorectalmucosa(12.5%),HP(59.6%),SSA(63.4%),TSA(73.9%) and CRC group(100%), therewere significant differences between HP,SSA,TSA and normal colorectal mucosa,CRCgroup. The methylation rate of Wif-1gene promoter were gradually increased innormal colorectal mucosa(12.5%),HP(34.5%),SSA(55.2%),TSA(78.9%) and CRCgroup(92.9%) as well. The rate was lower in HP(34.5%),SSA group(55.2%) than inCRC group(92.9%), while higher in SSA(55.2%),TSA group(78.9%) than in normalcolorectal mucosa(12.5%) and lower in HP group(34.5%) than in TSA(78.9%). Thesensitivity of SYBR Green PCR was53.2%, while the specificity was87.5%indetecting Wif-1methylation of serrated lesions. Abnormal positive rate of β-catenin andmethylation rate of Wif-1promoter were significantly correlated in serrated lessionsespecially in serrated adenomas.[Conclusion]1Immunohistochemistry results showed that abnormal positive rate of β-catenin wassignificant higher in serrated lesions, suggesting that β-catenin as a key of Wntsignaling pathway may be involved in progress of serrated cancerous.2SYBR Green PCR results showed that methylation rate of Wif-1gene was significanthigher in serrated lesions, suggesting that methylation of promoter region could activateWnt/β-catenin signaling pathway, may play an important role in development of SSA,progress of HP and occurrence of TSA. Methylation of Wif-1gene may be a molecularmarker, and detecting methylation status of Wif-1is expected to become one ofdiagnosis tool in serrated lesions.3There were significant relationships between methylation of Wif-1gene andexpression of β-catenin in serrated adenoma, suggesting that abnormal expression ofβ-catenin may be caused by methylation of Wif-1promoter. [Background and purpose]The occurrence of colorectal cancer is rising, which seriously threat to human health.Recent studies suggested that60%of colon cancers came from ordinary adenomas,35%from the serrated adenomas, while5%from Lynch syndrome. Serrated lesions are agroup of colorectal adenoma/polyps characterized morphologically by a serratedstructure of the crypt epithelium. Due to the adjustment mechanism of apoptosis isinhibited, epithelial cells continue to accumulate and convex to the glandular, thenformate micro-papillary within fold structure, which constitutes the serrated form.Serrated lesions were divided into hyperplastic polyps, sessile serrated adenoma/polypsand traditional serrated adenoma in WHO classification of tumors of the digestivesystem(2010). Depending on the type of mucus HPs were divided into microvesicula HP,goblet cellric HP and mucin poor HP.In recent years, in addition to the classic adenoma-carcinoma pathway, hyperplasticpolyps-serrated adenoma-adenocarcinoma pathway has been accepted gradually. Thereare two ways in the serrated pathway, one is that MVHP may be precancerous lesions ofSSA/P, because of high microsatellite instability (MSI-H) by BRAF mutations andMLH1methylation, then progress to cancer. The other is that GCHP as well as TSAcome from low microsatellite instability (MSI-L) through K-ras mutation and MGMTmethylation, then develop to cancer. The occurrence and progress of each HPs group isnot yet clear, still need more researches.The clinicopathological features of each HPs group and the expression of a set of colorectal cancer-related protein (including RUNX3of signal transduction, β-catenin ofWnt signaling pathway, p16of cell cycle regulation factor, MLH-1of mismatch repairgene, MGMT of DNA damage repair gene, CDX2of gastrointestinal embryonic geneticpathway and proliferation index Ki-67) in52cases of HPs were observed. To generalizethe clinicopathological diagnostic points of HPs and explore relationship betweenexpression of each antibody and occurrence as well as development of HPs, and screenhelpful antibodies in diagnosis of HPs.[Method]A total of4417cases of colorectal polyps from The Military General Hospital of BeijingPLA during a five-year period were retrospectively reviewed. The serrated lesions wereclassified based on WHO(2010) standards. Amongst4417colorectal polyps studied,586cases of serrated lesions were found. From these serrated lesions, a total of207cases of HPs were screened, including157cases of MVHP,48cases of GCHP and2cases of MPHP. The clinicopathological features of52cases of HP, including38casesof MVHP,12cases of GCHP and2cases of MPHP from above groups were obversed.Combined with41cases of SSA,23cases of TSA,24cases of nomal tissue and24casesof adenocarcinoma in colorectum, they were compared by immunohistochemical stain.The specimens were fixed in10%neutral formalin, embedded in paraffin, sliced4μmthick continuously and stained of HE. While immunohistochemistry with MaxVisiontwo-step method.[Results]HPs accounted for35.3%(207/586) of collected serrated lesions,40.6%(84/207)occurred in the left colon approximately, the diameter ranged from0.1to0.3cm. HPscan be divided into MVHP,GCHP and MPHP, mainly characterized by elongation of thecrypts with prominent serrations. The crypts were straight and proliferation was locatedin the lower third of the crypts, with serration developing in the more luminal aspects.The bases of the crypts were narrow and lined with undifferentiated cells or withinterspersed neuroendocrine cells. Immunohistochemical results showed that theexpression differences among HPs and the others had statistic significance in RUNX3,β-catenin,p16and Ki-67. MPHP had higher proliferative activity, and positiverate of MLH1in MVHP significantly differed from in SSA.[Conclusion]1HP is common, occurs in the left colon, often has a small size.2The morphological observations mainly characterized by elongation of the crypts withprominent serrations. The crypts were straight and proliferation was located in the lowerthird of the crypts. The bases of the crypts were narrow and lined with undifferentiatedcells or with interspersed neuroendocrine cells.3Immunohistochemistry results showed that abnormal expression of RUNX3,p16andβ-catenin may contribute to the diagnosis of HPs, while MPHP may have malignantpotential, should get the attention of clinicians and strengthen the follow-upmanagement of these patients. And MVHP may not be the precancerous lesion of SSA.