LAMN, and serrated lesion are two major appendiceal premalignant lesions, they morphologically resemble each other. Sometimes, when the lesion is small and is not as typical, it is difficult to distinguish them. We try to look for their differences from the immunohistochemical and genetic point of view.
Torlakovic and his colleagues used CK20 and Ki-67 immunohistochemistry to appreciated the aberrant maturation and proliferation of the crypts in serrated lesions[2]. In a large proportion of serrated lesions in our study, scattered CK20 positivity in the deep crypt was observed, which wasn’t different from Torlakovic's findings. This phenomenon was also observed by Andrew M. Bellizzi et al[9]. Of note, a significantly smaller fraction of LAMN also showed this staining pattern, which has not been previously reported. So, CK20 positivity in the deep crypt was charatrastic of serrated lesions in appendix.
In the present study, 6 in 13 of LAMN cases showed P53 scattered positive in deep crypt.This rate was relatively higher than results of Hara et all[10], Besides, all the P53 positive LAMNs patients were female and had concomitant PMP, which was in concordance with existing literatures, P53 over-expression in PMP of appendiceal origin was significantly related to female sex, spreading to the abdominal cavity, and worse survival for patient[11].In serrated lesions, P53 were all negative, even in cases with epithelial dysplasia, this result was the same as Yantiss et all[12], only 1 serrated adenoma showed more than 10% of the surface epithelium positive. This may imply that serrated lesions in appendix was mostly low grade, the reason might be they lack of mucus secretion and lower p53 expression.
Ki-67 was mostly expressed in deep crypt of LAMN (9/13) and serrated lesions (11/12), while four LAMNs and one serrated lesions have Ki-67 staining in surface epithelium as in conventional colon adenomas.
Different types of mucins expression with varying strength has been reported in serrated lesions and adenomas of the colon and rectum [13–15]. We therefore chose three types of mucins, including MUC6, MUC5AC, and MUC1. MUC6 was already showed to associate with morphologic serrated features of appendix in the study of Bellizzi et al [9]. Our result showed that LAMN and serrated lesions both can have MUC6 expression (5/13, 6/12), although only in focal area.
LAMN shared similarity with pancreatobiliary subtype of pancreatic intraductal papillary mucinous neoplasm (IPMN), which is usually positive for MUC5AC and MUC1 and negatively for MUC6 [16, 17]. MUC5AC and MUC1 were indeed highly expressed in LAMN in our cases. Besides, serrated lesions also have high MUC5AC expression and 6 of 12 serrated lesions have focal MUC6 expression as LAMN. This indicated that both LAMN and serrated lesions of appendix can have MUC5AC and focal MUC6 expression, and a combination of MUC5AC and MUC1 might help us to distinguish LAMN from other lesions of the appendix. The possible mechanism may be GNAS mutation, which was characteristic of LAMN[18], could induce MUC5AC and MUC2 expression in colorectal cancer cell lines, so in LAMN, MUC5AC is all positive in both crypt and surface epithelium. Focal MUC6 expression in both LAMN and serrated lesions may explain their morphologic similarities. Besides, in Mesa’s research[19], MMR deficiency in appendix neoplasms showed a correlation with MUC5AC and MUC6 expression. In our LAMN and serrated cases, 11/24 that have MUC5AC expression also have dMMR, and 6/11 that have focal MUC6 expression also have dMMR.
Serrated lesions of colon carcinoma tend to have higher rate of microsatellite instability[20]. In concordance with colon carcinoma, our result also showed that serrated lesions in appendix are prone to have dMMR than LAMN.7 of 12 serrated lesions have dMMR in the protein level including loss of MSH6, PMS2 and MLH1, although Direct analyses of representative MSI loci showed none of serrated lesions of the appendix were MSI-H. Yantiss et all [12]also reported that incomplete loss of MLH1 protein in serrated polyps of appendix was not accompanied by MSI-H. In our cases, three LAMNs have loss of MSH6 in the villous structure but no loss of MSH2, although this type was rarely reported in LAMN, only in MSI-high appendiceal carcinoma[21]. This indicated that LAMN can also have microsatellite instability, even though they are at an early stage.
KRAS mutation in LAMN was significantly higher than in serrated lesions, with 12 of 13 cases having different type of mutations. This is consisted with the previous reports[18, 12, 22, 23]. In their study, 94% cases of LAMN were KRAS mutated and the most frequent mutation type was 35G > A. 5 of 12 serrated lesions also have K-ras mutations, which indicated that LAMN and serrated lesions may share similar oncogenic pathways.
Several articles reported a considerable proportion of LAMNs harboring GNAS mutation[24, 18]. In our study, only 5 of 13 (38.5%) LAMN cases exists GNAS mutation. This rate was higher than the serrated lesions. This relatively low rate of GNAS mutation in LAMNs might be due to the low sensitivity of Sanger sequencing and limited exon coverage. Alakus and other researchers [24–27] also proposed that co-existing mutations of KRAS and GNAS were characteristic to LAMN and KRAS mutation occurs earlier in the course of tumorigenesis. This pathway was also shared by IPMN. In the present study, 8 cases, including both LAMN and serrated lesions, that have GNAS mutation, also have KRAS mutation co-exist. These may have indicated that GNAS mutation was prone to happen following the KRAS mutation.
BRAF mutation was only presented in 3/12 cases of serrated lesion. Rish K. Pai et al [28] found serrated lesions in appendix harbor more KRAS mutations instead of BRAF mutations, by which they regarded serrated lesions in appendix as a distinct entity from their counterparts in the colon, which was supposed to have more BRAF mutation.Other researchers[25] reported higher rate of BRAF mutation of the serrated lesion. Our result also indicated a slightly more KRAS mutation (5/12) in serrated lesions than BRAF mutation (3/12) in the appendix and our histological critieria was almost the same with them. BRAF mutation was only confined to serrated lesions in the present study, suggestting that BRAF could be used as a specific yet insensitive marker for serrated lesion in appendix. Similar as the colorectal counterparts, serrated lesion in appendix can have dMMR and BRAF mutation.
In conclusion, LAMN and serrated lesion each had their characteristic immunohistochemical expression and molecular mutations, LAMN was both MUC1 and MUC5AC positive and harbors both KRAS and GNAS mutation, and they can also have loss of MSH6 immunoexpression and also have high Ki67 index in the surface epithelium. Serrated lesions can also have dMMR, with MUC5AC and MUC6 focal expression and they were all p53 negative and they also harbor KRAS mutation. Combination of these different markers could asist the differentiation.