Expression of Annexin A10 in Serrated Polyps Predicts the Development of Metachronous Serrated Polyps

Hyperplastic polyps (HPs), which are non-neoplastic, compose the greatest proportion of lesions within the family of serrated polyps (SPs). The most recent World Health Organization classification of serrated colorectal lesions includes HP, sessile serrated polyp (SSP) with and without cytological dysplasia, and traditional serrated adenoma (TSA).1 A normal continuous and symmetric proliferation of the epithelium at the base of the crypts defines an HP. SSPs demonstrate abnormal cellular proliferation characterized by a proximally displaced proliferative zone, epithelial serrations, and characteristic distorted basilar crypt architecture with crypt dilation, branching, and lateral growth along the muscularis mucosa, which is the hallmark of SSP. Nuclear atypia, cytoplasmic eosinophilia, ectopic crypt foci, and serrated crypts are the reported characteristics of the rare TSA.2 Prior to the recognition that SSPs are the precursor of a substantial percentage of colorectal cancers (CRCs), most SSPs were diagnosed as HPs. However, molecular and genetic differences exist between these two types of polyps.2 SSPs frequently have a BRAF mutation, CpG island methylation, and occasional loss of MLH1 due to MLH1 promoter methylation, hallmarks of microsatellite unstable CRC2 and supporting the clinical observation linking SSPs to CRC.2, 3, 4 Recognition of the pathological characteristics differentiating SPs with premalignant potential from those without is essential in assessing CRC risk and guiding postpolypectomy surveillance recommendations.5, 6 However, the distinction between SPs can be challenging, even to the experienced gastrointestinal pathologist. As a major criterion for the diagnosis of SSP and distinction from HP is the basilar crypt morphology, complete polyp resection and well-oriented biopsies are needed but often not achieved.7, 8 Furthermore, concordance in the diagnosis of optimally oriented sections of SPs (HP, SSP, TSA) by pathologists with a special interest in gastrointestinal pathology is only moderate (k=0.55).7 Given these challenges, identifying molecular markers that more accurately predict the biological behavior of SPs and individuals at greatest risk of recurrent neoplasms would be of great clinical importance. RNA-sequencing studies identified many highly and differentially expressed genes in SSPs compared with adenomas and HPs. Annexin A10 (ANXA10) was among one of the highly expressed genes.9 ANXA10, member of the Annexin family, is a calcium-and-phospholipid-binding protein. It is implicated in multiple physiological processes, including growth regulation, cell division, apoptosis, and differentiation.10 Increased expression was reported in Barrett’s esophagus, oral cancers, and pancreatic cancer.11 Its role in CRC remains to be determined. A recent study12 demonstrated that ANXA10 gene and protein expression by immunohistochemistry (IHC) differentiated SSPs from HPs. High ANXA10 IHC expression had a sensitivity of 73% and a specificity of 95% in the diagnosis of an SSP. Additionally, ANXA10 showed to be highly expressed in serrated colon carcinoma13 compared with conventional colon cancer, perhaps proving valuable in the progression from SSP to colon cancer. Therefore, we conducted a study aiming at determining the utility of ANXA10 expression in baseline SPs in predicting patients at increased risk of metachronous SPs and/or adenomas. The study was approved by the Cleveland Clinic Institutional Review Board. Patients with serrated colorectal lesions reviewed in the Department of Pathology between 2006 and 2010 were identified through a natural language search. The electronic records of these patients were reviewed. Only patients with an SSP with or without dysplasia or an HP who had a complete baseline colonoscopy, no remaining polyps, and a follow-up colonoscopy by 2014 were included and analyzed. A gastrointestinal pathologist (R.K.P.) re-reviewed the SPs at baseline for histological confirmation. The diagnosis of SSP was based on the consensus criteria provided by an expert panel.5 The presence of at least one unequivocal architecturally distorted, dilated, and/or horizontally branched crypt was sufficient for a diagnosis of SSP. Individuals with TSA or a combination of serrated and adenomatous polyps at baseline colonoscopy were not included in the study group. We also excluded individuals with a history of colectomy, personal history of CRC, hereditary CRC syndrome (familial adenomatous polyposis, hereditary nonpolyposis CRC, and serrated polyposis syndrome), incomplete colonoscopy, or poor/inadequate bowel preparation. The primary objective was to determine the rate of development of SPs and adenomas on follow-up colonoscopy in relationship to baseline ANXA10 IHC expression. The secondary end points were the rates of advanced serrated neoplasia (defined as an SSP ≥10 mm, SSP with cytological dysplasia, TSA, or adenocarcinoma) and advanced adenoma (presence of villous component, size ≥10 mm, presence of high-grade dysplasia) in patients with SPs with high and low ANXA10 expression. 0—No crypts stained positive for ANXA10; 1—<5% of crypts stained positive for ANXA10; 2—5–25% of crypts stained positive for ANXA10; 3—26–50% of crypts stained positive for ANXA10; 4—51–75% of crypts stained positive for ANXA10; and 5—76–100% of crypts stained positive for ANXA10. On baseline colonoscopy, 54% of patients had only 1 SP while 24% had ≥3 SPs (Table 2). High ANXA10 expression was related to the number of baseline SPs (P=0.032). SSPs comprised 43% of the SPs with low ANXA10 expression and 92% of SPs with high ANXA10 expression (<0.001). Patients with high ANXA10 expression near universally had only SSPs (94%, P<0.001; Figure 1) and more numerous SSPs (P<0.001) The SPs with high ANXA10 expression were most often proximal (84.7% vs. 48.5%, P<0.001) and larger in size (7 vs. 5 mm, P=0.009). Patients with low ANXA10 expression were more likely to have exclusively HPs and more numerous HPs (P=0.003). Ninety-eight patients (55%) had polyps detected on follow-up colonoscopy, including 32% with ≥2 polyps (Table 3). SSPs were identified in 17% of patients with baseline polyps with high ANXA10 expression and 7.5% of patients with polyps with low ANXA10 expression (Figure 2, P=0.03). Almost all (95%) of the metachronous SSPs in patients with polyps with high ANXA10 expression were proximal in location while only 60% of SSPs with low ANXA10 expression were noted above the descending colon. The third of recurrent SPs in both groups (8/24) were in the same segment of the colon than the baseline SP. Four patients with polyps with high ANXA10 expression developed an SSP ≥10 mm or an SSP with dysplasia compared with 1 patient with polyps with low ANXA10 expression. There was no difference in the detection rate of adenomas on follow-up colonoscopy in patients with polyps with high or low ANXA10 expression, 18.8% vs. 19.4%, respectively (P=0.52). Five patients with polyps with high ANXA10 expression and two patients with polyps with low ANXA10 expression had an advanced adenoma on follow-up. In univariate analysis, patients with polyps with high ANXA10 expression were three times at higher risk of SSP recurrence (HR 2.7; P=0.048). Particularly, the recurrence risk of proximal SSPs was four times higher in this group of patient (HR=4; P=0.02). High ANXA10 expression remained significantly associated with higher risk of SSP recurrence after adjusting for gender, age, smoking, or family history of CRC. In the regression analysis, the association between high ANXA10 expression and risk of recurrent SSPs weakened (P=0.098) (Table 4). When the analysis was repeated using the baseline polyp diagnosis from the original pathologist, the association of high ANXA10 and SSP recurrence was stronger (P=0.054). In the present study, we found that patients with baseline SPs with high ANXA10 expression had a higher risk of SSP but not adenomas on follow-up colonoscopy compared with patients with SP with low ANXA10 expression. This association remained valid after adjustment for age, gender, smoking, or family history of CRC. The present large study is the first to demonstrate an association between high ANXA10 expression and metachronous SSPs, including proximal SSPs. We also found a non-significant 2.5-fold increase in large SSPs in the high vs. low ANXA10 cohort. Several recent studies14, 15, 16 indicate that ANXA10 is not only a marker of SSPs but more importantly a marker of the serrated pathway of CRC. Microsatellite unstable CRC expressing ANXA10 were significantly associated with CpG island methylator predictor status, MLH1 promoter gene methylation, and female predominance and were most often in the proximal colon, known features of the serrated carcinoma pathway. Our results indicate that ANXA10 expression in SPs is associated with a moderate risk of SSP recurrence in the proximal colon and a possible increased risk of metachronous advanced serrated lesions. These findings confirm the results of prior research and strongly suggest that ANXA10 expression might be maintained during the multistep transformation from SSP to carcinoma. Multiple studies7, 17 have shown variable SSP diagnosis rates among pathologists, up to 13-fold in one study suggesting perhaps that the use of an adjunct tool such as IHC for ANXA10 may improve the ability of pathologists to make the diagnosis of SSPs. In this large study, we observed that high ANXA10 expression was only associated with the development of SSPs but not adenomas on follow-up colonoscopy. In the multivariate analysis, the relationship between high ANXA expression and metachronous SSPs weakened (P=0.098), suggesting that ANXA10 may not be a better predictor of SSPs on follow-up compared with baseline histology interpreted by an expert pathologist. However, when we adjusted for the diagnosis from the original pathologist, the utility of ANXA10 was more compelling (P=0.054). It is likely that the association of ANXA10 expression and recurrent SSPs would only be strengthened using non-expert pathologists where greater variability in diagnosis exists. Despite having a fairly large number of patients with SPs at baseline, the number of patients in each subgroup is small and does not allow a comparison between SP recurrence in patients with ANXA10 low SSPs with ANXA high SSPs. Nonetheless, an important question remains unanswered. If not better than histology, is IHC for ANXA10 an adjunct to histopathology for the risk stratification of patients with SPs? Additional studies to elucidate the role of ANXA10 stain in clinical practice would be valuable in guiding surveillance colonoscopy in patients with SPs. ANXA10 was not associated with metachronous adenomas (P=0.52). Although this negative finding may be due to the small size of the cohort, previous research indicates that SPs and adenomatous polyps arise through different molecular and biological processes. Some authors suggested that SPs may flourish in a milieu where hypermethylation leads to increased serrated neoplasia.16 Therefore, based on observations acknowledging ANXA10 as a supportive marker of the serrated pathway, one might not anticipate to find an association with metachronous adenomas. To the best of our knowledge, the present study is the first study to evaluate the utility of ANXA10 expression in predicting the development of subsequent polyps. In our study, all SPs at baseline were re-reviewed by a single gastrointestinal pathologist and the majority of the SPs (75%) included in the analysis were SSP and proximal in location (71%), which are considered as high-risk lesions. Nevertheless, our study has several limitations. First, the primary end point of the study, recurrence of SSPs, is only a surrogate marker of CRC development and its true significance is still to be determined. Second, it is well known that the awareness and detection of SPs has improved over time. If so, it is possible that some serrated lesions detected on follow-up were missed rather than newly developed, which may decrease the predictive value of ANXA10 for metachronous lesions. Third, it is possible that some of the follow-up polyps were incompletely resected at baseline. Fourth, the pathology of serrated lesions were not re-reviewed on follow-up. We believe that this is less of a concern as the consensus criteria for the diagnosis of SSPs was utilized at our institution since 2011. Finally, the small number of patients included in the study and the small number of metachronous polyps may have diminished the power of the study to show statistical significance of the results. Albeit these limitations, our findings suggest that ANXA10 is a potential marker of “high-risk” patients with SSPs who are at considerable risk of recurrent SSPs, in particular proximal SSPs. Additional studies to confirm the role of ANXA10 expression in predicting metachronous neoplasia, particularly large and dysplastic serrated lesions, are needed. If additional studies confirm our findings, ANXA10 may be an adjunct molecular tool to stratify patients with SPs into high- and low-risk groups allowing personalized CRC surveillance. Given the minimal cost associated with performing an IHC stain, cost savings may occur if ANXA10 better stratifies patients and results in more efficient usage of colonoscopies. Guarantor of the manuscript: Carol A. Burke, MD. Specific authors contribution: Carole Macaron: collecting and interpreting the data and drafting the manuscript. Rocio Lopez: statistical analysis and interpreting the data. Rish K. Pai: planning, interpreting the data, and reviewing the manuscript. Carol A. Burke: planning, interpreting the data, and drafting the manuscript. All authors have reviewed and approved the final draft submitted. Financial support: This work was supported by American College of Gastroenterology Clinical Research Grant 2014. Potential competing interests: None.