Here, we evaluated the LGR5 expression profile of 30 CAC cases and 10 sporadic CRC cases and identified that LGR5 was less frequently expressed in CACs than in sporadic CRCs. Further, we found CAC with non-conventional morphology showed significantly lower LGR5 expression levels and nuclear translocation of beta-catenin than the conventional subtype of CAC.
LGR5 has been considered one of the most reliable crypt stem cell markers of CRCs, particularly those that arise through the APC-mutant pathway of tumorigenesis. LGR5 is reportedly overexpressed in human colorectal adenomas and cancers by immunohistochemistry [6, 17, 18] and ISH,  and Martin et al showed that the mRNA levels of LGR5 expressed in human CRCs is 10-fold expanded than normal intestinal crypt.  Jang et al recently showed that LGR5 mRNA positivity is observed in 68% of 788 human CRCs and suggested positive correlation with the chromosomal-instability pathway (characterized by left-sided location and nuclear β-catenin expression; representing an abnormal Wnt signal activation) and negative correlation with MSI, CIMP-high, and BRAF mutations.  In accordance with these findings, we found positive LGR5 expression in 90% of our sporadic CRC control group. However, only 30% of CAC were LGR5 positive. The latter finding is in agreement with the previous study by Yasuda et al reporting significantly less expression of reliable intestinal stem cell markers such as CD133,  OCT4  and NANOG  in CAC than sporadic CRC.  The molecular pathogenesis of CAC is different than that of sporadic CRCs with genomic changes that appear directly linked to the effects of ongoing inflammation and repeated mucosal injury in the IBD. [11, 12] In keeping with this, we recently reported CAC tends to show non-conventional tumor morphology and loose intestinal markers such as SATB2 and CDX2, and show aberrant gastric mucin expression,  Ishibashi et al described the expression profile of Atonal homolog-1 (ATOH1), a master transcription factor of the secretory lineage of intestinal epithelial cells, which retain their potential to revert to intestinal stem cells [24-26] in colitic mucosa. They identified both ATOH1 and LGR5 double positive tumor cells as well as LGR5 single positive tumor cells, suggesting that colitic tumors are mosaic and consisted of a heterogenous population of tumor stem cell.  Our data also support this interpretation given the heterogeneous distribution of LGR5 expression in the dysplastic and invasive components of some cases. While the exact mechanism of decreased LGR5 expression in CAC is uncertain, these data demonstrate that CACs have a unique expression profile of intestinal stem cell markers.
We found that despite only 40% of CACs in our cohort showing a conventional morphology, 89% of LGR5 positive CAC were conventional in appearance. These also tended to be located in the rectum and associated with nuclear beta-catenin staining. This raises the possibility that the minority of CACs in our cohort that showed conventional morphology might have arisen through the conventional APC pathway. However, as the majority of tumors had non-conventional morphology and were also LGR5 negative, our data provide support to other recent studies demonstrating that most CAC cases do not arise through the conventional APC pathway. [12, 28] Although it did not reach statistical significance, conventional type CACs trended toward a lower level of LGR5 expression compared to the control group of sporadic CRCs. Thus, even in tumors arising through more conventional molecular pathways in the setting of colitis, LGR5 expression might be affected by ongoing inflammation and repeated mucosal injury. This hypothesis is supported by other studies showing that Lgr5+ stem cells are highly sensitive to epithelial injury induced by radiation or colitis. [27, 29, 30]
Our results in human cancers do not entirely align with previous data from animal models of colitis. Kim et al reported increased Lgr5 expression in an AOM / DSS - colitis mouse model. They showed that Lgr5 expression is gradually increased as tumors developed with repeated colitis and reported all dysplastic lesions and cancers showed high Lgr5 expression.  This discrepancy could be due to differences between human CAC and the murine model. Interestingly, the figure showing tumor morphology in their report shows a more conventional morphology, implying that the murine model may simulate the carcinogenesis pathway in the conventional type of human CAC.
Data regarding any potential prognostic significance of LGR5 expression in sporadic CRC are mixed. Recent studies using RNA-ISH for LGR5 evaluation reported that LGR5 expression is an independent predictor of favorable outcome in CRCs. [15, 31]. On the contrary, a previous meta-analyses of immunohistochemical studies of LGR5 expression showed that high LGR5 expression is associated with shorter overall survival and disease free survival. [32, 33]
Kazama et al described the immunohistochemical expression profile of LGR5 in ulcerative colitis cases and showed increased LGR5 expression in dysplasia and CAC.  This result is contrary to our present study, but may be due to the different methods of LGR5 detection, as the reliability of antibodies against LGR5 remains uncertain, [35, 36] and most recent studies have used ISH to detect LGR5 expression.
One limitation of our cohort is the limited number of cases and the unavailability of clinical follow up data as well as further molecular analysis. Additional studies using a larger cohort will be needed to determine the relationship between LGR5 expression and clinical prognosis in human CACs.