Many carcinomas of the digestive system have been considered cancer stem cell-related diseases in recent years, including esophageal, gastric, pancreatic and colorectal cancer 
. In an effort to identify these small populations of cells, a number of cell surface proteins have been identified as CSC markers. Many studies have demonstrated experimentally that CD166 can enrich for CSC-like cells in a variety of cancers 
. Moreover, Levin et al 
have found that CD166 is expressed at low levels in differentiated intestinal cells but robustly expressed on the surface of cells within the stem cell niche at the base of a crypt, which strongly infers its relationship with stem cell properties. Although high expression of CSC markers are usually considered as a prognosis of poor outcome, several contradictions to this generalization exist in published studies on the putative CSC-marker CD166 
To investigate the basis for these contradictory conclusions, the staining methods among the studies in this meta-analysis were compared. Although the same antibody was used in each of these studies, the staining methods have varied between analyzing both membranous and cytoplasmic staining, whereas others take only membranous staining in consideration. Tachezy et al 
found that CD166 was predominantly expressed at the cell membrane and that cytoplasmic staining intensity was related to the intensity of membranous staining and did not occur in the absence of membranous staining; thus, only membranous staining was considered in their study. The other three studies in this meta-analysis that focused on membranous staining including colorectal and pancreatic cancer, all concluded that high CD166 expression is a positive marker for good prognosis 
. In contrast, the three studies in our analysis that analyze both membrane and cytoplasmic staining intensity propose that CD166 high expression contributes to poor clinical outcome 
. Interestingly, in oral, breast and ovarian carcinomas, decreased membranous and increased cytoplasmic expression of CD166 is also associated with worse prognosis 
CD166 has been demonstrated to participate in the metastatic cascade of cancer cells. CD166-mediated intercellular adhesion involves interactions between the amino terminal D1 domain of opposing receptor molecules on two cells and is strengthened by lateral oligomerization of neighboring molecules on the cell surface, which engage the membrane proximal domains D4 and D5 
. These data suggest that high CD166 expression could impede cancer cell release from a local lesion. Furthermore, it has been shown that CD166/ALCAM can be actively cleaved by ADAM17/TACE-mediated proteolysis 
. In ovarian cancer, pharmacologic inhibition of ADAM proteins, or specific silencing of ADAM17/TACE, hampered shedding of CD166 expressed on the cell surface. Interestingly, CD166/ALCAM can be translocated from the cell surface to the cytoplasm via a clathrin-dependent pathway. Specifically, soluble CD166/ALCAM (sALCAM) binds to scFv I/F8 to form a chimera, which induces endocytosis of the membrane-bound CD166/ALCAM. Recombinant sALCAM chimeric molecules inhibit the adhesive function of CD166/ALCAM through a competitive binding effect, which results in increased cancer cell motility 
. Van Kempen et al 
also found that disruption of CD166 self-interaction was associated with tumor cell motility and metastasis. These studies all suggest that CD166 shedding from the cell surface may predict tumor progression and poor prognosis.
Colon cancer is a classical model for tumor progression studies because of its natural development from crypt stem cells to adenomas to fully formed carcinomas 
; CD166 is highly expressed on the surface of crypt cells in this disease. However, both cell surface and cytoplasmic expression of CD166 is apparent in early adenoma formation in ApcMin/+
mice, human colorectal cancer and metastatic disease. Furthermore, only a subset of CD166 positive cells co-localize with the proliferation marker Ki67 
. These observations suggest that specific subcellular localization of CD166 could be used as a clinical prognostic marker because the loss of CD166 cell surface expression appears to be a precursor for tumor progression.
Part of the shedding CD166 would release into the tumor environment and circulation. A few studies have gone so far as to examine the shedding of CD166/ALCAM into blood serum. Two studies in this meta-analysis of esophageal and pancreatic cancer found a significant upregulation of CD166 in the blood serum of patients, but this observation only had a prognostic value in the esophageal cancer patients, as no significant correlation was found between elevated tissue expression and serum level in the pancreatic cancer patients 
. Klasingam et al. 
have described significantly high sALCAM in the blood serum of breast cancer patients, but no prognostic data were reported. Variation in sALCAM among studies may have multiple causes: sALCAM must pass through the barrier of tumor tissue and vascular endothelial cells to be flushed into the blood stream, and sequential sectioning has failed to establish a direct relationship between ADAM17/TACE and ALCAM 
. These data imply that the level of sALCAM in circulation is unstable and that it is inappropriate to use it for estimating prognoses.
Although the evidence addressed above may imply that cell surface expression of CD166 would be a positive prognostic marker and that the shedding of CD166, in other words, cytoplasmic CD166 expression would predict the reverse outcome, but in our stratified analysis, only cytoplasmic staining showed close relationship with poor prognosis, and the result of membrane staining of CD166 in unclear because two of the included studies provided significantly contradictory results 
. Recently, a colorectal cancer study may have provided a succinct method to assess the prognostic value of CD166 
. They found that an elevated mRNA level of CD166 was associated with poor outcome, yet intact membranous CD166 protein (co-localized extracellular and intracellular domain) is associated with improved outcome. With a novel method that stained the extracellular and intracellular domains of CD166 separately, they found that the extracellular domain of CD166 underwent shedding while the intracellular epitope remained. Thus, they concluded that shedding of the extracellular domain of CD166 correlated with patient outcome rather than loss of expression, which was previously considered the prognostic value of CD166. Unfortunately, the antibody applied in previous studies could not differentiate the subcellular epitope of CD166 in immunohistochemistry, making it difficult for a pathologist to accurately judge whether the protein was located on the cell surface, greatly affecting the prognostic capacity of CD166 expression.
This meta-analysis is subject to a few limitations. First, the number of studies included is relatively small, particularly for gastric and esophageal cancer. Second, 8 of the 9 studies were from Europe, including 7 from Germany, 1 from Switzerland and only one from Asia. Distinct site differences are believed to exist and could cause publication bias. Third, the criteria for determining positive or negative expression of CD166 varied among studies. Six studies only studied membranous CD166 expression while the rest also took cytoplasmic expression under consideration. Finally, although we tried to identify the disease free survival rate, these data were almost entirely missing from these studies. Most importantly, based on our meta-analysis of previous studies and systematic review of related articles indicates that the biological function of CD166 in tumor progression is complicated and that determining its subcellular location could be the key for accurate prognostic predictions. Thus, a more standardized staining method should be employed in future studies.