Here we report for the first time, to our knowledge, that immunoreactive DCAMKL-1 although minimally expressed in normal distal esophageal squamous mucosa, is markedly expressed in BE epithelium. Furthermore, an increased epithelial and stromal expression pattern was observed in patients with progression of dysplasia. Moreover, a marked increase in stromal DCAMKL-1 was observed in EAC. Despite the tremendous increase in EAC incidence over the past 3 decades, identification of the cellular origin of BE and the role of this cell during progression to EAC remains elusive. BE is a premalignant lesion detected in the majority of patients with EAC, conferring increased risk for cancer development 20
. EAC is associated with a very low rate of survival once detected clinically. BE progression is associated with increasing severity of dysplasia prior to the development of cancer 10
. Therefore, development of biomarkers that can assist in evaluating the risk of histologic progression toward EAC from BE are essential in limiting cancer development.
Surveillance programs are the mainstay for monitoring the progression of BE from no dysplasia to HGID. Generally, patients with HGID are offered a surgical option, after confirmation of the diagnosis by two expert pathologists 21
. Newer experimental ablative therapies may be an option for patients at high surgical risk or for those who decline surgery. The limitations of these approaches and the lack of medical therapy illustrate the need for additional techniques that estimate the risk of progression and confirm the presence of dysplasia either within the specimen or potentially in the bloodstream.
The recent emerging stem cell hypothesis of solid tumor cancers has only recently been explored in esophageal cancer 22
. The squamous epithelium of the normal esophagus undergoes metaplasia to form intestinal type mucosa in patients with BE. Recent challenges to this hypothesis suggest that the cell of origin may be a proximal gastric stem cell that migrates across the EG junction and crosses the squamocolumnar junction 23
. These stem cells then proliferate and give rise to the intestinal type epithelia known as Barrett’s epithelium. Recently, in animal models it has been shown that the Barrett’s epithelium may be driven by bone marrow derived stromal cells that convert to epithelium and promote aggressive growth of BE 23–25
. Thus, identification of the cell(s) of origin is key to gaining a more complete understanding of the molecular features of BE, including initiation and progression to EAC. In this manuscript we have developed immunohistochemical evidence of a unique cellular expression pattern of the novel intestinal stem cell marker DCAMKL-1. Although expressed in many gut tissues and pancreas, up regulation of DCAMKL-1 has been particularly noted in the stromal desmoplastic compartment in many solid tumors 16, 17
. Although DCAMKL-1 staining is primarily epithelial, there is also clear evidence in early BE, of distinct stromal expression. In addition to the epithelial and stromal staining, evidence of endothelial and blood vessel expression of DCAMKL-1 was observed in some patients. As BE progresses to dysplasia, an increase in stromal and epithelial DCAMKL-1 is observed. Many more patients with varying degrees of dysplasia need to be examined to confirm the present observations. Nevertheless, we contend that even in this small sample set there is a clear, statistically significant increase in the expression of DCAMKL-1.
These findings suggest that DCAMKL-1 is expressed in pre-malignant tissues, which makes it an intriguing candidate for investigation as a tissue specific biomarker for BE. If validated, it could potentially be used as a surrogate marker after diagnostic confirmation using conventional pathologic techniques. Finally, this marker could be used in the confirmation of focal lesion eradication following endoscopic ablative therapies. Endoscopic techniques have been developed recently to eradicate BE with HGID, and EAC with varying levels of success 26–28
. However, eradication does not always occur and its durability remains in question. Therefore, identification of cellular markers indicating the presence of neoplastic stem/progenitor cells either before or after endoscopic ablation would enhance the clinical and endoscopic management of individuals who undergo this therapy.
Previous efforts attempting to categorize patients at increased risk for progression to EAC using genetic markers have yielded limited results. These have included markers for tumor suppressor genes, aneuploidy, or tetraploidy. To date, there are no studies identifying an increased esophageal stem cell cohort during the progression from normal squamous mucosa through dysplasia to EAC. Identification of such a marker in serum could allow for a noninvasive assessment of general EAC risk in patients with gastroesophageal reflux disease.
Recently, epidermal growth factor receptor (EGFR) was reported to increase during the transition from BE to EAC29
. However, use of EGFR as a molecular marker for transition of BE toward EAC is problematic for several reasons. EGFR was noted to stain squamous tissue at a higher intensity than BE or EAC. The optimal biomarker would be either absent or low in the normal state prior to the development of metaplasia or cancer as a negative control, limiting potential sample contamination. In addition, EGFR was not observed to increase in the stroma during transition of BE toward EAC. The absence of increased stromal staining does not allow for assessment of the stromal compartment as seen with DCAMKL-1, decreasing the potential usefulness of EGFR as a biomarker.
We hypothesize that a gastrointestinal type stem cell originating from the proximal stomach, EG junction, or bone marrow may represent the precursor cell type for the intestinal metaplasia associated with BE and EAC. In nude mice, knockdown of DCAMKL-1 results in cessation of HCT-116-mediated tumor xenograft growth. Furthermore, reduction of DCAMKL-1 correlates with increased expression of the tumor suppressor microRNA Let-7a
and a reduction in oncogenic c-Myc RNA and protein 14
. These data support a functional role for DCAMKL-1 in cancer progression.
Using a series of tissue microarray slides obtained from the Tissue Array Network, we performed immunohistochemical analysis to investigate DCAMKL-1 protein levels in human patients. Given the potential for interactions between epithelial stem cells and stroma during tumor progression, we predict that DCAMKL-1 may play a key role in the initiation and progression of BE and EAC. Our observations of increased accumulation of DCAMKL-1 provide a potential mechanistic link between esophageal injury/inflammation and carcinogenesis risk in proximal gastric epithelial cells.
In order to further evaluate the expression patterns of stem/progenitor markers in BE, we evaluated mRNA expression of DCAMKL-1, Msi-1 and LGR5 in patients with BE compared to normal. Relative mRNA expression determined using quantitative real-time RT-PCR demonstrated upregulation of all of these mRNAs in patients with BE. These data provide strong support for the involvement of stem/progenitor proteins in BE.
Overall, these data present a detailed immunohistochemical analysis of the putative gastrointestinal stem cell marker, DCAMKL-1, in the distal esophagus of patients with BE and EAC. These findings suggest a potential candidate for further study to evaluate the role of stem/progenitor cells in BE initiation and progression to adenocarcinoma, as well as a novel hypothesis to explain the intestinal metaplasia associated with this pre-malignant condition.