Background
Once a rare malignancy, esophageal carcinoma is the sixth most frequent cause of death worldwide and the most rapidly rising cancer in the United States [
1,
2]. Esophageal adenocarcinoma (EAC) represents over 50% of all esophageal cancers in the western world, and the incidence of EAC arising from Barrett’s esophagus (BE) continues to increase at an alarming rate [
1,
3,
4]. Nearly 60% of cases are diagnosed at an advanced, incurable stage; however, if diagnosed early these cancers are highly curable with a combination of surgical, endoscopic and adjuvant therapies [
5]. Expected five-year survival rates for stage I disease approach 60% to 90%, highlighting the importance of developing effective screening tools to facilitate early diagnosis [
4,
6,
7].
The majority, if not all cases of EAC arise from a region of BE, a well-recognized premalignant condition and common complication of gastro-esophageal reflux disease (GERD) affecting 12% to 20% of patients suffering from reflux [
2,
5,
8,
9]. BE remains the strongest individual risk factor for development of EAC and the only known precursor lesion [
8]. Despite the presence of a well-defined histopathologic metaplasia-dysplasia-carcinoma sequence, current screening strategies for early detection have failed to reduce mortality from esophageal cancer in randomized prospective trials, and many studies have questioned their cost-effectiveness [
9,
10].
New sensitive tools are needed to overcome shortcomings of current screening and surveillance approaches for BE and esophageal cancer. Urinary metabolomics may offer one such potential for non-invasive screening of early tumor-associated perturbations in cellular metabolism. Identification of a discrete, urinary metabolomic signature associated with esophageal cancer and its precursor lesion, could allow for non-invasive screening in targeted, high-risk populations while helping to further elucidate underlying molecular pathogenesis and progression of disease. Furthermore, knowledge gained from metabolomics-based research could help advance personalized therapeutic approaches guided by early metabolic responses before phenotypic changes develop.
Currently, application of metabolomics-based techniques in the investigation of esophageal cancer remains limited. The majority of studies are serum-based and have included patients with late stage disease. The current case–control study sets out to compare the urinary metabolomic profiles of patients with early stage or locally advanced esophageal carcinoma and BE with healthy, age- and gender-matched controls. We hypothesize that global metabolite analysis of urine using NMR spectroscopy combined with statistical pattern recognition reductive techniques will reveal a characteristic metabolomic signature associated with esophageal carcinoma and its precursor lesion BE.
Discussion
Esophageal cancer is an aggressive malignancy with poor prognosis, in part due to delayed diagnosis. Nearly 50% of patients do not present until they are at an advanced, incurable stage; however, current surgical and adjuvant therapies offer high cure rates in early stage disease [
5]. For most of the twentieth century, squamous cell carcinoma accounted for the vast majority of esophageal cancers. In the past three decades, however, the overall incidence of EAC has increased at an alarming rate, and this has been accompanied by dramatic increases in mortality [
5]. While reasons for this steady progression in incidence remain largely unknown, they are thought to be secondary to increased rates of obesity and GERD. BE, affecting up to 20% of patients with GERD, is a well-recognized precursor lesion of EAC and carries a 30- to 125-fold increased risk of cancer development when compared with the general population [
25].
Despite the presence of a known, stepwise, metaplasia-dysplasia-carcinoma sequence of cancer progression providing the opportunity for early detection, screening and treatment, there is little evidence that current surveillance strategies have prevented deaths resulting from EAC [
25]. Traditionally, screening and surveillance strategies for EAC among patients with BE involve endoscopic detection with histopathologic confirmation of Barrett's metaplasia-dysplasia. Challenges impeding the effectiveness of current strategies aimed at early detection include the large number of patients with silent reflux harboring BE, the discontinuous nature of metaplastic and dysplastic epithelium when using a technique of random endoscopic biopsy and the absence of a well-validated risk stratification model capable of accurately and reliably identifying those individuals truly at risk of progression along the metaplasia-carcinoma sequence [
26]. Moreover, there exists a subset of patients who progress to high-grade dysplasia and cancer with no evidence of dysplasia on previous recent surveillance endoscopies, representing a cohort of patients who may, therefore, evade detection using current screening strategies [
26,
27].
Urinary metabolomics offers a novel and sensitive approach to simultaneously evaluating tumor-associated perturbations of multiple metabolic pathways and their downstream functional significance prior to the surfacing of gross phenotypic change. Application of metabolomic tools could therefore provide an opportunity for screening and early detection, molecularly-guided and personalized therapeutics as well as further interrogation of the molecular pathogenesis of esophageal cancer progression.
There remains a dearth of research examining the role of metabolomics as a potential diagnostic or screening tool in esophageal cancer and its precursor lesion, BE. The majority of studies have been serum- or tissue-based, limited in sample size and inclusive of patients with metastatic disease. As a biofluid, urine has a number of distinct advantages, namely related to ease of sample collection, storage, processing and reproducibility. Furthermore, urinary metabolomics avoids the issue of degraded spectral resolution resulting from high lipid and protein content, often a complicating feature in the analysis phase of serum studies.
Djukoivc
et al. used a targeted metabolite approach to investigate the potential role of nucleosides as cancer biomarkers in EAC [
28]. They uncovered a number of differences in nucleoside expression when comparing cancer patients to healthy controls. Using a serum-NMR based approach, Zhang
et al. were able to discriminate patients with EAC from healthy controls, in congruence with results of other serum studies of smaller sample size [
29‐
31]. They also found that patients with BE and high-grade dysplasia appeared to overlap with both cancers and controls; however, this analysis was limited to five patients. Moreover, patients with stage IV disease were included and, while a subgroup analysis was done, potential confounding effects of age and gender were not taken into account at the primary stage of analysis.
Using a representative sample of patients with early stage or locally advanced disease, we have identified a discrete and specific urinary metabolomic signature of esophageal cancer which is clearly distinguishable from healthy controls and exhibits strong predictive accuracy. Moreover, even at the pre-invasive stage of disease, metabolomic profiling accurately distinguishes patients with BE. Esophageal cancer is a highly curable malignancy when treated in its early stages. A non-invasive, highly predictive screening tool capable of early disease detection could, therefore, have significant impact on the management of esophageal cancer with the potential to drastically alter outcomes.
Biochemical interpretation of the altered patterns of metabolite expression when comparing esophageal cancers with controls must be made with caution since it is likely that the overall metabolite expression profile results from a convergence of changes occurring at the tumor microenvironment level and disturbances in overall global metabolism. We suspect that cancer-specific elevations of 2-aminobutyrate (a key intermediate in the biosynthesis of opthalmate which is a tripeptide analogue of glutathione) may be reflective of increased oxidative stress [
32,
33]. Cancer cells both
in vitro and
in vivo are thought to be under continuous oxidative stress, in part resulting from outgrowth of blood supply occurring with tumor proliferation [
34]. Esophageal cancers grow and spread rapidly, with lymph node metastases in nearly 20% of T1 lesions and 60% of T2 lesions [
5]. Likewise, cancer-specific elevations of hypoxanthine may represent an underlying enhanced capacity for DNA synthesis in association with tumor growth. Our findings of cancer-specific decreases of pantothenate, a precursor of coenzyme A and elevations of cis-aconitate, a tricarboxylic acid cycle intermediate, were in agreement with observations of Ikeda
et al. and may be a result of aggressive tumor growth with resulting increased energy demands [
29]. It is possible that elevated levels of fucose among cancer patients may be associated with subclinical hepatocellular injury from early metastatic disease, as this metabolite is often secreted in association with liver damage [
33].
Cachexia is present in up to 80% of patients with malignancies of the upper gastrointestinal tract and is a complicating feature in a significant proportion of patients with esophageal cancer [
35]. Significant elevations of pi-methylhistidine were observed among cancer patients and may be reflective of cachexia-induced skeletal muscle protein breakdown. Trends towards significance were observed with regard to cancer-specific elevation of a number of amino acid and amino acid derivatives (tyrosine, tryptophan, threonine, trigonelline, 4-hydroxyphenylacetate and isoleucine), possibly resulting from muscle wasting in these patients. In the early stages of malignancy, cachexia-associated muscle wasting is often an occult phenomenon [
12,
36]. A noninvasive means of detecting early muscle wasting in patients harboring occult or early malignancy could have significant clinical utility.
As expected, there was a larger degree of overlap when comparing the metabolite expression profiles of patients with EAC and BE compared to those of healthy controls. Barrett’s metaplasia was noted in over 50% of final surgical specimens resected in the setting of EAC. Accordingly, several key metabolites were common to the models discriminating esophageal cancers from healthy controls and BE. Metabolite differences observed when comparing patients with BE to healthy controls may in part be reflective of the hyperproliferative state known to characterize Barrett’s epithelium [
1,
26]. Relative decreases in the concentration in a number of metabolites among patients with BE (succinate, pantothenate, acetate, formate) may be reflective of this hyperproliferative state and a resultant overall increased cellular energy demand. Additionally, preferential use of branch chain amino acids, specifically leucine, is characteristic of stress states. Lower levels seen among patients with BE may have been reflective of the metabolic stress associated with low grade, chronic inflammation [
33]. At this stage, however these postulations remain speculative and further experimental
in vivo modeling is required to confirm their relevance. Of note, disease chronicity, extent and severity appeared to be well correlated with degree of metabolic derangement. The metabolic profiles of those patients with a longstanding history of BE (mean four years), those with long segment BE or those with low grade dysplasia (one patient) showed the greatest degree of discrimination from that of normal controls.
In an effort to explore whether the metabolic disturbances observed were, in fact, specific to esophageal cancer or reflective of common final pathways of global metabolic change associated with malignancy, the metabolomic profiles of patients with esophageal and pancreatic cancer were compared. Key discriminatory metabolites identified through VIP-analysis revealed several distinguishing patterns of metabolic expression. Acetone (an end-product of ketogenesis) and glucose, two key discriminatory metabolites elevated among patients with pancreatic cancer, are perhaps reflective of underlying diabetogenic disturbances associated with this malignancy. Hyperinsulinemia and peripheral insulin resistance are metabolic perturbations frequently observed in pancreatic cancer [
37,
38]. Elevated levels of 3-indoxylsulfate, another key discriminatory metabolite, were observed among patients with esophageal cancer. This may perhaps be reflective of the prominent role of oxidative damage in the malignant transformation of esophageal cancer as 3-indoxylsulfate acts to propagate oxidative stress by strongly decreasing circulating levels of glutathione, a key cellular antioxidant [
39,
40].
While the metabolite expression profiles of patients with esophageal and pancreatic cancer were clearly distinguishable, a region of overlap was evident. This area of overlap could be reflective of metabolic changes associated with shared common pathways of tumorigenesis including increased energy expenditure associated with tumor cell proliferation and growth, other molecular commonalities involved in tumor angiogenesis, invasion and distant spread as well as cachexia-associated muscle wasting. The metabolites most contributory to the overlapping region were related to energy metabolism and cellular proliferation (1-methylhistidine, o-acetylcarnitine) as well as those reflective of muscle wasting, including pi-methylhistidine, creatinine, and a number of amino acids and amino acid derivatives (leucine, tyrosine, isoleucine, valine, tryptophan and 4-hydroxyphenylacetate).
While these preliminary results are encouraging, a number of limitations must be acknowledged including small sample size. Future studies will include an external validation step to further confirm model validity using an independent cohort of patients not involved in model development. The predictive accuracy of reduced models containing only those key discriminatory metabolites identified here (metabolites with significant concentration differences or a VIP-parameter ≥1) will also be tested at this stage.
While the potential confounding effects of chemoradiation treatments were controlled for by collecting samples prior to any therapeutic intervention, one limitation of this study could be the potential confounding effect of medication. It was impossible, however, in a study of this size and design to control for this potential confounder given the wide range of medications being prescribed among enrolled patients. We suspect, however, that any effect was likely small and not relevant as considerable differences in the medication profiles were not apparent on initial analysis. Furthermore, while drug metabolites and drug vehicle constituents were excluded from analysis, it was not possible for us to control for the potential downstream effects of these medications on the expression of other metabolites. While this could be a potential confounder, we believe the effect is small and likely reasonably balanced between groups.
An additional limitation of this study could have resulted from a lack of control for dietary factors. However, when a number of exogenous metabolites were excluded from analysis, predictive accuracy was maintained. While dietary factors were not directly controlled for, we have no clear reason to believe there would be any major systematic dietary differences between these patients given that a large proportion of the patients with esophageal cancers were early stage and the patients with BE were otherwise medically fit. Furthermore, given that all cases and all controls were not consuming identical diets, it is more likely that any dietary effects were responsible for creating background noise, potentially obscuring further important, biological patterns. This issue could be addressed further in future studies, however, through the use of dietary records to account for any gross dietary differences.
Future studies should also integrate results from both serum and urine analysis, while using diverse analytical platforms such as gas-chromatography or liquid chromatography-mass spectrometry in order to gain a more complete assessment of the metabolic milieu associated with esophageal cancer and its precursor lesion, BE.
Authors' contributions
VWD participated in study design, carried out sample processing and spectroscopy, completed statistical analysis and drafted the manuscript. DES was involved in study design and coordination, and helped to draft the manuscript. DE participated in study design and helped to draft the manuscript. MBS conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.