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© 2002 American Society for Clinical Oncology Incipient Angiogenesis in Barrett’s Epithelium and Lymphangiogenesis in Barrett’s AdenocarcinomaByFrom the Cellular Signalling Group, Division of Biochemistry, Department of Biosciences, Viikki BioCenter, University of Helsinki; and Department of Cardiothoracic Surgery, Helsinki University Central Hospital; and Karyon Ltd, Viiki BioCenter, Helsinki, Finland. Address reprint requests to: Merja Auvinen, PhD, Division of Biochemistry, Department of Biosciences, Viikinkaari 5D, P.O. Box 56, Viikki BioCenter, FIN-00014 University of Helsinki, Finland; email: merja.auvinen{at}astrazeneca.com
PURPOSE: Barrett’s esophagus (BE), a precancerous condition for Barrett’s adenocarcinoma, is classically characterized by flames of salmon-colored mucosa extending into normal pale esophageal mucosa. This flaming is thought to be a consequence of continuous erosis of mucosa caused by chronic reflux. Another characteristic feature of Barrett’s adenocarcinoma patients is the frequent development of lymph node metastases. We addressed whether onset of angiogenesis occurs in BE and if the lymphatic system might provide a route for Barrett’s adenocarcinoma cells to infiltrate regular lymph nodes. PATIENTS AND METHODS: Fifteen surgically resected Barrett’s dysplasia or adenocarcinoma patients were included. Immunohistochemistry and a modified whole mount analysis were used. RESULTS: The incipient angiogenesis originates from the pre-existing vascular network in the lamina propria and infiltrates Barrett’s epithelium, giving its ominous salmon-red color. Barrett’s epithelium–specific goblet cells express vascular endothelial growth factor (VEGF)-A. The immature blood vessels show a relative absence of smooth muscle actin (SMA)-positive mural cells and express VEGF receptor (VEGFR)-2 and matrix metalloproteinase (MMP)-9 on their exterior. Coexpression of VEGF-C and its receptor VEGFR-3 on lymphatic vessels is demonstrated. CONCLUSION: BE is strongly neovascularized not eroded. This novel concept of a molecular mechanism of the origin of BE might emphasize why precancerous BE can give rise to the more cancerous dysplasia and Barrett’s adenocarcinoma stages. In addition, adenocarcinoma cells induce lymphangiogenesis. The new lymphangiogenic vessels might provide a systemic route for adenocarcinoma cells to invade circulation and induce lymph node metastasis.
BARRETT’S ESOPHAGUS (BE) is a precancerous condition of the lower esophagus in which the normal stratified squamous epithelium is replaced with specialized metaplastic columnar epithelium.1,2 The Barrett’s mucosa represents an epithelium type that is entirely different from the normal esophageal mucosa. Barrett’s epithelium can give rise to adenocarcinoma that frequently seeds lymph node metastases. BE is diagnosed in up to 20% of patients with documented chronic gastroesophageal reflux disease. Follow-up studies have shown that BE patients have a 30- to 125-fold increased risk of developing adenocarcinoma, which emerges at a rate of approximately one cancer per 100 patient years.3 The incidence of Barrett’s adenocarcinoma is the most prevalently increasing gastrointestinal tract cancer in the Western World. Diagnosis of Barrett’s adenocarcinoma is usually made late, and consequently, it is associated with poor prognosis. After cancer surgery, the 5-year survival is at best only approximately 20% to 30%.4 The formation of new capillaries from pre-existing ones, angiogenesis, occurs in pathologic tumor progression. Induction of angiogenesis is a discrete component of the tumor phenotype, one that is often activated during the early precancerous stages in the tumor progression.5 The importance of neovascularization for solid tumor growth is well recognized. The persistent new blood vessel growth, and thus increasing vascular density, permit tumor cell dissemination and metastasis.6,7 For cancers of epithelial origin, lymphatics are an important route to colonize lymph nodes. Lymphangiogenesis, the growth of new lymphatic vessels, may be an important phenomenon in tumor angiogenesis.8 So far, there are only a few reports describing putative lymphatic overgrowth in human tumors.9,10 In general, lymphatics do not penetrate into the tumor stroma because of the increased interstitial pressure.11 The classical endoscopic feature of BE is the presence of salmon pink mucosa.12 The border between the normal pale stratified epithelium and the Barrett’s intestinal metaplasia is flaming. It has been speculated that the flaming is caused by chronic reflux of gastric and/or duodenal juice injuring the mucosa. The injured mucosa is thought to be thinner, and therefore, the net-like blood vessels lining the normal esophageal mucosa would be visible. In the present study, we hypothesized, however, that the characteristic red flaming of BE might be caused by onset of early angiogenesis beneath the flat surface of mucosa and not due to erosion of the mucosa layer. It is recognized that early lymphatic spread of esophageal adenocarcinoma is unique when compared with other cancers of the gastrointestinal tract.13 The extent of lymph node metastasis is related to the depth of invasion of cancerous cells. The esophageal mucosa can be divided into the mucosa, submucosa, and muscularis propria layers.13 When the primary tumor reaches muscularis mucosa within submucosa, the first lymph node metastases can be detected.14 To our knowledge, this is the first report addressing the role of lymphangiogenesis related to the appearance of lymph node metastases in Barrett’s adenocarcinoma.
Patients and Specimens Barrett’s mucosa samples (and lymph nodes if positive) were collected from 15 surgically resected Barrett’s dysplasia4 and adenocarcinoma patients,11 previously defined by the presence of specialized columnar metaplasia in biopsy specimens from the esophagus, between March 1998 and November 2000 at the Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki, Finland. After resection, muscularis propria was cut off, and mucosa and the underlying submucosa/muscularis mucosa were kept intact. The surgical specimens (normal, metaplasia, dysplasia, in situ, and invasive adenocarcinoma and lymph nodes) were processed on the day of collection. This study was approved by the Ethics Committee of the Helsinki University Central Hospital. Written consent was obtained from the patients.
Immunohistochemistry and Quantification of Blood Vessels
Whole-Mount Analysis
Barrett’s Epithelium Is Neovascularized, not Eroded Specialized intestinal epithelium (glandular epithelium) is normally not found in the esophagus and is characterized by the presence of goblet cells.1,2,16 All the surgically resected normal esophagus and Barrett’s epithelium samples were first histopathologically confirmed. Figure 1A shows mucosa and lower submucosa layers of the normal esophageal mucosa, and Fig 1B shows that the goblet cells specific for Barrett’s epithelium were present in the submucosal layer.
To examine whether the extent of angiogenesis correlated with the occurrence of salmon pink mucosa, the characteristic macroscopic feature of the Barrett’s epithelium, blood vessels were highlighted by staining endothelial cells with mAb EN4 (stains both large and small vessels, including lymphatics) in paraffin sections (Fig 1C and 1D. The histopathology of Barrett’s epithelium differs from normal esophageal mucosa, eg, in Barrett’s mucosa microvessels span not only the submucosa but also through the mucosa layer (Fig 1D). Notably, the mucosa layer was not thinner (eroded) in Barrett’s epithelium (Fig 1D) than in normal esophagus (Fig 1C). Next, capillaries and microvessels were separately counted, both in mucosa and submucosa. The mean capillary counts were not increased in Barrett’s epithelium in comparison with normal esophageal mucosa. Fig 2 shows that the microvessel density, however, was double in the Barrett’s epithelium compared with normal esophageal mucosa. In advanced Barrett’s adenocarcinoma, the microvessel density was two- to three-fold in comparison with normal esophageal mucosa, but the number of capillaries remained unchanged.
To visualize onset of angiogenesis in Barrett’s epithelium, a modified whole mount staining technique for surgically resected patient samples was set up. This technique has previously been used only for mouse embryos15 and tissues.17-19 As shown in Fig 3A, the net-like blood vessels below the normal flat esophageal mucosa were stained with PAL-E, an antibody specific for human microvessels. Only a few of these pre-existing blood vessels penetrated into the mucosa, whereas new angiogenic microvessels infiltrated the whole Barrett’s epithelium. The remodeled vascular morphology with repeated twists, bends or turns, not seen in normal vessels was clearly visible (Fig 3B).
VEGF-Induced Tumor Angiogenesis Associated With Expression of VEGFR-2, MMP-2, and MMP-9 in Barrett’s Epithelium Next, we assessed the expression of well-established angiogenic markers in whole mounts of normal and Barrett’s esophageal mucosa patient specimens. A prime regulator of angiogenesis, VEGF-A, was expressed in Barrett’s epithelium (Fig 3D), whereas its receptor, VEGFR-2, was strongly expressed on endothelial cells of new angiogenic blood vessels, particularly on those vessels feeding the Barrett’s specialized intestinal mucosa (Fig 3F). Expression of VEGFR-1 was not, however, so evident (data not shown). Because MMPs have been shown to be implicated in angiogenesis,20,21 expression of MMP-2 and MMP-9 was determined in whole mounts. MMP-2 was expressed in normal mucosa (Fig 3G) and in Barrett’s epithelium (Fig 3H. In contrast, MMP-9 was expressed more in Barrett’s epithelium (Fig 3J) than in normal esophageal mucosa (Fig 3I). Interestingly, anti–MMP-2 (Fig 4A) and anti–MMP-9 antibodies (Fig 4C) showed distinct staining of blood vessels in Barrett’s adenocarcinoma, whereas in controls, this pattern was not visible (Fig 4B and 4D).
Blood Vessels Are Immature in BE-Malignant Progression A feature that distinguishes pre-existing vessels from newly formed vasculature might be the state of vessel maturation.22 To assess this, whole mounts and paraffin sections were stained for SMA to detect perivascular smooth muscle cells and pericytes.23 As expected, the muscularis mucosa layer within the submucosa was strongly stained in normal esophagus, Barrett’s epithelium, dysplasia, and adenocarcinoma stages of tumor progression (open arrows in Fig 5A to 5D). However, the overall structure of the muscularis mucosa became more fragmented during development of esophageal adenocarcinoma, as seen in whole mounts (Fig 5A to 5D. In addition, mature blood vessels were stained (closed arrows in Fig 5A to 5D).
By comparison of blood vessel staining by EN4 (stains all blood vessels; Fig 1C and 1D) and anti-SMA antibodies (stains the actin of mature vessels; Fig 5E to 5H) it became evident that the newly formed tumor vessels were immature and devoid of SMA. Quantification of this phenomenon showed a significant increase in the percentage of SMA-negative/EN4-positive vessels, 5% in BE, 25% in dysplasia, and 40% in adenocarcinoma; whereas, the blood vessels of normal esophageal mucosa were all SMA-positive (Fig 5E). Characteristic features of BE and its progression to adenocarcinoma are the disappearance of a clear border between the mucosa and the submucosa and the increasing volume of glandular epithelium inside the submucosa (Fig 1B). The possible association of this appearance of glandular epithelium with the onset of expression of angiogenic growth factors was analyzed. On paraffin sections, expression and secretion of VEGF-A by the goblet cells specific for glandular epithelium is shown (Fig 5J to 5L). Thus, in parallel with the progressive downregulation of SMA expression (Fig 5G and 5H), VEGF-A expression was upregulated (Fig 5K and 5L).
Lymphangiogenesis in Barrett’s Adenocarcinoma
Among the controversies concerning carcinoma of the cardia and esophagus is if they represent the same disease at different locations or not.24,25 The main macroscopic difference between cardia and esophagus is the net-like blood vessel structure lining the esophageal mucosa but not the cardia. In this study, we show that the characteristic flaming of BE is not a question of erosive esophagitis, a potentially serious condition associated with chronic reflux, but instead a consequence of incipient angiogenesis. Barrett’s epithelium derives its increased blood supply from the pre-existing vasculature in submucosal lamina propria and these new blood vessels occupy the mucosa layer, giving it its ominous salmon-pink color. The results indicate that BE is indeed an angiogenesis-dependent disease. BE is associated with an increased risk of esophageal adenocarcinoma, even when only a short segment is present.12,24 In breast cancer, melanomas, gliomas, and lung, bladder, and prostate cancers, the greater the degree of angiogenesis detected in a primary tumor, the worse the prognosis.6 A recent article shows the first immunohistochemical evidence of remodeling of Barrett’s mucosa by microvascular invasion,26,27 and, in our article, we confirm this result. First, CD31 staining shows the increase in the microvessel density both in the mucosa and in the underlying submucosa of Barrett’s epithelium. Second, using a whole mount staining technique for surgically resected Barrett’s patients, we show three-dimensional evidence of a highly abnormal neovasculature during an early stage of tumor development, particularly in the Barrett’s epithelium. The angioarchitecture within precancerous Barrett’s epithelium consists of new microvessels that are very small, deformed, containing tortuousities, twisted structures, blind ends, and abnormal branching characteristics (Figs 3B and 6B). In Barrett’s adenocarcinoma, however, significance of angiogenesis on prediction of disease prognosis remains to be established.28,29 Our results suggest a greater role for angiogenesis in BE progression than earlier recognized. However, any interpretations have to be taken cautiously because only 15 resectable Barrett’s dysplasia and adenocarcinoma patients were included in the present study. Our comparison of Barrett’s epithelium with normal esophageal mucosa indicated that VEGF-induced angiogenesis might be of importance for tumor progression. We show that Barrett’s specific glandular epithelium, characterized by goblet cells, secretes VEGF-A (Figs 3 and 5), in addition to a mixture of sialomucin and sulfated mucins.15 The receptor of VEGF-A, VEGFR-2, is strongly expressed on angiogenic blood vessels feeding the Barrett’s epithelium. In addition, MMP-2 and MMP-9 are expressed along angiogenic blood vessels, indicating a role for these proteases in matrix remodeling. The results together suggest an interesting functional interplay between angiogenic goblet cells and new invading blood vessels in neovascularization of Barrett’s epithelium. A feature that distinguishes quiescent nontumor endothelium from proliferating tumor endothelium is that a fraction of the tumor endothelium has not yet recruited smooth muscle cells and pericytes.22 These mural cells are incorporated within the basement membrane of a stable, mature vessel bed in lamina propria in normal esophagus. Our results show that an increasing fraction of tumor blood vessels were devoid of pericytes and smooth muscle cells and, therefore, are immature during Barrett’s epithelium progression. In parallel, VEGF-A expression was upregulated. It seems that a high level of VEGF-A can sustain immature blood vessels in the relative absence of pericytes and smooth muscle cells. This result is in concert with an earlier study of human prostate cancer whose immature blood vessels are dependent on androgen-mediated VEGF-A expression for survival.23 Notably, nearly 80% of the Barrett’s adenocarcinoma patients who proceed to surgery, have metastasis-positive lymph nodes. We show here that expression of the lymphangiogenic growth factor VEGF-C and its receptor VEGFR-3 are upregulated in Barrett’s metaplasia, dysplasia, and most strongly in adenocarcinoma. This agrees with the notion that when the invasion of esophageal cancer reaches the muscularis mucosa in submucosa, the first cases with positive lymph node metastasis can be observed.13,14 Comparison of staining of blood vessels with different antibodies in Fig 3A and 3B (PAL-E), Fig 6A to 6D (EN4), and Fig 6I to 6J (anti–VEGFR-3) reveals that VEGFR-3 becomes upregulated on the lymphatic vessels, which are morphologically clearly distinct from the angiogenic microcapillaries. Unfortunately, we could not quantify the number of lymphatic vessels during development of esophageal adenocarcinoma, as the anti–VEGFR-3 antibody did not stain paraffin or cryosections. In addition, the other often used anti–VEGFR-3 antibody does not distinguish lymphatic vessels from blood vessels on human cancer tissue sections.10 Despite this problem, the whole mount results indicate that induction of lymphatic vessel growth may occur in human cancers, as reported previously.9,10,30 Interestingly, in support of the recent finding that overexpression of VEGF-C in breast cancer cells increase intratumoral lymphangiogenesis in nude mice,31 lymphatic vessels were seen to penetrate the adenocarcinoma tumor stroma (Fig 6L). This could be instrumental for the metastatic process, and it is reasonable to assume that the thin-walled lymphatic vessels offer less resistance and more contact area for penetration of adenocarcinoma cells into the lymphatic system than blood vessels. Thus, tumor lymphangiogenesis may be an important phenomenon for the frequent lymph node metastasis in Barrett’s adenocarcinoma patients. The current clinical concern is that diagnosis of Barrett’s esophageal adenocarcinoma is usually made late. The clinical histology allows detection of low- or high-grade dysplasia, which are the only accepted criteria for identifying patients at high risk of adenocarcinoma development. Therefore, there has been a continuous search for biologic markers specific for Barrett’s adenocarcinoma progression to complement clinicopathology. So far, the molecular markers that could be of greater diagnostic value than the direct detection of dysplasia have been expected to be found on the genetically unstable Barrett’s adenocarcinoma cells12,32,33 or in the bloodstream.34 Our results, however, raise the possibility that the proliferating endothelial cells of new blood vessels might serve as an important additional source for angiogenic, diagnostic markers specific for the progression of Barrett’s epithelium.
Supported by grants from the Research Foundation of the Helsinki University Central Hospital (EVO), the Magnus Ehrnrooth Foundation, and the Paulo Foundation, Helsinki, Finland. We thank Tuomo Timonen, MD, Senior Lecturer, for his pathohistologic expertise.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
