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Barrett’s esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barrett’s esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barrett’s esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5.
Each year, more than 461,000 new cases of esophageal cancer are diag-nosed and more than 385,000 people die of this disease worldwide. Esophageal cancer is the ninth most commonly diagnosed cancer, but it is the fifth leading cause of death associated with cancer, as the symptomatic presentation of this cancer occurs late in the course of disease (http://info.cancerresearchuk.org/). In the Western world, 50% of esophageal cancers are adenocarcinoma (EAC), and the inci-dence of EAC is markedly increasing in these regions1.
EAC is a condition that is ideally suited to early detection using endoscopic surveillance programs because it has an identifiable pre-cancerous stage (Barrett’s esophagus). Treatment for pre-symptomatic EAC diagnosed using surveillance methods is associated with a mark-edly improved patient outcome2. In addition, there is an identifiable transition phase between Barrett’s esophagus and EAC known as high-grade dysplasia (HGD) that is associated with progression to EAC in up to 59% of patients within 5 years3, and it offers a stage at which cancer can be prevented using endoscopic interventions such as radio-frequency ablation4. Despite this fact, routine surveillance is under
dispute5 because it is costly, invasive and prone to sampling error, as HGD can be patchy, confined to very small areas and appear macro-scopically indistinguishable from non-dysplastic mucosa. Current surveillance regimens recommend quadrantic biopsies be taken every 2 cm within the Barrett’s segment6 to maximize the chance of detecting dysplasia. However, this method samples only a small frac-tion of the Barrett’s epithelium and may provide false reassurance to those individuals in whom dysplasia is present but is not detected7. This concern also means that many patients with Barrett’s esophagus without dysplasia, who are at low risk of progression to EAC, undergo unnecessary biopsies.
Endoscopic technologies aimed at improving the detection of dys-plasia are developing rapidly. However, none of these technologies has been adopted into routine clinical practice because of problems with high magnification and the associated small field of view, the need for expensive equipment and specialist interpretation, and, in some instances, a high false positive rate. These technologies include commercially available narrow band imaging8, confocal laser endo-microscopy9 and a number of optical biopsy techniques which remain research tools, such as angle-resolved low coherence interferometry10, Raman spectroscopy11, optical coherence tomography12 and elastic scattering spectroscopy13,14.
A concept that has emerged recently is to use knowledge of specific molecular and biochemical alterations occurring in cancer progres-sion to develop molecular imaging tools. For example, endoscopic molecular imaging using fluorescently labeled targeted peptides has been investigated for detection of colonic dysplasia15 and in Barrett’s esophagus ex vivo16; however, both of these studies used microscopic imaging techniques, which have a small field of view that would make it difficult to translate into routine clinical practice. Furthermore, those studies identified the targeting peptides in screening experi-ments by their binding, or lack thereof, to certain cell lines, and, there-fore, the molecular targets of these probes have not been identified.
Glycans are abundant and have large and diverse structures, and terminal glycan groups (such as sialyl LewisA and sialyl Tn antigen) have been shown to be altered in cancers such as those of the pancreas,
Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett’s esophagusElizabeth L Bird-Lieberman1, André A Neves2, Pierre Lao-Sirieix1, Maria O’Donovan3, Marco Novelli4, Laurence B Lovat5, William S Eng6, Lara K Mahal6, Kevin M Brindle2 & Rebecca C Fitzgerald1
1Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, UK. 2Cancer Research UK Cambridge Research Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK. 3Department of Pathology, Addenbrooke’s Hospital, Cambridge, UK. 4Department of Pathology, University College London, London, UK. 5Department of Gastroenterology, University College London, London, UK. 6Department of Chemistry, New York University, New York, New York, USA. Correspondence should be addressed to R.C.F. ([email protected]).
Received 9 November 2010; accepted 23 May 2011; published online 15 January 2012; doi:10.1038/nm.2616
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colon and stomach17–19. Glycans therefore have the potential to be used as a molecular target for the endoscopic imaging of mucosal surfaces. Unsupervised clustering of gene expression profiling data from 75 esophageal cancers20 (Gene Expression Omnibus GSE19417) highlighted a cluster of samples with overexpression of genes related to glycan synthesis (P = 0.0002 compared to the other groups) (Supplementary Fig. 1). Glycan changes can be detected using lectins, which are carbohydrate recognition proteins of non-immune origin that have specificity for particular glycan structures. Lectin probes have the advantage that their binding specificities are well estab-lished21 and their relatively large size (20–200 kDa) means that their binding is less likely to be affected by fluorescent labeling. Lectins are also abundant, making them relatively inexpensive to produce, and they are heat stable, stable at low pH and resistant to proteolysis22. Lectins are also attractive as probes because they are constituents of the normal human diet and, therefore, depending on the particular lectin and the concentration used, can have low toxicities.
We describe here a molecular imaging approach in which fluores-cence endoscopy and a fluorescently labeled lectin are used to detect the changes in glycan expression on the epithelial cell surface that accompany the transition from Barrett’s esophagus through dysplasia to EAC in situ.
RESULTSDysplasia alters glycan expression and lectin bindingWe analyzed gene expression profiling data from samples repre-sentative of the stages of progression that precede the development of EAC using gene set enrichment analysis to determine the point at which glycan pathways were upregulated. This analysis revealed four glycan pathways in which there were coordinated increases in the expression of genes encoding proteins involved in the biosynthe-sis and degradation of glycan structures in the progression to EAC (Fig. 1a). The pathway for glycan degradation was enriched during the metaplastic transition to Barrett’s esophagus, whereas the bulk of enrichment in the glycosphingolipid pathway lactoseries took place as Barrett’s esophagus progressed to LGD, and the neo lactoseries increased between LGD and HGD. These data indicate that coordi-nated changes in glycan expression begin before the emergence of EAC and, therefore, glycans have the potential to act as biomarkers for the detection of dysplasia and the identification of those individuals at risk of progression from Barrett’s esophagus to EAC.
We applied human esophageal samples across the progression sequence, independent from those of the gene expression profiling cohort, to a ratiometric lectin array23. An unsupervised clustering analysis revealed a group of lectins (Fig. 1b) with high binding to human esophagus, but which then decreased in binding during pro-gression to EAC (P < 0.000001). Because lectin array technology is new and has not often been used with human tissue, we applied
the same samples to two independent arrays. The evanescent lectin array24 confirmed significant changes in binding (P < 0.05) in the progression to EAC for four of the lectins within the cluster identi-fied above, and we therefore took these lectins forward for validation. These four lectins were Aspergillus oryzae lectin (AOL), Helix pomatia agglutinin (HPA), Trichosanthes japonica agglutinin-I (TJA-I) and Tritiicum vulgare agglutinin (WGA).
Validation of lectin binding changesWe performed histochemistry of the lectins in an independent cohort of biopsies (cohort 3 described in the Online Methods) taken from sub-jects known to have dysplasia within their Barrett’s segment (Fig. 2). TJA-I stained the stroma and, therefore, would not have been useful as an endoscopic imaging probe, and we therefore discarded it. WGA, HPA and AOL all showed a highly significant decreased binding (P < 0.001, P < 0.05 and P < 0.0001, respectively, using a Jonckheere-Terpstra test) in the progression to EAC, and we confirmed this statistical significance using analysis of variance with Bonferroni correction. WGA (from wheat germ) and AOL (from Aspergillus) showed the most significant changes in binding. However, as WGA is a normal dietary constituent in humans, we considered that this lectin had the greatest potential for use in vivo, and we therefore took it forward for further proof-of-principle work.
We obtained fluorescence images from whole biopsies incubated with WGA labeled with Alexa Fluor 680 (Fig. 3). As expected from the results of the lectin arrays and the lectin histochemistry, biopsies that contained dysplastic tissue showed lower fluorescence intensities,
a bSquamous esophagus Barrett’s esophagusLGDHGD
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Figure 1 Array data. (a) Gene set enrichment analysis of gene expression data (squamous esophagus (NE), n = 6; Barrett’s esophagus (BE), n = 21; LGD, n = 17; HGD, n = 13) revealed four glycan pathways that had a significant trend in their enrichment scores in the progression from squamous esophagus to HGD (Jonckheere-Terpstra P < 0.05). (b) An unsupervised clustering of lectin array (n = 78 lectins) data (squamous esophagus, n = 6; Barrett’s esophagus, n = 5; LGD, n = 5; HGD, n = 5) grouped all but one of the HGD samples together. A cluster of lectins (indicated by vertical brackets) was characterized by its members’ strong binding to the human samples. Within this cluster of lectins, there was significantly lower binding to the HGD samples compared to non-dysplastic Barrett’s esophagus.
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indicating that the fluorescently labeled WGA probe had the potential to allow macroscopic identification of molecular alterations using fluorescence endoscopy. Furthermore, because reflux-exposed esophagus is frequently inflamed, we assessed the degree of inflam-mation in these biopsies to ensure that it did not account for any alterations in WGA binding. Sixty-four percent of the biopsies had mild chronic inflammation and 20% had acute inflammation, but this inflammation occurred across all biopsy types and did not correlate with WGA binding.
We determined the specificity of the WGA binding in two ways. N-acetylglucosamine is the monosaccharide that shows the strongest competition with WGA25 for binding, and, at high concentrations, will displace it. We therefore performed a competition assay with N-acetylglucosamine and WGA. Then, as α-neuraminidase cleaves sialic acid residues, we compared whole-biopsy binding of WGA after incubation with α-neuraminidase to the binding of a paired biopsy that had not undergone α-neuraminidase digestion. Competition from N-acetylglucosamine (P = 0.03) and pre-incubation with α-neuraminidase (P = 0.01) resulted in significant decreases in the biopsy fluorescence of normal squamous esophageal tissue, which showed the highest WGA binding of all the biopsy types tested (Fig. 3).
Imaging WGA using a clinical fluorescence-capable endoscopeWe conducted proof-of-principle studies on four specimens from separate patients obtained immediately after resection. We per-formed these studies using an endoscope in a way that exactly mimics a clinical study in vivo. Briefly, we intubated esophagi from the proximal end with a clinical fluoresence endoscope. After we obtained baseline white-light and auto-fluorescence images, we sprayed the esophagi topically with fluorescein-labeled WGA and then imaged it endoscopically with excitation light between 395–475 nm and emission light between 500–630 nm using a charge-coupled device (CCD) camera (Online Methods). The white-light endoscopic images that we obtained using this method were indis-tinguishable from those obtained during clinical endoscopic imaging of the esophagus in situ using standard methods. We then opened the esophagus and imaged it using an IVIS camera, which permits the quantification of fluorescence using a color-coded intensity scale. We used a grid map to allow for co-registration of pathological changes detected using the fluorescence images from the endoscope and from the IVIS camera.
We specifically selected subjects for these studies who had macro-scopically visible Barrett’s esophagus; three subjects had early lesions and one had more advanced disease (Table 1). In the subject with advanced disease (OES2, stage T3N0), the WGA binding, and
therefore the fluorescence intensity, of the adenocarcinoma and the Barrett’s esophagus–associated dysplasia was lower than that of non-dysplastic Barrett’s esophagus and squamous mucosa. In samples from subjects OES1 and OES4, there were no visible lesions in the white light images, but WGA fluorescence highlighted extensive dysplastic areas in these samples (P = 0.0057, comparing EAC or dysplasia with areas of squamous or Barrett’s esophagus). The esophagectomy speci-men from subject OES1 had a 6-cm segment of Barrett’s esophagus containing macroscopically invisible residual HGD and focal intra-mucosal carcinoma after a previous endoscopic mucosal resection (Fig. 4). We detected differences in WGA binding using the endo-scope as well as with the IVIS camera. By overlaying a grid onto the endoscopic and ex vivo images, the alterations in WGA binding could be co-registered with the histopathological assessments. There was a highly significant statistical correlation between WGA fluorescence and the degree of dysplasia (P = 0.0002). Areas of HGD within the esophagus showed low WGA binding. The scar from the previous endoscopic mucosal resection (EMR) (2 cm specimen removed endoscopically) also showed low WGA binding. The contrast-to- background ratio obtained was greater than 10, and the signals from areas of HGD were more than 10 times lower than from the sur-rounding normal tissue. Inflammation can be a confounding factor in endoscopic imaging; however, using standard histopathological criteria, there was no correlation between the degree of inflam-mation and the WGA fluorescence pattern (see Supplementary Fig. 2 for the OES1 inflammation map; P = 0.82 and P = 0.68 for
LGD
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Figure 2 Fluorescent lectin histochemistry validation (cohort of n = 80 biopsies). (a–c) The data in each column are from a single lectin: WGA (a), HPA (b) and AOL (c). The top row shows representative examples of lectin histochemistry for WGA-555 (yellow) (a, top), HPA-488 (green) (b, top) and AOL-555 (yellow) (c, top); a DAPI counterstain highlights the cell nuclei in blue (magnification, ×20). The second row of images shows the corresponding sequential section stained with H&E for each lectin. Data in the graphs are means ± s.e.m. staining for each lectin, with the n number of samples shown below each bar in the graphs. The top row of graphs shows the staining for the apical epithelial membrane (the part of the cell membrane that lines the esophageal lumen). The bottom row of graphs shows the staining of the epithelial mucous globules (the intracellular aggregations of mucus). The P value represents the Jonckheere-Terpstra test for trend. *P < 0.05 by ANOVA with Bonferroni correction, **P < 0.01, ***P < 0.001.
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chronic and acute inflammation, respectively). Similar correlations between histopathology and WGA fluorescence are shown for the EMR specimen (subject OES4) in Supplementary Figure 3. Subject OES3 had HGD with no visible lesion, which was confirmed on two separate occasions at endoscopic surveillance. This subject opted for esophagectomy rather than endoscopic therapy with radiofrequency ablation. We detected no WGA binding abnormality in the ex vivo endoscopic examination of this subject. Histopathological examina-tion of the entire resected esophagus revealed a single subsquamous focus of HGD (<1 mm in diameter), which was therefore inacces-sible to WGA. The rest of the Barrett’s esophagus sample was non-dysplastic. Therefore, this subject served as a negative control. The mean signal-to-background ratio (SBR) (for dysplasia compared with squamous) for the three informative specimens was 5.2 ± 3.9 (s.d.), with a signal-to-noise ratio (SNR) (average signal in the imaging field outside the specimen or instrument noise) of 30.3 ± 15.1 (s.d.).
DISCUSSIONWe have shown that glycosylation patterns are candidate biomark-ers for detecting disease progression in Barrett’s esophagus and that
these biomarkers can be detected using currently available fluores-cence endoscopes and fluorescently labeled lectins that are sprayed on to the mucosal surface of the tissue. An advantage of using fluo-rescently labeled lectins as compared to other molecular imaging techniques that are based on peptide-targeting agents15,16 is that we selected a specific target of known function and showed its relevance in gastrointestinal tract malignancy. In addition, the SBR >5 (with an SNR > 30) that we obtained in this study of Barrett’s dysplastic tissue is considerably higher than those obtained using other large targeted molecular imaging agents reported in the literature. Values for the SBR of less than 2 are commonly obtained after systemic admin-istration of antibody-based agents26,27, which have slow clearance
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Figure 3 Representative whole-biopsy fluorescence. Fresh biopsies were labeled at the bedside with WGA that was labeled with Alexa Fluor 680. The color-coded intensity scale is shown beneath the fluorescence images. (a) Biopsies subsequently shown to contain dysplasia had lower whole-biopsy fluoresence than non-dysplastic biopsies (P = 0.01 by Wilcoxon signed rank test). Depiction of the overall biopsy fluoresence is shown at the top, examples of the relevant histology are shown at the bottom (magnification, ×10), and the graph shows a boxplot representing the mean ± s.e.m. of the data, with the whiskers indicating the total range of the data. (b) A glucosamine competition assay shows that glucosamine removes almost all the fluorescently labeled lectin (P = 0.03 by Wilcoxon signed rank test). The images show representative examples of overall biopsy fluoresence before (−) and after (+) incubation with N-acetylglucosamine, and the graph shows the data for all biopsies (n = 5). (c) Treatment with α-neuraminidase produced a significant decrease in WGA labeling (P = 0.01 by Wilcoxon signed rank test). The images show representative examples of overall biopsy fluorescence without (−) and with (+) incubation with α-neuraminidase, and the graph shows the data for all biopsies (n = 6). *P < 0.05 by Wilcoxon signed rank test.
Table 1 Fluorescence imaging of resected esophageal lumen tissue ex vivo labeled with WGA and Alexa Fluor 488 using the IVIS 200 cameraOES1 OES2 OES3 OES4 Mean
Indication Intramucosal Ca + HGD Poor diff EAC HGD Intramucosal Ca + HGD N/A
Regions of interest were identified in each specimen based on differential levels of fluorescence and were subsequently assessed by histopathology. Intramucosal Ca, intramucosal carcinoma; EMR, endoscopic mucosal resection; N/A, not applicable.aValues are the mean fluorescence intensity ×10−5 of three readings (± s.d.). bThe signal-to-background fluorescence ratio of squamous esophagus and Barrett’s esophagus. cThe signal-to-background fluorescence ratio or Barrett’s esophagus and dysplasia. The lesions were staged by endoscopic ultrasound (EUS) prior to surgery and then again by the findings in the pathological specimen following surgery using standard staging systems. The T stage represents the degree of tumor spread, N represents nodal involvement and M identifies metastatic spread. X denotes if this information is not determined.
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kinetics and, thus, long washout times (2–8 d)28. Using fluorescence confocal microendoscopy and a targeted peptide sprayed onto the tissue, researchers from a previous study15 reported an SBR between normal and adenomatous epithelial tissue in the colon of 17.9 ± 4.2 (with an SNR of 9.3 ± 0.9). Another study16 also recently proposed a peptide for detecting dysplasia in Barrett’s esophagus that was selected by phage display technology and validated on histologi-cal specimens using fluorescence stereomicroscopy. However, that peptide showed only a twofold higher binding to dysplasia than to normal squamous esophagus.
Although glycan expression has been known to be altered in cancer and these changes are known to be associated with a poor progno-sis19,21,29, there is a paucity of data about changes in glycan expression in esophageal EAC. We have shown that EAC behaves similarly to other carcinomas and is associated with coordinated changes in gene expression in specific glycan pathways (especially the glycosphin-golipid synthetic pathways) that occur during different stages of disease progression (for example, the transition from squamous to Barrett’s esophagus and the development of dysplasia). Changes in glycan expression during the transition to dysplasia offer potential biomarkers to identify tissue that is on the pathway to EAC.
The study of resected esophagi highlights the different clinical sce-narios in which molecular imaging with WGA could be useful. In two subjects, flat dysplastic lesions in a Barrett’s segment were invisible
with conventional white-light endoscopic images. In such individuals, lectin imaging would be valuable in delineating the area of concern for biopsies and for EMR. In another subject, in which WGA fluorescence highlighted no abnormality, resection (which was arguably unneces-sary) revealed a <1 mm diameter area of dysplasia. Molecular imag-ing may have helped with counseling this patient toward endoscopic ablative therapy or monitoring, which would have had substantially less comorbidity compared to esophagectomy. Compared with the current random biopsy protocol, fluorescence imaging with WGA has the potential to delineate the extent of disease and therefore help inform the choice of treatment.
Using glucosamine competition measurements and removal of sialic acid residues using neuraminidase, we confirmed that WGA binds specifically to sialic acid and that there is a decrease in sialic acid expression in the apical membrane and superficial epithelium in the progression to EAC. This decrease may be the result of sialic acid residues becoming hidden by the addition of other groups, such as through O-acetylation30. Although we restricted our endoscopic stud-ies to WGA because of the limited availability of resected esophagi and the potential clinical acceptability of WGA, it would be interesting to further evaluate AOL, which also showed a significant reduction in binding in the metaplasia-dysplasia-carcinoma sequence.
The toxicity of WGA is a key consideration if it is to be used as a targeted imaging agent in the clinic. The concentration of WGA we
Figure 4 Whole-organ imaging ex vivo. (a) Images taken with an endoscope. White-light image (left), imaging fluorescence at 490–560 nm before the application of WGA (middle) and imaging fluorescence at 490–560 nm after application of WGA and Alexa Fluor 488 (right). The areas of low WGA binding appear in purple. The dashed white line is placed longitudinally along the posterior wall of the esophagus to facilitate orientation between the different images, and the numbers 7, 8 and 9 refer to the y coordinates on the reference grid in b. White-light imaging of the lower esophagus revealed no macroscopic abnormalities such as ulcers or nodules, and before WGA application, we detected no appreciable differences in mucosal auto-fluorescence; however, after incubation with WGA, differences in lectin binding were evident, in which high binding is represented by a green signal and low binding is represented by a purple signal on the pseudocolor image. (b) Grid showing the pathological diagnostic map (color coded, with darker color representing a worsening grade of dysplasia) of each block made from the resection specimen. This same grid can be compared with the endoscopic and IVIS fluorescence images in a, on the right, and d. The dashed line represents the longitudinal axis along the posterior wall of the esophagus. (c) The same specimen after being opened longitudinally along the anterior border of the esophagus is shown with the overlying grid from b. (d) The WGA fluorescence signal from the esophageal specimen taken with the IVIS 200 camera. The pink arrow marks an area of artifact from the exposed submucosal tissue, and the blue arrow indicates the site of a previous endoscopic mucosal resection (outlined with a dashed gray box). The specimen was cut into 11 transverse sections (rows labeled 1–11) and each of these was further divided into 8 areas (columns labeled A–H) by the pathologist to allow mapping. (e) Examples of the histological appearance (magnification, ×40) at various coordinates from the grid. From left to right, the images show non-dysplastic Barrett’s esophagus, LGD and two examples of HGD. The corresponding grid reference is given at the bottom of each image.
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used in the experiments reported here was far below the concen-trations shown to cause toxicity in animal models (>0.05 mg ml−1), during which the animals were also exposed to WGA for much longer periods of time than the exposure times we used here (multiple hours compared to 10 min, respectively). We do not expect significant cel-lular uptake of WGA, as this effect is reported to take between 1 h and 4 h31. Nevertheless, any potential toxicity in using WGA should be minimized, and, therefore, we propose that after image acquisi-tion, glucosamine should be applied in vast excess (1,000-fold molar excess) to the lectin concentration to compete with and force off lectins bound to the mucosal surface.
Another key consideration before such a technique can be applied clinically is the delivery mechanism. The technique that we have developed involves local application of the fluorescently labeled lectin using a spray catheter introduced into the biopsy channel of the endoscope. The lectins would be present for up to 10 min to permit visualization and collection of targeted biopsies before the application of glucosamine to wash off the bound lectin.
The study has several potential limitations. Lectin binding is reduced in the context of dysplasia, whereas ideally one might select an imaging agent that shows an increased signal from diseased regions compared to normal tissue (a ‘positive contrast agent’). There is how-ever a potential benefit of using a negative contrast agent such WGA. A positive contrast agent may lose specific signal as a result of con-founding factors diminishing specific binding and, thus, increasing the chance that dysplasia would be missed. A negative contrast agent such as WGA will result in all areas of low binding being biopsied, minimizing the risk of clinically important dysplasia being missed. We used a fluorescein-based probe, as it has a proven safety profile and can be detected using currently available endoscopes; however, the use of longer wavelength fluorophores would minimize interference from tissue autofluoresence. Alternatively, a multispectral endoscope could be used to suppress tissue autofluorescence32,33. This study was restricted to measurements on tissues ex vivo. Although we examined segments of excised esophagus with a fluorescence endoscope in a manner similar to that of a clinical examination, we did not immedi-ately histologically sample areas identified with fluorescence endos-copy. In addition, we investigated only a small number of esophagi in this manner. The next step is to test this technique in vivo in subjects with dysplasia and intramucosal carcinoma and to perform real-time histological sampling. This in vivo information could then be used to determine the sensitivity and specificity of the technique in target-ing dysplastic lesions compared to current practice. In conclusion, lectins are relatively cheap and nontoxic imaging probes that have the potential to be used in conjunction with conventional fluorescence endoscopes to screen for the presence of dysplasia in the context of Barrett’s esophagus to help guide patient management.
METhODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/.
Note: Supplementary information is available on the Nature Medicine website.
ACKNOWLEDgMENtSThis work was supported by a research fellowship from GlaxoSmithKline awarded to E.L.B.-L. The authors thank J. Ong, C. Peters, S. Hilborne, E. Moore, N. Shannon, B. Spencer, R. Cayado-Lopez, B. Haynes, J. Gray, W. Howat, J. Harris and S. Reichelt for their advice and support and the Moritex Corporation for lending the evanescent lectin array equipment (Glycostation). This work was supported by a Medical Research Centre core grant (R.C.F.), the Cambridge
Experimental Medicine Centre (R.C.F.), the National Institute for Health Research Cambridge Biomedical Research Centre (R.C.F.), a Cancer Research UK core grant (K.M.B.) and a Clinical Research Fellowship from GlaxoSmithKline (E.L.B.-L.). The work undertaken in the Department of Chemistry, University of New York was supported by a New York University startup fund and by the US National Institutes of Health (W.S.E. and L.K.M.). The work undertaken at UCLH/UCL was supported in part by funding from the Department of Health’s NIHR Biomedical Research Centre’s funding scheme and also by a grant from Cancer Research UK to the Experimental Cancer Research Centre, UCL. The views expressed in this publication are those of the authors and not necessarily those of the UK Department of Health.
AUtHOR CONtRIBUtIONSE.L.B.-L. performed the experimental work and data analysis with help from A.A.N., P.L.-S. and L.K.M. M.O. and M.N. performed histopathological analyses. L.K.M. and W.S.E. applied prepared samples for the ratiometric array, and L.B.L. provided samples. R.C.F. and K.M.B. conceived of and supervised this work. The manuscript was written by E.L.B.-L. and R.C.F.
COMPEtINg FINANCIAL INtEREStSThe authors declare no competing financial interests.
Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
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ONLINE METhODSHuman tissue. After obtaining ethical committee approval (07/H0305/52, 01/149 and 01/0039) and informed consent from the study participants, tissue was collected from patients at Addenbrooke’s Hospital (Cambridge) and University College Hospital (London) between 1999 and 2010. Clinical diagnoses of cancer and dysplasia were performed by two independent patho-logists and were reviewed by an expert gastrointestinal histopathologist (M.O.). Concomitant inflammation was reported using standard histopathological criteria. The patients had a male to female ratio of 4.7 and an average age of 67 years (range, 45–82 years), and the mean length of the subjects’ Barrett’s segments was 6.8 cm (range, 2–15 cm). For all subjects, one single biopsy was used per subject per study, but a given biopsy may have had more than one grade of dysplasia, in which case each grade was scored separately.
Cohort 1. The progression gene expression array consisted of squamous esophagus (n = 6), Barrett’s esophagus (n = 21), LGD (n = 17) and HGD (n = 13) from fresh frozen tissue.
Cohort 2. The lectin array used fresh frozen tissue from squamous esopha-gus (n = 6), Barrett’s esophagus (n = 5), LGD (n = 5) and HGD (n = 5).
Cohort 3. The histochemistry validation cohort used n = 80 paraffin-embedded biopsies from subjects with HGD or an intramucosal carcinoma within a Barrett’s segment, which was confirmed on two separate endoscopies.
Cohort 4. Ex vivo lectin-staining biopsies (squamous esophagus, n = 8; Barrett’s esophagus, n = 18; LGD, n = 3; HGD, n = 4) were stained with lectin at the bedside. Whole-esophageal work (n = 4) was performed using freshly resected tissue obtained after surgery or EMR.
Gene set enrichment analysis. Specific glycan pathways identified from the Kyoto Encyclopedia of Genes and Genomes were applied to progression expression array data (Gene Expression Omnibus GSE19417). Gene set enrich-ment analysis software version 2.0 (Broad Institute) was used to calculate the normalized enrichment scores.
Lectin arrays. Samples from cohort 2 were used for the lectin arrays23,24. Homogenized samples were sonicated (three 5-s pulses at 70% amplitude; Sonics, Vibra-Cell), followed by ultracentrifugation at 5 °C, 100,000g for 1 h. The pellet was resuspended in 0.1 M Na2CO3, pH 9.3, and the protein con-centration was determined. A 1:1 ratio of Cy3 or Cy5 dye (GE Life Sciences) was added for each milligram of protein.
The slides were scanned using a GenePix 4100A fluorescent slide scan-ner (Molecular Devices), and the data were extracted using GenePix Pro 5.1 software (Molecular Devices). Median normalized values for each spot were used. Replicate data were tested statistically using the Grubbs’ outlier method. The Yang method was used to calculate the dye-bias–corrected ratios. Cluster 3.0 with Java TreeView (http://rana.lbl.gov/EisenSoftware.htm) was used to create the hierarchical clustering map.
Lectin histochemistry. Slides were deparaffinized, placed in a humidified incubation chamber, and 5 µg ml−1 of lectin was applied at 37 °C for 15 min, and then the slides were washed in water and mounted with Prolong Gold antifade with DAPI (Invitrogen).
Ex vivo imaging. Biopsies were incubated at the bedside with WGA and Alexa Fluor 680 conjugate (Invitrogen) at 5 µg ml−1 for 15 mins, washed in PBS, fixed in 4% paraformaldehyde, scanned using an IVIS 200 camera (Caliper Life Sciences), paraffin embedded and cut for review by a gastrointestinal pathologist.
Esophagectomy imaging. The endoscope (Evis Lucera gastrointestinal video-scope GIF Type FQ260Z with a xenon light source CLV-260SL) was passed through the esophageal lumen immediately after resection and after air-tight stapling of the stomach end, and air insufflation was used. Baseline white-light and auto-fluorescent images were obtained. After topical application of 20 ml of 10 µg ml−1 WGA and Alexa Fluor 488 (Invitrogen) (wavelength of excitation (λex) = 488 nm; wavelength of emission (λem) = 519 nm) for 10 min, excitation light of 395–475 nm was applied through the endoscope, and the fluorescence emission was detected between 500–630 nm using a CCD camera. The fluores-cence emission, as well as the red and green reflectances, were used to produce a pseudocolor image. The esophagus was then opened longitudinally along the most anterior border to allow for imaging with the IVIS 200 camera, which allows for fluorescence imaging. Samples were illuminated from above using a tungsten halogen light source and the emitted fluorescence was detected using a cooled CCD camera. A grid was applied to the endoscopic fluorescent image and to the image of the opened specimen so that the coordinates of any areas with altered fluorescence could be recorded and co-localized with the histol-ogy. The fresh specimen was then fixed and processed as per normal clinical protocols. Transverse blocks were taken for histological examination throughout the Barrett’s segment for dysplasia mapping using the same reference grid as the one applied to the fluorescent images.
Competition assays were performed over 12 h at 5 °C with 131 µM n-acetyl-d-glucosamine (Sigma-Aldrich), and binding specificity was assessed on adjacent paired biopsies by elimination of sialic acid residues after incuba-tion with 0.2 µg ml−1 α(2→3,6,8,9) neuraminidase (Sigma-Aldrich) for 12 h at 5 °C in one sample from the pair and then incubating both samples in the paired biopsies with WGA and Alexa Fluor 680, as described above.
Statistics. The Jonkheere-Terpstra test was used to test for trend, with P < 0.05 being regarded as statistically significant. Analysis of variance with Bonferroni cor-rection was used to correct for multiple hypothesis testing. For paired data that was not normally distributed, a Wilcoxon signed rank was applied. For ex vivo experi-ments, a two-tailed t test with a Mann-Whitney correction was applied for small sample size. The Spearman two-tailed non-parametric test was used for correlation measurements between inflammation, dysplasia and fluorescence values.
