Dig Dis Sci DOI 10.1007/s10620-014-3204-5
ORIGINAL ARTICLE
White Opaque Substance Visualized Using Magnifying Endoscopy with Narrow-Band Imaging in Colorectal Epithelial Neoplasms Takashi Hisabe • Kenshi Yao • Kentaro Imamura • Hiroshi Ishihara • Fumihito Hirai • Toshiyuki Matsui Akinori Iwashita
•
Received: 13 December 2013 / Accepted: 3 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Background The presence of a white opaque substance (WOS) on magnifying endoscopy (ME) with narrow-band imaging (NBI) has been reported for gastric epithelial neoplasms, but the presence of WOS in colorectal epithelial neoplasms has not been investigated. Aims The purpose of this study was to determine whether WOS is present in colorectal epithelial neoplasms and to clarify its clinical significance. Methods A total of 590 colorectal epithelial neoplasms from 368 consecutive patients were retrospectively analyzed using prospectively collected data. Presence or absence of WOS in colorectal epithelial neoplasms was recorded based on the findings of ME with NBI. Results White opaque substance was present in 236 of the 590 (40 %) colorectal epithelial neoplasms. Compared with WOS-negative patients, WOS-positive patients showed significantly larger tumors (p \ 0.0001) and significantly more tumors in the proximal colon (p = 0.0003). WOS was more frequently present in carcinomas (66.0 %) than in adenomas (31.8 %; p \ 0.0001). WOS was also more frequent in submucosal carcinomas (75.9 %) than in intramucosal carcinomas (59.0 %; p = 0.0380). Conclusions This study confirmed the presence of WOS in colorectal epithelial neoplasms, and prevalence increased with the progression of cancer, from adenoma to T. Hisabe (&) K. Yao H. Ishihara F. Hirai T. Matsui Department of Gastroenterology, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-8502, Japan e-mail: [email protected] K. Imamura A. Iwashita Department of Pathology, Fukuoka University Chikushi Hospital, Chikushino, Japan
carcinoma and from intramucosal carcinoma to submucosal carcinoma. Keywords Cancer Colorectal epithelial neoplasm Magnifying endoscopy Narrow-band imaging White opaque substance
Introduction New fatty acid synthesis is known to occur actively in cancer cells, with fatty acid synthase (FASN) and other lipid-metabolizing enzymes facilitating the proliferation and migration of cancer cells [1, 2]. However, how they are isolated and converted to promote malignancy remains unclear. Various lipid-metabolizing enzymes, such as monoacylglycerol lipase (MAGL) [3], and lipid dropletassociated PAT-proteins [4] are reportedly overexpressed in cancer cells. The meaning of the roles played by lipid metabolism has been a focus of attention in research into the pathology of cancer cells. Narrow-band imaging (NBI) with magnifying endoscopy (ME) has been widely applied clinically in endoscopic diagnosis of the digestive tract in recent years [5–8]. NBI utilizes an optical filter with narrow bandwidths of two different central wavelengths (415 and 540 nm). NBI with ME can clearly visualize both the subepithelial microvascular architecture and the microsurface structure. Yao et al. [9] first reported that a white opaque substance (WOS) within the gastric neoplastic epithelium was obscuring the subepithelial microvascular pattern, and suggested that this WOS could offer a new optical sign for discriminating adenoma from carcinoma when using ME with NBI. Furthermore, Yao et al. [10] demonstrated that WOS represents accumulated lipid droplets in the
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superficial part of epithelial neoplasms within the stomach. When lipid droplets within the superficial epithelium strongly reflect projected light, the projected light cannot reach the hemoglobin in microvessels, and strong backward scattering of light is recognized as white coloration by the human eye. The presence of WOS has been reported for neoplastic lesions of the stomach and duodenum [11], but has not previously been identified in the colon. This study investigated whether WOS is present in colorectal epithelial neoplasms and examined its clinical significance.
Histopathological Investigations Resected specimens were mounted on a plate and sliced at 2-mm intervals after overnight fixation in 20 % buffered formalin solution. These specimens were embedded in paraffin and cut into 5-lm-thick tissue sections. The sections were subsequently stained with hematoxylin and eosin (H&E stain). Histopathological diagnosis was made based on World Health Organization criteria [13], and tumors were classified into three groups: (1) adenoma; (2) intramucosal carcinoma; or (3) submucosal carcinoma. Definition of WOS
Materials and Methods Participants in this study comprised patients with colorectal adenoma or early colorectal carcinoma that had been examined by ME with NBI and that was endoscopically or surgically resected at Fukuoka University Chikushi Hospital between January 2009 and December 2012. Lesions that could not be visualized using ME with NBI and nonneoplastic lesions that did not require treatment were excluded. All patients provided written, informed consent for colonoscopic examinations. This study was approved by the Medical Ethics Committee of Fukuoka University Chikushi Hospital.
We defined WOS as a white substance within the colorectal superficial epithelium that obscured the subepithelial microvascular pattern. The presence or absence of WOS was investigated under maximal magnification (Fig. 1). When WOS was identified in any part of the lesion, the lesion was assessed as WOS-positive. Outcome Measures The primary endpoint was to determine whether WOS is present in colorectal neoplasms. A secondary endpoint was to investigate differences in clinicopathological characteristics between WOS-positive and WOS-negative subjects.
Endoscopic Specifications and Procedures
Statistical Analysis
All endoscopic procedures were performed using an electronic endoscopy system (Evis-Lucera Spectrum; Olympus Optical, Tokyo, Japan) with magnifying colonoscopy (CFH260AZI, PCF-Q240ZI; Olympus Optical). A soft black hood attachment was mounted at the tip of the scope. The lesion is visualized employing incremental movements of the tip of the endoscope to bring the image into focus, with a distally attached soft black hood to stabilize the tip of the endoscope without causing mucosal injury [7]. All patients were given 2–3 l of polyethylene glycol-electrolyte solution starting in the morning on the day of examination as preparation. When a colorectal epithelial lesion was detected during non-magnifying observation with whitelight imaging, the tumor surface was washed and immediately examined by ME with NBI. All endoscopic findings were analyzed retrospectively by two endoscopists (T.H. and Y.K.), each with more than 15 years of experience in the field of colonoscopy and more than 500 cases assessed by NBI with ME. In the case of differences in diagnosis, the final evaluation of endoscopic findings was made by the consensus agreement of the two endoscopists. Macroscopic classification was made according to the updated Paris classification [12].
Mean values were compared using Student’s t test. Comparison of prevalences between two groups was made using the Chi-square test or Fisher’s exact test. Values of p \ 0.05 were considered statistically significant. SPSS version 11.5J software (SPSS, Chicago, IL, USA) was used for all statistical analyses.
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Results A total of 590 colorectal epithelial neoplasms from 368 consecutive patients were investigated in this study. WOS was present in 236 of these 590 colorectal epithelial neoplasms (40 %) based on the findings of ME with NBI (Fig. 2). Comparisons of clinical features of colorectal epithelial neoplasms between WOS-positive and WOSnegative subjects are shown in Table 1. Mean tumor size was significantly greater in WOS-positive subjects (17.6 mm) than in WOS-negative subjects (11.2 mm; p \ 0.0001). By location, the proportion of tumors was significantly greater in the proximal colon (p = 0.0003) and significantly smaller in the distal colon (p \ 0.0001) in WOS-positive patients. The endoscopic macroscopic
Dig Dis Sci
A Fig. 1 Magnifying endoscopy with narrow-band imaging finding. a Morphology of the partially positive WOS shows a well-organized and symmetrical distribution. b The enlarged picture of a black
B square part revealing WOS (arrow) within the neoplastic epithelium obscures the subepithelial microvascular pattern
Fig. 2 Magnifying endoscopy with narrow-band imaging finding. a Tubular adenoma. WOS is absent and regular microvascular pattern are clearly visualized. b Tubular adenoma. Morphology of the WOS shows a well-organized and symmetrical distribution of a regular reticular pattern. c Well-differentiated adenocarcinoma (intramucosal carcinoma). Morphology of the WOS shows a disorganized and asymmetrical distribution of irregular speckled pattern. d Tubular adenoma. Morphology of the WOS shows a dense pattern
classification in WOS-negative patients included significantly greater proportions of 0-Ip, Isp, and Is lesions (p = 0.0234). On histopathological investigation, WOS was more frequently present with carcinoma (66.0 %, 93/141) than with adenoma (31.8 %, 143/449; p \ 0.0001). WOS was also more frequently present with submucosal carcinoma
(75.9 %, 44/58) than with intramucosal carcinoma (59.0 %, 49/83; p = 0.0380). Among adenomas, WOSpositive cases comprised 107 cases of tubular adenoma, 34 cases of villous adenoma, and two cases of traditional serrated adenoma. WOS-negative cases comprised 269 cases of tubular adenoma, 33 cases of villous adenoma, and four cases of traditional serrated adenoma. No significant
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Dig Dis Sci Table 1 Clinical features of colorectal neoplasia WOS-positive (n = 236)
WOS-negative (n = 354)
p value
Gender Male/female Age (mean ± SD) Tumor size (mean ± SD)
Table 2 Presence of WOS between adenoma and carcinoma according to the tumor diameter Tumor size (mm)
Adenoma
Carcinoma
p value
\10
39/205 (19.0 %)
14/21 (66.7 %)
\0.0001
170/66
233/121
0.1120
10–19
61/168 (36.3 %)
44/70 (62.9 %)
0.0002
66.1 ± 10.8 years
65.8 ± 10.5 years
0.8911
20–29
21/42 (50.0 %)
22/31 (71.0 %)
0.0719
C30
22/34 (64.7 %)
13/19 (68.4 %)
0.075
\0.0001
17.6 ± 14.0 mm
11.2 ± 8.1 mm
Proximal colon
109 (46.2 %)
111 (31.4 %)
0.0003
Distal colon
70 (29.7 %)
162 (45.8 %)
\0.0001
Rectum
57 (24.2 %)
81 (22.9 %)
Tumor location
Adenoma (n = 143)
0.7208
Macroscopic classification 0-Ip, 0-Isp, 0-Is 0-IIa, IIc, IIa ? IIc
Table 3 Comparison in WOS-positive adenoma and cancer p value
Tumor location 175 (74.2 %) 61 (25.8 %)
302 (85.3 %) 52 (14.7 %)
0.0234 0.5485
SD standard deviation
Proximal colon
78 (54.5 %)
31 (33.3 %)
0.0197
Distal colon
42 (29.4 %)
28 (30.1 %)
0.3262
Rectum
23 (16.1 %)
34 (36.6 %)
0.6379
119 (83.2 %)
56 (60.2 %)
0.0729
24 (16.8 %)
37 (39.8 %)
0.6619
Macroscopic classification 0-Ip, 0-Isp, 0-Is
difference in histological type was seen. Among carcinomas, WOS-positive cases comprised 73 cases of well-differentiated adenocarcinoma and 17 cases of moderately differentiated adenocarcinoma, while WOS-negative cases included 41 cases of well-differentiated adenocarcinoma and ten cases of moderately differentiated adenocarcinoma. No significant differences in histological type were evident. WOS was more frequently present with carcinoma than with adenoma for both lesions B10 mm (66.7 vs. 19.0 %, respectively; p \ 0.0001) and lesions 10–19 mm in diameter (62.9 vs. 36.3 %, respectively; p = 0.0002) (Table 2). Investigation of the tumor site in WOS-positive cases showed WOS in the proximal colon significantly more with adenoma (54.5 %) than with carcinoma (33.3 %; p = 0.0197) (Table 3).
Discussion The present study is the first to demonstrate WOS with colorectal epithelial neoplasms. Yao et al. [9, 10] have already reported that the morphology of WOS as visualized by ME with NBI offers a new optical marker for differentiating between gastric low-grade dysplasia and gastric high-grade dysplasia/early carcinoma, providing an alternative to microvascular architecture. However, investigations with regard to the presence of WOS in the colorectum on endoscopic observation have been lacking. Furthermore, WOS has not yet been described in NBI classifications proposed for the diagnosis of neoplastic lesions of the colon [5, 6, 14–16]. Finding that the microvascular pattern of neoplastic colon lesions is indistinct on ME with NBI is
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Cancer (n = 93)
0-IIa, IIc, IIa ? IIc
Fig. 3 Histopathological findings of the biopsied specimen from the neoplasia (Oil Red O staining, 9200). By Oil Red O staining, numerous round or oval amorphous lipid droplets are demonstrated within the neoplastic epithelium alone (reprinted with permission from Ref. [10])
not uncommon. In the present study, the presence of WOS in the superficial part of the mucous membrane was shown to be the cause of difficulty in visualizing the microvascular pattern. With regard to the properties of WOS, Yao et al. [10] demonstrated the accumulation of lipid microdroplets, as visualized by Oil Red O staining, in the superficial part of WOS-positive gastric epithelial neoplasms (Fig. 3). Furthermore, Ueo et al. [17] reported the accumulation of vacuoles 0.1–4 lm in size under
Dig Dis Sci
immunoelectron microscopy. From the perspectives of size and immunoreaction to adipophilin, these vacuoles were speculated to represent lipid droplets composed of triglycerides. Since Warburg [18] first proposed that cancer may be a disease defined by aberrant metabolism, scientists have sought to define the specific biochemical pathways enabling previously normal cells to take on deviant tumorigenic characteristics [19]. New fatty acid synthesis occurs actively in cancer cells, and lipid-metabolizing enzymes facilitate the proliferation and migration of these cells. However, the biochemical pathways that regulate lipolysis in cancer cells remain poorly understood. Nomura et al. [3] reported that the enzyme MAGL was highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promote migration, invasion, survival, and in vivo tumor growth. Mutoh et al. [20] examined fatty change/lipid accumulation in intestinal mucosa and polyps in Min mice using Oil Red O staining and electron microscopy. A number of large lipid droplets were found in the epithelium of the upper part of polyps, and low-density lipoprotein receptor (LDLR) was overexpressed in the area where lipid droplets were observed. That study indicated that lipid accumulation and overexpression of LDLR in polyp epithelium may contribute to polyp development. Lipid metabolism is thus clearly important in the pathology of cancer and its growth and development. The present study is significant in that it shows the possibility of observing lipid droplets accumulated in neoplasms of the colorectal epithelium in vivo. This investigation demonstrated that WOS is present in the colon just as in the stomach, and found that significantly more WOS-positive patients had tumors that were large, located in the proximal colon, superficial flat-type lesions, and colorectal carcinomas. Furthermore, WOS was more frequently present in carcinoma than in adenoma for lesions B10 mm in diameter. WOS might thus provide a useful index for differentiating adenoma from cancer for small neoplasms of the colorectal epithelium. In the present study, differences with the stomach were also seen in clinical background characteristics. WOS was more frequently present with gastric adenoma (78 %) than with gastric carcinomas (43 %) [9]. However, WOS was more frequent with colorectal carcinoma (66.0 %) than with colorectal adenoma (31.8 %). The reason for this is unclear, but differences in the absorption and production of lipid droplets in various organs [21] may be one factor contributing to differences in WOS development in the stomach and colon. The stomach is constantly exposed to dietary fat, and absorption of lipid droplets into tumor epithelium is thought to have a large impact on the development of WOS. On the other hand, short-chain fatty
acids (SCFAs) are produced by commensal bacteria in the colon through anaerobic bacterial fermentation of unabsorbed carbohydrates and dietary fibers [22]. These SCFAs are mainly produced in the proximal colon, which may be why WOS-positive patients in the present study showed WOS in the proximal colon. Tumors in the left and right colon are known to display different molecular biological characteristics [23–25]. Hereditary non-polyposis colorectal cancer and sessile serrated adenoma/polyp appear more commonly in the right colon, while microsatellite instability is seen with a higher frequency in the right colon [26, 27]. Such background factors may also play roles in the differences in the WOS-positive rate. However, the fact that colorectal carcinoma and SSAP were not significantly apparent in the right colon among the present subjects also suggests that the higher rate of WOS in the right colon was due to SCFA. In addition, the colon is exposed to less dietary fat than the stomach, and the development of WOS in the colon is speculated to reflect an increased lipid droplet-production capacity in tumor cells. Matsubara et al. [28] reported that adipophilin was expressed primarily on the basal side of colorectal cancer cells forming polarized tubular structures, and adipophilin was expressed in well- or moderately differentiated adenocarcinoma, but not in the adjacent normal colonic mucosa or poorly differentiated adenocarcinoma. Adipophilin levels were significantly elevated, even in patients with localized early cancer. They speculated that expression of adipophilin might be induced during the process of early colorectal carcinogenesis. In the present study, WOS was more frequently present with colorectal carcinoma than with colorectal adenoma and also with submucosal carcinoma than with intramucosal carcinoma. The frequency of WOS increased together with the progression of colon cancer. Some limitations to this study must be considered. A retrospective study design was used, and we could not clarify whether WOS in the colorectum represented accumulations of lipid droplets, as for WOS in the stomach. However, Matsubara et al. [28] reported that adipophilin was expressed primarily on the basal side of colorectal cancer cells, and since the shapes of endoscopically observed WOS were the same in both the stomach and colon, the WOS was considered likely to represent lipid droplets. We are currently conducting a prospective histopathological investigation of whether WOS in colon epithelial tumors consists of lipid droplets, and we should be able to clarify this issue in the very near future (UMIN000011220). The endoscopic findings of WOS in the present study seem likely to prove useful in the diagnosis of neoplastic lesions of the colon, facilitating elucidation of the growth and development of colorectal epithelial neoplasms.
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In conclusion, the presence of WOS in colorectal epithelial neoplasms has been demonstrated for the first time. Incidence of this finding increased with the progression of cancer, from adenoma to carcinoma and from intramucosal carcinoma to submucosal carcinoma. Conflict of interest
None.
References 1. Kuhajda FP, Jenner K, Wood FD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci. 1994;91:6379–6383. 2. Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer. 2007;7:763–777. 3. Nomura DK, Long JZ, Niessen S, Hoover HS, Ng SW, Cravatt BF. Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis. Cell. 2010;140:49–61. 4. Straub BK, Herpel E, Singer S, et al. Lipid droplet-associated PAT-proteins show frequent and differential expression in neoplastic steatogenesis. Mod Pathol. 2010;23:480–492. 5. Sano Y, Ikematsu H, Fu KI, et al. Meshed capillary vessels by use of narrow-band imaging for differential diagnosis of small colorectal polyps. Gastrointest Endosc. 2009;69:278–283. 6. Kanao H, Tanaka S, Oka S, Hirata M, Yoshida S, Chayama K. Narrow-band imaging magnification predicts the histology and invasion depth of colorectal tumors. Gastrointest Endosc. 2009;69:631–636. 7. Yao K, Anagnostopoulos GK, Ragunath K. Magnifying endoscopy for diagnosing and delineating early gastric cancer. Endoscopy. 2009;41:462–467. 8. Ezoe Y, Muto M, Uedo N, et al. Magnifying narrowband imaging is more accurate than conventional white-light imaging in diagnosis of gastric mucosal cancer. Gastroenterology. 2011;141:2017–2025. 9. Yao K, Iwashita A, Tanabe H, et al. White opaque substance within superficial elevated gastric neoplasia as visualized by magnification endoscopy with narrow-band imaging: a new optical sign for differentiating between adenoma and carcinoma. Gastrointest Endosc. 2008;68:574–580. 10. Yao K, Iwashita A, Nambu M, et al. Nature of white opaque substance in gastric epithelial neoplasia as visualized by magnifying endoscopy with narrow-band imaging. Dig Endosc. 2012;24:419–425. 11. Tanaka M, Usuda K, Ookura Y, et al. Significance of magnifying endoscopy in diagnosis of duodenal elevated lesions [in Japanese with English abstract] Stomach Intestine 2003;38:1709–1720. 12. Endoscopic Classification Review Group. Update on the Paris classification of superficial neoplastic lesions in the digestive tract. Endoscopy. 2005;37:570–578.
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13. Hamilton SR, Aaltonen LA, eds. World Health Organization classification of tumours: pathology and genetics of tumours of the digestive system. Lyon: IARC Press; 2000:104–119. 14. Wada Y, Kashida H, Kudo S, Misawa M, Ikehara N, Hamatani S. Diagnostic accuracy of pit pattern and vascular pattern analysis in colorectal lesions. Dig Endosc. 2010;22:192–199. 15. Hisabe T, Yao K, Beppu T, et al. Validity of the usefulness of microvascular architecture and microsurface structure using magnifying endoscopy with narrow-band imaging in the colorectal neoplasm. Ann Gastroenterol. 2013;26:45–51. 16. Hayashi N, Tanaka S, Hewett DG, et al. Endoscopic prediction of deep submucosal invasive carcinoma: validation of the NarrowBand Imaging International Colorectal Endoscopic (NICE) classification. Gastrointest Endosc. 2013;78:625–632. 17. Ueo T, Yonemasu H, Yada N, et al. White opaque substance represents an intracytoplasmic accumulation of lipid droplets: immunohistochemical and immunoelectron microscopic investigation of 26 cases. Dig Endosc. 2013;25:147–155. 18. Warburg O. On the origin of cancer cells. Science. 1956;123:309–314. 19. Benjamin DI, Cravatt BF, Nomura DK. Global profiling strategies for mapping dysregulated metabolic pathways in cancer. Cell Metab. 2012;16:565–577. 20. Mutoh M, Komiya M, Teraoka N, et al. Overexpression of lowdensity lipoprotein receptor and lipid accumulation in intestinal polyps in min mice. Int J Cancer. 2009;125:2505–2510. 21. Heiden MGV, Cantly LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–1033. 22. Ropert A, Cherbut C, Roze´ C, et al. Colonic fermentation and proximal gastric tone in humans. Gastroenterology. 1996;111:289–296. 23. Hawkins N, Norrie M, Cheong K, et al. CpG island methylation in sporadic colorectal cancers and its relationship to microsatellite instability. Gastroenterology. 2002;122:1376–1387. 24. Samowitz WS, Curtin K, Ma KN, et al. Prognostic significance of p53 mutations in colon cancer at the population level. Int J Cancer. 2002;99:597–602. 25. An B, Kondo Y, Okamoto Y, et al. Characteristic methylation profile in CpG island methylator phenotype-negative distal colorectal cancers. Int J Cancer. 2010;127:2095–2105. 26. Aaltonen LA, Peltoma¨ki P, Mecklin JP, et al. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res. 1994;54:1645–1648. 27. Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology. 2010;138:2088–2100. 28. Matsubara J, Honda K, Ono M, et al. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Epidemiol Biomarkers Prev. 2011;20:2195–2203.
ORIGINAL ARTICLE
White Opaque Substance Visualized Using Magnifying Endoscopy with Narrow-Band Imaging in Colorectal Epithelial Neoplasms Takashi Hisabe • Kenshi Yao • Kentaro Imamura • Hiroshi Ishihara • Fumihito Hirai • Toshiyuki Matsui Akinori Iwashita
•
Received: 13 December 2013 / Accepted: 3 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Background The presence of a white opaque substance (WOS) on magnifying endoscopy (ME) with narrow-band imaging (NBI) has been reported for gastric epithelial neoplasms, but the presence of WOS in colorectal epithelial neoplasms has not been investigated. Aims The purpose of this study was to determine whether WOS is present in colorectal epithelial neoplasms and to clarify its clinical significance. Methods A total of 590 colorectal epithelial neoplasms from 368 consecutive patients were retrospectively analyzed using prospectively collected data. Presence or absence of WOS in colorectal epithelial neoplasms was recorded based on the findings of ME with NBI. Results White opaque substance was present in 236 of the 590 (40 %) colorectal epithelial neoplasms. Compared with WOS-negative patients, WOS-positive patients showed significantly larger tumors (p \ 0.0001) and significantly more tumors in the proximal colon (p = 0.0003). WOS was more frequently present in carcinomas (66.0 %) than in adenomas (31.8 %; p \ 0.0001). WOS was also more frequent in submucosal carcinomas (75.9 %) than in intramucosal carcinomas (59.0 %; p = 0.0380). Conclusions This study confirmed the presence of WOS in colorectal epithelial neoplasms, and prevalence increased with the progression of cancer, from adenoma to T. Hisabe (&) K. Yao H. Ishihara F. Hirai T. Matsui Department of Gastroenterology, Fukuoka University Chikushi Hospital, 1-1-1 Zokumyoin, Chikushino, Fukuoka 818-8502, Japan e-mail: [email protected] K. Imamura A. Iwashita Department of Pathology, Fukuoka University Chikushi Hospital, Chikushino, Japan
carcinoma and from intramucosal carcinoma to submucosal carcinoma. Keywords Cancer Colorectal epithelial neoplasm Magnifying endoscopy Narrow-band imaging White opaque substance
Introduction New fatty acid synthesis is known to occur actively in cancer cells, with fatty acid synthase (FASN) and other lipid-metabolizing enzymes facilitating the proliferation and migration of cancer cells [1, 2]. However, how they are isolated and converted to promote malignancy remains unclear. Various lipid-metabolizing enzymes, such as monoacylglycerol lipase (MAGL) [3], and lipid dropletassociated PAT-proteins [4] are reportedly overexpressed in cancer cells. The meaning of the roles played by lipid metabolism has been a focus of attention in research into the pathology of cancer cells. Narrow-band imaging (NBI) with magnifying endoscopy (ME) has been widely applied clinically in endoscopic diagnosis of the digestive tract in recent years [5–8]. NBI utilizes an optical filter with narrow bandwidths of two different central wavelengths (415 and 540 nm). NBI with ME can clearly visualize both the subepithelial microvascular architecture and the microsurface structure. Yao et al. [9] first reported that a white opaque substance (WOS) within the gastric neoplastic epithelium was obscuring the subepithelial microvascular pattern, and suggested that this WOS could offer a new optical sign for discriminating adenoma from carcinoma when using ME with NBI. Furthermore, Yao et al. [10] demonstrated that WOS represents accumulated lipid droplets in the
123
Dig Dis Sci
superficial part of epithelial neoplasms within the stomach. When lipid droplets within the superficial epithelium strongly reflect projected light, the projected light cannot reach the hemoglobin in microvessels, and strong backward scattering of light is recognized as white coloration by the human eye. The presence of WOS has been reported for neoplastic lesions of the stomach and duodenum [11], but has not previously been identified in the colon. This study investigated whether WOS is present in colorectal epithelial neoplasms and examined its clinical significance.
Histopathological Investigations Resected specimens were mounted on a plate and sliced at 2-mm intervals after overnight fixation in 20 % buffered formalin solution. These specimens were embedded in paraffin and cut into 5-lm-thick tissue sections. The sections were subsequently stained with hematoxylin and eosin (H&E stain). Histopathological diagnosis was made based on World Health Organization criteria [13], and tumors were classified into three groups: (1) adenoma; (2) intramucosal carcinoma; or (3) submucosal carcinoma. Definition of WOS
Materials and Methods Participants in this study comprised patients with colorectal adenoma or early colorectal carcinoma that had been examined by ME with NBI and that was endoscopically or surgically resected at Fukuoka University Chikushi Hospital between January 2009 and December 2012. Lesions that could not be visualized using ME with NBI and nonneoplastic lesions that did not require treatment were excluded. All patients provided written, informed consent for colonoscopic examinations. This study was approved by the Medical Ethics Committee of Fukuoka University Chikushi Hospital.
We defined WOS as a white substance within the colorectal superficial epithelium that obscured the subepithelial microvascular pattern. The presence or absence of WOS was investigated under maximal magnification (Fig. 1). When WOS was identified in any part of the lesion, the lesion was assessed as WOS-positive. Outcome Measures The primary endpoint was to determine whether WOS is present in colorectal neoplasms. A secondary endpoint was to investigate differences in clinicopathological characteristics between WOS-positive and WOS-negative subjects.
Endoscopic Specifications and Procedures
Statistical Analysis
All endoscopic procedures were performed using an electronic endoscopy system (Evis-Lucera Spectrum; Olympus Optical, Tokyo, Japan) with magnifying colonoscopy (CFH260AZI, PCF-Q240ZI; Olympus Optical). A soft black hood attachment was mounted at the tip of the scope. The lesion is visualized employing incremental movements of the tip of the endoscope to bring the image into focus, with a distally attached soft black hood to stabilize the tip of the endoscope without causing mucosal injury [7]. All patients were given 2–3 l of polyethylene glycol-electrolyte solution starting in the morning on the day of examination as preparation. When a colorectal epithelial lesion was detected during non-magnifying observation with whitelight imaging, the tumor surface was washed and immediately examined by ME with NBI. All endoscopic findings were analyzed retrospectively by two endoscopists (T.H. and Y.K.), each with more than 15 years of experience in the field of colonoscopy and more than 500 cases assessed by NBI with ME. In the case of differences in diagnosis, the final evaluation of endoscopic findings was made by the consensus agreement of the two endoscopists. Macroscopic classification was made according to the updated Paris classification [12].
Mean values were compared using Student’s t test. Comparison of prevalences between two groups was made using the Chi-square test or Fisher’s exact test. Values of p \ 0.05 were considered statistically significant. SPSS version 11.5J software (SPSS, Chicago, IL, USA) was used for all statistical analyses.
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Results A total of 590 colorectal epithelial neoplasms from 368 consecutive patients were investigated in this study. WOS was present in 236 of these 590 colorectal epithelial neoplasms (40 %) based on the findings of ME with NBI (Fig. 2). Comparisons of clinical features of colorectal epithelial neoplasms between WOS-positive and WOSnegative subjects are shown in Table 1. Mean tumor size was significantly greater in WOS-positive subjects (17.6 mm) than in WOS-negative subjects (11.2 mm; p \ 0.0001). By location, the proportion of tumors was significantly greater in the proximal colon (p = 0.0003) and significantly smaller in the distal colon (p \ 0.0001) in WOS-positive patients. The endoscopic macroscopic
Dig Dis Sci
A Fig. 1 Magnifying endoscopy with narrow-band imaging finding. a Morphology of the partially positive WOS shows a well-organized and symmetrical distribution. b The enlarged picture of a black
B square part revealing WOS (arrow) within the neoplastic epithelium obscures the subepithelial microvascular pattern
Fig. 2 Magnifying endoscopy with narrow-band imaging finding. a Tubular adenoma. WOS is absent and regular microvascular pattern are clearly visualized. b Tubular adenoma. Morphology of the WOS shows a well-organized and symmetrical distribution of a regular reticular pattern. c Well-differentiated adenocarcinoma (intramucosal carcinoma). Morphology of the WOS shows a disorganized and asymmetrical distribution of irregular speckled pattern. d Tubular adenoma. Morphology of the WOS shows a dense pattern
classification in WOS-negative patients included significantly greater proportions of 0-Ip, Isp, and Is lesions (p = 0.0234). On histopathological investigation, WOS was more frequently present with carcinoma (66.0 %, 93/141) than with adenoma (31.8 %, 143/449; p \ 0.0001). WOS was also more frequently present with submucosal carcinoma
(75.9 %, 44/58) than with intramucosal carcinoma (59.0 %, 49/83; p = 0.0380). Among adenomas, WOSpositive cases comprised 107 cases of tubular adenoma, 34 cases of villous adenoma, and two cases of traditional serrated adenoma. WOS-negative cases comprised 269 cases of tubular adenoma, 33 cases of villous adenoma, and four cases of traditional serrated adenoma. No significant
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Dig Dis Sci Table 1 Clinical features of colorectal neoplasia WOS-positive (n = 236)
WOS-negative (n = 354)
p value
Gender Male/female Age (mean ± SD) Tumor size (mean ± SD)
Table 2 Presence of WOS between adenoma and carcinoma according to the tumor diameter Tumor size (mm)
Adenoma
Carcinoma
p value
\10
39/205 (19.0 %)
14/21 (66.7 %)
\0.0001
170/66
233/121
0.1120
10–19
61/168 (36.3 %)
44/70 (62.9 %)
0.0002
66.1 ± 10.8 years
65.8 ± 10.5 years
0.8911
20–29
21/42 (50.0 %)
22/31 (71.0 %)
0.0719
C30
22/34 (64.7 %)
13/19 (68.4 %)
0.075
\0.0001
17.6 ± 14.0 mm
11.2 ± 8.1 mm
Proximal colon
109 (46.2 %)
111 (31.4 %)
0.0003
Distal colon
70 (29.7 %)
162 (45.8 %)
\0.0001
Rectum
57 (24.2 %)
81 (22.9 %)
Tumor location
Adenoma (n = 143)
0.7208
Macroscopic classification 0-Ip, 0-Isp, 0-Is 0-IIa, IIc, IIa ? IIc
Table 3 Comparison in WOS-positive adenoma and cancer p value
Tumor location 175 (74.2 %) 61 (25.8 %)
302 (85.3 %) 52 (14.7 %)
0.0234 0.5485
SD standard deviation
Proximal colon
78 (54.5 %)
31 (33.3 %)
0.0197
Distal colon
42 (29.4 %)
28 (30.1 %)
0.3262
Rectum
23 (16.1 %)
34 (36.6 %)
0.6379
119 (83.2 %)
56 (60.2 %)
0.0729
24 (16.8 %)
37 (39.8 %)
0.6619
Macroscopic classification 0-Ip, 0-Isp, 0-Is
difference in histological type was seen. Among carcinomas, WOS-positive cases comprised 73 cases of well-differentiated adenocarcinoma and 17 cases of moderately differentiated adenocarcinoma, while WOS-negative cases included 41 cases of well-differentiated adenocarcinoma and ten cases of moderately differentiated adenocarcinoma. No significant differences in histological type were evident. WOS was more frequently present with carcinoma than with adenoma for both lesions B10 mm (66.7 vs. 19.0 %, respectively; p \ 0.0001) and lesions 10–19 mm in diameter (62.9 vs. 36.3 %, respectively; p = 0.0002) (Table 2). Investigation of the tumor site in WOS-positive cases showed WOS in the proximal colon significantly more with adenoma (54.5 %) than with carcinoma (33.3 %; p = 0.0197) (Table 3).
Discussion The present study is the first to demonstrate WOS with colorectal epithelial neoplasms. Yao et al. [9, 10] have already reported that the morphology of WOS as visualized by ME with NBI offers a new optical marker for differentiating between gastric low-grade dysplasia and gastric high-grade dysplasia/early carcinoma, providing an alternative to microvascular architecture. However, investigations with regard to the presence of WOS in the colorectum on endoscopic observation have been lacking. Furthermore, WOS has not yet been described in NBI classifications proposed for the diagnosis of neoplastic lesions of the colon [5, 6, 14–16]. Finding that the microvascular pattern of neoplastic colon lesions is indistinct on ME with NBI is
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Cancer (n = 93)
0-IIa, IIc, IIa ? IIc
Fig. 3 Histopathological findings of the biopsied specimen from the neoplasia (Oil Red O staining, 9200). By Oil Red O staining, numerous round or oval amorphous lipid droplets are demonstrated within the neoplastic epithelium alone (reprinted with permission from Ref. [10])
not uncommon. In the present study, the presence of WOS in the superficial part of the mucous membrane was shown to be the cause of difficulty in visualizing the microvascular pattern. With regard to the properties of WOS, Yao et al. [10] demonstrated the accumulation of lipid microdroplets, as visualized by Oil Red O staining, in the superficial part of WOS-positive gastric epithelial neoplasms (Fig. 3). Furthermore, Ueo et al. [17] reported the accumulation of vacuoles 0.1–4 lm in size under
Dig Dis Sci
immunoelectron microscopy. From the perspectives of size and immunoreaction to adipophilin, these vacuoles were speculated to represent lipid droplets composed of triglycerides. Since Warburg [18] first proposed that cancer may be a disease defined by aberrant metabolism, scientists have sought to define the specific biochemical pathways enabling previously normal cells to take on deviant tumorigenic characteristics [19]. New fatty acid synthesis occurs actively in cancer cells, and lipid-metabolizing enzymes facilitate the proliferation and migration of these cells. However, the biochemical pathways that regulate lipolysis in cancer cells remain poorly understood. Nomura et al. [3] reported that the enzyme MAGL was highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promote migration, invasion, survival, and in vivo tumor growth. Mutoh et al. [20] examined fatty change/lipid accumulation in intestinal mucosa and polyps in Min mice using Oil Red O staining and electron microscopy. A number of large lipid droplets were found in the epithelium of the upper part of polyps, and low-density lipoprotein receptor (LDLR) was overexpressed in the area where lipid droplets were observed. That study indicated that lipid accumulation and overexpression of LDLR in polyp epithelium may contribute to polyp development. Lipid metabolism is thus clearly important in the pathology of cancer and its growth and development. The present study is significant in that it shows the possibility of observing lipid droplets accumulated in neoplasms of the colorectal epithelium in vivo. This investigation demonstrated that WOS is present in the colon just as in the stomach, and found that significantly more WOS-positive patients had tumors that were large, located in the proximal colon, superficial flat-type lesions, and colorectal carcinomas. Furthermore, WOS was more frequently present in carcinoma than in adenoma for lesions B10 mm in diameter. WOS might thus provide a useful index for differentiating adenoma from cancer for small neoplasms of the colorectal epithelium. In the present study, differences with the stomach were also seen in clinical background characteristics. WOS was more frequently present with gastric adenoma (78 %) than with gastric carcinomas (43 %) [9]. However, WOS was more frequent with colorectal carcinoma (66.0 %) than with colorectal adenoma (31.8 %). The reason for this is unclear, but differences in the absorption and production of lipid droplets in various organs [21] may be one factor contributing to differences in WOS development in the stomach and colon. The stomach is constantly exposed to dietary fat, and absorption of lipid droplets into tumor epithelium is thought to have a large impact on the development of WOS. On the other hand, short-chain fatty
acids (SCFAs) are produced by commensal bacteria in the colon through anaerobic bacterial fermentation of unabsorbed carbohydrates and dietary fibers [22]. These SCFAs are mainly produced in the proximal colon, which may be why WOS-positive patients in the present study showed WOS in the proximal colon. Tumors in the left and right colon are known to display different molecular biological characteristics [23–25]. Hereditary non-polyposis colorectal cancer and sessile serrated adenoma/polyp appear more commonly in the right colon, while microsatellite instability is seen with a higher frequency in the right colon [26, 27]. Such background factors may also play roles in the differences in the WOS-positive rate. However, the fact that colorectal carcinoma and SSAP were not significantly apparent in the right colon among the present subjects also suggests that the higher rate of WOS in the right colon was due to SCFA. In addition, the colon is exposed to less dietary fat than the stomach, and the development of WOS in the colon is speculated to reflect an increased lipid droplet-production capacity in tumor cells. Matsubara et al. [28] reported that adipophilin was expressed primarily on the basal side of colorectal cancer cells forming polarized tubular structures, and adipophilin was expressed in well- or moderately differentiated adenocarcinoma, but not in the adjacent normal colonic mucosa or poorly differentiated adenocarcinoma. Adipophilin levels were significantly elevated, even in patients with localized early cancer. They speculated that expression of adipophilin might be induced during the process of early colorectal carcinogenesis. In the present study, WOS was more frequently present with colorectal carcinoma than with colorectal adenoma and also with submucosal carcinoma than with intramucosal carcinoma. The frequency of WOS increased together with the progression of colon cancer. Some limitations to this study must be considered. A retrospective study design was used, and we could not clarify whether WOS in the colorectum represented accumulations of lipid droplets, as for WOS in the stomach. However, Matsubara et al. [28] reported that adipophilin was expressed primarily on the basal side of colorectal cancer cells, and since the shapes of endoscopically observed WOS were the same in both the stomach and colon, the WOS was considered likely to represent lipid droplets. We are currently conducting a prospective histopathological investigation of whether WOS in colon epithelial tumors consists of lipid droplets, and we should be able to clarify this issue in the very near future (UMIN000011220). The endoscopic findings of WOS in the present study seem likely to prove useful in the diagnosis of neoplastic lesions of the colon, facilitating elucidation of the growth and development of colorectal epithelial neoplasms.
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In conclusion, the presence of WOS in colorectal epithelial neoplasms has been demonstrated for the first time. Incidence of this finding increased with the progression of cancer, from adenoma to carcinoma and from intramucosal carcinoma to submucosal carcinoma. Conflict of interest
None.
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