ORIGINAL ARTICLE
New Classification of Gastric Pit Patterns and Vessel Architecture Using Probe-based Confocal Laser Endomicroscopy Zhen Li, PhD,* Xiu-Li Zuo, PhD,* Chang-Qing Li, PhD,* Zhi-Yan Liu, PhD,w Rui Ji, PhD,* Jun Liu, PhD,* Jing Guo, MD,* and Yan-Qing Li, PhD*
Goals: To propose a new probe-based confocal laser endomicroscopy (pCLE) classification of gastric pit patterns and vessel architecture, and to assess the accuracy and interobserver agreement. Background: pCLE is a newly developed endoscopic device that allows the application of laser microscopy with any conventional endoscope and mosaic imaging. Study: A total of 291 pCLE videos from 32 patients were recruited in phase I to establish the new pCLE image classification in the stomach. Eligible patients were then prospectively investigated by pCLE using the newly established classification system. All patients were examined first with high-definition endoscopy followed by pCLE at 7 standardized locations and endoscopic-suspected lesions. Targeted biopsies were performed with precise matching of pCLE recordings. Results: The sensitivity and specificity of type 2b pit pattern for predicting atrophic gastritis were 88.51% and 99.19%, respectively. The sensitivity and specificity of type 2c pit pattern for predicting intestinal metaplasia were 92.34% and 99.34%, respectively. The overall sensitivity and specificity of type 3 pit pattern or vessel architecture for predicting neoplasia were 89.89% and 99.44%, respectively. The interobserver agreement was “substantial” (kappa = 0.70) for the differentiation of neoplasia versus nonneoplasia. Conclusions: The new pCLE classification system in the stomach correlates well with specific pathologic conditions and is reproducible by multiple investigators. Multicenter researches are warranted to further validate its value in clinical practice. Key Words: probe-based confocal laser endomicroscopy, pit pattern, vessel architecture, gastric cancer, intraepithelial neoplasia
(J Clin Gastroenterol 2016;50:23–32)
E
arly detection of gastric cancer and precancerous gastric lesions is essential for each Gastrointestinal (GI) endoscopy. However, most of these lesions lack distinctive
Received for publication July 25, 2014; accepted January 6, 2015. From the *Department of Gastroenterology, Qilu Hospital, Shandong University; and wDepartment of Pathology and Pathophysiology, Shandong University School of Medicine, Jinan, China. Y.-Q.L. receives research funding from: The National Natural Science Foundation of China, No. 81330012; the Shandong Province Science and Technology Committee, No. 2013GSF11833. Z.L. receives research funding from the National Natural Foundation of Science of China, No. NSFC81301264. The remaining authors declare that they have nothing to disclose. Reprints: Yan-Qing Li, PhD, No. 107, Wenhuaxi Road, Jinan 250012, China (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
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endoscopic characteristics, which make it difficult for accurate differentiation of malignant from benign lesions. During recent decades, advancements in endoscopic technologies including magnifying endoscopy, narrow-band imaging, and virtual chromoendoscopy have significantly improved the diagnostic performance of early gastric neoplasia and preneoplastic changes.1–3 Confocal laser endomicroscopy (CLE) is a novel endoscopic system that can provide real-time microscopic observation of the GI mucosa. Currently, 2 kinds of endomicroscopes are available including endoscope-based CLE system (eCLE; Pentax, Tokyo, Japan) and probebased confocal laser endomicroscopy (pCLE) system (Cellvizio, Mauna Kea Technologies, Paris, France). The eCLE system was built by the integration of a confocal laser microscope at the distal tip of a conventional endoscope. Whereas pCLE is a newly developed endoscopic device that allows the application of laser microscopy with any conventional endoscope and mosaic imaging. To date, numerous studies have evaluated the utility of pCLE in various GI diseases, including Barrett’s esophagus, gastric intestinal metaplasia, superficial gastric neoplasia, colorectal polyps, ulcerative colitis, and pancreaticobiliary disease.4–9 Moreover, recent data also explored its potential application in the field of molecular imaging.10 Our previous studies using endoscope-based CLE system have established and assessed its diagnostic value for gastric intestinal metaplasia, intraepithelial neoplasia, and gastric carcinoma.11–13 We have also reported a classification of gastric pit patterns using eCLE, which classified the gastric pit patterns into 7 types (type A to G), and demonstrated that the patterns of gastric pits identified by eCLE correlated well with histologic findings.14 However, these studies were based upon confocal images from eCLE, and may suffer from potential challenges accordingly. For instance, pCLE has a smaller field of view and higher image acquisition rate as compared with eCLE, and the image differences caused by these technical issue may render those published eCLE criteria under further assessment by using pCLE. In 2011, Wallace et al15 proposed a state-of-the-art classification for the differentiation between normal and pathologic states in GI diseases using pCLE, which is named the “Miami classification system.” Later, a singlecenter study by Bok et al6 showed that, by using the “Miami classification system,” pCLE have a sensitivity of 90.6% and a specificity of 90.9% for the diagnosis of superficial gastric neoplasias. However, the “Miami classification system” only briefly depicted endomicroscopic changes using pCLE for different gastric diseases, including normal stomach, gastritis, gastric dysplasia, and www.jcge.com |
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TABLE 1. A New pCLE Classification of Gastric Pit Patterns and Vessel Architecture
Categories
pCLE Appearance
Distribution Area
pCLE Image
Gastric pit patterns Type 1
Regular pits with wide/round/slit-like openings
Normal cardia/corpus/antrum
Regular pits with elongated openings, increased fluorescence in stroma
Inflammatory gastric mucosa
b
Reduced pits with dilated openings
Atrophic mucosa
c
Appearance of goblet cells with dark mucin
Intestinal metaplastic mucosa
Mild to moderate irregular pits with variable width of the epithelial lining
Low-grade intraepithelial neoplasia
Prominent distorted pits with irregular epithelial lining
High-grade intraepithelial neoplasia
Type 2 a
Type 3 a
b
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TABLE 1. (continued) Categories
pCLE Appearance
c
Appearance of atypical glands/dispersion of irregular dark cells
Vessel architecture Type 1 Regular capillaries with normal caliber, anfractous/honeycomb-like/coil-shaped
Distribution Area
Differentiated/poorly differentiated adenocarcinoma
Normal cardia/corpus/antrum
Type 2
Increased capillaries with elevated leakage
Inflammatory gastric mucosa
Type 3
Irregular capillaries with heterogenous leakage/ dilated caliber
Neoplastic gastric mucosa
adenocarcinoma. These parameters still lack prospective validation, and do not include the description of vessel architecture for different pathologic conditions. Furthermore, considering the fact that pCLE has the priority for blood flow because of its increased image acquisition rate, it would be desirable to specify the distinctive capillary changes for different lesions in pCLE images. However, to date, there has been limited data concerning the application of pCLE in the stomach, and no investigation has yet focused on the characterization and classification of gastric pit patterns and vessel architecture using pCLE. Therefore, the aims of the present study were to propose a new pCLE classification of gastric pit patterns and vessel architecture, and to assess the accuracy and interobserver agreement of this new pCLE classification system for diagnosing various premalignant and malignant changes in the stomach. Copyright
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pCLE Image
MATERIALS AND METHODS Phase I: Establishment of the New Classification A total of 291 pCLE videos from 32 patients were recruited in phase I to establish the new pCLE image classification in the stomach. This data set consisted of 40 inflammation, 18 atrophic gastritis, 25 intestinal metaplasia, 2 low-grade intraepithelial neoplasia (LGIN), 1 high-grade intraepithelial neoplasia (HGIN), 1 differentiated adenocarcinoma, and 2 poorly differentiated adenocarcinoma. The endoscopic procedure were carried out as described in the prospective study (phase II). All 291 confocal videos were openly evaluated by 3 experienced endomicroscopists (Y.-Q.L., X.-L.Z., C.-Q.L.) and 1 GI pathologist (Z.-Y.L.). A new classification of gastric pit patterns and vessel architecture using pCLE (Cellvizio, Mauna Kea Technologies) was developed based
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FIGURE 1. Normal and inflammatory gastric mucosa. A, Probe-based confocal laser endomicroscopy (pCLE) image of normal cardia: regular pits with wide openings (type 1). B, pCLE image of normal gastric mucosa with fundic glands: round gastric pits with round openings (type 1). C, pCLE image of normal gastric mucosa with pyloric glands: continuous short rod-like pits with slit-like openings (type 1). D, pCLE image of cardiac mucosa with chronic inflammation: aggregation/branching of enlarged pits (type 2a). E, pCLE image of corporal mucosa with chronic inflammation: elongated pits with thread-like openings (type 2a). F, pCLE image of antral mucosa with chronic inflammation: tortuosity/branching of elongated pits (type 2a).
on the comparison between in vivo pCLE images and corresponding histologic findings from targeted biopsy specimens, histologic description for each state, and with reference to previously published researches.11–14 This new pCLE classification in the stomach consists of 3 types of pit patterns with 7 subtypes (type 1, type 2a-c, type 3a-c), and 3 types of vessel architecture (type 1, type 2, type 3). Type 1 pit patterns and vessel architecture represent normal gastric mucosa, type 2a pit patterns and type 2 vessel architecture indicate inflammation, type 2b pit pattern shows atrophic gastritis, type 2c pit pattern shows intestinal metaplasia, and type 3 pit pattern and vessel architecture indicate gastric neoplasia. Details of the pCLE classification system in the stomach were summarized in Table 1. Figures 1–4 represent confocal images and corresponding histology of the new pCLE classification system in the stomach.
Phase II: Prospective Study From April to September 2013, consecutive patients were recruited prospectively for pCLE examinations at Qilu Hospital, Shandong University. Inclusion criteria were: (1) patients with dyspeptic symptoms and aged 40 years and above; or (2) patients with H. pylori infection; or (3) patients with known premalignant or malignant gastric lesions (including atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and gastric adenocarcinoma). Exclusion criteria were: (1) patients with a history of GI surgery; (2) patients with contraindications to pCLE
examination, such as coagulopathy, pregnancy, breast feeding, or allergy to fluorescein sodium; (3) patients refused to provide informed consent. This study was approved by the Ethics Committee of Qilu Hospital, Shandong University (clinicaltrials.gov NCT01896310), and was conducted in accordance with the revised Declaration of Helsinki (2008).
pCLE Endomicroscopic visualizations were achieved using a pCLE (Cellvizio, Mauna Kea Technologies). The pCLE probe can be used in conjunction with any endoscope, and enables rapid image acquisition at a scan rate of 12 frames per second. It has a depth of scanning of 60 mm, a lateral resolution of 1 mm, a field of view of 240240 mm, and 1000 magnification. Confocal images can be displayed in real-time on a standard personal computer screen that is connected to the laser scanning unit of pCLE miniprobe. A specially designed software (Cellvizio Viewer) allows immediate sequences display, real-time image reconstruction and postprocedure analysis of all the captured pCLE videos. Fluorescein sodium (5 mL, 10%) was applied intravenously as the contrast agent before pCLE scanning.
Endoscopic Procedure All patients underwent high-definition endoscopic examinations using a Pentax EG-2990i endoscope (Pentax, Japan) with an image resolution of 1.3 mega pixels. After
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FIGURE 2. Gastric atrophy and intestinal metaplasia. A, Probe-based confocal laser endomicroscopy (pCLE) image showed glandular atrophy with decreased gastric pits and markedly dilated opening (arrows) (type 2b). B, Corresponding histologic results confirmed the appearance of atrophic gastritis (H&E original magnification 400). C, pCLE image showed dark goblet cells (arrows) within the columnar epithelium (type 2c). D, Corresponding histologic results confirmed gastric intestinal metaplasia of the mucosa (H&E original magnification 400).
FIGURE 3. Gastric intraepithelial neoplasia and adenocarcinoma. A, Probe-based confocal laser endomicroscopy (pCLE) image showed mild to moderate irregular pits with variable width of the epithelial lining (arrows) (type 3a). B, Corresponding histology specimen showed a low-grade intraepithelial neoplasia (H&E original magnification 200). C, pCLE image showed prominent distorted pits (arrows) with irregular epithelial lining (type 3b). D, Corresponding histology specimen showed a high-grade intraepithelial neoplasia (H&E original magnification 200). E, pCLE image showed the appearance of atypical/naive glands (arrows) with dark epithelium (type 3c). F, Corresponding histology specimen showed a differentiated adenocarcinoma (H&E original magnification 400). G, pCLE image showed loss of pits, appearance of irregular dark cells (arrows) (type 3c). H, Corresponding histology specimen showed a poorly differentiated adenocarcinoma (H&E original magnification 400).
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intravenous injection of 5 mL 10% fluorescein sodium, the pCLE probe was passed through the accessory channel of the endoscope, and placed in gentle contact with the gastric mucosa. Endomicroscopic imaging was achieved at all endoscopic-suspected lesions and 7 standardized locations in the stomach for each patient. The 7 standardized locations included the following sites: gastric cardia within 1 cm below the gastroesophageal junction, fundus, incisura angularis, middle portion of the greater curvature of the corpus, lesser curvature of the corpus about 4 cm proximal to the angulus, greater and lesser curvature of the antrum within 2 to 3 cm from the pylorus. All endoscopic procedures were performed by 1 experienced endoscopist (C.-Q.L.). Finally, targeted biopsies were performed with precise matching of pCLE recordings.
Although the endoscopist was informed that the study population was enriched and included patients with dyspeptic symptoms above 40 years and patients undergoing surveillance endoscopy, he has no access to any patients’ previous endoscopic and pathologic diagnoses before endoscopy. All endoscopic procedures were conducted under the supervision of a study coordinator (J.G.).19 Three months after endoscopic examination, all pCLE recordings were reassessed by the same endoscopist (C.Q.L.) who made the in vivo diagnosis. During this offline review, the endoscopist was blinded to the patients’ clinical, endoscopic, and histologic data, and endomicroscopic categorization was made according to the same pCLE classification used for in vivo diagnosis.
Histologic Evaluation
Agreement Evaluation
All specimens were evaluated by an expert GI pathologist (Z.-Y.L.) who was blinded to the patients’ pCLE findings. Histologic diagnosis was made according to the updated Sydney System for the classification of gastritis, the Vienna criteria for neoplasia, and the WHO classification of tumors (digestive system).16–18
To assess the interobserver agreement, pCLE video recordings of all endoscopic lesions examined in phase II were randomized and offline evaluated by 3 independent endomicroscopists (X.-L.Z., R.J., J.L.) who were blinded to the endoscopic findings and histology. Endomicroscopic diagnoses were also made according to the newly developed classification system in the stomach (Table 1). These pCLE videos were reevaluated by the 3 endomicroscopists after a 7-day interval to calculate the intraobserver agreement. All pCLE sequences were displayed in their original format (.mkt, Mauna Kea Technologies, Paris, France).
pCLE Videos Assessment Real-time pCLE diagnosis was made by the performing endoscopist (C.-Q.L.) during the procedure based on the newly developed classification system (Table 1).
FIGURE 4. Different types of vessel architecture in the stomach. A, Vessel architecture in the normal cardia: anfractous capillaries with regular shapes surrounding gastric pits (type 1). B, Vessel architecture in the normal corpus: honeycomb-like capillaries surrounding gastric pits (type 1). C, Vessel architecture in the normal antrum: coil-shaped capillaries surrounding gastric pits (type 1). D, Vessel architecture in the inflammatory gastric mucosa: increased capillaries with elevated leakage (type 2). E, Vessel architecture in neoplastic gastric mucosa: irregular capillaries with heterogenous leakage/dilated caliber (type 3). The arrows indicate the microvessels.
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Statistical Analysis Statistical analysis was performed by using SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the diagnosis of atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and adenocarcinoma were calculated for in vivo pCLE diagnosis and offline pCLE diagnosis. And the accuracy results stated in each section represent a comparison of the pattern type versus all other types. w2 test was used for comparisons. P < 0.05 (2-tailed) was considered as statistically significant. The interobserver agreement was estimated using the kappa value with 95% confidence intervals (CIs). The interpretation of kappa values was as follows: values > 0.80 were regarded as excellent, 0.6 to 0.8 substantial, 0.4 to 0.6 moderate, 0.2 to 0.4 fair, values < 0.2 indicating poor agreement. Kappa values for different gastric pit patterns and vessel architecture were calculated using the STATA 9.0 software package (StataCorp LP, College Station, TX). The study report was in accordance with the standards for reporting studies of diagnostic accuracy.20
Patient Characteristics A total of 244 patients were enrolled in phase II of this study according to predefined inclusion and exclusion criteria. Four patients were further excluded because of severe esophageal stricture after insertion of the endoscope. Therefore, 240 patients were finally included for data analysis. The mean age of patients was 57 years (range, 27 to 83 y), and 48.33% were women. Clinical indications included 175 patients with dyspeptic symptoms and aged 40 years and above, 34 patients with H. pylori infection, and 31 patients with known premalignant or malignant gastric lesions. Clinical characteristics of these patients were summarized in Table 2. We collected 1878 pCLE video sequences, ranging from 7 to 10 videos per patient.
Comparison of pCLE Classifications and Histologic Diagnosis A total of 198 lesions in the stomach were identified in phase II of this study. Histopathology demonstrated 61 TABLE 2. Patients’ Characteristics in This Study
Phase I
Phase II
32 19/13 50 (19-75) 26
240 124/116 57 (27-83) 198
11 4 5 2 1 1 2
61 20 28 25 9 13 42
HGIN indicates high-grade intraepithelial neoplasia; LGIN, low-grade intraepithelial neoplasia.
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inflammatory, 20 atrophic gastritis, 28 intestinal metaplasia, 25 LGIN, 9 HGIN, 13 differentiated adenocarcinoma, 42 poorly differentiated adenocarcinoma (of which 9 were signet-ring cell carcinoma). Among the 1680 standardized locations, histologic diagnosis revealed 372 normal mucosa, 986 inflammation, 128 atrophic gastritis, and 194 intestinal metaplasia (Table 2). Table 3 shows the comparison results of this new pCLE classifications and histologic diagnosis on per-location analyses (including 198 lesions and 1680 standardized locations). During real-time pCLE examination, the sensitivity and specificity of type 2b pit pattern for predicting atrophic gastritis were 88.51% (95% CI, 82.37%-92.70%) and 99.19% (95% CI, 98.65%-99.52%), respectively. The sensitivity and specificity of type 2c pit pattern for predicting intestinal metaplasia were 92.34% (95% CI, 88.08%-95.16%) and 99.34% (95% CI, 98.81%-99.63%), respectively. Overall sensitivity and specificity of type 3 pit pattern or type 3 vessel architecture for predicting neoplasia were 89.89% (95% CI, 81.89%-94.59%) and 99.44% (95% CI, 98.97%-99.70%), respectively.
Offline pCLE Diagnosis
RESULTS
Patients (n) Sex (male/female) (n) Mean age (range) (y) Macroscopic lesions (n) Histologic diagnosis Inflammation Atrophic gastritis Intestinal metaplasia LGIN HGIN Differentiated adenocarcinoma Poorly differentiated adenocarcinoma and signet-ring cell carcinoma
New Classification in the Stomach Using pCLE
The offline pCLE diagnostic results of different histologic status were as follows: atrophic gastritis with a sensitivity of 89.86% and a specificity of 99.25%; intestinal metaplasia with a sensitivity of 93.69% and a specificity of 99.40%; and the non-neoplasia versus neoplasia with a sensitivity of 92.13% and a specificity of 99.50%. The accuracy for offline pCLE diagnosis of neoplasia was 99.15%. There was no difference between the accuracies of in vivo and offline diagnosis of neoplasia (P = 0.610). Table 4 shows the results of sensitivity, specificity, accuracy, and positive and negative predictive values for offline pCLE diagnosis of various gastric abnormalities.
Interobserver and Intraobserver Agreement The interobserver agreements on different gastric pit patterns (3 types) and vessel architecture (3 types) were “substantial” with kappa values of 0.63 (95% CI, 0.60-0.66) and 0.64 (95% CI, 0.61-0.70), respectively. The intraobserver agreements were all “excellent” for the differentiation among 3 types of gastric pit patterns (mean kappa = 0.90) and vessel architecture (mean kappa = 0.94). We also investigated the agreement values on each subtype of gastric pit patterns, which showed an interobserver agreement of 0.54 (95% CI, 0.51-0.55) and an intraobserver agreement of 0.93 (0.94, 0.92, 0.92), respectively. When using the type 3 pit pattern or type 3 vessel architecture for the differentiation between neoplasia versus non-neoplasia, the interobserver agreement was “substantial” with a kappa value of 0.70 (95% CI, 0.630.71), and the intraobserver agreement was “excellent” with a mean kappa value of 0.94 (0.92, 0.94, 0.96).
DISCUSSION This study proposed a new classification system of gastric pit patterns and vessel architecture specifically designed for probe-based CLE device. Prospective study including 240 patients proved that this new classification system has high accuracies for the diagnoses of atrophic gastritis, intestinal metaplasia, and gastric neoplasia.
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TABLE 3. Comparisons Between Real-time pCLE Diagnosis and Histologic Diagnosis on Per-location Diagnosis
CLE Diagnosis Histopathology Normal Inflammation AG IM LGIN HGIN DA PDA Total
Normal
Inflammation
AG
IM
LGIN
HGIN
DA
PA
Total
372 101 — — — — — — 473
— 937 12 4 1 — — — 954
— 3 131 10 1 — — — 145
— 5 4 205 2 — — — 216
— — 1 3 20 1 — — 25
— — — — 1 7 1 — 9
— — — — — 1 12 1 14
— 1 — — — — — 41 42
372 1047 148 222 25 9 13 42 1878
AG indicates atrophic gastritis; DA, differentiated adenocarcinoma; HGIN, high-grade intraepithelial neoplasia; IM, intestinal metaplasia; LGIN, lowgrade intraepithelial neoplasia; PDA, poorly differentiated adenocarcinoma and signet-ring cell carcinoma; pCLE, probe-based confocal laser endomicroscopy.
Furthermore, a substantial interobserver agreement and an excellent intraobserver agreement was achieved for the differentiation among different types of pit patterns and vessel architecture, and when it reduced to a dichotomous diagnosis of non-neoplasia and neoplasia, the interobserver agreement was also substantial with a higher kappa value. Although our previous study has reported a classification of gastric pit patterns using endoscope-based CLE system,14 it did not describe the endomicroscopic features of vessel architecture for various histologic changes, and the pit pattern classifications covered only normal, inflammatory, atrophic, intestinal metaplastic, and cancerous changes in the antrum and corpus. Whereas the new classification system in the stomach proposed in our current study correlated well with specific pathologic conditions, and firstly included the description of gastric cardia, the characteristics of LGIN and HGIN, and different vessel architectural changes in pCLE videos. Moreover, the interobserver and intraobserver agreements were also assessed for different types and subtypes of gastric pit patterns and vessel architecture in this study, which further substantiated its validity for clinical application. Most patients with gastric adenocarcinoma die during the first year of the disease even if referred to aggressive and costly therapies due to diagnosis at late stages. Therefore, one of the aims of this new pCLE classification was an accurate identification of premalignant gastric conditions
or lesions, namely atrophic gastritis, intestinal metaplasia, and intraepithelial neoplasia. Although previous studies have investigated the diagnostic ability of endoscope-based CLE for identifying atrophic gastritis,14,21 intestinal metaplasia and intraepithelial neoplasia,11,12 it is still not clear whether similar results could be achieved by using pCLE, given the fact that the pCLE system slightly lags in resolution and penetration depth compared with eCLE. Our present study showed that comparable results can be achieved with the 2 endomicroscopic modalities (pCLE and eCLE) in diagnosing various gastric lesions, including atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and adenocarcinoma. Of interest, the diagnostic specificity of pCLE in this study was much higher than previous researches. This was due to the large number of standardized biopsies with histologic diagnosis of inflammation and normal gastric mucosa, most of which can be easily recognized and correctly diagnosed using pCLE. Our earlier study showed that eCLE can reliably differentiate noncancerous gastric lesions from cancer/HGIN lesions using a 2-tiered eCLE classification system.13 In this study, we found that using the new pCLE classification system in the stomach, pCLE also enables an accurate in vivo differentiation between neoplastic and non-neoplastic gastric lesions. As shown in Table 3, macroscopic gastric lesions with LGIN and intestinal metaplasia may not easily be distinguished from each other in some cases.
TABLE 4. Offline pCLE Diagnosis of Various Gastric Abnormalities in Phase II
Abnormality AG IM LGIN HGIN DA PDA Neoplasia (IN + CA)
Sensitivity [% (CI)] 89.86 93.69 84.00 88.89 92.31 97.62 92.13
(83.95-93.76) (89.69-96.21) (65.35-93.60) (56.50-98.01) (66.69-98.63) (87.68-99.58) (84.64-96.14)
Specificity [% (CI)] 99.25 99.40 99.78 99.89 99.89 99.95 99.50
(98.72-99.56) (98.89-99.67) (99.45-99.92) (99.61-99.97) (99.61-99.97) (99.69-99.99) (99.05-99.74)
PPV [% (CI)] 91.10 95.41 84.00 80.00 85.71 97.62 90.11
(85.36-94.72) (91.76-97.49) (65.35-93.60) (49.02-94.33) (60.06-99.59) (87.68-99.58) (82.26-94.71)
NPV [% (CI)] 99.13 99.16 99.78 99.95 99.95 99.95 99.61
(98.58-99.47) (98.59-99.50) (99.45-99.92) (99.70-99.99) (99.70-99.99) (99.69-99.99) (99.19-99.81)
Accuracy [% (CI)] 98.51 98.72 99.57 99.84 99.84 99.89 99.15
(97.85-98.97) (98.11-99.14) (99.16-99.78) (99.53-99.95) (99.53-99.95) (99.61-99.97) (98.62-99.47)
AG indicates atrophic gastritis; CA, adenocarcinoma; CI, confidence interval; DA, differentiated adenocarcinoma; HGIN, high-grade intraepithelial neoplasia; IM, intestinal metaplasia; IN, intraepithelial neoplasia; LGIN, low-grade intraepithelial neoplasia; NPV, negative predictive value; PDA, poorly differentiated adenocarcinoma and signet-ring cell carcinoma; pCLE, probe-based confocal laser endomicroscopy; PPV, positive predictive value.
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This is especially true for complex intestinal metaplasia with foveolar hyperplasia, or hyperchromatic metaplastic mucosa with only slightly altered glandular architecture. In phase II of this study, we compared the accuracy of in vivo real-time pCLE diagnosis with that of the offline review made by the same endoscopist. Our results showed that the offline pCLE diagnosis tended to be more accurate than the real-time one, but this was not statistically significant. During real-time diagnosis, the endoscopist can view the patients’ endoscopic videos and the pCLE images simultaneously, but due to the speed at which pCLE videos were displayed and the constriction of time in clinical practice, details such as a few goblet cells, or local epithelial stratification may be overlooked. Whereas during the offline review process, the endoscopist have enough time to freeze individual frames and is able to assess the recorded pCLE images in detail multiple times, which may be helpful to increase the diagnostic accuracy as compared with realtime pCLE diagnosis. However, our results showed that there was no difference in the accuracy for the diagnosis of neoplasia between real-time and offline pCLE diagnosis. In practical terms, these results suggest that real-time pCLE might be a useful tool for the surveillance of patients at high risk of gastric neoplasia, and aid to on-site management of certain patients with HGIN/cancer. Of note, our current study furthermore demonstrated the reliability of this new pCLE classification system in the stomach by performing interobserver and intraobserver agreement evaluations among 3 independent investigators. To the best of our knowledge, our study was the first to assess the interobserver and intraobserver agreements of pCLE for the interpretation of various gastric abnormalities including atrophic gastritis, intestinal metaplasia, LGIN, HGIN, differentiated, and poorly differentiated adenocarcinoma. Bok et al6 performed the interobserver agreement calculation for pCLE in the diagnosis of superficial gastric neoplasia and achieved an excellent kappa value of 0.931. Pittayanon et al22 reported an “almost perfect” agreement among 5 experienced readers for the detection of gastric intestinal metaplasia. These findings were in line with our present study supporting the reliability of pCLE for diagnosing premalignant and malignant gastric lesions. As the primary outcome of this study was to establish a new pCLE classification of gastric pit patterns and vessel architecture, virtual chromoendoscopy (eg, i-Scan, NBI, etc.) was not performed at the time of high-definition endoscopy. However, prior studies revealed that narrowband imaging could diagnose gastric neoplasia with high accuracies, and the digital enhancement of i-Scan improved the image quality of magnification endoscopy.1,2,23 Therefore, further studies should be performed to assess diagnostic accuracies of gastric neoplasia by using the combination of virtual chromoendoscopy and pCLE. There are several limitations to this study. First, the endomicroscopist who performed the in vivo real-time pCLE diagnosis was inevitably affected by the endoscopic appearance of the lesion. Yet our offline evaluation results that were made only by review of the pCLE videos were comparable with in vivo diagnosis of neoplasia. Second, the performing endoscopist is experienced in pCLE manipulation and endomicroscopic images interpretation, which may optimize images acquisition and diagnostic accuracies. Therefore, further studies for learning curve evaluations are needed to see whether this new pCLE classification system in the stomach can be learned rapidly by a wide range of GI Copyright
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specialists. Third, this study recruited a selected population referred to our endoscopic center, thus the results of this study should be confirmed in a larger, multicenter trial. In conclusion, the new pCLE classification system in the stomach correlates well with specific pathologic conditions. A high accuracy with pCLE was demonstrated for predicting atrophic gastritis, intestinal metaplasia, and gastric neoplasia during either real-time or offline settings. The satisfactory interobserver and intraobserver agreements demonstrated in this study substantiated the reliability of this new pCLE classification of gastric pit patterns and vessel architecture. However, multicenter researches are warranted to further validate its value in clinical practice. REFERENCES 1. Pimentel-Nunes P, Dinis-Ribeiro M, Soares JB, et al. A multicenter validation of an endoscopic classification with narrow band imaging for gastric precancerous and cancerous lesions. Endoscopy. 2012;44:236–246. 2. Yao K, Doyama H, Gotoda T, et al. Diagnostic performance and limitations of magnifying narrow-band imaging in screening endoscopy of early gastric cancer: a prospective multicenter feasibility study. Gastric Cancer. 2014;17:669–679. 3. Pohl J, May A, Rabenstein T, et al. Computed virtual chromoendoscopy: a new tool for enhancing tissue surface structures. Endoscopy. 2007;39:80–83. 4. Pohl H, Rosch T, Vieth M, et al. Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett’s oesophagus. Gut. 2008;57:1648–1653. 5. Lim LG, Yeoh KG, Srivastava S, et al. Comparison of probebased confocal endomicroscopy with virtual chromoendoscopy and white-light endoscopy for diagnosis of gastric intestinal metaplasia. Surg Endosc. 2013;27:4649–4655. 6. Bok GH, Jeon SR, Cho JY, et al. The accuracy of probe-based confocal endomicroscopy versus conventional endoscopic biopsies for the diagnosis of superficial gastric neoplasia (with videos). Gastrointest Endosc. 2013;77:899–908. 7. Buchner AM, Shahid MW, Heckman MG, et al. Comparison of probe-based confocal laser endomicroscopy with virtual chromoendoscopy for classification of colon polyps. Gastroenterology. 2010;138:834–842. 8. Watanabe T. Efficacy of probe-based confocal laser endomicroscopy for surveillance in ulcerative colitis endomicroscopy for ulcerative colitis surveillance. Endoscopy. 2011;43:374. 9. Meining A, Shah RJ, Slivka A, et al. Classification of probebased confocal laser endomicroscopy findings in pancreaticobiliary strictures. Endoscopy. 2012;44:251–257. 10. Nakai Y, Shinoura S, Ahluwalia A, et al. Molecular imaging of epidermal growth factor-receptor and survivin in vivo in porcine esophageal and gastric mucosae using probe-based confocal laser-induced endomicroscopy: proof of concept. J Physiol Pharmacol. 2012;63:303–307. 11. Guo YT, Li YQ, Yu T, et al. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo: a prospective study. Endoscopy. 2008;40:547–553. 12. Li Z, Yu T, Zuo XL, et al. Confocal laser endomicroscopy for in vivo diagnosis of gastric intraepithelial neoplasia: a feasibility study. Gastrointest Endosc. 2010;72:1146–1153. 13. Li WB, Zuo XL, Li CQ, et al. Diagnostic value of confocal laser endomicroscopy for gastric superficial cancerous lesions. Gut. 2011;60:299–306. 14. Zhang JN, Li YQ, Zhao YA, et al. Classification of gastric pit patterns by confocal endomicroscopy. Gastrointest Endosc. 2008;67:843–853. 15. Wallace M, Lauwers GY, Chen Y, et al. Miami classification for probe-based confocal laser endomicroscopy. Endoscopy. 2011;43:882–891. 16. Dixon MF, Genta RM, Yardley JH, et al. Classification and grading of gastritis. The updated Sydney System. International
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Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161–1181. 17. Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut. 2000;47:251–255. 18. Hamilton SR, Aaltonen LA. World Health Organization classification of tumors Pathology and genetics of tumors of the digestive system. 2000Lyon, France: International Agency for Research on Cancer (IARC) Press. 38–52. 19. Kato M, Kaise M, Yonezawa J, et al. Autofluorescence endoscopy versus conventional white light endoscopy for the detection of superficial gastric neoplasia: a prospective comparative study. Endoscopy. 2007;39:937–941.
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20. Bossuyt PM, Reitsma JB, Bruns DE, et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. BMJ. 2003;326:41–44. 21. Wang P, Ji R, Yu T, et al. Classification of histological severity of Helicobacter pylori-associated gastritis by confocal laser endomicroscopy. World J Gastroenterol. 2010;16:5203–5210. 22. Pittayanon R, Rerknimitr R, Wisedopas N, et al. Flexible spectral imaging color enhancement plus probe-based confocal laser endomicroscopy for gastric intestinal metaplasia detection. J Gastroenterol Hepatol. 2013;28:1004–1009. 23. Li CQ, Li Y, Zuo XL, et al. Magnified and enhanced computed virtual chromoendoscopy in gastric neoplasia: a feasibility study. World J Gastroenterol. 2013;19:4221–4227.
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New Classification of Gastric Pit Patterns and Vessel Architecture Using Probe-based Confocal Laser Endomicroscopy Zhen Li, PhD,* Xiu-Li Zuo, PhD,* Chang-Qing Li, PhD,* Zhi-Yan Liu, PhD,w Rui Ji, PhD,* Jun Liu, PhD,* Jing Guo, MD,* and Yan-Qing Li, PhD*
Goals: To propose a new probe-based confocal laser endomicroscopy (pCLE) classification of gastric pit patterns and vessel architecture, and to assess the accuracy and interobserver agreement. Background: pCLE is a newly developed endoscopic device that allows the application of laser microscopy with any conventional endoscope and mosaic imaging. Study: A total of 291 pCLE videos from 32 patients were recruited in phase I to establish the new pCLE image classification in the stomach. Eligible patients were then prospectively investigated by pCLE using the newly established classification system. All patients were examined first with high-definition endoscopy followed by pCLE at 7 standardized locations and endoscopic-suspected lesions. Targeted biopsies were performed with precise matching of pCLE recordings. Results: The sensitivity and specificity of type 2b pit pattern for predicting atrophic gastritis were 88.51% and 99.19%, respectively. The sensitivity and specificity of type 2c pit pattern for predicting intestinal metaplasia were 92.34% and 99.34%, respectively. The overall sensitivity and specificity of type 3 pit pattern or vessel architecture for predicting neoplasia were 89.89% and 99.44%, respectively. The interobserver agreement was “substantial” (kappa = 0.70) for the differentiation of neoplasia versus nonneoplasia. Conclusions: The new pCLE classification system in the stomach correlates well with specific pathologic conditions and is reproducible by multiple investigators. Multicenter researches are warranted to further validate its value in clinical practice. Key Words: probe-based confocal laser endomicroscopy, pit pattern, vessel architecture, gastric cancer, intraepithelial neoplasia
(J Clin Gastroenterol 2016;50:23–32)
E
arly detection of gastric cancer and precancerous gastric lesions is essential for each Gastrointestinal (GI) endoscopy. However, most of these lesions lack distinctive
Received for publication July 25, 2014; accepted January 6, 2015. From the *Department of Gastroenterology, Qilu Hospital, Shandong University; and wDepartment of Pathology and Pathophysiology, Shandong University School of Medicine, Jinan, China. Y.-Q.L. receives research funding from: The National Natural Science Foundation of China, No. 81330012; the Shandong Province Science and Technology Committee, No. 2013GSF11833. Z.L. receives research funding from the National Natural Foundation of Science of China, No. NSFC81301264. The remaining authors declare that they have nothing to disclose. Reprints: Yan-Qing Li, PhD, No. 107, Wenhuaxi Road, Jinan 250012, China (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
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endoscopic characteristics, which make it difficult for accurate differentiation of malignant from benign lesions. During recent decades, advancements in endoscopic technologies including magnifying endoscopy, narrow-band imaging, and virtual chromoendoscopy have significantly improved the diagnostic performance of early gastric neoplasia and preneoplastic changes.1–3 Confocal laser endomicroscopy (CLE) is a novel endoscopic system that can provide real-time microscopic observation of the GI mucosa. Currently, 2 kinds of endomicroscopes are available including endoscope-based CLE system (eCLE; Pentax, Tokyo, Japan) and probebased confocal laser endomicroscopy (pCLE) system (Cellvizio, Mauna Kea Technologies, Paris, France). The eCLE system was built by the integration of a confocal laser microscope at the distal tip of a conventional endoscope. Whereas pCLE is a newly developed endoscopic device that allows the application of laser microscopy with any conventional endoscope and mosaic imaging. To date, numerous studies have evaluated the utility of pCLE in various GI diseases, including Barrett’s esophagus, gastric intestinal metaplasia, superficial gastric neoplasia, colorectal polyps, ulcerative colitis, and pancreaticobiliary disease.4–9 Moreover, recent data also explored its potential application in the field of molecular imaging.10 Our previous studies using endoscope-based CLE system have established and assessed its diagnostic value for gastric intestinal metaplasia, intraepithelial neoplasia, and gastric carcinoma.11–13 We have also reported a classification of gastric pit patterns using eCLE, which classified the gastric pit patterns into 7 types (type A to G), and demonstrated that the patterns of gastric pits identified by eCLE correlated well with histologic findings.14 However, these studies were based upon confocal images from eCLE, and may suffer from potential challenges accordingly. For instance, pCLE has a smaller field of view and higher image acquisition rate as compared with eCLE, and the image differences caused by these technical issue may render those published eCLE criteria under further assessment by using pCLE. In 2011, Wallace et al15 proposed a state-of-the-art classification for the differentiation between normal and pathologic states in GI diseases using pCLE, which is named the “Miami classification system.” Later, a singlecenter study by Bok et al6 showed that, by using the “Miami classification system,” pCLE have a sensitivity of 90.6% and a specificity of 90.9% for the diagnosis of superficial gastric neoplasias. However, the “Miami classification system” only briefly depicted endomicroscopic changes using pCLE for different gastric diseases, including normal stomach, gastritis, gastric dysplasia, and www.jcge.com |
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TABLE 1. A New pCLE Classification of Gastric Pit Patterns and Vessel Architecture
Categories
pCLE Appearance
Distribution Area
pCLE Image
Gastric pit patterns Type 1
Regular pits with wide/round/slit-like openings
Normal cardia/corpus/antrum
Regular pits with elongated openings, increased fluorescence in stroma
Inflammatory gastric mucosa
b
Reduced pits with dilated openings
Atrophic mucosa
c
Appearance of goblet cells with dark mucin
Intestinal metaplastic mucosa
Mild to moderate irregular pits with variable width of the epithelial lining
Low-grade intraepithelial neoplasia
Prominent distorted pits with irregular epithelial lining
High-grade intraepithelial neoplasia
Type 2 a
Type 3 a
b
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TABLE 1. (continued) Categories
pCLE Appearance
c
Appearance of atypical glands/dispersion of irregular dark cells
Vessel architecture Type 1 Regular capillaries with normal caliber, anfractous/honeycomb-like/coil-shaped
Distribution Area
Differentiated/poorly differentiated adenocarcinoma
Normal cardia/corpus/antrum
Type 2
Increased capillaries with elevated leakage
Inflammatory gastric mucosa
Type 3
Irregular capillaries with heterogenous leakage/ dilated caliber
Neoplastic gastric mucosa
adenocarcinoma. These parameters still lack prospective validation, and do not include the description of vessel architecture for different pathologic conditions. Furthermore, considering the fact that pCLE has the priority for blood flow because of its increased image acquisition rate, it would be desirable to specify the distinctive capillary changes for different lesions in pCLE images. However, to date, there has been limited data concerning the application of pCLE in the stomach, and no investigation has yet focused on the characterization and classification of gastric pit patterns and vessel architecture using pCLE. Therefore, the aims of the present study were to propose a new pCLE classification of gastric pit patterns and vessel architecture, and to assess the accuracy and interobserver agreement of this new pCLE classification system for diagnosing various premalignant and malignant changes in the stomach. Copyright
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pCLE Image
MATERIALS AND METHODS Phase I: Establishment of the New Classification A total of 291 pCLE videos from 32 patients were recruited in phase I to establish the new pCLE image classification in the stomach. This data set consisted of 40 inflammation, 18 atrophic gastritis, 25 intestinal metaplasia, 2 low-grade intraepithelial neoplasia (LGIN), 1 high-grade intraepithelial neoplasia (HGIN), 1 differentiated adenocarcinoma, and 2 poorly differentiated adenocarcinoma. The endoscopic procedure were carried out as described in the prospective study (phase II). All 291 confocal videos were openly evaluated by 3 experienced endomicroscopists (Y.-Q.L., X.-L.Z., C.-Q.L.) and 1 GI pathologist (Z.-Y.L.). A new classification of gastric pit patterns and vessel architecture using pCLE (Cellvizio, Mauna Kea Technologies) was developed based
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FIGURE 1. Normal and inflammatory gastric mucosa. A, Probe-based confocal laser endomicroscopy (pCLE) image of normal cardia: regular pits with wide openings (type 1). B, pCLE image of normal gastric mucosa with fundic glands: round gastric pits with round openings (type 1). C, pCLE image of normal gastric mucosa with pyloric glands: continuous short rod-like pits with slit-like openings (type 1). D, pCLE image of cardiac mucosa with chronic inflammation: aggregation/branching of enlarged pits (type 2a). E, pCLE image of corporal mucosa with chronic inflammation: elongated pits with thread-like openings (type 2a). F, pCLE image of antral mucosa with chronic inflammation: tortuosity/branching of elongated pits (type 2a).
on the comparison between in vivo pCLE images and corresponding histologic findings from targeted biopsy specimens, histologic description for each state, and with reference to previously published researches.11–14 This new pCLE classification in the stomach consists of 3 types of pit patterns with 7 subtypes (type 1, type 2a-c, type 3a-c), and 3 types of vessel architecture (type 1, type 2, type 3). Type 1 pit patterns and vessel architecture represent normal gastric mucosa, type 2a pit patterns and type 2 vessel architecture indicate inflammation, type 2b pit pattern shows atrophic gastritis, type 2c pit pattern shows intestinal metaplasia, and type 3 pit pattern and vessel architecture indicate gastric neoplasia. Details of the pCLE classification system in the stomach were summarized in Table 1. Figures 1–4 represent confocal images and corresponding histology of the new pCLE classification system in the stomach.
Phase II: Prospective Study From April to September 2013, consecutive patients were recruited prospectively for pCLE examinations at Qilu Hospital, Shandong University. Inclusion criteria were: (1) patients with dyspeptic symptoms and aged 40 years and above; or (2) patients with H. pylori infection; or (3) patients with known premalignant or malignant gastric lesions (including atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and gastric adenocarcinoma). Exclusion criteria were: (1) patients with a history of GI surgery; (2) patients with contraindications to pCLE
examination, such as coagulopathy, pregnancy, breast feeding, or allergy to fluorescein sodium; (3) patients refused to provide informed consent. This study was approved by the Ethics Committee of Qilu Hospital, Shandong University (clinicaltrials.gov NCT01896310), and was conducted in accordance with the revised Declaration of Helsinki (2008).
pCLE Endomicroscopic visualizations were achieved using a pCLE (Cellvizio, Mauna Kea Technologies). The pCLE probe can be used in conjunction with any endoscope, and enables rapid image acquisition at a scan rate of 12 frames per second. It has a depth of scanning of 60 mm, a lateral resolution of 1 mm, a field of view of 240240 mm, and 1000 magnification. Confocal images can be displayed in real-time on a standard personal computer screen that is connected to the laser scanning unit of pCLE miniprobe. A specially designed software (Cellvizio Viewer) allows immediate sequences display, real-time image reconstruction and postprocedure analysis of all the captured pCLE videos. Fluorescein sodium (5 mL, 10%) was applied intravenously as the contrast agent before pCLE scanning.
Endoscopic Procedure All patients underwent high-definition endoscopic examinations using a Pentax EG-2990i endoscope (Pentax, Japan) with an image resolution of 1.3 mega pixels. After
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FIGURE 2. Gastric atrophy and intestinal metaplasia. A, Probe-based confocal laser endomicroscopy (pCLE) image showed glandular atrophy with decreased gastric pits and markedly dilated opening (arrows) (type 2b). B, Corresponding histologic results confirmed the appearance of atrophic gastritis (H&E original magnification 400). C, pCLE image showed dark goblet cells (arrows) within the columnar epithelium (type 2c). D, Corresponding histologic results confirmed gastric intestinal metaplasia of the mucosa (H&E original magnification 400).
FIGURE 3. Gastric intraepithelial neoplasia and adenocarcinoma. A, Probe-based confocal laser endomicroscopy (pCLE) image showed mild to moderate irregular pits with variable width of the epithelial lining (arrows) (type 3a). B, Corresponding histology specimen showed a low-grade intraepithelial neoplasia (H&E original magnification 200). C, pCLE image showed prominent distorted pits (arrows) with irregular epithelial lining (type 3b). D, Corresponding histology specimen showed a high-grade intraepithelial neoplasia (H&E original magnification 200). E, pCLE image showed the appearance of atypical/naive glands (arrows) with dark epithelium (type 3c). F, Corresponding histology specimen showed a differentiated adenocarcinoma (H&E original magnification 400). G, pCLE image showed loss of pits, appearance of irregular dark cells (arrows) (type 3c). H, Corresponding histology specimen showed a poorly differentiated adenocarcinoma (H&E original magnification 400).
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intravenous injection of 5 mL 10% fluorescein sodium, the pCLE probe was passed through the accessory channel of the endoscope, and placed in gentle contact with the gastric mucosa. Endomicroscopic imaging was achieved at all endoscopic-suspected lesions and 7 standardized locations in the stomach for each patient. The 7 standardized locations included the following sites: gastric cardia within 1 cm below the gastroesophageal junction, fundus, incisura angularis, middle portion of the greater curvature of the corpus, lesser curvature of the corpus about 4 cm proximal to the angulus, greater and lesser curvature of the antrum within 2 to 3 cm from the pylorus. All endoscopic procedures were performed by 1 experienced endoscopist (C.-Q.L.). Finally, targeted biopsies were performed with precise matching of pCLE recordings.
Although the endoscopist was informed that the study population was enriched and included patients with dyspeptic symptoms above 40 years and patients undergoing surveillance endoscopy, he has no access to any patients’ previous endoscopic and pathologic diagnoses before endoscopy. All endoscopic procedures were conducted under the supervision of a study coordinator (J.G.).19 Three months after endoscopic examination, all pCLE recordings were reassessed by the same endoscopist (C.Q.L.) who made the in vivo diagnosis. During this offline review, the endoscopist was blinded to the patients’ clinical, endoscopic, and histologic data, and endomicroscopic categorization was made according to the same pCLE classification used for in vivo diagnosis.
Histologic Evaluation
Agreement Evaluation
All specimens were evaluated by an expert GI pathologist (Z.-Y.L.) who was blinded to the patients’ pCLE findings. Histologic diagnosis was made according to the updated Sydney System for the classification of gastritis, the Vienna criteria for neoplasia, and the WHO classification of tumors (digestive system).16–18
To assess the interobserver agreement, pCLE video recordings of all endoscopic lesions examined in phase II were randomized and offline evaluated by 3 independent endomicroscopists (X.-L.Z., R.J., J.L.) who were blinded to the endoscopic findings and histology. Endomicroscopic diagnoses were also made according to the newly developed classification system in the stomach (Table 1). These pCLE videos were reevaluated by the 3 endomicroscopists after a 7-day interval to calculate the intraobserver agreement. All pCLE sequences were displayed in their original format (.mkt, Mauna Kea Technologies, Paris, France).
pCLE Videos Assessment Real-time pCLE diagnosis was made by the performing endoscopist (C.-Q.L.) during the procedure based on the newly developed classification system (Table 1).
FIGURE 4. Different types of vessel architecture in the stomach. A, Vessel architecture in the normal cardia: anfractous capillaries with regular shapes surrounding gastric pits (type 1). B, Vessel architecture in the normal corpus: honeycomb-like capillaries surrounding gastric pits (type 1). C, Vessel architecture in the normal antrum: coil-shaped capillaries surrounding gastric pits (type 1). D, Vessel architecture in the inflammatory gastric mucosa: increased capillaries with elevated leakage (type 2). E, Vessel architecture in neoplastic gastric mucosa: irregular capillaries with heterogenous leakage/dilated caliber (type 3). The arrows indicate the microvessels.
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Statistical Analysis Statistical analysis was performed by using SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the diagnosis of atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and adenocarcinoma were calculated for in vivo pCLE diagnosis and offline pCLE diagnosis. And the accuracy results stated in each section represent a comparison of the pattern type versus all other types. w2 test was used for comparisons. P < 0.05 (2-tailed) was considered as statistically significant. The interobserver agreement was estimated using the kappa value with 95% confidence intervals (CIs). The interpretation of kappa values was as follows: values > 0.80 were regarded as excellent, 0.6 to 0.8 substantial, 0.4 to 0.6 moderate, 0.2 to 0.4 fair, values < 0.2 indicating poor agreement. Kappa values for different gastric pit patterns and vessel architecture were calculated using the STATA 9.0 software package (StataCorp LP, College Station, TX). The study report was in accordance with the standards for reporting studies of diagnostic accuracy.20
Patient Characteristics A total of 244 patients were enrolled in phase II of this study according to predefined inclusion and exclusion criteria. Four patients were further excluded because of severe esophageal stricture after insertion of the endoscope. Therefore, 240 patients were finally included for data analysis. The mean age of patients was 57 years (range, 27 to 83 y), and 48.33% were women. Clinical indications included 175 patients with dyspeptic symptoms and aged 40 years and above, 34 patients with H. pylori infection, and 31 patients with known premalignant or malignant gastric lesions. Clinical characteristics of these patients were summarized in Table 2. We collected 1878 pCLE video sequences, ranging from 7 to 10 videos per patient.
Comparison of pCLE Classifications and Histologic Diagnosis A total of 198 lesions in the stomach were identified in phase II of this study. Histopathology demonstrated 61 TABLE 2. Patients’ Characteristics in This Study
Phase I
Phase II
32 19/13 50 (19-75) 26
240 124/116 57 (27-83) 198
11 4 5 2 1 1 2
61 20 28 25 9 13 42
HGIN indicates high-grade intraepithelial neoplasia; LGIN, low-grade intraepithelial neoplasia.
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inflammatory, 20 atrophic gastritis, 28 intestinal metaplasia, 25 LGIN, 9 HGIN, 13 differentiated adenocarcinoma, 42 poorly differentiated adenocarcinoma (of which 9 were signet-ring cell carcinoma). Among the 1680 standardized locations, histologic diagnosis revealed 372 normal mucosa, 986 inflammation, 128 atrophic gastritis, and 194 intestinal metaplasia (Table 2). Table 3 shows the comparison results of this new pCLE classifications and histologic diagnosis on per-location analyses (including 198 lesions and 1680 standardized locations). During real-time pCLE examination, the sensitivity and specificity of type 2b pit pattern for predicting atrophic gastritis were 88.51% (95% CI, 82.37%-92.70%) and 99.19% (95% CI, 98.65%-99.52%), respectively. The sensitivity and specificity of type 2c pit pattern for predicting intestinal metaplasia were 92.34% (95% CI, 88.08%-95.16%) and 99.34% (95% CI, 98.81%-99.63%), respectively. Overall sensitivity and specificity of type 3 pit pattern or type 3 vessel architecture for predicting neoplasia were 89.89% (95% CI, 81.89%-94.59%) and 99.44% (95% CI, 98.97%-99.70%), respectively.
Offline pCLE Diagnosis
RESULTS
Patients (n) Sex (male/female) (n) Mean age (range) (y) Macroscopic lesions (n) Histologic diagnosis Inflammation Atrophic gastritis Intestinal metaplasia LGIN HGIN Differentiated adenocarcinoma Poorly differentiated adenocarcinoma and signet-ring cell carcinoma
New Classification in the Stomach Using pCLE
The offline pCLE diagnostic results of different histologic status were as follows: atrophic gastritis with a sensitivity of 89.86% and a specificity of 99.25%; intestinal metaplasia with a sensitivity of 93.69% and a specificity of 99.40%; and the non-neoplasia versus neoplasia with a sensitivity of 92.13% and a specificity of 99.50%. The accuracy for offline pCLE diagnosis of neoplasia was 99.15%. There was no difference between the accuracies of in vivo and offline diagnosis of neoplasia (P = 0.610). Table 4 shows the results of sensitivity, specificity, accuracy, and positive and negative predictive values for offline pCLE diagnosis of various gastric abnormalities.
Interobserver and Intraobserver Agreement The interobserver agreements on different gastric pit patterns (3 types) and vessel architecture (3 types) were “substantial” with kappa values of 0.63 (95% CI, 0.60-0.66) and 0.64 (95% CI, 0.61-0.70), respectively. The intraobserver agreements were all “excellent” for the differentiation among 3 types of gastric pit patterns (mean kappa = 0.90) and vessel architecture (mean kappa = 0.94). We also investigated the agreement values on each subtype of gastric pit patterns, which showed an interobserver agreement of 0.54 (95% CI, 0.51-0.55) and an intraobserver agreement of 0.93 (0.94, 0.92, 0.92), respectively. When using the type 3 pit pattern or type 3 vessel architecture for the differentiation between neoplasia versus non-neoplasia, the interobserver agreement was “substantial” with a kappa value of 0.70 (95% CI, 0.630.71), and the intraobserver agreement was “excellent” with a mean kappa value of 0.94 (0.92, 0.94, 0.96).
DISCUSSION This study proposed a new classification system of gastric pit patterns and vessel architecture specifically designed for probe-based CLE device. Prospective study including 240 patients proved that this new classification system has high accuracies for the diagnoses of atrophic gastritis, intestinal metaplasia, and gastric neoplasia.
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TABLE 3. Comparisons Between Real-time pCLE Diagnosis and Histologic Diagnosis on Per-location Diagnosis
CLE Diagnosis Histopathology Normal Inflammation AG IM LGIN HGIN DA PDA Total
Normal
Inflammation
AG
IM
LGIN
HGIN
DA
PA
Total
372 101 — — — — — — 473
— 937 12 4 1 — — — 954
— 3 131 10 1 — — — 145
— 5 4 205 2 — — — 216
— — 1 3 20 1 — — 25
— — — — 1 7 1 — 9
— — — — — 1 12 1 14
— 1 — — — — — 41 42
372 1047 148 222 25 9 13 42 1878
AG indicates atrophic gastritis; DA, differentiated adenocarcinoma; HGIN, high-grade intraepithelial neoplasia; IM, intestinal metaplasia; LGIN, lowgrade intraepithelial neoplasia; PDA, poorly differentiated adenocarcinoma and signet-ring cell carcinoma; pCLE, probe-based confocal laser endomicroscopy.
Furthermore, a substantial interobserver agreement and an excellent intraobserver agreement was achieved for the differentiation among different types of pit patterns and vessel architecture, and when it reduced to a dichotomous diagnosis of non-neoplasia and neoplasia, the interobserver agreement was also substantial with a higher kappa value. Although our previous study has reported a classification of gastric pit patterns using endoscope-based CLE system,14 it did not describe the endomicroscopic features of vessel architecture for various histologic changes, and the pit pattern classifications covered only normal, inflammatory, atrophic, intestinal metaplastic, and cancerous changes in the antrum and corpus. Whereas the new classification system in the stomach proposed in our current study correlated well with specific pathologic conditions, and firstly included the description of gastric cardia, the characteristics of LGIN and HGIN, and different vessel architectural changes in pCLE videos. Moreover, the interobserver and intraobserver agreements were also assessed for different types and subtypes of gastric pit patterns and vessel architecture in this study, which further substantiated its validity for clinical application. Most patients with gastric adenocarcinoma die during the first year of the disease even if referred to aggressive and costly therapies due to diagnosis at late stages. Therefore, one of the aims of this new pCLE classification was an accurate identification of premalignant gastric conditions
or lesions, namely atrophic gastritis, intestinal metaplasia, and intraepithelial neoplasia. Although previous studies have investigated the diagnostic ability of endoscope-based CLE for identifying atrophic gastritis,14,21 intestinal metaplasia and intraepithelial neoplasia,11,12 it is still not clear whether similar results could be achieved by using pCLE, given the fact that the pCLE system slightly lags in resolution and penetration depth compared with eCLE. Our present study showed that comparable results can be achieved with the 2 endomicroscopic modalities (pCLE and eCLE) in diagnosing various gastric lesions, including atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and adenocarcinoma. Of interest, the diagnostic specificity of pCLE in this study was much higher than previous researches. This was due to the large number of standardized biopsies with histologic diagnosis of inflammation and normal gastric mucosa, most of which can be easily recognized and correctly diagnosed using pCLE. Our earlier study showed that eCLE can reliably differentiate noncancerous gastric lesions from cancer/HGIN lesions using a 2-tiered eCLE classification system.13 In this study, we found that using the new pCLE classification system in the stomach, pCLE also enables an accurate in vivo differentiation between neoplastic and non-neoplastic gastric lesions. As shown in Table 3, macroscopic gastric lesions with LGIN and intestinal metaplasia may not easily be distinguished from each other in some cases.
TABLE 4. Offline pCLE Diagnosis of Various Gastric Abnormalities in Phase II
Abnormality AG IM LGIN HGIN DA PDA Neoplasia (IN + CA)
Sensitivity [% (CI)] 89.86 93.69 84.00 88.89 92.31 97.62 92.13
(83.95-93.76) (89.69-96.21) (65.35-93.60) (56.50-98.01) (66.69-98.63) (87.68-99.58) (84.64-96.14)
Specificity [% (CI)] 99.25 99.40 99.78 99.89 99.89 99.95 99.50
(98.72-99.56) (98.89-99.67) (99.45-99.92) (99.61-99.97) (99.61-99.97) (99.69-99.99) (99.05-99.74)
PPV [% (CI)] 91.10 95.41 84.00 80.00 85.71 97.62 90.11
(85.36-94.72) (91.76-97.49) (65.35-93.60) (49.02-94.33) (60.06-99.59) (87.68-99.58) (82.26-94.71)
NPV [% (CI)] 99.13 99.16 99.78 99.95 99.95 99.95 99.61
(98.58-99.47) (98.59-99.50) (99.45-99.92) (99.70-99.99) (99.70-99.99) (99.69-99.99) (99.19-99.81)
Accuracy [% (CI)] 98.51 98.72 99.57 99.84 99.84 99.89 99.15
(97.85-98.97) (98.11-99.14) (99.16-99.78) (99.53-99.95) (99.53-99.95) (99.61-99.97) (98.62-99.47)
AG indicates atrophic gastritis; CA, adenocarcinoma; CI, confidence interval; DA, differentiated adenocarcinoma; HGIN, high-grade intraepithelial neoplasia; IM, intestinal metaplasia; IN, intraepithelial neoplasia; LGIN, low-grade intraepithelial neoplasia; NPV, negative predictive value; PDA, poorly differentiated adenocarcinoma and signet-ring cell carcinoma; pCLE, probe-based confocal laser endomicroscopy; PPV, positive predictive value.
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This is especially true for complex intestinal metaplasia with foveolar hyperplasia, or hyperchromatic metaplastic mucosa with only slightly altered glandular architecture. In phase II of this study, we compared the accuracy of in vivo real-time pCLE diagnosis with that of the offline review made by the same endoscopist. Our results showed that the offline pCLE diagnosis tended to be more accurate than the real-time one, but this was not statistically significant. During real-time diagnosis, the endoscopist can view the patients’ endoscopic videos and the pCLE images simultaneously, but due to the speed at which pCLE videos were displayed and the constriction of time in clinical practice, details such as a few goblet cells, or local epithelial stratification may be overlooked. Whereas during the offline review process, the endoscopist have enough time to freeze individual frames and is able to assess the recorded pCLE images in detail multiple times, which may be helpful to increase the diagnostic accuracy as compared with realtime pCLE diagnosis. However, our results showed that there was no difference in the accuracy for the diagnosis of neoplasia between real-time and offline pCLE diagnosis. In practical terms, these results suggest that real-time pCLE might be a useful tool for the surveillance of patients at high risk of gastric neoplasia, and aid to on-site management of certain patients with HGIN/cancer. Of note, our current study furthermore demonstrated the reliability of this new pCLE classification system in the stomach by performing interobserver and intraobserver agreement evaluations among 3 independent investigators. To the best of our knowledge, our study was the first to assess the interobserver and intraobserver agreements of pCLE for the interpretation of various gastric abnormalities including atrophic gastritis, intestinal metaplasia, LGIN, HGIN, differentiated, and poorly differentiated adenocarcinoma. Bok et al6 performed the interobserver agreement calculation for pCLE in the diagnosis of superficial gastric neoplasia and achieved an excellent kappa value of 0.931. Pittayanon et al22 reported an “almost perfect” agreement among 5 experienced readers for the detection of gastric intestinal metaplasia. These findings were in line with our present study supporting the reliability of pCLE for diagnosing premalignant and malignant gastric lesions. As the primary outcome of this study was to establish a new pCLE classification of gastric pit patterns and vessel architecture, virtual chromoendoscopy (eg, i-Scan, NBI, etc.) was not performed at the time of high-definition endoscopy. However, prior studies revealed that narrowband imaging could diagnose gastric neoplasia with high accuracies, and the digital enhancement of i-Scan improved the image quality of magnification endoscopy.1,2,23 Therefore, further studies should be performed to assess diagnostic accuracies of gastric neoplasia by using the combination of virtual chromoendoscopy and pCLE. There are several limitations to this study. First, the endomicroscopist who performed the in vivo real-time pCLE diagnosis was inevitably affected by the endoscopic appearance of the lesion. Yet our offline evaluation results that were made only by review of the pCLE videos were comparable with in vivo diagnosis of neoplasia. Second, the performing endoscopist is experienced in pCLE manipulation and endomicroscopic images interpretation, which may optimize images acquisition and diagnostic accuracies. Therefore, further studies for learning curve evaluations are needed to see whether this new pCLE classification system in the stomach can be learned rapidly by a wide range of GI Copyright
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New Classification in the Stomach Using pCLE
specialists. Third, this study recruited a selected population referred to our endoscopic center, thus the results of this study should be confirmed in a larger, multicenter trial. In conclusion, the new pCLE classification system in the stomach correlates well with specific pathologic conditions. A high accuracy with pCLE was demonstrated for predicting atrophic gastritis, intestinal metaplasia, and gastric neoplasia during either real-time or offline settings. The satisfactory interobserver and intraobserver agreements demonstrated in this study substantiated the reliability of this new pCLE classification of gastric pit patterns and vessel architecture. However, multicenter researches are warranted to further validate its value in clinical practice. REFERENCES 1. Pimentel-Nunes P, Dinis-Ribeiro M, Soares JB, et al. A multicenter validation of an endoscopic classification with narrow band imaging for gastric precancerous and cancerous lesions. Endoscopy. 2012;44:236–246. 2. Yao K, Doyama H, Gotoda T, et al. Diagnostic performance and limitations of magnifying narrow-band imaging in screening endoscopy of early gastric cancer: a prospective multicenter feasibility study. Gastric Cancer. 2014;17:669–679. 3. Pohl J, May A, Rabenstein T, et al. Computed virtual chromoendoscopy: a new tool for enhancing tissue surface structures. Endoscopy. 2007;39:80–83. 4. Pohl H, Rosch T, Vieth M, et al. Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett’s oesophagus. Gut. 2008;57:1648–1653. 5. Lim LG, Yeoh KG, Srivastava S, et al. Comparison of probebased confocal endomicroscopy with virtual chromoendoscopy and white-light endoscopy for diagnosis of gastric intestinal metaplasia. Surg Endosc. 2013;27:4649–4655. 6. Bok GH, Jeon SR, Cho JY, et al. The accuracy of probe-based confocal endomicroscopy versus conventional endoscopic biopsies for the diagnosis of superficial gastric neoplasia (with videos). Gastrointest Endosc. 2013;77:899–908. 7. Buchner AM, Shahid MW, Heckman MG, et al. Comparison of probe-based confocal laser endomicroscopy with virtual chromoendoscopy for classification of colon polyps. Gastroenterology. 2010;138:834–842. 8. Watanabe T. Efficacy of probe-based confocal laser endomicroscopy for surveillance in ulcerative colitis endomicroscopy for ulcerative colitis surveillance. Endoscopy. 2011;43:374. 9. Meining A, Shah RJ, Slivka A, et al. Classification of probebased confocal laser endomicroscopy findings in pancreaticobiliary strictures. Endoscopy. 2012;44:251–257. 10. Nakai Y, Shinoura S, Ahluwalia A, et al. Molecular imaging of epidermal growth factor-receptor and survivin in vivo in porcine esophageal and gastric mucosae using probe-based confocal laser-induced endomicroscopy: proof of concept. J Physiol Pharmacol. 2012;63:303–307. 11. Guo YT, Li YQ, Yu T, et al. Diagnosis of gastric intestinal metaplasia with confocal laser endomicroscopy in vivo: a prospective study. Endoscopy. 2008;40:547–553. 12. Li Z, Yu T, Zuo XL, et al. Confocal laser endomicroscopy for in vivo diagnosis of gastric intraepithelial neoplasia: a feasibility study. Gastrointest Endosc. 2010;72:1146–1153. 13. Li WB, Zuo XL, Li CQ, et al. Diagnostic value of confocal laser endomicroscopy for gastric superficial cancerous lesions. Gut. 2011;60:299–306. 14. Zhang JN, Li YQ, Zhao YA, et al. Classification of gastric pit patterns by confocal endomicroscopy. Gastrointest Endosc. 2008;67:843–853. 15. Wallace M, Lauwers GY, Chen Y, et al. Miami classification for probe-based confocal laser endomicroscopy. Endoscopy. 2011;43:882–891. 16. Dixon MF, Genta RM, Yardley JH, et al. Classification and grading of gastritis. The updated Sydney System. International
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Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161–1181. 17. Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut. 2000;47:251–255. 18. Hamilton SR, Aaltonen LA. World Health Organization classification of tumors Pathology and genetics of tumors of the digestive system. 2000Lyon, France: International Agency for Research on Cancer (IARC) Press. 38–52. 19. Kato M, Kaise M, Yonezawa J, et al. Autofluorescence endoscopy versus conventional white light endoscopy for the detection of superficial gastric neoplasia: a prospective comparative study. Endoscopy. 2007;39:937–941.
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20. Bossuyt PM, Reitsma JB, Bruns DE, et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. BMJ. 2003;326:41–44. 21. Wang P, Ji R, Yu T, et al. Classification of histological severity of Helicobacter pylori-associated gastritis by confocal laser endomicroscopy. World J Gastroenterol. 2010;16:5203–5210. 22. Pittayanon R, Rerknimitr R, Wisedopas N, et al. Flexible spectral imaging color enhancement plus probe-based confocal laser endomicroscopy for gastric intestinal metaplasia detection. J Gastroenterol Hepatol. 2013;28:1004–1009. 23. Li CQ, Li Y, Zuo XL, et al. Magnified and enhanced computed virtual chromoendoscopy in gastric neoplasia: a feasibility study. World J Gastroenterol. 2013;19:4221–4227.
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