In the diagnosis of gastric mucosal lesions by endoscopy, it is important to distinguish between tumors and non-tumors. Pathological evaluation by biopsy is the gold standard for the diagnosis of gastric epithelial tumors. In recent years, with the increase in the incidence of cardiovascular and cerebrovascular diseases, the number of patients taking antithrombotic drugs has been growing [10, 14]. Moreover, most inexperienced endoscopists usually take multiple biopsies during the operation, which will aggravate the tissue damage. Therefore, it’s imperative to find a method to keep the number of biopsies during endoscopy as low as possible. Studies have confirmed that ME-NBI can significantly improve the diagnostic performance for gastric mucosal lesions and reduce the number of biopsies when diagnosing tumors [10]. At present, the VS classification system is commonly used to diagnose gastric cancer under the ME-NBI. For applying the VS classification system to determine whether the suspected lesion is cancerous, the DL needs to be clarified first [8]. However, not all suspected lesions have clear DL and/or IMVP/IMSP [5, 10]. When the DL and/or IMVP/IMSP are indeterminate, the accuracy of the VS classification system in diagnosing gastric cancer will decrease. Under this circumstance, the diagnosis mainly depends on the lesion biopsy. So how can we maintain a high accuracy rate of diagnosing gastric cancer without increasing the number of biopsy when the DL and/or IMVP/IMSP can not be determined?
In the early stage of this study, we first discovered that gastric cancer lesions showed pink color change under ME-NBI (Fig. 1 and Figure S1), and named it as "pink pattern". Cancerous lesions are often accompanied by vascular changes. Abnormal blood vessels can also have reddish-brown color changes under the ME-NBI. Therefore, when the pink pattern appears in the cancerous area, it is easy to be confused with the color change of abnormal blood vessels in the same area, leading to the pink pattern is overlooked at the ME-NBI examination. So it’s necessary to get rid of the vascular interference to recognize the pink pattern in the suspected lesions under ME-NBI. Later, the pathological diagnosis was performed on the suspected lesions that showed pink pattern under ME-NBI, it was found that the nucleus-to-plasma ratio of the gastric epithelial cells in cancerous area increased (Fig. 2 and Figure S2). For this reason, we speculated that the pink pattern in the cancerous area under the ME-NBI may be related to the change of nucleus-to-plasma ratio in gastric epithelial cells of the same area. But the key point to link the pink pattern and the nucleus-to-plasma ratio of gastric epithelial cells was still unknown.
The NBI has two light bands with center wavelengths of 415nm (blue light) and 540nm (green light) [15]. The dual-wavelength light band is confined to mucosa and does not penetrate the submucosal tissue [15], so the microstructure of the mucosal surface can be clearly visualized. Based on the above characteristics of the NBI light band, we tried to find the link point between the pink pattern and the nucleus-to-plasma ratio by analyzing the color features of crypt epithelium on the mucosal surface in NBI image and the epithelial cell layer in pathological image. In the pathological image, the nucleus looks dark while the cytoplasm is lighter under HE staining. The nucleus-to-plasma ratio increases when the epithelial cells become cancerous, and the color of cancerous area become darker correspondingly under HE staining (Fig. 2 and Figure S2). Thus the change in nucleus-to-plasma ratio of epithelial cells can be represented by the color changes of these cells. If the color change trend of cancerous epithelial cells in the HE-stained pathological images is consistent with the color change trend of cancerous lesions in the ME-NBI images, it can be confirmed that the increased nucleus-to-plasma ratio of gastric cancer epithelial cells is highly correlated with the pink pattern of the cancerous lesions in the ME-NBI image. So the color change is considered as the key point to link the pink pattern and the nuclear-to-plasma ratio of cancerous epithelial cells.
In order to verify the pink pattern of the cancerous lesions in the ME-NBI images is highly correlated with the increase of nucleus-to-plasma ratio in gastric cancer epithelial cells, the color features of ME-NBI images(Fig. 3) and HE-stained pathological images(Fig. 4) on gastric cancerous mucosal surface were extracted and quantized. Then the cosine similarity between the color values of the ME-NBI image and the HE-stained pathological image on gastric cancerous mucosal layer was calculated, and the results showed that the pink color change trend of the cancerous lesions in ME-NBI images was consistent with the color change trend of cancerous epithelial cells in HE-stained pathological images (Fig. 5, Table 3), suggesting that the pink pattern of ME-NBI image on the cancerous lesions is greatly related to the increased nucleus-to-plasma ratio of gastric cancer epithelial cells. Hence, the presence of a pink pattern is a warning sign that can be part of a preoperative assessment strategy for early gastric cancer in clinical practice.
Subsequently, the stored images captured by endoscope with NBI were evaluated independently by 12 endoscopists using VS classification system (test 1) and VS classification system plus pink pattern (test 2) respectively to assess the effect of performing ME-NBI after taking pink pattern into account. As far as overall diagnostic performance, the median AUC, accuracy and specificity of test 1 were 79.0%, 78.7% and 90.0% in all endoscopists, respectively, while the sensitivity was only 67.4%, which was similar to that in previous studies[5, 10]. However, when the “pink pattern” was taken into account for the ME-NBI diagnosis (test 2), the median values of AUC, accuracy, sensitivity and specificity increased to 87.3%, 87.0%, 78.4% and 95.0% (Table 4 and Fig. 7a), meaning that the cancer diagnostic capability of ME-NBI was significantly improved. Then these endoscopists were assigned to junior and experienced groups respectively according to the cases of endoscopy examinations (< 10000, junior; ≥10,000, experienced). In the two subgroups, the AUC, accuracy, sensitivity and specificity of test 2 were significantly higher than those obtained from test 1(Fig. 7b and 7c). In addition, test 2 significantly improved the PPV and NPV compared with test 1 in all endoscopists, junior and experienced groups respectively (Table 4, Fig. 7). It has great significance in clinical practice for that the examination with high PPV and NPV can enable endoscopists to determine which lesion needs pathological diagnosis. These results have the potential to make so-called “optic biopsy” come true. Next, we calculated the Fleiss’ kappa value of all endoscopists, junior and experienced groups in test 1 and test 2 to evaluate the agreement of the endoscopic diagnosis level among the participants. The interpretation of kappa value for all endoscopists, junior and experienced groups in test 1 and test 2 was at least moderate, especially the interpretation of all endoscopists and experienced group was improved to substantial agreement in test 2(Table 5). The moderate or higher diagnostic agreement among participants is mainly due to the fact that the endoscopists all come from the same department and use similar diagnostic algorithm, which guarantees the consistency of test.
Table 5
Fleiss’ kappa value of tests 1 and 2 in 12 endoscopists
Endoscopists
|
Test 1
|
Test 2
|
Fleiss’ kappa
|
Agreement
|
Fleiss’ kappa
|
Agreement
|
All(n = 12)
|
0.51
|
Moderate
|
0.65
|
Substantial
|
Junior(n = 6)
|
0.47
|
Moderate
|
0.58
|
Moderate
|
Experienced(n = 6)
|
0.60
|
Moderate
|
0.76
|
Substantial
|
Fleiss’ kappa values were evaluated by R with lpSolve and irr packages. Kappa values of 0.01 to 0.20, 0.21 to 0.40, 0.41 to 0.60, 0.61 to 0.80, and 0.81 to 1.00 were defined as slight agreement, fair agreement, moderate agreement, substantial agreement, and almost perfect agreement, respectively.
At the same time, from the results of this study, we devised a provisional strategy for differentiated EGC diagnosis under ME-NBI by taking the pink pattern as a supplement to the VS classification system (Fig. 8). Briefly, gastric endoscopic examination is taken by using conventional white-light imaging (C-WIL) firstly. When a suspicious lesion is detected, the endoscope should be switched to ME-NBI to differentiate cancer and non-cancer. The key step to diagnose gastric cancer and non-cancer is to determine the DL between the suspicious lesion and the background mucosa. If a DL is absent, it’s easy to diagnose the suspicious lesion as noncancerous. If a DL is indeterminate, the pink pattern can be taken into account and diagnosis of a cancerous lesion can probably be made. If a DL is clearly visible, the subsequent presence of an IMVP and IMSP should be determined. If IMVP and/or IMSP are identified within the DL, the suspicious lesion can be diagnosed as gastric cancer; if IMVP and/or IMSP are absent, the lesion can be determined as noncancerous. But if IMVP and/or IMSP are indeterminate within the DL, the pink pattern is also helpful to diagnose EGC. By the way, our study has some limitations. First, this study was retrospective and did not involve real-time assessment, the endoscopic diagnosis was just performed by using stored images. The selection bias might occur when the stored images were selected for the two diagnostic tests. Second, we recruited the 12 endoscopists only from one institution to perform diagnostic tests, which might easily lead to the participant bias. Third, the number of lesions and endoscopists in our study was small, and this could result in insufficient power in evaluating diagnostic performance. To overcome these limitations, in future study, we will conduct on-site real-time evaluation of newly detected and undiagnosed gastric lesions. Furthermore, larger number of cases and endoscopists from multiple centers are required in future study to evaluate the diagnostic performance of pink pattern for EGC.