Role of Confocal Laser Endomicroscopy in Early Detection of Upper Gastrointestinal Malignancy in High Risk Patients

Background: Upper gastrointestinal malignancies are a major global health burden. Early diagnosis of upper gastrointestinal premalignant and malignant lesions is crucial for improving prognosis and reducing morbidity and mortality. The purpose of this study was to investigate the diagnostic accuracy of confocal laser endomicroscopy (CLE) in detecting upper gastrointestinal premalignant and early malignant lesions in high-risk patients, as well as diagnosing patients with inconclusive white light endoscopy (WLE) and histopathology results. Methods: It was a cross-sectional study that included ninety (n = 90) high-risk patients with inconclusive diagnoses of upper gastrointestinal lesions on WLE and WLE-based biopsy histopathology. These patients underwent CLE, and the definitive diagnosis was confirmed using CLE and CLE-target biopsy histopathology. Diagnostic accuracy was determined by comparing the sensitivity, specificity, predictive values, and accuracy between the procedures. Result: The mean patient age was 47.43 ± 11.18 years. CLE and target biopsy confirmed that 30 (33.3%) patients had normal histology, while 60 (66.7%) patients were diagnosed with gastritis, gastric intestinal metaplasia, high-grade dysplasia, adenocarcinoma, Barrett’s esophagus, and squamous cell carcinoma of the esophagus. The results of CLE were superior to those of WLE in terms of diagnostic parameters. Additionally, CLE demonstrated nearly similar results in sensitivity (98.33%), specificity (100%), positive predictive value (100%), negative predictive value (96.77%), and accuracy (98.89%) when compared to CLE-target biopsy. Conclusion: CLE showed higher diagnostic accuracy in differentiating normal, premalignant and malignant lesions. It effectively diagnosed patients who initially had inconclusive WLE and/or biopsy results. Furthermore, early detection of upper gastrointestinal premalignant or malignant lesions may improve prognosis and reduce morbidity and mortality.

Introduction premalignant lesions and the progression of gastric carcinoma is therefore crucial for the prompt detection and treatment (Ge et al., 2018). The intestinal metaplasia is typically diagnosed through a pathological examination of a biopsy specimen using white light endoscopy (WLE). However, it is time-consuming, fails to identify mucosal changes, and increases the likelihood of random biopsies (Sobrino-Cossío et al., 2018).
Barrett's esophagus, which develops as a result of long-standing gastroesophageal reflux disease, requires vigilant surveillance due to a significant 30-50 fold increase in the risk of esophageal adenocarcinoma. WLE with random biopsies may overlook certain lesions in Barrett's esophagus, as intraepithelial neoplasia or early adenocarcinoma can present in patchy segments (Neumann et al., 2012). Recently, a latest endoscopic procedure, Confocal Laser Endoscopy (CLE), has emerged for diagnosing various gastrointestinal disorders (Tomizawa et al., 2022). Early detection of Barrett's esophagus, gastric cancer, and other upper gastrointestinal malignancies using CLE significantly improves prognosis, reduces healthcare costs, and has a substantial impact on mortality reduction (Canakis et al., 2022).
This study aims to determine the diagnostic accuracy of CLE in detecting premalignant and early malignant lesions in the upper gastrointestinal tract among high-risk patients. Additionally, it aims to assess the utility of CLE in diagnosing patients with inconclusive results from white light endoscopy (WLE) and pathology results.

Study design and patients selection
This prospective cross-sectional study was conducted at the PNS Diagnostic and Research Institute of Gastroenterology and Hepatology, Liaquat University of Medical and Health Sciences (LUMHS), Jamshoro, Pakistan, from January to December 2021. The minimum sample size of n=87 was calculated using the online sample size calculator OpenEpi (https://www.openepi. com/SampleSize/SSPropor.htm) considering the reported prevalence of 6% for upper gastrointestinal malignancies missed during endoscopy (Januszewicz et al., 2022), a 95% confidence level, and a design effect of 1. Ninety (n = 90) patients undergoing CLE were recruited through non-probability consecutive sampling. These patients had previously undergone white light endoscopy (WLE) with clinical suspicion of premalignant or early malignant lesions, but had negative histopathological results from WLE-based biopsy. Written informed consent was obtained from all participants, and a comprehensive medical history, clinical examination, and investigations were conducted. The study adhered to the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines (Bossuyt et al., 2015), and a study flow diagram is presented in Figure 1.
• High-risk patients with alarming symptoms and/or family history with symptoms suggestive of upper gastrointestinal malignancies, but a negative or failed biopsy result of WLE and a strong suspicion of disease.

Exclusion criteria
• Allergic to the fluorescein dye used in the procedure.
• Patients with acute gastrointestinal bleeding, gastrectomy, advanced gastrointestinal malignancies, diagnosed cases of premalignant and malignant lesions of the upper gastrointestinal tract.
• Patients with bleeding disorders, serious cardiopulmonary disorders, and renal insufficiency.
• Pregnant and lactating females.
• Patients not willing to participate.

Procedure
Initially, all patients were given Inj. fluorescein 0.5 cc I/V as a test dose to check for an allergic reaction to the injected dye. In the absence of any allergic reaction, a complete dose of contrast agent, fluorescein 2.5 cc to 5 cc I/V, was administered. After sedation (Inj. Midazolam 2 cc I/V), a probe CLE (Cellvizio; Mauna Kea Technologies, Paris, France) was performed by a highly skilled gastroenterologist, and endoscopic diagnosis was established through real-time images. Biopsies were taken from the targeted lesions and sent for histopathology. The histopathologists, with over 5 years of experience, were blinded to the clinical information and results of the index tests. Digital images of different sites were recorded and reviewed by an endoscopic expert who was blinded to the patient's medical history and diagnostic information. Patients were kept under observation for an hour after the procedure to check vitals and ensure stability. The lesions on CLE were diagnosed according to the Miami classification (Wallace et al., 2011).

Stastical analysis
The data was analyzed using SPSS v.23. Baseline characteristics of patients are presented as frequency/ percentage or mean ± SD for categorical and continuous variables, respectively. To determine the diagnostic accuracy, the procedure results were compared and analyzed for sensitivity, specificity, predictive values, and accuracy.
Sensitivity and specificity were used to analyze the accuracy of the index test in comparison with the reference standard test. Sensitivity was defined as the test's ability to correctly identify patients with upper gastrointestinal lesions, while specificity was defined as the test's ability to correctly identify patients without lesions. Based on the results of the index test compared to the reference standard test, patients were categorized as true positive (correctly identified with upper gastrointestinal lesions), true negative (correctly identified without upper gastrointestinal lesions), false positive (incorrectly identified with upper gastrointestinal lesions), or false negative (incorrectly identified without upper gastrointestinal lesions).
Positive predictive value (PPV) was used to indicate the accuracy of a positive test result, calculated by dividing true positives by the sum of true positives and false positives. Negative predictive value (NPV) measures the accuracy of a negative test result, obtained nine (98.8%) patients had a history of proton pump inhibitor (PPI) medication, whereas 87 (96.6%) patients by dividing true negatives by the sum of true negatives and false negatives. The diagnostic accuracy of the test was defined by its ability to correctly identify or rule out upper gastrointestinal lesions. The calculations were performed using MedCalc statistical software.
First, diagnostic parameters were calculated to compare the diagnostic accuracy of the index test WLE with the reference standard test CLE. Second, the results of WLE combined with WLE-based biopsy were taken as the index test, and diagnostic accuracy parameters were calculated by comparing them with the reference standard test of CLE combined with CLE-target biopsy. Third, the results of CLE as an index test were compared with CLE-target biopsy as the reference standard test.

Patient Characteristics
Baseline characteristics are presented in Table 1. The mean patient age was 47.43 ± 11.18 years. In total, there were 60 males (66.7%) and 30 females (33.3%). Eighty

Accuracy of WLE, CLE, and Histopathology
We compared the accuracy between (i) WLE and CLE, (ii) WLE combined with WLE-based biopsy and CLE combined with CLE-target biopsy, and (iii) CLE and CLE-target biopsy. As shown in Table 3, with CLE as the standard test, WLE sensitivity was 100%; however, the test was not specific and had the lowest accuracy. When comparing WLE and CLE along with specific biopsy histopathology reports, the sensitivity, specificity, and accuracy increased. With WLE combined with WLE-based biopsy, there were 27 (30%) misdiagnosed Barrett's oesophagus with high grade dysplasia 06 (6.7) Squamous cell carcinoma of oesophagus 06 (6.7)

Discussion
Advanced endoscopic imaging technologies enhance the ability to detect dysplastic lesions with high accuracy; however, they do not replace the conventional histological diagnosis (Maione et al., 2022). Even though the time and cost required to use the new CLE technology must be considered, its impact on the differential diagnosis of benign and malignant conditions is substantial (Kim et al., 2022). In our study, we found that the CLE is an appropriate diagnostic approach since it is far superior to conventional WLE in terms of diagnostic accuracy. In addition, random biopsies through WLE may not accurately identify all dysplastic regions, as it samples only 4-5% of the mucosa (Maione et al., 2022). CLE was designed to acquire higher resolution and magnification imaging, referred to as "optical biopsies," of the gastrointestinal mucosa's histopathology. The significance of CLE lies in its ability to precisely and accurately identify normal tissue architecture. Moreover, CLE requires fewer samples as it utilizes microscopically targeted "smart" biopsies, resulting in a higher yield. CLE can be employed to visualize luminal structures such as the esophagus, stomach, and colon, as well as ductal structures like the pancreatic and bile ducts. This optimization of endoscopic diagnosis through CLE leads to a reduction in unnecessary resections, avoids repeated biopsies, and ultimately mitigates the risks and financial burden associated with repetitive indiscriminate endoscopic exams (Lerner et al., 2022). The most significant use of CLE in the esophagus is for surveillance and interpretation of suspicious lesions in Barrett's esophagus. In a meta-analysis of 14 studies involving 843 individuals, CLE was found to improve the diagnostic yield for dysplasia/cancer by 34% in Barrett's esophagus (Qumseya et al., 2013). In contrast, conventional WLE has been shown to frequently overlook early malignancies in Barrett's esophagus (Tang et al., 2021). Consistent with our finding, Kim Y et al. (2022) found that probe-based CLE exhibited significantly higher sensitivity, accuracy, and negative predictive value compared to WLE/M-NBI. Moreover, CLE demonstrated superior diagnostic and predictive capabilities for residual gastric carcinoma when compared to WLE/M-NBI.
In another study, conventional biopsies misclassified 12 out of 54 lesions. However, CLE accurately diagnosed 11 of these 12 lesions. The accuracy of conventional biopsy for diagnosing adenocarcinoma was 85.2%, and 91.7% for cancer differentiation. However, 8 out of the 32 adenocarcinomas were misclassified. On the other hand, CLE achieved an accuracy of 90.7% for diagnosing adenocarcinoma and 75.9% for cancer differentiation. Three of the 32 adenocarcinomas were misclassified by CLE. Notably, there was no difference in adenocarcinoma diagnosis between real-time and offline images (Bok et al., 2013). In our study, CLE alone proved to be sufficient for detecting lesions and distinguishing between normal tissue and premalignant/malignant lesions. It yielded diagnostic test results that were comparable to CLEtargeted biopsy histopathology. On the other hand, isolated WLE demonstrated higher sensitivity, but when combined with WLE-based biopsy reports, it exhibited improved specificity, predictive values, and accuracy. We were unable to report the specificity of isolated WLE due to our stringent patient selection criteria, which only included high-risk patients with initial negative biopsies. Besides the small sample size, another limitation of this study was its single study center design.
In conclusion, CLE showed higher diagnostic accuracy in differentiating normal, premalignant and malignant lesions. It effectively diagnosed patients who initially had inconclusive WLE and/or biopsy results. Furthermore, early detection of upper gastrointestinal premalignant or malignant lesions may improve prognosis and reduce morbidity and mortality.

Funding
This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ethical approval
The study was approved by the Ethical Review Committee of LUMHS, Jamshoro (Approval No. LUMHS/REC/-880, Dated: 12-03-2020). The study was performed in accordance with the ethical standards as laid down in the Declaration of Helsinki.

Consent to participate
Written informed consent was obtained from all individual participants included in the study.

Consent for publication
Written informed consent was obtained from all individual participants in the study for publication of their data.

Availability of data and material
The data of the manuscript will be provided on the request, by the corresponding author.