In Vivo Analysis of Gastro-Intestinal and Urothelial Lesions

Number: 0783

Table Of Contents

Policy

Scope of Policy

This Clinical Policy Bulletin addresses in vivo analysis of gastro-intestinal and urothelial lesions.

1. Experimental, Investigational, or Unproven

   Aetna considers the following interventions experimental, investigational, or unproven because the effectiveness of these approaches has not been established:

   1. In-vivo analysis of colorectal polyps (e.g., chromoendoscopy, confocal laser (fluorescent) endomicroscopy (including Cellvizio probe-based confocal laser endomicroscopy), endocytoscopy, EVIS EXERA 160A System, fiberoptic analysis, multi-band imaging, narrow-band imaging optical chromocolonoscopy, Optical Biopsy System, Pentax Confocal Laser System, and WavSTAT™ Optical Biopsy System);
   2. Elastic-scattering spectroscopy for evaluation of colonic polyps;
   3. Endoscopic retrograde cholangio-pancreatography (ERCP) with optical endomicroscopy for evaluation of biliary lesions (including strictures)
   4. Confocal laser endomicroscopy (including Cellvizio probe-based confocal laser endomicroscopy) for the following indications (not an all-inclusive list):
      1. Assessment of safe surgical margins during laryngectomy
      2. Bronchoscopic evaluation of broncho-alveolar lavage components
      3. Confirmation of low-grade dysplasia and surveillance of Barrett’s esophagus
      4. Detection of colorectal mucosa microinflammation in individuals with irritable bowel syndrome
      5. Detection of neoplasia in biliary strictures associated with primary sclerosing cholangitis
      6. Diagnosis and histologic grading of bladder cancer
      7. Diagnosis and monitoring of urogenital schistosomiasis
      8. Diagnosis and staging of lung cancer
      9. Diagnosis of acute cellular rejection in lung transplant recipients
      10. Diagnosis of clinical complete response after chemoradiation for rectal cancer
      11. Diagnosis of early-stage gastric cancer or early-stage ovarian cancer
      12. Diagnosis of functional dyspepsia (leaky gut disorder)
      13. Diagnosis of head and neck squamous cell carcinoma (SCC) including laryngeal and pharyngeal SCC
      14. Diagnosis of indeterminate biliary strictures and pancreatic lesions
      15. Diagnosis of liver cancer
      16. Diagnosis of prostate cancer
      17. Diagnosis of vocal cord lesions
      18. Diagnosis, prediction of disease course, and therapeutic responses of inflammatory bowel disease (Crohn's disease and ulcerative colitis)
      19. Differentiation of benign and malignant gallbladder polyps
      20. Differentiation of colorectal polyps during routine colonoscopy
      21. Differentiation of parenchymal lung diseases from normal alveoli
      22. Evaluation of depth of invasion in colorectal lesions
      23. Evaluation of epithelial barrier function and presence of gastro-esophageal reflux disease
      24. Evaluation of esophageal neoplasia
      25. Evaluation of pancreatic cysts
      26. Evaluation of the tumor vasculature in gastric and rectal carcinomas
      27. Intra-operative diagnosis and resection of glioblastoma or other central nervous system tumors
      28. Intra-operative use during surgery for Hirschsprung's disease and allied disorders
      29. Management of upper tract urothelial carcinoma.

Background

In the United States, colorectal cancer is the third most common cancer diagnosed among men and women and the second leading cause of death from cancer.  Early stage colorectal cancers and pre-cancerous adeomatous lesions can be visualized and treated endoscopically.  However, complications of colonoscopic polypectomy include hemorrhage (reported incidence of 0.2 % to 3 %) and perforation (reported incidence of 0.5 % to 3 %).  In addition, biopsy and polypectomy are frequently performed for lesions that carry a low risk of malignancy in the colon.  Thus, the ability to determine colorectal polyp pathology by endoscopy could potentially reduce the risks of polypectomy and improve the diagnosis of early colonic neoplasia. 

A number of imaging technologies are under development that have the potential to enhance detection of colorectal neoplasia.  Recent technology has led to the development of colonoscopes that are specifically designed to further improve visualization, including wide angle of view and high resolution.  Traditionally, white light colonoscopy (WLC) is used whereby the light source provides red, green and blue wavelength light sequentially by a rotation filter.  However, flat lesions are often difficult to identify with WL and diagnosis is usually dependent on random biopsies.  Newer colonoscopes equipped with non-white light imaging capabilities have been designed to enhance image contrast and potentially improve the visualization of lesions.  Some of these methods include fiberoptic analysis, narrow band imaging, and confocal fluorescent endomicroscopy. 

The Optical Biopsy System (SpectraScience, Inc., Minneapolis, MN) includes a laser light, a long optical fiber that the light passes through and a computer that analyzes the light given off by suspicious lesions.  It received premarket approval (PMA) through the U.S. Food and Drug Administration (FDA) as an adjunct to lower gastrointestinal endoscopy for the evaluation of polyps less than 1 cm in diameter that the physician has not already elected to remove.  The device is only to be used in deciding whether such polyps should be removed (which includes submission for histological examination).  The FDA approval was based on an un-published prospective, non-randomized phase II study.  According to the information submitted by the manufacturer to the FDA, patients (n = 101) who had at least one polyp identified from a prior endoscopic examination, underwent another standard colonoscopy using the Optical Biopsy System.  When a polyp was identified, the physician documented whether they thought the polyp was adenomatous or hyperplastic based on the visual assessment and whether they would remove the polyp.  The optical fiber was placed on a region of the polyp and the tissue's auto-fluoresence was evaluated from three different locations on the polyp.  The effectiveness of the Optical Biopsy System was characterized by its ability to correctly identify adenomatous polyps as adenomatous and to correctly identify hyperplastic polyps as hyperplastic either alone or in conjunction with the physician assessment.  The sensitivity and specificity of the physician assessment alone were 82.7 % and 50 %, respectively, whereas, the combined sensitivity and specificity were 96.3 % and 33 %, respectively.  The study demonstrated that the Optical Biopsy System, when used as an adjunctive tool, will increase the number of adenomatous polyps that are biopsied (i.e., increase the sensitivity of physician assessment), but will also increase the number of hyperplastic polyps that are biopsied (i.e., decrease the specificity of physician assessment).  Optimal methodologies for the endoscopic detection of dysplastic colonic lesions have yet to be outlined; thus, it is not clear what additional benefits this device would have over simply increasing the number of polyps chosen for biopsy through visual inspection.  In addition, it is not known whether the performance of the Optical Biopsy System would be different by physicians who are less experienced and skilled than those who participated in the phase II clinical study. 

Pilot studies have demonstrated the safety and feasibility of polyp detection using narrow-band imaging (NBI) (Su et al, 2006; Rastogi et al, 2007; Tischendorf et al, 2007; East et al, 2008).  Evaluating NBI studies, however, has been difficult because of the lack of standardization between NBI systems (Emura et al, 2008). 

The Evis Exera Xenon Light Source CLV-160A (Olympus Medical Systems, Melville, NY) is a video processing system that received pre-marketing clearance from the FDA through the 510(k) process.  It is intended for endoscopic diagnosis, treatment and video observation and has an optional filter which allows the user to select either standard WL illumination or NBI.

Rogart et al (2008) compared WL with NBI for the differentiation of colorectal polyps in vivo during colonoscopy.  Standard WL colonoscopy was performed with the Olympus 180-series colonoscopes.  Each detected polyp was first characterized by WL and then by NBI.  Modified Kudo pit pattern and vascular color intensity (VCI) were recorded, and the histology was predicted.  Endoscopists were given feedback every 2 weeks.  Main outcome measurements were overall accuracy and sensitivity and specificity of endoscopic diagnosis by using WL alone and with NBI, as well as improvement in endoscopists' performance.  A total of 265 polyps were found in 131 patients.  Diagnostic accuracy was 80 % with NBI and 77 % with WL (p = 0.35).  Narrow-band imaging performed better than WL in diagnosing adenomas (sensitivity 80 % versus 69 %, p < 0.05).  Non-adenomatous polyps were more likely to have a light VCI compared with adenomas (71 % versus 29 %, p < 0.001).  During the second half of the study, NBI accuracy improved, from 74 % to 87 %, and out-performed an unchanged WL accuracy of  79 % (p < 0.05).  Overall, NBI was not more accurate than WL in differentiating colorectal polyps in vivo; however, once a learning curve was achieved, NBI performed significantly better.  The authors concluded that further refinements of an NBI pit-pattern classification and VCI scale are needed before broad application to clinical decisions regarding the necessity of polypectomy can be made.

Rex (2009) evaluated the ability of the Olympus Exera 180 high-definition colonoscope (Olympus America, Inc., Center Valley, PA) with NBI to predict colorectal polyp histology.  A library of 320 endoscopic photographs with correlated histologic information were used to identify endoscopic features associated with adenomatous and hyperplastic histology.  These features were tested in a prospective study of 451 consecutively identified colorectal polyps.  Polyps were observed endoscopically and assigned a designation of high or low confidence.  The primary end points were the predictive value of high-confidence endoscopic interpretations of adenoma and hyperplastic histology for polyps greater than 5 mm in size.  Endoscopic predictions of adenoma and hyperplastic histology were made with high confidence for 80 % and 83 % of cases, respectively.  High-confidence predictions were more likely than low-confidence predictions to be correct (p < 0.001).  High-confidence predictions of adenoma and hyperplastic histology were correct for 91 % and 95 %, respectively, of polyps greater than 5-mm in size.  The author concluded that the introduction of confidence levels to the endoscopic interpretation of colorectal polyp histology allowed sufficient accuracy for the use of the Exera narrow-band imaging system in the identification of distal hyperplastic polyps that do not need resection, as well as to plan post-polypectomy surveillance without pathologic evaluation of polyps 5-mm in size or smaller.

In a prospective randomized study, Adler et al (2008) evaluated NBI versus conventional colonoscopy for adenoma detection.  Eligible patients presenting for diagnostic colonoscopy were randomly assigned to undergo wide-angle colonoscopy using either conventional high-resolution imaging or NBI during instrument withdrawal.  The primary outcome parameter was the difference in the adenoma detection rate between the 2 techniques.  A total of 401 patients were included (mean age of 59.4 years, 52.6 % men).  Adenomas were detected more frequently in the NBI group (23 %) than in the control group (17 %), however, the difference was not statistically significant (p = 0.129).  When the two techniques were compared in consecutive subgroups of 100 study patients, adenoma rates in the NBI group remained fairly stable, whereas these rates steadily increased in the control group (8 %, 15 %, 17 %, and 26.5 %, respectively).  Significant differences in the first 100 cases (26.5 % versus 8 %; p = 0.02) could not be maintained in the last 100 cases (25.5 % versus 26.5 %, p = 0.91).  The authors concluded that the increased adenoma detection rate of NBI colonoscopy were not statistically significant and whether the increasing adenoma rate in the conventional group was caused by a training effect of better polyp recognition on NBI remains speculative.

In a randomized controlled trial (RCT), Kaltenback et al (2008) compared NBI versus WLC.  Patients were randomly assigned to undergo a colonoscopic examination using NBI or WLC.  All patients underwent a second examination using WLC as the reference standard.  The primary end point was the difference in the neoplasm miss rate, and secondary outcome was the neoplasm detection rate.  Patients who underwent tandem colonoscopy (n = 276) experienced no significant difference of miss or detection rates between NBI or WLC.  Of the 135 patients in the NBI group, 17 patients (12.6 %; 95 % confidence interval [CI]: 7.5 to 19.4 %) had a missed neoplasm, as compared with 17 of the 141 patients (12.1 %; 95 % CI: 7.2 to 18.6 %) in the WLC group with a miss rate risk difference of 0.5 % (95 % CI: -7.2 to 8.3); 130 patients (47 %) had at least 1 neoplasm.  Missed lesions with NBI showed similar characteristics to those missed with WLC.  All missed neoplasms were tubular adenomas, the majority (78 %) was less than or equal to 5.0 mm and none were larger than 1 cm (1-sided 95 % CI: up to 1%).  Non-polypoid lesions represented 35 % (13/37) of missed neoplasms.  The authors concluded that NBI did not improve the colorectal neoplasm miss rate compared to WLC and that the neoplasm detection rates were similar using NBI or WLC.

It is not known whether NBI improves patient outcomes.  Studies that compared NBI to WLC reported similar neoplasm detection rates and there is a lack of standardization between NBI systems making it difficult to interpret the studies. 

Confocal endomicroscopy is a new endoscopic technique that provides microscopic images of cellular morphology in the gastro-intestinal tract during ongoing endoscopy.  The peer-reviewed literature for confocal endomicroscopy consists of small non-randomized safety and feasibility studies (Odagi et al, 2007; Hsiung et al, 2008; Watanable et al, 2008).  This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer, however, there is insufficient evidence of its effectiveness.

According to a review by Anandasabapathy (2008) on emerging optical techniques for the detection of colorectal neoplasia, auto-fluorescent imaging and NBI are "red flag" techniques which enhance visualization of mucosal change(s) and complementary technologies, such as confocal endomicroscopy and endocytoscopy provide subcellular imaging.  However, it is unclear how these techniques impact clinical outcomes.  Tissue biopsy is considered the gold standard for histopathological diagnosis; furthermore, optimal methodologies for the endoscopic detection of dysplastic colonic lesions have yet to be outlined.

A systematic review by van den Broek et al (2009) found that narrow band imaging had high sensitivity and specificity for the differentiation of neoplastic from non-neoplastic colon polyps when used by experienced endoscopists, and that its accuracy was comparable to chromoendoscopy.  A critique of this review by the Centre for Reviews and Dissemination (CRD, 2009) found, however, that this systematic review suffered from a number of limitations, which means that these findings should be interpreted with some caution.  The systematic evidence review identified 6 studies (n = 1,222 patients) meeting inclusion criteria that assessed the detection of neoplasia, including 4 RCTs and 2 tandem design studies (that compared the 2 techniques back to back).  The CRD noted that one randomized study was not included in the meta-analysis.  The CRD observed that eleven studies (n = 866 patients) on the differentiation of lesions were included in a table in the systematic evidence review but only 9 (n = 770) were included in the analysis and quality assessment table.  One study assessed both and so contributed data to each analysis.  The CRD noted that results of the quality assessment for the detection studies were not reported.  The systematic evidence review found that studies on the differentiation of lesions all fulfilled items on use of an appropriate reference standard and avoidance of disease progression, partial verification, differential verification and incorporation bias.  Items relating to test details, reference standard details, test bias and review bias were poorly reported.  Only 4 studies included an appropriate patient spectrum and only 3 reported sufficient details of selection criteria.  The systematic evidence review found that the proportion of patients with at least 1 adenoma detected by narrow-band imaging was similar to the proportion detected by white-light endoscopy (pooled odds ratio 1.19, 95 % CI: 0.86 to 1.64; 3 RCTs) as was the mean number of adenomas detected (relative ratio of means 1.23, 95 % CI: 0.93 to 1.61; 3 RCTs).  In the 2 observational studies, the adenoma miss rates of white-light endoscopy were 40 % (29/72) and 46 % (21/46) for each study.  Regarding the use of narrow band imaging in the differentiation of lesions, the review excluded 3 studies on the diffentiation of lesions from the metaanalysis as they included highly selected patient groups and were therefore thought to be biased.  Based on the remaining 6 studies, the pooled sensitivity of narrow-band imaging for the differentiation of neoplastic compared to non-neoplastic colon polyps was 92 % (95 % CI: 89 to 94) and pooled specificity was 86 % (95 % CI: 80 to 91).  Five studies also reported on the accuracy of chromoendoscopy.  Pooled sensitivity was reported to be 91% (95 % CI: 83 to 96) and pooled specificity was 89 % (95 % CI: 83 to 93).  Four studies provided data on inter-observer agreement.  Kappa values ranged from 0.48 to 1.0, suggesting moderate to excellent agreement.  The authors of the systematic review concluded that narrow-band imaging showed high sensitivity and specificity for the differentiation of neoplastic from non-neoplastic colon polyps when used by experienced endoscopists, and that its accuracy was comparable to that of chromoendoscopy.  In a critique of this systematic evidence review, the CRD (2009) found only limited study details reported, and that further details, especially in relation to the patients included in the studies, would have helped to assess the generalizability of findings.  The CRD noted that not all studies reported to have fulfilled inclusion criteria and summarized in tables contributed to the analysis, and that the reasons for this are unclear.  The CRD also noted that heterogeneity was not formally assessed or investigated in this metaanalysis.  The CRD stated that the conclusions of this meta analysis should be interpreted with some caution, due to the unclear generalizability of findings and the fact that some studies were excluded from the analysis without justification.

Benes and Antos (2009) examined the correlation between the results of an optical biopsy system and the histopathology report of the physical biopsy specimens of the same polyps removed at colonoscopy.  Paired optical and physical biopsies were performed on 55 polyps with complete polypectomy of the same tissue.  A total of 53 adenomatous polyps and 2 hyperplastic polyps were identified by the hospital pathologist.  The optical biopsy system identified 52 polyps as suspect (adenomatous) and 2 as non-suspect (hyperplastic).  One villous adenoma could not be optically analyzed due to friability.  The authors concluded that the WavSTAT Optical Biopsy System provides accurate information to the gastroenterologist to assist in distinguishing between hyperplastic and adenomatous polyps.  However, the impact of this technology on health outcomes is unclear.

The American Society for Gastrointestinal Endoscopy (ASGE, 2008) published a technology status evaluation report regarding NBI.  It stated that NBI may enhance the diagnosis and characterization of mucosal lesions in the gastrointestinal tract, especially as an adjunct to magnification endoscopy; however, standardization of image characterization, further image pathology correlation and validation, as well as the impact of these technologies on patient outcomes are needed before endorsing the use of NBI in the routine practice of gastrointestinal endoscopic procedures. 

Kahi et al (2010) noted that high-definition chromoscopy is used to increase the yield of colonoscopy for flat and depressed neoplasms; however, its role in average-risk patients undergoing routine screening remains uncertain.  This study compared high-definition chromo-colonoscopy with high-definition white light colonoscopy for average-risk colorectal cancer screening.  Average-risk patients referred for screening colonoscopy at 4 United States medical centers were randomized to high-definition chromocolonoscopy or high-definition white light colonoscopy.  The primary outcomes, patients with at least 1 adenoma and the number of adenomas per patient, were compared between the 2 groups.  The secondary outcome was patients with flat or depressed neoplasms, as defined by the Paris classification.  A total of 660 patients were randomized (chromocolonoscopy: 321, white light: 339).  Overall, the mean number of adenomas per patient was 1.2 +/- 2.1, the mean number of flat polyps per patient was 1.4 +/- 1.9, and the mean number of flat adenomas per patient was 0.5 +/- 1.0.  The number of patients with at least 1 adenoma (55.5 % versus 48.4 %, absolute difference 7.1 %, 95 % CI: -0.5 % to 14.7 %), p = 0.07), and the number of adenomas per patient (1.3 +/- 2.4 versus 1.1 +/- 1.8, p = 0.07) were marginally higher in the chromocolonoscopy group.  There were no significant differences in the number of advanced adenomas per patient (0.06 +/- 0.37 versus 0.04 +/ -0.25, p = 0.3) and the number of advanced adenomas less than 10 mm per patient (0.02 +/- 0.26 versus 0.01 +/- 0.14, p = 0.4).  Two invasive cancers were found, 1 in each group; neither was a flat neoplasm.  Chromocolonoscopy detected significantly more flat adenomas per patient (0.6 +/- 1.2 versus 0.4 +/- 0.9, p = 0.01), adenomas less than 5 mm in diameter per patient (0.8 +/- 1.3 versus 0.7 +/- 1.1, p = 0.03), and non-neoplastic lesions per patient (1.8 +/- 2.3 versus 1.0 +/- 1.3, p < 0.0001).  The authors concluded that high-definition chromocolonoscopy marginally increased overall adenoma detection, and yielded a modest increase in flat adenoma and small adenoma detection, compared with high-definition white light colonoscopy.  The yield for advanced neoplasms was similar for the 2 methods.  The authors concluded that these findings do not support the routine use of high-definition chromocolonoscopy for colorectal cancer screening in average-risk patients.  The high adenoma detection rates observed in this study may be due to the high-definition technology used in both groups.

Neumann and colleagues (2011) noted that endocytoscopy (EC) enables in-vivo microscopic imaging at 1,400-fold magnification, thereby allowing the analysis of mucosal structures at the cellular level.  In contrast to fluorescence imaging with confocal laser endomicroscopy (CLE), which allows analysis of mucosal structures up to 250 μm in depth, EC is based on the principle of contact light microscopy and only allows visualization of the very superficial mucosal layer.  These researchers systemically reviewed the feasibility and diagnostic yield of EC for in-vivo diagnosis of diseases.  A systematic search of the literature on diagnostic interventions in the gastro-intestinal tract using EC was performed by searches in MEDLINE, Current Contents, PubMed, cross-references and references from relevant articles using the search terms "endocytoscopy", "endocytoscope", "magnification endoscopy", "endocytoscopic imaging", "virtual histology" and "optical biopsy".  Only full manuscripts and case reports published in English were included.  A total of 29 relevant reports were identified.  Endocytoscopy was feasible to detect esophageal squamous cell cancer with sensitivity, specificity and accuracy of 95 %, 84 % and 82 %, respectively. Moreover, EC reached excellent sensitivity and specificity for in- vivo diagnosis of colon polyps (91 % and 100 %, respectively).  Other diagnostic applications of EC included diagnosis of Barrett's esophagus, Helicobacter pylori, celiac disease and small cell lung cancer.  No serious complications of EC have yet been reported.  The authors concluded that endocytoscopy is a safe and effective new endoscopic imaging technique to obtain in-vivo histology and guided biopsies with high diagnostic accuracy.  Therefore, endocytoscopy has the potential to facilitate both diagnosis and patient management.

Yeung and Mortensen (2011) stated that conventional white-light endoscopy is currently the gold standard for the detection and treatment of colorectal polyps.  However, up to 20 % of polyps may be missed on initial examination, especially flat and small mucosal lesions.  These investigators reviewed the literature reporting on the use of new advances in endoscopic visualization.  Literature searches were performed on PubMed using the terms "chromoendoscopy", "narrow-band imaging" (NBI), "auto-fluorescence imaging" (AFI), "Fujinon Intelligent Color Enhancement" (FICE), "i-Scan colonoscopy", "zoom colonoscopy" and "confocal laser endomicroscopy".  They focused on systematic reviews, national guidelines and RCTs written in English.  Studies were assessed for methodological quality using QUADAS.  Prospective studies assessing new technology were also reviewed.  Further publications were identified from reference lists.  Chromoendoscopy increases the detection of neoplastic polyps compared with conventional colonoscopy.  Narrow-band imaging avoids the use of additional dyes and enhances the vascular network of capillaries surrounding the crypts, increasing the adenoma detection rate and the ability to distinguish between neoplastic and non-neoplastic lesions.  Fujinon Intelligent Color Enhancement, AFI and i-Scan are new developments that improve tissue contrast.  Zoom endoscopy may be combined with different modalities to help further characterize colonic lesions.  Confocal laser endomicroscopy provides live in-vivo high-resolution optical sections of tissue and may be particularly useful in the surveillance of patients with long-standing ulcerative colitis, reducing the number of random biopsies.  The authors concluded that although there is mounting evidence that these new technologies are superior to conventional endoscopy, current guidelines are limited.  Moreover, they stated that further large-scale RCTs comparing these modalities in different patient subpopulations are warranted.

In a RCT and meta-analysis of published studies, Sabbagh et al (2011) examined if NBI improve detection of colorectal polyps.  Eligible adult patients presenting for screening or diagnostic elective colonoscopy were randomly allocated to undergo conventional colonoscopy or NBI during instrument withdrawal by 3 experienced endoscopists.  For the systematic review, studies were identified from the Cochrane Library, PUBMED and LILACS and assessed using the Cochrane risk of bias tool.  These investigators enrolled a total of 482 patients (62.5 % females), with a mean age of 58.33 years (standard deviation [SD] of 12.91); 241 into the intervention (NBI) colonoscopy and 241 into the conventional colonoscopy group.  Most patients presented for diagnostic colonoscopy (75.3 %).  The overall rate of polyp detection was significantly higher in the conventional group compared to the NBI group (RR 0.75, 95 % CI: 0.60 to 0.96).  However, no significant differences were found in the mean number of polyps (MD -0.1; 95% CI: -0.25 to 0.05), and the mean number of adenomas (MD 0.04 95 % CI: -0.09 to 0.17).  Meta-analysis of studies (regardless of indication) did not find any significant differences in the mean number of polyps (5 RCT, 2,479 participants; WMD -0.07 95 % CI: -0.21 to 0.07; I2 68 %), the mean number of adenomas (8 RCT, 3,517 participants; WMD -0.08 95 % CI: -0.17 to 0.01; I2 62 %) and the rate of patients with at least 1 adenoma (8 RCT, 3,512 participants, RR 0.96 95 % CI: 0.88 to 1,04; I2 0 %).  The authors concluded that NBI does not improve detection of colorectal polyps when compared to conventional colonoscopy.

In a Cochrane review, Nagorni et al (2012) compared standard- or high-definition white light colonoscopy (WLC) with NBI colonoscopy for detection of colorectal polyps.  These investigators searched The Cochrane Library, MEDLINE, and EMBASE to August 2011.  They scanned bibliographies of relevant publications and wrote to experts for additional trials.  Two authors independently applied the inclusion criteria and extracted the data to all potential studies without blinding.  Authors extracted data independently.  Trials with adequate randomization, allocation concealment, and complete outcome data reporting, as well as without selective outcome reporting or other bias were classified as having a lowest risk of bias.  Random-effects and fixed-effect meta-analyses were conducted.  These researchers identified 11 randomized trials comparing WLC with NBI for detection of colorectal polyps.  A total of 8 randomized trials with 3,673 subjects provided data for the analyses.  There was no statistically significant difference between WLC (standard-definition and high-definition pooled) and NBI for the detection of patients with colorectal polyps (6 trials, n = 2,832, RR 0.97, 95 % CI: 0.91 to 1.04), patients with colorectal adenomas (8 trials, n = 3,673, RR 0.94, 95 % CI: 0.87 to 1.02), or patients with colorectal hyperplastic polyps (2 trials, n = 645, RR 0.87, 95 % CI: 0.76 to 1.00).  Number of patients with at least 1 colorectal adenoma was not significantly different between WLC and NBI group irrespective of adenoma size (less than 5 mm: RR 0.95, 95 % CI: 0.84 to 1.08, I(2) = 56 %; 6 to 9 mm: RR 1.06, 95 % CI: 0.81 to 1.39, I(2) = 0 %; greater than or equal to 10 mm: RR 1.06, 95 % CI: 0.77 to 1.45, I(2) = 0 %).  Number of patients with at least 1 colorectal polyp, or colorectal adenoma was significantly lower in the standard-definition WLC group compared to NBI group in fixed-effect meta-analysis (RR 0.87, 95 % CI: 0.78 to 0.97, I(2) = 78 %; RR 0.87, 95 % CI: 0.77 to 0.99, I(2) = 0 %, respectively), but not significantly different in random-effects meta-analysis (RR 0.86, 95 % CI: 0.68 to 1.10, I(2) = 78 %).  There was no statistically significant difference between high-definition WLC and NBI in the number of patients with at least 1 colorectal polyp or colorectal adenoma (RR 1.10, 95 % CI: 0.95 to 1.28; RR 0.87, 95 % CI: 0.77 to 0.99, I(2) = 0 %, respectively).  The authors concluded that they could not find convincing evidence that NBI is significantly better than high-definition WLC for the detection of patients with colorectal polyps, or colorectal adenomas.  Moreover, they found evidence that NBI might be better than standard-definition WLC and equal to high-definition WLC for detection the patients with colorectal polyps, or colorectal adenomas.

In a meta-analysis, Dinesen and colleagues (2012) examined if use of NBI enhances the detection of adenomas.  Meta-analyses were conducted of 7 studies using NBI for adenoma detection rate.  MEDLINE, Embase, PubMed, and Cochrane databases were searched by using a combination of the following terms: "colonoscopy", "NBI", and "electronic chromoendoscopy".  There was a total of 2,936 patients in the NBI studies.  Prospective, randomized trials of NBI versus standard WLC were conducted.  These researchers excluded spray chromoendoscopy studies as well as studies of inflammatory bowel disease and polyposis syndromes.  Main outcome measures were adenoma and polyp detection rates and the number of polyps and adenomas detected per person.  There was no statistically significant difference in the overall adenoma detection rate with the use of NBI or WLC (36 % versus 34 %; p = 0.413 [relative risk 1.06; 95 % CI: 0.97 to 1.16]), and there was no statistically significant difference in polyp detection rate by using NBI or WLC (37 % versus 35 %; p = 0.289 [relative risk 1.22; 95 % CI: 0.85 to 1.76]).  When the number of adenomas and polyps per patient was analyzed, no significant difference was found between NBI and WLC (0.645 versus 0.59; p = 0.105 and 0.373 versus 0.348; p = 0.139 [weighted mean difference 0.19; 95 % CI: 0.06 to 0.44], respectively).  The authors concluded that NBI did not increase adenoma or polyp detection rates.

Carignan and Yagi (2012) stated that new optical technologies are capable of identifying early pathology in tissues or organs in which cancer is known to develop through stages of dysplasia, including the esophagus, colon, pancreas, liver, bladder, and cervix.  These diagnostic imaging advances, together as a field known as optical endomicroscopy, are based on confocal microscopy, spectroscopy-based imaging, and optical coherence tomography (OCT), and function as "optical biopsies," enabling tissue pathology to be imaged in-situ as well as in real time without the need to excise and process specimens as in conventional biopsy and histopathology.  Optical biopsy techniques can acquire high-resolution, cross-sectional images of tissue structure on the micron scale through the use of endoscopes, catheters, laparoscopes, and needles.  Since the inception of these technologies, dramatic technological advances in accuracy, speed, and functionality have been realized.  The current paradigm of optical biopsy, or single-area, point-based images, is slowly shifting to more comprehensive microscopy of larger tracts of mucosa.  With the development of Fourier-domain OCT, also known as optical frequency domain imaging or, more recently, volumetric laser endomicroscopy, comprehensive surveillance of the entire distal esophagus is now achievable at speeds that were not possible with conventional OCT technologies.  Optical diagnostic technologies are emerging as clinically useful tools with the potential to set a new standard for real-time diagnosis.  New imaging techniques enable visualization of high-resolution, cross-sectional images and offer the opportunity to guide biopsy, allowing maximal diagnostic yields and appropriate staging without the limitations and risks inherent with current random biopsy protocols.  However, the ability of these techniques to achieve widespread adoption in clinical practice depends on future research designed to improve accuracy and allow real-time data transmission and storage, thereby linking pathology to the treating physician.

Neumann et al (2012) evaluated the clinical utility of CLE in patients with Crohn's disease (CD) and examined if disease activity can be graded using CLE.  Consecutive patients with and without CD were enrolled.  The colonic mucosa was examined by standard white-light endoscopy followed by CLE.  The features seen on CLE were compared between CD patients and controls.  A total of 76 patients with CD were screened, of whom 54 patients were included in the present study.  Eighteen patients without inflammatory bowel disease (IBD) served as controls.  A significantly higher proportion of patients with active CD had increased colonic crypt tortuosity, enlarged crypt lumen, micro-erosions, augmented vascularization, and increased cellular infiltrates within the lamina propria.  In quiescent CD, a significant increase in crypt and goblet cell number was detected compared with controls.  Based on these findings, these investigators proposed a Crohn's Disease Endomicroscopic Activity Score (CDEAS) for assessing CD activity in-vivo.  The authors concluded that CLE has the potential to significantly improve diagnosis of CD compared with standard endoscopy.  These findings should be evaluated in future prospective trials to assess the value of this newly developed CLE score for prediction of disease course and therapeutic responses.

In a randomized controlled trial, Kiesslich et al (2007) evaluated the value of combined chromoscopy and endomicroscopy for the diagnosis of intra-epithelial neoplasia.  The authors concluded that endomicroscopy based on in-vivo histology can determine if ulcerative colitis lesions identified by chromoscopy should undergo biopsy examination, thereby increasing the diagnostic yield and reducing the need for biopsy examinations.  Thus, chromoscopy-guided endomicroscopy may lead to significant improvements in the clinical management of ulcerative colitis (UC).

Buchner et al (2010) noted that probe-based CLE (pCLE) allows in-vivo imaging of tissue at micron resolution.  Virtual chromoendoscopy systems, such as Fujinon intelligent color enhancement and narrow band imaging, also have potential to differentiate neoplastic colorectal lesions.  The authors concluded that confocal endomicroscopy demonstrated higher sensitivity with similar specificity in classification of colorectal polyps.  Moreover, they stated that these new methods may replace the need for ex- vivo histological confirmation of small polyps, but further studies are needed.

In a feasibility study, Shahid et al (2012a) evaluated the accuracy of pCLE and NBI for prediction of histology.  The authors concluded that pCLE demonstrated higher sensitivity in predicting histology of small polyps compared with NBI, whereas NBI had higher specificity.  When used in combination, the accuracy of pCLE and NBI was extremely high, approaching the accuracy of histopathology.  Together, they may reduce the need for histological examination.  However, these researchers stated that further studies are needed to evaluate the role of these techniques, especially in the population-based colon cancer screening.

In a prospective, blind, pilot study, Shahid et al (2012b) estimated and compared the accuracy of virtual chromoendoscopy (VCE) and pCLE for detection of residual neoplastic tissue at the site of prior endoscopic mucosal resection (EMR).  The authors concluded that confocal endomicroscopy significantly increased the sensitivity for detecting residual neoplasia after colorectal EMR compared with endoscopy alone.  When confocal endomicroscopy is used in combination with VCE, the accuracy is extremely high, and sensitivity approaches that of histopathology.  Together, they may reduce the need for histologic examination and allow a highly accurate on-table decision to treat again or not, thus avoiding unnecessary repeat procedures.  The main drawbacks of this study were its small sample size, lack of power, involvement of highly experienced pCLE experts.

In a meta-analysis, Wanders et al (2013) established the sensitivity, specificity, and real-time negative predictive value of 3 types of narrowed spectrum endoscopy (NBI, image-enhanced endoscopy [i-scan], and Fujinon intelligent chromoendoscopy [FICE]), CLE, and auto-fluorescence imaging for differentiation between neoplastic and non-neoplastic colonic lesions.  The authors concluded that all endoscopic imaging techniques other than auto-fluorescence imaging could be used by appropriately trained endoscopists to make a reliable optical diagnosis for colonic lesions in daily practice.  Moreover, they stated that further research should be focused on whether training could help to improve negative predictive values.

In a systematic review and meta-analysis, Su et al (2013) assessed the effectiveness of CLE for discriminating colorectal neoplasms from non-neoplasms and its contributing factors.  The authors concluded that CLE is comparable to colonoscopic histopathology in diagnosing colorectal neoplasms, and is better in conjunction with conventional endoscopy.  An endoscopy-based rather than a probe-based modality would be optimal in the application of CLE. The Centre for Reviews and Dissemination (2014) reviewed the meta-analysis by Su et al. and concluded: “Given the high variation between trials and potential limitations in the methods used to synthesize the data, it is difficult to assess the reliability of the findings and the authors' conclusions and recommendations for practice should be interpreted with caution.”

Ladabaum et al (2013) prospectively evaluated real-time optical biopsy analysis of polyps with NBI by community-based gastroenterologists.  These investigators first analyzed a computerized module to train gastroenterologists (n = 13) in optical biopsy skills using photographs of polyps.  Then they evaluated a practice-based learning program for these gastroenterologists (n = 12) that included real-time optical analysis of polyps in-vivo, comparison of optical biopsy predictions to histopathologic analysis, and ongoing feedback on performance.  Twelve of 13 subjects identified adenomas with greater than 90 % accuracy at the end of the computer study, and 3 of 12 subjects did so with accuracy greater than or equal to 90 % in the in-vivo study.  Learning curves showed considerable variation among batches of polyps.  For diminutive recto-sigmoid polyps assessed with high confidence at the end of the study, adenomas were identified with mean (95 % CI) accuracy, sensitivity, specificity, and negative-predictive values (NPVs) of 81 % (73 % to 89 %), 85 % (74 % to 96 %), 78 % (66 % to 92 %), and 91 % (86 % to 97 %), respectively.  The adjusted odds ratio for high confidence as a predictor of accuracy was 1.8 (95 % CI: 1.3 to 2.5).  The agreement between surveillance recommendations informed by high-confidence NBI analysis of diminutive polyps and results from histopathologic analysis of all polyps was 80 % (95 % CI: 77 % to 82 %).  The authors concluded that in an evaluation of real-time optical biopsy analysis of polyps with NBI, only 25 % of gastroenterologists assessed polyps with greater than or equal to 90 % accuracy.  The NPV for identification of adenomas, but not the surveillance interval agreement, met the ASGE-recommended thresholds for optical biopsy.  Moreover, they stated that better results in community practice must be achieved before NBI-based optical biopsy methods can be used routinely to evaluate polyps

In a prospective, multi-center study, Repici et al (2103) examined if NBI is able to predict colonoscopy surveillance intervals and histology of distal diminutive polyps according to ASGE criteria.  Consecutive patients undergoing colonoscopy in 5 centers were included.  Participating endoscopists were required to pass a before-study qualifying examination.  Histology of polyps that were less than 10 mm was predicted at NBI and assigned a designation of high or low confidence.  Accuracy of high-confidence NBI prediction for polyps less than or equal to 5 mm in predicting surveillance intervals and NPV for adenomatous histology in the recto-sigmoid colon were compared with the ASGE thresholds (90 % agreement, 90 % NPV).  A total of 278 patients (mean age of 63 years; 58 % male) were enrolled.  At colonoscopy, 574 (97.3 %) polyps less than 10 mm (429 less than or equal to 5 mm, 60 % adenomatous) were retrieved for histologic analysis.  Sensitivity, specificity, positive-predictive value (PPV) and NPV, and accuracy of high confidence-NBI predictions for adenomatous histology in lesions less than or equal to 5 mm were 90 %, 88 %, 89 %, 89 %, and 89 %, respectively.  High-confidence characterization of polyps less than or equal to 5 mm predicted the correct surveillance interval in 92 % to 99 % of cases, according to the American and European guidelines; NPV of high-confidence NBI for adenomatous histology for the recto-sigmoid colon lesions less than or equal to 5 mm was 92 %.  The authors concluded that high-confidence prediction of histology for polyps less than or equal to 5 mm appears to be sufficiently accurate to avoid post-polypectomy histologic examination of the resected lesions as well as to allow recto-sigmoid hyperplastic polyps to be left in place without resection.  The main drawback of this study was that only experienced endoscopists were included.

An UpToDate review on “Colorectal cancer surveillance in inflammatory bowel disease” (Peppercorn and Odze, 2014) states that “Chromoendoscopy involves the topical application of stains or pigments to improve tissue localization, characterization, or diagnosis during endoscopy.  A least one controlled trial suggested that staining with methylene blue enhanced the ability to detect the extent of inflammatory changes and identify intraepithelial neoplasia in patients with ulcerative colitis undergoing surveillance.  Other studies have suggested that staining with indigo carmine permitted better detection of dysplasia.  The clinical implications and generalizability of these findings require further clarification …. Narrow band imaging (NBI) is a high-resolution endoscopic technique that enhances the fine structure of the mucosal surface without the use of dyes.  NBI is not recommended for surveillance in patients with IBD as it has not demonstrated a benefit in the detection of dysplasia as compared with white light endoscopy or chromoendoscopy.  In one study that compared the performance of NBI with chromoendoscopy, 44 patients with colitis of eight years or greater disease duration underwent screening colonoscopy with NBI, followed by chromoendoscopy.  NBI detected significantly fewer lesions as compared with chromoendoscopy (102 versus 131); however, most missed lesions were not dysplastic.  NBI also detected fewer dysplastic lesions as compared with chromoendoscopy (20 versus 23), although the difference was not statistically significant in this small study”.

Guidelines from the Association of Coloproctology of Great Britain and Ireland (Willilams et al, 2013) concluded: “Other methods of surface and lesion examination, as well as endoscopic staging, are currently research tools or not currently sufficiently sensitive or specific to be widely recommended .... Optical coherence tomography and confocal laser endoscopy (CLE) are being evaluated.  A recent review and meta-analysis of CLE suggests that this modality offers comparable diagnostic accuracy to colonoscopic histopathology in colorectal neoplasia [citing Su et al, 2013].  This offers the possibility of in vivo real-time optical biopsy in the colorectum. I-Scan is a new modality launched by Pentax (Hoya Corporation, Japan) to enhance lesions difficult to visualize by WLE [white light endoscopy].  There is, as yet, little literature on its value in colorectal neoplastic characterization of malignant change”.

Wu et al (2014) reported on a meta-analysis of the accuracy of CLE in diagnosing Barrett's esophagus (BE)-associated neoplasia by pooling data of existing trials. Databases including PubMed, EMBASE, the Cochrane Library, the Science Citation Index and momentous meeting abstracts were searched and evaluated by two reviewers independently.  Meta-analysis was performed.  Pooling data were conducted in a fixed effect model or a random effects model.  A total of 8 studies involving 709 patients and 4,008 specimens were analyzed.  In a per-patient analysis, the pooled sensitivity of CLE for detection of neoplasia was 89 % (95 % CI: 0.80 to 0.95), and the specificity was 75 % (95 % CI: 0.69 to 0.81).  The area under the curve under the summary receiver operating characteristic was 0.9472.  In a per-location analysis, the pooled sensitivity of CLE for detection of neoplasia was 70 % (95 % CI: 0.65 to 0.74), and the specificity was 91 % (95 % CI: 0.90 to 0.92).  The area under the curve under the summary receiver operating characteristic was 0.9509.  The authors stated that CLE is a reasonable, promising modality for management of patients with BE; more prospective trials need doing to determine whether it is superior to traditional method in diagnosing BE-associated neoplasia. 

Guidelines from the Society for Thoracic Surgeons (Fernando et al, 2009) stated that "advanced endoscopic imaging technologies, such as narrow-band imaging, auto-fluorescence, and confocal laser endo-microscopy have been used in attempts to improve detection of dysplasia.  Another approach is the use of vital stains, such as methylene blue, acetic acid, or indigo carmine, which can help direct and reduce the number of biopsies required to detect HGD with a segment of Barrett’s.  These promising modalities have not currently demonstrated superiority to existing biopsy protocols".

Guidelines on Barrett's esophagus from the American Gastroenterological Association (AGA) (Wang et al, 2008) stated that narrow band imaging, auto-fluorescence imaging, chromoendoscopy, optical coherence tomography, and confocal laser endomicroscopy are promising, but "there is not sufficient evidence at this time to recommend the use of these imaging systems on a routine clinical basis".  More recently, an AGA position statement onn BE stated that "we suggest against requiring chromoendoscopy or advanced imaging techniques for the routine surveillance of patients with Barrett’s esophagus at this time".  This was a weak recommendation, based upon low quality evidence.

Guidelines on endoscopy for BE from the ASGE (2012) stated: "Adjuncts to white-light endoscopy used to improve the sensitivity for the detection of BE and dysplastic BE include chromoendoscopy, electrical enhanced imaging, magnification, and confocal endoscopy.  These techniques are still in development and are discussed in detail elsewhere".

British Society of Gastroenterology guidelines on BE (Fitzgerald et al, 2014) stated: "Advanced imaging modalities, such as chromoendoscopy or ‘virtual chromoendoscopy’, are not superior to standard white light endoscopy in Barrett’s esophagus surveillance and are therefore not recommended for routine use (Recommendation grade A)".

Dik and colleagues (2014) noted that up to 25 % of polyps and adenomas are missed during colonoscopy due to poor visualization behind folds and the inner curves of flexures, and the presence of flat lesions that are difficult to detect.  These numbers may however be conservative because they mainly come from back-to-back studies performed with standard colonoscopes, which are unable to visualize the entire mucosal surface.  In the past several years, new endoscopic techniques have been introduced to improve the detection of polyps and adenomas.  The introduction of high-definition (HD) colonoscopes and visual image enhancement technologies have been suggested to lead to better recognition of flat and small lesions, but the absolute increase in diagnostic yield seems limited.  Cap-assisted colonoscopy and water-exchange colonoscopy are methods to facilitate cecal intubation and increase patients comfort, but show only a marginal or no benefit on polyp and adenoma detection.  Retro-flexion is routinely used in the rectum for the inspection of the dentate line, but withdrawal in retro-flexion in the colon is in general not recommended due to the risk of perforation.  In contrast, colonoscopy with the Third-Eye Retroscope may result in considerable lower miss rates compared to standard colonoscopy, but this technique is not practical in case of polypectomy and is more time consuming.  The recently introduced Full Spectrum Endoscopy colonoscopes maintains the technical capabilities of standard colonoscopes and provides a much wider view of 330 degrees compared to the 170 degrees with standard colonoscopes.  Remarkable lower adenoma miss rates with this new technique were recently demonstrated in the first randomized study.  Nonetheless, more studies are needed to determine the exact additional diagnostic yield in clinical practice.  The authors concluded that optimizing the efficacy of colorectal cancer screening and surveillance requires HD colonoscopes with improved virtual chromoendoscopy technology that visualize the whole colon mucosa while maintaining optimal washing, suction and therapeutic capabilities, and keeping the procedural time as low and patient discomfort as optimal as possible.

Schachschal et al (2014) prospectively examined if the high accuracy for endoscopic polyp diagnosis as reported by reference centers can be reproduced in routine screening colonoscopy.  A total of 10 experienced private practice endoscopists had initial training in pit patterns.  Then they assessed all polyps detected during 1,069 screening colonoscopies.  Patients (46 % men; mean age of 63 years) were randomly assigned to colonoscopy with conventional or latest generation HD-TV instruments.  The main outcome measure was diagnostic accuracy of in-vivo polyp assessment (adenomatous versus hyperplastic).  Secondary outcome measures were differences between endoscopes and reliability of image-based follow-up recommendations; a blinded post-hoc analysis of polyp photographs was also performed.  A total of 675 polyps were assessed (461 adenomatous, 214 hyperplastic).  Accuracy, sensitivity and specificity of in-vivo diagnoses were 76.6 %, 78.1 % and 73.4 %; size of adenomas and endoscope withdrawal time significantly influenced accuracy.  Image-based recommendations for post-polypectomy surveillance were correct in only 69.5 % of cases.  Post-hoc analysis of polyp photographs did not improve accuracy.  The authors concluded that in everyday practice, endoscopic classification of polyp type is not accurate enough to abandon histopathological assessment and use of latest generation colonoscopes does not improve this.  Image-based surveillance recommendations after polypectomy would consequently not meet guideline requirements.

Louie and colleagues (2014) stated that the high-resolution micro-endoscope (HRME) is a novel imaging modality that allows real-time epithelial imaging at subcellular resolution.  Used in concert with any standard endoscope, this portable, low cost, "optical biopsy" technology has the ability to provide images of cellular morphology during a procedure.  This technology has been the subject of a number of studies investigating its use in screening and surveillance of a range of gastro-intestinal neoplasia, including esophageal adenocarcinoma, esophageal squamous cell cancer, colorectal neoplasia, and anal neoplasia.  These studies have shown that HRME is a modality that consistently provides high specificity, NPV, and accuracy across different diseases.  In addition, they have illustrated that HRME users can be relatively easily trained in a short period of time, and that users have demonstrated solid inter-rater reliability.  These features make HRME a potential complement to HD white-light imaging, NBI, and other red flag technologies in facilitating real-time clinical diagnosis, endoscopic therapy, and margin determination.  These researchers stated that further clinical validation is needed to determine whether this translates to reduced procedure times, pathology costs, and follow-up procedures.  Finally, the HRME has a relatively simple design compared with other similar technologies, making it portable, simple to maintain, and low cost.  This may allow the HRME device to function in both advanced care settings as well as in places with less resources and specialized support systems.  The authors concluded that the HRME device has shown good performance along with low cost and portable construction, and its application in different conditions and settings has been promising.

In a large, retrospective study, Mooiweer et al (2015) examined if implementing chromoendoscopy can increase the detection of dysplasia.  Patients with UC and CD undergoing colonoscopic surveillance between January 2000 and November 2013 in 3 referral centers were identified using the patients' medical records.  In recent years, the use of HD chromoendoscopy was adopted in all 3 centers using segmental pan-colonic spraying of 0.1 % methylene blue or 0.3 % indigo carmine (chromoendoscopy group).  Previously, surveillance was performed employing WLE with random biopsies every 10 cm (WLE group).  The percentage of colonoscopies with dysplasia was compared between both groups.  A total of 440 colonoscopies in 401 patients were performed using chromoendoscopy and 1,802 colonoscopies in 772 patients using WLE.  Except for a higher number of CD patients with extensive disease and more patients with a first-degree relative with colorectal cancer (CRC) in the chromoendoscopy group, the known risk factors for IBD-associated CRC were comparable between both groups.  Dysplasia was detected during 48 surveillance procedures (11 %) in the chromoendoscopy group as compared with 189 procedures (10 %) in the WLE group (p = 0.80).  Targeted biopsies yielded 59 dysplastic lesions in the chromoendoscopy group, comparable to the 211 dysplastic lesions detected in the WLE group (p = 0.30).  The authors concluded that despite compelling evidence from randomized trials, implementation of chromoendoscopy for IBD surveillance did not increase dysplasia detection compared with WLE with targeted and random biopsies.

Yamada et al (2015) stated that their recent prospective study found equivalent accuracy of magnifying chromoendoscopy (MC) and endoscopic ultrasonography (EUS) for diagnosing the invasion depth of CRC; however, whether these tools show diagnostic differences in categories such as tumor size and morphology remains unclear.  These researchers conducted detailed subset analysis of the prospective data.  In this multi-center, prospective, comparative trial, a total of 70 patients with early, flat CRC were enrolled from February 2011 to December 2012, and the results of 66 lesions were finally analyzed.  Patients were randomly allocated to primary MC followed by EUS or to primary EUS followed by MC.  Diagnoses of invasion depth by each tool were divided into intra-mucosal to slight sub-mucosal invasion (invasion depth less than 1,000 μm) and deep sub-mucosal invasion (invasion depth greater than or equal to 1,000 μm), and then compared with the final pathological diagnosis by an independent pathologist blinded to clinical data.  To standardize diagnoses among examiners, this trial was started after achievement of a mean κ value of greater than or equal to 0.6 which was calculated from the average of κ values between each pair of participating endoscopists.  Both MC and EUS showed similar diagnostic outcomes, with no significant differences in prediction of invasion depth in subset analyses according to tumor size, location, and morphology.  Lesions that were consistently diagnosed as Tis/T1-SMS or greater than or equal to T1-SMD with both tools revealed accuracy of 76 to 78 %.  Accuracy was low in borderline lesions with irregular pit pattern in MC and distorted findings of the third layer in EUS (MC, 58.5 %; EUS, 50.0 %).  The authors concluded that MC and EUS showed the same limited accuracy for predicting invasion depth in all categories of early CRC.  They stated that since the irregular pit pattern in MC, distorted findings to the third layer in EUS and inconsistent diagnosis between both tools were associated with low accuracy, further refinements or even novel methods are still needed for such lesions.

The ASGE Technology Committee’s guideline on “Electronic chromoendoscopy” (Manfredi et al, 2015) states that “Electronic chromoendoscopy technologies provide image enhancement and may improve the diagnosis of mucosal lesions.  Although strides have been made in standardization of image characterization, especially with NBI, further image-to-pathology correlation and validation are required.  There is promise for the development of a resect and discard policy for diminutive adenomas by using electronic chromoendoscopy; however, before this can be adopted, further community-based studies are needed.  Further validated training tools for NBI, FICE, and i-SCAN will also be required for the use of these techniques to become widespread”.

Tang and associates (2018) noted that colonoscopy is routinely performed for CRC screening but lacks the capability to accurately characterize precursor lesions and early cancers; HRME is a low-cost imaging tool to visualize colorectal polyps with subcellular resolution.  These researchers presented a computer-aided algorithm to evaluate HRME images of colorectal polyps and classify neoplastic from benign lesions.  Using histopathology as the gold standard, clinically relevant features based on luminal morphology and texture were quantified to build the classification algorithm.  They demonstrated that adenomatous polyps could be identified with a sensitivity and specificity of 100 % and 80 % using a 2-feature linear discriminant model in a pilot test set.  The authors concluded that the classification algorithm presented here offered an objective framework to detect adenomatous lesions in the colon with high accuracy and could potentially improve real-time assessment of colorectal polyps.

Klenske and co-workers (2018) noted that dye-less chromoendoscopy is an emerging technology for colorectal polyp characterization.  In a prospective, 3-phased study, these investigators examined if the newly introduced I-scan optical enhancement (OE) can accurately predict polyp histology in-vivo in real-time.  A total of 84 patients with 230 diminutive colorectal polyps were included.  During the first 2 study phases, 5 endoscopists examined if analysis of polyp color, surface and vascular pattern under i-scan OE can differentiate in-vivo between adenomatous and hyperplastic polyps.  Finally, junior and experienced endoscopists (JE, EE, each n = 4) not involved in the prior study phases made a post-hoc diagnosis of polyp histology using a static i-scan OE image database.  Histopathology was used as a gold-standard in all study phases.  The overall accuracy of i-scan OE for histology prediction was 90 % with a sensitivity, specificity, PPV and NPV of 91 %, 90 %, 86 % and 94 %, respectively.  In high confidence predictions, the diagnostic accuracy increased to 93 % with sensitivity, specificity, PPV and NPV of 94 %, 91 %, 89 % and 96 %.  Colonoscopy surveillance intervals were predicted correctly in greater than or equal to 90 % of patients.  In the post-hoc analysis, EE predicted polyp histology under i-scan OE with an overall accuracy of 91 %.  After a single training session, JE achieved a comparable diagnostic performance for predicting polyp histology with i-scan OE.  The authors concluded that the histology of diminutive colorectal polyps can be accurately predicted with i-scan OE in-vivo in real-time.  Furthermore, polyp differentiation with i-scan OE appeared to require only a short learning curve.  These researchers stated that in the future, additional multi-center studies including comparative analysis of optical diagnosis with i-scan OE between expert and non-expert endoscopists are needed before this approach can be implemented into daily endoscopic routine.  These investigators noted that although i-scan OE showed a promising potential in the characterization of colorectal polyps, the direct clinical applicability has yet to be further evaluated in larger studies.  Specifically, it is unclear if optical diagnosis with i-scan OE indeed resulted in actual cost savings.  In this regard, it might be speculated that the cost savings which are conferred by optical diagnosis and associated reduced pathology costs are counter-balanced by the over-estimation of polyp histology (i.e., that hyperplastic polyps are predicted to exhibit adenomatous histology) with i-scan OE and the subsequently narrower colonoscopy intervals that is associated with this over-estimation when surveillance is based on optical diagnosis.  It can be argued that it might be better from an individual patient's stand-point to have every polyp found also reviewed by a pathologist to ensure they are screened at an appropriate interval rather than following a narrower colonoscopic surveillance.  In this regard, it has also be taken into account that patient acceptance of optical diagnosis still appears to be limited.

The authors stated that potential limitations should also be discussed; one of which was the fact that during the first 2 study phases, only experienced endoscopists used i-scan OE for histology prediction.  Although the findings in the 3rd phase indicated that accurate optical diagnosis of polyp histology could be accomplished by inexperienced endoscopists, it was still clear that the findings of the accurate assessment of polyp histology during ongoing endoscopy needs to be verified in separate studies involving also non-tertiary referral centers or non-teaching hospitals as well as gastroenterologists in private practice.  This aspect appeared to be of certain importance since a recent study with i-scan (without optical enhancement) including 10 private practice endoscopists found that the endoscopic classification of polyp type was not accurate enough to abandon histopathology.  Furthermore, a wash-out phase of 14 days might have been too short and hence might have directly influenced the decision-making process when attempting an optical diagnosis by the junior endoscopists.

Mason and colleagues (2019) noted that accurate, real-time, endoscopic risk stratification of colorectal polyps would improve decision-making and optimize clinical efficiency.  Technologies to manipulate endoscopic optical out-puts can be used to predict polyp histology in-vivo; however, it remains unclear how accuracy has progressed and whether it is sufficient for routine clinical implementation.  These researchers carried out a meta-analysis by searching Medline, Embase, and the Cochrane Library.  Studies were included if they prospectively deployed an endoscopic optical technology for real-time in-vivo prediction of adenomatous colorectal polyps; polyposis and IBD were excluded.  Bayesian bivariate meta-analysis was performed, presenting 95 % CI.  A total of 102 studies using optical technologies on 33,123 colorectal polyps were included.  Digital chromoendoscopy differentiated neoplasia (adenoma and adenocarcinoma) from benign polyps with sensitivity of 92.2 % (90.6 % to 93.9 % CI) and specificity of 84.0 % (81.5 % to 86.3 % CI), with no difference between constituent technologies (narrow-band imaging, Fuji intelligent Chromo Endoscopy, iSCAN) or with only diminutive polyps.  Dye chromoendoscopy had sensitivity of 92.7 % (90.1 % to 94.9 % CI) and specificity of 86.6 % (82.9 % to 89.9 % CI), similarly unchanged for diminutive polyps.  Spectral analysis of auto-fluorescence had sensitivity of 94.4 % (84.0 % to 99.1 % CI) and specificity of 50.9 % (13.2 % to 88.8 % CI).  Endomicroscopy had sensitivity of 93.6 % (85.3 % to 98.3 % CI) and specificity of 92.5 % (81.8 % to 98.1 % CI).  Computer-aided diagnosis had sensitivity of 88.9 % (74.2 % to 96.7 % CI) and specificity of 80.4 % (52.6 % to 95.7 % CI).  Prediction confidence and endoscopist experience alone did not significantly improve any technology.  The only subgroup to demonstrate a NPV for adenoma above 90 % was digital chromoendoscopy, making high confidence predictions of diminutive recto-sigmoid polyps.  Chronologic meta-analyses showed a falling NPV over time; and a significant publication bias existed.  The authors concluded that this novel approach to meta-analysis demonstrated that existing optical technologies are increasingly unlikely to allow safe "resect and discard" strategies and that step-change innovation may be needed.  A "diagnose and leave" strategy may be supported for diminutive recto-sigmoid polyps diagnosed with high confidence; however, limitations exist in the evidence base for this cohort.

Confocal Laser Endomicroscopy

Ypsilantis et al (2015) stated that evaluation of the adequacy of EMR of gastro-intestinal lesions remains challenging by use of conventional endoscopy.  Confocal laser endomicroscopy is a novel imaging technique, designed to provide in-vivo histology, and facilitate diagnosis with real-time intervention.  These researchers undertook a systematic review of the available literature, exploring the role of CLE in assuring completeness of EMR of gastro-intestinal lesions.  The number of pertinent studies is very limited, including only 1 RCT and 2 prospective comparative case-series studies.  Per-lesion meta-analysis showed that the sensitivity of CLE for detection of residual neoplasia was 91% (95 % CI: 82.5 % to 96 %) with specificity of 69 % (95 % CI: 61 % to 77 %), with significant heterogeneity noted in all outcomes.  The authors concluded that the evidence underpinning the usefulness of CLE in ensuring adequate EMR of gastro-intestinal neoplasia is currently very weak, with limited promising results related to gastric and colorectal polyp resections.

In a pilot study, Tontini and colleagues (2015) evaluated the effectiveness of CLE to differentiate between UC and CD.  This was a prospective study involving consecutive patients with a well-established diagnosis of UC or CD who underwent colonoscopy with fluorescein-aided confocal imaging.  Overall, 79 patients were included (40 CD, 39 UC).  Confocal laser endomicroscopy findings in patients with CD, showed significantly more discontinuous inflammation (87.5 % versus 5.1 %), focal cryptitis (75.0 % versus 12.8 %), and discontinuous crypt architectural abnormality (87.5 % versus 10.3 %) than in UC (p < 0.0001).  Conversely, UC was associated with severe, widespread crypt distortion (87.2 % versus 17.5 % in CD), decreased crypt density (79.5 % versus 22.5 %), and frankly irregular surface (89.7 % versus 17.5 %; p < 0.0001 for all comparisons).  Statistically significant differences were not seen for heavy, diffuse lamina propria cell increase or mucin preservation.  No granulomas were visible.  Based on these findings, a CLE scoring system was developed that revealed excellent accuracy (93.7 %) when compared with the historical clinical diagnosis and the histopathological gold standard.  The authors concluded that CLE could visualize several disease-specific microscopic features, which are conventionally used in standard histopathology to differentiate between UC and CD.  However, because of the limited penetration depth of CLE, sub-mucosal details or granulomas were not visible.  The new scoring system may allow in-vivo diagnosis of UC or CD.

Rasmussen et al (2015) systematically reviewed current indications and perspectives of CLE for IBD.  Available literature was searched systematically for studies applying CLE in CD or UC.  Relevant literature was reviewed and only studies reporting on original clinical data were included.  Next, eligible studies were analyzed with respect of several parameters such as technique and clinical aim and definitions of outcomes.  Confocal laser endomicroscopy has been used for a wide range of purposes in IBD covering assessment of inflammatory severity, prediction of therapeutic response and relapse and adenoma surveillance in patients with UC.  Methods for measurement of the histological changes ranged from subjective grading to objective quantification analyzed by computer-aided models.  The studies derived their conclusions from assessment of histological features such as colonic crypts, epithelial gaps and epithelial leakiness to fluorescein.  The authors concluded that the technique remains an experimental but emerging tool for assessment of IBD.  They noted that CLE is the only method that enables in-vivo functional assessment of the intestinal barrier function; however, the literature displays great heterogeneity and no single approach has been validated and reproduced to a level of general acceptance.

Sumiyama (2017) stated that methodology for the diagnosis and staging of early gastric cancer (EGC) has improved in Japan since the development of the gastro-camera and determination of a definition of EGC.  Imaging technology has been steadily evolving in the endoscopy field.  Improvements in the resolution of standard endoscopy images used in screening and surveillance provide greater opportunities to find gastric cancer earlier.  Image enhancement endoscopy (IEE), such as narrow band imaging (NBI), highlights mucosal structures and vascularity . In particular, when NBI is used with magnifying endoscopy, it reveals fine details of subtle superficial abnormalities of EGC that are difficult to recognize using standard white light endoscopy (WL).  IEE-assisted magnifying endoscopy has improved the accuracy of the differentiation of superficial gastric cancer as well as delineation of the diseased mucosa.  The advanced imaging technology enables precise assessment of the risk of lymph node metastasis of EGC and is widely used to determine indications for endoscopic treatment.  It is not an over-statement to say that this has become the basis for the current development and dissemination of endoscopic treatments.  Moreover, the resolution of endoscopic imaging has been up-graded to the microscopy level by the development of endomicroscopy, including endocytoscopy and CLE.  Endomicroscopy allows real-time histological analysis of living tissue during routine endoscopy and may reduce the number of biopsies needed to reach the correct diagnosis, minimizing the risk of sampling errors.

Horiguchi and associates (2018) noted that EGC found after Helicobacter pylori (Hp) eradication often displays non-tumorous regenerative epithelium and/or maturated tumorous epithelium overlying the cancerous tissue, which may confuse endoscopic and histologic diagnosis.  Probe-based CLE (pCLE) enables in-vivo real-time optical biopsy.  These researchers compared the diagnostic yields for these EGC cases using conventional white light endoscopy (WL), magnifying endoscopy with NBI (ME-NBI), pCLE, and endoscopic biopsy; thee investigators also compared the accuracy of the horizontal extent diagnosis between ME-NBI and pCLE.  This study enrolled 30 patients with 36 EGC lesions after successful Hp eradication.  The diagnostic yields of WL, ME-NBI, pCLE, and endoscopic biopsy were prospectively compared.  Four points of cancerous margins (oral, anal, anterior, and posterior sites) were also prospectively evaluated with M-NBI and pCLE to determine the horizontal extent of the EGC.  The diagnostic yield was significantly higher with pCLE than with WL and endoscopic biopsy (97 versus 72 %, 97 versus 72 %, p = 0.0159, 0.0077, respectively), whereas it did not differ from ME-NBI (88.9 %, p = 0.371).  The height of non-tumorous regenerative epithelium or maturated atypical glands was 104.7 ± 34.2 μm in the pCLE-positive cases, whereas it was 188.3 ± 27.1 μm in a pCLE-negative case (p = 0.0004).  The diagnostic accuracy of the horizontal margin of EGC was significantly higher with pCLE than with ME-NBI (92 versus 70 %, p = 0.0159).  The authors concluded that pCLE may be helpful for the diagnosis of ambiguous ECGs found after Hp eradication because it enabled real-time scanning throughout the lesion and the detection of subsurface microstructure.

Chene and co-workers (2017) noted that it has recently been postulated that most ovarian cancers have a tubal origin.  The identification of pre-invasive tubal lesions would be of great interest in the early diagnosis of ovarian cancer.  Optical biopsy has been developed and validated in the detection of pre-cancerous lesions (such as Barrett's esophagus).  In a prospective study, these researchers evaluated the feasibility of optical biopsy (e.g., confocal laser endomicroscopy) in the study of fallopian tubes during laparoscopy.  They also described the images in benign pre-malignant and malignant tubes with a histopathological and immuno-histochemical (p53 and Ki67 expressions) correlation.  A total of 40 patients undergoing laparoscopic salpingectomy for benign conditions (benign hysterectomy), prophylactic conditions (BRCA mutation) or in case of pelvic cancers were included after obtaining informed and signed consent prior to surgery.  The optical biopsy was performed on the fimbria of each tube in-vivo and ex-vivo.  A correlation was made with the histopathological and immuno-histochemical analysis.  The feasibility of optical biopsy was always confirmed during laparoscopy.  The optical biopsy iconography revealed different images in benign tubal epithelium (well-defined black and grey structure), in adenomatoid tumor (tortuous architectural organization), in STIC pre-cancerous lesion (enlarged, irregular and pleomorphic cells, dilated and distorted vessels) and in tubal metastasis of high grade serous ovarian cancer (dark neoplastic cells irregular in size and shape).  The authors concluded that optical biopsy may be the first emerging mini-invasive technology that could detect tubal lesions and may be considered as a promising tool in the early detection of ovarian cancer. 

Breda and colleagues (2018) stated that despite the recent growing interest in the conservative management of upper tract urothelial carcinoma (UTUC), the diagnostic process is still a challenge for the risk of tumor under-grading.  Real-time CLE provides in-vivo microscopic images of tissues using a low-energy laser light source.  These researchers described their initial experience with CLE for the real-time characterization of UTUC.  A total of 14 flexible ureteroscopies (f-URS) were performed at the authors’ institution with CLE for UTUC.  Lesions were pre-operatively identified with computed tomography (CT)-intravenous urography.  Cellvizio system was used during f-URS to perform CLE on the targeted lesions.  Biopsies were then performed.  Surgeon's CLE readings (low-grade/high-grade/carcinoma in-situ [CIS]) were documented in the operation notes.  A dedicated genito-urinary pathologist, blinded to the surgeon reading, examined all specimens.  A 3rd person collected prospectively the CLE readings and the histopathological reports.  Cohen's Kappa analysis was performed to test inter-observer agreement.  The mean diameter of tumors using CT scan was 26 mm (range of 5 to 50 mm).  In 8 patients, CLE allowed to obtain images compatible with low-grade UTUC, in 5 patients with high-grade UTUC, and in 1 case with CIS.  These investigators found correspondence between the CLE images and the final histopathological results in 7/7 cases of low-grade UTUC (100 %), in 5/6 cases of high-grade UTUC (83 %), and in 1/1 case of CIS (100 %).  Substantial agreement was found at inter-observer agreement (k = 0.64) between CLE and histological reading.  No complications and/or limitations related to the use of CLE were recorded.  The authors concluded that CLE is a promising new technology in providing a reliable real-time histological characterization of UTUC lesions.  Ideal targets might be UTUC patients potentially candidates for conservative management.

Zhang and colleagues (2017) evaluated the diagnostic value of CLE in detection of gastric cancer (GC), gastric intraepithelial metaplasia (GIM), and gastric intraepithelial neoplasia (GIN) lesions.  PubMed, the Cochrane Library, and Wangfang databases were searched to include eligible articles about CLE in detection of gastric lesions.  After study selection, quality assessment and data extraction conducted by 2 reviewers independently, meta-analysis was performed by Meta-Disc 1.4.  The pooled sensitivity and specificity was calculated, receiver operating characteristic (ROC) curve was constructed, and the area under ROC curve (AUC) was calculated.  A total of 23 studies evaluating the diagnostic value of CLE were included.  For the diagnosis of GC lesions, the pooled sensitivity, specificity, and AUC were 91 % (88 to 94 %), 99 % (99 to 99 %), and 0.9513, respectively.  For the diagnosis of lesions, the pooled sensitivity, specificity, and AUC were 92 % (90 to 94 %), 97 % (96 to 98 %), and 0.9774, respectively.  For the diagnosis of GIN lesions, the pooled sensitivity, specificity and AUC were 81 % (75 to 85 %), 98 % (97 to 98 %), and 0.9204, respectively.  The authors concluded that CLE could provide an accurate diagnosis with high sensitivity and specificity for GC, GIM, and GIN lesions.  Moreover, they stated that these findings should be confirmed by well-designed, multi-centered, randomized controlled, and double-blinded trials with large samples.

Brunckhorst and colleagues (2019) noted that current optical diagnostic techniques for malignancies are limited in their diagnostic accuracy and lack the ability to further characterize disease, leading to the rapidly increasing development of novel imaging methods within urology.  In a systematic review, these investigators evaluated the literature for novel imagining modalities, in the detection and staging of urological cancer and examined their effectiveness via their utility and accuracy.  These researchers stated that CLE was examined in 3 studies meeting inclusion criteria.  Studies had a combined 39 UTUC patients and were largely conducted to assess feasibility of differentiation between low- and high-grade tumors; IV fluorescein prior to the procedure was used in both, with 2 utilizing a commercially available system (Cellvizio system).  Two studies claimed that CLE was able to differentiate between benign/malignant and low-/high-grade tissue without further no quantitative data for diagnostic accuracy.  One study assessed correspondence between CLE images and biopsy results, identifying this as 100 % for low-grade lesions, 83 % for high-grade and 100 % for in-situ disease in a limited cohort of 14 patients.  However, no formal sensitivity or specificity was provided with no study discussing cost.  Unclear significance of patient selection bias was revealed in all studies with concerns regarding histopathology as the reference standard.  These investigators also noted that CLE for bladder cancer has generated interest due to its ability to assess tissue at a cellular level intra-operatively, this, having potential for intra-operative grading and improving diagnostic accuracy.  However, at present, there is little objective data to show this with predominant feasibility of use demonstrated.  Evidence is needed to support its predominant clinical applicability within grading of disease that would improve its clinical utility.  Furthermore, as with OCT, CLE also requires identification of a suspicious lesion prior to utilization of a probe for assessment.  Thus, its combination with other modalities should be conducted to examine if this improves its diagnostic accuracy, and, therefore, clinical utility.  Confocal laser endomicroscopy does, however, benefits from the availability of widespread commercial systems available that improves its potential for diffusion if improvements in diagnostic accuracy are proven.  At present though its use is still limited, not yet demonstrating to be a useful tool in widespread clinical practice.  The authors concluded that due to current limitations in diagnosis of urological malignancies, numerous additional optical imaging modalities have been developed and assessed for the detection of neoplastic disease and to provide increased real-time information to guide intra-operative decisions.  OCT for bladder cancer and photodynamic diagnosis for upper tract malignancy demonstrated the largest potential.  However, at present, both still lack the evidence base needed for translation into routine clinical practice.  Further large and well-designed trials are needed for these modalities to evaluate not only their overall and staging diagnostic accuracies, but also how to best utilize them.  Other modalities such as CLE and auto-fluorescence for bladder cancer and NBI for upper tract disease also demonstrated potential but are at an earlier stage of their investigation.

Bronchoscopic Evaluation of Broncho-Alveolar Lavage Components

Zirlik and associates (2018) noted that in many studies, CLE has proven to be a useful tool in pulmonology; nevertheless, the application in this field is still experimental.  These researchers demonstrated the identification of bronchoalveolar lavage (BAL) components applying CLE, using a dye.  In 21 patients with various underlying diseases a bronchoscopy with BAL was performed.  As in routine clinical practice, BAL fluid (BALF) was analyzed in terms of cytologic, virologic, and microbiologic aspects.  To one fraction of BALF, these investigators added acriflavine.  After centrifugation CLE was applied and the video sequences were analyzed by an experienced investigator.  Using CLE, BALF components (such as alveolar macrophages or leucocytes) could be easily identified.  A further sub-division of leucocytes (neutrophilic, eosinophilic granulocytes, and lymphocytes) was not possible.  Analogous to conventional cytology, a precise distinction of lymphocyte sub-population (cd 4/cd 8 ratio) was not feasible.  In terms of quantification, this is still the application field of flow cytometry and immunohistochemistry.  The authors concluded that using CLE, alveolar macrophages and leucocytes in stained BALF could be differentiated independent of smoking status.  They stated that further studies should be initiated in order to sub-classify leucocytes in eosinophilic, neutrophilic granulocytes, and lymphocytes, which is important for routine clinical practice.  These researchers stated that CLE has not been firmly established in pulmonology.  Indeed, encouraging data in this field are increasing.  In this study, these investigators showed that BALF components (alveolar macrophages and leucocytes) are differentiable by CLE-based analysis, offering a new diagnostic approach.

Confirmation of Low-Grade Dysplasia and Surveillance of Barrett’s Esophagus

Shah and associates (2018) stated that for surveillance of Barrett's esophagus (BE), the current standard of random 4-quadrant biopsies misses 10 - to 50 % of esophageal neoplasms, and does not permit real-time decision-making.  Probe-based confocal laser endomicroscopy (pCLE) permits real-time in-vivo histologic assessment of esophageal mucosa during upper endoscopy.  Prospective studies comparing the accuracy of pCLE to 4-quadrant biopsies in routine clinical practice are lacking.  In this study, consecutive patients with BE underwent high definition white light endoscopy (HD-WLE) and narrow-band imaging (NBI) followed by pCLE and targeted biopsy or mucosal resection.  Four-quadrant biopsies were obtained during the same session.  Baseline variables, real-time pCLE interpretation, and histology results were prospectively recorded.  Blinded expert review of pCLE sequences and histology specimens was performed.  A sample size of 64 patients was calculated a priori based on 3 % estimated prevalence of high-grade dysplasia (HGD) or cancer.  A total of 66 patients were included in the study.  The prevalence of HGD or cancer was 4.55  %.  Both real-time and blinded pCLE correctly identified all cases of cancer.  For the primary outcome, real-time pCLE was 98 % specific but only 67 % sensitive for HGD/cancer compared to non-blinded pathologist interpretation.  For HGD and cancer, inter-observer agreement was substantial between real-time and blinded endomicroscopists (kappa = 0.6); pCLE identified dysplasia in 75 % of cases where both blinded and un-blinded pathology interpretation was low-grade dysplasia (LGD).  The authors concluded that pCLE demonstrated high specificity for detecting dysplasia and cancer, but the relatively low sensitivity and lack of incremental benefit over HD-WLE and NBI may limit its utility in routine surveillance of BE.  Moreover, they stated that pCLE may have a role in confirming LGD in real-time before eradication therapy; but further study is needed to validate pCLE for this specific indication.

The authors stated that a drawback of this study was that investigators were not required to strictly adhere to Miami criteria, because the aim was to assess accuracy in routine clinical practice.  These investigators did not use validated criteria to distinguish HGD from LGD during real-time pCLE interpretation, and there was significant disagreement between blinded and un-blinded pathologists.  Although inclusion of subjects was limited to a single tertiary Veterans Affairs (VA) medical center, the demographics of these patients closely resemble those of BE patients in the community setting.  These researchers did not collect information to calculate “per optical biopsy” accuracy because the objective was to assess “per patient accuracy” as suggested by the ASGE PIVI.  Only 3 patients in this study had HGD or cancer, which had implications for estimating predictive values of pCLE.  However, these findings were well within the sample size estimates, and highlighted the cost-effectiveness barriers that any imaging technology faces when used for routine BE surveillance.

Diagnosis of Acute Cellular Rejection in Lung Transplant Recipients

Keller and colleagues (2019) stated that acute cellular rejection (ACR) in lung transplant recipients requires demonstration of peri-vascular lymphocytic infiltration in alveolar tissue samples from trans-bronchial biopsies (TBBs).  Probe-based CLE (pCLE) allows in-vivo observation of alveolar, vascular, and cellular microstructures in the lung with potential to identify ACR.  In a prospective, blinded, multi-center observational study, these researchers identified pCLE findings in patients with ACR diagnosed histopathologically by TBB.  Lung transplant recipients undergoing diagnostic bronchoscopies within 1 year post-transplant for suspected ACR had pCLE video imaging obtained immediately prior to tissue sampling via TBB.  Findings of 2 pCLE criteria, abundant alveolar cellularity and peri-vascular cellularity (PVC), were assessed by 4 investigators familiar with pCLE and compared to histopathologic criteria of ACR to derive sensitivity, specificity, area under the receiver operating characteristic curve (ROC), and accuracy.  Inter-observer agreement was assessed by calculating intra-class coefficient and Fleiss κ.  Findings were analyzed before and after a consensus meeting of investigators on interpreting images.  A total of 30 pCLE procedures were performed on 24 patients, 8 showing ACR in TBB.  Diagnostic performance and inter-observer agreement using pCLE to identify PVC were significantly higher than those of abundant alveolar cellularity (p < 0.01).  The number of blood vessels identified with PVC on pCLE was significantly correlated with histopathologic activity grading of ACR (p < 0.01); PVC agreement among investigators significantly improved after consensus meeting (p < 0.01).  The authors concluded that when found on pCLE, PVC was a feasible and reproducible criterion for assessment of ACR in-vivo, but there is a learning curve for image interpretation.  These preliminary findings need to be validated by well-designed studies.

Furthermore, an UpToDate review on “Evaluation and treatment of acute lung transplant rejection” (Pilewski, 2008) does not mention confocal laser endomicroscopy as a management tool.

Diagnosis and Histologic Grading of Bladder Cancer

Liem and colleagues (2020) stated that cystoscopy enables the visualization of suspicious bladder lesions but lacks the ability to provide real-time histopathologic information; CLE is a probe-based optical technique that can provide real-time microscopic images.  This high-resolution optical imaging technique may enable real-time tumor grading during cystoscopy.  These researchers validated and adapted CLE criteria for bladder cancer diagnosis and grading.  A total of 73 patients scheduled for transurethral resection of bladder tumor(s) were included; CLE imaging was performed intra-operatively prior to en bloc resection.  Histopathology was the reference standard for comparison.  Three independent observers evaluated the CLE images to classify tumors as low- or high-grade urothelial carcinoma (UC), or benign lesions.  Inter-observer agreement was calculated with Fleiss kappa analysis and diagnostic accuracy with 2×2 tables.  Histopathology of 66 lesions (53 patients) revealed 25 low-grade UCs, 27 high-grade UCs, and 14 benign lesions.  For low-grade UC, most common features were papillary configuration (100 %), distinct cell borders (81 %), presence of fibro-vascular stalks (79 %), cohesiveness of cells (77 %), organized cell pattern (76 %), and monomorphic cells (67 %).  A concordance between CLE-based classification and histopathology was found in 19 cases (76 %).  For high-grade UC, pleomorphic cells (77 %), indistinct cell borders (77 %), papillary configuration (67 %), and disorganized cell pattern (60 %) were the most common features.  A concordance with histopathology was found in 19 cases (70 %).  In benign lesions, the most prevalent features were disorganized cell pattern (57 %) and pleomorphic cells (52 %), and a concordance with histopathology was found in 4 cases (29 %).  The authors concluded that the CLE criteria enabled identification of UC; CLE features correlated to histopathologic features that may enable real-time tumor grading.  However, flat lesions remained difficult to classify.  These preliminary findings need to be validated by well-designed studies.

The authors stated that a drawback of this study was the impossibility to identify discriminating CLE features for benign lesions and CIS due to heterogeneity of benign lesions and the small number of both benign lesions and CIS. In addition, heterogeneity within bladder tumors may be another drawback.  Considering the limited field of view of the probe (240 μm), only a fraction of the tumor surface was imaged.  Thus, the recorded image sequence may give a biased view with regard to the whole tumor, and might be responsible for discrepancies between CLE-based classification and histopathology.  Additionally, variability in CLE image quality could impede CLE image evaluation.  Specifically, at the start of this study, there was a learning curve with regard to probe stabilization.  Movement artefacts could have contributed to the 14 % non-diagnostic rate of CLE images.  Lastly, despite a wash-out time of several weeks to months, a recall bias might still exist for the urologists who predicted the tumor grade based on WLC images.  However, this bias would have led to an over-estimation; hence, the actual concordance of the WLC-based diagnoses with histopathology should be even lower.

Wu and colleagues (2020) examined the diagnostic efficacy of CLE in detection of bladder cancer.  These researchers carried out a systematic literature search on CLE in diagnosing bladder cancer in PubMed, Embase, and the Cochrane Library databases.  A bi-variate meta-regression model was used for meta-analysis to evaluate the pooled diagnostic value of CLE.  A total of 5 eligible studies involving 302 lesions were available for this meta-analysis.  In a per-lesion analysis, pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and summary receiver-operating curve (SROC) area under the curve (AUC) of CLE for malignant lesions were 0.90 (95 % CI: 0.85 to 0.94), 0.72 (95 % CI: 0.59 to 0.82), 3.20 (95 % CI: 2.14 to 4.79), 0.14 (95 % CI: 0.09 to 0.21), 23.27 (95 % CI: 11.71 to 46.25), and 0.91 (95 % CI: 0.89 to 0.94), respectively.  For low-grade urothelial carcinomas, pooled sensitivity, specificity, PLR, NLR, DOR, and AUC for CLE were 0.72 (95 % CI: 0.57 to 0.84), 0.87 (95 % CI: 0.77 to 0.93), 5.48 (95 % CI: 3.12 to 9.62), 0.32 (95 % CI: 0.20 to 0.50), 17.19 (95 % CI: 8.01 to 36.89), and 0.85 (95 % CI: 0.82 to 0.88), respectively.  For high-grade urothelial carcinomas, pooled sensitivity, specificity, PLR, NLR, DOR, and AUC for CLE were 0.82 (95 % CI: 0.62 to 0.92), 0.84 (95 % CI: 0.73 to 0.91), 4.96 (95 % CI: 2.58 to 9.54), 0.22 (95 % CI: 0.09 to 0.52), 22.49 (95 % CI: 5.33 to 94.85), and 0.89 (95 % CI: 0.86 to 0.91), respectively.  The authors concluded that CLE is a promising endoscopy technique for real-time tumor grading of bladder cancer.

Diagnosis of Prostate Cancer

Swaan and colleagues (2018) noted that focal therapy for prostate cancer has been proposed as an alternative treatment to whole-gland therapies in selected men to diminish side effects in localized prostate cancer.  As nowadays imaging cannot offer complete prostate cancer disease characterization, multi-core systematic biopsies are recommended (trans-rectal or trans-perineal).  Optical imaging techniques such as CLE and OCT allow in-vivo, high-resolution imaging.  Moreover, they can provide real-time visualization and analysis of tissue and have the potential to offer additive diagnostic information.  This study has 2 separate primary objectives.  The first is to assess the technical feasibility and safety of in-vivo focal imaging with CLE and OCT.  The second is to identify and define characteristics of prostate cancer and normal prostate tissue in CLE and OCT imaging by comparing these images with the corresponding histopathology.  In this prospective, in-vivo feasibility study, needle-based CLE and OCT imaging will be performed before trans-perineal template mapping biopsy or radical prostatectomy.  First, CLE and OCT will be performed in 4 patients (2 for each imaging modality) undergoing trans-perineal template mapping biopsy to assess the feasibility and safety of CLE and OCT.  If proven to be safe and feasible, CLE and OCT will be performed in 10 patients (5 for each imaging modality) undergoing radical prostatectomy; CLE and OCT images will be analyzed by independent, blinded observers; CLE- and OCT-based qualitative and quantitative characteristics and histopathology will be compared.  The study complies with the IDEAL (Idea, Development, Exploration, Assessment, Long-term study) stage 2a recommendations.  At present, the study is enrolling patients and results and outcomes are expected in 2019.  The authors concluded that CLE and OCT are promising optical imaging techniques that can visualize and analyze tissue structure, possible tumor grade, and architecture in real time.  They can potentially provide real-time, high-resolution microscopic imaging and tissue characteristics of prostate cancer in conjunction with magnetic resonance imaging (MRI) or trans-rectal ultrasound fusion-guided biopsy procedures.  This study will provide insight into the feasibility and tissue-specific characteristics of CLE and OCT for real-time optical analysis of prostate cancer.

Diagnosis of Vocal Cord Lesions

Goncalves and colleagues (2017) evaluated the reliability and limitations of CLE for diagnosing lesions of the vocal cords and differentiating malignant from non-malignant lesions.  During micro-laryngoscopy, the vocal cords were scanned by pCLE.  The video recordings were analyzed and compared with the histological results.  A total of 31 representative images were extracted and presented to 4 medical professionals (blinded examiners) for assessment.  The accuracy for the category malignant/non-malignant ranged between 58.1 % and 87.1 %.  Overall inter-rater reliability was 0.29.  Sensitivity ranged between 45.5 and 100 %, specificity between 60.0 and 100 %, PPV between 38.5  and 100 % and NPV between 66.7 and 100%.  The authors concluded that CLE is a promising method for the non-invasive diagnosis of vocal cord lesions in-vivo, but factors such as small penetration depth, not available contrast media for the nuclei and subjective analyses of the images limited, at the moment, its diagnostic value.

Differentiation of Colorectal Polyps during Routine Colonoscopy

In a prospective study, Belderbos and associates (2017) noted that pCLE is used to differentiate between neoplastic and non-neoplastic colorectal polyps during colonoscopy.  These researchers evaluated the accuracy of 2 endoscopists starting to use real-time pCLE for differentiation of colorectal polyps and to determine the NPV for neoplasia in polyps of less than or equal to 5 mm.  Patients undergoing colonoscopy in a tertiary hospital were included in this trial.  After a training session, 2 colonoscopists assessed 50 polyps between August 2012 and April 2014.  They sequentially used NBI and real-time pCLE to differentiate non-adenomatous, adenomatous, and carcinomatous polyps during colonoscopy.  Histologic diagnosis by a GI pathologist was the gold standard.  Results were compared to post-hoc pCLE by a panel of gastroenterologists and pathologists.  The accuracy of real-time pCLE was 76 %, compared to 73 % for NBI, and was not significantly different between the first 50 cases (74 %) and the last 50 cases (78 %, p  = 0.64).  The accuracy in polyps of greater than 5 mm was 87 % versus 59 % in polyps of less than or equal to 5 mm (p  = 0.04) and increased from 45 % (13/29) in poor quality images to 86 % (44/51) in fair quality images and 95 % (19/20) in good quality images (p  < 0.01).  The post-hoc pCLE accuracy was 62 %.  The NPV for polyps of less than or equal to 5 mm was 58 % for real-time pCLE and 54 % for post-hoc pCLE.  The authors concluded that although a fair accuracy of real-time pCLE for differentiation of colorectal polyps can be achieved within 50 cases, low NPV and difficulty in obtaining high-quality pCLE images hampered implementation in routine clinical practice.  Moreover, they stated that in the current era of high-definition endoscopes and digital chromoendoscopy, the additional value of using pCLE in the colon is likely to be limited.

The authors stated that the small sample size was a drawback of this study and the results should therefore be interpreted with caution.  They also noted that the learning curve for the interpretation of pCLE images was probably already completed within the sample size of this study, but the learning curve to obtain pCLE images might not.  This was however not just a drawback of this study, but it was also an outcome, reflecting a difficulty of the technique.  Of note, the long inclusion period might have prolonged the learning curve and was therefore another drawback of this study.  In addition, the brief hands-on training that was provided to both endoscopists might have been insufficient, although it was performed in accordance with the standard Cellvizi.  instruction.  Based on this, it may well be that the requirement of a longer training period was a potential limitation of pCLE implementation.  In this study, these investigators did not use a cap attached to the colonoscope, which may help to stabilize the probe.  Another limitation of this study was the relatively large sizes and high rate of neoplasia of the polyps, which probably affected the accuracy of real-time diagnoses.  To assess the use of pCLE in a resect-and-discard strategy, investigation of small polyps is necessary.  This was not the primary aim of this study, but these researchers included more than 60 % small polyps.  Sensitivity and NPV for detecting neoplasia in small polyps were quite low and a larger sample size probably would not have contributed essentially to the outcome.

Evaluation of Depth of Invasion in Colorectal Lesions

In a pilot study, Abe and co-workers (2018) examined the diagnostic yield of pCLE in the evaluation of depth of invasion in colorectal lesions.  Patients with colorectal lesions eligible for either endoscopic treatment or surgery were enrolled in the study.  Tumor's depth of invasion was classified as mucosal or slight submucosal (M-SM1) and deep submucosal invasion or deeper (SM2 or deeper).  White light endoscopy (WLE), magnifying narrow band imaging (M-NBI), and magnifying chromo-endoscopy (M-CE) were used to assess colorectal lesions, and pCLE was used to identify tumor's features related to SM2 or deeper.  The diagnostic classification of depth of invasion was obtained by correlating pCLE findings with histology results (on-site diagnosis).  All colorectal lesions were stratified by a second endoscopist who was blinded to any clinical and histological information with the use of WLE, M-NBI, M-CE, and pCLE (off-line review).  A total of 22 colorectal lesions were analyzed: 7 were adenoma, 10 intra-mucosal cancer, and 5 SM2 or deeper cancer.  With respect to pCLE findings, loss of crypt structure was seen in all SM2 or deeper cancers and only in 1 M-SM1 lesion.  Sensitivity, specificity, and accuracy of WLE, M-NBI, and M-CE in off-line review were 60/94/86, 60/94/86, and 80/94/91 %, respectively.  Sensitivity/specificity/accuracy of pCLE in off-line review were 80/94/91 %, respectively.  The inter-observer agreement of pCLE between on-site diagnosis and off-line review was 0.64 (95 % CI: 0.27 to 1.0).  The authors concluded that pCLE may represent a useful tool to evaluate the depth of invasion in colorectal lesions.

Furthermore, National Comprehensive Cancer Network’s clinical practice guidelines on “Colon cancer” (Version 2.2018) and “Rectal cancer” (Version 2.2018) do not mention confocal laser endomicroscopy as a management tool.

Evaluation of Esophageal Neoplasia

Kollar and associates (2018) noted that pCLE enables real-time histopathological assessment during endoscopic procedures to evaluate epithelial and sub-epithelial structures with a 1,000x magnification.  It may be used in various localizations not only in the digestive tract, but its role in clinical practice is still a matter of discussion.  The main advantages of pCLE compared to standard biopsies may be:

1. real-time diagnosis;
2. which may be done by the endoscopist; and
3. a larger evaluated area compared to standard biopsies. 

In theory, pCLE has the potential to eliminate the need for biopsy.  However, pCLE could not replace standard biopsies at this time, among others, standard forceps biopsies are presently more cost-effective.  These investigators stated that pCLE may be used to enhance the diagnostic arsenal and improve mucosal visualization and evaluation in patients with BE, with visible esophageal lesions and in patients undergoing surveillance endoscopy after endoscopic treatment of BE-related neoplasia.  Probe-based CLE requires sufficient training and use of validated classifications systems.  At present, the majority of endoscopic centers do not use pCLE routinely and no guidelines recommend its routine use for patients with different esophageal diseases, although pCLE is (in selected indications) reimbursed in some countries.

Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Esophageal and esophagogastric junction cancers” (Version 2.2018) does not mention confocal laser endomicroscopy as a management tool.

Evaluation of Pancreatic Cysts / Lesions

Li and co-workers (2018) noted that increases in the quality as well as utilization of cross-sectional imaging have led to rising diagnoses of pancreatic cystic lesions (PCL).  Accurate pre-surgical diagnosis enables appropriate triage of PCLs.  Unfortunately, current diagnostic approaches have sub-optimal accuracy and may lead to unnecessary surgical resections or missed diagnoses of advanced neoplasia.  Additionally, early detection represents an opportunity for intervention to prevent the progression to pancreatic adenocarcinoma.  These investigators systematically reviewed the current literature on CLE and molecular biomarkers in the evaluation of PCLs.  Confocal laser endomicroscopy is a novel technology that allows for real-time in-vivo microscopic imaging with multiple clinical trials identifying characteristic endomicroscopy findings of various pancreatic cystic lesions.  DNA-based molecular markers have also emerged as another diagnostic modality as the pattern of genetic alternations present in cyst fluid can provide both diagnostic and prognostic data.  The authors proposed that both techniques can be utilized to improve patient outcomes.  Moreover, these researchers stated that prospective multi-center studies are needed to determine how to integrate endoscopic ultrasound (EUS)-guided needle-based CLE and molecular analysis into existing management protocols and clinical practice.  In clinical practice, these technologies may especially be applied in the setting of cases with diagnostic uncertainty in order to improve accuracy and allow for appropriate risk stratification.  They also noted that expertise in these technologies may not be widespread and referral to centers with experience may be necessary.

Napoleon and co-workers (2019) stated that needle-based CLE (nCLE) enables observation of the inner wall of PCLs during an EUS-guided fine-needle aspiration (EUS-FNA).  In a prospective, multi-center, validation study, these researchers evaluated the diagnostic performance of nCLE for large, single, non-communicating PCLs using surgical histopathology or EUS-FNA cytohistopathology as a reference diagnosis.  From April 2013 to March 2016, consecutive patients referred for EUS-FNA of indeterminate PCLs without evidence of malignancy or chronic pancreatitis were prospectively enrolled at 5 centers.  EUS-FNA and nCLE were performed and cystic fluid was aspirated for cytohistopathological and carcinoembryonic antigen (CEA) analysis.  The diagnostic performance of nCLE was assessed against the reference standard and compared with that of EUS and CEA.  This study was registered on ClinicalTrials.gov (NCT01563133).  A total of 206 patients underwent nCLE and 78 PCLs (mean size of 40 mm, range of 20  to 110 mm) had reference diagnoses (53 pre-malignant and 25 benign PCLs).  Post-procedure pancreatitis occurred in 1.3 % of the patients.  nCLE was conclusive in 71 of the 78 cases (91 %).  The sensitivity and specificity of nCLE for the diagnosis of serous cystadenoma, mucinous PCL, and pre-malignant PCL were all greater than or equal to 0.95 (with 95 % CI: 0.85 to 1.0).  The AUROC was significantly larger for nCLE than for CEA or EUS.  The authors concluded that nCLE had excellent diagnostic performance that surpassed that of CEA and EUS for the diagnosis of large, single, non-communicating PCLs.  The nCLE procedure should be considered in patients with indeterminate PCLs to ensure a more specific diagnosis.

Krishna and colleagues (2020) noted that imaging patterns from EUS-guided nCLE have been associated with specific PCLs.  In a prospective study, these researchers compared the accuracy of EUS with nCLE in differentiating mucinous from non-mucinous PCLs with that of measurement of CEA and cytology analysis.  A total of 144 consecutive patients with a suspected PCL (greater than or equal to 20 mm) who underwent EUS with fine-needle aspiration of pancreatic cysts from June 2015 through December 2018 at a single-center were included in this trial; 65 patients underwent surgical resection.  Surgical samples were analyzed by histology (reference standard).  During EUS, the needle with the mini-probe was placed in the cyst, which was analyzed by nCLE.  Fluid was aspirated and analyzed for level of CEA and by cytology.  These investigators compared the accuracy of nCLE in differentiating mucinous from non-mucinous lesions with that of measurement of CEA and cytology analysis.  The mean size of dominant cysts was 36.4 ± 15.7 mm and the mean duration of nCLE imaging was 7.3 ± 2.8 mins.  Among the 65 subjects with surgically resected cysts analyzed histologically, 86.1 % had at least 1 worrisome feature, based on the 2012 Fukuoka criteria.  Measurement of CEA and cytology analysis identified mucinous PCLs with 74 % sensitivity, 61 % specificity, and 71 % accuracy.  EUS with nCLE identified mucinous PCLs with 98 % sensitivity, 94 % specificity, and 97 % accuracy.  nCLE was more accurate in classifying mucinous versus non-mucinous cysts than the standard method (p < 0.001).  The overall incidence of post-procedure acute pancreatitis was 3.5 % (5/144); all episodes were mild, based on the revised Atlanta criteria.  The authors concluded that analysis of cysts by nCLE identified mucinous cysts with greater accuracy than measurement of CEA and cytology analysis; EUS with nCLE can be used to differentiate mucinous from non-mucinous PCLs.

Commenting on the afore-mentioned study by Krishna et al (2019), Adler (2019) stated that “This study demonstrates the value of CLE for the evaluation of patients with pancreatic cystic lesions.  CLE has been commercially available to gastroenterologists for over a decade, with both scope-based and (as in this study) probe-based platforms, but despite many studies showing the value of this technology, it has not been widely adopted, mostly due to concerns about procedure times, cost, and reimbursement.  While the current findings are very encouraging, until CLE becomes inexpensive, easy to use, and widely available I suspect it will remain a niche approach”.

Chin and associates (2021) noted that the prevalence of pancreatic cystic lesions (PCLs) has increased recently due to the increased use of cross-sectional abdominal imaging and the aging population.  Current diagnostic techniques are inadequate to distinguish between PCLs that require surgery, close surveillance, or expectant management.  This has resulted in increased morbidity from both inappropriately aggressive and conservative management strategies; however, nCLE has allowed microscopic examination and visual delineation of the surface epithelium of PCLs.  Landmark studies in the past 10 years had correlated nCLE and histological findings and identified characteristics differentiating various types of PCLs.  Subsequent studies have confirmed the high diagnostic yield of nCLE and its diagnostic utility in PCLs with an equivocal diagnosis.  Moreover, nCLE has been shown to improve the diagnostic yield of PCLs.  This will aid in avoiding unnecessary pancreatic surgery, which is associated with significant risks of morbidity and mortality.  The early detection of high-grade dysplasia in PCLs will provide early surgical treatment and improve outcomes for pancreatic cancer.  Despite the high upfront cost of nCLE, the improved diagnostic accuracy and resultant appropriate management have resulted in improved cost effectiveness.  Refining the procedure technique and limiting the procedure length have significantly improved the safety of nCLE.  Th authors concluded that a structured training program and device improvements to allow more complete mapping of the pancreatic cyst epithelium will be crucial for the widespread adoption of this promising technology.

The authors stated that the use of nCLE is limited by the presence of a significant learning curve.  The surmounting of this learning curve requires not only understanding the technical aspect of setting up the device but also the ability to interpret the image sequences.  While the manufacturer offers online learning modules to help the endosonographer’s learning process, further formal training initiatives are needed.  Another challenge of nCLE is that it can only examine approximately 50 % of the cyst wall lining due to the trajectory of the FNA needle precluding the examination of the cyst wall behind it; therefore, the result may not be completely representative of the pathology of the PCL given that the cyst wall epithelium is known to be heterogenous.

Saghir and colleagues (2021) stated that nCLE is a procedure in which an AQ-Flex nCLE mini-probe is passed via an EUS-FNA needle into a pancreatic lesion to enable sub-surface in-vivo tissue analysis.  In a systematic review and meta-analysis, these researchers examined the effectiveness of EUS-guided nCLE for the diagnosis of pancreatic lesions.  They carried out a comprehensive search of several databases and conference proceedings, including PubMed, Embase, Google-Scholar, Medline, Scopus, and Web of Science databases (earliest inception to March 2020).  The primary outcomes assessed the pooled rate of diagnostic accuracy for nCLE; and the secondary outcomes examined the pooled rate of sensitivity, specificity, PPV, NPV, and adverse events (AEs) of nCLE to diagnose pre-malignant/malignant pancreatic lesions.  A total of 11 studies on 443 patients were included in this analysis.  The pooled rate of diagnostic accuracy of EUS nCLE was 83 % (95 CI: 79 to 87; I2 = 0).  The pooled rate of sensitivity, specificity, PPV and NPV of EUS nCLE were 85.29 % (95 % CI: 76.9 to 93.68; I2 = 85 %), 90.49 % (95 % CI: 82.24 to 98.74; I2 = 64 %), 94.15 % (95 % CI: 88.55 to 99.76; I2 = 68 %), and 73.44 % (95 % CI: 60.16 to 86.72; I2 = 93 %), respectively.  The total AEs rate was 5.41 % (± 5.92) with post-procedure pancreatitis being the most common AE at 2.28 % (± 3.73).  The authors concluded that this study highlighted the rate of diagnostic accuracy, sensitivity, specificity, and PPV for distinguishing pre-malignant/malignant lesions.  These researchers stated that pancreatic lesions need to be further defined with more validation studies to characterize CLE diagnosis criteria and to evaluate its use as an adjunct to EUS-FNA.

Evaluation of the Tumor Vasculature in Gastric and Rectal Carcinomas

Spessotto and colleagues (2017) stated that pCLE is an imaging technique that can perform gIendomicroscopy at subcellular resolution.  These researchers examined the use of pCLE to evaluate tumor angiogenesis in rectal and gastric cancers.  A total of 35 consecutive patients with gastric and 91 with rectal carcinomas underwent endoscopy and pCLE during the same examination.  Vascular assessment was based on vessel shape and size, vessel permeability and blood flow, and allowed the creation of an angiogenic score ranging from 0, for normal vasculature, to 4, for aberrant vasculature.  A significant difference for the presence of vessels with large diameter and defective blood flow was found between rectal and gastric cancers.  Overall, rectal cancers displayed a higher angiogenic score compared to gastric cancers.  Conventional therapy induced a striking reduction in the angiogenic score only in rectal cancer patients.  The authors concluded that these findings suggested that the pCLE technology was suitable for the evaluation of the tumor microvasculature abnormalities.  Thus, the real-time assessment of the vasculature status may represent a promising approach to predict the efficacy of the treatments and improve the clinical management of patients with gastric or rectal carcinomas.  They stated that the results indicated that pCLE has the potential to generate a significant impact and there is a tangible possibility of translating the information gathered into clinical practice.

Elastic-Scattering Spectroscopy

In a pilot study, Rodriguez-Diaz et al (2015) evaluated the potential of elastic-scattering spectroscopy (ESS) for differentiating neoplastic from non-neoplastic polyps during colonoscopy.  A total of 83 patients undergoing screening/surveillance colonoscopy were included in this analysis; ESS spectra of 218 polyps (133 non-neoplastic, 85 neoplastic) were acquired during colonoscopy.  Spectral data were correlated with the classification of biopsy samples by 3 gastro-intestinal (GI) pathologists.  High-dimensional methods were used to design diagnostic algorithms.  Main outcome measure was the diagnostic performance of ESS.  Analysis of spectra from polyps of all sizes (n = 218) resulted in a sensitivity of 91.5 %, specificity of 92.2 %, and accuracy of 91.9 % with a high-confidence rate of 90.4 %.  Restricting analysis to polyps smaller than 1 cm (n = 179) resulted in a sensitivity of 87.0 %, specificity of 92.1 %, and accuracy of 90.6 % with a high-confidence rate of 89.3 %.  Analysis of polyps 5 mm or smaller (n = 157) resulted in a sensitivity of 86.8 %, specificity of 91.2 %, and accuracy of 90.1 % with a high-confidence rate of 89.8 %.  The authors concluded that the findings of this pilot study indicated that ESS permits accurate, real-time classification of polyps as neoplastic or non-neoplastic; ESS is a simple, low cost, clinically robust method with minimal impact on procedure flow, especially when integrated into standard endoscopic biopsy tools.  Performance on polyps 5 mm or smaller indicated that ESS may, in theory, achieve Preservation and Incorporation of Valuable Endoscopic Innovations performance thresholds.  They stated that ESS may one day prove to be a useful tool used in endoscopic screening and surveillance of colorectal cancer.  The main drawbacks of this study were its sample size and retrospective validation used to obtain performance estimates.

In a prospective, analytic study, Grillone and colleagues (2017) evaluated the usefulness of ESS as a diagnostic adjunct to frozen section analysis in patients with diagnosed squamous cell carcinoma of the oral cavity.  Subjects for this single-institution, institutional review board (IRB)-approved study were recruited from among patients undergoing surgical resection for squamous cell cancer of the oral cavity.  A portable ESS device with a contact fiberoptic probe was used to obtain spectral signals; 4 to 10 spectral readings were obtained on each subject from various sites including gross tumor and normal-appearing mucosa in the surgical margin.  Each reading was correlated with the histopathologic findings of biopsies taken from the exact location of the spectral readings.  A diagnostic algorithm based on multi-dimensional pattern recognition/machine learning was developed.  Sensitivity and specificity, error rate, and area under the curve were used as performance metrics for tests involving classification between disease and non-disease classes.  A total of 34 subjects were enrolled in the study; 176 spectral data point/biopsy specimen pairs were available for analysis.  ESS distinguished normal from abnormal tissue, with a sensitivity ranging from 84 % to 100 % and specificity ranging from 71 % to 89 %, depending on how the cut-off between normal and abnormal tissue was defined (i.e., mild, moderate, or severe dysplasia).  There were statistically significant differences in malignancy scores between histologically normal tissue and invasive cancer and between non-inflamed tissue and inflamed tissue.  The authors concluded that this was the first study to evaluate the effectiveness of ESS in guiding mucosal resection margins in oral cavity cancer.  ESS provided fast, real-time assessment of tissue without the need for pathology expertise; it appeared to be effective in distinguishing between normal mucosa and invasive cancer and between "normal" tissue (histologically normal and mild dysplasia) and "abnormal" tissue (severe dysplasia and carcinoma in-situ) that might require further margin resection.  Moreover, they stated that further studies, however, are needed with a larger sample size to validate these findings and to determine the effectiveness of ESS in distinguishing visibly and histologically normal tissue from visibly normal but histologically abnormal tissue.

Rodriguez-Diaz et al (2022) noted that colonoscopic screening and surveillance for CRC could be made safer and more efficient if endoscopists could predict histology without the need to biopsy and perform histopathology on every polyp.  Elastic-scattering spectroscopy, by means of fiberoptic probes integrated into standard biopsy tools, could evaluate, both in-vivo and in real-time, the scattering and absorption properties of tissue related to its underlying pathology.  In a prospective study, these researchers examined the potential of ESS to predict polyp pathology accurately.  They obtained ESS measurements from patients undergoing screening/surveillance colonoscopy using an ESS fiberoptic probe integrated into biopsy forceps.  The integrated forceps were used for tissue acquisition, following current standards of care (SOC), and optical measurement.  All measurements were correlated to the index pathology.  A machine-learning (ML) model was then applied to measurements from 367 polyps in 169 patients to prospectively examine its predictive performance.  The model achieved sensitivity of 0.92, specificity of 0.87, NPV of 0.87, and high-confidence rate (HCR) of 0.84 for distinguishing 220 neoplastic polyps from 147 non-neoplastic polyps of all sizes.  Among 138 neoplastic and 131 non-neoplastic polyps of 5 mm or less, the model achieved sensitivity of 0.91, specificity of 0.88, NPV of 0.89, and HCR of 0.83.  The authors concluded that findings of this study demonstrated that ESS is a viable endoscopic platform for real-time polyp histology, especially for polyps of 5 mm or less.  These researchers stated that they believe that ESS continues to be a promising tool for real-time histology (RTH) assessment of colorectal polyps and that their continued efforts in developing this technology will provide a clinically friendly, minimally obtrusive endoscopic tool for in-situ CRC management.

These investigators stated that future work will focus on addressing the limitations of this trial.  First, as in any approach using ML, ongoing acquisition of labeled data will serve to enrich the development and validation of prediction models based on ESS spectral measurements.  Second, these researchers plan to further analyze the capability of ESS in predicting the histology of sessile serrated adenomas/polyps (SSAPs).  These types of polyps were excluded from the training process because of their under-representation in their data set as well as the inherent challenge presented by sessile serrated polyps.  While morphologically similar to hyperplastic polyps, SSAPs are clinically managed as pre-malignant because of an increased risk of future neoplasia.  A positive finding in this study was that ESS classified the majority of these lesions as “neoplastic”.  Additional studies with a larger representation of this specific pathology grouping as well as other unique pathologies of interest, would serve to develop more robust prediction models and improve histology assessments across all pathology groupings.  Finally, this study is the result of prospective data analysis, not in-situ predictions.  To this end, these investigators are designing clinical studies to examine ESS performance with prediction made in-situ and in real-time using the Preservation and Incorporation of Valuable Endoscopic Innovations (PIVI) thresholds as benchmarks, with a re-engineered system that will be more compact, user-friendly, and easily deployable than predecessor technologies.

Endoscopic Retrograde Cholangio-Pancreatography (ERCP) with Optical Endomicroscopy for Evaluation of Biliary Lesions

Yoon and Brugge (2013) noted that differentiating between malignant and benign bile duct strictures is often challenging.  Endoscopic retrograde cholangio-pancreatography (ERCP) with brush cytology and/or endo-biliary forceps biopsy is routinely performed.  Advanced cytologic methods such as fluorescence in-situ hybridization (FISH) or digital image analysis increases the sensitivity of cytology.  Endoscopic ultrasonography enables detailed examination of tissues surrounding the bile duct stricture and offers the advantage of fine-needle aspiration.  Intra-ductal ultrasonography enables detailed evaluation of bile duct wall layers, and cholangioscopy offers direct visualization of the bile duct lesions.  Novel techniques of probe-based CLE (pCLE) and OCT have introduced the era of in-vivo histology.

Moon et al (2014) stated that new technological developments in ERCP for diagnosis and treatment have been slow to progress.  However, several informative study results were presented during the 2014 Digestive Disease Week in specific ERCP areas (e.g., prevention of post-ERCP pancreatitis using non-steroidal anti-inflammatory drugs and pancreatic duct stenting).  Novel and interesting study results regarding pre-operative stent selection for peri-ampullary tumors, metal stents for hilar stricture or for prevention of duodenal reflux, and intra-ductal biliary tumor ablation using photodynamic therapy or radiofrequency ablation were discussed.  Study results presented at the meeting regarding single-operator cholangioscopy using the SpyGlass system or direct per-oral cholangioscopy have indicated the possibility of future development.  The authors noted that results using per-oral pancreatoscopy and CLE for biliary lesions (including strictures) were also presented.

Slivka and colleagues (2015) noted that characterization of indeterminate biliary strictures (IDBSs) remains problematic.  Tissue sampling is the criterion standard for confirming malignancy but has low sensitivity.  Probe-based CLE showed excellent sensitivity in a registry; however, it has not been validated in a prospective study.  In a prospective, international, multi-center study, these researchers validated pCLE in real time during ERCP for IDBSs.  A total of 136 patients with IDBSs were included in this analysis.  Investigators provided a presumptive diagnosis based on the patient history, ERCP impression, and pCLE during the procedure before and after tissue sampling results were available.  A presumptive diagnosis also was made separately by a blinded investigator during ERCP and after tissue sampling to estimate care without pCLE.  Follow-up was at least 6 months.  Main outcome measures included accuracy, sensitivity, and specificity during ERCP alone, ERCP with pCLE, and ERCP with pCLE and tissue sampling.  A total of 112 patients were evaluated (71 with malignant lesions).  Tissue sampling alone was 56 % sensitive, 100 % specific, and 72 % (95 % CI: 63 % to 80 %) accurate; pCLE with ERCP was 89 % sensitive, 71 % specific, and 82 % (95 % CI: 74 % to 89 %) accurate.  After tissue sampling returned, strictures could be characterized with 88 % (95 % CI: 81 % to 94 %) accuracy.  The authors concluded that pCLE provided a more accurate and sensitive diagnosis of cholangiocarcinoma compared with tissue sampling alone.  They stated that incorporation of pCLE into the diagnostic armamentarium of patients with indeterminate biliary strictures may allow for a more accurate assessment, potentially reducing delays in diagnosis and costly repeat testing.  These preliminary findings need to be validated by well-designed studies.

Tabibian et al (2015) stated that endoscopic evaluation of IDBSs has evolved considerably since the development of flexible fiberoptic endoscopes over 50 years ago; ERCP was introduced nearly 10 years later and has since become the mainstay of therapy for relieving obstruction of the biliary tract.  However, long-standing methods of ERCP-guided tissue acquisition (i.e., biliary brushings for cytology and intra-ductal forceps biopsy for histology) have demonstrated disappointing performance characteristics in distinguishing malignant from benign etiologies of IDBSs.  The limitations of these methods have thus helped drive the search for novel techniques to enhance the evaluation of IDBSs and thereby improve diagnosis and clinical care.  These modalities include, but are not limited to, EUS, intra-ductal ultrasound, cholangioscopy, confocal endomicroscopy, and OCT.  However, there is currently insufficient evidence that confocal endomicroscopy used in conjunction with ERCP provides better health outcomes.

An UpToDate review on “Endoscopic retrograde cholangiopancreatography: Indications, patient preparation, and complications” (Loperfido and Costamagna, 2015) does not mention the adjunctive use of optical endomicroscopy/confocal endomicroscopy.

Robles-Medranda (2016) stated that CLE permits in-vivo microscopy evaluation during endoscopic procedures.  It can be used in all the parts of the GI tract including the esophagus, stomach, small bowel, colon, biliary tract via ERCP and pancreas via EUS.  Many studies showed a high correlation of results between CLE and histopathology in the diagnosis of GI lesions; with accuracy in about 86 % to 96 %.  Despite histopathology remains the gold-standard technique for final diagnosis of any diseases; a considerable number of misdiagnosis rate could be present due to many factors (e.g., interpretation mistakes, biopsy site inaccuracy, or number of biopsies).  Theoretically; the diagnostic accuracy of CLE could help in a daily practice to improve diagnosis and treatment of the patients.  However, it is still not routinely used in the clinical practice due to many factors (e.g., cost of the procedure, lack of codification and reimbursement in some countries, absence of standard of care indications, availability, physician image-interpretation training, medico-legal problems, and the role of the pathologist).  The authors stated that these limitations are relative, and solutions could be found based on new researches focused to solve these barriers.

Confocal Laser Endomicroscopy in the Detection of Colorectal Mucosa Microinflammation in Individuals with Irritable Bowel Syndrome

Robles-Medranda and colleagues (2020) noted that irritable bowel syndrome (IBS) is considered to be a functional disease, but recent data indicated measurable organic alterations.  In a prospective, controlled, non-randomized, single-blind study, these researchers examined the presence of colorectal mucosa microinflammation in-vivo via probe-based CLE (pCLE) and histological evaluation in IBS patients.  pCLE images and targeted biopsy of each colon segment obtained during colonoscopies of IBS patients and controls were analyzed for inflammatory changes.  Biopsies were classified using the Geboes scale, and the odds ratio (OR) and overall diagnostic accuracy were calculated.  During the 15-month study period, a total of 37 patients were allocated to each group.  The mean age was 53.1 ± 14.3 years; 64.9 % were women.  Signs of colonic mucosa inflammation were evident on 65.8 % of pCLE images from IBS patients compared to 23.4 % of images from controls (OR 6.28; 4.14 to 9.52; p < 0.001).  In total, 20/37 patients had microinflammation via pCLE in greater than or equal to 3 colon segments in the IBS group, compared to 1/37 in the control group.  A Geboes score of greater than 0 was attributed to 60.8 % of biopsies from patients in the IBS group compared to 27.5 % of biopsies from the control group.  The sensitivity, specificity, PPVs, NPVs, observed and inter-rater agreement of pCLE-detected inflammatory changes in IBS using histology as gold standard were 76 %, 91 %, 76 %, 91 %, 86.5 %, and 66.8 %, respectively.  The authors concluded that patients with IBS have a 6-fold higher prevalence of colorectal mucosa microinflammation than healthy controls.  These researchers stated that pCLE might be a reliable method to detect colorectal mucosa microinflammation in IBS patients.  These researchers hoped that this study will encourage research in the field of microinflammation in the colons of patients with IBS.  They stated that further prospective, multi-center studies with larger cohorts are needed to validate and ensure the utility of pCLE in mapping inflammation in the colonic mucosa in patients with IBS.  Furthermore, it would be interesting to examine pCLE inflammatory patterns by IBS subtypes and IBS severity of symptoms and determine the stage after initial symptom onset at which pCLE can accurately detect inflammation in IBS patients.

The authors stated that this study was limited by the small IBS population and its single-center nature.  Although the Geboes score was developed for judging ulcerative colitis inflammation, they used this score to assess inflammation in the colonic mucosa of patients with IBS.  In addition, an objective measurement of IBS severity using validated scales was not performed in this trial.  The present study showed the potential benefit of pCLE for identifying signs of microinflammation in each segment of the colon in patients with IBS in addition to histopathological deviations and opened a discussion regarding evaluating other specific treatments, such as budesonide and mesalazine, for IBS patients with documented low-grade microinflammation.

Confocal Laser Endomicroscopy in the Diagnosis of Indeterminate Biliary Strictures and Pancreatic Lesions

Martínek and colleagues (2020) noted that an accurate histopathological diagnosis of indeterminate biliary strictures and pancreatic lesions is challenging because of insufficient quality of tissue specimen taken during ERCP (brush cytology), cholangioscopy (biopsies) or endosonography (EUS, fine-needle aspiration biopsy [FNAB]).  Confocal laser endomicroscopy (CLE) allows virtual histopathological diagnosis with the potential to either replace or increase the diagnostic yield of standard histopathological diagnosis in patients presenting with biliary strictures and pancreatic lesions.  In a prospective, pilot study, these researchers examined the diagnostic yield of standard histopathology compared to CLE in patients referred for cholangioscopy or for EUS of the pancreas; and assessed the cost of CLE in these indications.  CLE was performed (during cholangioscopy or EUS), followed by standard tissue sampling.  CLE-based diagnosis was compared with standard histopathology/cytology.  CLE probe was introduced through the working channel of the cholangioscope or through the FNAB needle.  A total of 23 patients were enrolled (12 women, mean age of 61 years); 13 patients underwent cholangioscopy and 10 patients underwent EUS.  Cholangioscopy: CLE diagnosed correctly all 4 malignant strictures (histology 2 of them only as 2 patients had insufficient quality of the tissue specimen).  Agreement between standard histopathology and CLE was achieved in 85 %.  EUS: All 3 cases of pancreatic cancer were correctly diagnosed by both CLE and FNAB.  All remaining (pre-malignant and benign) lesions were also correctly diagnosed by both methods.  The cost of CLE examination was higher compared to FNAB but comparable with tissue sampling during digital cholangioscopy.  The authors concluded that CLE demonstrated sufficient diagnostic accuracy in patients with indeterminate biliary strictures or pancreatic lesions and, thus, might improve diagnostic accuracy or even replace standard histopathology in these indications.

Mi et al (2022) stated that pCLE, also known as optical biopsy, is a new endoscopic technique that provides real-time magnification of 1,000 × microscopic tissue information to diagnose indeterminate biliary strictures.  Tissue sampling by ERCP is routinely carried out to examine indeterminate biliary strictures.  In a meta-analysis, these researchers examined the accuracy of pCLE and tissue sampling by ERCP in the diagnosis of indeterminate biliary strictures.  A total of 18 articles were identified from 2008 to 2021 via Embase, PubMed, Web of Science, and Cochrane library databases.  The summary estimates for the pCLE diagnosis of indeterminate biliary strictures were: sensitivity 0.88 (95 % CI: 0.84 to 0.91); specificity 0.79 (95 % CI: 0.74 to 0.83); and DOR of 24.63 (95 % CI: 15.76 to 38.48).  The summary estimates for tissue sampling by ERCP diagnosis for indeterminate biliary strictures were: sensitivity 0.54 (95 % CI: 0.49 to 0.59); specificity 0.96 (95 % CI: 0.94 to 0.98); and DOR of 11.31 (95 % CI: 3.90 to 32.82).  The area under the sROC curve of pCLE diagnosis of indeterminate biliary strictures was 0.90 higher than 0.65 of tissue sampling by ERCP.  The authors concluded that pCLE is a better approach than tissue sampling by ERCP for the diagnosis of indeterminate biliary strictures by providing real-time microscopic images of the bile ducts.  Moreover, these researchers stated that pCLE is expensive compared to conventional ERCP, and future studies are needed to confirm the cost-effectiveness.  They stated that the pCLE has the potential to overcome the limitations inherent in tissue sampling by ERCP by providing real-time microscopic images of the bile ducts to make an accurate diagnosis of indeterminate biliary strictures.

The authors stated that this study had several drawbacks.  First, as most of the included studies were retrospective studies, and there was potential selective bias needing further prospective studies to confirm.  Second, the poor quality of some of the included studies may affect the findings of the meta-analysis.  Third, some patients in some of the included studies were followed-up rather than pathology as the gold standard.

Confocal Laser Endomicroscopy in the Diagnosis of Liver Cancer

Zheng and colleagues (2020) noted that accurate identification of tumor tissues and their margins are still challenging for conventional clinical imaging methods during liver cancer surgery.  In a preliminary study, dual-band CLE combined with image mosaic was used to guide liver cancer surgery.  In the experiments with mice bearing orthotropic liver tumor, CLE can accurately detect the tumors and identify their margins with 2 excitation wavelengths of 488 nm and 660 nm by clinically available dyes fluorescein sodium (FS) or indocyanine green (ICG).  The mosaic CLE images enlarged the imaging field and detected the liver tumor margins more accurately.  Normal liver tissues fluorescence intensity of CLE images was significantly higher than that of tumor tissues in the same tumor-bearing mice (p < 0.0001).  The authors concluded that the findings of this preliminary study showed that dual-band CLE imaging is a promising method to identify liver tumor tissues and margins, which has the prospect of clinical application and aids in achieving intra-operative radical resection.

Confocal Laser Endomicroscopy in the Differentiation of Parenchymal Lung Diseases from Normal Alveoli

Bondesson and colleagues (2020) stated that pCLE provides real time videos of auto-flourescent elastin structures within the alveoli.  With it, multiple changes in the elastin structure due to different diffuse parenchymal lung diseases (DPLDs) have previously been described.  However, these evaluations have mainly relied on qualitative evaluation by the examiner and manually selected parts post-examination.  These researchers developed a fully automatic method for quantifying structural properties of the imaged alveoli elastin and carried out a preliminary examination of their diagnostic potential.  A total of 46 patients underwent pCLE, of which 38 were divided into 4 groups categorizing DPLDs; 8 patients were imaged in representative healthy lung areas and used as control group.  Alveolar elastin structures were automatically segmented with a trained machine learning algorithm and subsequently evaluated with 2 methods developed for quantifying the local thickness and structural connectivity.  The automatic segmentation algorithm performed generally well and all 4 patient groups showed statistically significant differences with median elastin thickness, SD of thickness and connectivity compared to the control group.  The authors concluded that alveoli elastin structures could be quantified based on their structural connectivity and thickness statistics with a fully-automated algorithm and initial results highlighted its potential for distinguishing parenchymal lung diseases from normal alveoli.

The authors stated that this study had several drawbacks.  The primary factors were the absence of a histological comparison to pCLE snapshots from the same regions in the lungs resulting in some uncertainty as to whether pCLE could offer “optical biopsy”.  Although the automatic segmentation’s performance was satisfactory on most snapshots a larger measurement set was needed to examine its capabilities on different DPLDs and still unseen descriptors by the machine learning algorithm.  Given that the snapshots were taken from a very small fraction of the whole lung this could lead to a poor morphological consistency of the evaluation method.  However, this was mitigated already by the use of a guiding tool such as high resolution computer tomography (HR-CT) to select representative areas of diseased tissue.  Variability can also be decreased by utilizing average parameter values generated from multiple snapshots of different representative areas for every patient.  Since pCLE is an in-vivo imaging technique complementing bronchoscopy, it does not suffer the same increase of risk associated with invasive techniques such as biopsy and could be used to examine multiple sections.  So far, procedures performed in multiple studies have shown no severe side effects other than minor bleeding in 3 out of 42 patients making it ideal for longitudinal studies.  As the presented method evaluates 3D structures on 2D images, unattached structures such as macrophages in the alveolar space could potentially overlap and appeared to be part of the structure of interest.  Furthermore, the work-flow relied on the operating pulmonologist to ensure a proper orientation of the probe relative to the area of interest.  The 1st problem was partly mitigated in this study by training the machine learning method to distinguish background structures from structures of interest, but more data would likely be needed to test the model’s generality.  These researchers stated that future studies can further incorporate macrophages as a 3rd segmentation class to expand on the analysis potential of the method.  Consideration can also be taken by the performing pulmonologists if structures are overlapping or probe orientation needs adjustment when selecting snapshot.  Validation cohorts would be needed to confirm the clinical relevance of this method.  In this initial study, these investigators have focused on the distinction between normal and DPLD alveoli due to the small sample size.  A next step would be to establish respective value ranges for different types of pathologies aiming to further evaluate the presented methods’ diagnostic potential.

Confocal Laser Endomicroscopy for the Assessment of Safe Surgical Margins During Laryngectomy

In a pilot study, Sievert and colleagues (2021) examined the feasibility of intra-operative assessment of safe margins with CLE during planned partial or total laryngectomy.  A total of 8 patients with confirmed larynx squamous cell carcinoma (SCC) and planned partial or total laryngectomy were included in this study in March 2020.  Two head and neck surgeons and 1 pathologist were asked to classify carcinoma or healthy epithelium in a sample of 94 representative sequences (5.640 images), blinded to the histological results (H&E staining).  Healthy mucosa areas showed epithelium with cells of uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture.  CLE optical biopsy of SCC demonstrated a disorganized arrangement of variable cellular morphology.  These investigators calculated an accuracy, sensitivity, specificity, PPV, and NPV of 80.1 %, 72.3 %, 87.9 %, 85.7 %, and 76.1 %, respectively.  A distinct transition between healthy appearing tissue and suspicious lesions could also be detected.  The authors concluded that CLE can be easily integrated into the intra-operative setting, generate real-time, in-vivo microscopic images of the larynx for evaluation and demarcation of cancer.  Moreover, these researchers stated that if validated in further studies, CLE could eventually contribute to a less radical approach by enabling a more precise evaluation of the cancer margin.

Confocal Laser Endomicroscopy for the Detection of Neoplasia in Biliary Strictures Associated with Primary Sclerosing Cholangitis

Tanisaka and associates (2020) noted that the accurate diagnosis of biliary strictures remains problematic.  In a prospective, single-center study, these researchers examined the use of pCLE under the direct view of per-oral cholangioscopy (POCS) for the diagnosis of biliary strictures; consecutive patients with biliary strictures were included.  These investigators examined sensitivity, specificity, and accuracy to diagnose malignancy for ERCP alone, POCS, pCLE under the direct view of POCS, and tissue sampling under the direct view of POCS.  A total of 30 patients (17 with malignant lesions) were prospectively enrolled.  ERCP alone showed 88.2 % sensitivity, 46.2 % specificity, and 70 % (95 % CI: 52.1 % to 83.3 %) accuracy.  POCS showed 100 % sensitivity, 76.9 % specificity, and 90 % (95 % CI: 74.4 % to 96.5 %) accuracy.  pCLE under the direct view of POCS showed 94.1 % sensitivity, 92.3 % specificity, and 93.3 % (95 % CI: 78.7 % to 98.8 %) accuracy.  Tissue sampling under the direct view of POCS showed 82.4 % sensitivity, 100 % specificity, and 90 % (95 % CI: 74.4 % to 96.5 %) accuracy.  The authors concluded that pCLE under the direct view of POCS provided highly accurate and sensitive characterization of biliary strictures and showed the potential for more diagnostic reliability and reduction of delays in diagnosis.  Moreover, these researchers stated that further investigations with larger study populations are needed to compare with direct view and fluoroscopy guidance of pCLE.

The authors stated that this study had several drawbacks.  First, this was a single-center study, and the number of patients was small (n = 30) to show the accuracy and usefulness of pCLE under the direct view of POCS.  These investigators intended to report the feasibility of pCLE under the direct view of POCS as soon as possible.  Although it showed good outcomes, more cases or a larger, multi-center studies are needed.  Second, tissue sampling under fluoroscopy and other cytology such as brush cytology were not carried out in this trial.  These modalities are currently used for stricture characterization.  These researchers should have used and compared the results of these modalities with those of tissue sampling under the direct view of POCS.  Third, the Miami classification was used in this study; however, the Paris classification should be used in the future.  Furthermore, the authors did not compare pCLE under the direct view of POCS to pCLE under fluoroscopic guidance with ERCP

Han and colleagues (2021) stated that patients with primary sclerosing cholangitis (PSC) and dominant biliary strictures carry increased risk for the development of cholangiocarcinoma.  Although ERCP-based techniques including brush cytology and intra-ductal biopsy sampling represent 1st-line tissue sampling methods for dominant strictures, sensitivity is low; pCLE offers microscopic-level imaging of sub-epithelial biliary mucosa.  Because data regarding the use of pCLE in PSC are limited, these investigators examined its diagnostic performance in dominant strictures in a prospective, multi-center study.  ERCP with pCLE was carried out with use of the Miami classification (2+ criteria for malignant diagnosis) and Paris classification.  Final malignant diagnoses required histopathologic confirmation, and benign diagnoses required a minimum of 1 year of follow-up without development of cancer.  A total of 59 patients (mean age of 49 years; 59 % men) with 63 strictures were included in the study.  Stricture locations included the common bile duct (31.7 %), bifurcation (22.2 %), and common hepatic duct (19 %); 7 patients (11.9 %) were found to have cholangiocarcinoma.  The sensitivity and specificity of pCLE was 85.7 % (95 % CI: 42.1 to 99.6) and 73.1 % (95 % CI: 58.9 to 84.4), respectively.  Within specific stricture locations, the highest sensitivity was observed at the bifurcation (100 %; 95 % CI: 2.5 to 100) and the right hepatic duct (100 %; 95 % CI: 29.2 to 100).  The lowest sensitivities were observed at the common bile duct (25 %; 95 % CI: 5.5 to 57.2) and the left hepatic duct (28.6 %; 95 % CI: 3.7 to 70.9).  The authors concluded that pCLE had a high sensitivity in detecting cholangiocarcinoma, but technical aspects of the probe may limit evaluation in the common bile duct and left hepatic duct.  Moreover, these researchers stated that further investigation is needed to examine the role of pCLE in the algorithm of excluding neoplasia in biliary strictures associated with PSC.

Confocal Laser Endomicroscopy for the Diagnosis of Clinical Complete Response After Chemoradiation for Rectal Cancer

Safatle-Ribeiro and colleagues (2021) stated that neoadjuvant chemo-radiotherapy (nCRxt) followed by radical surgery is the optimal treatment for advanced rectal adenocarcinoma.  Patients with clinical complete response (cCR) may be followed closely without immediate surgery.  Probe-based confocal laser endomicroscopy is a real-time in-vivo method that allows acquisition of optical biopsies with 1,000 times magnification, evaluating both epithelial and vascular patterns.  These researchers examined the role of pCLE in the diagnosis of cCR after nCRxt for advanced rectal adenocarcinoma; pCLE was carried out in 47 patients with locally advanced rectal adenocarcinoma (T3/T4, or N+) who underwent nCRxt (5-fluorouracil, 5,040 cGy).  A total of 27 (57.5 %) patients were men, and the mean age was 62.8 years; 37 had partial response (PR) confirmed by pCLE; 10 (21.3 %) patients had good endoscopic response and presented small ulcer (n = 5) or residual scar (n = 5).  After nCRxt, the essential features to differentiate malignancy from post-radiation alterations at pCLE were the presence of irregular crypts, budding, back-to-back glands, cribriform pattern, increased vessel/crypt ratio, and fluorescein leakage.  A scoring system was created considering these epithelial and vascular features, with high accuracy for differentiating patients with complete response from those with residual neoplasia (p < 0.00001).  pCLE sensitivity, specificity, PPV, NPV, and accuracy were 100 %, 71.4 %, 95.2 %, 100 %, and 95.7 %, respectively.  The authors concluded that pCLE evaluation of epithelial and vascular features may improve the diagnosis of cCR and may alter patient management; and pCLE might be valuable for identifying patients with advanced rectal cancer who will benefit from watch and wait strategy, avoiding immediate surgical treatment.

Confocal Laser Endomicroscopy for the Diagnosis of Functional Dyspepsia (Leaky Gut Disorder)

Nojkov and associates (2020) stated that duodenal epithelial barrier impairment and immune activation may play a role in the pathogenesis of functional dyspepsia (FD).  In a pilot study, these researchers examined the duodenal epithelium of patients with FD (n = 16) and healthy individuals (n = 18)for detectable microscopic structural abnormalities.  They employed esophagogastroduodenoscopy (EGD) enhanced with duodenal CLE and mucosal biopsies in patients with FD and healthy controls.  Blinded CLE images analysis evaluated the density of epithelial gaps (cell extrusion zones), a validated endoscopic measure of the intestinal barrier status.  Analyses of the biopsied duodenal mucosa included standard histology, quantification of mucosal immune cells/cytokines, and immunohistochemistry for inflammatory epithelial cell death called pyroptosis.  Trans-epithelial electrical resistance (TEER) was measured using Ussing chambers.  Epithelial cell-to-cell adhesion proteins expression was evaluated by real-time polymerase chain reaction (RT-PCR).  Patients with FD had significantly higher epithelial gap density on CLE in the distal duodenum than that of controls (p = 0.002).  These mucosal abnormalities corresponded to significant changes in the duodenal biopsy samples of patients with FD, compared with controls, including impaired mucosal integrity by TEER (p = 0.009) and increased number of epithelial cells undergoing pyroptosis (p = 0.04).  Reduced TEER inversely correlated with the severity of certain dyspeptic symptoms.  Furthermore, patients with FD demonstrated altered duodenal expression of claudin-1 and interleukin-6.  No differences in standard histology were found between the groups.  The authors concluded that they reported for the 1st time in patients with FD, distal duodenal structural changes detectable on pCLE, confirmed the presence of duodenal epithelial barrier impairment using an ex-vivo model, and reported evidence of increased duodenal pyroptosis.  These findings introduced pCLE as a potential diagnostic biomarker, capable of identifying patients with FD with impaired duodenal epithelial structure and function.  These researchers stated that additional larger studies are needed to further define the pCLE characteristics of the duodenal mucosa in patients with FD.  Based on these findings, it is tempting to speculate that in a subset of patients with FD, environmental or luminal factors may cause junctional protein damage, leading to impaired barrier integrity and entry of luminal irritants/chemicals into the mucosa.  The resulting epithelial immune activation and cell death could lead to increased epithelial gaps, enabling mucosal microbial trespass and ultimately, alternations in gut function and sensation; further studies are needed to validate this hypothetical conceptual model.

The authors concluded that this pilot study had several drawbacks.  First, these investigators enrolled a relatively small number of patients (n = 16) from a single tertiary care center.  This could have led to under-estimation or over-estimation of the outcomes evaluated and may influence the generalizability of these findings to patients from primary or secondary care settings.  Although these researchers tried to control for potential environmental confounders such as medications and diet, it was possible that differences in environmental exposures could have affected these findings.  Second, not every subject completed every aspect of the study.  Performance of EGD with CLE, followed by acquisition of multiple duodenal biopsies and immediate tissue processing created practical challenges that made it impossible to conduct all aspects of the study in every subject.  This trial was powered to detect a difference in CLE; thus, secondary measurements in this exploratory study may not reach 80 % power and require further investigation.  Furthermore, this study was purely observational and cannot discern whether the duodenal epithelial barrier disruption was a cause or effect of FD.  In addition, these researchers could not say whether the increased gap density by pCLE and/or abnormalities on tissue-based analyses could predict response to specific treatments for FD.  These investigators stated that these preliminary findings require confirmation in larger, well-controlled clinical trials.

Tack and colleagues (2021) stated that for up to the past 20 years, pathophysiological research in FD focused on gastric sensorimotor dysfunction underlying symptom generation.  Recent pathophysiological research has focused on low-grade inflammation in the duodenal mucosa.  Emerging evidence confirms a loss of mucosal integrity in the duodenum in FD, and this was confirmed in a CLE study demonstrating altered mucosal barrier function and pyroptosis.  The authors concluded that this technique may help to establish underlying mechanisms and evaluate novel therapeutic approaches to FD.  They noted that new diagnostic tools such as pCLE offers the potential to evaluate epithelial gaps as biomarker for subgroups of FD patients, with a specific underlying pathophysiology and as a tool to evaluate and develop current or novel therapeutic approaches in FD.

Confocal Laser Endomicroscopy for the Diagnosis of Head and Neck Squamous Cell Carcinoma

Sievert and colleagues (2021) stated that CLE, with a magnification of up to 1,000 ×, offers the possibility to visualize intercellular spaces in-vivo.  These investigators provided an overview of the current research on CLE in the diagnosis of the head and neck SCC (HNSCC).  They carried out a systematic bibliographic research in the following online databases: PubMed, Medline, Thompson Reuters Web of Science, SPIE using the following keywords: confocal laser endomicroscopy, CLE, endomicroscopy, head and neck, larynx.  Evaluation of the scientific relevance according to defined criteria.  All studies were analyzed concerning the clinical application, clinical findings, and computer-aided data processing.  The authors concluded that the recently published data suggested that CLE has a high potential to improve the diagnosis of malignant mucosal lesions in the upper aerodigestive tract.

Sievert and colleagues (2022) noted that CLE is an optical imaging technique that allows in-vivo, real-time, microscope-like images of the upper aerodigestive tract's mucosa.  The assessment of morphological tissue characteristics for the correct differentiation between healthy and malignant suspected mucosa requires strict evaluation criteria.  These researchers validated an 8-point score for the correct assessment of malignancy.  They performed confocal laser endomicroscopy between March and October 2020 in 13 patients resulting in 197 sequences (11.820 images) originated from the marginal area of pharyngeal and laryngeal carcinomas.  Specimens were taken at corresponding locations and analyzed in H&E staining as a standard of reference.  A total of 6 examiners examined the sequences based on a scoring system; they were blinded to the histopathological examination.  The primary endpoints were sensitivity, specificity, and accuracy; and secondary endpoints were inter-rater reliability and receiver operator characteristics.  Healthy mucosa showed epithelium with uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture; CLE of malignant cells demonstrated a disorganized arrangement of variable cellular morphology.  These investigators calculated an accuracy, sensitivity, specificity, PPV, and NPV of 83.2 %, 81.3 %, 85.5 %, 86.7 %, and 79.7 %, respectively, with a κ-value of 0.64, and an area under the curve of 0.86.  The authors concluded that the findings of this study confirmed that this scoring system was applicable in the laryngeal and pharyngeal mucosa to classify benign and malignant tissue.  A scoring system based on defined and reproducible characteristics could aid in translating this experimental method to broad clinical practice in head and neck diagnosis.  Moreover, these researchers stated that CLE is a promising imaging technology that may improve the non-invasive characterization of HNSCC.  A standardized classification system could improve diagnostic accuracy and consistency of results among examiners.

The authors stated that CLE has, however, as a method with experimental status, limitations intrinsic to this technology and independent of the classification system used.  As the probe-based CLE has only a penetration depth of 55 to 65 um, it is not possible to evaluate the deeper margins in muscle and fascia.  Indeed, mucosal epithelium is at its thinnest (floor of the mouth) over 100 um, which formally makes the differentiation between carcinoma in-situ and invasive carcinoma, as well as infiltration of submucosa, muscle and fascia very difficult.  Direct evaluation of these deeper layers with CLE will need to be examined in the future.  These investigators stated that CLE has a considerable potential to aid intra-operative characterization of mucosal regions in the head and neck, dependent on further validation in clinical trials.

Dittberner and associates (2021) noted that white-light endoscopy and microscopy combined with histological analysis is currently the mainstay for intra-procedural tissue diagnosis during pan-endoscopy for head and neck cancer.  However, taking biopsies leads to selection bias, ex-vivo histopathology is time-consuming, and the advantages of in-vivo intra-operative decision-making cannot be used.  These investigators stated that CLE has the potential for a rapid and histological assessment in the head and neck operating room.  Between July 2019 and January 2020, a total of 13 patients (69 % men, median age of 61 years) with newly diagnosed head and neck cancer (T3/T4: 46 %) underwent fluorescein-guided pan-endoscopy.  CLE was carried out from both the tumor and margins followed by biopsies from the CLE spots.  The biopsies were processed for histopathology.  The CLE images were ex-vivo classified blinded with a CLE cancer score (DOC score).  The classification was compared to the histopathological results.  Median additional time for CLE during surgery was 9 mins.  A total of 2,565 CLE images were taken (median CLE images: 178 per patient; 68 per biopsy; evaluable 87.5 %).  The concordance between histopathology and CLE images varied between the patients from 82.5 % to 98.6 %.  The sensitivity, specificity, and accuracy to detect cancer using the classified CLE images was 87.5 %, 80.0 %, and 84.6 %, respectively.  The PPV and NPV were 87.0 % and 80.0 %, respectively.  The authors concluded that CLE with a rigid hand-held probe was easy and intuitive to handle during pan-endoscopy.  As next step, the high accuracy of ex-vivo CLE image classification for tumor tissue suggested the validation of CLE in-vivo.  This will evolve CLE as a complementary tool for in-vivo intra-operative diagnosis during pan-endoscopy.  These researchers also noted that the presented accurate ex-vivo classification results have to be validated in further in-vivo studies.  They stated that CLE appeared to be a versatile technology enabling a more precise intra-operative tumor staging by better evaluation of the tumor margins.  These preliminary findings from a pilot study need to be validated by well-designed studies.

Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Head and neck cancers” (Version 3.2021) does not mention confocal laser endomicroscopy as a management tool.

Confocal Laser Endomicroscopy for the Diagnosis and Monitoring of Urogenital Schistosomiasis

Xie and colleagues (2021) stated that urogenital schistosomiasis remains a major global challenge.  Optimal management of this infection depends upon imaging-based assessment of sequelae.  Although established imaging modalities such as US, X-ray, MRI, NBI, and CT have been used to determine tissue involvement by urogenital schistosomiasis, newer refinements in associated technologies may lead to improvements in patient care.  Moreover, application of investigational imaging methods such as CLE and 2-photon microscopy in animal models of urogenital schistosomiasis are likely to contribute to the understanding of this infection's pathogenesis.

Confocal Laser Endomicroscopy for the Diagnosis and Staging of Lung Cancer

Wijmans and associates (2019) stated that diagnosing lung cancer in the absence of endobronchial abnormalities is challenging; nCLE enables real-time microscopic imaging of cells.  These researchers examined the feasibility and safety of using nCLE for real-time identification of lung cancer.  In patients with suspected or proven lung cancer scheduled for EUS, lung tumors and mediastinal lymph nodes were imaged with nCLE before fine-needle aspiration (FNA) was carried out.  nCLE lung cancer characteristics were identified by comparison with pathology.  Multiple blinded raters validated CLE videos of lung tumors and mediastinal nodes twice.  EUS-nCLE-FNA was carried out in 22 patients with suspected or proven lung cancer in whom 27 lesions (6 tumors, 21 mediastinal nodes) were evaluated without complications; 3 nCLE lung cancer criteria (dark enlarged pleomorphic cells, dark clumps and directional streaming) were identified.  The accuracy of nCLE imaging for detecting malignancy was 90 % in tumors and 89 % in metastatic lymph nodes.  Both inter-observer agreement (mean κ = 0.68, 95 % CI: 0.66 to 0.70) and intra-observer agreement (mean ± S.D. κ = 0.70±0.15) were substantial.  The authors concluded that real-time lung cancer detection by endosonography-guided nCLE was feasible and safe.  Moreover, these researchers stated that pending technological improvements and confirmation of their findings, these results showed the potential of nCLE as a guidance tool to identify the optimal area for sampling and as a real-time feedback technique for diagnostic, staging and treatment procedures in lung cancer.  Integration of nCLE into the recently developed robotic bronchoscopic navigational technology may result in an optimal real-time bronchoscopic lung cancer detection tool.

The authors stated that a drawback of this study was the selection of the video sequences.  In the same fashion as most published validation studies of CLE in other organs, not all collected video material was shown to make it feasible for the 7 raters to evaluate the nCLE videos in a limited amount of time.  These researchers stated that for future lung cancer studies, they recommended that the needle probe be moved slowly forwards for optimal image quality.  In their experience, a backwards motion led to obscured imaging due to the presence of erythrocytes from damaged vessels.  The time to image a lymph node with nCLE over the full diameter in a single plane was approximately 10 s, including fanning the needle with a slightly changed angle to increase the imaged area. 

Kramer and colleagues (2022) noted that diagnosing peripheral lung cancer with the bronchoscope is challenging with near miss of the target lesion as major obstacle; nCLE enables real-time microscopic tumor visualization at the needle tip (smart needle).  These investigators examined the feasibility and safety of bronchoscopic nCLE imaging of suspected peripheral lung cancer and examined if nCLE imaging would allow real-time discrimination between malignancy and airway/lung parenchyma.  Patients with suspected peripheral lung cancer based on PET/CT scan underwent radial endobronchial ultrasound (rEBUS) and fluoroscopy-guided flexible bronchoscopy.  After rEBUS lesion detection, an 18-G needle loaded with the CLE probe was inserted in the selected airway under fluoroscopic guidance.  The nCLE videos were obtained at the needle tip, followed by aspirates and biopsies.  The nCLE videos were reviewed and compared with the cytopathology of the corresponding puncture and final diagnosis.  A total of 5 blinded raters validated nCLE videos of lung tumors and airway/lung parenchyma twice.  The nCLE imaging was carried out in 26 patients; no adverse events (AEs) occurred.  In 24 patients (92 %) good-to-high quality videos were obtained (final diagnosis; lung cancer n = 23 and organizing pneumonia n = 1).  The nCLE imaging detected malignancy in 22 out of 23 patients with lung cancer.  Blinded raters differentiated nCLE videos of malignancy from airway/lung parenchyma (280 ratings) with a 95 % accuracy.  The inter-observer agreement was substantial (κ = 0.78, 95 % CI: 0.70 to 0.86) and intra-observer reliability excellent (mean ± SD, κ = 0.81 ± 0.05).  The authors concluded that bronchoscopic nCLE imaging of peripheral lung lesions was feasible, safe and allowed real-time lung cancer detection.  Blinded raters accurately distinguished nCLE videos of lung cancer from airway/lung parenchyma, showing the potential of nCLE imaging as real-time guidance tool.

The authors stated that this study had several drawbacks.  These researchers carried out nCLE imaging in a limited number of selected patients with relatively large lesions and a high prevalence of lung cancer.  Although nCLE imaging provided high-quality microscopic images in a real-time fashion, its use is inevitably dependent on the guidance tool, such as rEBUS, electromagnetic navigation and robotic bronchoscopy, to bring the needle to the target area.  Whether feasibility and outcomes of nCLE imaging in larger, multi-center cohorts with smaller lesions without a bronchus sign and different (benign) pathologies will be similar, has to be investigated.  Another drawback was the selection of video material used in the validation set.  Like most published CLE studies, these researchers had to make a selection to make the validation sessions feasible in time.  Consequently, limited videos were included in the validation set to validate each individual airway/lung parenchyma criterium.  They noted that for future studies, it would be interesting to prospectively use the nCLE malignancy and airway/lung parenchyma criteria to evaluate the number of needle re-positioning based on the nCLE guidance, in order to examine the added value of nCLE imaging to adequately sample the peripheral lung tumor.  Furthermore, will it be of key importance to develop nCLE criteria of benign (e.g., granulomas, hamartomas) pathologies in order to make a clear distinction with malignant lung lesions.

Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Non-small cell lung cancer” (Version 5.2021) does not mention confocal laser endomicroscopy as a management tool.

Confocal Laser Endomicroscopy for Distinguishing Benign and Malignant Gallbladder Polyps

Tang and colleagues (2020) stated that in the past 10 years, CLE has emerged as a new endoscopic imaging modality for real-time in-vivo histological examination at the microscopic level.  In a single-case report, these researchers used CLE for the first time to examine the morphology of cholesterol polyps as well as the different parts of normal gallbladder mucosa.  A 57-year old woman was diagnosed by US with a polyp of 21 mm in the gallbladder wall.  She consented to polyp removal by laparoscopic choledochoscopy.  During laparoscopic cholecystectomy combined with choledochoscopic polyp resection, CLE was used to observe the morphology of the polyp surface cells.  The appearance of the mucosa and micro-vessels in various parts of the gallbladder were also observed under CLE.  Through comparison between post-operative pathology and intra-operative CLE diagnosis, the reliability of intra-operative CLE diagnosis was confirmed.  Based on their practice, CLE should be prioritized in the diagnosis of gallbladder polyps.  The authors concluded that compared with traditional histological examination, CLE has several advantages; they believed that CLE has great potential in this field.

Confocal Laser Endomicroscopy for Use During Surgery for Hirschsprung's Disease and Allied Disorders

Harada and colleagues (2021) noted that they previously demonstrated that the human enteric nervous system (ENS) can be visualized with CLE following topical application of cresyl violet using surgically resected intestine specimens.  This study reported the 1st in-vivo visualization of the human ENS with CLE using local cresyl violet staining.  These researchers examined the technical feasibility and clinical efficiency of CLE in patients with Hirschsprung's disease and allied disorders in-vivo.  Confocal laser endomicroscopy was carried out in-vivo in 2 patients to confirm the presence of the ENS during surgery in patients with Hirschsprung's disease and allied disorders.  Cresyl violet was gently injected from the serosal side into the muscular layer of the intestine, and scanning was carried out within 30 mins.  Thereafter, the scanned intestines were resected, and the visualized area of the specimens was pathologically evaluated.  The ganglion cell nuclei and the ENS network were clearly visualized intra-operatively in both cases.  The morphological findings were similar to the pathological findings of the ENS in both cases although the period of visibility was brief.  The authors concluded that this study was the 1st to report ENS visualization using CLE in-vivo and demonstrated the technical feasibility of visualizing the ENS in patients with Hirschsprung’s disease and allied disorders.  These researchers stated that although much still remains to be done, they believe that success of ENS visualization in 2 live patients was a very important 1st step for the future, and this innovative, minimally-invasive, CLE navigation technique will contribute to improving conventional surgery for Hirschsprung’s disease and allied disorders.

Confocal Laser Endomicroscopy for Evaluation of Epithelial Barrier Function and Presence of Gastro-Esophageal Reflux Disease

Mutha and colleagues (2022) noted that epithelial barrier function (EBF) disruption is a principal mechanism underlying gastro-esophageal reflux disease (GERD).  These researchers examined if 2 novel technologies, probe-based CLE (pCLE) and mucosal integrity testing (MIT), could evaluate EBF.  They prospectively enrolled patients undergoing upper endoscopy for refractory GERD or non-GERD conditions.  Patients underwent esophagogastroduodenoscopy, pCLE, MIT, esophageal biopsy at 2 cm and 6 cm above the esophago-gastric junction (EGJ), and wireless pH testing.  To examine EBF in-vitro, biopsied specimens were mounted in a mini-Ussing chamber, 1-ml of fluorescein was instilled on the mucosal side, and concentration of fluorescein on the serosal side was measured at 3 hours.  These investigators enrolled 54 subjects (28 GERD, 26 non-GERD based on Lyon consensus criteria).  In-vivo permeability evaluated by pCLE did not differ significantly between GERD versus non-GERD patients and did not correlate with in-vitro permeability.  Mean MIT at 2 cm was lower in GERD compared to non-GERD (1,914 versus 3,727 ohms).  MIT correlated inversely with in-vitro permeability at 2 cm and at 6 cm.  Using a predictive model that used slope and intercept of MIT at 2 cm and 6 cm, sensitivity and specificity of MIT at identifying GERD was 76 % and 72 %, respectively.  The authors concluded that pCLE did not differentiate GERD versus non-GERD and did not correlate with EBF measured in-vitro.  On the other hand, MIT may be more promising as it differentiated GERD versus non-GERD and correlated with EBF measured in-vitro.

Confocal Laser Endomicroscopy for Intra-Operative Diagnosis and Resection of Glioblastoma or Other Central Nervous System Tumors

Acerbi and colleagues (2020) stated that CLE allowing intra-operative near real-time high-resolution cellular visualization is a promising method in neurosurgery.  These researchers prospectively examined the accuracy of a new-designed miniatured CLE (CONVIVO system) in giving an intra-operative first-diagnosis during glioblastoma removal.  Between January and May 2018, a total of 15 patients with newly diagnosed glioblastoma underwent fluorescein-guided surgery.  Two biopsies from both tumor central core and margins were harvested, dividing each sample into 2 specimens.  Biopsies were firstly intra-operatively ex-vivo analyzed by CLE, subsequently processed for frozen and permanent fixation, respectively.  Then, a blind comparison was carried out between CLE and standard permanent section analyses, checking for CLE ability to provide diagnosis and categorize morphological patterns intra-operatively.  Blindly comparing CONVIVO and frozen sections images, these investigators obtained a high rate of concordance in both providing a correct diagnosis and categorizing patterns at tumor central core (80 % and 93.3 %, respectively) and at tumor margins (80 % for both objectives).  Comparing CONVIVO and permanent sections, concordance resulted similar at central core (total/partial concordance in 80 % and 86.7 % for diagnosis and morphological categorization, respectively) and lower at tumor margins (66.6 % for both categories).  Time from fluorescein injection and time from biopsy sampling to CONVIVO scanning was 134 ± 31 min (122 to 214 mins) and 9.23 mins (1 to 17 mins), respectively.  Mean time needed for CONVIVO images interpretation was 5.74 mins (1 to 7 mins).  The authors concluded that high rate of diagnostic/morphological consistency found between CONVIVO and frozen section analyses suggested the possibility to use CLE as a complementary tool for intra-operative diagnosis of ex-vivo tissue specimens during glioblastoma surgery.  Moreover, these researchers stated that future studies are clearly needed to confirm these preliminary findings, and to eventually extend such a standardized, prospective and blinded-to-permanent section study in an in-vivo model, aiming to confirm the potentiality of such new CLE system in helping during intra-operative diagnosis in central nervous system (CNS) tumors, and, more importantly, in identifying small residual tissue at the surgical cavity, with a possible impact on extent of resection.

The authors stated that this study had several drawbacks.  First, the relatively small number of patients (n = 15) enrolled may affect the further generalization of these findings to larger cohorts.  Furthermore, from a technical point of view, the amount of time needed from sodium fluorescein (SF) injection to image interpretation was relatively high (134 ± 31 mins), with peaks up to 214 mins.  This was surely related to the application in this study of the same protocol of SF injection (i.e., at the time of patient intubation) that these investigators were extensively applying for fluorescein-guided resection of CNS tumors, and to the ex-vivo nature of the study.  Given the clear and demonstrated inverted correlation that exists between time from contrast injection to images interpretation and readability of the pictures (less time, clearer images), this aspect may have partially affected readability of CONVIVO images, especially if such time was summed up to the time needed for subsequent CONVIVO images interpretation (mean of 5.74 mins).  This drawback could be partially overcome by a totally in-vivo setting, which needs a dedicated sterile sheath covering the CONVIVO probe, allowing for a direct tumor bed analysis, surely much closer to the SF injection time.  These researchers noted that in previous in-vivo published series, performed with other confocal prototypes, or with CONVIVO system only in animals, the quality of the image related to the improved background fluorescence, was much higher.  Furthermore, other authors suggested to use different protocols of SF injection, right before in-vivo CLE analysis, with an impact on image quality.  however, as these investigators considered SF a significant intra-operative adjunct to improve tumor visualization and resection, and as these researchers stressed the importance of using the right SF injection timing and dosage to obtain a good discrimination between tumor and normal peritumoral parenchyma, they wanted to examine if the same protocol could provide similar results in terms of “microscopic” discrimination by using CLE.  Another drawback linked to the ex-vivo sampling and scanning was the small field of views of the confocal imaging, that could limit the identification of important structures that were on the contrary identified on standard histology and frozen section analyses, due to the possibility to enlarge the area of interest in the slide.  However, also in this case, in-vivo analysis could partially address this drawback, by performing multiple virtual biopsies in closer areas of the tumor bed or the brain-tumor interface, enlarging the area of tissue evaluation.  Moreover, due to the prospective nature of the study, with the pathologist who analyzed a priori the CONVIVO images, being blind to the subsequent permanent section analysis, the authors hypothesized the presence of possible misinterpretations of the CONVIVO images, that could eventually be reduced if a posteriori analysis would have been performed.

Hohne and associates (2021) stated that CLE is an established tool in basic research for tissue imaging at the level of microstructures.  Miniaturization and refinement of the technology have made this modality available for operative imaging with a hand-held device.  Sufficient image contrast is provided by the pre-operative application of fluorescein sodium.  These investigators reported their first experiences in a clinical case series using the new CLE.  Handling, operative workflow, and visualization of the CLE were critically evaluated in 12 cases of different CNS tumors. Three different imaging positions in relation to the tumor were chosen: the tumor border (I), tumor center (II), and perilesional zone (III).  Respective diagnostic sampling with H & E staining and matching intra-operative neuro-navigation and microscope images were provided.  CLE was found to be beneficial in terms of high-quality visualization of fine structures and for displaying hidden anatomical details.  The handling of the device was good, and the workflow was easy.  The authors concluded that handling ergonomics and image acquisition are intuitive.  The endomicroscope allowed excellent additional visualization of microstructures in the surgical field with a minimally invasive technique and could improve safety and clinical outcomes.  The new confocal laser endomicroscope is an advanced tool with the potential to change intra-cranial tumor surgery.  Imaging of these microstructures is novel, and research with comparative validation with traditional neuropathological assessments is needed.  Moreover, these researchers stated that fluorescein sodium and CONVIVO CLE are safe and feasible tools; further prospective evaluations are needed to help neurosurgeons determine the validity of CLE and to confirm its promising potential.

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