Barrett’s esophagus (BE) is an important condition given its significant premalignant potential and dismal five-year survival outcomes of advanced esophageal adenocarcinoma. It is therefore suggested that patients with a diagnosis of BE undergo regular surveillance in order to pick up dysplasia at an earlier stage to improve survival. Current “gold-standard” surveillance protocols suggest targeted biopsy of visible lesions followed by four quadrant random biopsies every 2 cm. However, this method of Barrett’s surveillance is fraught with poor endoscopist compliance as the procedures are time consuming and poorly tolerated by patients. There are also significant miss-rates with this technique for the detection of neoplasia as only 13% of early neoplastic lesions appear as visible nodules. Despite improvements in endoscope resolution these problems persist. Chromoendoscopy is an extremely useful adjunct to enhance mucosal visualization and characterization of Barrett’s mucosa. Acetic acid chromoendoscopy (AAC) is a simple, non-proprietary technique that can significantly improve neoplasia detection rates. This topic highlight summarizes the current evidence base behind AAC for the detection of neoplasia in BE and provides an insight into the direction of travel for further research in this area.

The incidence of esophageal cancer is increasing[1], representing the ninth most common cancer in the United Kingdom. Seven thousand and eight hundred people are diagnosed with the condition every year, and it accounts for 5% of all cancer deaths in the United Kingdom[2]. It is well recognized that Barrett’s esophagus (BE) is a significant risk factor for the development of esophageal adenocarcinoma (EAC) and is present in 1.6% of the general population[3] and in up to 20% of patients with gastroesophageal reflux[4].

BE is defined as an esophagus in which any portion of the normal distal squamous epithelial lining has been replaced by metaplastic columnar epithelium, which is clearly visible endoscopically above the gastro-esophageal junction[5]. It is universally recognized that the presence of intestinal metaplasia (IM) confers an increased risk of developing Barrett’s-related EAC and that IM is present in the vast majority of long-segment Barrett’s[6]. The development of Barrett’s EAC is postulated to occur in a progressive fashion from IM to low grade dysplasia (LGD) to high grade dysplasia (HGD) and then EAC. The annual rate of transformation into EAC in patients with non-dysplastic BE is estimated to be between 0.07% and 0.82%[7-9]. However, the annual rate of progression from LGD to HGD or EAC is as high as 8.8% as demonstrated by the recent SURF trial[10] and from HGD to EAC is 12% to 40%[11,12]. The aim of endoscopic surveillance is to alter the natural history of the disease by identifying neoplasia at an earlier stage and thus instituting curative endoscopic therapy.

Established surveillance protocols suggest taking targeted biopsies of visible lesions and random four quadrant biopsies (4QBS) every 2 cm (Cleveland protocol) which reportedly proffers the maximum yield of dysplasia in comparison with other biopsy protocols[13]. However, there are several drawbacks to this technique. With only 13% of early neoplastic lesions appearing as visible nodules[14], a significant proportion of Barrett’s neoplasia is not visible on high-definition white-light endoscopy alone, with reported sensitivity in the range 40%-64% and specificity 98%-100%[15]. These non-visible neoplastic foci can occupy areas as small as 0.5 cm^2[16]. Unsurprisingly, there is a significant miss-rate with 4QBS. Studies comparing 4QBS with surgical resection specimen have shown that 41%-66% of dysplastic lesions are missed by 4QBS[17,18]. The total mucosal surface sampled with 4QBS is equivalent to 0.5 cm² equating to sampling of only 3.5% of an average-length BE. 4QBS are notoriously poorly adhered to[19], with worse adherence for longer segments, further compounding miss-rates. In addition, 4QBS are time-consuming and poorly tolerated by patients. The cost of processing 4QBS is significant, with each cassette of tissue costing £58.90 ($90.61) to process[20].

These pitfalls in surveillance have prompted evaluation of more effective techniques to improve the diagnostic accuracy for the detection of IM and early Barrett’s neoplasia, the most promising of which is acetic acid chromoendoscopy (AAC). This review aims to summarize the current evidence for AAC in BE and provide insight into the direction of travel for further research in this area.

The use of acetic acid (AA) in the digestive tract was first reported by Guelrud and Herrera[21], to aid in the identification of small islands of BE following ablative therapy. The technique was derived from gynecology where AA instilled onto the cervix has been used to highlight dysplastic areas during screening for cervical intraepithelial neoplasia[22]. When AA is sprayed onto squamous epithelium, there is an acetowhitening reaction caused by masking of the submucosal capillaries and increasing opacity of the mucosal surface[23]. As AA (pH 2.5-3.0) infiltrates through the multi-layered squamous epithelium it is neutralized, which protects the subepithelial stroma and vasculature[24]. In contrast, when sprayed on Barrett’s epithelium, at low concentrations (1%-3%), AA initially eliminates the superficial mucus layer by breakage of glycoprotein disulphide bonds. The unbuffered acid then causes a reversible acetylation of cellular proteins and a change in the spatial properties of nuclear and cytoplasmic proteins, initially causing an acetowhitening reaction that highlights the surface pattern (Figure 1). With the disruption of the mucus layer, AA reaches stromal capillaries causing vascular congestion, leading to focal erythema but this is hidden under the acetowhite mucosa and only becomes visible after the loss of acetowhitening (LAW). This focal redness due to LAW was first described, by the Portsmouth group in 2010[2], as a strong predictor of neoplasia. The exact mechanism remains unclear but it is believed that the difference in acetowhitening reaction between non-neoplastic and neoplastic mucosa is due to the difference in the nucleocytoplasmic ratio between non-neoplastic and neoplastic cells. The low cytoplasmic content of neoplastic cells allows them to lose acetowhitening quicker than non-neoplastic cells. This reaction leads to focal erythema - a pathognomic sign of neoplasia with AAC.

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Figure 1 Acetic acid mechanism of action. A: Non-dysplastic Barrett’s with HDWL; B: Non dysplastic BE following AAC (Olympus Lucera ELITE processor, GIFHQ290 gastroscope).

The diagnosis of Barrett’s esophagus, according to American society guidelines[25], is defined as the presence of esophageal IM. As IM is not readily identifiable by white light endoscopy, this diagnosis is made based on histology. Efforts have been made to visually identify IM by means of enhanced endoscopy. AA coupled with magnification endoscopy has been shown to accurately identify IM[21]. Guelrud et al[21] classified the surface pattern of Barrett’s mucosa into 4 categories: (1) round pits; (2) reticular (circular or oval pits); (3) villous (fine villiform appearance without visible pits); and (4) ridged (thick villi with convoluted, cerebriform appearance without visible pits).

They found that Pattern I corresponded to fundic or cardiac type without IM and Patterns II, III, an IV each corresponded to IM with increasing sensitivities. The overall accuracy of AA with magnification endoscopy for the diagnosis of IM was 92.2%. These findings were reliably replicated by Toyoda et al[26] and Fortun et al[27].

A recent meta-analysis by Coletta et al[28] evaluated the use of AA for the detection of IM and HGD/EAC in patients with BE using histology as the reference standard. A total of 13 prospective studies (1690 patients) were included in the meta-analysis. Eight of the 13 studies, provided data on the diagnosis of IM. For the characterization of IM, the pooled sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) for all the included studies (8 studies, 516 patients) were 0.96 (95%CI: 0.83-0.99), 0.69 (95%CI: 0.54-0.81), 3.0 (95%CI: 2.0-4.7) and 0.06 (95%CI: 0.01-0.26), respectively. No significant sources of heterogeneity were identified on subgroup analysis. AA may be helpful for the exclusion of specialized IM, however, histological confirmation remains critical due to low specificity (0.69). In our view, this is clinically not relevant when dealing with long-segment BE as presence of specialized IM would not alter surveillance intervals.

Use of AA to aid identification of IM in BE is important in the stratification of surveillance intervals[5]. However, the overriding utility of AA is the identification and characterization of Barrett’s neoplasia. There is a growing body of evidence to support the use of AA in this setting.

In 2006 Réaud et al[29], furthered Guelrud’s work aiming to define the neoplastic appearances of BE following 6% AA dye spray and magnification endoscopy. In their study of 28 patients, they noted that patients with HGD on biopsy displayed mucosal architectural disorganization and hypervascularity - a phenomenon previously identified by Rey et al[30] in 2003. Using these parameters, they demonstrated a positive predictive value (PPV) of 75% for neoplasia. Camus et al[31] identified similar features when combining AA with FICE.

In their study of 62 patients in 2006, Fortun et al[27] examined whether the combination of magnification endoscopy and 3% AA could improve diagnostic accuracy in patients with BE. Patients underwent a repeat endoscopy having recently undergone surveillance endoscopy (mean 7 mo prior). Barrett’s neoplasia was identified in 9 patients: 5 LGD, 1 HGD and 3 EAC. The main drawback from this study is that the index endoscopy was used as a control, raising the question as to whether the neoplasia detected was de-novo or previously missed, with the total number of neoplasias being small. At the same time, Yagi et al[32] reported that Barrett’s EAC was associated with an irregular granular pattern or a minute grain-like pattern following 1.5% AA dye spray and magnification endoscopy.

A year later Vázquez-Iglesias et al[33] reported on their prospective study of 100 patients undergoing Barrett’s surveillance, 13 of whom had neoplasia, using 3% AA and non-magnification endoscopy. They proposed the following mucosal classification: (1) normal pattern: uniform reticulum along entire columnar-lined esophagus; and (2) abnormal pattern: rough or irregular reticulum.

Applying these characteristics, they demonstrated 100% sensitivity and 97.7% specificity (PPV 86% NPV 100%) for the detection of early neoplasia. with the false positives arising in 2 patients; one with esophagitis, the other with an esophageal ulcer.

These results were a significant improvement on those reported by Mayinger et al[34] in 2006 who reported sensitivities for neoplasia recognition in the range 55.5% to 82.4% in endoscopists trained in interpretation of AA enhanced magnification endoscopy. The same study also demonstrated extremely low inter- and intra-observer agreement for the technique.

The Wiesbaden group first reported their experiences of AAC for neoplasia detection in Barrett’s in 2007[35]. They performed a prospective randomized crossover tandem endoscopy study examining 57 patients with a history of Barrett’s neoplasia with AAC or virtual chromoendoscopy, using Fujinon Intelligent Chromoendoscopy (FICE), 4-6 wk apart. The patients had a known history of Barrett’s neoplasia (discrete mucosal alteration/ macroscopically occult lesions/ prior endoscopic treatment for neoplasia). Targeted biopsy of visible abnormalities was performed along with 4QBS. In 24 patients neoplasia was identified with the AAC achieving an 87% sensitivity. There are however, limitations with this study in that combined biopsies (targeted plus 4QBS) were used as the reference standard not surgical resection specimens. The study population was neoplasia-enriched in a tertiary center and thus results may not reflect the true performance of AAC in the community, surveillance population.

Longcroft-Wheaton et al[36] from Portsmouth reported on their cohort of patients undergoing Barrett’s examination with AAC with strikingly similar results. The study design was similar to the Wiesbaden group with 190 procedures performed in 119 patients. After esophageal cleansing, with a 50 mL solution containing 40 mL of water, 5 mL of 10% N-acetylcysteine and 5 mL of simeticone, patients underwent conventional white light endoscopy followed by 2.5% AAC. The Barrett’s segment was assessed for the following features: (1) surface pattern: ridged, villous, round, irregular; (2) vascular pattern: regular or irregular; and (3) acetowhitening reaction: No loss of acetowhitening or focal early loss of acetowhitening.

Dysplastic Barrett’s was defined endoscopically as (Figure 2): (1) irregular surface patterns AND/OR; (2) increased vascularity or irregular vessels AND/OR; and (3) focal, early loss of acetowhitening was present.

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Figure 2 Dysplastic Barrett’s was defined endoscopically. A: Barrett’s with HDWL; B: Same patient note dysplasia only visible post AAC with early loss of acetowhitening (Olympus Lucera ELITE processor, GIFHQ290 gastroscope).

Targeted biopsies were performed followed by 4QBS (unless area already sampled with targeted biopsy). Again, the combination of targeted and 4QBS was used as the reference for final histological diagnosis.

Seventy-eight procedures were performed in patients with no prior neoplasia history (low-risk group) and 112 procedures were performed in patients referred with a history of neoplasia (high-risk group). Neoplasia was histologically confirmed in 88/190 procedures: 21/88 EAC (T1a/b), 51/88 HGD, 16/88 LGD. AAC targeted biopsy demonstrated a sensitivity of 95.5% and specificity 80% for neoplasia detection. Significant correlation between the in vivo diagnosis of neoplasia and final histology was noted (r = 0.98). There was a 2.5-fold increase in visible neoplasia detection with AAC as compared to white light alone (P = 0.001). The limitations of this study are similar to those of the Wiesbaden group: single center, expert endoscopist with a dysplasia-enriched population. What these studies cannot answer is how AAC would perform in the surveillance population where dysplasia prevalence is much lower and how AAC performs in non-expert hands.

Another factor limiting the use of AAC is the additional skills required to interpret surface and vascular patterns and their subjective nature. To that end the Portsmouth group sought to develop an objective tool using the duration of acetowhitening for the diagnosis of neoplasia[2]. One hundred and thirty-two patients underwent 2.5% AAC with targeted biopsies of neoplasia, followed by 4QBS. Time taken to lose acetowhitening effect was measured and analyzed for metaplasia, HGD and EAC. In cases of cancer, acetowhitening was lost in a median of 23 s (range 3-81 s), for HGD the median was 53 s (range 4-288 s). In non-dysplastic Barrett’s median time was 311 s (range 14-992). They proved the concept of focal loss of acetowhitening (LAW) as a very effective tool in distinguishing metaplasia from HGD and HGD from EAC. The time differences to lose acetowhitening were statistically significant (P < 0.05). In order to further refine the tool, the authors plotted a receiver operating characteristic and determined that a time of 142 s yielded the optimum sensitivity of 98% and specificity of 84%. The benefit of this tool is that it provides endoscopists an objective measure of neoplasia, avoiding subjective interpretation of mucosal and vessel patterns. This is clinically very relevant as this phenomenon can be universally applied, regardless of endoscope manufacturer or definition, and requires minimal training. Their results reach the ASGE PIVI (preservation and incorporation of valuable endoscopic innovations) criteria[37] (sensitivity ≥ 90%, NPV ≥ 98% and specificity > 80%), reaching these thresholds eliminates the need for random 4QBs.

In the meta-analysis by Coletta et al[28], 9 studies (1379 patients) looked at AAC for the diagnosis of HGD/EAC. The pooled sensitivity, specificity, LR+, LR- was 0.92 (95%CI: 0.83-0.97), 0.96 (95%CI: 0.85-0.99), 25.0(95%CI: 5.9-105.3) and 0.08 (95%CI: 0.04-0.18), respectively. Subgroup analysis did not identify significant sources of heterogeneity. The results highlight, high sensitivity 92% and specificity 96% for AAC in the diagnosis of HGD/EAC.

The advent of advanced endoscopic imaging technologies such as NBI, FICE and i-scan have improved the identification and characterization of neoplastic lesions, but these technologies require significant financial investment. Therefore, the role of AA in the surveillance population is of great interest as a potentially cost-effective, accurate and non-proprietary tool for improving dysplasia detection.

The evidence for the use of AAC in the detection and characterization of Barrett’s neoplasia is compelling. The large studies from the Portsmouth and Wiesbaden groups demonstrate that experts are able to meet the ASGE PIVI criteria[40] (sensitivity ≥ 90%, NPV ≥ 98% and specificity > 80%) and are thus able to justifiably dispense with 4QBS. The technique is cheap and can be universally applied, regardless of endoscope manufacturer. However, further data from a well-powered randomized controlled trial are required before completely abandoning 4QBS and it may be that the modified Portsmouth protocol provides optimum results for cost-effective Barrett’s surveillance.