Guidelines on Diagnosing and Managing Barrett's Esophagus
Guidelines on Diagnosing and Managing Barrett's Esophagus
When Barrett's oesophagus is detected at endoscopy and confirmed by histopathological findings, this diagnosis should be discussed with the patient in the clinic, so that patient preference can be taken into account. Patients should receive an early outpatient appointment (ideally within 4–6 weeks) to discuss the implications of this diagnosis with a physician with a clinical interest in Barrett's. Discussion should include the low but significant cancer risk, possible lifestyle changes, whether or not there is an indication for endoscopic surveillance, and the therapeutic options if dysplasia is detected (endoscopic and surgical). Family history for Barrett's oesophagus and OAC should also be recorded. If there is still uncertainty about a diagnosis of Barrett's that requires further work up, this should be clearly explained to the patient to avoid confusion. Written information should be provided for the patient to take away using BSG (see online supplementary appendix 4) or other approved materials such as from MacMillan CancerBACUP (http://www.macmillan.org.uk/Cancerinformation/Cancertypes/Oesophagusgullet/Pre-cancerousconditions/Barrettsoesophagus.aspx) or H-CAS (http://www.h-cas.org/barretts.asp).
Before seeking informed consent for surveillance, the diagnosis of Barrett's oesophagus should have been confirmed on endoscopic and histopathological grounds based on the criteria above. Because of the recent advancement in the endoscopic treatment of HGD and mucosal adenocarcinoma, it is no longer appropriate to restrict surveillance to patients who are fit, and willing, to undergo oesophagectomy. In addition, radiotherapy and/or chemo-radiotherapy may be treatment options in patients with more advanced disease who are deemed not fit for surgery. However, the patient should be fit for repeated endoscopy procedures and endoscopic therapy if HGD or early cancer is detected. Very few studies have used the performance status (PS) to correlate patient fitness with the outcome of endoscopic therapy for GI early cancers. Endoscopic therapy can be safely performed in patients with Eastern Cooperative Oncology Group PS 0–2. Therefore it is reasonable to consider endoscopic surveillance in patients with PS 0–2, provided that the estimated patient life expectancy is sufficiently long for the individual to benefit from surveillance if dysplasia or early cancer were detected.
If surveillance is thought to be clinically indicated, then the clinician should discuss with the patient the possible benefits of surveillance in detecting early-stage tumours and improving cancer survival. However, this discussion should also mention the lack of randomised controlled data to prove the benefits of surveillance, and clinicians must also emphasise to the patient that the actual risk of death from oesophageal cancer is small. Furthermore, the disadvantages of endoscopy surveillance should also be discussed, including the small risks of the procedure and the associated psychological morbidity. For example, in an American study conducted in a population of Veterans with a diagnosis of Barrett's, more than half of the patients missed their follow-up endoscopy, suggesting that not all patients are willing to adhere to surveillance programmes. Clinicians should also emphasise that, as with any monitoring programme, there is a failure rate, in that surveillance cannot guarantee to detect every tumour that may develop. There are no clear data to support how best to impart this complex information, and more work in this area is warranted.
Patients should have early access to an outpatient clinic to be informed about a new diagnosis of Barrett's oesophagus and to have an initial discussion about the pros and cons of surveillance with written information provided (Recommendation grade C).
For a given patient, whether or not surveillance is indicated should be determined on the basis of an estimate of the likelihood of cancer progression and patient fitness for repeat endoscopies, as well as patient preference (Recommendation grade C).
Technological advancement with new-generation charge coupled devices has allowed the routine use of high-resolution endoscopy (HRE), which produces images with resolutions ranging from 850 000 to more than one million pixels. HRE allows fine definition of the mucosal layer for the recognition of subtle superficial abnormalities, with theoretical advantage in the recognition of dysplasia and Barrett's oesophagus-related early neoplasia. It is the opinion of the experts that HRE, in conjunction with careful cleaning of the mucosal surface of mucus, saliva and food debris, is the minimum standard for the evaluation of patients with known Barrett's oesophagus; however, to date, there is no randomised trial comparing conventional endoscopy with HRE in Barrett's oesophagus dysplasia detection (Evidence grade IV). In an RCT, HRE performed equally compared with chromoendoscopy and narrow band imaging (NBI) in the overall diagnosis of dysplasia (Evidence grade Ib). Mucolytic agents (eg, 4–10% N-acetylcysteine) or antifoaming agents (eg, simethicone) can be used to disperse excess mucus and bubbles. There is also evidence that longer inspection times during assessment with white light endoscopy is associated with an increased detection rate for HGD and early cancer (Evidence grade III). This should be taken into account when planning how much time to allocate for endoscopic surveillance of very long segments of Barrett's, particularly those longer than 10 cm.
Although transnasal endoscopy has been shown to be accurate in the diagnosis of Barrett's oesophagus (Evidence grade Ib), the randomised studies performed so far either included a small number of patients, or were performed in a low-risk population. Furthermore, it should be noted that the biopsy specimens taken with these endoscopes are significantly smaller, and this may increase sampling bias and hamper the interpretation of dysplasia. Therefore there is currently a lack of robust data to recommend transnasal endoscopy in routine Barrett's oesophagus surveillance.
HRE should be used in Barrett's oesophagus surveillance (Recommendation grade C).
There is insufficient evidence to recommend transnasal endoscopy as a replacement for transoral endoscopy (Recommendation grade C).
Advanced endoscopic imaging has been investigated to increase the detection of both IM and dysplasia in Barrett's oesophagus with the aim to help target biopsies (Table 7).
Chromoendoscopy uses dyes to enhance endoscopic detection. Methylene blue (MB) is a vital dye actively absorbed by columnar intestinal-type cells and has been used to improve the yield of IM in Barrett's oesophagus (Evidence grade III). In a historical cohort, Sharma and coworkers found significant enrichment of IM in MB-targeted biopsy samples compared with random samples (Evidence grade III). The detection rate of IM and dysplasia during MB chromoendoscopy has been investigated in a number of randomised and cohort studies with conflicting data (Table 7). A recent meta-analysis has found no incremental yield of both IM and dysplasia with MB chromoendoscopy compared with standard endoscopy with random samples (Evidence grade Ia). It should also be noted that MB may damage DNA, which, coupled with the lack of evidence for efficacy, suggests that its use cannot be recommended (Evidence grade III).
Indigo carmine (IC) is a contrast agent that allows detailed inspection of the mucosal pattern in combination with magnification endoscopy. A prospective multicentre study found that the ridged/villous pattern had a 71% sensitivity for IM, while the irregular/distorted pattern had an 83% sensitivity and an 88% specificity for HGD/early cancer (Evidence grade III). The limitation of IC chromoendoscopy is the need for high magnification with consequent narrow field of view. Only one randomised trial has evaluated IC chromoendoscopy for detection of dysplasia in Barrett's, but failed to find an increased rate of dysplasia compared with high-resolution white light endoscopy (Evidence grade Ib).
The value of acetic acid (AA) to improve the diagnostic yield of surveillance endoscopy has also been studied. AA induces intracellular protein denaturation, with swelling of the mucosal surface and enhancement of the architecture. Randomised cross-over studies have produced contradictory results on the diagnostic yield of AA-enhanced magnification endoscopy for IM (Evidence grade Ib). AA-enhanced magnification endoscopy has been shown to have a higher dysplasia yield in Barrett's oesophagus surveillance, with 24% of patients having histological upgrade compared with a previous standard endoscopy with random biopsies performed in a non-specialist centre (Evidence grade III). In a large single-centre prospective study, Pohl et al found that AA-targeted biopsies had a sensitivity of 96.7% and a specificity of 66.5% for a diagnosis of HGD/early cancer. A single-centre retrospective cohort study has showed significantly increased dysplasia yield (p=0.001) compared with standard endoscopy with random biopsies (Evidence grade III). The same group showed that histology on AA-targeted biopsies was more cost-effective than the Seattle protocol in a high-risk population. More data are needed to decide on the usefulness of this technique.
With recent technological advancements, 'virtual chromoendoscopy' has become available, which allows chromoendoscopy without the use of dyes. This is based on light filters (NBI, Olympus) or post-image acquisition processing (i-scan, Pentax and FICE, Fujinon). The most extensively studied 'virtual chromoendoscopy' technique in Barrett's oesophagus is NBI, which highlights the mucosal pattern and the superficial vasculature. A number of different classifications have been proposed to describe mucosal pits in non-dysplastic and dysplastic Barrett's, which yielded high diagnostic accuracy (Evidence grade III). When NBI was compared with standard imaging techniques, one prospective tandem study showed an incremental diagnostic yield for dysplasia in the per-patient analysis (Evidence grade IIa), and two additional studies reported an increased dysplasia detection only in the per-biopsy analysis (Evidence grade Ib). A meta-analysis of eight studies has found that NBI has a sensitivity and specificity of 96% and 94%, respectively, for the diagnosis of HGD, and 95% and 65%, respectively, for the diagnosis of IM. However, the interobserver agreement for the interpretation of the NBI images is only moderate. Overall, despite the finding that NBI performed by an expert endoscopist may increase the targeted yield of dysplasia, it also transpires that high-resolution endoscopy alone is sufficient to maximise dysplasia detection on a per-patient basis.
Autofluorescence imaging (AFI), which exploits endogenous fluorophores excited by short wavelengths, has been studied in the context of Barrett's oesophagus. Initial single-centre cohort studies showed that AFI can improve the diagnostic yield of dysplasia compared with standard endoscopy, but with a false-positive rate as high as 80% (Evidence grade III). To overcome this, AFI has been incorporated into an HRE-NBI scope with magnification, also known as endoscopic trimodal imaging (ETMI). Although an initial multicentre non-randomised feasibility study showed that ETMI increased the diagnostic yield for dysplasia from 63% to 90% compared with standard endoscopy with random biopsies (Evidence grade III), this was not confirmed in two subsequent multicentre randomised studies, where ETMI only improved the diagnostic yield of dysplasia in the per-biopsy analysis (Evidence grade Ib). Overall, these studies showed that, in selected high-risk cohorts of patients, ETMI does not allow the requirement for random biopsies to be abandoned. Further studies in low-risk patients will inform whether AFI can have a role in reducing the number of biopsies without loss of diagnostic accuracy.
Other imaging techniques that have showed some value in Barrett's oesophagus include confocal laser endomicroscopy, spectroscopy and optical coherence tomography; however, further studies are needed to clarify whether they can improve diagnostic accuracy during Barrett's oesophagus surveillance.
In the future, molecular imaging may improve our imaging armamentarium to increase dysplasia detection. Molecular imaging exploits fluorescently labelled molecules that bind with different affinity to dysplastic compared with non-dysplastic cells. Two types of compound have been studied so far. In a proof-of-principle study, Li and colleagues identified a 7-amino acid peptide that binds an OAC cell line more avidly than a non-dysplastic Barrett's oesophagus cell line, and they confirmed the differential binding in surgical specimens of OAC ex vivo. Similarly, Bird-Lieberman and coworkers identified a natural lectin (wheat germ agglutinin) that differentially binds surface glycoproteins of dysplastic and non-dysplastic cells and used an autofluorescence endoscope in surgically resected oesophagi to validate the ex vivo findings. In vivo studies are needed to validate these techniques.
Advanced imaging modalities, such as chromoendoscopy or 'virtual chromoendoscopy', are not superior to standard white light endoscopy in Barrett's oesophagus surveillance and are therefore not recommended for routine use (Recommendation grade A).
To find dysplasia, endoscopists have generally relied on the directed sampling of any visible lesions, which may be aided by enhanced endoscopic visualisation tools as discussed above, together with systematic, four-quadrant biopsies every 2 cm according to the so-called 'Seattle protocol'. A prospective study has demonstrated a significant increase in the detection of early lesions through the introduction of such a protocol (Evidence grade III). However, adherence to this protocol is limited and ranges from 10% to 79%, with poorer adherence for longer segments, and failure to adhere to the protocol has been shown to result in a significantly lower rate of dysplasia detection. Overall, although intense and time-consuming, the multiple biopsies involved in the Seattle protocol have been demonstrated to be safe when performed by experienced endoscopists. Another limitation of this technique is the high cost generated by processing multiple biopsies, but this still seems justified at the current time in the absence of an alternative. Future RCTs will need to compare cost-effectiveness of the standard practice with alternative techniques such as histology on targeted biopsy samples guided by conventional or virtual chromoendoscopy.
Adherence to a quadrantic, 2 cm biopsy protocol in addition to sampling any visible lesions is recommended for all patients undergoing surveillance. This should also apply to long segments (Recommendation grade B).
In the previous BSG guidelines published in 2005, the recommended surveillance interval for non-dysplastic Barrett's was every 2 years. However, given the recent data suggesting that the overall risk of malignant conversion is lower than previously thought, we recommend that the interval should be lengthened in line with other guidelines. We therefore advocate a new surveillance strategy whereby the managing clinician synthesises the endoscopic and histopathological findings to tailor the surveillance interval on a more individual basis (figure 3). A degree of variation in this interval is permitted, which may be influenced by the presence of risk factors for the development of cancer.
To summarise, in practical terms, short segments of columnar epithelium with no IM have an extremely low risk of malignant conversion (~0.05% per annum) (Evidence grade III). For these patients, it is recommended to repeat the endoscopy once in 3–5 years time to confirm the findings and account for sampling and measurement error. If there is doubt, the endoscopy could be repeated sooner. If two good-quality endoscopies, each with a minimum number of four oesophageal biopsies where possible, confirm a short segment (<3 cm) with gastric metaplasia only, then discharge is encouraged, as the risks of endoscopy probably outweigh the benefits. In selected cases with a strong personal risk profile for OAC (see recommendation on screening), continued endoscopic surveillance can be considered.
For patients with Barrett's oesophagus shorter than 3 cm, without IM or dysplasia, a repeat endoscopy with quadrantic biopsies is recommended to confirm the diagnosis. If repeat endoscopy confirms the absence of IM, discharge from surveillance is encouraged, as the risks of endoscopy probably outweigh the benefits (Recommendation grade C).
There is evidence that the risk of cancer progression correlates significantly with the length of the Barrett's segment, such that segments shorter than 3 cm have a lower cancer incidence (Table 5). Therefore, in view of the recent evidence supporting a lower cancer risk in non-dysplastic Barrett's oesophagus than previously thought, it is reasonable for patients with short segments containing IM to have a longer endoscopic surveillance interval than patients with long segments. We propose a range of 3–5 years to allow the clinician to tailor surveillance on the perceived individual cancer risk.
Patients with Barrett's oesophagus shorter than 3 cm, with IM, should receive endoscopic surveillance every 3–5 years (Recommendation grade C).
For longer segments (>3 cm), a shorter surveillance interval is more appropriate. This is regardless of the presence of IM, since it is noted, that in long segments, IM is almost always present, but can be missed due to sampling error. We propose a range (between 2 and 3 years), which may be informed by the individual risk factors and patient and physician preference. Because of the poor adherence to the surveillance biopsy protocol for long segments of Barrett's oesophagus, consideration should be given to refer patients with a very long segment (>10 cm) to tertiary referral centres for endoscopic surveillance, as suggested also in the new Dutch guidelines (personal communication).
Patients with segments of 3 cm or longer should receive surveillance every 2–3 years (Recommendation grade C).
Pathological Features and Reporting of Dysplasia. Online supplementary appendix 3 shows histological examples of Barrett's with different degrees of dysplasia.
There are very few studies that investigated reporting of dysplasia in Barrett's oesophagus. Two studies examined the Vienna classification and found a degree of agreement among pathologists that was at best moderate for HGD, fair for LGD, and poor for indefinite for dysplasia. The approach to reporting upper GI tract neoplasia differs significantly in certain parts of the world and this has led to inconsistency in the terminology used and hence inconsistent data on incidence and clinical progression outcomes. The most recent recommendations by the WHO state that dysplasia should be graded as either low or high grade. The revised Vienna classification for GI mucosal neoplasia attempts to standardise diagnostic terminology into biologically similar groupings with scores of 1–5 depending on the presence or absence of dysplasia or malignancy.
Revised Vienna Classification and Dysplasia Subtypes. 1. Negative for Dysplasia: This includes normal epithelium, metaplastic epithelium showing reactive or regenerative changes, and mucosa showing reactive/regenerative changes including nuclear enlargement, nuclear hyperchromasia and prominent nucleoli.
2. Indefinite for Dysplasia: This category is used for cases where the morphological features between true dysplasia and regenerative/inflammatory atypia are blurred. It is important to appreciate that this diagnosis may in fact mean that the patient has features suspicious of HGD, but not enough certainty is present to warrant this call. This may be due to technical factors, such as poor staining, poor orientation, cross cutting or denuded surface epithelium, or to severe active inflammation or ulceration leading to marked atypia, precluding a confident diagnosis of dysplasia. In other cases, the epithelium appears abnormal, but the features are not sufficiently well developed to justify a definite diagnosis of dysplasia. Features favouring dysplasia are the presence of an abrupt transition from normal to atypical epithelium, together with nuclear pleomorphism, atypical mitoses and loss of nuclear polarity. Evidence of 'surface maturation'—that is, loss of the cytological atypia seen in the deeper glands as the mucosa matures into the surface epithelium—is often taken as the best marker to favour regeneration rather than dysplasia, although this is also not invariably true (eg, crypt dysplasia described below). Explicit mention in the pathology report of the reason justifying this diagnosis can be useful to aid patient management.
3. Low-grade Dysplasia: In LGD, glandular architecture is relatively preserved and the diagnosis is made on the basis of cytological atypia.
Morphological patterns of LGD:
4a. HGD (Incorporating Carcinoma in Situ): The distinction between HGD and LGD is largely based on the presence of architectural changes in conjunction with more marked nuclear atypia. These changes may be accompanied by complex architectural changes including a papillary or villous surface (although villiform change may also be seen in reactive epithelium), in conjunction with branching, complex budding or back-to-back 'crowding' arrangements. Intraluminal papillae, bridges or cribriform patterns are also seen. There are increased numbers of atypical mitoses on upper levels of crypts, together with mucin depletion and a loss of nuclear polarity. Of note, HGD can be accompanied by acute inflammation and should not be downgraded in its presence.
Morphological Patterns of HGD:
Crypt Dysplasia: Significant cytological atypia in the crypt bases with surface maturation has been reported in up to 7.3% of cases of Barrett's. Previously, this would have been regarded as either 'negative for dysplasia' or 'indefinite for dysplasia', as the atypia does not reach the surface epithelium. It seems likely, however, that crypt dysplasia represents an early stage in the development of dysplasia, and the atypia is highly likely to progress up to the surface over time and so warrants recognition. Crypt dysplasia stands out as a focus that is distinctly different from the surrounding crypts and can appear as low-grade or high-grade cytological atypia. Although the dysplasia can be of the 'adenomatous' or 'non-adenomatous' round cell type, the most common features are nuclear enlargement, loss of polarity, marked pleomorphism with irregular shapes and sizes, nuclear crowding, increased mitotic activity and goblet cell dystrophy. Crypt dysplasia should not be diagnosed purely on the basis of stratification and hyperchromasia in the absence of significant nuclear pleomorphism, as these changes may often be seen in regenerative cryptal epithelium. p53 immunohistochemistry may be a helpful adjunct for the assessment of crypt dysplasia. Crypt dysplasia should be reported according to the degree of dysplasia present. If there is uncertainty, then the 'indefinite' category may be appropriate. Some pathologists favour putting cases with high-grade features into an 'at least low grade' category in view of the likely early nature of the lesion and the implications of a high-grade diagnosis; however, cases of isolated crypt dysplasia are probably best managed as low grade until further data become available.
4b. Intramucosal Carcinoma (Including Suspicious for Invasive Carcinoma): Intramucosal carcinoma is a lesion in which neoplastic cells have penetrated the basement membrane and invaded the lamina propria or muscularis mucosae, but without invasion into the submucosa. However, histological recognition of lamina propria invasion may be difficult because of the absence of objective criteria. Patterns of lamina propria invasion that are used by gastrointestinal pathologists include sheets of neoplastic cells, abortive angulated glands, a never-ending/anastomosing gland pattern, a highly complex cribriform arrangement of glands, tightly packed small tubular glandular arrays, and single-cell infiltration. Recognition of each of these patterns is somewhat subjective, with κ statistics varying between 0.21 and 0.47, suggesting poor or, at best, moderate agreement. If definitive submucosal invasion is in question, the term 'suspicious of invasive carcinoma' can be used.
5. Submucosal Invasion by Adenocarcinoma: Unequivocal invasion of submucosa or deeper structures often accompanied and characterised by a desmoplastic response in the tissue stroma to invasive tumour cords/acini.
Given the important management implications for a diagnosis of dysplasia, we recommend that all cases of suspected dysplasia are reviewed by a second GI pathologist, with review in a cancer centre if intervention is being considered (Recommendation grade C).
Given the difficulties associated with the management of the 'indefinite for dysplasia' category, all such cases should also be reviewed by a second GI pathologist, and the reasons for use of the 'indefinite for dysplasia' category should be given in the histology report in order to aid patient management (Recommendation grade C).
Dysplasia Reporting and Reproducibility. It has long been recognised that there is inter- and intra-observer variability in the diagnosis of GI tract dysplasia. This relates to differentiating between HGD and intramucosal adenocarcinoma, HGD and LGD and also distinguishing between regenerative changes and LGD. In the case of definite dysplasia, this is because these divisions involve unnatural cut-offs along a biological/histological continuum. Studies have shown that the prediction of progression of oesophageal dysplasia is improved if at least two expert pathologists agree on the diagnosis and increases further when a greater number of pathologists concur with the diagnosis. For practical reasons, and in day-to-day diagnostic practice, a diagnosis of dysplasia in the setting of Barrett's should be corroborated by a second pathologist with a specialist GI interest. The Royal College of Pathology recommends that 'double' reporting of a diagnosis of HGD in the upper GI tract should be mandatory and this has been confirmed by consensus statements agreed by Barrett's international experts. We have extended this consensus reporting to all grades of dysplasia.
Aids to Histological Diagnosis of Dysplasia and p53 Immunostaining. Of all the putative experimental molecular markers, the one with the greatest body of evidence and which can also be applied in the routine clinical setting is immunohistochemistry for nuclear p53. Although the p53 positivity rate in Barrett's oesophagus dysplasia is variably reported in the literature, ranging from 50% to 89%, when positive it can improve interobserver agreement for reporting dysplasia and can be a powerful predictor of progression, with an OR between three and eight in different studies (Table 6). In a study from Skacel et al, who analysed factors predictive of progression in patient with LGD, p53 immunostaining positivity and 100% agreement among three GI pathologists on LGD diagnosis correlated with the risk of progression, suggesting that p53 might improve interobserver agreement. This was replicated in a later study. Interpretation of p53 immunostaining can be problematic and poorly reproducible subject to variation in methodology and interobserver variation. Notwithstanding this, some pathologists find staining for p53 of use, especially in distinguishing between atypical reactive proliferation (indefinite for dysplasia) and true LGD. Low background wild-type p53 expression is often seen in nuclei of normal columnar and basal layers of squamous mucosa, which is a useful baseline to identify the overexpression pattern typical of dysplasia. Overexpression is generally a consequence of mutations that stabilise the inactivated protein. However, not all p53 mutations lead to stabilisation of a mutated inactive p53 protein. A study performed in non-small cell lung cancer showed that, as opposed to missense mutations, the majority of null mutations did not lead to p53 overexpression. In such cases, mutation is expected to lead to failed translation of the protein. In fact, an absent pattern of p53 immunostaining, when compared with normal wild-type background, is now recognised as an abnormal pattern which also occurs in dysplasia as a result of silencing mutations of the p53 gene. Online supplementary appendix 3 shows immunohistochemical examples of Barrett's with overexpression and loss of p53.
The addition of p53 immunostaining to the histopathological assessment may improve the diagnostic reproducibility of a diagnosis of dysplasia in Barrett's oesophagus and should be considered as an adjunct to routine clinical diagnosis (Recommendation grade C).
Practicalities of Endosopic Surveillance
Patient Selection and Informed Consent
When Barrett's oesophagus is detected at endoscopy and confirmed by histopathological findings, this diagnosis should be discussed with the patient in the clinic, so that patient preference can be taken into account. Patients should receive an early outpatient appointment (ideally within 4–6 weeks) to discuss the implications of this diagnosis with a physician with a clinical interest in Barrett's. Discussion should include the low but significant cancer risk, possible lifestyle changes, whether or not there is an indication for endoscopic surveillance, and the therapeutic options if dysplasia is detected (endoscopic and surgical). Family history for Barrett's oesophagus and OAC should also be recorded. If there is still uncertainty about a diagnosis of Barrett's that requires further work up, this should be clearly explained to the patient to avoid confusion. Written information should be provided for the patient to take away using BSG (see online supplementary appendix 4) or other approved materials such as from MacMillan CancerBACUP (http://www.macmillan.org.uk/Cancerinformation/Cancertypes/Oesophagusgullet/Pre-cancerousconditions/Barrettsoesophagus.aspx) or H-CAS (http://www.h-cas.org/barretts.asp).
Before seeking informed consent for surveillance, the diagnosis of Barrett's oesophagus should have been confirmed on endoscopic and histopathological grounds based on the criteria above. Because of the recent advancement in the endoscopic treatment of HGD and mucosal adenocarcinoma, it is no longer appropriate to restrict surveillance to patients who are fit, and willing, to undergo oesophagectomy. In addition, radiotherapy and/or chemo-radiotherapy may be treatment options in patients with more advanced disease who are deemed not fit for surgery. However, the patient should be fit for repeated endoscopy procedures and endoscopic therapy if HGD or early cancer is detected. Very few studies have used the performance status (PS) to correlate patient fitness with the outcome of endoscopic therapy for GI early cancers. Endoscopic therapy can be safely performed in patients with Eastern Cooperative Oncology Group PS 0–2. Therefore it is reasonable to consider endoscopic surveillance in patients with PS 0–2, provided that the estimated patient life expectancy is sufficiently long for the individual to benefit from surveillance if dysplasia or early cancer were detected.
If surveillance is thought to be clinically indicated, then the clinician should discuss with the patient the possible benefits of surveillance in detecting early-stage tumours and improving cancer survival. However, this discussion should also mention the lack of randomised controlled data to prove the benefits of surveillance, and clinicians must also emphasise to the patient that the actual risk of death from oesophageal cancer is small. Furthermore, the disadvantages of endoscopy surveillance should also be discussed, including the small risks of the procedure and the associated psychological morbidity. For example, in an American study conducted in a population of Veterans with a diagnosis of Barrett's, more than half of the patients missed their follow-up endoscopy, suggesting that not all patients are willing to adhere to surveillance programmes. Clinicians should also emphasise that, as with any monitoring programme, there is a failure rate, in that surveillance cannot guarantee to detect every tumour that may develop. There are no clear data to support how best to impart this complex information, and more work in this area is warranted.
Patients should have early access to an outpatient clinic to be informed about a new diagnosis of Barrett's oesophagus and to have an initial discussion about the pros and cons of surveillance with written information provided (Recommendation grade C).
For a given patient, whether or not surveillance is indicated should be determined on the basis of an estimate of the likelihood of cancer progression and patient fitness for repeat endoscopies, as well as patient preference (Recommendation grade C).
Endoscopic Assessment
Technological advancement with new-generation charge coupled devices has allowed the routine use of high-resolution endoscopy (HRE), which produces images with resolutions ranging from 850 000 to more than one million pixels. HRE allows fine definition of the mucosal layer for the recognition of subtle superficial abnormalities, with theoretical advantage in the recognition of dysplasia and Barrett's oesophagus-related early neoplasia. It is the opinion of the experts that HRE, in conjunction with careful cleaning of the mucosal surface of mucus, saliva and food debris, is the minimum standard for the evaluation of patients with known Barrett's oesophagus; however, to date, there is no randomised trial comparing conventional endoscopy with HRE in Barrett's oesophagus dysplasia detection (Evidence grade IV). In an RCT, HRE performed equally compared with chromoendoscopy and narrow band imaging (NBI) in the overall diagnosis of dysplasia (Evidence grade Ib). Mucolytic agents (eg, 4–10% N-acetylcysteine) or antifoaming agents (eg, simethicone) can be used to disperse excess mucus and bubbles. There is also evidence that longer inspection times during assessment with white light endoscopy is associated with an increased detection rate for HGD and early cancer (Evidence grade III). This should be taken into account when planning how much time to allocate for endoscopic surveillance of very long segments of Barrett's, particularly those longer than 10 cm.
Although transnasal endoscopy has been shown to be accurate in the diagnosis of Barrett's oesophagus (Evidence grade Ib), the randomised studies performed so far either included a small number of patients, or were performed in a low-risk population. Furthermore, it should be noted that the biopsy specimens taken with these endoscopes are significantly smaller, and this may increase sampling bias and hamper the interpretation of dysplasia. Therefore there is currently a lack of robust data to recommend transnasal endoscopy in routine Barrett's oesophagus surveillance.
HRE should be used in Barrett's oesophagus surveillance (Recommendation grade C).
There is insufficient evidence to recommend transnasal endoscopy as a replacement for transoral endoscopy (Recommendation grade C).
Use of Chromoendoscopy and Advanced Endoscopic Imaging
Advanced endoscopic imaging has been investigated to increase the detection of both IM and dysplasia in Barrett's oesophagus with the aim to help target biopsies (Table 7).
Chromoendoscopy uses dyes to enhance endoscopic detection. Methylene blue (MB) is a vital dye actively absorbed by columnar intestinal-type cells and has been used to improve the yield of IM in Barrett's oesophagus (Evidence grade III). In a historical cohort, Sharma and coworkers found significant enrichment of IM in MB-targeted biopsy samples compared with random samples (Evidence grade III). The detection rate of IM and dysplasia during MB chromoendoscopy has been investigated in a number of randomised and cohort studies with conflicting data (Table 7). A recent meta-analysis has found no incremental yield of both IM and dysplasia with MB chromoendoscopy compared with standard endoscopy with random samples (Evidence grade Ia). It should also be noted that MB may damage DNA, which, coupled with the lack of evidence for efficacy, suggests that its use cannot be recommended (Evidence grade III).
Indigo carmine (IC) is a contrast agent that allows detailed inspection of the mucosal pattern in combination with magnification endoscopy. A prospective multicentre study found that the ridged/villous pattern had a 71% sensitivity for IM, while the irregular/distorted pattern had an 83% sensitivity and an 88% specificity for HGD/early cancer (Evidence grade III). The limitation of IC chromoendoscopy is the need for high magnification with consequent narrow field of view. Only one randomised trial has evaluated IC chromoendoscopy for detection of dysplasia in Barrett's, but failed to find an increased rate of dysplasia compared with high-resolution white light endoscopy (Evidence grade Ib).
The value of acetic acid (AA) to improve the diagnostic yield of surveillance endoscopy has also been studied. AA induces intracellular protein denaturation, with swelling of the mucosal surface and enhancement of the architecture. Randomised cross-over studies have produced contradictory results on the diagnostic yield of AA-enhanced magnification endoscopy for IM (Evidence grade Ib). AA-enhanced magnification endoscopy has been shown to have a higher dysplasia yield in Barrett's oesophagus surveillance, with 24% of patients having histological upgrade compared with a previous standard endoscopy with random biopsies performed in a non-specialist centre (Evidence grade III). In a large single-centre prospective study, Pohl et al found that AA-targeted biopsies had a sensitivity of 96.7% and a specificity of 66.5% for a diagnosis of HGD/early cancer. A single-centre retrospective cohort study has showed significantly increased dysplasia yield (p=0.001) compared with standard endoscopy with random biopsies (Evidence grade III). The same group showed that histology on AA-targeted biopsies was more cost-effective than the Seattle protocol in a high-risk population. More data are needed to decide on the usefulness of this technique.
With recent technological advancements, 'virtual chromoendoscopy' has become available, which allows chromoendoscopy without the use of dyes. This is based on light filters (NBI, Olympus) or post-image acquisition processing (i-scan, Pentax and FICE, Fujinon). The most extensively studied 'virtual chromoendoscopy' technique in Barrett's oesophagus is NBI, which highlights the mucosal pattern and the superficial vasculature. A number of different classifications have been proposed to describe mucosal pits in non-dysplastic and dysplastic Barrett's, which yielded high diagnostic accuracy (Evidence grade III). When NBI was compared with standard imaging techniques, one prospective tandem study showed an incremental diagnostic yield for dysplasia in the per-patient analysis (Evidence grade IIa), and two additional studies reported an increased dysplasia detection only in the per-biopsy analysis (Evidence grade Ib). A meta-analysis of eight studies has found that NBI has a sensitivity and specificity of 96% and 94%, respectively, for the diagnosis of HGD, and 95% and 65%, respectively, for the diagnosis of IM. However, the interobserver agreement for the interpretation of the NBI images is only moderate. Overall, despite the finding that NBI performed by an expert endoscopist may increase the targeted yield of dysplasia, it also transpires that high-resolution endoscopy alone is sufficient to maximise dysplasia detection on a per-patient basis.
Autofluorescence imaging (AFI), which exploits endogenous fluorophores excited by short wavelengths, has been studied in the context of Barrett's oesophagus. Initial single-centre cohort studies showed that AFI can improve the diagnostic yield of dysplasia compared with standard endoscopy, but with a false-positive rate as high as 80% (Evidence grade III). To overcome this, AFI has been incorporated into an HRE-NBI scope with magnification, also known as endoscopic trimodal imaging (ETMI). Although an initial multicentre non-randomised feasibility study showed that ETMI increased the diagnostic yield for dysplasia from 63% to 90% compared with standard endoscopy with random biopsies (Evidence grade III), this was not confirmed in two subsequent multicentre randomised studies, where ETMI only improved the diagnostic yield of dysplasia in the per-biopsy analysis (Evidence grade Ib). Overall, these studies showed that, in selected high-risk cohorts of patients, ETMI does not allow the requirement for random biopsies to be abandoned. Further studies in low-risk patients will inform whether AFI can have a role in reducing the number of biopsies without loss of diagnostic accuracy.
Other imaging techniques that have showed some value in Barrett's oesophagus include confocal laser endomicroscopy, spectroscopy and optical coherence tomography; however, further studies are needed to clarify whether they can improve diagnostic accuracy during Barrett's oesophagus surveillance.
In the future, molecular imaging may improve our imaging armamentarium to increase dysplasia detection. Molecular imaging exploits fluorescently labelled molecules that bind with different affinity to dysplastic compared with non-dysplastic cells. Two types of compound have been studied so far. In a proof-of-principle study, Li and colleagues identified a 7-amino acid peptide that binds an OAC cell line more avidly than a non-dysplastic Barrett's oesophagus cell line, and they confirmed the differential binding in surgical specimens of OAC ex vivo. Similarly, Bird-Lieberman and coworkers identified a natural lectin (wheat germ agglutinin) that differentially binds surface glycoproteins of dysplastic and non-dysplastic cells and used an autofluorescence endoscope in surgically resected oesophagi to validate the ex vivo findings. In vivo studies are needed to validate these techniques.
Advanced imaging modalities, such as chromoendoscopy or 'virtual chromoendoscopy', are not superior to standard white light endoscopy in Barrett's oesophagus surveillance and are therefore not recommended for routine use (Recommendation grade A).
Biopsy Protocol
To find dysplasia, endoscopists have generally relied on the directed sampling of any visible lesions, which may be aided by enhanced endoscopic visualisation tools as discussed above, together with systematic, four-quadrant biopsies every 2 cm according to the so-called 'Seattle protocol'. A prospective study has demonstrated a significant increase in the detection of early lesions through the introduction of such a protocol (Evidence grade III). However, adherence to this protocol is limited and ranges from 10% to 79%, with poorer adherence for longer segments, and failure to adhere to the protocol has been shown to result in a significantly lower rate of dysplasia detection. Overall, although intense and time-consuming, the multiple biopsies involved in the Seattle protocol have been demonstrated to be safe when performed by experienced endoscopists. Another limitation of this technique is the high cost generated by processing multiple biopsies, but this still seems justified at the current time in the absence of an alternative. Future RCTs will need to compare cost-effectiveness of the standard practice with alternative techniques such as histology on targeted biopsy samples guided by conventional or virtual chromoendoscopy.
Adherence to a quadrantic, 2 cm biopsy protocol in addition to sampling any visible lesions is recommended for all patients undergoing surveillance. This should also apply to long segments (Recommendation grade B).
Frequency of Surveillance for Non-dysplastic Barrett's Oesophagus
In the previous BSG guidelines published in 2005, the recommended surveillance interval for non-dysplastic Barrett's was every 2 years. However, given the recent data suggesting that the overall risk of malignant conversion is lower than previously thought, we recommend that the interval should be lengthened in line with other guidelines. We therefore advocate a new surveillance strategy whereby the managing clinician synthesises the endoscopic and histopathological findings to tailor the surveillance interval on a more individual basis (figure 3). A degree of variation in this interval is permitted, which may be influenced by the presence of risk factors for the development of cancer.
To summarise, in practical terms, short segments of columnar epithelium with no IM have an extremely low risk of malignant conversion (~0.05% per annum) (Evidence grade III). For these patients, it is recommended to repeat the endoscopy once in 3–5 years time to confirm the findings and account for sampling and measurement error. If there is doubt, the endoscopy could be repeated sooner. If two good-quality endoscopies, each with a minimum number of four oesophageal biopsies where possible, confirm a short segment (<3 cm) with gastric metaplasia only, then discharge is encouraged, as the risks of endoscopy probably outweigh the benefits. In selected cases with a strong personal risk profile for OAC (see recommendation on screening), continued endoscopic surveillance can be considered.
For patients with Barrett's oesophagus shorter than 3 cm, without IM or dysplasia, a repeat endoscopy with quadrantic biopsies is recommended to confirm the diagnosis. If repeat endoscopy confirms the absence of IM, discharge from surveillance is encouraged, as the risks of endoscopy probably outweigh the benefits (Recommendation grade C).
There is evidence that the risk of cancer progression correlates significantly with the length of the Barrett's segment, such that segments shorter than 3 cm have a lower cancer incidence (Table 5). Therefore, in view of the recent evidence supporting a lower cancer risk in non-dysplastic Barrett's oesophagus than previously thought, it is reasonable for patients with short segments containing IM to have a longer endoscopic surveillance interval than patients with long segments. We propose a range of 3–5 years to allow the clinician to tailor surveillance on the perceived individual cancer risk.
Patients with Barrett's oesophagus shorter than 3 cm, with IM, should receive endoscopic surveillance every 3–5 years (Recommendation grade C).
For longer segments (>3 cm), a shorter surveillance interval is more appropriate. This is regardless of the presence of IM, since it is noted, that in long segments, IM is almost always present, but can be missed due to sampling error. We propose a range (between 2 and 3 years), which may be informed by the individual risk factors and patient and physician preference. Because of the poor adherence to the surveillance biopsy protocol for long segments of Barrett's oesophagus, consideration should be given to refer patients with a very long segment (>10 cm) to tertiary referral centres for endoscopic surveillance, as suggested also in the new Dutch guidelines (personal communication).
Patients with segments of 3 cm or longer should receive surveillance every 2–3 years (Recommendation grade C).
Histopathological Diagnosis of Dysplasia
Pathological Features and Reporting of Dysplasia. Online supplementary appendix 3 shows histological examples of Barrett's with different degrees of dysplasia.
There are very few studies that investigated reporting of dysplasia in Barrett's oesophagus. Two studies examined the Vienna classification and found a degree of agreement among pathologists that was at best moderate for HGD, fair for LGD, and poor for indefinite for dysplasia. The approach to reporting upper GI tract neoplasia differs significantly in certain parts of the world and this has led to inconsistency in the terminology used and hence inconsistent data on incidence and clinical progression outcomes. The most recent recommendations by the WHO state that dysplasia should be graded as either low or high grade. The revised Vienna classification for GI mucosal neoplasia attempts to standardise diagnostic terminology into biologically similar groupings with scores of 1–5 depending on the presence or absence of dysplasia or malignancy.
Revised Vienna Classification and Dysplasia Subtypes. 1. Negative for Dysplasia: This includes normal epithelium, metaplastic epithelium showing reactive or regenerative changes, and mucosa showing reactive/regenerative changes including nuclear enlargement, nuclear hyperchromasia and prominent nucleoli.
2. Indefinite for Dysplasia: This category is used for cases where the morphological features between true dysplasia and regenerative/inflammatory atypia are blurred. It is important to appreciate that this diagnosis may in fact mean that the patient has features suspicious of HGD, but not enough certainty is present to warrant this call. This may be due to technical factors, such as poor staining, poor orientation, cross cutting or denuded surface epithelium, or to severe active inflammation or ulceration leading to marked atypia, precluding a confident diagnosis of dysplasia. In other cases, the epithelium appears abnormal, but the features are not sufficiently well developed to justify a definite diagnosis of dysplasia. Features favouring dysplasia are the presence of an abrupt transition from normal to atypical epithelium, together with nuclear pleomorphism, atypical mitoses and loss of nuclear polarity. Evidence of 'surface maturation'—that is, loss of the cytological atypia seen in the deeper glands as the mucosa matures into the surface epithelium—is often taken as the best marker to favour regeneration rather than dysplasia, although this is also not invariably true (eg, crypt dysplasia described below). Explicit mention in the pathology report of the reason justifying this diagnosis can be useful to aid patient management.
3. Low-grade Dysplasia: In LGD, glandular architecture is relatively preserved and the diagnosis is made on the basis of cytological atypia.
Morphological patterns of LGD:
LGD generally shows an 'adenomatous' cytological appearance (resembling the dysplastic changes associated with adenomatous polyps of the colon) in which nuclei are elongated (pencil shaped), slightly enlarged and hyperchromatic with inconspicuous nucleoli. There may be mild pleomorphism, mucin depletion, mild loss of polarity, nuclear crowding, and stratification of nuclei up to three-quarters of the height of the cell, but not touching the luminal surface. Mitoses and apoptotic debris may be seen on the surface or in the upper portions of the glands. Evidence of loss of 'surface maturation'—that is, presence of cytological atypia seen in the deeper glands—into the surface epithelium is often taken as the best marker to distinguish true dysplasia from regenerative atypia; however, in the presence of ulceration, regenerative surface epithelium may also closely mimic LGD.
A 'non-adenomatous' (foveolar) type composed of small round cells with abundant cytoplasm may occasionally be seen. Although this is less well characterised, cells with nuclear/cytoplasmic ratios <50% are probably best put into this low-grade category.
4a. HGD (Incorporating Carcinoma in Situ): The distinction between HGD and LGD is largely based on the presence of architectural changes in conjunction with more marked nuclear atypia. These changes may be accompanied by complex architectural changes including a papillary or villous surface (although villiform change may also be seen in reactive epithelium), in conjunction with branching, complex budding or back-to-back 'crowding' arrangements. Intraluminal papillae, bridges or cribriform patterns are also seen. There are increased numbers of atypical mitoses on upper levels of crypts, together with mucin depletion and a loss of nuclear polarity. Of note, HGD can be accompanied by acute inflammation and should not be downgraded in its presence.
Morphological Patterns of HGD:
'Adenomatous' cytological appearance: nuclei are elongated, pencil shaped, enlarged, hyperchromatic and show crowding and stratification up to the luminal surface of the cells. The distinction between the upper end of 'low grade' and 'high grade' dysplasia can be subjective.
'Non adenomatous', which includes the term foveolar dysplasia: cells have a cytological appearance characterised by rounded nuclei showing marked nuclear enlargement and marked atypia with increased nuclear/cytoplasmic ratios, irregular nuclear membranes (including angular edges), coarse chromatin, and prominent or irregular nucleoli. The foveolar type may have a more bland appearance, comprising small round nuclei with conspicuous nucleoli. The grading of this variant is less well characterised; however, the nuclear/cytoplasmic ratio appears to be more important for grading, with high ratios (nuclei involving >50% of cell) being put into a high-grade category.
Crypt Dysplasia: Significant cytological atypia in the crypt bases with surface maturation has been reported in up to 7.3% of cases of Barrett's. Previously, this would have been regarded as either 'negative for dysplasia' or 'indefinite for dysplasia', as the atypia does not reach the surface epithelium. It seems likely, however, that crypt dysplasia represents an early stage in the development of dysplasia, and the atypia is highly likely to progress up to the surface over time and so warrants recognition. Crypt dysplasia stands out as a focus that is distinctly different from the surrounding crypts and can appear as low-grade or high-grade cytological atypia. Although the dysplasia can be of the 'adenomatous' or 'non-adenomatous' round cell type, the most common features are nuclear enlargement, loss of polarity, marked pleomorphism with irregular shapes and sizes, nuclear crowding, increased mitotic activity and goblet cell dystrophy. Crypt dysplasia should not be diagnosed purely on the basis of stratification and hyperchromasia in the absence of significant nuclear pleomorphism, as these changes may often be seen in regenerative cryptal epithelium. p53 immunohistochemistry may be a helpful adjunct for the assessment of crypt dysplasia. Crypt dysplasia should be reported according to the degree of dysplasia present. If there is uncertainty, then the 'indefinite' category may be appropriate. Some pathologists favour putting cases with high-grade features into an 'at least low grade' category in view of the likely early nature of the lesion and the implications of a high-grade diagnosis; however, cases of isolated crypt dysplasia are probably best managed as low grade until further data become available.
4b. Intramucosal Carcinoma (Including Suspicious for Invasive Carcinoma): Intramucosal carcinoma is a lesion in which neoplastic cells have penetrated the basement membrane and invaded the lamina propria or muscularis mucosae, but without invasion into the submucosa. However, histological recognition of lamina propria invasion may be difficult because of the absence of objective criteria. Patterns of lamina propria invasion that are used by gastrointestinal pathologists include sheets of neoplastic cells, abortive angulated glands, a never-ending/anastomosing gland pattern, a highly complex cribriform arrangement of glands, tightly packed small tubular glandular arrays, and single-cell infiltration. Recognition of each of these patterns is somewhat subjective, with κ statistics varying between 0.21 and 0.47, suggesting poor or, at best, moderate agreement. If definitive submucosal invasion is in question, the term 'suspicious of invasive carcinoma' can be used.
5. Submucosal Invasion by Adenocarcinoma: Unequivocal invasion of submucosa or deeper structures often accompanied and characterised by a desmoplastic response in the tissue stroma to invasive tumour cords/acini.
Given the important management implications for a diagnosis of dysplasia, we recommend that all cases of suspected dysplasia are reviewed by a second GI pathologist, with review in a cancer centre if intervention is being considered (Recommendation grade C).
Given the difficulties associated with the management of the 'indefinite for dysplasia' category, all such cases should also be reviewed by a second GI pathologist, and the reasons for use of the 'indefinite for dysplasia' category should be given in the histology report in order to aid patient management (Recommendation grade C).
Dysplasia Reporting and Reproducibility. It has long been recognised that there is inter- and intra-observer variability in the diagnosis of GI tract dysplasia. This relates to differentiating between HGD and intramucosal adenocarcinoma, HGD and LGD and also distinguishing between regenerative changes and LGD. In the case of definite dysplasia, this is because these divisions involve unnatural cut-offs along a biological/histological continuum. Studies have shown that the prediction of progression of oesophageal dysplasia is improved if at least two expert pathologists agree on the diagnosis and increases further when a greater number of pathologists concur with the diagnosis. For practical reasons, and in day-to-day diagnostic practice, a diagnosis of dysplasia in the setting of Barrett's should be corroborated by a second pathologist with a specialist GI interest. The Royal College of Pathology recommends that 'double' reporting of a diagnosis of HGD in the upper GI tract should be mandatory and this has been confirmed by consensus statements agreed by Barrett's international experts. We have extended this consensus reporting to all grades of dysplasia.
Aids to Histological Diagnosis of Dysplasia and p53 Immunostaining. Of all the putative experimental molecular markers, the one with the greatest body of evidence and which can also be applied in the routine clinical setting is immunohistochemistry for nuclear p53. Although the p53 positivity rate in Barrett's oesophagus dysplasia is variably reported in the literature, ranging from 50% to 89%, when positive it can improve interobserver agreement for reporting dysplasia and can be a powerful predictor of progression, with an OR between three and eight in different studies (Table 6). In a study from Skacel et al, who analysed factors predictive of progression in patient with LGD, p53 immunostaining positivity and 100% agreement among three GI pathologists on LGD diagnosis correlated with the risk of progression, suggesting that p53 might improve interobserver agreement. This was replicated in a later study. Interpretation of p53 immunostaining can be problematic and poorly reproducible subject to variation in methodology and interobserver variation. Notwithstanding this, some pathologists find staining for p53 of use, especially in distinguishing between atypical reactive proliferation (indefinite for dysplasia) and true LGD. Low background wild-type p53 expression is often seen in nuclei of normal columnar and basal layers of squamous mucosa, which is a useful baseline to identify the overexpression pattern typical of dysplasia. Overexpression is generally a consequence of mutations that stabilise the inactivated protein. However, not all p53 mutations lead to stabilisation of a mutated inactive p53 protein. A study performed in non-small cell lung cancer showed that, as opposed to missense mutations, the majority of null mutations did not lead to p53 overexpression. In such cases, mutation is expected to lead to failed translation of the protein. In fact, an absent pattern of p53 immunostaining, when compared with normal wild-type background, is now recognised as an abnormal pattern which also occurs in dysplasia as a result of silencing mutations of the p53 gene. Online supplementary appendix 3 shows immunohistochemical examples of Barrett's with overexpression and loss of p53.
The addition of p53 immunostaining to the histopathological assessment may improve the diagnostic reproducibility of a diagnosis of dysplasia in Barrett's oesophagus and should be considered as an adjunct to routine clinical diagnosis (Recommendation grade C).
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