is essential for treatment plans,

follow-up visits, and therapeutic

interventions.

Three Layers of Paint. Photograph courtesy of Craig Damlo. www.soapboxrocket.com.

Surgical and Molecular of Barrett Sherma Zibadi, MD, PhD, and Domenico Coppola, MD

Background: Patients with Barrett esophagus (BE) are predisposed to developing and . Adenocarcinoma, which is associated with BE, is the most common type of esophageal tumor and, typically, it has an aggressive clinical course and a high rate of mortality. Methods: The English-language literature relating to tumor epidemiology, etiology, and the of BE was reviewed and summarized. Results: The role of pathologists in the diagnosis and pitfalls associated with grading Barrett dysplasia is addressed. Current molecular testing for Barrett neoplasia, as well as testing methods currently in develop- ment, is discussed, focusing on relevant tests for diagnosing tumor types, determining prognosis, and assessing therapeutic response. Conclusions: Grading is essential for developing appropriate treatment plans, follow-up visits, and therapeutic interventions for each patient. Familiarity with current molecular testing methods will help physicians correctly diagnose the and select the most appropriate therapy for each of their patients.

Introduction tinal are also defined as Barrett mucosa.1 Barrett mucosa refers to a metaplastic process in- Barrett esophagus (BE) is more common in men duced by the acid-peptic content of the and has a male:female ratio of 3:1 to 7:1.2 Although that then erodes the esophageal squamous mucosa. BE can present at any age, its peak incidence oc- During endoscopy, changes of any length recognized curs in the sixth decade of life and it occurs more as columnar-type mucosa in the distal esophageal ep- frequently in whites (80% of patients) than patients ithelium that, on biopsy, are confirmed to have intes- of other races.3 Patients with BE are predisposed to esophageal adenocarcinoma (EAC; 5-year survival rate of 14%–22%), which has increased in incidence From the Department of Pathology and Oncological Sciences, Uni- during the last 30 years.4,5 versity of South Florida Morsani College of Medicine (SZ), and the Guidelines for diagnosis and surveillance inter- Departments of Anatomic Pathology (DC), Chemical Biology and Molecular Medicine, and Tumor Biology, H. Lee Moffitt Cancer vals for patients diagnosed with BE have been es- Center & Research Institute, Tampa, Florida. tablished and developed by the American College of Submitted July 8, 2014; accepted September 11, 2014. Gastroenterology (Table 1).1 Address correspondence to Domenico Coppola, MD, Department of Anatomic Pathology, Moffitt Cancer Center, 12902 Magnolia Etiology and Pathogenesis Drive, Tampa, FL 33612. E-mail: [email protected] Typically, patients acquire BE as a result of gastro- No significant relationships exist between the authors and the 6,7 companies/organizations whose products or services may be esophageal reflux. The factors putting patients at referenced in this article. risk for BE are several and include duodenogastric

April 2015, Vol. 22, No. 2 Cancer Control 177 Table 1. — ACG Guidelines for BE of BE varies between 14% and 40%,26 and 7% to Dysplasia Diagnosis Follow-Up 15% of patients will have EAC at the time of initial 27 None present 2 EGDs with biopsy in 1 y EGD every 3 y diagnosis of BE. Low grade Highest grade on repeat EGD 1-y interval with biopsies within 6 mo until no Pathology Expert pathology dysplasia (× 2) Grossly, the esophagogastric junction in BE is dis- confirmation placed by areas of velvety, gastric-type mucosa that 11 High grade Mucosal irregularity Continued 3-mo macroscopically appear salmon in color. These ar- Repeat EGD with biopsies surveillance or eas of gastric-type mucosa proximally extend from to rule out esophageal intervention their typical location. The area of BE may be ulcerat- within 3 mo based on results and outcomes ed or contain islands of residual, whitish squamous Expert pathology confirmation mucosa surrounded by pink-colored, gastric-type Invasive Expert pathology confirmation Esophagectomy mucosa (Fig 2). It is possible that the health care adenocarcinoma professional may be unable to distinguish the ap- ACG = American College of Gastroenterology, BE = Barrett esophagus, EGD = esophagogastroduodenoscopy. pearance of simple BE — grossly and endoscopi- 28 Adapted from Macmillan Publishers Ltd: Wang KK, Sampliner RE; Practice Param- cally — from BE with early cancer and dysplasia. eters Committee of the American College of Gastroenterology. Updated guidelines With regard to its location, BE characteristically in- 2008 for diagnosis, surveillance and therapy of Barrett’s esophagus, Am J Gastro- enterol, 103(3):788-797, copyright 2008. volves the lower one-third of the esophagus, but it may involve the middle and upper esophagus as it reflux, which is a delay in the clearance of acid juice progresses.11 from the esophagus, a resting pressure in the sphinc- Previously, BE was histologically indicated by the ter of the lower esophagus less than the optimal rate, presence of intestinal metaplasia or the gastric, cardia, and the presence of hiatal hernia.8-11 or oxyntic type of columnar mucosa in the esopha- Other etiological factors include postgastrectomy gus.29,30 However, in the modern era of endoscopy, bile reflux12 and esophageal injury (eg, lye ingestion, the American Gastroenterological Association requires tobacco use).13,14 Alcohol intake does not promote the presence of unequivocal intestinal metaplasia for BE15 and Helicobacter pylori has a protec- there to be a diagnosis of BE.31,32 tive effect, particularly if Helicobacter possesses the The nonintestinalized, gastric-type mucosa was CagA gene.16 In infants, BE may arise from congen- previously thought to have no potential for malig- ital rests of gastric columnar reacting to nant transformation; however, in recent years, small gastroesophageal reflux.5,17 (presumably early) EACs arising in the absence of The way in which normal squamous epi- intestinal metaplasia have been reported.33-35 Thus, thelium transitions into specialized columnar these findings suggest that the identification of gob- epithelium is not yet clear.18 However, based let cells as a requirement for diagnosing BE should on the theory of restitution and replication pro- be challenged.26-38 For example, the requirement no posed by Jankowski et al,19 after the acid-pep- longer exists in Japan, which was the first country tic content of the stomach erodes the parabasal to propose the existence of malignant potential for and superficial layers of the squamous mucosa nonintestinalized BE.35,38 In recent years, this view (Fig 1A), the basal progenitor cells move into de- has been supported by study findings indicating that nuded areas.5 In the setting of persistent gas- –containing epithelium has similar DNA troesophageal reflux, these multipotential stem abnormalities as metaplastic esophageal columnar cells become resistant to acid and bile, selective- epithelium without goblet cells.39,40 ly differentiating into columnar epithelium that Although goblet cell metaplasia is not always an secretes mucin (Fig 1B).5,19 By contrast, the up- obligate precursor of , it is important to ward movement of the columnar epithelium from point out that the recognition of goblet cell metapla- the stomach could metaplasia due to wound sia remains the most important criterion in clinical healing of the ulcerated mucosa.5 practice for the routine histological recognition and diagnosis of BE. Cancer Risk In BE, the intestinalized mucosa rarely has a vil- BE is a premalignant condition associated with EAC liform appearance and Paneth cells, absorptive cells, and dysplasia.20,21 During the last several decades, and intestinal-type endocrine cells are typically absent the incidence of EAC has risen.21-23 Approximately because of the incomplete form of metaplasia pres- 10% to 20% of patients with symptomatic gastro- ent. Uncommonly, the complete form of metaplasia esophageal reflux who undergo endoscopy have presents in conjunction with the usual incomplete BE.24 However, many patients with BE are asymp- form of metaplasia. tomatic.25 The prevalence of dysplasia in the setting Shaped like barrels, goblet cells have a distended

178 Cancer Control April 2015, Vol. 22, No. 2 Bile A Acid

Superficial compartment Desquamating cells

Parabasal compartment

Capillaries

Basal compartment Positively labeled nuclei/cells by autoradiography Submucosa Papillae region Basal region Smooth muscle

B Bile Acid

Superficial compartment Desquamating cells

Parabasal compartment

Capillaries

Positively labeled nuclei/cells Basal compartment by autoradiography

Submucosa Papillae region Basal region Smooth muscle

C Bile Acid Rapidly dividing mucin-secreting cells

Superficial compartment Desquamating cells

Parabasal compartment

Capillaries

Positively labeled nuclei/cells Basal compartment by autoradiography

Submucosa Papillae region Basal region Smooth muscle

Fig 1A–C. — (A) Basal progenitor cells move into denuded areas after the gastric acid-peptic content from the stomach has eroded the squamous mucosa. (B, C) Persistent gastroesophageal reflux causes multipotential stem cells to differentiate into columnar, mucin-secreting epithelium resistant to acid and bile. Reprinted from Am J Pathol, 154(4), Jankowski JA, Wright NA, Meltzer SJ, et al, Molecular evolution of the metaplasia-dysplasia-adenocarcinoma sequence in the esophagus, 965-973, Copyright 1999, with permission from Elsevier.

April 2015, Vol. 22, No. 2 Cancer Control 179 which it arises and within the superficial layer of ep- ithelium is the defining characteristic of dysplasia.41-43 Criteria have been established for grading dysplasia in BE44 that consider a combination of features, such as gland architecture, the presence of , ulceration, or erosion, cytological features, and the level of maturation of the surface epithelium.28

Negative When cases of BE are negative for dysplasia, the un- derlying glands will not be as mature as the surface epithelium.30 The nuclear:cytoplasmic ratio is lower than that of the deeper glands.30 Lamina propria is abundantly present between the glands.30 Cytologically, nuclear polarity is retained, the nuclear membrane is smooth, and the nucleoli are inconspicuous. In the event of prominent inflammation, reactive changes will be present but surface maturation is maintained.44,45

Indefinite A diagnosis of indefinite for dysplasia is used for cases with worrisome changes but not diagnostic for dysplasia, typically because of the presence of marked inflammation. Normal glandular architec- ture may be observed but crowding will be present, and, cytologically, nuclear hyperchromasia, nuclear membrane irregularities, and increased mitoses at Fig 2. — Gross appearance of BE as a velvety, salmon-colored mucosa the base of the glands (but with surface maturation) extending above the gastroesophageal junction. BE = Barrett esophagus. may be present. Nuclear polarity is not lost in cases cytoplasm filled with acidic mucin and can be identi- of indefinite for dysplasia. Such a diagnosis may also fied using hematoxylin and eosin stain, and, if neces- be used in the presence of tangential embedding that sary, also highlighted with either Periodic acid–Schiff does not allow for the assessment of the superficial or Alcian blue (pH 2.5) stain.11 portion of the glands.44,46

Role of Pathologists Low Grade The role of pathologists when dealing with esoph- In cases of BE with low-grade dysplasia (LGD), the agogastric biopsy or resection is twofold: mucosal surface is similar to the underlying glands, 1. Identification of BE: The pathologist must which reveal nuclear stratification and nuclear polarity differentiate between nonmetaplastic (Fig 3).30 Glandular crowding can be seen, but the columnar epithelium (eg, hiatal hernia, lamina propria is still present between the glands. The ectopic gastric mucosa), intestinal nuclei are elongated, hyperchromatic, and have irregu- metaplasia of the cardia, and true BE. lar contours and sustain a moderate increase in mitotic 2. Identification and grading of dysplasia: activity. The nuclear:cytoplasmic ratio will be mildly This task will affect patient treatment. increased. In the setting of LGD, minimal inflammation When dealing with endoscopic mucosal resection may be present and nucleoli are not prominent.45-47 specimens, pathologists are responsible for assessing the resection margins. If invasive cancer is present, High Grade then pathologists are also required to report the dis- In the setting of BE with high-grade dysplasia (HGD), tance of the invasive carcinoma from the deep mar- surface maturation is lacking; glandular distortion gin, the presence of angiolymphatic , and, if and glandular crowding are both present with a min- possible, the level of invasion within the muscularis imal amount of lamina propria between the glands.47 mucosa, submucosa, or both. The glands may become dilated with luminal necrot- ic debris. The nuclei display marked enlargement, Dysplasia anisocytosis, , hyperchromasia, mem- The presence of the neoplastic epithelium confined brane irregularity, and prominent nucleoli. The mitotic within the basement membrane of the gland from figures are numerous and the nuclear:cytoplasmic

180 Cancer Control April 2015, Vol. 22, No. 2 observing these findings.48,49

Clinical Significance One study found that HGD was a marker of meta- chronous or synchronous adenocarcinoma in ap- proximately 40% of cases.50 In another study, Weston et al51 found that HGD progressed to adenocarcinoma or multifocal HGD in 53% of cases.

Pitfalls Associated With Grading The grading of dysplasia in BE is subjective due to sampling errors as well as interobserver and intraob- server variations. In addition, the biological behavior of the lesion and its degree of dysplasia may not correlate.34,52 However, the task of grading dysplasia in BE is important and can sometimes have crucial therapeutic implications (see Table 1).1 For this rea- son, biopsy findings in cases of BE, particularly in the setting of HGD and carcinoma, should be reviewed by a second pathologist with expertise in gastrointestinal pathology.53 Morphological and molecular data suggest that dysplasia in BE begins at the base of the crypts.54 Crypt dysplasia is thought to progress into the re- maining portion of the crypt and surface epithelium.55 Histologically, crypt dysplasia may exhibit the same Fig 3. — BE with low-grade dysplasia. The glands exhibit mild architectural cytological changes characteristic of LGD or, rarely, distortion and are lined by elongated, stratified, and hyperchromatic nuclei have high-grade cytological features with very large with retained polarity (hematoxylin and eosin, × 200). BE = Barrett esophagus. nuclei, a large nuclear:cytoplasmic ratio, irregular nu- clear membranes, nuclear polarity loss, and eosino- philic cytoplasm. One study had good interobserver agreement when diagnosing crypt dysplasia.55

Intramucosal Carcinoma In intramucosal adenocarcinoma, the neoplastic cells are limited to the mucosa (Fig 5); however, these cells will make their way to the basement membrane and

Fig 4. — BE with high-grade dysplasia. The epithelium is composed of cells containing enlarged, polygonal, and hyperchromatic nuclei that ex- hibit prominent nucleoli. Polarity is lost (hematoxylin and eosin, × 200). BE = Barrett esophagus. ratio will be large. Nuclear polarity is lost (Fig 4). Inflammation will be minimal. In the setting of HGD, the presences of superficial ulceration, cytologically high-grade nuclei, solid nests of dysplastic cells with multiple secondary lumina, and/or dilated dysplastic tubules containing granular eosinophilic and nuclear Fig 5. — BE with intramucosal carcinoma showing single cells and debris may indicate an unsampled invasive carcino- a syncytial arrangement of the cells infiltrating the lamina propria. ma. Additional biopsies should be performed when BE = Barrett esophagus.

April 2015, Vol. 22, No. 2 Cancer Control 181 into the muscularis mucosae or the lamina propria, example, following mucosal injury, the mucosa rapidly but they do not go beyond this point.5 Histologically, heals by restitution. During this phase, metaplastic an effacement of the architecture of the lamina propria clones with specialized intestinal metaplasia propa- can be seen,28 as well as back-to-back microglands, gate in the presence of persistent gastroesophageal re- small clusters, or infiltrating single cells.56 A syncy- flux, followed by the loss of cell-cycle checkpoints. In tial growth pattern may also be observed. Typically, addition, because of increasing proliferation, genomic is absent or not completely developed, instability is thought to be associated with slow clonal perhaps accounting for the poor intraobserver repro- expansion.19,63,64 The inhibition of occurs ducibility in the diagnosis of this entity.45,46 late and only in cases of HGD. Altering cell adhesion can precede invasive cancer,65 and the accumulation of Invasive Adenocarcinoma genetic errors that follows may be associated with the Invasive adenocarcinoma has a variable gross ap- generation of multiple, transformed cell clones, thus pearance and size (≤ 10 cm) and typically involves expanding the number of altered cells with metastatic the distal one-third of the esophagus. Tumor cells or angiogenic potential.19 infiltrate beyond the muscularis mucosa in the setting of invasive adenocarcinoma and oftentimes evoke a Aberrant p53 desmoplastic reaction.5 The microscopic pattern is Several studies have observed alterations in p53 mostly of the intestinal form, however diffuse, and during the progression of BE.66-68 Typically, aberrant signet ring cell or mixed types can be observed. The p53 is accumulated in the nucleus of the neoplas- tumor cells may produce mucin, and the tumor itself tic cells, particularly in cases of HGD (Fig 6). When will generally be well to moderately differentiated. they were detected by polymerase chain reaction single-strand conformation polymorphism, the p53 Intestinal Metaplasia of the Cardia molecular alterations coincided with the level of p53 Intestinal metaplasia of the cardia has a prevalence protein overexpression detected by immunohisto- of 6% to 25%.57,58 Generally in this setting, the cardia chemistry in Barrett adenocarcinoma and BE-HGD contains areas of intestinal metaplasia with goblet but were discordant in BE-LGD.68 cells and a normal Z line. H pylori infection, gastro- Currently, the only accepted predictor for pro- esophageal reflux, and carditis can all be causes for gression is a histological diagnosis of LGD; its pre- intestinal metaplasia of the cardia. Patients with intes- dictive value is low.69,70 However, the immunohisto- tinal metaplasia of the cardia are at increased risk for chemistry status of p53 has diagnostic value and can gastric cardia cancer; however, this type of intestinal help predict neoplastic progression in patients with metaplasia may go into regression if its cause is treat- BE. Using more than 12,000 biopsy samples from ed; furthermore, research suggests that this condition 635 patients, including those with BE who developed is not typically associated with dysplasia.57,59 HGD, those with invasive adenocarcinoma, and those without neoplastic progression, Kastelein et al70 stud- Endoscopic Mucosal Resection ied the protein expression of p53 via immunohisto- Gastroesophageal endoscopic mucosal resection is chemistry. The researchers found that 49 patients (8%) a procedure to remove dysplastic or carcinomatous developed HGD or invasive adenocarcinoma during mucosa from the stomach, esophagus, or both.52 It the follow-up period.70 The results of the study in- is performed on low-risk adenocarcinomas using ei- dicated that patients with a diagnosis of LGD had a ther an injection- or cap-assisted endoscopic mucosal resection. Low-risk adenocarcinoma is defined as a stage 1A tumor no larger than 20 mm in size with- out vascular or lymphatic invasion and a histological grade of G1 or G2. Following endoscopic mucosal resection, a com- plete tumor response was reported in 99% of patients after a follow-up time of 36.7 months.60 Recurrent or metachronous lesions were present in 11% of patients and the 5-year survival rate was 98%.61,62

Molecular Alterations Jankowski et al19 highlight restitution, cell cycle and apoptosis, adhesion, DNA instability, and invasion as potential factors in the molecular pathways associated Fig 6. — p53 immunostaining of Barrett mucosa showing strong nuclear with the progression of BE to adenocarcinoma. For positivity and negativity in high- and low-grade dysplasia, respectively.

182 Cancer Control April 2015, Vol. 22, No. 2 positive predictive value for neoplastic progression of Table 2. — FISH Probe Sets 70 15%. Conversely, patients with LGD and concurrent Probe Fluorophore altered p53 expression had a positive predictive value Set 70 Spectrum Spectrum Spectrum Spectrum of 33%. Thus, these data show that p53 alterations Red Green Aqua Gold are a better predictor of tumor progression than BE I LSI 9p21 LSI 5p15 CEP 9 LSI 5q21–22 70 with LGD. (P16) (APC) II CEP Y LSI 17q11.2–12 CEP 17 LSI 17p13.1 ERBB2 Amplification and Overexpression (ERBB2a) (P53) 71 Using a tissue microarray, Hu et al looked at II CEP Y LSI 17q11.2–12 CEP 17 LSI 17p13.1 34 cases of BE, 18 cases of LGD, 15 cases of HGD, and (ERBB2a) (P53) 116 cases of EAC to study ERBB2 (formerly known as aFormerly known as HER2/neu. HER2 or HER2/neu) amplification using chromogenic CEP = centromere enumeration, FISH = fluorescence in situ hybridization, LSI = locus-specific indicator. in situ hybridization. They found ERBB2 amplification Reprinted from J Mol Diagn, 8(2), Brankley SM, Wang KK, Harwood AR, et al, in 21 of the cases of EAC (18.1%), ERBB2 overexpres- The development of a fluorescence in situ hybridization assay for the detection of dysplasia and adenocarcinoma in Barrett’s esophagus, 260-267, Copyright 2006, sion in another 14 cases of EAC (12.1%), and ERBB2 with permission from Elsevier. protein overexpression in 1 case of HGD (6.7%).71 All of the cases of LGD and BE had negative results for were similar in all cases of BE, which clustered to- fluorescence in situ hybridization (FISH) and immu- gether and were different from the EAC cases (also nohistochemistry.71 clustered together).75 The researchers also differenti- A recent study looked at the immunohistochemi- ated adenocarcinoma from squamous cell carcinoma cal expression of ERBB2 in BE, BE with dysplasia, and and signet ring cell carcinoma. adenocarcinoma.72 The overexpression of ERBB2 in a In another study, Helm et al76 analyzed microar- significant number of HGD cases was an unexpected rays so that they could obtain expression profiles finding that may have clinical implications. The low from individual genes based on biopsy samples expression level of ERBB2 in LGD was a novel find- (9 cases of EAC/BE from which 3 had arisen). ing and may indicate the role of ERBB2 in the early To use as a reference point, the researchers pooled stages of Barrett .72 samples of BE from 6 patients with BE but without cancer or dysplasia. Their findings showed that in- Molecular Testing creasing changes in gene expression were related Molecular testing has been proposed to accurately to the progression from BE to EAC. The early loss detect and grade dysplasia and adenocarcinoma in of gene function that governs differentiation began patients with BE.73 Among these tests, FISH can be prior to such histological change, whereas a gain in performed on endoscopic esophageal brushings, gene function was associated with the progression of which can be used to sample a large diseased area invasiveness and remodeling.76 of the esophagus. In a study by Brankley et al,74 Using DNA microarrays, Hao et al77 focused various probe combinations were used to select a on the contributions of the stroma to the neo- combination of FISH probes specific for identify- plastic process, studying the gene expression ing dysplasia and carcinoma in the setting of BE profiles of EAC or BE specimens obtained via bi- (Table 2). A total of 170 brushing specimens from opsy and the associated normal duodenum and 138 patients with BE were examined. Biopsy results esophagus among 17 volunteers. The research- were used as the gold standard. The authors found ers found a unique expression profile for each that probes to 8q24, 9p21, 17q11.2, and 20q13.2 tissue. Both EAC and BE had a unique set of detected LGD with sensitivity and specificity rates 37 stromal genes distinct from those found in normal of 70% and 89%, respectively.74 The same probes tissue.77 Collagen 5A2 and periostin were validated were also able to identify HGD with sensitivity and by in situ hybridization. No differences in gene specificity rates of 84% and 93%, respectively, and expression were seen between long- and identify adenocarcinoma with sensitivity and spec- short-segment-type BE.77 The authors concluded ificity rates of 94% and 93%, respectively.74 Thus, that stromal gene changes precede transformation. these probes have been proposed for testing in prospective clinical trials. Ultrasensitive Complementary DNA Selaru et al75 used global gene expression pro- Microarray Chip filing to study BE and using DNA In 2007, Ito et al78 introduced an ultrasensitive cDNA microarrays. They examined 13 esophageal resections microarray chip for gene-expression profiling to be or biopsies, including 7 cases of BE and 6 cases of used in preoperative esophageal cancer biopsies EAC, using a microarray platform containing 8,000 that requires 0.1 to 0.01 µg of total ribonucleic acid complementary DNA (cDNA) clones. Gene profiles (RNA). However, no RNA amplification is needed.

April 2015, Vol. 22, No. 2 Cancer Control 183 Nucleus Cytoplasm the personalized management of Barrett esophagus. RNase III Dicer Pathologists are essential for planning the most ap- Pre-miRNA TRBP propriate follow-up period needed for each patient. RAN- The advent of molecular testing for Barrett esopha- GTP gus also provides new tools for selecting the most Exportin 5 Mature-miRNA appropriate therapy for each patient. RISC DGCR8 References Pri-miRNA Drosha Imperfect 1. Wang KK, Sampliner RE; Practice Parameters Committee of the Perfect complementarity RNase III complementarity American College of Gastroenterology. Updated guidelines 2008 for diag- Polymerase II, III nosis, surveillance and therapy of Barrett’s esophagus. Am J Gastroenterol. 2008;103(3):788-797. Cap AAAAAA 5’ 3’ Cap ORF AAAA 2. Rashid F, Khan RN, Iftikhar SY. Probing the link between oestrogen receptors and oesophageal cancer. World J Surg Oncol. 2010;8:9. 5’ 3’ Translational 3. Pohl H, Welch HG. 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