Clinical Services

Barrett's BioStrat® Assay

Barrett's Esophagus - Background

It is estimated that there will be 18,170 new cases and approximately 15,450 deaths from esophageal cancer in the United States during 2014.1 Approximately 80% of these tumors will be esophageal adenocarcinoma (EA) and the remaining 20% will be squamous cell carcinoma. The majority, if not all, of EA are thought to arise in patients with Barrett's esophagus (BE), a pre-neoplastic condition caused by metaplasia of the normal squamous mucosa of the distal esophagus into specialized intestinal mucosa containing goblet cells.2 BE is caused by chronic gastroesophageal reflux disease (GERD), a disorder that affects more than 20 million Americans on a daily basis. Six to fourteen percent of people with chronic GERD will develop BE. The incidence of EA in patients with BE has been reported to be approximately 0.5%-1.0% per year and the lifetime cancer risk for patients with BE is about 5%.3-5

The evolution to esophageal adenocarcinoma from BE is generally accepted to be a multistep progression as follows: 1) intestinal metaplasia (IM) of the normal stratified squamous epithelium, 2) low-grade dysplasia (LGD), 3) high-grade dysplasia (HGD) and 4) EA. The incidence of EA is increasing more rapidly than any other malignancy in the United States.6 Because EA has a poor prognosis unless detected and treated at its earliest stages, the guidelines from gastrointestinal organizations have recommended that patients with BE undergo periodic endoscopic surveillance with biopsies.7 However, the histologic diagnosis of dysplasia has several shortcomings, which include: 1) limited sampling of affected mucosa, 2) extensive time required to obtain the recommended number of biopsies leading to lack of compliance, 3) poor pathologic interobserver reproducibility, and 4) relatively poor ability of histological findings to predict which patients are likely to progress to adenocarcinoma.8-10 It has been estimated that endoscopic surveillance protocols that utilize four-quadrant biopsies every cm only sample about 1-2% of the affected mucosa. This limited sampling may lead to false negative pathology results or to under-staging (e.g., pathology results showing only IM or LGD in a patient who has HGD or EA). Thus, there is a need for more accurate methods for distinguishing HGD and EA from LGD/IM/Normal and LGD from Normal/IM patient samples.

Recently, studies have demonstrated the ability of fluorescence in situ hybridization (FISH) to detect chromosomal alterations in cells collected from endoscopic brushings and have identified probe sets useful for the detection of dysplasia and esophageal adenocarcinoma, as well as differentiate low-grade dysplasia from high-grade dysplasia/adenocarcinoma in patients with Barrett's esophagus.11-13

Introduction to Barrett's BioStrat® Assay

The Barrett's BioStrat® Assay provides a method for the detection of cytogenetic evidence of Barrett's associated neoplasia in cytology (esophageal brushing) specimens and in formalin-fixed, paraffin-embedded esophageal tissue specimens of patients undergoing surveillance endoscopy for Barrett's esophagus. It consists of fluorescently labeled DNA probes to 8q24, 20q13, 17q12 and 9p21 loci containing the C-MYC, ZNF217, HER2/neu and P16 genes, respectively.

When hybridized and visualized, these probes provide quantitative information on DNA alterations relating to the detection of active and/or potential Barrett's associated neoplasia and the differentiation of low-grade dysplasia from high-grade dysplasia/adenocarcinoma. With the assay result and taking into account other standard diagnostic procedures, the physician and patient can make more informed decisions regarding the surveillance and management of Barrett's esophagus.

Criteria for Barrett's BioStrat® Abnormality

A specimen is considered "positive" for FISH-associated evidence of esophageal dysplasia or adenocarcinoma if at least one of the following criteria is met:

  1. ≥4 cells showing gains for 2 or more loci (8q24, 20q13, and 17q12); or
  2. ≥5% cells showing gains for a single locus (8q24, 20q13, 7p12 or 17q12); or
  3. ≥6% of the cells with homozygous loss of the 9p21 locus; or
  4. ≥11% of the cells with any combination of homozygous and hemizygous 9p21 loss.

In addition, FISH results can be classified into 'low-risk' and 'high-risk' groups for prognostic analysis.14

  1. Low-risk FISH:
    1. Negative-FISH result; or
    2. Positive-FISH result
      1. Gains for a single locus (low-grade dysplasia)
      2. 9p21 loss (low-grade dysplasia)
  2. High-risk FISH:
    1. Positive-FISH result
      1. Gains for multiple loci (high-grade dysplasia/adenocarcinoma)

Brushing specimens taken from patients with low-grade dysplasia tend to have high percentages of cells with 9p21 (P16) loss but relatively low percentages of cells with genetic loci gains. In contrast, brushing specimens taken from patients with high-grade dysplasia and adenocarcinoma have high percentages of cells with 8q24 (C-MYC), 20q13 (ZNF217), and 17q12 (HER2/neu) gains.

These findings are consistent with studies that have shown that 9p21 (P16) loss occurs early in the development of Barrett's associated neoplasia and that the progression of low-grade dysplasia to high-grade dysplasia is accompanied by a significant increase in chromosomal instability and the appearance of aneuploidy.15-17

Barrett's BioStrat® Images

The following examples of patient specimens demonstrate both the clinical utility of the Barrett's BioStrat® Assay as well as its simplicity of interpretation.

BioStrat Figure 1 Figure 1: Normal result observed in an esophageal cell after hybridization with the HER2/neu (17q12), P16 (9p21), MYC (8q24) and ZNF217 (20q13) FISH probe showing two 17q12 (green), two 9p21 (red), two 8q24 (aqua) and two 20q13 (gold) signals.
BioStrat Figure 2 Figure 2: Abnormal result observed in an esophageal cell after hybridization with the HER2/neu (17q12), P16 (9p21), MYC (8q24) and ZNF217 (20q13) FISH probe showing three 17q12 (green), four 9p21 (red), four 8q24 (aqua) and two 20q13 (gold) signals.

Sensitivity and Specificity for Detection of Barrett's Related Neoplasia

The FISH probe set consisting of locus-specific probes to 8q24, 9p21, 17q12 and 20q13 has demonstrated high sensitivity and specificity for the detection of Barrett's associated neoplasia and to differentiate high-grade dysplasia/adenocarcinoma from low-grade dysplasia (Table 1).12


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Barrett's Related Neoplasia at the Molecular Level

In a recent study, Fritcher et al. found that eighty-five percent of patients with a FISH result showing multiple loci gains had HGD or EA at the time of the initial biopsy, and the remaining 15% progressed to HGD or EA within 6 months.14 For patients with 9p21 loss, 43% had HGD or EA on the initial biopsy and another 24% progressed within 4 months. In comparison, only 13% of patients with a negative FISH result had HGD/EA on the original biopsy, with an additional 18% progressing to HGD/EA after 22 months. This analysis indicates that regardless of the original biopsy diagnosis, patients with FISH results showing multiple loci gains are at highest risk for progression to HGD/EA, followed by patients with 9p21 loss and single locus gain.

Likewise, patients presenting with positive FISH results and a negative endoscopy/biopsy termed an "anticipatory positive" result will have HGD/EA detected ~70 to 80% sooner as compared to a negative FISH and negative endoscopy/biopsy result.

An anticipatory positive test serves to appropriately heighten suspicion and increase surveillance efforts in selected patients where HGD/EA appears missing by histological methods. For the patient, this means that the chance of catching the cancer earlier is greatly improved.

The Barrett's BioStrat® Advantage

Barrett's BioStrat® analysis of esophageal brush cytology specimens offers many advantages as a method to detect genetic markers, including simplicity, lower cost, and the potential to sample a larger area of the Barrett's esophagus epithelium when compared with taking random biopsies. The Barrett's BioStrat® Assay may be used not only to detect LGD, HGD, and EA but also to discriminate patients with high-grade dysplasia/adenocarcinoma from those with low-grade dysplasia. This distinction is critical from a patient care perspective because a diagnosis of high-grade dysplasia is often the point at which aggressive corrective procedures such as esophagectomy are considered.

In addition, the ability to stratify the risk of progression of Barrett's metaplasia to adenocarcinoma may permit more effective targeting of repeated endoscopy for patients at particularly high risk of progression.

Combined with other standard surveillance methods, the BioStrat® Assay provides valuable information regarding the molecular events surrounding the development of dysplastic change and progression to adenocarcinoma further assisting physicians and patients make more informed decisions regarding the surveillance and management of Barrett's esophagus.


Please contact BioVantra Client Support Center at (866) 301-0960 to arrange for Barrett's BioStrat® Assay testing. Our experienced Client Support Team can assist with:

  • Barrett's BioStrat® Assay Requisition
  • Sample requirements
  • Logistics and specimen transportation
  • Testing methodology
  • Report delivery, status or interpretation
  • Expert oncology and pathology consultations
  • Requests for BioVantra literature and scientific references

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  1. Siegel R, Ma J, Zou Z and Jemal A: Cancer statistics, 2014. CA Cancer J Clin. 64: 9-29, 2014.
  2. Haggitt RC: Barrett's esophagus, dysplasia, and adenocarcinoma. Hum Pathol. 25: 982-93, 1994.
  3. Cameron AJ, Ott BJ and Payne WS: The incidence of adenocarcinoma in columnar-lined (Barrett's) esophagus. N Engl J Med. 313: 857-9, 1985.
  4. O'Connor JB, Falk GW and Richter JE: The incidence of adenocarcinoma and dysplasia in Barrett's esophagus: report on the Cleveland Clinic Barrett's Esophagus Registry. Am J Gastroenterol. 94: 2037-42, 1999.
  5. Drewitz DJ, Sampliner RE and Garewal HS: The incidence of adenocarcinoma in Barrett's esophagus: a prospective study of 170 patients followed 4.8 years. Am J Gastroenterol. 92: 212-5, 1997.
  6. Devesa SS, Blot WJ and Fraumeni JF, Jr.: Changing patterns in the incidence of esophageal and gastric carcinoma in the United States. Cancer. 83: 2049-53, 1998.
  7. Sampliner RE: Updated guidelines for the diagnosis, surveillance, and therapy of Barrett's esophagus. Am J Gastroenterol. 97: 1888-95, 2002.
  8. Cameron AJ and Carpenter HA: Barrett's esophagus, high-grade dysplasia, and early adenocarcinoma: a pathological study. Am J Gastroenterol. 92: 586-91, 1997.
  9. Reid BJ, Haggitt RC, Rubin CE, Roth G, Surawicz CM, Van Belle G, Lewin K, Weinstein WM, Antonioli DA, Goldman H et al.: Observer variation in the diagnosis of dysplasia in Barrett's esophagus. Hum Pathol. 19: 166-78, 1988.
  10. Spechler SJ: Dysplasia in Barrett's esophagus: limitations of current management strategies. Am J Gastroenterol. 100: 927-35, 2005.
  11. Brankley SM, Wang KK, Harwood AR, Miller DV, Legator MS, Lutzke LS, Kipp BR, Morrison LE and Halling KC: The development of a fluorescence in situ hybridization assay for the detection of dysplasia and adenocarcinoma in Barrett's esophagus. J Mol Diagn. 8: 260-7, 2006.
  12. Rygiel AM, Milano F, Ten Kate FJ, Schaap A, Wang KK, Peppelenbosch MP, Bergman JJ and Krishnadath KK: Gains and amplifications of c-myc, EGFR, and 20.q13 loci in the no dysplasia-dysplasia-adenocarcinoma sequence of Barrett's esophagus. Cancer Epidemiol Biomarkers Prev. 17: 1380-5, 2008.
  13. Falk GW, Skacel M, Gramlich TL, Casey G, Goldblum JR and Tubbs RR: Fluorescence in situ hybridization of cytologic specimens from Barrett's esophagus: a pilot feasibility study. Gastrointest Endosc. 60: 280-4, 2004.
  14. Fritcher EG, Brankley SM, Kipp BR, Voss JS, Campion MB, Morrison LE, Legator MS, Lutzke LS, Wang KK, Sebo TJ et al.: A comparison of conventional cytology, DNA ploidy analysis, and fluorescence in situ hybridization for the detection of dysplasia and adenocarcinoma in patients with Barrett's esophagus. Hum Pathol. 39: 1128-35, 2008.
  15. Reid BJ, Blount PL, Feng Z and Levine DS: Optimizing endoscopic biopsy detection of early cancers in Barrett's high-grade dysplasia. Am J Gastroenterol. 95: 3089-96, 2000.
  16. Krishnadath KK, Tilanus HW, van Blankenstein M, Hop WC, Teijgeman R, Mulder AH, Bosman FT and van Dekken H: Accumulation of genetic abnormalities during neoplastic progression in Barrett's esophagus. Cancer Res. 55: 1971-6, 1995.
  17. Reid BJ, Haggitt RC, Rubin CE and Rabinovitch PS: Barrett's esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Gastroenterology. 93: 1-11, 1987.

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