J Breast Cancer.  2010 Sep;13(3):267-274.

Branched-chain Assay for ER, PR, and HER2 RNA Levels is a Useful Adjunct in the Evaluation of ER, PR, and HER2 in Breast Cancer

• Yim HW
• Song BJ
• Jung SS
• Kim HJ
• Choi YJ
• Lee KY
• Lee A

Abstract

PURPOSE
The status of estrogen receptor (ER), progesterone receptor (PR) and HER2 is critical in patients undergoing breast cancer treatment. We performed this study to evaluate the QuantiGene Reagent System as a clinical test for detecting ER, PR, and HER2 RNA levels in formalin-fixed, paraffin-embedded (FFPE) tissue.
METHODS
The RNA levels of ER, PR and HER2 were measured using the QuantiGene2.0 assay in FFPE tissue from breast cancer patients (n=40) and compared with ER and PR immunohistochemistry results and HER2 fluorescence in situ bybridization (FISH) results.
RESULTS
When the cut-off values for ER, PR, and HER2 RNA levels were 5.0, 7.2, and 50, respectively, the sensitivity, specificity, and positive and negative predictive value of the QuantiGene 2.0 Assay were 96.6%, 90%, 96.7%, and 90%, for ER; 89.7%, 81.8%, 92.9%, and 75% for PR; and 83.3%, 96.4%, 90.9%, and 93.1% for HER2, respectively. The Allred scores for ER and PR as well as the HER2 FISH ratio were correlated with RNA levels (p=0.046, r=0.32; p<0.001, r=0.61; p<0.001, r=0.75, respectively).
CONCLUSION
We demonstrated that the ER, PR, and HER2 RNA levels as measured by the QuantiGene 2.0 assay were reproducible and correlated well with immunohistochemistry and FISH results. Measuring ER, PR, and HER2 RNA levels from formalin-fixed, paraffin-embedded tissue using the QuantiGene 2.0 assay, which was a relatively simple technique easily performed in a usual laboratory, appeared to a helpful adjunct in determining the status of ER, PR, and HER2 in breast cancer.

Keyword

erbB-2 receptor; Estrogen receptors; Immunohistochemistry; Progesterone receptors; RNA

MeSH Terms

Breast
Breast Neoplasms
Estrogens
Fluorescence
Humans
Immunohistochemistry
Receptor, erbB-2
Receptors, Estrogen
Receptors, Progesterone
RNA
Sensitivity and Specificity
Estrogens
RNA
Receptor, erbB-2
Receptors, Estrogen
Receptors, Progesterone

Figure

• 

  Figure 1 A receiver operating curve for estrogen receptor (ER) (A), progesterone receptor (PR) (B), and HER2 (C) was drawn to identify the cut-off value for negative vs. positive with the highest sensitivity and specificity.

• 

  Figure 2 The estrogen receptor (ER)/progesterone receptor (PR) Allred scoring showed a positive correlation with normalized ER/PR RNA levels (A, B). The HER2/CEP17 ratio showed a strong positive correlation with normalized HER2 RNA levels (C).

Reference

1. Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, et al. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med. 2000. 124:966–978.

2. Goldhirsch A, Glick JH, Gelber RD, Coates AS, Thürlimann B, Senn HJ. Meeting highlights: international expert consensus on the primary therapy of early breast cancer 2005. Ann Oncol. 2005. 16:1569–1583.

3. Early Breast Cancer Trialists' Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet. 1998. 351:1451–1467.

4. Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 2007. 25:5287–5312.

5. Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999. 17:1474–1481.

6. Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD. Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems. J Clin Pathol. 2000. 53:125–130.

7. Canales RD, Luo Y, Willey JC, Austermiller B, Barbacioru CC, Boysen C, et al. Evaluation of DNA microarray results with quantitative gene expression platforms. Nat Biotechnol. 2006. 24:1115–1122.

8. Elbeik T, Surtihadi J, Destree M, Gorlin J, Holodniy M, Jortani SA, et al. Multicenter evaluation of the performance characteristics of the bayer VERSANT HCV RNA 3.0 assay (bDNA). J Clin Microbiol. 2004. 42:563–569.

9. Stenman J, Orpana A. Accuracy in amplification. Nat Biotechnol. 2001. 19:1011–1012.

10. Urdea MS, Horn T, Fultz TJ, Anderson M, Running JA, Hamren S, et al. Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucleic Acids Symp Ser. 1991. 24:197–200.

11. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007. 131:18–43.

12. Bast RC Jr, Ravdin P, Hayes DF, Bates S, Fritsche H Jr, Jessup JM, et al. 2000 update of recommendations for the use of tumor markers in breast and colorectal cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol. 2001. 19:1865–1878.

13. Dowsett M, Bartlett J, Ellis IO, Salter J, Hills M, Mallon E, et al. Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres. J Pathol. 2003. 199:418–423.

14. Owens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 2004. 5:63–69.

15. Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol. 2009. 27:1323–1333.

16. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Comparison of fluorescence in situ hybridization and immunohistochemistry for the evaluation of HER-2/neu in breast cancer. J Clin Oncol. 1999. 17:1974–1982.

17. Perez EA, Suman VJ, Davidson NE, Martino S, Kaufman PA, Lingle WL, et al. HER2 testing by local, central, and reference laboratories in specimens from the North Central Cancer Treatment Group N9831 intergroup adjuvant trial. J Clin Oncol. 2006. 24:3032–3038.

18. Lal P, Salazar PA, Hudis CA, Ladanyi M, Chen B. HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring. Am J Clin Pathol. 2004. 121:631–636.

19. Wang J, Shen L, Najafi H, Kolberg J, Matschinsky FM, Urdea M, et al. Regulation of insulin preRNA splicing by glucose. Proc Natl Acad Sci U S A. 1997. 94:4360–4365.

20. Price JE, Polyzos A, Zhang RD, Daniels LM. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res. 1990. 50:717–721.

21. Tan M, Yao J, Yu D. Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities. Cancer Res. 1997. 57:1199–1205.

22. Taylor CR. FDA issues final rule for classification and reclassification of immunochemistry reagents and kits. Am J Clin Pathol. 1999. 111:443–444.

23. Collins LC, Botero ML, Schnitt SJ. Bimodal frequency distribution of estrogen receptor immunohistochemical staining results in breast cancer: an analysis of 825 cases. Am J Clin Pathol. 2005. 123:16–20.

24. Umemura S, Itoh J, Itoh H, Serizawa A, Saito Y, Suzuki Y, et al. Immunohistochemical evaluation of hormone receptors in breast cancer: which scoring system is suitable for highly sensitive procedures? Appl Immunohistochem Mol Morphol. 2004. 12:8–13.

25. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. Expression of the 21 genes in the Recurrence Score assay and tamoxifen clinical benefit in the NSABP study B-14 of node negative, estrogen receptor positive breast cancer. J Clin Oncol. 2005. 23:16 Suppl. abstract #510.

26. Vondracek M, Weaver DA, Sarang Z, Hedberg JJ, Willey JC, Wärngård L, et al. Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen-immortalized oral keratinocytes. Int J Cancer. 2002. 99:776–782.

27. Gleaves CA, Welle J, Campbell M, Elbeik T, Ng V, Taylor PE, et al. Multicenter evaluation of the Bayer VERSANT HIV-1 RNA 3.0 assay: analytical and clinical performance. J Clin Virol. 2002. 25:205–216.

28. Knudsen BS, Allen AN, McLerran DF, Vessella RL, Karademos J, Davies JE, et al. Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues. J Mol Diagn. 2008. 10:169–176.

29. Paik S, Kim C, Wolmark N. HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med. 2008. 358:1409–1411.

30. Lyng MB, Laenkholm AV, Pallisgaard N, Ditzel HJ. Identification of genes for normalization of real-time RT-PCR data in breast carcinomas. BMC Cancer. 2008. 8:20.