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Bimodal Population or Pathologist Artifact?

Department of Pathology, Yale University School of Medicine, New Haven, CT

Gina G. Chung

Department of Oncology, Yale University School of Medicine, New Haven, CT

Robert L. Camp

Department of Pathology, Yale University School of Medicine, New Haven, CT

Barbara Burtness

Fox Chase Cancer Center (BB), Philadelphia, PA

To the Editor:

In a recent Comments and Controversies article Dr Schnitt¹ makes a series of observations regarding the variability and lack of reproducibility in the common methods for assessment of estrogen receptor (ER) expression in the current clinical practice in breast cancer. Although he compares the value of immunohistochemistry favorably to radio ligand binding assays (LBA), he notes that the continuity of expression seen in the LBA has generally not been reproduced in the largest population-based studies.^2,3 Schnitt then asks whether this is a biologic phenomenon that should alert us to be more attentive in our selection of endocrine therapy, or whether the fault is in the IHC assay. He goes on to cite a number of articles that illustrate substantial nonreproducibility in IHC ER assays with particular emphasis on work with variable sensitivity. In particular, he cites the work of Umemura et al⁴ showing good correlation between a low sensitivity IHC assay with the biochemical assays, but the correlation was diminished with a highly sensitive IHC test.

We share Schnitt's concern regarding the IHC test and have recently reported the potential for artifacts when using assays with dynamic range that is insufficient for the protein being measured.⁵ However, techniques are available that provide accurate and reproducible measurement of protein expression. AQUA (version 1.2; HistoRx, New Haven, CT) is a fluorescence-based method for quantitative assessment of in situ protein concentration with accuracy comparable to an enzyme-linked immunosorbent assay, without loss of spatial information.⁶ AQUA, combined with the use of standard curves produced from well-characterized cell lines prepared as tissue microarrays demonstrates both the reproducibility and dynamic range of ER expression (Fig 1). Then once the range is appreciated, large cohorts can be tested to address the bimodality question. In our cohort of 650 patients, scored by the Hscore method (intensity X area),⁷ we observed a bimodal pattern similar to the works cited by Schnitt (Fig 2). On the contrary, AQUA-based quantitative analysis of the same cohort (calculated as the average of five unique histospot AQUA scores) produces a continuous pattern similar to that seen in the biochemical assays (Fig 3).

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Cell lines that are grown, then formalin fixed and paraffin embedded into tissue microarrays are used as standard controls. Each read is a four-fold redundancy the average score is shown with error bars showing the SE of the mean. The choice of cell lines was suggested by work by DeFazio et al.9 Although the exact concentration for each line has not yet been determined, AQUA (version 1.2; HistoRx, New Haven, CT) scores have been shown to be linear with concentration in previous works.5,10

View larger version (11K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. The frequency distribution of Hscores from a cohort of 651 patients which were used for the construction of the tissue microarrays for the quantitative analysis in Figure 3. More than one half of the patients fall into the category of 0 or 300. The distribution is clearly bimodal.

View larger version (9K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. The frequency distribution by average AQUA (version 1.2; HistoRx, New Haven, CT) scores of estrogen receptor in five histospots from a cohort of 651 patients shows no evidence of bimodality.

Even if we could count every molecule accurately every time, does it matter? Schnitt suggests that it does and that the LBA data and limited data with IHC suggest we should attempt to do so. But given the limitations of both assays, can that be done? We would argue that the data herein, along with reproducible standard curves using cell lines should allow the best of both worlds—accurate quantification and analysis of tiny tissue specimens. However, our data are currently limited to disease-specific survival outcomes, which may not be predictive of response to therapy. We are in the process of looking at a series of ongoing prospective trials, including a Tamoxifen trial collected by Richard Love, a Tamoxifen trial conducted by the Southern and Southeastern Swedish Cancer Groups, in collaboration with Lisa Rydén, and National Surgical Adjuvant Breast and Bowel Project B-14, in collaboration with Soon Paik. When these studies are complete we may be able to provide a more definitive answer to Dr Schnitt.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment: N/A Leadership: N/A Consultant: David Rimm, HistoRx; Gina G. Chung, HistoRx; Robert Camp, HistoRx Stock: David Rimm, HistoRx; Robert Camp, HistoRx Honoraria: N/A Research Funds: N/A Testimony: N/A Other: N/A

ACKNOWLEDGMENTS

Supported by grants from the National Institutes of Health (D.L.R., R.L.C.).

REFERENCES

1. Schnitt SJ: Estrogen receptor testing of breast cancer in current clinical practice: What's the question? J Clin Oncol 24:1797-1799, 2006[Free Full Text]

2. 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 123:16-20, 2005[CrossRef][Medline]

3. Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et al: Immunohistochemistry of estrogen and progesterone receptors reconsidered: Experience with 5,993 breast cancers. Am J Clin Pathol 123:21-27, 2005[CrossRef][Medline]

4. Umemura S, Itoh J, Itoh H, et al: Immunohistochemical evaluation of hormone receptors in breast cancer: Which scoring system is suitable for highly sensitive procedures? Appl Immunohistochem Mol Morphol 12:8-13, 2004[Medline]

5. McCabe A, Dolled-Filhart M, Camp RL, et al: Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis. J Natl Cancer Inst 97:1808-1815, 2005[Abstract/Free Full Text]

6. Camp RL, Chung GG, Rimm DL: Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med 8:1323-1327, 2002[CrossRef][Medline]

7. McCarty KS Jr, Szabo E, Flowers JL, et al: Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res 46:4244s-4248s, 1986[Medline]

8. Camp RL, Dolled-Filhart M, King BL, et al: Quantitative analysis of breast cancer tissue microarrays shows that both high and normal levels of HER2 expression are associated with poor outcome. Cancer Res 63:1445-1448, 2003[Abstract/Free Full Text]

9. deFazio A, Chiew YE, Sini RL, et al: Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines. Int J Cancer 87:487-498, 2000[CrossRef][Medline]

10. Dolled-Filhart M, McCabe A, Giltnane J, et al: Quantitative in situ analysis of {beta}-catenin expression in breast cancer shows decreased expression is associated with poor outcome. Cancer Res 66:5487-5494, 2006[Abstract/Free Full Text]

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