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Expression of the hormone receptors (estrogen receptor (ER) and progesterone receptor (PR)) as well as overexpression / amplification of the human epi-dermal growth factor receptor-2 (HER2) have not only been identified as important prognostic factors among patients with breast cancer. They have also been characterized as essential predictive factors for benefit derivable from endocrine treatment or immunologic therapies directed against HER2, respectively. Hence, reliable determination of hormone receptor and HER2 status among patients with breast cancer has become of utmost importance. The need for developing and promoting standard methodologies for the assessment of the individual disease phenotype has previously been acknowledged by publication of novel guidelines for HER2 testing. For HER2 testing, these guidelines have been developed in 2007 by the American Society of Clinical Oncology (ASCO) in conjunction with the American College of Pathology (CAP). Recently, ASCO/CAP have acknowledged the need for standardized assessment of hormone receptor status.
We asked two experts in the field of breast pathology whether the new guidelines on hormone receptor testing would change current practice of hormone receptor status measurement in Germany and how they should be viewed in face of novel developments regarding gene expression based multigene predictive factors.
Cornelia Liedtke, Münster and Oleg Gluz, Wuppertal
Hammond ME, Hayes DF, Dowsett M, et al.: American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer. J Clin Oncol 2010: DOI 10.1200/JCO.2009.25.6529 [Epub ahead of print]
Purpose: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. Methods: The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance. Results: to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in preanalytic variables, thresholds for positivity, and interpretation criteria.
The new ASCO/CAP guidelines  for hormone receptor testing provide a summary of the current standards that are needed for correct and reproducible testing of hormone receptors. The main recommendations are 1) tumors with at least 1% positive tumor nuclei for estrogen receptor (ER) and/or progesterone receptor (PR) should be designated hormone receptor positive; 2) internal (adjacent normal epithelium) and external (e.g. endometrial tissue) controls should be evaluated routinely, 3) fixation time should be between 6 and 72 h in neutral buffered formalin, 4) laboratories should participate in national proficiency testing programs.
These standardized guidelines, as well as the similar guidelines for HER2 testing , are part of national and international initiatives that are currently changing diagnostic clinical pathology. As a result, standardization and quality control in histopathology need to reach the level that is achieved in laboratory medicine and clinical chemistry. Traditional pathology was based on standard H and E stained sections and – in some cases – additional immunohistochemical evaluation of tumor markers that were used, for example, to clarify the origin of a tumor. These markers were typically analyzed as a marker panel and interpretation was based on the experience of the pathologist in a combination of the H and E morphology as well as the results of the antibody panel.
With the focus on more individual therapeutic approaches, the role of immunohistochemical markers has changed. As these predictive biomarkers are a prerequisite for therapy, there is a need for increased reproducibility, quantification and exact results for every single case. This is particularly important in breast cancer, where determination of hormone receptors and HER2 is a basis for the decision on adjuvant therapies. Fortunately, these developments have been recognized by the national pathological societies and the recommendations that are included in the ASCO/CAP guidelines are already standard in most institutions in Germany. National quality control programs have been established for immunohistochemical stainings [3,4,5,6].
It should be noted that the diagnostic environment for biomarker testing on oncology is currently rapidly changing . As it is possible to evaluate mRNA biomarkers in routine FFPE tissue, new tests are in preparation or already on the market to evaluate biomarkers on the mRNA level . These assays have the advantage that a quantitative assessment of multiple markers in small tissue samples is possible. The disadvantage is that these tests are not linked to morphological alterations. As a consequence, it is essential to integrate molecular markers on the mRNA level into the routine diagnostic pathology workflow. Morphology-based immunohistochemical as well as mRNA-based molecular testing could then be used as complementary methods to assess the biomarker status of the tumor tissue with higher reproducibility.
In the ASCO/CAP guidelines it is stated that image analysis is a desirable method for quantification of the percentage of tumor cells that are immunoreactive. However, it is also reported that controversy exists on how image analysis should be implemented at this time, as standards of system performance have not been developed. Given the new options on quantitative assessment of molecular markers by gene expression analysis, is it very likely that for the future there will be an increased pressure to provide a similar degree of quantification also for immunohistochemistry, which will only be possible using standardized image analysis approaches. Therefore, image analysis systems need to be validated in samples from clinical studies  to determine which cutoff values and methodological parameters lead to an optimal prediction of clinical outcome based on quantitative immunohistochemistry.
In summary, the ASCO/CAP guidelines provide a solid basis for state-of-the-art biomarker testing. New molecular technologies investigating gene expression as well as quantitative approaches to immunohistochemistry need to be integrated into validated combined diagnostic systems that will be essential for the pathology lab of the future.
Breast cancer in recent years has functioned as a pioneer tumour setting the stage for a new era of diagnostics and therapy in oncology. Estrogen (ER) and progesterone (PR) receptors followed by HER2 provided the first examples for targeted therapy and marked the beginning of the age of personalized medicine. Determination of ER and PR by immunohistochemistry (IHC) has almost completely replaced extract based methods because it can easily be integrated in standard histopathological procedures with no need of complicated fresh tissue logistics. Also, correlation to the number of tumour cells and their viability, admixture of normal, noninvasive and stromal cells is possible. But IHC suffers from lack of standardization and reproducibility appears to be suboptimal with up to 20% discordant results between local and central testing . In a non-centralized test setting standardization can only be achieved by adherence of pathology institutes to guidelines and quality control . The ASCO/CAP consented guidelines on ER and PR try to meet this requirement and parallel those on HER2 published 3 years earlier, in particular with regard to tissue fixation and test validation [3, 4].
ER positive has been defined as >1% labelled invasive tumour cells regardless of the staining intensity , because retrospective analysis of 14 studies on endocrine treatment provided evidence for this threshold. For PR the same threshold is valid and it was concurred that independent predictive information on endocrine responsiveness comes from PR testing. In most instances an ER-/PR+ status, however, may be due to inaccurate IHC. Proportion of positively labelled cells together with intensity should be reported, scores (Allred, Remmele) may be provided but are not mandatory. The guidelines leave it open what percentage of ER+ cells constitutes a strongly positive tumour which may be controlled by endocrine therapy only without need of chemotherapy . No general recommendation is given on routine ER/PR testing of DCIS which may be useful in individual patients. Whereas breast recurrences are recommended for fresh ER/PR testing no statement on metastases can be found in the guidelines. Discrepant ER/PR status between primary and metastasis occurs in up to 20% . Another critical issue in the guidelines is that in case of multiple tumours only one of the tumours (preferably the largest) is considered sufficient for ER/PR assessment .
Because of optimal fixation core biopsies are regarded as superior to resection specimens for testing as long as comparison with the resection specimens shows identity of tumour type and grade. Since discordance rates of 9–12% were reported between IHC and RNA expression signatures such as the recurrence score, it was concluded that a recommendation would be premature .
Pathologists should apply internal controls, e.g. normal duct, which demonstrate a spectrum of weakly to strongly stained cells and external on-slide positive and negative controls, which might include cell lines with known receptor content. In case that one of both controls is not sufficient, and no normal cells are represented in the tumour tissue or normal cells fail to demonstrate a heterogeneous labelling from weak to strong, the test can not be interpreted and has to be repeated. In addition external quality assurance methods to ensure ongoing accuracy in ER/PR testing are recommended, such as accreditation and at least bi-annual participation in proficiency testing programs with at least 10 challenges. Although it summarizes mostly what appears to be self-evident and at least the German proficiency testing program reveals ER/PR testing to be far less problematic than HER2 , the guideline has the merit to provide for the first time common standards on ER/PR testing in pathology.