ER, PR and HER2 Tests: Section 6.c.
CONTENTS:
6.8 ‘Predictive’ Breast Immunohistochemistry (IHC): Hormone Receptor Positive Status: ER and PR
6.8.1 Hormone Receptor Positive Testing: ER and PR
6.8.2 How is ER and PR Expression by IHC Quantified?
6.9 ‘Predictive’ Breast Immunohistochemistry (IHC): HER2
6.9.1 FDA Approved Tests for HER2
6.9.2 How is HER2 Expression Quantified?
6.9.3 2013 ASCO/CAP Guidelines for HER2 Testing
6.9.4 Limitations of HER2 Testing
Forward to section 6D triple negative tests. Back to 6B about Immunohistochemistry
6.8 ‘Predictive’ Breast Immunohistochemistry (IHC): ER and PR
A predictive factor is capable of providing information on the likelihood of response to a given therapy. With the development of new cancer biomarkers that may direct patient treatment, the analysis of predictive biomarkers using IHC has become the basis of ‘targeted therapy’ or ‘personalized medicine.’
The predictive IHC (immunohistochemistry) markers in breast pathology include hormone receptor positive factors such as:-
Figure 6.14
Estrogen Receptor (ER) Testing using IHC (brown).
Progesterone Receptor (PR) Testing using IHC (brown). (x20)
6.8.1 Hormone Receptor Positive Testing: ER and PR
The estrogen receptor (ER) was first identified in the 1960’s and with the progesterone receptor (PR) became recognized as a ‘predictive’ marker for which women with breast cancer would respond to hormone treatment.
Ligand binding assays (LBA) using frozen breast tumor tissues were an early detection method for assessing hormone receptor positive cancers.
In the last three decades, the mammographic screening program has led to a decrease in the size of detected breast cancers and an increase in the use of tumor sampling by core needle biopsy (CNB).
The availability of specific antibodies that recognize ER in formalin-fixed, paraffin-embedded (FFPE) tissue is the basis for the development of immunohistochemical (IHC) assays to detect ER retrospectively in small specimens.
According to clinical studies, IHC can determine ER status and is also predictive of patient response to endocrine therapy. The ability of ER status as determined by IHC to predict hormonal therapy response is superior to that of ER status as determined by biochemical methods.
The use of IHC to assess the ER and PR status of breast cancers in FFPE tissue sections is now a routine part of pathology practice worldwide. So, the caveat for these visual, IHC methods is that optimal fixation and a high standard of method quality assurance (QA) are necessary.
6.8.2 How is ER and PR Expression by IHC Quantified?
Most Pathology testing labs use IHC for Estrogen receptors (ER) and Progesterone receptors (PR). If the tissue is hormone receptor-positive, ER and PR are present, this confirms that the cancer cells growth responds to the hormones estrogen and/or progesterone.
Not all Pathology laboratories use the same method for analyzing the results of the ER and PR IHC. A variety of scoring systems are in place:-
i. The ‘H’ score:
The ‘H’ score assess the percentage of tumor cells with cell membrane staining. So, the pathologist grades the cells as ‘weak,’ ‘moderate’ or ‘strong.’ The scores for the grades are then added together to give an overall maximum score of 300 and a cut-off point of 100 to distinguish between a ‘positive’ and ‘negative.’
ii. The ‘quick’ score:
Determines the percentage or range of cells staining from 1 to 4 and overall intensity as 1 to 3. The scores are again added to give a maximum score of 7.
iii. The Allred score:
Nowadays, the Allred score replaces the early ‘scoring systems’. So, ER-positive tumors have ⩾10% positive cells and ER-negative tumors have 1 – 9% positive cells. A modification has been to expand the lower end of the percentage of cells staining, giving a range of 1 to 5 and a maximum of 8. ER positivity is defined as score >2 (Allred, 1998).
Because ER staining is present in normal breast epithelial cells, the pathologist only assesses invasive tumor cells.
6.9 ‘Predictive’ Breast Immunohistochemistry (IHC): HER2
HER2 stands for ‘human epidermal growth factor receptor-2.’ HER2 is a trans-membrane tyrosine kinase receptor expressed by between 15 % to 20 % of invasive breast cancers.
Other abbreviations for HER2 include:
- c-erbB-2
- Her2/neu
- HER-2
- HER-2/neu
‘HER2 positivity’ refers to the tumor cells having extra copies of the HER2 gene and/or increased levels of expression of the HER2 protein. HER2-positive tumors grow more rapidly than HER2-negative tumors.
The reason that HER2 testing is done is to determine which patients may benefit from HER2-targeted therapy (see Section 8), such as:
- trastuzumab (Herceptin®);
- lapatinib (Tykerb®);
- pertuzumab (Perjeta®) and
- T-DM1 (Kadcyla®).
These targeted treatments can improve survival in patients with HER2-positive invasive breast cancer.
Figure 6.15 Immunohistochemistry for HER2:
Shows (brown) staining of the cells of a moderately
differentiated invasive ductal carcinoma. (HER2 IHC x 60)
6.9.1 FDA Approved Tests for HER2
The U.S. Food and Drug Administration (FDA) has approved two main HER2 testing methods; immunohistochemistry (IHC) and in-situ hybridization (ISH).
Immunohistochemistry (IHC) detects the HER2 protein that is present on the cell membrane of breast cancer cells.
In-situ hybridization (ISH) testing measures how many copies of the HER2 gene are present inside breast tumor cells. For over a decade now pathologists use in-situ hybridization testing, non-isotopic (NISH) and fluorescence in situ hybridization (FISH).
The original FDA testing guidelines focused on IHC and fluorescence in-situ hybridization (FISH). The updated FDA guidelines add recommendations for a newer diagnostic technique known as ‘bright-field’ or light microscopy in-situ hybridization (ISH).
Bright-field or light microscopy ISH evaluates amplification of the HER2 gene and can use a regular light microscope rather than a fluorescent microscope. This test allows the Pathologist to assess the morphology of the tumor on microscopy, together with cell (nucleus) staining for HER2 gene expression. The light microscopy technique may reduce some testing variability because this technique can identify the invasive component of the tumor.
Figure 6.16 A. Immunohistochemistry for HER2
tumor cell membrane
staining. B. In-situ Hybridization (ISH) for HER2 – shows nuclear localisation.
Figure 6.17 HER2 Testing using fluorescence
in-situ hybridization (FISH)
6.9.1 How is HER2 Expression Quantified?
The main problems in interpretation of HER2 IHC are in intra-observer and inter-observer variation in interpretation. If the results of HER2 testing with IHC are ‘equivocal,’ the pathologist may request fluorescence in-situ hybridization (FISH) or in-situ hybridization (ISH).
The IHC test for HER2 status uses a score of 0 to 3+:-
- A score of 0 to 1+ is ‘HER2 negative.’
- If the score is 2+, it is ‘borderline.’
- A score of 3+ is ‘HER2 positive.’
>0 means that no staining is seen or membrane-staining is <10% of the invasive cancer cells;
1+ has faint membrane-staining in >10% of cells.
2+ staining is weak to moderate complete membrane-staining in >10% of cells or <30% with strong, complete membrane-staining.
3+ is strong, complete membrane-staining in >30% of cancer cells.
HER2 over-expression is more common in high-grade invasive breast cancers of Grade 2 or Grade 3. Unlike ER, IHC staining should not be present in normal breast.
6.9.3 2013 ASCO/CAP Guidelines for HER2 Testing
In 2013, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) produced and published their updated guidelines on HER2 testing. These guidelines replace the last update from 2007 (Wolff et al., 2013).
The 2013 ASCO/CAP guidelines are now being implemented to improve the testing for overexpression of human epidermal growth factor receptor 2 (HER2) in patients with invasive breast cancer.
ASCO and CAP undertook a systematic review of the literature as the basis for their recommendations. The results of this review have implications for diagnostic testing of breast cancer and also for the provision of HER2-directed targeted therapy in certain patients (Wolff et al.,2013)
The 2013 ASCO/CAP HER2 Testing Guidelines
- Always test HER2 status on newly diagnosed, invasive breast cancers (primary site and/or metastatic site).
- Ensure that at least one tumor sample is tested for either HER2 protein expression (immunohistochemistry [IHC] assay) or (in-situ hybridization [ISH assay]) for HER2 gene amplification.
- Discuss the role of HER2-targeted therapy if the HER2 test result is positive and if there is no apparent histopathologic discordance with HER2 testing.
Delay the decision to recommend HER2-targeted therapy if the HER2 test result is equivocal. - Mandatory re-testing should be done on the same specimen, using the alternative test if the initial HER2 test result is equivocal, or on an alternative specimen.
- Do not administer HER2-targeted therapy if the HER2 test result is negative. If there is apparent histopathologic discordance with the HER2 test result, additional HER2 testing should be considered.
- Report a HER2 test result as indeterminate if technical issues prevent one or both tests (IHC and ISH) from being done in a tumor specimen, or prevent the test (or tests) from being reported as positive, negative, or equivocal.
- Confirm that the testing laboratory conforms to standards set for accreditation by CAP or an equivalent accreditation authority.
- In rare cases, it may be difficult to know for sure if the result is positive or negative. If additional testing on other tissue specimens is not possible, pathologists and oncologists should consider all available clinical data on the patient prior to recommending HER2-targeted therapy.
In 2013, Wolff and colleagues developed a clinical algorithm for evaluation of HER2 protein expression by immunohistochemistry on the invasive component of the breast cancer specimen.
Figure 6.18 2013 ASCO/CAP Clinical Algorithm for HER2 Testing.
From: Wolff et al., 2013. J Clin Oncol.
6.9.4 Limitations of HER2 Testing
Before the implementation of the first 2007 ASCO/CAP HER2 testing guidelines, the number of patients with ‘equivocal’ HER2 test results was rather large.
Since 2007, the quality of HER2 testing has improved; the frequency of ‘equivocal’ and inaccurate results has decreased. These improvements are believed to be due in part to the implementation of the testing guidelines since 2007.
With the increasing number of laboratories performing HER2 testing, uniform standards and quality control are mandatory.
The updated 2013 ASCO/CAP guidelines contain even more detailed recommendations on what physicians should discuss with their patients regarding HER2 status; reasons for HER2 testing; types of tests used; interpretation of test results, and any potential need for re-testing in the case of disease recurrence (Wolff et al., 2013).
References
Allred, D.C., Bustamante, M.A., Daniel, C.O., Gaskill, H.V., Cruz, A.B. Jr. (1990). Immunocytochemical analysis of estrogen receptors in human breast carcinomas: evaluation of 130 cases and review of the literature regarding concordance with biochemical assay and clinical relevance. Arch Surg 125, 107–113. (Retrieved November 19th 2014): https://www.ncbi.nlm.nih.gov/pubmed/1688490
Wolff, A.C., Hammond, E.H., Schwartz, J.N., et al. (2007). 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 131, 18–43. (Retrieved November 20th 2014): https://www.ncbi.nlm.nih.gov/pubmed/19548375
Patient Information
Breast Cancer Org.. HER2 Status. (Retrieved January 7th2014): http://www.breastcancer.org/symptoms/diagnosis/her2
Web MD. Breast Cancer Health Center: Types of Breast Cancer: ER Positive, HER2 Positive, and Triple Negative. (Retrieved January 6th2014):
Forward to section 6D triple negative tests. Back to 6B about Immunohistochemistry
