Molecular diagnostics for determination of HER2 status in breast cancer

Assaying the type I receptor tyrosine kinase HER2 is of increasing benefit in determining patient prognosis, eligibility for Herceptin therapy and selection of both hormonal and chemotherapeutic therapy. Evidence from correlation of test results with quantitative p185^HER2 expression levels, response to Herceptin therapy and retrospective analysis of patient outcome supports the conclusion that fluorescence in situ hybridization (FISH)-based methods, particularly with reference to chromosome 17, provide the most precise and accurate means of determining HER2 status. In addition, the low interobserver variability and high reproducibility of the FISH technique suggest that use of FISH for HER2 status determination will increase rapidly. However, this method is not widely available at present, therefore current guidelines recommend screening by immunohistochemistry, backed by rigorous quality controls and FISH testing of equivocal cases. Further training in FISH and expansion of FISH-based diagnostic services may provide significant benefit in future patient management if current evidential trends are continued.     

HER2 testing in the UK: further update to recommendations

These guidelines update the previous UK HER2 testing guidelines and have been formulated to give advice on methodology, interpretation and quality assurance to ensure that HER2 testing results are accurate, reliable and timely with the expansion of testing to all patients with breast cancer at the time of primary diagnosis. The recommendations for testing are the use of immunohistochemistry but with analysis of equivocal cases by in situ hybridisation to clarify their HER2 status or the use of frontline fluorescence in situ hybridisation (FISH) testing for those laboratories wishing to do so; the inclusion of a chromosome 17 probe is strongly recommended. Laboratories using chromogenic or silver in situ hybridisation should perform an initial validation against FISH. For immunohistochemistry and in situ hybridisation there must be participation in the appropriate National External Quality Assurance scheme.     

Recommendations for HER2 testing in the UK

Determining the HER2 status of breast carcinomas is a prerequisite for the use of the monoclonal antibody trastuzumab (Herceptin), which has recently been licensed for the treatment of metastatic disease. This necessitates a test based on archival material. The preferred analyses are immunohistochemistry with fluorescent in situ hybridisation (FISH) as a follow up test for ambiguous results. Guidelines have been developed for standardised, well controlled procedures for the provision of reliable results. A group of three reference laboratories has been established to provide advice, quality assurance, and materials, where needed.     

Immunochemistry evaluation of HER2 status in infiltration breast cancer: technical protocol and interpretation guidelines

In Europe, patients who may benefit from Herceptin((R)) (an HER2 targeted drug) are currently selected by immunohistochemistry (IHC). Reliable detection of HER2 status is essential to the appropriate usage of Herceptin(R), because its specificity is limited to tumours overexpressing HER2. It is essential that the IHC evaluation of the HER2 status of a mammary carcinoma be optimized and reliable. This technical paper reviews the different steps of the IHC technique, the controls and, the rules for interpretation. The sensitivity of the IHC technique must be adjusted so as not to produce false negatives or false positives. As opposed to other methods, it can be carried out whatever the fixation conditions of the tissues. The interpretation of the immunostains also requires training; it is fraught with problems for intermediate positivities. The ideal score to evaluate HER2 status has not yet been defined. It will thus be necessary to report the percentage of stained cells, the intensity of the staining, and, in respect to Herceptin((R)) treatment, the HercepTest scoring system (recommended in the package insert). Once acquired, this knowledge must be perpetuated by the observation of rules of good technical practice (internal and external controls, quality assurance programs). FISH should be used for complementary assessment of 2+ cases (on condition that they have not been fixed in Bouin's liquid) and for the calibration of the IHC technique.     

HER2 testing in the UK: recommendations for breast and gastric in-situ hybridisation methods

These guidelines supplement existing guidelines on HER2 testing by immunohistochemistry and in-situ hybridisation(ISH) methods in the UK. They provide a specific focus on aspects of guidance relevant to HER2 ISH testing methods, both fluorescent and chromogenic. They are formulated to give advice on methodology, interpretation and quality control for ISH-based testing of HER2 status in common tumour types, including both breast and gastric tumours. The aim is to ensure that all ISH-based testing is accurate, reliable and timely.     

HER2 testing in metastatic breast cancer – Is reflex ISH testing necessary on HER2 IHC-equivocal (2+) cases?

Current guidelines recommend HER2 testing on all primary invasive breast cancers and re-biopsy at disease relapse. The discordance rate between HER2-negative primaries and HER2 IHC2+ metastases that are ISH-amplified is unknown. We hypothesize that the majority of such cases are non-amplified. ISH testing is time-consuming and resource-intensive, and there may be situations where it is unnecessary. A retrospective review of IHC2+ metastatic lesions assessed with ISH at our center from 2013 to 2021 was undertaken. 105 cases were identified after exclusion of cases missing HER2 results, with primaries of unconfirmed origin, and cases of synchronous primary and metastatic disease. IHC and ISH results were recorded with detailed slide review of discordant cases. 91/105 metastases had HER2-negative primaries (87%). A metastasis was significantly more likely to be HER2-negative when the primary was HER2-negative (93%) versus positive (43%) (p < 0.0001). 54/91 primaries were IHC2+/ISH-non-amplified, and 50/54 (93%) corresponding metastases had identical results. Of the 37 HER2-negative primaries that were IHC0/1+, 35 (95%) corresponding metastases were ISH-non-amplified. Six metastases in cases with HER2-negative primaries were discordant. Characteristics of metastases suggesting ISH testing was warranted to assess for discordance included IHC heterogeneity, morphological discordance, increased staining of moderate intensity, and ER/PR discordance. One or more of these factors were present in all discordant metastases. Our results suggest selective ISH testing on HER2 IHC2+ breast cancer metastases in the context of HER2-negative primary disease may be appropriate when there is careful review of the IHC. Validation of our findings awaits further studies with larger sample sizes.     

HER2 testing recommendations in Australia

HER2 is the target of a new treatment for metastatic breast cancer using the humanised monoclonal antibody trastuzumab (Herceptin). Since only around 20% of breast cancers carry the overexpressed HER2 receptor protein to which this treatment is directed, patient selection is very important in determining eligibility for the drug. Currently, immunohistochemistry and fluorescence in situ hybridisation are the main tests used for HER2 detection, and these testing recommendations have been developed based on national and international data.     

External quality assurance of HER2 fluorescence in situ hybridisation testing: results of a UK NEQAS pilot scheme

Background and AIMS: Trastuzumab provides clinical benefit for advanced and early breast cancer patients whose tumours over-express or have gene amplification of the HER2 oncogene. The UK National External Quality Assessment Scheme (NEQAS) for immunohistochemical testing was established to assess and improve the quality of HER2 immunohistochemical testing. However, until recently, no provision was available for HER2 fluorescence in situ hybridisation (FISH) testing. A pilot scheme was set up to review the performance of FISH testing in clinical diagnostic laboratories. METHODS: FISH was performed in 6 reference and 31 participating laboratories using a cell line panel with known HER2 status. RESULTS: Using results from reference laboratories as a criterion for acceptable performance, 60% of all results returned by participants were appropriate and 78% either appropriate or acceptable. However, 22.4% of results returned were deemed inappropriate, including 13 cases (4.2%) where a misdiagnosis would have been made had these been clinical specimens. CONCLUSIONS: The results of three consecutive runs show that both reference laboratories and a proportion of routine clinical diagnostic (about 25%) centres can consistently achieve acceptable quality control of HER2 testing. Data from a significant proportion of participating laboratories show that further steps are required, including those taken via review of performance under schemes such as NEQAS, to improve quality of HER2 testing by FISH in the "real world".     

Impact of the updated 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on Human Epidermal Growth Factor Receptor 2 (HER2) gene testing in invasive breast cancers: A single center study

The study aimed to evaluate the impact of the updated 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on Human Epidermal Growth Factor Receptor 2 (HER2) testing in invasive breast cancer compared with previous 2013 guidelines. Between Jan 2014 and May 2020, 3364 consecutive invasive breast carcinomas with concurrent HER2 immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) results were retrospectively reviewed for HER2 status. Both 2013 and 2018 testing criteria were applied to establish the HER2 status. The testing algorithms involved testing of invasive breast carcinomas by IHC, with equivocal results being reflexed to FISH assays. Concordance rate improved from 92.7% to 94.1% in the non-equivocal IHC cases with the 2018 guidelines. Comparing 2013 versus 2018 criteria, HER2 non-amplified cases increased significantly from 73.7% (n = 2478) to 76.8% (n = 2585), HER2 amplified cases remained similar from 23.4% (n = 789) to 23.2% (n = 779) while equivocal cases decreased from 2.9% (n = 97) to 0% with the new guidelines. Thus, 107 cases (3.2%) were reclassified from HER2 equivocal (n = 97) and amplified (n = 10) to non-amplified with the updated 2018 guidelines. Under the 2018 criteria, a total of 259 cases (7.7%) belonged to the uncommon categories (groups 2 to 4), with group 3 being the most frequent (4.6%), followed by group 4 (2.9%) and group 2 (0.2%). Implementation of 2018 guidelines resulted in a significant increase in HER2 non-amplified cases, mainly due to the abolishment of the equivocal FISH group. This has helped resolve the clinical practice dilemma by providing a more definitive HER2 gene status.     

HER2 testing in gastric cancer

Molecular therapies targeting HER2 are part of the established drug armamentarium in breast carcinoma. Now the ToGA trial, an international multicenter phase III clinical study, involving 24 countries globally, has shown that the anti-HER2 humanized monoclonal antibody Trastuzumab is effective in prolonging survival in HER2-positive carcinoma of the stomach and the gastroesophageal junction (GEJ). Similarly to breast carcinoma, >20% of gastric cancers show HER2 overexpression and/or amplification, and this percentage increases to 33% in GEJ tumors. Thus, as in breast carcinoma, pathologists are now asked to evaluate HER2 status in gastric carcinoma samples. As validated in the ToGA trial, the HER2 testing criteria that must be used in evaluating both gastric carcinoma biopsies and surgical specimens significantly differ from those routinely applied in breast carcinoma. The main variations with regard to the pattern of reactivity in HER2-expressing cells are as follows: the completeness of membrane staining is not a "conditio sine qua non" and the number of stained cells necessary to consider a case as positive is different. We must also take note of the much more frequent heterogeneity of HER2 positivity in gastric cancer compared with breast carcinoma and the less stringent correlation between HER2 amplification and protein overexpression that is observed in gastric carcinoma, where more than 20% of cases may carry HER2 amplification, although of low level, without HER2 expression. In these patients, in the ToGA trial, there was no apparent benefit from adding Trastuzumab to chemotherapy: for this reason the European Medicines Agency, while approving usage of Trastuzumab for metastatic adenocarcinoma treatment, indicated HER2 testing by immunohistochemistry as first evaluation assay, followed by fluorescence in situ hybridization in 2+ equivocal cases. HER2 testing in gastric carcinoma is a new field, opening several opportunities: for patients with gastric cancer, this is a new promising therapeutic option; for pathologists, strengthening our role in therapy selection and emphasizing our duty of providing accurate and reproducible HER2 testing results; for all interested in understanding the biology of gastric and GEJ cancer and in discovering new possible molecular therapy targets.     

HER2 testing in the UK: consensus from a national consultation

OBJECTIVE: To gain an understanding of current attitudes among oncologists and pathologists to prospective HER2 testing in breast cancer and to gauge whether a national consensus exists regarding extent and quality of testing. DESIGN: Qualitative study, with semi-quantitative components, using emailed questionnaires and open-ended discussion documents. PARTICIPANTS: 186 relevant specialists, including 76 breast oncologists and 99 pathologists, representing all but three of the UK cancer networks. RESULTS: A strong consensus was seen in favour of universal, non-selective testing for HER2 at the point of breast cancer diagnosis. Similarly, an overwhelming majority of participants agreed that, to optimise the quality of test results, all laboratories undertaking HER2 testing should be CPA-accredited, participate in the recognised national external quality assessment scheme (UK NEQAS), and carry out a formal annual audit of its testing service. A further recommendation that testing be restricted to laboratories undertaking a minimum 250 tests per annum for immunohistochemistry and 100 tests per annum for in situ hybridisation techniques met with majority support. However, this was not a clear consensus; a significant minority of participants favoured continued use of local services falling short of these criteria. CONCLUSION: This study was successful in gauging national specialist opinion regarding the extent and quality assurance of HER2 testing in the UK.     

Standardization of HER2 testing: results of an international proficiency-testing ring study.

Human epidermal growth factor receptor 2 (HER2) positivity in breast cancer is a prognostic factor regarding tumor aggressiveness and a predictive factor for response to trastuzumab (Herceptin). Early and accurate HER2 testing of all breast cancer patients at primary diagnosis is essential for optimal disease management. Routine HER2 tests, such as immunohistochemistry and fluorescence in situ hybridization (FISH), are subject to interlaboratory variation, and validation by laboratory proficiency testing is important to improve standardization. This study compared immunohistochemistry and FISH testing between five international pathology reference centers. Each center evaluated 20 immunohistochemistry and 20 FISH breast cancer specimens in five testing rounds. In each round, one center selected two sets of four different invasive tumor specimens (set A for immunohistochemistry and set B for FISH) and sent samples to the other four centers in a blinded manner, while retaining samples for its own evaluation. Results were analyzed by an independent coordinator. With immunohistochemistry, there were no differences between the five centers for any of the specimens at the level of diagnostic decision (positive or negative HER2 status). However, differences between laboratories were observed in immunohistochemistry scoring. Of the 20 specimens, four were scored as negative (0/1+) and five as positive (3+) in all centers; eight were negative or equivocal (2+), and three positive or equivocal. After FISH retesting of nine of the 11 equivocal immunohistochemistry cases, consensus was achieved in 15 of 18 (83%) specimens. FISH analysis of set B specimens resulted in consensus between centers in 16 of 20 (80%) specimens (six negative and 10 positive). All four discordant FISH specimens were scored as having HER2:CEP17 ratios within the range 1.7-2.3 by at least one center. Equivocal immunohistochemistry and borderline FISH cases are difficult to interpret, even for highly experienced and validated laboratories, highlighting the need for quality-control procedures.     

Update of the GEFPICS' recommendations for HER2 status determination in breast cancers in France

In Europe, patients who may benefit from an HER2 targeted drug are currently selected by immunohistochemistry (IHC). In situ hybridization (ISH) techniques should be used for complementary assessment of ambiguous 2+ IHC cases and for the calibration of the IHC technique. Eligibility to an HER2 target treatment is defined by an HER2 positive status being IHC test 3+ or 2+ amplified. Reliable detection of HER2 status is essential to the appropriate usage of HER2 targeted drugs because its specificity is limited to tumors overexpressing HER2. It is essential that the IHC evaluation of the HER2 status of a mammary carcinoma is optimized and reliable. This GEFPICS' guidelines look over the different steps of the IHC technique, the controls and, the rules for interpretation. Once acquired, this knowledge must be perpetuated by the observation of rules of good technical practice (internal and external controls, quality assurance programs).     

Carrier testing in minors: a systematic review of guidelines and position papers

The objective of this article is to review all published normative ethical and clinical guidelines concerning the genetic carrier testing of minors. The databases Medline, Philosopher's Index, Biological Abstracts, Web of Science, and Google Scholar were searched using keywords relating to the carrier testing of children. We also searched the websites of the national bioethics committees indexed on the websites of WHO and the German Reference Center for Ethics in the Life Sciences, the Human Genetics Societies of various nations indexed on the website of the International Federation of Human Genetics Societies and related links, and the national medical associations indexed on the website of the World Medical Association. We retrieved 14 guidelines emanating from 24 different groups. All guidelines advanced the following preferences: (1) carrier testing should not be performed in children, and (2) testing should be deferred until the child can give proper informed consent to be tested. The guidelines varied in three areas: (a) the role of genetic services in ensuring that children are informed about their carrier status and associated risks when they are older; (b) exceptions to the general rule of withholding or deferring carrier testing; and (c) the communication of incidentally discovered carrier status. In the absence of compelling reasons, carrier testing of a child can reasonably be deferred until the child has the intellectual capacity needed to discern if and when to be tested.     

Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: a technical review with interpretive guidelines

Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2 -driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control. The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing.     

Human epidermal growth factor receptor 2 testing in gastric carcinoma: issues related to heterogeneity in biopsies and resections

Lee S, de Boer W B, Fermoyle S, Platten M & Kumarasinghe M P
(2011) Histopathology 59 , 832–840 Human epidermal growth factor receptor 2 testing in gastric carcinoma: issues related to heterogeneity in biopsies and resections Aims: To assess human epidermal growth factor receptor 2 (HER2) status and heterogeneity using immunohistochemistry (IHC) and silver in‐situ hybridization (SISH) in gastric carcinoma and dysplasia, and to correlate HER2 status between biopsy and resection specimens of gastric carcinoma. Methods and results: Immunohistochemistry for HER2 was performed in 178 cases of gastric carcinoma, and SISH in cases showing at least 1+ reaction. HER2 positivity [European Medicines Agency (EMA) guidelines] was identified in 20.2% of carcinomas and 12.9% of high‐grade dysplasia, and HER2 heterogeneity noted in 50% and 33% of these cases, respectively. IHC negative/positive reactivity and SISH results were concordant in 96.2%. SISH amplification was seen in 35.3% of IHC 2+ and in a case with previously unrecognized staining pattern. Concordance of IHC HER2 status on biopsies and gastrectomies was seen in 74.1%. False negative IHC results on either the biopsy or gastrectomy were seen in 19.4% of HER2 amplified cases. Conclusions: Human epidermal growth factor receptor 2 status in gastric carcinoma is comparable to previous studies with good concordance between IHC and SISH; all IHC 2+ and unusual patterns should be assessed with ISH studies; heterogeneity of tumour HER2 overexpression/amplification is common with possible implications for HER2 testing; and HER2 overexpression appears sufficiently specific to be considered a potential diagnostic biomarker of dysplasia.     

Impact of polysomy 17 on HER2 testing of invasive breast cancer patients

Background: Current American Society of Clinical Oncology/College of American Pathologists guidelines define HER2-positive tumors as those with > 6 HER2 genes per nucleus or those with HER2/CEP17 (chromosome 17) ratio > 2.2. These guidelines are potentially contradictory in tumors with polysomy of chromosome 17. The current study was performed to determine the impact of polysomy 17 on the interpretation of HER2 testing of invasive breast carcinomas. Methods: Chromosome 17 copies and HER2 gene status were identified by fluorescent in situ hybridization in 384 cases with invasive breast cancer, and the corresponding HER2 expression was obtained by immunohistochemistry stain. Results: The average CEP17 copy number for the group was 2.1 (range, 1.0-12.4). Forty-eight cases (13.8%, 48/348) were identified as chromosome 17 polysomy with CEP 17 copy number ≥ 3. Ninety-two (26.4%) cases had > 6 copies of HER2 per nucleus, and 92 cases (26.4%) qualified as HER2 gene amplified using the HER2/CEP17 ratio (> 2.2) guideline. Polysomy 17 showed poorly positive correlations with both HER2 gene copy number and HER2 overexpression (P < 0.01, r = 0.338 and 0.271, respectively). The distribution of clinicopathologic parameters of Polysomy 17 tumors was more similar to HER2 negative than HER2 positive tumors. Conclusions: Polysomy 17 is a crucial cause of equivocal HER2 testing results by FISH, depending on which criterion (ratio vs. absolute number) is used for interpretation. Polysomy 17 cannot be an independent predictive factor for HER2 gene amplification or protein overexpression.     

The clinical evaluation of HER‐2 status: which test to use?

Accurate determination of the status of the type I receptor tyrosine kinase HER-2 in breast carcinomas provides significant insight into patient prognosis and may also inform selection of chemotherapeutic and hormonal treatments. At present, however, the single most important application of HER-2 testing is in the selection of patients for treatment with targeted therapies such as Herceptin. Although, based on current literature, fluorescence in situ hybridization (FISH) detection of HER-2 gene amplification may provide more accurate information in this context, this method is not yet widely available. Therefore, screening by immunohistochemistry (IHC) for HER-2 protein, backed by rigorous quality controls and FISH testing of equivocal cases with intermediate staining intensity, remains the current practice. In laboratories with highly standardized testing and quality assurance procedures, this protocol appears highly effective. Improvements in fixation procedures, standardization of antibodies, and use of automated image analysis may all increase the precision of IHC testing. However, on the basis of current data, there is a case to be made for the wider implementation of FISH testing to determine HER-2 status in breast cancer.     

Impact of the 2018 ASCO/CAP HER2 guidelines update for HER2 testing by FISH in breast cancer

Recently, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the guidelines on HER2 testing for invasive breast cancer. Little is known about the impact of the guidelines update. We aimed to study the impact of the 2018 ASCO/CAP HER2 testing guidelines update. We compared the HER2 FISH results interpreted by 2013 and 2018 ASCO/CAP guidelines in 331 cases of invasive breast cancers. We also analyzed the pathological features and clinical outcomes of these cases. In comparing to the 2013 ASCO/CAP guidelines, the HER2 negative rate was increased significantly from 62.5% to 75.8%(P?<?0.05), and 13.3% changed from equivocal to negative by the 2018 guidelines. Our findings indicate that the guidelines update significantly increased the rate of negative results. The reclassification of the equivocal results by the 2018 guidelines is the main reason for this change. Patients with HER2 equivocal results were associated with larger tumor size and higher Ki67 index than those with negative results, while clinical outcomes were similar between them.     

Predictive and prognostic biomarker testing in invasive breast cancer

Surgical pathologists play an important role in ordering and interpreting biomarker testing for prognostic and predictive purposes. In invasive breast cancer, these biomarkers include hormone receptors, HER2 expression, multi-gene expression assays, checkpoint inhibitor staining, single gene mutation status, and genomic findings. Additionally, there are tests that predict response to “tumor-agnostic” drugs targeting gene-specific alterations or protein expression changes, regardless of tumor type. The assays employed to assess these biomarkers range from immunohistochemistry to RNA expression to next-generation sequencing. Each assay has specific indications and technical pitfalls. The pathologist must select the correct specimen, choose the area for microdissection, and consider cellularity and tissue adequacy. Often, the pathologist will also provide interpretation of the results. This review will provide an overview of these biomarker tests in breast cancer, describe their indications and utilization, and list some of their challenges. It will serve as a practical reference for pathologists in their integral role in patient care.