The clinical importance of amplification of human epidermal growth factor receptor gene HER-2/neu (also known as c-erbB-2, now HER2) in breast cancer was recognised in 1987.1 Since then numerous studies have found that either HER2 gene amplification or protein over expression predicts for poorer prognosis.2 There is also some evidence of a relationship between HER2 and response of cancers to chemotherapy and endocrine therapies.3^–5 However, following licensing of the humanised anti-HER2 monoclonal antibody trastuzumab (Herceptin) establishing tumour HER2 status became necessary for guiding trastuzumab’s clinical use. The identification of reliable diagnostic tests for HER2 gene amplification and overexpression became the subject of much research,6^–11 with evaluation and comparison of in situ hybridisation (ISH) and immunohistochemistry respectively (IHC).

Early trials of its use in patients with metastatic disease showed trastuzumab to improve time to progression and prolong survival as a single agent but particularly in combination with chemotherapy.12 13 This resulted in the drug being endorsed by the UK National Institute for Clinical Excellence (NICE) for treatment of metastatic disease.14

More recently, multiple prospective randomised trials15^–18 have shown that adjuvant trastuzumab reduces the risk of recurrence and mortality in patients with early stage breast cancer. NICE have endorsed its use as a treatment option for women with early stage HER2 positive (either HER2 protein expression or gene amplification) breast cancer following surgery and chemotherapy (neoadjuvant or adjuvant) with or without radiotherapy.19

There is therefore now a need to provide accurate and timely HER2 testing for patients with breast cancer at the time of primary diagnosis to enable discussion of appropriate adjuvant therapy, so resulting in a marked expansion of HER2 testing services.

These guidelines update the previous UK HER2 testing guidelines20 and have been formulated to give advice on methodology and quality assurance for testing to ensure that HER2 testing results are accurate and reliable, regardless of the test that is used. Authorship is based on representation of existing UK groups with substantial experience in HER2 assay methodology and performance, quality assurance, guideline production and establishing performance standards. These guidelines aim to provide (a) guidance to laboratories offering or considering offering a HER2 evaluation service, (b) information on internal and external quality assurance and (c) a basis for establishing service standards for use by professional and National Health Service groups in the UK. NICE has referred to the need to adhere to such published guidelines and this document serves to update the existing guidance. It does not seek to provide an extensive evidence base; this can be obtained from the National Comprehensive Cancer Network (NCCN) task force report on HER2 testing21 and the recent American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines.22 The aim is to be concise and practical.

GENERAL POINTS

Fixation

The IHC and ISH testing which form the basis of clinical tests of HER2 status require optimally fixed samples. Surgical specimens should be sliced promptly after excision into 5–10 mm slices and placed in a large volume of fixative (at least twice the volume of the specimen) to ensure rapid and even fixation.23 Buffered formalin should be used for fixation; fixatives containing alcohol can result in immunoreactivity of non-neoplastic epithelium.24 Fixation of surgically excised tumours should ideally be for 24 h to 48 h to obtain adequate penetration and optimal cross-linking.25 Variation in immunostaining between the periphery and centre of tumours can be due to a fixation gradient. Adequate fixation of small biopsies may be achieved in shorter time periods (6–8 h).

Samples

Needle core biopsies (NCBs)

NCBs are now frequently used for non-operative diagnosis of breast cancer, and should be used for patients who will receive neoadjuvant therapy to ensure pre-treatment testing. They should result in more rapid and even fixation of tissues than surgical specimens. Fixation duration and processing schedules may vary within and between laboratories, and may have an effect on required duration of antigen retrieval/enzyme digestion. Crush and edge artefacts can cause problems in interpretation, particularly of IHC. If there are any concerns about interpretation then fluorescence in situ hybridisation (FISH) should be undertaken. Small studies of HER2 testing of NCB and whole tumours have shown comparability,26 27 but larger series are required to be certain of full concordance. However, assessment of NCB can enable results to be available more rapidly for therapeutic discussions and planning at multi disciplinary meetings.

Excision specimens

Whole tissue sections are required: if there is only a small amount of invasive disease in a NCB, if invasive disease is intimately admixed with in situ or only identified in the excised specimen, if NCBs cannot be interpreted, or if there is only a cytology sample. The potential gradient effects of suboptimal fixation must be considered in interpretation of staining.

Tissue microarrays

These have a good level of concordance with standard tumour sections.28 29 They are of value for quality assurance and inter-laboratory comparisons,30 but currently there is insufficient evidence to support their use for diagnostic work.

Recurrent disease/metastases

The small numbers in published comparisons, plus the differences in samples assessed between studies (eg, comparison of local lymph node metastases with primary tumours, comparison of distant metastases with primary tumours) make evaluation as to whether there are changes in HER2 status with disease progression difficult to evaluate.31 32 There is evidence from a small number of cases of development of HER2 amplification and/or overexpression with acquired tamoxifen resistance.33 Assessment of distant metastases is, therefore, appropriate if samples are available. There is no evidence base to indicate whether or not HER2 status of tumours changes in patients who are receiving or have received trastuzumab. Further data are required to determine whether such a change occurs and, if so, at what level of frequency, and whether there is any clinical significance to such a change.

Sections

Deterioration in antigenicity can occur once sections from paraffin blocks have been put onto slides,34 and in view of this a time period of no more than 6 weeks between sectioning and staining has been recommended for IHC.22 If sections have been dipped in paraffin wax and then stored this time period can be extended, but there should be relevant controls to ensure that deterioration has not occurred. There are no published data on the effect of section storage on ISH, however it is recommended that storage of cut sections from controls or samples for over 12 months should be avoided.20

Controls

The inclusion of controls is essential to ensure test accuracy. These should reflect the range of cut-off points including a known negative and a 2+ case (see below for scoring).

Cell line preparations containing samples of known HER2 status are valuable.35 In addition, tissue whose HER2 status has been validated by more than one method, should be included as controls in all assay runs. Some centres include controls on each slide.

Methods

The recommended methods for determination of HER2 status evaluate the level of HER2 protein (IHC) or assess whether there is gene amplification (ISH). IHC is widely used;6^–11 it can be affected by fixation and requires standardisation of methodology and monitoring of scoring procedures as detailed in these guidelines. Equivocal (2+) cases require evaluation by FISH to determine their HER2 status.

FISH is used extensively for the evaluation of gene amplification.6^–11 It requires similar approaches to standardisation and monitoring of methodology. Dual probe FISH, with HER2 and chromosome 17 probes, determines whether the amplification of HER2 gene is independent of increases in chromosome 17 copies, which is common in breast carcinoma,36 and is thus a significant advantage. There are FISH assays that use a single probe targeted to HER2 in HER2 status is based on gene copy number alone without reference to chromosome 17. Cases with borderline copy numbers (4–6) require assessment of chromosome 17 on a parallel section or testing with a dual probe FISH assay to accurately determine the HER2 status.

Chromogenic ISH (CISH), which does not rely on fluorescent microscopy, is being used increasingly.37^–39 It can give comparable results to FISH and also requires standardisation and monitoring. At present it uses a single HER2 probe, which is detected by a peroxidase reaction; a chromosome 17 probe is used on a parallel section, and use of this is advocated. It is essential for cases with borderline copy numbers (4–6 signals). It is important that HER2 signals can be accurately counted. There are new developments making use of silver detection (SISH) to give distinct, more easily countable signals that can be assessed by automated systems.40 Again, use of chromosome 17 probe is advocated.

Other methods that can be used to determine HER2 status include PCR, real time quantitative PCR and enzyme linked immunoabsorbant assay (ELISA),41 42 but these should presently only be applied in the research setting.

Workload

There is evidence from pilot studies from two of the adjuvant trials and other studies that have compared local and central HER2 testing results that there are discordances between local and reference laboratories.43^–46 Such findings support the importance of larger volume, experienced laboratories for HER2 testing.46 Laboratories with lower volumes using IHC have a higher frequency of false positive (IHC+/FISH–) results.45 Laboratories providing a testing service should be carrying out a minimum of 250 assays each year for IHC and 100 cases each year for FISH, and if these levels are not reached a reference laboratory service should be used. Laboratories undertaking testing should be defined by each UK cancer network to ensure that there is larger volume, quality assured, central testing.

Evaluation

For assessment of IHC and ISH, training and experience in interpretation of histological features of breast tissue are essential. Before undertaking evaluation of HER2 consultant histopathologists should receive relevant training. HER2 status should only be reported on the invasive portion of a tumour. Amplification or overexpression of HER2 in in situ disease is common but should not be reported in the context of HER2 as a predictive marker for trastuzumab.

Following training and with experience, biomedical scientists and clinical scientists can assess IHC and ISH, with consultant histopathology support for interpretation of complex histology. The training requirements and qualifications should be determined by the relevant professional bodies.

Image analysis systems for IHC and ISH are being evaluated40 47 48 and may provide an adjunct to manual scoring in the future.

Quality assurance

All clinical laboratories in the UK using assays for HER2 as predictive tests must participate in appropriate external quality assurance programmes (EQA) such as those run by the UK National External Quality Assessment Scheme (NEQAS). The schemes are open to laboratories worldwide. There are schemes for IHC (UK NEQAS-ICC) and ISH (FISH and CISH; UK NEQAS-HER2 FISH). Participating laboratories test UK NEQAS-ICC cell-line sections on a quarterly basis and return them to the organising centre. They are evaluated by a panel of four expert assessors with the median taken as the final score.49 For FISH, data returned by the participating laboratories is compared to the data for the same sections from six reference laboratories, which is used to define an acceptable range.50

Systems are in place to identify and rectify suboptimal performance. UK NEQAS-ICC approaches all UK laboratories that achieve an inappropriate result, and provides advice for improvement. Any participants achieving an inappropriate result at the subsequent assessment runs are issued with a warning letter. Further technical advice and training is provided, with all attempts made to assist the laboratory. Failure of the laboratory to improve with accrual of a total of four successive inappropriate scores will result in the laboratory being removed from the scheme register and being reported to the Chairman of the National Quality Assurance Panel (NQAAP). This may ultimately result in the laboratory concerned losing its Clinical Pathology Accreditation (CPA) status for this test.

UK NEQAS-HER2 FISH has been established more recently.50 The scheme is now live and uses the same approaches as UK NEQAS-ICC regarding improvement of quality. Initial findings show that many laboratories demonstrate acceptable quality control of FISH testing for HER2, but that others need to assess procedures and improve. The number of laboratories participating in the CAP HER-2 FISH proficiency testing has increased over 5 years.51 Survey data has identified the need for standardisation of cut-off values.

At the present time there are no QA schemes to assess proficiency in interpretation although these are being considered. Virtual systems and the use of tissue microarrays (TMAs) are methods being explored.

Audit

This is needed as well as EQA to ensure that laboratories are maintaining standards. Audit of internal positive controls (cell lines and tissues) is necessary for checking test consistency. Audit of the frequency of overall results, as well as the distribution of IHC scores (0 to 3+), with comparison between IHC and FISH when both are performed, is of value for assessing over- and under-assessment of results and for implementing necessary changes. Intra- and inter-observer variation of interpretation should be performed for all staff undertaking evaluation. Within a network audit of results can be invaluable to highlight local issues, eg, fixation differences between different hospitals. Comparisons on a national basis can also be of value for standardisation.

IMMUNOHISTOCHEMISTRY

The Food and Drug Administration (FDA) approved commercial assay system HercepTest (Dako, Ely, UK) has been shown to give generally reliable results when tested on cells of known HER-2 positive/gene amplification status49 and has been found to give consistent quality results by UK NEQAS-ICC.52 The Ventana Pathway system (FDA approved; Ventata, Cambridge, UK) now uses clone 4B540 and gives comparable results. The polyclonal antiserum A0485 (Dako) and monoclonal antibody clones CB11 (Novocastra, Newcastle, UK) and TAB250 (Zymed, San Francisco, California, USA) are the other most widely used alternatives. Test conditions should be standardised, which ever assay is used.

Factors that are particularly important in achieving accuracy are antigen retrieval53 and rinse steps.54 Membrane reactivity of any adjacent normal breast tissue is an indicator of excess antigen retrieval and tests should be repeated or assessed with great care. In general, it has been found that accurately controlled water bath retrieval at 98°C using 0.1 M citrate buffer at pH 6.0 is the optimal method for several assays.52

Validation

Test conditions should be optimised so that distinct strong complete membrane staining will identify those cases with gene amplification (FISH positive). Validation can be achieved by testing a representative sample of cases assessed as 0/1+, 2+ and 3+ using a previously validated assay, with comparison with ISH data where appropriate. At least 100 cases should be compared.

Scoring

Only membrane staining of invasive tumour should be considered. The scoring method recommended is shown in table 1. Samples scoring 0/1+ are classed as negative. Equivocal 2+ samples require further analysis by another system to check amplification status

The percentage of cells with strong membrane staining to be considered 3+ and therefore positive has been increased to >30% in line with other recommendations.22 The effect of this on workload and resources will be minimal as few cases are seen with between 10% and 30% of invasive tumour showing strong complete membrane staining. There will, however, be a small number of cases where, as a result of the change in definition, they will be classified as 2+ (equivocal) and ISH undertaken, which previously would have been categorised as 3+/positive with IHC.

There are a range of artefacts that can affect interpretation such as edge artefacts (commoner in small biopsies), fixation variation and excess antigen retrieval.53 Cytoplasmic staining is not considered. If there is variability of staining due to these factors that cannot be solved by modification of IHC methodology then ISH should be performed. The testing algorithm is shown in fig 1. Inter-observer variation in assessment of staining can lead to misclassification of HER2 status.49 Agreement can be excellent for 0/1+ and 3+ but is poorer for 2+, even among expert pathologists.54

• Download figure
• Open in new tab
• Download powerpoint

Figure 1 Recommended testing algorithm for immunohistochemistry (IHC).

Quality assurance

Unstained sections containing four breast cancer cell lines are circulated by UK NEQAS. These are SK-BR3 which shows 3+ immunoreactivity and has HER-2 amplification, MDA-MB-453 which scores 2+ immunohistochemically and has HER-2 amplification, MDA-MB-175 which shows 1+ reactivity and is not HER2 amplified and MDA-MB-231 which is negative (score 0), and not amplified.52 Staining should fit with the expected pattern, with no excessive cytoplasmic staining. In addition counterstaining should not be excessive as this may mask low levels of reactivity. Suboptimal staining can be due to excessive antigen retrieval causing damaged cell morphology, the presence of excessive cytoplasmic staining and excessive counterstain.

Problems

These most commonly relate to the following.

Technical

• Fixation: inadequate fixation may result in variability of staining throughout the tissue section. If no other tissues samples are available for repeat IHC testing then undertaking ISH is recommended.

• Excessive antigen retrieval: this can result in damaged cell morphology, cytoplasmic staining, staining of normal breast epithelium. It is recommended that the test is repeated, adjusting times of retrieval.

• Antibody or commercial kit batch variation: it is important to check and audit controls carefully in order to detect this.

• Excessive nuclear counterstaining.

• Failure of standardisation of assay procedures can result in either under- or overstaining.

Interpretive

• Crush/edge artefacts, particularly in NCB: this can result in staining which is difficult to assess and to score reliably. It is recommended that retesting on other samples is performed or that ISH is undertaken.

• Interpretation: those undertaking interpretation of staining must be aware of potential technical problems in order to avoid pitfalls in assessment. If there is doubt regarding the appropriate score of a case, especially between 1+/2+ and 2+/3+ a low threshold for either repeat testing or assessment of amplification status is advised, in order to prevent under- or over-assessment of HER2 status.

FISH

At present there are three FISH based assays which are approved for HER2 testing in the USA: the PathVysion HER2 FISH test (Abbott-Vysis, Downers Grove, Illinois, USA; also approved for predicting anthracycline sensitivity); the INFORMHER2 FISH system (Ventana Medical Systems) which is only approved for assigning prognosis; and the PharmDx HER2 FISH (Dako Cytomation) which is approved for prediction of trastuzumab sensitivity.

The majority of laboratories in the UK use commercially available probes and systems, all of which give good performance. The PathVysion System (Abbott UK, Maidenhead, Berkshire, UK) comprises two fluorescently labelled probes complementary to centromeres of either HER2 gene or chromosome 17. The probe for chromosome 17 serves as an internal control as well as a marker of polysomy (additions) or monosomy (deletions) of the chromosome. Pharm Dx (Dako) is also a dual probe system. Conversely the INFORM system (Ventana) has only a HER2 probe so chromosome 17 alterations (particularly polysomy 17) are not determined by this method.

Factors such as tissue digestion, hybridisation and washing steps should all be standardised, and it is essential that protocols are strictly followed.

Scoring

It is advisable to use a serial section stained with haematoxylin and eosin (H&E) to locate invasive areas of carcinoma to be scored, and to avoid areas of ductal carcinoma in situ, which should not be included.

Counting is targeted to areas of tissue sections with a large proportion (>50%) of nuclei showing signals. The number of chromosome 17 (Ch 17) and HER2 signals are counted in 20 to 60 non-overlapping invasive cancer cell nuclei, using at least three distinct tumour fields or more if there is heterogeneity. Over-digested, mechanically damaged and truncated nuclei should be excluded from counting. The mean HER2 to Ch17 copy ratio is then calculated. For single probe analysis the HER2 gene copy number is recorded. Images of FISH results should be retained as a record.

A HER2/Ch17 ratio of ⩾2.00 has been considered to be HER2 positive, and this cut-off has been used in adjuvant trials. No clinical trial data are available to determine the appropriate cut off for single probe ISH. However, the equivocal category has been introduced51 which is defined as a HER2/Ch17 ratio between 1.80 and 2.20, or average gene copy number between 4.0 and 6.0 for single HER2 probe. The Vysis PathVysion Kit recommends that a ratio between 1.80 and 2.20 should be interpreted with caution since it is near the cut-off and this should be indicated in the report. The optimal approach to improving accuracy in this range is to increase the number of cells counted to 60–120 and/or repeat the test. A ratio of 1.80–1.99, after counting further cells and/or repeating the test, should be reported as borderline but not amplified and include a clear statement that the carcinoma is regarded as HER2 negative. A ratio of 2.00–2.20 should be reported as borderline but amplified, and therefore regarded as HER2 positive. Data on the response of patients to trastuzumab whose cancers fall within the borderline amplified category are not available; a statement to this effect can be included in reports. The recommended HER2 FISH testing algorithm is shown in fig 2.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2 Recommended testing algorithm for in situ hybridisation (ISH).

Polysomy

Aberrations of chromosome 17 (aneusomy) are common in breast cancer.36 These may be deletions or gains (polysomy). Such aberrations have a critical impact on the assessment and reporting of HER2 gene amplification. Therefore measurement of chromosome copy number in conjunction with HER2 is critically important. The frequency of polysomy varies between studies, which largely relates to how cases were selected for analysis and IHC scores and the use of different cut-offs to define polysomy scores.36 55^–58 Ideally, polysomy should be defined by analysis of normal breast in the same sample, since nuclear truncation and hybridisation efficiency can result in disomic cells exhibiting <2 chromosome copies.36 Using this approach Watters et al36 identified a range from 1.35 to 1.85 in normal so considered polysomy to be >1.86 copies. Tubbs et al58 have defined polysomy as 3.0 or more copies in at least 80% of cancer cells, and observed it in 3% of cases. Ma et al56 used different cut-offs and reported a high level (>3.76 Ch17 copy number)of polysomy in around 7% of cases with a low level (2.26–3.75 Ch17 copy number) in around 35%. They and others55 (who used Ch17 copy number of three or more) suggest that polysomy 17 might lead to protein overexpression in the absence of HER2 gene amplification, although this is not a universal finding.36 Further validation of how to define polysomy would be advantageous, including comparison with normal. Laboratories undertaking FISH should be aware of polysomy and the differences in definition between studies.

COMPARISON OF IHC AND FISH

Key questions are as follows.

• Which test is more accurate at detecting overexpression of HER2 in clinical breast cancer?

• What is the degree of concordance between IHC and FISH?

• Does the degree of concordance vary between laboratories?

• Do the assays differ in their ability to predict response to trastuzumab in the metastatic and/or the adjuvant setting?

Few studies have assessed the performance of FISH and IHC in breast cancers previously characterised for HER2 status using other methodologies. Two studies8 9 concluded that FISH more accurately reflected HER2 expression in diagnostic specimens. Analysis of data from three initial UK reference laboratories10 showed a high degree of concordance: tumours with 0/1+ IHC reactivity were FISH positive in 0.7% of cases, those scoring 2+ to be FISH positive in 48% and those with 3+ scores to be FISH positive in 94% of cases. The percentage of tumours with 2+ reactivity that were FISH positive varied, however, between the three centres from 30 to 82%. Other series24 59 have reported that 3 to 4% of IHC 0/1+ and 89 to 92% of IHC 3+ cases were FISH positive. Between 17%11 and 24%58 of IHC 2+ cases have been found to show HER2 gene amplification. This confirms the need to assess HER2 gene amplification status when IHC is assessed as 2+.

Comparison of local and central testing for two of the adjuvant trials43 45 showed that the concordance rate between smaller local and reference laboratories was better for FISH than IHC. Data from the use of trastuzumab in the metastatic setting13 60 61 suggest that there may be a higher overall response rate in patients with HER2 FISH positive rather than FISH negative cancers. However, there are still insufficient data on comparison of IHC and FISH in the prediction of benefit in the adjuvant setting, and given the predicted number of cases with discordant results evidence is unlikely to emerge in the near future. Very little information is available regarding benefits of trastuzumab to patients with tumours that are IHC 3+ but FISH non-amplified, for cancers that fall into the borderline/equivocal FISH category (ratio 1.8–2.2 as noted above) and for patients with cases with chromosome 17 polysomy. Therefore it is still difficult to determine which assays are better at predicting benefit from anti-HER2 therapy.

EXPECTED FREQUENCIES

Frequencies will differ markedly depending on whether the cases tested are selected due to the presence of metastatic disease, because they have aggressive features, or represent all new breast cancer, including screen-detected lesions. Recent UK audit data of IHC (data not shown) have shown a lower proportion of HER2 positive breast cancers than previously identified in the literature when HER2 testing was largely concentrated on cases that developed metastatic disease (table 2)

Although there will be variations in HER2 positivity rates between centres relating to grade distribution, percentage of screen-detected invasive cancers, age distribution and percentage of cases tested with metastatic disease, this reflects the distribution from testing all new invasive breast cancers and provides an approximate level to expect.

SUMMARY OF RECOMMENDATIONS

• Fixation: optimal prompt fixation in formalin for 6 to 48 h, dependent on size of specimen, is critical, particularly for IHC.

• Samples: NCB are more rapidly fixed and can enable results to be available for multidisciplinary discussions, but can be difficult to assess with staining artefacts seen with IHC. Excision specimen assessment is preferable if there is DCIS with a small focus of invasion or NCBs are small with little invasive tumour present. Recurrent tumour/distant metastasis should be assessed, if tissue available.

• Workload: laboratories should test a minimum of 250 cases by IHC and/or 100 ISH per year and be designated by each UK cancer network.

• Availability of results: timely results for clinical decision making; ideally should be available for multi-disciplinary meetings.

• Controls: tumour samples with negative, equivocal and positive results should be included in every IHC/ISH assay run, with cell lines for IHC kits.

• Quality assurance: all laboratories performing HER2 testing by IHC, ISH or both methods must participate in the relevant UK NEQAS schemes. These have strict criteria for ensuring ongoing accuracy, and appropriate action is taken when quality fails to meet NEQAS criteria.

• Audit: the same control samples should be included over multiple assays in order to highlight problems quickly. Cell line/tissue of borderline reactivity is crucial for identifying relatively minor changes in the assay. There should be ongoing audit of the frequency of results and distribution of IHC scores, with comparison with FISH when performed. Intra- and inter-observer interpretation variation should be checked.

• IHC: >30% invasive cancer cells with complete strong membrane staining is regarded as HER2 positive. All 2+ cases require assessment by ISH (see fig 1). IHC can be affected by fixation, requires standardisation of methodology, use of validated antibodies and monitoring of scoring.

• FISH: amplification of invasive cancer is considered to be HER2/Ch 17 ratio ⩾2.00 or HER2 gene copy number >6.0 (see fig 2). FISH requires standardisation of methodology and use of validated assays. Use of a dual probe for HER2 and chromosome 17 is strongly advocated.

• Borderline: cases with HER2/Ch 17 ratios between 1.80 and 2.20 should have additional cells scored to allow a diagnostic decision to be reached. Thereafter a HER2/Ch 17 ratio of 1.80–1.99 should be reported as borderline not amplified and HER2 negative, and a ratio of 2.00–2.20 as borderline amplified and HER2 positive.

• CISH: CISH can give comparable results to FISH and is being used increasingly. CISH requires similar standardisation and monitoring as FISH.

In summary, the UK recommendations for testing are:

• Use of immunohistochemistry but with analysis of equivocal cases by ISH to clarify their HER2 status.

Or:

• Use of frontline FISH testing for those laboratories wishing to do so; the inclusion of a chromosome 17 probe is strongly advocated.

• Laboratories using CISH/SISH must perform an initial validation against FISH.

• For IHC and ISH there must be participation in the appropriate NEQAS scheme.