Preanalytical Measures
Specimens
HER2 status should be assessed in all invasive primary breast carcinomas and in recurrent and metastatic tumours whenever biopsy tissue is available. Bilateral carcinomas, histologically distinct ipsilateral carcinomas or widely separated carcinomas considered to be separate synchronous primary tumours should each be assessed. It is deemed reasonable not to assess multiple ipsilateral tumours if they are histologically similar and colocated in the same quadrant/region of the breast. There is no consensus on testing residual invasive tumour following neoadjuvant therapy, although some recommend this approach. Retesting non-responding stable or progressive HER2-negative tumours particularly high-grade tumours or those with a long time period between preoperative biopsy and excision may be considered but cannot be recommended routinely in view of the lack of evidence.
Excellent concordance between core biopsy and surgical specimens has been shown using immunohistochemistry (IHC) and in situ hybridisation (ISH). In the majority of UK centres, HER2 testing is performed on the diagnostic needle core biopsy specimens, mainly to ensure timely availability of results at the time of postoperative multidisciplinary team (MDT) treatment planning discussion and also to enable consideration for neoadjuvant treatment use which is increasingly used for operable cases. Although assessment of HER2 status on needle core biopsy is recommended and no repeat on excision specimens is needed if the test is clearly positive or negative, performing/repeating the assay on incisional or excisional surgical specimens should be considered if:
(1) the core biopsy is not available (ie, there is only a cytology sample); or (2) there is a possibility that the HER2 test on the core biopsy is unreliable or unrepresentative of the tumour identified in the resection specimen as follows:
HER2 assessment is uninterpretable on the core due to technical artefacts (ie, suboptimal processing or staining) or there is doubt about the core biopsy handling.
The core biopsy HER2 status remains in the equivocal category after IHC and ISH; for example, repeat assessment is advised if the core biopsy was scored as 2+ on HER2 IHC with borderline negative ISH (ratio of number of HER2 to chromosome 17 centromere copies of 1.8–1.99 or HER2 gene copy number is 4–6).
Invasive tumour on the core is too small for reliable assessment, or if invasive disease is intimately admixed with in situ carcinoma, or only identified in the excision specimen. There is insufficient data to define the amount of invasive tumour tissue in core biopsy sufficient for analysis; however this can be left to the reporting pathologist's discretion.
If the tumour in the resection specimens is morphologically distinct from that in the core biopsy, for example of a clearly different histological type or histological grade (eg, low grade on the core and high grade on the excision, but not just reflecting minor difference in the mitotic count or proportion of solid areas). A repeat may also be undertaken on concurrent metastatic nodal disease if it is morphologically distinct from the primary breast tumour.
If the core biopsy staining is heterogeneous and shows a focus of strong HER2 positivity in <10% of the area of the invasive carcinoma in the core biopsy, HER2 testing should be repeated on the excision specimen. If this pattern is detected on the excision specimen, a different tumour block or a nodal metastasis can be tested, to determine the percentage of positive/amplified tumour present in a larger tumour sample.
Fine needle aspirates from primary breast carcinoma are not suitable for assessment of HER2 status as the distinction between invasive and in situ disease cannot be made on such samples. However, if fine needle aspiration (FNA) is the only material available, or in metastatic disease, some evidence indicates that ISH is reliable for assessing HER2 status in liquid-based and cell block preparations. In the case of metastatic bone lesions that require HER2 assessment, it should be noted that decalcification techniques have the potential to influence immunohistochemical assessment in a detrimental manner and such decalcified samples should be tested with ISH methods.
Fixation and Processing
Good fixation of specimens used for HER2 testing should be ensured and the cold ischaemic time (time from removal from the patient to placing in fixative (cold ischaemic time)) should be as short as possible, certainly less than 1 h. Formalin fixed, paraffin embedded tumour tissue samples are appropriate for assay. Tumours samples should be fixed in buffered formalin and embedded in paraffin wax; fixatives containing alcohol can result in staining of normal tissue and use of Bouin's fixative will preclude testing by fluorescence in situ based methods. Other methods of tissue fixation can also adversely affect antigen reactivity. At least 6 h fixation is recommended for core biopsies. Surgical specimens should be incised as soon as possible through the carcinoma to allow initial penetration of fixative and then sliced into 5–10 mm slices to ensure rapid penetration and even fixation. Tissue should be placed in an adequate volume (ideally 10:1; fixative:tissue) of fixative for at least 24 h and not more than 72 h Centres using rapid fixation and processing must validate their methodology for HER2 assessment.
Sections should be stained within 1–2 days of cutting and drying. Excessive section drying time has also been shown to cause a loss of HER2 expression and it is therefore recommended that freshly cut sections are either dried at 60°C for 1 h or 37°C overnight (http://www.ukneqasicc.ucl.ac.uk/neqasicc.shtml).