HER-2: Its molecular biology and testing by immunochemistry and FISH

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St Vincents Hospital
Sydney, NSW


The treatments available for managing breast cancer have recently been augmented by the arrival of Herceptin, a monoclonal antibody that targets a subset of breast cancers over-expressing the specific HER-2 antigen. This treatment has relatively few side-effects and has been shown to assist patients with metastatic carcinoma, and is now being trialled in earlier disease. The drug is expensive and of no use in patients whose cancers do not express the HER-2 antigen. Therefore it is essential that a reliable and as inexpensive as possible test be readily available and used to identify women who will benefit. There has been an international and Australian debate as to which test to use, when, who should perform the test, and of course, who should pay. In addition, an Australian study has addressed the question as to whether routine surgical histopathology can at least triage breast carcinomas to reduce the numbers of cases that need specialised immunoperoxidase or fluorescent in situ hybridisation testing for the HER-2 antigen.

The human epidermal growth factor receptor-2 (HER-2) has been found to be over-expressed in approximately 10-30% of breast cancers, due to gene amplification in almost all cases. HER-2 over-expression is a poor prognostic factor for patients with both axillary lymph node-negative and positive breast cancer, and predicts for a likely response to specific chemotherapy regimes. Since 1998 HER-2 assessment has become vitally important for selecting patients with metastatic breast cancer who are eligible for treatment with trastuzumab (Herceptin). Herceptin has shown significant benefit in the treatment of patients with advanced disease whose tumours strongly over-express HER-2 and/or show HER-2 gene amplification with a response less likely in those patients whose tumours only weakly or do not over-express HER-2. Although testing is now being performed in laboratories worldwide, there remains a number of unanswered questions concerning who to test, when to perform the test, which technique to employ, which reagents to use and how to assess and express the test result?

It is essential to accurately diagnose HER-2 status to avoid false negatives that deny patients the opportunity to receive Herceptin, and to avoid false positives that waste an expensive drug and raise false hopes in the patient.

There is debate as to whether HER-2 status should be systematically evaluated in every primary carcinoma at the time of initial diagnosis, or whether testing should occur at the time of relapse, which incurs a lesser cost. Testing at the time of diagnosis allows for the selection of an appropriate adjuvant therapy such as anthracycline containing chemotherapy which may have greater efficacy in HER-2 positive tumours, or for the selection of patients for current trials which are using Herceptin in the adjuvant setting. It also maximises accuracy of immunohistochemistry (IHC) since tissue fixation and time in paraffin block are at their shortest, minimising protein denaturing and loss of antigen. There is no significant difference in the HER-2 status of primary and metastatic tumours of the breast.

There is considerable debate comparing the benefits of centralised testing for both IHC and fluorescent in situ hybridisation (FISH). Centralised testing provides economies of scale, reduced costs, standardisation of methodologies, procedures and reagents, and allows for increased experience to be gained by a group of pathologists. But local testing, as long as it is supported by quality assurance programs and training for technical and pathologist staff, avoids problems with fixation artefacts because fixation and antigen retrieval can be standardised locally.

The testing is currently performed mainly on archival material because in Australia federal government funding is only available for the treatment of metastatic disease, but increasingly some laboratories are testing all breast carcinomas at the time of initial diagnosis.

The two main methods used in the routine, clinical, diagnostic setting for HER-2 are IHC, which tests for protein production and membrane HER-2 receptor, and FISH which tests for gene amplification in the nucleus. IHC is routine technology for many laboratories and allows for rapid testing of large numbers of cases with minimal infrastructure setup costs. But there has to be rigid quality-control testing conditions and there is considerable interobserver variation in test interpretation because the HercepTest requires assessment of the intensity of staining of the cancer cells, as well as a percentage of cells that are positive. There are a large number of commercially available kits and over 20 antibodies available with varying recommended methods of antigen retrieval, tissue fixation and sensitivities and specificities, leading to varying rates of HER-2 over-expression in breast cancer of between two and 60%. This has been highlighted recently in the US in a College of American Pathology (CAP 2001) study where up to 9% of laboratories using IHC recorded a negative carcinoma as 2 or 3+, while a FISH survey by the College showed all laboratories correctly found no amplification in two breast cancers.  Specific HER-2 test kits such as HercepTest minimise laboratory variables by including all reagents, detailed instructions on the specific technique and a scoring system to assess and express the results.  But the reading of the test remains quantitative rather than qualitative and requires considerable training. The majority of cancers in which HER-2 over-expression is weakly positive by IHC and assessed as 2+, fail to show amplification of the HER-2 gene on FISH retesting. As a result, most authors now advocate that a score of 2+ by IHC should be regarded as an equivocal test result requiring FISH.

FISH employs fluorescence microscopy which is a complex and specialised technique not commonly available in most pathology laboratories, and it is a relatively expensive test for routine use. There are significant infrastructure costs including an immunofluorescent microscope, and each case takes considerable time to read by the skilled pathologist, who needs to recognise morphology, eg the nuclei of 60 infiltrating carcinoma cells need to be counted while avoiding intraduct carcinoma. In our Australian experience in the Roche-funded centralised laboratory, there has been a high rate of technically non-assessable tumours varying markedly between laboratories involved in the study, ranging from zero in one laboratory to 70% in another. Most of these were due to the lack of specific HER-2 hybridisation signal, reflecting poor tissue fixation possibly due to variations in time in formalin and paraffin embedding temperatures, or other adverse processing variables, such as the use of Carnoy’s solution to defat specimens.

Other HER-2 testing methods such as chromogen in situ hybridisation for gene amplification and assays of ‘down-stream’ molecules such as tyrosine kinase are being developed, and may yet provide a more accurate, reliable and reproducible technique for routine use. In the meantime, however, if Herceptin is shown to have clinical benefit as adjuvant therapy as a result of prospective, randomised clinical trials currently underway, there will be pressure on laboratories to test all breast cancers at first presentation.

Recently in Australia, pathologists from 13 laboratories associated with the HER 2000 International Study correlated pathology features of cancers, including the size, type, histological grade and lymph node status, with the frequency of HER-2 over-expression assessed by IHC using the HercepTest and the HER-2 DNA Probe Kit to re-test the equivocal cancers by FISH. This kit includes an internal control probe to the centromere of chromosome 17 as well as the HER-2 probe, labelled with SpectrumGreen and SpectrumOrange respectively, to control for tumour ploidy and potential processing problems. Interobserver variation in assessing HER-2 over-expression on IHC was examined by a slide circulation scheme as a quality assurance measure.

Some 1,144 of the 1,536 cancers (74.5%) assessed did not over-express HER-2. Unequivocal over-expression  (3+ by IHC) was seen in 186 cancers (12%) and an equivocal result (2+ by IHC) was seen in 206 cancers (13%). Of the 156 IHC 3+ cancers for which complete data was available, 149 (95.5%) were ductal NST and 152 (97%) were histological grade 2 or 3.  Only one of 124 infiltrating lobular carcinomas, a pleomorphic variant (0.8%) showed HER-2 over-expression. None of the 49 ‘special types’ of carcinoma, which are grade 1 and usually oestrogen and progestogen positive, showed HER-2 over-expression.  Retesting by FISH of a proportion of the IHC 2+ cancers showed that only 25 (23%) of those assessable exhibited HER-2 gene amplification, but 46 of the 47 IHC 3+ cancers (98%) were confirmed as showing gene amplification. The highest yield of HER-2 over-expressing carcinomas is seen in the grade 3 NST subgroup in which 24% are positive by IHC.

Various testing guidelines and algorithms have been produced largely based on IHC as a first-line test with FISH as a confirmatory test to try to overcome concerns regarding the reliability of IHC. Cancers which are HER-2 2+ on IHC show no gene amplification in FISH in 77% of cases, and the 2+ category should be regarded as equivocal. The Australian study suggests that it may be possible to triage breast cancers so that HER-2 testing is only performed on carcinomas with a significant risk of HER-2 over-expression. The strong correlation between HER-2 3+ and infiltrating duct carcinoma NST, grades 2 and 3, has also been found by other groups, suggesting a predominant HER-2 positive phenotype.  Triaging using grading and typing does require training pathologists and quality assurance programs.

The HER-2000 International Study was supported by grants from F Hoffmann-La Roche LTD and from Dako Corporation.

This paper was presented at the 3rd Asian International Academy of Pathology Meeting in Bangkok, January 2003.

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