The LeicaSelection of therapeutic strategies according to the presence or absence of specific biological markers relies on the availability of accurate and reliable laboratory testing, highlighting the need for standardisation of tissue handling, biomarker assay procedures and accurate interpretation of results. Despite being labour-intensive and technically challenging, fluorescence in situ hybridisation (FISH) assays remain the gold standard for HER2 assessment. Traditionally, HER2 FISH has been performed manually and has lacked a standardised approach, making it subject to inter-laboratory and interoperator variability. Automation of the staining process addresses a number of challenges associated with HER2 FISH testing, providing a straightforward, labour-saving method to aid standardisation and improve consistency.
HER2 FISH system is a fully automated, quantitative FISH assay for use on the Leica BOND system, employing the PathVysion HER2/CEP17 DNA FISH probes (supplied by Abbott Molecular). The Department of Pathology at the Royal College of Surgeons in Ireland (RCSI) Education and Research Centre has been using Leica’s BOND staining platforms for automation of immunohistochemistry (IHC) staining for five years, and evaluated the performance of the Leica HER2 FISH system compared with its own in-house technique based on the Poseidon ERBB2 probe (Kreatech).
Trial design
A parallel study was performed on a cohort of 100 archived samples of known HER2 protein and/or gene status, comprising: 25 IHC 0/1+ samples, 25 IHC 2+ samples with a FISH ratio <2.0, 25 IHC 2+ samples with a FISH ratio >2.0, and 25 IHC 3+ samples. A mixture of resection specimens and core biopsies was chosen, and all samples were stained in parallel using the laboratory’s in-house technique and the Leica HER2 FISH system. In addition, the same 100 samples were analysed manually by the Heart of England NHS Foundation Trust, Birmingham, using the Abbott PathVysion HER2 DNA probe kit. A total of 20 cells were counted per case using a ratio scoring method, with an additional 20 cells counted in borderline cases (ratios 1.80–2.19).
Results
Staining quality
As can be seen in Figure 1, there are readily apparent differences between the staining achieved with each method. Although the colours used for each probe set are the same, the size, signal intensity and level of digestion vary between the techniques. The level of digestion observed with the Leica HER2 FISH system is significantly lower than either the manual PathVysion technique or the Poseidon method, with overall good preservation of cell morphology. This can be very helpful for localising specific cells or identifying cell clusters from haematoxylin and eosin (H&E)-stained sections, without compromising overall signal quality (Fig 2). In some samples, non-specific green signals could also be seen; however, these were present in all three techniques, and the non-specific signal intensity was not sufficiently high to interfere with interpretation.
Concordance
Concordance between the three staining methods was high (Table 1), demonstrating that staining quality for the new Leica method is equivalent to manual techniques. All cases showing 2X2 discordance had ratio scores in the equivocal range (1.80–2.19) for each method. Table 2 shows the mean and range of HER2 FISH ratio scores for each technique. All three methods produced a similar mean value for 0/1+, 2+ (<2.00) and 2+ (>2.00) cases, and it was noted that the Leica HER2 FISH system provided more consistent, standardised staining with reduced process variation compared to either of the manual techniques.
Routine implementation
Based on the results of the retrospective study, the fully automated Leica HER2 FISH system was introduced as the standard technique for HER2 FISH staining in the RCSI laboratory. As it was already using a Leica BOND-III platform for automated IHC staining, implementation required very few changes to laboratory practices, with no modifications to sample preparation, counterstaining or interpretation protocols.
Introduction of fully automated HER2 FISH staining has led to improvements in the laboratory’s productivity and the turnaround time for some results. Since its implementation in May 2011, over 250 cases have been assessed, with no repetition of staining required. A major benefit of automation is the reduced hands-on time required for staining of samples. Using the manual technique, it would take approximately three hours of staff time to perform HER2 FISH staining using the laboratory’s in-house technique, compared with just 30 minutes to prepare the same number of samples for automated staining. Elimination of this laborious and time-consuming manual method has also led to reduced turnaround times for some cases, as the laboratory no longer has to wait for sufficient samples before performing the assay in batches.
Importantly, the time saving offered by the automated method allows more staff time for the interpretation of stained slides or to perform other tasks. This additional capacity offers the potential for expansion of HER2 FISH testing from the current level of around 300 cases per year up to 1000 cases a year, which is an important consideration given the current trend towards centralisation of molecular testing services.
Conclusions
The Leica HER2 FISH system provides a straightforward, quantitative FISH staining protocol that is easy and cost-effective to implement for users of BOND series instruments. The fully automated Leica HER2 FISH system provides equivalent signal quality to manual staining while saving time and reducing variability.