Quality assurance for HER2 receptor evaluation

All UK clinical laboratories utilising immunohistochemical assays or in situ hybridisation to assess HER2 status as a predictive or prognostic marker must participate in an appropriate external quality assurance (EQA) programme, such as that run by the UK National External Quality Assessment Scheme for Immunocytochemistry and in situ hybridisation (UK NEQAS ICC and ISH).

8.5.1 Distribution of EQA material

UK NEQAS ICC and ISH have nearly 300 participants for their breast HER2 IHC module, of which 72 are from the UK. The assessments take place on a quarterly basis, with unstained formalin fixed and paraffin processed carcinoma cell lines distributed used in quality control assessments. The cell lines have been updated from those initially used^123,124 and consist of breast carcinoma cells showing the full range of diagnostic membrane staining (see Table 11) and have been further characterised for gene status using FISH. UK NEQAS ICC and ISH data clearly indicates that commercial kits produce more reproducible staining than those from ‘home-brew’ methods. Information on kits that work well is available from the UK NEQAS website and journal (www.ukneqasiccish.org.

Table 11: The cell lines used by the UK NEQAS ICC and ISH EQA schemes Cell line HER2 status by IHC¹ HER2 status by FISH²

SK-BR-3 3+ Amplified

MDA-MB-453 2+ Borderline: amplified to amplified

MDA-MB-175 1+ Not amplified

MDA-MB-231 Negative Not amplified

Participating laboratories are requested to stain the UK NEQAS ICC and ISH section using their usual clinical methodology. As well as the UK NEQAS ICC and ISH slide, participants are also requested to submit their own in-house control material for assessment. In-house control material should ideally include 3+, 2+ and 1+/0 invasive breast cancer cases and show good morphological preservation, which is paramount to gauge the sensitivity of the HER2 IHC test. However, it has become quite apparent that as patient tumour size and respective biopsies become smaller laboratories are finding it difficult to source appropriate invasive control material. It is therefore acceptable by UK NEQAS ICC and ISH to submit DCIS in-house tissue that has the expected range (3+, 2+ and 1+/0) of membrane staining.

8.5.2 Assessment procedure

The HER2 IHC slides are evaluated by a panel of 4 expert assessors using an adapted method initially devised by the Clinical Trials Assay. Due to the nature of cell lines, where cell line viability can be from 30–90%, the expected level of membrane staining is initially controlled by cutting and testing every 50th section. Furthermore, the UK NEQAS ICC and ISH has devised an EQA specific algorithm for scoring cell lines and in-house control sections (Table 12), in order to provide enhanced feedback to participants on the sensitivity/specificity of their test. This is illustrated further below.

Assessors score the cell lines using the guidelines above and a participant must achieve the expected level of staining for each of the four cell lines. An ‘acceptable’ score is awarded when more than 3 out of the 4 assessor are in agreement, a ‘borderline pass’ is awarded when two out of the four assessors find the staining to be appropriate and an

‘unacceptable’ score is awarded when more than three out of the four assessors find the staining to be unacceptable, which could be due a) staining being stronger than expected, leading to false positive staining in a clinical situation; b) staining being weaker than expected, possibly leading to false negative staining in a clinical situation; or c) uninterpretable, due to reasons shown in the table above.

8.5.3 Poor performance monitoring

The UK NEQAS ICC and ISH also have a duty to monitor the performance of all UK clinical laboratories performing breast HER2 IHC testing. Because of the direct impact that the results of assays for hormonal receptors have on patient management, more stringent performance monitoring mechanisms are employed. Furthermore, as of September 2010 the National Quality Advisory Panel (NQAAP) has made it mandatory for EQA schemes to use a ‘traffic light’ system for the grading of all its UK participants. Table 13 highlights the traffic-light system used in the breast hormonal receptor module. The poor performance criteria are applicable only to the UK NEQAS ICC and ISH distributed cell lines and monitoring covers the five most recent assessment periods. Although in-house sections are not part of the poor performance monitoring system, in-house material scores may also be used to gauge overall performance status.

Table 12: The cell lines scoring system used by the UK NEQAS ICC EQA scheme Cell line Score Acceptable level/s

of staining during level of membrane staining is deemed acceptable for this cell line.

MDA-MB-453 2+ 2+ or 1+/2+ or 2+/3+ i) 1+/2+: Staining is slightly weaker than expected when compared to the 'gold standard' stained sections.

ii) 2+/3+: Staining is slightly stronger than expected when compared to the 'gold standard' stained sections MDA-MB-175 1+ 1+ or 0/1+ or 1+/0 i) 0/1+: Staining is more towards the

weaker end of 1+ staining but still acceptable.

ii) 1+/0: Staining is more towards the weaker end of 1+ staining but still acceptable.

MDA-MB-231 Negative 0 or 0/1+ or 1+/0 0/1+ or 1+/0 = Cells are starting to show very weak membrane staining U = uninterpretable scores

Assessors may also give a score of 'U' indicating that the cell lines/tissue sections were 'uninterpretable’ due to the reasons set out below. A score of U/x, e.g. U/3+ or U/2+ or U/1+ or U/0 indicates a borderline uninterpretable scores indicating that the staining is just about readable and further improvements are required.

Table 13: The traffic-light system used to summarise performance in the breast hormonal receptor module

Status When triggered Monitoring procedure GREEN 1 underperformance (1

score = No) over 5 runs on NEQAS ICC slides

No issues with poor performance. Participant will be offered assistance to improve

AMBER 2 underperformances (2 scores = No) over 5 runs on NEQAS ICC slides

Participant and Head of Department will be notified of continued underperformance and will be sent a ‘Warning letter’ indicating that they are close to being deemed a poor

performer and to contact the scheme director.

The scheme Director will then provide advice and assistance on how the laboratory

concerned might improve their results RED 3 underperformances (3

scores = No) over 5 runs on NEQAS ICC slides

Participant and Head of Department will be notified that they have been deemed a ‘poor performer’ and to contact the scheme Director to discuss the situation. The scheme is also obliged to refer the laboratory to NQAAP