The slide to be stained for HER-2 comprised the following six tissues:

* HER-2 immunohistochemical score (see table below) as achieved by using the two FDA approved kits and antibodies (HercepTest™, Dako and PATHWAY^®, Ventana) in NordiQC reference laboratories.
** HER-2 gene/chromosome 17 Ratio achieved by using HER2 FISH pharmDX™ Kit, Dako.

All carcinomas were fixed 24 - 48 h in 10 % neutral buffered formalin.

IHC scoring system according to the guidelines given by ASCO/CAP:

Criteria for assessing a HER-2 staining as optimal included:

• A clear and unequivocal immunohistochemical staining marked as score 0 or 1+ in the breast ductal carcinomas no. 1 & 2.

• A clear and unequivocal immunohistochemical staining marked as score 2+ in the breast ductal carcinoma no 3.
• A clear and unequivocal immunohistochemical staining marked as score 1+ in the breast ductal carcinoma no 4.
• A clear and unequivocal immunohistochemical staining marked as score 3+ in the breast ductal carcinoma no 5 & 6.
• No or only a weak cytoplasmic reaction that did not affect the interpretation of the true membranous HER-2 reaction.

A staining was assessed as good, if the HER-2 gene amplified tumour no. 5 & 6 showed a 2+ reaction. (An equivocal 2+ IHC staining should always be analyzed by FISH according to the ASCO/CAP guidelines and the national guidelines in Denmark, Norway and Sweden).

A staining was assessed as borderline if the signal-to-noise ratio was low, e.g., because of moderate cytoplasmic reaction, excessive counterstaining or excessive retrieval hampering the interpretation.

A staining was assessed as poor in case of false negativity (i.e., the 3+ tumours and the 2+ tumour with gene amplification showing a 1+/0 reaction) or false positivity (i.e., the 0/1+ tumours without gene amplification showing a 3+ reaction).

Results
114 laboratories participated in this assessment. 72 % achieved a sufficient mark. In table 1 the antibodies (Abs) used and marks are summarized.

Table 1. The IHC systems/Abs used and the scores given

1) Proportion of sufficient stains (optimal or good), 2) Proportion of sufficient stains with optimal protocol settings only, see below.

FDA approved systems

PATHWAY^® rmAb clone 4B5 (Ventana): 30 out of 33 (91%) obtained an optimal mark. The protocols giving an optimal result were all based on HIER using Cell Conditioning 1 mild or standard and an incubation time of 12-32 min in the primary Ab and iView or ultra View as the detection system. Using these protocol settings all of 31 (100 %) laboratories produced a sufficient staining.

HercepTest™ (Dako): 33 out of 47 (70%) obtained an optimal mark. The protocols giving an optimal result were typically based on HIER for 40 min using water bath at 97 - 99°C and an incubation time of 30 min in the primary Ab. Using these protocol settings 33 out of 43 (75 %) laboratories produced a sufficient staining.

CE IVD approved systems

Oracle™ (Leica) mAb clone CB11: Both of two obtained an optimal mark. These protocols used HIER in Bond Epitope Retrieval Solution 1 for 25 min. and the mAb clone CB11 in a Ready-To-Use format and an incubation time for 30 min. 

Abs in in-house systems

pAb A0485: 9 out of 15 (60 %) obtained an optimal mark. All protocols resulting in an optimal staining were based on HIER using Citrate pH 6 (2/4)*, EDTA/EGTA pH 8 (2/2)*, Tris-EDTA/EGTA pH 9 (1/4), Target Retrieval Solution (TRS) pH 9 (EnVision FLEX TRS high pH, Dako, (2/2) or TRS pH 6.1 (FLEX TRS low pH, Dako, (2/2). The pAb A0485 was typically diluted in the range of 1:200-1:1.000. Using these settings 10 out of 13 (77 %) obtained a sufficient staining marked optimal or good.
* (number of optimal results/number of laboratories using this buffer)

mAb CB11: 2 out of 7 (29 %) obtained an optimal mark. The optimal protocols were based on HIER using Tris-EDTA/EGTA pH 9 (1/3) and Citrate pH 6 (1/1) as HIER buffer. The Ab was typically diluted was 1:200-500. Using these settings 3 out of 4 (75 %) obtained a sufficient staining optimal or good.

Comments
In this assessment the prevalent feature of an insufficient staining was a too weak or a false negative reaction, which particularly and most critical was observed as a 0 or 1+ reaction in the HER-2 gene amplified breast carcinoma no. 3. This tumour was shown to be IHC 2+ in the NordiQC reference laboratories using HercepTest™, Dako and PATHWAY^®, Ventana and showed a low level of HER-2 gene amplification with a ratio of 2.4 – 2.8. The weak or negative reaction was seen in 88% of the insufficient results (28/32) whereas 12 % (4/32) of the insufficient results were caused by a false positive staining and/or a poor signal-to-noise ratio.

Grouped together, the FDA approved and CE IVD labelled IHC systems gave a total pass rate of 85 % (66 out of 78 laboratories following the vendors protocol recommendations and guidelines obtained a sufficient staining), while the pass rate for an in-house system was 50 % (16 out of 32 laboratories).

This was the 8th NordiQC HER-2 assessment. As illustrated in table 2, the two FDA approved systems have given a superior pass rate compared to the in-house HER-2 protocols.
As shown in Fig. 1. the average pass rate in 8 runs was 94 % for PATHWAY^® (Ventana, rmAb clone 4B5), 82 % for HercepTest™ (Dako) and 41 % for in-house protocols.

Figur 1. Pass rate through 8 HER-2 assessments

The slight decline in the overall pass rate for HER-2 in the current run was mainly caused by a lower pass rate of HercepTest™, which in this run was 70 % compared to 89 % in the previous run B6. The decline was in part caused by four laboratories not following the protocol guidelines from Dako (HIER was shortened or performed in a microwave oven instead of a calibrated water bath). For the remaining 10 laboratories obtaining an insufficient result with HercepTest ™ no plausible cause could be identified.

Scoring consensus

The laboratories were requested to send in their own scores (0, 1+, 2+, 3+) on the stained sections. For 82 out of 106 laboratories (77 %) returning the slip, the scores on all the tissues in the multi-tissue sections were in concordance with the scores given by the NordiQC assessor group. This is an improvement from 58 % and 62 % in run B6 and B7 respectively.
A sufficient staining combined with an interpretation in concordance with the NordiQC assessors was seen in 61% (65 out of 106).

Conclusion
The FDA approved HER-2 systems HercepTest™ (Dako) and PATHWAY^® rmAb clone 4B5 (Ventana), and the CE IVD labelled assay Oracle™ (Leica) were in this assessment the most reliable methods for the semi-quantitative IHC determination of HER-2 protein expression. The inclusion of 2+ tumours (from run B5  onwards) in the assessment material is essential to evaluate the IHC HER-2 performance and the robustness of the protocols used by the participants.

Figures
Figs. 1a and 1b – optimal staining results, same protocol
Figs. 2a and 2b – insufficient staining results – false negative, same protocol
Figs. 3a and 3b – insufficient staining results – false positive, same protocol.