The slide to be stained for HER-2 comprised the following 5 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 HER-2 FISH pharmDX™ Kit, Dako

*** Staining varied through the tissue block.

All carcinomas were fixed for 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 1+/2+ in the breast carcinoma no 3.

• A clear and unequivocal immunohistochemical staining marked as score 2+ in the breast ductal carcinoma no 4.

• A clear and unequivocal immunohistochemical staining marked as score 3+ in the breast ductal carcinoma no 5.

• 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 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) and the other breast carcinomas showed a reaction pattern as described above.

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 (e.g. the 3+ tumour and the 2+ tumour with gene amplification showed a 1+ reaction) or false positivity (e.g. the 0, 1+ and 2+ tumours without gene amplification showed a 3+ reaction).

Results
136 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 assessment marks 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® / CONFIRM™ rmAb clone 4B5 (Ventana): 38 out of 40 (95 %) obtained an optimal mark. The protocols giving an optimal result were all based on HIER using Cell Conditioning 1 mild or standard, 1 lab used MWO and Tris-EDTA pH 9. The incubation time for the primary Ab was in the range of 8 - 32 min and as detection kit either iView or UltraView was used. Using these protocol settings all of 40 (100 %) laboratories produced a sufficient staining.

HercepTest™ (Dako): 34 out of 49 (70%) obtained an optimal mark. The protocols giving an optimal result were based on HIER for 40 min using water bath at 96 - 99°C and an incubation time of 25-30 min in the primary Ab. Using these protocol settings 38 out of 47 (81 %) laboratories produced a sufficient staining.

CE IVD approved systems

Oracle™ (Leica) mAb clone CB11: 1 out of 3 obtained an optimal mark. The optimal protocol used HIER in Bond Epitope Retrieval Solution 2 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: 7 out of 19 (37 %) obtained an optimal mark. All protocols resulting in an optimal staining were based on HIER using either Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako) (2/3)*, Cell Conditioning 1 (BenchMark, Ventana) (2/3), Citrate pH 6 (1/9), Tris-EDTA/EGTA pH 9 (1/2) or EDTA/EGTA pH 8 (1/1). The pAb A0485 was typically diluted in the range of 1:400-1:1.000 depending on the total sensitivity of the protocol employed. Using these settings 7 out of 11 (64 %) obtained a sufficient staining marked optimal.
* (number of optimal results/number of laboratories using this buffer)

rmAb SP3: 7 out of 15 (47 %) obtained an optimal mark. The optimal protocols were based on HIER using either Tris-EDTA/EGTA pH 9 (2/6)*, Cell Conditioning 1 (BenchMark, Ventana) (2/3) or Citrate pH 6 (3/5) as HIER buffer. The Ab was typically diluted in the range of 1:20-200 depending on the total sensitivity of the protocol employed. Using these settings 8 out of 13 (62 %) obtained a sufficient staining (optimal or good).

Comments
In this assessment and in concordance with the previous HER-2 assessments, 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. 4. This tumour was shown to be IHC 2+ in the NordiQC reference laboratories using both HercepTest™, Dako, and PATHWAY®, Ventana, and showed a low level of HER-2 gene amplification (ratio of 2.5 – 2.9). The weak or false negative reaction was seen in 63 % of the insufficient results (24/38) whereas 27 % (14/38) of the insufficient results were due to a false positive staining and/or a poor signal-to-noise ratio. The weak, insufficient results were seen both when kits like the HercepTest™, Dako, and Oracle™, Leica, were used and when in-house protocols were used. The false positive stains and poor signal-to-noise ratios were virtually only seen when an in-house protocol was applied. The mAb clone 3B5 and the mAb clone cocktail 3B5 + e2-4001 both gave a strong granular cytoplasmic reaction in the majority of the neoplastic cells in all the specimens in the multitissue block hampering the interpretation of the specific membranous reaction. All 6 protocols based on these two Abs gave an insufficient result.

Grouped together, the FDA approved and CE IVD labelled IHC systems gave a pass rate of 86 % (79 out of 92 laboratories), while the pass rate for an in-house system was 43 % (19 out of 44 laboratories).

This was the 9th NordiQC HER-2 assessment. As illustrated in Fig. 1, the two FDA approved systems have almost constantly given a superior pass rate compared to the in-house HER-2 protocols.
As shown in Fig. 1. the average pass rate in the 9 runs was 94 % for PATHWAY® (Ventana, rmAb clone 4B5), 81 % for HercepTest™ (Dako) and 41 % for in-house protocols.

Figur 1. Pass rate through 9 HER-2 assessments

In this HER-2 assessment the over-all pass rate of 72 % was exactly the same as obtained in the previous assessment, run B7 2009. Many new laboratories participated in the current HER-2 assessment for the first time. For the 35 laboratories participating for the first time, the pass rate was 60 %, whereas the pass rate for the 101 laboratories participating in both run B7 and B8, the pass rate was 76 %.

Scoring consensus
The laboratories were requested to send in their own scores (0, 1+, 2+, 3+) on the stained sections. For 81 out of the 126 laboratories (64 %) 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. A sufficient staining combined with an interpretation in concordance with the NordiQC assessors was seen in 77 % (75 out of 98), which was a significant improvement from 61 % in run B7.

Conclusion
The two FDA approved HER-2 systems HercepTest™ (Dako) and PATHWAY® rmAb clone 4B5 (Ventana), were in this assessment the most reliable methods for the semi-quantitative IHC determination of HER-2 protein expression. The inclusion of the 2+ tumours (from run B5 onwards) with and without HER-2 gene amplification 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.