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.           3) mAb: mouse monoclonal antibody, rmAb: rabbit monoclonal antibody, pAb: polyclonal antibody.

CE-IVD / FDA approved systems:
PATHWAY® rmAb clone 4B5 (Ventana): 20 out of 21 (95 %) protocols were assessed as optimal. The protocols giving an optimal result were typically based on Heat Induced Epitope Retrieval (HIER) in Cell Conditioning 1 (CC1), mild or standard in the BenchMark XT or Ultra. The incubation time for the primary Ab was in the range of 16 – 32 min. and either iView, UltraView or OptiView were used as the detection kit. Using these protocol settings 20 out of 20 (100%) laboratories produced a sufficient staining (optimal or good).

CONFIRM™ rmAb clone 4B5 (Ventana): 6 out of 8 (75 %) protocols were assessed as optimal. The protocols giving an optimal result were typically based on HIER in CC1 mild in the BenchMark XT or Ultra. The incubation time for the primary Ab was typically in the range of 12 – 32 min. and UltraView was used as the detection kit. Using these protocol settings 6 out of 6 (100%) laboratories produced a sufficient staining.

HercepTest™ SK001 (Dako): 5 out of 8 (63 %) protocols were assessed as optimal. The protocols giving an optimal result were typically based on HIER in HercepTest epitope retrieval solution at 95 - 99°C for 40 min in a water bath or PT Link and an incubation time of 20-30 min in the primary Ab. Using these protocol settings 8 out of 8 (100 %) laboratories produced a sufficient staining.

HercepTest™ K5207 (Dako): 3 out of 5 (60 %) protocols were assessed as optimal. The protocols giving an optimal result were typically based on HIER in HercepTest epitope retrieval solution at 95 - 99°C for 40 min in a water bath or PT Link and an incubation time of 30 min in the primary Ab. Using these protocol settings 4 out of 5 (80 %) laboratories produced a sufficient staining.


Abs for in-house systems:
rmAb SP3: 2 out of 3 (67 %) protocols were assessed as optimal. All protocols resulting in an optimal staining were based on HIER using either Bond Epitope Retrieval Solution 2 (BERS2; Bond, Leica) (1/1)* or Citrate pH 6.7 (1/1)*. The rmAb SP3 was diluted in the range of 1:50-60 depending on the total sensitivity of the protocol employed. Using these settings 2 out of 2 laboratories produced an optimal staining.
*number of optimal results/number of laboratories using this buffer).

pAb A0485: 2 out of 4 (50 %) protocols were assessed as optimal. All protocols resulting in an optimal staining were based on HIER using Target Retrieval Solution pH 6.1 (Dako) (2/3). The pAb A0485 was diluted in the range of 1:200-1:300 depending on the total sensitivity of the protocol employed. Using these settings 2 out of 2 laboratories produced an optimal staining.


Comments
In this second run for HER-2 IHC in the gastric cancer pilot module a pass rate of 96 % was obtained. Only 2 out of 51 laboratories obtained an insufficient mark due to a false negative result.
The false negative reaction was in particular and most critically observed as a 0/1+ IHC reaction in the HER-2 gene amplified gastric carcinoma no. 8 shown to be IHC 2+ in the NordiQC reference laboratories using both HercepTest™, Dako, and PATHWAY®, Ventana, with a low level of HER-2 gene amplification (HER-2/chr17 ratio 2.5). No reason for the insufficient result could be identified, as the applied protocol settings were similar to the protocols giving the optimal staining results.
The two most widely used assays for HER-2 PATHWAY®, Ventana and HercepTest™, Dako showed an almost identical membrane staining reaction in all the carcinomas. However, in the carcinoma no. 5 a moderate to strong cytoplasmic and nuclear staining reaction was seen with PATHWAY® complicating the interpretation of the specific membrane staining (1+), whereas this tumour showed a staining reaction easily interpreted as 1+ with HercepTest™ (Figs. 3a & 3b).
It was noteworthy that all 7 out of 7 protocols based on an in-house HER-2 system gave a sufficient result. In run G1 only 8 out of 12 protocols (75 %) based on an in-house HER-2 system gave a sufficient result.

Scoring consensus
The laboratories were requested to send in their own scores (0, 1+, 2+, 3+) on the stained sections. For 24 out of the 44 laboratories (55 %) 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 57 % (24 out of 42). The relative low scoring consensus most likely is related to the modified interpretation guidelines compared to the well established guidelines for HER-2 IHC within breast pathology. The most frequent discrepancies were related to the carcinoma no. 2 (HER-2, 2+, non-amplified) and the carcinoma no. 8 (HER-2, 2+, amplified). The carcinomas no. 5 and 10 giving an aberrant non-membranous staining pattern were by many laboratories scored as 2+. This was accepted as all equivocal results must be retested with another assay.

This was the second run for HER-2 IHC in the gastric cancer pilot module and a similar pass rate compared to the first run was obtained as seen in table 2.

Table 2: Proportion of sufficient results for HER-2 IHC in the two NordiQC runs performed

Conclusion
In both the G1 and G2 assessment, a very high pass rate for HER-2 IHC was seen. Both the CE-IVD / FDA approved HER-2 systems and in-house HER-2 systems could be used to obtain an optimal staining result and no significant difference regarding the pass rates could be identified between in-house systems or CE-IVD / FDA approved systems. The inclusion of the 2+ tumours with and without HER-2 gene amplification is essential to evaluate the HER-2 IHC performance and the robustness of the protocols used by the participants. Training in scoring is highly warranted and image analysis assisted scoring has to be taken in consideration to improve and facilitate the interpretation.

The gastric cancer pilot module will not be implemented as a module in 2012.