The slide to be stained for PR comprised the following five tissues:
 

*PR-positivity and intensity as characterized by NordiQC reference laboratories using the mAb clone 16 (Leica/Novocastra) and the rmAb clone 1E2 (Ventana).

All tissues were fixed in 10% neutral buffered formalin for 24 – 48 hours and processed according to Yaziji et al. (1).

Criteria for assessing a PR staining as optimal were:

• A moderate to strong, distinct nuclear staining reaction of both the columnar and basal squamous epithelial cells and most of the stromal cells (with the exception of endothelial cells and lymphoid cells) in the uterine cervix.

• An at least weak to moderate distinct nuclear staining reaction in the appropriate proportion of the neoplastic cells in the breast ductal carcinomas no. 3, 4 & 5.

• No nuclear staining reaction of the neoplastic cells in the breast carcinoma no. 2 and no more than a weak cytoplasmic staining reaction in cells with a strong nuclear staining reaction - for the mAb clone PgR636 a moderate to strong cytoplasmic staining reaction in the columnar epithelial cells of the uterine cervix was accepted.

258 laboratories participated in this assessment. 87 % achieved a sufficient mark. Table 1 summarizes antibodies (Abs) used and marks.

Table 1. Abs and assessment marks for PR, run B14

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

Following protocol parameters were central to obtain an optimal staining result:

Concentrated antibodies
mAb clone 16: Protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using either Target Retrieval Solution (TRS) pH 9 (3-in-1) (Dako) (5/5)*, TRS pH 9 (Dako) (2/2), TRS low pH 6.1 (Dako) (1/1), Cell Conditioning 1 (CC1; Ventana) (2/2), Bond Epitope Retrieval Solution 2 (BERS 2; Leica) (5/6), BERS 1 (Leica) (2/3), Tris-EDTA/EGTA pH 9 (1/3) or Citrate pH 6 (3/4) as the retrieval buffer.
The mAb was typically diluted in the range of 1:75-1:800. Using these protocol settings 25 of 25 (100 %) laboratories produced an optimal staining result.
* (number of optimal results/number of laboratories using this reagent)

mAb clone cocktail 16 + SAN27: The protocol giving an optimal result was based on HIER using CC1 (Ventana) (1/1) as the retrieval buffer. The dilution was 1:200.

mAb clone1A6: The protocol giving an optimal result was based on HIER using Tris-EDTA/EGTA pH 9 (1/1) as the retrieval buffer. The dilution of the primary Ab was 1:50.

mAb clone PgR 636: Protocols giving an optimal result were all based on HIER using either TRS pH 9 (3-in-1) (Dako) (12/12), TRS pH 9 (Dako) (7/10), TRS low pH 6.1 (Dako) (1/1), CC1 (Ventana) (1/2), BERS 2 (Leica) (1/3), BERS 1 (Leica) (1/1), Tris-EDTA/EGTA pH 9 (10/13) or Citrate pH 6 (4/5) as the retrieval buffer.
The mAb was typically diluted in the range of 1:100-1:800 depending on the total sensitivity of the protocol employed. Using these protocol settings 45 of 45 (100 %) laboratories produced a sufficient staining result (optimal or good).

mAb clone PgR 1294: Protocols giving an optimal result were based on HIER using Tris-EDTA/EGTA pH 9 (1/1) or Citrate pH 6 (1/1) as the retrieval buffer.
The mAb was diluted in the range of 1:1.250-1:5.000 depending on the total sensitivity of the protocol employed. Using these protocol settings 2 of 2 (100 %) laboratories produced an optimal staining.

rmAb clone EP2: The protocol giving an optimal result was based on HIER using Citrate pH 6 as the retrieval buffer. The rmAb was diluted 1:100.

rmAb clone SP2: Protocols giving an optimal result were based on HIER using either CC1 (Ventana) (1/1) or Citrate pH 6 (1/1) as the retrieval buffer.
The rmAb was diluted in the range of 1:100-1:300 depending on the total sensitivity of the protocol employed. Using these protocol settings 2 of 2 (100 %) laboratories produced an optimal staining.

rmAb clone Y85: Protocols giving an optimal result were based on HIER using Citrate pH 6 (2/3) as the retrieval buffer. The rmAb was diluted in the range of 1:50-1:200 depending on the total sensitivity of the protocol employed. Using these protocol settings 3 of 3 (100 %) laboratories produced a sufficient staining result (optimal or good).

Ready-To-Use antibodies
mAb clone 16 (prod. no. PA0312, Leica/Novocastra): Protocols giving an optimal result were based on HIER using BERS 2 (Leica), 15 min incubation of the primary Ab and Bond Polymer Refine Detection (DS9800) as detection system. Using these protocol settings 5 of 5 (100 %) laboratories produced an optimal staining result (optimal or good).

mAb clone PgR 636 (prod. no. IR/IS068, Dako): Protocols giving an optimal result were typically based on HIER in PT-Link (heating time for 10-20 min at 95 - 98°C) using TRS pH 9 (3-in-1) (Dako) or TRS pH 9 (Dako) as HIER buffer, 15-30 min incubation of the primary Ab and EnVision Flex or EnVision Flex+ (K8000/K8002) as the detection system. Using these protocol settings 32 of 33 (97 %) laboratories produced a sufficient staining result.

mAb clone PgR 1294 (prod. no SK310/K4071, Dako): Protocols giving an optimal result were based on HIER in a Pressure Cooker using Citrate pH 6, 20-30 min incubation of the primary Ab and EnVision (SK310/K4071) as detection system. Using these protocol settings 3 of 3 (100 %) laboratories produced a sufficient staining result (optimal or good).

rmAb clone 1E2 (prod.no 790-2223/4296, Ventana): Protocols giving an optimal result were typically based on HIER using mild or standard CC1 8-60 min incubation of the primary Ab and iView (760-91, Ventana), UltraView (760-500, Ventana) or OptiView (760-700, Ventana) as detection system. Using these protocol settings 88 out of 114 (77 %) laboratories produced a sufficient staining result.

The most frequent causes of insufficient stainings were:

- Less successful primary antibody
- Low sensitivity of the staining protocol
- Protocols giving a false positive staining reaction (no single cause was identified)

In this assessment the prominent feature of an insufficient staining was characterized by a false positive staining reaction in > 10 % of the neoplastic cells of the breast carcinoma no. 2, which by the reference laboratories and the vast majority of the participating laboratories was classified as being PR negative. A false positive result was seen in 24 of the 34 insufficient staining results (69 %) and in the remaining insufficient results a false negative or a general too weak staining reaction was seen.
The false positive staining was only seen when the rmAb clone 1E2 applied as RTU (Ventana) was used, whereas as all other Abs and RTU systems consistently gave a negative staining result in this tumour. It was not possible to identify any single parameter (e.g. excessive HIER, prolonged Ab incubation time, lot-to-lot variations of the primary Ab or use of high sensitive detection systems). It must be considered if the breast carcinoma no. 2 expressed low levels of PR and that the positive staining was revealed by the use of a very sensitive protocol. However, based on the fact that, both 1) the majority of protocols based on the same Ab and identical protocol settings and 2) all other Abs (with equal or superior sensitivity in the remaining cores), gave a completely negative staining of the neoplastic cells of the breast carcinoma (no. 2), the positive staining reaction should be considered to be false.

NordiQC is in contact with Ventana to elucidate the aberrant staining pattern. In this context it has to be mentioned that 22 of the 24 protocols giving a false positive staining reaction was based on slightly modified protocol settings compared to the guidelines given by Ventana/Roche for the Ready-To-Use product – typically prolonged incubation time in the primary Ab.

The remaining insufficient staining results were primarily caused by a too low sensitivity of the protocol, e.g. too low concentration of the primary Ab or a protocol based on a RTU format applied to a staining system/platform for which the product was not calibrated.

The mAb clones 16 and PgR 636 from Leica/Novocastra and Dako respectively gave a very high pass rate both as a concentrate or as a Ready-To-Use system and provided the expected PR positivity in all the included tissues. For the mAb clone PgR 636 an intracytoplasmic staining reaction in the columnar epithelial cells of the uterine cervix was accepted, as this did not complicate the interpretation in the breast carcinomas.

Controls
As observed in the previous assessments of PR, uterine cervix is an appropriate positive control for evaluation of the sensitivity of PR staining: With an optimal protocol almost all columnar epithelial cells, the majority of basal squamous epithelial cells and most of the stromal cells must show a strong and distinct nuclear staining with only a minimal cytoplasmic reaction. No staining must be seen in endothelial cells and lymphocytes. As negative control it is recommendable to include tonsillar tissue, in which no nuclear staining should be seen.

Effect of external assessment
This was the 7th assessment of PR in NordiQC. An increase in the proportion of sufficient results was seen compared to the two latest runs for PR as shown in figure 1.

Figure 1 – pass rate in the NordiQC assessments for PR


The improvement has been obtained despite many laboratories participated for the first time and the improvement most likely is influenced by many factors inclusive the access to and extended use of optimized antibodies both as Ready-To-Use systems and as concentrates for the demonstration of PR.

1. Yaziji H, Taylor CR, Goldstein NS, Dabbs DJ, Hammond EH, Hewlett B, Floyd AD, Barry TS, Martin AW, Badve S, Baehner F, Cartun RW, Eisen RN, Swanson PE, Hewitt SM, Vyberg M, Hicks DG; Members of the Standardization Ad-Hoc Consensus Committee.
Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry.
Appl Immunohistochem Mol Morphol. 2008 Dec;16(6):513-20. PubMed PMID: 18931614.

Conclusion
The mAb clones 16 and PgR 636 were in this assessment the most robust Abs for PR. Both had a high pass rate and could be applied either as concentrate or as a Ready-To-Use system.
In this assessment a false positive staining reaction of the rmAb clone 1E2 was the prominent feature of an insufficient staining result. Uterine cervix is an appropriate positive control. Most of the columnar epithelial cells, basal squamous epithelial cells and most of the stromal cells must show a strong and distinct nuclear staining reaction. Lymphocytes and endothelial cells must be negative.