slide to be stained for p53 comprised:
1. Liver, 2. Colon, 3. Adenocarcinoma (colon), 4. Serous carcinoma (ovary),
5. Endometrioid carcinoma (endometrium), 6. Fallopian tube.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a p53 staining as optimal included:
A strong, distinct nuclear staining of ≥ 75 % of the neoplastic
cells of the ovarian serous carcinoma and ≥ 50 % of the neoplastic
cells of the colon adenocarcinoma.
A strong, distinct nuclear staining in 5 – 20 % of the neoplastic
cells of the endometrioid carcinoma.
No or only a weak nuclear staining in scattered basal epithelial
cells in the colon and the fallopian tube.
No staining in the liver.
93 laboratories submitted stains. At the assessment 18 achieved
optimal marks (19 %), 45 good (49 %), 27 borderline (29 %) and 3
poor marks (3 %).
The following Abs were used:
mAb clone DO-7 (Dako; n=70, Novocastra, n=5; Ventana, n=4; NeoMarkers,
n=1; Biocare Medical, n=1)
mAb clone DO-1 (Calbiochem, n=1; Dianova, n=1; Novocastra, n=1)
mAb clone BP53-12 (NeoMarkers, n=1; Novocastra, n=1)
mAb clone DO-7 + BP53-12 (NeoMarkers, n=2)
mAb clone Bp-53-11 (Ventana, n=1)
rmAb SP5 (NeoMarkers, n=3)
pAb NCL-p53-CM1 (Novocastra, n=1)
Optimal staining for p53 in this assessment was obtained with the
mAb clone DO-7 (14 out of 81), mAb clone DO-1 (2 out of 3), the mAb
clone BP53-12 (1 out of 2) and the mAb cocktail DO-7 + BP53-12 (1
out of 2).
DO-7: The protocols giving an optimal result were
all based on heat induced epitope retrieval (HIER) with
Tris-EDTA/EGTA pH 9 or Cell Conditioning1 (BenchMark, Ventana) as
HIER buffer. The mAb was typically diluted in the range of 1:100 –
1:1,000 depending on the total sensitivity of the protocol employed,
or as a Ready-To-Use (RTU) product. Using these protocol settings 34 out
of 44 (77 %) laboratories produced a sufficient (optimal or good) staining.
DO-1: The protocols giving an optimal result were
all based on HIER using Citrate pH 6 as HIER buffer. The Ab was
diluted in the range of 1:30 – 1:400 depending on the total
sensitivity of the protocol employed. Using these protocol settings
3 out of 3 (100 %) laboratories produced a sufficient staining.
BP53-12: The protocol giving an optimal result
was based HIER using Citrate pH 6 as HIER buffer. The Ab was diluted
DO-7 + BP53-12: The protocol giving an optimal
result was based on HIER using Bond Epitope Retrieval Solution 2
(Bond, Vision Biosystems) as HIER buffer. The Ab was diluted 1:200.
Using these settings 1 out of 2 laboratories produced a
The most frequent causes of insufficient staining were:
- Too low concentration of the primary antibody
- Too high concentration of the primary antibody
- Less successful primary antibody
The prevalent feature of an insufficient staining
was a weak/false negative staining, which was observed in 20 out of
the 30 insufficient stains. The majority of the laboratories was
capable to demonstrate p53 in the ovarian serous carcinoma, whereas
the reaction in the colon adenocarcinoma and endometrial carcinoma
was too weak or false negative.
However, also a false positive nuclear staining was observed in 10
out of the 30 insufficient stains, and was typically characterized
by a moderate reaction in virtually all the epithelial cells of the
fallopian tube and the majority of the benign epithelial cells of
The high proportion of both false negative and false positive stains
for p53 indicates that the immunohistochemical protocol for p53 has
to be carefully calibrated to be used as a diagnostic tool and that the
identification of a reproducible positive and negative control is
crucial to validate the threshold of the protocol for p53.
In this assessment colon adenocarcinoma and normal colon was the
most reliable control, in which only scattered basal epithelial
cells in the mucosa of the normal colon should display a weak
reaction, while the majority of the neoplastic cells of the colon
adenocarcinoma should display a strong nuclear staining.
Only antibodies raised to both wild type and mutant p53 gave
sufficient results, while all 3 laboratories using the rmAb clone
SP5 raised to wild type p53 achieved an insufficient mark showing a
too weak or false negative staining.
As no normal tissue is appropriate for control, the laboratory
should use a multitissue block including tumour with varying
expression of p53.
The mAb clones DO-7, DO-1, BP53-12 and the mAb cocktail
BP53-12 are all useful for the demonstration of p53. HIER is
mandatory to obtain an optimal result. Concentration of the primary
Ab should be carefully calibrated.