|
 The
slide to be stained for progesterone receptor
(PR)
comprised:
1. Uterine cervix, 2. Ductal breast carcinoma, PR negative,
3. Ductal breast carcinoma, PR 40-60 % positive, 4. Ductal breast
carcinoma, PR 80–100 % positive. The positivity of
the 3 ductal breast carcinomas was verified in 4 reference IHC
laboratories.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a PR staining as optimal included:
-
A strong and distinct nuclear staining of
the columnar epithelial cells, the basal squamous epithelial cells
and the stromal cells in the uterine cervix.
-
A moderate to strong and distinct nuclear
staining of the ductal breast carcinomas no. 3 and 4 in accordance
with the PR status.
-
No nuclear staining of the PR negative
ductal breast carcinoma no. 2 – only epithelial cells in remnants
of normal glands should show a positive reaction.
81 laboratories participated in the
assessment. 39 achieved optimal marks (49 %), 21
good (26 %), 19 borderline (23 %) and 2 (2 %) poor marks.
The following Abs were used:
mAb clone PgR 636 (Dako, n=39)
mAb clone 16 (Novocastra, n=12; Ventana, n=9)
mAb clone 1A6 (Novocastra, n=5; Ventana, n=1)
mAb clone PR-1 (Immunovision, n=2)
mAb clone PgR 1294 (Dako, n=2)
mAb clone PR 88 (Biogenex, n=1)
mAb clone hPRa 2+hPRa 3 (NeoMarkers, n=1)
rmAb clone 1E2 (Ventana, n=5)
rmAb clone SP2 (NeoMarkers, n=4)
Optimal staining for PR
in this assessment was obtained with the mAb clones PgR 636, 16,
PR 1294 and the rmAb clones 1E2 and SP2 in the following settings:
PgR 636: the
protocols giving an optimal result were all based on heat induced
epitope retrieval (HIER) using either Tris-EDTA/EGTA pH 9 (18
out of 29) or Citrate pH 6,0 (3 out of 7). The clone PgR
636 was used in the range of 1:20 – 800 depending on the total
sensitivity of the protocol employed. Using these protocol settings
28 out of 39 laboratories (72 %) produced a sufficient staining
(optimal or good), 21 of them were optimal (54 %).
16:
the protocols giving an optimal result were based on HIER using either Cell Conditioning 1 (CC1
Ventana, 5 out of 12), Tris-EDTA/EGTA pH 9 (4 out of 5) or
Bond Epitope Retrieval Solution 2 (BERS 2, Vision BioSystems, 1
out of 1) as the HIER buffer. The clone 16 could both be used
as a Ready-To-Use product and as a concentrate diluted in the range
of 1:100 – 500 depending on the total sensitivity of the protocol
employed. Using these protocol settings 14 out of 16 laboratories
(88 %) produced a sufficient staining (optimal or good), 10 of
them were optimal (63 %).
PgR 1294:
the protocols giving an optimal result were performed according to the protocol of the
PR-PharmDx kit (Dako) based on HIER (Pressure cooker) in
Target Retrieval Solution and a RTU Ab. 2 out of 2 using this kit
obtained an optimal mark.
1E2:
the protocols giving an optimal result were all based on HIER in Cell
Conditioning 1 (CC1, Ventana) and a RTU Ab. Using these protocol
settings 4 out of 5 produced a sufficient staining, all 4 assessed
as optimal (80 %).
SP2:
the protocols giving an optimal result were all based on HIER in either Cell
Conditioning 1 (CC1, Ventana 1 out of 1) or Tris-EDTA/EGTA pH
9 (1 out of 3). The clone SP2 was diluted 1:100. Using
these protocol settings 2 out of 4 produced a sufficient staining
both assessed as optimal (50 %).
The most frequent causes of an insufficient
staining were:
- Less successful primary antibody
- Too low or too high concentration of the primary antibody
- Inappropriate epitope retrieval
In this assessment (and in concordance with the observation in the
previous PR assessment, run 10, 2004) almost all laboratories were
able to demonstrate PR in the ductal breast carcinoma no. 4 with
80-100 % positivity and a strong staining intensity, whereas the
prevalent feature of the insufficient staining was a too weak or
false negative staining of the ductal breast carcinoma no. 3 with
40-60 % positivity and only a moderate staining intensity.
A too weak or false negative staining was seen in 90 % of the
insufficient results (19 out of 21), while in 9 % (2 out of 21) a too
strong staining and false positive PR staining reaction was seen.
The majority of the Abs used in the assessment showed almost the
same staining pattern except the mAb clone 1A6, which did not label
the nuclei of the basal squamous epithelium in the cervix, while the
other layers showed a positive cytoplasmic staining. The other
Abs showed a distinct nuclear reaction in the basal cells and no
or only minimal cytoplasmic reaction of the squamous epithelial
cells. At the same time the clone 1A6 seemed to have a lower
affinity of PR in the breast ductal carcinomas as fewer cells in
general were labelled with this clone.
The uterine cervix seemed to be an appropriate control for the
evaluation of the sensitivity of the PR staining. In the protocols
giving an optimal PR staining of the ductal breast carcinomas the
majority of the basal squamous epithelial cells of the cervix showed a distinct
nuclear reaction. In the protocols giving an insufficient PR
staining of the ductal breast carcinomas the basal cell layer only
showed a focal or a negative staining.
PR was also assessed in
run 10, 2004, where 79 laboratories
participated out of which 30 % (24 laboratories) obtained an
insufficient mark. Each of these was given a specific
recommendation to improve their protocol. 18 of the laboratories submitted a new PR stain
in run B2. 14 of them followed the recommendations to change their protocol and 11 improved
from insufficient to either good or optimal (79 %). 4 laboratories did not follow the
recommendations and none of these obtained a sufficient staining in
run B2.
Conclusion
The clones PgR 636, 16, PgR 1294,
1E and SP2 all seem to be robust Abs for the demonstration of PR. HIER is mandatory to obtain an
optimal result. The concentration of the primary Ab should be
carefully calibrated and the uterine cervix seems to be an
appropriate control tissue for this calibration as the basal squamous epithelial cells should show a distinct nuclear reaction
with minimal cytoplasmic reaction. |
