 The
slide to be stained for HER-2 comprised:
|
1. Cell line BT474 |
(Amplified)* |
|
2. Cell line JIMT-1** |
(Amplified) |
|
3. Cell line MCF-7 |
(Not amplified) |
|
4. Breast ductal carcinoma*** |
(Not amplified) |
|
5. Breast ductal carcinoma |
(Amplified) |
|
6. Breast ductal carcinoma |
(Amplified) |
|
7. Breast ductal carcinoma |
(Not amplified) |
*Amplification of the HER-2 gene demonstrated by FISH and CISH.
**The cell line JIMT-1 was not used in the assessment due to
technical problems in the preparation of a homogenous representative
cell population through out the cell pellet used for the
multi-tissue block.
*** All of the four carcinomas were selected as to reveal an
immunohistochemically strong membranous staining in genetically
amplified cases and none or only a weak staining in non-amplified
cases, as found in two reference laboratories.
The immunohistochemical HER-2 staining was assessed by NordiQC with
reference to the HER-2 amplification status indicated above.
Criteria for assessing a HER-2 staining as optimal included:
-
A staining corresponding score 3+¤ in cell line no. 1. and the two
breast ductal carcinomas no. 5 and 6.
-
A staining corresponding score 0 or 1+ in the cell line no. 3 and
the two breast ductal carcinomas no. 4 and 7.
-
No staining of normal breast glands.
-
A cytoplasmic staining that did not interfere with the
interpretation of the membranous staining.
¤The immunohistochemical
scoring system used:
|
Score 0 |
No staining is observed or
cell membrane staining is observed in less than 10% of the tumour
cells. |
|
Score 1+ |
A faint perceptible
membrane staining can be detected in more than 10% of the tumour
cells. The cells are only stained in part of their membrane. |
|
Score 2+ |
A weak to moderate
complete membrane staining is observed in more than 10% of the
tumour cells. |
|
Score 3+ |
A strong complete membrane
staining is observed in more than 10% of the tumour cells. |
68
laboratories submitted stains. At the assessment 28 achieved
optimal marks (41
%), 22 good (32
%), 8
borderline (12
%) and 10
(15
%) poor marks.
The following Abs/kits were used:
mAb clone 3B5 (NeoMarkers, n=1)
mAb clone 10A7 (Novocastra, n=1)
mAb clone CB11 (Novocastra, n=4; NeoMarkers, n=1)
mAb clone TAB250 (Zymed, n=1)
mAb clone SP3 (NeoMarkers, n=3)
pAb 28-0004
(Zymed, n=1)
pAb A0485 (Dako, n=11)
Herceptest K5204, K5205, K5206, K5207 (Dako, n=37)
Pathway 760-2694 (Ventana, n=8)
The table gives the results related to the Abs/kits
| |
Marks |
|
Abs/kits applied: |
Optimal |
Good |
Borderline |
Poor |
|
mAb clone 3B5 |
0 |
1 |
0 |
0 |
|
mAb clone 10A7 |
0 |
0 |
0 |
1 |
|
mAb clone CB11 |
1 |
1 |
1 |
2 |
|
mAb clone TAB250 |
0 |
0 |
0 |
1 |
|
mAb clone SP3 |
0 |
1 |
0 |
2 |
|
pAb 28-0004 |
1 |
0 |
0 |
0 |
|
pAb A0485 |
3 |
1 |
4 |
3 |
|
Herceptest K5204/K5205/K5206/K5207 |
22 |
13 |
2 |
0 |
|
Pathway 760-2694 |
1 |
5 |
1 |
1 |
Using the mAb clone CB11 an optimal stain was obtained with HIER in
a microwave oven with citrate pH 6 as heating buffer and the mAb
diluted 1:50.
Using the pAb 28-0004
optimal stain was obtained with HIER in a
microwave oven using citrate pH 7.2 as heating buffer and the mAb
diluted 1:200.
Using the pAb A0485 optimal stains were obtained with HIER either in
a pressure cooker with citrate pH 6 as heating buffer or the
Benchmark immunostainer (Ventana) with either CC1 or CC2 as heating
buffer, and the mAb diluted 1:40 - 1:500 depending on the
sensitivity of the protocol applied.
Using Herceptest and Pathway the procedure was performed accordingly
to the instructions from the companies.
The most frequent causes of insufficient staining were (often in
combination):
- Less successful primary Abs
- Wrong calibration of the primary Ab giving both false negative and
false positive reactions
- Excessive retrieval
The prevalent feature of an inappropriate staining was either a too
weak or false negative reaction in one of the breast carcinomas with
gene amplification (decreasing the score from 3+ to 2+ or 1+), or a
too strong and false positive reaction in one of the breast
carcinomas without gene amplification (increasing the score from
0/1+ to 2+ or 3+).
In this assesment the two FDA approved systems Herceptest (Dako) and
Pathway (Ventana) were the best methods to determine the level of
the HER-2 protein expression in relation to the gene amplification
status. A total of 41
out of 45 laboratories (91
%) obtained a
sufficient staining (optimal or good) for HER-2 using one of these
systems. Using an in-house staining system with a self established
level of sensitivity and specificity only 9
out of 24 (38
%)
obtained a sufficient staining. For instance, the pAb A0485 from
Dako was used both with the Herceptest and as
an in-house method. The
proportions of sufficient stains were 95 % and 36
%, respectively.
In the assessment the laboratories were requested to send in their
own scores for their HER-2 stains. 57 out of 66 laboratories
followed the request. For 33 laboratories (58 %) the submitted
scores were in concordance with those given by the NordiQC
assessors, while for 24 laboratories (42%) a discrepancy was
observed. In 6 cases the discrepancy was related to the scoring of
the cell lines and in 18 cases it was related to the scoring of the
breast ductal carcinomas. Typically the two breast carcinomas (no. 5
and 6) with gene amplification and HER-2 protein 3+ over expression
was scored as 2+ and the breast carcinoma no. 7 without gene
amplification and HER-2 protein expression 0-1+ was scored as 2+. It
is noteworthy that 14 out of 52 laboratories (27 %), which produced
a sufficient HER-2 staining, had an inappropriate interpretation. An
optimal staining result in combination with a correct interpretation
was seen in 17 out of 57 laboratories (30%).
Conclusions
The FDA approved immunohistochemical HER-2 systems Herceptest and
Pathway seems to be more reliable methods for the detection of HER-2
protein over expression than in-house methods.
Training in the interpretation of HER-2 stains is needed for a
large number of the laboratories. |
