 The
slide to be stained for HER-2 comprised:
| |
IHC* |
FISH* |
| |
HER-2 Score
(0, 1+, 2+, 3+) |
Mean HER-2
Copy number |
|
1. Cell line SK-BR3 |
3+ |
Clusters > 3 |
|
2. Cell Line MDA-MB-453 |
2+ |
2.4 |
|
3. Cell Line MDA-MB-175 |
1+ |
1.3 |
|
4. Cell Line MDA-MB-231 |
0 |
1.1 |
|
5. Breast ductal carcinoma |
3+ |
Clusters > 3 |
|
6. Breast ductal carcinoma |
3+ |
Clusters > 3 |
|
7.
Breast ductal carcinoma |
1+/2+** |
1.3 |
|
8.
Breast ductal carcinoma |
1+ |
1.5 |
|
9.
Breast ductal carcinoma |
0 |
1.1 |
* Verified by 4 NordiQC reference
laboratories. ** The score varied through the block.
The stains were primarily assessed with respect to the capability of the laboratories to
identify and determine the level of the HER-2 protein expression
in the histological specimens corresponding to the gene status. The
staining of the additional slide containing 4 cell lines (provided
by UK NEQAS) was used to evaluate if they could be included for future HER-2
quality control.
The IHC scoring system was as
follows:
|
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. |
Criteria for assessing a HER-2 IHC staining as
optimal included:
- A 3+ staining of the breast ductal carcinomas no.
5 and 6.
- A 1+ or 2+ staining of the breast ductal carcinoma no 7.
- A 1+ staining of the breast ductal carcinoma no 8.
- No staining of the breast ductal carcinoma no. 9
and normal breast glandular tissue.
- In cells with a membraneous reaction, no more than a faint cytoplasmic reaction
that did not affect the interpretation of the former.
70 laboratories participated in the assessment.
37 laboratories achieved an optimal staining (53 %), 15 good (21 %),
14 borderline (20 %) and 4 (6 %) poor staining.
Table 1. Scores given for each
system/Ab (histological specimens).
| |
Score
(tissue slide) |
| |
Optimal |
Good |
Borderline |
Poor |
| FDA approved systems: |
|
|
|
|
| HercepTest K5204,
K5206, K5207, SK001 (Dako, n=34) |
23 |
8 |
2 |
1 |
| PATHWAY 790-2991,
800-2996 rmAb clone 4B5 (Ventana, n=15) |
12 |
2 |
1 |
0 |
| PATHWAY 760-2694 mAb
CB11 (Ventana, n=2) |
0 |
0 |
2 |
0 |
| Abs in in-house systems:
|
|
|
|
|
| pAb clone A0485 (Dako,
n=10) |
1 |
3 |
5 |
1 |
| mAb clone CB11
(NeoMarkers, n=1) |
0 |
0 |
1 |
0 |
| rmAb clone SP3
(NeoMarkers, n=2; Diagnostic Biosystems, n=1) |
0 |
0 |
1 |
2 |
| mAb clone e2-4001+3B5
(NeoMarkers, n=2) |
0 |
0 |
2 |
0 |
| mAb clone TAB250
(Zymed, n=1) |
0 |
1 |
0 |
0 |
| mAb clone 3B5
(NeoMarkers, n=1) |
0 |
1 |
0 |
0 |
| SISH system* |
|
|
|
|
| INFORM
HER2 DNA Probe (Ventana, n=1) |
1 |
0 |
0 |
0 |
Total |
37 |
15 |
14 |
4 |
* Silver In Situ
Hybridization system, accepted as equivalent to IHC.
As shown in Table 1, with an FDA approved system,
35 out of 51 (69%) obtained an optimal mark, while with an in-house
(home made) system only 11% (2 out of 19) obtained this.
HercepTest (Dako): 23 out of 34 (68%)
obtained an optimal mark. The procedure was in all cases performed
according to the
company's instructions, including Heat Induced Epitope Retrieval
(HIER) in a water bath. With these settings
31 out of 34 (91%) of the laboratories obtained a sufficient
(optimal or good) mark.
PATHWAY rmAb clone 4B5 (RTU)
(Ventana): 12 out of 15 (80%) using
this newly FDA-approved system obtained an optimal mark. The optimal protocols were all
based on HIER in either Cell Conditioning 1 pH 8,4
(CC1; Benchmark, Ventana)(11 out of 13) or EDTA/EGTA pH 8 (1 out
of 2). With CC1 as HIER buffer all of 14 laboratories
(100%) obtained a sufficient mark.
Using one of these two systems 92% was
marked as sufficient.
pAb A0485 (Dako): 1 out of 10 (10%) obtained an optimal mark.
The Ab was used with a dilution of 1:250 and Cell Conditioning 2 pH
6 (Benchmark, Ventana) was used as HIER buffer. 4 out of 10 (40%)
using A0485 obtained a sufficient mark.
Grouped together, using one of the six in-house systems, 6 out
of 18 (33%) of
the stains obtained a sufficient mark.
INFORM HER2 DNA Probe (SISH)(Ventana): One laboratory used
this method and was marked optimal.
The FISH results were used as reference and the SISH slide was
scored according to the instructions from the vendor.
The most frequent causes of insufficient
stains were (often in combination):
- Less successful primary Ab
- Wrong calibration of the primary Ab
- Excessive retrieval
The prevalent feature of an inappropriate staining was typically a
too weak or false negative reaction in the breast carcinomas with
gene amplification, or a false positive reaction in one of the breast carcinomas without
gene amplification as well as in the normal
breast glandular epithelium.
The laboratories were asked to send in their scores on the
multitissue section and the cell line stains.
For 38 out of 67 laboratories returning the slip (57 %) the scores
on the multitissue sections were in concordance with those given by
the NordiQC assessors.
In total 29 out of 67 laboratories (43 %) both had an appropriate
staining of the multitissue slide and an interpretation in
concordance with the NordiQC assessor group.
As regards the staining of the cell lines, the overall result was almost identical
with that of the staining of the tissue sections: 53 out of 69 slides returned (77%) were marked as sufficient compared to 74%
of the tissue
sections. However, even though the cell lines are processed similarly
to tissues in terms of fixation etc., the concordance (sufficient v.
insufficient) was not
total, as seen in the table.
| |
Tissues |
| Cell
lines |
Optimal |
Good |
Borderline |
Poor |
| Optimal |
31 |
5 |
4 |
0 |
| Good |
3 |
7 |
3 |
0 |
| Borderline |
0 |
1 |
5 |
2 |
| Poor |
2 |
2 |
2 |
2 |
For 57 out of 69 laboratories (83%) the
tissue and cell line stains were
concordant (i.e., both sufficient or both insufficient), while in 12 (17%), a discrepancy was
observed. Even though the mounting of the two materials on separate slides
may have contributed to this discrepancy (suggesting lack of
reproducibility in the laboratory), other explanations must be
looked for, such as the nature of the material, i.e., cell cultures
behave differently from tissue in the HER-2 stain. However, we found
no particular pattern in the discrepancy that could allow for an
explanation.
The laboratories' scoring
of the cell lines gave a higher concordance with the
NordiQC assessors (86% v. 57%) suggesting that the staining pattern
of the cell lines are easier to
interpret than that of the tissue slides.
Overall, the proportion of sufficient HER-2 stains
on the histological slides is about the same as in
Run
B2, 2006, i.e about 75%. In contrast
to previous assessments virtually all the laboratories adhered
strictly to the protocol settings described in the FDA approved
HER-2 systems, which made these laboratories improve their
performance, increasing the proportion of sufficient stains from 86% to 92%. In contrast, laboratories using in-house (home made)
systems performed worse, as the proportion of sufficient stains
declined from 58% in Run B2 to 33% in the current run.
Conclusion
The two FDA approved HER-2 systems HercepTest, Dako and the PATHWAY
rmAb clone 4B5, Ventana were also in this assessment the most
reliable methods for the semi-quantitative IHC determination of
HER-2 protein expression. In-house immunohistochemical systems give
unacceptable low performance and should be abolished. Training in
scoring is still highly warranted.
It is not settled if cell lines
(without concomitant histological slides) are sufficiently reliable
for quality assurance of HER-2 staining. |
