slide to be stained for
1. Esophagus, 2. Breast lobular carcinoma in situ (LCIS)*, 3-4.
Breast ductal carcinoma NOS, 5. Breast hyperplasia.
* In the accompany letter, the tumour
erroneously was marked breast ductal carcinoma in situ (DCIS).
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a CK-HMW staining as optimal included:
A strong and distinct cytoplasmic
staining of all the squamous epithelial cells of the esophagus
throughout all the cell layers.
A strong and distinct cytoplasmic
staining of the basal/myoepithelial cells in the breast hyperplasia
and the breast LCIS.
A negative (or very weakly positive)
staining in the neoplastic cells of LCIS
and breast ductal carcinomas no. 3 & 4, while the
basal/myoepithelial cells in remnants of normal ducts should show a
103 laboratories submitted stains. 6 laboratories used an
inappropriate Ab (such as CK-LMW or CK-PAN). Of the remaining 97
laboratories 10 achieved optimal marks (11%), 13 good (13%), 63
borderline (65 %) and 11 poor marks (11 %).
The following Abs were used:
Producer and number
CK 1, 5, 10, 14
Dako, n=63; Ventana, n=4; NeoMarkers/Thermo, n=1; Enzo, n=1; BioCare, n=1
CK 5, 6
Dako, n=13; Ventana, n=1
Novocastra/Leica, n=6; Vector, n=1
Novocastra/Leica, n=3, NeoMarkers/Thermo, n=1;
XM26 & LL002
CK 5, 14
Optimal staining for CK-HMW in this assessment was obtained with the
mAb D5/16 B4 (4 out of 14), XM26 (3 out of 7),
LL002 (3 out of 5) and XM26 & LL002 (1 out of 1).
All optimal protocols were based on heat induced epitope retrieval
D5/16 B4: The HIER buffers used were Tris-EDTA/EGTA pH 9.0
(1/6)*; Cell Conditioning1 (BenchMark, Ventana) (1/5), Bond Epitope
Retrieval Solution 2 (Bond, Leica) (1/2) or Target
Retrieval Buffer pH 9, (Dako) (1/1). The mAb was typically
diluted in the range of 1:50 – 1:200 depending on the total
sensitivity of the protocol employed. With these settings 7 out of
10 (93 %) laboratories produced a sufficient staining (optimal or
* (number of optimal results/number of
laboratories using this buffer)
XM26: The HIER buffers used were Tris-EDTA/EGTA pH 9.0 (2/2)
or Citrate pH 6.0 (1/3). The mAb was typically diluted in the range
of 1:100 – 1:300 depending on the total sensitivity of the protocol
employed. With these settings 3 out of 3 (100 %) laboratories
produced a sufficient staining.
LL002: The HIER buffers used were Bond Epitope Retrieval
Solution 2 (Bond, Leica) (1/1), Cell Conditioning1
(BenchMark, Ventana) (1/1) or Target Retrieval Buffer pH 9,
(Dako) (1/1). The mAb was typically diluted in the range of 1:30 –
1:300 depending on the total sensitivity of the protocol employed or
as a Ready-To-Use Ab. With these settings 3 out of 3 (100 %)
laboratories produced a sufficient staining.
XM26 & LL002: The HIER buffer used was Target Retrieval
Solution pH 6.1 (Dako). The mAb was diluted 1:100.
The most frequent causes of insufficient staining were:
- Less successful primary Ab (clone 34BE12 – 65 out of 70 protocols
gave an insufficient staining)
- Too low concentration of the primary Ab
- Insufficient HIER – usage of Citrate pH 6.0 and/or too short
efficient HIER time
In this assessment the prevalent feature of an insufficient staining
was a strong cytoplasmic staining of the neoplastic cells in the
breast carcinomas no. 2 and 4 hampering the identification of the
myoepithelial cells in the normal glands and LCIS. This reaction
pattern was only seen with the mAb clone 34BE12 and observed in 60
out of 70 protocols using the clone. In only 5 out of 70 protocols
the mAb clone 34BE12 gave a sufficient staining assessed as good.
The remaining 5 protocols were assessed as insufficient due to a
weak or a false negative staining. The clone 34BE12 reacts with CK
1, 5, 10, 14 but also with a yet unidentified CK-subtype. HIER
seemed to amplify both the specific reaction and the cross reaction,
whereas protelolytic pre-treatment either as a single pre-treatment
or in combination with HIER seemed to reduce the cross reactivity.
56 out of 57 stains (98 %) based on HIER were assessed as
insufficient, compared to 3 out of 10 (70 %) of the stains based on
a combined proteolysis and HIER. 2 out of 3 of the stains based on
proteolysis without HIER were assessed as insufficient. Due to this
cross reaction the utility of the clone to demonstrate myoepithelial
cells in breast pathology has previously been questioned, both in
NordiQC (CK-HMW run 16 and CK5 run 12) and in the literature (Lacroix-Triki
et al., Virchows Arch 2003; 442:548–554), whereas the clone is
excellent in for the demonstration of basal cells in prostate –
providing that HIER is used (CK-HMW run 16).
In the literature clone 34BE12 has been described to be a valuable
marker together with E-Cadherin to differentiate between lobular
carcinoma (E-Cadherin negative and 34BE12 positive) and ductal
carcinoma (E-Cadherin positive and 34BE12 negative). However this
application was not the scope of the assessment, as the main task
was to demonstrate myoepithelial cells and NordiQC did only evaluate
the laboratories performance in that diagnostic application as the
multi block was constructed with focus on the identification of CK-HMW
in myoepithelial cells.
Esophagus is recommended control for CK-HMW: All squamous epithelial
cells must show a strong staining without background reaction. In
contrast to anti-CK5 or CK5/6 antibodies (clone XM26 or D5/16 B4), the
anti-CK14 antibody (LL002) left focally the intermediate and
superficial cells negative in the esophagus muoca, even in optimal
protocols. This reaction pattern has also been shown in the previous
assessments. It was also observed that anti-CK14 left the normal
luminal breast epithelial cells unstained compared to anti-CK5 and
anti-CK5/6 - see Fig. 6a and 6b.
With the mAb CK5/6 clone D5/16 B4 caution should be taken in
interpretating the staining, as the antibody is an ascites format
giving the Mouse Ascites Golgireaction (MAG) in tissue from patients
with blood group A (http://www.nordiqc.org/Techniques/Primary_antibody.htm).
This was the third assessment of CK-HMW. No direct
comparison can be made with the previous assessments as these were related to
the general module and the assessment criteria and composition of
the multitissue blocks were designed for these purposes. However, it shall be stressed that in run 16, 2006, it was concluded that the
clone “34BE12 may cross react with an unidentified CK
particularly seen in breast (secretory cells and carcinomas). For
this reason 34BE12 cannot be recommended in breast pathology”.
The mAb clones XM26, D5/16 B4 and LL002 are all
well performing Abs for CK-HMW and should be the preferred choice
for the demonstration of CK-HMW in breast myoepithelial cells. HIER
is mandatory. The mAb clone 34BE12 can not be recommended for the
demonstration of CK-HMW in breast myoepithelial cells due to an
excessive cross reaction with other epithelial cells.