Assessors’ report for cIQc Run 8: panCK, LMWCK, HMWCK, CK20, CK7

RUN8 included testing for cytokeratin typing, including panCK, low molecular weight cytokeratin

(LMWCK), high molecular weight cytokeratin (HMWCK), CK20, and CK7.

Please note that it is essential to submit your protocols together with stained slides. RUN6 showed that

many laboratories did not readily include that information. Submission of protocols is essential as it

enables participants to adjust their methods if their success rate/level is not clinically acceptable. By

submitting your good protocols, you help other laboratories to improve their methods.

Assessor comments on panCK

Comparison of panCK IHC is shown in Figure 1 at the end of this document. The most important

finding regarding panCK testing is that about half of laboratories did not demonstrate cytokeratins in

hepatocytes or proximal renal tubules, and subsequently, tumors with low expression of CK8-18 were

not detected. For those laboratories that use AE1/AE3 cocktail, this may mean that they only

demonstrate cytokeratins detected by one Ab from the cocktail, not both. Alternatively, some keratins,

like CK8 (which is detected by AE3 component only of AE1/AE3 cocktail) will be detected only after

appropriate antigen retrieval (HIER). This illustrates a common problem in optimizing protocols for

cocktail antibodies. It is critical to use positive controls that will detect if any of the components of the

Ab cocktail is not working properly. For AE1/AE3 or other pan-keratin cocktails, benign liver tissue

and kidney are perfect. Optimal staining is demonstrated in Reference Laboratory, but also in many

other laboratories.

Assessor comments on LMWCK

Comparison of LMWCK IHC is shown in Figure 2 at the end of this document. Similar to panCK

results, many participants did not detect cytokeratins in hepatocytes or proximal tubules of the kidney.

Again, proper selection of positive controls to include liver and kidney tissue would immediately solve

this problem in daily practice. Of note, some results disclosed false-negative + false-positive

(endogenous biotin), which in daily practice may interfere with interpretation of results in both control

material and real test samples.

Assessor comments on HMWCK

Comparison of HMWCK IHC is shown in Figure 3 at the end of this document. HMWCK is used here

to refer to CK5 and CK14, which are expressed by basal layer of the stratified squamous epithelia.

CK5 and CK14representing the primary - constitutive - high molecular weight CKs of stratified

epithelia, and therefore, it is not very challenging to detect HMWCK in epidermis. Reference

Laboratory identified weak reactivity in distal tubules (or collecting ducts) and/or HL of the kidney

and surface mucous cells of gastric mucosa, as well as in several adenocarcinomas included in the

TMA design. All scores are based on comparison with reference laboratory results. The results were

interpreted as poor if no convincing staining was demonstrated in any of these tissues or if there was a

combination of very weak staining and false positive reactions. However, it is stated by some that these

normal tissues do NOT express CK5/14. It has been claimed that there is an unwanted cross reactions

when using clone 34BE12, because it also detects an epitope probably located to CK19 and that

34BE12 must not be used for the detection of HMWCK (see www.nordiqc.org). However, some

published literature (see Roland Moll, Markus Divo and Lutz langbein. The Human Keratins: Biology

and Pathology. Histochem Cell Biol 2008;129:705-733) states that sparse cells expressing CK5/14 are

detected in ductal epithelia of parenchymatous organs including bile ducts, pancreatic ducts, and renal

collecting ducts, while CK10 is found in gastric foveolar epithelium, as well as in most villus- and

surface-lining cells and also in scattered cells in crypts. While several of tissue cores were missing

from the slides (especially for laboratories which laboratory code is smaller than 125), the alternative

tissue was often present and enabled scoring. The results were not scored if any of the critical

indicators of the staining result were missing. Reference laboratory will report its methods (selection of

the clone and antigen retrieval) in the follow up to this discussion to resolve this discrepancy. For all

practical purposes, it should be considered that reference laboratory results stain certain tissues (as

shown here) when sensitive methods needed in clinical practice are used. Cross-reactivity with other

CK epitopes is not excluded. A systematic study of cross-reactivity with various CK epitopes is needed

by using methods like Western blot and absorption/blocking studies. Please send us your comments

and suggestions regarding HMWCK staining.

Assessor comments on CK20

Comparison of CK20 IHC is shown in Figure 4 at the end of this document. CK20 is very selectively

distributed in human body. For this test, it was expected to detect strong expression of CK20 in small

intestine, partly in gastric mucosa, and variably in several tumors included in the TMA block. Most

laboratories produced excellent results. For typically expected distribution of various cytokeratins, see

http://www.nordiqc.org/Epitopes/Cytokeratins/CK-table.htm with NordiQC table "Cytokeratins - Moll

types in normal cells".

Assessor comments on CK7

Comparison of CK7 IHC is shown in Figure 5 at the end of this document. Kidney is also very good

tissue to include in the design for CK7 positive control. There is moderate expression of CK7 in distal

tubules and at the same time, the proximal tubules will disclose endogenous biotin false-positive

reaction if present. Bile ducts show very strong staining, but hepatocytes should be completely

negative. Hepatocytes would also reveal endogenous biotin reaction if present. Small intestine contains

rare epithelial cells (endocrine cells?) that show weak expression of CK7 and may be useful as positive

control in daily runs. Pancreas is also very good for positive controls since ductal system including the

centroacinar cells are stained, while the acinar cells are unstained. Centroacinar staining is rather weak

and therefore, may be used for calibration.

Figure 1. Composite image of panCK staining.

Figure 2. Composite image of LMWCK staining.

Figure 3. Composite image of HMWCK staining.

Figure 4. Composite image of CK20 staining.

Figure 5. Composite image of CK7 staining.