Cytokeratin 7 (MSVA-607R)

Cytokeratin 7 (CK7), also termed keratin 7 (KRT7) is a basic high molecular weight Type II cytokeratin encoded by the KRT7 gene located 12q12-q13. It forms intermediate filaments that primarily shape the cytoskeleton of specific epithelial, mostly glandular cells. In these cells, CK7 is part of the cytoskeletal scaffold which contributes to the cell architecture and provides the cells with the ability to withstand mechanical stress. Because of its differential expression in carcinomas from different origins, CK7 immunohistochemistry can be used as an aide for defining the origin of cancer tissues.

Cytokeratin 7 staining pattern in Normal Tissues with antibody MSVA-607R (images are shown in our “Normal Tissue Gallery”)

These findings are largely comparable to the data described in the Human Protein Atlas (Tissue expression Cytokeratin 7). Of note, images represented in the protein atlas also show heterogeneous staining in endometrium  and prostate glands.

Suggested positive tissue control: Pancreas: CK7 staining is at least weak to moderate cytoplasmic intercalating ducts and strong in large pancreatic ducts.

Suggested negative tissue control: Pancreas: CK7 staining should be absent in all acinar cells.

Normal tissue gallery

KRT7 is expressed in a wide range of tumors, most of which are derived from KRT7 positive precursor cells. The highest frequency of KRT7 positivity is seen in carcinomas of the ovary, thyroid, urothelium, breast, and endometrium, chromophobe and papillary kidney cancer, adenocarcinomas of the pancreas, lung, esophagus, and the stomach as well as in mesothelioma. Many other tumor types can – at least occasionally – show KRT7 expression.

The TCGA findings on Cytokeratin 7 RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

Cancer tissue gallery

Cytokeratin 7 (MSVA-607R) publication summary

Relevant publication: Dum et al.: “Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers” Exp Mol Pathol 2022 Apr 4; 126:104762 Online ahead of print

A total of 14’110 tumors from 120 different tumor categories were successfully analyzed for CK 7 by using the following protocol:  Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 9 Target Retrieval Solution buffer. MSVA-620R at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent). This protocol was also used for all CK7 immunostainings depicted in our tumor and normal tissue galleries.

At least one case with a positive CK7 immunostaining was seen in 89 (74.2%) and at least one case with a strong CK7 staining was seen in 72 (60%) of 120 tumor categories. The distribution of positive staining results is shown in an “organ-systematic” (Figure 1) and in a “ranking order” figure (Figure 2) below (images based on data from Dum et al). Data on associations with histopathological and clinical parameters of tumor aggressiveness in several cancer types are also summarized below (Figure 3; based on data described by Dum et al).

Because CK7 is often used in combination with CK20, additional figures show ranking orders of tumors according to their CK7 positivity rate among CK20 negative (Figure 4) and among CK20 positive tumors (Figure 5). For completeness, a figure showing a ranking order of tumor entities according to their rate of CK7 and CK20 “double-negativity” is also given (Figure 6).

Authors conclusions on diagnostic utility with respect to the distinction of benign versus malignant (Dum et al):

• not applicable

Authors conclusions on diagnostic utility with respect to the distinction of different tumor entities (Dum et al):

• Cytokeratin 7 staining is seen in 59 of 85 different epithelial tumor entities (69%).
• The diagnostic utility of CK7 mostly comes from its rather rare expression in the clinically important adenocarcinomas derived from the colorectum (7.6% pos; 2% strong) and the prostate (9.4%–30.5% pos; 0.2% strong) while other adenocarcinomas of the GIT showed positivity rates between 61.7 and 82.1% (gastric cancer) and 86% (esophageal cancer) and other relevant adenocarcinomas had even higher rates such as ductal carcinomas of the pancreas (99%).

Authors conclusions on prognostic/predictive role of CK7 expression (Dum et al.):

• High CK7 expression was linked to unfavorable prognostic features in adenocarcinomas of the stomach (High CK7 expression in stomach cancer is aberrant because these tumors are derived from CK7 negative precursor cells).
• Low expression of CK7 was linked to unfavorable features in breast cancer of no special type (NST), renal cell carcinoma, and in urothelial carcinoma (Low CK7 expression in these cancers is aberrant because these tumors are derived from CK7 positive precursor cells).

Because CK7 is often used in combination with CK20, additional figures show ranking orders of tumors according to their CK7 positivity rate among CK20 negative (Figure 4) and among CK20 positive tumors (Figure 5). For completeness, a figure showing a ranking order of tumor entities according to their rate of CK7 and CK20 “double-negativity” is also given (Figure 6). Dum et al. summarize these findings as follows:

• CK20+/CK7+ is most commonly seen in mucinous ovarian cancer (63%) and in various types of non-invasive and invasive urothelial carcinomas (>45%) but also occurs at significant frequency in several other – mostly gastrointestinal – tumors.
• CK20-/CK7+ predominates in non-mucinous ovarian, breast, thyroid, kidney, endometrium, and pancreatic cancer, adenocarcinoma of the lung, and in mesotheliomas but also occurs in >50% of non-colorectal GIT adenocarcinomas.
• CK20+/CK7- is rather rare and predominates in colorectal adenocarcinomas and Merkel cell carcinomas but also occurs at relevant frequency in other tumors, most of which are derived from the GIT.
• CK20-/ CK7- is frequent in prostate cancer, seminoma, clear cell renal cell carcinoma, neuroendocrine tumors, adrenal tumors, squamous cell carcinomas as well as in mesenchymal and hematopoietic neoplasms.

Data from the publication: “Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers”. Published by Dum et al. Summarized in own graphics:

Figure 1. CK 7 staining in cancer (“organ-systematic”; according to Dum et al.)

Figure 2. CK 7 staining in cancer (“ranking list”; according to Dum et al.)

Figure 3. CK7 staining and tumor phenotype (according to Dum et al.)

Figure 4. CK7 staining in CK20 negative cancers (“ranking list”; according to Dum et al.)

Figure 5. CK7 staining in CK20 positive cancers (“ranking list”; according to Dum et al.)

Figure 6. CK7 negative and CK20 negative cancers (“ranking list”; according to Dum et al.)

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein. Accordingly, multiple different protocols can generate identical staining results.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 9 Target Retrieval Solution buffer. Apply MSVA-607R at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

Agilent / Dako – Autostainer Link 48

Pretreatment in PT-Link for 30 minutes at 95°C (pH high); FLEX peroxidase blocking for 5 minutes (room temperature), MSVA-607R 1:75 for 20 minutes (room temperature), FLEX+ mouse/rabbit (LINKER) for 15 minutes (room temperature), horseradish peroxidase (HRP) for 20 minutes (room temperature), FLEX DAB+Sub-Chromo for 10 minutes (room temperature), FLEX hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, and a longer incubation time of FLEX+LINKER result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Leica – BOND RX

Dewax at 72°C for 30 seconds; Pretreatment in Bond Epitope Retrieval Solution (ER2 – EDTA pH9) for 20 minutes at 100°C; Peroxidase blocking for 5 minutes (room temperature), MSVA-607R 1:75 for 30 minutes (room temperature), Post primary (rabbit anti mouse) for 8 minutes (room temperature), Polymer (goat anti rabbit) for 8 minutes (room temperature), mixed DAB refine for 10 minutes (room temperature), hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of Post primary and or the Polymer result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Roche – Ventana Discovery ULTRA

Pretreatment for 64 minutes at 100°C (pH 8,4); CM peroxidase blocking for 12 minutes (room temperature), MSVA-607R 1:25 for 40 minutes at 36°C, secondary antibody (anti-rabbit HQ) for 12 minutes at 36°C, anti-HQ HRP for 12 minutes at room temperature, DAB at room temperature, hematoxylin II at room temperature for 8 minutes, bluing reagent at room temperature for 4 minutes.

These images depict staining results obtained by the protocol described above. It is of note, that the Ventana machines generally require higher antibody concentrations than other commonly used autostainers because the antibodies are automatically diluted during the procedure. Various other protocols can result in an identical result as shown above. A longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of secondary antibody and or the anti-HQ HRP result in stronger staining, potentially at the cost of more background staining.

Impact of pH

The strongest KRT7 staining by MSVA-607R is obtained at a pH 9,0. However, pH 7,8 results in only a slight reduction of the staining intensity as compared to pH9. We thus consider pH7,8 as optimal for manual staining because of the better tissue preservation at pH7,8 than at pH 9,0.

• As the literature is partly confusing, the diagnostic utility of KRT7 expression analysis (in combination with KRT20 analysis) should be investigated in a large cohort of tumors from different entities.
• The biologic/clinical significance of aberrant CK7 expression in cancers needs to be evaluated (for example: what are the specific properties of CK7 negative pancreatic cancers or CK7 positive colorectal cancers?)

Specificity of MSVA-607R is documented by strong positive staining in cell types that are well documented to express CK7 such as epithelial cells of various glandular tissues and absence of staining in all tissues known to not express CK7 including tissues notorious for non-specific IHC background such as proximal tubules of the kidney, colon mucosa, and the skin. 

Normal tissue gallery