1

THE USE OF CYTOKERATIN 19 (CK19) IMMUNOHISTOCHEMISTRY IN LESIONS OF THE

PANCREAS, GASTROINTESTINAL TRACT AND LIVER

Richa Jain MBBS, MD

Sandra Fischer, MD

Stefano Serra, MD

Runjan Chetty MB BCh, FRCPath, DPhil

Department of Pathology, University Health Network/University of Toronto, Toronto,

Canada.

Correspondence:

Dr Runjan Chetty

Department of Pathology

University Health Network

The Toronto General Hospital

200 Elizabeth Street, 11th Floor, Eaton Wing

Toronto, Ontario

M5G 2C4, Canada.

Tel: 1-416-340-3661

Fax: 1-416-340-5517

Email: runjan.chetty@uhn.on.ca
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Abstract

Cytokeratin immunostaining forms the bedrock of the immunohistochemical evaluation of tumors.

Cytokeratin 19 (CK19) belongs to a family of keratins that are normally expressed in the lining of

the gastroenteropancreatic and hepatobiliary tracts. CK19 immunohistochemistry has been used

successfully in thyroid tumors to recognize papillary carcinomas for some time. However, its use

in the pancreas, liver and gastrointestinal tract (GIT) has only recently come to the fore. The

purpose of this review is to look at the use of CK19 immunohistochemistry in tumors occurring at

these sites.

CK19 has been shown to be an independent prognostic factor for pancreatic neuroendocrine

tumors, especially the insulin-negative tumors. CK19 positive tumors are associated with poor

outcome irrespective of the established pathological parameters such as size, mitoses,

lymphovascular invasion and necrosis. It is recommended that CK19 be part of the

immunohistochemical panel in the work-up of pancreatic endocrine tumors. CK19 is positive in

the vast majority of neuroendocrine tumors occurring in the rest of the GIT, except rectal tumors,

which are negative.

In the liver, CK19 is of prognostic value in hepatocellular carcinomas (HCC) and is of use in

distinguishing cholangiocarcinoma from HCC. It can also be employed to highlight native

ductules in the liver and helps separate conditions like focal nodular hyperplasia from hepatic

adenoma.

The vast majority of adenocarcinomas in the GIT and pancreas are CK19 positive.

KEY WORDS: Cytokeratin 19, neuroendocrine tumors, cholangiocarcinoma,

gastrointestinal tract, pancreas, liver.

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Introduction:

Cytokeratins or simply, keratins are filamentous proteins that belong to a category of intermediate

filaments that have diverse functions such as in the formation the cell cytoskeleton by

participating in a network of 10-12 nm wide intermediate filaments. They thus, impart mechanical

strength to the cells, participate in the response to stress, cell signaling and apoptosis by playing

a role in movement of organelles and substrates within the cell [1]. Cytokeratins are constituted

by a group of 20 proteins ranging from 40 to 70 kDa and have been grouped into: type I

comprising cytokeratins 9-20, and type II consisting of cytokeratins 1-8. The type I proteins are

acidic proteins of low molecular weight, whereas type II proteins are basic proteins of high

molecular weight.

Cytokeratin 19 (CK19) belongs to the type 1 group of cytokeratins with a molecular weight

ranging form 40-56kD [2]. CK19 is normally expressed in ductal epithelium (bile ducts, pancreas

and renal collecting tubules) and in the mucosa of the gastrointestinal tract [3]. The use of CK19

immunohistochemistry in diagnostic pathology has been mainly to confirm epithelial

immunophenotype in undifferentiated appearing tumors or establish biliary/pancreatic/renal

ductular origin, usually as part of a larger panel of markers.

The expression of a particular cytokeratin is dependent on the epithelial cell type, extent of

differentiation of the cell and development of the tissue being examined [2]. During

malignant transformation of epithelial cells, the cytokeratin profiles tend to be maintained,

thus allowing cytokeratin to be employed as a potential tumor marker [3, 4].

With regard to tumors, CK19 is expressed in squamous carcinomas of the head and neck, more

than 50% of renal cell carcinomas and tumors arising from stratified squamous epithelium [3].

With regards to the gastrointestinal tract, the vast majority of adenocarcinomas are CK19

positive, including cholangiocarcinomas [3, 5]. Neuroendocrine tumors (including neuroendocrine

carcinomas) are said to be frequently positive for CK19 [3, 6].

The role of CK19 as a prognostic marker has been applied to papillary thyroid carcinomas [7],

hepatocellular carcinomas [8, 9] and colorectal adenocarcinoma [10]. The use of CK19 in

endocrine pathology has been advocated recently in pancreatic neuroendocrine tumors, but its
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use in the remainder of the gastrointestinal tract and liver tumors has not been explored

extensively and information is somewhat limited at this juncture.

CK19 antibodies:

The two main clones of CK19 antibody (BA17 and RCK108) that are commercially available,

detect slightly different epitopes of the CK19 molecule. Most studies have used the RCK108

clone, which is thought to be more reliable than the BA17 clone [11]. However, the BA17 clone

tends to stain more cells and with greater intensity [11, 12]. Whichever antibody is utilized, it is

important to recognize which antibody is employed which may explain any differences in the

extent and intensity of staining that can be seen. As with all antibodies, comparison to built-in

positive control tissues and external positive control slides, provide a good guideline in assessing

staining with CK19.

The BA17 and RCK108 antibodies are available from Santa Cruz Biotechnology and

require microwave antigen retrieval.

CK19 staining patterns:

Basically, 2 staining patterns have been encountered with CK19. Cytoplasmic with membrane

decoration (Fig 1) is probably the most frequently encountered pattern. Cytoplasmic staining

usually predominates, while membrane staining often coexists and in many instances, only part of

the cell membrane is illuminated. The second staining pattern noted is paranuclear dot-like

accentuation (Fig 2). While this is seen with and without cytoplasmic/membrane staining, dot-like

CK19 staining is not associated with any significant pathological parameter nor defines a subset

of tumors. This pattern may reflect Golgi accentuation or collapse of the cytoskeleton.

CK19 expression in pancreatic carcinomas

Most pancreatic ductal adenocarcinomas are CK19 positive, as is the lining of the pancreatico-

biliary tree (see below, section on liver) [3].

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CK19 expression in pancreatic endocrine tumors

Various histopathological parameters like tumor size, necrosis, lymphovascular invasion,

extension into peripancreatic fat, mitoses and hormonal status of the tumor have been put

forward as putative markers of aggressive behavior in pancreatic endocrine tumors (PETs). Only

lymph node and/or liver metastases have been shown to be unequivocal markers of more

aggressive tumor behavior.

As a result of the ongoing search for a marker with predictive value, CK19 has been shown by

several investigators to correlate with an aggressive phenotype [11-15]. CK19 correlates

significantly with lymph node and liver spread, mitoses, Ki-67 labeling index, and lymphovascular

and perineural invasion. Also, in one of the studies, there was a distinct trend for smaller and

insulin positive PETs to be CK19 negative, which is in keeping with the known favorable outcome

of this particular subtype of PET [13]. Deshpande et al also demonstrated that although CK 19

positivity, lymphovascular and perineural invasion, mitoses, Ki-67 and necrosis were all

independent prognostic factors, CK19 had the best predictive value [14]. In this study, the 5-year-

survival of all CK19 negative cases was 100%, while that of CK19 positive patients was only

47%. Schmitt et al have also confirmed the predictive value of CK 19 and WHO 2004 criteria in

PETs [15]. La Rosa et al found CK 19 to be positive in malignant PETs and the predictive value

was more specific when the CK19 antibody clone RCK108 was used [12]. However, they found

CK 19 to be relatively insensitive in comparison to Ki-67 as an independent marker of poor

prognosis. Jonkers et al found CK19 to be the most optimal marker for tumor-related death in all

non-insulin PETs. They go on to say that they feel that CK19 is a sub-optimal marker of disease-

free survival in insulin-producing PETs [11]. It is well known that insulin-producing PETs tend to

be more indolent and this is probably why CK19 is of little or no value in this subset of PET.

The slight differences in results between various studies could be because of the difference in

antibodies used in the study and/or due to the method of antigen retrieval.

The reason why CK19 correlates with worse outcome is not well known but it is postulated that

CK19 is expressed in stem cells located in pancreatic ducts. With development of the endocrine

islets, they lose CK19 positivity while the ducts retain the capacity to express CK19. Acquisition
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of CK19 positivity by the endocrine cells is thought to be an indicator of a more ductal

immunophenotype, which is known to have more aggressive behavior with an adverse outcome.

Thus, there does appear to be a role for including CK19 in the immunohistochemical panel of

markers in the work-up PETs. It is advocated that CK19 positive tumors that are non-insulin

producing and lacking the known pathological parameters of aggression (lymphovascular

invasion, lymph node and liver metastases), should be flagged as tumors with potential for

aggressive behavior and warrant more careful clinical follow-up.

CK 19 expression in appendiceal neuroendocrine tumors

Alsaad and colleagues investigated the role of CK 19 in appendiceal neuroendocrine tumors. CK

19 was expressed in all goblet cell carcinoids (mucin-producing neuroendocrine tumours) and in

80% of so-called classical carcinoids, regardless of lymphovascular, perineural invasion, mitoses

or Ki-67 labeling index [16]. Hence, no correlation of CK 19 expression or pattern of expression

with the tumor behavior or clinicopathologic parameters was seen [16]. The nosology of goblet

cell carcinoids is debated, but because of its potential for more aggressive behavior compared to

traditional “carcinoid” tumors, several believe that it is a variant of adenocarcinoma while others

favor it being a true amphicrine carcinoma [16]. Despite this, CK19 expression in goblet cell

carcinoids is not too unexpected given its morphologic and immunophenotypic resemblance to

conventional adenocarcinoma [17, 18]. Somewhat surprisingly, the vast majority of classical

carcinoids of the appendix were also CK19 positive. These tumors are morphologically indolent

appearing, and if CK19 is a marker of aggressive cases, then there is no correlation between

morphology and CK19 expression.

CK19 expression in the remainder of gastrointestinal tract neuroendocrine tumors

There is very limited information on CK19 immunoexpression in neuroendocrine tumors of the

tubular GIT. We recently evaluated 48 neuroendocrine tumors from GIT that were distributed as

follows: stomach (7), duodenum (5), jejunum (1), ileum (23), ileocecal (3), cecal (1), sigmoid

colon (1), rectosigmoid (1) and rectum (6) [19]. Using the WHO classification, the cases were
 7

stratified as follows: 13 were well differentiated neuroendocrine tumors with benign behavior, 3

well differentiated with uncertain behavior, 25 were categorized as well differentiated, low-grade

malignant neuroendocrine tumors and the remaining 7 as poorly differentiated neuroendocrine

carcinomas.

Forty-four of the 48 GI neuroendocrine tumors were CK19 positive, a statistically significant

finding. As with PETs CK19 immunostaining was observed in membrane and cytoplasmic

locations and 4 cases paranuclear dot-like accentuation in addition to membrane and cytoplasmic

staining. Interestingly, the staining pattern of metastases to the lymph nodes and liver

recapitulated the CK19 staining of the primary gut tumors in extent and intensity.

Similar to the appendix, CK19 immunostaining did not correlate with any clinicopathological

parameter and was expressed in cases irrespective of site, size, mitotic count, and invasiveness.

Furthermore, dot-like CK19 staining also did not correlate with any parameter. Of note, was the

fact that the weakest CK19 immunolabeling was encountered in neuroendocrine tumors of the

distal large bowel (rectum and rectosigmoid) [19]. This may be analogous to their lack of

chromogranin immunoreactivity, although the reason for the decreased CK19 immunoexpression

is not apparent. The CK19 antibody used in this study was against clone BA17and this may

account for the high incidence of CK19 encountered in this study [19].

CK19 expression in GIT carcinomas

Gastric cancer: The vast majority (up to 90%) of gastric cancers are CK19 immunoreactive [20]

[21]. Hence, CK19 has no use either as a diagnostic or prognostic marker in gastric cancers.

Colorectal cancer: Like gastric cancers, most colorectal carcinomas stain with CK19 [3].

Thus, there is no real value in using CK19 in the evaluation of adenocarcinomas from the GIT

and pancreas. It does not yield any information and is not as diagnostically useful as CK7 and

CK20.
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CK19 expression in the liver

CK19 expression in the liver is a marker of biliary differentiation and progenitor cell phenotype

and is also a promising prognostic marker for hepatocellular carcinoma (HCC).

CK19 as a marker of biliary and hepatocyte progenitor cell lineage

In the normal adult human liver CK19 is expressed in the bile duct epithelium, together with

keratins 7 and 8. Mature hepatocytes express only keratins 8 and 18 [3, 22]. This profile is

achieved through changes in the cellular phenotype of hepatocyte precursor cells (hepatoblasts

or progenitor cells) during embryonic life [22, 23]. In its early stage of development, the embryonic

liver is composed of hepatoblasts, which express CK8, 18 and 19. Intrahepatic biliary epithelial

cells develop from the hepatoblasts surrounding the portal vein branches. The phenotypic change

from hepatoblast to cholangiocyte involves the expression of keratin 7 and other markers [22]. In

contrast, hepatocytes develop from the precursor cells not in contact with the perivenous

mesenchyme by losing CK19, at the same time acquiring further parenchymal cell markers [22,

23]. In the adult liver, hepatic progenitor cells (HPCs) reside in the bile ductules and the canals of

Hering that are localized in the interface between the portal tracts and the periportal parenchyma

[24]. These are small epithelial cells with an oval nucleus, and scant cytoplasm that are

immunoreactive for CK7, 8, 18, and 19, chromogranin A and for the rat oval cell marker OV-6 [24,

25]. HPCs can differentiate towards the biliary and hepatocytic lineages in response to various

types of stress or injury, including viral hepatitis, acute and chronic obstruction of extrahepatic bile

ducts, primary biliary cirrhosis, sepsis, primary sclerosing cholangitis, alcoholic liver disease, and

recovery from massive liver necrosis [26]. Differentiation towards the biliary lineage occurs via

ductular reaction, i.e. formation of reactive bile ductules [25, 27, 28]. Hepatocytic differentiation

leads to the formation of intermediate hepatocyte-like cells. These are polygonal cells with a size

intermediate between that of HPCs and hepatocytes. The epithelial lining of the reactive ductules

expresses both CK7 and CK19, whereas intermediate cells do not show immunoreactivity for

CK19 [24].

Immunohistochemistry for biliary markers, including CK7 or CK19, is a very useful

complementary tool to evaluate loss of native interlobular bile ducts in liver diseases associated
 9

with ductopenia (e.g. primary biliary cirrhosis, primary or secondary sclerosing cholangitis,

Alagille’s syndrome, post-liver transplantation chronic rejection, ischemic cholangitis, toxic/drug

injury, idiopathic vanishing bile duct syndrome) [29].

Because CK19 is differentially expressed in hepatocytes and cholangiocytes, it also represents a

helpful marker in the differential diagnosis of reactive and neoplastic liver conditions. Focal

nodular hyperplasia (FNH), which essentially represents a hepatocellular hyperplastic response

to a vascular abnormality, typically shows fibrous septa with large arteries and ductular reaction.

The reactive ductules can be demonstrated by CK7 and CK19 immunostaining [30]. This feature

is especially useful to differentiate FNH from hepatocellular adenoma (HA) (See Fig 4A &B). HAs

which are composed mostly by mature hepatocytes, are negative or show only weak staining for

CK19, as they typically have no or rare bile ductules [30]. The cells that are immunoreactive for

CK19 in HA show the phenotype of HPCs and intermediate hepatocyte-like cells, and they are

present in a “starry sky” pattern on a background of mature hepatocytes [24, 25, 31].

Likewise, HPCs and intermediate hepatocyte-like cells can be identified in up to 50% of small

dysplastic foci, which are likely the precursor lesions of HCC [25].

Diffuse and strong immunopositivity for CK19 is usually not present in HCC [32, 33]. However,

focal expression for CK7 and CK19 in a considerable proportion of HCC has been shown by

several studies [25, 34, 35]. Typically, CK19 is detected in isolated or discrete clusters of small

tumor cells in 10% to 27% of HCC [33, 34]. Distinguishing HCC from cholangiocarcinoma (CC)

can be problematic, often requiring the use of immunohistochemistry. Because CK19 stains most

CCs, positivity for this antibody is very effective in separating HCC from CC [32, 33]. Unlike

CK19-positive HCC, the staining pattern in CC is moderate and diffuse [36].

Combined hepatocellular-cholangiocarcinoma (HCC-CC) is a rare distinct malignant liver

neoplasm displaying hepatocellular and cholangiocellular components [21, 37-40]. Typically, the

cholangiocellular component shows diffuse immunopositivity for CK19. The diagnosis has to be

supported by morphology and mere presence of biliary-type markers such as CK19 and CK7 is

not sufficient to establish the diagnosis of combined HCC-CC [32].

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Combined HCC-CC may also contain a variable proportion of small tumor cells with HPC

immunophenotype with several studies suggesting that these tumors result from bidirectional

differentiation of neoplastic progenitor cell populations [21, 37, 39, 41, 42]. Tumors composed of

small cells with a phenotype intermediate between hepatocytes and cholangiocytes and

simultaneously expressing CK7, 8,18 and 19 have been called “liver tumor of intermediate (dual

hepatocyte-cholangiocyte) phenotype” [21] (Fig 5 A & B. Likewise, the so called

cholangiolocellular carcinoma (CLC) which is thought to originate from the ductules/canals of

Hering is composed predominantly by small cuboidal cells arranged in small anastomosing

glands separated by abundant hyalinized or edematous stroma [42]. Areas of HCC- and/or CC-

like pattern can sometimes be identified focally in a small portion of the tumor. CLC tumor cells

show strong cytoplasmic reactivity for CK19 and other progenitor cell markers [42].

Hepatoblastoma is the most common pediatric liver tumor with epithelial components resembling

embryonal and fetal liver cells [26, 43]. Cells resembling fetal or immature biliary can also be

present [44, 45]. Mixed or teratoid hepatoblastomas contain one or more mesenchymal

components (e.g. osteoid, cartilage, neural tissue) in addition to the embryonic and fetal epithelial

cells. The wide range of epithelial and mesenchymal lines of differentiation seen in

hepatoblastoma suggests that this tumor originates from a pluripotent stem cell [26, 43-46]. In

hepatoblastoma, CK19 immunopositivity is often present in isolated epithelial tumor cells in fetal

areas, with embryonal areas showing moderate staining for CK19 in cells in tubular structures

[46]. The majority of tumor cells in small cell undifferentiated hepatoblastoma express CK19 [46].
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CK19 as a prognostic marker of HCC

A few studies have suggested that hepatocellular carcinomas expressing progenitor cell or

ductular markers like CK19 have a more aggressive clinical course [8, 9, 35, 37, 47-51]. In one

study, immunopositivity for CK19 and OV-6 in HCC with lymph node metastasis was significantly

higher than in HCC without lymph node involvement [47]. In the same study, the CK19-positive

HCC group also had a shorter median survival. Other studies have shown that CK19 is a

significant predictor of overall survival, and early postoperative recurrence [8, 9, 48].

Conclusions

CK19 is a robust immunohistochemical antibody in general but its use as a diagnostic and/or

prognostic marker for neuroendocrine tumors of the gastroenteropancreatic system is limited at

this juncture. There is good evidence to suggest that CK19 predicts behavior in a subset of

PETs, i.e., the non-insulin subtypes. It has been shown to be an independent predictive factor

that is superior to the traditional pathological parameters that are used when assessing be

endocrine tumors. However, it is prudent not to rely solely on a single immunohistochemical

marker to predict biologic behavior. At this stage, CK19 should be used in conjunction with the

accepted pathologic criteria contained in the WHO classification, and the clinician could be

alerted to possible aggressive behavior from an otherwise “benign” appearing PET.

Other than the pancreas, there does not appear to be any merit in using CK19 immunostaining in

the evaluation of GIT neuroendocrine tumors, as the vast majority of cases are positive.

With regards to carcinomas of the GIT, CK19 stains the vast majority of cases and cannot be

used to discriminate between different sites in the GIT.

In HCC, CK19 has a role in determining those with an adverse prognosis and we advocate that

CK19 is part of the routine immunohistochemical panel for the evaluation of liver tumors.
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Legends to Figures

Figure 1:

The typical staining pattern seen with CK19: intense cytoplasmic labeling of a neuroendocrine

tumor of the ileum. (anti-CK19, clone BA 17 X 400).

Figure 2:

In addition to cytoplasmic immunostaining membrane staining is also membrane staining. Tumor

cells within a lymphovascular space display both cytoplasmic and membrane staining. (anti-

CK19, clone BA 17 X 400).

Figure 3:

Prominent dot-like CK19 accentuation, most like corresponding to Golgi body staining, is also

seen in occasional pancreatic neuroendocrine tumors. (anti-CK19, clone BA 17 X 400).

Figure 4 A & B:

CK 19 immunostaining showing strong positivity in ductules in focal nodular hyperplasia (A), and

no staining in a case of hepatic adenoma (B).

Figure 5 A & B:

Combined hepatocellular-cholangiocarcinoma. There is a desmoplastic tubulo-glandular

cholangiocarcinoma focus (top), adjacent to trabecular hepatocellular carcinoma (bottom) (A).

(H&E x 20).

(B) Immunohistochemistry using anti-CK19 antibody highlights the cholangiocarcinoma

component (anti-CK19, clone BA 17 X 20).