Malignancy of
Keratin 19 positive
hepatocellular
carcinomas in dogs
and cats.
Karin van der Bent
3154505
Supervisor:
Dr. Bart Spee
Content
Content...................................................................................................... 2
Abstract ..................................................................................................... 3
Introduction ............................................................................................... 4

Liver tumors ....................................................................................... 4

Keratins ............................................................................................. 5

Liver progenitor cells ........................................................................... 6
Markers ..................................................................................................... 7

BMI-1 and EZH2 ................................................................................. 7

NF2 and NF2-P ................................................................................... 7

Laminin1 and CD29 ............................................................................. 7

Glypican3........................................................................................... 8

Platelet derived growth factor receptor alpha (PDGFRα) ........................... 8

Metastasis suppressor 1 (MTSS1) ......................................................... 8

S100A6 (Calcyclin) .............................................................................. 8

MAC 387 ............................................................................................ 9
Hypotheses .............................................................................................. 10
Material and Methods ................................................................................. 11

Samples .......................................................................................... 11

Immunohistochemistry ...................................................................... 12

Staining protocol............................................................................... 13

Sample grading ................................................................................ 15

Sample staging ................................................................................. 16

Viability assay .................................................................................. 17
Results .................................................................................................... 18

Liver tumors ..................................................................................... 18

Sample grading and staging ............................................................... 19

Hepatocellular tumors ....................................................................... 20

BMI-1 and EZH2 ............................................................................... 21

NF2 and NF2-P ................................................................................. 22

Laminin1 and CD29 ........................................................................... 23

Glypican3......................................................................................... 23

PDGFRα ........................................................................................... 24

MTSS1 ............................................................................................. 25

S100A6 ........................................................................................... 25

MAC387 ........................................................................................... 26
Conclusion................................................................................................ 27
Discussion ................................................................................................ 28
References ............................................................................................... 30
Acknowledgements .................................................................................... 32
Appendix .................................................................................................. 33
2
Abstract
A study by van Sprundel et al. has shown that 12% of canine hepatocellular
carcinomas (HCCs) expressed Keratin 19 (K19). These K19 positive tumors are
more invasive and metastasize faster compared to K19 negative tumors. In this
study we looked at the malignancy of K19 positive HCCs compared to K19
negative HCCs by staging and grading and looking at the difference in expression
of metastasis markers. Immunohistochemistry was performed for K19 on 145
liver tumor samples. Of these samples a selection of six K19 negative and five
K19 positive tumors were stained for metastasis markers: BMI-1, EZH2, NF2,
NF2-P, Laminin1, CD29, Glypican3, PDGFRα, MTSS1, S100A6 and MAC387.
Results: In our study, 20% of all hepatocellular carcinomas expressed Keratin 19.
These positive tumors are poorly differentiated, show pleiomorfism,
multinucleated cells, and mitosis and often metastasize. While K19 negative
tumors still resemble healthy liver, are well encapsulated and do not metastasize.
Metastasis markers BMI-1, Laminin1, PDGRFα, S100A6 and MAC387 are higher
expressed in K19 positive HCCs compared to K19 negative HCCs. There was a
lower expression in K19 positive HCCs, compared to K19 negative HCCs, of NF2
and NF2-P. Conclusion: Keratin 19 positive hepatocellular carcinomas are more
malignant compared to keratin 19 negative carcinomas and keratin 19 positive
hepatocellular carcinomas express many metastasis markers.
3
Introduction
 Liver tumors
In dogs and cats primary hepatic tumors are rare.17,23 The frequency of hepatic
tumors, excluding adenomas, in dogs varies from 0.6% to 1.3% of all tumors 22
and in cats it is 1.0 to 2.9%.28 In humans hepatocellular carcinomas is the third
leading cause of death of all cancer mortality in the world.1 This means that
primary liver tumors are more common in humans compared to dogs and cats.
A difference between humans and these animals is that in dogs and cats there is
no immediate cause for the tumors, while in humans, liver diseases can result
into tumors.17,26 In man, chronic liver diseases, originating from viral infections
can lead to tumors in the liver.26 Another common cause for Hepatocellular
carcinomas is aflatoxin. This is a fungal toxin, which is common on peanuts and
rice.18
There are different kinds of hepatic tumors. Nodular hyperplasia is not a tumor,
but is a nodule which consists of an increased number of normal to vacuolated
hepatocytes with an increased number of mitotic figures. It is very common in
older dogs, but the cause is unknown. Nodular hyperplasia is a benign condition
that does not cause clinical illness.17
The hepatic tumors consist of hepatocellular adenoma, hepatocellular carcinoma,
cholangiocellular carcinoma and carcinoid. Hepatocellular adenoma and carcinoma
are tumors originated from the hepatocytes. The adenoma is a benign tumor that
most often occurs as a single, well capsulated mass. Cholangiocellular carcinoma
originates from the bile duct. Both hepatocellular and cholangiocellular
carcinomas metastasize early in their development. The most common sites for
the tumor cells to metastasize to are the lymph nodes, lung and peritoneum. The
carcinoid is a tumor derived from the neural cells.17
4
 Keratins
Keratins are noncovalently associated heteropolymers and a subgroup of the
intermediate filament (IF) proteins. In total there are five subgroups of IF
proteins, of which keratins form the largest. IF proteins are one of the three
major filament cytoskeletal protein networks that maintain cellular structure and
cell integrity. The other two protein networks are actin microfilaments and
tubulin.19
Keratins can be divided into two types. Of the epithelial cell-specific keratins,
Keratin9 (K9) through K20 are of type I, and K1 through K8 are of type II. Each
epithelial cell expresses at least one type I and one type II keratin. 19 In the liver,
adult hepatocytes express only K8 and K18, but hepatic progenitor cells also
express K19. Cholangiocytes express K19 and K7.19,29,33
Hepatocytes normally do not have K19, however, several hepatocellular
carcinomas do show the expression of K19. They have a normal typical
hepatocellular carcinoma growth pattern and morphologic appearance, but
several reports have indicated that patients with the K19 marker have a poorer
prognosis compared to patients without this marker. This poorer prognosis is with
and without treatment.19,29,33
This is why it is thought that keratin 19 positive tumors are more malignant and
that their expression of metastasis markers is different compared to K19 negative
HCCs. In our study we stained for different markers in K19 positive and K19
negative hepatocellular carcinomas to find out if they have a different marker
expression.
5
 Liver progenitor cells
Liver progenitor cells (LPC) are liver-specific adult stem cells that are activated
when the mature hepatocytes and/or cholangiocytes are damaged or inhibited in
their replication. Liver progenitor cells are capable in developing into hepatocytes
or cholangiocytes, depending on which cells are damaged the most. Liver
progenitor cells exist in the canals of Hering; these are peripheral branches of the
biliary tree. When the LPCs are activated during liver disease, there is an
increased number of liver progenitor cells and intermediate hepatocytes around
the portal tract. The ductular appearance of activated LPCs is called ductular
reaction.10
In several studies, activated LPCs were considered target populations for tumor
growth and several tumors have been shown to have LPCs characteristics.10
Some hepatocellular carcinomas indeed show characteristics of the liver
progenitor cells, which includes the expression of keratin 19 (K19).10,33 (fig. 1)
K19
positivity
Figure 1: Possible cancer phenotypes originating from a progenitor cell.28 Progenitor cells (LPCs) can
give rise to hepatocytes and cholangiocytes which, in turn, could give rise to hepatocellular tumors or
cholangiocellular tumors. However, since LPCs are present in the majority of liver diseases, and
depending on their stage of differentiation, they could give rise to HCCs or CCs as well as mixed
phenotypes.
Taken together, the origin of K19 positive HCCs remain unclear and could
originate from liver progenitor cells (Maturation arrest theory). But it is also
possible that they develop from cholangiocytes or that they dedifferentiate from
mature hepatocytes (Dedifferentiation Theory). (fig. 2)
Figure 2: dedifferentiation theory, Possible way hepatocellular tumors can originate.27
6
Markers
In this study a wide variety of markers is used which have been proven to be
markers of malignancy in human tumors (B.Spee, personal communication) or
have been proven as malignancy markers in other types of tumors. These
markers are described below.
 BMI-1 and EZH2
BMI-1 and EZH2 are proteins of the polycomb group (PcG). They prevent changes
in cell identity by repressing the transcription of several genes.26,30 They do this
by forming multiprotein complexes (PcG bodies) and organize chromatin in an
inaccessible structure that cannot bind transcription factors. EZH2 is one of the
essential proteins for BMI-1 recruitment to the PcG bodies.30
Several studies revealed that an altered expression of the polycomb group
proteins result into the formation of tumors.26,30,35 HCCs with a high expression of
BMI-1 and EZH2 also show a more malignant character. How this works is still
unclear.30,35
In our study we expect BMI-1 and EZH2 to be higher in keratin 19 positive
tumors.
 NF2 and NF2-P
NF2 or Merlin is a member of the ezrin-radixin-moesin (ERM) family. In healthy
hepatocytes NF2 is on the membrane interacting with the extracellular matrix.
This way it decreases the proliferation rate and inhibits cell growth. This means
NF2 could function as a tumor suppressor.16,24
But NF2 can also have a different function. When it is in the cytoplasm interacting
with proteins there, it can function as a growth stimulator.16
Previous studies have shown that when NF2 is lacking in cells, tumors, like
hepatocellular carcinomas, can develop.2,16
This is why we think that in K19 positive tumors, NF2 is not present.
Phosphorylated NF2 (NF2-P) is normally present in the nucleus of quiescent cells.
Like NF2, it functions as a growth inhibitor.16
We expect NF2-P to be absent in the nucleus in K19 positive hepatocellular
carcinomas.
 Laminin1 and CD29
Laminin is one of the major components of the basement membrane. There are
distinct isoforms of laminin detected. Laminins can have different effects. Certain
fragments can inhibit the metastatic activity and some can enhance the
metastatic activity.20 Laminin1 is important for the epithelial development in
embryonic tissue. In hepatocellular carcinoma it is involved with the progression
of the tumor.7 In normal liver, there is barely any laminin detected. In HCC
however, laminin is continuously present.32
In our study, we stained for laminin1 in keratin 19 positive and keratin 19
negative tumors. Because we expect the K19 positive tumors to be more
malignant, we expect laminin1 to be higher expressed in those tumors.
CD29 is a subunit of integrins. Integrins are adhesion molecules which mediate
cell-matrix interactions.20,32 CD29 is a subunit of an integrin that is a specific
receptor for laminin (α6β1). This integrin is not present in healthy liver. But, like
laminin, it is present in hepatocellullar carcinomas in a continuous pattern on the
membrane, in accordance whith the location of laminin.6,32
In our study we expect CD29 to be higher expressed in keratin 19 positive tumors
compared to keratin 19 negative tumors, because they are the receptors of
laminin1.
7
 Glypican3
Glypican3 is a member of the glypican family of heparan-sulfate proteoglycans. It
is anchored to the cell surface through a glycosyl-phosphatidylinositol anchor.
Glypican3 plays an important role in cell proliferation in embryonic tissue. In adult
tissue it is down regulated, except for the lung, mesothelium and the ovarian
epithelium.11 Different studies found that glypican3 is higher expressed on the
mRNA and the protein level in hepatocellular carcinomas with a worse
prognosis.5,11,29
We expect glypican3 to be highly expressed in K19 positive HCC, in comparison to
other studies.
 Platelet derived growth factor receptor alpha (PDGFRα)
PDGFRα is a member of the class III receptor tyrosine kinase family. PDGFRs play
many critical roles in embryonic and postnatal development. PDGFRα assists in
proliferation, morphogenesis, angiogenesis or epithelial-mesenchymal interactions
during the development of many tissues.31
PDGFs may also be involved in growth stimulation of tumors. PDGFRα is
associated with tumor cell proliferation, progression or angiogenesis.31
In an earlier study in humans, researchers showed that in immature hepatocytes
PDGFRα is much higher in the cytoplasm and the membrane than in adult healthy
liver tissue. They also stained hepatocellular carcinomas, where a big percentage
of the tumors revealed a higher level of PDGFRα, in the cytoplasm and on the
membrane, compared to healthy liver tissue.15
In our study we expect that when a tumor is more aggressive, it has more
PDGFRα in the cytoplasm and on the membrane. So we think that keratin 19
positive tumors, of which we think are more aggressive, have higher levels of
PDGFRα compared to keratin 19 negative tumors.
If PDGFRα is indeed higher in more aggressive tumors, it could be a marker to
use for treatment. There is a protein-tyrosine kinase inhibitor on the market
called Imatinib. This kinase inhibitor binds to and inhibits PDGFRα as well as CKIT tyrosine kinases by interfering with their downstream processes. 25
To see if this really works on PDGFRα, we performed a viability assay on a HCClike cell-line called HepaRG treated with different concentrations Imatinib.
HepaRG cells are tumor cells of early hepatoblasts, which resemble K19 positive
HCCs and also express PDGFRα.
 Metastasis suppressor 1 (MTSS1)
MTSS1 is an actin-binding protein that plays a role in the regulation of cell
proliferation.12 Researchers have shown in other types of cancer that reduction of
MTSS1 in tumor cells, may contribute to growth, development and metastases of
the tumor.12,13,21
We expect that in keratin 19 positive tumors there is less MTSS1 expression
compared to keratin 19 negative tumors.
 S100A6 (Calcyclin)
S100A6 is a low molecular weight calcium binding protein and a member of the
S100 family.9,34 This family can interact with effector proteins and regulate
enzyme activities, cell growth and differentiation and calcium homeostasis. The
exact role of S100A6 is not yet clearly defined, but it is thought to play many
roles in the healthy body.34 The protein is also up regulated in a variety of tumors.
In previous studies, tumors with elevated S100A6 invade faster in surrounding
tissue and have a higher metastatic ability.9,34
In our study we think S100A6 is elevated in K19 positive tumors, because of the
malignant character of these tumors.
8
 MAC 387
MAC 387 is not a metastasis marker, but it is a marker to measure the
macrophage activity in the hepatocellular carcinomas. This gives an indication of
the amount of inflammation in the tumor and in its surroundings. MAC 387 stains
the macrophages/monocytes, which are infiltrating in or reactive with the
surrounding tissue.14 MAC 387 is a molecule, which is present in the cytoplasm of
monocytes and macrophages.8
9
Hypotheses

Keratin 19 positive hepatocellular carcinomas are more malignant
compared to keratin 19 negative carcinomas.

Keratin 19 positive hepatocellular carcinomas express many metastasis
markers.
o Higher expression of markers:
 BMI-1
 EZH2
 Laminin1
 CD29
 Glypican3
 PDGFRα
 S100A6
 MAC 387
o Lower expression of markers:
 NF2
 NF2-P
 MTSS1
10
Material and Methods
 Samples
The liver research group has collected the hepatic tumor samples we used for this
study. The samples came from Valuepath the Netherlands, Faculty of Veterinary
Medicine of Utrecht University, the University of Zurich and the University of
Berlin.
The tissue samples were collected by resections and biopsies from the liver and
were fixed in formalin and embedded in paraffin. For our study we only used the
resection blocks. The blocks were cut in 5 µm thick slices and mounted on poly-L
lysine or silane coated slides.
In total we had 86 new samples for staining, from 86 patients diagnosed with
liver tumors. Of these patients, there are 67 dogs and 19 cats.
We included data from a previous study, performed by Renee van Sprundel. This
makes it a total of 145 patients. Of these 145 patients, 113 are dogs and 32 are
cats.
All the samples were stained with haematoxylin and eosin at the pathology
department of pathology at Utrecht University for histology.
Immunohistochemistry was performed for K19 on all liver tumor samples. BMI-1,
EZH2, NF2, NF2-P, Laminin1, CD29, Glypican3, PDGFRα, MTSS1, S100A6 and
MAC387 were stained with immunohistochemistry on a selection of six K19
negative and five K19 positive tumors. Immunohistochemistry of the metastasis
markers had to be optimized prior to staining on the selection of tumors.
All samples were examined by a certified veterinary pathologist, Dr. T.S.G.A.M.
van den Ingh and feed-back was provided on all samples.
11
 Immunohistochemistry
Immunohistochemistry is a staining method in which antibodies are used to make
specific antigens visible.3
There are two different methods that can be used with immunohistochemistry,
the direct method or the indirect method.
The direct method (fig.3) is the oldest technique, where an enzyme-labeled
primary antibody reacts with an antigen in the tissue. Subsequent the substratechromogen is added and the reaction sequence is complete. 3
Figure 3: direct staining method.3
In the indirect method (fig.4), an unconjugated primary antibody binds to the
antigen first. An enzyme-labeled secondary antibody directed against the primary
antibody is then applied, followed by the substrate-chromogen.3
Figure 4: indirect staining method.3
The indirect method has an advantage by being more versatile, because the
secondary antibody can be used with multiple primary antibodies, and this
method is more sensitive.3
In our study we only used the indirect method with primary antibodies made in
rabbit or mouse. This means that the secondary antibodies had to be directed
against rabbit or mouse. An enzyme was attached to the secondary antibody. This
enzyme reacted to the substrate-chromogen, which made the antigens visible.
12
 Staining protocol
Slides were deparrafinized by placing slides in a xylene bath twice for 5 minutes.
Then the slides were rehydrated in an alcohol series for 5 minutes each (96%
alcohol (2x), 80%, 70%, 60% and 30%). Prior to use slides were washed once in
milli-Q (highly prurified demi-water) for 5 minutes. After the rehydration, the
tissue underwent an antigen retrieval. This was done either by enzymatic antigen
retrieval with Proteinase K (for 10 min at room temperature), or heat-induced by
means of Tris-EDTA or citrate buffer for 40 min at 96oC in a water-bath (the
specifics for each antibody are listed in table 1). Slides were washed in phosphate
buffered saline (PBS) or tris buffered saline (TBS) (see table 1) including tween
(twice for 2 minutes) and the endogenous peroxidase was blocked by DAKO ready
to use enzyme block for 10 minutes at room temperature. After this step, slides
were washed in PBS/TBS with tween (three times for 5 minutes). Background
staining was reduced by incubating slides in 10% goat serum for 30 minutes at
room temperature. The goat serum was diluted in PBS/TBS without tween. After
removing the goat serum, the primary antibody was incubated on the slides. The
antibodies were diluted in Antibody Diluent (DAKO), and stayed on the slides for 1
hour at room temperature or over-night at 4oC. Slides were washed in PBS/TBS
with tween three times for 5 minutes, and the secondary antibody was incubated
on the slides for 45 minutes at room temperature. The secondary antibody used
was the labeled polymer Envision (Dako, Glostrup, Denmark), either goat anti
Rabbit or goat anti Mouse depending on the primary antibody (see table 1). After
this slides were washed three times for 5 minutes, but this time in PBS/TBS
without tween. After washing the chromogen diaminobenzidine (DAB) (DAKO)
was placed on the slides for 5 minutes at room temperature. This reacts with the
peroxidase label on the secondary antibody and makes a visible brown color. The
remaining DAB was washed off in milli-Q, three times for 5 minutes. Slides were
counterstained with hematoxylin (DAKO) for 10 seconds. Afterwards slides were
washed in running tab water for 10 minutes. Then slides were dehydrated in an
alcohol series followed by xylene (60% alcohol, 70%, 80%, 96% (twice) and
xylene (twice)) for 5 minutes each. Finally slides were mounted in permanent
mounting media Vectamount (VECTOR). (All protocols for each antibody are in
the appendix)
13
Antibody
Antigen
retrieval
Antibody
dilution
Over Night
/1 hour
Secondary
antibody
Washing
medium
K19
Proteinase K
1:100
1 hour RT
Anti Mouse
TBS
BMI-1
Tris-EDTA
(40min)
1:80
Over Night
Anti Mouse
PBS
EZH2
Citrate (30min)
1:500
1 hour RT
Anti Rabbit
PBS
NF2
Proteinase K
1:500
1 hour RT
Anti Rabbit
PBS
NF2-P
Citrate (30min)
1:750
Over Night
Anti Rabbit
PBS
Laminin1
Proteinase K
1:50
Over Night
Anti Rabbit
PBS
CD29
Citrate (40min)
1:100
Over Night
Anti Mouse
PBS
Glypican3
Citrate (40min)
1:100
Over Night
Anti Mouse
PBS
PGDFRα
Tris-EDTA
(40min)
1:100
Over Night
Anti Rabbit
PBS
MTSS1
Tris-EDTA
(40min)
1:100
Over Night
Anti Rabbit
PBS
S100A6
Tris-EDTA
(40min)
1:100
Over Night
Anti Mouse
PBS
MAC 387
Proteinase K
1:1000
Over Night
Anti Mouse
PBS

Table 1: staining-specifics for each antibody.
RT= Room temperature
14
 Sample grading
Edmondson and Steiner have developed a grading system for hepatocellular
carcinomas. This was based on the principle that the tumors that look worse will
grow faster and progress more rapidly to cause death of the patient.4 Van
Sprundel has modified the Edmondson and Steiner grading system to be applied
in the veterinary sciences. This is a four category grading system, based on:
 Cell morphology (anisocytosis)
 Nuclear morphology (anisokaryosis) and nucleoli morphology
 Presence of multinucleated tumor cells
 Mitotic activity28
To place the tumors in different groups of malignancy, a pathologist
microscopically examined the slides. The slides were graded for their appearance.
The slides got three different grades, which were later counted to define in which
group of the grading system the tumor was placed. The slides were scored on a
scale with a range from zero to three for the amount of pleiomorfism of the
tumor. When there was no pleiomorphism the tumor scored zero and when there
was a lot of pleiomorphism the tumor scored three.
The multinucleated cells were either absent or present, and could therefore score
zero or one. Mitotic activity had a range from zero to three, where zero is no
mitotic figures and three is many mitotic figures.
When the three grades are summed, there are four different grades in which the
tumor can be classified. 28
 Grade 0: 0
 Grade 1: 1-2
 Grade 2: 3-4
 Grade 3: 5-6
15
 Sample staging
With staging, the extent or severity of the tumor can be described. This is done
by looking at the original tumor and the extent of the spread of the tumor in the
body. Tumor cells can grow and divide without control. In addition they can
invade in healthy tissue and enter the bloodstream or lymphatic system. When
the cells are in the bloodstream they can go to other organs and infiltrate and
grow there, this is called metastasis.
There are many systems to stage a tumor. A commonly used system is the TNM
system. TNM system is based on the extent of the tumor (T), the extent of spread
to the lymph nodes (N) and the presence of metastasis (M).4
In this study we used summary staging, which is only based on the spread of the
tumor through the body. It is a modified three category staging system and is
based on the invasion and spread of the tumor.
The tumors are staged in three categories:
 Stage 0: Macroscopically there is only one tumor process in the liver
and/or the tumor is microscopically well circumscribed or encapsulated.
There are no indications for metastases.
 Stage 1: Microscopically the tumor has spread beyond the original site to
the adjacent tissue and/or vessels or microsatellites can be seen and/or
macroscopically multiple tumor processes are present in the liver.
 Stage 2: The tumor has spread from the primary site to the lymph nodes
and/or other organs. 28
The tumors were all microscopically examined to determine in which group they
had to be placed. Also the anamneses of the patients and findings of the vets was
important to determine whether or not the tumor has spread to other organs.
16

Viability assay
Viability was determined from a K19 positive cell line called HepaRG
treated with Imatinib.
Material:
- HepaRG cells. These are cells of early hepatoblast tumors of humans. These
cells resemble K19 positive HCC cells and also express PDGFRα.
- Imatinib, methanesulfonate salt (I-5508 LC Laboratories)
CAS 220127-57-1
Mw: 589.71
Dissolve 295 mg in 5 ml Hanks (100mM), filter sterilize
- MTT solution: 5mg/ml MTT (Thiazolyl Blue Tetrazolium Bromide) in PBS.
Solution must be filter sterilized before use.
- Dimethyl Sulfoxide (DMSO)
- Culture Media: William’s Medium E (WME) supplemented with 10% Fetal Calf
Serum (FCS), Penicillin-Streptomycin solution (PS) and additives.
Protocol:
Day 1:
1. Plate out HepaRG cells at 10,000 cells per well in a 96 well plate (100µL
total volume) under normal culture conditions
2. Prepare a dilution series of Imatinib in Hanks using 4-fold serial dilutions.
3. After 4 hours, add 10µL of Imatinib solutions to their respective wells (n=6
per concentration). Add 10µL Hanks to the zero measurement.
4. Return to culture stove and incubate for 24 hours.
Day 2:
1. Remove cultures from incubator into laminar flow hood or other sterile
working area.
2. Add 20µL of 5mg/ml MTT to each well. Include one set of wells with MTT
but no cells (control). All should be done aseptically.
3. Incubation times should be consistent when making comparisons.
4. Incubate for 2 hours at 37°C in culture stove.
5. Carefully remove media. Do not disturb cells and do not rinse with PBS.
6. Add 50µL DMSO.
7. Cover with tinfoil an agitate cells on orbital shaker for 15 minutes.
8. Read absorbance at 590 nm with a reference filter of 620 nm.
Imatinib concentration used:
We used 10 different dilutions. Each dilution was put on 6 wells. We used
10,000nM, 2,500nM, 630nM, 156nM, 39nM, 9.8nM, 2.4nM, 0.6nM, 0.2nM and
0nM.
17
Results
 Liver tumors
Together with a veterinary pathologist (TvdI) all 145 samples were examined.
Of 113 dog samples, 17 did not have a tumor or a tumor that did not originate
from the liver. This leaves us with a total of 96 liver tumors for our study.
We have 13 nodular hyperplasia, 70 hepatocellular tumors, 7 cholangiocellular
carcinomas and 6 carcinoids. (Table 2)
In cats 12 of the 32 samples did not have a liver tumor or the diagnosis was
inconclusive. This makes it a total of 20 tumors we used in our study. Of those, 1
was a nodular hyperplasia, 8 hepatocellular tumors, 4 cholangiocellular tumors
and 7 carcinoids. (Table 2)
Number
in Dogs
% of total
liver
tumors
Number
in Cats
% of total
liver
tumors
Nodular hyperplasia
13
13.5%
1
5%
Hepatocellular adenoma
56
58.3%
8
40%
Hepatocellular carcinoma
14
14.6%
-
-
Cholangiocellular carcinoma
7
7.3%
4
20%
Carcinoid
6
6.3%
7
35%
Liver tumor
Table 2: Number and percentage of liver tumors examined microscopically.
The hepatocellular tumors can be divided in adenomas and carcinomas. A
difference between these tumors can be the expression of Keratin 19. The
carcinomas can have a high expression of Keratin 19, while the adenomas have
(almost) no expression of keratin 19. The prevalence of K19 positivity in
hepatocellular tumors is in table 3.
Number
in Dogs
% of HCCtumors
Hepatocellular adenoma (K19-)
56
80%
Hepatocellular carcinoma (K19+)
14
20%
Hepatocellular tumor
Table 3: Number and percentage of Hepatocellular tumors.
18
 Sample grading and staging
With the grading and staging system a difference can be made in malignancy
between K19 positive and K19 negative hepatocellular tumors.
Hepatocellular carcinomas are mostly graded in higher groups compared to the
adenomas (table 4). This means that in carcinomas there is more pleiomorphism,
there are more multinucleated cells present and there is a higher mitotic activity
compared to adenomas.
With staging, there is also a difference between these two groups. Hepatocellular
adenomas are mostly stained in group 0. This means that there is only one tumor
process in the liver and/or the tumor is microscopically well circumscribed or
encapsulated. Carcinomas are all staged in group 1 or 2. This means they are
metastasized within the liver or to other organs. (table 4)
Hepatocellular tumor
Dog
Dog
Grading
Staging
Normal liver
0
0
Hepatocellular adenoma
1: n=45
0
2: n=11
3: n=0
Hepatocellular carcinoma
1: n=1
1&2
2: n=3
3: n=10
Table 4: grading and staging of Hepatocellular tumors.
Malignancy can also be seen in the hepatocytes staining. We stained all
hepatocellular tumors for hepatocyte markers (HepParR1). This revealed that in
K19 negative tumors, hepatocyte markers were highly expressed. But in K19
positive tumors, the hepatocyte markers were not stained. This means that K19
positive tumor cells are poorly differentiated and do not resemble hepatocytes
anymore.
19
 Hepatocellular tumors
In figure 5A a representative image is shown of a hepatocellular adenoma, which
is well encapsulated. Figure 5B is the Keratin 19 staining of this tumor and shows
that this tumor does not express keratin 19. Figure 6A is a representative image
of a hepatocellular carcinoma. It shows that it is invasive in the surrounding
tissue and not encapsulated. Figure 6B is the Keratin 19 staining, and shows that
K19 is highly expressed in this tumor.
A
B
Figure 5: A: H&E staining in hepatocellular adenoma, 4x. B: K19 staining in hepatocellular
adenoma, 10x.
A
B
Figure 6: A: H&E staining in hepatocellular carcinoma, 4x. B: K19 staining in hepatocellular
carcinoma, 10x.
20
 BMI-1 and EZH2
BMI-1 is in all five K19 positive tumors highly expressed in the cytoplasm. In
contrast to the six K19 negative HCCs, where there is little BMI-1 present. Figures
7A and 7B show the most representative images of these results.
The expression of EZH2 is not different in both types of tumors (fig. 7C&D)
A
B
C
D
Figure 7: A: BMI-1 staining in K19 negative tumor, 20x. B: BMI-1 staining in K19 positive
tumor, 20x. C: EZH2 staining in K19 negative tumor, 20x. D: EZH2 staining in K19 positive
tumor, 20X.
21
 NF2 and NF2-P
NF2 is positively stained in the membranes of the tumor cells of all keratin 19
negative hepatocellular tumors (fig. 8A). In all keratin 19 positive tumors there is
no staining on the membranes(fig. 8B).
NF2-P is stained in the nuclei in the K19 negative tumors. Figure 8C shows an
example of a K19 negative tumor with this staining. In contrast to the K19
positive tumors, where there is no staining in the nuclei, as shown in figure 8D.
A
C
B
D
A
Figure 8: A: NF2 staining in K19 negative tumor, 20x. B: NF2 staining in K19 positive
tumor, 20x. C: NF2-P staining in K19 negative tumor, 20x. D: NF2-P staining in K19
positive tumor, 20X.
22
 Laminin1 and CD29
In the K19 positive tumors, laminin1 was highly expressed, while K19 negative
tumors showed no expression of laminin1 in the tumor cells. Figure 9A and 9B are
an example of a K19 negative and a K19 positive tumor.
For CD29 there is no difference in staining between all K19 positive and K19
negative HCCs. (fig. 9C&D). In these tumors the endothelium is stained positive.
This could be a different integrin which is stained, however this needs to be
determined.
A
B
C
D
Figure 9: A: Laminin1 staining in K19 negative tumor, 20x. B: Laminin1 staining in K19
positive tumor, 20x. C: CD29 staining in K19 negative tumor, 20x. D: CD29 staining in
K19 positive tumor, 20X.
 Glypican3
For glypican3 we do not have results which correlate to previous studies. We
found no positive staining in any of the tumors.
23
 PDGFRα
For PDGFRα there is a big difference between the six K19 negative tumors and
the five K19 positive tumor. In the K19 positive tumors PDGFRα is higher
expressed compared to the K19 negative tumors. Figures 10A and B show
representative images of a K19 negative tumor, with little PDGFRa expression,
and a K19 positive tumor, with a high expression of PDGFRa.
A
B
Figure 10: A: PDGFRα staining in K19 negative tumor, 20x. B: PDGFRα staining in K19
positive tumor, 20x.
In the Viability assay we found HepaRGs are highly sensitive for Imatinib with an
IC50 of 80nM (fig. 11). This means that half of the HepaRG cells had died after 24
hours of treatment with Imatinib at a concentration of 80nM.
Viability after Imatinib treatment (HepaRG)
Percentage viability (%)
120
100
80
60
40
20
0
0,1
1,0
10,0
100,0
1000,0
Concentration Imatinib (nM)
Figure 11: Result of the viability assay. HepaRG cells were treated with different
concentrations of Imatinib for 24 hours. Cell viability was determined with MTT. The
untreated cells were set to 100 percent viable.
24
 MTSS1
In K19 negative tumors, MTSS1 is less expressed compared to K19 positive
tumors. This is shown, in representative images of these tumors, in figures 12A
and 12B.
A
B
Figure 12: A: MTSS1 staining in K19 negative tumor, 20x. B: MTSS1 staining in K19
positive tumor, 20x.
 S100A6
S100A6 is highly expressed in the cytoplasm of all stained K19 positive tumors
(fig. 13B). While in the stained K19 negative tumors there seems to be a little
expression of S100A6 in the cytoplasm (fig. 13A). As shown in figures 13A and
13B, the expression of S100A6 is much less in K19 negative HCCs compared to
K19 positive HCCs.
A
B
Figure 13: A: S100A6 staining in K19 negative tumor, 20x. B: S100A6 staining in K19
positive tumor, 20x.
25
 MAC387
In the K19 negative HCCs (fig. 14A) there are almost no macrophages present in
the tumors, although a few can be seen circulating in the blood vessels. In the
K19 positive tumors there are a lot of macrophages within the tumor (fig. 14B).
A
B
Figure 14: A: MAC387 staining in K19 negative tumor, 4x. B: MAC387 staining in K19
positive tumor, 4x.
26
Conclusion

Keratin 19 positive hepatocellular carcinomas are more malignant
compared to keratin 19 negative carcinomas.
Keratin 19 positive hepatocellular tumors are poorly differentiated, show
pleiomorphism, multinucleated cells, and mitosis and often metastasize. While
Keratin 19 negative hepatocellular tumors still resemble healthy liver, is well
encapsulated and does not metastasize.
This that K19 positive hepatocellular carcinomas are more malignant
compared to K19 negative hepatocellular carcinomas. means

Keratin 19 positive hepatocellular carcinomas express many
metastasis markers.
Compared to K19 negative hepatocellular carcinomas, Keratin 19 positive
hepatocellular carcinomas have a higher expression of BMI-1, Laminin1,
PDGFRα, S100A6 and MAC387.
There was a lower expression in K19 positive hepatocellular carcinomas,
compared to K19 negative hepatocellular carcinomas, of NF2 and NF2-P.
Overall, keratin 19 is a highly sensitive marker for aiding in the grading and
staging of hepatocellular tumors. In addition, due to the high similarity between
dogs and man based on these results, the dog possess as a relevant model for
new therapies against malignant forms of hepatocellular carcinoma.
27
Discussion
In this study a multitude of malignancy markers were linked to a more malignant
subtype of hepatocellular carcinomas. This subtype can be easily detected by a
single marker (keratin 19). Here we confirm that HCCs which poses this marker
scale higher in staging and grading systems and express several metastasis
markers including BMI-1, NF2, NF2-P, laminin1, PDGFRα, S100A6 and MAC387.
Although the initial setup was to determine these markers in dogs and cats, the
amount of tumors obtained from cats was not enough to proof or disproof our
based hypothesis. Therefore the majority of the results in this report are based on
dogs. This said, almost all markers of malignancy in the dog were optimized on
our tumor sections and confirmed the higher malignancy of the keratin 19
positive tumors.
In our study we found a prevalence of K19 positive hepatocellular carcinomas of
20% of all hepatocellular tumors. In a previous study done by van Sprundel et al.
12% was found. The number of tumors included in the studies can explain the
difference. We included 70 hepatocellular tumors, while in the previous study only
34 tumors were included.
Staging the tumors was not always possible. This was because of the little
information of the heritage of some tumors. Sometimes histology could tell us a
little more, but when it was not clear, we did not include the tumor in the results.
A number of markers did not show a difference in expression between the K19
positive and the K19 negative hepatocellular carcinomas.
CD29 had the same expression in both K19 negative and K19 positive tumors.
There was some endothelium staining. We expected CD29 on the cell membrane
of epithelial cells and not in the endothelium. A possible explanation for the
endothelial staining is that another integrin has been stained. However this
remains to be determined.
EZH2 was also equally expressed in K19 positive and K19 negative tumors. There
was in both kind of tumors little EZH2 present. This has to be re-examined. It
could be that EZH2 is not higher expressed in K19 positive HCC, or it could be
due to the staining.
Glypican3 was not stained in all samples, except for one. Because of a previous
study, by van Sprundel et al., we know glypican3 is higher expressed in K19
positive HCCs. We did find that in one sample, but the rest of the samples did not
have any staining at all. These samples have to be stained again to show the
specificity of the antibody. Although the same antibody and manufacturer was
used, the antibody did have another lot number compared to the older antibody
that did work.
MTSS1 was higher expressed in the K19 positive tumors compared to the K19
negative tumors. MTSS1 is a metastasis suppressor, so is expected to be higher
in the K19 negative tumors. This staining needs to be re-examined. In the K19
positive HCC the staining can be a very strong background staining.
In the viability assay, we found an IC50 of 80nM. In other studies an IC50 of
400nM was found. These studies were done on other kind of tumors, for a longer
period of time (3-4 days). This means that our hepatic cells are more sensitive to
Imatinib; so lower doses can be used to attack the tumor.
Before Imatinib can be used as a treatment for hepatocellular carcinomas, further
studies have to be done.
28
MAC 387 was highly expressed in the K19 positive hepatocellular carcinoma. This
can be because of the inflammation of the surrounding tissue. It can be that in
and around the fast growing tumor cells become stressed and spread
inflammatory signals.
This study provided the basis of sub classify hepatocellular carcinomas based on
malignancy by using a single marker. This could lead to new diagnosing and
treatment options in veterinary medicine and provide the basis for treatment
options of HCC in man.
29
References
1. Altekruse, S.F., McGlynn, K.A. & Reichman, M.E. (2009) Hepatocellular
carcinoma incidence, mortality, and survival trends in the united states from
1975 to 2005. Journal of clinical oncology. 27(9): 1485-1491.
2. Benhamouche, S., Curto, M., Saotome, I., Gladdeu, A.B., Liu, C.H.,
Giovannini, M. & McClatchey, A.I. (2010) Nf2/Merlin controls progenitor
homeostasis and tumorigenesis in the liver. Genes & Development. 24: 17181730.
3. Boenisch, T. Handbook immunochemical staining methods. 3rd ed.
4. Burt, D., Portmann, C., Ferrel, D., (2007) MacSween’s Pathology of the liver.
Churchill Livingstone Elsevier
5. Capurro, M., Wanless, I.R., Sherman, M., Deboer, G., Shi, W., Miyoshi, E. &
Filmus, J. (2003) Glypican-3: a novel serum and histochemical marker for
hepatocellular carcinoma. Gastroenterology. 125(1): 89-97.
6. Carlori, V., Mazzocca, A., Pantaleo, P., Cordella, C., Laffi, G. & Gentilini, P.
(2001) The integrin a6b1, is necessary for the matrix-dependent activation of
FAD an MAP kinase and the migration of human hepatocarcinoma cells.
Hepatology 34(1): 42-49.
7. Ekblom, P., Lonai, P. & Talts, J.F. (2003) Expression and biological role of
laminin-1. Matrix biology 22(1): 35-47.
8. Karakucuk, I., Dilly, S. A. & Maxwell, J. D. (1989), Portal tract macrophages
are increased in alcoholic liver disease. Histopathology; 14: 245–253.
9. Komatsu, K., Kobune-Fujiwara, Y., Andoh, A., Ishiguro, S., Hunai, H., Suzuki,
N., Kameyama, M., Murata, K., Miyoshi, J., Akedo, H., Tatsuta, M. &
Nakamura, H. (2000). Increased expression of S100A6 at the invading fronts
of the primary lesion and liver metastasis in patients with colorectal
adenocarcinoma. British Journal of Cancer. 83(6): 769-774.
10. Komuta, M., Spee, B., Vander Borght, S., de Vos, R., Verslype, C., Aerts, R.,
Yano, H., Suzuki, T., Matsuda, M., Fujii, H., Desmet, V.J., Kojiro, M. &
Roskams, T. (2008) Clinicopathological study on cholangiolocellular carcinoma
suggesting hepatic progenitor cell origin. Hepatology. 47(5):1544-1556.
11. Kwack, M.H., Choi, B.Y. & Sung, Y.K. (2006) Cellular changes resulting from
forced expression of Glypican-3 in hepatocellular carcinoma cells. Molecules
and Cells. 21(2): 224-228.
12. Liu, K., Wang, G., Ding, H., Chen, Y., Yu, G. & Wang, J. (2010)
Downregulation of metastasis suppressor 1 (MTSS1) is associated with nodal
metastasis and poor outcome in Chinese patients with gastric cancer. BMC
Cancer. 10: 428.
13. Loberg, R.D., Neeley, C.K., Adam-Day, L.L., Fridman, Y., St. John, L.N.,
Nixdorf, S., Jackson, P., Kalikin, L.M. & Pienia, K. (2005) Differential
expression analysis of MIM (MTSS1) splice variants and a functional role of
MIM in prostate cancer cell biology. International Journal of Oncology. 26(6):
1699-1705
14. McGuinness, P.H., Painter, D., Davies, S. & McCaughan G.W. (2000) Increases
in intrahepatic CD68 positive cells, MAC387 positive cells, and
proinflammatory cytokines (particularly interleukin 18) in chronic hepatitis C
infection, Gut; 46:260-269.
15. Monga, D.K., Sodhi, D., Micsenyi, A. & Monga, S.P.S. (2004) Aberrant PDGFRa
signaling in human hepatocellular cancers. Proc Amer Assoc Cancer Res. 45
16. Morrison, H., Sherman, L.S., Legg, J., Banine, F., Isacke, C., Haipek, C.A.,
Gutmann, D.H., Ponta, H. & Herrlich, P. (2001) The NF2 tumor suppressor
gene product, merlin, mediates contact inhibition of growth through
interactions with CD44. Genes & Development. 15: 968-980.
17. Nelson, R.W. & Couto, C.G. (2003) Small animal internal medicine, 3rd ed.
Mosby
30
18. Novak, K. (2003) Cancer susceptibility: diet-related cancers: HCC and
Aflatoxin. Medscape general medicine. 5(1)
19. Omary, M.B., Ku, N. & Tiovola, D.M. (2002) Keratins: Guardians of the liver.
Hepatology. 35(2): 251-256.
20. Ozaki, I., Yamamoto, K., Mizuat, T., Kajihara, S., Fukushima, N., Setoguchi,
Y., Morito, F. & Sakai, T (1998) Differential expression of laminin receptors in
human hepatocellular carcinoma. Gut 43: 837-842.
21. Parr, C. & Jiang, W.G. (2009) Metastasis suppressor 1 (MTSS1) demonstrates
prognostic value and anti-metastatic properties in breast cancer. European
Journal of Cancer 45(9): 1673-1683.
22. Patnaik, A.K., Hurvitz, A.I. & Lieberman, P.H. (1980) Canine Hepatic
Neoplasms: A Clinicopathologic Study. Vet. Pathol. 17: 553-564.
23. Patnaik, A.K., Hurvitz, A.I., Lieberman, P.H. & Johnson, G.F. (1981) Canine
Hepatocellular Carcinoma. Vet. Pathol. 18: 427-438.
24. Rong, R., Surace, E.I., Haipek, C.A., Gutmann, D.H. & Ye, K. (2004) Serine
518 phosphorylation modulates merlin intramolecular association and binding
to critical effectors important for NF2 growth suppression. Oncogene. 23:
8447-8454.
25. Sanford, M. & Scott, L.J. (2010) Imatinib: as adjuvant therapy for
gastrointestinal stromal tumour. Drugs. 70(15): 1963-1972.
26. Sasaki, M., Ikeda, H., Itatsu, K., Yamaguchi, J., Sawada, S., Minato, H., Ohta,
T. & Nakanuma, Y. (2008) The overexpression of polycomb group proteins
BMI1 an EZH2 is associated with the progression and aggressive biological
behaviour of hepatocellular carcinoma. Laboratory Investigation. 88: 873882.
27. Sell, S. (1993) The role of determined stem-cells in the cellular lineage of
hepatocellular carcinoma. International journal of developmental biology. 37:
189-201.
28. van Sprundel ET verslag (2008) clinicopathological characteristics and
prognostic markers in canine and feline primary liver tumors.
29. van Sprundel, R.G.H.M., van den Ingh, T.S.G.A.M., Desmet, V.J.,
Katoonizadeh, A., Penning, L.C., Rothuizen, J. & Spee, B. (2010) Keratin 19
marks poor differentiation and a more aggressive behaviour in canine and
human hepatocellular tumours. Comparative Hepatology. 9:4.
30. Steele, J.C., Torr, E.E., Noakes, K.L., Kalk, E., Moss, P.A., Reynolds, G.M.,
Hubscher, S.G., van Lohuizen, M., Adams, D.H. & Young, L.S. (2006) The
polycomb group proteins, BMI-1 and EZH2, are tumour-associated antigens.
British Journal of Cancer. 95: 1202-1211.
31. Stock, P., Monga, D., Tan, X., Micsenyi, A., Loizos, N. & Monga, S.P.S. (2007)
Platelet-derived growth factor receptor-a: a novel therapeutic target in human
hepatocellular cancer. Mol. Cancer. Ther. 6(7): 1932-1941.
32. Torimura, T., Ueno, T., Kin, M., Sakisaka, S., Tanikawa, K. & Sata, M. (1999)
B1-Integrin mediates cell adhesion, haptotaxis, and chemokinesis of
hepatoma cells. Med Elecron Microsc. 32: 2-10.
33. Uenishi, T., Kubo, S., Yamamoto, T., Shuto, T., Ogawa, M., Tanaka, H.,
Tanaka, S., Kaneda, K. & Hirohashi, K. (2003) Cytokeratin 19 expression in
hepatocellular carcinoma predicts early postoperative recurrence. Cancer Sci.
94(10): 851-857.
34. Vimalachandran, D., Greenhalf, W., Thompson, C., Luttges, J., Prime, W.,
Campbell, F., Dodson, A., Watson, R., Crnogorac-Jurcevic, T., Lemoine, N.,
Neoptolemos, J. & Costello, E. (2005) High nuclear S100A6 (Calcyclin) is
significantly associated with poor survival in pancreatic cancer patients.
Cancer Res 2005. 65(8): 3218-3225.
35. Wang, H., Pan, K., Zhang, H., Weng, D., Zhou, J., Li, J., Huang, W., Song, H.,
Chen, M. & Xia, J. (2008) Increased polycomb-group oncogene BMI-1
expression correlates with poor prognosis in hepatocellular carcinoma. J.
Cancer Res. Clin. Oncol. 134: 535-541.
31
Acknowledgements
I first of all would like to thank my supervisor Bart Spee, he helped us a lot
during those three months.
I also would like to thank Ted van den Ingh, for watching and examining all those
hundreds of slides.
And of course, I want to thank Hideyuki Kanemoto, for cutting almost all the
slides and the enormous amount of time he put in to doing that.
Before I forget someone I want to thank everyone from the liver department, for
helping when needed. And all the students, for help and mostly for the nice time.
And finally I want to thank my partner in this three month project, Lesley-Anne
de Groot.
32
Appendix
Two step IHC protocol Keratin 19

Deparaffinize and rehydrate sections in a series of:

Proteinase-K (Dako)
15 min. RT

Rinse in TBS/T buffer solution
2 x 2 min.

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in TBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 K19 – #29 – 1:100 (mouse)
60 min. RT

Rinse the sections in TBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti mouse HRP
45 min. RT

Rinse the sections in TBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
33
Two step IHC protocol NF2-P

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot citrate buffer pH 6.0, 98oC
30 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 NF2-P – #56 – 1:750 (rabbit)
4oC O/N

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti Rabbit HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
34
Two step IHC protocol Glypican-3

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot citrate buffer pH 6.0, 98oC
40 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 Glypican-3 – #39 – 1:100 (mouse)
4oC O/N

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti mouse HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
35
Two step IHC protocol NF2

Deparaffinize and rehydrate sections in a series of:

Proteinase-K

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 NF2 – #44 – 1:500 (rabbit)
60 min. RT

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti rabbit HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
(Dako)
10 min. RT
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
36
Two step IHC protocol LAMININ1

Deparaffinize and rehydrate sections in a series of:

Proteinase-K

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 Laminin1 – #45 – 1:50 (rabbit)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti rabbit HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
(Dako)
10 min. RT
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
37
Two step IHC protocol CD29

Deparaffinize and rehydrate sections in a series of:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’

Antigen retrieval in 10 mM hot citrate buffer pH 6.0, 98oC
40 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 CD29 - #25 - 1:100 (mouse)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti mouse HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
38
Two step IHC protocol MAC387

Deparaffinize and rehydrate sections in a series of:

Proteinase-K

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 MAC387 – #37 – 1:1000 (mouse)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti mouse HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
(Dako)
10 min. RT
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
39
Two step IHC protocol PDGFRa

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot TE buffer pH 9.0, 98oC
40 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use dual endogenous enzyme block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 PDGFRA- #47 - 1:100 (rabbit)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti Rabbit HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
40
Two step IHC protocol MTSS1

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot TE buffer pH 9.0, 98oC
40 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use dual endogenous enzyme block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 MTSS1- #48 - 1:100 (rabbit)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti Rabbit HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
41
Two step IHC protocol S100A6

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot TE buffer pH 9.0, 98oC
40 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use dual endogenous enzyme block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 S100A6- #32 - 1:100 (mouse)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti Mouse HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
42
Two step IHC protocol BMI-1

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot TE buffer pH 9.0, 98oC
40 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use dual endogenous enzyme block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 BMI-1- #24 - 1:80 (mouse)
O/N at 4°C

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti Mouse HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
43
Two step IHC protocol EZH2

Deparaffinize and rehydrate sections in a series of:

Antigen retrieval in 10 mM hot citrate buffer pH 6.0, 98oC
30 min.
Cool down at room temperature (still in hot buffer outside water bath) 30 min.

Rinse in PBS/T buffer solution

Inhibit endogenous peroxidase activity by incubating the slides in 10 min. RT
Dako Ready to use peroxidase block

Rinse in PBS/T buffer solution
3 x 5 min.

Incubate in 10 percent normal goat serum
30 min. RT

Incubate with (in ab-diluent, DAKO)
 EZH2 – #46 – 1:500 (rabbit)
60 min. RT

Rinse the sections in PBS/T buffer solution
3 x 5 min.

Incubate in Envision Goat anti Rabbit HRP
45 min. RT

Rinse the sections in PBS
3 x 5 min.

Incubate the sections in freshly made DAB substrate (result: brown) 5 min.

Rinse the sections in mQ
3 x 5 min.

Counterstain the sections haematoxylin QS-Dako
10 sec.

Rinse sections in running tapwater
10 min.

Dehydrate section and cover in vectamount:
Xylene: 2x5’ ; Alc. 96% 2x; Alc. 80% ; Alc. 70%; Alc. 60%; Alc. 30% (each step 5’);
MQ 5’
2 x 2 min.
5’ 60% alcohol; 5’ 70% alcohol; 5’ 80% alcohol; 5’ 96% alcohol; 5’ 96% alcohol; 5’
100% alcohol; 2x5’ xylene.
44