RIGA STRADIŅŠ UNIVERSITY
IIze Strumfa
CYCLOOXYGENASE-2 PROTEIN EXPRESSION IN
GASTROOESOPHAGEAL TUMOURS - STUDY OF A
COMPLEX INTERPLAY OF BIOLOGICAL AND
TECHNOLOGICAL FACTORS
Summary of PhD Thesis
Speciality - pathology
Riga - 2005
BACKGROUND
Within the several last years, the role of the enzyme cyclooxygenase (COX) in
carcinogenesis has caused great interest. COX catalyzes the rate-limiting steps for the
conversion of arachidonic acid to prostaglandins and thromboxanes. Two COX
isoenzymes have been definitely identified: COX-1 which is primarily constitutively
expressed and COX-2, an inducible form which can be up-regulated by, for example,
mitogenic stimuli, growth factors, cytokines, and carcinogens (DuBois et al., 1998).
Whilst the incidence of fundic and distal gastric cancer has fallen over the last 50
years, there has been a dramatic increase in adenocarcinomas of the cardia and the
oesophagus, particularly in the West (Corley and Buffler, 2001). At the same time,
there has been a marked increase in the incidence of gastrooesophageal reflux disease
associated with columnar cell metaplasia of the distal oesophagus (Barrett's
oesophagus), a lesion which predisposes to oesophageal dysplasia and
adenocarcinoma (Cameron et al., 1985). Whilst the genetic events leading to
development of cancers of the cardia and oesophagus are poorly understood (Mueller
et al, 2000; van Dekken et al., 2001), there is evidence implicating COX in their
development.
Epidemiologic, laboratory animal and clinical studies provide strong evidence that
use of COX inhibitors (such as aspirin and other non-steroidal anti-inflammatory
drugs, NSAIDs) reduces the risk of intestinal and other cancers (Thun et al., 1991;
Baron, 2003; Funkhouser and Sharp, 1995; Farrow et al., 1998; Coogan et al.; 2000).
Increased expression of COX-2 in vitro has been shown to have a number of cellular
effects including increasing proliferation, reducing apoptosis (Tsujii and Dubois,
1995), promoting angiogenesis (Jones et al., 1999), and increasing invasive/metastatic
potential (Tsujii et al., 1997), all of which may contribute to gastrointestinal
carcinogenesis. Experimental data from the last decade has shown that: (i) COX-2
expression is increased in a number of malignant and premalignant tissues (Kandil et
al., 2001; Zimmermann et al., 1999; Morris et al, 2001; Saukkonen et al., 2001) and
(ii) genetic modulation or specific inhibition of COX-2 expression affects tumour
yield in laboratory animals (Oshima et al., 1996; Liu et al., 2001; Hu et al, 2004),
indicating that inhibition of COX-2 may be relevant for the prevention or treatment of
human gastrointestinal cancers.
The evidence implicating COX-2 in the pathogenesis of carcinomas of the
oesophagus and proximal stomach comes from several sources. Epidemiological
studies have shown a reduction in the incidence of oesophageal (Corley et al., 2003)
and gastric (Baron and Sandler, 2000) carcinoma in persons taking NSAIDs. Ex vivo
experiments using a Barrett's oesophagus (BE) culture model have shown that the
constituents of gastroesophageal reflux, including acid and bile, can regulate COX-2
expression (Shirvani et al., 2000). It has recently been shown in an animal model of
BE-related cancer, that use of selective and non-selective COX-2 inhibitors inhibits
inflammation, COX-2 activity, and the development of reflux-induced
adenocarcinoma (Buttar et al., 2002).
Histological studies show that COX-2 is expressed in gastric and oesophageal
cancers, and in their precursor lesions metaplasia and dysplasia. However, reported
rates vary widely. Using immunohistochemistry (111C), epithelial COX-2 expression
is seen in the oesophagus in 54.2% - 90.7% of squamous cancers (Zimmermann et al.,
1999; Wang et al., 2002) and in 78% - 100% of adenocarcinomas (Zimmermann el
al., 1999; Morris et al., 2001). Expression in oesophageal carcinoma precursors is
even more variable, being seen in 0% - 75% cases of intestinal metaplasia
(Zimmermann et al., 1999; Morris et al., 2001) and in 41% - 100% cases of dysplasia
(Morris et al., 2001; Kandil et al., 2001). In the stomach, COX-2 cytoplasmic staining
is reported in 42.6% - 100% of carcinomas and 44.4-100% of comparable
premalignant lesions (Saukkonen et al., 2001; Lim et al., 2000).
Most previous studies have concentrated on a qualitative or at best semiquantitative
analysis of COX-2 expression in tumours. This reflects the general problems involved
in IHC. However, attempts to use molecular methods of mRNA or protein
quantification - e.g. based on whole tissue extracts - are hampered by the
heterogeneous pattern of COX-2 expression in both tumour (epithelial) and nonneoplastic lesional tissue components such as stroma, vessels, inflammatory cells
(Lim et al., 2000; van Rees et al.; 2002; Komhoff et al., 2000). This imprecision
makes it difficult to determine the prognostic significance of COX-2 expression in
premalignant gastrointestinal lesions. The exact location of COX-2 expression in
tumours is also of importance in order to understand better its role in oncogenesis. For
example, it is still unclear whether COX-2 acts via PGs directly on epithelial cells to
promote neoplasia, or whether other cell types in the stroma are involved, perhaps
with a paracrine effect.
Thus, well-standardized immunohistochemical technique for the direct visualisation
of COX-2 protein with exact estimation of technical bias would be significant as a tool
for further studies of the COX-2 role in carcinogenesis and as background in the
evaluation of already published controversial data. Such technique gains even greater
significance as ongoing studies and meta-analysis will be necessary to clarify the
protective and therapeutic role of COX-2 inhibitors in oncology.
Aim: to study the role of cyclooxygenase-2 (COX-2) in upper gastrointestinal tract
tumourigenesis, with emphasis on oesophageal and gastric adenocarcinoma,
premalignant lesions of the oesophagus (Barrett's oesophagus) and gastrointestinal
stromal tumours, as well as to study the relevant technological factors.
Tasks:
1. To identify the confounding technological factors in COX-2 IHC and to describe in
detail the influence of these factors on the results.
2. On the basis of experimental evidence by total test, to set up optimal protocol of the
immunohistochemical investigation of COX-2 protein expression in different tissues.
3. To determine the frequency, intensity and pattern of COX-2 protein expression in
oesophageal adenocarcinomas, Barrett's oesophagus and GIST.
4. To estimate the informativily of total test procedure and apply it on clinically
important set of primary antibodies.
Laboratory basis
The presented work was carried out in the Institutes of Pathology, P.Stradin's
Clinical university hospital and Aarhus University Hospital, Aarhus, Denmark.
Scientific and practical diagnostic novelty
1. The first comparative description of the results of COX-2 protein expression by 8
primary antibodies and 7 HIER protocols will provide reasonable background for any
future studies in COX field. The importance of the data is emphasized by the revealed
tremendous technology-caused differences in COX-2 expression rate and pattern,
concerning both malignant and benign tissues. The detailed pattern in dependence on
antibody and HIER has not been published thus the data have world-wide
significance.
2. COX-2 expression in GISTs is described, together with the description of
technology-related influences. As Medline search for such data does not provide
relevant information, the data are of world-wide significance.
3. COX-2 expression in Barrett's oesophagus is described.
4. The dependence of COX-2 protein expression levels on the age of archival tissue
blocks is described. As there is lack of semi-quantitative studies of IHC results as a
time function, the data will have world-wide significance.
5. Protocols for the visualisation of 48 antigens are adjusted for local tissue material
gaining improved accuracy and positive economic effect. The data cannot be obtained
from literature as are completely dependant on local peculiarities of fixation and other
technological variations. 29 antigens targeted by standardized IHC technologies were
approved by the Agency of Health Statistics and Medical Technologies of Latvia as
new medical technologies in Latvia.
Personal input
The author has performed the whole work on the technologic investigation of
COX-2 protein immunohistochemical expression, the COX-2 protein expression in
Barrett's oesophagus, oesophageal and gastric adenocarcinomas and GISTs, including
archive search, staining, evaluation and scoring, re-evaluation, data analysis. During
the setup of immunohistochemical technologies, 80% of the tested procedures and the
whole evaluation were carried out by the author. The help of qualified technician was
available during obtaining microtome sections and performing 20% of the
immunostains. The author was personally involved in the
preparing
data
for
evaluation
and
certification
of
immunohistochemical technologies and the Immunohistochemical laboratory.
the
Structure of the PhD Thesis
The PhD Thesis is in English. It has the classic structure, consisting of Introduction,
Literature review, Materials and Methods, Results, Discussion, Conclusions and List
of references, in total 2.11 pages. The Thesis includes 22 tables, thorough illustrative
material of 22 microphotographs and assembled microphotographs as well as
analytically illustrative material of 43 graphs. In total, 67 figures illustrate the findings
and conclusions. The list of references includes 312 sources.
MATERIALS
The following tissues were retrieved from the archives of the Institutes of
Pathology, P.Stradin's Clinical University Institute and University Hospital, Aarhus
University, Denmark.
For the technological investigation of COX-2 IHC, blocks containing formalinfixed, paraffin-embedded (FFPE) tumour tissue as well as the normal components of
the wall of gastrointestinal hollow organs were selected from 5 resected oesophageal
and gastric adenocarcinomas. Three cases of resected squamous cell cancer were
included in limited technological studies. Tissues of 8 GISTs and 8 tissue blocks,
representing normal full-thickness gastric wall were retrieved as well. COX-2
expression in malignant epithelial tumours was studied in 21 resected oesophageal
and gastric cancer (5 cases from the years 2002-2003; 16 - from 1982-1991). In order
to study COX-2 expression in Barrett's oesophagus, 668 relevant biopsies were
retrieved by SNOMED search using combined codes for intestinal metaplasia and
oesophageal mucosa through years 1981 -1991.
Appropriate tissues were retrieved in order to perform standardization of IHC as a
novel technology (48 cases), to determine the frequency of the necessity to use IHC
and to estimate the diagnostic value of IHC (36 cases).
The tissues were fixed in 10% neutral buffered formalin, sampled widely during
the grossing of operation material, processed and embedded in paraffin blocks
(Hopwood, 2002; Anderson and Bancroft, 2002).
Ethical concerns in obtaining tissue materials
The present work does not give rise to ethical problems. All the above mentioned
tissue materials have been removed for diagnostic or curative purposes. No additional
investigations were performed on patients since all analyses were carried out on
tissues previously removed for routine diagnostic purposes and which have been
retrieved from the archives of participating pathology institutes. No human
experimentation was involved.
METHODS
4-micromctrc-thick sections of the FFPE tissues were cut with automatic
microtome on Hislobond glass slides (Menzel Glasser, Germany) and stained with
haematoxylin-eosin (HE) for screening ((iambic and Wilson, 2002). BE and cardiac
intestinal metaplasia (C1M) were diagnosed as presence of goblet cells by Alcian
blue-PAS stain at pH 2.6. The location of metaplastic changes within the oesophagus
was determined by the presence of squamous epithelium adjacent to the intestinal
metaplasia (1M). The cardiac location of IM was identified by the morphology of the
cardiac mucosa, presenting with loose mucous glands and cystic changes in
occasional glands. The endoscopic records were correlated with the microscopy.
In order to study expression of COX-2 protein and to diagnose GISTs reliably as
well as to create background for wide spectrum of scientific and clinical
immunohistochemical studies, a new specialized branch of the Pathohistological
laboratory was developed in the Institute of Pathology, P.Stradin's Clinical University
Hospital within the frames of the presented work. The immunohistochemical
technologies were standardized and submitted to the Agency of Health Statistics and
Medical Technologies of Latvia (Veselības Statistikas un Medicīnas Tehnoloģiju
Aģentūra) where the technologies were approved and the Immunohistochemical
laboratory was certified. The frequency of use and diagnostic informativity of IHC
was additionally analysed in the model of chest wall and pleural tumours.
The standardization of IHC including the diagnostics of GISTs was carried out by
the total test, implying that all concerned primary antibodies (DakoCytomation,
Glostrup, Denmark) at full set of dilutions were tested upon different protocols of
antigen retrieval. Different incubation time was tested instead of dilution for N-series
of reagents (DakoCytomation). The bound primary antibody was detected by LSAB2
(DakoCytomation).
For the technological investigation of COX-2 IHC, the staining patterns and
intensity produced by 8 commercially available anti-COX-2 primary antibodies were
characterized on consecutive sections of the same tissue blocks, thus limiting the
differences to primary antibody and antigen retrieval. Using serial sections from the
same blocks, sampling, fixing, processing and biological differences were excluded.
The primary anti-COX-2 antibodies were: 1) monoclonal mouse antibody
(catalogue number 160112; Cayman Chemical, Ann Arbor, MI, USA), obtained by
immunisation with a synthetic peptide corresponding to amino acids 580-599 of
human COX-2; 2) affinity-purified polyclonal rabbit anti-murine antibody (cat.nr.
160126; Cayman Chemical), known to be reactive with human COX-2, obtained by
immunisation with a peptide corresponding to amino acids 584-598 of murine COX-2;
3) polyclonal rabbit antibody (cat. nr. PG 27b; Oxford Biomedical research, Oxford,
Michigan, USA), obtained by immunisation with a synthetic peptide
corresponding to a distinct C-terminal region of human COX-2; 4) affinity-purified
rabbit antibody (cat. nr. 18516; ImmunoBiological Laboratories (IBL) Co, LTD,
(Gunma, Japan), obtained by immunisation with a synthetic peptide corresponding to
a part of human COX-2; 5) monoclonal mouse antibody, clone 131114 (cat. nr. 1021
1; IBL), obtained by immunisation with a synthetic peptide corresponding to a part of
human COX-2; 6) monoclonal mouse antibody, clone 41112 (cat. code NCL-COX-2;
Novocastra Laboratories Ltd., Newcastle upon Tync, UK), obtained by immunisation
with a prokaryotic recombinant protein corresponding to amino acids 38-163 of the Nterminal domain of human COX-2; 7) polyclonal rabbit antibody (cat. nr. sc-7951;
Santa Cruz Biotechnology, Inc., Heidelberg, Germany), obtained by immunisation
with a recombinant protein corresponding to amino acids 50-111 mapping near the
amino terminus of human COX-2; and 8) affinity-purified goat antibody (cat. nr. sc1745; Santa Cruz Biotechnology), obtained by immunisation with a synthetic peptide
mapping at the carboxy terminus of human COX-2.
Four primary anti-COX-2 antibodies were additionally investigated in GIST model:
1) monoclonal mouse antibody, Cayman Chemical; 2) polyclonal rabbit antibody, cat.
nr. PG 27b; Oxford Biomedical research; 3) polyclonal rabbit antibody, cat. nr. sc7951; Santa Cruz Biotechnology; 4) affinity purified goat antibody, cat. nr. sc-1745;
Santa Cruz Biotechnology.
Two-micrometre-thick serial sections of the FFPE tissues were cut on
electrostatically charged glass slides SuperfrostRPlus (Menzel-Glasser, Germany) and
incubated in 60°C for 1 hour to ensure tissue adhesion to slides. Deparaffinisation and
rehydration was carried out by routine treatment in xylene and graded ethanol.
Endogenous peroxidase activity was blocked by immersing slides in 0.5% hydrogen
peroxide (Bie&Berntsen, Rodovre, Denmark) in methanol for 10 min. After rinse in
TBS for 5 min., slides were subjected to antigen retrieval.
In total test all primary antibodies at full set of dilutions were tested upon 6
protocols of heat-induced antigen retrieval (HIER); treatment in microwaves 3x5 min.
in basic (TEG, pH 9.0, Tris base l0mM/L, EGTA 0.5 mM/L, Bie&Berntsen) or acidic
(CIT, pH 6.0, tri-Sodium citrate dihydrate 5 mM/L, di-Sodiumhydrogencitrat
sesquihydrate 5mM/L, Bie&Berntsen) buffer, incubation in preheated TEG or CIT
buffer at 90°C-30 min. or at 60°C-14 hours. After HIER, the slides were allowed to
cool at room temperature (RT) for 20 min. in the HIER buffer. IHC staining without
HIER was also performed.
The slides were encircled with DakoCytomation pen (DakoCytomation A/S,
Glostrup, Denmark) and transferred to magnetic immunostaining trays (CellPath plc,
Newtown, UK). After rinse with TBS (pH 7.6, Tris buffered saline, THAM-HC1 50
mMl/L, NaCl 150mM/L) buffer for 5 min., the incubation with primary antibodies
was carried out at RT for 60 min. The following dilutions of primary antibodies were
tested: Cayman Mab 160112, 1:50, 1:100, 1:200, 1:400; Cayman AP-ab 160126,
1:100, 1:200, 1:500; PG 27b, 1:100, 1:200, 1:500, 1:1000; both IBL AP-ab 18516 and
Mab, clone 131114, 1:25 and 1:50; Novocastra Mab, clone 4H12, 1:50, 1:100, 1:200;
Santa Cruz Pab sc-7951, 1:50, 1:100, 1:200, 1:500; Santa Cruz AP-ab sc-1745, 1:100,
1:200, 1:500, 1:1000. DakoCytomation antibody diluent S0809 was used to dilute the
primary antibodies. After incubation, slides were washed 3x3min.with TBS buffer.
The relevant horse radish peroxidase-labelled polymer EnVision+ (K4001 antimouse, K4003 antirabbit; DakoCytomation) conjugated with secondary antibody
was applied for 30 min. in RT to detect bound mouse or rabbit antibodies. After rinse
with TBS (3x3min.), 3,3'-diaminobcnzidine (S3000, DakoCytomation) solution was
applied for 10 min. Slides were washed and counterstained in haematoxylin for 3 min.
After colour development in lap water, slides were coverslipped, using Aquatex
(Merck 1.08562, Merck, Darmstadt, Germany). For Santa Cruz AP-ab sc-1745 of goat
origin, following substitutions were made. The slides were preincubated with serum
(4 drops of concentrated goat serum, Vectaslain Elite ABC kit, Vector Laboratories,
Inc., Burlingame, CA, USA; diluted in 10 ml TBS buffer) for 5 min. immediately
before the application of the primary antibody. The detection of primary antibody was
carried out by secondary anti-goat antibody (3 drops of concentrated secondary
antibody and 3 drops of concentrated serum, Vectastain Elite ABC kit, Vector
Laboratories, Inc.; diluted in 10 ml TBS buffer) for 30 min. That was followed by
ABC complex (3 drops of reagent A and 3 drops of reagent B, Vectastain Elite ABC
kit, Vector Laboratories, Inc.; diluted in 10 ml TBS buffer) for another 30 min. The
washing, colour development, counterstaining and coverslipping steps were
performed as described before. Negative control slides were included in each run.
The COX-2 protein expression was evaluated semiquantitatively in 20 elements
(adenocarcinoma, undifferentiated areas of cancer, single-invasive cancer cells, signet
ring cancer cells, squamous epithelium, parietal, chief, foveolar cells, mucous glands,
IM, dysplastic cylindrical epithelium, plasma cells, macrophages, fibroblasts, erosions
or ulcers, nerves and neurons, smooth muscle, endothelium, background and cell
nuclei). Each of them was scored as negative, 0; weakly positive, 1; moderately
intensively staining, 2; intensively positive, 3; brightly reacting, 4. If the staining in
the element under concern was uneven within a slide, each field was scored separately
and its area was evaluated in percents. The resulting score was used to characterize
the staining of a morphologic structure element (cell type or tissue) from one case in a
whole slide under definite technologic conditions. The resulting score of each
morphologic element was calculated as sum of all products of score and percentage
per slide. Summary scores were used to characterize the staining of a particular
morphologic structure element under definite technologic conditions and were
calculated as sum of resulting scores for this cell type from all cases, stained by one
primary antibody at definite and the same dilution, implementing particular antigen
retrieval. Total cancer score was designed to characterize the IHC COX-2 expression
in cancer encountering all grades and was calculated as sum of scores characterizing
adenocarcinomatous complexes and undifferentiated areas or single invasive cells
under definite technologic conditions. The final cancer score was
calculated as the difference between total cancer score and background score, For
verification, the evaluation and scoring were performed twice.
The optimal visualisation protocol was performed on oesophageal and gastric
tumours as well as on biopsies containing RH and C1M. The evaluation was
performed in 5-degree scale as described above. In the biopsies from BE and C1M, 16
elements were scored independently: squamous epithelium, parietal, chief, foveolar
cells, mucous glands, intestinal metaplasia, dysplasia, plasmatic cells, macrophages,
fibroblasts, erosions or ulcers, nerves and neurons, smooth muscle, endothelium,
background and cell nuclei. The scale of real staining intensity was reflected in the
scores of IM, squamous epithelium, macrophages.
RESULTS
Practical results: setting the Immunohistochemical laboratory
During the first stage of the work, the Immunohistochemical laboratory was
organised in the Institute of Pathology, P.Stradin's Clinical University Hospital.
In the result of total test procedure, standardized concentrations and incubation time
for 48 primary antibodies were detected. This set of technologies is intended for the
following indications: 1) determination of the histogenesis of an anaplastic tumour,
allowing to define the tumour as belonging to carcinoma, lymphoma, sarcoma,
melanoma or glial tumour group; 2) differential diagnostics of soft tissue tumours, 3)
diagnostics and differential diagnostics of GISTs; 4) subtyping of lymphomas; 5)
analysis of the histogenesis and most probable site of origin in case of metastatic
tumour; 6) differentiation between primary and secondary tumours of central nervous
system; 7) discrimination between primary and secondary lung adenocarcinoma; 8)
subtyping of primary lung tumours; 9) diagnostics and differential diagnostics of
primary pleural tumours; 10) diagnostics of neuroendocrine tumours; 11) evaluation
of prognostic factors in breast cancer; 12) exact evaluation of the spread of malignant
tumour, including evaluation of the depth of invasion, microscopic extension to the
resection line, presence of micrometastases or microscopic residual disease; 13)
facilitation of the differential diagnostics between hyperplastic processes and cancer
of the prostate; 14) evaluation of renal biopsies in cases of diffuse renal diseases. The
standardization was carried out for FFPE tissues by the use of LSAB, ABC and
EnVision visualisation systems.
29 antigens targeted by standardized IHC technologies were approved by the
Agency of Health Statistics and Medical Technologies of Latvia as new medical
technologies in Latvia. The Immunohistochemical Laboratory with complete
technological set underwent certification as a branch of the Pathohistological
laboratory, Institute of Pathology, P. Stradin's Clinical University Hospital.
Informativity of the total test procedure
In order to reach the highest standard of IHC visualisation, the staining protocol
must be individualized in accordance to the local specifity. Carrying out the
standardization of IIIC visualisation of 48 antigens by total test, the optimal IHC
protocol for 44/48 antigens (91.6%) was found to be different from the guidelines. 31
primary antibodies were obtained as concentrated reagents. 1-or 24 of 31 antibodies
(77.4%), both the optimal dilution and incubation time was found to be different from
the guidelines. The optimal dilution was 1.25-10 times greater than the guidelines.
These results are of huge practical importance and provide the evidence of the
informativity of the total test procedure.
There was a trend to correlation between isotype of the primary antibody and
results of standardization. 17/18 (94.4%) of the monoclonal antibodies whose working
dilution was raised after total test procedure belonged to IgG class. In contrast, only
4/5 (80.0%) of monoclonal antibodies, whose working concentration was not
changed, belonged to IgG. The relative frequency of IgM in those groups was 5.6%
and 20%, respectively. The relative frequency of polyclonal antibodies also was
greater in the unchanged group: 2 of 7 (28.6%) vs. 4 of 22 (18.2%).
Economic effect of the total test
IHC is indispensable as it brings diagnostic and prognostic information that cannot
be obtained by other medical technologies. The technological variables are of the
utmost importance as they are directly related to the quality of visualisation and thus
to the obtained data. However, the economical side also has to be evaluated. The
expenses of IHC consist of the expenses for the primary antibodies, the visualisation
system, accessory reagents and (he work load of the medical staff. The primary
antibody is one of the most important and most expensive components.
Assuming that the price of 1 mL of concentrated antibody is P, the recommended
dilution 1: r, the standardized optimal dilution according to the results of the total test
is found to be 1: s and 1 slide necessitates 250 microlitres of the working dilution, the
expenses for primary antibody per slide are ER = P / (4 × r) if the recommended
dilutions are used and ES = P / (4 × s) if the standardized dilutions are used. The
economic effect per slide (EE) is:
EE = ES - ER → EE=P / (4 × r) - P / (4 × s) → EE=P × (s - r) / (4 × s × r)
The economic effect was obtained on 75.9%) of primary antibodies. It ranged from
4.67 LVS to 141.69 LVS per slide (mean 51.7 LVS per slide; median 42.77 LVS per
slide; standard deviation 39.28 LVS per slide). For all but 1 of the restandardized
antibodies (95.5%), the economic effect was equal to the residual expenses or
exceeded those, thus saving more than 50% of the IHC budget for primary antibodies.
Informativity of IHC
Informativity of IHC was screened in a model of 36 neoplasms of the pleura and
chest wall. In 10 cases, exact diagnosis was reached by HE. This group comprised 3
lipomas (intramuscular, subpleural, and subcutaneous), extra abdominal desmoid,
neurofibroma, 2 chondrosarcomas (grade II and IIB), fibrous dysplasia of rib,
osteoma and osteosarcoma. In 6 cases, definite diagnoses were suspected, but IHC
was necessary to prove the histogenesis of the tumour. Five cases presented as small
blue cell tumours of the chest wall. Despite examination of multiple tissue blocks, no
exact diagnosis was reached by HE. IHC determined the diagnoses. 2 tumours
represented primitive neuroectodermal tumour (PNET) of chest wall (Askin's
tumour). The remaining 3 neoplasms were secondary - non-Hodgkin's lymphoma,
small cell cancer, and recurrent corticomedullary thymoma. The general diagnoses of
the secondary neoplastic processes were made by 3 primary antibodies. In contrast,
diagnostics of PNET necessitated visualisation of 12 antigens: cytokeralin (CK),
vimentin, CD45, S-100, desmin, CD34, CD31, NSE, synaptophysin (Syn), kappa,
lambda, CD30. The tumour cells diffusely, strongly expressed Vim in the cytoplasm,
but were completely devoid of CK, CD45, CD30, kappa, lambda, desmin, CD34 and
CD31. Syn was moderately intensively expressed in the cytoplasm of a fraction of
tumour cells. S-100 was focally positive in the nuclei and cytoplasm of the tumour
cells. Expression of NSE in the malignant cells was diffuse, intense, cytoplasmic.
In 11 cases, solitary fibrous tumour of the pleura (SFPT) was suspected. However,
in all cases fibrous mesothelioma had to be excluded and 1 tumour had to be
differentiated from other high-grade sarcomas. In 4 cases, the diagnostics was
complicated as macroscopic origin from the pleura was not evident due to giant size
(28 cm in diameter) of the tumour, dissemination throughout the chest wall in
combination with giant intrapulmonary mass, and intrapulmonary location (2 cases).
Panel of anti- CD34, CD31, thrombomodulin, desmin, S-100, BCL-2, and
pancytokeratin resulted in diagnosis. All SFPTs were CD34 positive. The CD34
expression was cytoplasmic, intense (7/11 cases; 63.6%) or moderately intense
(27.4%). The CD34 reactivity was lower in tumours with higher amount of thick,
wavy collagen fibres and lower volume of tumour cell cytoplasm. The rate of BCL-2
expression was 8/11 (72.7%). BCL-2 reactivity pattern was cytoplasmic as well. The
tumours were invariably negative for CD31, thrombomodulin (TM) and CK. Small
focus (5% of cells) of desmin positivity was noted in 1/11 cases (9.1%). All the
tumours were negative for S-100 protein; however, occasional S-100-positive tumour
infiltrating macrophages were present in 1 case (9.1%).
In 4 cases, mesothelioma was suspected; however, metastatic cancer could not be
excluded by HE, mucicarmin and PAS stains. IHC confirmed the histogenesis. CK,
high molecular weight cytokeratin, epithelial membrane antigen, vimentin,
carcinoembryonic antigen, cytokeratin 5/6, TM, mesothelial antigen HMBE-1 and
CD34 were searched for. The diagnostically most useful positive stains were the IHC
of TM and CK5/6 that were positive in 4/4 cases. Both of them were remarkable for
the lack of background and clear-cut specific pattern after appropriate standardization.
The characteristic pattern for TM was thick membranous line, for CK 5/6 cytoplasmic reactivity. Both of them were expressed focally within a slide or case.
HMBE-1 was detected in 2/4 cases.
Only 30.3% (10/33) of the cases of primary chest wall and pleural tumours could
be reliably diagnosed on the grounds of HE alone. IHC was necessary to confirm the
diagnosis in 18.2% cases and mandatory in 51.5% (17/33) cases of primary pleural
and chest wall tumours, including all pleural neoplasms and tumours presenting with
small blue cell pattern.
Thus, the first stage of the work resulted in selling up laboratory with standardized
technologies and proved informativity.
The dependence of COX-2 protein expression on technological factors
The COX-2 protein expression was studied on consecutive sections from the same
tissue blocks thus excluding biological, fixation, processing differences. Highly
different patterns were found with different antibodies and protocols.
Monoclonal mouse antibody. Cayman Chemical (Cayman Mab)
Cayman Mab showed a characteristic granular cytoplasmic staining in the positive
cancer cells. This pattern was shift to diffuse cytoplasmic if no HIER was employed.
The nuclei were negative in protocols that yielded optimal staining of cancer cells.
Undifferentiated cancer was intensively positive. The highest total cancer score was
obtained after antigen retrieval in TEG in microwaves (MW). The retrieval in CIT
buffer always resulted in lower cancer score than HIER in TEG. MW treatment
yielded higher cancer scores than low temperature HIER. The background with
Cayman Mab was slight. The optimal protocol as reflected by the highest final cancer
score (table 1) included antigen retrieval in TEG MW and incubation in 1:100 diluted
primary antibody for lhr in RT.
Analysing COX-2 protein expression in squamous epithelium by Cayman Mab,
qualitatively different distribution patterns were observed with different HIER
modalities. After HIER in TEG MW moderately intense cytoplasmic staining was
observed in basal layer, and weak granular perinuclear - in prickle cell layer. In
reactive squamous epithelium adjacent to or overlying cancer weak to moderate
suprabasal reactivity predominated. If no HIER was applied, moderately intense
positivity was observed in prickle layer and parakeratosis, but basal layer was
negative. Thus, although squamous epithelium was reactive with Cayman Mab upon
all protocols, the results showed significant qualitative and quantitative differences.
Cayman Mab consistently brightly stained parietal cells. The pattern was diffuse
cytoplasmic with more intense intracytoplasmic large dots and segments of circles
that were situated paranuclearly or confluent with nuclear or cellular membrane and
probably represented canalicular system. The cytoplasm of chief cells was stained
upon cancer-effective protocols. Cayman Mab focally, moderately intensively stained
atrophic mucous glands. In foveolar cells, mucus and nuclei were negative, but weak
to moderate reactivity was present in the basal cytoplasm, with relatively highest
intensity after TEG MW. Sharply demarcated foci of supranuclear and subnuclear
cytoplasmic positivity in IM and dysplasia were restricted to HIER in MW.
Plasma cells were positive with Cayman Mab, most intensively after HIER in TEG
MW. Macrophages in lamina propria, submucosa and cancer stroma reacted
intensively. The highest scores were obtained with any kind of HIER in TEG (table
2). HIER in CIT was less effective but reactivity of macrophages was relatively less
influenced by the pH 1 of HIER buffer than the reactivity of plasma cells. Intensity of
COX-2 expression in fibroblasts in submucosa or desmoplastic stroma correlated
strongly with the reactivity in macrophages. Cayman Mab showed intense
cytoplasmic reactivity in fibroblast- and macrophage-like cells in erosions and ulcers.
The reactivity was so intense that it was preserved with less efficient antigen retrieval
or high dilution of primary antibody, when other elements are already negative. These
slides (TEG 90°C, 1:400) showed the "erosion only" pattern. The staining in smooth
muscle was of low intensity except no-retrieval technique that resulted in focal or
general moderate positivity. Tiny cytoplasmic granules were found in the cytoplasm
of smooth muscle fibres. Endothelium was mostly unreactive with Cayman Mab.
Neuronal bodies were slightly more intense as nerve fibres.
Cancer stain was more dependent on the amount of the absorbed energy that
plasma cell score and macrophage score was least sensitive to the temperature
regimen. All scores, their relations and staining pattern changed if no HIER is
applied, effect, that cannot be explained solely by higher background.
Polyclonal anti-COX-2, Oxford Biomedical research (PG 27b)
In adenocarcinoma, PG 27b showed unique "supranuclear cleaved dot" pattern compact or blending cluster of intensively stained grains at the apical side of the
nucleus. The reactivity was highly heterogeneous. In solid cancer, the granules were
small and evenly distributed in the cytoplasm. Partial membranous or apical intense or
moderate reactivity in cancer cells was case-dependant. In squamous cell cancer, the
few positive cells showed intense diffuse cytoplasmic reactivity. The most effective
antigen retrieval for this antibody was heating in TEG at 90°C for 30 min. (table 1).
Treatment in MW, as well as prolonged low temperature HIER at 60°C yielded less
cancer reactivity. CIT was significantly less effective than TEG. PG 27b showed
almost no background. The optimal protocol for PG 27b included HIER in TEG 90°C
and overnight incubation in diluted primary antibody (1:500) at 4°C.
In squamous epithelium, with overnight incubation in 1:500 at 4°C after HIER in
TEG 90°C, moderate granular cytoplasmic reactivity was noted in squamous
epithelium adjacently to cancer. Remote epithelium showed weak unspecific
positivity in the upper layers.
Occasional weak dot-like reactivity was observed in parietal cells. Occasional
diffuse reaction was observed in the basal part of gastric corpus glands: chief cell-rich
area. A combination of 2 staining patterns was observed in the foveolar epithelium
with PG 27b. The most common pattern was diffuse staining of foveolar mucus.
Another pattern was focal presence of supranuclear dots in foveolar epithelium. PG
27b did not react with glandular mucous epithelium. The characteristic supranuclear
dot-like reactivity was observed in the deep IM. No mucus reactivity in IM was
observed in contrast to foveolar epithelium. The positive dots in dysplastic surface
epithelium were blurred.
Thus, PG 27b showed markedly different epithelial patterns - the most
characteristic, supranuclear cleaved dot; apical or membranous in cancer cells; highly
heterogeneous intense cytoplasmic in squamous cell cancer; diffuse small cytoplasmic
granules in diffuse cancer; and diffuse, weak or moderate, probably unspecific
reactivity in foveolar but not colonic mucus.
The stromal reactivity with PG 27b was limited, of diffuse cytoplasmic type, when
present. Macrophages and fibroblasts in erosions were intensively positive (table 2).
Small subset of plasma cells showed cytoplasmic reactivity. No endothelial or nerve
reactivity with PG 27b was observed. The reactivity of smooth muscle did not exceed
the background.
Affinity purified anti-COX-2, IBL (IBL AP-ab)
The epithelial pattern of IBL AP-ab was diffuse cytoplasmic, consistently
combined by recognition of subset of mitoses. A larger subset of atypical mitoses was
stained although reactivity in normal-appearing mitoses also was found. IBL AP-ab
showed unique trait-the reactivity with mitotically active cells. The optimal protocol
included HIER in MW in TEG buffer, incubation with IBL AP-ab at dilution 1:25 for
60 min. at RT. The impact of the pH of HIER buffer was especially marked for this
antibody (table 1). Diffuse cancer reacted weaker than adenocarcinoma, signet cells
were negative. The cancer reactivity colocalised with Cayman Mab, but was weaker
and more influenced by pH of HIER buffer. IBL AP-ab showed variable, mainly
moderate levels of background in dependence on the mode of HIER.
After HIER in TEG MW, intact squamous epithelium showed weak paranuclear or
central cytoplasmic accentuation. In reactive areas, basal layer was weakly positive
but prickle cell layer showed moderate cytoplasmic positivity. Without HIER, only
reactive squamous epithelium showed weak cytoplasmic positivity in prickle layer
and upwards, but the basal layer and remote squamous epithelium was negative. This
difference was in accordance with weaker sensitivity as reflected also by lower total
cancer scores (TEG MW 17.8 vs. no HIER, 11.54). Although CIT MW resulted in
low total cancer score, the weak reactivity in squamous epithelium was partly
preserved.
Parietal cells showed moderately intense finely granular cytoplasmic reactivity that
became intense in some parietal cells in the upper part of glands. Occasional weak
reactivity in chief cells was found. The reactivity of IBL AP-ab with the foveolar
epithelium was variable between cases and protocols. After HIER in TEG MW, there
was a trend to weak or moderate basal cytoplasmic reactivity in the proliferative area,
with negative surface epithelium and mucus (4/4). After HIER in CIT, the reactivity
decreased to weak or disappeared. HIER in TEG 90°C resulted in greater variability
between cases. The variability decreased again with TEG 60°C and protocol without
HIER. Although low temperature HIER in TEG caused greater variability, the
positive results colocalised in all TEG protocols. The correlation was not preserved if
no HIER was applied: completely different cases became negative
and positive, respectively. All TEG-based HIER protocols, HIER in CIT MW and
staining without HIER resulted in focal cytoplasmic reactivity in the mucous glands.
IM showed a combination of focal cytoplasmic reactivity and brightly staining
subgroup of endocrine-like cells. The cytoplasmic reactivity was best evident after
HIER in TEG MW or staining without HIER but it was not present after CIT 60"C.
Dysplastic surface epithelium locally moderately intensively reacted after HIER in
TEG MW and staining without HIER. Endocrine-like cells were best seen if HIER
was performed in TEG 90"C or in TEG 60°C, and were less intense in TEG MW, CIT
90°C or CIT 60°C. This subpopulation was not evident after HIER in CIT MW and
staining without HIER. Thus, omitting HIER lead to qualitatively different pattern in
the aspect of IM: although the intensity of cytoplasmic reactivity was relatively high,
the subpopulation of brightly positive cells was not evident; result that is not
consistent with different sensitivity only.
IBL AP-ab reacted with plasma cells (table 2). The pattern was mostly perinuclear,
but cytoplasmic positivity occurred after TEG MW and in the highly reactive subset.
IBL AP-ab recognised macrophages and fibroblasts although the reactivity was less
intense as with Cayman Mab. HIER in TEG retrieved most reactivity. Macrophage
and fibroblast-like cells in erosions showed intense cytoplasmatic reactivity.
Endothelium was consistently weakly positive after TEG MW, consistently negative
after HIER with CIT MW. Without antigen retrieval, the reactivity was variable, from
no staining in 2/5 cases to moderately intense positivity in 2/5 cases. The reactivity of
smooth muscle was weak to moderate. Nerves were mildly or moderately reactive.
Monoclonal mouse anti-COX-2, IBL (IBL Mab)
The characteristic trait of IBL Mab was extremely low reactivity with epithelia. It
showed weak traits of cancer reactivity (scores between 0 and 1, weak) if HIER was
done in TEG MW or if staining was performed without HIER. Other protocols
yielded completely negative cancer scores (table 1).
In the squamous epithelium, only parakeratosis was weakly positive in resting
epithelium after no HIER or TEG MW. The upper prickle layer and parakeratosis in
reactive squamous epithelium were intensively positive, if no HIER was applied.
Without antigen retrieval, parietal cells were very weakly positive (3/3). This
reactivity disappeared after treatment in MW. The reactivity in chief cells was weakto-moderate, without clear-cut correlation with the type of antigen retrieval. IBL Mab
did not stain mucous glandular epithelium. Foveolar epithelium was mostly
unreactive as well except for 1/3 cases after HIER in CIT MW. In this slide, a subset
of endocrine-like small cells was highlighted in foveolar proliferative zone. The
pattern was granular cytoplasmic; nucleus in these cells was situated apically. IM and
dysplasia were consistently negative.
In contrast to the lack of definite reactivity in epithelia, intense reactivity was
present in a subset of plasma cells after HIER in TEG MW in all cases. After HIER
in TEG MW, bright reactivity in fibroblast-like cells was detected in small erosions
(table 2).
Macrophages and fibroblasts apart from erosions did not show reliable reactivity.
Endothelium and smooth muscle were mostly negative. Nerve cells and fibres showed
weak reactivity in 4/5 cases after 1 HER in TEG and in 3/5 cases if no antigen
retrieval was performed.
The general pattern for 1BL antibody included cancer negativity in combination
with bright reactivity of macrophage and fibroblast-like cells in erosions and a subset
of plasma cells.
Affinity-purified polyclonal antibody, Cayman Chemical, (Cayman AP-ab)
This antibody showed a combination of cytoplasmic, membranous and apical
reactivity in cancer cells. The most effective HIER for cancer testing was TEG 90°C
(table 1). Cayman AP-ab reacted with diffuse cancer although the reaction was
estimated weaker as with Cayman Mab. This may be due to background or lack of
recognisable pattern.
In the squamous epithelium, basal layer was weakly positive, and prickle cell layer
showed weak perinuclear and nuclear reaction after HIER in TEG MW. After HIER
in TEG 90°C, basal layer showed moderate cytoplasmic positivity, but prickle cells weak cytoplasmic reaction with slight perinuclear accentuation. Squamous epithelium
was negative after HIER in CIT MW, CIT 90°C, TEG or CIT 60°C. If no HIER was
applied, the basal layer was negative, but prickle cell layer in the areas of squamous
epithelium overlying cancer still showed weak granular cytoplasmic reactivity with
perinuclear accentuation.
Parietal cells were intensively reactive with Cayman AP-ab in all protocols. The
highest scores were reached with TEG MW and TEG 90°C. At higher dilutions, dots
on the background of paler positive or even negative cytoplasm became evident, the
pattern analogous to that of Cayman Mab. At higher dilutions, the gradient also
became evident: the staining was more intense in those parietal cells that were
situated in the deep part of gastric gland, the reverse as for IBL AP-ab. There was no
definite evidence of chief cell positivity with Cayman AP-ab.
After TEG MW, 2/3 cases showed weak staining in basal cytoplasm of foveolar
cells, but not mucus. After HIER in TEG 90°C the reactivity was higher reaching
moderate degree. There was a trend to reactivity in proliferative zone. Cayman AP-ab
did not react with mucous glandular epithelium. IM was positive after HIER in TEG
MW and 90°C. However, both nuclear and cytoplasmic reactivity was present,
suspicious for unspecific reaction. No reliable reactivity in dysplastic epithelium was
observed.
Most of plasma cell reactivity was obtained after TEG 90°C, closely followed by
TEG MW (table 2). TEG 60°C was significantly less effective; HIER in CIT yielded
very low results. Protocol without HIER gave results comparable to those after CIT
retrieval. The same case as with Cayman Mab and IBL was most intensively reactive.
The reactivity of macrophages, fibroblasts and cells in erosions was
intense
cytoplasmic. Again, using high dilutions (TEG 90°C 1:500), it was possible to
observe "erosion only" pattern. Consistent bright reactivity was observed in nerve
fibres and cells. The reactivity was highest after TEG 90"C, closely followed by TEG
MW. Retrieval in CIT was always less effective than in TEG. Muscular reactivity was
mostly weak with 1 HER. The endothelial reactivity was up to moderate, and thus
higher than with Cayman Mab, PG 27B, 1151. Ap-ab or 1BL Mab. It was the highest
at TEG 90°C and persistent weak at TEG MW and no HIER. None of the tested
protocols showed good cancer reactivity without endothelial staining.
Novocastra monoclonal antibody (NC Mab)
The reactivity pattern with NC Mab in cancer cells was diffuse cytoplasmic with
frequent nuclear reactivity. Small foci of Cayman Mab type reactivity pattern with
"supranuclear cobblestone" were noted. Diffuse cancer and signet ring cells were
mostly unreactive. NC Mab stained secretions in the lumina of cancerous complexes
in higher intensity than the cancer cells. This antibody followed the general rule of
greater sensitivity after HIER in TEG. Low temperature HIER was more effective
than treatment in MW (table 1). The antibody was almost unreactive if no HIER was
used. NC Mab showed high background scores after HIER in TEG 90°C and TEG
60°C. However, due to high cancer reactivity, the final cancer scores for these
protocols were high.
In squamous epithelium, after HIER in TEG 90°C or TEG 60°C there was intense
perinuclear reactivity in basal and prickle cell layer. In squamous epithelium
overlying cancer, the stain was intense cytoplasmic with negative borders, mainly
suprabasally and in prickle cell layer. CIT 90°C, CIT 60°C and TEG MW showed the
same pattern with weaker expression. CIT MW resulted in very weak reaction.
Without HIER, the squamous epithelium was negative as were most other structures.
Parietal cells were reactive with NC Mab upon all protocols. The highest - intense
reactivity was obtained with TEG 60°C. TEG and CIT 90°C resulted in moderate
intensity, TEG MW and CIT 60°C - in moderate to weak, CIT MW and the protocol
without HIER - in weak. The reactivity of parietal cells was preserved even in CIT
MW, CIT 60°C, and no HIER protocols, especially standing out in the background of
overall negativity in the last case. Chief cell showed marked reactivity with NC
antibody. It was intense after TEG in all regimens, less with CIT-based retrieval, but
absent, if no HIER was applied.
The general pattern of foveolar reactivity with NC Mab was basal cytoplasmic
staining in proliferative area. The surface epithelium was negative or weaker reactive.
Foveolar mucus was consistently negative. The staining was comparably intense and
consistent if HIER was performed in TEG (in any temperature regimen), CIT 90°C or
in CIT 60°C. The reactivity was estimated as moderate or intense, evaluating the basal
(subnuclear) cytoplasm. 2/3 cases showed weak reactivity if HIER was not applied or
was performed in CIT MW. In glands, mucus was unreactive. Focal moderate
cytoplasmic reaction was evident in basal cytoplasm, except for the protocols without
HIER or with HIER in CIT 60°C or CIT MW when the reaction
was weak, limited to small foci and 1/4 case. NC Mab stained 1M locally, most
intensively after HIER in TEG 90°C or 60°C, followed closely by TEG MW. TEG
90°C resulted in sharply demarcated foci of reactivity in dysplastic surface
epithelium.
Although plasma cells were positive upon some protocols, the reactivity was weak
to moderate (table 2). Relatively higher scores were gained with TEG 90°C, TEG
60°C, and TEG MW in decreasing order. HIER in CIT and protocols without HIER
gave negative results. Reactivity of macrophages and fibroblasts with NC Mab was
generally weak, reaching moderate degree only after HIER in TEG at 60°C. In sharp
contrast with the previously described antibodies, erosions were unreactive upon all
protocols.
TEG at 90°C 60°C resulted in consistent weak to moderate endothelial staining.
TEG MW resulted in consistent weak endothelial staining in 4/5 cases. The protocols
that have not been effective for cancer resulted in least endothelial stain - CIT MW,
CIT 60°C, CIT 90°C, and no HIER. NC Mab was at least weakly reactive with nerve
fibres and neurones after TEG MW. Neurones showed more intense reactivity. TEG
90°C and TEG 60°C resulted in the maximal reactivity for this antibody that was
moderate in average. The neuronal bodies were intensely reactive after TEG 90°C.
CIT MW and no HIER resulted in areactivity. CIT 60°C and CIT 90°C result in
consistent weak reaction in the nerve fibres. Muscle reactivity was moderate or higher
with TEG 90°C and TEG 60°C and moderate after TEG MW and CIT 90°C. CIT MW
and no HIER resulted in much less reactivity in conjunction with bad general
sensitivity. For NC Mab, cancer reactivity correlated with muscular. A trend towards
uneven muscular reactivity with higher scores at lamina muscularis mucosae, vascular
muscle, and muscle adjacent to invasive cancer complexes was noted.
Santa Cruz anti-COX-2 antibody sc-7951
The cancer reactivity was pale, diffuse cytoplasmic, highly heterogeneous. The lack
of recognisable pattern burdened the discrimination between true reactivity and
background. Most reactivity was retrieved by incubation in basic buffer at 90°C for
30 min. (table 1). Sc-7951 had generally low background scores, but low final cancer
scores due to low cancer reactivity.
Sc-7951 showed also different patterns in epithelia: in squamous epithelium, there
was cell border accentuation in prickle layer; small cuboidal cells were positive in
foveolar proliferative area; intercellular lines in dysplastic surface epithelium were
noted.
Squamous epithelium after HIER in TEG 90°C showed weak to moderate
membranous positivity in prickle cell layer and occasional weak perinuclear
accentuation. The basal layer was negative. Intraepithelial mononuclear cells (IEM)
were moderately cytoplasmically positive. The pattern was preserved in CIT 90°C.
TEG MW resulted in weak membranous and focal weak perinuclear reaction in
prickle cell layer. 1KM showed moderate cytoplasmic reactivity. Other protocols
resulted in either negative results or undefined pattern.
Intensity of staining in parietal cells was case-dependant. In the best-reactive case,
the pattern included moderate cytoplasmic reactivity, membranous accentuation and
occasional dot-and ring-like structures reminiscent of those seen with Cayman Mab
antibody. Although there were fewer differences among protocols than among cases,
TEG MW and TEG 90"C retrieved more reactivity.
Foveolar epithelium was mostly unreactive with sc-7951. In 1/4 cases a subset of
small cubical cells was identified in the foveolar proliferative area. The reactivity of
these cells was moderate after HIER in TEG 90°C, weak after HIER in TEG MW or
CIT 90°C, weak and limited to rare cells after HIER in C1T MW or staining without
HIER. HIER in 60°C did not highlight this subset of cells. Mucous glands were
mostly negative with sc-7951. In 2/3 cases, there was focal weak reactivity in IM:
cytoplasmic reactivity was observed in deeper parts of metaplastic glands that
contained also a subpopulation of brighter positive round cells. The reactivity of
dysplastic surface epithelium was limited to intercellular lines and paranuclear
vacuoles, possibly serum leaks.
Plasma cells were weakly or focally moderately positive with this antibody (table
2). TEG 90°C and TEG MW were the most effective. HIER in CIT or protocols
without HIER resulted in pale reactivity. Reactivity of macrophages and fibroblast did
not exceed moderate. No cytoplasmic positivity was observed in cells located
adjacently to erosions or subepithelially. Sc-7951 resulted in weak reactivity in neural
fibres almost independently of the mode of antigen retrieval. Smooth muscle mostly
stained weakly, endothelium showed inconsistent weak stain.
Santa Cruz affinity purified anti-COX-2 antibody sc-1745
Sc-1745 showed 2 staining patterns in cancer cells: trend to membranous
enhancement and diffuse cytoplasmic reactivity. The most efficient HIERs for sc1745 were TEG MW, TEG 90°C. Sc-1745 reacted with diffuse cancers relatively
weaker than Cayman Mab but had the highest reactivity with signet-ring cells. It
showed moderate background scores. The final cancer scores were high for HIER in
MW or 90°C (TEG more effective that CIT), and TEG 60°C (table 1).
In squamous epithelium, after HIER in TEG MW, moderate to intense cytoplasmic
reactivity with basal membrane accentuation was present in the basal layer. In CIT,
the pattern was preserved; possibly due to lower background, focal perinuclear
positivity was discernible. After TEG 90°C, in addition to intense positivity in the
basal layer weak to moderate paranuclear reactivity was also observed in the prickle
cell layer. The epithelium adjacent to cancer reacted less intensively than the remote
epithelium. After HIER in CIT 90°C and TEG 60°C, reactive squamous epithelium
became negative. CIT 60°C and protocol without HIER resulted in negative
squamous epithelium.
Parietal cells were weakly reactive with sc-1745 after TEG based HIER (MW, 2/2;
TEG 90°C, 2/2; TEG 60°C; 1/2) or without HIER. CIT-based retrieval resulted in
loss of this reactivity. Background was difficult to exclude. The most intense
positivity within the gastric corpus glands was situated around the lumen in the deep
part of gland. The localisation corresponded to the apical cytoplasm of chief cells.
Intense reactivity of such type was achieved by HIER in TEG 90°C and TEG 60°C,
moderate - by CIT 90°C. HIER in MW resulted in mostly weak reaction, but CIT
60"C and no HIER- in negative reaction. Intensively positive small round cells were
noted in normal gastric glands. A characteristic trait of sc-1745 was trend to diffuse,
total reactivity in foveolar epithelium. It was intense after HIER in TEG, moderate
with trend to proliferative zone after HIER in CIT. The foveolar epithelium was
mostly negative (2/3 cases) if HIER was not performed. IM was totally positive with
sc-1745 as well. The positivity was equally intense in the basal and upper part of
metaplastic glands (surface more intense as deep part after HIER in TEG, 60"C) and
extended to dysplastic surface epithelium. The intensity was highest after HIER in
TEG. A subpopulation of endocrine-like positive cells was evident also in the basal
part of metaplastic glands after HIER in 90°C.
Plasma cells were moderately reactive (table 2). Intense reactivity was reached in
1/5 by TEG MW; 3/5 TEG 90°C, 1/5 TEG 60°C. CIT-based HIER was less
effective. The highest results for an individual case were reached by TEG MW in
2/5 cases and TEG 90°C in 3/5 cases. Staining was cytoplasmic with occasional
Golgi complex or peripheral accentuation.
Mostly moderate reactivity of macrophages and fibroblasts and intense reactivity
in erosions was observed with this antibody. Neural reactivity was present. Sc-1745
showed high scores for smooth muscle in all protocols. Focal accentuation in lamina
muscularis mucosae and in muscle in ulcer bed was noted.
Table 1. Total cancer reactivity, background score and resulting cancer score
Primary
Antigen
retrieval
1
TEG
MW
CIT
MW
TEG
90°C
CIT
90 °C
Score
Cayman
Mab
2
PG
27b
IBL AP- IBL
Mab
ab
5
antibody
Cayman
AP-ab
NC
Mab
Sc7951
Sc-1745
3
4
6
7
8
9
10
TCR
19.2
6.7
17.8
0.7
13.75
15.1
5.77
14.70
BS
0
0
2.5
5
9.5
5.5
2
0
RCS
19.2
6.7
15.3
-4.3
4.25
9.6
3.77
14.7
TCR
17.1
1.81
4.23
0
2.58
2.35
1.32
10.91
BS
0
0
0.5
0
1
1
0.54
0
RCS
17.1
1.81
3.73
0
1.58
1.35
0.74
10.91
TCR
16.2
15.19
14.55
0
17.9
22.55
7.04
21.12
BS
2.5
0.75
1.5
0
11
9
2.56
3
RCS
13.7
14.44
13.05
0
6.9
13.55
4.48
18.12
TCR
15.6
6.45
4.31
0
6.25
12.40
4.40
13.14
BS
2.5
0
0
0
1.5
5
2.6
1
RCS
13.1
6.45
4.31
0
4.75
7.40
1.8
12.14
1
TEG
60°C
2
TCR
BS
RCS
TCR
3
17.2
2.5
14.7
10.4
4
5.51
0
5.51
2.51
5
17.20
4.5
12.7
1.05
6
0.1
0
0.1
0
CIT
60°C
BS
0
0
0
10.4
17.2
15
2.51
1.57
0
2.2
1,57
RCS
NO HIER TCR
BS
RCS
7
7
1
6
1.3
8
21.85
10
11.85
5.15
9
4.15
2
2.15
2.50
10
16.87
4.5
12.37
7
0
1
1
0
2
1.05
11.54
3.5
0
0.8
1
0.3
8.2
7
4.15
1.74
0
2.50
0.8
0
5
2.55
3.5
8.04
-0.2
1.2
1.74
0.8
-0.95
Sc7951
Sc1745
Table 2. Stromal COX-2 reactivity expressed as summary scores
Cell/
Antigen
structu
retrieval
re
1
2
Plasma TEG MW
cells
CIT MW
TEG 90°C
CIT 90°C
TEG 60°C
CIT 60°C
No HIER
Smoot TEG MW
h
muscle CIT MW
TEG 90°C
CIT 90°C
TEG 60°C
CIT 60°C
No HIER
Endoth TEG MW
elium
CIT MW
TEG 90°C
CIT 90°C
TEG 60°C
CIT 60°C
No HIER
Nerves TEG MW
CIT MW
TEG 90°C
CIT 90°C
Caym PG
an
Mab 27b
3
10.8
2.5
6.25
1.7
5
0
3.3
4.2
3.3
2.5
3.3
1.7
0.8
5.8
1.7
0
1.7
0
0
0
2
10
5.4
6.7
8.3
4
0
0
0
0
0
0
0
0
0
0.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Primar antibody
y
IBL
Caym NC
IBL
AP-ab Mab an AP Mab
5
6
7
8
9
10
4.5
0.5
4.5
1.5
8.5
2
6
6.5
3
5.75
4.25
6.5
3.75
8.5
5
0
5.5
2.5
7.5
0.5
5
6.75
4.25
7.00
5.00
15.5
4.5
1
0
6.5
0
8
1
0
0
0
0
0
4
1
0
0
0
0
0
1
3.5
0
0
0
10.2
2.75
13.5
2.5
5
0.75
3.25
3.5
1
4.5
1
1
0
7.5
5.5
1
9.5
1.5
3.5
0
5.5
12
7.5
13
9
5
0
8
1
7
0
0
9
1
11.7
9.75
13
6.5
1
3.5
0
8
3
8.5
0.5
0.5
6.5
1.75
8.5
6.5
5.5
1
6
2.5
5.25
0
0.5
1.5
1.5
5.5
6
0.5
0
1.5
2
0
4.5
1
2.5
0.5
0
4
3
4.5
4.75
7.25
2.5
8
2.5
4
0.5
1.75
10.7
6.5
13.5
7
13.7
10.2
7
7
2
8.5
5
8.5
0
4
7.5
5.6
9.75
9.1
1
2
Nerves TEG 60°C
CIT 60°C
No HIER
TEG MW
Macro CIT MW
phages TEG 90°C
CIT 90°C
TEG 60°C
CIT 60°C
No HIER
TEG MW
Fibrob CIT MW
lasts
TEG 90°C
CIT 90°C
TEG 60°C
CIT 60°C
No HIER
Erosio TEG MW
ns
CIT MW
TEG 90°C
CIT 90°C
TEG 60°C
CIT 60°C
No HIER
3
6.7
5
8.3
14.2
10.8
14.6
11.2
15
5
10
14.2
9.2
15
10.8
15
5.8
10
15
15
15
15
15
15
15
4
0
0
0
1.5
1
1
0
0
0
0
0.5
0
0
0
0
0
0
13.3
7.5
17.5
5
12.5
0
6.25
5
10.0
4.50
6.5
10.5
5.5
9.5
4.5
13
4.5
5.5
11
4.5
9.5
4.5
13
4.5
7
16.7
11.7
9.17
6.25
17.5
2.5
12.5
6
0
0
2.5
2
0
1
0
0.25
0
0.5
1.5
0
0
0
0
0
0
17.5
5
6.25
2.19
15
3.75
6.9
7
9.5
7.5
8.5
10
3
12
4
7.5
2
5.5
8.5
3.5
12.5
4
7.5
1.5
6
14.4
3.75
16.3
8.75
10
2.5
3.75
8
8.5
5
1
3.5
0
6.5
2
8
0
0
4
0
4
2
8.5
0
0
0
0
0
0
0
0
0
9
3
0
1
6
3
8
1.75
3.5
0
0
4.5
3
7
1
0.5
0
0
0
0
0
0
0
0
0
10
5.5
4.50
5
6.5
2.5
9
5
7.5
5
3
5.5
2
6.5
5
6
1
3
3.4
10
17.5
10
18.7
6.25
4.4
Abbreviations: Cayman Mab, monoclonal mouse antibody (catalogue number
160112; Cayman Chemical, Ann Arbor, MI, USA); PG 27b, polyclonal rabbit
antibody (cat. nr. PG 27b; Oxford Biomedical research, Oxford, Michigan, USA; IBL
AP-ab, affinity-purified rabbit antibody (cat. nr. 18516; ImmunoBiological
Laboratories (IBL) Co, LTD, Gunma, Japan); IBL Mab, monoclonal mouse antibody,
clone 13H14 (cat. nr. 10211; IBL); Cayman AP-ab, affinity-purified polyclonal rabbit
anti-murine antibody (cat.nr. 160126; Cayman Chemical), known to be reactive with
human COX-2; NC Mab, monoclonal mouse antibody, clone 4H12 (cat. code NCLCOX-2; Novocastra Laboratories Ltd., Newcastle upon Tyne, UK); sc-7951,
polyclonal rabbit antibody (cat. nr. sc-7951; Santa Cruz Biotechnology, Inc,
Heidelberg, Germany); sc-1745, affinity-purified goat antibody (cat. nr. sc-1745;
Santa Cruz Biotechnology); TEG, antigen retrieval in basic TEG buffer, pH 9.0; CIT,
antigen retrieval in citrate buffer, pH 6.0; MW, antigen retrieval performed in
microwave oven 3x5 min.; 90°C, antigen retrieval performed by incubation in
preheated HIER buffer at 90°C for 30 min.; 60°C, antigen retrieval performed by
incubation in preheated HIER buffer at 60°C for 14 hours; no HIER, staining
performed without antigen retrieval.
The quantitative estimates of COX-2 expression on the same cases implying no
biological variations among the material tested with each antibody differ largely. The
final cancer scores vary from positive to negative values the last meaning that cancer
reactivity is less than background even in the cancer-optimal protocol. The
quantitative differences are accompanied with qualitative differences. The staining of
benign tissues also is dependant on the primary antibodies. There is no correlation
between intensity of reactivity in those structures and cancer. The qualitative
characteristics of the staining in benign tissues also differ in dependence from the
primary antibody. Thus, the differences cannot be explained by different sensitivity
only.
The outcome of staining was influenced by HIER in comparison to no retrieval
technique, by pH of HIER buffer and heating regimen during HIER. The dominant
influencing factor for each cell type or morphologic structure by each primary
antibody was identified. For Cayman MAb, PG 27b, sc-7951, sc-1745, either
temperature regimen during HIER or pH of HIER buffer can be more important in
accordance to the cell type (table). IBL AP-ab and IBL Mab are mostly influenced by
pH. NC Mab is more influenced by pH, Cayman AP-ab by temperature. All scores
are influenced by 2 groups of HIER factors, each predominating in accordance to the
primary antibody. The variability of the most important HIER factor, as evident
between antibodies and scores of cell type or morphologic structure, suggest no
homogeneity of epitopes.
GISTs: diagnostics and COX-2 expression as biological and technical variable
For diagnostics of GIST, IHC panel consisting of antibodies against CD117, CD34,
CD31, desmin, S-100 and cytokeratins AE1/AE3 was standardized by total test
within the first stage. The optimal protocol included HIER in MW for 3x5 min. at
maximum power in TEG buffer, pH 9.0, followed by 20 min. cooling in the same
HIER buffer at RT. The incubation time was 60 min. at room temperature for CD117,
desmin, S-100, Ki-67, cytokeratin AE1/AE3 and 30 min. for CD34 and CD31. The
following dilutions were used: CD 117, 1:400; CD34, 1:1; CD31, 1:1; desmin, 1:200;
S-100, 1:4000; Ki-67, 1:100; CK AE1/AE3, 1:200.
Six of the IHC-confirmed GISTs were of spindle cell type, while 2 were epitheloid.
Fascicular architecture, palisades, micro cystic degeneration and vacuole-like micro
cysts were consistently present. One of the tumours showed weak diffuse positivity of
desmin, cytokeratins AE1/AE3, S-100 that disappeared after biotin blocking or switch
in visualisation systems from LSAB2 to EnVision TM+ (DakoCytomation). The
positive interpretation of CD117 and CD34 was unequivocal due to widespread
cytoplasmic (8/8) positivity in tumour cells and lack of background. Heterogeneous
expression of desmin, S-100, CD31 and cytokeratin AE1/AE3 due to entrapped
benign tissue was observed. Five of eight GISTs had entrapped desmin-positive
benign smooth muscle - the muscular layer of vascular wall (4/8) and/or fascicles of
the lamina muscularis propria (3/8). The presence of
heterologous S-100 positive elements was found in 5/8 cases or 62.5%: tumourinfiltrating activated macrophages (4/8) or entrapped nerve fibres (4/8). Endothelial
expression of CD31 was consistent. In all cases, the described elements could be
recognised by the morphological structure, sharp contrast with tumour cells, and
generally smooth outline, if tissue structure was concerned.
The tissues from morphologically intact areas of the gastric wall were consistently
positive for all of the investigated antigens. However, the reactivity in all cases by all
antibodies was limited to specific cells or tissue structures. GDI 17 was expressed in
lamina propria and submucosal mast cells (8/8). The predominant pattern of mast cell
reactivity was strong membranous, or weak cytoplasmic with strong membranous
accentuation, rarely cytoplasmic. The Cajal cells in intermuscular nerve plexus also
were invariably CD117 positive but few. CD34 reactivity was consistent in the
endothelium of blood vessels and lymphatics, perivascular and perineural fibroblasts.
CD31 stained the endothelial lining of blood vessels. S-100 was strongly positive in
activated macrophages and nerve fibres (8/8). Desmin was expressed in lamina
muscularis propria, lamina muscularis mucosae and muscular layer of the vascular
wall. In 5/8 cases splitting of lamina muscularis mucosae was present, leading to
disorganised desmin-positive fascicles of smooth muscle within the mucosa. Ki-67
nuclear expression was invariably present in the proliferative zone of epithelium,
lymphoid follicles and scattered stromal cells. Cytokeratin AE1/AE3 was expressed in
surface and glandular epithelium.
Six GISTs were selected for technological and biomedical investigation of COX-2
protein. Four of the tumours were of spindle cell type, while 2 were epitheloid. The
results were highly dependant on the primary antibody. The summary tumour score
was highest for sc-1745, reaching 19.7. The summary tumour score for Cayman Mab
was high as well, reaching 14.3. In contrast, the summary tumour score for sc-7951
was 3.6, and for PG 27b - 1.2. The data were in parallel with the number of tumours
showing at least moderate (grade 2) general reactivity: 6/6 for sc-1745 and 5/6 for
Cayman Mab, but none if tested by other 2 primary anti-COX-2 antibodies. The total
background score was 1.0 for the Cayman Mab, 0 for PG 27b, 2.0 for sc-7951 and 1.0
for sc-1745. Cayman Mab showed the highest final tumour score, followed by sc1745. Thus, Cayman Mab has the best specifity characteristics. The calculations are in
accordance with the observation that Cayman Mab showed the most consistent
staining pattern with large cytoplasmic granules and invariably negative nuclei. The
general quality of PG 27b IHC was superior as reflected by characteristic
supranuclear dot pattern in colonic epithelium in control slides. However, the staining
in GIST cells was focal, weak, finely granular cytoplasmic. Sc-7951 had a trend to
general background due to consistent intense unspecific cross-reactivity with a plasma
component. The reactivity was not abolished by qualitative peroxidase block and was
not present in red blood cells. The myxoid areas and microcysts showed the highest
level of background. The reactivity in the tumour cells was finely granular
cytoplasmic. Sc-1745 had a trend to general background as well. The reactivity
pattern was diffuse cytoplasmic.
There was no correlation between the location of tumour, predominant pattern
(epitheloid versus spindle cell), extent of oedema and specific COX-2 reactivity with
any of anti-COX-2. The reactivity in the mitotically most active areas was not
different from inactive ones. Palisades did not differ in staining intensity or pattern.
There was no correlation and no colocalisation between the expression of COX-2 and
CD117, CD34, desmin, S-100 or cytokeratin.
Immunohistochemical expression of COX-2 protein in Barrett's oesophagus
198 blocks containing 668 biopsies coded as oesophageal were retrieved and
analysed. After Alcian blue-PAS stain, 80 blocks containing 309 biopsies of BE,
defined as IM adjacent to squamous epithelium, were identified. Another 27 blocks
corresponding to 103 biopsies contained CIM. In total, 412 biopsies representing
oesophageal or cardiac IM were identified.
Analysing the informativity of Alcian blue-PAS, pH 2.6 stain (AB-PAS), the
following data were obtained. The AB-PAS prevented hyperdiagnostics of IM in
11/482 or 2.3% of biopsies and hypodiagnostics in 44/482 or 9.1% of biopsies. In HEstained slides, magnification 40 times was necessary to find and identify (within the
frames of method informativity) goblet cells, the mainstay of the diagnosis of
intestinal metaplasia, in 8.7% of cases, magnification 100 times - in 65.4%, 400 times
- in 25.9% of cases. In contrast, in AB-PAS stained slides, goblet cells were
definitively identified and differentiated under magnification 40 times in 65.4% of
cases, under magnification 100 times in 34.6% of cases. The mean time for
diagnosing IM on HE stained slides was 47.5 sec (median 39 sec, mode 35 sec, range
183 sec: 14 sec. - 197 sec, standard deviation 29.1 sec, standard error 2.6 sec). The
mean time for diagnosing IM in AB-PAS stained serial sections of the same tissue
blocks was 24.8 sec. (median 22 sec, mode 23 sec, range 108 sec: 4 sec. - 112 sec,
standard deviation 14.1 sec, standard error 1.3 sec).
COX-2 IHC was performed on 102 blocks of BE and CIM. In 25 cases (24.5%), no
COX-2 expression in IM metaplasia was found. In the remaining 75.5% of intestinal
metaplasia cases, focal granular cytoplasmic COX-2 protein expression was observed,
mostly in the deepest proliferating metaplastic epithelium. The positive scores below
1 were observed in 20 cases (23.5%), between 1 and 2 in 43 cases (50.6%o), over 2 in
22 cases (259%). No nuclear reactivity was observed. There was trend to diminishing
values in the biopsies from the earlier years, and statistically significant
(p=0.039<0.05) difference between frequency of COX-2 completely negative IM
cases in the time periods 1981-1987 and 1988-91, respectively.
In squamous epithelium, focal specific granular cytoplasmic or paranuclear
reactivity was observed in the basal layer. Peculiar unspecific immunoreactivity in the
upper layers of the squamous epithelium coinciding with PAS-negative foci of
swollen cells was frequent.
Plasma cells were definitely identified in 55 immunoslides. In 21 case (38.2%)
plasma cells showed focal weak (gradel) membranous reactivity. In 5 cases (9.1%)
plasma cells showed consistent weak reactivity, in 2 cases (3.6%) - moderately
intense reactivity. Membranous type of expression was consistent. Plasma cells were
negative in 27 cases or 49.1%. The mean reactivity of plasma cell was 0.35 in blocks
from 1981 - 1991 but 2.16 in blocks from 2002 - 2003.
The macrophage and fibroblast reactivity in the erosions and ulcers was only
partially present in comparison with recent blocks. 21 erosions were identified in the
study group blocks from 1981 - 1991. 4 (19.0%) erosions were COX-2 negative, 6
showed COX-2 expression in rare cells only. COX-2 reactive fibroblasts were found
in 5 erosions, being the dominant COX-2 positive cell type in 1 case. The intensity of
fibroblast reactivity was 3 in 4/5 and 4 in 1/5 cases. Cytoplasmic reactivity was
observed in fibroblasts. Macrophages were reactive in 16 cases, in 4 of those together
with fibroblasts. In 6 cases only rare COX-2 expressing macrophages were observed.
COX-2 expression in macrophages was cytoplasmic in 15, membranous in 1 case.
The intensity was of grade 1 in 2 cases, grade 2 in 6 cases, grade 3 in 7 erosions and
grade 4 in 1 case. In contrast, in recent blocks (2002 - 2003) consistent intense
reactivity of macrophages and fibroblasts was demonstrated.
COX-2 expressing macrophages in lamina propria were identified with certainty in
79 cases. In 58 cases the COX-2 expression was membranous, in 9 cases cytoplasmic
and in 12 cases subpopulations showing membranous and cytoplasmic type of
expression were identified.
The decrease of COX-2 protein expression in macrophages along with the aging of
blocks within the period 1981-1991 and over longer time period was found to be
linear function of time. The changes of the COX-2 scores in IM and squamous
epithelium in relation to the aging of blocks were demonstrable. The weak trend
towards correlation of COX-2 expression in different cell types can be attributed to
similar influence of the aging changes.
Endothelial reactivity was analysed in distended capillaries that were identified
with certainty in the lamina propria or submucosa. Endothelium was mostly negative.
Among 48 cases, focal weak endothelial reactivity was observed in 2 cases (4.2%).
The smooth muscle fibres of lamina muscularis mucosae or within lamina propria
were included in 30 immunoassayed cases. Among these, smooth muscle fibres were
negative in 5 cases (16.7 %), but showed focal weak reactivity in 21 case (70 %),
consistent weak reactivity in 4 cases (13.3 %). The characteristic reactivity was in the
form of tiny cytoplasmic granules.
Immunohistochemical expression of COX-2 protein in oesophageal and gastric
carcinoma
In blocks from 1981-1990, the reactivity of adenocarcinomas, grade 2-3, was
highly uneven. Although the intensity of COX-2 immunopositivity in a single
positive cell was not less than moderate, the percentage of reactive cells was
estimated to be from 10% to 100%. The total cancer score ranged from 0.2 to 3
degrees (mean cancer score, 0.9 degrees). The average reactivity of grade 3
adenocarcinomas was 1.44 degrees. No COX-2 expression was found in signet cell
cancers. The average reactivity of high grade dysplasia was 2.5 degrees. In blocks
from 2002-2003, the same protocol of 1HC resulted in mean cancer score 3.84 and
undifferentiated cancer score 2.13. The main difference between the immunostained
sections of older and new blocks was the distribution of reactivity. Assuming that loss
of immunoreactivity due to aging is not dependant on the grade of cancer, the COX-2
expression was found to be highest in the early stages of malignant process and lower
in high-grade cancers.
DISCUSSION
Technology of immunohistochemistry
IHC is a sensitive and specific method that allows determining the presence or
absence of a particular molecule in a specific cell or tissue structure that can be
identified visually. However, as the technology is aimed at recognition of a specific
structure by such a sensitive process as the epitope-antibody interaction is, tissue
fixation, processing (Werner et al., 2000) and other stages of the routine laboratory
work largely influence it. Most of these phases are not unified yet; therefore
standardization has become the mainstay of immunohistochemical investigation in
research and diagnostics (O'Leary, 2001; Pileri et al., 1997). The application of each
reagent necessitates the use of total test procedure, testing each antibody at several
concentrations and several incubation times. If other technological variations are
possible, each of those must be checked for all other variables. Although it might
seem time consuming, the procedure can reveal major technology-based differences in
results (Chan, 1998).
By total test an optimal protocol for each from 48 antigen was found that results in
optimal signal-to-noise ratio finally leading to specific procedure, cost-effectiveness
and easily analysable results thus even saving time. IHC technologies for visualisation
of 29 antigens were approved by the Agency of Health Statistics and Medical
Technologies as novel medical technologies in Latvia. Thus, total test is scientifically
and experimentally justified way to start a new technology.
The optimal staining conditions were found to be different from the recommended
for 91.6% of antigens. For 75.9 % of concentrated antibodies, the optimal dilution was
found to be 1.25 - 10 times greater than recommended, due to more effective antigen
retrieval in basic buffer and slightly increased incubation time. The increased
dilutions of the primary antibody resulted in qualitatively better visualisation at lower
regular expenses. The economic effect was 4.67 - 141.69 LVS per slide (mean 51.7
LVS per slide; standard deviation 39.28 LVS per slide). For all but 1 antibodies, the
economic effect was at least equal to the residual expenses or even exceeds those, thus
saving more than 50% of the IHC budget for primary antibodies. Indeed, the total test
results in a rare, still scientifically justified combination of higher quality and higher
cost-effectiveness and provides possibility to introduce up-to-dated economic thinking
into the best traditions of evidence-based medicine.
Informativity of immunohistochemistry
A number of studies have substantiated the informativity of IHC (Taylor, 2000). In
an early study of more than 100 anaplastic tumours, the initial, He-based diagnosis
of carcinoma or lymphoma was revised in approximately 50% of cases following
basic 1HC. In a study of 200 consecutive cases, it was concluded that IHC contributed
to the diagnosis in almost 50% and was confirmatory in the remainder. In analysis of
557 poorly differentiated round cell or spindle cell tumours that could not be
diagnosed on the basis of HE, IHC provided a definitive diagnosis in 70% of the
former and 92% of the latter (Taylor, 2000). Our data are in agreement with the
conclusion that IHC is necessary and provides irreplaceable information. 1HC was
necessary to confirm the diagnosis in 18.2% cases and mandatory in 51.5% (17/33)
cases of primary pleural and chest wall tumours, including all pleural neoplasms and
tumours presenting with small blue cell pattern. Thus, randomly selected field of
pathology showed an important role of IHC as a general diagnostic technology.
The most common modus operandi is to select stains from a panel that have proven
to be of value in a particular diagnostic area. The other approach would be to use an
algorithm with sequential panels of selected stains that introduces logic into the
process but may compromise the turnaround time (Taylor, 2000). The second
approach is tested in the Immunohistological laboratory, P.Stradin's Clinical
university hospital at 2001-2002. The prerequisite for its functional behaviour in
terms of reasonable turnover time is the presence of highly motivated staff that is able
to perform stain and evaluation of specimens, proceeding immediately to the next
step. Such a degree of motivation seems to be dependant either on personal idealism
or high salary. The first is a qualitative factor that is not liable to guaranteed everyday
use. The last is not possible in the present economic situation and seems reasonable in
large laboratory with general 24-hour-working process as the logical input making
diagnostic process more scientific must withstand large economic impact in
combination with questionable gain in diagnostic accuracy.
There is an important ongoing shift in emphasis in the use of IHC from a primary
focus on cell and tissue markers as an aid to the recognition and classification of
tumours to the demonstration of cell products, receptors or oncogenes of possible
prognostic value. The precision required for this latter approach places new and more
exacting demands on our ability to perform IHC in a reproducible manner (Taylor,
2000).
Immunohistochemical diagnostics of GIST
The immunohistochemical analysis of gastric wall by the antibodies, included in
the diagnostic panel of GISTs, revealed consistently positive normal cells and tissues.
Awareness of these elements and their immunophenotype is mandatory in order to
avoid false positive diagnosis of a mesenchymal neoplasm. On the basis of these
results, a list of endogenous positive controls for GIST IHC can be recommended,
namely, mast cells for CD117, endothelium for CD31 and CD34, smooth muscle for
desmin, macrophages or nerves for S-100. Sampling tumour tissue together with the
overlying mucosa or adjacent tissue can be recommended on the basis of the
presented results. The presence of endogenous controls will exclude the necessity to
use positive control slide with beneficial economic consequences.
According to the literature, desmin expression in true GISTs is uncommon (1-2%
of cases) and limited to small foci (Fletcher el al., 2002). The obtained data are
analogous, no true desmin reactivity was found in the cells of the analysed GISTs.
However, 2 possible sources of diagnostic difficulties were found. The first is related
to the presence of endogenous biotin, which was found in 1/8 of the analysed
tumours. The clue to the identification of that problem is presence of diffuse weak
reactivity with most of antibodies and disappearance of that reactivity with either
biotin blocking or switch to biotin-prone visualisation technologies such as polymeric
system EnVision +. Another possible source of overestimation of desmin expression
is wrong identification of entrapped normal smooth muscle that can be part of
vascular wall or lamina muscularis propria. It has to be noted that vascular wall can
be partially deformed or destroyed by the invading tumour. However, even in this
case, the benign nuclear morphology that contrast with tumour cells, and the sharp
contrast between tumour cells and the fascicles of smooth muscle allow the correct
identification. Although lamina muscularis mucosae could be entrapped in a tumour
that invades mucosa, this was not observed, possibly because of the small volume of
this tissue structure.
Around 5% of GISTs express S-100 protein. Again, high endogenous biotin
content or wrong identification of the positive elements can result in false positive
estimation. The most common S-100 positive elements within a GIST were nerve
fibres and activated macrophages. It has to be underlined that careful standardization
is of utmost importance for S-100 in order to reach the described specifity. Presence
of CD31 in endothelial cells is a consistent phenomenon. If the reactivity is not
present in the tumour cells, there is no proof of vascular neoplasm. Similarly, CD34 is
invariably expressed in endothelium, perivascular fibroblasts and perineural
fibroblast-type cells. Such reactivity does not suggest the diagnosis of GIST.
Finally, as shown by our results and literature data, CD117 is expressed not only in
other neoplasms, but also in normal tissue elements. Mast cells and Cajal cells are
typically positive in the wall of gastrointestinal organs. Such reactivity, limited to
normal cells and structures, must not be interpreted as an evidence of GIST. It is
recommended to pay attention to the location of positive reaction and careful
identification of any positive tissue structure as already suggested by Seidal et al.,
2001. The immunohistochemical reaction must not be characterized by the bare terms
"positive" or "negative". Instead, the specific location of the concerned reactivity must
be recorded and analysed. If this rule is observed, IHC is highly reliable tool in GIST
diagnosis.
Expression of cyclooxygenasc-2 protein
COX-2 has become an object of intense investigations as it may be involved in
important steps of carcinogenesis, may be a target of cancer prevention or treatment,
or a biomarker of cancer risk. In order to obtain analysable data, standardized and
comparable technologies are necessary. COX-2 might be up-regulated in different
types of cells; therefore IHC has a strong preference over methods that require
analysis of homogenized whole tissue as it demonstrates clearly the cells and
structures responsible for COX-2 over expression. The presented data also justify
independent COX-2 expression in different tissues, as metaplastic intestinal-type
epithelium, squamous epithelium and macrophages. This emphasizes the necessity to
identify the COX-2 expressing cell populations in each case.
The main problem in COX-2 IHC has been the huge qualitative and quantitative
differences in the published data. Although methodological diversity has been
suspected as a cause of widely divergent results, a limited number of studies have
been devoted to this question (Garewal et al., 2003; Shi et al., 2002; Saukkonen et al.,
2001). In the present work, the staining patterns and intensity of 8 primary anti-COX2 antibodies upon 7 protocols of antigen retrieval were analysed in accordance to total
test principle. The tests were performed on serial sections of the same 5 cases. Thus
variables as different fixation, processing, selection bias were excluded.
The reactivity of cancers was strongly dependent on the primary antibody. The
choice of antigen retrieval influenced the sensitivity. Although this may be partially
compensated by different scoring systems and cut-off values, this might also change
the percentage of estimated-as-positive cases in qualitative studies and significantly
influence the analysis of COX-2 expression at levels that are close to the sensitivity
threshold.
The optimal HIER that provided highest cancer reactivity with less background
was different for each antibody: microwave treatment in TEG for Cayman Mab, IBL
AP-ab and Mab. HIER at 90°C - 30 min. in TEG ensured effective visualisation with
PG 27b and sc-1745, relatively optimal with sc-7951 and Cayman AP-ab. It was
effective for Novocastra Mab, along with HIER in TEG at 60°C for 12 hours. HIER
in CIT was consistently less sensitive than HIER in TEG. It may give first impression
of higher specifity due to more limited reactivity.
After identification of the optimal antigen retrieval and dilution for each primary
antibody, the efficiency of cancer staining still differed significantly. After correction
of the cancer score for the background and nuclear reactivity, the highest values of
resulting cancer score were obtained for Cayman Mab, followed by sc-1745, IBL APab and PG 27b. PG 27b was remarkable for very low background. Sc-1745 showed
high background score that included also nuclear reactivity; however, the resulting
value was high due to intensive cancer reactivity. The resulting scores of IBL AP-ab
and Novocastra Mab were close to PG 27b. However, these antibodies showed more
background, making the work and evaluation more difficult. The resulting scores for
Cayman AP-ab and sc-7951 were low, but for IBL Mab - negative. The last value
reflected the lack of epithelial reactivity with IBL Mab.
The influence of the primary antibody upon staining in the undifferentiated cancer
component was analysed, comparing the staining in this component to that in
adenocarcinomatous complexes. Diffuse cancers showed the highest relative
reactivity with Cayman Mab, sc-7951, Cayman AP-ab, IBL AP-ab. PG 27b showed
the weakest reactivity, even complete negativity of undifferentiated component and
single-invasive cells if the protocol was not the most sensitive. Novocastra Mab also
relatively weakly reacted with the undifferentiated cancer. The absolute reactivity in
diffuse cancers was the highest with Cayman Mab due to overall high sensitivity.
Signet ring cells also reacted differently with different primary anti-COX-2
antibodies. This cell type was completely negative with sc-7951, IBL AP-ab, and IBL
Mab antibodies and showed no specific reaction with PG 27b. Cayman antibodies
showed occasional weak to moderate membranous reaction with TEG MW, TEG
90"C and CIT 90°C. Novocastra Mab showed such membranous reaction only after
HIER in TEG 90°C that was the most sensitive protocol for this antibody. In contrast,
sc-1745 reacted with signet ring cells after any HIER, showing moderately intense
reaction in the peripheral rim of cytoplasm but not mucus.
Our findings that confirm COX-2 expression in diffuse cancers by different
antibodies are in accordance with published data. COX-2 presence in diffuse cancers
is shown by mRNA and cell culture studies. COX-2 mRNA was expressed both by
intestinal and diffuse type adenocarcinomas, analyzing by Northern blot hybridization
and reverse transcription PCR (Ristimaki et al., 1997). COX mRNA was found by
PCR in 4 of five gastric cell lines. The positive cell lines were from intestinal (2) and
diffuse (2) cancer (Uefuji et al., 2001). No correlation was found between COX-2
mRNA level and histological type - intestinal vs. diffuse (Ohno et al., 2001). It has to
be concluded that COX-2 expression in high-grade cancers is possible, but not all
primary anti-COX-2 antibodies will identify the reactivity even if the protein is
present and would be detected by other primary antibodies. Thus, choice of primary
antibody is the critical decision in studies of relation between COX-2 expression and
tumour grade. This choice will also influence studies that concern COX-2 expression
as prognostic factor, as prognosis is known to be influenced by tumour grade.
The fact that different primary antibodies have different affinity to high-grade
component, does not exclude possibility of biologically different level of COX-2
expression. In fact, our findings would strongly support those studies that have found
such difference between cancers by an antibody that is reactive with undifferentiated
cancers. This is shown by Saukkonen et al. Using Cayman Mab, COX-2 positivity
after scoring was detected in 58% of the intestinal type gastric adenocarcinomas and
6% of diffuse type tumours (Saukkonen et al., 2001). Also, by Cayman Mab positive
reaction occurred more frequently in intestinal (77.9%) than in diffuse (46.2%) or
mixed (45.5%) types of cancer (Joo et al, 2002). However, use of the same antibody
has yielded also opposite results. IHC by Cayman Mab overnight at 4°C without
HIER resulted in cytoplasmic and nuclear membrane staining in cancer cells (72.5%,
if positivity defined as relevant staining in more than 50% of tumour cells) without
correlation between COX-2 expression and location of the tumour (cardia vs. gastric
remnant vs. corpus and antrum) or histopathological grading (Kawabe et al., J Surg
Oncol, 2002). These findings might be attributed to omission of HIER as our data
show less difference in the reactivities of adenocarcinomatous and undifferentiated
components in such conditions in contrast to TEG MW protocol. As already shown,
detailed knowledge of COX-2 IHC technology is necessary in order to avoid
estimating technology-based differences as biological differences.
Not only was the intensity, also the pattern of reactivity was antibody-dependent.
The Cayman Mab showed characteristic granular cytoplasmic staining with
moderately large granules and peripheral attenuation. The most characteristic pattern
of PG 27b in adenocarcinoma was supranuclear cleaved dot. In squamous cell cancer
this antibody was reacting differently upon the same antigen retrieval and
immunostaining conditions showing highly heterogeneous diffuse cytoplasmic
reactivity. IBL AP-ab stained cancer diffusely cytoplasmatically, in combination with
marked positivity in a subset of mitoses. IBL Mab was almost unreactive with cancer
cells. Cayman AP-ab showed combination of cytoplasmic, apical and membranous
reactivity. Novocastra Mab was characterized by diffuse cytoplasmic staining and
small foci of peripheral attenuation reminiscent of Cayman Mab. Sc-7951 showed
highly heterogeneous diffuse cytoplasmic staining. Sc-1745 was characterized by
diffuse cytoplasmic reactivity and trend to membranous enhancement. These
differences demonstrate that the epitopes recognised by different antibodies are
located in different ultrastructural units.
Two of the investigated antibodies were directed against amino terminus of human
COX-2.: Novocastra Mab and sc-7951. Both of them showed diffuse cytoplasmic
stain. Although diffuse cytoplasmic reactivity was a part of the typical pattern for IBL
AP-ab, Cayman AP-ab and sc-1745, it as the only pattern was limited to abs against
amino terminus. As it is described further, those antibodies had in common also the
lack of reactivity in erosions. The sensitivity in cancer staining was higher for
Novocastra Mab, but heterogeneity - for sc-7951.
The antibodies against carboxyl terminus, however, show large variability in
patterns. It might be hypothesized that COX-2 antibodies are produced mainly against
synthetic peptides therefore the real tertiary or quaternary structure of COX-2
molecule interferes with binding to some epitopes.
Another possible explanation for the different results obtained by carboxy-terminus
detecting COX-2 antibodies is based of COX-3 hypothesis.
In 1999, NSAID-induced (sic!) COX activity was described with different
characteristics. The localization of NSAID-induced COX was switched from nuclear
and microsomal membranes to the cytoplasm. The NSAID-induced COX protein was
totally reactive with anti-COX-2 (Merck Frosst Labs, Pointe Claire, Canada) in
Western blot. The COX-2b hypothesis stating that COX-3 is a variant of COX-2 that
included the COX-2 specific carboxyl terminus was based on these findings
(Simmons el al., 1999). In 2002, a sequence for the proposed COX-3 was published
that could be attributed to a clone of the COX-1 gene with alternative splicing,
resulting in the retention of intron-1 in the mature mRNA. Translation results in a
COX isozyme with an N terminal extension due to intron 1 and the retained signal
peptide. The authors describe 65 kDa protein from human aorta as COX-3 and 53 kDa
proteins as PCOX-la. PCOX-1 is identical to COX-3 except for a deletion in a
catalytic domain of the protein corresponding to exons 5-8. It lacked detectable COX
activity. A 50kDa protein was detected only by COX-1 monoclonal antibody and was
named PCOX-1b (Chandrasekharan et al., 2002).
If both results describe the same molecule, it is COX variant with N-terminal
extension precluding the reactivity with N-terminal recognizing anti-COX-2 and
retained reactivity with some anti-COX-2. This may interfere with COX-2 IHC if
some anti-COX-2 recognize it and include into the result but others do not recognize.
Specific elements can be better highlighted with particular anti-COX-2 antibodies.
These have to be analysed separately.
Cayman Mab reacted strongly with parietal cells, staining cytoplasm and especially
canalicular system. Occasional weak reactivity with PG 27b was observed; the pattern
was dot-like, reminiscent either of the typical supranuclear dotted line or fragmentary
staining of canalicular system as the last was highlighted by Cayman Mab in the form
of large dots and rings. IBL AP-ab resulted in finely granular cytoplasmatic stain with
accentuation toward the upper part of gastric glands. IBL Mab gave negative results.
Cayman AP-ab showed trend towards the same pattern as with Cayman's Mab, but it
was less clearly evident. The gradient with Cayman AP-ab was reverse as with IBL
AP-ab - the staining was stronger in those parietal cell situated deeply in gastric
glands. Novocastra Mab reacted with parietal cells as well. Sc-7951 stained parietal
cell weakly and there was marked variability between cases. Moderate cytoplasmic
reactivity and dot-and-ring-like structures similarly to the picture obtained by Cayman
Mab were observed occasionally, in spite to low sensitivity of this antibody to COX-2
expression in cancers. Sc-1745 reacted with parietal cells weakly, in the level of
background. Thus, 5/8 antibodies reacted with parietal cells and 3 of those showed the
same pattern - moderate to intense cytoplasmic reactivity with strong accentuation of
canalicular system. 2 of those antibodies were monoclonal, directed towards carboxyl
terminus and amino terminus of COX-2 respectively. The results might suggest true
COX-2 presence in parietal cells. This is in accordance with findings of McCarthy et
al., who described consistent strong parietal cell reactivity in Helicobacter pylori
infected men by IHC, using Cayman's polyclonal rabbit anti-human IgG antibody at
dilution 1:100 incubating overnight at 4°C, without HIER (McCarthy, 1999). In
another study, using rabbit polyclonal anti-human-COX-2 (ImmunoBiological
Laboratories, Fujioka, Japan) at 4°C overnight after HIER at 90°C for 9 min.,
sporadic staining was observed in normal fundic glandular cells in all cases. The
immunostaining in normal mucosa was in accordance with the magnitude of the
results of an immunoblot (Uefuji et.al., 1998).
A possibility of cross-reaction might be considered. The previous studies have
analysed mostly a possibility of cross-reaction with COX-1. McCarthy et al. observed
no cross-reaction with COX-1 in Western blot, and mentioned that no cross-reactivity
with sheep seminal vesicle COX-1 was found by the manufacturer (McCarthy et al.,
1999). Strong parietal cell immunoreactivity for COX-2 along with COX-1 was found
by Jackson et al. COX-2 was detected by polyclonal anti-human antibody, Cayman
Chemical (catalogue number 160107) after HIER in citrate buffer, 102°C-12 min.
However, specifity of IHC was confirmed by antigen absorption, and COX-2 and
COX-1 expression was confirmed by Western blot, thus suggesting rather presence of
both isoforms than cross-reactivity (Jackson et al., 2000). These data do not allow
ascribing all the detected COX immunoreactivity in parietal cells to COX-1.
The canalicular system as the target of COX-2 detecting antibodies was consistent
with the findings in immunoelectron microscopy. COX-1 and COX-2 in gastric
parietal cells were found in smooth endoplasmic reticulum and canalicular
membranes but not in the nucleus or interior of cytoplasmic organelles such as
mitochondria (Jackson et al., 2000).
There are also seemingly opposite results. In Helicobacter pylori infected tissues,
antrum contained more COX-2 than body by mRNA analysis and Western blot. The
incidence and severity of acute and chronic inflammation was not significantly
increased in antrum compared with corpus (Fu et al., 1999).
Functional importance of COX-2 expression in parietal cells can be hypothesised,
both in normal and pathological processes. Pausawasdi et al. have found that isolated
"resting" canine gastric parietal cells in culture expressed low levels of COX-2
mRNA (by Northern blot) and protein (by Western blot, using polyclonal anti-COX-2
antibody, Cayman Chemical). Stimulation with carbachol but not gastrin, histamine
and combinations of those induced marked expression of both COX-2 mRNA and
protein. The induction of COX-2 was mediated through muscarinic receptor M3
activating Ca2+, NF-kappaB and protein kinase C dependent pathway with partial
involvement of p38 kinase. This might be relevant to clinical processes as
prostaglandins not only promote gastrointestinal mucosal defence, but also inhibit
gastric acid secretion. Cholinergic neurotransmitters are released in the inflamed
gastrointestinal mucosa, so M3-dependent induction of COX-2 gene expression in
parietal cells is possible in inflammation. Parietal cells are capable to produce growth
factors, in particular, transforming growth factor alpha, so these cells might be an
important mechanism for the paracrine regulation of gastric epithelial cell growth and
differentiation (Pausawasdi et al., 2002).
Loogna at al. described strong cytoplasmic expression of COX-2 in the lower
portion of rat glandular corpus epithelium by IHC, using polyclonal murine antiCOX-2 antibody, Cayman Chemical. COX-2 expression in corpus epithelium was
found in control animals, but it was increased in N-methyl-N-nitro-Nnitrosoguanidine treated animals and in the group exposed to Helicobacter pylori and
bile. Most of staining was found in the corpus, while preneoplastic changes occurred
in the antrum. There was a correlation between the area of COX-2 expression in
corpus and atrophy and goblet cell metaplasia in antrum (Loogna et al, 2002).
Although the COX-2 expression may differ in different species (Kargman el al.,
1996), these findings highlight a possible role of parietal cell COX-2 expression in
carcinogenesis.
In conclusion, positive IHC staining for COX-2 in gastric corpus is described by
different methods. The work of Pausawasdi confirms this finding by showing that
parietal cells are able to express high levels of COX-2 without morphological
changes, upon M3 stimulation that is possible in inflamed stomach. Together with
finding of Loogna that area of COX-2 expression in corpus correlates with the degree
of atrophic and metaplastic changes in antrum, it is possible to speculate that parietal
cells participate in precancerogenesis producing growth factors. However, the causal
relationships in this process are not proved. Another independent finding partially
contradicts this possibility by the fact that levels of COX-2 in gastric antrum is equal
to that in corpus; in the absence of significant inflammatory or neoplastic processes
limited to antrum (Fu et al., 1999). However, if all the reactivity demonstrated by
Cayman Mab is true, such a "balance" may be achieved with strong COX-2
expression in parietal and chief cells in corpus, and moderate COX-2 expression in
cells of mucosal immune system, stromal cells, metaplastic and dysplastic epithelium
in antrum. There are several possibilities: 1) COX-2 is easily up-regulated in corpus
and some antibodies are sufficiently sensitive to demonstrate this. 2) Some anti-COX2 antibodies exhibit cross-reaction with COX-1. McCarthy's, Saukkonen's and
Cayman's data argue against this. This probability is not high. 3) Some antibodies
show significant cross-reactivity with unidentified protein. This might be caused by
choice of non-specific epitope as the immunogen. This probability also is not high as
this reactivity is not limited to antibodies from the same source. 4) Cross reaction
occurs with a member of so-called COX-3 group.
The differences in staining pattern might be due to conformational changes in
COX-2 molecule, e.g., PG 27 recognise COX-2 in "early stage of its life", in
endoplasmic reticulum close to Golgi complex, but it does not recognise "old" COX-2
in parietal cells.
Chief cells were markedly positive with Novocastra Mab and reactive with Cayman
Mab in lower intensity as parietal cells, but mostly negative with Cayman AP-ab.
There was no convincing reactivity with IBL AP-ab and Mab. If the most sensitive
protocol was applied, PG 27b resulted in weak dot-like cytoplasmic granularity in the
basal part of corpus glands. Occasional weak reactivity in basal part of corpus glands
was found with sc-7951. The cells situated in deep part of corpus glands were apically
positive with sc-1745. Thus, 2/8 antibodies demonstrate chief cell reactivity, another
2/8 show reactivity in the basal part of corpus glands that was difficult to localize to
particular cell type. However, the location suggest chief cell as the COX-2 source in
these cases. Chief cell reactivity might have the same causes and functional role as the
COX-2 protein expression in parietal cells.
Using HIER that resulted in the highest cancer score, foveolar epithelium showed
characteristic reactivity with Cayman Mab. The pattern was basal cytoplasmic stain in
foveolar proliferative zone without the supranuclear dotted line that was focally
evident in intestinal metaplasia and/or dysplasia. The same trend to reactivity mainly
in the proliferative zone was observed with IBL AP-ab, Cayman AP-ab and
Novocastra Mab. Although a characteristic trait of sc-1745 was the diffuse, total
reactivity in foveolar epithelium, trend to more intense stain in proliferative zone was
noted after HIER in CIT. IBL Mab and sc-7951 showed different pattern: subset of
positive endocrine-like cells in proliferative area on background of otherwise negative
foveolar epithelium. This type of reactivity was remarkable for 2 peculiarities presence of epithelial reactivity with IBL Mab and higher efficiency of HIER in CIT
MW than TEG MW for IBL Mab. A combination of 2 patterns was observed in the
foveolar epithelium with PG 27b. The more common was diffuse reactivity with
foveolar mucus that was variable between cases and HIER protocols. Another, rarer
pattern was the focal presence of supranuclear dots in foveolar epithelium upon a
sensitive protocol. It is interesting that foveolar reactivity independently of pattern
was mostly present in the proliferative area (by 7/8 antibodies). The correlation
between proliferation and COX-2 expression is described previously (Ohike and
Morohoshi, 2001). So, the reactivity limited to the proliferative zone seems to be true.
The reactivity of non-dysplastic surface epithelium with sc-1745 might be
equalized to consistent expression in biopsy studies; this might signify a crossreactivity, or, less probably, consistent expression of COX-2.
Two types of COX-2 immunoreactivity were identified in mucous glands. In
contrast to the findings in foveolar epithelium, these patterns were not mutually
exclusive. One of those patterns was focal cytoplasmic staining without nuclear and
mucus reactivity, mostly evident in atrophic mucosa. Cayman Mab stained atrophic
glands focally, moderately intensively. PG 27b generally did not react with mucous
glandular epithelium. The characteristic supranuclear dot-like reactivity, however,
was observed in atrophic epithelium beneath intestinal metaplasia. Most of protocols,
namely, all TEG-based HIER protocols, HIER in CIT MW and staining without
HIER resulted in focal cytoplasmic reactivity in the mucous glands with IBL AP-ab.
Focal moderate cytoplasmic reaction with Novocastra Mab was evident in basal
cytoplasm, except for the protocols without HIER or with slide incubation in CIT
60°C or in CIT MW when the reaction was weak, limited to small foci in 1/4 case.
Atrophic mucous glands were positive with sc-1745. A subset of endocrine-like cells
was highlighted by IBL AP-ab in the mucous glands, most effectively after HIER in
MW. There was also a subset of small round intensively positive cells with sc-1745.
IBL Mab did not stain mucous glands. Cayman AP-ab showed weak focal reactivity
only after HIER in TEG MW (2/3), 90°C (3/3), 60°C (1/3), and protocol without
HIER. Mucous glands were mostly negative with sc-7951: only 1/4 case showed
sharply demarcated foci of weak reactivity if HIER in TEG, MW or 90°C was
applied.
The IHC COX-2 expression in mucous glands thus is dependant on primary
antibody. The reactivity is weak and dependent also on the antigen retrieval.
Focal COX-2 expression in mucous glands on the background of atrophic gastritis
might be possible in the light of Correa's cascade. The nature of positively staining
cell subpopulation is not clear; these differ from similar subpopulation in the foveolar
proliferative zone by location and by antibodies that recognize these cells. COX-2
brightly positive subpopulation in foveolar proliferative zone is recognized by 1BL
AP-ab and sc-7951, but the glandular subpopulation is demonstrated by IBL Mab and
sc-1745.
6/8 of primary antibodies demonstrated focal COX-2 expression in intestinal
metaplasia and dysplasia. Consistent negativity was found with IBL Mab that was in
accordance with the lack of epithelial reactivity with this antibody. No reliable
reactivity in dysplastic and metaplastic epithelium was observed with Cayman AP-ab,
possibly due to lower sensitivity of this antibody in comparison to Cayman Mab,
demonstrated both by lower cancer scores in our results and by the published data
(Saukkonen et al., 2001). Focal sharply demarcated supranuclear and subnuclear basal
cytoplasmic reactivity with Cayman Mab in IM and dysplasia occurred after HIER in
MW. The reactivity of PG 27b with IM was most consistent after HIER in TEG 90°C.
All other HIER protocols resulted in less positive area, less intensity and lower
number of positive cases. The dot-like reactivity was most marked in the basal part of
metaplastic glands. No mucus reactivity in IM was observed in contrast to foveolar
epithelium. IM showed a combination of focal cytoplasmic reactivity and bright
staining in a subgroup of endocrine-like cells with IBL AP-ab. These cells were best
seen after low temperature HIER in TEG, less in TEG MW and low temperature
HIER in CIT, but were not evident after HIER in CIT MW and staining without
HIER. The cytoplasmic reactivity was best evident after HIER in TEG MW or
staining without HIER and was absent after CIT 60°C. Dysplastic surface epithelium
focally moderately intensively reacted after HIER in TEG MW and staining without
HIER. IM and dysplasia was negative with sc-7951 in 1 case. In other 2 cases, there
was focal weak reactivity in metaplastic that contrasted with the total negativity of
foveolar epithelium. Within the study the recognition of weakly positive areas was
facilitated by colocalisation of positive areas with more contrasting slides, obtained by
use of other anti-COX-2 antibodies. In IM, cytoplasmic reactivity was observed in
deeper parts of metaplastic glands that contained also a subpopulation of brighter
positive round cells. The reactivity of dysplastic surface epithelium in 2/3 cases was
limited to intercellular lines and paranuclear vacuoles, presumably serum leaks.
Novocastra Mab stained IM focally. Although the intensity of reactivity in metaplastic
epithelium was only slightly stronger after HIER in TEG 90°C in comparison with
TEG 60°C and TEG MW, TEG 90°C resulted in sharply demarcated foci of reactivity
in dysplastic surface epithelium. IM was totally positive with sc-1745. The positivity
was equally intense in the basal and upper part of metaplastic glands and extended to
dysplastic surface epithelium. The intensity was highest after HIER in TEG. Surface
stained more intensively than deep part after HIER in TEG 60°C. A subpopulation of
endocrine-like positive cells was present in the basal part of metaplastic glands
after HIER in 90°C. Thus, contrasting
subpopulations of COX-2 positive cells in 1M were recognized by IBL AP-ab sc7951, sc-1745.
COX-2 expression in 1M and dysplasia has been described (Lim et al., 2000; Sung
et al., 2000, van Roes et al., 2002) and pathogenetically is substantiated as IM and
dysplasia arc parts of Correa cascade leading to cancers that are COX-2 positive at
least in a subset. However, there was significant variability by different primary
antibodies on the same tissue. The focal, sharply demarcated reactivity as seen with
Cayman Mab, IBL AP-ab, sc-7951, Novocastra Mab and PG 27b seem more probable
as the trend to total staining with sc-1745. The differences in the predominant location
of COX-2 expression (basal with PG 27b, predominantly superficial with sc-1745
upon those protocols that recognize such gradient) again point to reactivity with
different epitopes and possibility of cross-reactivity. Our data show that differences in
the COX-2 expression in precancerous processes can be explained with technical
factors only. Examples of such differences are studies by Lim et al. and van Rees et
al. By IHC with Santa Cruz polyclonal goat antibody at dilution 1:50 overnight at 4°C
after microwave treatment in citrate buffer, consistent positivity was found in IM and
adenomatous epithelium (Lim et al., 2000). Using Cayman Mab at dilution 1 TOO at
4°C overnight after HIER in MW in CIT buffer 10 min., the results were: reactive
epithelium 7% negative, 71% +, 21% ++; IM 70%+ and 30% ++; low-grade dysplasia
20%+, 30%++, 50+++; high-grade dysplasia 11% +, 33% ++ 55% +++. By RT-PCR,
there were elevated COX-2 mRNA levels in IM-containing biopsies, if compared to
paired non-metaplastic biopsies (van Rees et al., 2002).
With Cayman Mab, squamous epithelium showed moderate reactivity in basal layer
and weak perinuclear in prickle cell layer. In reactive epithelium, the positivity
"raised" to suprabasal layer. Interestingly, the pattern was qualitatively different
without HIER - inverted, with basal negativity. Reactivity of squamous epithelium
was difficult to demonstrate with PG 27b: it was limited to most sensitive protocols
and reactive areas. With IBL AP-ab antibody, basal layer stained weaker than prickle
cell layer, and reactive areas stronger than remote epithelium. IBL Mab stained only
parakeratosis weakly, after no HIER or TEG MW. The upper prickle layer and
parakeratosis in reactive areas were intensively positive, if no HIER was applied.
With Cayman AP-ab, basal layer and prickle cell layer were weakly positive after
HIER in TEG MW. After low temperature HIER in TEG 90°C, basal layer showed
moderate cytoplasmic positivity, but prickle cells - weak cytoplasmic reaction with
slight perinuclear accentuation. If no HIER was applied, the basal layer was negative,
but prickle cell layer in reactive areas showed weak granular cytoplasmic reactivity
with perinuclear accentuation. Novocastra Mab after optimal HIER (TEG in 90°C or
60°C) stained intensively perinuclearly the basal and prickle cell layer, and suprabasal
and prickle cell layer in reactive epithelium. In this case, no HIER resulted in
areactivity of the squamous epithelium. Sc-7951 after the optimal HIER TEG 90°C
resulted in weak to moderate membranous/cell border positivity in prickle cell layer
and occasional weak perinuclear accentuation, but the basal layer was negative. With
sc-1745, alter HIER in TEG MW, moderate to intense cytoplasmic reactivity was
present in the basal layer with basal membrane accentuation. In CIT, the pattern was
preserved; possibly due lo lower background, focal perinuclear positivity was
discernible that might be confluent with the general background-like stain after TEG
MW. After TEG 90°C, in addition to intense positivity in the basal layer weak to
moderate paranuclear reactivity was also observed in the prickle cell layer. The
epithelium adjacent lo cancer showed only weak expression - less intense than in the
remote epithelium.
HIER vs. no HIER changed not only intensity, but also the pattern of expression
with Cayman Mab and AP-ab. Basal reactivity was acquired and dominated only after
HIER. Basal layer showed higher COX-2 expression with Cayman Mab, Cayman APab, Novocastra Mab and sc-1745, but less - with IBL AP-Ab, IBL Mab and sc-7951.
As expected from the diverse patterns in relation to technological changes, the
literature data about COX-2 expression in squamous epithelium are controversial.
COX-2 was weakly expressed the basal layer of the normal oesophageal mucosa by
IHC using monoclonal antibody from Cayman Chemical with overnight incubation at
4°C. Results of IHC in general were confirmed by preabsorption and Western blot in
selected cases (Kawabe et al., 2002). In human keratinocyte differentiation, COX-2
gene expression showed increasing intensity in the upper part of the epidermis with
less expression in the basal layer (Leong et al., 1996). If COX-2 expression correlates
with the proliferative activity, higher expression in basal layer and reactive areas
might be expected. It is evident that great variety of COX-2 IHC staining patterns
exists. The main emphasis is on illustration of the differences between the distribution
of epitopes recognised by different antibodies.
COX-2 expression in macrophage- and fibroblast-like cells in erosions is a welldescribed phenomenon (Saukkonen et al, 2001; Jackson et al, 2000; Han et al, 2003).
In our experience, this reactivity was not manifested by all primary antibodies.
Cayman Mab, PG 27b, IBL AP-ab, IBL Mab, Cayman AP-ab, sc-1745 were reactive.
The erosions were negative with Novocastra Mab and sc-7951, both directed against
amino terminus of COX-2. The reactivity, when present, was intense, therefore with
some antibodies, e.g., Cayman Mab, it was almost independent of the type of antigen
retrieval. With others, e.g., sc-1745, the general rule of less positivity after HIER in
CIT and staining without HIER was followed. For sc-1745 and PG 27b, the score
were mainly influenced by pH. Although results by Cayman AP-ab and IBL abs were
also influenced by pH, there was also effect of temperature saturation, namely loss of
reactivity at low temperature HIER for Cayman AP-ab and saturation at TEG MW or
60°C for IBL AP-ab and MAb. The relative input sequence of factors that influence
the degree of erosion-related COX-2 reactivity is only partially the same as for cancer
reactivity degree.
Reactivity of macrophages and fibroblastss apart from erosions was found with
most antibodies, although in different degree. PG 27b and IBL Mab were almost
unreactive. The intensity of COX-2 expression in macrophages and fibroblasts was
paralleling each other, but not correlating with the reactivity in erosions. The last
finding is demonstrated brightly by PG 27b and 1BL Mab that show marked reactivity
in erosions. The COX-2 score in macrophages was mostly influenced by the choice of
the primary antibody and by pi 1 of 1 HER buffer, but less - by temperature regimen
of HIER. The published data arc controversial, again. In Helicobacter pylori infected
men, spotty positivity in macrophages in the germinal centres was demonstrated by
Cayman polyclonal rabbit anti-human IgG antibody at dilution 1:100 incubating
overnight at 4"C, without 1 HER (McCarthy et al., 1999). Occasional macrophage and
myofibroblast reactivity for COX-2 was detected by polyclonal anti-human antibody
(catalogue number 160107, Cayman Chemical) after HIER in citrate buffer, 102°C-12
min. (Jackson et al., 2000). By Cayman Mab overnight at 4°C without HIER,
inflammatory mononuclear cells, fibroblasts occasionally stained (Kawabe et al., J
Surg Oncol, 2002). In contrast, by rabbit polyclonal anti-human-COX-2
(Immunobiological Laboratories, Fujioka, Japan) at 4°C overnight after HIER at 90°C
for 9 min., no expression of COX-2 was observed in inflammatory mononuclear cells
or fibroblasts (Uefuji et al, 1998). Also, the tumour stroma outside the areas of
epithelial injury was found to be negative, using Cayman Mab at dilution 1:50
overnight after HIER in citrate buffer, MW, 2.5 min in 800 W and 15 min in 440 W
(Saukkonen et al., 2001).
COX-2 expression in plasma cells was found with all antibodies, suggesting true
COX-2 presence. In literature, spotty positivity in lamina propria plasma cells of
Helicobacter pylori infected men was described (McCarthy et al., 1999).
Endothelial reactivity with Cayman Mab was slight and focal, revealed only by
HIER in TEG MW or at 90°C. Endothelium was unreactive with PG 27b. The
reactivity with IBL AP-ab was markedly dependant on antigen retrieval -endothelium
was consistently weakly positive after TEG MW, negative after CIT MW.
Interestingly, staining without HIER resulted in dispersed results, including no
endothelial reactivity in 2/5 cases and moderate reaction in another 2/5 cases. With
IBL Mab, endothelium was mostly negative, with the exception of weak positivity in
1/5 cases after HIER in TEG MW and in the protocol without HIER (the same
protocols as for neural and muscular positivity). The endothelial reactivity was the
highest with Cayman AP-ab. It was present with Novocastra Mab and sc-1745:
mostly after TEG 90°C and TEG 60°C, less after TEG MW. Sc-7951 reacted focally
weakly. At least focal endothelial reactivity is present with most of antibodies and is
in accordance with the following literature data. Under normal conditions, cultured
human gastric endothelial cells expressed COX-1 and low levels of COX-2 by
Western blot and reverse transcriptase PCR. COX-2 expression was induced in
angiogenesis models by stimulating endothelial cell with phorbol ester or plating them
on basement membrane matrix (Hull et al., 1999). Increased COX-2 immunostaining
in endothelial cells was seen at the rim of ulcers (Jackson et al., 2000). No COX-2
was detected in the existing vasculature in normal tissues, but moderate to intense
COX-2 expression was consistently observed in the blood vessels in all cancers
studied and in the endothelium overlying and immediately adjacent to
the fibrofatty lesion, if studied by IHC, and verified by Western blot and quantitative
RT-PCR (Koki et al., 2002). Thus, according to literature data, unstimulated
endothelium in preformed blood vessels or in normal culture conditions expresses
predominantly COX-1 (Hull et al., 1999). In contrast, stimulated endothelial cells in
culture (Hull et al., 1999), rim of ulcers (Jackson el al., 2000), adjacent to
atherosclerotic fibro fatty lesion or in tumour stroma (Koki el al., 2002) over express
COX-2, as demonstrated by Western blot, reverse transcriptase polymerase chain
reaction and 1HC. Although focal COX-2 expression in endothelial cells seems to be
biological phenomenon, consistent positivity might signify a cross reactivity, possibly
with COX-1.
The reactivity in smooth muscle was variable with different antibodies. The
literature data are controversial, again. In the present work very low reactivity of
smooth muscle with Cayman Mab was found, except if staining was performed
without HIER. No reliable reactivity was observed with PG 27b. IBL AP-ab antibody
resulted in weak to moderate reactivity. It was higher without HIER than with TEG
MW, in contrast to cancer reactivity. There was no reliable reactivity with IBL Mab
antibody. Cayman AP-ab showed mostly weak reaction after HIER, but it was higher
if no antigen retrieval was performed. Sc-7951 stained muscle weakly. Novocastra
Mab and sc-1745 were the main "muscle reactors". Literature data suggest consistent
presence of COX-1 in smooth muscle, along with focal induction of COX-2 in the
sites of injury (Komhoff et al., 2000). However, other authors mentioned positive
reaction in smooth muscle as internal positive control (Ohno et al., 2001; Lim et al.,
2000; Kawabe et al., 2002). The last recommendation included sc-1745 but was not
specific for it. The present experimental and literature data result in two hypothetic
possibilities - either sc-1745 display high sensitivity or cross-reaction with a protein
that is present in smooth muscle fibres. At the level of present knowledge, smooth
muscle positivity with sc-1745 reflects the general quality of IHC as smooth muscle
cells is consistently reactive with this particular antibody. However, it does not
provide any data about specifity of sc-1745 and cannot be used as general internal
control for COX-2 visualisation with other primary antibodies.
Reactivity with nerve fibres was found with most of tested antibodies, except for
PG 27b in all protocols and IBL Mab in most of protocols (5/7). The most intense
reactivity was observed with Cayman AP-ab (figure), closely followed by the most
sensitive protocols for Cayman Mab, sc-1745 and Novocastra Mab.
Primary anti-COX-2 antibodies
Cayman Mab was characterized by high sensitivity as reflected by high total and
final cancer scores, low background that was eradicated by lowering concentration of
the primary antibody, and well-defined pattern, consistent with the ultrastructural
findings: by EM IHC, COX-2 was found on the luminal surfaces of the endoplasmic
reticulum and nuclear envelope (Tatsuguchi et al, 2000, Gut). Cayman Mab has been
previously evaluated by Saukkonen et al. This group has characterized this antibody
as specific based on preadsorption using the antigenic peptides. Cayman Mab at
dilution 1:50 after HIER in microwaves, 2.5 min. at 800 W followed by 15 min. at
440W, stained 7/8 intestinal type gastric adenocarcinoma specimens, which had been
shown previously to express elevated levels of Cox-2 mRNA. Immunoreactivity
obtained by the Mab and by the Cayman AP-ab colocalized and correlated in
intensity.
Saukkonen et al. have tested also polyclonal antibody PG 27b. At dilution 1:1000 it
stained the known COX-2 positive tumours only weakly or gave high background
staining. The characteristic pattern of dotted line was found with this antibody as well
as with polyclonal 160106 and 160116, Cayman Chemical. Preadsorption using the
antigenic peptides reduced the positivity of the polyclonal antibodies only partially:
the diffuse cytoplasmic positivity was completely blocked, dotted line was not
blocked.
In a study comparing monoclonal antibody from Cayman Chemical in dilution
1:250 and PG 27 in dilution 1:200, after HIER in microwaves for 10 min. in high
power, preheating citrate buffer to 95°C, different staining patterns were found.
Cayman Mab did not react with normal colonic epithelium, but stained tubular
adenoma cells in diffuse cytoplasmatic pattern, as well as subsurface myofibroblasts.
No staining in inflammatory cells or smooth muscle was observed. PG 27 reacted with
a discrete area near the nucleus on the apical side of epithelial cell both in the normal
crypt, surface epithelium and adenoma. Cytoplasm of scattered stromal cells showed
positive staining. At higher magnification, diffuse granular stain was seen in the
cytoplasm of the epithelial cells. A light granular stain occurred in cytoplasm of
muscularis mucosa and vascular endothelium (Garewal et al, 2003).
According to these literature data, it might seem that PG 27b reactivity is wider
than that of Cayman Mab and includes normal colonic epithelium. Our data did not to
confirm this - reactivity is coincident or wider for Cayman Mab in diffuse cancer.
Cayman Mab reacts with normal colon if the concentration is not very low and
antigen retrieval is sensitive. Literature data also confirms presence of low level of
COX-2 in colonic epithelium. Low-level constitutive COX-2 was detected in colonic
epithelium by IHC, verified by Western blot and quantitative RT-PCR (Koki et al,
2002). Normal colonic epithelium expressed low levels of COX-2 mRNA (Lim et
al.,). Thus, data from independent studies, using different methods, confirmed
presence of low level of COX-2 in non-neoplastic colonic epithelium. Our findings
with Cayman Mab are in accordance. Normal colonic epithelium was also reactive
with Novocastra Mab. So, the described reactivity of PG 27b with colonic epithelium
does not seem to be unspecific. Cayman Mab in the report of Garewal et al. was
unreactive due to high dilution and HIER in citrate that in our experience consistently
retrieves less reactivity than HIER in basic buffer.
Our data confirm the finding that low temperature HIER is more effective than
treatment in MW for PG 27b (Shi et al, 2002). It is also more effective for Novocastra
Mab and can be used for sc-1745. However, MW treatment is beneficial for "strong
reactors" Cayman Mab and IBL AP-ab and "weak reactors" sc-7951 and Cayman APab. It seems that COX-2 molecule it not destroyed by boiling but some
epitopes might be changed. This also emphasize that COX-2 antibodies recognize
different epitopes and thus may exhibit also different cross-reactivity. The fact is
emphasized also by lack of correlation in the staining intensity of different structures.
IBL AP-ab is described by Ohno et al. and Uefuji et al. Intense diffuse cytoplasmic
immunoreactivity (using anti-COX-2 1:20 - 90 min. no HIER) was observed in
tumour cells. Stroma and normal gastric glands were negative, but smooth muscle
was used as internal control (Ohno et al., 2001). By IHC using rabbit polyclonal antihuman-COX-2 (Immunobiological Laboratories, Fujioka, Japan) at 4°C overnight
after HIER at 90°C for 9 min., sporadic staining was observed in the normal fundic
and metaplastic glandular cells in all cases. IHC demonstrated diffuse cytoplasmic
and perinuclear stain in cancer cells. No expression of COX-2 was observed in
inflammatory mononuclear cells or fibroblasts (Uefuji et al., 1998).
The most characteristic trait of IBL Mab was the almost complete lack of epithelial
reactivity. According to literature data and results obtained with other antibodies, the
reactivity in erosion and subset of plasmatic cells might be true, but represent only
part of the reactivity shown by other antibodies.
Cayman AP-ab has been tested by Saukkonen et al. As with the Cayman Mab, all
of the staining in tumour cells was blocked by the antigenic peptides but not by
smooth muscle actin peptide. No staining was observed in the unspecific areas that
were positive with die nonpurified polyclonal antibodies. Immunoreactivity obtained
by the Cayman Mab and by the AP-ab colocalized and correlated in intensity. The
signal intensity of the Mab was stronger than that of AP-ab.
Sc-1745 has been tested in the study of Saukkonen et al., as well. At concentration
1:1000 it stained the tumours only weakly or gave high background staining.
COX-2 in GIST
The 4 anti-COX-2 antibodies revealed significant differences in the staining
intensity, distribution, variability and even pattern. As the testing was performed on
the same tissues, it is evident that the differences are generally technological. This is
in agreement with literature data that present markedly different results of
immunohistochemical COX-2 protein expression in comparable tissue structures
(Zimmerman et al., 1999; Morris et al., 2001; Lim et al., 2000; Saukkonen et al.,
2001). However, the presented data prove the role of technology exactly and evidently
as the reactivity of all antibodies is determined and assessed on the same tissues.
83.3% of GISTs (5 of 6) showed at least moderately intense cytoplasmic granular
positive reaction, if IHC was performed by Cayman Mab. This is in agreement with
the results of Sheehan et al. (2003), who have found positive expression of COX-2
protein in 80% of GISTs by comparable IHC technology.
The staining obtained by different primary anti-COX-2 antibodies was qualitatively
and quantitatively different as seen by different total cancer scores and different
expression patterns.
The Cayman Mab provided the most consistent results. It had strict staining pattern
- granular cytoplasmic - that is an important criterion of the specifity of an antibody.
PG 27b exhibits peculiar reactivity pattern (Garewal el at., 2003) but generally did not
stain GISTs or reacted weakly. Sc-1745 was characterized by widespread reactivity
with different tissues (Lirn et al., 2000). This, along with the marked muscular
reactivity suggests lack of specifity (Komhoff et al.) Sc-7951 exhibited crossreactivity with a serum component that compromised the quality of staining and the
ease of evaluation, especially in oedematous areas, common in GISTs.
Basing on the markedly different staining with 4 anti-COX-2 antibodies, it may be
hypothesised that COX-2 has either complex or unusually dynamically changing
tertiary or quaternary structure. These higher-level structures could not be
encountered if immunization was carried out by peptide fragments that probably
reflected only the primary and secondary structure of the epitope. The differences in
staining intensity and pattern cannot be explained by hypothetic biochemical
similarity between any part of COX-2 molecule and some other protein. There is no
correlation in staining pattern and intensity between those three primary antibodies
that are directed towards closely related epitopes in the carboxyl terminus of COX-2
molecule. Alternatively, COX-3 hypothesis has to be considered.
As no correlation was observed between COX-2 expression and morphological
structure of a tumour, it can be concluded that COX-2 is essential for the development
of the tumour. Thus, GIST can be added to the growing list of COX-2-dependant
tumours that possibly could be treated by blocking of this molecule, or prevented by
pharmacological COX-2 inhibition or eradication of the conditions that cause
pathological COX-2 over expression.
COX-2 expression in Barrett's oesophagus
The study has resulted in several important findings. Firstly, the COX-2 expression
in Barrett's oesophagus was identified and distribution of reactivity obtained. The
pattern and location of immunoreactivity also was described.
The aging changes were described analysing the COX-2 expression in
macrophages, IM and squamous epithelium. Qualitative changes in the COX-2
immunoreactivity pattern in plasmatic cells also were described. The COX-2
expression in erosions was found to be sensitive to the aging of blocks and data were
provided.
CONCLUSIONS
1. Standardization is an essential part of the complex technology of IHC. It must be
performed by total test procedure in accordance to local characteristics of
morphological material with following gain in quality and beneficial economic effect.
Total test assured optimal quality of the IHC that resulted in higher probability of
reaching the correct diagnosis in biologically complex cases and pathologist -friendly
analysis. Testing different technological variations on local material
provided important irreplaceable information that could not be obtained from
literature as it was dependant on the local peculiarities. This information changed the
methodology significantly for high fraction (91.6% in the presented experience) of
antigens.
2. The total test results in a rare, still scientifically justified combination of higher
quality and higher cost-effectiveness. The economic effect of 4.67- 141.69 LVS is
obtained on 75.9% of primary antibodies. The economic effect mostly is equal to the
residual expenses or even exceeds those, thus saving more than 50% of the IHC
budget for primary antibodies.
3. IHC is highly informative technology in biomedical research and diagnostic
work. IHC was confirmatory in 18.2% cases and mandatory in 51.5% (17/33) cases of
primary pleural and chest wall tumours, including all pleural neoplasms and tumours
presenting with small blue cell pattern. Randomly selected field of pathology showed
an important role of IHC as a general diagnostic technology.
4. As COX-2 protein is found in different cell types, immunohistochemistry for the
detection of COX-2 protein is the optimal tool for enrolling patients into preventive
trials. However, technical variations, using different anti-COX-2 primary antibodies
are enormous. The quantitative estimates of COX-2 expression on the same cases
implying no biological variations among the material tested with each antibody still
differ largely. The total cancer score is variable in dependence from the antibody.
Quantitative differences are accompanied with qualitative differences. The final
cancer scores vary from positive to negative values the last meaning that cancer
reactivity is less than background even in the cancer-optimal protocol. The impact of
technological factors upon the evaluation of COX-2 expression is larger than the
influence of biological factors. Detailed knowledge of COX-2 IHC technology is
necessary in order to avoid estimating technology-based differences as biological
differences.
5. The COX-2 protein expression is influenced not only by the choice of primary
anti-COX-2 antibody but also the mode of antigen retrieval. The important variables
in HIER are pH of HIER buffer and temperature regimen. Although HIER in CIT is
characterised by consistently lower sensitivity than HIER in TEG, each of the
mentioned variables may have the largest impact in dependence from the primary
antibody and concerned tissue structure. The variability of the most important HIER
factor, as evident between antibodies and scores of cell type or morphologic structure,
suggest no homogeneity of epitopes.
6. Monoclonal anti-COX-2, Cayman Chemical showed a characteristic granular
cytoplasmic staining in the positive cancer cells. Stromal reactivity was evident best if
HIER was performed in basic buffer. Cancer stain was more dependant on the amount
of the absorbed energy that plasma cell or macrophage score. All scores, their
relations and staining pattern changes if no HIER is applied, effect, that cannot be
explained solely by higher background.
7. PG 27b showed markedly different epithelial patterns - the most characteristic,
supranuclear cleaved dot; apical or membranous in cancer cells; highly
heterogeneous intense cytoplasmic in squamous cell cancer; diffuse small cytoplasmic
granules in diffuse cancer; and diffuse, weak or moderate reactivity in foveolar but
not colonic mucus. The stromal reactivity with PG 27b was limited. Macrophages and
fibroblasts in erosions were intensively positive. The most effective antigen retrieval
for this particular antibody was heating in basic buffer at 90"C for 30 min.
8. The epithelial pattern of IBL, AP-ab was diffuse cytoplasmic, consistently
combined by recognition of subset of mitoses. IM showed a combination of focal
cytoplasmic reactivity and brightly staining subgroup of endocrine-like cells.
Omitting HIER lead to qualitatively different pattern in the aspect of IM: although the
intensity of cytoplasmic reactivity was relatively high, the subpopulation of brightly
positive cells was not evident; result that is not consistent with different sensitivity
only.
9. The general pattern for IBL monoclonal anti-COX-2 includes cancer negativity
in combination with bright reactivity of macrophage and fibroblast-like cells in
erosions and a subset of plasma cells.
10.Affinity-purified polyclonal antibody, Cayman Chemical showed a combination
of cytoplasmic, membranous and apical reactivity in cancer cells. Consistent bright
reactivity was observed in nerve fibres and neurons. The reactivity pattern with NC
Mab in cancer cells was diffuse cytoplasmic with frequent nuclear reactivity. Diffuse
cancer was mostly unreactive. The cancer reactivity with Santa Cruz polyclonal antiCOX-2, sc-7951 was pale, diffuse cytoplasmic, highly heterogeneous. The lack of
recognisable pattern burdened the discrimination between true reactivity and
background.
ll. Sc-1745 showed 2 staining patterns in cancer cells: trend to membranous
enhancement and diffuse cytoplasmic reactivity. A characteristic trait of sc-1745 was
trend to diffuse, total reactivity in foveolar epithelium, IM and smooth muscle in all
protocols. As the width of reactivity was not confirmed by the reactivity with other
anti-COX-2 antibodies, unspecific cross-reaction must be considered.
12.COX-2 expression in high-grade cancers is possible, but not all primary antiCOX-2 antibodies will identify the reactivity even if the protein is present and would
be detected by other primary antibodies. Thus, choice of primary antibody is the
critical decision in studies of relation between COX-2 expression and tumour grade.
This choice will also influence studies that concern COX-2 expression as prognostic
factor, as prognosis is known to be influenced by tumour grade.
13. GISTs can be reliably diagnosed by IHC. The recommended diagnostic panel
includes CD117, CD34, CD31, desmin, S-100, cytokeratin AE1/AE3 and Ki-67. The
morphologic features of GIST in routine histological stains may be confusing,
mimicking epithelial, neuroendocrine, neural or muscular tumours. Pseudoschwannomatous palisades are typical for GIST.
14. Although the primary antibodies, included in the diagnostic panel, have distinct
informative value in the identification of the histogenesis of mesenchymal tumours,
they react also with particular normal cells. Such tissue elements may also be
entrapped within a tumour. Awareness of this reactivity is mandatory in order to avoid
over diagnosis or diagnostic confusion and can provide endogenous positive controls
with beneficial economic consequences. The recommended endogenous positive
control elements in the diagnostics of GIST include mast cells for CD117,
endothelium for CD31 and CD34, macrophages or nerves for S-100, vascular smooth
muscle or lamina muscularis mucosae or lamina muscularis propria for desmin,
nuclei of the epithelial cells in the proliferative zone for Ki-67.
15.The pattern of immunohistochemical COX-2 expression in GIST is specific for
each primary antibody - cytoplasmic granular with the monoclonal mouse antibody
from Cayman Chemical, weak finely granular with polyclonal rabbit antibodies PG
27b (Oxford Biomedical research) and sc-7951 (Santa Cruz Biotechnology), diffuse
cytoplasmic with affinity purified goat antibody sc-1745 (Santa Cruz Biotechnology).
The incidence of COX-2 reactivity in GISTs is highly dependant on the primary
antibody. The majority of GISTs (83.3%) express COX-2 protein by
immunohistochemical visualisation with Cayman monoclonal anti-COX-2. There is
no correlation between the expression of COX-2 and various biological factors, as the
tumour localisation, cell type, morphological structure, particular architectural details,
total or partial immunophenotype, degree of mitotic activity. Thus, over expression of
COX-2 in GISTs may be an essential detail and an underlying mechanism of
tumorigenesis.
16.COX-2 expression is observed in 75% of Barrett's oesophagus and cardiac
intestinal metaplasia. The immunohistochemical expression of COX-2 protein in
intestinal metaplasia in the model of Barrett's oesophagus and cardiac intestinal
metaplasia, squamous epithelium, macrophages, plasma cells and erosions diminishes
with increase of the time period elapsed between biopsy and analysis. The remaining
reactivity is linear function of time. The "aging" of blocks cause not only quantitative
but also qualitative changes in COX-2 expression.
17.COX-2 expression correlates inversely with the grade of neoplasia. According
to the obtained data, COX-2 is up-regulated early in the cancerogenesis of oesophagus
and cardia, and may be lost in high-grade cancers due to progressive genomic
instability.
PRACTICAL RECOMMENDATIONS
1. Starting IHC, total test is strongly recommended.
2. The presented work has resulted in protocols for immunohistochemical
visualisation of 48 antigens (table 3) in FFPE tissues by LSAB, ABC or EnVision
systems. These protocols provide background in further standardization of IHC on
local material and can be recommended as a starting point for standardization, when
testing local pathomorphological material. Such focused standardization could result
in even higher cost-effectiveness due to lower number of variations.
Table 3. Standardized IH technologies
Nr.
1.
1
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Antigen
2
CK AE1/A 1-3
CK 7
CK20
CK 8, LMW
CK 5/6
CK HMW
Mesothelial cell
Epithelial antigen
EMA
CEA
TTF-1
SuAA
Vimentin
Desmin
Actin
S-100 protein
GFAP
ChrA
Synaptophysin
NSE
21. AKTH
22. CD45
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
CD3
CD20
CD30
CD31
CD34 class II
CD68
CD117
Melanosome
protein
Ki-67
ER alpha
PR
Thrombomodulin
BCL2
P53 protein
Primary antibody characteristics
3
MM-AH,cloneAEl/AE3,codcM3515
MM-AH, clone OV-TL 12/30, code M7018
MM-AH, clone Ks20.8, code M7019
MM-AH, clone 35betal H11, code M0631
MM-AH,cloneD5/16B4,code M7237
MM-AH, clone 34betaE12, M0630
MM-AH, cloneHBME-l, code M3505
MM-AH, clone Ber-EP4, code M0804
MM-AH, clone E29, code N1504
MM-AH, clone II-7, code Nl586
MM, clone 8G7G3/1, code M3575
MM-AH, clone PE-10, code M4501,
MM, clone 3B4, code M7020
MM, clone D33, code M0760
MM-AH, clone HHF35, code M0635
PR, code Z0311
PR, code Nl 506
PR-AH, code N1535
PR-AH, cat. code N1566
MM-AH, clone BBS/NC/VI-H14, code
N1557
PR, cat. code Nl 531
MM-AH, clones 2B11 and PD7/26, code
M0701
MM-AH, clone PC3/ 188A, code M7193
MM-AH, clone L26, code M0755
MM-AH, clone Ber-H2, code M0751
MM-AH, clone JC70A, code N1596
MM-AH,clone QBEnd 10,codeN 1632
MM-AH, clone PG-M1, M0876
PR-AH CD117, code A4502
MM-AH, clone HMB-45, code M0634
MM-AH, clone MIB-1, code M7240
MM-AH, clone 1D5, code N1575
MM-AH, clone 1A6, code N1595
MM, clone 1009, code M0617
MM-AH, clone 124, code M0887
MM-AH, clone DO-7, code M7001
D
4
1:200
1:800
1:200
1:200
1:100
1:400
1:50
1:800
1:1
1:1
1:100
1:400
1:200
1:200
1:200
1:4000
1:1
1:1
1:1
1:1
T
5
60
60
60
60
60
60
60
60
8
8
60
60
60
60
60
60
8
8
8
5
1:1
1:200
8
60
1:200
1:1000
1:50
1:1
1:1
1:800
1:400
1:200
60
60
60
30
30
60
60
60
1:100
1:1
1:1
1:50
1:400
1:400
60
60
60
60
60
60
1
37.
2
Amyloid A
38. Alpha chains, Ig
39. Gamma chains, Ig
40. Mi chains, lg
41.
42.
43.
44.
45.
Kappa chains, Ig
Lambda chains, Ig
HP
CF Clq
CF C3c
46. CF C4c
47. Fibrinogen
48. vWf
3
4
5
MM-AH, clone mc 1, code M0759
PR-AH, code N1507
PR-AH, code N1508
1:400
1:1
1:1
60
8
PR-AH, code N1509
PR-AH, code Nl510
PR-AH, code N1513
PR, code B0471
PR-AH, code A0136
PR-All, code A0062
PR-AH, code A0065
PR-AH, code A0080
PR-AH, code N1505
1:1
1:1
1:1
1:400
1:400
1:800
1:800
1:400
1:1
8
8
8
8
60
60
60
60
60
5
Abbreviations: Nr., number of the sequence in the table; D, optimal dilution as
determined by total test; T, incubation time; min., minutes; CK, cytokeratin; MMAH, monoclonal mouse antibody against human antigen; code, catalogue code,
DakoCytomation; LMW, low molecular weight; HMW, high molecular weight;
EMA, epithelial membrane antigen; CEA, carcinoembryonic antigen; TTF-1,
thyroid transcription factor-1; MM, monoclonal mouse antibody; SuAA, surfactant
apoprotein A; S-100, S-100 protein; PR, polyclonal rabbit antibody; GFAP, glial
fibrillary antigen protein; ChrA, chromogranin A; PR-AH, polyclonal rabbit
antibody against human antigen; NSE, neuron specific enolase; ACTH,
adrenocorticotropin; CD, cellular determinant; ER, estrogen receptor; PR,
progesteron receptor; Ig, immunoglobulin; HP, Helicobacter pylori; CF,
complement fraction; vWf, von Willebrandt factor.
3. As COX-2 is expressed in many cell types, IHC is the optimal tool for the COX-2
analysis as it allows direct visual identification of the positive elements.
4. However, major differences in the results can be obtained by purely technological
differences therefore the choice of appropriate protocol is of utmost importance.
Based on our results, the following protocol is optimal.
Formalin-fixed paraffin-embedded tissues were cut at 3-micrometer-thickness on
electrostatically charged Histobond glass slides (Menzel Glasser, Germany) and
incubated in 60°C for 1 hour to ensure tissue adhesion to slides. Deparaffinisation
and rehydration was carried out by routine treatment in xylene and graded ethanol.
Endogenous peroxidase activity was blocked by 0.5% hydrogen peroxide in
methanol for 10 minutes. Antigen retrieval was achieved with HIER in domestic
microwave for 3x5 min. at maximum power in TEG buffer (Tris-EGTA buffer, pH
9.0), followed by 20 min. cooling in the same HIER buffer at RT. The incubation
with primary antibody was carried out using Cayman Mab at dilution 1:100 for 60
min. at room temperature. After the incubation with the primary antibodies, slides
were rinsed with
TBS buffer (Tris buffered saline, THAM-HC1 50mM/L, NaCl 150mM/L, pH 7.6),
2x5 min. A commercially available horseradish peroxidase-linked polymer-based
visualisation system EnVisionTM + (DakoCytomation, cat. code 4001) was used for
detection of bound mouse primary antibody, incubating the slides in a humid chamber
for 30 minutes, with following rinse in TBS as previously. 3,3'-diaminobenzidine
(DakoCytomation, cat. code S3000) solution was used as the chromogenic substrate
(10 min). Slides were rinsed in water and counterstained in haematoxylin for 3 min.
After colour development in tap water (5 min.) slides were coverslipped using
aqueous mounting medium Paramount (DakoCytomation, cat. code S3025). Positive
and negative control slides were included in each run.
PUBLICATIONS
1. Immunohistochemical Expression of Cyclooxygenase-2 in Archival Tissues
from the Upper Gastrointestinal Tract: The Influence of Biological and
Technical Variables on Staining Using Commercial Antibodies. I. Strumfa, H.
Hager, B. Norgard, H. T. Sorensen, S.J. Hamilton-Dutoit. In press.
2. Immunohistochemical expression of cyclooxygenase-2 in gastrointestinal
stromal tumours: a complex interplay of biological and technical factors. L.
Strumfa, L. Feldmane, G. Volanska. Accepted for publication in the Scientific
Proceedings of Riga Stradiņš University, 2004.
3. Standardization and diagnostic value of immunohistochemistry in
gastrointestinal stromal tumours. I. Strumfa, L. Feldmane, G. Volanska.
Accepted for publication in the Scientific Proceedings of Riga Stradins
University, 2004.
4. Intrathoracic PNET in an elderly patient with a history of solid tumour. L
Strumfa, U. Kopeika, L. Feldmane, J. Basko. Accepted for publication in the
Acta Chirurgica Latviensis.
5. Diagnostic value of immunohistochemistry in primary pleural and chest wall
tumours. I. Strumfa, L. Feldmane, G. Volanska. Accepted for publication in
the Scientific Proceedings of Riga Stradins University, 2004.
6. Giant intrathoracic tumour associated with hypoglycemia [Hipoglikēmisks sindroms
gigantiska intratorakāla audzēja gadījumā]. K. Ducena, S. Šteina, A. Štifts, V.
Pīrāgs, I. Strumfa, J. Basko. "For You, Colleagues" [„Jums, Kolēģi"],
06/2004, 72-5.
7. Gastrointestinal stromal tumour - up-to-dated concept and diagnostics in
Latvia [Gastrointestināls stromāls tumors - potenciāli ārstējama audzēja
mūsdienīga koncepcija un diagnostika Latvijā]. I. Strumfa, L. Feldmane, G.
Volanska. The Scientific Proceedings of Riga Stradins University: 2003, 381388, 2004, Riga, Latvija.
8. Primary giant cell tumour of bone in the lung. 1. Strumfa, L. Feldmane, G.
Ambalovs, J. Basko. European Respiratory Journal, 2002, Vol. 20, Suppl. 38, 467s.
9. Malignant mesothelioma of pleura. J. Basko, R. Henina, J. Lejnieks, J. Fridlenders,
1. Strum fa. European Respiratory Journal, 2002, Vol. 20, Suppl 38 40s.
l0. Pathoinorphological changes and (heir dynamics (1993-2001) in the upper part of
the digestive tract of Chernobyl clean-up workers residing in Latvia. 1. Strumfa, L.
Feldmane, M. Eglite, E. Churbakova. Proceedings of the Latvian Academy of
Sciences, 2002, Vol. 56, N. 3, 114-120.
1 1.Malignant paraganglioma of the anterior mediastinum. 1. Strumfa, A. Pirtnieks, L.
Feldmane, J Bashko. Proceedings of the 3rd Baltic congress of Oncology, 2002,
Lithuania, 116.
12.Relation between occupational lung diseases and primary malignancies of lung and
pleura in the population of Latvia. I. Strumfa, J. Bashko, L.Feldmane, A. Lange, A.
Pirtnieks, M. Eglite. Proceedings of the 3rd Baltic congress of Oncology, 2002,
Lietuva, 123.
13.Extended pathomorphology diagnostic possibilities in the Institute of Pathology, P.
Stradin's Clinical University hospital [Diagnostisko iespēju paplašināšanas
P.Stradiņa Klīniskās universitātes slimnīcas Patoloģijas institūtā]. L. Feldmane, L
Strumfa. "For You, Colleagues" [Jums, kolēģi; 4/2002, 55-57.
14.Asbestos in Latvia. M. Eglite, J. Jekabsone, I. Lielpetere. In: "The Asbestos
Legacy", Vol. 23 of "The Sourcebook on Asbestos Diseases: Medical. Legal and
Enginecering Aspects", ed. by Peters G. A., Peters B. G., USA: Lexis Nexis Group,
2001,409-423.
15. The quality of work in the Institute of Pathology, P. Stradin's Clinical University
hospital [Darba kvalitātes pacēlums BO VAS "P.Stradiņa Klīniskā Universitātes
slimnīca" Patoloģijas institūtā]. L. Feldmane, I. Strumfa. Quality [Kvalitāte],
5/2001, 18-19.
16.Histological microdissection of complex neoplastic tissue - a new metod for
genetic and proteonomic analysis in cancer diagnostic and research. A Technical
Advance. I. Lielpetere, L.M. Gjerdrum, St. Hamilton - Dutoit. The Proceedings of
the 2nd World Congress of Latvian Scientists [2. Pasaules Latviešu zinātnieku
kongresa tēžu krājums], 2001, 406.
17.The use of tissue microdissection in the genetic analysis of tumours [Kompleksu
audu histoloģiskā lāzera mikrodissekcija - jaunas ģenētiskas analīzes iespējas
audzēju diagnostikā un pētījumos]. I. Lielpētere, L. M. Gjerdrum, St. Hamilton Dutoit. The Proceedings of the 4th World Congress of Latvian doctors [Pasaules
latviešu ārstu 4. kongresa tēzes], 2001, 115-116.
18.The pathomorphology of the upper gastrointestinal mucosa in Chernobyl clean-up
workers [Morfoloģiskas izmaiņas gremošanas trakta augšdaļā Černobiļas AES
katastrofas seku likvidētājiem: ainas dinamika un saistība ar kompleksām izmaiņām
organismā]. I. Lielpētere, L. Feldmane, E. Čurbakova, M. Eglīte. The Proceedings
of the 4th World Congress of Latvian doctors [Pasaules latviešu ārstu 4. kongresa
tēzes], 2001, 116-117.
19.Laser-Assisted Microdissection of Membrane-Mounted Paraffin Sections for
Polymerase Chain Reaction Analysis: Identification of Cell Populations Using
lmmunohistochemistry and In situ Hybridization. L. M. Gjerdrum, I. Lielpētere, St.
Hamilton - Dutoit. The Journal of Molecular Diagnostics, 2001, Vol.3 (3):105-110.
PRESENTATIONS IN CONGRESSES AND CONFERENCES
1. The pathology in Latvia and abroad. I. Strumfa. The Conference of P. Stradin's
Clinical University Hospital, 27.05.2004.
2. GIST. I. Strumfa. The Conference of the Latvian Association of surgeons,
23.05.2003.
3. GIST. I. Strumfa. The Conference of the Latvian Centre of Oncology, 28.03.03.
4. Lung cancer: morphological and immunohistochemical study. I. Strumfa. The
Conference of the Latvian Association of pneumonologists, 20.03.2003.
5. Immunohistochemistry in the diagnostics of gastrointestinal diseases. I. Strumfa.
The Conference of the Latvian Association of Gastrointestinal endoscopy,
26.02.2003.
6. Pathomorphological diagnostics of celiac disease. I. Strumfa. The Conference of the
Latvian Association of Gastrointestinal endoscopy, 26.02.2003.
7. The novelties in the morphological diagnostics of thoracic pathology. I. Strumfa.
The Conference of P. Stradin's Clinical University Hospital, 20.02.2003.
8. The pitfalls in diagnostics and differential diagnostics of gastric and intestinal
pathology. I. Strumfa. The Conference of the Clinics of Internal Diseases, P.
Stradin's Clinical University Hospital, 18.02.2003.
9. The morphology of gastrointestinal lymphomas. I. Strumfa. The Conference of P.
Stradin's Clinical University Hospital, 19.09.2002.
l0. Primary giant cell tumour of bone in the lung. I. Strumfa, L. Feldmane, G.
Ambalovs, J. Basko. 12th European Respiratory Society Annual Congress,
Stockholm, Sweden, 14.-18.09.2002.
11.Malignant mesothelioma of pleura. J. Basko, R. Henina, J. Lejnieks, J.
Fridlenders, I. Strumfa. 12th European Respiratory Society Annual Congress,
Stockholm, Sweden, 14.-18.09.2002.
12.The pleural pathology: morphological and immunohistochemical study. I.
Strumfa. The Conference of the Latvian Association of pneumonologists,
30.05.2002.
13.Malignant paraganglioma of the anterior mediastinum. I. Strumfa, A. Pirtnieks,
L. Feldmane, J Bashko. 3rd Baltic congress of Oncology, 02-04.05.2002, Vilnius,
Lithuania.
14. Relation between occupational lung diseases and primary malignancies of lung
and pleura in the population of Latvia. I. Strumfa, J. Bashko, L. Feldmane, A.
Lange, A. Pirtnieks, M. Eglite. 3rd Baltic congress of Oncology, 03.05.2002,
Vilnius, Lithuania.
15. Case reports in the Conferences of the P. Stradin's Clinical University Hospital:
99 cases. I. Strumfa 2001.-10.2004.
16. Histological microdissection of complex neoplastic tissue - a new metod for
genetic and proteonomic analysis in cancer diagnostic and research. A Technical
Advance. I. Lielpetere, L.M. Gjerdrum, St. Hamilton - Dutoit. The 2nd World
Congress of Latvian Scientists, 14.08.2001. 1
17. The use of tissue microdissection in the genetic analysis of tumours. I. Lielpetere,
L. M. Gjerdrum, St. Hamilton - Dutoit. The 4" World Congress of Latvian
doctors, 22.06.2001.
18.The pathomorphology of the upper gastrointestinal mucosa in Chernobyl clean-up
workers. I. Lielpetere, L. Feldmane, E. Churbakova, M. Eglite. 'The 4th World
Congress of Latvian doctors, 20.-22.06.2001.
19. Immunohistochemistry in diagnostics and research. I. Lielpetere. The Conference
of P. Stradin's Clinical University Hospital, 31.05.2001.
20.The morphology of gastric mucosa in Chernobyl clean-up workers. I. Lielpetere,
L. Feldmane, E. Churbakova. AML/RSU Scientific conference, 25.02.2000.