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141
International Journal of Academic Medicine and Pharmacy (www.academicmed.org)
ISSN (O): 2687-5365; ISSN (P): 2753-6556
TO CORRELATE THE EXPRESSION OF CD97, CD55
& CCK-AR WITH GRADE AND STAGE OF
GALLBLADDER CANCER
Mohd. Anwar1, Priya Singh2, Sachin Kumar3, Sharique Ahmad4, Parul
Gupta5, Saba Naziya6
1Senior Resident, Department of Transfusion Medicine, SGPGI, Lucknow, Uttar Pradesh, India
2Senior Resident, Dr RMLIMS, Uttar Pradesh, India.
3Senior Resident, Department of Pulmonary and critical care Medicine, King George’s Medical
University, Lucknow, Uttar Pradesh, India.
4Professor, Department of Pathology, Era's Lucknow Medical College and Hospital, Era University,
Lucknow, Uttar Pradesh, India.
5Professor, LNCT Medical College Bhopal Madhya Pradesh. India.
6Junior Resident, Department of Pathology, Era's Lucknow Medical College and Hospital, Era
University, Lucknow, Uttar Pradesh. India.
Abstract
Background: India is a high incidence region for gallbladder carcinoma
accounts for 9-11% burden of gall bladder carcinoma worldwide. In the recent
years, in order to understand the pathogenesis and progression of gall bladder
cancer in a better way, emphasis is being laid on assessing its link with various
molecular and genetic markers. Material and Method: In this case control study
we target that on the co-expression of CD97, CD55 and CCK-AR for any
diagnostic and prognostic significance in the Gall bladder cancers with Grade and
Staging. Expression of CD97, CD55 & CCK-AR with in all non-neoplastic and
neoplastic lesions of gall bladder was evaluated and compared. Out of all cases
expression of CD97, CD55 & CCK-AR was significantly higher in malignant
cases as compared to benign cases. Results: Expression, intensity and score of
CCK-AR were found in order mucinous adenocarcinoma, papillary
adenocarcinoma and adenocarcinoma respectively. Conclusion: Out of 56 cases
expression of CD97, CD55 & CCK-AR was significantly higher in malignant
cases as compared to benign cases. Expression, intensity and score of CCK-AR
were found in order mucinous adenocarcinoma, papillary adenocarcinoma and
adenocarcinoma respectively.
INTRODUCTION
Gallbladder(GB) cancer is very aggressive and
untreatable neoplasm representing the commonest
malignancy of biliary tract.[1] In North India
population, it is the 3rd most common malignancy in
female with an overall 5-year survival of <5%. No
adjuvant chemotherapy is widely accepted due to the
toxic effect of drug, resistance and less efficacy and
therefore surgical approach is followed for cure. Both
environmental and epidemiology factors play a
critical role in cancer developing in gallbladder
cancer, best illustrate by chronic inflammation and
cholelithiasis.[2] In the recent years, in order to
understand the pathogenesis and progression of gall
bladder cancer in a better way, emphasis is being laid
on assessing its link with various molecular and
genetic markers. Recently Wu et al.[3] Expression of
CD55 and CD97 in primary gallbladder cancer and
their prognostic significance in a Chinese population.
CD97 is expressed in series of epithelial cancer and
also normal tissues. It is a member of epidermal
growth factor seven span transmembrane (EGF TM7)
families with adhesive properties.[4] CD97 exists in a
variety of splice forms, each of which binds CD55
with different affinity. CD97 is expressed in many of
human neoplasm like stomach, colon, thyroid and
brain.
CD97 expression shows an invasive phenotype and
also correlate with tumor grade, metastatic spread,
lymph node invasion, and overall prognosis. CD97
have role in tumor invasion, signaling and novel
therapeutic target.[5] Similarly, decay accelerating
factor (DAF/CD55) is a major negative regulator of
the complement cascade expressed by cells to prevent
complement mediated destruction and was the first
known ligand of CD97. CD55 binds to the small
CD97 isoforms and this interaction is calcium
dependent and mediated by short consensus repeat
(SCR) domain of CD55 and EGF like domain of
CD97.[6,7,8,9] Surrounding inflammation leads to
upregulation of CD55 expression among epithelial
RESEARCH
Received : 19/04/2022
Received in revised form : 25/07/2022
Accepted : 02/08/2022
Keywords:
Gall bladder cancer,
Cholelithiasis,
CD97, CD55, CCK-AR.
Corresponding Author:
Dr. Sharique Ahmad,
Email: diagnopath@gmail.com
ORCID: 0000-0002-9637-8838
DOI: 10.47009/jamp.2022.4.3.32
Source of Support: Nil,
Conflict of Interest: None declared
Int J Acad Med Pharm,
2022; 4 (3); 141-147
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International Journal of Academic Medicine and Pharmacy (www.academicmed.org)
ISSN (O): 2687-5365; ISSN (P): 2753-6556
and endothelial cells. Co- localization of CD97 with
CD55 appear to play an important role in malignant
state.[10] Regulatory peptide hormones are
Cytokeratin and gastrin having a wide range of
physiological actions in gastrointestinal tract.
Cytokeratin have shown growth stimulation in
several neoplasms and their receptors have been
found to be expressed in variety of human neoplastic
tissues.[11,12] Further, CCK is a gall bladder modulator
having motility by activating CCK-AR distributed on
gall bladder smooth muscle cells and abnormal
processing of the CCK-AR gene is associated with
the gall bladder lesions.[13,14] A search of biological
markers associated with advanced stage of tumor
progression is necessary for early diagnosis and a
discovery of a therapeutic target. Hence, in present
study we target that on the co-expression of CD97,
CD55 and CCK-AR for any diagnostic and
prognostic significance in the GB cancers.
Aim
To evaluate and compare the expression of CD97,
CD55 & CCK-AR in non-neoplastic and neoplastic
lesions of gall bladder cancer.
The expression of these markers was compared and
correlated with respect to grade and stage of cancer.
MATERIALS AND METHODS
A case control study with total sample size of 56
patients which included cholecystectomy specimens
received in the pathology department Era’s Lucknow
Medical College and Hospital, Lucknow. It
comprised 28 cases of gallbladder adenocarcinoma
and 28 randomly selected cases of cholelithiasis
associated chronic cholecystitis. Tissue sections were
fixed in 10% buffered formalin and embedded in
paraffin wax and retrieved from our archives, re-cut,
stained with hematoxylin & eosin and reviewed by 3
pathologists using a light microscope. In gallbladder
cancer cases, tumor differentiation was assessed.
Tumors were divided into 3 groups regarding
differentiation (well, moderate and poor). The
immunohistochemistry was applied for CD97, CD55,
CCK-AR on formalin fixed paraffin embedded
tissues. The results were evaluated according to the
intensity of staining pattern of the scoring system
used for breast cancer, as there is no standard scoring
system for gall bladder cancer. Intensity of staining
was graded as, Grade 0- Negative, Grade 1- Weak,
Grade 2-Moderate, Grade 3 -Strong. The percentage
of cells showing staining was graded as: -None - 0,
Grade 1 - <1%, Grade 2 -1%.
10%, Grade 3 -11%-33%, Grade 4 -34%- 66%, Grade
5 - >66%. Total staining score was calculated by
adding the intensity score and the percentage score as
Negative-0, Weak = + (2), Moderate=2+ (3-5),
Strong=3+ (6-8).
RESULTS
Expression of CD97, CD55 & CCK-AR within 56
non-neoplastic and neoplastic lesions of gall bladder
was evaluated and compared.
[Table 1] shows the histopathological diagnosis of
study population.
Table 1: Histopathological Diagnosis of Study
Population
Diagnosis
No. of cases
Percentage
Cholelithiasis
28
50.00
Adenocarcinoma
20
35.71
Mucinous adenocarcinoma
5
8.93
Papillary adenocarcinoma
3
5.36
Out of 56 cases of gall bladder lesions,
histopathological diagnosis of 28 (50.0%) was
Cholelithiasis, only 3 (5.36%) patients were
diagnosed as Papillary adenocarcinoma, 5 (8.93%) as
mucinous adenocarcinoma and rest 20 (35.71%) as
adenocarcinoma. All the cases of cholelithiasis were
classified as Benign lesions (50.0%) and rest of the
cases were classified as Malignant lesions (50.0%).
Age of patients in the present study ranged between
21 and 70 years, median age of patients was 42 years
and mean age of patients was 42.45±14.56 years. In
the present study age of 50.0% patients was up to 40
years and of rest 50.0% were aged above 40 years.
All the patients with benign lesions were lower aged
i.e. 21-30 years (64.28%) and 31-40 years (35.72%)
while all the patients with malignant lesions were
aged above 41 years and this was found to be
statistically significant (p<0.001). Out of 56 cases of
gall bladder lesions, 47 (83.93%) were females and
rest 9 (16.07%) were males. Male to female ratio in
the present study was 0.19. Though proportion of
males was higher among benign (21.43%) as
compared to malignant (10.71%) but this difference
was not significant. All the patients of gall bladder
lesions were subjected to USG investigation,
Cholelithiasis was seen in majority of the patients
(n=46; 82.14%) cases. All the benign cases had
cholelithiasis whereas a total of 10 out of 28
(35.72%) cases with malignancy did not have
cholelithiasis. Statistically, this difference was
significant (p<0.001).
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International Journal of Academic Medicine and Pharmacy (www.academicmed.org)
ISSN (O): 2687-5365; ISSN (P): 2753-6556
Table 2: Histopathological Grade of Malignant Cases
(n=28)
Grade
No. of cases
Percentage
Well differentiated
19
67.86
Moderately differentiated
4
14.29
Poorly differentiated
5
17.86
Out of 28 malignant cases, histopathological grade of
19 (67.86%) cases was Well differentiated, of 5
(17.86%) cases was Poorly differentiated and rest of
the cases were diagnosed as Moderately
differentiated (n=4; 14.29%).
Table 3: Stage wise distribution of Malignant Cases
(n=28)
Stage
No. of cases
Percentage
Stage I
7
25.0
Stage II
15
53.6
Stage III
6
21.4
Majority of patients were Stage II (n=15; 53.6%),
followed by Stage I (25.0%) while least common
stage was Stage III (n=7; 24.1%). Difference in CD97
expression among malignant (82.86%) and benign
lesions (82.14%) was not found to be statistically
significant. Mean Expression of CD97 was found to
be significantly higher among cases with malignant
lesion (2.61±1.03) as compared to benign lesions
(1.50±0.97). Mean Intensity of CD97 was found to be
significantly higher among cases with malignant
lesion (1.89±0.79) as compared to benign lesions
(1.18±0.72). Mean Score of CD97 was found to be
significantly higher among cases with malignant
lesion (5.36±2.88) as compared to benign lesions
(2.11±1.45). Based on the direction of assessment,
CD97 total score was evaluated for prediction of
malignancy of gall bladder lesion at a cut-off with a
larger value indicating positive result. Area under
curve findings were 0.851 (indicating a projected
accuracy of 85.1%) for CD97 total score. On
evaluating CD97 total score, a cut off value ≥3.50
was predicted to be 78.6% sensitive and 75.0%
specific. Cut- off value of CD97 total score with high
sensitivity was >1.50 which was 92.9% sensitive and
35.7% specific while at a cut off value with high
specificity was ≥5.00 which was 46.4% sensitive but
100.0% specific. Difference in CD55 expression
among malignant (82.86%) and benign lesions
(82.14%) was not found to be statistically significant.
Mean expression of CD55 was found to be
significantly higher among cases with malignant
lesion (2.57±1.07) as compared to benign lesions
(1.50±0.96). Mean Intensity of CD55 was found to be
significantly higher among cases with malignant
lesion (1.93±0.77) as compared to benign lesions
(1.18±0.72). Mean Score of CD55 was found to be
significantly higher among cases with malignant
lesion (5.36±2.88) as compared to benign lesions
(2.11±1.45). Based on the direction of assessment,
CD55 total score was evaluated for prediction of
malignancy of gall bladder lesion at a cut-off with a
larger value indicating positive result. Area under
curve findings were 0.851 (indicating a projected
accuracy of 85.1%) for CD55 total score. On
evaluating CD55 total score, a cut off value ≥3.50
was predicted to be 78.6% sensitive and 75.0%
specific. Cut- off value of CD55 total score with high
sensitivity was >1.50 which was 92.9% sensitive and
35.7% specific while at a cut off value with high
specificity was ≥5.50 which was 46.4% sensitive but
100.0% specific. CCK-AR expression among
malignant cases (85.71%) was significantly higher as
compared to Benign cases (39.29%). Mean
expression of CCK- AR was found to be significantly
higher among cases with malignant lesion
(2.54±1.40) as compared to benign lesions
(0.32±0.46). Mean Intensity of CCK-AR was found
to be significantly higher among cases with
malignant lesion (1.96±1.03) as compared to benign
lesions (0.32±0.46). Mean Score of CCK-AR was
found to be significantly higher among cases with
malignant lesion (4.50±2.30) as compared to benign
lesions (0.64±0.91). Based on the direction of
assessment, CCK- AR total score was evaluated for
prediction of malignancy of gall bladder lesion at a
cut-off with a larger value indicating positive result.
Area under curve findings were 0.895 (indicating a
projected accuracy of 89.5%) for CCK-AR total
score. On evaluating CCK-AR total score, a cut off
value ≥2.50 was predicted to be 82.1% sensitive and
96.4% specific. Cut-off value of CCK-AR total score
with high sensitivity was >1.50 which was 85.7%
sensitive and 78.6% specific while at a cut off value
with high specificity was ≥3.50 which was 75.0%
sensitive but 100.0% specific.
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ISSN (O): 2687-5365; ISSN (P): 2753-6556
Table 4: Comparison of CD55, CD97 and CCK-AR Total Scores for different carcinoma types
Adeno- carcinoma
(n=20)
Mucinous adeno-
carcinoma (n=5)
Papillary adeno-
carcinoma (n=3)
Kruskal Wallis Test
Mean
SD
Mean
SD
Mean
SD
H
‘p’
CD97
Expression
2.65
1.18
2.40
0.55
2.67
0.58
0.815
0.665
Intensity
1.95
0.83
2.00
0.71
1.33
0.58
2.735
0.255
Score
5.85
3.22
4.60
1.34
3.33
0.58
4.232
0.121
CD55
Expression
2.60
1.23
2.80
0.45
2.00
0.00
2.381
0.304
Intensity
2.00
0.79
1.60
0.89
2.00
0.00
2.030
0.362
Score
5.85
3.22
4.20
1.64
4.00
0.00
3.339
0.188
CCK-AR
Expression
2.15
1.42
3.80
0.45
3.00
1.00
7.245
0.027
Intensity
1.60
0.99
3.00
0.00
2.67
0.58
11.362
0.003
Score
3.75
2.27
6.80
0.45
5.67
1.15
10.873
0.004
Among different markers, CCK-AR was found to have a significant association with different carcinoma types,
with mean values of expression scores, Intensity scores and total scores being in order mucinous adenocarcinoma
followed by papillary adenocarcinoma and adenocarcinoma respectively.
Table 5: Comparison of CD55, CD97 and CCK-AR Total scores for different histopathological grades
Well differentiate d
(n=19)
Moderately
differentiate d (n=4)
Poorly differentiate d
(n=5)
Kruskal Wallis Test
Mean
SD
Mean
SD
Mean
SD
H
‘p’
CD97
Expression
2.16
0.90
3.50
0.58
3.60
0.55
13.941
0.001
Intensity
1.68
0.82
2.00
0.00
2.60
0.55
6.292
0.043
Score
3.95
1.87
7.00
1.15
9.40
2.61
15.369
<0.001
CD55
Expression
2.11
0.94
3.50
0.58
3.60
0.55
13.750
0.001
Intensity
1.74
0.81
2.00
0.00
2.60
0.55
6.104
0.047
Score
3.95
1.87
7.00
1.15
9.40
2.61
15.369
<0.001
CCK-AR
Expressio n
2.89
1.41
1.50
1.29
2.00
2.89
5.568
0.062
Intensity
2.00
1.11
1.50
1.00
2.20
2.00
1.357
0.507
Score
4.89
2.40
3.00
2.16
4.20
4.89
3.853
0.146
With increasing grade there was a significant increase in expression of CD97 and CD55 markers but not for CCK-
AR.
Table 6: Comparison of Sensitivity and Specificity of different markers for malignancy (Based on Positive Expression)
Marker
TP
FP
FN
TN
Sens
Spec
PPV
NPV
CD97
26
2
23
5
92.9
17.9
53.1
71.4
CD55
26
2
23
5
92.9
17.9
53.1
71.4
CCK-AR
24
4
11
17
85.7
60.7
68.6
81.0
CD97 vs CD55: 2=0.000; p=1.000(NS) CD97 vs CCK-AR: 2=11.5; p=0.009(Sig) CCK-AR vs CD55: 2=11.5;
p=0.009(Sig)
CD97 and CD55 had higher sensitivity as compared to CCK-AR while CCK-AR had higher specificity as
compared to CD-97 and CD-55 and the difference among these was significant statistically.
Table 7: Comparison of CD55, CD97 and CCK-AR Total scores for different histopathological grades
Stage I (n=7)
Stage II (n=15)
Stage III (n=7)
Kruskal Wallis Test
Mean
SD
Mean
SD
Mean
SD
H
‘p’
CD97
Expression
2.29
0.49
2.40
1.18
3.50
0.55
7.794
0.020
Intensity
1.71
0.76
1.73
0.80
2.50
0.55
5.413
0.067
Score
3.71
1.25
4.73
2.40
8.83
2.71
11.405
0.003
CD55
Expression
2.29
0.49
2.27
1.16
3.67
0.52
10.027
0.007
Intensity
2.00
0.58
1.67
0.82
2.50
0.55
5.736
0.057
Score
4.43
1.13
4.27
2.34
9.17
2.40
11.897
0.003
CCK-AR
Expression
2.71
1.38
2.60
1.59
2.17
0.98
1.288
0.525
Intensity
2.00
1.00
1.87
1.19
2.17
0.75
0.097
0.953
Score
4.71
2.21
4.47
2.67
4.33
1.63
0.430
0.807
With increasing stage there was a significant increase in expression and score of CD97 and CD55 markers but not
for CCK-AR.
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International Journal of Academic Medicine and Pharmacy (www.academicmed.org)
ISSN (O): 2687-5365; ISSN (P): 2753-6556
DISCUSSION
One of the reasons for poor outcome related with gall
bladder carcinoma is the fact that it is diagnosed at an
advanced stage. The commonly performed
histopathological evaluation is a cumbersome task
and has a limited prognostic value. Keeping in view
of the emerging role of biological markers in
evaluation of gall bladder carcinoma, the present
study was planned to study the expression of CD97,
CD55 and CCK-AR with grade and stage of gall
bladder cancer in order to mainly evaluate its role as
a diagnostic and prognostic marker. For this purpose,
a study was carried out in which a total of 56 cases
(28 cholelithiasis and 28 gall bladder carcinoma)
were enrolled. The sampling was done using a
purposive sampling design as incidence of gall
bladder carcinoma is quite low ranging from 0.5-
1.5% cases undergoing cholecystectomy,[15] and
hence it was difficult get the adequate number of gall
bladder carcinoma cases in a cross-sectional or
prospective evaluation. In present study, all the cases
of gall bladder carcinoma were adenocarcinoma.
These findings are in consonance with the
epidemiological studies that show that
adenocarcinoma is the major type of gall bladder
cancer found in nearly 75-85% of total gall bladder
cancer cases.[16] Among different subtypes of
adenocarcinoma non-papillary adenocarcinoma is
most common in present study to 20/28 (71.4%) of
cases were non-papillary adenocarcinoma while 5/28
(17.9%) were mucinous adenocarcinoma and 3/28
(10.7%) were papillary adenocarcinoma. With
respect to expression of different biological markers,
expression of CD97 was seen in 82.14% of benign
and 92.86% of malignant cases. Statistically, this
difference was not significant. Compared to this, Wu
et al.3 and Meng et al. (2017),[17] both absence of
expression or a weak expression were treated at par
as absence of CD97 expression. In present study, in
order to increase the sensitivity of CD97, weak
expression was also categorically accepted as
expression, however, the overall discriminant
classification of expression was done on the basis of
combined quick score derived as product of
expression and intensity and through receiver
operator characteristic curve analysis derived a cut-
off value >3.50 i.e. 3 to be 78.6% sensitive and 75%
specific in differentiating malignant from benign gall
bladder disease. The findings of present study are
promising from the point of view that they were able
to transform the non- discriminant role of CD97 into
a discriminant one.[18] However, the method of CD97
expression used in present study is being employed
for the first time and hence validation of same is
essential.
In present study, the role of CD55 marker was also
evaluated in terms of categorical expression,
expression scores, intensity and combined scores. As
far as categorical expression was concerned, it failed
to discriminate between benign and malignant gall
bladder diseases, however, expression scores,
intensity and combined scores of malignant cases
were found to be significantly higher as compared to
that in benign cases. In present study, CD55
expression was seen in 82.14% of benign and 92.86%
of malignant cases. Compared to this Wu et al.[3] in
their study evaluated only malignant cases and found
the CD55 expression in 90/138 (65.2%) cases.
However, the criteria for positive expression was
relatively strict in their study, comparable to quick
score 4 or above. Meng et al,[17] (2017) too in their
study found CD55 expression in 70.4% of
cholangiocarcinoma patients. A comparison of CD55
expression between benign and malignant cases was
observed in endometrium tissue by Murray et al,[19]
who assessed the expression of CD55 in terms of
optical density in quantitative terms and found a
significant difference between malignant and benign
lesions. In present study too, using a semi
quantitative approach we were able to differentiate
between malignant and benign lesions. Subsequently,
in order to derive a cut-off value for discrimination
between malignant and benign lesions with the help
of CD55 expression scores (quick scores) we
performed a receiver operator characteristic curve
analysis and derived an area under curve value of
0.851. The analysis provided a cut-off value of >3.50
(>3) which showed to have a projected sensitivity and
specificity of 78.6% and 75% respectively. The Area
under curve value derived for CD97 and CD55
showed a similar trend, thus showing that the two
markers are generally complementary. CD55 is
identified as a ligand to CD976 and binding of CD97
to its ligand might be responsible for development of
cancer. Meng et al,[17] in their study showed
coexpression of CD55 and CD97 in 47/71 (66.2%) of
their malignant cases. In their study, independent
expression of CD97 and CD55 was seen in 7/71
(9.9%) and 3/71 (4.2%) cases only.
In present study we did not perform any such analysis
as we had performed a semi quantitative analysis
instead of a qualitative assessment performed by
Meng et al,[17] however the close area under curve
values and projected sensitivity and specificity values
of CD97 and CD55 the possibility of coexpression
cannot be ruled out. CCK is a potent modulator of
gall bladder motility by activating CCK-AR
distributed on gall bladder smooth muscle cells and
abnormal processing of the CCK-AR gene is
associated with the gall bladder lesions.[20] In present
study, CCK-AR expression was seen in 39.29% of
cholelithiasis (benign) cases as compared to 85.71%
of gall bladder cancer (malignant) cases. Similar to
present study, Rai et al,[21] too compared CCK-AR
expression between gallstone disease and gall
bladder cancer patients and found CCK-AR
expression in 44.1% of gall stone disease and 76.6%
of gall bladder cancer patients, thereby showing a
significant difference between two.[22,23,24] Xu et
al,[25] in their study also showed that CCK
polymorphism increases the gall bladder
susceptibility. Kazmi et al,[22] in their study showed
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ISSN (O): 2687-5365; ISSN (P): 2753-6556
that the CCK-AR expression is affected by the age
and aggressiveness of gall bladder disease.
Unfortunately, there are limited studies on this
biomarker using the evaluation method similar to
ours. However, its potential in causation and
progression of gall bladder cancer is accepted by a
number of workers.[17,21,22,23] In one such study
evaluating expression of CCK-AR between normal,
gallstone and gall bladder cancer disease, the
expression levels were found to be 62.5%, 86.6% and
52.6% respectively for normal, gallstone and gall
bladder cancer respectively.[23] Thus showing higher
expression in gallstone disease as compared to that in
malignant disease, as observed in present study.
In present study, on performing ROC analysis, we
obtained area under curve value of 0.895 and at
projected cut-off for quick score >2.50(>2) the
projected sensitivity and specificity of CCK-AR in
distinguishing malignant from the benign cases was
82.1% and 96.4% respectively. This finding shows
that CCK-AR probably has a higher utility in
distinguishing benign from malignant cases. Most of
the literature on CD97, CD55 and CCK-AR
biomarkers has revolved around their prognostic
value and not as a diagnostic marker and these
assessments were mainly limited to assessment
within the malignant group only. In present study too,
we studied the association of these biomarkers for
different carcinoma types, histological grades and
clinical stages. When evaluating the usefulness of
three biomarkers under study (CD97, CD55 and
CCK-AR) for differentiation among different
carcinoma types, we found that only CCK-AR
expression, intensity and quick scores had a
significant difference in mean values among different
types of carcinoma. It was seen that mean values
were maximum for mucinous adenocarcinoma and
minimum for non-papillary adenocarcinoma.
However, CD97 and CD55 did not show any such
association. On evaluating the literature, we did not
find any study exploring and reporting any such
association, however, given the higher potential of
mucinous adenocarcinoma to metastasize, the
increased levels of CCK-AR in mucinous carcinoma
cannot be ruled out. With respect to association with
histological grades, in present study, both CD97 and
CD55 showed a significant increase in expression,
intensity and quick scores when evaluating them in
well differentiated, moderately differentiated and
poorly differentiated grades. A similar association
between CD97 and CD55 expression with
histological grade has also been reported by Meng et
al,[17] who reported CD97 expression to be 28.6%,
77.3% and 90.0% respectively in histological grade
1, 2 and 3 respectively and CD55 expression to be
28.6%, 65.9% and 95.0% respectively for the
corresponding histopathological grades. In another
study Wu et al,[3] also showed a significant increasing
expression of both CD97 and CD55 with the
increasing histopathological grade. In present study,
although we could not find a significant difference in
mean expression, intensity and quick scores of CCK-
AR, however, Rai et al,[21] in their study found that
CCK-AR levels in poorly differentiated form were
significantly lower as compared to that in moderately
differentiated cases. However, on evaluating the
association of expression of three biomarkers being
studied with the clinical stage, the only significant
association was seen for CD97 expression and quick
scores only which showed a significantly increase
with increasing clinical stage of cancer. On the other
hand, Meng et al,[17] in their study did not evaluate
the association of CD97 and CD55 with clinical
stage, however, on evaluation this association with
tumor size they did not find a significant association
between tumor size and CD97 or CD55 expression.
However, Wu et al,[3] in their study similar to our
study also reported a significant association between
increased expression of CD97 and CD55 with
increasing stage of cancer. In present study, although
we could not derive a significant association between
clinical stage and CCK-AR expression, however, Rai
et al,[21] in their study reported a significant difference
in CCK-AR expression between TNM stage II and III
but failed to find out significant difference between
stage II and IV and III and IV.
There were three major limitations in present study,
first was evaluation of association of CD97, CD55
and CCK-AR biomarkers between benign and
malignant group. In fact, diagnostic role of these
markers is not much discussed rather their prognostic
role or association with clinicopathological variables
predictive of outcome has been evaluated so far.
Hence, we did not have ample evidence to compare
the results of present study with earlier studies with
respect to discriminant role of CD97, CD55 and
CCK-AR in distinguishing benign from malignant
gall bladder carcinoma cases. Secondly, the sample
size for gall bladder cancer group was too small,
owing to which assessments with clinical stage,
histopathological grade and carcinoma type, though
performed, could not provide exactly comparable
results as depicted in previous studies owing to fewer
number of cases for each grade, stage and cancer
type. Moreover, we have limited number of clinical
variables available. The third major limitation was
availability of limited data depictive of prognosis and
clinicopathological profile of patients. In present
study we did not have data related with lymph node
involvement and other relevant parameters. In view
of these limitations further studies should be
conducted on a larger sample size with inclusion of
more variables and survival as an outcome.
CONCLUSION
Out of 56 cases expression of CD97, CD55 & CCK-
AR was significantly higher in malignant cases as
compared to benign cases. Expression, intensity and
score of CCK-AR were found in order mucinous
adenocarcinoma, papillary adenocarcinoma and
adenocarcinoma respectively. This association was
supported statistically. Sensitivity, specificity, PPV,
147
International Journal of Academic Medicine and Pharmacy (www.academicmed.org)
ISSN (O): 2687-5365; ISSN (P): 2753-6556
NPV of CD97 and CD55 were found to be similar,
while sensitivity of CD97 and CD55 was higher as
compared to CCK-AR and specificity. PPV and NPV
of CCK-AR was higher as compared to CD97 and
CD55. With increasing grade and stage there was a
significant increase in expression of CD97 and CD55
markers but not for CCK-AR.
REFERENCES
1. Piehler JM, Crichlow RW. Primary carcinoma of the
gallbladder. Surg Gynecol Obstet. 1978;147(6):929-42.
2. Rakić M, Patrlj L, Kopljar M, Kliček R, Kolovrat M, Loncar
B, et al. Gallbladder cancer. Hepatobiliary Surg Nutr.
2014;3(5):221-6. doi: 10.3978/j.issn.2304-3881.2014.09.03.
3. Wu LC, Chen LT, Tsai YJ, Lin CM, Lin CY, Tian YF, et al.
Alpha-methylacyl coenzyme A racemase overexpression in
gallbladder carcinoma confers an independent prognostic
indicator. J Clin Pathol. 2012;65(4):309-14. doi:
10.1136/jclinpath-2011-200489.
4. Kwakkenbos MJ, Kop EN, Stacey M, Matmati M, Gordon S,
Lin HH, et al. The EGF-TM7 family: a postgenomic view.
Immunogenetics. 2004;55(10):655-66. doi: 10.1007/s00251-
003-0625-2.
5. Safaee M, Clark AJ, Ivan ME, Oh MC, Bloch O, Sun MZ, et
al. CD97 is a multifunctional leukocyte receptor with distinct
roles in human cancers (Review). Int J Oncol.
2013;43(5):1343-50. doi: 10.3892/ijo.2013.2075.
6. Hamann J, Vogel B, van Schijndel GM, van Lier RA. The
seven-span transmembrane receptor CD97 has a cellular
ligand (CD55, DAF). J Exp Med. 1996;184(3):1185-9. doi:
10.1084/jem.184.3.1185.
7. Liu J, Miwa T, Hilliard B, Chen Y, Lambris JD, Wells AD, et
al. The complement inhibitory protein DAF (CD55)
suppresses T cell immunity in vivo. J Exp Med.
2005;201(4):567-77. doi: 10.1084/jem.20040863.
8. Niu M, Xu S, Yang J, Yao D, Li N, Yan J, Zhong G, Song G.
Structural basis for CD97 recognition of the decay-
accelerating factor CD55 suggests mechanosensitive
activation of adhesion GPCRs. J Biol Chem.
2021;296:100776. doi: 10.1016/j.jbc.2021.100776.
9. Lublin DM, Atkinson JP. Decay-accelerating factor:
biochemistry, molecular biology, and function. Annu Rev
Immunol. 1989;7:35-58. doi:
10.1146/annurev.iy.07.040189.000343.
10. Li L, Spendlove I, Morgan J, Durrant LG. CD55 is over-
expressed in the tumour environment. Br J Cancer.
2001;84(1):80-6. doi: 10.1054/bjoc.2000.1570.
11. Thomas RP, Hellmich MR, Townsend CM Jr, Evers BM. Role
of gastrointestinal hormones in the proliferation of normal and
neoplastic tissues. Endocr Rev. 2003;24(5):571-99. doi:
10.1210/er.2002-0028.
12. Baldwin GS, Shulkes A. CCK receptors and cancer. Curr Top
Med Chem. 2007;7(12):1232-8. doi:
10.2174/156802607780960492.
13. Ivy AC, Oldberg A. A hormone mechanism for gallbladder
contraction and evacuation. Am J Physiol 1928; 86: 599-613.
14. Miller LJ, Holicky EL, Ulrich CD, Wieben ED. Abnormal
processing of the human cholecystokinin receptor gene in
association with gallstones and obesity. Gastroenterology.
1995;109(4):1375-80. doi: 10.1016/0016-5085(95)90601-0.
15. Lai CH, Lau WY. Gallbladder cancer--a comprehensive
review. Surgeon. 2008;6(2):101-10. doi: 10.1016/s1479-
666x(08)80073-x.
16. Brandt-Rauf PW, Pincus M, Adelson S. Cancer of the
gallbladder: a review of forty-three cases. Hum Pathol.
1982;13(1):48-53. doi: 10.1016/s0046-8177(82)80138-x.
17. Meng ZW, Liu MC, Hong HJ, Du Q, Chen YL. Expression
and prognostic value of soluble CD97 and its ligand CD55 in
intrahepatic cholangiocarcinoma. Tumour Biol.
2017;39(3):1010428317694319. doi:
10.1177/1010428317694319.
18. Nabhan F, Ringel MD. Thyroid nodules and cancer
management guidelines: comparisons and controversies.
Endocr Relat Cancer. 2017;24(2):R13-R26. doi:
10.1530/ERC-16-0432.
19. Murray KP, Mathure S, Kaul R, Khan S, Carson LF, Twiggs
LB, et al. Expression of complement regulatory proteins-CD
35, CD 46, CD 55, and CD 59-in benign and malignant
endometrial tissue. Gynecol Oncol. 2000;76(2):176-82. doi:
10.1006/gyno.1999.5614.
20. Miller LJ, Holicky EL, Ulrich CD, Wieben ED. Abnormal
processing of the human cholecystokinin receptor gene in
association with gallstones and obesity. Gastroenterology.
1995;109(4):1375-80. doi: 10.1016/0016-5085(95)90601-0.
21. Rai R, Tewari M, Kumar M, Singh TB, Shukla HS.
Expression profile of cholecystokinin type-A receptor in
gallbladder cancer and gallstone disease. Hepatobiliary
Pancreat Dis Int. 2011;10(4):408-14. doi: 10.1016/s1499-
3872(11)60069-6.
22. Kazmi HR, Chandra A, Baghel K, Singh A, Nigam J, Parmar
D, et al. Differential expression of cholecystokinin A receptor
in gallbladder cancer in the young and elderly suggests two
subsets of the same disease? Biomed Res Int.
2014;2014:625695. doi: 10.1155/2014/625695.
23. Faridi MS, Jaiswal MS, Goel SK. Expression of CCK
Receptors in Carcinoma Gallbladder and Cholelithiasis: A
Pilot Study. J Clin Diagn Res. 2015;9(7):PC04-7. doi:
10.7860/JCDR/2015/12697.6152.
24. Rustagi T, Dasanu CA. Risk factors for gallbladder cancer and
cholangiocarcinoma: similarities, differences and updates. J
Gastrointest Cancer. 2012;43(2):137-47.
25. Xu HL, Hsing AW, Vogtmann E, Chu LW, Cheng JR, Gao J,
et al. Variants in CCK and CCKAR genes to susceptibility to
biliary tract cancers and stones: a population-based study in
Shanghai, China. J Gastroenterol Hepatol. 2013;28(9):1476-
81. doi: 10.1111/jgh.12278.
