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Early View Article: Online published version of an accepted article before publication in the
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Journal Name: International Journal of Hepatobiliary and Pancreatic Diseases (IJHPD)
Type of Article: Original Article
Title: Expression of Her-2/neu receptor in Carcinoma Gallbladder correlates significantly
with advanced tumour stage
Authors: Farzana Ashai, Aadil Ashraf, Arshad Rashid, Bilal Musharaf Banday, Sunita
Bhalla, Shashi Dhawan
doi: To be assigned
How to cite the article: Ashai F, Ashraf A, Rashid A, Banday B.M, Bhalla S, Dhawan S,
Expression of Her-2/neu receptor in Carcinoma Gallbladder correlates significantly with
advanced tumour stage International Journal of Hepatobiliary and Pancreatic Diseases
(IJHPD) Forthcoming 2015.
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TYPE OF ARTICLE: Original Article 1
2
TITLE: Expression of Her-2/neu receptor in Carcinoma Gallbladder correlates 3
significantly with advanced tumour stage 4
5
AUTHORS: 6
Author Information: Farzana Ashai, DNB1; Aadil Ashraf, MD2; Arshad Rashid, MS, 7
FNB (MAS)3*; Bilal Musharaf Banday, MD2; Sunita Bhalla, MD4; Shashi Dhawan, 8
MD4. 9
10
AFFILIATIONS: 11
1Senior Resident, Department of Pathology, Sheri Kashmir Institute of Medical 12
Science Medical College, Srinagar – 190015. (Current Affiliation) 13
2Post Graduate Scholar, Department of Pathology, Sir Ganga Ram Hospital, 14
Rajender Nagar, New Delhi – 110060, India. (At the time of Study) 15
3Senior Resident, Department of Pathology, Government Medical College, Srinagar 16
– 190010. 17
4Medical Officer, ASYM District Hospital, Budgam, Health Services Kashmir – 18
190014. 19
Senior Consultant, Department of Pathology, Sir Ganga Ram Hospital, Rajender 20
Nagar, New Delhi – 110060, India. 21
22
CORRESPONDING AUTHOR DETAILS 23
24
Dr Arshad Rashid 25
G – 22, Shah Anwar Colony, Hyderpora, Srinagar (INDIA) - 190014 26
Email: [email protected]; 27
Phone number: +918494095736 28
29
Short Running Title: Her-2/neu in Gallbladder Cancer. 30
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31
Guarantor of Submission : The corresponding author is the guarantor of 32
submission 33
34
TITLE: Expression of Her-2/neu receptor in Carcinoma Gallbladder correlates 35
significantly with advanced tumour stage 36
37
ABSTRACT 38
39
Aims 40
Overexpression of Her-2/neu is associated with increased disease recurrence and 41
worse prognosis in many cancers. The aim of this study was to correlate Her-2/neu 42
positivity with the histopathologic features in carcinoma gallbladder. 43
44
Methods 45
This study was conducted on 54 cholecystectomy specimens of carcinoma 46
gallbladder in a superspeciality referral center from January 2007 to July 2011. A 47
gross evaluation of tumor, study of histopathology and immunohistochemical staining 48
was done for Her-2/neu. The relative frequency of Her-2/neu positivity was scored 49
and correlated with other histological prognostic parameters of the tumour. 50
51
Results 52
Maximum cases in our study were in stage T2 at the time of diagnosis and most of 53
them were moderately differentiated carcinoma. Her-2/neu was strongly positive in 5 54
cases (9.25%) of gallbladder cancer and 14 cases (25.92%) showed moderate 55
positivity. There was a lot of heterogeneity in the expression of Her-2/neu showing 56
1+ and 2+ positivity in different areas. No correlation was seen between 57
immunohistochemical staining pattern and tumour grade, but frequency of Her-2/neu 58
positive cases was highest in advanced stage (p = 0.042). 59
60
61
62
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Conclusion 63
Her-2/neu expression occurs in gallbladder cancer, especially in advanced stage 64
disease and its therapeutic targeting seems promising. 65
66
Keywords: Gallbladder Cancer, Her-2/neu, Expression, Histopathology, Prognosis. 67
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TITLE: Expression of Her-2/neu receptor in Carcinoma Gallbladder correlates 94
significantly with advanced tumour stage 95
96
INTRODUCTION 97
Gallbladder cancer (GBC), the commonest biliary malignancy, was first described in 98
1777 by a Viennese researcher Maximilian de Stoll [1]. Its incidence parallels the 99
prevalence of gall stone disease; large and long-standing gall stones being 100
associated with a higher risk of GBC [2]. It has a very poor prognosis with only about 101
32 percent five-years survival rate for lesions confined to the gallbladder mucosa that 102
drops to about 10 percent, for more advanced stages. GBC is very common in Chile, 103
Northern India, Japan and Korea [3]. Adenocarcinoma accounts for 80- 90% of 104
cases. There are a number of risk factors that may increase the chances of 105
developing gallbladder cancer that include gallstones and inflammation, polyps, 106
porcelain gallbladder, obesity and S almonella typhi infection of gallbladder [4]. 107
Her-2/neu (also known as Erb B-2) stands for "Human Epidermal growth factor 108
Receptor 2. Overexpression of this receptor in breast cancer is associated with 109
increased disease recurrence and worse prognosis [5]. Overexpression also occurs 110
in other cancer such as ovarian cancer, stomach cancer, and biologically aggressive 111
forms of uterine cancer, such as uterine serous endometrial carcinoma [6-8]. There 112
are few but conflicting reports dealing with the clinical significance of expression of 113
Her-2/neu in GBC. Moreover, reported immunohistochemical (IHC) over expression 114
ranges from 5% to 70% in GBC [9-13]. The aim of this study was to correlate Her-115
2/neu positivity with the histopathologic features in carcinoma gallbladder. 116
117
MATERIALS AND METHODS 118
Material comprised of resected specimens of carcinoma gallbladder examined in the 119
Department of Pathology, Sir Ganga Ram Hospital, New Delhi from January 2007 to 120
July 2011. This study included both retrospective as well as prospective 121
components. The study comprised of fifty-four cases of GBC found during this period 122
out of 1851 cholecystectomy specimen. A gross evaluation of tumor was done in the 123
resected specimens. After the preliminary study of all Hematoxolin & Eosin stained 124
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sections, IHC staining was done for Her-2/neu in malignant tumour along with non-125
tumorous area. The relative frequency of Her-2/neu positivity was scored and 126
correlated with other histological prognostic parameters of the tumour. Areas that 127
were not involved by the tumour were also studied for Her-2/neu and external 128
positive and negative controls were put while performing IHC staining in each batch. 129
Controls were taken from the known cases of the breast specimen. Her-2/neu was 130
graded as per criteria defined by the American Society of Clinical Oncology and the 131
College of American Pathologists [14]. 132
The data thus collected was compiled and analyzed using SPSS version 21 for Mac 133
(IBM Corporation, 2012). Qualitative variables were expressed as proportions in 134
percentages. The association between variables was calculated for 95% confidence 135
intervals by using “Chi square test”. A P-value < 0.05 was taken as significant. For 136
quantitative data, mean and standard deviation was calculated. An approval for this 137
study was obtained from the Institutional Ethical Committee. 138
RESULTS 139
Fifty-four cases in 1851 cholecystectomy specimens were diagnosed as carcinoma 140
of gallbladder (2.9%). Out of these, 22 cases (1.1%) were diagnosed after 141
cholecystectomy in routine histopathology without prior clinical diagnosis. Male to 142
female ratio was 1:4.5 and the youngest patient was a 27 year-old-woman. Twenty-143
six patients (48 %) had calculi in the gallbladder. Most of the cases (98%) were 144
adenocarcinoma and only 2% were adenosquamous carcinoma. 145
Pyloric metaplasia, intestinal metaplasia and dysplasia were seen in 39 (72%), 32 146
(59%) and 24 cases (44.44%) respectively. Maximum cases in our study were in 147
stage T2 at the time of diagnosis and most of them were moderately differentiated 148
carcinoma. Carcinomas detected incidentally were mostly in the early stages of 149
cancer (T1). Her-2/neu was strongly positive (3+) in 5 cases (9.25%) and moderately 150
positive (2+) in14 cases (25.92%). There was a lot of heterogeneity in the expression 151
of Her-2/neu showing 1+ and 2+ positivity in different areas. No correlation was seen 152
between IHC staining pattern and tumour grade [Table 1], but frequency of Her-153
2/neu positive cases was highest in advanced stage (p = 0.042) [Table 2]. 154
155
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DISCUSSION 156
GBC is the most common cancer of the biliary tract and has a particularly high 157
incidence in Chile, Japan, and northern India. Its incidence steadily increases with 158
age and women are affected two to six times more often than men [3]. High 159
incidence of GBC in our region and its poor prognosis necessitates a closer look at 160
the molecular changes for evolving an effective early diagnostic and therapeutic 161
strategy. Expression of HER-2/neu has been intensively studied in different tumor 162
entities and has led to the use of targeted therapy with specific inhibitors or 163
antibodies of these receptors in colorectal, breast, lung as well as head and neck 164
cancer. Its expression as potential therapeutic targets has been reported in various 165
tumors [6-8]. For biliary tract carcinoma, data for HER-2/neu overexpression have 166
been presented in mostly small patient cohorts. Its overexpression and amplification 167
has been found in a range between 5% and 76% in biliary tract cancers [9-13]. 168
Targeted therapy with the anti-HER-2/neu antibody in breast cancer is effective 169
when the HER2/neu receptor is overexpressed [5]. However, its study in 170
gallbladder carcinoma is limited. 171
In the present study histo-pathological examination of 54 gallbladder carcinoma 172
cases was done in our institute and it was correlated with Her-2/neu IHC staining. 173
The youngest patient in our study was 27 years and the oldest was 86 years. To the 174
best of our knowledge, the youngest patient of carcinoma gallbladder reported in 175
literature is a 15-year-old girl reported by Khandelwal et al in their study of 150 cases 176
of GBC [15]. The mean age of presentation in our series was 57.31 years. The 177
maximum numbers of patients were in the seventh decade (33.33 %) followed by 178
fifth decade (29.3%). 179
The association between cholelithiasis and gallbladder cancer has been known since 180
1861. However, it is seen that only 0.5%-1% of cases with gallstone disease develop 181
carcinoma of gallbladder [16,17]. In the study by Mohan et al 1% of 182
cholecystectomies performed for cholelithiasis showed early carcinoma [18]. We 183
found 2.9% of gallbladder cancer out of a total of 1851 cholecystectomies, out of 184
which 22 (1.1%) were incidental carcinoma. In our study, 26 patients (48 %) of 185
gallbladder cancer had calculi in the gallbladder. 186
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Fifty-three of our cases (98%) were adenocarcinoma and one case (2%) was 187
adenosquamous carcinoma. Levin et al [19] and Rajagopalan et al [20] reported over 188
85% to 90% percent of gallbladder carcinoma histologically as adenocarcinoma in 189
their studies. Twenty-two cases (41%) in our study were diagnosed incidentally. Out 190
of which seven cases (32%) were in stage T1, twelve cases (55%) in stage T2 and 191
three cases (13%) were in stage T3. On the other hand, out of 32 previously 192
diagnosed cases of gallbladder carcinoma, 24 cases (75%) were in stage T2 and 4 193
cases (12.5%) each in stage T1 and T3. It reflects the fact that most of the cases 194
diagnosed incidentally are in the early stages of cancer (T1). Shih et al reported 195
incidentally discovered GBC during cholecystectomy in 53 patients out of 107 196
patients of GBC (50%) at Johns Hopkins Hospital in a 10-year study period [21]. 197
They concluded that patients who were found to have gallbladder carcinoma 198
incidentally during cholecystectomy had a significant increase in survival when 199
compared with those who were admitted electively with a known diagnosis. In our 200
study, 36 (66.66%) of the cases were in stage T2; eleven (20.37%) in stage T1, and 201
seven (13%) in stage T3. Memon et al reported 61% gallbladder cancers in clinical 202
stage IV, 22% in stage III and 17% in stage II at the time of presentation [22]. 203
Her-2/neu was strongly positive in 5 cases (9.25%) of gallbladder cancer in our 204
study, out of which 4 were moderately differentiated adenocarcinoma and one was 205
poorly differentiated adenocarcinoma. This finding was in accordance with the 206
findings of Puhalla et al who found expression of HER-2/neu in 13% of the patients of 207
GBC with IHC staining [23]. Similar IHC pattern was seen between the tumour cells 208
and dysplastic cells. Adjacent normal mucosa was negative for Her-2/neu staining in 209
all except one case in our study. Kawamoto et al reported positive overexpression of 210
Her-2/neu in GBC in 31.2% of specimens by IHC method [9]. 211
Only few studies of overexpression of Her-2/neu in gallbladder cancer have been 212
reported from India. Chaube et al investigated the role of Her-2/neu in the 213
carcinogenesis of gallbladder by IHC and found 25% of cases positive for Her-2/neu 214
expression [24]. In their study, positivity decreased with increasing grade of 215
gallbladder carcinoma. They have concluded that decreasing over expression of c-216
erbB-2 with increasing grade suggest that c-erbB-2 is likely to play a lesser role in 217
tumour progression. In our study we did not find any such correlation. However, 218
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there seems to be some correlation of IHC staining pattern and tumour stage. There 219
were 11 cases in stage T1, 36 cases in stage T2 and 7 cases in T3 stage. Out of 220
these case 3+ positivity was shown by 2 cases (28.57%) in stage T3; three cases 221
(8.33%) in stage T2 and none in stage T1. Hence the frequency of Her-2/neu 222
positive cases was highest in advanced stage (p = 0.042). Study with larger number 223
of cases is required to further substantiate this finding. 224
In our study, one case each of well differentiated and a poorly differentiated tumour, 225
showed sharply defined 3+ positivity adjacent to 1+ positive area. Also there were 226
few cases in which focal 2+ positive areas were present adjacent to predominant 1+ 227
positive area. This suggests that even in a single case of GBC, different staining 228
patterns of Her-2/neu can be seen which highlights the importance of adequate 229
sampling of the tumour for Her-2/neu immuno-staining. To the best of our 230
knowledge, such a heterogeneous pattern of immuno-staining of Her-2/neu has 231
never been reported in the literature prior to this study. 232
Also equivocal IHC staining (2+) cases are an important part of the ongoing 233
discussion. Next to IHC staining to evaluate Her-2/neu protein overexpression, a 234
second line gene amplification test is generally deemed necessary for these cases 235
[25]. In a study done by Kawamoto et al 26.7% of equivocal immunohistochemistry 236
were Flourescent In situ Hybridisation (FISH) positive in GBC [9]. Hence, if we had 237
done the gene amplification in equivocal cases (2+) of GBC, which included 14 238
cases (25.92%) in our study, the percentage of Her-2/neu positivity would have gone 239
still higher; however, it was not done as it was outside the protocol of the study. 240
241
CONCLUSION 242
In view of aggressive nature of gallbladder carcinoma and lack of good therapeutics, 243
there is an urgent need for identification of cancer specific cellular targets that form 244
the basis of translational therapeutics in future. One of the important conclusions of 245
our study is that as many cases of GBC express Her-2/neu, its therapeutic targeting 246
seems to be promising. 247
248
CONFLICT OF INTEREST 249
NOT GIVEN 250
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251
AUTHOR’S CONTRIBUTIONS 252
Farzana Ashai 253
Group1: Conception and design, Acquisition of data, Critical revision of the article 254
and Final approval of the version to be published. 255
Aadil Ashraf 256
Group2: Analysis and interpretation of data, Drafting the article, Critical revision of 257
the article and Final approval of the version to be published. 258
Arshad Rashid 259
Group3: Analysis and interpretation of data, Drafting the article, Critical revision of 260
the article and Final approval of the version to be published. 261
Bilal Musharaf Banday 262
Group4: Analysis and interpretation of data, Drafting the article, Critical revision of 263
the article and Final approval of the version to be published. 264
Sunita Bhalla 265
Group5: Conception and design, Acquisition of data, Critical revision of the article 266
and Final approval of the version to be published. 267
Shashi Dhawan 268
Group5: Conception and design, Acquisition of data, Critical revision of the article 269
and Final approval of the version to be published. 270
271
ACKNOWLEDGEMENTS 272
NOT GIVEN 273
274
REFERENCES 275
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Cancer Inst 1980;65:1209-14. 277
2. Vitetta L, Sali A, Little P, Mrazek L. Gallstones and gall bladder carcinoma. Aust 278
N Z J Surg 2000;70:667–73. 279
3. Lai CH, Lau WY. Gallbladder cancer – a comprehensive review. Surgeon 280
2008;6:101-10. 281
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4. Mittal R, Jesudason MR, Nayak S. Selective histopathology in cholecystectomy 282
for gallstone disease. Indian J Gastroenterol 2010;29:32-36. 283
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oncogene is frequently amplified in squamous cell carcinoma of the uterine 290
cervix. Cancer Res 1994;54:637-639. 291
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gallbladder cancer. Pathol Res Pract 1993;189:283–292. 304
12. Kamel D, Paakko P, Nuorva K, Vahakangas K, Soini Y. p53 and c-erbB-2 protein 305
expression in adenocarcinomas and epithelial dysplasias of the gall bladder. J 306
Pathol 1993;170:67–72. 307
13. Kim YW, Huh SH, Park YK, Yoon TY, Lee SM, Hong SH. Expression of the c-308
erb-B2 and p53 protein in gallbladder carcinomas. Oncol Rep 2001;8:1127–1132. 309
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15. Khandelwal M, Khandelwal C. Surgery for advanced gall bladder cancer. In: 314
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345
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TABLES 346
347
Table 1: Correlation of immunohistochemical staining with tumour differentiation 348
349
Differentiation Negative 1+ 2+ 3+ Total
Well
differentiated
1 9 (one with
focal 3+
positivity)
1 Nil
(0%)
11
Moderately
differentiated
7 4 12 4
(14.81%)
27
Poorly
Differentiated
2 12 1 (with focal
3+
positivity)
1
(6.25%)
16
Total 10
(18.51%)
25(46.29%
)
14(25.92%) 5 (9.25%) 54
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
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Table 2: Correlation of immunohistochemical staining with staging (p < 0.05) 367
368
Staging Negative 1+ 2+ 3+ Total
T1 1 9 (1 with focal 3+
positivity)
1 Zero 11
T2
7
13
13 (one with focal 3+
positivity)
3(8.33%)
36
T3
2
3
Zero
2(28.57%)
7
Total
10
25
14
5(9.25%)
54
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
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FIGURE LEGENDS 387
Figure 1: Photomicrograph showing strong and diffuse 3+ positivity of Her-2/neu in 388
Gallbladder Carcinoma (100X). 389
Figure 2: Photomicrograph showing focal 3+ Her-2/neu positivity in a predominant 1+ 390
area of gallbladder cancer (100X). 391
392
FIGURE 393
394
395
Figure 1: Photomicrograph showing strong and diffuse 3+ positivity of Her-2/neu in 396
Gallbladder Carcinoma (100X). 397
398
399
400
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401
Figure 2: Photomicrograph showing focal 3+ Her-2/neu positivity in a predominant 1+ 402
area of gallbladder cancer (100X). 403
404
405
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