|Year : 2021 | Volume
| Issue : 4 | Page : 687-692
|Expression of VEGF-A, HER2/neu, and KRAS in gall bladder carcinoma and their correlation with clinico-pathological parameters
Pomilla Singh1, Shyam Lata Jain2, Puja Sakhuja3, Anil Agarwal4
1 Bharati Vidyapeeth Deemed to be University Medical College, Pune, Maharashtra; Department of Pathology, Maulana Azad Medical College, New Delhi, India
2 Department of Pathology, Maulana Azad Medical College, New Delhi, India
3 Department of Pathology, GB Pant Institute of Post Graduate Medical Education and Research, New Delhi, India
4 Department of GI Surgery, GB Pant Institute of Post Graduate Medical Education and Research, New Delhi, India
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|Date of Submission||23-Mar-2020|
|Date of Decision||27-Apr-2021|
|Date of Acceptance||26-May-2021|
|Date of Web Publication||20-Oct-2021|
| Abstract|| |
Background: Gall bladder carcinoma (GBC) is a multi-factorial disease, involving multiple genetic alterations. The present pilot study aims to explore some of the molecular pathways, by studying immunohistochemical (IHC) expression of biomarkers (HER2/neu, KRAS, and VEGF) in GBC with their correlation with various clinicopathological parameters. Aim of the Study: To study the expression of prognostic biomarkers (HER2/neu, KRAS and VEGF-A) in GBC and their correlation with clinico-morphological parameters. Materials and Methods: This prospective study was conducted over a period of 2 years. The study group included tissue of GBC (29) reported as malignant on histopathology and cholecystitis as a control group (29) for histopathological evaluation and IHC expression of above markers. Results: HER2/neu was expressed in 27.5% cases, and KRAS in 51.6%; however, both showed no association with tumor type, stage and grade. No association was found in KRAS expression and dysplasia. Vascular Endothelial Growth Factor - A (VEGF-A) was expressed in 86.1% cases, of which strong positivity was seen in 48.27%; it showed significant association with tumor stage (P value-0.027, Fishers' exact test), hence possibly suggesting its role in tumor progression; though no association was found in VEGF expression with tumor type and grade. No significant association was seen with vascular and tumor invasion also. Conclusion: The results suggest that the VEGF-A expression may be used as a potential prognostic biomarker in GBC.
Keywords: Cancer biomarkers, gall bladder carcinoma, HER2/neu, KRAS, VEGF-A
|How to cite this article:|
Singh P, Jain SL, Sakhuja P, Agarwal A. Expression of VEGF-A, HER2/neu, and KRAS in gall bladder carcinoma and their correlation with clinico-pathological parameters. Indian J Pathol Microbiol 2021;64:687-92
|How to cite this URL:|
Singh P, Jain SL, Sakhuja P, Agarwal A. Expression of VEGF-A, HER2/neu, and KRAS in gall bladder carcinoma and their correlation with clinico-pathological parameters. Indian J Pathol Microbiol [serial online] 2021 [cited 2022 Aug 14];64:687-92. Available from: https://www.ijpmonline.org/text.asp?2021/64/4/687/328549
| Introduction|| |
Carcinoma of gall bladder (GBC) is the fifth most common cancer of digestive tract and the most common malignancy of the biliary tract. It has an annual incidence of 2.5% and mortality rate of 2.75%. Like other neoplasms, GBC is also a multi-factorial disease, involving multiple genetic alterations.
The present study is a pioneer study, and aims to explore molecular pathways in GBC by immunohistochemical (IHC) expression of various biomarkers (HER2/neu, KRAS, VEGF-A). Targeted therapies against HER2/neu and VEGF have been successfully tried in many other cancers (e.g., colon, pancreas, lung, and breast); though not validated yet, in future these novel biomarkers could be considered to treat GBC which would be superior to conventional cytotoxic drugs.
| Materials and Methods|| |
The study was conducted in a tertiary care in northern India. Out of approximately 200 clinico-radiologically suspected cases of GBC, 29 histopathologically proven cases of GBC were included in the study. Patients with metastasis to GB and who received chemotherapy/radiotherapy for GBC were excluded from the study. The control group included tissue sections of 29 cholecystitis cases, to serve as controls for IHC study. The approval from ethics committee was obtained on 15 Sep 2014.
Detailed clinical history and examination in all cases were recorded. The resected specimens were studied for gross findings (i.e., shape, size, wall thickness, tumor size and site, presence of stones). Morphologically distinct areas of each tumor were identified and tissue sections of 5 μm were taken. The routine paraffin slides (H and E stain) were studied for tumor type, stage, grade, lympho-vascular, and/perineural invasion and dysplasia. Grading and Tumor Nodes Metastases (TNM) staging was done as per established criteria (AJCC, 8th ed).
IHC analysis of selected markers (A panel of markers were studied (HER2/neu, KRAS, VEGF-A) for the study was performed on paraffin-embedded tissue sections. Tissue sections were deparaffinized in xylene and then re-hydrated with sequential washes of 100%, 70%, and 50% ethanol. Endogenous peroxidase activity was inhibited with 3% hydrogen peroxidase. For antigen retrieval, slides were placed in 50 mM citrate buffer pH 6.0 to unmask the epitopes at 95°C. Microwave antigen retrieval was performed for unmasking of antigens. Tissue sections were then incubated with specific antibodies for 1 hour at room temperature. Slides were rinsed with Tris buffer (pH 7.4) for three times followed by treatment with polymer based secondary antibody kit with 3'3 diaminobenzidine tetra hydrochloride (DAB), as substrate. All sections were counterstained with 0.1% Hematoxylin. After dehydration tissue sections were fixed with permanent mounting medium and covered with glass cover slips. Positive and negative controls were included in each run. immunohistochemical characteristics of each morphologically distinct area of the tumors were evaluated. The scoring was done as mentioned:
KRAS expression was scored (cytoplasmic stain) as 1-4: 1+ (<10% positive stain)-negative; 2+ (>10-<25% positive stain), 3+ (>25-<50% positive stain); 4+ (>50% positive stain).
HER 2/neu expression was scored (membrane staining) as 0-3: [0 no staining, 1 (negative): None/faint and focal membrane positivity (≥ 30% of neoplastic cells); 2 (positive): Weak but definitive entire membrane staining (≥ 30% of neoplastic cells); 3 (positive): Strong staining of the entire membrane (≥ 30% of neoplastic cells)].
VEGF-A expression was scored by combining the percentage (%) of cells stained positive and intensity of cytoplasmic and membrane staining as illustrated in [Table 1].
Histological features were correlated with IHC and interrelation of above markers was studied.
Data entered in MS EXCEL sheet were analyzed by SPSS software. The results were expressed as % and proportions. Quantitative variables were expressed as mean ± SD. The statistical analysis of qualitative variables was performed by Chi square test/Fisher's exact test.
| Results|| |
The patients in study group (29) ranged in age from 20 to 74 years (mean-52.27 years); 24 patients were females (80%), and only five were males (20%) with F: M = 4.8:1. Most patients (50%) presented with pain in right hypochondrium, followed by jaundice (5%) and symptoms of cholelithiasis (33%). The patients with cholecystitis had a mean age of 48.31 years, with age ranging from 32 to 58 years. Twenty five of them were females and 4 males, F:M ratio of 6.2:1. The most common symptom (83%) reported was chronic intermittent pain in right upper abdomen, and epigastrium. Most common radiological finding was calculus (69%), followed by increased wall thickness (23%).
Histopathological findings- A. Tumor type- 27/29 (93%) cases were adenocarcinoma, one of which was mucinous type [Figure 1], whereas rest had no specific type. Two (6.89%) were adenosquamous carcinoma [Figure 1]. B. Tumor grade- 22/29 (81.4%) cases were moderately differentiated, four (14.8%) well differentiated and, one (3.7%) was poorly differentiated. C. Tumor stage - as per AJCC staging for GBC, 7 (24.13%) were in stage 1; 11 (37.93%) stage 2a, 1 case (3.44%) stage 2b; 3 (10.3%) stage 3a; 5 (17.24%) stage 3b, 1 case each (3.44%) in stage 4a and 4b.
|Figure 1: (a) Focal squamoid differentiation with adenocarcinoma. (b) Mucin pools in case of mucinous adenocarcinoma; (H and E 250×)|
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Vascular invasion noted in four cases (13.8%) and neural invasion in six cases (20.7%). Metaplasia - antral metaplasia in 9 (31%) cases, Dysplasia - associated cholecystitis in five (17.2%) while xantho-granulomatous cholecystitis in five cases (17.2%).
[Table 2] describes the correlation of HER2/neu, KRAS and Vascular Endothelial Growth Factor (VEGF) with type, grade and staging, dysplasia of GBC. HER2/neu was expressed in 27.5% of cases [Figure 2] and did not show statistically significant association with tumor type, grade, or stage. KRAS showed expression in 51.6% cases [Figure 3]; it also showed no statistically significant association with tumor type, grade, or stage. No significant association was established between expression of KRAS with dysplasia. VEGF-A was expressed in total 86.1% cases including low positive (37.9%) cases and high positive (48.27%) cases [Figure 4]. It was significantly associated with tumor stage (P value 0.027, Fisher's exact test), though, it did not show any association with tumor type and grade. There was no significant association of VEGF with vascular/neural invasion. Expression of biomarkers showed no statistically significant interrelationship. Expression of these biomarkers was also studied in cases of cholecystitis (controls). HER2/neu and VEGF showed complete absence of expression. Two cholecystitis cases weakly expressed KRAS, in <10% cells, regarded negative.
|Table 2: Correlation of HER2/neu, KRAS, and VEGF expression with tumor type, stage, grade, dysplasia, chronic cholecystitis, vascular and neural invasion (n=29)|
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|Figure 2: (HER2/neu): (a) Weak focal membranous positivity in <30% cells (1+); (b) weak but definitive membranous staining in >30% cells (2+); (c and d) strong and complete membranous staining >30% cells (3+); (400×)|
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|Figure 3: (KRAS): (a) Cytoplasmic positivity in <10% cells (1+); (b) positivity in >10-<25% cells (2+); (c) Positivity in >25-<50% cells (3+); (d) positivity in >50% cells (4+); (400×)|
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|Figure 4: (VEGF): (a) Focal weak to moderate cytoplasmic positivity <50% cells (score <5); (b) intense cytoplasmic positivity in >50% cells (score >5)|
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| Discussion|| |
VEGF-A was overall expressed in 86.2% cases in the present study (29) of GBC; whereas it was not expressed in cholecystitis controls. VEGF overexpression was observed in 34% of the cases in a study done by Doval et al. Their results did not confirm the overexpression reported earlier and showed a decreased rate of expression. Zhang et al found expression of VEGF-A in 65.4% (51/78) of GBC and correlated it with tumor differentiation. However, other studies found VEGF-A expression in 81.69% GBC and correlated with poor prognosis. The significant overexpression of VEGF-A in tumors and its relationship with advanced stages suggest that this biomarker plays an important role in GBC progression.
In present study, we found a statistically significant positive relationship between its expression and Tumor Nodes Metastases (TNM) stage (P = 0.027). However, no statistically significant association was found with tumor grade in the present study, though there are some reports which have shown correlation with tumor grade., Though a study showed that VEGF-A expression in GBC tissues is correlated with histologic differentiation and is an independent prognostic factor. Our study observed no association of VEGF-A with vascular and neural invasion.
HER2/neu was expressed in 8/29 cases (27.5%) of GBC, of which most were moderately differentiated adenocarcinoma (4), followed by well differentiated (2) and adeno squamous (1). A recent study quotes to have 14% overexpression of HER2/neu, its two-thirds (21/28) of HER-2/neu positive cases well-to-moderately differentiated (G ≤ 2). Her2/neu positivity in 24% cases (6/25) was observed in a study, with no significant correlation with various clinicopathological parameters other than liver involvement. The author observed trend of decreasing Her2/neu expression with increasing grade. While the current study did not show any association with tumor grade, stage or type. In another study, HER2/neu was overexpressed in 4% cases and was not correlated with any of the clinicopathological factors, similar to the present study. Considering both 3+ and 2+ staining as overexpression, HER2/neu overexpression was seen in 13.4% cases of GBC and was found frequent in well differentiated and high stage tumors, i.e., stage II onward tumors in a study by Kumari et al. Roa et al. found overexpression of HER2/neu in 12.8% of cases more frequent in the advanced cancers and in the better differentiated tumors, but the difference was nonsignificant, whereas another researcher observed overexpression in 36.67% (22/60) tumor cases along with 5% (3/60) control cases, HER2/neu overexpression significantly correlated with sex, T-stage, nodal spread and distant metastasis (P < 0.05). [Table 3] consolidates and compares VEGF and HER2/neu expression on IHC in GBC in our study with other studies.
KRAS on IHC was expressed in 51.6% cases in the present study, and showed no association with tumor type, stage, and grade. No significant association of KRAS was seen with dysplasia and cholecystitis which is in concordance with the study by Ajiki et al. where the researcher found KRAS mutations in 30/51(59%) cases of GBC. In the present study, KRAS was studied by IHC and not by molecular techniques. Nagahashi et al. studied GBC in Japanese and Hungarian population and reported a KRAS mutation in just 1/42 (2.4%). A study from Chile found KRAS mutation only 1/21 (4.8%) GBC cases. A more recent study from China detected a KRAS gene mutation in just 2/75 (2.7%) GBC. In a study from US the authors found 2/29 (6.9%) with KRAS mutation and none with a BRAF mutation. To conclude, the present study suggest that the VEGF-A expression in GBC may be a potential prognostic biomarker; however, needs further larger studies to explore and validate its role in carcinogenesis; if results are significant this biomarker can be used in future to select patients who may benefit by anti-VEGF targeted therapy. Bevacizumab, a humanized monoclonal antibody against VEGF-A, is the first anti-angiogenic molecular targeting agent (approved by US-Food and Drug Administration) for the treatment of advanced colorectal cancer. The drug Avastin, has been approved for advanced lung, kidney, and brain cancers, and it is under evaluation in clinical trials to determine its effcacy and safety in other malignancies, including biliary tract carcinomas., Aflibercept is under various phase III studies for ovarian cancer, non–small cell lung cancer, and prostate cancer. Anti-VEGF therapy may be a promising strategy for the treatment of advanced GBC patients too.
Her2/neu was chosen to study because of its established role in the most common female malignancy, i.e., breast carcinoma, along with cancers of gastrointestinal tract. GBC being proportionally high in female patients, the research was intended to unfold some hidden molecular pathway in GBC, if any. VEGF was selected since anti VEGF therapy is already under clinical trials for many GI carcinomas. While KRAS (HRAS, KRAS, NRAS) is the most common oncogenes in human cancer, the mutations permanently activating RAS are found in 20–25% of all human tumors and up to 90% in certain types of cancer (pancreatic cancer). KRAS gene mutation being found in gall bladder dysplasia at an incidence similar to that in carcinomas incites a research into its role.
Financial support and sponsorship
Department of pathology, Maulana Azad Medical College, New Delhi.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Wullstein C, Woeste G, Barkhausen S, Gross E, Hopt UT. Do complications related to laparoscopic cholecystectomy influence the prognosis of gallbladder cancer? Surg Endosc 2002;16:828-32.
Amin MB, Edge SB, Green FL, Byrd DR, Brookland RK, Washington MK, et al
. AJCC Cancer Staging Manual. 8th
ed. New York: Springer; 2017. p. 79-94.
Doval DC, Azam S, Sinha R, Batra U, Mehta A. Expression of epidermal growth factor receptor, p53, Bcl2, vascular endothelial growth factor, cyclooxygenase-2, cyclin D1, human epidermal receptor-2 and Ki-67: Association with clinicopathological profiles and outcomes in gallbladder carcinoma. J Carcinog 2014;13:10.
] [Full text]
Zhang L-Q, Xu X-S, Wan Y, Song S-D, Wang R-T, Chen W, et al
. Prognostic implications of estrogen receptor 1 and vascular endothelial growth factor A expression in primary gallbladder carcinoma. World J Gastroenterol 2015;21:1243-50.
Letelier P, Garcia P, Leal P, Ili C, Buchegger K, Riquelme I, et al
. Immunohistochemical expression of vascular endothelial growth factor A in advanced gallbladder carcinoma. Appl Immunohistochem Mol Morphol 2014;22:530-6.
Quan ZW, Wu K, Wang J, Shi W, Zhang Z, Merrell RC. Association of p53, p16, and vascular, endothelial growth factor protein expressions with the prognosis and metastasis of gallbladder cancer. J Am Coll Surg 2001;193:380-3.
Batmunkh E, Shimada M, Morine Y, Imura S, Kanemura H, Arakawa Y, et al
. Expression of hypoxia inducible factor-1 alpha (HIF-1alpha) in patients with the gall bladder carcinoma. Int J Clin Oncol 2010;15:59-64.
Sun XN, Cao WG, Wang X, Wang Q, Gu BX, Yang QC, et al
. Prognostic impact of vascular endothelial growth factor-A expression in resected gall bladder carcinoma. Tumour Biol 2011;32:1183-90.
Jain P, Goyal S, Chauhan G, Majumdar K, Ali S, Sakhuja P, et al
. HER-2/neu over expression in gall bladder adenocarcinoma: A quest for potential therapeutic target. Indian J Pathol Microbiol 2020;63:214-20.
] [Full text]
Pujani M, Makker I, Makker A, Jetley S, Goel MM. Expression of human epidermal growth factor receptor (Her 2/neu) and proliferative marker Ki-67: Association with clinicopathological parameters in gallbladder carcinoma. Asian Pac J Cancer Prev 2016;17:3903-9.
Kumari N, Kapoor VK, Krishnani N, Kumar K, Baitha DK. Role of C-erbB2 expression in gall bladder cancer. Indian J Pathol Microbiol 2012;55:75-9. [Full text]
Roa I, de Toro G, Schalper K, de Aretxabala X, Churi C, Javle M. Overexpression of the HER2/neu gene: A new therapeutic possibility for patients with advanced gallbladder cancer. Gastrointest Cancer Res 2014;7:42-8.
Singh A, Mishra PK, Saluja SS, Talikoti MA, Kirtani P, Najmi AK. Prognostic significance of HER-2 and p53 expression in gallbladder carcinoma in North Indian patients. Oncology 2016;91:354-60.
Ajiki T, Fujimori T, Onoyama H, Yamamoto M, Kitazawa S, Maeda S, et al
. K-ras gene mutation in gall bladder carcinomas and dysplasia. Gut 1996;38:426-9.
Nagahashi M, Ajioka Y, Lang I, Zatonski WA, Lowenfels AB, Boyle P. Genetic changes of p53, K-ras, and microsatellite instability in gallbladder carcinoma in high-incidence areas of Japan and Hungry. World J Gastroenterol 2008;14:70-5.
Wistuba II, Sugio K, Hung J, Kishimoto Y, Virmani AK, Roa I, et al
. Allele-specific mutations involved in the pathogenesis of endemic gall bladder carcinoma in Chile. Cancer Res 1995;55:2511-5.
Rashid A, Ueki T, Gao YT, Houlihan PS, Wallace C, Wang BS, et al
mutation, p53 overexpression, and microsatellite instability in biliary tract cancers. Clin Cancer Res 2002;8:3156-65.
Pai RK, Mojtahed K, Pai RK. Mutations in the RAS/RAF/MAP kinase pathway commonly occur in gall bladder adenomas but are uncommon in gall bladder adenocarcinomas. Appl Immunohistochem Mol Morphol 2011;19:133-40.
Ichihara E, Kiura K, Tanimoto M. Targeting angiogenesis in cancer therapy. Acta Med Okayama 2011;65:353-62.
Lubner SJ, Mahoney MR, Kolesar JL, Loconte NK, Kim GP, Pitot HC, et al
. Report of a multicentre phase II trial testing a combination of biweekly bevacizumab and daily erlotinib in patients with unresectable biliary cancer: A phase II Consortium study. J Clin Oncol 2010;28:3491-7.
Wiedmann MW, Mossner J. Molecular targeted therapy of biliarytract cancer–results of the first clinical studies. Curr Drug Targets 2010;11:834-50.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]
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