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Year : 2019  |  Volume : 8  |  Issue : 3  |  Page : 151-158

Glucose transporter 1 expression in bladder carcinoma and its association with human epidermal growth factor receptor-2 and Ki-67

1 Department of Pathology, Government Medical College, Kottayam, Kerala, India
2 Department of Urology, Government Medical College, Kottayam, Kerala, India

Date of Web Publication9-Dec-2019

Correspondence Address:
Dr. Renu Thambi
Department of Pathology, Government Medical College, Kottayam, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sjhs.sjhs_52_19

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Objective: To assess the expression of glucose transporter 1 (GLUT1) in urothelial carcinomas of urinary bladder and its relationship with human epidermal growth factor receptor-2 (HER2) protein and Ki67 proliferation index. Materials and Methods: Forty-three patients with urothelial carcinoma of bladder were included in the present study. Transurethral resection or cystectomy specimens of tumor were evaluated by histopathology. Grading and staging of these tumors were done followed by immunohistochemistry using antibodies against GLUT1, HER2, and Ki67. Results: Strong expression of GLUT1 score and Ki67 index were seen in higher grades and tumor node metastasis stages of urothelial carcinoma (P < 0.05). In the current study, HER2 overexpression was not significantly associated with tumor grade and stage (P > 0.05). Conclusions: GLUT1 expression showed moderate correlation with HER2 and good correlation with Ki67 index in urothelial carcinoma. Further studies may be needed to assess the role of GLUT1 in prognosis and therapy.

Keywords: Glucose transporter 1, human epidermal growth factor receptor-2, Ki67, urothelial carcinoma

How to cite this article:
Sainulabdeen S, Thambi R, Sundaram S, Bhat S. Glucose transporter 1 expression in bladder carcinoma and its association with human epidermal growth factor receptor-2 and Ki-67. Saudi J Health Sci 2019;8:151-8

How to cite this URL:
Sainulabdeen S, Thambi R, Sundaram S, Bhat S. Glucose transporter 1 expression in bladder carcinoma and its association with human epidermal growth factor receptor-2 and Ki-67. Saudi J Health Sci [serial online] 2019 [cited 2021 Jan 21];8:151-8. Available from: https://www.saudijhealthsci.org/text.asp?2019/8/3/151/272443

  Introduction Top

Urinary bladder cancer is the 10th most common form of cancer worldwide, more common in men with respective incidence and mortality rates of 9.6 and 3.2 per 100,000; which is about four times those of women globally.[1],[2] According to the data from recent National Cancer Registry Program, Bengaluru 2016, the overall incidence rate of the urinary bladder cancer in India is 3.8 (per 100,000 annually), frequent in males (3.77) than females (0.82). Important risk factors include smoking and occupational hazard in leather and textile industries. The marked difference in the incidence of smoking among Indian males and females (74% vs. 22%) compared to what is reported in the literature might have contributed to the differences in the Indian figures of urothelial carcinoma.[3]

Presenting symptoms are painless hematuria, flank pain, and passing of blood clots.[1] Treatment for nonmuscle invasive bladder carcinoma is transurethral resection and intravesical immunotherapy with Bacillus Calmette–Guerin with regular surveillance cystoscopy. In muscle invasive cases, radical cystoprostatectomy with lymph node dissection is the treatment of choice.[4] Among the several prognostic factors, tumor grade and tumor node metastasis (TNM) stage are 2 independent factors.

Cancer cells need more glucose for aerobic glycolysis which provides them with intermediate molecules that are used for synthesis of cell constituents (Warburg effect). Glucose transporter family (GLUT) regulates glucose uptake, and GLUT1 is the first to be described and overexpressed in many malignant tumors.[5],[6] Carcinoma in situ, noninvasive, and invasive urothelial carcinomas express GLUT1 which is in contrast to negative GLUT1 expression in normal bladder mucosa. The expression of GLUT1 detected by immunohistochemistry (IHC) is associated with aggressive behavior of the tumor.[7],[8],[9],[10],[11]

The human epidermal growth factor receptor-2 (HER2) contributes to physiologic mechanisms of cell proliferation by an intrinsic tyrosine kinase activity. HER2 can be assessed by IHC and gene amplification. HER2 assessment is an essential prerequisite for management, prognosis, and prediction of the response to targeted therapies in carcinoma breast. Expression of HER2 in urothelial carcinoma showed varied results in different studies.[12],[13],[14],[15],[16] A positive HER-2 in combination with other markers such as CK20 and p53 is useful in differentiating carcinoma in situ from reactive atypia in bladder.[17] Positive HER2 status in bladder urothelial carcinoma is associated with aggressive behavior and provides prognostic information regarding recurrence and mortality.[14],[16] The significance of HER2 assessment in targeted therapy of bladder tumors is under evaluation.

Ki67 is a nonhistone nuclear protein which is a useful proliferation marker in malignant tumors. There is a good correlation between Ki67 labeling index and well-known prognostic factors such as grade and stage of urothelial carcinoma. High Ki-67 expression is associated with poor recurrence-free/progression-free/overall/cancer-specific survival outcome. Rare studies have documented contradictory results with Ki 67.[18] The current study was done to evaluate the expression of GLUT1 in bladder urothelial carcinoma and its association with HER2 and Ki67 index.

  Materials and Methods Top

Forty-three bladder tumors diagnosed by biopsy, transurethral resection, and cystectomy specimens during the study period from January to December 2017 were included.

The 2016 WHO guidelines were followed for classification, tumor staging, and grading. Two pathologists reviewed the cases, and representative tumor area was selected for IHC with polyclonal antibodies for GLUT1, HER2, and Ki67. Pressure cooker-aided antigen retrieval and standard immunoperoxidase ABC technique were used. The sections were dewaxed, hydrated and antigens retrieved into TRIS-EDTA buffer at pH 8.5–9.5 for 20 min. After being cooled to room temperature, sections were washed in distilled water for 5 min, followed by washing in TRIS saline buffer at pH 7.6 for 5 min. The sections were then quenched in H<Subscript>2</Subscript>O<Subscript>2</Subscript> for 10–15 min to block endogenous peroxidase activity which was followed by 2 TRIS saline buffer washes and protein block for 10 min. The primary antibody was applied for 1 h followed by 2 TRIS saline buffer washes and application of secondary antibody for 30 min. This was followed by 2 TRIS saline buffer washes. The chromogen used was 3, 39-diaminobenzidine tetrachloride for 5 min followed by wash in distilled water. The slides were then counterstained lightly with hematoxylin, dehydrated, and mounted. Positive and negative controls and wherever applicable internal controls were used. All cases were evaluated by 2 individual pathologists, and when the results were different, agreement was reached on the expression after careful discussion.

GLUT 1 expression given as immunoreactive score (IRS) was assessed by the level of membraneous staining: 0 - absent, 1 - mild, 2 - moderate, 3 - marked; and percentage of positive cells graded on a semiquantitative scale as 0(no staining), 1 (1%–10%) 2 (11%–25%), 3 (26%–50%), 4 (51%–80%), and 5(>80%) membrane positivity. GLUT1 IRS was calculated as level of staining X percentage of positive cells. The IRS negative: 0, weak: 1–4, moderate: 5–9, and strong: 10–15.

HER2 protein expression was assessed by the membranous staining; intensity and percentage of staining and scored on a scale of 0 − 3+ using ASCO scoring system. Only tumors with score 3+ are categorized as positive. A score of 1+ was defined as barely perceptible membrane staining in >10% of cells, a score of 2+ was defined as weak-to-moderate complete membrane staining present in >10% of tumor cells, and a score of 3+ was defined as strong complete membrane staining in >30% of tumor cells. Cytoplasmic staining was considered nonspecific and hence negative. Ki-67 protein showed nuclear positivity and scoring done after studying 1000 cells and expressed as percentage. The expression of these 3 markers was correlated with histopathological grading and invasiveness of bladder carcinoma.

Statistical analysis was performed with SPSS version 22 was used for statistical analysis. IBM SPSS Statistics V22.0. A descriptive analysis of all collected variables was performed. The correlation between expression of GLUT1, HER2, Ki67 and tumor grade and TNM stage were assessed with a significant P < 0.05.

  Results Top

Among the 43 patients, age ranged from 33 to 86 years with a median age of 65 years and majority (95.3%) were males which is comparable to most other studies.[4] Invasive urothelial carcinoma was diagnosed in 90.7% of the cases (39 patients). The lamina propria was invaded in 24 cases, and muscle invasion was seen in 15 cases. Low-grade and high-grade urothelial carcinomas were seen in 48.8% and 51.2%, respectively.

GLUT1-IRS assessed in the abovementioned method was grouped into weak, moderate, and strong, of which 55.8% cases showed strong expression. The GLUT1score was significantly associated with the grade of tumor (P < 0.001) and TNM stage (P < 0.05) [Table 1]. HER2 positivity was taken as score: 3+ and was seen in 4 of 43 tumors (9.3%). 39 cases showed a score 0−2 + were taken as HER2 negative (90.7%). HER2 expression was not significantly associated with the grade and TNM stage of urothelial carcinoma (P > 0.05) [Table 2]. The Ki67 value in low-grade tumors showed a mean of 11.2 ± 5.9% and in high-grade tumors 55.2 ± 19.2% which was statistically significant (P < 0.001). Ki67 value was significantly associated with TNM stage of urothelial carcinoma (P < 0.05) [Table 3]. Expression of GLUT1 correlated well with Ki67 value (P < 0.001) and showed moderate correlation with HER2 (Spearman rank correlation was 0.338). HER2 expression did not significantly correlate with Ki67 value in our study. GLUT1, HER2, and Ki67 expression as compared to the H and E sections are provided in photomicrographs [Figure 1] and [Figure 2].
Table 1: Association of glucose transporter 1 scores with the grade and tumor-node-metastasis stage of urothelial carcinoma

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Table 2: Association of human epidermal growth factor receptor-2 overexpression (presence versus absence) with different variables in the study

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Table 3: Association of Ki 67 proliferation index with the grade and tumor-node-metastasis stage of Urothelial carcinoma

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Figure 1: Hematoxylin and eosin (H and E, ×200) and IHC markers, ×400-glucose transporter 1, human epidermal growth factor receptor-2, and Ki67 in Ta and T1 (low and high grade) bladder urothelial carcinoma

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Figure 2: Hematoxylin and eosin (H and E, ×200) and IHC markers, ×400-glucose transporter 1, human epidermal growth factor receptor-2, and Ki67 in T2 (low and high grade) bladder urothelial carcinoma

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  Discussion Top

In the present study, we systematically evaluated 43 cases of urothelial carcinomas both invasive and noninvasive. GLUT1 over expression based on the intense membrane positivity was strong in high grade and stage bladder tumors which is comparable with that of previous studies. Membrane positivity for GLUT1 was noted in urothelial and squamous cell carcinomas while adenocarcinoma showed cytoplasmic GLUT1 positivity.[8]

Ki67 is an IHC marker present on actively proliferating cells, and it is a predictive factor of cancer cell proliferation which correlates with poor prognosis in several types of cancers. Ki67 index is assessed microscopically and expressed as percentage positive cells. Many studies have opted for a cutoff value of 20% (wide range 5%–55% in different studies) as low/high expression which correlated with tumor prognosis.[18],[19],[20] In our study, when we correlated the percentage positive value of Ki 67 expression, it showed statistically significant association with tumor grade and stage, and this was in concordance with the other studies.[5] The recurrence rate and poor survival rate in patients with overexpression of Ki67 mark its importance as an independent prognostic marker.

The GLUT1 IRS and the Ki-67 proliferative index significantly correlated (P < 0.001) in our study. Both showed strong association with increasing grade and stage of urothelial carcinoma (P < 0.05) which was similar to other studies.[7],[10],[11]

Various studies have documented HER2 overexpression to be associated with aggressive behavior and tumor recurrence in urothelial carcinomas.[13],[14],[21],[22] HER2 over-expression (score 3+) was observed in 9.3% cases in our study which was toward the lower end as is observed in most studies ranging from <10% to 81%.[12],[13],[14],[15],[16] The possible reasons could be the choice and concentrations of antibodies, variability in the interpretation and stratification criteria, and technical procedures involved in IHC.[12],[13],[14] Different scoring systems are available for HER2 and most authors used criteria of Wolff et al. reported for breast carcinoma (ASCO scoring system). The correlation between the use of IHC and fluorescence in situ hybridization for HER2 gene amplification is not well established for bladder carcinomas.[14],[15],[16] The current study did not show a significant association between HER2 overexpression and grade and TNM stage of urothelial carcinoma (P > 0.05). It was observed that HER2 was overexpressed in high-grade urothelial carcinomas, and this observation was similar to that by El Ochi et al.[16] Extensive necrosis seen in high-grade carcinoma may be a reason for negative HER2 expression in some cases. GLUT1-IRS and HER2 expression showed moderate correlation in our study (Spearman rank correlation = 0.338). Unlike breast and stomach carcinomas where HER2 targeted therapy is effective, the clinical trials are unsuccessful in bladder carcinoma.[23] The reason for this discrepancy is unclear and thought to be the complex molecular landscape of HER2 alteration which requires further research into its relevance as driver gene mutation during oncogenesis.[23],[24] The present study is limited by its retrospective nature, small sample size, and by the lack of gene amplification for HER2 assessment which may have contributed to lack of association between HER2 and tumor grade and stage of urothelial carcinoma.

  Conclusions Top

We have used a standardized scoring system to demonstrate that the GLUT1 protein expression pattern is significantly associated with increasing malignant potential (tumor grade and stage). Ki 67 proliferation index also showed significant association with tumor grade, stage, and correlated well with GLUT1 IRS in urothelial carcinomas. HER2 overexpression in urothelial carcinomas was 9.3%, and it showed moderate correlation with the GLUT1 IRS. These findings suggest the role of GLUT1 and Ki 67 as useful prognostic markers for urothelial bladder carcinomas. Further evaluations on larger sample size are needed to assess the role of HER2 in urinary bladder carcinoma.


The research project was funded by the State Board of Medical Research, Government Medical College, Kottayam, Kerala, India. We sincerely thank Mrs. Kalakumari, research assistant for meticulously doing the IHC.

Financial support and sponsorship

The research project was funded by the State Board of Medical Research, Government Medical College, Kottayam, Kerala, India.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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