| Abstract|| |
Aims: This study aims to explore the utility of GATA binding protein 3, a zinc finger transcription factor, expression in genitourinary carcinoma, especially urothelial carcinoma. Settings and Design: It is a prospective study where 74 consecutive cases of urothelial carcinoma along with 10 cases each of prostatic adenocarcinoma (PC) and conventional clear cell renal cell carcinoma were included between August 2016 and January 2017. Methods and Materials: All the cases were histopathologically evaluated and immunohistochemically stained for GATA binding protein 3. Only nuclear positivity was considered as positive. Immunoreactivity score for GATA expression was calculated based on the staining intensity as well as percentage. Statistical Analysis Used: The statistical analysis was done using Statistical Package for Social Sciences Version 15.0 statistical analysis software. P value of <0.05 was considered statistically significance. Results: GATA3 expressions were seen in 77% of the cases of urothelial carcinoma, whereas none of the clear cell renal cell carcinoma and prostatic adenocarcinoma cases was GATA3 positive. GATA3 expression significantly correlated with histological grade and muscle invasion with a weaker or negative expression in high-grade muscle invasive tumor as compared to low-grade and noninvasive neoplasm. Significantly weaker expression of GATA3 was found in cases with severe nuclear pleomorphism, mitosis >10/10 hpf, presence of necrosis, and tumor-infiltrating lymphocytes. No significant change in the status of GATA3 expression was seen in follow-up cases between initial Transurethral resection of bladder tumor (TURBT) and post-recurrence TURBT or radical cystectomy specimens. Conclusions: GATA3 as a sensitive and specific marker for urothelial carcinoma can be effectively used to exclude other genitourinary malignancies, PC, and renal cell carcinoma, at metastatic site. This marker can also be effectively used in predicting the probable grade and invasion in biopsy material with poor morphological characteristics, thereby helping in appropriate management in such cases.
Keywords: Carcinoma, expression, GATA-3, genitourinary, urothelial
|How to cite this article:|
Agarwal H, Babu S, Rana C, Kumar M, Singhai A, Shankhwar SN, Singh V, Sinha RJ. Diagnostic utility of GATA3 immunohistochemical expression in urothelial carcinoma. Indian J Pathol Microbiol 2019;62:244-50
|How to cite this URL:|
Agarwal H, Babu S, Rana C, Kumar M, Singhai A, Shankhwar SN, Singh V, Sinha RJ. Diagnostic utility of GATA3 immunohistochemical expression in urothelial carcinoma. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 May 23];62:244-50. Available from: http://www.ijpmonline.org/text.asp?2019/62/2/244/255808
| Introduction|| |
As per the Indian cancer registry data, urinary bladder cancer (UBC) is the ninth most common cancer of all cancer cases in men, with the male:female ratio of 8.6:1, which is more than the worldwide ratio of about 3.5:1., Most of the urothelial cancers (UC) are superficial at initial diagnosis, and as many as 70% are characterized by a prolonged clinical course over which patient experiences multiple recurrences following local resection without tumor progression. In contrast, a smaller but significant percentage of patients present with tumors that have an aggressive clinical course over a short period. Hence, UBC can be considered as one of costliest diseases, which requires lifelong surveillance.,
Detection of grade as well as muscle invasion is of great consequence as it highly influences the management and overall prognosis. Clinical staging is determined by transurethral resection (TURB) of all visible lesions followed by histopathological evaluation for accurate assessment of depth of tumor invasion, proper staging, and grading. Hence, histopathology plays a crucial part in directing the therapy and gauging the prognosis in patients with BC. Unfortunately, it is subjective to individual variability and has thus had poor specificity. Compounding the problem are technical issues, including crush and cautery artifacts brought about by the biopsy procedure itself, as well as distortion of the anatomical landmarks caused by prior biopsy or intravesical therapy.
UC also frequently presents as a tumor of unknown origin and can display a wide range of morphological patterns posing a diagnostic challenge; moreover, invasive high-grade urothelial carcinomas can be difficult to differentiate from other high-grade carcinomas as the morphology of high-grade urothelial carcinomas is not always specific. Hence, it may be difficult to distinguish invasive urothelial carcinoma from other genitourinary lesions such as prostatic and renal carcinoma.,
To overcome these issues and help in proper staging, grading, and to increase specificity at metastatic site of this disease, many immunohistochemical markers have been studied in the past including p63, cytokeratin 7, cytokeratin 20, uroplakin, placental S100 (S100P), and thrombomodulin, but none of them have proven to be effective.,, Since these markers are also expressed in a significant number of other epithelial malignancies, such as breast, ovary, and lung, their specificity as well as role at metastatic site remain limited.
GATA-3 is also known as GATA-binding protein 3 and transacting T-cell-specific transcription factor and has been known to be associated with cancer progression in several cancers like breast and colorectal carcinoma., It is only recently being recognized as an urothelial-associated immunohistochemical marker. There have been few studies done to study expression in different grade, stage, and histological variants or urothelial carcinoma with variable results.,,,, However, to the best of our knowledge, no such study has been done in Indian population so far.
With a hypothesis that the immunohistochemical expression of GATA-3 in UC and its correlation with clinical and histopathological parameters can have diagnostic, therapeutic, as well as prognostic implication along with site specificity, this study aims to identify the utility of GATA-3 expression in genitourinary carcinoma, especially urothelial carcinoma.
| Materials and Methods|| |
This study included 94 genitourinary tumors received between August 2016 and January 2017 in the Department of Pathology of our institute. The clinical details of the patients were collected from the case sheets. The study was approved by the Institutional Ethical Committee.
Previous biopsies of cases in follow-up when available were also included.
Exclusion criteria: All poorly preserved and inadequate specimens were excluded from this study. Patients in which proper clinical and radiological details were missing were also excluded from the study.
Tissue specimens were collected and immediately fixed in 10% formalin, processed, and later, paraffin embedded blocks were prepared. About 3–4-μm thick sections were taken from each block and stained by normal hematoxylin and eosin stain for histopathological examination.
All the cases were evaluated by two independent pathologists and reported as per the WHO/ISUP Classification 2016.
About 3–4 μm thick sections were taken from formalin fixed paraffin embedded block from each case. GATA3 immunohistochemical staining was performed on 3-aminopropyltriethoxysilane-coated slides. Staining and evaluation using specific Rabbit monoclonal antibody to GATA3 (Clone no. EPR16651, 1:300 dilution, abcam) were done. Human neuroblastoma tissue served as a positive control [Figure 1]. For negative control, primary antibody was omitted while performing immunohistochemical staining. Both positive and negative controls were included in every batch of Immunohistochemistry (IHC) staining.
|Figure 1: (a) Hematoxylin and eosin stain (400×), and (b) immunohistochemical staining for GATA3 in neuroblastoma case used as positive control|
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Immunohistochemical staining evaluation
The slides were examined at 400× magnification. Only nuclear staining was considered positive. The percentage of tumor cells labeled by GATA3 was scored as follows:
- Score 0: No tumor cells stained
- Score 1: 1–10%
- Score 2: 11–50%
- Score 3: 51–80%
- Score 4: 81–100%
The staining intensity of tumor cells labeled by GATA3 was scored as follows:
- Staining Score 0: No tumor cells stained
- Staining Score 1: Weak
- Staining Score 2: Moderate
- Staining Score 3: Strong.
Finally, immunoreactivity score for GATA3 expression was calculated by multiplying the number representing the percentage of immunoreactive cells by the number representing staining intensity and the cases were categorized in four groups [Figure 2] and shown in [Table 1].
|Figure 2: Groups of GATA3 immunohistochemical staining (200×): (a) negative (Group I), (b) weakly positive (Group II), (c) moderately positive (Group III), and (d) strongly positive (Group IV)|
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The statistical analysis was done using Statistical Package for Social Sciences Version 15.0 statistical analysis software. The values were represented in number (%) and mean ± SD. Chi-square test and Wilcoxon signed rank statistics were used as required. P value of <0.05 was considered statistically significance.
| Results|| |
The study included 94 genitourinary tumors, of which 74 were UC, 10 were clear cell renal cell carcinoma (CCRCC), and 10 were prostatic adenocarcinoma (PCa). The mean age of UC patient was 55.9 years (range of 21–83 years). There was male predominance with M–F = 7.2:1. Approximately 10% patients were aged ≤40 years; however, most common age group was 40–60 years (60.81%). Demographic, clinical, risk factors, and radiological details were available for all cases [Table 2].
|Table 2: Patient demography details, clinical presentation, risk factors, and radiological findings|
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GATA3 expression was analyzed on paraffin-embedded tissue section as per the immunoreactivity score. When positive, GATA3 staining was nuclear within clusters of malignant cells and no cytoplasmic staining was noted. The details of the group along with the study population are mentioned in [Table 3].
|Table 3: Distribution of study population according to GATA3 expression (n=74)|
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GATA3 expression was seen in 77% of the cases of urothelial carcinoma, whereas none of the CCRCC and PCa cases were GATA3 positive [Figure 3]. GATA3 had a sensitivity of 78.7% and specificity of 100% for urothelial carcinoma.
|Figure 3: GATA3 immunohistochemical staining in other genitor-urinary malignancies: (a) renal cell carcinoma hematoxylin and eosin stain (200×), (b) corresponding negative GATA3 immunohistochemical expression, (c) prostatic adenocarcinoma (200×), and (d) corresponding negative GATA3 immunohistochemical expression|
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The incidence of hematuria was found to be significantly higher among cases with positive GATA3 expression (P = 0.015). There was no significant association between GATA3 expressions and other signs/symptoms, age, gender, risk factors, size, site of lesion, or perivesical spread.
GATA3 expression also significantly correlated with histological grade (P < 0.001) and muscle invasion (P = 0.005) such that the low-grade and noninvasive tumors had moderate-to-strong expression as opposed to high-grade and invasive tumor which had a weak or no expression [Figure 4] and [Figure 5].
|Figure 4: (a) Hematoxylin and eosin (100×) and corresponding, (b) negative GATA3 immunohistochemical staining in a case of high-grade muscle invasive urothelial carcinoma, (c) hematoxylin and eosin (100×) and corresponding, and (d) weakly positive GATA3 immunohistochemical staining in a case of high-grade muscle invasive urothelial carcinoma|
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|Figure 5: (a) Hematoxylin and eosin (100×) and corresponding, (b) moderately positive GATA3 immunohistochemical staining in a case of low-grade noninvasive urothelial carcinoma, (c) hematoxylin and eosin (100×) and corresponding, (d) strongly positive GATA3 immunohistochemical staining in a case of low-grade noninvasive urothelial carcinoma|
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When GATA3 expression was correlated with other histopathological parameters, significant association was found with nuclear pleomorphism, mitosis, necrosis, and tumor-infiltrating lymphocytes. Weak or negative GATA3 expression was seen with marked nuclear pleomorphism and increased mitotic activity (>10 mitosis/10 high power field, necrosis, and tumor-infiltrating lymphocytes). Though lymphovascular invasion was found to be present in higher proportion of Group I (41.18%) as compared to Group II (20.00%), Group III, and Group IV (16.67% each), this difference was not found to be statistically significant (P = 0.240) [Table 4].
|Table 4: Intergroup comparison of GATA3 expression with histopathological examination findings of study population|
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No significant change in the status of GATA3 expression was seen in follow-up cases between initial TURBT and post-recurrence TURBT or radical cystectomy specimens. Majority of patients survived (n = 67; 90.54%) and rest (7; 9.46%) expired during the period of study. All these seven patients succumbed to their disease. Three had high-grade muscle invasive UC and remaining three had lamina invasive but with signet ring, plasmacytod, or sarcomatoid differentiation. Proportion of survived patients was higher among Group III (94.44%) and Group IV (95.83%) as compared to Group I (88.24%) and Group II (80.00%), but this difference was not found to be statistically significant.
| Discussion|| |
GATA is a family of transcription factors whose members bind the DNA sequence (A/T) GATA (A/G) in the promoters of genes to directly activate or repress expression of target genes. GATA family members have been further subdivided into two subgroups; GATA1, GATA2, and GATA3, which are often, but not exclusively, associated with hematopoietic lineages and nervous system development, and GATA4, GATA5, and GATA6, which are commonly associated with mesoderm-derived and endoderm-derived tissues/organs., In particular, GATA3 also plays an important role in the development and differentiation of tissues including the luminal glandular epithelial cells of the breast,,, parathyroid gland, adipose tissue, kidney, sympathetic nervous system,,, and lens fiber cells of the eye. Mutations, loss of expression, or overall expression of GATA factors have all been associated with a broad variety of cancers in humans, including leukemia, breast cancers, gastrointestinal cancers, and others.
GATA3 is one of the most recently recognized urothelial-associated immunohistochemical markers which has been reported to be useful for assisting in the differential diagnosis of urothelial carcinomas. In 2007, Higgins et al. were the first to investigate the expression of GATA3 as marker for transitional epithelium and urothelial carcinoma and suggested to be highly specific for urothelial carcinoma. Morphology of invasive high-grade urothelial carcinoma is not always specific and may morphologically mimic other high-grade metastatic tumors, like PC, renal cell carcinoma (RCC), and squamous cell carcinoma. It is very important to differentiate as the treatment and management vary according to the primary malignancy.,
Few investigators investigated the utility of GATA3 immunohistochemical expression in various tumors including urothelial carcinoma and concluded that it is a sensitive marker for breast carcinoma and UC.,,, Few studies in past have very well-demonstrated lack of GATA3 expression in CCRCC.,, Later in 2014, Nilda Gonzalez Riobon studies GATA3 expression in different types of renal tumors, including CCRCC, as well as metastatic RCC. They showed an overall lack of GATA3 expression in primary and metastatic RCCs and hence supported a role for GATA3 in the differential diagnosis of primary renal masses and a potential utility in the interrogation of metastatic tumors of unknown primary in the presence of a renal mass. Chang et al. in 2012 in their study demonstrated that none of the high-grade PC showed GATA3 immunoexpression; however, weak positivity was seen in some of the basal of benign prostatic tissue. Our results are also the same such that all the PCs or RCC were negative for GATA3, suggesting it to be a specific marker for UC. Hence, this marker should be routinely used in immunohistochemical panel in assessment of a high-grade carcinoma of genitourinary tract when the primary site is not certain. It is also know that GATA3 expresses similar immunostaining properties in both primary and metastatic sites.
So far few studies have been done showing variable expression of GATA3 in urothelial carcinoma ranging from 63% to up to 93%. In our study, the GATA3 expression was seen in 77% of the cases which was in accordance with many of the previous studies done. None of the studies so far have seen any association of GATA3 expression with age, gender, or clinical signs and symptoms. However, we found that hematuria was the most common presenting symptom in the study population and its incidence was found to be significantly higher in cases showing GATA3 expression. Mohammed et al. have found that high GATA3 expression was associated with larger tumor size in invasive urothelial carcinomas, which is not seen in any other study including ours.
The prognostic significance of GATA3 in urothelial neoplasm was first studied by Miyamoto et al., where they found that loss of GATA3 was associated with high-grade and/or muscle invasive tumors, whereas strong expression was an independent predictor of poor prognosis. Statistically significant correlation (P < 0.001) was also found between histological grade and GATA3 expression pattern in our study as well. It was seen that 100% of low-grade tumors were moderate-to-strongly immunoreactive for GATA3 as compared to 70% of high-grade tumors showing only weak positivity. Rest of the high-grade tumors did not show any reactivity. Almost similar results have been shown by other investigators except for Hoang et al. and Higgins et al., where the less number of low-grade tumor UC and high-grade UC, respectively, showed GATA3 expression.,,,,,,,,,,,,,,,,,,,, In our study, 66% of muscle invasive tumors showed GATA3 positivity (weak positivity) in comparison to 87% of nonmuscle invasive tumors (noninvasive and lamina invasive). It was also seen that all the cases of noninvasive UC and majority of lamina invasive UC showed moderate-to-strong intensity of GATA3 expression. Thus, loss of GATA3 expression was observed in muscle invasive urothelial tumors which are in concordance with the finding of Miyamoto et al. Apart from grade and invasion, we also found a significant statistical association of weak or absent GATA3 expression with other histopathological parameter like marked nuclear pleomorphism (P = 0.002), >10 mitosis per 10 high power field (P < 0.001), presence of necrosis (P = 0.019), and presence of tumor-infiltrating lymphocytes (P = 0.046). No study so far has documented any such association.
In our study, recurrence was found in only 9/74 (12%) patients. Change in the GATA3 expression between initial TURBT and post-recurrence TURBT/radical cystectomy was also studied in these cases. However, no statistically significant association was established between GATA3 expression and recurrence. Similar finding was also seen in study by Miyamoto et al. where GATA3 levels were not associated with recurrence. Moreover, no significant change in GATA3 expression of follow-up patients after recurrence was seen as compared to their GATA3 status in initial TURBT. Majority of patients (90%) in our study showed survival during the first year of diagnosis and no significant association could be established between GATA3 expression and survival. However, this may be due to our shorter study duration and small sample size. Larger studies may be necessary to obtain a significant correlation in this variable.
Hence, this is the first study from India that confirms GATA3 as a sensitive and specific marker for urothelial carcinoma which can be effectively used to exclude other genitourinary malignancies, PC, and RCC, at metastatic site. In addition, intensity of GATA3 expression decreases with high-grade, invasion, increasing nuclear pleomorphsim, as well as mitosis, presence of necrosis, and tumor-infiltrating lymphocytes. There is no association between GAT 3 expressions with recurrence or survival.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, King Geroge's Medical University, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]