Expression of transient receptor potential melastatin 4 in differential diagnosis of eosinophilic renal tumors
- Authors:
- Published online on: September 10, 2021 https://doi.org/10.3892/mco.2021.2393
- Article Number: 230
Abstract
Introduction
Renal cell carcinoma accounts for approximately 2-3% of adult cancers, and the rate of its incidental diagnosis is increasing with the increased use of imaging methods. Numerous diagnostic, predictive and prognostic biomarkers have been studied for the diagnosis, treatment planning, and follow-up. Especially the differentiation of eosinophilic kidney tumors has become more challenging with newly defined tumors and the need for different immunohistochemical and molecular-genetic studies for differential diagnosis has increased day by day (1,2). The marker thought to help in the differentiation of these tumors is TRPM4 (transient receptor potential cation channel subfamily M4), a protein localized on the cell membrane.
In mammals, there are 28 TRP (transient receptor potential) channels, which are cation-selective and located on the cell membrane. Although this protein superfamily has 6 subgroups, one of the most important is TRP that contains Melastatin (3). TRPM (transient receptor potential cation channel subfamily M) has eight subgroups, most of which are permeable to bivalent cations, while TRPM4 and TRPM5 are channels that are impermeable to calcium (Ca+2) but only permeable to monovalent cations. When the amount of cytosolic ATP decreases and Ca+2 level increases, the TRPM4 channel is activated (4) and indirectly help regulate intracellular Ca+2 through the regulation of cell membrane potential. These ion channels and Ca+2 are responsible for cell proliferation, apoptosis, and differentiation to maintain cell homeostasis (5). The expression of TRPM4 has been evaluated in cancer of several organs, especially the prostate and colon (6,7). However, there is no clear data regarding the place of the kidney in the differential diagnosis of eosinophilic tumors.
The aim of our study was to analyze the staining pattern of TRPM4 antibody in common eosinophilic kidney tumors such as eosinophilic clear cell renal cell carcinoma (CCRCC), Chromophobe Renal Cell Carcinoma (ChRCC), Renal Oncocytoma (RO) and Papillary Renal Cell Carcinoma Type 2 (PRCC2), and to determine whether it can be a diagnostic marker.
Materials and methods
Patient
The study included a total of 112 patients, including 97 patients diagnosed with eosinophilic CCRCC, ChRCC, P2RCC, RO and 15 patients with P1RCC from the partial and radical nephrectomy specimens studied at Bezmialem Vakif University Faculty of Medicine between January 2014 and 2019. This study is a retrospective study made of paraffin blocks. The presence of TRPM4 could also be demonstrated by Western Blot analysis; however, Western Blot analysis could not be performed in this study as there were no fresh tissues of the cases. The study was approved by the ethics committee board of Bezmialem University.
Immunohistochemistry
In the present study, hematoxylin and eosin (H&E) stained preparations were re-evaluated, and block selection was made for immunohistochemical staining. Two-micron thick slides were taken from the paraffin blocks prepared from formalin-fixed specimens obtained from the primary tumor. On these slides, TRPM4 (monoclonal mouse antibody; Novus Biologicals, USA Clone OTI10H5) immunohistochemistry was performed by being diluted at a ratio of 1/100 in 2 h of incubation in Ventana Benchmark Ultra and Bench Mark XT devices.
Scoring of immunoreactivity
The extent of TRPM4 staining was evaluated semi-quantitatively and interpreted as 0, negative; 1, focal positive (1-10%); and 2, diffuse positive staining (more than 10%). Staining patterns were evaluated as membranous-cytoplasmic and basolateral. Staining patterns and their extent were compared with tumor types, demographic data, and cytokeratin 7 (CK7) staining status.
Statistical analysis
The statistical analysis was evaluated using the IBM SPSS 22.0 statistical software package. The distribution of categorical variables was evaluated with the Chi-square test. Descriptive statistics were expressed as standard deviation, frequency, and percentage. A P-value of <0.05 was considered to indicate a statistically significant difference.
Results
Patient data and histopathological parameters
Of the 112 patients included in the study, 33 (29.5%) had eosinophilic CCRCC, 35 (31.3%) had eosinophilic ChRCC, 8 (7.1%) had P2RCC, 21 (18.8%) had RO, and 15 (13.4%) had P1RCC. Of the patients, 46 (41.1%) were female and 66 (58.9%) were male, with a mean age of 57.8 years. The clinical and pathological parameters of the patients are summarized in Table I.
TRPM4 staining rates and distributions in CCRCC, ChRCC, RO and P2RCC tumors
The staining ratios of the tumors with TRPM4 are presented in Table II. Among the 97 eosinophilic tumors, 2 (4.9%) of the 41 patients with diffuse staining for TRPM4 were CCRCC, 15 (36.6%) were ChRCC, 20 (48.8%) were RO, and 4 (9.8%) were P2RCC. While a significant result was obtained between the RO and ChRCC and CCRCC and P2RCC patients (P=0.043), there was no significant result between the RO and ChRCC patients (P=0.065).
While TRPM4 staining was diffuse membranous in the collecting ducts of the healthy kidney, different staining patterns were observed in the oncocytoma and eosinophilic RCC subtypes. Staining was observed in 57 of the patients. Membranous staining was observed in 50 patients and basolateral staining in 7 patients. Of the patients with membranous staining, 26 (52%) had ChRCC, 21 (42%) had RO, 3 (6%) had P2RCC. Membranous staining was not observed in any of the CCRCC patients, and there was a statistically significant difference between the RO and ChRCC patients (P<0.005). There were 2 (28.6%) CCRCC, 1 (14.3%) ChRCC, and 4 (57.1%) P2RCC patients with basolateral staining. In terms of P1RCC, 1 (6.7%) patient had negative staining, 3 (20%) patients had focal, and 11 (73.3%) patients had diffuse staining. Of the P1RCC patients, 11 (73.3%) had basolateral and 3 (20%) had membranous staining (Fig. 1).
TRPM4 staining (negative, focal, diffuse staining) of the patients were compared with CK7 immunohistochemistry, which is used for the diagnosis and differentiation of tumors. CK7 and TRPM4 staining by the diagnoses of the tumors are summarized in Fig. 2. In CCRCC, focal staining was observed in 5 (15%) cases with CK7 and diffuse staining in 2 (4.9%) cases with TRPM4. In RO, focal staining was observed in 13 (61.9%) cases with CK7, diffuse staining in 1 (4.7%) case, diffuse staining in 20 (86.9%) cases with TRPM4, and focal staining in 1 (8.6%) case. ChRCC had diffuse staining in 34 (97.1%) cases with CK7, focal staining in 1 (2.8%) case, diffuse staining in 15 (42.8%) cases with TRPM4, and focal staining in 12 (34.2%) cases. In P2RCC, diffuse staining was observed in 3 (37.5%) cases with CK7 and focal staining in 3 (37.5%) cases, while diffuse staining was observed in 4 (50%) cases with TRPM4 and focal staining in 3 (37.5%) cases.
Discussion
Ca+2 dependent signaling pathways are associated with proliferation, migration, invasion, metastasis, and apoptosis of tumor cells (8,9). TRPM4 is a monovalent nonselective cation channel activated by decreased ATP level and increased Ca+2 in case of hypoxia, the membrane is depolarized, and the voltage-dependent calcium channel is blocked by the Na+ current, decreasing the permeation of Ca+2 (10).
The expression of TRPM4 has been studied in multiple sclerosis (11), subarachnoid haemorrhage (12), and cerebral infarction (13), but there are very few studies on the relationship between the tumor and TRPM4(14). It has recently been evaluated in prostate (15), bladder (16), colorectal cancer (17), cervical cancer (18), large B-cell lymphoma (19), and liver cancer (20). It has been determined that it is higher in the tumor in the prostate compared to healthy tissues, and its expression increases (21,15) in the transition from prostatic intraepithelial neoplasia to prostatic carcinoma, and high levels of TRPM4 are associated with recurrence after prostatectomy (22). It has been found to be highly expressed in cervical cancer and large B-cell lymphoma compared to reactive tissues (18,19). No difference has been found in the expression of TRPM4 between carcinoma of the bladder and healthy tissue (16). In our study, diffuse membranous staining was detected in healthy tissue and oncocytoma, while loss of expression was observed in CCRCC and different rates of staining were observed in ChRCC and P2RCC. In addition to healthy tissue, its staining in RO and ChRCCs suggests that it may be related to the same origin.
Over the past two decades, there have been many morphological, immunohistochemical and prognostic innovations in renal tumors (23). Among tumors with eosinophilic cytoplasm, there are new entities such as SDH-deficient RC, eosinophilic solid and cystic RCC, Warthin-like papillary RCC, and low grade oncocytic tumor in addition to ChRCC, RO, P2RCC, eosinophilic CCRCC (2,23). There are a limited number of cases regarding these tumors, and studies are ongoing in terms of both diagnosis and prognosis. It is important to differentiate the oncocytoma, which accounts for approximately 10% of renal tumors, from malignant tumors, especially in needle biopsies. In addition to morphological findings, CK7 is the most important marker in differentiating ChRCC, another CD117-positive eosinophilic tumor (2,24). While the staining pattern of CK7 is negative or focal positive in RO, it is usually diffuse positive in ChRCC. In our study, the staining of TRPM4 expression was diffuse positive in oncocytoma, unlike CK7. But negative, focal positive and diffuse positive stainings were detected in ChRCC. Besides morphological findings in the differentiation between eosinophilic CCRCC and ChRCC, the use of stains such as CK7, CD117 and Carbonic Anhydrase IV along with TRPM4 will be supportive in the differentiation.
Papillary RCC has an incidence rate of 15% and consists of type 1 with amphophilic cytoplasm and type 2 with eosinophilic cytoplasm (25). TRPM4 generally showed diffuse basolateral staining in P2RCC, and the staining pattern was completely different from other eosinophilic tumors included in the study. Among papillary RCCs, P2RCC has worse prognosis than P1RCC, and it is important to differentiate between them. Diffuse positive basolateral staining was usually seen for TRPM4 in both of them. Although TRPM4 is not a marker that can be used to differentiate between P1RCC and P2RCC, it is interesting that basolateral staining is dominant in papillary structuring.
There are publications in the literature showing an increase in the expression of TRPM4 in prostate, cervix, and large cell lymphoma. In these studies, there are arguments that suppression of TRPM4 can prevent tumor growth and metastasis (26). Wong and Hussain (27) also found TRPM4 to be associated with poor prognostic parameters in breast carcinoma. However, in this study, different expressions of TRPM4 were observed in malignant tumors of the kidney, while diffuse strong expression was remarkable in healthy tissue and oncocytoma, a benign tumor. In this study, TRPM4 was studied only for diagnostic purposes, although no interpretation could be made regarding the treatment or prognosis in RCCs, it is thought that TRPM4 can be used together with other immunohistochemical markers in eosinophilic renal tumors.
In this study, it was concluded that TRPM4 was useful in the differentiation of eosinophilic kidney tumors. The extent of staining along with CK7 may be helpful in the differentiation between common oncocytoma and chromophobe RCC, and for the diagnosis of papillary RCC, it can be helpful in cases of difficulty with different staining patterns. In this respect, its reliability can be increased to a more significant level with other markers and large series.
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
GÇ, PY and NŞ conceived and designed the study. GÇ, NŞ, and BD acquired the data. GÇ and ZG analyzed and interpreted the data. GÇ, NŞ and ZG confirm the authenticity of the raw data. GÇ and NŞ wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethical Committee of Bezmialem Vakif University Hospital (Istanbul, Turkey; approval no. 11-202), and written informed consent was obtained from each patient.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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