Medicine
®
OBSERVATIONAL STUDY
BSND and ATP6V1G3: Novel Immunohistochemical Markers for Chromophobe Renal Cell Carcinoma Kazuya Shinmura, MD, PhD, Hisaki Igarashi, Hisami Kato, Kenji Koda, MD, PhD, Hiroshi Ogawa, MD, PhD, Seishiro Takahashi, MD, PhD, Yoshiro Otsuki, MD, PhD, Tatsuaki Yoneda, MD, PhD, Yuichi Kawanishi, PhD, Kazuhito Funai, MD, PhD, Tatsuya Takayama, MD, PhD, Seiichiro Ozono, MD, PhD, and Haruhiko Sugimura, MD, PhD
Abstract: Differentiating between chromophobe renal cell carcinoma (RCC) and other RCC subtypes can be problematic using routine light microscopy. This study aimed to identify novel immunohistochemical markers useful for a differential diagnosis between chromophobe RCC and other RCC subtypes. We selected 3 genes (including BSND and ATP6V1G3) that showed specific transcriptional expression in chromophobe RCC using expression data (n ¼ 783) from The Cancer Genome Atlas (TCGA) database. A subsequent immunohistochemical examination of 186 RCCs obtained in our patient series resulted in a strong diffuse positivity of BSND and ATP6V1G3 proteins (both of which are involved in the regulation of membrane transport) in all the chromophobe RCC specimens (23/23 cases, 100%) but not in the clear cell RCC specimens (0/153 cases, 0%) or the papillary RCC specimens (0/10 cases, 0%). BSND and ATP6V1G3 protein expressions were also detected in renal oncocytoma (13/14 cases, 92.9%) and in the distal nephron, including the collecting duct, in the normal kidney. A computational analysis of TCGA data suggested that DNA methylation was involved in the differential expression pattern of both genes among RCC subtypes. Finally, an immunohistochemical analysis showed lung carcinomas were negative (0/85 cases, 0%) for the expression of both proteins. These results suggest that BSND and ATP6V1G3 are excellent novel immunohistochemical markers for differentiating between chromophobe RCC and other subtypes of RCC, including clear cell and papillary RCCs. (Medicine 94(24):e989) Abbreviations: RCC = renal cell carcinoma, RNA-seq = RNAsequencing, TCGA = The Cancer Genome Atlas, TMA = tissue microarray, TSS = transcription start site.
Editor: Ricardo S. Macarenco. Received: January 8, 2015; revised: May 4, 2015; accepted: May 18, 2015. From the Department of Tumor Pathology (KS, HI, HK, HS); ResearchEquipment Center (YK); Department of Surgery 1 (KF); Department ofUrology(TT, SO), Hamamatsu University School of Medicine, Hamamatsu; Department of Pathology, Fujieda Municipal General Hospital,Fujieda (KK); Division of Pathology, Seirei Mikatahara General Hospital, Hamamatsu (HO, ST); and Department of Pathology (YO); Department of Urology(TY),Seirei Hamamatsu General Hospital, Hamamatsu, Japan. Correspondence: Kazuya Shinmura, Department of Tumor Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192, Japan (. e-mail: [email protected]). This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare (21–1), the Japan Society for the Promotion of Science (25460476), the Ministry of Education, Culture, Sports, Science and Technology (221S0001), and the Smoking Research Foundation. The authors have no conflict of interests to disclose. Copyright # 2015 Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN: 0025-7974 DOI: 10.1097/MD.0000000000000989
Medicine
Volume 94, Number 24, June 2015
INTRODUCTION
C
hromophobe renal cell carcinoma (RCC) is a subtype of RCC and constitutes approximately 5% of renal neoplasms.1 Patients with chromophobe RCC have a better prognosis than those with conventional clear cell RCC, and more than 90% of patients with chromophobe RCC are alive 5 years after surgery;2–4 however, aggressive features and distant metastasis of chromophobe RCC can occur.5 Hoffmann et al6 previously examined sites of distant metastases in chromophobe RCC patients with initial M0 disease who had experienced metastasis during their follow-up period and found that the lung is the most frequent site of distant metastasis but that metastases of chromophobe RCC can also be found in other tissues. Histologically, there are 2 major variant types, referred to as the classic (alternatively, typical) variant and the eosinophilic variant in the chromophobe RCC.7,8 Although representative cases of chromophobe RCC can be easily distinguished from other renal epithelial tumors based on morphologic features, difficulties in making a proper diagnosis are sometimes encountered in histologically borderline cases; for example, a classic variant of chromophobe RCC might be confused with clear cell RCC, and its eosinophilic variant might be confused with clear cell RCC, papillary RCC, or renal oncocytoma. Therefore, immunohistochemistry is often used to assist in the differential diagnosis of chromophobe RCC in such cases.7,9 So far, the frequencies of immunoreactivity for CK7, CD117 (C-Kit), parvalbumin, CDH1 (E-Cadherin), CLDN7 (Claudin 7), CA2 (Carbonic Anhydrase II), EMA, CD82 (KAI1), EPCAM (Epithelial Cell Adhesion Molecule), and CDH16 (KSP-Cadherin) have been reported to be higher in chromophobe RCC than in clear cell RCC and/or papillary RCC,10–21 and some antibodies against these antigens are used in practical pathological diagnosis. However, since a subset of chromophobe RCCs that are immunohistochemically negative for these markers and a subset of clear cell RCCs and papillary RCCs that are positive for these markers have also been reported,10–21 the results of immunohistochemical analyses are not always reliable. Microarray analysis has been used as an analytical method for measuring the level of mRNA expression for more than a decade, despite some limitations such as the occurrence of hybridization artifacts and a low dynamic range.22,23 Recently, however, the RNA-sequencing (RNA-seq) method has been developed, enabling the aforementioned limitations of microarray analysis to be overcome. Moreover, this method also has an advantage over microarray analysis in terms of some issues, such as the detection of low abundance transcripts, the differentiation of isoforms, and its dynamic range.24 Thus, the use of the RNA-seq method might lead to the identification of novel cancer-related changes in mRNA expression. Therefore, to identify novel reliable immunohistochemical markers that are useful for distinguishing between chromophobe RCC and other www.md-journal.com |
1
Medicine
Shinmura et al
subtypes of RCC, such as clear cell and papillary RCCs, we globally compared the processed RNA-seq expression data from 66 cases of chromophobe RCC, 519 cases of clear cell RCC, and 198 cases of papillary RCC that were included in The Cancer Genome Atlas (TCGA)25 and found 3 candidate genes including BSND and ATP6V1G3. Next, we examined the expression statuses of these genes in 200 primary renal tumors and 85 primary lung carcinomas, and found that BSND and ATP6V1G3 were highly sensitive and specific markers of chromophobe RCC. Our study suggests that evaluating the expression levels of BSND and ATP6V1G3 could be of great value for distinguishing between chromophobe RCC and other subtypes of RCC.
Volume 94, Number 24, June 2015
primary lung carcinoma cases, composed of 44 cases of squamous cell carcinoma of the lung and 41 cases of adenocarcinoma of the lung, which had undergone surgery at Hamamatsu University Hospital (Japan) were used for the TMA block. To explain in further detail, the block was prepared by transferring a cylinder of 3-mm diameter from each of the paraffinembedded tissue samples using a microarrayer (KIN-1; Azumaya, Tokyo, Japan), as previously described.29 All the cases used for the immunohistochemical staining were listed in Table 1. This study was conducted with the approval of the Institutional Review Board (IRB) of Hamamatsu University School of Medicine.
Immunohistochemical Staining MATERIALS AND METHODS Collection of Publicly Available TCGA Data Gene expression data and DNA methylation data for a total of 783 RCC cases, composed of 66 chromophobe RCC cases, 519 clear cell RCC cases, and 198 papillary RCC cases (TCGA public data available in April 2014), were collected from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga). Gene expression data for bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, stomach adenocarcinoma, and thyroid carcinoma were also collected from the TCGA data portal. The expression data were obtained as processed RNA-seq data in the form of RNA-seq by Expectation Maximization or in the form of Reads Per Kilobase of exon Model per million mapped reads.26,27 The RNA-seq by Expectation Maximization or Reads Per Kilobase of exon Model per million mapped reads expression value for each gene was divided by that of the TBP gene, which is a control housekeeping gene,28 to compare the expression levels. DNA methylation data obtained using the Infinium HumanMethylation450 platform (Illumina, Inc, CA) were shown as the b value.25
Preparation of Tissue Microarray (TMA) Blocks Paraffin-embedded tissue samples from a total of 200 primary renal tumor cases, composed of 23 chromophobe RCC cases, 153 clear cell RCC cases, 10 papillary RCC cases, and 14 oncocytoma cases, that had undergone surgery at Hamamatsu University Hospital (Japan), Fujieda Municipal General Hospital (Japan), Seirei Mikatahara General Hospital (Japan), or Seirei Hamamatsu General Hospital (Japan) were collected, and 190 cases of them were used for the TMA block. In addition, paraffin-embedded tissue samples from a total of 85
Sections of paraffin blocks were used for immunohistochemical staining with an automatic immunohistochemical stainer, the HISTOSTAINER (Nichirei Bioscience, Tokyo, Japan). Briefly, the sections were deparaffinized, rehydrated, and boiled at 968C for 40 minutes in TE solution (pH 9.0) for antigen retrieval. Endogenous peroxidase activity was blocked by incubation for 5 minutes in a 3% hydrogen peroxide solution. Next, the sections were incubated with a rabbit anti-BSND polyclonal antibody (1:1000; Sigma–Aldrich, St. Louis, MO), a rabbit anti-ATP6V1G3 polyclonal antibody (1:2000; Sigma– Aldrich), or a rabbit anti-FBN3 polyclonal antibody (1:100; Sigma–Aldrich) for 30 minutes at room temperature. After washing, the sections were incubated for 30 minutes at room temperature with an amino acid polymer conjugated with goat antirabbit IgG and horseradish peroxidase (Histofine Simple Stain MAX-PO Kit; Nichirei, Tokyo, Japan). The antigen– antibody complex was visualized with 3,30 -diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. The staining intensity for BSND and ATP6V1G3 were graded for each specimen as follows: negative, weakly positive, or strongly positive. Additionally, proportion of positive cells for each specimen in the immunostaining was grouped into 3 categories as follows: none (
®
OBSERVATIONAL STUDY
BSND and ATP6V1G3: Novel Immunohistochemical Markers for Chromophobe Renal Cell Carcinoma Kazuya Shinmura, MD, PhD, Hisaki Igarashi, Hisami Kato, Kenji Koda, MD, PhD, Hiroshi Ogawa, MD, PhD, Seishiro Takahashi, MD, PhD, Yoshiro Otsuki, MD, PhD, Tatsuaki Yoneda, MD, PhD, Yuichi Kawanishi, PhD, Kazuhito Funai, MD, PhD, Tatsuya Takayama, MD, PhD, Seiichiro Ozono, MD, PhD, and Haruhiko Sugimura, MD, PhD
Abstract: Differentiating between chromophobe renal cell carcinoma (RCC) and other RCC subtypes can be problematic using routine light microscopy. This study aimed to identify novel immunohistochemical markers useful for a differential diagnosis between chromophobe RCC and other RCC subtypes. We selected 3 genes (including BSND and ATP6V1G3) that showed specific transcriptional expression in chromophobe RCC using expression data (n ¼ 783) from The Cancer Genome Atlas (TCGA) database. A subsequent immunohistochemical examination of 186 RCCs obtained in our patient series resulted in a strong diffuse positivity of BSND and ATP6V1G3 proteins (both of which are involved in the regulation of membrane transport) in all the chromophobe RCC specimens (23/23 cases, 100%) but not in the clear cell RCC specimens (0/153 cases, 0%) or the papillary RCC specimens (0/10 cases, 0%). BSND and ATP6V1G3 protein expressions were also detected in renal oncocytoma (13/14 cases, 92.9%) and in the distal nephron, including the collecting duct, in the normal kidney. A computational analysis of TCGA data suggested that DNA methylation was involved in the differential expression pattern of both genes among RCC subtypes. Finally, an immunohistochemical analysis showed lung carcinomas were negative (0/85 cases, 0%) for the expression of both proteins. These results suggest that BSND and ATP6V1G3 are excellent novel immunohistochemical markers for differentiating between chromophobe RCC and other subtypes of RCC, including clear cell and papillary RCCs. (Medicine 94(24):e989) Abbreviations: RCC = renal cell carcinoma, RNA-seq = RNAsequencing, TCGA = The Cancer Genome Atlas, TMA = tissue microarray, TSS = transcription start site.
Editor: Ricardo S. Macarenco. Received: January 8, 2015; revised: May 4, 2015; accepted: May 18, 2015. From the Department of Tumor Pathology (KS, HI, HK, HS); ResearchEquipment Center (YK); Department of Surgery 1 (KF); Department ofUrology(TT, SO), Hamamatsu University School of Medicine, Hamamatsu; Department of Pathology, Fujieda Municipal General Hospital,Fujieda (KK); Division of Pathology, Seirei Mikatahara General Hospital, Hamamatsu (HO, ST); and Department of Pathology (YO); Department of Urology(TY),Seirei Hamamatsu General Hospital, Hamamatsu, Japan. Correspondence: Kazuya Shinmura, Department of Tumor Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192, Japan (. e-mail: [email protected]). This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare (21–1), the Japan Society for the Promotion of Science (25460476), the Ministry of Education, Culture, Sports, Science and Technology (221S0001), and the Smoking Research Foundation. The authors have no conflict of interests to disclose. Copyright # 2015 Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN: 0025-7974 DOI: 10.1097/MD.0000000000000989
Medicine
Volume 94, Number 24, June 2015
INTRODUCTION
C
hromophobe renal cell carcinoma (RCC) is a subtype of RCC and constitutes approximately 5% of renal neoplasms.1 Patients with chromophobe RCC have a better prognosis than those with conventional clear cell RCC, and more than 90% of patients with chromophobe RCC are alive 5 years after surgery;2–4 however, aggressive features and distant metastasis of chromophobe RCC can occur.5 Hoffmann et al6 previously examined sites of distant metastases in chromophobe RCC patients with initial M0 disease who had experienced metastasis during their follow-up period and found that the lung is the most frequent site of distant metastasis but that metastases of chromophobe RCC can also be found in other tissues. Histologically, there are 2 major variant types, referred to as the classic (alternatively, typical) variant and the eosinophilic variant in the chromophobe RCC.7,8 Although representative cases of chromophobe RCC can be easily distinguished from other renal epithelial tumors based on morphologic features, difficulties in making a proper diagnosis are sometimes encountered in histologically borderline cases; for example, a classic variant of chromophobe RCC might be confused with clear cell RCC, and its eosinophilic variant might be confused with clear cell RCC, papillary RCC, or renal oncocytoma. Therefore, immunohistochemistry is often used to assist in the differential diagnosis of chromophobe RCC in such cases.7,9 So far, the frequencies of immunoreactivity for CK7, CD117 (C-Kit), parvalbumin, CDH1 (E-Cadherin), CLDN7 (Claudin 7), CA2 (Carbonic Anhydrase II), EMA, CD82 (KAI1), EPCAM (Epithelial Cell Adhesion Molecule), and CDH16 (KSP-Cadherin) have been reported to be higher in chromophobe RCC than in clear cell RCC and/or papillary RCC,10–21 and some antibodies against these antigens are used in practical pathological diagnosis. However, since a subset of chromophobe RCCs that are immunohistochemically negative for these markers and a subset of clear cell RCCs and papillary RCCs that are positive for these markers have also been reported,10–21 the results of immunohistochemical analyses are not always reliable. Microarray analysis has been used as an analytical method for measuring the level of mRNA expression for more than a decade, despite some limitations such as the occurrence of hybridization artifacts and a low dynamic range.22,23 Recently, however, the RNA-sequencing (RNA-seq) method has been developed, enabling the aforementioned limitations of microarray analysis to be overcome. Moreover, this method also has an advantage over microarray analysis in terms of some issues, such as the detection of low abundance transcripts, the differentiation of isoforms, and its dynamic range.24 Thus, the use of the RNA-seq method might lead to the identification of novel cancer-related changes in mRNA expression. Therefore, to identify novel reliable immunohistochemical markers that are useful for distinguishing between chromophobe RCC and other www.md-journal.com |
1
Medicine
Shinmura et al
subtypes of RCC, such as clear cell and papillary RCCs, we globally compared the processed RNA-seq expression data from 66 cases of chromophobe RCC, 519 cases of clear cell RCC, and 198 cases of papillary RCC that were included in The Cancer Genome Atlas (TCGA)25 and found 3 candidate genes including BSND and ATP6V1G3. Next, we examined the expression statuses of these genes in 200 primary renal tumors and 85 primary lung carcinomas, and found that BSND and ATP6V1G3 were highly sensitive and specific markers of chromophobe RCC. Our study suggests that evaluating the expression levels of BSND and ATP6V1G3 could be of great value for distinguishing between chromophobe RCC and other subtypes of RCC.
Volume 94, Number 24, June 2015
primary lung carcinoma cases, composed of 44 cases of squamous cell carcinoma of the lung and 41 cases of adenocarcinoma of the lung, which had undergone surgery at Hamamatsu University Hospital (Japan) were used for the TMA block. To explain in further detail, the block was prepared by transferring a cylinder of 3-mm diameter from each of the paraffinembedded tissue samples using a microarrayer (KIN-1; Azumaya, Tokyo, Japan), as previously described.29 All the cases used for the immunohistochemical staining were listed in Table 1. This study was conducted with the approval of the Institutional Review Board (IRB) of Hamamatsu University School of Medicine.
Immunohistochemical Staining MATERIALS AND METHODS Collection of Publicly Available TCGA Data Gene expression data and DNA methylation data for a total of 783 RCC cases, composed of 66 chromophobe RCC cases, 519 clear cell RCC cases, and 198 papillary RCC cases (TCGA public data available in April 2014), were collected from the TCGA data portal (https://tcga-data.nci.nih.gov/tcga). Gene expression data for bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, liver hepatocellular carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, stomach adenocarcinoma, and thyroid carcinoma were also collected from the TCGA data portal. The expression data were obtained as processed RNA-seq data in the form of RNA-seq by Expectation Maximization or in the form of Reads Per Kilobase of exon Model per million mapped reads.26,27 The RNA-seq by Expectation Maximization or Reads Per Kilobase of exon Model per million mapped reads expression value for each gene was divided by that of the TBP gene, which is a control housekeeping gene,28 to compare the expression levels. DNA methylation data obtained using the Infinium HumanMethylation450 platform (Illumina, Inc, CA) were shown as the b value.25
Preparation of Tissue Microarray (TMA) Blocks Paraffin-embedded tissue samples from a total of 200 primary renal tumor cases, composed of 23 chromophobe RCC cases, 153 clear cell RCC cases, 10 papillary RCC cases, and 14 oncocytoma cases, that had undergone surgery at Hamamatsu University Hospital (Japan), Fujieda Municipal General Hospital (Japan), Seirei Mikatahara General Hospital (Japan), or Seirei Hamamatsu General Hospital (Japan) were collected, and 190 cases of them were used for the TMA block. In addition, paraffin-embedded tissue samples from a total of 85
Sections of paraffin blocks were used for immunohistochemical staining with an automatic immunohistochemical stainer, the HISTOSTAINER (Nichirei Bioscience, Tokyo, Japan). Briefly, the sections were deparaffinized, rehydrated, and boiled at 968C for 40 minutes in TE solution (pH 9.0) for antigen retrieval. Endogenous peroxidase activity was blocked by incubation for 5 minutes in a 3% hydrogen peroxide solution. Next, the sections were incubated with a rabbit anti-BSND polyclonal antibody (1:1000; Sigma–Aldrich, St. Louis, MO), a rabbit anti-ATP6V1G3 polyclonal antibody (1:2000; Sigma– Aldrich), or a rabbit anti-FBN3 polyclonal antibody (1:100; Sigma–Aldrich) for 30 minutes at room temperature. After washing, the sections were incubated for 30 minutes at room temperature with an amino acid polymer conjugated with goat antirabbit IgG and horseradish peroxidase (Histofine Simple Stain MAX-PO Kit; Nichirei, Tokyo, Japan). The antigen– antibody complex was visualized with 3,30 -diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. The staining intensity for BSND and ATP6V1G3 were graded for each specimen as follows: negative, weakly positive, or strongly positive. Additionally, proportion of positive cells for each specimen in the immunostaining was grouped into 3 categories as follows: none (
