
==== Front
Investig Clin Urol
Investig Clin Urol
ICU
Investigative and Clinical Urology
2466-0493
2466-054X
The Korean Urological Association

39249924
10.4111/icu.20230318
Original Article
Basic/Translational Research
Immunophenotypic and molecular changes during progression of papillary urothelial carcinoma
https://orcid.org/0000-0002-4097-4710
Kim Bohyun 1
https://orcid.org/0000-0002-4586-5062
Kim Kwangsoo 2
https://orcid.org/0000-0001-6332-5396
Yang Sunah 2
https://orcid.org/0000-0002-1969-8360
Moon Kyung Chul 345
1 Department of Pathology, Konkuk University Medical Center, Konkuk University School of Medicine, Seoul, Korea.
2 Transdisciplinary Department of Medicine & Advanced Technology, Seoul National University Hospital, Seoul, Korea.
3 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
4 Department of Pathology, Seoul National University Hospital, Seoul, Korea.
5 Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea.
Corresponding Author: Kyung Chul Moon. Department of Pathology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea. TEL: +82-2-740-8380, FAX: +82-2-743-5530, blue7270@snu.ac.kr
9 2024
22 8 2024
65 5 501510
22 9 2023
11 1 2024
01 7 2024
© The Korean Urological Association
2024
The Korean Urological Association
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Purpose

Urothelial carcinoma has various molecular subtypes, each with different tumor characteristics. Although it is known that molecular changes occur during tumor progression, little is known about the specifics of these changes. In this study, we performed transcriptional analysis to understand the molecular changes during tumor progression.

Materials and Methods

Formalin-fixed, paraffin-embedded tumor tissues were obtained from 12 patients with muscle-invasive bladder cancer (MIBC). The invasive and non-invasive papillary areas were identified in papillary urothelial carcinoma specimens. Immunohistochemistry (IHC) and mRNA sequencing were performed for each tumor area.

Results

Patients with CK5/6-negative and CK20-positive non-invasive papillary areas were selected and classified into the IHC switch subgroup (CK5/6-positive and CK20-negative in the invasive area) and the IHC unchanged subgroup (CK5/6-negative and CK20-positive in the invasive area) according to the IHC results of the invasive area. We identified differences in the mRNA expression between the non-invasive papillary and invasive areas of the papillary MIBC tissue samples. In both the non-invasive papillary and invasive areas, the IHC switch subgroup showed basal subtype gene expression, while the IHC unchanged subgroup demonstrated luminal subtype gene expression.

Conclusions

The non-invasive papillary area showed a gene expression pattern similar to that of the invasive area. Therefore, even if the non-invasive papillary area exhibits a luminal phenotype on IHC, it can have a basal subtype gene expression depending on the invasive area.

Graphical Abstract

Bladder cancer
Cytokeratin
Immunohistochemistry
Molecular evolution
Urinary bladder neoplasms
National Research Foundation of Korea https://doi.org/10.13039/501100003725 2018R1D1A1B07045763
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pmcINTRODUCTION

Bladder cancer (BC) is the 7th most common cancer and the 9th most common cause of death worldwide [1]. Approximately 75% of the cases are superficial stage BC with no muscularis propria invasion, while the remaining 25% are advanced stage BC with muscularis propria invasion [2]. Non-muscle-invasive bladder cancer (NMIBC) and early-stage BC are associated with improved survival rates [3]. Therefore, NMIBC is usually managed by transurethral resection of the bladder (TURB), intravesical chemotherapy, and Bacillus Calmette–Guérin instillation. Conversely, muscle-invasive bladder cancer (MIBC) is managed with radical cystectomy, pelvic lymph node dissection, and cisplatin-based neoadjuvant chemotherapy. Advanced or metastatic BC is managed with cisplatin or gemcitabine/carboplatin-based chemotherapy, and immunotherapy [345]. Because tumor staging according to the invasion layer and lymph node status is the most important histopathological prognostic factor, MIBC has a poor prognosis, unlike NMIBC [5]. Urothelial carcinoma (UC) is largely segregated into papillary urothelial carcinoma (pUC), which displays papillary growth, and invasive urothelial carcinoma (iUC), which exhibits invasive growth, and they have different pathogeneses. In a recent study, pUC was shown to originate from intermediate cells that form the middle layer of the urothelium, whereas iUC originates from basal cells located in the basal layer of the urothelium [6].

Recently, UC has been categorized into basal and luminal subtypes based on gene expression patterns using next-generation sequencing analysis [678910]. Cytokeratin (CK)5/6, mainly positive in the basal subtype, and CK20, mainly positive in the luminal subtype, are the representative markers used for distinguishing between them [891112]. Differences in prognosis and treatment strategies have been reported depending on the molecular subtype [1314]. The response to chemotherapy and immunotherapy also differs according to the molecular subtype [15161718]. Additionally, it is known that the CK5/6-positive basal subtype has a worse prognosis in MIBC [1920]. Conversely, the CK20-positive luminal subtype has a worse prognosis in NMIBC [2122].

Furthermore, the phenomenon of molecular subtype change has been reported [23]. In non-invasive papillary tumors with a luminal phenotype, the luminal phenotype may be maintained, or the basal phenotype may appear during invasion. It has not been confirmed what happens during the molecular phenotype change process or whether the immunohistochemistry (IHC) phenotype change is accompanied by a molecular subtype change.

In this study, based on the fact that among pUC cases, the UC molecular subtypes exhibiting aggressive behavior differ according to invasion depth, MIBC cases were reviewed to identify the tumor-invasive and non-invasive papillary areas. IHC results were analyzed for each area, and RNA was extracted. Through this process, we aimed to uncover the gene expression profiles of the non-invasive papillary and invasive areas. In addition, we grouped cases into an IHC profile switch subgroup and an IHC profile unchanged subgroup and analyzed their transcriptional characteristics.

MATERIALS AND METHODS

1. Patient selection and clinicopathological review

For immunohistochemical analysis, archival formalin-fixed paraffin-embedded (FFPE) tissue blocks from 109 papillary MIBC tumor samples were selected from TURB bladder specimens at the Department of Pathology, Seoul National University Hospital over a 6-year period (January 2015 to December 2020). The number 109 represents the total number of cases diagnosed as pUC with proper muscle invasion from TURB specimens received between 2015 and 2020. Clinicopathological data were acquired from the medical records. Grading and staging were based on the 2022 World Health Organization (WHO) classification [24] and the American Joint Committee on Cancer 8th TNM staging system [25], respectively. H&E-stained slides were reassessed to confirm the non-invasive papillary and invasive areas in the pUC samples. The IHC unchanged subgroup comprised 14 cases, and the IHC switch subgroup included seven cases. Among these, only a few cases contained sufficient tumor tissue for RNA analysis; therefore, six cases were selected to match the number of cases in each subgroup. Overall, the two subgroups (IHC switch and IHC unchanged) consisted of 12 muscle-invasive pUC specimens (n=6, each) (Fig. 1).

This study was approved by the Institutional Review Board of Seoul National University Hospital (approval number: H-2008-125-1149). The review board approved a waiver of informed consent.

2. Immunohistochemistry

IHC staining for CK5/6 (1:100, D5/16 B4, research resource identifier [RRID]: AB_2281083; Dako), CK20 (1:50, Ks 20.8, RRID: AB_2133718; Dako), and CK14 (1:300, LL002, RRID: AB_1159418; Cell Marque) was performed using an automatic immunostainer (BenchMark XT; Ventana Medical Systems), following the manufacturer’s instructions. Through H&E-stained slides review, the invasive and non-invasive papillary areas of the pUC were identified, and IHC for CK5/6, CK20, and CK14 was performed on the corresponding parts. Based on the distribution of the invasive and non-invasive papillary areas on the H&E-stained slides, IHC analysis was performed on one or two FFPE tissue blocks per case. In previous studies, a 20% cut-off value was used for the interpretation of IHC markers in UC’s molecular classification. However, in this study, a more stringent criterion was applied for clear distinction. Positive expression was defined as showing positive cytoplasmic expression in more than two-thirds of tumor cells within each tumor area, while negative expression was defined as the presence of positive stained tumor cells in less than 1% of each tumor area. All positively stained tumor cells exhibited moderate to strong staining intensity, and staining intensity was not used as a criterion for classifying positive or negative.

3. Transcriptional analysis and data processing

Using H&E-stained and IHC slides, the invasive and non-invasive papillary areas were identified, and a series of consecutive unstained slides (FFPE blocks cut in 10-µm-thick sections) were cut. Using a microscope, the tumor areas on the unstained slide were marked, and mRNA was extracted from each area. To prevent contamination, tumor areas located at least 4 mm apart were selected. DNA contamination was removed using DNase, and cDNA libraries were prepared using the SureSelectXT RNA Direct Reagent Kit (Agilent). For mRNA with poly A tails, the RNA was purified using an mRNA purification kit. Purified RNA was randomly fragmented for sequencing using short reads. The cleaved RNA fragments were converted into cDNA by reverse transcription, and different adaptors were attached to both ends of the cDNA fragments and ligated. After sufficient polymerase chain reaction (PCR) amplification, sequencing was performed following a size selection process. Fragments with an insertion size of 200–400 bp were selected for sequencing. Sequence analysis was performed using the NovaSeq 6000 Sequencing System (RRID: SCR_016387; Illumina) and NovaSeq 6000 S4 Reagent Kit (Illumina). We conducted a quality control analysis of the raw reads obtained through sequencing using the FastQC v0.11.7 program. We generated and inspected basic statistics, including overall read quality, total bases, total reads, and GC content (%). To reduce bias in the analysis, we performed a preprocessing step to remove low-quality reads and artifacts such as adaptor sequences, contaminant DNA, and PCR duplicates. We trimmed bases at the start or end of reads using the Trimmomatic 0.38 program for Illumina paired-end or single-end data when the quality fell below the specified threshold. The preprocessed reads were mapped to the reference genome (UCSC hg19) using the HISAT2 program to create aligned reads. Using the information of aligned reads based on the reference genome, and transcript assembly was performed using the StringTie program. Raw data were trimmed, the mean of M-values normalized, and mRNA expression data presented as read counts per million of TMM (trimmed mean of M-values)-normalized counts and transformed into log 2 volume values for analysis.

4. Bioinformatic analysis

Differentially expressed genes (DEGs) between the two groups were identified with an adjusted p-value <0.05 and an absolute fold change >2 as the cut-off. The Z-score for each gene expression was calculated by subtracting the mean gene expression of the samples and dividing by the standard deviation of the samples’ gene expression. The first step involves converting the RNA-Seq by expectation—maximization-normalized read count values for each gene into log values. Subsequently, the mean and standard deviation of the log values for each gene across all samples were calculated. Finally, each log value was converted into a z-score as follows: (specific gene log value-mean of log values of all samples)/standard deviation of log values of all samples. Functional analysis of DEGs was conducted by Gene Ontology (GO).

RESULTS

1. Subgroups of papillary urothelial carcinoma based on immunohistochemical staining for CK5/6 and CK20 and patient characteristics

Based on the fact that patients with NMIBC and upper tract UC (UTUC) have a worse prognosis in subgroups with low CK5/6 and high CK20 expression; first, cases showing CK5/6-negative and CK20-positive expression on IHC in the non-invasive papillary area were selected. Subsequently, cases showing an IHC profile switch, such as a CK5/6-positive and CK20-negative expression in the invasive area, were classified as the IHC switch subgroup. Cases with maintained IHC profiles, such as CK5/6-negative and CK20-positive expression in the invasive area, were classified as the IHC unchanged subgroup (Fig. 2). Twelve cases with clearly distinguishable non-invasive papillary and invasive areas and distinct differences in the IHC expression were selected. Additionally, CK14 IHC was positive in the invasive area in three out of six cases in the IHC switch subgroup and negative in all areas of the remaining cases. The median age of the patients was 74 years (range, 45–83 years), and the male-to-female ratio was 7:5. All cases were stage pT2 and WHO/International Society of Urological Pathology (ISUP) high grade. The follow-up period ranged from 4 to 94 months, with a median follow-up of 40 months. During the follow-up period, two cases of disease recurrence were confirmed, but there was no distant metastasis or disease-specific death. The clinicopathologic characteristics of the 12 patients are summarized in Table 1.

2. Gene expression of the signature markers

We compared the mRNA expression among the IHC switch subgroup non-invasive papillary, IHC switch subgroup invasive, IHC unchanged subgroup non-invasive papillary, and IHC unchanged subgroup invasive tumor areas (Fig. 3). The expression of basal-type markers was most enriched in IHC switch subgroup invasive, IHC switch subgroup non-invasive papillary, IHC unchanged subgroup non-invasive papillary, and IHC unchanged subgroup invasive tumor areas. The p63 pathway gene expression, involved in controlling basal-type gene expression, was highly expressed in the IHC switch subgroup invasive and non-invasive papillary areas. When comparing the expression of cell adhesion-associated genes, gap junction, epithelial integrin, and hemidesmosome-related genes highly expressed in the basal type [10] were also highly expressed in both tumor areas of the IHC switch subgroup. The expression patterns of luminal markers clustered strongly within the IHC unchanged subgroup non-invasive papillary and invasive tumor areas. Urothelial differentiation markers, which share gene sets with luminal type markers, were also highly expressed in the IHC unchanged subgroup non-invasive papillary and invasive tumor areas. The peroxisome proliferator-activator receptor (PPAR) pathway gene, activated in the luminal type, was highly expressed in the IHC unchanged subgroup non-invasive papillary and invasive tumor areas. A tight junction-associated gene, highly expressed in the luminal type, was also highly expressed in both tumor areas in the IHC unchanged subgroup [10]. Late cell cycle marker genes were highly expressed in the IHC unchanged subgroup non-invasive papillary and invasive tumor areas. The expression of p53 target genes was most enriched in the IHC unchanged subgroup invasive, the IHC switch subgroup invasive, the IHC unchanged subgroup non-invasive papillary, and the IHC switch subgroup non-invasive papillary areas, in that order. Epithelial-mesenchymal transition (EMT)-associated genes were highly expressed in the IHC unchanged subgroup invasive areas. The Z-score was calculated by comparing the expression level of a gene in a given sample to the expression level of that gene across all samples. The absolute value of the Z-score was then classified as follows: 1+ if it was between 0–0.5, 2+ if it was between 0.5–1, and 3+ if it was greater than 1 (Table 2). In this table, the expression level of mRNA is compared to the average value, not the absolute value. Therefore, even if a specific gene is expressed sufficiently, it may appear to be expressed relatively less if it is expressed much higher in other cases.

3. Functional analysis and Gene ontology terms

In the IHC switch subgroup, between non-invasive papillary and invasive areas, GO terms related to cell motility, cell proliferation, cell growth, signaling pathway, and angiogenesis were found to be enriched. The related DEGs (ABCA12, ANK2, APOE, BOC, ADAMTS12, CALCR, CAVIN3, COL1A1, FN1, FBLN5, IGF1, MMP2, NTRK3, POSTN, SOX11, etc.) were identified (Fig. 4).

In the IHC unchanged subgroup, the enriched GO terms between non-invasive papillary and invasive areas were ion transport, and positive regulation of developmental processes.

In non-invasive papillary areas, GO terms related to cell motility, proliferation, differentiation, cell death, and signaling pathways were enriched between the IHC switch and the IHC unchanged subgroup. The related DEGs (PTPRT, SELL, CCL18, FAT3, PLA2G7, PTN, P2RY6, MDM2, etc.) were also identified.

In the invasive area, the top GO terms were related to cell motility, proliferation, cell growth, differentiation, cell death, and signaling pathways between the IHC switch and the IHC unchanged subgroup. The related DEGs (KRT16, CYP24A1, KRT23, FZD4, DSC3, MMP7, MDM2, etc.) were identified.

DISCUSSION

In the present study, we analyzed urinary bladder pUC tissue samples, performed IHC and mRNA sequencing analyses, and identified differences in mRNA expression between non-invasive papillary and invasive tumor areas. We compared the IHC switch and unchanged subgroups at the mRNA level to define the relationship between the CK5/6 and CK20 IHC phenotype switch and biological activity.

In MIBC, the basal subtype with high KRT5 expression was reported to show poor prognosis, whereas in NMIBC, the luminal subtype with high KRT20 expression showed adverse outcomes. In non-muscle-invasive UTUC, patients with low CK5/6 and high CK20 expression demonstrated a poor prognosis [26]. In a study conducted only on papillary urothelial neoplasms, an increase in CK20 and loss of CD44 were associated with a high tumor grade and stage [27]. In a study comparing the molecular subtypes of primary BC lesions and matched lymph node metastatic lesions, discordance of the molecular subtype was found in approximately 18% of cases [28]. Additionally, another study revealed the molecular changes accompanying the progression from NMIBC to advanced BC [23]. Similarly, in a study targeting urothelial carcinoma in situ (CIS), a phenotype switch from luminal to basal was observed during the progression from CIS to invasive tumors, although the participants were limited to those with invasive UC with CIS, excluding pUC [29]. A high KRT20 expression phenotype is related to aggressive behavior in non-invasive papillary UC based on previous studies. Therefore, it is meaningful to spatially distinguish between non-invasive papillary and invasive tumor areas and to identify the phenotypic changes that occur during invasion in pUC.

In the IHC switch subgroup, analyzing the DEGs between non-invasive papillary and invasive areas showed that most DEGs upregulated in the invasive area were related to cell migration, motility, proliferation, and signaling pathways.

However, unlike in the IHC switch subgroup, comparing non-invasive papillary and invasive areas in the IHC unchanged subgroup showed that GO terms related to major cellular function were not enriched.

In addition, the expression levels of basal and luminal subtype signature genes were analyzed in the four tumor areas. Although we expected to see significant differences in the expression of various signature genes between the non-invasive papillary and invasive areas of the IHC switch subgroup, the gene expression patterns of the non-invasive papillary area of the IHC switch subgroup were similar to the basal subtype expression in the invasive area. The non-invasive papillary area already has basal-subtype gene expression, which can be interpreted as a luminal subtype but based on the IHC CK profile. The molecular subtype of the invasive area may be luminal or basal, even if the non-invasive papillary tumor area is CK20-positive and CK5/6-negative. The non-invasive papillary area of the IHC unchanged subgroup also showed luminal subtype signature gene expression patterns similar to those in the invasive area of the IHC unchanged subgroup.

Interesting results were obtained from heat maps of genes involved in the identified biological processes. Both the IHC unchanged subgroup non-invasive papillary and invasive areas showed high expression of late cell cycle marker genes known to be associated with disease progression in UC [1030]. This is consistent with the high expression of late cell cycle marker genes in a tumor class with luminal-like characteristics in a study targeting early-stage NMIBC. However, late cell cycle marker gene expression was remarkably low in the invasive area of the IHC switch subgroup, corresponding to the phenotype of the MIBC basal subtype, which is known to have a poor prognosis. This may be because the study only included cases that progressed from pUC to invasive tumors. In previous studies, since the participants were selected without distinction, there must have been many cases that progressed from urothelial CIS to invasive tumors. Therefore, the tumor areas showing basal-like expression in our study may have different characteristics from existing basal subtype tumors, which could be the cause of the discrepancy. Both the IHC unchanged subgroup non-invasive papillary and invasive areas showed high expression of EMT-associated genes.

Moreover, the expression of p53 target genes was enriched in the invasive areas of the IHC switch and IHC unchanged subgroups. In the non-invasive papillary area, there were differences in the expression levels of key genes involved in several biological processes between the IHC switch and the IHC unchanged subgroup. Since these areas are both non-invasive papillary areas with the same CK5/6-negative and CK20-positive IHC profiles, we did not expect several DEGs to show statistically significant differences between them. In this study, the IHC unchanged subgroup, which had luminal-like characteristics, showed enrichment in active processes related to aggressiveness. This aligns with the results of a previous study that revealed the CK20-positive and CK5/6-negative groups are at high risk of non-muscle-invasive UTUC [26]. Thus, the non-invasive papillary area has the potential for aggressive tumor behavior, and this characteristic extends to the invasive area. Research on the molecular classification of UC is actively underway, but there is no explicit indication of which specific part should be collected from UC tissue samples for IHC or mRNA sequencing analysis. It was necessary to determine which tumor area could represent the entire tumor due to intertumoral heterogeneity. Our study revealed that the invasive area of pUC accurately reflects both immunohistochemical and molecular characteristics.

Unlike the IHC results, gene expression alterations were not observed, and each non-invasive papillary area showed mRNA expression similar to the invasive area. In the non-invasive papillary area, there was a discrepancy between gene expression and immunophenotype. Several studies have revealed the molecular changes that occur when non-invasive tumors progress to invasive tumors. A study that revealed a switch from the luminal to the basal phenotype during progression to invasion in urothelial CIS was conducted in flat urothelial lesions. This study only used IHC without analyzing the gene expression [29]. Another study that showed the molecular subtype changes during the progression from NMIBC to advanced BC secured urinary bladder tissue samples longitudinally according to the chronological sequence [23].

This study has certain limitations. It utilized strict IHC scoring criteria and focused mainly on a relatively small cohort of 12 cases. Although the clustering of cases within each subgroup was successful, the limited number of study subjects restricts the ability to draw definitive conclusions. More research will be necessary, including mRNA sequencing analysis using a larger cohort and survival analysis between each subgroup.

CONCLUSIONS

Our study is the first to observe differences in mRNA expression between the non-invasive papillary and invasive areas of a single papillary MIBC tissue sample. It can be inferred that even a non-invasive tumor area has the potential to show a gene expression pattern related to the molecular subtype or aggressive behavior of the entire tumor, based on our results. A non-invasive papillary area may have basal-like characteristics, even if it shows a luminal-like phenotype on IHC.

Fig. 1 Overview images of the IHC switch subgroup and IHC unchanged subgroup. IHC, immunohistochemistry.

Fig. 2 Representative H&E stained (×200) and immunohistochemical (×200) image of the IHC switch subgroup and IHC unchanged subgroup. (A) The non-invasive papillary area of the IHC switch subgroup tumor exhibits CK20-positive and CK5/6-negative immunohistochemical profiles. (B) The invasive area of the IHC switch subgroup tumor exhibits CK20-negative and CK5/6-positive immunohistochemical profiles. (C) The non-invasive papillary area of the IHC unchanged subgroup tumor exhibits CK20-positive and CK5/6-negative immunohistochemical profiles. (D) The invasive area of the IHC unchanged subgroup tumor exhibits CK20-positive and CK5/6-negative immunohistochemical profiles. IHC, Immunohistochemistry.

Fig. 3 Heatmap of the signature gene expression. The IHC switch subgroup exhibited high expression of a basal-type marker, while the expression of a luminal-type marker, late cell cycle marker, and EMT-associated marker were relatively low. Conversely, the IHC unchanged subgroup showed low expression of basal-type markers, while the expression of luminal-type markers, late cell cycle markers, and EMT-associated markers were high. IHC, Immunohistochemistry; EMT, epithelial-mesenchymal transition.

Fig. 4 Functional analysis: Gene ontology terms using differentially expressed genes. Gene Set Enrichment Analysis (GSEA) is an analytical technique used to determine the activation level of a given gene set in particular data. Gene ontology and biological pathway (GOBP) is a type of gene set used as a reference in analysis. In this figure, the results indicate that the GOBP showed significant p-values and high normalized enrichment scores (NES) through GSEA analysis. IHC, Immunohistochemistry.

Table 1 Clinicopathologic findings for 12 patients with papillary urothelial carcinoma

	IHC switch (n=6)	IHC unchanged (n=6)	
Age (y)	76 (47–83)	72 (45–81)	
Sex, male:female	4:2	3:3	
WHO/ISUP grade			
	Low grade	0	0	
	High grade	6	6	
Local recurrence	1	1	
Distant metastasis	0	0	
Values are presented as median (range) or number only.

IHC switch: IHC switch subgroup, IHC unchanged: IHC unchanged subgroup.

IHC, Immunohistochemistry; WHO, World Health Organization; ISUP, International Society of Urological Pathology.

Table 2 Gene expression of the signature markers

	IHC switch-papillary	IHC switch-invasive	IHC unchanged-papillary	IHC unchanged-invasive	
Basal type marker	Inc(2+)	Inc(3+)	Dec(3+)	Dec(2+)	
p63 target	Inc(2+)	Inc(3+)	Dec(3+)	Dec(3+)	
Luminal type marker	Dec(3+)	Dec(3+)	Inc(3+)	Inc(3+)	
PPAR pathway	Dec(3+)	Dec(2+)	Inc(2+)	Inc(3+)	
Urothelial differentiation	Dec(2+)	Dec(3+)	Inc(3+)	Inc(3+)	
p53 target	Dec(3+)	Inc(2+)	Dec(2+)	Inc(3+)	
Immune marker	Dec(3+)	Inc(2+)	Inc(1+)	Inc(3+)	
EMT	Dec(3+)	Inc(1+)	Inc(2+)	Inc(3+)	
Cell adhesion	-	-	-	-	
Early cell cycle	Inc(3+)	Inc(2+)	-	-	
Late cell cycle	Dec(3+)	Dec(3+)	Inc(3+)	Inc(3+)	
IHC switch-papillary: IHC switch subgroup_non-invasive papillary, IHC switch-invasive: IHC switch subgroup_invasive, IHC unchanged-papillary: IHC unchanged subgroup_non-invasive papillary, IHC unchanged-invasive: IHC unchanged subgroup_invasive.

IHC, Immunohistochemistry; PPAR, peroxisome proliferator-activator receptor; EMT, epithelial-mesenchymal transition; Dec, decrease; Inc, increase.

CONFLICTS OF INTEREST: The authors have nothing to disclose.

FUNDING: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07045763).

AUTHORS’ CONTRIBUTIONS: Research conception and design: Kyung Chul Moon and Bohyun Kim.

Data acquisition: Bohyun Kim and Kyung Chul Moon.

Statistical analysis: Bohyun Kim, Kwangsoo Kim, and Sunah Yang.

Data analysis and interpretation: Bohyun Kim, Kwangsoo Kim, and Sunah Yang.

Drafting of the manuscript: Bohyun Kim and Kyung Chul Moon.

Critical revision of the manuscript: Bohyun Kim, Kwangsoo Kim, Sunah Yang, and Kyung Chul Moon.

Editing of the manuscript: Bohyun Kim, Kwangsoo Kim, Sunah Yang, and Kyung Chul Moon.

Obtaining funding: Kyung Chul Moon.

Administrative, technical, or material support: Kwangsoo Kim and Kyung Chul Moon.

Supervision: Kyung Chul Moon.

Approval of the final manuscript: Bohyun Kim, Kwangsoo Kim, Sunah Yang, and Kyung Chul Moon.
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