Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease

Abstract

End-stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of 2 major histopathological types: clear-cell RCCs (ESRD-ccRCCs) and acquired cystic disease (ACD)-associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. Here, we investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD-ccRCCs and 7 ACD-associated RCCs to identify their molecular alterations and cellular origins. Targeted sequencing of 409 cancer-related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD-ccRCCs harbored frequent VHL mutations, while ACD-associated RCCs did not. CNA analysis showed that ESRD-ccRCCs had a frequent loss of chromosome 3p while ACD-associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD-ccRCCs had methylation profiles similar to those of sporadic ccRCCs, while ACD-associated RCCs had profiles similar to those of papillary RCCs. Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD-ccRCCs and ACD-associated RCCs had high expression of proximal tubule cell marker genes, while chromophobe RCCs had high expression of distal tubule cell/collecting duct cell marker genes. In conclusion, ESRD-ccRCCs and ACD-associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these 2 histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron.

Keywords: ACD-RCC; epigenetics; hemodialysis; kidney cancer; renal cell carcinoma.

FIGURE 1

Representative histological findings of RCCs subjected to this study. A, ESRD‐ccRCC (ESRD‐ccRCC9). B, sporadic ccRCC (sporadic ccRCC4). C‐E, ACD‐associated RCCs (C: ACD‐RCC6; D: ACD‐RCC7; E: ACD‐RCC5). F, papillary RCC type II (PRCC6). ESRD‐ccRCCs (A) and sporadic ccRCCs (B) showed similar histological appearance. Both were composed of alveolar architectures of tumor cells with clear cytoplasm. Also, there were interposing fine sinusoid‐like vascular networks. ACD‐associated RCCs (C, D) and PRCCs (F) showed a morphological resemblance, namely papillotubular architectures. At the same time, ACD‐associated RCCs had unique features such as (C) frequent microcystic spaces (arrows), (D) intratumoral hemorrhage, and (E) deposition of calcium oxalate crystals (arrowheads)

FIGURE 2

Genome‐wide analysis of copy number alterations in RCCs arising in ESRD. A, Genome‐wide copy number alterations in 9 ESRD‐ccRCCs and (B) 7 ACD‐associated RCCs. The 9 ESRD‐ccRCCs and 7 ACD‐associated RCCs consistently had a loss of chromosome 3p and a gain of chromosome 16, respectively

FIGURE 3

Genome‐wide DNA methylation analysis in RCCs arising in ESRD. A, Hierarchical clustering analysis and (B) UMAP using 1000 probes with the highest SD among all specimens and samples throughout the genome. ESRD‐ccRCCs clustered together with sporadic ccRCCs, and ACD‐RCCs clustered with PRCCs. C, Hierarchical clustering analysis and (D) UMAP using 1000 probes with the highest SD in TSS200 CGIs. These probes failed to classify the histopathological types. E, Hierarchical clustering analysis and (F) UMAP using 1000 probes with the highest SD in gene bodies. ESRD‐ccRCCs clustered together with sporadic ccRCCs, and ACD‐RCCs clustered with PRCCs

FIGURE 4

Analysis of gene expression of marker genes of cellular origin in RCCs arising in ESRD. A, Expression levels of 3 proximal tubule cell markers (HNF1a, HNF4a, SLC17A3). These were highly expressed in ESRD‐ccRCCs, ACD‐RCCs, sporadic ccRCCs, and PRCCs. B, Expression levels of 2 distal tubule cell/collecting duct cell markers (CLDN8, SLC4A1). These were highly expressed in ChRCCs. *P < .05

FIGURE 5

Immunohistochemistry of marker genes of cellular origin in RCCs arising in ESRD. Representative immunohistochemical findings of 2 proximal tubule cell markers (HNF1a, and HNF4a) and one distal tubule cell/collecting duct cell marker (CLDN8). ESRD‐ccRCC (ESRD‐ccRCC7) and ACD‐associated RCC (ACD‐RCC2), along with sporadic ccRCC (sporadic ccRCC5) and PRCC (PRCC8), had high expression of HNF1a and HNF4a and lacked CLDN8 expression. In contrast, ChRCC (ChRCC3) had high CLDN8 expression and lacked expression of HNF1a and HNF4a. Noncancerous, noncancerous6. Scale bars indicates 50 µm