蛋白质组学筛出 ELOC 突变型肾细胞癌的潜在标志物：SCIN 与 SORT1Proteomic Analysis Identifies Potential Biomarkers of ELOC-Mutated Renal Cell Carcinoma

Scope: PMC open-access full text (CC BY-NC-ND 4.0) — abstract, introduction, materials and methods, results and discussion, Table 1 and Figures 1–5 with captions. Supplementary material, acknowledgments, conflict statements and references are not included.

Abstract

ELOC-mutated renal cell carcinoma (RCC) is a rare tumor with only ∼40 cases reported to date; it shares a molecular background with clear cell RCC (ccRCC) in terms of hypoxia-inducible factor-alpha (HIF-α) protein accumulation. ELOC-mutated RCC is characterized by prominent leiomyomatous stromal growth and a more indolent clinical course compared with ccRCC. In our previous study, whole-genome sequencing of 102 ccRCC cases identified 5 cases of ELOC-mutated RCC. In the present study, we conducted proteomic and immunohistochemical analyses on up to 13 Japanese ELOC-mutated RCCs, including 8 previously reported cases, to elucidate its distinct molecular mechanisms and identify biomarkers that may be useful in distinguishing ELOC-mutated RCC from ccRCC. Proteomic profiling revealed that molecules, including cytokeratin 7, scinderin (SCIN), and sortilin 1 (SORT1), were significantly overexpressed in ELOC-mutated RCC compared with ccRCC. Notably, SCIN and SORT1 emerged as novel potential diagnostic biomarkers for distinguishing ELOC-mutated RCC from ccRCC. The analysis further suggested that ELOC-mutated RCC relies more on oxidative phosphorylation and less on glycolysis than ccRCC. This metabolic shift may be linked to the relative depletion of NAD+ due to the low expression of NAPRT and QPRT. In addition, we observed geographic variation in the disease frequency among different cohorts, with a higher frequency in Japan. Our findings provide novel insights into the pathogenesis of ELOC-mutated RCC and highlight SCIN and SORT1 as potential supportive biomarkers.

INTRODUCTION

ELOC-mutated renal cell carcinoma (RCC) has been classified as a distinct entity by the World Health Organization (WHO),¹ with only ∼40 reported cases, including our previous report, to date.^2–10 This rare molecular subtype is characterized by biallelic inactivation of the ELOC, leading to the accumulation of HIF-α proteins and the consequent development of tumors.³ This mechanism is similar to that of clear cell RCC (ccRCC). Tumors with ELOC mutations generally lack biallelic VHL inactivation and have a low frequency of mutations in PBRM1, SETD2, and BAP1, which are common in ccRCC.¹¹ Furthermore, ELOC-mutated cases frequently lack specific somatic copy number alterations (SCNAs), such as the gain of chr.5q and loss of chr.3p, chr.9, and chr.14, which are typical in ccRCC.¹¹ ELOC-mutated RCC is associated with a better clinical prognosis than that of ccRCC, generally producing smaller histologically low-grade tumors, according to the WHO/International Society of Urologic Pathologists grading system.^7,11 ELOC-mutated RCC is distinguished by the separation of its nodules by a prominent smooth muscle stroma, known as a fibromuscular band, and focal papillary architecture. Immunophenotypically, ELOC-mutated RCC is characterized by patchy to diffusely labeled cytokeratin 7 (CK7) and box-shaped diffuse positive staining for carbonic anhydrase 9 (CAⅨ).^7,11 Although gene and protein expression studies are limited, the mammalian target of rapamycin (mTOR) pathway was reported to be downregulated in ELOC-mutated RCC.¹¹ However, the molecular mechanisms underlying the unique histologic features of this tumor remain largely unclear, and no useful diagnostic biomarkers have been reported.

In our previous study, we identified 5 ELOC-mutated RCC cases among 102 ccRCC cases.⁸ In addition, 8 Japanese cases of ELOC-mutated RCC have been reported previously.³ Here, we represent the genomic, transcriptomic, proteomic, and immunohistochemical characteristics of ELOC-mutated RCC and compare them with those of ccRCC.

MATERIALS AND METHODS

Patients and Tissue Specimens

We adhered to the ethical standards of both the institutional and national research committees, as well as the 1964 Declaration of Helsinki and its subsequent amendments. This study received approval from the Research Ethics Committee of the National Cancer Centre (approval number: G2008-03). Written informed consent was obtained from all patients for the publication of their data (including 3 or more indirect identifiers, such as exact age, sex, medical center attended, or rare diagnoses), as well as for the collection of samples, their analysis, and the deposition of sequencing data.

This study involved 102 patients diagnosed with ccRCC at the National Cancer Centre Hospital (Tokyo, Japan). Details regarding the participant’s sex, number, and age can be found in Supplementary Data 1, Supplemental Digital Content 1, http://links.lww.com/PAS/C373 and the previous study.⁸ After surgical resection, all specimens were immediately stored in liquid nitrogen, and germline control specimens were obtained from adjacent normal kidney tissues. Tumors harboring biallelic ELOC inactivation and exhibiting concordant morphologic features without biallelic VHL inactivation were classified as ELOC-mutated RCC.

DNA Extraction and Whole-Genome Sequencing

Genomic DNA was extracted from both tumors and matched normal tissues. Libraries with an insert size of 350 bp were prepared from 1 μg sonicated DNA using a TruSeq DNA PCR-free kit (Illumina, San Diego, CA). Libraries were sequenced on a NovaSeq. 6000 instrument (Illumina) using paired-end reads of 150 bp. The sequencing reads were aligned to the human reference genome (GRCh37) using BWA-MEM version 0.7.8¹² for both tumor and nontumor samples. To process the data, we used SAMtools version 1.9 and an in-house program to remove potential PCR duplicates and generate pileup files. The criteria for base selection included a mapping quality score of ≥20 and a base quality score of ≥10.

Genomic Analyses

We performed mutation calling using our in-house program to minimize false positives, as previously described.^13,14 FACETS version 0.6.0¹⁵ was used for copy number analysis with the following parameters: “snp. nbhd =500,” “preProcSample’s val =50,” and “procSample’s Cval =300.” The definitions of amplification, gain, homozygous deletions, loss of heterozygosity, and whole-genome doubled samples are provided in a prior study.⁸

Mutational Signature Analysis

Mutational signature analysis was performed using FitMS (https://signal.mutationalsignatures.com/analyse2) with the GEL-kidney-SBS signal-mutational signatures from the organ-specific signature.¹⁶

RNA Extraction and RNA Sequencing

RNA sequencing was performed using a HiSeq. 2500 instrument (Illumina). Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA). Paired-end reads were aligned using the Bowtie version 0.12.7,¹⁷ applying the “–v 3” and “-a” options to permit mismatches of up to 3 and to detect all multiple hits, respectively. Transcripts per million (TPM) values were calculated after selecting the best hits with the correct distances and orientations.

Immunohistochemistry

Immunohistochemistry (IHC) for CK7, CAIX, scinderin (SCIN), and sortilin 1 (SORT1) was performed in 13 ELOC-mutated RCC cases. In addition, GPNMB IHC was performed in 8 cases. Deparaffinized sections were autoclaved in a target retrieval solution (pH 9.0) for antigen retrieval. The primary antibodies used were as follows: anti-CAIX (clone EP161, 1:400, rabbit monoclonal; Epitomic, catalog number not applicable), anti-CK7 (clone OV-TL 12/30, 1:500, mouse monoclonal; Agilent Technologies, Santa Clara, CA, M7018), anti-SCIN (1:100, rabbit polyclonal; Proteintech, 11579-1-AP), anti-SORT1 (1:1000, rabbit polyclonal; Abcam, ab16640), and anti-GPNMB (Human Osteoactivin/GPNMB antibody, 1:400; R&D Systems). Antibodies were diluted in Antibody Diluent (Agilent Technologies). The IHC analysis was conducted automatically using AutostainerLink 48 (Agilent Technologies).

Immunohistochemical staining was independently evaluated by at least 2 expert pathologists. A positive score was calculated by multiplying the proportion of positive tumor cells (positive rate) by the staining intensity. Staining intensity was graded as 1 for weak and 3 for strong; for example, a tumor with strong staining in 80% of cells received a score of 3×0.8. When staining intensity varied within the sample (eg, weak to strong), the average intensity value was used for calculation. To obtain an internally validated estimate of discrimination while minimizing small-sample instability, we used stratified 5-fold cross-validation repeated 50 times. In each split, folds preserved the class ratio. A fixed random seed was set for reproducibility. Receiver operating characteristic (ROC) curves were generated using the roc function in the pROC package in R.

Proteomic Analysis

Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on 4 samples from each patient (a total of 32 samples; sample IDs: ELOC-mutated RCC samples KI111, KI236, KI275, and KI279, and ccRCC samples KI235, KI237, KI257, and KI263). Peptides from biotinylated protein fractions were prepared for LC-MS. Peptide samples were extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections. Tissue sections of 3 μm were cut from the FFPE tissue blocks using a microtome and deparaffinated with xylene, ethanol, and distilled water. The deparaffinized samples were suspended in EasyPep lysis buffer (Thermo Fisher Scientific) and homogenized with a handheld homogenizer. After incubation at 95°C for 30 minutes, sonication was performed using a Bioruptor with 30-second on/off cycles at “High” power for a total of 30 minutes. Acetone was precipitated following the EasyPep protocol. The resulting pellet was resuspended in 1.4 M guanidine and subjected to carbamidomethylation. Peptide digestion was performed using Lys-C and trypsin after resuspension. After centrifugation, the supernatant was collected, and the protein concentration was determined using the bicinchoninic acid assay. The peptides (500 ng) were purified using EVOTIP (EVOSEP). Purified peptides were analyzed using an LC-MS system equipped with an Evosep One LC system (Evosep) coupled to an astral mass spectrometer (Thermo Fisher Scientific). The mobile phases consisted of 0.1% formic acid in water and 0.1% formic acid in 99.9% acetonitrile. Data-independent acquisition was conducted using a 2Th isolation window, covering a mass range of 380 to 1000 m/z. Separation was performed using an 8 cm Evosep column with 60 samples/day. The data were analyzed using Dia-NN version 1.8, using the human UniProt database for protein identification. The false discovery rate threshold for peptide identification was set at 1%.

Gene Set Enrichment Analysis

Gene set enrichment analysis (GSEA) was conducted using the h.all.v2023.2.Hs.symbols.gmt gene set with GSEA version 4.3.2. Significant pathways were identified on peptide ion intensities or TPM. The number of permutations was set to 1000, along with the “gene_set” type. A cutoff q-value of 0.15 was established for determining significance.

RESULTS

Frequency of ELOC-Mutated RCC and Mutation Spots at the ELOC Gene

In addition to our previous genomic study,⁸ ELOC mutations have been reported in various large-scale genome studies,^2,3,7,18 likely in the analysis of a random selection of ccRCC cases. We compared the frequencies of these ELOC mutations across large-scale studies (Fig. 1A). ELOC-mutated RCC was detected in 3.80% (ELOC-mutated RCC: 13 cases, ccRCC: 342 cases) of the Japanese cohort, whereas other cohorts showed a frequency of <1%. This discrepancy in the incidence of malignant tumors may be attributable to variations in germline mutations or exposure to exogenous mutagens.^19,20 However, we did not find any association in ELOC-mutated RCC with any specific mutational signatures when compared with ccRCC or any high-frequency germline mutations (Supplementary Fig. 1A, Supplemental Digital Content 2, http://links.lww.com/PAS/C374).

Thirty-nine ELOC mutations were recorded in 37 ELOC-mutated RCC cases worldwide.^2–7,9,10 Elongin C, encoded by the ELOC gene, comprises 112 amino acids and contains 2 regions that interact with the VHL protein.²¹ All but one ELOC-mutated RCC case exhibited alterations within these 2 regions. Amino acid substitutions occurred more frequently at residues 71 to 80, particularly Y79C and Y79S (Fig. 1B). In Japanese cases, 7 patients harbored a missense mutation at codon 236, leading to the Y79C substitution frequently observed in ELOC-mutated RCC (Fig. 1B). Notably, no nonsense mutations were identified in the present study or in previous reports.

Genomic Aberrations in ELOC-Mutated RCC

We compared the nonsynonymous mutations and the statuses of chr.3p and chr.8q in ELOC-mutated RCC with those in ccRCC (Fig. 1C). Eight cases were excluded due to the inability to assess SCNAs, leaving 94 cases for genomic analysis (Supplementary Data 1, Supplemental Digital Content 1, http://links.lww.com/PAS/C373). Among these, 5 cases harbored an ELOC mutation and loss of chr.8q21.11, the locus containing the ELOC gene (Fig. 1C). None of these cases showed biallelic VHL inactivation, and 2 exhibited loss of chr.3p, where the VHL gene is located—a common alteration in ccRCC (Fig. 1C, D). We further analyzed mutations in genes frequently altered in ccRCC, including PBRM1, SETD2, and BAP1, using data from Sato et al (Fig. 1D).² No PBRM1 nor SETD2 mutations were observed, and only 2 ELOC-mutated RCC cases carried BAP1 mutations; however, the difference was not statistically significant (Fig. 1D). While ccRCC typically harbors frequent mutations in several chr.3p genes, including VHL, PBRM1, SETD2, and BAP1, ELOC-mutated RCCs did not exhibit recurrent mutations on chr.8, excluding ELOC (data not shown). Because some tumors with mTOR pathway-related mutations are histologically similar to ELOC-mutated RCC,^22,23 we also examined these genes. Seven of 94 cases carried mutations in the mTOR pathway (TSC1 mutation, n=1; TSC2 mutation, n=1; MTOR mutation, n=5). VHL mutations were present in 6 of these, and all showed loss of chr.3p; these tumors were classified as ccRCC.

Somatic Copy Number Aberration of ELOC-Mutated RCC

We examined the SCNAs across ELOC-mutated RCC genomes (Fig. 2A). Deletion of the region from chr.8p23.3 to chr.8q24.23, which harbors ELOC, MYC, and E2F5, occurred more frequently in ELOC-mutated RCC than in ccRCC. Conversely, deletions in the chr.3p26.3-chr.3p14.1 region and gains in the chr.5q23.3-chr.5q35.3 were detected less often in ELOC-mutated RCC, unlike in ccRCC (Fig. 2A). Deletions of chr.9 and chr.14, typically seen in progressive ccRCC,⁸ were detected in one case and none of the ELOC-mutated cases, respectively. The total number of SCNAs and deletions across the ELOC-mutated RCC genome was not significantly different from that in ccRCC (Fig. 2B). In contrast, ELOC-mutated RCC showed fewer copy number gains than did ccRCCs (P=2.89E-03). Among the cases, the one diagnosed with pT3a had the highest number of deletions but showed no copy number gains.

Clinicopathologic Features of ELOC-Mutated RCC

We analyzed the clinicopathologic data of patients with RCC harboring ELOC mutations. The Japanese patients were aged 42 to 77 years, with a mean age of 62 years (Supplementary Data 2, Supplemental Digital Content 3, http://links.lww.com/PAS/C375). In the Japanese cases, we observed 11 cases of pT1 and 2 cases of pT3a, most of which were nonprogressive (Supplementary Data 2, Supplemental Digital Content 3, http://links.lww.com/PAS/C375). However, one patient with pT3a experienced postoperative recurrence of lung metastasis.

Our 5 ELOC-mutated RCC cases displayed a multinodular appearance, creating a nodular gross configuration (Fig. 3A and Supplementary Fig. 2A–D, Supplemental Digital Content 4, http://links.lww.com/PAS/C376). Histologically, all these cases had prominent leiomyomatous stroma, so-called fibromuscular bands (Fig. 3B, C). The tumor cytoplasm was voluminous and clear, with various architectural patterns (Fig. 3D-F). The tumors featured a mix of solid alveolar, tubular, branching tubular, and cystic architectures, which were very similar to those of ccRCC (Table 1). In contrast, 2 patients presented with a pure papillary architecture, an occurrence that is rare in ccRCC. Immunohistochemical studies have shown that ELOC-mutated RCC exhibits varying levels of CK7 expression across cases, with only 4 cases being negative for CK7.^6,7,9 In the Japanese cases, 11 cases were positive for CK7, while 3 were almost negative (Fig. 3G and Table 1). All ELOC-mutated RCC cases demonstrated strong and diffuse positivity for CAⅨ, a target gene of HIF, with expression surrounding the entire cell membrane instead of the typical cup-like pattern in clear cell papillary renal cell tumor (ccpRCT) (Fig. 3H and Table 1). We additionally evaluated GPNMB immunohistochemistry in 8 ELOC-mutated RCC cases. Seven cases were completely negative, whereas 1 case showed weak positivity (Table 1).

All cases showed box-shape pattern.

N.D. indicates not done; pap, papillary; Sol Alv, solid alveolar; Tu/BTu, tubular/branching tubular.

Biomarkers Useful for Distinguishing ELOC-Mutated RCC From ccRCC

To identify potential diagnostic biomarkers, we performed LC-MS-based proteomic analysis on 32 samples derived from 4 ELOC-mutated RCC cases and 4 ccRCC cases, 4 samples per case. This analysis revealed several proteins with significantly higher expression in ELOC-mutated RCC than in ccRCC (Fig. 4A). Among them, SCIN and SORT1 were significantly overexpressed in ELOC-mutated RCC, a finding that was corroborated by transcriptomic data, which also showed significantly higher expression levels in ELOC-mutated RCC (Fig. 4A, B).

We next evaluated the diagnostic utility of SCIN and SORT 1 through IHC in 13 ELOC-mutated RCC cases and 20 ccRCC cases. Both proteins were positive in the cell membrane of distal tubule cells, but not in those of proximal tubule cells. They were almost negative or only focally weakly positive in ccRCC, whereas ELOC-mutated RCC demonstrated distinct membranous positivity (Fig. 4C, D, and Supplementary Data 3, Supplemental Digital Content 5, http://links.lww.com/PAS/C377). We performed IHC for 6 ccpRCT cases, and all cases were positive for these proteins. (Supplementary Fig. 3A–C, Supplemental Digital Content 6, http://links.lww.com/PAS/C378). Diagnostic performance was assessed using both the positivity rate and a composite score combining positivity rate and staining intensity (Supplementary Data 3, Supplemental Digital Content 5, http://links.lww.com/PAS/C377). Both proteins showed high sensitivity and specificity under repeated stratified 5-fold cross-validation, supporting their potential utility as biomarkers for distinguishing ELOC-mutated RCC from ccRCC (Fig. 4E).

Metabolic System in ELOC-Mutated RCC

Although ELOC-mutated RCC is reportedly less dependent on the mTOR pathway than ccRCC,¹¹ the differences in their molecular features remain poorly understood. To address this, we performed GSEA using proteomic data from both tumor types. GSEA revealed that protein expression of MYC target genes was upregulated in ccRCC (Fig. 5A). In contrast, both MYC gene expression and MYC target gene expression were downregulated in ELOC-mutated RCC (Fig. 5B, C). The MYC genes are located on chr.8q, and all ELOC-mutated cases showed loss of heterozygosity (LOH) at this locus. While HIF-1α acts to suppress MYC and HIF-2α enhances its activity,^24,25 both factors were expressed in ELOC-mutated RCC (Supplementary Fig. 4A and B, Supplemental Digital Content 7, http://links.lww.com/PAS/C379). E2F5, which is also located on chr.8q and promotes cell proliferation in various cancer types,^26–28 was relatively downregulated (Fig. 5D, E).

GSEA further indicated enrichment of myogenesis, angiogenesis, and several metabolic pathways, including oxidative phosphorylation (OXPHOS) and fatty acid metabolism, were enhanced in ELOC-mutated RCC (Fig. 5A). Given that both ELOC-mutated RCC and ccRCC share a common molecular background characterized by HIF-α accumulation,³ their tumor environments are likely subject to similar hypoxic conditions. Under hypoxia, cancer cells generally shift toward glycolysis for adenosine triphosphate production.²⁹ However, analysis of glycolytic enzymes revealed that several, including PGM1, ENO1, PKLR, and SDS, were expressed at lower levels in ELOC-mutated RCC (Fig. 5F), suggesting a preference for OXPHOS over glycolysis.

To investigate the basis of this metabolic shift, we examined pathways involved in NAD+ metabolism. During glycolysis, NAD+ is reduced to NADH, whereas in OXPHOS, NADH is oxidized back to NAD+.³⁰ NAD+ is mainly produced through the Preiss-Handler, kynurenine, and salvage pathways. Expressions of nicotinate phosphoribosyltransferase (NAPRT) (involved in the Preiss-Handler pathway) and quinolinate phosphoribosyltransferase (QPRT) (associated with the kynurenine pathway) were relatively low in ELOC-mutated RCC (Fig. 5G).³¹

DISCUSSION

In this study, we performed genomic, transcriptomic, and proteomic analyses, along with IHC, in up to 13 ELOC-mutated RCC cases. Our research highlighted this rare histologic subtype, which has not been extensively studied in terms of its molecular pathology. The findings from this study may enhance our understanding of this type of tumor. However, due to its rarity, the sample size was limited and comprised exclusively of Japanese patients; therefore, further validation with larger cohorts will be necessary.

Considering previous studies in conjunction with our research, a total of 13 ELOC-mutated RCC cases have been reported in Japan, suggesting that this tumor type may be more prevalent among Japanese individuals. This trend was determined by comparing different cohorts with a limited number of cases. In addition, these studies were not necessarily performed using identical sequencing platforms and analytical pipelines, although both Japanese cohorts showed a relatively higher frequency of ELOC-mutated RCC. Exposure to mutagenic factors or germline mutations may contribute to these differing incidence rates. However, mutational signature analysis restricted to Japanese RCC cases did not identify any specific mutagenic process associated with the ELOC mutation, whereas SBS1 has been reported to contribute to non-Japanese ELOC-mutated carcinoma cases. Furthermore, no germline mutations were found to correlate with the ELOC mutation. Due to the limited sample size in our study, we could not determine the causes underlying the more frequent occurrence of this molecular subtype in the Japanese population.

Among the nonsynonymous mutations, biallelic VHL inactivation was not found in all cases of ELOC-mutated RCC, indicating that these are mutually exclusive mutations. ccRCC often harbors several mutations adjacent to the VHL gene, such as PBRM1, SETD2, and BAP1 on chr.3p.² However, these gene mutations were rarely observed in ELOC-mutated RCC, as in previous studies.^3,7,11 No recurrent gene mutations, excluding the ELOC mutation on chr.8q, were also found in this study. Biallelic inactivation of the VHL gene alone is considered insufficient for acquiring malignant potential,³² and several additional mutations are necessary to develop ccRCC.³³ Further investigations are needed to elucidate the precise role of ELOC inactivation in tumorigenesis. Notably, no nonsense or frameshift mutations have been reported, while hot spot mutations, such as Y79C, on the ELOC gene have been observed. This pattern suggests that retention of some elongin C functions is essential for tumor development.³ Regarding SCNAs, ELOC-mutated RCC exhibited a lower frequency of copy number gains than that of ccRCC, consistent with the previous study,¹⁰ whereas the frequency of deletions did not differ significantly between the two tumor types. Among the ELOC-mutated cases, the case with pT3a showed a higher level of LOH than did the other ELOC-mutated cases. This suggested that LOH may play a more critical role in tumor progression in ELOC-mutated RCC than copy number gain.

Leiomyomatous stroma was observed in all cases, consistent with the previously reported characteristics of ELOC-mutated RCC.^7,11 Although CK7-positive immunostaining has attracted attention for its differential expression in ccRCC,^7,11 Three cases in our cohort were almost negative, suggesting that CK7-negative findings alone cannot rule out ELOC-mutated RCC. LC-MS proteomics highlighted several candidates in ELOC-mutated RCC; SCIN and SORT1 were selected for IHC validation based on antibody quality and availability. SCIN is an actin-severing and -capping protein that regulates remodeling of the cortical actin cytoskeleton,³⁴ and SORT1, a type I transmembrane receptor of the Vps10p-domain family, functions in intracellular protein sorting, endocytosis, and ligand trafficking.³⁵ Dysregulation of the expression of these proteins has been implicated in cancer cell motility and metastatic potential.^36,37 Immunohistochemical evaluation demonstrated that both proteins showed high sensitivity and specificity, suggesting that they may serve as useful diagnostic biomarkers in clinical practice, similar to CK7 in differential diagnosis between ELOC-mutated RCC and ccRCC. Although the model performed well in internal cross-validation, the small sample size necessitates validation in larger, independent cohorts to establish generalizability. Moreover, the relationship between these proteins and ELOC mutation remains unclear, and additional studies are needed to elucidate the molecular mechanisms underlying their elevated or preserved expression in ELOC-mutated RCC. Because ccpRCT was positive for these proteins, they are unlikely to be informative for distinguishing ELOC-mutated RCC from ccpRCT. In addition, GPNMB was negative in most ELOC-mutated RCC cases examined in this study, with only one case showing weak positivity. In this study, we did not evaluate a separate comparator group of RCC with fibromatous stroma harboring TSC/MTOR pathway alterations.²³ In our cohort, cases with TSC/MTOR pathway alterations also harbored VHL alterations and were therefore interpreted as ccRCC; thus, further investigation of this differential diagnostic setting is warranted.

We investigated the molecular mechanisms underlying the distinguished histology in ELOC-mutated RCC. We propose that the downregulation of MYC target genes and a metabolic shift toward OXPHOS contribute to the indolent growth observed in ELOC-mutated RCC. Downregulation of MYC target genes was likely caused by the loss of chr.8q, which contains the MYC gene (Fig. 5A-C). Additionally, HIF-1α antagonizes MYC, while HIF-2α enhances its activity.^24,25 Both genes were expressed in ELOC-mutated RCC (Supplementary Fig. 4A, B, Supplemental Digital Content 7, http://links.lww.com/PAS/C379), suggesting that HIF-1α could contribute to the downregulation of MYC target genes. The reliance on OXPHOS was likely induced by relative NAD+ depletion due to the low expression of NAPRT and QPRT. Chr.8q loss may create a contradictory situation where tumor cells cannot rely on glycolysis, even under HIF-1α accumulation, as it also encodes NAPRT. Consistent with these notions, ccRCC with chr.8q loss exhibits histology similar to that of ELOC-mutated RCC,⁷ and chr.8q gains have been associated with poor prognosis across various cancer types.^38–40 The stromal cells in ELOC-mutated RCC do not exhibit uncontrolled proliferation, unlike the tumor cells, and their growth is likely a reactive process driven by various factors. Indeed, we observed upregulation of several cytokines, such as BMP4, PDGFA, and TGFA, in ELOC-mutated RCC (Supplementary Fig. 5A-C, Supplemental Digital Content 8, http://links.lww.com/PAS/C380). Given the growth speeds of the stroma and tumor cells, the indolent tumor behavior may also contribute to the formation of the fibromuscular bands. Although ELOC-mutated RCC possesses distinct molecular features that promote leiomyomatous stromal growth, it remains possible that some ccRCC cases may acquire similar histologic features through reactive stromal responses.

In conclusion, the features of ELOC-mutated RCC have been rarely described, and our studies highlighted the distinctive molecular mechanism compared with that of ccRCC. These findings could contribute to the development of improved diagnostic strategies for ELOC-mutated RCC, particularly in distinction from ccRCC. However, the rarity of this tumor type resulted in a limited sample size in our study. Therefore, further larger-scale, international studies are required to validate these findings.