Recent molecular biological studies on chRCC have provided profound insights into the development and evolution of these tumors. The following section will therefore discuss findings from genome, epigenome, transcriptome and proteome analyses concerning the unique features of chRCC and their potential new clinical significance.

The genome of sporadically occurring chRCC often shows aneuploidy. Most of the non-dedifferentiated chRCC presented losses of chromosomes of type 1, 2, 6, 10, 13 and 17. Changes in the karyotypes of chromosomes 3, 5, 8, 9, 11, 18 and/or 21 are observed in 12% to 58% of cases [16]. These aneuploidies are observed significantly more frequently in the histologically classic subtype of chRCC than in the eosinophilic subtype, indicating further genomic differences between these two variants of chRCC [16].

Furthermore, it is assumed that additional genomic alterations occur in chRCC cells with increasing degrees of dedifferentiation [11]. While well-differentiated cells of classical chRCC are characterized by hypodiploidy, dedifferentiated cells usually exhibit a completely diploid or even hyperdiploid chromosome set with two or more copies of chromosomes 1, 2, 6, 10, 13, 17 and 21 [11].

This finding suggests that the dedifferentiated tumor cells develop from the classic tumor cells of chRCC. In this regard, it appears that the monosomes present in the histologically classic tumor cells are duplicated through amplification to generate the dedifferentiated tumor cells [11].

In addition, the analysis of genetic changes shows that the TP53 gene is frequently mutated in chRCC, leading to a loss of function of p53 as a consequence [16]. These mutations can be broadly interpreted as reduced protection of the genome by the "guardian of the genome", TP53.

Notably, if a TP53 mutation is found in classical tumor cells within a tumor, the same mutation is also observed in the dedifferentiated tumor cells [11]. This TP53 mutation is hemizygous in classical tumor cells and homozygous in the corresponding dedifferentiated tumor cells [11]. A similar observation can be made for the PTEN mutation in chRCC on chromosome 10. It is therefore assumed that TP53 and PTEN mutations precede the aforementioned amplification and arise before the development of dedifferentiation in tumor cells [11].

In addition, mutations in the MTOR, NRAS, TSC1 and TSC2 genes have previously been described [16]. However, according to the 2022 WHO classification, renal tumors with alterations in the mTOR pathway, particularly biallelic loss of TSC1 or TSC2, are now classified as eosinophilic solid and cystic renal cell carcinoma (ESC-RCC), a distinct entity characterized by mTORC1 pathway activation rather than being considered a subtype of chRCC [17].

Alongside aneuploidies, increased genomic rearrangements to recurrent structural breakpoints in the TERT promoter region have been found in chRCC. These effects are functionally associated with increased TERT expression and, as a result, increased transcriptional activity of the promoter [16]. Since TERT has important functions in telomere maintenance and DNA repair, it is assumed that these TERT-associated rearrangements and dysregulations could contribute to tumorigenesis [16].

Another important aspect is mutations in the genes of the HER family ("human epidermal growth factor receptors"), which consists of HER1, HER2, HER3 and HER4. Weng et al. have shown that loss of expression of the HER2 gene is frequently observed in chRCC. This could be explained by the localization of the HER2 gene on chromosome 17q12 and the frequently observed chromosomal deletion of this chromosomal segment [18]. However, loss of expression of HER2 could not be statistically associated with tumor progression or aggressiveness [18].

Furthermore, chromosomal rearrangements in the HER1 and HER3 genes have been observed in chRCC, which were attributed to the insertion of an unknown gene [18]. As a result, altered gene functions and abnormal transduction activities with possible effects on tumor induction and tumor progression are discussed [18].

Additionally, alterations in the mitochondrial DNA of chRCC have been detected, with the MT-ND5 gene being affected [16]. This gene is important for the functionality of complex 1 of the respiratory chain [16]. Complex 1 of the respiratory chain was altered in 18% of the cases reported by Davis et al. Table 2.

The epigenome encompasses a wide range of mechanisms that regulate gene expression independently of changes in the DNA sequence. In this regard, epigenetic modifications and changes in interactions between epigenome and genome may be significantly involved in the process of tumorigenesis [19]. According to the stem cell hypothesis, these epigenetic changes occur in somatic cancer stem cells and can be passed on to daughter cells, promoting carcinogenesis and tumor growth [20]. Such changes in the epigenome have been described for chRCC, where they may contribute to tumor development and progression, as discussed in the following section.

Epigenetic modifications involving DNA methylation and demethylation play important roles in carcinogenesis [19]. Tumor cells have been shown to exhibit both DNA hypermethylation and simultaneous global hypomethylation in different genomic regions [19]. These DNA hypomethylations occur earlier and are more strongly associated with chromosomal instability [21]. Hypermethylation occurs through DNA methyltransferases in CpG islands, which are more frequently found in the promoter regions of tumor suppressor genes, leading to the inactivation of these genes [19, 21]. Overall, global hypomethylation results in genetic instabilities, while hypermethylation leads to the inactivation of tumor suppressor genes [19]. These processes have a significant influence on carcinogenesis.

In this context, Faraj Tabrizi et al. reported that increased DNA methylation of the cadherin3 gene (CDH3) may be associated with higher tumor stages and higher degrees of differentiation in clear cell RCC [22]. Future research could explore changes in the expression of CDH3 and DNA methylation of CDH3 in chRCC to draw conclusions about potential correlations with the extensive atypia of chRCC and their tumor behavior.

Another important component of the epigenome is microRNAs (miRNAs), which are non-coding RNA molecules approximately 19–25 nucleotides long [23]. MiRNAs play a important role in post-transcriptional gene regulation by binding to messenger RNA (mRNA), thereby influencing gene expression [23]. In this context, certain genes coding for miRNAs may be overexpressed, causing them to act as oncogenes that promote tumor progression. Conversly, downregulation of tumor-suppressive miRNAs can promote carcinogenesis. Thus, we hypothesize that the loss of chromosomes 1, 2, 6, 10, 13 and 17 may also lead to the loss of genes encoding miRNAs with tumor suppressive functions. This could result in downregulation of such miRNAs, thereby promoting tumor progression in chRCC. Given the limited research on miRNAs in chRCC, further studies are necessary to improve the understanding of these molecules and to develop potential miRNA-based therapeutic approaches for the treatment of chRCC.

Another important component of the epigenome is long noncoding RNAs (lncRNAs), which consist of more than 200 nucleotides and can also regulate gene expression [24]. Tumor-specific lncRNAs in chRCC have been significantly associated with tumor progression and tumor-specific survival [24]. High expression of the lncRNAs COL18A1-AS1, BRE-AS1, SNHG7, TMEM51-AS1, C21orf62-AS1 and LINC00336, along with low expression of LINC00882, are significantly positively associated with tumor-specific overall survival [24] (see Table 3).

According to the "competitive endogenous RNA" (ceRNA) hypothesis, lncRNAs, pseudogene RNAs and mRNAs communicate with each other by binding to the same miRNA through so-called "miRNA response elements" (MREs), thereby competing for the miRNA [24, 25]. The main idea of this hypothesis is that miRNAs are blocked by the MRE-mediated binding of lncRNAs and pseudogene RNAs. As a result, these miRNAs can no longer bind to mRNAs, leading to a shift in the regulation of gene expression.

Overall, this demonstrates that characteristic lncRNAs, miRNAs and pseudogene RNAs play a central role in tumorigenesis and in the tumor biology of chRCC. They should, therefore, be considered as key research targets for therapeutic and diagnostic strategies in the future.

The transcriptome comprises the entire spectrum of genes transcribed into RNA within a cell at a given time, allowing conclusions about the activity and gene expression profiles of tumors such as chRCC. In addition to immunohistochemical methods, transcriptomic differences can be analyzed to facilitate the differentiation between eosinophilic-type chRCC and RO for clinical diagnosis.

To this end, Satter and Tran et al. have developed a signature model consisting of 30 genes that exhibit significantly different expression levels in chRCC and RO. This model can distinguish the extensive transcriptomic differences between the two tumors with an accuracy of 97.8% [26].

This model, termed COGS (chromophobe and oncocytoma related gene signature), includes the AP1M2 gene, which encodes a subunit of the clathrin-associated adapter protein complex 1 and thus assumes important functions in protein sorting in the trans-Golgi network and in endosomes [27]. These findings indicate that both tumor types exhibit fundamentally different properties and follow different pathways of protein sorting.

Additionally, PLCL1 and PLCL2 show differential expression in chRCC and RO. PLCL1 encodes proteins involved in phospholipid-based intracellular signaling cascades, whereas PLCL2 is thought to regulate Ins(1,4,5)P53 in the endoplasmic reticulum [27]. Another gene within the COGS is ITGB3, which encodes a protein of the FTS/ Hook/ FHIP complex and may thereby facilitate vesicle transport via the homotypic vesicular protein sorting complex [27]. These expression alterations in PLCL1, PLCL2 and ITGB3 could could potentially influence tumor development and tumor progression.

A further important gene for differentiating these tumor types is the BSPRY gene, which regulates epithelial calcium transport by inhibiting TRPV5 activity [27]. Altered BSPRY expression could thus contribute to the reduced TRPV5 expression observed in renal cell carcinomas and may be associated with the altered expression of the vitamin D receptor [28]. Disruptions in calcium homeostasis and impaired vitamin D signaling could therefore be significantly involved in tumor development and tumor progression in chRCC.

This gene signature model holds promise as a complementary tool to immunohistochemistry for the clinical diagnosis of chRCC and offers a high degree of diagnostic certainty. However, further validation studies are still required.

In addition to the transcriptome, the proteome can also provide insights into tumor biology. Proteomics encompasses the entirety of proteins present in a cell and is used to identify the characteristic properties of certain tissues and tumors.

Both chRCC and RO exhibit alterations in protein amino acid, lipid and carbohydrate metabolism compared to normal adjacent tissue [29]. Furthermore, dysregulations in mitochondrial metabolism have been observed [29]. The proteomes of chRCC and RO share greater similarities with each other than with normal adjacent tissue, which aligns with their phenotypic and histologic resemblance [29].

This is particularly evident in the fact that proteins involved in gluconeogenesis, such as fructose-1,6-bisphosphatase and pyruvate carboxylase, and proteins involved in fatty acid synthesis, such as alcohol dehydrogenase 1b, are significantly downregulated in both tumors compared to normal adjacent tissue [29, 30]. Additionally, amino acid metabolism is significantly downregulated in both tumors, as demonstrated by the reduced expression of argininosuccinate synthetase, phosphoglycerate dehydrogenase and glutamate oxaloacetate transaminase 1 and 2 [29]. This downregulation of amino acid metabolism, and the associated deficiency of amino acids such as arginine or glycine supports the hypothesis of amino acid auxotrophy in these tumors [29, 31]. Consequently, tumor cells must obtain these essential amino acids from the normal surrounding tissue [29, 31]. It is plausible that normal adjacent tissue compensates for these metabolic alterations by upregulating the amino acid metabolism.

Nevertheless, chRCC also exhibits important unique characteristics. Firstly, proteins involved in oxidative phosphorylation pathway are less dysregulated in chRCC compared to RO [29]. Additionally, proteins from other mitochondrial metabolic pathways, including those of the respiratory chain, show significantly greater dysregulateion in RO than in chRCC [29].

These findings indicate distinct mitochondrial metabolic pathways and substantial mutations in mitochondrial DNA [29].

A significantly higher number of mutations in mitochondrial genes in RO results in a large proportion non-functional mitochondria. Their function may be partially compensated by the import of functional mitochondria from the normal surrounding tissue into the respective tumor cells [32,33,34]. However, these mutations lead to an accumulation of defective mitochondria within the cells, suggesting a correlation between chronic metabolic disorder and impaired autophagy [32].

These mitochondrial defects and disruptions in cellular respiration contribute to chronic metabolic disorders in RO, leading to the activation of AMP kinase and the downregulation of mTOR [32]. Activation of AMP kinase during transient energy crises normally leads to the degradation of the Golgi apparatus in order to inhibit trafficking and conserve energy [32]. This Golgi apparatus is subsequently rebuilt, allowing the cells to resume growth. However, in RO cells, persistent activation of AMP kinase results in the irreversible disassembly of the Golgi apparatus, leading to impaired autophagy due to ongoing metabolic dysfunction and chronic energy shortages [32]. Inhibition of complex 1 of the respiratory chain plays a crucial role in this process. Ultimately, these mechanisms contribute to the slower growth rate of RO cells [32].

In contrast, PTEN and P53 mutations in chRCC attenuate AMP kinase activation, thereby preserving Golgi function and autophagy [32]. These findings suggest a comparatively faster growth rate of chRCC.