Integrated bioinformatics analysis reveals novel PANoptosis biomarkers and therapeutic response in clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC. Apoptosis, pyroptosis, and necroptosis are key regulatory processes in carcinogenesis. Accumulating evidence indicated significant crosstalk among three forms of cell death, which is termed as the PANoptosis. However, the connection between PANoptosis and ccRCC remains uncertain. Here, we utilized the TCGA and GEO database to explore distinct PANoptosis patterns based on 62 PANoptosis genes and investigated the clinical, biological, and immune cell infiltration characteristics of PANoptosis patterns. Then, we identified prognosis-related genes from PANoptosis patterns and developed a scoring system that effectively predicts clinical outcomes of ccRCC patients. We also explored the expression of key regulators to confirm these identifications. Immunological analyses revealed a positive correlation between risk score and M0 type macrophages, activated mast cells, follicular helper T cells, and regulatory T cells. Finally, the risk model demonstrated the ability to predict drug sensitivity for ccRCC, such as sorafenib, rapamycin and pazopanib. In conclusion, our findings offer novel insights into the role of PANoptosis in ccRCC and identify potential targets for controlling ccRCC.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10238-025-01800-1.

Introduction

Renal cell carcinoma (RCC) is a common malignant tumor in the urinary system and has high incidence rates in Eastern Asia [1–3]. Clear cell RCC (ccRCC) is the predominant form of RCC, characterized by diverse molecular features and poor prognosis³. Therefore, detecting high risk patients, predicting prognosis and drug sensitivity is urgently needed for ccRCC patients.

Cell death is a highly conserved process linked to cell proliferation and tissue homeostasis. The unbalance between cell death and proliferation especially exemplifies in the development of tumor. Therefore, dysregulation of cell death could be a driver of several diseases, such as tumor and autoimmune disease [4–6]. Advances in our understanding of tumor cell death have identified various types and mechanisms of cell death, which is generally classified into unregulated necrosis and programmed cell death (PCD), including apoptosis, pyroptosis, and necroptosis [7]. Although these forms of PCD were initially considered independent, growing evidence suggests significant crosstalk among them [8]. PANoptosis is a distinct and physiologically significant form of inflammatory programmed cell death that combines molecular features of pyroptosis, apoptosis, and necroptosis [9]. However, the majority of PANoptosis research has been performed with innate immune cells. In tumor-related studies, PANoptosis plays a potential role in predicting prognosis, sensitivity to drugs and the correlation with tumor microenvironment (TME) in gastric cancer and prostate cancer [10, 11]. Despite extensive research on PANoptosis in other tumors, its role in ccRCC development remains unclear [12, 13]. Therefore, exploring PANoptosis in ccRCC is essential, as it could identify novel therapeutic targets for ccRCC.

Here, we investigated the potential correlation between ccRCC characteristics and PANoptosis and evaluated its predictive value for patient risk, prognosis, immune cell infiltration and therapeutic response using bioinformatics analysis and in vitro experiment. Our findings indicated that PANoptosis markers had potential to predict the molecular, prognosis and immune characteristics of ccRCC, which could also serve as indicators for therapeutic response.

Methods and materials

Data collection

Gene expression and clinical data for 539 ccRCC and 72 normal samples were sourced from the TCGA database. Gene expression data and clinical information for 71 ccRCC samples were sourced from the GEO database, including dataset GSE29609. Samples lacking survival data were excluded. Somatic mutation and copy number variation (CNV) data were sourced from TCGA database. The “limma” package was employed to normalize gene expression data and transform FPKM to TPM values.

We compiled a list of PANoptosis genes by downloading related genes from the Human Molecular Signatures Database (MSigDB) and the AmiGO 2 database. After obtaining all the PANoptosis genes, we removed overlapping genes and identified the PANoptosis gene list, resulting in a list of 106 PANoptosis regulators (Table. S1).

Identification of differentially expressed genes

The “limma” package was utilized to identify differentially expressed PANoptosis genes, which were then visualized using "ggplot2" in the TCGA KIRC and GEO datasets. PANoptosis genes with differential expression were identified based on the criteria: |logFC|> 1, p value < 0.05, and FDR < 0.05.

Functional analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted using “clusterProfiler” package. Prognosis-related regulators were determined through univariate Cox analysis, applying a significance level of p < 0.05. Unsupervised clustering analysis was performed on the GSE29609 cohort and TCGA KIRC dataset using the "ConsensusClusterPlus" package with 1000 iterations [14]. The “GSVA” package was employed for single-sample gene set enrichment analysis (ssGSEA). The "GSVA" package was utilized to perform gene set variation analysis, evaluating biological pathway differences among various PANoptosis clusters [15]. We utilized the “c2.cp.kegg.v7.4-symbols” gene set from the MSigDB database [16]. An adjusted p < 0.05 was used as threshold. Principal component analysis (PCA) confirmed the expression patterns of PANoptosis genes, and the “pheatmap” package depicted the clinicopathologic features across various groups.

Estimation of tumor microenvironment and tumor mutation burden

The ESTIMATE algorithm was employed to assess stromal and immune scores in tumor samples utilizing data from the TCGA and GSE29609 databases. Tumor mutation burden (TMB) was analyzed using the “maftools” R package with tumor somatic mutation data. The CIBERSORT algorithm was employed to quantify tumor-infiltrating immune cells. The drug sensitivity prediction was conducted via "pRRophetic" R packages with the IC₅₀ used as an indicator of drug sensitivity.

Development and validation of the PANoptosis-related gene signature

To quantify the PANoptosis-based gene expression cluster of each tumor sample, the PANoptosis risk score was established. Initially, we identified intersected differentially expressed genes (DEGs) from the PANoptosis-based gene expression clusters. LASSO and multivariate Cox regression analyses were employed to develop the PANoptosis-based gene signature. The PANoptosis-related risk score is determined by summing the products of each regulator's expression level and its respective coefficient. Patients were divided into high and low-risk groups according to the median risk value via “survminer” package. The accuracy of the PANoptosis-based gene signature was assessed using the K-M curves and ROC curves.

Cell lines, RNA extraction and quantitative real-time PCR

The human renal proximal tubular epithelial cell line HK2 and human RCC cell lines (A498, Caki-1, ACHN, 786-O, and 769-P) were sourced from the ATCC. A498 and ACHN cells were maintained in MEM (Pricella). 786-O and 769-P cells were maintained in RPMI 1640 (Pricella). Caki-1 cells were maintained in McCoy’s 5A (Pricella). HK2 cells were grown in DMEM/F12. All medium supplemented with 10% FBS. Cells were cultured at 37 °C with 5% CO₂. RNA was isolated utilizing the Total RNA Extraction Kit (Vazyme). Reverse transcription was conducted using the reverse-transcription kit from Vazyme. Quantitative PCR (qPCR) was performed on the Bio-Rad detection system.

Statistical analysis

The Kruskal–Wallis test assessed the significance of differences among three or more groups. The correlation coefficient between immune cell counts and PANoptosis-related regulator expression levels was assessed using Spearman's correlation analysis. The "maftools" package was utilized to illustrate the mutation landscape of tumor cohorts. Statistical analysis of qPCR data was analyzed using raw Ct values. Statistical analyses were conducted using R (v4.2.3), with a two-tailed significance threshold of p < 0.05.

Results

Genetic variation and interaction of PANoptosis gene in ccRCC

Firstly, we established a PANoptosis gene list comprising 106 genes sourced from two databases, including 27 pyroptosis genes, 72 apoptosis genes, and 7 necroptosis genes. We analyzed the transcriptomic profiles of these genes in ccRCC and normal samples from TCGA to estimate the expression patterns of all PANoptosis regulators (Fig. 1A). Then, the CNV and somatic mutations were estimated among the selected PANoptosis genes in TCGA KIRC samples. Notably, 64 of 336 samples (19.05%) existed the somatic mutations of PANoptosis genes. Specifically, 19 out of 106 PANoptosis genes existed somatic mutations (Fig. 1B). Further analysis revealed that CNV frequency was predominantly duplications, with high frequencies observed in the loci of UNC5A, CD14, TICAM2 and TNFSF10 (Fig. 1C). The chromosomal locations of CNV alterations were mapped (Fig. 1D). These results suggest that genetic variations in PANoptosis genes are common and varied between tumor and normal tissues in ccRCC, potentially affecting tumor burden and survival outcomes.

Fig. 1.

The expression pattern and characteristics of genetic and variation of PANoptosis genes in ccRCC. A. Transcriptomic profile of 106 PANoptosis genes in ccRCC and normal samples. B. Mutation frequency of 106 PANoptosis regulators in 336 ccRCC patients. Numbers on the right part represent mutation frequency. C. CNV alteration frequency of 106 PANoptosis regulators in 336 ccRCC cohort. The height of each column represents the alteration frequency. The amplification frequency and deletion frequency are represented by red dot and the green dot separately. D. The location of CNV alterations of 106 PANoptosis genes on different chromosomes. E. The interaction network of 106 PANoptosis genes in the ccRCC cohort.*. p < 0.05; **. p < 0.01; ***. p < 0.001

Then, we investigated the prognostic value and potential clinical relevance of all PANoptosis regulators. Our analysis revealed 62 PANoptosis regulators significantly associated with patient’s prognosis. Using univariate Cox analysis and Pearson correlation analysis, we determined that 40 genes (64.5%) were prognosis-favorable, while 22 genes (35.5%) were prognosis-unfavorable (Fig. 1E).

Characteristics of PANoptosis patterns mediated by PANoptosis genes and its clinical relevance

Based on the expression of the 62 PANoptosis genes, we performed unsupervised clustering analysis on ccRCC patients, identifying two patterns: PANoptosis clusters A and B, comprising 118 and 451 cases, respectively (Fig. 2A). The PCA analysis demonstrated distinct features of transcriptome profiles between the two clusters (Fig. 2B). K-M analysis revealed improved survival in PANoptosis cluster B relative to cluster A (Fig. 2C).

Fig. 2.

Identification of the PANoptosis patterns. A. The ccRCC cohort was divided into two PANoptosis patterns by consensus clustering. B. PCA analysis indicated the distinct difference of distribution between PANoptosis clusters. C. The Kaplan–Meier analysis indicated the overall survival for the two PANoptosis clusters. D. Unsupervised clustering of 62 PANoptosis genes in different PANclusters. E. GSVA enrichment analysis depicting the biological processes in two PANoptosis clusters. F. the principle immune cells enriched in two PANoptosis clusters. *. p < 0.05; **. p < 0.01; ***. p < 0.001

We generated a heat map illustrating the correlation between PANoptosis clusters and clinical characteristics by integrating clinical data from TCGA and GEO datasets. Figure 2D shows that PANoptosis cluster B is associated with younger patients and tumors of lower malignant potential, with 45.7% of patients in cluster A and 39.9% in cluster B having stage III/IV tumors. Cluster B exhibited higher expression levels of PANoptosis regulators (Fig. 2D).

To investigate the biological processes associated with two PANoptosis patterns, we analyzed the enriched pathways in the two subtypes using GSVA. As illustrated in Fig. 2E, we found that cluster B was associated with oncogenic signaling pathways, including renal cell carcinoma and apoptosis pathways, while cluster A was linked to metabolism-related processes (Fig. 2E). To confirm the critical role of key infiltrating immune cells in diverse PANoptosis clusters, ssGSEA analysis was conducted. These findings revealed that cluster A showed increased enrichment of CD56 bright, dim natural killer cells and T helper cells, whereas cluster B was mainly enriched with innate immune cells, such as mast cells and neutrophils (Fig. 2F).

Biological characteristics of the three PANoptosis-based gene patterns

We first determined 91 intersected PANoptosis-based DEGs among two PANoptosis clusters (Fig. 3A, Table. S1). Then, we performed GO and KEGG enrichment analyses to assess the functional characteristics of these DEGs. GO analysis indicated a significant enrichment in the positive regulation of tumor necrosis factor production (Fig. 3B, Table. S2). The KEGG analysis identified the HIF-1 signaling and renal cell carcinoma pathways (Fig. 3C). ccRCC often involves VHL gene loss of function mutations or epigenetic silencing, resulting in frequent activation of the HIF-signaling pathway. This activation induces a metabolic switch that enhances angiogenesis and promotes tumorigenesis. Our results further verified the pivotal role of PANoptosis in the development of ccRCC.

Fig. 3.

Construction of PANoptosis scoring system. A. The transcriptomic profile of 91 intersected differentially expressed genes among two PANoptosis clusters. B, C. Representative characteristics of the GO enrichment and KEGG pathways. D. unsupervised clustering analysis was conducted to construct the genomic cluster based on the prognosis-related genes. E. PCA analysis indicated the distinct difference of distribution between PANoptosis-based gene expression clusters. F. The Kaplan–Meier analysis indicated the overall survival for three PANoptosis-based gene expression clusters. G. Unsupervised clustering of prognosis-related genes in different PANoptosis-based gene expression clusters. H. The transcriptomic profile of PANoptosis genes among two PANoptosis clusters

Using two identified PANoptosis clusters, we conducted univariate Cox regression analysis on the TCGA KIRC and GSE29609 databases, revealing 82 PANoptosis-related regulators associated with prognosis (Table. S3). We extracted these genes for unsupervised clustering analysis to enhance the genomic cluster construction. Using the optimal k of 3, we identified three genomic clusters, labeled as PANoptosis-based gene expression clusters A, B, and C (Fig. 3D). PCA confirmed differences among the clusters as well (Fig. 3E). K-M analysis revealed that cluster A had the worst prognosis, while cluster B showed the favorable outcome (Fig. 3F).The heat map demonstrated that cluster A was enriched with metastatic tumors and advanced grades (Fig. 3G). Differential gene analysis further verified the PANoptosis-based gene signatures (Fig. 3H). These results validated that different PANoptosis patterns occur in ccRCC.

Construct and evaluate the model of the PANoptosis-based regulators

To further quantify and evaluate PANoptosis-based gene patterns, we established the PANoptosis-related risk model by dividing the ccRCC cohort into training (n = 278) and test (n = 278) sets. Using LASSO analysis and multivariate Cox analysis, 19 regulators were identified and a 12-gene risk model was further established (Fig. S1A, Table. S4). We divided each dataset into low and high-risk groups based on median risk scores. K-M analysis indicated that patients in high-risk group had poorer prognosis (Fig. 4A). Furthermore, correlation analysis revealed a negative relationship between risk score and prognosis (Fig. 4C-4E). The model's predictive capability was validated via ROC analysis, resulting in AUC values of 0.849, 0.815, and 0.865 for one, three, and five years, respectively (Fig. 4I).

Fig. 4.

Construction and evaluation of the PANoptosis-based gene patterns. A, B. The Kaplan–Meier analysis for the OS of patients in the high- and low-risk group among training and validation groups. C-E. The expression and distribution of risk scores of each regulator in the PANoptosis-related risk model among training groups. F–H. The expression and distribution of risk scores of each gene in the PANoptosis-related risk model among validation groups. I, J. ROC curves of training cohort and validation cohort. K. Alluvial diagram shows the differences in PANoptosis cluster, PANoptosis-based gene expression cluster and PANoptosis-related risk score. L, M. Differential analysis of risk scores among PANoptosis cluster and PANoptosis-based gene expression cluster. N. the differential analysis of selected PANoptosis genes between low-risk and high-risk groups

We also produced and inspected an alluvial diagram to the relationship between PANoptosis clusters, gene expression clusters, risk scores, and patient survival. These results suggested that most of the ccRCC patients in the PANoptosis-based gene expression cluster B were marked with low PANoptosis-related risk score and showed better survival condition. (Fig. 4K). In addition, differential analysis confirmed significant differences in risk scores among these clusters (Fig. 4L, M). We also conducted a differential analysis of selected PANoptosis genes between low and high-risk groups. These finding demonstrated that PANoptosis regulators have an impact on patient risk (Fig. 4N).

To valid the above results, we conducted the K-M analysis, assessed risk score distribution and analyzed the ROC curve. These findings revealed that high-risk patients exhibited worse survival outcomes than low-risk patients (Fig. 4B). The risk scores distribution indicated that patients with high-risk scores exhibit worse survival outcomes (Fig. 4F-4H). The ROC curve indicated that the twelve-gene signature effectively predicted OS, achieving AUC values of 0.634, 0.639, and 0.628 at one, three, and five years, respectively (Fig. 4J). Afterward, the qPCR analysis revealed that six regulators (including RGS5, RGL1, SLC40A1, TLR4, EGFR and ZNF366) mRNA were highly expressed in RCC cells. Other genes were significantly down-regulated in RCC cells (Fig. 5A-L). These results were consistent with former results (Fig. 3A).

Fig. 5.

Experimental verification of four genes in the prognostic signature. A–L. mRNA expression levels of twelve genes were evaluated using qRT–PCR in ccRCC cell lines. *. p < 0.05; **. p < 0.01; ***. p < 0.001

Immune relevance and pharmacological analysis of the risk model

To further verify the relationship between major immune cells and the PANoptosis-related risk score, we performed the Spearman correlation analysis. Our analysis identified a positive correlation between the risk score and the presence of M0 type macrophages, activated mast cells, CD8⁺ T cells, follicular helper T cells, and regulatory T cells (Fig. 6A-6F). These findings align with previous results [17, 18]. While an increase in CD8⁺ T cells was noted alongside the risk score, prior research indicates that higher CD8⁺ T cell density correlates with poorer outcomes in RCC [19]. In contrast, M2 macrophages, resting mast cells, monocytes, and resting CD4⁺ memory T cells showed a negative correlation with the risk score.

Fig. 6.

The characteristic of immune infiltration and drug sensitivity based on the risk model. A-K. The correlation analysis between immune cells and the PANoptosis-related risk score. L. The correlation analysis between immune cells and twelve PANoptosis-based regulators. M. The correlation analysis between ESTIMATE scores and the PANoptosis-related risk score. N. The tumor stemness correlation analysis shows the relationship between the PANoptosis-related risk score and stemness scores. O-R. The relationship between sensitivity to therapeutic drugs and risk score. *. p < 0.05; **. p < 0.01; ***. p < 0.001

We also explored the relationship between 12 regulators in the risk model and major immune cells. The results indicated that most native immune cell was negatively correlated with the 12 PANoptosis-based regulators, except for the resting memory CD4 + T cells (Fig. 6L). Then, we analyzed the correlation between ESTIMATE scores and the risk score, which indicate that ccRCC patients with high immune scores exhibit increased risk (Fig. 6M). Tumor stemness correlation analysis revealed a positive correlation between risk score and tumor stemness score (Fig. 6N).

We further assessed whether the risk signature had ability to predict drug sensitivity for ccRCC. The pRRophetic algorithm was used to predict the relationship between drug sensitivity and risk score. The study revealed significant variations in sensitivity to 93 chemotherapeutic and targeted drugs between high and low risk groups. The IC₅₀ values of 43 therapeutic drugs were elevated in the high-risk group, whereas 50 drugs exhibited increased IC₅₀ values in low-risk group (Fig. S1-3). We also identified several targeted drugs commonly used for renal cell carcinoma. Patients with high risk exhibit increased tolerance to sorafenib, rapamycin, and gefitinib (Fig. 6O-6Q). However, the sensitivity to pazopanib is high in high-risk group (Fig. 6R).

Discussion

ccRCC is essentially a metabolic disease characterized by a reprogramming of energetic metabolism [20–24]. In particular, the metabolic flux through glycolysis is partitioned [25–27], and mitochondrial bioenergetics and OxPhox are impaired, as well as lipid metabolism [25, 28–31]. In this scenario, it has been shown that apoptosis, pyroptosis, and necroptosis are important regulator of cell metabolism and regulate many biological characteristics of renal cancer stem cells [32, 33]. In addition, RCC is one of the most immune-infiltrated tumors [34–36]. Emerging evidence suggests that the activation of specific metabolic pathway has a role in regulating angiogenesis and inflammatory signatures [37, 38]. Features of the tumor microenvironment heavily affect disease biology and may affect responses to systemic therapy [39–43].

Programmed cell death (PCD) is a conserved pathway playing an essential role in the immune response, encompassing apoptosis, pyroptosis, and necroptosis [44]. Apoptosis, a well-studied type of cell death, was associated with both the inhibition and progression of tumor cells [45]. Pyroptosis, a type of PCD mediated by gasdermin, is characterized by progressive cell expansion until cytomembrane ruptures [46]. Cytokines, such as IL-1β, IL-18, released after pyroptosis facilitate infiltration, induce cell death and inhibit tumorigenesis [47, 48]. Necroptosis is another regulated cell death in caspase-independent process. It has been reported that necroptosis engages in complex interplay with pyroptosis and apoptosis [49]. Many studies had found that necroptosis could serve as an alternative mode of PCD that have potential role in overcoming apoptosis resistance and trigger antitumor immunity [49, 50]. Given the crosstalk among these PCD pathways, a dynamic molecular interaction is believed to guide cell death events, introducing the concept of PANoptosis [8, 51].

The PANoptosis was first proposed by Kuriakose T et al. in 2016. The researchers found that deletion of ZBP1 could suppress PANoptosis, which suggests ZBP1 could trigger PANoptosis [8]. Currently, many studies found that PANoptosis could occur in tumor patients. In colorectal cancer, the loss of NFS1 could synergize with oxaliplatin to trigger PANoptosis by elevating intracellular levels of ROS [52]. In addition, ADAR1 suppresses ZBP1-mediated PANoptosis, which could promote tumorigenesis in colorectal cancer and melanoma by interacting with Zα2 domain of ZBP1 [53]. However, the effect of PANoptosis on ccRCC has not yet been analyzed.

Here, we investigated the changes in PANoptosis genes in ccRCC. The mutations of PANoptosis regulators occurred relatively infrequently in ccRCC. We then investigated the prognostic value of PANoptosis genes and found that 62 PANoptosis regulators correlated with patient prognosis. In 2022, Pan et al. reported that PANoptosis regulators could predict prognosis and immunotherapy response [11]. This study identified two PANoptosis patterns based on prognosis-related genes. Utilizing the GSVA, we determined the biological processes related to these PANoptosis patterns. The PANoptosis cluster A was associated with metabolism-related processes, such as arachidonic acid metabolism and xenobiotics metabolism. Moreover, the PANoptosis cluster B was associated with oncogenic signaling pathways, including renal cell carcinoma pathways. Li et al. reported that tumor cells could manipulate metabolic regulation to achieve growth advantage or to escape from apoptosis and cell death [54]. Therefore, whether aberrant metabolism contributes to the worse prognosis of patients in PANoptosis cluster A is deserved to be further determined.

Afterward, we identified 91 intersected DEGs among two PANoptosis clusters and determined 82 prognosis-related regulators among selected DEGs. In order to predict the outcome of ccRCC, we constructed the risk model. The K-M curve, distribution of risk scores and time-dependent ROC curve revealed that patients in high-risk group tended to have worse prognosis than patients in the low-risk group. qPCR analysis corroborated the bioinformatics results. We also evaluated the immune relevance. The M0 type macrophages, activated mast cells, CD8⁺ T cells, follicular helper T cells and regulatory T cells were positively correlated with risk score. In addition, ccRCC patients in high-risk group inclined to have high immune scores with ESTIMATE analysis. Previous studies have suggested that regulatory T cells are prevalent in various tumors and could serve as an immunosuppressive regulator of immune responses [55]. Moreover, we found a positive correlation between regulatory T cells and the risk score, consistent with previous results. Although CD8⁺ T cell was relatively high in high risk group, previous study indicated that increased CD8⁺ T cell density is associated with a worse outcome in RCC, including metastatic lesions and primary tumors [56]. Finally, we investigated the relationship between risk score and pharmacological response. These results suggested that risk score could greatly predict pharmacological response in ccRCC patients. However, it should be noted that this study is primarily based on data mining. While our results provide potential mechanistic insights, further experimental validation using independent clinical specimens is essential to confirm the biological and translational relevance of these findings.

To our knowledge, this is the first report concerning the molecular and immune characteristics of PANoptosis pattern in ccRCC. Nevertheless, the underlying mechanism of PANoptosis in ccRCC is still unknown. Our next step will be to further verify the function and mechanism of PANoptosis regulators via both in vivo and in vitro experiments.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

B.Z, Q.OY and K.L designed the study. S.P.W. and T.Y.J. collected and analyzed the data. J.F., J.C.W., and Y.H.D. interpreted the data. B.Z, Q.OY and K.L drafted the manuscript. X.B.L., X.M., and X.Z revised the manuscript. All authors reviewed and approved the manuscript for publication.

Funding

The authors have not disclosed any funding

Data availability statement

Data is provided within the manuscript or supplementary information files.

Declarations

Conflict of Interest

The authors declare no competing interests.

Ethical Approval

None.