Abstract

   Xp11.2 translocation renal cell carcinoma (tRCC), involving
   transcription factor E3 (TFE3) gene fusions, is a rare and aggressive
   RCC variant when present in adults and has been recently recognized as
   a unique entity in RCC. Biomarkers and treatment guidelines do not
   exist for patients with aggressive Xp11.2 tRCC. The aim was to identify
   and evaluate therapeutic biomarkers for aggressive Xp11.2 tRCC. RNA
   sequencing was performed using formalin-fixed, paraffin-embedded
   tissues from 11 adult patients with clinical T1N0M0 Xp11.2 tRCC,
   including three patients with aggressive characteristics (recurrence or
   cancer-specific death after nephrectomy). Thirty genes were
   differentially expressed between the aggressive and non-aggressive
   groups, even after adjustment, and were associated with KEGG pathways
   related to the aggressiveness of Xp11.2 tRCC. PIK3R2, involved in
   various KEGG pathways, including the PI3K/AKT/mTOR pathway, was
   overexpressed in the Xp11.2 tRCC cell lines UOK120 and UOK146. The PI3K
   pathway inhibitor LY294002 showed a significant therapeutic benefit.
   This study provides the first candidate biomarker, PIK3R2, for
   aggressive clinical T1N0M0 Xp11.2 tRCC. Furthermore, this study is the
   first to recommend a targeted drug, LY294002, for aggressive Xp11.2
   tRCC based on the molecular pathophysiology.

   Keywords: renal cell carcinoma, Xp11.2, translocation, TFE3, biomarker,
   PIK3R2

1. Introduction

   Renal cell carcinoma (RCC) involving translocations at Xp11.2, referred
   to as Xp11.2 translocation RCC (tRCC), is a rare subtype and has gained
   significant clinical interest in the last two decades [[34]1,[35]2],
   after its inclusion as a new distinct entity in the 2004 World Health
   Organization (WHO) classification of renal tumors [[36]3]. tRCC has
   been subclassified as belonging to the microphthalmia transcription
   factor-associated family tRCC in the updated 2016 WHO classification
   based on improvements in genetic profiling technology [[37]4]. Xp11.2
   tRCC is predominantly reported in children, accounting for about
   one-third of pediatric RCC cases [[38]5,[39]6]. However, tRCC is rare
   in adults, accounting for 1.6–5.0% for adult RCC cases [[40]7,[41]8].
   This low incidence might be due to morphological overlap with clear
   cell RCC (ccRCC) and papillary RCC, resulting in its misdiagnosis
   [[42]8,[43]9].

   Numerous fusion partners in Xp11.2 tRCC have been identified. PRCC,
   ASPL/ASPSCR1, and SFPQ are the most common partners, and many novel
   genes are still being discovered [[44]9,[45]10]. The ASPL/ASPSCR1-TFE3
   fusion type is associated with unfavorable outcomes [[46]7] and shares
   the same chromosomal breakpoint as alveolar soft part sarcoma, which is
   refractory to chemotherapy [[47]11]. The PRCC-TFE3 fusion type is
   associated with nodal involvement; however, patients can be
   disease-free for at least 3 years after lymph node dissection [[48]7].

   There are only a few case reports and observational studies of systemic
   therapies for advanced Xp11.2 tRCC, most of which focused on sunitinib
   [[49]1]. Systemic treatment guidelines for advanced Xp11.2 tRCC are
   currently unavailable owing to the rarity of the disease as well as the
   poor understanding of its molecular pathophysiology [[50]1]. In this
   study, tissue samples were analyzed to develop molecular biomarkers for
   aggressive Xp11.2 tRCC and evaluated the value of these biomarkers for
   determining the appropriate systemic therapy.

2. Materials and Methods

2.1. Patients and Tissues

   For this retrospective study, data were gathered from patients with
   Xp11.2 tRCC who underwent radical nephrectomy between January 2008 and
   December 2018 and had available formalin-fixed, paraffin-embedded
   (FFPE) tissues. Tumors sized ≤7 cm (clinical T1 stage) without
   synchronous metastasis on images (N0M0 stage) were included. Patients
   with Xp11.2 tRCC pathologically confirmed by TFE3 immunohistochemistry
   staining and fluorescence in situ hybridization (FISH) assay were
   included. Immunohistochemistry (IHC) was performed on FFPE sections
   with a TFE3 antibody; tumors were considered TFE3-positive when
   labeling was moderate to strong (2+ to 3+) in more than 10% of cells.
   Tumors with non-diffuse (<10% of cells) and weak labeling (1+) were
   excluded. A dual-color, break-apart FISH assay was performed with
   samples from patients who had positive TFE3-IHC results on FFPE tissue
   microarray slides. Patients were divided into aggressive and
   non-aggressive Xp11.2 tRCC groups. An aggressive tumor was defined as a
   tumor exhibiting recurrence or cancer-specific death.

2.2. Tissue Preparation

   A urologic pathologist reviewed all FFPE sections of Xp11.2 tRCC
   patients, which were obtained from the archives of the Department of
   Pathology at Yonsei University College of Medicine (Seoul, Korea).
   Identification of non-tumor elements was based on hematoxylin and
   eosin-stained slides. The samples were cut into 20 μm sections and
   transferred to an extraction tube.

2.3. RNA Extraction and Sequencing

   Briefly, RNA was extracted from 20 μm sections of FFPE tissues using an
   FFPE RNeasy Kit (Qiagen, Gaithersburg, MD, USA), and 100 ng of RNA was
   used to construct a sequencing library using a SureSelectXT RNA Direct
   Library Preparation Kit (Agilent Technologies, Santa Clara, CA, USA)
   according to the manufacturer’s protocols.

   The sequencing libraries were quantified using a Kapa Biosystems
   Library Quantification Kit for Illumina Sequencing platforms according
   to the quantitative polymerase chain reaction (qPCR) Quantification
   Protocol Guide (Kapa Biosystems, Wilmington, MA, USA; Cat# KK4854) and
   were qualified using TapeStation D1000 ScreenTape (Agilent
   Technologies, Santa Clara, CA, USA; Cat# 5067-5582). Indexed libraries
   were then subjected to paired-end sequencing (2 × 101 bp) using the
   NovaSeq 6000 system (Illumina, San Diego, CA, USA) by Macrogen (Seoul,
   Korea).

2.4. RNA Sequencing (RNA-Seq) Analysis and Identification of Differentially
Expressed Genes

   Paired-end sequencing reads from cDNA libraries (101 bp) were
   generated, and the trimmed reads were aligned to the human reference
   genome, hg19 (GRCh37), using HISAT (v2.1.0). The transcriptome assembly
   of known transcripts, novel transcripts, and alternative splicing
   transcripts was processed.

   Transcript abundance and gene expression levels were calculated as read
   counts (i.e., number of reads mapped to a gene) or fragments per
   kilobase of exon per million fragments mapped (FPKM) per sample. To
   identify differentially expressed genes (DEGs) between aggressive and
   non-aggressive Xp11.2 tRCC groups, genes with more than one sample with
   a zero read count were pre-filtered. Expression levels of 35,993 RefSeq
   genes and 19,594 genes were retained.

   For DEGs, an unsupervised hierarchical clustering analysis was
   performed, with the complete linkage method and Euclidean distances as
   a measure of similarity. A principal component analysis (PCA) was
   performed to reduce the dimensionality of the dataset by transforming
   it into a new set of variables summarizing the data features. All data
   analyses and the visualization of DEGs were conducted using R.3.5.1
   (available online: [51]www.r-project.org (accessed on 23 December
   2021)).

2.5. Fusion Type Analysis

   Fusion genes from RNA-Seq data were identified using FusionCatcher and
   Arriba. FusionCatcher searches for novel/known somatic fusion genes,
   translocations, and chimeras from RNA-Seq data. In brief, FusionCatcher
   filters and trims low-quality reads and then finds fusion junctions
   with at least two genes (both in the ENSEMBL database) using known exon
   and intron information. Arriba is a command-line tool for the detection
   of gene fusions from RNA-Seq data. It is based on the ultrafast STAR
   aligner.

2.6. KEGG Analysis

   A KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of
   DEGs was performed based on the KEGG pathway (available online:
   [52]https://www.genome.jp/kegg/ (accessed on 26 December 2021))
   database. A sorted heatmap of KEGG enrichment results was generated to
   visualize significant pathways filtered by a p-value of <0.05, as
   determined by the Fisher’s exact test.

2.7. Xp11.2 tRCC Cell Lines

   tRCC cell lines with the two most common gene fusions, ASPL- and
   PRCC-TFE3, were obtained. UOK120 and UOK146 were previously derived
   from tumors excised from patients with Xp11.2 tRCC who were treated at
   the National Cancer Institute (NCI, Bethesda, MD, USA) and harbored
   PRCC-TFE3 gene fusions [[53]12]. The UOK120 and UOK146 lines were
   obtained from and authenticated by the NCI (Bethesda, MD, USA). S-TFE,
   which was established from patients with tRCC with ASPL-TFE3 gene
   fusions [[54]13], was obtained from the Riken Cell Bank (Tsukuba,
   Japan). These cells were cultured in Dulbecco’s modified Eagle’s medium
   or Roswell Park Memorial Institute 1640 medium, supplemented with 10%
   fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Waltham, MA,
   USA) and 1% penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO,
   USA), at 37 °C in a humidified atmosphere containing 5% CO[2].

2.8. Treatment Regimens

   The anticancer drugs sunitinib, sorafenib, everolimus, and LY294002 (a
   PI3K pathway inhibitor) were purchased from Selleckchem (Houston, TX,
   USA).

2.9. Cell Proliferation Assay

   Cells were seeded at 3000–5000 cells per well in 96-well culture
   plates. The next day, the cells were treated with anticancer drugs at
   the desired concentration in culture media. At 24–72 h after treatment,
   cell viability was assessed by a CCK-8 assay (Dojindo Laboratories,
   Kumamoto, Japan), in accordance with the manufacturer’s instructions.
   Absorbance was detected at 450 nm using a VersaMax Microplate Reader
   (Molecular Devices, Sunnyvale, CA, USA).

2.10. Reverse Transcription-qPCR Analysis

   To detect PIK3R2 expression, total RNA was extracted using the RNeasy
   Mini Kit (Qiagen Inc., Valencia, CA, USA). A complementary DNA template
   was synthesized from 1 μg of RNA using iNtRon Maxime RT PreMix (iNtRON
   Biotechnology, Seongnam, Korea), and qPCR was performed using the
   StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA,
   USA). The reaction conditions were as follows: predenaturation at 95 °C
   for 10 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 20
   s, and extension at 72 °C for 34 s, with a total of 40 cycles. U6 small
   nuclear RNA was used as the internal reference. The qPCR data were
   analyzed using the 2^−ΔΔCT method. These procedures were repeated three
   times.

2.11. Wound-Healing Assay

   Cells were cultured to confluence in 6-well plates. When the cells
   reached 100% confluence, a scratch was produced by a sterile pipette
   tip. The cells were rinsed two times in phosphate-buffered saline and
   treated with 10 µM LY294002 in culture media. Images of cells were
   obtained under an inverted microscope at 0 and 24 h. ImageJ was used to
   calculate the healing area, and the healing rate (expressed as a
   percentage) was calculated as follows: (initial scratch width—existing
   scratch width)/initial scratch width × 100.

2.12. Invasion Assay

   In vitro Matrigel invasion assays were performed using the CytoSelect ™
   24-Well Cell Invasion Assay Kit (Cell Biolabs, San Diego, CA, USA)
   following the manufacturer’s instructions. Cells were treated with
   phosphate-buffered saline or LY294002 (10 µM) and plated on the upper
   chamber (2–5 × 10^4 cells/well) using serum-free medium. For the
   assays, 10% FBS-containing medium was placed in the lower chambers as a
   chemoattractant. After incubation at 37 °C for 24 h, invading cells on
   the bottom surface of the membrane were stained with crystal violet,
   and cells passing through the Matrigel in each group were counted as an
   indicator of invasion ability.

2.13. Western Blot Analysis

   To detect PIK3R2 expression, proteins were extracted with
   radioimmunoprecipitation assay buffer (Cell Signaling Technology,
   Danvers, MA, USA), and the amount of protein was determined using a
   bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Equal
   amounts of denatured proteins were separated by sodium dodecyl
   sulfate–polyacrylamide gel electrophoresis, transferred to a
   polyvinylidene fluoride membrane, and blocked with 5% bovine serum
   albumin in tris-buffered saline with Tween 20. Samples were incubated
   with antibodies to PIK3R2 (1:2000; Santa Cruz Biotechnology, Dallas,
   TX, USA) and β-actin (1:5000, Cell Signaling Technology) at 4 °C
   overnight and subsequently with horseradish peroxidase-conjugated
   secondary antibody (Santa Cruz Biotechnology). To detect reactive
   bands, the membranes were examined using the ECL Prime Western Blotting
   Detection System (GE Healthcare, Amersham, UK) and LAS-3000 (Fujifilm,
   Tokyo, Japan).

2.14. Statistical Analysis

   Statistical analyses were performed using GraphPad Prism version 8.0
   (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS version 23.0 (IBM
   Corp., Armonk, NY, USA). All results are expressed as means ± standard
   deviation (s.d.) unless otherwise indicated. The Student’s t-test was
   used for data that followed a normal distribution, as determined by the
   Shapiro–Wilk normality test. If the dataset did not follow a normal
   distribution, the Mann–Whitney U test was used. All statistical tests
   were two-tailed, and p < 0.05 was considered significant.

3. Results

3.1. Baseline Characteristics

   Clinicopathologic characteristics of patients with clinical T1N0M0
   Xp11.2 tRCC at presentation (n = 11) are summarized in [55]Table 1. The
   mean age and tumor size were 45.1 ± 18.2 years and 4.9 ± 1.7 cm,
   respectively. Among 11 patients, three patients had recurrence or
   cancer-specific death and were assigned to the aggressive Xp11.2 tRCC
   group. Age, tumor size, or World Health Organization/International
   Society of Urologic Pathologists grade did not differ significantly
   between patients with aggressive Xp11.2 tRCC and those with
   non-aggressive Xp11.2 tRCC. All three patients in the aggressive group
   exhibited tumor recurrence after nephrectomy within a mean follow-up
   period of 20.7 months. One patient exhibited cancer-specific death
   after 11.0 months of follow-up. Survival time did not differ
   significantly between the aggressive and non-aggressive Xp11.2 tRCC
   groups (p = 0.290).

Table 1.

   Clinicopathological characterization of the study population.
   Aggressive (Y/N) Patient ID Sex Age (year) Tumor Size (cm) WHO/ISUP
   Grade Outcome ^a Recurrence-Free Time ^b (m) Recurrence Site Death
   (Y/N) Survival Time ^c (m)
   N Pt0-1 F 21 2.5 2 ned N 111
   N Pt0-2 F 41 3.9 3 ned N 112
   N Pt0-3 M 56 2.2 3 ned N 97
   N Pt0-4 F 73 4.9 2 ned N 63
   N Pt0-5 F 29 5.3 3 ned N 67
   N Pt0-6 M 54 3.2 2 ned N 54
   N Pt0-7 F 30 6.0 3 ned N 58
   N Pt0-8 F 46 7.0 3 ned N 25
   Y Pt1-1 F 68 5.7 3 pd 42 Lung N 93
   Y Pt1-2 M 21 6.5 3 pd 2 Lung, Lymph nodes, liver Y 11
   Y Pt1-3 M 57 6.8 3 pd 18 Lung, Lymph nodes, liver N 40
   [56]Open in a new tab

   ^a ned, no evidence of disease; pd, progression of the disease; ^b
   recurrence-free time was defined as the time from nephrectomy until the
   first time that the patient was known to have recurrence; ^c survival
   time was defined as the time from nephrectomy until death or the last
   time that the patient was known to be alive; WHO, World Health
   Organization; ISUP, International Society of Urologic Pathologists.

3.2. RNA-Seq Analysis

   The RNA-seq analysis generated 10,256 × 10^6 base pairs (bp) from
   Xp11.2 tRCC cases with aggressive characteristics and 10,521 × 10^6 bp
   from Xp11.2 tRCC cases without aggressive characteristics. In total,
   101 × 10^6 reads from Xp11.2 tRCC cases with aggressive characteristics
   and 104 × 10^6 reads in Xp11.2 tRCC cases without aggressive
   characteristics were mapped. There were no differences in the number of
   reads between the two groups, and 19,594 genes were identified.

3.3. DEGs, Fusion Types, and KEGG Pathway Enrichment

   In total, 1609 genes were differentially expressed with fold changes ≥2
   and p < 0.05, including 603 upregulated genes and 1006 downregulated
   genes in Xp11.2 tRCCs with aggressive characteristics compared to
   matched Xp11.2 tRCCs without aggressive characteristics ([57]Figure 1).
   In total, 91 genes were strongly upregulated or downregulated in
   patients with aggressive tRCCs and retained significance even after
   adjustment. Among these, 77 genes were protein-coding genes, and four
   genes were long non-coding RNAs; the others were pseudogenes. Clear
   clustering was observed, distinguishing between the aggressive and
   non-aggressive Xp11.2 tRCC groups, as determined by PCA ([58]Figure 1).

Figure 1.

   [59]Figure 1
   [60]Open in a new tab

   (A) Unsupervised hierarchical clustering analysis (yellow, high
   relative expression; blue, low relative expression) of patients with
   aggressive Xp11.2 translocation renal cell carcinoma (tRCC). (B)
   Principal component analysis. The first and second principal component
   scores in patients with aggressive Xp11.2 tRCC (n = 3, red) versus
   matched patients with non-aggressive Xp11.2 tRCC (n = 8, blue) are
   plotted.

   Among 11 patients, fusion types were identified for five patients.
   Among patients with aggressive tRCC, Pt1-2 and Pt1-3 harbored the
   ASPL-TFE3 gene fusion and RBM10-TFE3 gene fusion, respectively. For
   patients with non-aggressive tRCC, Pt0-4, Pt0-6, and Pt0-8 harbored the
   SFPQ-TFE3 gene fusion.

   A KEGG pathway enrichment analysis revealed that the DEGs were mainly
   enriched in metabolic pathways, olfactory transduction, neuroactive
   ligand–receptor interaction, herpes virus 1 infection, pathways in
   cancer, and the PI3K-Akt signaling pathway ([61]Figure 2A). The top 20
   significant pathways associated with the aggressiveness of Xp11.2 tRCC
   ([62]Figure 2A) involved genes that were significantly associated with
   the aggressiveness of Xp11.2 tRCC, including 14 upregulated and 16
   downregulated genes ([63]Table 2 and [64]Figure 2B). Among the 30 genes
   that were significantly associated with the aggressiveness of Xp11.2
   tRCC, PIK3R2 was involved in the most known pathways (i.e., 11
   pathways). PIK3R2 expression levels in aggressive Xp11.2 tRCC (27.44 ±
   4.87 FPKM) were significantly higher than those in non-aggressive
   Xp11.2 tRCC (14.15 ± 2.45 FPKM) (adjusted-p > 0.04 by the
   Benjamini–Hochberg algorithm).

Figure 2.

   [65]Figure 2
   [66]Open in a new tab

   KEGG pathway analysis. (A) Top 20 significant KEGG pathways and their
   functional classification by five main categories (A, cellular
   processes; B, environmental information processing; C, genetic
   information processing; D, metabolism; and E, organismal systems). The
   x-axis indicates the number of genes annotated under the indicated
   pathway. The y-axis indicates the names of KEGG pathways. The bar
   indicates the −log10 (p-value). (B) Genes significantly associated with
   the aggressiveness of Xp11.2 translocation renal cell carcinoma and the
   number of related KEGG pathways for each gene. KEGG, Kyoto Encyclopedia
   of Genes and Genomes.

Table 2.

   Significantly upregulated/downregulated genes in clinical T1N0M0 Xp11.2
   translocation renal cell carcinoma with versus without aggressive
   characteristics (recurrence or cancer-specific death).
   Upregulated Downregulated
   Gene Symbol Gene Name Log[2] Fold Change (Aggressive/Non-Aggressive) #
   Of Related Pathways Gene Symbol Gene Name Log[2] Fold Change
   (Aggressive/Non-Aggressive) # Of Related Pathways
   KRT10 Keratin 10 2.09 1 ZNF662 Zinc Finger Protein 662 −2.56 1
   PIK3R2 Phosphoinositide-3-Kinase Regulatory Subunit 2 2.22 11 RARB
   Retinoic Acid Receptor Beta −2.76 1
   PCYT2 Phosphate Cytidylyltransferase 2, Ethanolamine 2.37 1 ATP1B2
   ATPase Na+/K+ Transporting Subunit Beta 2 −3.36 2
   GALE UDP-Galactose-4-Epimerase 2.48 2 PDE7B Phosphodiesterase 7B −3.73
   1
   GPX1 Glutathione Peroxidase 1 2.55 2 KCNMB4 Potassium Calcium-Activated
   Channel Subfamily M Regulatory Beta Subunit 4 −5.54 1
   MIF Macrophage Migration Inhibitory Factor 2.64 1 NAT2
   N-Acetyltransferase 2 −6.45 3
   SHC1 SHC Adaptor Protein 1 2.66 1 OR56A5 Olfactory Receptor Family 56
   Subfamily A Member 5 −6.91 1
   DTYMK Deoxythymidylate Kinase 2.71 1 OR2T7 Olfactory Receptor Family 2
   Subfamily T Member 7 −7.47 1
   ENO1 Enolase 1 2.93 4 OR10A2 Olfactory Receptor Family 10 Subfamily A
   Member 2 −8.75 1
   TALDO1 Transaldolase 1 3.00 3 ZNF479 Zinc Finger Protein 479 −9.17 1
   GSS Glutathione Synthetase 3.30 2 GK2 Glycerol Kinase 2 −9.30 1
   TYMP Thymidine Phosphorylase 3.42 2 OR6C70 Olfactory Receptor Family 6
   Subfamily C Member 70 −9.67 1
   HLA-DRB5 Major Histocompatibility Complex, Class II, DR Beta 5 9.81 4
   OR10H2 Olfactory Receptor Family 10 Subfamily H Member 2 −9.89 1
   PTGDS Prostaglandin D2 Synthase 23.73 1 OR2L3 Olfactory Receptor Family
   2 Subfamily L Member 3 −12.53 1
   ATP6V1C2 ATPase H+ Transporting V1 Subunit C2 −12.56 2
   OR5K2 Olfactory Receptor Family 5 Subfamily K Member 2 −16.55 1
   [67]Open in a new tab

   #, number.

3.4. PIK3R2 Expression in tRCC Cell Lines

   Reverse transcription-qPCR and Western blotting were performed to
   detect the expression of PIK3R2 in tRCC cell lines (S-TFE, UOK120, and
   UOK146) ([68]Figure 3). PIK3R2 expression levels were significantly
   higher in UOK120 and UOK146 than in S-TFE (p < 0.05, [69]Figure 3).

Figure 3.

   [70]Figure 3
   [71]Open in a new tab

   Expression of PIK3R2 in Xp11.2 translocation renal cell carcinoma
   (tRCC) cells. (A) Expression of PIK3R2 mRNA in tRCC cells detected by
   quantitative real-time polymerase chain reaction. (B) Relative PIK3R2
   protein expression. Western blotting was used to detect the expression
   of PIK3R2 protein in tRCC cells. * p < 0.01.

3.5. Effects of Various Treatments on tRCC Cell Proliferation

   After treatment with LY294002, sunitinib, sorafenib, and everolimus in
   Xp11.2 tRCC cell lines (S-TFE, UOK120, and UOK146), the viability of
   each cell line was measured every 24 h until 72 h post-treatment.

   After treatment with LY294002, cell viability was lower for UOK120 and
   UOK146 than S-TFE (p < 0.01) ([72]Figure 4A). Sunitinib also decreased
   the proliferation rates more substantially for UOK120 and UOK146 than
   for S-TFE (p < 0.05) ([73]Figure 4B); however, the effect was not as
   highly significant as that of LY294002. In contrast, sorafenib resulted
   in a greater reduction in the rate of proliferation of S-TFE than
   UOK120 (p < 0.05) or UOK146 (p < 0.01) ([74]Figure 4C). After treatment
   with everolimus, viability was significantly lower for UOK120 and
   UOK146 than for S-TFE (p < 0.05) ([75]Figure 4D).

Figure 4.

   [76]Figure 4
   [77]Open in a new tab

   Therapeutic efficacy of LY294002, sunitinib, sorafenib, and everolimus
   in Xp11.2 translocation renal cell carcinoma (tRCC) cells.
   Proliferation curves for Xp11.2 tRCC cells treated with (A) LY294002,
   (B) sunitinib, (C) sorafenib, or (D) everolimus for 24, 48, and 72 h.
   (E) UOK146, (F) UOK120, and (G) S-TFE cell viability after LY294002,
   sunitinib, sorafenib, and everolimus treatment for 48 h. * p < 0.01, **
   p < 0.05.

   As shown in [78]Figure 4E, UOK146 cell viability was significantly
   lower following LY294002 treatment than following sunitinib, sorafenib,
   or everolimus treatment (p < 0.01). In terms of UOK120 cell viability,
   LY294002 treatment was more effective than sunitinib treatment (p <
   0.01); sorafenib and everolimus also resulted in significantly lower
   cell proliferation rates than those observed after sunitinib treatment
   (p < 0.05) ([79]Figure 4F). The proliferation of S-TFE was more
   effectively reduced by sorafenib than by other treatments (p < 0.01),
   and sunitinib was the second most effective treatment ([80]Figure 4G).

3.6. Effects of LY294002 on the Migration and Invasion Ability of tRCC Cell
Lines

   After LY294002 treatment, the migration ability and number of invasive
   cells of UOK120 and UOK146 were significantly lower than those of S-TFE
   (p < 0.01) ([81]Figure 5).

Figure 5.

   [82]Figure 5
   [83]Open in a new tab

   Effects of LY294002 (a PI3K pathway inhibitor) on aggressive Xp11.2
   translocation renal cell carcinoma (tRCC) cells. (A) The cell scratch
   test was used to detect the effect of PI3K inhibitor treatment on the
   migration of Xp11.2 tRCC cells. The migration ability of UOK120 and
   UOK146 was significantly inhibited by treatment with 10 µM LY294002
   (×10). (B) An invasion assay was used to detect the effect of PI3K
   inhibitor treatment on the invasion of Xp11.2 tRCC cells. The invasive
   ability of UOK120 and UOK146 was significantly inhibited by treatment
   with 10 µM LY294002 (×10). * p < 0.01.

4. Discussion

   The therapeutic biomarker PIK3R2 was identified for aggressive Xp11.2
   tRCC, and the efficacy of a novel therapy (i.e., the PI3K pathway
   inhibitor LY294002) was evaluated according to this biomarker. This
   study is the first to suggest a targeted agent for Xp11.2 tRCC
   treatment based on a therapeutic biomarker determined by scientific
   rationale, with an understanding of the molecular pathophysiology and
   verification in several tRCC cell lines.

   Owing to the low prevalence of Xp11.2 tRCC, treatment guidelines are
   not available [[84]14]. Xp11.2 tRCC is mainly diagnosed in children,
   and its diagnosis is rarely reported in the adult population
   [[85]7,[86]9,[87]15,[88]16]. Although its prevalence in adults is low,
   the presentation is more advanced and aggressive
   [[89]17,[90]18,[91]19]. The optimal treatment option for aggressive
   Xp11.2 tRCC remains undetermined [[92]7]. Accordingly, guidelines for
   systemic therapy in Xp11.2 tRCC are urgently needed. Although several
   targeted agents have been approved for the treatment of advanced RCC
   and are used in all RCC subtypes, clinical trials, for the most part,
   have focused on the most common subtype, ccRCC [[93]20]. Furthermore,
   no studies have evaluated the therapeutic efficacies of current
   systemic therapies in Xp11.2 tRCC cell lines. This is the first study
   to validate the therapeutic efficacy of currently available targeted
   agents in Xp11.2 tRCC cell lines.

   No clinical trials have suggested the use of targeted agents in Xp11.2
   tRCC, and most are based on observational studies, mainly using
   sunitinib [[94]1]. Choueiri et al. have reported the effect of
   anti-vascular endothelial growth factor (VEGF) therapies (sunitinib,
   sorafenib, and monoclonal anti-VEGF antibodies) in 15 patients with
   metastatic Xp11.2 tRCC, with objective response rates of 66.7%
   [[95]20]. Additionally, Solano et al. reported a case demonstrating the
   therapeutic efficacy of pazopanib, a similar anti-VEGF agent [[96]1].
   Alternatively, some studies have suggested that the mammalian target of
   rapamycin (mTOR) inhibitors is a treatment option for Xp11.2 tRCC
   [[97]21]. Malouf et al. reported an objective response rate of 33.0%
   for an mTOR inhibitor in patients with metastatic Xp11.2 tRCC [[98]22].
   Additionally, the therapeutic benefits of mTOR inhibitors have been
   demonstrated in a patient with Xp11.2 tRCC who did not show a favorable
   response to first-line VEGF inhibitor treatment [[99]21]. Based on the
   current literature, targeted molecular therapies, including VEGF
   receptor inhibitors and mTOR inhibitors, have therapeutic advantages
   over immunotherapy in Xp11.2 tRCC [[100]23]. We evaluated and compared
   the therapeutic efficacy of sunitinib, sorafenib, and everolimus, which
   are known to be effective in treatments of Xp11.2 tRCC in addition to
   LY294002, the PI3K pathway inhibitor, which targets the overexpression
   of PIK3R2 in aggressive Xp11.2 tRCC patients.

   In the present study, sunitinib and sorafenib, which are VEGFR
   inhibitors, demonstrated a significantly better efficacy in the S-TFE
   cell line, which has the ASPL-TFE3 gene fusion, than in the UOK120 and
   UOK146 cell lines, which have the PRCC-TFE3 gene fusion. In contrast,
   LY294002 and mTOR inhibitors demonstrated better therapeutic efficacy
   in the UOK120 and UOK146 cell lines than in the S-TFE cell line. In
   addition to the difference in fusion genes, UOK120 and UOK146 cell
   lines have elevated levels of PIK3R2, which is the potential target of
   LY294002, unlike the S-TFE cell line. Therefore, LY294002 has the
   greatest therapeutic benefit in UOK120 and UOK146 cell lines.
   Additionally, PI3K/AKT/mTOR signaling pathway dysregulation is
   frequently identified in patients with RCC, and mTOR inhibitors,
   including everolimus and temsirolimus, are effective in metastatic
   ccRCC [[101]24]. mTOR inhibitors also have therapeutic effects in adult
   patients with Xp11.2 tRCC with aggressive courses [[102]21].

   Although the mechanism underlying the effects of VEGF-targeted
   therapies in Xp11.2 tRCC is largely unknown, a few studies have
   provided insight into the mechanism underlying the effects of mTOR
   inhibitors [[103]25]. In particular, phosphorylated 4EBP1 acts as a
   critical factor for integrating upstream oncogenic signals in the
   PI3K/AKT/mTOR signaling pathway and drives proliferative downstream
   signaling [[104]25]. Our results also revealed a significant
   association of the PI3K/AKT/mTOR signaling pathway with aggressive
   Xp11.2 tRCC. Dysregulation of the PI3K/AKT/mTOR signaling pathway,
   which contributes to various biological processes, such as apoptosis,
   metabolism, cell proliferation, and cell growth, frequently occurs in
   cancer [[105]26]. In RCC, the PI3K/AKT/mTOR signaling pathway serves an
   important role in tumorigenesis [[106]24]. Song et al. reported that a
   decrease in the expression of the target gene PIK3R2 influences the
   PI3K/AKT/mTOR signaling pathway, thereby suppressing the proliferation,
   migration, and invasive abilities of a human lung cancer cell line
   [[107]26]. PIK3R2, which encodes the p85ß regulatory subunit of PI3K
   (phosphatidylinositol-3-kinase), is frequently overexpressed in
   melanoma, breast, and squamous cell lung carcinoma, and its expression
   level increases during tumor progression [[108]27]. The role of PIK3R2
   as an oncogene has not been investigated in RCC, especially in tRCC.
   Our results provide the first evidence for the overexpression of PIK3R2
   in aggressive Xp11.2 tRCC and show that suppressing PIK3R2 by LY294002
   in Xp11.2 tRCC cell lines eventually leads to a decrease in
   proliferation, migration, and invasion abilities, similar to
   observations in lung cancer cell lines. Additionally, everolimus
   demonstrated a better therapeutic efficacy than sunitinib in the UOK120
   and UOK146 cell lines in the present study. This can be explained by
   the overexpression of PIK3R2 in these cell lines, since the activation
   of PIK3R2 increases PI3K/AKT/mTOR signaling pathway activity, leading
   to the overexpression of mTOR.

   In exploring the association between the fusion type and oncological
   outcomes, Komai et al. demonstrated different biological behaviors
   among different fusion types of Xp11.2 tRCC [[109]7]. In this previous
   study, four patients, including two with the ASPL-TFE3 gene fusion, had
   an unfavorable prognosis, and one patient with the PRCC-TFE3 gene
   fusion with nodal involvement remained disease free for 3 years after
   lymph node dissection [[110]7]. The two patients with the ASPL-TFE3
   gene fusion both developed visceral metastases, and one patient died of
   the disease [[111]7]. The ASPL-TFE3 fusion seems to have a worse
   prognosis, with frequent lymph node metastasis; however, it is not
   clear whether the fusion partner has prognostic value. In the present
   study, although not all fusion types were identified, associations
   between those that were identified and the prognosis were consistent
   with previous results. For patients with aggressive tRCC, the ASPL-TFE3
   gene fusion and RBM10-TFE3 gene fusion were found. Along with
   ASPL-TFE3, the RBM10-TFE3 gene fusion is also associated with an
   aggressive course. Kato et al. reported that among 10 patients, two
   patients died, and three patients developed recurrence [[112]28]. We
   detected SFPQ-TFE3 gene fusion in three patients with non-aggressive
   tRCC. Argani et al. reported seven cases of SFPQ-TFE3 gene fusion
   Xp11.2 tRCC, where all cases were confined to the kidney, and only one
   case showed recurrence [[113]29]. The SFPQ-TFE3 gene fusion seems to
   have a more favorable prognosis than the ASPL-TFE3 gene fusion
   [[114]29], which is consistent with our findings.

   In terms of the PRCC-TFE3 gene fusion, oncological outcomes are
   controversial. Although Komai et al. reported a favorable prognosis for
   the PRCC-TFE3 gene fusion and poor oncological outcomes for the
   ASPL-TFE3 gene fusion [[115]7], Argani et al. reported two cases of the
   PRCC-TFE3 gene fusion with metastatic disease [[116]29]. In our study,
   although the PRCC-TFE3 gene fusion was not identified in patient
   samples, aggressiveness was associated with the PRCC-TFE3 gene fusion.
   The S-TFE cell line had the ASPL-TFE3 gene fusion, while the UOK120 and
   UOK146 cell lines had the PRCC-TFE3 gene fusion and showed PIK3R2
   overexpression, which was associated with the aggressiveness of Xp11.2
   tRCC. Therefore, the gene fusion types show a difference in the
   expression of PIK3R2. However, a direct comparison between studies is
   difficult for a few reasons. First, among four cases in a study by
   Komai et al., one patient was 15 years old, while other studies
   excluded patients younger than 18 years [[117]30]. Many studies have
   reported a difference in oncological outcomes between adults and
   children [[118]30]; thus, the inclusion of adults and children in one
   study would cause a selection bias. Second, Komai et al. reported a
   tumor more than 7 cm in size [[119]7]; in contrast, only cases with
   tumor sizes <7 cm were included in the present study. Previous studies
   of the tumor size in Xp11.2 tRCC have revealed that a larger tumor size
   (p < 0.01) is associated with aggressiveness [[120]30]. However, in the
   present study, the tumor size did not significantly differ between the
   aggressive and non-aggressive Xp11.2 tRCC groups. This might be because
   the study population was limited to those with clinical stage T1.
   Third, previous studies have included cases with nodal metastasis at
   the time of initial diagnosis, including Komai et al. [[121]7]. Nodal
   disease without distant metastasis has a favorable short-term prognosis
   after surgery alone in patients with Xp11.2 tRCC [[122]7,[123]16]. The
   present study included patients who developed nodal metastasis after
   surgery, for whom the initial presentation was at the clinical stage
   T1N0M0. Although it was not within the scope of the present study,
   future studies are needed to evaluate the correlation between fusion
   type and biological behavior in patients with Xp11.2 tRCC, including
   clinical T1N0M0 Xp11.2 tRCC.

   The natural history of Xp11.2 tRCC is poorly understood; however,
   growing evidence indicates that adult Xp11.2 tRCC is clinically
   aggressive and often presents at an advanced stage, with poor outcomes
   [[124]31]. Argani et al. reported that 50% of 28 adult Xp11.2 tRCC
   cases presented with stage 4 disease [[125]9]. Qu et al. reported that
   26.7% of patients had recurrence and 30.0% of patients died of cancer
   in a study of 30 adult patients with Xp11.2 tRCC [[126]31]. Kuthi et
   al. reported that 33% of patients died from cancer-related causes in a
   cohort of 28 patients; however, three children were included in this
   cohort [[127]32]. Similarly, there were three aggressive cases (27.3%;
   including patients with recurrence or cancer-related death) among the
   11 cases in the present study. These data indicate that Xp11.2 tRCC has
   the same recurrence and mortality rates as ccRCC [[128]33]. Although
   the clinical behavior of Xp11.2 tRCC has been reported to be more
   aggressive in adults than in children [[129]31], this might be due to
   relative indolent course of Xp11.2 tRCC in children.

   Xp11.2 tRCCs are typically considered as a malignancy primarily found
   in pediatric patients, with very low incidence in adult populations
   [[130]7,[131]34]. However, being recognized as a distinct entity, the
   prevalence of Xp11.2 tRCC in adult populations is increasing, and its
   behavior is considered aggressive compared to pediatric onset Xp11.2
   tRCC, which has been reported as a benign clinical course [[132]34].
   Therefore, a treatment strategy is urgently needed. Based on the
   molecular understanding of pathophysiology of aggressive Xp11.2 tRCC,
   we have suggested novel therapy for advanced Xp11.2 tRCC, for which
   systemic treatment guidelines are currently unavailable. We believe
   that our study will be the cornerstone of the research on novel
   therapeutic approaches for Xp11.2 tRCC.

   The major strength of the present study was the inclusion of a
   relatively large sample size, with long-term follow-up periods.
   However, there were some limitations as well. First, there is the
   possibility of selection bias. This study only included Korean patients
   treated at a single institution. However, the results were supported by
   cancer cell lines developed in the USA and Japan. Second, the RNA-Seq
   technique has poor sensitivity in detecting fusion genes; thus, the
   fusion partner genes could not be fully explored [[133]35]. The
   identification of fusion partner genes associated with PIK3R2
   expression should be a focus of future research. Third, international
   guidelines now suggest the use of immune-based combination treatments
   as the first-line systemic treatment for metastatic disease, based on
   the results of a series of randomized phase III trials
   [[134]36,[135]37]. However, due to the difficulty of developing
   appropriate humanized mouse models that mimic the human immune system
   and inferior therapeutic benefit of immunotherapy over VEGF receptor
   inhibitors and mTOR inhibitors in Xp11.2 tRCC patients, we have not
   evaluated the therapeutic efficacy of immune-based combination
   treatments. However, since immunotherapy is emerging as the main
   treatment for metastatic RCC, future studies will incorporate
   immunotherapy by using humanized mouse models.

5. Conclusions

   This study is the first to report a therapeutic biomarker for
   aggressive Xp11.2 tRCC in clinically localized T1 stage, where the
   prognosis is generally good. PIK3R2, which is highly expressed in
   patients with aggressive Xp11.2 tRCC, is a biomarker for the use of a
   novel therapy, LY294002. More data are needed to determine the best
   treatment methods and prognostic markers for patients with Xp11.2 tRCC,
   including international multicenter studies with large sample sizes.

Acknowledgments