Abstract

   Cancer‐testis (CT) genes are a group of genes restrictedly expressed in
   testis and multiple cancers and can serve as candidate driver genes
   participating in the development of cancers. Our previous study
   identified a number of CT genes in nongerm cell tumors, but their
   expression pattern in testicular germ cell tumor (TGCT), a cancer type
   characterized by less genomic alterations, remained largely unknown. In
   this study, we systematically investigated the expression pattern of CT
   genes in TGCT samples and evaluated the transcriptome difference
   between TGCT and normal testis tissues, using datasets from the UCSC
   Xena platform, The Cancer Genome Atlas (TCGA) and the Genotype‐Tissue
   Expression (GTEx) project. Pathway enrichment analysis and survival
   analysis were conducted to evaluate the biological function and
   prognostic effect of expressed CT genes. We identified that 1036
   testis‐specific expressed protein‐coding genes and 863 testis‐specific
   expressed long noncoding RNAs (lncRNAs) were expressed in TGCT samples,
   including 883 CT protein‐coding genes and 710 CT lncRNAs defined
   previously. The number of expressed CT genes was significantly higher
   in seminomas (P = 3.48 × 10^−13) which were characterized by frequent
   mutations in driver genes (KIT, KRAS and NRAS). In contrast, the number
   of expressed CT genes showed a moderate negative correlation with the
   fraction of copy number altered genomes (cor = −0.28,
   P = 1.20 × 10^−3). Unlike other cancers, our analysis revealed that
   96.16% of the CT genes were down‐regulated in TGCT samples, while CT
   genes in stem cell maintenance related pathways were up‐regulated.
   Further survival analysis provided evidence that CT genes could also
   predict the prognosis of TGCT patients with both disease‐free interval
   and progression‐free interval as clinical endpoints. Taken together,
   our study provided a global view of CT genes in TGCT and provided
   evidence that CT genes played important roles in the progression and
   maintenance of TGCT.

   Keywords: cancer‐testis gene, stem cell maintenance, survival,
   testicular germ cell tumor

1. INTRODUCTION

   Cancer‐testis (CT) genes are predominantly expressed in normal testis
   tissues and a wide type of cancers.[54]1 They were first named as
   Cancer‐testis Antigens (CTAs) since early defined CT genes can encode
   immunogenic antigens which evoke immune responses in cancer
   patients.[55]2, [56]3 Because of the immunogenicity and restricted
   expression pattern in normal tissues, CTAs are commonly considered as
   ideal immunotherapy targets for cancer therapy.[57]3, [58]4, [59]5
   Recently, CT genes have also been proved that contribute to various
   neoplastic phenotype features,[60]3 and can be driver candidates
   participating in the process of tumorigenesis.[61]6 In our recent work,
   we performed a systematic identification of CT genes by integrating
   gene expression data of 24 normal tissues and tumor samples from 19
   cancer types and defined 876 new CT genes.[62]6 However, all previous
   studies investigated CT genes in nongerm cell tumors, leaving a blank
   for testicular germ cell tumor (TGCT).

   Testicular cancer is the most commonly diagnosed malignancy of adult
   males among Americans,[63]7 while TGCT accounts for more than 95% of
   testicular cancers.[64]8 The main types of TGCT are seminomas and
   non‐seminomas, and non‐seminomas consist of either undifferentiated
   (embryonal carcinoma) or differentiated (teratoma, yolk sac tumor, and
   choriocarcinoma) histologic subtypes.[65]9 In a recent study, Shen et
   al identified that TGCTs were characterized by a lack of genomic
   alterations, deficiency of methylation level and high aneuploidy,[66]10
   which highlighted the importance of transcriptome alteration in the
   development of TGCT. Considering the special expression pattern, the
   transcriptome alteration of CT genes may participate in the development
   of TGCT. Interestingly, some CT genes have previously been reported to
   be expressed in TGCT samples and have a potential function in
   tumorigenesis, such as CTAG1B [67]11 and the MAGE families MAGEA1,
   MAGEA2, MAGEA3, and MAGEA4.[68]12, [69]13, [70]14, [71]15 Although
   attracting more attention, CT genes in the process of TGCT development
   have not been well characterized.

   In this study, to make a comprehensive description of CT genes in TGCT,
   we first described the expression pattern of CT protein‐coding genes
   and CT long noncoding RNAs (CT‐lncRNAs) in TGCT samples. Then we
   conducted differential expression analysis between normal and malignant
   testis samples to provide a global view of the expression alterations
   of CT genes between these samples. Additionally, we performed pathway
   enrichment analysis to investigate the function of these altered CT
   genes, as well as survival analysis to evaluate the prognostic effect
   of CT genes. Overall, our study provided evidence that CT genes may
   play important roles in the progression and maintenance of TGCT.

2. METHODS

2.1. Quantification expression data of testicular germ cell cancer samples
used in this study

   To comprehensively explore the expression pattern of our previous
   defined testis‐specific expressed genes (TSGs)[72]6 in TGCT, we
   retrieved quantification expression data of 150 TGCT samples from the
   University of California Santa Cruz (UCSC) Xena website
   ([73]https://xenabrowser.net/datapages/).[74]16 The expression data
   were quantified as raw read counts. In our previous study,[75]6 we
   analyzed the transcriptome data of normal tissues (24 different organs
   from 175 individuals) from the Genotype‐Tissue Expression (GTEx)
   project ([76]https://www.gtexportal.org/home/) and used specificity
   measure to classify all human genes (50 016) into six categories
   (C1‐C6) based on their expression pattern, including high confidence
   testis‐specific expressed protein coding genes (C1) and noncoding genes
   (C2), moderate confidence testis‐specific expressed protein‐coding
   genes (C3) and noncoding genes (C4), low‐confidence testis‐specific
   expressed genes (C5), genes with transcripts exhibiting a
   testis‐specific expression pattern (C6a), and genes without transcripts
   exhibiting a testis‐specific expression pattern (C6b). In this study,
   only 1061 protein‐coding genes and 1184 long noncoding RNAs (lncRNAs)
   exhibited testis‐specific expression pattern with high confidence (C1
   and C2 groups) as well as a median expression greater than 0.5
   fragments per kilobase of exon model per million reads mapped (FPKM) in
   normal testis samples were included.[77]6 According to GENCODE v19
   annotation data, lncRNAs were reclassified into 6 biotypes,[78]17
   including three prime overlapping ncRNA (lncRNAs located within the 3'
   UTR of protein‐coding genes), antisense (lncRNAs overlapping any
   protein‐coding genes on the opposite strand), lincRNA (long intergenic
   noncoding RNA with a length greater than 200 bp), sense intronic
   (lncRNAs located within the intron of any protein‐coding gene), sense
   overlapping (lncRNAs within any protein‐coding gene within its intron
   on the coding strand), and processed transcript (transcripts without an
   open reading frame). Testis‐specific expressed genes exhibited
   expression (normalized read counts >5) in at least 1% TGCT samples were
   defined as expressed.

2.2. Differential gene expression analysis

   To further explore the expression difference of TSGs in malignant
   (TGCT) and normal testis samples, we performed differential expression
   analysis by using data from The Cancer Genome Atlas (TCGA) project
   ([79]https://xenabrowser.net/datapages/) and the GTEx portal.
   Expression data of 174 normal testes were obtained from GTEx Analysis
   V7 (dbGap Accession phs000424.v7.p2,
   [80]https://www.gtexportal.org/home/datasets) and quantified as raw
   read counts. Differential expression analysis was performed with R
   package DESeq2. The batch effect was removed by sva package and raw
   read counts data was normalized by DESeq2. The magnitude (log2
   transformed fold change) and significance (P‐value) of differential
   expression between groups were calculated, and genes with
   |log2foldchange|≥1 and Benjamini–Hochberg false discovery rate (FDR)
   adjusted P < 0.05 were considered as significant.

2.3. Pathway enrichment analysis

   To investigate the potential function of altered TSGs in TGCT samples,
   we performed pathway enrichment analysis based on GO Biological Process
   Ontology gene sets with the R Bioconductor package clusterProfiler. As
   most lncRNAs were not included in the pathway annotation data, only
   protein‐coding genes were included in this analysis. TSGs with
   significantly altered expression in TGCT samples from that in normal
   testis tissues were included in this analysis (|log2foldchange|≥1 and P
   [fdr] < 0.05). Pathway with enrichment P < 0.01 was considered as
   significant.

2.4. Survival analysis

   The clinical and follow‐up information of 134 TGCT samples were
   obtained from a previous TCGA study[81]18 (Table [82]S1). As for the
   few disease‐specific survival and overall deaths of TGCT patients, we
   used disease‐free interval (DFI) and progression‐free interval (PFI) as
   clinical outcome endpoints.[83]18 We performed a multivariate Cox
   proportional hazards regression model to calculate the Hazard Ratio
   (HR), the 95% confidence interval (95% CI), and P‐values with
   adjustments for age and tumor stage. The Kaplan‐Meier (K‐M) method was
   used to create the survival plots and the log‐rank test was used to
   compare the difference in survival curves.

2.5. Statistical analysis

   Wilcoxon rank‐sum test was performed to compare the number of expressed
   CT genes in different groups. Spearman rank‐sum test was used to
   evaluate the association between the expression level of CT genes and
   the stemness scores, the fraction of altered genomes, and immune cells
   level in 133 samples. The stemness scores, the fraction of altered
   genomes and immune cells information were obtained from a previous
   study.[84]19 General statistical analyses were performed with R
   software (R version 3.2.2).

3. RESULTS

3.1. Characterization of cancer‐testis genes in testicular germ cell tumor

   We first investigated the expression pattern of TSGs in 150 TGCT
   samples from the TCGA project. In total, 97.64% (1036/1061) of our
   previously defined testis‐specific expressed protein‐coding genes (C1
   group) and 72.89% (863/1184) testis‐specific expressed noncoding RNAs
   (C2 group) were expressed (normalized read counts >5) in at least 1%
   TGCT samples (Figure [85]1A, Table [86]S2), including 883 CT genes and
   710 CT‐lncRNAs defined in other 19 cancer types[87]6, [88]20 (Table
   [89]S2).

Figure 1.

   Figure 1
   [90]Open in a new tab

   Classification of CT genes and the association with patient
   characteristics. A, The number of expressed testis‐specific expressed
   genes and CT genes. B, The number of expressed CT genes in 2 histologic
   subtypes. C, Association of the number of expressed CT genes and 3
   significantly mutated genes of TGCT. D, Association of the number of
   expressed CT genes and fraction of copy number altered genomes in TGCT
   samples. E, Association of the number of expressed CT genes and
   expression level of immune cells in TGCT samples

   In an attempt to explore the relationship between CT genes and clinical
   characteristics in TGCT samples, we performed subgroup analysis and
   identified that the number of expressed CT genes was significantly
   higher in the seminoma histologic subtype than other non‐seminoma
   subtypes (Median[seminomas] = 867.5, Median[non‐seminomas] = 764;
   P = 3.48 × 10^−13) (Figure [91]1B). Further differential expression
   analysis between 68 seminomas and 65 nonseminomas revealed that
   366 TSGs showed elevated expression in the seminomas and 165 were
   decreased. Interestingly, although spermatogenesis‐related genes shared
   a similar expression level in both two groups of patients, meiotic cell
   cycle‐related genes were specifically elevated in the seminoma type
   (Table [92]S3‐S4). Additionally, we identified that CT genes were more
   prone to be expressed in samples with mutations in the only 3
   significantly mutated genes identified previously,[93]10 including KIT
   (Median[Mut‐type(MT)] = 871, Median[Wide‐type(WT)] = 804,
   P = 9.18 × 10^−4), KRAS (Median[MT] = 867, Median[WT] = 806,
   P = 1.49 × 10^−2), and NRAS (Median[MT] = 876, Median[WT] = 820,
   P = 3.99 × 10^−2) (Figure [94]1C). However, the number of expressed CT
   genes showed a negative moderate correlation with the fraction of copy
   number altered genomes (cor = −0.28, P = 1.20 × 10^−3) (Figure [95]1D).

   As CT genes were commonly considered as immunotherapy targets, we
   further evaluated the association of expressed CT genes with the
   estimated expression of 6 types of immune cells (B cells, CD4 T cells,
   CD8 T cells, neutrophils, macrophages and dendritic cells) in 133 TGCT
   samples. Interestingly, we identified moderate associations between the
   number of expressed CT genes and the expression level of 5 types of
   immune cells, including B cells (cor = 0.43 P = 1.98 × 10^−7), CD4 T
   cells (cor = 0.33, P = 9.58 × 10^−5), CD8 T cells (cor = 0.28,
   P = 1.29 × 10^−3), neutrophils (cor = 0.20, P = 1.80 × 10^−2), and
   dendritic cells (cor = 0.24, P = 5.07 × 10^−3) (Figure [96]1E).
   Additionally, further co‐expression analysis revealed that the number
   of expressed CT genes were also significantly associated with
   PD‐1/PD‐L1 gene expression (PD‐1: cor = 0.47, P = 1.10 × 10^−8; PD‐L1:
   cor = 0.23, P = 8.58 × 10^−3; Figure [97]S1).

3.2. A comprehensive comparison of cancer‐testis genes in malignant and
normal testis samples

   As TSGs were highly expressed in both malignant (TGCT) and normal
   testis samples, we then evaluated the differential expression of TSGs
   in 174 normal testis samples and 150 TGCT samples. Interestingly, we
   identified that 96.16% (1826/1899) of the TSGs genes were
   differentially expressed between malignant and normal testis samples
   (Figure [98]2A, Table [99]S5) while 53.48% (6637/12411) of the genes
   without testis‐specific expression pattern were differentially
   expressed (Figure [100]2B). Additionally, as the majority of the
   differential expressed TSGs (98.69%, 1802/1826) were down‐regulated in
   TGCT patients (Figure [101]2A), only 54.26% (3601/6637) of the
   differential expressed genes without testis‐specific expression pattern
   were down‐regulated in TGCT (Figure [102]2B).

Figure 2.

   Figure 2
   [103]Open in a new tab

   A, Differential expression analysis of testis‐specific expressed genes.
   Volcano plot displayed differentially expressed TSGs in TGCT and normal
   testis samples. B, Differential expression analysis of genes without
   testis‐specific expression pattern. Volcano plot displayed differential
   expressed genes without testis‐specific expression pattern in TGCT and
   normal testis samples. C, Differential expression analysis of
   testis‐specific expressed genes. Differential expression pattern of
   DPPA2 and IGF2BP3 in TGCT and normal testis samples

   Of the 1802 down‐regulated TSGs in TGCT samples, 951 were
   protein‐coding genes, and 851 were lncRNAs, including 125 classic CT
   genes, such as the MAGE family (MAGEA1: log2FC = −4.04,
   P = 1.65 × 10^−115; MAGEA3: log2FC = −3.18, P = 3.75 × 10^−55; MAGEA4:
   log2FC = −1.98, P = 1.64 × 10^−18; MAGEA8: log2FC = −1.74,
   P = 1.30 × 10^−39; MAGEA10: log2FC = −2.56, P = 4.87 × 10^−55; MAGEA11:
   log2FC = −3.63, P = 4.28 × 10^−193 and MAGEA12: log2FC = −2.24,
   P = 3.34 × 10^−25) and HORMAD family (HORMAD1: log2FC = −3.45,
   P = 6.84 × 10^−185 and HORMAD2: log2FC = −7.43, P < 2.20 × 10^−16). The
   24 up‐regulated TSGs including 22 protein‐coding genes and 2 lncRNAs,
   of which 2 were classic CT genes (DPPA2: log2FC = 2.35,
   P = 1.71 × 10^−112 and IGF2BP3: log2FC = 1.57, P = 8.58 × 10^−107)
   (Figure [104]2C, Table [105]S5).

3.3. Stem cell maintenance genes are up‐regulated in testicular germ cell
cancer

   To evaluate the potential function of differential expressed
   testis‐specific protein‐coding genes, we performed pathway enrichment
   analysis with GO Biological Process Ontology dataset. The result
   revealed that the down‐regulated TSGs in TGCT were significantly
   enriched in 2 pathways, including spermatogenesis (P = 1.90 × 10^−4)
   and male gamete generation (P = 1.90 × 10^−4) pathways (Figure [106]3A,
   Table [107]S6); while the up‐regulated testis‐specific protein‐coding
   genes in TGCT samples were involved in translational regulation and
   stem cell maintenance related pathways, such as negative regulation of
   translation (P = 9.04 × 10^−8), regulation of translation
   (P = 1.07 × 10^−5), stem cell population maintenance
   (P = 9.11 × 10^−6), somatic stem cell population maintenance
   (P = 8.88 × 10^−4), and stem cell proliferation (P = 8.88 × 10^−4)
   (Figure [108]3A, Table [109]S7). Interestingly, although the number of
   expressed CT genes was significantly higher in the seminoma group with
   KIT, KRAS and NRAS mutations, expression of 4 genes in stem cell
   maintenance related pathways were significantly elevated in the
   nonseminoma group and samples that without driver gene mutations
   (Figure [110]S2).

Figure 3.

   Figure 3
   [111]Open in a new tab

   Pathway enrichment analysis of differentially expressed testis‐specific
   expressed genes. A, Dotplot depicts the enriched pathways of
   differentially expressed testis‐specific expressed protein‐coding genes
   in TGCT samples compared with normal testis samples. B, Heatmap depicts
   the correlation of testis‐specific expressed protein‐coding genes in
   stem cell maintenance related pathways with the stemness signatures

   As 6 of 9 stem cell maintenance related pathways included in GO
   Biological Process Ontology dataset were over‐represented in
   testis‐specific expressed protein‐coding genes, we further evaluated
   the association of 22 up‐regulated testis‐specific expressed
   protein‐coding genes with the stemness scores. Interestingly, 16 of the
   22 up‐regulated testis‐specific expressed protein‐coding genes showed
   strong positive associations with both epigenetic‐related and
   expression‐related stemness signatures (Cor ≥ 0.2, P [adj] < 0.05)
   (Figure [112]3B, Table [113]S8).

3.4. Survival outcome analysis

   To evaluate the prognostic effect of TSGs in TGCT patients, we
   performed survival analysis with DFI and PFI as the clinical endpoints
   as recommended in the previous study.[114]10 In this analysis, we used
   30 days past diagnosis as a duration of the primary treatment interval.
   The baseline and follow‐up information of the patients were shown in
   Table [115]S1. The median DFI event time was 27.03 months, and the
   median PFI event time was 28.18 months. Wilcoxon rank‐sum analysis
   revealed that four TSGs exhibited significantly higher expression in
   high‐stage (III, IV) TGCT patients, while 59 TSGs showed higher
   expression level in low‐stage (I, II) TGCT patients (P < 0.01). Of the
   59 TSGs that showed negative correlations with TGCT progression, 45
   were protein‐coding genes, including 6 classic CT genes (PIWIL2:
   median[high] = 619.45, median[low] = 1425.66, P = 7.79 × 10^−3; CCDC36:
   median[high] = 28.89, median[low] = 59.83, P = 8.37 × 10^−3; TFDP3:
   median[high] = 1.26, median[low] = 22.92, P = 4.24 × 10^−3; FBXO39:
   median[high] = 7.26, median[low] = 19.30, P = 2.64 × 10^−3; CXorf48:
   median[high] = 2.14, median[low] = 5.07, P = 7.96 × 10^−3 and HEMGN:
   median[high] = 0.00, median[low] = 2.13, P = 1.06 × 10^−3).
   Interestingly, 2 of the stem cell population maintenance related genes
   showed a positive correlation with the progression of TGCT samples,
   including LIN28A (median[high] = 18045.24, median[low] = 12731.15,
   P = 0.03) and IGF2BP1 (median[high] = 17224.03, median[low] = 12036.74,
   P = 0.03) (Figure [116]4A, Table [117]S9).

Figure 4.

   Figure 4
   [118]Open in a new tab

   Survival analysis of testis‐specific expressed genes. A, Association of
   testis‐specific expressed genes with clinical tumor stages. B‐C,
   Kaplan‐Meier plot depicts the association of testis‐specific expressed
   genes with TGCT prognosis statuses

   With the multivariate Cox proportional hazards regression model, we
   identified a total of 48 TSGs that were associated with DFI, 38 were
   associated with PFI and 20 were consistently associated with both
   clinical ends (P < 0.01) (Table [119]S10). Of the 20 survival‐related
   TSGs, 2 were previously reported CT genes, including TSGA10 and AKAP4.
   TGCT patients with higher expression of TSGA10 had a significantly
   worse survival than those with low expression when using both PFI
   (HR = 4.05, P = 2.99 × 10^−4) and DFI (HR = 5.66, P = 1.20 × 10^−4) as
   clinical endpoints after controlling for age and tumor stage (Figure
   [120]4B, Table [121]S10). However, an inverse association was observed
   for AKAP4 as higher expression of this gene was associated with a
   better survival of TGCT (PFI: HR = 0.33, P = 3.88 × 10^−3; DFI:
   HR = 0.28, P = 4.70 × 10^−3) (Figure [122]4C, Table [123]S10).
   Interestingly, the stem cell maintenance related gene LIN28A was also
   associated with a better survival of TGCT (PFI: HR = 0.36,
   P = 5.81 × 10^−3; DFI: HR = 0.32, P = 6.64 × 10^−3) (Table [124]S10).

4. DISCUSSION

   CT genes are a class of genes predominantly expressed in the germ cells
   of testis and are ectopically reactivated in various types of tumor
   cells. Accumulating evidence suggested that this group of genes can
   also serve as driver candidates in the development of cancer.[125]3
   However, previous studies focused mainly on nongerm cell tumors
   although some classic CT genes have also been found in testicular germ
   cell tumors.[126]11, [127]12 Interestingly, TGCT is a kind of cancer
   characterized by genomic de‐methylation and a low genomic alteration
   rate,[128]10 which indicates the important role of transcriptome
   alteration during tumorigenesis. Therefore, systematically
   investigation of CT genes in TGCT could unravel the role of this group
   of genes in the development of TGCT. In this study, we performed a
   comprehensive description of CT genes in TGCT and provided the first
   evidence that CT genes play important roles in the progression and
   maintenance of TGCT.

   Of the two most common types of TGCT, although seminomas are less
   aggressive,[129]8 we observed a significant enrichment of expressed CT
   genes in this type of cancer. Interestingly, consistent with our
   previous hypothesis that de‐methylation was an important mechanism for
   the reactivation of CT genes in cancers,[130]6 seminomas are also a
   group of cancers that lack of DNA methylation.[131]10 Thus, the
   globally de‐methylated genomes of seminoma cells could serve as the
   epigenetic mechanism for CT genes expression in TGCT.[132]6 However, in
   contrast to non‐germ cell solid cancer,[133]6 CT genes in TGCT are
   prone to be expressed in samples with driver genes (KIT, KRAS and NRAS)
   mutations, which may be attributed to the altered aneuploidy and
   genomic instability in these samples. Additionally, we also observed a
   positive association between the number of expressed CT genes with
   different types of immune cells. Recently, there is accumulating
   evidence confirming the association between immune biomarkers and the
   response to anti‐PD‐1/L1 therapy across several solid tumors.[134]21,
   [135]22 In this study, we found that PD‐1/PD‐L1 expression was
   positively correlated with the number of expressed CT genes, suggesting
   that CT genes represent a potential marker for effective PD‐1/L1
   immunotherapy.

   During the tumorigenesis of the testis, we identified that the
   expression level of most CT genes in spermatogenesis pathway was
   de‐regulated, while the expression of genes in the maintenance of stem
   cells was up‐regulated. As TGCT is a cancer type derived from immature
   germ cells that retain expression of pluripotency genes,[136]9 further
   correlation analysis provided additional evidence that CT genes
   up‐regulated in TGCT samples showed strong associations with the
   stemness scores. Stemness is defined as the ability of self‐renewal and
   differentiation for normal cells, as well as the shared characteristic
   with cancer cells.[137]23 Recently, increasing evidence suggested that
   the development of cancer cells involves the acquisition of
   stem‐cell‐like phenotypes and loss of differential features, which may
   lead to the progression and distant metastasis of cancer cells.[138]23,
   [139]24, [140]25 Interestingly, we identified that LIN28A and IGF2BP1,
   the essential members in stem cell maintenance, were also associated
   with advanced tumor stages in addition to the oncogenic effect in
   tumorigenesis.[141]26, [142]27 Thus, we proposed that the dysregulation
   of stemness‐related CT genes in TGCT may not only lead to the
   initiation of tumorigenesis and oncogenic de‐differentiation, but also
   contribute to the progression of testicular germ cells.

   Consistent with our hypothesis, further survival analysis revealed that
   22 genes were associated with the prognosis of TGCT, including 2
   previously known CT genes. TSGA10, with the highest expression level in
   elongating spermatids and localized in the fibrous sheath of mature
   sperm, has previously been reported to be a biomarker in brain
   tumors[143]28, [144]29 and regulated the development of esophageal
   squamous cell carcinoma.[145]30 AKAP4, as a kinase anchor protein, has
   been reported to contribute to the development of cervical cancer and
   ovarian serous carcinoma.[146]31, [147]32 In this current study, we
   first provided evidence for the prognostic effect of TSGA10 and AKAP4
   in the progression of TGCT, which may serve as potential treatment
   targets. Additionally, we identified that although some CT genes have
   previously been reported to be oncogenic, they could also predict a
   better survival with either DFI or PFI as the endpoint. LIN28A is a
   highly conserved RNA binding protein and has emerged as an oncogenic
   driver by blockading of let‐7 micro‐RNA biogenesis in many
   cancers[148]33, [149]34, [150]35; however, in this study, we identified
   that LIN28A was associated with a better prognosis of TGCT, which is
   different from the other cancers types.[151]36, [152]37, [153]38
   Nevertheless, there also exists some limitations in this study. As for
   the small sample size used in this study as well as no independent
   validations, further studies are warranted to validate the findings.

   In summary, we identified a total of 1899 testis‐specific expressed
   genes (1036 testis‐specific protein‐coding genes and 863
   testis‐specific long noncoding genes), including 1593 CT genes (883 CT
   genes and 710 CT‐lncRNAs) in TCGA TGCT samples. By integrating
   expression from both normal and malignant testis samples, we proposed
   that CT genes up‐regulated in TGCT were primarily involved in stem cell
   maintenance and could serve as prognostic factors. This founding
   greatly broadens our understanding of CT genes in testicular germ‐cell
   tumors.

CONFLICT OF INTEREST

   The authors declare no conflict of interest.

Supporting information


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ACKNOWLEDGMENTS