The testicular germ cell tumor proteome

Testis cancer constitutes approximately 1% of cancer in males. Tumors of germ cell origin account for approximately 95% of all testis cancers. The prognosis for testis cancer is excellent following proper management. More than 90% are cured with a >95% five-year survival rate. Pathology plays a key role in the management of patients with testicular tumors by allowing for accurate classification of tumors to provide the prognostic parameters needed for optimizing decisions regarding treatment and follow-up.

Testis cancer is divided into two major categories: seminoma and non-seminomatous germ cell tumors. Seminomas account for approximately 45% of all germ cell tumors and are characterized histologically by evenly spaced and relatively large uniform tumor cells with distinct cell borders. Tumor cell nuclei are often centrally localized and show distinct nuclear membranes and one or two distinct nucleoli. The characteristic tumor stroma in seminoma is built up by a delicate fibrovascular network with thin collagenous septa containing variable amounts of small lymphocytes. Of the non-seminomatous tumors, embryonal carcinoma accounts for 15-30% and represents the second most frequent pure type of testicular cancer. Embryonal cancer displays an acinar, tubular, papillary or solid growth pattern with areas of necrosis, hemorrhage and fibrosis. Tumor cells are highly pleomorphic with large, irregular nuclei and indistinct cell borders.

Here, we explore the testicular germ cell tumor proteome using TCGA transcriptomics data and antibody-based protein data. 36 genes are suggested as prognostic based on transcriptomics data from 133 patients; 28 genes are associated with unfavorable prognosis and 8 genes are associated with favorable prognosis.

TCGA data analysis

In this metadata, study we used data from TCGA where transcriptomics data was available from 133 males with seminoma or non-seminomatous germ cell tumors. Most of the patients (129 patients) were still alive at the time of data collection. The stage distribution was i) 55 patients ii) 12 patients iii) 14 patients is) 45 patients and 7 patients with missing stage information.

Unfavorable prognostic genes in testicular germ cell tumor

For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. There are 28 genes associated with an unfavorable prognosis in testicular germ cell tumor. In Table 1, the top 20 most significant genes related to an unfavorable prognosis are listed.

TBXAS1 is a gene associated with unfavorable prognosis in testicular germ cell tumor. The best separation is achieved by an expression cutoff at 8.3 fpkm which divides the patients into two groups with 88 % 5-year survival for patients with high expression versus 100 % for patients with low expression (p-value: 9.05e-4 ). Immunohistochemical using an antibody targeting TBXAS1 (HPA031257) shows a differential expression pattern in testicular germ cell tumor samples.

p<0.001
TBXAS1 - survival analysis
TBXAS1 - high expression
TBXAS1 - low expression

Table 1. The 20 genes with highest significance associated with an unfavorable prognosis in testicular germ cell tumor.

Favorable prognostic genes in testicular germ cell tumor

For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients. There are 8 genes associated with a favorable prognosis in testicular germ cell tumor, listed in Table 2.

NDUFV3 is a gene associated with a favorable prognosis in testicular germ cell tumor. The best separation is achieved by an expression cutoff at 7.1 TPM which divides the patients into two groups with 100 % 5-year survival for patients with high expression versus 88 % for patients with low expression (p-value: 6.73e-4). Immunohistochemical staining using an antibody targeting NDUFV3 (HPA030427) shows a differential expression in testicular germ cell tumor samples.

p<0.001
NDUFV3 - survival analysis
NDUFV3 - high expression
NDUFV3 - low expression

Table 2. The 8 genes with significant association with a favorable prognosis in testicular germ cell tumor.

The testicular germ cell tumor transcriptome

The transcriptome analysis shows that 74% (n=14933) of all human genes (n=20162) are expressed in testicular germ cell tumor. All genes were classified according to the testicular germ cell tumor-specific expression into one of five different categories, based on the ratio between mRNA levels in testicular germ cell tumor compared to the mRNA levels in the other 16 analyzed cancer tissues.

Figure 1. The distribution of all genes across the five categories based on transcript abundance in testicular germ cell tumor as well as in all other cancer tissues.

640 genes show some level of elevated expression in testicular germ cell tumor compared to other cancers (Figure 1). The elevated category is further subdivided into three categories as shown in Table 3.

Table 3. The number of genes in the subdivided categories of elevated expression in testicular germ cell tumor.

Additional information

Non-seminomatous tumors are further classified as pure or mixed tumors. For mixed tumors, all included components must be defined and reported along with an estimation of the extent of each component. Tumors containing both seminomatous and non-seminomatous components are regarded as non-seminomatous germ cell tumors for treatment purposes.

Embryonal carcinoma is a relatively undifferentiated germ cell tumor from which the other more differentiated components are derived. These non-seminomatous components include;

• Endodermal sinus tumor (yolk sac tumor), typically characterized by the presence of Schiller-Duval bodies (resembling a central capillary lined by flattened layers of tumor cells),
• Choriocarcinoma, representing extra-embryonic development,
• Teratoma, representing embryonic development. Teratomas can in turn be composed of both immature and mature components representing cell types and structures from all embryonic germ layers. Schiller-Duval bodies are said to resemble a glomerulus. They have a mesodermal core with a central capillary, all lined by flattened layers of both visceral and parietal cells.

The distinction between different tumor types and components within a testicular tumor is based on microscopical examination and in addition to morphology, immunohistochemistry provides important information. Commonly used antibodies for the differential diagnostics of these tumors include D2-40, OCT 3/4, hCG and AFP in addition to CD30 and markers of intermediate filaments, e.g. cytokeratin and vimentin.

Relevant links and publications

Uhlen M et al., A pathology atlas of the human cancer transcriptome. Science. (2017)
PubMed: 28818916 DOI: 10.1126/science.aan2507

Cancer Genome Atlas Research Network et al., The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. (2013)
PubMed: 24071849 DOI: 10.1038/ng.2764

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Djureinovic D et al., The human testis-specific proteome defined by transcriptomics and antibody-based profiling. Mol Hum Reprod. (2014)
PubMed: 24598113 DOI: 10.1093/molehr/gau018

Histology dictionary - Testis cancer