Genomic features of mediastinal germ cell tumors: a narrative review

Introduction

Testicular cancer is the most prevalent solid tumor in young men aged between 15 and 45 years. Generally, testicular cancer accounts for approximately 1% of all male tumors, and Germ cell tumors (GCTs) make up 95% of tumors arising from testes (1,2). Seminoma and nonseminoma are the main histologic subtypes of GCTs, with nonseminoma being more aggressive than seminoma (3). Around 5% of GCTs originate from extragonadal sites, mainly from the anterior mediastinum, followed by the retroperitoneum, pineal gland, and suprasellar regions and the most common histologic subtype of primary mediastinal GCTs (PMGCTs) is nonseminoma (70–80%) (4,5). Mediastinal seminomas are slow-growing tumors, but they can be bulky by the time they are diagnosed due to their indolent course (6). Most seminoma cases have elevated levels of beta human chorionic gonadotropin (hCG) and lactate dehydrogenase (LDH) at the time of diagnosis. Conversely, non-teratoma nonseminoma (NS)-PMGCTs are aggressive tumors with a poor prognosis and an estimated 5-year overall survival rate of around 40–50%. Aside from seminomas, they also have elevated levels of alfa-fetoprotein (AFP) which always shows the existence of a nonseminoma component. Poor prognosis is mainly related to the lower sensitivity of these tumors to cisplatin-based chemotherapy and the limited success of salvage second-line therapies, including high-dose chemotherapy with autologous bone marrow transplant that has not majorly affected the survival of NS-PMGCTs (7).

Better approaches to treat these patients are needed. There have been small-scale clinical researches about PMGCTs so far, which could not generate reliable information. Thanks to The Genome Cancer Atlas (TGCA) project, the molecular background responsible for cancer development provided crucial insights about disease and translational potential for rare diseases. Multiomics data (genome, proteome, transcriptome, epigenome, microbiome) may provide actionable data for biomarkers discovery to better classify the patients and to create a personalized treatment approach for each patient. For PMGCTs, the mechanism of lower sensitivity to chemotherapy remains unclear. However, recent data have demonstrated a higher prevalence of TP53 mutations in cisplatin-resistant GCTs and NS-PMGCTs, suggesting loss of P53 function as a possible mechanism responsible for the poor response of the disease to treatments (8,9). There might be other possible mechanisms to explore for a better understanding of this rare disease.

Multi-omics studies in GCTs are scarce and are even more limited in PMGCTs, for which more studies are warranted. This review summarizes the available data describing the molecular characteristics of PMGCTs compared to testicular GCTs (TGCT), highlighting the most relevant prognostic factors and possibly uncovering some molecular pathways which could be helpful for future patients’ treatment selection and implementation. We present the following article in accordance with the Narrative Review reporting checklist (available at https://med.amegroups.com/article/view/10.21037/med-22-4/rc).

Methods

Relevant studies published between 1982 and 2021 were identified via a PubMed and Google Scholar search using different combinations of the following terms: “mediastinal germ cell tumors (MGCTs)”, “prognostic biomarkers”, “genomic alterations”, “clinical features”, “prognostic factors”. Additional papers were identified by reviewing reference lists of relevant publications. Publications with relatively low reliability and which were not written in English were excluded. Data were extracted based on their relevance to the topic. More details of the method are shown in Table 1.

PMGCTs

While the incidence of primary gonadal GCTs has been consistently rising in the last three decades, extragonadal GCT incidence has remained steady overtime (10). PMGCTs account for 1–3% of all germ cell malignancies and for 15% of adult anterior mediastinal cancers. Thymoma, thymic carcinoma, teratoma, thyroid goiter, sarcomas, and lymphomas are considered in the differential diagnosis of anterior mediastinal masses. High levels of LDH, AFP, and/or beta hCG are a meaningful tools to suspect PMGCTs. While AFP is mostly positive in embryonal carcinoma, teratoma and yolk sac tumors, higher beta hCG levels are detected in seminomas, choriocarcinomas, and embryonal carcinomas. Other immune markers such as placental alkaline phosphatase (PLAP) (can be detected in yolk sac tumors and seminoma), leukocyte common antigen (LCA) (lymphoma), desmin/vimentin (sarcomas) and CD30 (embryonal carcinoma) can be helpful in the differential diagnosis, especially by immunohistochemistry and in challenging cases for the diagnosis.

The most accredited mechanism of extragonadal GCTs pathogenesis consists of a defective migration of primordial germ cells along the urogenital ridge or reverse migration of germ cells from testes following the nerve roots (11,12).

Seminomas are usually diagnosed at an older age than nonseminoma testicular GCTs (NS-TGCTs) (median age at diagnosis 34 vs. 30, P=0.02), and cryptorchidism is more common in patients with seminoma (P=0.008) (13). Isochromosome 12p [i(12p)] is the widespread shared cytogenetic abnormality in gonadal and mediastinal GCTs and is equally represented in seminomas and nonseminomas (4,14). Seminomas are highly sensitive to cisplatin-based chemotherapy and radiotherapy (RT) and this sensitivity is not affected by the primary tumor location. Therefore, seminoma PMGCT (S-PMGCT) and seminoma TGCT (S-TGCT) share the same prognosis established by the International Germ Cell Consensus Classification (IGCCC) (15).

NS-PMGCTs include teratoma, yolk sac tumor, choriocarcinoma, and/or embryonal carcinoma. Pure or mixed yolk sac tumors are the most common NS-PMGCTs subtypes, while embryonal carcinoma is the less frequent. The prognosis of pure mature primary mediastinal teratoma is excellent and these tumors are usually successfully managed with surgery. However, non-teratoma NS-PMGCTs have a poorer prognosis than their gonadal counterpart. Although PMGCTs and primary gonadal testis cancer share common histologic and clinical characteristics, PMGCTs have distinct features, including association with Klinefelter’s syndrome, hematologic malignancies (HMs), and distinct molecular features (16-19).

Molecular features of GCT precursors

Progenitor germ cells (PGCs) express pluripotency markers such as OCT3/4 and NANOG and have low DNA methylation levels. As these cells are essential for normal germline development, delay or block of the PGCs maturation steps can trigger the development of germ cell neoplasia in situ (GCNIS) (20). These lesions are precursors of invasive malignant GCTs. The presence of i(12p) in GCNIS is associated with a faster progression to invasive GCT. However, how exactly the amplification of the genes located in the chromosome 12p participates in the pathogenesis of GCTs remains unclear (21).

Seminomas resemble PGC and GCNIS in terms of histology, methylation, and gene expression features. The common precursor of nonseminoma GCTs (NSGCTs) is embryonal carcinoma, originating from epigenetic reprogramming of GCNIS or seminomas. As embryonal carcinomas are characterized by pluripotency of embryonic stem cells, they can differentiate in yolk sac tumors, choriocarcinoma, and teratoma (20,22).

Although chromosomal abnormalities are commonly seen in GCNIS and GCTs, somatic genomic mutations are infrequent, especially in early GCNIS, and they slightly increase with the patient’s age. Therefore, most somatic TGCT mutations are passenger mutations that accumulate with the patient’s age, usually after the early onset of the disease (23).

Genomic landscape of TGCT and PMGCT

Genomics of TGCTs

TGCTs are characterized by a very low tumor mutational burden of approximately 0.5 mutations per Mb (0.50 in seminoma and 0.49 in nonseminoma), the second-lowest mutational burden in solid malignancies after Ewing sarcoma (0.3/Mb). TGCTs have high CG>TA transitions [27% of single-nucleotide variants (SNVs)], TA>CG transitions (23%) and CG>AT transversions (31%) (13,23,24).

Copy number alterations (CNAs) have been described in 47% of the nonseminoma patients and are mainly associated with high tumor aneuploidy, which is however not well established in seminoma patients (25). This high prevalence of aneuploidy is most likely induced by tetraploidization followed by chromosome loss. The most common chromosome change in TGCTs is gain of chromosome 12p, observed in 80% of cases, which is considered pathognomonic of TGCT. Studies mapping the 12p region have failed to identify the central TGCT driver genes. Other chromosome changes, including chromosomes 7, 8, 21, 22, and X gain and loss of chromosome Y, have been described in 25–40% of TGCTs patients. Seminomas have less frequent copies of 11q, whereas nonseminomas have fewer copies of chromosomes 8, 9, 15, 19, 22. Like TGCTs, PMGCTs are mostly aneuploid with large-scale CNAs, including chromosome 12p and X amplification and deletion of the Y chromosome (26-28).

The fibrous sheath interacting protein 2 (FSIP2), located in 2q32.1, is the most frequently copy number altered gene in TGCTs, and its focal amplification has been observed in >15% of cases. Interestingly, dysplasia of the fibrous sheath and mutations in AKAP4 (which binds to FSIP2) have been associated with male infertility, a well-known risk factor for TGCT (23,29).

Overall, 44 risk loci for TGCT have been described in genome-wide association studies (GWAS). The most frequent risk locus for TGCT is 12q21, including the KITLG gene that codifies for the c-KIT ligand. This receptor and its substrate have a crucial role in regulating germ cells, and the c-KIT pathway is a central driver in TGCT (30). c-KIT mutations occur mainly in exon 17 and rarely in exon 11, and they are more frequent in seminoma than in nonseminoma (18–25% of cases vs. 2%, respectively) (31,32). In fact, seminoma and NSGCTs present slightly different genomic alterations. Other than c-KIT, the most frequent gene alterations described in S-TGCTs are KRAS (14%) and NRAS (4%) mutations. In NSGCTs, c-KIT and KRAS mutations are reported only in a small proportion of patients. Moreover, while seminomas are usually hypertriploid, nonseminomas have lower ploidy and more frequent i(12p) than seminomas. Overall, tumor mutational burden is comparable in seminomas and NSGCTs (13,33,34).

Compared with c-KIT-mutated seminomas, c-KIT-wild-type seminomas exhibited unique features, including significant lymphocyte infiltration, lack of global DNA methylation, decreased CNAs, i(12p) gain, and a more frequent association with cryptorchidism. Moreover, c-KIT-mutated seminomas have a lower rate of KRAS mutations, and conversely, c-KIT wild-type seminomas have increased copies of KRAS (P=0.0007) (13,35). However, the biological relevance of these alterations is unclear, and despite the high prevalence of activating c-KIT mutations and overexpression, clinical trials using imatinib, a c-KIT inhibitor, in platinum-resistant GCT patients failed to demonstrate relevant activity (36).

Epigenetics of TGCTs

DNA methylation patterns differ among histology subtypes of GCTs. While embryonal carcinoma demonstrates a widespread methylation pattern at non-canonical cytosine sites (e.g., CpA, CpT, CpC), known as CpH sites, other NSGCT subtypes (teratoma, yolk sac, or mixed GCTs) have a stroma-like methylation profile (soma and extra-embryonal lineages) (37,38). DNA methyltransferase is overexpressed in NSGCTs, and preclinical and early phase I studies have demonstrated that their inhibition increases cisplatin activity in cisplatin refractory TGCT cells (39). Methylation is associated with epigenetic silencing of tumor suppressors such as BRCA1, RAD51C, MGMT, and RASSF1A in NSGCTs. Thirty-five percent of non-embryonal carcinoma NSGCT cases have BRCA1 and RAD51C promoter methylation, suggesting a possible role for PARP inhibitors in these patients (13,40). Moreover, specific miRNA clusters have been extensively described in GCTs, and their role as biomarkers of the disease is currently under investigation. However, the specific target genes whose transcription is regulated by these miRNAs are still unclear (41-43).

Cisplatin resistant GCTs

Cisplatin-resistant or refractory GCTs are characterized by an increased DNA repair function to prevent chemotherapy-induced apoptosis. Most of the cisplatin-sensitive TCGT cell lines are unable to assemble Rad51 foci, which indicates a defect in initiating homologous recombination (HR) repair and explains the high sensitivity to cisplatin. On the other hand, platinum resistance is likely related to high TP53 mutation rates and/or high tumor mutational burden (TMB) (33,40,44).

Recent studies have shown that mutations of TP53, PIK3CA, AKT1, FGFR3, BRAF V600E, RAS, and FGFR3 are correlated to cisplatin resistance. KRAS and NRAS mutations have been described in 8/11 (72.7%) and 3/4 patients (75%) of cisplatin-resistant NSGCTs (but not in seminoma) (33,45,46). In a patient, who has both PMGCT and AML-M7 has shown to have TP53 and PTEN mutations in both tumor types which were reported to be associated with treatment resistance and poor prognosis (47).

RAC1, a member of the GTPase family, has analogous parts with RAS and plays a role in cell growth, migration (Sertoli cells), and therapy resistance in TGCCTs. Although RAC1 mutation is rare, 5% was the highest rate declared so far in the general GCT tumor cohort (33,48).

Genomic landscape of PMGCTs

The genomic landscape of PMGCTs is characterized by higher TMB and specific pathogenic alterations in known oncogenes or oncosuppressors involved in cancer development and progression compared to TGCTs. The most common mutations observed in PMGCTs are: TP53 (46%), c-KIT (18%), KRAS (18%), PTEN (%11), NRAS (4%), and PIK3CA (4%). NS-PMGCT presents a higher TMB than NS-TGCTs. In a small retrospective study, 11.4% of the NS-PMGCTs harbored ≥10 mutations per Mb vs. 4.66% of the NS-TGCT (8,26,49).

In general, PMGCTs have a greater likelihood of having yolk sac differentiation and TP53 alterations. Multivariate analysis considering the IGCCC prognostic risk factors demonstrated that TP53/MDM2 alterations are independent negative prognostic factors for progressive disease after first-line cisplatin-based chemotherapy in NS-PMGCTs (8,50). In a retrospective study, TP53 alterations were found in 72.2% of NS-PMGCT (13 of 18 patients), while none of the S-PMGCT patients harbored a TP53 mutation. In the same study, MYCN amplification was described in 5 of 104 (4.8%) patients with cisplatin resistance and was almost mutually exclusive with TP53/MDM2 mutations (51). TP53 and MDM2 are under the direct transcriptional regulation of MYCN. Small molecules (i.e., Nutlin) targeting the MDM2-TP53 interaction were demonstrated to sensitize testicular cells to cisplatin chemotherapy and induce apoptosis (52).

When compared to NS-TGCTs and seminomas (both gonadal and extragonadal), NS-PMGCTs have higher genomic alterations in TP53 (P<0.0001), PIK3CA pathway (PIK3CA, mTOR, PTEN, AKT1/2) (P<0.0001), and cell-cycle regulation genes (CCND1/2/3, CDK4/6, CDKN2A/B, RB1) (P=0.0004). Other common tumor genomic alterations, including RAS-RAF (KRAS, NRAS, HRAS, BRAF), RTK (ERBB2, PDGFRA, KIT, MET, FGFR 1/2/3), and DDR (BRCA1/2, ATM, CHEK2, MUTYH), are not significantly different between NS-PMGCTS and S-PMGCTS and TGCTS (8).

The genomic profile described above could explain the worse prognosis observed in NS-PMGCTs versus TGCTs and S-PMGCTs. As the prognosis of NS-PMGCTs remains poor even in the modern era, new drugs and more effective first-line or salvage therapy regimens are needed. All the clinical trials using single-agent immune checkpoint inhibitors in cisplatin-resistant GCTs have failed to demonstrate a clinically meaningful activity (53). Of note, all these studies were conducted on unselected patients. Since NS-PMGCTS are among the GCTs with the highest TMB, even if single-agent immunotherapies did not show meaningful effects, future studies evaluating immunotherapies combined with platin-based chemotherapy and novel therapies such as demethylating agents could be studied in this rare subset. In this aspect, the comprehensive analysis of current data will help to provide a theoretical background for further research and explore druggable targets. It is also possible to explore biomarkers related to therapy response for immunotherapies.

Association of PMGCTs with HMs and risk of malignant transformation

HMs are seen in approximately 2–3% of NS-PMGCT patients (54). The HMs can be simultaneously diagnosed with NS-PMGCTs (in 31% of the cases) or developed after the initial diagnosis of NS-PMGCTs (in 46% of the cases). The most common HM associated with NS-PMGCT is acute megakaryoblastic leukemia (AML-M7), followed by other types of AML, chronic myeloid leukemia, myelodysplasia, malignant mastocytosis, essential thrombocytopenia, and malignant histiocytosis. Interestingly, the accompanying HM cells are positive for i(12p) (shown in about 47% of PMGCT related HMs) and other GCTs specific mutations such as TP53, KRAS, and PTEN, suggesting that they derive from a common GCT progenitor.

Conversely, the lack of typical alterations for primary HMs such as MLL rearrangements or FLT3 mutation further indicates a different etiopathogenesis of these malignancies (55). Moreover, in PMGCTs with associated HMs, TP53 is the most frequently altered gene described in 90% of the patients and correlated to therapy resistance and poor prognosis (56,57). Additionally, KRAS/NRAS alterations are seen in 63% of PMGCT with concomitant HM, while PTEN mutation rates are similar between PMGCTs with or without HM (57,58).

Some studies have recently suggested that the HM cells arise from resistant vasculogenic areas selected by chemotherapy. Although these theories unveil certain aspects of the HM associated with PMGCTs, further studies are needed to clarify the molecular associations of these tumors (59).

Besides HMs, somatic-type malignancies such as rhabdomyosarcoma, primitive neuroectodermal tumor (PNET), adenocarcinoma, and Wilms tumor can also be seen in association with PMGCTs and NS-TGCTs (60,61). Most of these tumors are believed to arise from pluripotent teratoma cells. Analysis of tumor samples showed chromosome 12p amplification, i(12p), and identical patterns of loss of heterozygosity in almost all of the somatic malignancies associated to GCTs which suggest a common origin. Interestingly, tumor mutations specific of the transformed malignancies have also been observed, such as t(11;12) characteristic of the PNET (61-63). As these tumors exhibit poor responses to cisplatin-based chemotherapy, surgery is the first and most effective treatment. The chemotherapy regimen should be selected on the base of the somatic transformation (64).

Conclusions

In summary, NS-PMGCTs have a distinct genomic landscape characterized by a higher TMB and higher prevalence of P53 mutations than TGCTs and S-PMGCT which could explain their lower sensitivity to cisplatin chemotherapy and poorer prognosis. Although some studies have unveiled recurrent molecular alterations in PMGCTs, few are suitable for targeted therapy. Due to the rarity of PMGCTs, the design of clinical trials in this patient population is extremely challenging. Data sharing and the creation of international collaborations would be helpful to have a better understanding of the molecular drivers of these tumors to improve patients selection and care.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editors (Margaret Ottaviano and Giovannella Palmieri) for the series “New Treatments and Novel Insights of Thymic Epithelial Tumors and Mediastinal Germ Cell Tumors” published in Mediastinum. The article has undergone external peer review.

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://med.amegroups.com/article/view/10.21037/med-22-4/rc

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://med.amegroups.com/article/view/10.21037/med-22-4/coif). The series “New Treatments and Novel Insights of Thymic Epithelial Tumors and Mediastinal Germ Cell Tumors” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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