New analysis of atypical spermatocytic tumours reveals extensive heterogeneity and plasticity of germ cell tumours†

Testicular germ cell tumours (TGCTs) derived from immature (type I) and pluripotent germ cell neoplasia in situ (GCNIS, type II) are characterised by remarkable phenotypic heterogeneity and plasticity. In contrast, the rare spermatocytic tumour (SpT, type III), derived from mature spermatogonia, is considered a homogenous and benign tumour but may occasionally present as an anaplastic or an aggressive sarcomatoid tumour. While various oncogenic processes had been proposed, the precise mechanism driving malignant progression remained elusive until the molecular characterisation of a series of atypical SpTs described in a recent issue of The Journal of Pathology. The emerging picture suggests the presence of two distinct trajectories for SpTs, involving either RAS/mitogen‐activated protein kinase pathway mutations or a ploidy shift with secondary TP53 mutations and/or gain of chromosome 12p, the latter known as pathognomonic for type II GCNIS‐derived TGCTs. Here, we discuss the implications of these findings, seen from the perspective of germ cell biology and the unique features of different TGCTs. The evolving phenotype of SpTs, induced by genomic and epigenetic changes, illustrates that the concept of plasticity applies to all germ cell tumours, making them inherently heterogenous and capable of significant transformation during progression. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

No conflicts of interest were declared.

Testicular germ cell tumours
Testicular germ cell tumours (TGCTs) differ markedly from somatic cancers because the neoplastic origin is a germ cell, designed by nature to preserve genomic integrity and capable of undergoing meiotic cell divisions to form haploid gametes.These constraints explain the unusual lack of driver mutationsbut frequent aneuploidy, often involving whole chromosome armsobserved in TGCTs.Another unique feature of germ cells is their totipotency, which likely gives rise to the extraordinary phenotypic heterogeneity of germ cell tumours, ranging from a mass of immature germ cells to mixtures of variably differentiated somatic tissues, including even extra-embryonic elements, such as yolk sac or placental trophoblast.
It is well established that different stages of germ cell maturation give rise to distinct types of TGCTs, occurring within a wide age range and associated with different prognostic, molecular, and histological features (Figure 1).TGCTs can occasionally be detected in young children, as type I TGCTs without a known precursor lesion (Figure 1), but are most frequently found among young menfrom puberty to around 40-45 years of age, although older age at diagnosis is not uncommon.The TGCTs of young men, which are classified as type II TGCTs, are derived from germ cell neoplasia in situ (GCNIS), which resemble arrested and transformed fetal germ cells (primordial germ cells or gonocytes) that failed to mature into spermatogonia (Figure 1).How GCNIS transforms into invasive TGCTs (seminoma or non-seminoma) is not completely understood, but the current hypothesis stipulates that at puberty, GCNIS cells acquire adaptive genomic and epigenetic changes, including polyploidisation and amplification of selected chromosome arms.Among these, the gain or polysomy of chromosome 12 (chr 12), often manifested as isochromosome i(12p), is the most frequent genomic aberration, likely providing a survival advantage.During progression, type II TGCTs can acquire second hits in genes like KIT (seminoma) and occasionally TP53 [1].
In older menwith median age at diagnosis around 52-56 yearsa different, much rarer, TGCT type can occur: spermatocytic tumour (SpT), also known as spermatocytic seminoma or TGCT type III (Figure 1).SpT is not associated with GCNIS and originates from spermatogonia, which after puberty clonally expand and progressively become aneuploid [2,3].Due to their rarity (<1% of all testicular tumours), SpT pathogenesis and genetic landscape have remained elusive.At the beginning of the 21st century, the first molecular studies of SpTs demonstrated the presence of a recurrent amplification of chromosome 9p (chr 9p), pointing to increased copy numbers of DMRT1 as a likely driver event [4].Subsequent studies identified large genomic rearrangements, confirming recurrent amplification of chr 9p, but also chr 20 and/or loss of chr 7, pointing to a mechanism disrupting the mitosis-meiosis transition and leading to progressive polyploidisation [2].
The presence of this aberration is considered responsible for the distinctive and diagnostic morphological features of SpTs, which are characterised by cells of different sizes, with small, intermediate, and large nuclei.Rare gain-of-function point mutations were identified within components of the RAS/mitogen-activated protein kinase (MAPK) pathway (i.e.HRAS, NRAS, and FGFR3) in a subset of SpTs, which promote the proliferation and survival of spermatogonia but apparently not invasiveness, so the clinical course remains indolent and the patients are usually diagnosed at a relatively older age [2,5].Notably, gains of chr 12p were not detected in these studies.

Novel evidence on molecular mechanisms involved in malignant progression of spermatocytic tumours
The clinical course of SpTs is benign in most patients, but 7% have poor prognosis.It was noted earlier that some tumours had somewhat different histological features with homogenous intermediate cell size, named anaplastic tumours, but the biological significance of this difference was unclear because only a small fraction of these tumours was associated with metastasis.The majority of SpTs that metastasise transform into sarcomatoid tumours (most often rhabdomyosarcoma) [6].
The molecular basis of this rare malignant progression of SpTs had remained poorly understood until the indepth characterisation of 25 SpT samples, including five malignant SpTs (mSpT), from the multicentre study presented recently in The Journal of Pathology by Gupta et al [7].The authors analysed these rare samples using a variety of approaches that include sequencing of a large panel of known oncogenes, DNA methylation, and SNP arrays, as well as fluorescence in situ hybridisation (FISH) for i(12p) and immunohistochemistry.The study was able to clarify the molecular events involved during malignant progression of SpTs by identifying the likely oncogenic drivers and associated phenotypic changes.Based on their analysis, Gupta et al [7] confirmed the existence of two genomic subgroups of SpTs in their series: (1) tumours carrying recurrent oncogenic point mutations in the RAS/MAPK pathway (i.e.HRAS, NRAS, and BRAF) with predominantly diploid genomes, which occurred in relatively older patients, and (2) tumours with global ploidy shift and absence of recurrent driver mutations.The four sarcomatoid SpTs belong to the second group and are characterised by the acquisition of two well-known oncogenic events: pathogenic TP53 mutations and/or gain of 12p (Figure 1).The presence of TP53 mutations may explain the drastic morphological change observed in these tumours, from germ cell-like to connective tissue-like sarcomas, which is a known feature of tumours in patients with Li-Fraumeni syndrome (heterozygous germline TP53 mutations).

Is there a relationship between SpTs and GCNIS-derived TGCTs?
The detection of 12p gain is interesting and in line with earlier case reports on the sporadic presence of i(12p) in mSpTs, previously referred to as a 'hybrid' tumour [8].Gupta et al [7] suggested that mSpTs with a gain of 12p represent an intermediate state between type II and type III TGCTs.Although such a scenario cannot be excluded, caution should be taken.Even though i(12p) is considered a genomic marker of GCTs, gain of 12p is not unique to GCTs and has been detected in other tumour types.The short arm of chr 12 contains not only genes encoding pluripotency factors, such as NANOG, but also proliferation-inducing factors, i.e. cyclin D2 and KRAS.Hence, the apparent gain of 12p in anaplastic mSpTs could be caused by the progressive growth advantage conferred to germ cells carrying amplified copies of chromosome 12.
Another interesting observation made by Gupta et al concerns the epigenetic features of SpTs.A previous study [9] reported a very heterogenous pattern of DNA methylation, with seemingly random mixtures of hypo-or hyper-methylated cells.In contrast, the mSpTs analysed by Gupta et al [7] showed global hypomethylation, reminiscent of that described in seminomas (TGCT type II) but with a different imprinting pattern typical of post-pubertal (mature) germ cells.How this low methylation level is maintained or acquired in the absence of the embryonic factors present in GCNIS and seminoma is worth exploring.
Interestingly, Gupta et al described a patient (case 7) with a RAS/MAPK-mutant SpT (NRAS p.G12A mutation) whose testis contained a typical GCNIS adjacent to the SpT [7].The question arises as to whether there is a relationship between these two components, but the answer is not simple.GCNIS has never, so far, been described in connection with SpTs [3,4].Strikingly, the age at diagnosis of this patient (39 years) is within the typical age range of the first appearance of seminomas.Therefore, it remains possible that the finding is coincidental because the metachronous presence of two different malignancies is not a rare event, but we cannot exclude the possibility that the SpT in this patient grew from a GCNIS or an early seminoma tumour cell.A possible mechanism of such unusual transformation towards a more mature germ cell type could include epigenetically induced downregulation of pluripotency factors with upregulation of DMRT1, which is expressed in a subset of GCNIS cells and is likely required for the transition towards meiosiscompetent cells [10].The acquisition of a secondary RAS/MAPK mutation in this situation could promote the growth of a SpT-like tumour.However, this remains speculative and based on observations from a single individual, and further studies and additional cases should provide an opportunity to test these alternative hypotheses.

Concluding remarks
The comprehensive analysis presented by Gupta et al [7] of a series of 25 SpTs uncovered fascinating new concepts concerning the biology of TGCTs.The pathobiology of TGCTs never stops to surprise, and the identification of different subtypes of SpTs, with distinct prognostics, is a further illustration of the extreme heterogeneity and phenotypic plasticity of TGCTs, which derive from germ cells at different stages of maturation.Interestingly, the acquired genomic changes, such as gain of i(12p) and/or TP53 mutations, seem to give a growth (or survival) advantage to germ cells regardless of their maturation stageshining new light on the basic biology of male germ cells.Finally, better delineation of the progression of SpTs from indolent to aggressive will likely open new diagnostic and therapeutic opportunities in the near future.

Figure 1 .
Figure 1.Schematic illustration of different types of testicular germ cell tumour (TGCT) and their likely origin from germ cells at different stages of development.Type I TGCTs are rare neoplasms that derive from early fetal germ cells, although the specific precursor has not been identified, and are diagnosed primarily in young children.Type II TGCTs are derived from arrested and transformed gonocytes or primordial germ cells, which persist in the adult testis as germ cell neoplasia in situ (GCNIS).Type II TGCTs are by far the most common type of TGCTs and give rise to either seminoma or non-seminoma found in young and adult men.Type III TGCTs originate from post-pubertal spermatogonia and give rise to spermatocytic tumours (SpT) found in older men.The conventional SpT is benign and phenotypically characterised by cells of different sizes (with small, medium, and large nuclei) and exhibit increasing aneuploidy.Occasionally, an anaplastic SpT characterised by cells of the same size can form.Ploidy shift is observed, and the tumour can acquire an aggressive and malignant behaviour often by sarcomatoid transformation.Figure created with BioRender.com.