Introduction

Primary mediastinal germ cell tumours (PMGCTs) are extragonadal germ cell tumours (GCTs) that arise in the mediastinum and account for up to 2–5% of all GCTs. They account for up to 15% of mediastinal tumours in adults and approximately 20% in children.^{1, 2} The patients often present with large mediastinal masses. These are most commonly located in the anterior (prevascular) mediastinum (60–70%), and cause symptoms by compression of surrounding anatomic structures.^{1, 3-5}

The diagnosis is made by histopathological analysis, correlation with radiographic images and serum levels of tumour markers such as lactate dehydrogenase (LDH), alpha-fetoprotein (AFP) and β-subunit of human chorionic gonadotropin (β-hCG).^{6, 7} Therefore, a primary gonadal GCT or other primary extragonadal GCTs (e.g. in the retroperitoneum) must be excluded by clinical and radiographic investigations.⁸

PMGCTs share many similarities with their gonadal counterparts, with some histological, immunohistochemical and molecular, but also biological, differences which are discussed in the following review.

PMGCTs are divided into Types I and II tumours based on different age distributions and different underlying genetic alterations.^9-12 Type I PMGCTs typically occur in infants and young children before puberty and include primary mediastinal teratomas (PMTER) with mature or immature elements, primary mediastinal yolk sac tumour (PMYST, which are thought to develop from immature teratomas during development), as well as mixed tumours composed of PMTER and PMYST.^{13, 14} Notably, pure PMTERs (‘Type I mediastinal teratomas’) occur not only in children but comprise almost all mediastinal teratomas in adult women and rarely occur in adult men.^{15, 16} The chromosome 12 alterations characteristic of Type II PMGCTs are missing in Type I PMGCTs. Type II PMGCTs develop in adolescents and adult men and encompass seminomas (PMSEM) and primary mediastinal non-seminomatous germ cell tumours (PMNSGCTs). PMNSGCTs comprise embryonal carcinoma (PMEC), PMTER, PMYST, and choriocarcinoma (PMCC).¹⁷ Furthermore, Type II PMGCTs also comprise mixed tumours (with or without a teratomatous componenta teratomatous component) and PMNSGCTs with somatic-type solid malignancies or associated hematological malignancies. Type II PMGCTs show the same characteristic molecular alteration as Type II gonadal GCTs; that is, gain of material chromosome 12p, most often an isochromosome 12p i(12p).^{15, 18-21}

In addition, Type II PMNSGCTs have a worse prognosis than primary gonadal GCTs,²² probably due to larger tumour masses at the time of presentation, compression of vital anatomical structures,²³ and higher rates of TP53 mutations, as well as a higher prevalence of somatic-type malignancies or the unique feature of associated hematological malignancies.^{24, 25}

Epidemiology

Type I PMGCTs usually occur in infants and children under the age of 8 years, with a gender-equal distribution, and only comprise PMTERs and PMYSTs, as well as mixed PMGCTs with PMTER and PMYST components.²⁶ However, pure Type I-like PMTERs are virtually the only PMGCTs occurring in adolescent and adult women. They can also very rarely be detected in adult men and might be intermediate types between Types I and II PMGCTs.^{10, 22, 26}

Type II PMGCTs occur almost exclusively in male patients > 8 years and show a male predominance, with a sex ratio of 9:1.²⁷ Most cases are diagnosed in young men between the age of 25 and 35 years (age range: 0–80 years).²⁸ The incidence of Type II PMGCTs in men increases after puberty and decreases in elderly men.¹⁷ They can comprise histological subtypes in pure or mixed forms.^{9, 16, 29} In female adolescents and adults, only sporadic case reports of pure or mixed Type II PMGCTs (e.g. PMEC, PMYST, PMCC) are on record.^30-32

Aetiology

The only known risk factor for PMGCTs in male patients is Klinefelter syndrome (KS), a chromosomal alteration with an XYY/XXYY genotype.³³ KS has been diagnosed in approximately 8–33% of male patients with PMGCTs,³⁴ and people with KS have a 67-fold increased risk for developing a PMGCT.^{33, 35-39} KS patients are usually young, with an age range between 4 and 31 years, and can be afflicted by Types I and II PMGCTs.³⁴

Pathogenesis

The pathogenesis of PMGCTs is not clear. They are thought to arise from primordial germ cells, which remain in the mediastinum during embryonic development.^{9, 12, 40} Primordial germ cells develop in the yolk sac and migrate to the gonadal ridge, where they find their physiological niche.⁴¹ Some primordial germ cells may find other niches (e.g. the thymus), where epigenetic reprogramming occurs.¹¹ Surviving primordial germ cells in different stages of epigenetic reprogramming may eventually develop either into Types I or II PMGCTs.¹¹ PMGCTs show the same chromosomal alterations as their gonadal counterparts, but there are differences between Types I and II PMGCTs (see ‘Molecular alterations’ section).

Clinical presentation

Most PMGCTs develop in the anterior mediastinum^{1, 5} and only rarely in the posterior (paravertebral) mediastinum^42-45 or the middle (visceral) mediastinum.⁴⁶ Patients with PGMCTs can be asymptomatic for a long time and usually present with symptoms such as chest pain, chronic cough, dyspnoea and/or dysphagia. These symptoms are caused by large mediastinal masses,⁶ that can rarely also lead to pericarditis⁴⁷ or seizures.⁴⁸ Other severe symptoms include haemoptysis,⁴⁹ superior vena cava syndrome⁵⁰ or cardiac thrombosis.⁵¹ Few cases show systemic symptoms, including fever, nausea and hoarseness.⁵² Trychoptysis is a rare but pathognomonic symptom for PMTER that occurs when bronchial walls are perforated by teratomatous elements.⁵³ Recently, a case of meningomyelocele associated with a mature teratoma has been described in the posterior mediastinum.⁴² Metastases of primary testicular or retroperitoneal GCTs must be ruled out. One study with 100 cases of testicular GCTs with distant metastases (all with prior retroperitoneal lymph node metastases) showed that metastases to the mediastinum occurred in 7% of cases.⁵⁴

Radiographic findings

PMGCTs present as large masses, and their size can vary from < 3 to 25 cm.^{6, 46, 52, 55} There are some imaging clues suggestive of specific histological subtypes. Pure seminomas tend to be homogenous, whereas PMNSGCTs are heterogenous and teratomas are usually multicystic in more than 90% of cases.^{52, 56} The presence of fat or bone within the tumour is highly suggestive for a teratoma.^{57, 58} FDG-PET/CT is helpful to detect occult primary tumours or metastases.^{59, 60}

Serum tumour markers

Patients with PMGCTs have the same serum tumour markers as testicular GCTs. LDH is increased in all histological subtypes, but is neither sensitive nor specific, as it is elevated in all tumours or conditions with prominent cell damage.^{6, 7} However, elevated LDH serum levels have an adverse prognostic significance and predict a worse outcome in patients of the ‘good prognostic’ group.^{6, 61}

Elevation of AFP in a young male with a mediastinal mass is highly suspicious for a PMYST. If a biopsy shows PMSEM in the presence of high or rising AFP serum level, the tumour should be considered a PMNSGCT and treated accordingly. However, some rare non-GCT tumours, such as NUT carcinomas, can also lead to elevated AFP levels.⁶² Elevation of β-hCG must be significant (> 1000 U/l) to indicate PMCC.^{6, 8} Low levels of β-hCG can also be detected in cases of pure seminomas or other PMNSGCTs that contain isolated β-hCG-producing syncytiotrophoblasts.^{6, 7, 63}

In testicular GCTs, circulating microRNAs in serum probes were found to have a higher sensitivity (84.7%) and specificity (99%) than AFP and β-hCG together (about 60%).^{64, 65} Before their use in routine diagnostics and monitoring of patients with PMGCTs can be recommended, further clinical studies are needed.⁶⁶

Macroscopic examination

PMSEMs have a lobulated contour and cut surface. The cut surface shows a white-to-tan colour with solid areas, cystic changes, and areas of necrosis.⁶⁷ PMECs show infiltrative growth and often have a haemorrhagic and necrotic cut surface.⁶⁸ Mature PMTERs are well-circumscribed, with prominent cystic areas filled with sebum and hair. The cut face can show fat, cartilage, teeth or bone structures.^{52, 56, 68} Immature PMTERs are more solid tumours with fleshy or fibrous consistency and haemorrhagic or necrotic areas. PMYSTs are solid tumours with a soft texture and a grey-to-whitish cut surface.⁶⁹ PMCCs are fragile tumours that show infiltrative growth and prominent haemorrhagic and necrotic areas.^{70, 71} Because surgical resection of a PMGCT usually occurs after initial chemotherapy, massive regressive changes such as fibrosis and necrosis may predominate.⁷²

Histological subtypes

Histopathological analysis is performed on tissue obtained by core needle biopsy (in most cases) or surgical biopsy (e.g. during mediastinoscopy or mediastinotomy). Due to the vast morphological spectrum of PMGCTs and the broad spectrum of differential diagnoses, PMGCT classification based on limited core needle biopsy material is highly challenging. To compound the difficulty, this material might be the only viable tissue in PMGCT patients that is available for analysis. Thymic remnants in small biopsies are easily misinterpreted as epithelial neoplasms.

With few but typical exceptions (e.g. in teratomas), PMGCTs show the same morphological and immunohistochemical features as their gonadal counterparts. Furthermore, there are some morphological differences between Types I and II PMGCTs, especially with regard to teratomas (see below).¹⁶ PMECs and PMCCs rarely occur as pure tumours and are seen in mixed PMGCTs.^{9, 17}

For the determination of the germ cell origin of a tumour, the Spalt-like transcription factor 4 (SALL4) is the most sensitive marker with homogenous strong nuclear staining.^{21, 73-75} SALL4 stains nearly 100% of PMECs and PMYSTs and > 90% of PMSEMs.^{21, 67, 76, 77} PMCCs show variable staining for SALL4 because syncytiotrophoblasts usually lack SALL4 staining. Of note, SALL4 is usually absent in mature teratomas.^{74, 76} SALL4 can be positive in some non-GCT-tumours (see ‘Differential diagnosis and diagnostic pitfalls’ section). Lin-28 homologue A (LIN28) can be used as an additional germ cell marker,^{21, 78} but placental-like acidic phosphatase (PLAP) has limited specificity.^{79, 80}

Primary mediastinal seminoma

PMSEMs consist of polygonal or epithelioid medium-sized tumour cells with distinct cell borders, clear to slightly eosinophilic cytoplasm and central to eccentric nuclei with vesicular chromatin and prominent eosinophilic nucleoli. They show a sheet-like and solid or lobular growth pattern, usually forming nests of tumour cells separated by thin fibrous septa (Figure 1A–B). There can be prominent lymphocytic infiltrates with florid lymphoid hyperplasia^{81, 82} or granulomatous reactions (Figure 1A) mimicking granulomatous diseases and effacing tumour cells.^{83, 84} Contrary to their gonadal counterpart, up to 8% of PMSEMs show cystic changes due to the induction of tumour-associated multilocular (secondary) thymic epithelial cysts.⁸⁵ In some cases, single intermingled syncytiotrophoblasts can be present, causing elevated serum β-hCG.^{63, 86} Prominent fibrosis as a sign of regression, well known in testicular GCT,⁸⁷ has only been described in few cases of PMGCTs with seminomatous components.^88-90

Immunohistochemical markers for PMSEMs (Figure 1C–H) are SALL4, OCT3/4, SOX17 and CD117.^{19, 21, 67, 76} They are also positive for keratins, usually in a dot-like perinuclear staining pattern, which is different from testicular SEM.^{21, 91} Other markers that are frequently positive are D2-40²¹ and TdT.²¹ Stemness markers such as NANOG⁷⁶ and PLAP¹⁹ can also be positive. A recent study has shown that there are single FOXA2-positive cells in a small percentage of testicular seminomas. This is seen as a possibility that seminomas have the potential for FOXA2-driven extra-embryonic differentiation.⁹² However, in order to investigate this hypothesis in PMGCTs, further analysis is necessary.

Primary mediastinal non-seminomatous germ cell tumours

Primary mediastinal embryonal carcinoma

PMECs usually occur in mixed PMGCTs and rarely as pure tumours.⁹³ They consist of large tumour cells that show a glandular/tubular, papillary or solid growth pattern (Figure 2A). The polygonal tumour cells have a clear to eosinophilic or slightly basophilic cytoplasm with indistinct cell borders. Overlapping nuclei with prominent eosinophilic nucleoli are characteristic (Figure 2B). Atypical mitoses are frequent. Necrosis, sometimes of comedo-type, is often detected. Furthermore, prominent haemorrhagic areas are found. These should be sampled for choriocarcinoma components, but single or small groups of syncytiotrophoblasts not fulfilling the criteria for choriocarcinoma are quite frequent (about 30%).⁹³

Immunohistochemically, PMECs show positive staining for SALL4, OCT3/4, SOX2, CD30, keratins, NANOG and PLAP (Figure 2C–F).^{21, 55, 76, 94} Intermingled syncytiotrophoblasts can be detected by using β-hCG staining.

Primary mediastinal teratoma

PMTERs consist of pluripotent cells that differentiate into mature (adult-type teratoma) or immature (embryonal/fetal-type teratoma) tissues resembling somatic tissue structures of all three germ layers. Both tissue types can coexist in the same tumour. PMTERs often arise in the anterior mediastinum, often inside the thymus, seldom inside the pericardium^{95, 96} and only rarely in the posterior mediastinum.^{42-44, 97} Remnant thymic tissue adjacent to PMTERs is therefore a common finding.

Mature PMTERs can encompass epidermal epithelium with dermal appendages forming cysts, gastrointestinal mucosa, pancreatic acini, smooth or skeletal muscle, cartilage, bone, nerves and brain tissue (Figure 3A–D). Atypia can be seen in Type II PMTERs. In particular, different degrees of atypia of cartilage have been described in Type II PMTERs similar to those seen in chondrosarcomas.⁹⁸ PMTERs in Type I PMGCTs often show organoid structures (e.g. dermoid cysts) and lack atypia.¹⁶ Pancreatic tissue in an organoid formation is typical for PMTERs, but not a common finding in testicular teratomas. The diagnostic term ‘fetus in fetu’ has been proprosed for organoid structures that show the complex combination of a spinal cord with neural tube development and metamerisation, symmetric development and ‘synchronised’ organ development with organs in the same state of maturation.⁹⁹

Immature PMTERs usually show embryonic-type neuroectodermal tissue such as neuroepithelial (multilayered) tubules and different types of rosettes. Furthermore, blastemal-like stromal cells, mesenchymal and primitive cartilage, bone, rhabdomyoblasts, immature glands and fetal lung tissue can also be present (Figure 3E). According to the 5th edition of the WHO classification of thoracic tumours, there is still no grading system for immature PMTERs compared to retroperitoneal or ovarian teratomas due to lacking data, but it should be implemented.^{13, 100}

The use of immunohistochemistry in PMTERs is not useful, as different types of tissues stain for many different markers that are positive in other PMGCT subtypes (e.g. SALL4, LIN28, SOX2, GATA3, FOXA2, GPC3, AFP, keratins).

Primary mediastinal yolk sac tumour

PMYSTs resemble tissue of the yolk sac, the allantois and/or extra-embryonic mesenchyme. They show histological diversity with a wide variety of different growth patterns, including a microcystic/reticular, macrocystic, glandular–alveolar, endodermal sinus, myxomatous, hepatoid, enteric, polyvesicular–vitelline, solid and spindle pattern, which can occur side by side in the same tumour.^{20, 21, 77, 93}

In areas with microcystic/reticular pattern (Figure 4A,B), flat to cuboidal cells with scant and clear cytoplasm form a network of small cystic areas. Pseudopapillary groups of cells are found in areas with endodermal sinus pattern. Schiller-Duval bodies (SDBs) are pathognomonic for YSTs in general (Figure 4B, inlet).¹⁰¹ SDBs are glomeruloid-like structures with an inner (visceral) tumour cell component surrounding a central capillary and an outer (parietal) tumour-cell component, lining an empty space between both. The hepatoid pattern is characterised by polygonal cells with abundant eosinophilic cytoplasm.^{102, 103} Cysts lined by flat to cuboidal tumour cells surrounded by a dense fibrotic stroma characterise the polyvesicular–vitelline pattern. Solid pattern PMYSTs show areas of dense tumours cells with different degrees of atypia, sometimes with highly pleomorphic cells (Figure 4C).^{20, 21}

Some PMYSTs can show intracytoplasmic or extracellular hyaline globules (Figure 4B) that are positive for periodic acid-Schiff¹⁰⁴ or extensive basement membrane deposition (parietal PMYST).¹⁰⁵ Furthermore, single intermingled syncytiotrophoblasts can be found, causing an elevation of serum β-hCG levels.²²

Positive immunohistochemical stainings include SALL4 (Figure 4D), LIN28 and keratins.^{21, 76-78} FOXA2, glypican-3 and AFP are used to identify PMYST components in PMGCTs (Figure 4E–G). FOXA2 is the most sensitive marker for the detection of PMYSTs because it stains tumour cells irrespective of growth patterns, while AFP is only positive in up to 70% of PMYSTs and sometimes only very focally.^{21, 106, 107} Furthermore, PMYSTs can be positive for GATA3 (Figure 4H),^{21, 108} as well as SOX17 and CD117, especially in cases with solid patterns.^{20, 21} Notably, GATA3 is also positive in PMCCs. Other sensitive and specific markers for YSTs including PMYSTs are HNF-1β and ZBTB16.^{109, 110} PMYSTs can also stain positive for CDX2, which must be considered in the differential diagnosis between PMYSTs and primary thymic (enteric-type) adenocarcinomas or metastases to the mediastinum.¹¹¹ PMYSTs are negative for OCT3/4, NANOG, SOX2 and D2-40.

Primary mediastinal choriocarcinoma

PMCCs usually occur in mixed Type II PMGCTs, rarely in a pure form.⁵² They consist of cytotrophoblasts and multinucleated syncytiotrophoblasts, which show a solid or sheet-like growth pattern, often in the form of a bilaminar, plexiform pattern resembling the trophoblastic component of placental villi without the typical stromal component (Figure 5A). Cytotrophoblasts are polygonal cells with clear to eosinophilic cytoplasm and oval-to-round nuclei with prominent nucleoli. Syncytiotrophoblasts are large cells with eosinophilic cytoplasm and multiple hyperchromatic nuclei with prominent nucleoli (Figure 5B, inlet). In addition, PMCCs are closely associated with vascular structures that they infiltrate.

PMCCs show positive immunohistochemical stainings for SALL4,¹¹² GATA3 and keratins. Cytotrophoblasts stain positive for p63 and syncytiotrophoblasts for β-hCG, glypican-3¹¹³ and α-inhibin (Figure 5C–F).¹¹⁴ They are negative for OCT3/4, PLAP, AFP, CEA, CD30 and vimentin.

PMGCTs with somatic-type solid malignancies

Somatic-type solid malignancies (STSMs) are malignant neoplasms that occur in up to 30% of Type II PMNSGCTs, but not in Type I PMGCTs.^{115, 116} Most arise from teratomatous elements of Type II PMGCTs and only very rarely from Type I PMGCTs.¹⁶ They can resemble a variety of malignant somatic neoplasms, such as sarcomas or carcinomas.^{117, 118} STSMs most commonly develop in male patients in the second or third decade of life, with a range from 8 to 76 years.^{24, 116, 119-121} STSM components share molecular alterations with the accompanying PMGCT components, especially alterations of chromosome 12 such as i(12p).^{20, 122-125} STSMs may occur simultaneously at the time of diagnosis or successively in residual tumours (e.g. with or without prior chemotherapy) or in tumours at metastatic sites.

The current diagnostic criteria for STSMs required by the 5th edition of the WHO classification of thoracic tumours is an overgrowth of sufficient size, occupying an area of at least a ×4 microscopic field.¹⁷ However, for the sake of uniformity, we recommend using as a criterion at least 5 mm in diameter for the diagnosis of STSMs, because this was proposed by the 5th edition of the WHO classification of tumours of the urinary tract and male genital organs as the diagnostic criteria of STSMs.¹²⁶ Furthermore, the pathological report should include the percentage of STSMs in a PMGCT because of its prognostic significance.²⁴

The most frequent STSMs are sarcomas, especially rhabdomyosarcomas (60%) and angiosarcomas,^{24, 127} but also leiomyosarcomas, malignant peripheral nerve sheath tumours and primitive neuroectodermal tumours (PNETs) can be seen.¹⁶ The current WHO of Tumours of the urinary tract and male genital organs changed the name of PNET to embryonic-type neuroectodermal (ENET) tumour because of their similarity to paediatric embryonal tumours of the central nervous system. Although this has not yet been adopted for PNETs in PMGCTs,¹²⁸ it is reasonable to use the same nomenclature. Carcinomas often resemble intestinal adenocarcinoma,^129-134 adenosquamous and squamous cell carcinomas,¹³⁵ but also neuroendocrine neoplasms such as neuroendocrine carcinomas (Figure 3F) or neuroendocrine tumours.^136-139 Other STSMs include glioblastomas.^{16, 140} The immunohistochemical pattern of STSMs is similar to that of the corresponding somatic neoplasms, often with loss of GCT markers.

Levy et al. described a case series with a new entity called vasculogenic mesenchymal tumour (VMT).¹⁴¹ Their cases of 55 patients were associated with yolk sac tumour components, and all specimens were analyzed after chemotherapy. A few similar cases have been published previously, associated with PMTERs independent of prior chemotherapy.^142-145 Recently, a case report also showed a VMT combined with a PMSEMs without prior chemotherapy.¹⁴⁶ VMT consists of neoplastic vascular and stromal components that show different grades of atypia in both components (low- and high-grade VMT). Immunohistochemically, vascular components stain positive for CD31, CD34, ERG and D2-40, and stromal components were positive for smooth muscle actin, desmin, and caldesmon.¹⁴¹ Typical PMYSTs markers such as glypican-3 were positive only in a few cases. A case report by Fujii et al. showed different results with positive stainings for glypican-3 in vascular components of a VMT.¹⁴⁷ Furthermore, VMT might be a precursor lesion for angiosarcomas or other sarcomas and show a high association with associated hematological malignancies.¹⁴¹ Pathologists need to recognise this entity in initial biopsies, but also in the biopsies from patients after treatments.

PMGCTs with associated haematological malignancies

Type II PMGCTs with associated haematological malignancies (HMs) are somatic-type malignancies that show a secondary malignant haematolymphoid neoplasm with similar molecular alterations, indicating a common origin.^{141, 148-151} HMs have only been described in PMGCR (2–3%) but not in GCT of other primary sites,^{152, 153} and have been reported only in male patients.¹⁵⁴ An association with Klinefelter syndrome has been reported in 10–20% of HMs.^{38, 155, 156} Neoplastic haematopoietic stem cells can be found in stromal components of PMYSTs in cases with HM.¹⁴⁹

The HM and PMGCT component, most often PMYST but also immature PMTER and mixed PMNSGCT, share similar molecular alterations, and the HM develops independently of a prior chemotherapy. In particular, i(12p) is the most common alteration found in both subsets in 48% of cases,^{124, 148, 157-159} but HM also show other alterations determining the subtype of HM, such as del(5q) or trisomy 8.¹²⁴ Other molecular alterations include mutations of TP53, KRAS or PTEN.^{160, 161} A chemotherapy-induced PMGCT-independent malignant haematopoietic neoplasm has to be ruled out.^{124, 148, 151, 153, 159, 162}

Many different types of HM have been described, including acute myeloid leukaemia,^{6, 148, 151, 159, 163, 164} acute lymphatic leukaemia,^{159, 165, 166} myeloproliferative neoplasms (e.g. chronic myeloid leukaemia, essential thrombocytosis, primary myelofibrosis)¹⁵⁹ or myelodysplasia (e.g. with blast excess),^{148, 151, 158, 159, 167} as well as malignant histiocytosis,^{150, 151, 155, 159, 168, 169} histiocytic sarcoma,¹⁷⁰ malignant mastocytosis,¹⁷¹ and granulocytic sarcoma,^{172, 173} with acute megakaryoblastic leukaemia (AML-M7) being the most common HM.^{6, 174} The diagnostic criteria and immunohistochemical markers are identical, as in their somatic counterparts.

Differential diagnosis and diagnostic pitfalls

PMCGT subtypes, especially PMYSTs with different morphological patterns, can mimic each other, and may have overlapping immunohistochemical markers. The combined use of the above-mentioned immunohistochemical markers is therefore helpful to clearly differentiate these neoplasias in most cases (Figure 6),²¹ even though there is no single specific marker for each PMGCT subtype.

Thymic epithelial tumours (e.g. thymomas, thymic carcinomas), lymphomas, neurogenic tumours and metastases of carcinomas are the most important differential diagnoses that have to be investigated and excluded.¹⁷ In addition, benign diseases such as thyroid goitres, cysts and granulomatous reactions can also cause tumoral masses in the mediastinum. There are some morphological and immunohistochemical pitfalls that pathologists need to be aware of.

First, high-grade carcinomas can mimic solid growth patterns of PMECs, PMYSTs and PMCCs. Fetal adenocarcinomas of the lung and hepatoid carcinomas are particularly difficult to differentiate from YST because of overlapping morphology and immunohistochemical features. Fetal adenocarcinomas can have a clear cytoplasm and hepatoid carcinomas can mimic hepatoid YST. Moreover, both neoplasms can be positive for SALL4, glypican-3, FOXA2, CDX2 and even AFP.^{175, 176} Consequently, investigation of molecular differences and clinical aspects must be evaluated for the final diagnosis.

Secondly, rare entities such as thoracic SMARCA4-deficient undifferentiated tumour can also mimic PMGCTs. The tumour cells may have a pale cytoplasm with medium-sized nuclei and prominent nucleoli, and may also stain positive for germ cell markers such as SALL4 and SOX2, with variable positivity for keratins.^{177, 178} They can be diagnosed by loss of SMARCA4/BRG1 immunohistochemically or additionally with molecular analysis.¹⁷⁷

Thirdly, NUT carcinomas can be a diagnostic pitfall because they can imitate PMGCTs such as PMEC if the biopsy does not contain cells with squamous differentiation. Furthermore, NUT carcinomas can rarely express AFP and elicit elevated AFP serum levels and commonly show strong nuclear SOX2 staining.⁶² SALL4 expression is helpful in this setting, as it is rare and usually only focal in NUT carcinomas.^{21, 179, 180} Conversely, NUT staining can be potentially misleading, as germ cell tumours can express NUT focally.¹⁸¹ As a result, the differential diagnosis between PMGCTs and NUT carcinoma can sometimes need a broad spectrum of immunohistochemical markers or even molecular studies which address the typical NUTM1-fusions of NUT carcinomas.¹⁸²

Fourthly, PMSEMs in particular show positive immunohistochemical markers, which are also positive in other neoplasias, such as CD117 in rare thymomas and most thymic carcinomas¹⁸³ or melanomas, D2-40 in lymphatic or mesothelial neoplasms^{184, 185} and TdT in lymphoblastic lymphomas.^{21, 186, 187} Furthermore, rare synchronous PMSEMs and thymic neoplasms have been described.^188-191 For pure PMTERs, the main differential diagnosis encompasses bronchiogenic cysts^{192, 193} and multilocular thymic cysts (MTCs).^{84, 194} There is also the possibility of a mixed PMTER with MTC.¹⁹⁵

Molecular alterations

Staging

No official staging system for PMGCTs has been acknowledged so far by the AJCC/UICC. Moran and Suster proposed a staging system based on the analysis of 322 cases.^{27, 209} Their proposal is limited, however, by the fact that it depends upon surgical resection specimens without pretreatment, while current practice relies upon pre-operative chemotherapy as state-of-the-art (see ‘Therapy’ below).

Therapy

The first-line therapy of PMGCTs consists of primary chemotherapy and subsequent surgical resection of remaining tumour tissue, although there is no level 1 evidence due to lacking randomised clinical trials.²¹⁰

Chemotherapy encompasses bleomycin, etoposide and cisplatin (BEP), as in testicular GCTs.^{7, 28} PMSEMs are usually treated with three cycles of BEP or four cycles of EP if bleomycin is not feasible, while PMNSGCTs are treated with three to four cycles of BEP.²⁸ Recent studies which aimed to reduce severe lung damage (e.g. pneumonitis and fibrosis) suggest that the use of ifosfamide instead of bleomycin in combination with etoposide and cisplatin can reduce the high risk of surgical and postoperative morbidity and mortality.²¹⁰

Resection of PMSEMs after chemotherapy is optional, depending upon whether or not vital residual tumour is suspected. Small PMSEMs can also be treated with initial surgery and adjuvant chemotherapy. In contrast, surgical removal of PMNSGCTs is mandatory after chemotherapy, and post-chemotherapeutic surgery with complete resection of the tumour mass is essential for long-term survival, as vital tumour cells might still exist or somatic-type solid malignancies might have developed.^{6, 154, 211} Pathologists should therefore sample the tumour tissue thoroughly and report the amount of vital tumour cells, although there is currently no established system to indicate the degree of regression.

In cases of PMGCTs that are refractory to conventional chemotherapy, radical surgical intervention is necessary as radiation therapy is only helpful for brain metastases. Patients with recurring diseases and persistently elevated serum tumour markers may be eligible for high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Some patients show a good response to this treatment, but the majority still has a poor response, and surgical resection of residual tumours is indicated.^212-214

Prognosis and clinical outcome

The prognosis and clinical outcome varies widely between the different PMGCT subtypes. Type I PMGCTs are considered benign, whereas Type II PMGCTs are malignant neoplasms, except for pure mature PMTERs, contrary to testicular teratomas. Pure mature PMTERs (adult-type) are benign tumours that only need therapy (surgery) when compression of mediastinal structures (e.g. vessels, heart) occurs.^{15, 16} Immature teratomas tend to pursue a slightly more aggressive clinical course.^{222, 223} A specific condition in which teratomas show clinical complications is the growing teratoma syndrome (GTS). GTS describes the growth of a teratoma component in a mixed PMGCT after chemotherapy in spite of falling serum tumour markers.²²⁴ In these cases, surgical resection is recommended^{224, 225} and sometimes necessary, as GTS might lead to cardiovascular deterioration.^{224, 226}

The development of a STSM or an associated HM usually implies a very poor outcome. Patients with STSMs show a median overall survival of 9 months, with few cases with progression-free survival (PFS) of more than five years,^{24, 120, 227} while patients with HMs have a median overall survival (OS) time of 6 months with no patients surviving longer than 2 years.^{121, 228} Complete surgical resection is the therapy of choice, because STSMs and HMs show resistance to PMGCT-directed chemotherapeutic schemes and might need HDCT,²²⁹ where maintenance with oral etoposide therapy might be transiently effective.²³⁰

PMSEMs show a much better long-term PFS and OS in comparison to PMECs, PMYSTs and PMGCTs, with approroximately 95%, respectively, because of their good response to CT and radiation.^{117, 211, 231-233} A worse prognosis is associated with visceral metastases or more than two metastatic sites.²¹¹ Long-term PFS in high risk PMGCTs is approximately 35–45% for PMNSGCTs,^{211, 231, 232, 234, 235} where PMYSTs have a better prognosis than PMECs and PMCCs.²³⁶ PMCCs have the worst prognosis, because they often show haematogenous spread and distant metastases at the time of diagnosis.^{93, 237-239} Care must be taken, as histological subtypes might differ between primary and metastatic sites.²⁴⁰

It has been proposed that an evaluation of serum tumour markers after the first dose of CT should be performed because their normalisation is a favourable prognostic factor.²⁴¹ An intensified CT regimen in patients with insufficient decline of serum tumour markers should follow, because these patients showed a higher PFS in 3-year interval.²⁴²

In post-CT resected PMGCTs, the assessment of vital tumour cells is essential. Favourable prognostic factors for a good outcome include complete surgical resection, the absence of metastases and fewer than 10% vital tumour cells, together with ‘good prognostic group’ features, according to the International Germ Cell Cancer Collaborative Group.^{154, 211, 243} Immunohistochemical analysis is often required to identify vital tumour cells between fibrosis, necrosis and inflammation. Vital teratomatous components can be found in approroximately 30–40% of tumours.^{7, 244-246} Another indicator of poor prognosis is a TP53 mutation,²⁴⁷ as it is linked to cisplatin resistance.²⁰⁴

Conclusion

The diagnosis of PMGCTs is usually made on small biopsies and has to take a wide range of differential diagnoses (e.g., thymic neoplasias, lymphomas, etc.) into account. Types I and II PMGCTs show differences in age distribution, morphology, molecular characteristics, and clinical behavior (Figure 6). PMGCTs with STSMs or associated HMs make the spectrum even more complex. The clinical correlation and the assessment of tumour markers such as AFP and β-hCG is therefore essential in patients with anterior (prevascular) mediastinal masses. Histopathological analysis should include the detection of characteristic morphological patterns and the additional use of suitable immunohistochemical marker panels. In challenging cases of Type II GCTs, detecting i(12p) can be very helpful. In the future, methylation assays may also prove useful in difficult cases, especially in tumours with STSMs and HMs. Finally, pending further studies, initial molecular analyses might be helpful in detecting prognostic factors associated with poor treatment response or potential therapeutic targets.

Funding information

F.B. is supported by the Wilhelm Sander-Stiftung (grant numbers 2016.041.1, 2016.041.2, 2016.041.3).

Conflicts of interest

The authors confirm that there are neither conflicts of interest nor competing interests.