Most GCTMs are accidentally detected by imaging examinations. Patients may be either asymptomatic or symptomatic, with common symptoms such as chest pain, cough, dyspnea, fever, night sweats, and weight loss^[26]. A mediastinal mass has various possible etiologies; besides GCTs, it could be other tumors like thymic tumors, lymphoma, or metastatic carcinoma, or non-neoplastic diseases such as thyroid goiter, thymic cysts, or aortic aneurysms. Because treatment differs, all mediastinal masses should undergo a comprehensive evaluation process, which typically includes imaging, laboratory/molecular tests, and histopathological assessment, to guide treatment decisions [Figure 1].

For mediastinal nodules found in physical examination, especially asymptomatic, small anterior mediastinal nodules less than 3 cm in diameter, benign cysts are the main form, and most of the nodules remain unchanged during follow-up^[27]. To avoid unnecessary surgery, it is recommended to determine the type of a nodule based on the imaging characteristics of contrast-enhanced chest computed tomography (CT) and magnetic resonance imaging (MRI). Serum biomarker tests can be used to supplement to the diagnosis of suspected cases. Patients considered to have benign tumors should undergo regular follow-up to observe changes in the nodules.

Contrast-enhanced chest CT is the main imaging modality for evaluating mediastinal tumors. It can reveal the tumor’s morphology, blood supply, and relationship to surrounding vasculature^[28]. Three-dimensional reconstruction can be performed if necessary. For suspected vascular tumors or mediastinal tumors that invade or compress major vessels, angiography can be used for further evaluation^[7,29]. Additionally, MRI can reveal the relevant characteristics of the lesion by showing its morphology, border, cystic or solid nature, relationship with adjacent tissues/organs, and etc., which helps differentiate thymic hyperplasia, cysts, and malignant thymic neoplasms. For patients with contraindications to iodine-based contrast agents, MRI can serve as an alternative method for assessing mediastinal tumors^[7,29]. Positron emission tomography-CT (PET-CT) can be used for systemic evaluation in patients with mediastinal tumors, helping to determine the presence of recurrent or metastatic lesions. It is also a relatively effective tool for staging and assessing treatment response^[30,31]. One study indicated that PET-CT also has utility in assisting with the differential diagnosis of benign versus malignant lesions and in distinguishing between mediastinal SGCT and NSGCT^[32].

Consensus 2: Patients with mediastinal tumors found by physical examination or by accident are recommended to routinely receive contrast-enhanced chest CT/MRI scans and serum biomarker tests. If a tumor is assessed as benign, a CT/MRI re-examination is recommended to be performed after 3-6 months, and once every 1-2 years thereafter. Patients with suspected malignancies, abnormal serum biomarkers, and/or symptoms are recommended to receive the tissue biopsy as soon as possible to clarify the nature of the lesion. PET-CT may be considered for patients with mediastinal tumors that are difficult to accurately assess by conventional CT/MRI, or those who need a systemic examination and assessment. (Strongly recommended)

All male patients with suspected GCTMs should undergo testicular ultrasonography to rule out a primary gonadal tumor^[7]. Similarly, ovarian ultrasonography should be performed in suspected female patients. Some tumors may lead to inspiratory flow limitation, so pulmonary function tests are required for patients scheduled for surgery. If necessary, additional cardiopulmonary exercise testing may also be performed with reference to the preoperative assessment of lung surgery^[33]. For tumors suspected to originate from other sites, corresponding imaging examinations should be performed, for example, radioactive iodine scanning to assess activity in an intrathoracic goiter and a ^99mTc-isotope scan for a possible retrosternal parathyroid adenoma^[7].

Consensus 3: Any suspected GCTM should undergo adequate clinical and imaging examination to exclude the possibility of mediastinal metastasis from gonadal or retroperitoneal tumors or metastatic carcinoma from non-mediastinal primary sites. For tumors suspected to originate from other origins, specific imaging examinations of the relevant tissues and organs are routinely recommended. (Strongly recommended)

Although most of the mediastinal diseases are benign, there is still a possibility of a variety of malignant diseases, especially in patients who have already developed symptoms. Therefore, all mediastinal masses where the possibility of malignancy cannot be excluded should be subject to tissue biopsy. It is useful in obtaining tumor samples to determine the nature of the lesion through pathological examination. Options for diagnostic tissue biopsy include transdermal core needle biopsy, transbronchial needle aspiration, mediastinoscopy, mediastinotomy, and thoracoscopic surgery^[38]. For patients with suspected lymphoma, surgery is not routinely preferred due to the significant tumor heterogeneity, and tissue biopsy is an indispensable diagnostic method^[39]. When lymphoma is suspected, image-guided core needle biopsy with multiple passes is the preferred initial approach over fine-needle aspiration to ensure adequate tissue for IHC and flow cytometry. However, for resectable lesions that are highly suspected to be thymoma based on clinical manifestations and imaging features, biopsy should be avoided due to the risk of tumor capsule disruption and potential seeding. Sufficient tissue specimens for pathological and molecular examinations should be obtained in a single biopsy attempt. Additionally, all patients with confirmed GCTMs should undergo karyotype analysis to determine the presence of concomitant genetic syndromes^[40].

Consensus 5: Patients with mediastinal tumors where the possibility of malignancy cannot be excluded by typical imaging features and serum biomarkers are routinely recommended to receive tissue biopsy to carefully determine the nature of the lesion through pathological examination. An appropriate biopsy method should be selected based on the needs, and sufficient tissue specimens required for pathological examination and molecular testing should be obtained in a single biopsy attempt. If a CT-guided biopsy is performed, specimens containing necrotic margins should be adequately sampled. (Strongly recommended)

Consensus 6: Patients with pathologically confirmed GCTMs should be subjected to karyotype analysis to determine the presence of concomitant genetic syndromes. (Strongly recommended)

Different subtypes of GCTMs exhibit distinct pathological and molecular characteristics that facilitate their specific classification^[41]. Special attention should be taken for mixed GCTM, as non-teratomatous components may regress after neoadjuvant therapy, potentially inducing "growing teratoma syndrome", where the residual teratoma overgrows^[42]. Therefore, it is necessary to collect sufficient samples during the pathological examination, carefully observe for the existence of other GCT components, and make the judgment in combination with treatment history to exclude the possibility of mixed GCTMs.

Consensus 7: Any suspected GCTM should be adequately sampled, and the possibility of mixed GCTM should be carefully judged in combination with the treatment history. (Strongly recommended)

In the differential diagnosis, the emphasis on evidence of pathology, immunohistochemistry (IHC), and molecular detection varies with different subtypes of GCTMs. For mediastinal teratoma, since there are no specifically elevated serum biomarkers, the differential diagnosis mainly depends on tissue biopsy and pathological examination. Histologically, mature mediastinal teratoma is manifested as phenotypically well-differentiated tissues deriving from one of the three germinal layers (e.g., skin with skin appendages, teeth or bone, and cartilage), with components such as keratinized or non-keratinized squamous epithelium, skin appendages (e.g., hair, sweat glands, and sebaceous glands), respiratory epithelium, fat, and cartilage commonly observed, and a high incidence of pancreatic tissues^[43]. Other lesions that need to be differentiated from mediastinal teratomas include multilocular thymic cysts^[44] and somatic-type malignant tumors^[45]. The former shows xanthogranulomatous inflammation and myofibroblastic inflammation, while the latter often presents as well-circumscribed nodules with significant cellular atypia and invasive growth.

Compared to benign mediastinal teratoma, organoid morphology is less common, while cellular atypia is more common in malignant mediastinal teratoma. A study on 34 cases of GCTMs provided some pathological characteristics that could aid in the diagnosis of benign or malignant mediastinal teratoma^[46]: (1) Compared to benign teratoma, organoid structures were relatively rare in malignant teratoma (83% vs. 27%), with no pancreatic differentiation; (2) The incidence of glial tissue was also higher in malignant teratoma (11% vs. 63%); (3) If cartilage was present, it could be used to assess less obvious atypia in the epithelium, where malignant tumors feature frequent multinucleation, proliferation, and apoptosis. Additionally, comparative genomic hybridization techniques can also assist in the differential diagnosis of mediastinal GCTs. Mature teratomas have a normal genetic profile with no 12p abnormalities^[47], whereas malignant mediastinal GCTs often exhibit gains/losses of chromosomal arms^[48]. For malignant GCTM patients under 8 years old, their genomic features are more similar to those of testicular GCTs of the same age. Common genomic variations observed include gains of 1q, 3, and 20q, as well as losses of 1p, 4q, and 6q. Conversely, adolescent and adult patients commonly show gains of 12p. Other reported genome variations include loss of chromosome 13 and gain of chromosome 21/X^[47]. Universal thresholds for genomic variations such as i(12p) have not yet been standardized for mediastinal GCTs.

Consensus 8: Any suspected mediastinal teratoma found in pathological examination should be adequately sampled and determined for the benign or malignant nature in combination with treatment history, and be subjected to differential diagnosis with multilocular thymic cysts and somatic-type malignant tumors to avoid misdiagnosis. Although the differential diagnosis of mediastinal teratomas is primarily based on pathological morphology, IHC is routinely recommended as an adjunctive test to prevent missing occult components. For immature mediastinal teratoma with uncertain differential diagnosis, supplemental 12p fluorescence in situ hybridization (FISH) or reverse transcription-polymerase chain reaction (RT-PCR) testing can be considered to assist in determining germ cell origin. (Strongly recommended)

Mediastinal SGCTs should be subjected to differential diagnosis with other GCTMs or mediastinal tumors such as thymic epithelial tumors, mediastinal B-cell lymphomas, and metastatic melanomas through histomorphology and IHC. Particularly, metastatic SGCTs from the testis are prone to misdiagnosis due to similar features. The possibility of primary testicular seminoma should be excluded based on clinical history, physical examination, and imaging features^[49]. Tumor cells of mediastinal SGCTs are arranged in sheets or nested form, with a homogeneous cell morphology, and are characterized by unique fibrovascular septa and lymphocytic infiltration. It is worth noting that inflammation can mask tumor cells. Additionally, scattered multinucleated syncytiotrophoblast cells may also appear in mediastinal SGCTs, so choriocarcinoma should not be diagnosed unless mononuclear cytotrophoblast cells are present. Unlike thymic carcinoma, mediastinal SGCT presents as discrete epithelioid cells that express CD117 in IHC^[43]. Mediastinal SGCTs with positive CAM5.2 are prone to being misdiagnosed as carcinoma, so differential diagnosis is required in combination with OCT3/4 and 12p tests^[50]. Besides IHC, some molecular characteristics are also considered valuable for the auxiliary diagnosis of mediastinal SGCTs. A retrospective study found that TP53 mutations occurred only in mediastinal NSGCTs but not in mediastinal SGCTs (72.2% vs. 0%)^[51]. Two other studies pointed out that there were differences in frequencies of p53 expression (31% vs. 77%), KIT exon 17 mutations (50% vs. 0%), and KRAS mutations (8% vs. 15%) between mediastinal SGCTs and testicular SGCTs, suggesting that they could assist in the differentiation of primary and metastatic SGCTs^[52,53].

Consensus 9: Mediastinal SGCTs should be subjected to differential diagnosis with other GCTMs or mediastinal tumors through histomorphology and IHC, and at least 3 biomarkers: CD117, OCT3/4, and SOX2 (negative) should be tested. Meanwhile, the possibility of primary testicular seminoma should be excluded based on clinical history, physical examination, and imaging features. If necessary, additional genetic testing may be performed to assist in the diagnosis. (Strongly recommended)

YSTs exhibit diverse morphologies. Common patterns include reticular, myxomatous, endodermal sinus, polyvesicular-vitelline, and solid, while rarer patterns include hepatoid, spindle cell, enteric, and endometrioid. IHC is a crucial method for the differential diagnosis of YSTs. Co-expression of AFP, glypican-3 (GPC3), and spalt-like transcription factor 4 (SALL4) can help differentiate YSTs from metastatic carcinoma^[54]. However, due to the lack of specificity of the above biomarkers, a comprehensive diagnosis is still needed based on pathological morphology, clinical factors, and imaging features, such as identifying typical YST areas like Schiller-Duval bodies. Other tumor subtypes that are easily confused and require differential diagnosis include: primary thymic carcinoma, which can be differentiated by positive CD5; seminoma, which can be differentiated by positive OCT3/4, CD117, and D2-40, and by negative GPC3^[45]; embryonal carcinoma, which can be differentiated by positive CD30 and OCT3/4, and cell atypia; sarcoma, which can be differentiated by negative keratin staining and positive staining of mesenchymal markers; multilocular thymic cyst, which can be differentiated by the absence of YST cells^[41]. It is important to note that YSTs may contain focal syncytiotrophoblast cells, which should not be over-interpreted as choriocarcinoma.

Embryonal carcinomas are solid, adenoid, or papillary in arrangement, with significant cellular atypia, and are accompanied by extensive necrosis. They should be carefully differentiated from other GCTs and malignant tumors. Compared with SGCTs, embryonal carcinoma exhibits more cellular atypia and more blurred cell borders, often with common positive CD30^[41]. Compared with YSTs, embryonal carcinoma shows a relatively greater degree of cellular atypia, but typical PAS-positive hyaline bodies (a characteristic of YST) can still be observed in some pediatric cases^[34]. Similar to choriocarcinoma, embryonal carcinoma shows large syncytial cells^[55] and an appliqué pattern of degenerative change at the periphery of nests^[9]. Compared with thymic carcinoma or other metastatic carcinomas, although the cellular atypia is similar, IHC can effectively assist in the differential diagnosis^[41]. Finally, compared to other hematological malignancies such as anaplastic large-cell lymphoma, both CD30 and epithelial membrane antigen (EMA) can be positive, so epithelial structures such as glandular or papillary morphology should be carefully observed for differential diagnosis^[41].

For choriocarcinoma, a “biphasic cell” population of syncytiotrophoblast cells and cytotrophoblast cells, together with the villous-like spatial configuration formed by multinucleated cells surrounding mononuclear cells, is its key characteristic. It can be easily confused with pleomorphic carcinoma of the lung, as both exhibit multinucleated and syncytial cells, with the production of hCG. Characteristics such as multiple tumor masses invading the lung, a second population of cytotrophoblast cells, and a younger age of onset are helpful in diagnosing choriocarcinoma^[56]. Additionally, characteristics such as the absence of significant atypia, a biphasic cell population, and hCG staining are necessary for differentiation from thymic carcinoma.

Consensus 10: The diagnosis of mediastinal NSGCTs should be based on the comprehensive consideration of IHC and histomorphology, clinical factors, and imaging features, and differential diagnosis should be performed with other GCTs, malignant tumors, or metastatic carcinoma. For YSTs, at least 3 biomarkers: AFP, GPC3, and SALL4, should be tested; for embryonal carcinoma, at least 3 biomarkers: CD30, OCT3/4, and SOX2, should be tested; for choriocarcinoma, at least hCG should be tested; if necessary, tests for other markers should be added. (Strongly recommended)

Common genetic variations in GCTM include mutations or deletions of TP53, activating mutations of KRAS/NRAS, loss of PTEN, and etc.^[57]. These variations may be helpful for the differential diagnosis of subtypes and primary sites of GCT. Among them, common gene mutations in mediastinal NSGCT include: TP53 (46%), KIT (18%), KRAS (18%), PTEN (11%), NRAS (4%), and PIK3CA (4%)^[58,59]. Compared with testicular GCT and SGCT (both gonadal and extragonadal), mediastinal NSGCT has a higher frequency of mutations in TP53, PIK3CA pathways (e.g., PTEN, PIK3CA), and cell cycle-related genes (e.g., CCND1/2/3, CDK4/6, CDKN2A/B, RB1), while the frequency of gene mutations in RAS-RAF (e.g., KRAS, NRAS), RTK (e.g., KIT), and DNA damage response (e.g., BRCA1/2, ATM, CHEK2, MUTYH) pathways does not differ significantly^[58,60].

Teratoma with somatic-type malignancies refers to GCT containing non-germ cell-derived malignant components such as sarcoma or carcinoma. Common sarcomas include rhabdomyosarcoma and angiosarcoma. There are also case reports of colon adenocarcinoma, glioblastoma, melanoma, carcinoid, neuroendocrine carcinoma, and other concomitant cancers^[41]. Although these cells retain their respective morphology and phenotype, their mutational and methylation profiles are close to those of GCT^[61,62] and differ significantly from the molecular characteristics of the primary tumor^[63]. The concomitant somatic-type malignancies generally retain chromosome 12p aberrations^[64,65], which is a key factor in distinguishing teratoma with somatic-type malignancies from metastatic cancer. Furthermore, for adenocarcinoma components that are easily confused with embryonal carcinoma, IHC for embryonal carcinoma-specific CD30 and OCT3/4 may aid in differential diagnosis^[66].

Consensus 11: Teratoma with somatic-type malignancies should be differentiated from metastatic carcinoma through 12p FISH/RT-PCR testing. If any adenocarcinoma component is detected, IHC for CD30 and OCT3/4 is recommended to differentiate from embryonal carcinoma. (Strongly recommended)

For GCT with associated hematological malignancy, differential diagnosis should be mainly performed with chemotherapy-related hematological malignancies^[17]. The former has a clonal isochromosome 12p [i(12p)], and the hematological malignancy typically appears earlier, with a median time of only 4.8 months^[67], whereas chemotherapy-related hematological malignancies typically occur 25-60 months after chemotherapy^[45].

Consensus 12: GCT with associated hematological malignancy should be differentiated from chemotherapy-related hematological malignancies through 12p FISH/RT-PCR testing and considering the time of onset. (Strongly recommended)

Tables 1 and 2 outline the IHC and molecular markers useful for the differential diagnosis of GCTM, respectively^[60,68]. IHC markers specific to lymphoma (e.g., CD30) and epithelial cells [e.g., Cytokeratin (CK)] can be used to rule out tumors of other origin. Stem cell markers such as NANOG, OCT3/4, SALL4, and SOX2 are crucial for the differential diagnosis of GCT subtypes. Specifically, SGCT generally expresses KIT and OCT3/4, but SOX2 is negative; YST generally expresses AFP, GPC3, and SALL4; Embryonal carcinoma generally expresses OCT3/4 and SOX2; hCG is specifically expressed in choriocarcinoma^[41,60,68]. To accurately diagnose the subtypes of GCTM, a combination of pathological morphology and at least 3 IHC markers is required to obtain a reliable diagnosis^[68]. Furthermore, novel biomarkers have been confirmed to have been confirmed to have value in the diagnosis of specific subtypes of GCT, such as glial cell line-derived neurotropic factor receptor alpha-1 (GFRA-1) specifically expressed in immature teratoma^[69], CAM5.2 positive in mediastinal SGCT but not in testicular SGCT^[70], LIN28 positive in SGCT and YST but negative in teratoma^[71], and HNF1-β and ZBTB16 specifically expressed in YST^[72,73].

Consensus 13: IHC is important for determining the pathological subtypes of GCTMs. All patients with suspected GCTMs are recommended to be tested for AFP, CD117, CK, GPC3, hCG, OCT3/4, SALL4, and SOX2. When the differential diagnosis is still uncertain, testing of relevant novel biomarkers may be considered. (Strongly recommended)

Molecular testing is also a key to differential diagnosis of GCTM. Chromosome 12p abnormalities, such as i(12p) or increased 12p copy number, are classic molecular characteristics of GCT. i(12p) is associated with the strong tumor aggressiveness of GCT. Genes located in the short arm of this chromosome may be associated with the occurrence and development of GCT, including KRAS, Cyclin D2, FGF6, and etc., but the pathogenic mechanism is still unclear^[60]. Except in pediatric and benign GCTs^[74,75], 12p abnormalities are prevalent in malignant GCTs, regardless of subtype^[76,77]. For cases where the origin of germ cells is difficult to determine, additional testing of 12p status may be considered to differentiate mediastinal GCT from other mediastinal tumors such as thymoma and lymphoma, and to differentiate primary mediastinal tumors from metastatic gonadal tumors^[68,78,79].

Consensus 14: For cases where germ cell origin is uncertain, additional 12p FISH/RT-PCR testing should be considered to differentiate mediastinal GCTs from other mediastinal tumors such as thymoma and lymphoma, and to distinguish primary mediastinal tumors from metastatic gonadal tumors. (Strongly recommended)

GCTM has a worse prognosis than GCT occurring in other sites (5-year PFS rate of 35% vs. 77% and 5-year OS rate of 40% vs. 82% for mediastinal NSGCT compared to testicular GCT), and mediastinal SGCT has a better prognosis than mediastinal NSGCT (5-year PFS rate of 80% vs. 35% and 5-year OS rate of 85% vs. 40%)^[10,87]. For pediatric patients, GCTM also has a worse prognosis compared to other sites (5-year OS rate of 49% vs. 89%^[141]), but patients with malignant GCTM have a relatively better prognosis compared to adults, with a 5-year survival rate of approximately 60%^[142]. It should be noted that patients with certain subtypes of GCTM have an extremely poor prognosis. For example, the median cancer-specific survival is only 12 months for choriocarcinoma and embryonal carcinoma^[141], whereas the median survival is only 6 months for GCTM patients with associated hematological malignancies^[17].

Prognostic evaluation is helpful in guiding treatment decision for GCTM. The International Germ Cell Cancer Collaborative Group (IGCCCG) classification is a classic prognostic model for GCT^[143]. For NSGCT, primary mediastinal tumor, elevated AFP/hCG/LDH, and visceral metastasis outside the lung (e.g., liver, bone, brain) are independent factors for poor prognosis. For SGCT, visceral metastasis other than the lung is the main adverse prognostic factor. By combining the aforementioned prognostic factors, GCT patients can be effectively stratified according to prognosis. Subsequent studies have further identified other factors for more precise patient prognosis stratification. A study found that in the IGCCCG poor-prognosis subgroup, patients with primary mediastinal tumors and lung metastases experienced worse outcomes, while patients without visceral metastases had a relatively better prognosis^[144]. Another study also indicated that in IGCCCG high-risk NSGCT, a subset of patients with a better prognosis can be identified based on the presence of an embryonal carcinoma component, high Ki-67 expression, low apoptotic protein levels, and low p53 expression^[145]. For GCTM, Moran and Suster established a staging system based on lesion location and extent of tumor involvement, which can further subdivide the prognosis of patients on the basis of pathological subtypes^[146]. Other reported factors associated with the prognosis of GCTM include age, tumor size, number of metastatic sites, completeness of surgical resection, presence of complete tumor necrosis after chemotherapy, and ratio of neutrophil to lymphocyte^[93,147-151]. For teratomas not covered by IGCCCG, studies have shown that the presence of digestive tract tissue, immature neuroectodermal cells, or somatic malignant transformation suggests a higher tumor aggressiveness^[152-154].

Consensus 23: The IGCCCG classification can be utilized for prognostic stratification of GCT patients, but there remains room for optimization. TP53 mutations, expression of Ki-67, and the ratio of neutrophil to lymphocyte may also contribute to the prognosis of GCTM patients. (Recommended)

Because patients with a history of GCTM are at higher risk of subsequent AML-M7 development^[14] and have a 10% chance of developing metachronous testicular cancer (for male patients) within 10 years^[155], long-term follow-up after treatment is recommended for all GCTM patients. Serum biomarkers such as AFP and hCG are important for post-treatment monitoring and can be used to assess the presence of residual tumor after surgery and the risk of postoperative recurrence^[156]. Nonetheless, these biomarkers perform poorly in identifying residual tumor after chemotherapy^[157]. Studies have found that miR-371-373 and miR-302 clusters are widely expressed in malignant GCTs and are potential biomarkers for dynamic monitoring of tumor burden and recurrence^[158]. A case report has shown that although elevated serum AFP made the differential diagnosis more challenging in a patient with mediastinal NUT carcinoma, miR-371a-3p levels were negative, suggesting a high specificity of this biomarker^[159].

Consensus 24: For mediastinal SGCT with a good prognosis, it is recommended to perform annual CT/MRI examination, testicular/ovarian ultrasound, and serum biomarker testing as follow-up after surgical therapy until 10 years postoperatively. For mediastinal NSGCT with a poor prognosis, it is recommended to perform CT/MRI examinations, testicular/ovarian ultrasound, and serum biomarker testing every 6 months as follow-up within 3 years after surgical therapy, then change to annual re-examination. Special attention should be paid to evaluating the occurrence of hematological malignancies. If necessary, testing of miRNA levels may be considered as a means of monitoring tumor status. (Strongly recommended)