Molecular Pathology and Targeted Therapies for Personalized Management of Central Nervous System Germinoma

Intracranial germinomas are rare tumours, usually affecting male paediatric patients. They frequently develop in the pineal and suprasellar regions, causing endocrinological disturbances, visual deficits, and increased intracranial pressure. The diagnosis is established on magnetic resonance imaging (MRI), serum and cerebrospinal fluid (CSF) markers, and tumour stereotactic biopsy. Imaging techniques, such as susceptibility-weighted imaging (SWI), T2* (T2-star) gradient echo (GRE) or arterial spin labelling based perfusion-weighted MRI (ASL-PWI) facilitate the diagnosis. Germinomas are highly radiosensitive tumours, with survival rates >90% in the context of chemoradiotherapy. However, patients with resistant disease have limited therapeutic options and poor survival. The aim of this review is to highlight the genetic, epigenetic, and immunologic features, which could provide the basis for targeted therapy. Intracranial germinomas present genetic and epigenetic alterations (chromosomal aberrations, KIT, MAPK and PI3K pathways mutations, DNA hypomethylation, miRNA dysregulation) that may represent targets for therapy. Tyrosine kinase and mTOR inhibitors warrant further investigation in these cases. Immune markers, PD-1 (programmed cell death protein 1) and PD-L1 (programmed death-ligand 1), are expressed in germinomas, representing potential targets for immune checkpoint inhibitors. Resistant cases should benefit from a personalized management: genetic and immunological testing and enrolment in trials evaluating targeted therapies in intracranial germinomas.


Introduction
Intracranial germ cell tumours (ICGCT) are rare tumours that primarily affect children and adolescents, with a male predominance, accounting for 3.6% of brain tumours in Western Europe and reaching a higher incidence of 15.4% in Japan [1][2][3]. A comparative study between Japanese and American populations regarding ICGCT revealed a different distribution of the tumours. More cases of basal ganglia involvement were present in the Japanese, whereas more bifocal (synchronous pineal and suprasellar) locations in western

Biological Markers
Different biomarkers have been studied in order to establish the optimum diagnosis. A relative correlation between serum and cerebrospinal fluid (CSF) biomarkers and the tumour's histological category was set. Alpha-fetoprotein (AFP) is elevated in embryonal carcinomas and teratomas, whereas choriocarcinomas and germinomas secrete β-hCG [47,48]. However, germinomas have an inconsistent secretion of β-hCG [49,50]. There is no clear cut-off for hCG levels to distinguish germinomas from mixed germ tumours, but it is considered that pure germinomas produce no or mild levels of CSF β-hCG (<50 mUI/mL), in the latter cases being classified as high risk and requiring a more aggressive chemotherapy regimen [37,51]. A study conducted on 80 germ cell tumours revealed a sensitivity of 78.9% and a specificity of 96.6% for CSF tumour markers (with a cut-off of 50 IU/L for ß-HCG and 25 ng/mL for AFP). Marker positive germinomas, as well as marker negative NGGCT have been reported [52].
Other biomarkers associated with germinomas include elevated lactate dehydrogenase (LDH) and placental alkaline phosphatase (PLAP). These biomarkers, in combination with the radiological finding of a pineal mass, could provide a high suspicion of the histological subtype, especially in cases of heterogeneous tumours, and may represent a way of monitoring the treatment response [53]. Aihara et al. discovered that the CSF PLAP level is a specific marker for pure germinomas, which can provide a reliable diagnosis of intracranial germinoma, in the absence of a histopathological examination [54]. However, Chiba et al. propose that CSF PLAP levels, with a cut-off value of 8 pg/mL, also correlate with the germinoma component in the context of mixed GCT [55]. The diagnostic algorithm of CNS germinomas, including the main serum/CSF biomarkers, is shown in Figure 1.
with the radiological finding of a pineal mass, could provide a high suspicion of the histological subtype, especially in cases of heterogeneous tumours, and may represent a way of monitoring the treatment response [53]. Aihara et al. discovered that the CSF PLAP level is a specific marker for pure germinomas, which can provide a reliable diagnosis of intracranial germinoma, in the absence of a histopathological examination [54]. However, Chiba et al. propose that CSF PLAP levels, with a cut-off value of 8 pg/mL, also correlate with the germinoma component in the context of mixed GCT [55]. The diagnostic algorithm of CNS germinomas, including the main serum/CSF biomarkers, is shown in Figure 1.

Radiological Characteristics
On standard MRI, germinomas appear as heterogeneous tumours in T1/T2-weighted imaging, in 40% of the cases, and the uptake of the gadolinium can be either homogeneous (47%) or heterogeneous (53%) [56]. Relevant images of a pure pineal germinoma are shown in Figure 2.

Radiological Characteristics
On standard MRI, germinomas appear as heterogeneous tumours in T1/T2-weighted imaging, in 40% of the cases, and the uptake of the gadolinium can be either homogeneous (47%) or heterogeneous (53%) [56]. Relevant images of a pure pineal germinoma are shown in Figure 2.
Multiple imaging studies have been conducted to provide a better characterization of germinomas. For example, Inoue et al. showed that 90% of the patients with pineal germinomas presented a cardioid-shape tumour image on the axial MRI views, due to its progression pattern on both sides of the third ventricle, concluding that this was a specific aspect for pure pineal germinoma [31]. Awa et al. described two significant features differentiating pineal germinomas from NGGCT: peritumoural edema thicker than 5 mm (peritumoural area with T2 hyperintensity) and bithalamic extension [57]. T2* (T2-star) sequence is generally used to obtain a better characterization of intratumoural/intraventricular/cerebral microhaemorrhage, iron deposits, and calcifications [58]. Susceptibility-weighted imaging (SWI) or T2* gradient echo (GRE) technique can be used for better differentiation between pure germinoma and NGGCT in the pineal region: 93% of the germinomas present iso-or hyperintensity, whereas NGGCT are hypointense compared to the healthy brain [56]. Another imaging technique, such as the arterial spin labelling based perfusion-weighted MRI (ASL-PWI) could be used in differentiating germinomas from NGGCT, based on lower values of relative tumour blood flow encountered in germinomas [59]. Calcification can be present in both germinomatous and non-germinomatous pineal tumours [56].
Suprasellar germinomas seem to develop from the tuber cinereum and median eminence, infiltrating the infundibulum [60]. Therefore, an isolated thickened pituitary stalk may be the first radiological appearance of a hypothalamo-hypophyseal germinoma [61,62]. However, they have a delay in diagnosis of a median of 1.4 years, due to insidious onset of symptomatology and MRI findings, often suggestive of inflammation (lymphocytic hypophysitis), pituitary adenomas, and secondary neoplasms, with radiological appearance similar to germinomas [63][64][65]. Nonetheless, GCT represent 66.7% of widened pituitary stalk causes in paediatric population, whereas germinomas represent the second etiology (21-31%) of enlarged pituitary stalk in adults [62,66]. Cases of diabetes insipidus followed by the occurrence of germinoma during the MRI follow-up have been described, highlighting the importance of imaging re-examination or endoscopic biopsy (with higher sensitivity compared to imaging studies) [38,61]. Usually, pituitary stalk infiltration is reversible following adequate treatment [60]. Multiple imaging studies have been conducted to provide a better characterization of germinomas. For example, Inoue et al. showed that 90% of the patients with pineal germinomas presented a cardioid-shape tumour image on the axial MRI views, due to its progression pattern on both sides of the third ventricle, concluding that this was a specific aspect for pure pineal germinoma [31]. Awa et al. described two significant features differentiating pineal germinomas from NGGCT: peritumoural edema thicker than 5 mm (peritumoural area with T2 hyperintensity) and bithalamic extension [57]. T2* (T2-star) sequence is generally used to obtain a better characterization of intratumoural/intraventricular/cerebral microhaemorrhage, iron deposits, and calcifications [58]. Susceptibil- Figure 2. MRI features of intracranial germinoma in a teenage patient: (a) pineal germinoma: heterogeneous contrast enhancement on axial gadolinium-enhanced T1-weighted image, with a tendency to cardioid shape; (b) pineal germinoma: heterogeneous contrast enhancement on sagittal gadolinium-enhanced T1-weighted image; (c) postoperative disseminated disease: bilateral nodular enhancement of anterior horns of the lateral ventricles on axial gadolinium-enhanced T1-weighted image; (d) postoperative disseminated disease: nodular enhancement of the hypothalamus, optic chiasm, mammillary bodies, and corpus callosum on sagittal gadolinium-enhanced T1-weighted image.
Basal ganglia and thalamus germinomas may present variable neuroimaging features (cystic lesion, peritumoural oedema, calcification, intratumoural haemorrhage, contrast enhancement, ipsilateral cerebral atrophy) that may impede reaching the correct diagnosis. Nonetheless, ipsilateral hemiatrophy seems to be a characteristic feature of basal ganglia and thalamus germinomas, which may differentiate them from other tumour types [44].
Surprisingly, the number of lesions detected on the MRI does not represent a poor prognosis factor and does not correlate with the overall survival in the setting of an appropriate treatment protocol [67].

Biopsy
In the management of germinomas, most studies recommend stereotactic biopsy for a definite diagnosis. Balossier et al. have shown that the histopathological diagnosis for pineal biopsies is more accurate with stereotactic procedures than with endoscopic procedures (93.7% vs. 81.1%) [68]. However, several studies emphasized the importance of endoscopic diagnosis, since in patients with pineal germinoma and DI, metastatic lesions to the third ventricular floor are more frequently identified by direct endoscopy than initially diagnosed by MRI [37,38]. Accordingly, biopsy-diagnosed pineal germinoma in DI patients should be classified as disseminated disease, even in the absence of MRI evidence [38]. Still, a retrospective multicentre study evaluated the necessity of performing biopsy in patients with bifocal tumour, diabetes insipidus, and negative tumour markers. The study included 91 patients with available histologic diagnosis, of which 92% were pure germinomas and germinomas with syncytiotrophoblastic giant cells, concluding that a tumour biopsy is recommended to ensure the proper diagnosis [69]. Another retrospective study revealed that in cases of pituitary germinoma suspected on MRI, the biopsy can reveal another pathology in 22% of the tumours [70]. Moreover, a fairly recent technique that combines endoscopic biopsy with endoscopic ventriculostomy, using a single trajectory, is considered safe and could become an alternative for the dual procedure in pineal germinomas [71].

Histological Diagnosis
Macroscopically, germinomas are solid, soft, grey-white, homogenous tumours; however, they can rarely present areas of haemorrhage, necrosis, or cystic components. They can be variably encapsulated or poorly circumscribed and infiltrative. Microscopically, they consist of large primordial germ cells (undifferentiated cells), with clear, abundant PAS+ cytoplasm, large, round nuclei, and prominent nucleoli; occasionally syncytiotrophoblastic giant cells may be present [72]. The cells have high mitotic activity and are organized in sheets, lobules, or nests patterns, separated by fibrovascular septae filled with lymphocytic infiltrates. Occasionally, the lymphoplasmacellular reaction is so robust that the granulomatous inflammation can obscure the tumour cells [5]. Therefore, a characteristic histopathological feature of germinoma is the "two-cell pattern": a massive immune cell population, with a high lymphocytic predominance, dispersed between tumour cells [73].
Immunohistochemistry (IHC) is further used to provide the histological diagnosis. The membrane immunoreactivity for C-kit (transmembrane protein with tyrosine kinase activity), CD30 (tumour necrosis factor receptor), and D2-40 (podoplanin) aid in differentiating germinomas from embryonal carcinoma and yolk sac tumours [5]. The nucleus is usually reactive for OCT 3/4 (octamer binding transcription factor 3/4), SALL4 (sal-like protein 4), UTF1 (undifferentiated embryonic cell transcription factor 1), NANOG (transcription factor in embryonic stem cells), and ESRG (embryonic stem cell-related gene protein), whereas ribosomes are positive for LIN28 (RNA-binding protein LIN28) [74,75]. Although PLAP is a distinctive marker of primordial cells, its expression is less consistent, being detected in 82% of germinomas. On the other hand, C-kit and OCT 3/4 are more sensible, with 100% staining among germinoma cells. When the germinoma also contains a syncytiotrophoblastic component, these cells are positive for hCG, human placental lactogen (HPL), CD 30, and CK AE1/3 (cytokeratin AE1/3) [72]. A summary of IHC staining and representative histological images from intracranial germinomas are shown in Table 2 and Figures 3 and 4. Table 2. Immunohistochemical staining in pure germinomas and germinomas with STGC.

Staging
Though there are no standard staging criteria for germinomas, a modified Chang staging system is usually used, based on aspect imaging and serum/CSF markers. Localized disease on MRI (no evidence of metastasis) and negative CSF cytology are classified as M0, while intracranial/spinal metastasis or positive cytology classify the tumour as disseminated disease M+. Bifocal disease is defined by synchronous pineal and pituitary tumours [76]. Accordingly, an M1 stage presumes positive CSF cytology for tumour cells, and an M2 metastatic germinoma is defined by intracranial nodular seeding (except bifocal disease). The presence of spinal metastases includes the patient in the M3 stage and

Staging
Though there are no standard staging criteria for germinomas, a modified Chang staging system is usually used, based on aspect imaging and serum/CSF markers. Localized disease on MRI (no evidence of metastasis) and negative CSF cytology are classified as M0, while intracranial/spinal metastasis or positive cytology classify the tumour as disseminated disease M+. Bifocal disease is defined by synchronous pineal and pituitary tumours [76]. Accordingly, an M1 stage presumes positive CSF cytology for tumour cells, and an M2 metastatic germinoma is defined by intracranial nodular seeding (except bifocal disease). The presence of spinal metastases includes the patient in the M3 stage and metastases outside CNS in the M4 stage [77].

Genetic Approach
Germinomas have immunohistochemical and molecular alterations similar to testicular seminoma, suggesting a common pathogenesis. One theory regarding the origin of germinomas postulates that these tumours develop from primordial germ cells (PGCs) that mismigrated in the midline structures during early embryogenesis. This hypothesis is sustained by the global hypomethylation of germinoma cells, an epigenetic hallmark of PGCs, and by the presence of specific germ cells markers (c-Kit, Oct-3/4, and Nanog). Another theory proposes that germinomas arise from a pluripotent braincell, via KIT gene mutations [73]. There is little molecular data regarding germinoma's development, considering the low incidence of this type of a tumour. Although rare, several familial cases of intracranial germinomas have been described, prompting further genetic studies regarding their tumourigenesis mechanism [78][79][80][81]. Fukushima et al. proposed that MAPK and/or PI3K pathway alterations, DNA hypomethylation, and chromosomal abnormalities represent a triad involved in the pathogenesis of pure germinomas [82].
A characteristic feature of germinomas that differentiates them from other ICGCT involves the methylation profile, germinomas presenting DNA hypomethylation, an acknowledged cause of genomic instability [82]. DNA methylation is a process normally involved in the epigenetic reprogramming of the germline during development, and, therefore, aberrant methylation patterns may have a significant role in the aetiology of germ cell tumours [83]. In a cohort of 54 germinomas, hypomethylation and KIT staining by immunohistochemistry were detected in 100% of the cases, underlining the resemblance to primordial germ cells [14]. Moreover, patients with chromosomal abnormalities (Down's and Klinefelter syndrome) have been reported to develop CNS germinomas [84,85]. Chromosomal gains and losses are frequently encountered in germinomas (as presented in Table 3) [14,80,86,87]. Chromosomal aberrations such as gain of 2q and 8q and loss of 5q, 9p/q, 13q, and 15q are associated with a worse prognosis [7]. Wang et al. hypothesize that meiosis errors are involved in the pathogenesis of germinomas since 90% of the cases present chromosomal instability [87].
The most frequent genes involved in the pathogenesis of these tumours are the KIT and RAS genes, encountered in up to 40% and respectively 34.6% of germinomas (Table 4). Interestingly, KIT and RAS mutations are described as mutually exclusive in 97-100% of cases [14,[86][87][88].    Gain of function mutations of KIT proto-oncogene generate a constitutive activation of the KIT protein, which consequently activates signal transduction molecules via MAPK (mitogen-activated protein kinase) or PI3K (phosphoinositide 3-kinase) pathways, resulting in increased cell proliferation, migration, and apoptosis resistance ( Figure 5) [86].
However, so far, no correlations have been established between the KIT gene mutation and the expression of the KIT protein or clinical parameters (tumour location, size, and prognosis) [90,91]. The frequency and type of KIT mutation are differently distributed in the population: 5.9% in the germinomas encountered in the Chinese population (missense mutation in exon 11), whereas 23-25% in the Japanese patients (75% affecting the exon 17 and 25% the exon 11, others involving exons 2, 13) [89][90][91]. Another study mentions exon 10 variant (c.1621A>C) as the most common encountered in germinomas, other KIT exonic variants including exon 2 (c.251C>T), exon 11 (c.1658A>G), exon 13 (c.1965T>A), exon 17 (c.2447A>T) [92]. Nevertheless, 27.4% of CNS GCT have no detectable KIT mutations, even though KIT expression by IHC is high, suggesting implication of other mechanisms [88]. For example, CBL is a tumour suppressor gene involved in the process of down-regulation of the KIT receptor, and its mutation causes sustained KIT activation. Somatic mutations in the CBL gene are frequently encountered in ICGCT and represent another cause of KIT overexpression in germinomas [87,88].
The PI3K/AKT and MAPK pathways seem to be widely implicated in the pathogenesis of germinoma, being present simultaneously in 83% of tumour cells [14]. MAPK pathway alterations are more frequent in germinomas than NGGCT and have a tendency to correlate with a better prognosis, in comparison with PI3K pathway mutations. Upregulation of MAPK pathway by somatic point mutations represents the dominant genetic alteration in germinomas (64.3% of cases) [88]. These cases are more frequent in male patients and seem to be associated with an elevated serum HCG [7]. A case of 16p11.2 microdeletion was associated with the presence of bifocal germinoma, presumably due to deletion of MAPK3 gene [93]. Amplification of 12p involves the KRAS gene (a component of MAPK pathway) and seems to be playing an important role in germinomas, similar to testicular germ cell tumours [14]. Furthermore, NF-1, a negative regulator of MAPK pathway, can present mutations in both germinomas and NGGCT [88].
Gain of function mutations of KIT proto-oncogene generate a constitutive activation of the KIT protein, which consequently activates signal transduction molecules via MAPK (mitogen-activated protein kinase) or PI3K (phosphoinositide 3-kinase) pathways, resulting in increased cell proliferation, migration, and apoptosis resistance ( Figure 5) [86].  Upregulation of PI3K pathway is the second genetic event involved in germinoma pathogenesis, MTOR gene being frequently mutated [88]. MTOR mutation promotes cell proliferation via mTORC1 and cell survival via mTORC2 and AKT. These effects were downregulated by pp242, an MTOR inhibitor, underlining the therapeutic prospects in germinoma [88]. Basal ganglia germinomas appear to frequently present PI3K/mTOR pathway mutations and chromosomal losses (1p, 3p/q, 4p, 9p/q, 10p/q, 11p, 13q, 18p/q, 19p/q, and 20p) [7]. Therefore, blockade therapy targeting these pathways may represent an alternative for germinomas that fail to respond to the standard regimens.
Compared to NGGCT, germinomas present with overexpression of genes within 4q13.3-4q28.3 and genes involved in self-renewal mechanisms (see Table 3) that have the capacity to induce dedifferentiation of matured somatic cells to pluripotent embryonic stem cells [94]. Takayasu et al. suggest that gene mutation analysis using CSF circulating tumour DNA is also a feasible study method in germinomas [95]. Genes reported in at least two distinct studies, and their genetic alterations are presented in Table 5.  Recently, microRNAs (miRNAs) have been evaluated as possible biomarkers for various pathologies. MiRNAs are small, noncoding RNA molecules, involved in posttranscriptional gene regulation. GCT are associated with up-regulation of the miR-371~373 and miR-302 clusters, disregarding tumour site, age, or histopathologic type [96]. MiR-371a-3p was identified as a reliable marker in the differential diagnosis between germinoma and Langerhans cell histiocytosis, granting an early detection in cases where imaging studies and serum/CSF work-up are inconclusive [97]. A prospective observational cohort study is currently recruiting patients to evaluate whether miRNA 371 can be used as a prognostic marker for the risk of GCT recurrence [98]. MiR-142-5p and miR-146a are upregulated in the paediatric CNS germinoma, the former inversely correlating with NRP1 (Neuropilin 1), SVIL (Supervillin), and PDGFRA (Platelet Derived Growth Factor Receptor Alpha) and the latter with RUNX1T1 (RUNX1 Partner Transcriptional Co-Repressor 1) and THRB (Thyroid Hormone Receptor Beta) [94]. Low et al. observed a persistent correlation between KIT and downregulation of MiR-221-3p, although further studies are needed to validate this association and evaluate its clinical implications. Downregulation of miR-503 is also encountered in germinomas (Table 6) [92]. Table 6. Up and downregulation of miRNAs in GCT.

Immunological Approach
Other factors seem to be involved in the pathophysiology of germinomas, tumour immune microenvironment being one of them [73]. Paradoxically, the large immune infiltrate encountered in germinomas seems to have no antitumour effect, and several studies aimed to evaluate the role of the programmed death receptor 1 (PD-1)/programmed death receptor 1 ligand (PD-L1) pathway, after it has been reported that PD-L1 is expressed by testicular seminomas, the gonadal counterpart to CNS germinomas [100]. The interaction between the ligand PD-L1, expressed by tumour cells, and PD-1 localised on activated lymphocytes induces T-cell anergy and downregulates the immune response. A small study of 8 patients with intracranial germinoma showed in all patients PD-L1 staining of the tumour cells and PD-1 expression of the tumour-infiltrating lymphocytes (TILs) and found a correlation between the immunosuppressive microenvironment and the growth of the tumours [101]. Takami et al. evaluated the tumour immune microenvironment in 32 germinoma cases, concluding that tumour cells have a 73.5% positivity for PD-L1, while the majority of infiltrating, stained immune cells are PD-1 positive (93.8%), making germinomas a proper candidate for immunotherapy [102]. The high expression of CD4 T helper lymphocytes correlates with a good prognosis, while great levels of nitric oxide synthase 2 produced by myeloid-derived suppressor cells and macrophages associates with a shorter progression free survival (PFS), possibly by generating immune tolerance [102]. Although there are some discrepancies between studies (Table 7), the role of PD-1/PD-L1 pathway in germinomas warrants further investigation and may offer new potential therapeutic perspectives.

Current Management
The treatment of intracranial germinomas is multidisciplinary, including surgery, chemotherapy, radiotherapy (RT), and endocrine therapy. Germinomas are distinctively radiosensitive, with overall survival rates of over 90%, with radiation therapy alone [6,22,105,106]. Chemotherapy alone can induce complete remissions in 84% of the cases, but the long-term efficacy has been proven to be unsatisfactory, with high rates of morbidity and mortality, only 50% of the patients being treated successfully by this method [107][108][109]. Therefore, the standard treatment is represented by a combination of chemotherapy (Carboplatin/Cisplatin and Etoposide ± Ifosfamide) and radiotherapy [110,111]. Nevertheless, surgical resection is commonly performed as the first therapeutic option. The largest multicentre analysis of pituitary germinomas (SEER-Surveillance, Epidemiology, and End Result program) showed that chemotherapy was used more frequently in paediatrics, whereas surgery being applied in the adult population [112].
However, given the high radiosensitivity of germinomas, the extensive tumour resection is not necessary for a complete response, the surgery approach being currently limited to the treatment of hydrocephalus and a tumour biopsy in order to obtain a histological diagnosis. Obstructive hydrocephalus represents a severe complication and requires appropriate management. Until recently, increased intracranial pressure was relieved by ventriculoperitoneal shunts. However, it was discovered that this technique involves a high risk of peritoneal metastasis of the primary GCT and that patients require a thorough follow-up with CT scans of the abdomen to detect metastatic disease [113]. Nowadays, hydrocephalus can be treated by performing endoscopic ventriculostomy, without the risk of peritoneal metastasis [37]. Different techniques have been proposed for the approach, combining third ventriculostomy (ETV) with endoscopic biopsy. Usually, posterior third ventricle tumours are approachable through two trajectories, necessitating two burr holes or one "compromised" burr hole. Roth et al. performed this combined technique through a single burr hole, using a rigid endoscope to perform the ETV, followed by a flexible one to retrieve the biopsy sample, with favourable results. The benefits of this technique are represented by the necessity of a single burr hole, a better visualization of the tumour offered by the rigid endoscope, and a better access offered by the flexible one [114]. Moreover, a combined intervention for pineal tumours, using a single burr hole and a rigid endoscope for both ETV and biopsy, was successfully performed. The larger forceps of the rigid endoscope have the advantage of obtaining larger samples in comparison with flexible endoscopes [71,115]. Supracerebellar infratentorial approach, performed by microsurgery or endoscopic techniques, is a feasible alternative in cases of pineal tumours. The endoscopic approach offers a better visualization of the tumour, a wider range of motion of surgical instruments, a shorter duration of the procedure, a quick recovery, and fewer complications [116,117]. Second-look surgery should be considered in patients with residual tumour or high serum/CSF markers after an appropriate treatment protocol. In these instances, it is possible that the tissue sample was insufficient, containing only the germinomatous component of a tumour with a mixed histology [118,119].
The treatment for localized disease may consist of either craniospinal irradiation (CSI) alone, or chemotherapy and reduced-field radiotherapy. Whole ventricular irradiation (WVI) is recommended, as the ventricles and periventricular areas represent a frequent site of relapse [76,120]. Most studies recommend chemoradiotherapy (CRT), to avoid whole brain irradiation (WBI) and CSI and the adverse effects of radiotherapy. In addition, Zhang et al. suggest that limited radiotherapy represents a feasible treatment strategy for bifocal germinomas, without metastasis, as well [121]. This method of treatment ensured the achievement of long-term survival rates as high as 95-97% [122,123]. In addition, a prospective multicentre cohort study revealed that relapse rates could be reduced by adapting the RT volume as follows: whole ventricle radiotherapy (WVRT) for localized pineal/suprasellar lesions, whole-brain radiotherapy (WBRT) for localized basal ganglia/thalamus, and CSI for disseminated disease [124]. Moreover, in an analysis of 253 cases of CNS germinomas, Jennings et al. reported that signs and symptoms suggestive of hypothalamic-pituitary axis dysfunction should steer the treatment towards CSI and systemic chemotherapy [125]. Optimal RT dosage and field inclusion are still under debate. Selected prospective studies evaluating different treatment regimens (doses of chemotherapy and radiotherapy), and their conclusions are summarized in Table 8, and retrospective studies are resumed in Appendix A Table A1. We conducted a PubMed search for English written articles regarding intracranial germinomas, published between 1996-2021. The articles were selected based on a combination of search terms (germinoma, intracranial, CNS, germ cell tumour, pineal, suprasellar, bifocal, paediatric, treatment, radiotherapy, chemotherapy). We analysed studies evaluating solely germinomas, as well as studies evaluating a wider range of germ cell tumours, provided germinomas were included.
As far as the radiotherapy techniques are concerned, proton beam therapy (passively scattered proton therapy and spot scanning proton therapy) delivers lower doses of radiation to the healthy tissue surrounding the tumour, sparing greater volumes of the temporal lobes and hippocampus than intensity-modulated radiotherapy (IMRT) [126].  ±Surgery-3 gross total/3 partial resections, 10 biopsies for pure germinomas; 1 gross total/3 partial resections, 7 biopsies for 11 β-HCG secreting germinoma Localised disease: Etoposide (100 mg/m 2 ) + Cisplatin (20 mg/m 2 ) 5 consecutive days every 4 weeks −4 cycles after partial resection/biopsy −3 cycles after total/subtotal resection    Moreover, there appears to be a correlation between the ventricles volume and the dose of irradiation received by the healthy brain tissue (at least 12 Gy) after WVI and the boost phase of the treatment with IMRT (the smaller the ventricles, the smaller the dose of irradiation of the brain) [131]. Recently, gamma knife radiosurgery has been proposed as an integrated therapy in the management of pineal tumours. As far as germinomas are concerned, stereotactic radiosurgery (SRS) seems to be an effective boost local treatment that warrants the administration of a smaller dose of CSI, in order to minimize the adverse effects in the long-term, as well as a suitable treatment for tumour recurrence. Pineal germinomas treated with SRS alongside with standard adjuvant treatment showed a 20-year local control rate and survival of 80% [132]. Patients with metastatic pineal germinoma have a bad prognosis, with a 65.8% rate of long term survival and an increased risk of death represented by diffuse cerebral subarachnoid or leptomeningeal dissemination [26].
The principle of radiotherapy is to irradiate all the tumour cells disseminated in the ventricular system, by covering the cerebrospinal fluid pathway sufficiently. However, whole ventricle and boost irradiation of the tumours situated in the midline structures of the brain also deliver a heavy dose of radiation to the temporal lobes and the hippocampus, structures involved in learning and memory, causing neuropsychological deficits and endocrine dysfunctions [126,129,[133][134][135]. These are frequently encountered in highdose regimens, suggesting that strategies that limit cranial irradiation and the use of simultaneous integrated boost techniques may reduce the adverse effects associated with radiotherapy, having a beneficial impact on the quality of life of the patients [136][137][138][139]. A retrospective multi-centre cohort study evaluated the long-term toxicity of the treatment in 112 ICGCT, including 94 germinoma patients. Neurocognitive dysfunction was the most common adverse effect, while suprasellar/hypothalamic tumours and cisplatin treatment were associated with a high risk of hypopituitarism (36.1%) and ototoxicity (39.2%) respectively [140]. Hypopituitarism induced by radiotherapy is encountered in 90% of the cases after radiation treatment of the germinomas located in the neurohypophyseal region, but it also develops after treatment of germinomas located in other sites of the CNS [33]. However, a recent retrospective study, including 49 intracranial germinomas, concluded that hypopituitarism is mainly caused by the tumour and that radiotherapy has no further impairment on the pituitary function [141].
Restoration of endocrine deficiencies has been described in patients treated with chemotherapy alone, suggesting that delaying radiotherapy, especially in young patients with germinomas, may represent a treatment modality [142,143]. With early diagnosis of hypopituitarism and a suitable hormone replacement therapy, most patients achieve an average adult height [21]. Preoperative DI represents a positive predictor factor for the necessity of postoperative long-term hormonal replacement with desmopressin [144]. Secondary malignancies, such as glioblastoma, meningioma, thyroid carcinoma, acute lymphoblastic/myeloid leukaemia, and B-cell lymphoma, have been reported, highlighting the necessity of vigilant follow-up [145,146].
The patient follow-up consists in regular clinical evaluations (neurologic, endocrine, visual, and hearing assessments), tumour markers (serum and/or CSF AFP and β-HCG), and MRI scans. Tumour markers and imaging examinations should be evaluated simultaneously, since the diagnosis of relapsed disease can be established either on the presence of elevated markers or on the appearance of a new lesion [129,147]. Currently, there is no consensus regarding the superior sensitivity between serum and CSF markers. Initial evaluation shall be carried out 1-2 months after treatment completion and every 4-6 months thereafter, for the first 2-5 years, followed by annual assessments [51,76,148,149].

Future Perspectives
Ongoing molecular research aims to shed light upon germinoma's underlying genomic and epigenetic mechanisms and their impact on the tumour evolution and treatment response. In this regard, molecular targeted therapy, such as selective tyrosine kinase inhibitors (TKI), may achieve promising results in cases of intracranial germinomas pre-senting KIT mutations that are resistant to standard CRT. Treatment with TKI has been proposed, considering their efficiency in cases of gastrointestinal stromal tumours (GIST) harbouring KIT mutations. Imatinib, a selective TKI, showed efficacy in cases of GISTs resistant to conventional treatment that presented exon 9 and 11 mutation [150]. Similarly, in CNS germinomas, KIT exons 11 and 17 are most frequently mutated, followed by exons [2,10,13,14,87,89,90,92]. Therefore, it can be presumed that germinomas harbouring exon 11 mutations are candidates for Imatinib therapy. KIT mutation V560D (Val560Asp), reported in one case of CNS germinoma, is likely to respond to Imatinib, as was previously reported in cases of GIST and mastocytosis harbouring the same mutation [89]. Ripretinib, another KIT inhibitor, is effective in cases of Imatinib resistant GISTs presenting D816V (Asp816Val), a mutation in exon 17 also encountered in CNS germinomas [151]. Furthermore, Dasatinib, a tyrosine kinase inhibitor (TKI) that crosses the blood-brain barrier, was evaluated in a retrospective review including five patients with CNS pure germinomas. However, despite the multimodal treatment, four patients finally experienced disease progression, and the efficacy of Dasatinib was not demonstrated [152]. Nevertheless, an ongoing phase I/II trial (NCT00788125) evaluates the treatment response to Dasatinib in combination with Ifosfamide, Carboplatin, and Etoposide of various tumours, including extragonadal germ cell tumours [153].
Other therapeutic options include MAPK and AKT/mTOR inhibitors. Ichimura et al. evaluated on cell cultures the effect of pp242 (Torkinib), an inhibitor of both MTOR complexes (mTORC1 and mTORC2), on two germinoma MTOR mutations (M2327I and L2334V). They discovered that Torkinib inhibits in a dose-dependent manner the phosphorylation, cell migration, and growth induced by the mutated MTOR [88]. These results confirm that the mutations analysed are pathogenic and suggest that Torkinib warrants further evaluation as a potential targeted molecular agent in resistant CNS germinomas. Targeted inhibition of RAS/MAPK pathway may play a role in recurrent or resistant tumours and warrants further investigation.
Immune checkpoint blockade, with PD-1/PDL-1 inhibitors (Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab), represents another potential alternative therapy for resistant cases, considering the presence of a large immune infiltrate and the reported high expressions of PD-1 and PD-L1. Zschäbitz et al. reported a partial response after 15 cycles of Pembrolizumab in a patient with metastatic pituitary germinoma that did not respond to Sunitinib and second-line chemotherapy [154]. A phase II study is currently recruiting patients to evaluate the response of resistant pineal germinomas to Durvalumab (PD-L1 inhibitor) in combination with Tremelimumab (CTLA-4 inhibitor) [155].
In order to select the most appropriate course of treatment, the patients require a personalized management. Relapsed/resistant cases may be enrolled in clinical trials evaluating targeted therapies in CNS germinomas. They should also benefit from genetic and immunological testing, to discover whether they present pathogenic mutations or express immunological markers. Based on these results, the patient may receive the appropriate treatment, in accordance with each genotype (tyrosine kinase inhibitors/MTOR inhibitors/immune checkpoint blockade). Considering the young age of the patients and the radiation side effects, it is also important to offer the best quality of life possible. In this regard, association between targeted therapy and standard CRT may permit dose reduction or even RT elimination.

Conclusions
Intracranial germinomas are rare tumours mainly affecting the paediatric population. Although CRT has favourable results, research has been carried out in the endeavour to minimize the RT dosage and field, to provide the best quality of life for these patients. Nevertheless, resistant/relapsed tumours are therapeutically challenging. These cases may require genetic and immunological testing to identify patients that may benefit from personalized targeted therapy. Ongoing clinical trials aim to evaluate the efficiency of molecular targeted therapy in these cases. Informed Consent Statement: To publish this paper, written informed consent has been obtained from the participating patient (whose histological and imaging MRI images were presented as examples in this article).

Conflicts of Interest:
The authors declare no conflict of interest.