Background
Testicular germ cell tumors are among the most common solid neoplasms in young-adult males. Their overall good prognosis puts them on the top of most curable solid cancers, with survival rates above 85–90% [
1]. Around 85% and 70–75% of stage I seminoma and non-seminoma patients, respectively, are cured with orchiectomy alone [
2‐
5], meaning that a substantial amount of patients can be safely followed-up using surveillance, which is indeed being increasingly adopted [
6]. However, still a subgroup of these patients relapses, most frequently during the first 2 years after initial diagnosis, and requires further treatments, which possibly lead to morbidity and mortality [
7,
8]. For this reason, there is an urgent need for predictive biomarkers to assess the risk of stage I patients, and accurately discriminate those truly benefiting from adjuvant treatment to prevent relapses, from those that can safely be followed using surveillance to avoid early and late side effects of additional treatments on individuals most likely becoming long-term cancer survivors [
9‐
11].
This risk stratification of stage I patients has so far relied on clinicopathological parameters, predominantly being vascular invasion and the amount of embryonal carcinoma (for non-seminomas) [
12,
13] and size and
rete testis invasion (for seminomas) [
14]. With the exception of vascular invasion for non-seminomas, the prognostic power of the other biomarkers to guide treatment decisions is still under debate [
14,
15]. Vascular invasion is the most discriminative biomarker so far, even in multivariable analyses [
12,
13]. Recently, we confirmed the value of vascular invasion assessment in a surveillance cohort of stage I non-seminoma patients [
16]. Moreover, we demonstrated that all patients depicting simultaneously lymph vessel and blood vessel invasion developed relapse; possibly, if validated, this should further identify high-risk patients. Overall, accurate pathological assessment is key since overdiagnosis (commonly observed in vascular invasion assessment by less experienced centers) may result in overtreatment [
17,
18].
Other biomarkers have been studied for their prognostic/predictive value in testicular germ cell tumors, including MIB-1, CXCL12, CXCR4 and beta-catenin [
19‐
22]. However, none has been introduced in the clinic yet, possibly since results among studies were not consistent or reflecting the variability among study designs. MECA-79 is another biomarker shown to be involved in antitumor responses in some malignancies, although so far not been explored in testicular germ cell tumors [
23‐
25]. Also, for TEX19, a cancer testis antigen present in normal adult testis and involved in proliferation of several cancer types and of germ cells [
26], its expression profile in testicular germ cell tumors has not been demonstrated yet. In this work we aim to assess the prognostic value of biomarkers related to proliferation (MIB-1, TEX19) and to the surrounding immune microenvironment (CXCL12, CXCR4, beta-catenin and MECA-79) in a cohort of stage I testicular germ cell tumor patients undergoing surveillance, including their impact in patient outcome, and to compare their performance to the classical histopathological variable vascular invasion.
Discussion
Testicular germ cell tumors are diagnostically challenging, especially because optimal pathological assessment of the primary tumor is key to determine the best therapeutic approach, predominantly in stage I disease [
32]. In fact, accurate discrimination of high vs. low risk stage I patients is essential, in order to facilitate treatment decisions and avoid unnecessary side effects from chemotherapy and radiation as well as surgery. Assessment of this risk status has been largely dependent on histopathological variables, with vascular invasion being the most consistent prognostic tool for predicting disease relapse [
22]. However, disagreements in scoring vascular invasion exist, more evident between peripheral and centralized centers with expertise on germ cell tumors, and these could result in over- and undertreatment [
17,
18,
33]. Surveillance strategies are increasingly being employed in the approach to testicular germ cell tumor patients, given the outstanding cure rates of stage I disease with orchiectomy alone [
6]. Still, there is a need of adjunctive biomarkers to complement the value of vascular invasion and other clinicopathological data.
Our work re-confirms the prognostic value of clinicopathological variables regarding disease relapse of stage I testicular germ cell tumor patients. Vascular invasion significantly associated with relapse in the overall cohort and significantly influenced relapse-free survival (including in multivariable analysis), and this impact was maintained when considering only non-seminoma patients, but not for seminoma patients, in line with previous studies [
34,
35]. Presence of embryonal carcinoma is also associated with poorer relapse-free survival. This is in line with previous knowledge which unequivocally support the predictive value of vascular invasion assessment in non-seminoma patients [
5,
12,
36‐
46] and further suggest amount of embryonal carcinoma as adjunctive in this context [
5]. For seminoma, however, other variables are advanced to help in clinical decision-making: tumor size and
rete testis invasion. In our series we did not however find these variables to be informative regarding disease recurrence; despite the limited number of patients included, this is in line with the systematic review of Boormans et al. [
14] showing that the prognostic power of these features is still poor and cannot be reliably and blindly used to determine therapeutic action. Remarkably, elevation of HCG significantly associated with vascular invasion and it also significantly impacted relapse-free survival, even when adjusting for vascular invasion effect. It has been suggested that HCG elevations in testicular germ cell tumors (both seminomas and non-seminomas) positively regulate the vascular endothelial growth factor (VEGF), leading to vessel neoformation, which explains both the association with vascular invasion status and the poor prognostic value of this finding [
47,
48].
The role of MIB-1 scoring as a prognostic marker in testicular germ cell tumors has been debatable, also because of different methodologies and cutoffs used in its assessment. While some studies demonstrated higher immunoexpression scores associating with poor prognosis (using the 40 and 70% cutoffs) [
30,
31], more recent studies were not able to validate these cutoffs and reported this immunoexpression to be overall non-informative in a large cohort of stage I non-seminomas on surveillance, especially when adjusting to vascular invasion [
21]. The distribution of MIB-1 staining proportions among these studies were also variable, possibly due to the different antibodies applied. In our work we could also not validate the 40 and 70% cutoffs. However, patients with > 50% scores experienced significantly poorer relapse-free survival, although the effect was again lost when adjusting for vascular invasion status. Importantly, when focusing solely on the subset of patients without vascular invasion, MIB scoring of more than 50% identified a group of patients with a worse clinical outcome. This finding, highlighted in Fig.
3, constitutes a novelty compared to previous studies mentioned above, and deserves validation in much larger cohorts to confirm its clinical usefulness.
TEX19 is a recently characterized player within the family of cancer testis antigens, which are normally expressed in the germ cell compartment of the normal testis, but also in a wide range of cancers, with current evidence pointing towards an oncogenic function of these proteins [
49]. Planells-Palop et al. [
26] revealed that TEX19 regulates proliferation and analysis of The Cancer Genome Atlas database shows it associates with distinct clinical outcomes in several tumor models. The authors evidenced cytoplasmic staining for the marker in basal cells within seminiferous tubules, which seemed to correspond to a subpopulation of Sertoli cells. This was in line with findings of Zhong et al. and did not support the role of this marker as a cancer testis antigen [
50]. However, the former authors also evidence, by immunofluorescence, foci of nuclear expression of TEX19 in MAGE-A1-positive cells, corresponding to spermatogonia [
26], a finding our study confirms by use of immunohistochemistry, as depicted in Supplementary Fig.
1. This supports the advanced role of TEX19 in germ cells, and not only in Sertoli cells, thereby fulfilling the classification of a cancer testis antigen. In addition, they demonstrate that this protein can accumulate both in the cytoplasm and nucleus of several cell lines, and describe staining in the nuclei of the germ cell tumor cell line NTera-2 (representative of a non-seminoma), contrasting to the absence of nuclear staining for this marker in our cohort of 70 stage I primary tumors. NTera-2 cells can easily develop signs of neural differentiation, which could affect the subcellular location of the protein. In our cohort we did find a weak cytoplasmic staining for TEX19 in a minority of primary tumors (i.e., 14 non-seminomas and one seminoma). The biological meaning of this finding is still unknown but indicates TEX19 may not behave solely as a cancer testis antigen. This biomarker was indeed advanced as contributing to tumor cells proliferation; in our series of stage I patients its expression did not, however, significantly affect clinical outcome, not did it impact disease-relapse.
The CXCL12/CXCR4 axis plays a role during male germ cell development, with primordial germ cells expressing CXCR4 and migrating towards locations with high CXCL12 content [
51,
52]. In this line, there is rationale for exploring these markers in germ cell tumors, since they are developmental cancers [
53,
54]. In two studies from the same group [
21,
22] it was shown that a CXCL12 gradient was able to stimulate migration of germ cell tumor cells [
22], and that non-seminoma stage I patients on surveillance with higher CXCL12 immunoexpression in tumor cells exhibited significantly better relapse-free survival, independently of vascular invasion status [
21]. Authors hypothesized that the constant supply of CXCL12 might abrogate a gradient that is necessary to trigger migration. Our work, despite of using similar cutoff values, could not reproduce these findings, in line with recent observations by Fankhauser et al. [
19]. However, all studies, including ours, demonstrate that CXCL12 is almost exclusively found in non-seminomas, and we confirmed this on an in silico analysis of The Cancer Genome Atlas (TCGA) database (Supplementary Fig.
6,
p < 0.0001). So, we hypothesize that CXCR4/CXCL12 axis activation should be relevant for TGCT tumorigenesis. Indeed, Gilbert et al. demonstrated that CXCR4 expression led to activation of the PI3K-AKT and MEK-ERK pathways, constituting additional data showing the relevance of activated RAS pathway in TGCT tumorigenesis [
22]. Given the known influence of the microenvironment in testicular germ cell tumors [
28] we hypothesized that CXCL12 expression in stromal/inflammatory cells might also influence the final tendency for tumor cells to migrate and invade. Also, besides autocrine CXCL12 expression, peri-tumor immune cells may be inducing CXCL12 expression in the tumor cells they encase, in an attempt to prevent dissemination; this has not been explored before. Indeed, we found that non-seminoma patients with CXCL12 positivity in surrounding stromal/immune cells showed a significantly improved relapse-free survival, which was able to discriminate two risk groups within patients showing vascular invasion (
p = 0.015). This finding has not been reported in previous works and deserves further validation. We believe that the high expression levels of CXCL12 in these cells immediately surrounding the tumor nests may also abrogate the necessary gradient for triggering migration of tumor cells towards other CXCL12-rich distant locations.
The receptor CXCR4 is present at the cell membrane, can be sequestered into the cytoplasm, but recent evidence also showed that it can translocate to the nucleus. Accurate detection of nuclear CXCR4 depends greatly on the antibody used and its ability to specifically target this relevant epitope [
55]. Nuclear expression of CXCR4 has been shown to impact prognosis in several tumor models, associating both with poorer and improved outcome [
56‐
58]. To the best of our knowledge, this has not been addressed in germ cell tumors. In our work, cases with exclusively CXCR4 nuclear immunoexpression were significantly more frequent in seminomas and significantly associated with better relapse-free survival. However, again, the effect was not maintained in multivariable analysis. We hypothesize that the absence of CXCR4 at the membrane renders the cell insensitive to any available CXCL12 gradient, and so these tumors may not migrate and invade.
Beta-catenin was demonstrated by Chovanec et al. as a poor prognostic factor in testicular germ cell tumors; the mechanism implied to be related to a suppressed immune microenvironment [
20]. Using the same approach of the authors and applying a combined score of intensity and percentage of stained cells, we confirmed that the immunoexpression pattern is significantly lower in seminomas when compared to non-seminomas. Also, the authors did not find a significant impact of beta-catenin expression on relapse-free survival, including when stratifying the analysis for seminomas or non-seminomas only. However, they included both gonadal and extra-gonadal tumors, several disease stages and patients with metastatic disease receiving adjuvant treatment. In our more strict stage I surveillance setting we did find higher beta-catenin immunoexpression to be significantly associated with the event of relapse and a tendency for patients with higher expression to show overall poorer relapse-free survival (
p = 0.045 and
p = 0.056, respectively), which corroborates the immune-suppressed microenvironment initially described by the authors [
20].
MECA-79 was documented to be ectopically expressed in tumor cells of gastric cancer patients (28% of the cases), which associated with poor prognostic features and poorer disease-specific survival [
23]. Its expression has not been assessed in germ cell tumors to date. In our study, we found MECA-79 not to be expressed in tumor cells of both seminomas and non-seminomas. However, we specifically concentrated on the clinical setting of stage I disease on surveillance, with good prognosis, so it is possible that its expression is restricted to higher stage, advanced disease, like in the reported study on gastric cancer [
23]. Moreover, MECA-79 has been used as a marker of the so-called high endothelial venules, which occur naturally in tertiary lymphoid organs and lymph nodes [
59]. These specialized vessels are frequently found in solid tumors (demonstrated in colon, breast, lung and ovarian carcinoma and in malignant melanoma) and are thought to facilitate tumor infiltration by lymphocytes, contributing to better prognosis [
60]. In malignant melanoma, for instance, a high density of such vessels was associated with tumor regression and good prognostic features [
24]. Our work shows no presence of such MECA-79-positive vessels in all testicular germ cell tumor samples included (in the presence of staining of the positive control – human tonsil – depicted in Supplementary Fig.
4), indicating a limited role of this assessment as a predictive biomarker in this tumor model.
One of the limitations of our work is its retrospective nature and the relative small cohort size. However, the power is the true surveillance cohort of 70 stage I patients (partly already described in [
16]), avoiding confounding factors related to any treatment except initial orchiectomy. Also, and because our cases derive from several institutions across the Netherlands, our findings do not reflect a selection bias due to inclusion of patients from a single center. Moreover, the classical prognostic histopathological variables already known to be informative in predicting relapse were re-confirmed in this work, validating our cohort.
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