Purpose: Up to 50% of patients diagnosed with stage I nonseminomatous germ cell tumors (NSGCTs) harbor occult metastases. Patients are managed by surveillance with chemotherapy at relapse or adjuvant treatment up front. Late toxicities from chemotherapy are increasingly recognized. Based on a potential biologic role in germ cells/tumors and pilot data, our aim was to evaluate tumor expression of the chemokine CXCL12 alongside previously proposed markers as clinically useful biomarkers of relapse.

Experimental Design: Immunohistochemistry for tumor expression of CXCL12 was assessed as a biomarker of relapse alongside vascular invasion, histology (percentage embryonal carcinoma), and MIB1 staining for proliferation in formalin-fixed paraffin-embedded orchidectomy samples from patients enrolled in the Medical Research Council's TE08/22 prospective trials of surveillance in stage I NSGCTs.

Results: TE08/TE22 trial patients had a 76.4% 2-year relapse-free rate, and both CXCL12 expression and percentage embryonal carcinoma provided prognostic value independently of vascular invasion (stratified log rank test P = 0.006 for both). There was no additional prognostic value for MIB1 staining. A model using CXCL12, percentage embryonal carcinoma, and VI defines three prognostic groups that were independently validated.

Conclusions: CXCL12 and percentage embryonal carcinoma both stratify patients' relapse risk over and above vascular invasion alone. This is anticipated to improve the stratification of patients and identify high-risk cases to be considered for adjuvant therapy. Clin Cancer Res; 22(5); 1265–73. ©2015 AACR.

Translational Relevance

Patients diagnosed with stage I nonseminomatous germ cell tumors face a choice between surveillance with treatment at relapse or up-front adjuvant therapy. Although adjuvant therapy is effective, it may be unnecessary and long-term effects of chemotherapy are increasingly recognized. Histologic evidence of vascular invasion is currently used to target patients for adjuvant therapy, but improved markers for risk of relapse are required. Embryologically, primordial germ cells use CXCR12/CXCR4 and KITLG/KIT signaling to migrate to the developing gonads. Previously, we showed that CXCL12 stimulates migration of germ cell tumor cells in a CXCR4-dependent manner and that tumor cell expression of CXCL12 was associated with reduced risk of metastatic relapse. Here, we validate this finding in a large series of samples from patients that underwent surveillance within prospective clinical trials and propose CXCL12 expression and percentage of embryonal carcinoma as clinically useful biomarkers to assist in stratifying patients for adjuvant therapy.

Although overall considered rare cancers, testicular germ cell tumors (TGCTs) are the most common solid malignancy to affect young adult Caucasian males. They are divided into seminomas that resemble primordial germ cells or nonseminomas (NSGCTs) that exhibit embryonal or extra-embryonal patterns of differentiation (1). A total of 60% of NSGCTs present with stage I disease (confined to the testes), and this proportion is increasing (2). About 15% to 50% of such patients may harbor micrometastatic disease and will relapse without further treatment (3, 4).

Assuming protocols are closely adhered to (5), immediate adjuvant treatment (one to two cycles of bleomycin, etoposide, and cisplatin—BEP; ref. 6) or surveillance with chemotherapy as salvage both have excellent rates of cure. Chemotherapy may have significant long-term effects, including cardiovascular disease (7, 8), second malignancies (9, 10), Reynauds syndrome, neuropathies, fertility, and emotional disorders. For this reason, the routine use of adjuvant chemotherapy has been criticized in some quarters. Adjuvant retroperitoneal lymph node dissection is an alternative adjuvant strategy but with associated potential complications and morbidities these patients would also benefit from improved risk stratification (11–13).

Histologic evidence of vascular invasion (VI) is the only validated histologic prognostic factor currently used to define risk of relapse in clinical stage I NSGCTs (3, 4), notwithstanding the distinct role that plasma tumor markers play in assessing disease state. Tumors with VI have relapse rates of up to 50% and patients may be offered adjuvant chemotherapy reducing subsequent relapses to approximately 2% (6). In the absence of VI, around 15% patients relapse and surveillance may be a more reasonable option. More accurate stratification of patients for likelihood of relapse would improve patient management, decreasing the risk of unnecessary treatment with associated side-effects and reducing risks and costs associated with overtreatment and excess imaging (14).

A systematic review (4) identified the percentage of embryonal carcinoma (%EC) within the primary tumor and the proliferation marker MIB1 as promising markers for relapse. Both are continuous variables and studies used differing cutoffs with variable levels of risk prediction. Univariate odds ratios for relapse range from 2.8 to 9 (4, 15–18). However, %EC and VI were correlated and in multivariate analysis the prognostic effect of %EC diminished (4). In a subsequent study, 77% of metastasizing tumors showed MIB1 staining in >70% cells, equivalent to an odds ratio of 3.18 (95% CI, 1.51–6.65). However, these data derive from a series of 195 patients after retroperitoneal lymph node dissections and not strictly a surveillance population (19).

Considering novel molecular markers, TGCTs resemble primordial germ cells (PGC; ref. 20) that physiologically utilize KITLG/KIT and CXCL12/CXCR4 for migration and survival during embryologic development (21–23) and in the maintenance of the spermatogonial stem cell niche (24). Both signaling pathways are implicated in the malignant counterpart; KIT is strongly implicated in testicular tumorigenesis with both discrete amplification and activating mutations described (20) and TGCT express the receptor CXCR4 that can mediate invasive migration toward its ligand, CXCL12 (25, 26). Importantly, NSGCTs can express CXCL12 in an autocrine fashion and in a pilot study containing a high proportion of EC cases (n = 80) of TGCTs this was associated with reduced risk of relapse (25). Here we investigate this feature further for clinical utility alongside VI, %EC, and MIB1 staining and in addition to other clinicopathologic characteristics.

Two recent clinical trials conducted by the Medical Research Council (MRC) investigated surveillance strategies in clinical stage I NSGCTs and provide a unique large cohort of well-characterized stage I NSGCT patients managed on prospective protocols. TE08 (NCT00003420; ref. 27) compared two frequencies of CT scanning during surveillance of stage I NSGCTs. TE22 (NCT00045045; ref. 28) investigated the ability of a baseline FDG PET scan to distinguish patients at lower risk of relapse who might safely be managed by surveillance. In total, 501 patients were managed by surveillance, with a relapse rate of 21% (103 patients). A total of 130 of 501 patients (26%) had VI, although in an unselected population this would be closer to 50%.

To refine treatment stratification in patients diagnosed with stage I NSGCTs, we set out to investigate and validate CXCL12, %EC, and MIB1 as biomarkers prognostic for relapse. Additional evidence of a prognostic effect for CXCL12 over and above the previous pilot data (25) was first sought using a tissue microarray (TMA) comprising representative cores from 59 patients with stage I NSGCTs managed with surveillance. We then investigated the markers MIB1, CXCL12, and %EC in the samples from patients with stage I NSGCTs managed by surveillance in the MRC TE08/TE22 clinical trials. Finally, we validated a combined prognostic model using VI, CXCL12, and %EC in the previous cohort of samples (25).

Patients and tumor samples

This study has national research ethics committee approval (09/MRE00/30) and complies with the REMARK guidelines for biomarker studies (29). Samples from patients with stage I NSGCTs managed by surveillance were collected from the Medical Research Council (MRC) trials TE08 (27, 28). Specifically, these were patients diagnosed with stage I NSGCTs (negative tumor markers and CT scan confirming stage I) and enrolled postoperatively into a randomized study of two alternate imaging surveillance protocols (TE08) or in the case of TE22, undergoing FDG-PET imaging followed by surveillance if negative, to assess the negative predictive value of this scan. Formalin-fixed paraffin-embedded (FFPE) tumor blocks were available for 200/501 (40%) cases; 139 from TE08 and 61 from TE22.10 of the 61 TE22 patients were not eligible for this study as 7 were PET positive and received adjuvant chemotherapy and 1 was PET negative but received adjuvant chemotherapy at the patient's request. Two cases were lost to follow up. The final trial samples consisted of material from 190 patients (Table 1). Importantly, this cohort was representative of the overall trial sample set with a relapse-free rate of approximately 78% at 2 years after orchidectomy (Supplementary Fig. S1). Complete tumor cases were retrieved from each patient, and a full set of hematoxylin and eosin (H&E) sections from tumor for each case were examined by a board-certified histopathologist. Representative tumor material, to include all significant areas of pathology, was selected from each case. VI was assessed as previously described (16). In addition, sections from a TMA containing 0.6-mm-diameter cores were available from Princess Margaret Hospital, Toronto (JS) representing primary tumors from 59 patients with stage I NSGCTs managed by surveillance and a minimum follow-up of 2 years (Supplementary Table S1). Finally, TMAs comprising material from 80 patients with stage I nonseminomatous germ cell tumors (NSGCTs) managed with surveillance at the Royal Marsden Hospital (RMH, previously described in ref. 25) were rescored as per the below by a pathologist (D. Berney) blinded to outcomes.

Table 1.

Histologic subtype, %EC, tumor marker status, and immunohistochemistry for CXCL12 and MIB1 for 190 stage I NSGCT primary tumors from patients treated in the TE08/TE22 clinical trials

Vascular invasion
NoYesTotal
N (%)N (%)N (%)
Histology 
 Pure EC 23 (19%) 18 (27%) 41 (22%) 
 Mixed NSGCTs 79 (64%) 39 (57%) 118 (62%) 
 Yolk sac 8 (7%) 3 (4%) 11 (6%) 
 Differentiated teratoma 9 (7%) 1 (2%) 10 (5%) 
 Other type 3 (3%) 7 (10%) 10 (5%) 
EC presence 
 EC absent 29 (26%) 9 (14%) 38 (22%) 
 EC present 83 (74%) 56 (86%) 139 (79%) 
 Not known 10 13 
% EC optimal categories 
 ≤25% 60 (54%) 20 (31%) 80 (45%) 
 26%–99% 29 (26%) 27 (41%) 56 (32%) 
 100% 23 (20%) 18 (28%) 41 (23%) 
 Not known 10 13 
AFP pre-orchidectomy 
 Normal 53 (43%) 25 (37%) 78 (41%) 
 Raised 69 (57%) 43 (63%) 112 (59%) 
HCG pre-orchidectomy 
 Normal 65 (53%) 29 (43%) 94 (50%) 
 Raised 57 (47%) 39 (57%) 96 (50%) 
CXCL12 (≤1% = weak/absent) 
 Insufficient tumor 7 (6%) 1 (2%) 8 (4%) 
 Absent/weak 26 (21%) 11 (16%) 37 (20%) 
 Moderate/strong 89 (73%) 56 (82%) 145 (76%) 
MIB1 staining 
 Weak 37 (32%) 8 (12%) 45 (25%) 
 High 77 (68%) 57 (88%) 134 (75%) 
 Not assessable 11 
Total 122 (64%) 68 (36%) 190 (100%) 
Vascular invasion
NoYesTotal
N (%)N (%)N (%)
Histology 
 Pure EC 23 (19%) 18 (27%) 41 (22%) 
 Mixed NSGCTs 79 (64%) 39 (57%) 118 (62%) 
 Yolk sac 8 (7%) 3 (4%) 11 (6%) 
 Differentiated teratoma 9 (7%) 1 (2%) 10 (5%) 
 Other type 3 (3%) 7 (10%) 10 (5%) 
EC presence 
 EC absent 29 (26%) 9 (14%) 38 (22%) 
 EC present 83 (74%) 56 (86%) 139 (79%) 
 Not known 10 13 
% EC optimal categories 
 ≤25% 60 (54%) 20 (31%) 80 (45%) 
 26%–99% 29 (26%) 27 (41%) 56 (32%) 
 100% 23 (20%) 18 (28%) 41 (23%) 
 Not known 10 13 
AFP pre-orchidectomy 
 Normal 53 (43%) 25 (37%) 78 (41%) 
 Raised 69 (57%) 43 (63%) 112 (59%) 
HCG pre-orchidectomy 
 Normal 65 (53%) 29 (43%) 94 (50%) 
 Raised 57 (47%) 39 (57%) 96 (50%) 
CXCL12 (≤1% = weak/absent) 
 Insufficient tumor 7 (6%) 1 (2%) 8 (4%) 
 Absent/weak 26 (21%) 11 (16%) 37 (20%) 
 Moderate/strong 89 (73%) 56 (82%) 145 (76%) 
MIB1 staining 
 Weak 37 (32%) 8 (12%) 45 (25%) 
 High 77 (68%) 57 (88%) 134 (75%) 
 Not assessable 11 
Total 122 (64%) 68 (36%) 190 (100%) 

Abbreviations: AFP, alpha fetoprotein; HCG, human chorionic gonadotrophin.

Sectioning, histology review, and staining

Sections were stained with H&E and assessed to ensure adequate tumor material. Sections were deparaffinized prior to staining for CXCL12 (Antibody 79018, 1:100; R&D Systems), including positive (tonsillar crypt) and negative controls as previously described (25) and MIB1 (Antibody M7240, 1:100; Dako). Antibodies were visualized using the Bond Polymer Refine Kit (Leica Biosystems). Immunostaining was performed on a Bond max automated immunostainer.

Scoring and categorization

H&E slides were scored for %EC (by D. Berney) as a continuous variable, and then additionally grouped as described in previous studies or a new data-derived grouping for subsequent analysis. Immunostaining was scored by two independent histopathologists (K. Thway and I. Chandler) recording intensity of staining as 0 to 3 (absent, weak, medium, and high intensity) and % cells staining positive. Samples where scores differed were reviewed and a consensus obtained. Scores were categorized as absent/weak CXCL12 if <1% cells across the whole tumor stained positive for CXCL12. A second exploratory analysis was also performed classifying <10% cells staining for CXCL12 as CXCL12 absent/weak. Analysis for MIB1 was performed separately using both intensity and % cells positive using cutoffs described in the previous studies, i.e., ≥70% and ≥40% (15–18) as well as additional exploratory analyses.

Statistical methods

The primary outcome measure was relapse-free rate, measured from the date of orchidectomy to the date of relapse confirmation, with relapse-free patients censored on the date last known to be alive. Relapse-free rates on Kaplan–Meier survival curves were compared by the log-rank test, with an initial assessment of the independence of %EC, CXCL12, and MIB1 over VI determined by log-rank tests stratified for VI. Subsequently, a proportional hazards regression model was fitted to adjust for baseline clinical variables, VI, %EC, MIB1, and CXCL12 staining, using forward and backwards stepwise selection. χ2 tests were used to investigate the association of %EC and CXCL12/MIB1 staining with clinicopathologic variables.

TMA CXCL12 expression and outcome

To investigate CXCL12 as a marker for relapse prior to application to the clinical trial sample sets, we first studied the TMA representing 59 cases. A total of 25 of 59 cases (42.4%) demonstrated moderate/strong expression of CXCL12 cells of which three had relapsed (RFR 88.0%). Of the 34 patients with absent/weak staining for CXCL12, 11 had relapsed (RFR 67.6%, log-rank test P = 0.68). Although not reaching statistical significance, the rates of CXCL12 expression and subsequent relapse were consistent with previous data (25) and analysis of CXCL12 expression was taken forward to TE08/TE22 samples.

TE08/22—CXCL12 and relapse

Samples representing 182/190 samples from patients in the TE08 and TE22 trials were assessable for CXCL12 staining (Table 1; Fig. 1A–D). Using <1% as the cutoff, 37 (20.3%) tumors were classified as absent/weak with the other 145 (79.7%) moderate/strong (as scored by two pathologists, κ = 0.465, P < 0.001). In an exploratory analysis, a <10% cutoff was also assessed and produced a similar performance (Supplementary Table S2 and Supplementary Fig. S2). Therefore, either cutoff may be used. There was no association between CXCL12 and VI, the presence of seminomatous elements or raised markers pre-orchidectomy. There was, however, a strong association with the presence of EC, which was more prevalent in those with absent/weak staining (75.7% vs. 30.3% of those with moderate/high staining, χ2P < 0.001). The log-rank test shows evidence of a prognostic impact, alone, and stratified by VI (P = 0.006, Table 2) for CXCL12 with reduced relapse-free rate in the absent/weak group (Fig. 2A). In VI-positive patients, CXCL12 further stratified relapse rates; 56 patients with VI but moderate/strong staining had a 2-year RFR of 62.4% versus 11 patients with VI and absent/weak staining for CXCL12 where a RFR of 27.3% was observed (95% CI, 1%–53.6%).

Figure 1.

Representative staining of stage I nonseminomatous germ cell tumor samples. Immunohistochemistry for CXCL12 staining (A) negative (B), <10%, (C) ∼30%, and (D) 100% positive. E, hematoxylin and eosin staining showing a combined seminomatous (bottom of photomicrograph) and nonseminomatous tumour (top) composed of less than 10% EC. F, tumor composed of 25% EC and 75% yolk sac tumor. The yolk sac and EC are intermingled in a polyembryomatous fashion, mimicking the earliest stages of embryonic development. G, tumor entirely composed of EC. H, EC showing 75% positivity for MIB1 immunohistochemistry (scale bar, 100 μm).

Figure 1.

Representative staining of stage I nonseminomatous germ cell tumor samples. Immunohistochemistry for CXCL12 staining (A) negative (B), <10%, (C) ∼30%, and (D) 100% positive. E, hematoxylin and eosin staining showing a combined seminomatous (bottom of photomicrograph) and nonseminomatous tumour (top) composed of less than 10% EC. F, tumor composed of 25% EC and 75% yolk sac tumor. The yolk sac and EC are intermingled in a polyembryomatous fashion, mimicking the earliest stages of embryonic development. G, tumor entirely composed of EC. H, EC showing 75% positivity for MIB1 immunohistochemistry (scale bar, 100 μm).

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Figure 2.

A, relapse-free survival data for 182 patients with stage I NSGCTs managed by surveillance with the MRC TE08 and TE22 clinical trials, stratified by VI, and immunohistochemistry for CXCL12 in combination with the presence/absence of histologic VI (stratified log-rank test P = 0.006). B, relapse-free survival data for 177 patients with stage I NSGCTs managed with surveillance stratified by %EC and VI, illustrated using optimal cutoffs for %EC.

Figure 2.

A, relapse-free survival data for 182 patients with stage I NSGCTs managed by surveillance with the MRC TE08 and TE22 clinical trials, stratified by VI, and immunohistochemistry for CXCL12 in combination with the presence/absence of histologic VI (stratified log-rank test P = 0.006). B, relapse-free survival data for 177 patients with stage I NSGCTs managed with surveillance stratified by %EC and VI, illustrated using optimal cutoffs for %EC.

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Table 2.

Univariate and stratified log-rank test results for factors of interest in 177 cases with complete data

Number of patients2-year RFR95% CILog-rank P valueStratified (by VI) log-rank P value
Vascular invasion 
 Absent 112 88.3 (82.2–94.4) <0.001 n/a 
 Present 65 58.3 (46.0–70.6)   
CXCL12 
 Moderate/high, no VI 87 90.7 (84.6–96.8)   
 Moderate/high, VI 55 63.7 (50.6–76.8)   
 Subtotal mod/high 142 80.4 (73.7–87.1) 0.078 0.009 
 Absent/weak, no VI 25 80.0 (64.3–95.7)   
 Absent/weak, VI 10 30.0 (1.6–58.4)   
 Subtotal absent 35 65.7 (50.0–81.4)   
MIB1 
 Weak, no VI 35 91.4 (82.2–99.9)   
 Weak, VI 100.0 (39.8–99.9)   
 Subtotal weak 42 92.8 (85.0–99.9) 0.007 0.045 
 High, no VI 76 86.7 (79.1–94.3)   
 High, VI 56 53.7 (40.4–67.0)   
 Subtotal high 132 73.0 (65.4–80.6)   
EC (present/absent) 
 Absent, no VI 29 96.3 (89.2–99.9)   
 Absent, VI 66.7 (35.9–97.5)   
 Subtotal absent 38 89.2 (79.2–99.2) 0.096 0.243 
 Present, no VI 83 85.4 (77.8–93.0)   
 Present, VI 56 56.8 (43.5–70.1)   
 Subtotal present 139 74.3 (66.9–81.7)   
EC (optimal categories) 
 ≤25% EC, no VI 60 94.9 (89.2–99.9)   
 ≤25% EC, VI 20 68.1 (46.9–89.3)   
 Subtotal ≤25% 80 88.4 (81.3–95.5)   
 26%–99% EC, no VI 29 93.1 (83.9–99.9)   
 26%–99% EC, VI 27 57.3 (38.1–76.5) <0.001 0.006 
 Subtotal 26–99% 56 76.4 (65.2–87.6)   
 100% EC, no VI 23 63.6 (43.4–83.8)   
 100% EC, VI 18 50.0 (26.9–73.1)   
 Subtotal 100% 41 57.5 (42.2–72.8)   
Number of patients2-year RFR95% CILog-rank P valueStratified (by VI) log-rank P value
Vascular invasion 
 Absent 112 88.3 (82.2–94.4) <0.001 n/a 
 Present 65 58.3 (46.0–70.6)   
CXCL12 
 Moderate/high, no VI 87 90.7 (84.6–96.8)   
 Moderate/high, VI 55 63.7 (50.6–76.8)   
 Subtotal mod/high 142 80.4 (73.7–87.1) 0.078 0.009 
 Absent/weak, no VI 25 80.0 (64.3–95.7)   
 Absent/weak, VI 10 30.0 (1.6–58.4)   
 Subtotal absent 35 65.7 (50.0–81.4)   
MIB1 
 Weak, no VI 35 91.4 (82.2–99.9)   
 Weak, VI 100.0 (39.8–99.9)   
 Subtotal weak 42 92.8 (85.0–99.9) 0.007 0.045 
 High, no VI 76 86.7 (79.1–94.3)   
 High, VI 56 53.7 (40.4–67.0)   
 Subtotal high 132 73.0 (65.4–80.6)   
EC (present/absent) 
 Absent, no VI 29 96.3 (89.2–99.9)   
 Absent, VI 66.7 (35.9–97.5)   
 Subtotal absent 38 89.2 (79.2–99.2) 0.096 0.243 
 Present, no VI 83 85.4 (77.8–93.0)   
 Present, VI 56 56.8 (43.5–70.1)   
 Subtotal present 139 74.3 (66.9–81.7)   
EC (optimal categories) 
 ≤25% EC, no VI 60 94.9 (89.2–99.9)   
 ≤25% EC, VI 20 68.1 (46.9–89.3)   
 Subtotal ≤25% 80 88.4 (81.3–95.5)   
 26%–99% EC, no VI 29 93.1 (83.9–99.9)   
 26%–99% EC, VI 27 57.3 (38.1–76.5) <0.001 0.006 
 Subtotal 26–99% 56 76.4 (65.2–87.6)   
 100% EC, no VI 23 63.6 (43.4–83.8)   
 100% EC, VI 18 50.0 (26.9–73.1)   
 Subtotal 100% 41 57.5 (42.2–72.8)   

TE08/22—%EC and relapse

A total of 177 of 190 patients were assessable for %EC (Table 1; Fig. 1E–G). This showed a bimodal distribution, with clusters at 0% and 100% and a relatively even spread of the remaining values between these levels. %EC was significantly higher in patients with VI (median 70% vs. 20%, Mann–Whitney test P = 0.013), and also in those with absent/weak CXCL12 intensity (medians 100% vs. 20% for moderate/strong, P < 0.001) and with presence of MIB1 staining (medians 50% vs. 10%, P = 0.012).

%EC was assessed as in previous reports as a continuous variable, a binary variable (presence/absence) and applying previously reported cutoffs (<45%, 46%–70%, >70%; above or below 50%; ref. 4). In addition, to better reflect the unusual distribution of %EC, a data-derived categorization was investigated, formed by dividing the data initially into approximate quintiles (0%, 1%–25%, 26%–75%, 76%–99%, 100%), collapsing groups with similar relapse-free rates to create an “optimal” categorization (≤25%, 26%–99%, 100%). With the exception of presence/absence of EC, higher %EC was associated with higher relapse rates (Fig. 2B, Table 2), independent of VI, for all categorizations.

TE08/TE22—MIB1 and relapse

A total of 179 cases were assessable for MIB1 staining; 45 (25.1%) were MIB1 weak on both intensity and proportion of cells staining (Table 1; Fig. 1H). There was a significant association of both MIB1 intensity and proportion of cells staining for MIB1 with decreasing likelihood of the tumor containing seminomatous elements (Mann–Whitney test P < 0.001) and increasing likelihood of VI (Mann–Whitney test P = 0.004 for intensity and P < 0.001 for % cells staining).

There was no evidence of prognostic value for MIB1 staining intensity (log-rank test for trend P = 0.26) nor for the proportion of cells staining positive for MIB1, either when analyzed as per previous reports (≥70% and ≥40%), as quartiles or using a log-rank test for trend. In contrast to previous studies, only 5/179 patients (3%) had MIB1 staining in >70% of cells. The main distinction observed was between the 45 samples with weak versus any staining for MIB1 (Table 2). Analyzing the samples in this binary fashion (MIB1 positive or negative) has a prognostic effect (univariate analysis), which was reduced after stratification for VI (Table 2).

TE08/TE22 multivariate analyses

Multivariate analyses were performed on the 177 patients with complete data to assess the additional prognostic value of these factors over and above the presence/absence of VI and clinical variables, specifically VI (yes/no), histology type, seminomatous elements present/absent, age (continuous variable), alpha feto protein raised pre-orchidectomy (yes/no), human chorionic gonadotrophin raised pre-orchidectomy (yes/no), CXCL12 expression, MIB1 staining (high/weak), %EC (as a continuous variable), EC present/absent, and %EC categorized according to previous studies (<45%, 46%–79%, >80%; <50% vs. ≥50%; ref. 4) and %EC categorized according to the optimal cutoffs for this dataset (<25%, 26%–99%, and 100%).

Both forward and backward stepwise model selection procedures were used, which all led to a model (Supplementary Table S3) including only VI and %EC (continuous variable). Dropping EC as a continuous variable but keeping all the other variations, the model includes only VI and %EC, using the “optimal” categorization (Fig. 2B). However, if %EC is used as previously reported (4), then VI and CXCL12, but not %EC, are retained as independent variables (Fig. 2A).

Components of both groupings have potential clinical utility in defining subsequent relapse risk, and as such we also present a combined model (Fig. 3A) using three prognostic groups. Specifically, these are (i) VI negative, <100% EC and any CXCL12, (ii) VI negative, 100% EC any CXCL12 or VI positive, any EC and mod/high CXCL12, and (iii) VI positive, any EC, absent/weak CXCL12 with 2-year RFRs of 94.3% (95% CI, 89.4%–99.2%), 63.9% (52.9%–74.9%), and 30% (1.6%–58.4%), respectively (log-rank X2 = 38.6 on 2.df, P < 0.001).

Figure 3.

A, relapse-free survival data for 177 patients with stage I NSGCTs managed with surveillance according to data-derived risk groups. B, relapse-free survival data for an independent cohort of 80 patients with stage I NSGCTs managed with surveillance stratified by the risk grouping derived in A.

Figure 3.

A, relapse-free survival data for 177 patients with stage I NSGCTs managed with surveillance according to data-derived risk groups. B, relapse-free survival data for an independent cohort of 80 patients with stage I NSGCTs managed with surveillance stratified by the risk grouping derived in A.

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Validation of model combining VI, CXCL12, and %EC on RMH cohort

Staining characteristics of the cohort of 80 RMH patients managed with surveillance mirror those of the TE08/TE22 patients and are detailed in Table 3. Overall this cohort experienced a 2-year relapse-free rate (RFR) of 60% (95% CI, 59%–71%). For patients with VI this was 47% (31%–63%) as opposed to 69% without (54%–83%). CXCL12 expression was prognostic as previously described with tumors absent/weak for CXCL12 having a 2-year RFR of 34% (18%–50%), whereas tumors with moderate/high staining for CXCL12 having an RFR of 80% (68%–92%); HR, 0.24 (95% CI, 0.11–0.49); P < 0.001. CXCL12 expression retained additional prognostic value over and above VI (stratified log rank P < 0.001). Finally, %EC was also prognostic in this cohort, with the 2-year RFR ranging from 79% (61–87) for patients with ≤25% EC, through 64% (47%–82%) with 26%–99% EC and 48% (31%–66%) in cases that were 100% EC (P = 0.04). Using the combined model developed above, the three risk groupings have 2 RFR of 81.5% (66.8–96.2) for patients that were VI negative and %EC < 100, 64.5% (47.6–81.4) for those that were VI negative and 100% EC or VI positive and moderate/high expression of CXCL12 and a 2-year RFR of just 27.3% (8.7–45.9) for those that had evidence of VI but absent/weak expression of CXCL12 (Fig. 3B, log rank Mantel–Cox <0.001).

Table 3.

Tumor characteristics (VI, CXCL12, and %EC) for 80 stage I NSGCT RMH patients managed by surveillance

No vascular invasionVascular invasion +Total
N (%)N (%)N (%)
EC% 
 ≤25% 14 (33.3%) 5 (13.9%) 19 (24.4%) 
 26%–99% 13 (31.0%) 15 (41.7%) 28 (35.9%) 
 100% 15 (35.7%) 16 (44.4%) 31 (39.7%) 
CXCL12 intensity 
 0 11 (26.2%) 19 (50.0%) 30 (37.5%) 
 1 2 (4.8%) 3 (7.9%) 5 (6.3%) 
 2 9 (21.4%) 5 (13.2%) 14 (17.5%) 
 3 20 (47.6%) 11 (28.9%) 31 (38.8%) 
CXCL12 
 Absent/low 13 (31.0%) 22 (57.9%) 35 (43.8%) 
 Moderate/high 29 (69.0%) 16 (42.1%) 45 (56.3%) 
Proposed grouping 
 No VI, %EC <100 27 (64.3%) 0 (0.0%) 27 (33.8%) 
 No VI and 100% EC or VI and mod/high CXCL12 15 (35.7%) 16 (42.1%) 31 (38.8%) 
 VI and absent/low CXCL12 0 (0.0%) 22 (57.9%) 22 (27.5%) 
Totals 42 (100%) 38 (100%) 80 (100%) 
No vascular invasionVascular invasion +Total
N (%)N (%)N (%)
EC% 
 ≤25% 14 (33.3%) 5 (13.9%) 19 (24.4%) 
 26%–99% 13 (31.0%) 15 (41.7%) 28 (35.9%) 
 100% 15 (35.7%) 16 (44.4%) 31 (39.7%) 
CXCL12 intensity 
 0 11 (26.2%) 19 (50.0%) 30 (37.5%) 
 1 2 (4.8%) 3 (7.9%) 5 (6.3%) 
 2 9 (21.4%) 5 (13.2%) 14 (17.5%) 
 3 20 (47.6%) 11 (28.9%) 31 (38.8%) 
CXCL12 
 Absent/low 13 (31.0%) 22 (57.9%) 35 (43.8%) 
 Moderate/high 29 (69.0%) 16 (42.1%) 45 (56.3%) 
Proposed grouping 
 No VI, %EC <100 27 (64.3%) 0 (0.0%) 27 (33.8%) 
 No VI and 100% EC or VI and mod/high CXCL12 15 (35.7%) 16 (42.1%) 31 (38.8%) 
 VI and absent/low CXCL12 0 (0.0%) 22 (57.9%) 22 (27.5%) 
Totals 42 (100%) 38 (100%) 80 (100%) 

Using samples from patients with stage I NSGCT managed by surveillance on prospective clinical trials, we have demonstrated that CXCL12 expression and %EC in the primary tumor are both predictors of relapse independently of VI. Importantly, samples obtained for this study were representative of the total trial populations (∼78% relapse-free rate at 2 years, by which point most relapses have usually occurred) and hence represent patients with stage I NSGCTs in general. This provides prognostic information of potential clinical relevance over and above the presence/absence of histologic evidence of VI and is able to identify patients at low, moderate, or very high risk of relapse (Table 2; Supplementary Tables S2 and S3). However, %EC and CXCL12 are strongly inversely correlated and in multivariate analyses one or the other—but not both—are selected in addition to VI, depending on how %EC is analyzed. This gives two alternative prognostic models, as illustrated in Fig. 2A and B. Although the multivariate analysis does not support a model containing VI with both %EC and CXCL12, there are unique characteristics of each combination that an ideal model would combine, specifically identification of a very high-risk group (VI positive, CXCL12 absent) and identification of VI-negative patients who have a prognosis closer to that of VI positive (VI negative, 100% EC). We therefore derived an exploratory model that combines these elements into three prognostic groups (Fig. 3A). Using a cohort of cases previously characterized for CXCL12 (25), through the additional inclusion of %EC scores and VI we demonstrate the validity of this model (Fig. 3B).

As shown previously (4), %EC is a subjective analysis. CXCL12 expression (<1% or <10% of cells staining positive as thresholds to classify as CXCL12 absent) together with VI is a potentially more reproducible approach to determining prognosis, and one that identifies a small group of patients with distinctly poorer prognosis.

Adjuvant chemotherapy is highly effective treatment and even one cycle, currently being tested in a UK phase II single arm study (BEP 111), can reduce the risk of recurrence to <4% (11, 13, 30, 31). This use of adjuvant BEP has been criticized for exposing a high proportion of patients who would not go on to relapse to intensive chemotherapy. Refining the current risk model (utilizing lymphovascular invasion alone) would therefore be of clinical utility, allowing adjuvant therapy to be focused on those at highest risk. Our analysis in this cohort supports our previous study in showing that CXCL12 immunohistochemistry can add valuable additional prognostic information to the model based on VI, particularly in identifying a small cohort of patients with a very high risk of relapse. It also in identifies groups for whom surveillance, potentially using reduced intensity follow-up (at least to the reduced frequency arm used in TE08; ref. 27) may be most appropriate, and those suitable for either surveillance or adjuvant therapy depending on personal preferences. Identifying high-risk patients for minimal effective adjuvant therapy will ultimately reduce long-term side-effects for the stage I NSGCT population as a whole.

The prognostic value of MIB1 was analyzed in a number of ways looking at intensity and percentage of cells both separately and combined and including previously used categorizations (15–18, 32). Although patients with weak staining have a better prognosis than those with high MIB1 staining, MIB1 staining was associated with VI and in multivariate analysis does not add clear independent prognostic value.

The prognostic value of CXCL12 expression demonstrated here is consistent with a growing body of evidence for the CXCL12/CXCR4 axis supporting the male germ cell niche and the metastatic spread of cancers, including this possibility in germ cell tumors (20, 22, 24, 25, 26, 33). CXCR4 expression is associated with invasion and metastases in a range of tumor types where lower levels of CXCL12 in tumors predict a reduced risk of metastatic dissemination (34, 35). Furthermore, in breast cancer, lower levels of plasma CXCL12 appear to be associated with increased risk of metastatic relapse (36). That autocrine expression of CXCL12 consistently reduces the subsequent risk of relapse in stage I NSGCTs (independent from histologic VI) suggests the abrogation of a chemokine gradient (toward CXCR4) might prevent extravasation into the vascular compartment and/or invasion at metastatic sites. To this end, assessment of stromal and/or plasma CXCL12 might provide additional prognostic information. The apparent association with histologic subtypes of TGCT requires further work, aligned with a better understanding of how these tumours develop from the in situ counterpart. Further investigations will inform on these and other potential mechanisms of dissemination and relapse (37) in NSGCT patients.

As a paradigm of protecting future quality of life in an essentially curable disease, prospective study is recommended (potentially also investigating novel imaging strategies in an effort to minimize radiation exposure, e.g., MRI) to further validate the prognostic value of CXCL12 and/or %EC expression in addition to the presence/absence of histologic VI. This is anticipated to lead to the ability to identify patients diagnosed with stage I NSGCTs at high risk of relapse to be considered for adjuvant therapy while others are safely surveilled.

D.M. Berney reports receiving speakers bureau honoraria from Sanofi. No potential conflicts of interest were disclosed by the other authors.

Conception and design: D.C. Gilbert, I. Chandler, R. Huddart, J.M. Shipley

Development of methodology: D.C. Gilbert, I. Chandler, R. Huddart

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Thway, I. Chandler, D. Berney, J. Sweet, R. Huddart, J.M. Shipley

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.C. Gilbert, K. Thway, R. Gabe, S.P. Stenning, R. Huddart, J.M. Shipley

Writing, review, and/or revision of the manuscript: D.C. Gilbert, K. Thway, I. Chandler, D. Berney, R. Gabe, S.P. Stenning, J. Sweet, R. Huddart, J.M. Shipley

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Al-Saadi, I. Chandler, R. Gabe, S.P. Stenning

Study supervision: D.C. Gilbert, R. Huddart, J.M. Shipley

The authors thank Philippa Jones for help in performing the immunohistochemistry, Brenda Summersgill and Ewa Aladowicz for organizing samples, and the pathology departments across the United Kingdom for their assistance in retrieving tumor blocks. This study was supported by the National Cancer Research Institute (NCRI) Testis Cancer Clinical Studies Group.

This study was funded by the Medical Research Council (MRC) Biomarkers Grant G0801477. D. Berney is supported by the Orchid charity. We acknowledge NHS funding to the NIHR Biomedical Research Centre at The Royal Marsden and The Institute of Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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