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Clinical Chemistry 54: 1840-1843, 2008. First published September 11, 2008; 10.1373/clinchem.2008.108548
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(Clinical Chemistry. 2008;54:1840-1843.)
© 2008 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Free β-Subunit of Human Chorionic Gonadotropin in Serum Is a Diagnostically Sensitive Marker of Seminomatous Testicular Cancer

Anna Lempiäinen1,a, Ulf-Håkan Stenman1, Carl Blomqvist2 and Kristina Hotakainen1

Departments of1 Clinical Chemistry and 2 Oncology Helsinki University Central Hospital and Helsinki University, Helsinki, Finland.

aAddress correspondence to this author at: Helsinki University Central Hospital/Biomedicum Helsinki, Room A419a, Haartmaninkatu 8, PO Box 700, FIN-00029. Fax +358-9-47171731; e-mail anna.lempiainen{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: We studied whether measurement of the free β subunit of human chorionic gonadotropin (hCGβ) in serum offers additional diagnostic information compared to determination of intact hCG alone in testicular cancer.

Methods: We determined hCG and hCGβ with ultrasensitive assays in 94 serum samples obtained preoperatively, 22 samples obtained during relapse, and 3687 samples obtained during routine follow-up of 351 patients with testicular tumors.

Results: In preoperative samples, isolated increases of hCGβ were seen in 40% of the samples from seminoma patients (n = 42) and in 8% of those from patients with nonseminomatous testicular cancer (NSGCT) (n = 51). Both markers were increased in 12% of the seminoma and 71% of the NSGCT patients and were within reference intervals in 43% of the seminoma and 20% of the NSGCT patients. Specific determination of hCGβ increased the frequency of marker-positive seminomas from 17% to 57% and of marker-positive relapses from 32% to 59% (n = 22). Theoretically, about 40% of marker-positive seminomas and relapses would have been missed with an assay measuring hCG and hCGβ together. Preoperative hCG and hCGβ concentrations correlated with stage, tumor histology, and disease-related mortality. Additionally, hCGβ correlated with tumor size.

Conclusions: hCGβ is a diagnostically sensitive marker for testicular cancer. In patients with seminomatous testicular cancer, hCGβ is superior to hCG, and in some NSGCT patients it provides additional information.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Testicular cancer is the most common cancer in men age 15–35 years. The cure rate of low-stage disease is about 95%, and to avoid long-term toxicity, primary treatment is often limited to surgery (1). Without adjuvant therapy, however, relapses are fairly common, and patients need careful monitoring for 5–10 years. Thus there is a need for markers that enable early detection of relapse.

Either {alpha}-fetoprotein (AFP)1 or human chorionic gonadotropin (hCG) in serum is increased in about 80% of the patients with nonseminomatous testicular cancer (NSGCT), but seminomas are by definition AFP-negative, and hCG is increased in only about 15%–20% of the cases (2). However, 30%–40% of seminoma patients have been found to have increased serum concentrations of the free β-subunit of hCG (hCGβ) (3)(4)(5).

We studied whether separate measurements of hCGβ and hCG offer additional information in the diagnosis and follow-up of testicular cancer compared to the determination of hCG or simultaneous detection of hCG and hCGβ together.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and samples
We studied 94 preoperative serum samples (seminoma, n = 42; NSGCT, n = 51; and unclassified germ cell cancer, n = 1), 22 samples obtained during relapse (seminoma, n = 4; NSGCT, n = 18), and 3687 samples obtained during routine follow-up from 351 patients with testicular tumors treated between 1990 and 2003 at the Helsinki University Central Hospital. Data regarding clinical characteristics (see Supplemental Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol54/issue11) were retrieved from patient charts in 2006 and were reevaluated by A.L. and C.B. to unify staging according to the International Union Against Cancer classification (6). Germ cell tumors with an extratesticular primary site (n = 4) were not staged. The local ethics committee approved the study.

serum determinations
hCG in serum was determined by a time-resolved immunofluorometric assay (AutoDelfia, Perkin-Elmer Wallac) according to the manufacturer’s instructions. The assay measures only intact hCG but not hCGβ (7)(8). The functional detection limit for hCG is 1.5 pmol/L (0.5 IU/L), and the upper reference limit in males is 6 pmol/L (2 IU/L) (9). Because testicular cancer is often associated with testicular malfunction, which may cause a moderate increase in the serum concentration of hCG (10), a cutoff concentration of 15 pmol/L (5 IU/L) was used.

hCGβ was determined by an ultrasensitive in-house immunofluorometric assay by use of monoclonal antibody (MAb) 9C11 as the capture antibody and MAb 1B2 as the detection antibody. MAb 9C11 detects only hCGβ and not intact hCG, whereas 1B2 detects both forms. The limit of quantification of the assay was 0.5 pmol/L, and the upper reference limit was 2 pmol/L (see Supplemental Text 1 in the online Data Supplement).

Preoperative AFP concentrations measured with the Delfia assay (Perkin-Elmer Wallac) were retrieved from patient charts (reference interval 1–9 IU/L).

statistical analyses
Statistical analysis was performed using SPSS (version 14.0) software. The association of preoperative serum hCG and hCGβ concentrations with tumor type, location of the primary tumor, stage (Kruskal–Wallis), tumor size (Kendall tau-b), vascular invasion, tunica albuginea invasion, relapse, treatment resistance, and disease-related mortality (Mann–Whitney U-test) were analyzed. All tests were 2-sided, and P values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
preoperative samples
In seminomatous testicular cancer, the preoperative serum concentration of hCGβ was increased in 22 (52%) of 42 patients and that of hCG in 7 patients (17%). In 5 patients (12%) both hCG and hCGβ were increased, in 17 (40%) only hCGβ was increased, and in 2 patients (5%) only hCG was increased. In 18 patients (43%) both markers were negative (Fig. 1 ). If the cutoff for hCG was lowered to 3 pmol/L (1 IU/L), hCGβ was the only increased marker in 16 (38%) of 42 seminoma patients.


Figure 1
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  		Figure 1. Preoperative serum concentrations of hCGβ (y axis) and hCG (x axis) in patients with seminomatous (A) and nonseminomatous (B) testicular cancer.

The solid lines mark the upper reference limits and the short dashed lines indicate a concentration of 3 pmol/L (1 IU/L) on the x axis and the upper reference limit for the hCG + hCGβ assay (15 pmol/L or 5 IU/L) on the y axis.

In NSGCT, the preoperative serum concentration of hCGβ was increased in 40 (78%) of 51 patients and that of hCG in 37 patients (73%). Both markers were increased in 36 patients (71%) and were within reference intervals in 10 patients (20%). Isolated increase of hCGβ was seen in 4 patients (8%) and of hCG in 1 patient (2%) (Fig. 1Up ). Two NSGCT patients had hCG concentrations of 3–15 pmol/L (1–5 IU/L). The preoperative concentration of AFP was determined in 39 of 51 NSGCT patients; it was increased in 30 patients (77%) and within reference intervals in 9 patients (23%). Both hCG and AFP were increased in 22 (56%) of 39 patients, hCG alone in 6 patients (8%), and AFP in 8 patients (21%).

The preoperative concentrations of hCG and hCGβ correlated strongly (P < 0.0005), whereas hCGβ also correlated with disease stage (P < 0.0005) and disease-related mortality (P = 0.025, n = 3), tumor size (P < 0.0005), and tumor type (P = 0.018), with the highest concentrations occurring in choriocarcinoma patients and the lowest in seminoma patients. HCG was associated with disease stage (P < 0.0005), disease-related mortality (P = 0.028), and tumor type (P < 0.0005), being highest in choriocarcinoma and lowest in seminoma. HCGβ and hCG correlated with disease stage in NSGCT patients (P = 0.013 for both).

detection of relapse
At relapse, an isolated increase in hCGβ was seen in 1 of 4 seminoma patients and 5 (28%) of 18 NSGCT patients. Both hCG and hCGβ were increased in 1 of 4 seminoma patients and 6 of 18 NSGCT patients. Both markers remained negative in 2 (50%) of seminoma patients and 7 (39%) of NSGCT patients, and no patients had an isolated increase of hCG at the time of relapse. Thus, the separate determination of hCGβ increased the number of marker-positive relapses from 7 to 13 (46%), or if a cutoff of 3 pmol/L (1 IU/L) was used for hCG, from 8 to 13 (38%). AFP was increased in 4 (44%) of 9 patients and hCG was concomitantly increased in 3 of these patients.

follow-up samples
A slightly increased hCGβ (2–8.5 pmol/L) without signs of relapse was observed in 28 (1.1%) of 2540 follow-up samples from 19 (5.6%) of 340 patients. We reassayed 23 samples available for control by using a buffer with higher concentration of blocking antibody. For these samples the concentration was within the reference interval in 22 samples and was 2.1 pmol/L in 1 sample.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some earlier studies have shown that in seminoma patients hCGβ may be increased without an increase of hCG (3)(4)(5), but separate determinations of hCG and hCGβ are seldom used clinically. We used specific assays to study the utility of hCGβ and hCG in the diagnosis and monitoring of germ cell tumors of the testis. The results confirm that hCGβ is a better diagnostic marker for testicular cancer than hCG. This finding is of particular value in seminoma patients, and in 40% of these individuals hCGβ was the only increased marker. Thus, the frequency of marker-positive seminomas increased from 17% to 57%. In NSGCT, the concentrations of hCG and hCGβ were mostly concordant, but an isolated increase of hCGβ (8%) or hCG (2%) was seen in a few patients.

Because both hCG and hCGβ can be increased in patients with testicular cancer, assays recognizing both of these together have been recommended for monitoring of this disease (3)(4)(11). Most currently available hCG assays are of the so-called total hCG assay type. However, because the upper reference limits for intact hCG (15 pmol/L or 5 IU/L) and hCG + hCGβ (17 pmol/L) are much higher than that for hCGβ (2 pmol/L) (9), tumors causing only a moderate increase of serum hCGβ will not be detected with an assay measuring hCG and hCGβ together. Theoretically, if an assay measuring hCG and hCGβ together would have been used in the present study, 10 (42%) of 24 marker-positive seminomas, 3 (8%) of 40 marker-positive NSGCTs, and 5 (38%) of 13 marker-positive relapses would have been missed, confirming earlier results (5)(12).

In our study the frequency of isolated increases of serum hCGβ was higher than in previous reports (3)(4)(5)(13)(14)(15), particularly in seminoma patients. In a recent study, an isolated increase of hCGβ was detected in 37% of the seminoma patients and in 3% of NSGCT patients, whereas hCG was detected in 50% of the seminoma patients and in 82% of the NSGCT patients (5). The high frequency of increased hCG values observed in that study is most likely explained by more advanced disease stages in the study population, especially in seminomatous testicular cancer, i.e., 44% of stage II and III disease, compared to 7% in present study.

In our study the frequency of falsely increased values of hCGβ (1.1%) was lower than the 2.5% expected on the basis of the upper reference limit. In most of these samples the result was within reference intervals when the sample was reassayed several years later. This change may be explained by decay of factors causing nonspecific interference during sample storage (16). In most of these cases, results for the next follow-up samples were within reference intervals. Slightly increased hCGβ values do not necessarily indicate pathological conditions and thus it is important not to initiate cancer treatment on the basis of a single increased value (16)(17).

We conclude that hCGβ is a useful diagnostic marker for testicular cancer and that in seminomas hCGβ is much more useful than hCG. In most cases, the increase of hCGβ was only moderate and therefore approximately 40% of marker-positive seminomas and of relapses would have been missed by an assay measuring hCG and hCGβ together. Thus separate determination of hCGβ provides clinically valuable information.


   Acknowledgments
 
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: A. Lempiäinen received funding from the Finnish Cancer Foundation, Finska Läkaresällskapet, and Wilhelm och Else Stockmanns Stiftelse; and C. Blomqvist, from the Finnish Cancer Society.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We would like to thank Taina Grönholm for expert technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: AFP, {alpha}-fetoprotein; NSGCT, nonseminomatous testicular cancer; hCG, human chorionic gonadotropin; hCGβ, free β-subunit of hCG; MAb, monoclonal antibody.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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