University Department of Growth and Reproduction (GR-5064), Rigshospitalet Blegdamsvej 9, DK-2100 Copenhagen, Denmark
1 To whom correspondence should be addresed. Email: chh{at}dadlnet.dk
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Abstract |
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Key words: carcinoma in situ testis/intratubular germ cell neoplasia/testicular germ cell cancer/testicular intraepithelial neoplasia/TFAP2C
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Introduction |
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The diagnosis of CIS is established by surgical biopsy, performed in selected at-risk patients (e.g. with a history of cryptorchidism) or where the suspicion has arisen after clinical examination (atrophic testes) and ultrasonography (microlithiasis or a very irregular echo pattern) (Rorth et al., 2000). However, CIS is usually asymptomatic, therefore very few patients are diagnosed at this stage. In the semen of patients with testicular cancer, morphologically abnormal cells may be seen although the morphology of seminal cells other than sperm cells are poorly preserved (Czaplicki et al., 1987
). We have previously demonstrated that CIS cells could be found in semen samples of patients with testicular tumours, as they are present nearly always in seminiferous tubules in the vicinity of a tumour (Giwercman et al., 1988a
). Some CIS markers available at that time (e.g. M2A) have been used to detect CIS cells in semen by immunohistochemistry (Giwercman et al., 1988b
; Meng et al., 1996
). In another approach, in situ hybridization with chromosomal probes was used to demonstrate CIS cells in semen, taking advantage of their aneuploid DNA content and occasional presence of the isochromosome i(12p) (Giwercman et al., 1990
; Meng et al., 1998
). However, both methods were time-consuming and false-negative or false-positive results were frequent, due to a partial degradation of cells and cell surface antigens in semen. Thus, these methods were not sufficiently reliable to be used for diagnostic purposes in the clinical setting.
In a recent genome-wide gene expression profiling study of CIS cells (Almstrup et al., 2004), we have identified a number of genes expressed in CIS and also in embryonic stem cells and fetal gonocytes but not in adult germ cells, thus providing many possible new markers for CIS. One such gene was TFAP2C (mapped to chromosome 20q13.2), which encodes the transcription factor activator protein-2 (AP-2
) (Williamson et al., 1996
). AP-2
is involved in vertebrate embryogenesis and is required within the extra-embryonic lineages for early post-implantation development (Auman et al., 2002
). We established AP-2
as a novel marker for fetal gonocytes and neoplastic germ cells, including testicular CIS, with a role in pathways regulating cell differentiation and a possible involvement in oncogenesis (Hoei-Hansen et al., 2004
).
The fact that AP-2 protein was not expressed in normal adult reproductive tract but was abundant in nuclei of CIS and tumour cells, which are better protected than the cytoplasm from degradation in semen due to structural strength, prompted us to analyse the value of AP-2
for detection of CIS and/or tumour cells in ejaculates. Having already tested a relatively small series of patients and controls, we have detected AP-2
-positive cells in a presumed control subject, subsequently confirmed by surgical biopsy, harbouring CIS in one of his testicles. We report this case and present a preliminary evaluation of the new procedure, which has the potential to become a diagnostic assay.
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Case report |
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Materials and methods |
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Freshly delivered semen samples were centrifuged (5 min at 1500 rpm) and cellular sediments were collected, after a routine semen analysis had been performed. A semen sample from each patient was diluted if necessary, divided in portions of 100 µl, centrifuged on a microscope slide with 400 µl phosphate-buffered saline buffer, dried, fixed for 10 min in formalin, washed and dried again. The immunocytochemical staining was performed with the monoclonal anti-AP-2 antibody (6E4/4:sc-12762; Santa Cruz Biotechnology Inc., USA) using a standard indirect peroxidase method, as previously described (Hoei-Hansen et al., 2004
), except for small modifications tested using control semen samples spiked with AP-2
-positive seminoma or CIS cells (Figure 1A). For a negative control, another centrifuge sample was incubated with a dilution buffer, and for a positive control a centrifuge sample of AP-2
-positive seminoma cells was used. The evaluation was performed independently by two investigators (C.H.H. and E.R.M.), who had no prior knowledge of the diagnosis. Staining was scored using an arbitrary 05 scale based on the intensity and morphological resemblance of stained elements to CIS or tumour cells: score 0: no staining; 1: negative with some unspecific particles stained; 2: trace reaction in small fragments of nucleus-resembling structures; 3: clear staining in part of a nucleus, with correct morphology; 4: clear staining in a whole nucleus, with correct morphology; 5: two or more cells with score 4. Scores 02 were classified as negative, while scores 35 were classified as positive. Statistical analysis of data was performed using the Confidence Interval Analysis programme, version 2.0, developed by D.G.Altman, D.Machin, T.N.Bryant and M.J.Gardner.
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Results |
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Preliminary assessment of the immunocytological assay
Because of this encouraging event, we have decided to report here the value of the AP-2 immunoassay in a preliminary manner, having analysed the first 100 subjects. The results are summarized in Table I and examples are shown in Figure 1. We detected AP-2
-positive cells in five of 12 patients harbouring testicular neoplasia, giving a sensitivity of our assay of 42%. However, after addition of the newly diagnosed control patient with CIS, the sensitivity is 46% (95% CI, 2371). As lifetime risk of TGCT (and assumed prevalence of CIS) is
1% in the male population in Denmark, and probably even higher among subfertile men, we anticipated a case of testicular neoplasia after investigating
100 normal subjects or subfertile patients. Accordingly, we detected one in a total of 85 young controls (excluding seven patients cured from testicular cancer), which is 1.2% (95% CI 0.26.4), and, as expected, this case was among the subfertile patients.
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Discussion |
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The study is an example of a rapid clinical application of new knowledge provided by gene expression profiling, a tool of basic research. We have demonstrated unequivocally that the immunocytological AP-2 staining is able to detect CIS cells in semen. We were able to detect AP-2
-stained cells in approximately half the analysed patients harbouring testicular neoplasia, which is encouraging, but a more accurate estimate will be available from a larger series of patients. We are currently trying to improve the intensity of staining and are testing the value of adding additional markers on serial centrifuging. In addition, the speed of scoring may be improved by using an automated scanning microscope. The results of ongoing studies in a wider panel of patients and controls will confirm whether this assay should be offered widely to patients at risk for TGCT as a method of non-invasive screening for CIS. Such screening could be performed, for example, at andrology and fertility clinics in young men with atrophic testes, history of cryptorchidism or who need an assisted reproduction technique. If diagnosis of testicular cancer is made at the pre-invasive CIS stage, the patient can be offered the most gentle and optimal treatment.
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Acknowledgements |
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References |
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Auman HJ, Nottoli T, Lakiza O, Winger Q, Donaldson S and Williams T (2002) Transcription factor AP-2gamma is essential in the extra-embryonic lineages for early postimplantation development. Development 129, 27332747.
Czaplicki M, Rojewska J, Pykalo R and Szymanska K (1987) Detection of testicular neoplasms by cytological examination of seminal fluid. J Urol 138, 787788.[ISI][Medline]
Giwercman A, Marks A and Skakkebaek NE (1988a) Carcinoma-in-situ germ-cells exfoliated from seminiferous epithelium into seminal fluid. Lancet 1(8584), 530.[Medline]
Giwercman A, Marks A, Bailey D, Baumal R and Skakkebaek NE (1988b) A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis. Acta Pathol Microbiol Immunol Scand 96, 667670.
Giwercman A, Hopman AH, Ramaekers FC and Skakkebaek NE (1990) Carcinoma in situ of the testis. Detection of malignant germ cells in seminal fluid by means of in situ hybridization. Am J Pathol 136, 497502.[Abstract]
Einhorn LH (1997) Testicular cancer: an oncological success story. Clin Cancer Res 3, 26302632.[Abstract]
Hoei-Hansen CE, Nielsen JE, Almstrup K, Brask Sonne S, Graem N, Skakkebaek NE, Leffers H and Rajpert-De Meyts E (2004) Transcription factor AP-2 is a developmentally regulated marker of testicular carcinoma in situ and germ cell tumors. Clin Cancer Res 10, 85218530.
Huyghe E, Matsuda T and Thonneau P (2003) Increasing incidence of testicular cancer worldwide: a review. J Urol 170, 511.[ISI][Medline]
Meng FJ, Zhou Y, Skakkebaek NE, Marks A and Giwercman A (1996) Detection and enrichment of carcinoma-in-situ cells in semen by an immunomagnetic method using monoclonal antibody M2A. Int J Androl 19, 365370.[ISI][Medline]
Meng FJ, Zhou Y, Giwercman A, Skakkebaek NE, Geurts van Kessel AD and Suijkerbuijk RF (1998) Fluorescence in situ hybridization analysis of chromosome 12 anomalies in semen cells from patients with carcinoma in situ of the testis. J Pathol 186, 235239.[CrossRef][ISI][Medline]
Rorth M, Rajpert-De Meyts E, Andersson L, Dieckmann KP, Fossa SD, Grigor KM, Hendry WF, Herr HW, Looijenga LH, Oosterhuis JW and Skakkebaek NE (2000) Carcinoma in situ in the testis. Scand J Urol Nephrol Suppl 20S, 166186.
Skakkebaek NE (1972) Possible carcinoma-in-situ of the testis. Lancet 2, 516517.[CrossRef][ISI][Medline]
Vogelzang NJ, Scardino PT, Shipley WU and Coffey DS (eds) (2000) Comprehensive Textbook of Genitourinary Oncology. 2nd edn. LippincottWilliams & Wilkins, Philadelphia.
Von der Maase H, Giwercman A and Skakkebæk NE (1986) Radiation treatment of carcinoma in situ of testis. Lancet I(8481), 624625.
Williamson JA, Bosher JM, Skinnaler A, Sheer D, Williams T and Hurst HC (1996) Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors. Genomics 35, 262264.[CrossRef][ISI][Medline]
Submitted on December 2, 2004; resubmitted on December 23, 2004; accepted on December 30, 2004.