A subfertile patient diagnosed with testicular carcinoma in situ by immunocytological staining for AP-2{gamma} in semen samples: Case report

C.E. Hoei-Hansen1, E. Rajpert-De Meyts, E. Carlsen, K. Almstrup, H. Leffers and N.E. Skakkebaek

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


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The incidence of testicular cancer is rising. Despite a high cure rate, efforts should be made to obtain diagnosis at the pre-invasive intratubular carcinoma in situ (CIS) stage, as the disease is potentially lethal and treatment has severe side-effects, especially regarding reproductive function. CIS diagnosis is presently only possible by a surgical biopsy of the testis. Immunocytological staining for transcription factor activator protein (AP-2{gamma}), previously identified as a marker for neoplastic germ cells, was performed in centrifuged samples of ejaculates obtained from 104 andrological patients, including patients with testicular cancer and subfertility. Cells positive for AP-2{gamma} were found only in semen samples from patients diagnosed a priori with testicular neoplasms and, surprisingly, in a 23 year old control subject with oligozoospermia and no symptoms of a germ cell tumour. Testicular biopsies performed during the follow-up of this patient revealed widespread CIS in one testicle, thus proving a potential diagnostic value of the new marker. For the first time, a patient without clinical symptoms of testicular neoplasia was diagnosed at the pre-invasive CIS stage using a new, simple method based on immunocytological staining of a semen sample for AP-2{gamma}, a novel marker for CIS. The value of this method for diagnostic use in the clinic requires further careful validation in a large series of patients and controls, but the preliminary results are promising.

Key words: carcinoma in situ testis/intratubular germ cell neoplasia/testicular germ cell cancer/testicular intraepithelial neoplasia/TFAP2C


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Testicular germ cell tumour (TGCT) is the commonest malignancy in young Caucasian males, with a lifetime risk of ~0.5–1% (Huyghe et al., 2003Go). TGCT of young adults originate from a common precursor, the carcinoma in situ (CIS) cell (Skakkebaek, 1972Go), also known as intratubular germ cell neoplasia. Despite the high cure rate of testicular cancer, efforts should be made to obtain diagnosis at the pre-invasive CIS stage, as the disease is potentially lethal, especially in a subset of poor-prognosis patients with non-seminomas, who frequently present with disseminated disease, or in patients with relapsed testis cancer, which is often refractory to chemotherapy (Vogelzang et al., 2000Go). CIS or a unilateral tumour confined to one testicle (stage I) are treated by surgery alone. However, while the presence of a tumour is a medical emergency, a patient with CIS can be given a few weeks to fulfil his desire for fatherhood or store several samples in a semen bank. More advanced testicular cancer requires chemotherapy, which is in most cases successful (Einhorn, 1997Go) but has severe side-effects, especially regarding reproductive function. If CIS is diagnosed in a patient with a contralateral tumour or if CIS is bilateral (very rare) the patient usually can be treated with a low-dose irradiation (von der Maase et al., 1986Go), which results in sterility but preserves, at least partially, testicular endocrine function.

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., 2000Go). 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., 1987Go). 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., 1988aGo). Some CIS markers available at that time (e.g. M2A) have been used to detect CIS cells in semen by immunohistochemistry (Giwercman et al., 1988bGo; Meng et al., 1996Go). 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., 1990Go; Meng et al., 1998Go). 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., 2004Go), 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{gamma}) (Williamson et al., 1996Go). AP-2{gamma} is involved in vertebrate embryogenesis and is required within the extra-embryonic lineages for early post-implantation development (Auman et al., 2002Go). We established AP-2{gamma} 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., 2004Go).

The fact that AP-2{gamma} 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{gamma} 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{gamma}-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.


    Case report
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 


    Materials and methods
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The local ethics committee approved the study and participants gave written informed consent. In most cases the patients were enrolled on the occasion of semen cryopreservation before treatment or during andrological work-up. The testicular cancer group included 12 patients prior to orchidectomy with a subsequently verified germ cell neoplasm (five seminomas, four non-seminomas, one mixed TGCT and two unilateral CIS, which had not proceeded to an invasive tumour). To control for specificity of the assay, we included several control groups: testicular cancer patients after operation (n=7, none had contralateral CIS), 12 patients with non-germ cell tumours or other diseases (one benign Leydig adenoma, one malignant lymphoma, two Hodgkin's disease, one glioblastoma, one Wegener's granulomatosis, one Crohn's disease, one bone tumour, one torsio testis, one pituitary tumour, one Klinefelter, one XYY male), patients referred for semen analysis because of subfertility (16 with normozoospermia and 43 with oligozoospermia), and a group of healthy controls (recruited among apparently healthy young men attending our department for other projects).

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{gamma} antibody (6E4/4:sc-12762; Santa Cruz Biotechnology Inc., USA) using a standard indirect peroxidase method, as previously described (Hoei-Hansen et al., 2004Go), except for small modifications tested using control semen samples spiked with AP-2{gamma}-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{gamma}-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 0–5 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 0–2 were classified as negative, while scores 3–5 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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Figure 1. Examples of immunocytological and immunohistological stainings. (A) Semen sample from a control patient spiked with carcinoma in situ (CIS) cells that are strongly stained with AP-2{gamma}. (B1) AP-2{gamma}-positive CIS cell in the semen sample of the subfertile case patient diagnosed with CIS by this assay. (B2, B3) Examples of AP-2{gamma}-stained cells in the case patient, higher magnification. (C1, C2) Examples of AP-2{gamma}-stained cells in a patient with known testicular cancer. (D) Histological features of the biopsy of the left testis of the case patient with AP-2{gamma} staining and (E) placental alkaline phosphatase (PLAP) staining showing CIS cells both adjacent to the tubular membrane and in the lumen. (F) Semen sample from a control patient, with no AP-2{gamma}-positive cells. Scale bar = 25 µm.

 

    Results
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
At the evaluation of one of the first series of semen samples, we detected six AP-2{gamma}-positive cells in a sample from one of the control participants (Figure 1B). Since our assay was not yet validated, we invited the patient to our department for further clinical examination. He was a 23 year old man who presented for routine semen analysis due to a 11/2 year long pregnancy wish, with a female factor in the infertility problem. History was uneventful, except for parotitis in childhood. Clinical evaluation revealed normal virilization and normal genitalia. The testes had normal consistency and measured 15 ml (right) and 12 ml (left, normal range 15–35 ml) by orchidometry. Ultrasound examination showed a right testicle of 11.5 ml with echo-score 2 (slightly irregular), and a left testicle of 6.9 ml with echo-score 3 (irregular with small echogenic points). The karyotype was normal 46,XY, with no Y chromosome microdeletions. Semen analysis revealed a sperm concentration of 1.03 x 106/ml with a normal volume and reduced motility. Serum reproductive hormones were in the normal range. The presence of one small testis and a very poor semen quality in combination with the finding of AP-2{gamma}-stained cells constituted an indication for bilateral open testicular biopsies, which were performed in agreement with the patient. The right-sided biopsy demonstrated normal testicular tissue, whereas the left-sided biopsy revealed numerous tubules containing CIS, which was confirmed with placental-like alkaline phosphatase (PLAP, a commonly used CIS marker) and AP-2{gamma} staining (Figure 1D, E). The patient was advised to undergo unilateral orchidectomy, prior to which semen was cryopreserved. The couple is currently awaiting a child, which was conceived without assisted reproduction.

Preliminary assessment of the immunocytological assay
Because of this encouraging event, we have decided to report here the value of the AP-2{gamma} 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{gamma}-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, 23–71). 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 0.2–6.4), and, as expected, this case was among the subfertile patients.


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Table I. Summary and preliminary evaluation of the results of the transcription factor activator protein (AP-2{gamma}) staining of semen samples from the various patient categories and control subjects

 

    Discussion
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This is the first report of the diagnosis of a germ cell neoplasm at the CIS stage in a semen sample in a young patient with a suspicion of infertility, who—if not for the inclusion in our study of AP-2{gamma}—would most probably have been diagnosed much later, perhaps only after an overt tumour had developed. We are still in the process of further testing of the new assay but present here preliminary evaluation. It is especially noteworthy that we did not detect false-positive staining among the healthy controls and patients with other cancers, suggesting a high specificity of the assay. The large proportion of negative results in the testicular cancer patients could be due to a small volume of the semen sample used for the test (50–300 µl out of total normal volume of 3–5 ml, depending on concentration and number of samples), thus stressing the need either for repeated analysis or for larger volume analysed. These conditions will be assessed in an ongoing study of a larger number of patients. Negative results could also be due to compression from the tumour causing obstruction of seminiferous tubules resulting in few or no CIS cells shed into semen. The pressure by a tumour should not be a factor in patients with incipient germ cell neoplasm, therefore we anticipate a somewhat higher sensitivity if the assay is used as a diagnostic procedure. Moreover, considering the variability of the number of CIS cells present in the tissue adjacent to a tumour, the sensitivity of our assay is reasonably good, as nearly half of the patients harbouring testicular germ cell neoplasm have been detected in this preliminary study. However, the real predictive value of this assay (both positive and negative) can only be assessed on a large number of subjects with careful evaluation of all variables which are very important and potentially may influence the results, such as the abstinence time, the sample volume, as well as the inter-sample and inter-observer variation during the scoring of stained samples.

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{gamma} staining is able to detect CIS cells in semen. We were able to detect AP-2{gamma}-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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank all the participating patients for their cooperation, and K.Lambrethsen, L.Andersen, T.Adelfest, M.Simonsen, H.Kistrup and S.Soultanova for excellent technical assistance. This study was supported by The Danish Cancer Society, The Svend Andersen Foundation, The Copenhagen University Hospital and The Danish Medical Research Council.


    References
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Almstrup K, Hoei-Hansen CE, Wirkner U, Blake J, Schwager C, Ansorge W, Nielsen JE, Skakkebaek NE, Rajpert-De Meyts E and Leffers H (2004) Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling. Cancer Res 64, 4736–4743.[Abstract/Free Full Text]

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, 2733–2747.[Abstract/Free Full Text]

Czaplicki M, Rojewska J, Pykalo R and Szymanska K (1987) Detection of testicular neoplasms by cytological examination of seminal fluid. J Urol 138, 787–788.[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]

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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, 497–502.[Abstract]

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Submitted on December 2, 2004; resubmitted on December 23, 2004; accepted on December 30, 2004.