APMIS 99: 586-594, 1991

Placental-like alkaline -phosphatase as a marker of carcinoma-in=situ of the testis. Comparison with monoclonal antibodies M2A and 4S9F -

ALEKSANDER GIWERCMAN’, LISBETH CANTELL’ and ALEXANDER MARKS* ‘University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark, and ’Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada

Giwercman, A., Cantell, L. & Marks, A. Placental-like alkaline phosphatase as a marker of carcinomain-situ of the testis. Comparison with monoclonal antibodies M2A and 43-9F. APMIS 99: 586-594, 1991. In an immunohistochemical study of 59 routinely processed tissue specimens from 48 adult testes with isolated carcinoma-in-situ (CIS) changes and of 66 specimens from adult testes without neoplasia, placental-like alkaline phosphatase (PlAP) was shown to be a reliable marker of CIS cells preceding the development of a testicular tumour. Thus, a positive reaction was encountered in all 36 biopsies treated with formaldehyde, or Bouin’s or Stieve’s fluid. However, only 11 of 23 specimens fixed with Cleland’s fluid were immunoreactive for PlAP. None of the non-malignant components of seminiferous tubules, including the large abnormal spermatogonia, reacted with the antibody against PlAP. Besides the antibody against PlAP, monoclonal antibodies M2A and 43-9F were tested on CIS specimens fixed with the above-mentioned fixatives. In the 17 specimens fixed with Stieve’s or Bouin’s fluid, a positive reaction was obtained in all sections with all three antibodies tested. However, for each antibody at least two specimens gave a weak staining reaction. When all three immunostainings were performed, in each case at least one of them gave a moderate or strong reaction, thus making CIS cells easily detectable. In the samples fixed with Cleland’s fluid, a negative reaction was found in one to three specimens, depending on the antibody used. However, at least one of the three antibodies gave a positive reaction if all three immunostainings were applied. In only one of the formaldehydefixed paraffin specimens did CIS cells react with the monoclonal antibody 43-9F, whereas M2A gave no positive reaction at all if this method of fixation was used. Thus, the sensitivity of the immunohistochemical staining procedure in the detection of CIS is dependent on the fixative used and increases when immunostainings with all three markers are performed simultaneously. Key words: CIS testis; immunohistochemical markers; PlAP; fixation methods. Aleksander Giwercman, University Dept. of Growth and Reproduction, Rigshospitalet 5064, Blegdamsvej 9, DK 2100, Copenhagen, Denmark.

Placental-like alkaline phosphatase (PIAP) is a well-known marker of testicular germ cell tumours ( Wahren et al. 1979, Uchida et al. 1981, Jacobsen & Nmgaard-Pedersen 1984). PlAP has also been demonstrated in carcinoma-in-situ (CIS) germ cells of the human

Received September 13, 1990. Accepted December 20, 1990.

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testis (Beckstead 1983, Jacobsen & NergaardPedersen 1984, Hustin et al. 1987, Koide et al. 1987, Manivel et al. 1987, Burke & Mostofi: 1988, Nistal et al. 1989). However, most of these studies focused on CIS found in the vicinity of testicular germ cell tumours (Jacobsen & Nm-gaard-Pedersen 1984, Hustin et al. 1987, Manivel et al. 1987). Only anecdotal reports on PlAP in isolated CIS changes preceding tumour development have been pub-

IMMUNOHISTOCHEMICAL MARKERS OF CIS TESTIS

lished (Beckstead 1983, Koide et al. 1987, Skakkebak et al. 1987, Burke & Mostofi 1988, Nistal et al. 1989). Despite the morphological resemblance between CIS cells occurring in an isolated histopathological pattern and those located close to a tumour, an antigenic diversity, for instance due to a different degree of differentiation, cannot be excluded. From the diagnostic point of view, however, marker studies of the former category of CIS cells seem to be by far the most interesting. Testicular tumours can be prevented if the malignancy is diagnosed at the CIS stage (Skakkebak 1972, Skakkebak et al. 1982) but in routine histological preparation the diagnosis of this early neoplasia can easily be overlooked (Pryor et al. 1983, Koide et al. 1987, Schiitte 1988). Additionally, in some cases, large abnormal spermatogonia, frequently occurring in testicular biopsies from infertile men and in maldescended gonads but not fulfilling the morphological criteria of malignant germ cells, can be misinterpreted as being CIS cells (Holstein et al. 1987). Regarding evaluation of the diagnostic value of an immunohistochemical PlAP staining, it remains to be seen whether these nonmalignant abnormal spermatogonia are PlAPpositive or not. Furthermore, previous studies have revealed that, in a proportion of testicular specimens, the antibody against PlAP does not react with CIS cells or the reaction is only encountered in a very small proportion of CIS cells, which may be overlooked (Jacobsen & Nmgaard-Pedersen 1984, Hustin et al. 1987, Manivel et al. 1987). Similar observations were made when other markers of CIS cells, the monoclonal antibodies (MAbs) M2A or 43-9F, were used in an immunohistochemical staining procedure (Giwercman et al. 1988a, Giwercman et al. 1990). Simultaneous use of a panel of antibodies might improve the diagnostic sensitivity of the immunohistochemical staining procedure. Nearly all studies on the detection of PlAP in CIS cells have been performed on formaldehyde-treated tissue. However, for the preservation of testicular morphology, other fixatives such as Stieve’s, Bouin’s or Cleland’s fluid are recommended (Rowley & Heller 1966, Skakkebak et al. 1982, Schiitte 1988). Little information is available on the influence of the different routinely used fixation methods on the de-

tectability of PlAP and other immunological markers in CIS germ cells. In this study of a total of 59 specimens from 48 testes with CIS not accompanied by a tumour and 66 biopsies from testes without malignancy, we therefore focused on the following topics: 1) PlAP immunoreactivity in CIS cells occurring without any evidence of a tumour; 2) The suitability of different fixatives for immunohistochemical staining with antibody against PlAP and the MAbs M2A and 43-9F; 3 ) The sensitivity of an immunohistochemical staining procedure in the diagnosis of CIS when simultaneous stainings with use of the three antibodies are performed and finally 4) Immunoreactivity for PlAP in the non-malignant abnormal spermatogonia. MATERIALS AND METHODS Testicular biopsies We included 59 consecutive routinely processed tissue specimens from 48 adult testes from 48 men with CIS without a macroscopically visible tumour. The diagnosis of CIS was made at our laboratory during the period October 1970 to March 1987 (Table 1) and was based on the morphological criteria of CIS cells (Skakkebak 1972). The 48 men (age: 2 2 4 8 years) had the biopsy performed during screening for CIS due to infertility, cryptorchidism or unilateral testicular cancer. In order to investigate the influence of different fixatives on antigen expression, in four cases orchidectomy specimens were also included as the testis was removed due to CIS (Table 1). For the same reason, for three men, two biopsy specimens, treated with different fixatives, were tested (Table 1). Additionally, 66 biopsies from previously malde-

TABLE 1. Fixation methods f o r preparation of paraffin sections of CIS specimens No. of testes Fixative No. of specimens 22 S 22 17 C 17 1 B 1 1 L 1 4* s, C 8 1 s, L 2 2** S. C. B. L 8 Total: 48 59 S - Stieve’s fluid; C - Cleland’s fluid; B - Bouin’s fluid; L 4% buffered formaldehyde; * - 2 biopsies and 2 orchidectomy specimens; ** - orchidectomy specimens. ~

~~

~

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scended adult testes (Giwercman et al. 1989) without any microscopical sign of malignancy were studied. In 18 of these 66 specimens, routine histological examination revealed the presence of non-malignant abnormal spermatogonia, whereas the spermatogenesis was qualitatively normal in the remaining 48 testes. Finally, two frozen specimens with CIS were tested. Testicular biopsies were performed surgically under local anaesthesia, as previously described (Bruun et al. 1987). Fixation and tissue preparation All specimens from testes without CIS were fixed in Stieve's fluid (Hg2Cl, [4.8 g], 40 YOformaldehyde [20 ml], acetic acid [4 ml] and H 2 0 [76 ml]). The tissue from gonads with CIS was treated with one of the following fixatives, and the number of specimens included and the duration as well as temperature of fixation are stated in brackets: Stieve's fluid (n = 29; 24-72 h, 20 "C), Cleland's fluid (n = 23; 6-12 h, 4 "C), Bouin's fluid (n=3; 6-12 h, 4°C) or 4% buffered formaldehyde (Lillie's fluid) (n =4; 12 h, 4 "C). Thus, for biopsies from 41 testes only one fixation method was applied. In the remaining seven cases, two or more fixatives were tested (Table 1). After fixation, the tissue was dehydrated and embedded in paraplast. The tissue blocks were stored at room temperature or at 4°C for up to 16 years. For immunohistochemical stainings, 4 pm thick sections were prepared. Cryostat sections of the two frozen specimens were prepared and fixed in 4% buffered formaldehyde for 15 min at 4 "C. Antibodies For each of the four fixatives, the concentration of the three primary antibodies giving an optimal specific staining/background ratio was determined in experiments preceding this study. Polyclonal rabbit antibody against PlAP (DAKOpatts, Copenhagen, Denmark) was diluted according to the method of fixation: Stieve's and Bouin's: 1 : 100 & 1 : 200; Cleland's: 1 : 200 & 1 : 300; Lillie's 1 : 50 & 1 : 100. MAb M2A was raised against a human ovarian adenocarcinoma cell line HEY (Bailey et al. 1986) and was shown to react against dysgerminomas, seminomas (Bailey et al. 1986) and CIS germ cells (Giwercman et al. 1988a) as well as immature Sertoli cells (Baumal et al. 1989), but apparently not against other types of malignant and non-malignant tissue (Bailey et al. 1986). This MAb was shown to recognize a yet uncharacterized carbohydrate epitope on the cell surface. MAb M2A was used at a 1 : 400 and a 1 : 800 dilution regardless of the fixation method. MAb 43-98 was raised against a squamous cell lung carcinoma cell line RH-SLC-L1 1 (Petitjohn et al. 1987). Recently the epitope recognized by the

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MAb 43-9F was shown to be an Lea-X oligosaccharide determinant (MGrtensson et al. 1988). This MAb was tested at a final dilution of 1 : 20 and 1 : 40. All 59 specimens with CIS and 66 specimens without neoplasia were tested with the antibody against PlAP. For staining with the two MAbs, 14 of the Stieve-fixed, eight of the Cleland-fixed, four of the formaldehyde-fixed and three of the Bouin-fixed samples with CIS were available. Only MAbs M2A and 43-9F were used for staining of formaldehyde-fixed cryostat sections as these antibodies did not react well with CIS cells on formaldehyde-fixed paraffin sections. Immunohistochemical procedure Prior to the immunohistochemical staining, paraffin sections were deparaffinized. The specimens were tested without as well as with proteolytic pretreatment. For proteolytic pretreatment, 0.1% Pronase, Type VII (Sigma) diluted in TRIS buffer, was used (20°C, 5 min). Subsequently, an identical procedure was used for paraffin as well as frozen sections. Thus, human serum diluted 1 : 3 was applied for 30 min at 20 "C, followed by one of the primary antibodies in the dilutions mentioned above. All incubations with the primary antibody were performed overnight at 4 "C. The immunoperoxidase PAP technique was applied for staining with the polyclonal antibody against PlAP. As second layer, swine anti-rabbit antibody (DAKOpatts, Copenhagen, DK) diluted 1 : 30 (20 "C, 30 min) was used, followed by rabbit peroxidase-antiperoxidase complex (DAKOpatts, Copenhagen, DK) at a dilution of 1 : 50 (20"C, 30 min). The biotin-streptavidin method was used for detection of the 2 MAbs. Biotinylated rabbit anti-mouse antibody (Zymed, San Francisco, Ca) (20 "C, 30 min) and finally horseradish peroxidase-conjugated streptavidin-biotin complex (DAKOpatts, Copenhagen, DK) (20°C, 30 min) were applied. All antibodies were diluted in 0.05 M TRIS-buffered saline, pH 7.4. In all cases, the colour reaction was developed in 3,3'-diaminobenzidine and the slides were counterstained with Mayer's haematoxylin. For negative control, TRIS buffer was substituted for the primary antibody. Evaluation All slides were evaluated by light microscopy (magnification: x 125 and x 312). A positive colour reaction was recognized as a brownish staining of cells. A semiquantitative four grade scoring system was used for evaluation of the intensity of the immunohistochemical staining. "0" indicated no staining and if the staining was weak with less than 10% of CIS cells reacting positively a score of "+" was given. Moderate staining with 1&75% CIS cells reacting was scored as "+ +", whereas very strong staining

IMMUNOHISTOCHEMICAL MARKERS OF CIS TESTIS

with a positive reaction in more than 75% of CIS cells was designated “ ”. The colour reaction was easily seen in preparations given a or “ ” score, whereas a careful microscopic examination was necessary in cases with a score. All slides were evaluated blindly by the same investigator

+++

+++

“+ +”

“+”

(A.G.).

RESULTS For all three antibodies, the colour reaction was mainly located on the cell membrane of CIS cells with only sparse cytoplasmatic staining (Fig. 1). The results will be given separately according to the fixative used. St ie ve ’sfixative CIS: A positive reaction with the antibody against PlAP was encountered in all 29 specimens (Fig. 1). In five of them the reaction was rather weak (+), whereas in the remaining 24 a moderate (n = 13) or strong reaction (n = 1 1) was found. Also with the two MAbs, M2A and 43-9F, a positive reaction was encountered in all of the 14 specimens tested. A comparison of results obtained in specimens when all three antibodies were applied is shown in Table 2. Approximately the same number of specimens exhibiting a strong (+ +), moderate (+ +) or weak (+) reaction was found for each antibody

+

tested. In some cases the staining intensity varied from for one antibody to for one of the other markers. However, if immunohistochemical staining was performed with all of the three antibodies on separate slides of the same biopsy, at least one of the preparations exhibited a moderate or strong reaction, which made the CIS cells easy to identify (Table 2). Non-malignant tissue: No staining of Sertoli cells, spermatogonia, spermatocytes, spermatids or Leydig cells was encountered. Also the abnormal large spermatogonia were PlAP-negative (Fig. 2).

+

+++

Cleland’s fixative PlAP-positive CIS cells were found in 11 of the 23 specimens treated with this fixative. The results for the eight biopsies tested with all three antibodies are shown in Table 2. Generally, for each of them, an absence of staining of CIS cells was encountered in one or more specimens. However, if all three antibodies were applied on separate slides of the same specimen, CIS cells were positively stained in at least one of the preparations. Bouin ’sfixative In all three cases, CIS cells reacted with each of the antibodies used. However, a varying proportion of cells reacting was found (Table 2).

Fig. 1. CIS pattern. Stieve’s fixative. Immunoperoxidase staining for PlAP. Counterstaining with haematoxylin. Note the well-preserved morphology of positively reacting CIS (C) cells and the colour reaction mainly corresponding to the cell membrane. N o staining of Sertoli cell(s) and in a tubule with spermatogenesis and without CIS (N). Bar - 25 Fm.

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TABLE 2. Results of immunohistochemical staining of adult CIS germ cells with three different antibodies applied on paraffin sections fixed in Stieve’s, Cleland’s, Lillie’k or Bouin’s fluid Fixative Specimen Antibody No. anti-P1AP M2A 43-9F Stieve 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cleland 15 0 16 0 0 17 18 0 19 20 21 22 0 + 0 Lillie 23 0 0 24 0 0 25 0 0 26 +++ 0 ++ Bouin 27 28 29 + + + More than 75% of CIS cells positively stained; + + Positive reaction in 1(r-75% of CIS cells; Staining reaction in some CIS cells, but not more than 10%; 0 No staining.

+++ +++ +++ +++ +++ + ++ + ++ ++ ++ ++ ++ +++ ++ +++ ++ ++ ++ ++ +++ +++ +++ +++ +++ +++

++ ++ +++ +++ ++ ++ +++ ++ + +++ ++ +++ ++ ++ +++ +++ +++ ++ ++ ++

+++ ++ +++ +++ ++ ++ +++ + ++ + ++ ++ +++ +

+ + + ++ +

++ +++ +++ + + + ++

+

4% buffered formaldehyde On paraffin sections, a strong reaction was found in all four cases tested with the antibody against PIAP; however, the morphology of CIS cells was not well preserved as compared to the other three fixatives (Fig. 3). No staining of CIS cells was seen when MAb M2A was used as primary antibody, whereas MAb 43-9F gave a moderate staining in one of the four specimens investigated. However, in the two formaldehyde-fixed cryostat sections, CIS cells stained strongly with both MAbs. Generally, in paraffin sections, the intensity

590

of staining did not depend on the duration or temperature of storage. No change in staining intensity was obtained by proteolytic pretreatment. DISCUSSION This study including testicular biopsies from 48 adult testes with CIS confirmed that PlAP is a sensitive immunohistochemical marker of CIS germ cells preceding the development of an invasive testicular tumour. Additionally, we found that the sensitivity of this marker is comparable with that of MAbs M2A and 43-9F in tissue fixed with Bouin’s, Cleland’s or Stieve’s fluid. In specimens processed in formaldehyde, the antibody against PlAP is superior to the 2 MAbs. Previous histochemical (Beckstead 1983) as well as immunohistochemical (Jacobsen & Norgaard-Pedersen 1984, Hustin et al. 1987, Koide et al. 1987, Manivel et al. 1987, Burke & Mostofi 1988, Nistal et al. 1989) studies have demonstrated the presence of PlAP in CIS germ cells. However, these reports were mainly based on CIS tissue adjacent to a tumour and the reports on PlAP immunoreactivity in isolated CIS changes include relatively small numbers ( < 10) of specimens (Beckstead 1983, Koide et al. 1987, Manivel et al. 1987, Burke & Mostofi 1988, Nistal et al. 1989). Despite the morphological resemblance between CIS cells adjacent to invasive cancer and those preceding a tumour, some antigen diversity might exist due to a different degree of differentiation. Thus, PlAP seems to be expressed more consistently in seminomas than in non-seminomas (Uchida et al. 1981, Jacobsen & Norgaard-Pedersen 1984). In all 48 patients with CIS, the early neoplasia was found without signs of a testicular tumour. It is now evident that adult CIS, in most if not all cases, progresses to invasive cancer (Skakkebak et al. 1982, von der Maase et al. 1986). Additionally, development of seminoma has been observed in a 20-year-old man in whom the diagnosis of CIS was established before puberty (Miiller et al. 1984). As testicular cancer can be prevented if the neoplasia is diagnosed at the CIS stage (Skakkebak et al. 1982, von der Maase et al. 1987), the diagnosis of CIS in testicular biopsy should not be overlooked.



IMMUNOHISTOCHEMICAL MARKERS OF CIS TESTIS

Fig. 2. A large abnormal non-malignant spermatogonia (A) and a normal spermatogonia (N) in a maldescended testis. Stieve’s fixative. Staining as in Fig.1. No positive reaction for PlAP. Bar 25 pm.

Fig. 3. CIS. Formaldehyde fixation. Immunostaining for PlAP. Note the morphology of CIS ( C ) cells and cells in tubules without CIS (N) not as well preserved as in Fig. 1. and in Fig. 2. Bar - 25 pm.

However, CIS changes may be missed in routine histological preparations (Pryor et al. 1983, Koide et al. 1987, Burke & Mostoj? 1988, Schutte 1988). Immunohistochemical staining may thus be of significant diagnostic value. The reliability of this procedure is strengthened by lack of PlAP expression in the large benign abnormal

spermatogonia which are often found in gonads from men with a history of cryptorchidism and from infertile males, and may be misinterpreted as being CIS cells (Holstein et al. 1987). Additionally, none of the other components of seminiferous tubules were PlAP-positive. Thus, we could not confirm the findings of Hustin et al. 59 1

GIWERCMAN el al.

(1987) and of Paiva et al. (1983), who found a positive reaction for PlAP in non-malignant germ cells. Using more sensitive electrophoretic methods of detection, Hofmann et al. (1989) recently found that normal testes contain no, or only trace amounts of, PlAP activity. In this study, only non-malignant tissue treated with Stieve’s fixation was included. The choice of fixative is of importance for the diagnosis of CIS. Thus, CIS cells may be difficult to recognize morphologically in formaldehyde-treated specimens (Fig. 3). Therefore, fixatives such as Cleland’s, Bouin’s or Stieve’s fluid are recommended for the preparation of testicular biopsies (Rowley & Heller 1966, SkakkebRk et al. 1982, Schutte 1988). The fixation procedure has previously been shown to be of importance for the preservation of different tissue antigens (Jacobsen & Jacobsen 1984). Our study showed that the choice of fixative is also of importance for immunohistochemical detection of PlAP in routinely processed specimens. Whereas a positive reaction was only obtained in 11 of 23 specimens fixed in Cleland’s fluid, PlAP immunoreactivity was present in all of the remaining 36 cases treated with one of the other three fixatives. Recently 2 MAbs, M2A (Giwercman et al. 1988a) and 43-9F (Giwercman et al. 1990), were reported to be suitable for immunohistochemical detection of CIS germ cells. MAb M2A was also used to identify malignant germ cells in seminal fluid (Giwercman et al. 1988b). The sensitivity of these two markers seems to be equal to that of PlAP in biopsies not treated with formaldehyde (Table 2). Our data indicate that the diagnostic sensitivity of an immunohistochemical procedure may be increased by use of three of the immunohistochemical markers. Thus, in Stieve- or Bouin-fixed specimens, all of the 17 biopsies tested exhibited a strong or moderate staining reaction in at least one preparation if stainings with all three antibodies were performed. Similarly, no false-negative results were obtained in specimens treated with Cleland’s fixative if staining for all three of these markers was performed (Table 2). This study and two previous studies (Giwercman et al. 1988a; Giwercman et al. 1990) have demonstrated that, in testicular tissue of adults, the 2 MAbs are specific for CIS germ cells as no staining of other cellular components 592

was obtained. In children, the MAb M2A is also a marker for immature Sertoli cells (Baumal et al. 1989), whereas MAb 43-9F seems not to react with prepubertal cells in the testes, at least in children aged 4 years and over (Giwercman et al. 1990). MAb M2A gave no reaction at all and MAb 43-9F only moderate staining in one of the four cases of formaldehyde-fixed paraffin sections. This preparation procedure was previously shown not to destroy the 43-9F reactivity in lung cancer cells (Olsson et al. 1984), but the findings are in accordance with results of immunohistochemical stainings of larger series of formaldehyde-fixed specimens with CIS adjacent to testicular tumours (Giwercman et ul. 1990, Giwercman & Visjeldt unpublished data). The 2 MAbs, 43-9F and M2A, recognize different carbohydrate epitopes on the cell membrane of CIS cells. Such carbohydrates may be bound to ceramides as glycolipids or to cell surface proteins as glycoproteins (Hakomori 1985). A strong positive reaction was obtained with both MAbs in formaldehyde-fixed cryostat sections. Formaldehyde, unlike the other three fixatives, does not bind the lipids. Our findings indicate that the loss of antigenicity in formaldehydetreated paraffin sections is due to alcohol dehydration prior to embedding rather than to the effect of the fixative. Thus, in CIS germ cells, the M2A and the 43-9F epitope might be mainly bound to lipids of the cell membrane and are therefore dissolved and removed during dehydration, whereas, in lung cancer cells, the 43-9F epitope is apparently expressed on glycoproteins and is thereby resistant to ethanol dehydration. Similar findings have previously been made for other carbohydrate epitopes (Garin-Chesa & Rettig 1989). The presence of trace amounts of PlAP has been demonstrated in normal testicular tissue (Hofmann et al. 1989); however, the concentration is apparently too low to allow immunohistochemical detection. The association between the high expression of PlAP in CIS germ cells and the aetiology and pathogenesis of testicular neoplasia is as yet unclear. It has been postulated that the overexpression of PlAP might, at least partly, be due to a polysomy of chromosomes 1 and 2, which harbour the genes encoding for alkaline phosphatase (Hqfmann et al. 1989). However, PlAP is also a marker of

IMMUNOHISTOCHEMICAL MARKERS OF CIS TESTIS

early foetal gonocytes (McKay et al. 1953, Hustin et al. 1987), and it has therefore been proposed that CIS germ cells are, in fact, malignant gonocytes (Skakkebek et al. 1987). In conclusion, this study demonstrates that immunohistochemical staining for PlAP is a reliable tool in the detection of CIS cells in routinely processed adult testicular biopsies performed during screening for early testicular neoplasia. The best combination of well-preserved morphology and antigenicity is obtained if Stieve's or Bouin's fluid is used as fixative. The other two markers of CIS germ cells, MAbs M2A and 43-9F, are as reliable as PlAP in detection of CIS cells, and a combination of the three markers may even improve the sensitivity of the immunohistochemical procedure. Formaldehyde-fixed paraffin sections are the one exception. In such preparations, only staining for PlAP is of diagnostic value.

8.

9.

10.

1 1.

12.

This study was supported by grants from the Danish Cancer Society (88-100) and the Daell Foundation. 13.

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Placental-like alkaline phosphatase as a marker of carcinoma-in-situ of the testis. Comparison with monoclonal antibodies M2A and 43-9F.

In an immunohistochemical study of 59 routinely processed tissue specimens from 48 adult testes with isolated carcinoma-in-situ (CIS) changes and of 6...
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