Carcinoma In Situ Cells Research Articles

Detection and enrichment of carcinoma-in-situ cells in semen by an immunomagnetic method using monoclonal antibody M2A.

Carcinoma-in-situ (CIS) is a precursor of germ cell cancers in the testis. Early detection and treatment of CIS can prevent the development of invasive cancer. We have developed an immunomagnetic approach for enrichment of CIS cells from semen. Immunomagnetic beads were coated with monoclonal antibody (mAb) M2A, which is specific for seminoma and CIS. In preliminary mixing experiments, cultured human HEY cells, which express the M2A surface antigen, were added to semen samples and recovered selectively by incubation with M2A-coated immunomagnetic beads followed by magnetic activated cell sorting (MACS). Application of this procedure to ejaculates from five subjects with CIS and 22 control subjects with no evidence of testicular neoplasia resulted in the preferential recovery of a population of large round cells from a 0.5 ml semen sample obtained from CIS subjects (median 1 x 10(4), range 2 x 10(3) to 2 x 10(4)) relative to controls (median 2 x 10(2); range 0 to 10(3)), with no overlap between the two groups (p < 0.001). These results suggest that a subpopulation of large round cells recovered from the semen of CIS patients by incubation with M2A-coated immunomagnetic beads and MACS are CIS cells. Immunomagnetic enrichment of CIS cells in semen will facilitate the definitive identification of exfoliated CIS cells in ejaculates and could be an important first step in a non-invasive screening procedure for testicular CIS.

Alterations of the p53 tumor suppressor gene in carcinoma in situ of the testis.

Carcinoma in situ (CIS) is regarded as the precursor of all histologic variants of testicular germ cell tumors except spermatocytic seminoma. For a variety of human malignancies, alterations of the p53 tumor suppressor gene have been identified as prognostic factors for a poor clinical course. Discussions of the occurrence of p53 gene alterations in testicular carcinoma have been controversial. Immunohistochemical detection of the p53 oncoprotein has been reported in four of eight CIS cell areas adjacent to mature teratoma. The majority of investigations have failed to demonstrate p53 gene alterations on the DNA level in testicular carcinoma specimens. However, the genetic analysis of testicular carcinoma is complicated by the histologic variety of tumors, resulting in a mixture of subtypes undergoing moleculargenetic analysis. In the present study, CIS cells identified in normal testicular tissue adjacent to different testicular tumors were examined for alterations of the p53 tumor suppressor gene. The authors believed that the detection of p53 alterations in CIS of the testis would not only support the idea of malignant potential in CIS but might also demonstrate the involvement of the p53 tumor suppressor gene in the development of germ cell cancers. CIS cells were identified in the normal testicular tissue adjacent to 18 seminomatous and nonseminomatous germ cell tumors by histopathologic criteria and the immunohistochemical staining reaction for placental-like alkaline phos phase, a highly specific marker for testicular CIS. About 20-50 CIS cells per tumor were collected by a microdissection technique and were subjected to RNA. SSCP analysis and additional DNA-sequence analysis. In 12 of 18 cases (66%), RNA-SSCP analysis of the microdissected CIS cells revealed mutational band shifting at the p53 gene locus. In 7 of 18 cases (39%). the results of SSCP analysis were confirmed by DNA sequencing. DNA-sequence analysis revealed missense point mutations in the p53 gene in four cases (exon 5, codons 158, 170, and 176; exon 6, codon 213) and silent mutations in two cases (exon 5, codon 178; exon 6, codon 213). In one case, the identical missense point mutation (exon 5, codon 176) was detected in the CIS cells and also in the associated germ cell tumor. In two of six cases, different mutations were found in the CIS cells and the testicular tumor. In three cases in which DNA sequencing revealed point mutations in CIS cells, alterations of the p53 gene could not be detected in the examination of the associated tumor specimen. These findings appear to support the concept that malignant biologic characteristics are present in the in situ stage of testicular germ cell tumors. This is the first report demonstrating monoclonal development of a manifested testicular carcinoma from associated CIS cells on the basis of a certain mutational event in the p53 gene sequence. The involvement of p53 gene alterations in precursor cells of testicular carcinoma appears likely.

Investigation of carcinoma in situ cells of testis by quantification of argyrophilic nucleolar organizer region associated proteins (AgNORs).

The silver staining which specifically stains argyrophilic proteins (AgNORs) in interphase nuclei was applied to paraffin sections of 24 testicular specimens with carcinoma in situ (CIS). AgNOR area per nucleus was quantified by a computerized image analyser. Significant quantitative differences were found between CIS, Sertoli cells, and spermatogonia (P = 0.0001), with median values of 10.3, 2.8, and 1.4 microns2 in the three cell types, respectively. A Sertoli cell index (SCI), defined as the ratio between AgNORs in CIS or spermatogonia and Sertoli cells, was shown to be significant in the differential diagnosis of CIS cells from spermatogonia when 1.0 was used as the cut-off value (CIS > 1; spermatogonia < 1). Furthermore, CIS associated with non-seminoma was found to have a significantly higher level of AgNORs than CIS associated with pure seminoma (P < 0.01), indicating that subclonal variation in transformation potential might be present within morphologically identical CIS of the testis. It remains to be seen whether quantification of AgNORs in isolated CIS could be used to predict transformation of CIS into seminoma or non-seminoma.

Heterogeneity of expression of immunohistochemical tumour markers in testicular carcinoma in situ: pathogenetic relevance

Testicular carcinoma in situ (CIS) is the precursor of germ cell tumours in adults, except for spermatocytic seminoma. The mechanism of the progression from premalignant CIS to invasive and overt tumours is largely unknown. There are currently two main hypotheses: one is that CIS can progress directly to either seminoma or nonseminoma; according to the other, seminoma is the intermediate stage between CIS and nonseminoma. CIS cells express several tumour antigens, such as placental-like alkaline phosphatase (PLAP), TRA-1-60, or the c-kit proto-oncogene protein product (Kit), which are present to varying degrees in the invasive germ cell tumours. In this study, CIS cells adjacent to either pure or combined tumours were examined by double immunohistochemical staining for simultaneous expression of TRA-1-60 (typical for embryonal carcinoma) and either Kit (expressed by seminomas) or PLAP (found mainly in seminomas, but also in some cases of nonseminoma). Marked differences in the expression of these antigens were found among CIS cells, especially those adjacent to mixed tumours. We conclude that CIS is a phenotypically heterogeneous lesion, and that the CIS cells, despite identical morphology and close spatial localization, may be in different stages of progression. The results lend support to the hypothesis that CIS can progress directly to both seminomatous and nonseminomatous tumours.

IMMUNOREACTIVE NEURON-SPECIFIC ENOLASE (NSE) IS EXPRESSED IN TESTICULAR CARCINOMA-IN-SITU

Neuron-specific enolase (NSE) is a well-known marker of tumours that have neuroendocrine origin. High levels of NSE have also been described in various types of testicular germ cell neoplasms, particularly in seminomas. To evaluate the presence of NSE in testicular carcinoma-in situ (CIS), a preinvasive stage of testicular germ cell tumours, a panel of CIS tissue specimens was examined. Fifteen of 18 (83 per cent) CIS samples showed immunohistochemical staining with anti-NSE monoclonal antibody. Immunoreactivity has also been found in overt testicular germ cell tumours, including seminomas, non-seminomas, and a mixed germ cell tumour. As the co-existence of high NSE production and gene amplification of N-myc has been reported in some tumours, including germ cell tumours, the expression of the protein product of N-myc was also examined in this study, but only sporadic cases showed N-myc staining. These results are evidence against a relationship between NSE and N-myc in testicular germ cell tumours. The high expression of NSE in CIS and overt germ cell tumours may be due to the increased gene dosage effect associated with the overrepresentation of isochromosome 12p.

Over expression of the P53 Oncoprotein in Carcinoma in Situ of the Testis

Carcinoma in situ (CIS) is regarded as the precursor lesion of testicular germ cell tumors. In adults CIS cells have also been described within the normal testicular tissue adjacent to mature teratomas with a frequency of 52-88%. These CIS-cells can be identified by immunohistochemical staining for "placental like alkaline phosphatase" (PLAP). In four of eight patients with mature teratomas CIS was identified by immunohistochemistry for "PLAP" in atrophic testicular tubules adjacent to the tumors. In three of these patients cells of CIS were positive for the p53 oncoprotein, indicating a mutational inactivation of the p53 tumor suppressor gene. The malignant potential of mature teratomas, which do occur without histological signs of malignancy, may therefore be associated with the occurrence of CIS within the histologically normal appearing testicular tissue adjacent to the mature tumor. Immunohistochemical positivity for the p53 protein in CIS cells of some patients may indicate that the mutational inactivation of the p53 tumor suppressor gene could be involved early in the development of testicular germ cell tumors.

Immunohistochemical expression of embryonal marker TRA-1-60 in carcinoma in situ and germ cell tumors of the testis.

Testicular cancer is preceded by the noninvasive stage of carcinoma in situ (CIS). According to a recent hypothesis, testicular CIA cells are germ cells transformed in fetal life. The idea of an embryonal origin of testicular germ cell neoplasia would be strengthened by the finding of antigenic similarity between fetal germ cells, CIS cells, and invasive testicular germ cell tumors. Monoclonal antibody (MoAb) TRA-1-60 raised against a human embryonal carcinoma cell line was immunohistochemically tested on 21 fetal gonads (11 male gonads and 10 female gonads; 11th-24th week of gestation). In addition, TRA-1-60 was tested on tissue from 27 testes with CIS, 11 testes with invasive testicular cancer, and 24 adult and 4 infant testicular control specimens. Expression of TRA-1-60 was found in germ cells of six female and two male fetal gonads. In addition, 26 of 27 adult human testes with CIS, 7 of 8 seminomas, and 3 of 3 embryonal carcinomas were TRA-1-60 positive. The study demonstrated an antigenic link between fetal germ cells, cells of CIS and seminomas, and embryonal carcinomas. The results provided additional evidence for the hypothesis that testicular neoplasia arises during early fetal life and CIS cells are malignant fetal gonocytes.

A new classification of germ cell tumours of the testis.

The partially conflicting British and WHO classifications of testicular tumours are confusing and fail to give due prominence to their histogenesis from carcinoma in situ (CIS). Spermatocytic seminoma does not originate from CIS and should be renamed 'spermatocytoma'. All other germ cell tumours are gonocytomas arising from CIS cells which have many of the characteristics of the gonocytes of the early embryo and fetus. Gonocytomas are subdivided into seminoma, if they retain their gonocyte characteristics, or teratogenic gonocytoma if the tumour stem cell has differentiation potential. A new category of anaplastic germ cell tumour is intermediate between seminoma and teratogenic gonocytoma. The teratogenic gonocytoma is an epiblastoma if no differentiation is manifest, but may differentiate along embryonic lines into teratoma or may show extraembryonic differentiation towards yolk sac tumour or choriocarcinoma. The new classification uses unambiguous terminology and is midway between the British and WHO classifications. Those familiar with either of these classifications should have no difficulty in adapting to the new proposals, and the new classification should help others to acquire a better understanding of the biology and pathology of testicular germ cell tumours.

The possible role of sex hormones in the development of testicular cancer.

The peak incidence of testicular cancer in young men suggests that gestational development and events during early infancy and puberty are important in the pathogenesis of the disease. There are two potentially significant events: the transformation of fetal germ cells into carcinoma-in-situ cells (CIS) and later conversion of CIS cells into fully invasive cancer. Several hypotheses suggest an endocrinological background to testicular neoplasia. Based on epidemiological and experimental evidence, the possible role of oestrogens, androgens and gonadotrophins is discussed in this review. The role of Sertoli cells and the importance of interplay between endocrine and paracrine factors is also stressed.

Further Practical Experiences in the Recognition and Management of Carcinoma in situ of the Testis

99 biopsies from the contralateral testis in patients with unilateral germ cell tumor were investigated using the semithin section technique. Four cases (4%) revealed a carcinoma in situ (CIS) pattern. Two patients underwent a local radiation (20 Gy), 2 patients received combination chemotherapy (cisplatin, etoposide and bleomycin; PEB). No tumor cells were found in control biopsies 4-8 months after therapy. Both biopsy specimens taken from radiated patients lacked also germ cells. In contrast, 1 patient who was treated with PEB and also another 1 presenting with a teratocarcinoma of apparently retroperitoneal origin and unilateral CIS revealed germ cells after chemotherapy. The present data suggest radiation therapy to be the first line treatment for CIS-bearing testes. In order to get informations about the distribution of CIS cells in the affected testes, one or two biopsies were additionally taken from macroscopically unsuspicious tissue surrounding various solid germ cell tumors (74 patients). 56 cases (76%) revealed CIS. Even considering the possibility of missing CIS, a screening biopsy is actually the only method for detecting early manifestations of germ cell tumors and should be performed routinely in contralateral testes of patients with germ cell tumors.

DETECTION OF GONADAL CARCINOMA IN SITU IN CHILDHOOD AND IMPLICATIONS FOR MANAGEMENT

Cells with the characteristic features of carcinoma in situ (CIS) were detected in histological sections of the gonads of three of seven children with clinical syndromes associated with a high risk of gonadal neoplasia. It is suggested that early detection of these cells may be useful in resolving problems of management, including gonadectomy and decisions about sex of rearing. Early detection of CIS cells may have a place in the management of the undescended testis.

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 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 formaldehyde-fixed 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.

Carcinoma in situ of the testis in infertile men. A histological, immunocytochemical, and cytophotometric study of DNA content.

Of 723 infertile men (128 with a history of cryptorchidism) whose testes were biopsied at the outer lateral face of the testis, five presented carcinoma in situ (CIS) in one testis. These testes were removed, serially sectioned, and examined by light microscopy. In order to evaluate whether only one or two biopsies are sufficient to diagnose CIS, before sectioning the testes four biopsies were taken at the anterior face, posterior face, superior pole, and inferior pole of the testis, respectively. Two of the five men had undergone orchiopexy in infancy and the testis contained tubules with Sertoli cells and isolated spermatogonia. CIS was also present in some tubules that were principally located near the rete testis. Of the four simulated biopsies, only that performed at the posterior face of the testis revealed CIS. The other three infertile men showed tubules with complete, although reduced, spermatogenesis, and tubules lined by Sertoli cells only. CIS was found in both types of tubules. These tubules with CIS formed lobules that extended throughout the testicular parenchyma. Most simulated biopsies performed in these three testes showed CIS. The average nuclear DNA content of CIS cells was about 4c in all testes. This content was similar both in tubules with complete spermatogenesis and in tubules with Sertoli cells only.

Management of carcinoma‐in‐situ of the testis

Carcinoma-in-situ (CIS) of the testis progresses to invasive cancer within 5 years in 50% of cases, and therefore requires therapeutic intervention. CIS is probably eradicated by intensive cancer chemotherapy but this is too toxic for the management of non-invasive disease. Eight patients with unilateral testicular cancer and contralateral CIS received localized irradiation (20 Gy in ten fractions of 2 Gy) to the remaining testis: after 3 months the CIS cells had disappeared and 'Sertoli-cell-only' tubules were found. LH and FSH levels were elevated but testosterone levels remained fairly constant. Localized irradiation should be considered as the treatment of CIS in the contralateral testis of testicular tumour patients unless chemotherapy is indicated for the primary tumour. Unilateral orchidectomy is recommended for unilateral CIS associated with infertility or testicular maldescent. Localized testicular irradiation should now be considered for bilateral disease. Patients with the androgen insensitivity syndrome should normally be treated with bilateral orchidectomy, but irradiation may be useful in selected cases.

Placental alkaline phosphatase in testicular germ cell tumours and in carcinoma-in-situ of the testis. An immunohistochemical study.

Recently, placental alkaline phosphatase (PLAP) has been suggested as a tumour marker in patients with seminomas (S), since elevated serum levels of PLAP were found with high frequency in these patients. The present immunoperoxidase study of 33 testicular germ cell tumours was undertaken to localize PLAP in the various types of these tumours as well as in the carcinoma-in-situ (CIS) pattern. Eighteen out of 19 (95%) S were PLAP positive compared to nine out of 14 (64%) non-seminomas (NS). In the NS the positive staining reaction was localized to tumour components of embryonal carcinoma (EC) in six cases, of choriocarcinoma (CC) in one and of S in two, while components of yolk sac tumour and teratoma were PLAP negative. The number of positively stained cells in S was much higher than in EC. The staining reaction was pronounced in the syncytiotrophoblast of CC and in some syncytiotrophoblast-like cells present in S. The staining reaction product was mainly confined to the cell membrane in the positive tumour types. In 20 out of 24 cases with CIS various numbers of CIS cells were PLAP positive, while PLAP was not found in normal germinal epithelium. Sixty three per cent of S patients had serum values above 1.0 micrograms/l, while such values occurred in 21% of NS patients. The tissue staining pattern for PLAP was found to correspond to the preoperative serum value. On the basis of these findings it is concluded that PLAP may be a useful marker in patients with S. Serum levels of PLAP may be used diagnostically in patients with testicular tumours and for monitoring therapy and detection of recurrences in patients with S. For optimal utility of this marker, determinations of serum profiles of PLAP are recommended. Finally, demonstration of PLAP in CIS indicates a functional relationship between CIS and S supporting the hypothesis that CIS is the precursor state of these tumours.