Isoelectric focusing of alpha-L-fucosidase from human embryonal carcinoma.

Isoelectric focusing profiles of alpha-L-fucosidase recovered from human embryonal carcinoma (EC) and seminoma were compared with those of mouse germ cell tumor-derived stem cells and some human somatic cell neoplasms in an attempt to determine whether EC cells could be used as a source of human basic isoelectric forms of the enzyme previously identified in normal and malignant mouse embryonic cells. alpha-L-Fucosidase activity in all human tumors was associated with isoelectric forms in the isoelectric point (pl) range between approximately 4.5 and 7, corresponding to the range seen in normal human tissues. The basic isoelectric forms (pl values, 7.5-9.5) that predominate in the embryonic isoelectric focusing pattern in mouse were not found in human neoplasms. The present data illustrate another difference between human and mouse EC and show that mouse EC cells are not a complete replica of equivalent human tumor cells.

Significant improvement of the survival of seminoma cells in vitro by use of a rat Sertoli cell feeder layer and serum-free medium.

Seminoma cell lines, essential to the study of the biology of seminoma, do not exist. Tissue culture conditions for establishing such cell lines have to be developed. Under conventional culture conditions, seminoma cells usually die within the first 3 days after plating. The enhanced survival of rat gonocytes when cocultured with rat Sertoli cells in serum-free medium suggests that seminoma cells, the neoplastic counterparts of gonocytes, might benefit from the same conditions. Indeed, when cocultured with rat Sertoli cells in a serum-free medium, viable seminoma cells could be demonstrated on the 11th day of culture. This result is a significant improvement over the results with conventional methods.

Alkaline phosphatase isozymes in human testicular germ cell tumors, their precancerous stage, and three related cell lines.

The four known isozymes of the human alkaline phosphatase (ALP) were detected by isoelectric focusing in extracts of various types of germ cell tumors, three related cell lines, and their precancerous elements (atypical germ cells). In seminoma, placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (PLAP-like) could be separated by isoelectric focusing following isolation by immunoaffinity. The occurrence of both isozymes in seminoma could explain partial heat sensitivity and variation in electrophoretic patterns of the seminoma isozyme frequently observed upon starch gels, in comparison to the normal placental phenotype. The four ALP isozymes are produced not only in germ cell tumors, but already in precancerous tissues. Quantitative analysis showed that the amount of the four isozymes varies in parallel in the tumors tested. Maximal expression was found in seminoma. The relation between ALP gene overexpression and gene amplification by polyploidy of chromosomes 1 and 2 in these lesions is discussed. On the other hand, the ectopic expression of intestinal alkaline phosphatase and PLAP associated with overexpression of PLAP-like in tumor cells as well as in their precancerous stage indicates gene activation by some unknown mechanisms, probably a regulatory process affecting the three tissue-specific ALP genes simultaneously.

Placental alkaline phosphatase as a tumor marker for seminoma.

A sensitive and specific enzyme-linked immunoabsorbent assay was used in a retrospective study of serum levels of placental alkaline phosphatase (PLAP) in testicular cancer. Sixteen of 28 men with active seminoma had elevated PLAP levels, and 71% had elevated levels of either PLAP, human chorionic gonadotropin, or both. Only four of 22 men with active nonseminomatous cancer had elevated PLAP levels, and the levels were normal in all control patients, including 33 men apparently cured of testicular cancer. In six of ten serial studies, PLAP levels provided information not otherwise available that would have been useful clinically, and the levels never were elevated inappropriately. Our data suggest that PLAP is a clinically useful serum tumor marker for seminoma.

Simultaneous detection of all four alkaline phosphatase isoenzymes in human germ cell tumors using reverse transcription-PCR.

We have developed a reverse transcription-PCR method that clearly distinguishes between the RNA transcripts of all four alkaline phosphatase (AP) genes. If compared to the methods used up to the present, the main advantages of the reverse transcription-PCR method presented are its specificity and high sensitivity. The germ cell AP and the placental AP, which are the two most closely related AP isoenzymes (98% homology), can clearly be distinguished without any interference by other AP isoenzymes. An enhanced expression of AP isoenzymes has been reported for various tumors. The examination of the pattern of AP isoenzyme expression in a specific tumor and the corresponding tissue of origin enables discrimination between eutopically and ectopically expressed isoenzymes and thus represents an important tool in the elucidation of AP isoenzymes as potential tumor markers. The pattern of AP expression in 15 germ cell tumors, 2 germinal epithelia adjacent to seminoma, 2 cell lines of germ cell tumor origin (Tera-1 and BeWo), and 5 normal testes was studied. In comparison to normal testes, in all seminomatous germ cell tumors eutopic expression of germ cell AP and ectopic expression of tissue-nonspecific AP were demonstrated. In both samples of pure embryonal carcinoma and in the embryonal carcinoma cell line, the transcription of all four mRNAs was shown. These results indicate that the expression of the isoenzymes depends on the degree of differentiation of a tumor and that a simultaneous up-regulation of all AP isoenzymes in all types of germ cell tumors does not exist.

Alternating cycles of PVB and BEP in the treatment of patients with advanced seminoma.

33 patients (median age 39 years) with advanced seminoma were treated with 4 courses of alternating cisplatin-containing chemotherapy PVB/BEP (cisplatin, vinblastine and bleomycin; bleomycin, etoposide and cisplatin). Patients were classified as stage IIC (n = 7), IID (n = 9), III (n = 13) and IV (n = 4). 8 had had prior radiotherapy; 9 had an elevated beta human chorionic gonadotropin (beta HCG). 30 patients were evaluable for response and 33 for toxicity. During chemotherapy 3 patients died, 1 due to malignant disease, another due to a cardiac arrest, and 1 patient of a bleomycin pneumonitis. 13 (43%) had a complete remission and 17 (57%) had a clinical partial remission (residual radiographic mass). At a median follow-up of 28 months (range 16-88), 3 patients relapsed, 6-8 months after entry. After completion of therapy there were 2 deaths, 1 due to bleomycin pneumonitis and 1 neither tumour nor treatment related. 26 of 33 (79%) patients achieved a continuously disease-free status. Leucocytopenia and thrombocytopenia of WHO grade 3/4 occurred in, respectively, 32/33 (97%) and 20/33 (61%) of the patients. This study shows that alternating PVB/BEP in this group yields comparable response rates with non-alternating schedules but at the expense of considerable toxicity.

Is placental alkaline phosphatase (PLAP) a useful marker for seminoma?

The usefulness of placental alkaline phosphatase (PLAP) as a tumour marker was assessed in 1578 serum samples from 236 patients with seminoma. Smoking habits were known for all but 7 patients (22 samples). Smoking was associated with significantly higher mean levels of PLAP in disease-free patients (28.8 [S.E. 2.1] U/l vs. 15.9 [1.3] U/l in non-smokers). Mean PLAP levels were higher in patients with active disease (78.6 [23.5] U/l in non-smokers and 47.2 [18.5] U/l in smokers). The median values showed a similar trend. However, there was considerable overlap between the various groups and differences between mean and median values indicated that PLAP values were distributed asymmetrically. The predictive value of PLAP as a tumour marker was consequently much less than superficial inspection of these values might suggest. In 97 patients on surveillance, only 2 out of 11 patients who relapsed had elevated PLAP at the time of clinically detectable relapse. With the upper limit of normal PLAP quoted by our laboratory (35 U/l), specificity and sensitivity were, respectively, 88% and 45% (all patients) and 96% and 47% (non-smokers). The sensitivity and specificity of PLAP were assessed in more detail for a series of threshold values (normal vs. abnormal) with a graphical method. Only in non-smokers did PLAP seem useful and even in this group the positive predictive value of an "abnormal" test may be low; less than 50% in clinically relevant circumstances. Serum PLAP assay cannot usefully stand alone as a marker for seminoma and its routine estimation contributes little to follow-up.

Alkaline phosphatase histochemistry in human germ cell neoplasms.

Alkaline phosphatase is a useful and reliable marker of germ cell neoplasia that has been almost completely overlooked in the recent medical literature and in the practice of surgical pathology. Its presence in immature germ cells and in germ cell cancers was noted as early as 1953, but a systematic study of its use in the diagnosis and classification of germ cell tumors has not appeared in the literature. Using a recently developed plastic embedding technique combined with enzyme histochemistry, a large series of germ cell tumors and gonadal specimens were examined for the presence of alkaline phosphatase. The neoplastic germ cells in all cases of seminoma and embryonal carcinoma showed strong plasma membrane positivity for alkaline phosphatase. Choriocarcinomas (gestational and nongestational) and mature teratomas were negative. These findings suggest that the alkaline phosphatase reaction is a useful adjunct in the diagnosis of germ cell cancers.

Angiotensin-I-converting enzyme in germinomas.

Angiotensin-I-converting enzyme (ACE) was detected in 18 germinomas, both of testicular or extratesticular localization, and studied by immunohistochemical methods using specific polyclonal antibodies and by enzyme activity measurements. ACE was also detected in normal human germ cells. On the other hand, it was not present in other types of testicular tumors. Biochemical studies and immunohistochemical findings suggest that at least part of the enzyme is membrane bound. Plasma ACE levels appeared to be normal, indicating that measurement of plasma ACE levels in germinomas would be of little help for the diagnosis and follow-up of patients. However, the apparent specificity of ACE detection in germinomas among germ cell tumors might help in histologic diagnosis, especially for tumors of extragonadal localization.

Placental alkaline phosphatase immunohistochemistry of intratubular malignant germ cells and associated testicular germ cell tumors.

Two hundred three testicular germ cell tumors were studied immunohistochemically for the presence of placental alkaline phosphatase (PLAP). Special emphasis was placed on the pattern and incidence of positive staining of intratubular malignant germ cells (ITMGCs) adjacent to tumors. 99% of cases with adjacent ITMGCs showed a positive staining reaction in some or all IT-MGCs present. Other germ cell elements showed at least a focal positive staining reaction in the following proportions: seminomas, 96%; embryonal carcinomas, 96%; yolk sac tumors, 25%; mature teratomas, 5%; immature teratomas, 4%; choriocarcinomas, 45%; and syncytiotrophoblasts, 43%. The staining pattern for seminomas tended to be diffuse, whereas for embryonal carcinomas the staining pattern was more focal. Yolk sac tumors stained inconsistently for PLAP and a positive reaction was limited to a small percentage of cells. Syncytiotrophoblasts, singly or in choriocarcinomas, also showed variable positivity. These results corroborate the fact that PLAP is a sensitive marker for ITMGC, seminoma, and embryonal carcinoma.

Isoenzymes of alkaline phosphatases in seminomas. An immunohistochemical and biochemical study.

Placental alkaline phosphatase (the PLAP-like isoenzyme) and liver alkaline phosphatase (LAP) were demonstrated immunohistochemically by use of monoclonal antibodies in the tumor cells of twelve seminomas and one seminoma metastasis. Intestinal alkaline phosphatase (IAP) was not found. The PLAP-like and LAP enzymes showed high catalytic activities compared to normal testis. This is the first occasion that LAP has been demonstrated by immunochemistry in seminoma cells. The results suggest that demonstration of these tumor enzymes may be useful markers for seminomas in histopathological specimens.

Serum lactate dehydrogenase isoenzyme 1 in patients with advanced testicular cancer.

Abnormal levels of serum lactic dehydrogenase-1 (LD-1) activity have been observed in 81% (34/42) of patients with stage III germ cell malignancy of the testis. The criteria for evaluation the electrophoretic isoenzyme patterns of these patients were, as follows: For criterion 1 elevations the LD-1 value in absolute units was greater than 52.0 U/I with the LD-1/Total LD ratio greater than 37.2%. Criterion 2 elevations had absolute values of LD-1 less than 52.0 U/I, but the LD-1/Total LD ratio was greater than 37.2%. For criterion 3 elevations of LD-1, the absolute value was greater than 52 U/I and the LD-1/Total LD ratio was less than 37.2% activity with the LD-5/LD-1 ratio less than 0.5; or when the LD-5/LD-1 ratio was greater than 0.5 but the LD-1 is equal to or greater than the LD-2. The frequency of LD-1 elevation correlated well with the extent of the disease (stage II-B-1 and 2, 50%; stage III-B-3, 86%; stage III-B-4, 91%; stage III-B-5, 93%). LD-1 elevation occurred in groups I, II, IV and V histopathologic cell types (Dixon and Moore Classification) and there did not appear to be any correlation between the histologic cell type and the frequency of elevation of LD-1. Interpretation of LD-1 activity only on the basis of its relative ratio to the total LD value (criterion 1 and 2) identified a total of 28 patients (67%). A criterion 3 elevation was demonstrated in 6 (14%) additional patients. All patients with persistent elevations or recurrent elevations of LD-1 have shown progressive or recurrent disease and patients with no clinical evidence of disease have demonstrated normal LD-1 values. In those patients with elevated LD-1 activity, serial measurements of serum. LD-1 isoenzyme reflect the response of the patient to therapy.

Immunohistochemical localization of lactate dehydrogenase isoenzyme 1 in germ cell tumors of the testis.

In this study, antiserum to lactate dehydrogenase isoenzyme 1 (LD 1) was used to determine immunohistochemical patterns of localization in a variety of germ cell neoplasms of the testis. All 29 seminomas and teratocarcinomas and four of seven embryonal carcinomas were positive for LD 1. These results suggest that elevated serum LD 1 levels noted in patients with germ cell neoplasms may be derived directly from neoplastic tissue and explain the relationship between serum LD 1 levels and tumor burden. A variety of neoplasms from other organs also were evaluated in order to determine the specificity of LD 1 staining for testicular tumors.

Isoenzymes of alkaline phosphatase in germinoma cells.

Utilizing 18 germinomas, the characteristics of isoenzymes of alkaline phosphatase (ALP) in germinoma cells were examined by light and electron microscopic enzymohistochemical and immunohistochemical techniques and biochemical methods including inhibition tests, electrophoresis and electrosyneresis. ALP in germinoma cells was generally located on the cell surface. In a few germinoma cells, however, ALP was seen not only on the cell membrane but also in the endoplasmic reticulum. Enzymohistochemically as well as biochemically, ALP in germinoma cells was little sensitive to L-phenylalanine, moderately sensitive to L-homoarginine and somewhat resistant to heat in its inhibition tests. Immunohistochemical examinations and electrosyneresis showed the presence of the placental type of ALP in germinoma cells and biochemical analyses revealed that the heat-stable component of ALP in germinoma cells was consistent with the D-variant of the placental type of ALP. Therefore, germinoma cells possessed mixed isoenzymes of ALP, consisting mainly of the liver/bone type and a small amount of D-variant of the placental type on the cell surface. The prominent expression of ALP in germinoma cells may be due to the enhanced expression of gonadal genes active in the germinoma genome.

Dysgerminoma and serum lactic dehydrogenase levels.

In this study, the author reports the levels of serum lactic dehydrogenase in patients with ovarian dysgerminoma. The results of previous studies have shown that serum lactic dehydrogenase is elevated in patients with primary ovarian carcinoma but not in those with other pelvic tumors, benign or malignant. In the present study, serum lactic dehydrogenase levels were highly elevated before patients were treated for ovarian dysgerminoma and sharply reduced to normal levels after treatment. This finding may demonstrate an important tumor marker in the diagnosis and treatment of ovarian dysgerminoma.

Catalase determination in various etiologic forms of Wilms' tumor and gonadoblastoma.

We have previously mapped the gene coding for catalase to 11p13 by gene dosage analysis. Deletion of this chromosomal region causes aniridia, mental retardation, and predisposition to Wilms' tumor (WT). In the present study, 22 patients with various etiologic forms of WT and/or aniridia were investigated. The catalase (CAT) level and karyotype were examined in order to determine the linkage and the gene ordering on chromosome number 11 of the different loci involved. The CAT concentration was normal in the 19 cases without detectable chromosomal abnormalities.

Enzymatic heterogeneity of seminomas.

Heterogeneity of placental-like alkaline phosphatase (PLAP-like enzyme) in seminoma was studied. PLAP-like enzyme from seminoma tissues was separated into three areas with different proportions between tumors, while PLAP and PLAP-like enzymes in normal testes were separated into two areas on the basis of hydrophobicity. By use of lectin affinity chromatography, PLAP-like enzyme in seminoma revealed extra sugar chains compared to PLAP, indicating heterogeneity of the carbohydrate moiety. However, the glycosylation patterns were found to be essentially similar between seminoma and normal testis. On isoelectric focusing, differences in migration patterns of PLAP-like enzyme were revealed between seminoma and normal testis as well as between PLAP-like enzyme and PLAP. The differences in charge were mainly due to differences in sialylation of the molecules. The complex pattern on isoelectric focusing was not altered by neuraminidase treatment, indicating a considerable charge heterogeneity within the population of PLAP-like enzyme molecules from seminoma.

Placental alkaline phosphatase as a tumor marker for primary intracranial germinoma.

A sensitive enzyme-linked immunosorbent assay (ELISA) was used in a retrospective study of placental alkaline phosphatase (PLAP) levels in serum, cerebrospinal fluid (CSF), and intratumoral cyst fluid in primary intracranial germinoma. The ELISA showed no cross-reactivity with intestinal alkaline phosphatase except in very high concentrations, after samples had been heat-treated. Three patients with germinoma were studied for serum PLAP levels and in all the levels were elevated (3.78, 0.52, and 2.11 IU/liter). Two of the germinoma patients were studied for PLAP levels in the CSF, and both had elevated levels (0.83 and 9.83 IU/liter). The intratumoral cyst fluid in one case of germinoma was tested for PLAP and the level was found to be very high (603 IU/liter). These PLAP levels decreased concomitantly with the reduction in tumor size during irradiation. Serum PLAP levels were measured in 40 control adult male individuals and in the CSF of 20 nonpregnant patients with subarachnoid hemorrhage. The upper normal limits were 0.20 and 0.11 IU/liter in the serum and the CSF, respectively. All PLAP levels measured in the serum of patients with various brain tumors were 0.18 IU/liter or less. This study strongly suggests that PLAP is a clinically useful tumor marker for primary intracranial germinoma.

Elevated serum angiotensin converting enzyme levels in metastatic ovarian dysgerminoma.

A case of a 32-year-old XY genotype female is described, presenting with mediastinal and abdominal lymphadenopathy and associated with an elevated serum angiotensin I converting enzyme (SACE) level. Lymph node histology showed a malignant dysgerminoma of ovarian origin. Combined chemotherapy led to a radiological regression of the lymphadenopathy and coincided with a decrease in SACE concentration. The authors suggest that SACE may be a marker for disseminated germinoma tumours and may be useful for monitoring treatment.

Lack of evidence for aromatase expression in human ovarian epithelial carcinoma.

It is controversial whether ovarian epithelial carcinoma possesses steroidogenic enzymes. We investigated aromatase expression in ovarian epithelial carcinoma, and compared it with the normal ovary and placenta. Samples were obtained from an ovarian carcinoma cell line SK-OV-3, ovarian tumour tissues from four patients with epithelial carcinoma and one patient with dysgerminoma. Aromatase enzymatic activity was measured in microsome fractions by quantitating 3H2O released from [1-3H] androstenedione and [3H]oestrone converted from [1,2,6,7-3H] androstenedione. Aromatase messenger ribonucleic acid (mRNA) was determined by reverse transcription-polymerase chain reaction (RT-PCR) using oligonucleotide primers synthesized according to the published human aromatase gene sequence. No aromatase activity was detected in either of two mucinous cystadenocarcinoma specimens or in SK-OV-3 cells, while aromatization proceeded with apparent Michaelis-Menten kinetics in the normal ovaries and placentas. The apparent Km value was 200 nmol/L for the ovary. Aromatase mRNA was detected in dysgerminoma, and the normal ovary and placenta, but not in any of three mucinous cystadenocarcinoma specimens, one serous cystadenocarcinoma specimen and SK-OV-3 cells. These results for both enzyme activity and gene expression suggest that the human ovarian epithelial carcinoma lacks aromatase. The demonstration of absence of aromatase gene expression raises the possibility that aromatase activity in ovaries bearing epithelial carcinoma may be associated with hyperplastic stromal rather than tumour cells.