Longitudinal CA125 detection of sporadic papillary serous carcinoma of the peritoneum.

In this case, rising longitudinal levels of CA125 were significant in the detection of peritoneal papillary serous carcinoma, and led to the indication for surgery. Rising longitudinal CA125 has been shown to be important in the detection of ovarian cancer, but little data exist as to its relevance for the detection of peritoneal papillary serous carcinoma. This rare primary tumor is usually associated with women at high risk for epithelial ovarian carcinoma, although this patient was not at high risk as she had no familial breast or ovarian cancer history.

Tumor markers in screening for ovarian cancer.

Tumor markers are used for multiple purposes in clinical care, including screening asymptomatic subjects, differential diagnosis of symptomatic patients, treatment planning, prognosis during and immediately following treatment, and monitoring for recurrence. Generally, tumor markers have found most clinical utility in monitoring for recurrence of disease (1). Bast and coinvestigators discovered CA125 in 1979 using monoclonal antibody techniques (2), and subsequently demonstrated its utility in monitoring treatment and recurrence of ovarian cancer (3). CA125 is the most widely used ovarian tumor marker, and is currently approved in the United States for monitoring of disease to determine if second-look surgery is required. Tumor markers have not gained wide acceptance for early detection of disease with the one exception of PSA for prostate cancer in the U.S. The lack of acceptance is mainly because of the difficult hurdles a screening strategy must overcome, and few tumor markers have shown sufficient promise in overcoming these hurdles to put them to the test in a randomized controlled trial. Because of the low incidence of most cancers, sample sizes for prospective randomized screening trials are huge, so that sufficient numbers of disease specific events occur by the end of the trial. The significant costs entailed in clinical trials of this size imply that only very promising approaches to screening warrant prospective investigation. For ovarian cancer, three large trials are underway, two trials are planning to randomize 120,000 women followed for 7-8 yr (4,5), and an NCI trial will randomize 74,000 women followed for 16 yr (6).

Use of a second-generation CA125 assay in gynecologic patients.

OBJECTIVE: To determine the correlation between the widely used CA125 assay and a second-generation assay, CA125 II, in a large population of women to determine the utility of measuring CA125 levels in the diagnosis of benign gynecologic disease. METHODS: A total of 822 patients who presented to a gynecology clinic completed a medical questionnaire and underwent a physical examination. Blood samples were obtained to determine serum CA125 levels. Hospital charts were reviewed, and diagnoses were correlated with serum CA125 levels as measured by the CA125 and CA125 II assays. RESULTS: CA125 and CA125 II assay values were highly correlated within four subject categories: age, ethnicity, smoking history, and menstrual category. Mean values decreased with increasing age but were unchanged across ethnic groups and cigarette-smoking status. When means were compared for results of the second-generation and current assays, little difference was found in the age, ethnicity, and smoking categories. Mean serum CA125 values on the CA125 II assay exceeded those on the CA125 assay when obtained during the menstrual and follicular/luteal phases of the menstrual cycle, but they did not differ in anovulatory, pregnant, and menopausal women or in those patients taking hormonal medications. CA125 levels were elevated on both assays in women with fibroids and were higher on the CA125 II assay than on the CA125 assay in women with benign ovarian cysts. CONCLUSIONS: Serum CA125 levels obtained using the CA125 II assay are closely correlated with those obtained using the more familiar CA125 assay. In the diagnosis of benign gynecologic disease, the usefulness of a single serum level measured with the newer CA125 II assay was the same as that of a single CA125 assay level.

Toward an optimal algorithm for ovarian cancer screening with longitudinal tumor markers.

Stored samples from women in the Stockholm screening study were reassayed for CA125II (Centocor, Malvern, PA) and OVX1. The postmenopausal women older than age 50 without ovarian cancer were randomly split into a training set to develop a screening test based on longitudinal marker levels and a second set to validate the test. The CA125II data from each woman is summarized by the slope and intercept from a linear regression of log(CA125II) on time since first sample. The slope versus the intercept for the training set and the ovarian cancer cases formed a bivariate scatter plot. A curve was drawn on the scatter plot that separated most of the women with ovarian cancer from all other women; it delineated a screening test. The specificity of this test was examined on the validation set with a specificity of 99.8%. Bayes' theorem was used to calculate the risk of ovarian cancer (ROC) based on the intercept, slope, and assay variability. It is important to account for assay variability because it can produce large slopes over short periods of time. The maximum risk, which identified 83% (5 of 6) of the ovarian cancers detected within a year of last assay, was applied as a test to the training set and confirmed a high specificity of 99.7%. With this specificity and sensitivity, the ROC algorithm using the CA125II assay has an estimated positive predictive value of 16%, substantially greater than the positive predictive value based on a single assay. Further study is planned to confirm the sensitivity of this approach.

Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs).

Primary human ovarian surface epithelial (HOSE) cells were immortalized by a retroviral vector (LXSN-16E6E7) expressing HPV-E6E7 open reading frames (ORF). Immortalizations of primary ovarian epithelial cells were achieved in three of three attempts. Detailed analysis was carried out in one line, HOSE 6-3, selected on the basis of its epithelial morphology. The immortalized line (HOSE 6-3) was nontumorigenic in nude mice when examined at subculture number 20. Cytogenetic analysis confirmed its human origin and detailed karyotypic analysis revealed a mixed karyotype made up of about 60% of diploid and 40% of near-tetraploid cells. Clonal chromosomal aberration was observed in a subpopulation of cells involving a ring chromosome number 9. Immunofluorescence and two-dimensional gel electrophoresis revealed the presence of vimentin and several species of cytokeratin (K7, K8, K18, K19). The profile of the cytoskeletal filaments of HOSE 6-3 cells is largely identical with that of normal ovarian epithelial cells before immortalization. The immortalized ovarian epithelial cells have a lower sensitivity to TGF-beta 1 inhibition compared to normal ovarian epithelial cells. The immortalized line, HOSE 6-3, has altered growth properties including a higher proliferation rate, plating efficiency, and saturation density. The establishment of a continuous line of human ovarian epithelial cells may provide an in vitro model for study of carcinogenesis in human ovarian cancers.

Evidence for a multifocal origin of papillary serous carcinoma of the peritoneum.

Histopathological evidence suggests that papillary serous carcinoma of the peritoneum (PSCP) may be multifocal in origin. Utilizing a PCR based method to detect tandem repeat polymorphisms in formalin fixed tissue, loss of heterozygosity at eight loci on chromosomes 1, 3, 4, and 17 was studied in six cases of PSCP. Loss of heterozygosity was assessed at between 5 and 11 tumor sites/patient. Allelic losses at 4 loci (1q32-qter, 3p14.3-21.1, 17q12, 17q21.3-23) were noted. Three cases demonstrated a different pattern of allelic loss at various anatomic sites within the same patient. In an additional case, a mutation of the p53 gene, detected by quantitative PCR followed by single-strand conformation polymorphism analysis, was detected in only 2 of 5 tumor sites. The pattern of allelic loss and the mutational pattern of the p53 gene varied at tumor sites within the same patient in 4 of 6 cases of PSCP. These findings are consistent with histopathological evidence that PSCP is multifocal in origin.

Molecular cloning of differentially expressed genes in human epithelial ovarian cancer.

A DNA-fingerprinting approach has been adapted to detect differentially expressed genes in human ovarian carcinoma. This method is based on the use of arbitrary primers to generate fingerprints from total RNA isolated from normal ovarian epithelial cells and ovarian carcinoma cells by polymerase chain reaction (PCR). Using this method, we cloned two cDNA fragments (DOC-1 and DOC-2) which were present in normal ovarian surface epithelial cells but consistently absent in all of the ovarian cancer cell lines from the differential display. In addition, we also identified a cDNA fragment (LF4.0) which is overexpressed in most of the tumor cell lines and tumor tissues in comparison to the normal ovarian surface epithelial cells. The differential expression of the genes in the tumor cell lines as well as in the tumor tissues was also confirmed by Northern analysis. The clone DOC-2, which is a 800-bp cDNA fragment, has one open reading frame suggesting that the gene may be translated. Assuming that this frame is the sense strand, we generated both sense and antisense riboprobe for in situ mRNA hybridization. Only the antisense DOC-2 riboprobe revealed a hybridization signal which was restricted to the human surface ovarian epithelium. The potential functional roles of these genes is now under investigation.

Mutation of K-ras protooncogene in human ovarian epithelial tumors of borderline malignancy.

The mutation of K-ras protooncogene was examined in 44 cases of borderline ovarian epithelial tumors and 18 cases of invasive ovarian carcinomas. In borderline tumors, K-ras mutations are a common feature, having been found in 21 of 44 cases (48%). Twenty of the 21 mutations were identified at codon 12, and one was identified at codon 13. A detailed analysis of the mutation pattern of K-ras revealed a close association with the histological cell types of the tumor. Mutation of K-ras was detected at a higher frequency in mucinous borderline tumor (identified in 12 of 19 cases) compared to serous borderline tumor (identified in 9 of 25 cases). K-ras mutation was also detected in invasive mucinous and serous ovarian carcinomas, hence supporting the notion that borderline ovarian tumors may represent a pathological continuum between benign and frankly invasive diseases.

Molecular genetic evidence of a unifocal origin for human serous ovarian carcinomas.

The hypothesis that ovarian cancer is multifocal in origin was examined using molecular genetic techniques. Patterns of allelic deletion on chromosome 17 were studied in 16 informative cases of Stage III serous epithelial ovarian carcinoma. DNA was extracted from specimens collected from the omentum and both ovaries, and the specific alleles and chromosomal loci involved in the deletion were identified and compared. In all cases, the patterns of allelic deletion were identical for the tumors that had been collected from different sites in the same patients. In addition, 4 of the 16 cases were heterozygous for the hypoxanthine phosphoribosyl transferase (HPRT) gene on the X-chromosome and were examined for methylation status. In all 4, the same parental allele of the HPRT gene was methylated in tumor cells collected from both ovarian and omental sites, suggesting that the patterns of inactivation of the X-chromosome are identical. This pattern of allelic deletion and HPRT-gene methylation in tumor samples collected from different sites implies that ovarian carcinomas have a unifocal origin.

Unifocal origin of advanced human epithelial ovarian cancers.

Ovarian cancers are often diagnosed at a late stage, after the cancer cells have spread to extraovarian sites. Failure to diagnose these tumors earlier may reflect the lack of symptoms and the need for a sensitive, reliable screening test. Alternatively, this can be explained by the hypothesis that some of the extraovarian tumor implants do not represent metastatic spread from the primary cancer but instead are multiple primary tumors developing simultaneously in the peritoneal epithelium. If this is the case, some patients with advanced ovarian cancer may never have had a stage I disease, making early detection theoretically impossible. In this study, we examined the mutational pattern of the p53 gene in 9 patients with epithelial ovarian cancers using tissue collected from different sites within the same patient. In all 9 cases, the mutational pattern of the p53 gene was identical in cancer cells from different sites within the same patient, strongly suggesting that these ovarian tumors were of unifocal origin and that cancer tissues collected from different sites are derived from a single origin.

The role of cytoreductive surgery in the management of stage IV epithelial ovarian carcinoma.

Patients with Stage IV epithelial ovarian carcinoma are generally treated in the same manner as are patients with disease confined to the abdomen--cytoreductive surgery followed by combination chemotherapy. Between 1980 and 1990, 35 women with histologically or cytologically documented Stage IV ovarian carcinoma were treated in this fashion. Sixteen women (45%) underwent optimal initial cytoreductive surgery, defined as less than 2 cm maximum residual disease. Eleven of the 19 women undergoing suboptimal initial procedures underwent interval cytoreduction after two to four cycles of chemotherapy, with 7 achieving an optimal status after the interval procedure. Overall, 23 of 35 patients (66%) were successfully cytoreduced to less than 2 cm either initially or at an interval procedure. Thirty-one of the 35 patients received combination regimens containing platinum as part of their initial therapy. Kaplan-Meier survival curves demonstrated no significant difference in survival between those groups of women cytoreduced intervally or initially, or between those groups of women optimally cytoreduced at some point during their initial therapy and those who were not. The 5-year survival for the entire group was less than 5%, with no significantly prolonged survival seen in those patients undergoing successful cytoreduction.

Preoperative serum CA-125 levels in borderline tumors of the ovary.

Preoperative serum CA-125 levels were evaluated in 38 patients who underwent primary surgery for epithelial ovarian tumors of borderline malignancy at the Brigham and Women's Hospital and Massachusetts General Hospital between 1981 and 1990. Surgical staging was Stage I in 25 (66%) patients, Stage II in 2 (5%) patients, Stage III in 10 (26%) patients, and Stage IV in 1 (3%) patient. The mean sizes of mucinous and serous ovarian tumors were 21.9 and 10.3 cm, respectively (P = 0.0002). All 13 patients (100%) with mucinous tumors had Stage I disease, while 12 (50%) of 24 patients with serous tumors were Stage I. Combining all cell types, 10 (40%) of 25 patients with Stage I disease had an elevated preoperative CA-125 level, while 2 (100%) of 2, 9 (90%) of 10, and 1 (100%) of 1 patient with Stage II, III, and IV disease, respectively, had increased preoperative levels. Among patients with serous tumors, 3 (25%) of 12 Stage I patients had an elevated preoperative CA-125 level, while 11 (92%) of 12 Stage II-IV tumors had elevated levels (P less than 0.001). These data suggest that preoperative CA-125 level correlates with stage of disease in patients with serous borderline ovarian tumors.

Prospective evaluation of serum CA 125 levels for early detection of ovarian cancer.

Detection of ovarian cancer at an early stage should reduce the mortality associated with this disease. Through the Stockholm Population Registry, 5550 apparently healthy women were enrolled in a study designed in part to define the use of the CA 125 radioimmunoassay (RIA) as an initial test for early detection of ovarian cancer. Women whose CA 125 levels were elevated and an equal number of age-matched controls with normal levels were followed by means of pelvic examinations, transabdominal sonography, and serial CA 125 determinations. Of the 175 women with high CA 125 levels, six were found to have ovarian cancer: two each in stages IA, IIB, and IIIC. Of those with normal-range CA 125 levels, three had ovarian cancer as identified through the Swedish Cancer Registry; all three were under 50 years of age. Ovarian cancer was diagnosed on laparotomy in six of the women age 50 or over. Using thresholds of 30 and 35 U/mL, the rates of specificity for the CA 125 RIA were 97 and 98.5%, respectively, for women age 50 or older, and 91 and 94.5%, respectively, for those younger than 50 years of age. Thus, the specificity of the CA 125 RIA is adequate in postmenopausal women to undertake a larger study to determine whether screening using CA 125 influences survival of patients with ovarian cancer.

Analysis of cytotoxicity of 131I-labelled OC125 F(ab')2 on human epithelial ovarian cancer cell lines.

Monoclonal antibody (mAb) OC125 detects the cell surface-antigen CA125, which is expressed in more than 80% of epithelial ovarian cancers but not in normal adult ovaries. Its high specificity and binding affinity makes OC125 a potential candidate for use in radioimmunotherapy (RIT) in patients with recurrent ovarian cancer. Initial biodistribution studies using radiolabelled specific mAbs have demonstrated significant increase in tumor uptake of dose as compared to radiolabelled irrelevant antibody. We report here an isodose comparison of the cytotoxicity of 131I-labelled OC125 F(ab')2, 131I-labelled nonspecific protein and external beam irradiation using a cesium-137 gamma source. Enhancement of cytotoxity due to the specific binding of the mAb could only be observed when a critical activity of 131I localized at the cell membrane. At a specific activity labelling of less than 4.1 mCi/mg, the antigen specificity of OC125 does not contribute to cell kill. Using a specific activity of 10.2 mCi/mg, the relative biological effectiveness of 131I-labelled OC125 (F(ab')2 was increased by a factor of 5 compared with external-beam X-ray therapy, and the specificity of mAb OC125 was found to enhance the cytotoxicity of the radioimmunoconjugate (RIC) by a factor of 2.7. This low value is in accordance with previously reported theoretical calculations for long range, low-LET isotopes and may be one of the reasons why RIT using 131I has severe limitations. In conclusion, it is necessary to maximize the specific activity of RICs with low-LET isotopes such as iodine-131 in order to maximize the ratio of the dose delivered specifically by membrane-bound mAb versus free-floating nonspecific protein.

Involvement of p53 gene in the allelic deletion of chromosome 17p in human ovarian tumors.

Previous reports have shown that one copy of the chromosome 17 was frequently lost in human ovarian cancers (1). The position of the allelic deletion has not been mapped and involvement of p53 gene has not been determined. In this study, we have shown that in human ovarian carcinoma, the commonest region of allelic loss in chromosome 17p is 17p 13.3 (65%) and 17p13.1 (63.7%; 6 out of 9 informative cases). Allelic loss was also observed at region 17p12 - 11.1 but at a lower frequency (38.6% to 37.5%). The pattern of allelic loss of p53 gene was consistent in both primary and secondary metastatic tumors of the same patient. No gross rearrangement of p53 was however observed at the remaining allele using Southern blot analysis. Allelic loss of p53 gene was closely associated with 17p 13.3, the terminal portion of chromosome 17p. The high frequency of allelic loss of p53 gene in ovarian carcinomas conformed with recent findings in cancers of colon, breast, lung and brain suggesting inactivation of p53 gene play a rate limiting step in pathogenesis of human malignancies.

Coordinate elevation of serum markers in ovarian cancer but not in benign disease.

Effective screening for occult ovarian cancer will require a strategy that is both sensitive and specific. Preliminary data suggest that CA 125 is elevated at diagnosis in a majority of patients with ovarian cancer. Although CA 125 is sufficiently specific to prompt its evaluation as one component of a strategy to detect ovarian cancer in postmenopausal women, a further improvement in specificity would facilitate cost-effective screening. In an attempt to develop a more specific screening strategy, multiple markers were assayed in a panel of sera from 47 patients with ovarian cancer and in a separate panel of sera from 50 individuals with benign disease whose serum CA 125 levels exceeded 35 U/ml. Among the patients with ovarian cancer, elevations of CA 125 (greater than 35 U/ml) were observed in 91%, CA 15-3 (greater than 30 U/ml) in 57%, TAG 72 (greater than 10 U/ml) in 49%, placental alkaline phosphatase (PLAP) in 25%, human milk fat globule protein (HMFG) 1 in 77%, HMFG2 in 62%, and NB/70K in 57%. Among the 50 sera selected from patients with benign disease, CA 125 was more than 35 U/ml in 100% and more than 65 U/ml in 42%. Among those patients with benign disease and elevated CA 125, NB/70K was elevated in 62%, HMFG1 in 26%, and HMFG2 in 12%, whereas TAG 72 and CA 15-3 were elevated in only 6% and 2%, respectively. In addition PLAP appeared promising; elevated enzyme levels were not found in the benign disease group. Among patients with ovarian cancer with CA 125 levels more than 35 U/ml, either TAG 72 or CA 15-3 was elevated in 77%. In the false-positive group, only 6% had elevations of one or the other marker. The CA 125 levels in cancer patients were, however, substantially greater than in patients with benign disease. If sera from patients with ovarian cancer were diluted to a range comparable to that found in benign disease, at least one of the two confirmatory tests was elevated in 63% of the samples from the malignant cases. Consequently, use of CA 15-3 and TAG 72 in combination with CA 125 can increase the apparent specificity of the CA 125 assay for distinguishing malignant from benign disease. Prospective studies will be required to test critically whether the use of additional serum markers in combination with the CA 125 assay would contribute to the specificity of a cost-effective screening strategy for ovarian cancer.

Effect of differentiation agents on expression of CA 125, alkaline phosphatase, and cytokeratins in human ovarian adenocarcinoma cells (OVCA 433).

A number of chemical agents have been found to influence the proliferation, morphology, enzymatic activity, and antigen expression of neoplastic cells toward a more differentiated phenotype. We studied the effects of differentiating agents retinoic acid, sodium butyrate, and dibutyryl cyclic AMP on the expression of the tumor-associated antigen CA 125 and several biochemical markers of differentiation in cultured OVCA 433 ovarian cancer cells. Treatment of OVCA 433 cells with these agents for 96 hr reduced cellular proliferation and altered cellular morphology. Quantitation of cell surface CA 125 using flow cytometry revealed that CA 125 expression was reduced by 35-50%. The amount of CA 125 antigen shed into the culture media was reduced to a similar degree. In addition, differentiation inducers markedly enhanced cellular alkaline phosphatase activity and induced the expression of a 65-67-kDa cytokeratin. These findings provide support for the induction of a more differentiated phenotype by these agents.

Human anti-murine antibody responses in ovarian cancer patients undergoing radioimmunotherapy with the murine monoclonal antibody OC-125.

Human anti-murine antibody (HAMA) responses were monitored in 23 patients with recurrent or persistent epithelial ovarian carcinoma undergoing single-dose intraperitoneal radioimmunotherapy (RIT) with the murine monoclonal antibody OC-125. Sera of patients receiving escalating doses of OC-125 F(ab')2 (10-70 mg) radiolabeled with 18 to 141 mCi of iodine-131 were assayed for HAMA by a protein A-based radioimmunoassay. Overall, 70% of patients (16/23) developed HAMA within 10 to 46 days (median = 29) postinfusion, with peak values (23 +/- 6 to 325 +/- 10 micrograms/ml) at 32 to 102 days (median = 38). HAMA was undetectable prior to infusion in all cases and persisted up to 76 weeks. Of patients receiving a dose of 123 mCi or less, 80% (16/20) developed HAMA, whereas in the 140-mCi group, none of the three patients had detectable levels. Two patients in the 140-mCi group demonstrated dose-limiting bone marrow toxicity (severe thrombocytopenia and neutropenia). It is concluded that a single intraperitoneal dose of monoclonal antibody leads to a high incidence of HAMA production. The results also suggest that the likelihood of HAMA formation in patients who either had undergone recent chemotherapy or had received the highest dose of the radioimmunoconjugate is reduced. These observations may be of significance in designing multiple-dose therapy trials as HAMA has been demonstrated to decrease antibody-to-tumor binding and may potentially increase renal, hepatic, and hematologic toxicity associated with radioimmunotherapy.