Detection of chromosomal aberrations by fluorescence in situ hybridization in cervicovaginal biopsies from women exposed to diethylstilbestrol in utero.

Epidemiologic studies have associated estrogens with human neoplasms such as those in the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17beta-estradiol [17beta-E(2)]) and synthetic (diethylstilbestrol [DES]) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17beta-E(2) treatment of mice results in increased nuclear DNA content of cervicovaginal epithelium that precedes histologically evident neoplasia. In order to determine whether this effect was associated with chromosomal changes in humans, the frequencies of trisomy of chromosomes 1, 7, 11, and 17 were evaluated by the fluorescence in situ hybridization (FISH) technique in cervicovaginal tissue from 19 DES-exposed and 19 control women. The trisomic frequencies were significantly elevated in 4 of the 19 (21%) DES-exposed patients. One patient presented with trisomy of chromosomes 1, 7, and 11, while trisomy of chromosome 7 was observed in one patient. There were two patients with trisomy of chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was not observed in the cervicovaginal tissue taken from control patients. These data suggest that DES-induced chromosomal trisomy may be an early event in the development of cervicovaginal neoplasia in humans.

Analysis of 96 patients with advanced ovarian carcinoma treated with high-dose chemotherapy and autologous stem cell transplantation.

The purpose of this study was to identify characteristics significant to survival and progression-free survival in patients with advanced ovarian cancer receiving high-dose chemotherapy. In all, 96 patients received autologous stem cell transplantation. Regimens included paclitaxel with carboplatin (PC), topotecan, melphalan, cyclophosphamide (TMC) and cyclophosphamide, BCNU, thiotepa (CBT). At the time of transplantation, 43% of patients were in clinical CR, 34% were in clinical PR, 18% had progressive disease and 5% had stable disease. There were no treatment-related deaths. The 6-year survival by Kaplan-Meier was 38%. For patients who received transplantation for remission consolidation, the 6-year survival was 53% with a PFS of 29%. On univariate analysis, the CBT regimen, clear cell histology and disease status other than CR prior to treatment were statistically significant adverse prognostic factors. This analysis has demonstrated that patients in clinical remission are most likely to benefit from autologous transplantation, with the exception of patients with clear cell histology. The TMC combination appeared to be superior to the PC and CBT combinations. Comparative studies of different consolidation approaches will be necessary to determine if autologous transplantation is the preferred treatment for this patient population.

Thiotepa in combination with cisplatin for primary epithelial ovarian cancer: a phase II study.

The objectives of this phase II protocol were: 1) to determine the clinical activity of thiotepa combined with cisplatin in suboptimally debulked advanced epithelial ovarian carcinoma as first-line chemotherapy, 2) to determine by surgery the response after 6 courses of chemotherapy, and 3) to identify the regimen's qualitative and quantitative toxicities. Patients with FIGO stage IIIC or IV epithelial ovarian cancer were eligible to receive cisplatin (50 mg/m2) followed by thiotepa (40 mg/m2) on an every 4-week schedule. Patients showing no evidence of disease after six cycles of chemotherapy underwent surgical reassessment. Thirty-one patients were evaluable for toxicity and response. Myelosuppression was the major toxicity and hematologic toxicities prompted all dose reductions. No growth factor support was given in this trial. Thirty-nine percent of patients (12/31) had a clinical complete response. Of these, 16% (5/31) had complete pathologic response and 19% (6/31) had partial pathologic response. One long-term survivor declined reassessment laparotomy. Including the 16% of patients with a partial response, the overall response rate was 55% (17/31). Five patients are currently alive 8 years after enrollment. Median survival was 16.8 months for all patients, 21.5 months for patients with partial response, and 60.8 months for patients with complete pathologic response. A normalization or >50% decrease in CA125 level occurred in 93% of patients. This study indicates that first-line treatment with thiotepa and cisplatin produces significant long-term responses when tumors are sensitive. Such treatment is a reasonable option when paclitaxel is not available.

Desmoplastic small round cell tumor: report of a case presenting as a primary ovarian neoplasm.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is an intra-abdominal malignancy that typically has extensive peritoneal spread at the time of diagnosis. We report a case of DSRCT with involvement of the ovary and omentum as well as an elevated CA-125 level at presentation. CASE: A 23-year-old female presented to another institution with a pelvic mass and a CA-125 level of 140 U/ml. During tumor reductive surgery the right ovary, omentum, and liver were found to be involved. Initial histologic examination favored an undifferentiated small cell carcinoma of the ovary. The patient received two cycles of Taxol and cisplatin chemotherapy and was referred to the University of Texas M. D. Anderson Cancer Center. Upon review of the pathology material at the time of the referral, a diagnosis of DSRCT was made. Despite two additional cycles of chemotherapy, the tumor progressed, and the patient returned home. CONCLUSION: DSRCT may mimic an ovarian primary tumor by presenting with involvement of the ovary and an elevated CA-125 level, and should be included in the differential diagnosis of ovarian neoplasms in young patients.

Expression of cell-cycle mediators in ovarian cancer cells after transfection with p16(INK4a), p21(WAF1/Cip-1), and p53.

OBJECTIVE: The purpose of this study was to determine whether transfection of ovarian cancer cell lines with recombinant adenoviral vectors containing wild-type p16(INK4a), p21(WAF1/Cip-1), and p53 caused growth inhibition and induction of apoptosis. We also measured the expression of the cell-cycle mediators Bax, Bcl-2, pRb, and mdm-2. METHODS: We introduced the wild-type p16(INK4a), p21(WAF1/Cip-1), and p53 genes into the ovarian cancer cell lines SK-OV-3 (p16(INK4a) and p53 null) and OVCA-420 (p16(INK4a) and p53 wild-type) by adenoviral transfection. Cell growth inhibition was measured over a 10-day period. Induction of apoptosis was tested for both cell lines 48 h after cell transfection. Expression of cell-cycle mediators was evaluated by Western blot analysis and densitometry. RESULTS: Growth inhibition was documented after transfection with p16(INK4a), p21(WAF1/Cip-1), and p53 in both SK-OV-3 cells and OVCA-420 cells. Apoptosis was greatest in SKOV-3 cells after transfection with p53. A significant expression of Bax was only seen in the SKOV-3 cells transfected with p53. The bcl-2 protein was poorly expressed in both cell lines. Expression of pRb was suppressed in OVCA-420 cells transfected with p16(INK4a) and p21(WAF1/Cip-1). Infection with Adp16(INK4a) and Adp53 led to an increase in the level of mdm-2 in the SK-OV-3 cell line only. CONCLUSIONS: In the ovarian cancer cell lines studied, cell growth was inhibited after transfection with p16(INK4a), p21(WAF1/Cip-1), and p53. Cell cycle arrest was highest with p53 transfection. The expression of pro-apoptosis proteins was primarily a function of p53 expression.

Biomarker modulation in a nonhuman rhesus primate model for ovarian cancer chemoprevention.

OBJECTIVE: The objective of this study was to explore whether a nonhuman primate model could be developed to test drugs for the prevention of ovarian cancer. METHODS: Nineteen adult female Rhesus macaques were given fenretinide (4HPR), oral contraceptives (OCP), the combination (4HPR + OCP), or no medication for 3 months. Exploratory laparotomy was done pre- and postdrug to assess intermediary biomarkers of neoplastic phenotype, proliferation, response pathways, and growth-regulatory and metabolic markers. Fluorescence emission spectra were plotted for each group pre- and postdrug and means were overlaid on these plots and normalized. Fluorescence intensities were compared using the 2-tailed Student t test, (P = 0.1-0.01). RESULTS: All monkeys tolerated drugs and surgeries without difficulty. Histochemical markers showed no significant trend. However, fluorescence spectroscopy showed increased intensity at 450 nm excitation, 550 nm emission correlating with increased FAD presence. The 4HPR group (P = 0.01) showed higher intensity than the OCP group (P = 0.05-0.07) when compared with the controls. Decreased emission was seen at 350 nm excitation, 450 nm emission correlating with decreased NAD(P)H presence. The OCP group showed the largest change (P < 0.01), and the control group showed the smallest change. CONCLUSIONS: The nonhuman primate is an excellent model to test drug effect on the ovarian surface epithelium and merits additional study. Fluorescence spectroscopy was the most sensitive marker for drug activity and the apparent increase in NAD and FAD in the 4HPR group is consistent with the effect of 4HPR observed in cell culture. The differences between the OCP and the 4HPR groups suggest a different mechanism of activity of these drugs.

High-dose topotecan, melphalan, and cyclophosphamide (TMC) with stem cell support: a new regimen for the treatment of advanced ovarian cancer.

OBJECTIVE: The goal of this study was to determine the optimal dose of topotecan when used in combination with high-dose melphalan and cyclophosphamide (TMC), and to assess the toxicity and efficacy of the regimen in patients with advanced ovarian cancer. METHODS: Fifty-three patients with persistent or recurrent ovarian cancer were treated. Disease status at study entry included: platinum-sensitive recurrent disease (15 patients), platinum-resistant or refractory recurrent disease (15 patients), positive second-look surgery (16 patients), failure to achieve a primary clinical complete response (CR) (7 patients). Following stem cell mobilization and collection, patients were given cyclophosphamide 1 g/m(2)/day on Days -6, -5, -4; melphalan 70 mg/m(2)/day on Days -3, -2; and topotecan at escalating doses from 1.25 to 4.0 mg/m(2)/day on Days -6 to -2. Peripheral blood stem cells were infused on Day 0. RESULTS: The optimal topotecan dose selected for future trials was 4.0 mg/m(2)/day x 5 days. The regimen had acceptable toxicity with no regimen-related death. Toxicity (Bearman toxicity criteria) was limited mostly to grade 1-2 mucositis and diarrhea. The overall response rate of patients with measurable or evaluable disease was 93%. Median survival has not yet been reached, but with a median follow up of 18 months (range: 11-37) 77% of patients are alive. CONCLUSION: With a topotecan dose of 4.0 mg/m(2)/day x 5 days, the TMC regimen has acceptable toxicity and produces high response rates. In the setting of ovarian cancer, high-dose chemotherapy should be administered only as part of well-designed clinical trials. TMC should be considered a potential regimen for future randomized trials in patients with advanced ovarian cancer.

Hypersensitivity reactions and the utility of oral and intravenous desensitization in patients with gynecologic malignancies.

OBJECTIVES: The aims of this study were to characterize hypersensitivity reactions to chemotherapy in patients with gynecologic malignancies and to determine the utility of oral and intravenous desensitization. METHODS: We retrospectively reviewed patients with hypersensitivity reactions identified by direct physician query and by review of charts with ICD9 code E933.1 (Adverse Effect Anti-Neoplastic). RESULTS: Thirty-two patients were identified: 27 with ovarian cancer, 4 with primary peritoneal cancer, and 1 with cervical cancer. Nine patients experienced hypersensitivity reactions during the primary regimen and 23 during chemotherapy for recurrent disease. Hypersensitivity occurred following an average of nine courses. Hypersensitivity occurred secondary to paclitaxel (10) carboplatin (16), cisplatin (4), bleomycin (1), and paclitaxel/carboplatin combination therapy (1). Patients had previously received the agent in 93.8% of carboplatin reactions, in 54.5% of paclitaxel reactions, and in all other agent reactions. Hypersensitivity reactions most commonly included flushing, dyspnea/bronchospasm, back pain, chest discomfort, pruritus, erythema, and nausea and occasionally included alterations in blood pressure or pulse rate. Reactions were successfully treated in 96.9% of patients by interrupting the infusion and administering steroids, antihistamines, benzodiazepines, nebulized beta-agonists, and/or pressors. Seventeen patients underwent desensitization, one to two agents, with 94% success. Nine of ten patients had successful iv desensitization, and 8/10 patients had successful oral desensitization. One failure on the oral regimen had previous successful iv desensitization. CONCLUSIONS: Hypersensitivity reactions to chemotherapeutic agents do not necessarily require exclusion of a compound from the treatment regimen. Intravenous and oral desensitization protocols are useful for successful and safe administration of paclitaxel and platinum compounds in patients with prior hypersensitivity reactions.

Rhesus macaque model for ovarian cancer chemoprevention.

PURPOSE: The objective of the study reported here was to explore whether a nonhuman primate model could be developed for chemoprevention of ovarian cancer. METHODS: An initial feasibility trial was done with three monkeys to determine tolerance for these drugs and for acquisition of surgical ovarian biopsy specimens. In the study, 19 female adult Macacca mulatta (rhesus macaques) were given fenretinide (4HPR) oral contraceptive (OCP), the combination of 4HPR+OCP, or no medication for three months. Laparotomy was performed before and after drug administration, and ovarian biopsy specimens were obtained to evaluate the potential for this animal as a model for ovarian cancer chemoprevention, as well as evaluating fluorescence spectroscopy and other potential biomarkers for ovarian cancer prevention studies. RESULTS: The monkeys tolerated the drugs, surgeries, and acquisition of multiple ovarian biopsy specimens with resultant minimal morbidity. On initial data analysis, fluorescence spectroscopy was the marker that appeared the most promising. CONCLUSIONS: On the basis of results of this study, this model merits further investigation. The rhesus monkey is an excellent candidate for a nonhuman primate model for ovarian cancer chemoprevention.

Intensive-dose ifosfamide and etoposide with filgrastim for cytoreduction before peripheral blood stem cell collection in patients with advanced ovarian cancer.

OBJECTIVE: To evaluate the antitumor activity and toxic effects of intensive-dose ifosfamide plus etoposide with filgrastim given as stem cell mobilization therapy before high-dose chemotherapy for recurrent or persistent ovarian cancer. METHODS: We studied 32 patients with epithelial ovarian cancer who had a positive second-look laparatomy or recurrent disease. Ifosfamide was given at 10 g/m2 (total dose) by continuous infusion over 72 h; etoposide was given at 150 mg/m2 in 2-h infusions every 12 h during the same 72-h period; and filgrastim was given at 10 microg/kg/day subcutaneous injection from day 5 through completion of stem cell harvest. RESULTS: Nine (64%) of the 14 patients assessed responded to the treatment. The target stem cell dose was achieved with a median of 1 apheresis (range 1-5 aphereses). Nonhematologic toxicity was limited to grade 2 nephrotoxicity in one patient and grade 2 hepatic toxicity in three patients. CONCLUSIONS: In this patient group, intensive-dose ifosfamide plus etoposide with filgrastim was well tolerated and produced antitumor activity.

Posttherapy surveillance of women with cervical cancer: an outcomes analysis.

OBJECTIVE: The aim of this study was to develop a surveillance program that optimizes clinical outcome following primary treatment of women with cervical cancer. METHODS: The records of 1096 patients with FIGO stage IB cervical cancer treated from 1983 to 1993 were retrospectively reviewed. Recurrence was analyzed by site, presence or absence of symptoms, method of detection, and survival. Univariate and multivariate analyses using a Cox proportional hazards model were performed. RESULTS: One hundred thirty-three patients (13%) developed recurrent disease. Of these, 114 were symptomatic and 19 were asymptomatic at the time of recurrence. Thirty-seven patients recurred in the central pelvis, 21 each in the lung or pelvic wall, 22 in nodes, and 35 in other sites. The median disease-free interval was 17 months for symptomatic patients and 16 months for asymptomatic patients. The median survival from initial diagnosis was 31 months for symptomatic and 83 months for asymptomatic patients (P = 0.001). The median survival from recurrence was 11 months for symptomatic and 42 months for asymptomatic patients (P < 0.001). Multivariate analysis revealed that symptom status at time of recurrence was a significant predictor of survival, even when known prognostic factors were considered (P < 0.001). All asymptomatic pelvic recurrences were diagnosed by pelvic exam; all asymptomatic pulmonary recurrences were detected by chest radiographs. Pap smears did not detect a single asymptomatic recurrence. CONCLUSIONS: Posttherapy surveillance programs are directed toward asymptomatic patients in whom early detection of recurrence may impact survival. These data indicate that a subset of women may benefit from surveillance. A model for surveillance is proposed.

High-dose ifosfamide and etoposide with filgrastim for stem cell mobilization in patients with advanced ovarian cancer.

High-dose chemotherapy combined with autologous peripheral blood stem cell transplantation has shown promise as treatment for recurrent or persistent epithelial ovarian cancer. We evaluated the stem cell mobilization regimen of high-dose ifosfamide plus etoposide in 32 patients with epithelial ovarian cancer, who had a positive second-look laparatomy or recurrent disease. Ifosfamide was given at 10 g/m2 by continuous i.v. from days 1 to 3. Etoposide was given at 150 mg/m2 every 12 h for six doses on days 1-3. Filgrastim was given at 10 microg/kg/d s.c. from day 5 until the completion of peripheral blood stem cell harvest. Fourteen of 32 patients had measurable or evaluable disease before mobilization therapy and were assessed for response. In nine (64%) of the 14 patients, treatment response was demonstrated, and these patients received a second cycle of mobilization therapy. The target CD34+ cell dose (>8 x 106 cells/kg) was achieved with a median of one apheresis (range 1-5). A median of 25.1 (range 8.0-122.5) x 106 CD34+ cells/kg body weight was collected. Non-hematologic toxicity was limited to grade 2 renal dysfunction in one patient and grade 2 hepatic dysfunction in three patients. In this patient group, high-dose ifosfamide plus etoposide with filgrastim support was well tolerated, lead to successful stem cell harvest and had antitumor activity.

Outcome and reproductive function after conservative surgery for borderline ovarian tumors.

OBJECTIVE: To study reproductive function and disease outcome in women with borderline ovarian tumors who were treated with conservative surgery. METHODS: Patients with borderline ovarian tumors were identified from institutional databases. Patients were eligible if they had pathologically confirmed borderline ovarian tumors, no prior sterilization, no history of radiation therapy, retained their uterus and ovarian tissue, and were younger than age 45. Information was acquired by retrospective medical record review and patient interview. RESULTS: Forty-three patients met the eligibility criteria. The median age was 25 years, with a range of 15-39 years. Twenty-six patients had serous tumors, and 17 had mucinous tumors. Fifteen had stage I disease, three had stage III, and 25 were unstaged. Follow-up was available for all patients (median, 5.7 years). Twenty-nine remained disease-free, and 14 developed a new primary lesion/recurrence, with a median time to recurrence of 39.3 months. Recurrence was more frequent in patients treated with ovarian cystectomy than in those treated with oophorectomy alone (58% compared with 23%) (P <.04). After treatment, 29 of 36 patients (81%) retained normal menstrual cycles, and 12 of 24 patients attempting pregnancy conceived 25 pregnancies. Most patients were highly satisfied with conservative surgery. CONCLUSION: Conservative surgery remains a therapeutic option in selected patients with borderline ovarian tumors. Although the rate of new lesion/recurrence is relatively high, especially in those treated with ovarian cystectomy, mortality from cancer remains low. Many patients who desire pregnancy are able to conceive and deliver healthy offspring after conservative surgery.

A phase I trial of intravenous melphalan, paclitaxel, and cisplatin plus granulocyte-colony stimulating factor in patients with suboptimal advanced epithelial ovarian carcinoma or peritoneal carcinoma.

BACKGROUND: The primary purpose of this study was to establish the maximum tolerated dose (MTD) of intravenous melphalan in combination with paclitaxel and cisplatin plus granulocyte-colony stimulating factor (G-CSF) in patients with suboptimal advanced epithelial ovarian carcinoma or primary peritoneal carcinoma. METHODS: Patients with suboptimal (>2 cm residual tumor) Stage III or Stage IV epithelial ovarian carcinoma or peritoneal carcinoma were eligible for this Phase I study. In the first stage of the study, the doses of paclitaxel and cisplatin were fixed at 135 mg/m(2) and 75 mg/ m(2), respectively, and the dose of intravenous melphalan was escalated in consecutive cohorts of 3-6 patients depending on toxicity. The planned dose escalation levels of melphalan were 6 mg/m(2), 10 mg/m(2), and 14 mg/m(2). In the second stage of the study, the doses of cisplatin and melphalan were fixed at 75 mg/m(2) and the MTD level, respectively, and the dose of paclitaxel was escalated. The planned dose escalation levels of paclitaxel were 150 mg/m(2), 175 mg/m(2), 200 mg/m(2), 225 mg/m(2), and 250 mg/m(2). G-CSF was administered for 12-19 days with each cycle, and cycles were repeated every 4 weeks for a total of 6 cycles. Other end points included clinical or surgical response, progression free survival, and survival. RESULTS: Between January 1993 and May 1996, 34 women with untreated advanced stage epithelial ovarian carcinoma or primary peritoneal carcinoma were treated with 192 cycles of therapy. The MTD of melphalan was 10 mg/m(2), with the dose-limiting toxicity being thrombocytopenia. Paclitaxel was escalated to a dose level of 200 mg/m(2) with a toxicity rate of < 33%. The clinical response rate was 80% in 29 patients with measurable disease. Of 11 patients who underwent second-look surgery, 5 (45%) had a surgical pathologic complete response. The median progression free survival was 16.8 months and the median survival was 32.8 months. CONCLUSIONS: The combination of intravenous melphalan, paclitaxel, and cisplatin was found to have acceptable toxicity and good activity. A Phase II study of this combination appears to be warranted.

Outcome and reproductive function after chemotherapy for ovarian dysgerminoma.

PURPOSE: To review the outcome for all patients with ovarian dysgerminoma treated at the M.D. Anderson Cancer Center who received bleomycin, etoposide, and cisplatin (BEP) and to assess the menstrual and reproductive function of those who received conservative treatment. PATIENTS AND METHODS: Clinical information was abstracted from the medical record. Patients completed a detailed questionnaire about menstrual and reproductive function; those who did not return the questionnaire were interviewed by telephone. RESULTS: Twenty-six patients were identified as having been treated with BEP chemotherapy for pure ovarian dysgerminoma from January 1984 to January 1998. Their median age was 19.5 years (range, 7 to 32 years). Sixteen patients underwent fertility-sparing surgery in the form of unilateral salpingo-oophorectomy. At a median follow-up time of 89 months, 25 (96%) of the 26 patients remained continuously disease-free. One patient apparently developed a second primary dysgerminoma in her remaining ovary after BEP and was clinically disease-free after further treatment. Of the 16 patients who underwent fertility-sparing surgery, one was lost to follow-up when she was pregnant, and one was still premenarchal. Of the remaining 14 patients, 10 (71%) maintained their normal menstrual function during and after chemotherapy, and 13 (93%) had returned to their prechemotherapy menstrual pattern at the time of the questionnaire. Five pregnancies have occurred thus far, and two patients have had difficulty conceiving. CONCLUSION: Most patients with metastatic dysgerminoma can expect cure with maintenance of normal reproductive function when treated with conservative surgery and BEP chemotherapy.

Mature results of a pilot study of pelvic radiotherapy with concurrent continuous infusion intra-arterial 5-FU for stage IIIB-IVA squamous cell carcinoma of the cervix.

PURPOSE: To evaluate the long-term results of continuous infusion intra-arterial 5-fluorouracil (CI IA 5-FU) given with concurrent pelvic radiotherapy (RT) for FIGO stage IIIB-IVA carcinoma of the cervix. METHODS AND MATERIALS: Between 1965 and 1974, 27 patients with extensive FIGO Stage IIIB (22 patients) or Stage IVA (5 patients) squamous cell carcinoma of the cervix were treated with CI IA 5-FU and RT. Twenty-one patients (78%) had bilateral pelvic wall involvement, 25 (93%) had massive tumors (> or =8 cm in diameter), 7 (27%) had involvement of the lower one-third of the vagina, and 15 (56%) presented with hydronephrosis. All patients underwent routine clinical staging, transperitoneal para-aortic lymph node dissection, and bilateral hypogastric artery catheter placement. 5-FU was continuously infused at a dose rate of 10 mg/kg/day on Days 1-15 of RT. The median dose of 5-FU was 376 mg/m2/day (range 270-692). All patients received concurrent pelvic RT to a median dose of 50 Gy at 2.0 Gy per fraction. Only 4 patients received intracavitary RT. The median follow-up of surviving patients was 190 months. RESULTS: The overall 5-year survival rate was 37%. For the 22 patients with FIGO Stage IIIB disease, the 5-year survival rate was 41%. The survival rate for 18 patients treated with only external beam radiation and chemotherapy for Stage IIIB disease was 33%. Four of 10 patients treated with only 50 Gy of external beam radiation and CI IA 5-FU were long-term survivors. Acute complications, including hematologic toxicity and skin reactions, were severe, with 1 death from neutropenic sepsis. Severe late complications were only observed in patients treated with > or =60 Gy of external beam radiation. CONCLUSIONS: While this series is small, the fact that 4 patients with massive Stage IIIB tumors survived after a total radiation dose of only 50 Gy suggests that RT with CI IA 5-FU deserves further study. Modifications in dose, technique, and route of administration should reduce toxicity, and the addition of intracavitary RT should improve the local effectiveness of combined treatment.

Growth suppression of human ovarian cancer cell lines by the introduction of a p16 gene via a recombinant adenovirus.

OBJECTIVE: The cell cycle regulatory protein p16 (CDKN2/cyclin dependent kinase 4 inhibitor/multiple tumor suppressor-1) causes cell cycle arrest at the G1 checkpoint by inhibiting activity of cyclin D-CDK4 complexes. The purpose of this study is to assess the effect of introduction of the p16 gene into two ovarian cancer cell lines via a recombinant adenoviral vector (Ad5CMV-p16). METHODS: Cells lines used were SKOV3, which has a p16 deletion, and OVCA420, which has normal p16. Transduction efficiency was established by infecting cells with an adenovirus containing the Escherichia coli beta-galactosidase gene (Ad5CMV-beta-gal) at multiplicity of infection from 0 to 1000 and staining for X-gal. Cells were infected with Ad5CMV-p16 and cell growth was assessed by counting cells every other day for up to 7 days. Western blotting was done to assess for p16 expression after infection. Fluorescence-activated cell sorting after staining with propidium iodide was done to assess the effect of p16 on the cell cycle. RESULTS: The SKOV3 cell line was transduced with the adenovirus at a slightly lower MOI than the OVCA420 cell line. Growth of the Ad5CMV-p16-infected cells was suppressed 75-80% by cell count in both cell lines and caused morphologic changes of the cells consistent with apoptosis. The p16 protein expression was seen to increase within 24 h after introduction of the p16 gene. G1 arrest of cells occurred beginning 24 h after introduction of the p16 gene. CONCLUSIONS: These results suggest that Ad5CMV-p16 may be further studied as a potential therapeutic agent for ovarian cancer as introduction of the p16 gene into ovarian cancer cell lines causes a G1 arrest and attenuation of growth, regardless of the endogenous p16 status of the cells.

Vaccine implications of folate binding protein, a novel cytotoxic T lymphocyte-recognized antigen system in epithelial cancers.

The immune system can be efficiently stimulated and targeted to specific antigens expressed exclusively or preferentially by experimental cancers. The foremost limitations to extending this vaccine technology to the prevalent epithelial-derived cancers are the lack of: (a) identified tumor-associated antigens recognized by cellular immunity; (b) antigens expressed on the majority of tumor cells during disease progression; and (c) immunogenic CTL epitopes. To date, only HER-2/neu has been shown to be the source of naturally occurring, MHC-restricted, CTL-recognized peptides in epithelial tumors. In this study, we demonstrate that the human high-affinity folate binding protein (FBP), which is a source of antigenic peptides recognized in ovarian cancer, is also recognized in breast cancer. Both immunodominant E39 (FBP, 191-199) and subdominant E41 (FBP, 245-253) epitopes are presented by HLA-A2 in these cancers. These peptides are efficient at amplifying the response of tumor-associated lymphocyte populations in terms of lytic function, enhanced proliferation, and specific IFN-gamma release. On a per cell basis, tumor-associated lymphocytes stimulated with the FBP peptides exhibit enhanced cytotoxicity not only against peptide-loaded targets but also against FBP-expressing epithelial tumors of different histologies. Furthermore, FBP peptides induced E39-specific CTLs and E39- and E41-specific IFN-gamma and IP-10 secretion in certain healthy donors. The broad distribution of FBP among >90% of ovarian and endometrial carcinomas, as well as 20-50% of breast, lung, colorectal, and renal cell carcinomas, along with pronounced differential overexpression in malignant tissues compared with the extremely limited expression in normal epithelium, suggests the exciting potential of a widely applicable FBP-based vaccine in epithelial cancers.

Folate binding protein peptide 191-199 presented on dendritic cells can stimulate CTL from ovarian and breast cancer patients.

Tumor associated lymphocytes (TAL) isolated from malignant ascites cultured in media containing interleukin-2 show antitumor responses. These antitumor responses are mediated by cytotoxic T lymphocytes (CTL) which recognize antigen in the context of MHC molecules using T cell receptors. CD8+ CTL recognize peptide epitopes processed from cellular proteins in the context of MHC class I molecules. These peptides have a restricted length of 8-11 amino acids. The folate binding protein (FBP) is overexpressed in over 90% of ovarian and 20-50% of breast cancers. We recently found that FBP is the source of antigenic peptides recognized by a number of these CTL-TAL. This indicated that FBP peptides are antigenic in vivo for ovarian and breast CTL-TAL. To define FBP immunogenicity, a peptide defining the epitope E39 (FBP, 191-199) was presented by PMBC derived dendritic cells (DC) from healthy donors isolated by the CD14 method to ovarian and breast CTL-TAL. Stimulation of ovarian and breast CTL-TAL by E39 pulsed DC (DC-E39), in the presence of IL-2, rapidly enhanced or induced E39 specific CTL activity. This E39-responder population consisted of cells expressing TCR V beta 9, V beta 13, and V beta 17 families, based on the increase in the percentages of these families in DC-E39 versus DC-NP stimulated TAL. Characterization of immunogenic tumor antigens and of cytokine requirements for induction of functional antitumor effectors may be important for future cancer vaccine developments.

Ovarian cancer-associated lymphocyte recognition of folate binding protein peptides.

BACKGROUND: Tumor-associated lymphocytes (TAL) isolated from ovarian cancer patients contain cytotoxic T lymphocytes (CTL) capable of recognizing specific HLA/peptide complexes on tumor cells leading to tumor cell lysis. Currently, HER2/neu, overexpressed in only 30% of breast and ovarian cancers, is the only known source of CTL-recognized peptides in epithelial cancers. Therefore, we have investigated peptides derived from folate binding protein (FBP), which is over-expressed in more than 90% of ovarian cancers and in the majority of other epithelial tumors. METHODS: TAL were isolated from the malignant ascites of four consecutive HLA-A2+ ovarian cancer patients and incubated in IL-2. Initial chromium-release assays were performed within 1 week. T2 cells, incubated with peptide, were used to reconstitute T cell epitopes. The FBP sequence was interrogated for HLA-A2 binding peptides, and five were synthesized (E37-41). RESULTS: Freshly cultured, unstimulated ovarian TAL recognize peptides derived from FBP. These peptides are presented in the context of HLA-A2, and are specifically recognized in a HLA class I-restricted fashion. TAL recognition of these reconstituted T cell epitopes is concentration dependent. Furthermore, the FBP peptides are shown by cold target inhibition studies to be naturally processed and presented antigens. CONCLUSIONS: FBP peptides are recognized by freshly isolated TAL from ovarian cancer patients, suggesting in vivo expression and sensitization. Because FBP is over-expressed 20-fold in most adenocarcinomas, these peptides may be used in a widely applicable peptide-based vaccine for epithelial tumors.