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A trial of cisplatin (P) and cyclophosphamide (C) ± WR-2721 (WR) in advanced ovarian carcinoma
SOCIETY
80
OF GYNECOLOGIC
16. Evidence of Tumor Heterogeneity in Cervical Cancers Lymph Nodes as Determined by Flow Cytometry. H.
and Metastatic
NGUYEN,B. U. SEVIN, H. AVERETTE, J. PERRAS,P. GANJEI,* R. RAMOS, D. DONATO, AND M. PENALVER, Division of Gynecologic Oncology and *Department of Pathology, University of Miami School of Medicine, Miami, Florida 33101.
Tumor heterogeneity is well documented in experimental conditions. However, it is difficult to prove in patients. The goal is to investigate the prevalence of tumor heterogeneity in cervical cancers and metastatic lymph nodes by DNA ploidy determination. Paraffin-embedded tissues of 98 radical hysterectomy patients for stage IB-IIA cervical cancers were used. There were 47 metastatic pelvic nodes from 27 patients. Two 20qm tissue sections were dewaxed with xylene, rehydrated serially with 100, 95, 70, and 50% ethanol, and washed in phosphatebuffered saline (PBS). Enzyme degradation was performed by incubation with 1 ml of 0.5% pepsin (pH 1.5) at 37°C for 1 hr. After washing with PBS, cells were subjected to dual-parameter flow cytometry. Formalin-fixed human lymphocytes were processed through the same steps and used as control. Microscopic slides were made from adjacent tissue sections for histologic confirmation. Three-dimensional graphs generated from Easy-88 computer were used to delineate different tumor populations. DNA index was calculated for each tumor population. The assay had an average CV of 4.5%. Tumor populations with a DNA index difference of at least 0.15 were used to define heterogeneity. Among 69 patients with negative pelvic nodes, there were 8 or 11.6% cases of tumor heterogeneity. Of the 27 patients with metastatic pelvic nodes, 13 or 48.1% had evidence of heterogeneity in the primary tumor. Meanwhile, 17 or 36.2% of 47 metastatic lymph nodes exhibited patterns of tumor heterogeneity. The mean DNA index difference of various tumor populations was 0.29 2 0.13. In 2 patients, we analyzed five to six representative paraffin blocks from primary tumors and were able to correlate DNA ploidy of metastatic lymph nodes to the primary tumors. Thus, it appeared that tumor heterogeneity was common in cervical cancers. Presence of tumor heterogeneity in the primary tumor was associated with a fourfold increase in risk of metastatic disease in the lymph nodes. 17. A Trial
of Cisplatin in Advanced
(P) and Cyclophosphamide Ovarian Carcinoma. G.
(C)
+ WR-2721
KEMP, P. ROSE, W. MCCULLOCH, AND P. SCHEIN,Eastern Virginia Medical School, Norfolk, Virginia 23507; University of Massachusetts, Worcester, Massachusetts 01605; and U.S. Bioscience, West Conshohocken, Pennsylvania 19428. (WR)
A randomized trial is being conducted to determine if pretreatment with WR (910 mg/m’) reduces the toxicity of c&platinum (100 mg/m’) and cyclophosphamide (1000 mg/m’) without loss of anti-tumor activity in patients with Stage III/IV epithelial ovarian carcinoma. To date, 58 patients have been randomized to CP and 63 to CP plus WR with 65 having completed six cycles. Patient characteristics are similar in the two treatment groups. Fifteen of 58 (26%) CP patients were hospitalized for neutropenic fever/sepsis as compared to 5 of 63 (8%) CP plus WR patients (P =
ONCOLOGISTS-ABSTRACTS 18. Cyclosporin Enhancement of Cisplatin Chemotherapy with Refractory Gynecologic Cancer: A Gynecologic Group Study. A. MANETTA, M. BERMAN, J. BOYLE,
in Patients Oncology
P. J. DISAIA, S. LENTZ, S. Y. LIAO, D. MUTCH, AND L. SLATER, University of California, Irvine, California 92717.
Cyclosporin (CsA) has been demonstrated to reverse resistance to several antineoplastic agents including cisplatin in vitro. One of the mechanisms implicated in cisplatin-related resistance is enzymatic enhancement of DNA repair. Studies in cisplatin-resistant cell lines have revealed increases in C-fos, c-H-ras, dTMP synthase and DNA polymerase beta gene expression. Treatment with CsA prior to cisplatin decreases gene expression and reverses in vitro resistance, restoring cisplatin cytotoxicity to the level of the sensitive parent cell line. The purpose of this phase I trial was to study the potential clinical application of CsA modulation of cisplatin and to establish a tolerable dose of CsA when combined with a standard dose of cisplatin of 75 mg/m*. A course of therapy consisted of two CsA infusions over 2 hr each, 24 hr apart, with cisplatin given 6 hr following the first dose. Treatment was repeated every 21 days. CsA was studied in a phase I fashion at five different levels, from 1 to 5 mg/kg per dose. Twenty patients (ovarian cancer 17, cervix cancer 2, vaginal cancer 1) received a total of 81 courses of therapy. All patients had received extensive chemotherapy containing cisplatin. Grade 4 nephrotoxicity was seen in four patients (4/20) treated with 1 mg/kg (one), 2 mg/kg (one), and 5 mg/kg (two) of CsA, respectively. The patient treated at the 1 mg/kg level was a partial clinical responder and was able to tolerate 6 courses. The patient at the 2 mg/kg level had received I4 prior courses of cisplatin and was able to tolerate only 2 additional courses prior to developing a grade 4 renal toxicity. The two patients receiving 5 mg/kg developed a grade 4 toxicity after 2 courses of chemotherapy. Additionally, one patient receiving 2 mg/kg CsA had a grade 3 gastrointestinal toxicity after 7 courses of chemotherapy. Two patients achieved a CR (2/20) and three additional had PR for a total response rate of 25% (5/20). The two women who achieved CRs started treatment with symptomatic ascites, one of whom also had multiple pulmonary lesions that were no longer evident after 3 courses of therapy. Currently both women with CRs are NED 6 months following discontinuation of CsA. We conclude that CsA at a dose of 4 mg/kg/day given in 2 consecutive days, in association with 75 mg/m* of cisplatin, can be given safely. The response rate observed is encouraging given the extensive prior chemotherapy received by all patients. A phase II study utilizing this combination is ongoing. 19. Synergistic
Activity in Vitro of Low-Dose Radiation, Necrosis Factor in Ovarian Cancer Cells. M.
Cisplatin,
and
DUDZINSKI, M. KETI~NG, M. CHUN, W. MCGUIRE, AND J. CURRIE, Johns Hopkins University, Baltimore, Maryland 21205. Tumor
Ovarian carcinoma is sensitive to a variety of therapeutic modalities including platinum-based chemotherapy and radiotherapy. Cure rates, however, continue to be poor and recent attempts at enhancing the therapeutic effect by combined modality therapy have demonstrated synergistic activity of cisplatin (CDDP) and recombinant human tumor necrosis factor (TNF). We examined the cytotoxic effect of the addition of low-dose radiation to CDDP, TNF, and CDDP plus TNF in an ovarian cancer cell line (OVCAR-3). OVCAR3 cells were plated in 96-well plates at a density of 11,ooO cells per well and incubated for 24 hr. Each plate was then divided into four sections: (1) nontreated and nonirradiated control, (2) irradiated only, (3) chemotherapy only, and (4) chemotherapy plus radiation. Radiation doses of 0 to 300 rad in 50rad increments, CDDP doses of 10m6 to 10m4 mg/ml, and 100 units of TNF were administered. Cytotoxicity was determined by crystal violet assay at 5 days. Statistical analysis was performed by Student t test. A statistically significant synergistic effect was demonstrated with the combination of radiation + CDDP, radiation + CDDP + TNF, and CDDP
80
OF GYNECOLOGIC
16. Evidence of Tumor Heterogeneity in Cervical Cancers Lymph Nodes as Determined by Flow Cytometry. H.
and Metastatic
NGUYEN,B. U. SEVIN, H. AVERETTE, J. PERRAS,P. GANJEI,* R. RAMOS, D. DONATO, AND M. PENALVER, Division of Gynecologic Oncology and *Department of Pathology, University of Miami School of Medicine, Miami, Florida 33101.
Tumor heterogeneity is well documented in experimental conditions. However, it is difficult to prove in patients. The goal is to investigate the prevalence of tumor heterogeneity in cervical cancers and metastatic lymph nodes by DNA ploidy determination. Paraffin-embedded tissues of 98 radical hysterectomy patients for stage IB-IIA cervical cancers were used. There were 47 metastatic pelvic nodes from 27 patients. Two 20qm tissue sections were dewaxed with xylene, rehydrated serially with 100, 95, 70, and 50% ethanol, and washed in phosphatebuffered saline (PBS). Enzyme degradation was performed by incubation with 1 ml of 0.5% pepsin (pH 1.5) at 37°C for 1 hr. After washing with PBS, cells were subjected to dual-parameter flow cytometry. Formalin-fixed human lymphocytes were processed through the same steps and used as control. Microscopic slides were made from adjacent tissue sections for histologic confirmation. Three-dimensional graphs generated from Easy-88 computer were used to delineate different tumor populations. DNA index was calculated for each tumor population. The assay had an average CV of 4.5%. Tumor populations with a DNA index difference of at least 0.15 were used to define heterogeneity. Among 69 patients with negative pelvic nodes, there were 8 or 11.6% cases of tumor heterogeneity. Of the 27 patients with metastatic pelvic nodes, 13 or 48.1% had evidence of heterogeneity in the primary tumor. Meanwhile, 17 or 36.2% of 47 metastatic lymph nodes exhibited patterns of tumor heterogeneity. The mean DNA index difference of various tumor populations was 0.29 2 0.13. In 2 patients, we analyzed five to six representative paraffin blocks from primary tumors and were able to correlate DNA ploidy of metastatic lymph nodes to the primary tumors. Thus, it appeared that tumor heterogeneity was common in cervical cancers. Presence of tumor heterogeneity in the primary tumor was associated with a fourfold increase in risk of metastatic disease in the lymph nodes. 17. A Trial
of Cisplatin in Advanced
(P) and Cyclophosphamide Ovarian Carcinoma. G.
(C)
+ WR-2721
KEMP, P. ROSE, W. MCCULLOCH, AND P. SCHEIN,Eastern Virginia Medical School, Norfolk, Virginia 23507; University of Massachusetts, Worcester, Massachusetts 01605; and U.S. Bioscience, West Conshohocken, Pennsylvania 19428. (WR)
A randomized trial is being conducted to determine if pretreatment with WR (910 mg/m’) reduces the toxicity of c&platinum (100 mg/m’) and cyclophosphamide (1000 mg/m’) without loss of anti-tumor activity in patients with Stage III/IV epithelial ovarian carcinoma. To date, 58 patients have been randomized to CP and 63 to CP plus WR with 65 having completed six cycles. Patient characteristics are similar in the two treatment groups. Fifteen of 58 (26%) CP patients were hospitalized for neutropenic fever/sepsis as compared to 5 of 63 (8%) CP plus WR patients (P =
ONCOLOGISTS-ABSTRACTS 18. Cyclosporin Enhancement of Cisplatin Chemotherapy with Refractory Gynecologic Cancer: A Gynecologic Group Study. A. MANETTA, M. BERMAN, J. BOYLE,
in Patients Oncology
P. J. DISAIA, S. LENTZ, S. Y. LIAO, D. MUTCH, AND L. SLATER, University of California, Irvine, California 92717.
Cyclosporin (CsA) has been demonstrated to reverse resistance to several antineoplastic agents including cisplatin in vitro. One of the mechanisms implicated in cisplatin-related resistance is enzymatic enhancement of DNA repair. Studies in cisplatin-resistant cell lines have revealed increases in C-fos, c-H-ras, dTMP synthase and DNA polymerase beta gene expression. Treatment with CsA prior to cisplatin decreases gene expression and reverses in vitro resistance, restoring cisplatin cytotoxicity to the level of the sensitive parent cell line. The purpose of this phase I trial was to study the potential clinical application of CsA modulation of cisplatin and to establish a tolerable dose of CsA when combined with a standard dose of cisplatin of 75 mg/m*. A course of therapy consisted of two CsA infusions over 2 hr each, 24 hr apart, with cisplatin given 6 hr following the first dose. Treatment was repeated every 21 days. CsA was studied in a phase I fashion at five different levels, from 1 to 5 mg/kg per dose. Twenty patients (ovarian cancer 17, cervix cancer 2, vaginal cancer 1) received a total of 81 courses of therapy. All patients had received extensive chemotherapy containing cisplatin. Grade 4 nephrotoxicity was seen in four patients (4/20) treated with 1 mg/kg (one), 2 mg/kg (one), and 5 mg/kg (two) of CsA, respectively. The patient treated at the 1 mg/kg level was a partial clinical responder and was able to tolerate 6 courses. The patient at the 2 mg/kg level had received I4 prior courses of cisplatin and was able to tolerate only 2 additional courses prior to developing a grade 4 renal toxicity. The two patients receiving 5 mg/kg developed a grade 4 toxicity after 2 courses of chemotherapy. Additionally, one patient receiving 2 mg/kg CsA had a grade 3 gastrointestinal toxicity after 7 courses of chemotherapy. Two patients achieved a CR (2/20) and three additional had PR for a total response rate of 25% (5/20). The two women who achieved CRs started treatment with symptomatic ascites, one of whom also had multiple pulmonary lesions that were no longer evident after 3 courses of therapy. Currently both women with CRs are NED 6 months following discontinuation of CsA. We conclude that CsA at a dose of 4 mg/kg/day given in 2 consecutive days, in association with 75 mg/m* of cisplatin, can be given safely. The response rate observed is encouraging given the extensive prior chemotherapy received by all patients. A phase II study utilizing this combination is ongoing. 19. Synergistic
Activity in Vitro of Low-Dose Radiation, Necrosis Factor in Ovarian Cancer Cells. M.
Cisplatin,
and
DUDZINSKI, M. KETI~NG, M. CHUN, W. MCGUIRE, AND J. CURRIE, Johns Hopkins University, Baltimore, Maryland 21205. Tumor
Ovarian carcinoma is sensitive to a variety of therapeutic modalities including platinum-based chemotherapy and radiotherapy. Cure rates, however, continue to be poor and recent attempts at enhancing the therapeutic effect by combined modality therapy have demonstrated synergistic activity of cisplatin (CDDP) and recombinant human tumor necrosis factor (TNF). We examined the cytotoxic effect of the addition of low-dose radiation to CDDP, TNF, and CDDP plus TNF in an ovarian cancer cell line (OVCAR-3). OVCAR3 cells were plated in 96-well plates at a density of 11,ooO cells per well and incubated for 24 hr. Each plate was then divided into four sections: (1) nontreated and nonirradiated control, (2) irradiated only, (3) chemotherapy only, and (4) chemotherapy plus radiation. Radiation doses of 0 to 300 rad in 50rad increments, CDDP doses of 10m6 to 10m4 mg/ml, and 100 units of TNF were administered. Cytotoxicity was determined by crystal violet assay at 5 days. Statistical analysis was performed by Student t test. A statistically significant synergistic effect was demonstrated with the combination of radiation + CDDP, radiation + CDDP + TNF, and CDDP