Purified, recombinant human 7-interferon (rlFN-7) was tested at clinically achievable doses for direct and indirect antiproliferative activity against human tumor cell lines using a Clonogenic assay. One-h treatment with rlFN-7 showed direct dose depend ent inhibition of tumor colony growth in cell lines established from human melanoma, myeloma, renal cell, and cervical can cers. Longer treatments resulted in suppression of ovarian and breast carcinoma clonogenicity. In order to test for indirect antiproliferative effects of rlFN-7, feeder cells were included in a separate agarose underlayer in the cloning assay. These feeder cells included mouse peritoneal macrophages, U-937 (human histiocytic lymphoma cell line), and adherent cells from human malignant ascites specimens. Colony growth of ovarian carci noma and melanoma cell lines was stimulated by each of these feeder cell types. Cultures containing mouse peritoneal macro phages or U-937 cells showed the same antiproliferative re sponses to rlFN-7 as did control cultures without feeder cells. In contrast, human adherent ascites cells (>80% macrophages) became strongly inhibitory to tumor colony growth when treated with rlFN-7. These results suggest that human tumor associated macrophages may become tumoricidal under the influence of rlFN-7, producing a diffusable substance in agarose culture which causes the observed antiproliferative effects on tumor cells.

A toxicity, dose, and schedule study of intraperitoneal (ip) cisplatin and α-interferon was conducted as a salvage therapy for patients with persistent, advanced epithelial ovarian cancer after primary systemic therapy with cisplatin-combination chemotherapy. Twenty-four patients were entered into this prospective, nonrandomized phase I–II trial conducted at two institutions following a uniform protocol. Cisplatin doses were escalated from 45 to 90 mg/m2, and α-interferon doses were escalated from 10 to 50 × 106 IU. At protocol entry, 4 (16%) patients had microscopic residual disease at second-look laparotomy, 5 (21%) had minimal residual disease <5 mm, 7 (30%) had residual disease 5–20 mm, and 8 (33%) had bulky residual disease >20 mm. Toxicity was acceptable overall. Hematopoietic toxicity included a grade 3 total white cell count in 12% of courses when the cisplatin dose was equal to or >60 mg/m2. Renal toxicity was modest with grade 2 toxicity in 20% of courses with a cisplatin dose >60 mg/m2. Gastrointestinal toxicity, especially nausea and vomiting was seen in most courses; however, it was grade 3 and dose-limiting in >30% of courses with cisplatin 75–90 mg/m2. General malaise, fever, flu-like symptoms, chills, and myalgias were seen in most courses, but it was dose-limiting (grade 3) toxicity in 6–11% of cycles when the dose of interferon was 25–50 × 106 IU. There was no grade 4 toxicity. Thus, the maximum tolerated dose (MTD) of the combination is 60 mg/m2 cisplatin and 25 × 106 IU interferon. Eighteen patients were evaluable for response, 15 of whom had responded to prior cisplatin therapy and 3 had not. Of the 10 patients evaluable for clinical response, one patient (10%) achieved a complete response (CCR), and one (10%) had a partial response (PCR). The progression free interval (PFI) and survival were 11 months and 19 months, respectively, for the CCR patient, 6 and 11 months, respectively, for the PCR patient, and the mean survival for nonresponders was 8 months. The other 8 patients underwent a reassessment laparotomy: 2 (25%) achieved a complete pathologic response (CPR), and 3 (38%) had a partial pathologic response (PPR). Both pathologic responses were in patients with minimal residual disease <5 mm. Mean PFI for patients with CPR was 17 months, one patient died at 24 months, and one is alive at 38 months; PFI and survival for PPR patients was 9 and 18 months, respectively, and the mean survival of nonresponders was 12 months. These data justify a phase II trial using an intraperitoneal regimen with the MTD of 25 × 106 IU of α-interferon and 60 mg/m2 cisplatin.

Effects of combination treatment with human recombinant alpha-2b interferon (IFN-α2b) and gamma interferon (IFN-γ) and sequencing of the combination on colony formation of human tumor cells were studied in a human tumor clonogenic assay (HTCA) with or without ascites-associated macrophages (AAM). Five different human tumor cell lines were studied. Three of the five cell lines (ovarian cancer cell line BG-1, cervical cancer cell line ME-180, and melanoma cell line SK-MEL 28) were sensitive to both IFNs.Cervical cancer cell line CaSki was sensitive to IFN-α2b but resistant to IFN-γ. Endometrial cancer cell line HEC-1A was resistant to both IFNs. Synergistic interaction was observed in BG-1 and SK-MEL 28 with a combination of the IFNs. ME-180 did not exhibit a positive interaction, in spite of its sensitivity to each IFN. CaSki and HEC-1A also did not exhibit a positive combined interaction at clinically achievable concentrations. One sequential combination method (method 1: IFN-α2b → IFN gamma with a 24-h interval) resulted in a similar antitumor effect as the simultaneous combination. A reversed sequential method (method 2: IFN-γ → IFN-α2b with a 24-h interval) was less effective in three of the five cell lines. In BG-1, AAM enclosed in the lower layer markedly enhanced the antitumor effect of combined IFNs as well as each IFN alone. The antitumor effect with method 1 was significantly greater than that achieved with simultaneous combination or combination according to method 2 in the presence of AAM (P<0.01). These results suggest that (1) a synergistic antitumor effect of IFN-α2b and IFN gamma is demonstrable in selected types of tumors, depending upon the sensitivity of each tumor cell line to both IFNs; (2) optimal scheduling for the direct antitumor effect of combined IFNs seems to be long-term exposure of cells to the IFN, the cells being treated with both IFNs either simultaneously or sequentially (IFN-α2b preceding IFN-γ); and (3) AAM potentiate the antitumor effect of IFNs either alone or in combination. Finally, IFN-α2b may have some priming effects for the indirect effect of IFN gamma mediated through AAM in certain tumor cells.

Eleven patients with untreated advanced squamous cell carcinoma of the cervix (FIGO Stage IIIB and IVA, or positive paraaortic nodes) were treated with cisplatin 50 mg/m2 every 3 weeks for six courses prior to irradiation therapy (RT). Six patients completed six courses of cisplatin prior to RT with one complete and two partial responses (PR). Five other patients did not complete cisplatin therapy; two progressed after two and three courses, one had a PR after three courses and refused further therapy, one refused treatment after two courses and one elected to begin RT after one course. Two patients had reversible nephrotoxicity. No other major toxicity was noted. Six patients had stem cell assay prior to treatment and assay results were predictive of cisplatin response in five. Only one patient remains alive at 44+ months. Cisplatin chemotherapy prior to irradiation therapy is feasible and without substantial toxicity but is unlikely to be of benefit in patients with advanced cervical cancer.

Combinations of recombinant interferon alfa2 (IFN-α2) with doxorubicin, 4′-epidoxorubicin, 4′-deoxydoxorubicin, or 4-demethoxydaunorubicin were tested for antiproliferative activity against a panel of human tumor cell lines in a human tumor clonogenic assay. The histologies of the cell lines were ovary, cervix, breast, and melanoma. Each of the cytotoxic compounds showed dose-dependent antiproliferative effects against each of the cell lines, and the results indicated that doxorubicin derivatives were consistently more potent than the parent drug. In all instances, 4-demethoxydaunorubicin was the most potent derivative, requiring 2–20 times less drug to inhibit 70% of tumor colony formation. Combinations of IFN-α2, with doxorubicin or its derivatives may show additive or synergistic antiproliferative activity against certain tumor cell lines. The ovarian carcinoma cell line, BG-1, responded synergistically to each of the four compounds in combination with IFN-α2. The cervical carcinoma cell line, CaSki, and the breast carcinoma line, MCF-7, responded to the combinations in a manner best described as additive. In the melanoma line, SK-Mel-28, the drugs were found to be subadditive or even antagonistic. While the potency of the anthracyline derivatives ranked consistently across the different cell lines, the synergistic interaction with IFN-α2 is a cell line-specific phenomenon unrelated to sensitivity to either anthracyclines or interferon.

The in vitro antiproliferative activity of human recombinant interferon-gamma (IFN-gamma) was tested against human tumor cells in vitro in combination with doxorubicin, cisplatin, or vinblastine. Using a human tumor clonogenic assay (HTCA), IFN-gamma alone showed dose-dependent inhibition of colony growth in six or seven human tumor cell lines as well as in each of nine fresh ovarian tumor specimens. The combination of IFN-gamma and either doxorubicin or cisplatin showed additive antiproliferative effects against all the cell lines with the exception of an IFN-gamma-resistant endometrial cancer cell line (HEC-1A). In combination with vinblastine, IFN-gamma rarely had an additive effect.

Both in vitro and in vivo studies have demonstrated antiproliferative effects of interferon alfa-2b (Intron A; Schering-Plough) when tested with human tumor cells. A clonogenic assay has been widely used to determine its direct antiproliferative effects on human tumor cells in vitro using colony reduction as a reproducible endpoint. As a single agent, interferon alfa-2b shows maximum tumor cell colony reduction when used in high concentrations with continuous cell exposure. Short-term exposure to interferon alfa-2b does not produce significant tumor cell colony reduction. Clonogenic assays have also been used to test combinations of interferon alfa-2b with cytotoxic drugs. Variations in drug scheduling, sequencing and concentrations have indicated the best combinations which maximize tumor cell colony reduction. Combinations of interferon alfa-2b with doxorubicin, cisplatin, vinblastine, melphalan and cyclophosphamide have been shown to have at least additive and occasionally synergistic antiproliferative effects.

In vitro and in vivo studies have both shown synergistic cytotoxic effects of combined recombinant human interferon alpha-2 (rIFNα2) and standard chemotherapeutic drugs. The Human Tumor Clonogenic Assay has been used in this study to test the cytotoxic effects of rIFNα2 alone and in combination with eight cytotoxic agents. Schedule-dependent and concentration-related cytotoxicity of the individual drugs and combinations have been assessed. The greatest enhancement of cytotoxic activity is seen when rIFNα2 is used in combination with either doxorubicin (DoX), cis-platin, or vinblastine. Human tumor cell lines from carcinomas of the ovary, cervix, breast, kidney, and melanomas as well as biopsies from 14 patients have been tested with combinations of rIFNα2 and DoX. Results of these studies show that the maximal antiproliferative effect of rIFNα2 is related both to the concentration utilized as well as to prolonged duration of cell exposure; DoX cytotoxicity is maximized by prolonged cell exposure time to the drug. Using clues from the in vitro data to design a clinical study, a protocol has been written, using sequential rIFNα2 and DoX to treat advanced solid tumors. Patients receive a simultaneous IM injection of 10 mil U/m2 rIFNα2 plus 10 mil U/m2 IV over 30 min. One-half hour later a two hour infusion of DoX (20 mg/m2) is given. Three induction courses are administered one week apart, followed by a two week rest. Patients having stable disease or a clinical response are continued on maintenance therapy every two weeks. Seventy-five patients have been entered on study, including 16 ovarian, 13 cervical, 12 renal, 11 gastrointestinal, 5 breast, 4 lung carcinomas, 4 melanomas, 4 sarcomas, and 6 miscellaneous tumor types. Only one patient has been dropped from the study for Grade IV toxicity-temperature to 107°F with hypotension. Clinical responses to date have been seen in 5 patients with cervical, 4 ovarian, one pancreatic carcinoma and one sarcoma. Continued follow-up is in progress. The regimen is well tolerated and shows some clinical efficacy.

Seventeen patients with advanced, previously treated malignancies were entered into a phase I trial utilizing recombinant DNA produced alpha 2 leukocyte interferon (rIFN-alpha 2). Sixteen patients were evaluable. Patients were to receive rIFN-alpha 2 by either the I.V. or I.M. route for 35 consecutive days. The dosage was identical by both routes, and patients were escalated from 3 X 10(6) to 10 X 10(6) to 30 X 10(6) to 50 X 10(6) and to 100 X 10(6) I.U. every 7 days. No patient was able to tolerate the consecutive treatment protocol as planned. Dose-limiting toxicity was a flu-like syndrome in 10 patients and was usually associated with a fall in performance status. Confusion resulted in study withdrawal for five patients, four receiving rIFN-alpha 2 by the I.M. route. Hematologic and liver function abnormalities were common, usually transient, and not associated with clinical sequelae. One patient with non-Hodgkin's lymphoma showed substantial improvement; otherwise, all had stable or progressive disease. Pharmacologic studies indicated substantial serum levels at doses greater than or equal to 10 X 10(6) I.U. regardless of route. No consistent changes in NK activity, lymphocyte subpopulations, or immunoglobulin levels were noted, and no patient developed antibodies to rIFN-alpha 2. The dose and schedule used here indicate that high levels of serum rIFN-alpha 2 activity are obtainable by either the I.M. or I.V. route. Intermittent rather than daily dosage is more likely to be better tolerated and should be considered for phase II trials.

During the last decade, the treatment of ovarian cancer has changed markedly as the available armamentarium of cytotoxic drugs has improved. It remains apparent that surgical cytoreduction (debulking) procedures are useful, particularly in those cases where the largest mass of residual tumor can be reduced to less than 1 cm in size [1]. However, even more important than the degree of surgical debulking which can be achieved is the apparent improvement in clinical response rates seen with new innovations in chemotherapy. The Gynecologic Oncology Group (GOG) conducted a large randomized trial between 1976 and 1979 of suboptimal (> 3 cm tumor bulk) untreated ovarian cancers (Protocol #22), which showed that melphalan alone was as effective as the combination of cyclophosphamide and doxorubicin in the observed response rates and survival durations [2]. However, the subsequent GOG Protocol # 47 showed that the addition of cisplatin to cyclophosphamide and doxorubicin improved response rates from 46% to 71% with the median duration of survival increasing from 9.5 months to 15.0 months [3]. The fact, however, becomes apparent that a. survival duration of 15 months is not a cure, and the good benefits which cisplatin bestowed on patients with ovarian cancers are limited. The empiric choice of chemotherapeutic agents does not necessarily provide maximal benefit to each patient with ovarian cancer.