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Purpose: ONYX-015 is a genetically modified adenovirus with a deletion of the E1B early gene and is therefore designed to replicate preferentially in p53-mutated cells. A Phase II trial of intralesional ONYX-015 was conducted in patients with hepatobiliary tumors to determine the safety and efficacy of such a treatment., Experimental Design: All patients had biopsy-proven, measurable tumors of the liver, gall bladder, or bile ducts that were beyond the scope of surgical resection. Patients received intralesional injections of ONYX-015 at either 6 x 10(9) or 1 x 10(10) plaque-forming units/lesion up to a total dose of 3 x 10(10) plaque-forming units, and i.p. injections were allowed in patients with malignant ascites. The status of p53 was assessed by immunohistochemistry or Affymetrix GeneChip microarray analysis. Studies were conducted for viral shedding and for the presence of antiadenoviral antibodies before and after the injection of ONYX-015. Patients were assessed for response and toxicity., Results: Twenty patients were enrolled, and 19 patients were eligible. Half of the patients had primary bile duct carcinomas. Serious toxicities (> grade 2) were uncommon and included hepatic toxicity (three patients), anemia (one patient), infection (one patient), and cardiac toxicity (one patient, atrial fibrillation). Sixteen patients were evaluable for response. Among these evaluable patients, 1 of 16 (6.3%) had a partial response, 1 of 16 (6.3%) had prolonged disease stabilization (49 weeks), and 8 of 16 (50%) had a >50% reduction in tumor markers. Of the 19 eligible patients, 18 (94.7%) had specimens available for p53 analysis. Fifteen of these 18 patients (83.3%) had evidence of p53 mutation by one or both methods, although the methods correlated poorly. Viral shedding was confined to bile (two of two patients) and ascites (four of four patients). Pretreatment adenoviral antibodies were present in 14 of 14 patients and increased by 33.2% after ONYX-015 treatment., Conclusions: Intralesional treatment with ONYX-015 in patients with hepatobiliary tumors is safe and well tolerated, and some patients had evidence of an anticancer effect. The high incidence of p53 mutations in these tumors makes this a logical population in which to test this therapy but precludes definitive evaluation about the necessity of a p53 mutation for ONYX-015 clinical activity.

Purpose: ONYX-015 is a chimeric, E1B-deleted adenovirus designed to replicate preferentially in p53-deficient tumor cells; however, little is understood about its actual replication potential in human tumors. We hypothesized that replication of a late viral gene, hexon, would demonstrate replication of virus in human tissues., Experimental Design: In the course of a clinical trial, a patient with paired abdominal wall implants from a primary gall bladder carcinoma was injected with ONYX-015, 1 x 10(10) viral particles/lesion, followed by sequential excision of the lesions at 37 h and 7 days. Tissue sections were analyzed for evidence of viral replication., Results: In situ Reverse transcription-PCR was used to measure expression of hexon. Strong signals were obtained in gland-forming tumor cells both at 37 h and at 7 days. Signal was predominantly observed in the cytoplasm. The signal was also observed in adjacent normal stromal cells. Analysis of p53 status of the tumor by immunohistochemistry and Affymetrix Genechip demonstrated an inactivating mutation in p53. Routine H&E staining of the tumor sections revealed no evidence of necrosis at 37 h or 7 days after injection of virus. Presence of viral protein at both 37 h and 7 days was confirmed by immunohistochemistry using antibodies directed against hexon, penton, and fiber proteins., Conclusions: Evidence for replication of hexon confirms that ONYX-015 is not only present but capable of replicating in tumor cells up to 1 week after intralesional injection and that replication is not confined to p53-mutated tumor cells.

Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), a novel 3-substituted indolinone compound, binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb, an increase in caspase-3 activation (5-84%), and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents.

Colon carcinoma cells overexpress c-myc due to defective Wnt signaling, but only patients whose tumors have an amplified c-myc gene show improved disease-free and overall survival in response to 5-fluoruracil (5FU). Here we show that in two colon carcinoma cell lines that do not have an amplified c-myc gene but differ in their p53 status, high c-myc levels can be further elevated by introducing a c-myc expression vector. Whereas sensitivity to low serum-induced apoptosis was imposed on the parental lines independent of p53 status and was unaffected by further elevation of c-myc, sensitivity to 5FU-induced apoptosis was dependent on both the higher c-myc levels due to the expression vector and wild-type p53 function. The elevated c-myc levels led to higher c-myc transactivation activity in the p53 wild-type cell line, but not in the mutant p53 cell line. The requirement for both elevated c-myc and p53 for 5FU sensitivity was confirmed using antisense c-myc and pifithrin-alpha, a specific inhibitor of p53. Finally, the in vitro data predicted that only patients with both amplified c-myc and wild-type p53 in their primary tumors would be responsive to 5FU-based therapy, which was borne out by analysis of tumors from 135 patients entered into a Phase III clinical trial of 5FU-based adjuvant therapy. The data provide significant insight into mechanisms that establish colon tumor cell sensitivity to 5FU, clearly demonstrate the necessity of exercising caution in considering combining novel strategies that target elevated c-myc with standard 5FU-based therapy, and suggest alternative therapeutic strategies that target c-myc and/or p53 mutations in the treatment of colon cancer.

Interleukin-12 (IL-12) is a pleiotropic cytokine with proinflammatory, immunoregulatory, antitumor, and antimetastatic properties. It plays a crucial role in the development of the Th1 response and subsequent interferon-gamma production and enhancement of cell-mediated cytotoxicity. Recently, IL-12 has been used as an experimental therapy for cancer. Given the multiple immunomodulatory properties of IL-12, there are potential concerns associated with its clinical use. Of special interest are the possible side effects of IL-12 therapy in patients with autoimmune diseases, especially those that are T cell mediated, such as rheumatoid arthritis (RA). We present a case of severe RA exacerbation caused by treatment with IL-12 for metastatic cervical cancer. This is the first reported case of RA flare caused by exogenous IL-12.

The cancer treatment-related diarrhea caused by acute graft-versus-host disease (GVHD) and chemotherapeutic agents, particularly fluoropyrimidines and irinotecan, significantly affects patient morbidity and mortality. The mechanisms causing cancer treatment-related diarrhea are not fully understood, but histopathologic evidence points to a multifactorial process that causes an absorptive and secretory imbalance in the small bowel. Cancer treatment-related diarrhea could be life-threatening, yet assessment and treatment are not currently standardized. Several clinicians participated in a closed roundtable meeting to review the mechanisms of chemotherapy-induced diarrhea (CID) and GVHD-induced diarrhea, management issues in cancer treatment-induced diarrhea, and pharmacologic approaches to treatment. The meeting produced a proposal for new treatment guidelines and an algorithm, which include the use of octreotide for the management of CID- and GVHD-induced diarrhea. The development of diarrhea assessment guidelines that expand on the current National Cancer Institute criteria and allow for better patient management was also proposed.

The gene expression levels of the nucleoside cleavage enzyme/angiogenic factor thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor, were measured by quantitative reverse transcription-PCR in 38 pretreatment biopsies of colorectal tumors from patients who were subsequently treated with 5-fluorouracil (5-FUra) and leucovorin (LV). The range of TP gene expression (relative mRNA levels) in those tumors nonresponsive to 5-FUra was much broader than that of the responding tumors. In contrast to in vitro studies that had shown that an increased intracellular level of TP potentiates the activity of 5-FUra by converting it to the more cytotoxic nucleoside form 5-fluoro-2'deoxyuridine, tumors with the highest basal TP expressions were nonresponders to 5-FUra/LV therapy. The mean TP mRNA level in the nonresponding tumors was 2.6-fold higher than that of the responding patients. We had shown previously that high expression of thymidylate synthase (TS), the target enzyme of 5-FUra, was also a predictor of nonresponse to 5-FUra (L. Leichman et al, J. Clin. Oncol., 15: 3223-3229, 1997). TP and TS expressions were found to be independent variables in these tumors, so that low expression levels of both TS and TP in tumors predicted a very high response rate (11 of 14) to 5-FUra/LV as well as a significantly longer survival, whereas none (0 of 24) of the patients with high expression of either TP or TS were responders.

We have used pulsed-field gel electrophoresis to examine 5-fluorouracil (5FU)-induced DNA double-strand breaks (DSBs), both with and without modulation by IFN-alpha2a (IFNalpha), in HT29 human colon adenocarcinoma cells. Although 24-h treatment with either 10 microM 5FU or 500 units/ml IFNalpha did not result in significant DNA fragmentation, the combination of 5FU + IFNalpha resulted in a significant increase in DNA DSBs versus either drug alone (P < 0.05). The pattern of fragmentation induced by treatment with 5FU + IFNalpha was compared to that induced by gamma-radiation, which generates lesions at random sites, digestion with NotI restriction endonuclease, which cleaves at the specific sequence 5' ellipsis GCGGCCGCellipsis 3', and HhaI restriction endonuclease, which cleaves at the specific sequence 5'ellipsis GCGCellipsis 3'. 5FU + IFNalpha resulted in a specific pattern characterized by the accumulation of fragments of <3 Mb in the absence of fragments of >3 Mb, which differed from that of gamma-radiation and restriction endonuclease digestion. Because neither morphological nor DNA fragmentation characteristic of apoptosis was observed after 5FU + IFNalpha treatment, the nonrandom pattern of DSBs that was observed did not appear to be the result of the initiation of programmed cell death within these cells.

The enzyme/cytokine thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) has diverse functions within cells, including the regulation of steady-state thymidine levels, the conversion of the cancer chemotherapeutic agent 5-fluorouracil (FUra) to an active metabolite, and the mediation of angiogenesis in normal and malignant cells. Although the levels of TP/PD-ECGF vary substantially among different tissues and are generally found to be elevated in tumors, little is known about the control of its expression in vivo in humans. In this study, peripheral blood mononuclear cells were obtained from patients prior to and during treatment with IFN and FUra and analyzed for TP/PD-ECGF expression. Sixteen of 21 patients (76%) exhibited an average 3-4-fold increase of TP/PD-ECGF protein levels after treatment with either IFN-alpha or-beta, with the remaining patients having either a decrease (four patients) or no change (one patient) at the sampling times examined. Expression in vivo increased rapidly within 1-2 h of IFN treatment and remained elevated for up to 48 h after its administration. The increase in TP/PD-ECGF protein was accompanied by a concomitant increase in TP/PD-ECGF mRNA levels. TP/PD-ECGF mRNA expression in cells in vitro was induced by IFN but not by pharmacologically relevant concentrations of FUra, suggesting that the IFN was responsible for the induction seen in the patients. This study demonstrates that IFN induces TP/PD-ECGF expression in vivo by regulation of the level of mRNA expression.

Human colonic cancer is associated with multiple genetic deletions, mutations, and alterations in gene expression; in contrast, gene amplification has not been recognized as a prominent characteristic of human colonic tumors. Although the c-myc gene is overexpressed in approximately 70% of human colonic cancers, previous studies have not detected frequent gene amplification or rearrangement of c-myc in these tumors, although such amplification has been reported in chemically induced rodent colon cancer and quantitative analysis of gene copy number has shown the gene to be amplified at a low level in mucinous and poorly differentiated human colon carcinomas. Using rigorously controlled blot methodology, we have established that the c-myc gene, located at 8q21, exhibited amplification of 87% to 35-fold in 7 of 10 human colonic carcinoma cell lines. This was highly significant even at a low level of amplification in HT29 cells (P < 0.0001). Cytogenetic analysis by G-banding did not detect aneuploidy involving chromsome 8q, suggesting that the amplification for the c-myc gene on 8q was relatively specific, and this was consistent with a lack of amplification detected for the c-mos gene on 8q24, which was assayed similarly. The same methodology then revealed amplification of c-myc from 1.5-fold to 5-fold in 32% of tumors from 149 patients entered into a multi-institutional Phase III study of adjuvant therapy for colon cancer. c-myc status was not related to time to recurrence or death, but low levels of c-myc amplification identified a subset of patients who showed a statistically significant increase in disease-free survival, and a corresponding trend to longer overall survival, in response to adjuvant therapy with 5-fluorouracil plus levamisole. Presence of c-myc amplification was not related to incidence of p53 mutations.

Substantial increases in both the understanding of the cellular mechanisms of actions of interferon (IFN) and in its clinical use in cancer have occurred in recent years. The efficacy of interferon for the treatment of select malignancies has been established, and IFN-&agr; and IFN-&bgr; have been approved by the Food and Drug Administration for multiple clinical indications. IFN-&agr; increased median survival and relapse-free survival in patients with locally advanced melanoma when used as adjuvant therapy and had modest activity against advanced disease. In other tumors where studies indicated that IFN lacked direct therapeutic activity, clinical trials suggested that it increased the antitumor activity of cytotoxic chemotherapeutic agents when used in combination therapy. IFN has substantial activity in chronic myelogenous leukemia, increasing survival in patients in early chronic phase when compared with conventional chemotherapy, and has some activity in non-Hodgkin's lymphoma in combination with cytotoxic agents. Recent molecular and pharmacologic studies defining cellular receptor activation, signal transduction pathways, and biochemical modulating activities of interferon have yet to be fully incorporated into clinical development. Further preclinical advances along with the expanding identification of potentially clinically sensitive tumors make it likely that the use of IFN in cancer chemotherapy will continue to grow.

Clinical studies have demonstrated that the combination of 5-fluorouracil (FUra) and IFN-alpha has activity in the treatment of advanced colorectal cancer. Treatment of human colon carcinoma cells with IFN caused a 5-fold increase in the level of thymidine phosphorylase (TP) mRNA and an 8-fold increase in TP enzyme activity. Since TP catalyzes the first step in the direct conversion of FUra to deoxyribonucleotides, its induction by IFN is a potential biochemical mechanism for the modulation of the antitumor activity of FUra. In contrast to the activity measured in cell extracts, however, thymine utilization by intact cells was increased less than 2-fold by IFN, suggesting that the metabolic activation of FUra by TP in the IFN-treated cells was similarly suboptimal. This was likely due to a rate-limiting amount of cosubstrate for TP, and in this study, a series of 5-substituted 2'-deoxyuridine analogues were synthesized and tested as potential deoxyribose donors for TP. One of the compounds, the novel pyrimidine analogue 5-ethoxy-2'-deoxyuridine (EOdU), was found to be a substrate for the transferase reaction of TP, to have little or no direct cytotoxicity, to selectively increase the cellular levels of 5-fluoro-dUMP, to enhance the inhibitory effect of FUra on thymidylate synthase activity, and to potentiate the cytotoxicity of FUra and IFN in human colon carcinoma cells. EOdU was tested in vivo against HT-29 cells grown as xenografts in nude mice. The combination of EOdU+FUra+IFN-alpha 2a produced tumor regressions and a significantly greater delay in tumor growth when compared to FUra+IFN-alpha 2a, FUra+EOdU, or FUra or IFN used alone; tumors were 72% smaller in the EOdU+FUra+IFN-alpha 2a-treated animals compared to the saline control group. A comparable antitumor effect was also found when a related nucleoside analogue, 5-propynyloxy-2'-deoxyuridine, was used with FUra+IFN, and it also showed modulating activity when used with only FUra. The antitumor activity of the three agent combination (nucleoside+IFN+FUra) was comparable to that of a higher dose of FUra used alone, but it was substantially less toxic to the animals than the higher dose of FUra, indicating that the modulating agents improved the therapeutic index of FUra.(ABSTRACT TRUNCATED AT 400 WORDS)

alpha-Interferon (IFN alpha) potentiates the cytotoxicity of 5-fluorouracil (FUra) in vitro, and the combination has clinical efficacy in advanced colorectal cancer. We have reported previously an IFN alpha-mediated elevation in cellular FdUMP levels accompanied by the stimulation of thymidine phosphorylase (TP) activity in extracts from HT-29 human colon carcinoma cells treated with IFN alpha. We have now found that this effect of IFN alpha can be measured in vivo as an increase in thymine incorporation in intact cells. The increase was only 3-fold, however, compared to the 12-fold increase seen in TP activity in cell extracts. This suggested that the cosubstrate for TP, deoxyribose-1-phosphate, was rate limiting in the cells. Since the synthetic pathway of TP can also proceed via a transferase reaction, natural and modified deoxyribonucleosides were tested as deoxyribosyl donors. TP activity was measurable in cell extracts using deoxyinosine as cosubstrate with either thymine or FUra, although activity was only 10% of that measured with deoxyribose-1-phosphate. The pyrimidine analogue 5-propynyloxy-2'-deoxyuridine (PO-dUrd) had 15% of the maximal TP activity in cell extracts and also increased thymine incorporation in intact cells 10-fold. Both 2'-deoxyinosine and PO-dUrd potentiated the cytotoxicity of FUra by 8-11-fold. IFN alpha potentiated the cytotoxicity of FUra by 1.8-fold, and the combination of IFN alpha and PO-dUrd produced a 25-fold increase in the cytotoxicity of FUra. Neither the corresponding analogue riboside, 5-propynyloxyuridine, nor the analogue base, 5-propynyloxyuracil, had any effect on FUra cytotoxicity. There was a significant correlation between the ability of a nucleoside and/or IFN alpha combination to increase thymine incorporation and to reduce the 50% inhibitory concentration for FUra. IFN alpha and PO-dUrd also potentiated the inhibition by FUra of thymidylate synthase activity. These findings suggest that the use of a deoxyribonucleoside to provide the rate limiting cosubstrate would complement the stimulation of TP by IFN alpha, and together they should further enhance the antitumor activity of FUra.

alpha-Interferon (IFN-alpha) enhances the activity of 5-fluorouracil in patients with advanced colorectal carcinoma. Preclinical evidence suggests a similar potential role for IFN-alpha combined with cyclophosphamide, doxorubicin (Adriamycin, Adria Laboratories, Columbus, OH), and 5-fluorouracil (CAF) in advanced adenocarcinoma of the breast. To determine a maximum tolerated dose of IFN-alpha that could be combined with CAF and that did not compromise CAF dose intensity and to determine the effect of IFN-alpha on the pharmacokinetics of doxorubicin, a phase I study of IFN-alpha plus CAF was performed by the Eastern Cooperative Oncology Group. Nine patients with advanced breast cancer received CAF (cyclophosphamide at 100 mg/m2/day p.o. on days 1-14, doxorubicin at 30 mg/m2 and 5-fluorouracil at 500 mg/m2 i.v. bolus on days 1 and 8) plus IFN-alpha (1 milliunit/m2, n = 6, or 2 milliunits/m2, n = 3) given s.c. on days 1, 3, 5, and 8 (1 h prior to the doxorubicin and 5-FU injection on days 1 and 8) of each cycle every 28 or more days. Escalation of the IFN-alpha dose occurred in cohorts of 3-6 patients if a dose-limiting toxic event (neutropenic fever, platelet nadir of < 25,000/microliters, > 2-week treatment delay, or a > 50% dose reduction in day 8 CAF) occurred during the first two cycles in 0 of 3 or 1 of 6 patients. During cycle 1, IFN-alpha was omitted on day 1, and multiple plasma samples were drawn on day 1 (without IFN-alpha) and day 8 (with IFN-alpha) after each doxorubicin injection and were analyzed for plasma doxorubicin concentration. The maximum tolerated dose of IFN-alpha by our criteria was 1 milliunit/m2, and neutropenia was the predominant toxic effect that precluded IFN-alpha dose escalation. The dose intensity of CAF achieved with IFN-alpha was identical to that for CAF alone observed in prior studies. IFN-alpha had no significant effect on the pharmacokinetics of doxorubicin, although 3 of 7 patients studied had reduced doxorubicin clearance, ranging from 32% to 69%. Alternative CAF drug delivery schedules (all drugs given i.v. every 3-4 weeks) that are more amendable to hematopoietic growth factor support may be more suitable to combine with higher doses of IFN-alpha that may produce modulation.

Fluorouracil (FUra) is the most active agent in advanced colorectal carcinoma, and this activity can be enhanced by various modulating agents both in vitro and in vivo. To determine whether interferon (IFN) is capable of augmenting the cytotoxic and cytokinetic effects of FUra, combinations of FUra and IFN alpha, -beta, and -gamma were tested against 2 human colon cancer cell lines in vitro. In a clonogenic assay, IFN alpha and -beta, at concentrations that produced less than 1 log cell kill, significantly increased the cytotoxic effects of FUra in both cell lines. IFN gamma also enhanced the cytotoxic effects of FUra, but unlike IFN alpha and -beta, only at the highest concentrations tested. Median effects analysis demonstrated that all 3 IFNs exhibited synergy with FUra. Combinations of IFNs were no more effective at modulating FUra activity than single agent IFN. Flow cytometric studies indicated that these effects did not correlate with cytokinetic alterations. Only the combination of FUra and IFN beta produced cytokinetic effects different from those of FUra alone. Incubation with IFN alpha or IFN gamma for 24 h resulted in only modest cytokinetic alterations, and they did not modify the effects of FUra. These results indicate that IFN is capable of increasing the cytotoxic actions of FUra and that this is separable from any cytokinetic effects produced by the interferons.

The combination of interferon (IFN) and conventional chemotherapeutic agents offers a promising therapeutic approach for the treatment of cancer. However, there is as yet no consensus on optimal strategies for combining this family of compounds with other cancer therapies. While in vitro studies have demonstrated both direct cytotoxic and cytokinetic effects for IFN, a more interesting role derives from its ability to synergistically potentiate the activity of a wide variety of cytotoxic agents against multiple human and rodent tumors, both in vitro and in animal models. The interaction between IFN and cytotoxic agents in vitro is complex and depends not only on the choice of cytotoxic agent but also on the concentrations, ratios, duration, and sequence of exposure to the two drugs. Preliminary data suggest that some combinations are not merely additive but rather that IFN may biochemically modulate the cellular uptake or metabolism of the cytotoxic agent resulting in synergistic antineoplastic activity. In vivo interactions between IFN and cytotoxic agents involve an additional layer of complexity because of the potential effects of the biological agent on the host immune system and drug-metabolizing enzymes. Furthermore, IFN may have a protective effect on normal host tissues which theoretically could allow for the delivery of higher doses of cytotoxic agents. The results of early clinical trials using combinations of IFN with chemotherapeutic agents have generally been disappointing. This may be due to the inability of preclinical models to accurately predict the clinical situation or alternatively from a failure to incorporate information on dose, scheduling, and sequence of drug administration into clinical trials. Preliminary clinical studies with IFN-alpha and the fluorinated pyrimidine, 5-fluorouracil, in patients with advanced colorectal carcinoma suggest that IFN may enhance the effects of the antimetabolite. Confirmatory trials are in progress. Further trials designed to exploit the preclinical experience with combinations of IFN and cytotoxic agents are warranted.

We have previously shown that the combination of 5-fluorouracil (5-FUra) and recombinant alpha-2a-interferon (rIFN-alpha-2a) produced objective responses in 23 of 32 (63%) previously untreated patients with advanced colorectal carcinoma. Because in vitro data suggest that rIFN-alpha-2a modulates the cytotoxic effects of 5FUra in a concentration-dependent manner, a phase I clinical trial was initiated to determine the maximum tolerated dose of rIFN-alpha 2a when administered in combination with 5FUra. A total of 27 patients with advanced colorectal carcinoma were enrolled. The median age was 64 years, and the median performance status was 1. A total of 18 patients had no prior chemotherapy and 19 no prior 5FUra. 5FUra was administered at 750 mg/m2/day by continuous i.v. infusion for 5 days, followed by weekly bolus therapy. rIFN-alpha 2a was administered at 6, 9, 12, 15, or 18 x 10(6) units s.c. beginning on day 1. The dose-limiting toxicity of this regimen was fatigue, resulting in a decrease in performance status, and this was the only toxicity that correlated with increasing dose of rIFN-alpha 2a. Eastern Cooperative Oncology Group grade 3-4 toxicities included leukopenia (6), thrombocytopenia (2), anemia (4), stomatitis (4), diarrhea (4), neurological (2), infection (2), and allergy (2). Three quarters of the patients required interruption of therapy or dose reductions of either 5FUra or rIFN-alpha 2a for toxicity. Among the patients with measurable disease who were previously untreated with 5FUra, 5 of 9 at the lowest dose levels achieved an objective response, including one pathological complete responder, whereas 0 of 9 at the three highest dose levels responded. Among patients previously treated with 5FUra, only 1 achieved an objective response. We conclude that the maximum tolerated dose of rIFN-alpha 2a, when administered with 5FUra as above, is 15-18 x 10(6) units; however, the efficacy of this regimen does not appear to be related to the dose intensity of rIFN-alpha 2a, and future regimens should employ a lower dose, intermittent schedule of rIFN-alpha 2a, which may be better tolerated.

Acquired resistance to chemotherapeutic agents is an important clinical problem. One preclinical model, termed multidrug resistance (MDR), is characterized by a complex phenotype of cross-resistance to biochemically unrelated antineoplastic agents, the presence of a high-molecular-weight membrane glycoprotein, and impaired accumulation of drug. To determine whether MDR is mediated in part by altered cyclic 3',5'-adenosine monophosphate (cAMP) levels, the effect of incubation with the adenylate cyclase agonist, forskolin, was investigated in the murine sarcoma S180 cell line and two MDR variants (A5-.8, A5-2.5). Basal cAMP levels in sensitive and MDR lines were not significantly different (range, 0.15 +/- 0.05 to 0.31 +/- 0.09 pmol/mg protein); however, 1-h incubation with forskolin, 10 microM, elevated intracellular cAMP 2-fold in the parent line and 43- and 35-fold in the variants. The adenylate cyclase agonists, prostaglandin E2 and cholera toxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine had no significant effect on cAMP levels. To determine the effect of forskolin on doxorubicin-induced cell lethality, S180 and MDR lines were incubated with doxorubicin plus forskolin for 1 h and cloned in soft agar. Coincubation with forskolin partially reversed doxorubicin resistance in the MDR lines in a dose-dependent fashion. To determine whether this effect was mediated solely by elevation of intracellular cAMP, the inactive 1,9-dideoxy analogue of forskolin (DF) was used. Incubation with DF resulted in no elevation of cAMP levels in the sensitive or resistant cell lines; however, DF also partially reversed doxorubicin resistance in the MDR variants. Furthermore, coincubation of the A5-2.5 cell line with doxorubicin and 8-bromo cAMP, 1 mM, did not result in reversal of resistance to doxorubicin. To determine whether the reversal of resistance by the diterpenes was associated with alteration of doxorubicin transport, uptake and efflux of [14C]doxorubicin were measured. Coincubation with both forskolin and DF, 10 microM, enhanced [14C]doxorubicin uptake in the resistant cells, while drug efflux was significantly affected only in the cell line exhibiting intermediate resistance. Since both forskolin and its inactive analogue are effective in partially reversing resistance to doxorubicin and augmenting anthracycline uptake, a mechanism other than elevation of cAMP is most likely responsible.

The bisdioxopiperazine (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)-propane (ICRF 187) abrogates doxorubicin cardiotoxicity in every mammalian species tested, but its effect on doxorubicin antitumor activity remains poorly understood. In order to better define the anthracycline-bisdioxopiperazine interaction, the ability of murine sarcoma S180 cells to form colonies in soft agar and their capability to proliferate in microtiter wells were assayed after exposure to drug at varying doses and schedules. Incubation of cell suspensions for 1 h with doxorubicin, 0.1 microgram/ml, with or without (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane, 80 micrograms/ml, produces additive cytotoxicity for the combination. Prolonged incubation (24 h) with the same drugs produces synergistic cytotoxic and antiproliferative effects at 1- and 2-log order reductions in dose. These studies indicate that the antineoplastic activity of the single agents doxorubicin and (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane is enhanced when the drugs are used in combination, and that this phenomenon is highly dose and schedule dependent.

Biological therapy is currently being investigated in the treatment of a number of malignancies. The hypothesis for the use of this therapeutic modality involves an attempt to stimulate an already existent but perhaps suboptimal immune response to foreign protein, including tumor. Immunologic therapy appears to work best against small-volume disease, as indicated from animal studies. This condition is potentially achievable in advanced ovarian cancer, where surgery is capable of producing multi-log reduction in tumor mass, and thus immunotherapy may be an option in this disease. The attraction of biologic therapy in patients with ovarian cancer is the potential to treat relatively localized but often chemotherapy-resistant disease. In cervical cancer, the rationale for the use of interferon is somewhat different in that this disease may be a manifestation of a virally induced proliferative lesion. Thus, the antiviral properties of interferon are being investigated in both limited and advanced cervical cancer. Both of these hypothesis have pre-clinical data to support them. This paper presents the pre-clinical and clinical work currently available for consideration of future use.