Search

A major goal in the development of chemopreventive agents has been to develop markers that reflect the underlying process of carcinogenesis and which are modulatable by the agent under study. An important application of such markers will be to select cohorts that are at elevated risk for cancer development, which should allow use of smaller sample sizes in definitive phase III trials as well as shorter duration (and lower cost), without loss of statistical power. Susceptibility and surrogate end-point biomarkers are particularly important in this respect. Intermediate markers are probably best assessed in terms of proportionate rather than relative risk. The systematic development of difluoromethylornithine for use in chemoprevention against human cancer has involved pilot, phase IIa and IIb trials using participants with prior colonic polyps as the study group. A unique feature of the phase IIa study was the use of a dose de-escalation design which allowed selection of the lowest effective non-toxic dose of difluoromethylornithine. The phase IIb trial now in progress is using a combination of sulindac with difluoromethylornithine; the rationale for selection of markers for this study and for a randomized phase III registration trial is discussed. We also review the findings in phase I and IIa trials of Bowman-Birk inhibitor concentrate, in which patients with measurable oral leukoplakia are the study group.

Difluoromethylornithine (DFMO) is an investigational chemopreventive agent that inhibits ornithine decarboxylase (ODC) activity, lowers cellular polyamine concentrations, and decreases cell proliferation in vivo and in vitro. In five subjects we have compared the polyamine concentrations in rectal mucosal biopsies and in exfoliated buccal mucosal cells (EBM) before and after DFMO treatment to assess the suitability of EBM as an easily accessible marker tissue for DFMO suppression of polyamine synthesis in the rectal mucosa. One month of 3 g/m2/day of DFMO treatment caused a statistically significant decrease in putrescine and spermidine concentrations in rectal mucosa biopsy specimens but not in EBM samples. ODC activity in EBM was high (approximately 1 mumol/min/mg protein), resistant to DFMO inhibition (Ki = 4200 microM), dependent on GTP concentration (maximal at 0.1 mM), and was reduced concomitantly with bacterial concentration by antiseptic mouthwashing. Bacteria adherent to EBM were visible by electron microscopy. Forty bacterial colonies/ng protein were culturable from washed EBM samples. Oral bacteria preclude the use of EBM samples as a marker tissue of DFMO effect in the rectal mucosa, but oral DFMO therapy is effective in depleting polyamines in rectal mucosa.

A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.

Retinoids, including isotretinoin, have demonstrated antiproliferative and antineoplastic activity in laboratory and clinical trials. In a phase II trial, 25 patients with extensive mycosis fungoides were evaluated for response to isotretinoin. There was a 44% (11 patients) objective clinical response rate with three clinical complete responses without concomitant evidence of pathologic clearing of the disease. An additional 24% (six patients) showed a minor degree of clinical improvement. The median time to response was two months (range, 0.5 to eight months) and the median response duration was eight months or longer (range, one to 25 months). Chronic toxic reactions consisted primarily of drying of the skin and mucous membranes and resulted in dose reduction in the majority of patients. It is concluded that isotretinoin produces significant clinical benefit to some patients with mycosis fungoides.

Pimozide, a potent neuroleptic which inhibits the release of pituitary releasing factors and is an effective dopamine antagonist, was administered to 30 patients with previously treated metastatic melanoma. Six patients were inevaluable because of poor drug tolerance (two), disease progression within 1 week and death within 2 weeks (three), and death from other causes (one). Among the 24 evaluable patients, two had complete response, two had partial response, and two had disease stabilization. Responses were observed in soft tissue, lymph nodes, liver, and lung. Toxic effects consisted of extrapyramidal manifestations in nine patients and malaise in seven. Pimozide has activity in patients with previously treated metastatic melanoma (17% response rate in evaluable patients) and merits consideration of further study in combination regimens.

13-cis-Retinoic acid (13-CRA) is a synthetic analog of vitamin A effective reversing preneoplastic lesions in both humans and animals. To study its physiochemical properties and disposition kinetics, we developed a rapid, sensitive, and precise high-performance liquid chromatography assay for 13-CRA in biological samples. This assay system resulted in a clear separation of 13-CRA from all-trans-retinoic acid and retinol and had a detection limit of 20 ng/ml plasma. Recovery was 89 +/- 6% (S.D.) at equivalent physiological concentrations with a precision of 8%. To study the disposition kinetics in humans, 13 patients received a p.o. bolus of 13-CRA and had blood samples collected at timed intervals. For the 10 patients studied on the first day of 13-CRA administration, the mean time to peak plasma concentration was 222 +/- 102 min. Interpatient peak 13-CRA plasma concentrations were found to be variable, suggesting irregular gastrointestinal absorption. Beta-Phase t 1/2 was approximately 25 hr. The prolonged terminal-phase plasma half-life may represent biliary excretion and enterohepatic circulation.

We investigated the effect of the synthetic vitamin A derivative isotretinoin (13-cis-retinoic acid) on advanced cancers in 103 patients and on preneoplastic lesions in five patients. Six of 14 patients with squamous cell epithelial cancers had objective regressions of skin or subcutaneous metastases. Three of five patients with preneoplastic lesions had objective responses. The major dose-limiting toxic effects were reversible dermatitis, emotional lability, and headaches. We conclude that the growth of some squamous cell epithelial malignancies can be inhibited by isotretinoin and suggest that other retinoids should be evaluated as antitumor agents.

Eighteen patients with myelodysplastic syndrome received 13-cis-retinoic acid (1.0-mg/kg/day starting dose with 0.5-mg/kg increment escalations) in a phase I-II trial. Two partial responses involving the erythroid series were observed in four patients with primary refractory anemia with ring sideroblasts. One of two patients with chronic myelomonocytic leukemia also achieved a partial response. No other responses were found in the remaining patients, which included eight with refractory anemia with excess blasts. In six patients drug toxicity necessitated termination of the trial. Four patients had unexpected drug-induced thrombocytopenia; three of these had low platelet counts before treatment. Two of the six patients had other toxic effects. Further studies are warranted to evaluate the effectiveness of 13-cis-retinoic acid in patients with refractory anemia with ring sideroblasts and chronic myelomonocytic leukemia. At moderate doses significant toxic effects, including thrombocytopenia, are not uncommon.

The successful development of a soft agar clonogenic assay for human tumor stem cells provides an in vitro technique with a high degree of accuracy for predicting in vivo clinical response to standard anticancer drugs. We used this system to conduct an "in vitro phase II trial" in human ovarian cancer and melanoma. This approach can potentially identify active phase I--II drugs suitable for treatment of given tumor types for specific patients and eliminates the need to subject patients (who would be predicted not to respond) to toxic side effects. In vitro sensitivity for new agents was operationally defined as at least a 70% reduction of tumor colony-forming units (TCFU) at concentrations which are readily achievable pharmacologically. The new agents AMSA and vindesine (as well as vinblastine) appeared to have activity in melanoma, while PALA and thymidine were inactive. Pentamethylmelamine, mitomycin C, methyl-GAG, and AMSA were relatively ineffective in ovarian cancer. Vinblastine and vindesine had definite activity. The human tumor stem cell assay may thus provide the basis for a useful alternative to the current clinical phase II testing approach for identifying antitumor activity of new agents. Validation of this concept with correlative in vitro and in vivo phase II trials of new agents in patients with tumor types predicted to be sensitive is clearly warranted.

Human malignant melanoma can form tumor colonies in soft agar, and at least two major morphological variants can be identified. Host cells play an important role in human melanoma colony formation, and their effects on the colony variants are selective. Neuroendocrine compounds also modulate expression of the melanoma TCFU pigmentary variants. This system appears suitable for evaluation of the response of melanoma TCFUs to chemotherapy, retinoids, heat, and radiation.

Both interferon-alpha (IFN-alpha) and alpha-difluoromethylornithine (DFMO) have shown modest activity as single-agent therapy in the treatment of malignant melanoma. Several investigators have demonstrated true synergism in vitro of the combination of DFMO and IFN-alpha against human tumor cells, including melanoma. We have investigated this combination in 17 patients with malignant melanoma in a Phase I trial. Patients were treated with 4 or 6 g/m2/day of oral DFMO in 3 divided doses for 11 days, followed by a 3-day rest period. Concomitant administration of 1.5, 3.0, 6.0 or 9.0 x 10(6) U/m2 IFN-alpha intramuscularly was given. The maximum tolerated dose was 4 g/m2/day of DFMO plus 6 x 10(6) U/m2/day of IFN-alpha. Dose-limiting toxicity occurred in 3 of 3 patients receiving 9 x 10(6) U/m2 IFN-alpha and consisted of leukopenia, fatigue, and weight loss. Other toxicities were mild and included reversible hearing loss, diarrhea, nausea, and vomiting. Three responses were seen, including one partial response (PR) of soft tissue metastases, one PR of lung and liver, and one complete response of liver metastases without clearance of carcinomatous meningitis. A Phase II trial has been initiated based on these encouraging results.

A phase II trial was conducted to determine the clinical activity of amsacrine (m-AMSA) in patients with heavily pretreated solid tumors, myeloma, and lymphoma at the University of Arizona Cancer Center. Additionally, m-AMSA was evaluated at other Southwest Oncology Group institutions in breast cancer, myeloma, melanoma, and oat cell cancer of the lung. At a dose of 120 mg/m2 given iv every 28 days, 12 partial responses were observed in 221 patients evaluable for response. Some antitumor activity was observed in breast cancer (four responses of 65 patients), non-Hodgkin's lymphoma (three of nine), Hodgkin's disease (two of five), and sarcoma (two of 15). A partial response was also documented in one of two patients with cervical cancer. Among the 135 patients treated at the University of Arizona who were extensively evaluated for toxic effects, only myelosuppression and anemia were seen in a significant number of patients. At this dose and schedule, 29% of patients developed leukopenia of less than 3000 cells/mm3, 16% developed a thrombocytopenia of less than 100,000 cells/mm3, and 29% had an acute fall in hemoglobin of greater than or equal to 2 g/100 ml. In addition, two patients suffered grand mal seizures which were not clearly drug-related. These results suggest that further study of m-AMSA in lymphoma, sarcoma, and cervical cancer is warranted.

Laryngeal papillomatosis often involves a relentless growth of papillomas on the vocal cords, requiring repeated excisions to maintain an adequate airway. Because of its antiproliferative effects on epithelial tissues, 13-cis-retinoic acid (0.5-2.0 mg/kd/day p.o.) was used in five patients whose disease was poorly controlled by laser beam surgery. Control of disease for 24+, 5+, and 12 months has been achieved in three of the patients, with two complete and one partial responses. Side effects of treatment were mild and rapidly reversible, following a 25-50% reduction in drug dose.

A phase I trial was conducted of the vitamin A derivative beta-all-trans-retinoic acid (vitamin A acid; TRA), delivered via a collagen sponge and cervical cap for mild or moderate intraepithelial cervical neoplasia. On the basis of known skin and mucosal membrane toxicity, a concentration of 0.05% TRA in a cream-based vehicle was selected as the starting dose and was escalated later with the use of a modified Fibonacchi scale. The delivery device and the TRA were changed daily for 4 days, and side effects were assessed on days 1, 2, 3, 4, 8, and 30 by clinical and colposcopic examination. Vaginal, cervical, and systemic toxicity were evaluated in 35 patients. No dose-related systemic effects were found; mild cervical inflammation increased in many patients at higher doses. Unacceptably high vaginal toxicity was reached at a TRA concentration of 0.484%. A concentration of 0.372% TRA is recommended for use in phase II trials in mild and moderate cervical intraepithelial neoplasia.

Retinoids are effective suppressors of the phenotypic development of cancer in many animal systems, whether the process is initiated by chemical, physical or viral carcinogens. Cases of cervical intraepithelial neoplasia are excellent for studying the effectiveness of retinoids as chemopreventive agents because the process can be closely followed by serial colposcopic and pathologic (cytology or biopsy) means and changes in the condition safely monitored. We have previously conducted a phase I study of trans-retinoic acid (Tretinoin) given topically by a collagen sponge and cervical cap. A dose of 0.372% was selected for phase II trial. We have treated 20 patients with topical retinoic acid, and a complete response with total regression of disease was obtained in 50%. Systemic and cervical side effects were mild and vaginal side effects moderate but tolerable. These results provide a clinical basis for a randomized, double-blind phase III study to definitely answer the question of whether retinoic acid is an effective chemopreventive agent for cervical cancer.