Clinical, pharmacologic, and immunologic effects of 2'-deoxycoformycin.

Clinical, pharmacologic, and immunologic effects of 2'-deoxycoformycin (dCF) were evaluated in 15 patients with advanced malignancies. Toxicity was less severe with a low dose (4 mg/m2) of dCF, but this dose still resulted in suppression of cellular adenosine deaminase activity, skin test reactivity, and lymphocyte responses to mitogens. Improvement in cutaneous T cell lymphoma plaques was seen after dCF. Further investigations of antitumor efficacy with the use of this low dosage schedule should continue in patients with hematologic neoplasms, and additional preliminary studies of the combination of an adenosine deaminase inhibitor with an adenosine analog should also be considered.

Phase I trial of the combination of 6-methylmercaptopurine riboside and 5-fluorouracil.

6-Methylmercaptopurine riboside (MMPR) and 5-fluorouracil (5-FU) were administered sequentially to 12 patients in a phase I clinical trial. Toxicities included mild nausea and vomiting, as well as reversible leukopenia and thrombocytopenia. Maximal accumulation of 6-methylmercaptopurine ribonucleoside 5'-monophosphate (MMPR-P), the active metabolite of MMPR, in patients' erythrocytes occurred between 2 and 6 h after the administration of MMPR and the degree of accumulation was dose-related. At 96 h after MMPR administration, MMPR-P was still detectable in patients' erythrocytes. Although no clinical responses were documented, a modified dosage schedule of these drugs should be pursued based on the pharmacokinetic data obtained.

Randomized comparison of tamoxifen versus diethylstilbestrol in estrogen receptor-positive or -unknown metastatic breast cancer: a Southeastern Cancer Study Group trial.

In a randomized study 115 postmenopausal women with advanced breast cancer who were estrogen receptor-unknown or -positive were treated initially with tamoxifen or diethylstilbestrol (DES). Their pretreatment characteristics showed no significant difference. The frequency of response was identical with tamoxifen and DES, showing a complete response rate of 2% versus 2% and a partial response rate of 4% versus 8%, respectively; stable disease was present in 78% versus 73% of the patients, respectively. The median time to disease progression (5 vs 6 months) and median survival depending on initial hormone therapy (34 vs 35 months) were identical for tamoxifen and DES, respectively. Gastrointestinal toxicity was more frequent and more severe with DES than tamoxifen. Responses were seen with withdrawal of each agent and on crossover to the alternative agent. Our conclusions are that: DES and tamoxifen are equally effective in treating metastatic breast cancer in the postmenopausal patient who is estrogen receptor-positive or -unknown; withdrawal and crossover responses are seen with both agents; side effects are minimal but more frequent with DES; and on the basis of cost-effectiveness DES is the preferable agent.

Tissue analysis of N-methylformamide: organ distribution.

This report describes a gas chromatographic procedure, utilizing a packed column and flame ionization detector, suitable for the quantitative measurement of N-methylformamide (N-MF) in tissue samples. N-MF is a polar solvent that induces the maturation of cancer cells in vitro and, in vivo, exhibits antitumor activity with human tumors xenografted in nude (athymic) mice. Therapeutic monitoring is essential as toxicology studies have shown this compound to be hepatotoxic. N-MF is currently undergoing phase 1 clinical trials as an anticancer drug. This method of tissue analysis was developed to aid in the understanding of N-MF disposition and distribution in a murine model. The data thus generated may help predict the clinical behavior of this drug.

Polar solvents: a novel class of antineoplastic agents.

It has been known for many decades that certain murine and human cancers can spontaneously mature to benign tissue. These observations have stimulated investigators to attempt to induce a state of more normal or benign differentiation in cancer cells using biologic substances or chemicals. Polar solvents including dimethylsulfoxide, dimethylformamide, and monomethylformamide have proven to be good inducers of maturational events in murine and human cancer cells. Moreover, several laboratories have demonstrated that polar solvents inhibit the growth of human tumor xenografts in nude mice. These findings have resulted in the entry of monomethylformamide into phase I clinical trials in America and Europe. Preclinical work further suggests that polar solvents may be useful agents in combination with conventional treatment modalities. The use of drugs such as monomethylformamide that can convert neoplastic cells to benign cells rather than kill the tumor cells represents an important conceptual departure from standard cytotoxic chemotherapy. The use of maturational-agent therapy should be considered as an important new development in the design of cancer treatment protocols.

Nude mouse model of the melanosis syndrome.

The occurrence of diffuse hyperpigmentation secondary to a xenografted human malignant melanoma was observed in nude mice. The patient from whom this cell line was established developed cutaneous hyperpigmentation after his disease became disseminated. Light and electron microscopy studies were performed on skin and organ biopsy specimens from the hyperpigmented mice. These studies indicated that this melanoma-associated melanosis was secondary to the release of pigment granules from the xenografted tumors and to the uptake of these granules by macrophages throughout the body, including those located in the dermis.

Human tumor cell heterogeneity and metastasis.

Heterogeneity within malignant neoplasms, although described for many years by pathologists, has only recently been extensively studied in the laboratory. It is now accepted that most tumors are composed of subpopulations of cells that differ in many phenotypic characteristics including the ability to form a metastasis. Cells with the capacity to metastasize are the ones most likely to prove lethal to the patient since clinicians can often control the primary neoplasm with surgery or radiotherapy. In this report the process of metastasis is discussed, those aspects of tumor cell heterogeneity that are relevant to this process are reviewed, intrapatient tumor heterogeneity is explored, and future preclinical studies are evaluated that may be useful in designing treatment strategies for patients with disseminated malignancies.

Response of a human colon adenocarcinoma (DLD-1) to X irradiation and mitomycin C in vivo.

Mice hosting a heterogeneous human colon xenograft tumor produced by subcutaneous injection of the DLD-1 tumor cell line were treated either with x irradiation alone, with mitomycin C alone (4 mg/kg), or with x irradiation given two hours after intraperitoneal injection of mitomycin C (4 mg/kg). Radiation alone produced a dose dependent delay in the time needed for tumors to regrow to twice their size at the time of irradiation, and in the mice receiving mitomycin C plus x irradiation, an additional growth delay equivalent to that produced by 3-3.5 Gy of X rays was seen at all X ray dose levels. As the DLD-1 tumor xenografts do not appear to possess a significant hypoxic fraction, we conclude that the two agents are acting in a simple additive cytotoxic manner by the killing of oxic tumor cells.

Characterization of two metastatic subpopulations originating from a single human colon carcinoma.

Two separate cell lines originating from distinct metastatic deposits in a patient with a primary colonic carcinoma have been established both in vivo and in vitro. One metastasis, OM-1, was found in the omentum, and the other, HOT-3, was located on the ovary. These two metastases differ in several significant characteristics, including growth kinetics and the production of carcinoembryonic antigen by the cultured cells. OM-1 xenograft tumors contain about 8-fold more colonic mucin antigen than do HOT-3 heterografts. Similarities also exist. Both cell lines contain extra chromosomes in the A group, and both are missing chromosomes in Pair 14 of the D group and in Pair 18 of the E group. Xenograft tumors from these two metastases contain equivalent amounts of carcinoembryonic antigen and nonspecific cross-reacting antigen. These metastases developed through a process of progression and natural selection that occurred during the course of the patient's disease. Thus, the cell lines established from them provide material which may be used to study functional intraneoplastic diversity.

Selection of metastatic variants from heterogeneous tumor cell lines using the chicken chorioallantoic membrane and nude mouse.

The chicken chorioallantoic membrane was used to select variant tumor cell subpopulations from the murine melanoma B16-BL6 and the rat glioma C6 cell lines. Tumor cells were deposited on the chicken chorioallantoic membrane of eggs 10 days postfertilization. Upon hatching, chickens were autopsied, and organs were removed, minced, and implanted s.c. in C57BL/6J mice (for melanoma) or nude mice (for glioma). A glioma growing s.c. from a chicken lung implant metastasized to the liver of the recipient nude mouse, and a melanoma growing s.c. from a chicken liver implant metastasized to the lung of its murine host. The s.c. melanoma contained distinct black and gray areas. Cell lines were established from the s.c. glioma (C6-V-1), from a metastasis of the C6-V-1 tumor (C6-V-2), and from the black and gray regions of the melanoma. Marked differences in lung colonization were seen 14 days after 1 X 10(5) parent BL6, Black, or Gray cultured cells were injected by tail vein into C57BL mice. In four separate experiments, fewer than 15 lung foci per mouse were found when BL6 cells were injected, whereas 100 to several hundred lung melanoma colonies per mouse were observed when Black or Gray cells were inoculated. Four of 18 nude mice bearing the s.c. C6-V-1 glioma developed liver metastases; no metastases have been observed in 15 nude mice bearing the s.c. parent C6 glioma. Significant differences in sensitivities to antineoplastic drugs were demonstrated between parent and variant glioma cell lines. The 33-fold increase in sensitivity to vincristine determined for C6-V-1 cells compared to parent C6 cells was particularly striking. Results suggest that the use of the chicken chorioallantoic membrane in situ, together with the nude mouse, might provide a method suitable for the selection and isolation of aggressive variants in heterogeneous human tumors.

Biochemical pharmacology and toxicology of formycin alone and in combination with 2'-deoxycoformycin (pentostatin).

The toxicology and pharmacology of formycin both as a single agent and combined with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF) were examined in outbred Swiss mice heterozygous for the nude gene (nu/+). The LD10 for formycin alone given on a daily x 5 schedule was 21 mg/kg. When the animals were pretreated with 1 mg/kg of dCF 1 hour prior to each dose of formycin, toxicity was approximately doubled, ie, LD10 was reduced to 10 mg/kg. Death was associated with hepatic toxicity in both treatment regimens; suppression of leukocyte counts was mild except at doses greater than the LD10. Formycin nucleotides were detected by high-performance liquid chromatography in the livers of mice treated with formycin either alone or combined with dCF. When isolated rat hepatocytes were incubated for 2 hours with either formycin or dCF plus formycin, analog nucleotides accumulated in the cells. Cellular ATP decreased to below the limits of detection, whereas a large peak corresponding to formycin-5'-triphosphate was present. This replacement of cellular ATP by formycin-5'-triphosphate may help explain the hepatic toxicity observed.

A therapeutic monitoring assay for N-methylformamide in human serum and urine.

This report describes a gas chromatographic procedure using a packed column and a flame ionization detector, which is suitable for the therapeutic monitoring of N-methylformamide (N-MF). N-MF is a polar compound that induces cancer cell maturation in vitro and exhibits antitumor activity with human tumors xenografted in nude mice. Toxicology studies with mice have shown this compound to be hepatotoxic. N-MF is currently undergoing Phase I clinical trial as an anticancer drug at this institution. In concert with its clinical trial, a method for the analysis of N-MF in biological fluids has been developed to study its pharmacokinetics. This method involves direct injection of urine or acetone-deproteinized serum onto a Chromosorb 103 column, using N,N-diethylformamide as an internal standard.

Hemodynamic effects of potentially useful antineoplastic agents.

Adenosine (Ado) and Ado analogues produce multiple hemodynamic effects including coronary vasodilation, bradycardia, alterations in left ventricular contractility, and peripheral vasodilation or vasoconstriction depending on the vascular bed. The intact anesthetized Sprague-Dawley rat was examined in relation to electrocardiogram and blood pressure alterations induced by a series of potentially useful antineoplastic agents that are purine or pyrimidine analogues as part of a preclinical evaluation of these agents. The drugs tested were arabinosyladenine and its 5'-monophosphate derivative arabinosyladenine-5'-monophosphate (ara-AMP), the 2-fluoro derivative of ara-AMP, the pyrazolo[4,3-d]pyrimidines (formycin and formycin B), 8-azaadenosine, 6-methylmercaptopurine riboside, tricyclic nucleoside-5'-monophosphate, 5-fluorouracil, arabinosylcytosine, and 3-deazauridine. Those Ado analogues subject to deamination by adenosine deaminase (ADA) were also studied in the intact Sprague-Dawley rat after pretreatment with the ADA inhibitor 2'-deoxycoformycin. The results indicate that these agents have significant hemodynamic effects and should alert clinicians to potential adverse reactions when infusing these drugs.

Inhibition of the growth of human colon cancer xenografts by polar solvents.

The effects of N,N-dimethylformamide (DMF) and N-methyl-formamide (NMF) on the growth of two human colon cancer cell lines xenografted in nude mice were assessed. Toxicological studies with mice heterozygous for the nu/nu gene showed that with both compounds the limiting organ toxicity was hepatic. The 10% lethal doses for DMF and NMF given i.p. daily for 21 days were 2219 and 374 mg/kg, respectively. Nude mice (10/group) received s.c. transplants of HCT-15 or DLD- 2 human colon cancer cells. Mice were treated i.p. with the approximate 10% lethal doses of either DMF (daily for 21 days) or NMF (daily for 19 days) or with 0.9% NaCl solution when tumors became palpable. With the HCT-15 tumor, a growth inhibition of 65% was obtained using DMF compared to 0.9% NaCl solution-treated controls. Two independent experiments with DLD-2 demonstrated that DMF effected growth inhibitions of 45 and 67%. NMF treatment produced 48 and 75% growth inhibitions of HCT-15 and DLD-2 tumors, respectively. Weight loss of groups of treated mice in all experiments was between 2 and 14%, within the acceptable range for 10% lethal drug doses. Results indicate that some human cancer xenografts respond to the polar solvent DMF and to its metabolite NMF and that DMF may be acting at least in part by its metabolism to NMF. Furthermore, the data should alert clinical investigators to the possibility of hepatotoxicity when polar solvents are tested in Phase I clinical trials.

Biochemical pharmacology and toxicology of 8-azaadenosine alone and in combination with 2'-deoxycoformycin (pentostatin).

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF). The LD10 (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. x 5) schedule was 30 mg . kg-1 . day-1. When the animals were pretreated with 0.1 mg . kg-1 . day-1 of dCF, the LD10 dose was reduced to 10 mg . kg-1 . day-1 x 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the LD10 dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 mumoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 mumoles/g of cells when 8-azaAdo was used in combination with dCF (1 microgram/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.

Heterogeneity of cancer cells from a single human colon carcinoma.

The human colon carcinoma cell line DLD-1, established from tumor tissue obtained from a 45 year old white man with an adenocarcinoma of the sigmoid colon, was studied from the perspective of tumor heterogeneity. The karyotype and morphology of cells from an early passage DLD-1 culture, as well as the histologic features of both the original tumor and neoplasms produced by inoculation of athymic nude mice with DLD-1 cells, indicated that both the DLD-1 cell line and the original tumor were heterogeneous. Two clones were isolated from the DLD-1 line; they differed in their morphology, karyotype, and cloning efficiency in soft agar. Furthermore, when cells from each clone were injected into athymic mice, histologically distinct tumors were produced. Various analyses showed that the two cloned lines were representative of the two subpopulations predominantly responsible for the heterogeneity of the original neoplasm. In vitro drug screening results demonstrated that the two cloned lines have differential sensitivities to chemotherapeutic agents. The parent DLD-1 human colon carcinoma cell line and its two cloned subpopulations provide material for the study of various aspects and implications of human cancer cell heterogeneity.