BACKGROUND:Verrucous carcinoma is distinguished by controversy regarding appropriate diagnosis and treatment. This study provides a contemporary survey of demographics, patterns of care, and outcome for this disease in the United States. METHODS:The National Cancer Data Base had 2350 cases of verrucous carcinoma of the head and neck diagnosed between 1985 and 1996. Statistical procedures included chi-square analyses, Student t tests, and relative survival. RESULTS:Tumors originated most frequently in the oral cavity (55.9%) and larynx (35.2%). Although most patients were male (60.0%), oral cavity tumors were more common among older females. The most prevalent treatment was surgery alone (69.7%), followed by surgery combined with irradiation (11.0%) and irradiation alone (10.3%). For oral cavity tumors, surgery alone was more common among early (85.8%) than advanced cases (56.9%); a larger proportion of advanced disease received radiation alone or surgery and irradiation combined. Most laryngeal tumors were treated with surgery (60.3% for early and 55.6% for advanced disease), but a higher proportion received radiation alone or surgery combined with radiation compared with oral cavity cases. Five-year relative survival was 77.9%. For localized disease, survival after surgery was 88.9% compared with 57.6% after irradiation. CONCLUSIONS:Demographic differences implicate different mechanisms of carcinogenesis for verrucous carcinoma arising in the oral cavity and the larynx. Although selection bias may account for the differences observed, patients receiving initial treatment with surgery had better survival than those treated with irradiation, especially for cases originating in the oral cavity.

Cyclopentenyl cytosine (CPEC) is cytotoxic to several tumor cell lines. CPEC inhibits CTP synthesis resulting in depletion of cytidylate pools. The aim of this study was to examine CPEC's cytotoxic and antitumor activity in vitro and in vivo against human colon carcinoma HT-29, and to relate its action on CTP synthesis. CPEC exhibits potent cytotoxicity in vitro to HT-29 cells with an LC50 (concentration that is lethal to the survival of 50% cell colonies) of 2.4 μM and 0.46 μM following 2 h and 24 h exposure, respectively. Incubation of cells with CPEC for 2 h resulted in a dose-dependent decrease in cytidylate pools. The in vivo antitumor activity of CPEC in athymic mice transplanted subcutaneously (s.c) with 3 million HT-29 cells was examined. Antitumor activity of CPEC was elucidated in early-staged tumor, wherein CPEC (1.5 mgkg, QD × 9 or 3 mgkg, QOD × 9) was administered intraperitoneally (i.p.) 24 h after tumor implantation and it resulted in a significant reduction in tumor weight to 48% of control. The effect of CPEC on established solid tumors in vivo was examined in athymic mice transplanted s.c. 14 days earlier with HT-29 cells and treated i.p. with 1.5 mgkg CPEC, QD × 5 for 4 courses, with a 10 day-interval between courses. This treatment resulted in a significant reduction in tumor weight (72%) in the treated group. HPLC analysis of HT-29 tumor obtained from mice after treatment with CPEC showed a depletion of the CTP concentration reaching a nadir at 8 h. In conclusion, the present studies demonstrate potent antitumor activity of CPEC against freshly transplanted and established human colon carcinoma which can be corroborated with the drug's biochemical actions.

Tiazofurin exhibits antitumor activity in murine and human tumor cells. In a recent phase I/II trial in patients with end-stage leukemia, tiazofurin showed good response; however, repeated treatment resulted in clinical resistance to the drug. To elucidate the mechanisms of resistance in human leukemic cells, two variants of human myelogenous leukemia K652 cells resistant to tiazofurin were developed by drug-selection pressure. Compared to a concentration producing 50% cell proliferation reduction that was 9.1 microM in sensitive cells, the resistant variants displayed concentrations producing 50% cell proliferation reductions of 12 and 16 mM. The activity of the target enzyme, IMP dehydrogenase, was not altered in the resistant cells. Studies on tiazofurin metabolism revealed that resistant variants formed < 10% of the active metabolite, thiazole-4-carboxamide adenine dinucleotide. This correlated with the activity of NAD pyrophosphorylase, the enzyme that synthesizes thiazole-4-carboxamide adenine dinucleotide, which was reduced to 10% in the resistant lines. Concurrently, the activity of thiazole-4-carboxamide adenine dinucleotide phosphodiesterase was elevated in the refractory cells. Compared to the sensitive counterpart, the levels of GMP and NAD were lower in the resistant lines. Guanine salvage activity was decreased in the resistant cells. Basal dGTP and dATP concentrations were elevated in the resistant line; nevertheless, tiazofurin incubation decreased dGTP levels in only the sensitive cells. Although there was no difference in the Km of tiazofurin transport or efflux, the Vmax of uptake of the drug was reduced in the resistant lines. Sensitive and resistant cells exhibit similar cytotoxicity to agents which do not share the mechanism of action of tiazofurin, suggesting that refractory cells are still sensitive to other standard antileukemic drugs.

Tiazofurin, 2‐β‐d‐ribofuranosylthiazole‐4‐carboxamide, is cytotoxic to murine and human tumor cells. In earlier Phase‐I/‐II trials performed in other centers in patients with solid tumors, the drug was given mainly as a 10‐min bolus or as a continuous i.v. infusion for 5 days. These protocols were associated with serious side effects, including neurotoxicity, pleuropericarditis, and occasional myelosuppression. In our study, 26 patients with end‐stage leukemia were treated with tiazofurin with I‐hr daily i.v. infusions, resulting in lower incidence and less severity of side effects. In this group, 7 attained complete remission and 7 showed hematologic responses. Out of 12 evaluable patients with myeloid blast crisis of chronic granulocytic leukemia, 10 (83%) responded to therapy, with 6 attaining complete response. We present pharmacokinetic parameters of our clinical study and examine some of the reasons for the lower toxicity found in our trials. In leukemic patients during and after infusion at doses of 1,100, 2,200 and 3,300 mg/m 2 tiazofurin peak plasma concentrations were 245, 441 and 736 μM, respectively, values one‐half of those calculated from other reports with a 10‐min bolus administration. In our I ‐hr infusion method, biphasic pharmacokinetics were noted with αt 1/2 and βt 1/2 of 0.5 and 6.2 hr, and tiazofurin was eliminated at a faster rate than in previous trials with continuous infusion. The area under the curve with our I ‐hr infusion was 52% of that reported for the same dose given by continuous infusion. Our I ‐hr infusion method and prompt and effective treatment of side effects enabled us to administer higher doses and larger total amounts of tiazofurin in longer treatment cycles than in any previous trials elsewhere. Tiazofurin therapy using I ‐hr infusion may be feasible for other carefully selected types of malignancies.

Tiazofurin is effective in treating end-stage leukemic patients (Tricot et al., Cancer Res 49:3696-3701, 1989). In sensitive tumors, the active metabolite of tiazofurin, TAD, potently inhibits IMP dehydrogenase activity, resulting in reduced guanylate pools. To elucidate tiazofurin activity in human solid tumors, we examined its activity in human colon carcinoma HT-29. Tiazofurin exhibited an LC50 of 35 microM in cultured HT-29 cells. Incubation of HT-29 cells with 100 microM tiazofurin for 2 h resulted in TAD formation (9.3 nmol/g cells) and in a 64% decrease in GTP pools. For biochemical and chemotherapy studies, athymic nude mice were transplanted s.c. with HT-29 cells. Twenty-four days later, mice were injected i.p. with tiazofurin (500 mg/kg); 6 h later, tumors were removed and analyzed. These tumors formed 17 nmol/g of TAD with decreased GTP pools (56%). To study oncolytic activity, transplanted mice were treated 24 h later with tiazofurin (500 mg/kg, once a day for 10 days). To examine the effectiveness of tiazofurin in established tumors, the drug was administered to mice 14 days after tumor implantation (500 mg/kg, once a day for 5 days, course repeated 4 times with a 10-day rest). Both treatment schedules resulted in significant antitumor activity. This study illustrates the potential usefulness of tiazofurin in treating human colon carcinoma.

Tiazofurin is an oncolytic nucleoside analog that has shown therapeutic activity in end-stage acute non-lymphocytic leukemia and in chronic granulocytic leukemia in blast crisis. Tiazofurin is anabolized to the active metabolite, TAD, which inhibits IMP dehydrogenase activity, leading to a reduction in guanylate pools and to the cessation of neoplastic cell proliferation. The drug exhibits potent cytostatic and cytotoxic activity against hepatoma 3924A cells in culture. In growth-inhibition and clonogenic assays, the 50% inhibitory concentration of tiazofurin was 3.8 and 4.2 µm, respectively. Dipyridamole, an inhibitor of nucleoside transport, curtails the salvage of nucleosides and bases for nucleotide biosynthesis. Dipyridamole exhibited cytotoxicity against hepatoma 3924A cells, with an LC50 of 24 µm and an IC50 of 29 µm being recorded. A combination of tiazofurin and dipyridamole provided synergistic cytotoxicity in hepatoma 3924A cells in culture. This synergistic activity was dependent on the order of addition of the drugs. Simultaneous addition of the two drugs produced antagonism, whereas preincubation of cells with tiazofurin or dipyridamole followed by addition of the second drug resulted in synergy. TAD concentrations were significantly higher (129% and 135%) in cells that had been pretreated with tiazofurin or dipyridamole before the addition of the second agent as compared with cells that had been treated simultaneously (113%). These studies indicate the importance of the order of the addition of drugs to obtain a synergistic response in combination chemotherapy and suggest the need for a careful selection of drug modulation in clinical trials of tiazofurin and dipyridamole.

An overview was presented of our approach of inhibition of de novo and salvage pathways in pyrimidine and purine metabolism. 1.1. Combination of acivicin, an inhibitor of de novo biosynthesis, and dipyridamole, a transport inhibitor, provided synergistic cytotoxicity in hepatoma and colon carcinoma cells.2.2. AZT, a competitive inhibitor of the salvage enzyme, thymidine kinase, and 5-FU or MTX provided synergistic cytotoxicity in hepatoma 3924A. In human colon carcinoma HT-29 cells AZT and methotrexate yielded synergistic cytotoxicity and thymidine and hypoxanthine together provided protection from the action of these drugs.3.3. These observations are significant because in rat hepatoma 3924A and in human cell lines HT-29, HL-60 and K562 thymidine kinase activity was 16- to 67-fold higher than that of dTMP synthase. Therefore, inhibition of dTMP synthase activity alone may provide poor responses because the salvage pathways can circumvent this block.4.4. In leukemic patients treated with tiazofurin, an inhibitor of IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis, and with allopurinol, which inhibits GPRT activity through raising plasma hypoxanthine levels, synergistic therapeutic results were obtained. The responses in sensitive patients entailed a decrease in IMP dehydrogenase activity and GTP concentration in leukemic cells and down-regulation of the ras and myc oncogenes. The down-regulation of the ras oncogene by tiazofurin through the decrease of GTP concentration has now been shown in K562, HL-60 and hepatoma cells and in patients with chronic granulocytic leukemia in blast crisis. Tiazofurin may be useful in studies on selective depression of the expression of the ras oncogene.5.5. In 27 consecutive patients 50% responded positively to tiazofurin treatment. From this group, 10 out of 12 patients (83%) with chronic granulocytic leukemia in blast crisis responded to tiazofurin treatment.

2-β-D-Arabinofuranosylthiazole-4-carboxamide and 2-β-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H. N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc ( the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of 15 pmol and linearity up to 3 nmol. The coefficient of variation was 0.6 +/- 0.1% for retention time and 2 +/- 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m2 (12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 microM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m2 (21.1 mM) were 42.8, 26.1, and 1.4 microM respectively.

New light was thrown on the action of tiazofurin in the treatment of end-stage leukemic patients and in leukemic cells in tissue culture. 1. In a population of 21 consecutive patients 50% responded to tiazofurin treatment, confirming the usefulness of this therapy in end-stage leukemia. 2. In leukemic patients treated with tiazofurin and allopurinol reciprocal action was manifested in the increase in hypoxanthine and the decrease in uric acid concentrations in the plasma. On discontinuation of allopurinol, hypoxanthine levels steeply declined but uric acid concentration increased slowly, taking days to reach pretreatment level. 3. With a new and sensitive method the concentration of the active metabolite of tiazofurin, TAD, was measured in the mononuclear cells of tiazofurin-treated patients. Approximately 5 to 13% of the plasma tiazofurin level was observed as TAD in the mononuclear cells. This TAD concentration was sufficient to account for the inhibition of IMP DH in these cells. 4. Tiazofurin or retinoic acid caused differentiation of HL-60 leukemic cells and inhibition of cell proliferation. 5. By treating leukemic cells incubated with tiazofurin or retinoic acid also with guanosine it was elucidated that the mechanism of the two drugs differed since only the tiazofurin effects were counteracted by guanosine. 6. Tiazofurin and retinoic acid together in HL-60 cells provided synergistic impact on differentiation and cytotoxicity. 7. Tiazofurin resulted in down-regulation of the expression of ras and myc oncogenes in three systems: K562 human erythroleukemic cells, rat hepatoma 3924A cells and human HL-60 leukemia cells. 8. Because both tiazofurin and retinoic acid are licensed drugs, their potential use in combination chemotherapy may have clinical relevance in the treatment of end-stage leukemia where our earlier studies have demonstrated the usefulness of tiazofurin.

The impact of tiazofurin on inhibition of IMP dehydrogenase was discussed at the clinical and molecular levels. 1. Evidence was provided for the role of IMP dehydrogenase and guanylates in the expression of the neoplastic program in cancer cells with particular relevance to human leukemic cells. 2. The argument for expecting an impact of tiazofurin in human myelocytic cells was provided. 3. Similarity of the kinetics of human leukemic cell IMP dehydrogenase to the rat hepatoma enzyme was documented. 4. New evidence was provided for the role of salvage in chemotherapy and the function of hypoxanthine in inhibiting guanine salvage. 5. The action of tiazofurin and retinoic acid was reported in HL-60 leukemic cells. 6. The effect of tiazofurin and retinoic acid on proliferation and cytotoxicity was outlined for hepatoma 3924A cells. 7. The effect of guanine on induced differentiation by tiazofurin and retinoic acid was examined. 8. Biochemical basis was provided for the lack of development of resistance in patients treated with tiazofurin. 9. Presumptive evidence was provided that tiazofurin treatment induced differentiation of leukemic cells in the patients. 10. The molecular biology of tiazofurin-induced differentiation in K-562 cells was reviewed with the possible relevance to clinical treatment that tiazofurin might also act through down-regulation of ras oncogene.