The molecular epidemiology of p53 gene mutations in human breast cancer.

The P53 tumor-suppressor gene is an advantageous tool for analyzing the molecular epidemiology of cancer. We describe the utility of the P53 gene as a 'mutagen test' and a prognostic indicator in breast cancer. Aspects of study design and methodology are discussed. Two major conclusions emerge: (1) there is an extraordinary diversity of mutational patterns among cohorts, hinting that the unique biology of mammary cells results in exposure to high doses of a diversity of ingested lipophilic mutagens; and (2) mutations in the P53 gene predict poor outcome in breast cancer.

Novel pattern of P53 mutation in breast cancers from Austrian women.

Since mutagens produce an extraordinary diversity of mutational patterns, differential mutational exposures among populations are expected to produce different patterns of mutation. Classical epidemiological methods have been successful in implicating specific mutagens in cancers such as those of lung and skin in which one mutagen predominates. In breast cancer, however, no mutagens have been implicated in an unequivocal manner. In an attempt to facilitate epidemiological studies, we have been studying the pattern of p53 gene mutations in breast cancers from multiple populations with high and low breast cancer incidences. We previously reported that breast cancers from Midwest United States, predominantly rural Caucasian women, have a different pattern of p53 gene mutation from populations of Western European women. Herein, we analyze patterns of p53 mutations from Graz, Austria, another population with a high incidence of breast cancer. Among the 60 Austrian breast cancers analyzed, 14 (23%) have a p53 gene mutation in exons 5-9 or in adjacent splice junctions. Analysis of the patterns of mutation shows differences between the "Western European" profile and the Austrian and Midwest United States groups (P = 0.027 and 0.024, respectively). The Austrian pattern is characterized by a high frequency of A:T-->T:A transversions (P = 0.006). The presence of distinct patterns of mutation among the limited number of analyzed populations of Western European origin supports the idea that differential mutagenic exposure and/or genetic differences contribute to breast cancer mutagenesis among geographically distinct Caucasians of Western European origin.

Levamisole potentiation of fluorouracil antiproliferative activity mimicked by orthovanadate, an inhibitor of tyrosine phosphatase.

BACKGROUND: Levamisole is an effective antihelminthic drug with immunomodulatory and anticancer activities in model systems. Combined with fluorouracil (5-FU) as adjuvant treatment following resection of Dukes' stage C colon carcinomas, levamisole significantly reduces mortality. However, neither 5-FU nor levamisole alone has a significant effect on survival in this patient group. Previously, we noted that in vitro levamisole potentiated the antiproliferative activity of 5-FU. PURPOSE: Because levamisole is known to inhibit alkaline phosphatases and has been reported to inhibit dephosphorylation of some membrane phosphoproteins, we studied the effects of levamisole analogues and of chemically unrelated inhibitors of phosphatases for their ability to potentiate 5-FU inhibition of tumor cell line proliferation in vitro. METHODS: Human cancer cell lines were exposed to drugs alone or in combination with 5-FU. Antiproliferative activity was measured by determining the extent of reduction of colony formation by the cell lines in test plates compared with control plates. RESULTS: We found that potentiation of 5-FU cytotoxicity by levamisole and by p-hydroxytetramisole, a metabolite of levamisole, is mimicked by orthovanadate, an inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine and threonine phosphatases, Furthermore, l-p-bromotetramisole, a synthetic analogue of levamisole that is 10-fold more potent in inhibition of alkaline phosphatase than levamisole, potentiates the antiproliferative activity of 5-FU to a greater extent than d-p-bromotetramisole, a stereoisomer of l-p-bromotetramisole with little antiphosphatase activity. CONCLUSION: Inhibition of tyrosine phosphatases may be responsible for the potentiation by levamisole of the inhibitory activity of 5-FU in vitro. IMPLICATIONS: Inhibition of dephosphorylation of regulatory phosphoproteins may be related to the therapeutic efficacy of the combination of levamisole and 5-FU in the adjuvant treatment of colon carcinoma and may underlie at least some of the multiple effects of levamisole on immune parameters.

Pattern of p53 gene mutations in breast cancers of women of the midwestern United States.

BACKGROUND: Mutation in the p53 gene is the most common genetic lesion in human cancers. The pattern of mutation in the p53 gene differs among cancers and may be a useful epidemiological tool for identification of factors contributing to carcinogenesis. PURPOSE: Our purpose was to determine if the pattern of p53 mutation in breast carcinomas in our population of women residing in the midwestern region of the United States is similar to the pattern of p53 mutation in breast cancers in patients from other regions of the United States and Europe and in other epithelial tumors. METHODS: With a technique we recently developed for the analysis of p53 mutations in genomic DNA from tumor cell clusters in touch preparations of solid tumors, we sequenced exons 5-9 and adjacent splice junctions of the gene in 44 breast cancers. Cells from each tumor were also stained with three monoclonal antibodies which recognize different epitopes of the p53 protein. RESULTS: We detected p53 mutations in 14 (32.6%) of 44 breast carcinomas. Only half of the mutations were missense changes. The other half included five microdeletions (three producing frame-shifts), one single-base substitution generating a stop codon, and one single-base substitution generating a splice junction abnormality. Nuclear expression of p53 antigen was present in eight of 44 cancers, including six with hemizygous missense mutations in the p53 gene. CONCLUSIONS: The pattern of p53 mutations in our breast cancer population differs from that reported in breast cancer populations by other investigators in which most p53 mutations were missense. Among 14 mutations in our population, at least five drastically altered the structure of p53, suggesting that a recessive mechanism of inactivation of the p53 gene may be more common than in other populations. IMPLICATIONS: Differences in the pattern of p53 mutation in breast cancers in Midwestern women and in breast cancers in other populations may reflect selection bias or small sample sizes currently available. However, our data are compatible with the possibility that an endogenous or exogenous factor influences p53 carcinogenesis in some women with breast cancer in the Midwest to a greater extent than in other regions of the United States and Europe.

Direct sequencing from touch preparations of human carcinomas: analysis of p53 mutations in breast carcinomas.

A new technique for characterizing somatic mutations in very small samples of cellularly heterogeneous human cancer tissue was developed and tested using mutations in the p53 gene in breast carcinomas as a model system. The technique combines touch preparation of specimens to obtain homogeneous clusters of carcinoma cells free of normal cells with a nested pair of polymerase chain reaction (PCR) amplifications of DNA to increase the amount of target gene sequence sufficiently to permit direct sequencing of the p53 gene. Touch preparations of fresh or previously frozen tissue from human adenocarcinomas derived from several organs were stained, and clusters of 10-50 malignant cells were transferred by pipette into microfuge tubes for PCR amplification. Exons 5-9 of the p53 gene, which contain the major mutational hot spots associated with most human cancers, were sequenced by the following steps: 1) two rounds of PCR amplification using DNA Taq polymerase and two sets of oligonucleotide primers, the second set being nested within the segment amplified by the first set and having attached T7 and SP6 phage promoter sequences, 2) transcription of the amplified DNA sequences with T7 and SP6 RNA polymerases, and 3) dideoxy sequencing of single-stranded RNA transcripts with reverse transcriptase and with additional oligonucleotide primers to achieve specificity for this unique region of the genome. The utility of this approach is illustrated by our success in detecting and analyzing point mutations in cell clusters from four of 11 primary adenocarcinomas of the human breast.

Disposition of bisantrene in humans and rabbits: evidence for intravascular deposition of drug as a cause of phlebitis.

The investigational antitumor agent bisantrene (9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride) causes frequent local complications of phlebitis and thromboses in patients receiving the drug by peripheral venous infusion. Bisantrene pharmacokinetics was studied in five patients. Plasma elimination was biphasic with t1/2 alpha of 65 min and t1/2 beta of 1142 min; the mean apparent volumes of distribution of the central compartment and peripheral compartments were 185 and 1662 liters/sq m, suggesting extensive uptake, binding, or deposition of drug. Total body clearance was 735 ml/min/sq m, and 11.3% of drug was excreted in urine. One hr after bisantrene (260 mg/sq m) at 1 mg/ml in 5% dextrose was infused into the marginal ear vein of a rabbit, the vein was congested with blood and contained 2.1 mg precipitated bisantrene. After 24 hr, the vein was clotted and contained 1.18 mg precipitated drug. Precipitation of bisantrene appears to be related to the low solubility of the drug at physiological pH. Maximum solubility of bisantrene in human and rabbit serum was 12.7 micrograms/ml. Intravascular precipitation of bisantrene may be responsible for phlebitis and thromboses in humans receiving the drug by i.v. infusion.

Metabolic conversion of indicine N-oxide to indicine in rabbits and humans.

Indicine N-oxide, a pyrrolizidine alkaloid N-oxide that exhibits antitumor activity without some of the toxic effects associated with other pyrrolizidine alkaloids, is metabolized to indicine in rabbits and humans. Indicine can be detected in the plasma and is excreted in the urine in a dose-dependent manner following the i.v. administration of indicine N-oxide. The p.o. administration of indicine N-oxide leads to an increased plasma concentration and an increased urinary excretion of indicine. The hepatic microsomal fraction and the gut flora both catalyze the anaerobic reduction of indicine N-oxide to indicine in vitro. Whole-animal studies suggest that the gut flora play a major role in the metabolic reduction of indicine N-oxide by the rabbit. Indicine N-oxide is not actively excreted in the bile, and it is probable that indicine N-oxide finds its way into the gut by passive diffusion following i.v. administration. Neomycin and erythromycin, which reduce the number of anaerobic bacteria in the gut, lead to decreased plasma levels and a decreased urinary excretion of indicine.

Pharmacological characterization of teroxirone, a triepoxide antitumor agent, in rats, rabbits, and humans.

Teroxirone is an experimental triepoxide antitumor agent currently undergoing evaluation in clinical trials. We have developed an assay based on derivatization with diethyldithiocarbamate followed by normal-phase high-performance liquid chromatographic analysis. When 14C-labeled teroxirone is administered to rabbits by rapid i.v. infusion, plasma disappearance of parent drug is very rapid (t1/2 less than 5 min), while plasma 14C-labeled drug equivalents are eliminated at a much slower rate (t1/2 greater than 60 min). Twenty-four-hr urinary recovery of parent drug is less than 1%, while recovery of 14C total radioactivity is 60 to 70%. Rapid plasma elimination (t1/2 less than 5 min) and total body clearance (greater than 5 liters/min) are observed following rapid i.v. administration of teroxirone to humans. When teroxirone is administered to humans at constant rates of infusion, plateau concentrations are rapidly achieved and maintained during infusion. Plasma concentrations rapidly decrease upon cessation of infusion. Less than 1% parent drug is recovered in 24-hr urine. Teroxirone is relatively stable in fresh human plasma and whole blood. Teroxirone is metabolized by rat liver, but not lung, microsomal preparations by an NADPH-independent pathway. Epoxide hydrolysis metabolites are detected in microsomal incubations, and cyclohexene oxide inhibits teroxirone metabolism, suggesting that epoxide hydrase may be responsible for teroxirone biotransformation. Cytotoxicity of teroxirone against continuous human tumor cell lines is abolished in the presence of 9000 X g rat liver supernatant preparations but partially restored when cyclohexene oxide is added to incubation mixtures.

Disposition and metabolism of pentamethylmelamine and hexamethylmelamine in rabbits and humans.

The disposition and metabolism of pentamethylmelamine (PMM) and hexamethylmelamine (HMM) were studied in the rabbit, and the disposition of PMM was studied in humans. Parent compound and metabolites were identified by thin-layer chromatography, gas chromatography, and gas chromatography/mass spectrometry analyses. Plasma elimination in both species following i.v. administration of each drug was best described by a two-compartment open model. Both compounds were extensively demethylated with less than 1% of the total dose administered recovered in the urine over 24 hr. The areas under the plasma time-concentration curves of PMM and HMM following p.o. administration to rabbits were 5 and 25% of the areas following i.v. administration. Gastrointestinal absorption was rapid and efficient with 75 to 89% of drug equivalents recoverable in the urine after p.o. administration of [ring-14C]PMM or [ring-14C]HMM to rabbits. Reduced bioavailability of PMM and HMM p.o. appears to be a consequence of rapid metabolism presumably in the liver.

Phase I study of difluoromethylornithine in combination with recombinant alpha 2a-interferon.

24 patients with advanced, histologically proven cancer were treated with difluoromethylornithine 2.25 g/m2 orally every 6 h for the first 7 days of each 4-week treatment cycle. These patients also received daily i.m. doses of recombinant human alpha 2a-interferon (IFN) on Days 3 through 7 of each cycle. IFN doses of 3, 6, 12, 24, 36, and 48 X 10(6) units/m2 have been studied utilizing three patients at each daily dose level. Three additional patients have been observed at each of the two highest doses for better toxicity definition. This combination produced slight transient declines in leukocyte and platelet counts and transient rises in serum aspartate aminotransferase; however, these changes were no more pronounced at the higher IFN doses than at daily doses of 6 X 10(6) units/m2. Mild nausea and vomiting occurred in most patients and mild diarrhea also was common at all IFN dose levels. Chills, fever, myalgia, lethargy and fatigue, and anorexia were also observed at all IFN doses; however, lethargy and fatigue (lassitude) seemed to be the major factor which limited patient tolerance of IFN to 48 X 10(6) units/m2 daily. No ototoxicity was identified clinically or audiometrically and no life-threatening toxicity has occurred. Initial Phase II studies in melanoma are currently in progress.

Phase 1 trial and assay of rubidazone (NSC 164011) in patients with advanced solid tumors.

A new high-pressure liquid chromatographic method was developed for the simultaneous determination of rubidazone and daunorubicin in human plasma at concentrations as low as 60 ng/ml. Clinical toxicity and the stability of rubidazone were studied in nine patients with advanced solid tumors. Rubidazone was administered by i.v. infusion over 1 hr on a single day every 4 weeks. Moderate leukopenia was the dose-limiting toxicity in four of six patients treated at 150 mg sq/m. Assay of rubidazone in plasma samples obtained after administration of rubidazone showed that the drug was stable for at least 7 hr.

Phase I and clinical pharmacological evaluation of a parenteral hexamethylmelamine formulation.

Hexamethylmelamine has been evaluated in single agent and combination regimen studies for many years, but only following p.o. administration. Pharmacological studies in animals and humans have shown that systematic availability of parent drug following p.o. administration is relatively low and variable due to extensive first-pass metabolism rather than due to poor absorption. Two Phase I clinical trials, with accompanying pharmacokinetic studies, have been conducted by using a parenteral formulation in which hexamethylmelamine was prepared by Intralipid 10%. The parenteral formulation was well tolerated by all patients receiving hexamethylmelamine by 1-day and by daily for 5-days schedules. Nausea and vomiting were the dose-limiting toxicities. Maximally tolerated doses on the 1-day and daily for 5-days schedules were approximately 850 mg/m2 and 630 mg/m2/day, respectively. No responses were observed in either study. Following i.v. administration of 540 mg/m2 hexamethylmelamine, plasma elimination was best described by a three-compartment open model with terminal half-life, total body clearance, and steady-state volume of distribution values of 10.4 h, 0.75 liter/min/m2 and 460 liters/m2, respectively. Twenty-four h urinary recoveries of parent drug were less than 1% for all patients. Accumulation of hexamethylmelamine during the 5-day treatment at 945 mg/m2 suggested possible saturation of parent drug elimination at that dose. Phase II studies are currently under way with the parenteral formulation of hexamethylmelamine.

Phase I trial of parenteral 6-thioguanine given on 5 consecutive days.

For almost 30 years, 6-thioguanine (6-TG) has been administered p.o. for treatment of various human cancers, especially leukemias, even though the systemic availability of the drug given p.o. is known to be low and highly variable. Parenterally administered 6-TG has been studied in detail in humans only on a single-day intermittent schedule, although multiple-day intermittent schedules are known to produce maximal cytotoxic effects in several animal species. To develop a multiple-day regimen for parenteral 6-TG therapy, we carried out a dose-seeking and pharmacokinetic study of the drug given i.v. daily for 5 days in patients with various refractory advanced solid tumors. Dose-limiting myelosuppression without other significant toxicity occurred at 55-65 mg/m2 daily for 5 days. After i.v. administration at 65 mg/m2, the mean peak plasma concentration of 6-TG ranged from 6-10 microM. These concentrations are 8-300 times greater than peak plasma concentrations of 6-TG in plasma reported to occur after p.o. administration at 100 mg/m2. We suggest that the antitumor activity of 6-TG be reassessed against human cancers in regimens of i.v. administration on multiple-day intermittent schedules.

Expression of p53 and 17p allelic loss in colorectal carcinoma.

Mutations in the p53 gene are the most common genetic changes in cancer thus far. Many p53 mutations result in a protein product having a prolonged half-life compared to wild-type p53. The mutant protein is frequently detectable immunohistochemically, whereas the wild-type p53 present in normal cells is not. We examined 90 colorectal carcinomas for increased expression of p53 using 3 p53 specific monoclonal antibodies, PAb1801, PAb421, and PAb240. Overall, 70% of the colorectal carcinomas stained for p53. Each tumor's DNA was also assessed for loss of heterozygosity on chromosome 17p, the location of the p53 gene. Of those tumors that reacted with the anti-p53 antibodies, 76% showed loss on chromosome 17p. Tumors with loss of heterozygosity on 17p generally stained with all 3 antibodies, whereas those without loss tended to stain with just one antibody, typically PAb240. Fifteen tumors were examined for the presence of specific p53 mutations. A total of 10 mutations were found, 6 were missense and 2 were deletions, and all but one of the tumors with missense mutations stained for p53.

p53 gene mutations inside and outside of exons 5-8: the patterns differ in breast and other cancers.

Most studies of mutations in the p53 tumor suppressor gene in tumors have examined only exons 5-8. Our laboratory previously found 64 mutations in exons 5-8 of the p53 gene in 194 primary breast cancers. Herein, we report 18 additional mutations found outside of exons 5-8. Mutations are present in exons 4, 9 and 10, and flanking splice junctions, but not in the promotor region or in exons 1, 2, 3 and 11. No missense mutations are found outside of exons 5-8. Instead, there is a predominance of frameshift mutations with lesser numbers of nonsense and splice site mutations. In contrast, the majority of mutations in exons 5-8 in this sample are missense changes and all of these are at amino acids that are identical in the 11 known p53 sequences that represent about 1.6 billion years of evolutionary divergence. The difference in mutational pattern between these two regions of the p53 gene is due to a lack of missense mutations and inframe microdeletions outside of exons 5-8. A review of our database of p53 mutations (De Vries et al., in preparation) shows that the patterns of mutation inside and outside of exons 5-8 differ in other types of cancers as well. The paucity of missense mutations in exons 2-4 and 9-11 in breast and other cancers (even at amino acids identical throughout p53 gene evolution) suggest that at least some missense mutations result in a phenotype other than malignant transformation. These data also illustrate the importance of examining identical exons when comparing the pattern of p53 gene mutations in different populations.

Molecular epidemiology of breast cancers in northern and southern Japan: the frequency, clustering, and patterns of p53 gene mutations differ among these two low-risk populations.

Comparison of acquired mutations in the p53 tumor suppressor gene can illuminate factors contributing to carcinogenesis among cancer cohorts. Japan has an ethnically homogeneous population with a low incidence of breast cancer. Previously we reported an unusual frequency, allelic status, and clustering of mutations in breast cancers from the northern part of the main Japanese island. To extend these findings, exons 2-11 and adjacent intronic sequences were analysed in tumors of women from northern (Hokkaido) and southern (Tokushima) Japan. The frequency of breast cancers with p53 gene mutations in the Hokkaido group is the highest reported (81%) while that in Tokushima (28%) is similar to most other populations. Thirteen of the 19 mutations (68.4%) in the Hokkaido cohort were heterozygous, an unusually high frequency for p53 mutations in any tumor type. There were three missense mutations at codon 175, a known hotspot for alterations in the p53 gene, and three missense mutations at codon 179, a rare site for p53 changes. In addition, the patterns of p53 gene mutation differed between the two Japanese cohorts (P=0.04). The multiple differences in acquired p53 mutations suggest unsuspected biological differences among breast cancers in northern and southern Japan. In addition, the high frequency of p53 mutations in breast cancers from Hokkaido predicted a poorer prognosis for this population which was confirmed on examination of mortality data.

Pilot study of a continuous five-day intravenous infusion of etoposide concomitant with cisplatin in selected patients with advanced cancer.

In view of experimental and clinical data suggesting enhanced antiproliferative efficacy from a continuous infusion of etoposide (VP-16) concomitant with cisplatin (CDDP), we performed a dose-seeking study of a five-day infusion of VP-16, 30 mg/m2/24 h and CDDP, 18.5 to 25 mg/m2/24 h. Among eight patients with advanced solid tumors, dose-limiting toxicities were leukopenia and thrombocytopenia. There were no clinically significant renal, neurologic, or otologic sequelae. Nausea and vomiting were mild and transient. Although the trial was not designed as a phase II assessment, we observed three objective regressions among three patients with advanced gastric adenocarcinoma who had failed prior chemotherapy. The total CDDP dose is similar to five-day single agent studies, yet the total VP-16 dose is approximately 24% to 50% less than a five-day single agent VP-16 dose. The recommended regimen for continuous 120-hour infusion is VP-16, 30 mg/m2/24 h and CDDP, 20 mg/m2/24 h, repeated at monthly intervals.

Phase I study of recombinant leukocyte A human interferon combined with BCNU in selected patients with advanced cancer.

Interferons manifest diverse immunomodulatory and antiproliferative characteristics. Since their spectrum of toxicities includes primarily fatigue and anorexia rather than the myelosuppression concomitant with cytotoxic therapies, it is conceptually appealing to combine both modalities. We conducted a phase I trial among 18 patients using the combination of leukocyte A recombinant interferon (IFN-rA) and BCNU. The intramuscular (IM) IFN-rA dose for the initial 12 patients was 12 X 10(6) U/m2 three times a week for an anticipated duration of 12 weeks. Among these patients, we escalated the monthly intravenous (IV) BCNU dose from 50 mg/m2 to 150 mg/m2. Six subsequent patients received IFN-rA 12 X 10(6) U/m2, days 1 to 3, and BCNU 150 mg IV on day 3 of each monthly cycle. Dose-limiting toxicities from both regimens were fatigue and myelosuppression. We recognize the limitation of small sample sizes. Nevertheless, in the absence of a significant number of life-threatening toxicities, it appears that near maximal doses of BCNU and concomitant IFN-rA can be administered with safety in an outpatient setting.

Rapid and efficient screening for p53 gene mutations by dideoxy fingerprinting.

Dideoxy fingerprinting (ddF) is an efficient method for detecting single base and other sequence changes in PCR-amplified DNA segments. This screening method is a hybrid between single-strand conformation polymorphism analysis (SSCP) and Sanger dideoxy sequencing. It involves a Sanger sequencing reaction with one dideoxynucleotide followed by non-denaturing gel electrophoresis. We are using ddF to screen for mutations in the p53 tumor suppressor gene in primary breast cancers. ddF detected more than 100 mutations in different regions of the gene, including all types of single-base mutations and microdeletions/microinsertions of various sizes. Furthermore, ddF reliably detected heterozygous mutations, if the region of interest was screened in both directions. In a blinded, prospective study, ddF detected all 25 mutations within exons 4-10 and adjacent flanking intronic regions previously found by direct sequencing. ddF was also useful in scoring two common polymorphisms within the p53 gene. Guidelines for preventing false-positive and false-negative results are summarized.

Cancer immunotherapy: current status of treatment with interleukin 2 and lymphokine-activated killer cells.

In recent years, the medical community has witnessed a growing interest in the use of adoptive immunotherapy in patients with malignant lesions refractory to standard treatments. Systemic administration of interleukin 2, in combination with the adoptive transfer of a patient's own activated immune cells, has resulted in objective regression of several types of advanced cancers. Pronounced regression of tumor has also been observed with use of systemic interleukin 2 alone. This ability to augment the immune defense system of the host against cancer has stimulated intense clinical and laboratory investigations.