A phase II study of oral eniluracil/fluorouracil in patients with anthracycline-refractory or anthracycline- and taxane-refractory advanced breast cancer.

BACKGROUND: Eniluracil is an effective inactivator of dihydropyrimidine dehydrogenase, the initial and rate limiting enzyme in the catabolism of fluorouracil. The current study was done to determine the objective tumour response of a 28-day oral regimen of eniluracil-fluorouracil in patients with advanced breast cancer. PATIENTS AND METHODS: This was a multicentre, phase II study in patients with anthracycline-refractory (AR) or anthracycline- and taxane-refractory (ATR) advanced breast cancer. Oral eniluracil (10 mg/m2) and fluorouracil (1 mg/m2) were taken twice daily for 28 days of each 35-day treatment course. RESULTS: In this study, 106 patients received treatment: 62 patients in the AR stratum and 44 patients in the ATR stratum. The objective tumour response rate in the intent-to-treat population was 18% (95% confidence intervals (CI): 11%-27%), including three complete responses. The response rate was similar in both strata: 19% in the AR and 16% in the ATR stratum. The overall median duration of response was 23.6 weeks. The treatment was well tolerated with a low incidence of grade 3 or 4 toxicities that were considered attributable to study medication. CONCLUSION: Treatment with oral eniluracil-fluorouracil was well tolerated by patients with advanced breast cancer. The efficacy data were comparable with those of other published studies in this group of refractory patients.

Low-dose oral fluorouracil with eniluracil as first-line chemotherapy against advanced breast cancer: a phase II study.

PURPOSE: Eniluracil (776C85) is an effective inactivator of dihydropyrimidine dehydrogenase that allows continuous low-dose oral fluorouracil (5-FU) to be given with predicable oral bioavailability. We have assessed this as first-line oral chemotherapy for patients with advanced/metastatic breast cancer. PATIENTS AND METHODS: Patients with histologically proven, locally advanced or metastatic breast cancer without previous chemotherapy for advanced disease were entered onto this open-label phase II study. Patients received oral 5-FU 1.0 mg/m(2) with eniluracil 10 mg/m(2), both given twice daily for the first 28 days of each 35-day cycle, continuing until disease progression or unmanageable toxicity. RESULTS: Thirty-three patients were entered, with a median age of 53 years. Sixteen partial responses were seen in twenty-nine assessable patients (55%; 95% confidence interval, 37% to 73%), including responses in four (40%) out of 10 patients who had received prior adjuvant 5-FU. Seven patients had stable disease for at least 3 months with symptom improvement. Median response duration was 14 months (range, 10 to 18+ months). Toxicity was low. There were only two episodes of drug-related grade 3 nonhematologic toxicity (diarrhea and infection), and only 6%, 3%, and 3% of patients developed granulocytopenia, thrombocytopenia, and neutropenic sepsis, respectively. Mild (grade 1/2) diarrhea occurred in 39% of patients, hand-foot syndrome in 15%, nausea in 27%, and mucositis in 18%. Toxicity-associated delay and dose reduction occurred in only 2% and 5% of courses, respectively. CONCLUSION: First-line treatment with the combination of oral 5-FU and eniluracil has high activity in patients with advanced breast cancer comparable with the most active conventional cytotoxic agents but with strikingly less toxicity.

Abnormalities of the RB1 and DCC tumor suppressor genes: uncommon in human pancreatic adenocarcinoma.

Evidence of RB1 allele loss was found in only 6% of pancreatic cancers, and we found no significant sequence abnormalities nor loss of RB protein expression in a panel of tumors and cell lines. Using reverse transcription-polymerase chain reaction and Southern blot analysis, we found no evidence for loss of DCC expression in pancreatic cancer cell lines, and allele loss only rarely in tumor biopsies. These findings suggest that abnormalities of RB1 and DCC are unlikely to play a major role in pancreatic carcinogenesis.

Antisense oligonucleotides directed against p53 have antiproliferative effects unrelated to effects on p53 expression.

Antisense oligonucleotides targeting p53 have been hailed as a potentially new technique for treating patients with cancer, and there have been encouraging reports of good patient tolerance in vivo and of antiproliferative effects in vitro. However, evidence is lacking that these oligonucleotides are acting via an antisense interaction to modulate p53 expression. We examined a phosphorothioate antisense oligonucleotide, directed against exon 10 of the TP53 gene, and a chimaeric phosphorothioate-phosphodiester oligonucleotide directed against the p53 translation initiation codon. Both failed to specifically suppress p53 protein production in a cell-free assay system or to have any effect on mutant p53 expression by human pancreatic cancer cell lines. Antiproliferative effects were apparent, especially with the phosphorothioate antisense oligonucleotide, but this was independent of the p53 status of the cells (mutant, wild-type or absent) and also occurred with the control (sense and randomised) oligonucleotides. The most dramatic antiproliferative effects were seen with the 'control' phosphorothioate oligonucleotides. These findings suggest that the antiproliferative effects of some antisense oligonucleotides may be unrelated to expression of the gene they have been designed to target.

Loss of the retinoblastoma susceptibility gene (RB1) is a frequent and early event in prostatic tumorigenesis.

Loss of the RB1 gene is an important event in the initiation and progression of many tumours. Prostate tissue from 43 patients with prostate cancers and ten with benign prostatic hypertrophy (BPH) were studied for loss of heterozygosity of the RB1 gene. Four intragenic polymorphic loci were studied with two techniques. These were restriction fragment length polymorphism (RFLP). Southern blotting and hybridisation with the p123m1.8 and p68RS2.0 probes (to introns 1 and 17 respectively) and also the polymerase chain reaction (PCR) to amplify loci within introns 17 and 20. Protein product (pRB) expression was determined by immunohistochemistry using the NCL-RB antibody in nine patients with cancer and four patients with BPH. Loss of heterozygosity was found in 24 out of 40 (60%) informative patients with cancer. Loss of RB1 occurred with a similar frequency in early-stage and low-grade cancers as in more advanced cancers. Loss of RB1 was also found in one patient with BPH. Expression of pRB was completely absent from seven cancers and markedly reduced in the other two, while nuclear pRB staining was always present in areas of BPH, whether alongside cancer-containing tissue or with BPH alone. We conclude that loss of RB1 is an early event in prostatic tumorigenesis.

Retroviral transduction of Philadelphia-positive chronic myeloid leukemia cells with a human mutant p53 cDNA and its effect on in vitro proliferation.

Alterations in the tumor suppressor gene p53 are associated with the pathogenesis of blastic transformation of chronic myeloid leukemia (CML), but their exact role, particularly their relationship with the chimeric protein p210BCR/ABL, is poorly defined. Point mutations in p53 have been found in some cases of blast crisis and CML blastic cell lines, but it is not clear whether complete inactivation of p53 tumor suppressor function, with or without the production of a mutant protein, can by itself trigger the process of blastic transformation. By using retroviral gene transfer, we showed that the introduction of a mutant human p53 cDNA into hematopoietic progenitor cells from patients with CML in chronic phase, which already contain p210BCR/ABL, could promote their proliferation in vitro, and occasionally even lead to the growth of factor-independent colonies. We conclude that a mutant p53 may act in synergy with p210BCR/ABL and promote the survival and proliferation of CML hematopoietic stem and progenitor cells in vitro.

Direct growth stimulation of normal human epithelial cells by mutant p53.

We developed a high-titer amphotropic retroviral vector that expresses mutant (Ala143) human p53 to test directly the response of genetically normal human epithelial cells to p53 mutation. Contrary to our prediction, we found that in pancreatic epithelium (whose tumors display a high frequency of p53 mutation) but not in thyroid (whose tumors show an exceptionally low mutation frequency), expression of mutant p53 induced a dramatic, though self-limiting, proliferative response. This result questions the assumption that p53 mutation is relevant only to the later stages of tumorigenesis.

The epidermal growth factor receptor in human pancreatic cancer.

The epidermal growth factor receptor (EGFR) and its ligands are thought to be important in the control of proliferation of many epithelial systems, including the exocrine pancreas. Abnormalities in expression of two of the known ligands of the EGFR, transforming growth factor alpha and epidermal growth factor, occur frequently in ductal adenocarcinoma of the human pancreas. We have examined an archival series of cases of pancreatic pathology for expression of the EGFR using the anti-EGFR antiserum 12E and found that there is almost ubiquitous overexpression of EGFR in pancreatic cancer and in chronic pancreatitis. Southern blot analysis showed no evidence of amplification or rearrangement of the EGFR gene. We conclude that an autocrine loop involving the EGFR system may be involved in the genesis of both neoplasia and reactive hyperplasia of pancreatic ductal epithelium.

Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer.

The tumour suppressor gene p53 has been found to be mutated or inactivated at high frequency in several common human tumours. We have examined a series of exocrine pancreatic carcinomas for over-expression of mutant forms of p53 by immunohistochemistry with a panel of specific antibodies. We found immunodetectable p53 in 13 of 22 (60%) frozen pancreatic cancers and seven of 13 pancreatic cell lines. One of the antibodies, CM1, recognises p53 in formalin-fixed, paraffin-embedded archival material and using this reagent we found immunodetectable p53 in 28 of 124 (23%) pancreatic cancers. We have successfully demonstrated the presence of point mutations by direct sequencing of genomic DNA extracted from archival tissue showing CM1 immunoreactivity. We conclude that p53 activation is an important event in human pancreatic tumorigenesis and that the CM1 antibody can detect a proportion of cases of overexpression of mutant p53 in archival pathological material.

Transforming growth factor alpha and epidermal growth factor in human pancreatic cancer.

Overexpression of the epidermal growth factor receptor (EGFR) has been reported as an important molecular abnormality in human pancreatic cancer. There is in vitro evidence that simultaneous overproduction of one of its ligands, transforming growth factor alpha (TGF-alpha), might result in an autocrine loop with an increased proliferation signal. We analysed by immunocytochemical staining a retrospective series of human pancreatic cancers, chronic pancreatitis, and normal fetal and adult pancreatic tissues for the presence of TGF-alpha and epidermal growth factor (EGF). Ductal epithelial cells showed TGF-alpha immunoreactivity in both normal tissue and chronic pancreatitis, and 95 per cent of tumours showed strong immunoreactivity. In contrast, EGF immunoreactivity was not found in normal pancreas, but was expressed in 12 per cent of pancreatic carcinomas. Well-defined areas of EGF immunoreactivity in exocrine ducts showing reactive changes in pancreatitis might represent a benign response to tissue damage similar to that previously described in the gastric mucosa.

Activating point mutations of the gsp oncogene in human thyroid adenomas.

The gene for the alpha polypeptide chain (alpha s) of the heterotrimeric G protein Gs can be activated to the putative oncogene gsp by specific point mutations at codons 201 and 227. Such mutations have been reported in 40% of human growth hormone-secreting pituitary adenomas and in a single autonomously functioning thyroid adenoma. We examined an archival series of 45 differentiated human thyroid tumors by polymerase chain reaction amplification and oligonucleotide hybridization to identify point mutations at each of the affected codons. Successful amplification was achieved in 38 cases, and activating mutations were identified in 5 of 13 (38%) autonomously functioning adenomas, but in none of 16 nonfunctioning adenomas, six papillary carcinomas, or three follicular carcinomas. Our results confirm that the gsp oncogene is involved in the pathogenesis of autonomously functioning tumors but do not support a role in other thyroid tumors.

The DNA content of Purkinje cells in mammals.

Nerve cells have generally been assumed to have a diploid DNA content, typical of non-dividing somatic cells. However several reports have suggested that certain nerve cells types, notably Purkinje cells of the cerebellum, are polyploid. Other studies have contradicted these findings, stating Purkinje cells to be diploid. In this paper we reinvestigate the DNA status of Purkinje cells, in a variety of mammalian species. Cell DNA content is measured on tissue smears by Feulgen microspectrophotometry. Results show that for all species examined by us, Purkinje cells have, without exception, a DNA content comparable to that of somatic cells. A critical appraisal of the techniques used in those studies claiming a tetraploid DNA content for Purkinje cells leads us to believe our findings to be correct.

Post-mortem changes in human central nervous tissue and the effects on quantitation of nucleic acids and enzymes.

A study of post-mortem changes in human central nervous tissue has shown that within 100 h of death, no significant change occurs in the amount of nerve cell DNA and nucleolar RNA nor in some membrane-associated enzymes such as succinate dehydrogenase, NADH and NADPH diaphorase, and cytochrome oxidase. Low molecular weight RNA species, probably transfer and messenger RNA are quickly lost, but there is little alteration in ribosomal RNA content. Cytoplasmic enzymes show variable changes; phosphofructokinase activity is rapidly decreased; hexokinase is unaltered but lactate dehydrogenase, pyruvate kinase and glucose-6-phosphate dehydrogenase initially show increases in activity which subsequently decline. Oxygen uptake diminishes quickly. These findings indicate that mechanical alterations in cell structure, following death, render organelles physiologically ineffective long before any significant changes in certain constituent biochemicals are detected. This report emphasizes the great importance necessary in the selection of appropriately time matched post-mortem tissues if accurate comparative studies of many of the cells constituents are to be made.

Cytophotometric mapping of neuronal changes in senile dementia.

Results of a cytophotometric study have shown a widespread reduction in cytoplasmic RNA of nerve cells. It appears, therefore, that although certain aspects of the symptomatology of senile dementia may be accounted for by lesions in particular anatomical sites, the main part of the neurological disturbance is related to more broadly based changes in nerve cell metabolism affecting much, if not all, of the CNS.

Neuromelanin and RNA in cells of substantia nigra.

We have found that with accumulation of neuromelanin granules within cell bodies of neurones of the human substantia nigra there is a reduction in cytoplasmic RNA and a decrease in nucleolar volume. These observations imply a gradual decrease in the functional capacity of the cell such that eventually, the cell is unable to produce sufficient protein to maintain its metabolic economy with atrophy and death ensuing. This reduction in protein synthesis may result from the mechanical displacement and disruption of the endoplasmic reticulum by the accumulated pigment granules.

Variations in melanin content with age in the human substantia nigra.

Evidence is presented here that the decrease in mean melanin content we have measured in old age is due, not to a general decline in melanin in all cells, but rather to a selective loss of those nerve cells containing greatest amounts of pigment. This cell loss is likely to be caused by the physical affects of pigment accumulation, per se, upon capacity for protein synthesis, as marked by losses of cytoplasmic RNA and nucleolar shrinkage in melanin rich cells.