A clinical study assessing the tolerability and biological effects of infliximab, a TNF-alpha inhibitor, in patients with advanced cancer.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective. PATIENTS AND METHODS: Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = 20) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, IL-6 and C-reactive protein (CRP). RESULTS: Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient. CONCLUSIONS: Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.

A consensus DNA-binding site for the androgen receptor.

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5'-GGA/TACANNNTGTTCT-3', similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +1 position in the spacer and for A and T nucleotides in the 3'-flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonucleotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element.

Identification of residues in the estrogen receptor that confer differential sensitivity to estrogen and hydroxytamoxifen.

We have generated mutant mouse estrogen receptors which differ in their sensitivity to estrogen and the antiestrogen 4-hydroxytamoxifen. Mutation of the glycine at position 525 and the methionine and/or serine at positions 521/522 virtually abolishes the ability of the receptor to bind estradiol and stimulate transcription. In contrast, the mutant receptors retain the partial agonist activity exhibited by the wild-type receptor in the presence of 4-hydroxytamoxifen. The mutations do not affect the expression and DNA-binding activity of the receptor, but do abolish the estrogen-induced increase in the mobility of the receptor-DNA complex observed with the wild-type receptor. Other mutant receptors that were able to bind and stimulate transcription in the presence of estradiol also failed to show the agonist-induced increase in the mobility of the receptor-DNA complex, suggesting that it is unlikely to reflect the formation of a hormone-dependent transcriptional activation function.

Environmentally persistent alkylphenolic compounds are estrogenic.

We show that a number of alkylphenolic compounds, used in a variety of commercial products and found in river water, are estrogenic in fish, birds, and mammals. 4-Octylphenol (OP), 4-nonylphenol, 4-nonylphenoxycarboxylic acid, and 4-nonylphenoldiethoxylate were each capable of stimulating vitellogenin gene expression in trout hepatocytes, gene transcription in transfected cells, and the growth of breast cancer cell lines. The most potent of the chemicals is OP, which was able to stimulate these biological responses to a similar extent as 17 beta-estradiol itself, albeit at a 1000-fold greater concentration. The action of alkylphenols is mediated by the estrogen receptor, as their effects depended on its presence and was blocked by estrogen antagonists. OP, 4-nonylphenol, and 4-nonylphenoxycarboxylic acid appear to possess intrinsic estrogenic activity, because they compete for binding to the estrogen receptor. Moreover, it is likely that they interact with a similar region of the hormone-binding domain as 17 beta-estradiol, because the mutant receptor G-525R, which is defective in estrogen binding, is also insensitive to OP. Like 17 beta-estradiol, OP is capable of stimulating the activity of both transcriptional activation functions, TAF-1 and TAF-2, in the receptor, as judged by analyzing the activity of the wild-type and mutant receptors in transiently transfected cells. The significance of our results will depend to a large extent on the degree of exposure of wildlife and humans to these estrogenic alkylphenolic compounds.

Effect of ligand binding and DNA binding on the structure of the mouse oestrogen receptor.

We have investigated the effects of ligand and DNA binding on the structure of the oestrogen receptor by performing limited proteolysis and analysing DNA binding activity by gel shift analysis. The effects of oestradiol, 4-hydroxytamoxifen and ICI 164,384 have been examined and we have found that despite differences in the DNA binding activity or relative mobility of the receptor-DNA complex we were unable to detect differences in the cleavage pattern produced by trypsin, chymotrypsin, Staphylococcus aureus V8, papain or elastase. Inhibition of DNA binding by ICI 164,384 was lost in receptor fragments that lacked the hormone binding domain. In contrast to the full-length receptor, proteolytic fragments produced by chymotrypsin differed in their ability to bind to an oestrogen response element (ERE) vs a thyroid response element (TRE). Evidence is presented that this difference can be accounted for by the inability of fragments lacking the hormone binding domain to dimerise on a TRE.

Identification of phosphorylation sites in the mouse oestrogen receptor.

Phosphorylation sites in the mouse oestrogen receptor, expressed in COS-1 cells in the presence of 17 beta-oestradiol, have been mapped by solid phase microsequencing. The receptor was first radio-labelled with [32P]orthophosphate and a number of 3H- or 14C-labelled amino acids, immunopurified and then tryptic peptides were separated by thin layer chromatography or high performance liquid chromatography. Amino acid sequence analysis indicated that Ser-122, Ser-156, Ser-158 and Ser-298 were phosphorylated. The substitution of Ser-122 and Ser-298 with alanine had a negligible effect on the transcriptional activity of the receptor in transfected cells. However, a reduction of transcriptional activity was observed when Ser-122 was mutated in the context of mutations in a putative amphipathic alpha-helix involved in AF-2 activity. Thus a region of AF-1 that encompasses Ser-122 appears to interact with AF-2 in the full-length receptor.

Sex-hormone-binding globulin and breast cancer risk.

We have measured serum concentrations of SHBG in 5000 women over the age of 35. In both premenopausal and postmenopausal women who were not suffering from cancer or other diseases or taking drugs likely to affect SHBG, the protein decreased with increasing weight and was lower in single nulliparous women than in married nulliparous women or parous women. In premenopausal women, SHBG was higher in women with late menarche. These findings suggest that diminished SHBG concentrations may be associated with the increased risk for breast cancer conferred by nulliparity and early menarche.

Centrifugal ultrafiltration-dialysis for non-protein-bound oestradiol in blood: importance of the support.

The percentage of non-protein-bound oestradiol in serum or other fluid measured by centrifugal ultrafiltration-dialysis was critically dependent on the supporting medium used to collect the ultrafiltrate. When either Whatman No. 1 filter paper discs, Whatman GF/D glass fibre discs or Celite were used, the ratios of 3H-steroid to [14C]glucose on the support were found to change with centrifugation time. This occurred when serum or protein-free filtrates were analysed and was eliminated when silanised Celite was employed as the support. It was also observed that when serum was analysed, the [3H]/[14C] ratio inside the cell increased linearly with centrifugation time and this was ascribed to the small changes in volume which occurred inside the cell as ultrafiltrate accumulated on the support. It was concluded that the correct time to sample the ratio of [3H]/[14C] inside was before centrifugation.

Diurnal variations in prolactin and growth hormone levels in normal premenopausal vegetarian and omnivorous women.

Diurnal levels of plasma prolactin and growth hormone were measured in 47 normal premenopausal women, 26 of whom were established vegetarians and 21 of whom were omnivorous controls. There were no differences between the median prolactin levels of the two groups during a 24-hour period of investigation. When the groups were further subdivided into ovolactovegetarians and vegans, still no differences were found in prolactin secretion. In contrast, the vegetarian women exhibited elevation of growth hormone levels during the period of study, which was particularly marked at midnight and 10 A.M.

Urinary hydroxyproline and prognosis in human breast cancer.

The excretion of urinary hydroxyproline has been measured before mastectomy in 342 patients presenting with breast cancer for the first time to Guy's Hospital. The first 106 women were maintained on a gelatine-free diet whilst the remainder were on unrestricted diet. In both dietary groups hydroxyproline levels or the ratio of hydroxyproline to urinary creatinine were not related to pathological stage or histological grade. The time between initial presentation and subsequent bone metastases was negatively and significantly associated with hydroxyproline excretion (P less than 0.05) and the ratio of hydroxyproline to creatinine (P less than 0.01) in women on a gelatine-free diet. A similar, but not significant, trend was observed in patients on unrestricted diet. Although hydroxyproline excretion was related to the time to onset of bone metastases the amount of hydroxyproline excreted by these patients was not significantly different from patients who had recurrences at sites other than bone or patients who were disease-free up to 5 years after initial diagnosis. The conclusion is that hydroxyproline is of little value in the early detection of bone metastases.

The binding of blood-borne estrogens in normal vegetarian and omnivorous women and the risk of breast cancer.

Serial blood samples were taken at two-hour intervals over a 24-hour period from 25 premenopausal vegetarians (12 vegans and 13 ovolactovegetarians) and from 21 omnivorous controls. All members of the former group had been on a vegetarian diet for a minimum of three years. The mean proportion of estradiol unbound to blood proteins was similar in both vegetarians (1.26%) and meat eaters (1.16%). However, the amount bound to albumin was significantly raised in vegetarians (50.1% vs. 43.1%, p less than 0.009), whereas that bound to sex hormone-binding globulin (SHBG) was correspondingly lower (48.7% vs. 55.8%, p = 0.01). Mean levels of SHBG were similar in vegetarians (59.9 nmole/l) and omnivores (62.0 nmole/l), as was the total amount of free fatty acid (0.42 mmole/l for both). Within the vegetarian group, no differences were detected between vegans and ovolactovegetarians.

Binding of oestradiol to blood proteins and aetiology of breast cancer.

A prospective study of 5,000 women has shown that, compared to controls, those who subsequently developed breast cancer had a higher proportion of their blood oestradiol in the non-protein-bound and albumin-bound fractions (the bio-available fraction) and a lower proportion in the sex-hormone-bound fraction. The increased proportion of bio-available oestradiol was partly due to a lower concentration of sex-hormone-binding globulin. Weight was excluded as a confounding factor.

Effect of prednisolone on hormone profiles during primary endocrine treatment of advanced breast cancer.

Various plasma hormones were measured in 13 pre- and 20 post-menopausal women with advanced breast cancer before and for 12 months after ovarian irradiation or during continuous administration of tamoxifen at a dose of 10 mg twice a day, respectively; some patients received additional prednisolone at a dose of 5 mg twice a day. These patients were taken from a larger clinical trial which demonstrated a higher response to primary endocrine therapy when prednisolone was added. Levels of dehydropiandrosterone sulfate were depressed in patients receiving prednisolone, confirming adrenal suppression. Estradiol levels were reduced in all patients, while luteinizing hormone and follicular stimulating hormone increased after ovarian irradiation, but all three hormones fell during tamoxifen administration; no further changes were caused by the addition of prednisolone. Prolactin and thyroxine remained constant throughout the study. There were no differences between responders and nonresponders in hormone profiles or in changes in the profiles after treatment.