R. Charles Coombes

Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study

BACKGROUNDnBone-marrow micrometastases have been found in patients with primary breast cancer. We report long-term follow-up of women with primary breast cancer, diagnosed between 1981 and 1986, who had multiple aspirates taken at the time of initial surgery.nnnMETHODSn350 women with primary breast cancer were examined immunocytochemically with antibody to epithelial membrane antigen. We investigated associations with various prognostic factors as well as the effect of micrometastases on relapse-free survival and overall survival.nnnFINDINGSnAt median follow-up of 12.5 years, 151 patients had metastatic disease and 136 patients had died from breast cancer. 10-year relapse-free and overall survival were 43.9% (95% CI 33.4-54.7) and 44.9% (34.2-55.9) in patients with micrometastases, and 62.7% (56.5-68.6) and 65.7% (59.4-71.5) in patients without micrometastases at presentation (p<0.001). For relapse-free survival and overall survival, allowing for tumour size, lymph-node status, and vascular invasion, the effect of micrometastases decreased and was no longer significant, with a hazard ratio of 1.09 (0.74-1.61) for relapse-free survival and 1.21 (0.84-1.75) for overall survival.nnnINTERPRETATIONnThe presence of bone-marrow micrometastases in patients with primary breast cancer is associated with a shorter relapse-free survival and overall survival, but is not an independent prognostic factor. This immunocytochemical technique may be of value in patients for whom pathological tumour size and lymph-node status are unavailable (ie, patients receiving primary medical treatment).

POSSIBLE ROLE FOR VITAMIN D IN CONTROLLING BREAST CANCER CELL PROLIFERATION

By means of an immunocytochemical method the 1.25-dihydroxyvitamin D [1.25(OH)2D] receptor status of tumours from 136 patients with primary carcinoma of the breast was determined. Patients with receptor-positive tumours had significantly longer disease-free survival than those with receptor-negative tumours (Chi2 = 4.01, p less than 0.05). 1.25(OH)2D3 inhibits the proliferation of several established human breast cancer cell lines in vitro. Effects of 1.25(OH)2D3 on breast tumour growth in vitro were assessed by means of the nitrosomethylurea-induced rat mammary tumour model of hormone-responsive breast cancer. Treatment of tumour-bearing animals with 0.1 microgram of the synthetic analogue, 1 alpha-hydroxyvitamin D3, three times weekly produced significant inhibition of tumour progression. Taken together, these studies suggest that the levels of 1.25(OH)2D occurring in vivo may exert an inhibitory effect on receptor-positive tumours. Further studies are required to evaluate the role of vitamin D metabolites in the treatment of human malignant disease.

EB1089 : a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro

EB1089 is a novel vitamin D analogue which has been tested for its effects on breast cancer cell growth in vitro, using the established human breast cancer cell line MCF-7, and in vivo on the growth of established rat mammary tumours. Both EB1089 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited MCF-7 cell proliferation with the synthetic analogue being at least an order of magnitude more potent than the native hormone. In vivo anti-tumour effects were investigated using the N-methyl-nitrosourea-induced rat mammary tumour model. Oral treatment with EB1089 was tested at three doses. With the lower dose, significant inhibition of tumour growth was seen in the absence of a rise in serum calcium. The same dose of 1,25-(OH)2D3 had no effect on tumour growth but caused hypercalcaemia. With the higher dose of EB1089, striking tumour regression was seen although serum calcium rose. This report demonstrates that EB1089 possess enhanced anti-tumour activity coupled with reduced calcaemic effects relative to 1,25-(OH)2D3 and thus may have therapeutic potential as an anti-tumour agent.

Effects of synthetic vitamin D analogues on breast cancer cell proliferation in vivo and in vitro.

Calcipotriol (MC903) is a novel vitamin D analogue which effects cellular differentiation and proliferation in vitro and has reduced effects on calcium metabolism in vivo. In the present study its in vitro activity was evaluated using the MCF-7 breast cancer cell line, and its effects on calcium metabolism and mammary tumour growth were measured in vivo in adult female rats. Calcipotriol was compared to the natural metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3] and its synthetic analogue 1 alpha hydroxycholecalciferol [1 alpha(OH)D3]. Both calcipotriol and 1,25(OH)2D3 produced significant inhibition of MCF-7 cell proliferation at a concentration of 5 x 10(-11) M. Intraperitoneal administration of calcipotriol to normal female rats showed that the analogue was 100-200 times less active than 1,25(OH)2D3 in raising serum calcium concentration and urinary calcium excretion. Anti-tumour activity of the vitamin D analogues was investigated in vivo using the nitrosomethylurea-induced rat mammary tumor model. Rats, maintained on a low calcium diet, were treated with 1 alpha(OH)D3 (0.25 and 1.25 micrograms/kg). Both doses produced a response rate of 25% but hypercalcaemia developed. Treatment with calcipotriol (50 micrograms/kg) of rats maintained on a normal laboratory diet caused inhibition of tumour progression (response rate 17%) without the development of severe hypercalcaemia. This study supports the concept that vitamin D derivatives may inhibit breast cancer cell proliferation in vivo.

Phase I and pharmacokinetic study of D-limonene in patients with advanced cancer

Purpose: d-Limonene is a natural monoterpene with pronounced chemotherapeutic activity and minimal toxicity in preclinical studies. A phase I clinical trial to assess toxicity, the maximum tolerated dose (MTD) and pharmacokinetics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. Methods: A group of 32 patients with refractory solid tumors completed 99 courses of d-limonene 0.5 to 12u2009g/m2 per day administered orally in 21-day cycles. Pharmacokinetics were analyzed by liquid chromatography-mass spectrometry. Ten additional breast cancer patients received 15 cycles of d-limonene at 8u2009g/m2 per day. Intratumoral monoterpene levels were measured in two patients. Results: The MTD was 8u2009g/m2 per day; nausea, vomiting and diarrhea were dose limiting. One partial response in a breast cancer patient on 8u2009g/m2 per day was maintained for 11 months; three patients with colorectal carcinoma had prolonged stable disease. There were no responses in the phase II study. Peak plasma concentration (Cmax) for d-limonene ranged from 10.8u2009±u20096.7 to 20.5u2009±u200911.2u2009μM. Predominant circulating metabolites were perillic acid (Cmax 20.7u2009±u200913.2 to 71u2009±u200929.3u2009μMu200a), dihydroperillic acid (Cmax 16.6u2009±u20097.9 to 28.1u2009±u20093.1u2009μMu200a), limonene-1,2-diol (Cmax 10.1u2009±u20098 to 20.7u2009±u20098.6u2009μMu200a), uroterpenol (Cmax 14.3u2009±u20091.5 to 45.1u2009±u20091.8u2009μMu200a), and an isomer of perillic acid. Both isomers of perillic acid, and cis and trans isomers of dihydroperillic acid were in urine hydrolysates. Intratumoral levels of d-limonene and uroterpenol exceeded the corresponding plasma levels. Other metabolites were trace constituents in tissue. Conclusions:d-Limonene is well tolerated in cancer patients at doses which may have clinical activity. The favorable toxicity profile supports further clinical evaluation.

An important role for BRCA1 in breast cancer progression is indicated by its loss in a large proportion of non‐familial breast cancers

The presence of BRCA1 protein was determined immunohistochemically in normal and benign breast biopsies, non‐familial breast carcinomas and breast carcinomas from one or more individuals from 8 BRCA1 families. Strikingly, little staining was detected in breast carcinomas from BRCA1 families, regardless of the position or type of mutation, whereas strong immunostaining was observed in 28/28 of non‐malignant breast biopsies. Furthermore, BRCA1 staining was reduced in non‐familial breast carcinomas, since loss of nuclear BRCA1 staining was evident in 19% of non‐familial breast carcinomas whilst a similar proportion (20%) showed absence of either cytoplasmic or nuclear BRCA1 staining. Statistical analysis indicates that breast cancer is characterised by a reduction in levels of nuclear BRCA1 in familial (p < 0.001) and non‐familial breast cancer (p = 0.001). In non‐familial breast cancer absence of nuclear BRCA1, but not cytoplasmic BRCA1, is more common in high grade breast carcinomas (p = 0.03) and in patients with evidence of lymph node involvement (p = 0.05). Correlation between the absence of BRCA1 protein with high grade is consistent with previous findings of a correlation between mutations in the BRCA1 gene and high grade. Our findings provide new evidence in support of BRCA1 as a tumour suppressor protein in non‐familial breast cancer. Int. J. Cancer (Pred. Oncol.) 79:334–342, 1998.

A preliminary clinical study of gossypol in advanced human cancer.

SummaryA total of 34 patients with advanced cancer were given weekly or daily escalating doses of oral gossypol, a cottonseed-oil constituent showing evidence of antineoplastic activity in pre-clinical studies. No major adverse events occurred and there was no evidence of haematological or biochemical disturbance. As determined by dose escalation in 17 patients, the dose-limiting toxicity was emessis in 16 patients. There was no evidence of tumour regression in any of the 20 patients assessed for response. We conclude that gossypol is safe but unlikely to be clinically useful in patients with advanced cancer.

Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

BACKGROUNDnActivating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a liquid biopsy.nnnMETHODSnWe developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR).nnnRESULTSnNine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans.nnnCONCLUSIONSnEarly identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease.

Oral 4-hydroxyandrostenedione, a new endocrine treatment for disseminated breast cancer

SummaryThirty-one post-menopausal female patients, with locally advanced or disseminated breast cancer were treated with the aromatase inhibitor 4-hydroxyandrostenedione given orally at a dose of 500 mg daily. Twenty-nine patients had assessable disease. Eight patients (28%) had objective evidence of partial response and six remain in remission 7–10 months later. A further four patients (14%) had stabilisation of disease and 11 patients (37%) had progressive disease in spite of treatment. Plasma oestradiol levels were measured throughout therapy in 16 patients and were lowered to 53%±8% of baseline levels within 7 days of commencing 4-hydroxyandrostenedione.With regard to toxicity, one patient developed a transient skin rash and another patient some facial swelling. A further patient developed a transient leucopaenia and treatment was therefore discontinued. Twenty-seven of the 30 evaluable patients (90%) experienced no side effects. These results indicate that oral administration of 4-hydroxyandrostenedione is an acceptable new treatment for post-menopausal women with disseminated breast cancer.

Prognostic significance of aromatase and estrone sulfatase enzymes in human breast cancer

The aromatase and estrone sulfatase enzymes are important sources of local synthesis of biologically active estrogens in human breast cancer. Significant intratumoral aromatase activity was detected in 91/145 (63%) of tumors and estrone sulfatase was detected in 93/104 (89%) of tumors. There was no relationship between aromatase activity and tumor size, site, nodal status, menopausal status or estrogen receptor status. There was a significant correlation between the aromatase activity and histological grade, with an excess of aromatase-positive in the high grade tumors (P = 0.03). There was a marginally inverse correlation between the aromatase activity and time to relapse (P < 0.1), a significant correlation between aromatase activity and survival after relapse (P < 0.05) but not with overall survival (P > 0.1). Intratumoral estrone sulfatase activity was not significantly correlated to any putative prognostic factors, nor with time to relapse nor overall survival time.