Frances S. Kenny

Gamma linolenic acid with tamoxifen as primary therapy in breast cancer

Gamma linolenic acid (GLA) has been proposed as a valuable new cancer therapy having selective anti‐tumour properties with negligible systemic toxicity. Proposed mechanisms of action include modulation of steroid hormone receptors. We have investigated the effects of GLA with primary hormone therapy in an endocrine‐sensitive cancer. Thirty‐eight breast cancer patients (20 elderly Stage I‐II, 14 locally advanced, 4 metastatic) took 8 capsules of oral GLA/day (total = 2.8 g) in addition to tamoxifen 20 mg od (T+GLA). Quality and duration of response were compared with matched controls receiving tamoxifen 20 mg od alone (n = 47). Serial tumour biopsies were taken to assess changes in oestrogen receptor (ER) and bcl‐2 expression during treatment. GLA was well tolerated with no major side effects. T+GLA cases achieved a significantly faster clinical response (objective response vs. static disease) than tamoxifen controls, evident by 6 weeks on treatment (p = 0.010). There was significant reduction in ER expression in both treatment arms with T+GLA objective responders sustaining greater ER fall than tamoxifen counterparts (6‐week biopsy p = 0.026; 6‐month biopsy p = 0.019). We propose GLA as a useful adjunct to primary tamoxifen in endocrine‐sensitive breast cancer. The effects of GLA on ER function and the apparent enhancement of tamoxifen‐induced ER down‐regulation by GLA require further investigation. Int. J. Cancer 85:643–648, 2000.

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival.

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P=0.0061), whereas cyclin D1 mRNA overexpression was not (P=0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P=0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P=0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P=0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P=0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age >50 years (P=0.0001), postmenopausal status (P=0.0008), lymph node negativity (P=0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.

BRCA1 expression levels predict distant metastasis of sporadic breast cancers

The role of BRCA1 in progression of sporadic breast cancers has to date been equivocal, although preliminary studies on small numbers of samples have suggested an association between expression levels of this gene and acquisition of an invasive phenotype. We have further reasoned that loss of oestrogen receptor positivity may have a detrimental effect on BRCA1 expression. In order to test this hypothesis and extend earlier investigations we have applied a sensitive RT‐PCR procedure to determine the associations between BRCA1 expression and a variety of clinical parameters in a sample cohort derived from sporadic breast tumour specimens. We have established that BRCA1 and ER mRNA expression are closely associated (p=0.013), indicating a possible functional relationship between these 2 genes. We have further identified an association between low levels of BRCA1 expression and acquisition of distant metastasis in sporadic disease (p=0.019). In light of our findings, we suggest that suppression of BRCA1 has a role to play in progression of a significant fraction of sporadic breast cancers and may additionally prove to be a useful, novel, prognostic marker for this disease type. Int. J. Cancer (Pred. Oncol.) 84:258–262, 1999.

Long-term follow-up of elderly patients randomized to primary tamoxifen or wedge mastectomy as initial therapy for operable breast cancer

One hundred and thirty-one fit elderly patients (> 70 years) presenting with operable breast cancer (< 5 cm) between 1982 and 1987 were randomized to receive primary tamoxifen 20 mg b.d. (n = 66) or wedge mastectomy (n = 65) as sole initial therapy, unselected by oestrogen receptor status. Outcome measures were comparison of loco-regional control, metastasis and overall survival. With median follow-up of 145 months there is no statistical difference in breast cancer specific survival or rate of metastasis. Local control, however, has been significantly better with surgery - 38% of mastectomy patients having developed local recurrence by 10 years compared to 81% with local progression on primary tamoxifen (P < 0.0001 Wilcoxon-Gehan statistic). Of the latter 50% have since required mastectomy in order to achieve local control. In conclusion, primary tamoxifen or wedge mastectomy are comparable treatments in terms of metastasis and overall survival but a high local failure rate on tamoxifen suggests that optimal management of the fit elderly should include surgery.

A randomised trial of mastectomy only versus tamoxifen for treating elderly patients with operable primary breast cancer-final results at 20-year follow-up.

A recent Cochrane review of trials involving elderly women with operable primary breast cancer showed no significant difference in overall survival between surgery (±adjuvant tamoxifen) and primary endocrine therapy using tamoxifen. We report the final results of a randomised pilot trial comparing primary tamoxifen and wedge mastectomy as initial treatment in this population. One hundred and thirty-one women >70 years with early operable primary breast cancer (<5 cm), unselected for oestrogen receptor (ER), entered the trial in 1982-1987. Sixty-eight patients were allocated to tamoxifen only and 67 to wedge mastectomy only, as primary treatment. At 20 years of follow-up, the median time to local failure was significantly shorter in the tamoxifen arm though approximately one-fifth of patients in this group did not develop local failure requiring mastectomy. There was no difference in regional recurrence, distant metastases or overall survival between the mastectomy and tamoxifen arms. In this small study, primary endocrine therapy achieved local control in 30% of those surviving at 5 years and 20% at 10 years, unselected for ER. The primary therapy used did not significantly affect regional recurrence, incidence of distant metastases or overall survival. Primary endocrine therapy should certainly be considered in those patients with ER positive tumours and who are unfit (based on life expectancy) for or refuse surgery.

Change in Expression of ER, bcl-2 and MIB1 on Primary Tamoxifen and Relation to Response in ER Positive Breast Cancer

Pre-treatment oestrogen receptor (ER) expression in breast cancer predicts for rate of response to endocrine therapy but not for the quality or duration of response (DofR). ER is known to be down-regulated by anti-oestrogens. This study has tested the hypothesis that the degree of down-regulation of ER and the ER-regulated marker bcl-2 are associated with the quality and duration of tamoxifen response. 80 patients with ER+ve breast cancer (H-score ≥10) receiving primary tamoxifen (n=51 Stage I–II elderl; n=29 Stage III) underwent sequential tumour biopsies for immunocytochemical assessment of ER, bcl-2 and the proliferation marker MIB1. Median follow-up is 45 months. By 6-months on therapy three patients had attained complete response (CR), 27 partial response (PR); 44 static disease (SD) and six progression (PD) by UICC criteria. Greater decrease in ER and bcl-2 H-score from pre-treatment to 6 weeks (p=0.035, p=0.037) and ER and bcl-2 H-score from pre-treatment to 6 months (p=0.058, p=0.036) were significantly associated with better quality of response (CR/PR vs SD/PD). Greater 6-week and 6-month reduction in bcl-2 H-score (p=0.041, p=0.036) and 6-week reduction in MIB1 (p=0.013) were significantly correlated with longer DofR. This study demonstrates that greater down-regulation of ER and the ER-regulated protein bcl-2 on primary tamoxifen are significantly associated with a better quality of response and bcl-2 and the proliferation marker MIB1 a longer duration of response in ER+ve breast cancer.

Endocrine response and resistance in breast cancer: A role for the transcription factor Fos

We have previously demonstrated that elevated Fos expression may be important in de novo endocrine resistance in breast cancer. However, changes in Fos expression during endocrine response and subsequently on acquisition of resistance are unknown. This study immunocytochemically monitors Fos protein within sequential biopsies from primary human breast cancer patients obtained pre‐treatment (T1), during tamoxifen therapy (T2, T3) and on disease progression (T5), examining in parallel proliferation [i.e., MIB1 (Ki67) immunostaining, mitotic activity], cellularity and endocrine response. Significantly diminished Fos, proliferation and cellularity were observed after 6 weeks of therapy in patients exhibiting a better quality and/or duration of response, while modest Fos increases and a maintained proliferation and cellularity were seen in poorer responders. Decreases in Fos, proliferation and cellularity at 6 months similarly hallmarked better responders. We confirmed a significant association between de novo resistance and elevated Fos and proliferation. Additionally, however, these parameters increased at the time of disease relapse over pre‐treatment and “on therapy” values. Our data indicate that tamoxifen response involves a reduction in both tumor cell proliferation and cell survival, potentially entailing diminished Fos protein expression in better‐responding patients. Our data are also supportive of elevated Fos expression being involved in the departure from endocrine control inherent in both primary and acquired resistance. Int. J. Cancer (Pred. Oncol.) 84:54–61, 1999.

Effect of dietary GLA+/−tamoxifen on the growth, ER expression and fatty acid profile of ER positive human breast cancer xenografts

Gamma linolenic acid (GLA) possesses a number of selective anti‐tumour properties including modulation of steroid receptor structure and function. We have investigated the effect of dietary GLA on the growth, oestrogen receptor (ER) expression and fatty acid profile of ER+ve human breast cancer xenografts. Experimental diets A, B, C, D were commenced after subcutaneous implantation of 40 female nude mice with the MCF‐7 B1M cell line (Group A = control diet: B = control diet + GLA supplement: C = control diet + tamoxifen: D = control diet + GLA + tamoxifen; 10 mice/group). The mice were terminated when tumour cross‐sectional area reached 250 mm2. ER H‐scores were assessed by immunohistochemical assay and fatty acid profiles by gas‐liquid chromatography of termination tumour samples. Groups C and D displayed significantly slower tumour growth (p =.0002, p =.0006) with trend for slower growth in B (p =.065) compared to control Group A. ER was significantly reduced in all groups compared to A (p <.0001) with Group D (combined therapy) displaying markedly lower ER expression than with either therapy alone (p =.0002). There were significantly raised levels of tumour GLA and metabolites in the two groups (B and D) receiving GLA (p <.0001). This xenograft model of ER+ve breast cancer has demonstrated significantly lower tumour ER expression in those groups receiving GLA, an effect which appears to be additive to the reduced ER expression resulting from tamoxifen alone. The effects of GLA on ER function and the possibility of synergistic inhibitory action of GLA with tamoxifen via enhanced down‐regulation of the ER pathway require further investigation.