Juliette Murray

Letrozole Suppresses Plasma Estradiol and Estrone Sulphate More Completely Than Anastrozole in Postmenopausal Women With Breast Cancer

PURPOSE To compare the effects of anastrozole and letrozole on plasma estradiol (E2) and estrone sulfate (E1S) levels. PATIENTS AND METHODS Fifty-four postmenopausal women with estrogen receptor-positive breast cancer receiving aromatase inhibitors (AIs) as part of their adjuvant therapy were randomly assigned to receive either 3 months of anastrozole (1 mg) followed by 3 months of letrozole (2.5 mg), both given orally once daily, or 3 months of the opposite sequence. Blood was taken at the same time and the same day of the week from each patient, before and after 3 months of each drug, and plasma levels of E2 and E1S were determined using highly sensitive radioimmunoassays. RESULTS There were 27 patients in each group. The mean age of the patients was 63 years (range, 49 to 83 years). Baseline E2 levels ranged from 3 pmol/L to 91 pmol/L with a mean of 25.7 pmol/L. Only one of 54 (2%) patients had an E2 value >or= 3 pmol/L after receiving letrozole, versus 20 of 54 (37%) patients after receiving anastrozole (P < .001). Extrapolation revealed a mean E2 level after anastrozole treatment of 2.71 pmol/L (range, 2.38 to 3.08 pmol/L). Following letrozole, it was 1.56 pmol/L (range, 1.37 to 1.78 pmol/L). Mean residual E2 was 10.1% for anastrozole and 5.9% for letrozole. Residual E1S levels were 4.6% for anastrozole and 2.0% for letrozole (P = .001). CONCLUSION Letrozole reduces plasma E2 and E1S levels to a significantly greater extent than anastrozole in postmenopausal women taking AIs as part of their adjuvant therapy for hormone receptor-positive breast cancer.

Changes in breast cancer transcriptional profiles after treatment with the aromatase inhibitor, letrozole.

Objective The aim of the study was to identify changes in tumour expression profiling associated with short-term therapy of breast cancer patients with letrozole. Experimental design Microarray analysis was performed on RNA extracted from paired tumour core biopsies taken before and after 14 days of treatment with letrozole (2.5 mg/daily) in 58 patients. Changes in expression profile were identified by three different approaches on the basis of frequency of changes, magnitude of changes and significance analysis of microarray. Results No single gene was consistently changed by therapy in all cases. Fifty-two genes, however, were downregulated and 36 upregulated in at least 45 of the 58 cases. In terms of quantitative change, 46 genes showed at least a median 1.5-fold change in expression. Significance analysis of microarray identified 62 genes that were significantly changed by therapy (P<0.0001, 56 downregulated and six upregulated). All three approaches showed that greater numbers of genes were downregulated rather than upregulated. Merging data produced a total of 143 genes, which were subject to gene ontology and cluster analysis. The ontology of the 91 downregulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, upregulated genes were associated with organ development, connective tissue extracellular matrix regulation and inflammatory response. Cluster analysis segregated the patients into four groups differing in patterns of gene expression. Conclusion Genes have been identified which either change markedly or consistently in breast cancer after 14 days treatment with letrozole. These are new important data in understanding letrozoles molecular mechanism of action in breast cancers.

Aromatase inhibitors: Cellular and molecular effects☆

Marked cellular and molecular changes may occur in breast cancers following treatment of postmenopausal breast cancer patients with aromatase inhibitors. Neoadjuvant protocols, in which treatment is given with the primary tumour still within the breast, are particularly illuminating. In Edinburgh, we have shown that 3 months treatment with either anastrozole, exemestane or letrozole produces pathological responses in the majority of oestrogen receptor (ER)-rich tumours (39/59) as manifested by reduced cellularity/increased fibrosis. Changes in histological grading may also take place, most notably a reduction in mitotic figures. This probably reflects an influence on proliferation as most tumours (82%) show a marked decrease in the proliferation marker, Ki67. These effects are generally more dramatic than seen with tamoxifen given in the same setting. Differences between aromatase inhibitors and tamoxifen are also apparent in changes in steroid hormone expression. Thus, immuno-staining for progesterone receptor (PgR) is reduced in almost all cases by aromatase inhibitors, becoming undetectable in many. This contrasts with effects of tamoxifen in which the most common change on PgR is to increase expression. Changes in proliferation occur rapidly following the onset of exposure to aromatase inhibitors. Thus, neoadjuvant studies with letrozole in which tumour was sampled before and after 14 days and 3 months treatment show that decreased expression of Ki67 occur at 14 days and, in many cases, the effect is greater at 14 days than 3 months. These early changes precede evidence of clinical response but do not predict for it. However, this study design has allowed RNA analysis of sequential biopsies taken during the neoadjuvant therapy. Based on clustering techniques, it has been possible to subdivide tumours into groups showing distinct patterns of molecular changes. These changes in tumour gene expression may allow definition of tumour cohorts with differing sensitivity to aromatase inhibitors and permit early recognition of response and resistance.

DCIS and aromatase inhibitors

In patients with hormone receptor positive DCIS tamoxifen reduces recurrence rates by almost 50%. Few data are available with aromatase inhibitors from randomised studies. In the ATAC study there were three DCIS lesions in the anastrozole arm and four in the tamoxifen arm in the women with ER positive invasive cancer. In the MA17 study which randomised patients to up to 5 years of letrozole or placebo there was only one DCIS event in the contralateral breast in patients taking letrozole and five on placebo. There were also four patients in this study who had DCIS in the conserved breast on placebo and none in the letrozole treated group. The few clinical data that are available therefore suggest the aromatase inhibitors are likely to be effective in DCIS. A histological review of a study of 206 postmenopausal women with invasive oestrogen receptor positive breast cancer who were randomised as part of a 14 day preoperative study to receive 2.5mg of letrozole or 1mg of anastrozole identified 27 patients with 28 pairs of tumours in whom there was sufficient ER positive DCIS in invasive cancer in the initial core biopsy and in the subsequent surgery specimen, to evaluate for PgR activity and proliferation. Within the DCIS both aromatase inhibitors significantly reduced PgR expression and both drugs also produced a significant fall in proliferation. There was a moderate degree of agreement between the fall in PgR in both the invasive cancer and DCIS (Kappa=0.5; p=0.0013) and between the fall in proliferation and between the invasive and in situ components (correlation coefficient=0.68; p<0.001). This study has shown significant effects of aromatase inhibitors on DCIS indicating that these agents are therapeutically active in this condition.

Neoadjuvant endocrine therapy models.

Neoadjuvant therapy is therapy administered before surgical intervention and while the tumor remains in the breast. It may be given to treat large, locally advanced tumors, with the aim of shrinking them and thus making their surgical excision either simply possible or less radical. Most neoadjuvant therapy is chemotherapy, but adjuvant endocrine therapy is increasingly used in hormone-sensitive tumors; for example, those responsive to tamoxifen. Repeat biopsies aimed at assessing response to treatment--for example, by examining estrogen receptor status or markers of proliferation in tumor tissue--may be taken during the course of adjuvant therapy. In this chapter, the essential protocols associated with designing neoadjuvant trials are described, methods of assessing response to neoadjuvant therapy are detailed, and various approaches to collecting appropriate clinical samples and their assessment are presented.

Surgical issues surrounding use of aromatase inhibitors

There are important surgical issues related to the use of the third generation aromatase inhibitors in both the neoadjuvant and adjuvant settings. Neoadjuvant hormone therapy is effective at downstaging tumours, particularly large tumours initially thought to be inoperable or requiring mastectomy. Randomised trials have shown that the newer aromatase inhibitors letrozole and anastrozole increase the numbers of women who are suitable for breast-conservation compared with tamoxifen, and that letrozole is superior to tamoxifen in terms of clinical response. Aromatase inhibitors are most effective in ER-rich tumours and are clinically and biologically effective in both HER2 positive and negative tumours, whereas HER2 positive tumours show a level of resistance to tamoxifen. In neoadjuvant studies comparing aromatase inhibitors with tamoxifen, the duration of use has been 3-4 months, by which time any response is usually evident but longer treatment periods produce continued shrinkage and response. The re-excision rate following breast conservation surgery after neoadjuvant hormone therapy is favourable compared with the rates following immediate wide local excision. Local recurrence rates are acceptable in patients undergoing neoadjuvant therapy and breast-conserving surgery providing post-operative radiotherapy is given. Adjuvant aromatase inhibitors, as well as having an effect on metastatic disease and survival, reduce local and regional recurrence.

A randomised study of the effects of anastrozole (A), letrozole (L) and exemestane (E) on bone turnover.

Abstract #1145 Background: Aromatase inhibitors (AIs) reduce circulating oestrogen which increases bone turnover. L decreases circulating oestrogen levels to a greater degree than A. This study compares the effects of the non-steroidal aromatase inhibitors, A and L and the steroidal inactivator, E on bone turnover.
 Patients and Methods: This was an open, randomized pharmacodynamic study. Bone turnover markers were measured in 162 postmenopausal women with invasive ER +ve breast cancer. As part of their adjuvant hormone therapy, patients were randomized to receive either:
 16 weeks of A, 16 weeks of L or 16 weeks of E
 Fasting blood and urine samples were collected at entry and after 12 and 16 weeks of each drug. Bone remodelling releases breakdown products of type 1 collagen including the terminal peptide fragments N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx). An increase in these indicates bone resorption. Increases in type 1 pro-collagen peptides (N-terminal – PINP) and bone specific alkaline phosphatase (ALP) indicate bone formation. These were measured as was parathyroid hormone (PTH) which is an indirect measure of overall bone turnover.
 Results: A vs E vs L A, E and L significantly increased the bone turnover markers - PINP, CTX and bone ALP.
 E showed a greater change in bone turnover but there were no significant differences between the drugs.
 E resulted in a greater increase in PINP and uNTX compared to the non-steroidal AIs however these were of borderline significance.
 PINP, CTX and bone ALP all have significantly higher than zero percentage changes, regardless of drug or drug type.
 Conclusions: AIs cause a significant increase in both bone breakdown and bone formation.
 There is no evidence to suggest E has less effects on bone turnover than A or L. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1145.

Abstract P3-06-35: Association of estrogen receptor (ER) levels and prediction of antiproliferative effect of hormone therapy (HT) in lower ER-expressing tumors

Intro Accurate measurement of ER in early stage invasive breast cancer (EBC) is important to identify patients likely to benefit from HT. While immunohistochemistry (IHC) is the most common method to quantify ER, other methods can also accurately measure ER, such as RT-PCR. ER is one of the genes included in the RT-PCR based 21-gene Recurrence Score assay (Onco type DX ® , Genomic Health, Redwood City, CA) and is also reported separately as a single gene expression. Additionally, the association between ER expression by RT-PCR (ER-PCR) and tamoxifen benefit has been reported by Kim, et al (2011). A recent study reported that patients with ER levels Aim The study aims are: (1) To correlate quantification of ER in EBC as assessed by Allred Score (AS) and ER as measured by RT-PCR in the 21-gene assay; (2) To describe changes in ER, Recurrence Score, and measures of proliferation after 2wks of an aromatase inhibitor (AI); (3) To perform exploratory analyses of factors associated with changes in proliferation. Methods 55 postmenopausal EBC patients with lower ER (AS 2-7) were treated with 2wks of an AI followed by wide excision. All patients had a 21-gene assay on a pre-and post-treatment (Tx) sample. Proliferation was measured by both Ki67 by IHC (in 45 patients) and by the proliferation gene group score (PGS) in the RT-PCR based 21-gene assay (in all patients). Proliferation response was defined by a 20% relative decrease in Ki67 or a decrease in PGS. Changes in proliferation were correlated with AS, ER-PCR and Recurrence Score result. Results The Table shows the correlation of AS with ER-PCR measured in the pre-Tx (r=0.83) samples. 94% of AS (2-3) patients and 56% of AS (4-5) were ER(-) by RT-PCR There was a significant change (pre to post) in the average Ki67 level (18% to 11%; p Conclusions • Results confirm earlier reports showing substantial disagreement in ER measured by IHC vs RT-PCR in patients with lower ER-expressing tumors • The clinical implications are that a substantial number of patients with low ER by IHC may have little to no benefit from HT • The 21-gene assay may be useful in selecting patients likely to benefit from HT • Further studies in larger cohorts are required to confirm these findings. Citation Format: J Michael Dixon, Arran Turnbull, Lorna Renshaw, Megan P Rothney, Cynthia A Loman, Laura Arthur, Jeremy S Thomas, Oliver Young, Juliette Murray, Linda Williams, Amy P Sing, David Cameron. Association of estrogen receptor (ER) levels and prediction of antiproliferative effect of hormone therapy (HT) in lower ER-expressing tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-35.

Effects of letrozole and anastrozole on low ER expressing invasive breast carcinomas: results from a randomised trial.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1065 Introduction: Changes in proliferation as measured by Ki67 occur within 14 days of starting treatment with an aromatase inhibitor and have been shown to be predictors of long term outcome. The biological effect of different aromatase inhibitors on low ER expressing cancers has not been studied. This study compared changes in proliferation in breast cancers expressing low levels of ER (Allred score 2-5) following 14 days of treatment with anastrozole or letrozole. Materials and methods: Thirty six postmenopausal women with invasive breast cancer, classified as estrogen receptor (ER) poor by an Allred score of 2-5 on initial core biopsy, were enrolled into a randomised pre-operative trial of 14 days treatment with either 2.5 mg of letrozole or 1 mg of anastrozole. Paired biopsy tissue was available for all patients: [15 who received anastrozole and 21 letrozole] with sufficient invasive cancer present for analysis. Assessment included ER (Allred score) and proliferation (% tumor cells Ki67 positive) by immunohistochemistry. Results are presented as means (SEM) and medians (range). Due to the non-normality of the data, pre- and post-treatment Ki67 values were transformed on the log scale. Change between pre- and post- treatment scores was calculated as the difference in the logged scores; analysis is by t-test and Fishers Exact Test. All tests are two-sided. Results were back-transformed in order to preserve the original scale. Results: Proliferation measured by Ki67: Anastrozole reduced tumour cell proliferation from baseline in 10/15 cancers from a mean of 31.0% (7.1) to 20.8% (6.1). The median and mean reductions in Ki67 were 37% (range -94.0% to 92.6%) and 38% (13.9) from baseline. This equates to an average fall of 2.6 times from baseline, p=0.006, by analysis of the transformed data. Letrozole reduced proliferation from baseline in 17/21 cancers from a mean of 39.0% (7.3) to 25.3% (6.4). The median and mean reductions were 67% (range -115.6% to 99.6%) and 46% (10.6) from baseline. This equates to an average fall of 3.13 times from baseline, p=0.0002, by analysis of the transformed data. There was no evidence of a significant difference between drugs in reduction of proliferation, the number of cases showing a fall in proliferation, or the absolute Ki67 index at 14 days. Only 3/15 (anastrozole) and 2/21 (letrozole) tumours had post-treatment absolute Ki67 indices of ≤1%. There were no significant differences in changes in proliferation between tumours with ER score 2-3 vs score 4-5. Conclusions: 1. Both anastrozole and letrozole significantly reduced proliferation in invasive cancers with low ER expression. 2. There were no evident significant differences between drugs. 3. These data suggest that aromatase inhibitors are of value even in low-ER expressing cancers, either on their own or perhaps as part of combined therapy. 4. Analysis of more patients and of biological response according to molecular phenotype is underway. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1065.

A randomised study comparing the effects of anastrozole (A), letrozole (L), exemestane (E) and tamoxifen (T) on coagulation.

Abstract #1132 Background:
 Aromatase inhibitors (AIs) reduce circulating oestrogens and so should have either no or a positive effect on coagulation . This study compares the effects of the non-steroidal aromatase inhibitors, A and L and the steroidal inactivator, E on coagulation.
 Patients and Methods:
 This was an open, randomized pharmacodynamic study. Plasma coagulation factors were measured in 120 postmenopausal women with invasive ER +ve breast cancer. As part of their adjuvant hormone therapy, patients were randomized to receive upfront therapy with either:
 16 weeks of A, 16 weeks of L or 16 weeks of E.
 Fasting blood samples were collected at entry and after 12 and 16 weeks of each drug. AI patients were then switched to T and further samples measured after 8 months on T.
 Plasminogen activator inhibitor (PAI) antigen, von Willebrand9s Factor (vWF) antigen, antithrombin III (AT111), protein C, protein S total, protein S free, activated protein C, resistance (APCR), factor VIII and fibrinogen were measured.
 Results:
 Results expressed as % change from baseline.
 E vs A vs L
 Protein C. E caused a significant fall compared to A and L (-15.75 (-21.48, -10.02) vs -3.80 (-10.77, 3.16) vs -3.63 (-10.50, 3.25)) respectively. p = 0.008.
 ATIII. E caused a significant fall from baseline (-8.55 (-13.33, -3.79))
 Protein S Free. E + A caused a significant increase from baseline (E 6.90 (1.98, 11.82), A 7.36 (1.37, 13.34).
 Steroidal vs non-steroidal:
 Protein C. A significantly greater fall was seen with E than A+L (-15.75 (-21.45, -10.05) vs -3.71 (-8.58, 1.15)) p = 0.002.
 vWF A+L caused a significant increase from baseline (6.52 (0.66, 12.38) but this was not statistically different to E 5.02 (-1.88, 11.91)).
 ATIII A significant decrease from baseline was seen with A + L, -4.54 (-8.64, -0.43) but no difference from E 8.55 (13.31, -3.70) p=0.21.
 Protein S Free There was a significant increase from baseline in both groups (A+L: 5.70 (1.47, 9.92), E: 6.90 (1.98, 11.81), but no difference between the groups p=0.72.
 T effect:
 AT111 and protein C levels fell significantly on T whereas protein S free increased on T. Prior A, E or L had no significant impact on post-tamoxifen results.
 Conclusions:
 No significant differences in any coagulation factors were seen between A and L
 E caused a significant fall in protein C and ATIII.
 The non-steroidal AIs caused a significant increase in vWF.
 T caused a significant fall in protein C and ATIII and a significant increase in protein S free.
 No AI significantly influenced post-tamoxifen results.
 These drugs have significant effects on coagulation which helps to explain their clinical effects on thrombotic and thromboembolic disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1132.