689
Endocrine and Targeted Manipulation of Breast Cancer: Proceedings of the Sixth Cambridge Conference Supplement to Cancer
Predicting Response and Resistance to Endocrine Therapy Profiling Patients on Aromatase Inhibitors
William R. Miller, PhD, DSc1 Alexey Larionov, MD1 Thomas J. Anderson, FRCPath1 John R. Walker, PhD2 Andreas Krause, PhD3 Dean B. Evans, PhD3 J. Michael Dixon, MD1
Selection for endocrine therapy requires the identification of markers that accurately predict response/resistance. In this report, the authors review their published work and abstract results from an unpublished study to illustrate the potential of RNA microarrays from sequential tumor biopsies from patients who were offered neoadjuvant endocrine therapy treatment to identify the molecular signatures associated with tumor sensitivity/resistance. Clinical response was assessed by serial ultrasound measurements in postmenopausal women with large, primary, estrogen receptor-rich breast cancers who received neoadjuvant
1
Breast Research Group, University of Edinburgh, Edinburgh, United Kingdom.
treatment with letrozole for 3 months. Tumor RNA from biopsies that were taken
2
to determine expression profiles. Classic estrogen-dependent genes and markers
Genomics Institute of the Novartis Research Foundation, San Diego, California. 3
Novartis Pharma AG, Basel, Switzerland.
before and after 14 days of treatment was hybridized on Affymetrix U133A chips of proliferation were changed with treatment in most tumors but were poorly associated with clinical response (they frequently were changed in letrozole-resistant tumors). Differential expression patterns could be used to identify heterogeneity in clinically resistant tumors. The results indicated that molecular profiling of early changes with letrozole treatment offers the opportunity to dis-
William R. Miller received educational grants from AstraZeneca, Novartis, and Pfizer and served as a member of Advisory Boards for Novartis and Pfizer. Andreas Krause is a former full-time employee of Novartis. Dean B. Evans is a full-time employee of Novartis and a shareholder in Novartis. J. Michael Dixon received an educational grant and honoraria from Novartis Pharma. Presented at Endocrine and Targeted Manipulation of Breast Cancer: Proceedings of the Sixth Cambridge Conference, April 30–May 1, 2007, Cambridge, Massachusetts. Address for reprints: William R. Miller, PhD, DSc, Breast Cancer Research Group, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU Scotland, United Kingdom; Fax: (011) 44 131 537 2449; E-mail:
[email protected]. ac.uk Received July 30, 2007; revision received September 26, 2007; accepted October 2, 2007.
ª 2007 American Cancer Society
tinguish between clinically responsive and nonresponsive tumors and provides important information about the heterogeneity of endocrine resistance. Cancer 2008;112(3 suppl):689–94. 2007 American Cancer Society.
KEYWORDS: genetic expression, neoadjuvant therapy, clinical response/resistance, letrozole, breast neoplasm, estrogen receptor.
T
hird-generation aromatase inhibitors, such as letrozole, have an established role in the treatment of hormone-sensitive postmenopausal breast cancer.1–7 The drugs are specific, in that their only primary endocrine effect is to inhibit the aromatase enzyme8 and reduce endogenously synthesized estrogen.8-10 In hormone-dependent breast cancers, estrogen deprivation may cause cell death, reduced proliferation, and tumor regression. However, this does not occur in all tumors, and there is a need to identify molecular markers of response so that treatment may be given selectively. In this report, we review some published work and abstract results from an unpublished study from our own research to define early changes in the expression of estrogen deprivation/proliferation markers.
DOI 10.1002/cncr.23187 Published online 10 December 2007 in Wiley InterScience (www.interscience.wiley.com).
690
CANCER Supplement
February 1, 2008 / Volume 112 / Number 3
MATERIALS AND METHODS Patients Patients were postmenopausal women who presented to the Edinburgh Breast Unit with large, primary, estrogen receptor (ER)-positive breast cancer. All provided informed consent to be included in the study, which had been approved by the local ethics committee (2001/W/BU/09 and 2001/W/BU/10). A consecutive series of 80 patients was recruited excluding patients who had known multifocal tumors or tumors of special histological type. Patients received neoadjuvant treatment for 3 months with letrozole (Femara; 2.5 mg daily). Core biopsies were taken with a 14-gauge needle before and after 10 to 14 days of treatment. Clinical response was based on reductions in tumor volumes, as determined from ultrasound measurements over 3 months. Reduction in volume >50% was taken as evidence of clinical response.
Tissue Processing and RNA Extraction Biopsies were snap-frozen immediately and stored in liquid nitrogen. Frozen sections were taken to confirm the presence of cancerous tissue. Only samples in which the malignant component comprised 20% of the section area were used in further analyses. Biopsies were pulverized using U2 microdismembranator U (Braun Biotech). Total RNA was extracted by using TRI-reagent (Sigma) and was purified further on RNeasy mini columns (Qiagen). RNA quantity and quality were verified on a Bioanalyser 2100 (Agilent).
RNA Amplification and Reverse Transcription RNA (500 ng) was subject to 2 rounds of amplification.11 Briefly, the first round of amplification used nonbiotinylated ribonucleotides and the Ambion MegScript kit (Ambion, Austin, Tex). This complementary RNA (cRNA) was converted to doublestranded DNA, and biotinylated cRNA was generated by using the Enzo kit. Microarray Hybridization and Data Normalization Biotinylated cRNA was fragmented and added to Affymetrix HG_U133A chips (Affymetrix, Santa Clara, Calif) as described in the standard protocol outlined in the Gene Chip Expression Analysis Technical Manual (Affymetrix). After hybridization, microarrays were washed with a custom GNF chip washer and were scanned with an Affymetrix 3000 laser scanner. Expression values as read out from Affymetrix CEL
TABLE 1 Genes Classically Associated With Either Estrogen Dependence or Proliferation* Gene Estrogen markers KIAA0101 TFF3 SERPINA 3 IRS-1 TFF1 Proliferation markers CDC-2 Cyclin B1 CKS-2 TYMS PCNA
Cases (58 patients)
Decrease (median fold)
SAM (P value)
54 (2nd) 45 (43rd) 45 (43rd) 48 (14th) 50 (60th)
2.28 (3) 1.71 (14) 1.96 (6) 1.50 (27) 2.61 (1)
