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Published OnlineFirst March 14, 2013; DOI: 10.1158/1078-0432.CCR-12-1000 Clinical Cancer Predictive Biomarkers and Personalized Medicine Research Molecular Profiling of Aromatase Inhibitor–Treated Postmenopausal Breast Tumors Identifies Immune-Related Correlates of Resistance Anita K. Dunbier1,2,5, Zara Ghazoui1,2, Helen Anderson1,2, Janine Salter1, Ashutosh Nerurkar1, Peter Osin1, Roger A'hern3, William R. Miller4, Ian E. Smith1, and Mitch Dowsett1,2 Abstract Purpose: Estrogen withdrawal by treatment with aromatase inhibitors is the most effective form of endocrine therapy for postmenopausal estrogen receptor–positive (ERþ) breast cancer. However, response to therapy varies markedly and understanding of the precise molecular effects of aromatase inhibitors and causes of resistance is limited. We aimed to identify in clinical breast cancer those genes and pathways most associated with resistance to aromatase inhibitors by examining the global transcriptional effects of AI treatment. Experimental Design: Baseline and 2-week posttreatment biopsies were obtained from 112 postmen- opausal women with ERþ breast cancer receiving neoadjuvant anastrozole. Gene expression data were obtained from 81 baseline and 2-week paired samples. Pathway analysis identified (i) the most prevalent changes in expression and (ii) the pretreatment genes/pathways most related to poor antiproliferative response. Results: A total of 1,327 genes were differentially expressed after 2-week treatment (false discovery rate < 0.01). Proliferation-associated genes and classical estrogen-dependent genes were strongly downregulated whereas collagens and chemokines were upregulated. Pretreatment expression of an inflammatory signature correlated with antiproliferative response to anastrozole and this observation was validated in an inde- pendent study. Higher expression of immune-related genes such as SLAMF8 and TNF as well as lymphocytic infiltration were associated with poorer response (P < 0.001) and validated in an independent cohort. Conclusions: The molecular response to aromatase inhibitor treatment varies greatly between patients consistent with the variable clinical benefit from aromatase inhibitor treatment. Higher baseline expression of an inflammatory signature is associated with poor antiproliferative response and should be assessed further as a novel biomarker and potential target for aromatase inhibitor-treated patients. Clin Cancer Res; 19(10); 2775–86. Ó2013 AACR. Introduction inhibitors is the most effective form of endocrine therapy, Approximately 80% of human breast carcinomas present but response to them varies markedly (1–3). Mechanisms of as estrogen receptor a–positive (ERþ). In postmenopausal resistance appear to be multiple but are poorly character- In vitro women, estrogen withdrawal by treatment with aromatase ized. studies of acquired resistance to estrogen deprivation have identified several putative mechanisms which largely involve growth factor–related signal trans- Authors' Affiliations: 1Royal Marsden Hospital; 2Breakthrough Breast duction pathways (4–7) but there is limited clinical evi- 3 Cancer Research Centre, Institute of Cancer Research; Cancer Research dence to support these. It is also notable that these models UK Clinical Trials and Statistics Unit, Section of Clinical Trials, Institute of Cancer Research, London; 4Breast Research Group, University of Edin- do not involve an assessment of the contribution of human burgh, Edinburgh, United Kingdom; and 5Department of Biochemistry, stromal elements. University of Otago, Dunedin, New Zealand The presurgical/neoadjuvant setting provides an excep- Note: Supplementary data for this article are available at Clinical Cancer tionally valuable scenario for linking biology to clinical Research Online (http://clincancerres.aacrjournals.org/). response and to study mechanisms of resistance; within this A.K. Dunbier, Z. Ghazoui, and H. Anderson contributed equally to this work. setting, the proliferation marker Ki67 is a validated phar- Corresponding Author: Anita K. Dunbier, Department of Biochemistry, macodynamic indicator of response to endocrine therapy University of Otago, PO Box 56, Dunedin 9016, New Zealand. Phone: (8–10). Treatment-dependent changes in sequential mea- 643479-9258; Fax: 643479-7738; E-mail: [email protected] surements of Ki67 in neoadjuvant trials of endocrine ther- doi: 10.1158/1078-0432.CCR-12-1000 apy in postmenopausal women (IMPACT, p024, and Ó2013 American Association for Cancer Research. Z1031; refs. 9, 11, 12) have all revealed differences or lack www.aacrjournals.org 2775 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 14, 2013; DOI: 10.1158/1078-0432.CCR-12-1000 Dunbier et al. Materials and Methods Translational Relevance Patient samples Most postmenopausal women with estrogen receptor Fourteen-gauge core-cut tumor biopsies were obtained (ER)–positive breast cancer receive an aromatase inhib- from 112 postmenopausal women with stage I to IIIB ERþ itor at some stage during their treatment. However, early breast cancer before and after 2-week anastrozole responsiveness to aromatase inhibitors varies greatly. monotherapy in a neoadjuvant trial (17, 18). Patient demo- Previous studies of resistance mechanisms have been graphics are shown in Supplementary Table S1. Tissue was conducted in cell lines and have highlighted the impor- stored in RNAlater at À20C. Two 4-mm sections from the tance of signal transduction pathways, but these studies core were stained with hematoxylin and eosin (H&E) and omit the stromal influences that are increasingly recog- examined by a pathologist (A. Nerurkar and P. Osin) to nized to have a major influence on tumor biology. In confirm the presence of cancerous tissue, assess histopa- contrast, we report genome-wide expression profiling of thology, and the presence or absence of lymphocytic infil- 81 ER-positive breast carcinomas including stromal tis- tration. Tumors were deemed to be positive for lymphocytic sues before and during treatment with an aromatase infiltration if intraepithelial mononuclear cells could be inhibitor in the neoadjuvant setting. We identify an seen within tumor cell nests or in direct contact with tumor inflammatory signature as the strongest correlate of poor cells. Biopsies in which lymphocytic infiltrate could be seen antiproliferative response and confirm this finding in an without direct contact to tumor cells were not considered to independent set of tumors as well as using gross lym- have lymphocytic infiltration. Samples were assessed blind- phocytic infiltration as an alternative measure of inflam- ly by both pathologists and the concordance rate was 81%. matory activity. This work provides a potential new Discordant samples were then reassessed by A. Nerurkar avenue for drug development and for identifying who was blinded to the result of the initial assessment and patients with a reduced likelihood of response to aro- the more frequent of the 3 assessments was used in the matase inhibition. analysis. Total RNA was extracted using RNeasy (Qiagen). RNA quality was checked using an Agilent Bioanalyser: samples with RNA integrity values of less than 5 were excluded from further analysis. ER (H-score) and Ki67 (% cells positive) values by centralized immunohistochem- of differences that were parallel to recurrence-free survival istry were already available (17). (RFS) differences in the equivalent adjuvant trials (ATAC, An additional 71 stored sections from paraffin-embed- BIG 1-98, and MA27; refs. 1, 13, 14), respectively. In ded, formalin-fixed core-cut biopsies were obtained from a addition, Ki67 of patients after 2 weeks’ treatment predicts subset of patients from the IMPACT trial who received RFS more closely than pretreatment values (8). neoadjuvant anastrozole or tamoxifen (15). Sections were Nearly all patients on an aromatase inhibitor show H&E-stained, and the presence or absence of detectable reduction in Ki67 expression, suggesting that some benefit lymphocytic infiltration was assessed by a pathologist (A. is derived, although this may be modest (9). This contin- Nerurkar). Ki67 data were obtained from previous analysis uous marker of response is well-suited to assessment of of these patients (8, 9). mechanisms of resistance that are also likely to have a nonbinary effect. This may explain why changes in Ki67 have proven to be better predictors of benefit from endo- Gene expression analysis and data preprocessing crine therapy than clinical response during neoadjuvant RNA amplification, labeling, and hybridization on endocrine therapy (15, 16). HumanWG-6 v2 Expression BeadChips (Illumina) were Gene expression profiling of tumor biopsies before and conducted according to the manufacturer’s instructions at during treatment has the potential to enable the identifica- a single Illumina BeadStation facility. Tumor RNA of suf- tion of the most important genes/pathways involved in the ficient quality and quantity was available to generate expres- response to estrogen deprivation therapy and the pretreat- sion data from 104 pretreatment biopsies and 85 two-week ment determinants of response and resistance. Availability biopsies (Supplementary Fig. S1). Data were extracted using of comprehensive expression datasets, as provided here, will BeadStudio software and normalized with variance-stabi- allow the evaluation of candidate genes from experimental lizing transformation (VST) and Robust Spline Normaliza- research
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  • Supplementary Information

    Supplementary Information

    Osa et al Supplementary Information Clinical implications of monitoring nivolumab immunokinetics in previously treated non– small cell lung cancer patients Akio Osa, Takeshi Uenami, Shohei Koyama, Kosuke Fujimoto, Daisuke Okuzaki, Takayuki Takimoto, Haruhiko Hirata, Yukihiro Yano, Soichiro Yokota, Yuhei Kinehara, Yujiro Naito, Tomoyuki Otsuka, Masaki Kanazu, Muneyoshi Kuroyama, Masanari Hamaguchi, Taro Koba, Yu Futami, Mikako Ishijima, Yasuhiko Suga, Yuki Akazawa, Hirotomo Machiyama, Kota Iwahori, Hyota Takamatsu, Izumi Nagatomo, Yoshito Takeda, Hiroshi Kida, Esra A. Akbay, Peter S. Hammerman, Kwok-kin Wong, Glenn Dranoff, Masahide Mori, Takashi Kijima, Atsushi Kumanogoh Supplemental Figures 1 – 8 1 Osa et al Supplemental Figure 1. The frequency of nivolumab-bound T cells was maintained in patients who continued treatment. Nivolumab binding in CD8 and CD4 T cells was analyzed at two follow-up points, as indicated, in fresh peripheral blood from three representative cases from protocol 1 that continued treatment. 2 Osa et al Supplemental Figure 2. Long-term follow-up of nivolumab binding to T cells from fresh whole blood. Nivolumab binding was followed up in fresh peripheral blood from an additional case, Pt.7. 3 Osa et al Supplemental Figure 3. Long-term duration of nivolumab binding is due to sustained circulation of residual nivolumab in plasma. (A) PBMCs acquired from Pt.8 and 9 at pretreatment (pre PBMCs) and after a single dose (post 1 PBMCs) were cultured in regular medium without nivolumab (top and middle). Pre PBMCs were also incubated with 10 µg/ml nivolumab in vitro before the cultures were started (bottom). Nivolumab binding status was monitored at the indicated time points.