RESPONSE: Re: Limits of Predictive Models Using Microarray Data for Breast Cancer Clinical Treatment Outcome

CORRESPONDENCE Re: Limits of Predictive Models Using Microarray Data for Breast Cancer Clinical Treatment Outcome The ability to accurately predict outcome to tamoxifen therapy would advance the management of breast cancer. Tamoxifen acts through the estrogen receptor (ER), and ER expression levels are currently the best predictors of response. Last year, Ma et al. (1) reported a novel biomarker of a two-gene expression ratio (HOXB13 to IL17BR) in 60 patients with ER-positive early-stage breast cancer treated with adjuvant tamoxifen. However, recently, Reid et al. (2) failed to validate the performance of this two-gene predictor. We would like to point out three problems with the study of Reid et al. (2): 1) cohort selection, 2) definition of response, and 3) primer design. 1) HOXB13 and IL17BR were originally identified as clinically relevant factors in a discovery subset that included lymph node–negative (53%) and lymph node–positive (47%) patients (1). This finding was further tested and validated in 145 ER-positive lymph node–negative patients only (1,3). As discussed by Simon in the editorial (4), this latter subset of patients was not comparable with the discovery set studied by Ma et al. (1). The study by Reid et al. of mainly lymph node–positive patients (78%) (2), although clinically less relevant, is justifiable but failed to confirm the study of Ma et al. (1). 2) Ma et al. (1) stated that in the adjuvant setting “the two-gene HOXB13: IL17BR ratio might predict a tumor’s response to tamoxifen but also its intrinsic aggressiveness” but that the two-gene ratio levels appeared not to be associated with aggressiveness. However, neither Ma et al. (1) nor Reid et al. (2) could determine the response to tamoxifen therapy because their cohorts lacked a randomized nontreated control group. The only possible conclusion is that the gene ratio is a prognostic factor in this subgroup of patients. Criteria for response to tamoxifen therapy have been defined for patients with advanced disease, with measurable disease as the endpoint and with approximately half of the ER-positive relapsed patients not responding to treatment (5). We have recently reported (6) our genome-wide screening in ER-positive tumors from 112 relapsed patients with clearly defined types of response from the start of firstline tamoxifen. In this report, we identified 81 differentially expressed genes that contained neither HOXB13 nor IL17BR. However, the ratio of HOXB13 to IL17BR showed a statistically significant association with tamoxifen response when we analyzed our microarray data by the t test, the Mann-Whitney test, or the area under the receiver operating characteristic curve, whereas a trend was observed with logistic regression (Table 1). 3) Reid et al. (2) determined expression levels of both genes with quantitative reverse transcription–polymerase chain reaction. The IL17BR primer set that they used, however, is located in the 5′end region (from exons 1 to 2), whereas Ma et al. (1) used primers in the 3′-end region (in the last exon) of IL17BR. Our concern is that, because of the presence of splice variants—two of which have been described [National Center for Biotechnology Information Entrez Gene identification number 55540 (7)]—and of the difference in efficiency of cDNA synthesis between the 3′ and 5′ ends, the actual IL17BR levels measured by the two groups may not be comparable. To overcome this problem, we recommend that primers be used that are, if not identical, then at least compatible, in assays of the two-gene predictor. To summarize, our data show that the HOXB13-to-IL17BR ratio does predict response to tamoxifen. In addition, for a proper validation of the performance of the two-gene predictor, or any predictor, we recommend that an adequately sized patient cohort be used that is clinically equivalent to the original cohort. For the analyses of the predictor, we recommend that an identical assay or at least an assay that has been proven to be equivalent be used. MAURICE P. H. M. JANSEN JOHN A. FOEKENS JAN G. M. KLIJN ELS M. J. J. BERNS REFERENCES (1) Ma XJ, Wang Z, Ryan PD, Isakoff SJ, Barmettler A, Fuller A, et al. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 2004;5:607–16. Table 1. Association and discrimination of expression data (log2-transformed) obtained from cDNA microarrays of 112 estrogen receptor–positive primary breast tumors from relapsed patients treated with first-line tamoxifen therapy* Test t test P value Mean difference (95% CI of the difference) Mann-Whitney P value (95% CI of P value) AUC P value AUC (95% CI of AUC) Logistic regression P value Odds ratio (95% CI) HOXB13 IL17BR HOXB13:IL17BR .257 −0.219 (−0.601 to 0.162) .093 0.306 (−0.052 to 0.664) .050 −0.525 (−1.051 to –0.0001) .202 (0.194 to 0.210) .164 (0.157 to 0.171) .038 (0.034 to 0.042) .202 0.57 (0.46 to 0.68) .168 0.424 (0.32 to 0.51) .042 0.612 (0.53 to 0.72) .251 1.26 (0.85 to 1.85) .121 0.70 (0.45 to 1.10) .059 1.31 (0.99 to 1.75) *Comparison of gene expression levels between patients showing an objective response (n = 52) (i.e., complete [n = 12] or partial remission [n = 40]) or progressive disease (n = 60) after first-line tamoxifen therapy. SPSS, release 11.0.1, was used for statistical analysis. All P values are two-sided. The t test was based on unequal variances. Receiver-operating characteristic curve analysis resulted in AUC with the null hypothesis: AUC = 0.5 and alternative AUC ≠ 0.5. CI = confidence interval; AUC = area under the curve. Journal of the National Cancer Institute, Vol. 97, No. 24, December 21, 2005 CORRESPONDENCE 1851 (2) Reid JF, Lusa L, De Cecco L, Coradini D, Veneroni S, Daidone MG, et al. Limits of predictive models using microarray data for breast cancer clinical treatment outcome. J Natl Cancer Inst 2005;97:927–30. (3) Sgroi DC, Haber DA, Ryan PD, Ma XJ, Erlander MG. RE: A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 2004;6:445. (4) Simon R. Development and validation of therapeutically relevant multi-gene biomarker classifiers. J Natl Cancer Inst 2005;97:866–7. (5) Hayward JL, Rubens RD, Carbone PP, Heuson JC, Kumaoka S, Segaloff A. Assessment of response to therapy in advanced breast cancer. A project of the programme on clinical oncology of the International Union against Cancer, Geneva, Switzerland. Eur J Cancer 1978;14:1291–2. (6) Jansen MP, Foekens JA, van Staveren IL, Dirkzwager-Kiel MM, Ritstier K, et al. Molecular classification of tamoxifen-resistant breast carcinomas by gene expression profiling. J Clin Oncol 2005;23:732–40. (7) Tian E, Sawyer JR, Largaespada DA, Jenkins NA, Copeland NG, Shaughnessy JD. Evi27 encodes a novel membrane protein with homology to the IL17 receptor. Oncogene 2000;19:2098–2109. NOTES Affiliation of authors: Department of Medical Oncology, Erasmus MC–Daniel den Hoe (...truncated)