Clinical significance of the nuclear receptor co-regulator DC-SCRIPT in breast cancer: an independent retrospective validation study

The protocol to study biological markers associated with disease outcome was approved by the medical ethics committee of the Erasmus Medical Center (Rotterdam, The Netherlands) (MEC 02.953). This retrospective study used 1,505 M0 (no metastasis) and 32 M1 (with metastasis) blind-coded freshly frozen primary tumor tissues of female patients with primary operable breast cancer from 1978 through 2000. The study was performed in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in The Netherlands [10], and consent was not required. Wherever possible, the study has been reported in accordance with the Reporting Recommendations for Tumor Marker Prognostic Studies guidelines [11]. The primary breast tumors were from patients with detailed clinical follow-up as previously described [12‐14]. ER protein status was determined by routine ligand-binding assays or enzyme immunoassays [15], and ESR1, ESR2, and PGR mRNA status was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) [14, 16, 17]. Follow-up, tumor staging, and response to therapy were defined by standard International Union Against Cancer (Geneva, Switzerland) classification criteria [18] and applied previously by Foekens and colleagues [19]. All 1,537 patients underwent breast-conserving lumpectomy (44%) or modified mastectomy (56%). Of the 1,505 patients included for the evaluation of tumor aggressiveness, 462 lymph node-positive patients (31%) were treated with adjuvant systemic therapy, 207 patients received hormonal therapy, 233 chemotherapy, and 22 combination therapy. Disease recurrence occurred in 836 patients, and 703 developed a distant metastasis. The median follow-up time of patients alive was 90 months (range of 4 to 260 months).

Eight hundred thirty-seven patients had no involved nodes and did not receive systemic adjuvant therapy. Of these 837 LNN patients, 383 had a disease relapse, 300 developed a distant metastasis, and 273 died during follow-up.

Of the 703 patients who developed a distant metastasis, 296 ER-positive patients, including the 32 M1 patients, received hormonal therapy as first-line therapy for advanced disease. Clinical benefit of first-line tamoxifen treatment was observed in 185 patients. Median follow-up time for treatment of advanced disease was 38 (4 to 120) months. Two hundred nineteen patients had died at the end of the follow-up. None of these patients had received prior adjuvant hormonal therapy, whereas 19% received prior adjuvant chemotherapy. A more detailed description of the patients and their therapy is given in the Supplementary materials and methods (Additional file 1). Patient and tumor characteristics combined with DC-SCRIPT mRNA expression and clinical outcome are listed in Table 1.

Tissue processing, RNA isolation, cDNA synthesis, and quantitative RT-PCR were performed as previously described [16]. Real-time quantitative PCRs were performed in a 25-μL reaction volume in an M×3000P™ Real-Time PCR System (Agilent, Amsterdam, The Netherlands). In addition to an SYBR-based assay to detect a 129-base pair (bp) DC-SCRIPT transcript covering exon 4 to 5 (forward primer: 5'-AAAGTCAAGCATGGAGTCATG-3'; reverse primer: 5'-GCTTCTGAGAGAGGTCAAAG-3'), a commercially available Taqman Gene Expression Assay from Applied Biosystems (Nieuwerkerk aan den IJssel, The Netherlands) covering exon 3 to 4 and generating a 62-bp product was used (Hs00403536_m1, RefSeq NM_152625.1). DC-SCRIPT levels were readily detected with both assays, and data generated with these assays correlated significantly (Spearman's rho = 0.87; P < 0.0001). We therefore performed our analyses on the real-time RT-PCR data generated with the Taqman assay, which is generally considered to be more specific. Intron-spanning primer sequences for the three reference genes - that is, hydroxymethylbilane synthase (HMBS), hypoxanthine-guanine phospho-ribosyltransferase (HPRT1), and β-2-microglobulin (B2M) - and for ESR1, ESR2, PGR, and real-time PCR conditions for these SYBR-based assays were as described previously [16, 17]. Forty rounds of amplification were performed, and fluorescent signals of the Taqman probe or SYBR green signal were used to generate cycle threshold (Ct) values from which mRNA expression levels were calculated. Ct values of HPRT1 and B2M were adjusted to the higher HMBS Ct values. Next, the expression levels of DC-SCRIPT were normalized against the average expression levels of the three reference genes as follows: mRNA target = 2^{(mean Ct reference genes - mean Ct target)} [16].

Primary tumor tissue was processed as described previously [16]. To assess the amount of invasive tumor cell nuclei relative to the amount of surrounding stromal cells, 5-μm sections were cut for hematoxylin-and-eosin staining before, during, and after the sections were cut for RNA isolation. Only specimens with at least 30% invasive tumor cell nuclei were included in this study.

The relationship between DC-SCRIPT and patient and tumor characteristics was investigated with the use of non-parametric methods (Spearman rank correlations for continuous variables and Wilcoxon rank-sum for dichotomized or Kruskal-Wallis test for ordered variables). To reduce skewness, DC-SCRIPT levels were transformed with the Box-Cox transformation. DC-SCRIPT levels were dichotomized with the previously identified 66.7% high versus 33.3% low cutoff for DC-SCRIPT [7]. To test for an association with tumor aggressiveness and the time to progression during first-line therapy, Cox regression analysis was applied on the Box-Cox-transformed and dichotomized DC-SCRIPT mRNA levels. The hazard ratio (HR) and its 95% confidence interval were computed to correlate the expression levels with DFS, MFS, OS, and PFS, respectively. In multivariable analysis, Cox proportional hazards models for DFS, MFS, OS, and PFS were applied to test DC-SCRIPT levels added to models with traditional factors. The proportional hazards assumptions were checked with Schoenfeld residuals. The analyses were stratified if necessary. The models for DFS, MFS, and OS for LNN patients who had not received adjuvant systemic therapy included age, menopausal status, tumor size, grade, and ESR1 and PGR mRNA levels. Survival curves were generated with the method of Kaplan and Meier. The log-rank test was used to test for differences between survival curves. Logistic regression was used for the association of DC-SCRIPT with clinical benefit. Computations were performed with the STATA statistical package, release 11.0 (STATA Corp., College Station, TX, USA) and SPSS 15.0 (SPSS Inc., Chicago, IL, USA). All P values are two-sided, and a P value of less than 0.05 was considered statistically significant.

In analogy with our previous study, DC-SCRIPT mRNA expression was readily detected by quantitative RT-PCR in five normal breast tissues taken adjacent from tumor tissue and five prophylactic breast tissues (median [interquartile]: 0.063 [0.015] and 0.054 [0.035], respectively), whereas median levels were over 8-fold lower (P < 0.05) in 1,505 invasive breast tumors (0.0069 [0.0074]). Table 1 shows the median expression levels and interquartile ranges of DC-SCRIPT transcripts and relation with patient and tumor characteristics for these 1,505 patients who were evaluable for prognosis. DC-SCRIPT levels were positively associated with tumor grade and ESR1, PGR, and ESR2 steroid hormone receptor expression level and negatively associated with invasive epithelial tumor cell content and tumor size. In addition, ESR2 was more highly expressed in tumors with a higher percentage of stromal cells (786 tumors with 30% to 70% invasive epithelial cells), and ESR1 was more highly expressed in tumors with a high percentage of invasive epithelial cells (719 tumors with at least 70% invasive epithelial cells) (P < 0.001) (data not shown). High levels of DC-SCRIPT were found in breast tumors with a ductal carcinoma in situ (DCIS) component or infiltrating lobular carcinoma compared with infiltrating ductal carcinomas (both P < 0.01). Of 308 LNN tumors, intrinsic subtyping data were available [20]. In these tumors, basal-like tumors had the lowest levels and normal-like breast tumors expressed significantly higher levels of DC-SCRIPT compared with the other intrinsic subtypes (P < 0.001; Figure S1 in Additional file 2). Furthermore, luminal A tumors expressed higher levels of DC-SCRIPT and ESR2 but lower levels of ESR1 compared with luminal B tumors (median levels in luminal A versus luminal B: 0.0078 and 0.056 for DC-SCRIPT [P = 0.003], 0.0095 and 0.0023 for ESR2 [P < 0.001], and 6.1 and 13.6 for ESR1 [P < 0.001]). This may be explained at least partly by the fact that, in this cohort of 308 LNN tumors, the luminal B tumors contained a higher percentage of invasive epithelial cells (mean ± standard deviation [SD]: 77% ± 9% for the n = 64 luminal B tumors versus 67% ± 12% for the n = 71 luminal A tumors).

In the analyses including all 1,505 M0 patients, increasing levels of DC-SCRIPT mRNA were significantly associated with favorable DFS, MFS, and OS (HR 0.78, 0.74, and 0.77, respectively; all P < 0.001). To test for a relation between DC-SCRIPT mRNA levels and tumor aggressiveness (that is, the natural course of the disease without the confounding effect of systemic adjuvant therapy), we restricted our next analyses of MFS to those 837 LNN disease patients who had not received (neo)adjuvant systemic therapy. The significant relationships of DC-SCRIPT as a continuous variable in these univariate analyses justified the use of the previously identified cut point that dichotomized the cohort in 33.3% of the patients with low levels and 66.7% of patients with high levels of DC-SCRIPT mRNA in their primary tumors [7]. In univariate analysis, high levels of DC-SCRIPT were significantly associated with a favorable prognosis (HR 0.55; P < 0.001) (Table 2). When added to a multivariable base model for LNN disease - which included the traditional prognostic factors of age, menopausal status, grade, and PGR - stratified by ESR1 and tumor size to meet the proportional hazards assumption, the association of DC-SCRIPT with MFS remained highly significant (HR 0.60; P < 0.001) (Table 2). Adding ESR2 to the model did not significantly affect the prognostic value of DC-SCRIPT in these analyses (Table 2).

Because the proportional hazards assumptions were violated by ESR1 and tumor size and because DC-SCRIPT is a transcriptional co-regulator of nuclear receptors - including the, for breast cancer biologically relevant, steroid hormone receptors - we next explored its prognostic value as continuous variable in subgroups of tumors stratified by steroid hormone receptor status and tumor size (Table 3 and Figure 1). Subdividing the 837 primary LNN tumors into ESR1-positive and -negative [14] showed that increasing levels of DC-SCRIPT were, in univariate and multivariable analyses, associated with good prognosis only for the patients with ESR1-positive tumors. Subdividing these LNN tumors at the median level of ESR2 into high and low revealed that, in contrast to ESR1, increasing levels of DC-SCRIPT were, in both univariate and multivariable analyses, associated with good prognosis only for patients with primary tumors with low levels of ESR2. With respect to tumor size, in univariate and multivariable analyses, increasing levels of DC-SCRIPT were associated with good prognosis only for pT1 (small tumor without lymphatic/vascular invasion) tumors and not for larger tumors. These and additional exploratory Cox univariate analyses are summarized in Table 3. The prognostic value of DC-SCRIPT is visualized in Kaplan-Meier curves (Figure 1) as a dichotomized variable in these biologically relevant LNN ESR1-negative (Figure 1a) and -positive (Figure 1b) and LNN ESR2-high (Figure 1d) and -low (Figure 1e) subsets in combination with patients with pT1 primary tumors (Figure 1c, f).

DC-SCRIPT expression levels were evaluated in 296 hormone-naïve ER-positive primary breast tumors from patients whose relapse was treated with first-line tamoxifen monotherapy. These patients had not received (neo)adjuvant endocrine systemic treatment. In univariate analyses, no statistically significant associations were observed between DC-SCRIPT as transformed continuous variable and PFS or clinical benefit after start of first-line treatment with tamoxifen (HR = 1.08 [0.99 to 1.18], P = 0.07 and odds ratio = 0.88 [0.74 to 1.05], P = 0.16, respectively).