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Because dexamethasone remains a key component of myeloma therapy, we wished to examine the impact of baseline and relapse expression levels of the glucocorticoid receptor gene NR3C1 on survival outcomes in the context of treatment with or without thalidomide.
We investigated the clinical impact of gene expression profiling (GEP)–derived expression levels of NR3C1 in 351 patients with GEP data available at baseline and in 130 with data available at relapse, among 668 subjects accrued to Total Therapy 2 (TT2).
Low NR3C1 expression levels had a negative impact on progression-free survival (PFS, HR=1.47; p=0.030) and overall survival (OS, HR=1.90; p=0.002) in the no-thalidomide arm. Conversely, there was a significant clinical benefit of thalidomide for patients with low receptor levels (OS, HR=0.54, p=0.015; PFS, HR=0.54, p=0.004), mediated most likely by thalidomide’s up-regulation of NR3C1. In the context of both baseline and relapse parameters, post-relapse survival (PRS) was adversely affected by low NR3C1 levels at relapse in a multivariate analysis (HR=2.61, p=0.012).
These findings justify the inclusion of NR3C1 expression data in the work-up of patients with myeloma as it can significantly influence the choice of therapy and, ultimately, OS. The identification of an interaction term between thalidomide and NR3C1 underscores the importance of pharmacogenomic studies in the systematic study of new drugs.
Our Total Therapy 2 (TT2) protocol was a randomized phase-III trial evaluating the impact of the up-front addition of thalidomide to a multi-agent chemotherapy and high-dose melphalan program supported by tandem autotransplants.(1, 2) The long median overall survival (OS) of 10 years among 668 patients accrued to this protocol affords the unique opportunity to examine the contributions to clinical outcomes of added thalidomide in the context of baseline clinical and tumor-specific molecular variables and salvage strategies employed.
Because dexamethasone remains an important component of myeloma therapy, we have studied and reported on the prognostic implications of the glucocorticoid receptor gene NR3C1, which is upregulated by both dexamethasone and thalidomide following test-dosing of both agents.(3) Here, we examine the impact of baseline and relapse NR3C1 expression levels on survival outcomes in the context of randomization to control or thalidomide treatment in TT2. As this required the availability of gene expression profiling (GEP) data of purified plasma cells, our analysis was limited to 351 TT2 patients with such baseline information and to 130 who had GEP data obtained at the time of relapse.
Protocol details and clinical outcomes have been reported previously.(1, 2) In brief, 668 patients with newly diagnosed multiple myeloma received two cycles of intensive melphalan-based chemotherapy, each supported by autologous hematopoietic stem-cell transplantation. A total of 323 were randomly assigned to receive thalidomide from the outset until disease progression or undue adverse effects, and 345 did not receive thalidomide. Patients who were initially randomized to not receive thalidomide (control arm) had the opportunity to be treated with a thalidomide -based regimen after relapse.
All patients signed an informed consent acknowledging the investigational nature of the protocol and agreeing to the ongoing research investigations. The protocol and its revisions were approved by the Institutional Review Board at our institution, which also received annual follow-up reports. Approximately 80% of all patient records have been audited by an independent team of investigators. Due to its randomized trial design and grant support from the National Institutes of Health, a Data and Safety Monitoring Board was convened annually to review the protocol.
GEP samples were obtained as previously described,(4) and both GEP-defined risk(5) and molecular subgroup designations were determined(6) in addition to NR3C1 expression levels and GEP-derived TP53 deletion status.(7) To guard against bias, the subsets of patients with and without baseline GEP data were compared. This revealed no differences in progression-free survival (PFS), OS, or post-relapse survival (PRS) (p=0.17, p=0.40, p=0.11, respectively data not shown). There were no differences in prognostic features between the GEP and no GEP baseline groups, such as age, albumin, B2M, CA. The analysis is based on data with a cut-off date of March 16, 2012.
OS and PFS were measured from the time of protocol enrollment. Events included death from any cause for OS and death, relapse, or progression for PFS. PRS was measured from time of relapse until death. Responses were defined according to International Myeloma Working Group criteria.(8) Kaplan-Meier statistical methods were employed for OS, PFS, and PRS plots, and the log-rank test was used for comparisons.(9) Cox regression modeling(10) was used to determine which baseline and relapse parameters significantly affected the aforementioned endpoints. Variables included in multivariate models were selected using stepwise selection techniques, requiring a significance level of 0.10 for entry into the model and 0.05 to remain. CR was defined according to the IMWG criteria (8)
NR3C1 expression was defined as gene expression of the probe 261321_s_at on the Affymetrix U133Plus2 microarray. There was no significant difference between the five probes representing the NR3C1 gene. All samples were ordered according to their NR3C1 expression level and then divided in 3 equal groups of 117 patients each with low (895 to 3124), mid (3136 to 4284) and high (4301 to 12,158) NR3C1 expression. NR3C1 expression groups (low, mid, high) at relapse were defined using cutoffs of <= 2280 and >=3885. We also evaluated the effect of NR3C1 groups at relapse defined by baseline cutoffs. This approach however performed worse than using NR3C1 groups defined by gene expression at relapse and thus was excluded from further analysis (data not shown).
Baseline GEP data has previously been published and deposited in the NIH Gene Expression Omnibus (GEO, National Center for Biotechnology Information [NCBI], http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE2658. Relapse GEP data presented in this manuscript have been deposited in the NIH GEO under the accession number GSE38627.
Table 1 compares patient characteristics according to NR3C1 levels. Patients with low NR3C1 expression were more likely to present with low albumin, high LDH or to have high-risk features such as cytogenetic abnormalities or GEP-defined high risk. There was also a preponderance of GEP-defined Cyclin D2 (CD-2) and Proliferation (PR) molecular subgroups in the low NR3C1 group, whereas the Hyperdiploid (HY), Low bone disease (LB) and MAF/MAFB (MF) subgroups (6) were under-represented. There were fewer patients with an amplification of chromosome 5q to which NR3C1 maps. Among patients randomized to the control arm, OS improved with the transition from low to mid or high NR3C1 expression; no difference was noted in PFS between high- and mid-expression groups. Among patients randomized to thalidomide, OS and PFS were NR3C1-expression-neutral (Supplemental Figure 1). When examined within NR3C1 tertiles, thalidomide benefited both OS and PFS in the low-expression group. PFS but not OS was extended by thalidomide in patients the mid-expression group whereas, in patients with high expression of NR3C1, no difference was observed between thalidomide and control arms (Supplemental Figure 2).
This observation suggested an interaction between NR3C1 expression levels and treatment arms. The presence of an interaction describes a situation in which the effects of two variables on a third are not simply additive. Thus, the presence of an interaction term would imply that the effect of thalidomide on survival outcomes varies as a function of NR3C1 expression level. This was further examined and validated for both OS and PFS. In the control arm, low NR3C1 expression significantly increased the hazard of death to 1.90 (p=0.002) compared with mid- and high receptor levels, whereas, in the thalidomide arm, NR3C1 expression did not affect OS (HR=1.01, p=0.972) (Figure 1A). This trend was also seen for PFS where, in the control arm, low NR3C1 expression significantly increased the hazard of progression or death to 1.47 (p=0.030); in the thalidomide arm, low NR3C1 did not significantly impact PFS (HR=1.13, p=0.552) (Figure 1B). CR frequency and CR duration were not affected by NR3C1 levels (data not shown).
Univariate analysis of survival outcomes across treatment arms showed that many of the well-established prognostic features, including levels of β2-microglobulin (B2M), albumin, creatinine, lactate dehydrogenase (LDH), and hemoglobin, as well as metaphase cytogenetic abnormalities (CA), GEP-defined high-risk designation (5), deletion of TP53 and the GEP defined MMSET/FGFR3 (MS) subgroup (6) affected both PFS and OS adversely (Table 2, Panel A). LB and HY subgroup designation had a favorable effect on PFS and OS respectively. Low NR3C1 expression conferred inferior OS, whereas randomization to thalidomide prolonged PFS. On multivariate analysis, across treatment arms (Table 2, Panel B), the presence of CA, elevated B2M, GEP-defined TP53 deletion, high-risk status in the 70-gene model (11) and MS subgroup designation adversely affected the OS and PFS. Elevated LDH had an adverse effect on OS only. Patients with low NR3C1 expression who were randomized to thalidomide (interaction term) had improved OS with a HR of 0.63 (p=0.007).
We further investigated the effect of cumulative thalidomide dose on survival. For this we calculated the cumulative thalidomide dose from protocol enrollment until the start of maintenance therapy. OS and PFS were measured from the beginning of maintenance therapy. Patients on the thalidomide arm, who received a cumulative dose that was greater than the median showed a trend towards a better PFS compared to patients who received equal or less than the median dose (p = 0.083), and was significantly better than receiving no thalidomide at all on the control arm (p = 0.002), with 5-year survival estimates of 63%, 53% and 42% respectively. There was no significant difference between low cumulative thalidomide dose and the control arm (p=0198). There was also no significant difference in OS between the three groups (supplemental figure 3). The effect on PFS was even more pronounced when the analysis was limited to the patients with low expression of NR3C1 (PFS: 73% vs. 53% vs 44%, logrank p-value= 0.05; OS: not significant; data not shown)
In addition to initial PFS, PRS is an important component in determining the total length of OS. Salvage regimens are depicted in Supplemental Table 1. There was no difference between the treatment arms. With an overall median PRS of 3.4 years, there was no difference related to the initial treatment randomization when examined for all patients (Figure 2A) or in relation to type of salvage therapy (Supplemental Figure 4). Examining PRS in the subset of patients with available NR3C1 data at baseline or at relapse an adverse PRS trend was apparent for patients randomized to thalidomide (Figure 2B). We investigated the impact of NR3C1 expression levels at relapse on PRS. PRS shortened progressively as the NR3C1 levels decreased from high to mid to low levels (Figure 3). For the 88 patients with available baseline and relapse GEP data we also examined PRS in the context of both baseline and relapse NR3C1 levels. Patients maintaining low NR3C1 levels from baseline to relapse and those transitioning from mid/high expression to low expression had the shortest PRS duration. Patients with high levels at relapse had the longest PRS regardless of NR3C1 expression levels at baseline (Supplemental Figure 5). However, due to the small sample number in each group this last observation needs to be considered with some caution.
We also examined PRS in the context of potentially relevant prognostic baseline and relapse variables. On univariate analysis, whether taken at relapse or baseline, low NR3C1 levels imparted short and high NR3C1 levels longer PRS (Table 3, Panel A). In addition, many standard and newer genetic variables affected PRS. Age >=65yr, elevated baseline B2M or LDH, GEP high-risk designation at baseline and relapse, MS subgroup classification at baseline, PR subgroup classification at baseline and relapse and deletion of TP53 were associated with shorter PRS. CD-2 subgroup classification at baseline and HY classification at relapse were prognostically favorable. Adjusting for all individually significant baseline and relapse variables in a multivariate regression analysis, low NR3C1 expression levels at relapse imparted inferior while relapse HY subgroup designation conveyed superior PRS (Table 3, Panel B). GEP-defined high-risk status, whether examined at baseline or relapse, both conferred poor PRS.
Glucocorticoids, such as dexamethasone, have marked anti-myeloma activity (12) and have been shown to act synergistically with most other anti-myeloma agents, justifying their use in combination regimens.(13–16) However, only approximately one-half of newly diagnosed patients respond to single-agent dexamethasone and CRs are infrequently observed.(17, 18) Thus, steroid-resistant tumor cell subpopulations exist both de novo and, in higher proportion at the time of relapse.(19, 20)
Most glucocorticoid hormone effects are mediated by the glucocorticoid receptor. Although there is only one known gene encoding this receptor, NR3C1 (located on chromosome 5q31.3), several receptor isoforms result from alternative splicing.(21, 22) Poor clinical responses to glucocorticoid therapy associated with low expression of the receptor have been previously reported for multiple myeloma (23) and other malignancies.(24) This correlation can be reproduced in vitro with glucocorticoid-resistant myeloma cell lines.(25) Comparing gene profiles of glucocorticoid-sensitive myeloma cells (MM1.S) with those that are glucocorticoid-resistant (MM1.RE and MM1.RL) revealed a significant reduction in NR3C1 mRNA, which was correlated with decreased expression of glucocorticoid receptor protein and glucocorticoid resistance.(22)
The favorable survival effects of high NR3C1 expression levels are consistent with a good prognosis linked to gains of chromosome 5q31.3.(26) This matches our observation that amplification of 5q, identified by a virtual GEP-based karyotyping model (11) is significantly overrepresented in the group of patients with middle or high expression of NR3C1. In a previous analysis of myeloma GEP among patients enrolled in the TT3 protocol,(27) high NR3C1 levels were associated with superior and low levels with inferior survival outcomes. We also noted that cumulative glucocorticoid dosing during induction therapy extended both OS and PFS significantly when NR3C1 expression was low; this favorable survival effect seen for thalidomide but was not observed with bortezomib.(28)
Analyzing gene expression profiles after short-term exposure to various agents in vivo, we found that NR3C1 was upregulated by both dexamethasone and thalidomide.(3) In the current study, we show that added thalidomide in the TT2 protocol neutralizes the inferior prognosis of patients with low glucocorticoid receptor gene expression, implying that thalidomide compensates for the deleterious effect of low NR3C1 expression by inducing up-regulation of this receptor and that failure of exogenous glucocorticoids to induce apoptosis in tumor cells with insufficient glucocorticoid receptor levels can be reversed by the addition of thalidomide.
Due to the study design we cannot evaluate the effect of thalidomide alone on PFS and OS and, therefore, are not able to determine whether the survival benefit for low NR3C1 patients was due to the combination of thalidomide and dexamethasone or could have been achieved with thalidomide alone, nor can we evaluate the effect of different doses of dexamethasone on survival. Rajkumar et al. recently showed improved short-term OS for patients treated with low-dose dexamethasone plus lenalidomide compared to high-dose dexamethasone plus lenalidomide.(29) The doses of glucocorticoids used in TT2 compare to the high dose arm in the study by Rajkumar et al. It is conceivable that patients with high NR3C1 expression at baseline derive little benefit from the high dose of glucocorticoid and would possibly have done just as well if a lower dose was used. Conversely, using a lower dose of dexamethasone in patients with low NR3C1 expression levels would have had a detrimental effect as high doses of glucocorticoid most likely partially overcome the adverse effect of low NR3C1 expression levels.
In the current study, thalidomide extended OS and PFS in patients presenting with low baseline NR3C1 expression levels. Among the patients treated with thalidomide, higher cumulative doses resulted in a significantly better landmarked PFS compared to no thalidomide and showed a trend towards significance in the comparison between high and low cumulative thalidomide doses, suggesting a dose dependent effect. However this could also be a reflection of other myeloma related factors causing intolerance to thalidomide, thus making it de-facto a more aggressive disease. In addition, relapse GEP features impacted PRS, with low NR3C1 levels and GEP-defined high-risk having adverse roles and the HY subclass designation having a favorable impact. The latter most likely reflect the better prognosis of patients with gains of chromosome 5 and which coincides with mid- and high NR3C1 expression as mentioned earlier.
We believe our findings justify the inclusion of NR3C1 expression data, or other means of glucocorticoid receptor quantification, in the work-up of patients. For example, if patients with high NR3C1 expression levels do not benefit from upfront thalidomide (and possibly newer immune-modulatory derivatives), reserving these agents for treatment of relapse, when NR3C1 levels are lower, may prolong overall survival.
Glucocorticoids have remained an important component of myeloma therapy, owing to their independent activity and synergism with most other agents currently in use. Results of our Total Therapy 2 trial, randomizing approximately half the patients to receive thalidomide, show that overall and progression-free survival are linked to expression levels of the nuclear glucocorticoid receptor, NR3C1, with low expression imparting poorer outcomes in the control (no thalidomide) arm, which was overcome by the addition of thalidomide. In contrast, myeloma with high NR3C1 levels did not benefit from the addition of thalidomide. Should these findings also apply to second- and third-generation thalidomide analogues (lenalidomide, pomalidomide), their application in high-NR3C1 myeloma should be reserved for salvage therapy as relapse is often associated with a decrease or even loss of NR3C1 expression.
Support: CA 55819-15 from the National Cancer Institute, Bethesda MD, US
CONFLICT OF INTEREST DISCLOSURES
JDS is co-founder of Myeloma Health LLC and owns stock in the company; he receives royalties from Novartis, Genzyme, and Myeloma Health, and he is a paid consultant to Novartis, Myeloma Health, Genzyme, Array BioPharma, Onyx, Millennium and Celgene.
BB has received research funding from Celgene and Novartis. He is a consultant to Celgene and Genzyme. He has received speaking honoraria from Celgene and Millennium. Dr. Barlogie is a co-inventor on patents and patent applications related to use of GEP in cancer medicine.
SZU has served as a consultant to Celgene, Millennium, and Onyx. He has received research funding from Celgene and Onyx, and speaking honoraria from Celgene and Millennium.
AUTHORSHIP CONTRIBUTIONSConceptualized work: CJH, JS, JDS, FvR, JE, BB
Wrote paper: CJH, JS, JDS, SU, FvR, BB
Contributed patients and performed clinical research: FvR, BN, SW, YA, SU, BB
Supervised and discussed gene expression profiling analyses: JDS
Performed statistical analyses: JS, AH, JC,EH
Provided data management support: CB, NP