Multiple myeloma (MM) is a hematologic tumor characterized by accumulation of monoclonal plasma cells (PCs) in the bone marrow (BM) producing antigen-specific immunoglobulins. The transcription factor X box binding protein 1 (XBP1), the interferon regulatory factor 4 (IRF4) and the transcriptional repressor B lymphocite-induced maturation protein 1 (BLIMP1) are essential to drive physiological plasmacytic differentiation.1,2 XBP1 is particularly required for the last stages of B-cell differentiation into PC, and, consistently, XBP1-deficient mice display normal B-lymphocite development up to germinal center, but are unable to produce PCs.2 High mRNA levels of IRF4, BLIMP1 and XBP1 have been detected in malignant PC and are negative prognostic factors in patients treated with standard chemotherapy or thalidomide.3,4 Lenalidomide seems to overcome the negative prognostic impact of IRF4 overexpression, due to its rapid downregulation following treatment. Bortezomib induces better responses in patients with high levels of XBP1.5,6
We assessed the prognostic role of gene-driven plasmacytic differentiation in a large cohort of patients treated with bortezomib. RNA expression of three genes involved in PCs differentiation was investigated in purified PCs (CD138 BM fraction) of well-characterized patients with newly diagnosed MM. One hundred and fifty-one patients enrolled in two multicenter clinical trials (the PAD-MEL100-LP-L and the VMP-VMPT) were assessed.7,8 PCs were purified using anti-CD138-coated magnetic MicroBeads and AutoMACS Pro Separator (Miltenyi Biotech GmbH, Germany) following manufacturer specifications. Gene expression was investigated on isolated PCs with more than 90% of purity assessed by flow cytometry. RNA was extracted using the DNA/RNA Purification Kit (Norgen, Thorold, Canada). Complementary DNA was produced using High capacity cDNA RT Kit (Applied Biosytem, Foster Ciy, CA, USA). Quantitative PCR to measure RNA expression of XBP1, IRF4 and BLIMP1 was performed with the Abi Prism 7900HT (Applied Biosystems, Carlsbad, CA, USA) using a relative quantification based on ΔΔCt approach and GUSB (β-glucuronidase) as housekeeping gene. All RNA determinations were performed using the following assays: Hs00231936_m1 (XBP1), Hs01056533_m1 (IRF4), Hs00153357_m1 (BLIMP1). Patients were divided according to gene expression using the median value as cut off. Response to therapy and clinical outcome were assessed following IMWG criteria.9 The progression-free survival (PFS) and overall survival (OS) were estimated by the Cox proportional hazard model, comparing the risk factors by the Wald test; best response was treated as a time-dependent variable. Patients’ characteristics were compared by the Fisher’s exact test for the categorical variables and the Mann-Whitney test for the continuous ones. All reported P-values were two-sided, at the conventional 5% significance level. Data were analyzed as of January 2013 by SPSS 21.0.0 and R 3.0.1 package survivalROC.
No differences in base-line β2-microglobulin and albumin levels have been observed between patients according to gene expression. A higher proportion of patients with high XBP1 had ISS I (n=24) compared to patients with low XBP1 (n=12, P=0.03). No differences in FISH karyotype were observed between patients with high and low XBP1 expression.
Though a recent study found an association between XBP1 RNA expression levels and response to treatment in patients receiving bortezomib-based therapy,6 in our study, no correlation between XBP1 expression and response to bortezomib-containing regimens was observed. Patients achieving a complete response had median XBP1 RNA expression (8.14; QR 4.68–13.76) similar to that of patients obtaining very good partial response (8.73; QR 3.56–12.72), partial response (8.26; IQR 3.82–9.93), or stable disease (7.68; IQR 4.11–11.12). The discrepancy between our study and the previous study6 may be due to differences in the: i) inclusion criteria (previously untreated versus relapsed patients, respectively); and ii) interval between BM investigation and start of bortezomib treatment (short vs. heterogeneous, respectively).
In our study, the 3-year PFS was 59% for patients with high XBP1 RNA expression and 28% for patients with low XBP1 (P=0.001), translating into a higher 3-year OS probability (86% vs. 74%; P=0.067) for patients with high XBP1 levels. High IRF4 RNA expression identified patients with better PFS (51% vs. 36% respectively; P=0.008) but with only slightly and not significantly improved OS (85% vs. 75% respectively; P=0.484) (Figure 1). No differences in PFS (P=0.444) and OS (P=0.529) differences have been observed according to BLIMP1 RNA expression. Similar results were obtained when only patients receiving the same treatment were analyzed even if no statistical significance was reached due to the low number of events in each subgroup.
In univariate analysis, response to therapy, XBP1 expression and IRF4 expression were the main predictors of PFS. Response to therapy also significantly correlated with OS, while XBP1 expression almost reached statistical significance (Table 1). In Cox multivariate analysis, response to therapy, XBP1 and IRF4 expression were shown to be independent predictors of PFS.
Although no correlation between XBP1 RNA expression and response to therapy was found, our results evidenced that patients with high XBP1 expression who received bortezomib-based therapy have a better outcome. Bortezomib inhibits the proteasome activity and induces apoptosis determining reduction of protein degradation and accumulation of misfolded proteins. In MM, the amount of immunoglobulin production (controlled also by XBP1) correlates with bortezomib sensitivity and XBP1 RNA decreases after bortezomib administration.10 Bortezomib is more effective in patients with high XBP1 expression, probably due to its key role in the unfolded protein response and in immunoglobulin production, suggesting that bortezomib could reduce protein degradation leading to immunoglobulin accumulation and finally to cell damage.
High expression of IRF4 was associated with poor prognosis in MM patients treated with standard chemotherapy, but lenalidomide can overcome its negative prognostic impact.3–5 IRF4 is one of the target genes of lenalidomdie and is necessary for the drug activity. Our study highlighted the prognostic role of IRF4 in MM patients receiving bortezomib, suggesting that all novel drugs can overcome the negative impact of high IRF4 expression.
High XBP1 showed to be a marker of improved outcome in MM patients treated with bortezomib. The combination of XBP1 expression and response to therapy further predicts better clinical outcome. Additional analyses are required to confirm these data in an independent cohort, to clarify the action of novel drugs on genes involved in plasmacytic differentiation and to evaluate the opportunity to include drugs targeting this pathway in the myeloma therapy.
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