Expression of tumor-associated genes in multiple myeloma patients during high-dose chemotherapy and auto-SCT

Summary

High-dose therapy enables further improvement in the outcome of multiple myeloma (MM) patients. However, it is still necessary to determine prognostic factors that may influence treatment results and provide additional criteria for the precise selection of treatment approaches. There are several tumor-associated genes (MAGE, LAGE, GAGE, PRAME) that are over-expressed in different malignancies including MM. These genes are believed to modulate cancer properties and should be taken into account during treatment. Their significance as prognostic factors is under investigation.

The aim of our study was to analyze the expression levels of PRAME, WT1, and XIAP genes in MM patients at diagnosis and during high-dose chemotherapy followed by auto-SCT and therapy of proteasome inhibitors.

Our study included 34 primary MM patients; all of them gave informed consent. The median age was 48 years (range 31–62). IgG MM was diagnosed in 28 cases, IgA MM in 2 and light chain MM in 4. Initial treatment consisted of 3 cycles of VAD. If CR or PR were not achieved, the treatment was changed to bortezomib at 1.3 mg/m² on days 1,4,8, and 11 and dexamethasone (dex) at 40 mg daily on days 1–4 (4–6 cycles). If CR or PR was attained, stem cell mobilization was performed with cyclophosphamide 6 mg/m²+G-CSF. Melphalan at 200 mg/m² was given before auto-SCT. Investigation was performed before therapy (n=34), after VAD (n=20), after bortezomib (n=10), and after auto-SCT (n=9). Results were normalized against the expression of the ABL gene, which was used as an internal control.

In primary MM patients, PRAME gene expression was found in 62% (n=21), WT1 in 20% (n=7) of patients, all of who were PRAME positive. Median expression levels were 0.3% (0.001–132%) for PRAME and 0.01% (0.002–2.54%) for WT1. In primary MM patients the XIAP expression was found in 100%. The meaning of XIAP/ABL*100% varied between the range of 5 to 5382% (median 28%). Of the MM patients, 41% (n=14) demonstrated XIAP hyperexpression (XIAP/ABL*100%>40%). We subdivided MM patients into two groups according to XIAP/ABL*100%, meaning that I<40%, II>40%. In the control group of healthy donors (n=9) PRAME gene expression was found in 50% (median expression 0.003%), WT1 in 12% (median 0.0017%), and XIAP in 62% (median 2%). PRAME, WT1, and XIAP expression did not correlate with tumor bulk and was independent of the levels of M–protein, beta-2M, and albumin.

In the group with primary XIAP hyperexpression (II) we observed that the decrease of XIAP expression paralled tumor reduction (from more then 40% to 5–20%). By contrast, in group I the XIAP gene expression increased right after chemotherapy initiation to extremely high levels of 2425%. However, after high-dose melphalan and auto-SCT, the XIAP level significantly decreased (22–157%) along with the attainment of CR or very good PR. The level of the XIAP gene expression changed depending on the response of the bortezomib treatment. In the MM patients who achieved CR+PR after 4–6 courses of bortesomib+dex (n=6), the expression of XIAP reduced from 11–325% (median 66%) to 1–123% (median 20%). In the MM patients with a poor response after 4–6 courses of bortesomib+dex (n=4), we observed an increased expression of XIAP from 16–127% (median 36%) to 22–528% (median 121%).

The expression of PRAME significantly decreased after 3 VAD cycles to 0.001–207% (n=10): at the moment of auto-SCT it was 0.001–6.1% (n=6) and after auto-SCT it was 0.004–4.9% (n=9). However, for WT1we observed an increase of WT1 expression after 3 VAD cycles to 0.004–0.07% (n=5); at the moment of auto-SCT it was 0.001–0.4% (n=6), and after auto-SCT it was 0.0193–2.03% (n=7). In the patients with high primary PRAME expression (>median expression) the frequency of CR+PR was significantly lower than in PRAME–negative primary patients and in patients with low (<median expression) primary PRAME expression (27% vs 69). It should be stressed that during treatment in a small number of initially negative PRAME and WT1 gene patients, we demonstrated detection by PCR. We detected the appearance of gene expression during therapy at low levels in 1 of 13 initially negative PRAME patients (6.1%) and in 8 of 26 initially negative WT1 patients (range of level 0.01–1.03%). The detection of gene expression did not correlate with disease status. All these patients achieve CR+VGPR. In one of the secondary positive patients – who acquired PRAME and WT1 – relapse occurred, with high and low expression of PRAME (104%), WT1 (0,62%), and XIAP (1264%).

Conclusion

The expression of the PRAME gene was found in 62% of primary patients, and the level of PRAME decreased with tumor reduction. A high expression level of PRAME turned out to be a factor of unfavorable prognosis. The expression of WT1 was found in 20% of MM patients – all of who were PRAME positive. WT1 expression increased during treatment in a small group of patients. Some initially negative patients acquired PRAME and WT1 expression during treatment, but clinical relevance of this is so far unclear. In the MM patients at diagnosis, the level of XIAP expression was high. The decrease of XIAP expression correlates with the chemotherapy and proteasome inhibitor treatment efficicacy. XIAP expression comes to the normal values at the time of CR and PR achievement.

Keywords

multiple myeloma, prognostic factors, tumor associated genes