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only Cyclin D1 and Cyclin D2 protein levels were strongly correlated with the respective CCND1 and CCND2 mRNA levels (Figure 4D). We observed a modest correlation for Aiolos, Calnexin and DDX21 proteins with their respective mRNA.
Although the number of proteins analyzed was limited, we examined the correlation between the levels of the dif- ferent proteins. A positive correlation was observed between most of the protein pairs (Online Supplementary Figure S2). We confirmed the previously described relation- ship between Ikaros/Aiolos and c-myc.19 Additionally, c- myc protein expression was positively correlated with Cereblon, Calnexin, and RIPK1, and negatively correlated with DDX21 (Online Supplementary Figure S2). We found that protein levels of Cereblon, Ikaros and Aiolos, all of which are required for the activity of immunomodulatory drugs, were correlated with each other (Online Supplementary Figure S2).
The potential association between the expression of pro- teins and mRNA tested in the study and chromosomal abnormalities was also explored. We confirmed the well- established pattern of CCND1/Cyclin D1 and CCND2/Cyclin D2 expression in t(11;14) and t(4;14) (Online Supplementary Figure S3). A lower level of expression of PSME1 and RIPK1 proteins in MM with 1q gains, and a higher level of IKZF1 mRNA expression in MM with t(11;14) were also observed.
Influence of mRNA and protein levels on survival of myeloma patients
Since clinical data were available for 43 MM patients, 24 enrolled in GEM 2010 and 19 in BenVelPres clinical trials, we also performed survival analysis for proteins and mRNA using PFS as the endpoint (Table 2). Cereblon and Ikaros were the only proteins able to predict PFS. Interestingly, mRNA levels of CRBN and IKZF1 were not associated with prognosis (Figure 5). Accordingly, patients with a high level of Cereblon protein had a longer PFS than those with a low level (50.4 versus 16.3 months, P<0.001). Similarly, high levels of Ikaros protein were associated with longer PFS (45.1 versus 17.8 months, P<0.01). The levels of two mRNA were associated with longer PFS: a high level of PSME1 (50.4 versus 23.5 months, P<0.05) and a low level of XAF1 (20.3 versus 45.1 months,P<0.05).
Since Cereblon, Ikaros and Aiolos are involved in the mechanism of action of immunomodulatory drugs, and only GEM2010 patients were treated with lenalidomide, we examined whether the prognostic value of these pro- teins was influenced by the type of treatment. Indeed, high levels of Cereblon and Ikaros were both associated with longer PFS only in patients who received immunomodulatory drugs and not in those treated with other drugs (Figure 6).
ABD
C
Figure 3. Optimization of protein expression quantification. For each protein the standard curve was generated using the sample with the strongest signal to prepare the serial dilutions. Each dilution was run in a separate capillary. The sample result for Aiolos is visualized as a virtual blot (A) or as peaks (B). The standard curve established the linear range for each protein (C). The sample result for Aiolos together with the respective GAPDH for each sample was run in separate capillaries (D). Each peak area was quantified and Aiolos was normalized with respective to GAPDH, as shown in the example.
haematologica | 2018; 103(5)