Effect of Continuous Lenalidomide Treatment on Copy Number Alteration and Cytogenetic Abnormalities in Multiple Myeloma Cells

In the last years the role of clonal selection and evolution has been underlined in the progression of multiple myeloma (MM). The potential role of anti-myeloma drugs on clonal selection is currently under investigation. Continuous Lenalidomide (LEN) treatment is becoming the backbone of MM therapy either in young patients as maintenance after stem cell transplantation or in newly diagnosed elderly patients, however the potential role of Len in the clonal selection and the emerging of resistant clones are unknown and has been explored in this study.

The experimental approach used was firstly to analyze the sub-clonal evolution in human myeloma cell lines (HMCLs) (JJN3, KMS-12 and RPMI-8226) after long term treatment with low dose of LEN (2 µM) or vehicle (DMSO), mimic the continuous treatment regimens of MM patients, collecting sample at starting point (T0), 1 month and 2 months for single nucleotide polymorphism (SNP)+Comparative Genomic Hybridization (CGH) arrays and fluorescence in situ hybridization (FISH) analyses. Secondly to confirm data by FISH analysis on purified CD138⁺ plasma cells (PCs) from MM patients treated continuously with LEN.

In all collected samples both copy number alterations (CNAs) and loss of heterozygosity (LOH) regions have been detected, ranging from few Kb to more than 10 Mb. These alterations were not mainly specific for LEN treatment being also detected in DMSO treated samples. Moreover, FISH analysis of IGH locus translocations and of cMYC amplification/translocations revealed that the mutational pattern reported at T0 did not change after LEN treatment in the analyzed HMCLs. In the combined CGH+SNP arrays few regions with CNAs and LOH seemed to be specific only in LEN treated samples. In particular, in the RPMI-8226, we detected the presence of a LOH in the 5p region (chr5:14547524-36790726) at T0 but, in the same region, we reported a mosaic duplication in the Len treated samples at 1 month (log ratio: 0,45) and 2 months (log ratio: 0,46) as compared to the vehicle. In this chromosome (Chr) 5p region different genes are coded, including S-phase kinase-associated protein 2 (SKP2) an oncogene known to be involved in tumor polyploidy in cancer cells and in Bortezomib resistance of MM cells. Interestingly our recent data obtained in the LEN resistant cells JJN3 indicated that SKP2 was down-regulated overcoming LEN resistance by hypoxia inducible factor (HIF)-1α knock-out.

Based on these data, we performed FISH with SKP2 probe on 5p13.2 locus (RP11-RP23 Empire Genomics) and a control probe on 5p11 locus in RPMI-8226 and in JJN3 samples. FISH analysis revealed in the LEN resistant cell line JJN3 at T0 the coexistence of multiple clones with different degrees of chr5 polysomy, presumably reflecting different ploidy levels, which are not modified by LEN treatment, in all the time point considered. This justified CGH+SNP array data reporting an amplification of SKP2 locus (JJN3 T0 logratio: 1,295; JJN3 DMSO logratio:1,205; JJN3 LEN logratio:1,12), related to chr5 polysomy. Moreover FISH revealed that RPMI-8226 at T0 showed a single clone with chr 5 tetrasomy with SKP2 deletion on two 5p13.2 locus, presumably deriving from the diploid clone with monoallelic SKP2 deletion, reflecting LOH as reported by the CGH+SNP array. After the long-term treatment with either LEN or DMSO, RPMI-8226 developed different sub-clones with different degrees of Chr5 polysomy, maintaining SKP2 deletion suggesting that LEN had not a significant impact on their development. To confirm these observations we analyzed CD138⁺ cells from 4 MM patients (2 newly diagnosed and 2 relapsed) treated for almost 1 year with LEN and dexamethasone that developed LEN resistance. FISH analyses on bone marrow PCs revealed that 3 of 4 patients showed Chr5 trisomy, coupled with trisomy of Chr9 and Chr15, defining them in the hyperdiploid group; this cytogenetic architecture was not altered at the moment of relapse during LEN treatment. In conclusion, our data suggest that long-term continuous LEN treatment did not induce a clonal MM cell selection and that a possible relationship between the amount of the SKP2 locus and the polyploid level may exist in MM cells. Indeed the potential relationship between SKP2 status and LEN resistance deserve further investigation.