Enrichment Of Embryonal Stem Cell Signature and Persistent Genomic Complexity In Residual Disease Following Pomalidomide Therapy In Myeloma

Current therapy of myeloma including combination with IMID(R) immunomodulatory agent such as pomalidomide (POM) leads to high rates of clinical responses in patients with advanced multiple myeloma without translating to cures. Therefore, there is a need to better characterize the nature of residual disease after anti-myeloma therapy. POM has shown clinically promising efficacy in relapsed myeloma but nearly all patients eventually progress. In order to better understand the nature of residual disease, we compared baseline CD138+ myeloma tumor cells from 3 patients with those remaining after completing 2 cycles of therapy with POM (2-4 mg/day) and dexamethasone (40 mg/week). CD138+ plasma cells were isolated from the bone marrow before and after POM therapy and analyzed with gene expression profiling (GEP), and whole exome sequencing (WES). Principal component analysis (PCA) on the GEP data (pre vs. post POM treatment) revealed that in contrast to baseline samples, the ones with residual disease were clustered together, and this was further confirmed with unsupervised hierarchical clustering. Analysis of differentially expressed genes revealed nearly 600 differentially modulated genes, including some involved in the immune system regulation, inflammatory pathways and stem cells. Gene set enrichment analysis (GSEA) identified enrichment of distinct gene-sets/pathways including- transcriptional targets regulated by core embryonal stem (ES) cell factors SOX2 and NANOG. Analysis of genes in the core ES signature (Nat Genet. 2008; 40: 499) revealed that nearly 80% of these ES genes were enriched in the residual disease after treatment with POM. Whole exome sequencing has emerged as a powerful tool to dissect the genomic complexity in cancer. Genomic DNA from bone marrow derived CD138+ tumor cells before and after POM therapy was captured on the NimbleGen 2.1M human exome array and subjected to 74 base paired-end reads on the Illumina HiSeq instrument as described previously (Proc Natl Acad Sci U S A. 2009; 106:19096). Sequence reads were mapped to the reference genome (hg19) using the ELAND program. Reads outside the targeted sequences were discarded and statistics on coverage were collected from the remaining reads using perl scripts. ELAND was also used for indel detection. For matched normal/germline and CD138+ tumor pairs, somatic mutations were called by comparing reference and non-reference reads from the matched pair by Fisher’s exact test with tumor-specific thresholds determined from approximation of the null distribution. WES analysis of baseline (pre therapy) samples identified a median of 36 protein altering coding mutations per sample. Importantly, the degree of mutational load was very comparable between baseline and residual disease (post therapy), and nearly 80% of the mutations detected in the residual disease were also observed at baseline. These data suggest that residual disease following therapy in myeloma is characterized by high level of genomic complexity similar to that observed at baseline. However in spite of the genetic heterogeneity and complexity at baseline, the residual CD138+ plasma cells converge to a distinct signature enriched in a transcriptional program associated with embryonal stem cell genes known to be targets of SOX2 and NANOG. Drug resistant/residual CD138+ tumor cells in myeloma therefore show transcriptional profiles previously implicated in cancer stem cells. Targeting stemness-associated genes may be essential to effectively treat residual disease in myeloma.