Cereblon expression in multiple myeloma: not ready for prime time

One of the major recent advances in the biology of multiple myeloma (MM) is the discovery of Cereblon (CRBN) as a potential target for immunomodulatory drugs (IMiDs) activity (Zhu et al, 2011). The use of lenalidomide and dexamethasone (Len-Dex) as frontline therapy might become one of the future standards of care in the treatment of symptomatic MM. Therefore, identification of a biological test able to predict individual patients’ response to lenalidomide is a major challenge, especially in the dawning world of personalized medicine. Recently Heintel et al (2013) reported on a moderate correlation between baseline CRBN expression and clinical response in patients treated with upfront Len-Dex (r = 0 48), CRBN expression being additionally associated with that of CTNNB1 (r = 0 7). These interesting results, providing two possible predictive markers of clinical response, have however to be carefully evaluated before drawing final conclusions, and we would like to discuss at least three important issues in this setting. Firstly, and this point is indeed well addressed by the authors, it is commonly admitted that biological analyses in MM patients have to be performed on sorted CD138 plasma cells. Yet Heintel et al (2013) explored results on sorted MM plasma cells in only six out of 49 patients. Because of the potential role of lenalidomide on CRBNexpressing microenvironmental cells, it will be necessary to clarify the issue of the type of samples that should be used to best assess CRBN expression in patients. It however seems quite obvious that the CD138 population of plasma cells would be the key target of such therapy and therefore that in which CRBN expression should be assessed. Secondly, the complexity of CRBN physiology should be considered. In order to evaluate CRBN expression in patients, Heintel et al (2013) used a real time reversetranscription polymerase chain reaction (RTqPCR) Taqman assay designed on exons 8–9 junction, which is different from the exons 10–11 junction design used by Broyl et al (2012). Other Taqman predesigned probes spanning different exon junctions are also currently available (Life Tech, Grand Island, NY, USA) but standardization of RTqPCR has not been proposed so far. Moreover, as do most of the genes in the human genome, CRBN undergoes mRNA alternative splicing (http://genome.ucsc.edu) and several isoforms have been described in MM (Gandhi et al, 2012). We performed overlapping RT-PCR designed on all CRBN exons in sorted CD138 plasma cells from 19 MM patients (purity > 90%). We showed multiple bands on gel in addition to expected size band (‘full-length’ CRBN-001/004) in virtually all of these patients (Fig 1A). By sequencing these RT-PCR products and alignment on a public genome database, we confirmed the presence of frequent alternative spliced transcripts including CRBN-002, lacking exon 10, and several other in frame alternative transcripts not described in Ensembl (http://www.ensembl.org) to date and that we named CRBN D8, CRBN D 8, 10 and CRBN D 7,8,10 as they had lost exon 8 only, exons 8 and 10 or exons 7, 8 and 10, respectively (Fig 1B). In some patients, these alternative spliced variants accounted for more than 50% of total CRBN (data not shown). This result shows that the design of the RTq-PCR test chosen to assess CRBN expression level is critical and could yield variable results. Evaluation of the CRBN splicing profile of each patient should therefore be considered prior to performing RTqPCR because the latter might yield irrelevant data if the exon assessed by the RTqPCR has been lost, and particularly if exon 10 has been removed (CRBN -002) as it contains a portion of the IMiD-binding domain (Ito et al, 2010; LopezGirona et al, 2012). Thirdly, because both CRBN and CNNTB1 genes are located on chromosome 3 (3p26 2 and 3p21, respectively), and given that chromosome 3 trisomy is the most frequent hyperdiploidy (HY) in MM (Kumar et al, 2012), we looked at the impact of HY with respect to other molecular MM subgroups for CRBN and CNNTB1 expression levels. Publicly available Affymetrix (Santa Clara, CA, USA) gene expression levels of 414 newly diagnosed MM patients enabled us to assess CRBN expression in each of the subgroups, designed according to Zhan et al (2006) (Fig 2). Of note, none of the patients completely lacked CRBN or CNNTB1 expression. Yet, HY patients significantly expressed higher levels of CRBN than all other subgroups (Kruskall–Wallis P < 0 0001), together with a trend for a higher level of CNNTB1 expression (P = 0 07). Comparison of CRBN and CNNTB1 expression between HY patients and all other subgroups revealed a significantly higher level of both genes in HY patients (Mann– Whitney P < 0 0001 and P = 0 006, respectively; Fig 2A). We also confirmed in this cohort of newly diagnosed patients a direct correlation between CRBN and CNNTB1 expression (P < 0 001; r = 0 52, Spearman test; Fig 2B). Given the wellknown better prognosis and survival observed in HY patients and the beneficial effect of odd chromosomes trisomy in high-risk MM patients, further investigations of CRBN expression in relation to the response to IMiDs should also