PF572 ANALYSES OF EXPRESSION LEVELS, MUTATIONS, AND METHYLATION STATUS OF CRBN‐RELATED GENES PRE‐ AND POST‐LENALIDOMIDE TREATMENT IN MULTIPLE MYELOMA
Authors: Tachita T. , Kinoshita S. , Ri M. , Aoki S. , Asano A. , Kanamori T. , Totani H. , Asao Y. , Narita T. , Ito A. , Kusumoto S. , Komatsu H. , Iida S. 
Published: June 2019

Background:

Lenalidomide (Len) plays an essential role in the treatment of multiple myeloma (MM). Len binds to cereblon (CRBN) and alters the substrate specificity of the CRL4CRBN E3 ubiquitin ligase. This alteration results in ubiquitination and degradation of many proteins, such as the transcription factors IKZF1, IKZF3, and nuclear transport protein KPNA2. Although several studies have investigated the expression levels of CRBN and its substrates in primary MM samples, the association between their expression levels and treatment outcomes has not been elucidated.

Aims:

In this study, we investigated the association between the expression levels of CRBN pathway genes and the efficacy of Len plus dexamethasone (Ld) therapy. Furthermore, the mutation status of CRBN pathway genes and the methylation status of the CRBN promoter sequence were explored in a fraction of patients both pre‐ and post‐Ld therapy.

Methods:

[Gene expression] A total of 47 bone marrow (BM) specimens from relapsed MM patients were collected just prior to Ld therapy in our hospital. Among them, 25 paired BM samples were obtained both pre‐ and post‐Ld therapy, and 22 samples were derived from patients with refractory disease to Ld. After purifying the primary MM cells from the BM specimens, mRNA levels of CRBN, IKZF1, IKZF3, CUL4A, and KPNA2 were quantified using real‐time RT‐PCR. The relationship between their expression levels and clinical outcomes, including best response, progression‐free survival (PFS), and overall survival (OS) was analyzed. Alterations in the expression levels of these genes were also compared between pre‐ and post‐Ld samples.

[Mutation] In 22 paired samples of BM‐derived genomic DNA, targeted amplicon sequencing was performed on a MiSeq (Illumina) sequencer to detect mutations in the complete coding exons of 5 genes: CRBN, IKZF1, IKZF3, CUL4A, and TP53. The average depth of sequencing coverage was 903 × .

[Methylation] In 22 post‐Ld samples of BM‐derived genomic DNA, methylation screening of the CRBN promoter was performed by methylation‐specific PCR.

Results:

CRBN pathway gene expression levels were not significantly associated with best response, PFS, or OS. However, the ratio of IKZF1/CRBN expression was significantly lower in poor Ld responders (SD + PD, n = 15) than in good responders (CR + VGPR + PR, n = 32) (P = 0.01). Conversely, patients with a higher ratio of KPNA2/CRBN had significantly shorter PFS and OS times than those with a lower ratio (Fig A, P = 0.01). The ratio of IKZF1/CRBN tended to be negatively correlated with the ratio of KPNA2/CRBN (Fig B, Spearman's rank correlation coefficient = −0.2311; p = 0.1223. Pearson correlation coefficient = −0.2986; p = 0.0438). There was no association between the ratios of IKZF3/CRBN and CUL4A/CRBN with the efficacy of Ld therapy.

In the 22 paired samples, no mutations were observed in CRBN, IKZF1, or CUL4A, whereas somatic mutations were identified post‐Ld in TP53 and IKZF3 in 2 samples and 1 sample, respectively. None of the samples showed hypermethylation of the CRBN promoter.

Summary/Conclusion:

In the current study, we demonstrated that a lower ratio of IKZF1/CRBN and a higher ratio of KPNA2/CRBN predicted poor outcomes of Ld therapy. Notably, a negative correlation was observed between the ratio of IKZF1/CRBN and that of KPNA2/CRBN. In clinical sample analysis, neither CRBN pathway gene mutations nor hypermethylation of the CRBN promoter was associated with lower expression levels of the CRBN gene. Thus, these results might provide us with a better understanding of the sensitivity of MM to Len treatment.

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