Cyclin D1 Overexpression In Clonal Plasma Cells In Systemic AL Amyloidosis Is Associated with Differential Expression of Protein Quality Control Genes and Bias In Clonal Germline IgV_L donor Gene Use

Abstract 4043

Cyclin D1 (CCND1) overexpression in AL plasma cells (PC) is associated with patient characteristics such as production of free immunoglobulin (Ig) light chains (FLC) without an intact M-protein (that is, without partner Ig heavy chains), increased cardiac biomarkers and shorter survival (Amyloid 2010;17(S1):61a; Blood 2008;111:4700; Haematologica 2009;94:380). The molecular ramifications of CCND1 overexpression within AL PC clones have not been described. To study these associations, we used CD138+ AL PC from 69 untreated AL patients at diagnosis for (1) gene expression profiling (GEP, Affymetrix U133A 2.0) (n=16), (2) qRT-PCR to validate GEP findings (n=53), and (3) clonal IgV_L germline donor gene identification (n=69) by established methods (Blood 2008;111:549; Blood 2001;98:714). By GEP, all cases displayed significant overexpression of the appropriate isotypic IgV_L constant region gene, confirming the preponderance of clonal AL PC. Five cases were CCND1^hi and 11 CCND1^lo, and a supervised analysis of CCND1^hi vs CCND1^lo transcriptomes showed that in CCND1^hi PC among the most down-regulated genes were IGHG1, IGHG3 and CCND2 while among the most up-regulated ones (after CCND1) were FAM129A, WARS, SEC63, PDIA6 and SEL1L. By RT-PCR all 53 cases used for qRT-PCR displayed prominent amplification of spliced and unspliced XBP1, confirming PC derivation. By qRT-PCR, median CCND1 expression was 1.51 (range, 0–19.36) with 27 cases above (CCND1^hi) and 26 below the median (CCND1^lo) with clear-cut quartile differences (25% 0.02, 75% 4.78). We examined PDIA6 and SEL1L expression by qRT-PCR, and found that both correlated with CCND1 expression (PDIA6, P=0.018, r=0.452; SEL1L, P=0.038, r=0.395). In addition, PDIA6 and SEL1L values above and below the CCND1 median differed significantly (P=0.01, P=0.04). The genes up-regulated in CCND1^hi cases are involved in endoplasmic reticulum (ER) and protein control processes: WARS in protein production, FAM129A in autophagy, SEC63 in ER protein transport, PDIA6 in catalysis of disulfide bonds and SEL1L in modifying misfolded proteins and channeling them to cytosolic proteasomes. We then identified the clonal IgV_L germline donor genes in the CCND1^hi (n=32) and CCND1^lo (n=37) AL PC clones. We knew that CCND1^hi clones displayed biased Ig light chain restriction with 10/12 κ and 22/57 λ cases being CCND1^hi (p=0.009, Fisher's exact). Surprisingly, we also identified biased λ family use as only 6/27 λ1 and λ2 cases were CCND1^hi compared to 16/30 λ3 and λ6 cases (P=0.03). Overall these results confirm that CCND1^hi AL PC clones express significantly higher levels of important ER protein quality control genes than CCND1^lo clones, possibly due to CCND1^hi AL PC clones adapting to the production of FLC without partner Ig heavy chains. Moreover, CCND1^hi AL PC clones display a biased clonal IgV_L germline donor gene repertoire, raising questions about the origin of CCND1^hi clones since germline gene selection is an early and CCND1 overexpression likely a late event in malignant clonal PC emergence.