Increased Calreticulin Expression in Clonal Plasma Cells Is Associated with Complete Response to Melphalan and Autologous Stem Cell Transplant in Systemic Light-Chain Amyloidosis.

Systemic light-chain amyloidosis (AL) is a protein deposition disorder and a monoclonal plasma cell disease. Treatment of AL has focused on the reduction or elimination of the clonal plasma cells that make amyloid-forming light chains. High-dose melphalan and stem cell transplant (MEL SCT) is an effective therapy for selected AL patients. At 3 months post-SCT, response of the clonal plasma cell disease can be reliably scored; the achievement of a complete response (CR= immunofixation negative) is usually associated with extended survival while no response (NR= < 50% reduction) is not. Many factors likely contribute to this distribution of responses. In order to identify factors specific to clonal plasma cells, gene expression profiles (GEP; Affymetrix U133 PLUS 2.0) were obtained using FACS-sorted CD138+/DAPI- plasma cells (>95% pure) from untreated AL patients before SCT. Supervised analyses were then performed based on responses at 3 months post-SCT, comparing the CR (n=4) and NR (n=5) groups. The basic analysis was a t-test, filtering genes for differential expression at p<0.01, and those that passed were subject to EASE analysis in order to identify gene ontology families over-represented on the gene list. A secondary filtering selected only genes with two-fold difference in expression between CR and NR, average expression of at least 1000, and inclusion in an EASE group that had a p<0.05. Five genes were identified as over-expressed in the CR group: WARS, SHMT2, IARS, CALR, and PSMB4. Our initial validation studies, using purified baseline clonal plasma cells from 11 patients and baseline marrow biopsy specimens from 25 patients, have focused on calreticulin (CALR) and tryptophanyl tRNA synthetase (WARS). By real-time polymerase chain reaction studies with GAPDH and normal plasma cell controls, the expression levels of CALR were significantly lower in NR patients (p=0.02) while differences in WARS expression did not achieve significance (p=0.25). By immunohistochemical (IHC) staining for CALR in marrow biopsies, a CALR IHC index was developed, with scoring of the fraction of plasma cells expressing CALR (over total CD138 positivity) and intensity of CALR staining (both scored 0, 1, 2 or 3; the scores were multiplied to compute the index, range 0–9). CALR-stained marrows were scored blindly. Patients with CALR index of 9 had an 88% likelihood of achieving CR while those with scores of 0–6 had a 39% chance (Fisher’s exact test, p=0.046). In sum, calreticulin expression in clonal plasma cells from AL patients is associated with the response to MEL SCT. Further studies are on-going to determine the basis for this association.