Elevated Cyclin D1 (CCND1) Expression in the Plasma Cells of Patients with Systemic AL Amyloidosis at Diagnosis Is Associated with Higher Brain Natriuretic Peptide (BNP) Levels, Increased Plasma Cells and Significantly Poorer Survival.

The clonal plasma cells in AL can be considered malignant because they contain cytogenetic abnormalities, including t(11;14) in 40% to 50% of cases (Blood 2001;98:2266; 2008;111:4700; Haematologica 2009;94:380). By gene expression profiling (GEP) they are also reported to overexpress CCND1 (Blood 2005;105:794). To assess the frequency and significance of differences in CCND1 expression in plasma cells from AL patients at diagnosis, we evaluated the differences in CCND1 expression in CD138+ plasma cells from newly diagnosed patients. We then correlated these differences with features of both the deposition and clonal plasma cell diseases, and with treatments, outcomes and overall survival.

Newly diagnosed AL patients were evaluated and CD138+ cells selected and used for GEP with Affymetrix U133 PLUS2.0 arrays (Affymetrix; Santa Clara, CA) or qRT-PCR as previously described (JNCCN 2007;5:179; Blood 2008;111:549). qRT-PCR was performed using TaqMAN Gene Expression Assays with CCND1 (Hs00277039_m1) and RPLP0 (Hs99999902_m1) primers and probes (Applied Biosystems; Foster City, CA) and 2 myeloma cell lines as positive and negative controls (KMS-12BM, RPMI 8226) on an Mx 3000P platform and related software (Stratagene; La Jolla, CA). The comparative Ct method was used and the amount of target was normalized to the control (2^-ΔΔCt) assuming an efficiency of 2. Statistical analyses were performed with GraphPad PRISM (GraphPad; La Jolla, CA).

CD138+ cells from 58 newly diagnosed AL patients were evaluated. With GEP (n=16), CCND1 median log_e-transformed quantitative expression levels were 11.08 (range, 9.6-11.55) in five versus 4.163 (3.875-5.447) in eleven patients (P < 0.01). With qRT-PCR (n=42), relative CCND1 expression levels were 4.21 (1.76-17.24) in twenty versus 0.014 (0-0.99) in twenty-two patients (P < 0.0001). Overall, 43% (25/58) of patients were CCND1+ and did not differ from CCND1- patients with respect to organ involvement, troponin I, urine total protein, creatinine or alkaline phosphatase levels. The median BNP of CCND1+ patients was 252pg/ml (range 20-1880), significantly higher than that of CCND1- patients (109pg/ml (5-4210), P < 0.05). CCND1+ patients tended to have more plasma cells (13% versus 8%, P = 0.06), higher serum free light chain levels (23.3 versus 14mg/dl, P = 0.12) and more kappa clones (6/25 versus 2/33, P = 0.06), and had fewer intact immunoglobulin M-proteins (4/25 versus 22/33, P < 0.01). There were no differences in frequencies of treatment with stem cell transplant or oral melphalan/dexamethasone or in the rates of complete hematologic responses. CCND1+ patients survived a median of only 27 months compared to 60 months for CCND1- patients (P = 0.02) and had a risk of death of 2.86 (CI 1.18-6.94).

Significant differences in CCND1 expression in the plasma cells of AL patients at diagnosis can be appreciated by GEP and qRT-PCR and clearly define two groups, CCND1+ and CCND1-, that differ in baseline BNP levels and features of the clonal plasma cell disease. Despite similar treatments and initial responses, CCND1+ patients have poorer overall survival, possibly due to asymptomatic cardiac involvement at diagnosis and low-level persistence of clonal plasma cells despite treatment, hypotheses that merit prospective evaluation.

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