A Gene Expression Based Proliferation Index as Independent Prognostic Factor in Multiple Myeloma.

BACKGROUND. The proliferation-rate of primary myeloma cells is a strong adverse prognostic factor in various trials, but not routinely assessed, partially due to effort in obtaining it.

AIM. As gene-expression profiling is increasingly considered a standard diagnostics in myeloma, we investigated the possibility to develop a prognostically relevant gene-expression based proliferation index (GPI).

PATIENTS AND METHODS. Gene expression was determined by Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. The GPI was derived by selecting genes associated with proliferation (in terms of gene ontology) differentially expressed in proliferating malignant (human myeloma cell lines) and benign (plasmablastic) cells compared to non-proliferating, non-malignant cells (normal plasma cells and memory B-cells). The GPI comprises the sum of the expression values of 50 genes (ASPM, AURKA, AURKB, BIRC5, BRCA1, BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDC25C, CDC6, CDCA8, CDKN3, CEP55, CHEK1, CKS1B, CKS2, DLG7, ESPL1, GINS1, GTSE1, KIAA1794, KIF11, KIF15, KIF20A, KIF2C, KNTC2, MAD2L1, MCM10, MCM6, MKI67, NCAPD3, NCAPG, NCAPG2, NEK2, NPM1, PAK3, PCNA, PGAM1, PLK4, PTTG1, RACGAP1, SMC2, SPAG5. STIL, TPX2, ZWINT). Proliferation of primary myeloma cells was assessed by propidium iodinestaining (n=67). Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3).

RESULTS. In the two groups, 39 and 32 percent of primary myeloma cells show a GPI above the median plus three standard deviations of normal bone marrow plasma cells, respectively. The GPI is significantly higher in advanced- compared to early-stage myeloma (P=.001) and in patients harboring a gain of 1q21 (n=95, P<0.001). It correlates significantly with proliferation as determined by propidium iodine in primary myeloma cells (r_s=.52, P<.001, n=67). The GPI as continuous variable is significantly predictive for event-free survival (EFS, n=120, P<.001, n=345, P<.001, respectively) and overall survival (OAS, n=345, P<.001) in patients treated with high-dose chemotherapy, independent of serum-β2-microglobulin (B2M) or ISS-stage. A GPI above the median (GPI^high) delineated significantly inferior EFS (n=168, 41.6 vs. 26 months, P=.04, HR 1.57, CI [1.02,2.42]; n=345, 68.6 vs. 45.2 months, HR 1.55, CI [1.16,2.09], P=.003) and OAS (n=345, P<.001) in two independent cohorts of patients undergoing high-dose chemotherapy. By using B2M above 3.5 mg/l and GPI as staging variables, four groups with difference in median EFS (n=345, B2M <3.5mg/l, GPI^high/low 76.1 months; B2M < 3.5mg/l, GPI^high 62.4 months, B2M ≥3.5mg/l, GPI^low 41.8 months, B2M ≥3.5mg/l, GPI 36.1 months, P<.001) and OAS can be delineated.

CONCLUSION. The GPI

• represents a validated tool for the assessment of proliferation in multiple myeloma patients,

• allows a risk stratification in terms of proliferation either alone or in combination with B2M or ISS, respectively, and

• has the potential to be used within a risk adapted targeting of anti-proliferative treatment.