Elevated Expression Levels of Adrenomedullin Trend with Suboptimal Treatment Response and Inferior Survival of Newly Diagnosed Multiple Myeloma Patients

Introduction: Increased angiogenesis is a hallmark feature of multiple myeloma (MM) resulting in poor clinical outcomes of MM patients. Current knowledge suggests that tumor- stroma interactions result in an imbalance between pro-angiogenic and anti- angiogenic modulators which favors vascular development and tumor growth. Adrenomedullin (AM) is a multifunctional peptide which mainly regulates vasodilation and maintains vascular integrity but is also implicated in the growth of several solid tumors. It has been shown that adrenomedullin is expressed in a hypoxia-dependent and hypoxia-independent manner by human myeloma cell lines and that it enhances MM-driven angiogenesis mainly by stimulating endothelial cell proliferation and endothelial tube formation. However, the clinical impact of AM remains unknown.

Methods: We evaluated the expression of adrenomedullin in 32 newly diagnosed multiple myeloma (NDMM) patients and 20 age and sex-matched healthy donors. Diagnosis of MM was based on International Myeloma Working Group consensus criteria. Patients received either VCD or VRD based regiments. Response was evaluated after five cycles of induction therapy. All samples were taken at the time of diagnosis, prior treatment initiation. Bone marrow aspirates were collected in EDTA-containing tubes and immediately processed. Bone marrow mononuclear cells (BMMNCs) were separated using density gradient separation with Ficoll, total RNA was extracted from BMMNCs and then total RNA was reverse transcribed into cDNA. RT q-PCR was carried out in a 36 well Rotor Gene Q using CYBR green 1 as fluorescence dye. ACTB was used as housekeeping gene. Primers were as follows: for ADM F: TTGTCCTCCCCTATTTTAAGACG, R: CTTCCACACAGGAGGTAATCAGTC and for ACTB F:TTTTTGTCCCCCAACTTGA, R: TGGCTGCCTCCACCCA. Gene expression levels were analyzed with the Livak method.

Results: To establish the differential expression of AM in MM, we first compared AM levels of the ADM gene between NDMM patients and healthy donors. AM mRNA abundance was 10-fold higher in the NDMM group compared to the HD group (P < 0.0001). We, then, categorized patients into two groups based on the expression levels of AM. The median DCT value of the NDMM population served as cut-off point. The first group (High) comprised of patients with elevated expression measures of AM (n=16, median DCT 4.6, range 2.7-5.8) and the second one (Low) comprised of patients with lower measures (n=16, median DCT 7.8, range 6.1-10.3). The 2 groups did not differ in age, sex, percentage of bone marrow infiltration, ISS stage, R2-ISS stage and were equally treated either with VRD-based or VCD-based regiments. The Overall response rate (≥ PR) was 56% (n = 9/16) for the high group and 81% (n = 13/16) for the low group. After a median follow up period of 23 months (range 1-57 months), 14 (87%) and 8 (50%) patients from the high and low expression group, respectively, have relapsed or died with a median time to progression or death of 16 (range 1-42) and 16.5 (range 1-55) months respectively. Kaplan-Meier curves were used to calculate probability of survival. The median estimated overall survival for patients with elevated AM measures was 29.5 months compared to 55 months for patients with lower AM measures (logrank HR = 2.1, 95% CI of ratio 0.7-6.1, P = 0.1).In univariate analysis, age (HR = 1.6, P = 0.02), LDH levels (HR = 1.01, P = 0.002), b2-microglobulin levels (HR = 1.15, P = 0.03), R2-ISS stage 3 (HR = 2.4, P = 0.4), R2-ISS stage 4 (HR = 5.4, P = 0.1) and levels of AM (for every DCT reduction by one unit; HR = 1.2, P = 0.1) were factors affecting survival. In multivariate analysis, age (HR = 1.1, P = 0.001) and R2-ISS stage 4 (HR = 21.8, P = 0.01) were factors predicting poor survival whereas elevated levels of AM (expressed as reduced DCT values) increased the risk of death (for every DCT reduction by one unit; HR = 1.1, p = 0.4). Pearson and Spearman correlation analyses did not show any correlation of AM DCT values with age, LDH, b2-microglobulin, ISS and R²-ISS implying that the observed differences between the 2 groups are probably due to AM expression and not a result of other confounding factors.

Conclusion: This is the first exploratory study that evaluated the prognostic potential of AM in NDMM patients. Our preliminary findings indicate that elevated levels of AM trend with suboptimal treatment response and inferior survival of NDMM patients.

No relevant conflicts of interest to declare.