Biomarkers of Cardiotoxicity Among Multiple Myeloma Patients Subsequently Treated with Proteasome Inhibitor Therapy

BACKGROUND:  Cardiovascular (CV) events are a known complication to proteasome inhibitor therapy in myeloma. Underlying mechanisms are unknown.  We recently completed an investigator initiated, single institution Phase II study of high dose carfilzomib (56mg/m2) in patients with relapsed/refractory MM (NCT01351623).  Among 42 response evaluable patients, 11 patients (25%) developed treatment-emergent heart failure of any grade, and 5 patients (11%) developed severe heart failure requiring mechanical ventilation. We undertook a study to identify potential biomarkers that may point to underlying mechanisms of CV events among multiple myeloma patients treated with carfilzomib therapy.

METHODS:  We performed a nested case-control study with 7 patients who experienced a CV event on our high dose carfilzomib study and had pre-treatment (baseline) plasma stored and 19 case matched controls treated on the same study who did not have a CV event. We screened for 90 proteins known to be associated with CV disease using O-linked glycosylation. We used the Proseek Multiplex CVD I 96x96 platform which is based on the Proximity Extension Assay (PEA) technique. PEA is a 96-plex immunoassay that allows high throughput detection of protein biomarkers in liquid samples. For each biomarker, a matched pair of antibodies linked to unique oligonucleotides (proximity probes) binds to the respective protein target. Upon binding, the unique proximity probes can hybridize to each other and subsequently be detected and quantified by real-time PCR. Mean biomarker levels were compared using a t-test. False discovery rate (FDR) was used for multiple comparisons adjustment.

RESULTS:  Using samples collected prior to initiation of carfilzomib therapy, in an agnostic statistical model we identified the following four proteins to have altered levels in myeloma patients who developed CV events (p=0.002-0.004, unadjusted; p=0.089, after FDR correction): matrix metalloproteinase-1 (MMP-1, heparin-binding EGF-like growth factor (HB-EGF), TNF-related apoptosis-inducing ligand (TRAIL), and myoglobin (MB). Myeloma patients who developed CV events had 37% lower MMP-1, 15% lower MB, and 4% lower HB-EGF, while TRAIL was 7% higher in patients who developed CV events. Matrix metalloproteinases are a family of proteolytic enzymes responsible, among other functions, for myocardial extracellular protein degradation. Interestingly, several MMP species, including MMP-1, have been identified within the human myocardium and are thought to be dysregulated in congestive heart failure.  HB-EGF is a mitogenic and chemotactic glycoprotein that is essential for maintaining normal cardiac function and is known to play an important role in myocardial remodeling.

CONCLUSIONS:  We found that there was a trend towards lower MMP-1, HB-EGF, and MB levels and higher TRAIL levels in patients with CV events while receiving proteasome therapy.  MMP-1 appears to be the most promising potential biomarker based on our data. Our study supports further investigation of these proteins as potential biomarkers for patients at risk of CV events when treated with carfilzomib.

Table 1.

	CV event	No CV event
	N=7	N=19
CKD Proteins1	Mean (SD)	Mean(SD)	Unadjusted P-value	Adjusted P-value
MMP_1	1.7 (0.5)	2.7 (0.9)	0.002	0.089
HB_EGF	6.9 (0.2)	7.2 (0.3)	0.004	0.089
TRAIL	8 (0.3)	7.5 (0.5)	0.004	0.089
MB	5 (0.5)	5.9 (0.8)	0.004	0.089
HSP_27	2.2 (0.3)	2.7 (0.8)	0.032	0.528
PDGF_subunit_B	4 (0.7)	5 (1.5)	0.036	0.528
CD40_L	3.4 (0.6)	4.2 (1.2)	0.042	0.533
EGF	3.7 (0.9)	4.7 (1.4)	0.053	0.592
CX3CL1	5.9 (0.2)	5.6 (0.6)	0.092	0.895
TRAIL_R2	4.2 (0.4)	4.6 (0.6)	0.101	0.895

1.      Proteins are listed based on the p-value associated with the difference between patients who did and did not have CV events, with lowest p-value on the top.  The top 10 biomarkers are shown.