Tissue Factor Pathway Inhibitor 2 (TFPI2) Is Commonly Methylated in Multiple Myeloma

Tissue Factor Pathway Inhibitor 2 (TFPI2) is a member of the Kunitz-type serine proteinase inhibitor family that regulates extracellular tissue homeostasis by inhibiting matrix metalloproteinases. Transcriptional silencing of TFPI2 due to DNA methylation of the CpG island of its promoter has been reported in several tumors and has been suggested to facilitate tumor cell invasion and angiogenesis. We investigated the DNA methylation status of TFPI2 in multiple myeloma (MM) patients as part of a program of epigenetic profiling of multiple myeloma.

Genomic DNA extracted from two human MM cell lines (U-266 and RPMI-8226) and bone marrow aspirate samples from a well chracterized series of MM patients was modified by sodium bisulphite using the EZ DNA methylation kit, ZymoResearch. Bisulfite modified DNA was used as a template for Methylation Specific PCR with primers specific for unmethylated and methylated alleles. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNA was included in each experiment. Chi-square test, logistic regression analysis and unpaired t-test were used to investigate associations between gene methylation and ISS stage, presence of extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), renal failure, serum albumin and beta (2)-microglobulin level. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates.

Forty six patients with MM (26 male, 20 female; mean age 64) and two human MM cell lines were studied. According to the International Staging System (ISS) 21 patients were classified as stage I, 10 stage II and 15 as stage III disease. Methylation in the CpG island of the promoter of TFPI2 was detected in 30 patients (65%, 95%CI= 52,6–78,7%) and in both cell lines (100%). Patients with methylated TFPI2 had significantly elevated beta 2 microglobulin levels (5553 vs 3787 mg/L mean values, p=0.0002) but we did not detect a statistically significant difference in overall survival among patients with methylated versus non methylated TFPI2. No other relevant correlations were detected.

TFPI2 is commonly methylated in MM patients. Transcriptional silencing of TFPI2 may play a role in the mechanism of myelomatogenesis and its methylation status warrants further evaluation as a diagnostic co-marker for this disease.

No relevant conflicts of interest to declare.