The online version of this article (doi:10.1186/s13045-014-0105-1) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
AB, DH, NZ, HL and BK designed the research. AB performed the experiments. AS, SL, DH and BK performed gene expression profiling and analysed the data. AB, MS, DHe and DH performed the statistical analysis. AB, DH and NZ wrote the paper. All authors reviewed and approved the final version of the manuscript.
Insulin like growth factor binding protein 7 (IGFBP7) is a secreted protein binding insulin like growth factor 1 (IGF-1), insulin, vascular endothelial growth factor A (VEGFA), and activin A. It antagonizes bone morphogenetic proteins and is involved in the tumour propagation of solid as well as haematological malignancies. Its role in multiple myeloma (MM) is not defined so far. We therefore aim here to investigate its prognostic and pathophysiological role in MM.
The clinical significance of IGFBP7 gene expression was investigated by gene expression profiling in two independent cohorts (n = 948) of newly-diagnosed MM patients. Methylation of the IGFBP7 promoter was analysed by pyrosequencing and treatment of MM cell lines with 5-aza-2-deoxycytidine. The impact of IGFBP7 on MM cells was studied by CCK-8 assay, BrdU assay and flow cytometry, respectively. IGFBP7 expression in bone marrow stromal cells (BMSCs) was studied by quantitative RT-PCR. For osteoblast development, immortalized and primary human BMSCs were cultured in osteogenic differentiation medium for 7–14 days in the presence of recombinant human IGFBP7 and/or activin A.
Median IGFBP7 expression is significantly lower in CD138-purified plasma cells from individuals with MGUS and MM, compared to normal bone marrow plasma cells. IGFBP7 gene expression in MM cells is regulated by methylation, shown by pyrosequencing and exposure to demethylating agents (5-aza-2-deoxycytidine). High expression of IGFBP7 in MM cells is associated with adverse survival in two independent cohorts of 247 and 701 newly-diagnosed MM patients treated with high-dose therapy and autologous stem cell transplantation. IGFBP7 is associated with prognostically adverse chromosomal aberrations (t(4;14) and gain of 1q21), MMSET expression, and higher myeloma cell proliferation. In vitro, IGFBP7 overcomes activin A induced osteoblast suppression and promotes osteogenesis. MM cells downregulate IGFBP7 in stromal cells, possibly contributing to the osteoblast suppression found in MM. Conversely, higher IGFBP7 expression is associated with a lower probability of myeloma bone disease.
Our data indicate that IGFBP7 expression is a marker for a specific methylation pattern in myeloma, linked to translocation t(4;14) associated MMSET expression, showing clinical features of adverse prognosis with absence of myeloma bone disease.
Additional file 1: Table S1. BMP antagonist expression in CD138+ purified cells. Bold indicates differential expression in MM compared to healthy donor bone marrow plasma cells (BMPCs).13045_2014_105_MOESM1_ESM.doc
Additional file 2: Table S2. BMP antagonist expression pattern in whole bone marrow (WBM) samples. Bold indicates differential expression.13045_2014_105_MOESM2_ESM.doc
Additional file 3: Figure S1. EFS and OS in the LR cohort when the same fraction of patients defined as “high” IGFBP7 expressers in the HM-cohort (18.6%) was designated as high expressers in the LR-cohort. High IGFBP7 expression was associated with adverse event-free (P<0.001) and overall survival (P<0.002) by applying this criterion.13045_2014_105_MOESM3_ESM.pdf
Additional file 4: Figure S2. IGFBP7 expression is associated with high risk cytogenetics. Gene expression analysis of the HM-patient cohort revealed that high IGFBP7 expression levels were associated with poor risk cytogenetic markers including (A) translocation t(4;14) and (C) amplification 1q21. Moreover, a trend was found regarding the more frequent presence of deletion 13q in patients with high IGFBP7 expression values (D). No association was observed for (B) translocation t(11;14) and (E) deletion 17p. Grey data points indicate an absent, black data points a present Affymetrix detection call.13045_2014_105_MOESM4_ESM.pdf
Additional file 5: Figure S3. IGFBP7 expression is elevated in MMSET defined molecular subgroups of myeloma. IGFBP7 expression was analysed in two publically available GEP datasets published by (A) Zhan et al.  and (B) Agnelli et al. . Both datasets classified MM cases into distinct molecular subgropus based on microarray experiments. (A) IGFBP7 expression was significantly elevated in the MMSET (MS) overexpressing subgroup compared to five other GEP-defined subgroups. In addition, IGFBP7 expression tended to be elevated in the MS subgroup compared to the t(11;14) associated cyclin D1 (CD1) overexpressing samples. The other subgoups were defined by a proliferation (PR), low bone involvement (LB), hyperdiploid (HY), t(6;14) associated cyclin D (CD2) and MAF (MF) expression based GEP profile. (B) Analysis of the dataset from Agnelli et al.  confirmed overexpression of IGFBP7 in the MMSET defined molecular subgroup of MM (TC4). The other subgroups were defined by cyclin D overexpression (TC1), hyperdiploid status (TC2) and MAF overexpression (TC5.) TC3 was defined by the lack of association with any of the other subgroups. Asterisks indicate statistical significance compared with the other molecular subgroups (* P < 0.05). Horizontal lines represent median IGFBP7 expression with interquartile range.13045_2014_105_MOESM5_ESM.pdf
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