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Blood, Vol. 114, Issue 1, 128-143, July 2, 2009
Induction of angiogenesis by normal and malignant plasma cells
Blood Hose et al.
114: 128
Supplemental materials for: Hose et al
Calculation of the gene expression based proliferation index
The gene expression based proliferation index is calculated as follows. In brief, genes are selected based on genes over-expressed in proliferating cells (malignant: human myeloma cell lines (HMCLs), benign: polyclonal plasmablastic cells (PPCs)) compared to non-proliferating cells (normal bone marrow plasma cells (BMPCs) and memory B cells (MBCs)). Here, four comparisons between the groups are made (i) HMCLs vs. MBCs, ii) HMCLs vs. BMPCs, iii) PPCs vs. BMPCs and iv) PPCs vs. MBCs) by a onesided t-test, with the alternative hypothesis being that expression values of HMCLs and PPC values are greater compared to BMPCs and MBCs in each comparison. P-values are permutation-adjusted regarding a family wise error rate with an α-level of 0.025. To adjust for comparing each group twice, the α-level is halved to 0.0125. Only genes being statistically significant in each of the 4 comparisons are retained for the index. To select biologically (in terms of proliferation) relevant genes, only genes matching with the gene-ontology term “cell proliferation” or “cell cycle” were retained. Thus, 50 genes (57 probesets) represent the final index. For genes with more than one probeset per gene, the probeset with the highest variance within the TG is selected. The index is calculated as follows: As proliferation-genes determined as stated above are over-expressed by definition, the individual gene expression based proliferation index for each sample is calculated as the sum of expression values of each of the 50 genes in an individual sample. For genes not expressed as judged by PANP, the expression level of the respective gene is defined as 0.
Files in this Data Supplement:
- Table S1. Clinical data for patients undergoing high-dose chemotherapy and autologous stem cell transplantation (PDF, 58.3 KB) -
Clinical patient data for age, serum-β2-microglobulin, and plasma cell infiltration in the training group (TG), the validation group (VG), and the Arkansas-data. Median value and range are given. NA, not available; ISS, International Staging System.
- Table S2. Overview over patient populations, subpopulations and samples used (PDF, 65.1 KB) -
GEP, gene expression profiling; MMC, multiple myeloma cells; HM-group, Heidelberg/Montpellier-group; TG, training group; VG, validation group; MBC, memory B cells; BMPC, bone marrow plasma cells; MGUS, monoclonal gammopathy of unknown significance; HMCL, human myeloma cellline; WBM, whole bone marrow; ND, normal donor; MM, multiple myeloma; dce-MRI, dynamic contrast-enhanced magnetic resonance imaging; iFISH, interphas efluorescence in situ hybridization; MCG, metaphase cytogenetics; HDT, high-dose chemotherapy.
- Table S3. List of 402 genes used for the analysis (PDF, 156 KB) -
Genes are exploratively divided into (A) pro-, (B) anti-angiogenic, and (C) no information available (NI). Genes retained in the analysis (present in at least one sample within the training and validation group) are depicted in orange.
- Table S4. Differential gene expression for the training group. Differential gene expression between bone marrow plasma cells and memory B cells within the training group (PDF, 422 KB) -
Genes with differential expression between normal bone marrow plasma cells (BMPC) and memory B cells (MBC) as determined by EB-statistics and Benjamini-Hochberg correction for multiple testing of genes expressed at least once in the training and validation group. (A1) Pro-and (A2) anti-angiogenic genes. Differential gene expression between (B) normal and malignant plasma cells and (C) between early and late stage myeloma within the training group. Genes with differential expression between normal (BMPC) and malignant plasma cells (MMC) as determined by EB-statistics and Benjamini-Hochberg correction for multiple testing of genes expressed at least once in the training and validation group. (B1) Pro- and (B2) anti-angiogenic genes. (C1) Pro-and (C2) anti-angiogenic genes differentially expressed between early stage (MGUS+MMI) and late stage (MMII+MMIII) myeloma. Differential gene expresion between bone marrow plasma cells and plasma cells from patients suffering from monoclonal gammopathy of unknown significance within the training group.Genes with differential expression between normal bone marrow plasma cells (BMPC) and plasma cells from patients with monoclonal gammopathy of unknown significance (MGUS) as determined by EB-statistics and Benjamini-Hochberg correction for multiple testing of genes expressed at least once in the training and validation group. (C3) Pro- and (C4) anti-angiogenic genes.
- Table S5.PAM based predictor (PDF, 211 KB) -
APAM-based predictor for “being”(A) memory B cell or normal or malignant plasma cell or myeloma cell line (MBC/BMPC/MM/HMCL), and (B) whole bone marrow from normal donors or myeloma patients (ND-WBM/MM-WBM), respectively, was created. For the first, in the training group, all MBC, BMPC and HMCL are predicted correctly. Three MMC samples are predicted as BMPC. In the validation group, all MBC and HMCL are predicted correctly, whereas 2 MMC are predicted as BMPC and 2 as HMCL, respectively. Out of 7 BMPC, 4 are predicted as MMC. In the latter case, all 7 ND-WBM samples are predicted correctly. 6/57 MM-WBM samples are predicted as ND-WBM. Shown are in each case the genes (probe sets) comprising the predictor (A1, B1), the cross validated error rate on the training (A2, B2), and the validation group (A3).
- Figure S1. Expression of pro- and anti-angiogenic genes within the training group (JPG, 879 KB)
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Shown is the expression of (A) the pro-angiogenic genes VEGFA, ADM, and IGF-1, (B) the anti-angiogenic genes TIMP1, TIMP2, and PF4, and (C) the aberrantly expressed genes HGF, CTGF, and TGFA as well as MET, IL-15, and ANG within the training group. For the validation group, see Fig. 2. (D) The unsupervised hierarchical clustering shows BMPCs (depicted in blue) clustering together in a sub-branch within the MMCs (depicted in white). All HMCLs (depicted in orange) clustering together with the MBCs (depicted in light blue) in a separate branch each. For the validation group, see Fig. 2D.
- Figure S2. Correlation between qRT-PCR and gene expression profiling (JPG, 354 KB)
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Expression of VEGF, TGFA, CTGF, and ADM as determined by quantitative real-time PCR (shown are −dCt-values) plotted against microarray values. (A) VEGF (rs=-0.45, P=.2), (B) TGFA (rs=-0.42, P=.2), (C) CTGF (rs=-0.9, P=<.001), and (D) ADM (rs=-0.84, P=.004).
- Figure S3. Unsupervised hierarchical clustering (JPG, 37.5 KB)
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The dendrogramm shows four arms: all whole bone marrow samples from normal donors (ND-WBM; depicted in light green) fall in one arm together with 6 myeloma patients (MM-WBM; depicted in white). The MM-WBM shows three separate arms.
- Figure S4. Globaltest model (JPG, 654 KB)
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By using Goeman’s globeltest, a significant association of the angiogenic gene expression (signature) could be found for event-free (EFS) and over-all survival (OS) within the validation group and the Arkansas-data. (A) Training group (n=48). (A1) EFS (P=.08), (A2) OS (P=.1). (B) Validation group (n=159). (B1) EFS (P<.001), (B2) OS (P=.03). (C) Arkansas-data (n=345). (C1) EFS (P<.001), (C2) OS (P=.001).
- Figure S5. Globaltest model including the presence of t(4;14) and the gene expression based proliferation index as co-variables (JPG, 659 KB)
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(A) Training group (n=48). (A1) EFS (P=.6), (A2) OS (P=.2). (B) Validation group containing only patients with information on t(4;14), (n=124). (B1) EFS (P=.5), (B2) OS (P=.2). (C) Globaltest on the reduced validation group (see above, n=124) without co-variables showing still a significant association with (C1) EFS (P<.001) and (C2) OS (P=.05).
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