Gene Expression Profiling of Isolated Mesenchymal and Osteoblastic Cells Exhibits a Different Pattern of Expression in Multiple Myeloma Patients as Compared to Healthy Subjects: Potential Relationship with the Presence of Bone Lesions.

Abstract Gene expression alterations occurring in the bone microenvironment cells and their potential relationships with the occurrence of bone lesions in multiple myeloma (MM) patients have never been investigated. In this study, we have isolated both mesenchymal (MSC) and osteoblastic (OB) cells, without in vitro differentiation, from bone biopsies obtained by iliac crest of 24 MM patients, 7 MGUS subjects and 8 healthy donors (N) who underwent orthopedics surgery. Bone status was evaluated in all MM patients by total X rays scan and MRI for the spine. Firstly, we evaluated cell proliferation in relationship with growth substrate (bone and glass) and cell phenotype by flow cytometry and immunohistochemistry. We found that both MSC and OB cells have higher cell doubling rate in MM patients as compared to N. Higher expression of alkaline phosphatase and Runx2 was observed in OB as compared to MSC cells in both N and MM patients without osteolytic lesions, but not in osteolytic ones. We performed a gene expression profiling analysis of isolated MSC and OB cells using GeneChip® Affymetrix HG-U133A oligonucleotide arrays. An unsupervised analysis of the most variable genes across the dataset generated a hierarchical clustering with the two major branches containing respectively MSC and OB samples. A multiclass analysis of N, MGUS and MM patients identified 33 differentially expressed probe-set (specific for 27 genes) in MSC cells, and 19 differentially expressed probe-set (13 genes) in OB, and the identified transcripts mainly characterized N versus MM and MGUS samples. A supervised analysis between N and MM samples identified 65 probes (56 genes: 17 up-regulated and 39 down-regulated) differentially expressed in MSC and 35 probes (29 genes, 12 up-regulated and 17 down-regulated) in OB. Notably, genes encoding the Homeobox class proteins, such as HOXB2-6-7, were up-regulated in both MSC and OB of MM patients as compared to N. As regards the bone status, a total of 60 probe-sets (3 up-regulated and 57 down-regulated genes) were found differentially expressed in MSC from osteolytic vs. non-osteolytic MM patients, whereas MGUS-MSC exhibited an intermediate transcriptional profile between osteolytic and non-osteolytic MM patients. A distinct pattern of gene expression profiling was also observed in MSC versus OB when osteolytic and non-osteolytic MM patients were compared (26 vs. 94 differentially expressed probe-sets, respectively), including transcription factors related to MSC osteogenic differentiation belonging to Runx2 pathway (HEY1) or Wnt and BMP signaling On the other hand, few genes were found differentially expressed in OB cells in relationship with the presence of bone lesions. In conclusion, we identified a distinctive transcriptional fingerprint in isolated MSC and OB cells of MM patients as compared to N subjects, which mainly correlated with cell proliferation. Moreover, a different gene expression profile was observed in MSC cells of MM patients according to the presence/absence of bone lesions, highlighting the critical role of the block of the osteogenic differentiation.

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Bone Microenvironment Cells Show a Different Pattern of Gene Expression Profiling in Relationship with the Presence of Osteolytic Bone Lesions in Multiple Myeloma Patients

Blood ◽

10.1182/blood.v112.11.2740.2740 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2740-2740

Author(s):

Katia Todoerti ◽

Gina Lisignoli ◽

Simona Colla ◽

Paola Storti ◽

Luca Agnelli ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Differentially Expressed ◽

Bone Lesions ◽

Bone Microenvironment ◽

Bone Status ◽

Transcriptional Alterations ◽

Cellular Types

Abstract Multiple myeloma (MM) is a plasma cell malignancy with the high capacity to induce osteolytic bone lesions. Whereas previous studies identified genes overexpressed by MM cells related to the bone status, the occurrence of transcriptional alterations in the bone microenvironment cells in the relationship with the bone involvement has not yet investigated. To clarify this issue, in this study we have analyzed the gene expression profiling of mesenchymal (MSC) and osteoblastic (OB) cells obtained from MM patients (n=24; osteolytic n=10; non-osteolytic n=14) in relationship with the presence or absence of osteolytic bone lesions. MGUS subjects (n=7) and healthy donors (n=8) were also included in the study as controls. Both MSC and OB were isolated from trabecular bone biopsies without in vitro differentiation. The presence of potential contaminating cells was excluded by FACS analysis in both MCS and OB, testing CD3, CD14, CD20 and CD138 antigens, as well as the expression of CD105 and CD146; the osteoblast-related markers Osteocalcin, Alkaline Phosphatase, Collagen I and Runx2 were evaluated in OB in comparison with MSC. Thereafter a gene expression profiling analysis of isolated MSC and OB cells was performed using GeneChip® HG-U133A oligonucleotide arrays. The obtained data were validated by real time PCR. Using conventional hierarchical clustering, the unsupervised analyses performed of the whole dataset generated a dendrogram clearly distinguishing MSC and OB cellular types. When considering MSC and OB dataset separately, a preferential clustering in relation to the presence of osteolytic bone lesions was observed for MSC but not OB samples. A supervised multi class analysis identified a total of 84 probe sets differentially expressed in MSC with an intermediate transcriptional profile in MGUS-MSC between osteolytic and non osteolytic MM patients. A supervised analysis performed on MSC MM samples revealed a total of 49 probe-sets (36 up-regulated and 9 down-regulated genes) as differentially expressed in osteolytic vs. non-osteolytic patients. Specifically, genes belonging to Wnt signaling as WNT6 and extracellular matrix structure as decorin (DCN) were found to be down-regulated in osteolytic as compared to and non-osteolytic MSC. Interestingly, no significantly modulated genes were found by comparing osteolytic and non-osteolytic OB samples. Finally, we performed two distinct supervised analyses by comparing the two cellular types (MSC and OB) in the two groups of MM patients in relationship with the bone status. A distinct transcriptional pattern was observed in MSC versus OB between osteolytic and non-osteolytic MM patients (52 vs. 21 differentially expressed probe-sets, respectively), mainly involving cell-cycle realted genes. Our results highlight that in MM bone microenvironment MSC rather than OB show transcriptional alterations in relationship with the presence of osteolytic bone lesions in MM patients.

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Mesenchymal and Osteoblastic Cells Isolated from Multiple Myeloma Patients Reveal a Different Behavior, Phenotype and Gene Expression Profiling in Relationship with the Presence of Osteolytic Bone Lesions.

Blood ◽

10.1182/blood.v108.11.3511.3511 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3511-3511

Author(s):

Nicola Giuliani ◽

Gina Lisignoli ◽

Katia Todoerti ◽

Sara Tagliaferri ◽

Cristina Manferdini ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Proliferation ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Osteoblast Differentiation ◽

Osteoblastic Cells ◽

Bone Lesions ◽

Facs Analysis ◽

Bone Status

Abstract Studies of gene expression performed on multiple myeloma (MM) cells have leaded to identify molecules able to inhibit osteoblast differentiation. Whereas potential alterations occurring in the bone microevironment cells in MM patients are not completely elucidated. To clarify this issue we have developed a method to direct isolate mesenchymal cells (MSC) and osteoblastic cells (OB) without in vitro differentiation from trabecular bone biopsies obtained by iliac crest of MM patients (n°=24) with or without osteolytic bone lesions. Bone status was evaluated in all MM patients by total X rays scan and NMR for the spine. MSC and OB isolated from trabecular bone of healthy donors underwent to orthopedics surgery was used as controls. Cell proliferation in relationship with growth substrate (bone and glass) was evaluated in isolated MSC (osteolytic n°=9, non-osteolityc n° 15) and OB cells (osteolytic n°=9, non-osteolityc n°=11) as well as immunophenotype by FACS analysis, protein pattern by immunohistochemical staining and ELISA assay and finally gene expression profiling by microarray (Affimetrix). First the presence of potential contaminating cells was excluded by FACS analysis in all the samples tested being both MCS and OB obtained negative for CD3, CD14, CD20 and CD138 antigens. We found that cell proliferation was significantly higher in MSC as compared to OB in MM patients and that both MSC and OB cells have higher cell doubling rate as compared to controls. Immunophenotype reveals a different pattern of expression of the chemokine receptors CXCR4, CXCR5, CCR6 in MSC and OB in the different group of patients. Higher alkaline phosphatase (AP) expression was observed in OB versus MSC in non-osteolitic patients but not in osteolityc ones. In line with these observations we found that the expression of the osteoblast transcrition factor Runx2/CBFA1 was higher in MSC obtained from non-osteolytic patients as compared with osteolytic ones. Hierarchical clustering by unsupervised analysis of gene expression profiles (Affymetrix U133A chips) identified two major cluster branches containing respectively MSC and OB cells, with subgroups correlated with the bone status. Following supervised analysis, a total of 121 probe-set were found differentially expressed in MSCs from patients with/without osteolytic lesions (57 up-regulated and 64 down-regulated) Distinct patterns of gene expression profiling were observed in MSCs versus Obs when osteolityc or non-osteolytic patients were compared. Interestingly, markers and transcription factors known to be specific for osteoblast cells were up-regulated in OB versus MSC in non-osteolytic patients but not in osteolytic ones. Notably, a significant downregulation of Runx2 and AP-1 related pathways was observed in OB of osteolytic MM patients as compared to non-osteolytic ones. In conclusion in this study for the first time we have identified a different pattern of growth, phenotype and gene expression in isolated MSC and OB cells in relationship with the bone status of MM patients highlighting the critical role of the block of osteoblast differentiation and the involvement of the related signature pathways.

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A New Molecular Classification of Multiple Myeloma Using Gene Expression Profiling and Fluorescence In Situ Hybridisation as Predictor for Event Free Survival.

Blood ◽

10.1182/blood.v104.11.73.73 ◽

2004 ◽

Vol 104 (11) ◽

pp. 73-73 ◽

Cited By ~ 3

Author(s):

Dirk Hose ◽

Jean-Francois Rossi ◽

Carina Ittrich ◽

John deVos ◽

Axel Benner ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Molecular Classification ◽

Differentially Expressed ◽

Event Free Survival ◽

Free Survival ◽

Cytogenetic Aberrations

Abstract AIM was to establish a new molecular classification of Multiple Myeloma (MM) based on changes in global gene expression attributable to cytogenetic aberrations detected by interphase FISH (iFISH) in order to (i) predict event free survival (EFS) and (ii) investigate differentially expressed genes as basis for a group specific and risk adapted therapy. PATIENTS AND METHODS. Bone marrow aspirates of 105 newly diagnosed MM-patients (65 trial (TG) / 40 independent validation group (VG)) and 7 normal donors (ND) were CD138-purified by magnetic activated cell sorting. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). CCND1 and CCND2 expression was verified by real time RT-PCR. iFISH was performed on purified MM-cells using probes for chromosomes 11q23, 11q13, 13q14, 17p13 and the IgH-translocations t(4;14) and t(11;14). Expression data were normalised (Bioconductor package gcrma) and nearest shrunken centroids (NSC) applied to calculate and cross validate a predictor on 40 patients of the TG with a comprehensive iFISH panel available combined with CCND overexpression. Differentially expressed genes were identified using empirical Bayes statistics for pairwise comparison. RESULTS. Overexpression of a D-type cyclin (D1 or D2) was found in 61/65 patients with MM compared to ND. CCND3 overexpression only appeared concomitantly with CCND2 overexpression. Four groups could be distinguished: (1.1) CCND1 (11q13) overexpression and trisomy 11q13, (1.2) CCND1 overexpression and translocations involving 11q13 i.e. t(11;14), (2.1) CCND2 overexpression without 11q13+, t(11;14), t(4;14), (2.2) CCND2 overexpression with t(4;14) and FGFR3 upregulation. A predictor of 6 to 566 genes correctly classifies all 40 patients of the TG (estimated cross validated error rate 0%). An independent VG of 40 patients was used. Genes with highest scores in NSC are: (1.1) CCND1, ribosomal proteins (e.g. RPL 28, 29), GPX1, CCRL2, (1.2) CCND1, TGIF, and NCAM (non-overexpression), (2.1) CCND2, (2.2) FGFR3, WHSC1, CCND2, IRTA2, SELL, and MAGED4. Distribution of clinical parameters (i.e. β2M, Durie Salmon stages, ISS) was not significantly different between the groups. The distribution of del(13)(q14q14) was (1.1) 31.5%, (1.2) 37.5%, (2.1) 37.5% and (2.2) 100%. (p<0.01). I.e. HGF, DKK1, VCAM, CD163 are differentially expressed between all 4 groups and ND (adjusted p<0.001). The groups defined by the predictor show a significantly different EFS after autologous stem cell transplantation according to the GMMG-HD3 protocol (median: (1.1) 18 / (1.2) not reached (no event) / (2.1) 22 / (2.2) 6 months; log-rank-test: p=0.004). CONCLUSION. CCND1 or CCND2 overexpression is nearly ubiquitous in MM and attributable to defined cytogenetic aberrations. Gene expression and iFISH allow a molecular classification of MM which can be predicted by gene expression profiling alone. Groups in the classification show a distinctive pattern in gene expression as well as a different EFS interpretable as risk stratification and indicator of therapeutic targets.

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IMiD-induced gene expression profiling predicts multiple myeloma prognosis

The Lancet Haematology ◽

10.1016/s2352-3026(17)30153-9 ◽

2017 ◽

Vol 4 (9) ◽

pp. e406-e407

Author(s):

Sinto Sebastian

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Gene Expression Profiling ◽

Expression Profiling

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Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients

Blood ◽

10.1182/blood-2009-12-261032 ◽

2010 ◽

Vol 116 (14) ◽

pp. 2543-2553 ◽

Cited By ~ 171

Author(s):

Annemiek Broyl ◽

Dirk Hose ◽

Henk Lokhorst ◽

Yvonne de Knegt ◽

Justine Peeters ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Plasma Cells ◽

Medical Science ◽

Protein Tyrosine Phosphatases ◽

Molecular Classification ◽

Newly Diagnosed ◽

Expression Of Genes

Abstract To identify molecularly defined subgroups in multiple myeloma, gene expression profiling was performed on purified CD138+ plasma cells of 320 newly diagnosed myeloma patients included in the Dutch-Belgian/German HOVON-65/GMMG-HD4 trial. Hierarchical clustering identified 10 subgroups; 6 corresponded to clusters described in the University of Arkansas for Medical Science (UAMS) classification, CD-1 (n = 13, 4.1%), CD-2 (n = 34, 1.6%), MF (n = 32, 1.0%), MS (n = 33, 1.3%), proliferation-associated genes (n = 15, 4.7%), and hyperdiploid (n = 77, 24.1%). Moreover, the UAMS low percentage of bone disease cluster was identified as a subcluster of the MF cluster (n = 15, 4.7%). One subgroup (n = 39, 12.2%) showed a myeloid signature. Three novel subgroups were defined, including a subgroup of 37 patients (11.6%) characterized by high expression of genes involved in the nuclear factor kappa light-chain-enhancer of activated B cells pathway, which include TNFAIP3 and CD40. Another subgroup of 22 patients (6.9%) was characterized by distinct overexpression of cancer testis antigens without overexpression of proliferation genes. The third novel cluster of 9 patients (2.8%) showed up-regulation of protein tyrosine phosphatases PRL-3 and PTPRZ1 as well as SOCS3. To conclude, in addition to 7 clusters described in the UAMS classification, we identified 3 novel subsets of multiple myeloma that may represent unique diagnostic entities.

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