Characterization of Oncogene Dysregulation in Multiple Myeloma by Combined FISH and RT-PCR Analyses.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5056-5056
Author(s):  
Shenxian Qian
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Target Genes ◽  
Expression Profiles ◽  
Youden Index ◽  
Rt Pcr ◽  
Chi Square ◽  
Pcr Product ◽  
Fish Samples ◽  
Chi Square Test

Abstract Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), and t(14;16)(q32;q23), respectively. The analysis was performed by RT-PCR from purified plasma cell populations from 57 MMs and we compared the results with the presence of translocations as assessed by dual-color FISH. A t(4;14) was found in 11MMs, t(11;14) in 9 MMs, t(6;14) in 5 MM, and t(14;16) in 4 MMs. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM) and MAF (1 MM) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. IGH-MMSET hybrid transcripts were found in 10 of the 57 (17.7%) MM samples. There was complete concordance between the findings of RT-PCR and FISH analyses of the MM samples, with 19.2% (11/57) t(4; 14) detected by FISH. Samples were separated further into three major groups based on the size of the RT-PCR product. The 1064bp, 438bp, and 275bp of IGH-MMSET were found in 7, 2, and in 1 sample, respectively. We then screened all 57 MM samples for the expression of FGFR3 using RT-PCR, with primers amplifying the 283bp fragments. Specific transcripts were detected in 11 (19.2%) samples that validate the t(4; 14) from cytogenetic studies. In the remaining 46 MM patients without t(4; 14), and 10 normal bone marrow controls, the FGFR3 amplified transcript was barely detectable. Only one patient sample without t(4; 14) revealed detectable levels of FGFR3 expression. Thus, RT-PCR assay for FGFR3 expression can detect all cases with evident or cryptic t(4; 14) translocation (P< 0.01). Using the primers corresponding to 7–10 exon in 11 cases of MM patients with overexpression of FGFR3, we directly sequenced the FGFR3 cDNA fragments amplified by PCR. Polymorphism (GGC>GGT) was detected in nine of the 11 patients. This polymorphism was tightly associated with higher expression of FGFR3. No FGFR3 mutations were found in the remaining 2 MM patients with overexpression of FGFR3. Our data indicate that RT-PCR is a sensitive and reliable method for the detection of FGFR3 and IGH-MMSET. Translocation t(4; 14) in MM detected by FISH can be validated by RT-PCR method. We examined our result by the Chi-Square test and revealed 90% sensitivity and 100% specificity. The Youden Index remains 0.9. This rapid and reliable detection of FGFR3 and IGH-MMSET overexpression may have practical clinical utility in the analysis and monitoring of the disease in MM patients with t(4; 14). Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.

A Trangenic Mouse Model of Plasma Cell Malignancy Shows Cytogenetic and Gene Expression Profiles Similar to Human Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 619-619
Author(s):  
Kristin Boylan ◽  
Mary A. Kvitrud ◽  
Brian G. Van Ness
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Chromosomal Abnormalities ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Rt Pcr ◽  
Mouse Tumors ◽  
Human Multiple Myeloma

Abstract Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. Human plasma cell tumors are genetically diverse, with no single chromosomal abnormality defining the disease, however, dysregulation of the genes c-myc and bcl-xl are both commonly observed. We have previously shown that targeted expression of c-myc and bcl-xl transgenes in mouse plasma cells produces malignancy which displays features of human myeloma such as localization of tumor cells to the bone marrow and lytic bone lesions. Tumors are also present at extramedullary sites (Cheung et al., J. Clin. Invest.113: 1763, 2004). Tumors rapidly develop (median 16 weeks) in 100% of mice, and can be adoptively transferred to syngeneic controls using as few as 1 million tumor cells to produce tumors in as few as 10 days. Adoptive transfer of similar cell numbers from younger double transgenic mice, without evidence of malignancy, results in increased tumor latency (>8 weeks) or the absence of tumor formation, suggesting that an accumulation of genetic changes is required for tumor development. In order to understand the specific genetic alterations required for tumor progression and for localization of tumors to the bone marrow vs extramedullary sites, we have undertaken a detailed analysis of plasma cell tumors in myc/bcl-xl mice and have begun to compare them with human multiple myeloma. Analysis of cell surface markers shows the majority of tumors have a plasmablast phenotype, expressing CD138+, B220+, CD38+, and CD19+. This result is confirmed by RT-PCR for B cell and plasma cell specific markers Pax5, Xbp1 and Blimp1, which can be detected in tumor samples. In addition, transcripts for Mip1α, EZH2, and Dusp6, genes shown to be upregulated in human myeloma, can also be detected in the mouse myc/bcl-xl tumors. Spectral karyotype analysis of metaphase chromosomes from primary tumor cell cultures demonstrates that a variety of chromosomal abnormalities are present in mouse tumors, including trisomies and translocations, similar to what is observed in human myeloma. The most frequently aberrant chromosomes are 12 and 16, followed by chromosomes 1 and 4. Interestingly, two common sites for translocations were identified; 12F which corresponds to the mouse immunoglobulin heavy chain locus, and 4D, which corresponds to a genomic region containing genes for plasma cell tumor susceptibility (Bliskovsky et al., PNAS100:14982, 2003). Further characterization of these translocations are being done to identify the precise breakpoints involved, and analysis of gene expression by RT-PCR and microarray analysis will be correlated to specific chromosomal abnormalities. Additionally, global gene expression profiles from myc/bcl-xl tumor cell cultures have been compared to existing profiles of human myeloma (Zhan et al., Blood99: 1745, 2002). Our preliminary comparison of gene expression profiles from myc/bcl-xl tumors to human myeloma tumors with high myc expression show the mouse tumors are more similar to human tumors than to normal plasma cells. These data suggest the myc/bcl-xl mouse tumors are similar to a subset of human myelomas, and will provide insight into the specific genes and pathways underlying human disease.

Interleukin-6–dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family–independent survival pathway closely associated with Stat3 activation

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 242-251 ◽  
Author(s):  
Katja Brocke-Heidrich ◽  
Antje K. Kretzschmar ◽  
Gabriele Pfeifer ◽  
Christian Henze ◽  
Dennis Löffler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Multiple Myeloma ◽  
Interleukin 6 ◽  
Target Genes ◽  
B Lymphocyte ◽  
Expression Profiles ◽  
B Cell Lymphoma ◽  
Myeloma Cells ◽  
Survival Pathway

Abstract Interleukin 6 (IL-6) is a growth and survival factor for multiple myeloma cells. As we report here, the IL-6–dependent human myeloma cell line INA-6 responds with a remarkably rapid and complete apoptosis to cytokine withdrawal. Among the antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of apoptosis regulators, only myeloid cell factor-1 (Mcl-1) was slightly induced by IL-6. Overexpression studies demonstrated, however, that IL-6 does not exert its survival effect primarily through this pathway. The IL-6 signal transduction pathways required for survival and the target genes controlled by them were analyzed by using mutated receptor chimeras. The activation of signal transducer and activator of transcription 3 (Stat3) turned out to be obligatory for the survival of INA-6 cells. The same held true for survival and growth of XG-1 myeloma cells. Gene expression profiling of INA-6 cells by using oligonucleotide microarrays revealed many novel IL-6 target genes, among them several genes coding for transcriptional regulators involved in B-lymphocyte differentiation as well as for growth factors and receptors potentially implicated in autocrine or paracrine growth control. Regulation of most IL-6 target genes required the activation of Stat3, underscoring its central role for IL-6 signal transduction. Taken together, our data provide evidence for the existence of an as yet unknown Stat3-dependent survival pathway in myeloma cells.

Characterization of Oncogene Dysregulation in Multiple Myeloma by Combined FISH and DNA Microarray Analyses.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4844-4844
Author(s):  
Antonino Neri ◽  
Sonia Fabris ◽  
Luca Agnelli ◽  
Michela Mattioli ◽  
Luca Baldini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Dna Microarray ◽  
Plasma Cell ◽  
Target Genes ◽  
Expression Profiles ◽  
Molecular Cytogenetic ◽  
Igh Locus ◽  
Microarray Analyses

Abstract Chromosomal translocations involving the immunoglobulin heavy chain (IGH@) locus and variuos partner loci are frequently associated with multiple myeloma (MM). We investigated the expression profiles of FGFR3/MMSET, CCND1, CCND3, MAF and MAFB genes, respectively involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23) and t(14;20)(q32;q12), in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) using DNA microarray analysis, and compared the results with the presence of translocations as assessed by dual-color FISH or RT-PCR. The t(4;14) was found in six MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in one MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in one PCL. The translocations were associated with the spiked expression of target genes in all cases. Furthermore, gene expression profiling allowed the identification of putative translocations dysregulating CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased levels of MMSET expression were found in one MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.

Gene Expression Profiling and Real-Time PCR Analyses Make It Possible To Identify Novel Potential Cancer-Testis Antigens in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1793-1793
Author(s):  
Maud Condomines ◽  
Dirk Hose ◽  
Thierry Reme ◽  
John de Vos ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Real Time ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Rt Pcr ◽  
Normal Tissues ◽  
Present Call ◽  
The Mean ◽  
Cancer Testis

Abstract The identification of novel tumor-associated antigens is critical for the development of immunotherapeutic strategies. Cancer-testis (CT) antigens represent attractive targets due to their restricted pattern of expression. More than 90 CT genes have been previously classified into four categories according to their expression profiles: testis-restricted (expression in testis and tumor samples only), “tissue restricted” (mRNA detected in 2 or fewer non-gametogenic tissues), “differentially expressed” (mRNA detected in three to six non-gametogenic tissues), and “ubiquitously expressed”. Among those, we previously reported that 18 CT genes were expressed by primary myeloma cells (MMC) of more than 10% of patients with multiple myeloma (MM). This study aimed at finding novel putative CT genes expressed in MM using cDNA microarray analysis and real-time RT-PCR validation. Gene expression profiles of 5 testis samples, 64 MMC, 7 normal memory B cell (MB), 7 normal bone marrow plasma cell samples and 23 normal tissue samples available on a public database were obtained using Affymetrix U133AB microarrays. Out of 45000 probe sets of Affymetrix U133 AB chips, we selected 16982 probe sets which had a “Present” Affymetrix Call in MMC of at least 6/64 patients and in 3/5 testis samples. In order to select genes with a similar pattern of expression than the known CT genes, we developed 4 independent filters making it possible to keep a high number of known CT genes while decreasing the total number of probe sets. Firstly, 2514 of 16982 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MB > 2.5. Secondly, 541 of these 2514 probe sets had a Present call in less than 7 of the 23 normal tissues. Thirdly, 333 of these 541 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MMC with an Absent call > 2.5. Fourthly, we removed genes whose expression profiles were discordant with different probe sets or discordant with data of the literature. The final probe set list contains 88 probe sets which include 13 of 18 known CT genes reported in MM, thus resulting in a 190-fold enrichment. The expression in 13 normal tissues and in MM samples of 21 out of these 75 putative novel CT genes was investigated by real time RT-PCR. Seven genes were ubiquitously expressed or poorly expressed in MMC samples and further deleted. According to the previously defined CT gene categories, we found one novel “testis-restricted” (TEX14), 8 “tissue-restricted” and 5 “differentially expressed” CT genes. Immunogenicity of one gene product - IGSF11 - was already demonstrated in other cancers by identifying a T-cell epitope. Two genes - NLGN4X and FAM133A - are located in X chromosome and 2 genes - CTNNA2 and FAM133A - are expressed only in brain and testis. In conclusion, by analyzing gene expression patterns with Affymetrix microarrays, we found 75 novel putative CT antigen candidates expressed in MMC of 10 to 100% of patients. Real time RT-PCR validation made it possible to confirm the CT status of 14 genes out of the 21 tested. Further studies are warranted to determine their immunogenicity.

scholarly journals Characterization of Differential Gene Expression in Adrenocortical Tumors Harboring β-Catenin (CTNNB1) Mutations

2011 ◽  
Vol 96 (7) ◽  
pp. E1206-E1211 ◽  
Author(s):  
Julien Durand ◽  
Antoine Lampron ◽  
Tania L. Mazzuco ◽  
Audrey Chapman ◽  
Isabelle Bourdeau
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Adrenal Glands ◽  
Expression Profiles ◽  
Genetic Alterations ◽  
Nuclear Accumulation ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Adrenocortical Tumors

Abstract Background: Mutations of β-catenin gene (CTNNB1) are frequent in adrenocortical adenomas (AA) and adrenocortical carcinomas (ACC). However, the target genes of β-catenin have not yet been identified in adrenocortical tumors. Objective: Our objective was to identify genes deregulated in adrenocortical tumors harboring CTNNB1 genetic alterations and nuclear accumulation of β-catenin. Methods: Microarray analysis identified a dataset of genes that were differently expressed between AA with CTNNB1 mutations and wild-type (WT) tumors. Within this dataset, the expression profiles of five genes were validated by real time-PCR (RT-PCR) in a cohort of 34 adrenocortical tissues (six AA and one ACC with CTNNB1 mutations, 13 AA and four ACC with WT CTNNB1, and 10 normal adrenal glands) and two human ACC cell lines. We then studied the effects of suppressing β-catenin transcriptional activity with the T-cell factor/β-catenin inhibitors PKF115-584 and PNU74654 on gene expression in H295R and SW13 cells. Results: RT-PCR analysis confirmed the overexpression of ISM1, RALBP1, and PDE2A and the down-regulation of PHYHIP in five of six AA harboring CTNNB1 mutations compared with WT AA (n = 13) and normal adrenal glands (n = 10). RALBP1 and PDE2A overexpression was also confirmed at the protein level by Western blotting analysis in mutated tumors. ENC1 was specifically overexpressed in three of three AA harboring CTNNB1 point mutations. mRNA expression and protein levels of RALBP1, PDE2A, and ENC1 were decreased in a dose-dependent manner in H295R cells after treatment with PKF115-584 or PNU74654. Conclusion: This study identified candidate genes deregulated in CTNNB1-mutated adrenocortical tumors that may lead to a better understanding of the role of the Wnt-β-catenin pathway in adrenocortical tumorigenesis.

scholarly journals A Robust Gene selection Method for Microarray-based Cancer Classification

2010 ◽  
Vol 9 ◽  
pp. CIN.S3794 ◽  
Author(s):  
Xiaosheng Wang ◽  
Osamu Gotoh
Keyword(s):  
Gene Expression ◽  
Feature Selection ◽  
Gene Selection ◽  
Information Gain ◽  
Expression Profiles ◽  
Feature Selection Method ◽  
Gene Expression Profiles ◽  
Molecular Classification ◽  
Selection Method ◽  
Chi Square

Gene selection is of vital importance in molecular classification of cancer using high-dimensional gene expression data. Because of the distinct characteristics inherent to specific cancerous gene expression profiles, developing flexible and robust feature selection methods is extremely crucial. We investigated the properties of one feature selection approach proposed in our previous work, which was the generalization of the feature selection method based on the depended degree of attribute in rough sets. We compared the feature selection method with the established methods: the depended degree, chi-square, information gain, Relief-F and symmetric uncertainty, and analyzed its properties through a series of classification experiments. The results revealed that our method was superior to the canonical depended degree of attribute based method in robustness and applicability. Moreover, the method was comparable to the other four commonly used methods. More importantly, the method can exhibit the inherent classification difficulty with respect to different gene expression datasets, indicating the inherent biology of specific cancers.

scholarly journals Classify Hyperdiploidy Status of Multiple Myeloma Patients Using Gene Expression Profiles

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e58809 ◽  
Author(s):  
Yingxiang Li ◽  
Xujun Wang ◽  
Haiyang Zheng ◽  
Chengyang Wang ◽  
Stéphane Minvielle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Expression Profiles ◽  
Gene Expression Profiles

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Relationship of Bone Marrow Plasma Cell Morphology with Cytogenetic Abnormalities in Patients with Multiple Myeloma

2021 ◽  
Author(s):  
Vikram Narang ◽  
Maneet Luthra ◽  
Avantika Garg ◽  
Amit Dhiman ◽  
Neena Sood
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Descriptive Analysis ◽  
Cross Sectional Study ◽  
Chi Square ◽  
Morphological Pattern ◽  
Cross Sectional ◽  
Cytogenetic Abnormalities ◽  
Cytogenetic Studies ◽  
Chi Square Test

Introduction: Cytogenetics has become an integral part of Multiple Myeloma (MM) diagnosis and prognostication. A combination of conventional cytogenetics and interphase Fluorescence In Situ Hybridization (FISH) is currently used to stratify tumours into high, intermediate and standard risk disease. Aim: To compare the morphological details of plasma cells with cytogenetic abnormalities. Materials and Methods: The present retrospective cross sectional study was conducted at Department of Pathology Dayanand Medical College and Hospital, Ludhiana in three and a half year duration (1st January 2014 to 30th June 2017). All the diagnosed MM patients in whom cytogenetic was available were included and descriptive analysis was done using Chi-Square test and relevant statistical analysis using SPSS 21 version. Correlation was done with various morphological pattern (plasmacytic, plasma blastic). Results: Cytogenetic studies were performed on 42 cases using FISH technique (n=31, 81.6%) and GTG (Giemsa) banding (n=4, 10.5%). Three (7.9%) patients were tested with both methods. In the present study, all the patients (n=2,100%) with plasmablastic morphology who got tested with cytogenetics had del13q14.3 and none of the patients with normal genome (n=22) had plasmablastic morphology. Conclusion: Morphologic patterns of plasma cells and cytogenetic studies correlate well and can together help in better prognostication of MM patients.

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