From cryptic chromosomal lesions to pathologically relevant genes: Integration of SNP-array with gene expression profiling in myelodysplastic syndrome with normal karyotype

Abstract In AML, the majority of patients <60 years of age will enter remission but at least 50% will subsequently relapse, therefore the monitoring of minimal residual disease (MRD) during treatment has become an important issue. Current molecular markers for MRD are mainly limited to the RT-PCR detection of the fusion genes resulting from recurrent translocations which paradoxically are mostly limited to favourable risk groups who are least likely to relapse leaving the majority of the patients, including those with a normal karyotype, without a molecular marker suitable for monitoring. Two hundred and twenty patients have been assessed by gene expression profiling using Affymetrix U133A chips and the data analysed with the aim of identifying novel MRD markers for patients who do not currently have a suitable marker. As an initial “proof of principle”, we have identified possible MRD markers for patients with either t(15;17), t(8;21) or inv(16) and correlated with changes in expression of these markers with clinical changes as measured by established molecular MRD markers (PML-RARα or WT1). Of the expression profile from 22,283 probe sets in 29 cases of t(15;17), 20 genes were identified which had at least a two fold over expression which was unique to the t(15;17) subgroup. Of these several of the probe sets were related to the same gene, but from the reduced gene list 2 (HGF and ILGF binding protein) were selected for quantitation by quantitative PCR. Similarly the expression profile identified 20 genes which were unique to the 15 cases of t(8;21), and 20 genes which were unique to the 19 cases of inv(16). These included ETO and MYH11 representing the respective 3′ end of the respective fusion transcript. Three other genes (PRAME, POU4F1, and IL5RA) were selected for the t(8;21) cases and ST18, CLIP-170 and MNI for the inv(16) cases. When the relative quantitative expression of each of these “unique” genes was correlated with the expression of the established markers of minimal residual disease (PML-RARα or WTI) there was good correlation. These data suggest that gene expression profiling can identify ‘unique’ genes which can be used to develop specific markers for minimal residual disease monitoring for a larger proportion of cases of AML than is currently available.

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P073 Gene expression profiling in the myelodysplastic syndrome: patients with intermediate 1 versus higher risk

Leukemia Research ◽

10.1016/s0145-2126(07)70143-1 ◽

2007 ◽

Vol 31 ◽

pp. S79-S80

Author(s):

A. Kracmarova ◽

H. Bruchova ◽

M. Belickova ◽

J. van Delft ◽

J. Cermak ◽

...

Keyword(s):

Gene Expression ◽

Myelodysplastic Syndrome ◽

Gene Expression Profiling ◽

Expression Profiling

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Gene expression profiling of the bone marrow mononuclear cells from patients with myelodysplastic syndrome

Oncology Reports ◽

10.3892/or.14.5.1189 ◽

2005 ◽

Author(s):

Jun Qian ◽

Zixing Chen ◽

Wei Wang ◽

Jiannong Cen ◽

Yongquan Xue

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Mononuclear Cells ◽

Bone Marrow Mononuclear Cells

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Amplification of 6p Associated with Familial CLL

Blood ◽

10.1182/blood.v116.21.2432.2432 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2432-2432

Author(s):

Jennifer R Brown ◽

Bethany Tesar ◽

Megan Hanna ◽

Megan Ash ◽

Stacey M Fernandes ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Second Generation ◽

Somatic Mutations ◽

Risk Allele ◽

Conflicts Of Interest ◽

Snp Array ◽

High Density ◽

A Genome

Abstract Abstract 2432 Chronic lymphocytic leukemia (CLL) is one of the most familial of all cancers but the genetic basis of this heritability remains poorly characterized. Families with very strong inheritance of CLL have been described in the literature, and recently the occurrence of CLL in one such family was associated with a polymorphism in the DAPK gene. Here we report the genomic characterization of a family in which CLL appears to be inherited in a Mendelian autosomal dominant manner. Within this family, five of eleven siblings of the first generation were affected, and one of those affected siblings had five children, of whom three were also affected (the second generation). The children of the second generation are currently aged 20–30 and hence too young to know whether they will develop CLL. We performed high-density single-nucleotide polymorphism (SNP) array analysis and gene expression profiling on tumor and germline DNA from four of the offspring of the second generation, as well as six of their children. Analysis of the SNP array data revealed a significant germline amplification of 6p, spanning 0–720 Mb and encompassing a known copy number variant (CNV) region but significantly larger than the CNV region. This amplification was found in both affected individuals with samples available from the second generation, and was transmitted by each of them to one of their two children in the third generation. This amplification was absent from the two unaffected members of the second generation, their children, or any of the other 189 individuals with CLL who were analyzed in our high-density SNP array dataset. None of the unaffected individuals with or without the amplification had evidence of monoclonal B cell lymphocytosis (MBL) by highly sensitive flow cytometry. These unaffected individuals also lacked any PCR-detectable oligoclonal or monoclonal immunoglobulin heavy chain gene rearrangement suggestive of MBL. The region of amplification contains four protein-coding genes: EXOC2, DUSP22, HUS1B and IRF4. We sequenced the coding regions of these four genes and the 5` and 3` UTRs of IRF4 in all family members, but found no somatic mutations in this family. All four genes were also sequenced in 92 other familial CLLs, identifying no somatic mutations. We then analyzed our gene expression profiling data to assess whether any genes in this region were altered in the affected individuals with the amplification. This analysis revealed a significant 1.74X increase in IRF4 expression in the CLLs with the amplification compared to those without (q value < 0.001). By Western blotting, we confirmed that IRF4 protein was increased approximately two-fold in amplified compared to non-amplified samples. These data suggest that the amplification may target IRF4, which has been previously implicated in CLL by a genome wide association study that identified a tag SNP in its 3` UTR as a CLL risk allele. Further analysis of our SNP data demonstrated allele specific amplification in this region, and mass-spectrometric genotyping confirmed enrichment of the CLL risk allele in the individuals with amplification. We conclude that amplification of IRF4 carrying the risk allele for CLL appears likely to be the culprit predisposing to CLL in this family. Disclosures: No relevant conflicts of interest to declare.

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O-21 Gene expression profiling of thebone marrow mononuclear cells from patients with myelodysplastic syndrome

Leukemia Research ◽

10.1016/s0145-2126(05)80020-7 ◽

2005 ◽

Vol 29 ◽

pp. S7-S8

Author(s):

Z. Chen ◽

J. Qian ◽

W. Wang ◽

J. Cen ◽

Y. Xue

Keyword(s):

Gene Expression ◽

Myelodysplastic Syndrome ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Mononuclear Cells

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Gene expression profiling in myelodysplastic syndrome after SPARC overexpression associated with Ara-C

Oncology Reports ◽

10.3892/or.2015.4139 ◽

2015 ◽

Vol 34 (4) ◽

pp. 2072-2082 ◽

Cited By ~ 3

Author(s):

QING NIAN ◽

ZHIQIANG ZHANG ◽

CHUNMEI WEI ◽

XINGYI KUANG ◽

XINGYONG WANG ◽

...

Keyword(s):

Gene Expression ◽

Myelodysplastic Syndrome ◽

Gene Expression Profiling ◽

Expression Profiling

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Gene Expression Profiling Predicts Outcome in De Novo Acute Myeloid Leukemia (AML) with Normal Karyotype: Results of Children’s Oncology Group (COG) Study POG #9421.

Blood ◽

10.1182/blood.v108.11.1915.1915 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1915-1915

Author(s):

Norman J. Lacayo ◽

Maureen O’Brien ◽

Shweta Jain ◽

Soheil Meshinchi ◽

Ron Yu ◽

...

Keyword(s):

Gene Expression ◽

Clinical Outcome ◽

Gene Expression Profiling ◽

Expression Profiling ◽

De Novo ◽

Gene List ◽

Normal Karyotype ◽

Classification Error ◽

Classification Error Rate ◽

Number Of Genes

Abstract We previously reported a 36% event-free survival (EFS) estimate for patients with normal karyotype (NK) on the COG study POG #9421 (n=144). In addition, we hypothesized that gene expression profiling would identify signatures linked to clinical outcome and useful for retrospective risk determination. Bone marrows in a subset of patients with NK (n=58) were analyzed using a 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. Prediction analysis for microarrays (PAM) was used to find genes that identified samples associated-with and unassociated-with events (relapse or death); after analyzing 28,711 genes with PAM we chose a 727-gene cluster that differentiated patients with NK on the basis of clinical outcome (cumulative classification error rate 19%). The analysis was biased for a larger number of genes in order to obtain a more biologically informative gene pathways analysis. Significance analysis of microarrays (SAM) on the PAM output identified 633 genes (false-discovery rate of 0%) that differed significantly between the event-associated and event-unassociated samples. Spearman based hierarchical clustering on these genes yielded 2 clusters with statistically significant different event-free survivals: 65% (n=24) for the event-unassociated curve and 23% (n=34) for the event-associated curve with P=0.01. The patients in these clusters did not differ at diagnosis for WBC (70K vs. 100K/microL with P=0.19) and age (10.1 vs. 9.9 yrs with P=0.83) by unpaired t-test; or for sex (P=0.11) and FLT3-ITD status (P=0.76) by Fisher’s exact test. The gene list (GenBank #) and fold-change in gene expression from SAM output were analyzed using Ingenuity Pathway Analysis software (Ingenuity™ Systems, Mountain View, CA). Canonical pathways identified 33 networks associated with event-unassociated outcome using 302 eligible genes that included: underexpressed CDC73, RAD50, SPARC, PTPN12, MXD1, TNF, ABCA1, STAT4, CCNA1, TNF, BCL2A1, JUN, BCL6 and AREG; and overexpressed RUNX3, FKBP9, FKBP8, MAP2K2, CHES1, HOXA11, HRK, CDK6, MGA, MAPK3, ABL1, HDAC7A, SMARCC2, SYK, MXD4, CDC42. Several of these genes have been previously reported to be associated with improved outcome in AML. However, two of these genes (MXD4 and MXD1) previously not identified as related to outcome in AML, but identified in our analysis in two highly interacting networks related to the MYC gene, result in a difference in EFS of 51% vs. 24% (P=0.04), suggesting that a smaller number of genes may be predictive of outcome. Conclusion: Risk assignment for patients with NK may be feasible by analyzing a limited number of genes. We will validate these findings by correlating gene expression results with quantitative real-time PCR. Prospective validation of this strategy in clinical trials may be warranted.

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Gene Expression Profiling and FLT3 Status Correlate with Outcome in De Novo Acute Myeloid Leukemia (AML) with Normal Karyotype: Results of Children’s Oncology Group (COG) Study POG #9421.

Blood ◽

10.1182/blood.v106.11.2372.2372 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2372-2372

Author(s):

Norman Lacayo ◽

Soheil Meshinchi ◽

Susana Raimondi ◽

Chitra Saraiya ◽

Maureen O’Brien ◽

...

Keyword(s):

Gene Expression ◽

Clinical Outcome ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Gene List ◽

Normal Karyotype ◽

Error Rates ◽

Pcr Analysis ◽

Classification Error ◽

Wild Type

Abstract The event-free survival (EFS) estimate for patients with normal karyotype (NK) on COG study POG #9421 (n=144) was 36%. We previously reported a subgroup of patients (n=68) with AML and NK that could be divided into 2 groups whose clinical outcomes correlated with abnormalities of FLT3 [internal tandem duplications (ITD) or activating loop mutations]. EFS estimates were 13% for patients with mutant FLT3 and 61% for children with wild-type FLT3 (P=0.01). We hypothesized that gene expression profiling would identify signatures that are linked to clinical outcome and can be used for risk determination. Cytogenetic testing was carried out in clinical laboratories at the institutions in which AML was diagnosed and then centrally reviewed. We analyzed bone marrow from 45 patients with NK on 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. FLT3 status (mutant or wild type) was determined by RT-PCR analysis of RNA from these 45 samples (exon 11 for ITDs, exon 17 for point mutations): 18 expressed mutant FLT3, 27 expressed wild-type FLT3. Using prediction analysis for microarrays (PAM) to find the minimum number of genes that identified samples associated with and unassociated with events (relapse or death), we identified a 128 gene cluster that differentiated patients with NK on the basis of clinical outcome (classification error rates were 15% for samples associated with events and 12.5% for event-unassociated samples). Significance analysis of microarrays (SAM) identified, with a false-discovery rate of 1.25%, 82 genes in the cluster whose expression differed significantly between the event-associated samples and the event-unassociated samples. Hierarchical clustering based on these 82 genes yielded 2 signatures: one event-associated and one event-unassociated. FLT3 Status Event-Associated Signature Event-Unassociated Signature Wild-type EFS=44% (n=15) EFS=90% (n=12) Mutant EFS=7% (n=13) EFS=60% (n=5) The median WBC counts at the time of diagnosis were 68 x 109/L in the event-associated group and 61 x 109/L in the event-unassociated group (P=0.27). The gene list and d-scores from SAM analysis were analyzed using Ingenuity Pathway Analysis software (Ingenuity™ Systems, Mountain View, CA). Canonical pathways associated with poor outcome included apoptosis signaling (↑BCL2A1, ↓BAK1), ERK/MAP signaling (↑RAC2), cell cycle (↓ABL1), SAPK/JNK signaling (↑RAC2, ↓CDC42), integrin signaling (↑RAC2, ↓BCAR3, ↓ABL1, ↓CDC42), and IL6 signaling (↑IL6R). We conclude that risk assignment for patients with NK can be more precisely determined by combining FLT3 analysis and gene expression profiling. Such an approach identified 4 distinct groups with different outcomes. We will validate these findings by analyzing additional diagnostic samples with normal karyotype. Prospective validation of this strategy in clinical trials may be warranted.

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A20 Gene Deregulation In Waldenstrom's Macroglobulinemia.

Blood ◽

10.1182/blood.v116.21.3628.3628 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3628-3628

Author(s):

Stephanie Poulain ◽

Rachid Aijjou ◽

Natacha Broucqsault ◽

Salomon Manier ◽

Agnes Daudignon ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Real Time ◽

Expression Profiling ◽

Copy Number ◽

Research Funding ◽

Snp Array ◽

Fish Analysis ◽

Nfkb Pathway ◽

A20 Gene

Abstract Abstract 3628 Background. Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells and monoclonal IgM gammopathy. The deletion 6q is the most frequent cytogenetic aberration in WM and it has been suspected that this region harbours a tumour suppressor gene of pathogenic significance for WM. Comparative genomic hybridization (CGH) array delineated several minimal deleted regions (MDR) on 6q and pointed out key regulator genes of the NFKB pathway involved in these deletions, including A20 gene. The zinc finger protein A20 has been characterized as dual inhibitor of NFKB activation and was recently described as a tumor suppressor gene in lymphoma. However, the mechanisms of A20 deregulation are not fully understood in WM. We aimed to study the mechanisms of A20 deregulation in WM using gene expression profiling (GEP) and single nucleotide polymorphism (SNP) based arrays, a powerful high resolution method allowing both the detection of loss of heterozygosity (LOH) and copy number alteration (CNA) analysis in the same experiment. Methods. We have studied BM samples from 42 patients (pts) with WM (31 males, mean age: 67 years). Conventional karyotype study was analyzed following stimulation with IL2 and DSP30 of BM cells. FISH analysis was performed to detect deletion 6q21 (MYB probe). DNA and RNA were extracted following BM CD19 B cell negative selection. Quantification of copy number of A20 gene was performed using real time PCR, RNAse P gene was used as reference gene. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA in 31 pts. Paired samples (B cells/normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities in 29 patients. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA were identified using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Gene expression profiling (GEP) was performed in 11 pts using Affymetrix U133A arrays. Gene-expression intensity values were log transformed, normalized and analyzed using GeneSpring GX software. Results. SNP array identified deletion 6q in 9/31 (29%) patients, confirmed by FISH analysis. All cases were monoallelic deletion. In 90% of cases, deletion 6q was associated with others genetic abnormalities. In one case, we observed a loss of 6q combined with a gain of 6p. Deletion 6q was associated with gain of chromosome 4 in 2 cases, and with deletion of 13q in 3 cases. The MDR was located on chromosome 6q21-6q25. This MDR contained several candidate genes included always deletion of A20 gene, located on 6q23, and was confirmed by real time DNA PCR quantification in all cases. Overall, the frequency of A20 gene deletion in our entire WM cohort (N=42 pts) was 29.2% (12/42), determined by real time DNA PCR of the copy number of A20 gene. In patient without deletion 6q, we looked at potential deregulated mechanisms of A20 by LOH with no CNA, e.g. UPD (uniparental disomy). We have not seen any UPD targeting the A20 gene. We then looked at A20 gene expression by GEP, and found no significant variation of A20 gene expression related to A20 monoallelic deletion as compared to pts with two copies, reflecting probably the existence of other mechanisms of A20 gene deregulation. This result was consistent with the absence of clear difference in genome-wide expression profiling in pts with 6q deletion and those without the deletion. As A20 is a key player in the negative feedback loop regulation of NFKB, we also looked at the gene expression of a set of genes involved in NFKB pathway. The presence or absence of A20 deletion did not influence the gene expression profiling of this NFKB pathway gene set. Conclusion. We described a high frequency of deletion of A20 gene. In most cases, deletion of A20 was associated with other genetic abnormalities. A20 deletion was not associated with a significant signature by GEP. Additional studies are needed to understand the cellular consequences of A20 deletions in the pathogenesis of WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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