Gene expression profiling and copy number analysis to identify predictive molecular markers in breast cancer: Successful use of formalin fixed paraffin embedded tissue (FFPE)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 574-574
Author(s):  
M. Y. Iddawela ◽  
Y. Wang ◽  
R. Russell ◽  
G. Cowley ◽  
M. El-Sheemy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Treatment Response ◽  
Expression Profiling ◽  
Copy Number ◽  
Predictive Markers ◽  
Copy Number Analysis ◽  
Significant Differential Expression ◽  
Response To Chemotherapy ◽  
Copy Number Changes

574 Background: FFPE is a valuable and widely available resource for translational research which to date has been under-used due to technical limitations. Improvement in technology has enabled genome-wide analysis of FFPE samples. We have assessed gene expression and copy number changes in the same cohort of breast cancers to identify markers or pathways important in prediction of treatment response. Methods: FFPE tissues from patients treated with neoadjuvant adriamycin/cyclophosphamide followed by taxanes in a clinical study were used. Gene expression profiling was assessed using the cDNA mediated annealing selection and ligation assay using the cancer panel which assess 502 genes (DASL assay, Illumina). Data was analysed using BeadStudio software. Copy number changes were assessed using the Molecular inversion probe assay with the 50K SNP panel (Affymetrix, California) and analysed using Nexus software (Biodiscovery). Results: Gene expression profiling was carried out on 44 samples. 12/44 (27%) patients had a pathological complete response (pCR) following chemotherapy. Significant differential expression of genes between pCR and non-pCR cancers were shown. TNFRSF5, CTSD, BCL3, ARNT, BIRC3, TGFBR1, MLLT6, and EVI2A were over-expressed and COL18A1, FGF12, IGFBP1 and NOTCH4 which were down-regulated in cancers that have a pCR (p ≤ 0.01). Copy number changes were assessed in 33 samples and comparison of copy number changes in pCR vs. non-pCR showed gains in regions 6q22, 21q21, 4p14, 4q21, 4p14, and loss at 11q11 (p ≤ 0.01). Three regions containing microRNA coding sequences, mir130a (11q11) mir142 (17q23) and mir21 (17q23) showed significant loss among pCR tumours (p < 0.05). Conclusions: This feasibility study shows that FFPE can be used for gene expression and copy number analysis which is a useful tool for the discovery of predictive markers for treatment response in neoadjuvant treatment trials. The role of TNFRSF5, microRNA 21/130a/142, and 11q11 loss should be further investigated as predictive markers of response to chemotherapy. [Table: see text]

Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines

2007 ◽  
Vol 46 (10) ◽  
pp. 895-908 ◽  
Author(s):  
Claudia Zanazzi ◽  
Remko Hersmus ◽  
Imke M. Veltman ◽  
Ad J.M. Gillis ◽  
Ellen van Drunen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Malignant Mesothelioma ◽  
Expression Profiling ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Mesothelioma Cell ◽  
Copy Number Changes

High Resolution Genomic Analysis In CLL Demonstrates Genomic Stability In Untreated Patients and Novel Markers of Progression In Treated Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2426-2426
Author(s):  
Jennifer R Brown ◽  
Megan Hanna ◽  
Bethany Tesar ◽  
Lillian Werner ◽  
Hazel Reynolds ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Resolution ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Copy Number ◽  
Genomic Analysis ◽  
Copy Number Analysis ◽  
Trisomy 12 ◽  
Very High ◽  
13Q Deletion

Abstract Abstract 2426 Chronic lymphocytic leukemia is the most common leukemia of adults but still incurable. Prognosis at diagnosis is widely variable, and the key cytogenetic abnormalities determined by FISH remain one of the best predictors of prognosis and treatment response. We therefore undertook very high resolution genomic analysis of 161 CLLs with matched germline samples using Affymetrix 6.0 SNP arrays, in an effort to identify additional predictors of prognosis, and have also performed gene expression profiling on most of this patient cohort. The median age at diagnosis for the cohort was 55 (31–79), and the median time to sampling was 4.6 months (0.5–291). 22% of the cohort was previously treated, with an additional 21% of patients receiving treatment during the follow-up period, for a total of 43% treated, with a median time from diagnosis to treatment of 41 months (0.4-161.2 months). The genomic data were analyzed both by GISTIC, which identifies significant deletions and amplifications based on analysis of the frequency and amplitude of each aberration in the tumor samples alone, as well as by paired copy number analysis of each tumor and its cognate germline, using Birdseed, PLINK and PennCNV. Our results show that the CLL genome is overall quite stable, with a median of only one acquired copy number aberration per sample, excluding rearrangements at the immunoglobulin gene loci. GISTIC analysis on the entire population identified the known common CLL abnormalities at frequencies that would be expected in a largely untreated cohort: 57% del 13q, 6.2% deletion 11q, 5.0% deletion 17p, and 12% trisomy 12. The presence of two or more acquired copy number aberrations (CNAs) of any type was associated with a significantly shorter time to first therapy (p<0.0001). A higher number of CNAs was strongly associated with deletions of 11q or 17p, but the predictive power of a higher number of CNAs was still present in those CLLs without deletions 11q or 17p. Detailed analysis of 13q deletion revealed no association of longer deletions or homozygous deletions with time to first therapy. However, any additional somatic copy number aberration in addition to 13q deletion significantly reduced the time to first therapy, making it comparable to non-13q patients. In order to identify genetic markers of progression, we compared treated to untreated patients using GISTIC. This analysis revealed that untreated patients showed peaks largely limited to deletion 13q and trisomy 12. Treated patients however showed three additional significant peaks: a deletion peak at 8p, as well as significant amplification peaks at 3q26.32 and 8q24.21. The deletion peak at 8p was observed in 8 of 161 samples tested (5.0%), and was large, with a common region of deletion spanning 11.0–29.6 Mb. Six of eight of these patients were untreated at the time of sampling but had a very short time to treatment thereafter, independent of whether they had coexistent deletions of 17p or 11q, suggesting that this deletion carries a very poor prognosis in itself. Another notable region of amplification was found on 3q26.32 in nine patients (9/161 or 5.6%). Although many of these amplified regions were large, three of the nine patients carrying this amplification demonstrated focal somatic amplification of the final exon of PIK3CA, the alpha catalytic subunit of PI3K. Finally the amplification on 8q24 was present in 6 of 161 CLLs (3.7%), two of which were focal and amplified only the gene desert region previously implicated in CLL risk by genome-wide association study and located approximately 335 kb centromeric to MYC. Analysis of gene expression profiling comparing patients with and without amplifications demonstrated upregulation of MYC mRNA expression and alteration of downstream targets of MYC in samples with amplification. We conclude that very high resolution copy number analysis with matched germline comparison in CLL reveals a quite stable genome in untreated patients, and identifies amplifications of 3q26 and MYC at 8q24 as progression events. Disclosures: No relevant conflicts of interest to declare.

Gene Expression Profiling Reveals Fundamental Differences between Leukemic Stem Cells (Lin-CD34+CD38−) and Mature Blasts (Lin−CD34+CD38+) of Acute Myeloid Leukaemia (AML) Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1377-1377
Author(s):  
Kazem Zibara ◽  
Daniel Pearce ◽  
David Taussig ◽  
Spyros Skoulakis ◽  
Simon Tomlinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Populations ◽  
Leukemic Stem Cells ◽  
Significant Differential Expression ◽  
New Genes

Abstract The identification of LSC has important implications for future research as well as for the development of novel therapies. The phenotypic description of LSC now enables their purification and should facilitate the identification of genes that are preferentially expressed in these cells compared to normal HSC. However, gene-expression profiling is usually conducted on mononuclear cells of AML patients from either peripheral blood and/or bone marrow. These samples contain a mixture of blasts cells, normal hematopoietic cells and limited number of leukemic stem cells. Thus, this results in a composite profile that obscure differences between LSC and blasts cells with low proliferative potential. The aim of this study was to compare the gene expression profile of highly purified LSC versus leukemic blasts in order to identify genes that might have important roles in driving the leukemia. For this purpose, we analyzed the gene expression profiles of highly purified LSCs (Lin−CD34+CD38−) and more mature blast cells (Lin−CD34+CD38+) isolated from 7 adult AML patients. All samples were previously tested for the ability of the Lin−CD34+CD38− cells but not the Lin−CD34+CD38+ fraction to engraft using the non-obese diabetic/severe combined immuno-deficiency (NOD-SCID) repopulation assay. Affymetrix microarrays (U133A chip), containing 22,283 genes, were used for the analysis. Comparison of Lin-CD34+CD38- cell population to the Lin−CD34+CD38+ cell fraction showed 5421 genes to be expressed in both fractions. Comparative analysis of gene-expression profiles showed statistically significant differential expression of 133 genes between the 2 cell populations. Most of the genes were downregulated in the LSC-enriched fraction, compared to the more differentiated fraction. Gene ontology was used to determine the categories of the up-regulated transcripts. These transcripts, which are selectively expressed, include a number of known genes (e.g., receptors, signalling genes, proliferation and cell cycle genes and transcription factors). These genes play important roles in differentiation, self-renewal, migration and adhesion of HSCs. Among the genes showing the highest differences in expression levels were the following: ribonucleotide reductase M2 polypeptide, thymidylate synthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II, CDC20, nucleolar and spindle associated protein 1, Rac GTPase activating protein 1, leukocyte immunoglobulin-like receptor, proliferating cell nuclear antigen, myeloperoxidase, cyclin A1 (RRM2, TYMS, ZWINT, CTSG, AZU1, TOP2A, CDC20, NUSAP1, RACGAP1, LILRB2, PCNA, MPO, CCNA1). Some transcripts detected have not been implicated in HSC functions, and others have unknown function so far. This work identifies new genes that might play a role in leukemogenesis and cancer stem cells. It also leads to a better description and understanding of the molecular phenotypes of these 2 cell populations. Hence, in addition to being a more efficient way to further understand the biology of LSC, this should also provide a more efficient way of identifying new therapeutics and diagnostic targets.

The potential role of IGF1r in young adult patients with gastroinetstinal stromal tumor (GIST)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 10562-10562
Author(s):  
M. Pantaleo ◽  
A. Astolfi ◽  
M. Di Battista ◽  
P. Paterini ◽  
D. Santini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Young Adult ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Copy Number ◽  
Adult Patients ◽  
The Other ◽  
Gene Copy ◽  
Gastric Gist ◽  
Array Analysis

10562 Background: The insulin-like growth factor 1 receptor (IGF1r) is a tyrosine kinase receptor that plays a key role in the growth of normal tissues. The aberration of IGF system has been found in many cancers. Some interesting results about IGF1r were published on GISTs. However, until now the real role on the pathogenesis of this disease and its clinical implications still needs to be defined. Methods: We studied IGF1r in 8 patients affected by gastric GIST. Seven patients underwent surgery at diagnosis, whereas one patient was operated after imatinib and sunitinib treatment. Two patients were young (< 30 years old), and other patients ranged between 54 and 85 years. IGF1r was studied as gene expression profiling performed with Affymetrix GeneChip HG-U133 Plus 2.0 arrays, as genomic copy number with SNP array analysis Affymetrix Genome Wide Human SNP 6.0 arrays, and with western blotting (WB) usinganti-IGF-IRβ (Santa Cruz Biotechnology). Results: The unsupervised analysis of gene expression profiling of our patients merged with a data set from a gastric GIST identified two groups with different regulation of IGF1r (FDR threshold to 0.2%). In particular, IGF1r was up-regulated in the two youngest patients (28 and 30 years-old). The SNPs array analysis gene copy number showed that none of the patients bore IGF1r amplification. The quantitative analysis of protein level by WB again showed that only the two youngest patients had an over-expression of IGF1r. In all the other patients the WB analysis was negative. Conclusions: These results suggest that IGF1r seems to be a novel signalling pathway other than KIT and PDGFRA in a subset of GISTs. The young adult patients had a strongly different molecular background in comparison to the other older ones. The correlation between IGF1r and mutational status of KIT and PDGFRA, clinical outcome, treatments responsiveness as well as its role as potential target deserves to be further investigated. No significant financial relationships to disclose.

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