Correlations of EGFR mutations and increases in EGFR and HER2 copy number to gefitinib response in a retrospective analysis of lung cancer patients

<p>Abstract</p> <p>Background</p> <p>Gefitinib, a small molecule tyrosine kinase inhibitor of the Epidermal Growth Factor Receptor (<it>EGFR</it>), has shown limited efficacy in the treatment of lung cancer. Recognized clinical predictors of response to this drug, specifically female, non-smoker, Asian descent, and adenocarcinoma, together suggest a genetic basis for drug response. Recent studies have addressed the relationship between response and either sequence mutations or increased copy number of specific receptor tyrosine kinases. We set out to examine the relationship between response and the molecular status of two such kinases, <it>EGFR </it>and <it>HER2</it>, in 39 patients treated with gefitinib at the BC Cancer Agency.</p> <p>Methods</p> <p>Archival patient material was reviewed by a pathologist and malignant cells were selectively isolated by laser microdissection or manual recovery of cells from microscope slides. Genomic DNA was extracted from 37 such patient samples and exons 18–24, coding for the tyrosine kinase domain of <it>EGFR</it>, were amplified by PCR and sequenced. <it>EGFR </it>and <it>HER2 </it>copy number status were also assessed using FISH in 26 samples. Correlations between molecular features and drug response were assessed using the two-sided Fisher's exact test.</p> <p>Results</p> <p>Mutations previously correlated with response were detected in five tumours, four with exon 19 deletions and one with an exon 21 missense L858R point mutation. Increased gene copy number was observed in thirteen tumours, seven with <it>EGFR </it>amplification, three with <it>HER2 </it>amplification, and three with amplification of both genes. In our study cohort, a correlation was not observed between response and <it>EGFR </it>mutations (exon 19 deletion p = 0.0889, we observed a single exon 21 mutation in a non-responder) or increases in <it>EGFR </it>or <it>HER2 </it>copy number (p = 0.552 and 0.437, respectively).</p> <p>Conclusion</p> <p>Neither mutation of <it>EGFR </it>nor increased copy number of <it>EGFR </it>or <it>HER2 </it>was diagnostic of response to gefitinib in this cohort. However, validation of these features in a larger sample set is appropriate. Identification of additional predictive biomarkers beyond <it>EGFR </it>status may be necessary to accurately predict treatment outcome.</p

Circos: An information aesthetic for comparative genomics

We created a visualization tool called Circos to facilitate the identification and analysis of similarities and differences arising from comparisons of genomes. Our tool is effective in displaying variation in genome structure and, generally, any other kind of positional relationships between genomic intervals. Such data are routinely produced by sequence alignments, hybridization arrays, genome mapping, and genotyping studies. Circos uses a circular ideogram layout to facilitate the display of relationships between pairs of positions by the use of ribbons, which encode the position, size, and orientation of related genomic elements. Circos is capable of displaying data as scatter, line, and histogram plots, heat maps, tiles, connectors, and text. Bitmap or vector images can be created from GFF-style data inputs and hierarchical configuration files, which can be easily generated by automated tools, making Circos suitable for rapid deployment in data analysis and reporting pipelines

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis. Introduction Chromosomal translocations to the immunoglobulin heavy chain (IGH) or light chain (IGL or IGK) loci, and less frequently to non-IG loci represent well known mechanisms of oncogene activation in B-cell neoplasms. 1,2 Many of the genes deregulated by chromosomal translocations in these neoplasms are involved in important cellular processes like cell-cycle control (CCND1, CCND3, BCL6, and CDK6), apoptosis (BCL2), proliferation (MYC), or signal transduction (BCL3 and MALT1). Methods Patient samples The features of B-cell malignancies showing aberrations in the TERT region (5p1) are listed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The study was performed as part of the &quot;Molecular Mechanisms in Malignant Lymphoma&quot; Network Project for which approval from the Institutional Review Board of the Medical Faculity of the Christian-Albrechts-University Kiel has been obtained. FISH Fluorescence in situ hybridization (FISH) was performed on fixed cells from bone marrow, peripheral blood, or lymph node cell suspensions as described previously. 7 Generation of FISH probes as well as FISH procedures are described in supplemental Methods. A list of FISH probes used is shown in supplemental Array CGH to custom-designed arrays The microarrays were designed using the eArray software from Agilent (https://earray.chem.agilent.com/earray/) and the 4 ϫ 44k format. The experimental procedures were performed according to the manufacturer&apos;s instructions. Arrays were scanned with the GenePix4000B Scanner (Axon Instruments) and log ratios obtained with the comparative genomic hybridization (CGH) Analytics Version 3.5.14 software (Agilent). All microarray data are available to be viewed at ArrayExpress under accession number E-MEXP-2675 (http://www.ebi.ac.uk/miamexpress/). Quantitative reverse transcription PCR RNA was extracted from tumor cell-containing samples and control tissue, using the RNeasy Mini Kit (QIAGEN) and transcribed into cDNA with the QuantiTect Rev. Transcription Kit (QIAGEN). TERT transcripts were amplified and detected as described elsewhere. 8 TRAP assay The PCR-based telomeric repeat amplification protocol (TRAP) assay was performed as described previously. 9,10 Protein from tumor cell containing samples and control tissue was isolated according to Kim et al. 11 Additional information about materials and methods is available in supplemental Methods. Results and discussion By FISH screening of B-cell neoplasms for translocations affecting the IGH locus we observed a cytogenetically cryptic translocation t(5;14)(p15;q32) involving the IGH locus in 3 cases of chronic lymphocytic leukemia (CLL) and one case of precursor B-cell acute lymphoblastic leukemia (ALL; The IGH locus harbors strong transcriptional enhancers, which can activate oncogenes by translocation on both der(14) and der(5) chromosomes 12 (supplemental To investigate the frequency of rearrangements in the TERT-CLPTM1L region we screened additional cases by FISH (supplemental None of the additional 6 cases with translocations detected by FISH showed juxtaposition to any of the 3 IG loci. Cytogenetically, in 4 of the 6 variant translocations (ie, non-IG), 7p11, 9q31ϳ33, 10q25 and 19p13 were identified as partners of 5p15. In a splenic marginal zone lymphoma (SMZL, case 5) with t(5;7)(p15.33;p11), FISH and tiling array CGH again confirmed a breakpoint centromeric to TERT with loss of CLPTM1L similar to that in the t(5;14)-positive CLL mentioned above ( The inactivation of CLPTM1L by deletion and disruption in 3/10 cases with break at the TERT-CLPTM1L region and also the fact that in precursor B-cell ALL with IGH translocation oncogene activation has always been assigned to the der14 chromosome 13 (supplemental To investigate the effect of the identified chromosomal aberrations on TERT expression, we performed qRT-PCR in cases with t(5;14)/TERT-IGH juxtaposition (n ϭ 3; 1318 NAGEL et al BLOOD, 26 AUGUST 2010 ⅐ VOLUME 116, NUMBER 8 We next assessed whether the up-regulation of TERT mRNA was also associated with increased telomerase activity. Using the TRAP assay, cases of CLL (cases 1 and 6) and MCL (cases 8 and 9) with chromosomal breakpoints at the TERT-CLPTM1L locus showed markedly higher telomerase activity compared with appropriate disease and tissue controls ( Taken together, we show that the TERT-CLPTM1L region in 5p15 is recurrently targeted by chromosomal translocations in B-cell neoplasms. Based on the finding that CLPTM1L is recurrently lost or disrupted by the mentioned aberrations, it does not seem to contribute to the process of lymphomagenesis. Interestingly, germ line sequence variants in the TERT-CLPTM1L region were recently shown to be associated with predisposition for several cancer types. 14-17 All 6 additional translocations and both amplifications identified herein were of somatic origin as cells without the aberration were always present. The pattern of IGH translocations as well as the increased TERT expression and telomerase activation in cases with 5p15 translocations strongly suggest TERT to be the candidate gene involved in lymphomagenesis. Telomerase activity, which is clearly detectable in up to 90% of human tumors, including hematologic neoplasias, but not in most normal somatic cells, is in part explained by changes in chromatin structure or amplification of the TERT locus

Correlations of mutations and increases in and copy number to gefitinib response in a retrospective analysis of lung cancer patients-0

<p><b>Copyright information:</b></p><p>Taken from "Correlations of mutations and increases in and copy number to gefitinib response in a retrospective analysis of lung cancer patients"</p><p>http://www.biomedcentral.com/1471-2407/7/128</p><p>BMC Cancer 2007;7():128-128.</p><p>Published online 13 Jul 2007</p><p>PMCID:PMC1952070.</p><p></p>ur with in-frame exon 19 deletions impacting L747-A750, four with a variety of exon 20 point mutations, and one with an exon 21 point mutation, L858R. Two previously documented synonymous polymorphisms were detected in this study, G2607A in exon 20 (rs10251977) and T2955C in exon 23 (rs17290643). Amino acid numbering is from the initial methionine residue of the protein isoform a (NCBI accession NP_005219)