Determination Of Gene Copy Number Research Articles

Quality Control of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel.

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype.

Microbially influenced corrosion and rust tubercle formation on sheet piles in freshwater systems.

The extent of how complex natural microbial communities contribute to metal corrosion is still not fully resolved, especially not for freshwater environments. In order to elucidate the key processes, we investigated rust tubercles forming massively on sheet piles along the river Havel (Germany) applying a complementary set of techniques. In-situ microsensor profiling revealed steep gradients of O2 , redox potential and pH within the tubercle. Micro-computed tomography and scanning electron microscopy showed a multi-layered inner structure with chambers and channels and various organisms embedded in the mineral matrix. Using Mössbauer spectroscopy we identified typical corrosion products including electrically conductive iron (Fe) minerals. Determination of bacterial gene copy numbers and sequencing of 16S rRNA and 18S rRNA amplicons supported a densely populated tubercle matrix with a phylogenetically and metabolically diverse microbial community. Based on our results and previous models of physic(electro)chemical reactions, we propose here a comprehensive concept of tubercle formation highlighting the crucial reactions and microorganisms involved (such as phototrophs, fermenting bacteria, dissimilatory sulphate and Fe(III) reducers) in metal corrosion in freshwaters.

Quantitative PCR from human genomic DNA: The determination of gene copy numbers for congenital adrenal hyperplasia and RCCX copy number variation.

Quantitative PCR (qPCR) is used for the determination of gene copy number (GCN). GCNs contribute to human disorders, and characterize copy number variation (CNV). The single laboratory method validations of duplex qPCR assays with hydrolysis probes on CYP21A1P and CYP21A2 genes, residing a CNV (RCCX CNV) and related to congenital adrenal hyperplasia, were performed using 46 human genomic DNA samples. We also performed the verifications on 5 qPCR assays for the genetic elements of RCCX CNV; C4A, C4B, CNV breakpoint, HERV-K(C4) CNV deletion and insertion alleles. Precision of each qPCR assay was under 1.01 CV%. Accuracy (relative error) ranged from 4.96±4.08% to 9.91±8.93%. Accuracy was not tightly linked to precision, but was significantly correlated with the efficiency of normalization using the RPPH1 internal reference gene (Spearman's ρ: 0.793-0.940, p>0.0001), ambiguity (ρ = 0.671, p = 0.029) and misclassification (ρ = 0.769, p = 0.009). A strong genomic matrix effect was observed, and target-singleplex (one target gene in one assay) qPCR was able to appropriately differentiate 2 GCN from 3 GCN at best. The analysis of all GCNs from the 7 qPCR assays using a multiplex approach increased the resolution of differentiation, and produced 98% of GCNs unambiguously, and all of which were in 100% concordance with GCNs measured by Southern blot, MLPA and aCGH. We conclude that the use of an internal (in one assay with the target gene) reference gene, the use of allele-specific primers or probes, and the multiplex approach (in one assay or different assays) are crucial for GCN determination using qPCR or other methods.

In silico analysis enabling informed design for genome editing in medicinal cannabis; gene families and variant characterisation.

BackgroundCannabis has been used worldwide for centuries for industrial, recreational and medicinal use, however, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. This study proposes and develops an in silico best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis.ResultsA large dataset of reference genomes was accessed and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variance and highly polymorphic gene sequences exist in the genome making genome editing using CRISPR, Zinc Fingers and TALENs technically difficult. Evaluation of allele or additional gene copies was determined through nucleotide and amino acid alignments with comparative sequence analysis performed. From determined gene copy number and presence of SNPs, multiple online CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways to create knockouts for further investigation. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites.ConclusionsUsing this framework, the approach has wider applications to all plant species regardless of ploidy number or highly homologous gene sequences.Significance statementUsing this framework, a best-practice approach to genome editing is possible in all plant species, including cannabis, delivering a comprehensive in silico evaluation of the cannabinoid pathway diversity from a large set of whole genome sequences. Identification of SNP variants across all genes could improve genome editing potentially leading to novel applications across multiple disciplines, including agriculture and medicine.

High-Resolution Characterization of KIR Genes in a Large North American Cohort Reveals Novel Details of Structural and Sequence Diversity.

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies.

Creation and validation of models to predict response to primary treatment in serous ovarian cancer

Nearly a third of patients with high-grade serous ovarian cancer (HGSC) do not respond to initial therapy and have an overall poor prognosis. However, there are no validated tools that accurately predict which patients will not respond. Our objective is to create and validate accurate models of prediction for treatment response in HGSC. This is a retrospective case–control study that integrates comprehensive clinical and genomic data from 88 patients with HGSC from a single institution. Responders were those patients with a progression-free survival of at least 6 months after treatment. Only patients with complete clinical information and frozen specimen at surgery were included. Gene, miRNA, exon, and long non-coding RNA (lncRNA) expression, gene copy number, genomic variation, and fusion-gene determination were extracted from RNA-sequencing data. DNA methylation analysis was performed. Initial selection of informative variables was performed with univariate ANOVA with cross-validation. Significant variables (p < 0.05) were included in multivariate lasso regression prediction models. Initial models included only one variable. Variables were then combined to create complex models. Model performance was measured with area under the curve (AUC). Validation of all models was performed using TCGA HGSC database. By integrating clinical and genomic variables, we achieved prediction performances of over 95% in AUC. Most performances in the validation set did not differ from the training set. Models with DNA methylation or lncRNA underperformed in the validation set. Integrating comprehensive clinical and genomic data from patients with HGSC results in accurate and robust prediction models of treatment response.

An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard

Nowadays, Pichia pastoris is a well-known yeast for the production of recombinant proteins. The yield of protein production tightly depends on the copy number of the gene of interest into the host chromosome. Real-time PCR has been used as a high throughput method for molecular detection of gene copy number. In light of determining an absolute gene copy number, the reliability of the qPCR quantification standard is a major issue and it can be a potential source of errors in the final results. Since the literature on this issue is inconclusive, we set out to find a reliable quantification method that allows comparing results in different laboratories. We generated standard curves for two genomic loci (5′UTR AOX1 and ARG4) and for plasmid DNA carrying hGM-CSF coding sequence. These data was used to calculate the integrated hGM-CSFcDNA copy number in a recombinant P. pastoris clone. In our expriments the 5′UTR AOX1 gene showed a more accurate quantification standard, based on more efficient amplification and better reproducibility. The results obtained in this study showed that the differences in terms of structure and length between circular plasmid and linear gDNA could be the source of significant differences in the pattern of DNA amplification.

Application of digital PCR with chip-in-a-tube format to analyze Adenomatous polyposis coli (APC) somatic mosaicism

BackgroundOver the past decade, digital PCR (dPCR) technology has significantly improved, and its application in clinical diagnostics is rapidly advancing. The Clarity™ dPCR platform, which employs the chip-in-a-tube format to broaden its range of applications, has been used to determine gene copy number. However, detection of mutations in human samples, the most demanding task in clinical practice, has not yet been reported using this platform. MethodsThe Clarity™ dPCR platform was used to detect somatic Adenomatous polyposis coli mosaicism c.834+2T>C, which had been identified using next-generation sequencing (NGS) technology in a patient with sporadic familial adenomatous polyposis. In addition, we were able to determine the size of the dPCR product. ResultsThe mutation rate in the peripheral blood of the patient calculated using the dPCR platform was 13.2%. This was similar to that determined using NGS (12.7%). In contrast, in healthy donors, the mutation rate was <0.1%. Furthermore, it was confirmed that the dPCR product size was consistent with its theoretical value. ConclusionOur results show that the dPCR platform with the chip-in-a-tube format is suitable for the analysis of mosaicism and enables the validation of the dPCR product size.

OR28 High-throughput KIR sequencing by NGS: 500,000 registry samples genotyped at allelic resolution

Aim The human killer-cell immunoglobulin-like receptor (KIR) family of genes is a key regulator of natural killer cell activity. Several studies have indicated that KIR genotypes affect hematopoietic stem cell transplantation outcome and the evidence is rising that the extensive allelic diversity discovered in KIR genes may be an important component to consider. However, high-resolution characterization of KIR genotypes has so far been applied only to smaller cohorts. Therefore, we developed a KIR genotyping approach based on NGS that would be cost-effective and amendable for high-throughput application. Methods We amplified KIR exons 3, 4, 5, 7, 8 and 9, targeting one to two exons per PCR reaction but multiplexed across all KIR genes. Amplicons of 2 × 3760 samples were combined for sequencing on Illumina HiSeq 2500 instruments together with amplicons for HLA and blood groups. The reads were mapped against the described KIR alleles (IPD-KIR Release 2.6). Based on the read coverage, we estimated the genomic copy number of each sequence feature. These sequence copy calculations enabled precise allele calling and in addition empowered us to determine gene copy numbers. Results After successful validation, we applied this workflow to genotype 500,000 registry samples. Despite the focus on throughput and cost efficiency, we achieved mostly allotype (3 digit) resolution with the exception of certain allotypes differing only in the short non-targeted exon regions. In addition, certain phasing ambiguities remained. We report the observed allele diversity and the relative abundance of alleles for a mainly Caucasian cohort. Conclusions We demonstrate that high-resolution KIR genotyping is feasible using a very cost-efficient workflow. This approach enables KIR genotyping for population genetics, disease association studies and other applications requiring large cohorts. As of October 2016 we have been applying this workflow to the analysis of all volunteer samples registering with DKMS as potential donors for hematopoietic stem cell transplantation. High-resolution KIR genotyping results will thus become available to search coordinators and may soon be used as an additional selection criterion to improve transplantation outcome.

Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane.

Droplet digital PCR combined with the low copy ACT allele as endogenous reference gene, makes accurate and rapid estimation of gene copy number in Q208 A and Q240 A attainable. Sugarcane is an important cultivated crop with both high polyploidy and aneuploidy in its 10Gb genome. Without a known copy number reference gene, it is difficult to accurately estimate the copy number of any gene of interest by PCR-based methods in sugarcane. Recently, a new technology, known as droplet digital PCR (ddPCR) has been developed which can measure the absolute amount of the target DNA in a given sample. In this study, we deduced the true copy number of three endogenous genes, actin depolymerizing factor (ADF), adenine phosphoribosyltransferase (APRT) and actin (ACT) in three Australian sugarcane varieties, using ddPCR by comparing the absolute amounts of the above genes with a transgene of known copy number. A single copy of the ACT allele was detected in Q208 A , two copies in Q240 A , but was absent in Q117. Copy number variation was also observed for both APRT and ADF, and ranged from 9 to 11 in the three tested varieties. Using this newly developed ddPCR method, transgene copy number was successfully determined in 19 transgenic Q208 A and Q240 A events using ACT as the reference endogenous gene. Our study demonstrates that ddPCR can be used for high-throughput genetic analysis and is a quick, accurate and reliable alternative method for gene copy number determination in sugarcane. This discovered ACT allele would be a suitable endogenous reference gene for future gene copy number variation and dosage studies of functional genes in Q208 A and Q240 A .

Loop ileostomy-mediated fecal stream diversion is associated with microbial dysbiosis

ABSTRACTLoop ileostomy is an effective procedure to protect downstream intestinal anastomoses. Ileostomy reversal surgery is often performed within 12 months of formation but is associated with substantial morbidity due to severe post-surgical complications. Distal ileum is deprived of enteral nutrition and rendered inactive, often becoming atrophied and fibrotic. This study aimed to investigate the microbial and morphological changes that occur in the defunctioned ileum following loop ileostomy-mediated fecal stream diversion. Functional and defunctioned ileal resection tissue was obtained at the time of loop-ileostomy closure. Intrapatient comparisons, including histological assessment of morphology and epithelial cell proliferation, were performed on paired samples using the functional limb as control. Mucosal-associated microflora was quantified via determination of 16S rRNA gene copy number using qPCR analysis. DGGE with Sanger sequencing and qPCR methods profiled microflora to genus and phylum level, respectively. Reduced villous height and proliferation confirmed atrophy of the defunctioned ileum. DGGE analysis revealed that the microflora within defunctioned ileum is less diverse and convergence between defunctioned microbiota profiles was observed. Candidate Genera, notably Clostridia and Streptococcus, reduced in relative terms in defunctioned ileum. We conclude that Ileostomy-associated nutrient deprivation results in dysbiosis and impaired intestinal renewal in the defunctioned ileum. Altered host-microbial interactions at the mucosal surface likely contribute to the deterioration in homeostasis and thus may underpin numerous postoperative complications. Strategies to sustain the microflora before reanastomosis should be investigated.

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

Objectives: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. Methods: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. Results: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. Conclusions: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

Use of quantitative PCR technique for determining gene copy number in the genome of Beauveria bassiana transformant

This study describes the use of quantitative PCR (qPCR) to determine gene copy number in Beauveria bassiana genome. This procedure has been improved by generating a reference strain to calibrate PCR amplification efficiency and using a γ-tubulin gene as internal control. The reference strain was constructed by introducing one copy of phosphinothricin resistance gene and two copies of green fluorescent protein gene into the wild-type strain. Finally, this improved procedure was validated via determining the gene copy number in the previously characterized transformants, and displays a great potential in rapid detection of gene copy number in the genome of transformant.

QKAT: a high-throughput qPCR method for KIR gene copy number and haplotype determination.

Killer cell immunoglobulin-like receptors (KIRs), expressed on natural killer cells and T cells, have considerable biomedical relevance playing significant roles in immunity, pregnancy and transplantation. The KIR locus is one of the most complex and polymorphic regions of the human genome. Extensive sequence homology and copy number variation makes KIRs technically laborious and expensive to type. To aid the investigation of KIRs in human disease we developed a high-throughput, multiplex real-time polymerase chain reaction method to determine gene copy number for each KIR locus. We used reference DNA samples to validate the accuracy and a cohort of 1698 individuals to evaluate capability for precise copy number discrimination. The method provides improved information and identifies KIR haplotype alterations that were not previously visible using other approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0358-0) contains supplementary material, which is available to authorized users.

Droplet digital PCR-aided screening and characterization of Pichia pastoris multiple gene copy strains.

Pichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real-time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real-time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two-copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542-1551. © 2015 Wiley Periodicals, Inc.

Development and validation of a high throughput, closed tube method for the determination of haemoglobin alpha gene (HBA1 and HBA2) numbers by gene ratio assay copy enumeration-PCR (GRACE-PCR).

BackgroundDeletions of the α-globin genes are the most common genetic abnormalities in the world. Currently multiplex Gap-PCRs are frequently used to identify specific sets of common deletions. However, these assays require significant post-amplification hands on time and cannot be used to identify novel or unexpected deletions. The aim of the current study was to develop a rapid screening test for the detection of all deletions of the α-globin genes that can be integrated into a high volume clinical laboratory workflow.MethodsA gene ratio assay copy enumeration (GRACE) PCR method was developed by simultaneous amplification of targets in the α-globin genes (HBA1 and HBA2) and the chloride channel voltage sensitive 7 (CLCN7) reference gene. A novel application of High Resolution Melting (HRM) analysis then allowed rapid determination of α-globin gene copy numbers. The assay was validated using 105 samples with previously determined and 62 samples with unknown α-globin genotypes.ResultsThe GRACE-PCR assay detected abnormal α-globin gene copy numbers in 108 of the 167 samples evaluated. The results were consistent with those from a commercial reverse hybridization assay and no allele drop out was observed.ConclusionsWe have successfully developed and validated a GRACE-PCR screening test for the detection of deletions and duplications of the α-globin genes. The assay is based on copy number determination and has the ability to detect both known and novel deletions of the α-globin genes. It is a closed tube technique; consequently the risk of amplicon contamination is negligible. Amplification, detection and analysis can be completed within one hour, making it faster, cheaper and simpler than other existing tests and thus well suited as a rapid first step in a clinical laboratory workflow.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-015-0258-y) contains supplementary material, which is available to authorized users.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase (MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides (W1, MF4I, INU1A, αpre, HFBI) were chosen to be analyzed by SignalP 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector pGAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4I, INU1A, apre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/mL, while those mediated by MF4I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/mL, respectively. The maximum MAN activity reached 347.5 U/mL with 4 gene copies mediated by MF4I. These results indicate that replacing the signal peptide α-factor with MF4I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Analysis of Copy Number Variation by Pyrosequencing® Using Paralogous Sequences.

The determination of gene copy numbers of highly similar genes is difficult with conventional PCR-based methods. However, by amplification of similar genes in the same PCR reaction followed by Pyrosequencing(®), one may distinguish the genes based on a single-nucleotide difference. The ratio between the peak heights of gene-specific nucleotides obtained in the Pyrosequencing reaction may thereby be used to calculate the relative copy numbers of target genes. This method is easy and cost effective compared to other methods, and allows for the determination of copy numbers of genes that were previously difficult to achieve.

Abstract 3734: Development of a nanostring copy number assay for a customized 55 gene panel using challenging formalin-fixed paraffin-embedded (ffpe) tumor samples

Abstract Introduction: Gene amplification/deletion is a common tactic employed by tumor cells to proliferate uncontrollably. Multiple methods have been developed to determine gene copy numbers, such as FISH, qPCR, and, more recently, Next Generation Sequencing (NGS). However, these methods have limitations: FISH and NGS are very labor intensive; and FISH and qPCR can detect only a limited number of targets. Therefore, it is imperative to develop methods that are more user-friendly and have increased multiplexing capabilities to detect copy number variations (CNV). Using a novel digital barcode technology, NanoString Technologies (Seattle, WA) has developed the nCounter® technology to meet these needs. This new technology will be especially valuable after the recent FDA approval of the ProsignaTM assay, a breast cancer gene signature assay for subtype classification to help guide treatment decisions. In the BioPharma group at Genoptix, Inc., Carlsbad, CA, we have started to assess the nCounter technology for copy number determinations in a CLIA laboratory setting. Methods: A cancer gene CNV panel composed of 55 genes was custom designed and evaluated. The CNV results of a subset of genes generated from our gene panel using the nCounter technology were compared with the results generated from qPCR, digital droplet PCR (ddPCR), FISH, publications, and NGS. Furthermore, assay robustness was tested by using different amounts and quality of input FFPE DNA. An initial comparison on the CNVs obtained from genomic DNA digested by Alu1 enzyme or sonicated by Bioruptor® Pico (Diagenode Inc, Denville, NJ). Finally, the effects of different DNA isolation methods on copy number determinations were also evaluated. Results: Overall, the copy numbers generated using nCounter technology were concordant with results from the abovementioned methods as analyzed by comparing selected genes with known CNVs. A wide range of FFPE DNA input (200ng-4ug) could be tolerated by the assay. Based on a limited number of samples, DNA isolation methods, such as Qiagen, Promega Maxwell® CSC, or Roche Cobas®, have minimal effect on copy numbers. Conclusions: With the short hands-on time, relative high level of automation, and no amplification requirements, nCounter technology is able to generate CNVs in good agreement with other established CNV methods. This new 55 cancer gene CNV assay may be useful in clinical trials to assist in patient stratification and care. Citation Format: Tingdong Tang, Wenge Shi, Loretta Hipolito, Julie Mayer, Jelveh Lameh, Shabnam Tangri, Reinhold Pollner. Development of a nanostring copy number assay for a customized 55 gene panel using challenging formalin-fixed paraffin-embedded (ffpe) tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3734. doi:10.1158/1538-7445.AM2014-3734

The three receptor tyrosine kinases c-KIT, VEGFR2 and PDGFRα, closely spaced at 4q12, show increased protein expression in triple-negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer with poor prognosis and no targeted therapy available. Receptor tyrosine kinases (RTKs) are emerging targets in anticancer therapy and many RTK-inhibiting drugs are currently being developed. The aim of this study was to elucidate if there is a correlation between the protein expression of three RTKs c-KIT, VEGFR2 and PDGFRα, their gene copy number, and prognosis in TNBC compared to non-TNBC.MethodsTumor tissue samples from patients diagnosed with primary breast cancer were stained with immunohistochemistry (IHC) for protein assessment, and with fluorescence in situ hybridization (FISH) for gene copy number determination. Breast cancer mortality (BCM), measured from the date of surgery to death, was used as endpoint.ResultsThe cohort included 464 patients, out of which 34 (7.3%) had a TNBC. High expression of the three RTKs was more common in TNBC compared to non-TNBC: c-KIT 49% vs. 10% (P<0.001), PDGFRα 32% vs. 19% (P = 0.07) and VEGFR2 32% vs. 6% (P<0.001). The odds ratio (OR) of c-KIT, VEGFR2 and PDGFRα positivity, adjusted for tumor characteristics, was 6.8, 3.6 and 1.3 times higher for TNBC than for non-TNBC. 73.5% of the TNBC had high expression of at least one of the three investigated receptors, compared to 30.0% of the non-TNBC (P<0.001). Survival analysis showed no significant difference in BCM for TNBC patients with high vs. low c-KIT, PDGFRα or VEGFR2 protein expression. 193 (42%) tumors were evaluated with FISH. No correlation was seen between increased gene copy number and TNBC, or between increased gene copy number and high protein expression of the RTK.Conclusionc-KIT, VEGFR2 and PDGFRα show higher protein expression in TNBC compared to non-TNBC. Further investigation clarifying the importance of these RTKs in TNBC is encouraged, as they are possible targets for anticancer therapy.