Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number

Background: Human papilloma virus (HPV)–negative oropharyngeal squamous cell carcinomas (OPCs) have a poorer prognosis and best management is an unmet need. We studied the prognostic role of epidermal growth factor receptor (EGFR) and PIK3CA amplifications and TP53 functional status. Methods: Between 1992 and 2000, 90 consecutive patients with OPCs were treated with surgery, followed by radiotherapy in case of high-risk pathologic features. Of those, 73 cases were HPV-negative and therefore were selected for molecular analysis ( PIK3CA and EGFR fluorescent in situ hybridization [FISH] analysis and TP53 mutation analysis). Results: FISH analyses of EGFR and PIK3CA were successfully conducted on 69 and 63 of 73 tumor samples, respectively. EGFR alterations were detected in 43% of patients but just 7% showed amplification. Seven cases (11%) carried PIK3CA amplification and 18 (29%) gene gain or high polysomy. TP53 was detected as nonfunctional in 24 of 67 (36%) successfully analyzed cases. Both univariable and multivariable analysis showed statistically significantly worse disease-free survival (DFS) for patients with PIK3CA disomy compared to those with gene gain or high polysomy. No differences in overall survival or DFS for EGFR and TP53 alteration were evident. The combined evaluation of PIK3CA and TP53 showed that PIK3CA gene copy number gain separated a population with better outcome, defining an overall worse prognosis population (disomy) now clearly further divided according to TP53 functional status. Conclusion: PIK3CA gene copy number increase is associated with a favorable clinical outcome in HPV-negative OPCs treated with surgery ± postoperative radiotherapy. In patients without PIK3CA alteration, TP53 nonfunctional mutations are associated with poor prognosis.

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Determination of CYP2D6 Gene Copy Number by Pyrosequencing

Clinical Chemistry ◽

10.1373/clinchem.2004.043182 ◽

2005 ◽

Vol 51 (3) ◽

pp. 522-531 ◽

Cited By ~ 50

Author(s):

Erik Söderbäck ◽

Anna-Lena Zackrisson ◽

Bertil Lindblom ◽

Anders Alderborn

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Relative Quantification ◽

Sequence Context ◽

Cyp2d6 Gene ◽

Equivalent Region ◽

Peak Pattern ◽

Single Set

Abstract Background: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. Methods: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing™ technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. Results: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0–4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. Conclusions: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

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Digital PCR for determination of cytochrome P450 2D6 and sulfotransferase 1A1 gene copy number variations

Drug Discoveries & Therapeutics ◽

10.5582/ddt.2017.01057 ◽

2017 ◽

Vol 11 (6) ◽

pp. 336-341 ◽

Cited By ~ 3

Author(s):

Yutaro Motoi ◽

Kazufumi Watanabe ◽

Hiroyuki Honma ◽

Yousuke Tadano ◽

Hiroshi Hashimoto ◽

...

Keyword(s):

Cytochrome P450 ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Copy Number Variations ◽

Gene Copy ◽

Cytochrome P450 2D6 ◽

Sulfotransferase 1A1 ◽

P450 2D6

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Automated Analysis of Protein Expression and Gene Amplification within the Same Cells of Paraffin-Embedded Tumour Tissue

Analytical Cellular Pathology ◽

10.1155/2010/157926 ◽

2010 ◽

Vol 33 (2) ◽

pp. 105-112 ◽

Cited By ~ 3

Author(s):

Timo Gaiser ◽

Lissa Berroa-Garcia ◽

Ralf Kemmerling ◽

Aparajita Dutta ◽

Thomas Ried ◽

...

Keyword(s):

Protein Expression ◽

Copy Number ◽

Simultaneous Detection ◽

Gene Copy Number ◽

Gene Copy ◽

Fish Analysis ◽

Tissue Sections ◽

Automated Algorithm ◽

Significant Difference ◽

Copy Number Changes

Background: The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH) negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell.Methods: Paraffin-embedded tissue sections of colorectal cancers were stained for CD133 expression. IHC images were acquired and image coordinates recorded. Slides were subsequently hybridized with fluorescently labeled DNA probes. FISH images were taken at the previously recorded positions allowing for direct comparison of protein expression and gene copy number signals within the same cells/tissue areas. Relocation, acquisition of the IHC and FISH images, and enumeration of FISH signals in the immunophenotyped tumour areas were done in an automated fashion.Results: Automated FISH analysis was performed on 13 different colon cancer samples that had been stained for CD133; each sample was scored for MYC, ZNF217 and Chromosome 6 in CD133 positive and negative glands. From the 13 cases four (31%) showed amplification for the MYC oncogene and seven of 13 (54%) cases were amplified for ZNF217. There was no significant difference between CD133 positive tumour and CD133 negative tumour cells.Conclusion: The technique and algorithm presented here enables an easy and reproducible combination of IHC and FISH based on a novel automated algorithm using relocation and automated spot counting.

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Human creatine kinase: Isolation and sequence analysis of cDNA clones for the B subunit, development of subunit specific probes and determination of gene copy number

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(87)91427-6 ◽

1987 ◽

Vol 144 (3) ◽

pp. 1116-1127 ◽

Cited By ~ 37

Author(s):

Gerardo Villarreal-Levy ◽

Tony S. Ma ◽

Sandra A. Kerner ◽

Robert Roberts ◽

M. Benjamin Perryman

Keyword(s):

Creatine Kinase ◽

Sequence Analysis ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Cdna Clones ◽

B Subunit

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The relationship of K-ras mutations and EGFR gene copy number to outcome in patients treated with Erlotinib on National Cancer Institute of Canada Clinical Trials Group trial study PA.3

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.4521 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 4521-4521 ◽

Cited By ~ 6

Author(s):

M. J. Moore ◽

G. da Cunha Santos ◽

S. Kamel-Reid ◽

K. Chin ◽

D. Tu ◽

...

Keyword(s):

Clinical Trials ◽

Mutation Analysis ◽

National Cancer Institute ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Fish Analysis ◽

Exon 2 ◽

Ras Mutation ◽

Egfr Gene

4521 Background: The National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) PA.3 study compared treatment with the EGFR tyrosine kinase inhibitor erlotinib to placebo in patients with advanced stage pancreatic carcinoma who were receiving gemcitabine. This study demonstrated a significant advantage in survival [HR=0.82; P<0.04] and in progression free survival [HR=0.77; P<0.004] for patients who received erlotinib [Moore MJ et al, J Clin Oncol 2007]. While high-level expression of EGFR is common in pancreatic cancer, EGFR protein expression level as evaluated by immunohistochemistry did not predict which patients might benefit from erlotinib. In non- small cell lung cancer (NSCLC) where erlotinib has shown a survival benefit; the use of EGFR copy number measured by FISH is a better predictive factor for benefit, and patients whose tumor had a K-ras mutation do not respond nor benefit. Methods: The NCIC-CTG PA.3 trial had a sample size of 569 and NCIC.CTG has collected 280 archival formalin-fixed and paraffin embedded tumor biopsy or primary resection specimens from patients randomized on study. Many were fine needle aspirates and a total of 181 had sufficient tissue to allow molecular analysis. We performed a K-ras mutation analysis by PCR of exon 2 followed by sequencing, with negative results being validated by DHPLC. EGFR gene copy number was analysed by fluorescent in situ hybridization. Both methods were performed on all tumor samples adequate for analysis. Results: Overall 146 cases were suitable for K-ras mutation analysis and preliminary results have identified K-ras mutation in 90 patients [76%] and wild type KRAS in 29 patients; the remainder are pending. A total of 118 cases are suitable for EGFR FISH analysis, results have been obtained in 73 patients, and the remaining cases are pending. The correlations between K-ras mutational status and EGFR copy number, and outcomes [survival and disease control] in patients randomized to both erlotinib and placebo will be presented. [Table: see text]

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