Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

Kim, Mi Kyeong; Hwang, Hai Li; Park, Seong Yeol; Lee, Kwang Man; Park, Won Cheol; Kim, Han Seong; Um, Tae Hyun; Hong, Young Jun; Lee, Jin Kyung; Joo, Sun Young; Seoh, Ju Young; Song, Yeong Wook; Kim, Soo Youl; Kim, Yong Nyun; Hong, Kyung Taek

doi:10.1371/journal.pone.0069414

Cited by 7 publications

(13 citation statements)

References 36 publications

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“…To investigate the correlation, the relative drug sensitivities were determined in NCI-60 cells showing copy-gain drug-target genes (Figure 1A). In our previous study on copy number gain of drug-target genes in NCI-60 cells [10], eight genes, namely FAK , MYC , EGFR , ERBB2 , FGFR1 , MET , IGF1R , and MAP2K2 , showed copy gain in NCI-60 cells. In the present study, we treated specific targeted drugs to cells showing copy gain of the corresponding target genes.…”

Section: Resultsmentioning

confidence: 99%

“…A modified real competitive PCR (mrcPCR) was performed to detect the FAK -copy-gain of breast cancer cell lines as previously described [10].…”

Section: Methodsmentioning

confidence: 99%

“…( A ) Scheme for screening of copy-gain predictive markers. The copy numbers of 24 drug-target genes in an NCI-60 cancer panel had previously been measured by mrcPCR [10]. For eight copy-gain genes in the NCI-60 cells, their targeted drugs were employed for the selection of predictive markers.…”

Section: Figurementioning

confidence: 99%

“…We have reported copy-gain genes in NCI-60 cells employing a modified real competitive PCR (mrcPCR) method [10]. In that study, we found that eight drug-target genes including FAK (or PTK2 ), MYC , EGFR , ERBB2 , FGFR1 , MET , IGF1R , and MAP2K2 gained copies in NCI-60 cells.…”

Section: Introductionmentioning

confidence: 99%

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FAK-Copy-Gain Is a Predictive Marker for Sensitivity to FAK Inhibition in Breast Cancer

Kim

et al. 2019

Cancers

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Background: Cancers with copy-gain drug-target genes are excellent candidates for targeted therapy. In order to search for new predictive marker genes, we investigated the correlation between sensitivity to targeted drugs and the copy gain of candidate target genes in NCI-60 cells. Methods: For eight candidate genes showing copy gains in NCI-60 cells identified in our previous study, sensitivity to corresponding target drugs was tested on cells showing copy gains of the candidate genes. Results: Breast cancer cells with Focal Adhesion Kinase (FAK)-copy-gain showed a significantly higher sensitivity to the target inhibitor, FAK inhibitor 14 (F14). In addition, treatment of F14 or FAK-knockdown showed a specific apoptotic effect only in breast cancer cells showing FAK-copy-gain. Expression-profiling analyses on inducible FAK shRNA-transfected cells showed that FAK/AKT signaling might be important to the apoptotic effect by target inhibition. An animal experiment employing a mouse xenograft model also showed a significant growth-inhibitory effect of F14 on breast cancer cells showing FAK-copy-gain, but not on those without FAK-copy-gain. Conclusion: FAK-copy-gain may be a predictive marker for FAK inhibition therapy in breast cancer.

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Section: Resultsmentioning

confidence: 99%

“…A modified real competitive PCR (mrcPCR) was performed to detect the FAK -copy-gain of breast cancer cell lines as previously described [10].…”

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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FAK-Copy-Gain Is a Predictive Marker for Sensitivity to FAK Inhibition in Breast Cancer

Kim

et al. 2019

Cancers

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“…The 34 cell lines of NCI-60 cells, except for CALU6, were acquired from the National Cancer Institute (MTA No. 2702–09), as noted in our previous study [16]. CALU6 was purchased from Korean Cell Bank.…”

Section: Methodsmentioning

confidence: 99%

False-negative errors in next-generation sequencing contribute substantially to inconsistency of mutation databases

et al. 2019

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BackgroundMore than 11,000 laboratories and companies developed their own next-generation sequencing (NGS) for screening and diagnosis of various diseases including cancer. Although inconsistencies of mutation calls as high as 43% in databases such as GDSC (Genomics of Drug Sensitivity in Cancer) and CCLE (Cancer Cell Line Encyclopedia) have been reported, not many studies on the reasons for the inconsistencies have been published. Methods: Targeted-NGS analysis of 151 genes in 35 cell lines common to GDSC and CCLE was performed, and the results were compared with those from GDSC and CCLE wherein whole-exome- or highly-multiplex NGS were employed.ResultsIn the comparison, GDSC and CCLE had a high rate (40–45%) of false-negative (FN) errors which would lead to high rate of inconsistent mutation calls, suggesting that highly-multiplex NGS may have high rate of FN errors. We also posited the possibility that targeted NGS, especially for the detection of low-level cancer cells in cancer tissues might suffer significant FN errors.ConclusionFN errors may be the most important errors in NGS testing for cancer; their evaluation in laboratory-developed NGS tests is needed.

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Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells

Erbe

Wang

Gallenberger

et al. 2016

Natural Killer Cells

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Natural Killer cells (NK cells) are one of the main effector immune cells involved in Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). Upon recognition of cell bound IgG antibodies, which occurs through Fc gamma receptors (FCGRs) expressed on the cell surface of NK cells, NK cells become activated and lyse target tumor or infected cells. The FCGRs, FCGR3A and FCGR2C, expressed on the surface of NK cells have single nucleotide polymorphisms (SNPs) that result in differential activity of NK cells. In addition to SNP genetic variation within each of these genes, the FCGRs are subject to copy number variation (CNV), which leads to variable protein expression levels on the cell surface. Studies have found that FCGR genotype for FCGR3A and FCGR2C is associated with variation in the response to immunotherapy. Due to high sequence homology within FCGR3 and FCGR2 families, there are difficulties associated with genotyping these specific receptors related to cross-amplification of non-targeted FCGRs. To improve specificity for both FCGR3A and FCGR2C, Rnase-H primers (RH primers) were designed to amplify specifically FCGR3A (while not co-amplifying FCGR3B) and FCGR2C (while not co-amplifying FCGR2B). In addition, fluorescently labeled Locked Nucleic Acid (LNA) probes provide additional precision for determination of the SNPs within both FCGR3A and FCGR2C. For CNV determination, separate fluorescently labeled probes for FCGR3A, and for FCGR2C, can be used with the same RH primers for each gene. These probes can be combined in the same well with control primers/probe for a known diploid gene and used to calculate the copy number of both FCGR3A and FCGR2C. Here we provide new detailed methodology that allows for the specific amplification of these FCGRs in a single PCR reaction, allowing for genotyping of both the SNPs and CNVs using Real Time PCR.

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Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

Cited by 7 publications

References 36 publications

FAK-Copy-Gain Is a Predictive Marker for Sensitivity to FAK Inhibition in Breast Cancer

FAK-Copy-Gain Is a Predictive Marker for Sensitivity to FAK Inhibition in Breast Cancer

False-negative errors in next-generation sequencing contribute substantially to inconsistency of mutation databases

Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells

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