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Methylation analysis of MLH1 using droplet digital PCR and methylation sensitive restriction enzyme
abstracts and 8.3 mut/Mb (TruSight assay and WES, respectively) to assign TMB High and Low statuses, overall percent agreement (OPA) was 85%. Conclusions: The tumor-only TruSight assay showed high accuracy in detecting biomarkers across a range of solid tumors. The assay showed a good correlation in TMB score and agreement in TMB status with tumor-normal WES in a collection of NSCLC samples. Legal entity responsible for the study: The authors. Funding: Illumina. Disclosure: I. Deras: Shareholder / Stockholder / Stock options, Full / Part-time employment:
1411P
Methylation analysis of MLH1 using droplet digital PCR and methylation sensitive restriction enzyme
C. De Rop1, G. Beniuga2, J. Radermacher2, K. Dahan3, P. Vannuffel1 1 Molecular Biology Department, Institut de Pathologie et de Ge´ne´tique, Gosselies, Belgium, 2Anatomical Pathology Department, Institut de Pathologie et de Ge´ne´tique, Gosselies, Belgium, 3Clinical Genetics Department, Institut de Pathologie et de Ge´ne´tique, Gosselies, Belgium Background: Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes being MLH1 and MSH2 the most commonly mutated. By contrast MLH1 inactivation as the result of promoter methylation is strongly indicative of a sporadic cancer, providing LS exclusion criteria for 85% of high microsatellite instability (MSI-H) tumours. Here we present a cost effective strategy enabling to test methylation of MLH1 promoter without bisulfite conversion and with minimal DNA quantity requirement. Methods: After macrodissection from HE stained slides, DNA was extracted from FFPE tissue sections of colorectal carcinomas. A droplet digital PCR (ddPCR) was performed using two reactions mix. A 270-bp region of the MLH1 promoter (chr3:36993151-36993420) is amplified in the presence/absence of HinP1I, a methylation sensitive restriction enzyme, targeting 3 CpG islands. Analysis was made by the QuantaSoftTM (Bio-Rad) software which calculates amplicon concentrations between the 2 reactions to obtain a percentage of MLH1 methylation. Sensitivity of the technique was assigned to 5% of methylation with a minimal DNA concentration of 5 ng. Results: Methylation analysis by ddPCR was compared to pyrosequencing combined with bisulfite conversion for 65 samples and a 100% concordance was obtained. Moreover 10 samples were analysed by ddPCR and MethylLight RealTime-PCR with the same concordance. After validation, the technique was implemented in the clinical diagnosis and, in one year, out the 79 MMR-deficient colorectal carcinomas analysed, 60 (76 %) were MLH1-methylated tumours. Conclusions: This ddPCR sequencing combining methylation sensitive restriction enzyme is a cost-effective strategy, requiring less technical turn around time and minimal DNA quantity as compared to standard analysis. Moreover, this technique could be further used for other promoter methylation analysis (such as MGMT) and on circulating tumoral DNA. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
1412P
Analytical performance of the resolution-HRD plasma assay used to identify mCRPC patients with biallelic disruption of DNA repair genes for treatment with niraparib
I. Pekker1, L. Lim1, J.S. Simon2, M. Gormley2, Z. Li3, J. Pollak1, K. Potts1, S. Watford1, J. Posey1, P. Chan1, K. Urtishak2, K. Garg1, A. Hosseini1, M. Li1 1 Diagnostics, Resolution Bioscience, Kirkland, WA, USA, 2Oncology Diagnostics, Janssen R&D/Johnson & Johnson Pharmaceutical R&D, Raritan, NJ, USA, 3Diagnostics, Resolution Bioscience, Kirkland, WA, USA Background: Metastatic castration-resistant prostate cancer (mCRPC) patients with DNA repair gene defects (DRD) have a shorter life expectancy than patients without
v576 | New Diagnostic Tools
DRD and may benefit from treatment with PARP inhibitors. Niraparib is a highly selective PARP inhibitor, with activity against PARP-1/2 DNA-repair polymerases. Detection of DRD in cell free DNA (cfDNA) isolated from blood is minimally invasive and of special benefit to mCRPC patients, including patients without accessible lesions. The assay would also have the advantage of a shorter turnaround time (TAT) than genotyping of tissue. However, using cfDNA to identify biallelic disruption of DNA-repair genes is technically challenging. Methods: Resolution-HRD identifies patients with biallelic pathogenic alterations of the ATM, BRCA1, BRCA2, BRIP1, CHEK2, FANCA, HDAC2, or PALB2 genes, by targeted NGS sequencing of cfDNA. Analytical performance of Resolution-HRD was validated using cfDNA from mCRPC patient plasma, cfDNA from healthy donor plasma, and contrived samples with a wide spectrum of technically challenging genetic aberrations. Results: The LOD95 at a cfDNA input level of 40 ng ranged from 0.2 to 1.37 for SNVs and indels, and 6-12 for CNL. APA for intra-run and inter-run studies at the 1X LOD was 95% and 95% respectively. No false-positives were detected in any samples from healthy donors (N ¼ 60). Resolution-HRD has been validated to give consistent results across the 10-75 ng input range. Resolution-HRD is used to identify patients for enrollment in the GALAHAD Phase II Efficacy and Safety Study (64091742PCR2001) of Niraparib in Men with mCRPC and DNA-Repair Anomalies. As of April 2019, over 2000 patients are tested successfully (0.88% failure rate) with a median TAT of 8.6 days (range 5-12 days). Conclusions: The analytical performance of the Resolution-HRD assay offers highly sensitive, specific and robust test results, and meets analytical requirements for clinical applications. This test is currently being evaluated in several clinical trials for prospective identification of mCRPC patients with DRD for treatment with niraparib. Legal entity responsible for the study: Resolution Bioscience. Funding: Janssen Research and Development. Disclosure: I. Pekker: Shareholder / Stockholder / Stock options, Full / Part-time employment: resolution bioscience. L. Lim: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment, Officer / Board of Directors: Resolution Bioscience. J.S. Simon: Leadership role, Shareholder / Stockholder / Stock options: Janssen. M. Gormley: Shareholder / Stockholder / Stock options, Full / Part-time employment: Janssen. Z. Li: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. J. Pollak: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. K. Potts: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. S. Watford: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. J. Posey: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. P. Chan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. K. Urtishak: Shareholder / Stockholder / Stock options, Full / Part-time employment: Janssen. K. Garg: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. A. Hosseini: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. M. Li: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Resolution Bioscience.
1413P
Results of a global external quality assessment scheme for EGFR testing on liquid biopsy
N. Normanno1, J. Fairley2, M. Cheetham3, M.G. Denis4, E. Dequeker5, C. Keppens5, F. Fenizia1, S. Patton3, E. Rouleau6, E. Schuuring7, K. Van Casteren5, Z. Deans2 1 Translational Research, Istituto Nazionale Tumori – I.R.C.C.S - Fondazione Pascale, Naples, Italy, 2GENQA, UKNEQAS, Edinburgh, UK, 3EMQN, EMQN, Manchester, UK, 4 Biochemistry Department, CHU du Nantes - Hoˆtel-Dieu, Nantes, France, 5Quality, University Hospitals Leuven - Campus Gasthuisberg, Leuven, Belgium, 6Genetics, Gustave Roussy - Cancer Campus, Villejuif, France, 7Pathology, University Hospital Groningen (UMCG), Groningen, Netherlands Background: Cell free DNA (cfDNA) testing of EGFR mutations is widely employed in lung cancer patients. Liquid biopsy testing is highly challenging due to the low level of mutant DNA present with normal DNA. Therefore, cfDNA testing requires quality assessment to ensure patient safety. The international external quality assessment (EQA) provider consortium, IQNPath has delivered a second successful EQA run to determine the standard of cfDNA testing for EGFR mutations. Methods: Five European EQA providers (AIOM, EMQN, ESP, Gen&Tiss, UKNEQAS), under the umbrella of IQNPath, collaborated to deliver the assessment during 2018-19 to a total of 310 laboratories from 44 countries. A panel of bespoke manufactured plasma samples with varying EGFR mutations at a range of allelic frequencies were validated by a range of methodologies prior to distribution to ensure stability and reproducibility. The EQA samples were supplied for testing and reporting according to laboratory routine protocols. Peer reviewed criteria was applied to assess the standard of genotyping and reporting. Results: Of the 310 laboratories that had joined the program, 270 submitted the results within the established deadline. Preliminary analysis of the data submitted by participating laboratories showed that low allelic frequency samples were the most challenging and some methods did not detect these mutations. Reporting of such cases often did not address the risk that tumour DNA may have not been tested and limitations of the testing performed was not addressed when reporting the result. The final results of the EQA scheme will be presented at the meeting. Conclusions: The variability in the standard of genotyping and reporting highlights the need for EQA in this field and educational guidance to ensure the delivery of high-quality clinical service where testing of cfDNA is the only option for clinical management.
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz257.006/5576758 by guest on 26 October 2019
Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. T. Du: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Zhao: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. N. Haseley: Full / Part-time employment: Illumina. A. Yazdanparast: Full / Part-time employment: Illumina. T. Jiang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. A. Mentzer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. A. Purdy: Shareholder / Stockholder / Stock options, Full / Parttime employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. B. Crain: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Echegaray: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. D. Lee: Full / Part-time employment: Illumina. J. Lee: Full / Part-time employment: Illumina. J. Silhavy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. K. O’Brien: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Vijayaraghavan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Garcia: Full / Part-time employment: Illumina. R. Haigis: Shareholder / Stockholder / Stock options, Full / Parttime employment: Illumina. T. Pawlowski: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. J. Dockter: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina.
Annals of Oncology
1411P
Methylation analysis of MLH1 using droplet digital PCR and methylation sensitive restriction enzyme
C. De Rop1, G. Beniuga2, J. Radermacher2, K. Dahan3, P. Vannuffel1 1 Molecular Biology Department, Institut de Pathologie et de Ge´ne´tique, Gosselies, Belgium, 2Anatomical Pathology Department, Institut de Pathologie et de Ge´ne´tique, Gosselies, Belgium, 3Clinical Genetics Department, Institut de Pathologie et de Ge´ne´tique, Gosselies, Belgium Background: Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes being MLH1 and MSH2 the most commonly mutated. By contrast MLH1 inactivation as the result of promoter methylation is strongly indicative of a sporadic cancer, providing LS exclusion criteria for 85% of high microsatellite instability (MSI-H) tumours. Here we present a cost effective strategy enabling to test methylation of MLH1 promoter without bisulfite conversion and with minimal DNA quantity requirement. Methods: After macrodissection from HE stained slides, DNA was extracted from FFPE tissue sections of colorectal carcinomas. A droplet digital PCR (ddPCR) was performed using two reactions mix. A 270-bp region of the MLH1 promoter (chr3:36993151-36993420) is amplified in the presence/absence of HinP1I, a methylation sensitive restriction enzyme, targeting 3 CpG islands. Analysis was made by the QuantaSoftTM (Bio-Rad) software which calculates amplicon concentrations between the 2 reactions to obtain a percentage of MLH1 methylation. Sensitivity of the technique was assigned to 5% of methylation with a minimal DNA concentration of 5 ng. Results: Methylation analysis by ddPCR was compared to pyrosequencing combined with bisulfite conversion for 65 samples and a 100% concordance was obtained. Moreover 10 samples were analysed by ddPCR and MethylLight RealTime-PCR with the same concordance. After validation, the technique was implemented in the clinical diagnosis and, in one year, out the 79 MMR-deficient colorectal carcinomas analysed, 60 (76 %) were MLH1-methylated tumours. Conclusions: This ddPCR sequencing combining methylation sensitive restriction enzyme is a cost-effective strategy, requiring less technical turn around time and minimal DNA quantity as compared to standard analysis. Moreover, this technique could be further used for other promoter methylation analysis (such as MGMT) and on circulating tumoral DNA. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
1412P
Analytical performance of the resolution-HRD plasma assay used to identify mCRPC patients with biallelic disruption of DNA repair genes for treatment with niraparib
I. Pekker1, L. Lim1, J.S. Simon2, M. Gormley2, Z. Li3, J. Pollak1, K. Potts1, S. Watford1, J. Posey1, P. Chan1, K. Urtishak2, K. Garg1, A. Hosseini1, M. Li1 1 Diagnostics, Resolution Bioscience, Kirkland, WA, USA, 2Oncology Diagnostics, Janssen R&D/Johnson & Johnson Pharmaceutical R&D, Raritan, NJ, USA, 3Diagnostics, Resolution Bioscience, Kirkland, WA, USA Background: Metastatic castration-resistant prostate cancer (mCRPC) patients with DNA repair gene defects (DRD) have a shorter life expectancy than patients without
v576 | New Diagnostic Tools
DRD and may benefit from treatment with PARP inhibitors. Niraparib is a highly selective PARP inhibitor, with activity against PARP-1/2 DNA-repair polymerases. Detection of DRD in cell free DNA (cfDNA) isolated from blood is minimally invasive and of special benefit to mCRPC patients, including patients without accessible lesions. The assay would also have the advantage of a shorter turnaround time (TAT) than genotyping of tissue. However, using cfDNA to identify biallelic disruption of DNA-repair genes is technically challenging. Methods: Resolution-HRD identifies patients with biallelic pathogenic alterations of the ATM, BRCA1, BRCA2, BRIP1, CHEK2, FANCA, HDAC2, or PALB2 genes, by targeted NGS sequencing of cfDNA. Analytical performance of Resolution-HRD was validated using cfDNA from mCRPC patient plasma, cfDNA from healthy donor plasma, and contrived samples with a wide spectrum of technically challenging genetic aberrations. Results: The LOD95 at a cfDNA input level of 40 ng ranged from 0.2 to 1.37 for SNVs and indels, and 6-12 for CNL. APA for intra-run and inter-run studies at the 1X LOD was 95% and 95% respectively. No false-positives were detected in any samples from healthy donors (N ¼ 60). Resolution-HRD has been validated to give consistent results across the 10-75 ng input range. Resolution-HRD is used to identify patients for enrollment in the GALAHAD Phase II Efficacy and Safety Study (64091742PCR2001) of Niraparib in Men with mCRPC and DNA-Repair Anomalies. As of April 2019, over 2000 patients are tested successfully (0.88% failure rate) with a median TAT of 8.6 days (range 5-12 days). Conclusions: The analytical performance of the Resolution-HRD assay offers highly sensitive, specific and robust test results, and meets analytical requirements for clinical applications. This test is currently being evaluated in several clinical trials for prospective identification of mCRPC patients with DRD for treatment with niraparib. Legal entity responsible for the study: Resolution Bioscience. Funding: Janssen Research and Development. Disclosure: I. Pekker: Shareholder / Stockholder / Stock options, Full / Part-time employment: resolution bioscience. L. Lim: Leadership role, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment, Officer / Board of Directors: Resolution Bioscience. J.S. Simon: Leadership role, Shareholder / Stockholder / Stock options: Janssen. M. Gormley: Shareholder / Stockholder / Stock options, Full / Part-time employment: Janssen. Z. Li: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. J. Pollak: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. K. Potts: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. S. Watford: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. J. Posey: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. P. Chan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. K. Urtishak: Shareholder / Stockholder / Stock options, Full / Part-time employment: Janssen. K. Garg: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. A. Hosseini: Shareholder / Stockholder / Stock options, Full / Part-time employment: Resolution Bioscience. M. Li: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Resolution Bioscience.
1413P
Results of a global external quality assessment scheme for EGFR testing on liquid biopsy
N. Normanno1, J. Fairley2, M. Cheetham3, M.G. Denis4, E. Dequeker5, C. Keppens5, F. Fenizia1, S. Patton3, E. Rouleau6, E. Schuuring7, K. Van Casteren5, Z. Deans2 1 Translational Research, Istituto Nazionale Tumori – I.R.C.C.S - Fondazione Pascale, Naples, Italy, 2GENQA, UKNEQAS, Edinburgh, UK, 3EMQN, EMQN, Manchester, UK, 4 Biochemistry Department, CHU du Nantes - Hoˆtel-Dieu, Nantes, France, 5Quality, University Hospitals Leuven - Campus Gasthuisberg, Leuven, Belgium, 6Genetics, Gustave Roussy - Cancer Campus, Villejuif, France, 7Pathology, University Hospital Groningen (UMCG), Groningen, Netherlands Background: Cell free DNA (cfDNA) testing of EGFR mutations is widely employed in lung cancer patients. Liquid biopsy testing is highly challenging due to the low level of mutant DNA present with normal DNA. Therefore, cfDNA testing requires quality assessment to ensure patient safety. The international external quality assessment (EQA) provider consortium, IQNPath has delivered a second successful EQA run to determine the standard of cfDNA testing for EGFR mutations. Methods: Five European EQA providers (AIOM, EMQN, ESP, Gen&Tiss, UKNEQAS), under the umbrella of IQNPath, collaborated to deliver the assessment during 2018-19 to a total of 310 laboratories from 44 countries. A panel of bespoke manufactured plasma samples with varying EGFR mutations at a range of allelic frequencies were validated by a range of methodologies prior to distribution to ensure stability and reproducibility. The EQA samples were supplied for testing and reporting according to laboratory routine protocols. Peer reviewed criteria was applied to assess the standard of genotyping and reporting. Results: Of the 310 laboratories that had joined the program, 270 submitted the results within the established deadline. Preliminary analysis of the data submitted by participating laboratories showed that low allelic frequency samples were the most challenging and some methods did not detect these mutations. Reporting of such cases often did not address the risk that tumour DNA may have not been tested and limitations of the testing performed was not addressed when reporting the result. The final results of the EQA scheme will be presented at the meeting. Conclusions: The variability in the standard of genotyping and reporting highlights the need for EQA in this field and educational guidance to ensure the delivery of high-quality clinical service where testing of cfDNA is the only option for clinical management.
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz257.006/5576758 by guest on 26 October 2019
Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. T. Du: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Zhao: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. N. Haseley: Full / Part-time employment: Illumina. A. Yazdanparast: Full / Part-time employment: Illumina. T. Jiang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. A. Mentzer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. A. Purdy: Shareholder / Stockholder / Stock options, Full / Parttime employment: Illumina; Shareholder / Stockholder / Stock options: Bristol-Myers Squibb. B. Crain: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. C. Echegaray: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. D. Lee: Full / Part-time employment: Illumina. J. Lee: Full / Part-time employment: Illumina. J. Silhavy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. K. O’Brien: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Vijayaraghavan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. R. Garcia: Full / Part-time employment: Illumina. R. Haigis: Shareholder / Stockholder / Stock options, Full / Parttime employment: Illumina. T. Pawlowski: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. J. Dockter: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina.
Annals of Oncology