- Email: [email protected]
Results of a global external quality assessment scheme for EGFR testing on liquid biopsy
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:
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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
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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
abstracts
Annals of Oncology Legal entity responsible for the study: IQNPath. Funding: IQNPath. Disclosure: All authors have declared no conflicts of interest.
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Clinical impact of plasma next-generation sequencing (NGS) in advanced non-small cell lung cancer (aNSCLC)
Background: NGS provides genetic information with potential impact on clinical practice. Plasma NGS has the advantage to reduce the need to repeat biopsy and provide information about tumor heterogeneity. Methods: Since March 2018 we prospectively screened aNSCLCs consecutively referring to our institution for potential eligibility to VISION trial (NCT02864992). All the patients (pts) were previously screened for EGFR/ALK/ROS1 sensitizing alterations according to standard methods and positive cases were excluded. NGS was performed R covering 73 genes including all somatic alterations recwith METex14 Guardant360V ognized as potential targets by NCCN. A parallel cohort of pts was also analysed with NGS in tissue by using METex14 OncomineTM Focus Assay (MolecularMD) covering 59 genes. All identified druggable genetic alterations were tested for confirmation with a different method. Results: We included 159 pts, 91 (57%) male, 37 (23,3%) smokers and 81 (50,9%) former smokers. Histology was: 144 adenocarcinoma, 7 squamous cell carcinoma, two sarcomatoid and 6 large cell/undifferentiated. 129 (81%) cases were analyzed in plasma and 63 (49%) had tissue NGS results for comparison. Median number of detected genetic alterations was 2 and maximum number was 17. No alterations were found in 14 cases (11%). Two of them were then retested and became positive. We found 34 (26%) potentially druggable genetic alterations and three of them showed discordant results between tissue and plasma. Among all druggable genetic alterations, till now we have treated 12 pts with targeted agents and six had already radiological response evaluable: five of them obtained control of disease. 18 pts with druggable alterations had already received immunotherapy (IT) and only two of them obtained objective response: METex14 mutation and RET rearrangement. We also found 5 cases of KRASSTK11 co-mutations, three were treated with IT and no response was recorded. In parallel, 89 (56%) cases were analyzed in tissue and 44 (49%) were evaluable for NGS. Ten (23%) potentially druggable genetic alterations were found. Conclusions: Plasma NGS was feasible and provided additional information: new druggable genetic alterations were found and potential impact of NGS on response to IT emerged. Legal entity responsible for the study: Istituto Oncologico Veneto, IOV IRCCS. Funding: Merck KGaA. Disclosure: All authors have declared no conflicts of interest.
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Feasibility study of a ctEGFR prototype assay on the fully automated IdyllaTM platform
M. Reijans1, S.V. Gestel1, E.D. Haes1, C. Vandesteene1, J.M. Castro Gomez1, C. Gouedard2, S. Patera2, S. Murray3, G.G. Maertens1 1 R&D, Biocartis, Mechelen, Belgium, 2BioPath Innovations SA, Athens, Greece, 3 BioMarker Solutions Ltd, London, UK Background: To predict the response to EGFR tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients, formalin-fixed paraffin-embedded (FFPE) tumor tissue is routinely tested for the presence of somatic mutations in the epidermal growth factor receptor (EGFR) gene. Sufficient tumor tissue is not always available and ctEGFR testing from plasma is an alternative approach for the detection of EGFR mutations. Therefore, a fast and fully automated ctEGFR assay with minimal hands-on time should allow laboratories to quickly generate EGFR testing results. Methods: IdyllaTM (Biocartis) is a fully integrated molecular diagnostics platform that combines speed and ease of use with high sensitivity and high multiplexing capabilities. In terms of ctDNA testing, it overcomes the time-consuming step of ctDNA extraction from plasma. After insertion of 2 ml of plasma into the cartridge, the complete process of ctDNA extraction, real-time PCR, data analysis and reporting is fully automated. The ctEGFR prototype assay allows the detection of 49 mutations including insertions and deletions in exons 18, 19, 20 and 21. The results obtained by the ctEGFR prototype assay were compared with NGS (sensitivity 2-5%). Results: Sixty-four NSCLC samples were tested with both assays. Overall, 34 mutations were detected by NGS and confirmed by the ctEGFR prototype assay. In 33 samples,
Volume 30 | Supplement 5 | October 2019
ment: Biocartis. E.D. Haes: Full / Part-time employment: Biocartis. C. Vandesteene: Full / Part-time employment: Biocartis. J.M. Castro Gomez: Full / Part-time employment: Biocartis. C. Gouedard: Full / Part-time employment: BioPath Innovations SA. S. Patera: Full / Part-time employment: BioPath Innovations SA. S. Murray: Full / Part-time employment: BioMarker Solutions Ltd. G.G. Maertens: Full / Part-time employment: Biocartis.
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Enhanced access to EGFR molecular testing in NSCLC using a cell-free DNA tube for liquid biopsy
T.E. May1, S.A. Scudder2, S.J. Joshi1, M. Kohlmann3, N. Shrestha3, N. Lee3, J.A. Højbjerg4, J. Lai5, A.T. Madsen6, M.S. Clement4, P. Meldgaard7, M. Tsourounis3, B. Sørensen8, A. Kohlmann9, P. O’Donnell1, H. Halait1 1 Development Department, Roche Molecular Systems Inc – USA, Pleasanton, CA, USA, 2 Roche Sequencing Solutions, Roche Molecular Systems Inc - USA, Pleasanton, CA, USA, 3 Development, AstraZeneca PLC, Gaithersburg, MD, USA, 4Clinical Biochemistry, Aarhus University, Aarhus, Denmark, 5Clinical Operations, Roche Molecular Systems, Inc, Pleasanton, CA, USA, 6Oncology, Aarhus University, Aarhus, Denmark, 7Clinical Oncology, Aarhus University, Aarhus, Denmark, 8Pathology, Aarhus University, Aarhus, Denmark, 9Research, AstraZeneca PLC, Gaithersburg, MD, USA, Background: The clinical utility of non-invasive liquid biopsy and ability to accurately detect EGFR mutations in circulating-tumor DNA of patients with NSCLC has been well demonstrated. This expands the pool of patients eligible for molecular testing. The V cobas EGFR Mutation Test v2 (cobas test) is a real-time PCR test for qualitative and semi-quantitative detection of 42 EGFR mutations in DNA from tissue and circulating free DNA (cfDNA) from K2 EDTA plasma. Blood collected in K2 EDTA requires plasma be separated within 8 hours, which can be a barrier to molecular testing and thus impact treatment decisions. The Roche Cell-Free DNA Collection Tube (Roche cfDNA) stabilizes blood up to 8 days, allowing greater flexibility in transportation and time to plasma separation. Here the suitability of plasma from Roche cfDNA tubes for use with the cobas test is demonstrated. Methods: Test performance with Roche cfDNA plasma was verified with NSCLC patient specimens or surrogate samples (sheared cell line DNA in healthy donor plasma). Correlation was tested using paired draws in K2 EDTA and Roche cfDNA tubes for 51 NSCLC patients and 20 healthy donor surrogate samples. Limit of detection (LoD) and linearity established with K2 EDTA plasma were verified with Roche cfDNA plasma. Reproducibility was assessed using surrogate samples at two levels with multiple operators, Roche cfDNA tube lots, instruments, days and sites. Blood storage conditions were established at an external laboratory with 6 NSCLC patient specimens. Results: Compared to K2 EDTA plasma, Roche cfDNA plasma had a PPA, NPA and OPA of 100.0% with the cobas test. LoD for EGFR mutation detection in Roche cfDNA plasma was verified as 100 cp/mL. Linearity for EGFR mutations in Roche cfDNA plasma was verified. The reproducibility study had a call agreement of 98.6%. Blood in Roche cfDNA tubes was stable for up to 8 days at 18-25 C with one excursion of up to 24 hours at 15-30 C prior to separation. V Conclusions: Use of the Roche cfDNA tube with the cobas EGFR Mutation Test v2 provides the flexibility to store blood for up to 8 days prior to separation, with equivalent performance to K2 EDTA plasma, and facilitates use of liquid biopsy for NSCLC patients needing molecular testing. Legal entity responsible for the study: Roche Molecular Systems Inc. Funding: AstraZeneca PLC. Disclosure: T.E. May: Full / Part-time employment: Roche Molecular Solutions. S.A. Scudder: R
R
Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Solutions. S.J. Joshi: Full / Part-time employment: Roche Molecular Solutions. M. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca, PLC. N. Shrestha: Full / Parttime employment: Roche Molecular Solutions. N. Lee: Full / Part-time employment: Roche Molecular Solutions. J. Lai: Full / Part-time employment: Roche Molecular Systems Inc. M. Tsourounis: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. A. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. P. O’Donnell: Full / Part-time employment, Spouse / Financial dependant: Roche Molecular Systems. H. Halait: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Systems. All other authors have declared no conflicts of interest.
doi:10.1093/annonc/mdz257 | v577
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L. Bonanno1, A. Pavan2, A. Ferro2, L. Calvetti3, S. Frega1, G. Pasello1, G. Aprile3, V. Guarneri4, P.F. Conte2 1 Medical Oncology 2, Istituto Oncologico Veneto IRCCS, Padua, Italy, 2Surgery Oncology and Gastroenterology, Universit a degli Studi di Padova, Padua, Italy, 3 Medical Oncology, Ospedale San Bortolo di Vicenza, Vicenza, Italy, 4Surgery, Oncology and Gastroenterology, Istituto Oncologico Veneto IRCCS, Padua, Italy
NGS detected no mutation. The ctEGFR prototype assay detected 7 additional mutations in this cohort. Retesting with the cobas EGFR Mutation Test v2 confirmed the presence of these mutations. Analytical sensitivity was assessed for 20 mutations using plasma spiked with synthetic targets. Analytical sensitivities ranging from 1 to 4% were obtained for the tested mutations. Inclusivity was demonstrated for 49 mutations in total. The average turnaround time of a run was <2h 40 min and the hands-on time for the assay was <2 min. Conclusions: This study shows that the IdyllaTM platform enables the development of a prototype ctEGFR assay with high sensitivity and ease of use combined with a fast turnaround time for the testing of 49 relevant EGFR mutations in 2 ml of plasma from NSCLC patients. Legal entity responsible for the study: Biocartis. Funding: Biocartis. Disclosure: M. Reijans: Full / Part-time employment: Biocartis. S.V. Gestel: Full / Part-time employ-
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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
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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
abstracts
Annals of Oncology Legal entity responsible for the study: IQNPath. Funding: IQNPath. Disclosure: All authors have declared no conflicts of interest.
1414P
Clinical impact of plasma next-generation sequencing (NGS) in advanced non-small cell lung cancer (aNSCLC)
Background: NGS provides genetic information with potential impact on clinical practice. Plasma NGS has the advantage to reduce the need to repeat biopsy and provide information about tumor heterogeneity. Methods: Since March 2018 we prospectively screened aNSCLCs consecutively referring to our institution for potential eligibility to VISION trial (NCT02864992). All the patients (pts) were previously screened for EGFR/ALK/ROS1 sensitizing alterations according to standard methods and positive cases were excluded. NGS was performed R covering 73 genes including all somatic alterations recwith METex14 Guardant360V ognized as potential targets by NCCN. A parallel cohort of pts was also analysed with NGS in tissue by using METex14 OncomineTM Focus Assay (MolecularMD) covering 59 genes. All identified druggable genetic alterations were tested for confirmation with a different method. Results: We included 159 pts, 91 (57%) male, 37 (23,3%) smokers and 81 (50,9%) former smokers. Histology was: 144 adenocarcinoma, 7 squamous cell carcinoma, two sarcomatoid and 6 large cell/undifferentiated. 129 (81%) cases were analyzed in plasma and 63 (49%) had tissue NGS results for comparison. Median number of detected genetic alterations was 2 and maximum number was 17. No alterations were found in 14 cases (11%). Two of them were then retested and became positive. We found 34 (26%) potentially druggable genetic alterations and three of them showed discordant results between tissue and plasma. Among all druggable genetic alterations, till now we have treated 12 pts with targeted agents and six had already radiological response evaluable: five of them obtained control of disease. 18 pts with druggable alterations had already received immunotherapy (IT) and only two of them obtained objective response: METex14 mutation and RET rearrangement. We also found 5 cases of KRASSTK11 co-mutations, three were treated with IT and no response was recorded. In parallel, 89 (56%) cases were analyzed in tissue and 44 (49%) were evaluable for NGS. Ten (23%) potentially druggable genetic alterations were found. Conclusions: Plasma NGS was feasible and provided additional information: new druggable genetic alterations were found and potential impact of NGS on response to IT emerged. Legal entity responsible for the study: Istituto Oncologico Veneto, IOV IRCCS. Funding: Merck KGaA. Disclosure: All authors have declared no conflicts of interest.
1415P
Feasibility study of a ctEGFR prototype assay on the fully automated IdyllaTM platform
M. Reijans1, S.V. Gestel1, E.D. Haes1, C. Vandesteene1, J.M. Castro Gomez1, C. Gouedard2, S. Patera2, S. Murray3, G.G. Maertens1 1 R&D, Biocartis, Mechelen, Belgium, 2BioPath Innovations SA, Athens, Greece, 3 BioMarker Solutions Ltd, London, UK Background: To predict the response to EGFR tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients, formalin-fixed paraffin-embedded (FFPE) tumor tissue is routinely tested for the presence of somatic mutations in the epidermal growth factor receptor (EGFR) gene. Sufficient tumor tissue is not always available and ctEGFR testing from plasma is an alternative approach for the detection of EGFR mutations. Therefore, a fast and fully automated ctEGFR assay with minimal hands-on time should allow laboratories to quickly generate EGFR testing results. Methods: IdyllaTM (Biocartis) is a fully integrated molecular diagnostics platform that combines speed and ease of use with high sensitivity and high multiplexing capabilities. In terms of ctDNA testing, it overcomes the time-consuming step of ctDNA extraction from plasma. After insertion of 2 ml of plasma into the cartridge, the complete process of ctDNA extraction, real-time PCR, data analysis and reporting is fully automated. The ctEGFR prototype assay allows the detection of 49 mutations including insertions and deletions in exons 18, 19, 20 and 21. The results obtained by the ctEGFR prototype assay were compared with NGS (sensitivity 2-5%). Results: Sixty-four NSCLC samples were tested with both assays. Overall, 34 mutations were detected by NGS and confirmed by the ctEGFR prototype assay. In 33 samples,
Volume 30 | Supplement 5 | October 2019
ment: Biocartis. E.D. Haes: Full / Part-time employment: Biocartis. C. Vandesteene: Full / Part-time employment: Biocartis. J.M. Castro Gomez: Full / Part-time employment: Biocartis. C. Gouedard: Full / Part-time employment: BioPath Innovations SA. S. Patera: Full / Part-time employment: BioPath Innovations SA. S. Murray: Full / Part-time employment: BioMarker Solutions Ltd. G.G. Maertens: Full / Part-time employment: Biocartis.
1416P
Enhanced access to EGFR molecular testing in NSCLC using a cell-free DNA tube for liquid biopsy
T.E. May1, S.A. Scudder2, S.J. Joshi1, M. Kohlmann3, N. Shrestha3, N. Lee3, J.A. Højbjerg4, J. Lai5, A.T. Madsen6, M.S. Clement4, P. Meldgaard7, M. Tsourounis3, B. Sørensen8, A. Kohlmann9, P. O’Donnell1, H. Halait1 1 Development Department, Roche Molecular Systems Inc – USA, Pleasanton, CA, USA, 2 Roche Sequencing Solutions, Roche Molecular Systems Inc - USA, Pleasanton, CA, USA, 3 Development, AstraZeneca PLC, Gaithersburg, MD, USA, 4Clinical Biochemistry, Aarhus University, Aarhus, Denmark, 5Clinical Operations, Roche Molecular Systems, Inc, Pleasanton, CA, USA, 6Oncology, Aarhus University, Aarhus, Denmark, 7Clinical Oncology, Aarhus University, Aarhus, Denmark, 8Pathology, Aarhus University, Aarhus, Denmark, 9Research, AstraZeneca PLC, Gaithersburg, MD, USA, Background: The clinical utility of non-invasive liquid biopsy and ability to accurately detect EGFR mutations in circulating-tumor DNA of patients with NSCLC has been well demonstrated. This expands the pool of patients eligible for molecular testing. The V cobas EGFR Mutation Test v2 (cobas test) is a real-time PCR test for qualitative and semi-quantitative detection of 42 EGFR mutations in DNA from tissue and circulating free DNA (cfDNA) from K2 EDTA plasma. Blood collected in K2 EDTA requires plasma be separated within 8 hours, which can be a barrier to molecular testing and thus impact treatment decisions. The Roche Cell-Free DNA Collection Tube (Roche cfDNA) stabilizes blood up to 8 days, allowing greater flexibility in transportation and time to plasma separation. Here the suitability of plasma from Roche cfDNA tubes for use with the cobas test is demonstrated. Methods: Test performance with Roche cfDNA plasma was verified with NSCLC patient specimens or surrogate samples (sheared cell line DNA in healthy donor plasma). Correlation was tested using paired draws in K2 EDTA and Roche cfDNA tubes for 51 NSCLC patients and 20 healthy donor surrogate samples. Limit of detection (LoD) and linearity established with K2 EDTA plasma were verified with Roche cfDNA plasma. Reproducibility was assessed using surrogate samples at two levels with multiple operators, Roche cfDNA tube lots, instruments, days and sites. Blood storage conditions were established at an external laboratory with 6 NSCLC patient specimens. Results: Compared to K2 EDTA plasma, Roche cfDNA plasma had a PPA, NPA and OPA of 100.0% with the cobas test. LoD for EGFR mutation detection in Roche cfDNA plasma was verified as 100 cp/mL. Linearity for EGFR mutations in Roche cfDNA plasma was verified. The reproducibility study had a call agreement of 98.6%. Blood in Roche cfDNA tubes was stable for up to 8 days at 18-25 C with one excursion of up to 24 hours at 15-30 C prior to separation. V Conclusions: Use of the Roche cfDNA tube with the cobas EGFR Mutation Test v2 provides the flexibility to store blood for up to 8 days prior to separation, with equivalent performance to K2 EDTA plasma, and facilitates use of liquid biopsy for NSCLC patients needing molecular testing. Legal entity responsible for the study: Roche Molecular Systems Inc. Funding: AstraZeneca PLC. Disclosure: T.E. May: Full / Part-time employment: Roche Molecular Solutions. S.A. Scudder: R
R
Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Solutions. S.J. Joshi: Full / Part-time employment: Roche Molecular Solutions. M. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca, PLC. N. Shrestha: Full / Parttime employment: Roche Molecular Solutions. N. Lee: Full / Part-time employment: Roche Molecular Solutions. J. Lai: Full / Part-time employment: Roche Molecular Systems Inc. M. Tsourounis: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. A. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. P. O’Donnell: Full / Part-time employment, Spouse / Financial dependant: Roche Molecular Systems. H. Halait: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Systems. All other authors have declared no conflicts of interest.
doi:10.1093/annonc/mdz257 | v577
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz257.008/5576775 by guest on 24 October 2019
L. Bonanno1, A. Pavan2, A. Ferro2, L. Calvetti3, S. Frega1, G. Pasello1, G. Aprile3, V. Guarneri4, P.F. Conte2 1 Medical Oncology 2, Istituto Oncologico Veneto IRCCS, Padua, Italy, 2Surgery Oncology and Gastroenterology, Universit a degli Studi di Padova, Padua, Italy, 3 Medical Oncology, Ospedale San Bortolo di Vicenza, Vicenza, Italy, 4Surgery, Oncology and Gastroenterology, Istituto Oncologico Veneto IRCCS, Padua, Italy
NGS detected no mutation. The ctEGFR prototype assay detected 7 additional mutations in this cohort. Retesting with the cobas EGFR Mutation Test v2 confirmed the presence of these mutations. Analytical sensitivity was assessed for 20 mutations using plasma spiked with synthetic targets. Analytical sensitivities ranging from 1 to 4% were obtained for the tested mutations. Inclusivity was demonstrated for 49 mutations in total. The average turnaround time of a run was <2h 40 min and the hands-on time for the assay was <2 min. Conclusions: This study shows that the IdyllaTM platform enables the development of a prototype ctEGFR assay with high sensitivity and ease of use combined with a fast turnaround time for the testing of 49 relevant EGFR mutations in 2 ml of plasma from NSCLC patients. Legal entity responsible for the study: Biocartis. Funding: Biocartis. Disclosure: M. Reijans: Full / Part-time employment: Biocartis. S.V. Gestel: Full / Part-time employ-