- Email: [email protected]
Feasibility study of a ctEGFR prototype assay on the fully automated Idylla™ platform
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/mdz257.010/5576797 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-
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/mdz257.010/5576797 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-