Tom Morrison, Bryan Lo, Sophie J. Deharvengt, Nick Lazaridis, and Gregory J. Tsongalisd. (2024). Internal Standards for Limit Controls and Absolute Abundance Measurement of Oncogenic Fusions and Mutations. JALM 175

  • As more clinical decision-making becomes dependent on accurate NGS molecular profiling of DNA and RNA, the introduction of robust quality control measures becomes imperative. We introduce a method, SNAQTM-SEQ IS, to alleviate much of the variability in interpretation of VAFs, especially when used in the liquid biopsy testing of ctDNA/RNA. Incorporation of synthetic IS into each sample allows direct calculation of molecules of the variants per ml of plasma for any sample, on any panel, on any NGS platform. These IS are an appropriate set of reference materials (standards) to allow for comparability across multiple minimal residual disease assays and studies and across a longitudinal timeline to monitor disease progression or response to therapy.

Willey, J.C., Morrison, T.B., Austermiller, B., Crawford, E.L., Craig, D.J., Blomquist, T.M., Jones, W.D., Wali, A., Lococo, J.S., Haseley, N., Richmond, T.A., Novoradovskaya, N., Kusko, R., Chen, G., Li, Q.-Z., Johann, D.J., Deveson, I.W., Mercer, T.R., Wu, L. and Xu, J., (2021). Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA. Cell Reports Methods, 1(7), p.100106.

  • This study reports a targeted next-generation sequencing (NGS) method that will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. Use of internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position in each sample. True-positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

Gong, B., Li, D., Kusko, R., Novoradovskaya, N., Zhang, Y., Wang, S., Pabón-Peña, C., Zhang, Z., Lai, K., Cai, W., LoCoco, J.S., Lader, E., Richmond, T.A., Mittal, V.K., Liu, L.-C., Johann, D.J., Willey, J.C., Bushel, P.R., Yu, Y., Xu, C., Chen, G., Burgess, D., Cawley, S., Giorda, K., Haseley, N., Qiu, F., Wilkins, K., Arib, H., Attwooll, C., Babson, K., Bao, L., Bao, W., Lucas, A.B., Best, H., Bhandari, A., Bisgin, H., Blackburn, J., Blomquist, T.M., Boardman, L., Burgher, B., Butler, D.J., Chang, C.-J., Chaubey, A., Chen, T., Chierici, M., Chin, C.R., Close, D., Conroy, J., Cooley Coleman, J., Craig, D.J., Crawford, E., del Pozo, A., Deveson, I.W., Duncan, D., Eterovic, A.K., Fan, X., Foox, J., Furlanello, C., Ghosal, A., Glenn, S., Guan, M., Haag, C., Hang, X., Happe, S., Hennigan, B., Hipp, J., Hong, H., Horvath, K., Hu, J., Hung, L.-Y., Jarosz, M., Kerkhof, J., Kipp, B., Kreil, D.P., Łabaj, P., Lapunzina, P., Li, P., Li, Q.-Z., Li, W., Li, Z., Liang, Y., Liu, S., Liu, Z., Ma, C., Marella, N., Martín-Arenas, R., Megherbi, D.B., Meng, Q., Mieczkowski, P.A., Morrison, T., Muzny, D., Ning, B., Parsons, B.L., Paweletz, C.P., Pirooznia, M., Qu, W., Raymond, A., Rindler, P., Ringler, R., Sadikovic, B., Scherer, A., Schulze, E., Sebra, R., Shaknovich, R., Shi, Q., Shi, T., Silla-Castro, J.C., Smith, M., López, M.S., Song, P., Stetson, D., Strahl, M., Stuart, A., Supplee, J., Szankasi, P., Tan, H., Tang, L., Tao, Y., Thakkar, S., Thierry-Mieg, D., Thierry-Mieg, J., Thodima, V.J., Thomas, D., Tichý, B., Tom, N., Garcia, E.V., Verma, S., Walker, K., Wang, C., Wang, J., Wang, Y., Wen, Z., Wirta, V., Wu, L., Xiao, C., Xiao, W., Xu, S., Yang, M., Ying, J., Yip, S.H., Zhang, G., Zhang, S., Zhao, M., Zheng, Y., Zhou, X., Mason, C.E., Mercer, T., Tong, W., Shi, L., Jones, W. and Xu, J., (2021). Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions. Genome Biology, 22(1), p.109.

  • This comprehensive study from the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

Deveson, I.W., Gong, B., Lai, K., LoCoco, J.S., Richmond, T.A., Schageman, J., Zhang, Z., Novoradovskaya, N., Willey, J.C., Jones, W., Kusko, R., Chen, G., Madala, B.S., Blackburn, J., Stevanovski, I., Bhandari, A., Close, D., Conroy, J., Hubank, M., Marella, N., Mieczkowski, P.A., Qiu, F., Sebra, R., Stetson, D., Sun, L., Szankasi, P., Tan, H., Tang, L., Arib, H., Best, H., Burgher, B., Bushel, P.R., Casey, F., Cawley, S., Chang, C.-J., Choi, J., Dinis, J., Duncan, D., Eterovic, A.K., Feng, L., Ghosal, A., Giorda, K., Glenn, S., Happe, S., Haseley, N., Horvath, K., Hung, L.-Y., Jarosz, M., Kushwaha, G., Li, D., Li, Q.-Z., Li, Z., Liu, L.-C., Liu, Z., Ma, C., Mason, C.E., Megherbi, D.B., Morrison, T., Pabón-Peña, C., Pirooznia, M., Proszek, P.Z., Raymond, A., Rindler, P., Ringler, R., Scherer, A., Shaknovich, R., Shi, T., Smith, M., Song, P., Strahl, M., Thodima, V.J., Tom, N., Verma, S., Wang, J., Wu, L., Xiao, W., Xu, C., Yang, M., Zhang, G., Zhang, S., Zhang, Y., Shi, L., Tong, W., Johann, D.J., Mercer, T.R., Xu, J., and SEQC2 Oncopanel Sequencing Working Group, (2021). Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology. Nature Biotechnology, 39(9), pp.1115–1128.

  • Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

Craig, D. J., Morrison, T., Khuder, S. A., Crawford, E. L., Wu, L., Xu, J., Blomquist, T. M., & Willey, J. C. (2019). Technical advance in targeted NGS analysis enables identification of lung cancer risk-associated low frequency TP53, PIK3CA, and BRAF mutations in airway epithelial cells. BMC Cancer, 19(1), 1–14.

  • Use of SNAQ-SEQ to identify lung cancer risk associated variants at low (<1%) VAF. Application of SNAQ-SEQ to measure mutations in the 0.05–1.0% VAF range enabled identification of an AEC somatic mutation field of injury associated with lung cancer risk. A biomarker comprising TP53, PIK3CA, and BRAF somatic mutations may better stratify individuals for optimal lung cancer screening and prevention outcomes.

Craig, D.J., Blomquist, T.M., Crawford, E.L., Xu, J., Wu, L., Morrison, T. and Willey, J.C., (2019). Poster: Abstract #G009 Use of Synthetic Internal Standards to Measure Very Low Frequency TP53, PIK3CA, and BRAF.

Craig, D.J., Crawford, E.L., Xu, J., Blomquist, T.M., Wu, L., Morrison, T. and Willey, J.C., (2019). Poster: Novel method for NGS analysis of actionable mutations in circulating tumor DNA specimens: Improved quality control and 20-fold lower sequencing required

Morrison, T., Austermiller, B., Lazaridis, nick, Holshouser, C., Smith-Moore, C., O’Connell, K., Bernhardt, P., Fowler, V., Scott, M., Thomas, P. and Hoover, N., (2019). Poster: NIIMBL Adventitious Agent Detection by NGS. NIIMBL National Meeting 2019. Washington DC.

deAbreu, F., Deharvengt, S., Austermiller, B., Morrison, T., Tsongalis, G. and Lefferts, J., (2018). Poster: Spike in NGS Controls for Copy Number Assessment and Improved LOD and VAF Confidence. AMP 2018 Annual Meeting. San Antiono, Texas.

Willey, J.C., Blomquist, T.M., Crawford, E.L., Xu, J., Wu, L. and Morrison, T., (2018). Abstract 1623: Inter-laboratory harmonization of next generation sequencing somatic mutation assays for cancer response prediction. Clinical Research. Proceedings: AACR Annual Meeting 2018; April 14-18, 2018; Chicago, IL. American Association for Cancer Research.pp.1623–1623.

Yeo, J., Crawford, E. L., Zhang, X., Khuder, S., Chen, T., Levin, A., Blomquist, T. M., & Willey, J. C. (2017). A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells. BMC Cancer, 17(1), 1–10.

  • Clinical data sets leveraging the discriminating potential of SNAQ-SEQ RNA controls to identify lung cancer risk in high-risk patients but with healthy bronchial epithelial cells.

Blomquist, T., Crawford, E. L., Yeo, J., Zhang, X., & Willey, J. C. (2015). Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing. Biomolecular Detection and Quantification, 5, 30–37.

  • Seminal publication on utility of SNAQ-SEQ as a control for stochastic and technical errors in NGS. In targeted NGS, synthetic competitive IS control for stochastic sampling at input of both target into library preparation and of target library product into sequencer, and control for qualitative errors generated during library preparation and sequencing. These controls enable accurate clinical diagnostic reporting of confidence limits and limit of detection for copy number measurement, and of frequency for each actionable mutation.

Yeo, J., Crawford, E.L., Blomquist, T.M., Stanoszek, L.M., Dannemiller, R.E., Zyrek, J., De Las Casas, L.E., Khuder, S.A. and Willey, J.C., (2014). A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples. PLoS ONE, 9(2), p.e89395.

Blomquist, T. M., Crawford, E. L., Lovett, J. L., Yeo, J., Stanoszek, L. M., Levin, A., Li, J., Lu, M., Shi, L., Muldrew, K., & Willey, J. C. (2013). Targeted RNA-Sequencing with Competitive Multiplex- PCR Amplicon Libraries. PLoS ONE, 8(11).

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