Catherine Huang

PK assays for antibody–drug conjugates: case study with ado-trastuzumab emtansine

BACKGROUND Antibody-drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory guidelines and standard industry best practices for developing strategies for bioanalytical PK assays are still being established. RESULTS A PK assay strategy was developed that included comprehensive novel reagent and assay characterization approaches for the ADC ado-trastuzumab emtansine (T-DM1). CONCLUSION The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development.

An interlaboratory study of complex variant detection

Next-generation sequencing (NGS) is widely used and cost-effective. Depending on the specific methods, NGS can have limitations detecting certain technically challenging variant types even though they are both prevalent in patients and medically important. These types are underrepresented in validation studies, hindering the uniform assessment of test methodologies by laboratory directors and clinicians. Specimens containing such variants can be difficult to obtain; thus, we evaluated a novel solution to this problem in which a diverse set of technically challenging variants was synthesized and introduced into a known genomic background. This specimen was sequenced by 7 laboratories using 10 different NGS workflows. The specimen was compatible with all 10 workflows and presented biochemical and bioinformatic challenges similar to those of patient specimens. Only 10 of 22 challenging variants were correctly identified by all 10 workflows, and only 3 workflows detected all 22. Many, but not all, of the sensitivity limitations were bioinformatic in nature. We conclude that Synthetic controls can provide an efficient and informative mechanism to augment studies with technically challenging variants that are difficult to obtain otherwise. Data from such specimens can facilitate inter-laboratory methodologic comparisons and can help establish standards that improve communication between clinicians and laboratories.

Abstract 4758: Use of highly multiplexed reference materials to facilitate validation of a clinical NGS tumor fusion RNA assay

Introduction: Next Generation Sequencing assays for detection of tumor RNA fusions must undergo rigorous validation before clinical implementation. Validations include assessment of the assay9s accuracy, precision, reproducibility and limits of detection or reportable range. Obtaining samples with all needed variants is difficult and time consuming. When found, they are often in limited quantities such that repeated testing for precision and reproducibility studies is not possible. Sample heterogeneity or lack of characterization further complicates interpretation of results. This study demonstrates how highly characterized, uniformly manufactured reference materials can facilitate clinical NGS assay validation. Methods: An RNA panel representing decreasing levels of 16 different RNA fusions and exon skipping events compared to the total cellular RNA was generated. GM24385 cell line was engineered to contain the fusion RNAs then formalin fixed, and the total RNA was extracted using the Maxwell RSC FFPE RNA kit. The fusion RNA was serially diluted into total RNA from similarly processed non-engineered GM24385 cells to create a panel with decreasing levels of each fusion. The panel was characterized by fusion-specific, TaqMan digital PCR assays to obtain a “truth set” on which to compare NGS results. Each panel member was tested in triplicate on three separate days as part of a validation study. Results: Precision and reproducibility were assessed quantitatively through analysis of the NGS unique reads. The intra-run precision varied among fusion targets with many targets having %CV of less than 15%, while a few targets had significantly higher variability between replicates. The linearity of the assay was good, with R-squared value greater than 0.95. Lower limits of detection for each fusion target were estimated using the digital PCR data and were different for different fusion targets; for example, detection of EML4-ALK fusion was five-fold more sensitive than detection of CD74-ROS1 fusion. Conclusions: Manufactured reference materials can supplement validation studies and their uniformity and digital PCR characterization allows greater insight into assay performance such as limits of detection than use of remnant specimens alone. The sensitivity of the NGS panel used was not the same for different fusion targets and, and underscores the need for highly multiplexed reference materials and for labs to test all clinically important fusions during validation Citation Format: Catherine Huang, Subit Barua, Deepika Philkana, Russell Garlick, Bharathi Anekella, Helen Fernandes. Use of highly multiplexed reference materials to facilitate validation of a clinical NGS tumor fusion RNA assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4758.

Abstract 3825: Digital PCR-characterized, highly multiplexed, oncology RNA fusion reference materials: Performance on multiple NGS platforms

Introduction: Genomic structural alterations are increasingly actionable for targeted therapeutics and personalized medicine. Molecular diagnostics are rapidly being introduced for detection of fusion RNAs by highly multiplexed next-generation sequencing assays. However, reference materials to aid in the development, optimization and validation of these assays are lacking. We developed Seraseq FFPE Fusion RNA Reference Material to fill this unmet need and show that this material is compatible across a wide range of NGS assay platforms. Methods: Biosynthetics were used for transcription of clinically actionable RNA fusions, including fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. Sixteen (16) in vitro transcribed RNAs were introduced into GM24385 reference cell line (The 1000 Genomes Project, Coriell). The cells were collected, fixed in formalin, and total RNA was isolated. Digital PCR with TaqMan® chemistry was used to determine the target Fusion RNA copies per microliter. Use of fusion-specific digital PCR provides an orthogonal method of verifying transcript levels and serves as the “ground truth” for the abundance of each RNA. NGS testing of the purified RNA used the ArcherDx FusionPlex™ CTL Panel, QIAGEN QIAseq Targeted RNAscan Panel, and the Thermo Fisher Oncomine Focus Assay. Results: All sixteen (16) fusions present in the prototype were detected as expected on each NGS platform. Results among the three different NGS platforms were generally concordant, although the reads across the fusion junctions did vary slightly among NGS assays and with digital PCR results. However, all methods indicated that the reference material gave low positive results, similar to a patient sample, and that the single reference material could serve as a positive control for detection of sixteen different fusions, which represent a variety of different solid tumor types. Conclusions: Seraseq FFPE RNA Fusion Reference Material allows simultaneous evaluation of detection for sixteen fusions observed in a variety of solid tumors, both common and rare. It provides a consistent, unlimited supply of QC materials particularly valuable for difficult to find rare fusions. The reference material generates low positive results on three leading assays, and this is important to truly challenge the assay system. This material is handled identically to a patient sample from extraction through analysis, and verifies performance at levels expected for patient samples. Citation Format: Catherine Huang, Yves Konigshofer, Lequan Nguyen, Rajeswari Vemula, Praveena Kamineni, Deepika Philkana, Ekta Jaiswal, Bharathi Anekella. Digital PCR-characterized, highly multiplexed, oncology RNA fusion reference materials: Performance on multiple NGS platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2017-3825

Abstract 1392: Development of highly multiplexed, whole process reference materials for monitoring oncology RNA fusions

Introduction: Genomic structural alterations are increasingly actionable for targeted therapeutics and personalized medicine. Next-generation sequencing methods are increasingly used to detect these fusion RNAs in FFPE material. However, quality control materials for these assays are lacking, and validation materials for rare fusions are frequently not available. We developed a novel FFPE Fusion RNA Reference Material to fill this unmet need. Methods: Highly multiplexed RNAs were designed which contain multiple fusion targets observed predominantly in solid tumors including ALK, RET, ROS1 FGFR3, and NTRK1 fusions as well as PAX-PPARG fusion and ETV6-NTRK3 fusion. These RNAs were transcribed in vitro and contained a 5’ guanosine cap and a poly-adenosine tail to improve intracellular stability. The RNA was introduced into GM24385 reference cell line (The 1000 Genomes Project, Coriell). After recovery from transfection, the cells were collected and fixed in formalin. Digital PCR with TaqMan chemistry was used to determine the average number of synthetic RNA transcripts per cell. These transfected cells were then mixed with non-transfected GM24385 cells to achieve a consistent “low positive”, formulation. The cell mixture was embedded in a paraffin block and 10 micron sections were produced. Quality control was performed by extracting RNA and testing using the ArcherDx FusionPlex™ Lung Thyroid Panel and the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel. Results: All twelve (12) fusions present in the prototype were detected as high confidence calls on the ArcherDx Lung Thyroid panel. Whereas embedded cell lines (with genomic mutations) give extremely high positive results (typical results are thousands of reads across the fusion junction), Seraseq™ FFPE Fusion RNA Reference material gave low positive results, similar to patient samples. The reads spanning the fusion junction ranged from 38 to 396. The Ion AmpliSeq™ RNA Fusion Lung Cancer panel assays only 6 of the fusions in the Seraseq reference material due to their assay design; however, all assayed fusions were appropriately detected. Conclusions: Highly multiplexed reference materials in FFPE format are needed for quality control of NGS-based detection of oncology RNA fusions. Seraseq FFPE RNA Fusion Reference Material allows simultaneous evaluation of detection for twelve fusions observed in a variety of solid tumors, both common and rare. It generates low positive results on two leading assays, and this is important to truly challenge the assay system and verify performance at levels expected for patient samples. Citation Format: Catherine E. Huang, Yves Konigshofer, Lequan Nguyen, Bharathi Anekella. Development of highly multiplexed, whole process reference materials for monitoring oncology RNA fusions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1392.