Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients’ T cells are transduced in vitro using lentiviral or retroviral vectors containing a ...CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to ∼50 copies/μg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 101–106 copies/200 ng genomic DNA (R2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R2 = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials.
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Highly specific, sensitive, and quantitative analytical assays are needed to monitor the in vivo cellular kinetics of CAR-T cells in the patient after infusion. In this study, Davis and colleagues describe the validation results of a novel qPCR assay designed to specifically identify the tisagenlecleucel CAR transgene in patient samples.
BackgroundBoth proteins (e.g., PD-L1 IHC) and tumor mutation burden (NGS-based) are known to independently predict clinical response to anti-PD-1/PD-L1 therapies. In a meta-analysis of tumor ...specimens from 8135 patients treated with PD-1/PD-L1 blockers, multiplex fluorescence immunohistochemistry (mFIHC) had significantly higher diagnostic accuracy than PD-L1 IHC, tumor mutational burden (TMB), or gene expression profiling alone in predicting clinical response1 or equivalent to a multimodality approach (e.g., PD-L1IHC + TMB). While the benefits of combining mFIHC (tumor-immune interplay) and NGS approaches in selection of patients for next generation immunotherapies is appealing, tumor tissue is a key limiting factor for multimodality analyses in clinical trials. To address this critical limitation, we developed a novel approach for sequential profiling of tumor and immune cell interactions by 7-parameter mFIHC assays, followed by analyses of nucleic acid extracted from same tissue sections.MethodsFormalin-fixed paraffin-embedded (FFPE) tumor tissue and cell line blocks were sectioned, and then stained using mFIHC followed by isolation of nucleic acids, or direct isolation of total nucleic acids. NanoString, qPCR, and NGS were performed on isolated nucleic acids. Nucleic acid quality, transcript abundance, and TMB scores were compared before and after mFIHC staining.ResultsmFIHC revealed a broad range of immune cell phenotypes and spatial interactions, including T cells, B cells, NK cells, monocytes, neutrophils, and their functional status. Isolation of testable quantities of DNA from mFIHC treated slides was achieved when using a DNA-only isolation method, and TMB scores were robust across tested conditions. Cell phenotypes identified by mFIHC were compared to TMB scores across the tested samples. Following mFIHC treatment, RNA yields were reduced relative to the non-mFIHC treated replicates, but still sufficient for optimal input into a 770-target NanoString gene expression panel. However, for mFIHC treated samples, transcript levels were not distinguishable from background for the assessed targets.ConclusionsIn summary, integrating mFIHC testing and TMB analysis on the same samples allows for comprehensive biomarker evaluation. The real world benefits of the combined approach will be described in upcoming clinical trials.ReferenceLu, et al., Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade, a systematic review and meta-analysis. JAMA Oncology 2019; 5(8):1195–1204
Abstract
Introduction
Myeloid neoplasms are a complex class of disorders characterized by a range of genotypes. Accurate, precise, and rapid identification of these genotypes is crucial in giving the ...right drug to the right patient at the right time. In this study, we present a comparison of three Next-Generation Sequencing (NGS) panels targeting genes associated with myeloid cancers. We evaluated assay performance as a function of sensitivity and specificity of somatic variant callings, sequencing metrics, and general workflow, for the analytically validated Illumina TruSight Myeloid Sequencing Panel (TSM), the new Illumina AmpliSeq for Illumina Myeloid Panel (IA), and the ThermoFisher Oncomine™ Myeloid Research Assay (TA).
Methods
Thirty-two libraries were prepared across all three panels. Samples included a mix of Horizon Discovery and SeraCare myeloid DNA controls, a well-characterized Hapmap sample, and six clinical DNAs isolated from peripheral blood of patients with myeloid disorders. Libraries were prepared according to manufacturer protocols, and quantified by Qubit Flourometer. For Illumina panels, libraries were manually prepared, normalized, pooled, and sequenced on Illumina’s MiSeq instruments. For the TA panel, libraries were generated, pooled, templated, and loaded onto sequencing chips by the IonChef, then sequenced on the S5xL system. Primary data analysis was completed using cloud-based bioinformatic solutions followed by manual annotation.
Results
Hands-on time varied for the assays, with the TA panel requiring just 30 minutes compared to four to eight hours for the TSM and IA protocols. Time to results from DNA dilution to variant calls was comparable, at three days for the TSM panel and four days for the IA and TA protocols. The TSM assay exhibited the highest average coverage, but also the greatest amplicon dropout. Both the IA and TA panels exhibited similar coverage uniformity and read depths. Detection of expected variants in control samples down to 5% allele frequency was robust for all assays, however both the IA and TA panels contained increased regions of poor sequencing quality relative to the TSM panel. No false positives were found in the somatic-variant negative Hapmap sample NA12878, and concordance of variant calls for the clinical samples from the IA and TA assays to the validated TSM assay was greater than 95%.
Conclusions
In the complex landscape of myeloid neoplasms, NGS is an invaluable tool for the diagnosis and management of the diseases. Overall, the three sequencing panels evaluated herein provided accurate genotype information for actionable targets in myeloid cancers. Although results for the assays were similar, the short time-to-data for the TSM assay, increased coverage and detection of low frequency variants for the IA assay, and fully-automated option for the TA assay should all be considered when determining the optimal panel for a given need.
Citation Format: Sarah Johnson, Nathan Riccitelli, Reinhold Pollner. Comparison of commercially available myeloid next-generation sequencing assays abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1693.
Abstract
Introduction. Acute Myeloid Leukemia (AML) is the most common acute Leukemia among adults with a poor overall prognosis. With over 20,000 cases in the US alone, about a third of AML patients ...pertain to an important oncogenic driver composed of internal tandem duplication (ITD) somatic mutations in exons 14 and 15 of the FMS-like tyrosine kinase 3 (FLT3) gene. Detecting the presence of measurable residual disease in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. Yet, despite its importance, the ability to detect FLT3-ITD mutations is hampered by the limited sensitivity of conventional PCR-based assays in general and in Next Generation Sequencing-based assays, in part due to the difficulty to align ITD-containing reads to the reference genome.
Methods. In order to facilitate the analysis of AML samples, we developed FLT3-Explorer, a comprehensive bioinformatics pipeline composed of open source software, combined with in-house algorithms to accurately detect low levels of FLT3-ITD mutations. The input of the pipeline is unmapped reads in fastq format. The pipeline then searches for either ITD sequences previously observed in our extensive database of historical data, or a de-novo detection procedure is used to determine mutation sequences of variable lengths within the FLT3 gene. To assess the performance of FLT3-Explorer, we considered a cohort of clinical samples from bone marrow and peripheral blood, as well as cell lines. For sample collection, genomic DNA was isolated, and polymerase chain reaction was performed using primers flanking exons 13 and 15 to amplify the FLT3 gene. DNA was then sequenced using NGS Illumina sequencing. Results were validated using fragment size analysis by Capillary Electrophoresis assays. Also, to determine the limits of detection, various dilutions were performed on cell lines. Furthermore, to assess the performance of our pipeline, we performed a comparison with other FLT3-ITD programs publicly available.
Summary. Results from our analysis showed that our method identifies FLT3-ITD mutations of variant allele fraction as low as 0.003%. Also, we found that our pipeline can detect as many FLT3-ITD mutants as other methods, or more, when the mutant signal is considerably low.
Conclusions. FLT3-Explorer constitutes a powerful solution for low FLT3-ITD detection signal of mutant sequences, of diverse lengths, that can be used on FLT3-ITD AML patients to track disease progression and relapse.
Citation Format: Christian Laing, Wenge Shi, Reinhold Pollner. FLT3-Explorer: A bioinformatics pipeline for detecting low signal in FLT3 internal tandem duplications in acute myeloid leukemia abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2475.
Abstract
Introduction
Surgical biopsy or excisional resection is a standard practice in oncology diagnosis that requires a detectable tumor mass and can be highly invasive, limiting its role in the ...very early detection, as well as ongoing monitoring efforts, of genetic aberrations relevant to tumor biology. In contrast to surgical biopsies, liquid biopsies provide a readily accessible source of circulating tumor DNA, albeit in a highly diluted state that necessitates a very sensitive and robust method of detection. With recent advents in molecular barcoding and bioinformatics, next-generation sequencing (NGS) has emerged as a platform for probing this liquid oncology space. Herein, we evaluate the ability of the Oncomine™ cell-free DNA (cfDNA) Breast, Colon, and Lung NGS panels to reliably detect clinically relevant genomic alterations in a large background of non-mutated genetic material.
Methods
A total of 197 individual libraries were prepared across all three cfDNA panels according to the manufacturer protocols. Libraries were manually normalized, pooled, and loaded onto the ThermoFisher IonChef/S5xL system for bead-templating and sequencing. Samples included a mix of commercially available cfDNA controls, plasma samples, and cell lines spiked into healthy donor plasma, and were selected to cover a wide range of clinically relevant mutations and mimic the conditions inherent to real patient specimens. Isolation of cfDNA was performed on both Qiagen and Kingfisher platforms. Raw variant call files were generated using IonReporter and processed through a custom filtering pipeline to generate the final results.
Results
Nucleic acid isolations yields were comparable for cfDNA samples isolated using the Qiagen or Kingfisher extraction methods, with both yielding a 40-60% DNA recovery rate when known DNA quantities were spiked into plasma background. Library yield mapped to NGS panel size, and the greatest variability was observed for the Colon cfDNA panel. The sensitivity for all Oncomine™ cfDNA assays exceeded 92%, and was highly dependent on sample input, with robust detection at the 0.1% allele frequency requiring up to 50 ng of input cfDNA. A single false positive was present in one HapMap sample analyzed with the breast cfDNA panel.
Conclusion
The Oncomine™ cfDNA panels provide accurate detection of variants in cfDNA down to 0.1% variant allele frequency. However, increased DNA inputs are required to achieve this highly-sensitive level of detection. Although sensitivity is reduced at lower inputs, the ability to generate high-quality libraries from as little as 10 ng DNA ensures sufficient material can be obtained for sequencing from even low-yielding samples. Overall, these results indicate that the Oncomine™ cfDNA assays can aid in the very early diagnosis and monitoring of tumor populations via minimally-invasive blood-based diagnostics.
Citation Format: Nathan Riccitelli, Nancy Valencia, Ashley Beams, Sarah Johnson, Reinhold Pollner. Progress toward non-invasive oncology diagnostics: Evaluation of cfDNA next-generation sequencing assays abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2282.
Abstract
Introduction: Rapidly advancing single cell genomics technology enables uncovering cell heterogeneity missed by bulk sequencing/analysis. Multi-omic single cell sequencing has ability to ...assess up to a hundred cell surface proteins, hundreds of transcripts to whole transcriptome, or TCR/BCR gene rearrangement in thousands of cells. Here we report deep cell immune profiling of PBMC by assessing selected cell surface proteins by AbSeq and targeted immune response genes using two single cell sequencing platforms.
Experimental procedures: For targeted RNASeq on 10xGenomics Chromium platform, droplets containing single PBMC cell/bead were generated. After cDNA synthesis, libraries with genes of interest were generated using BD Rhapsody Human Immune Response Targeted Panel. For BD Rhapsody multi-omic single cell analysis, PBMC were stained using tagged AbSeq antibodies and sample specific cell tag antibodies. Then viable cells after incubation with cell viability dyes were counted on BD Rhapsody scanner. Some samples were processed for removal of apoptotic cells using EasySep Dead Cell Removal (Annexin V) Kit (Stem Cell Tech). Labeled PBMC from multiple subjects were pooled and loaded to a BD Rhapsody chip. Followed by cell capture beads loading and lysis, mRNA and antibody tags were captured. cDNA was synthesized and libraries containing genes of interest were generated using Human immune response panel along with AbSeq and sample tag libraries. Sequencing was performed on NextSeq 500 (Illumina). Data analysis from 10xGenomics were conduct using its Cell Ranger software. Data analysis from BD Rhapsody workflow were conduct using Seven Bridges Genomics Pipeline and SeqGeq (FlowJo). For comparison, PBMC were also profiled for general major cell populations by flow cytometry.
Summary and Conclusions: Targeted RNASeq using both single cell sequencing platforms enables deep cell profiling with benefits of lower cost and higher sensitivity compared with whole transcriptome RNASeq. The results for general cell populations generated by single cell sequencing are comparable to those generated by flow cytometry, however, single cell sequencing classifies sub-populations in greater details based on gene expression markers. Including tagged AbSeq antibodies to assess cell surface proteins in the workflow further helps classification in the case of post-transcriptional regulation, while identifying alternatively spliced isoforms (CD45RO and CD45RA) elusive by RNASeq. One of the challenges is the requirement for viable cells. Although some apoptotic cells could be removed, less viable cells leftover could interfere single cell sequencing and thus careful data QC bioinformatics is warranted. Overall, multi-omic single cell sequencing could enable deep cell profiling and assessing patient samples has great potential to provide biological insights and identify predictive biomarkers.
Citation Format: Wenge Shi, Christian Laing, Jane Gao, Kerri Burns, Shyam Sarikonda, Reinhold Pollner, Hua Gong. Multi-omic single cell sequencing for deep cell immune profiling and identification of potential biomarkers for cell therapy and immunotherapy abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4290.
Abstract
Purpose
Formalin-fixed, paraffin-embedded (FFPE) tumor tissue, while standard for biological preservation, can significantly impact nucleic-acid quality via degradation and cross-linking. ...The low quality of FFPE-treated samples, coupled with the presence of native small molecules inhibitors in various tissue types, yields significant challenges for PCR-based technologies. Alternatively, NanoString RNA assays combine straightforward workflows with multiplexed detection of up to 800 targets via hybridization-based chemistry that is not reliant on enzymatic processes, and thus may offer advantages in a melanin-rich environment. For utilization in a melanoma disease setting, we evaluated the impact of different melanin-levels in FFPE samples on the performance of NanoString mRNA and miRNA assays
Methods
One FFPE cell line block was sectioned and spiked with zero or a range of biologically-relevant levels of melanin, both before and after total nucleic acid isolation with the Qiagen AllPrep FFPE procedure. Total RNA content was measured by NanoDrop and fluorescent-based methods. Abundance of individual RNA molecules was assessed by the NanoString IO360 gene expression assay and the NanoString Human v3 miRNA panel.
Results
Melanin concentration was observed to directly impact quantification results, with higher concentrations correlating to elevated estimates of total RNA yield by NanoDrop, but reduced RNA by fluorescent-based measurement. Different melanin concentrations did not impact RNA expression measurements in the enzyme-free IO360 gene expression assay. For the v3 miRNA assay, however, increased melanin concentration resulted in significant reduction in miRNA signal, but as observed with the IO360 assay, did not affect endogenous mRNA transcript detection. The reduction in miRNA levels was significantly greater for the samples spiked with melanin post-extraction compared with those aliquots spiked prior to isolation. Tumor inflammation signature (TIS) scoring was not impacted by elevated melanin levels.
Conclusions
NanoString hybridization reactions exhibited minimal sensitivity to melanin content. Conversely, NanoString assays requiring enzymatic processing yielded substantially reduced counts at elevated melanin concentrations. Isolation procedures were effective in removing lower levels of melanin, but could not fully mitigate melanin impact at all tested concentrations. Significantly, classification of tumor immune-environment by NanoString was not affected by melanin, potentially making TIS scoring an attractive tool for biomarker analysis of checkpoint inhibitor therapies in melanoma.
Citation Format: Alexis Kurmis, Eileen Kelly, Nancy Valencia, Justin Wahl, Christian Laing, Nathan Riccitelli, Reinhold Pollner. Effects of melanin on NanoString gene and miRNA expression assays for utilization in biomarker analysis for melanoma abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1568.
Abstract
Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) ...internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.
Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity.
Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation.
Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy.
Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2299.
Exceptional clinical responses produced by the first chimeric antigen receptor T CAR‐T cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next ...generation CAR‐T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these “living” drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR‐T therapies. Herein, three main areas for monitoring CAR‐T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR‐T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit‐for‐purpose tools and help to more rapidly advance the development of next generation CAR‐T therapies.
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