The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal ...tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.
Using isolated genomic DNA from bone marrow aspirates or whole-blood samples, PCR primers flanking exons 14 and 15 of the FLT3 gene were designed and highly diverse NGS libraries were generated. These libraries were then sequenced by Illumina's sequencing-by-synthesis method. The bioinformatics approach we used identifies unique FLT3-ITD mutations of varying length along with wild type sequences and calculates a mutant allelic frequency. The assay was validated using clinical samples to assess accuracy and reproducibility. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% but was capable of detecting FLT3-ITD mutations at a level as low as 0.003%.
We next validated the assay using clinical samples from patients with FLT3-ITD AML. The negative prognostic impact of FLT3-ITD mutations can be mitigated in part when an FLT3 inhibitor is administered in combination with induction chemotherapy, as demonstrated in CALGB10603/RATIFY (N Engl J Med. 2017;377:454). It was reported in this study that patients treated with an FLT3 inhibitor combined with chemotherapy followed by allogeneic transplant in first remission had better overall survival than their counterparts in the control arm. One hypothesis for this outcome is that the FLT3-inhibitor-treated patients had a lower leukemic burden prior to transplant. As a pilot test of this concept, we used our MRD assay on a series of bone marrow aspirate samples collected from 10 patients with newly diagnosed FLT3-ITD AML. The patients were selected to be as uniform as possible. All patients had intermediate-risk karyotype, a detectable FLT3-ITD mutation by conventional PCR, and mutated NPM1. All patients received cytarabine-based intensive induction and achieved morphologic first remission with a single course of chemotherapy. Finally, all patients underwent allogeneic transplant in first remission. The sample analyzed for MRD was the first collected after remission induction, 5-8 weeks after the start of therapy. The investigators performing the MRD assay were blinded to the clinical data. Four patients received chemotherapy alone, while 6 were treated with chemotherapy (7+3) plus an FLT3 inhibitor. In all patients' remission samples, the MRD assay identified the FLT3-ITD mutation that precisely matched the one observed in the original diagnostic specimen. This demonstrates the sensitivity of the assay (all samples had a detectable mutation), and the unique length of each patient's mutation confers a degree of specificity not achievable with MRD detection methods that focus on other AML-associated mutations. Supporting our hypothesis was the observation that patients treated with FLT3 inhibitors had MRD levels lower than those in patients treated with chemotherapy alone (Figure).
Our results help establish the role of NGS-based MRD assays for the clinical management of FLT3-ITD AML. This assay could be used to define the depth of remission, identify persistent disease, and help guide decision making in the use of FLT3 inhibitors as continuation therapy. This study provides validation of the clinical utility of our MRD assay, which will be used to analyze the remission samples from patients in the ongoing phase 3, randomized, double-blind, placebo-controlled QuANTUM-First clinical trial, in which patients with newly diagnosed FLT3-ITD AML are randomized to receive either the highly potent and selective FLT3 inhibitor quizartinib or placebo in combination with chemotherapy, followed by single-agent quizartinib as continuation therapy.
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Shi:Novartis: Employment, Equity Ownership; Daiichi Sankyo: Other: Provide clinical trial testing services. Chang:Daiichi Sankyo: Employment. Laing:Novartis: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Berisha:Daiichi Sankyo: Employment. Adams:Johns Hopkins University: Employment. Ding:Navigate BP: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Nakamaru:Daiichi Sankyo: Employment. Lameh:Navigate BioPharma Inc,: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Pollner:Navigate BioPharma Inc.: Employment. Kobayashi:Daiichi Sankyo: Employment.
Abstract
Introduction: Gene amplification/deletion is a common tactic employed by tumor cells to proliferate uncontrollably. Multiple methods have been developed to determine gene copy numbers, such ...as FISH, qPCR, and, more recently, Next Generation Sequencing (NGS). However, these methods have limitations: FISH and NGS are very labor intensive; and FISH and qPCR can detect only a limited number of targets. Therefore, it is imperative to develop methods that are more user-friendly and have increased multiplexing capabilities to detect copy number variations (CNV). Using a novel digital barcode technology, NanoString Technologies (Seattle, WA) has developed the nCounter® technology to meet these needs. This new technology will be especially valuable after the recent FDA approval of the ProsignaTM assay, a breast cancer gene signature assay for subtype classification to help guide treatment decisions. In the BioPharma group at Genoptix, Inc., Carlsbad, CA, we have started to assess the nCounter technology for copy number determinations in a CLIA laboratory setting.
Methods: A cancer gene CNV panel composed of 55 genes was custom designed and evaluated. The CNV results of a subset of genes generated from our gene panel using the nCounter technology were compared with the results generated from qPCR, digital droplet PCR (ddPCR), FISH, publications, and NGS. Furthermore, assay robustness was tested by using different amounts and quality of input FFPE DNA. An initial comparison on the CNVs obtained from genomic DNA digested by Alu1 enzyme or sonicated by Bioruptor® Pico (Diagenode Inc, Denville, NJ). Finally, the effects of different DNA isolation methods on copy number determinations were also evaluated.
Results: Overall, the copy numbers generated using nCounter technology were concordant with results from the abovementioned methods as analyzed by comparing selected genes with known CNVs. A wide range of FFPE DNA input (200ng-4ug) could be tolerated by the assay. Based on a limited number of samples, DNA isolation methods, such as Qiagen, Promega Maxwell® CSC, or Roche Cobas®, have minimal effect on copy numbers.
Conclusions: With the short hands-on time, relative high level of automation, and no amplification requirements, nCounter technology is able to generate CNVs in good agreement with other established CNV methods. This new 55 cancer gene CNV assay may be useful in clinical trials to assist in patient stratification and care.
Citation Format: Tingdong Tang, Wenge Shi, Loretta Hipolito, Julie Mayer, Jelveh Lameh, Shabnam Tangri, Reinhold Pollner. Development of a nanostring copy number assay for a customized 55 gene panel using challenging formalin-fixed paraffin-embedded (ffpe) tumor samples. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3734. doi:10.1158/1538-7445.AM2014-3734
Abstract
Introduction: There is an urgent need for deep targeted sequencing for detection of varieties of somatic aberrations, including single nucleotide variants (SNV), insertions/deletions ...(indels), loss of heterozygosity (LOH), and copy number variants (CNV) in heterogeneous FFPE cancer patient specimens. We have developed a Next Generation Sequencing (NGS)-based assay for deep sequencing of a number of selected oncogenes and tumor suppressor genes for testing of clinical breast cancer samples. The panel includes those genes, which are commonly mutated in cancers (e.g. breast and ovarian cancer), based on The Cancer Genome Atlas studies and are potentially indicative of responses to experimental compounds undergoing clinical trials. Importantly, the assay can be used in FFPE samples which pose challenges because of high variability in quality and often suboptimal quantities.
Methods: To minimize FFPE sample input requirement, we optimized multiple assay steps. DNA was extracted from macrodissected FFPE tissue, and randomly fragmented. The DNA library was constructed and subject to target capture and enrichment before being sequenced on the Illumina MiSeq® systems. To analytically qualify the assays, we tested well-characterized cell lines and reference HapMap cell lines. Furthermore, to confirm the performance of the NGS results in clinical FFPE samples, we compared the results generated using orthogonal methodologies, e.g. SNVs using a custom Ion AmpliSeq™ panel (Life Technologies) on Ion Personal Genome Machine® (PGM), or CNVs using real-time PCR assays or custom Nanostring nCounter® CNV assays (NanoString Technologies). To establish the lower limit of detection, we used mixed DNA samples from FFPE cell lines and from HapMap DNA and further tested Horizon FFPE reference DNA containing mutations at defined allele frequencies.
Results: For FFPE DNA extraction, we selected Cobas® FFPE kit (Roche), and Maxwell® CSC DNA FFPE kit (Promega) for higher yield and better purity. For library construction, we chose a KAPA streamlined library preparation kit. For target capture, we used custom xGEN® DNA probes (Integrated DNA Technologies) for improved flexibility in assay optimization, reduced reagent cost, and minimizing batch-to-batch variability. We were able to detect SNVs for >99% of exons selected down to a 5% sensitivity level using about 100 ng FFPE DNA input. Indels, CNV and LOH could also be detected reliably.
Conclusions: We developed a targeted NGS assay that could be used in clinical trials and meets the dual challenge of limited DNA amounts and/or variable quality often associated with FFPE clinical specimens and the detection of low level mutations. Considering the recent recognition of Illumina NGS platform by FDA, this approach can represent a path forward to assist in cancer patient assessment, clinical trial recruitment and care.
Citation Format: Wenge Shi, Christine Chin, Tingdong Tang, Loretta Hipolito, Preethi Srinivasan, Derek Chiang, David Peng, Emmanuelle Di Tomaso, Shabnam Tangri, Jelveh Lameh, Reinhold Pollner. Development of a clinical targeted next generation sequencing test for challenging formalin-fixed paraffin-embedded (FFPE) cancer samples. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2014-1892
Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics.
Luminescent oxygen channeling assay (LOCI(TM)) latex ...particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types.
In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets.
This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.
The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two α1(IV) and one α2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear ...run-on analysis and mRNAs by RNase protection assay indicates the involvement of transcriptional as well as post-transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady-state levels α1(IV)/ α2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post-translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.
The cDNA and protein sequences of the N‐terminal 60% of the α2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides ...and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both α chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the α2(IV) chain as compared to the α1(IV) chain. 21 of these additional residues form a disulfide‐bridged loop within the triple helix which is unique among all known collagens.
Presently, growth-based tests are used for the detection and quantitation of microbiological contaminants in the environment. These tests take a minimum of 24 h to generate a result, which ...compromises the ability to take the most appropriate action. This report describes a rapid test for Enterococcus in recreational water as an indicator of faecal contamination. This method involves (1) isolation and lysis of the target organism, (2) purification of ribosomal RNA (rRNA) from the lysate and (3) amplification and detection of the purified rRNA. rRNA is used as the target since, in contrast to DNA, there are hundreds to thousands of copies in the cell. The rRNA is purified from the lysate by target capture onto magnetic microspheres, which removes interfering substances present in the sample. The rRNA is then quantitated using transcription-mediated amplification (TMA) with real-time homogeneous detection of amplicon using a fluorescent oligonucleotide probe. Compared to polymerase chain reaction (PCR) amplification, TMA is isothermal, more rapid, and ideally suited to RNA detection. The test described here demonstrates sensitive detection and quantitation of enterococci over a wide dynamic range with a high level of analytical specificity. The latter is particularly important for accurate and relevant monitoring both for protecting public health and for source tracking. Many conventional microbiological tests are time-consuming, exhibit limited dynamic range and are known to lack specificity. This assay demonstrates the advantages achievable by the application of TMA of rRNA targets to current environmental testing challenges.
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