NGS-based Method Research Articles

Validation of Next-Generation Sequencer for 24-Chromosome Aneuploidy Screening in Human Embryos.

Next-Generation Sequencing (NGS) is the latest approach for preimplantation genetic diagnoses (PGD). The purpose of this study was to standardize and validate an NGS method for comprehensive chromosome screening and to investigate its applicability to PGD. Embryo biopsy, whole-genome amplification, array comparative genomic hybridization (aCGH), and semiconductor sequencing were employed. A total of 204 whole-genome-amplified products were tested with an NGS-based protocol, from which 100 samples were used for standardization and to evaluate the quality of the results produced by this new technique. The remaining 104 samples tested by NGS were previously analyzed by using the aCGH protocol to determine the sensitivity and specificity of this new technique. In total, 4896 chromosomes were assessed, out of which 196 carried a copy number imbalance. NGS sensitivity and specificity for calling aneuploidy was 100%. This is the first study reporting preclinical validation and accuracy assessment of the Ion Torrent Personal Genome Machine (PGM) NGS-based comprehensive chromosome screening method using blastomeres and blastocysts. The NGS proved to be a robust methodology and is ready for clinical application in reproductive medicine, with the major advantage of low cost and enhanced precision when compared with other technologies used for comprehensive chromosome screening.

P179 Multi-center study on automating holotype HLA on a Biomek 4000

Aim Holotype HLA is a powerful NGS-based method for HLA genotyping being adopted by HLA laboratories over legacy technologies such as SSO and Sanger SBT for its increased accuracy, resolution, repeatability and high-throughput nature. The elimination of human and random error is a desirable attribute of any diagnostic test and the implementation of a liquid handling system can significantly reduce both sources of error in an NGS workflow. Development of a protocol that streamlines multiple modules of the NGS process was the goal of this multi-center study. Methods The Beckman Coulter Biomek 4000 automated liquid handler is a small footprint solution for pre- and post-PCR processes in NGS-based HLA typing. During development, the protocol was optimized through comparison of multiple data points at measurable stopping points to provide a customized modular liquid handling solution for any HLA lab. Results Technologists and scientists from three sites performed multiple runs of 24 samples for 11 HLA loci using the Holotype HLA 24/11 configuration automated on the Biomek 4000 to generate data on the accuracy, specificity, repeatability and reproducibility of an automated Holotype HLA solution. Conclusion Data obtained from the three centers were analyzed using Omixon HLA Twin and assessed for accuracy, reproducibility, and allele assignment ambiguities to demonstrate that high-throughput NGS-based HLA typing is achievable in under 48 h from gDNA to genotype using the Beckman Coulter Biomek 4000 modular based NGS protocol for Holotype HLA 24/11.

Mining sequence variations in representative polyploid sugarcane germplasm accessions

BackgroundSugarcane (Saccharum spp.) is one of the most important economic crops because of its high sugar production and biofuel potential. Due to the high polyploid level and complex genome of sugarcane, it has been a huge challenge to investigate genomic sequence variations, which are critical for identifying alleles contributing to important agronomic traits. In order to mine the genetic variations in sugarcane, genotyping by sequencing (GBS), was used to genotype 14 representative Saccharum complex accessions. GBS is a method to generate a large number of markers, enabled by next generation sequencing (NGS) and the genome complexity reduction using restriction enzymes.ResultsTo use GBS for high throughput genotyping highly polyploid sugarcane, the GBS analysis pipelines in 14 Saccharum complex accessions were established by evaluating different alignment methods, sequence variants callers, and sequence depth for single nucleotide polymorphism (SNP) filtering. By using the established pipeline, a total of 76,251 non-redundant SNPs, 5642 InDels, 6380 presence/absence variants (PAVs), and 826 copy number variations (CNVs) were detected among the 14 accessions. In addition, non-reference based universal network enabled analysis kit and Stacks de novo called 34,353 and 109,043 SNPs, respectively. In the 14 accessions, the percentages of single dose SNPs ranged from 38.3% to 62.3% with an average of 49.6%, much more than the portions of multiple dosage SNPs. Concordantly called SNPs were used to evaluate the phylogenetic relationship among the 14 accessions. The results showed that the divergence time between the Erianthus genus and the Saccharum genus was more than 10 million years ago (MYA). The Saccharum species separated from their common ancestors ranging from 0.19 to 1.65 MYA.ConclusionsThe GBS pipelines including the reference sequences, alignment methods, sequence variant callers, and sequence depth were recommended and discussed for the Saccharum complex and other related species. A large number of sequence variations were discovered in the Saccharum complex, including SNPs, InDels, PAVs, and CNVs. Genome-wide SNPs were further used to illustrate sequence features of polyploid species and demonstrated the divergence of different species in the Saccharum complex. The results of this study showed that GBS was an effective NGS-based method to discover genomic sequence variations in highly polyploid and heterozygous species.

Prognostic value of sequencing-based minimal residual disease detection in patients with multiple myeloma who underwent autologous stem-cell transplantation.

BackgroundMost patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper response are required.Patients and methodsWe retrospectively analyzed 125 patients with MM who underwent high-dose melphalan plus autologous stem-cell transplantation (ASCT) to detect MRD in autograft/bone marrow (BM) cells using a next-generation sequencing (NGS)-based method and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR).ResultsNGS-based method was applicable to 90% and this method had at least one to two logs greater sensitivity compared to ASO-PCR. MRD negative by NGS [MRDNGS(−)] (defined as <10−6) in post-ASCT BM cases (n = 26) showed a significantly better progression-free survival (PFS) (96% at 4 years, P < 0.001) and overall survival (OS) (100% at 4 years, P =0.04) than MRDNGS(+) in post-ASCT BM cases (n = 25). When restricting the analysis to the 39 complete response cases, patients who were MRDNGS(−) (n = 24) showed a significantly better PFS than those that were MRDNGS(+) (n = 15) (P =0.02). Moreover, MRDNGS(−) in post-ASCT BM cases (n = 12) showed significantly a better PFS than MRDNGS(+) cases (n = 7) where MRD was not detected by ASO-PCR (P = 0.001). Patients whose autografts were negative by NGS-based MRD assessment (<10−7) (n = 19) had 92% PFS and 100% OS at 4 years post-ASCT. Conversely, the NGS-based MRD positive patients who received post-ASCT treatment using novel agents (n = 49) had a significantly better PFS (P = 0.001) and tended to have a better OS (P= 0.214) than those that were untreated (n = 33).ConclusionsLow level MRD detected by NGS-based platform but not ASO-PCR has significant prognostic value when assessing either the autograft product or BM cells post-ASCT.

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

BackgroundCytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material.MethodsWe established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species.ResultsAs a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice.ConclusionsPlant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

Investigation of Genetic Defects in Severe Combined Immunodeficiency Patients from Turkey by Targeted Sequencing

Primary immunodeficiencies (PIDs) represent a large group of disorders with an increased susceptibility to infections. Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiencies (PIDs) with marked T-cell lymphopenia. Investigation of the genetic aetiology using classical Sanger sequencing is associated with considerable diagnostic delay. We here established a custom-designed, next-generation sequencing (NGS)-based panel to efficiently identify disease-causing genetic defects in PID patients and applied this method in SCID patients of Turkish origin with previously undefined genetic aetiology. We used HaloPlex enrichment technology, a targeted, NGS-based method which was designed to diagnose patients with SCID and other PIDs. Our HaloPlex panel included a total of 356 PID-related genes, and we searched disease-causing mutations in 19 Turkish SCID patients without a genetic diagnosis. The coverage of targeted regions ranged from 97.47% to 99.62% with an average of 98.31% for all patients. All known SCID genes were covered with a percentage of at least 97.3%. We made a genetic diagnosis in six of 19 (33%) patients, including four novel disease-causing mutations identified in RAG1, JAK3 and IL2RG, respectively. We showed that this NGS-based method can provide rapid genetic diagnosis for patients suffering from SCID, potentially facilitating clinical treatment decisions.

Abstract P6-07-08: The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples

Abstract Background: Approximately 70% of newly diagnosed breast cancers express estrogen receptor alpha (ERα), and are treated with agents that block ER signaling. Acquired mutations in ESR1, the gene that encodes ERα, have been associated with resistance to aromatase inhibitor therapy in patients with ER positive metastatic breast cancer (ER+ mBC). The most frequently occurring ESR1 mutations are clustered between amino acids 536 to 538 within the ligand binding domain (LBD), although limited data exists characterizing the full mutation profile in a large number of breast cancer samples. Methods: We surveyed the Foundation Medicine dataset of 7590 primary and metastatic breast cancer tumor samples for ESR1 short variants and copy number alterations. Hormone receptor status was unavailable, therefore two assumptions were made to provide an estimate of prevalence in the ER+ HER2- population: 70% of the tumor samples are from ER+ HER2- patients, and all ESR1 mutations from non-HER2 amplified metastatic sites are from ER+ HER2- patients. In a separate cohort of 48 ER+ mBC patients, circulating tumor DNA (ctDNA) was analyzed for ESR1 mutations using the BEAMing method by Sysmex and with Foundation Medicine's sequencing assay, FoundationACT (Assay for Circulating Tumor DNA). Results: The prevalence of mutations in ER+ HER2- breast cancer was estimated to be 22% in samples from metastatic sites but less than 3% in samples from primary sites. ESR1 amplification was rare in samples from both primary and metastatic disease sites at 1.3% and 2.0% respectively. A total of 153 unique short variants of known and unknown status were identified. In addition to hotspot mutations at 537 and 538, previously undescribed rare mutations were identified throughout the entire length of the LBD, although 10 alterations at amino acids 380, 463, 536, 537, and 538 account for 86% of all ESR1 mutations in the ER+ HER2- metastatic sites. We also characterized the overlap of ESR1 alterations with commonly altered and clinically relevant genes in breast cancer, including PIK3CA mutations and HER2 amplification, and we report here a landscape of co-occurring alterations. In the cohort of patient samples where ctDNA was analyzed, BEAMing and FoundationAct assays both detected ESR1 mutations in 19 out of 48 samples, and overall concordance of mutation status (wild-type vs mutant) was 100%. A total of 51 individual mutations were detected with the BEAMing assay, 42 of which were detected with the FoundationACT assay. Seven mutations that were undetected by FoundationACT had mutant allele frequencies less than 0.1%. Ten ESR1 mutations were detected only by FoundationACT, 9 of which are not covered with the BEAMing assay. Alterations in PIK3CA, CDH1, TP53, ERBB2, and other breast cancer relevant genes were also detected with FoundationACT. Conclusions: Understanding the mutational landscape of ESR1 and co-occurring alterations is important for diagnostic development in conjunction with the clinical development of novel anti-endocrine therapies. Our data demonstrate a large spectrum of mutations in the LBD in addition to known hotspot mutations. In addition, the FoundationACT assay offers a robust NGS-based method to screen for mutations in ctDNA that is highly concordant with digital PCR methods. Citation Format: Spoerke JM, Schleifman E, Clark TA, Young G, Nahas M, Kennedy M, Young L, Chmielecki J, Otto GA, Lipson D, Wilson TR, Gendreau S, Lackner MR. The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-08.

Accurate detection of segmental aneuploidy in preimplantation genetic screening using targeted next-generation DNA sequencing

To evaluate the sensitivity and specificity of segmental aneuploidy detection as a function of segmental copy number variant (CNV) size using EmbryVu, a targeted next-generation DNA sequencing (NGS) assay. We have previously described a targeted NGS-based method for preimplantation genetic screening (PGS) that has been validated for accurate detection of full chromosome aneuploidy (Gole et al., 2016). The EmbryVu test leverages the FAST-SeqS NGS technology to target tens of thousands of sites across the genome and therefore has the potential to detect sub-chromosomal or segmental aneuploidy. To measure the specificity and sensitivity of this test with respect to segmental aneuploidy, we first tested cell line-derived samples known to contain specific segmental CNVs of varying sizes as well as known negative samples. Next, to evaluate performance with respect to segmental CNVs falling in parts of the genome for which samples were not available, we developed and implemented a simulation strategy that enables genome-wide assessment of automated segmental calling sensitivity and specificity. Cell-line derived genomic DNA from 138 cell lines with known segmental duplication/deletions ranging in size from 0.1 to ∼80 Mb and 170 known normal samples were run through the EmbryVu assay and the resulting molecular karyotype calls were compared to the previously reported karyotypes. Next, using data derived from trophectoderm biopsies from embryos that were classified as normal on both EmbryVu and microarray comparative genomic hybridization, we simulated segmental duplications of varying sizes across the entire genome and evaluated the frequency at which these duplications were called. For the cell-line derived samples, 112 of the 115 expected segmental copy number variants of size at least 10 Mb were called, yielding a sensitivity estimate of 0.974 (0.926,0.991). A total of 6 samples were falsely classified as aneuploid, yielding a specificity estimate of 0.965 (0.925,0.984), an estimate that was very similar to that obtained in our embryo-based simulation studies - 0.968 (0.909, 0.989). In addition, our simulations illustrate that while there do exist positions in the genome where segmental detection sensitivity is reduced, these regions often coincide with heterochromatic and/or highly repetitive regions that are likely problematic with most PGS technologies. The EmbryVu PGS test, using the FAST-SeqS targeted NGS technology, is capable of accurately detecting sub-chromosomal changes of 10 Mb and larger with an analytic sensitivity of >97% and a specificity that is comparable to that noted in our previous validation study. Comprehensive genome-wide simulations further expand upon our understanding of the performance of this test.

Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy

Conventional PCR methods combined with linkage analysis based on short tandem repeats (STRs) or Karyomapping with single nucleotide polymorphism (SNP) arrays, have been applied to preimplantation genetic diagnosis (PGD) for spinal muscular atrophy (SMA), an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild-type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) is a new method allowing embryo selection by a one-step next-generation sequencing (NGS) procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion (carriers) from the wild-type (normal) ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos (homozygous deletion), which can be regarded as probands for linkage analysis, in case that the affected family member is absent. In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.

Quantitative Assessment of Circulating Clonal IG-VDJ Sequences in Plasma of Follicular Lymphoma at Diagnosis Is Highly Predictive of Progression Free Survival (PFS)

Background:Recent advances in next-generation sequencing (NGS) techniques enable the quantitation of circulating tumor DNA (ctDNA) encoding the clonal rearranged V(D)J immunoglobulin (IG) receptor gene sequence. The aims of this study were 1) To evaluate the clonal heterogeneity of follicular lymphoma (FL) and its variations between tumor and plasma at diagnosis in patients (pts) included in PRIMA trial, and 2) To assess the prognostic value of the level of ctDNA at diagnosis on PFS (median follow up of 6 years).Methods:Using the NGS-based immunosequencing method (Adaptive Biotechnologies, South San Francisco, CA), the lymphoma clonotype was first established in the tumor biopsy using locus-specific primer sets for IGH-V, IGH-D and IGK rearrangements. Clonotypes regarded as originating from the lymphoma clone were present at a frequency greater than 5%. The lymphoma-derived sequences identified in the biopsy sample were then used as targets to assess the presence of ctDNA in plasma samples. The quantity and frequency of the ctDNA in plasma is calculated relative to the total number of reads in the sample and defined as lymphoma clonotype molecules per million diploid genomes. For 34 FL pts from the PRIMA trial, both tumor biopsy and plasma samples at diagnosis were available.Results:One tumor clonotype or more could be detected in 29 pts (85%) in the tumor diagnostic sample (DNA isolated from fresh frozen tissue). Indeed, with the IGH-V assay, between 1 to 3 different clonotypes were identified in 23 pts. With the IGH-D assay, 1 clonotype was detected in 2 pts. With the IGK assay, 1 to 2 different clonotypes were detected in 17 pts. The tumor clonotypes were detected in diagnostic plasma samples in 25 out of the 29 pts (83%). Moreover, in 14 pts we detected clones in the plasma samples that are related (with point mutations in the V(D)J sequence) to the highest frequency index clone identified in the tumor sample. In half of the cases, we observed a different repartition of the subclonal populations between tumor and plasma samples at diagnosis: for these cases the highest frequency clone in the tumor was not the highest frequency clone in the plasma.Among the 24 pts with an IGH clonotype (IGH-V or IGH-D assay), ctDNA could be detected in 19 cases. We could not find a correlation between peripheral blood dissemination and the presence of a stereotyped sequence or an N-glycosylation motif within the CDR3 region of IGH clonotypes. Interestingly, in one case, two different related clones were detected at high frequency in tumor sample and the putative parental clone, not detected in the tumor sample, was detected in plasma sample as the highest frequency clone.The level of ctDNA ranged from 0 to 345,000/million diploid genomes in the 29 plasma samples (median 39,720). The presence of ctDNA was correlated with bone marrow involvement (p=.005), but not with FLIPI score, LDH level, anemia, bulky disease, presence of circulating lymphoma cells (detected by morphology or flow examination), Ann Arbor stage or β2microglobulin level. The absolute level of ctDNA in diagnostic samples did not correlate with any of the clinical characteristics. We assessed the prognostic value of the level of ctDNA at diagnosis. Pts (14/29) with higher levels of ctDNA at diagnosis (>40,000/million diploid genomes) had shorter PFS than patients with lower levels of ctDNA (median 15.7 m vs NR, p=.027, Fig 1). In an exploratory multivariate Cox regression model including FLIPI score, bulky disease and β2-microglobuline level, high ctDNA level was the only factor significantly associated with a worse PFS (HR 4.2, p=.039). In the observation arm, pts with high ctDNA levels had a median PFS of only 11.6 months vs NR (p=.01). Whereas in the maintenance arm, the prognostic value of elevated ctDNA levels was erased (p=.6).Conclusion:Using the NGS-based immunosequencing method, we were able to show a variation in clonal heterogeneity between tumor biopsy and plasma samples obtained at diagnosis. The poor prognostic value of elevated ctDNA levels in plasma at diagnosis appeared higher than other clinical parameters in a multivariate cox regression model. These findings need to be validated in larger cohorts. [Display omitted] DisclosuresCarlton:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:adaptive biotech: Employment, Other: stockholders. Salles:Roche: Honoraria, Research Funding.

Assessment of Central Nervous System Involvement in Pediatric Acute Lymphoblastic Leukemia Patients Using Next-Generation Sequencing Method

Background: Successful outcome in childhood acute lymphoblastic leukemia (ALL) relies upon appropriate central nervous system (CNS)-directed therapy for treatment of subclinical or overt CNS leukemia. Patients with leukemia blasts detected in the cerebrospinal fluid (CSF) at diagnosis have poorer survival compared with patients without blasts in the CSF and require intensified intrathecal therapy to avert a higher rate of relapse. Traditionally, CSF assessment is performed by morphological analysis of CSF smears and the blasts confirmed by immunohistochemistry or flow cytometry. The confirmation tests are done at few institutions and rely on subjective operator judgment for identification of leukemic blasts. A precise definition of CNS involvement is necessary to avoid over- or under- treatment. Next-generation sequencing (NGS) is currently being used for minimal residual disease (MRD) assessment in lymphoid malignancies (Faham et al., Blood 2012). In this study, we assessed whether the NGS method could be used to detect leukemic clonotypes in CSF samples from 89 newly diagnosed pediatric ALL patients.Methods: Diagnostic bone marrow samples and paired CSF samples were obtained from 89 patients. CSF samples were collected in one tube and fractionated for Wright stained cytospins (1ml), TdT immunohistochemistry (1ml), and the remainder (3-5ml) for NGS-based MRD studies (Adaptive Biotechnologies, South San Francisco, CA). Briefly, using universal primer sets, we amplified variable, diversity, and joining gene segments from immunoglobulin (Ig) heavy chain (IGH), Ig kappa chain (IGK), and T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) loci from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then quantitated in CSF (cell pellet and supernatant) samples obtained at diagnosis. A quantitative and standardized measure of leukemic clonotype level per million leukocytes in each follow-up sample was determined.Results: Diagnostic bone marrow samples from 89 pediatric ALL patients were used to identify the tumor-specific clonotypes. At least one tumor-specific clonotype was identified in 86/89 (97%) of the patients. The IGH-VDJ assay was the most frequent gene rearrangement: at least one IGH-VDJ clonal rearrangement was detected in 66 of the diagnostic ALL samples. TRD was the second most informative receptor with a clonal rearrangement found in 43 patients, followed by TRG in 38 patients, IGK in 31 patients, TCB in 16 patients and IGH-DJ in 13 patients. The level of tumor burden associated with each leukemic clonotype was then assessed in CSF samples drawn at diagnosis from each of the 86 patients. All 40 CNS1 patients had no detectable or minimal (<10) leukemic cell equivalents. Of the 24 patients with CNS2 but TDT negative status, only 2 had >10 leukemic cell equivalents in the CSF. By contrast, 8 of 20 (40%) patients with CNS2 and TDT positive status and both patients with CNS3 had CSF with >10 leukemic cell equivalents (Figure 1).Conclusions: This study demonstrates that the NGS-based method can be used to assess CNS involvement in pediatric patients with ALL. Based on these results, NGS-based CNS assessment will be used for patient stratification in future clinical trials for pediatric ALL. [Display omitted] DisclosuresZheng:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Carlton:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

BCR-ABL Mutations in Chronic Myeloid Leukemia (CML) Patients (pts) with Failure and Warning to First- and Second-Line Tyrosine Kinase Inhibitor (TKI) Therapy: What Is the Advantage of Next-Generation Sequencing (NGS) over Conventional Sequencing?

BCR-ABL Mutations in Chronic Myeloid Leukemia (CML) Patients (pts) with Failure and Warning to First- and Second-Line Tyrosine Kinase Inhibitor (TKI) Therapy: What Is the Advantage of Next-Generation Sequencing (NGS) over Conventional Sequencing?

Abstract 4916: Development of a NGS-based method for EGFRvIII detection: sequence analysis of the junction

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary tumor in humans. One of the most common mutations in GBMs is an interstitial deletion in the epidermal growth factor receptor (EGFR), EGFRvIII, which occurs at a frequency of ∼30%. EGFR is a transmembrane tyrosine kinase receptor and the EGFRvIII mutant is characterized by a deletion of 267 amino acids in the extracellular domain leading to ligand independent constitutive activation. The deletion of exons 2-7 leads to an in-frame deletion in EGFR with a novel glycine residue at the junction. The amino acid at the junction of exons 1 and 2 is a valine, making the novel transcript an attractive target for immunotherapy. A custom next generation sequencing (NGS) based assay and bioinformatic pipeline have been developed in our laboratory to detect EGFRvIII from RNA extracted from formalin fixed paraffin embedded tissue. The targets include the exon 1-2 boundary (wild type), the exon 1-8 boundary (EGFRvIII), amplification of various sized RNA fragments to determine RNA degradation and bioavailability, and expression levels of three housekeeping genes. Following cDNA synthesis multiplex PCR of all targets are captured simultaneously for the sequencing library with NGS performed on the Illumina MiSeq. The output from the bioinformatics pipeline includes the sequence and number of reads from the wild-type and mutant, ratio of EGFRvIII reads with respect to total EGFR sequenced, expression of three housekeeping genes and relative amount of bioavailable EGFR RNA. This assay was validated through comparison of NGS sequence results with an established qRT-PCR to detect normal and mutant EGFR. Negative controls from normal brain (temporal lobe excisions from epilepsy patients) and adipose tissue (a tissue with high expression of EGFR) were used to determine whether low-level exon 1-8 fusions from mis-splicing were detectable in normal tissue (Figure 1). Twenty five GBM specimens were sequenced, with 8/25 positive for EGFRvIII (Figure 2), and confirmed by RT-PCR. In addition to detection of the EGFRvIII mutant, relative expression of EGFR is detected in this assay, and when taken together with EGFR amplifications detected by routine NGS panels, we can determine whether the EGFRvIII is present on the amplified or unamplified allele and whether additional mutations are detectable. Detection of EGFRvIII utilizing NGS improves the precision of mutant detection to better serve CART-EGFRvIII clinical trial to ensure the target is present. The NGS assay provides the EGFRvIII/wild-type ratio, relative expression levels for EGFR and EGFRvIII and evaluation of RNA degradation in a single assay. Figure 1A. Baseline in normal samples. EGFRvIII ratio in 18 “normal” brain and 11 adipose tissue samples, plotted without (top panel) and with (bottom panel) a EGFRvIII positive sample. Figure 2. EGFRvIII ratio in 25 GBM samples. Cutoff for EGFRvIII positive is EGFRvIII ratio of 0.3 (30%). Citation Format: Jianhua Zhao, Shrey Sukhadia, Alan Fox, David Lieberman, Barnett Li, Robert D. Daber, Matthew C. Hiemenz, David B. Roth, Maria Martinez-Large, Arati Desai, Donald M. O'Rourke, Marcela V. Maus, Jennifer JD Morrissette. Development of a NGS-based method for EGFRvIII detection: sequence analysis of the junction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4916. doi:10.1158/1538-7445.AM2015-4916

Abstract 5237: Monitoring minimal residual disease by urinary or plasma circulating tumor DNA of KRAS mutation burden in colorectal cancer patients with resectable liver metastases

Abstract Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer deaths. Over half of patients with CRC will develop liver metastases. Surgical resection, in combination with systemic therapies, greatly improves long-term outcomes, and around 40% of patients with resected liver limited disease are alive 5 years after diagnosis. While tumor staging and radicality of surgery are commonly used for prognostic assessment, better non-invasive markers are needed for monitoring chemo-responsiveness, following minimal residual disease (MRD), and guiding complex treatment decisions in these patients. This study evaluated the utility of quantitating KRAS mutation burden in urinary and plasma ctDNA as a means of monitoring MRD in surgical CRC patients with liver limited metastases. Methods: We developed a novel, NGS-based method for enrichment and quantitative detection of KRAS mutations in urinary and plasma ctDNA. A blinded retrospective study was conducted on 20 Stage I-IV CRC patients, 15 of whom had undergone curative or palliative intent surgical resection of primary tumor or liver metastases in combination with various systemic therapies. Results: Archived, matched urine and plasma samples were collected from 20 patients with KRAS positive primary tumor. For operable patients, specimens were collected prior to surgery, during and immediately after surgery, plus additional time points post-surgery. A total of 193 plasma and urine samples archived for 3-5 years were tested. All 101 of 101 plasma samples (100%) and 79 of 92 urine samples (86%) had sufficient DNA and were deemed evaluable. In a blinded analysis, a correct KRAS mutation that correlated with KRAS mutation in tissue was identified in 95% of evaluable baseline plasmas (19 of 20) and 92% of evaluable baseline urines (11 of 12). In one patient, a KRAS mutation distinct from that identified in the tumor biopsy was detected consistently across all serial ctDNA samples. Overall, we observed a clear correlation and highly comparable fold change between plasma and urinary ctDNA KRAS levels on treatment. Significantly, in all patients with curative intent surgery, ctDNA KRAS levels were undetectable in urine or plasma after surgery. In contrast, in 8 of 10 patients with incomplete, palliative surgery, the ctDNA KRAS signal remained detectable or increased after surgery. Further correlation between ctDNA KRAS and clinical outcomes will be discussed. Conclusion: We demonstrate for the first time that quantitative changes of mutational KRAS burden in plasma and urinary ctDNA are highly correlated. We further demonstrate clinical applicability of urinary ctDNA KRAS analysis for monitoring quantitatively, with single molecule sensitivity, the MRD post-surgery for CRC patients with liver resectable metastases. Supported by Grant no. 13660. Citation Format: Vlada Melnikova, Jason C. Poole, Cecile Rose T. Vibat, Lucie Benesova, Barbora Belsanova, Saege Hancock, Latifa Hassaine, Errin Samuelsz, Timothy T. Lu, Mark G. Erlander, Marek Minarik. Monitoring minimal residual disease by urinary or plasma circulating tumor DNA of KRAS mutation burden in colorectal cancer patients with resectable liver metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5237. doi:10.1158/1538-7445.AM2015-5237

T-cell clonality assessment by next-generation sequencing improves detection sensitivity in mycosis fungoides

T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction-based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF.

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing.

BackgroundHLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.ResultsWe developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.ConclusionsIn this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users.

Targeted next-generation sequencing for the genetic diagnosis of dysferlinopathy

Dysferlinopathy comprises a group of autosomal recessive muscular dystrophies caused by mutations in the DYSF gene. Due to the large size of the gene and its lack of mutational hot spots, analysis of the DYSF gene is time-consuming and laborious using conventional sequencing methods. By next-generation sequencing (NGS), DYSF gene analysis has previously been validated through its incorporation in multi-gene panels or exome analyses. However, individual validation of NGS approaches for DYSF gene has not been performed. Here, we established and validated a hybridization capture-based target-enrichment followed by next-generation sequencing to detect mutations in patients with dysferlinopathy. With this approach, mean depth of coverage was approximately 450 fold and almost all (99.3%) of the targeted region had sequence coverage greater than 20 fold. When this approach was tested on samples from patients with known DYSF mutations, all known mutations were correctly retrieved. Using this method on 32 consecutive patient samples with dysferlinopathy, at least two pathogenic variants were detected in 28 (87.5%) samples and at least one pathogenic variant was identified in all samples. Our results suggested that the NGS-based screening method could facilitate efficient and accurate genetic diagnosis of dysferlinopathy.

Minimal Residual Disease Detection Using High-Throughput Sequencing Predicts Clinical Outcome in Patients with Pediatric B-Lineage Acute Lymphoblastic Leukemia

Background: Minimal residual disease (MRD) assessment at the end of induction is critical for risk stratification of B-lineage acute lymphoblastic leukemia (B-ALL) patients. There are multiple techniques for molecular MRD assessment, including next-generation sequencing (NGS) and allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin (Ig) or T-cell receptor rearrangements. Leukemia-specific rearrangements can be identified in >97% of B-ALL patients using the NGS-based LymphoSIGHT™ method, while ASO-PCR can typically identify leukemia-specific rearrangements in only 80-90% of B-ALL patients. In this report, we assessed the ability of the NGS method to predict event-free survival (EFS) in a cohort of B-ALL patients who were inevaluable by ASO-PCR.Methods: All patients were treated between 2005-2011 on a clinical trial conducted by the Dana-Farber Cancer Institute (DFCI) ALL Consortium (05-001) on which high levels of end-induction MRD (≥10-3) led to intensified treatment. The 37 pediatric B-ALL patients included in this study were inevaluable by ASO-PCR, and therefore this cohort represents a subset of patients in whom a decision to intensify therapy per protocol could not be made. Archived DNA samples from diagnosis and Day 32 were provided for MRD assessment using the NGS method. Using universal primer sets, we amplified the immunoglobulin (IGH-VDJ, IGH-DJ and IGK) and T cell receptor (TRB, TRD, TRG) loci in diagnostic bone marrow samples. Leukemia-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell and/or T-cell repertoire. The presence of leukemia-specific clonotypes was then assessed in the end-induction (Day 32) sample. NGS MRD assessment was performed blinded to all clinical and ASO-PCR information on these patients. The association of NGS MRD results and clinical outcome (EFS) was examined, and significance assessed using the log-rank test.Results: Of 37 patients who were inevaluable for end-induction MRD by ASO-PCR, leukemia-specific clonotypes were successfully identified in 35 (95%) by the NGS method. The use of a threshold of 10-3, which is the ASO-PCR threshold used for risk-stratification on DFCI protocols, demonstrated significantly improved EFS between the MRD negative and MRD positive groups (p<0.0001) (Figure 1A). A similar analysis with a threshold of 10-4 also demonstrated significantly improved EFS between the MRD negative and MRD positive groups (p<0.0001) (Figure 1B).Conclusions: This study demonstrates the prognostic significance of NGS-based MRD assessment in pediatric patients with B-ALL. This NGS-based assay led to successful assessment of MRD in 95% of patients who were inevaluable by ASO-PCR. The NGS method represents an alternative approach for clinical MRD monitoring that could fundamentally improve the ability to identify high-risk B-ALL patients and potentially improve their outcome. [Display omitted] [Display omitted] DisclosuresZheng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique

▪IntroductionMRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background.AimsTo develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints.MethodsTwo round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files.For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution.ResultsWe sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed.DiscussionWe present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks.The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis.Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-76: Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool

Abstract Circulating tumor DNA (ctDNA) analysis requires absolute single molecule sensitivity, since only a few copies of the mutation may be present per blood draw. Recent developments in next-generation sequencing (NGS) sample preparation have improved the relative sensitivity to detect &amp;lt;0.01% variants in background. However, when the total number of input mutant molecules falls below 10, these improvements have not translated into the absolute sensitivity required to sequence these rare templates. Here we develop and present SPLITSEQ, a novel microfluidics and NGS-based method that combines 0.01% fractional sensitivity with absolute single copy input sensitivity in an excess of background DNA. We provide multiplexed performance data on mutations in KRAS codons 12 and 13 and BRAF V600E, targets that are currently being developed as circulating predictive and prognostic biomarkers in colorectal and other cancers. Using reconstituted DNA solutions from mutant and normal cell lines ranging from 0.01% to 10% mutated allele fractions, we detected the sequence of approximately 0.2, 2, and 20 nominal mutated KRAS and BRAF inputs total in 10 000X background. Single copy numbers were supported by droplet digital PCR (ddPCR) data, an orthogonal single-molecule sensitive technique. Lastly, we confirm the specificity of the technique by screening wild-type DNA only and finding a false positive rate significantly lower than ddPCR. Moreover, in comparison with digital PCR, SPLITSEQ is easily amenable to high levels of multiplexing and detects non-recurrent mutations in tumor suppressors along the length of the sequence. Since the only requirement is a single PCR-amplifiable template, SPLITSEQ fills an unmet need in ctDNA detection, providing biomarkers from hereto unanalyzable liquid biopsies containing rare mutants. Citation Format: Ronald Lebofsky, Jordan Madic, Adrien Saliou, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Jean-Yves Pierga. Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool. [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 LB-76. doi:10.1158/1538-7445.AM2014-LB-76