Next-generation Sequencing Kit Research Articles

Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the ‘separating’ and ‘merging’ strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Assessing the performance and utility of targeted next-generation sequencing for screening and genotyping of human papillomaviruses

A next-generation sequencing (NGS)-based Ezplex HPV NGS kit (SML Genetree, Seoul, Korea) was used for human papillomavirus (HPV) screening. Of 885 cervical swab samples, HPV was detected in 162 samples. High-risk HPVs were detected in 82 samples, and other types of HPV were detected in 13 samples (HPV86, 71, 102, 91, and 114). At the read depth ≥ 500, NGS results exhibited 100 % agreement among repeated tests. HPV NGS results were compared with those of real-time PCR assays, Anyplex HPV28 (Seegene, Seoul, Korea) (n = 383) and Cobas HPV (Roche, Mannheim, Germany) (n = 64); concordances were 92.4 % and 95.0 %, respectively. Sanger sequencing of discordant results (n = 13) produced compatible results with those of HPV NGS. Pap smear abnormalities were detected in 31 patients (3.5 %), and 19 patients had high-risk HPV. Using HPV NGS for screening, rare HPV subtypes were detected, and quantitative values were obtained as read depth.

B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing

Abstract Background High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods. Methods A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda). Results Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results. Conclusion NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.

Next-Generation Sequencing for Screening Analysis of Cystic Fibrosis: Spectrum and Novel Variants in a South-Central Italian Cohort.

The incidence of cystic fibrosis (CF) and the spectrum of cystic fibrosis transmembrane conductance regulator (CFTR) gene variants differ among geographic regions. Differences in CF carrier distribution are also reported among Italian regions. We described the spectrum of the CFTR variants observed in a large group of subjects belonging from central-southern Italy. We also provide a predictive evaluation of the novel variants identified. CFTR screening was performed in a south-central Italian cohort of 770 subjects. We adopted a next-generation sequencing (NGS) approach using the Devyser CFTR NGS kit on the Illumina MiSeq System coupled with Amplicon Suite data analysis. Bioinformatics evaluation of the impact of novel variants was described. Overall, the presence of at least one alternative allele in the CFTR gene was recorded for 23% of the subjects, with a carrier frequency of CF pathogenic variants of 1:12. The largest sub-group corresponded to the heterozygous carriers of a variant with a conflicting interpretation of pathogenicity. The common CFTR p.(Phe508del) pathogenic variants were identified in 37% of mutated subjects. Bioinformatics prediction supported a potential damaging effect for the three novel CFTR variants identified: p.(Leu1187Phe), p.(Pro22Thr), and c.744-3C > G. NGS applied to CF screening had the benefit of: effectively identifying asymptomatic carriers. It lies in a wide overview of CFTR variants and gives a comprehensive picture of the carrier prevalence. The identification of a high number of unclassified variants may represent a challenge whilst at the same time being of interest and relevance for clinicians.

Corded and hyalinized endometrioid endometrial carcinoma with high-grade features: a clinicopathological and TCGA-based molecular analysis.

Corded and hyalinized endometrioid carcinoma (CHEC) typically shows low-grade features and "no specific molecular profile" (NSMP). This study aimed to perform a clinicopathological and molecular characterization of endometrial CHEC with high-grade features. Immunohistochemistry for cytokeratin AE1/AE3, e-cadherin, β-catenin, estrogen receptor, progesterone receptor, p53, p16, and mismatch repair proteins was performed. A next-generation sequencing kit was used to assess POLE, POLD1, APC, MLH1, MSH2, MSH6, PMS2, MUTYH, EPCAM, and CTNNB1. Molecular groups, i.e., POLE-mutant, mismatch repair deficient (MMRd), p53-abnormal, and NSMP, were assigned according to the TCGA classifier. Six high-grade endometrial CHECs were identified. The mean age was 57.5years; 5/6 cases were uterine-confined. Five cases showed a diffusely and markedly atypical corded component and a MMRd or p53-abnormal signature; additional features included single-cell keratinization, necrosis, osteoid or myxoid/chondro-myxoid matrix, foci of anaplasia, and nuclear β-catenin expression. The remaining case showed a low mitotic count and a NSMP phenotype, with focal bizarre cells in an otherwise classical CH endometrioid carcinoma. All cases showed variably reduced expression of epithelial markers and hormone receptors in the corded component. No mutations were found in any of the analyzed genes. In conclusion, high-grade CHECs are a heterogeneous subset of biphasic endometrial carcinoma which show similarities and differences with classical CHEC and carcinosarcoma. These cases often show MMRd or p53-abnormal signatures.

925P Analytical performance of a next-generation sequencing (NGS) assay kit for assessing homologous recombination deficiency (HRD) from solid tumor samples

HRD status is predictive of response to PARP inhibitors (PARPi) in ovarian cancer. Developing reliable methods to assess HRD status is important to enable genomic profiling of tumor types with PARPi indications. Here we describe the analytical performance of the TruSight™ Oncology 500 HRD (TSO 500 HRD) research assay, an in-development NGS kit for assessing HRD using DNA from solid tumor samples. The TSO 500 HRD assay was developed by adding an HRD probe pool targeting genome-wide single nucleotide polymorphisms (SNPs) to the TruSight Oncology 500 (TSO 500) assay to enable Genomic Instability Score (GIS) assessment. For each sample, the DNA library was split into two hybridization reactions, one with TSO 500 probes and the other with HRD probes. Both libraries were pooled for sequencing with 8 samples per NextSeq™ 550 run. The GIS algorithm was licensed from Myriad, re-implemented as part of the DRAGEN Bio-IT software suite and integrated into the TSO 500 analysis workflow. Forty nanograms of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples was used as assay input. Samples were also tested with a reference assay as the orthogonal test. Testing of serial dilutions of 8 ovarian cancer samples showed the limit of detection for small variant calling in BRCA1/2 genes is at 5-10% variant allele frequency (VAF), for large rearrangement variant calling in BRCA1/2 genes is at 30-50% VAF, and for GIS is at 30%-40% tumor content. The false positive rate for GIS status and BRCA variants is 0%. Testing of ovarian cancer samples and control samples with multiple operators, reagent lots, and library preparation start days showed >95% concordance in BRCA variant calling and GIS status. The GIS scores were highly concordant with the reference method assay (R = 0.98). The results were robust on both NextSeq 550 and NovaSeq™ 6000 with SP flowcells. The TSO 500 HRD assay demonstrated high analytical sensitivity and specificity in detecting BRCA variants and GIS status for HRD status assessment in ovarian cancer samples. TSO 500 HRD enables high resolution comprehensive genomic profiling alongside HRD assessment to maximize biomarker detection from limited amount of sample.

BRCA testing on buccal swab to improve access to healthcare and cancer prevention: a performance evaluation

ObjectiveBRCA1/2 (BRCA) genetic testing allows patients with high-grade serous ovarian cancer to receive appropriate medical management with molecular target therapy and prevention strategies. Most of the BRCA sequencing methods use...

Identification of 8-Digit HLA-A, -B, -C, and -DRB1 Allele and Haplotype Frequencies in Koreans Using the One Lambda AllType Next-Generation Sequencing Kit.

BackgroundRecent studies have successfully implemented next-generation sequencing (NGS) in HLA typing. We performed HLA NGS in a Korean population to estimate HLA-A, -B, -C, and -DRB1 allele and haplotype frequencies up to an 8-digit resolution, which might be useful for an extended application of HLA results.MethodsA total of 128 samples collected from healthy unrelated Korean adults, previously subjected to Sanger sequencing for 6-digit HLA analysis, were used. NGS was performed for HLA-A, -B, -C, and -DRB1 using the AllType NGS kit (One Lambda, West Hills, CA, USA), Ion Torrent S5 platform (Thermo Fisher Scientific, Waltham, MA, USA), and Type Steam Visual NGS analysis software (One Lambda).ResultsEight HLA alleles showed frequencies of ≥10% in the Korean population, namely, A*24:02:01:01 (19.5%), A*33:03:01 (15.6%), A*02:01:01:01 (14.5%), A*11:01:01:01 (13.3%), B*15:01:01:01 (10.2%), C*01:02:01 (19.9%), C*03:04:01:02 (11.3%), and DRB1*09:01:02 (10.2%). Nine previous 6-digit HLA alleles were further identified as two or more 8-digit HLA alleles. Of these, eight alleles (A*24:02:01, B*35:01:01, B*40:01:02, B*55:02:01, B*58:01:01, C*03:02:02, C*07:02:01, and DRB1*07:01:01) were identified as two 8-digit HLA alleles, and one allele (B*51:01:01) was identified as three 8-digit HLA alleles. The most frequent four-loci haplotype was HLA-A*33:03:01-B*44:03:01:01-C*14:03-DRB1*13:02:01.ConclusionsWe identified 8-digit HLA-A, -B, -C, and -DRB1 allele and haplotype frequencies in a healthy Korean population using NGS. These new data can be used as a representative Korean data for further disease-related HLA type analysis.

Plasma-based longitudinal mutation monitoring as a potential predictor of disease progression in subjects with adenocarcinoma in advanced non-small cell lung cancer

BackgroundIdentifying and tracking somatic mutations in cell-free DNA (cfDNA) by next-generation sequencing (NGS) has the potential to transform the clinical management of subjects with advanced non-small cell lung cancer (NSCLC).MethodsBaseline tumor tissue (n = 47) and longitudinal plasma (n = 445) were collected from 71 NSCLC subjects treated with chemotherapy. cfDNA was enriched using a targeted-capture NGS kit containing 197 genes. Clinical responses to treatment were determined using RECIST v1.1 and correlations between changes in plasma somatic variant allele frequencies and disease progression were assessed.ResultsSomatic variants were detected in 89.4% (42/47) of tissue and 91.5% (407/445) of plasma samples. The most commonly mutated genes in tissue were TP53 (42.6%), KRAS (25.5%), and KEAP1 (19.1%). In some subjects, the allele frequencies of mutations detected in plasma increased 3–5 months prior to disease progression. In other cases, the allele frequencies of detected mutations declined or decreased to undetectable levels, indicating clinical response. Subjects with circulating tumor DNA (ctDNA) levels above background had significantly shorter progression-free survival (median: 5.6 vs 8.9 months, respectively; log-rank p = 0.0183).ConclusionLongitudinal monitoring of mutational changes in plasma has the potential to predict disease progression early. The presence of ctDNA mutations during first-line treatment is a risk factor for earlier disease progression in advanced NSCLC.

AB0013 HLA ASSOCIATION WITH SYSTEMIC SCLEROSIS (SSc) IN NORTH INDIAN POPULATION AND FAMILIAL INHERITANCE PATTERNS

Background:It is widely believed that SSc develops in an individual with a permissive genetic makeup.Genetic influences have long been suspected to impact SSc. In families with a history of SSc, the incidence of disease can range from 1.5 to 1.7% (1). There are several reports of familial occurrence and certain alleles of the HLA system have been associated with the disease (2).No Indian data pertaining to genetic basis of systemic sclerosis is present. Understanding the genetic basis of the disease will help us in defining the biomarkers of the disease in the population that can help in early diagnosis and prognosis.Objectives:To study HLA association with Systemic sclerosis (SSc) in North Indian Population and its genetic susceptibility to familial systemic sclerosis.Methods:A total of 150 SSc patients diagnosed by following ACR and EULAR criteria and 150 control subjects, were genotyped for HLA-A, B, DRB1, DQB1 loci by Luminex® 200 Instrument (USA). The association of alleles with disease susceptibility was tested by Chi-square test and Fisher’s exact test.HLA Typing for HLA class I (A, B, C) and II(DR,DQ,DP) for familial study of systemic sclerosis in 2 families was performed by Next Generation Sequencing(NGS) with illumina MiniSeq using MIA FORA NGS Kits from IMMUCOR. Antinuclear patterns (ANA) and specific antibodies were detected by indirect Immunofluorescence and Immunoblot (Euroline, Germany).Results:Strong disease associations were observed for haplotypes A*24(OR=1.7;< 0.02), A*32(OR=2.8;< 0.02), B*35(OR=1.7;< 0.03), DRB1*11(OR=2.1;< 0.007). The reduced frequencies of haplotypes A*68(P< 0.05), DRB1*10(P< 0.05), DRB1*12 (P<0.00) among patients suggested a protective association. There was no statistical association found with HLA DQB*1.Through NGS we observed that in the 1stfamily haplotypes HLA –A*11, 32, 24; B* 51, 55, 35; C*-14, 04; DRB1*15, 04; DQB1*05, 03; DPB1*04, 26 appears in affected family members with serological abnormalities.In the 2ndfamily both mother and daughter had same set of haplotypes except DQB1 with serological abnormalities. The haplotypes DPB1*04 was present in all the diseased individuals of both the families (Fig. 1 and table 1).Table 1.NGS HLA typing reportABCDRB1DQB1DPB1F111 2435 1504 0415 1505 0502 26F211 3251 5514 0415 0405 0304 04F311 2435 5504 0415 1505 0526 04F432 1151 1514 0415 0405 0302 04F524 3335 4404 0715 0705 0226 14F611 2435 5504 0415 1505 0504 26F711 2435 5504 0415 1505 0504 26F824 3251 3514 0404 1503 0526 04F911 3251 5514 0415 0405 0304 04F1011 3344 5207 1211 0702 0304 13F1111 3344 5207 1211 0703 0304 13Fig. 1Conclusion:The risk alleles A*24, 32; B*35; DRB1*11 were found to be associated with North Indian cohort of SSc, while the protecting alleles were A*68; DRB1*10, 12.These risk alleles were present in the SSc affected family members and the protective alleles were absent in the same. Surprisingly, even healthy members carried the same risk alleles but did not manifest the disease or have serological evidence of the same. We have not excluded occurrence of disease at a later age, as presently the healthy siblings are young. Thus our study indicates that though HLA association are found with SSc but many other factors like HLA (HLA *C, DPB1*) or non HLA genes as wells as epigenetic factors might also play a role in disease manifestation and severity.

Decentralization of Next-Generation RNA Sequencing-Based MammaPrint® and BluePrint® Kit at University Hospitals Leuven and Curie Institute Paris

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting.

A work lifetime of passion! Is it worth the time, money, stress and relationship changes when you disagree with hierarchy?

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting.

Sensitivity of next-generation sequencing assays detecting oncogenic fusions in plasma cell-free DNA

ObjectivesPlasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility. Materials and methods: Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360™(G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling. ResultsOf 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360. ConclusionFocusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.

Abstract P4-08-25: Decentralized beta testing of MammaPrint and BluePrint NGS kit at University Hospitals Leuven and Curie Institute Paris

Abstract Background Many countries restrict patient material exchange to central diagnostic laboratories abroad, limiting access to assays like MammaPrint® (MP) and BluePrint® (BP). Both assays are microarray-based, with MP being prognostic for distant recurrence and BP for molecular subtyping of breast cancer (Luminal-, HER2-, and Basal-type). To increase accessibility, decentralization is required with Next Generation Sequencing (NGS) being the preferred testing platform given that most diagnostic laboratories have the technology in place. The aim of this beta testing study is to validate a previously developed and centrally validated MP and BP NGS kit for RNA samples in two large tertiary academic hospitals in Europe. Patients and Methods Patients with early breast cancer diagnosed at the Multidisciplinary Breast Center at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Patients with bilateral breast cancer or presenting with more than 3 positive lymph nodes were excluded. Only patients with invasive ductal and invasive lobular carcinoma were included. Twenty tissue sections were cut from formalin-fixed, paraffin-embedded (FFPE) blocks; 10 tissue sections were analyzed at the local site using the MP and BP NGS kit, and 10 tissue sections were analyzed at Agendia using the same kit and procedure, as well as with the golden standard method (gene expression microarrays). Targeted RNA sequencing of the 70 MP and 80 BP signature genes was performed on Illumina MiSeq instruments. The raw NGS data generated at the local test sites was sent through a secure file transfer protocol server to Agendia for interpretation and comparison with microarray and NGS performed in the Agendia laboratories. We aimed for a minimum concordance rate between MP and BP outcome of 90% between each local site and Agendia's centralized site. Results In this study, 116 early breast cancer patients were included (73 from University Hospitals Leuven and 43 from Curie Institute). Out of these patients, 52% were MP Low Risk and 48% MP High Risk according to microarray. The patients had a BP luminal, HER2 or basal subtype in respectively 83%, 9% and 8%. Concordance between MP microarray obtained from Agendia and MP NGS obtained from the local sites was 91.4%. Concordance between MP High and Low Risk classification between NGS Leuven versus NGS Agendia was 92.1% and between NGS Curie versus NGS Agendia 95.3%. For BP subtype outcomes, the results from microarray versus NGS for all patients combined from both local sites gave a 98.3% concordance and NGS Agendia versus NGS from each local site gave a 100% concordance. Conclusion The MP and BP NGS kit was successfully validated in a decentralized setting, showing high concordance between results obtained at three different sites. There was a clear benefit of having well-trained NGS experienced diagnostic technical teams. The MP and BP NGS kit the first FFPE targeted RNA sequencing based multigene signature for breast cancer care, will provide a high and equal standard of MP and BP gene expression testing for breast cancer in a decentralized setting. Citation Format: Slembrouck L, Laurent C, Delahaye LJ, Mittempergher L, Vanden Bempt I, Vander Borght S, Darrigues L, Vliegen L, Sintubin P, Raynal V, Bohec M, Reyes C, Rapinat A, Helsmoortel C, Jongen L, Hoste G, Neven P, Wildiers H, Smeets A, Nevelsteen I, Punie K, Van Nieuwenhuysen E, Han S, Salomon AV, Faron EL, Cynober T, Witteveen AT, Neijenhuis S, Glas AM, Reyal F, Floris G. Decentralized beta testing of MammaPrint and BluePrint NGS kit at University Hospitals Leuven and Curie Institute Paris [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-25.

PS02.083: CIRCULATING CELL FREE TUMOR DNA FOR DISEASE MONITORING AFTER NEOADJUVANT CHEMORADIOTHERAPY FOR ESOPHAGEAL CANCER: PROOF-OF-PRINCIPLE

Abstract Background An active surveillance approach has been proposed for patients with a clinically complete response (cCR) after neoadjuvant chemoradiotherapy (nCRT) in esophageal cancer (SANO trial). To justify renouncing surgical resection, patients with residual disease after nCRT should be accurately identified. However, substantial residual disease (TRG3–4) cannot be detected in 10% of patients with current diagnostic tests (preSANO trial). Circulating cell free tumor DNA (ctDNA) potentially improves detection of residual malignancy after nCRT and could be used for disease monitoring. The objective of this study was to investigate the feasibility of using ctDNA as biomarker for disease status after nCRT in esophageal cancer. Methods Twelve typical patients from the preSANO trial with variable pathological responses to nCRT were included. Blood was drawn and processed pretreatment. The feasibility of detecting TP53 mutations in baseline tumor biopsies was investigated using a next generation sequencing (NGS) panel. Subsequently, baseline blood samples of patients in whom specific TP53 mutations could be identified in baseline tumor biopsies or the surgical resection specimen were analyzed for ctDNA using cell free DNA NGS kits with single molecule barcoding (Oncomine Thermo Fisher). Results Baseline biopsy samples were available in 8 out of 12 patients. In 7 of these 8 patients (88%) specific TP53 mutations could be identified in their baseline biopsies. In 11 out of 12 patients (92%) specific TP53 mutations could be identified in baseline biopsies or the resection specimen. Eight of these 11 mutations were potentially detectable by the Oncomine panel. The panel detected TP53 mutational ctDNA in 4 of these 8 samples (50%). Conclusion Specific and clonal TP53 mutations can be identified in pretreatment biopsy samples and in surgical resection specimens of patients with esophageal cancer. These mutations can be matched to ctDNA identified in blood samples. Hence, ctDNA analyses in blood samples can potentially be used for disease monitoring during active surveillance and for disease monitoring in follow-up after surgical resection. Disclosure All authors have declared no conflicts of interest.

P008 Ashi validation with holotype HLA for high resolution typing

Aim Clinical validation of Next Generation sequencing(NGS) for HLA typing has been a high priority for many HLA laboratories worldwide, giving the opportunity to assess the correct allele matching of high-resolution genotypes to improve transplantation outcomes and decrease the rate of GvHD.Holotype HLA (Omixon Inc.) is an optimized, pre-configured assay and software combination product that provides comprehensive gene characterization of multiple HLA loci for sequencing on the Illumina platform. The goal of this study is to provide a validation of high-resolution HLA typing using Omixon’s NGS kits. Methods To test the robustness of the NGS protocol for HLA typing by Omixon Holotype HLA Assay and validate its use in our lab, we will type 96 DNA samples from peripheral blood using an 11-locus kit configuration to genotype HLA class I and Class II genes. All DNA samples have been previously typed by Sanger sequencing. HLA typing will be assigned using Omixon’s HLA Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Inter-laboratory reproducibility will be assessed with 20 samples from Children’s Hospital of Philadelphia, Immunogenetics Laboratory, as a courtesy from Dr. Dimitri Monos. The validation will follow the Minimum Guidelines for Validation of NGS for HLA standards from American Society for Histocompatibility and Immunogenetics (ASHI). Results Sequence concordance will be assessed, where allele calls (1st and 2nd fields) from each sample will be compared with those obtained by Sanger sequencing. Reproducibility between technologists will be also assessed. We expect to demonstrate the advantages of the NGS-based HLA genotyping over SSO and SBT techniques, as it may provide credible and more informative HLA genotyping with the first passage without any reflexive testing. Conclusions It’s expected fully concordance of sequencing results for allele call for first and second field. Concluding this validation will provide the confidence that HLA typing by NGS is ready to be introduced in our laboratory. According to our best knowledge, this will be the first validation using Omixon Holotype Assay in Brazil, and the second laboratory in Brazil to validate NGS following the ASHI standards.

P088 Haplotype analyses of classical HLA genes from families

Aim HLA alleles are observed in specific haplotypes because of linkage disequilibrium between particular alleles. HLA typing using Next Generation Sequencing (NGS) platforms allows generating nearly full-length gene sequences and detecting novel alleles. NGS technologies also permit high-throughput HLA typing for many samples in a cost effective manner. To establish HLA haplotypes from families for the 17th International HLA and Immunogenetics Workshops (IHIW), we developed the Family HLA Haplotype (FamHLAHap) software that compares offspring and parents’ HLA genotypes and builds haplotypes in an automated fashion. To optimize and validate the software, we generated haplotypes from 200 quartet and 1500 trio nuclear families. Methods The quartet families include both parents and two children, and occasional extended family members. A trio contains a child who is diagnosed with multiple sclerosis (MS) and two unaffected parental controls. DNA sequencing library was prepared using MIA FORA NGS kits, and sequenced on either MiSeq or NextSeq platforms. HLA types were obtained using MIA FORA software. The FamHLAHap software reports HLA haplotypes according to the order of genes along the chromosome to facilitate identifying potential meiotic recombination, and also compares to the previously established haplotypes. Results We built haplotypes from a total of 1700 families using the FamHLAHap software. Typing of DPB1 locus by NGS-shotgun sequencing and Sanger often results in ambiguous genotypes because of lack of phasing between exons 2 and 3. In the present study virtually all ambiguities were resolved through the analyses of segregation of sequences. Conclusions We have shown the suitability of the FamHLAHap software to efficiently build haplotypes from NGS data in a large number of families included in the 17th IHIW. Understanding haplotype structures allows delineating evolutionary pathways of the HLA region that include both divergent and consequent evolution. The haplotypes generated from family study provide valuable resources for hematopoietic stem cell donor match prediction, and identification of haplotypes associated with autoimmune diseases.

HLA genotyping using the Illumina HLA TruSight next-generation sequencing kits: A comparison.

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

OR37 Haplotype analyses of classical HLA genes from quartet families

HLA alleles are observed in specific haplotypes, due to linkage disequilibrium (LD) between particular alleles. Haplotype frequencies for alleles have been established for specific ethnic groups and racial categories from population studies. HLA typing using Next Generation Sequencing (NGS) platforms allows generating full-length gene sequences, obtaining four-field HLA types and detecting novel alleles. NGS technologies also permit high-throughput HLA typing for large numbers of samples in a cost effective manner. Aim To establish HLA haplotypes with four-field resolution from quartet (two children and two parents) families. Methods We selected quartet families from our clinical database. Extended family members, aunt, uncle, cousin and grandparents, were also included if available. HLA types generated using SBT, SSP and SSO were reviewed for selecting subjects. Two children were selected if they did not share two HLA haplotypes. DNA was processed using MIA FORA NGS kits, and sequenced on either MiSeq or NextSeq sequencer. HLA types were obtained using MIA FORA 2.0 software. HLA haplotypes were built by comparing children’s and parents’ HLA types. The HLA haplotypes were reported according to the order of genes along the chromosome to identify potential meiotic recombination, and were also compared to the previously established haplotypes. Results Of 100 selected families, we sequenced 50 families. We built 200 haplotypes consisting of four-field HLA types from these. We developed automated haplotype building software. It was feasible to resolve almost all ambiguities including phase ambiguities and to generate completely phased full-length gene sequences from the consensus sequences generated from these studies. Conclusions The haplotypes generated from quartet families will be important resources for hematopoietic stem cell donor match prediction and play a role as reference haplotypes for expanding a collection of HLA haplotypes.

Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting.

291 Background: A challenge in the analysis of circulating tumor cells (CTC) is their scarcity, and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs can be aggregated into close contact to interact and grow in culture. In this study, we demonstrated the ability to aggregate CTCs and perform next generation sequencing (NGS) to detect somatic mutations from renal and prostate cancers. Methods: Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then removed of microbeads, then magnetized by incubation with NanoShuttle (NS, Nano3D), a magnetic nanoparticle assembly, and printed into spheroids in 384-well plates. After 4 d of growth, the cells were lysed and DNA was amplified by whole genome amplificaiton (WGA) with the NGS kit (Fluxion) and quantified via qPCR. Targeted libraries were sequenced using the PGM (ThermoFisher) sequencing instrument; data was analyzed using a customized variant calling/filtering pipeline based on standard Ion Reporter alignment tools and VarSeq for variant filtering and functional interpretation. Results: CTCs were successfully aggregated using magnetic 3D bioprinting, and grew over 4 d. We then demonstrated the detection of somatic variants within a majority of the samples, for both prostate and renal cancers. Using the commercial Oncomine, we found a median of 5 COSMIC variants (32 total) per sample using cell cultures. Conclusions: We successfully developed methods to aggregate CTCs with magnetic 3D bioprinting, expanded them, and then detected somatic mutations using NGS.