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Tumor mutational burden (TMB), measured by exome or panel sequencing of tumor tissue or blood (bTMB), is a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint pathways. However, significant variability in TMB measurement has been observed. We developed contrived bTMB reference materials using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines to support calibration and alignment across laboratories and platforms. Contrived bTMB reference materials were developed using genomic DNA from lung tumor cell lines blended into donor-matched lymphoblastoid cell lines at 0.5% and 2% tumor content, fragmented and size-selected to mirror the size profile of circulating cell-free tumor DNA with TMB scores of 7, 9, 20, and 26 mut/Mb. Variant allele frequency (VAF) and bTMB scores were assessed using PredicineATLAS and GuardantOMNI next-generation sequencing assays. DNA fragment sizes in the contrived reference samples were similar to those found within patient plasma-derived cell-free DNA, and mutational patterns aligned with those in the parental tumor lines. For the 7, 20, and 26 mut/Mb contrived reference samples with 2% tumor content, bTMB scores estimated using either assay aligned with expected scores from the parental tumor cell lines and showed good reproducibility. A bioinformatic filtration step was required to account for low-VAF artifact variants. We demonstrate the feasibility and challenges of producing and using bTMB reference standards across a range of bTMB levels, and how such standards could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories.

Background Highly characterized reference materials are required to expand noninvasive prenatal testing (NIPT) for low incidence aneuploidies and microdeletions. The goal of this study was to develop reference materials for the development of next generation circulating cell-free DNA (ccfDNA) assays. Methods This was a prospective study of pregnancies complicated by positive prenatal genetic screening. ccfDNA was isolated from maternal plasma and amplified. Lymphoblastoid cell lines were prepared from maternal peripheral blood mononuclear cells and fetal cord blood cells. Cells were Epstein-Barr virus immortalized and expanded. Amplified DNA and to a limited extent formulated lymphoblastoid-derived ccfDNA was tested in SNP-based and chromosome counting (CC) based massively parallel sequencing assays. Results Enrolled cases included fetuses with: T21 (2), T18 (1), T18-XXX (1), XYY (1), microdeletions (1), and euploid (2). Three lymphoblastoid cells lines were prepared. Genomic DNA was extracted from cell lines and fragmented to simulate ccfDNA. ccfDNA isolation yielded about 2000 usable genome equivalents of DNA for each case for amplification. Although the sonicated genomic DNA derived from lymphoblastoid cell lines did not yield results compatible with NIPT assays, when blinded, NIPT platforms correctly identified the amplified ccfDNA isolated from blood in the majority of cases. Conclusions This study showed that maternal blood samples from pregnancies complicated by common chromosomal abnormalities can be used to generate materials for the development and evaluation of NIPT assays.

Mutations in the tumor suppressor BRCA1 and BRCA2 genes can cause cell damage that significantly increases the risk of developing breast, ovarian, prostate, and pancreatic cancers. Large genomic rearrangements (LGRs) are defined as deletions, duplications or insertions; often involving a significant portion of an exon. Usually pathogenic, they have been reported to account for up to 27% of the BRCA1 and 5% of BRCA2 disease-causing mutations with a strong founder effect accounting for about 1/3 of all cancer diagnoses in some populations. Accurate detection of a BRCA1/2 pathogenic variants has immense impact on clinical management of disease including eligibility for PARP inhibitor therapy. However, these LGRs are frequently missed by PCR-based methods and NGS assays that do not detect partial or complete exon losses or gains. Given the difficulty in detecting LGRs, there is a need for improvement of BRCA1/2 testing algorithms including reference materials that incorporate challenging LGRs to support NGS assays that analyze for these mutations. Biosynthetic DNA constructs bearing clinically relevant BRCA1/2 variants including deletions up to 500 bp spanning 2 exons and duplications up to 170 bp, were mixed with purified genomic DNA from the GM24385 human reference cell line at 10% and 50% variant allele frequency (VAF) to represent somatic and inherited BRCA disease states, respectively. The same variants were engineered into GM24385 cells at the desired VAF (>5%) and the cells were formalin fixed and paraffin embedded using a proprietary method to mimic preserved tumor biopsies. In both materials, VAF was measured using digital PCR. The prepared purified gDNA and FFPE materials were sent to multiple clinical laboratories and analyzed using various NGS assays to assess variant detection performance and ensure that the product was compatible with clinical testing workflows. Digital PCR confirmed the presence of all 20 BRCA1 and BRCA2 variants within a 20% range of the target VAF in the purified gDNA material and between 11-23% in FFPE. NGS results were variable but confirmed the presence of most variants. A few variants were detected at very low levels but reported as not detected by the bioinformatic software due to limit of detection. We have successfully developed a reference material to support both BRCA1 and BRCA2 inherited and somatic genetic testing, providing laboratories and assay developers with a tool to challenge and optimize their assay’s detection and quantitation of pathogenic variants by NGS Provided as full process in FFPE format or in purified gDNA, these reference materials include 11 challenging LGRs, which are useful in evaluating the ability of different NGS platforms to detect such variants. This study has highlighted the need for reference materials that contain these challenging genomic alterations to improve genetic testing and ultimately, support PARP inhibitor treatment selection. Citation Format: Dana Ruminski Lowe, Benedicta Forson, Maria Cowen, Matthew G. Butler, Yves Konigshofer, Melissa Berenger, Krystyna Nahlik, Dianren Xia, Catherine Huang, Russell Garlick, Bharathi Anekella. Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6109.

Homologous recombination deficiency (HRD) arises when defects in DNA repair pathways occur, leading to genomic instability. HRD status is an emerging therapeutic biomarker; NGS assays that measure it can be used to stratify ovarian and breast cancer patients and determine eligibility for clinical trials, and PARP inhibitor- and platinum-based therapies. Seraseq FFPE HRD reference materials were developed to cover a range of genomic instability scores (GIS) to help NGS HRD assay validation and development. Here we discuss a multi-site evaluation across various platforms of our high-positive, low-positive, and negative HRD reference materials. Tumor cell lines were characterized by sequencing and evaluated in collaboration with several IVD partners. Three breast cancer cell lines (along with their SNP-matched normal cell lines) were selected based on their GIS. Tumor cells were blended with their SNP-matched normal cells to achieve ~65% tumor content. Biosynthetic DNA containing mutations in homologous recombination repair (HRR) genes (ATM, BRIP1, RAD51C, RAD51D) were added to the high-positive and negative reference materials targeted at >5% variant allele frequency (VAF) and measured by digital PCR. Formalin fixed paraffin embedded (FFPE) blocks were made and each block was tested for yield per curl using both the Qiagen QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit for extraction and Qubit dsDNA HS kit for concentration analysis. DNA quality was assessed using an Agilent gDNA ScreenTape Assay for the TapeStation. Whole genome shotgun sequencing was performed on extracted DNA using LGC NxSeq AmpFREE Low DNA Library Kit and Roche KAPA UDI adapters and Illumina NextSeq 2000 P1 flow cell. HRD status was evaluated by external collaborators with multiple NGS and microarray assays, including the Illumina TSO500 HRD RUO assay, the SOPHiA DDM HRD Solution and the OncoScan CNV Array A breast cancer cell line with a GIS of ~75 was selected as the HRD high-positive reference material, a second breast cancer cell line with a GIS of ~60 was selected as HRD low-positive, and a third breast cancer cell line with a GIS of ~30 was selected as HRD negative. Representative DNA yields per 10-micron section (determined using the HRD positive FFPE curls extracted by Qiagen QIAamp method) were 165 ± 28 ng/curl. Digital PCR confirmed the presence of the 8 HRR mutations (in 4 genes) at levels >5% VAF. GIS varied for each material across assays, but HRD status was consistent, confirming the wide applicability of the new reference materials We have developed the Seraseq HRD reference materials to meet the needs of laboratories looking to analyze HRD in cancer patient samples. These reference materials facilitate standardization and quality control in HRD testing by clinical labs for current and new PARP inhibitor treatment stratification in expanded patient populations that may include those with WT BRCA1/2 genes Citation Format: Dana Ruminski Lowe, Robert M. Whiting, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Indira Chivukula, Krystyna Nahlik, Dianren Xia, Russell Garlick, Bharathi Anekella. Multi-site evaluation of FFPE homologous recombination deficiency reference materials. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6110.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Next Generation Sequencing (NGS) is an important technology to identify genetic changes involved in lymphoid malignancies. Genome-level understanding of these changes can aid in diagnosis, prognosis, and therapy selection. Cancer biopsies are often preserved by formalin-fixed, paraffin-embedding (FFPE) procedures which provide long-term preservation but introduce damage to nucleic acids that are present in tissue including double strand breaks, nicks, and oxidation. The gold standard for Lymphoma diagnosis is surgical removal of the lymph node making FFPE the preferred sample format for analysis. To address the need for high quality reference materials for tumor profiling assays for these patients, we developed two multiplexed, biosynthetic reference materials: the Seraseq® FFPE Lymphoma DNA Reference Material and the Seraseq® Lymphoma DNA Mutation Mix. Both contain 26 plasmids containing important SNVs, INDELs, and gene fusions in lymphoid disease. For the FFPE format, plasmids were pooled at equal concentrations and introduced into the GM24385 reference human cell line. Engineered cells were diluted to achieve desired allele frequencies (AFs) as determined by digital PCR (dPCR). The cells were processed with a proprietary FFPE protocol that mimics damage in patient samples. The QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit were used for DNA isolation. DNA was quantified by the Qubit dsDNA HS Assay and quality assessed by the Agilent TapeStation. AFs were determined by dPCR for QIAamp-extracted DNA and a custom ArcherDX VariantPlex NGS panel for DNA extracted by both kits. DNA yield was similar, 193±44 ng (QIAamp) and 124±11 ng (Maxwell); however, a higher DNA integrity number was observed with the QIAamp kit (6.1 vs 2.7). The average % AF by dPCR was 9.6±1.8 (QIAamp) (target ≥5%). The average % AF by NGS was 7.6±2.8 (QIAamp) and 3.3±1.1 (Maxwell). For the DNA mix format, the same plasmids were pooled and blended with total purified genomic DNA from GM24385 cells. The average % AF was 8.4±0.6 by dPCR (target 5-10%) and 8.2±1.6 for the ArcherDX VariantPlex NGS panel. Overall, there was good concordance between AFs measured by dPCR and NGS for the DNA and FFPE formats. We noted that overall sample performance may be impacted by the FFPE DNA extraction method used. In conclusion, these reference materials allow for monitoring of a broad range of somatic mutations and gene fusions which can aid testing laboratories in accurately detecting and quantifying various genetic events in Lymphoid patient samples. The FFPE format can serve as an extraction control in assay development, while the DNA mix format allows laboratories to go directly into NGS library preparation and dPCR. A complementary circulating tumor DNA (ctDNA) version of these reference materials would aid laboratories in detecting minimal residual disease in patient blood resulting in less invasive lymphoid testing and therapeutic response monitoring. Citation Format: Tonya N. Watkins, Benedicta Forson, Dana Ruminski Lowe, Yves Konigshofer, Catherine Huang, Omoshile Clement, Bharathi Anekella. Comprehensive NGS-based reference materials for variant detection in lymphoid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 756.

Here we present data on the characterization and implementation of a set of reference materials for the standardization of homologous recombination deficiency (HRD) assessment. The safety and effectiveness profile of a drug can be improved by using it in patients where that drug is most likely to be effective. While PARP inhibitors are indicated for use in some patients with ovarian, breast, pancreatic, and prostate cancer, patients with other cancers that are deficient in homologous recombination repair (HRR) might also benefit from their use. However, determining whether a cancer is HRD-positive is neither trivial nor precise. While clinical trials have shown that patients with deleterious mutations in BRCA2 and BRCA1 are most likely to benefit from PARP inhibitors, determining that a given BRCA mutation (or set of such mutations) is in fact deleterious is frequently not straightforward. Therefore, assays are now looking for signs of HRD, such as genomic instability consistent with non-homologous end joining (NHEJ) being used for the repair of double-stranded breaks in DNA as opposed to HRR. For example, some assays look at the combination of loss of heterozygosity (LOH) and large-scale state transitions (LST) across chromosomes as well as telomeric allelic imbalance (TAI) when determining whether a cancer is HRD-positive, while other assays use alternative approaches. How those characteristics are measured, integrated, and distilled into a final determination of whether HRD is present or absent varies, which can create uncertainty around treatment options and enrollment into clinical trials. This also makes the path of follow-on companion diagnostics challenging because perfect agreement between imprecise measurements is unlikely. In order to enable more standardized reporting of HRD and to enable the assessment of HRD assays, we created a set of characterized reference materials composed of HRD negative, borderline, and positive tumor/normal matched cell lines that were analyzed using both array-based and next generation sequencing-based assays in order to characterize chromosomal changes across their genomes and obtain HRD scores. Because of differences in the results, assay imprecision should be taken into account in clinical trials that implement cutoffs in order to establish safety data for patients who are biomarker-negative but may be biomarker-positive in a future assay, which is similar to what is done for complementary diagnostics. Citation Format: Yves Konigshofer, Matthew G. Butler, Krystyna Nahlik, Dana Ruminski Lowe, Catherine Huang, Omoshile Clement, Russell K. Garlick. The challenge of standardizing the measurement of an imprecise biomarker like HRD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1024.

TMB is a biomarker with potential for predicting positive patient response to immune checkpoint inhibitors. TMB measurements can be determined using genomic DNA extracted from FFPE-preserved tissue biopsy samples. However, assessment of TMB from a liquid biopsy, or blood TMB (bTMB), is an attractive alternative clinical diagnostic tool that would allow clinicians and patients to avoid the invasive challenge of tissue biopsies. Accurately measuring bTMB with targeted gene panels has been problematic, thus we have developed the new Seraseq® bTMB reference materials at various mutational burden levels from genomic DNA extracted from tumor-derived and SNP-matched normal cell lines. Somatic variants were identified in each cell line by whole exome sequencing (WES). TMB assessments were made by an in-house developed TMB bioinformatics pipeline based on recommendations by the Friends of Cancer Research (FoCR) TMB Harmonization Consortium. DNA from tumor and normal cell lines were blended at 0%, 0.5%, and 2% tumor fractions and fragmented to approximate the size of cell free DNA (cfDNA). The resulting bTMB mixes were enriched with the TruSight™ Oncology 500 ctDNA Assay (2x151 bp), in triplicate, and sequenced on a NovaSeq™ 6000. Blood TMB scores were determined using the DRAGEN™ TruSight Oncology 500 ctDNA Analysis Software v1.1. The observed bTMB scores for the tumor-containing materials were slightly lower in the TF 0.5% mix versus the TF 2% mix due to variants moving below the limit of detection of the TSO500 ctDNA assay (Table 1). As is the case with patient samples and clonal hematopoiesis, there are background variants in 0% tumor content material that may elevate apparent bTMB scores, thus adjusted bTMB scores are shown. The Seraseq Blood TMB Score reference materials provide a tumor-normal matched blood TMB control to aid development, validation and QC of cfDNA NGS assays for accurate determination of bTMB Scores. Table 1. Average bTMB scores and adjusted bTMB scores for each mix. Mix Average bTMB Score (mutations/megabase ± 1 SD) Adjusted bTMB Score (mutations/megabase ± 1 SD) Score 7 0% 7.5 ± 1.7 0 Score 7 0.5% 13.1 ± 2.6 5.6 ± 3.1 Score 7 2% 17.9 ± 1.3 10.4 ± 2.1 Score 13 0% 4.6 ± 0.5 0 Score 13 0.5% 18.7 ± 2.1 14.1 ± 2.2 Score 13 2% 24.6 ± 0.8 20.0 ± 0.9 Score 20 0% 7.5 ± 1.4 0 Score 20 0.5% 26.0 ± 2.3 18.5 ± 2.7 Score 20 2% 35.6 ± 1.0 28.1 ± 1.7 Score 26 0% 6.0 ± 0.5 0 Score 26 0.5% 20.7 ± 5.5 14.7 ± 5.5 Score 26 2% 30.4 ± 1.5 24.4 ± 1.9 Citation Format: Dana Ruminski Lowe, Benedicta Forson, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Omoshile Clement, Russell K. Garlick, Bharathi Anekella. Development of blood TMB (bTMB) reference materials for validation of cfDNA-based targeted NGS assays that measure tumor mutational burden (TMB) in patient blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 537.

Monitoring measurable residual disease (MRD) via liquid biopsy is a promising method for catching early stage cancer and disease relapse long before traditional diagnostics, which generally require significant disease progression for detection. Assays in development include those that target patient-specific variants and fixed panels for all patients. Regardless, detection of variant allele frequencies at extremely low levels, well below the limit of detection of typical circulating tumor DNA (ctDNA) assays, presents a challenge that can be surmounted with well-designed reference materials that allow for assessment of sensitivity and specificity. We developed the Seraseq® ctDNA MRD Panel kit composed of 4 tumor fractions at decreasing levels to meet validation needs of both patient-specific and fixed panel targeted cfDNA NGS MRD assays. Genomic DNA from tumor and their SNP-matched normal cell lines, characterized by whole exome sequencing (WES), was fragmented to approximate the size of circulating cell free DNA (ccfDNA). This DNA was blended at tumor fractions (TF) of 0.5%, 0.05%, and 0.005% and biosynthetic DNA fragments containing more common and clinically relevant variants were spiked in at similar TF levels. A normal (0% tumor) ctDNA reference material was produced for comparison and identification of background variants. The VAFs for the biosynthetic variants were determined by digital PCR (BioRad QX200) and targeted cfDNA NGS assay (ArcherDx LiquidPlex) in the 0.5% TF mix. Somatic variants from the tumor DNA were assessed using a custom Agilent SureSelect XT HS2 assay targeting ~100 variants in the MRD panel mix using 50 ng as input. Variants were called at 1 observation. All biosynthetic variants were detectable in the TF 0.5% mix at anticipated VAFs (dPCR: 0.43% ± 0.12% and NGS: 0.33% ± 0.15%). Somatic VAFs of the ctDNA MRD panel mixes were measured at expected levels although not all variants were detected below 0.05% due to sampling. Agilent SureSelect XT HS2 libraries had ~25% incorporation efficiency and ~ 72% on-target. The 0.5% and 0.05% mixes showed evidence of more of the somatic variants at 1 or more copies than the other samples. The 0.005% mix showed evidence of more of the somatic variants than the 0% mix. The Seraseq ctDNA MRD Panel Mixes have been designed to provide a broad range of DNA mutations from tumor-normal matched cell lines and spike-in variants to aid sensitivity and specificity when applied to MRD evaluation of patient-specific and fixed panel cfDNA NGS MRD assays. Citation Format: Dana Ruminski Lowe, Benedicta Forson, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Omoshile Clement, Russell K. Garlick, Bharathi Anekella. Reference materials for ctDNA-based measurable residual disease (MRD) assay development and validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2219.

Here we describe new reference materials (RMs) for liquid biopsies that are designed to mimic the methylation found in circulating cell-free DNA (ccfDNA). Methylation is an important epigenetic modification that influences cellular differentiation and gene expression. Recently, liquid biopsies have started to incorporate analyses of epigenetic modifications for screening purposes to detect cancer-derived DNA in the blood. Additionally, epigenetic modifications are being used to also assign a tissue of origin to the cancer to direct confirmatory diagnostic procedures. Obtaining sufficient amounts of ccfDNA for assay development, validation, and proficiency testing is difficult. Furthermore, methods of analyzing the epigenetic modifications, such as bisulfite conversion, can damage a significant fraction of the input material; but, failing to carry them out to completion can result in an over-estimation of methylation. To address the challenges associated with assessing the methylation of ccfDNA, we created and characterized three types of RMs. The first type of RM is a fragmented genome of the GM24385 reference cell line that is used to create a set of RMs with different degrees of methylation (0%, ~25%, ~50%, ~75% and close to 100%). The exact degree of methylation was assigned by digital PCR using assays for heterozygous SNPs, where one allele is both a site for CpG methylation and a recognition site for a methylation-sensitive restriction enzyme while the other allele is not. The second type of RM is an amplified ccfDNA sample that has been methylated similarly but also reproduces the fragment lengths and genomic representation biases of ccfDNA. The degree of methylation was assigned by digital PCR, but only a subset of assays was useful because not all SNPs were heterozygous. The third type of RM is an amplified ccfDNA sample that has been processed to mimic the methylation found in the original ccfDNA. Analyses of the RMs by whole genome sequencing (WGS) showed that, while bisulfite conversion and/or the amplification of the resulting dU-containing DNA appeared to lead to significant biases in genomic representation, the resulting data was generally in agreement with the amount of methylation assigned by digital PCR. Additionally, it was possible to prepare an RM that generally reproduces the methylation found in ccfDNA; although, this was limited to individual molecules having either no or close to full methylation, which is unlikely to be the case for all molecules of ccfDNA in vivo. Citation Format: Jayanthi Ramprakash, Matthew G. Butler, Russell K. Garlick, Yves Konigshofer. Novel reference materials for the analysis of methylation in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3376.

Background Tumor mutational burden (TMB), as measured by exome or panel sequencing of tumor tissue (tTMB) or blood (bTMB), has been identified as a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint inhibitors (e.g. PD-1, PD-L1, CTLA-4). However, significant variability in TMB measurement has been reported due to differences in pre-analytical and laboratory methods, panel size, number of genes covered and bioinformatics pipelines. Reference standards have been proposed and evaluated for tTMB analysis by the Friends of Cancer Research (FoCR) to enable harmonization and standardization across different tTMB panel providers. Reference standards for bTMB are likely to be even more important given the unique challenges and higher sensitivity required for bTMB assays. Methods Contrived bTMB reference materials with 0.5% and 2% tumor content were developed using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines fragmented and size-selected to mimic cell-free DNA with TMB scores of 7, 9, 20 and 26 mut/Mb. Mutation coverage, mutant allele frequency (MAF) and bTMB scores were assessed using the PredicineATLAS and GuardantOMNI next-generation sequencing (NGS) platforms. Results The DNA fragment size for the contrived samples was similar to naturally occurring circulating cell-free tumor DNA and mutation patterns were aligned with those from parental tumor lines. As anticipated, low frequency artefactual MAF variants were observed, requiring removal by bioinformatic filtration. For samples with 2% tumor content, standards for 7, 20 and 26 mut/Mb were found to have as-expected bTMB scores across both evaluation platforms, with good reproducibility, following removal of low frequency MAFs. Results for 0.5% tumour content were also promising, although with greater variability in post-filtration bTMB scores observed. Conclusions The findings demonstrate it is feasible to produce bTMB reference standards across a range of bTMB levels. The data highlight the importance of data filtration to account for underlying low MAFs in such cell-line derived samples and that this reference material can control for variant sensitivity though not variant specificity. bTMB reference standards reported here could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories, thus improving the accuracy of testing to aid treatment decisions in oncology. Acknowledgements The study was funded by AstraZeneca. Medical writing support, which was in accordance with Good Publication Practice (GPP3) guidelines, was provided by Rachel Cicchelli, PhD, of Cirrus Communications (Macclesfield, UK), an Ashfield company, and was funded by AstraZeneca.

Immune Checkpoint Inhibitor (ICI) therapies continue to revolutionize treatment of advanced malignancies. However, identification of individuals who respond to ICI treatment remains challenging. Along with programmed death ligand 1(PD-L1 aka CD274) expression and microsatellite instability, tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase of genome, has emerged as a promising biomarker for differentiating responders from non-responders, especially in non-small cell lung cancer. Although promising, TMB is not a perfect biomarker for ICI efficacy with some individuals responding conversely to expectations. The imperfection of TMB as a prognostic biomarker may result from it being a proxy measurement of potential neoantigens. Indeed, all mutations that contribute to TMB will not be expressed as neoantigens and not all neoantigens will illicit immune responses. In refining ICI response prediction, tumor abundance of nonsynonymous mutations has aided in neoantigen prediction. In order to begin development of quality reference materials for neoantigen prediction, we have characterized the transcriptomes of four tumor cell lines that were previously used to formulate TMB reference materials. Sequencing libraries were prepared with the TruSeq Stranded Total RNA with Illumina Ribo-Zero Plus rRNA Depletion Kit and Invitrogen Collibri Stranded RNA Library Prep Kit from Illumina with Collibri H/M/R rRNA Depletion Kit. The resultant transcriptome libraries were sequenced on an Illumina NextSeq2000 platform and relative transcript counts were determined. Broad ranges of expression were observed among transcripts containing nonsynonymous mutations in each of the tumor cell lines tested. These datasets will serve as benchmarks for others attempting to combine TMB and tumor abundance in predicting neoantigen potential. Citation Format: Matthew G. Butler, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Reference materials for tumor mutational burden transcriptome profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 471.

Purpose: Whole transcriptome RNA-sequencing (RNA-Seq) has emerged as one of the most effective methods for detection of genomic rearrangements in cancer. Enrichment for poly(A) + RNA as part of library preparation is a standard method to select for mRNAs, but ribosomal RNA depletion and exon capture methods may be preferred for samples of suboptimal RNA quality, such as FFPE. Regardless of the selection and library preparation procedure, RNA-Seq for clinical use requires well characterized reference materials for assay quality control and fusion detection verification. However, reference materials developed for targeted NGS panels performed sub-optimally on RNA-Seq. This study was undertaken to design, optimize and improve RNA-Seq performance with a novel transcriptome-scale RNA Fusion reference material. Procedure: Seraseq® Fusion RNA Mix is highly multiplexed with 18 clinically relevant fusion RNAs and is designed for targeted NGS panels. The biosynthetic fusions are targeted to 60 copies per nanogram of total RNA from GM24385 human reference cell line and contain poly-A tail of ~37 templated A's. We tested whether increasing the length of the poly-A tail would improve the efficiency of binding to an oligo dT column and improve performance in total RNA-Seq NGS assays that use poly(A) + selection. E. coli Poly(A) Polymerase was used to extend tails, and efficiency of RNA recovery from oligo-dT columns was assessed visually by gel analysis. The fusion RNAs (with standard or lengthened poly-A tails) were mixed with total RNA from GM24385 human reference cell line. Since GM24385 is a lymphoid cell line, highly abundant immune transcripts may reduce the prevalence of the fusion RNAs in the selected RNAs. Therefore, mixtures were made at the normal concentration (~60 copies/ng, or 1X) as well as at 3-fold and 10-fold concentrations. Fusion detection was performed using Illumina TruSeq Stranded mRNA(polyA) kit and Illumina HiSeq, run in 2 × 150bp mode. Results: The proportional recovery was improved with the longer (>100 A's) tail length. This translated to improvement in NGS detection with longer poly-A tail length giving from 10% to 50% more fusion reads. Increasing the Fusion RNA concentration led to a proportion increase in fusion reads per million total reads. The increase from 5 fusion reads/million resulted in more robust and consistent fusion detection. Conclusions: These studies indicate that reference materials similar to the Seraseq Fusion RNA mix reference material, but with greater concentration of Fusion RNAs (3 - 10 fold higher concentration) and with longer polyA tail length will be applicable quality control materials for whole transcriptome RNA-Seq NGS assays. These novel reference materials may aid clinical labs in developing sensitive new RNA-Seq Fusion detection assays and tuning their bioinformatics pipeline to more precise selection of therapeutic strategies for patients. Citation Format: Catherine Huang, Matthew G. Butler, Yves Konigshofer, Jessica Dickens, Rajeswari Vemula, Omoshile Clement, Bharathi Anekella. Optimizing Performance of Whole Transcriptome RNA-Seq Reference [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2232.

Tumor mutational burden (TMB) is a measurement of the somatic mutations acquired by a tumor genome that cause nonsynonymous changes in proteins. TMB is a biomarker that serves as a proxy for the potential to produce neoantigens that will be recognized by T cells. Despite some reports to the contrary, TMB has successfully categorized the likelihood that an individual will respond positively to immune checkpoint inhibitors in some clinical trials. Although most TMB measurements are made from genomic DNA extracted from formalin-fixed and paraffin-embedded tissue sections, some diagnostic tests measure TMB in circulating tumor DNA (ctDNA) extracted from blood samples; an approach referred to as blood TMB (bTMB). Blood TMB is an attractive biomarker since it bypasses many of the obstacles encountered with resected and hard to biopsy tumors. Only a handful of diagnostic laboratories measure bTMB and their analytic validations rely on hard to obtain specimens acquired from ongoing drug trials or tissue repositories. This specimen scarcity impedes in depth analytic assay validation and the generation of harmonized bTMB data. For bTMB measurements to become routine, robust, and accessible to all diagnostic laboratories, we developed contrived patient-like bTMB reference materials. Blood TMB reference materials were formulated by blending and sizing genomic DNA purified from tumor and corresponding normal cell lines by proprietary methodology. Fragment analysis demonstrated that the bTMB reference materials mimic the size distribution typical of purified ctDNA. Sequencing by a targeted gene panel showed the bTMB reference materials had variant allele frequencies (VAFs) characteristic of ctDNA. For example, bTMB reference materials produced with either 2.0 or 0.5% tumor content had mean VAFs of approximately 1.0 and 0.3 %, respectively. Additional sequencing of bTMB reference materials by other gene panels demonstrated comparable results. Evaluation of contrived bTMB reference materials with various cancer gene panels will assist in the standardization of the measurement of bTMB, an emerging biomarker for clinical utility of immune checkpoint inhibitor therapy. Citation Format: Matthew G. Butler, Yves Konigshofer, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella, Russell Garlick. Development of reference material for blood tumor mutational burden measurement [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1982.

Here we describe reference materials for measurable residual disease (MRD) assay design and validation as well as initial data from testing. The analytical validation of liquid biopsy-based assays that attempt to monitor for the disappearance and reemergence of cancer can be challenging due to the need for reference materials that allow for the assessment of sensitivity and specificity at variant allele frequencies (VAFs) that can be over an order of magnitude below those that can be detected reliably by typical circulating tumor DNA (ctDNA) assays. At such low VAFs, a given plasma sample may only contain limited somatic variants - and only at a few copies - which is why some MRD assays focus on monitoring only patient-specific somatic variants and do not analyze other parts of the genome. However, this also means that validation should include the steps needed to monitor patient-specific somatic variants. Therefore, we designed our reference materials for a tumor/normal workflow as a set of three components. First, one cell line is a source of normal DNA that can be used to assess specificity. Second, a germline SNP-matched cell line provides hundreds to thousands of additional somatic variants. Third, blends of fragmented and sized DNA are used to mimic circulating cell-free DNA (ccfDNA) and serve as the input for MRD assays at VAFs from 0.1% to 0.01% (one mutant copy per 3.5 to 35 ng of ccfDNA) and at 0%. As a proof of concept, we analyzed three cell lines for somatic variants by Whole Exome Sequencing (WES). Using the resulting data, digital PCR assays were designed and two different error corrected NGS assays were customized to measure some of the identified variants. Based on past experiments, A>G (T>C) and C>T (G>A) changes had the highest background in NGS data, so these were generally avoided. The tumor and normal samples were used to assess the performance of a given customized assay to measure a variant. Digital PCR was used to verify VAFs in the ccfDNA format, which proved challenging for 0.01% given the expectation of approximately one positive droplet per assay well. NGS was then used to look for the variants using around 50 ng of input per reaction. While the detection of 0.1% VAFs appeared to be feasible with customized off-the-shelf assays, 0.01% was difficult given the need to measure a variant-containing region over 10,000 times to observe a somatic variant - if it was included in the library - and to measure each starting molecule around 10 times for error correction, which required a depth of greater than 100,000 for 20 of several hundred identified candidate variants. While we did not evaluate the reference materials with emerging clinical MRD assays and focused our initial testing on customized off-the-shelf assays, these reference materials should enable the design, validation, and evaluation of MRD assays. Citation Format: Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella, Russell K. Garlick. Reference materials for measurable residual disease (MRD) monitoring [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1978.

Background Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test. Methods Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory. Results Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed. Conclusion This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications.

Determining and using the most effective and safest treatment is of great importance in cancer disease management. Checkpoint inhibitors that target immune regulatory molecules such as PD-1, PD-L1, and CTLA-4, have successfully improved PFS and OS - but only in some patients and for some cancers. In the case of PD-1 and PD-L1 inhibitors, it is believed that PD-L1 expression by a solid tumor allows it to escape attack from the immune system and that by inhibiting the PD-L1/PD-1 interaction immune evasion is no longer possible. This may then cause some of the mutations that give rise to expressed neoantigens to act as targets for T cells and allow for the gradual elimination of the tumor. Since the risk for developing autoimmune adverse events is not insignificant with the use of checkpoint inhibitors, determining which patients are likely to respond favorably to their use is imperative. Similarly, expanding approved uses to new indications also benefits from the identification of populations that are most likely to show improvement in PFS and OS - generally, through clinically-validated biomarkers. Current indications for the use of PD-1 and PD-L1 inhibitors rely on cancer type and several biomarkers. One of these is the expression (or level of expression) of PD-L1 on the tumor. Another - usually in colorectal cancer - is the presence of microsatellite instability (MSI), which indicates that DNA is not being copied with high fidelity and resulting mutations may lead to the emergence of potential neoantigens. While MSI can be assessed by the PCR amplification of several loci, the presence or absence of neoantigens cannot, and they can also be created in the absence of MSI. This has given rise to a potential biomarker - tumor mutational burden (TMB) - that is an assessment of the number of relevant mutations in a tumor. TMB measurement is challenging since different targeted next generation sequencing (NGS) panels look at different regions and percentages of the genome and use different criteria as to what constitutes a relevant mutation. Presently, there is poor correlation between different TMB assays at mutation levels that may be relevant for a companion diagnostic where patients may be denied treatment. Here, we describe the characterization of a panel of reference materials for the assessment, harmonization, and improvement of TMB measurements by NGS assays. The panel is comprised of cancer cell lines and their SNP-matched normals. DNA from unfixed cells was used to establish a baseline truth set of somatic mutations and germline SNPs, and DNA from FFPE cells was used to determine how TMB assessment is affected by fixation artifacts - especially, at lower variant allele frequencies. We present data from high coverage whole exome sequencing - the current gold standard in TMB assessment - and from targeted NGS panels that are more typical of what may be used clinically. We show that accurate measurements at what may be clinically relevant TMB cutoffs remain a challenge. Citation Format: Matthew G. Butler, Yves Konigshofer, Lequan Nguyen, Shikha Kalotra, Omo Clement, Russell K. Garlick, Bharathi Anekella. Improving and standardizing TMB assay performance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3167.

Measurements of circulating tumor DNA (ctDNA) hold great potential to detect residual disease, therapeutic response monitoring and tumor evolution. However, there are many technical challenges including trace levels of circulating cell free DNA 1-10 ng/ml in plasma (300-3,000 genome equivalents/ml), DNA fragment size 170 bps found in the circulation, low variant allelic fractions ~ 0.1% and the need to detect all variant types SNVs, insertions, deletions, CNVs and structural variants. CANCER-ID partners comprise a consortium of experts, companies and institutions for blood -based biomarker validation, assay development, clinical sciences and bioinformatics. This CANCER-ID ring trial was established to help standardize complex ctDNA diagnostic assays by testing a highly complex reference material. The trial looks to improve concordance and to evaluate analytical performance by collecting metrics covering the entire workflow from sample preparation through variant reporting. The common reference material, Seraseq ctDNA Complete, used in the trial is human genomic DNA background material from the Genome-in-a-bottle consortium mixed with synthetic DNA sized to ~170 bp and formulated in artificial plasma. The material contains 25 variants per sample including 12 SNVs (8 genes), 5 deletions (3 genes), 2 insertions (2 genes), 3 CNVs (3 genes) and 3 fusion pairs (3 genes). The variant allelic fractions range from 0.1% to 5% for the SNVs, INDELS and ~ 1 -6 additional copy number for ERBB2, MET and MYC genes. Interlab and intralab testing was done on Roche AVENIO targeted Assay, Agilent SureSelect XT custom assay, QiaSeq Targeted DNA panel, Qiagen GeneRead QIAAct Lung Assay, one clinically validated digital droplet assay and one research digital droplet PCR assays and Agena Bioscience’s UltraSEEK Lung Panel. Data analysis, peer comparison and reporting of common and unique QC metrics was accomplished through the cloud based, analytical QC software iQ NGS QC Management. The results demonstrate that a common full process reference material can be used to compare distinctly different assays formats at different levels of input DNA by mutations. For the 1% AF material, the BRAF V600 variant was detected by all assay formats and the cv’s ranged from 12%-52% or on average for all variants measured by the assay ranged from 11% to 46%. The study furthermore shows that peer review analytical QC software is useful in establishing standardization of ctDNA assays. This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID). Citation Format: Dan Brudzewsky, Rita Lampignano, Sabrina Weber, Alexander Sartori, Sumitra Mohan, Yves Konigshofer, Lorn Davis, Dominic Rothwell, Ed Schuuring, Russell K. Garlick, Thomas Schlange, Ellen Heitzer. Multicenter evaluation of circulating tumor DNA assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 438.

e14746 Background: Next Generation Sequencing based assays are designed to detect genomic aberrations in a limited number of target regions. However, there is a need for accurate measurement of tumor mutational burden (TMB) as low as 4 to as high as 50. As TMB assessment is added to various targeted panels, consistent results between panels are required to advance the broad use of this biomarker. Properly designed reference materials aid measurement standardization and are required to demonstrate assay concordance. We developed reference materials that vary in TMB score, tumor content 5-50% and are prepared in FFPE format. Methods: Seven lung and two breast tumor cell lines as well as matched “normal” lymphoblastoid cell lines were expanded in cell culture. Genomic DNA (gDNA) from each cell line was extracted. Tumor/normal mixes were made by mixing DNA and by embedding cells in FFPE blocks. Whole exome sequencing (WES) results were obtained using Agilent SureSelectXT for library construction and an Illumina Novaseq for sequencing. The Friends of Cancer Research TMB consensus method for analyzing WES data was used to filter variants and calculate TMB scores. Results: The cell lines were grown at large scale to produce extractable gDNA. 100% gDNA tumor, 30% gDNA tumor mixes and 30% FFPE cell line mixes were prepared. Preliminary results show that a clinically-relevant range of TMB values ranging from 4 to 35 mutations per million bases. The several thousand mutations that were observed across the lines were found in a variety of genes, which may explain why TMB in targeted panels is influenced by the specific target regions. Also, the initial results show that 30% cell line mixes showed similar TMB results to 100% gDNA. Conclusions: Our approach with wide ranging TMB values as tumor normal mixes is flexible and can be used to test different tumors and assays. For this study we established WES as the ground truth measurement for comparison to other assay formats and obtained comparison data from other panels. This approach also allows laboratories to test additional variables including formalin fixation, sample extraction, gene panel size, target regions, sequencing depth, filtering and limits of detection.

Introduction: Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We describe a method that combines a kittable and efficient wet-bench workflow with accurate dry-bench analytics to reduce costs and turnaround time, and is relevant to clinical research and patient testing. Methods: We developed a comprehensive, targeted NGS technology to enable the ultra-sensitive detection of variants from liquid biopsy samples. Input DNA molecules from Seraseq™ ctDNA v2 reference materials (SeraCare) and >50 matched FFPE and plasma samples were uniquely tagged with a random molecular barcode (MBC), amplified in an efficient PCR protocol, and sequenced using a targeted panel covering >500 somatic mutation hotspots. Sequence-ready libraries were prepared from input DNA within 9 hours. Data were analyzed with a robust bioinformatics pipeline tailored to the library chemistry to correct for multiple background errors. Variants were identified with a site-specific model, which effectively eliminated recurring non-biological aberrations that remained despite MBC-facilitated error-suppression. We verified variant calls in plasma by Droplet Digital™ PCR (Bio-Rad). To evaluate the limit of detection, healthy control and mutation-positive plasma admixtures were prepared and sequenced. Results: Input template molecules were efficiently recovered. The median number of unique MBCs across all amplicons was >90% of expectation based on input DNA quantities for both the tumor-associated plasma and Seraseq™ ctDNA v2 material. SNVs and indels were recognized with >90% sensitivity and PPV at 0.5% allele frequency (AF) in the Seraseq™ ctDNA mutation mixes. In both neat plasma samples and plasma admixtures, we correctly identified tumor mutations down to ~0.1% AF while maintaining a low false-positive rate (sensitivity and PPV remained high for AF ≥0.1%). Reference ctDNA material and tumor-associated plasma had analogous template amplification and variant frequency rates. Conclusions: A fast, efficient, and sensitive NGS panel approach was developed and evaluated, and it demonstrated the reliable and specific detection of rare variants in liquid biopsy specimens. The method is able to distinguish a low frequency ctDNA signal from the overwhelming background noise in plasma cell-free DNA using a streamlined workflow and purpose-built bioinformatics pipeline. This technology may provide an easy-to-use, high-performance, and adaptable NGS diagnostic framework for disease detection and therapeutic intervention monitoring. Citation Format: Jessica L. Larson, Liangjing Chen, Lando Ringel, Blake Printy, Farol L. Tomson, Yves Konigshofer, Sarah Statt, Joseph K. Kaplan, Shobha Gokul, Jeffrey Shelton, Gary J. Latham, Brian C. Haynes. A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5574.

The accurate diagnosis and monitoring of cancer, using circulating tumor DNA (ctDNA), is a major challenge, given the low concentration and complexity of the target molecules. Suitable reference materials are required for clinical laboratories to achieve the high levels of measurement assurance and reproducibility. The methods and instruments for ctDNA assays are rapidly evolving to improve the sensitivity and specificity, resulting in a challenge to compare these different assays for the measurement of the diverse classes of ctDNA cancer biomarkers. To address this goal, a new reference material was developed consisting of 40 cancer mutations in a background of wild-type DNA at six different allele fractions (2%, 1%, 0.5%, 0.25%, 0.125, and 0 %). The cancer mutations include single nucleotide variants, insertions, deletions, and two structural variations that were selected for their clinical importance and challenges for next generation sequencing (NGS) methods. The reference material is formulated in a synthetic plasma matrix at a concentration of 25 ng/mL, levels that are consistent with patient samples. The DNA has been processed to accurately simulate the size of patient ctDNA, resulting in a similar conversion rate of the DNA for next generation sequencing methods. The samples were sent to five different laboratories involved in ctDNA measurements. Digital PCR assays for the variants were developed to confirm the allele fractions by several laboratories. When absorbance, fluorescent dye binding, and PCR methods were used to quantify the extracted ctDNA reference material, the results were significantly different, indicating the need for standard methods and reference materials to ensure reproducible results of this critical step. Different NGS methods (including targeted amplicons, hybrid selection, and SiMSen-seq) and instruments were used by the labs to analyze the refence materials at the different allele fractions. The results showed that the assays had significant differences in the ability to detect classes of variants at different concentrations. This new reference material was shown to be useful to benchmark the sensitivity and selectivity of different NGS methods and bioinformatic pipelines. This multi-laboratory assessment clearly demonstrates that the new reference material is highly valuable tool to ensure the reproducibility of ctDNA measurements including the sample extraction, analytical, and bioinformatic steps. Citation Format: Erica Stein, Hua-Jun He, Kenneth D. Cole, Russell K. Garlick, Yves Konigshofer, Tony E. Godfrey, Michael G. Goggins, Michael Borges, Margaret Gulley, Mickey Williams, Chris Karlovich, Corinne Camalier, Lynn Sorbara, Matthew R. Young, Sudhir Srivastava. Multi-laboratory assessment of a new reference material for quality assurance of circulating tumor DNA measurements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3657.

The limited quantities of ccfDNA in plasma can make it difficult to assess the sensitivities and specificities of circulating tumor DNA (ctDNA) assays because there is often insufficient material to confirm the presence or absence of particular mutations by orthogonal assays. Additionally, there have been reports of discordance between different ctDNA assays with patient samples at lower variant allele frequencies (VAF), but the limited amount of material is a bottleneck to investigating the causes of discordance. To overcome this, we developed a method to amplify nanograms of ccfDNA from donors to microgram quantities. As an initial proof of concept, we amplified ccfDNA from pregnant female donors that were known to be carrying either a normal fetus or one with chromosomal abnormalities. These samples were analyzed externally at commercial Non-Invasive Prenatal Testing (NIPT) laboratories and were found to be compatible with both chromosome counting and SNP-based testing, and fetal fraction could also be assessed. In order to evaluate the suitability of amplified ccfDNA for ctDNA assays, we split ccfDNA samples so that part was analyzed as-is by a given ctDNA assay and part was analyzed after amplification. The same amplified material was also analyzed with four different ctDNA assays on different NGS platforms in order to assess concordance between assays in shared regions. Good concordance was seen between variants and their VAFs identified in native and amplified ccfDNA, although increasing amounts of discordant background errors were observed at lower VAF. With the Archer Reveal ctDNA 28 assay, obtaining more unique fragments was associated with obtaining more low VAF background errors that could be offset by obtaining more replicate reads of a given unique fragment. Discordant variants were often C>T (or the corresponding G>A) that can be generated by cytosine deamination, which can occur by heating the sample during PCR for library construction. In conclusion, we have developed a method for amplifying limited amounts of ccfDNA that generates sufficient donor ccfDNA-like material for analysis by multiple assays. This should enable the development, improvement and validation of assays that analyze ccfDNA - and especially ctDNA - because sufficient material can be generated in order to assess sensitivity and specificity using orthogonal assays. Additionally, this should also allow for concordance studies that attempt to analyze the same material at multiple sites and with multiple assays. Citation Format: Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Katherine Bianco, Karl G. Sylvester, Bharathi Anekella, Russell K. Garlick. Donor-derived circulating cell-free DNA (ccfDNA) reference materials for concordance studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5569.

Introduction: Copy number variation (CNV), including gene amplification and deletion, can be a key driver of oncogenesis. Pathogenic CNVs are often associated with unfavorable prognosis and drug resistance, therefore detection of these types of genetic changes will be important for personalized treatment. Many tumor profiling workflows can detect CNVs in addition to somatic mutations, but well-characterized reference materials for CNVs are not widely available, which makes it difficult to assess accuracy and sensitivity. CNV content of cell lines can be variable and may change with passages, the genomic background may not be diploid, and germline and somatic variants may not be well characterized. Remnant patient samples often lack characterization and the volumes needed for such characterization and larger validation studies. Therefore, we developed and evaluated Seraseq CNV reference materials to serve as accuracy, precision, and limit-of-detection (LOD) controls for tumor profiling assay development and validation. By providing a consistent source of purified DNA with stable CNV content, these reference materials allow for repeatable assessment of clinical diagnostic assays targeting CNVs. Methods: Seraseq CNV reference materials were prepared by mixing genomic DNA with additional copies of target genes from the well-characterized cell line GM24385. Copies of ERBB2, FGFR3, and MYC were combined with genomic DNA to simulate CNVs found in breast cancer, while sequences for EGFR, MET, and MYCN were used to mimic lung and brain cancer CNVs. Three levels of amplification were targeted: +3, +6, and +12 additional copies. The copy number of each gene was precisely quantified using digital PCR and confirmed on several NGS-based assays internally and externally. Results & Conclusions: Testing revealed CNVs of each target gene were close to the expected copy number. Good correlation was observed between digital PCR data and NGS data generated internally with the Archer VariantPlex Solid Tumor assay. In addition, internal and external NGS data showed correlation and broad assay compatibility. While CNVs observed clinically can be upwards of 50 additional copies and more easily detectable, LOD controls such as those described here are imperative in evaluating assay performance. Collectively, the data demonstrate the utility of biosynthetic Seraseq CNV reference materials, which serve as superior alternatives to DNA from cell lines or patient-derived material. Citation Format: Dana Ruminski Lowe, Jessica Dickens, Catherine Huang, Yves Konigshofer, Bharathi Anekella. Development of well characterized breast, lung, and brain cancer copy number variation reference materials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4540.

gammadelta T cells contribute to host immune competence uniquely. This is most likely because they have distinctive antigen-recognition properties. While the basic organization of gammadelta T-cell receptor (TCR) loci is similar to that of alphabeta TCR loci, there is a striking difference in how the diversity of gammadelta TCRs is generated. gammadelta and alphabeta T cells have different antigen-recognition requirements and almost certainly recognize a different set of antigens. While it is unclear what most gammadelta T cells recognize, the non-classical major histocompatibility complex class I molecules T10 and T22 were found to be the natural ligands for a sizable population (0.2-2%) of murine gammadelta T cells. The recognition of T10/T22 may be a way by which gammadelta T cells regulate cells of the immune system, and this system has been used to determine the antigen-recognition determinants of gammadelta T cells. T10/T22-specific gammadelta T cells have TCRs that are diverse in both V gene usage and CDR3 sequences. Their Vgamma usage reflects their tissue origin, and their antigen specificity is conferred by a motif in the TCR delta chain that is encoded by V and D segments and by P-nucleotide addition. Sequence variations around this motif modulate affinities between TCRs and T10/T22. That this CDR3 motif is important in antigen recognition is confirmed by the crystal structure of a gammadelta TCR bound to its ligand. The significance of these observations is discussed in the context of gammadelta T-cell biology.

It is unclear what the antigen recognition determinants of γδ T-cell receptors (TCRs) are. Compared with immunoglobulin and αβ TCRs, γδ TCRs have the highest potential CDR3 diversity generated by VDJ recombination. However, γδ T-cell reactivities seem to segregate with V gene usage, which has been taken to suggest that rearrangement has little role in generating different antigen specificities. During the past year, the CDR3 regions were found to determine the antigen specificities of T10- and T22-reactive γδ TCRs, a surface protein complex was identified as a ligand for human phosphoantigen-reactive γδ T cells, and the first co-crystal structure of a γδ TCR bound to its ligand was reported. These advances warrant a fresh look at γδ T-cell antigen recognition.

Before the onset of autoimmune destruction, type 1 diabetic patients and an animal model, the nonobese diabetic (NOD) mouse, show morphological and functional abnormalities in target organs, which may act as inciting events for leukocyte infiltration. To better understand these abnormalities, but without the complications associated with lymphocytic infiltrates, we examined genes expressed in autoimmune target tissues of NOD/severe combined immunodeficient (scid) mice and of autoimmune-resistant C57BL/6/scid mice. Our results suggest that the NOD genetic background may predispose them to diabetic complications, including insulin resistance in the absence of high circulating glucose levels and without autoimmune destruction of their β cells. Several of these genes lie within known type 1 and 2 diabetes loci. These data suggest that the NOD mouse may be a good candidate to study an interface between type 1 and type 2 diabetes.

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

An assay’s ability to detect the presence of a KRAS driver mutation is critical when attempting to determine whether a tumor may respond to anti-EGFR therapy. Currently, there are two FDA approved companion diagnostics that can be used to assess whether there are mutations in KRAS codons 12 and 13, and both include their own internal controls. However, there is a need for external reference materials that allow laboratories to validate such assays - especially, at levels near their limits of detection (LODs). We describe the design of the Seraseq™ FFPE Tumor KRAS Reference Material. This reference material is composed of 7 different 5-micron FFPE curls that contain the 7 different KRAS mutations detected by the companion diagnostics: G12A, G12C, G12D, G12R, G12S, G12V and G13D. Each curl contains a mixture of KRAS wildtype GM24385 cells and a KRAS mutant cell line in sufficient quantity for most assays. KRAS mutant frequencies of about 5% were targeted and subsequently verified by digital PCR. A 5% mutant frequency was selected because it is near the LOD for the existing companion diagnostics, and is close to the minimum reported frequency of many next generation sequencing (NGS)-based assays that test for somatic variants. The reference material was evaluated using different methods, which include digital PCR, the Roche cobas® KRAS Mutation Test, the QIAGEN therascreen® KRAS RGQ PCR Kit and several NGS-based assays. Testing revealed general compatibility with these assays in that sufficient DNA could be extracted, and concordance of the results with the expected calls was high. For example, apparent DNA yields by Nanodrop with the cobas assay exceeded the required amount by more than 3-fold and all 7 curls led to a “mutation detected” result in duplicate testing. Interestingly, not all variants were detected with the therascreen assay, which may be due to some variants being present at slightly below the claimed LODs, however DNA yields were sufficient and the correct variants were identified. Finally, variant frequencies obtained by NGS were consistent with those observed by digital PCR. We conclude that the Seraseq FFPE Tumor KRAS Reference Material may be useful as part of KRAS codon 12/13 assay validation. Citation Format: Russell Garlick, Yves Konigshofer, Farol L. Tomson, Dale Yuzuki, Bharathi Anekella. Multiple platform evaluation of KRAS codon 12/13 reference material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4651. doi:10.1158/1538-7445.AM2017-4651

We developed and assessed a novel ctDNA reference material that was designed with an emphasis on commutability. Possessing qualities that are well-matched with clinical specimens is critical for a reference material, however many existing methods of preparing ctDNA analogues result in material that is not commutable for certain assays. For example, sonication of genomic DNA yields fragments across a broad range of sizes that must be used at greater input amounts relative to native cell-free DNA (cfDNA) - even after enzymatic end-repair to reduce damage. This can affect the utility of the reference material for assays that depend on the ligation of adapters to double-stranded DNA, such as many hybrid/capture-based Next Generation Sequencing (NGS) assays. If higher input amounts are not used, then the LOD and precision of such an assay may appear substantially poorer than expected. At the same time, sonicated DNA has been shown to be acceptable for amplicon-based ctDNA assays. We created commutable ctDNA reference material by starting with genomic DNA from GM24385 cells and mixing with synthetic variant-containing DNA sequences at defined ratios. The use of GM24385 cells allows for the comparison of detected variants against the NIST RM 8391 high confidence variants. The use of synthetic DNA permits addition of large numbers of different cancer-associated variants at defined allele frequencies. Fragmentation was followed by size-selection in order to obtain ctDNA-like sizes. Additional steps were performed to increase the amount of output material from the size-selection step. The general method of preparing the reference material is also compatible with native cfDNA, such as that isolated from cancer patients. The reference material was submitted to several testing laboratories along with similar sonicated material. When tested using hybrid/capture-based assays, variant detection was superior in the novel reference material compared to sonicated DNA when similar amounts of material were used as the input. We therefore conclude that our new reference material has greatly improved commutability compared to existing materials composed only of sonicated DNA. Citation Format: Yves Konigshofer, Farol L. Tomson, Matthew Ryder, Russell Garlick, Bharathi Anekella. A commutable circulating tumor DNA (ctDNA) reference material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2017-2738

Introduction: Genomic instability is a hallmark of cancer, and copy number variation (CNV), including gene duplication and deletion, can be a key driver of oncogenesis. Detection of CNV is needed for personalized treatment, and may involve microarray analysis or Next Generation Sequencing. Often, DNA from patient-derived cell lines is used as positive control material in these assays. However, these materials are often poorly characterized, may change over time, and have additional confounding genetic alterations. Here we describe the development of new reference materials that are highly characterized by digital PCR and can aid in the validation and calibration of new diagnostic assays for CNV. Methods: The GM24385 reference cell line has been extensively characterized by the Genome in a Bottle project1 and originates from a participant in the Personal Genome Project. DNA from this cell line was used to make a library of full-length gene inserts. The library had >12X coverage of the GM24385 genome and an average insert size of ~135 Kb. PCR based screening was used to identify and isolate full length MYC, FGFR3 and ERBB2 genes. Next Generation Sequencing, using Illumina Nextera library preparation kits and a MiSeq instrument, was used to verify the gene of interest. The genomic DNA and gene inserts were then formulated into reference materials, in purified DNA format to amplify the copy numbers of these key genes. Digital PCR was used to control the mixing and verify the number of copies of genes and/or mutations within the reference material. Results: The sequence confirmed genes were formulated into GM24385 genomic DNA at levels consistent with 5X and 10X amplification. Testing on the ArcherDx VariantPlex™ Solid Tumor Kit indicated that each amplification could be detected at the expected level. Conclusions: Highly characterized and consistently manufactured reference materials for copy number detection are currently lacking. The described Seraseq CNV Reference Materials provide proof of concept for a biosynthetic, engineered material that uses digital PCR as an orthologous reference method and provides a “ground truth” copy number level for key oncogenes. Citation Format: Catherine Huang, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Highly multiplexed and precisely calibrated reference materials for copy number variation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 738. doi:10.1158/1538-7445.AM2017-738

γδ T lymphocytes in the intestinal intraepithelial layer (γδ IELs) are thought to contribute to immune competence, but their actual function remains poorly understood. Here we used DNA microarrays to study the gene expression profile of γδ IELs in aYersiniainfection system to better define their roles. To validate this approach, mesenteric lymph node CD8+αβ T cells were similarly analyzed. The transcription profiles show that, whereas lymph node CD8+αβ T cells must be activated to become cytotoxic effectors, γδ IELs are constitutively activated and appear to use different signaling cascades. Our data suggest that γδ IELs may respond efficiently to a broad range of pathological situations irrespective of their diverse T cell antigen receptor repertoire. γδ IELs may modulate local immune responses and participate in intestinal lipid metabolism, cholesterol homeostasis, and physiology. This study provides a strong basis for further investigations of the roles of these cells as well as mucosal immune defense in general.

Full process controls and validation materials for next generation sequencing (NGS)-based molecular diagnostics in Oncology that mimic a biopsy sample and are renewable and scalable have been elusive. Patient-derived Xenografts (PDXs), which are similar in morphology and somatic mutations to original tumors, may present a solution to this pressing need. In order to evaluate the abilities of PDXs to serve as full process controls and validation materials, we had PDXs that contained established driver mutations KRAS G12C analyzed by clinical laboratories using a variety of different NGS-based assays. The resulting data was analyzed for genetic homogeneity as well as for the presence of mouse-derived variants. PDXs were found to be generally compatible with NGS-based assays and similar variant frequencies of driver mutations were reported. High concordance was also observed in variant frequencies of PDXs at passage 5 that were derived from the same tumor but grown in different mice. For example, the observed differences in over 90% of variants and all driver mutations were not statistically significant. In amplicon-based assays, which represent the majority of current assays that look at mutational hotspots, mouse-derived variants were rare. Typically, variant data was usable without any further processing. In hybrid/capture-based assays, mouse-derived variants were common because of the lower binding stringency used by these assays. Without further processing, this manifested in slightly lowered variant frequencies for driver mutations - where wild-type “reference” sequences are often conserved between species and thus inflate the number of reference sequences - and greatly elevated numbers of total variants. However, by processing the data at the FASTQ stage using in-house developed software, it was possible to effectively remove mouse-derived variants in a manner that would be compatible with patient testing as well. In conclusion, we show that PDXs are compatible with NGS-based assays and can be used to control the full process from the point where the Pathologist first fixes the tissue. Those containing driver mutations that have been previously characterized in cancer also enable laboratories to control and validate their assays by showing that they can consistently detect these mutations. Citation Format: Yves Konigshofer, Bharathi Anekella, Russell Garlick. Patient-derived xenografts as validation material for NGS assays: Analysis of genetic homogeneity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1385.

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

Background: High-density lipoprotein (HDL) is believed to be protective against coronary heart disease (CHD). HDL is comprised of different subfractions. Of these, HDL 2b is believed to be the most important in preventing CHD. Current methods for HDL subfraction measurements are not standardized and often exhibit poor analytical performance, which can limit their usefulness in clinical practice. Methods: We have developed a microfluidic Lab-on-Chip electrophoresis system for serum HDL subfraction measurements. Linear polymers are used as a sieving buffer for HDL particle separation. Nine samples and two controls can be analyzed in 30 min. The percent HDL 2b that comprises total high-density lipoprotein cholesterol (HDL-C) is reported. Results: Samples with HDL-C of 0.259–2.072 mmol/L could be evaluated for percent HDL 2b with coefficient of variations (%CVs) below 8%. Total precision was typically below 1.5% and linearity was observed between 8% and 32% HDL 2b. Measurements were not affected by many therapeutic and biological compounds. Consistent results were obtained by three laboratories and showed r2 values between 0.93 and 0.95. Conclusions: The assay allows for easy and reproducible measurement of percent HDL 2b. The experimental procedure and small size of the instrumentation needed for measurement make it viable in clinical settings. Clin Chem Lab Med 2010;48:883–9.

Background: Alterations in high-density lipoprotein (HDL) subfractions, especially in the HDL2b subfraction, have been reported in type 2 diabetes mellitus (T2DM). However, new methods for convenient and reliable quantitation of HDL2b are yet to be developed.Methods: Thirty-eight patients with T2DM were enrolled and age-, sex- and body mass index (BMI)-matched controls were selected from the same population. A microfluidic chip method was employed to analyse serum HDL subfractions.Results: The microfluidic chip method revealed a significant reduction in HDL2b and its ratio to total HDL in T2DM patients. There was a reverse correlation for total HDL and HDL2b, and its ratio with triglycerides, homeostasis model assessment-insulin sensitivity index (HOMA-IS) and insulin resistance index (HOMA-IR).Conclusions: We have shown a reduction of HDL2b and its ratio to total HDL by a novel chip method in T2DM patients. The significant correlation between HDL2b and HOMA-IS and HOMA-IR may have further predictive valu...

Background: It has been shown that low concentrations of high-density lipoprotein cholesterol (HDL-C) are associated with non-alcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM). HDL2b, a major subfraction of high-density lipoprotein (HDL), is more significantly correlated with coronary heart disease (CHD) compared with total HDL. In this study, we analysed HDL2b in a cohort of Chinese T2DM subjects with or without NAFLD. Methods: A highly sensitive and reliable microfluidic chip method was adopted to measure HDL2b. In total, 48 T2DM patients with NAFLD diagnosed by a B-ultrasound were enrolled from our Beijing Community Pre-Diabetes (BCPD) study cohort. A total of 48 age and gender matched diabetic controls without NAFLD were selected from the same population. Results: Clinical characteristics and serum biochemical analyses demonstrated a significantly increased body mass index (BMI), waist circumference, homeostasis model assessment-insulin resistant index (HOMA-IR), total cholesterol (TC), and triglyceride (TG) concentrations in the NAFLD group. However, the concentrations of HDL2b and its ratio to total HDL in NAFLD patients was decreased, compared with controls (p

Background: High-density lipoprotein (HDL) subtractions are among the new emerging risk factors for atherosclerosis. In particular, HDL 2b has been shown to be linked to cardiovascular risk. This study uses a novel microfluidics-based method to establish HDL 2b clinical utility using samples from the Prospective Cardiovascular Muenster (PROCAM) Study. Methods: Method performance was established by measuring accuracy, precision, linearity and inter-site precision. Serum samples from 503 individuals collected in the context of the PROCAM study were analyzed by electrophoresis on a microfluidics system. Of these, 251 were male survivors of myocardial infarction (cases), while 252 individuals were matched healthy controls. HDL cholesterol, HDL 2b concentration and HDL 2b percentage were analyzed. Results: This novel method showed satisfactory assay performance with an inter-site coefficient of variance of

gammadelta T cells contribute to host immune competence uniquely. This is most likely because they have distinctive antigen-recognition properties. While the basic organization of gammadelta T-cell receptor (TCR) loci is similar to that of alphabeta TCR loci, there is a striking difference in how the diversity of gammadelta TCRs is generated. gammadelta and alphabeta T cells have different antigen-recognition requirements and almost certainly recognize a different set of antigens. While it is unclear what most gammadelta T cells recognize, the non-classical major histocompatibility complex class I molecules T10 and T22 were found to be the natural ligands for a sizable population (0.2-2%) of murine gammadelta T cells. The recognition of T10/T22 may be a way by which gammadelta T cells regulate cells of the immune system, and this system has been used to determine the antigen-recognition determinants of gammadelta T cells. T10/T22-specific gammadelta T cells have TCRs that are diverse in both V gene usage and CDR3 sequences. Their Vgamma usage reflects their tissue origin, and their antigen specificity is conferred by a motif in the TCR delta chain that is encoded by V and D segments and by P-nucleotide addition. Sequence variations around this motif modulate affinities between TCRs and T10/T22. That this CDR3 motif is important in antigen recognition is confirmed by the crystal structure of a gammadelta TCR bound to its ligand. The significance of these observations is discussed in the context of gammadelta T-cell biology.