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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.

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.

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.

Tumor profiling often begins with a tissue biopsy that is formalin fixed and paraffin embedded (FFPE), a process that introduces various kinds of damage in the nucleic acids found in the tissue, however FFPE reference materials that closely mimic the damage profile of patient samples are lacking. Depurination, depyrimidination, deamination, oxidation, nicks, and double strand breaks may be found in DNA extracted from FFPE tissue, despite the use of extraction kits that attempt to repair some of this damage. We have developed a formalin-damaged, multiplexed biosynthetic reference material, Seraseq® FFPE Tumor DNA, to mimic this type of damage found in patient samples to create a more patient-like control. Biosynthetic DNA containing 24 cancer variants of interest were pooled in equivalent concentrations and introduced into the GM24385 reference cell line (Coriell) to simulate SNVs, indels, and structural rearrangements. Full length, allele-specific copies of ERBB2, MET, and MYC were also introduced to create copy number amplifications (CNV). Engineered cells were diluted to achieve desired allele frequencies and copy numbers as determined by digital PCR. The cells were processed with a proprietary FFPE protocol that mimics the damage seen in patient samples. Various FFPE extraction kits were used to isolate DNA and the amount of damage was assessed using the KAPA hgDNA Quantification & QC Kit. Allele frequencies and copy numbers were determined by digital PCR, the Archer VariantPlex Solid Tumor assay, and the Illumina TruSight Tumor 170 assay. DNA extractions of the FFPE reference material with the Qiagen QIAamp DNA FFPE Tissue Kit and the Promega Maxwell RSC FFPE DNA Kit yielded similar amounts (>200 ng per curl). All 24 variants were detected close to the 5-10% targeted allele frequency range and CNVs were within 5-10 total copies by dPCR. Similar results were observed with the TST170 assay. The quality as measured by Q120 bp/Q41 bp ratio using the KAPA hgDNA Quantification & QC Kit of the FFPE extracted damaged DNA was reduced by ~33% compared to non-damaged DNA extracted from FFPE, closer to the quality seen for some patient samples while still maintaining good amplifiability in downstream processes. The Seraseq FFPE Tumor DNA Reference Material performs as designed in digital PCR, amplicon-based, and hybrid capture-based NGS assays. It can be used in full process assay development and validation, bioinformatics pipeline optimization, and as a positive control in clinical research. The biosynthetic manufacturing approach allows for multiplexing of customizable variants and adjustable DNA damage. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Matthew G. Butler, Omoshile Clement, Bharathi Anekella. Development and performance of a formalin-damaged multiplexed DNA tumor mutation FFPE reference material [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 1322.

The large range of genetic aberrations and genes involved Myeloid malignancies, clonal diseases of hematopoietic progenitor cells which can lead to accumulation of immature blast cells in the bone marrow and peripheral blood, make Next Generation Sequencing (NGS) assays a cost effective and sensitive way to determine genetic changes. Understanding the genetic changes which lead to the clonal proliferation can aid in both determining prognosis and therapy selection. Consequently, there are an increasing number of NGS-based oncology tests available for somatic mutation detection in patients with hematological malignancies. However, reference materials that contain the relevant genetic blood cancer mutations are lacking. We designed a purified RNA reference material that contains nine RNA fusions two different ETV6-ABL1 transcripts, including BCR-ABL1, MYST3-CREBBP, RUNX1-RUNX1T1, and PML-RARα which are important in myeloid cancers. Conversely, a purified DNA reference material was designed to include 23 somatic variants included two FLT3 internal tandem duplications. The biosynthetic RNA or DNA constructs were mixed with either total RNA or purified genomic DNA from GM24385 reference human cell line. Digital PCR was used to quantify the variant sequences to determine the abundance of the fusion RNAs and the allele frequencies of the somatic mutations. NGS testing on a variety of targeted NGS assays was then performed to show compatibility. The Seraseq Myeloid RNA Mix was tested by digital PCR to confirm that each of the nine fusions in the reference samples are present at approximately 100 fusion copies per nanogram of total RNA. The FusionPlex Myeloid Kit for Illumina (ArcherDx) as well as the Oncomine Myeloid Panel (Thermo Fisher) showed positive detection for all fusions present in the reference samples. In addition, the Seraseq Myeloid DNA Mix was also tested by digital PCR to confirm the variant allele frequencies were on target at the 5%, 10% or 15% VAF. While, dPCR allele specific assays could not be obtained for CEBPA mutations, NGS testing of the reference samples by VariantPlex Core Myeloid Kit for Illumina (ArcherDx) and Oncomine Myeloid Panel (Thermo Fisher) confirmed the presence of these mutations at the expected levels, as well as all of the other variants in the DNA reference samples. In conclusion, SeraCare has developed highly multiplexed RNA and DNA-based reference materials for evaluating Myelogenous disorders by NGS. The reference materials allow monitoring of a broad range of somatic mutations and gene fusions which can aid optimization and verification of detection limits for NGS-based Myeloid disease assay testing, and provide laboratories greater assurance in their ability to correctly detect and quantify various types of genetic events in diseased patient samples. Citation Format: Farol Lovell Tomson, Praveena Kamineni, Catherine Huang, Jessica Dickens, Dan Brudzewsky, Omoshile Clement, Ram Santhanam, Bharathi Anekella. NGS-based reference materials for fusion and somatic variant detection in myeloid cancers [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 4315.

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.

Introduction: Gene fusions, usually the result of chromosomal rearrangements, are frequently associated with many cancer types, and hence clinically actionable, making fusion detection an important part of cancer disease management. We developed a new, optimized version of the Seraseq® Fusion RNA Reference Material, and demonstrate its consistent performance across different NGS enrichment assays, sequencing coverage depths, bioinformatics pipelines, and RNA mass inputs from a multi-lab investigation (Sites A, B, C, D & E). Methods: Biosynthetic DNA was used for transcription of 18 RNA fusions, including more common fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. The in vitro transcribed RNAs were mixed with total RNA extracted from GM24385 reference cell line (The 1000 Genomes Project, Coriell). Digital PCR with TaqMan® chemistry was used to determine the target fusion RNA concentration and serve as the “truth” data set for comparison to NGS, which can be variable depending on input, assay, and bioinformatics. The fusion-total RNA mix was analyzed by five external laboratories; it was tested using the ArcherDx FusionPlex™ Solid Tumor Panel (Site A, Site B, Site C), the ArcherDx FusionPlex™ CTL Panel (Site C), the ArcherDx FusionPlex™ Lung Panel (Site B), a custom ArcherDx FusionPlex™ Panel (Site D), and the TruSight Tumor 170 Panel (Site E). Results: All eighteen (18) fusions in the new Seraseq Fusion RNA Mix v4 reference standard were detected as expected on each NGS platform with an average of greater than 85% of on-target reads across all assays. Even at inputs as low as 20 ng, all 18 fusions were typically detected above fusion-calling thresholds. In general, the results for individual fusions among the different NGS panels and among replicates were concordant, with observed variance in reads across some fusion junctions between assays and replicates. Within FusionPlex assay results, the average percent of reads supporting the fusion call across all fusions was about 63%, regardless of input (a range between 20 to 250 ng). Collectively, the multi-lab results confirm that the Seraseq® Fusion RNA Mix v4 reference standard is compatible with both amplicon and hybridization-capture based NGS assays. Conclusions: Seraseq RNA Fusion Mix v4 has broad NGS assay compatibility and allows for reliable and simultaneous detection of 18 clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this reference standard for targeted NGS assay development, assay validation, bioinformatics pipeline optimization, and as positive controls in clinical NGS RNA fusion assays. The biosynthetic manufacturing approach produces reference materials that provide consistent results for a wide variety of common and rare gene fusions. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella. Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study [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 1709.

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.

HIV-1 Integrase (IN) is an essential enzyme for viral replication. The discovery of beta-diketo acids was crucial in the validation of IN as a legitimate target in drug discovery against HIV infection. In this study, we discovered a novel class of IN inhibitors using a 3D pharmacophore guided database search. We used S-1360 (1), the first IN inhibitor to undergo clinical trials, and three other analogues to develop a common feature pharmacophore hypothesis. Testing this four-featured pharmacophore against a multiconformational database of 150,000 structurally diverse small molecules yielded 1,700 compounds that satisfied the 3D query. Subsequently, all 1,700 compounds were docked into the active site of IN. On the basis of docking scores, Lipinski's rule-of-five, and structural novelty, 110 compounds were selected for biological screening. We found that compounds that contain both salicylic acid and a 2-thioxo-4-thiazolidinone (rhodanine) group (e.g. 5-13) showed significant inhibitory potency against IN, while the presence of either salicylic acid or a rhodanine group alone did not. Although some of the compounds containing only a salicylic acid showed inhibitory potency against IN, none of the compounds containing only rhodanine exhibited considerable potency. Of the 52 compounds reported in this study, 11 compounds (5, 6, 8, 10-13, 32-33, 51, and 53) inhibited 3'-processing or strand transfer activities of IN with IC(50) < or = 25 microM. This is the first reported use of S-1360 and its analogues as leads in developing a pharmacophore hypothesis for IN inhibition and for identification of new compounds with potent inhibition of this enzyme.

The binding of metal ions to heteroatomic centers of biomolecules has been utilized as a probe of metal ion effects in living systems. This article focuses on the effect of N-coordination by transition metals, especially Pt(II), Co(III), Cr(III), on isotopic C(2)−H or C(8)−H exchange of imidazoles, thiazoles, and purines. The usual reactivity trend, protonated ≫ metalated ≫ neutral substrate, is excepted for Cr(III)/1-methylimidazole, where Cr(III) activates stronger than H+. An interplay of factors is considered, including metal-to-ligand back-bonding, electronic structure of metal ions, and differences in crystal field stabilization energy.

The detritiation kinetics of the Cr(III) complexes 1 and 3-6 have been studied in aqueous buffers at 35°C, using the liquid scintillation counting technique. Results for 5 and 6 provide benchmark kinetic data for N-T/N-H exchange under the experimental conditions of the study and have aided in the delineation of N-T/N-H from C-T/C-H exchange in the parallel reactions observed experimentally. Curved first-order plots obtained for 1, 3, and 4 were treated to yield rate constants for two parallel exchange reactions kobsA and kobsB for the "fast" and "slow" processes, respectively. The "fast" process has been assigned to C(2)-H exchange in 1, competing N-H and C(2)-H exchange in 3, and N-H exchange in 4. In all cases, the "slow" process is associated with C(4,5)-H exchange. Identification of exchange sites in 1, 3, and 4 was made possible by the consideration of the results of an IR spectroscopic study of hydrogen-deuterium exchange, comparison of the extent of 3H incorporatation in different complexes in the tritiation experiments, and a careful analysis of the exchange kinetic data. Analysis of the rate data indicates that Cr(III) significantly enhances C(2)-H exchange in 1 and 3, while C(4,5)-H exchange, hitherto reported in the literature only under drastic reaction conditions, was observed for 1, 3, and 4 under the mild conditions of the present study. Quantitation of the effect of Cr(III) coordination on 3H exchange in imidazole-type nuclei was achieved fully in 1; giving kM+ values of 6 × 103 and 7 × 102 M-1s-1 for C(2)-H and C(4,5)-H exchange, respectively. Using the literature value for kH+, the second-order rate constant for C(2)-H exchange under H+ catalysis, 2.9 × 102 M-1s-1, it follows that Cr(III) is ca. 20 times better as a catalyst for C(2)-H exchange in 1-methylimidazole than H+, providing the first example of a metal ion being more effective than a proton in these processes. Comparison of the results obtained with 1 with literature results for 2 shows a very large (ca. 3 × 105-fold) difference in the catalytic activities of Cr(III) and Co(III), favouring the former. The dichotomy in the effectiveness of the two metal ions in catalyzing 3H exchange in the imidazole nucleus has been ascribed to differences in (i) extent of Mn+—N bond polarization (and the consequent effect on ligand C-H acidity); (ii) electronic configuration; (iii) crystal field stabilization and activation energies; and (iv) importance of metal-ligand π back-bonding. The study highlights the diversity of factors and complexity of interactions involved in determining the role of metal ions in biological systems, especially where such processes involve complex formation between metal ions and heterocyclic fragments of biomolecules.Key words: metal ion-biomolecule interactions, methylimidazole, isotopic hydrogen exchange, catalysis by chromium.

A molecular mechanics (MM3(96)) force field is reported for modeling metal complexes of amides in which the amide is coordinated through oxygen. This model uses a “points-on-a-sphere” approach whic...

This report describes the synthesis and characterization of Pt(II)-bis(guanosine)(R-phen) complexes (where R=H (phen) or 2,9-dimethyl (dmphen)), as a model for bifunctional covalent adduct formation between antitumor (dichloro)Pt(II)amine complexes and DNA bases. The 1 H and 13 C NMR spectra (in D 2 O) of the isolated product in the reaction of the corresponding [(dichloro)(R-phen)Pt(II)] complex, with 2 mole equivalents of guanosine, reveal formation of a cis -bis(guanosine)(phen)Pt(II) ( 1 ) and a trans -bis(guanosine)-(dmphen)(Cl)Pt(II) ( 2 ). The 1 H NMR spectrum of 1 reveals a mixture of head-to-tail ( htt ) and head-to-head ( hth ) rotamers that do not interconvert on the NMR time scale (200 and 400 MHz, 298–343 K). For complex 2 the formation of a trans -bis(guo) complex with monocoordinated dmphen ligand is in accord with recent findings in analogous reactions of [Pt(dmphen)Cl 2 ] with alkenes, alkynes and aryl phosphine ligands. It is noteworthy that the room-temperature 1 H NMR spectrum of 2 in D 2 O showed no intramolecular process due to ‘flipping’ of the mono-coordinated dmphen ligand; this is in contrast to observations with trans -bis(PPh 3 ) complex (in CD 2 Cl 2 ,RT) previously reported. The NMR spectral data of the two complexes have been re-examined and the different behavior is discussed.

This study describes the synthesis and characterization of two new compounds, i.e. [Pt(en)(DMSO)Cl][Pt(DMSO)Cl3] (2) and [Pt(bn)2]Cl2 (3), which were isolated from reactions of cis-[Pt(DMSO)2Cl2] (1) with 1,2-diaminethane (en) and 1,4-diaminobutane (bn), respectively. The complexes 2 and 3 were characterized by NMR, IR and elemental analysis, and their molecular structures have been determined by X-ray crystallographic analysis. Compound 2 crystallizes in the monoclinic space group P21/n with a = 8.093(1),b = 7.599(2), c = 28.041(5) A, β = 91.09(2)° and Z = 4 (dinuclear units. The structure consists of hydrogen bonded pairs of [Pt(en)(DMSO)Cl]+ cations and [Pt(DMSO)Cl3]− anions with the PtPt distance of 3.655(1) A. The five-membered ring of the Pt-1,2-diaminoethane ligand has a C1-envelope conformation. Also observed is the normal trans influence of coordinated DMSO, as reflected in significantly longer Pt-ligand bonds trans to this ligand. Compound 3 crystallizes in the orthorhombic space group Pnma with a = 11.231(3), b = 8.045(2), c = 16.998(3) A and Z = 4 . The structure is composed of the bis(1,4-diaminobutane) platinum(II) cation and two uncoordinated chloride ions all located in special positions in the unit cell; the Pt and both chloride ions lie on the mirror plane perpendicular to the y-axis. Each of the two bn ligands is statistically disordered across a mirror plane over two conformations with 50% occupancy, while the seven-membered chelate ring of the Pt-bn adopts a twist-chair conformation. The cations with a unique ‘spider-like’ shape are hydrogen bonded to chloride ions in a three-dimensional network in the crystal lattice.

A kinetic study of hydrogen−deuterium (H/D) exchange in Pt(II)−1-methylimidazole complexes has been performed in D2O/NaOD solution, at 60 °C, by means of 1H NMR spectroscopy. Isotopic exchange has been observed at C(2)−H, C(4)−H, and C(5)−H of the imidazole moiety. Kinetic data analysis of H/D exchange in the complexes cis-[Pt(NH3)2(MeIm)2]Cl2 (3), trans-[Pt(NH3)2(MeIm)2]Cl2 (4), [Pt(en)(MeIm)2]Cl2 (5), and [Pt(MeIm)4](ClO4)2 (6) revealed that Pt(II) enhances C(2)−H exchange in the coordinated 1-methylimidazole (MeIm) by ca. 102 to 103, relative to the neutral substrate. However, it is ca. 104−105 times less effective compared to H+ and CH3+. In complex 5, C(5)−H exchange is ca. 6 times faster than C(4)−H exchange, in contrast with expectations based on an inductive/field effect by Pt(II). The observation of the relative reactivity order, C(5)−H exchange > C(4)−H exchange in 5, is discussed by consideration of contributing resonance structures of the intermediates formed upon abstraction of C(4)−H and C(5...

Hydrogen–deuterium exchange of the carbon-bound protons of thiazole coordinated to PtII in complexes 1, 2 and 3, has been measured in aqueous buffer solutions, at 60 °C, by 1H NMR spectroscopy. Analysis of the rate data indicates that PtII enhances exchange of C(2)–H by a factor of ca. 106, with respect to the neutral substrate, but is ca. 103–104 times less effective compared to H+ or Et+. Exchange of C(5)–H is enhanced ca. 6 × 103 by PtII relative to the neutral heterocycle; an effect much less than that for C(2)–H exchange. We also report the first quantitative measurement of C(4)–H exchange in a thiazole moiety, as observed in 1 under moderate conditions (pD 8.69 and 60 °C, k2= 2.54 dm3 mol–1s–1). The C(5)–H exchange process in complex 1 is moderately faster than C(4)–H exchange, a result opposite to that expected on the basis of an inductive/field effect by PtII. This is ascribed to the enhanced stabilization of the carbanionic intermediate at C(5) by the adjacent sulfur atom which is absent in the carbanion at C(4). This factor overrides the inductive/field effect of PtII, and accounts for the order of reactivity observed in this study, C(2)–H C(5)H > C(4)–H.

The binding of metal ions to heteroatomic centers of biomolecules has been utilized as a probe of metal ion effects in living systems. This article focuses on the effect of N-coordination by transition metals, especially Pt(II), Co(III), Cr(III), on isotopic C(2)−H or C(8)−H exchange of imidazoles, thiazoles, and purines. The usual reactivity trend, protonated ≫ metalated ≫ neutral substrate, is excepted for Cr(III)/1-methylimidazole, where Cr(III) activates stronger than H+. An interplay of factors is considered, including metal-to-ligand back-bonding, electronic structure of metal ions, and differences in crystal field stabilization energy.

Hydrogen-deuterium exchange of the carbon-bound C(8)-H protons of the inosine residues in tetrakis(inosine)platinum(ii) chloride, S, with Pt binding at N(7), was studied in aqueous buffer solutions at 60 degrees C by (1)H NMR spectroscopy. The kinetics at all four C(8) sites as a function of pD of the D(2)O/OD(-) medium was measured through the disappearance of the C(8)-H signal, which yielded the pseudo first-order rate constant for exchange, k(obs). Plots of k(obs)versus [OD(-)] showed curvature reminiscent of saturation type kinetics and indicative of competitive deprotonation of N(1)-H sites. In contrast, the analogous N(1)-methylated cis-bis(1-methylinosine)diammineplatinum(ii) chloride leads to a linear k(obs)versus [OD(-)] plot. The potentiometrically determined macroscopic composite N(1)-H ionization constant was further dissected into the successive microscopic N(1)-H acidity constants of the four inosine residues of the complex S. The k(obs) values were also deconvoluted into individual rate constants k(ex) (i.e.k(0), k(1), k(2), k(3) for exchange of the successively deprotonated inosine moieties, S, S(1), S(2), S(3), it being assumed that S(4) where all four inosine ligands are deprotonated at N(1) is unreactive ("immunized") to exchange. The k(ex) values show a progressive attenuation in Pt activation of H-D exchange along the series, k(0), k(1), k(2), k(3). The k(ex) data thus generated, together with the deconvoluted individual pK(a) values allow the construction of the plot, log k(ex) [C(8)-H] vs. pK(a) [N(H)-1]. Remarkably, this plot exhibits good linearity (R(2) = 0.99), which accords this as a linear free energy relationship (LFER). The large negative slope value (-2.3) of this LFER reflects the high sensitivity of transmission of electron density from the ionized N(1) via Pt and/or through space to the remaining C(8)-H sites. This is to our knowledge the first instance in which a LFER is generated through modulation of a structure in a single molecule. One can anticipate that this approach may lead to: (1) predicting N-H acidity; (2) C-H H-D exchange susceptibility in a range of metal-biomolecule complexes; (3) their carbon acidity.