ALBERT

Description: Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Description: DNA standards are a critical resource in diagnostic and research labs. Positive and negative controls are essential for validating the sensitivity and specificity of next generation sequencing (NGS) and other genetic assays. Several commercial DNA standards have been widely used to validate clinical oncology assays. However, as genomic technologies have become more sensitive, the metrics defining what a "gold standard" entails have not been carefully re-evaluated. Cell culture and synthetic chemical means of generating DNA standards have the potential to artificially introduce mutations. Cell lines are often exposed to supraphysiologic levels of reactive oxygen. Oligonucleotide synthesis has an error rate orders of magnitude above biological levels. This background may not interfere with detection of clonal or sub clonal variants with moderately sensitive assays, but it will obscure the presence of very rare mutations when assessing with extremely sensitive ones and it will decrease apparent assay performance. In this study, we applied Duplex Sequencing (DS), an extremely sensitive NGS technology with an error rate below one-in-ten-million, to characterize widely used commercial myeloid DNA standards. Duplex sequencing is an error correction method that independently sequences and compares the two strands of original DNA molecules to enable error correction. The cross-strand comparison eliminates errors caused by DNA damage, early cycle PCR errors and various technical errors that escape standard NGS and other error-corrected NGS methods. We performed DS on two commercial myeloid leukemia DNA standards and an in-house control DNA. Commercial standard A (CS-A) is constructed of a mix of DNA from multiple mutation-containing cell lines. Commercial standard B (CS-B) is constructed of synthetic mutant DNA molecules mixed into DNA from a single well-characterized cell line. The control DNA was obtained from the donated apheresis product of a healthy 25-year old never-smoker. We used a hybrid capture panel covering 15 variants with a 5%-40% expected variant allele frequency (VAF) in CS-A, and 12 variants with a 5%-15% expected VAF in CS-B. CS-A, CS-B and control DNA were sequenced to maximum Duplex depths of 12,256x, 15,844x and 38,535x, respectively. In the two commercial DNA standards, all targeted variants were detected by DS. The majority of variants were within +/- 1.5-fold from the expected VAF. For CS-A, the correlation of DS vs. vendor target VAF was strong (r2 = 0.96). For CS-B, the correlation between DS and vendor target VAF was very weak (r2 = 0.07), although was better correlated to vendor-reported ddPCR and conventional NGS (r2 = 0.49 and 0.61). We also performed DS on a specially designed in-house standard, comprised of apheresis-derived control DNA with a series of mutant cell lines spiked at low frequencies from 1/100 to 1/100,000, that we sequenced to more than 1 million-fold Duplex depth. 9/9 mutations were detected down to a target VAF of 10-5 with r2 = 0.96 for DS vs. target values. We performed DS on a second in-house mutation dilution series which included the CS-A standard and other samples diluted into apheresis control DNA, and detected 9/9 mutations down to a target VAF of 4x10-5 with r2 = 0.93. The overall mutation frequencies (number of unexpected mutations / total number of base-pairs sequenced) of CS-A, CS-B and the control were 1.6x10-6, 2.1x10-6 and 4.7x10-7, respectively. The in-house mutation mixes were both 5.4x10-7. CS-A carried 96 low-frequency clonal variants (≥2 counts,

Description: Acute myeloid leukemia (AML) is a challenging disease to treat: most patients achieve remission after induction chemotherapy, but the majority eventually relapse. Minimal residual disease (MRD) after initial treatment is the best predictor of relapse and is thus a critical metric around which to develop new treatments. However, conventional MRD diagnostics, including cytology and flow cytometry, are of variable sensitivity and often only perform well in specialty centers, and there is no gold standard. Molecular tests developed to measure trace MRD in other hematological malignancies (i.e. CML) are high-resolution but assay a single, universally-present mutation, while many different genetic drivers exist in AML and these are spread among dozens of genes. Virtually every AML patient harbors a unique combination of mutations, making it difficult to design an effective universal assay. As such, most reported molecular AML MRD assays are either sensitive for mutations that are only found in a narrow subset of patients, or can screen many potential sites of mutation, but with low sensitivity. Here we present a broadly applicable Duplex Sequencing-based AML MRD assay that can readily detect mutant allele frequencies (MAF) below 1/10,000 across a large panel of genes, and below 1/100,000 in a focused panel. Conventional next generation sequencing (NGS) introduces errors during amplification and sequencing, creating a background of artifactual noise that obscures true mutations present below ~1%. Duplex Sequencing improves accuracy 〉100,000-fold through a molecular tagging approach whereby both strands of each original DNA duplex are ligated with a unique molecular barcode and amplified such that the reads generated from each strand can be related back to their unique original duplex. Reads can also be distinguished from those of their mate strand, thus the two strands of each DNA duplex can be compared and any discrepant nucleotide positions are discounted as errors. Our complete AML panel targets 151 exons or hotspot codons in 29 genes with a 59 kilobase (kb) hybrid-capture footprint. This region comprises loci containing single-nucleotide and short indel mutations found in approximately 90% of adult AMLs. A mean Duplex error-corrected sequence depth of 10,837 and a maximum Duplex depth of 14,967 was obtained across these targets from a single library preparation using 250 ng of sheared leukocyte DNA (Fig. 1). Duplex depth can be readily increased by preparing additional Duplex libraries from the same source DNA to achieve proportionally higher sensitivity for rarer variants. This stands in contrast to conventional NGS where, beyond a modest level, an increase in depth simply increases the number of background errors identified (Fig. 2A). We simulated low-level residual disease by mixing control DNA from a healthy young blood donor with DNA from 9 human cell lines harboring known AML mutations at dilutions from 1:100 to 1:100,000 (Table 2). The genomic loci of these 9 mutations in NRAS, KRAS and TP53 were captured with a small 1 kb probe panel. This mixture was sequenced to a mean Duplex depth of 〉1,000,000-fold, with the highest and lowest MAFs shown in Fig. 2B. All were close to expected frequencies (r2=0.96) with MAF as low as 6x10-6 (Fig. 3). As proof of specificity, we examined all coding nucleotide positions (excluding the 9 expected variants) and identified only 241 background variant counts out of 414,452,402 total Duplex BP, for an aggregate mutation frequency of 5.8x10-7, consistent with the estimated background of normal human aging. Our Duplex Sequencing-based AML MRD assay is flexible, broadly applicable and extremely sensitive. The assay is easily implemented using standard NGS equipment and automated cloud-based analysis software. The ~90% of AML patients served by this SNV-focused panel can be expanded to nearly 100% with complementary indel detection via targeted NGS RT-PCR. Optionally, when a patient's mutation profile from time-of-diagnosis is known, MRD testing can focus exclusively on those targets using a subset of pre-validated probes to reduce sequencing cost. Improved MRD testing will facilitate accurate prognostication, better selection among treatment options, and could serve as a surrogate endpoint in clinical trials to bring new treatments to patients faster. We are currently evaluating Duplex Sequencing MRD tests in both retrospective and prospective clinical trials. Disclosures Higgins: TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Buckley:CTI Biopharma: Employment; TwinStrand Biosciences: Consultancy. Radich:TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Employment, Equity Ownership.

Description: Hematopoietic cell transplantation (HCT) is used in the treatment of hematologic malignancies (HM). Clonal hematopoiesis of indeterminate potential (CHIP) is the age-associated process whereby healthy individuals accumulate low-frequency clones driven by mutations in genes recurrently mutated in myeloid cancers; CHIP is associated with an increased risk of HM and cardiovascular disease. It has been hypothesized that CHIP clones present in an HCT donor might disproportionately proliferate in the recipient due to a relative growth advantage during immune reconstitution. Here we use Duplex Sequencing (DS) to investigate, years after HCT, whether the clonal makeup of CHIP in prior donors differs from that of the donor-derived hematopoietic system long after allogeneic transfer to a recipient. DS is an ultra-sensitive NGS error-correction technology that relies on sequencing of the individual paired strands of original source DNA molecules to dramatically reduce technical background compared to other NGS methodologies. Here we performed DS on 10 pairs of donor/recipient peripheral blood DNA samples collected 7-46 years post-transplant, as well as DNA from a healthy young control. We used a sequencing panel comprising 29 genes recurrently mutated in acute myeloid leukemia (AML) and generated mean DS molecular depth of 27,580x per sample, with maximum DS depth reaching 45,402x. We detected an average of 356 and 283 total variants per donor and recipient, respectively, and 462 total variants in the control. These included 291 single-count exonic variants (SEV) in the control and an average of 215 SEVs in donors (Fig. A). To enrich for CHIP (clonal) variants, we filtered out single-count (non-clonal) events, as well as intronic (non-splice site) variants and germline SNPs. Whereas the young control had only 5 clonal exonic variants (CEV), meaning nearly all of the variants in the control were SEVs, SNPs or intronic (Fig. A,B), HCT donors and recipients respectively averaged 44 and 38 CEVs. 91% of CEVs were non-synonymous (Fig. B), which is higher than expected by chance. The average age of the donors was 36 years old at the time of HCT (range 12-84), and the oldest donor (M1) had the most CEVs. 24/29 genes in the panel exhibited CEVs in the control or HCT donors (Fig. C). DNMT3A, TET2, BRINP3 (FAM5C) and ASXL1 had the greatest number of CEVs and this imbalance was not explained purely by gene size. The young control sample had an overall somatic mutation frequency (MF) of 4.7x10-7. HCT donors and recipients had average MFs of 1.0x10-6 and 1.7x10-6, respectively. 154 CEVs were shared in at least 1 donor-recipient pair, with the majority present at variant allele frequencies (VAFs) 10-fold, all in DNMT3A, TET2 and RAD21. Only 1 CEV decreased 〉10-fold: TP53 R273H (4.9x10-4 to 4.2x10-5). Analysis of the same 10 donor-recipient pairs using a commercial sequencing service found that, with a VAF cutoff of 2% and filtering for SNPs, only 7 shared CEVs could be detected that increased or decreased in any recipient. Our ultra-high accuracy DS data indicates that the structure of CHIP is more polyclonal than has been previously reported. In a handful of older healthy donors we detected dozens of concurrent CHIP mutations, with a relative gene distribution nearly identical to that previously reported in studies on thousands of patients using less sensitive method. We observed a similar age-dependent effect. Furthermore, our findings indicate that the clonal dynamics of CHIP clones between HCT donors and recipients are substantially more complex than previously known. Nearly all somatic variants shared between HCT donors and recipients were observed at

Description: Background: Some previous studies in Ph+ ALL suggested that T315I mutations of the ABL1 KD are present in many pts prior to tyrosine kinase inhibitor (TKI) treatment. However, these reports used RT-PCR to generate complementary DNA (cDNA) prior to analysis, which may introduce random errors and lead to false positive results. We hypothesized that ultrasensitive DS of genomic DNA (gDNA) would more accurately detect low-level pretreatment ABL1 mutations in Ph+ ALL because it does not rely upon error prone RT-PCR. Methods: DS compares the nucleotide sequences of each strand of double-stranded molecules, improving the accuracy of conventional next-generation sequencing by more than 10,000-fold. DS of exons 4-10 of ABL1 was performed to an average molecular depth of 〉10,000x. A mixture of known ABL1 variants at varying low variant allelic frequencies (VAFs) versus a negative control were used to determine sensitivity and specificity of DS. DS of ABL1 was performed on 64 pts with newly diagnosed Ph+ ALL prior to receiving frontline hyper-CVAD plus a TKI (imatinib, n=5; dasatinib, n=38; ponatinib, n=21). DS was also performed on cDNA samples from RT-PCR to assess the RT-induced error rate. On RNA from relapse samples, the KD (codons 221 through 500) of BCR-ABL1 was sequenced by the Sanger method, using a nested PCR approach, with a detection limit of 10-20%. Results: A total of 115 pretreatment ABL1 mutations were detected by DS, with ≥1 mutation detected in 47/64 pts (73%). These mutations were generally present at very low levels (median VAF 0.008% [range, 0.004%-0.649%]). Eleven TKI resistance (TKI-R) mutations (10% of all detected mutations) were identified by DS in 7 pts (Table 1). Eighteen pts (28%) relapsed; however, none of the pts with TKI-R mutations relapsed, despite 5 pts receiving a TKI at least intermediately resistant to the detected mutation. Using Sanger sequencing, TKI-R mutations were detected at relapse in 9 pts (T315I, n=6; F317I, V229L and V338G, n=1 each). None of these TKI-R mutations was detected by DS in pretreatment samples. Together, these results suggest that low-level pretreatment ABL1 KD mutations are not clinically meaningful in Ph+ ALL. To validate the DS methodology, we examined its sensitivity and specificity using 6 gDNA samples containing ABL1 KD mutations (9 different SNVs, including 6 TKI-R) mixed with control gDNA from a healthy young individual for predicted VAFs of 1/250 to 1/25,000. Mutations were identified by DS with 100% sensitivity down to VAF

Description: The formation of the Southern Hemisphere spiral jet is investigated using observations over a 40-yr period. It is found that between late March and early April, the upper-tropospheric westerly jet in the Southern Hemisphere undergoes a transition from an annular structure in midlatitudes to a spiral structure that extends from 20° to 55°S. The transition to the spiral structure is initiated by the formation of a subtropical jet, localized in the central Pacific. The inception of the jet spiral is completed with the formation of a band of northwest-to-southeast-oriented zonal winds, which is connected to both the subtropical and the polar-front jets. This band, referred to as the tilting branch, arises from momentum flux convergence associated with breaking Rossby waves. As such, the direction of the wave breaking determines the direction of the jet spiral; an anticyclonic wave breaking, associated with equatorward wave dispersion, establishes a jet spiral that turns cyclonically toward the pole. This formation mechanism of the jet spiral is supported by a set of calculations with an idealized numerical model. These model calculations indicate that the jet spiral is obtained only if the model’s localized subtropical jet is sufficiently strong, and if the latitude of the polar-front jet is sufficiently higher than that of the subtropical jet. The calculations also indicate that the spiral jet is a transient solution, implying that the lack of spiral structure during the austral winter may be caused by the zonal wind field reaching a new statistically steady state.

Description: Sensitive and specific detection of measurable residual disease (MRD) after treatment in pediatric acute myeloid leukemia (AML) is prognostic of relapse and is important for clinical decision making. Mutation-based methods are increasingly being used, but are hampered by the limited number of common driver gene mutations to target as clone markers. Additional targets would greatly increase MRD detection power. However, even in cases with many AML-defining mutations, it is the limited accuracy of current molecular methods which establishes the lower bounds of sensitivity. Here we describe an ultrasensitive approach for disease monitoring with personalized hybrid capture panels targeting hundreds of somatic mutations identified by whole genome sequencing (WGS), and using extremely accurate Duplex Sequencing (DS) in longitudinal samples. In a pilot cohort of 13 patients we demonstrate detection sensitivities several orders of magnitude beyond currently available single locus testing or less accurate sequencing. With multi-target panels, overall power for MRD detection is cumulative across sites. For example, if a patient has MRD at a true frequency of 1/30,000, sequencing a single mutant site to 10,000x molecular depth would be unlikely to detect MRD. However, sequencing 10 sites each to 10,000x would effectively total 100,000x informative site depth, increasing power to 〉95%. However, standard sequencing assays are insufficiently accurate to achieve this theoretical limit of detection (LOD). DS enables accurate detection of individual variants to