Phase 1 dose-escalation study of SEA-CD40: a non-fucosylated CD40 agonist, in advanced solid tumors and lymphomas.

BACKGROUND: SEA-CD40 is an investigational, non-fucosylated, humanized monoclonal IgG1 antibody that activates CD40, an immune-activating tumor necrosis factor receptor superfamily member. SEA-CD40 exhibits enhanced binding to activating FcγRIIIa, possibly enabling greater immune stimulation than other CD40 agonists. A first-in-human phase 1 trial was conducted to examine safety, pharmacokinetics, and pharmacodynamics of SEA-CD40 monotherapy in patients with advanced solid tumors and lymphoma. METHODS: SEA-CD40 was administered intravenously to patients with solid tumors or lymphoma in 21-day cycles with standard 3+3 dose escalation at 0.6, 3, 10, 30, 45, and 60 µg/kg. An intensified dosing regimen was also studied. The primary objectives of the study were to evaluate the safety and tolerability and identify the maximum tolerated dose of SEA-CD40. Secondary objectives included evaluation of the pharmacokinetic parameters, antitherapeutic antibodies, pharmacodynamic effects and biomarker response, and antitumor activity. RESULTS: A total of 67 patients received SEA-CD40 including 56 patients with solid tumors and 11 patients with lymphoma. A manageable safety profile was observed, with predominant adverse events of infusion/hypersensitivity reactions (IHRs) reported in 73% of patients. IHRs were primarily ≤grade 2 with an incidence associated with infusion rate. To mitigate IHRs, a standardized infusion approach was implemented with routine premedication and a slowed infusion rate. SEA-CD40 infusion resulted in potent immune activation, illustrated by dose dependent cytokine induction with associated activation and trafficking of innate and adaptive immune cells. Results suggested that doses of 10-30 µg/kg may result in optimal immune activation. SEA-CD40 monotherapy exhibited evidence of antitumor activity, with a partial response in a patient with basal cell carcinoma and a complete response in a patient with follicular lymphoma. CONCLUSIONS: SEA-CD40 was tolerable as monotherapy and induced potent dose dependent immune cell activation and trafficking consistent with immune activation. Evidence of monotherapy antitumor activity was observed in patients with solid tumors and lymphoma. Further evaluation of SEA-CD40 is warranted, potentially as a component of a combination regimen. TRIAL REGISTRATION NUMBER: NCT02376699.

Species comparison: human and minipig PBMC reactivity under the influence of immunomodulating compounds in vitro.

Considering the similarities between swine and humans, it is a logical consequence to use swine as a translational model in research and drug development, including non-clinical safety. Here, we compared the reactivity of peripheral blood mononuclear cells (PBMCs) from humans and minipigs under the influence of different compounds in vitro. We conducted a flow cytometry-based proliferation assay that focused on the T-cell response to three different stimuli: concanavalin A (ConA), phytohemagglutinin-L (PHA-L), and staphylococcal Enterotoxin B (SEB). Furthermore, four approved immunosuppressive drugs-abatacept, belatacept, rapamycin, and tofacitinib-which are used for the treatment of rheumatoid arthritis or rejection in transplant recipients, were combined with the different stimuli. This allowed us to study the effect of suppressive drugs in comparison with the different stimuli in both species. We examined proliferating T cells (CD3+) and investigated the presence of TCR-αß+ and TCR-γδ+ T cells. Differences in the response of T cells of the two species under these various conditions were evident. CD4+ T cells were more activated within humans, whereas CD8+ T cells were generally more abundant in swine. The effectiveness of the used humanized antibodies is most likely related to the conserved structure of CTLA-4 as abatacept induced a much stronger reduction in swine compared with belatacept. The reduction of proliferation of rapamycin and tofacitinib was highly dependent on the used stimuli. We further investigated the effect of the immunosuppressive compounds on antigen-specific restimulation of pigs immunized against porcine circovirus 2 (PCV2). Treatment with all four compounds resulted in a clear reduction of the proliferative response, with rapamycin showing the strongest effect. In conclusion, our findings indicate that the effectiveness of suppressive compounds is highly dependent on the stimuli used and must be carefully selected to ensure accurate results. The results highlight the importance of considering the response of T cells in different species when evaluating the potential of an immunomodulatory drug.

Characterization of the immune system of Ellegaard Göttingen Minipigs - An important large animal model in experimental medicine.

Interest in Ellegaard Göttingen Minipigs (EGMs) as a model in experimental medicine is continuously growing. The aim of this project is to increase the knowledge of the immune system of EGMs as information is still scarce. Therefore, we studied the postnatal maturation of their immune system from birth until 126 weeks of age. For the first 26 weeks of the study, animals were kept under pathogen-reduced conditions (SPF) and afterwards under conventional housing conditions. The development of the immune system was analyzed by monitoring changes in total numbers of leukocytes and lymphocytes of ten individuals and the composition of leukocyte populations by multi-color flow cytometry (FCM). We followed the presence of monocytes using monoclonal antibodies (mAbs) against CD172a+ and CD163+ and B cells based on the expression of CD79a. NK cells were distinguished as CD3-CD16+CD8α+/dim cells and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells. T-cell receptor (TCR) γδ T cells were defined by the expression of TCR-γδ and different subsets were determined by their CD2 and perforin expression. TCR-αß T cells were classified by their CD8ß+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8ß++ T cells and CD8α together with CD27 for CD4+ T cells. We clearly detected a postnatal development of immune cell composition and identified phenotypes indicative of differentiation within the respective leukocyte subsets. Examination of the development of the antigen-specific immune system after transfer to different distinct housing conditions and after vaccination against common porcine pathogens such as porcine circovirus 2 (PCV2) revealed a markedly increased presence of more differentiated CD8+ and CD4+ T cells with central and effector memory T-cell phenotypes. To complement the findings, a PCV2 vaccine-specific antigen was used for in vitro restimulation experiments. We demonstrated antigen-specific proliferation of CD4+CD8α+CD27+ central and CD4+CD8α+CD27- effector memory T cells as well as antigen-specific production of TNF-α and IFN-γ. This study of postnatal immune development defines basic cellular immune parameters of EGMs and represents an important milestone for the use of EGMs for immunological questions in experimental medicine.

Accurate detection of subclonal variants in paired diagnosis-relapse acute myeloid leukemia samples by next generation Duplex Sequencing.

Mutations characterize diverse human cancers; there is a positive correlation between elevated mutation frequency and tumor progression. One exception is acute myeloid leukemia (AML), which has few clonal single nucleotide mutations. We used highly sensitive and accurate Duplex Sequencing (DS) to show now that AML, in addition, has an extensive repertoire of variants with low allele frequencies, < 1%, which is below the accurate detection limit of most other sequencing methodologies. The subclonal variants are unique to each individual and change in composition, frequency, and sequence context from diagnosis to relapse. Their functional significance is apparent by the observation that many are known variants and cluster within functionally important protein domains. Subclones provide a reservoir of variants that could expand and contribute to the development of drug resistance and relapse. In accord, we accurately identified subclonal variants in AML driver genes NRAS and RUNX1 at allele frequencies between 0.1% and 0.3% at diagnosis, which expanded to comprise a major fraction (14-53%) of the blast population at relapse. Early and accurate detection of subclonal variants with low allele frequency thus offers the opportunity for early intervention, prior to detection of clinical relapse, to improve disease outcome and enhance patient survival.

Transcriptome Profiling of Porcine Naïve, Intermediate and Terminally Differentiated CD8+ T Cells.

The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8+ CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8ß+ T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8ß+CD27+CD11alow), intermediate differentiated (CD8ß+CD27dimCD11a+), and terminally differentiated cells (CD8ß+CD27-CD11ahigh). Cells were analyzed in ex vivo condition as well as upon in vitro stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8+ T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (IL7-R, CCR7, SELL, TCF7, LEF1, BACH2, SATB1, ZEB1 and BCL2) and late (KLRG1, TBX21, PRDM1, CX3CR1, ZEB2, ZNF683, BATF, EZH2 and ID2) stages of CD8+ T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8+ T-cell subsets, respectively. Intermediate differentiated CD8+ T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (GNLY, PRF1, GZMB, FASL, IFNG and TNF) were highly expressed in terminally and intermediate differentiated CD8+ T-cell subsets, while naïve CD8+ T cells lacked expression even after in vitro stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8+ T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail.

Species selection for nonclinical safety assessment of drug candidates: Examples of current industry practice.

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. METHODS: In several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

l-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. l-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. l-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating l-citrulline. METHODS: In several repeat-dose toxicity studies, plasma l-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma l-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline l-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma l-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of l-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute l-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in l-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute l-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in l-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of l-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of l-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of l-citrulline as a small intestine biomarker.

Intra-host changes in Kaposi sarcoma-associated herpesvirus genomes in Ugandan adults with Kaposi sarcoma.

Intra-host tumor virus variants may influence the pathogenesis and treatment responses of some virally-associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has to date been explored with sequencing technologies that possibly introduce more errors than that which occurs in the viral population, and these studies have only studied variable regions. Here, full-length KSHV genomes in tumors and/or oral swabs from 9 Ugandan adults with HIV-associated KS were characterized. Furthermore, we used deep, short-read sequencing using duplex unique molecular identifiers (dUMI)-random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors to ~10-9/base as well as afforded accurate determination of KSHV genome numbers sequenced in each sample. KSHV genomes were assembled de novo, and rearrangements observed were confirmed by PCR and Sanger sequencing. 131-kb KSHV genome sequences, excluding major repeat regions, were successfully obtained from 23 clinical specimens, averaging 2.3x104 reads/base. Strikingly, KSHV genomes were virtually identical within individuals at the point mutational level. The intra-host heterogeneity that was observed was confined to tumor-associated KSHV mutations and genome rearrangements, all impacting protein-coding sequences. Although it is unclear whether these changes were important to tumorigenesis or occurred as a result of genomic instability in tumors, similar changes were observed across individuals. These included inactivation of the K8.1 gene in tumors of 3 individuals and retention of a region around the first major internal repeat (IR1) in all instances of genomic deletions and rearrangements. Notably, the same breakpoint junctions were found in distinct tumors within single individuals, suggesting metastatic spread of rearranged KSHV genomes. These findings define KSHV intra-host heterogeneity in vivo with greater precision than has been possible in the past and suggest the possibility that aberrant KSHV genomes may contribute to aspects of KS tumorigenesis. Furthermore, study of KSHV with use of dUMI provides a proof of concept for utilizing this technique for detailed study of other virus populations in vivo.

Single-Molecule Sequencing Reveals Patterns of Preexisting Drug Resistance That Suggest Treatment Strategies in Philadelphia-Positive Leukemias.

Purpose: Sequential treatment with targeted therapies can result in complex combinations of resistance mutations in drug targets. This mutational complexity has spurred the development of pan-target inhibitors, i.e., therapies for which no single target mutation can cause resistance. Because the propensity for on- versus off-target resistance varies across cancer types, a deeper understanding of the mutational burden in drug targets could rationalize treatment outcomes and prioritize pan-target inhibitors for indications where on-target mutations are most likely.Experimental Design: To measure and model the mutational landscape of a drug target at high resolution, we integrated single-molecule Duplex Sequencing of the ABL1 gene in Philadelphia-positive (Ph+) leukemias with computational simulations.Results: A combination of drug target mutational burden and tumor-initiating cell fraction is sufficient to predict that most patients with chronic myeloid leukemia are unlikely to harbor ABL1 resistance mutations at the time of diagnosis, rationalizing the exceptional success of targeted therapy in this setting. In contrast, our analysis predicts that many patients with Ph+ acute lymphoblastic leukemia (Ph+ ALL) harbor multiple preexisting resistant cells with single mutants. The emergence of compound mutations can be traced to initial use of an ABL1 inhibitor that is susceptible to resistance from single point mutations.Conclusions: These results argue that early use of therapies that achieve pan-inhibition of ABL1 resistance mutants might improve outcomes in Ph+ ALL. Our findings show how a deep understanding of the mutational burden in drug targets can be quantitatively coupled to phenotypic heterogeneity to rationalize clinical phenomena. Clin Cancer Res; 24(21); 5321-34. ©2018 AACR.

Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations.

Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules. We also highlight major applications for low-frequency mutation detection in science and medicine, describe emerging methodologies and provide our vision for the future of DNA sequencing.

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.

The influence of subclonal resistance mutations on targeted cancer therapy.

Clinical oncology is being revolutionized by the increasing use of molecularly targeted therapies. This paradigm holds great promise for improving cancer treatment; however, allocating specific therapies to the patients who are most likely to derive a durable benefit continues to represent a considerable challenge. Evidence continues to emerge that cancers are characterized by extensive intratumour genetic heterogeneity, and that patients being considered for treatment with a targeted agent might, therefore, already possess resistance to the drug in a minority of cells. Indeed, multiple examples of pre-existing subclonal resistance mutations to various molecularly targeted agents have been described, which we review herein. Early detection of pre-existing or emerging drug resistance could enable more personalized use of targeted cancer therapy, as patients could be stratified to receive the therapies that are most likely to be effective. We consider how monitoring of drug resistance could be incorporated into clinical practice to optimize the use of targeted therapies in individual patients.

Sequencing small genomic targets with high efficiency and extreme accuracy.

The detection of minority variants in mixed samples requires methods for enrichment and accurate sequencing of small genomic intervals. We describe an efficient approach based on sequential rounds of hybridization with biotinylated oligonucleotides that enables more than 1-million-fold enrichment of genomic regions of interest. In conjunction with error-correcting double-stranded molecular tags, our approach enables the quantification of mutations in individual DNA molecules.

Detecting ultralow-frequency mutations by Duplex Sequencing.

Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 10(7) wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence 'families' that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1-3 d.

Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.

Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G → T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G → A and T → C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.

A substitution in the fingers domain of DNA polymerase δ reduces fidelity by altering nucleotide discrimination in the catalytic site.

DNA polymerase δ (Pol δ) is one of the major replicative DNA polymerases in eukaryotic cells, catalyzing lagging strand synthesis as well as playing a role in many DNA repair pathways. The catalytic site for polymerization consists of a palm domain and mobile fingers domain that opens and closes each catalytic cycle. We explored the effect of amino acid substitutions in a region of the highly conserved sequence motif B in the fingers domain on replication fidelity. A novel substitution, A699Q, results in a marked increase in mutation rate at the yeast CAN1 locus, and is synthetic lethal with both proofreading deficiency and mismatch repair deficiency. Modeling the A699Q mutation onto the crystal structure of Saccharomyces cerevisiae Pol δ template reveals four potential contacts for A699Q but not for A699. We substituted alanine for each of these residues and determined that an interaction with multiple residues of the N-terminal domain is responsible for the mutator phenotype. The corresponding mutation in purified human Pol δ results in a similar 30-fold increase in mutation frequency when copying gapped DNA templates. Sequence analysis indicates that the most characteristic mutation is a guanine-to-adenine (G to A) transition. The increase in deoxythymidine 5'-triphosphate-G mispairs was confirmed by performing steady state single nucleotide addition studies. Our combined data support a model in which the Ala-to-Gln substitution in the fingers domain of Pol δ results in an interaction with the N-terminal domain that affects the base selectivity of the enzyme.

Implications of genetic heterogeneity in cancer.

DNA sequencing studies have established that many cancers contain tens of thousands of clonal mutations throughout their genomes, which is difficult to reconcile with the very low rate of mutation in normal human cells. This observation provides strong evidence for the mutator phenotype hypothesis, which proposes that a genome-wide elevation in the spontaneous mutation rate is an early step in carcinogenesis. An elevated mutation rate implies that cancers undergo continuous evolution, generating multiple subpopulations of cells that differ from one another in DNA sequence. The extensive heterogeneity in DNA sequence and continual tumor evolution that would occur in the context of a mutator phenotype have important implications for cancer diagnosis and therapy.

Detection of ultra-rare mutations by next-generation sequencing.

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.