Publications by authors named "Lawrence A Loeb"

121 Publications

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195;

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
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http://dx.doi.org/10.1073/pnas.2023373118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346827PMC
August 2021

Rare Mutations in Cancer Drug Resistance and Implications for Therapy.

Clin Pharmacol Ther 2020 09 10;108(3):437-439. Epub 2020 Jul 10.

Departments of Pathology and Biochemistry, University of Washington School of Medicine, Seattle, Washington, USA.

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http://dx.doi.org/10.1002/cpt.1938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484911PMC
September 2020

Extensive subclonal mutational diversity in human colorectal cancer and its significance.

Proc Natl Acad Sci U S A 2019 Dec 5. Epub 2019 Dec 5.

Department of Oncology, Georgetown University Medical Center, Washington, DC 20007.

Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (10) and accuracy (<10). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10 The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10).
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http://dx.doi.org/10.1073/pnas.1910301116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936702PMC
December 2019

A high-resolution landscape of mutations in the super-enhancer in normal human B cells.

Proc Natl Acad Sci U S A 2019 12 20;116(49):24779-24785. Epub 2019 Nov 20.

Department of Pathology, University of Washington, Seattle, WA 98195;

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the SE alone. Within the SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10 Mutational spectra show a predominance of C > T/G > A and A > G/T > C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA polymerase η, respectively, in mutagenesis in normal B cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signatures SBS85, SBS37, and SBS39 were found in the SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.
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http://dx.doi.org/10.1073/pnas.1914163116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900602PMC
December 2019

Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue over a Century of Human Lifespan.

Cell Rep 2019 07;28(1):132-144.e3

Department of Pathology, University of Washington, Seattle, WA 98195, USA. Electronic address:

High-accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low-frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low-frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging, and great care must be taken to distinguish tumor-derived from age-associated mutations in high-sensitivity clinical cancer diagnostics.
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http://dx.doi.org/10.1016/j.celrep.2019.05.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639023PMC
July 2019

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

Clin Cancer Res 2018 11 24;24(21):5321-5334. Epub 2018 Jul 24.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.

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. 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. 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. 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214777PMC
November 2018

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

Nat Rev Genet 2018 05 26;19(5):269-285. Epub 2018 Mar 26.

Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA.

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.
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http://dx.doi.org/10.1038/nrg.2017.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485430PMC
May 2018

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

Proc Natl Acad Sci U S A 2017 08 10;114(35):9415-9420. Epub 2017 Aug 10.

Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195;

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.
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http://dx.doi.org/10.1073/pnas.1709166114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584456PMC
August 2017

Evolutionary dynamics and significance of multiple subclonal mutations in cancer.

DNA Repair (Amst) 2017 08 9;56:7-15. Epub 2017 Jun 9.

Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington School of Medicine, Seattle, WA, 98195 USA. Electronic address:

For the last 40 years the authors have collaborated on trying to understand the complexities of human cancer by formulating testable mathematical models that are based on mutation accumulation in human malignancies. We summarize the concepts encompassed by multiple mutations in human cancers in the context of source, accumulation during carcinogenesis and tumor progression, and therapeutic consequences. We conclude that the efficacious treatment of human cancer by targeted therapy will involve individualized, uniquely directed specific agents singly and in simultaneous combinations, and take into account the importance of targeting resistant subclonal mutations, particularly those subclones with alterations in DNA repair genes, DNA polymerase, and other genes required to maintain genetic stability.
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http://dx.doi.org/10.1016/j.dnarep.2017.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551892PMC
August 2017

Mutational spectra of aflatoxin B in vivo establish biomarkers of exposure for human hepatocellular carcinoma.

Proc Natl Acad Sci U S A 2017 04 28;114(15):E3101-E3109. Epub 2017 Mar 28.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;

Aflatoxin B (AFB) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB, and, as such, is an early detection metric for AFB-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.
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http://dx.doi.org/10.1073/pnas.1700759114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393230PMC
April 2017

Homozygosity for the WRN Helicase-Inactivating Variant, R834C, does not confer a Werner syndrome clinical phenotype.

Sci Rep 2017 03 9;7:44081. Epub 2017 Mar 9.

INMEGEN, National Institute of Genomic Medicine, Periferico Sur No.4809, Col. Arenal Tepepan, Del. Tlalpan Mèxico, D.F, C.P. 14610, Mexico.

Loss-of-function mutations in the WRN helicase gene cause Werner syndrome- a progeroid syndrome with an elevated risk of cancer and other age-associated diseases. Large numbers of single nucleotide polymorphisms have been identified in WRN. We report here the organismal, cellular, and molecular phenotypes of variant rs3087425 (c. 2500C > T) that results in an arginine to cysteine substitution at residue 834 (R834C) and up to 90% reduction of WRN helicase activity. This variant is present at a high (5%) frequency in Mexico, where we identified 153 heterozygous and three homozygous individuals among 3,130 genotyped subjects. Family studies of probands identified ten additional TT homozygotes. Biochemical analysis of WRN protein purified from TT lymphoblast cell lines confirmed that the R834C substitution strongly and selectively reduces WRN helicase, but not exonuclease activity. Replication track analyses showed reduced replication fork progression in some homozygous cells following DNA replication stress. Among the thirteen TT homozygotes, we identified a previously unreported and statistically significant gender bias in favor of males (p = 0.0016), but none of the clinical findings associated with Werner syndrome. Our results indicate that WRN helicase activity alone is not rate-limiting for the development of clinical WS.
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http://dx.doi.org/10.1038/srep44081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343477PMC
March 2017

Richmond T. Prehn: In Memoriam (1922-2016).

Authors:
Lawrence A Loeb

Cancer Res 2017 02;77(3):593-594

Department of Pathology, University of Washington, Seattle, Washington.

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http://dx.doi.org/10.1158/0008-5472.CAN-16-3328DOI Listing
February 2017

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency.

Proc Natl Acad Sci U S A 2016 09 19;113(36):10151-6. Epub 2016 Aug 19.

Department of Dermatology, University of California, San Francisco, CA 94143;

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.
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http://dx.doi.org/10.1073/pnas.1610020113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018765PMC
September 2016

Human Cancers Express a Mutator Phenotype: Hypothesis, Origin, and Consequences.

Authors:
Lawrence A Loeb

Cancer Res 2016 04;76(8):2057-9

Department of Pathology, University of Washington School of Medicine, Seattle, Washington. Department of Biochemistry, University of Washington School of Medicine, Seattle, Washington.

The mutator phenotype hypothesis was postulated more than 40 years ago. It was based on the multiple enzymatic steps required to precisely replicate the 6 billion bases in the human genome each time a normal cell divides. A reduction in this accuracy during tumor progression could be responsible for the striking heterogeneity of malignant cells within a tumor and for the rapidity by which cancers become resistant to therapy. Cancer Res; 76(8); 2057-9. ©2016 AACRSee related article by Loeb et al. Cancer Res. 1974;34:2311-21.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004728PMC
April 2016

Decreased Mitochondrial Mutagenesis during Transformation of Human Breast Stem Cells into Tumorigenic Cells.

Cancer Res 2016 08 17;76(15):4569-78. Epub 2016 May 17.

Department of Pathology, University of Washington, Seattle, Washington. Department of Biochemistry, University of Washington, Seattle, Washington.

Rare stochastic mutations may accumulate during dormancy of stem-like cells, but technical limitations in DNA sequencing have limited exploring this possibility. In this study, we employed a recently established deep-sequencing method termed Duplex Sequencing to conduct a genome-wide analysis of mitochondrial (mt) DNA mutations in a human breast stem cell model that recapitulates the sequential stages of breast carcinogenesis. Using this method, we found significant differences in mtDNA among normal stem cells, immortal/preneoplastic cells, and tumorigenic cells. Putative cancer stem-like cell (CSC) populations and mtDNA copy numbers increased as normal stem cells become tumorigenic cells. Transformed cells exhibited lower rare mutation frequencies of whole mtDNA than did normal stem cells. The predicted mtDNA rare mutation pathogenicity was significantly lower in tumorigenic cells than normal stem cells. Major rare mutation types in normal stem cells are C>T/G>A and T>C/A>G transitions, while only C>T/G>A are major types in transformed cells. We detected a total of 1,220 rare point mutations, 678 of which were unreported previously. With only one possible exception (m10342T>C), we did not find specific mutations characterizing mtDNA in human breast CSCs; rather, the mitochondrial genome of CSCs displayed an overall decrease in rare mutations. On the basis of our work, we suggest that this decrease (in particular T>C/A>G transitions), rather than the presence of specific mitochondrial mutations, may constitute an early biomarker for breast cancer detection. Our findings support the hypothesis that the mitochondrial genome is altered greatly as a result of the transformation of normal stem cells to CSCs, and that mtDNA mutation signatures may aid in delineating normal stem cells from CSCs. Cancer Res; 76(15); 4569-78. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004738PMC
August 2016

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

Proc Natl Acad Sci U S A 2016 May 5;113(21):6005-10. Epub 2016 May 5.

Department of Pathology, University of Washington, Seattle, WA 98195;

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.
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http://dx.doi.org/10.1073/pnas.1601311113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889384PMC
May 2016

Exploring the implications of distinct mutational signatures and mutation rates in aging and cancer.

Genome Med 2016 Mar 17;8(1):30. Epub 2016 Mar 17.

Department of Pathology, University of Washington, Seattle, WA, 98195, USA.

Signatures of mutagenesis provide a powerful tool for dissecting the role of somatic mutations in both normal and pathological processes. Significantly, cancer genomes are dominated by mutation signatures distinct from those that accumulate in normal tissues with age, with potentially important translational implications.
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http://dx.doi.org/10.1186/s13073-016-0286-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797182PMC
March 2016

Tobacco Causes Human Cancers--A Concept Founded on Epidemiology and an Insightful Experiment Now Requires Translation Worldwide.

Authors:
Lawrence A Loeb

Cancer Res 2016 Feb;76(4):765-6

Department of Pathology, University of Washington School of Medicine, Seattle, Washington. Department of Biochemistry, University of Washington School of Medicine, Seattle, Washington.

The recognition that tobacco smoke is carcinogenic led to the most significant and successful effort at reducing cancer incidence in human history. A major milestone of this effort was the publication in Cancer Research by Wynder and colleagues, which demonstrated the ability of tobacco tars to produce tumors in mice. This study provided a powerful link between the epidemiology of cancer and mechanisms of carcinogenesis. This commentary asserts that we have a moral obligation to translate our success in reducing lung cancer in the United States to the 1.25 billion smokers throughout the rest of the world. See related article by Wynder et al., Cancer Res 1953;13:855-64.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0149DOI Listing
February 2016

The influence of subclonal resistance mutations on targeted cancer therapy.

Nat Rev Clin Oncol 2016 06 20;13(6):335-47. Epub 2015 Oct 20.

Departments of Biochemistry and Pathology, University of Washington, 1959 Northeast Pacific Street, Box 357705, Seattle, WA 98195, USA.

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.
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http://dx.doi.org/10.1038/nrclinonc.2015.175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838548PMC
June 2016

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

PLoS One 2015 25;10(8):e0136216. Epub 2015 Aug 25.

Department of Pathology, University of Washington, Seattle, Washington, United States of America; Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549069PMC
May 2016

Sequencing small genomic targets with high efficiency and extreme accuracy.

Nat Methods 2015 May 6;12(5):423-5. Epub 2015 Apr 6.

Department of Pathology, University of Washington, Seattle, Washington, USA.

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.
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http://dx.doi.org/10.1038/nmeth.3351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414912PMC
May 2015

Accuracy of Next Generation Sequencing Platforms.

Next Gener Seq Appl 2014;1

Department of Biostatistics, University of Washington, USA.

Next-generation DNA sequencing has revolutionized genomic studies and is driving the implementation of precision diagnostics. The ability of these technologies to disentangle sequence heterogeneity, however, is limited by their relatively high error rates. A Several single molecule barcoding strategies have been propose to reduce the overall error frequency. A Duplex Sequencing additionally exploits the fact that DNA is double-strand, with one strand reciprocally encoding the sequence information of its complement, and can eliminate nearly all sequencing errors by comparing the sequence of individually tagged amplicons derived from one strand of DNA with that of its complementary strand. This method reduces errors to fewer than one per ten million nucleotides sequenced.
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http://dx.doi.org/10.4172/jngsa.1000106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331009PMC
January 2014

Detecting ultralow-frequency mutations by Duplex Sequencing.

Nat Protoc 2014 Nov 9;9(11):2586-606. Epub 2014 Oct 9.

1] Department of Pathology, University of Washington, Seattle, USA. [2] Department of Biochemistry, University of Washington, Seattle, USA.

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.
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http://dx.doi.org/10.1038/nprot.2014.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271547PMC
November 2014

Cancer: One cell at a time.

Nature 2014 Aug 30;512(7513):143-4. Epub 2014 Jul 30.

1] Department of Pathology, University of Washington, Seattle, Washington 98195-7750, USA. [2] Department of Biochemistry, University of Washington.

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http://dx.doi.org/10.1038/nature13650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287243PMC
August 2014

Sphingosine, a modulator of human translesion DNA polymerase activity.

J Biol Chem 2014 Aug 13;289(31):21663-72. Epub 2014 Jun 13.

From the Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195,

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.
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http://dx.doi.org/10.1074/jbc.M114.570242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118125PMC
August 2014

A rapid assay for measuring nucleotide excision repair by oligonucleotide retrieval.

Sci Rep 2014 May 8;4:4894. Epub 2014 May 8.

Departments of Pathology and Biochemistry, University of Washington, Seattle, Washington 98195-7705, USA.

Nucleotide excision repair (NER) excises bulky DNA lesions induced by mutagens and carcinogens. The repair process includes recognition of DNA damage, excision of a short patch of nucleotides containing the damaged base, re-synthesis of a new DNA strand and ligation of the nicks to restore the sequence integrity. Mutation or aberrant transcription of NER genes reduces repair efficiency and results in the accumulation of mutations that is associated with the development of cancer. Here we present a rapid, sensitive and quantitative assay to measure NER activity in human cells, which we term the Oligonucleotide Retrieval Assay (ORA). We used oligonucleotide constructs containing the UV-damaged adduct, cyclobutane pyrimidine dimer (CPD), to transfect human cells, and retrieved the oligonucleotides for quantification of the repaired, CPD-free DNA by real-time quantitative PCR. We demonstrate that ORA can quantify the extent of NER in diverse cell types, including immortalized, primary and stem-like cells.
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http://dx.doi.org/10.1038/srep04894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013936PMC
May 2014

Clonal expansions and short telomeres are associated with neoplasia in early-onset, but not late-onset, ulcerative colitis.

Inflamm Bowel Dis 2013 Nov;19(12):2593-602

*Department of Pathology, †Department of Medicine, ‡Department of Biostatistics, and §Division of Gastroenterology, University of Washington, School of Medicine, Seattle, Washington; ‖Division of Anatomic Pathology, University of Utah, Salt Lake City, Utah; and ¶Division of Public Health Science, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Background: Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case-control study.

Methods: This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics.

Results: Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity.

Conclusions: Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields.
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http://dx.doi.org/10.1097/MIB.0b013e3182a87640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885330PMC
November 2013

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

PLoS Genet 2013 26;9(9):e1003794. Epub 2013 Sep 26.

Department of Pathology, University of Washington, Seattle, Washington, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1003794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784509PMC
March 2014

APOBEC3B mutagenesis in cancer.

Nat Genet 2013 Sep;45(9):964-5

Recent evidence has implicated APOBEC3B as a source of mutations in cervical, bladder, lung, head and neck, and breast cancers. APOBEC enzymes normally function in innate immune responses, including those that target retroviruses, suggesting links between mutagenesis, immunity and viral infection in the process of cancer development.
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http://dx.doi.org/10.1038/ng.2736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965181PMC
September 2013

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers.

Transl Oncol 2013 Aug 1;6(4):458-69. Epub 2013 Aug 1.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA ; Department of Surgery, University of Washington Medical School, Seattle, WA ; Department of Surgery, University of Nebraska Medical School, Omaha, NE.

Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730021PMC
http://dx.doi.org/10.1593/tlo.13238DOI Listing
August 2013
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