authors@Fred Hutch

Kocarnik, Jonathan

January 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
Identifying Which Smartphone App Features Aid 
Smoking Cessation 
January 19, 2015 
      JM Kocarnik 
Cigarette smoking kills six million people worldwide each year, but is an addictive behavior that can 
be difficult to stop.  No wonder, then, that there are over 400 smartphone apps available to help 
people quit smoking.  Despite their ubiquity, little research has evaluated whether these apps are 
successfully aiding smoking cessation, or what features of the app are the most useful.  To explore 
this issue, Drs. Jaimee Heffner and Jonathan Bricker, along with colleagues in the Public Health 
Sciences Division, sought to evaluate the features of their recently piloted SmartQuit app.  As 
reported in The American Journal of Drug and Alcohol Abuse, the authors found that only two of the 
most commonly used features were prospectively associated with quitting in the pilot group. 
Smartphone apps for smoking cessation are certainly in demand, with an average of 780,000 
downloads per month.  Whether these apps successfully help people quit smoking, however, is still 
under investigation.  In order to help guide future development in the most productive way, the 
authors developed and evaluated their own smoking cessation app, called SmartQuit (see 
figure).  "By studying how users engage with specific app features and whether use of these features 
predicts successful smoking cessation," said lead author Dr. Heffner, "we are identifying the basic 
building blocks needed to create effective interventions on the smartphone app platform, which 
reaches millions of smokers each year and has the potential to transform treatment delivery."  
First, "we developed the first smartphone app for smoking cessation based on Acceptance and 
Commitment Therapy (ACT)," said Dr. Heffner.  This type of therapy focuses on increasing the 
user’s willingness to accept the physical, mental, and emotional challenges of quitting, while also 
encouraging commitment to engage in values-based behavior change.  The app uses these 
principles through features that help enhance the user’s motivation to quit, provide the skills needed 
to accept the urge to smoke without acting on it, and aid coping skills for when smoking slips 
occur.  These principles are accompanied by other features based on elements of cognitive 
behavioral therapy (CBT), which helps develop skills for avoiding or controlling smoking 
triggers.  These CBT features help the user to set goals, track their smoking, view progress, and get 
positive reinforcement and social support. 
January 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
The authors evaluated the effectiveness of these techniques by tracking usage of 41 different app 
features, and then comparing this use against whether users successfully quit 
smoking.  Surprisingly, only 2 of the top 10 most-used features were prospectively associated with 
30 days abstinence at the 60 day follow-up: viewing the quit plan and tracking the practice of letting 
urges pass.  One less-used feature, tracking ACT skill practice, was also associated with 
cessation.  In describing these findings, Dr. Heffner recalls a quote attributed to Einstein: "What is 
popular is not always right." 
Continued Dr. Heffner, "we think that the message contained in that quote is critically important not 
just for this particular app, but for the broader field of smartphone apps for smoking cessation, where 
there has been greater emphasis on designing apps that are appealing to users rather than on 
developing apps grounded in behavioral theory and with demonstrated effectiveness as a cessation 
aid.  Appeal and effectiveness are both important and, as we demonstrated in this study, they don’t 
necessarily go hand-in-hand."  The findings from this study are thus extremely valuable for ensuring 
that future iterations of health intervention apps are useful. 
To that end, the authors are now focused on updating the app and continuing to evaluate its 
performance.  "These results have informed the revision of our SmartQuit app (now available 
publicly)," said senior author Dr. Bricker, "and we will be testing the revised app starting January 
15th with employees of several companies through a Life Sciences Discovery Grant."   Additional 
future work will also allow the authors to experiment for causal relationships between feature usage 
and smoking cessation.  Said Dr. Heffner, "this would provide the most accurate and rigorous test of 
the each feature’s contribution to successful quitting."  If successful, these apps could certainly 
generate a large public health impact. 
Other PHS researchers contributing to this project were Dr. Roger Vilardaga and Ms. Laina Mercer.  
 
Heffner JL, Vilardaga R, Mercer LD, Kientz JA, Bricker JB. 2015. Feature-level analysis of a novel 
smartphone application for smoking cessation. Am J Drug Alcohol Abuse. 41(1):68-73. doi: 
10.3109/00952990.2014.977486 
See also:  
Bricker JB, Mull KE, Vilardaga R, Kientz JA, Mercer LD, Akioka K, Heffner JL.  2014. Randomized, 
controlled pilot trial of a smartphone app for smoking cessation using acceptance and commitment 
therapy. Drug and Alcohol Dependence, 143, 87-94 
 
January 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
 
 
Image provided by Dr. Jaimee Heffner 
Example screen of the SmartQuit app, which helps users track their 
progress towards their smoking cessation goals. 
 


English

Identifying Which Smartphone App Features Aid Smoking Cessation

Frost, Sophia

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Plastic Fantastic! Biopolymers Efficiently Boost T-
Cell Therapy 
February 16, 2015 
     SHL Frost 
Solid tumors can be difficult to remove completely through standard surgery, depending on their 
location and invasive characteristics. This forces physicians and scientists to explore other therapies 
in order to reduce the risk of relapse by eliminating as many malignant cells as possible. One such 
alternative treatment under development is called adoptive cell therapy, and uses tumor-reactive 
immune cells (T cells) to specifically locate and kill diseased cells. Although promising, the technique 
is in many cases hampered by inefficient tumor homing and poor survival of injected T cells. This 
concern prompted Drs. Sirkka and Matthias Stephan in the Clinical Research Division to investigate 
established surgical protocols in search of "a simple tool" that could increase the clinical efficacy of T 
cell therapy. What they found was biodegradable polymer implants, currently in use for local delivery 
of chemotherapeutic agents to brain cancer patients. The drawback of this technique is 
the passive release of drugs from the polymer, making it hard for the chemotherapeutics to reach 
tumor cells that are more than a few millimeters away from the implant. Consequently, the resulting 
overall survival among treated patients has only been improved to a limited degree. 
Nevertheless, the Stephan Lab saw potential in these polymers as vehicles for T cell delivery and 
generated customized macroporous scaffolds from the US Food and Drug Administration (FDA)-
approved polysaccharide polymerized alginate. To test their effectiveness, the investigators used 
an in vivo mouse breast cancer resection model that mimics tumor recurrence after surgery, and 
embedded the developed T cell-containing scaffolds directly into the cavities from which tumors had 
been incompletely removed. The results were clear: neither intravenous nor intracavitary injection of 
tumor-reactive T cells in solution produced any substantial survival advantages; similarly, 
prestimulation of immune cells with various antibodies and interleukin 15 superagonist improved the 
outcome only modestly. Biopolymer-delivered T cells, on the other hand, effectively reduced the 
relapse rate to zero. 
"Once implanted, T cells actively emigrate deep into the surrounding tumor, where they robustly 
expand (up to 400-fold in our studies), and even infiltrate tumor-draining lymph nodes," explained Dr. 
Matthias Stephan, whose results are hot off the press in the highly ranked scientific journal Nature  
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
Biotechnology. The key to the Stephan Lab's success lay in making the scaffolds bioactive, which 
means providing an adhesive coating to support cell egress and stimulatory signals to trigger 
proliferation. This was achieved through integration of collagen-mimetic peptides and porous 
antibody-coated microparticles infused with T cell growth factors, called interleukins. 
The striking effectiveness of the new technique was further studied through bioluminescence 
imaging, showing equally impressive improvements in targeting of tumor sites and proliferation in 
vivo compared with directly injected T cells. Lastly, significant increases in overall survival and time 
to relapse were also demonstrated in a mouse model of late-stage ovarian cancer. 
"We demonstrate for the first time that launching cancer-fighting immune cells from polymeric 
devices can safely and effectively prevent relapse and provide an effective treatment option for 
inoperable tumors," Dr. Stephan said, adding that "implemented in the clinic, biomaterial-supported T 
cell implants could maximize the success of tumor surgery, and spare patients from repetitive 
operations, extended hospital stays, and rounds of radiation or chemotherapy." 
The novel approach shows great potential for resolving two issues that have restricted the clinical 
use of T cell therapy against solid tumors: efficiency and practicality. Compared with adoptive cell 
therapy, which demands time-consuming and expensive procedures for isolation and in 
vitro expansion of the infused T cells, in addition to irradiation and/or immunosuppressing 
pretreatment regimens, the biodegradable scaffolds incorporate all key activation and proliferation 
factors. The released T cells can attack malignant target cells directly, without requiring impairment 
of the patient's own immune system before therapy. 
Encouraged by these advances, the investigators are now continuing their research to include 
delivery of genetically reengineered T cells and other tumor-fighting immune cells, such as natural 
killer cells or invariant natural killer T cells, or even combinations of cell types. "We are also 
developing various injectable polymeric compositions for the minimally invasive implantation of 
active depots of anticancer T cells into poorly accessible or inoperable tumors (e.g., under 
ultrasound guidance)," Dr. Stephan said, adding that their ultimate goal is to implement biomaterial-
supported T cell implants in the clinic, in collaboration with clinical investigators at Fred Hutch. 
Stephan SB, Taber AM, Jileaeva I, Pegues EP, Sentman CL, Stephan MT. 2015. Biopolymer 
implants enhance the efficacy of adoptive T-cell therapy. Nat Biotechnol 33(1):97-101. 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
 
Images provided by Dr. Matthias Stephan 
The photographs show biopolymer scaffolds being 
hydrated and loaded with tumor-targeting T cells. The 
schematic diagram demonstrates the action of a T cell-
containing scaffold implanted at a tumor site; green dots 
represent stimulatory microspheres incorporated into the 
polymer framework, which trigger proliferation and 
migration of the T cells (shown in blue) into the surrounding 
tissue as the scaffolding material biodegrades. 
 
 


Plastic Fantastic! Biopolymers Efficiently Boost T-Cell Therapy

Neves, Alexandre

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
UPF1 DUX NMD in Facioscapulohumeral 
Muscular Dystrophy 
February 16, 2015 
    A Neves 
Next generation sequencing has revolutionized the field of human genetics, and discovery of 
disease causing mutations continues at an accelerated pace. However, for some human diseases, 
knowledge of the genetic mutation is not sufficient to resolve disease etiology. One such example is 
facioscapulohumeral muscular dystrophy (FSHD), an adult-onset muscular dystrophy characterized 
by progressive weakness in a subset of skeletal muscle groups of the upper body, namely ones in 
the face, shoulders and upper arms. Previous studies showed that FSHD is caused by defective 
epigenetic repression of a macrosatellite array in a region of chromosome 4, which results in mis-
expression of DUX4 in skeletal muscle. DUX4 is a testis-specific transcription factor that activates 
genes involved in germline development, but its mis-expression is toxic to muscle cells. However, 
how DUX4 expression is regulated in normal and pathologic states remained poorly understood. 
A new Fred Hutch collaboration between the labs of Stephen Tapscott (Human Biology and Clinical 
Divisions) and Robert Bradley (Basic Sciences and Public Health Sciences Divisions), spearheaded 
by graduate student Qing Feng and published in eLife, describes a novel feedback loop between a 
quality control pathway called nonsense-mediated decay (NMD) and DUX4. "Our findings indicate 
that regulated RNA degradation contributes to the expression and downstream toxicity of DUX4, and 
also suggest a new mechanism for post-transcriptional autoregulation of transcription factors," said 
Dr. Bradley. 
The study began by reexamining previous data from the Tapscott lab that had used deep 
sequencing of RNA to determine both coding and non-coding RNAs induced by high levels of DUX4 
(Young et al., 2013). This analysis revealed that increased DUX4 levels resulted in increased 
abundance of many coding RNA isoforms with premature stop codons (nonsense mutations) 
upstream of splice junctions, which are predicted to be NMD substrates. To explore whether 
increased levels of NMD substrates were linked to reduced NMD efficiency, the authors compared 
relative levels of a heterologous NMD substrate (β-globin open reading frame with or without a 
premature stop codon) and found that DUX4-expressing myoblasts had two-fold higher levels of the 
β-globin NMD reporter. Globally, DUX4 expression increased levels of predicted NMD substrates for 
all classes of splicing events examined. 
February 16, 2015 SCIENCE SPOTLIGHT 
 
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Next, the authors pursued the mechanism of NMD inhibition following DUX4 expression. Because 
DUX4 expression led to up-regulation, rather than down-regulation, of most NMD factors, a pattern 
reminiscent of UPF1 knockdown cells, the authors hypothesized that protein levels of UPF1, a 
central component of the NMD machinery, might be divorced from its mRNA levels. To test this, the 
authors measured UPF1 levels, as well as levels of NMD factors that were transcriptionally up-
regulated in DUX4-expressing myoblasts (SMG7 and UPF3B) and found that only UPF1 was 
robustly down-regulated. The tight coupling between DUX4 protein production, decreased UPF1 
levels, and increased levels of NMD substrates revealed by time-course analysis suggested that low 
levels of UPF1 and possibly other NMD factors, contributed to inefficient NMD in DUX4-expressing 
myoblasts. To test if DUX4 expression leads to UPF1 degradation, the researchers inhibited the 
proteasome (with MG-132) in DUX4-expressing or control myoblasts and found that UPF1 levels 
were restored in DUX4-expressing, but not control cells. The authors next tested whether DUX4 
itself was an NMD substrate, as it contains a constitutively spliced intron within its 3’ untranslated 
region (3’ UTR). To test this, the authors knocked down UPF1 in FSHD myoblasts and then 
differentiated them into myotubes to induce DUX4 expression. Indeed, DUX4 mRNA expression was 
four-fold higher in UPF1 knockdown cells. 
A characteristic feature of FSHD is the variegated nature of DUX4 expression in muscle cells, 
meaning that only a small percentage of FSHD nuclei are DUX4+. Intriguingly, UPF1 knockdown led 
to an increase in the fraction of DUX4+ nuclei (from 0.3 to 2.1%). Finally, the investigators showed 
that expressing DUX4 in FSHD myotubes led to a five-fold increase in the levels of endogenously 
transcribed DUX4 mRNA. These data led the authors to propose a double negative feedback loop 
where DUX4 indirectly stabilizes its own mRNA by inhibiting NMD. Overall, this study showed that 
impaired RNA quality control mechanisms might contribute to FSHD pathophysiology. "The next key 
step is to test whether these abnormalities in nonsense-mediated decay explain clinical aspects of 
FSHD, such as inflammation in affected muscles, that are not yet understood," concluded Dr. 
Bradley. 
Feng Q, Snider L, Jagannathan S, Tawil R, van der Maarel SM, Tapscott SJ, Bradley RK. 2015. A 
feedback loop between nonsense-mediated decay and the retrogene in facioscapulohumeral 
muscular dystrophy. eLife, 4. 
Young JM, Whiddon JL, Yao Z, Kasinathan B, Snider L, Geng LN, Balog J, Tawil R, van der 560 
Maarel SM, Tapscott SJ. 2013. DUX4 binding to retroelements creates promoters that are 561 active 
in FSHD muscle and testis. PLoS Genet 9: e1003947. 
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Robert Bradley 
Schematic of potential double-negative 
feedback loop between DUX4 and NMD, 
in which DUX4 inhibits NMD and NMD 
degrades DUX4 mRNA. 
 


UPF1 DUX NMD in Facioscapulohumeral Muscular Dystrophy

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Dietary Macronutrient Intake Impacts Plasma 
Adiponectin Levels 
April 20, 2015 
    JM Kocarnik 
Over time, chronically elevated systemic inflammation contributes to many chronic diseases, 
including cardiovascular disease, cancer, and type 2 diabetes.  Dietary fat intake has long been 
assumed to contribute to chronic systemic inflammation, but the effects of dietary macronutrient 
composition on inflammatory biomarkers have not been clearly established.  While dietary fat might 
increase inflammation through increased fat mass, inflammatory effects independent from changes 
in body weight have also been suggested.  In order to better measure these associations, Drs. 
Xiaoling Song, Mario Kratz, and colleagues in the Public Health Sciences Division evaluated 
changes in inflammatory biomarkers among participants on different diets in a randomized controlled 
feeding study.  As recently reported in the European Journal of Nutrition, the researchers found that 
adiponectin concentrations were substantially lowered in individuals consuming a low-fat high-
carbohydrate diet compared to those consuming a moderate-fat diet. 
Systemic inflammation can be measured through circulating biomarkers such as C-reactive protein 
(CRP), interleukin-6 (IL-6), and the soluble tumor necrosis factor receptors I and II (sTNFRI and 
sTNFRII).  In addition to these four biomarkers of systemic inflammation, adiponectin levels can also 
be used as a marker of adipose tissue inflammation.  An adipocyte-derived hormone with anti-
inflammatory and insulin-sensitizing effects, adiponectin is inversely associated with 
adiposity.  Increased body fat is associated with low-grade chronic adipose tissue inflammation.  As 
this inflammation suppresses the secretion of adiponectin from adipocytes, decreased adiponectin 
levels can be considered a marker of increased adipose tissue inflammation.  
To evaluate the effects of dietary macronutrient composition on inflammation biomarker 
concentrations, the researchers conducted a secondary analysis of samples available from the Fatty 
Acids Marker Evaluation (FAME) study.  This randomized controlled feeding trial consisted of 92 
individuals assigned to one of three diets.  Two of these were eucaloric diets, consisting of sufficient 
calories to maintain body weight, while a third diet provided 33% fewer calories.  These diets differed 
in macronutrient content (see figure), consisting of either moderate-fat (36% fat, 46% carbohydrate) 
or low-fat high-carbohydrate (18% fat, 64% carbohydrate) intake.  Plasma biomarker concentrations 
were measured at baseline, and then again after 6 weeks of the assigned diet. 
April 20, 2015 SCIENCE SPOTLIGHT 
 
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After 6 weeks on these diets, the researchers found no differences in these biomarkers for systemic 
inflammation.  For adipose tissue inflammation, the authors did find that adiponectin concentrations 
tended to be increased in those who lost weight, as expected.  After adjusting for weight change, 
however, the authors found that adiponectin levels were significantly reduced, by about 10%, in 
those eating low-fat diets compared to the moderate-fat diet (p=0.008).  "This may be of concern 
because plasma adiponectin concentrations are inversely associated with the risk of type 2 diabetes, 
coronary heart disease, hypertension, and a variety of cancers," said lead author Dr. Xiaoling Song. 
This surprising finding should motivate future research of long-term macronutrient intake on 
inflammatory biomarkers.  Said Dr. Song, "it is not clear whether the reduction in fat intake per se, 
the increase in carbohydrates, or a shift in the fat-to-carbohydrate ratio was the cause of the 
decreased adiponectin concentrations.  This again demonstrates the difficulty in studying the effects 
of specific dietary components. However, although there were differences in carbohydrate quality 
between diets (i.e. the amount of fiber and added sugars were higher in the low-fat diet than those in 
the moderate-fat diet), our diets mimicked the carbohydrate choices that free-living individuals are 
likely to make when consuming moderate vs. low fat diets in the real world. Therefore, our results 
are relevant to real world situations, and do raise a new concern for the health effects of consuming 
very-low fat diets." 
Other researchers from the Public Health Sciences Division contributing to this project were Ms. 
Yvonne Schwarz, Pamela Yang, and Xiaojun Hu, as well as Drs. Mark Kestin and Johanna Lampe. 
 
Citation:  
Song X, Kestin M, Schwarz Y, Yang P, Hu X, Lampe JW, Kratz M. 2015. A low-fat high-
carbohydrate diet reduces plasma total adiponectin concentrations compared to a moderate-fat diet 
with no impact on biomarkers of systemic inflammation in a randomized controlled feeding 
study. Eur J Nutr. doi: 10.1007/s00394-015-0841-1 [Epub ahead of print]. 
 
 
 
 
April 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
 
 
Image provided by Dr. Xiaoling Song 
Macronutrient content (percent of total energy intake) in FAME 
Study diets. 
 


Dietary Macronutrient Intake Impacts Plasma Adiponectin Levels

Zentner, Gabriel

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Doxorubicin Gives Active Promoters a Break 
April 20, 2015 
    GE Zentner 
Doxorubicin is a widely used anticancer drug, and while its mechanism of action has been 
understood for decades, the reason for its selective toxicity to cancer cells has been the subject of 
much debate.  Doxorubicin and related drugs, called anthracyclines, insert between DNA bases 
(intercalate) causing a positive torsion of the double helix.  This alteration of DNA structure can trap 
enzymes including Topoisomerase II (TopoII), which relieves torsional stress by generating DNA 
double-strand breaks (DSBs) and subsequently ligating the cleaved strands.  It has been shown that 
Doxorubicin traps TopoII following cleavage but before re-ligation, leading to cell killing, but it is not 
known if this, or overall inhibition of TopoII and subsequent failure to relieve torsional stress, is 
Doxorubicin's mechanism of selective cancer cell killing.  Previously, postdoctoral fellow Dr. Fan 
Yang in the lab of Dr. Steven Henikoff (Basic Sciences Division), in collaboration with Dr. 
Christopher Kemp (Human Biology and Public Health Sciences Divisions), found that doxorubicin 
increases nucleosome turnover around active gene promoters (Yang et al., 2013).  This led to the 
hypothesis that, by causing increased exposure of DNA through histone turnover, doxorubicin and 
other anthracyclines might cause increased levels of DNA DSBs within active promoters. 
To map the positions of DSBs genome-wide, the authors used a protocol known as BLESS (direct in 
situ breaks labeling, enrichment on streptavidin and next-generation sequencing).  They first 
performed BLESS in mouse squamous cell carcinoma (SCC) cells treated with various doses of the 
Doxorubicin as well as the TopoII poison Etoposide.  Treatment of cells with a dose of Doxorubicin 
ten times below the therapeutic dose resulted in a slight increase in DSBs relative to untreated cells, 
while treatment of cells with therapeutic doses of Doxorubicin and Etoposide led to massive 
increases in DSBs.  The authors then sequenced DNA from control cells, cells treated with 
Doxorubicin and Etoposide, and cells treated with a low dose of a second anthracycline, Aclarubicin, 
that does not poison TopoII. 
Analysis of the distribution of spontaneous DSBs (that is, DSBs not caused by drug treatment) 
revealed preferential enrichment around gene promoters, with highly expressed genes displaying 
more DSBs.  This suggested that spontaneous DSBs were coupled to gene transcription.  Drug 
treatment resulted in increased levels of DSBs at promoters in a pattern similar to that seen in 
untreated cells, indicating that anthracyclines and Etoposide induce DSBs at regions of the genome 
that are susceptible to transcription-induced damage. 
April 20, 2015 SCIENCE SPOTLIGHT 
 
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Mammalian promoters are frequently contained with CpG islands, which are regions of the genome 
containing a large proportion of C-G base pairs but low levels of DNA methylation.  The authors thus 
asked if there were differences in DSB levels at CpG versus non-CpG.  They first analyzed DSBs at 
all CpG islands and found that CpG islands were protected from DSBs regardless of their genomic 
location.  Consistent with this observation, they found that CpG-island promoters displayed lower 
levels of DSBs than non-CpG promoters.  Further analysis revealed that this distinction was not due 
to differences in sequence composition, and the authors thus concluded that CpG islands are 
protective against DSBs. 
While analysis of DSB levels revealed increases relative to control with all drug treatments, it was 
not possible for the authors to compare between samples due to data normalization parameters.  To 
address this, the authors used a 'spike-in' method, wherein a fixed amount of yeast DNA was added 
to each mouse sample.  Using this approach, they found that both low and therapeutic doses of 
Doxorubicin produced equivalent increases of DSBs, while a therapeutic dose of Etoposide caused 
higher levels of DSBs than a low dose.  Interestingly, a low dose of Aclarubicin caused even higher 
levels of DSBs than the therapeutic dose of Doxorubicin. 
Combined with the authors' previous results, the work presented in this study indicates that 
anthracyclines cause increased nucleosome turnover at gene promoters, leading to increased DNA 
exposure and subsequent breakage.  The similar patterns of DSBs induced by three drugs are 
striking, given that there are notable differences in their mechanisms: Doxorubicin intercalates into 
DNA and traps TopoII after DSB production, Etoposide traps TopoII and causes single-strand 
breaks, and Aclarubicin intercalates into DNA but does not trap TopoII.  As all three drugs exert 
effects on DNA torsion, this is likely the common denominator in their similar effects on nucleosome 
turnover and DSB production. 
 
Yang F, Kemp CJ, Henikoff S. 2015. Anthracyclines induce double-strand DNA breaks at active 
gene promoters. Mutat Res 773:9-15. 
See also: Yang F, Kemp CJ, Henikoff S. 2013. Doxorubicin enhances nucleosome turnover around 
promoters. Curr Biol23(9):782–787. 
 
 
 
April 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
 
 
Image from Wikipedia 
Structural representation of two Doxorubicin molecules intercalating 
into DNA. 
 


Doxorubicin Gives Active Promoters a Break

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Y Male Sex Chromosomes Degenerate 
May 18, 2015 
     A Neves 
In many animals, sex is determined by the presence of sex chromosomes. In humans, males have 
an X and a Y whereas females have two X chromosomes. Despite significant differences that are 
apparent under the microscope, both the X and Y evolved from non-sex chromosomes (autosomes; 
chromosomes 1-22 in humans). However, it remains unclear how the Y became dissimilar from the 
X. For instance, the Y can no longer appreciably swap genetic material with the X, a property that 
evolved over the last 180 million years and is known as recombination suppression. This property is 
thought to be important for the Y chromosome to retain its sex-determining genes. But many 
questions remain unanswered regarding the sequence of events that led to recombination 
suppression, DNA sequence degeneration (the Y is much smaller than the X), and mechanisms that 
restore gene dosage balance (genes on the X have one copy in males and two in females). 
The threespine stickleback fish has emerged as a powerful model to study vertebrate sex 
chromosome evolution due to the ability to raise it in the laboratory together with the fact that it has a 
XY chromosome system that has evolved relatively recently, 13-16 million years ago. A new Fred 
Hutch study from Dr. Catherine Peichel’s Laboratory (Human Biology and Basic Sciences Divisions), 
led by post-doctoral fellow Dr. Michael White and published in Molecular Biology and Evolution, used 
high-throughput sequencing of DNA and RNA of both female and male fish to infer sequence 
evolution and dosage compensation. "The threespine stickleback has been an important model in 
the study of sex chromosome evolution. In this study we provide the first detailed sequence-based 
characterization of molecular evolution across the X and Y chromosomes of the threespine 
stickleback", said Dr. White. 
Previous work in the Peichel lab (Ross and Peichel, 2008) provided cytogenetic evidence that the Y 
chromosome structurally diverged from the X through 3 inversions and a large deletion (6Mb). The 
new report reanalyzed these regions with higher resolution and separated the inversions into a more 
recent evolutionary group while the deletion represented an older group. Strikingly, some genes 
within the deletion were maintained on the Y, and these were found to be under purifying selection, a 
form of natural selection that preserves gene function. Since DNA sequence divergence occurs at 
different rates on the X and Y chromosomes, the authors compared the sex chromosomes from 
threespine sticklebacks to the sex chromosomes of the ninespine stickleback, a related species,  
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
because the latter independently evolved a XY chromosome system from a different autosomal 
ancestor. This comparison revealed an elevated rate of evolution of the X chromosome, particularly 
for genes that did not have a functional counterpart on the Y. To test whether dosage compensation 
evolved in threespine sticklebacks, the investigators used RNA sequencing to monitor expression 
levels of X-linked genes with corresponding genes in the ninespine sticklebacks. This analysis 
uncovered that within the older region, genes with a single copy in males exhibited decreased 
expression, arguing against a global dosage compensation mechanism. In support of this, genes 
with two copies in males had X-biased expression caused by reduced expression of the Y-linked 
copy. Finally, the authors found that Y-linked genes that were retained under purifying selection 
were enriched for genes predicted to have a role in intracellular transport. 
"Although much progress has been made understanding the evolution of heteromorphic sex 
chromosomes, it remains unclear whether dosage compensation evolves in males in response to 
gene loss on the Y chromosome. Recent evidence suggests dosage imbalances in the 
heterogametic sex are only resolved at the most haploinsufficient genes but this has not been widely 
studied. Our results strengthen these conclusions. In the oldest region of the threespine stickleback 
Y chromosome, we found a handful of genes that were still expressed from the Y chromosome. 
These genes were under strong purifying selection and were enriched for dosage sensitive 
functions. These patterns have only been recently shown in the well-studied mammalian XY sex 
chromosome system. Our results provide additional evidence in a vertebrate system that global 
dosage compensation may not be a common route to restore gene expression imbalance in the 
heterogametic sex," summarized Dr. White. 
 
White MA, Kitano J, Peichel CL. 2015. Purifying selection maintains dosage-sensitive genes during 
degeneration of the threespine stickleback Y chromosome. Mol Biol Evol. msv078, Epub ahead of 
print. 
See also: 
Ross, JA, Peichel CL. 2008. Molecular cytogenetic evidence of rearrangements on the Y 
chromosome of the threespine stickleback fish. Genetics, 179: 2173-2182. 
 
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Michael White. 
Threespine stickleback males possess a young 
XY sex chromosome system at the early stages 
of differentiation. 
 


Y Male Sex Chromosomes Degenerate

Zentner, Gabriel E.

August 17, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
HP1E leaves a lasting impression on paternal 
chromosomes 
Aug. 17, 2015  
     GE Zentner 
 
 
 
 
 
Fluorescent imaging of DNA reveals 
the presence of a chromatin bridge 
(indicated by an arrow) in PEL embryos 
fathered by HP1E-depleted males. 
 
Image from the publication 
 
 
 
The cells of most animals contain two copies of each chromosome.  Notable exceptions are egg and 
sperm cells, which contain only one copy of each chromosome.  Thus, when an egg fuses with a 
sperm during fertilization, the resulting zygote contains two copies of each chromosome, one 
maternal and one paternal.  During development, each chromosome will duplicate and be 
segregated many times during mitosis.  The first zygotic division is notably complex, as both 
maternal and paternal chromosomes must go through drastic and different reorganization of their 
chromatin structures.  Paternal DNA is packaged with proteins called protamines, which condense 
DNA more tightly than the histone proteins used by the egg to condense maternal DNA.  Thus, 
paternal chromosomes must decondense, exchange these protamines for histones, recondense, 
and then recondense in time to for paternal and maternal chromosomes to divide in synchrony in the 
zygote.  While previous work has shown that maternal proteins, provided by the egg, are involved in 
facilitating the protamine-to-histone transition in the resulting zygote, it is much less clear how 
paternal proteins participate in this process. 
August 17, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
Previous work by postdoctoral fellow Dr. Mia Levine (now Assistant Professor of Biology at the 
University of Pennsylvania) and colleagues in the laboratory of Dr. Harmit Malik (Basic Sciences 
Division) demonstrated that a particular protein involved in the formation of condensed, 
transcriptionally silent chromatin (heterochromatin), called heterochromatin protein 1E (HP1E), is 
only expressed in testes.  Dr. Levine hypothesized that HP1E, given its testis-restricted expression 
and the well-known roles of HP1 family members in chromatin organization, was a strong candidate 
for a paternal protein that could influence the exchange of protamines for histones.  Dr. Levine, 
along with research technician Ms. Helen Vander Wende, thus studied the role of HP1E during 
paternal genome reorganization.  Not only did they find that HP1E is essential for successful 
completion of the first zygotic mitosis, but that the protein itself is not present during this mitosis. 
Instead, HP1E epigenetically primes paternal chromosomes for future faithful completion of mitosis. 
To dissect the role of HP1E during sperm maturation, the authors first examined the pattern of HP1E 
expression in testes.  They found that HP1E was localized to paternal chromosomes only during the 
zygotic exchange of protamines for histones, and not in mature sperm.  As HP1 proteins are well-
known organizers of heterochromatin, the authors asked if loss of HP1E might preferentially alter the 
expression of heterochromatic genes.  This analysis showed that 100% of heterochromatic genes 
altered by HP1E knockdown increased in expression, consistent with the general transcriptional 
silence of heterochromatin. 
The authors next assessed the role of HP1E in male fertility by generating HP1E-depleted male flies, 
which displayed prominent sterility.  However, HP1E-deficient fathers produced motile sperm 
capable of transferring to females, fertilizing eggs, and initiating embryogenesis.  Rather, all but 
0.5% of the embryos sited by HP1E-depleted fathers failed to hatch due to a chromosomal defect 
called a chromatin bridge, which results from incomplete chromosome segregation, followed by 
further defects in subsequent mitoses.  Thus, HP1E is a paternal effect lethal (PEL) gene, meaning 
that the genotype of the father influences the phenotype of the offspring. 
To further understand the requirement of HP1E for the fidelity of chromosome segregation, the 
authors followed the dynamics of paternal DNA prior to the first zygotic telophase.  Paternal DNA 
from PEL embryos completed the protamine-to-histone transition, recondensed, migrated toward 
maternal DNA, and entered the first mitosis just as in wild-type embryos.  However, paternal DNA 
failed to condense in synchrony with maternal DNA and failed to separate along the mitotic spindle, 
instead forming chromatin bridges. 
While tracking the fate of DNA from HP1E-depleted fathers, the authors noted that only a portion of 
the paternal genome is susceptible to HP1E deficiency.  Based on their observation that HP1E 
August 17, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
depletion upregulates heterochromatic genes, they hypothesized that the regions of the genome 
most vulnerable to HP1E loss might be long stretches of heterochromatin.  To test this, they 
performed fluorescent in situ hybridization (FISH) analysis of heterochromatic regions on all five fly 
chromosomes in PEL embryos.  The Y chromosome, which is completely heterochromatic, was 
trapped as a chromatin bridge in 94% of PEL embryos, while the maternal X was never 
bridged.  Autosomes were trapped at frequencies intermediate to those of the sex chromosomes. 
This work shows not only that HP1E is required for proper zygotic mitosis, but that it exerts this 
effect transgenerationally; that is, it is not present during the first mitosis but instead primes paternal 
chromosomes for faithful segregation in zygotes by acting earlier during sperm development.  Given 
that the heterochromatic Y chromosome was frequently trapped in chromatin bridges, it appears that 
HP1E somehow primes heterochromatic regions of the genome for proper condensation for the first 
zygotic mitosis. 
"Paternal effect lethal genes like HP1E provide a means to investigate the mysterious process of 
chromatin remodeling that prepares sperm chromatin to re-engage in mitosis. They also reveal the 
important role of such remodeling factors to coordinate mitosis between maternal and paternal 
chromosomes, even in the absence of cellular checkpoints that would normally police this 
coordination," said Dr. Malik. 
Levine MT, Vander Wende HM, Malik HS. 2015. Mitotic fidelity requires transgenerational action of a 
testis-restricted HP1. eLife 4:e07378. 
See also: Levine MT, McCoy C, Vermaak D, Lee YC, Hiatt MA, Matsen FA, Malik HS. 2012. 
Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin 
protein 1 (HP1) gene family. PLOS Genet 8:e1002729. 
Funding for this work was provided by the National Institutes of Health, the Howard Hughes Medical 
Institute, and the G. Harold and Leila Y. Mathers Foundation. 
 


HP1E leaves a lasting impression on paternal chromosomes

September 21, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
Survey helps identify diagnostic misconception 
in Latino study participants 
September 21, 2015 
     JM Kocarnik 
 
 
 
 
 
Biological specimens are increasingly collected 
and stored for population health research. 
 
Image provided by Dr. Sarah Knerr. 
 
 
 
 
Researchers conducting observational studies are increasingly collecting biological samples to help 
understand and improve population-level health and disease management.  While collectively useful, 
the results from any given participant's sample are generally not clinically meaningful, and so are 
generally not made available.  Despite this distinction, some research participants mistakenly expect 
that donating biological samples may benefit them personally by providing meaningful health 
information.  Identifying participants susceptible to such misconceptions would help ensure ethical 
recruitment in biospecimen research projects, particularly in underserved populations.  In a recent 
report in BMC Medical Ethics, Drs. Sarah Knerr and Rachel Ceballos in the Public Health Sciences 
Division describe a new survey instrument they developed to systematically measure conflation of 
biospecimen-based research and clinical care for use with Latino communities. 
Research participants’ expectations of receiving personal health benefits when participating in 
observational studies have been termed diagnostic misconception.  This is an extension of the 
concept of therapeutic misconception, most common in clinical trials, where trial participants 
misunderstand the different goals and principles governing research compared to clinical 
September 21, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
care.  These misunderstandings can alter participants' perceptions of the balance of potential 
benefits and burdens of research participation, which in turn can impact the effectiveness of the 
informed consent process and the decision whether to participate. "With the increasing focus on 
developing precision and patient-centered medicine the line between research and clinical care will 
likely get blurrier," said lead author Dr. Knerr.  "It is important for the research community to think 
through the implications of what this means for our participants." 
Of particular importance is evaluating the extent and impact of diagnostic misconception in 
communities that have limited health care access.  These populations may be more strongly affected 
by the negative consequences of misunderstandings about research and clinical care, which could 
damage trust in both doctors and researchers.  Furthermore, diagnostic misconception could 
potentially motivate project participation if health care is otherwise inaccessible due to false beliefs 
that research participation can substitute for medical care. Said Knerr, "We were inspired to create 
this instrument by observations we made analyzing data from a prior research study Dr. Ceballos 
conducted with Latinos living on the US-Mexico border.  During interviews about biomedical 
research participation, people often described 'getting checked' as a main benefit of providing a 
sample for research, even though our interview guide had initially explained the differences between 
providing samples for research versus clinical care." 
To enable future research on the causes and consequences of such misconceptions, the authors 
developed a quantitative survey instrument measuring conflation of observational biospecimen-
based research and clinical care. This involved developing 20 questions pertaining to participants’ 
understanding of the difference between observational research and clinical trials, the purpose of 
biospecimen-based research, and the perceived likelihood of benefiting personally from 
participation. By piloting these questions with 150 Latino adults in the lower Yakima Valley of 
Washington state and performing psychometric analyses, the authors identified the combination of 
questions that best measured conflation of research and clinical care. The authors also found that 
individuals with the highest scores on the final instrument, indicating the greatest conflation of 
biospecimen-based research and clinical care, were unemployed, spoke only Spanish, had no 
health insurance, had less access to traditional health care, and had good self-rated health.  Future 
biospecimen-based research studies may be able to modify their recruitment and informed consent 
processes in order to help clarify misunderstandings about research and clinical care, particularly for 
subgroups that may be more likely to have misconceptions about research. 
While a good first step, further research is needed to refine the instrument and assess its 
generalizability in more diverse communities and settings.  Said Dr. Knerr, "the most pressing 
September 21, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
questions that this work raises are ethical ones about research recruitment and informed decision-
making in communities that have poor access to health care, who are often also underrepresented in 
population-based biobanks and targeted for special recruitment efforts. How much do they know 
about biobanking and observational research? How likely are they to conflate research and clinical 
care and does this influence their decisions to take part in research studies? Is this coercive? What 
can we do about it?"  Answering these questions will be important for ensuring that all populations 
are able to appropriately participate and benefit from biospecimen research. 
  
Citation: 
Knerr S, Ceballos RM. 2015. Giving samples or "getting checked": measuring conflation of 
observational biospecimen research and clinical care in Latino communities. BMC Med Ethics. 
16:49. doi: 10.1186/s12910-015-0041-9. 
This work was supported by the National Cancer Institute under R25 CA92408 and U54 CA132381 
 
 
 


Survey helps identify diagnostic misconception in Latino study participants

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
Chimpanzee APOBEC3 proteins deter SIVs from 
any monkey business 
October 19, 2015 
     A Neves 
 
 
Illustration of the rationale 
behind the study of Drs. 
Michael Emerman, Lucie 
Etienne and colleagues. 
 
Image provided by Dr. 
Lucie Etienne. 
 
 
The current HIV-1 pandemic is a result of cross-species transmission of simian immunodeficiency 
viruses (SIVs) from chimpanzees to humans. Both SIV and HIV belong to family of viruses known as 
lentiviruses. To break the species barrier, a virus must adapt to a new suite of antiviral proteins 
collectively known as host restriction factors. While previous studies have documented the 
adaptation of lentiviruses from chimpanzees to humans, much less is known about the adaptive 
processes that enabled monkey SIVs to infect chimpanzees. Intriguingly, chimpanzees are infected 
by a single type of SIV (SIVcpz), despite being exposed to a wide variety of monkey SIVs through 
their predator-prey relationship hunting. A new Fred Hutch study from Dr. Michael Emerman’s 
Laboratory (Basic Sciences and Human Biology Divisions), led by former post-doctoral fellow Dr. 
Lucie Etienne, now at the International Center for Infectiology Research in Lyon, asked why 
chimpanzees are resistant to infection by most SIVs, including ones present in their monkey prey. 
This work was recently published in PLOS pathogens. 
The APOBEC3 family of host restriction factors potently blocks the lentiviral life cycle in the absence 
of the viral protein Vif. Vif is present in all primate lentiviruses and it inhibits APOBEC3 by targeting it 
for proteasomal degradation. Because adaptation of SIVcpz to chimpanzees involved the evolution 
of vif to counteract chimpanzee APOBEC3G, the authors hypothesized that APOBEC3G constituted 
a barrier to SIV infection. As a first step to test their hypothesis, they queried a panel of Vifs from ten 
October 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
SIV lineages for their ability to antagonize chimpanzee APOBEC3G. Most SIV Vifs were incapable of 
inhibiting APOBEC3G in single-round infectivity assays. In addition, known cases of cross-species 
transmissions were shown to occur when the virus’ Vif was capable of antagonizing APOBEC3G 
from their new host species. Next, the researchers tested the ability of the SIV Vif to counteract other 
APOBEC3 family members and found that most Vif proteins could antagonize APOBEC3F and 
APOBEC3H while only a few could counteract APOBEC3D and APOBEC3G. To determine whether 
these Vif-dependent restrictions also occurred in immune cells, the authors infected primary 
chimpanzee CD4+ T cells, the main target cells of HIV and SIV, with either no Vif or Vif from different 
SIV lineages. They demonstrated that vif was essential for replication in chimpanzee CD4+ T cells 
and that similar Vif-dependent restrictions were observed. Because APOBEC3 proteins mediate 
their function by inducing mutations in the viral genome, the investigators sequenced the vif DNA 
extracted from infected cells and found the characteristic G-to-A hypermutation in the absence of Vif, 
an effect that was counteracted by Vif from SIVcpz. Finally, the authors characterized the genetic 
variants of the APOBEC3 genes in bonobos and in different subspecies of chimpanzees by 
analyzing sequencing data from the Great Ape Genome Project. While many polymorphisms were 
present, none appeared to affect either their antiviral activity or their Vif-sensitivity. Thus, although 
bonobos and western chimpanzees do not appear to have any current lentiviral infections, these 
studies imply that the ancestors of all current chimpanzee species may have been selected for 
resistance to SIV infections. Said Dr. Etienne "Overall, our study highlights that the evolution of the 
APOBEC3 antiviral gene family may directly participate in the protection of a species against viral 
cross-species transmissions. In the long-term, this may have impact for human health in mostly two 
ways: (a) it helps to better understand which viruses may be more prone to cross the species barrier 
to humans and (b) it characterizes how natural defense proteins may be strong barriers against 
heterologous viruses, which may ultimately help the design of novel antiviral strategies against 
pathogenic viruses".  
Etienne L, Bibollet-Ruche F, Sudmant PH, Wu LI, Hahn BH, Emerman M 2015. The role of the 
antiviral APOBEC3 gene family in protecting chimpanzees against lentiviruses from monkeys. PLOS 
pathogens. 11(9):e1005149. 
This work was funded by the NIH and an amfAR Mathilde Krim Fellowship in Basic Biomedical 
Research. 
 
 


Chimpanzee APOBEC3 proteins deter SIVs from any monkey business

November 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
A DNA sequencing sleuth called CypherSeq 
November 15, 2015 
     A Neves 
 
CypherSeq error correction and detection of rare mutations. A) Base substitutions determined by next-generation 
sequencing with a quality filter. The sample contains four mutations at the sites shown in red, which were spiked into 
the sample at known ratios of one mutant copy in 100, 1000, 10,000, and 100,000 wild type copies. However, due to 
the high background of erroneous substitutions, the real mutations cannot be distinguished from sequencing errors. 
B) Base substitutions determined by sequencing with CypherSeq. The increased accuracy of CypherSeq removes 
the background, allowing accurate and quantitative identification of the four real mutations down to a frequency of ~1 
mutant in 100,000 copies. 
 
Image provided by Dr. Mark Gregory. 
Next-generation sequencing (NGS) technologies have transformed both basic and clinical research, 
as they have enabled the development of diverse applications ranging from pre-natal testing to 
cancer prognosis. However, there are a number of limitations that prevent NGS technologies from 
fulfilling their full potential. These include the inherent error of DNA sequencing instruments, the 
sequencing coverage depth and the amount of available sample DNA required. To address these 
limitations, post-doctoral fellows Drs. Mark Gregory and Jessica Bertout and collaborators in Dr. 
Jason Bielas’ Laboratory (Human Biology and Public Health Sciences Divisions), developed a new 
method called CypherSeq that markedly improved NGS-based detection of rare mutations. This 
exciting report was recently published in the journal Nucleic Acids Research. 
The investigators first designed the CypherSeq vector as a double-stranded barcoded vector that 
can be amplified in bacteria and contains all of the necessary sequences required to perform NGS. 
To delineate the power of the CypherSeq error correction algorithm, the researchers first applied the 
CypherSeq technology to a mixed DNA population with known ratios of both wild-type and four 
November 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
mutant versions of the tumor suppressor gene TP53. This analysis revealed that a single TP53 
mutant could be resolved among more than 10
5
 copies of wild-type TP53, which translates to a 
frequency of 2.4 x 10
-7
 mutations per base pair (see figure) and represents at least a 10-fold 
improvement over other NGS methods. Next, the authors addressed whether CypherSeq could be 
used for genome-wide assessment of both spontaneous and induced mutations. To this end, they 
grew liquid cultures of the baker’s yeast Saccharomyces cerevisiae with or without the mutagen 
ethyl methanesulfonate (EMS). After application of CypherSeq sequencing and computational error-
correction these samples revealed a base substitution frequency of 1.4 x 10
-6
 and 4.6 x 10
-6
 in 
untreated and EMS-treated cultures, respectively, demonstrating a clear increase due to treatment. 
Importantly, the additional mutations seen in the EMS-treated samples were nearly all C to T 
mutations, which match the known mutation signature of the EMS mutagen. As the EMS mutations 
in this experiment would only be present on a single genome within the yeast culture, the accurate 
detection of these mutations represent single cell resolution sequencing. Thus, CypherSeq is a more 
accurate DNA sequencing method that, for the first time, enables genome-wide calculations of 
mutations rates without resorting to single cell approaches.  
Finally, the authors demonstrated that CypherSeq can also efficiently enrich a sequencing library for 
specific genomic sites. Because the CypherSeq vector is circular, rolling circle amplification (RCA) of 
a biotinylated primer was used to generate single-stranded DNA concatemers, which could be 
further enriched with streptavidin affinity purification. Indeed, CypherSeq vectors containing TP53 
sequences in the background of randomly sheared genomic DNA were enriched 977-fold. Such 
enrichment, decreases off-target superfluous sequencing, permits deeper sequencing depth of 
targeted sites, and thus the detection of much rarer variants. In summary, CypherSeq’s highly 
sensitive error correction and RCA-based target enrichment strategies offer tantalizing possibilities 
for cancer diagnostics. Said Dr. Gregory: "Combining highly accurate sequencing with the ability to 
target specific regions of the genome is an important advance which will allow us to investigate rare 
mutations throughout the genome, at a resolution unmatched before. This technology should enable 
the development of a new class of sequencing-based clinical tests, such as noninvasive blood tests 
for early cancer diagnosis, identification of optimal therapeutic approaches, and monitoring of 
treatment response".  
Funding for this study was provided by the National Institutes of Health, the Department of Defense, 
the Canary Foundation, the Marsha Rivkin Center for Ovarian Cancer Research, and the Ellison 
Medical Foundation.    
November 15, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
Gregory MT, Bertout JA, Ericson NG, Taylor SD, Mukherjee R, Robins HS, Drescher CW, Bielas JH. 
2015. Targeted single molecule mutation detection with massively parallel sequencing. Nucleic 
Acids Res. doi: 10.1093/nar/gkv915. [Epub ahead of print.] 
 
 
 


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