authors@Fred Hutch

Zentner, Gabriel

March 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
NHR-49 Helps Germline-Less Worms Chew the 
Fat 
March 16, 2015 
     GE Zentner 
Numerous studies have demonstrated a tradeoff between reproduction and aging, as decreased 
fertility is associated with increased longevity in many species, from the nematode C. elegans to 
humans.  Despite this, reproductive fitness confers physiological benefits, highlighting a complex 
relationship between reproduction and aging.  One point of connection between reproduction and 
aging is fat metabolism, particularly lipid homeostasis.  For instance, obesity on one hand and low 
body weight on the other account for approximately 12% of female infertility, and reproductive 
senescence in mammals is associated with altered patterns of fat storage and weight gain.  In terms 
of aging, obesity is associated with increased risk of several age-associated diseases, including 
diabetes, cardiovascular disease, and cancer.  While lifespan and fertility are generally at odds, 
recent work has suggested that lipid metabolism is a point of contact between these two 
processes.  To better understand the effect of germline removal on lipid metabolism, research 
technician Shaw-Wen Chen in the lab of Dr. Carissa Olsen (Basic Sciences Division) and 
collaborators at the Universities of Pittsburgh, Pennsylvania, and California-San Francisco 
investigated the roles of nuclear hormone receptors (NHRs), transcriptional regulators that often bind 
lipids, in regulation of lifespan of C. elegans in which the germline has been removed.  "In order to 
begin to understand how reproduction influences lifespan, we used a combination of gene 
expression analysis and lipid biochemistry to identify a key transcription factor, nhr-49, involved in 
this regulation and explore its impact on fat metabolism," said Dr. Olsen. 
To identify NHRs involved in lifespan extension after germline loss, the authors performed a targeted 
RNAi screen of NHRs in C. elegans.  They identified 19 NHRs that, when knocked down, both 
shortened the lifespan of germline-less worms and prevented upregulation of a reporter gene whose 
expression is increased by germline ablation.  Strikingly, RNAi depletion of NHR-49 was associated 
with complete loss of this extended lifespan, a phenotype confirmed by lifespan analysis of nhr-
49 mutants.  Consistent with a role for NHR-49 in lifespan extension, the authors detected a robust 
increase in NHR-49 protein levels following germline loss.  Notably, it was also found that 
overexpression of NHR-49 in non-germline tissues also extended the lifespan of fertile worms to a 
modest extent. 
March 16, 2015 SCIENCE SPOTLIGHT 
 
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Using high-throughput RNA sequencing (RNA-seq) to identify NHR-49-regulated genes, the authors 
found that many genes involved in fatty acid oxidation and desaturation were increased.  RNAi 
knockdown of several of these NHR-49 targets reduced lifespan in germline-ablated worms, 
suggesting a key role for NHR-49 in mediating extended lifespan by controlling specific lipid 
metabolic pathways.  Consistent with this idea, loss of NHR-49 was associated with decreased fat 
storage and de novo lipid synthesis in germline-ablated worms, and nhr-49 mutants displayed age-
related depletion of fat stores.  Further experiments revealed a role for NHR-49 in desaturation of 
multiple fatty acids. 
This study delineates a molecular mechanism by which reduced fertility promotes extended 
longevity, wherein germline ablation reprograms lipid metabolism.  "Interestingly, we observed a role 
for nhr-49 in both fat synthesis and fat breakdown, and understanding how these two contradicting 
processes impact metabolism in a meaningful way will be the focus of future work," said Dr. Olsen.  
Ratnappan R, Amrit FR, Chen SW, Gill H, Holden K, Ward J, Yamamoto KR, Olsen CP, Ghazi A. 
2014. Germline signals deploy NHR-49 to modulate fatty-acid ß-oxidation and desaturation in 
somatic tissues of C. elegans. PLOS Genet 10(12):e1004829. 
 
 
Image from the publication 
(A) Expression of NHR-49 in wild-type N2 and (B) germline-null glp-
1 worms as assayed with a fluorescent reporter fusion. 
 


English

NHR-49 Helps Germline-Less Worms Chew the Fat

Pattacini, Laura

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Beyond Neutralization: Antibody-Mediated 
Cytotoxicity In HIV Control 
May 18, 2015 
       L Pattacini 
As HIV continues to spread despite improved prevention strategies, the only possibility to end the 
loss of lives caused by the epidemic is a vaccine that either prevents the infection or controls the 
progressive loss of immune cells that leads eventually to AIDS. The design of either of those 
vaccines requires a better knowledge of the natural mechanisms that block the virus at the entry site 
or its replication. Non-human primate studies have demonstrated the importance of neutralizing 
antibodies-that is antibodies that can recognize and bind to the virus and inhibit its function- in HIV 
prevention. Aside from neutralization, an important role is covered by antibody-dependent cellular 
cytotoxicity (ADCC), an immune mechanism through which an effector cell actively lyses an infected 
target cell whose membrane-surface antigens have been recognized by the antibody. 
In an article published in April on Cell Host & Microbe, Caitlin Milligan and coworkers from Julie 
Overbaugh's group in the Division of Human Biology at Fred Hutch addressed the question of 
whether ADCC is involved in reducing transmission or mortality rate in a cohort of neonates born 
from HIV+ mothers. Antibodies are passively transferred from mothers to their infants during 
pregnancy and thus infants born to HIV-positive mothers have HIV-specific antibodies at birth. This 
setting mimics that of a vaccine whereby HIV-specific antibodies are present at the time of virus 
exposure. The researchers compared the ADCC activity in a group of neonates that acquired the 
disease to one that did not. "We did observe that infants who remained uninfected had higher ADCC 
than those who became infected, however the relationship was not significant. This result does hint 
at a protective effect of ADCC antibodies in infection, but we may have lacked the power to detect a 
significant difference between the groups because of the small number of infected infants. Thus, 
larger studies will have to be conducted to determine whether or not pre-existing ADCC activity 
protects from infection", explains Ms. Milligan. 
When the researchers looked more closely at infected infants, they saw a higher survival rate in 
those with higher ADCC. Thus, passively acquired ADCC is important in controlling disease 
progression. Surprisingly, they did not observe the same pattern in maternal ADCC. "It was definitely 
surprising that we observed an association between infected infant passively acquired ADCC activity  
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
and survival, but the same association was not seen with maternal antibodies. Given that the infant 
ADCC activity was passively acquired from the mother we were expecting to see the same result 
with maternal samples. We have not yet clarified why the results were different but the difference 
may be due to timing of maternal antibody sampling or a difference in passive transfer of antibodies 
to the infant", continues Ms. Milligan, and then emphasizes another aspect of the finding: "This result 
also has larger implications for the mother-to-child transmission field as maternal antibodies are 
often used as a surrogate for the infant passively-acquired antibodies in studies - our result suggests 
that maternal antibodies may not always be an appropriate surrogate measure for infant passively 
acquired responses for some antibody functions such as ADCC." 
Furthermore, based on results from previous neutralizing antibody studies in macaques, the 
researchers hypothesized that a higher passive ADCC activity would mirror the de novo activity 
developed by infants after infection. Instead, their results showed no correlation between ADCC from 
transferred maternal antibodies and the de novo ADCC. The level of ADCC immediately after birth 
cannot be considered as an indication of de novo responses. 
In summary, said Ms. Milligan, "Overall, the results from our study suggest that pre-existing ADCC 
antibody activity may provide a therapeutic effect in individuals who become infected. And thus, 
eliciting ADCC-mediating antibodies by a therapeutic vaccine could be a desirable goal." 
 
 
Milligan C, Richardson BA, John-Stewart G, Nduati R, Overbaugh J. 2015. Passively Acquired 
Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity in HIV-Infected Infants Is Associated with 
Reduced Mortality. Cell Host Microbe, 17(4), 500-6. 
 
 
 
 
 
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
 
 
“Image provided by Caitlin Milligan” 
Schematic representation of the role of maternal antibody ADCC in 
infant born from HIV+ mothers that acquired the infection. 
 


Beyond Neutralization: Antibody-Mediated Cytotoxicity In HIV Control

June 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
A CRISPR Approach to Genetic Screening 
June 15, 2015 
     GE Zentner 
Identifying genes involved in biological processes is essential for understanding cellular and 
organismal function.  While the genomes of many organisms have been sequenced, the function of 
a large fraction of genes even in genomes as well-studied as human and mouse are 
unknown.  Perhaps the most widely used method to discover gene function is the forward genetic 
screen, in which chemical mutagens are used to cause random mutations throughout the genome, 
followed by tedious work to identify mutant animals and the mutated gene.  This process can take 
several years, even using modern high-throughput sequencing technology to facilitate mutation 
identification.  To develop a more efficient way to screen for gene function, research technician Arish 
Shah and colleagues in the lab of Dr. Cecilia Moens (Basic Sciences Division) turned to the CRISPR 
(clustered regularly interspaced short palindromic repeats) genome editing system.  CRISPR 
involves a nuclease called Cas9 that is targeted to specific DNA sequences by engineered single 
guide RNAs (sgRNAs).  The authors took advantage of this for genetic screening by expressing 
Cas9 and sgRNAs that cause small insertions or deletions (indels) in zebrafish.  "We show the utility 
of the system by examining 48 genes for their requirement in electrical synapse formation and find 
two new genes that are involved in the process. Our screen took ~1 month to complete, showing that 
this can greatly speed up discovery," said Dr. Adam Miller, a postdoctoral fellow involved in the 
study. 
The authors first assessed the potential of CRISPR to cause indels in single zebrafish genes by 
targeting a gene involved in retinal pigmentation and two genes involved in neuronal migration.  This 
was done by injecting sgRNAs and mRNA encoding Cas9 into one-cell zebrafish embryos.  Each 
gene disruption produced the expected phenotype, despite that fact that embryos were mosaic for a 
number of mutations due to a delay in Cas9 protein expression following injection. 
Having confirmed that CRISPR could be used to screen for known phenotypes in injected embryos, 
the authors next wanted to ask whether they could screen for new genes involved in a process of 
interest. The authors chose to screen for electrical synapse formation because little is known about 
the genes required for their formation. The authors used the Mauthner circuit (M) in the zebrafish 
spinal cord due to the accessibility of its electrical synapses. CRISPR targeting of the gjd1a gene, 
previously found to be required for M electrical synapse formation, with two separate sgRNAs 
resulting in a >95% decrease in synapse number in 95% and 60% of injected embryos.  This 
June 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
difference in efficiency was subsequently found to be a consequence of the sequence composition 
of the sgRNA target. 
To screen a large number of genes simultaneously, the authors tested the efficiency of pooling 
sgRNAs for a single injection with Cas9.  The most efficient gjd1a sgRNA was pooled with sgRNAs 
targeting five genes not involved in electrical synapse formation and injected.  All pool-injected 
embryos displayed loss of synapses, suggesting that pooling of sgRNAs is effective.  Following up 
on this, they designed a set of 48 sgRNAs targeting genes potentially involved in synapse 
formation.  These were injected in pools of six and eight, with each sgRNA present in two 
pools.  This led to six pools and a total of nine genes likely to be involved in 
synaptogenesis.  Injection of individual sgRNAs from the pools recapitulated the pooled phenotype 
in a subset of injections, and allowed the authors to identify two genes not previously implicated in 
electrical synapse formation. The genes were subsequently confirmed in stable mutant lines. From 
sgRNA synthesis to phenotypic screening of embryos injected with individual sgRNAs, the screen 
took 3 weeks.  Furthermore, sequencing of the target sites for each sgRNA showed that most 
targets were successfully disrupted. 
"Looking forward this approach can certainly be expanded to many different phenotypes," said Dr. 
Miller.  "Ongoing work in lab has found that the technique can be used to examine genes involved in 
neuronal migration, cell polarity, and cancer metastasis. Moreover, our screen of 50 genes was a 
pilot, and we hope to expand the screen to hundreds, perhaps even up to 1,000 genes in future 
work. CRISPR reverse genetic screening greatly increases the speed of doing genetics in a 
vertebrate system." 
 
Shah AN, Davey CF, Whitebirch AC, Miller AC, Moens CB. 2015. Rapid reverse genetic screening 
using CRISPR in zebrafish. Nat Methods 12(6):535-540. 
June 15, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
 
Image provided by Dr. Adam Miller 
Schematic of the screening strategy. A pool of sgRNAs and 
mRNA encoding Cas9 is injected into zebrafish eggs. The 
phenotype resulting from the sgRNA pool is then analyzed. 
Following this, individual sgRNAs from the pool and Cas9 are 
injected to identify the gene(s) responsible for the pool’s 
phenotype. 
 


A CRISPR Approach to Genetic Screening

June 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
Looking For Therapeutic Targets in Bacterial 
Vaginosis 
June 15, 2015 
   L Pattacini 
The human body hosts complex communities of microbes that differ at each body site.  These 
microbes can impact human physiology and health through production of metabolites that directly 
affect human cells or microbial community composition. The human vagina hosts a limited number of 
Lactobacillus species, but in the dysbiotic condition bacterial vaginosis (BV), the bacterial community 
shifts to complex communities of diverse anaerobes. 
BV is a highly prevalent condition in the United States, where it affects up to 29% of women and has 
been associated with numerous adverse outcomes including preterm birth, pelvic inflammatory 
disease, and elevated risks for sexually transmitted infections including HIV. It is unclear how BV-
associated bacteria mediate these increased health risks, and there have been few studies linking 
vaginal bacterial communities to detailed metabolite profiles. 
Identification of the metabolites present in the vagina of women affected by this condition may 
elucidate how different bacterial populations thrive, and this knowledge could be used for disrupting 
metabolic pathways present in deleterious bacteria. Dr. Srinivasan and coworkers from Dr. David 
Fredriks’ Laboratory (Vaccine and Infectious Disease Division) at Fred Hutch pursued a 
metabolomics-based approach wherein gas or liquid chromatography was coupled with mass 
spectrometry to measure hundreds of low molecular weight metabolites in vaginal fluid and to link 
these metabolites to particular bacteria detected by PCR. 
"The novelty of our study," explained Dr. Srinivasan, "is the breadth and depth of the approach, and 
the linking of important metabolites with concentrations of bacteria and clinical findings in women. 
BV is associated with strong metabolic signatures affecting multiple pathways including amino acid, 
carbohydrate and lipid metabolism, with significant differences in concentrations of 62% of 
metabolites detected in women with BV." The study indeed demonstrated a different pattern of 
metabolites in women with BV. Such metabolites correlated with the presence of BV-associated 
bacteria and were associated with characteristics distinctive of BV, such as an elevated pH, amine 
odor, vaginal discharge and presence of clue cells, which are vaginal epithelial cells covered by 
bacteria. Furthermore, antibiotic treatment resulted in the increase of metabolites prevalent in 
women not diagnosed with BV. 
June 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
Women with BV have metabolic profiles marked by much lower concentrations of amino acids and 
dipeptides, concomitant with higher levels of amino acid catabolites and polyamines, suggesting that 
BV-associated bacteria may use amino acids as carbon sources. In contrast, lactobacilli are known 
to metabolize sugars such as glycogen derived from the host and do not appear to rely on amino 
acids as their primary carbon source which are more elevated in women not diagnosed with BV. 
"This study has advanced our understanding of the functional status of bacterial communities in BV. 
Insights from this work may provide opportunities for developing novel approaches for treatment or 
prevention of BV," commented Dr. Srinivasan. Given the absence of a vaccine and a durable 
therapy for BV, these results give hope that the solution to this condition is getting closer. 
Srinivasan S, Morgan MT, Fiedler TL, Djukovic D, Hoffman NG, Raftery D, Marrazzo JM, Fredricks 
DN. 2015. Metabolic signatures of bacterial vaginosis. MBio 6(2): e00204-15. 
 
 
Image courtesy of Sue Bartlett 
Bacteria, metabolites and their associations in the human vagina. 
Bacteria in vaginal fluid from a woman with bacterial vaginosis (BV) 
are detected by fluorescence in situ hybridization in the top panel. A 
selection of metabolites in vaginal fluid (left) and a heat map of 
associations between metabolites and vaginal bacteria (right) are 
presented in the lower panels. 
 


Looking For Therapeutic Targets in Bacterial Vaginosis

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
The more is not always the merrier 
October 19, 2015 
     L Pattacini 
 
HIV-1 viral load measurements over the 
first year since HIV-1 diagnosis in the 
RV144 (left) and Step (right) trials. The 
mean viral load and associated 95% 
confidence interval at each time is 
shown for subjects with homogeneous 
vs. heterogeneous founding viral 
populations at the time of diagnosis. 
Means are estimated using models that 
adjust for baseline subject 
characteristics and assume that the 
effects of heterogeneity are constant 
over time. 
 
Image provided by Dr. Holly Janes. 
 
 
The term viral load denotes the number of HIV particles, quantified by detection of the viral RNA, in a 
milliliter of blood. This value increases soon after the infection then drops and stabilizes at a value 
termed viral set point. A lower set point correlates with a slower progression of the infection. Viral 
load set point, while being fairly stable for a single individual, varies across individuals, highlighting 
the importance of host characteristics in establishing it. This variability may be accounted for by 
host-virus interactions as well as by differences in characteristics of the viral infection, such as an 
infection with multiple HIV variants. While this is a rare event as compared to the infection with a 
single variant, it accounts for 20-35% new infections. 
To evaluate whether infection with multiple HIV founders is associated with a higher viral load, Dr. 
Holly Janes from the Vaccine and Infectious Disease Division at Fred Hutch and collaborators from 
other institutions in US and Thailand analyzed two sets of data, obtained from breakthrough 
infections in RV144 and Step vaccine efficacy trials. RV144, also known as the Thai trial, tested a 
vaccine designed to induce antibodies against the envelope (Env) protein of HIV, and recruited 
volunteers in Thailand. The vaccine evaluated in the Step study induces T cell responses and was 
tested on subjects recruited at several study sites located in the Americas. Two different measures 
of the diversity of the founder virus population at the time of HIV diagnosis were used, a categorical 
October 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
measure (homogeneous vs. heterogeneous virus population) and a continuous measure of env gene 
diversity.  
In the samples from the RV144 trial, a higher average viral load was observed in the subjects with 
heterogeneous viral infections as compared to those with homogeneous infections at the time of 
detection of HIV infection as well averaged over times within 1- and 2-years of infection diagnosis. 
Accordingly, a lower average CD4+ T cell count was observed in subjects with heterogeneous 
infections. Interestingly, although the associations with viral load at later time points were similar for 
the Step study, heterogeneous infections were not found to be correlated with a higher viral load at 
the time of infection and the average CD4+ T cell count was comparable in subjects with 
heterogeneous and homogeneous founding virus populations. The difference between the two 
studies was not explained by the differences between the vaccines.  
The study identified infection with multiple HIV founders as a factor potentially involved in the 
establishment of the viral set point. "This research suggests that a prophylactic intervention that 
reduced the number of founding viruses in a person who becomes HIV infected could have clinical 
benefit, and motivates measuring the number of founding viruses in future efficacy trials of these 
interventions" said Dr. Janes commenting on the results. Given the relevance that the viral set point 
has on the progression of the disease, the finding is important not only to better understand the 
progression of HIV infection, but it might have a clinical relevance. 
Funding: The research was supported by grants from NIAID and NIH. 
Janes H, Herbeck JT, Tovanabutra S, Thomas R, Frahm N, Duerr A, Hural J, Corey L, Self SG, 
Buchbinder SP, McElrath MJ, O'Connell RJ, Paris RM, Rerks-Ngarm S, Nitayaphan S, Pitisuttihum 
P, Kaewkungwal J, Robb ML, Michael NL, Mullins JI, Kim JH, Gilbert PB, Rolland M. 2015. HIV-1 
infections with multiple founders are associated with higher viral loads than infections with single 
founders. Nat Med. 21(10):1139-41. doi: 10.1038/nm.3932. 
 
 
 


The more is not always the merrier.

Herman, Jake

November 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
4+8=0: Synergy from defined cell populations in 
CAR-T-cell therapy 
November 15, 2015 
     J Herman 
 
 
 
 
 
T cell products derived from defined 
patients' T cell subsets have enhanced 
antitumor potency. (A) Bioluminescence 
imaging of tumor growth in mice that 
had received 1 million control T cells or 
CD19 CAR-T cells prepared from either 
unselected cells or from defined T cell 
subsets (CD4 naïve/CD8 central 
memory). (B) Frequency of transferred 
CD8+ T cells (human CD45+/CD8+) in 
peripheral blood of mice one week after 
T cell transfer. Flow cytometry data from 
mice with median T cell frequency are 
shown for each group. 
 
Image provided by Dr. Daniel 
Sommermeyer 
 
 
 
One of the difficulties in treating cancer is that tumors arise from a patient's own cells, which allows 
them to evade the human immune system more easily than infections by bacteria or viruses. 
However, there are few therapies that perform better than the natural human immune system, 
because of its long-term protection, which lasts years to decades. To this end, researches are hotly 
pursuing cancer therapies that enable patient immune cells to recognize cancer cells and elicit 
antitumor activities. One such approach is through T cells modified with a chimeric antigen receptor 
(CAR) that can recognize and target cancer cells with high specificity. One half of this fusion protein 
is the cytosolic domain of CD3ζ, which is the activating part of the T-cell receptor complex. The 
appropriate antigen-binding domain must be chosen to ensure the immune response against healthy 
tissue is minimal. In currently successful CAR-T-cell therapy CD3ζ is fused to the binding domain of  
November 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
an antibody that targets CD19. CD19 is expressed solely in healthy and malignant B cells and thus 
can target cancers while minimally damaging healthy hematopoietic stem cells and other healthy 
tissue. The DNA coding for this CAR is introduced into the genome of T cells isolated from patients 
or donors, and these CAR-T cells are then transferred into leukemic patients, with great success. 
Research efforts thus far have focused on optimizing CARs for increased potency; however, in a 
recent publication in Leukemia, Dr. Sommermeyer and his colleagues in the Riddell Laboratory 
(Clinical Research Division) instead focused on the properties of T cells that were being genetically 
modified.  
Historically T cells have been isolated from patients and donors by their unique expression of CD3, 
resulting in an extremely diverse population of T cells. Early in the differentiation process, T cells 
express both CD4 and CD8, however their expression becomes mutually exclusive during 
differentiation. Within both CD4 and CD8 lineages, there also exist naïve, effector, and memory T 
cells, which contribute uniquely to antitumor activity. Thus under current CAR-T-cell therapy, every 
patient has been treated with a different population of T cells. Researchers verified this by 
measuring the ratios of CD4 and CD8 T cells in patients and donors and found patients who had 
previously received cytotoxic chemotherapy were depleted of CD4 T cells. Moreover, there was a 
general decrease in the number of naïve T cells of either CD4 or CD8 lineages. In general, the 
populations varied dramatically among individuals, emphasizing the heterogeneity of CAR-T-cell 
therapy. 
To understand the contribution of each cell type towards antitumor activity, researches measured 
lysis of cancer cells and production of inflammatory cytokines. They observed that CD8 CAR-T cells 
performed robustly at lysing tumor cells, while CD4 CAR-T cells dramatically outperformed at 
cytokine production. Activity of CD4 CAR-T cells was further characterized by isolating three 
subtypes: naïve (4N) and two classes of memory cells, central (4CM) and effector memory (4EM). 
Using the same tests as above, as well as measuring survival in mouse leukemia models, the 
researchers found that 4N and 4CM cells had the most potent antitumor activity. The same panel of 
tests was performed on CD8 naïve (8N), central memory (8CM), and effector memory (8EM) T cells. 
CD8 CAR-T cells generally outperformed CD4 CAR-T cells, and among the subtypes, 8CM were 
more potent in vivo. Finally, combination of 8CM with each CD4 subtype was tested. 8CM in 
combination with either 4N or 4CM (1:1) demonstrated synergistic antitumor activity, whereby the 3 
times fewer mixed cells achieved the same antitumor activity of either population alone. When 
testing the persistence of CAR-T cells in the blood stream, significantly more CAR-T cells were 
detected when the 8CM+4N mix was used for treatment. These findings were then directly 
compared to the current clinical protocols using mouse models, and demonstrated that an undefined 
November 15, 2015 SCIENCE SPOTLIGHT 
 
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CAR-T-cell population extended lifespan 2-3 times, yet a mix of 8CM+4N resulted in full remission in 
all mice. 
The success of this work isn’t simply the increased antitumor activity, "This work demonstrates that 
certain T cell subsets are superior for adoptive therapy and that formulating optimal compositions 
can ensure uniform efficacy and dramatically reduce the cell doses needed to achieve tumor 
eradication, which improves the feasibility and lowers the cost of therapy. We are already using this 
approach in a clinical trial at Fred Hutch, which demonstrates that lower cell doses can also reduce 
toxicity", said Dr. Riddell. This exciting research helps remind us cancer therapeutics are in a new 
frontier beyond simply remissions, but onto reducing toxicity and cost of care. 
Funding for this research was provided by the National Institutes of Health, German Research 
Foundation, and the Leukemia and Lymphoma Society. 
Sommermeyer D, Hudecek M, Kosasih PL, Gogishvili T, Maloney DG, Turtle CJ, Riddell SR. 2015. 
Chimeric antigen receptor-modifed T cells derived from defined CD8
+
 and CD4
+
. Leukemia. doi: 
10.1038/leu.2015.247. [Epub ahead of print.] 
 
 


4+8=0: Synergy from defined cell populations in CAR-T-cell therapy

Kocarnik, Jonathan

March 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
Aspirin Use Reduces Colorectal Cancer Risk in 
Many Subgroups 
March 16, 2015 
    JM Kocarnik 
A large body of evidence suggests that the use of aspirin and other non-steroidal anti-inflammatory 
drugs (NSAIDs) reduces the risk of colorectal cancer.  However, because their use can also lead to 
side effects such as gastrointestinal bleeding, NSAID use is not recommended for colorectal cancer 
prevention among the general population.  Of great interest for researchers, then, is identifying 
subgroups of people for whom the benefits of colorectal cancer chemoprevention outweigh the risk 
of side effects.  To identify these groups, Ms. Wendy Wang and Dr. Emily White and colleagues in 
the Public Health Sciences Division evaluated the association between NSAID use and colorectal 
cancer risk across a range of subgroups with differing risk factors.  In a recent article in Cancer 
Epidemiology, Biomarkers, & Prevention, the authors report that the protective effect was consistent 
across these various subgroups, suggesting that the beneficial role of NSAIDs on colorectal cancer 
risk is unmodified by these additional risk factors. 
Chronic inflammation is an established risk factor for colorectal cancer, and NSAIDs are thought to 
lower this risk by inhibiting inflammatory pathways and reducing inflammation.  In addition to 
inflammation, there are a large number of other risk factors that have been associated with 
colorectal cancer risk.  These include factors such as obesity, physical inactivity, smoking, family 
history, and high consumption of red meat and alcohol.  While it is suspected that some of these risk 
factors might also impact risk by increasing inflammation, their interaction with NSAID use are 
unclear.  If NSAID use were to mitigate the increased risk due to some of these additional factors, 
then subgroups of people with higher risk could be more appropriately targeted for chemoprevention 
strategies. 
To identify such groups, the authors tested the interaction between NSAID use and these various 
risk factors on colorectal cancer risk using data from the VITamins and Lifestyle (VITAL) study.  This 
large cohort study of roughly 75,000 western Washington residents provided detailed prospective 
information on a large range of exposures.  Incident colorectal cancer cases were identified through 
the Surveillance, Epidemiology, and End Results (SEER) cancer registry.  Cox regression was used 
to evaluate the association between NSAID use in the 10 years prior to baseline and development of 
colorectal cancer during follow-up (average 8.4 years). 
March 16, 2015 SCIENCE SPOTLIGHT 
 
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Testing across these subgroups, the authors found that any type of NSAID use for at least 4 days a 
week for at least 4 years was associated with a 42% lower risk of colorectal cancer (p-trend < 
0.001).  This protective association was consistent among subgroups defined by additional risk 
factors, with reductions in risk ranging from 32% to 56% within each subgroup (p-trend < 
0.05).  Though not statistically significant, high NSAID use appeared to have a greater risk reduction 
among men, participants who were obese, and regular alcohol drinkers.  Further evaluations looking 
at cancer site and cancer stage also suggested that NSAID use was associated with a lower risk of 
proximal colon cancer compared to distal colon cancer, and distant stage colorectal cancer 
compared to local stage. 
"Our results suggest that use of aspirin or other NSAIDs has a generally beneficial role in colorectal 
cancer prevention, largely unmodified by other lifestyle factors," said lead author Ms. Wendy 
Wang.  "However, the differential effects by colorectal cancer subsites may indicate potential 
chemopreventive pathways of NSAID use. Our study, along with previous observational studies, did 
not have sufficient power to detect small differences in the effects of NSAIDs on colorectal cancer 
risk between subgroups of the population. Therefore, we aim to replicate this systematic evaluation 
of effect modifiers between NSAID use and colorectal cancer risk in the Genetic Epidemiology of 
Colorectal Cancer Consortium (GECCO), a large consortium of cohort and case-control studies for 
colorectal cancer." 
Other PHS researchers contributing to this project were Drs. Ulrike Peters and John Potter. 
 
 
Wang X, Peters U, Potter JD, White E. 2015. Association of Non-steroidal Anti-inflammatory Drugs 
with Colorectal Cancer by Subgroups in the VITamins And Lifestyle (VITAL) Study. Cancer 
Epidemiol Biomarkers Prev. pii: cebp.1253.2014. [Epub ahead of print] 
 
 
 
 
 
March 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Ms. Wendy Wang 
Generic non-steroidal anti-inflammatory drugs (NSAIDs) are readily 
accessible over the counter. 
 


Aspirin Use Reduces Colorectal Cancer Risk in Many Subgroups

June 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
Vitamin D Intake Lowers IL-6 Levels in the 
Context of Weight Loss 
June 15, 2015 
    JM Kocarnik 
Overweight or obesity is associated with an increase in chronic inflammation, which in turn 
is    associated with increased risk for several diseases, including various cancers.  Several studies 
have suggested that vitamin D deficiency may be associated with increased inflammation 
levels.  Being deficient in vitamin D also appears to be associated with higher levels of circulating 
inflammatory markers.  While weight loss is associated with reductions in inflammatory biomarkers, it 
is not known whether vitamin D supplementation might further boost this effect.  To evaluate this 
question, Drs. Catherine Duggan, Anne McTiernan, and colleagues in the Public Health Sciences 
Division evaluated data from a recent randomized clinical trial of vitamin D3 supplementation vs. 
placebo in the context of weight loss.  Reporting their results in Cancer Prevention Research, the 
authors found that vitamin D3 supplementation lowered IL-6 levels compared to placebo, but only 
among those who lost at least 5% of their baseline weight.  
Vitamin D is a steroid hormone that plays a significant role in calcium homeostasis and 
metabolism.  Humans obtain vitamin D primarily through dermal synthesis from sunlight, but also 
through dietary sources.  Vitamin D is commonly consumed as a dietary supplement, and in some 
countries is also added to various food products.  Though there is some debate on the cutoffs for 
deficiency and insufficiency, it is estimated that 50 to 60% of older populations worldwide are vitamin 
D deficient.  As both vitamin D deficiency and increased adiposity have been associated with higher 
inflammation levels, the authors were interested in evaluating whether vitamin D supplementation 
might help lower inflammation levels in those trying to lose weight. 
To do so, the authors utilized data from the Vitamin D diet and Activity (ViDA) clinical trial.  This trial 
randomized 218 healthy postmenopausal women who were vitamin D insufficient (serum 25-
hydroxyvitamin (25-OH) D level of 10-32 mg/mL) and overweight or obese, to receive either 2000 IU 
oral vitamin D3 supplementation/day or placebo.  Over one year of study, both study arms also 
received a lifestyle-based weight loss program of diet and exercise, with a goal of losing 10% of their 
baseline weight.  To measure the effect of this intervention on inflammatory biomarkers, the trial 
compared pre- and post-intervention serum measurements of leptin, adiponectin, the anti-
inflammatory marker IL-10, and the pro-inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-8.  
June 15, 2015 SCIENCE SPOTLIGHT 
 
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At the end of the study, the average serum 25-OH-D levels increased 13.6 ng/mL in the vitamin D 
arm, but decreased by an average of 1.3 ng/mL in the placebo arm (P < 0.0001).  While all of the 
inflammatory biomarker levels were reduced compared to baseline, these changes did not 
significantly differ between arms.  To explore further, the authors performed analyses stratified by 
the degree of weight loss.  Comparing those who lost at least 5% of their baseline weight to those 
who gained weight or did not lose weight, participants in the vitamin D arm had significantly greater 
decreases in IL-6 levels than those in the placebo arm (see figure).  
These results suggest that weight loss and vitamin D3 supplementation may act synergistically to 
reduce IL-6 levels, at least among postmenopausal women who are vitamin D insufficient and 
overweight/obese.  In vitro experiments have shown that the active form of vitamin D inhibits IL-6 
production, and some hypothesize that weight loss may also increase the bioavailability of vitamin D 
in different tissues.  Vitamin D supplementation did not, however, appear to lower other inflammatory 
cytokines beyond weight loss alone.  Future studies are needed to further explore these potential 
relationships between weight loss, vitamin D intake, and inflammation. 
Other PHS researchers contributing to this project were Drs. Jean de Dieu Tapsoba, Caitlin Mason, 
Ikuyo Imayama, Larissa Korde, and Ching-Yun Wang. 
 
Duggan C, Tapsoba JD, Mason C, Imayama I, Korde LA, Wang CY, McTiernan A. 2015. Effect of 
Vitamin D3 supplementation in combination with weight loss on inflammatory biomarkers in 
postmenopausal women: a randomized controlled trial. Cancer Prev Res pii: canprevres.0449.2014. 
[Epub ahead of print] 
 
 
 
 
 
 
 
June 15, 2015 SCIENCE SPOTLIGHT 
 
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Image provided by Dr. Jonathan Kocarnik 
Absolute change in IL-6 levels (pg/mL) from baseline to after a one-year 
weight loss intervention in the intervention arm (2000 IU/day vitamin D3) 
versus the placebo arm, overall and stratified by percentage of baseline 
weight lost. P-values are from covariate-adjusted generalized estimating 
equation models. 
 
 


Vitamin D Intake Lowers IL-6 Levels in the Context of Weight Loss

Frost, Sophia

September 21, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
BSCL therapy for ocular GVHD - a sight for sore 
eyes 
September 21, 2015 
     SHL Frost 
 
 
 
Lee eye subscale score over time. P-
values were 0.01, 0.005, and 0.02 after 
2 weeks, 4 weeks, and 3 months, 
respectively, as determined by paired t-
tests compared with baseline. 
 
Image provided by Dr. Yoshihiro 
Inamoto 
 
 
 
Graft-versus-host disease (GVHD) is a condition that may affect recipients of allogeneic 
hematopoietic stem cell transplants, with effects that range from manageable to life-threatening. 
Among patients with chronic GVHD, 40% to 60% develop symptoms in the eyes, referred to as 
ocular GVHD. All layers of the eye are at risk, and manifestations include milder "pink eye" 
(conjunctivitis) and dry eye syndrome, as well as more severe scarring or penetration of the 
conjunctiva and cornea. Keratitis - inflammation of the cornea - is the most common cause of eye 
pain, light sensitivity, and vision loss in affected patients. Although symptoms may be partly 
alleviated through suppression of the immune system, additional care is often required in the form of 
eye lubricants, immunosuppressive eye drops, and moisture-preservative eyewear. However, the 
efficacy may be limited depending on the clinical manifestation. 
An alternative treatment of severe ocular GVHD is fluid-ventilated gas-permeable (PROSE) lenses 
that cover the white of the eye (sclera), thereby providing a liquid "bandage" that protects the cornea. 
For reasons of accessibility and expense, PROSE lenses are unfortunately not available to many 
patients, but there are other options; bandage soft contact lenses (BSCLs) can be easily fitted and 
dispensed at a much lower cost, possibly providing relief of ocular GVHD symptoms. This 
hypothesis was tested by Drs. Yoshihiro Inamoto and Stephanie Lee from the Clinical Research 
Division in collaboration with a University of Washington ophthalmologist, Dr. Tueng Shen. The 
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results of this small clinical trial were recently published in Biology of Blood and Marrow 
Transplantation. 
A total of 19 patients were included in the analysis, all of whom had already tried most other 
conventional options, e.g. artificial tears, viscous ointment, cyclosporine eye drops, punctual plugs, 
steroid eye drops, and systemic immunosuppressants. None had used scleral lenses. At the time of 
enrollment, 14 of the 19 subjects had moderate ocular GVHD; 5 had severe ocular GVHD. The 
lenses were used continuously - even during nighttime - with exchange every two to four weeks. 
Treatment results were evaluated in terms of three scores: Lee eye subscale score, which is a 
validated symptom scale for chronic GVHD including 3 items (range 0 to 100); ocular surface 
disease index, which measures dry eye symptoms (range 0 to 100) using a validated survey 
consisting of 12 items; and a 11-point rating of eye symptoms recommended by the National 
Institutes of Health consensus (range 0 to 10). These standard questionnaires were completed by 
patients at enrollment and then after 2 weeks, 4 weeks, and 3 months; in all cases higher scores 
indicated worse symptoms. In addition, ophthalmological assessments were made at enrollment and 
at 2 weeks, and beyond that as medically indicated. 
The investigators reported statistically significant, clinically meaningful improvements in all 
measures. At enrollment, the mean Lee subscale score was 75.4, which was lowered to 63.2 after 2 
weeks of BSCL therapy, 61.8 at 4 weeks, and 56.3 at 3 months. For ocular surface disease index 
the corresponding mean scores were 54.5, 36.8, 32.9, and 35.6; the mean 11-point eye symptom 
rating decreased accordingly, from 7.11 at enrollment to 5.00, 4.37, and 3.94 at 2 weeks, 4 weeks, 
and 3 months, respectively. During the 3 months of study, relief of symptoms was experienced in 
47% to 68% of the participants; clarity of vision and corneal effects were also significantly improved, 
already after 2 weeks. Adverse events from BSCLs were mild, and did not cause patients to quit 
using the lenses to any great extent. 
The mechanism behind these dramatic effects is quite simple: by protecting the surface of the 
GVHD-injured cornea wounds are stabilized, facilitating the epithelial healing. Dr. Lee explained that 
an injured cornea is very bothersome, just like a scratch on the eye: "The bandage lens covers the 
area and protects it - like a band aid for the eye." Drs. Lee and Inamoto agree that the promising 
results need to be validated in a larger multicenter trial. "This was a very small study of 20 people 
who already had pretty severe eye GVHD," Dr. Lee said, who now wants to explore earlier 
intervention with BSCLs to see if it can help prevent permanent damage. In addition, Dr. Inamoto 
underlined that long-term safety and efficacy remain to be determined. Keep an eye out for these 
studies in the future. 
September 21, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
Inamoto Y, Sun Y-C, Flowers MED, Carpenter PA, Martin PJ, Li P, Wang R, Chai X, Storer BE, 
Shen TT, Lee SJ. 2015. Bandage soft contact lenses for ocular graft-versus-host disease. Biol Blood 
Marrow Transplant. [Epub ahead of print] 
The Chronic GVHD Consortium (U54CA163438) is part of Rare Diseases Clinical Research Network 
(RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), NCATS. The consortium is 
funded through collaboration between NCATS, and the National Cancer Institute. 
 
 
 


BSCL therapy for ocular GVHD - a sight for sore eyes

Zubair, Niha

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
New public resource to model impact of cancer 
biomarkers 
October 19, 2015 
     N Zubair 
 
Schematic of the Diagnostics Biomarker module. 
The virtual study population is subjected to two 
scenarios: all are tested for the diagnostic 
biomarker or none are tested. The resulting 
distribution of tailored versus standard therapy for 
each scenario is based on a comparative 
effectiveness (CE) study. CANTRANce uses 
additional data from the literature/other studies to 
project from the time of treatment to mortality. 
Image provided by Dr. Ruth Etzioni. 
 
Biomarkers assessed at the time of cancer diagnosis may help predict a patient's response to a 
specific treatment; such information can improve a patient's outcome and/or quality-of-life. Advances 
in genomic research over the past decade have produced a multitude of candidate "diagnostic 
biomarkers", which may disseminate into clinical practice long before survival data becomes 
available. In the interim, comparative effectiveness (CE) studies collect data on how a potentially 
diagnostic biomarker affects an intermediate endpoint, such as treatment recommendations. These 
studies often do not assess whether the biomarker reduces cancer mortality.  One way to address 
this question is to conduct a complementary modeling study that translates changes in an 
intermediate endpoint into projected effects on mortality, quality-of-life, and costs. However, the 
resources needed to conduct such complementary modeling studies are not always readily 
available. Drs. Jeanette Birnbaum, Ruth Etzioni, and colleagues in the Public Health Sciences 
Division have developed a system of statistical simulation models for Cancer Translation of 
Comparative Effectiveness Research (CANTRANce), to project the mortality impact of interventions 
given their effects on intermediate endpoints. In a recent article in Medical Decision Making, the 
authors introduce the Diagnostic Biomarker module of CANTRANce and use it to evaluate a 
diagnostic breast cancer biomarker for mortality, quality-of-life, and costs.  
 
CANTRANce uses an individual-level micro-simulation approach in which subjects transition through 
health states in continuous time. The Diagnostic Biomarker module of CANTRANce, presented in 
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the article, compares two scenarios: 1) all individuals receive a one-time testing intervention using 
the diagnostic biomarker and 2) no individuals receive testing. Based on primary or published data 
from a CE study, the model uses the distributions of the two possible treatments, tailored or 
standard, for each scenario. To then project mortality in each scenario, the model uses results of 
published prognostic studies; users can specify annual cost and utility weights to model cost and 
quality-of-life.  
 
To validate the Diagnostic Biomarker module, the authors conducted two case studies. Each case 
study used data from CE studies testing an intervention of a gene recurrence score (RS) for breast 
cancer on treatment recommendations: adjuvant chemotherapy plus hormone therapy (tailored) vs. 
hormone therapy alone (standard).  
 
The first case study compared CANTRANce's performance to that of a previously developed 
customized model by Reed and others. This case study used published CE data in which two 
medical oncologists made two treatment recommendations for node-negative, estrogen receptor 
(ER)-positive patients, with and without the patients' RS information. No individual-level data were 
available, but the Diagnostic Biomarker module can utilize summary statistics. Once all assumptions 
were matched, their model closely replicated the Reed model.  
 
In the second case study the authors carried out a novel projection of the long-term impact of the RS 
using individual-level data from a previously published CE study of how RS information affected 
treatment recommendations in node-negative ER-positive women. 
 
In both case studies the use of the RS decreased adjuvant chemotherapy (tailored treatment) 
among low-risk women and increased it among high-risk women. In addition, the models for both CE 
studies projected that testing with the RS increased life-years and quality-of-life at reasonable cost, 
but with substantial uncertainty. The models from the first case study projected greater benefit and 
costs than the second case study because of the different population risk distributions and the 
differences in the impact of the testing indicated by the two CE studies. According to the authors, 
"the interpretation and application of these projected outcomes should be considered in context of 
the original CE study population" and the quality of the input data.  
 
"This work provides a flexible and fast tool for projecting the impact of a new biomarker for treatment 
targeting on disease outcomes and costs," says Dr. Etzioni. The novelty of CANTRANce "is the 
automation of the tool so that anyone with data on how a treatment-targeting biomarker affects 
treatment patterns can use the tool to project the downstream and long-term impacts of the marker. 
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This work is part of a larger suite of automated tools for projecting the long-term and downstream 
impacts of cancer interventions from studies that evaluate the impact of these interventions on short-
term and intermediate outcomes." A user-friendly Windows application interface for CANTRANce, 
developed by Fred Hutch investigator Mark W. Mason, is downloadable to the public from 
http://www.fredhutch.org/en/labs/phs/projects/cantrance.html.  
  
Citation:  
Birnbaum JK, Ademuyiwa FO, Carlson JJ, Mallinger L, Mason MW, Etzioni R. 2015. Comparative 
effectiveness of biomarkers to target cancer treatment: modeling implications for survival and costs. 
Med Decis Making. doi: 10.1177/0272989x15601998. 
 
Funding for this study was provided by the National Cancer Institute and the National Institutes of 
Health. 
 
 
 


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