authors@Fred Hutch

Neves, Alexandre

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
A match.com for T cell receptor α and β chains 
October 19, 2015 
     A Neves 
 
Diagram of pairSEQ experiment. A pre-determined number of T cells are distributed between wells of a 96-well plate. 
The mRNA in each well in then extracted and cDNA is synthesized and amplified by primers that are specific to the T-
cell receptor genes. Well-specific barcodes allows for pooled sequencing and a computational framework maps each 
TCRA or TCRB sequence to its original well. Pairs of TCRA and TCRB sequences that share occupancy patterns are 
inferred to have come from the same T-cell clone. 
Image from the publication. 
T cells, a major subtype of white blood cells, are key to harnessing the potential of the immune 
system to fight cancer and other diseases. Current cancer immunotherapies seek to expand or 
reengineer T cells with the best cancer-fighting prowess. However, identifying the right T cell clones 
(a T-cell and all of its descendants) can be time and labor-intensive. T cells recognize their target 
cancer cell or pathogen through a T cell receptor (TCR), composed of two polypeptides, an α chain 
and a β chain. To identify T cell lineages it is important to know the identity of both the α and β 
sequences and which α and β chains pair together. Previous work from Dr. Harlan Robins (Public 
Health Sciences and Human Biology Divisions), and his collaborators at Adaptive Biotechnologies, 
developed a high throughput assay called immunoSEQ to sequence individual α and β chains. 
However, without resorting to single-cell methods, which are not easily scalable, it had not been 
previously possible to know which α and β chains occur together. Dr. Robins’ team has now 
developed and validated a clever high-throughput way of identifying α and β chain pairs, which 
enables reconstitution of specific TCRs for functional assays and therapeutic applications. This study 
was recently published in Science Translational Medicine. 
"We have spent many years sequencing single chains of the T cell receptor, which has allowed us to 
track individual T cells and develop diagnostics.  With this new technology, we can sequence the full 
October 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
TCR (both chains paired together) at high throughput.  This allows us to recreate the TCR for 
functional studies as well therapeutic use," said Dr. Robins. The α and β chains are encoded by two 
separate genes: TCRA (α) and TCRB (β). Because both TCRA and TCRB experience genetic 
rearrangements during T-cell development, their mRNA sequences are unique for each T-cell clone. 
The investigators thus reasoned that the TCRA and TCRB from a T-cell clone could be identified by 
first dividing a T-cell sample into different subsets, and then by tracking which TCRA and TCRB 
sequences co-occur in the same subsets. Each subset harbored a barcode, which enable the 
amplified receptor to be pooled for high-throughput sequencing. To validate their new method, called 
pairSEQ, the authors sequenced the TCRA and TCRB from 1 million peripheral blood mononuclear 
cells (PBMCs) from two healthy adults. The two samples were mixed in a single plate and the 
pairSEQ computational framework correctly assigned more than 3200 TCRA/TCRB pairs with less 
than 30 errors. Next, the authors asked whether pairSEQ could identify rare T-cell clones by 
increasing the number of T-cells per well (160,000), and they identified more than 362,528 TCR 
pairs at a false discovery rate of 1%, including 26,667 with frequencies of 1 in a million or lower. 
Finally, the researchers asked whether pairSEQ could be used to interrogate samples other than 
blood, such as tumor-infiltrating lymphocytes (TILs) present in solid tumors. To this end, they 
obtained samples from nine solid tumors and matched blood samples. As before, the authors first 
sequenced the TCRA and TCRB repertoires from each sample to determine how many cells to use 
for the pairSEQ assay. They then ran the pairSEQ assay on dissociated tumor cells as well as the 
PBMCs. The pairSEQ algorithm assigned 6172 pairs from the tumor sample and 14,123 from 
PBMCs, while maintaining an FDR of 1%. In summary, pairSEQ can accurately identify TCR pairs in 
multiple sample types and across a range of clonal frequencies using standard laboratory 
equipment, and therefore will have broad applicability in biomedical research.  
Howie B, Sherwood AM, Berkebile AD, Berka J, Emerson RO, Williamson DW, Kirsch I, Vignali M, 
Rieder MJ, Carlson CS, Robins HS. 2015. High-throughput pairing of T cell receptor alpha and beta 
sequences. Sci Transl Med. 7(301):301ra131. doi: 10.1126/scitranslmed.aac5624. 
 
 
 


English

A match.com for T cell receptor α and β chains.

November 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
Conscious uncoupling between FANCI and 
FANCD2 in DNA repair 
November 15, 2015 
     A Neves 
 
New model of the FA pathway activation. Non-phosphorylated, non-ubiquitinated FANCI plays a critical role in 
recruitment of the FA core complex at sites of DNA damage. ATR phosphorylates substrates including FANCI and 
FANCM. The FA core complex monoubiquitinates FANCD2 and FANCI following FANCI phosphorylation, and USP1 
deubiquitinates FANCD2 and FANCI. FANCI, FANCM, USP1, and BRCA1 positively regulate recruitment of FA core 
complex and FANCD2 at sites of interstrand crosslinks (ICLs). Monoubiuqitination of FANCD2 is required for 
retention of FANCD2– FANCI at sites of ICLs. Monoubiquitinated FANCD2 is also required for downstream events, 
such as recruitment of various nucleases (not depicted). For the regulation of FANCI phosphorylation/ 
dephosphorylation, ATR and unidentified phosphatase are involved. In this new model, the FANCI ubiquitination/ 
deubiquitination and phosphorylation/dephosphorylation cycle is important for proper activation of the FA pathway. In 
the old model, only monoubiquitinated and phosphorylated FANCI is considered an active form. 
Image provided Dr. Toshiyasu Taniguchi. 
The study of the Fanconi anemia (FA) pathway has helped elucidate the molecular basis for several 
biological processes, including DNA repair, protein modification and cancer susceptibility. FA is a 
clinically heterogeneous genetic disease that results in bone marrow failure, congenital skeletal 
defects as well as early onset of cancer. The importance of DNA repair was recently highlighted by 
the award of this year’s Nobel Prize in chemistry for mechanistic studies of DNA repair. Several FA 
genes have been identified and some of these are also breast and ovarian cancer susceptibility 
genes, such as BRCA1 and BRCA2. Eight FA proteins form a protein complex known as the FA core 
November 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
complex that is required to both detect and respond to DNA lesions. However, how the FA core 
complex is recruited to DNA lesions is not well understood. A new Fred Hutch study by the 
Taniguchi Laboratory (Human Biology and Public Health Sciences Divisions), led by former post-
doctoral fellow Dr. Maria Castella and published in PLOS Genetics, developed an improved method 
to visualize the FA core complex, which enabled a detailed study of its regulation. 
"The Fanconi anemia pathway regulates DNA repair and plays a critical role in determining 
sensitivity of cancer cells to commonly used chemotherapeutics, such as cisplatin", said Dr. 
Toshiyasu Taniguchi. The investigators first refined immunostaining methods to detect nuclear foci 
of FA core complex subunits revealing that the FA core complex foci formed during the S and G2 
phases of the cell cycle and relied on the whole FA complex. The FA core complex is an ubiquitin 
ligase that monoubiquitinates FANCD2 and FANCI. Because FANCD2 and FANCI form a complex 
and their monoubiquitination is required for both their DNA localization and efficient DNA repair, the 
investigators were surprised to find that FANCI, but not FANCD2, was required for FA core complex 
foci formation. This novel FANCI function required neither its monoubiquitination nor its 
phosphorylation. Next, the authors searched for additional factors required for the formation of FA 
core complex foci. To this end, they assayed FA foci in cells lacking one of several proteins known to 
be involved in the FA pathway. These experiments revealed that depletion of either BRCA1 or USP1 
resulted in marked decrease in the formation of FA core complex foci. USP1 is a deubiquitinase, so 
the authors asked whether FANCI was the relevant USP1 substrate. Consistent with this notion, they 
found that a non-ubiquitinatable version of FANCI (K523R mutant) partially rescued the formation of 
FA foci in USP1-deficient cells. In summary, a careful analysis of FA core complex formation 
revealed new regulatory mechanisms for FA core complex recruitment, including novel roles for 
several proteins (BRCA1, USP1 FANCI, ATR) that were previously thought to act in a more 
restricted manner. 
Said Dr. Taniguchi "It is believed that activation of this pathway requires monoubiquination and 
phosphorylation of FANCI. Unexpectedly, we found that non-phosphorylated, non-ubiquitinated 
FANCI plays a critical role in recruitment of the FA core complex at sites of DNA damage, 
suggesting that deubiquitination and dephosphorylation of FANCI protein is also critical for the 
function of the pathway. This finding changes our current view of the Fanconi anemia pathway."  
Funding for this study was provided by the National Institutes of Health and the Howard Hughes 
Medical Institute. 
 
November 15, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
Castella M, Jacquemont C, Thompson EL, Yeo JE, Cheung RS, Huang J-W, Sobeck A, Hendrickson 
EA, Taniguchi T. 2015. FANCI regulates recruitment of the FA Core complex at sites of DNA 
damage Independently of FANCD2. PLoS Genet. 11(10):e1005563. 
doi:10.1371/journal.pgen.1005563. 
 
 


Conscious uncoupling between FANCI and FANCD2 in DNA repair

Herman, Jake

December 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
Engineered T cells behind enemy lines in solid 
tumors 
December 20, 2015 
     J Herman 
 
 
 
 
 
Immunofluorescent microscopy 
demonstrates that T cells engineered 
to target MSLN infiltrate solid PDA 
tumors. Engineered donor T cells 
shown in red are in the immediate 
vicinity of MSLN expressing tumor 
cells shown in green. Engineered T 
cells can alternatively be detected 
using flow cytometry in PDA primary 
tumors as well as in metastatic 
lesions. 
 
Image provided by Dr. Ingunn 
Stromnes (Clinical Research Division). 
 
 
 
The human immune system recognizes non-native antigens and targets them for destruction. This 
mechanism recognizes viruses, bacteria, fungi, transplanted tissue, and even cancers. In cancer, 
transformed cells must escape detection by the immune system and continue proliferating to 
become clinically relevant tumors. Different cancers exploit unique mechanisms to prevent detection 
including: low mutation rates, physical barriers, expression of immunosuppressive proteins, 
prevention of antigen processing/presentation, and growth in immune privileged sites. These 
adaptations must be overcome when developing immunotherapies. Some of the first successful T 
cell based therapies surmounted the issues of physical barriers and immune privilege growth sites 
by targeting B cell malignancies, which do not form solid tumors and circulate in the same 
environment as T cells. On the heels of these successes in leukemia, scientists in the Greenberg 
December 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
and Hingorani Laboratories in the Clinical Research Division have demonstrated that engineered T 
cells are also effective against solid tumors. In a recent publication in Cancer Cell, these researchers 
engineered T cells to target pancreatic ductal adenocarcinoma (PDA) in a genetically engineered 
mouse model. Targeted immunotherapy nearly doubled the survival time in these preclinical studies. 
PDA is an incredibly difficult target for immunotherapy: it is genetically stable with few mutations or 
chromosomal rearrangements, builds stromal barriers that limit perfusion, and often expresses 
inhibitory T cell ligands. One of the keys to achieving success in this study was choosing the correct 
T cell antigen. Previous studies found mesothelin (MSLN), a cell adhesion protein that promotes 
invasiveness, to be an attractive target in PDA, and explored immunization against it. In this study, 
they verified in their mouse model of PDA that MSLN is rarely expressed in adult tissues and is 
consistently expressed in all stages of PDA. To isolate the highest affinity T cell receptor (TCR) 
against this native protein, mice were immunized with eight different peptides and T cells were 
isolated. After identifying the T cells with highest affinity for MSLN and elevated cytokine production, 
researchers sequenced the TCR expressed in these cells. 
With this high affinity TCR in hand, they found it functioned independent of the CD8 co-receptor, 
suggesting either CD8 or CD4 T cells could be engineered, however, only CD8 T cells were used in 
this study. Engineered T cells were injected intravenously into mice bearing spontaneous PDA 
tumors and were found to concentrate in the tumor compared to spleen. Once in the tumor region, 
the cells induced tumor apoptosis and loss of extracellular matrix –  one of the barriers to immune 
access to tumors. These results were observed eight days after infusion, yet after 28 days, while 
engineered T cells concentrated in the tumor, the total number of T cells had significantly decreased. 
Phenotypic and functional characterization of T cells 28 days after infusion demonstrated signs of 
immune dysfunction. Some level of repression is expected in cells with chronic TCR signaling, 
however, non-activated engineered T cells also experienced immunosuppression. This suggests the 
tumor microenvironment was contributing in part to these observations. 
The simple solution for the failure of engineered T cells to proliferate is to administer more cells, yet 
it is possible the first exposure may activate immune regulatory pathways in the tumor, rendering the 
second exposure useless. Surprisingly, when mice were dosed at 0 and 20 days, then analyzed 28 
days after initial injection the engineered T cell population in tumors was 10 fold higher than with the 
single dose. Moreover, the second dose of engineered T cells demonstrated minimal signs of 
immune supression compared to cells persisting from the first dose. Having learned from these 
studies, researchers enrolled PDA bearing mice in a preclinical trial administering engineered T cell 
infusions every two weeks. Under this regimen 62.5% of mice experienced a decrease in tumor 
December 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
volume, and overall survival time was nearly doubled. Moreover, there was a 20-30% decrease in 
metastatic disease and, as Dr. Stromnes, a research associate in the Greenberg Lab said, "We did 
not observe detectable clinical symptoms or histopathological evidence for toxicity with this 
approach." 
In order to move forward into human studies the team also identified a high-affinity TCR targeting the 
human sequence of MSLN. However, they are not stopping there. According to Dr. Stromnes, 
"Obviously, it is wonderful to be part of a team that will translate this work to patients as soon as 
possible. However, I am certain we can do better. We are currently testing ways to further engineer 
the T cells for sustained benefit, including co-opting factors in the immunosuppressive environment 
to paradoxically sustain T cell survival, persistence and function rather than interfere with it." Another 
possibility suggested by Dr. Stromnes is targeting multiple antigens, "The Greenberg lab already has 
T cells in clinical trials targeting Wilms' tumor antigen (WT1), and WT1 is also expressed in PDA." 
Likewise, work done with CAR T cells in the Riddell lab may also benefit affinity enhanced TCR 
therapy, "Modulating the T cell subsets that are engineered for therapy, including provision of CD4 
helper T cells, may also greatly improve upon efficacy of this approach." said Dr. Stromnes.  
Funding for this research was provided by the National Cancer Institute, Fred Hutch/UW Cancer 
Consortium, Giles W. and Elise G. Mead Foundation, The Safeway Foundation, Juno Therapeutics, 
the Cancer Research Institute, and the Jack and Sylvia Paul Estate Fund. 
Stromnes IM, Schmitt TM, Hulbert A, Brockenbrough JS, Nguyen HN, Cuevas C, Dotson AM, Tan X, 
Hotes JL, Greenberg PD, Hingorani SR. (2015). T Cells Engineered against a native antigen can 
surmount immunologic and physical barriers to treat pancreatic ductal adenocarcinoma. Cancer Cell. 
28(5):638-52. 
 
 


Engineered T cells behind enemy lines in solid tumors

Kocarnik, Jonathan

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Culturally Appropriate Support Groups for Latino 
Cancer Survivors 
February 16, 2015 
     JM Kocarnik 
Many cancer survivors face long-term social and psychological burdens that last far beyond their 
initial diagnosis and treatment.  While social support can help alleviate some of these burdens, this 
support is often less available to Latino cancer survivors due to linguistic, social, and cultural 
barriers.  As Latino cancer survivors often experience lower psychosocial well-being compared to 
non-Latino Whites, there is a need to develop culturally appropriate methods for providing adequate 
psychosocial support.  To address this gap, Dr. Rachel Ceballos and Mr. Nathan Marchello in the 
Public Health Sciences Division developed and tested a ten week Spanish language support 
group.  As reported in Supportive Care in Cancer, this approach is feasible and effective for 
improving several aspects of well-being in Latino cancer survivors. 
In addition to the physical and medical toll of their diagnosis, cancer survivors are often faced with a 
large number of social and psychological burdens even long after successful treatment.  To identify 
and help address some of these concerns, the authors first conducted focus groups and one-on-one 
interviews with Latino cancer survivors.  Importantly, these interviews were conducted by 
promotores, bilingual and bicultural lay health workers who provide health-related support, 
education, and advocacy for the Latino community.  These promotores worked with 29 Mexican 
American members of a rural Washington community to identify the needs and burdens of Latino 
cancer survivors.  These wide-ranging burdens included thoughts about the impact of cancer on the 
family, changes in body- and self-image, depression and isolation, fear of recurrence, stress, and 
lack of support.  
Based on these community-identified concerns, the authors developed a support group intervention 
protocol.  Ten weekly 90 minute sessions included topics like stress, nutrition, physical activity, body 
image, sexuality, medical advocacy, and social support.  Each session included components of 
didactic learning, dynamic group discussion, and an activity that explored behavioral strategies for 
the topic.  Led by promotores, these sessions additionally integrated cultural concepts such as 
personalismo (sharing of personal information) and familismo (importance of family) into the 
curriculum.  To also accommodate potential differences in how men and women address and 
discuss these topics, male and female specific support groups were held.  To evaluate the effect of 
the intervention on quality of life, the participants filled out pre- and post-session 
February 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
questionnaires.  Changes in distress were also assessed, through pre- and post-session reporting of 
stress and measurement of saliva cortisol levels.  
From follow-up participant focus groups, the feedback for the ten week session was overwhelmingly 
positive.  "Participants left the groups with a profound new sense of hope and a drive that was fueled 
with and in one another," said co-author Mr. Marchello.  "They shared on the various ways they have 
grown, such as 'I value life much more', 'I have a better relationship with my family', 'I make healthier 
choices in my eating habits', and 'I thought it was not going to be worth my time'."  Furthermore, 
comparing pre- and post-session surveys demonstrated a significant improvement in both functional 
and social well-being.  Distress and cortisol levels were also lower after the ten week program. 
Overall, the authors demonstrated that this targeted intervention strategy can be beneficial to Latino 
cancer survivors.  "With our support groups, these survivors are able to engage in the process of 
healing and finding their new normal in a way that otherwise would not have been available to them," 
said lead author Dr. Ceballos.  Furthermore, the near 100% participation rate suggests that such a 
program is both feasible and desired.  The authors point out that the promotores played a key role in 
this intervention, and should be strongly considered for any future implementation of these 
sessions.  Moving forward, additional work is needed to evaluate the improvements in stress and 
well-being in larger groups, and to ensure that this approach is generalizable to other Latino 
groups.   
"A study building on this project is already under way," said Dr. Ceballos.  "We are currently running 
a randomized control trial with a larger sample of Latinas who are survivors of breast and 
gynecological cancers.  This study involves more in-depth evaluation of psychosocial variables and 
biomarkers of distress and inflammation including cortisol, C-reactive protein, and telomere length. 
We have been building relationships with community medical organizations for some time keeping 
them abreast of our progress.  Assuming the current project is effective, future studies will focus on 
dissemination and sustainability in the current community and, potentially, other regions across the 
United States." 
Other PHS researchers contributing to this project were Ms. Rachel Malen, Ms. Genoveva Ibarra, 
and Ms. Monica Escareño. 
 
Ceballos RM, Molina Y, Malen RC, Ibarra G, Escareño M, Marchello N. 2015. Design, development, 
and feasibility of a spanish-language cancer survivor support group. Support Care Cancer. doi: 
10.1007/s00520-014-2549-9. 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
 
 
Images provided by Dr. Rachel Ceballos 
and Mr. Nathan Marchello, respectively 
Study authors Dr. Rachel Ceballos (top), 
Assistant Member in the Public Health 
Sciences Division, and Mr. Nathan 
Marchello (bottom), a Community Health 
Promoter in the Center for Community 
Health Promotion. 


Culturally Appropriate Support Groups for Latino Cancer Survivors

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Myod Crashes Neurod2’s Private Party 
May 18, 2015 
     A Neves 
The human body consists of hundreds of different cell types, each with a different form and function. 
A large body of evidence, including Nobel-prize winning work on generation of pluripotent stem cells, 
suggests that proteins known as transcription factors are master regulators of cell type identity that, 
by directly binding to DNA, function to switch on specific sets of genes in each cell type. One such 
transcription factor, MyoD, which was first discovered here at the Fred Hutch in the laboratory of the 
late Dr. Harold Weintraub almost 30 years ago, is a master regulator of muscle cells as it can 
convert non-muscle cells such as fibroblasts into skeletal muscle, in a process known as 
transdifferentiation. Since its discovery, other transcription factors with similar cell fate-determining 
functions have been identified. For instance, NeuroD2 can transdifferentiate cells into neurons. Both 
MyoD and NeuroD2 are part of a family of transcription factors that contain a basic-helix-loop-helix 
(bHLH) domain that is required for DNA binding and both bind to highly similar target sequences. 
Previous studies in Dr. Stephen Tapscott’s Laboratory (Clinical Research and Human Biology 
Divisions) used chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-
seq) to show that while MyoD and NeuroD2 can bind to shared DNA sites, they also bind DNA sites 
that are specific to each factor, termed private sites, that are highly correlated with cell type specific 
gene activation (Fong et al.,2012). 
However, it remained unclear which domains of MyoD and NeuroD2 were important for cell type 
determination. A new study by the Tapscott lab, led by research associate Dr. Abraham Fong and 
published in Cell Reports, addressed this question by generating a chimeric transcription factor 
wherein the bHLH domain of MyoD was replaced with the bHLH domain of NeuroD2. This chimera, 
MyoD (NeuroD2-bHLH), exhibited strong gene activation of a reporter containing a pair of NeuroD2 
private sites and weak activation of a reporter containing a pair of either one of the shared sites or of 
the MyoD private sites, similar to NeuroD2 itself. This gene activation pattern correlated with DNA-
binding affinity, as demonstrated by in vitro gel shift assays. 
Next, to assay the DNA-binding properties of the chimera in cells, the authors performed ChIP-seq 
experiments with NeuroD2, MyoD and MyoD (NeuroD2-bHLH) in a mouse embryonal carcinoma cell 
line (P19). Indeed, discriminative motif enrichment analysis showed that MyoD (NeuroD2-bHLH) 
acquired the DNA-binding properties of NeuroD2. Next-generation sequencing of RNA (RNA-seq) of  
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
P19 cells transduced with either NeuroD2, MyoD or MyoD (NeuroD2-bHLH), revealed that the 
chimera activated both neuronal and muscle genes. These studies were corroborated by examining 
both neuronal and muscle markers either in P19 cells or in zebrafish embyros that had been 
transduced or injected with the chimera, respectively. 
Finally, the authors addressed the question of why the chimera still bound to a subset of muscle 
genes. To this end, they performed discriminative motif enrichment analysis of the DNA region 
surrounding sites bound both by the chimera and MyoD, but not NeuroD2, which revealed an 
enrichment for binding sites for homeodomain transcription factors of the PBX/MEIS family. 
Intriguingly, a PBX/MEIS complex had been previously shown by the Tapscott lab to be required for 
full activation of 10% of MyoD target genes. Strikingly, P19 cells transduced with a modified chimera 
in which the PBX/MEIS interaction domain was mutated essentially behaved as a dedicated 
neurogenic factor and lost the ability to activate key muscle genes such as myogenin. 
"It was initially surprising to us that retargeting MyoD to the NeuroD2 binding sites was sufficient to 
convert it to a master regulator of neurogenesis, but now this helps us understand recent reports 
showing that spindle cell rhabdomyosarcomas have a mutation in MyoD that changes its binding 
specificity to Myc sites and makes it oncogenic. So by changing the MyoD DNA binding specificity 
we can change it from a myogenic factor to either a neurogenic factor or an oncogenic factor. This 
suggests a direct approach to tissue engineering as well as an understanding of how new 
transcriptional programs can arise during evolution", said Dr. Stephen Tapscott. In conclusion, this 
report showed that the DNA binding specificity of bHLH transcription factors largely determines their 
transcriptional output, which warrants similar studies of other transcription factor families to establish 
the generality of this concept. 
 
Fong AP, Yao Z, Zhong JW, Johnson NM, Farr GHr, Maves L, Tapscott SJ. 2015. Conversion of 
MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage. Cell Rep. 10,1937-1946. 
See also: 
Fong AP, Yao Z, Zhong JW, Cao Y, Ruzzo WL, Gentleman RC, Tapscott SJ. 2012. Genetic and 
Epigenetic Determinants of Neurogenesis and Myogenesis. Dev Cell. 22, 721-35. 
 
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Stephen Tapscott. 
The transcription factor MyoD normally determines muscle fate 
by binding to its private DNA sites (red). Swapping MyoD’s 
DNA-binding domain with a similar yet distinct domain in 
NeuroD2 converts MyoD into a neuron-determining factor, by 
retargeting the MyoD-NeuroD2 chimera to NeuroD2’s private 
DNA sites (blue). 
 


Myod Crashes Neurod2’s Private Party

Kocarnik, Jonathan M

July 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
Physical Activity before Colorectal Cancer 
Diagnosis Improves Survival 
July 20, 2015 
     JM Kocarnik 
Hazard ratio estimates and 95% 
confidence intervals for the association 
between pre-diagnostic physical activity 
level (at least 8.75 MET-hours/week 
versus less) and colorectal cancer 
specific survival. This relationship did 
not significantly vary when stratified by 
BRAF/KRAS mutation status (red), 
microsatellite instability status (green), 
cancer stage (blue), and tumor location 
(yellow). 
 
Image provided by Dr. Sheetal Hardikar 
 
Physical activity throughout life is beneficial for lowering the risk of developing or dying from several 
forms of cancer.  Consequently, guidelines for cancer survivors generally recommend at least 150 
minutes of moderate intensity or 75 minutes of vigorous exercise per week.  While there is 
consistent evidence that post-diagnostic physical activity improves both overall and colorectal 
cancer-specific survival, the evidence for pre-diagnostic physical activity is less clear.  To further 
explore this relationship, Drs. Sheetal Hardikar and Amanda Phipps in the Public Health Sciences 
Division evaluated the association between physical activity and colorectal cancer survival while 
accounting for potential epidemiologic and molecular sources of heterogeneity.  As recently reported 
in Cancer Epidemiology, Biomarkers & Prevention, the authors observed that individuals who were 
physically active before colorectal cancer diagnosis had significantly better survival than those who 
had been inactive or minimally active. 
Colorectal cancer is a heterogeneous disease, and recent work has identified molecular 
characteristics within the tumor that can differentiate different subtypes of disease.  These colorectal 
cancer subtypes have been shown to have differential associations with survival: the presence of 
somatic mutations in the BRAF or KRAS oncogenes has been associated with poorer survival, while 
July 20, 2015 SCIENCE SPOTLIGHT 
 
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the presence of microsatellite instability (MSI) has been associated with better survival.  Additionally, 
evidence has suggested that the association of other lifestyle factors, such as smoking, with 
colorectal cancer risk and survival may also vary according to these tumor markers.  Therefore the 
inconsistent associations previously seen for pre-diagnostic physical activity could have been due to 
those studies not accounting for these molecular characteristics. 
To evaluate this relationship, the authors analyzed pre-diagnostic physical activity and outcomes 
data in more than 1,300 colorectal cancer patients from the Seattle Colon Cancer Family 
Registry.  Participants were asked about their recreational physical activity during defined age 
periods, which were converted into standard metabolic equivalent of task hours per week (MET-
h/wk).  At least 8.75 MET-h/wk of physical activity was the threshold for meeting the recommended 
level of exercise.  Tumor markers were assayed from banked tissue samples.  After a median follow-
up time of 6.1 years, pre-diagnostic physical activity levels were evaluated for an association with 
both overall and colorectal cancer specific survival using Cox regression. 
The authors found that being physically active was associated with statistically significant 
improvements in both overall and colorectal cancer-specific survival.  Importantly, patients who 
reported at least 8.75 MET-/wk of pre-diagnostic physical activity had better survival regardless of 
their tumor-markers, cancer stage, or tumor site (see figure).  "Our results support these 
recommendations for colorectal cancer patients and suggest that physical activity in the years 
preceding cancer diagnosis may offer a survival benefit," said lead author Dr. Hardikar.  "This 
survival benefit did not appear to differ for patients with different molecular subtypes of disease." 
These results solidify the importance of maintaining sufficient levels of physical activity to not only 
reduce the risk of colorectal cancer, but also to improve prognosis should the disease 
develop.  Importantly this study shows that this holds true regardless of the molecular characteristics 
of the tumor, leading to broad generalizability of this finding.  "Further studies are needed to better 
understand the underlying mechanisms through which pre-diagnostic physical activity may impart 
survival benefit," said Dr. Hardikar.  "This may aid us to better inform physical activity 
recommendations to the public."  In the meantime, people have yet another reason to be physically 
active. 
Other PHS investigators contributing to this project were Drs. Polly Newcomb, Karen Makar, and 
John Potter. 
 
July 20, 2015 SCIENCE SPOTLIGHT 
 
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Citation:  
Hardikar S, Newcomb PA, Campbell PT, Win AK, Lindor NM, Buchanan DD, Makar KW, Jenkins 
MA, Potter JD, Phipps AI. 2015. Prediagnostic physical activity and colorectal cancer survival: 
overall and stratified by tumor characteristics. Cancer Epidemiol Biomarkers Prev. 24(7):1130-7. doi: 
10.1158/1055-9965.EPI-15-0039.  
 
 


Physical Activity before Colorectal Cancer Diagnosis Improves Survival

July 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
Small RNAs Can Pack a Punch in 
Facioscapulohumeral Dystrophy 
July 20, 2015 
    A Neves 
 
 
 
 
RNA-mediated epigenetic gene silencing of 
the D4Z4 repeats and the DUX4 gene 
embedded in the repeat. 
 
Image provided by Dr. Filippova 
 
 
 
Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disorder characterized by progressive 
weakness of skeletal muscle, most noticeably in muscles of the face (facio), back (scapula) and 
upper arms (humeral), and is estimated to affect almost one million people worldwide. FSHD is 
thought to be caused by mis-expression of DUX4, a homeobox transcription factor that is normally 
only expressed in testes but is misexpressed in FSHD skeletal muscle cells. The DUX4 gene resides 
within a macrosatellite repeat known as D4Z4, and the size of the D4Z4 array correlates with FSHD 
severity. For example, unaffected individuals have between 11 and 100 units of the array, whereas 
contraction of the array to between 1 and 10 units is linked to FSHD. However, the epigenetic 
mechanisms that repress DUX4 expression in normal muscle cells remain to be completely 
elucidated. A new Fred Hutch study by the Tapscott Laboratory (Human Biology and Clinical 
Research Divisions), led by Drs. Jong-Won Lim, Laurie Snider and Gala Filippova, and published in 
Human Molecular Genetics, showed that the endogenous RNAi pathway is required to silence the 
D4Z4 repeat array and DUX4 expression and that exogenous small interfering RNAs (siRNAs) that 
mimic endogenous D4Z4 small RNAs enhance epigenetic silencing of the D4Z4 array. "Our work 
July 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
suggested a potential mechanism by which the D4Z4 repeat (and the DUX4 gene imbedded in the 
repeat) is silenced by small RNAs generated from D4Z4 in the nonpathogenic condition. We expect 
therapeutic targeting strategies based on this mechanism would be effective for epigenetic silencing 
of DUX4, the FSHD causing gene, in FSHD condition," said Dr. Lim. 
The authors had previously identified bidirectional transcripts and small RNAs throughout the D4Z4 
region. To determine if such RNAs could modulate DUX4 expression, the researchers synthesized 
siRNAs that targeted not only the coding region of DUX4, but also the upstream non-coding region, 
often referred to as the promoter. Remarkably, the promoter siRNAs could suppress DUX4 
expression as potently as the coding region siRNAs, albeit with delayed kinetics. This delayed effect 
is consistent with a model in which promoter siRNAs repress DUX4 transcription, whereas coding 
region siRNAs degrade the DUX4 mRNA directly. Because siRNA-dependent transcriptional 
silencing is known to involve Argonaute (AGO) proteins and dimethylation of Histone H3 on lysine 9 
(H3K9me2), the authors examined both AGO recruitment and H3K9 methylation status by chromatin 
immunoprecipitation. This analysis revealed that promoter siRNAs increase the amount of AGO2 
and H3K9me2 at the D4Z4 repeats. 
To further probe the role of the RNAi pathway in the epigenetic silencing of DUX4, the authors 
knocked down DICER1, AGO1 and AGO2 in control myoblasts derived from unaffected individuals 
that had 13 D4Z4 repeats, which is in the low range of the non-FSHD population. Strikingly, the 
authors observed de-repression of both DUX4 and DUX4 target genes in cells that contained 13 
D4Z4 units. In contrast, knockdown of RNAi components in control cells obtained from unaffected 
individuals that contained 74 D4Z4 repeats did not result in DUX4 de-repression. Furthermore, 
knocking down AGO2 prevented promoter siRNAs from suppressing DUX4 expression in FSHD 
cells. Importantly, targeting non-coding RNAs upstream of DUX4 for degradation was not sufficient 
to de-repress DUX4, consistent with the proposed RNAi mechanism. 
"Although still not widely accepted, this evolutionarily conserved RNA-mediated silencing 
mechanism clearly plays an important role in keeping repeats and genes imbedded in the repeats 
under control in both normal development and disease,such as FSHD" said Dr. Filippova. Overall, 
this study demonstrated the role of the endogenous RNAi pathway in epigenetic repression of 
DUX4, and suggests that enhancing the activity of this pathway with exogenous siRNAs could be a 
valid therapeutic approach to silence DUX4 in FSHD. 
Lim J-W, Snider L, Yao Z, Tawil R, van der Maarel SM, Rigo F, Bennett CF, Filippova GN, Tapscott 
SJ. 2015. DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous 
July 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
siRNA suggests mechanisms and therapies for FSHD. Hum Mol Genet. pii: ddv206. DOI: 
10.1093/hmg/ddv206. Epub ahead of print. 
See also: 
Hewitt JE. 2015. Loss of epigenetic silencing of the DUX4 transcription factor gene in 
facioscapulohumeral muscular dystrophy. Hum Mol Genet. pii: ddv237. Epub ahead of print. 
This study was supported by funding from Friends of FSH Research and NIH NINDS P01NS069539. 
 
 


Small RNAs Can Pack a Punch in Facioscapulohumeral Dystrophy

August 17, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
Most cancer transcriptomes are bloated by 
widespread intron retention 
Aug. 17, 2015  
 
     JM Kocarnik 
 
 
 
 
 
 
 
 
 
A) Comparing RNA-seq read coverage for a 
portion of the CDK10 gene shows that some 
introns are more frequently retained in the 
tumor sample than the adjacent normal tissue. 
B) For each individual, the number of introns 
retained in the tumor sample (y-axis) is plotted 
against the number of introns retained in the 
matched normal sample (x-axis). Aggregating 
patients, most tumors showed much more 
frequent intron retention (left, middle), while 
breast cancer was the exception where normal 
tissue exhibited more common intron retention 
(right). 
 
Image provided by Dr. Heidi Dvinge 
 
 
 
 
 
Alternative splicing of specific genes can contribute to cancer initiation, progression, and 
metastasis.  With the advent of cancer genome sequencing, it was discovered that many tumor 
types contain high frequencies of somatic mutations affecting components of the RNA splicing 
machinery.  Additional evidence suggested that common RNA processing differences might 
distinguish between cancer and normal cells, but had not been systematically tested.  To evaluate 
this question, Drs. Heidi Dvinge and Robert Bradley in the Public Health Sciences and Basic 
Sciences Divisions performed a genome-wide search for signals of abnormal cancer-associated 
August 17, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
splicing.  As recently reported in Genome Medicine, the authors found that abnormal RNA splicing is 
a common characteristic of most cancers, even when the RNA splicing machinery appears 
unaltered.   
Genes are made up of protein-encoding exons interspersed with introns.  Normally, DNA is 
transcribed into RNA and then modified through a process that only retains the genetic information 
from the exons.  In this process, the cellular splicing machinery excises the introns out of the RNA 
copy and joins the ends of exons together.  "One of the surprising findings from the large-scale 
cancer genomics effort in the last few years has been the high frequency of mutations in the RNA 
processing machinery," said lead author Dr. Dvinge.  Errors in this process can result in the retention 
of introns, potentially leading to abnormal protein products with unknown consequences.   
To evaluate for systematic differences in RNA splicing, the authors utilized the comprehensive 
transcriptome data available from The Cancer Genome Atlas (TCGA).  This involved comparing 
patterns of RNA splicing between 800 tumor and patient-matched normal control samples, and 
across 16 distinct cancer types.  These cancers included 15 solid tumors as well as one liquid tumor 
(acute myeloid leukemia).  Using RNA-seq data from TCGA, for each tumor-normal pair the authors 
identified introns that were differentially retained between the samples (see figure).  These 
differences in intron retention were quantified and then compared among and across cancer 
types.  Said Dr. Dvinge, "we found that abnormal RNA splicing is a common feature of a wide range 
of primary tumors, even in the absence of DNA mutations or other obvious changes to the RNA 
processing machinery." 
Notably, 15 of the 16 cancer types showed a strong enrichment for increased retention of alternative 
introns within the tumor compared to the adjacent normal control tissue.  The one exception was 
breast cancer, which actually showed the opposite pattern.  The authors found that the majority of 
these retained introns were either specific to a particular cancer, or were found at low frequencies in 
multiple cancers.  At the same time, a handful of retained introns were shared across most cancer 
types, mostly within genes encoding RNA splicing and export factors.  These results suggest that the 
RNA splicing process may be commonly dysregulated in cancer, which may be useful for 
differentiating normal versus cancerous cells.   
While this study demonstrates that intron retention is a common occurrence in many tumor types, 
the functional consequences of this phenomenon remain to be explored.  "It remains to be seen 
whether the intron-containing transcripts are dead-end cellular products, or whether they contribute 
to the cancer proteome," said Dr. Dvinge.  "Regardless, analyzing the dysregulated RNA splicing 
August 17, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
events may help us to unravel why small molecules that inhibit splicing also have anti-tumor activity, 
or help design novel therapies targeting tumor cells." 
Citation:  
Dvinge H, Bradley RK. 2015. Widespread intron retention diversifies most cancer transcriptomes. 
Genome Med. 7(1):45. doi: 10.1186/s13073-015-0168-9.  
Funding for this study was provided by the National Cancer Institute (NCI), the Damon Runyon 
Cancer Foundation, and the Ellison Medical Foundation. 
 


Most cancer transcriptomes are bloated by widespread intron retention

Koch, Lori

October 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
What swine flu can tell us about the evolution of 
influenza 
October 19, 2015 
     L Koch 
 
 
Phylogenetic tree of swine (blue) 
and human (red) influenza 
nucleoprotein. 
 
Image provided by Heather 
Machkovech. 
 
 
Viruses are constantly evolving to escape destruction by host immune systems.  The host adaptive 
immune response uses antibodies as well as cytotoxic T-cells to combat viral antigens.  The T-cell 
response is more general because T-cells recognize more conserved epitopes, or specific parts, of 
viral antigens.  Despite an obvious pressure to evolve away from T-cell recognition, studies of 
human influenza virus have so far been unable to find any evidence for positive selection on 
epitopes that are recognized by T-cells compared to non-epitope residues. In order to further 
investigate this confusing observation, graduate student Heather Machkovech in the Laboratory of 
Dr. Jesse Bloom (Basic Sciences Division) conducted a comparative analysis of influenza viruses 
from human and swine lineages that was recently published in the Journal of Virology.  The authors 
reasoned that swine influenza is unlikely to be under selective pressure from human T-cells and 
therefore provides a control for analyzing the evolution of the human virus.  The idea that the 
evolution of epitopes on swine influenza viruses is random with respect to pressure from T-cells 
makes sense given the limited lifespan and thus limited number of infections pigs can 
experience.  Furthermore, despite this difference in selective pressure, there is high homology 
between human and swine influenza proteins that are recognized by T-cells so the comparison is 
straightforward and informative.    
 
To begin, the authors collected a list of epitopes that had been reported to elicit a response from 
CD8+ T-cells using Immune Epitope Database. Consistent with other reports, they found that the 
majority of epitopes that elicited responses were either in the nucleoprotein (NP) or the matrix 
protein (M1) of flu virus.  They further classified each residue of the proteins with a value reflecting 
October 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
how many unique epitopes that residue contributed to overall.  Using these data, they confirmed that 
conventional methods of identifying positive selection, that use the ratio of non-synonymous (dN) to 
synonymous mutations (dS) as a metric for selection, do not provide evidence for positive selection 
in the T-cell epitope sites in NP or M1.  Furthermore, they discovered that the number of non-
synonymous changes at each site was not very different between epitope and non-epitope sites, 
suggesting that the substitution rate is not faster at residues contributing to T-cell epitopes.  This can 
help explain the lack of evidence for change in dN/dS between epitope and non-epitope sites.   
 
Although they found no evidence for an elevated (>1) rate of mutation at epitope vs. non-epitope 
sites, the authors noted an interesting trend in their data.  The ratio of mutation rates at epitope vs. 
non-epitope sites is clearly higher in human vs. swine NP, with the human ratio being close to 1 and 
the swine closer to 0.5.  This suggests that epitope sites are actually evolving faster in human NP 
compared to swine NP, even though the underlying difference in mutation rate between non-epitope 
and epitope sites within each viral lineage is minimal.  To further test the statistical significance of 
this trend, they took into account the number of epitopes each residue contributed in a new statistic 
F, which they define as the average number of unique epitopes that are changed per 
substitution.  They found strong statistical support (p<0.0002) to reject the hypothesis that epitope-
changing mutations fix with equal probability between human and swine influenza virus.  Instead, the 
probability of observing epitope-changing mutations was significantly elevated for human vs swine 
influenza, across the whole phylogenetic tree and especially in the "trunk" of the tree.  Enrichment 
for epitope-altering mutations in the trunk of the phylogenetic tree of human influenza NP suggests 
that viruses that acquire those mutations have a wider evolutionary range.   
 
This work identifies the CD8+ T-cell arm of the human adaptive immune response as a driver for the 
evolution of human influenza and informs further studies aimed at treatment and prediction of 
influenza infectivity.  "Understanding how influenza escapes from our immunity is very important for 
determining which viral strains are likely to spread from year-to-year," said Dr. Jesse Bloom. 
"Studies in the past have focused mostly on how the virus escapes from the antibody arm of our 
immune system. Heather's work is the first to conclusively show that influenza also evolves to 
escape from the T-cell arm of the immune system."   In the future, research in the Bloom Lab aims 
"to establish the extent to which the T-cell escape mutations that we identify enable certain viral 
strains to spread more effectively in the human population."   
 
Machkovech HM, Bedford T, Suchard MA, Bloom JD.  2015.  Positive selection in CD8+ T-cell 
epitopes of influenza nucleoprotein revealed by a comparative analysis of human and swine viral 
lineages.  Journal of Virology. 89(22):11275-83. doi: 10.1128/JVI.01571-15. 
October 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 10 | Fred Hutchinson Cancer Research Center 
 
 
The research reported here was supported by the NIGMS and NIAID of the National Institutes of 
Health. 
 
 
 
 


What swine flu can tell us about the evolution of influenza

Pattacini, Laura

November 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
Time gives answers and raises questions 
November 15, 2015 
     L Pattacini 
 
 
Hazard ratios for HIV in vaccine 
versus placebo in men and women 
combined (purple), men (blue) and 
women (green) in Phambili (503) 
and Phambili (503) plus 503-S. 
 
Image provided by Dr. Zoe Moodie 
 
 
 
 
Time gives answers, but they are not always what we expect, and may raise more questions. This is 
the case for the HVTN 503-S study, designed to obtain further data on uninfected participants in the 
Phambili HIV vaccine trial. The Phambili trial, similarly to the Step trial, evaluated the safety and 
efficacy of an adenovirus 5 (Ad5) based vaccine, where the vector encoded for Gag, Pol and Nef 
proteins of HIV. Vaccinations in both studies were stopped after the Step study’s interim analysis 
met its pre-specified futility criteria.  
The Phambili trial, or HVTN 503, was run in a principally heterosexual population in South Africa, 
where clade C is the predominant HIV-1 circulating subtype, in a principally heterosexual population. 
At the time enrollment and vaccinations were stopped, Phambili had enrolled 800 HIV-1 uninfected 
participants who were subsequently unblinded to their treatment assignment and continued followed-
up to 42 months. The first analysis conducted after twenty-five months of follow-up showed a 
statistically non-significant increased risk of HIV acquisition in the vaccine arm as compared to the 
placebo arm. A subsequent analysis at the end of follow-up showed a significant increase in HIV 
acquisition in the vaccine arm. HVTN 503-S was designed to recall all uninfected participants for an 
additional HIV test, circumcision status evaluation and to expand information on behavioral risk.  
November 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
Dr. Zoe Moodie, a senior staff scientist in the Vaccine and Infectious Disease Division at Fred Hutch, 
reported the results obtained from the HVTN 503-S trial in a recent publication in the journal PLOS 
One produced in collaboration with HIV vaccine trial network (HVTN) investigators in South Africa 
and the United States. In the publication, she reported that in the combined Phambili and HVTN 503-
S follow-up period (seventy-two months average after enrollment), significantly more infections were 
detected in the vaccine arm, with an incidence rate of 4.7%, as compared to the placebo arm, for 
which the incidence rate was 3.0%. The vaccine to placebo hazard ratio (HR), adjusted for herpes 
virus -2 status at enrollment, was 1.52 in the combined Phambili and HVTN 503-S and 1.70 in 
Phambili alone. Thus, the follow-up study confirmed the results seen in the Phambili trial. Strikingly, 
the effect of vaccination on HIV-1 infection was more pronounced in men (HR=2.75, 95% CI=(1.49, 
5.06)) and less in women (HR=1.12, 95% CI=(0.73, 1.72)). There was no evidence that the effect of 
vaccination varied by baseline Ad5 status, age, study site or, in men, by circumcision status.  
One of the hypotheses to explain the increased HIV acquisition rate in the vaccine arm is that the 
vaccine would expand Ad5-specific memory CD4+ T cells for Ad5, therefore increasing the pool of 
possible target cells. Interestingly, while the Step study showed an effect of Ad5 serostatus on 
increased HIV acquisition in men in the vaccine arm, the Phambili study did not confirm this finding. 
Another hypothesis, although not verified, is that the vaccine led to changes in inflammation in men, 
but not in women. "It is difficult to explain the observed gender difference. The vaccine may have 
induced subclinical inflammation in men that contributed to the increased rate of HIV-1 acquisition 
whereas in women the subclinical inflammation didn't exceed the high levels already present due to 
genital inflammation. However, this hasn't been demonstrated- it remains a hypothesis" said Dr. 
Moodie. 
The study confirms some of the previous findings and raises more questions about which kind of 
immune response we want to induce and avoid in HIV-1 vaccine design. Hopefully the lessons 
learned will drive researchers to design a successful future trial. 
The research was supported by grants from the National Institutes of Health. 
Moodie Z, Metch B, Bekker L-G, Churchyard G, Nchabeleng M, Mlisana K, Laher F, Roux S, Mngadi 
K, Innes C, Mathebula M, Allen M, Bentley C, Gilbert PB, Robertson M, Kublin J, Corey L, Gray GE. 
2015. Continued Follow-Up of Phambili Phase 2b Randomized HIV-1 Vaccine Trial Participants 
Supports Increased HIV-1 Acquisition among Vaccinated Men. PLoS One. 10(9):e0137666. doi: 
10.1371/journal.pone.0137666. 


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