authors@Fred Hutch

Kocarnik, Jonathan

January 19, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
Sickle Cell Trait Associated With Increased Risk 
of Renal Disease 
January 19, 2015 
     JM Kocarnik 
Sickle cell disease is a hereditary blood disorder characterized by abnormal red blood cells that are 
prone to adopting a rigid, sickle-like shape.  An autosomal recessive condition, individuals with two 
copies of the sickle hemoglobin mutation have sickle cell disease, while individuals inheriting only 
one copy have sickle cell trait, which is generally considered benign.  While renal disease in the 
context of sickle cell disease is well established, the relationship between sickle cell trait and renal 
disease is less known.  In a recent report in the Journal of the American Medical Association, Dr. 
Alex Reiner in the Public Health Sciences Division led an effort to characterize the potential 
association between sickle cell trait and chronic kidney disease.  The results from this study suggest 
that sickle cell trait may be associated with higher risk of kidney disease in African Americans.  
The sickle cell mutation (rs334 in the HBB gene) is more common in individuals of African ancestry 
than other populations, and an estimated 1 in 12 African Americans are affected by sickle cell 
trait.  African Americans also have a disproportionately higher risk of chronic kidney disease and 
progression to end-stage renal disease.  While renal complications have been reported in those with 
sickle cell trait, the extent of this relationship is not known.  According to senior author Dr. Reiner, 
"our findings suggest that sickle cell trait appears to be one factor that contributes to the higher 
burden of kidney disease among that population." 
To characterize this relationship, the authors combined data from five large population-based, 
prospective cohort studies in the United States, totaling nearly 16,000 participants.  This allowed the 
researchers to evaluate the associations between sickle cell trait and several kidney function 
outcomes: chronic kidney disease, incident chronic kidney disease during follow-up, estimated 
glomerular filtration rate (eGFR), end stage renal disease, and albuminuria.  Compared to non-
carriers, the authors found that sickle cell trait carriers had significantly increased risk of chronic 
kidney disease, decline in eGFR, and albuminuria (see figure).  
Compared to non-carriers, those with sickle cell trait had a 57% increased risk of chronic kidney 
disease and an 86% increased risk of albuminuria.  These associations appeared to be independent 
of APOL1 risk variants, which have also been associated with chronic kidney disease progression in  
 
January 19, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
 
African Americans.  Several sensitivity analyses suggested these findings to be fairly robust.  "There 
was remarkable consistency of the results across the 5 studies, even though they were composed of 
different ages and sex distributions," said Dr. Reiner. 
Since sickle cell trait carriers are often identified at an early age, these findings could be potentially 
useful for developing early detection and intervention programs to improve outcomes.  More 
research is needed, however, in order to ensure the appropriate translation of these findings.  "There 
have been national pushes for screening of sickle cell trait in several different contexts (e.g., 
reproductive health; military or college athletics due to a possible increased risk of exercise-induced 
sudden death)," said Dr. Reiner, "but little guidance about what to do medically with such screening 
information.  Moreover, the current results DO NOT indicate that every single person with sickle cell 
trait is going to develop kidney disease.  While it is possible that sickle cell trait screening may 
ultimately be found to have utility for early detection of kidney dysfunction and whittling of health 
disparities faced by African Americans, further investigation will be necessary to yield specific 
recommendations." 
Overall, these findings suggest that sickle cell trait contributes to the racial disparity in chronic kidney 
disease.  "This study highlights the need for additional research into the health consequences of 
sickle cell trait," said Dr. Reiner.  "Future studies might evaluate possible associations between SCT 
and other chronic vascular-related diseases such as retinopathy, stroke and other conditions." 
 
Naik RP, Derebail VK, Grams ME, Franceschini N, Auer PL, Peloso GM, Young BA, Lettre G, 
Peralta CA, Katz R, Hyacinth HI, Quarells RC, Grove ML, Bick AG, Fontanillas P, Rich SS, Smith 
JD, Boerwinkle E, Rosamond WD, Ito K, Lanzkron S, Coresh J, Correa A, Sarto GE, Key NS, 
Jacobs DR, Kathiresan S, Bibbins-Domingo K, Kshirsagar AV, Wilson JG, Reiner AP. 2014. 
Association of sickle cell trait with chronic kidney disease and albuminuria in African 
Americans. JAMA. 312(20):2115-25. doi: 10.1001/jama.2014.15063. 
 
 
 
 
 
January 19, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 1 | Fred Hutchinson Cancer Research Center 
 
 
 
 
 
Image provided by Dr. Jonathan 
Kocarnik 
Odds ratio estimates for the 
association between sickle cell trait 
and renal disease outcomes, 
compared with non-carriers (eGFR = 
estimated Glomerular Filtration 
Rate). 


English

Sickle Cell Trait Associated With Increased Risk of Renal Disease

Frost, Sophia

March 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
From Specific to General, and Back Again 
March 16, 2015 
    SHL Frost 
Understanding the underlying mechanisms behind specific cancer types and other hereditary 
diseases is valuable not only for preventing and treating particular syndromes of interest, but also for 
adding general knowledge about cancer-promoting mutations overall. Considerable effort has been 
spent on sequencing the human genome to study how the order of nucleotides (As, Cs, Gs, and Ts) 
in our DNA code influences the likelihood of developing such inherited syndromes, but even if 
seemingly important individual mutations can be identified it is not always clear what part they play. 
Thus, combining specific case studies with general sequencing approaches informs our 
understanding of mutations that drive the malignant transformation. This premise was confirmed 
through a joint research effort undertaken by Fred Hutch scientists and external collaborators, led by 
the Clinical Research Division's Dr. Akiko Shimamura. The team studied a new hereditary syndrome 
featuring low blood platelet counts (thrombocytopenia) and a predisposition for various hematologic 
malignancies in multiple individuals from three families, with the aim of identifying common 
mutations. First-authored by a graduate student in the MD PhD program, Michael Zhang from the 
Shimamura Lab, their enlightening results were recently published in Nature Genetics. 
Through exome sequencing studies of a family of German and Native American ancestry with 
genetically undefined familial thrombocytopenia and cancer predisposition, the investigators 
identified a heterozygous hereditary variant in the ETV6 gene. "Heterozygous" signifies that only one 
of the two gene copies, coming from either the mother or the father, in a particular individual is 
mutated, as opposed to homozygous mutations which affect both gene copies. Targeted sequencing 
of individuals displaying similar disease characteristics (thrombocytopenia and hematologic 
malignancies) identified two additional families with germline heterozygous ETV6 mutations. These 
two families, of Scottish and African-American ancestry, respectively, each had a different 
hereditary ETV6 variant. 
These familial genetic studies suggest a vital role for the gene ETV6 in the formation, development 
and differentiation of blood cells, as well as in the promotion of malignancy. ETV6 encodes a protein 
that regulates expression of other genes, and the Shimamura lab demonstrated that 
this ETV6 function is impaired by mutations. The pinpointed gene was not an unknown player; large-
scale cancer genome sequencing efforts had previously demonstrated recurrent evidence for a  
 
March 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
 
potential role of somatic ETV6mutations in cancer, but whether ETV6 played an active role in 
malignant transformation had not previously been demonstrated. 
Dr. Shimamura elegantly summed up the new findings in three succinct sentences: "Acquired 
mutations in ETV6 were previously observed in cancers arising in the general population. Our study 
demonstrates that ETV6 mutations function to initiate or drive cancer development. Thus, the study 
of rare cancer predisposition syndromes advances our understanding of molecular pathways 
promoting cancer development in the general population." 
Besides increasing the fundamental understanding of driving forces behind cancer progression, 
identifying hereditary mutations improves diagnosis and medical care of individuals at risk. "The 
elucidation of genes driving malignancy is critical to our ongoing efforts to develop personalized 
approaches to the diagnosis and treatment of cancer in individual patients. Identification of genetic 
causes of cancer predisposition allows appropriate medical monitoring to initiate treatments early 
when therapies are most effective," said Dr. Shimamura, who also emphasized that genetic testing is 
helpful for making informed therapy choices and guiding donor selection for bone marrow 
transplants. 
Thus, the circle is closed; from defining a rare familial syndrome to understanding disease 
progression in the general population, and then back to personalized treatments for affected 
individuals. It's all in the code. 
 
Zhang MY, Churpek JE, Keel SB, Walsh T, Lee MK, Loeb KR, Gulsuner S, Pritchard CC, Sanchez-
Bonilla M, Delrow JJ, Basom RS, Forouhar M, Gyurkocza B, Schwartz BS, Neistadt B, Marquez R, 
Mariani CJ, Coats SA, Hofmann I, Lindsley RC, Williams DA, Abkowitz JL, Horwitz MS, King M-C, 
Godley LA, Shimamura A. 2015. Germline ETV6 mutations in familial thrombocytopenia and 
hematologic malignancy. Nat Genet. 47(2):180-5. 
 
 
 
 
 
March 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
 
 
Image provided by Dr. Akiko Shimamura 
Top: pedigree of a family with thrombocytopenia and cancer 
predisposition. Bottom: positions of germline and somatic mutations 
in ETV6, relative to the PNT oligomerization and ETS DNA-binding 
domains. Red highlights indicate germline alterations reported in 
this study. Somatic mutations described in the literature include 
alterations associated with a range of malignancies, as indicated. 
 


From Specific to General, and Back Again

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
A Ground-Breaking Method for Distinguishing 
Needles from Hay 
May 18, 2015 
     SHL Frost 
As scientists continue to elucidate differences between the genomes of cells in healthy and diseased 
conditions, therapies get progressively more targeted. The benefits are obvious: increased 
knowledge about disease states, including cancer-associated mutations and drug resistance, leads 
to greater possibilities for early diagnosis and efficient treatment. However, cells exhibit large 
mutational variations, leading to mixed populations of subclones that may respond differently to 
therapeutics and radiation. Although an individual cancer consists of billions of cells, conventional 
DNA sequencing approaches merely generate an "average" of the genomic DNA of most of the 
cells. Detection of rare mutations requires much more sensitive sequencing, but available 
techniques have been unable to sufficiently distinguish true variation from method-related errors. 
Worse yet, they are not just unsatisfactory for the task, they are also exceedingly expensive. 
To overcome these issues, Drs. Michael Schmitt and Jerry Radich from the Clinical Research 
Division teamed up with colleagues at the University of Washington to develop a sequencing 
approach based on a novel technique termed "duplex sequencing". In 2012, Dr. Schmitt published 
findings showing that results could be greatly improved by separately analyzing both strands of the 
DNA molecule. This was in contrast to current technologies, so called next-generation DNA 
sequencing, which look at a single strand, generating an unacceptable amount of technical errors in 
comparison to the rate of low-frequency mutations. Duplex sequencing overcomes those limitations 
by only scoring mutations that appear in the same position in both complementary DNA strands, 
ignoring amplification and sequencing errors found in just one strand. In a follow-up study, the Fred 
Hutch investigators combined this new approach with an iterative capturing method to enrich 
targeted genes by more than a million-fold. The result: jackpot. "Our approach improves the 
accuracy of DNA sequencing by 100,000-fold or more, and allows us to readily identify rare 
mutations that are undetectable by other approaches," Dr. Schmitt said. The technique is described 
in Nature Methods, demonstrating not only high capacity and accuracy, but also scaling ability for 
multiple targets. 
The field of oncology harbors numerous applications for this technique, according to Dr. Schmitt. 
Besides facilitating early cancer diagnosis and design of targeted treatments, another possibility is  
May 18, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
continuous screening of patients undergoing therapy. "For example, we could monitor for early 
resistance to a drug by monitoring the levels of cancer-associated mutations in blood, and then 
rapidly switch the patient to a new drug before their cancer progresses significantly," Dr. Schmitt 
said. However, there is more good news: the extreme accuracy of the technique allows sequencing 
of sample sizes as small as a single cell. The new technology could therefore be applied in settings 
where only tiny amounts of DNA are available, yet accuracy is critical, such as forensics and studies 
of extinct organisms (paleogenomics). 
Still, the dinosaurs will have to wait. The investigators are currently using the technique for exploring 
drug resistance mutations in blood cancers, and assessing genetic variability within various human 
cancer types. Methods are also under development for identification of occasional cancer cells that 
may persist after conventional treatment. To avoid relapse, patients who have those cells in their 
blood might need further aggressive treatment, whereas those who are free of circulating tumor cells 
could be treated less aggressively, with lower risks of negative side effects. 
The take-home message is clear: finding a needle in a haystack just got a million times easier. 
 
 
Schmitt MW, Fox EJ, Prindle MJ, Reid-Bayliss KS, True LD, Radich JP, Loeb LA. 2015. Sequencing 
small genomic targets with high efficiency and extreme accuracy. Nat Methods. 12(5):423-5. 
See also: Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB, Loeb LA. 2012. Detection of ultra-
rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A. 109(36):14508-13. 
 
 
 
 
 
 
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
 
 
 
Image provided by Dr. Michael Schmitt 
Duplex sequencing (lower panel) efficiently removed background 
errors, revealing a single point mutation that could not be discerned 
using conventional high-throughput sequencing (upper panel). 
 


A Ground-Breaking Method for Distinguishing Needles from Hay

Neves, Alexandre

June 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
GDNF as Collateral Damage of Prostate Cancer 
Chemotherapy 
June 15, 2015 
     A Neves 
The majority of prostate cancer deaths result from metastatic disease, which tends to initially 
respond to well to chemotherapeutic agents that damage DNA yet prostate cancers often acquire 
resistance. It is therefore important to understand the effects of DNA damaging agents in not only 
the prostate cancer cells themselves, but also in the benign cell types that surround the tumor, which 
are collectively referred to as the tumor microenvironment. Previous work in Dr. Pete Nelson’s 
Laboratory (Human Biology and Clinical Research Divisions) tested the hypothesis that treatment-
induced DNA damage in benign cells promote therapy resistance and tumor progression. This work 
identified over 40 secreted proteins that are induced in benign fibroblasts in response to 
chemotherapy with potential of affecting prostate cancer cells. The proteins collectively form the 
DNA damage secretory program (DDSP; Sun et al., 2012). 
A new Fred Hutch study led by former postdoctoral fellow Dr. Roland Huber in Dr. Nelson’s lab and 
published in Oncotarget addressed the functions one such protein, GDNF, in both fibroblasts and 
prostate cancer cells. GDNF, Glial Cell-derived Neurotrophic Factor, was first identified as a growth 
factor for neurons but is now known to promote proliferation of other cell types"I think the most 
important aspect of our work is that we learn how a tumor reacts to a treatment. Initial responses are 
often good in prostate cancer, but resistance and treatment failure are very common. So by 
understanding what happens during and after treatment will help us understand these active (non 
pre-existing) resistance mechanisms much better and could, mid to long term, lead to improved 
combination therapies," said Dr. Huber. 
The study began by confirming that both the expression and secretion of GDNF were elevated in 
prostate fibroblasts (PSC27) in response to a wide range of DNA-damaging agents (bleomycin, 
mitoxantrone, radiation and docetaxel). Importantly, GDNF expression was also elevated in cancer-
associated fibroblasts micro-dissected from men undergoing a clinical trial that combined 
mitoxantrone with docetaxel. Next, the authors examined the effects of GDNF in prostate fibroblasts 
which showed that GDNF exposure results both in increased stimulation of downstream protein 
kinases, such as SRC and ERK, and increased replicative potential. Curiously, fibroblasts from the  
 
June 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
 
bone marrow also secreted GDNF but did not exhibit the same downstream responses. Because 
GDNF is a secreted protein, the investigators tested the effect of GDNF exposure in a range of 
different prostate cancer cell lines, and found that the subset of cell lines that exhibited increased 
proliferation and invasive behavior also expressed GFRA1, a GDNF receptor. Strikingly, prostate 
cancer cells that were stimulated with GDNF were found to be resistant to DNA-damaging agents. 
To elucidate the gene expression program downstream of GDNF, the authors performed whole-
genome microarrays in prostate cancer cells and fibroblasts exposed to GDNF. This analysis 
revealed that GDNF stimulation was associated with reduced activity of the retinoblastoma tumor 
suppressor with concomitant increases in the activities of the transcriptional regulators E2F1 and 
androgen receptor (AR). "As previous studies have shown that E2F1 promotes AR activity, GDNF 
may provide a link for interactions between the tumor microenvironment and maintenance of the AR 
program, a crucial driver of prostate cancer growth, "said Dr. Nelson. "GDNF however is only one of 
probably hundreds of factors, so we do have a lot of work ahead of us to understand the collective 
effects of all these factors. I hope, that in the long run this will help to improve treatment success," 
said Dr. Huber. In summary, while it’s tempting to pursue therapeutics that inhibit GDNF, perhaps a 
more viable long-term strategy would be to target the DDSP more broadly in order to limit the 
treatment-induced damage to the tumor microenvironment. 
 
Huber RM, Lucas JM, Gomez-Sarosi LA, Coleman I, Zhao S, Coleman R, Nelson PS. 2014. DNA 
damage induces GDNF secretion in the tumor microenvironment with paracrine effects promoting 
prostate cancer treatment resistance. Oncotarget 6(4): 2134-47. 
See also: 
Sun Y, Campisi J, Higano C, Beer TM, Porter P, Coleman I, True L, Nelson PS. 2012. Treatment-
Induced Damage to the Tumor Microenvironment Promotes Prostate Cancer Therapy Resistance 
Through WNT16B. Nat Med 18(9):1359-68. 
 
 
 
 
June 15, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image by Dr. Alexandre Neves 
Prostate cancer chemotherapy induces DNA damage in both prostate cancer cells (red; 
PCC) and benign prostate fibroblasts (blue; BPF). BPF respond to DNA damage by 
secreting the Glial-cell Derived Neurotrophic factor (GDNF). GDNF, in turn, stimulates 
proliferation of both BPF and PCC and inhibits the tumor suppressor retinoblastoma 
protein (RB), while activating the oncogenic transcription factors E2F1 and androgen 
receptor (AR) in PCC. AR, activated by E2F1, promotes both survival and growth of 
PCC. 
 


GDNF as Collateral Damage of Prostate Cancer Chemotherapy

July 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
CGAT: a Vital Instrument in the Prognostic 
Toolbox for AML 
July 20, 2015 
     SHL Frost 
 
Kaplan-Meier curves showing the 
prognostic impact of cnLOH. Blue lines 
in A (% relapse after CR) and C (% 
survival) represent cnLOH; black lines 
represent no cnLOH. In B (% relapse 
after CR) and D (% survival), blue and 
black lines represent 13q cnLOH and 
no 13q cnLOH, respectively. 
Image provided by Christine Gronseth 
 
 
Normally, human cells contain two copies of each chromosome; one each from the mother and the 
father. In some cases, whole chromosomes or segments of chromosomes may appear to be from 
only one parent; although the copy numbers remain normal, the genetic variability is lost. This is 
called copy-neutral loss of heterozygosity (cnLOH), and can be identified using chromosome 
genomic array testing (CGAT). Detection of chromosomal DNA irregularities using genomic 
microarray techniques is generally important for diagnosis and prognosis of patients with acute 
myeloid leukemia (AML), but the World Health Organization does not currently include cnLOH 
among its criteria for classifying tumors of hematopoietic and lymphoid tissue. To elucidate its 
prognostic significance, Dr. Min Fang from Fred Hutch's Clinical Transplant Research Program 
(Clinical Research Division) initiated a study to correlate acquired clonal cnLOH in AML patients - 
which relatively often display the abnormality - with various covariates, mutations, risk classification, 
and outcome after treatment. The results were recently published in Cancer, with cytogenetics 
technologist Christine Gronseth at the Seattle Cancer Care Alliance (SCCA) as first author. 
July 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
A total of 112 AML patients who underwent chromosome genomic array testing (CGAT) at the SCCA 
were included in the study. DNA was taken from blood or bone marrow samples and analyzed using 
a high-density microarray, including detection of single-nucleotide polymorphisms. Of the 112 
patients, 40 had cnLOH; the remaining 72 comprised the control group. In the cnLOH cohort, 33 of 
40 had cnLOH involving a single chromosome arm; cnLOH involving 2, 3, and 4 chromosome arms 
were found in 5, 1, and 1 patient(s), respectively. The most common sites of cnLOH were at 11p and 
13q in this patient population. 
Overall, cnLOH did not significantly impact the clinical remission rate (60 % in patients with cnLOH, 
and 74 % in patients without; p = 0.14). On the other hand, presence of the abnormality was clearly 
correlated with disease recurrence (1.87-fold higher in patients with cnLOH; p = 0.02), with a striking 
6.64-fold higher relapse rate in patients with cnLOH in chromosome 13q (p = 0.002), also after 
adjustment for age, cytogenetics, and secondary versus de novo (without association to previous 
therapy) AML. The risk of death was consequently increased by a factor of 3.45 (p = 0.05) in this 
population. Further, the CGAT findings were correlated with results from molecular testing for gene 
mutations. Fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation data was 
available for 78 of the 112 patients, and the mutation was clearly associated with cnLOH in 13q. Six 
patients with 13q cnLOH were tested for FLT3; all of them were positive for FLT3-ITD. Patients with 
FLT3-ITD mutation with 13q cnLOH had shorter duration of remission and shorter overall survival 
than patients with the FLT3 mutation without 13q cnLOH (p < 0.01). 
The study is the first to demonstrate an association between a complex CGAT result (at least three 
CGAT abnormalities) and poor outcome, analogous to that of a complex karyotype (at least three 
clonal abnormalities) as determined using conventional cytogenetics. Importantly, 13 % of the 
patients had abnormalities detected by CGAT that would not have been found using standard 
techniques only. "CnLOH cannot be detected by conventional methods," Dr. Fang said. "Some of 
our providers have not yet fully embraced the usefulness of CGAT and are unsure about whether 
CGAT-identified abnormalities are equally significant as those detected by conventional cytogenetics 
and FISH," she continued, emphasizing her hope that this paper will highlight these concerns. 
According to the authors, CGAT may be most useful for AML patients with an intermediate 
cytogenetic risk, from the perspective of clinical practice. The risk is enriched in patients with normal 
cytogenetics but abnormal CGAT - a patient population with particularly uncertain prognosis, for 
which the benefits of allogeneic hematopoietic cell transplantation are difficult to predict, in relation to 
the treatment-associated risks. Identifying prognostic factors for this patient population is one of the 
future goals for the investigators, Christine Gronseth explained. 
July 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
Thus, the investigators found their answers: cnLOH has, indeed, prognostic significance in AML 
patients, and CGAT is useful for improving diagnosis and care. "Establishing the clinical utility of 
acquired clonal cnLOH in AML patients is an important key to personalized medicine," Gronseth 
said. "The detection of cnLOH is helpful not only to establish the patients' prognosis, but also to 
follow the disease status and progression," she added. 
Patients with cnLOH were few in the current study, and the prognostic effect of aberrations in 
individual chromosomes was therefore not easily quantifiable. The researchers are keen on 
continuing the investigations, and Gronseth staked out the future direction: "We have already begun 
analysis of cnLOH on specific chromosome arms and hope to conduct a multi-institutional 
collaboration to increase the study size." 
Gronseth CM, McElhone SE, Storer BE, Kroeger KA, Sandhu V, Fero ML, Appelbaum FR, Estey 
EH, Fang M. 2015. Prognostic significance of acquired copy-neutral loss of heterozygosity in acute 
myeloid leukemia. Cancer. DOI: 10.1002/cncr.29475. [Epub ahead of print] 
 


CGAT: a Vital Instrument in the Prognostic Toolbox for AML

August 17, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
HIV-1 superinfection results in broad polyclonal 
neutralizing antibodies 
Aug. 17, 2015 
     A Neves 
None of the four main epitopes on the HIV-1 envelope protein (CD4 binding site, N160 glycan in V1V2, N332 glycan 
in V3, and MPER; highlighted in warm colors and bold font), nor four additional sites (N276 glycan in C2, N611 glycan 
in C-C loop, N88 glycan in C1, K169 in V1V2; highlighted in blue colors and small font), are strongly targeted by 
neutralizing antibody responses in HIV-1 superinfected individuals from a Kenyan cohort. while the 4 main epitopes 
have been shown to be the dominant targets of broad and potent neutralizing antibody responses in individuals with a 
single infection from other cohorts, the absence of such a response in HIV-1 superinfected individuals suggests that 
their neutralizing antibody responses may be collectively mediated by diverse, polyclonal antibodies. 
 
Image provided by Dr. Valerie Cortez 
 
Development of a human immunodeficiency virus-1 (HIV-1) vaccine that elicits neutralizing 
antibodies (Nabs) that are both potent and broad remains a major public health goal. However, one 
of the main challenges of HIV-1 vaccine design is the diversity of HIV-1 subtypes that are currently 
circulating worldwide. One scenario that holds great potential to inform future vaccine design is HIV-
1 superinfection (SI), defined as one person receiving sequential infections from different source 
partners. Previous studies in Dr. Julie Overbaugh’s Laboratory (Human Biology Division) showed 
that a cohort of 12 superinfected women from Mombasa (Kenya) developed broader and more 
potent Nab responses than singly infected individuals (Cortez et al., 2012). However, it remains 
unclear whether SI and singly infected individuals differed in other ways, such as the specificity of 
the antibody response. A new Fred Hutch follow-up study by the Overbaugh lab, led by former 
graduate student Dr. Valerie Cortez and published in PLOS Pathogens, addressed this question by 
August 17, 2015 SCIENCE SPOTLIGHT 
 
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mapping the antibody response of 21 superinfected women onto epitopes (parts of proteins or other 
antigens that bind antibodies) of the HIV-1 Envelope protein.  
Previous studies on singly infected individuals with broad Nabs mapped the bulk of their neutralizing 
activity onto four major epitopes on the HIV-1 Envelope protein: the membrane proximal external 
region (MPER), the CD4 binding site, residues in the V1/V2 region, and the V3 loop (see figure). The 
study began by assaying post-SI plasma from all 21 women. MPER-specific antibodies were 
infrequently detected, and such antibodies appeared to arise over a year following SI. Next, the 
authors tested for CD4-binding site-specific antibodies in post-SI plasma by comparing binding with 
an Envelope core protein engineered to prominently display the CD4-binding site versus a mutant 
protein that harbored a deletion that abrogates the binding of CD4-specific antibodies. One out of the 
21 women had antibodies that bound the wild-type Envelope more strongly than the mutant, but this 
individual’s CD4-binding site-specific antibodies did not demonstrate neutralizing activity. Nabs that 
target either the V1/V2 region or the V3 loop require a glycan (a sugar moiety on a protein) at 
specific positions and comparisons of wild-type versus glycan deficient Envelopes failed to provide 
strong evidence for glycan-specific Nabs. 
 Beyond these four epitopes, several additional epitopes have been linked to newly characterized 
Nabs. To investigate further, the authors also engineered point mutations in these novel epitopes. 
Again, the antibodies targeted to the newly discovered epitopes did not appear to be major drivers of 
the SI response. Finally, the researchers employed computational analyses to corroborate the 
functional data, which confirmed that none of the 21 women developed a Nab response that could 
be ascribed with high confidence to a single known epitope. "Since none of neutralizing antibody 
responses from the 21 superinfected individuals strongly targeted any of the epitopes tested in our 
study, this may suggest that HIV-1 superinfection leads to a broad, polyclonal response that 
collectively mediates their ability to neutralize diverse HIV-1 envelopes Continual study of the virus-
antibody interplay in HIV-1 superinfected individuals may yield insights to how we can design 
sequential immunizations that elicit a diverse, polyclonal antibody response capable of recognizing 
globally diverse HIV-1 subtypes," said Dr. Cortez. In conclusion, a combination of functional and 
computational analyses enabled the authors to characterize the epitope targets of Nabs linked to SI 
for the first time.  
Cortez V, Wang B, Dingens A, Chen MM, Ronen K, Georgiev IS, McClelland RS Overbaugh J. 
2015. The broad neutralizing antibody responses after HIV-1 superinfection are not dominated by 
antibodies directed to epitopes common in single infection. PLoS Pathog, 11(7), e1004973. 
 
August 17, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
See also:  
Cortez V, Odem-Davis K, McClelland RS, Jaoko W, Overbaugh J. 2012. PLoS Pathog, 
8(3):e1002611. HIV-1 superinfection in women broadens and strengthens the neutralizing antibody 
response. 
Funding for this study was provided by the NIAID. 
 


HIV-1 superinfection results in broad polyclonal neutralizing antibodies

September 21, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
An improved method to detect low levels of 
methylated DNA 
September 21, 2015 
     SHL Frost 
 
Detection of decreasing amount of methylated 
NTRK3 in serial dilutions of methylated and 
unmethylated DNA standards containing 100%, 
20%, 4%, 0.8%, 0.16%, 0.032%, 0.006% and 0% of 
methylated DNA (20 ng DNA/sample), as shown by 
ddPCR analysis. Each sample was partitioned into 
an average of 15,000 droplets per well and 
replicated in 4 wells. The droplet counts (positive 
and negative) from all 4 replicates were combined 
for the final analysis. An event with fluorescence 
amplitude value >4,500 was considered a 
methylation-positive event (red line). The number of 
methylation-positive events decreased as the 
methylated DNA was progressively diluted with 
unmethylated DNA. Using ddPCR, it was feasible to 
detect and absolutely quantify as low as 0.032% of 
methylated NTRK3 
 
Image provided by Dr. Ming Yu 
 
The addition of methyl groups to DNA is a process that plays an important role in development and 
in certain diseases, through altering gene expression in cells. Abnormal DNA methylation patterns - 
either higher or lower levels compared with normal tissue - are associated with many human 
malignancies, including colorectal cancer. Interestingly, these alterations have been detected not 
only in colon cancer and colon polyps, the precursors to colorectal cancer, but also in the normal 
colon tissue in individuals with concurrent colorectal cancer, who are at high risk of colon polyps and 
metachronous (not synchronous) colorectal cancer. Thus, there is potential for methylated genes to 
be used as biomarkers for early detection of colorectal cancer. There is a problem, however: the 
aberrations are present at very low levels, and cannot be easily detected using current polymerase 
chain reaction (PCR) technologies. Dr. Ming Yu in the laboratory of Dr. William Grady in Fred 
Hutch's Clinical Research Division accepted this challenge, and set out to develop a more precise 
and sensitive method for detecting methylated DNA. In the September issue of Epigenetics, Drs. Yu, 
Grady, and colleagues describe their efforts to develop this method and their resulting conclusions. 
September 21, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
The investigators based their innovation on a conventional methylation-specific PCR method called 
MethyLight, but added the benefits of droplet digital PCR (ddPCR) - a new technology that enables 
precise detection of nucleic acid targets in clinical samples. Using colon cancer tissue samples and 
colon mucosa biopsy samples, the technique was tested for detecting infrequently methylated alleles 
- an application of ddPCR that had not been previously published. "In our study, we developed a 
ddPCR version of MethyLight assays and demonstrated that the resulting MethyLight ddPCR offered 
improvements in sensitivity with a much lower limit of detection, improved absolute quantification of 
methylated alleles, as well as increased precision in detecting infrequently methylated targets," Dr. 
Yu said. 
The investigators used the BioRad QX200 ddPCR system, which partitions each 20-µL PCR 
reaction into an average of 15,000 nanoliter droplets through water-oil emulsion. Essentially, this 
enables 15,000 simultaneous independent PCR reactions, with each droplet PCR-cycled to endpoint 
and read with a two-color fluorescence reader. Thus, it is possible to determine how many droplets 
are positive. Compared with conventional MethyLight PCR, the respective limits of quantification and 
detection were 25 and 20 times lower with MethyLight ddPCR, as determined through comparative 
analysis of methylated EVL and NTRK3 genes. Where the conventional technique was able to 
detect 1 methylated NTRK3 allele in a background of 125 unmethylated alleles, the new method 
detected 1 out of 3125. Moreover, low levels of EVL methylation were not detected in either of nine 
samples of normal colon mucosa from people with colorectal cancer using conventional MethyLight 
PCR, but MethyLight ddPCR selectively detected and absolutely quantified methylated EVL in all 
nine samples. 
The detection of infrequent methylation events in various clinical specimens is crucial, both for 
identifying cancer-associated epigenetic alterations that may be risk biomarkers, and for the early 
detection of colorectal cancer using blood or stool samples. "The ultimate goal for implementation of 
a sensitive detection tool like ddPCR Methylight is to determine its ability to detect methylated DNA 
in body fluid samples like blood, urine, or stool and to study normal tissue for evidence of field 
cancerization," explained Dr. Yu, who believes that ddPCR MethyLight is well suited to address 
many of the technical challenges associated with these applications, such as contaminating 
background DNA or the presence of various DNA polymerase inhibitors. 
With this study, the investigators have demonstrated proof of principle of the technique, which is 
currently being validated through further assessment of other risk biomarker candidates. Dr Yu is 
optimistic: "It will improve the sensitivity and precision for detecting infrequently methylated genes 
and will allow us to conduct studies to determine if methylated genes can be used as colon cancer 
September 21, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
risk biomarkers." In the long run, these biomarkers could facilitate and improve the effectiveness of 
personalized cancer prevention programs, hopefully decreasing the mortality of colorectal cancer - 
currently the second leading cause of cancer-related death in the United States. 
Yu M, Carter KT, Makar KW, Vickers K, Ulrich CM, Schoen RE, Brenner D, Markowitz SD, Grady 
WM. 2015. MethyLight droplet digital PCR for detection and absolute quantification of infrequently 
methylated alleles. Epigenetics. 10(9):803-9. 
The study was supported by the National Institutes of Health, a Burroughs Wellcome Fund 
Translational Research Award for Clinician Scientist, the Lattner Foundation, and R.A.C.E. Charities. 
 
 
 


An improved method to detect low levels of methylated DNA

September 21, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
The human microbiome in Barrett’s esophagus 
is hard to stomach 
September 21, 2015 
     A Neves 
 
 
 
(A) Diagram of the human upper 
gastrointestinal tract indicating regions 
sampled in the study. (B) Principal 
component analysis of phylogenetic 
similarity among samples collected from 
each participant at the four upper GI tract 
sites. Kaplan-Meier curves for participants 
within the cohort for (C) cancer incidence 
(n = 433, 39 infected) and (D) aneuploidy 
as measured by dna content flow 
cytometry (n = 337, 33 infected). 
 
Image provided by Ms. Gall 
 
It is now well known that in our bodies, microbial cells outnumber human cells 10 to 1. Furthermore, 
it has become increasingly clear that these microbial communities (collectively known as the human 
microbiome) have key roles in health and disease. For example, the bacterium Helicobacter pylori is 
well-known to cause gastric ulcers and stomach cancer in a subset of infected individuals. However, 
for unknown reasons, H. pylori infection is markedly lower in patients with esophageal 
adenocarcinoma (EAC). Important questions remain on the diversity of microbes present in the 
esophagus and stomach and how this diversity may be linked to H. pylori’s protective role in EAC. 
Such questions have remained unanswered in part, due to challenges associated with sampling the 
upper gastrointestinal (GI) tract. A recent Fred Hutch study from Dr. Nina Salama’s Laboratory 
(Human Biology and Public Health Sciences Divisions) in collaboration with Dr. Brian Reid’s 
Laboratory (Human Biology Division), led by graduate student Tina Gall and published in PLoS 
September 21, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
ONE, sought to characterize the human microbiome in the upper GI tract of a subset of individuals 
from the Seattle Barrett’s Esophagus Research Program (SBERP). 
"Our study was made possible by the great collaborative environment here at the Hutch. We relied 
on the expertise of Dr. Brian Reid, a leader in the Barrett’s Esophagus field, and utilized endoscopy 
samples that his group collected as part of the SBERP. Dr. Erick Matsen and his group in 
Computational Biology were instrumental in helping us analyze our complex genomic data. This 
allowed us to ask some fundamental questions like what is the best way to sample bacteria in the 
stomach and esophagus and do these distinct anatomical sites host the same or different bacteria? " 
said Ms. Gall. 
The study began by collecting four distinct samples from 12 SBERP participants. These were 
Barrett’s esophagus, normal esophagus, stomach antrum and stomach corpus (see figure). The 
authors found that mucosal brush sampling was the best method to enrich for bacterial diversity. To 
chart the microbial diversity in the sample, the authors amplified and sequenced 16S rRNA, a 
bacteria-specific nucleic acid. Principal component analysis of the sequencing data revealed that 
bacteria from the same study participant were more similar to each other than to samples from the 
same anatomical site in a different individual. In addition, the investigators found that bacteria 
belonging to the Streptococcus and Prevotella genera dominated the microbiome, both within study 
participants and across anatomical sites. Intriguingly, the ratio of Streptococcus to Prevotella varied 
among individuals and this ratio could be linked to Barrett’s esophagus risk factors, such as waist-to-
hip ratio. Finally, the authors examined H. pylori status in a larger SBERP cohort (n=433) and found 
that H. pylori infected individuals had decreased genomic instability, an important predictor of 
progression from Barrett’s esophagus to EAC. Said Ms. Gall " We hope to focus future studies on 
analyzing the microbial composition in the upper GI tract of H. pylori positive and negative 
individuals to determine whether the presence of H. pylori in the stomach and the esophagus 
influences colonization of these sites by specific bacteria that may be carcinogenic and promote the 
observed DNA damage". In conclusion, this study developed a new method for sampling the upper 
GI tract, which enabled the characterization of the microbial community in both the stomach and 
esophagus as well as provided an intriguing link between H. pylori status and genomic instability. 
Gall A, Fero J, McCoy C, Claywell BC, Sanchez CA, Blount PL, Li X, Vaughan TL, Matsen FA, Reid 
BJ, Salama NR. 2015. Bacterial composition of the human upper gastrointestinal tract microbiome is 
dynamic and associated with genomic instability in a Barrett's Esophagus cohort. PLoS One, 10(6), 
e0129055. 


The human microbiome in Barrett’s esophagus is hard to stomach

Pattacini, Laura

November 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
Timing it right 
November 15, 2015 
     L Pattacini 
 
 
 
 
Model for the proposed mechanism 
leading to establishment of ART 
resistant virus in post-transplant group. 
 
Image provided by Dr. Christopher 
Peterson. 
 
 
 
 
Last month’s spotlight edition featured a publication supporting safety and efficacy of maintaining 
regimens of combination antiretroviral therapy (ART) in subjects undergoing bone marrow 
transplantation. This month, a paper published by Fred Hutch scientists explains why it is important 
to maintain ART in the period around transplantation. There are multiple reasons to focus on this 
topic. First, recent improvements mean that HIV infection can be controlled by antiretroviral drugs; 
therefore, infected subjects are more likely to live longer and face additional health issues that may 
be related to HIV infection, antiretroviral therapy, or non-HIV related causes. Second, the only 
person who has so far been declared free from HIV was cured as a result of bone marrow 
transplantation. The marrow donor in this case was resistant to HIV infection, due to a genetic 
mutation of the HIV co-receptor. In the future, more cases similar to this might present; therefore, it 
is important to know when the therapy should be interrupted to evaluate whether the subject can 
control viral replication in the absence of therapy. A paper published this month on AIDS by Dr. 
Christopher Peterson, a staff scientist in Dr. Hans-Peter Kiem's Lab at Fred Hutch, shows the 
importance of carefully evaluating when ART can be interrupted after transplantation.  
The work utilizes the simian/human immunodeficiency virus (SHIV) model of HIV infection to 
understand the effects of starting antiretroviral treatment after bone marrow transplantation. Three 
November 15, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 11 | Fred Hutchinson Cancer Research Center 
 
groups were compared in the study: one that received a SHIV challenge but no transplantation, a 
second that received a SHIV challenge before bone marrow transplantation and the third that 
received a SHIV challenge after bone marrow transplantation. ART was initiated immediately after 
the SHIV challenge to control viremia and was found effective in reducing the viral load in the first 
two groups, but not in the third. In this group, the presence of resistance-linked mutations, especially 
in the pol gene, was detected by deep-sequencing analyses. To explain the reason of the elevated 
frequency of ART-resistance mutations, the researchers evaluated three possible mechanisms. 
Neither reduced bioavailability of the drugs due to intestinal damage caused by the myeloablative 
regimen, nor the prevalent infection of myeloid subsets that could serve as drug sanctuaries were 
affected by the myeloablative conditioning followed by the viral challenge. Therefore, the 
researchers explored the possibility that the depletion of CD4 and CD8+ T cells due to myeloablative 
and immune suppressive therapy allowed for the establishment of ART-resistant mutants. Indeed, 
the third group, where SHIV challenge followed transplantation, was characterized by depletion of 
naïve CD4+ and CD8+ T cells in the blood, while less marked differences were noted for memory T 
cells. Naive T cells rebound after ART initiation, but their expansion was not followed by a viral load 
decrease. Finally, the group was characterized by a low production of antibodies to SHIV. 
The clinical relevance of this study is summarized in the comment from Dr. Peterson: "In order to 
know whether or not a patient has been cured of HIV infection in future clinical trials, we will need to 
briefly take them off of their antiretroviral treatment to confirm that the virus does not come back; this 
is known as an analytical treatment interruption’. Understanding when to do this is particularly 
important in our work with patients who undergo stem cell transplantation and are 
immunosuppressed. Our study shows that these analytical treatment interruptions should not occur 
until the patient’s immune system has completely recovered from the transplant procedure or 
immunosuppressive therapy". 
The research was supported by grants from the National Institutes of Health. 
Peterson CW, Haworth KG, Polacino P, Huang M-L, Sykes C, Obenza WM, Repetto AC, Kashuba 
A, Bumgarner R, DeRosa SC, Woolfrey AE, Jerome KR, Mullins JI, Hu S-L, Kiem H-P. 2015. Lack of 
viral control and development of combination antiretroviral therapy escape mutations in macaques 
after bone marrow transplantation. AIDS. 29(13):1597-606.  
 
 


Timing it right

December 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
4EBP1 allows luminal prostate cancer cells to fly 
under the radar 
December 20, 2015 
     A Neves 
 
 
 
PTEN L/L prostate luminal epithelial cells can form tumors which are 
insensitive to inhibition of EIF4E activity in vivo. Representative 
immunofluorescence images of the anterior (AP), dorsal (DP), and 
ventral (VP) prostate in PTEN L/L prostate cancer mice with and without 
induction of the 4EBP1M transgene which inhibits eIF4E activity in a 
tissue specific manner in vivo. Basal epithelial cells (green, CK5+) are 
sensitive to inhibition of eIF4E activity, while luminal epithelial cells (red, 
CK8+) are resistant. 
 
Image provided by Dr. Andrew Hsieh. 
 
 
 
 
Whole-genome sequencing of various cancers has heralded a new era of targeted therapies and 
provides the tantalizing prospect of personalized medicine. These sequencing studies revealed that 
the PI3K-AKT-mTOR pathway is commonly altered in cancer and thus major efforts have been 
devoted to develop and test PI3K-AKT-mTOR inhibitors. Most, if not all, advanced prostate cancers 
harbor alterations in this pathway. While PI3K-AKT-mTOR pathway inhibitors have shown promise in 
preclinical studies, drug resistance often develops. However, it remains unknown if all types of 
epithelial cells present in normal or cancerous prostate glands are equally sensitive to such 
inhibitors. A new Fred Hutch study led by Dr. Andrew Hsieh (Human Biology and Clinical Research 
Divisions) leveraged a combination of a new prostate cancer mouse model, a clinical trial and 
detailed molecular studies to reveal that the protein synthesis inhibitor 4EBP1 is critically involved in 
prostate cancer initiation, progression and drug resistance. This study was recently published in the 
journal Science Signaling. 
December 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
The study began with an intriguing observation from a previous preclinical trial of an ATP-site mTOR 
inhibitor (MLN0128) in mice that develop prostate cancer due to prostate-specific deletion of the 
tumor suppressor PTEN (PTEN
L/L
 mice). The prostate gland is composed of two distinct epithelial 
cell types: luminal cells (CK8
+
) and basal cells (CK5
+
). Dr. Hsieh and his collaborators at UCSF 
observed tumors that resisted an 8-week course of treatment with MLN0128 were enriched in CK8
+
 
luminal cells, relative to CK5
+
 basal cells. To determine the molecular basis for this differential 
sensitivity to mTOR inhibitors, the authors queried the protein abundance of key components of the 
protein synthesis machinery, including eIF4A, eIF4E, eIF4G and 4EBP1. Western blotting of 
fluorescence activated cell sorting (FACS)-sorted basal or luminal cells obtained from PTEN
L/L
 mice 
showed that 4EBP1, an inhibitor of cap-dependent translation, was the only component of this 
machinery analyzed found to be enriched in both in normal and PTEN
L/L
 luminal cells compared to 
basal cells. Importantly, this increased 4EBP1 expression was likely functional as luminal cells 
exhibited the lowest rates or protein synthesis, as demonstrated by 
35
S methionine incorporation 
assays on FACS-sorted cells. 
Because 4EBP1 functions primarily by inhibiting the mTOR-regulated oncogene eIF4E, the authors 
sought to determine the role of 4EBP1 in vivo. To this end, they developed a novel genetic mouse 
model, wherein a doxycycline-inducible expression of a mTOR-insensitive mutant (4EBP1M) was 
combined with the PTEN
L/L
 model. Said Dr. Hsieh: "Historically it has been difficult to inhibit protein 
synthesis in a tissue specific inducible manner. We overcame this hurdle by developing a new 
prostate cancer mouse model called the PTEN
L/L
; 4EBP1M mouse. Here, PTEN loss leads to 
prostate cancer, and the addition of doxycycline to the drinking water induces a transgene, which 
can inhibit protein synthesis in vivo. Using this model we demonstrate that eIF4E mediated 
translation is essential for prostate cancer initiation and progression." Expression of 4EBP1M 
markedly decreased both the development and maintenance of prostate cancer, concomitant with an 
increase in apoptosis. Strikingly, 4EBP1M expression led to an enrichment of luminal cells, 
suggesting basal cells, but not luminal cells, require eIF4E for growth and survival. These findings 
along with in vitro molecular studies provide a mechanistic rationale for why tumor prone luminal 
epithelial cells are uniquely resistant to PI3K pathway inhibitors. 
Finally, the authors sought to determine whether increased 4EBP1 abundance also occurs in 
prostate cancer patients. Comparison of pre-treatment with post-treatment prostate cancer tissue 
from an ongoing clinical trial with a PI3K inhibitor (BKM120) revealed an enrichment of CK8
+
 luminal 
cells that expressed high levels of 4EBP1. In conclusion, this study showed that cell type-specific 
expression of the tumor suppressor 4EBP1 in luminal prostate cancer cells primes cells for either 
sensitivity or resistance to PI3K-AKT-mTOR inhibitors and may have utility in predicting drug 
December 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 12 | Fred Hutchinson Cancer Research Center 
 
responses in patients. "Our laboratory has discovered that protein synthesis rates of specific 
prostate epithelial cell types can tune them for resistance to an emerging line of PI3K pathway 
inhibitors," said Hsieh. Reflecting on the meaning of these findings, Hsieh said, "Many questions 
remain. If low protein synthesis leads to drug resistance, will more potent inhibitors of protein 
synthesis lead to drug sensitivity? What are the downstream translationally regulated transcripts that 
direct the therapeutic response and drug resistance?" 
Hsieh AC, Nguyen HG, Wen L, Edlind MP, Carroll PR, Kim W, Ruggero D. 2015. Cell type-specific 
abundance of 4EBP1 primes prostate cancer sensitivity or resistance to PI3K pathway inhibitors. Sci 
Signal. 8(403):ra116. doi: 10.1126/scisignal.aad5111. 
Funding for this work was provided by the National Institutes of Health, the Prostate Cancer 
Foundation, the V Foundation for Cancer Research and the Burroughs Wellcome Fund. 
 


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