The KRAS-Variant and miRNA Expression in RTOG Endometrial Cancer Clinical Trials 9708 and 9905
The KRAS-variant may be a genetic marker of risk for type 2 endometrial cancers. In addition, tumor miRNA expression appears to be associated with patient age, lymphovascular invasion and the KRAS-variant, supporting the hypothesis that altered tumor biology can be measured by miRNA expression, and that the KRAS-variant likely impacts endometrial tumor biology.
Published on: Mar 4, 2016
Transcripts - The KRAS-Variant and miRNA Expression in RTOG Endometrial Cancer Clinical Trials 9708 and 9905
The KRAS-Variant and miRNA Expression in RTOG
Endometrial Cancer Clinical Trials 9708 and 9905
Larissa J. Lee1., Elena Ratner2., Mohamed Uduman3, Kathryn Winter4, Marta Boeke2,
Kathryn M. Greven5, Stephanie King6, Thomas W. Burke7, Kelly Underhill8, Harold Kim9,
Raleigh J. Boulware10, Herbert Yu12,13, Vinita Parkash16, Lingeng Lu12, David Gaffney15, AdamP. Dicker11,
1 Department of Radiation Oncology, Brigham and Women’s Hospital/Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America, 2 Department of
Gynecologic Oncology, Yale University, New Haven, Connecticut, United States of America, 3 Interdepartmental Program in Computational Biology and Bioinformatics,
Yale University, New Haven, Connecticut, United States of America, 4 Statistical Center, Radiation Therapy Oncology Group (RTOG), Philadelphia, Pennsylvania, United
States of America, 5 Department of Radiation Oncology, Wake Forest University Baptist Medical Center, Winston-Salem, North Carolina, United States of America,
6 Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America, 7 Department of Gynecologic Oncology and
Reproductive Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 8 Department of Radiation Oncology, Benefis Sletten
Cancer Institute, Great Falls, Montana, United States of America, 9 Department of Radiation Oncology, Wayne State University Karamanos Cancer Center, Detroit,
Michigan, United States of America, 10 Radiation Oncology, South Carolina Oncology Associates, Columbia, South Carolina, United States of America, 11 Department of
Radiation Oncology, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, United States of America, 12 Department of Chronic Disease and Epidemiology,
Yale University, New Haven, Connecticut, United States of America, 13 Cancer Epidemiology Program, University of Hawaii Cancer Center, Honolulu, Hawaii, United States
of America, 14 Department of Therapeutic Radiology, Yale University, New Haven, Connecticut, United States of America, 15 Department of Radiation Oncology,
University of Utah Huntsman Cancer Hospital, Salt Lake City, Utah, United States of America, 16 Department of Pathology, Yale University, New Haven, Connecticut,
United States of America
Objective: To explore the association of a functional germline variant in the 39-UTR of KRAS with endometrial cancer risk, as
well as the association of microRNA (miRNA) signatures and the KRAS-variant with clinical characteristics and survival
outcomes in two prospective RTOG endometrial cancer trials.
Methods/Materials: The association of the KRAS-variant with endometrial cancer risk was evaluated by case-control analysis
of 467 women with type 1 or 2 endometrial cancer and 582 age-matched controls. miRNA and DNA were isolated for
expression profiling and genotyping from tumor specimens of 46 women with type 1 endometrial cancer enrolled in RTOG
trials 9708 and 9905. miRNA expression levels and KRAS-variant genotype were correlated with patient and tumor
characteristics, and survival outcomes were evaluated by variant allele type.
Results: The KRAS-variant was not significantly associated with overall endometrial cancer risk (14% controls and 17% type 1
cancers), although was enriched in type 2 endometrial cancers (24%, p = 0.2). In the combined analysis of RTOG 9708/9905,
miRNA expression differed by age, presence of lymphovascular invasion and KRAS-variant status. Overall survival rates at 3
years for patients with the variant and wild-type alleles were 100% and 77% (HR 0.3, p = 0.24), respectively, favoring the
Conclusions: The KRAS-variant may be a genetic marker of risk for type 2 endometrial cancers. In addition, tumor miRNA
expression appears to be associated with patient age, lymphovascular invasion and the KRAS-variant, supporting the
hypothesis that altered tumor biology can be measured by miRNA expression, and that the KRAS-variant likely impacts
endometrial tumor biology.
Citation: Lee LJ, Ratner E, Uduman M, Winter K, Boeke M, et al. (2014) The KRAS-Variant and miRNA Expression in RTOG Endometrial Cancer Clinical Trials 9708
and 9905. PLoS ONE 9(4): e94167. doi:10.1371/journal.pone.0094167
Editor: Shannon M. Hawkins, Baylor College of Medicine, United States of America
Received December 2, 2013; Accepted March 12, 2014; Published April 14, 2014
Copyright: 2014 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by NCI-NIH grant 5R01CA098346. This research was also supported by Radiation Therapy Oncology Group/American College
of Radiology (RTOG/ACR) Fellowship Program and RTOG grants U10 CA21661 and CCOP grant U10 CA37422 from the National Cancer Institute (NCI) and by NCI-NIH
grant 5R01CA098346. JW was supported by R01 CA131301-04, CA-157749-01A15 and 5R01CA131301-05. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Dr. Weidhaas and Dr. Slack discovered the KRAS-variant, which is patented by Yale University. This patent is called ‘‘Genetic Lesion
Associated with Cancer’’, and the number is 8,221,980. It has been licensed to a company, Mira Dx, that Dr. Slack and Dr. Weidhaas co-founded. Neither Dr. Slack
nor Dr. Weidhaas are employed or paid by Mira Dx, but act as unpaid scientific consultants. This does not alter the authors’ adherence to PLOS ONE policies on
sharing data and materials.
* E-mail: email@example.com
. These authors contributed equally to this work.
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The clinical and biologic heterogeneity of endometrial cancer
has been well recognized since the description of two major
subtypes by Bokhman in 1983. Type 1 endometrial cancer, the
endometrioid subtype, is associated with unopposed estrogen
exposure, chronic anovulation, nulliparity and obesity. Type 2
endometrial cancer includes the less common non-endometrioid
subtypes of uterine papillary serous and clear cell carcinoma.
Type 2 cancers are frequently seen in older women, arise in
atrophic endometrium, and are not estrogen responsive. The two-tiered
classification of endometrial cancer based on clinical and
pathologic factors is also supported at a molecular level. Further
molecular classification has recently been put forth by the
publication of The Cancer Genome Atlas. Loss of the tumor
suppressor PTEN has been reported in 30–50% of type 1
cancers[3–5], but is rarely observed in the serous or clear cell
subtypes. The proto-oncogene HER2/neu, a trans-membrane
growth factor receptor, is commonly amplified or over-expressed
in type 2 cancers[7,8]. Gene expression profiling by microarray
analysis has also confirmed the presence of distinct endometrial
cancer subtypes. Other characteristic molecular alterations
include microsatellite instability and mutation of beta-cate-nin[
11] in endometrioid endometrial carcinomas, and loss of E-cadherin
expression and p53 mutations in serous cancers.
Tumor acquired mutations in KRAS have been identified in 15–
30% of type 1 endometrial cancers but are rarely observed (0–5%)
in type 2 cancers. The prevalence of tumor-acquired KRAS
mutations has been variably associated with stage, grade and
survival in endometrial cancer[13–16]. KRAS mutations have also
been detected in endometrial hyperplasia, and may represent an
early event in tumorigenesis for type 1 endometrial cancers.
While adjuvant therapy for type 2 endometrial cancers often
involves chemotherapy, the role of combined chemotherapy and
radiation therapy (RT) for high-risk and advanced stage type 1
cancers is more controversial. Women with type 1 uterine cancers
with pathologic risk factors such as deep myometrial invasion, high
tumor grade, cervical invasion and/or pelvic-confined extrauter-ine
spread have a 15–30% risk of recurrence despite adjuvant
pelvic radiotherapy (RT) . The Radiation Therapy Oncology
Group (RTOG) conducted two prospective, multi-institutional
trials (9708 and 9905) that were designed to evaluate the feasibility
and efficacy of concurrent chemotherapy and post-operative RT
for women with International Federation of Gynecology and
Obstetrics (FIGO) Stages IC-IIIC, high-risk, type 1 endometrial
cancer. Given the reported response rates of 30–35% with
chemotherapy alone, these trials were conducted to evaluate the
safety and survival outcomes for a combined modality approach
for high-risk and advanced stage endometrial cancer. RTOG
9905, the randomized comparison of adjuvant chemoradiotherapy
versus RT alone, was closed early due to poor accrual and
therefore survival endpoints were not analyzed. Nevertheless,
tissue was collected for the exploration of novel biomarkers and
MicroRNAs (miRNAs) are a class of small non-coding RNA
that inhibit gene expression of downstream targets by binding
complementary sites in 39 untranslated regions (UTR) of
messenger RNA. As global gene regulators, miRNAs function as
a novel class of oncogenes or tumor suppressors depending on the
cellular context. Alterations in miRNA expression levels have been
implicated in oncogenesis as well as tumor biology in virtually all
cancers. Distinct miRNA expression signatures have also been
described for the type 1 and type 2 endometrial cancers,
supporting their ability to reflect inherent tumor biology. In
KRAS-Variant and miRNA in Endometrial Cancer
addition, inherited variants in miRNA binding sites in oncogenes
have been shown to predict cancer risk, tumor biology and altered
The first example of a functional miRNA binding site mutation
is a variant allele in the 39-UTR of KRAS (rs61764370), a germline
mutation found to disrupt let-7 binding, increase KRAS expres-sion[
21] and predict cancer risk[21–24]. This mutation has
additionally been associated with altered gene and miRNA
expression in tumors[21–23,25]. Interestingly, the KRAS-variant
predicts ovarian cancer risk in post-menopausal women, triple
negative breast cancer in pre-menopausal women, and
aggressive breast tumor biology in post-menopausal women with
a history of hormone replacement therapy, suggesting that
there is a likely impact of estrogen on the tumor-associated
function of the KRAS-variant. In addition, the KRAS-variant
appears to predict cancer biology in all cancers thus far studied,
including those for which it does not appear to predict increased
The objectives of this study were multifold: 1) to determine if the
KRAS-variant is associated with endometrial cancer risk; 2) to
evaluate the association of miRNA expression signatures with
clinical features in tumor specimens from RTOG 9708 and 9905;
and 3) to determine whether the KRAS-variant is associated with
endometrial cancer biology by evaluating both clinical and
miRNA expression associations in these same trials.
Materials and Methods
The subjects of the case-control study were Connecticut
residents diagnosed with primary endometrial cancer between
October 2004 and September 2008. Study staff of the Rapid Case
Ascertainment arm of the Yale Cancer Center visited the
Connecticut general hospitals to determine case eligibility and
identifying information. Controls were identified by random digit
dialing. Blood samples or saliva specimens were collected from
these study subjects. From collected specimens, DNA was isolated
using MagNA pure nucleic acid kit (Roche diagnostics, Indiana-polis,
IN) for the buffy-coat blood cells, and Oragene kit (DNA
Genotek Inc, Canada) for saliva samples. The presence of the
KRAS-variant was detected using a TaqMan PCR assay to identify
the wild-type (T) or variant (G) allele using allele-specific probes, as
previously described . Due to the minor allele frequency, the
heterozygous (TG) and homozygous (GG) forms were combined
for analysis to compare to the wild-type allele (TT).
Ethics Statement for Case-control Data
This protocol was approved by the Yale Human Investigations
Committee and State of Connecticut Department of Public Health
Human Investigation Committee. Certain data used in this study
were obtained from the Connecticut Tumor Registry in the
Connecticut Department of Public Health.
RTOG 9708 and 9905
RTOG 9708 was a single-arm phase II study of adjuvant RT
combined with cisplatin and paclitaxel chemotherapy, which
enrolled 45 patients with type I FIGO 1988 Stage IC-IIIC
endometrioid adenocarcinoma of the uterus. The successor trial,
RTOG 9905, was a randomized two-arm phase III study of
adjuvant RT with or without the same chemotherapy regimen,
which enrolled 42 patients. All patients enrolled in the two studies
started adjuvant radiation therapy within 8 weeks of surgery (total
abdominal hysterectomy and bilateral salpingo-oophorectomy).
Eligible patients had grade 2 or 3 adenocarcinoma with greater
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than 50% myometrial invasion, cervical stromal involvement or
pelvic-confined extrauterine disease and/or positive peritoneal
cytology. Patients with non-endometriod histology, such as
papillary serous or clear cell, were excluded from these studies.
Adjuvant treatment included pelvic RT to a dose of 45–50.4 Gy
followed by vaginal brachytherapy. Cisplatin (50 mg/m2) was
delivered concurrently on days 1 and 28. Following the completion
of RT, patients received 4 cycles of adjuvant cisplatin (50 mg/m2)
and paclitaxel (160–175 mg/m2) given every 4 weeks. Patients
were seen in follow-up every 4 months for 2 years, then every 6
months for 1 year and annually thereafter. A Pap smear and chest
X-ray were performed every year.
Ethics Statement for RTOG 9708 and 9908
Written informed consent was obtained from all patients prior
to enrollment. The clinical outcome and toxicity data for RTOG
9708 have been previously published [17,29]. RTOG 9905 was
terminated early due to poor accrual and follow up was
discontinued after at least 12 months of follow up for each patient.
Isolation of DNA and RNA and Testing of the KRAS-variant
Tumor specimens from paraffin-embedded formalin-fixed
blocks were microdissected after identification of areas with
sufficient tumor cellularity by a pathologist. Tumor sections were
de-paraffinized using Xylene and DNA was isolated for genotyp-ing
of the KRAS-variant as described above. Total RNA was
isolated from paraffin-embedded tumor specimens using the
Ambion Recover All kit (Life Technologies, Grand Island, NY)
per manufacturer’s instructions. miRNA was run on the ABI
Taqman TLDA platform. Samples were genotyped for the KRAS-variant.
The data was in Hardy-Weinberg equilibrium.
Statistical analysis was performed using the statistical software
JMP, v. 8.0.1 (SAS Institute, Cary, NC). For the case-control
analysis, the prevalence of the variant allele (TG/GG) was
compared for cases with endometrial cancer and population
controls using the Fisher exact test. Clinical, pathologic and
treatment characteristics of patients in the RTOG trials were
compared by allele type (wild-type vs. variant) using a t-test or the
Fisher exact test. Actuarial estimates of disease-free survival (DFS)
and overall survival (OS) were calculated using the Kaplan-Meier
method. The cumulative incidence method was used to estimate
the rates of local-regional failure (LRF) and distant failure (DF).
OS was calculated from the date of randomization until death
from any cause. DFS was defined as the interval from the date of
randomization until the development of distant metastases, local-regional
failure or death from any cause. LRF was defined as
primary recurrence or progression, vaginal recurrence and/or
nodal recurrence or progression. DF was defined as distant
metastases and/or para-aortic failure. Univariate analysis was
performed using Cox proportional hazards models. Estimated
prevalence rates for the variant allele were used for power
calculations. A type 1 error (a) of less than 0.05 was considered
For miRNA expression data, all pre-processing and statistical
analysis was performed in the statistical programming environ-ment
R, using customized functions and the Bioconductor limma
package. Each sample was normalized separately using the eight
endogenous control RNAs, and then the intensities were scaled
across the samples to have similar distributions. Statistically
significant changes in miRNA expression between samples
KRAS-Variant and miRNA in Endometrial Cancer
grouped by age, race, presence of lymphovascular space invasion,
DFS and KRAS-variant status were determined using limma with
p,0.05 adjusted by a Bonferroni correction, and an absolute fold-change
$2. Hierarchical clustering was based on Euclidean
distance metric and Ward’s linkage.
The KRAS-variant and Endometrial Cancer Risk
In the case-control study, to evaluate the role of the KRAS-variant
in predicting uterine cancer risk, allele data was obtained
for 583 population controls, 430 patients with type 1 endometrial
cancer and 37 patients with type 2 cancer. The prevalence of the
variant allele was 13.9% among controls and 16.5% for patients
with endometrial cancer (p = 0.29). As shown in Table 1, the
prevalence of the variant appeared higher in patients with type 2
uterine cancer of non-endometrioid histology (24.3%) compared
to type 1 endometrial cancer (16.7%) or population controls
(13.9%). However, none of the differences reached statistical
significance likely due to the small number of type 2 cancers in this
MiRNA Expression in RTOG 9708 and 9905
MiRNA expression patterns were evaluated from tumor
specimens of patients enrolled in RTOG 9708 and 9905 and
tested for association with clinical and pathologic characteristics.
We found no significant differences in miRNA expression between
samples from the two trials (data not shown), therefore the samples
were combined for the subsequent analysis. miRNA expression
patterns differed between tumors with lymphovascular invasion
(LVI) versus those without. Differentially expressed miRNAs (p
value,0.05 and fold-change.2) included miR-194, miR-192,
miR-203, miR-345, miR-30e-3p, miR-210 and miR-301 (Table
S1, Figure 1), all of which were overexpressed in tumors with
LVI present. Similarly, there were differences in miRNA
expression based on patient age: tumors in older women ($51
years) showed overexpression of miR-20b, miR-10a, and miR-187
and underexpression of miR-432 and let-7d (Table S2, Figure 2)
compared with tumors of younger women (,51 years).
Clinical Associations of the KRAS-variant in Trials RTOG
9708 and 9905
Allele data were available for 46 of 87 evaluable patients (53%)
enrolled in RTOG 9708 and 9905. The availability of allele data
was similar for the two trials, which included 22 patients (49%)
from 9708 and 24 patients (57%) from 9905. The prevalence of
the KRAS-variant was 22% overall, and was 18% (n =4) and 25%
(n= 6) from 9708 and 9905, respectively. The KRAS-variant was
found in only 1 of 11 (10%) women younger than 51 years of age
at the time of uterine cancer diagnosis, and in 9 of 35 (28%)
women who were older, although these prevalence rates were not
statistically different (p =0.41), likely due to small sample size.
The median follow-up time for patients with allele data in the
combined analysis was 29.3 months (min–max, 6.8–124.1
months). Patient characteristics, including age, ECOG perfor-mance
status, and race, were not significantly different between
women with and without the KRAS-variant (Table 2). The 1988
FIGO stage distribution, histology, depth of myometrial invasion
greater than 50% and the presence of LVI were also similar
between the groups.
The 3-year OS rates were 100% for the KRAS-variant patients
and 77% (95% confidence interval [CI]: 58%–87%) for the non-variant
patients (hazard ratio [HR] = 0.3, 95% CI: 0.04–2.29,
p = 0.24). One of 10 patients (10%) with the KRAS-variant had
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KRAS-Variant and miRNA in Endometrial Cancer
1. Prevalence of KRAS-variant in a control population and in patients with types 1 and 2 endometrial cancer.
KRAS-variant Control Type 1 cancer Type 2 cancer
Non-variant homozygotes 501 (86.1%) 360 (83.3%) 28 (75.7%)
Mutant/heterozygotes 81 (13.9%) 70 (16.7%) 9 (24.3%)
LRF and 2 (20%) had DF compared to 7 (19%) and 13 (36%),
respectively, among patients with the non-variant allele. The HRs
for LRF and DF were 0.45 (95% CI: 0.05–3.65, p = 0.45) and 0.44
(95% CI: 0.10–1.97, p = 0.28) for variant carriers. These trends
were not significant, perhaps because the study power was only
14–41% to detect a significant difference in the clinical endpoints
due to small sample size and limited follow-up.
MiRNA Expression and the KRAS-variant in RTOG 9708
Because the KRAS-variant has been previously shown to be
associated with altered gene as well as miRNA expression in
tumors, we evaluated the miRNA signatures from tumors with and
without the KRAS-variant. Furthermore, given that miRNA
signatures differed between tumors based on patient age (cutpoint
51 years), and that most patients with the KRAS-variant were post-menopausal,
we evaluated these differences only in tumors from
patients older than 51 years. We found significant differences in
miRNA signatures between tumors from patients with the KRAS-variant
and those without the KRAS-variant. These included lower
expression of miR-181b, miR-324–3p and miR-518b in KRAS-variant
patients’ tumors (Table 3, Figure 3).
This study represents the first analysis of the association of the
KRAS-variant with endometrial cancer. Although the KRAS-Table
Figure 1. MiRNA signatures differ in tumors with lymphovascular invasion (LVI). Yellow represents patients with LVI, and blue represents
patients without LVI.
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KRAS-Variant and miRNA in Endometrial Cancer
Figure 2. MiRNA signatures differ in tumors from women over and under 51 years of age. Yellow represents patients younger than 51
years of age, and blue represents patients equal to or older than 51 years of age.
variant was not a predictor of overall endometrial cancer risk, it
was enriched in patients with type 2 cancers, with a prevalence of
24.3%. In addition, in two prospective RTOG endometrial cancer
trials, miRNA signatures as well as the KRAS-variant were
associated with distinct clinical features. miRNA signatures were
different between tumors with or without LVI, between tumors in
pre- versus post-menopausal patients, and between KRAS-variant
patients and non-variant patients. Although RTOG 9708 and
9905 were not powered to detect statistically significant associa-tions
between the KRAS-variant and outcome, the trend was that
the KRAS-variant was associated with better outcome, although
confirmation of this hypothesis requires testing in a larger study
with more statistical power.
Although the association of miRNA expression and altered
clinical features of a tumor has been well documented, it may seem
surprising that the KRAS-variant, a germ-line, non-protein coding
sequence mutation that disrupts miRNA binding, predicts altered
miRNA signatures in a tumor and also alters prognosis. However,
the association of the KRAS-variant with altered miRNA signatures
has been found in every tumor in which it has been analyzed,
including lung cancer, melanoma, head and neck
cancer and triple negative breast cancer.
The association of the KRAS-variant with prognosis across
tumor types has also been widely reported. In a cohort of 344
patients with head and neck cancer, although an association with
cancer risk was not observed, variant carriers had a worse clinical
outcome when adjusted for age and stage (HR 1.6, 95% CI 1.0–
2.5) , with lower survival rates most pronounced among
patients with oral cavity cancer compared to pharyngeal and
laryngeal sites. Several studies have found that the KRAS-variant
predicts altered response to cetuximab in patients with metastatic
colorectal cancer[28,31]. In ovarian cancer, the KRAS-variant
predicts poor outcome due to platinum resistance. Platinum
resistance was also found in patients receiving chemotherapy alone
for metastatic head and neck cancer (Chung, submitted). Platinum
resistance is particularly interesting in the context of the RTOG
trials examined in this study, where a proportion of patients
received cisplatin delivered concurrently with radiation and in
conjunction with paclitaxel. One might hypothesize that the
addition of radiation to platinum agents might overcome the
associated resistance, a hypothesis that is currently being evaluated
in additional RTOG trials.
Among KRAS-variant positive and negative tumors in this study,
3 miRNAs were differentially expressed, including miR-181b,
miR-324 and miR-518b. In pre-clinical studies, miR-181b has
been shown to promote cellular proliferation and reduce apoptosis
in cervical cancer cells, mediate tumorigenesis through
STAT3, and induce gemcitabine resistance in pancreatic
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Table 2. Clinical and pathologic characteristics of 46 patients with allele data from RTOG trials 9708 and 9905.
Wildtype TT Variant TG or GG p-value
(n = 36) (n = 10)
Age Median (min–max) 61 years (36–81) 56 years (39–68) 0.16
Race White 29 (81%) 9 (90%) 0.66
Non-white 7 (19%) 1 (10%)
ECOG score 0 28 (78%) 8 (80%) 1.0
1–2 8 (22%) 2 (20%)
Histology Adenocarcinoma 31 (86%) 9 (90%) 0.74
Adenosquamous 3 (8%) 1 (10%)
Other 2 (6%) 0
Stage IB/IC/IIA/IIB 26 (72%) 6 (60%) 0.46
IIIA/IIIC 10 (28%) 4 (40%)
FIGO grade 1–2 19 (53%) 6 (60%) 0.73
3 17 (47%) 4 (40%)
MMI .50% 31 (86%) 8 (80%) 0.64
LVI 19 (53%) 5 (50%) 0.88
Key: min = minimum; max = maximum; ECOG score = Eastern Cooperative Oncology Group performance status score; FIGO = International Federation of Gynecology and
Obstetrics; MMI =myometrial invasion; LVI = lymphovascular invasion.
cancer cells. In human germ cell tumors, up-regulation of
miR-518b was associated with a cisplatin-resistant phenotype.
These studies may explain the improved outcomes seen in our
work that favored the KRAS-variant patients, as KRAS-variant
tumors had lower expression levels of miR-518b. In a study of
nasopharyngeal carcinoma, downregulation of miR-324 was
associated with radioresistance. In our study, KRAS-variant
tumors had lower levels of miR-324, although there was no clear
radioresistance as these patients had improved rates of local-regional
For a global view of what the differential miRNA expression
found in our tumors may represent, we used miR System, which
combines 7 algorithms and 2 validated databases to identify
potential gene targets of miRNAs and their function, as well as
pathway analysis using KEGG (Kyoto Encyclopedia of Genes and
Genomes). The target genes of miR-181b include MAP3K3
(mitogen-activated protein kinase 3, top hit), ESR1 (estrogen
receptor 1, a validated target), and KRAS, while those for miR-
518b include CTNNBIP1 (b-catenin interacting protein) and
MAP3K7IP3 (mitogen-activated protein kinase 7 interacting
protein 3). For pathway analysis of the enriched target genes,
differential expression of the miRNAs listed above were associated
with long-term potentiation, pathways in cancer, and endometrial
cancer, as well as the following signaling pathways: TGF-b, ErbB,
MAPK, and Wnt. The Wnt/b-catenin signaling pathway has been
shown to be dysregulated in 10–45% of endometrial cancers and is
intimately regulated by estrogen and progesterone. In
addition, KRAS is an important upstream mediator of the MAPK
pathway, and overexpression can lead to increased activation of
the RAF/MEK/MAPK pathway and thus promote tumorigene-sis.
Previous work has shown that the MAPK pathway is activated
in triple negative breast cancer with the KRAS-variant, and is
associated with lower estrogen signaling. These results and
others indicate that the role of the KRAS-variant in cancer risk and
biology may be dependent on hormonal environment such as
menopausal status, an ongoing topic of study.
We also performed pathway analysis for the 10 differentially
expressed miRNAs for tumors with and without lymphovascular
invasion. Of interest, predicted pathways involved in focal
adhesion, regulation of the actin cytoskeleton, gap junction and
adherens junction were top hits by KEGG, as well as the Wnt,
MAPK, TGF-b, and ErbB signaling pathways.
While our study was limited by the small sample size of the
RTOG trials we utilized, which provided insufficient statistical
power for us to develop solid conclusions regarding the potential
role of the KRAS-variant in predicting endometrial cancer risk or
outcome, we were able to see a potential association through
altered miRNA expression in tumors. In addition, we were able to
prospectively collect tissue samples in a cooperative group setting
and perform a translational research study, which has produced
Table 3. miRNA expression in KRAS-variant negative versus positive tumors.
ID logFC p-Value Adjusted p-Value m.e. KRAS Negative m.e. KRAS Positive
hsa-miR-181b –1.2486 0.0326 0.6593 1.9609 3.2095
hsa-miR-324-3p –1.1496 0.0432 0.6593 4.2346 5.3842
hsa-miR-518b –1.0684 0.0452 0.6593 6.5858 7.6542
Key: logFC = log fold change; m.e. = mean expression.
KRAS-Variant and miRNA in Endometrial Cancer
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Figure 3. MiRNA signatures differ between tumors with and without the KRAS-variant. Yellow represents patients without the KRAS-variant,
preliminary data worth validating in larger cohorts. These findings
further support the hypothesis that there are measurable ways to
assess the heterogeneity of endometrial cancer which could
provide important prognostic information in the future to enable
practitioners to more appropriately and individually direct patient
Table S1 Association between miRNA expression and
Table S2 Association between miRNA expression and
The cooperation of 28 Connecticut hospitals, including Charlotte
Hungerford Hospital, Bridgeport Hospital, Danbury Hospital, Hartford
Hospital, Middlesex Hospital, New Britain General Hospital, Bradley
Memorial Hospital, Yale/New Haven Hospital, St. Francis Hospital and
Medical Center, St. Mary’s Hospital, Hospital of St. Raphael, St. Vincent’s
Medical Center, Stamford Hospital, William W. Backus Hospital, Wind-ham
Hospital, Eastern Connecticut Health Network, Griffin Hospital,
Bristol Hospital, Johnson Memorial Hospital, Day Kimball Hospital,
Greenwich Hospital, Lawrence and Memorial Hospital, Milford Hospital,
New Milford Hospital, Norwalk Hospital, MidState Medical Center, John
Dempsey Hospital and Waterbury Hospital, in allowing patient access, is
gratefully acknowledged. We would like to thank Barbara Silver and
Corinne Doll for critical reading of this manuscript.
Conceived and designed the experiments: DG AD JW. Performed the
experiments: ER MB VP. Analyzed the data: MU KW L. Lee.
Contributed reagents/materials/analysis tools: HY L. Lu DG AD. Wrote
the paper: L. Lee JW. Involved in the inception of the RTOG 9708 and
9905 trials (and are included per RTOG publication policy): KG SK TB
KU HK RB.
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