KRAS rs61764370 is associated with HER2-overexpressed and poorly-differentiated breast cancer in hormone replacement therapy users: a case control study.

Abstract

A single nucleotide polymorphism located in the 3'-untranslated region of the KRAS oncogene (KRAS variant; rs61764370) disrupts a let-7 miRNA binding and was recently reported to act as a genetic marker for increased risk of developing human cancers. We aimed to investigate an association of the KRAS variant with sporadic and familial breast cancer and breast tumor characteristics.
Genotyping was accomplished in 530 sporadic postmenopausal breast cancer cases, 165 familial breast cancer cases (including N = 29, who test positive for BRCA1/2 mutations) and 270 postmenopausal control women using the flurogenic 5' nuclease assay. Information on hormone replacement therapy (HRT) use and tumor characteristics in sporadic breast cancer cases was ascertained from a postal questionnaire and pathology reports, respectively. Associations between the KRAS genotype and breast cancer or breast tumor characteristics were assessed using chi-square test and logistic regression models.
No evidence of association was observed between the KRAS variant and risk of sporadic and familial breast cancer - either among BRCA carriers or non-BRCA carriers. The KRAS variant was statistically significantly more often associated with human epidermal growth factor receptor 2 (HER2) - positive tumors and tumors of higher histopathologic grade. However, both associations were detected only in HRT users.
Our data do not support the hypothesis that the KRAS variant rs61764370 is implicated in the aetiology of sporadic or of familial breast cancer. In postmenopausal women using HRT, the KRAS variant might lead to HER2 overexpressed and poorly-differentiated breast tumors, both indicators of a worse prognosis.

Full text

Page 1

RESEARCH ARTICLEOpen Access
KRAS rs61764370 is associated with HER2-
overexpressed and poorly-differentiated breast
cancer in hormone replacement therapy users: a
case control study
Jasmina-Ziva Cerne1, Vida Stegel2, Ksenija Gersak1and Srdjan Novakovic2*
Abstract
Background: A single nucleotide polymorphism located in the 3’-untranslated region of the KRAS oncogene (KRAS
variant; rs61764370) disrupts a let-7 miRNA binding and was recently reported to act as a genetic marker for
increased risk of developing human cancers. We aimed to investigate an association of the KRAS variant with
sporadic and familial breast cancer and breast tumor characteristics.
Methods: Genotyping was accomplished in 530 sporadic postmenopausal breast cancer cases, 165 familial breast
cancer cases (including N = 29, who test positive for BRCA1/2 mutations) and 270 postmenopausal control women
using the flurogenic 5’ nuclease assay. Information on hormone replacement therapy (HRT) use and tumor
characteristics in sporadic breast cancer cases was ascertained from a postal questionnaire and pathology reports,
respectively. Associations between the KRAS genotype and breast cancer or breast tumor characteristics were
assessed using chi-square test and logistic regression models.
Results: No evidence of association was observed between the KRAS variant and risk of sporadic and familial
breast cancer - either among BRCA carriers or non-BRCA carriers. The KRAS variant was statistically significantly more
often associated with human epidermal growth factor receptor 2 (HER2) - positive tumors and tumors of higher
histopathologic grade. However, both associations were detected only in HRT users.
Conclusion: Our data do not support the hypothesis that the KRAS variant rs61764370 is implicated in the
aetiology of sporadic or of familial breast cancer. In postmenopausal women using HRT, the KRAS variant might
lead to HER2 overexpressed and poorly-differentiated breast tumors, both indicators of a worse prognosis.
Keywords: KRAS rs61764370, Breast cancer, Tumor characteristics, Hormone replacement therapy
Background
MicroRNAs (miRNAs) are a class of small non-coding
RNAs that function as negative gene regulators.
Depending on the degree of complementarity between
the miRNA and its target mRNA, miRNAs post-tran-
scriptionally regulate target gene expression by either
inhibiting mRNA translation or inducing mRNA degra-
dation [1]. Recent evidence has shown that impaired
miRNA expression or single nucleotide polymorphisms
(SNPs) that reside on miRNAs and/or miRNA-binding
sites correlate with various human cancers [2]. Depend-
ing on target mRNAs, miRNAs can function as tumor
suppressors or oncogenes [3].
The let-7 family of miRNAs plays an important role in
tumorigenesis by regulating the expression of multiple
oncogenes, including KRAS [4]. A germline SNP
rs61764370 is located in the 3’-untranslated region of
the KRAS oncogene and is referred to as the KRAS var-
iant. The KRAS variant was demonstrated to be func-
tional by disrupting a let-7 miRNA-binding site, and
therefore leading to increased KRAS levels in vitro [5].
The same group also identified the KRAS variant to be
* Correspondence: snovakovic@onko-i.si
2Department of Molecular Diagnostics, Institute of Oncology Ljubljana,
Zaloska 2, Ljubljana 1000, Slovenia
Full list of author information is available at the end of the article
Cerne et al. BMC Cancer 2012, 12:105
http://www.biomedcentral.com/1471-2407/12/105
© 2012 Cerne et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

associated with 2.3-fold increased risk for non-small-cell
lung cancer (NSCLC) among moderate smokers [5]. By
other report, tumors containing the KRAS variant allele
had lower let-7 levels, which has been associated with
shortened postoperative survival in NSCLC [6]. The pre-
sence of the KRAS variant was likewise associated with
poor prognosis in head and neck squamous cell carci-
noma as well as with the 2.5-fold increased risk of
developing epithelial ovarian cancer (EOC) [7,8]. The
variant allele was identified in 25% of non-selected EOC
cases and in 61% of EOC patients from hereditary breast
and ovarian cancer (HBOC) families previously consid-
ered uninformative for gene mutations [8]. However,
data from a subsequent meta-analysis excluded the pos-
sibility of an association between the KRAS variant and
a clinically significant risk of unselected, serous, familial
EOC, or EOC among women carrying deleterious muta-
tions in BRCA1 [9].
Since the KRAS variant was reported to be enriched in
ovarian cancer patients from HBOC families, the study
by Hollestelle and colleagues further investigated the
frequency of the KRAS variant in breast cancer families
[10]. As compared to the presence of the variant allele
among controls (17.3%), the increased frequency of the
KRAS variant was confirmed in breast cancer cases from
BRCA1 families (23.5%), but not among breast cancer
cases from BRCA2 (13.5%) or non-BRCA1/2 families
(15.8%) [10]. Another study found the KRAS variant to
act as a genetic marker for increased risk of developing
triple negative breast cancer in premenopausal women
(OR 2.31, 95% CI 1.26-4.22) [11].
On the basis of the current evidence, the purpose of
our study was to investigate the association of the KRAS
variant with sporadic and familial breast cancer risk
among Slovenian women. Furthermore, we aimed to
investigate the association of the KRAS variant with
breast tumor characteristics among Slovenian postmeno-
pausal sporadic breast cancer cases stratified by hor-
mone replacement therapy (HRT) use.
Patients and methods
Study population
Participants were those of our previous breast cancer
case-control study [12]. In brief, we enrolled postmeno-
pausal women, who were 50-69 years old and of Cauca-
sian ethnic origin. Cases diagnosed with invasive
primary breast cancer were enrolled from the Institute
of Oncology Ljubljana. Control women were randomly
recruited from the outpatient clinic records of the
Department of Obstetrics and Gynecology, University
Medical Centre Ljubljana during their routine gynecolo-
gic exams. Response rates and exclusion criteria for the
participants have been published previously [12].
The present analysis includes also a cohort of familial
breast cancer cases, who underwent genetic testing
between 2009-2011 at the Institute of Oncology
Ljubljana.
Informed written consent was obtained from all
women enrolled in the study. The study protocol was
approved by the National Medical Ethics Committee of
the Republic of Slovenia (No. 61/06/07).
Data collection
Women enrolled in breast cancer case-control study
were invited to participate through a postal question-
naire. Detailed questions were asked regarding sex hor-
mone intake, with an emphasis on HRT use. A color
chart displaying all preparations ever marketed in Slove-
nia was included in the questionnaire to aid recall.
Information was obtained on the duration of HRT use
(3 categories: less than 1 year, short-term use: 1 < 5
years, long-term use: 5 or more years) and regimen of
HRT use (estrogen therapy, estrogen plus progestin
therapy - there was no tibolone or other non-estrogen
user). Users of systemic and/or local route of HRT
administration were included in the analyses. HRT use
for less than 1 year was considered no use. Women
were considered postmenopausal if they had self-
reported their last menstrual bleeding being at least 12
months before the reference date or had undergone a
bilateral oophorectomy.
Information on tumor characteristics in sporadic
breast cancer cases was retrieved from pathology reports
in the patient’s medical records. Tumor grading was
performed according to the Nottingham scheme [13].
This grading method evaluates three parameters and
assigns a score of 1 to 3 for each parameter: tubule for-
mation (> 75% = 1, 10% to 75% = 2, < 10% = 3), nuclear
atypia (none = 1, moderate = 2, marked = 3), and num-
ber of mitoses per 10 high-power fields (HPF), based on
a HPF size of 0.274 mm2(< 10 mitoses = 1, 10 to 19
mitoses = 2, ≥ 20 mitoses = 3). The final Nottingham
histologic grade is based on the sum of scores of the
three parameters: 3, 4 or 5 = grade 1, 6 or 7 = grade 2,
8 or 9 = grade 3). Hormone receptor (HR) status (estro-
gen receptor (ER), progesterone receptor (PR)) was
ascertained using immunohistochemical (IHC) testing,
tumors with ≥ 10% nuclear staining were considered
positive for the respective antibody. HER2 protein
expression was determined by IHC using HercepTest™
(DAKO corp., CA, USA). HER2 gene amplification was
determined by dual-color fluorescent in situ hybridiza-
tion (FISH) using PathVysion®HER2 DNA probe kit
and Paraffin pretreatment kit (both Abbot-Vysis, Inc.,
Downers Grove, IL, USA). HER2 was considered posi-
tive when scored 3+ on the IHC staining and/or the
Cerne et al. BMC Cancer 2012, 12:105
http://www.biomedcentral.com/1471-2407/12/105
Page 2 of 7

Page 3

ratio of HER2 signal to chromosome 17 signal in 60
cells by FISH analysis scored > 2.2.
Genotyping
In sporadic breast cancer case patients, DNA was
extracted from archived paraffin-embedded non-malig-
nant breast tissues using HP PCR Template Preparation
Kit (Roche Diagnostics GmbH, Mannheim, Germany).
In familial breast cancer cases and controls, genomic
DNA was extracted from whole blood using the Flexi-
Gene DNA Kit 250 (Qiagen GmbH, Hilden, Germany).
Genotyping for the KRAS variant was carried out
using the 5’ nuclease assay (TaqMan; Applied Biosys-
tems, Werterstadt, Germany). The reaction employed
TaqMan Genotyping PCR Master Mix, forward primer
5-GCCAGGCTGGTCTCGAA-3, reverse primer 5-
CTGAATAAATGAGTTCTGCAAAACAGGTT-3,
probe-1 VIC-CTCAAGTGATTCACCCAC-MGB and
probe 2 FAM-CAAGTGATGCACCCAC-MGB, as pre-
viously described [7]. Analysis was performed using the
ABI PRISM®7900HT Sequence Detection System and
SDS 2.4 software (Applied Biosystems, Werterstadt, Ger-
many). Some of the samples (0.7%) failed to be geno-
typed due to poor DNA quality. Assay reliability was
assessed by random selection of 5% of samples in which
genotypes were confirmed by sequencing using the 3500
Genetic Analyzer (Applied Biosystems, Werterstadt,
Germany). Concordance rate was 100%.
Statistical analysis
Descriptive and summary statistics were used to
describe patient and breast tumor characteristics in the
dataset. Observed genotype frequencies were tested for
deviation from Hardy-Weinberg equilibrium with the
chi-square goodness-of-fit test. The homozygous wild-
type genotype, as determined by the more common of
the homozygous genotypes, served as a reference cate-
gory, with the heterozygous genotype and homozygous
variant genotypes being collapsed into one category.
Associations between the KRAS genotype and breast
tumor characteristics in relation to HRT use were
assessed using chi-square test. Odds ratios (ORs) for
breast cancer risk and the corresponding 95% confi-
dence intervals (CI) were calculated using logistic
regression analysis. All reported p values are two-sided
and considered statistically significant if p < 0.05. Ana-
lyses were performed using SPSS 19.0 software package
(SPSS, Chicago, IL, USA).
Results
The study population consisted of 530 postmenopausal
sporadic breast cancer cases, 165 familial breast cancer
cases (including N = 20, who test positive for BRCA1
mutations and N = 9, who test positive for BRCA2
mutations) and 270 postmenopausal control women
with no history of breast and/or ovarian cancer. The
mean age for sporadic breast cancer cases, familial
breast cancer cases and controls was 60.45 ± 5.84, 39.75
± 11.52 and 60.10 ± 5.85 years, respectively.
Genotype frequencies were close to those expected
under Hardy-Weinberg equilibrium in both cases and
controls (p > 0.05). No evidence of association was
observed between the KRAS variant and risk of sporadic
and familial breast cancer - either among BRCA carriers
or non-BRCA carriers. The KRAS variant allele was
detected in 17.2% of sporadic breast cancer cases and in
18.2% of all familial cases (10.3% of BRCA carriers -
10.0% of BRCA1 and 11.1% of BRCA2 carriers and in
19.9% of non-BRCA carriers). These frequencies were
not statistically significantly different from the preva-
lence of the variant allele in controls (17.8%, Table 1).
We further evaluated whether the KRAS variant
associates with a particular breast tumor characteristic.
Analyses were carried out for all sporadic breast cancer
cases, sporadic breast cancer cases using HRT (≥ 1 year
of HRT use) and sporadic breast cancer cases not using
HRT (0 < 1 years of HRT use).
Table 1 KRAS genotype frequencies in the study
population
Study
population
Genotype Frequency
N %
OR (95% CI)p
value
Controlswild (TT)221/269
(82.2)
48/269
(17.8)
1.0
variable (TG/
GG)
Sporadic cases wild (TT)434/524
(82.8)
90/524
(17.2)
0.96 (0.65-
1.40)
0.814
variable (TG/
GG)
Familial cases wild (TT)135/165
(81.8)
30/165
(18.2)
1.02 (0.62-
1.69)
0.929
variable (TG/
GG)
BRCA carriers wild (TT) 26/29 (89.7)0.53 (0.15-
1.83)
0.316
variable (TG/
GG)
3/29 (10.3)
BRCA1 carriers wild (TT)18/20 (90.0)0.51 (0.12-
2.28)
0.379
variable (TG/
GG)
2/20 (10.0)
BRCA2 carrierswild (TT) 8/9 (88.9)0.58 (0.07-
4.71)
0.606
variable (TG/
GG)
1/9 (11.1)
Non-BRCA
carriers
wild (TT) 109/136
(80.1)
27/136
(19.9)
1.14 (0.68-
1.93)
0.623
variable (TG/
GG)
Cerne et al. BMC Cancer 2012, 12:105
http://www.biomedcentral.com/1471-2407/12/105
Page 3 of 7

Page 4

Approximately one third of the sporadic breast cancer
cases (n = 157, 29.6%) were using HRT prior to diagno-
sis, 14.3% for short-term (1 < 5 years of HRT use) and
15.3% for long-term (≥ 5 years of HRT use). Among
HRT users, more than two thirds (n = 131, 71.2%) were
using combined estrogen plus progestin HRT (Table 2).
The vast majority of patients (n = 444, 84.9%) had
invasive ductal carcinoma, followed by invasive lobular
carcinoma (n = 62, 11.9%) and other special types of
carcinoma (n = 17, 3.2%) of which there were mucinous,
tubular, cribriform and medullary carcinomas. Distribu-
tions of other breast tumor characteristics are presented
in Table 3.
The prevalence of the KRAS variant was evenly dis-
tributed between HR+and HR-tumors (Table 4). The
KRAS variant was statistically significantly more often
associated with HER2+(42.9%) than HER2-(13.3%)
tumors and with tumors of higher histopathologic grade
- score 3 (28.6%) vs. score 1 and 2 (9.6%). However,
both associations were detected only in HRT users
(Tables 4 and 5).
To determine which of the three parameters (tubular
formation, nuclear atypia, mitotic index) contributed to
the higher histopathologic grade among HRT users car-
rying the variant allele, we evaluated the association of
the KRAS variant with the particular parameter of the
histopathologic grade among HRT users. KRAS variant
was statistically significantly more often associated with
tumors of marked nuclear atypia - score 3 (24.2%) vs.
score 1 and 2 (8.9%) and with tumors of higher mitotic
index - score 3 (28.9%) vs. score 1 and 2 (9.7%) (Table
6). On the other hand, the prevalence of the KRAS var-
iant was not statistically significantly different in regards
to what percent of the tumor formed normal duct struc-
tures (Table 6).
No statistically significant difference among KRAS
variant carriers and non-carriers was noted in the dis-
tribution of histologic type of the tumor, tumor size,
vascular invasion and lymph node involvement (data
not shown).
Discussion
The present study provides no evidence of association
between the KRAS variant and risk of sporadic and of
familial breast cancer - either among BRCA mutation
carriers or non-BRCA mutation carriers.
The lack of association between the KRAS variant and
sporadic postmenopausal breast cancer is in line with
the previous findings reported by Paranjape and collea-
gues [11]. Although the KRAS variant was significantly
associated with triple negative breast cancer in preme-
nopausal women, this association was not observed for
postmenopausal women regardless of HR and HER2 sta-
tus [11].
In contrast to Hollestelle and colleagues [10], who
found increased frequency of the KRAS variant among
cases from BRCA1 positive families, we did not observe
an association between the KRAS variant and either
cases from BRCA1, BRCA2 or non-BRCA breast cancer
families. The reason for this discrepancy might be insuf-
ficient power due to our medium-sized familial breast
cancer population, however, relatively narrow confidence
intervals suggest that the effect of a larger sample would
not be substantial. Furthermore, expansion of the num-
ber of BRCA1 mutation carriers by including other
family members in addition to the index cases in the
study by Hollestelle and colleagues did also not improve
significance, nor did the KRAS variant appear to modify
breast cancer risk for BRCA1 mutation carriers [10].
Since BRCA1 mutations have been consistently asso-
ciated with increased risk of triple negative breast can-
cer, Paranjape and colleagues evaluated whether the
observed association of the KRAS variant with premeno-
pausal triple negative breast cancer was only due to its
association with carriers of BRCA1 mutation. They
found no association between the KRAS variant and
BRCA1 mutations, however, the KRAS variant was asso-
ciated with a BRCA1 mutation-like gene expression sig-
nature [11]. This implies that there might be an
increased oncogenic risk in the presence of the KRAS
variant, but other mechanisms that uniquely down-
Table 2 Distribution of HRT use among sporadic breast cancer cases
Patients characteristicsSporadic breast cancer cases N (%)
HRT use
Nonusers: 0 < 1 years
Short-term users: 1 < 5 years
Long-term users: ≥ 5 years
Missing
373 (70.4)
76 (14.3)
81 (15.3)
0
Regimen of HRT*
Estrogen only
Estrogen plus progestin
Missing
53 (28.8)
131 (71.2)
2
* Among those who ever used HRT
Cerne et al. BMC Cancer 2012, 12:105
http://www.biomedcentral.com/1471-2407/12/105
Page 4 of 7

Page 5

regulate BRCA1 activity are assumed to be involved
[11,14]. Both studies [10,11], including our own, are
based on small-sized BRCA1 positive populations, there-
fore, validation in a larger cohort of BRCA1 mutation
carriers is warranted.
Since the association of the KRAS variant with triple
negative breast cancer risk reported by Paranjape and
colleagues was noted only for premenopausal women,
we carried out further analyses investigating the associa-
tion between the KRAS variant and breast tumor char-
acteristics in sporadic postmenopausal breast cancer
cases stratified by HRT use. This article shows for the
first time that the KRAS variant is more often associated
with HER2+tumors and tumors of higher histopatholo-
gic grade - the KRAS variant was enriched in tumors of
marked nuclear atypia and of higher mitotic index. Intri-
guingly, aforementioned associations were detected only
in HRT users. These findings support the notion that
there might be a meaningful interaction between the
KRAS variant and hormonal exposure.
We previously showed that tumors arising in women
taking HRT have a more favorable prognostic profile.
Tumors in HRT users were significantly smaller with
lower grade and lower mitotic index compared to
tumors in nonusers [15]. The present analysis, on the
other hand, revealed that HRT use in women carrying
the KRAS variant allele is associated with HER2 overex-
pressed and poorly-differentiated breast tumors, both
indicators of a worse prognosis. The plausable mechan-
ism for this switch caused by the KRAS genotype might
be the cross-talk between steroid hormone-, growth fac-
tor- and let-7 miRNA-directed pathways. It has been
postulated that aberrations in growth factor pathways
could dramatically influence steroid hormone action
[16]. The KRAS oncogene is an early player in many
growth factor signal transduction pathways and its over-
expression can lead to increased activation of the RAF/
MEK/mitogen activated protein kinase (MAPK) path-
way, which in turn phosphorylate and thereby activate
ERa [17]. Although there was not a substantial change
in KRAS mRNA, Paranjape and colleagues reported an
enrichment of both NRAS mutant and MAPK activation
signatures in breast tumors that had the KRAS variant
[11]. Another explanation could involve reduced expres-
sion of let-7 miRNA family members in KRAS variant-
associated breast tumors [6,11]. Indeed, recently identi-
fied compelling evidence demonstrated that let-7 miR-
NAs target ERa and thereby repress estrogen signaling
by causing a halt in cell proliferation and apoptosis [18].
Taken together, these observations suggest that due to
the KRAS variant-driven up-regulation of growth factor-
directed pathways and down-regulation of let-7
Table 3 Distribution of breast tumor characteristics
among sporadic breast cancer cases
Tumor
characteristics
Sporadic breast cancer cases
N (%)
Histologic type
Ductal
Lobular
Special-type
Missing
444 (84.9)
62 (11.9)
17 (3.2)
7
Tumor size
≤ 20 mm
21-50 mm
> 50 mm
Missing
349 (67.2)
152 (29.3)
18 (3.5)
11
Histopathologic grade
1
2
3
Missing
89 (17.1)
227 (43.6)
205 (39.3)
9
Tubular formation
1
2
3
Missing
33 (6.3)
143 (27.5)
344 (66.2)
10
Nuclear atypia
1
2
3
Missing
16(3.1)
267(51.3)
237(45.6)
10
Mitotic index
1
2
3
Missing
219(42.3)
141(27.2)
158(30.5)
12
Vascular invasion
No
Yes
Missing
403(79.3)
105(20.7)
22
Lymph node
involvement
No
Yes
Missing
288(57.5)
213(42.5)
29
HR status
ER-PR-
ER + and/or
PR+
Missing
83(15.7)
444(84.3)
3
HER2 status
HER2-
HER2+
Missing
449(87.7)
63(12.3)
18
Cerne et al. BMC Cancer 2012, 12:105
http://www.biomedcentral.com/1471-2407/12/105
Page 5 of 7