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A Further Investigation of Combined Mismatch Repair
and BRAFV600E Mutation Specific
Immunohistochemistry as a Predictor of Overall Survival
in Colorectal Carcinoma
Nathan Luey
1.
, Christopher W. Toon
1,2,3,4.
, Loretta Sioson
2
, Adele Clarkson
2,3
, Nicole Watson
2
,
Carmen Cussigh
2
, Andrew Kedziora
2
, Stuart Pincott
1,6
, Stephen Pillinger
1,6
, Justin Evans
1,6
,
John Percy
1,6
, Alexander Engel
1,4,5
, Margaret Schnitzler
1,5
, Anthony J. Gill
1,2,4,5
*
1Sydney Medical School, University of Sydney, Sydney, NSW, Australia, 2Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical Research, St
Leonards, NSW, Australia, 3Histopath Pathology, North Ryde, NSW, Australia, 4Sydney Vital Translational Research Centre, Royal North Shore Hospital, Pacific Highway, St
Leonards, NSW, Australia, 5Department of Anatomical Pathology, Royal North Shore Hospital, St Leonards, NSW, Australia, 6Department of Colorectal Surgery, Royal
North Shore Hospital, St Leonards, NSW, Australia
Abstract
Mutation specific immunohistochemistry (IHC) is a promising new technique to detect the presence of the BRAFV600E
mutation in colorectal carcinoma (CRC). When performed in conjunction with mismatch repair (MMR) IHC, BRAFV600E IHC
can help to further triage genetic testing for Lynch Syndrome. In a cohort of 1426 patients undergoing surgery from 2004 to
2009 we recently demonstrated that the combination of MMR and BRAFV600E IHC holds promise as a prognostic marker in
CRC, particularly because of its ability to identify the poor prognosis MMR proficient (MMRp) BRAFV600E mutant subgroup.
We attempted to validate combined MMR and BRAFV600E IHC as a prognostic indicator in a separate cohort comprising
consecutive CRC patients undergoing surgery from 1998 to 2003. IHC was performed on a tissue microarray containing
tissue from 1109 patients with CRC. The 5 year survivals stratified by staining patterns were: MMRd/BRAFwt 64%, MMRd/
BRAFV600E 64%, MMRp/BRAFwt 60% and MMRp/BRAFV600E 53%. Using the poor prognosis MMRp/BRAFV600E phenotype
as baseline, univariate Cox regression modelling demonstrated the following hazard ratios for death: MMRd/BRAFwt
HR = 0.71 (95%CI = 0.40–1.27), p = 0.31; MMRd/BRAFV600E HR = 0.74 (95%CI = 0.51–1.07), p = 0.11 and MMRp/BRAFwt
HR = 0.79 (95%CI = 0.60–1.04), p = 0.09. Although the findings did not reach statistical significance, this study supports the
potential role of combined MMR and BRAF IHC as prognostic markers in CRC.
Citation: Luey N, Toon CW, Sioson L, Clarkson A, Watson N, et al. (2014) A Further Investigation of Combined Mismatch Repair and BRAFV600E Mutation Specific
Immunohistochemistry as a Predictor of Overall Survival in Colorectal Carcinoma. PLoS ONE 9(8): e106105. doi:10.1371/journal.pone.0106105
Editor: William B. Coleman, University of North Carolina School of Medicine, United States of America
Received June 10, 2014; Accepted July 28, 2014; Published August 25, 2014
Copyright: ß2014 Luey 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.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This study was supported by funding from the Cancer Institute NSW as part of a translational research centre grant and internally by the department of
anatomical pathology Royal North Shore Hospital. No other funding support was received. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: affgill@med.usyd.edu.au
.These authors contributed equally to this work.
Introduction
The development of biomarkers to predict outcome after
definitive treatment of malignancy is an area of active research.
Despite literally thousands of biomarkers having been explored in
various cohorts, [1] very few have entered routine clinical practice.
Reasons for the failure to translate into clinical care include cost,
impracticality, lack of availability and failure of validation in
different cohorts or across diverse populations. [1] An ideal
biomarker would be inexpensive, readily deployable in the routine
clinical setting and add genuine prognostic information in addition
to that already provided by simple measures such as age, stage and
grade.
In many institutions patients with colorectal carcinoma (CRC)
undergoing surgery with curative intent are routinely offered reflex
immunohistochemistry (IHC) for the DNA mismatch repair
(MMR) proteins MLH1, PMS2, MSH2 and MSH6 in order to
triage formal molecular testing for Lynch Syndrome. [2] We
recently demonstrated, in a single institution cohort of patients
undergoing surgery for CRC at Royal North Shore Hospital
between calendar years 2004 and 2009, that the addition of
BRAFV600E mutation specific IHC to MMR IHC holds promise
as a biomarker for all cause survival. [3] This approach identifies
the poor prognostic group of mismatch repair proficient (MMRp)
BRAFV600E mutant CRC which accounted for 6.4% of CRC in
our previous study. [3] Because the presence of BRAFV600E
determination by either molecular means or IHC virtually
PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e106105
excludes Lynch Syndrome in mismatch repair deficient (MMRd)
CRC and is therefore commonly performed in many institutions
in MMRd CRC, [2] this approach requires minimal extra expense
and fits well into routine laboratory workflow.
In this study we sought to validate the combination of MMR
and BRAFV600E IHC as a prognostic marker in CRC by
examining its prognostic power in a different cohort – namely all
patients undergoing surgery for CRC at the same institution from
June 1998 to 2003.
Materials and Methods
Patients
We searched the database of the Department of Anatomical
Pathology, Royal North Shore Hospital, for all patients who
underwent surgery for CRC with curative intent from June 1998
to the end of calendar year 2003. During this period this
department provided a centralized pathology service for 2 major
quaternary centres with dedicated colorectal surgery units as well
as four community hospitals with general surgery units. Patients
treated endoluminally, with histologies other than adenocarcino-
ma or with tissue blocks unavailable for review were excluded. The
pathology reports of all cases were reviewed (and if necessary the
slides from cases were retrieved and reassessed) in order to stage
the tumours according to the AJCC 7
th
edition 2009 staging
system [4].
Immunohistochemistry
Tissue microarrays (TMAs) containing two 1 mm cores of
carcinoma were created. IHC for the MMR associated proteins
MLH1, PMS2, MSH2 and MSH6 was performed and interpreted
using standard and previously described methods. [6]
BRAFV600E mutation specific IHC was performed using a
commercially available mouse monoclonal antibody (clone VE1,
SpringBioscience, Pleasonton CA) using the same methods we
have previously described. [2,3,5] Briefly VE1 IHC was performed
using the Leica BondIII autostainer (Leica Microsystems, Mount
Waverley, VIC, Australia) used according to the manufacturer’s
protocol with alkaline antigen retrieval (solution ER2, VBS part
no: AR9640, Leica Microsystems) with the primary antibody
being used at a dilution of 1 in 80. BRAFV600E staining was
interpreted as positive if .20% of neoplastic cells stained
positively. The presence of definitive negative staining for any
one of the four MMR markers was interpreted as evidence of
mismatch repair deficiency (MMRd). MMR and BRAFV600E
IHC was interpreted by observers who were blinded to all other
clinical and pathological data.
Survival Data
Follow up data was obtained by examination of hospital medical
records and the hospital pathology database, assessment of records
from surgeons’ private rooms and examination from publicly
available death notices up to January 2014. Overall survival was
defined as the duration alive from time of definitive surgery. In
patients with metachronous CRCs, survival was taken from the
time of surgery for the first CRC with subsequent tumors (either
recurrences or second primary tumors) being excluded from
survival analysis.
Statistical analysis
Single variable p-values were computed using either the chi-
square test for categorical variables or the Mann-Whitney-U test
for scalar variables such as age at diagnosis. Five year survival
values were obtained via Kaplan Meier analysis for each of the
four MMR/BRAF IHC tumour phenotypes.
The effect of MMR/BRAF tumour IHC phenotype on overall
survival was explored using Cox regression proportional hazards
analysis, including a final model adjusted for gender, age at
diagnosis, anatomic location, histologic grade and overall stage.
A p-value of ,0.05 was taken as significant. All analysis was
performed using IBM SPSS statistics for MAC, Version 21.0 (IBM
Corp, Armonk NY USA, released 2012).
This study was approved by the Northern Sydney Local Health
District Human Research Ethics Committee under protocol 1201-
035 M. The ethics committee waived the need for consent to use
the archived formalin fixed paraffin embedded tissue blocks and to
access medical records on the basis that the study was only
performed on archived formalin fixed paraffin embedded tissue
removed during routine care many years previously. All patient
information was anonymized and de-identified prior to analysis.
Results
A total of 1109 colorectal carcinomas met inclusion criteria and
had cores available in the TMA sections. The clinical and
pathological details are presented in Table 1. Briefly, the median
age at diagnosis was 72 years, 49.2% were female, and 75% had
stage 2 or 3 disease. 856 patients were MMRp (85.9%) of which
133 (13.4% of the total) were MMRp/BRAFV600E mutant and
720 (72.5% of the total) were MMRp/BRAFwt. 144 were MMRd
(14.1%), of which 108 (10.9% of the total) were MMRd/
BRAFV600E and 32 (3.2% of the total) were MMR-deficient/
BRAF wild type.
Table 2 presents the overall survival figures for each of the four
MMR/BRAF phenotypes as determined by the Kaplan Meier
analysis. The 5-year survivals were 52.6% for MMRp/
BRAFV600E, 64.2% for MMRd/BRAFwt, 64.1% for MMRd/
BRAFV600E and 60.1% for MMRp/BRAFwt CRCs.
The univariate Cox regression survival function demonstrating
the crude (unadjusted) relationship between survival and MMR/
BRAF status is presented in Table 1 and Figure 1. Tumours
segregated according to their MMR/BRAF phenotype in a
consistent trend throughout the period of follow-up, with the
MMRp/BRAFV600E trending towards worse prognosis com-
pared to the other three phenotypes. Compared to the MMRp/
BRAFV600E phenotype, tumours displaying the MMRp/
BRAFwt phenotype tended towards significantly improved
survival with a hazard ratio of 0.79 (95%CI = 0.60–1.04,
p = 0.09). This effect was markedly diminished in the multivariate
model due to the dominant effects of gender, age at diagnosis and
tumour stage on overall survival (adjusted effect).
Discussion
The determination of BRAF mutation status by immunohisto-
chemistry has the significant advantages over molecular tech-
niques of being both inexpensive and fitting easily into standard
surgical pathology workflow. In laboratories where all CRCs
routinely undergo screening for Lynch Syndrome with MMR
IHC, the addition of BRAFV600E mutation specific immunohis-
tochemistry would simply be a matter of performing IHC for 5
rather than 4 markers entailing minimal extra labour or handling
costs and would have the added advantage of further triaging
molecular testing for Lynch Syndrome in MMRd CRC.
Therefore, if the addition of BRAFV600E mutation specific
IHC to all CRCs can be validated as a biomarker, there is real
potential that it may become the first prognostic biomarker for
CRC deployed into routine clinical practice.
BRAF and MMR in CRC
PLOS ONE | www.plosone.org 2 August 2014 | Volume 9 | Issue 8 | e106105
Table 1. Clinical and pathological features of 1109 consecutive patients with CRC.
Variable Count (%)
Single
Variable
p-value
Univariate
analysis HR
(95%CI),
p-value
Multivariate
analysis HR
(95%CI),
p-value
Gender 0.63
female 546 (49.2) 1.00 1.00
male 563 (50.8) 1.15 (0.96–1.37), 0.13 1.22 (1.00–1.50), 0.05
Age at diagnosis 72 (28–100) N/A 1.05 (1.04–1.05), ,0.01 1.05 (1.04–1.06), ,0.01
Anatomic location ,0.01
rectum 278 (25.5) 1.00 1.00
caecum 156 (14.3) 1.17 (0.86–1.59), 0.32 0.85 (0.60–1.21), 0.37
ascending colon 218 (20.0) 1.16 (0.89–1.50), 0.28 0.87 (0.64–1.18), 0.37
transverse colon 130 (11.9) 1.23 (0.91–1.67), 0.18 0.80 (0.55–1.15), 0.23
descending colon 48 (4.4) 0.99 (0.63–1.55), 0.97 0.90 (0.54–1.48), 0.67
sigmoid colon 260 (23.9) 1.19 (0.93–1.54), 0.17 1.04 (0.79–1.38), 0.78
Histologic grade ,0.01
low 835 (80.9) 1.00 1.00
high 197 (19.1) 1.39 (1.11–1.75), ,0.01 1.13 (0.87–1.47), 0.36
AJCC Stage ,0.01
I 207 (18.7) 1.00 1.00
IIA 295 (26.6) 0.28 (0.17–0.46), ,0.01 0.10 (0.05–0.20), ,0.01
IIB 54 (4.9) 0.35 (0.21–0.57), ,0.01 0.13 (0.06–0.25), ,0.01
IIC 15 (1.4) 0.95 (0.53–1.69), 0.86 0.36 (0.17–0.79), 0.01
IIIA 40 (3.6) 1.06 (0.48–2.33), 0.90 0.32 (0.12–0.81), 0.02
IIIB 321 (28.9) 0.31 (0.15–0.61), ,0.01 0.15 (0.06–0.34), ,0.01
IIIC 107 (9.6) 0.63 (0.39–1.01), 0.06 0.22 (0.11–0.44), ,0.01
IVA 39 (3.5) 1.49 (0.89–2.50), 0.13 0.59 (0.28–1.22), 0.16
IVB 5 (0.5) 2.23 (1.26–3.98), ,0.01 1.30 (0.60–2.83), 0.50
MMR IHC status ,0.01 N/A
proficient (MMRp) 856 (85.9) 1.00
deficient (MMRd) 140 (14.1) 0.90 (0.69–1.17), 0.42
BRAF IHC status ,0.01 N/A
wild type (BRAFwt) 774 (76.2) 1.00
mutant (BRAFV600E) 242 (23.8) 1.12 (0.91–1.38), 0.30
MMR/BRAF IHC phenotype ,0.01
MMRp/BRAFV600E 133 (13.4) 1.00 1.00
MMRd/BRAFwt 32 (3.2) 0.71 (0.40–1.27), 0.25 1.12 (0.61–2.06), 0.72
MMRd/BRAFV600E 108 (10.90 0.74 (0.51–1.07), 0.11 0.87 (0.58–1.31), 0.51
MMRp/BRAFwt 720 (72.5) 0.79 (0.60–1.04), 0.09 0.80 (0.60–1.08), 0.15
doi:10.1371/journal.pone.0106105.t001
Table 2. Overall survivals of each of the four MMR/BRAF phenotypes by Kaplan Meier actuarial analysis.
MMR/BRAF phenotype 5-year survival Mean survival
MMRp/BRAFV600E 52.6% 7.12 years (95%CI = 5.87–8.37)
MMRd/BRAFwt 64.2% 8.36 years (95%CI = 6.16–10.56)
MMRd/BRAFV600E 64.1% 8.08 years (96%CI = 7.04–9.40)
MMRp/BRAFwt 60.1% 8.06 years (95%CI = 7.62–8.50)
doi:10.1371/journal.pone.0106105.t002
BRAF and MMR in CRC
PLOS ONE | www.plosone.org 3 August 2014 | Volume 9 | Issue 8 | e106105
When we previously investigated the prognostic power of
combined MMR and BRAFV600E IHC in a group of 1426 CRC
from 2004 to 2009 [3], univariate analysis demonstrated that
MMRp/BRAFV600E CRCs had a statistically significantly worse
outcome compared to the other phenotypes (hazard ratio of 1.79
(95%CI = 1.24–2.60), (p),0.01). This result was negated in
multivariate analysis (hazard ratio of 1.10 (95%CI = 0.69–1.76),
(p) = 0.68) primarily due to the dominant effect of stage and age on
overall survival. In the current study, comprising CRCs from 1109
from the same institution resected from 1998 to 2003, MMRp/
BRAFV600E CRCs trended towards a worse prognosis compared
to all other tumour groups but failed to gain statistical significance
(MMRd/BRAFwt p = 0.31, MMRd/BRAFV600E p = 0.11 and
MMRp/BRAFwt p = 0.09). Whilst our findings support the
prognostic utility of the combination of MMR and BRAFV600E
IHC, the failure to achieve statistical significance indicates that
further studies in larger cohorts will be needed to validate this
approach. Ideally such further studies should be in truly
independent external cohorts (that is from other institutions)
rather than merely representing a preceding cohort from the same
institution as in this case.
To date 13 studies have directly compared the accuracy of
BRAF mutation status determination by IHC with molecular
techniques. In 11 studies BRAFV600E mutation specific IHC has
either outperformed or performed comparably to molecular
techniques [2,7,8,9,10,11,12,13,14,15,16] whereas in two studies
mutation specific IHC was found to be less reliable. [17,18] A fair
reading of the literature would support the approach taken by
Kuan et al, [12] that mutation specific IHC is reliable but requires
rigorous technical optimization and ongoing quality assurance
including the performance of molecular testing in equivocal cases.
Whilst this study was not intended or designed to assess the
accuracy of BRAFV600E mutation specific IHC we note that the
antibody has previously been proven to be extremely reliable in
our hands. [2] The overall rate of BRAF mutation as determined
by IHC in this study (23.8%) is in keeping with the 18.4%
incidence we reported in a similar cohort of consecutiveCRCs
from 2011 from the same institution tested by molecular means
alone [2].
Although there are limitations to this study, most importantly
that it did not represent a true external validation cohort but
rather a validation cohort from the same institution, our finding of
a trend towards survival differences amongst CRC when stratified
by BRAFV600E and MMR IHC status is very similar to that
which we have previously reported. [3] This provides cautious
support to the use of a combination of BRAFV600E and MMR
IHC as prognostic biomarkers in CRC. Ultimately similar studies
will need to be performed in large truly independent cohorts
before this approach can be considered validated. In the interim,
the combination of BRAFV600E and MMR IHC still has a clear
role in the triaging of patients with CRC encountered in routine
clinical practice for formal molecular testing for Lynch Syndrome.
Figure 1. Overall survival of patients with CRC stratified by MMR and BRAF status (Cox regression modelling).
doi:10.1371/journal.pone.0106105.g001
BRAF and MMR in CRC
PLOS ONE | www.plosone.org 4 August 2014 | Volume 9 | Issue 8 | e106105
[2,7,8,9,10,11,12,13,14,15,16] The strong likelihood that this
approach can also have the added benefit of predicting outcome
can be considered a likely downstream benefit of universal
screening for Lynch Syndrome by immunohistochemistry.
Author Contributions
Conceived and designed the experiments: AG CT. Performed the
experiments: NL AG CT CC LS AC NW. Analyzed the data: NL CT
AG. Contributed reagents/materials/analysis tools: NL CT LS AC NW
CC AK S. Pincott S. Pillinger JE JP AE MS AG. Contributed to the
writing of the manuscript: NL CT LS AC NW CC AK S. Pincott S.
Pillinger JE JP AE MS AG.
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