Immunohistochemistry of KRAS Protein in Colorectal Cancer

Dody Novrial

, Kamal Agung Wijayana

and Hanif Kun Cahyani

Department of Anatomical Pathology, Faculty of Medicine,Universitas Jenderal Soedirman, Purwokerto, Indonesia

Department of Surgery, Faculty of Medicine, Universitas Jenderal Soedirman Purwokerto, Indonesia

Faculty of Medicine, Universitas Jenderal Soedirman Purwokerto, Indonesia

Keywords: Ras gene aberration, KRAS mutation, Colorectal cancer.

Abstract: PCR assays are used for the detection of KRAS mutations in colorectal cancer (CRC). However, there are

some disadvantages in the clinical application such as the high-cost value and specific codon properties of

this method. Our study aimed to evaluate the effectivity of immunohistochemistry (IHC) in assessing ras

gene aberration in CRC before PCR testing. Paraffin-embedded tissue samples from 45 CRC patients were

stained immunohistochemically using anti-KRAS protein polyclonal antibody. KRAS protein expression was

assessed and correlated with clinicopathological features. A chi-square test was performed to evaluate the

results statistically. KRAS protein positivity was observed in 31.1% of cases. The positivity was correlated

with female predominance (p=0.03). No significant correlation between KRAS protein expression with age,

tumor topography, lymph node metastases, TNM staging, and tumor differentiation. In conclusion, IHC was

regarded as cost- effective, non-specific for codons, and may complement PCR in the detection of KRAS gene

mutation.

1 INTRODUCTION

Colorectal cancer (CRC) is one of the most common

cancer worldwide and is the second leading cause of

cancer deaths (Jemal et al., 2011). Sporadic CRC

frequently arises through the activation of oncogenes

such as KRAS and BRAF as well as inactivation of

tumor suppressor and mismatch repair genes

(Raskov, Pommergaard, Burcharth, & Rosenberg,

2014). KRAS mutation is one of the first alterations

that occur in colorectal tumorigenesis (Rajagopalan et

al., 2002). Mutation of KRAS occurs approximately in

14%-50% of CRC and frequently detected in codon

12 and codon 13 (Sammoud et al., 2012).

Anti-epidermal growth factor receptor (EGFR)

antibody such as cetuximab and panitumumab has

been approved for CRC treatment. It engaged with the

extracellular domain of EGFR, blocks ligand binding,

and leads to inhibition of downstream RAS-RAF-

MEK-ERK signaling pathway (Porru, Pompili,

Caruso, Biroccio, & Leonetti, 2018). However, this

therapy is not recommended for use in patients with

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KRAS mutation because of anti-EGFR antibody

resistance (De Roock et al., 2010). Therefore, KRAS

status becomes an important biomarker for patient

selection.

PCR is an established assay for KRAS mutation

detection since this analysis has favorable sensitivity

even in samples with low tumor cells (Cree, 2016).

However, there are some weaknesses of PCR assays

in the clinical setting such as high-cost value and

specific codon properties of this method.

Immunohistochemistry (IHC) has been suggested as

the prior screening method before genetic testing

(Wan Juhari et al., 2015). It has been a part of routine

service in most of the pathology laboratories which

not as expensive as molecular detection screening.

In the detection of MMR defects, IHC showed

high sensitivity and specificity compared to

molecular MSI-testing (Shia, 2008). However, in the

screening of ras gene aberration, several previous

studies revealed conflicting results (Elsabah & Adel,

2013; Piton, Borrini, Bolognese, Lamy, & Sabourin,

2015)

In this study, we evaluated the

Novrial, D., Wijayana, K. and Cahyani, H.

Immunohistochemistry of KRAS Protein in Colorectal Cancer.

DOI: 10.5220/0010487500470051

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 47-51

ISBN: 978-989-758-499-2

immunoexpression of KRAS proteins in CRC. We

discuss the result of KRAS immunohistochemistry

and its

relationship with some of the

clinicopathological features.

2 MATERIALS AND METHODS

This study was approved by the Research Ethics

Committee of Faculty of Medicine Universitas

Jenderal Soedirman. A total of 45 Formalin-fixed

paraffin-embedded (FPPE) tissue blocks of CRC

patients underwent colectomy were collected from

Margono Soekarjo Hospital and private pathology

laboratory from January to December 2019.

FPPE tissue blocks were subjected to staining by

immunohistochemical assays. Sections of FPPE

tissue blocks (4 µm) were transferred to positively

charged slides. Then, they were subjected to

deparaffinized, rehydration, and blocking with

hydrogen peroxide and antigen retrieval (Dako target

retrieval solution, citrate buffer pH 6.0) at 100

C for

10 minutes. After a short rinsed in phosphate-

buffered saline (PBS), slides then incubated overnight

at room temperature with primary antibody against

KRAS oncoprotein (orb53139 KRAS polyclonal

antibody: dilution 1:100, UK). Slides were washed

with PBS, then incubated for 30 minutes with labeled

secondary antibody. Product visualization (Dako)

was performed with diaminobenzidine substrate as

the chromogen. The slides were counterstained with

Mayer’s hematoxylin and washed once each with

distilled water and PBS. Finally, slides dehydrated

with ethanol, cleared in xylene, and mounted under a

coverslip.

Slides were evaluated under a light microscope by

pathologists subjectively. Sample with no primary

antibody added was used as a negative control. The

cytoplasmic expression of KRAS protein was assessed

using the previously established criteria of Allred et

al (Allred, Harvey, Berardo, & Clark, 1998). We

considered positive expression if the score of more

than 2 in tumor cells.

The corresponding clinical and pathological data

including sex, age, tumor topography, lymph node

metastases, TNM staging, and tumor differentiation

were obtained from a review of patient records.

Association between clinicopathological parameters

and KRAS protein immunostaining was statistically

examined by the Chi-square test, and p-value < 0.05

was considered significant.

3 RESULTS

Table 1 shows the clinicopathological features of

CRC cases. More than half of the patients were male.

The mean age of the patients was 52.2±12.5 years,

and most of them were >40 years old age. The

majority of the CRC were located on the distal colon,

and most of them were in stage 3 with well/moderate

differentiation.

Table 1: Association between KRAS protein expression

and clinicopathological features of CRC.

Features KRAS protein

expression

Positive

n (%)

Negative

n (%)

Sex 0.03

Male 5(35.7) 22(71)

Female 9(64.3) 9(29)

Age 0.63

≤40 3(21.4) 7(22.6)

>40 11(78.6) 24(77.4)

Topography 0.62

Proximal colon 5(35.7) 11(35.5)

Distal colon 9(64.3) 20(64.5)

Lymph Node

Status

0.15

Positive 7(50) 9(29)

Negative 7(50) 22(71)

Staging 0.15

Stage 2 7(50) 9(29)

Stage 3 7(50) 22(71)

Differentiation 0.69

Well and

moderate

14(100) 30(96.8)

Poor 0(0) 1(3.2)

Figure 1: Representative photomicrographs of KRAS

immunostaining in CRC tissues (A. Negative expression;

B. Positive expression; 400x magnification).

Positive KRAS protein expression was found in

14(31.1%) of cases (Figure 1). We found a significant

association between positive KRAS protein

expression and female predominance. Nevertheless,

our study did not reveal any statistical association

between KRAS protein expression with age,

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

topography, lymph node status, tumor staging, and

tumor differentiation.

4 DISCUSSION

Mutation of RAS proto-oncogenes is frequently

found in human cancer. In colorectal carcinogenesis,

mutation of KRAS leads to uncontrolled proliferation

and malignant transformation. Nowadays, the

determination of the KRAS mutation is mandatory for

treatment with anti-EGFR therapy in patients with

CRC. KRAS and BRAF mutations play a pivotal role

in colorectal carcinogenesis and are related to the

main resistance to anti-EGFR therapy (Larki et al.,

2017). Unfortunately, mutation of KRAS has only

been detected commonly in codon 12,13, and 61 (Lee

et al., 2017). Therefore, expanding RAS testing in

CRC to analyze more mutations may better predict

benefit from anti-EGFR therapy.

However, increase testing volume will increase

testing costs which may have economic implications.

Previous studies revealed that single standard KRAS

exon 2 testing was more than threefold costly than

single antibody IHC assay (Kircher, Mohindra, &

Nimeiri, 2015; Muirhead, Aoun, Powell, Juncker, &

Mollerup, 2010). Thus, the morphological study

followed by protein detection using IHC appears to

be an alternative to molecular screening. IHC of

KRAS might be useful as a prognostic and predictive

marker in CRC. KRAS positive protein expression

was associated with the disease aggressiveness of

CRC. There was a significantly reduced relapse-free

survival (RPS) in rectal cancer patients with KRAS

positive protein expression (Kanik, Gajjar, & Ghosh,

2018).

In the present study, we detected KRAS protein

expression in 31.1% of CRC cases. This result was

close to study in Europe (33%) (Piton et al., 2015) but

lower than study in Egypt (42.3%) (Elsabah & Adel,

2013). This KRAS IHC positivity was also close to a

study that revealed KRAS mutation in 32.8% of CRC

cases previously (Liu, Jakubowski, & Hunt, 2011).

However, other studies found mutation positivity

range between 14% to 50% (Sammoud et al., 2012).

These differences were reported because of several

weaknesses of mutation analysis such as codon

specific mutation sites, ethnic variations (Zhang et al.,

2015), diet, and lifestyle factors (Hughes, Simons,

van den Brandt, van Engeland, & Weijenberg, 2017).

Our data demonstrated the predominance of

KRAS protein expression in female CRC patients.

This finding was in line with several previous studies

that reported a correlation between mutated KRAS

and the female gender in CRC (Li et al., 2015; Tong

et al., 2014). However, other reports using IHC assays

did not demonstrate such a relationship (Kanik et al.,

2018; Sammoud et al., 2012). A possible explanation

for our result is likely complex and multifactorial

including lifestyle factors and the composition of gut

microbiota which varies among gender (Kostic et al.,

2011). Female sex hormones are related to colorectal

carcinogenesis by their effects on the production of

bile acid, bowel transit time, and bacterial

fermentation (Sammoud et al., 2012).

In this recent study, we did not find an association

between KRAS protein expression and the age of

patients. This is in keeping with some previous

reports both using IHC or molecular testing (Elsabah

& Adel, 2013; Rosty et al., 2013). However, another

study reported that ras p21 IHC overexpression was

relatively related to the advanced age of patients

(Sammoud et al., 2012). This difference result might

be caused by different classification of the patient's

age.

Besides, we did not discover any significant

relationship between KRAS protein expression and

other clinicopathological parameters such as

topography, lymph node status, stage, and

differentiation of the tumors. Our result was in

agreement with previous studies using either IHC

(Elsabah & Adel, 2013; Piton et al., 2015) or PCR

assays (Sammoud et al., 2012). Nevertheless, other

reports detected an association between mutant KRAS

with mucinous subtype and greater differentiation of

CRC (Zhang et al., 2015). Unfortunately, in our

study, we only had one sample of poorly

differentiated CRC, and showing negative KRAS

protein staining.

5 CONCLUSIONS

The prevalence of positive KRAS protein in CRC was

relatively the same as the prevalence of KRAS

mutation. However, the use of polyclonal antibody

which not specific to detect KRAS mutant became one

of the limitations of KRAS IHC assays. Therefore, the

development of a monoclonal antibody designed

against the mutated KRAS domain is necessary. This

could greatly assist the screening of CRC patients for

anti-EGFR therapies. In the future, IHC could

become a promising tool in diagnostic and prognostic

decisions.

Immunohistochemistry of KRAS Protein in Colorectal Cancer

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