SP174, NRAS Q61R Mutant-Specific Antibody, Cross-Reacts With KRAS Q61R Mutant Protein in Colorectal Carcinoma

Abstract

Context.

NRAS is a member of the RAS family oncoproteins implicated in cancer. Gain-of-function NRAS mutations were reported in a subset of colorectal cancers. These mutations occur at codons 12, 13, and 61 and are detected by molecular genetic testing. Recently, an antibody (clone SP174) became available to immunohistochemically pinpoint NRAS Q61R mutant protein. In malignant melanoma, NRAS Q61R mutant–specific immunohistochemistry was shown to be a valuable supplement to traditional genetic testing.

Objective.

To evaluate the significance of NRAS Q61R mutant–specific immunohistochemistry in a cohort of colorectal carcinomas.

Design.

A total of 1185 colorectal carcinomas were immunohistochemically evaluated with SP174 antibody. NRAS Q61R mutant–specific immunohistochemistry was validated by molecular genetic testing including Sanger sequencing, quantitative polymerase chain reaction (qPCR), and next-generation sequencing.

Results.

Twelve tumors showed strong SP174 immuno-reactivity. Sanger sequencing detected an identical c.182A>G substitution, causing NRAS Q61R mutation at the protein level, only in 8 SP174-positive cases. These results were confirmed by qPCR study. Subsequently, NRAS wild-type tumors with strong SP174 staining were evaluated by next-generation sequencing and revealed KRAS c.182A>G substitutions predicted to cause KRAS Q61R mutation. Review of colorectal carcinomas with known KRAS and NRAS genotype revealed that none of 62 wild-type tumors or 47 mutants other than Q61R were SP174 positive.

Conclusion.

SP174 immunohistochemistry allows sensitive detection of NRAS and KRAS Q61R mutants. However, molecular genetic testing is necessary to determine specifically which RAS gene is mutated.

The RAS proto-oncogenes (HRAS, KRAS, and NRAS) encode HRAS, KRAS 4A, KRAS 4B, and NRAS, 4 distinct but closely related and highly homologous proteins. These enzymes, located on the inner surface of the cell membrane, represent GDP/GTP-regulated switches that convey extracel-lular signals and play a central role in signal transduction, regulating cell proliferation, and apoptotic cell death.¹

In human cancer, gain-of-function RAS mutations are among the most common genetic changes with an estimated incidence around 20%. Yet, the frequency of RAS mutants widely depends on type of cancer. Amino acid residues G12, G13, and Q61 are main mutational “hotspots,” although other codons including 59, 117, and 146 could be affected.^2,3

Typically, mutational analysis of RAS oncogenes is done at the DNA level by using detection methods based on the polymerase chain reaction (PCR) amplification of mutational hotspots. The melting curve analysis, Sanger sequencing, pyrosequencing, and quantitative PCR (qPCR) assay are among the most commonly used strategies. However, recently, the mutant protein–specific antibodies have been introduced into immunohistochemistry as an alternative, robust tool for the detection of specific mutations in cancer.^4,5

Recently, a rabbit monoclonal antibody (clone SP174) against NRAS Q61R mutant protein has been made commercially available. Several studies successfully used immunohistochemistry with this antibody to assess NRAS Q61R mutant protein in primary and metastatic malignant melanomas. Because of its remarkable sensitivity and specificity, this immunohistochemical assay has been considered a valuable tool for routine detection of NRAS Q61R mutants in malignant melanoma.^6–8

In this study, the value of NRAS Q61R mutant–specific immunohistochemistry was evaluated in a large cohort of colorectal carcinomas.

MATERIALS AND METHODS

Study Material and Design

A total of 1185 anonymized colorectal carcinoma specimens from Europe and the United States were analyzed for this study. This cohort contained a series of previously characterized 110 adenocarcinomas with known NRAS and KRAS mutation status: 62 KRAS/NRAS wild type, 45 KRAS, and 3 NRAS mutant tumors. The latter included 1 NRAS Q61R mutant. Multitissue blocks were prepared as described.⁹ Immunostaining assays were performed in the Laboratory of Pathology (LP), National Cancer Institute, Bethesda, Maryland. DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues by following a previously reported procedure.¹⁰ In selected cases, tumor cell content was enriched by manual dissection. Screening for RAS mutations was completed independently in LP and in the Department of Molecular Diagnostics, Holycross Cancer Center, Kielce, Poland. Mutation analyses were done blindly without knowledge of the results from immunohistochemical studies. RAS mutation detection methods included Sanger sequencing, qPCR assay, and Ion Torrent (Life Technologies/Thermo Fisher Scientific, Waltham, Massachusetts) next-generation sequencing. The study protocol was approved by the Office of Human Subject Research (National Institutes of Health, Bethesda, Maryland).

Immunohistochemistry

Expression of the NRAS Q61R mutant protein was evaluated immunohistochemically by using a rabbit monoclonal antibody, clone SP174 (Spring Bioscience, Pleasanton, California). Immunostaining assays were performed on Leica Bond-Max automatic immunostainer (Leica, Bannockburn, Illinois) and Leica Refine Detection Kit. The primary antibody was incubated for 30 minutes, followed by epitope retrieval using Leica H2 buffer (25 minutes). The 1:200 dilution of primary antibody was selected as the lowest dilution yielding a strong signal in a series of positive controls (NRAS Q61R mutant malignant melanomas) while giving no staining in negative controls. The Leica polymer and postpolymer were each incubated for 30 minutes, followed by 10 minutes in diaminobenzidine, and a light hematoxylin counterstain. Immunohistochemical stains were scored arbitrarily by using a visual 4-level scale: negative (0); weakly (+1), moderately (+2), and strongly (+3) positive.

Molecular Studies

Sanger Sequencing.

NRAS and KRAS codon 61 sequences were PCR amplified and amplification products were evaluated by Sanger sequencing as previously reported.¹¹

Quantitative PCR Assay.

NRAS and KRAS mutation status was evaluated by RAS Mutation Analysis Kits NRAS-RT50 and KRAS-RT50 and analyzed by using Rotor-Gene Q Software version 1.7 according to the manufacturer’s instructions (EntroGen Inc, Woodland Hills, California). These tests detect spectrum of NRAS and KRAS substitutions leading to NRAS Q61H, -K, -L, and -R, and KRAS Q61H, -L, and -R mutations at the protein level.

Next-Generation Sequencing.

Next-generation sequencing was performed with the Ion Torrent next-generation sequencing platform and Ion AmpliSeq Cancer Hotspot Panel v2 Kit in accordance with the manufacturer’s instructions (Life Technologies/Thermo Fisher Scientific) and following a previously published detailed procedure.¹²

RESULTS

Screening of 1185 colorectal carcinomas revealed 12 tumors (in 1 case primary and metastatic lesion) with immunoreactivity to SP174, NRAS Q61R mutant-specific antibody. A strong (+3) diffuse cytoplasmic and granular membrane staining was documented in all SP174-positive tumors. Examples of SP174 immunohistochemistry are shown in Figure 1, A through D. All SP174-positive tumors were evaluated with Sanger sequencing. An identical single nucleotide substitution, NRAS c.182A>G, predicted to cause NRAS Q61R mutation at the protein level, was identified in 8 of 12 analyzed samples. Subsequently, a blinded qPCR study confirmed Sanger sequencing results. Next, NRAS wild-type tumors showing strong staining with SP174 antibody were evaluated by Ion Torrent next-generation sequencing. All cases revealed KRAS c.182A>G substitution predicted to cause KRAS Q61R mutation at the protein level. These results were confirmed by both qPCR assay and Sanger sequencing experiments in an independent manner. Representative examples of Sanger sequencing and next-generation sequencing are shown in Figure 2, A and B, and Figures 3 and 4.

Figure 1.

A through D, Two colorectal carcinomas: case 1 (A and B) and case 2 (C and D). Strong immunoreactivity to SP174, NRAS Q61R mutant-specific antibody (B and D) (hematoxylin-eosin, original magnifications ×200 [A] and ×100 [C]; immunohistochemistry, original magnifications ×200 [B] and ×100 [D]).

Figure 2.

A and B, Sanger sequencing of NRAS codon 61. NRAS c.182A>G substitution predicted to cause NRAS Q61R mutation in case 1 (A) and NRAS wild-type sequence in case 2 (B).

Figure 3.

Ion Torrent next-generation sequencing revealing KRAS c.182A>G substitution predicted to cause KRAS Q61R mutation in case 2.

Figure 4.

Sanger sequencing of KRAS codon 61 in case 2 revealing KRAS c.182A>G substitution and confirming results of Ion Torrent next-generation sequencing.

None of the NRAS/KRAS wild-type tumors and NRAS/KRAS mutants other than Q61R showed immunoreactivity with SP174 antibody. Results of immunohistochemical and genotyping studies are summarized in the Table.

Summary of Immunohistochemical and Molecular Genetic Studies of 1185 Colorectal Carcinomas

	Genotype
IHC SP174	KRAS					NRAS		KRAS and NRAS		Total No. of Tumors
	G12A (n = 2)	G13D	Q61R	Q61H (n = 4)	A146T (n = 4)	Q61H (n = 1)	Q61R	Wildtype	Mutation status unknown
	G12C (n = 6)			Q61L (n = 3)	A146V (n = 1)	Q61K (n = 1)
	G12D (n = 13)
	G12S (n = 1)
	G12V (n = 8)
Negative	30	3	0	7	5	2	0	62	1064	1173
Positive	0	0	4	0	0	0	8	0	0	12
										1185

Abbreviation: IHC, immunohistochemistry.

DISCUSSION

Approximately 40% of colorectal cancers carry KRAS mutations. By contrast, NRAS mutations are rather uncommon and could be identified in approximately 2% to 4% of analyzed tumors. A c.182A>G substitution in codon 61 leading to KRAS or NRAS Q61R mutation at the protein level is rare and has been reported in no more than 1% of colorectal carcinomas.^13–15

In vitro studies on genetically engineered mice demonstrated phenotypic differences between KRAS and NRAS mutants.¹⁶ In colorectal cancer, NRAS mutation correlates with less favorable clinical outcome and might require different treatment strategies.¹⁷ Thus, precise identification of mutation type is of major clinical importance.

Recently, the use of anti–epidermal growth factor receptor (EGFR) antibodies (cetuximab and panitumumab) has become a clinical standard in the treatment of metastatic colorectal cancer. Mutations affecting EGFR-signaling axis molecules including KRAS, NRAS, BRAF, and PIK3CA predict resistance to anti-EGFR therapies.¹⁸ The screening of these genes for specific mutations has been recommended before applying treatment with anti-EGFR agents.

This study revealed that immunohistochemistry with SP174 antibody did not allow identification of NRAS Q61R versus KRAS Q61R mutant protein. The immunoreactivity of SP174 antibody with both NRAS Q61R and KRAS Q61R mutant proteins has to be attributed to the high, almost 100% sequence homology of the amino-terminal catalytic domain (amino acids 1–165) of RAS proteins.¹⁹

Previous studies focused on malignant melanomas failed to address this matter because KRAS Q61R mutants are extremely rare in this type of cancer.^6–8 Catalogue of Somatic Mutations in Cancer (COSMIC) listed only 1 publication reporting KRAS c.182_183AA>GT double substitution predicted to cause Q61R mutation in a case of malignant melanoma.²⁰

Next-generation sequencing, as shown in this study, is a robust tool for the detection of RAS mutation in colorectal carcinomas.² However, availability of this technology is still restricted to research/academic centers. Also, in some cases, it may not be applicable because only a limited amount of tissue is available for testing. In such context, immunohistochemistry seems to be a valuable alternative approach.

This study showed that immunohistochemistry with SP174 antibody allowed identification of KRAS and NRAS Q61R mutant proteins. Although specificity and sensitivity of this test were 100%, differentiation between KRAS Q61R and NRAS Q61R was not possible. Thus, immunohistochemistry with SP174 antibody is a useful tool in the screening of colorectal carcinomas for RAS Q61R mutant proteins; however, molecular genetic confirmation is necessary to determine which RAS gene is mutated.