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NRASQ61R Mutation-specific Immunohistochemistry Also Identifies the HRASQ61R Mutation in Medullary Thyroid Cancer and May Have a Role in Triaging Genetic Testing for MEN2

Reagh, Jessica MBBS; Bullock, Martyn PhD; Andrici, Juliana MBBS, MPhil; Turchini, John MBBS; Sioson, Loretta BSc; Clarkson, Adele BSc; Watson, Nicole; Sheen, Amy BSc; Lim, Grace BMedSci; Delbridge, Leigh MD, FRACS; Sidhu, Stan PhD, FRACS; Sywak, Mark MMed Sci, FRACS; Aniss, Ahmad PhD; Shepherd, Phillip BMLSc; Ng, Daniel BSc, BMLSc, MSc, MHGSA; Oei, Paul MSc, MHGSA, FFSc (RCPA); Field, Michael MB, ChB, FRACP; Learoyd, Diana PhD, FRACP; Robinson, Bruce G. MD, FRACP; Clifton-Bligh, Roderick J. PhD, FRACP; Gill, Anthony J. MD, FRCPA

The American Journal of Surgical Pathology: January 2017 - Volume 41 - Issue 1 - p 75–81
doi: 10.1097/PAS.0000000000000740
Original Articles

A quarter of patients with medullary thyroid carcinoma (MTC) have germline mutations in the RET proto-oncogene indicating MEN2. Therefore genetic testing is recommended for all patients presenting with MTC. Approximately 40% of MTCs have somatic RET mutations. Somatic mutations in the RAS genes are the next most common driver mutations and appear to be mutually exclusive with germline RET mutation. The single most common somatic RAS mutation is HRASQ61R (c.182A>G), reported in 4.6% to 11% of all MTCs. Mutation-specific immunohistochemistry (IHC) initially developed to identify the NRASQ61R mutation in melanoma (clone SP174) has proven highly sensitive and specific. Because the amino acid sequences for the HRAS and NRAS proteins at codon 61 are identical, we postulated that SP174 IHC would also identify the somatic HRASQ61R mutation. IHC with SP174 was performed on a tissue microarray of 68 patients with MTC including 13 (22.8%) with molecularly confirmed MEN2. Seven (10.3%) MTCs demonstrated positive staining. Six of these patients had already undergone germline RET mutation testing as part of clinical care and were all confirmed to be wild type, excluding the diagnosis of MEN2. All SP174 immunohistochemically positive MTCs were proven to have HRASQ61R mutation (and lack KRASQ61R and NRASQ61R) by Sanger sequencing. All MEN2 patients showed negative staining. We conclude that IHC with SP174 is highly specific for the HRASQ61R mutation in MTC. Because current data suggest that this mutation is mutually exclusive with germline RET mutation, IHC may also have a role in triaging formal genetic testing for MEN2.

Departments of *Anatomical Pathology


Endocrine Surgical Unit

**Familial Cancer Clinic, Royal North Shore Hospital

Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical Research

§Hormones and Cancer Group, Cancer Genetics Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital, St Leonards

Sydney Medical School, University of Sydney, NSW, Australia

Auckland UniServices Sequenom Facility, Liggins Institute

#IGENZ Ltd, Auckland, New Zealand

Conflicts of Interest and Source of Funding: Supported by funding from the Cancer Institute NSW as part of a translational research center grant. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.

Correspondence: Anthony J. Gill, MD, FRCPA, Department of Anatomical Pathology, Royal North Shore Hospital, Pacific Highway, St Leonards, NSW, Australia 2065 (e-mail:

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.

Medullary thyroid cancer (MTC) accounts for 1% to 2% of all thyroid carcinomas1; however, it is responsible for a disproportionately high number of deaths.1,2 Importantly, approximately 25% of MTCs occur in the setting of the autosomal dominant hereditary cancer syndrome multiple endocrine neoplasia type 2 (MEN2).1,3,4 MEN2 is subdivided into MEN2A (accounting for 95% of cases) and the relatively rare MEN2B in which aggressive MTC commonly presents in young children and in addition to being associated with phaeochromocytoma is also associated with multiple mucosal neuromas, generalized ganglioneuromas, gastrointestinal symptoms, and multiple musculoskeletal abnormalities including typical facies, a marfanoid habitus, and ocular abnormalities.1,4 MEN2A is further subdivided into classical MEN2A (associated with phaeochromocytoma and/or hyperparathyroidism), MEN2A with chronic lichen amyloidosis, MEN2A with Hirchsprung disease, and familial MTC in which manifestations other than MTC are absent.1 Both MEN2A and MEN2B are caused by activating germline mutations in the RET proto-oncogene on chromosome 10q11.2, and germline mutations in RET are found in approximately 98% of patients who fulfil clinical criteria for MEN2 and familial MTC and from 3% to 7% of patients with clinically sporadic MTC.1,4–8 The identification of germline RET mutations in an index patient presenting with MTC facilitates cascade genetic testing in family members and permits risk reduction surgery, that is prophylactic thyroidectomy in asymptomatic carriers at a time suggested by phenotype-genotype correlations of the specific familial RET mutation and other clinical factors.1,4 Therefore it is currently recommended that all patients who present with MTC should be offered germline mutation testing for the RET proto-oncogene.1

In addition to the approximately 25% of MTC patients who have germline RET mutations (ie, MEN2), 40% of truly sporadic MTC patients will have somatic mutations in RET in their tumors usually in the absence of germline mutations.1,4,8–14 Recently, somatic driver mutations in the RAS genes (HRAS, KRAS, and NRAS) have been identified in a further 10% to 45% of sporadic MTCs.1,4,8–13 Interestingly, these somatic RAS mutations have been consistently demonstrated to be mutually exclusive with germline RET mutations.1,4,8–13 In fact, to date there has only been 1 reported case of a somatic RAS mutation coexisting with an RET mutation in MTC, and this was a somatic-only RET mutation.12 That is, on the basis of existing data if a somatic RAS mutation is found in association with MTC, then germline RET mutation can be considered very unlikely.

RAS mutations in MTCs occur most commonly in HRAS (73.9% of all RAS mutations), followed by KRAS (23.1%) and, rarely, NRAS (2.9%).9–13 The single most common RAS mutation is the missense mutation (c.182A>G) in codon 61 of HRAS, which results in the amino acid arginine replacing glutamine and is therefore known as the HRASQ61R mutation. The HRASQ61R mutation accounts for 49% of HRAS mutations and 36% of all RAS mutations.9–13 Put another way, depending on the populations assessed, the overall incidence of somatic HRASQ61R mutation in MTC ranges from 4.6% to 11%, and on the basis of current data the HRASQ61R mutation is not found in tumors from patients with MEN2.

There has recently been significant interest in mutation-specific immunohistochemistry (IHC) in routine surgical pathology practice. For example, we and others have demonstrated that mutation-specific IHC is highly sensitive and specific for the presence of the pathogenic BRAFV600E mutation in papillary thyroid carcinoma15,16 and in fact may outperform molecular testing for this somatic mutation in the routine clinical setting.15 Second to BRAFV600E, the second most common pathogenic mutation in melanoma is NRASQ61R, and considerable clinical importance is placed on identifying this mutation in melanoma in routine clinical practice.17 Recently, a novel commercially available mutation-specific rabbit monoclonal antibody, clone SP174, was developed primarily to identify the NRASQ61R in melanoma.18 Mutation-specific IHC with clone SP174 has consistently demonstrated excellent sensitivity and specificity for the presence of the NRASQ61R mutation in melanoma, and strong arguments have been made to include it as part of routine surgical pathology practice.18–23

While investigating the utility of NRASQ61R mutation-specific IHC with clone SP174 in colorectal carcinoma, we recently demonstrated that the antibody detects both the NRASQ61R mutation and the KRASQ61R mutation.24 This is not unexpected given that the amino acid sequence for both the KRAS and NRAS proteins at codon 61 are identical, as is the substitution that results from the RASQ61R mutation (CAA>CGA). Using similar logic we postulated that mutation-specific IHC with clone SP174 would also identify the HRASQ61R mutation in MTC, because, although there is a slightly different DNA base pair sequence at codon 61 in HRAS (CAG>CGG), the associated amino acid substitution is identical.

We therefore sought to investigate whether mutation-specific IHC with clone SP174 could be used to identify the HRASQ61R mutation (or perhaps the much rarer KRASQ61R or NRASQ61R mutations) in MTC. Given that RASQ61R mutations are mutually exclusive with RET germline mutations in MTC we also specifically sought to investigate whether IHC may have some potential to triage formal genetic testing for MEN2 in patients presenting with MTC.

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The computerized database of the Department of Anatomical Pathology, Royal North Shore Hospital, Sydney Australia was searched for cases of MTC undergoing surgical resection from June 1, 1998 to June 1, 2014. Inclusion criteria included the presence of sufficient tumor in archived formalin-fixed paraffin-embedded (FFPE) blocks to permit tissue microarray (TMA) construction. All cases underwent independent review by an experienced endocrine pathologist (A.J.G.) to confirm the diagnosis of MTC. A TMA was then constructed containing two 1-mm-thick cores of FFPE tumor tissue.

IHC with the rabbit monoclonal anti-NRASQ61R antibody (clone SP174, cat: M4742; Spring Bioscience Pleasanton, CA) was performed on the TMA using an automated staining platform, the Leica Bond III autostainer (Leica Biosystems, Mount Waverley, Vic., Australia), using previously described methods.24 Briefly, the primary antibody was used at a dilution of 1 in 50 after heat-induced antigen retrieval for 30 minutes at 97°C in the manufacturer’s alkaline retrieval solution (ER2, VBS part no: AR9640; Leica Microsystems). This antibody dilution and antigen retrieval conditions had previously been determined by optimizing the antibody on a group of melanomas with known mutation status for NRASQ61R.

Two pathologists (J.R. and A.J.G.) independently scored the IHC on the TMA sections. Cases with no staining in neoplastic cells were classified as negative. Cases with strong and diffuse staining of all neoplastic cells (arbitrarily classified as >80% of neoplastic cells) were classified as positive. Cases with faint nonspecific staining were classified as equivocal. MTCs that demonstrated either positive or equivocal staining on the TMA sections then underwent repeat IHC staining on whole sections.

All the MTCs that demonstrated confirmed positive staining on whole sections underwent molecular testing in search of the HRASQ61R, KRASQ61R, and NRASQ61R mutations by 2 orthogonal methods—both Sanger sequencing and multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry detection (MALDI-TOF PCR) assay. The tumors were also tested for the presence of somatic RET mutations by Sanger sequencing performed on FFPE tissue.

For MALDI-TOF PCR testing for the HRAS61R mutation, 10×5 μm FFPE sections of each tumor were macrodissected and underwent testing using the Sequenom MassArray platform with a panel designed to detect the following RAS mutations: HRASQ61R, HRASQ61L, and HRASQ61P.

For Sanger sequencing to identify RASQ61R mutations and RET mutations in tumor tissue, 10×5 μm FFPE sections of each tumor were macrodissected and placed in an eppendorf container with care being taken to prevent cross-contamination. Genomic DNA was extracted from the FFPE sections using a QIAamp DNA FFPE Tissue Kit (Qiagen), used according to the manufacturer’s instructions.

To identify RAS mutations, specific regions encompassing codon 61 within exon 2 of HRAS and 3 of KRAS and NRAS genes were amplified by PCR using HotStarTaq DNA polymerase (Qiagen). Sequences of the PCR primers and amplification conditions were as described by Moura et al12 PCR products were purified using Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI) according to manufacturer’s instructions. Each sample was sequenced for HRAS, KRAS, and NRAS using forward and reverse primers on an ABI PRISM 3700 platform (Applied Biosystems, Foster City, CA) (service provided by Australian Genome Research Facility, Sydney, Australia).

To identify somatic RET mutations in tumors by Sanger sequencing, the DNA extracted from the tumors that showed positive staining with SP174 (described above) underwent further targeted sequencing to identify mutations in selected hotspots of RET covering exons 10 (C609, C611, C618, C620), exon 11 (C630), exon 15 (C883), and exon 16 (C918). This was performed using the PCR protocol established for the diagnostic genetic testing carried out at the Cancer Genetics laboratory, Kolling Institute of Medical Research at the Royal North Shore Hospital, Sydney, Australia. PCR primer sequences and amplification conditions are available upon request.

The medical records of all patients in the cohort, regardless of the results of SP174 IHC, were searched to identify the results of germline RET mutation testing performed as part of routine clinical care. During the period of this study it was routine to offer all patients presenting with MTC germline mutation testing for RET covering exons 10 (C609, C611, C618, C620), exon 11 (C630), exon 15 (C883), and exon 16 (C918). The Northern Sydney Local Health District Human Research Ethics Committee approved this study.

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There were 68 patients with MTC who fulfilled the study criteria and had sufficient material in the TMA to permit interpretation of IHC and therefore became the study cohort. The average age was 56 years, median age 60 years and range 22 to 84 years. Thirty-four patients (50%) were female. When IHC with the SP174 antibody was performed on the TMA sections, 7 cases were scored as positive (n=6) or equivocal (n=1), and 61 cases were scored as negative. When IHC was performed on whole sections, definitive positive staining was found in all 7 positive or equivocal cases. The IHC-positive cases demonstrated diffuse strong cytoplasmic staining of all neoplastic cells illustrated in Figure 1. The diffuse and strong cytoplasmic pattern of staining was consistent in all positive cases regardless of the age of the blocks or the length of formalin fixation. Adjacent and entrapped non-neoplastic cells were IHC negative. There was complete interobserver concordance in the interpretation of the IHC. There were no statistically significant associations between the results of IHC staining and age, sex, or tumor size (Table 1).





The results of molecular testing on the 7 tumors that demonstrated positive IHC staining for RASQ61R are presented in Table 2. Sufficient DNA for Sanger sequencing could not be extracted from FFPE from 1 of the tumors despite repeated attempts. Sequencing of the remaining 6 tumors all confirmed the presence of the HRASQ61R (c.182A>G) mutation. These 6 tumors were also confirmed by Sanger sequencing to be wild type at codon 61 of KRAS and NRAS, excluding NRASQ61R or KRASQ61R mutations.



Results were available for MALDI-TOF PCR for all 7 IHC-positive cases and are summarized in Table 2. One case was initially thought to be wild type for HRAS at codon 61 by MALDI-TOF PCR. As this was discrepant with the results of Sanger sequencing, the Sanger trace was rechecked, and sequencing was repeated and confirmed to unequivocally demonstrate the HRASQ61R (c.182A>G) mutation. MALDI-TOF PCR was then repeated twice. On the third attempt an unequivocal HRASQ61R peak was identified. That is, this sample was confirmed to have the HRASQ61R mutation by MALDI-TOF PCR despite 2 initial false-negative tests. The estimated neoplastic cellularity of the macrodissected area that underwent MALDI-TOF PCR in this case was 20%. One case was thought to harbor the HRASQ61L mutation by MALDI-TOF PCR and not the HRASQ61R mutation. As this was discrepant with the results of Sanger sequencing, the initial Sanger trace was rechecked, and Sanger sequencing was repeated and confirmed to unequivocally demonstrate the HRASQ61R (c.182A>G) mutation—illustrated in Figure 2. This was therefore considered an error of MALDI-TOF PCR. The remaining 5 cases were all confirmed to harbor the HRASQ61R mutation by MALDI-TOF PCR. Interestingly, the case for which Sanger sequencing was not possible was also thought to harbor HRASQ61L as well as HRASQ61R. Unfortunately, this could not be verified by an orthogonal approach as sufficient DNA could not be extracted for Sanger sequencing.



Sanger sequencing of FFPE tissue for the mutation hotspots associated with MEN2A (609, 611, 618, and 620 in exon 10; codon 634 in exon 11) and MEN2B (codon 883 in exon 15 and codon 918 in exon 16) from all 6 tumors from which sufficient DNA could be extracted was negative for mutation.

As part of clinical care, a total of 57 of the 68 patients in the cohort (83.8%) had previously undergone formal genetic counseling and germline mutation testing on peripheral blood and had germline RET mutation results available. Six of the 7 patients with MTCs demonstrating positive IHC staining with SP174 had been tested clinically, and all 6 patients were confirmed to lack pathogenic germline RET mutations and had no personal or family history to suggest MEN2. The 1 patient who did not undergo germline mutation testing was considered to be at very low clinical risk of MEN2 (an incidental 2.5 mm medullary carcinoma in a 60-y-old man found in a completion thyroidectomy performed to facilitate treatment of a minimally invasive follicular carcinoma in the contralateral lobe) and was therefore presumed to be RET wild type on clinical grounds, an inference supported by the absence of an RET mutation on Sanger sequencing of FFPE tumor tissue. Thirteen (22.8%) of the 68 patients in the cohort had confirmed germline RET mutation as part of routine clinical care, and all of the associated MTCs showed negative IHC staining for SP174.

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In this paper we provide strong evidence that mutation-specific IHC with clone SP174 is highly specific for the presence of the HRASQ61R mutation in MTC. Of the 7 cases that demonstrated positive staining by IHC (10.3% of the cohort tested), 6 cases underwent successful Sanger sequencing, and all were confirmed to harbor the HRASQ61R mutation and to lack KRASQ61R and NRASQ61R mutations. The 1 case that could not be sequenced was confirmed to harbor the mutation when tested by MALDI-TOF PCR. That is, the specificity of IHC for the presence of HRASQ61R mutation in MTC is 100% in our study.

SP174 IHC has also been shown by other studies to be highly specific and sensitive for the presence of the NRASQ61R mutation in melanoma18–23 or either the NRASQ61R or the KRASQ61R in colorectal carcinoma.24,25 Although we caution that we did not sequence IHC-negative cases for the presence of RAS mutations, and therefore we cannot formally assess the sensitivity of SP174 IHC for RASQ61R mutations in this study, our incidence of HRASQ61R mutation of 10.3% is at the higher end of the expected range of 4.6% to 11% suggested by other studies.9–13 That is, on the basis of the molecular findings published in other studies, SP174 IHC is likely to also be very sensitive for the presence of HRASQ61R mutation in MTC. However, we emphasize that this study was not designed or intended to assess the sensitivity of IHC for the presence of the HASQ61R mutation. By extrapolating the results in melanoma and colorectal carcinoma, it is likely that SP174 IHC will also detect the rare KRASQ61R (2 reported cases)12,26,27 and NRASQ61R (1 reported case)9,27 mutation in MTC, but again we emphasize that we were unable to validate this hypothesis as none of the MTCs we sequenced carried these mutations.

Although we have demonstrated that SP174 is highly specific and likely to be sensitive for the presence of the HRASQ61R mutation in MTC, the question of whether there is a clinical benefit in performing this IHC in the routine clinical setting remains. Indeed, current guidelines do not recommend testing MTCs for RAS mutation in routine clinical practice.1 Although it is difficult to justify the expense of somatic RAS mutation testing in MTC by molecular means, the ready availability, rapid turnaround time, and low cost of IHC may alter the cost-benefit equation significantly. In our opinion, one potential role for SP174 IHC in MTC is either to triage molecular testing for germline RET mutation in the resource poor setting or to act as a cheap orthogonal method of checking the results of germline molecular testing for RET—at least for the fraction of cases that harbor the HRASQ61R mutation. Given that we have proven that SP174 IHC is highly specific for the RASQ61R mutation in MTC and others have demonstrated that the RASQ61R mutation is mutually exclusive with germline RET mutation,1,4,8–13 a strong argument can be made that if an MTC demonstrates positive staining with SP174 then MEN2 can be considered excluded or virtually excluded and genetic testing can be considered very low yield. This is further supported by the comparison with the clinical molecular testing performed in our cohort. All 13 confirmed MEN2 patients demonstrated negative SP174 staining, and all 7 IHC positive patients were confirmed to lack RET mutations—6 in germline DNA from blood as part of clinical testing and all 7 in FFPE tumor tissue.

Certainly in the resource poor setting it would be reasonable to not offer genetic testing for MTC patients with SP174 IHC-positive tumors who have no personal or family history to suggest MEN2. However, given the ready availability of RET mutation testing in most centers that treat MTC, and the potentially devastating consequences for families with undetected MEN2, most units including our own would not withhold mutation testing on the basis of IHC alone. Currently, most laboratories take a targeted approach to genetic testing for RET mutations in index patients with MTC by first sequencing mutation hotspots in the most commonly mutated codons and only proceeding to sequence the entire coding region if no RET mutation is identified or there is a discrepancy between the MEN2 phenotype and the expected genotype, and this approach is supported by recent guidelines.1 Therefore a reasonable cost-saving measure even in well-resourced centers may be to not proceed beyond hot spot mutation testing in patients with SP174 IHC-positive apparently sporadic MTCs.

Although to date no somatic RAS mutations have been reported in the presence of RET germline mutations, we caution that a single MTC with both a somatic RAS and somatic RET mutation has been reported.12 Therefore the identification of an RASQ61R mutation cannot be taken to completely exclude MEN2 in patients with other clinical features to suggest hereditary disease, and we would still recommend proceeding to formal genetic testing in patients considered clinically to be at high risk of hereditary disease regardless of IHC staining.

Historically, particularly before the widespread availability of molecular testing for RET, the presence or absence of C-cell hyperplasia played a role in triaging germline mutation testing for MEN2. It is now clear that, although C-cell hyperplasia is more common in patients with MEN2 and therefore may provide a clue to an underlying hereditary basis of disease, depending on criteria used C-cell hyperplasia may also occur at a significant incidence in patients without germline RET mutation.28,29 That is, the presence of classical nodular C-cell hyperplasia in the absence of MTC may be a relative indication for germline RET mutation testing but lacks specificity, and similarly the absence of C-cell hyperplasia in patients with MTC does not exclude the possibility of MEN2. For this reason current American Thyroid Association guidelines recommend germline RET mutation testing be offered to all MTC patients regardless of other clinical or pathologic features including C-cell hyperplasia.1 In this study we did not formally assess whole sections for the presence or absence of C-cell hyperplasia, although we did observe that non-neoplastic C-cells were consistently negative by SP174 IHC in cases in which adjacent tumors were positive for HRASQ61R mutation.

The clinical significance of RAS mutations compared with germline or somatic RET mutations is currently not completely clear; however, there is some evidence to suggest that MTCs associated with somatic RAS mutations may be less aggressive (summarized in Moura et al27). In this study we were unable to find statistically significant associations between the results of IHC staining and age, sex, or tumor size (Table 1), and at this stage we have insufficient data to link the MTCs to long-term follow-up. Therefore further studies are required to resolve whether SP174 IHC will have a role in risk stratification of sporadic MTC.

In recent years several tyrosine kinase inhibitors, including axitinib, cabozantinib, gefitinib, imatinib, motesanib, sorafenib, sunitinib, and vandetanib, have been evaluated for use in MTC, and vandetanib and carbozantinib have gained FDA approval for the treatment of patients with advanced progressive MTC.1 Although a recent meta-analysis has demonstrated the benefit of carbozantinib in patients with RET or RAS mutations, it may be that there are specific genotype-dependent rates of response for which the use of mutation-specific IHC could aid further understanding.30

In conclusion, we demonstrate that IHC with the rabbit monoclonal antibody SP174, originally developed to detect the NRASQ61R mutation in melanoma, is highly specific for the presence of the HRASQ61R mutation in MTC. This mutation was confirmed in 10.3% of unselected MTCs in our series. Given that the HRASQ61R mutation (and other RASQ61R mutations that may also be detected by this antibody) appear to be mutually exclusive with germline RET mutations, MTCs which show positive staining with this antibody are at very low risk of being associated with MEN2. Therefore IHC with clone SP174 may have a role in the resource-poor setting to triage genetic testing for MEN2 in patients presenting with MTC or may serve as a cheap orthogonal method for checking the results of molecular testing in the significant proportion of MTCs, which have somatic HRASQ61R mutation and are therefore very unlikely to be associated with germline RET mutation.

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medullary thyroid cancer; HRASQ61R; NRASQ61R; MEN2; SP174; RET

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