Introduction

MUTYH-associated polyposis (MAP) is an autosomal-recessive disease characterized by multiple colorectal adenomas and cancer [1]. Approximately 30% of patients with more than15 adenomas that do not carry pathogenic APC mutations are biallelic MUTYH mutation carriers [2]. MAP was first reported in a British family, in which three affected siblings were compound heterozygote for MUTYH mutations [1]. MUTYH acts together with OGG1 and MTH1 in the base excision repair (BER) system, a repair system to defend cellular DNA against the mutagenic effects of 7,8-dihydro-8-oxoguanine (8-oxoG) [2, 3]. 8-OxoG easily mispairs with adenine residues leading to G:C → T:A transversion mutations in the daughter strand [1, 4]. Normally, MUTYH is expressed in mitochondria and in the nuclei of human cells [5].

Identification of patients with biallelic MUTYH mutation-associated polyposis is important to target effective preventive measures for patients and their families, which may lead to reduction in CRC-related mortality. DNA mutation analysis can determine the possible genetic cause of polyposis. To avoid expensive, unnecessary, and time-consuming DNA mutation analyses, there is a need for a screening test to select individuals eligible for DNA mutation analysis.

Immunohistochemical analysis is a rapid and inexpensive method, useful for a wide range of diseases. In a recent study by Di Gregorio et al., immunohistochemical staining of MUTYH protein was performed to identify patients with MAP [6]. A specific pattern of staining for the MUTYH protein was seen; unlike in patients without MUTYH mutations, patients with biallelic MUTYH mutations showed absence of nuclear staining and segregation of immune reactivity in the cytoplasm (supranuclear staining), both in neoplastic and surrounding healthy mucosa [6]. Therefore, tissues from patients with and without mutations might be distinguished from each other. Consequently, IHC could be used to identify patients with MUTYH-associated polyposis. The aim of this study is to further investigate the possibilities of immunohistochemistry as a pre-screening test to select patients for MUTYH mutation analysis.

Materials and methods

The study included 20 samples from 19 patients, divided into three groups. Samples were collected in five different pathology laboratories in different hospitals in the Netherlands (Radboud University Nijmegen Medical Center; Rijnstaete Hospital, Arnhem; Amphia Medical Center, Breda; Jeroen Bosch Hospital, Den Bosch; MedischSpectrum Twente, Enschede).

Group 1 consists of five patients carrying biallelic MUTYH mutations (six samples from colorectal carcinoma or adenoma); an overview of the clinical features and mutations of these patients is given in Table 1. All patients were compound heterozygous for pathogenic mutations in MUTYH. Mutation analysis of MUTYH was performed, in the Leiden University Medical Center, as described by Nielsen et al. [7], with sequence analysis of exon 1 till 16. Group 2 consists of 11 patients with polyposis or CRC without detectable mutations in MUTYH (11 samples from adenoma or CRC). Group 3 consists of three patients carrying a monoallelic MUTYH mutation (with three samples from adenomas and normal mucosa).

Table 1 Clinical features of patients with MUTYH-associated polyposis, the type biallelic mutations, and the immunoreactivity pattern of MUTYH protein, using the Abcam antibody
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Immunohistochemical staining was performed on 4-μm-thick, formalin-fixed, paraffin-embedded tissue sections that were prepared on coated slides and dried for 30 min at 55°C. Tissue sections were deparaffinized in xylene and rehydrated with alcohol. Antigen retrieval was done by boiling in 10 mM citrate buffer (PH 6) for 10 min at 95°C. Endogenous peroxidase activity was blocked by exposing the slides to 3% H2O2 in methanol for 10 min. Then, sections were incubated with primary MUTYH antibody overnight at 4°C. Polyclonal MUTYH antibody (residues 531–546, Abcam, Cambridge, UK) at 1:300, primary polyclonal MUTYH antibody (residues 33–51, Calbiochem) at 1:1,600, and primary monoclonal MutYH antibody (clone 4D10, Abnova Corporation) at 1:200 were used. These dilutions were determined after examining several dilution series, to obtain the best results. Next, sections were incubated with Poly-HRP-GAM/R/R IgG for 30 min. Visualizing was done with DAB for 5 min. Nuclei were counterstained with hematoxylin. Slides were dehydrated, cleared in xylene, and mounted with micromount.

Normal immunoreactivity of the MUTYH protein was defined as the presence of nuclear and light cytoplasmic staining. Altered expression was considered when the cells showed disappearance of staining from the nucleus and instead showed supranuclear staining. Staining for MUTYH in the nucleus was evaluated by the scoring system Gao et al. reported: 0 = no staining; 1 = minimal to mild staining (10–50% section positive); 2 = strong staining (>50% section positive) [8]. Cytoplasmic staining was classified as present or absent.

Results

With the polyclonal antibody, adenomas and carcinomas of all patients with MUTYH biallelic mutations showed strong supranuclear cytoplasmic staining, without nuclear expression of protein (Table 1). Adjacent normal mucosa, in patients with biallelic MUTYH mutations, showed the same pattern of expression found in adenomas and carcinomas. As shown in Fig. 1, supranuclear cytoplasmic staining was localized at the apex of the colonocytes (a) or neoplastic cells (b). The 11 samples of colorectal tissue of patients without MUTYH mutations, incubated with the polyclonal antibody, showed several patterns (Table 2 and Fig. 2). Nine samples showed the supranuclear cytoplasmic staining described above of which five had weak and four no epithelial nuclear staining. Two samples did not show the supranuclear cytoplasmic staining; they showed weak nuclear and weak cytoplasmic staining. All three samples of colon tissue of patients with one MUTYH mutation showed supranuclear staining; additionally, two showed also nuclear staining.

Fig. 1
figure 1

Strong epithelial supranuclear cytoplasmic immunoreactivity and absence of nuclear expression of MUTYH protein in a normal mucosa (original magnification, ×500) and b carcinoma (original magnification, ×100) of patients with biallelic MUTYH mutations. Note nuclear staining in some stromal fibroblasts

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Fig. 2
figure 2

Limited epithelial supranuclear cytoplasmic immunoreactivity and occasional nuclear expression of MUTYH protein in a normal mucosa but strong supranuclear and absent nuclear staining in b adenoma of patients without biallelic MUTYH mutations. Occasional nuclear staining in stromal cells (original magnification, ×500)

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Table 2 Clinical features of patients without MUTYH-associated polyposis, the mutations tested, and the immunoreactivity pattern, using the Abcam antibody
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Diffuse cytoplasmic staining was observed in some samples either with or without MUTYH mutations; the intensity was always weak. From these results, we conclude that we could not differentiate between tissue with or without MUTYH mutations, while using the polyclonal antibody.

Two monoclonal antibodies were used to evaluate the MUTYH protein staining pattern as well. Samples incubated with the Calbiochem antibody showed strong nuclear and cytoplasmic staining for all tissue regardless whether MUTYH mutations were present. Samples incubated with the Abnova antibody showed no epithelial nuclear or cytoplasmic staining at all; however, nuclear staining was observed in stroma cells. This pattern was the same for tissue with and without MUTYH mutations.

Discussion

Using the commercially available polyclonal antibody for MUTYH, cytoplasmic staining in neoplastic cells does not discriminate MUTYH-mutated samples from unmutated cases; presence of nuclear staining excludes the MUTYH mutation, but is of limited specificity. Samples incubated with the Calbiochem or Abnova antibody showed a non-specific pattern since no differentiation was possible between tissue with and without MUTYH mutations. Consequently, the two other antibodies did not seem to work on formalin-fixed, paraffin-embedded tissue after using different pretreatments and dilutions. There are no indications that, with the present mutation analysis of MUTYH, mutations are being missed (reported by C.M.J. Tops, Center for Human and Clinical Genetics, Leiden University Medical Center).

There are just a few immunohistochemical studies of the MUTYH protein described. Di Gregorio et al. described that tissue of patients with biallelic MUTYH mutations showed absence of nuclear staining and segregation of immunoreactivity (supranuclear) in the cytoplasm [6]. Their hypothesis for this pattern was that the protein produced by the mutated gene could lack the capacity to transfer into the nucleus and remain trapped in the cytoplasm [6]. Our results confirm this finding, but importantly we show that this pattern of staining does not distinguish between tissue of patients with and without MUTYH mutations. Recently, O’Shea et al. published results more consistent with our own, showing MUTYH immunohistochemistry not discriminating controls, biallelic, and heterozygote MUTYH mutation carriers [9].

Koketsu et al. showed that loss of expression of the BER proteins, MUTYH, MTH1, and NTH1 occurs in sporadic colorectal cancer [10]. Nuclear MUTYH immunoreactivity was detected in only 57% of cases (46/81) [10]. They described that the presence of nuclear MUTYH expression showed a significant correlation with the T-stage of the tumor (p = 0.04) [10].

Further, it is not clear whether MUTYH protein is always expressed in the nucleus. Boldogh et al. showed that the majority of MUTYH protein was distributed in the cytoplasm, which is in agreement with a mitochondrial association of MUTYH, and that in only a small percentage (3–5%) of the cells MUTYH-specific fluorescence was also localized to the nuclei [11]. These findings are in contrast with the data of Tsai-Wu et al. which suggest that the MUTYH protein is mainly nuclear specific, based on their own polyclonal rabbit antibodies [12]. Recently, it was shown by Van Puijenbroek et al. that somatic KRAS2 mutation testing of carcinomas can successfully be used as a pre-screening test for germline MUTYH mutation analysis [13].

In conclusion, our results demonstrate that, in contrast with the findings of Di Gregorio et al., while using the same methods and two additional antibodies, cytoplasmic expression of the MUTYH protein is not specific for germline MUTYH mutation. At present, immunohistochemistry cannot be used in clinical practice to differentiate between colorectal tissue with and without MUTYH mutations.