Improved Molecular Classification of Serrated Lesions of the Colon By

Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E Ildiko Mesteri1,Gu¨ nther Bayer1, Jochen Meyer2,3, David Capper2,3, Sebastian F Schoppmann4, Andreas von Deimling2,3 and Peter Birner1,2

1Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria; 2Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Heidelberg, Germany; 3Clinical Cooperation Unit Neuropathology, German Cancer Research Centre, Heidelberg, Germany and 4Department of Surgery, Medical University of Vienna, Vienna, Austria

BRAF V600E mutation in serrated lesions of the colon has been implicated as an important mutation and as a specific marker for the serrated carcinogenic pathway. Recent findings point to microvesicular hyperplastic polyps that have similar histologic and molecular features to sessile serrated adenomas/polyps, as potential colorectal carcinoma precursors. The aim of this study was to evaluate BRAF V600E mutation status by immunohistochemistry in serrated lesions of the colon with regard to histomorphology. We investigated 194 serrated lesions of the colon, comprising 42 sessile serrated adenomas/polyps, 16 traditional serrated adenomas, 136 hyperplastic polyps and 20 tubular/tubulovillous adenomas (conventional adenomas) with the novel BRAF V600E mutation-specific antibody VE1. In addition, BRAF exon 15 and KRAS exon 2 status was investigated by capillary sequencing in selected cases. All sessile serrated adenomas/polyps (42/42, 100%), 15/ 16 (94%) traditional serrated adenomas and 84/136 (62%) hyperplastic polyps were VE1 þ . None of the VE1 À serrated lesions showed BRAF V600E mutation. Forty out of 42 (95%) sessile serrated adenomas/polyps displayed areas with microvesicular hyperplastic polyp-like features. In microvesicular hyperplastic polyps, VE1 positivity was significantly associated with nuclear atypia (P ¼ 0.003); however, nuclear atypia was also present in VE1 À cases. Immunostaining with VE1 allows not only the detection of BRAF V600E mutation but also the correlation with histomorphology on a cellular level in serrated lesions. VE1 enables a subclassifica- tion of microvesicular hyperplastic polyps according to the mutation status. This improved classification of serrated lesions including immunohistochemical evaluation of BRAF V600E mutation may be the key to identify lesions with higher potential to progression into sessile serrated adenoma/polyp, and further to BRAF V600E- mutated colorectal cancer. Modern Pathology (2014) 27, 135–144; doi:10.1038/modpathol.2013.126; published online 26 July 2013

Keywords: BRAF; colon; immunohistochemistry; morphology; serrated polyp

Colorectal cancer is one of the leading causes of A recent study suggested that colorectal cancers of cancer death worldwide. Although increased use of the proximal colon develop in a significantly shorter screening and surveillance, as well as detection and period of time than those in the distal colon, after the removal of conventional adenomas, has led to a a patient has had a negative colonoscopy.2,3 Thus, reduction in the incidence and mortality of this there is a need for improved and targeted identi- disease, this effect is mainly limited to distal fication of neoplastic precursor lesions in the colorectal cancer.1–5 However, difficulties remain proximal colon. in the prevention of proximal colorectal cancer. Colorectal cancer arises from several different types of precursor lesions. In the conventional pathway, carcinomas develop from adenomas via Correspondence: Professor P Birner, MD, MSc, Clinical Institute biallelic APC (adenomatous polyposis coli) muta- of Pathology, Medical University of Vienna, Wa¨hringer Gu¨ rtel tions6 and are reinforced by KRAS mutation. 18-20, 1090 Vienna, Austria. E-mail: [email protected] In the other major pathway, known as the serrated Received 8 November 2012; accepted 3 June 2013; published carcinogenic pathway, tumors develop from serrated online 26 July 2013 precursor lesions via BRAF mutations.7–9 Serrated www.modernpathology.org BRAF in serrated lesions of the colon 136 I Mesteri et al

precursor lesions consist of sessile serrated expression in serrated lesions of the colon in corre- adenomas/polyps (either with or without lation with morphology. The expression of BRAF cytologic dysplasia) and traditional serrated V600E protein in microvesicular hyperplastic adenomas.9–12 Serrated lesions constitute the polyps (with and without nuclear atypia), sessile main precursor lesion in at least one-third of serrated adenomas/polyps (with and without cytolo- all colorectal cancers.13,14 Progression results in gic dysplasia) and traditional serrated adenomas microsatellite-unstable colorectal cancer and should aid in subclassifying these lesions also possibly also CpG island-methylated microsatellite- according to their BRAF V600E mutation status stable carcinomas.3,9–12 Although consensus exists in addition to histomorphologic classification. In that sessile serrated adenomas/polyps and traditio- routine pathology, this would identify serrated nal serrated adenomas are colorectal cancer precur- lesions carrying the oncogenic BRAF V600E mutation, sor lesions,13 hyperplastic polyps were considered which might have an increased risk of progression to for decades to have no malignant potential. colorectal cancer. The implementation of a new This has been questioned in the past years and classification based on distinct molecular pheno- different subsets of hyperplastic polyps with dis- type could offer patients an individualized cancer tinct molecular profiles and potential to progress to prevention and therapy option. colorectal cancer have been identified.15 These findings implicate that BRAF V600E-mutated colorectal cancers have their origin in hyperplastic polyps classified morphologically as microvesicular Materials and methods 9,15,16 hyperplastic polyps. Should this be correct, a Tissue Samples refined classification of microvesicular hyperplastic polyps with regard to BRAF V600E mutation might Institutional review board approval was obtained for lead to an improved assessment of colorectal cancer this study. risk and guide patient management. Formalin-fixed, paraffin-embedded samples of In contrast to conventional adenomas where serrated, adenomatous and hyperplastic colon cytological changes have an important role in lesions were identified in the archives of the identifying dysplasia, diagnosis of the various Clinical Institute for Pathology of the Medical subclasses of serrated lesions is mainly based on University of Vienna (Vienna, Austria). Samples the architectural criteria.17,18 Owing to sampling that were improperly oriented or did not contain issues, poor specimen orientation and a significant sufficient amounts of tissue for further analysis were interobserver variation among pathologists,19–22 the excluded from the study. Before the immunohisto- differential diagnosis of microvesicular hyperplastic chemical evaluation with VE1 antibody, all tissue polyps vs sessile serrated adenomas/polyps can be samples were reviewed and histologically subclas- very challenging or even impossible. Especially, if sified on hematoxylin- and eosin-stained slides we consider that for the differential diagnosis of according to the current WHO scheme.27 microvesicular hyperplastic polyp vs sessile In brief, classification was based on the following serrated adenoma/polyp, the presence of the criteria: appropriate morphologic criteria in just one crypt Goblet-cell-rich hyperplastic polyp: comprises is considered as sufficient.13 pure goblet cells, straight crypts with proliferative It has been suggested that BRAF mutation is one of activity confined to the lower crypt, showing the important player in two of the five molecular minimal serration. subtypes of colorectal cancer according to Jass.23 Microvesicular hyperplastic polyp: comprises Currently, there are no established morphologic epithelial cells with hypermucinous microvesicular criteria that would identify oncogenic BRAF V600E cytoplasm only or mixed with goblet cells with mutation in serrated lesions. Until recently, maintained maturation towards the surface. Crypts assessment of BRAF V600E mutation status was are straight, dilatation/serration is confined in the not feasible in routine pathology, as sequencing of more luminal aspects, crypt bases are narrow with all serrated lesions is not suitable for everyday symmetrical proliferative activity and interspersed diagnostic pathology. In addition, direct sequencing neuroendocrine cells. might provide false-negative results owing to Sessile serrated adenoma/polyp: The criteria for dilution of the diagnostic mutation through a high the diagnosis, as suggested by an expert panel,13 was number of normal mucosa cells. In this regard, even the minimum of one crypt that clearly showed microdissection cannot give proper evidence of the dilatation and/or serration extending to the crypt distribution of cells with BRAF V600E mutation base, with or without branching (L-, inverted T- or within serrated lesions. anchor-shape) of the crypts and abnormal pro- The development of a novel, mutation-specific liferation (proliferative zone located on the side, antibody has enabled us to investigate the presence frequently asymmetrical) showing goblet cells or of V600E-mutated BRAF protein in formalin-fixed, gastric foveolar cells in the crypt basis. In addition, paraffin-embedded sections.24–26 The aim of this absence or presence of cytologic dysplasia were study was to evaluate BRAF V600E protein determined.

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Figure 1 Normal colonic mucosa: (a) hematoxylin and eosin (H&E), Â 100; (b) Immunostain VE1, Â 100. Goblet-cell-rich hyperplastic polyp: (c) H&E, Â 200; (d) Immunostain VE1, Â 200.

Traditional serrated adenoma: shows villiform Immunohistochemistry. A Benchmark ultraimmu- growth pattern with serration due to infolding, nostainer was used for immunohistochemistry budding and papillary tufting, composed of tall (Ventana, Tucson, AZ, USA). columnar cells with pencillated nucleus and hyper- Expression of V600E-mutated BRAF was evaluated eosinophilic cytoplasm with interspersed goblet in 3-mm-thick histological slides using the mono- cells. In addition, the grade of cytologic dysplasia clonal BRAF V600E mutation-specific antibody VE1 was determined. (provided by AvD, the antibody is commercially In total, 194 consecutive serrated lesions of the available from Spring Bioscience, Pleasanton, CA, colon were included in our study, these included: USA). Production and validation of this antibody has 42 sessile serrated adenomas/polyps (38 without been published in detail previously.28 Aspecimen cytologic dysplasia and 4 with cytologic dysplasia), was considered positive for BRAF V600E if distinct 16 traditional serrated adenomas with low-grade cytoplasmic staining reaction with the VE1 antibody dysplasia, 119 microvesicular hyperplastic polyps was present (samples of immunostaining are given and 17 goblet-cell rich hyperplastic polyp. Addi- in Figures 1–4).24,26 Nonspecific nuclear staining tionally for comparison, 20 tubular/tubulovillous without cytoplasmic signal was detected in the (conventional adenomas) with low-grade dysplasia surface epithelium of adjacent normal colon mucosa mainly from the right colon were also included. (Figure 1). This nuclear staining pattern has also Mucin-poor-type hyperplastic polyps were not been previously reported in brain metastases, but in found in our series. that study this type of staining showed no correla- Microvesicular hyperplastic polyps were evalu- tion with BRAF mutation status.26 ated for the degree of nuclear atypia according to the To exclude the possibility that nuclear staining schema of Torlakovic et al.17 In addition, sessile pattern with VE1 in normal mucosa corresponded to serrated adenomas/polyps were reviewed for the the presence of BRAF protein, cases with sufficient presence of areas revealing a morphology of material available from VE1 À hyperplastic polyps, microvesicular hyperplastic polyps. which showed nonspecific nuclear staining, were The classification was carried out by three immunostained with a pan-BRAF-antibody detect- independent pathologists (IM, GB and PB), with ing total (mutated and wild-type) BRAF (pBR1, consecutive consensus for each case. dilution 1:4).28 In these cases, only cytoplasmic, but

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Table 1 Results of VE1 immunohistochemistry in all (n ¼ 214) and KRAS sequencing in selected (n ¼ 41) samples

Diagnosis Localization BRAF VE1 þ KRAS

Sessile serrated adenoma/polyp All localizations (n ¼ 42) 42/42 (100%) 0/4 (0%) Right colon (n ¼ 32) 32/32 (100%) C. transversum (n ¼ 4) 4/4 (100%) Left colon (n ¼ 6) 6/6 (100%)

Traditional serrated adenoma All localizations (n ¼ 16) 15/16 (94%) 0/2 (0%) Right colon (n ¼ 2) 2/2 (100%) C. transversum (n ¼ 0) — Left colon (n ¼ 14) 13/14 (93%)

Hyperplastic polyp All localizations (n ¼ 136) 84/136 (62%) 2/31 (6%) Microvesicular hyperplastic polyp All localizations (n ¼ 119) 84/119 (71%) 2/20 (10%) Right colon (n ¼ 48) 39/48 (81%) C. transversum (n ¼ 18) 12/18 (67%) Left colon (n ¼ 53) 33/53 (62%)

Goblet-cell-rich hyperplastic polyp All localizations (n ¼ 17) 0/17 (0%) 0/11 (0%) Right colon (n ¼ 17) 0/17 (0%) C. transversum (n ¼ 0) — Left colon (n ¼ 0) —

Mucin-poor hyperplastic polyp All localizations (n ¼ 0) — — Tubulary/tubulovillous adenoma All localizations (n ¼ 20) 0/20 (0%) 1/4 (25%) Right colon (n ¼ 12) 0/12 (0%) C. transversum (n ¼ 2) 0/2 (0%) Left colon (n ¼ 6) 0/6 (0%)

no nuclear, staining was evident, indicating a lack of are results from two-tailed test, a P-value r0.05 was BRAF in the nucleus. The nuclear staining reaction considered as significant. did not interfere with the determination of BRAF status, as mutated BRAF protein is evident by strong cytoplasmic staining. Results

Sequence analysis of KRAS and BRAF. BRAF exon Table 1 provides details on polyp type, anatomic 15 and KRAS exon 2 (56 of 115) were investigated by localization and results of VE1 immunohistochemis- capillary sequencing after macrodissection of speci- try, as well as KRAS sequencing in selected samples. mens. Preparation of DNA and sequencing of BRAF All patients were Caucasian, of them 103 (48%) exon 15 was performed as described previously.24 female and 111 (52%) male, median age 63 years ± Briefly, a fragment spanning exon 2 of KRAS (range 27–92 years) and mean age (years) 62 11 (s.d.). (sense: 50-GCCTGCTGAAAATGACTGAA-30;antisense: 0 0 5 -AGAATGGTCCTGCACCAGTAA-3 ) was amplified Immunohistochemistry using 20 ng each of the respective forward and reverse primer. Primer design was based on In VE1 þ cases, a strong granular cytoplasmic accession number NG_007524.1. For PCR, 50 ng of staining of the epithelial cells was present. In sessile DNA and HotStar 2 Â PCR Master Mix (Qiagen, serrated adenoma/polyp without cytologic dyspla- Hilden, Germany) were used. PCR was performed in sia, a more intense staining was detected in the basal a total volume of 25 ml, and included initial half of the crypts compared with the superficial half. denaturation at 95 1C for 180 s, followed by 37 The intensity of the staining decreased continuously cycles with denaturation at 95 1C for 30 s, annea- from the base of the crypts towards the surface ling at 56 1C for 25 s and extension at 72 1C for 40 s. epithelium in sessile serrated adenoma/polyp, Two microliters of the amplification product were which was stronger than that in microvesicular submitted to bidirectional sequencing using the hyperplastic polyps. In traditional serrated adenoma BigDye Terminator v.3.1 Sequencing Kit (Applied and in sessile serrated adenoma/polyp with cytolo- Biosystems, Foster City, CA, USA). Sequences were gic dysplasia, a diffuse strong intense staining was determined using an ABI 3500 Genetic Analyzer present with consistent strong expression continu- (Applied Biosystems) and the Sequence-Pilot v. 3.4 ously from the base of the crypts up to the surface (JSI-Medisys, Kippenheim, Germany) software. epithelium. In all VE1 þ lesions (microvesicular hyperplastic polyps, traditional serrated adenomas, Statistics. For statistical analysis, w2 tests were sessile serrated adenomas/polyps), an abrupt transi- used as appropriate; SPSS 20.0 (IBM, Armonk, NY, tion could be observed between cells with and USA) was used for this purpose. All P-values given without BRAF V600E mutation.

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Table 2 VE1 positivity according to localization of lesions Table 4 Correlation of nuclear atypia in hyperplastic polyps with VE1 status Localization Diagnosis BRAF VE1 þ No nuclear Nuclear Right colon All Diagnosis atypia atypia Sessile serrated adenoma/polyp 32/32 (100%) Traditional serrated adenoma 2/2 (100%) Microvesicular hyperplastic polyp 44/119 (37%) 75/119 (63%) Microvesicular hyperplastic 39/48 (81%) (n ¼ 119) polyp VE1 À (n ¼ 35) 20/35 (57%) 15/35 (43%) Goblet-cell-rich hyperplastic 0/17 (0%) VE1 þ (n ¼ 84) 24/84 (29%) 60/84 (71%) polyp Tubulary/tubulovillous 0/12 (0%) Goblet-cell-rich hyperplastic 17/17 (100%) 0 (0%) adenoma polyp (n ¼ 17) VE1 À (n ¼ 17) 17/17 (100%) 0 (0%) Colon All VE1 þ (n ¼ 0) 0 (0%) 0 (0%) transversum Sessile serrated adenoma/polyp 4/4 (100%) Traditional serrated adenoma — Only minimal nuclear atypia (Grade 1 þ according Microvesicular hyperplastic 12/18 (67%) 17 polyp to Torlakovic et al ) and mild nuclear stratification Goblet-cell-rich hyperplastic — was present in 75 hyperplastic polyps (55%; 75 polyp microvesicular hyperplastic polyps, 0 goblet-cell rich Tubulary/tubulovillous 0/2 (0%) hyperplastic polyps). VE1 expression was seen in 84 adenoma (71%) of the microvesicular hyperplastic polyps, and 2 Left colon All 60 (71%) of them showed nuclear atypia (P ¼ 0.003, w Sessile serrated adenoma/polyp 6/6 (100%) test). Nevertheless, 15 (43%) of the 35 VE1 À Traditional serrated adenoma 13/14 (93%) microvesicular hyperplastic polyps also displayed Microvesicular hyperplastic 33/53 (62%) polyp nuclear atypia. No nuclear atypia was present in Goblet-cell-rich hyperplastic — goblet-cell-rich hyperplastic polyps (Table 4). polyp Tubulary/tubulovillous 0/6 (0%) adenoma Sequencing for BRAF and KRAS Mutations

Table 3 Sessile serrated adenoma/polyp showing microvesicular Sequencing for BRAF was performed in 30 cases, hyperplastic polyp-like features comprising 10 VE1 À (8 microvesicular hyperplastic polyps, 1 traditional serrated adenoma, 1 conven- Sessile serrated adenoma/polyp Number of % VE1 þ tional adenoma) and 20 VE1 þ samples (6 sessile (n ¼ 42) cases cases serrated adenomas/polyps, 9 microvesicular hyper- Microvesicular hyperplastic 40/42 100 plastic polyps, 5 traditional serrated adenoma), of polyp-like features which 1 microvesicular hyperplastic polyp showed Sessile serrated adenoma/polyp- 42/42 100 weak and heterogeneous immunostaining. like areas All VE1 À samples showed no BRAF exon 15 mutation. Of the 20 VE1 þ samples, 2 microvesicular hyperplastic polyps with a low number of One hundred and forty-one of 194 serrated lesions positive crypts were BRAF wild-type at direct (73%) showed specific cytoplasmic staining for sequencing, but after BRAF enrichment using a VE1: 100% sessile serrated adenomas/polyps, 94% V600E kit (Qiagen GmbH, Du¨ sseldorf, Germany), of traditional serrated adenomas and 62% of V600E mutations were evident. The remaining 17 hyperplastic polyps. Of the 136 hyperplastic polyps, VE1 þ samples (among them also the one with weak all 17 goblet-cell-rich hyperplastic polyps were immunostaining) showed V600E mutations at direct negative for VE1 and 84 (71%) of the 119 micro- sequencing. Interestingly, one VE1 weakly positive vesicular hyperplastic polyps were positive for VE1. microvesicular hyperplastic polyp showed BRAF No significant difference of VE1 expression was V600Q (c.1799_1800delinsAA) at sequencing. observed between microvesicular hyperplastic Sequencing for KRAS was performed in 41 polyps of the left colon compared with the right samples, of which 33 were VE1 À (17 microvesicu- colon (P40.05, w2 test; Table 2). lar hyperplastic polyp, 11 goblet-cell-rich hyper- All conventional adenomas were negative for VE1. plastic polyp, 1 traditional serrated adenoma, 4 Forty of 42 sessile serrated adenomas/polyps (95%) conventional adenoma) and 8 VE1 þ (4 sessile showed areas with histological features of micro- serrated adenoma/polyp, 3 microvesicular hyper- vesicular hyperplastic polyps and a continuous plastic polyp, 1 traditional serrated adenoma). In positive VE1 expression, displaying signi- VE1 À cases, 2 microvesicular hyperplastic polyp ficant correlation of microvesicular hyperplastic and 1 conventional adenoma showed KRAS exon 2 polyp-like features with BRAF V600E mutation mutations, whereas in the VE1 þ samples, no KRAS (Table 3). exon 2 mutations were found.

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Figure 2 Microvesicular hyperplastic polyp with mild nuclear atypia: (a) hematoxylin and eosin (H&E), Â 100; (b) Immunostain VE1 ( À ), Â 100; (c) H&E, Â 100; and (d) Immunostain VE1 ( þ ), Â 100.

Discussion and to colorectal cancer.9,15,16 Our findings, on one hand identifying (71%) microvesicular hyperplastic The development of a BRAF V600E mutation-speci- polyps with the oncogenic BRAF V600E mutation fic antibody has enabled us to investigate for the first and on the other hand the presence of VE1 þ areas time the distribution of BRAF V600E protein revealing the morphology of microvesicular hyper- in routine formalin-fixed, paraffin-embedded speci- plastic polyps in 95% of sessile serrated adenomas/ mens of serrated lesions of the colon. This approach polyps, would favor this theory. The observed allows a correlation of histomorphology with abrupt transition in all VE1 þ serrated lesions molecular pathological changes at the cellular between areas with and without gene mutation level and an immunohistochemical subclassifica- also indicates a clonal evolution of the BRAF tion of serrated lesions according to their BRAF V600E mutation. V600E mutation status. Generally, colorectal cancer is believed to arise on In this study, we propose a novel and refined the base of distinct precursor lesions and is classification for serrated lesions of the colon associated with different molecular pathways, that integrating histomorphology with immunohisto- is, mutation in the KRAS gene (mainly in exon 2) or chemical detection of BRAF V600E mutation. This in the BRAF gene (in the vast majority of cases the technique provides an objective and reproducible V600E mutation in exon 15).14,29–31 In our study, aid to identify serrated lesions with the oncogenic BRAF V600E mutation neither could be detected in BRAF V600E mutation. goblet-cell-rich hyperplastic polyps nor in BRAF V600E mutation is an early event in the conventional adenomas. The latter fact provides serrated neoplasia pathway.12 It has been anti- evidence that BRAF V600E mutation probably does cipated that microvesicular hyperplastic polyps are not have a major role in the conventional adenoma– the precursor lesions for the serrated pathway and carcinoma sequence, but in a subset of microsatel- might progress into sessile serrated adenoma/polyp lite-stable colorectal cancer, it has been shown that

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Figure 3 Sessile serrated adenoma/polyp with features of microvesicular hyperplastic polyp: (a) hematoxylin and eosin (H&E), Â 40; (b) immunostain VE1, Â 100. Sessile serrated adenoma/polyp without cytologic dysplasia: (c) H&E, Â 200; (d) immunostain VE1, Â 200.

BRAF V600E mutation does have a relevant role, is dysplasia are missing in most cases of sessile prognostically significant and correlates with poorer serrated adenomas/polyps. Therefore, high inter- survival.32,33 and even intraobserver variability among patho- Although consensus exists that sessile serrated logists is evident in the diagnosis of sessile serrated adenomas are precursors of BRAF V600E-driven adenomas/polyps,19–22 despite the formally clear serrated carcinoma pathway, the contribution of morphologic criteria.13,17 The need for a potential hyperplastic polyps is not unambiguously marker to identify microvesicular hyperplastic solved.9,13,15 These lesions are frequently found polyps with risk for progression in the serrated and ablated at routine colonoscopy. Their neopla- neoplasia pathway is also highlighted by our stic nature remains an issue of debate and many findings. Even though BRAF V600E mutation attempts have been made to find histomorphologic17 correlated with nuclear atypia and nuclear and immunomorphologic34 criteria to identify stratification, these morphologic changes were also among them the neoplastic lesions, in contrast to present in 43% of these cases in the absence of true hyperplastic lesions. In addition, the histo- BRAF V600E mutation. These findings suggest that logical diagnosis of serrated lesions often poses a morphology by itself is not a reliable criterion for difficult task to routine pathologists: biopsy speci- evaluating the presence of underlying molecular mens are often small and impossible to orientate. changes with regard to BRAF V600E mutation. However, proper specimen orientation is crucially Further problems appear by the fact, which was important for the differential diagnosis micro- also evident in our study, that serrated lesions with vesicular hyperplastic polyp vs sessile serrated the morphology of microvesicular hyperplastic adenoma/polyp, which is based on architectural polyps in one part of the lesion and with the changes including the crypt bases. In contrast to morphology of sessile serrated adenomas/polyps in conventional adenomas, cytological signs of the other part do exist.13 On one hand, this has been

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Figure 4 Sessile serrated adenoma/polyp with cytologic dysplasia: (a) hematoxylin and eosin (H&E), Â 100; (b) immunostain VE1, Â 100. Traditional serrated adenoma: (c) H&E, Â 100; (d) immunostain VE1, Â 100.

interpreted as evidence that sessile serrated rable higher risk of progression to malignancy. adenomas/polyps evolve from microvesicular Further clinical studies of patients with VE1 þ hyperplastic polyps or on the other hand that microvesicular hyperplastic polyps with microvesicular hyperplastic polyp features could appropriate follow-up data could possibly answer belong to the morphologic spectrum of sessile this question. serrated adenomas/polyps.13 This implicates that As BRAF V600E is also found frequently in there are borderline lesions35 and therefore no clear naevi,36–38 this finding indicates that non-malignant line can be drawn between the morphology of lesions with this mutation require a ‘second hit’ to microvesicular hyperplastic polyps and sessile become frankly malignant. This happens obviously serrated adenomas/polyps. It is likely that the only in a relatively small proportion of cases. The progression between microvesicular hyperplastic same scenario might be true in the development of polyp and sessile serrated adenoma/polyp is a colorectal cancer as well. In our study, BRAF V600E continuum with BRAF V600E mutation as an early mutation was encountered in a large percentage genetic event in the serrated neoplasia pathway. For (62%) of hyperplastic polyps almost equally this reason, it might generally be anticipated that frequent on both sides of the colon, whereas only microvesicular hyperplastic polyps with BRAF 5–20% of colorectal cancers show BRAF V600E V600E mutation are not innocent bystanders, but mutation and are located more frequently in the right indeed precursor lesions of sessile serrated colon.8,14,29,31 Therefore, the sole location of a lesion adenomas/polyps finding their end point in BRAF does not seem to be indicative of the underlying V600E-mutated colorectal cancer. It might also molecular pathway. However, the exact mechanisms be anticipated that serrated lesions including and host-related factors contributing to further microvesicular hyperplastic polyps with an already progressions should be addressed in subsequent existing BRAF V600E mutation have a conside- functional and clinical studies.

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In our opinion, this approach, based on immuno- 11 Hawkins NJ, Bariol C, Ward RL. The serrated neoplasia histochemical determination of BRAF V600E muta- pathway. Pathology 2002;34:548–555. tion, is a feasible technique in routine biopsy 12 Kambara T, Simms LA, Whitehall VL, et al. BRAF diagnostics, allowing unified, objective and repro- mutation is associated with DNA methylation in ducible classification of serrated lesions. This serrated polyps and cancers of the colorectum. Gut 2004;53:1137–1144. would overcome the problems of high interobserver 13 Rex DK, Ahnen DJ, Baron JA, et al. Serrated lesions of variability using the morphological criteria. Accord- the colorectum: review and recommendations from an ingly, microvesicular hyperplastic polyps should be expert panel. Am J Gastroenterol 2012;107:1315–1329. subclassified with regard to BRAF V600E mutation 14 Davies H, Bignell GR, Cox C, et al. Mutations of the status to identify those with BRAF V600E mutation BRAF gene in human cancer. Nature 2002;417: as possible precursor lesions of the serrated neopla- 949–954. sia pathway. Through this approach, a more elabo- 15 O‘Brien MJ. Hyperplastic and serrated polyps of the rate classification based on the interplay between colorectum. Gastroenterol Clin N Am 2007;36:947–968. histology and molecular pathology could offer 16 Kim KM, Lee EJ, Ha S, et al. Molecular features of patients an improved colorectal cancer prevention colorectal hyperplastic polyps and sessile serrated adenoma/polyps from Korea. Am J Surg Pathol strategy. 2011;35:1274–1286. 17 Torlakovic E, Skovlund E, Snover DC, et al. Morpho- logic reappraisal of serrated colorectal polyps. Am J Disclosure/conflict of interest Surg Pathol 2003;27:65–81. 18 Snover DC, Ahnen DJ, Burt RW, et al. 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