ORIGINAL ARTICLE
Immunohistochemistry for Annexin A10 Can Distinguish Sporadic From Lynch Syndrome–associated Microsatellite-unstable Colorectal Carcinoma Reetesh K. Pai, MD,* Bonnie L. Shadrach, MS,w Paula Carver, BS,w Brandie Heald, MS,z Jessica Moline, MS,z James Church, MD,y Matthew F. Kalady, MD,y Carol A. Burke, MD,8 Thomas P. Plesec, MD,w Keith K. Lai, MD,w David H. Gonzalo, MD,w and Rish K. Pai, MD, PhDw
Abstract: Differentiating sporadic microsatellite-unstable colorectal carcinoma due to MLH1 promoter hypermethylation from Lynch syndrome (LS)-associated tumors due to mutations in mismatch-repair proteins is time consuming, cost intensive, and requires advanced laboratory testing. A mutation in BRAF has been shown to be highly specific for sporadic tumors; however, a significant proportion of sporadic microsatellite-unstable tumors lack BRAF mutations. MLH1 promoter methylation analysis is subsequently used to differentiate LS and sporadic tumors, but both tests require specialized laboratories and are costly. Through previous gene expression profiling of serrated polyps, we identified annexin A10 as a protein highly expressed in sessile serrated adenomas/polyps. As these polyps give rise to the majority of sporadic microsatellite-unstable tumors, we evaluated the ability of annexin A10 expression to discriminate between LS and sporadic tumors. A marked increase in annexin A10 mRNA was observed in sporadic microsatellite-unstable tumors compared with LS tumors (378-fold increase, P < 0.001). Using immunohistochemistry, annexin A10 was expressed in 23/53 (43%) BRAF-mutated and 9/22 (41%) BRAF wild-type sporadic tumors. In contrast, only 3/56 (5%) LS tumors were positive for annexin A10 (P < 0.0001). One patient had a deleterious MSH2 mutation, and another had a variant of uncertain significance in MSH6. These 2 tumors could be easily distinguished from sporadic tumors using mismatchFrom the *Department of Anatomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; Departments of wAnatomic Pathology; yColorectal Surgery; zGenomic Medicine Institute; and 8Digestive Disease Institute, Cleveland Clinic, Cleveland, OH. R.K.P.: Study concept and design; acquisition of data; analysis and interpretation of data; critical revision of manuscript. B.L.S., B.H., J.M., J.C., M.F.K., C.A.B., T.P.P., K.K.L., and D.H.G.: Acquisition of data; critical revision of manuscript. P.C.: Acquisition of data. R.K.P.: Study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Rish K. Pai, MD, PhD, Department of Anatomic Pathology, Cleveland Clinic, 9500 Euclid Avenue, L25, Cleveland, OH 44195 (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
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repair protein immunohistochemistry. Only 1/28 (4%) LS tumors with loss of MLH1 was positive for annexin A10. This patient did not have a deleterious MLH1 mutation but rather germline promoter hypermethylation of MLH1. On the basis of these results, immunohistochemistry for annexin A10 may be a useful marker to distinguish sporadic from LS-associated microsatellite-unstable colon cancer. Key Words: Lynch syndrome, MLH1, annexin A10, sessile serrated polyp, BRAF (Am J Surg Pathol 2014;38:518–525)
L
ynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome and accounts for 2% to 3% of all colon cancers.1 The hallmark of LS is the presence of microsatellite instability (MSI) due to defective mismatch-repair (MMR) machinery. Germline mutations in 4 MMR proteins (MLH1, MSH2, MSH6, and PMS2) account for the majority of LS cases, with 80% of LS cases attributed to mutations in MLH1 and MSH2.2 More recently, deletions in EPCAM, a gene upstream of MSH2, have been shown to cause epigenetic inactivation of MSH2 in a subset of families.3,4 In addition, rare cases of LS have been reported in patients with germline promoter hypermethylation and silencing of MLH1.3 Initial screening for LS can be done in 2 ways: MSI testing using polymerase chain reaction (PCR) or immunohistochemistry for MMR proteins.5–8 Both methods are excellent for initial screening for LS, as B90% of tumors will demonstrate an MSI-H (MSI-H) phenotype and loss of 1 or more MMR proteins. Although LS almost always demonstrates an MSI-H phenotype, the majority (B75%) of MSI-H CRCs are not attributable to LS. These sporadic MSI-H tumors arise due to hypermethylation of the MLH1 promoter.9 Separating LS cases with loss of MLH1 expression due to germline defects from sporadic MSI-H cases due to promoter hypermethylation is challenging. Recent work has shed light on the origin of sporadic MSI-H tumors. These tumors often demonstrate Am J Surg Pathol
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hypermethylation of CpG islands (CIMP-high).10–13 CpG islands are present in the promoter regions of numerous genes (including MLH1), and when these islands are methylated, gene transcription is silenced. A V600E mutation in BRAF, a serine threonine kinase in the RAS/ RAF/MAPK pathway, is also common in these hypermethylated tumors. In a recent literature review of 20 studies that analyzed 663 sporadic MSI-H CRCs with known negative MMR mutation status, 46% were BRAF mutated (range, 21% to 79%).14 In the studies specifically analyzing those tumors with hypermethylation of the MLH1 promoter and loss of MLH1 expression, 63% were BRAF mutated.14 In contrast, the V600E hotspot mutation in BRAF is seen in 50% tumor were selected and manually microdissected. DNA was extracted using the High Pure PCR Template Preparation Kit (Roche Applied Science, Indianapolis, IN) according to manufacturer’s instructions as previously described. RNA was extracted with a High Pure RNA Paraffin Kit (Roche Diagnostics, Germany).
MSI Analysis MSI fluorescent PCR-based assay (MSI Analysis System; Promega, Madison, WI) was performed, which amplified 7 loci including 5 mononucleotide repeats (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeats (PentaC and PentaD). The fluorescently labeled amplified PCR products were analyzed by capillary gel electrophoresis on the ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). A tumor exhibiting 2 or more microsatellite-unstable markers was classified as MSI-H.
Immunohistochemistry MMR Protein Immunohistochemistry MSI-H tumors were subjected to immunohistochemical staining using anti-MLH1 (clone G168.15, 1:10; Biocare, Concord, CA), anti-MSH2 (clone FE11, 1:100; Biocare), anti-MSH6 (clone BC/44, 1:200, Biocare), and anti-PMS2 (clone A16-4, 1:200; BD Biosciences, San Jose, CA) where appropriate. The presence or absence of nuclear staining was evaluated in the lesional tissue. Loss of MMR protein expression was defined by lack of nuclear staining in the lesional tissue.
Annexin A10 Immunohistochemistry Rabbit polyclonal, affinity purified anti-ANXA10 (NBP1-90156, 1:250; Novus, Littleton, CO) was applied www.ajsp.com |
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for 1 hour at 371C.24 Secondary antibody (OmniMap anti-Rabbit HRP; Ventana, Tuscon, AZ) was applied for 32 minutes at 371C. The chromogenic substrate (ChromoMap DAB; Ventana Medical Systems) was applied for 8 minutes at 371C.
BRAF Mutation Analysis BRAF mutation analysis at codon 600 (V600E) was performed by a real-time PCR based on an allelic discrimination method previously described.22 Briefly, realtime PCR was performed using allele-specific primers designed to selectively amplify the wild-type (T1799) and mutant (A1799) BRAF alleles. The primer sequences were as follows: V, 50 -GTGATTTTGGTC TAGC TACTGT; E, 50 -CGCGGCCGGCCGCGGCGGTGATTTTGGTC TA GCTACCGA; and antisense, 50 -TAGCCTCAA TTCTTACCAT CCAC. PCR amplification and melting curve analysis were performed on an ABI 7500 (Applied Biosystems). After amplification, samples were subjected to a temperature ramp from 601C to 991C, increasing by 11C in each step. For wild-type samples, single peaks were observed at 781C, whereas samples containing mutant alleles produced an additional peak at 831C.
MLH1 Promoter Hypermethylation Analysis MLH1 promoter methylation was performed when a tumor lost expression of MLH1 and was BRAF wild type. For cases from the University of Pittsburgh Medical Center (n = 12), MLH1 promoter hypermethylation was performed at Mayo Clinic Laboratories (Rochester, MN) (test ID MLH1H). For cases from the Cleveland Clinic (n = 17), genomic DNA was treated with bisulfite according to the EZ DNA methylation kit (Zymo Research, Orange, CA). Bisulfite-converted DNA was amplified for MLH1 and COL2A1 (a methylation-independent reaction for normalization) using primers and probes as previously described.25 A constant reference sample consisting of M.SssI-methylated DNA was also amplified (Zymo Research). A COL2A1 standard curve was generated, and the input bisulfite-converted DNA was quantified. Next, the amount of methylated DNA for MLH1 in the sample and M.SssI reference DNA was determined. A percent methylated reference value was calculated using this equation: 100 (MLH1 amount/COL2A1 amount)sample/(MLH1 amount/COL2A1 amount)M.SssI-REFERENCE. A percent methylated reference value >10 was considered positive for hypermethylation.
Annexin A10 Quantitative Reverse Transcription PCR Analysis RNA was converted to cDNA, and quantitative reverse transcription PCR was performed using RNA-toCT 1-step kit (Life Technologies Corp.) on an ABI 7500 real-time PCR system. TaqMan Gene Expression Assays (Life Technologies Corp.) for ACTB (Hs01060665_g1) and ANXA10 (Hs01105012_m1) were used with the following reaction details: 951C for 10 minute, 40 cycles of 951C for 15 seconds, and 601C for 60 seconds. Relative
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gene expression values were calculated by the nnCt method. The nnCt method gives the amount of target gene expression normalized to an endogenous reference gene and relative to a calibrator sample (Human Reference Total RNA; Agilent Technologies). Pairwise comparisons were made using the Wilcoxon rank sum test.
Statistical Analysis Categorical variables were compared by w2 analysis. All statistical tests were 2-sided, with P < 0.05 considered statistically significant.
RESULTS Subject Demographics and Tumor Characteristics A total of 75 sporadic MSI-H tumors (from 75 individuals) and 56 LS-associated tumors (from 53 individuals) comprised the study population (Table 1). The sporadic cases were composed of 53 BRAF-mutated and 22 BRAF wild-type tumors that demonstrated hypermethylation of the MLH1 promoter. The LS cases were divided into definite (n = 32) and presumed LS (n = 24). Within the definite LS category, 19 tumors were from patients who harbored a deleterious germline MLH1 mutation. One definite LS patient had germline hypermethylation of the MLH1 promoter. Twelve definite LS patients had deleterious mutations in MSH2 (n = 11) or MSH6 (n = 1). Eight of the presumed LS tumors had immunohistochemical abnormalities in MLH1/PMS2 expression (6 lacked a BRAF mutation and promoter hypermethylation of MLH1, and 2 had a variant of uncertain significance in MLH1), and 16 had immunohistochemical loss of MSH2 and/or MSH6.
mRNA Expression of Annexin A10 in LS and Sporadic MSI-H CRCs To determine whether ANXA10 expression can differentiate between sporadic and LS-associated MSI-H carcinoma, ANXA10 mRNA was measured in sporadic (n = 13) and LS carcinomas (n = 5) by quantitative realtime reverse transcription PCR. There was a 378-fold increase in expression of ANXA10 in sporadic versus LS tumors (Fig. 1).
Immunohistochemical Expression of ANXA10 in LS and Sporadic MSI-H CRCs Immunohistochemical analysis for ANXA10 was performed on all 56 LS-associated CRCs and 75 sporadic MSI-H CRCs (Table 2). Only tumors with nuclear and cytoplasmic staining were considered to express ANXA10 (Fig. 2). Scattered positivity was also seen in some stromal and inflammatory cells, but this was easily distinguished from expression in tumor cells. ANXA10 was expressed in 32/75 (43%) sporadic MSI-H cases versus 3/ 56 (5%) LS-associated CRCs (P < 0.0001). Importantly, a similar proportion of BRAF wild-type and BRAF-mutated sporadic tumors were positive for ANXA10 (43% vs. 41%). Diffuse expression of ANXA10 expression was r
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TABLE 1. Characteristics of Sporadic MSI-H and LS-associated CRC
TABLE 2. Immunohistochemistry for ANXA10 on Sporadic and LS CRC ANXA10 Positive (n [%])
n [%] Sporadic MSI-H CRC (n = 75) Age (y) Female Proximal tumor location BRAF mutation BRAF WT/MLH1 promoter hypermethylation LS-associated MSI-H CRC (n = 56) Age (y) Female Proximal tumor location Definite LS MLH1 gene mutation MSH2 gene mutation MSH6 gene mutation MLH1 germline promoter hypermethylation Presumed LS PMS2 and MLH1 loss of expression, BRAF WT, no MLH1 promoter hypermethylation MLH1 variant of uncertain significance MSH2 variant of uncertain significance MSH6 variant of uncertain significance MSH2 and/or MSH6 loss of expression
Type of MSI-H CRC 73 49 (65) 71 (95) 53 (71) 22 (29) 50 24 (45) 34 (61) 19 11 1 1
(34) (19) (2) (2)
Sporadic cases BRAF mutated (n = 53) BRAF WT, MLH1 promoter hypermethylation (n = 22) Definite LS MLH1 mutation (n = 19) MLH1 germline promoter hypermethylation (n = 1) MSH2 mutation (n = 11) MSH6 mutation (n = 1) Presumed LS MLH1/PMS2 abnormal (n = 8) MSH2/MSH6 abnormal (n = 16)
23 (43) 9 (41) 0 (0) 1 (100) 1 (9) 0 (0) 0 (0) 1 (6)
WT indicates wild type.
6 (11) 2 1 1 14
(3) (2) (2) (25)
WT indicates wild type.
not seen in invasive tumors in contrast to the staining pattern in SSA/Ps.24 Rather, in ANXA10-positive cases, between 5% and 40% of tumors cells were positive (mean 10%). Although staining was focal, it was possible to
detect positive expression at low power. Given the focal expression of ANXA10, we performed immunohistochemistry on an additional tumor block from 20 CRCs (10 sporadic and 10 LS cases) that lacked ANXA10 expression. ANXA10 expression was not identified in any of the additionally stained tumor blocks indicating that testing additional tumor sections does not increase sensitivity. Two of the 3 ANXA10-positive LS tumors were from patients with immunohistochemical abnormalities in MSH2 and MSH6. One patient had a deleterious mutation in MSH2, and another had a variant of uncertain significance in MSH6. These tumors would easily be distinguished from sporadic MSH-H tumors through MMR immunohistochemical staining. The remaining positive LS tumor was from a patient with germline hypermethylation of the MLH1 promoter. Importantly, none of the 19 patients with a deleterious MLH1 mutation or 8 presumed LS patients with loss of MLH1 was positive for ANXA10.
DISCUSSION
FIGURE 1. Quantitative reverse transcription PCR for ANXA10 mRNA in LS-associated (n = 5) and sporadic MSI-H (n = 13) carcinomas. There was a 378-fold difference in mRNA expression between these 2 types of carcinoma (P < 0.0001). r
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Increased emphasis has been placed on screening all CRCs to identify individuals affected by LS.26,27 In order for universal screening to be effective, the screening algorithm must be robust and easily used by practicing pathologists in diverse clinical practices. Current screening algorithms use either immunohistochemistry for MMR proteins or PCR-based MSI testing.1,5,6 Abnormal MSH2 and/or MSH6 expression by immunohistochemistry is accepted as highly indicative of LS, and germline mutational analysis of MSH2 or MSH6 should be performed. However, by far the most common immunohistochemical pattern is loss of MLH1 and PMS2 expression. This pattern occurs in B85% of MMRdeficient CRCs. When there is loss of both PMS2 and MLH1, the challenge is to separate sporadic tumors from those caused by germline defects in MLH1. BRAF mutational analysis has shown to be specific for sporadic tumors; www.ajsp.com |
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FIGURE 2. A, A sporadic MSI-H, BRAF wild-type invasive adenocarcinoma arising from an SSA/P. B, ANXA10 expression is strong and diffuse in the precursor SSA/P, but focal in the invasive carcinoma. C and D, Although focal, ANXA10 expression can be identified at low power (C) as seen in this example of a sporadic MSI-H BRAF-mutated carcinoma. Higher-power examination demonstrates strong nuclear and cytoplasmic expression of ANXA10 in a subset of tumor cells (D). E, Scattered immunoreactivity was also seen in surrounding inflammatory cells and stroma in some cases; however, this was easily distinguished from expression in tumor cells. This tumor was MSI-H and harbored a mutation in BRAF. F, In this example of a sporadic BRAF wild-type MSI-H carcinoma, there were differences in ANXA10 expression between mucinous and nonmucinous components.
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however, not all sporadic tumors harbor mutations in BRAF.14 Furthermore, BRAF mutational analysis requires advanced molecular techniques that are performed by specialized laboratories. MLH1 promoter hypermethylation assays are also used to separate LS-associated CRCs from sporadic cancers; however, MLH1 promoter methylation testing is costly and is only being performed by a few large molecular pathology laboratories.20 Clearly, an easier method of separating sporadic from LS tumors in this setting is needed. In a recently published study, we analyzed the gene expression profile of hyperplastic polyps and SSA/Ps.24 Through these studies, we identified ANXA10 as a specific marker of SSA/Ps. We now recognize that SSA/Ps likely give rise to the majority of sporadic CIMP-high, MSI-H CRC on the basis of the following observations: (1) SSA/Ps are located predominantly in the proximal colon, a site preferentially involved by sporadic MSI-H tumors28; (2) synchronous SSA/Ps are more common in patients with proximal sporadic MSI-H CRC22; (3) SSA/ Ps can develop conventional features of dysplasia29; (4) SSA/Ps have been identified in an area that subsequently developed sporadic MSI-H CRC30; (5) SSA/Ps are seen immediately adjacent to some sporadic MSI-H CRCs22; and (6) SSA/Ps often have mutations in BRAF and demonstrate hypermethylation of some CpG islands.31–35 Given the importance of serrated precursors in the development of CIMP-high, MSI-H CRC, this pathway to CRC has been renamed the serrated neoplasia pathway.36–39 In contrast to sporadic MSI-H tumors, the majority of LS-associated CRCs do not arise from serrated precursors but rather arise from tubular and tubulovillous adenomas.21,23,40,41 Given the difference in precursor lesions, we hypothesized that ANXA10 may help differentiate sporadic from LS-associated MSI-H CRC. Indeed, 32/75 (43%) sporadic MSI-H tumors were positive for ANXA10 in contrast to 3/56 (5%) presumed and definite LS-associated carcinomas (P < 0.0001). Importantly, a similar percentage of BRAF-mutated and BRAF wild-type sporadic MSI-H tumors were positive for ANXA10. We had expected a higher percentage of sporadic tumors to be positive for ANXA10. One possible explanation is that ANXA10 expression decreases in the progression from SSA/P to SSA/P with cytologic dysplasia to invasive carcinoma. In our prior study, we did observe decreased ANXA10 expression in the cytologically dysplastic areas of SSA/Ps compared with the adjacent nondysplastic epithelium.24 Furthermore, in the few cases of invasive CRC with adjacent SSA/P precursor, the invasive carcinoma had decreased expression of ANXA10 (Figs. 2A, B). One potential drawback of these results is that ANXA10 positivity was focal, with the percentage of positive tumor cells ranging from 5% to 40% (mean 10%). Although the staining was focal, it was still possible to detect expression at relatively low power. We did not evaluate any biopsy specimens of CRC; however, the focal expression of ANXA10 observed in this study likely limits its utility in biopsies. r
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On the basis of these results, we propose that immunohistochemistry for ANXA10 can be useful in the screening algorithm for LS. ANXA10 immunohistochemistry is complimentary to BRAF mutational analysis, as a similar percentage of BRAF-mutated and BRAF wild-type tumors were positive for ANXA10. Furthermore, as ANXA10 can be measured by immunohistochemistry, it is less complicated and faster to perform compared with BRAF mutational analysis. A proposed LS screening algorithm using ANXA10 immunohistochemistry is shown in Figure 3. In a tumor with lack of MLH1 and PMS2 expression (B85% of MMR-deficient tumors), immunohistochemical analysis for ANXA10 is performed. The tumor must be carefully evaluated for ANXA10 expression given the focal expression seen in some tumors. If definitive expression is seen in the tumor, then it is very likely to be sporadic. If any uncertainty exists with regard to ANXA10 expression, then the tumor should be considered negative. If a tumor expresses ANXA10, and the clinical suspicion of LS is still high, germline MLH1 promoter methylation analysis may be considered given that the only LS tumor with MLH1 loss that was positive for ANXA10 came from a patient with germline promoter hypermethylation. If the tumor does not express ANXA10, then BRAF mutational analysis and MLH1 promoter methylation analysis (if the tumor is BRAF wild-type) should be performed. Recently, a BRAF V600E-specific antibody (clone VE1) was developed. Some studies have shown that this antibody can be a useful surrogate for BRAF mutational analysis with excellent concordance42–44; however, another study reported poor sensitivity with this antibody with only 35% of BRAF-mutated tumors demonstrating
FIGURE 3. Proposed screening algorithm for MMR-deficient CRCs that lack expression of MLH1 and PMS2. www.ajsp.com |
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strong expression.45 If the BRAF V600E-specific antibody does prove to be a viable alternative to mutational analysis, the proposed algorithm in Figure 3 can be modified so that immunohistochemical analyses for ANXA10 and BRAF V600E are performed at the same time in tumors that lack expression of MLH1 and PMS2. If the tumor is positive for 1 or both of these proteins, then one can reliably exclude the vast majority of sporadic MSI-H tumors using primarily immunohistochemical methods. In summary, we show that 43% of sporadic MSI-H tumors express ANXA10, a marker that we recently discovered to be expressed in SSA/Ps. In contrast, only 1 of 28 LS-associated tumors with loss of MLH1 expression was positive for ANXA10, and this tumor arose in a patient with germline MLH1 promoter hypermethylation. Importantly, no tumor from a patient with a deleterious MLH1 mutation expressed ANXA10. On the basis of these results, we propose that immunohistochemistry for ANXA10 can be useful in screening for LS. REFERENCES 1. Geiersbach KB, Samowitz WS. Microsatellite instability and colorectal cancer. Arch Pathol Lab Med. 2011;135:1269–1277. 2. Moreira L, Balaguer F, Lindor N, et al. Identification of Lynch syndrome among patients with colorectal cancer. JAMA. 2012;308:1555–1565. 3. Niessen RC, Hofstra RMW, Westers H, et al. Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer. 2009;48:737–744. 4. Ligtenberg MJL, Kuiper RP, Chan TL, et al. Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 30 exons of TACSTD1. Nat Genet. 2009;41:112–117. 5. Shia J, Tang LH, Vakiani E, et al. Immunohistochemistry as firstline screening for detecting colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome: a 2-antibody panel may be as predictive as a 4-antibody panel. Am J Surg Pathol. 2009;33:1639–1645. 6. Shia J. Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn. 2008;10:293–300. 7. Mojtahed A, Schrijver I, Ford JM, et al. A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas, skin sebaceous tumors, and gynecologic tract carcinomas. Mod Pathol. 2011;24:1004–1014. 8. Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 2005;352:1851–1860. 9. Bettington M, Walker N, Clouston A, et al. The serrated pathway to colorectal carcinoma: current concepts and challenges. Histopathology. 2013;62:367–386. 10. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004;4:988–993. 11. Ogino S, Nosho K, Kirkner GJ, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58:90–96. 12. Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–793. 13. Samowitz WS, Albertsen H, Herrick J, et al. Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology. 2005;129:837–845. 14. Parsons MT, Buchanan DD, Thompson B, et al. Correlation of tumour BRAF mutations and MLH1 methylation with germline
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mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification. J Med Genet. 2012;49:151–157. Deng G, Bell I, Crawley S, et al. BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res. 2004;10:191–195. Loughrey MB, Waring PM, Tan A, et al. Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer. Fam Cancer. 2007;6:301–310. Domingo E, Laiho P, Ollikainen M, et al. BRAF screening as a lowcost effective strategy for simplifying HNPCC genetic testing. J Med Genet. 2004;41:664–668. Rahner N, Friedrichs N, Steinke V, et al. Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome. J Pathol. 2008;214:10–16. Bettstetter M, Dechant S, Ruemmele P, et al. Distinction of hereditary nonpolyposis colorectal cancer and sporadic microsatellite-unstable colorectal cancer through quantification of MLH1 methylation by real-time PCR. Clin Cancer Res. 2007; 13:3221–3228. Gausachs M, Mur P, Corral J, et al. MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a costeffectiveness study. Eur J Hum Genet. 2012;20:762–768. Young J, Simms LA, Biden KG, et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol. 2001;159:2107–2116. Patil DT, Shadrach BL, Rybicki LA, et al. Proximal colon cancers and the serrated pathway: a systematic analysis of precursor histology and BRAF mutation status. Mod Pathol. 2012;25: 1423–1431. Walsh MD, Buchanan DD, Pearson S-A, et al. Immunohistochemical testing of conventional adenomas for loss of expression of mismatch repair proteins in Lynch syndrome mutation carriers: a case series from the Australasian site of the colon cancer family registry. Mod Pathol. 2012;25:722–730. Gonzalo DH, Lai KK, Shadrach B, et al. Gene expression profiling of serrated polyps identifies Annexin A10 as a marker of a sessile serrated adenoma/polyp. J Pathol. 2013;230:420–429. Ogino S, Kawasaki T, Brahmandam M, et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn. 2006;8:209–217. Hampel H. Point: justification for Lynch syndrome screening among all patients with newly diagnosed colorectal cancer. J Natl Compr Canc Netw.; 2010:597–601. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med. 2009;11:35–41. Torlakovic E, Skovlund E, Snover DC, et al. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol. 2003;27:65–81. Lash RH, Genta RM, Schuler CM. Sessile serrated adenomas: prevalence of dysplasia and carcinoma in 2139 patients. J Clin Pathol. 2010;63:681–686. Goldstein NS, Bhanot P, Odish E, et al. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol. 2003;119:778–796. O’Brien MJ, Yang S, Mack C, et al. Comparison of microsatellite instability, CpG island methylation phenotype, BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma end points. Am J Surg Pathol. 2006;30:1491–1501. Kambara T, Simms LA, Whitehall VLJ, et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut. 2004;53:1137–1144. r
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33. Spring KJ, Zhao ZZ, Karamatic R, et al. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology. 2006;131: 1400–1407. 34. O’Brien MJ, Yang S, Clebanoff JL, et al. Hyperplastic (serrated) polyps of the colorectum: relationship of CpG island methylator phenotype and K-ras mutation to location and histologic subtype. Am J Surg Pathol. 2004;28:423–434. 35. Yang S, Farraye FA, Mack C, et al. BRAF and KRAS Mutations in hyperplastic polyps and serrated adenomas of the colorectum: relationship to histology and CpG island methylation status. Am J Surg Pathol. 2004;28:1452–1459. 36. Sweetser S, Smyrk TC, Sinicrope FA. Serrated colon polyps as precursors to colorectal cancer. Clin Gastroenterol Hepatol. 2013;11:760–767. 37. Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology. 2010;138:2088–2100. 38. Rex DK, Ahnen DJ, Baron JA, et al. Serrated lesions of the colorectum: review and recommendations from an expert panel. Am J Gastroenterol. 2012;107:1315–1329.
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39. Huang CS, Farraye FA, Yang S, et al. The clinical significance of serrated polyps. Am J Gastroenterol. 2011;106:229–240. 40. Kim H, Jen J, Vogelstein B, et al. Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences. Am J Pathol. 1994;145:148–156. 41. Jass JR. Pathology of hereditary nonpolyposis colorectal cancer. Ann N Y Acad Sci. 2000;910:62–73. 42. Toon CW, Walsh MD, Chou A, et al. BRAFV600E immunohistochemistry facilitates universal screening of colorectal cancers for Lynch syndrome. Am J Surg Pathol. 2013;37:1592–1602. 43. Affolter K, Samowitz W, Tripp S, et al. BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma. Genes Chromosomes Cancer. 2013;52:748–752. 44. Sinicrope FA, Smyrk TC, Tougeron D, et al. Mutation-specific antibody detects mutant BRAF(V600E) protein expression in human colon carcinomas. Cancer. 2013;119:2765–2770. 45. Adackapara CA, Sholl LM, Barletta JA, et al. Immunohistochemistry using the BRAF V600E mutation-specific monoclonal antibody VE1 is not a useful surrogate for genotyping in colorectal adenocarcinoma. Histopathology. 2013;63:187–193.
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Immunohistochemistry for Annexin A10 Can Distinguish Sporadic From Lynch Syndrome–associated Microsatellite-unstable Colorectal Carcinoma Reetesh K. Pai, MD,* Bonnie L. Shadrach, MS,w Paula Carver, BS,w Brandie Heald, MS,z Jessica Moline, MS,z James Church, MD,y Matthew F. Kalady, MD,y Carol A. Burke, MD,8 Thomas P. Plesec, MD,w Keith K. Lai, MD,w David H. Gonzalo, MD,w and Rish K. Pai, MD, PhDw
Abstract: Differentiating sporadic microsatellite-unstable colorectal carcinoma due to MLH1 promoter hypermethylation from Lynch syndrome (LS)-associated tumors due to mutations in mismatch-repair proteins is time consuming, cost intensive, and requires advanced laboratory testing. A mutation in BRAF has been shown to be highly specific for sporadic tumors; however, a significant proportion of sporadic microsatellite-unstable tumors lack BRAF mutations. MLH1 promoter methylation analysis is subsequently used to differentiate LS and sporadic tumors, but both tests require specialized laboratories and are costly. Through previous gene expression profiling of serrated polyps, we identified annexin A10 as a protein highly expressed in sessile serrated adenomas/polyps. As these polyps give rise to the majority of sporadic microsatellite-unstable tumors, we evaluated the ability of annexin A10 expression to discriminate between LS and sporadic tumors. A marked increase in annexin A10 mRNA was observed in sporadic microsatellite-unstable tumors compared with LS tumors (378-fold increase, P < 0.001). Using immunohistochemistry, annexin A10 was expressed in 23/53 (43%) BRAF-mutated and 9/22 (41%) BRAF wild-type sporadic tumors. In contrast, only 3/56 (5%) LS tumors were positive for annexin A10 (P < 0.0001). One patient had a deleterious MSH2 mutation, and another had a variant of uncertain significance in MSH6. These 2 tumors could be easily distinguished from sporadic tumors using mismatchFrom the *Department of Anatomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; Departments of wAnatomic Pathology; yColorectal Surgery; zGenomic Medicine Institute; and 8Digestive Disease Institute, Cleveland Clinic, Cleveland, OH. R.K.P.: Study concept and design; acquisition of data; analysis and interpretation of data; critical revision of manuscript. B.L.S., B.H., J.M., J.C., M.F.K., C.A.B., T.P.P., K.K.L., and D.H.G.: Acquisition of data; critical revision of manuscript. P.C.: Acquisition of data. R.K.P.: Study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Rish K. Pai, MD, PhD, Department of Anatomic Pathology, Cleveland Clinic, 9500 Euclid Avenue, L25, Cleveland, OH 44195 (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
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repair protein immunohistochemistry. Only 1/28 (4%) LS tumors with loss of MLH1 was positive for annexin A10. This patient did not have a deleterious MLH1 mutation but rather germline promoter hypermethylation of MLH1. On the basis of these results, immunohistochemistry for annexin A10 may be a useful marker to distinguish sporadic from LS-associated microsatellite-unstable colon cancer. Key Words: Lynch syndrome, MLH1, annexin A10, sessile serrated polyp, BRAF (Am J Surg Pathol 2014;38:518–525)
L
ynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome and accounts for 2% to 3% of all colon cancers.1 The hallmark of LS is the presence of microsatellite instability (MSI) due to defective mismatch-repair (MMR) machinery. Germline mutations in 4 MMR proteins (MLH1, MSH2, MSH6, and PMS2) account for the majority of LS cases, with 80% of LS cases attributed to mutations in MLH1 and MSH2.2 More recently, deletions in EPCAM, a gene upstream of MSH2, have been shown to cause epigenetic inactivation of MSH2 in a subset of families.3,4 In addition, rare cases of LS have been reported in patients with germline promoter hypermethylation and silencing of MLH1.3 Initial screening for LS can be done in 2 ways: MSI testing using polymerase chain reaction (PCR) or immunohistochemistry for MMR proteins.5–8 Both methods are excellent for initial screening for LS, as B90% of tumors will demonstrate an MSI-H (MSI-H) phenotype and loss of 1 or more MMR proteins. Although LS almost always demonstrates an MSI-H phenotype, the majority (B75%) of MSI-H CRCs are not attributable to LS. These sporadic MSI-H tumors arise due to hypermethylation of the MLH1 promoter.9 Separating LS cases with loss of MLH1 expression due to germline defects from sporadic MSI-H cases due to promoter hypermethylation is challenging. Recent work has shed light on the origin of sporadic MSI-H tumors. These tumors often demonstrate Am J Surg Pathol
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hypermethylation of CpG islands (CIMP-high).10–13 CpG islands are present in the promoter regions of numerous genes (including MLH1), and when these islands are methylated, gene transcription is silenced. A V600E mutation in BRAF, a serine threonine kinase in the RAS/ RAF/MAPK pathway, is also common in these hypermethylated tumors. In a recent literature review of 20 studies that analyzed 663 sporadic MSI-H CRCs with known negative MMR mutation status, 46% were BRAF mutated (range, 21% to 79%).14 In the studies specifically analyzing those tumors with hypermethylation of the MLH1 promoter and loss of MLH1 expression, 63% were BRAF mutated.14 In contrast, the V600E hotspot mutation in BRAF is seen in 50% tumor were selected and manually microdissected. DNA was extracted using the High Pure PCR Template Preparation Kit (Roche Applied Science, Indianapolis, IN) according to manufacturer’s instructions as previously described. RNA was extracted with a High Pure RNA Paraffin Kit (Roche Diagnostics, Germany).
MSI Analysis MSI fluorescent PCR-based assay (MSI Analysis System; Promega, Madison, WI) was performed, which amplified 7 loci including 5 mononucleotide repeats (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeats (PentaC and PentaD). The fluorescently labeled amplified PCR products were analyzed by capillary gel electrophoresis on the ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). A tumor exhibiting 2 or more microsatellite-unstable markers was classified as MSI-H.
Immunohistochemistry MMR Protein Immunohistochemistry MSI-H tumors were subjected to immunohistochemical staining using anti-MLH1 (clone G168.15, 1:10; Biocare, Concord, CA), anti-MSH2 (clone FE11, 1:100; Biocare), anti-MSH6 (clone BC/44, 1:200, Biocare), and anti-PMS2 (clone A16-4, 1:200; BD Biosciences, San Jose, CA) where appropriate. The presence or absence of nuclear staining was evaluated in the lesional tissue. Loss of MMR protein expression was defined by lack of nuclear staining in the lesional tissue.
Annexin A10 Immunohistochemistry Rabbit polyclonal, affinity purified anti-ANXA10 (NBP1-90156, 1:250; Novus, Littleton, CO) was applied www.ajsp.com |
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for 1 hour at 371C.24 Secondary antibody (OmniMap anti-Rabbit HRP; Ventana, Tuscon, AZ) was applied for 32 minutes at 371C. The chromogenic substrate (ChromoMap DAB; Ventana Medical Systems) was applied for 8 minutes at 371C.
BRAF Mutation Analysis BRAF mutation analysis at codon 600 (V600E) was performed by a real-time PCR based on an allelic discrimination method previously described.22 Briefly, realtime PCR was performed using allele-specific primers designed to selectively amplify the wild-type (T1799) and mutant (A1799) BRAF alleles. The primer sequences were as follows: V, 50 -GTGATTTTGGTC TAGC TACTGT; E, 50 -CGCGGCCGGCCGCGGCGGTGATTTTGGTC TA GCTACCGA; and antisense, 50 -TAGCCTCAA TTCTTACCAT CCAC. PCR amplification and melting curve analysis were performed on an ABI 7500 (Applied Biosystems). After amplification, samples were subjected to a temperature ramp from 601C to 991C, increasing by 11C in each step. For wild-type samples, single peaks were observed at 781C, whereas samples containing mutant alleles produced an additional peak at 831C.
MLH1 Promoter Hypermethylation Analysis MLH1 promoter methylation was performed when a tumor lost expression of MLH1 and was BRAF wild type. For cases from the University of Pittsburgh Medical Center (n = 12), MLH1 promoter hypermethylation was performed at Mayo Clinic Laboratories (Rochester, MN) (test ID MLH1H). For cases from the Cleveland Clinic (n = 17), genomic DNA was treated with bisulfite according to the EZ DNA methylation kit (Zymo Research, Orange, CA). Bisulfite-converted DNA was amplified for MLH1 and COL2A1 (a methylation-independent reaction for normalization) using primers and probes as previously described.25 A constant reference sample consisting of M.SssI-methylated DNA was also amplified (Zymo Research). A COL2A1 standard curve was generated, and the input bisulfite-converted DNA was quantified. Next, the amount of methylated DNA for MLH1 in the sample and M.SssI reference DNA was determined. A percent methylated reference value was calculated using this equation: 100 (MLH1 amount/COL2A1 amount)sample/(MLH1 amount/COL2A1 amount)M.SssI-REFERENCE. A percent methylated reference value >10 was considered positive for hypermethylation.
Annexin A10 Quantitative Reverse Transcription PCR Analysis RNA was converted to cDNA, and quantitative reverse transcription PCR was performed using RNA-toCT 1-step kit (Life Technologies Corp.) on an ABI 7500 real-time PCR system. TaqMan Gene Expression Assays (Life Technologies Corp.) for ACTB (Hs01060665_g1) and ANXA10 (Hs01105012_m1) were used with the following reaction details: 951C for 10 minute, 40 cycles of 951C for 15 seconds, and 601C for 60 seconds. Relative
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gene expression values were calculated by the nnCt method. The nnCt method gives the amount of target gene expression normalized to an endogenous reference gene and relative to a calibrator sample (Human Reference Total RNA; Agilent Technologies). Pairwise comparisons were made using the Wilcoxon rank sum test.
Statistical Analysis Categorical variables were compared by w2 analysis. All statistical tests were 2-sided, with P < 0.05 considered statistically significant.
RESULTS Subject Demographics and Tumor Characteristics A total of 75 sporadic MSI-H tumors (from 75 individuals) and 56 LS-associated tumors (from 53 individuals) comprised the study population (Table 1). The sporadic cases were composed of 53 BRAF-mutated and 22 BRAF wild-type tumors that demonstrated hypermethylation of the MLH1 promoter. The LS cases were divided into definite (n = 32) and presumed LS (n = 24). Within the definite LS category, 19 tumors were from patients who harbored a deleterious germline MLH1 mutation. One definite LS patient had germline hypermethylation of the MLH1 promoter. Twelve definite LS patients had deleterious mutations in MSH2 (n = 11) or MSH6 (n = 1). Eight of the presumed LS tumors had immunohistochemical abnormalities in MLH1/PMS2 expression (6 lacked a BRAF mutation and promoter hypermethylation of MLH1, and 2 had a variant of uncertain significance in MLH1), and 16 had immunohistochemical loss of MSH2 and/or MSH6.
mRNA Expression of Annexin A10 in LS and Sporadic MSI-H CRCs To determine whether ANXA10 expression can differentiate between sporadic and LS-associated MSI-H carcinoma, ANXA10 mRNA was measured in sporadic (n = 13) and LS carcinomas (n = 5) by quantitative realtime reverse transcription PCR. There was a 378-fold increase in expression of ANXA10 in sporadic versus LS tumors (Fig. 1).
Immunohistochemical Expression of ANXA10 in LS and Sporadic MSI-H CRCs Immunohistochemical analysis for ANXA10 was performed on all 56 LS-associated CRCs and 75 sporadic MSI-H CRCs (Table 2). Only tumors with nuclear and cytoplasmic staining were considered to express ANXA10 (Fig. 2). Scattered positivity was also seen in some stromal and inflammatory cells, but this was easily distinguished from expression in tumor cells. ANXA10 was expressed in 32/75 (43%) sporadic MSI-H cases versus 3/ 56 (5%) LS-associated CRCs (P < 0.0001). Importantly, a similar proportion of BRAF wild-type and BRAF-mutated sporadic tumors were positive for ANXA10 (43% vs. 41%). Diffuse expression of ANXA10 expression was r
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TABLE 1. Characteristics of Sporadic MSI-H and LS-associated CRC
TABLE 2. Immunohistochemistry for ANXA10 on Sporadic and LS CRC ANXA10 Positive (n [%])
n [%] Sporadic MSI-H CRC (n = 75) Age (y) Female Proximal tumor location BRAF mutation BRAF WT/MLH1 promoter hypermethylation LS-associated MSI-H CRC (n = 56) Age (y) Female Proximal tumor location Definite LS MLH1 gene mutation MSH2 gene mutation MSH6 gene mutation MLH1 germline promoter hypermethylation Presumed LS PMS2 and MLH1 loss of expression, BRAF WT, no MLH1 promoter hypermethylation MLH1 variant of uncertain significance MSH2 variant of uncertain significance MSH6 variant of uncertain significance MSH2 and/or MSH6 loss of expression
Type of MSI-H CRC 73 49 (65) 71 (95) 53 (71) 22 (29) 50 24 (45) 34 (61) 19 11 1 1
(34) (19) (2) (2)
Sporadic cases BRAF mutated (n = 53) BRAF WT, MLH1 promoter hypermethylation (n = 22) Definite LS MLH1 mutation (n = 19) MLH1 germline promoter hypermethylation (n = 1) MSH2 mutation (n = 11) MSH6 mutation (n = 1) Presumed LS MLH1/PMS2 abnormal (n = 8) MSH2/MSH6 abnormal (n = 16)
23 (43) 9 (41) 0 (0) 1 (100) 1 (9) 0 (0) 0 (0) 1 (6)
WT indicates wild type.
6 (11) 2 1 1 14
(3) (2) (2) (25)
WT indicates wild type.
not seen in invasive tumors in contrast to the staining pattern in SSA/Ps.24 Rather, in ANXA10-positive cases, between 5% and 40% of tumors cells were positive (mean 10%). Although staining was focal, it was possible to
detect positive expression at low power. Given the focal expression of ANXA10, we performed immunohistochemistry on an additional tumor block from 20 CRCs (10 sporadic and 10 LS cases) that lacked ANXA10 expression. ANXA10 expression was not identified in any of the additionally stained tumor blocks indicating that testing additional tumor sections does not increase sensitivity. Two of the 3 ANXA10-positive LS tumors were from patients with immunohistochemical abnormalities in MSH2 and MSH6. One patient had a deleterious mutation in MSH2, and another had a variant of uncertain significance in MSH6. These tumors would easily be distinguished from sporadic MSH-H tumors through MMR immunohistochemical staining. The remaining positive LS tumor was from a patient with germline hypermethylation of the MLH1 promoter. Importantly, none of the 19 patients with a deleterious MLH1 mutation or 8 presumed LS patients with loss of MLH1 was positive for ANXA10.
DISCUSSION
FIGURE 1. Quantitative reverse transcription PCR for ANXA10 mRNA in LS-associated (n = 5) and sporadic MSI-H (n = 13) carcinomas. There was a 378-fold difference in mRNA expression between these 2 types of carcinoma (P < 0.0001). r
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Increased emphasis has been placed on screening all CRCs to identify individuals affected by LS.26,27 In order for universal screening to be effective, the screening algorithm must be robust and easily used by practicing pathologists in diverse clinical practices. Current screening algorithms use either immunohistochemistry for MMR proteins or PCR-based MSI testing.1,5,6 Abnormal MSH2 and/or MSH6 expression by immunohistochemistry is accepted as highly indicative of LS, and germline mutational analysis of MSH2 or MSH6 should be performed. However, by far the most common immunohistochemical pattern is loss of MLH1 and PMS2 expression. This pattern occurs in B85% of MMRdeficient CRCs. When there is loss of both PMS2 and MLH1, the challenge is to separate sporadic tumors from those caused by germline defects in MLH1. BRAF mutational analysis has shown to be specific for sporadic tumors; www.ajsp.com |
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FIGURE 2. A, A sporadic MSI-H, BRAF wild-type invasive adenocarcinoma arising from an SSA/P. B, ANXA10 expression is strong and diffuse in the precursor SSA/P, but focal in the invasive carcinoma. C and D, Although focal, ANXA10 expression can be identified at low power (C) as seen in this example of a sporadic MSI-H BRAF-mutated carcinoma. Higher-power examination demonstrates strong nuclear and cytoplasmic expression of ANXA10 in a subset of tumor cells (D). E, Scattered immunoreactivity was also seen in surrounding inflammatory cells and stroma in some cases; however, this was easily distinguished from expression in tumor cells. This tumor was MSI-H and harbored a mutation in BRAF. F, In this example of a sporadic BRAF wild-type MSI-H carcinoma, there were differences in ANXA10 expression between mucinous and nonmucinous components.
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however, not all sporadic tumors harbor mutations in BRAF.14 Furthermore, BRAF mutational analysis requires advanced molecular techniques that are performed by specialized laboratories. MLH1 promoter hypermethylation assays are also used to separate LS-associated CRCs from sporadic cancers; however, MLH1 promoter methylation testing is costly and is only being performed by a few large molecular pathology laboratories.20 Clearly, an easier method of separating sporadic from LS tumors in this setting is needed. In a recently published study, we analyzed the gene expression profile of hyperplastic polyps and SSA/Ps.24 Through these studies, we identified ANXA10 as a specific marker of SSA/Ps. We now recognize that SSA/Ps likely give rise to the majority of sporadic CIMP-high, MSI-H CRC on the basis of the following observations: (1) SSA/Ps are located predominantly in the proximal colon, a site preferentially involved by sporadic MSI-H tumors28; (2) synchronous SSA/Ps are more common in patients with proximal sporadic MSI-H CRC22; (3) SSA/ Ps can develop conventional features of dysplasia29; (4) SSA/Ps have been identified in an area that subsequently developed sporadic MSI-H CRC30; (5) SSA/Ps are seen immediately adjacent to some sporadic MSI-H CRCs22; and (6) SSA/Ps often have mutations in BRAF and demonstrate hypermethylation of some CpG islands.31–35 Given the importance of serrated precursors in the development of CIMP-high, MSI-H CRC, this pathway to CRC has been renamed the serrated neoplasia pathway.36–39 In contrast to sporadic MSI-H tumors, the majority of LS-associated CRCs do not arise from serrated precursors but rather arise from tubular and tubulovillous adenomas.21,23,40,41 Given the difference in precursor lesions, we hypothesized that ANXA10 may help differentiate sporadic from LS-associated MSI-H CRC. Indeed, 32/75 (43%) sporadic MSI-H tumors were positive for ANXA10 in contrast to 3/56 (5%) presumed and definite LS-associated carcinomas (P < 0.0001). Importantly, a similar percentage of BRAF-mutated and BRAF wild-type sporadic MSI-H tumors were positive for ANXA10. We had expected a higher percentage of sporadic tumors to be positive for ANXA10. One possible explanation is that ANXA10 expression decreases in the progression from SSA/P to SSA/P with cytologic dysplasia to invasive carcinoma. In our prior study, we did observe decreased ANXA10 expression in the cytologically dysplastic areas of SSA/Ps compared with the adjacent nondysplastic epithelium.24 Furthermore, in the few cases of invasive CRC with adjacent SSA/P precursor, the invasive carcinoma had decreased expression of ANXA10 (Figs. 2A, B). One potential drawback of these results is that ANXA10 positivity was focal, with the percentage of positive tumor cells ranging from 5% to 40% (mean 10%). Although the staining was focal, it was still possible to detect expression at relatively low power. We did not evaluate any biopsy specimens of CRC; however, the focal expression of ANXA10 observed in this study likely limits its utility in biopsies. r
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On the basis of these results, we propose that immunohistochemistry for ANXA10 can be useful in the screening algorithm for LS. ANXA10 immunohistochemistry is complimentary to BRAF mutational analysis, as a similar percentage of BRAF-mutated and BRAF wild-type tumors were positive for ANXA10. Furthermore, as ANXA10 can be measured by immunohistochemistry, it is less complicated and faster to perform compared with BRAF mutational analysis. A proposed LS screening algorithm using ANXA10 immunohistochemistry is shown in Figure 3. In a tumor with lack of MLH1 and PMS2 expression (B85% of MMR-deficient tumors), immunohistochemical analysis for ANXA10 is performed. The tumor must be carefully evaluated for ANXA10 expression given the focal expression seen in some tumors. If definitive expression is seen in the tumor, then it is very likely to be sporadic. If any uncertainty exists with regard to ANXA10 expression, then the tumor should be considered negative. If a tumor expresses ANXA10, and the clinical suspicion of LS is still high, germline MLH1 promoter methylation analysis may be considered given that the only LS tumor with MLH1 loss that was positive for ANXA10 came from a patient with germline promoter hypermethylation. If the tumor does not express ANXA10, then BRAF mutational analysis and MLH1 promoter methylation analysis (if the tumor is BRAF wild-type) should be performed. Recently, a BRAF V600E-specific antibody (clone VE1) was developed. Some studies have shown that this antibody can be a useful surrogate for BRAF mutational analysis with excellent concordance42–44; however, another study reported poor sensitivity with this antibody with only 35% of BRAF-mutated tumors demonstrating
FIGURE 3. Proposed screening algorithm for MMR-deficient CRCs that lack expression of MLH1 and PMS2. www.ajsp.com |
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strong expression.45 If the BRAF V600E-specific antibody does prove to be a viable alternative to mutational analysis, the proposed algorithm in Figure 3 can be modified so that immunohistochemical analyses for ANXA10 and BRAF V600E are performed at the same time in tumors that lack expression of MLH1 and PMS2. If the tumor is positive for 1 or both of these proteins, then one can reliably exclude the vast majority of sporadic MSI-H tumors using primarily immunohistochemical methods. In summary, we show that 43% of sporadic MSI-H tumors express ANXA10, a marker that we recently discovered to be expressed in SSA/Ps. In contrast, only 1 of 28 LS-associated tumors with loss of MLH1 expression was positive for ANXA10, and this tumor arose in a patient with germline MLH1 promoter hypermethylation. Importantly, no tumor from a patient with a deleterious MLH1 mutation expressed ANXA10. On the basis of these results, we propose that immunohistochemistry for ANXA10 can be useful in screening for LS. REFERENCES 1. Geiersbach KB, Samowitz WS. Microsatellite instability and colorectal cancer. Arch Pathol Lab Med. 2011;135:1269–1277. 2. Moreira L, Balaguer F, Lindor N, et al. Identification of Lynch syndrome among patients with colorectal cancer. JAMA. 2012;308:1555–1565. 3. Niessen RC, Hofstra RMW, Westers H, et al. Germline hypermethylation of MLH1 and EPCAM deletions are a frequent cause of Lynch syndrome. Genes Chromosomes Cancer. 2009;48:737–744. 4. Ligtenberg MJL, Kuiper RP, Chan TL, et al. Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 30 exons of TACSTD1. Nat Genet. 2009;41:112–117. 5. Shia J, Tang LH, Vakiani E, et al. Immunohistochemistry as firstline screening for detecting colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome: a 2-antibody panel may be as predictive as a 4-antibody panel. Am J Surg Pathol. 2009;33:1639–1645. 6. Shia J. Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn. 2008;10:293–300. 7. Mojtahed A, Schrijver I, Ford JM, et al. A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas, skin sebaceous tumors, and gynecologic tract carcinomas. Mod Pathol. 2011;24:1004–1014. 8. Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 2005;352:1851–1860. 9. Bettington M, Walker N, Clouston A, et al. The serrated pathway to colorectal carcinoma: current concepts and challenges. Histopathology. 2013;62:367–386. 10. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer. 2004;4:988–993. 11. Ogino S, Nosho K, Kirkner GJ, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58:90–96. 12. Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–793. 13. Samowitz WS, Albertsen H, Herrick J, et al. Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology. 2005;129:837–845. 14. Parsons MT, Buchanan DD, Thompson B, et al. Correlation of tumour BRAF mutations and MLH1 methylation with germline
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mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification. J Med Genet. 2012;49:151–157. Deng G, Bell I, Crawley S, et al. BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res. 2004;10:191–195. Loughrey MB, Waring PM, Tan A, et al. Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer. Fam Cancer. 2007;6:301–310. Domingo E, Laiho P, Ollikainen M, et al. BRAF screening as a lowcost effective strategy for simplifying HNPCC genetic testing. J Med Genet. 2004;41:664–668. Rahner N, Friedrichs N, Steinke V, et al. Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome. J Pathol. 2008;214:10–16. Bettstetter M, Dechant S, Ruemmele P, et al. Distinction of hereditary nonpolyposis colorectal cancer and sporadic microsatellite-unstable colorectal cancer through quantification of MLH1 methylation by real-time PCR. Clin Cancer Res. 2007; 13:3221–3228. Gausachs M, Mur P, Corral J, et al. MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a costeffectiveness study. Eur J Hum Genet. 2012;20:762–768. Young J, Simms LA, Biden KG, et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol. 2001;159:2107–2116. Patil DT, Shadrach BL, Rybicki LA, et al. Proximal colon cancers and the serrated pathway: a systematic analysis of precursor histology and BRAF mutation status. Mod Pathol. 2012;25: 1423–1431. Walsh MD, Buchanan DD, Pearson S-A, et al. Immunohistochemical testing of conventional adenomas for loss of expression of mismatch repair proteins in Lynch syndrome mutation carriers: a case series from the Australasian site of the colon cancer family registry. Mod Pathol. 2012;25:722–730. Gonzalo DH, Lai KK, Shadrach B, et al. Gene expression profiling of serrated polyps identifies Annexin A10 as a marker of a sessile serrated adenoma/polyp. J Pathol. 2013;230:420–429. Ogino S, Kawasaki T, Brahmandam M, et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn. 2006;8:209–217. Hampel H. Point: justification for Lynch syndrome screening among all patients with newly diagnosed colorectal cancer. J Natl Compr Canc Netw.; 2010:597–601. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genet Med. 2009;11:35–41. Torlakovic E, Skovlund E, Snover DC, et al. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol. 2003;27:65–81. Lash RH, Genta RM, Schuler CM. Sessile serrated adenomas: prevalence of dysplasia and carcinoma in 2139 patients. J Clin Pathol. 2010;63:681–686. Goldstein NS, Bhanot P, Odish E, et al. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol. 2003;119:778–796. O’Brien MJ, Yang S, Mack C, et al. Comparison of microsatellite instability, CpG island methylation phenotype, BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma end points. Am J Surg Pathol. 2006;30:1491–1501. Kambara T, Simms LA, Whitehall VLJ, et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut. 2004;53:1137–1144. r
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Annexin A10 and Screening for Lynch Syndrome
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