Utility of BRAF V600E Mutation-Specific Immunohistochemistry in Detecting BRAF V600E-Mutated Gastrointestinal Stromal Tumors

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1 AJCP / Original Article Utility of BRAF V600E -Specific Immunohistochemistry in Detecting BRAF V600E-Mutated Gastrointestinal Stromal Tumors Deepa T. Patil, MD, 1 Shuang Ma, PhD, 1 Mai Konishi, 2 Paula D. Carver, MS, 1 Marina Pukay, 3 Carol Beadling, PhD, 3 Christopher L. Corless, MD, PhD, 3 and Brian P. Rubin, MD, PhD 1 From the 1 Robert J. Tomsich Pathology and Laboratory Medicine Institute, Department of Pathology, Cleveland Clinic, Cleveland, OH; 2 Department of Pathology, Division of Molecular and Diagnostic Pathology, Shiga University of Medical Science, Shiga, Japan; and 3 Knight Diagnostic Laboratories, Oregon Health and Science University, Portland. Key Words: BRAF V600E; Immunohistochemistry; Gastrointestinal stromal tumor Am J Clin Pathol November 2015;144: ABSTRACT Objectives: As patients with BRAF V600E mutation respond to BRAF inhibitors, it is important to identify these mutations to stratify patients for the appropriate therapy. In this study, we evaluated the utility of a BRAF V600E allele-specific antibody in gastrointestinal stromal tumors (GISTs). Methods: BRAF V600E mutation-specific immunohistochemistry (negative, weak, or moderate/strong expression) and BRAF sequencing were performed on 38 consecutive GISTs diagnosed between January 2013 and April Results: GISTs from a cohort of 25 men and 13 women (mean age, 61 years; range, years) were localized to the stomach (18), small bowel (10), colon (three), rectum (two), and pelvis/omentum (five). Strong and diffuse cytoplasmic BRAF expression was noted in two (5%) of 38 cases, while eight (21%) of 38 cases showed weak staining, and 28 (74%) of 38 cases were negative. Both of the strongly positive cases arose in the stomach, occurring in a 42-year-old and a 47-year-old woman, respectively. The lesions measured 0.8 and 1 cm, showed spindle cell morphology, and had no risk of progressive disease by Miettinen criteria. Both cases showed heterozygous BRAF V600E, while no BRAF mutations were detected in cases with weak or negative BRAF expression. Conclusions: BRAF V600E mutation-specific immunohistochemistry is a highly sensitive and specific method for detecting BRAF-mutated GISTs. Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. It predominantly arises in the stomach and small bowel and demonstrates a spindled and/or epithelioid morphology. Nearly 85% of GISTs harbor oncogenic mutations in KIT or PDGFRA. 1 The most frequent KIT mutations occur in exon 11 (65%-70%) and exon 9 (20%), while exon 18 (6%) is the most common site for PDGFRA mutations. 2-4 The remaining 10% to 15% of GISTs that do not harbor KIT or PDGFRA mutation are designated as wild-type GISTs. Recent studies have shown that the subset of KIT/ PDGFRA wild-type GISTs is actually a heterogeneous group of tumors that often do not respond to tyrosine kinase inhibitors and harbor alterations in the succinate dehydrogenase (SDH) complex (SDH-deficient GISTs), 5-8 BRAF, 9 or NF1. 10 Identification of these alternative pathways of oncogenesis has not only provided an explanation for drug resistance but has also yielded new opportunities for targeted therapy, especially the use of BRAF inhibitors for BRAF-mutated GISTs. 11 The most common, activating BRAF mutation occurs in exon 15 and is a DNA base substitution of thymine for adenine (T to A) that converts valine to glutamic acid of amino acid residue 600 (BRAF V600E). A variety of molecular assays, such as Sanger sequencing, pyrosequencing, allelespecific real-time polymerase chain reaction (PCR), mass spectrometry based sequencing, and high-resolution melting curve analysis, have been used to detect BRAF V600E mutation. 12 More recently, a BRAF V600E mutant-specific monoclonal antibody (clone VE1) was introduced and used to detect the BRAF-mutant protein in formalin-fixed, paraffin-embedded tissue obtained from various malignancies, 782 Am J Clin Pathol 2015;144:

2 AJCP / Original Article including melanoma, thyroid cancer, and colorectal adenocarcinomas However, the utility of this BRAF V600E mutant-specific monoclonal antibody in the detection of BRAF-mutated GISTs has never been evaluated and forms the basis of this study. bidirectional Sanger sequencing as previously described. 19,20 All immunohistochemical analysis and genotyping were performed in a blinded manner. Results Materials and Methods Histologic Review Following approval by Cleveland Clinic s Institutional Review Board, consecutive GISTs diagnosed between January 2013 and April 2014 were culled from our database. In addition to documenting demographic data, we recorded the following parameters: location, size, primary/recurrent lesion, cell type (spindle, epithelioid, or mixed), mitotic activity, and immunoreactivity for CD117, CD34, and/or DOG1. Risk stratification for progressive disease was performed using the Miettinen risk assessment system. 1 Immunohistochemical Analysis Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue cut in 4-mm sections on the Ventana Benchmark Ultra automated immunostainer (Ventana Medical Systems [VMS], Tucson, AZ). Online deparaffinization was followed by online epitope retrieval using a high ph Tris-based solution (Ultra CC1; VMS) for 64 minutes at 100 C. The slides were incubated with the anti-braf V600E primary antibody (mouse monoclonal VE1; Spring Bioscience, Pleasanton, CA) at 1:175 dilution for 16 minutes at 37 C. Localization of the antigenantibody complex was achieved using the VMS OptiView DAB detection kit and VMS OptiView Amplification kit. Staining was scored as negative (no staining), weak (pale brown staining that was stronger than background staining in smooth muscle), or moderate/strong (intermediate to dark brown staining). Two authors (D.T.P. and B.P.R.) scored the slides, with discrepancies being resolved across the microscope. Genotyping DNA extraction was performed on formalin-fixed, paraffin-embedded tumor samples by macrodissecting tumor-rich areas from unstained paraffin sections. Selected exons of BRAF, 15 KIT, 9,11,13,17 and PDGFRA 12,14,18 were amplified by PCR and screened for sequence alterations by high-resolution melting curve analysis on an LC480 Lightcycler (Roche Applied Science, Penzberg, Germany). Primer sequences and PCR conditions are detailed in Data Supplement 1 (all supplemental materials can be found at All mutations were confirmed by Clinicopathologic Findings The cohort consisted of 38 GISTs (37 primary and one recurrent) resected from 25 men and 13 women. The mean age was 61 years (range, years). The tumors were localized to the stomach (18 [47%]), small bowel (10 [26%]), colon (three [8%]), rectum (two [5%]), and omentum/pelvis (five [13%]). Of the 38 cases, 35 were unifocal and three were multifocal. The mean size was 4.3 cm (range, cm). Most tumors showed spindled morphology (31 [82%]), followed by mixed epithelioid and spindle (four [11%]), and epithelioid (three [8%]) phenotype Table 1. All tumors were positive for CD117 (KIT) and DOG1. Comparison of BRAF Immunohistochemistry and Molecular Genetic Results Strong and diffuse cytoplasmic BRAF expression was observed in two (5%) of 38 cases, which were also CD117 (KIT) and DOG1 positive Image 1. Both the cases were localized to the stomach and were found incidentally in a 42-year-old and a 47-year-old woman who underwent sleeve gastrectomies for morbid obesity. The lesions measured 0.8 cm and 1 cm in the greatest dimension, displayed spindle cell morphology, and showed no risk of progressive disease as assessed by modified Miettinen risk assessment criteria (Image 1). No histologic features distinguished these tumors from the more common KIT-mutated tumors. Both patients are alive without evidence of disease after a follow-up duration of 7 months and 3 months. The rest of the cases either showed weak BRAF expression (eight [21%] of 38) Image 2 or were completely negative (28 [74%] of 38). Nonspecific granular staining was observed within the nuclei of small bowel epithelial cells or cytoplasm of parietal cells in 18 (86%) of 21 cases with adjacent overlying mucosa and within the cytoplasm of smooth muscle cells in 26 (93%) of 28 cases (Image 2). Genotyping was performed on all 38 cases, and the cases with both diffuse and strong BRAF protein expression harbored BRAF V600E mutations Figure 1. No BRAF mutations were detected in cases with weak or negative BRAF expression. Thus, the sensitivity and specificity of moderate to strong BRAF expression were 100%. The remaining cases with weak or negative BRAF expression harbored mutations in KIT exon 11 (19 of 38), exon 9 (five of 38), exon 17 (one of 38), or PDGFRA exon 18 (four of 38) Am J Clin Pathol 2015;144:

3 Patil et al / BRAF V600E Immunohistochemistry in Detecting BRAF-Mutated GISTs Table 1 Clinicopathologic Features, BRAF, and Molecular Analysis of Gastrointestinal Stromal Tumors Case No. Age, y/ Sex Site Size, cm Risk Stratification a Morphology BRAF Immunohistochemistry KIT PDGFRA BRAF V600E 1 63/M Omentum 0.1 No Spindle Weak Exon 11 WT WT 2 75/M Stomach 8 Low Spindle Weak WT Exon 18 WT 3 44/M Rectum 5 Low Spindle Negative Exon 11 WT WT 4 51/M Colon 1.5 No Spindle Negative Exon 11 WT WT 5 57/M Small bowel 7.8 Intermediate Spindle Negative Exon 9 WT WT 6 54/M Small bowel (jejunum) 2.2 Low Spindle and Weak Exon 9 WT WT epithelioid 7 48/F Stomach 0.7 No Spindle Negative Exon 11 WT WT 8 56/M Small bowel (ileum) 2 No Spindle Negative Failed Failed Failed 9 54/M Stomach 0.6 NA Spindle Negative Exon 11 WT WT 10 51/F Colon 2.5 High Spindle Weak Exon 11 WT WT 11 41/F Small bowel 1.5 Very low Spindle Negative Exon 11 WT WT 12 58/M Small bowel 4.2 Low Spindle Negative Exon 9 WT WT 13 42/F Stomach 0.8 No Spindle Strong WT WT Present 14 67/M Rectum 2 High Spindle Negative Exon 11 WT WT 15 49/F Pelvis/omentum (multifocal) 13.5 High Spindle and epithelioid Negative Exon 11 WT WT 16 73/M Pelvis/omentum 25 High Epithelioid Negative Failed Failed Failed 17 69/F Pelvis/omentum 5.1 High Spindle Negative Exon 11 WT WT 18 67/F Stomach 0.19 No Spindle Negative WT WT WT 19 71/M Small bowel (duodenum) 6 NA Spindle Weak Exon 17 WT WT 20 64/M Stomach 4.5 Very low Spindle Negative Exon 11 WT WT 21 88/M Pelvis/omentum 10 NA (posttreatment) Spindle Negative Exon 11 WT WT 22 45/F Stomach 1.4 Intermediate Spindle Weak Exon 11 WT WT 23 70/M Colon 3.5 High Spindle Weak Failed Failed Failed 24 66/M Stomach 0.3 No Spindle Negative Exon 11 WT WT 25 39/M Small bowel (jejunum) 5 High Spindle and Negative Exon 11 WT WT epithelioid 26 60/M Stomach 2.9 Very low Spindle Weak Exon 11 WT WT 27 52/M Small bowel (duodenum) 7.2 High Spindle Negative WT WT WT 28 64/M Stomach 1.7 None Spindle Negative WT Exon 18 WT 29 71/M Stomach 4 Very low Spindle Negative Exon 9 WT WT 30 77/F Stomach 0.5 No Spindle Negative Exon 11 WT WT 31 47/F Stomach 1 No Spindle Strong WT WT Present 32 77/M Stomach 5.8 Low Epithelioid Negative WT Exon 18 WT 33 67/F Stomach 3.2 Intermediate Spindle and Negative WT WT WT epithelioid 34 66/M Stomach 4.1 Very low Spindle Negative WT Exon 12 WT 35 70/F Stomach 7.5 NA (posttreatment) Epithelioid Negative WT Exon 18 WT 36 79/M Small bowel 6.5 Intermediate Spindle Negative Exon 11 WT WT 37 69/F Stomach 3.5 Very low Spindle Negative Exon 11 WT WT 38 39/M Small bowel 2 No Spindle Negative Exon 9 WT WT NA, not available; WT, wild type. a Risk stratification using modified Miettinen risk assessment criteria. 1 or exon 12 (one of 38). There were three examples of KIT/ PDGFRA wild-type GISTs, while three cases failed to yield meaningful results due to technical difficulties. Discussion Most GISTs harbor activating mutations in KIT or PDG- FRA genes, and depending on the genotype, KIT/PDGFRAmutated GISTs show a variable response to tyrosine kinase inhibitor therapy that targets KIT and PDGFRA KIT/ PDGFRA wild-type GISTs generally show poor responses to imatinib. A subset of these GISTs has been shown to harbor BRAF mutations. 9 BRAF belongs to the RAF family of serine/threonine protein kinases that is involved in cell cycle regulation and growth signaling via the mitogen-activated protein kinase pathway. Located downstream of KIT, BRAF is activated by the V600E mutation and supports KIT-independent growth of GIST cells. Gain-of-function BRAF mutations have been detected in several malignancies, including colonic adenocarcinoma, papillary thyroid carcinoma, 18 and melanoma, Am J Clin Pathol 2015;144:

4 AJCP / Original Article Image 1 BRAF V600E allele-specific antibody positive gastrointestinal stromal tumors (GISTs). Case 13 was a 0.8-cm gastric GIST from a 42-year-old woman (H&E, 20; inset, 200). Case 31 was a 1-cm gastric GIST from a 47-year-old woman (H&E, 200). Both tumors were composed of spindle cells and were diffusely positive for CD117 and DOG1 ( 200). Note the diffuse and strong granular cytoplasmic BRAF V600E expression in these lesions ( 200). Both lesions were confirmed to have a heterozygous BRAF V600E mutation. among others. Historically, these mutations have been detected using sequencing-based techniques. More recently, the BRAF V600E mutant-specific antibody VE1 became available for detecting BRAF mutations via immunohistochemistry.27 Studies that have evaluated this antibody in papillary thyroid carcinoma,28 melanoma,29,30 and pulmonary adenocarcinoma31 have demonstrated high sensitivity and specificity in detecting BRAF mutations. Am J Clin Pathol 2015;144:

5 Patil et al / BRAF V600E Immunohistochemistry in Detecting BRAF-Mutated GISTs A B C D Image 2 A, An example of a gastrointestinal stromal tumor with weak BRAF expression ( 200). This case was negative for BRAF V600E mutation. Nonspecific staining was observed within the nuclei of small bowel epithelial cells (B, 200), cytoplasm of parietal cells (C, 200), and smooth muscle cells (D, 200). However, false-positive17,32,33 and false-negative results16 in colonic adenocarcinomas have questioned their utility as a primary tool for establishing BRAF mutation status. As the utility of the VE1 antibody in detecting BRAF mutations in GISTs has never been evaluated, we decided to perform this systematic analysis on a series of consecutive GISTs diagnosed at the Cleveland Clinic. In our series, strong and diffuse BRAF expression was observed in two (5.3%) of 38 cases. Sequencing analysis confirmed the presence of BRAF V600E mutation in both cases, thus documenting 100% specificity and 100% sensitivity in detecting BRAF mutation using the VE1 antibody. Both these tumors were incidentally found within the stomach and measured 0.8 cm and 1.0 cm in the greatest dimension. Based on prior studies that have examined BRAF 786 Am J Clin Pathol 2015;144: Downloaded from mutations in GISTs (summarized in Table 2 ), most BRAFmutated tumors appear to originate within the small bowel and demonstrate spindle cell morphology. The stomach is an uncommon site of origin, and only four examples of gastric BRAF-mutated GISTs have been described thus far.9,34-36 Similar to our cases, all four cases of gastric BRAF-mutated GISTs reported in the literature demonstrated spindle cell morphology (Table 2). The tumor sizes (documented in three cases) and risk of progressive disease were 3 cm (very low), 4 cm (very low), and 10.5 cm (intermediate). Similar to prior studies, we did not find any histologic features that were distinctive of BRAF-mutated GISTs. The reported frequency of BRAF mutations among wild-type GISTs has ranged from three (5%) of 6136 to two (7%) of 28,9 nine (13%) of 70,35 and three (20%) of 15.34

6 AJCP / Original Article A T A C A G T G A A A T B T A C A G A G A AA T C T A C A G T G A A A T D T A C A G T G A A A T Figure 1 Sanger sequencing results for BRAF V600E mutation in case 13 (A) and case 31 (B) showing a heterozygous mutation (GTG>GAG). C, D, Examples of cases with wild-type BRAF harboring two GTG alleles. Arrows correspond to the V600 position in BRAF. Table 2 Summary of BRAF-Mutated Gastrointestinal Stromal Tumors Reported in the Literature Study Case No. Age, y/ Sex Site Size, cm Risk Stratification a Morphology KIT PDGFRA BRAF V600E Agaram et al /F Small intestine 10 High Spindle WT WT Positive 2 55/F Small intestine 10 Intermediate Spindle WT WT Positive 3 49/F Small intestine 9 High Spindle WT WT Positive Agaimy et al /M Stomach 4 Very low Spindle WT WT Positive 5 81/M Small intestine 4 Low Spindle WT WT Positive Hostein et al /M Small intestine 20 High Spindle WT WT Positive 7 38/M Small intestine 2.5 Low Spindle and epithelioid WT WT Positive 8 63/M Stomach NA NA Spindle WT WT Positive 9 78/M Stomach 3 Very low Spindle WT WT Positive 10 51/F Small intestine 2.5 High Spindle WT WT Positive 11 58/M Small intestine 2.5 Low Spindle and epithelioid WT WT Positive 12 58/M Small intestine 2.5 High Spindle WT WT Positive 13 41/M Small intestine 2.5 Low Spindle WT WT Positive 14 50/M Peritoneum 2.8 High Epithelioid WT WT Positive Daniels et al /F Esophagus 28 Low Spindle WT WT Positive 16 68/F Stomach 10.5 Intermediate Spindle WT WT Positive 17 60/F Small intestine 8 Intermediate Spindle WT WT Positive Miranda et al NA NA NA NA NA WT WT Positive 19 NA Small intestine NA High NA Exon 11 KIT WT Positive mutation b Current study 20 42/F Stomach 0.8 No Spindle WT WT Positive 21 47/F Stomach 1 No Spindle WT WT Positive NA, not available; WT, wild type. a Risk stratification using modified Miettinen risk assessment criteria. 1 b BRAF mutation in a gastrointestinal stromal tumor with concomitant KIT exon 11 mutation corresponding to a deletion of four amino acids (ΔV555-K558). In our series, a BRAF mutation was found in two (40%) of five KIT/PDGFRA wild-type GISTs. This higher rate is most likely a result of the smaller sample size and the type II error introduced therein. Nonetheless, determining the rate of BRAF mutations in wild-type GISTs was not the goal of this study. With one exception, 37 current and previous studies 9,35,36 have shown BRAF and KIT/PDGFRA mutations to be mutually exclusive genetic events in treatment-naive GISTs. Miranda et al 37 reported a BRAF mutation in a GIST with a concomitant KIT exon 11 deletion (ΔV555-K558). This tumor was located within the small bowel and was risk stratified to have a high risk of progressive disease. In vitro experiments performed by Miranda et al showed that in cell lines coexpressing an imatinib-sensitive mutant form of KIT and constitutively activated BRAF protein, imatinib was Am J Clin Pathol 2015;144:

7 Patil et al / BRAF V600E Immunohistochemistry in Detecting BRAF-Mutated GISTs able to inhibit KIT and its downstream signaling but not the extracellular signal regulated kinase 1/2 activation driven by mutationally activated BRAF. This suggests that mutant BRAF causes resistance to imatinib and furthermore that KIT/ PDGFRA wild-type tumors with BRAF mutations might be unresponsive to KIT inhibitors. A case reported by Falchook and colleagues 11 supports this view. A 60-year-old man with BRAF V600E GIST had disease recurrence while on adjuvant imatinib and again while on adjuvant sunitinib following a second surgery. Importantly, this patient s tumor did respond well to the BRAF inhibitor dabrafenib. 11 Given that there are now three therapies approved by the US Food and Drug Administration targeting BRAF V600E (vemurafenib, dabrafenib, and trametinib), screening for this mutation in KIT/ PDGFRA wild-type GISTs is clinically important. Nonspecific staining within the epithelial cells (nuclear in small bowel epithelial cells and cytoplasmic in parietal cells) and smooth muscle cells was observed in a subset of our cases (Image 2). This phenomenon has been previously reported and is believed to be a result of cross-reactivity with other epitopes present in normal tissues. 17,33,38,39 In summary, in addition to expanding the clinicopathologic profile of gastric BRAF-mutated GISTs, this study demonstrates the high sensitivity and specificity of VE1 antibody in detecting BRAF-mutated GISTs. This technique serves as an efficient tool to stratify patients with KIT/PDGFRA wild-type GISTs. We recommend using the VE1 antibody as a reflex test in routine practice, specifically in unresectable/ metastatic GISTs that are negative for KIT/PDGFRA mutation and that would benefit from medical therapy. Corresponding authors: Deepa T. 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