Distinct clinicopathological features in metanephric adenoma harboring BRAF mutation

BRAF mutation recently has been reported in metanephric adenoma. We sought to determine the clinical and morphologic features of BRAF-mutated metanephric adenoma and to correlate BRAF mutation with BRAF V600E immunohistochemical staining results. A series of 48 metanephric adenomas and 15 epithelial-predominant nephroblastomas were analyzed for the occurrence of BRAF mutation (BRAF V600E/V600E complex, BRAF V600D, BRAF V600K and BRAF V600R) using the BRAF RGQ PCR kit (Qiagen). Immunohistochemistry was performed using monoclonal mouse antibodies against p16INK4 and VE1 (Spring Bioscience), recognizing the BRAF V600E mutant protein. Forty-one of 48 cases (85%) showed BRAF V600E mutation; none of the other BRAF variants was detected. Of 41 BRAF-mutated metanephric adenomas, 33 showed positive VE1 immunostaining (sensitivity 80%, specificity 100%); in all cases we detected p16INK4 expression regardless of BRAF mutation status. All epithelial-predominant nephroblastomas were BRAF-wild-type and none expressed VE1. The following features were associated with BRAF V600E mutation: older patients (p=0.01), female predominance (p=0.005) and the presence of a predominantly acinar architecture (p=0.003). In summary, BRAF-mutated metanephric adenomas were associated with older age, female predominance, and the presence of a predominant acinar component. A subset (20%) of BRAF-mutated metanephric adenomas was not detected by VE1 immunostaining.


INTRODUCTION
Metanephric adenoma of the kidney is an uncommon benign neoplasm which is usually asymptomatic and discovered incidentally. These tumors mostly occur in middle-aged individuals, with a female predominance (2:1), although the age distribution is broad, ranging from children to the elderly [1][2][3][4]. On gross examination, metanephric adenomas are typically circumscribed, not-encapsulated, solid masses. Histologically, these neoplasms are typically composed of small epithelial cells arranged as tightly packed small acini. A hyalinized or edematous stroma is present occasionally. Psammoma bodies are common. The cells have scant cytoplasm, round nuclei, and variably-present nuclear grooves. However, metanephric adenomas may assume a variety of architectures and may thus present a diagnostic challenge to the pathologist. The main differential diagnostic considerations for metanephric adenoma are epithelialpredominant nephroblastoma in children and the solid variant of papillary renal cell carcinoma in adults. In challenging cases, immunohistochemistry and FISH techniques are helpful. With immunohistochemistry, metanephric adenomas usually label for WT1 and CD57 and are characteristically negative for CK7 and AMACR [5]. FISH can be used for analyzing chromosomes 7, 17 and Y. Metanephric adenoma lacks the gains of chromosome 7 and 17 and losses of Y that are typical of papillary renal cell carcinoma [6].
In this study, we correlated BRAF mutation, detected by molecular analysis, with BRAF V600E immunohistochemical staining in a series of 48 metanephric adenomas and 15 epithelial-predominant nephroblastomas. In addition, we sought to identify clinical and histopathological features of metanephric adenomas harboring BRAF mutation.

RESULTS
Of the 48 patients with metanephric adenoma, 31 were female and 17 were male (F:M ratio, 1.8:1). The median age at diagnosis was 54 years (range: 5 to 84 years) and the median size of the tumor was 4 cm (range from 1.1 to 8 cm) ( Table 1). Among the 15 epithelial-predominant nephroblastoma patients, 8 were female and 7 were male (F:M ratio, 1.1:1). The median age was 5 years (range, 8 weeks to 41 years). There were 3 tumors that occurred in adult patients (27,35, and 41 years, respectively), one female and two male ( Table 2).
Microscopic examination of metanephric adenomas showed neoplasms composed of small, uniform and overlapping epithelial cells with scant cytoplasm, inconspicuous nucleoli and essentially no mitotic figures. These cells were arranged in a variety of architectural patterns ( Figure 1). Simple tubules, acini and solid patterns were the most common. Papillary structures were present in 14 cases (29%) and were the predominant pattern (>50% of the tumor) in 3. Glomeruloid bodies were present in 14 cases (29%), and branching tubules were present in 24 cases (50%). Eighteen cases (38%) showed a variably thickened and usually discontinuous fibrous pseudocapsule. Fibrous septa that gave a vaguely multinodular appearance were recognized in 14 cases (29%). Twenty-eight tumors (58%) contained psammoma bodies, ranging from isolated and scattered to numerous. A few foamy macrophages were seen in only 3 cases (6%). A stromal component was virtually absent in 4 cases (8%). The remaining cases demonstrated edematous or hyalinized stroma or both (range from 5% to 40% of tumor volume).
BRAF V600E mutations were identified in 41 of 48 cases (85%) and none of the other BRAF mutation variants was detected. Of these, 30 patients were women and 11 were men (F:M, 2.7:1). The median age was 57 years (range from 5 to 84 years) and the greatest dimension ranged from 1.4 to 8 cm (median = 3.9 cm). Among the 7 BRAF-wild-type cases, there was a striking male predominance (F:M 1:6) (p=0.0055), and the patients tended to be younger (median 33, range from 10 to 74) (p=0.014). Tumor size in BRAF-wild-type cases (median = 3.5, range from 1.1 to 6.5) was similar to the BRAFwww.impactjournals.com/oncotarget mutated cases (p=0.71). Most BRAF-mutated cases exhibited a predominantly acinar architecture (p=0.003). Among 5 cases without this unique histologic feature, 4 were composed mostly of tubules and one mostly of papillae. The other morphological features were not associated with BRAF mutational status. None of epithelial-predominant nephroblastomas had BRAF mutation.
There was positive cytoplasmic immunolabeling for VE1 antibody in 33 of 41 (80%) metanephric adenomas with BRAF mutation ( Figure 2). All VE1 immunostaining positive cases showed BRAF V600E mutation, detected by Qiagen BRAF RGQ PCR kit. No nuclear staining was detected in any case. All cases of epithelial-predominant nephroblastoma were completely negative, correlating with their BRAF wild-type status. Positive immunolabeling

DISCUSSION
BRAF is an oncogene that normally functions as a regulator of cell division and differentiation through its role in the MAP kinase pathway. Mutations in this gene, which lead to constitutive activation of downstream signaling within this pathway, were most famously implicated in the development of melanoma [14]. Several specific pro-oncogenic mutations of BRAF have since been identified and implicated in the development of a variety of solid and hematopoietic neoplasms [14,[30][31][32]. The vast majority, however, are characterized by a thymineto-adenine transversion at exon 15, which results in an amino acid substitution of valine (V) for glutamic acid (E) at codon V600 (V600E) [14]. It is worth noting that clinical trials have recently emerged proposing targeted therapy for nonmelanoma cancers harboring BRAF mutations, demonstrating the important role of BRAFtesting [33].
Attention to the association of BRAF mutation with metanephric adenoma has been drawn by a few case reports and small series [7,8,10,11,34]. The current study, which tested 48 cases of metanephric adenoma for BRAF mutation, is the largest of its kind. Molecular testing demonstrated that 85% of these tumors harbored a BRAF mutation; a number in line with the findings of Choueiri et al. [7], who described this mutation in 26 of 29 cases (89%), but somewhat lower than the remaining cases in the literature, which cumulatively yielded a mutated BRAF in 22 of 24 cases (92%) ( Table 3). All the BRAF-mutated cases in this study carried the same V600E mutation, which again is in keeping with the findings of almost all prior studies [7,[9][10][11]34]. Of note, Udager et al. [8] identified BRAF V600D in 2 of 10 BRAF-mutated metanephric adenomas. The current study, which looked for the most common variants, including the V600D, did not find this or any other BRAF mutation variant. In this study we also provided clinical and morphologic features characteristic of each metanephric adenoma subset. We found that BRAF-mutated cases were associated with older age whereas BRAF wild-type metanephric adenomas presented earlier. Consistent with our findings, Choueiri et al. showed increased age (55 vs 33 years) in patients harboring a BRAF mutation [7]. A correlation between BRAF mutation status and tumor size has been proposed [7], though the current study did not show any size difference, a discrepancy that probably resulted from the limited number of BRAF-wild-type cases presented in the prior study. With regards to gender, this is the first study to demonstrate a strong male predominance in metanephric adenomas that are BRAF wild-type. Other studies have shown BRAF mutation in 3 of 3 and 4 of 5 male subjects respectively [7,8], however the present study, which included 17 males, is the largest to test for BRAF in a male cohort. Also, we included 4 pediatric cases (<12 years old) in our series. As in adults, BRAF mutation has been reported in pediatric metanephric adenomas [11]. For the first time, we have outlined distinct morphological features characteristic of BRAF-mutated and BRAF-wildtype metanephric adenomas. There was a predominance of acinar architecture (p=0.003) associated with BRAFmutated metanephric adenomas. The combination of solid architecture with psammoma bodies and background hyalinized stroma was found to occur with greater frequency in BRAF-wild-type cases, but the difference did not reach statistical significance. The presence of a specific and consistent mutation implies a potential role for immunohistochemistry, with the VE1 antibody, as a surrogate for molecular testing, particularly in instances where limited tissue is available or the molecular method for BRAF mutation detection is not accessible. Several studies have demonstrated excellent concordance between immunostaining and mutation status in a variety of other neoplasms [25][26][27][28][29]. In the current study, 33 of 41 BRAF-mutated cases were positive by immunohistochemistry. Despite it having been speculated that VE1 would be valuable diagnostically [9], this study demonstrated that VE1 antibody is a very specific (100%) but less sensitive (80%) marker for identifying BRAFmutated metanephric adenomas. Moreover, a subset (15%) of metanephric adenomas does not have BRAF mutation, prompting again careful use as a diagnostic tool. None of epithelial-predominant nephroblastomas was positive for BRAF mutation using either molecular or immunohistochemistry methods in current study. Previous investigations also found that nephroblastomas were negative for BRAF mutation by molecular assays [34,35].
Another aspect of the BRAF V600E mutation is its ability to act not only as an oncogene, but paradoxically, to induce cellular senescence. This has been well studied and documented in various BRAF-driven neoplasms [36][37][38][39][40][41]. One of the major markers shown to identify BRAF-induced senescence is p16 INK4 [36]. In light of the indolent clinical course and high frequency of BRAF mutation in metanephric adenomas, all cases were stained with the p16 INK4 antibody. We found that every metanephric adenoma was positive for this marker. The fact that even BRAF-wild-type tumors also exhibited positive staining with p16 INK4 suggests that mechanisms for oncogene-induced senescence independent of BRAF mutation exist. In addition, approximately half of the epithelial-predominant nephroblastomas were positive for p16 INK4 , illustrating again that alternative modes of senescence induction may be involved. Interestingly, p16 INK4 expression has been shown to correlate with good prognosis in nephroblastoma [42]. The mechanism by which some nephroblastomas evade the senescence pathways remains unknown [43].
In summary, we have identified distinct clinicopathologic patterns associated with BRAFmutated metanephric adenoma. These include older age, female predominance, and the presence of a prominent acinar component. A subset of BRAF-mutated metanephric adenomas (20%) was not detected by VE1 immunostaining. p16 INK4 immunostaining was uniformly positive in all metanephric adenomas.

Patients and samples
Forty-eight cases of metanephric adenoma and fifteen cases of epithelial-predominant nephroblastoma were collected from participating institutions. For each case of metanephric adenoma, the following morphologic features were recorded: the presence or absence of a pseudocapsule, foamy histiocytes, and fibrous septa; the relative proportion of stroma and its being either edematous or hyalinized; the presence and quantity of psammoma bodies; and the architectural patterns. With respect to architecture, specific patterns were recognized including tubules with or without complex branching, acini, glomeruloid structures (short, rounded papillae projecting into small cysts), solid-like (tightly packed acini with overlapping nuclei), and papillary. When present, the proportion of the tumor made up of each of these patterns was recorded as a percentage. A pattern that was greater than 50% within the tumor was considered predominant. This research was approved by the Institutional Review Board.

High resolution melting test for BRAF mutational analysis
The BRAF mutations were analyzed using realtime PCR-high resolution melting test. Areas of tumor in each case designated for testing were circled on hematoxylin and eosin-stained slides by a pathologist (LC). The DNA extractions were run using the BRAF RGQ PCR Kit (Qiagen, Valencia, CA), designed to detect somatic mutations of the BRAF gene using realtime polymerase chain reaction with the Rotor-Gene Q 5plex HRM instrument. DNA concentrations were analyzed by the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Using ARMS (Amplification Refractory Mutation System) and Scorpions technologies, the BRAF RGQ PCR Kit detects mutations at codon 600 of the BRAF oncogene against a background of wild type genomic DNA. The specific mutations detected by this assay are V600E/ V600E complex (V600E/Ec), V600D, V600K, and V600R. All procedures were performed according to the manufacturer's protocol.

Statistical analysis
Fisher's exact test was used to compare categorical data for clinicopathologic characteristics between BRAFmutated and BRAF-wild-type subgroups and Student's t test to compare continuous data. All P values were based on a two-sided hypothesis.