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Application of BRAF V600E mutation analysis for the diagnosis of metanephric adenofibroma.

To the Editor: In a recent issue of this journal, Udager et al1 demonstrated novel BRAF mutations in metanephric adenoma (MA) in addition to BRAF V600E mutations as had been previously described in approximately 90% of MAs. MA is thought to represent one end of a spectrum of rare metanephric neoplasms that include metanephric adenofibroma (MAF) and metanephric stromal tumor (MST).3,4 MAF is a benign biphasic tumor composed of varying proportion of epithelial and stromal components most commonly seen in children (mean age 10.2 y in patients with typical histologic features). In contrast to MAs, the biological nature and molecular characterization of the epithelial and stromal components of MAF have not been investigated. Currently, our knowledge of MAF and MST is based on pure morphology and limited immunophenotypic characterization. We recently encountered a tumor in an open kidney biopsy from a 10-year-old girl with a history of recurrent urinary tract infections who was found to have a solid and cystic lesion of her right upper pole kidney by imaging studies. She had no systemic manifestation, such as hypertension or polycythemia. A posterior approach was used to obtain 1.1 1.0 0.3 cm lesional tissue that was submitted for frozen section diagnosis with the intention of proceeding to nephrectomy if a diagnosis of Wilms tumor (WT) was rendered. Upon review of the histology, the tumor demonstrated a biphasic growth pattern (Figs. 1A–C). There was a 5mm well-circumscribed highly cellular epithelial nodule composed of packed tubules and papillary structures with calcifications typical of MA (Fig. 1B). There was a dominant moderately cellular stromal component composed of fibroblast-like spindle cells with entrapped native renal tubules surrounded by a loose fibromyxoid collarette (onion-skinning) typical of MST (Fig. 1C). Immunohistochemistry demonstrated positive staining of the epithelial component for WT-1, CD57, and cadherin 17, and negative staining for CK7 and AMACR, whereas the stromal component was focally positive for CD34 and WT-1.3–5 BRAF V600E mutation status was assessed separately in the epithelial (tumor cellularity 95%) and stromal (tumor cellularity 50%) components macrodissected from formalin-fixed paraffin-embedded sections (Fig. 1A). Initial screening by mutant allele–specific real-time polymerase chain reaction (PCR) with SYBR green that was carried out on the LightCycler 2.0 (Roche, Indianapolis, IN) demonstrated mutated BRAF products (as defined by a cycle threshold value [Ct]<35 in mutant PCRs) in both epithelial and stromal components (Fig. 1D). Post-PCR melting curve analysis demonstrated a single narrow melting peak for BRAF mutant– amplified reactions in both epithelial and stromal components (81.37± 0.061C), confirming specific amplification. The results of PCR were confirmed by direct Sanger sequencing (Fig. 1D). In addition, we evaluated 3 cases of MA and 3 cases of epithelial Wilms tumor (e-WT) for BRAF V600E mutation. All MAs harbored V600E mutation, whereas all e-WTs had wildtype BRAF. The immunohistochemical profile and molecular testing supported the morphologic diagnosis of MAF and reassured the clinical team that conservative management was appropriate. Subsequently, a partial nephrectomy was performed rather than complete nephrectomy for WT. The entirely submitted 3.0-cm tumor (Fig. 1E) contained only the stromal component similar to MST. In addition to the findings seen in the biopsy specimen, foci of sclerosis with intermixed cellular epithelioid stromal cells were present at the peripheral aspect of the lesion (Fig. 2A). No angiodysplasia, juxtaglomerular hyperplasia, or heterologous elements were present in the stromal component of the biopsy or excision specimen as has been previously described for MST and MAF. Angiodysplasia in the form of epithelioid transformation of medial smooth muscle has been described in approximately two thirds of MAF and the majority of MSTs but is not a constant finding.3,4 Juxtaglomerular cell hyperplasia is typically seen in pure MST rather than MAF, whereas heterologous elements can be seen in a subset of both tumors.3,4 For confirmation of the biopsy findings, molecular testing was performed on the excisional specimen (Fig. 1E) containing exclusively stromal component and was found to be positive for the BRAF V600E mutation. In contrast, sections composed entirely of non-neoplastic renal parenchyma were negative for the BRAF V600E mutation. The post-PCR melting curve of the stromal component demonstrated a similar peak to the biopsy samples, whereas a robust specific melting peak was absent in the non-neoplastic renal parenchyma. The PCR findings were also confirmed by Sanger sequencing. We subsequently performed BRAF V600E immunohistochemistry using the mutation-specific VE-1 monoclonal antibody (Ventana Medical Systems, Tucson, AZ) to correlate with the molecular findings. A melanoma and MA with a known BRAF V600E mutation were used as positive controls. There was moderate to strong positive staining in over half of the cells of the epithelial component but no significant staining of the spindled stromal component of the biopsy (Fig. 2B). However, sections of the excision specimen demonstrated variable weak to moderate positive staining that was most conspicuous in epithelioid stromal cells of the cellular foci (Figs. 2C, D). Our findings underscore the application of recently reported molecular LETTER TO THE EDITOR