title: The use of BRAF V600E mutation-specific immunohistochemistry in pediatric Langerhans cell histiocytosis.
abstract: BRAF p.V600E mutations are detected in greater than 50% of pediatric Langerhans cell histiocytosis (LCH) lesions. However, the use of mutation-specific BRAF V600E immunohistochemistry (IHC) as a surrogate for molecular testing in pediatric LCH is unknown. We tested the mutation-specific BRAF V600E monoclonal antibody (clone VE1) in formalin-fixed, paraffin-embedded LCH samples from 26 pediatric patients (14 males and 12 females, ages 7 mo-17 y) using allele-specific real-time polymerase chain reaction (PCR) with a limit of detection of 0.5% as the comparative gold standard. BRAF VE1 staining was scored for both intensity (0-3+) and percentage of immunoreactive tumor cells (0%-100%). BRAF VE1 immunoreactivity was determined using both lenient (≥1+, ≥1%) and stringent (≥2+, ≥10%) scoring criteria. Using lenient-scoring criteria, we found that the sensitivity and specificity of IHC compared with allele-specific real-time PCR were 100.0% and 18.2%, respectively. The poor specificity of lenient IHC analysis was attributable to weak, 1+ staining in both BRAF-mutated and wild-type LCH. Using stringent-scoring criteria, we found that specificity improved to 100.0% at the expense of sensitivity that decreased to 80.0%. Stringent scoring generated 3 false-negative results, but in all cases, neoplastic tissue comprised less than 5% of the stained section and/or the specimen was decalcified. In conclusion, highly sensitive molecular assays remain the gold standard for BRAF mutation analysis in LCH paraffin-embedded lesions. To avoid false-positive results, unequivocal VE1 staining of 2+ intensity in greater than or equal to 10% neoplastic histiocytes is required. However, negative VE1 results require additional studies to exclude false-negatives, and stringent-scoring criteria may not be optimal for scant or decalcified specimens.
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Leomar Y
authors.initials: LY
authors.lastname: Ballester
authors.affiliation: Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.|Division of Hematology and Oncology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Miguel D
authors.initials: MD
authors.lastname: Cantu
authors.affiliation: Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.|Division of Hematology and Oncology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Karen P H
authors.initials: KPH
authors.lastname: Lim
authors.affiliation: Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Sarabia
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.
authors.forename: Lizmery Suarez
authors.initials: LS
authors.lastname: Ferguson
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.
authors.forename: C
authors.initials: C
authors.lastname: Renee Webb
authors.affiliation: Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.|Division of Hematology and Oncology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Carl E
authors.initials: CE
authors.lastname: Allen
authors.affiliation: Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.|Division of Hematology and Oncology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: McClain
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Carrie A
authors.initials: CA
authors.lastname: Mohila
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Jyotinder N
authors.initials: JN
authors.lastname: Punia
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.|Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.
authors.forename: Angshumoy
authors.initials: A
authors.lastname: Roy
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.|Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.
authors.forename: Dolores H
authors.initials: DH
authors.lastname: López-Terrada
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.|Department of Pediatrics, Texas Children's Hospital, Houston, TX, USA.
authors.forename: M
authors.initials: M
authors.lastname: John Hicks
authors.affiliation: Department of Pathology, Texas Children's Hospital, Houston, TX, USA.|Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Kevin E
authors.identifier: http://orcid.org/0000-0002-6179-0937
authors.initials: KE
authors.lastname: Fisher
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D005260:Female; D006646:Histiocytosis, Langerhans-Cell / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008297:Male; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; LCH; V600E; VE1; immunohistochemistry; pediatric