Assessment of BRAF V600E (VE1) immunochemistry for the detection of BRAF V600E mutation in non-small cell lung carcinoma cytology specimens

Abstract

Background: Non-small cell lung carcinoma (NSCLC) patients with BRAF V600E-mutated tumors respond to targeted therapy. Testing for BRAF V600E is commonly performed with molecular methods; however, a mutation-specific VE1 antibody clone can provide an alternative testing option using immunohistochemistry (IHC) for practices using single-gene testing and in situations when the specimen is inadequate for molecular testing. This study evaluates the usefulness of VE1 IHC in screening for BRAF V600E mutations in NSCLC cytology specimens.

Methods: The authors retrospectively identified cytology cases with a diagnosis of NSCLC that had BRAF V600E IHC performed on cell block sections with the monoclonal VE1 antibody clone. The BRAF V600E IHC results were compared with those of molecular testing performed with an amplicon-based next-generation sequencing assay.

Results: There were 201 NSCLC cases evaluated. The VE1 IHC was positive in seven of seven BRAF V600E-mutated tumors (100%) and was negative in 158 of 158 nonmutated BRAF V600E tumors (100%). Thirty cases did not undergo molecular testing, primarily because of insufficient tissue or because molecular testing was performed on an alternative specimen. Six cases showed equivocal weak/focal staining: Two cases demonstrated BRAF V600E mutations, and four cases were negative by molecular testing.

Conclusions: This study suggests that BRAF V600E IHC can be used reliably to screen NSCLC cytology specimens, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining.

Keywords: BRAF; IHC; V600E; VE1; cytology specimens; immunochemistry; molecular testing; next-generation sequencing; non-small cell lung carcinoma.

Figure 1.

Non-small cell lung carcinoma cytology specimens with BRAF V600E immunostains. (A) Metastatic adenocarcinoma in a pleural fluid with a hematoxylin and eosin (H&E) stained cell block section, and (B) BRAF V600E immunostain, demonstrating strong, diffuse cytoplasmic staining in tumor cells. The concurrent molecular testing showed a BRAF V600E mutation. (C) Metastatic squamous cell carcinoma to a lymph node with a hematoxylin and eosin (H&E) stained cell block section, and (D) negative BRAF V600E immunostain. The concurrent molecular testing was negative for a BRAF V600E mutation.

Figure 2.

Metastatic lung adenocarcinoma to a lymph node with equivocal BRAF V600E immunostaining showing (A) weak but diffuse cytoplasmic staining of tumor cells; (B) Integrated Genome Viewer (IGV) showing a BRAF V600E mutation on the concurrent next-generation sequencing with a T>A missense mutation in codon 600.

Figure 3.

Metastatic lung adenocarcinoma to a lymph node with equivocal BRAF V600E immunostaining showing (A) membranous (edge artifact) and weak cytoplasmic staining in rare tumor cells; and (B) the concurrent next-generation sequencing showing wild-type sequence only and the absence of a BRAF V600E mutation.

Figure 4.

Metastatic adenocarcinoma to pleural fluid with (A) a negative BRAF V600E immunostain; (B) the concurrent next-generation sequencing (NGS) failed to detect a BRAF V600E mutation. However, a non-V600E missense mutation (G596R) is detected by NGS.

Figure 5.

Metastatic lung adenocarcinoma to bone with BRAF V600E immunostain showing focal nuclear staining, interpreted as negative. The concurrent molecular testing was negative for a BRAF V600E mutation. Non-specific nuclear staining may be a potential pitfall in interpretation of the BRAF V600E immunostain.