Decalcification can cause the failure of BRAF molecular analyses and anti‐BRAFV600E VE1 immunohistochemistry

Abstract

BRAF mutation detection is worthwhile for the management of patients with some advanced cancers. The tumor samples are sometimes difficult to analyze using DNA‐based molecular methods because of poor tumor DNA quality or quantity. Anti‐BRAFV600E VE1 immunohistochemistry (IHC) has been proposed as a valuable ancillary tool to analyze some “molecularly challenging“ tumor samples. In this technical study, we focused on its application in the field of decalcified tumor samples. We selected four patients with known BRAFV600E‐mutated cancer (3 metastatic melanomas and 1 hairy cell leukemia) and paired non‐decalcified/decalcified tumor samples. Molecular analyses failed in the four decalcified samples (3 bone metastases and 1 osteo‐medullar biopsy) with non‐contributive mutation status. Whereas non‐decalcified tumor samples were all positive using anti‐BRAFV600E VE1 IHC, the four decalcified samples were concluded negative. Because decalcified tumor samples are difficult to analyze from a molecular point of view, it is tempting to use IHC instead of DNA‐based methods searching for BRAFV600E mutations in these samples. Nevertheless, the decalcification process may also cause false‐negative results using VE1 IHC. Decalcified samples require specific and optimized IHC and molecular protocols and quality controls.

Comparison of Two Rapid Assays for the Detection of BRAF V600 Mutations in Metastatic Melanoma including Positive Sentinel Lymph Nodes

“…In total, 16 studies [16,17,[25][26][27][28][29][30][31][32][33][34][35][36][37][38] have already reported a comparison between the Idylla method and other molecular reference methods for the assessment of the BRAF V600E mutation in melanoma patients at the time of the publication (Table 5). Most of them used the CE-IVD Idylla TM BRAF Mutation Test with a concordance rate ranging from 96.2% to 100% when compared with NGS, Sanger sequencing, pyrosequencing, or digital PCR, confirming that the test is reliable.…”

“…In addition, nine previous studies reported results using the RT-PCR Idylla method and VE1 IHC for the diagnostic of BRAF mutations [28,29,33,35,[39][40][41][42][43]. Among these, four focused on melanoma samples [28,29,33,35].…”

“…In addition, nine previous studies reported results using the RT-PCR Idylla method and VE1 IHC for the diagnostic of BRAF mutations [28,29,33,35,[39][40][41][42][43]. Among these, four focused on melanoma samples [28,29,33,35]. the others included central nervous system tumors, colorectal cancers, ovarian tumors, hairy cell leukemia, and salivary gland tumors [39][40][41][42][43] (Table 6).…”

Évolution du statut braf dans le melanome : mythe ou Réalité ?

“…Le prélèvement pour lequel un faux-négatif a été donné par l'IHC correspondait à une métastase osseuse ayant nécessité une décalcification. Or l'impact de la décalcification a été étudié par Bourhis et al qui a constaté sur des échantillons jumelés non décalcifiés et décalcifiés mutés BRAFV600E, la disparition de l'immunommarquage sur les prélèvements décalcifiés [5].…”

“…Modele + ANNPAT-1642; No. of Pages 6 Annales de pathologie xxx (xxxx) xxx-xxx rapportant les excellentes performances diagnostiques de l'immunohistochimie, avec une sensibilité > 98 %, soit des performances supérieures à certaines approches de biologie moléculaire encore utilisées [5][6][7][8][9][10]. Dans des conditions pré-analytiques optimales, notamment une concentration abondante d'ADN, la fréquence allélique nécessaire à la détection d'une mutation est estimée à 20 % pour le séquenc ¸age direct SANGER, 10 % pour la PCR-HRM, 2 % pour le séquenc ¸age NGS et 0,0001 % pour la PCR digitale.…”