Department of Dermatology, CHU Bordeaux, Bordeaux, France.
PLoS One. 2013 Aug 20;8(8):e70826. doi: 10.1371/journal.pone.0070826. eCollection 2013.
BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF(V600) mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF(V600E) antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF(V600E) (45.2%), c.1799_1800TG>AA, BRAF(V600E2) (3.0%), c.1798_1799GT>AA, BRAF(V600K) (3.0%), c.1801 A>G, BRAF(K601E) (1.3%), c.1789_1790CT>TC, BRAF(L597S) (0.4%), c.1780G>A, BRAF(D594N) (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF(V600E/E2) mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF(V600E) mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF(V600E/E2) mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF(V600E) detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.
BRAF 抑制剂已证明可改善转移性黑色素瘤和 BRAF(V600)突变患者的总生存期。为了评估原发性和转移性部位 BRAF 肿瘤异质性，我们通过比较高分辨率熔解分析（HRM）与 Sanger 测序在平行盲法研究中，评估了抗 BRAF(V600E)抗体的免疫组化（IHC）在两种定位中的表现。共有 99 名晚期或转移性黑色素瘤（III 期或 IV 期）患者的 230 个样本分布为原发性黑色素瘤（n=88）和不同类型的转移性样本（n=142）。每种 BRAF 突变的患病率为 c.1799T>A，BRAF(V600E)（45.2%），c.1799_1800TG>AA，BRAF(V600E2)（3.0%），c.1798_1799GT>AA，BRAF(V600K)（3.0%），c.1801 A>G，BRAF(K601E)（1.3%），c.1789_1790CT>TC，BRAF(L597S)（0.4%），c.1780G>A，BRAF(D594N)（0.9%）。IHC 在 112 个携带 BRAF(V600E/E2)突变的样本中为阳性，在其他情况下为阴性。在 BRAF(V600E)突变病例的肿瘤细胞中，细胞质染色呈强阳性。它呈现出强烈的棕色，与黑素细胞的囊泡灰色细胞质色素沉着不同。两种技术之间的一致性为 96.4%。IHC 检测 BRAF(V600E/E2)突变的敏感性为 97.3%，特异性为 100%。我们的 IHC 和分子研究均表明黑色素瘤中 BRAF 状态在原发性和转移性部位之间具有同质性。这项研究还提供了证据表明，IHC 可能是检测 BRAF(V600E)的一种具有成本效益的一线方法。此后，应在阴性、模棱两可或无贡献的情况下使用分子技术。
