Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma

Abstract

Background: The Raf/MEK/ERK (mitogen activated protein kinase-MAPK) signal transduction cascade is an important mediator of a number of cellular fates, including growth, proliferation, and survival. The BRAF gene, one of the human isoforms of RAF, is activated by oncogenic Ras, leading to cooperative effects in cells responding to growth factor signals.

Aims: The aim of this study was to elucidate a possible function of BRAF in liver tumours.

Methods: Mutations of BRAF and KRAS were evaluated in 25 hepatocellular carcinomas (HCC) and in 69 cholangiocarcinomas (CC) by direct DNA sequencing analyses after microdissection. The presence of active intermediates of the MAPK pathway was assessed immunohistochemically. The results obtained were correlated with histopathological variables and patient survival.

Results: Activating BRAF missense mutations were identified in 15/69 CC (22%) and in one case of tumour surrounding liver. KRAS mutations were found in 31 of 69 (45%) CC examined and in two cases of tumour surrounding non-neoplastic liver tissue. In HCC, neither BRAF nor KRAS mutations were detected. All 31 CC with KRAS mutations had an intact BRAF gene. We failed to observe a correlation between BRAF or KRAS mutations and histopathological factors or prognosis of patients.

Conclusions: Our data indicate that BRAF gene mutations are a relatively common event in CC but not in HCC. Disruption of the Raf/MEK/ERK (MAPK) kinase pathway, either by RAS or BRAF mutation, was detected in approximately 62% of all CC and is therefore one of the most frequent defects in cholangiocellular carcinogenesis.

Figure 1

Microdissection of cholangiocarcinoma cells. The outlined areas (A) were microdissected (B) by the laser system (Palm MicrobeamSystem) and catapulted (C), as described in materials and methods.

Figure 2

Mutation analysis of the BRAF gene. Electropherograms of the DNA sequences of patient Nos 9, 54, and 58 (same patients as in tables 2 and 3 ▶ ▶).

Figure 3

Immunostaining of the active MAPK protein. (A) Perinuclear staining (red reaction product) of tumour cells (original magnification ×60). (B) Tumour No 51 in table 2 ▶: well differentiated cholangiocarcinoma with a strong staining of active MAPK protein (original magnification ×20). (C) Tumour No 4 in table 3 ▶: poorly differentiated cholangiocarcinoma with partial mucinous differentiation. Tumour cells were positive for active MAPK to a lesser extent compared with (B) (original magnification ×20).