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Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level
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Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level
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Journal Article
Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level
2012
Overview
Background:
The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as
BRAF
and
KIT
. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients.
Methods:
High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45
+
cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA
+
, CD45
−
, MART-1/gp100
+
). Single, isolated CTC were then subjected to
BRAF
and
KIT
mutational analysis.
Results:
CTC (HMW-MAA
+
, CD45
−
, MART-1/gp100
+
) were isolated from the blood of 11 patients and
BRAF
and
KIT
were sequenced in nine and four patients, respectively. The
BRAF
sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the
KIT
sequences were inconsistent in three patients. In addition, polyclonal
BRAF
mutations were identified in one patient and concomitant mutations in
BRAF
and
KIT
were identified in another patient.
Conclusion:
Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ Aged
/ Antigens
/ Biological and medical sciences
/ Biomedical and Life Sciences
/ Female
/ Genotype
/ Humans
/ Kinases
/ Male
/ Melanoma
/ Mutation
/ Neoplasm Proteins - genetics
/ Neoplastic Cells, Circulating - metabolism
/ Neoplastic Cells, Circulating - pathology
/ Oncology
/ Patients
/ Proto-Oncogene Proteins B-raf - blood
/ Proto-Oncogene Proteins B-raf - genetics
/ Proto-Oncogene Proteins c-kit - genetics
/ Tumors
