Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第945号）・境澤 香里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. British Journal of Cancer (2012) 106, 939-946. doi:10.1038/bjc.2012.12 www.bjcancer.com Published online 26 January 2012 (C) 2012 Cancer Research UKArticleBRITISH JOURNAL OF CANCER. 106(5):939-946 (2012)journal articl

Circulating Tumour DNA for Monitoring Treatment Response to Anti-PD-1 Immunotherapy in Melanoma Patients

Anti-programmed cell death-1 (anti-PD-1) antibody shows high therapeutic efficacy in patients with advanced melanoma. However, assessment of its therapeutic activity can be challenging because of tumour enlargement associated with intratumoural inflammation. Because circulating tumour DNA (ctDNA) correlates with tumour burden, we assessed the value of ctDNA levels as an indicator of tumour changes. Quantification of ctDNA (BRAFmutant or NRASmutant) levels by droplet digital PCR in 5 patients with BRAF or NRAS mutant melanoma during the treatment course showed dynamic changes corresponding to radiological and clinical alterations. In 3 cases in which the anti-PD-1 antibody was effective, ctDNA levels decreased within 2–4 weeks after treatment initiation. In 2 cases in which the anti-PD-1 antibody was ineffective, ctDNA levels did not decrease after treatment initiation. ctDNA could be a useful biomarker to predict early response to treatment in patients with advanced melanoma treated with anti-PD-1 immunotherapy

Melanoma with BRAF Mutation in Circulating Cell-free DNA despite no Mutation in the Primary Lesion: A Case Report

ArticleACTA DERMATO-VENEREOLOGICA.96(1):128-129(2015)journal articl

Thermal lens study in diode pumped N-g- and N-p-cut Nd:KGd(WO4)(2) laser crystals

A comparative study of thermal lensing effect in diode laser pumped N-g- and N-p-cut Nd:KGd(WO4)(2) (KGW) laser crystals was performed for laser emission polarized along the principle refractive axis, N-m. The thermal lens in the N-g-cut Nd: KGW was found to be weakly astigmatic with a positive refractive power for both the N-m- and N-p-directions. For N-p -cut Nd: KGW, strong astigmatism was observed and the refractive powers in the N-g- and N-m-directions had opposing signs. The degree of astigmatism was found to be considerably weaker for the N-g-cut Nd: KGW in comparison with the N-p-cut one: 0.35 dptr/(W/cm(2)) and 2.85 dptr/(W/cm(2)), respectively. The ratio of the thermal lens refractive powers in the planes parallel and perpendicular to the laser emission polarisation were measured as + 1.4 and -0.425 for N-g- and N-p-cut Nd: KGW respectively. (C) 2009 Optical Society of Americ