Figure 1
Figure 1. BRAFV600 genotyping. (A) 18-FDG PET-CT before the onset of treatment. (a) Fused PET-CT axial view of pulmonary nodules (standardized maximal uptake [SUVmax] = 12). (b) Left axillary mass (SUVmax = 11.9). 18-FDG PET-CT after 3 months of dabrafenib treatment. (c) Fused PET-CT showing no significant residual uptake in the lung. (d) Presence of a residual small axillary lymph node with decreased uptake (SUVmax = 4.5). The patient was classified as partial metabolic response according to the EORTC criteria (ie, >25% reduction of the SUVmax in the target lesions), with a decrease in SUVmax of 83% in the target lesions. 18-FDG PET-CT after 6 months of dabrafenib treatment at the time of LCS relapse. (e) Fused PET-CT showing a hypermetabolic pulmonary nodule (SUVmax = 6.3) and mediastinal lymph nodes (SUVmax = 8.7); (f) axillary nodal relapse (SUVmax = 9.6). (B) Light optic microscopy of the left axillary lymph node biopsy before and during treatment. (a) Hematoxylin-and-eosin (HE) staining showing a very pleomorphic proliferation composed of large tumor cells with folded nuclei, and numerous mitotic cells. (b) Tumor cells stained with an anti-CD1a antibody (clone O10; Dako); (c) most of the cells are langerin (CD207)-positive (clone 12D6; Novocastra). These features are characteristic of LCS. (d) Anti-BRAFV600E (clone VE1; Spring Biosciences) immunostaining is positive in tumor cells. (e) HE staining of a lymph node biopsy at day 10 of dabrafenib treatment, showing massive necrosis of tumor cells. (f) No cells stained positive with the anti-BRAFV600E mutation-specific antibody. Original magnification, ×400 in all sections. (C) Pyrosequencing of DNA extracted from lymph node biopsies. (a) Before treatment, the BRAFV600E mutation is present (mutated allele = 78%); (b) on day 10 of dabrafenib treatment, only wild-type BRAFV600 is observed (mutated allele = 1.4%); and (c) at the time of LCS recurrence, demonstrating the resurgence of the BRAFV600E mutation (mutated allele = 52%). (D) Expression of MAP3K8/COT protein in the pretreatment and relapsing tumor. HT-29 BRAFV600E colorectal cells that express both the long (1-467) and short (30-467) forms of COT are shown as a positive control. β-actin served as loading control. Extracellular signal-regulated kinase (ERK) expression and activation in relapsed LCS. (E) Tumor cells are intensively stained with an anti-phospho-ERK-1/2 antibody (clone MAPK-YT; Sigma) compared with pretreatment tumor cells. Original magnification, ×400. (F) Western blot analysis for phosphorylated ERK (pERK) and ERK (endogenous total ERK) in the pretreatment and relapsing tumors is shown. A375 BRAFV600E melanoma cells were used as a positive control.

BRAFV600 genotyping. (A) 18-FDG PET-CT before the onset of treatment. (a) Fused PET-CT axial view of pulmonary nodules (standardized maximal uptake [SUVmax] = 12). (b) Left axillary mass (SUVmax = 11.9). 18-FDG PET-CT after 3 months of dabrafenib treatment. (c) Fused PET-CT showing no significant residual uptake in the lung. (d) Presence of a residual small axillary lymph node with decreased uptake (SUVmax = 4.5). The patient was classified as partial metabolic response according to the EORTC criteria (ie, >25% reduction of the SUVmax in the target lesions), with a decrease in SUVmax of 83% in the target lesions. 18-FDG PET-CT after 6 months of dabrafenib treatment at the time of LCS relapse. (e) Fused PET-CT showing a hypermetabolic pulmonary nodule (SUVmax = 6.3) and mediastinal lymph nodes (SUVmax = 8.7); (f) axillary nodal relapse (SUVmax = 9.6). (B) Light optic microscopy of the left axillary lymph node biopsy before and during treatment. (a) Hematoxylin-and-eosin (HE) staining showing a very pleomorphic proliferation composed of large tumor cells with folded nuclei, and numerous mitotic cells. (b) Tumor cells stained with an anti-CD1a antibody (clone O10; Dako); (c) most of the cells are langerin (CD207)-positive (clone 12D6; Novocastra). These features are characteristic of LCS. (d) Anti-BRAFV600E (clone VE1; Spring Biosciences) immunostaining is positive in tumor cells. (e) HE staining of a lymph node biopsy at day 10 of dabrafenib treatment, showing massive necrosis of tumor cells. (f) No cells stained positive with the anti-BRAFV600E mutation-specific antibody. Original magnification, ×400 in all sections. (C) Pyrosequencing of DNA extracted from lymph node biopsies. (a) Before treatment, the BRAFV600E mutation is present (mutated allele = 78%); (b) on day 10 of dabrafenib treatment, only wild-type BRAFV600 is observed (mutated allele = 1.4%); and (c) at the time of LCS recurrence, demonstrating the resurgence of the BRAFV600E mutation (mutated allele = 52%). (D) Expression of MAP3K8/COT protein in the pretreatment and relapsing tumor. HT-29 BRAFV600E colorectal cells that express both the long (1-467) and short (30-467) forms of COT are shown as a positive control. β-actin served as loading control. Extracellular signal-regulated kinase (ERK) expression and activation in relapsed LCS. (E) Tumor cells are intensively stained with an anti-phospho-ERK-1/2 antibody (clone MAPK-YT; Sigma) compared with pretreatment tumor cells. Original magnification, ×400. (F) Western blot analysis for phosphorylated ERK (pERK) and ERK (endogenous total ERK) in the pretreatment and relapsing tumors is shown. A375 BRAFV600E melanoma cells were used as a positive control.

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