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Figure 1. BRAF V600E mutation analysis in PTC

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1 Figure 1. BRAF V600E mutation analysis in PTC
Figure 1. BRAF V600E mutation analysis in PTC. A, Percentage of mutated (mPTC, PTC CL, PTC FV, PTC TC) and wild-type (WT) samples. PTC CL, PTC-classic; PTC FV, PTC-follicular variant; PTC TC, PTC-tall cell variant. B, Hematoxylin and eosin sections of three representative tumors with BRAF V600E (PTC cases 67, 69, and 65, from left to right) in which the mutation is present in virtually all neoplastic cells. C, Hematoxylin and eosin sections of three representative tumors with BRAF V600E (PTC cases 24, 7, and 6, from left to right) in which the mutation is present in a minority of neoplastic cells. The boxes in panels B and C show the percentage of neoplastic cells in the tumor (Tumor Cell %), the percentage of BRAF V600E-mutated alleles after ASLNAqPCR (Mutated Allele %), and the percentage of BRAF V600E-mutated cells after the normalization to the estimated proportion of neoplastic cells within the tumor (Normalized Mutated Cell %). From: High-Sensitivity BRAF Mutation Analysis: BRAF V600E Is Acquired Early During Tumor Development but Is Heterogeneously Distributed in a Subset of Papillary Thyroid Carcinomas J Clin Endocrinol Metab. 2014;99(8):E1530-E1538. doi: /jc J Clin Endocrinol Metab | Copyright © 2014 by the Endocrine Society

2 Figure 2. Distribution of BRAF V600E-mutated neoplastic cells in PTC and mPTC. A and B, Percentage of BRAF V600E mutated cells before (A) and after (B) normalization to the estimated proportion of neoplastic cells within the tumor in all of the 85 PTC samples. Black bars, tumors greater than 1 cm; white bars, tumors 1 cm or less (mPTC). C and D, Scatter plots showing the percentage of BRAF V600E-mutated neoplastic cells, in all samples (C), and in the mPTC subset (D). Three groups of tumors are recognizable in panels C and D: tumors with less than 30% of BRAF V600E-mutated neoplastic cells, tumors with 30%–80% mutated neoplastic cells, and tumors more than 80% mutated neoplastic cells. Dotted lines indicate 30% and solid lines indicate 80% of BRAF V600E-mutated neoplastic cells, respectively (A–D). E and F, Diagrams showing the distribution of the number of cases according to the percentage of BRAF V600E-mutated neoplastic cells in all samples (E) and in the mPTC subset (F). From: High-Sensitivity BRAF Mutation Analysis: BRAF V600E Is Acquired Early During Tumor Development but Is Heterogeneously Distributed in a Subset of Papillary Thyroid Carcinomas J Clin Endocrinol Metab. 2014;99(8):E1530-E1538. doi: /jc J Clin Endocrinol Metab | Copyright © 2014 by the Endocrine Society

3 Figure 3. BRAF V600E mutational analysis using ASLNAqPCR, NGS, and immunohistochemistry with a BRAF V600E-specific antibody. A, Percentage of BRAF V600E-mutated neoplastic cells in 30 samples after parallel analysis with ASLNAqPCR, and NGS; the dotted line indicates 30% and the solid line indicates 80% of BRAF V600E-mutated neoplastic cells, respectively. B, Correlation between the percentage of BRAF V600E-mutated neoplastic cells established after ASLNAqPCR and NGS analysis (P = .0002, Spearman test). C, Box plot of the percentage of BRAF V600E-mutated neoplastic cells identified after ASLNAqPCR and NGS analysis: there is no statistical difference in the results obtained after ASLNAqPCR and NGS analyses (P = .1064, Wilcoxon signed rank test). D and E, Immunohistochemistry with a BRAF V600E-specific antibody of two representative PTCs with heterogeneous distribution of the BRAF-mutated protein (case 15, D; case 7, E). Mutational analysis of these samples performed by ASLNAqPCR showed a percentage of BRAF V600E-mutated cells after normalization to the estimated proportion of neoplastic cells within the tumor of 36% (case 15, D) and 22.5% (case 7, E), respectively. From: High-Sensitivity BRAF Mutation Analysis: BRAF V600E Is Acquired Early During Tumor Development but Is Heterogeneously Distributed in a Subset of Papillary Thyroid Carcinomas J Clin Endocrinol Metab. 2014;99(8):E1530-E1538. doi: /jc J Clin Endocrinol Metab | Copyright © 2014 by the Endocrine Society

4 Figure 4. Correlation of the percentage of BRAF V600E-mutated neoplastic cells with clinicopathological features. A–C, Tumor size (A): larger tumors tend to have a higher proportion of BRAF-mutated cells; the Spearman test did not reach statistical significance (P = .1121), but ANOVA with the Kruskal-Wallis test showed that tumor size and the proportion of BRAF V600E-mutated cells are statistically associated (P = .012). Patient age (B) (Spearman test, P = .4891; Kruskal-Wallis test, P = .9193) and tumor stage (C) (Spearman test, P = .3089; χ<sup>2</sup> tests, P > .05) are not statistically associated with the percentage of mutated neoplastic cells within the tumor. D and E, Percentage of BRAF V600E-mutated neoplastic cells in PTC cases with (N1) and without (N0) lymph node metastases: there is no statistical association (P = .7172, Mann-Whitney U test). From: High-Sensitivity BRAF Mutation Analysis: BRAF V600E Is Acquired Early During Tumor Development but Is Heterogeneously Distributed in a Subset of Papillary Thyroid Carcinomas J Clin Endocrinol Metab. 2014;99(8):E1530-E1538. doi: /jc J Clin Endocrinol Metab | Copyright © 2014 by the Endocrine Society

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