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Demographic data are shown in Table I. Ages ranged from 2.8 to 18 years (median, 13.6 years). Two patients had previously undergone thyroidectomy, whereas the remainder had thyroidectomy performed at our facility. Average tumor size was 2.18 cm (range, 3 mm to 4.2 cm). Five patients had papillary microcarcinoma, whereas the remainder had tumors > 1 cm. The average MACIS score was 5.1. Thirteen patients underwent cen- tral compartment neck dissection, nine underwent lat- eral neck dissection, including two who underwent bilateral neck dissections. Thirteen patients (68.4%) had metastases to the central neck, eight (42.1%) had lateral neck metastases, and five (26.3%) had pulmonary metas- tases. Two patients experienced regional recurrence. The BRAF V600E mutation was present in seven patients (36.8%). Eleven patients had classic PTC (including one with partial tall cell morphology), seven had a follicular variant of PTC, and one had an oncocytic variant. Seven of the 11 (63.6%) samples with classical PTC were BRAF V600E positive. All samples with variant pathology showed wild-type BRAF. PTC histology was significantly associated with the presence of the BRAF V600E mutation ( P 5 .013, Cramer’s effect size V 5 0.651). Similarly, FVPTC histol- ogy was negatively associated with the BRAF V600E mutation ( P 5 .017). There was no association of the fol- lowing variables with wild type or BRAF V600E (Table II): presence of lateral neck metastases (50.0% vs. 28.5%, P 5 .633), central neck metastases (75.0% vs. 57.1%, P 5 .617), pulmonary metastases (42% vs. 0%, P 5 .106), average tumor size (2.23 cm vs. 2.08 cm, t 5 0.176, P 5 .863), average age (12.9 years vs. 14.8 years, t 52 1.221, P 5 .239), lymphovascular invasion (77.8% vs. 60.0%, P 5 .580), extrathyroidal extension (62.5% vs. 60%, P 5 1.00), and incidence of papillary microcarcinoma (36.4% vs. 16.7%, P 5 .851). MACIS score approached significance (5.59 vs. 4.23, P 5 .087)

Fig. 1. Primers used the polymerase chain reaction to amplify exon 15 of the BRAF gene.

determine its association with aggressive disease charac- teristics. Better understanding of the relevance of this tumor marker in this population has possible implica- tions for adjuncts in diagnosis, treatment planning, and targeted therapy. MATERIALS AND METHODS Patients 0 to 18 years old were selected for a retrospective chart review if they underwent surgery for PTC at our institu- tion (Primary Children’s Medical Center, The University of Utah Hospital, Huntsman Cancer Hospital) between 1999 and 2012. Institutional review board approval was obtained (The University of Utah IRB 00057453). A retrospective chart review was performed using institu- tional electronic medical records. Patient demographic factors and disease characteristics (tumor size, lymphatic/distant metastases, surgical and adjuvant treatment rendered, lympho- vascular invasion, extrathyroidal extension, recurrence, histol- ogy) were obtained and kept in a secure patient database. Metastases, age at diagnosis, completeness of resection, inva- sion, and size of the tumor (MACIS) score was calculated. 15 Tumor samples for the study subjects were obtained from archived pathologic specimens. Formalin-fixed paraffin-embed- ded (FFPE) tissue blocks were used to prepare hematoxylin and eosin slides to identify areas of tumor cells. Aniline blue-stained slides were processed from adjacent slices of FFPE tissue, and microdissection of tumor cells was performed. A single patholo- gist ( A . M . A .) performed all tumor microdissection. DNA was then extracted using a standardized technique. 16 Exon 15 of the BRAF gene was amplified using polymerase chain reaction with primers as shown in Figure 1. After the ampli- fication, mutation status was determined by pyrosequencing using the Qiagen PyroMark Q24 pyrosequencer (Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions, as has been outlined previously. 17 Sequence analysis was performed using the Pyromark Q24 version 1.0.10 software in the allele quantification (AQ) analysis mode, using pyrograms as shown in Figure 2. The assay operates with a sensitivity of 5% of alleles. Statistical analysis was performed with SPSS software (IBM, Armonk, New York). Fischer exact test was used to mea- sure the association of the BRAF V600E mutation between binary variables (lateral and central neck metastases, pulmo- nary metastases, histology, lymphovascular invasion, extrathyr- oidal extension, recurrence). A two-tailed t test was used to measure association between the BRAF mutation and continu- ous data (tumor size, age, MACIS score). A review of the literature was performed by searching PubMed for “papillary thyroid carcinoma” and “BRAF” with lim- its applied for patients aged 0 to 18 years, as well as additional text search strings for “children” or “pediatric” or “adolescent.” Results of these relevant studies were summarized.

RESULTS Archived tumor specimens were available for 19 of 27 pediatric patients who initially fit inclusion criteria.

Fig. 2. Sequence analysis using the Pyromark Q24 version 1.0.10 software in the allele quantification analysis mode using pyrograms.

Laryngoscope 124: September 2014

Givens et al.: BRAF V600E and Pediatric Thyroid Carcinoma

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