Validation of BRAF V600E Mutation Using Qiagen Rotor-Gene Analysis System
Riyam Zreik, Eduardo Castro-Echeverry, Cary Chisholm, Daniel Smith, Jaeson Gildon, Scarlet Walker, Kimberly Walker, Lubna Sayage-Rabie, Arundhati Rao. Scott & White Memorial Hospital, Temple, TX
Background: Mutations in the BRAF oncogene are commonly seen in papillary thyroid carcinoma (PTC) and have been shown to be useful in cases of indeterminate cytology from thyroid fine needle aspirations (FNA). Mutation analysis can be performed on a variety of platforms with different diagnostic sensitivities. We present a validation study for BRAF mutational analysis of thyroid FNA samples on the newly introduced Qiagen Rotor-Gene Q platform which uses high resolution melting post-PCR analysis and compare it to results obtained by sequencing and final surgical specimen diagnosis.
Design: Ultrasound guided thyroid FNA samples were obtained. Non-enriched FNA samples were used. DNA was extracted from the specimens using the Roche MagNA Pure Compact extraction kit and was evaluated for the BRAF V600E mutation using the Qiagen PyroMark Q24 sequencer and Qiagen Rotor-Gene Q analysis system. The results were compared to the final surgical diagnosis (gold standard) to determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).
Results: 50 patients were identified for this study and 56 FNA samples were tested (Table 1). In this data set, 6 FNAs were indeterminate and 14 FNAs were diagnostic for PTC. 17 patients were diagnosed with PTC following total/partial thyroidectomy and 9 had metastatic disease. The FNA DNA quantity ranged from 0.34-32.07 ng/µL and the mutation prevalance ranged from 8%-34%.
|Sensitivity||14/19 (73.7%)||12/19 (63.2%)||17/19 (89.5%)|
|Specificity||37/37 (100.0%)||37/37 (100.0%)||34/37 (91.9%)|
|PPV||14/14 (100.0%)||12/12 (100.0%)||17/20 (85.8%)|
|NPV||37/42 (88.1%)||37/44 (84.1%)||34/36 (94.5%)|