Detection of BRAF Mutations Through Cell Transfer Technique
Detection of BRAF Mutations Through Cell Transfer Technique
This study was approved by the Indiana University Institutional Review Board. A computerized search of our anatomic pathology information system was performed for a 26-month period (January 2011 through February 2013). To have an adequate number of positive BRAF mutation cases, we selected thyroidectomy specimens with final diagnoses of PTC and two cases of follicular neoplasm that had corresponding FNA performed. Cases of benign thyroid lesions were also included.
In total, there were 30 FNA specimens from 30 patients, including six men and 24 women in our study. The patients' ages ranged from 26 to 84 years, with a median age of 45 years and a mean age of 45.7 years. Nodules were present in the left lobe in 15 cases, in the right lobe in 14 cases, and in the isthmus in one case. The final histologic diagnoses of 30 cases included 18 classic PTCs, four follicular variant PTCs, one follicular carcinoma, one follicular adenoma, and six benign nonneoplastic lesions.
The slides from direct smears of thyroid aspirates and histologic slides of the corresponding thyroid nodules were reexamined. Both cytologic and surgical pathology reports as well as pertinent clinical history and radiographic results were reviewed. One ethanol-fixed smear, one air-dried direct smear, and one corresponding FFPE block that contained lesional cells were selected for BRAF molecular testing. The areas on the direct smears containing abnormal cells were marked by a pathologist (H.H.W.), and photographs were taken from these areas, in which the tumor cells of interest were then collected by CTT and sent for molecular testing.
CTT was performed using clean technique as follows: (1) the coverslip was removed using fresh histologic-grade xylene (Fisher Scientific, Pittsburgh, PA), (2) a thin layer of Mount Quick media (Daido Sangyo, Tokyo, Japan) was spread uniformly over the top of the cellular material, (3) the slide was then placed in a 60°C oven for approximately 2 to 3 hours (or until hardened to the touch), (4) a Sharpie marker was used on the surface of the dried media to divide the slide into multiple areas of interest, (5) the slide was then placed into a clean Coplin jar of deionized water and submerged into a warm water bath at 45 ± 3°C for 30 minutes to 2 hours or until the media were soft enough to easily peel away from the slide, and (6) the media were cut along the marked areas, and each cut section was placed in an Eppendorf 2.0-mL safe-lock centrifuge tube and sent for molecular testing.
DNA extraction from FFPE tissue and cytologic specimens was performed using the Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). A microdissection was performed for the extraction of DNA from FFPE tissue. For the cytologic specimens, a modification from the manufacturer's recommendations was made. Samples were incubated at room temperature for 5 minutes with 1 mL xylene and then centrifuged at 15,000 rpm for 5 minutes. Xylene was removed from the pellet and followed by the ethanol wash recommended by the manufacturer. DNA concentration was determined using the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE).
Samples were analyzed by a Qiagen BRAF RGQ PCR run on the Rotor-Gene Q MDx (Qiagen) following the manufacturer's recommendations. Genomic DNA was used to detect five mutations in codon 600 of the BRAF gene. The real-time PCR detection system employed both Scorpions and Amplification Refractory Mutation System technologies (Qiagen) on the Rotor-Gene Q instrument. This assay can detect somatic mutations, including V600E, V600E complex, V600D, V600K, and V600R, although it is unable to differentiate the V600E and V600E complex mutations. Overall, this is a two-step procedure, with the first step being a control assay to assess the total BRAF DNA content in a sample and the second step combining both the mutation and control assays to determine the presence or absence of mutated DNA. The threshold at which the signal is detected above background is called the cycle threshold. Sample delta cycle threshold values are calculated as the difference between the mutation assay cycle threshold and the wild-type assay cycle threshold from the same sample. Samples are subsequently classified as "mutation positive" if they give a delta cycle threshold less than the stated cutoff value for the assay. If the delta cycle threshold is above the cutoff, the sample is classified as "mutation not detected." Appropriate positive and negative controls were run with each sample.
Materials and Methods
This study was approved by the Indiana University Institutional Review Board. A computerized search of our anatomic pathology information system was performed for a 26-month period (January 2011 through February 2013). To have an adequate number of positive BRAF mutation cases, we selected thyroidectomy specimens with final diagnoses of PTC and two cases of follicular neoplasm that had corresponding FNA performed. Cases of benign thyroid lesions were also included.
In total, there were 30 FNA specimens from 30 patients, including six men and 24 women in our study. The patients' ages ranged from 26 to 84 years, with a median age of 45 years and a mean age of 45.7 years. Nodules were present in the left lobe in 15 cases, in the right lobe in 14 cases, and in the isthmus in one case. The final histologic diagnoses of 30 cases included 18 classic PTCs, four follicular variant PTCs, one follicular carcinoma, one follicular adenoma, and six benign nonneoplastic lesions.
The slides from direct smears of thyroid aspirates and histologic slides of the corresponding thyroid nodules were reexamined. Both cytologic and surgical pathology reports as well as pertinent clinical history and radiographic results were reviewed. One ethanol-fixed smear, one air-dried direct smear, and one corresponding FFPE block that contained lesional cells were selected for BRAF molecular testing. The areas on the direct smears containing abnormal cells were marked by a pathologist (H.H.W.), and photographs were taken from these areas, in which the tumor cells of interest were then collected by CTT and sent for molecular testing.
Cell Transfer Technique
CTT was performed using clean technique as follows: (1) the coverslip was removed using fresh histologic-grade xylene (Fisher Scientific, Pittsburgh, PA), (2) a thin layer of Mount Quick media (Daido Sangyo, Tokyo, Japan) was spread uniformly over the top of the cellular material, (3) the slide was then placed in a 60°C oven for approximately 2 to 3 hours (or until hardened to the touch), (4) a Sharpie marker was used on the surface of the dried media to divide the slide into multiple areas of interest, (5) the slide was then placed into a clean Coplin jar of deionized water and submerged into a warm water bath at 45 ± 3°C for 30 minutes to 2 hours or until the media were soft enough to easily peel away from the slide, and (6) the media were cut along the marked areas, and each cut section was placed in an Eppendorf 2.0-mL safe-lock centrifuge tube and sent for molecular testing.
DNA Extraction
DNA extraction from FFPE tissue and cytologic specimens was performed using the Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). A microdissection was performed for the extraction of DNA from FFPE tissue. For the cytologic specimens, a modification from the manufacturer's recommendations was made. Samples were incubated at room temperature for 5 minutes with 1 mL xylene and then centrifuged at 15,000 rpm for 5 minutes. Xylene was removed from the pellet and followed by the ethanol wash recommended by the manufacturer. DNA concentration was determined using the NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE).
BRAF Mutations
Samples were analyzed by a Qiagen BRAF RGQ PCR run on the Rotor-Gene Q MDx (Qiagen) following the manufacturer's recommendations. Genomic DNA was used to detect five mutations in codon 600 of the BRAF gene. The real-time PCR detection system employed both Scorpions and Amplification Refractory Mutation System technologies (Qiagen) on the Rotor-Gene Q instrument. This assay can detect somatic mutations, including V600E, V600E complex, V600D, V600K, and V600R, although it is unable to differentiate the V600E and V600E complex mutations. Overall, this is a two-step procedure, with the first step being a control assay to assess the total BRAF DNA content in a sample and the second step combining both the mutation and control assays to determine the presence or absence of mutated DNA. The threshold at which the signal is detected above background is called the cycle threshold. Sample delta cycle threshold values are calculated as the difference between the mutation assay cycle threshold and the wild-type assay cycle threshold from the same sample. Samples are subsequently classified as "mutation positive" if they give a delta cycle threshold less than the stated cutoff value for the assay. If the delta cycle threshold is above the cutoff, the sample is classified as "mutation not detected." Appropriate positive and negative controls were run with each sample.
