Detection of BRAF Mutations Through Cell Transfer Technique
Detection of BRAF Mutations Through Cell Transfer Technique
Objectives To determine the utility of the cell transfer technique (CTT) for BRAF molecular testing on thyroid fine-needle aspiration (FNA) specimens.
Methods Polymerase chain reaction (PCR)–based BRAF molecular testing was performed on tissues obtained through CTT from both air-dried and ethanol-fixed direct smears of thyroid FNA specimens and then compared with the corresponding thyroidectomy formalin-fixed, paraffin-embedded (FFPE) tissues on 30 cases.
ResultsBRAF testing was successfully performed on 29 of 30 air-dried CTT, 27 of 30 ethanol-fixed CTT, and 27 of 30 FFPE tissues. The results exhibited 11, 13, and 13 BRAF mutations and 18, 14, and 14 wild types for the air-dried CTT, the ethanol-fixed CTT, and the FFPE tissues, respectively. The concordance rate was 96% between air-dried and ethanol-fixed CTT tissues, 88% between air-dried CTT and FFPE tissues, and 92% between ethanol-fixed CTT and FFPE tissues.
Conclusions PCR-based BRAF mutational testing can be reliably performed on the direct smears of the thyroid FNA specimens through the application of CTT.
Papillary thyroid carcinoma (PTC) accounts for 80% of all thyroid cancers in the United States, making it the most common thyroid malignancy. Fine-needle aspiration (FNA) biopsy has emerged as an important diagnostic tool in the workup of suspicious thyroid nodules. Papillary carcinoma, in particular, displays characteristic nuclear features that often allow for a cytologic diagnosis based on morphology alone. Some cases, however, have only a few of the diagnostic features or are of limited cellularity. These cases are often placed into the category of follicular lesions of undetermined significance (FLUS) or suspicious for malignancy.
In recent years, it has been shown that a mutation of the BRAF gene is the most common genetic alteration in PTC and is detected in 29% to 69% of PTCs. Most important, when dealing with primary thyroid neoplasms, BRAF mutations are found in PTC and in poorly differentiated or anaplastic thyroid carcinoma derived from PTC. Therefore, molecular techniques that can identify this BRAF mutation can be an important ancillary diagnostic tool in the diagnosis of PTC, especially in cases that would otherwise be classified as atypia of undetermined significance or suspicious for malignancy. In addition, numerous studies have shown that BRAF mutations in PTC are associated with characteristics that are predictive of tumor progression and recurrence such as extracapsular invasion, lymph node metastasis, and advanced tumor stage. Finally, with the advent of novel pharmaceutical agents that target the mitogen-activated protein kinase pathway that is abnormally activated by BRAF mutations, detection of BRAF mutations will likely have an impact on the treatment of PTC. Thus, analysis of specimens for the BRAF mutation can have diagnostic, prognostic, and therapeutic implications.
The use of formalin-fixed, paraffin-embedded (FFPE) cell blocks prepared from FNA specimens is generally considered a reliable source of tumor cells for molecular assessment as well as immunohistochemical studies that can detect BRAF mutations. In particular, thyroid FNA is commonly used for preoperational diagnosis because high vascularity of the thyroid tissue can cause complications with more invasive procedures such as core biopsy. Cell blocks, however, are not routinely made or sometimes lack adequate cellularity to perform these ancillary studies. At our institution, the cell transfer technique (CTT), using direct cytologic smears as a source of tumor cells, has proven to be a reliable method for performing immunohistochemical and molecular studies when cell blocks lack adequate cellularity. Our goal, therefore, was to establish CTT as a viable option for isolating tumor cells for ancillary BRAF mutation analysis via the polymerase chain reaction (PCR)–based platforms. To validate this technique, we concurrently tested corresponding FFPE tissue from follow-up surgical resection alongside tissue obtained from direct smears.
Abstract and Introduction
Abstract
Objectives To determine the utility of the cell transfer technique (CTT) for BRAF molecular testing on thyroid fine-needle aspiration (FNA) specimens.
Methods Polymerase chain reaction (PCR)–based BRAF molecular testing was performed on tissues obtained through CTT from both air-dried and ethanol-fixed direct smears of thyroid FNA specimens and then compared with the corresponding thyroidectomy formalin-fixed, paraffin-embedded (FFPE) tissues on 30 cases.
ResultsBRAF testing was successfully performed on 29 of 30 air-dried CTT, 27 of 30 ethanol-fixed CTT, and 27 of 30 FFPE tissues. The results exhibited 11, 13, and 13 BRAF mutations and 18, 14, and 14 wild types for the air-dried CTT, the ethanol-fixed CTT, and the FFPE tissues, respectively. The concordance rate was 96% between air-dried and ethanol-fixed CTT tissues, 88% between air-dried CTT and FFPE tissues, and 92% between ethanol-fixed CTT and FFPE tissues.
Conclusions PCR-based BRAF mutational testing can be reliably performed on the direct smears of the thyroid FNA specimens through the application of CTT.
Introduction
Papillary thyroid carcinoma (PTC) accounts for 80% of all thyroid cancers in the United States, making it the most common thyroid malignancy. Fine-needle aspiration (FNA) biopsy has emerged as an important diagnostic tool in the workup of suspicious thyroid nodules. Papillary carcinoma, in particular, displays characteristic nuclear features that often allow for a cytologic diagnosis based on morphology alone. Some cases, however, have only a few of the diagnostic features or are of limited cellularity. These cases are often placed into the category of follicular lesions of undetermined significance (FLUS) or suspicious for malignancy.
In recent years, it has been shown that a mutation of the BRAF gene is the most common genetic alteration in PTC and is detected in 29% to 69% of PTCs. Most important, when dealing with primary thyroid neoplasms, BRAF mutations are found in PTC and in poorly differentiated or anaplastic thyroid carcinoma derived from PTC. Therefore, molecular techniques that can identify this BRAF mutation can be an important ancillary diagnostic tool in the diagnosis of PTC, especially in cases that would otherwise be classified as atypia of undetermined significance or suspicious for malignancy. In addition, numerous studies have shown that BRAF mutations in PTC are associated with characteristics that are predictive of tumor progression and recurrence such as extracapsular invasion, lymph node metastasis, and advanced tumor stage. Finally, with the advent of novel pharmaceutical agents that target the mitogen-activated protein kinase pathway that is abnormally activated by BRAF mutations, detection of BRAF mutations will likely have an impact on the treatment of PTC. Thus, analysis of specimens for the BRAF mutation can have diagnostic, prognostic, and therapeutic implications.
The use of formalin-fixed, paraffin-embedded (FFPE) cell blocks prepared from FNA specimens is generally considered a reliable source of tumor cells for molecular assessment as well as immunohistochemical studies that can detect BRAF mutations. In particular, thyroid FNA is commonly used for preoperational diagnosis because high vascularity of the thyroid tissue can cause complications with more invasive procedures such as core biopsy. Cell blocks, however, are not routinely made or sometimes lack adequate cellularity to perform these ancillary studies. At our institution, the cell transfer technique (CTT), using direct cytologic smears as a source of tumor cells, has proven to be a reliable method for performing immunohistochemical and molecular studies when cell blocks lack adequate cellularity. Our goal, therefore, was to establish CTT as a viable option for isolating tumor cells for ancillary BRAF mutation analysis via the polymerase chain reaction (PCR)–based platforms. To validate this technique, we concurrently tested corresponding FFPE tissue from follow-up surgical resection alongside tissue obtained from direct smears.