Clinical utility of immunohistochemistry in Thyroid CancersJuly 17, 2018
The study used novel anti-BRAF V600E antibody (clone RM8) for detection of the BRAF V600E mutant protein in papillary thyroid cancers
Arvind Krishnamurthy, Vijayalakshmi Ramshankar, Kanchan Murhekar, Vidyarani Shyamsundar, Pavithra B Desai, Purvish Parikh
(Department of Surgical Oncology, Cancer Institute (WIA), Adyar, Chennai, India, Department Preventive Oncology Research, Cancer Institute (WIA), Adyar, Chennai, India, Department of Pathology, Cancer Institute (WIA), Adyar, Chennai, India, Department Oral Pathology, Balaji Dental College, Chennai, India, and Director of Precision Oncology, Asian Cancer Institute, Mumbai)
Thyroid cancer is the most common cancer of the endocrine system. The incidence in India is 13,801 new cases per 100,000 population, as per the GLOBOCAN data. The disease is rapidly increasing and there was an estimated prevalence of about 5539 cases in the year 2017. The major histological types of thyroid cancers are papillary, follicular, medullary and anaplastic thyroid cancers. Papillary thyroid cancers (PTC) and follicular thyroid cancers are generally well differentiated, indolent and highly curable with current treatment modalities. However a poorly differentiated thyroid cancer can progress to anaplastic thyroid cancer, which is considered to be one of the most aggressive and deadly histological types of thyroid cancers. A number of studies have established the association of the BRAF V600E with aggressive clinic-pathological characteristics of PTC. A few studies, including the author’s earlier study, have further suggested that the presence of mutant BRAF V600E portended an adverse prognosis in patients with PTCs. BRAF testing can be done by several methods including immunohistochemistry (IHC), Sanger sequencing, quantitative polymerase chain reaction (qPCR), mass spectrometry, real time PCR and next generation sequencing. In the present study, we have evaluated the presence of mutant BRAF V600E by conventional IHC in the formalin fixed paraffin section of patients treated for thyroid cancer using a mutation specific rabbit antibody RM8 and the results were compared to results obtained using molecular testing with qPCR, widely considered to be the current gold standard.
Background: Molecular markers are gaining increasing importance as diagnostic and prognostic tools in patients with well differentiated thyroid cancers and BRAF V600E mutation has received wide attention in this regard Aim: To evaluate the clinical value of immunohistochemistry (IHC) using anti-BRAF V600E antibody (clone RM8) for detection of the BRAF V600E mutant protein in formalin-fixed and paraffin-embedded tissues of patients with papillary thyroid carcinomas.
Materials and methods
Patients who were managed for well differentiated thyroid cancers (n=79) during the years 2005 and 2006 were included in the study. We evaluated the fidelity of the RM8 antibody specific for the BRAF V600E and compared its detection accuracy to real time polymerase chain reaction (PCR), which was taken as the gold standard.
Mutant BRAF V600E antibody was studied in 79 tissue sections, out of which 21 (26.5%) had staining for BRAF V600E in >20% of the tumour cells and these were considered positive. The BRAF staining was moderate in 10 (47.6%), strong in 9 (42.5%) and very strong in 2 (9.5%) of sections stained. There was a statistically significant concordance (P = 0.000) with quantitative PCR (qPCR) for BRAF mutant taken as standard. (Kappa agreement: 0.881) Further, the receiver operating characteristics (ROC) curve showed that IHC can be used as a comparable standard to the qPCR. The highest possible sensitivity of 92% and specificity of 92.6% could be achieved by considering the cytoplasmic positivity of >20% of cells with moderate to strong intensity (AUC = 0.923)
Our study has shown that BRAF V600E IHC can be done in a conventional manner using rabbit monoclonal antibody RM8 on formalin-fixed and paraffin-embedded tissues of patients with papillary thyroid carcinomas. With a comparable diagnostic accuracy to the gold standard qPCR testing and with an added advantage of being cost effective, this technology can be considered for use as a first-line method for detection of BRAF V600E mutations, especially in resource constrained settings.