New Assay Effectively Detects Circulating BRAFV600E in Patients With Papillary Thyroid Cancer

Carrie C. Lubitz, MD, MPH

The detection of BRAFV600E in patients with papillary thyroid cancer (PTC) using a blood-based assay was shown to be feasible in a cohort of patients undergoing thyroidectomy, according to a presentation by Carrie C. Lubitz, MD, MPH, at the 2014 Annual Meeting of the American Thyroid Association.

The objective of this research was to assess the feasibility and utility of this novel blood-based assay for identifying PTC patients with a high-risk tumor mutation. The researchers hypothesized that a blood BRAFV600E assay would be able to detect circulating BRAFV600E in patients with PTC and that the blood assay would correlate with tumor burden.

"BRAFV600E is an important driver of mutation in thyroid cancer and is found in half of patients with papillary thyroid carcinoma. Detection of this mutation in tumor DNA obtained cytologically or pathologically is useful in the work-up of nodules or for determining therapeutic options for using novel targeted therapies," explained Lubitz, an assistant professor of surgery at Harvard Medical School and a surgeon at Massachusetts General Hospital.

BRAFV600E is correlated with worse outcomes, including an increased risk of recurrence, and independently correlates with persistent PTC in patients with stage I/II disease and with mortality on univariate analysis. Therefore, knowledge of BRAF status is clinically actionable for surgical and adjuvant therapy. However, conventional BRAF testing requires a tissue sample.

"We have previously developed a novel, highly sensitive, and specific blood-based BRAFV600E assay for patients with melanoma," Lubitz said. This assay allowed for the detection of mutant BRAF in RNA from mixed populations with as few as 0.1% BRAFV600E-containing cancer cells. It was found that BRAFV600E levels decreased following BRAF inhibitor initiation, and blood BRAF was detectable prior to clinical or radiologic recurrence in stage IV patients.

For the study, blood samples were collected from 100 serial patients undergoing thyroidectomy for thyroid disease prior to undergoing surgery at Massachusetts General Hospital from September 2013 to July 2014, and for up to 4 weeks postoperatively. No patient had a history of colon cancer or melanoma. Investigators were blinded to both clinical and tissue-BRAF status. Circulating BRAFV600E levels were compared to BRAF mutation status from surgical pathologic assessment with conventional assays.

Circulating BRAFV600E levels were quantified for each blood sample, using a unique restriction enzyme site and a series of polymerase chain reaction (PCR) amplifications and wild-type BRAF digestions. Extracted RNA was then quantified using real-time PCR before beginning the assay to normalize all of the samples. Results were then analyzed using a receiver-operated curve and calculating the area under the curve. In determining a positivity criterion, the researchers sought to maximize the likelihood ratio (sensitivity/1-specificity) and considered the prevalence of "disease" (ie, BRAF positivity) and the consequences of false negative and false positive results.

Preoperative and postoperative blood samples were analyzed from 36 patients undergoing thyroidectomy for PTC (stages I-III) and 10 non-PTC patients (nodular hyperplasia [n = 6], Hurthie cell adenoma [n = 3], or medullary thyroid cancer [n = 1]). Mean tumor size was 1.9 ± 1.1 cm.

Tissue BRAFV600E results were positive for 24 (67%) of PTC patients and 2 (20%) of non-PTC patients. Using these data, a threshold value of 1.0 picogram was established as "positive" and separated the cohort into two distinct groups. Furthermore, tumor volume correlated with circulating tumor cell level.

One limitation of this study is that BRAF tissue status is considered to be the gold standard. Also, there is a potential for pathological missed or misdiagnosis, for example, in cases of coincident melanoma or colorectal cancer. Cytological BRAF testing was not done. Finally, postoperative and longitudinal follow-up would be needed for all patients, the researchers noted.

"Our findings confirm the feasibility of a novel blood assay in detecting circulating mutant BRAF in the blood of patients with papillary thyroid carcinoma. This rapid assay has potential as a more cost-effective method for diagnosis, surveillance in thyroglobulin-antibody positive patients postoperatively, and in assessing evidence of disease over time in the adjuvant setting, for example, during treatment with BRAF inhibitors," Lubitz concluded.

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