Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1

Abstract

Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.

Figure 1

Mass spectrometry (left panel) and Sanger sequencing (right panel) traces of representative samples with NRAS, AKT1 and PIK3CA mutations. Note that NRAS mutant and wild-type peaks are of comparable size, which is also reflected in the sequencing trace. By contrast, the mutant peaks in AKT1 and PIK3CA were small, and missed by Sanger sequencing. Subcloning and sequencing detected 8 of 27 (29%) and 2 of 24 (8%) clones to be mutated for AKT1 and PIK3CA, respectively. wt, wild type; mut, mutant; UEP, unextended primer.

Figure 2

A. Mutational frequency of BRAF, RET/PTC, NRAS, HRAS, KRAS, AKT1, PIK3CA in (A) 18 primary ATC, (B) 34 primary PDTC and (C) 23 RAIR FDG-PET positive PDTC. RAS is significant more prevalent than BRAF in primary PDTC (p=0.002) and BRAF is more prevalent than RAS in RAIR PET positive PDTC (p=0.04). RET/PTC rearrangements were analyzed in cases that were wild-type for BRAF and RAS.

Figure 3

Genotype of multiple tumor sites in a patient with radioactive iodine refractory, FDG-PET positive thyroid carcinoma. A: Histology of primary tumor showing a papillary thyroid carcinoma (PTC), tall cell variant (TCV). B: Histology of metastatic TCV-PTC to lung 15 months after diagnosis C: Histology of metastatic TCV-PTC to right supraclavicular lymph node that developed 10 years after removal of primary tumor. This high power view shows the tumor cells (arrow) to be tall (their height at least twice their width) with strong eosinophilic cytoplasm. D, E, F: Mass spectrometry traces for BRAF mutation from primary tumor (A), first (B) and second (C) recurrence, respectively. Note the mutant BRAF_T1799A peak (arrow). G: FDG-PET scan from the second recurrence showing PET positive lesion (arrow) in the right supraclavicular area corresponding to specimen C.