Background: Anaplastic thyroid carcinoma (ATC), the most aggressive type of thyroid cancer, has no effective therapy. Due to its dismal prognosis, it is vital to understand the genetic alterations of ATC and identify effective molecular targets. Targeted next-generation sequencing was performed to investigate the mutational profile of ATC using a massive parallel sequencing approach.

Methods: DNA from formalin-fixed, paraffin-embedded archival samples of 11 ATCs and normal matched pairs were used. A total of 48 genetic alterations were identified by targeted exome sequencing. These alterations were validated by mass spectrometric genotyping and direct Sanger sequencing.

Results: The most commonly mutated gene was BRAF, identified in 10 samples (91%), all showing the V600E point mutation. A KRAS point mutation was observed in the one sample (9%) without the BRAF^V600E mutation. All 11 ATCs harbored BRAF or RAS mutations, reflecting the possibility that differentiated thyroid carcinomas progress to ATCs after the accumulation of mutations. A loss of function mutation of TP53 was observed in eight samples (73%), a PIK3CA mutation was observed in two samples (18%), and a frameshift mutation of PTEN was observed in one sample (9%). Twenty-eight novel mutated genes were found that had not previously been associated with ATC. Of these, loss of function mutations of NF2, KMT2D, and PKHD1 were repeatedly seen in three samples (27%), two samples (18%), and two samples (18%), respectively. Using direct Sanger sequencing, two samples (18%) were also found with a RASAL1 mutation. KMT2D and RASAL1 mutations were significantly associated with shorter ATC patient survival.

Conclusions: This comprehensive analysis of ATCs using targeted massive parallel sequencing identified several novel mutations in ATCs, such as loss of function mutations of NF2 or KMT2D. Future studies are needed to confirm the role of these novel mutations as independent drivers of ATC development.