Table 1.
Clinical and pathological features of a pediatric cohort with papillary thyroid carcinoma (PTC).
Table 2.
Risk stratification and molecular markers.
Fig 1.
Gene fusions and SNV detection in PTC.
(A) Representative case of an RET rearranged gene with break apart probes by FISH in a Follicular variant PTC. Full arrow shows a normal RET gene (yellow signal) and the dotted arrow shows a rearranged RET gene (green and red separated signals). (B) Representative case of an ALK rearranged gene with break apart probes by FISH in a Follicular variant PTC. Full arrow shows a normal ALK gene (yellow signal) and the dotted arrow shows a rearranged ALK gene (green and red separated signals). Scale bars represent 50 μm at 1000x magnification. (C) Representative immunohistochemistry for BRAF V600E mutation in a Classic variant of PTC. Scale bar represents 50 μm at 400x magnification. (D) Representative Sanger sequencing chromatogram showing double peaks in c.1799A > T (p.V600E), indicating a BRAF V600E heterozygous mutation. Magenta arrow shows the double peak.
Fig 2.
Detection of NTRK alterations.
(A and B) Representative case of positive immunohistochemical staining for pan-TRK in a follicular variant of PTC. Scale bar represents 50 μm at 400x magnification. (C) Follicular variant PTC with NTRK3 rearranged gene by FISH with break apart probes. Full arrow shows a normal NTRK3 gene (yellow signal) and the dotted arrows show rearranged NTRK3 genes (green and red separated signals) (D) Follicular variant PTC with ETV6 rearranged gene by FISH with break apart probes. Full arrow shows a normal ETV6 gene (yellow signal) and the dotted arrow shows a rearranged ETV6 gene (green and red separated signals). Scale bars represent 50 μm at 1000x magnification.
Fig 3.
Proposed molecular algorithm following a complexity-increasing order of techniques for the assessment of molecular alterations in PTC in a public hospital from a developing country.
All PTC samples should be initially screened for pan-TRK and BRAF V600E proteins by immunohistochemistry (IHC). Positive cases for pan-TRK should be sequentially tested for NTRK3, NTRK1 and NTRK2 gene fusions, following NTRK genes fusion frequency by fluorescent in situ hybridization (FISH). When a NTRK3 fused gene is detected, a further FISH determination for ETV6 should be performed, since ETV6 is the most frequent fusion partner. BRAF V600E negative by IHC should be re-assessed by Sanger sequencing. All BRAF V600E and pan-TRK IHC negative cases should be sequentially tested for RET, ALK, MET and BRAF fusions, following the genes fusion frequency by FISH. Cases negative for all previous tests and those positive for NTRK3 but negative for ETV6 could be assessed by targeted next generation sequencing (NGS). Blue square refers to the PTC sample. Oval shapes represent different techniques and rounded-corner squares depict molecular targets. Teal color for IHC, violet for FISH, pink for direct Sanger sequencing and orange for NGS targeted sequencing. The green checkmark indicates an end-point in diagnosis.