Rearranged anaplastic lymphoma kinase (ALK) gene in adult-onset papillary thyroid cancer amongst atomic bomb survivors

Abstract

Background: We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAF(V600E)) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures.

Methods: The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription-polymerase chain reaction and 5' rapid amplification of cDNA ends (5' RACE).

Results: We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK rearrangements, being observed in 6 of 10 PTC cases with ALK rearrangements versus 2 of 15 cases with no ALK rearrangements. The six radiation-exposed cases of PTC harboring both ALK rearrangements and solid/trabecular-like architecture were associated with higher radiation doses and younger ages at the time of the A-bombing and at diagnosis compared to the other 19 PTC with no detectable gene alterations.

Conclusion: Our findings suggest that ALK rearrangements are involved in the development of radiation-induced adult-onset PTC.

FIG. 1.

(A) Diagram of the rearranged ALK gene. W and K regions indicate the regions spanning boundary of exons 19 and 20, and of exons 26 and 27 (kinase domain) in ALK, respectively. (B) Expression levels of W and K regions in ALK. The 5 μL of 40-cycle PCR products were electrophoresed on an 8% acrylamide gel. Sample I (lanes 2 and 3) indicates RT-PCR products of in-house control PTC. Samples II–V (lanes 4–11) reveal RT-PCR products of four exposed PTC with nondetected gene alterations; lanes with even numbers are cDNA fragments derived from the W region, and those with odd numbers are fragments derived from the K region. Lane 1 indicates pUC19-MspI digest for DNA size marker. (C) Screening of the rearranged ALK gene. The intensity ratio for selection of rearranged ALK was calculated using RT-PCR amplification with cDNA derived from 10 ng of total RNA of in-house control PTC for the K region, and with cDNA derived from 5 ng of total RNA of the same PTC for the W region as template. (D) Detection of expression of EML4-ALK fusion genes by RT-PCR. Lanes 3–7 indicate cDNA fragments of the EML4-ALK fusion gene in PTC cases from which the 3′-end fragments of exon 13 of EML4 were isolated by the SMART RACE method. Lanes 6 and 7 correspond to PTC in samples IV and III of (B), respectively. Lane 9 indicates cDNA fragments (49 bp) derived from the chimera gene that was formed by fusion of exon 20 of EML4 and exon 20 of ALK. Lanes 2 and 8 show H₂O for negative control, and Lane 1 shows pUC19-MspI digest for DNA size marker. ALK, anaplastic lymphoma kinase; PTC, papillary thyroid cancer; RT-PCR, reverse transcription–polymerase chain reaction; RACE, rapid amplification of cDNA ends.

FIG. 2.

Comparison of radiation dose distribution in PTC cases with (+, ◯) and without (−, □) rearranged ALK.

FIG. 3.

Epidemiological characteristics of PTC cases harboring both the rearranged ALK gene and solid/trabecular-like architecture, in relation to radiation dose (A), age at the time of atomic bombing (B), and age at the time of diagnosis (C).