In this study, we present a comparably large cohort of patients operated on for PTC, who were exposed to radioactive fallout in their childhood or as teenagers after the Chornobyl nuclear plant accident in 1986. The clinical features do not appear to be significantly different compared with other cohorts of PTC patients who were not exposed to radioactivity. Whether this cohort has a significantly different clinical course awaits follow-up; however, the time allowing for prognostic evaluation is presently too short. One weakness of the study is the lack of a control group in the experiments, in order to shed light over the specificity of the findings. A control group, however, would require recruitment from a totally different age group, or from another, noncontaminated, geographic area with a different demographic profile. Therefore, we decided not to include a control group in the experiments but to compare all the findings with existing data on similar cohorts found in the literature.
To further characterize the cohort, we have applied some established markers often used in the work-up of PTC patients. Some of these markers are summarized in , containing details of observations from published studies of postradiation PTCs and nonradiation-associated PTCs. The frequency of BRAF
mutation in the entire cohort was 37%, which is similar to many series of nonradiation PTC (35, 36)
. By contrast, BRAF
mutation has been less frequently observed in postradiation PTC, i.e. 4–24% (). It is worth noticing that BRAF
mutation was significantly underrepresented among the patients with PTC/CLT compared with PTC only, which is in accordance with the previous studies (37, 38)
. The finding may also reflect the facts that BRAF
mutation has been associated with more aggressive PTC (39, 40)
while the presence of CLT in PTC seems to lead to a better prognosis (41, 42)
. Thus, the fewer occurrences of BRAF
mutation in PTC/CLT patients may also be connected to good prognosis.
Comparison between published series of PTC.
In this study, we determined the proportion of BRAF
mutant alleles to be below 50% (mean 28%), which could be related to contamination of nontumor cells in the samples studied as well as intratumoral heterogeneity of the BRAF
mutation. The latter situation was recently shown by Guerra et al
. In our study, histopathological examination of all samples indicated a high PTC representativity with minor proportions of non-PTC cells. This was also true for the PTC/CLT cases in which large areas of lymphocytic infiltrations were not observed. Taken together with the sensitivity of the pyrosequencing (by which BRAF
1799T>A was observed in gDNA of a PTC after dilution), our observations would support intratumoral heterogeneity for BRAF
rearrangements in the form of RET/PTC1
were demonstrated in 29 and 6% respectively. While previous studies on RET/PTC
have reported highly varying frequencies from 5 to 87% (), postradiation cases have generally shown the highest frequencies. In comparison with these reports, our finding of 34% RET/PTC
positivity falls within the lower range of postradiation PTC and is comparable to the highest frequencies among nonradiation PTCs (15, 37, 44)
. However, with regard to the specific fusion type involved, we found RET/PTC1
to be five times more common than RET/PTC3
, which is in contrast to other reported postradiation PTCs but in agreement with nonradiation PTCs (45)
. Overall, the presence of RET/PTC
is usually considered to be a sign of poor prognosis in PTC. Moreover, RET/PTC
accompanied by a BRAF
1799T>A mutation is associated with a high risk of disease recurrence and metastases. In the current study, co-occurrence of RET/PTC
mutation was detected in four PTCs. Although the clinical features at surgery were not indicative of poor prognosis, these cases should be considered for close follow-up for early recognition of signs for PTC recurrence as reported in the literature (13, 39)
Moreover, different frequencies of RET/PTC1
were reported in childhood post-Chornobyl PTC. However, these studies showed significant variation of these genetic aberrations depending on the histopathological type of PTC. Thus, RET/PTC1
was associated with the classical and diffuse sclerosing variants of PTC, whereas RET/PTC3
was associated with the solid follicular type (46, 47)
. On the other hand, the solid follicular type of PTC is more common in pediatric patients, while the classical PTC is more common in adults (47)
. The patient's age is also an important factor for the BRAF 1799T>A
mutation, which is commonly found in adult patients, but is rare in childhood PTC, which is consistent with our finding (9, 10)
MIB-1 index is increasingly used in the immunohistochemical work-up of several cancer types. The MIB-1 MAB is directed toward the nuclear antigen Ki-67 and is used for identification of proliferative cells and areas of tumors with a high degree of proliferation. This index has been suggested to predict the prognosis in many cancer varieties, including PTC (19, 20, 21)
. PTC exhibits varying proportions of infiltrating lymphocytes, which was pronounced in the PTC/CLT entity and was less abundant in several PTC-only cases. As MIB-1 immunostaining targets proliferating cells, both proliferating lymphocytes and tumor cells will be stained, with associated risks of misclassification and false-positive or negative scoring as a consequence. To achieve optimal scoring conditions, we used double staining with MIB-1 and LCA in addition to regular MIB-1 staining of all cases. Typical examples of the result are illustrated in . Overall, lower MIB-1 proliferation index was revealed using the MIB-1/LCA-based analysis compared with MIB-1 only (mean 0.8 vs 1.5%; ). If substantiated in follow-up studies, the observations suggest that double staining of MIB-1 and LCA should be considered for use in clinical routine work-up of PTC instead of regular MIB-1-only analysis. Associations between MIB-1 proliferative index and clinical features were not observed. In the five cases with MIB-1 index above the ≥1.85% border applied in our previous studies (19, 20)
, signs of aggressive clinical features were not observed concerning histopathological and clinical features present at the time of surgery. However, in some previous publications, increased MIB-1 index in PTC has been associated with adverse outcome during follow-up (19, 20, 22)
. Possible prognostic implications of MIB-1 index in this cohort cannot be presently assessed given the lack of follow-up.
Expression of the antiapoptotic protein BCL2 was observed in the majority of PTCs. All normal thyroid tissue samples and 53/70 PTCs expressed BCL2, suggesting that BCL2 could have a protective role to prevent apoptosis in normal thyroid, which is partly lost in malignancy. In contrast to a previous study, no correlation was observed between BCL2 expression and MIB-1 index in PTC/CLT cases (48)
. However, a positive correlation was observed between BCL2
expression and BRAF
mutation. Although this correlation was not strong, it is consistent with the study by Preto et al
, showing inhibition of BCL2 in PTC cell lines treated with the BRAF and kinase inhibitor sorafenib. Moreover, the lack of association between clinical features and BCL2 expression in our cohort is consistent with the observations by Siironen et al
. Given that phosphorylation is needed for the antiapoptotic effect of BCL2 (50)
, further determination of phosphorylated BCL2
expression levels would add more information about the antiapoptotic status of PTC.
We observed significantly elevated expression of cyclin A in PTCs larger than 2
cm. Previous studies of this protein have reported overexpression in poorly differentiated and undifferentiated thyroid cancers, indicating a role in thyroid carcinoma de-differentiation (25)
. Our finding of an association between cyclin A expression and tumor size implies that cyclin A could have prognostic value in irradiation-associated PTC; however, much longer follow-up is required to prove or disprove this. Although the possible utility of cyclin A for routine clinical practice is presently unclear, the observed association warrants further investigation of cyclin A in relation to follow-up.
Cyclin D1 expression was detected in the majority of PTCs. This was not accompanied by regional amplification of the CCND1
locus, suggesting that cyclin D1 is deregulated at the transcriptional, translational, or posttranslational level. In our scorings, we have included nuclear expression of cyclin D1 as suggested elsewhere (34)
. However, we have also observed cytoplasmic staining, which could be explained by cytoplasmic sequestration of cyclin D1 due to inhibition of its transportation to the nucleus (51, 52)
. Elevated expression of cyclin D1 was found to be correlated with elevated MIB-1 index, an association that was also demonstrated by Alama et al
in meningioma. No other associations to clinical or pathological features were observed, which is in agreement with the previous studies (34, 54)
. However, others have reported that cyclin D1 overexpression may be a prognostic marker for PTC (55, 56)
In summary, we report a cohort of adult PTC patients exposed to the radioactive fallout from the Chornobyl accident during their childhood or as teenagers. Our results from genetic and molecular characterization suggest that this cohort is characterized by frequent BRAF 1799T>A mutation and RET/PTC1 rearrangement as well as low proliferation, which are partly overlapping and partly distinguishing from other reported cohorts of postradiation- and nonradiation-related PTC. Moreover, BRAF mutation was significantly underrepresented in the PTC/CLT group, and cyclin A expression was associated with tumor size in this entity. Long-term follow-up in this cohort will eventually identify possible effects on patient outcome in this patient group.