Telomere-related genomic instability in papillary thyroid cancers: a preliminary study

Papillary thyroid carcinoma (PTC) has two main histologic variants: classical-PTC (CL-PTC) and follicular variant PTC (FV-PTC). Recently, due to its similar features to benign lesions, the encapsulated FV-PTC variant was reclassified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Nonetheless, specific molecular signatures are not yet available. It is well known that telomere-related genome instability is caused by inappropriate DNA repair of dysfunctional telomeres and that mechanisms involved in the damaged telomere repair processing may led to detrimental outcomes, altering the 3D nuclear telomere and genome organization in cancer cells. This pilot study aimed to evaluate whether a specific nuclear telomere architecture might characterize NIFTP, potentially distinguishing it from other PTC histologic variants. Our findings demonstrate that 3D telomere profiles of CL-PTC and FV-PTC were different from NIFTP and that NIFTP more closely resembles follicular thyroid adenoma (FTA). NIFTP has longer telomeres than CL-PTC and FV-PTC samples and telomere length overlaps in NIFTP and FTA. There was no association between BRAF expression and telomere length in all tested samples. Our data showing that 3D nuclear telomere organization is altered differently in thyroid cancer variants, suggest that this parameter might guide clinical management of NIFTP. Although further investigations in a larger cohort of patients are necessary to corroborate our observations, telomere-related genomic instability might be of value in the diagnosis of NIFTP and allow for a more appropriate selection of the correct treatment.


Introduction
Thyroid cancer is one of the most common malignant endocrine neoplasms, and papillary thyroid carcinoma (PTC) constitutes approximately 80% of all thyroid cancer cases [1]. PTC consists of two main histological variants: classical-PTC (CL-PTC) and follicular variant PTC (FV-PTC).
FV-PTC is divided into two sub-groups based on two different morphological aspects: non-encapsulated and encapsulated nodules [2]. Recently, the encapsulated variant was reclassified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), because it shows features similar to non-malignant lesions [3]. The Cancer Genome Atlas (TCGA) study [4] has demonstrated a strong correlation between genetic alterations and histologic phenotypes, resulting in the identification of two distinct molecular subgroups: the BRAF V600E -like nodules, which show the true papillary architecture, and RAS-like nodules, with a follicular-pattern that includes follicular adenoma, follicular carcinoma, and FV-PTC [5] .Although no specific molecular signatures have been identified, the typical molecular profile of NIFTP is of RAS-like mutations [6]. Moreover, prevalence of TERT promoter mutations, mutations in the gene promoter of human telomerase reverse transcriptase, have been observed in advanced forms of PTC with a high level of recurrence and disease-specific mortality [7], and are not found in NIFTP.
It is well known that telomeres have an important role in preserving chromosomal stability and integrity. Undamaged telomeres prevent end-to-end fusions, degradation of the chromosome ends, and contribute to the adequate chromosome positioning within the nucleus [8]. Studies using quantitative 3D telomere imaging and quantitative analysis have shown that in contrast to normal cells, telomeres are organized in a distinct pattern within the 3D space of the normal cell nucleus, in contrast to cancer cells [9][10][11]. Recently, we highlighted the importance of telomere-related genomic instability in the tumorigenesis of PTC. By selecting PTC-derived cell lines characteristic of PTC tumors BRAF V600E -like subgroup, we demonstrated that cancer stem-like cells subpopulations (representing an early cancer-promoting subpopulation) had a trend toward a lower telomere shortening compared to the corresponding parental cells, representing the tumor bulk cells [12].The present pilot study was designed to explore the potential use of telomere "signatures" in the differentiation of the different PTC histotypes. In particular, considering that it is still not clear whether the biological features of NIFTP are similar to FTA or to FV-PTC, our findings suggest that the specific 3D pattern observed in NIFTP may provide an additional parameter in the differential diagnosis of indolent thyroid nodules and there by prevent unnecessary patient treatment.

NIFTP has a 3D profile similar to FTA and different from classical and follicular variants of PTC
To date, there is no specific molecular signature of NIFTP, since the available panels of mutations used to improve diagnostic accuracy of PTC are not optimal to differentiate NIFTP from CL-PTC and FV-PTC. In this context, we investigated the 3D telomere profiles of different PTC histotypes and FTA in search of specific characteristics that would differentiate between them from NIFTP. In our pilot study, we used fifteen FFPE thyroid neoplasms comprising CL-PTC (n=4), FV-PTC (n=3), NIFTP(n=3), and FTA (n=4). We compared their respective telomere parameters across and found a pattern differentiating FTA and NIFTP from FV-PTC and CL-PTC. FTA and NIFTP presented higher (↑) numbers of telomere signals, ↑ numbers of telomere aggregates, and ↑ total intensity when compared with FV-PTC and CL-PTC ( Figure 1 and Figure 2). The fact that classical and follicular variant PTC displayed lower numbers of telomeres ( Figure 1A) and shorter telomeres ( Figure 1C) when compared with NIFTP and FTA, suggested that the decrease in the number of telomeres might be a consequence of telomere shortening. In this case, fewer telomeric (TTAGGG) repeats would be available for probe binding. These results corroborate findings in a previous study that demonstrated the association of short telomeres with classic and follicular variant of PTC and of long telomeres with benign thyroid nodules [13][14]. However, the dot plot for telomere aggregates in Figure 1B shows ↑ numbers of telomere aggregates for FTA and NIFTP in comparison with FV-PTC and CL-PTC cases. Telomere aggregates are cluster of telomeres, found in close proximity that cannot be further resolved as separate entities at an optical resolution limit of 200 nm [15;16]. Some of the aggregates represent telomere fusions that can generate dicentric chromosomes and breakage-fusion-bridge cycles, leading to chromosome rearrangements and ongoing chromosome instability, usually found in FTC and FTA. This chromosome instability, results in the aneuploidy and telomere association seen in classical cytogenetic studies [17]. Our results seem to follow this trend.  TheTeloView® analysis allowed us to divide the telomeric signals into four quartiles based on their intensity, creating distributions based on the frequency of signals with a specific intensity. We identified four cell subpopulations based on their telomeric signal intensity in arbitrary units (a.u.):cells with very short telomeres (≤ 4000a.u.), cells with short telomeres (4001-7000 a.u), cell with medium telomeres (7001-13000 a.u), and cells with large telomeres(>13.000 a.u.). Interestingly, the frequency distribution of very short telomeres (≤ 4000 a.u) showed a significant difference between the histotypes FTA/NIFTP and CL-PTC/FV-PTC ( Figure 3). No significant differences were observed between FTA and NIFTP or between CL-PTC and PV-PTC. The figure 3 clearly shows that NIFTP parallels FTA telomere length, and that NIFTP has longer telomeres compared to classical and follicular variants of thyroid cancer. Our data clearly demonstrate that NIFTP has telomere signatures similar to FTA that is distinct from classical and follicular variants PTC. Although, NIFTP cannot be considered a benign lesion but rather a low-risk thyroid nodule, our results suggest and reinforce the idea that NIFTP are lesions more closely related to benign thyroid neoplasms, rather than to malignant lesions [18,19].

CL-PTC and FV-PTC
Considering that telomeres length in healthy tissue can vary greatly from one patient to another, we also analyzed the 3D telomere profiles in normal adjacent to tumor areas (NAT). In general, as we expected, most of the telomere parameters were different between NAT and tumor areas for FTA and NIFTP. However, for CL-PTC and FV-PTC cases, we observed the opposite.
Among the 3D telomeres parameters analyzed, we found that only the number of telomeres and total intensity in CL-PTC were significantly different between NAT and tumor areas, whereas in FV-PTC only the average intensity was different (Table 1 and Figure 4). The field effect theory has been invoked to explain this type of finding in different cancers. The theory suggests that molecular alterations develop in morphological normal-appearing epithelium tissue and contribute to the progression of carcinogenesis [20]. This seems to be the case for CL-PTC and FV-PTC.   Figure 2F). The lack of specific criteria for the evaluation of BRAFV600E expression in thyroid tumors, by immunofluorescence, and the inability to perform sequencing due to low amount of DNA, made it difficult to interpret this last result. More cases are required in order to validate our observations. Moreover, all cases were negative for RET rearrangements, which could be related to the limited number of PTC cases in our study, considering the low frequency (10% to 30%) of this molecular alteration in adult PTC [25].

Conclusions
Our data indicate that 3D telomere profiles appear to be a promising tool to distinguish CL-PTC and FV-PTC from NIFTP. We demonstrated that NIFTP has a similar 3D telomere organization to FTA and is clearly different from classical and follicular variant PTC. These preliminary findings are in line with the view that NIFTP are lesions closer to non-malignant thyroid nodules and confirmed that short telomeres are a feature of PTC. This is the first study of 3D nuclear telomere organization in PTC. It provides a strong rationale for additional studies in a larger cohort of thyroid lesions.
Moreover, if these data are confirmed, they may have diagnostic utility in facilitating the exact diagnosis of NIFTP, helping to predict the outcome of this tumor, and enabling patient-specific clinical management.

Patient Samples
This study received approval by the Research Ethics Board on human studies in Manitoba/Canada included in this study was composed of 3 (20%) men and 12 (80%) women ages 31 to 67, with a median age of 51 years. Surgical specimens were classified according to the World Health Organization classification (WHO) [26]. Clinical, pathological, and molecular characteristics of the 15 patients are shown in Table 2.

3D quantitative fluorescence in situ hybridization (Q-FISH)
Nuclei from 5-μm thyroid tissue sections underwent3D quantitative fluorescence in situ hybridization Q-FISH with Cy3-telomere probe-(TTAGGG)n (DAKO, Glostrup, Denmark). The hybridization procedure was performed as described previously with some modifications [27]. (telomeres) was used, while the exposure times for the DAPI (nuclei) varied for each sample. The constrained iterative algorithm was used for deconvolution [28]. Deconvolved images were analyzed using the TeloView® v1.03 software program (Telo Genomics Corp., Toronto, ON, Canada) [15]. TeloView® measures in each cell six different telomeric parameters -telomeric signal intensity (telomere length), number of telomeric signals, number of telomere aggregates, nuclear volume, a/c ratio, and distribution of telomeres relative to the nuclear periphery -generating specific 3D telomere profiles for each examined thyroid sample [12,29].

BRAF expression and RET/PTC rearrangements
BRAF V600E protein expression was detected using the anti-BRAF V600E (clone VE1) mutation-specific mouse monoclonal antibody (Abcam ab228461 Milano, Italy) in accordance with the protocol utilized in previous studies [30]. Images were obtained with an epifluorescence microscope (Olympus BX41) and charge-coupled device camera (Cohu), interfaced with the CytoVysion system (software 2.81 Applied Imaging, Pittsburg, PA, USA). In each section, tumor and normal area were evaluated, and ten randomly selected fields for each sample were acquired with a 100X objective. Sequencing-validated BRAF V600E mutation positive (7 samples) and negative (3 samples) PTC cases [31] were used as positive and negative controls, respectively. Normal thyroid tissue from the tumor-free contralateral lobe of five PTCs were used as control [32]. The Image J software (US National Institutes of Health, USA) was used to determine the fluorescence intensity as previously described [33]. The presence of RET/PTC variants was tested by FISH with home-made probes, using dual-colour break-apart strategy, as previously described [32]. The presence of RAS mutations was not investigated.

Statistical Analysis
To compare telomere parameters among samples with different histology nested factorial analysis of variance was used. We also compared telomeric features of cells from NAT and from tumor area for each patient with a different histology using the Wilcoxon rank sum tests.
Significance levels were set at p=0.05.