Identification of a Recurrent STRN/ALK Fusion in Thyroid Carcinomas

Thyroid carcinoma is the most common endocrine malignant tumor and accounts for 1% of all new malignant diseases. Among all types and subtypes of thyroid cancers that have been described so far, papillary thyroid carcinoma is the most frequent. The standard management treatment of these tumors consists of surgery, followed by radioiodine treatment in case of high risk of relapse. The most aggressive forms are commonly treated by chemotherapy, radiotherapy or experimental drug testing. We recently reported the case of a patient presenting an anaplastic thyroid carcinoma with lung metastases. Fluorescence in situ hybridization analysis allowed us to detect a rearrangement of the anaplastic lymphoma kinase (ALK) gene in both tumors. The patient was treated with crizotinib and presented an excellent drug response. We present here the subsequent investigations carried out to further characterize this genetic alteration and to assess the prevalence of ALK rearrangements in thyroid lesions. High resolution array-comparative genomic hybridization data complemented by RT-PCR and sequencing analyses, allowed us to demonstrate the presence of a STRN/ALK fusion. The STRN/ALK transcript consisted of the fusion between exon 3 of STRN and exon 20 of ALK. Subsequent screening of 75 various thyroid tumors by RT-PCR revealed that 2 out of 29 papillary thyroid carcinomas exhibited the same fusion transcript. None was detected in other types of malignant or benign thyroid lesions analyzed. These findings could pave the way for the development of new targeted therapeutic strategies in the treatment of papillary thyroid carcinomas and point to ALK inhibitors as promising agents that merit rapid evaluation.


Introduction
Thyroid carcinoma is the most common malignancy of the endocrine system and accounts for 1% of all new malignant diseases. Among the different thyroid cancer types described, papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) are the most frequent, accounting for 80-85% and 10-15% of thyroid malignancies, respectively [1][2].
PTC is a well-differentiated tumor defined by its characteristic aspect of the cell nuclei. Fifteen histological variants have been described in the World Health Organization (WHO) classification [1]. The prognosis for patients with PTC is good, with the 10-years survival rate being over 93% [3][4]. Some histological variants, as well as the presence of a poorly differentiated component, tend to show a more aggressive clinical behaviour.
FTC is a well-differentiated hypercellular tumor presenting a follicular differentiation pattern and lacking the nuclear features of PTC [1][2]. Follicular carcinomas are usually encapsulated and can be minimally to widely invasive. The 5-year survival rate is 80 to 98% for minimally invasive FTC and 38% for widely invasive tumors [5].
Anaplastic thyroid carcinoma (ATC) is a rare (1-2%) but very aggressive form of thyroid cancer. This highly malignant tumor is composed of undifferentiated cells along with necrosis and mitosis. ATC can be a de novo tumor or derive from PTC or FTC [2].
The standard management treatment for both PTC and FTC tumors is surgery followed by radioiodine if the risk of relapse is high or very high. In some cases, despite favorable outcomes late relapses are possible, and patients might die after local recurrence or metastases [6]. Moreover, about 10% of patients will experience advanced radioactive iodine refractory cancer with a poor prognosis and low response to chemotherapy [7][8]. Nevertheless, encouraging results have been obtained with the use of kinase inhibitors in the context of prospective trials [9]. In the case of the very malignant anaplastic thyroid carcinoma, with less than 20% of the patients surviving one year [10], there is urgent need for new therapeutic approaches. Although a multimodal approach was recently reported as being beneficial by Smallridge et al. [11], the elucidation of molecular events underlying carcinogenesis and the development of kinase inhibitors as potential therapeutic anticancer agents are emerging as promising strategies for the treatment of this malignant disease.
We recently reported the case of a 71-year-old woman presenting an anaplastic thyroid carcinoma with PTC component and lung metastases. A rearrangement of the anaplastic lymphoma kinase (ALK) gene was detected using FISH [12] both in the well-differentiated and anaplastic components of the thyroid tumor and in the anaplastic lung metastases. The patient was treated with crizotinib and presented an excellent response of .90% across all pulmonary lesions (criteria RECIST 1.1) that was confirmed at 6 months after therapy initiation [12]. These remarkable results provide new therapeutic options for these very poor carcinomas and potentially for all carcinomas presenting an ALK rearrangement.
We report here the subsequent identification of STRN gene as the ALK fusion partner and the screening via RT-PCR of a series of 75 thyroid carcinoma samples in order to test the presence of the STRN/ALK fusion.

Ethics statement
The samples used for this study were provided by the Biological Resources Centers of Institut Bergonié (CRB-IB) and the Academic Hospital of Angers (CRB-A) and were anonymized prior to research. The

Tumor samples and histological features
Tumor diagnosis was performed according to the World Health Organization classification [1]. The first tumors explored in this study consisted of an anaplastic thyroid carcinoma (ATC) with PTC component and its lung metastases (for case description see reference 12). FFPE material was available for both localizations. Normal thyroid tissue was also used in this study.
The screened series was composed of 75 thyroid tumors: 32 were papillary carcinomas (PTC), 11 follicular carcinomas (FTC), 2 poorly differentiated carcinomas (PDTC), 1 PDTC with a PTC component, 2 papillary type PDTC, 5 oncocytic carcinoma (OTC) and 25 benign tumors (Table S1). Among PTC, 16 corresponded to the classic type, 11 to a follicular variant, 1 to a solid variant and 1 to an oncocytic variant. Among FTC, 3 corresponded to the classic type and 8 to minimally invasive FTC. One FTC specimen corresponded to metastasis. Among benign tumors, 10 corresponded to follicular adenoma (FTA) and 15 to oncocytic adenoma (OTA). In 61 cases, frozen tissue was available and in the other 14 cases only fixed blocks of paraffin were available (Table S1).
For PCR control C1 sample, which corresponds to an Ewinglike sarcoma with a CIC-DUX4 translocation, was used.

RNA extraction and RT-PCR
RNA extraction from frozen or FFPE tissue and reverse transcription using random hexamers (RT) were performed as previously described [13]. A specific reverse transcription was also performed on samples using a reverse ALK primer (ALKex20R1) ( Table S2). For PCR, primers used were designed using the Primer 3 program (http://frodo.wi.mit.edu/primer3/) and are presented in Table S2. Control PCR were performed using STRN forward and reverse primers or ALK forward and reverse primers.
For fusion transcript detection, PCR were performed, on both classical RT and ALK specific RT, with different STRN forward primers and the ALK reverse primer: ALKex20R2 (Table S2). For FFPE samples only potential fusion between exons 3 to 7 of STRN with ALK exon 20 were screened because of the low quantity of RNA obtained. For frozen samples fusions between exons 3 to 18 of STRN and ALK exon 20 were screened (Table S2). PCR were performed on 50 ng of cDNA using AmpliTaqGoldH DNA polymerase (Applied Biosystems) with an annealing temperature of 60uC.

Genomic DNA extraction and PCR
Genomic DNA from a frozen sample was isolated using a standard phenol-chloroform extraction protocol. For PCR, primers were designed using the Primer 3 program (http:// frodo.wi.mit.edu/primer3/) and are presented in Table S3. All combinations of PCR were performed as previously described for RT-PCR on 50 ng of gDNA using AmpliTaqGoldH DNA polymerase (Applied Biosystems) with an annealing temperature of 60uC.
Sequencing PCR products were purified using an ExoSAP-IT PCR Purification Kit (GE Healthcare) and sequencing reactions were performed with the Big Dye Terminator V1.1 Kit (Applied Biosystems) according to the manufacturer's recommendations. Samples were purified using the Big Dye XTerminator Purification kit (Applied Biosystems) according to the manufacturer's instructions and sequencing was performed on a 3130xl Genetic Analyzer (Applied Biosystems).

Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed paraffin embedded 4 mm-thick tissues on a BenchMark Ultra instrument (Ventana). The primary antibody and dilution used in this study are as follows: rabbit monoclonal anti-ALK (clone D5F3; Cell Signaling Technology, Danvers, MA) applied at 1:50 and detected with Ultraview Universal DAB detection kit (Ventana). IHC pictures were taken using a Leitz DMRB microscope (Leica) and a DS-Ri1 camera (Nikon).

Fluorescence in situ hybridization
FISH assay was performed using the Histology FISH accessory kit (Dako) as previously described according to the manufacturers' instructions [14]. Interphase molecular cytogenetic studies using a commercially-available ALK probe (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe, Abbott Molecular) were performed on a 4-mm paraffin-embedded section. Nuclei were scored for non-rearranged patterns (red and green signals overlapping or close together) and unbalanced patterns (split red and green signals or single red signals) using a Nikon Eclipse 80i fluorescent microscope with appropriate filters. Pictures were captured using a Hamamatsu C4742-95 CCD camera and analyzed with the Genikon software (Alphelys). The positive threshold was defined as more than 15% of signals split and/or isolated red signal as previously described [15] in 100 tumor cells.

STRN/ALK fusion identification in an anaplastic thyroid carcinoma and in its lung metastases
We have recently reported a case of an ALK gene rearrangement detected by FISH both in an anaplastic thyroid carcinoma (ATC) with PTC component and in its lung metastases [12]. FISH revealed that 52% of primary thyroid tumor cells and 66% of lung metastasis carcinoma cells presented an unbalanced rearrangement of the ALK gene. The observation of this unbalanced rearrangement prompted us to perform array-CGH experiments. Previous array-CGH results, showed a deletion on chromosome 2 starting in the ALK gene and expanding from the region 2p23.2 to the region 2p22, in both localisations [12]. However the low CGH resolution did not allow us to perform a fine mapping of the breakpoints. For this purpose a new array-CGH experiment with a higher resolution was performed for the lung anaplastic carcinoma with adequate DNA quality and quantity. The tumor genomic profile displayed gains of the chromosomes 5, 8, 17q, 19p and losses of chromosomes 9, 18q, 21 and 22 ( Figure 1A). A deletion of approximately 7.7 Mb from the 2p23.2 region to the 2p22.2 could also be observed ( Figures 1A and B). A more detailed look at the deletion extremities on chromosome 2 revealed a breakpoint in the 39 end of the ALK gene ( Figure 1B). Most of the gene was lost whereas the last 39 probes in ALK and the 39 probes covering the adjacent ALK region were not deleted, these results being consistent with those obtained by FISH. The chromosome 2 deletion extended until the 59 end of the Striatin gene (STRN) ( Figure 1C). Since both genes (ALK and STRN) are oriented in the same direction on the same chromosome; we hypothesized that the deletion of the region between the two genes could lead to the fusion of the 59 part of the STRN gene with the 39 part of the ALK gene.
The log2 ratio values of CGH probes covering ALK and STRN regions allowed us to see that the deletion breakpoints were situated in ALK between the Agilent probes A_16_P00340892 located in intron 19 and A_16_P15598291 situated in intron 18 and STRN gene between the probes A_16_P00351573 in intron 3 and A_18_P13169357 located in intron 2 ( Figure 1D).
In order to prove that the STRN gene was the ALK partner, RT-PCR was performed on paraffin-embedded tumors (Table S2). First we could observe that only C1 sample was positive for ALK expression and that STRN was well-expressed in all studied samples (Figure 2A). Secondly regarding the STRN/ALK RT-PCR, a potential fusion RT-PCR product could only be obtained from the positive lung sample (Figure 2A). Therefore, in order to detect the fusion in the ATC tumor, we performed an ALK specific reverse transcription using an ALK reverse primer followed by PCR. A potential fusion transcript was detected both in ATC and in lung metastases ( Figure 2B). No RT-PCR product was observed neither in the control sample C1 nor in the normal thyroid tissue for the two experiments (Figures 2A and 2B). Sequencing of the RT-PCR products (ATC and lung samples) confirmed a fusion between exon 3 of STRN and exon 20 of ALK in both cases ( Figure 2C). The in-frame fusion transcript sequence was expected to contain 2562 nucleotides (including UTR regions) and the predicted protein to exhibit 701 amino acid residues ( Figure 2D).

STRN/ALK fusion screening in several histotypes of thyroid tumors
In order to test whether the STRN/ALK fusion is recurrent in thyroid tumors, 75 thyroid samples of various histotypes were screened by RT-PCR (Tables S1 and S2). Expression of the STRN/ALK fusion transcript was detected in two tumors (cases 2 and 5) among the 75 ( Figure 3A). Both cases presented the nucleotidic sequence previously observed in the ATC and lung metastases with the fusion between exon 3 of STRN and exon 20 of ALK ( Figure 3B).
As frozen material was available for case 5, we used PCR to search for the STRN/ALK fusion at the genomic level. A PCR product which measured 791 bp was obtained ( Figure 3C) and was constituted of 441 bp of STRN intron 3 at its 59end fused to 349 bp of ALK intron 19 at its 39end ( Figure 3D). One nucleotide, a cytosine, located at the junction overlapped in the sequences of both genes and might originate from either STRN or ALK. No PCR products were detected screening the two other positive cases with these primers and other primers closer to breakpoints (data not shown).
FISH was subsequently performed to confirm the presence of an ALK rearrangement in these two cases ( Figure 4A). Case 5 displayed 62% of cells with one fusion signal and separate green and red signals, whereas 20% showed one fusion signal and a red signal only. Case 2 exhibited 20% of cells with one or more fusion signals with separate green and red signals. It is widely accepted that both patterns (i.e. cells with one fusion signal and separate green and red signals, and cells with one fusion signal and a red signal only) correspond to rearranged ALK profiles.
ALK immunohistochemistry was also performed and we found an ALK overexpression in case 5 but none in case 2 ( Figure 4B).

Occurrence of STRN/ALK fusion in thyroid cancers
The two positive cases for STRN/ALK fusion were identified in conventional PTC (Table S1) revealing that 2/16 of conventional PTC harbor a rearrangement between STRN and ALK. The fusion was not observed in PTC variant forms (13 cases), in follicular thyroid carcinomas (11 cases), in poorly differentiated carcinomas (5 cases), in oncocytic thyroid carcinomas (5 cases) or in benign lesions including follicular thyroid adenoma (10 cases) and oncocytic thyroid adenoma (15 cases).

Discussion
The classification of thyroid carcinoma into different histotypes is essentially based on morphological and clinical features [1][2] and advances in molecular studies of these tumors have confirmed the relevance of this pre-existing classification. In PTC, the most frequent genetic alterations are the translocation of receptor tyrosine kinase genes RET (20 to 30%) and TRK (10%), point mutations of RAS family genes and BRAF mutations [1][2]. These alterations are generally mutually exclusive in PTC [2].
The present study was initiated following the detection of an ALK rearrangement in an ATC with PTC component and its lung metastases [12]. To date, there is only one publication on the presence of rearranged ALK gene in PTC [16]. Hamatani et al. described ALK rearrangement in 52.6% (10/19) of cases of a radiation exposed-patient cohort [16]. In six positive cases, EML4 was found as the ALK fusion partner whereas in the other four the ALK partner has not been identified yet. No rearrangement was observed in PTC patients with no radiation exposure.
High resolution array-CGH analysis and RT-PCR followed by sequencing allowed us to identify a fusion between the STRN exon 3 and the ALK exon 20 in the metastatic lung tumor. The same  fusion was identified in the ATC primary tumor. STRN gene encodes the striatin, a calmodulin-binding member of the WD repeat family of proteins, presenting a coiled-coil domain [17]. The anaplastic lymphoma kinase (ALK) is a protein tyrosine kinase from the insulin receptor subfamily [18]. The ALK gene was initially identified as a fusion partner of nucleophosmin in anaplastic large-cell lymphoma [19][20]. The STRN/ALK fusion observed by us may encode a predicted protein consisting of the  fusion of the N-terminal part of the STRN protein, retaining its coiled-coil domain with the C-terminal part of ALK protein, including its tyrosine kinase domain. Although ALK has been described to be fused to many different partners [18], the shared feature of all translocation products is the conservation of the ALK tyrosine kinase domain that is fused to a part of a protein displaying a coiled-coil domain [21][22].
To date, only two publications have described the implication of STRN in a translocation: in a chronic eosinophilic leukaemia case (CEL) [23] and in a non-small cell lung carcinoma (NSCLC) [24]. In the CEL case, the authors identified a fusion between the STRN exon 6 and a truncated PDGFRA exon 12. Similarly to the ALK gene, PDGFRA encodes a tyrosine kinase receptor and the predicted fusion protein retains both the coiled-coil domain of STRN and the tyrosine kinase domain of the receptor. Even though the STRN part implicated in the CEL case is larger than the one we report, the coiled-coil domain is conserved in both cases [23]. In the NSCLC case the STRN/ALK fusion resulted in the fusion of STRN exon 3 with the ALK exon 20, with the same nucleotidic sequence observed in our cases [24].
In order to see if the STRN/ALK fusion transcript could be observed in other thyroid carcinomas we have performed RT-PCR screening on 75 thyroid tumors (50 malignant and 25 benign). Two conventional PTC with a STRN/ALK fusion could be identified from the screening. Both of them presented the same sequence as the first ATC case and the previously described NSCLC [24]. It seems that in PTC and in NSCLC, the fusion occurs preferentially between the STRN intron 3 and the ALK intron 19. The precise DNA breakpoints were obtained for only one sample (case 5) and remain to be determined in other cases.
To independently confirm the presence of an ALK rearrangement in these two cases, FISH experiment using an ALK Breakapart probe was carried out. Interestingly distinct rearrangement patterns could be observed by FISH in these thyroid tumors. We could hypothesize that in the case of cells with one fusion signal and one red signal (no green signal, as in the described ATC case and its metastases, [12]) the fusion of the two genes could be due to a deletion of the chromosome 2 region between the two genes, while the pattern of one fusion signal with separate green and red signals (cases 2 and 5) could correspond to a translocation occurring between the two homologous chromosomes 2.
Concerning immunohistochemical (IHC) results, among the three ALK rearranged cases, case 2 was the only one not to show ALK positive staining, even if IHC and FISH were performed on the same tumor area (for PTC first case with lung metastases IHC results see reference [12]). ALK expression in ALK rearranged tumors is usually detectable by immunohistochemistry using ALK D5F3 clone, however false negative results might be obtained in some cases [25][26][27]. Also the fixator used was not formalin but ''Holland Bouin'' which could explain a false negative result. For this case we could not formally conclude if the absence of immunostaining corresponded to a false negative result or to a lack of protein expression, even if the latter is unlikely.
Morphologically, the two ALK-rearranged cases (cases 2 and 5) corresponded to conventional PTC. We did not observe either an anaplastic component as we did in the case previously reported, or a solid/trabecular like architecture as observed by Hamatani et al. in 60% of their positive cases [16]. However these two cases showed aggressive features with lymph nodes metastases at diagnosis.
The 75 thyroid tumors series study revealed that 2 among 16 conventional PTC exhibited a STRN/ALK fusion. Additionally, the fusion seems to be unique to PTC in this series since no transcript was detected in other malignant thyroid cases (11 FTC, 5 PDTC and 5 OTC) or in benign thyroid lesions (10 FTA and 15 OTA). However, the study was not representative of the thyroid carcinoma incidence. In order to assess the prevalence of ALKrearrangements, and particularly the STRN/ALK fusion occurrence in thyroid tumors more precisely, a larger batch of papillary thyroid carcinomas and anaplastic thyroid carcinomas will be screened via FISH and RT-PCR in the near future.
To conclude, this study offers new perspectives on the treatment of papillary thyroid carcinomas, especially in radioiodine refractory patients. The search of ALK rearrangements in thyroid cancers may be as important as in NSCLC and the efficiency of treatment by ALK inhibitors must be further evaluated.