IGF2 is a Potential Factor in RAI-refractory Non-medullary Thyroid Cancer

Background: Non-medullary thyroid cancer (NMTC) is the most frequent endocrine tumor with in most cases a good prognosis after thyroidectomy and 131 I radioactive iodide (RAI) ablation. In contrast, 30-40% of patients with metastatic NMTC are unresponsive to 131 I RAI treatment as a result of tumor dedifferentiation. Currently, underlying molecular mechanisms of NMTC dedifferentiation still remain elusive and predictive biomarkers are lacking. In the present study we therefore aim to identify molecular biomarkers in primary tumors that are associated with RAI refractoriness. Methods: A retrospective cohort of 63 NMTC patients, including all histological subtypes, was gathered for this study and consisted of RAI-sensitive differentiated NMTC patients (N=35) and RAI-refractory (poorly) differentiated NMTC patients (N=28). RAI sensitivity was dened as the response to RAI of residual disease after primary surgery (persistence and/or progression versus regression). Total DNA and RNA were extracted from archived formalin-xed paran embedded (FFPE) tumor tissues. Extensive intratumoral mutation proling, gene fusions analysis, TERT promoter mutation analysis and FFPE-compatible RNA sequencing were performed in all patients. In eight NMTC patients, available from an independent cohort, total circulating IGF2 concentrations were determined using ELISA before and after undergoing primary treatment. Results: RAI-refractory NMTC patients were diagnosed at an older age and displayed less favorable TNM staging as compared to RAI-sensitive NMTC patients. Genetic analyses revealed an increased mutational load in RAI-refractory NMTC, including mutations in AKT1, PTEN, TP53 and TERT promoter. Transcriptomic analyses revealed profound differential expression of insulin-like growth factor 2 (IGF2) with up to 100-fold higher expression in RAI-refractory NMTC as compared to RAI-sensitive NMTC cases. By ELISA we found signicant lower IGF2 plasma concentrations after surgery and subsequent 131 I RAI therapy in patients with NMTC compared to pretreatment baseline. Conclusions: Important clinical, genetic and transcriptomic differences between patients with RAI-sensitive NMTC and RAI-refractory NMTC were identied.


Background
Non-medullary thyroid cancer (NMTC) is the most frequent endocrine tumor with in most cases a good prognosis. Standard of care consists of hemi-thyroidectomy or total thyroidectomy often followed by 131 I radioactive iodide (RAI) ablation (1,2). Despite the increasing incidence of NMTC, less than 10% of patients with clinical disease will develop distant metastases. From this group, 30-40% of patients with metastatic NMTC are unresponsive to 131 I radioactive iodide (RAI) treatment, have a 10-year survival rate less than 10% and a mean life expectancy of 3-5 years. RAI refractoriness could be the result of a tumor dedifferentiation process. (3,4). There are currently no curative treatment options for these RAI-refractory tumors emphasizing the need for novel therapeutic options and molecular biomarkers (5)(6)(7)(8).
Several studies aimed to discover novel markers that predict RAI sensitivity. In this context, TERT promoter mutations have been proposed to assist identi cation of patients with poorly differentiated TC with high risk of RAI refractoriness (9). The same holds true for the assessment of sodium-iodide symporter (NIS) expression in circulating tumor cells (10). Also, prostate-speci c membrane antigen (PSMA) expression in tumor tissue has been suggested to contribute in the prediction of tumor aggressiveness and patient outcome. (11). The most intensively studied marker is Thyroglobulin (Tg).
Quantitative changes in Tg could re ect the response to RAI therapy or serve as diagnostic or prognostic tool. (12)(13)(14). However, current methods, such as serum Tg or PSMA expression measurements serve mostly as a diagnostic, predictive or prognostic tool. Therefore, the identi cation of NMTC patients at risk for RAI refractory disease is still an unmet medical need and additional markers need to be explored.
In the present study we aimed to identify molecular markers in primary tumors that are associated with RAI refractory disease. A retrospective cohort of 63 NMTC patients, including all histological subtypes, was collected for this study and consisted of 35 RAI-sensitive differentiated NMTC patients and 28 RAIrefractory (poorly) differentiated NMTC patients. Extensive intratumoral mutation pro ling, gene fusions analysis, TERT promoter mutation analysis and formalin-xed para n embedded-compatible RNA sequencing was performed in all patients. To validate potential circulating markers, an independent cohort of 8 NMTC patients was available.

Patient cohorts
Detailed pathology reports were collected of all NMTC patients diagnosed in Nijmegen and surrounding hospitals between 2000 and 2016 (1544 patients). We selected 35 RAI-sensitive NMTC patients and 28 RAI-refractory NMTC patients that underwent total or near-total thyroidectomy and showed residual disease after primary surgery. Patients with con rmed nodal metastases prior to primary surgery also underwent a modi ed radical lymph node dissection. RAI ablation of residual thyroid tissue was performed 4-6 weeks after surgery. All patients included in this study had residual disease after primary surgery as demonstrated by diagnostic RAI scintigraphy. If indicated, patients were repeatedly treated with RAI to reach remission. RAI sensitivity was de ned as a complete response to RAI therapy of histologically differentiated tumor lesions resulting in remission after the primary treatment by surgery and RAI ablation or (if indicated) after subsequent treatments with RAI for metastases with documented 131 I uptake. Remission was de ned as undetectable TSH stimulated Tg in the absence of anti-Tg antibodies and no evidence of loco-regional disease or distant metastasis on the whole-body iodide scans (WBS) and/or neck ultrasonographic examinations at 6-9 months after the last RAI treatment. The remission status was con rmed at the last follow-up visit. According to the RECIST criteria, RAI refractoriness was de ned as either new evidence of recurrent loco-regional disease or distant metastasis after successful primary therapy or progressive disease at least 6 months after primary treatment by surgery and RAI treatment preferably supported by presence of metastases that do not accumulate 131 I on the last post-therapy scan. Persistent disease was de ned as detectable Tg and/or evidence of locoregional disease or distant metastases. Of all selected NMTC patients, archived FFPE tissue specimens were collected for genetic, transcriptomic and protein analyses. Collection, storage and use of archival tissue and patient data were in compliance with the "Code of Proper Secondary Use of Human Tissue in the Netherlands" (http://www.fmwv.nl and www.federa.org). This study was approved by the Research Ethics Committee (CMO) under application 2015 − 1762 and followed the ethical guidelines of the CMO.
An independent cohort including eight consecutive newly diagnosed patients with NMTC (with and without metastases) that were therapy naïve and were planned to receive conventional primary treatment by surgery followed by RAI were included in which plasma insulin growth factor 2 (IGF2) concentrations were measured before surgery and 30 days after 131 I radioactive iodide therapy. CMO application: 2017-3628; NL62671.091.17; ClinicalTrials.gov Identi er: NCT03397238. Their results were compared to those of six gender and aged matched healthy volunteers.

Intratumoral mutation pro ling
Somatic mutations in human NMTC tumor tissue were detected by our in-house Cancer Hotspot Panel based on single-molecule molecular inversion probes (smMIPs), as described previously (15). Isolated STAR, a standard aligner that makes use of a reference genome (GRCh38), was selected for the alignment of the sequences. To quantify the alignments made in STAR the tool HTSeq was utilized. This produced "counts.txt" les that are easy to import into DESeq2, a package to carry out differential gene expression analysis. Raw RNA sequencing data are deposited in the GEO database under accession number GSE112202.

Plasma measurements
Plasma IGF2 and IGFBP2 concentrations were measured by ELISA according to manufacturer's instructions (R&D Systems, Inc. Minneapolis, USA).

Statistical methods
Statistical signi cance was tested with Student's T test, Mann Whitney U test, Wilcoxon matched-pairs signed rank test, when appropriate. P-values below 0.05 were considered statistically signi cant. For RNA sequencing data a false discovery rate of 0.05 was incorporated. All statistical tests were performed using GraphPad Prism 5.0.

Results
Primary tumors from RAI-refractory NMTC patients harbor a signi cant higher IGF2 RNA expression compared to RAI-sensitive NMTC patients Patient and tumor characteristics of 35 RAI-sensitive and 28 RAI-refractory NMTC patients from the cohort are listed in Table 1. RAI-refractory NMTC patients were diagnosed at an older age and displayed a less favorable TNM staging at diagnosis than RAI-sensitive patients. This showed to be signi cant (p < 0.001) ( Table 1). To assess the mutational status in these NMTC patients, intratumoral mutational pro ling, gene fusion analysis and TERT promoter mutational analysis were performed. There was a signi cantly higher proportion of tumors bearing TERT promoter mutations in RAI-refractory NMTC patients compared to RAI-sensitive patients (50% vs. 8.6%) ( Table 2). In contrast, more RAI-sensitive patients showed gene fusions compared to RAI-refractory patients. To gain insight into differentially expressed genes between tumors obtained from RAI-sensitive and RAI-refractory NMTC patients, whole transcriptomics analysis was performed using RNA sequencing. Subsequently, a heatmap was constructed to visualize expression values (Fig. 1A) and differential expression analysis was determined within the overall comparison of RAI-sensitive and RAI-refractory NMTC patients. IGF2 showed a signi cantly higher expression in RAI refractory NMTC patients compared to RAI sensitive NMTC patients. Figure 1B displays the datapoints of each individual patient also separated for the different histological subgroups indicating that the higher RNA expression of IGF2 in RAI refractory patients is independent of the tumor histology.  To further support the role of IGF2 in the pathogenesis of NMTC we investigated the IGF2 plasma concentration before and after treatment in an independent cohort, including eight newly diagnosed therapy-naïve patients with NMTC (4 males and 4 females, averaged 54.6 ± 16.5 years) scheduled for conventional treatment by surgery followed by RAI. Patients and tumor characteristics of this cohort are listed in Table 3. The IGF2 plasma concentrations in these patients were compared to those of six ageand gender-matched healthy volunteers (3 males and 3 females, averaged 51.7 ± 12.4 years). The average total IGF2 concentration before surgery (baseline) in NMTC patients showed higher (1.17*10 6 +/-2.6*10 5 pg/mL) compared to healthy volunteers (9.5*10 5 +/-1.7*10 5 pg/mL) although this difference did not reach statistical signi cance ( Fig. 2A). Thirty days after primary surgery the average IGF2 plasma concentration was signi cantly decreased (8.3*10 5 +/-1.7*10 5 pg/mL) compared to the level before surgery (Fig. 2B). To exclude interference between IGFBP2 and it's ligand IGF2 effecting the assay, we assessed the plasma IGFBP2 concentrations in the same samples. The IGFBP2 concentrations after therapy were not signi cantly different from those measured before surgery, which suggests an even stronger effect of treatment on the free IGF2 circulating concentrations (Fig. 2C).

Discussion
Obliterating tumor remnants after thyroidectomy by RAI therapy is of signi cant clinical importance in NMTC. Unresponsiveness to RAI is a major concern since RAI-refractory tumors usually respond poorly to alternative therapies. Apart from a lack of therapeutic options, markers predicting RAI refractoriness have so far not been identi ed. Therefore, we searched for differences between the molecular signatures obtained from primary RAI sensitive and RAI refractory NMTC samples. Interestingly, transcriptome data from RAI refractory NMTC patients and RAI sensitive NMTC patients revealed a signi cantly higher RNA expression of IGF2 in primary tumors of RAI refractory NMTC patients. Moreover, we show that the IGF2 plasma concentration in patients with NMTC signi cantly decreased after primary treatment by surgery and RAI.
The IGF2 gene is located on chromosome 11p15.5 and encodes for the IGF2 growth factor that has been shown to play an important role in the fetal embryonic development, growth and energy metabolism of mammals (16)(17)(18)(19)(20). Apart from this, IGF2 has also been reported to be involved in carcinogenesis. In several cancer types, increased expression of IGF2 has been linked to poor prognosis (21). In lung cancer patients, increased IGF2 protein expression in pleural effusion supernatants was associated with resistance to osimertinib treatment (22). Elevated levels of IGF2 mRNA in osteosarcoma cells were shown after chemotherapy resulting in preservation of these cells under chemotherapeutic stress (23). In a study investigating 445 gastrointestinal tumors, high protein expression of IGF2 in the tumor tissue was associated with a signi cant worse outcome (24). In colorectal cancer, overexpression of stromal-derived IGF2 has been shown to play a role in development and progression, whereas increased serum concentrations and tissue overexpression of IGF2 has been associated with metastasis (25)(26)(27). Moreover, in breast cancer tissues, IGF2 was found to be more potent than in normal breast tissue for activating insulin receptor (IR) autophosphorylation causing stimulation of cell growth (28). Finally, a study performed by Tominaga et al. described a positive feedback loop, IGF2-IGF1R-PI3K-ID1-IGF2, present in cancer stem-like cells causing cells to maintain in the stem cell state (29).
A few other studies suggest a role for IGF2 in the pathogenesis of NMTC. Differences in IGF2 mRNA expression between normal thyroid epithelial cells, thyroid adenoma and thyroid carcinoma were demonstrated using three pairwise comparisons with the GEO2R online tool (30). One research group published several studies in which they demonstrate the overexpression of IGF2 mRNA in undifferentiated thyroid cancer cell lines, poorly differentiated malignant thyrocytes, cancer thyrospheres and thyroid cancer specimens. This overexpression of IGF2 coincided with elevated expression of insulin receptor isoform A (IR-A) and insulin-like growth factor 1 receptor (IGF1R). They showed that this socalled IGF2/IR-A autocrine loop is associated with dedifferentiation and stem-like phenotypes, resembling RAI refractoriness (31)(32)(33)(34). In line with these ndings, we identi ed higher IGF2 expression in primary tumors from RAI refractory NMTC patients via RNA sequencing. For the rst time, our study also demonstrates a signi cant decrease of total IGF2 plasma concentration 30 days after primary therapy as compared to before surgery. Collectively these data suggest that IGF2 expression could have prognostic and therapeutic implications for several cancer types including NMTC, which strengthens the importance of our ndings and a potential role for IGF2 in acquiring RAI refractoriness.
The mechanism behind the increase in IGF2 expression in RAI refractory tumors demonstrated in this study is unknown. Previous studies have proposed several mechanisms to explain this increased expression causing therapy resistance. Wang et al. showed that IGF2 produced by cancer-associated broblasts (CAFs) induced autophagy in cancer cells post-radiation thereby promoting cancer cell recovery (35). Also, drug resistance in non-small cell lung cancer cells was observed, in icted by IGF2-AKT-Sox2-ABCB1 signaling in cancer cells co-cultured with CAFs (36). The survival of osteosarcoma cells, supported by IGF2, was dependent on enhanced autophagic ux and glutamine availability (23). In malignant rhabdoid tumors IGF2 has been shown to activate IGF1R and IR followed by activation of the PI3K-AKT and RAS-ERK pathways to promote proliferation and cell survival (37).
The present ndings have potential clinical and therapeutic consequences. Several studies have been performed where IGF2 has been proposed as a predictive or prognostic molecular marker (38)(39)(40). A limitation to the use of IGF2 as a molecular marker for RAI refractoriness or for a more aggressive tumor behavior could be the presence of an autocrine IGF signaling loop as described by . Measurement of serum IGF2 does not account for local IGF2 production in the tumors depending on an autocrine IGF2 signaling loop. Alternatively, measuring tumor IGF2 expression in the tissue available after thyroidectomy as we have shown in the study could be an potential option to be further explored (17).
Apart from serving as a molecular marker for RAI refractoriness, IGF2 could perhaps also be targeted therapeutically to overcome RAI refractoriness. Detecting increased circulating levels of IGF2 could be followed by targeting IGF2 or its receptors by using IGF monoclonal antibodies, IGF1R monoclonal antibodies or IGF1R/IR tyrosine kinase inhibitors. Multiple phase I/II trials in all types of cancers have been initiated using these therapeutic options, also in combination with chemotherapeutics and other drugs (17,41). Studies have shown that targeting the IGF1R combined with the IR pathway may increase therapy e cacy and prevents resistance to selective IGF1R antibodies or inhibitors (42,43). OSI-906, a dual inhibitor of the IGF1R and IR, has already shown antitumor activity in a phase I study and phase II studies in combination with other drugs are ongoing (44,45). In the case of NMTC using such inhibitors could perhaps be bene cial in combination with RAI to overcome RAI refractoriness.

Limitations
A few limitations of this study should be discussed. The retrospective design of our study and the criteria used for patient selection and RAI therapy indication possibly biased our results. Moreover, as mentioned earlier, the prevalence of distant metastasized NMTC is < 10%. Therefore, future studies will require larger sample sizes in addition to con rm our ndings from both the retrospective cohort as well as the independent cohort.

Conclusion
Page 12/16 In conclusion, important clinical, genetic and transcriptomic differences were identi ed between patients with RAI-sensitive NMTC and RAI-refractory NMTC. Interestingly, the tumor-promoting growth factor IGF2 showed a signi cantly higher expression in RAI refractory tumors. Plasma levels of IGF2 decreased following primary treatment in patients with NMTC. These ndings suggest that tumor-promoting growth factor IGF2 could be a potential factor in acquiring RAI refractoriness. Further studies in independent cohorts are needed to validate our ndings and elaborate on the mechanism behind the elevated IGF2 expression and RAI refractory in NMTC patients and to explore IGF2 as a potential therapeutic target. NCT03397238.

Consent for publication
Not applicable Availability of data and materials The raw RNA datasets generated and/or analyzed during the current study are available in the GEO database under accession number GSE112202.

Competing interests
The authors declare that they have no competing interests.