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

doi:10.21203/rs.3.rs-89212/v1

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

https://doi.org/10.21203/rs.3.rs-89212/v1

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Background: Non-medullary thyroid cancer (NMTC) is the most frequent endocrine tumor with in most cases a good prognosis after thyroidectomy and ¹³¹I radioactive iodide (RAI) ablation. In contrast, 30-40% of patients with metastatic NMTC are unresponsive to ¹³¹I 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 defined 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-fixed paraffin embedded (FFPE) tumor tissues. Extensive intratumoral mutation profiling, 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 significant lower IGF2 plasma concentrations after surgery and subsequent ¹³¹I 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 identified. Most notably an increased expression of IGF2 in RAI-refractory tumors. Plasma levels of IGF2 decreased post-therapy in NMTC patients from an independent cohort. These findings suggest that the tumor-promoting growth factor IGF2 has a potential role in acquiring RAI refractoriness.

Oncology

Biomedical Engineering

Pathology

non-medullary thyroid cancer

RAI refractoriness

insulin-like growth factor 2

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 ¹³¹I 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 ¹³¹I 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 (5–8).

Several studies aimed to discover novel markers that predict RAI sensitivity. In this context, TERT promoter mutations have been proposed to assist identification 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-specific 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 reflect the response to RAI therapy or serve as diagnostic or prognostic tool. (12–14). However, current methods, such as serum Tg or PSMA expression measurements serve mostly as a diagnostic, predictive or prognostic tool. Therefore, the identification 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 RAI-refractory (poorly) differentiated NMTC patients. Extensive intratumoral mutation profiling, gene fusions analysis, TERT promoter mutation analysis and formalin-fixed paraffin 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 confirmed nodal metastases prior to primary surgery also underwent a modified 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 defined 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 ¹³¹I uptake. Remission was defined 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 confirmed at the last follow-up visit. According to the RECIST criteria, RAI refractoriness was defined 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 ¹³¹I on the last post-therapy scan. Persistent disease was defined as detectable Tg and/or evidence of loco-regional 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 ¹³¹I radioactive iodide therapy. CMO application: 2017–3628; NL62671.091.17; ClinicalTrials.gov Identifier: NCT03397238. Their results were compared to those of six gender and aged matched healthy volunteers.

DNA and RNA isolation from FFPE tissues

For DNA isolation, 60 µm FFPE tissue slices were digested overnight at 56 °C in the presence of TET-lysis buffer (10 mM Tris/HCl pH 8.5, 1 mM EDTA pH 8.0, 0.01% Tween-20), 5% Chelex-100 (Bio-Rad, Hercules, CA, USA), 15 µg/mL GlycoBlue (Life Technologies, Carlsbad, CA, USA) and 400 µg proteinase K (Qiagen, Valencia, CA, USA), followed by inactivation at 95 °C for 10 minutes. For DNA isolation, the supernatant was transferred after centrifugation (20817 rcf), cooled on ice and precipitated in the presence of 100% EtOH and 1/33 volume 3M NaAc (pH 5.2). Pellets were washed with cold 70% EtOH, dissolved in 80 µL Tris-EDTA and DNA concentrations were determined using the Qubit Broad Range Kit (Thermo Fisher, Waltham, MA, USA). For RNA isolation, 60 µm FFPE tissue slices were digested overnight at 56 °C in the presence of 240 µl lysis buffer (Qiagen, Valencia, CA, USA) and 400 µg proteinase K. Next, supernatants were transferred after centrifugation (16,000 rpm) and mixed with RNA-Bee. Subsequently, RNA was isolated by phase separation with chloroform and precipitated by isopropanol, according to the manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA).

Intratumoral mutation profiling

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 DNA from FFPE tissues with a tumor cell percentage of > 60% was subjected to library preparation and clinically relevant regions were sequenced of the following genes: AKT1, BIRC3, BRAF, CHEK2,CTNNB1, CXCR4, EGFR, ERBB2, EZH2, GNA11, GNAQ, GNAS, H3F3A, H3F3B, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MSH2, MYD88, NRAS, PDGFRA, PIK3CA, SF3B1, SLC7A8 and ZNF2.

Gene fusion analysis

DNA/RNA was isolated from punched tumor tissue with a tumor cell percentage of > 50%. Gene fusion analysis was performed by Next Generation Sequencing (Archer FusionPlex CTL Panel) and data were analyzed by Archer Analysis software (version 5). Relevant fusions of the following target genes were sequenced: ALK (5'; exons 2, 4, 6, 10, 16–23, intron 19), AXL (3'; exons 18–20), BRAF ( 5'; exons 7–11, 3'; exons 7, 8, 10), CCND1 (5'; exons 1–4, 3'; exons 1, 2, 4), FGFR1 (5'; exons 2, 8–10, 17, 3'; exon 17), FGFR2 (5'; exons 2, 5, 7–10, 3'; exon 17), FGFR3 (5'; exons 3, 5, 8–10, 3'; exon 17, intron 17), MET (5'; exons 2, 4–6, 13, 14, 16, 17, 21, 3'; exon 2), NRG1 (5'; exons 1, 2, 3, 6), NTRK1 (5'; exons 2, 4, 6, 8, 10–13), NTRK2 (5'; exons 5, 7, 9, 11–17), NTRK3 (5'; exons 4, 7, 10, 13–16), PPARG (5'; exons 1, 2, 3, 5), RAF1 (5'; exons 4–7, 9–12), RET (5'; exons 2, 4, 6, 8, 9–14), ROS1 (5'; exons 2, 4, 7, 31–37), THADA (3'; exons 24–30, 36, 37). In addition, the FusionPlex-CTL hotspot panel also detects mutations in BRAF (exon 11, 15), HRAS, NRAS (exon 2 and 3, codon 12, 13, 61), KRAS (exon 2, 3 and 4, codon 12, 13, 61 and 146) and the EGFRvIII variant.

TERT promoter mutation analysis

TERT promoter mutations C228A, C228T (at position − 124 from translation start site) and C250T (-146 from translation start site) were detected by conventional PCR followed by Sanger sequencing. The TERT promoter region was amplified by the following M13-sequence extended primers: forward 5’- TGT-AAA-ACG-ACG-GCC-AGT-CCC-TTC-ACC-TTC-CAG-CTC-3’ and reverse 5’-CAG-GAA-ACA-GCT-ATG-ACC-AGC-GCT-GCC-TGA-AAC-TCG − 3’. DNA was amplified by AmpliTaq PCR 360 Gold Master Mix (Thermo Fisher, Waltham, MA, USA) and the following PCR program: 95 °C for 10 minutes followed by 95 °C for 30 seconds, 58 °C for 30 seconds and 72 °C for 1 minute (38 cycles) and a final step of 72 °C for 7 minutes. Subsequently, Sanger sequencing was performed with M13 primers and TERT promoter mutations were called by Sanger chromatogram software (Sequencher 4.8, Gene Codes Corp., Ann Arbor, MI, USA).

FFPE compatible RNA sequencing

Isolated RNA obtained from FFPE tissues with a tumor cell percentage of > 80% was processed for RNA sequencing by DNAse treatment, RNA demodification, RNA fragmentation (if necessary), first and second-strand cDNA synthesis and library preparation according to the Ovation SoLo RNA-Seq System (NuGEN, San Carlos, CA, USA). Subsequently, RNA sequencing was performed by Illumina NextSeq500 and analysis of the data was performed by the Centre for Molecular and Biomolecular Informatics (CMBI). 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” files 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 significance 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 significant. For RNA sequencing data a false discovery rate of 0.05 was incorporated. All statistical tests were performed using GraphPad Prism 5.0.

Primary tumors from RAI-refractory NMTC patients harbor a significant 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 significant (p < 0.001) (Table 1). To assess the mutational status in these NMTC patients, intratumoral mutational profiling, gene fusion analysis and TERT promoter mutational analysis were performed. There was a significantly 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 significantly 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.

Table 1

Clinical characteristics of RAI-sensitive and RAI-refractory NMTC patients.
Parameter		RAI-sensitive NMTC patients (N = 35)	RAI-refractory NMTC patients (N = 28)	P-value
Age at diagnosis (mean ± SD)		41.9 (± 18.9)	61.9 (± 10.2)	< 0.001
Gender		13 male, 22 female	16 male, 12 female	0.094
Histology	PTC	29	11	0.004
	FTC	4	10
	HCC (Hürthle)	1	5
	FVPTC	1	2
T-stage*	T1	12	3	< 0.001
	T2	12	4
	T3	8	7
	T4	3	14
N-stage*	N0	0	0	N/A
N-stage*	N1	35	28	N/A
M-stage*	M0	31	4	< 0.001
M-stage*	M1	4	24	< 0.001
Location of metastases	No metastases	31	4	< 0.001
	Lung	3	16
	Bone	0	2
	Lung and bone	1	5
	Lung, liver and muscle	0	1
Number of RAI treatments	0–1	22	6	0.017
	2	9	12
	> 2	4	10
Cumulative RAI dose (MBq, mean ± SD)		9806 (± 7127)	17240 (± 8955)	< 0.001
*Based on AJCC 8th edition of TNM classification
FTC = follicular thyroid cancer; FVPTC = follicular-variant papillary thyroid cancer; HCC = hürthle cell carcinoma; NMTC = non-medullary thyroid cancer; PTC = papillary thyroid cancer; RAI = radioactive iodide

Table 2

Genetic characteristics of RAI-sensitive and RAI-refractory NMTC patients.
Parameter:		RAI-sensitive NMTC patients (N = 35)	RAI-refractory NMTC patients (N = 28)	P-value
Oncogenic mutation status	BRAF V600E	15	9	0.435
	H/K/NRAS G12D / Q61R	4	5
	Other	CCDC6 : RET fusion (N = 4)	PTEN E242X, TP53 R158G (N = 1)
		CCDC6 : RET fusion, PIK3CA H1047R (N = 1)	PTEN E242X, TP53 P212fs (N = 1)
		SQSTM1 : NTRK3 fusion (N = 1)	NRAS Q61R, AKT1 E17L (N = 1)
		ETV6 :NTRK3 fusion (N = 1)	EML4 : NTRK3 fusion (N = 1)
		NCOA4:RET fusion (N = 1)	-
		PAX8 : PPARG fusion (N = 2)	-
	Unknown	6	10
TERT promoter mutation status	TERT Wild-type	32	14	< 0.001
TERT promoter mutation status	TERT C228T	3	14	< 0.001
RAI = radioactive iodide; NMTC = non-medullary thyroid cancer

NMTC patients show significantly lower circulating levels of IGF2 concentration after primary treatment

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 age- and 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*10⁶ +/- 2.6*10⁵ pg/mL) compared to healthy volunteers (9.5*10⁵ +/- 1.7*10⁵ pg/mL) although this difference did not reach statistical significance (Fig. 2A). Thirty days after primary surgery the average IGF2 plasma concentration was significantly decreased (8.3*10⁵ +/- 1.7*10⁵ 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 significantly different from those measured before surgery, which suggests an even stronger effect of treatment on the free IGF2 circulating concentrations (Fig. 2C).

Table 3

Clinical characteristics of NMTC patients from the independent cohort.
Patient number	Age at inclusion	Gender	Histology	TNM stage at diagnosis	RAI dose (mCi)
1	42	F	PTC	T1bN1M0	100
2	55	M	HCC (Hürthle)	T2mN0M1	200
3	49	M	PTC	T1bmN1aM0	100
4	65	M	PTC	T2N1bM0	200
5	78	M	HCC (Hürthle)	T4aN1bM0	200
6	50	F	PTC	T2mN1bMx	100
7	27	F	PTC	T3N1bM0	200
8	71	F	PTC	T1mN0M0	30
F = female; HCC = hürthle cell carcinoma;; M = male; NMTC = non-medullary thyroid cancer; PTC = papillary thyroid cancer; RAI = radioactive iodide

Obliterating tumor remnants after thyroidectomy by RAI therapy is of significant 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 identified. 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 significantly 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 significantly 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 (16–20). 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 significant 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 (25–27). 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 so-called IGF2/IR-A autocrine loop is associated with dedifferentiation and stem-like phenotypes, resembling RAI refractoriness (31–34). In line with these findings, we identified higher IGF2 expression in primary tumors from RAI refractory NMTC patients via RNA sequencing. For the first time, our study also demonstrates a significant 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 findings 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 fibroblasts (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, inflicted 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 flux 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 findings have potential clinical and therapeutic consequences. Several studies have been performed where IGF2 has been proposed as a predictive or prognostic molecular marker (38–40). 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 Vella and Malaguarnera (31–34). 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 efficacy 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 beneficial in combination with RAI to overcome RAI refractoriness.

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 confirm our findings from both the retrospective cohort as well as the independent cohort.

In conclusion, important clinical, genetic and transcriptomic differences were identified between patients with RAI-sensitive NMTC and RAI-refractory NMTC. Interestingly, the tumor-promoting growth factor IGF2 showed a significantly higher expression in RAI refractory tumors. Plasma levels of IGF2 decreased following primary treatment in patients with NMTC. These findings 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 findings 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.

ATC

anaplastic thyroid cancer ; FFPE:formalin-fixed paraffin embedded; FTC:follicular thyroid cancer; FVPTC:follicular-variant papillary thyroid cancer; HCC:hürthle cell carcinoma; IGF2:insulin-like growth factor 2; IGFBP2:insulin-like growth factor binding protein 2; NIS:sodium-iodide symporter; NMTC:non-medullary thyroid cancer; PET/CT:positron emission tomography / computer tomography; PSMA:prostate-specific membrane antigen; PTC:papillary thyroid cancer; RAI:radioactive iodide; smMIP:single-molecule molecular inversion probe; TERT:Telomerase reverse transcriptase; TG:thyroglobulin; TSH:thyroid stimulating hormone;

Ethics approval and consent to participate

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.

For the independent cohort: CMO application: 2017-3628; NL62671.091.17; ClinicalTrials.gov Identifier: 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.

Funding

T.S. Plantinga was supported by a Veni grant of the Netherlands Organization for Scientific Research (NWO; 016.136.065) and by the Alpe d’HuZes fund of the Dutch Cancer Society (KUN2014-6728).

Authors' contributions

Author contributions included the following: TC, MHT, MJ and TSP performed the experiments and the data analysis; WEC, HM performed the gene-fusion experiments and analysis. KR kindly provided the samples for the independent cohort. ACHE performed the pathological examinations of the tumor samples. TC, JWAS, RTN and TSP designed the study; TC, JWAS, RTN and TSP wrote the manuscript.

Acknowledgements

We would like to acknowledge the Centre for Molecular and Biomolecular Informatics (CMBI) for analysing the raw RNA sequencing data.

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

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Abstract

Figures

Background

Material And Methods

Patient cohorts

DNA and RNA isolation from FFPE tissues

Intratumoral mutation profiling

Gene fusion analysis

TERT promoter mutation analysis

FFPE compatible RNA sequencing

Plasma measurements

Statistical methods

Results

NMTC patients show significantly lower circulating levels of IGF2 concentration after primary treatment

Discussion

Limitations

Conclusion

Abbreviations

Declarations

References

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