Identification of novel genetic variants that increase risk and aggressiveness of paediatric thyroid cancer with impact in diagnosis and treatment

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Identification of genetic variants in patients with early-onset and/or familial thyroid cancer with impact in diagnosis and treatment

Publication . Pires, Carolina; Cavaco, Branca Maria Prudêncio Limón; Pojo, Marta; Leite, Valeriano; Matthiesen, Rune

Thyroid cancer is the most common malignancy of the endocrine system. Most thyroid cancers derive from the follicular cells, being designated as non-medullary thyroid carcinomas (NMTC). A subset of NMTC occurs in a familial form, known as familial non-medullary thyroid carcinoma (FNMTC), accounting for 5-15% of all NMTC cases. FNMTC is currently defined by the presence of follicular cell-derived thyroid carcinoma in at least three first-degree relatives, or by the existence of papillary thyroid carcinoma (PTC) in two or more first-degree relatives, in the absence of a history of previous radiation and inherited cancer syndrome. The family members frequently present benign lesions of the thyroid, such as follicular nodular disease (FND). Several candidate FNMTC susceptibility genes have already been reported, but these are mutated only in a small fraction of families. Thus, the molecular basis of FNMTC is still poorly understood, being regarded as a genetically heterogeneous disease. Recently, germline truncating mutations in DNA repair-related genes have been described in cases of thyroid cancer. In addition, activating mutations in BRAF and RAS oncogenes have been reported to be involved in the progression of familial thyroid tumours. Paediatric thyroid cancer is rare, representing less than 2% of all thyroid malignancies. Therefore, the biology and molecular genetics of these tumours are less well-characterized compared to those in adults, with a significant proportion of cases aetiology categorized as "dark matter". Gene fusions, such as those involving RET and NTRK genes, are more common in children than oncogenic point mutations. In fewer than 1% of thyroid cancer cases, malignant tumours may arise in ectopic thyroid tissue, as seen in cases of malignant struma ovarii. The genetic basis of these rare tumours remains largely unknown. Disease prevention, active surveillance, and precision therapies are key elements for reducing aggressive treatments, comorbidities, invasive surgeries, and the burden to the healthcare system. Therefore, the research of diagnostic and prognostic biomarkers, as well as targets for treatment, is essential for patient management, genetic counselling, and for the development and use of novel therapeutic approaches. This work aimed to further elucidate the epi/genetic mechanisms involved in the aetiology and progression of familial, paediatric, and rarer forms of thyroid cancer, through a combination of distinct approaches. In a first approach, 48 probands from FNMTC families were screened for germline variants in 94 cancer predisposition genes using next-generation sequencing (NGS). This study revealed likely pathogenic mutations in DNA repair genes (ATM, CHEK2, ERCC2, BRCA2, ERCC4, FANCA, FANCD2, FANCF, and PALB2) and in other hereditary cancer predisposing genes (DICER1, FLCN, PTCH1, BUB1B, and RHBDF2), segregating with the disease in at least two family members. Structural modelling and network analysis demonstrated disrupted protein interactions, suggesting a pivotal role for DNA repair pathway alterations in FNMTC susceptibility. MAPK activation was a common feature in tumour progression. Together, these findings xx highlighted the role of DICER1 in disease aetiology and broadened the genetic landscape of FNMTC by implicating rare germline variants in DNA repair genes and in additional candidate susceptibility genes previously unrelated to FNMTC. In the follow-up of this study, two germline CHEK2 missense variants (p.Glu321Ala and p.Ile157Thr), identified in two of the 48 FNMTC families, were further explored. CHK2 proteins (wild type and variants) were characterized using biophysical methods and molecular dynamics simulations. Overall, these variants were found to impair the structural and conformational stabilities and kinase activity of CHK2, compared to the wild-type protein. Moreover, these variants exhibited a higher propensity to form amyloid-like aggregates in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. In the third study, 38 radiation-unrelated paediatric thyroid cancer cases were stratified by risk, according to the American Thyroid Association (ATA) guidelines, and analysed by whole exome and transcriptome sequencing, DNA methylation arrays, targeted NGS, and Sanger sequencing. The results revealed a high prevalence of germline variants in cancer predisposition genes (CHEK2, RAD51C, ROS1, PIK3CA) and thyroid function-related genes (FOXE1, HHEX, DUOX1/2), particularly in high- and intermediate-risk cases, and identified DICER1 variants exclusively in low-risk patients. Additionally, somatic gene fusions involving RET and NTRK1/3 were predominantly observed in high-risk tumours, supporting patients’ eligibility for available targeted therapies. The presence of TERT promoter mutations co-existing with NTRK fusions in a subset of high-risk cases provided further insights into tumour progression. Transcriptomic and methylation analyses provided molecular support to the clinical ATA-based risk stratification, disclosed candidate biomarkers, and shed light on the biological drivers of tumour development and aggressiveness. Lastly, the role of the FOXE1 gene was investigated by Sanger sequencing in two families presenting with malignant struma ovarii (MSO) and FND (Family 1), or cleft palate and thyroid cancer (Family 2). Two distinct rare FOXE1 promoter variants were detected in the families. Functional studies in thyroid cell models revealed that these variants affected FOXE1 transcriptional activity and were associated with decreased FOXE1 expression in thyroid tumours. The case with MSO also carried a germline AXIN1 variant, which was linked to β-catenin cytoplasmic accumulation in the patient’s benign and malignant teratoma tissues, possibly due to the observed AXIN1 loss of heterozygosity in these specimens. These results suggested that germline FOXE1 variants might have had a role in cleft palate (in Family 2) and, together with the germline AXIN1 variant, in FND and thyroid ectopy (in Family 1), which, proceeded by AXIN1 somatic loss, and MAPK pathway activation, driven by the identified somatic BRAF and HRAS mutations, likely led to malignant transformation in the patients with MSO and thyroid cancer, respectively. In conclusion, this thesis identified novel candidate susceptibility genes involved in familial, paediatric, and other presentations of thyroid cancer. It highlights the relevance of genes involved in DNA repair pathways, particularly of CHEK2, thyroid function-related genes, and DICER1 in thyroid cancer predisposition, along with the oncogenic role of the MAPK signalling xxi cascade (RET/NTRK fusions, BRAF/RAS mutations) in tumour progression. These findings, together with the identification of disease-related methylation and expression profiles, contribute to a deeper understanding of the epi/genetic factors underlying these forms of thyroid cancer and, if supported by future studies in other cohorts, may provide a basis for molecular diagnosis, risk stratification, targeted therapies, and improve the clinical management of these patient

2025-03-10Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

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2020.07120.BD