Clinical Associations
Driver alterations were found to be associated with cervical metastases or N1 classified lymph node samples (P = 0.05, 19/23; Figure 4a). The presence of an activating kinase gene fusion correlated with higher ATA risk level (P = 0.027, 10/13 high-risk patients; Figure 4b) and multifocal disease pathology (P = 0.039, 13/22 multifocal tumors; Figure 4c). BRAF V600E  mutations were more frequent in smaller tumors (T1 and T2), while fusions were more frequent in larger tumors, T2 and higher (P = 0.001; Figure 4d).
Discussion :
Integrated DNA/RNA panel sequencing successfully identified frequent clinically-relevant alterations in a cohort of pediatric PTC patients enriched for metastatic and relapsed/refractory disease. Overall,BRAF V600E mutations were present in 19% of samples (6/31), consistent with other recent pediatric studies that have reported frequencies ranging from 17-61%1,11,14,17,30,31. In contrast with adult PTC, fusions were detected more than twice as frequently as BRAF V600E mutations in our cohort, with activating kinase fusions detected in 52% of samples tested (13/25). All but one of the driver alterations detected in this cohort were mutually exclusive (a single driver alteration per patient). This is consistent with our understanding of adult PTC development, in that these tumors usually arise from a single molecular driver whose alteration causes continuous activation of MAPK and PI3K signaling pathways6,7.
Fusion positive patients were more likely to be classified as ATA high-risk status and to have multifocal disease, findings that hold implications for future molecular testing. These patients are more likely to develop relapsed/recurrent disease requiring repeat surgery or RAI treatments. In comparison, patients with BRAF V600E mutations often presented with smaller primary tumors (T1 and T2) than those with fusion positive disease, although they frequently were found to have cervical lymph node metastasis at the time of diagnosis.
Employing the partner agnostic Oncology Research Panel (ArcherDx) enabled us to identify multiple unexpected fusions in our cohort. For example, three tumors were found to harbor fusions not known to be previously reported in PTC, including the patient with a novelVIM-NTRK3 fusion. A second patient was discovered to have PTC containing an EML4-NTRK3 fusion. This fusion has been identified in patients with infantile fibrosarcoma32,33, but not previously in PTC. Interestingly, this patient had a prior history of recurrent osteosarcoma and later developed malignant melanoma, and was found to have Li-Fraumeni Syndrome by targetedTP53 clinical testing; this was not detected in our analysis as the DNA panel testing was not successful. Finally, a MACF1-BRAFfusion was identified in a third patient who had previously been diagnosed with AML and received radiation prior to bone marrow transplant; this fusion is novel in PTC and has been rarely reported in low grade glioma 34.
Our cohort contained a number of individual patients with notable genomic and clinical findings. For instance, a patient with a history of short stature and delayed puberty was found to have tumor containing aPTPN11 hotspot mutation (p.S502T) with a variant allele frequency of 50%. Although a blood sample was not available for germline testing, this mutation has previously been detected in patients with Noonan’s syndrome 35. Another patient – the only case with two driver alterations detected - was found to have BRAF and AKT1 mutations at different variant allele frequencies (37% and 22%, respectively) in their tumor sample, suggesting that the AKT1 mutation was present in a sub-clonal population of the tumor.
Importantly, the vast majority of the alterations identified in our cohort are currently targetable with FDA-approved or investigational agents, including MAPK pathway inhibitors, PI3K pathway inhibitors, and other kinase inhibitors. Vemurafenib, a potent BRAF inhibitor that is specific for tumors with the BRAF V600E mutation, has shown antitumor efficacy in adults with progressive metastatic, RAI-refractoryBRAF V600E positive PTC36,37. While its use in pediatric patients has generally been limited, in part due to the difficulty of studying adequate numbers of pediatric PTC patients in clinical trials38, there are a number of case reports of children with BRAF V600E positive tumors whose disease responded to treatment39-41.
One recently FDA approved agent is larotrectinib, a highly selective small molecule inhibitor of the tropomyosin receptor kinase (TRK) proteins (encoded by kinase genes NTRK1 , NTRK2 , andNTRK3 ) which has demonstrated potent antitumor efficacy in both children and adults with TRK fusion positive tumors42,43. Notably, TRK fusion positive tumors comprised approximately 11% (4/36) of our cohort. Results from a phase I trial included two pediatric patients with TRK (NTRK1 and NTRK3 ) fusion-positive PTC; while these two patients could not be objectively evaluated by RECIST criteria (as they did not have measurable disease at enrollment), both patients remained on treatment without progression at the data cutoff point over 7 months later43.
Multiple targeted agents have been developed for adult patients with RET driven solid tumors. Sorafenib and lenvatinib, multi-kinase inhibitors that target RET , FLT1, KDR, FLT4 , PDGFRA, PDGFRB,and KIT , are FDA approved for adults with PTC and RETfusions 44. Sorafenib has been studied in a phase 2 trial in pediatrics but no PTC patients were enrolled on study45. A recent report of three pediatric patients with refractory PTC who demonstrated clinical improvement with lenvatinib suggests that this may also be of potential utility in relapsed or refractory patients46. Additionally, selpercatinib, an oral and selective investigational drug targeting RET kinase abnormalities, was recently FDA approved for patients ages 12 and older with metastatic or advanced RET -mutated medullary thyroid carcinoma or RET fusion positive (and RAI refractory) thyroid cancer 47. Selpercatinib will soon be available to relapsed pediatric patients through the National Cancer Institute and Children’s Oncology Group jointly sponsored Pediatric MATCH trial48.
Of the 20 cases which were evaluated by both DNA and RNA NGS panels, potentially clinically-relevant alterations were detected in 15 cases (75%). The frequency of driver alterations in metastatic and relapsed/refractory pediatric PTC, in combination with a steady increase in available molecularly targeted agents and the need to decrease morbidity from repeated surgeries and RAI treatments, has significant implications for the utility of molecular testing in this patient population. Our findings strongly support the inclusion of RNA testing in such analysis, especially in ATA high-risk patients where the diagnostic yield is particularly high. Given the potential therapeutic importance of identifying targetable gene fusions which are often characterized by diverse and novel gene partners, methods that enable partner-agnostic detection of fusion genes, such as anchored multiplex chemistry (used in this study to detect a novel VIM-NTRK3 fusion) or capture-based transcriptome sequencing should be preferred. At our center, we clinically test all relapsed and/or refractory pediatric patients with PTC using paired targeted DNA/RNA cancer gene panels as described above, and recommend upfront tumor testing in patients who are not amenable to conventional management, including RAI therapy, or who have symptomatic lung disease; however, a stepwise approach, in which such patients are first evaluated for BRAF V600E mutations, and if negative, undergo fusion testing, is a reasonable alternative.
In conclusion, our experience suggests that targeted DNA mutation and RNA fusion panel sequencing for pediatric patients with ATA high-risk PTC has the potential to be of clinical benefit, especially with the recent increase in available targeted agents for pediatric patients. We anticipate that as we continue to attempt to minimize morbidity associated with repeated RAI exposure and surgery for these patients, utilization of molecularly targeted agents in conjunction with current standard therapies will increase, particularly amongst patients with lung metastases and refractory disease. Additionally, as our cohort consisted of pre-treatment specimens obtained from thyroidectomy, further studies evaluating the degree of tumor evolution over time and necessity for re-biopsy will be needed.
Financial Support: Dr. Samara Potter is funded by a Paul Calabresi Scholar K12 Career Development Award, a St. Baldrick’s Foundation Fellowship Award, and the Gillson Longenbaugh Foundation. Mr. Raghu Chandramohan is funded by the Gillson Longenbaugh Foundation and the Cullen Foundation. Dr. Will Parsons is the recipient of a St. Baldrick’s Innovation Award.
Author Disclosure Statement : Dr. Potter serves as a consultant for Bayer Healthcare Pharmaceuticals.
Acknowledgements : We would like to acknowledge our patients and their families and the members of the Thyroid Tumor Program at Texas Children’s Hospital. We are grateful to the St. Baldrick’s Foundation, the Gillson Longenbaugh Foundation, and the Cullen Foundation for their financial support for this manuscript.
Data sharing: The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Legends:
TABLE 1. Demographic and clinical characteristics of pediatric PTC cohort
Figure 1. Flowchart demonstrating cohort selection and sequencing. PTC, papillary thyroid carcinoma.
Figure 2. Oncoprint illustrating genomic sequencing findings and selected clinical characteristics.
Figure 3. Fusions detected by RNA panel sequencing (A )NCOA4-RET (B ) CCDC6-RET (C )ETV6-NTRK3 (D ) VIM-NTRK3 (E )EML4-NTRK3 (F ) MACF1-BRAF . All fusions retain the protein kinase domain.
Figure 4. Distribution of driver alterations (gene fusions andBRAF V600E mutations) with various clinical characteristics, including (A) cervical lymph node metastases, (B) ATA risk level, (C) primary tumor focality, and (D) extent of primary tumor.
Supplemental Table S1. Cohort clinical and genomic data
Supplemental Figure 1. Copy number variant heatmap