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