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\begin{document}

\title{Integrated DNA and RNA sequencing reveals targetable alterations in
metastatic pediatric papillary thyroid carcinoma}



\author[1]{Samara Potter}%
\author[2]{Jacquelyn Reuther}%
\author[2]{Raghu Chandramohan}%
\author[2]{Ilavarasi Gandhi}%
\author[2]{Faith Hollingsworth}%
\author[3]{Hadi Sayeed}%
\author[3]{Horatiu Voicu}%
\author[1]{Nipun Kakkar}%
\author[1]{Koel Sen Baksi}%
\author[2]{Stephen Sarabia}%
\author[3]{Monica Lopez}%
\author[3]{Daniel Chelius}%
\author[2]{Ioanna Athanassaki}%
\author[1]{Priya Mahajan}%
\author[3]{Rajkumar Venkatramani}%
\author[3]{Norma Quintanilla}%
\author[2]{Dolores Lopez-Terrada}%
\author[3]{Angshumoy Roy}%
\author[2]{Donald Parsons}%
\affil[1]{Texas Children's Cancer Center}%
\affil[2]{Baylor College of Medicine}%
\affil[3]{Texas Children's Hospital}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Pediatric papillary thyroid carcinoma (PTC) is clinically
and biologically distinct from adult PTC. We sequenced a cohort of
clinically-annotated pediatric PTC cases enriched for high-risk tumors
to identify genetic alterations of relevance for diagnosis and therapy.
Methods: Tumor DNA and RNA were extracted from FFPE tissue and subjected
to next generation sequencing (NGS) library preparation using a custom
124 gene hybridization capture panel and the 75 gene Archer Oncology
Research Panel, respectively. NGS libraries were sequenced on an
Illumina MiSeq. Results: Thirty-six pediatric PTC cases were analyzed.
Metastases were frequently observed to cervical lymph nodes (29/36,
81\%), with pulmonary metastases less commonly found (10/36, 28\%).
Relapsed or refractory disease occurred in 18 patients (18/36, 50\%).
DNA sequencing revealed targetable mutations in 8 of 31 tumors tested
(26\%), most commonly BRAF p.V600E (n=6). RNA sequencing identified
targetable fusions in 13 of 25 tumors tested (52\%): RET (n=8), NTRK3
(n=4), and BRAF. Mutually-exclusive targetable alterations were
discovered in 15 of the 20 tumors (75\%) with both DNA and RNA analyzed.
Fusion positive PTC was associated with multifocal disease, higher tumor
staging, and higher American Thyroid Association (ATA) risk levels. Both
BRAF V600E mutations and gene fusions were correlated with the presence
of cervical metastases. Conclusions: Targetable alterations were
identified in 75\% of pediatric PTC cases with both DNA and RNA
evaluated. Inclusion of RNA sequencing for detection of fusion genes is
critical for evaluation of these tumors. Patients with fusion positive
tumors were more likely to have features of high-risk disease.%
\end{abstract}%



\sloppy


\textbf{Introduction:}

Papillary thyroid carcinoma (PTC) accounts for 90\% of thyroid
malignancies in children and is especially prevalent among females aged
15-19 years, in whom it is the second most common
malignancy\textsuperscript{1}. Pediatric PTC patients are more likely to
present with metastatic disease and have a higher likelihood of relapse
as compared to their adult counterparts\textsuperscript{2,3}. Standard
therapy for pediatric PTC most often involves total thyroidectomy (+/-
lymph node dissection) as well as radioactive iodine therapy (RAI) for
selected cases. While the prognosis for patients who have undergone
standard therapy is excellent, with a 15-year overall survival of 95\%
\textsuperscript{4}, there is significant morbidity secondary to
surgical procedures and radiation exposure \textsuperscript{5}.

Much of our knowledge regarding the genomic landscape of PTC can be
attributed to The Cancer Genome Atlas (TCGA) project, which
comprehensively analyzed 496 tumors and matched germline samples. This
study revealed that adult PTC is characterized by a relatively quiet
genome with mutually exclusive driving somatic genetic alterations in
the mitogen-activated protein kinase (MAPK) and phosphatidylinositol
3-kinase (PI3K) pathways\textsuperscript{6}. However, as only 5 of the
samples analyzed were from patients less than 19 years of age, the
extent to which these findings are applicable to childhood PTC was not
known. Instead, our understanding of the genomic landscape of pediatric
PTC and corresponding clinical correlations relies on data generated
from smaller pediatric cohorts analyzed by varying molecular methods.

These pediatric studies suggest that point mutations regularly found in
adult PTC, such as \emph{BRAF} V600E, are identified less frequently in
children, whereas gene fusions are more common\textsuperscript{1,7-9}.
Other studies note a correlation between the prevalence of oncogenic
alterations and patient age, attributed to the more frequent presence of
the \emph{BRAF} V600E mutation in older patients\textsuperscript{10}.
Unlike in adults, the presence of a \emph{BRAF} V600E mutation in
pediatric patients does not necessarily correlate with a more aggressive
phenotype\textsuperscript{11,12}. Rather, recent papers have suggested
that pediatric thyroid tumors driven by fusion genes, such as
\emph{RET/PTC1} and \emph{NTRK3-ETV6} , may be correlated with more
invasive disease\textsuperscript{13,14}.

Analysis from the Chernobyl nuclear accident helped delineate some of
the differences between radiation-induced and sporadic pediatric PTCs.
Ricarte-Filho \emph{et al} found that radiation-induced pediatric PTCs
have an increased prevalence of fusion oncogenes compared to sporadic
tumors, with the vast majority of these fusions targeting genes
(\emph{RET} , \emph{NTRK1} and \emph{NTRK3} , \emph{BRAF} ) that
activate the MAPK signaling pathway\textsuperscript{15}.
\emph{RET}rearrangements are also common in sporadic pediatric PTC,
however, with rates ranging from 17\% to 58\%\textsuperscript{13-16}.
Additionally, sporadic pediatric PTCs have been observed to have a
larger number of point mutations compared to radiation-induced PTCs,
particularly in\emph{BRAF} and \emph{RAS} genes\textsuperscript{15,17}.

While each of these studies has provided additional insight into the
nature of pediatric PTC, the molecular methods utilized have varied
significantly and largely relied on targeted analysis of known PTC
genes. We sought to perform more extensive DNA and RNA-based sequencing
of a cohort of clinically-annotated tumors, with a specific focus on
patients with metastatic, relapsed or refractory disease, in order to
identify genetic alterations that might inform improved diagnostic and
therapeutic approaches for these patients.

\textbf{Materials and Methods:}

\emph{Patients}

Potential cases of PTC were identified through review of the Texas
Children's Hospital pathology archives for the years 2005-2016, under a
Baylor College of Medicine Institutional Review Board-approved protocol
for clinical and genomic analysis of rare tumors. A total of 51 PTC
cases were identified for clinical review, with metastatic, relapsed,
and treatment-refractory cases prioritized for analysis (Figure 1).
Ultimately, 40 cases were selected, all of which were diagnostic
(pre-treatment) specimens obtained from either lobectomy or total
thyroidectomy. Of note, 14 of the patients included in this cohort were
also included in a prior study that included more targeted gene testing
(Supplemental Table S1)\textsuperscript{18}. Disease status was
classified using the American Joint Committee on Cancer TNM
classification system for differentiated thyroid carcinoma,
7\textsuperscript{th} edition, and risk level was assessed as outlined
in the American Thyroid Association (ATA) management guidelines for
children with differentiated thyroid cancer\textsuperscript{19}.

\emph{Tumor Sequencing}

A pathologist with specific expertise in thyroid tumors (N.M.Q.)
examined the hematoxylin and eosin-stained tumor slides to identify
representative areas of tumor suitable for molecular testing. DNA and
RNA were extracted from FFPE tumor specimens according to the
manufacturers' protocols utilizing the QIAamp DNA FFPE Tissue Kit
(Qiagen) and the RecoverAll Total Nucleic Acid Isolation Kit for FFPE
(Ambion), respectively. DNA and RNA were quantified by Qubit (Thermo
Fisher Scientific), with RNA undergoing an additional quality assessment
by the quantitative PCR assay PreSeq (ArcherDx).

Of the original 40 cases selected for sequencing, 4 cases were excluded
due to insufficient quantity and/or quality of nucleic acids. Therefore,
36 tumor samples with adequate quantity and quality of nucleic acids for
sequencing were utilized for NGS library preparation. DNA NGS libraries
were generated using a custom hybridization capture panel
(NimbleGen/Roche) designed to capture all coding exons of 124 genes
associated with pediatric solid tumors as well as the \emph{TERT} gene
promoter \textsuperscript{20}. RNA NGS libraries were generated using
the Oncology Research Panel (ArcherDx) that utilizes Anchored Multiplex
PCR chemistry to capture gene fusion events involving 75 recurrently
rearranged cancer genes\textsuperscript{21}. NGS libraries were
sequenced 4- to 6-plex on an Illumina MiSeq using V2 and V3 chemistry,
respectively, and de-mulitplexed using bcl2fastq v2.18.0.12 (Illumina),
achieving approximately 2.2-4.4 million sequencing reads per sample. The
resulting DNA FASTQ files were analyzed using a custom DNA
bioinformatics pipeline that includes alignment by BWA
v0.7.12\textsuperscript{22} (Wellcome Trust Sanger Institute) and
deduplication and QC analysis by Picard v2.9.2\textsuperscript{23}.
Variant calling was completed by NextGENe v2.4.1.2 (SoftGenetics) and
Platypus v0.8.1\textsuperscript{24} (Wellcome Trust Centre for Human
Genetics) for the detection of SNVs and short indels, with variant
annotation by VEP v.89\textsuperscript{25} (Ensembl). Copy number
analysis was performed using CNVkit v0.9.3\textsuperscript{26}. CNVkit
performs circular binary segmentation on the normalized GC and target
capture density bias corrected log2 difference of binned read depths,
using on-target (average bin-size = 300bp) and off-target reads (average
bin-size = 500,000bp), between the tumor sample and reference
pooled-normal peripheral blood samples to identify genome-wide regions
of CNVs. Gene-level deletions and amplifications were then derived using
these copy number segments. RNA FASTQ files were analyzed by the Archer
Analysis suite v4.1.0.6 (ArcherDx) with additional custom wrapper and
text-processing scripts for the detection of gene fusion events.

\emph{Statistical Analysis}

Fisher's exact test was performed to test for the association of tumor
alterations with specific clinical features. Samples were included in
this analysis if a driver alteration, defined as a \emph{BRAF} V600E
mutation or an oncogenic gene fusion, was identified. Samples sequenced
using only one methodology (DNA testing or RNA testing) and without an
identified driver alteration were not considered completely analyzed and
therefore not included in this analysis.

\textbf{Results} :

\emph{Demographic and Clinical Characteristics}

Our cohort consisted of 36 patients with pediatric PTC. Of those, 34
were diagnosed between ages 8-19 years, with the majority of patients
being 15-19 years (Table 1). The male-to-female ratio was 1:3.
Metastasis to cervical lymph nodes occurred in 29 of the 36 patients
(81\%), of which 10 also had lung metastases (28\%). Tumor relapse
post-surgery occurred in 18 of the patients (50\%). In addition, while
the majority of our cohort was comprised of patients without prior
history of irradiation, we did include 8 patients (22\%) who had
undergone radiation therapy, including 2 patients with a history of
Hodgkin lymphoma and 4 patients who received whole body irradiation
prior to bone marrow transplant. The time from prior radiation to
diagnosis of thyroid cancer ranged from 6 to 18.5 years (median 10.9
years). All patients underwent surgical resection of the thyroid gland,
and 34 of 36 received RAI (94\%). Tumors ranged in size between 0.2-7
cm, with a median size of 2.2 cm.

\emph{DNA Mutation Panel Testing}

DNA sequencing for mutation and copy-number profiling was successfully
completed for 31 cases (Figure 1), revealing 10 cancer gene mutations in
9 tumor samples (29\%; Figure 2). The most common mutation identified
was \emph{BRAF} p.V600E, found in six tumors (19\%) including one tumor
with a co-occurring \emph{AKT1} p.E17K mutation, a known hotspot driver
mutation in the PI3K pathway that has been identified in multiple cancer
types, including in PTC\textsuperscript{27-29}. Other alterations
identified included a \emph{PTPN11} hotspot mutation (p.S502T), a
frameshift mutation in \emph{PIK3R1} (p.Y29fs), and a nonsense mutation
in \emph{TP53} (p.Y163X). The genome-wide copy number profiles for these
tumor samples did not reveal high level amplifications or homozygous
losses (Supplemental Figure S1).

\emph{RNA Fusion Panel Testing}

RNA fusion panel testing was successfully completed for 25 cases (Figure
1), revealing fusions in 13 tumor samples (52\%; Figure 2). The majority
of these (n=8) were activating \emph{RET} kinase fusions, including the
well-known \emph{NCOA4-RET} fusions (also known as \emph{RET/PTC3} ) in
5 cases, and \emph{CCDC6-RET} (also known as \emph{RET/PTC1} ) in 3
tumors (Figure 3). Four other tumors harbored \emph{NTRK3} fusions,
including two \emph{ETV6-NTRK3} fusions, one \emph{EML4-NTRK3} fusion,
and a novel\emph{VIM-NTRK3} fusion in one sample. Finally, one tumor was
found to harbor a \emph{MACF1-BRAF} fusion. All fusion genes identified
retained the kinase domains of the oncogenic partners (\emph{RET} ,
\emph{NTRK3} ,\emph{BRAF} ).

\emph{Clinical Associations}

Driver alterations were found to be associated with cervical metastases
or N1 classified lymph node samples (\emph{P =} 0.05, 19/23; Figure 4a).
The presence of an activating kinase gene fusion correlated with higher
ATA risk level (\emph{P} = 0.027, 10/13 high-risk patients; Figure 4b)
and multifocal disease pathology (\emph{P} = 0.039, 13/22 multifocal
tumors; Figure 4c). \emph{BRAF V600E} ~mutations were more frequent in
smaller tumors (T1 and T2), while fusions were more frequent in larger
tumors, T2 and higher (\emph{P} = 0.001; Figure 4d).

\textbf{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,\emph{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\%\textsuperscript{1,11,14,17,30,31}. In contrast with adult PTC,
fusions were detected more than twice as frequently as \emph{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 pathways\textsuperscript{6,7}.

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 \emph{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
novel\emph{VIM-NTRK3} fusion. A second patient was discovered to have
PTC containing an \emph{EML4-NTRK3} fusion. This fusion has been
identified in patients with infantile
fibrosarcoma\textsuperscript{32,33}, 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 targeted\emph{TP53} clinical testing; this
was not detected in our analysis as the DNA panel testing was not
successful. Finally, a \emph{MACF1-BRAF}fusion 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 \textsuperscript{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
a\emph{PTPN11} 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 \textsuperscript{35}. Another patient -- the only
case with two driver alterations detected - was found to have
\emph{BRAF} and \emph{AKT1} mutations at different variant allele
frequencies (37\% and 22\%, respectively) in their tumor sample,
suggesting that the \emph{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 \emph{BRAF} V600E mutation, has shown
antitumor efficacy in adults with progressive metastatic,
RAI-refractory\emph{BRAF} V600E positive PTC\textsuperscript{36,37}.
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 trials\textsuperscript{38}, there are a number of
case reports of children with \emph{BRAF} V600E positive tumors whose
disease responded to treatment\textsuperscript{39-41}.

One recently FDA approved agent is larotrectinib, a highly selective
small molecule inhibitor of the tropomyosin receptor kinase (TRK)
proteins (encoded by kinase genes \emph{NTRK1} , \emph{NTRK2} ,
and\emph{NTRK3} ) which has demonstrated potent antitumor efficacy in
both children and adults with TRK fusion positive
tumors\textsuperscript{42,43}. 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 (\emph{NTRK1} and
\emph{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
later\textsuperscript{43}.

Multiple targeted agents have been developed for adult patients with RET
driven solid tumors. Sorafenib and lenvatinib, multi-kinase inhibitors
that target \emph{RET} , \emph{FLT1, KDR, FLT4} , \emph{PDGFRA,
PDGFRB,}and \emph{KIT} , are FDA approved for adults with PTC and
\emph{RET}fusions \textsuperscript{44}. Sorafenib has been studied in a
phase 2 trial in pediatrics but no PTC patients were enrolled on
study\textsuperscript{45}. 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 patients\textsuperscript{46}. 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 \emph{RET} -mutated medullary thyroid
carcinoma or \emph{RET} fusion positive (and RAI refractory) thyroid
cancer \textsuperscript{47}. Selpercatinib will soon be available to
relapsed pediatric patients through the National Cancer Institute and
Children's Oncology Group jointly sponsored Pediatric MATCH
trial\textsuperscript{48}.

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 \emph{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 \emph{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.

\textbf{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.

\textbf{Author} \textbf{Disclosure} \textbf{Statement} : Dr. Potter
serves as a consultant for Bayer Healthcare Pharmaceuticals.

\textbf{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.

\textbf{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:

\textbf{TABLE 1.} Demographic and clinical characteristics of pediatric
PTC cohort

\textbf{Figure 1.} Flowchart demonstrating cohort selection and
sequencing. PTC, papillary thyroid carcinoma.

\textbf{Figure 2.} Oncoprint illustrating genomic sequencing findings
and selected clinical characteristics.

\textbf{Figure 3.} Fusions detected by RNA panel sequencing (\textbf{A}
)\emph{NCOA4-RET} (\textbf{B} ) \emph{CCDC6-RET} (\textbf{C}
)\emph{ETV6-NTRK3} (\textbf{D} ) \emph{VIM-NTRK3} (\textbf{E}
)\emph{EML4-NTRK3} (\textbf{F} ) \emph{MACF1-BRAF} . All fusions retain
the protein kinase domain.

\textbf{Figure 4.} Distribution of driver alterations (gene fusions
and\emph{BRAF} 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.

\textbf{Supplemental Table S1.} Cohort clinical and genomic data

\textbf{Supplemental Figure 1.} Copy number variant heatmap

\textbf{Hosted file}

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