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\begin{document}

\title{Association of Bethesda category and molecular mutation in patients
undergoing thyroidectomy}



\author[1]{Marco Mascarella}%
\author[2]{Magdalena Peeva}%
\author[3]{Veronique-Isabelle Forest}%
\author[3]{Marc Pusztaszeri}%
\author[4]{Galit Avior}%
\author[3]{Michael Tamilia}%
\author[5]{Alex Mlynarek}%
\author[5]{Michael Hier}%
\author[5]{Richard Payne}%
\affil[1]{Research Institute of the McGill University Health Centre}%
\affil[2]{McGill University Faculty of Medicine}%
\affil[3]{Jewish General Hospital}%
\affil[4]{Hillel Yaffe Medical Center}%
\affil[5]{McGill University}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Objective: The aim of this study was to ascertain the relationship
between Bethesda category and molecular mutation of thyroid nodules in
patients undergoing thyroidectomy. Design: A retrospective cohort of
patients who underwent thyroidectomy following needle biopsy and
molecular profile testing was performed. Setting: Two tertiary care
academic hospitals. Participants: Consecutive patients with a dominant
thyroid nodule who underwent both USFNA and molecular profile testing
followed by thyroidectomy were included in the study. Main Outcome and
Measures: The main outcome was postoperative diagnosis of thyroid cancer
and aggressivity of disease based on histopathological variants, nodal
metastasis or extra-thyroidal extension. Associations between Bethesda
category, molecular mutation and postoperative pathology was assessed
using descriptive analysis and Chi-square testing. Results: 451 patients
were included. 95.9\% (93/97) of patients with a BRAFV600E mutation had
a Bethesda category V or VI (P\textless{}0.001), and all had confirmed
thyroid cancer on postoperative pathology. Those with H, K or N RAS or
EIF1AX mutations, gene expression profiling (GEP) or copy number
alterations showed an association with Bethesda categories III and IV
(P[?]0.01). Those with no identified molecular mutation had a lower
incidence of aggressive thyroid cancer compared to those with an
identified mutation (12.6\% vs 44.3\%, P\textless{}0.01). Conclusion:
BRAFV600E mutations were associated with thyroid cancer subtypes known
to be more aggressive. These findings may help thyroid specialists
better identify aggressive thyroid nodules associated with indeterminate
Bethesda categories.%
\end{abstract}%



\sloppy


\textbf{ABSTRACT}

\textbf{Objectives:} The aim of this study was to ascertain the
relationship between Bethesda category and molecular mutation of thyroid
nodules in patients undergoing thyroidectomy.

\textbf{Design:} A retrospective cohort of patients who underwent
thyroidectomy following needle biopsy and molecular profile testing was
performed.

\textbf{Setting:} Two tertiary care academic hospitals.

\textbf{Participants:} Consecutive patients with a dominant thyroid
nodule who underwent both USFNA and molecular profile testing followed
by thyroidectomy were included in the study.

\textbf{Main Outcome and Measures:} The main outcome was postoperative
diagnosis of thyroid cancer and aggressivity of disease based on
histopathological variants, nodal metastasis or extra-thyroidal
extension. Associations between Bethesda category, molecular mutation
and postoperative pathology was assessed using descriptive analysis and
Chi-square testing.

\textbf{Results:} 451 patients were included. 95.9\% (93/97) of patients
with a BRAFV600E mutation had a Bethesda category V or VI
(P\textless{}0.001), and all had confirmed thyroid cancer on
postoperative pathology. Those with H, K or N RAS or EIF1AX mutations,
gene expression profiling (GEP) or copy number alterations showed an
association with Bethesda categories III and IV (P[?]0.01). Those with no
identified molecular mutation had a lower incidence of aggressive
thyroid cancer compared to those with an identified mutation (12.6\% vs
44.3\%, P\textless{}0.01).

\textbf{Conclusion:} BRAFV600E mutations were associated with thyroid
cancer subtypes known to be more aggressive. These findings may help
thyroid specialists better identify aggressive thyroid nodules
associated with indeterminate Bethesda categories.

\textbf{Key Points:}

Almost all patients with a BRAFV600E mutation have a Besthesda
diagnostic category of V or VI. Patients with a RAS-type mutation were
categorized as Bethesda III or IV. Preoperative molecular testing of
dominant thyroid nodules may help guide management as patients with
BRAFV600E mutations are likely to have more aggressive disease. Patients
with no molecular mutation identified tended to have less aggressive
thyroid cancer.



\textbf{INTRODUCTION}

The widespread use of medical imaging has contributed to the increased
incidence of thyroid nodules detected and incidental thyroid cancer
diagnosed. {[}1{]} Studies suggest that the number of ultrasound guided
fine needles aspirations (USFNAs) performed has doubled and the number
of thyroid surgeries performed has increased by nearly 30\% over the
last decade. {[}1{]} Currently, USFNA is the standard diagnostic method
for thyroid nodules. Although USFNA specimens are categorized according
to the Bethesda classification, 24\%-57\% of thyroid USFNAs have an
indeterminate result. {[}2-4{]} As a consequence, thyroid lobectomy is
often performed for diagnostic and potentially therapeutic purposes. In
roughly 60\% of patients with indeterminate cytology, the thyroid nodule
is benign. {[}4{]} In these patients, molecular testing of thyroid
nodules has become an adjunct to USFNA biopsy to reduce the likelihood
of unnecessary diagnostic surgery. {[}5{]}

Mutations that are uncovered with molecular testing of thyroid nodules,
namely BRAF V600E or RAS-type mutations, can assist with uncovering the
phenotype of the nodule (benign versus malignant) as well as whether the
tumor may be an aggressive variant. Mutations of the BRAF V600E gene,
found in 35-70\% of papillary thyroid carcinomas (PTC), lead to the
activation of BRAF kinase and chronically stimulate the MAPK signaling
pathway. {[}7,8{]} The BRAF V600E mutation has been strongly associated
with classic and tall cell variant of PTC. {[}8{]} This mutation has
also been shown to correlate with aggressive tumor characteristics
including multifocality, extrathyroidal extension, regional or distant
metastases and an advanced tumor stage at presentation. {[}8-15{]}
Moreover, studies have shown that BRAF V600E mutations are associated
with tumor recurrence and poor prognosis. {[}14,15{]} Mutation in the
three RAS genes (HRAS, KRAS, and NRAS) are found in follicular adenomas
and follicular carcinomas, as well as in the follicular variant of PTC
and more recently for non-invasive follicular neoplasm with
papillary-like nuclear features (NIFTP). {[}7{]} RAS mutations result in
permanent RAS activation which chronically stimulate the MAPK and
PI3K/AKT signaling pathways. {[}7{]} They are associated
follicular-patterned neoplasms, with less prominent nuclear features of
PTC, more frequent encapsulation and a lower rate of lymph node
metastases than those with BRAF V600E mutations. {[}16-18{]} According
to a recent study, RAS mutations in malignant thyroid nodules, when
isolated, are associated with low-risk features and a favorable
prognosis. {[}16-18{]}

In this study, the association between Bethesda category and molecular
mutation assessed with ThyroSeq V3 or ThyGeNEXT in patients with thyroid
nodules undergoing thyroidectomy is explored to better tailor patient
diagnosis and treatment.

\textbf{METHODS}

A retrospective multicenter cohort study of patients who underwent
thyroid surgery at two tertiary care academic hospitals from January 1,
2016 to December 1, 2019 was performed. Consecutive patients with a
dominant thyroid nodule who underwent both USFNA and molecular profile
testing (ThyGeNEXT, Interpace Diagnostics, Parsippany, NJ, USA or
ThyroSeq v3, UPMC, Pittsburgh, PA, USA) followed by thyroidectomy were
included in the study. Indications for molecular profile testing of
thyroid nodules was for cytologically indeterminate Bethesda category
(III, IV, V) or discordance cytology results with ultrasound findings.
Patients who did not undergo thyroidectomy as well as pediatric patients
were excluded from the study. This study was approved by the Research
Ethics Committee at the tertiary care center (REB 2019-1532).

Clinical data including age, sex, Bethesda category of the dominant
nodule, and if a molecular mutation was identified were collected. In
addition, the extent of thyroidectomy performed (hemi, total or
completion thyroidectomy) and postoperative histopathology were noted.
Postoperative histopathology was classified as benign disease, malignant
or non-invasive follicular thyroid neoplasm with papillary-like nuclear
features (NIFTP). Furthermore, PTC of less than 10mm were classified as
microcarcinomas. Additionally, aggressive thyroid cancer was defined as
any evidence of thyroid carcinoma variants (tall cell, hobnail, solid,
diffuse sclerosing, anaplastic or poorly differentiated), clinical
evidence of extrathyroidal extension or central neck metastasis.

The STROBE guidelines were used for reporting data. Descriptive
statistics to characterize the cohort was performed. The association
between Bethesda category and type of molecular mutation of dominant
thyroid nodule on USFNA was compared using Chi-square testing. Logistic
regression was performed to quantify the measure of association between
Bethesda category and type of molecular mutation of the dominant thyroid
nodule. Also, comparison of Bethesda category and molecular mutation to
postoperative pathology was performed using Chi-square testing.

\textbf{RESULTS}

A total of 451 patients with a thyroid nodule undergoing USFNA,
molecular profile testing and surgery were included in the study.
Baseline patient characteristics are shown in Table 1. Most patients
were female and had a mean dominant thyroid nodule size of 2.1cm
(standard deviation of 1.1cm). 325 patients (72.1\%) underwent thyroid
lobectomy. The overall prevalence of malignancy, excluding cases of
NIFTP, was 78.5\% of the cohort. The prevalence of postoperative
malignancy by USFNA Bethesda category was 56.7\% (55/97) for Bethesda
III, 75.7\% (109/144) for Bethesda IV, 80.1\% (86/104) for Bethesda V
and 100\% for Bethesda VI lesions. 138 patients showed evidence of
aggressive thyroid cancer of which 79 had central neck lymph node
metastasis.

Select mutations of dominant thyroid nodules were strongly associated
with specific Bethesda categories (Table 2). Almost all BRAF V600E
mutations were associated with Bethesda V and VI (P\textless{}0.0001).
Only 4 of 97 (4.1\%) nodules with BRAF V600E mutations had a Bethesda
category III or IV, all of which were malignant on postoperative
pathology. RAS and EIF1AX mutations, copy number alterations and GEP
were strongly associated with Bethesda categories III and IV (P[?]0.01).
Figure 1 illustrates the above findings by displaying the frequency of
Bethesda categories among different molecular mutations. Thyroid nodules
with an identified molecular mutation were more likely to harbour
thyroid cancer. Specifically, nodules with a BRAF V600E, TERT or RET
mutation were most likely to harbour an aggressive thyroid cancer (Table
3).

Other identified molecular mutations were less associated with
aggressive disease. NIFTP was mostly associated with RAS-type mutations.
No molecular mutations were identified in 43.9\% of patients; of those
patients, 128 had a thyroid cancer. Overall, those with no identified
molecular mutation had a lower incidence of aggressive thyroid cancer
compared to those with an identified mutation (12.6\% vs 44.3\%,
P\textless{}0.01). When classified according to malignancy on
postoperative pathology, statistically significant associations to
Bethesda category by molecular mutation were identified in patients with
BRAF V600E, RAS, PTEN and EIF1AX mutations, copy number alterations and
GEP (Supplemental Table 1). The presence of an identified molecular
mutation on USFNA predicted postoperative malignancy.

\textbf{DISCUSSION}

Our study shows that thyroid nodules with BRAF V600E mutations are
strongly associated with Bethesda categories V and VI, whereas nodules
with a RAS or EIF1AX mutation, copy number alteration or GEP are more
commonly associated with Bethesda categories III and IV. Certain
mutations within dominant thyroid nodules, specifically those with a
BRAF V600E, TERT or RET mutation, were associated with thyroid cancer
subtypes known to be more aggressive. An increasing number of studies
have concluded that molecular testing of thyroid nodules has a
significant diagnostic as well as prognostic value and can help improve
management of patients. {[}19-25{]} Other studies, however, question the
clinical utility and cost-benefit analysis of molecular testing.
{[}20,24{]} The Afirma Gene Expression Classifier (Afirma\selectlanguage{ngerman}®, Veracyte,
Inc., South San Francisco, CA) measures mRNA transcript levels of 142
genes and has shown to have a high sensitivity of 91\% and a moderate
specificity of 68\%. {[}18,22{]} ThyGeNEXT (ThyGeNEXT®, Interpace
Diagnostics, PDI Inc, Parsippany, NJ) and ThyroSeq v3 (ThyroSeq®~joint
partnership between UPMC and CBLPath, Pittsburgh, PA) are two
comprehensive tests, designed to improve the overall accuracy of
molecular testing, with sensitivity of 89\% and 94\% and specificity of
85\% and 82\%, respectively. {[}20,24{]} The data regarding the clinical
utility of molecular testing in patients with a Bethesda V and VI
thyroid nodules remains unclear. {[}24-28{]} The value in molecular
testing for such thyroid nodules lies not in the fact that this tool can
predict malignancy. Further characterizing Bethesda V and VI thyroid
nodules with molecular testing can identify patients with higher-risk
disease (ex. presence of BRAF V600E, TERT) and may offer clinicians
better perioperative patient counselling and prognostication.

The American Association of Endocrine Surgeons 2020 guidelines strongly
recommend molecular testing as a diagnostic adjunct for cytologically
indeterminate thyroid nodules if the information gained will affect
clinical care. {[}20{]} Molecular testing may help clinicians with the
management of thyroid nodules by identifying the nature of indeterminate
lesions with mutations highly specific for malignancy such as BRAF
V600E, RET or TERT, leading to improved diagnostic accuracy and
potential reduction in the number of completion thyroidectomies
performed. {[}22-24{]} Moreover, the extent of surgery performed on
patients with a cytologic diagnosis of Bethesda category V or VI is
related to the risk of recurrence and determined by the tumor size,
radiographic features, presence of adenopathy and contralateral thyroid
disease. {[}20{]} In this study, 44\% of patients with a Bethesda
category V or VI nodule harboured a BRAF V600E mutation. Given the
relatively high prevalence of this mutation in Bethesda V/VI lesions,
identifying this mutation can stratify patients into a distinct higher
risk group. {[}8-15,22{]} Preoperative knowledge of a BRAF V600E
mutation can help clinicians recognize patients with high risk thyroid
nodule and potentially offer personalized patient counselling and
treatment planning, even in nodules of smaller size. In the four
patients with a less than 2 cm Bethesda III/IV thyroid nodule,
identification of a BRAF V600E mutation preoperatively allowed for a
total thyroidectomy with elective neck dissection to be performed. All 4
patients showed aggressive thyroid cancer features: 2 with central neck
metastasis and 2 with solid or tall cell variants of papillary thyroid
cancer.

The management of patients with thyroid nodules that are indeterminate
on cytology often consists of a diagnostic lobectomy or active
surveillance based on clinical factors including the nodule's size,
enlargement on ultrasound, suspicious ultrasound features, clinical and
family history, history of radiation exposure, and patient's
preferences. {[}5,20, 25{]} Yip et al. show that molecular testing of
thyroid nodules can also be useful in defining the extent of surgery for
patients with low risk mutations to thyroid lobectomy. {[}24{]}
Understanding that RAS-type mutations, which are shown to have a
favorable prognosis when isolated, are commonly associated with Bethesda
III and IV suggests that patients with an isolated RAS mutation may
potentially be more conservatively treated with lobectomy. {[}25{]} The
current results show that BRAF V600E mutations have been identified in
4.1\% of indeterminate nodules which highlights the need for molecular
testing to identify nodules of higher risk of malignancy. In this
context, it may be warranted for the thyroid specialist to consider
mutation testing analysis for specific higher-risk mutations such as
BRAF V600E, TERT or RET as a means to preoperatively risk stratify
patients. That being said, 128 patients did not have an identified
molecular mutation with 64.6\% proving to be malignant. In this subgroup
of patients, only 12.6\% of patients showed evidence of aggressive
variants of thyroid cancer or central neck metastasis and is
significantly lower in prevalence compared to patients with an
identified molecular mutation. Additional understanding of thyroid
carcinogenesis, presence of co-mutations and heterogeneity of nodules is
needed to better allow clinicians to predict risk of malignancy and
tumor aggressivity. {[}25{]}

Certain limitations to this study are related to the retrospective
design and inclusion of patients undergoing molecular testing and
thyroidectomy. By doing so, the overall prevalence of malignancy was
elevated due to the selection of cases with more suspicious disease.
Also, the indication for molecular profile testing was at the surgeon's
discretion. Expenses related to molecular profile testing of thyroid
nodules is currently not covered in the Quebec health system; this may
introduce a selection bias for patients who have the financial means to
access it. Lastly, validation with larger prospectively collected data
is needed to generalize results. That being said, the number of patients
included and analyzed in this study offers a measure of diagnostic
precision and clinical utility of using molecular test for thyroid
nodules.

\textbf{CONCLUSION}

Select molecular mutations in thyroid nodules were associated with
specific Bethesda diagnostic categories on cytology. BRAF V600E
mutations were mostly associated with Bethesda categories V and VI,
whereas RAS and EIF1AX mutations, copy number alterations, and GEP were
related to Bethesda categories III and IV. This information may guide
clinicians in the prediction of molecular mutations of thyroid nodules
and potentially improve management.



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