Pathology - Research and Practice 251 (2023) 154842
Contents lists available at ScienceDirect
Pathology - Research and Practice
journal homepage: www.elsevier.com/locate/prp
Functional loss of tumor suppressor genes detected by loss of
heterozygosity, but not driver mutations, predicts aggressive lymph node
status in papillary thyroid carcinoma
Sydney Finkelstein a, Venkata Arun Timmaraju a, Shabnam Samankan b, Quinn O’Malley c,
Danielle Kapustin c, d, Sarah Spaulding c, Monica Xing c, Ammar Matloob b, John Beute c,
Gabriella Seo c, Michael Saturno c, Lily Greenberg c, Lauren Wein c, Camilo Gonzalez-Velazquez c,
Scott Doyle e, Jonathan Levine a, Mark Urken c, d, Margaret Brandwein-Weber b, *
a
Interpace Diagnostics, Interpace Biosciences, Pittsburgh, PA, United States
Department of Pathology, Icahn School of Medicine, Mount Sinai Health System, New York, United States
c
THANC (Thyroid, Head and Neck Cancer) Foundation, 10 Union Square East, New York, NY 10003, United States
d
Department of Otolaryngology — Head and Neck Surgery, Icahn School of Medicine, Mount Sinai Health System, New York, NY, United State
e
Department of Pathology and Anatomical Sciences, University at Buffalo, Jacobs School of Medicine, Buffalo, NY, United States
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Papillary thyroid carcinoma
Loss of heterozygosity
LOH
ATA
Aggressive lymph nodes
Background: Recognizing aggressive tumor biology is essential to optimizing patient management for papillary
thyroid carcinomas (PTC). Aggressive lymph node (ALN) status is one feature that influences decision-making.
We evaluated genomic deletions in regions of tumor suppressor genes, detected by loss of heterozygosity
(LOH) analysis, to understand causal alterations linked to thyroid cancer aggressiveness and to serve as a mo­
lecular diagnostic biomarker for ALN status.
Methods: We analyzed 105 primary PTC enriched for patients with ALN (64% with, 36% without). We also
analyzed 39 positive lymph nodes (79% with, 21% without ALN). LOH was determined using a panel of 25
polymorphic microsatellite alleles targeting 10 genomic loci harboring common tumor suppressor genes.
Additionally, ThyGeNEXT® and ThyraMIR® assays were performed.
Results: LOH was detected in 43/67 primary PTC from patients with ALN status, compared with only 5/38
primary PTC without ALN (minimal metastatic burden) (P=0.0000003). This is further supported by post hoc
analyses of paired primary and metastatic samples. Paired samples from patients with ALN are more likely to
harbor LOH, compared to the ALN negative group (P=0.0125). Additionally, 12/31 paired samples from patients
with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor
samples. No association was seen between ALN and mutational, translocation, or microRNA data.
Conclusions: LOH detected in primary PTC significantly predicts ALN status. Analysis of paired primary and
metastatic samples from patients with / without ALN status further supports this relationship. The acquisition of
LOH at additional loci is common in lymph nodes from patients with ALN status.
Simple summary: A subset of patients with papillary thyroid carcinoma (PTC) will develop recurrent disease. One
known predictor of recurrence is the American Thyroid Association category “Aggressive Lymph Node” (ALN)
disease, considering metastatic burden. Loss of heterozygosity (LOH) — chromosomal loss in regions of tumor
suppressor genes — has yet to be investigated as a possible mechanism driving ALN status in PTC. The ability to
predict ALN status prior to surgery can guide the extent of surgery and postoperative treatment options. We
found that paired samples from patients with ALN are more likely to harbor LOH, compared to patients without
ALN disease. 38% of patients with ALN demonstrated additional or different LOH loci in metastatic samples
compared to the primary tumor samples. LOH complements current molecular analysis of thyroid cancer when
searching for evidence of aggressive biology.
* Corresponding author.
E-mail address: Margaret.brandwein@mountsinai.org (M. Brandwein-Weber).
https://doi.org/10.1016/j.prp.2023.154842
Received 24 August 2023; Accepted 30 September 2023
Available online 4 October 2023
0344-0338/© 2023 Elsevier GmbH. All rights reserved.
S. Finkelstein et al.
Pathology - Research and Practice 251 (2023) 154842
lymph node (ALN) status and long-term follow-up, from 2010 onward.
Aggressive lymph node (ALN) status considers metastatic burden and is
a more meaningful indicator of aggressive behavior, as compared to
binary lymph node status (negative or positive). It is defined as either
more than five positive lymph nodes, one or more positive lymph node
larger than 3 cm, or four or more positive lymph nodes with extranodal
extension (ENE). Inclusion criteria included availability of all pathology
slides and adequacy of tumor submission for histology. PTCs up to 4 cm
were routinely submitted in entirety. Recuts from corresponding
paraffin blocks were performed if slides were unavailable or the his­
tology was suboptimal. A cohort of 105 patient samples with known ALN
status (positive ALN= 67, negative ALN= 38) were submitted for blin­
ded molecular analysis. Primary thyroid cancer tissue was manually
microdissected from unstained 4-micron thick recut tissue sections of
the formalin-fixed paraffin-embedded tissue blocks under stereomicro­
scopic guidance (SZ-PT, Olympus microscopes) using the hematoxylineosin stained tissue section. Sufficient nucleic acid for mutational,
miRNA expression, and loss of heterozygosity (LOH) analysis was ob­
tained from manual microdissection of eight serial, four micron thick
unstained recut tissue sections. Based on the results of the tumor pri­
mary molecular analysis, a subset of formalin-fixed paraffin-embedded
positive lymph node samples from 39 patients (31 with ALN, 8 without
ALN) were subsequently randomly selected for post-hoc molecular
analysis.
1. Introduction
An understanding of cancer biological aggressiveness can be clini­
cally impactful in optimizing patient management, especially at the
outset of a cancer diagnosis, enabling more informed planning of sur­
gery and the use of other therapeutic modalities. A small subset of pa­
tients with well-differentiated thyroid carcinomas will pursue an
aggressive clinical course [1]. Early diagnosis for patients facing
recurrence and or metastatic spread is a critical means to achieve the
best outcome. The American Thyroid Association (ATA) has developed
the risk of recurrence (ROR) classification system for surgical and
postoperative decision paradigms [2]. The ATA-ROR system describes
aggressive lymph node (ALN) status in terms of the cumulative meta­
static burden, taking into account 1) the total number of positive lymph
nodes, 2) metastatic lymph node size, and 3) presence of extranodal
metastatic tumor invasion. Specifically, ALN status is defined as either
more than five positive lymph nodes, one or more positive lymph node
larger than 3 cm, or four or more positive lymph nodes with extranodal
extension. The ATA-ROR designation of ALN status has been shown to be
a more clinically meaningful indicator of aggressive behavior than
designating lymph node status as either negative or positive [2,3]. We
selected the ALN phenotype as the study outcome for two reasons.
Firstly, it is generally challenging to correlate biomarkers with adverse
outcome for PTC patients as initial local recurrence can develop decades
after primary treatment. A large cohort size with a long mean follow-up
is required to adequately power an outcome study. Therefore we framed
our investigation by enriching the study cohort with a known surrogate
for poor outcome, namely ALN status. Secondly, the finding of ALN
impacts therapeutic options. We sought to better understand the mo­
lecular basis of ALN phenotypes and, more particularly, to better un­
derstand its role based on the molecular changes detected in the primary
thyroid malignancy so this prognostic approach may be considered
when planning initial oncologic therapy.
Thyroid tumorigenesis involves the stepwise accumulation of mo­
lecular alterations that ultimately determine phenotypic expression
including tumor biological aggressiveness [4]. Genomic as well as epi­
genomic changes have been shown to contribute to thyroid cancer
development and progression [5]. While the greatest attention has
centered on thyroid cancer-associated gene families and oncogenic
translocations, we and others have shown that microRNA (miRNA)
classification, an important epigenomic controller of phenotypic
expression, together with defects in other cellular mechanisms play an
important causative role in the development, progression and clinical
expression of thyroid cancer [6–8]. Tumor suppressor gene (TSG) loss is
an important alteration that has been reported in thyroid cancers,
generally regarded to be a later event in cancer progression [9]. We and
others have previously shown that loss of heterozygosity (LOH) is
indicative of the second of the Knudsen’s two step inactivation of tumor
suppressor gene function [10]. Published data support the notion that
LOH tends to be associated with relatively greater thyroid cancer bio­
logical aggressiveness [9]. Here we explore the relationship between
LOH and ALN, which supports the hypothesis that the role of LOH in
chromosomal regions close to known tumor suppressor gene loci can be
a biomarker of thyroid cancer aggressive etiology. More specifically, we
show that detection of LOH in the primary thyroid malignancy is asso­
ciated with ALN status, in keeping with a causal role leading to
aggressive biology in this important subset of thyroid cancer patients.
2.1. Molecular analysis
Three molecular platforms were used for analysis in this study: 1)
next-generation sequencing (NGS, Illumina) genomic mutational panel,
detecting gene sequence variations and/or translocation mutations; 2)
miRNA classifier, which includes biomarkers for malignancy risk strat­
ification [9]; and 3) fragment analysis by means of capillary electro­
phoresis of TSG-related LOH. All molecular testing was performed using
standard clinical procedures for the ThyGeNEXT® mutation panel and
ThyraMIR® miRNA risk classifier commercial tests, and for LOH anal­
ysis at Interpace Diagnostics as previously described [6–14]. The
expanded mutation panel test (ThyGeNEXT) utilizes targeted NGS
(Illumina) to detect DNA-based gene mutation variants and mRNA
fusion transcripts, listed in Table 1. For a positive variant call, a spec­
imen was required to contain at least 3% BRAF V600E, 10% GNAS, or
5% other individual DNA variants in the panel. All samples required
predominance of PAX8 and NKX2.1 messenger RNA (mRNA) expression
to confirm thyroid follicular cell content. A minimum coverage of 1000
copies had to be met for each targeted genomic region or fusion product.
All mutational testing was performed in duplicate with the specific
sequence alteration confirmed in both replicates. Microdissected sam­
ples typically are composed of a chimeric mixture of cancers cells in
Table 1
Genomic and microRNA Targets for ThyGeNEXT and ThyraMIR Testing.
Expanded Mutation Panel (ThyGeNEXT)
2. Materials and methods
This study was approved by the Mount Sinai Health System (MSHS)
Institutional Review Board. Study inclusion criterion was patients
treated at MSHS with primary surgery for PTC with curative intent. All
eligible patients from 2017 onward were entered sequentially into this
study. In addition, personal patient databases from one of the authors,
MLU, were used to enrich this cohort for PTC patients with aggressive
DNA variant
Fusions (N) and mRNA
microRNA Risk Classifier
(ThyraMIR)
miRNA
BRAF†
ALK
GNAS
HRAS
KRAS
NRAS
PIK3CA
PTEN
RET‡
TERT promoter‡
BRAF (3)‡
ALK (2)
NTRK (8)
PPARg (5)
RET (14)‡
THADA (5)
NKX2.1
PAX8
TBP
USP33
miR-31–5p
miR-29b-1–5p
miR-138–1–3p
miR-139–5p
miR-146b-5p
miR-155
miR-204–5p
miR-222–3p
miR-375
miR-551b-3p
BRAF V600E is a strong driver mutation, while BRAF K601E is a weak driver
mutation
‡
Strong driver mutation
mRNA (messenger RNA)
†
2
S. Finkelstein et al.
Pathology - Research and Practice 251 (2023) 154842
which a subset of microdissected cancer cells contain these mutations
usually acquired later in tumorigenesis. Analytical validation of the
ThyGeNEXT assay has been previously reported [13]. The miRNA risk
classifier test (ThyraMIR) was performed using a validated panel of 10
specific miRNAs tested using quantitative reverse transcription poly­
merase chain reaction (RT-PCR) (QuantStudio) to evaluate miRNA
expression levels in relation to one another. The panel of miRNAs tested
is listed in Table 1.
LOH analysis was carried out on an analytical and clinical validated
panel of 25 polymorphic microsatellite alleles situated in close prox­
imity to common tumor suppressor genes (TSG) at 10 genomic loci [9].
LOH markers target the following genomic loci with corresponding
cancer genes: 1p (LMYC), 3p (VHL, OGG1), 5q (APC), 9p (CDKN2A,
CDKN2B), 10q (PTEN), 17p (TP53), 17q (RNF43), 18q (SMAD4, DCC),
21q (PSEN2) and 22q (NF2). LOH analysis was based on fragment
analysis separated by length using capillary electrophoresis (Applied
Biosystem) targeted polymorphic microsatellites located within or in
proximity of well characterized tumor suppressor genes [9]. Micro­
satellites were PCR amplified using flanking fluorescent labeled oligo­
nucleotides (Integrated DNA Technologies) according to manufacturer’s
recommendations (Applied Biosystems). LOH analysis utilized known
homozygous microsatellite allele controls assembled to meet pre­
determined alleles admixtures [9]. Specimens consisted of unstained
four-micron thick formalin-fixed paraffin-embedded tissue sections from
94 patients with primary carcinomas, and random subset of positive
lymph nodes from 34 patients. All sample slides were microdissected
under stereoscopic guidance followed by extraction of total nucleic acid
as previously described [11–14]. Analytical validation of LOH analysis
was based on known admixtures of polymorphic microsatellite alleles
using blood samples of subjects with previously determined non­
informative single alleles. Laboratory staff performing the molecular
analysis were performed blinded to ALN status and clinical patient
outcome.
2.3. Statistics
Chi-square tests (cell count ≥ 5) and Fisher’s exact tests (cell count <
5) were used to assess the relationship between categorical variables.
The Freeman-Halton extension of Fisher’s exact test was used to
compute two-tailed probabilities for distribution values in 2 × 3 con­
tingency tables. Kruskal–Wallis test and Wilcoxon rank-sum test were
performed to compare the continuous variables among three groups and
two groups, respectively. Kaplan–Meier outcome analyses, Log-rank
tests and Cox proportional hazards analyses were performed for
disease-free survival (DFS). All reported P values were two-sided; sta­
tistical significances were claimed at ≤ 0.05.
3. Results
3.1. Patient cohort and outcome
This cohort consists of 105 patients (38 men, 67 women), ages 16 –
82 (mean 44 years). As we are interested in predictive biomarkers of
ALN status, the cohort was deliberately enriched for patients of interest.
Thus, 67 patients (64%) were ALN positive, and 38 patients (36%) were
negative for ALN status (including cNx patients) for a ratio of 2:1. By
comparison, in our larger published cohort of 347 PTC patients, ALN
status represented 32% of the total cohort for a ratio of 1:2 [15]. Post
treatment disease status was known for 93 patients (mean follow-up 55
months): two patients manifested disease persistence in regional lymph
nodes (2 and 3 months) and 10 patients developed recurrence in
regional lymph nodes (12–60 months, mean 26). There were no cases of
biochemical recurrence only.
3.2. Molecular analysis including LOH determination
One or more TSG-related LOH mutations was detected in 48 of 105
patients (46%) (Table 2). All genomic loci tested (10 loci) were found to
harbor at least one detectable LOH mutation. The number of LOH
markers varied from one to four; 35 patients (73%) had one marker, nine
patients (19%) had two markers, two patients (4%) had three markers,
2.2. Outcomes
Patients were treated according to current American Thyroid Asso­
ciation (ATA) guidelines for extent of surgery and administration of
adjuvant radioiodine therapy [2]. Patients were monitored post­
operatively with ultrasound and biochemical parameters, in line with
ATA guidelines. Disease persistence is defined as newly recognized
disease less than six months after a total thyroidectomy. This reflects the
reality that suboptimal preoperative ultrasound or intraoperative eval­
uation may lead to incomplete surgery and therefore disease persistence.
New disease manifesting six months or later after total thyroidectomy is
classified as recurrence. For patients classified as recurrence-free, a
minimum of six months follow-up was required; disease-free patients
with fewer than six months of follow-up were excluded from outcome
analysis. The last available serum thyroid stimulating hormone (TSH),
thyroglobulin and thyroglobulin antibody titers were abstracted; pa­
tients with significantly elevated serum thyroid cancer markers, but no
pathological/radiographic evidence of recurrent PTC, were classified as
"biochemical recurrence." Kaplan–Meier analysis measured disease-free
survival from date of surgery to either first date of disease progression,
or last known disease-free date.
Clinicopathologic, demographic, and outcome data were stored in a
HIPAA (Health Insurance Portability and Accountability Act)-compliant
Microsoft Access database. The following collected data is part of this
analysis: gender, age, tumor size (centimeter (cm)), degree of extra­
thyroid extension (ETE: intrathyroidal, microscopic ETE, histologic
spread to strap muscles, or pT4 disease), extranodal extension (ENE),
ALN status, tumor multifocality, tumor necrosis, vascular invasion,
perineural invasion, and lymphatic tumor emboli.
Table 2
Summary of Molecular Data on 105 Primary Papillary Thyroid Carcinomas.
Mutational Status
None
BRAF V600E
BRAF V600E & TERT
CCDC6::RET
NCOA4::RET
ETV6::NTRK3
AGK::BRAF
KRAS Q61K
BRAF V600E &
PARG::BMS1
Failed
MicroRNA expression level
Level – 1
Level – 2
Level – 3
Specific chromosomal loci and
associated TSG
3p (VHL, OGG1)
17q (RNF43, NMEI1)
1p (RUNX3, CMM1, LMYC)
10q (PTEN, MXI1)
22q (NF2)
5q (MCC, APC)
9p (CDKN2A, CDKN2B)
17p (TP53)
18q (SMAD4, DCC)
21q (TFF1, PSEN2)
3
n
13
62
15
6
2
2
1
1
1
Loss of Heterozygosity Status
Primary PTC
n
No LOH
Any LOH
57
48
1 LOH detected
2 LOH detected
3 LOH detected
4 LOH detected
35
9
2
2
2
1
1
103
Frequency (n)
18
12
9
6
5
4
4
4
3
1
S. Finkelstein et al.
Pathology - Research and Practice 251 (2023) 154842
and two patients (4%) had four LOH markers (Table 2). Chromosomal
locus 3p was the most commonly affected genomic site for LOH (single
loss, n = 11, multiple losses, n = 7), followed by loss of 17q (single loss,
n = 7, multiple losses, n = 5) (Table 2). No LOH was detected in 57
(54%) of primary tumors for this 105-patient cohort.
Mutational change was frequent in this cohort enriched for aggres­
sive disease (Table 2). Not surprisingly, BRAF V600E-like, “strong”
driver type mutations, dominated mutation genotypes in the thyroid
follicular lining cell malignancies. BRAF V600E was the most common
mutation detected, either as the sole mutation 62/102 (61%) or as BRAF
V600E plus TERT promoter mutation (15/102, or 15%, Table 2). RET
fusions were present in eight patients (CCDC6::RET = 6, NCOA4::RET =
2). Remaining cases showed a variety of mutational changes and mRNA
fusions (AGK::BRAF, KRAS Q61K, NRAS, ETV6::NTRK3, and NTRK3 plus
TERT) (Table 2). Only one cancer manifested “weak” (RAS-like) driver
type mutations consisting of KRAS point mutations. Nine patients lacked
detectable mutational change in their primary thyroid cancers. Muta­
tional analysis failed in two cases; these were still included in this cohort
as LOH data is available for these patients.
miRNA risk classification indicated positive status in all but two
thyroid cancer samples (Table 2) in keeping with the neoplastic lesion
having undergone malignant transformation. Positive overexpression
recognizes two levels as previously described [6] consisting of a lower
state of overexpression associated with greater sensitivity for detection
of malignancy (Level-2) and a higher state of overexpression associated
with greater specificity for detection of malignancy (Level-3). All pa­
tients with positive ALN status showed Level-3 strong positive over­
expression of miRNA. Of note, all mutation negative thyroid cancers in
this series exhibited strong positive, Level-3 miRNA overexpression
(Table 2).
Table 3 demonstrates the relationship between LOH and phenotypic
features of aggressive biological behavior. LOH was detected in the
majority of primary tumors of patients with ALN status (43/67, 64%). In
contrast, LOH was uncommonly detected in primary tumors of patients
with non-ALN status (5/38, 13%). Therefore, LOH detected in primary
thyroid cancers is strongly predictive of ALN status (P=0.0000003). The
same was true for two components of ALN: extranodal extension
(P=0.000007), and metastases involving more than five lymph nodes
(P=0.000022). These data lead to post-hoc analysis of positive cervical
lymph node samples from 39 randomly selected patients (31 with ALN, 8
negative ALN status). Table 4 summarizes the paired primary cancer and
lymph node LOH and mutational data stratified for ALN status. Paired
primary and lymph node samples from patients with ALN more
commonly harbored LOH (19/31 or 61%), compared to ALN negative
group samples (1/8 or 13%, P=0.0125, two-tailed Fisher exact test).
Accumulation of additional or different LOH loci in positive lymph node
samples, compared with primary tumors, is common in the ALN+ group.
In contrast, the mutational driver status remained the same in paired
primary and metastatic samples in all but one ALN positive patient
(primary PTC no mutation, metastatic carcinoma BRAF V600E+,
Table 4). No association was seen with LOH status and patient age (cutpoint 50 years), gender, tumor size, perineural invasion, lymphatic
invasion, vascular invasion, tumor multifocality, necrosis, or degree of
extrathyroid extension on Student t-test.
BRAF V600E point mutation, with or without TERT promoter region
mutational change was acquired by the majority of thyroid cancers in
this series. Gene mutations were not associated with either LOH status,
ALN status, or extrathyroid extension, with respect to BRAF V600E point
mutation plus TERT promoter mutation versus BRAF V600E point mu­
tation alone, or versus other or no mutations. Positive status of the
miRNA-risk classifier, a general indicator of thyroid nodule malignant
transformation, did not associate with ALN status.
With respect to outcome prognostication, Kaplan–Meier analysis was
significant for decreased disease-free survival for BRAF V600E with
TERT promoter mutation versus BRAF V600E, (P=0.002, HR 16.01, 95%
CI 2.7, 92.9). Trends were seen for decreased disease-free survival and
LOH (P=0.09, HR 3.1, 95% CI 0.8, 11.9) and also for ALN status
(P=0.18, HR 2.6, 95% CI 0.64, 11.0) (Fig. 1). No associations wwere
found between time to recurrence and gender, patient age (cut point age
50), tumor size, extranodal extension, degree of extrathyroid extension,
or multifocal disease. Cox regression analysis was not significant.
4. Discussion
Prior studies on loss of heterozygosity alterations support the idea
that loss of tumor suppressor genes (TSG) are relatively late events in
thyroid cancer progression and correlate with increased biological
aggressiveness [9]. This is the first demonstration of the association
between LOH and ALN status (P=0.0000003). Subgroup analysis
confirmed that paired primary and positive lymph node samples from
patients with ALN more commonly harbored LOH (19/31 or 61%),
compared to ALN-negative (low metastatic burden) paired samples (1/8
or 13%, P=0.0125, two-tailed Fisher exact test). LOH is a marker of
genomic instability that impacts tumor suppressor genes. These data
support the role of LOH as a potential biological and prognostic marker
to identify the subset of thyroid cancers that are likely to show aggres­
sive biology and worse outcomes.
The BRAF V600E strong driver mutation is common in papillary
thyroid carcinoma (PTC) [4]. A landmark multicenter study of over 1,
800 PTC patients demonstrated that BRAF V600E was significantly
related to disease-specific mortality and synergistic with other adverse
features (older age, Stage IV, distant metastasis) [15]. High-risk sec­
ondary TERT promoter mutations can be present in a subgroup of
papillary thyroid carcinoma and are known predictors of lower
disease-free survival rates [16]. Here we demonstrate that LOH is
strongly associated with ALN status (Table 3), a known surrogate for the
outcome of disease progression. A variety of BRAF V600E-like, strong
driver mutations, were seen in this series, which was enriched for
aggressive thyroid malignancies (Table 2, Table 4). However, there was
no exclusive driver mutation that correlated with ALN. This supports the
idea that tumor suppressor gene loss is an additional event, independent
of driver mutation, associated with a surrogate marker of increased
thyroid cancer biological aggressiveness (ALN status). We also demon­
strate that the accumulation of additional or different LOH loci in
Table 3
Papillary Thyroid Carcinoma With/Without Aggressive Lymph Node Status (N = 105): LOH Status Within the Primary PTC.
LOH ≥ 2
LOH 1 locus
No LOH
Total
ALN
No ALN
p
ENE
No ENE
p
> 5 +LN
≤ 5 LN
12
31
24
67
1
4
33
38
0.000003
11
22
13
46
1
10
36
47
0.000007
12
30
24
6
4
31
37
N = Number of patients in each cell
LOH: Loss of heterozygosity
PTC: Papillary thyroid carcinoma
ALN: Aggressive lymph node status
ENE: Extranodal extension
LN: Lymph node
4
p
0.000022
S. Finkelstein et al.
Pathology - Research and Practice 251 (2023) 154842
Table 4
LOH and Mutational Analysis on Paired Primary Tumor and Positive Lymph Node Samples per ALN Category.
Aggressive Lymph Node Status (N = 31)
Primary PTC
LOH
status
10q
10q
17q
17q
17q
17q
17q
18q
1p 10q 17p 17q
1p, 3p
1p10q
1p17q
22q
3p
3p
3p
3p
5q
9q
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
Limited metastatic burden (N = 8)
+ LN
Driver
Mutation
BRAF V600E
BRAF V600E
ETV6:
:NTRK3
BRAF V600E
BRAF V600E
BRAF V600E
None
BRAF V600E TERT C228T
LOH status
BRAF V600E TERT C228T
BRAF V600E TERT C228T
BRAF V600E TERT C228T
CCDC6:
:RET
BRAF V600E
CCDC6:
:RET
BRAF V600E
CCDC6:
:RET
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E TERT C228T
None
None
None
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
1p 10q 17p 17q
5q 22q *
1p10q
1p17q
7p9p10q18q*
3p10q17q*
3p 17q *
17q 22q*
17q 22q*
17q
17q
18q
22q
9p17q*
3p17q*
3p 5q*
3p
5q 10q*
9p
17q*
17q*
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
No LOH
Primary PTC
Driver
Mutation
BRAF V600E
BRAF V600E
ETV6:
:NTRK3
BRAF V600E
BRAF V600E
BRAF V600E
None
BRAF V600E TERT C228T
LOH status
+ LN
LOH status
No LOH
No LOH
No LOH
Driver
Mutation
None
None
BRAF V600E
No LOH
No LOH
No LOH
No LOH
17q
BRAF
BRAF
BRAF
BRAF
BRAF
No LOH
No LOH
No LOH
No LOH
1p17q*
V600E
V600E
V600E
V600E
V600E TERT C228T
BRAF V600E TERT C228T
BRAF V600E TERT C228T
BRAF V600E TERT C228T
CCDC6:
:RET
BRAF V600E
CCDC6:
:RET
BRAF V600E
CCDC6:
:RET
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E TERT C228T
None
None
BRAF V600E*
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
No LOH
No LOH
No LOH
Driver
Mutation
None
None
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
TERT C228T
Legend
Metastatic carcinoma has acquired additional or different chromosomal losses, as compared to the primary carcinoma
LOH: Loss of heterozygosity.
PTC: Papillary thyroid carcinoma
ALN: Aggressive lymph node status
+LN: Positive lymph node
positive lymph node samples is common in the ALN+ group, as
compared with primary tumors, further supporting that these are later
events of cancer progression. In contrast, the mutational driver status
remained stable in paired primary and metastatic samples, for all but
one ALN positive patient (primary PTC no mutation, metastatic carci­
noma BRAF V600E+, Table 4).
The acquisition of LOH appears to differentially favor certain
genomic loci suggesting specific tumor suppressor genes that are pref­
erentially responsible for increased cancer aggressiveness (Table 2).
Chromosome 3p loss was the single most observed genomic locus
showing LOH. The interrogated region, 3p24, harbors several common
tumor suppressor genes including von Hippel Lindau (VHL) and 8-oxo­
guanine DNA Glycosylase (OGG1) genes. The former has been
described in thyroid malignancies [17] and the latter in head and neck
cancer; thus both are suitable candidates for potential driver mutations
responsible for increased biological aggressiveness. However our data
also supports the role of other cancer genes including those situated at
17q, 1p, 10q and other genomic loci. By expanding future molecular
analysis to search in more detail, we hope to address the presence, range
and specific genomic changes regarding this important issue.
A study limitation is the relatively small number of patients with
known outcome (93 patients, mean follow-up 55 months). On
Kaplan–Meier analyses, significance was seen only for the acquisition of
TERT promoter mutation with BRAF V600E, as compared to BRAF
V600E driver mutation alone. We were unable to achieve significance
for DFS and LOH or ALN status. In contrast, we were able to demonstrate
a strong association between ALN and decreased DFS (P=0.0001, HR
1.7, 95% CI 1.3, 2.2) by Kaplan–Meier analysis in a larger cohort of 347
patients with similar follow-up (mean 40.8 months) [18]. It is generally
challenging to correlate biomarkers with adverse outcome for PTC pa­
tients. First local recurrence can develop decades after primary treat­
ment. This justifies framing our investigation by enriching the study
cohort with a known surrogate for poor outcome, namely ALN status.
Future validation studies will test the hypothesis that LOH detection in
preoperative PTC specimensin addition to an expanded mutational
panel that includes known important events such as BRAF V600E and
TERT promoter mutations–can enhance the ability to predict disease
recurrence. This novel association requires additional study in an
increased patient cohort that is powered to query the impact of LOH
detection on outcome for patients with primary PTC.
In conclusion, the presence of LOH in primary PTC strongly predicts
ALN status, a known surrogate for aggressive behavior. The accumula­
tion of additional LOH hits is common within regional metastases of
patients with ALN status, while driver mutational status remains stable.
5
S. Finkelstein et al.
Pathology - Research and Practice 251 (2023) 154842
Fig. 1. Kaplan–Meier Curves. Y axis: Probability of locoregional recurrence-free survival. X axis: Time in months. Left – ALN status, P=0.18. Right – BRAF V600E
plus TERT promoter mutation versus BRAF V600E, P= 0.002. Bottom – LOH status, P= 0.09.
Levine, Mark Urken and Margaret Brandwein-Weber; Software, Venkata
Arun Timmaraju and Jonathan Levine; Supervision, Mark Urken and
Margaret Brandwein-Weber; Validation, Sydney Finkelstein, Venkata
Arun Timmaraju and Jonathan Levine; Writing – original draft, Sydney
Finkelstein, Mark Urken and Margaret Brandwein-Weber; Writing – re­
view & editing, Shabnam Samankan, Camilo Gonzalez-Velazquez, Scott
Doyle and Jonathan Levine.
Molecular diagnostics designed to show the presence or absence of LOH
mutational change could potentially predict ALN status and overall
aggressive biology of thyroid cancer based on sampling and analysis of
the primary malignancy. However, further work is necessary.
Ethics approval
The study was conducted according to the guidelines of the Decla­
ration of Helsinki and was approved by the Institutional Review Board of
Mount Sinai (IRB STUDY-18–01236: Molecular markers predictive of
aggressive and metastatic thyroid cancer). Last approval: 5/5/2023.
Declaration of Competing Interest
SDF, JL and VAT are employees of Interpace Diagnostics and own
limited stock in the company.
Consent to participate
Data Availability
Exempt from patient consent due to retrospective nature.
Upon request.
Consent for publication
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All authors have reviewed the manuscript and approve the content.
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CRediT authorship contribution statement
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