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Original Research
Identification of Genetic Alterations by
Circulating Tumor DNA in Leiomyosarcoma: A
Molecular Analysis of 73 Patients
Junaid Arshad,
1
Priscila Barreto-Coelho,
2
Emily Jonczak,
1
Andrea Espejo,
1
Gina D’Amato,
1,3
Jonathan C. Trent
1,3
1
Department of Medicine, Division of Medical Oncology, Miller School of Medicine, Jackson Memorial Hospital/
University of Miami, Miami, FL, USA
2
Department of Medicine, Division of Internal Medicine, Miller School of Medicine, Jackson Memorial Hospital/
University of Miami, Miami, FL, USA
3
Sylvester Comprehensive Cancer Center, Miami, FL, USA
Address correspondence to Jonathan Trent (jtrent@med.miami.edu)
Source of Support: None. Gina D’Amato serves on the advisory board for Blue print, Epizyme, and Bayer. Jonathan C. Trent
serves on the advisory board for Blueprint, Deciphera, Epizyme, and Daiichi. The other authors have nothing to disclose.
Based on a presentation at the American Society of Clinical Oncology 2019 Annual Meeting.
Received: February 10, 2020; Accepted: March 31, 2020.
Arshad J, Barreto-Coelho P, Jonczak E, Espejo A, D’Amato G, Trent J. Identification of genetic alterations by circulating tumor DNA
in leiomyosarcoma: A molecular analysis of 73 patients. J Immunother Precis Oncol. 2020; 3:64–68. DOI: 10.36401/JIPO-20-3.
&Innovative Healthcare Institute
ABSTRACT
Background: Leiomyosarcoma is a malignant mesenchymal tumor of cells of smooth muscle lineage arising
commonly in retroperitoneum, uterus, large veins, and the limbs. The genetics of leiomyosarcomas are complex and
there is very limited understanding of common driver mutations. Circulating tumor DNA (ctDNA) offers a rapid and
noninvasive method of next-generation sequencing (NGS) that could be used for diagnosis, therapy, and detection of
recurrence. Methods: ctDNA testing was performed using Guardant360, which detects single nucleotide variants,
amplifications, fusions, and specific insertion/deletion mutations in 73 genes using NGS. Results: Of 73 patients, 59
were found to have one or more cancer-associated genomic alteration. Forty-five (76%) were female with a median age
of 63 (range, 38–87) years. All samples were designated metastatic. The most common alterations were detected in
Tp53 (65%), BRAF (13%), CCNE (13%), EGFR (12%), PIK3CA (12%), FGFR1 (10%), RB1(10%), KIT (8%), and PDGFRA
(8%). Some of the other alterations included RAF1, ERBB2, MET, PTEN TERT, APC, and NOTCH1. Potentially
targetable mutations, by Food and Drug Administration–approved or clinical trials, were found in 24 (40%) of the 73
patients. Four patients (5%) were found to have incidental germline TP53 mutations. Conclusion: NGS of ctDNA
allows identification of genomic alterations in plasma from patients with leiomyosarcoma. Unfortunately, there is
limited activity of current targeted agents in leiomyosarcomas. These results suggest opportunities to develop therapy
against TP53, cell cycle, and kinase signaling pathways. Further validation and prospective evaluation is warranted to
investigate the clinical utility of ctDNA for patients with leiomyosarcoma.
Keywords: ctDNA, genetic alterations, leiomyosarcoma
INTRODUCTION
Leiomyosarcoma (LMS) is a malignant mesenchymal
tumor with smooth muscle cell differentiation. Because
these cells are present in all organs, LMS can arise
anywhere in the body, commonly in the retroperitone-
um, uterus, large veins, and the limbs. It accounts for
10% to 20% of all sarcomas and has been classically
reported as the most frequent soft tissue sarcoma subtype
together with liposarcoma.
[1,2]
Uterine LMSs are the
single largest site-specific group with an incidence of
0.64 per 100,000 women.
[3]
The overall incidence of LMSs increases with age and
peaks at the seventh decade of life. The presentation
differs from uterine LMS, which occurs in women in the
perimenopausal age group.
[3]
The differences among the
gender incidence depend on the primary tumor site;
retroperitoneal and inferior vena cava sites predominate
in women,
[4]
whereas noncutaneous soft tissue sites and
cutaneous LMS are more common in men.
[1,5]
There are
Journal of Immunotherapy and Precision Oncology 2020 | Volume 3 | Issue 2 | 64
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no specific diagnostic clinical features of soft tissue LMS
to distinguish from other soft tissue sarcomas. On
suspicion, the diagnostic and staging studies are per-
formed simultaneously with a biopsy to establish a
specific diagnosis.
[6]
The treatment and prognosis de-
pend on factors such as histological grade, tumor size,
and location, as well as the presence of localized or
metastatic disease. LMS outcomes are mostly dependent
on feasibility for surgical resection at an early stage with
wide margins. Once metastatic, systemic chemotherapy
such as doxorubicin 6ifosfamide or dacarbazine or
gemcitabine/docetaxel is used. The addition of chemo-
therapy offers a median progression-free survival (PFS) of
approximately 6 months and an overall survival (OS) of
10 to 15 months, representing an unmet need of further
therapeutic agents.
[1]
Pazopanib is a multikinase inhib-
itor approved for the second-line treatment in LMS; its
use has resulted in increased PFS but no OS benefit.
[7]
In the era of next-generation sequencing (NGS), there
is a paucity of available targets with therapeutic potential
in LMS. The exact pathophysiologic genetic framework
remains elusive because of the rarity of the disease,
limited NGS testing, and lack of universal standards.
Various studies have been published showing complex
heterogeneity with genomic instability associated with
defects in TP53 and ATM gene.
[1]
Circulating tumor DNA (ctDNA) offers a novel, rapid,
and noninvasive method of NGS that could be used for
diagnosis, therapy, and detection of recurrence. Similar
studies have been published in other soft tissue sarco-
mas, such as gastrointestinal stromal tumors (GIST).
[8]
ctDNA consists of small fragments of DNA, comprising
fewer than 200 nucleotides found in the bloodstream,
and is a marker of cancer cell turnover. There are no
oncogenic single nucleotide variants (SNVs) that char-
acterize LMS. Still, loss of tumor suppressors, including
TP53,RB1,andPTEN,arecommonlyseen,asare
multiple copy number alterations (CNAs). Most ctDNA
assays have been developed to detect SNVs that are
highly recurrent in many types of carcinomas. The lack
of recurrent SNVs in LMS poses a limitation for targeted
sequencing; however, the numerous CNAs characteristic
of this disease represent an ideal target for detection.
[9,10]
To date, this study is the largest evaluation of the genetic
landscape for looking at LMS using ctDNA.
PATIENTS AND METHODS
This is a retrospective observational study of 73 de-
identified patients from the Guardant Health Data base.
Patients with diagnosis of LMS were referred from
academic as well as community institutions. Patients
had blood samples collected between December 2014
and December 2018 and sent for analysis to the
Guardant Health Group.
The Guardant 360 is a commercially available NGS
panel for identifying SNVs in 73 genes, CNAs in 18
genes, and fusions, deletions and insertions in 23 genes.
ctDNA Isolation
The 10-mL blood samples were collected in Streck
tubes. The samples were then stored and shipped at
room temperature to Guardant Health (Redwood City,
CA, USA). The plasma was isolated by centrifugation of
blood by 1600gfor 10 minutes at 48C. Using the QIAamp
circulating nucleic acid kit (Qiagen, Hilden, Germany),
ctDNA was then extracted, concentrated, and size
selected using Agencourt Ampure XP beads (Beckman
Coulter,Brea,CA,USA),andquantifiedbyQubit
fluorometer (Life Technologies, Carlsbad, CA, USA).
ctDNA Sequencing
After isolation and oligo-nucleotide barcoding, ctDNA
digital sequencing library preparation was performed.
The library was then amplified and enriched for target
genes. Each base pair had a 15,000x average coverage
depth. After sequencing, algorithmic reconstruction of
the digitized sequencing signals were used to reconstruct
the ctDNA fragments.
[11]
The patient demographics and tumor characteristics
were obtained and subsequently stored in a secure
database for analysis.
Statistical Analysis
The patient and tumor characteristics were analyzed
by using simple descriptive statistics. The results of the
genetic alterations were reported in the form of a bar
graph.
RESULTS
A total of 73 patients were detected as having the
diagnosis of LMS from the formalin-fixed tissue samples.
Most of the patient cohort was composed of a female
population (76%) with a mean age of 63 years (range 38–
87 years). All patients were found to have metastatic
leiomyosarcoma.
The most common mutation found was the TP53,
present in 48 patients (65%), whereas KIT and PDGFRA
were the least frequent, each present in only 5 patients
(8%). In-between were BRAF and CCNE (present in 8
patients, corresponding to 13%), EGFR and PIK3CA (in 7
patients each, 12%), and FGFR1 and RB1 (6 patients,
10% of the sample). There were 14 patients with
variations of undetermined significance (VUS) (Figure 1).
The other alterations included RAF1,ERBB2,MET,
PTEN,TERT,APC, and NOTCH1. Twenty-four patients
(40%) had genomic alterations, detected by ctDNA,
potentially targetable by a Food and Drug Administra-
tion (FDA)-approved or clinical trial therapy according to
the European Society for Medical Oncology (ESMO) Scale
of Clinical Actionability for molecular targets (ESCAT)
and OncoKB (Table 2). There were 4 (5%) patients who
were found to have incidental germline TP53 mutations.
The various mutations are shown in the form a bar chart
in Figure 2.
Original Research 65
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There were a total of 90 samples. Of all the samples
collected and analyzed, alterations were detected in 73
ctDNA samples (.80%). After exclusion of VUS, 63
ctDNA samples harbored cancer-associated genomic
alterations (70%), and of these, 59 were found to have
1 or more cancer-associated genomic alterations.
The copy number variation count (the variation
between genomes in the number of copies of a genomic
region) was also described, being the highest among the
CCNE mutation. There were 14 insertions/deletions
among the Tp53 mutation and a total of 43 SNVs on
this same mutation.
DISCUSSION
LMSs are highly aggressive neoplasms with dismal
prognosis in the metastatic setting: PFS of only 6 months
andanOSofapproximately12to15months.
[12]
Regimens associated with survival benefit include doxo-
rubicin/ifosfamide
[13]
and gemcitabine/docetaxel.
[14]
The prognosis is even worse with second- and third-line
chemotherapy options. This accounts for the need of
new therapeutic options.
NGS offers detection of genetic alterations not only
responsible for diagnosis or the pathophysiology but also
provide information about therapeutic targets. The
deletions in canonical cancer genes are central to LMS
along with DNA repair enzymes.
[15]
ctDNA is a novel and
rapid method of NGS, especially useful when tissue
biopsy is not available or cannot be obtained. The
quantity of ctDNA varies among individuals and de-
pends on the type of tumor, its location and stage, and
the disease burden. The sensitivity of ctDNA is higher in
metastatic patients with high disease burden.
[11]
ctDNA
detection is useful in diagnosis, response assessment, and
disease progression.
[16,17]
Presented here is the largest study identifying the
genetic makeup of patients with metastatic LMS with the
use of ctDNA. Meanwhile, there have been a few studies
looking at the genetic framework from tissue-based NGS.
Lee et al
[15]
described the most frequent alterations
found in a series of 25 uterine and nonuterine LMS.
Frequently involved genes were Tp53,ATM,ATRX,EGFR,
and RB1, with the CNAs identified in 85% of cases.
Another study with whole genomic sequencing and
transcriptomic analysis showed that most of the alter-
ations were mutations in tumor suppressor genes
including Tp53,RB1,ATRX ,andATM;chemokine
receptors including FGFR2 and ALK; chromatin modifi-
ers, such as DNMT3A; and transcription regulators, such
as PAX3. Most of genes were associated with the copy
number amplification.
[18]
Cuppens et al
[7]
reported
common alterations in Tp53,RB1, and PTEN in uterine
LMSs.
Hemming et al
[10]
described the use of ctDNA in
patients with progressive LMS. Here similarly, the most
common alterations were deletions in Tp53 and Rb1. The
study also reported the correlation between higher levels
of ctDNA with tumor size and disease progression in
patients with LMS using 11 patients, 11 of 16 with active
LMS had detectable ctDNA, and their results suggest that
ctDNA may be useful as a biomarker for a subset of
uterine and extrauterine LMS. In our study, we were able
to evaluate the ctDNA of 73 patients with the diagnosis
of metastatic LMS. As previously discussed, use of ctDNA
detection for LMS is challenging because of the lack of
characteristic oncogenic SNVs. Most ctDNA assays were
developed to detect highly recurrent SNVs for many
types of carcinomas, and certain sarcomas.
[19,20]
We analyzed samples from 73 patients; 14 were found
to have VUS resulting in a mutation detection rate of
75%. The results of our study were consistent with the
prior studies showing frequent mutations in tumor-
Figure 1.—Consort flow diagram showing the number of patients with
alterations. VUS, variations of undetermined significance; Alt, alterations.
Table 1.—The percentage of patients with mutations and nature of mutations
Mutation Percent (Patients)
Copy Number
Variation Count
Insertion/Deletion
Count
Single Nucleotide
Variants Count
TP53 65 (48) - 14 43
BRAF 13 (8) 5 - 4
CCNE 13 (8) 8 - -
EGFR 12 (7) 4 - 4
PIK3CA Level 1 12 (7) 6 - 1
FGFR1 Level 4 10 (6) 3 - 3
RB1 10 (6) - 5 1
KIT 8 (5) 5 - -
PDGFRA 8 (5) 4 - 1
-, not detected.
66 Arshad et al: ctDNA in LMS
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suppressing genes including Tp53,BRAF, and Rb;SNV
was the most common alteration followed by the copy
number variation (CNV). There have been multiple prior
studies highlighting the importance of CNV in the
biology of LMSs. Despite the genetic heterogeneity, there
are some consistent DNA copy number changes detected
by comparative genetic hybridization and molecular
studies. Common changes include a high frequency of
losses in DNA copy numbers in 10q and 13q, gains in
17p, and the presence of tumor size–related alterations.
It is believed that changes in 10q, 13q, and 17p lead to
mutations in tumor suppressor genes, which may be
early changes in tumorigenesis. Studies have shown that
10q23 encodes for PTEN, 10q24–25 for MX11 tumor
suppressor genes. The MX11 gene negatively regulates c-
myc oncogene contributing to tumor development.
Changes in 13q encode genes such as RB1 at 13q14
and BRCA2 at 13q12, which are both tumor suppressor
genes. Similar gains in 17p are associated with Tp53-
related alterations.
[21,22]
ctDNA-detected genetic alter-
ations in our study are consistent with the prior studies.
Most of the alterations in the LMS do not have a
designated target available. This has been the ongoing
subject matter for most clinical trials. Tp53 is the most
commonlyalteredgeneinLMSandanattractive
pharmacologic target; however, there has been no
successful mutant Tp53 drug despite decade-long re-
search. There have been new advances, such as the
development of Tp53 reactivating molecules including
small peptide mutant Tp53 conformation stabilizers
(pCAPs), CTM,PEITC,ReACp53, and ZMC1. Although
these molecules have a different mechanism of action,
they all target a specific subset of Tp53 mutations, restore
the wild-type conformation and function of the protein,
and cause tumor regression. These molecules require
further studies to determine the clinical benefit.
[23]
A
pilot trial is under way to determine the use of
atorvastatin in Tp53 mutant and wild-type malignancies.
Similarly, other detected genes, such as BRAF,EGFR, and
PIK3CA, have available targeted agents approved for
other cancer types but have no clinical benefit in LMSs.
This can be explained with the concept of driver genes,
mutation types, and frequency and type of cancers.
Multiple clinical trials are under way to discover a
therapeutic target.
In other tumors, such as GIST, mutations detected on
ctDNA analysis, such as KIT/PDGFRA, have been found
relevant for diagnostic purposes as well as to predict
treatment responses.
[11]
Meanwhile, there are no avail-
able treatment targets in LMS; ctDNA in LMS can be
restricted to diagnosis related to the size cutoff 5 cm
irrespective of the disease progression, making it a
technical sensitivity threshold. This ctDNA detection
may help distinguish between leiomyoma and LMS.
Similarly, positive ctDNA results after completion of the
adjuvant treatment may help with detection of recur-
rence.
[10]
The detection of SNV has proven to be
beneficial in detecting resistant mutations and providing
important prognostic information. A highly specific
LMS-specific assay Cancer Personalized Profiling by deep
Sequencing (CAPP-Seq) has been designed for the SNVs,
indels, and CNAs. A similar publication described that
ultra-low pass whole genome sequencing assay based
ctDNA detection may have prognostic significance.
[24]
Although different tests have different sensitivities, the
test used in this publication appears to be helpful for
qualitative and quantitative ctDNA analysis, especially
useful in the setting of disease surveillance.
[25]
The retrospective design, lack of control arm, unavail-
ability of tumor characteristics, lack of sensitivity, and
Table 2.—The targetable mutations that are approved by the
Food and Drug Administration or used in the clinical trial
European Society for Medical Oncology Scale of Clinical
Accountability for molecular targets (ESCAT) and OncoKB
Mutation ESCAT/OncoKB, Tier
TP53 X
BRAF III
CCNE X
EGFR III
PIK3CA III
FGFR1 III
RB1 X
KIT III
PDGFRA III
RAF1 III
ERBB2 III
MET III
Figure 2.—Most commonly mutated genes
by ctDNA analysis of patients with metastatic
leiomyosarcoma (N¼73).
Original Research 67
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concordance data between ctDNA and the formalin-fixed
paraffin-embedded tissue are some of the limitations of
the study. Meanwhile, there have been advances in the
improvement of sensitivity of the assay; the use of
ctDNA is being prospectively studied in clinical trials in
LMSs as well as other sarcomas. A combination strategy
approach for targeted agents as well as the discovery of
newer targets is also under way.
CONCLUSION
In conclusion, NGS of ctDNA allows identification of
genomic alterations in plasma from patients with LMS.
Unfortunately, to this date there is limited activity of
targeted agents in LMS. Nevertheless, our results suggest
opportunities to develop therapy targeting TP53, cell
cycle, and kinase signaling pathways. On the other
hand, ctDNA in LMS has many potential clinical uses
regarding diagnosis, response to treatment, and evalua-
tion of the risk of recurrence. Further validation and
prospective evaluation are warranted to investigate the
clinical utility of ctDNA for patients with LMS.
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