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Identification of Genetic Alterations by Circulating Tumor DNA in Leiomyosarcoma: A Molecular Analysis of 73 Patients

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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.
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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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... The identification of molecular alterations can help develop therapy, targeting TP53, cell cycle, and kinase signaling pathways [120] ...
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... This strategy is worth exploring also in tumors not harboring a clear-cut gene driver like LMS. NGS of ctDNA allows identification of somatic and potentially germline genomic alterations in plasma from LMS patients [48,49]; however, further validation and prospective evaluation are warranted to investigate the clinical utility of ctDNA especially for LMS patients: (1) A Sarcoma Alliance for Research Through Collaboration (SARC)-funded pilot study is evaluating ctDNA as a biomarker of relapse-free survival and response to therapy in patients with high-grade, high-risk, localized LMS. (2) A SARC-supported study of ctDNA as biomarker of sarcoma response to chemotherapy in patients with metastatic LMS is currently being planned. ...
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Opinion statement Leiomyosarcoma is one of the most common subtypes of soft tissue sarcomas accounting for approximately 20% of sarcomas. As leiomyosarcoma patients frequently develop metastatic disease, effective systemic therapies are needed to improve clinical outcomes. The overall activity of the currently available conventional systemic therapies and the prognosis of patients with advanced and/or metastatic disease are poor. As such, the treatment of this patient population remains challenging. As a result, there is a clear unmet medical need, and designing and performing meaningful clinical studies are of utmost importance to improve the prognosis of this patient group. Therefore, the aim of this review is to briefly summarize state-of-the-art treatments for leiomyosarcoma patients and to describe trial characteristics needed for informative clinical studies.
... In a study of 73 LMS patients, 59 patients were found to have an alteration detected by the NGS panel. The most common alterations found by this panel were in TP53, BRAF, CCNE, EGFR, PIK3CA, FGFR1, RB1, KIT, and PDGFRA [42]. Unfortunately, most drugs targeting these alterations have not shown to be successful for the treatment of LMS until now. ...
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Sarcomas are rare tumors of bone and soft tissue with a mesenchymal origin. This uncommon type of cancer is marked by a high heterogeneity, consisting of over 70 subtypes. Because of this broad spectrum, their treatment requires a subtype-specific therapeutic approach. Tissue biopsy is currently the golden standard for sarcoma diagnosis, but it has its limitations. Over the recent years, methods to detect, characterize, and monitor cancer through liquid biopsy have evolved rapidly. The analysis of circulating biomarkers in peripheral blood, such as circulating tumor cells (CTC) or circulating tumor DNA (ctDNA), could provide real-time information on tumor genetics, disease state, and resistance mechanisms. Furthermore, it traces tumor evolution and can assess tumor heterogeneity. Although the first results in sarcomas are encouraging, there are technical challenges that need to be addressed for implementation in clinical practice. Here, we summarize current knowledge about liquid biopsies in sarcomas and elaborate on different strategies to integrate liquid biopsy into sarcoma clinical care.
... Potentially targetable mutations were found in 40% of the 73 patients. A total of 5% were incidentally found to have germline TP53 mutations [62]. ...
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Simple Summary In this position paper, we aim to summarize state-of-the-art treatments for patients with leiomyosarcomas in order to identify knowledge gaps and current unmet needs, thereby guiding the community to design innovative clinical trials and basic research and close these research gaps. This white paper arose from a leiomyosarcoma research meeting in October 2020 hosted by the National LeioMyoSarcoma Foundation (NLMSF) and Sarcoma Patients EuroNet (SPAEN). Abstract As leiomyosarcoma patients are challenged by the development of metastatic disease, effective systemic therapies are the cornerstone of outcome. However, the overall activity of the currently available conventional systemic treatments and the prognosis of patients with advanced or metastatic disease are still poor, making the treatment of this patient group challenging. Therefore, in a joint effort together with patient networks and organizations, namely Sarcoma Patients EuroNet (SPAEN), the international network of sarcoma patients organizations, and the National LeioMyoSarcoma Foundation (NLMSF) in the United States, we aim to summarize state-of-the-art treatments for leiomyosarcoma patients in order to identify knowledge gaps and current unmet needs, thereby guiding the community to design innovative clinical trials and basic research and close these research gaps. This position paper arose from a leiomyosarcoma research meeting in October 2020 hosted by the NLMSF and SPAEN.
... Achenbach et al. [7] provided us with a comprehensive and timely overview on some novel therapeutic targets, as well as early-phase clinical trials data, and the rationale for possible future regulatory approvals. In original research, Arshad et al. [8] shared their institutional experience of using circulating tumor DNA in more than 70 patients with advanced leiomyosarcomas, 40% of whom had tumors that harbored potentially targetable mutations by FDA-approved or clinical trials, thus opening potential treatment opportunities in an otherwise devastating group of diseases. Wainsztein and Chen [9] authored a comprehensive review highlighting potential advantages, risks, nuances, and clinical trial experiences in combining the use of various molecular targeted agents with immunotherapy to enhance our therapeutic armamentarium against soft tissue sarcomas. ...
Article
Background A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Questions/purposes Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? Methods This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. Results This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. Conclusion In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. Clinical Relevance These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.
Article
Background: The current treatment paradigm of imatinib-resistant metastatic gastrointestinal stromal tumor (GIST) does not incorporate KIT/PDGFRA genotypes in therapeutic drug sequencing, except for PDGFRA exon 18-mutant GIST that are indicated for avapritinib treatment. Here, ctDNA sequencing was used to analyze plasma samples prospectively collected in the phase III VOYAGER trial to understand how the KIT/PDGFRA mutational landscape contributes to tyrosine-kinase inhibitor (TKI) resistance and to determine its clinical validity and utility. Patients and methods: VOYAGER (N=476) compared avapritinib with regorafenib in patients with KIT/PDGFRA-mutant GIST previously treated with imatinib and 1 or 2 additional TKIs (NCT03465722). KIT/PDGFRA ctDNA mutation profiling of plasma samples at baseline and end-of-treatment was assessed with 74-gene Guardant360® CDx. Molecular subgroups were determined and correlated with outcomes. Results: 386/476 patients with KIT/PDGFRA-mutant tumors underwent baseline (pre-trial treatment) ctDNA analysis; 196 received avapritinib, and 190 received regorafenib. KIT and PDGFRA mutations were detected in 75.1% and 5.4%, respectively. KIT resistance mutations were found in the activation loop (A-loop; 80.4%) and ATP-binding pocket (ATP-BP; 40.8%); 23.4% had both. An average of 2.6 KIT mutations were detected per patient; 17.2% showed 4-14 different KIT resistance mutations. Of all pathogenic KIT variants, 28.0% were novel, including alterations in exons/codons previously unreported. PDGFRA mutations showed similar patterns. ctDNA-detected KIT ATP-BP mutations negatively prognosticated avapritinib activity, with a median progression-free survival (mPFS) of 1.9 versus 5.6 months for regorafenib. mPFS for regorafenib did not vary regardless of the presence or absence of ATP-BP/A-loop mutants and was greater than mPFS with avapritinib in this population. Secondary KIT ATP-BP pocket mutation variants, particularly V654A, were enriched upon disease progression with avapritinib. Conclusions: CtDNA sequencing efficiently detects KIT/PDGFRA mutations and prognosticates outcomes in patients with TKI-resistant GIST treated with avapritinib. ctDNA analysis can be used to monitor disease progression and provide more personalized treatment.
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Recent work confirms a bench-to-bedside approach that circulating tumor DNA is associated with outcome and objective response to chemotherapy in patients with advanced leiomyosarcoma. Liquid biopsies may be used for risk stratification in future trials guiding treatment decisions by identifying patients who are likely to benefit from chemotherapy. See related article by Madanat-Harjuoja et al., p. xxx
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Purpose: Leiomyosarcoma (LMS) is a soft tissue sarcoma characterized by multiple copy number alterations (CNAs) and without common recurrent single nucleotide variants. We evaluated the feasibility of detecting circulating tumor DNA (ctDNA) with next-generation sequencing in a cohort of patients with LMS whose tumor burden ranged from no evidence of disease to metastatic progressive disease. Patients and methods: Cell-free DNA in plasma samples and paired genomic DNA from resected tumors were evaluated from patients with LMS by ultra-low passage whole genome sequencing (ULP-WGS). Sequencing reads were aligned to the human genome and CNAs identified in cell-free DNA and tumor DNA by ichorCNA software to determine the presence of ctDNA. Clinical data were reviewed to assess disease burden and clinicopathologic features. Results: We identified LMS ctDNA in eleven of sixteen patients (69%) with disease progression and total tumor burden over 5 cm. Sixteen patients with stable disease or low disease burden at the time of blood draw were found to have no detectable ctDNA. Higher ctDNA fraction of total cell-free DNA was associated with increasing tumor size and disease progression. Conserved CNAs were found between primary tumors and ctDNA in each case, and recurrent CNAs were found across LMS samples. ctDNA levels declined following resection of progressive disease in one case and became detectable upon disease relapse in another individual patient. Conclusion: These results suggest that ctDNA, assayed by a widely available sequencing approach, may be useful as a biomarker for a subset of uterine and extrauterine LMS. Higher levels of ctDNA correlate with tumor size and disease progression. Liquid biopsies may assist in guiding treatment decisions, monitoring response to systemic therapy, surveying for disease recurrence and differentiating benign and malignant smooth muscle tumors.
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Background: New prognostic markers are needed to identify patients with Ewing sarcoma (EWS) and osteosarcoma unlikely to benefit from standard therapy. We describe the incidence and association with outcome of circulating tumour DNA (ctDNA) using next-generation sequencing (NGS) assays. Methods: A NGS hybrid capture assay and an ultra-low-pass whole-genome sequencing assay were used to detect ctDNA in banked plasma from patients with EWS and osteosarcoma, respectively. Patients were coded as positive or negative for ctDNA and tested for association with clinical features and outcome. Results: The analytic cohort included 94 patients with EWS (82% from initial diagnosis) and 72 patients with primary localised osteosarcoma (100% from initial diagnosis). ctDNA was detectable in 53% and 57% of newly diagnosed patients with EWS and osteosarcoma, respectively. Among patients with newly diagnosed localised EWS, detectable ctDNA was associated with inferior 3-year event-free survival (48.6% vs. 82.1%; p = 0.006) and overall survival (79.8% vs. 92.6%; p = 0.01). In both EWS and osteosarcoma, risk of event and death increased with ctDNA levels. Conclusions: NGS assays agnostic of primary tumour sequencing results detect ctDNA in half of the plasma samples from patients with newly diagnosed EWS and osteosarcoma. Detectable ctDNA is associated with inferior outcomes.
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Introduction: Gastrointestinal stromal tumor (GIST) is the most common malignant mesenchymal tumor of the gastrointestinal system. Multiple advances in the management of GIST from the discovery of KIT/PDGRA and other genetic alterations have led to the development of multiple tyrosine kinase inhibitors. Response assessment in GIST is determined with iRECIST (Response Evaluation Criteria in Solid Tumors), PERCIST (PET response criteria in solid tumors) or Choi criteria. Molecular genotyping of the tissue samples is the recent standard for diagnosis, treatment and response to treatment. Areas covered: Here we provide a brief overview of the history of the GIST, molecular sequencing, available treatment options and clinical trials, radiologic response assessment and the role of ctDNA in response evaluation. Expert opinion: Future GIST management is related to the development of sensitive assays to detect genetic alterations for initial diagnosis, treatment selection, monitoring the response to treatment, resistant mutations and predicting survival.
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PURPOSE GI stromal tumor (GIST) is the most common sarcoma of the GI tract. Management of patients with GIST is determined by KIT, PDGFRA, or other genomic alterations. Tissue-based next-generation sequencing (NGS) analysis is the standard approach for diagnosis, prognosis, and treatment selection. However, circulating tumor DNA (ctDNA)–based NGS is a novel and noninvasive alternative. METHODS ctDNA sequencing results were evaluated in blood samples from 243 de-identified patients within the Guardant360 database. Under an approved institutional review board protocol, a retrospective analysis was performed on 45 single-institution patients. RESULTS Of 243 patients, 114 (47%) were women, and the median age was 59 years (range, 17-90 years). Patients with no alterations and variations of uncertain significance were excluded. Of the 162 patients with known pathogenic mutations, KIT was the most common (56%), followed by NF (7%), PDGFRA (6%), PI3KCA (6%), KRAS (5%), and others (6%). Most tumors harbored an actionable KIT or PDGFRA mutation. Our institutional cohort (n = 45) had 16 (35%) KIT exon 11 mutations, 3 (6%) KIT exon 9 mutations, and 1 (2%) PDGFRA mutation detected on ctDNA. Resistance mutations were observed in KIT exon 17 (8 patients), exon 13 (3 patients), and in both (3 patients). Our comparison of ctDNA with tissue NGS revealed a positive predictive value (PPV) of 100%. Failure of concordance was observed in patients with localized or low disease burden. From the time of ctDNA testing, the median overall survival was not reached, whereas the median progression-free survival was 7 months. CONCLUSION ctDNA provides a rapid, noninvasive analysis of current mutations with a high PPV for patients with metastatic GIST. ctDNA-based testing may help to define the optimal choice of therapy on the basis of resistance mutations and should be studied prospectively.
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Circulating tumor DNA (ctDNA) is a promising noninvasive biomarker for hepatocellular carcinoma (HCC). In this study, we aimed to assess the diagnostic and prognostic value of ctDNA in HCC. Twenty-six operable HCC, 10 hepatitis and 10 cirrhosis patients were enrolled in this study. Treatment-naïve blood samples were collected from all patients, nevertheless resected tissue and postoperative blood samples were only collected from HCC patients. A custom-designed sequencing panel covering 354 genes was used to identify somatic mutations. Collectively, we identified 139 somatic mutations from 25 HCC baseline plasma samples (96.2%). TP53 (50.00%) was the most common mutant gene, and R249S was the most recurrent mutation (19.2%). Twenty-three patients (88.5%) carried at least one ctDNA mutation validated in matched tissue, and the driver mutations exhibited an advanced concordance than non-driver mutations (67.6% vs. 33.8%, P = 0.0002). For HCC patients, the number of mutations in ctDNA (R2 = 0.1682, P = 0.0375), maximal variant allele frequency (VAF) in ctDNA (R2 = 0.4974, P < 0.0001) and ctDNA concentration (R2 = 0.2676, P = 0.0068) were linearly correlated with tumor size. Multiple circulating cell-free DNA (cfDNA) parameters could be used in differentiating malignant lesions from benign lesions, and the performance was no less than blood alpha-fetoprotein (AFP). HCC patients with detectable mutation in postoperative plasma had a poor DFS than those without (17.5 months vs. 6.7 months, HR = 7.655, P < 0.0001), and postoperative cfDNA status (HR = 10.293, P < 0.0001) was an independent risk factors for recurrence. In conclusion, ctDNA profiling is potentially valuable in differential diagnosis and prognostic evaluation of HCC.
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Background & aims: Biomarkers are needed to identify patients at risk of tumor progression following chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of therapy. Methods: We performed deep sequencing (CAPP-Seq) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes, and fixed esophageal tumor biopsies collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy individuals (controls). We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6-8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) following chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy). Results: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA following chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P<.0001), formation of distant metastases (hazard ratio, 32.1; P<.0001), and shorter disease-specific survival times (hazard ratio, 23.1; P<.0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P=.03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P<.001 for comparison of either technique to combined analysis). Conclusions: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression.
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Sarcomas are connective tissue tumors accounting for only 1% of all adult malignancies. Leiomyosarcoma (LMS) is a sarcoma arising from smooth muscle cells, and accounts for 10-20% of all sarcomas. A subtype of LMS are those originating from the smooth muscle of blood vessels. Leiomyosarcoma of the inferior vena cava is a sarcomatous tumor, with less than 350 cases described in the literature. It carries a poor prognosis, with 5- and 10-year survival rates of 31.4% and 7.4%, respectively. We present a case of a 46-year-old female with no significant past medical history presented to the emergency department with mild abdominal pain and distention, early satiety, and weight loss for three weeks, found to have unresectable metastatic leiomyosarcoma of the inferior vena cava.
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Leiomyosarcomas (LMS) are malignant tumors of smooth-muscle origin that occur across age groups. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown, and few therapeutic options exist for LMS patients. To detect somatic mutations, copy number alterations, and structural rearrangements, we performed whole-exome and transcriptome sequencing of 49 and 37 LMS tumors, respectively, and performed integrative analysis. Recurrence analysis identified TP53, RB1, and ATRX as significantly mutated genes and various other cancer-associated genes mutated at low frequency, indicating substantial mutational heterogeneity. Copy number analysis revealed widespread chromosomal gains and losses and highly rearranged genomes in all tumors. Additionally, chromothripsis and whole-genome duplication were detected in 35% and 51% of cases, respectively. Principle component analysis and unsupervised hierarchical clustering of transcriptome data revealed three distinct subgroups of patients. Furthermore, we detected multiple non-recurrent fusion transcripts resulting from chromosomal rearrangements, many of which were predicted to result in loss of TP53 and RB1 function. In-depth analysis of these loci revealed protein-damaging microdeletions, intragenic or distal inversions, and exon skipping events as additional, previously unrecognized mechanisms of TP53 and RB1 disruption. Integration of whole-exome and transcriptome data demonstrated biallelic disruption of TP53 and RB1 in 92% and 94% of cases, respectively, and tumors with wildtype RB1 displayed loss of CDKN2A expression, overexpression of CCND1, or mutation of MAX resulting in CDK4 and CCND2 overexpression as alternative mechanisms of RB1 suppression. We also detected alternative lengthening of telomeres (ALT) in 78% of cases, and identified recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing novel insight into the genetic basis of this mechanism. Finally, most tumors displayed hallmarks of “BRCAness”, including alterations in homologous recombination DNA repair genes and enrichment of specific mutational signatures, and cultured LMS cells were sensitive towards olaparib and cisplatin. This comprehensive genomic and transcriptomic analysis has unveiled that LMS is characterized by mutational heterogeneity, genomic instability, near-universal inactivation of TP53 and RB1, and frequent whole-genome duplication. Furthermore, we have established that most LMS tumors rely on ALT to escape replicative senescence, and identified recurrent alterations in a broad spectrum of telomere maintenance genes. Finally, our findings uncover “BRCAness” as potentially actionable feature of LMS tumors, and provide a rich resource for guiding future investigations into the mechanisms underlying LMS development and the design of novel therapeutic strategies. Citation Format: Priya Chudasama, Sadaf Mughal, Mathijs Sanders, Daniel Hübschmann, Inn Chung, Aurélie Ernst, Bernd Kasper, Hans-Georg Kopp, Sebastian Bauer, Karsten Rippe, Benedikt Brors, Marcus Renner, Peter Hohenberger, Claudia Scholl, Stefan Fröhling. Integrative genomic and transcriptomic analysis of leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4336.