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Detection of Circulating Tumor DNA in Patients With Leiomyosarcoma With Progressive Disease

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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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Detection of Circulating Tumor DNA in Patients With
Leiomyosarcoma With Progressive Disease
Matthew L. Hemminga,b, Kelly S. Klegac, Justin Rhoadesd, Gavin Hae, Kate E. Ackerb,
Jessica L. Andersenb, Edwin Thaif, Anwesha Nagf, Aaron R. Thornerf, Chandrajit P. Rautg,
Suzanne Georgeb,*, and Brian D. Cromptonc,d,*
aDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
bCenter for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Harvard Medical School,
Boston, Massachusetts, USA.
cDana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston, Massachusetts,
USA.
Corresponding Authors: Suzanne George, MD, Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, 450
Brookline Ave, Boston, MA 02215; Phone: 617-632-5204; Fax: 617-632-3408; suzanne_george@dfci.harvard.edu; Brian D.
Crompton, MD, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, 450 Brookline Ave, Boston, MA 02215; Phone:
617-632-4468; Fax: 617-632-4410; briand_crompton@dfci.harvard.edu.
*Senior co-authors
AUTHOR CONTRIBUTIONS
Conception and design: Matthew L. Hemming, Kelly S. Klega, Brian D. Crompton, Suzanne George
Financial support: Brian D. Crompton, Suzanne George
Administrative support: Brian D. Crompton, Suzanne George
Provision of study material or patients: Chandrajit P. Raut, Matthew L. Hemming, Suzanne George
Collection and assembly of data: Matthew L. Hemming, Kelly S. Klega, Kate E. Acker, Jessica L. Andersen, Edwin Thai, Anwesha
Nag, Aaron R. Thorner, Brian D. Crompton, Suzanne George
Data analysis and interpretation: Matthew L. Hemming, Kelly S. Klega, Justin Rhoades, Gavin Ha, Brian D. Crompton, Suzanne
George
Manuscript writing: Matthew L. Hemming, Kelly S. Klega, Brian D. Crompton, Suzanne George
Final approval of manuscript: All authors
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Matthew L. Hemming
No relationship to disclose
Kelly S. Klega
No relationship to disclose
Justin Rhoades
No relationship to disclose
Gavin Ha
Patent application: WO2017161175A1
Kate E. Acker
No relationship to disclose
Jessica L. Andersen
No relationship to disclose
Anwesha Nag
No relationship to disclose
Aaron R. Thorner
No relationship to disclose
Chandrajit P. Raut
No relationship to disclose
Suzanne George
Consulting or Advisory Role: Blueprint Medicines, Deciphera Pharmaceuticals
Research Funding: Bayer, Pfizer, Novartis
Brian D. Crompton
No relationship to disclose
HHS Public Access
Author manuscript
JCO Precis Oncol
. Author manuscript; available in PMC 2019 February 19.
Published in final edited form as:
JCO Precis Oncol
. 2019 ; 2019: . doi:10.1200/PO.18.00235.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
dCancer Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
ePublic Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle,
Washington, USA.
fCenter for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA, USA.
gDepartment of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston,
Massachusetts, USA.
Abstract
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.
Keywords
Leiomyosarcoma; circulating tumor DNA; copy number alteration; liquid biopsy
Introduction:
Leiomyosarcoma (LMS) is a malignant neoplasm derived from smooth muscle that
represents one of the most common subtypes of soft tissue sarcoma1, 2. Though the vast
majority of LMS cases are sporadic, predisposing factors include Li-Fraumeni syndrome,
hereditary retinoblastoma and radiation exposure3, 4. There are no oncogenic single
nucleotide variants (SNVs) that characterize LMS, though loss of tumor suppressors
including
TP53
,
RB1
and
PTEN
are commonly seen, as are multiple copy number
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alterations (CNAs)5–8. LMS is frequently a clinically aggressive disease, and patients are at
high risk for local and metastatic relapse following initial complete resection9, 10. Efforts to
improve outcomes for patients would benefit from more reliable indicators of high-risk
disease and biomarkers of response to therapy.
Detection of circulating tumor DNA (ctDNA) has emerged as a new approach for identifying
oncogenic mutations, measuring disease burden, clinical prognostication, and assessing
tumor response to therapy11, 12. Most ctDNA assays have been developed to detect SNVs
that are highly recurrent in many types of carcinomas13. Though the lack of recurrent SNVs
in LMS limits efforts at targeted sequencing, the numerous CNAs characteristic of this
disease represent an ideal target for detection. A next-generation sequencing approach using
ultra-low passage whole genome sequencing (ULP-WGS) can detect CNAs in cell-free
DNA, indicating the tumor fraction of ctDNA present in blood14, 15. Previous studies have
shown that ctDNA can be detected in cell-free DNA samples sequenced at a minimum
coverage of 0.1X across the whole genome14. Sequencing for ULP-WGS uses the standard
Illumina next-generation sequencing platform without modifications or special adaptions.
The ichorCNA algorithm is used to detect megabase-scale CNAs from ULP-WGS data in
which ctDNA comprises as little as 3% of the total cell-free DNA extracted from a plasma
sample14.
In the present study, we evaluated plasma from patients with uterine and extrauterine LMS
for the presence of ctDNA using ULP-WGS. Paired resected tumors from each patient were
also sequenced when available (29/30 patients), enabling the identification and comparison
of CNAs between primary tumors and ctDNA. We related the tumor fraction of cell-free
DNA with the clinical status of the patient’s disease. Finally, we found that longitudinal
measurements of ctDNA declined with tumor resection in one patient and in another patient
became detectable at the time of disease recurrence. These results suggest that monitoring
ctDNA may have clinical utility in establishing the diagnosis, estimating prognosis,
measuring treatment response and performing surveillance for relapse in patients with LMS.
Patients and Methods:
Patients
Patients with LMS who had previously provided written consent for enrollment on an
institutional review board (IRB) approved sample banking and research protocol, including
collection of clinical data, and underwent surgery and treatment at the Brigham and
Women’s Hospital and Dana-Farber Cancer Institute were reviewed for inclusion in the
evaluated cohort. Inclusion required a diagnosis of LMS by pathologic review of a surgically
resected specimen. We identified 30 patients with at least one plasma sample, 29 of which
had a matched tumor sample available for profiling. We also identified an additional 8
patients with only tumor samples. Clinical, radiologic and pathologic data were obtained
from the medical record. All evaluated patients with plasma samples ultimately experienced
either local or metastatic disease recurrence.
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Sample Preparation
Patients had one or serial venous blood draws collected for research into EDTA tubes
(Beckton Dickinson), and plasma was isolated within four hours of collection and stored at
−80°C, as previously described16. A QIAamp Circulating Nucleic Acid Kit (Qiagen) was
used to isolate cell-free DNA from 3.4–5.0 mL of frozen plasma. Genomic DNA from LMS
tumors was isolated from formalin fixed paraffin embedded (FFPE) tissue using a QIAamp
DNA FFPE Tissue kit (Qiagen) or fresh frozen tumors using a QIAamp DNA Mini Kit
(Qiagen).
Ultra-Low Passage Whole Genome Sequencing and ctDNA Quantification
Extracted DNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher
Scientific). High molecular weight DNA contamination of the cell-free DNA was
determined by Bioanalyzer (Agilent) and size selection was performed if necessary
(AMPure XP beads, Agentcourt). DNA extracted from FFPE or fresh frozen tumors was
fragmented (Covaris) to 250 bp and purified with AMPure XP beads. Up to 40 ng of cell-
free DNA, 100 ng of DNA from fresh frozen tissue, and 200 ng of DNA from FFPE tissue
were used for KAPA Hyper library preparation (Kapa Biosystems). Libraries were assessed
for quality by Bioanalyzer followed by quantification using the MiSeq Nano flow cell
(Illumina). Barcoded libraries were pooled and sequencing was performed on a HiSeq 2500
in rapid run mode (Illumina) to a targeted coverage of 0.2X (actual range 0.15X-0.30X).
Seventeen of the 37 tumors were previously sequenced for a separate research project at
higher coverage (range 1.1–2.1X) following Bioruptor sonication (Diagenode) of
formaldehyde-fixed fresh frozen sample, with library preparation using a ThruPLEX DNA-
seq Kit (Rubicon) and sequencing on an Illumina NextSeq 500. This higher sequencing
coverage did not alter downstream analyses or data interpretation.
Sequencing results were demultiplexed, aligned, and processed using Picard, BWA
alignment17, and GATK tools18, 19. To assess for CNAs in tumor and cell-free DNA and
determine tumor fraction, or percentage of ctDNA in cell-free DNA, ichorCNA software was
utilized14 with manual curation of results as necessary to confirm tumor percentages.
Previous studies have demonstrated that this technique can be used to identify and quantitate
ctDNA constituting as little as 3% of the cell-free DNA in a sample14.
Copy Number Analysis
Copy number segments and estimates of tumor fraction and ploidy were generated by
ichorCNA. The log2 ratio values of the segments were adjusted for tumor fraction and
ploidy such that the data were consistent across samples. GISTIC2.020 was used to
determine gene-level copy number analyses. For copy neutral segments predicted by
ichorCNA, the log2 ratio was set to zero. The amplification/deletion log2 ratio threshold
used for GISTIC was 0.3. Significant gains and losses were determined with a false
discovery rate (FDR) of 0.25. This analysis was performed separately on tumor resection
samples (including the 8 samples for which plasma was not available) and on plasma
samples that were positive for detectable levels of ctDNA.
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Statistical Analysis
Group comparisons between LMS patients with Active or Indolent Disease at the time of
plasma collection was performed by non-parametric Mann-Whitney test, and Pearson
correlation coefficient between tumor fraction or cell-free DNA and tumor burden were
performed using GraphPad Prism version 7.0 (GraphPad Software). One was added to
integer values prior to log2 transformation to generate non-negative values for data
representation.
Results:
Detection of LMS ctDNA
We retrospectively identified thirty patients diagnosed with metastatic LMS who had plasma
banking performed at variable time points in their treatment history between January 2007
and November 2017. Two of these patients had plasma samples banked at two separate time
points, for a total of 32 plasma samples. The median age at diagnosis was 51, most patients
were female, the most common primary tumor location was the uterus (
n
=16) followed by
the retroperitoneum (
n
=8), and most tumors were originally of intermediate or high grade
(Table 1). Twenty-nine of the thirty patients had a tumor resection sample available for
comparison to cell-free DNA.
Reviewing clinical data at the time of plasma banking, the cases could be divided into Active
Disease or Indolent Disease groups based upon tumor volume and evidence of disease
progression. The Active Disease group was comprised of sixteen cases and was defined by
having a total tumor burden over 5 cm in greatest diameter and progressive disease at the
time of blood draw, based on imaging or clinical determination. In contrast, sixteen cases in
the Indolent Disease group had stable disease at the time of blood draw and/or tumor burden
less than 5 cm. Among the Active Disease group, eleven of sixteen, or 69%, of cases had
detectible ctDNA, including patients with both uterine and extrauterine primary tumors.
None of the samples obtained from the Indolent Disease group had detectible ctDNA (Fig.
1A).
When comparing the amount of ctDNA (measured as a fraction of total cell-free DNA) in
the plasma sample to the volume of tumor burden reported by CT scan, there was a
significant association between higher ctDNA levels and increasing tumor burden (Fig. 1B).
In contrast, there was no correlation between the amount of total cell-free DNA extracted
from the plasma sample and tumor burden (Fig. 1C). Several cases in the Indolent Disease
group of similarly high tumor burden produced no detectible ctDNA despite significant
tumor volume. Taken together, these data demonstrate that ctDNA is detectible using an
ULP-WGS approach in the majority of LMS patients with a tumor burden of >5 cm and
progressive disease.
CNA concordance between LMS tumors and ctDNA
In the eleven plasma samples with detectible levels of ctDNA, there was a high concordance
of CNAs between the tumor sample and ctDNA (Fig. 2A-D), with blood collection
occurring under two years apart from resection of the matched tumor specimen. In cases of
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high ctDNA with lower tumor fraction in the surgical sample, CNAs were more pronounced
in cell-free DNA (Fig. 2A). In contrast, with low tumor fraction many CNAs seen in the
surgical sample could not be readily identified (Fig. 2D-E). These results are consistent with
prior publications demonstrating agreement between plasma and tumor CNAs21 and further
underscores the diversity of chromosomal gains and losses seen across LMS tumors.
To characterize recurrent gene-level CNAs in tumor and cell-free DNA in this LMS cohort
we utilized GISTIC2.020. Overall, recurrent CNAs from tumors in this cohort are
remarkably similar to those found in prior studies5, 8. The most significantly amplified
genomic region was found on chromosome 17p which includes the transcriptional regulator
MYOCD
(copy gained in 20 of 37 tumors). This gene has previously been reported as
significantly amplified in LMS and associated with smooth muscle differentiation22.
Additional putative oncogenic and LMS-associated genes that were recurrently amplified
include
HDGF
in 20,
MYC
in 14,
CTHRC1
in 17,
TNFRSF19
in 10 and
MYH2
in 16 of 37
tumors (Fig. 3A). Copy gains in these genes were also observed in a portion of cell-free
DNA samples with detectable ctDNA (
MYOCD
in 4,
HDGF
in 6,
MYC
in 8,
CTHRC1
in 9,
TNFRSF19
in 3, and
MYH2
in 4 out of 11 ctDNA positive samples), however the number of
evaluable samples was too small for any copy gains of these genes to reach statistical
significance (Fig. 3B). From tumor samples, recurrent deletions in tumor suppressors were
found which are characteristic of LMS5, 8, 23, including
PTEN
deletions in 27,
RB1
in 27,
and
TP53
in 11 of 37 tumors profiled (Fig. 3C). Deletions involving these genes were also
detected in ctDNA (
PTEN
in 8,
RB1
in 6, and
TP53
in 2 of 11 ctDNA positive samples) but
sample size was too small for many of these deletions to reach significance (Fig. 3D). As
described in previous studies, gains and losses of these genes were caused by events that
ranged from focal (megabase scale) to whole-chromosomal copy-number alterations. Thus,
ULP-WGS is capable of detecting recurrent CNAs characteristic of LMS, and these methods
may be helpful in identifying gene-level CNAs in ctDNA.
ctDNA longitudinally correlates with disease status
To determine whether ctDNA levels change in agreement with longitudinal disease status we
evaluated serial plasma samples in two patients following either resection of localized LMS
or with disease recurrence following surgery. In the patient undergoing resection of locally
recurrent disease, ctDNA detected pre-operatively was eliminated 5 weeks following surgery
(Fig. 4A). In contrast, in the patient with disease recurrence 32 months following surgical
resection, the cell-free DNA tumor fraction increased from undetectable post-operatively to
detectable at the time of disease recurrence (Fig. 4B). These data demonstrate that ctDNA
correlates in individual patients with disease burden over time in this small cohort, and that
such liquid biopsies may represent a valuable diagnostic tool supporting evidence of LMS
recurrence or disease burden.
Discussion:
In the present study we analyzed cell-free DNA from the plasma of patients with LMS for
evidence of ctDNA. Stratifying patients by tumor burden and Active versus Indolent
Disease, we find that the majority of patients with Active Disease have detectable ctDNA. A
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significant association between tumor burden and the amount of ctDNA was identified,
suggesting that larger and actively growing tumors are more likely to release tumor DNA
into circulation. We were unable to detect ctDNA in any tumor under 5 cm in size,
regardless of evidence of disease progression, which may represent a technological
sensitivity threshold. Additionally, we were unable to detect ctDNA in patients with
significant disease burden but lacking evidence of disease progression. This presumably
arises from reduced levels of apoptosis or necrosis in tumors without active growth, which
may be exploited to determine the therapeutic benefit of anti-neoplastic therapies. DNA
from tumor resections and associated ctDNA demonstrated preserved CNAs, and recurrent
CNAs were found across LMS samples. We further found that ctDNA levels decline with
resection of disease and increase with disease recurrence in individual patients, indicating
that these methods may be useful for supporting a diagnosis of disease recurrence or
response to therapy.
Sequencing of SNVs in cell-free DNA has proven clinical utility in detecting resistance
mutations to targeted therapies and directing treatment strategies24. The emerging use of
whole genome sequencing to identify and quantify CNAs from tumor-derived DNA
circulating in cancer patients has been evaluated in a number of cancer types14, 25–27. This
non-invasive approach can be useful in the genomic characterization of malignancy and
provide diagnostic and prognostic information relevant for clinical care15, 21, 28.
In a recent study of patients with LMS, a highly customized LMS-specific assay (CAPP-
Seq) was designed for detection of selected SNVs and combined with CNA analysis to
identify and quantify ctDNA in patients with progressive disease29. In this study, the
investigators found that approximately 20% of patients (2 of 9) had tumors that did not have
somatic events detectable by the assay. In contrast, we found that tumors from 100% of
cases had CNAs detectable by ULP-WGS, suggesting that ULP-WGS may be more broadly
applicable for patients with LMS. Conversely, CAPP-Seq was able to detect ctDNA in 6 of 7
baseline samples of patients eligible for study (86%) and had a sensitivity of 68% among all
samples tested from their cohort. By comparison, our study detected ctDNA in 11 of 16
(69%) patients with active disease. Although these numbers are small, this suggests that
CAPP-Seq may have a higher sensitivity for ctDNA as might be expected given the deep
sequencing coverage used in this assay. It remains unclear what assay features will have the
highest clinical utility in sarcomas, and we expect that different assays may be beneficial for
different clinical indications. For example, our recent study in osteosarcoma demonstrated
that pre-treatment ctDNA levels detected by ULP-WGS are prognostic28 whereas a more
sensitive assay, such as CAPP-Seq, may be advantageous in the setting of disease
surveillance29.
There are several potential clinical uses of ctDNA in the diagnosis and management of LMS.
First, ctDNA may be able to differentiate benign smooth muscle neoplasms, such as
leiomyoma, from LMS30, 31. This knowledge would principally inform surveillance of
suspicious lesions and considerations of operative strategies for uterine tumors that could
potentially harbor LMS32. Second, there is significant clinical uncertainty regarding which
LMS patients derive benefit from adjuvant chemotherapy and radiation10, 33, 34. Should
ctDNA levels bear prognostic significance for tumors at highest risk of recurrence or
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identify the presence of residual disease, their measurement may help guide clinical
decisions regarding adjuvant therapies. Finally, ctDNA levels may be a useful indicator of
response to systemic therapy, and provide an early indication for switching or intensifying
treatment regimens used in this disease3, 35. Together with technological improvements in
sensitivity and throughput, these initial reports identifying ctDNA in LMS may quickly
evolve to transform clinical practice. Larger prospective studies will be needed to determine
the optimal approaches for adapting ctDNA assays to the clinical care of patients with LMS.
ACKNOWLEDGMENT
We are indebted to the patients and families who donated clinical samples that enabled this research.
SUPPORT
Support for this work was provided by the following sources: American Society of Oncology Conquer Cancer
Foundation Young Investigator Award (M.L.H.), NIH Grant K08 CA188073–01A1 (B.D.C.), Friends of Dana-
Farber Cancer Institute (B.D.C.), The Catherine England Leiomyosarcoma Fund (S.G) and The Jill Effect (S.G.)
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Fig 1.
Identification of ctDNA in plasma from LMS patients. (A) Plot of percent ctDNA in Active
Disease (
n
=16) and Indolent Disease (
n
=16) LMS groups. The Active Disease group
consists of patients with tumors >5 cm in size and with progressive disease at the time of
blood draw as indicated by CT scan or clinical report. The Indolent Disease group consists
of patients with tumors <5 cm in size and/or no evidence of disease progression by CT scan.
Groups were compared by Mann-Whitney test; ***,
P
< 0.0001. (B) Scatterplot of log2
tumor fraction and tumor burden. Labels indicate the Active Disease subgroup (red) with
tumor size >5 cm and progressive disease (
n
=16), or Indolent Disease subgroups (gray) with
tumor size >5 cm and stable disease (
n
=2, diamond), tumor size <5 cm and progressive
disease (
n
=4, triangle), and tumor size <5 cm and stable disease (
n
=10, circle). Tumor
burden was determined by adding the diameters of tumor lesions reported on CT scan at the
time of ctDNA assessment. The Pearson correlation coefficient between tumor fraction and
tumor burden is shown for all patients with measurable disease. (C) Scatterplot of log2 total
extracted cell-free DNA from all plasma samples (
n
=32) and tumor burden. The Pearson
correlation coefficient across all samples is shown.
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Fig 2.
Concordant CNAs in LMS tumors and ctDNA. (A-E) Copy number plots generated from
ULP-WGS sequencing of five representative tumor surgical (left panels) and plasma (right
panels) sample pairs. The x-axis indicates chromosome and y-axis copy number (log2 ratio).
The tumor fraction is indicated for each plot.
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Fig 3.
Recurrent CNAs in LMS. (A-D) GISTIC2.0 analysis identifying recurrent focal amplified
(A-B) and deleted (C-D) regions in LMS tumors (
n
=37, left panels) and cell-free DNA
samples with detectable ctDNA (
n
=11, right panels). LMS-associated genes and/or putative
oncogenes are labeled in
A
, while putative tumor suppressor genes are labeled in
C
. The x-
axis indicates chromosome and y-axis FDR. The green line indicates a FDR of 0.25.
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Fig 4.
LMS ctDNA correlates with changes in disease burden. (A-B) Change in tumor fraction in
the pre-operative and post-operative setting, with images indicating tumor burden at each
time point. (C-D) Change in tumor fraction following unifocal recurrence of disease, with
images indicating tumor burden at each time point. The interval between surgical date and
cell-free DNA collection is indicated.
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Hemming et al. Page 15
Table 1.
Clinical characteristics of 30 LMS cases with plasma samples. For tumor volume, progressive disease and ctDNA columns, assessment was performed at
the time of plasma collection. Two patients, LMS27 and LMS28, had serial plasma samples drawn, with data displayed in the table matching the time
point where ctDNA was detected. “NA” indicates that data were not reported during pathology review. HPF, high power fields; C, chest; A, abdomen; L,
liver; P, pelvis; ST, soft tissue including subcutaneous, muscle and paraspinal locations; B, bone.
ID Sex Age at Diagnosis Primary Tumor Location Primary Tumor Grade
Primary
Tumor
Mitotic
Rate Location of Metastases Tumor Volume >5cm Progressive Disease ctDNA Present
LMS1 F 49 Uterus High 10:1HPF C,A,L,P,B + + +
LMS2 F 60 Uterus NA 17:10HPF A,ST,B + + +
LMS3 F 42 Uterus Low 5:10HPF C - - -
LMS4 F 46 Uterus High 40:10HPF C,A,P + + +
LMS5 F 44 Uterus High NA C - - -
LMS6 F 50 Uterus NA 9:10HPF P - - -
LMS7 F 51 Uterus High >20:10HPF C - + -
LMS8 F 55 Retroperitoneum High 39:10HPF C,L,B,ST + + +
LMS9 M 70 Retroperitoneum High 22:10HPF C,A + + +
LMS10 F 61 Retroperitoneum Intermediate 5:10HPF C,A + + -
LMS11 F 58 Intraperitoneal NA 37:10HPF A,P + + +
LMS12 F 50 Uterus High >40:10HPF C,A,P + + -
LMS13 F 19 Intraperitoneal Low 4:10HPF A - - -
LMS14 F 24 Abdominal wall NA 23:10HPF C - + -
LMS15 F 59 Uterus Low 4:10HPF P - - -
LMS16 F 62 Uterus Intermediate 25:10HPF C,A,P + - -
LMS17 F 59 Uterus Intermediate 3:10HPF ST - - -
LMS18 F 52 Retroperitoneum High NA L,ST - + -
LMS19 F 85 Retroperitoneum High 10:10HPF C,A + + +
LMS20 F 38 Pelvis NA 17:10HPF P + + -
LMS21 F 59 Retroperitoneum NA 23:10HPF A,L,P + - -
LMS22 F 48 Uterus NA 20:10HPF A,P - - -
LMS23 M 30 Extremity High 33:10HPF C,L,A + + +
LMS24 M 48 Paratesticular Intermediate 20:10HPF C,L,A,ST + + +
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ID Sex Age at Diagnosis Primary Tumor Location Primary Tumor Grade
Primary
Tumor
Mitotic
Rate Location of Metastases Tumor Volume >5cm Progressive Disease ctDNA Present
LMS25 F 43 Retroperitoneum NA 8:10HPF C - - -
LMS26 F 47 Retroperitoneum High 29:10HPF L - + -
LMS27 F 73 Uterus High 52:10HPF P + + +
LMS28 F 48 Uterus High 46:10HPF A,P + + +
LMS29 F 53 Uterus NA 30:10HPF C + + -
LMS30 F 65 Uterus NA NA A + + -
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... Several studies show that ctDNA levels correlate with prognosis and that changes in ctDNA levels can be associated with response to therapy (7)(8)(9). In previous work, we demonstrated that ctDNA can be detected and quantified in patients with LMS and found that ctDNA levels correlate with tumor size and tracked longitudinally with changes in disease burden (10). However, this cohort was a small, clinically heterogenous population and associations with ctDNA levels and tumor burden were descriptive. ...
... In previous work, we have demonstrated that the detection of ctDNA in patients with LMS is feasible using a low-cost sequencing approach that identified the presence of segmental copy-number variants, characteristic of the somatic landscape of LMS, in cell-free DNA isolated from peripheral blood samples (10). Although this assay has limited sensitivity, being unable to detect ctDNA when constituting less than 3% of a cell-free DNA sample, this limit of detection has proven to be a convenient threshold for differentiating patients with high levels of ctDNA from those with low levels (10,15,16,21). ...
... In previous work, we have demonstrated that the detection of ctDNA in patients with LMS is feasible using a low-cost sequencing approach that identified the presence of segmental copy-number variants, characteristic of the somatic landscape of LMS, in cell-free DNA isolated from peripheral blood samples (10). Although this assay has limited sensitivity, being unable to detect ctDNA when constituting less than 3% of a cell-free DNA sample, this limit of detection has proven to be a convenient threshold for differentiating patients with high levels of ctDNA from those with low levels (10,15,16,21). In the case of osteosarcoma and Ewing sarcoma, the distinction between high and low levels of ctDNA is prognostic (21). ...
Article
Full-text available
Purpose We sought to determine whether the detection of circulating tumor DNA (ctDNA) in samples of patients undergoing chemotherapy for advanced leiomyosarcoma (LMS) is associated with objective response or survival. Methods Using ultra–low-passage whole-genome sequencing (ULP-WGS) of plasma cell-free DNA from patients treated on a prospective clinical trial, we tested whether detection of ctDNA evaluated prior to the start of therapy and after two cycles of chemotherapy was associated with treatment response and outcome. Associations between detection of ctDNA and pathologic measures of disease burden were evaluated. Results We found that ctDNA was detectable by ULP-WGS in 49% patients prior to treatment and in 24.6% patients after two cycles of chemotherapy. Detection of pretreatment ctDNA was significantly associated with a lower overall survival [HR, 1.55; 95% confidence interval (CI), 1.03–2.31; P = 0.03] and a significantly lower likelihood of objective response [odds ratio (OR), 0.21; 95% CI, 0.06–0.59; P = 0.005]. After two cycles of chemotherapy, patients who continued to have detectable levels of ctDNA experienced a significantly worse overall survival (HR, 1.77; 95% CI, 1–3.14; P = 0.05) and were unlikely to experience an objective response (OR, 0.05; 95% CI, 0–0.39; P = 0.001). Conclusions Our results demonstrate that detection of ctDNA is associated with outcome and objective response to chemotherapy in patients with advanced LMS. These results suggest that liquid biopsy assays could be used to inform treatment decisions by recognizing patients who are likely and unlikely to benefit from chemotherapy.
... Hemming et al. evaluated the utility of circulating tumor DNA (ctDNA) as a bloodbased biomarker in patients with leiomyosarcoma to detect progression or relapse of disease using the non-invasive liquid biopsy technique [16]. In 11 (69%) out of 16 patients who showed LMS progression and a primary tumor >5 cm in diameter, the ctDNA was detectable. ...
... In 11 (69%) out of 16 patients who showed LMS progression and a primary tumor >5 cm in diameter, the ctDNA was detectable. Additionally, in 16 patients with low grade disease, the liquid biopsy revealed no amounts of circulating ctDNA, in contrast with detection of high levels of ctDNA in patients with increasing tumor size [16]. Subsequently, the ctDNA was characterized as a remarkable biomarker in LMS, as it diminished with tumor resection but increased in tumor relapse. ...
Article
Full-text available
Soft tissue and bone sarcomas represent a group of aggressive neoplasms often accompanied by dismal patient prognosis, especially when distant metastases are present. Moreover, effective treatment can pose a challenge, as recurrences are frequent and almost half of patients present with advanced disease. Researchers have unveiled the molecular abnormalities implicated in sarco-mas' carcinogenesis, paving the way for novel treatment strategies based on each individual tumor's characteristics. Therefore, the development of new techniques aiding in early disease detection and tumor molecular profiling is imperative. Liquid biopsy refers to the sampling and analysis of pa-tients' fluids, such as blood, to identify tumor biomarkers, through a variety of methods, including qRT-PCR, qPCR, droplet digital PCR, magnetic microbeads and digital PCR. Assessment of circulating tumor cells (CTCs), circulating free DNA (ctDNA), micro RNAs (miRs), long non-coding RNAs (lncRNAs), exosomes and exosome-associated proteins can yield a plethora of information on tumor molecular signature, histologic type and disease stage. In addition, the minimal invasive-ness of the procedure renders possible its wide application in the clinical setting, and, therefore, the early detection of the presence of tumors. In this review of the literature, we gathered information on biomarkers assessed through liquid biopsy in soft tissue and bone sarcoma patients and we present the information they can yield for each individual tumor type.
... After segmentation, the genomic identification of significant targets in cancer (GISTIC) algorithm was applied to compare the gene-level CNAs. To determine the significant amplification and deletion regions, the significance threshold for the q-values was set to 0.25 [31]. We grouped the tissue, plasma, and peritoneal fluid samples separately and analyzed the recurrent regions of CNAs for each group. ...
Article
Full-text available
Endometrial cancer (EC) is the most common type of gynecological cancer. Studies comparing tumor gDNA and ctDNA isolated from the plasma and peritoneal fluid of EC patients are limited. Whole-exome sequencing and P53 immunohistochemistry of 24 paired tissue, plasma, and peritoneal fluid samples from 10 EC patients were performed to analyze somatic mutations, copy number alterations, microsatellite instability, and mutational signatures. Mutations in cancer-related genes (KMT2C, NOTCH2, PRKAR1A, SDHA, and USP6) and genes related to EC (ARID1A, CTNNB1, PIK3CA, and PTEN) were identified with high frequencies among the three samples. TP53 and POLE mutations, which are highly related to the molecular classification of EC, were identified based on several key observations. The ctDNA of two patients with negative peritoneal fluid presented TP53 mutations concordant with those in tissues. ctDNA from the plasma and peritoneal fluid of a patient with positive cytology harbored both TP53 and POLE mutations, although none were detected in tissues. Additionally, the patient presented with wild type P53 immunohistochemistry, with a focal “high” expression in a “low” wild type background. The tissues and peritoneal fluid of 75% EC patients showed concordant microsatellite instability. Furthermore, we observed strong mutational concordance between the peritoneal fluid and tumors. Our data suggest that the ctDNA from peritoneal fluid might be a suitable biomarker for identifying the mutational landscape of EC and could complement tumor heterogeneity.
... 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. ...
Article
Full-text available
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.
... Previous studies of ctDNA in patients with different types of sarcomas showed the feasibility of detecting tumor-derived genomic aberrations in circulation, but also revealed technological challenges that must be addressed to achieve sensitive detection of ctDNA. Our group and others have previously showed that tumor-derived copy number aberrations can be detected in plasma of patients with leiomyosarcoma (LMS) and leiomyoma (LM) [2][3][4]. In LMS patients, we also showed that the levels of ctDNA correspond with response to treatment, and that a combination approach that integrates detection of point mutations and copy number alterations substantially increases the number of molecular markers that can be tracked in plasma [2]. ...
Article
Full-text available
High-level amplification of MDM2 and other genes in the 12q13–15 locus is a hallmark genetic feature of well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS, respectively). Detection of this genomic aberration in plasma cell-free DNA may be a clinically useful assay for non-invasive distinction between these liposarcomas and other retroperitoneal tumors in differential diagnosis, and might be useful for the early detection of disease recurrence. In this study, we performed shallow whole genome sequencing of cell-free DNA extracted from 10 plasma samples from 3 patients with DDLPS and 1 patient with WDLPS. In addition, we studied 31 plasma samples from 11 patients with other types of soft tissue tumors. We detected MDM2 amplification in cell-free DNA of 2 of 3 patients with DDLPS. By applying a genome-wide approach to the analysis of cell-free DNA, we also detected amplification of other genes that are known to be recurrently affected in DDLPS. Based on the analysis of one patient with DDLPS with longitudinal plasma samples available, we show that tracking MDM2 amplification in cell-free DNA may be potentially useful for evaluation of response to treatment. The patient with WDLPS and patients with other soft tissue tumors in differential diagnosis were negative for the MDM2 amplification in cell-free DNA. In summary, we demonstrate the feasibility of detecting amplification of MDM2 and other DDLPS-associated genes in plasma cell-free DNA using technology that is already routinely applied for other clinical indications. Our results may have clinical implications for improved diagnosis and surveillance of patients with retroperitoneal tumors.
... In this patient cohort, the tumor burden ranged from no evidence of disease to progressive metastatic disease. The results showed that high levels of ctDNA were associated with an increase in tumor size and disease progression [66]. ...
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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.
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Leiomyosarcomas are soft tissue tumors that are derived from smooth muscle mainly in the pelvis and retroperitoneum. Percutaneous biopsy is paramount to confirm diagnosis. Imaging is necessary to complete clinical staging. Multimodal treatment should be directed by expert sarcoma multidisciplinary teams that see a critical volume of these rare tumors. Surgery is the mainstay of curative intent treatment; however due to its high metastatic progression, there may be a benefit for neoadjuvant systemic treatment. Adjuvant systemic treatment has no proven disease-free survival, and its main role is in the palliative setting to potentially prolong overall survival.
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Purpose: Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize LMS patient derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies. Experimental design: We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA-seq and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling. Results: We show that LMS PDX maintain the histologic appearance, copy-number alterations and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX co-cluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional CDK inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth. Conclusions: Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for LMS patients.
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Leiomyoma with bizarre nuclei (LM-BN) is a rare variant of leiomyoma with overall benign clinical course. It has histologic features showing focal or diffuse nuclear atypia surrounded by usual type leiomyoma. Uterine leiomyosarcomas (LMS) are a group of rare and aggressive malignancies with limited treatment options available. The potential association between LM-BN with LMS is largely unknown. In this study, we report 2 cases of uterine smooth muscle tumor with typical histologic and molecular evidence of LM-BN, which are associated with its progression to the malignant counterpart of LMS. We summarize the detailed histologic, morphologic, and genomic characteristics of these 2 sets of cases. Our findings suggest that LMS progressing from preexisting LM-BN can be one of the tumor pathogenesis pathways in uterine leiomyomas.
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Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.
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Sarcomas are a broad family of mesenchymal malignancies exhibiting remarkable histologic diversity. We describe the multi-platform molecular landscape of 206 adult soft tissue sarcomas representing 6 major types. Along with novel insights into the biology of individual sarcoma types, we report three overarching findings: (1) unlike most epithelial malignancies, these sarcomas (excepting synovial sarcoma) are characterized predominantly by copy-number changes, with low mutational loads and only a few genes (TP53, ATRX, RB1) highly recurrently mutated across sarcoma types; (2) within sarcoma types, genomic and regulomic diversity of driver pathways defines molecular subtypes associated with patient outcome; and (3) the immune microenvironment, inferred from DNA methylation and mRNA profiles, associates with outcome and may inform clinical trials of immune checkpoint inhibitors. Overall, this large-scale analysis reveals previously unappreciated sarcoma-type-specific changes in copy number, methylation, RNA, and protein, providing insights into refining sarcoma therapy and relationships to other cancer types. Genetic analysis of soft tissue sarcomas shows that they are characterized predominantly by copy-number changes and offers insights into the immune microenviroment to inform clinical trials of checkpoint inhibitors.
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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.
Article
Objective: Liquid biopsies are being rapidly used in adult cancers as new biomarkers of disease. Circulating tumor DNA (ctDNA) levels have been reported to be proportional to disease burden, correlate with treatment response, and predict relapse. However, little is known about how frequently ctDNA is detectable in pediatric patients with solid tumors. Therefore, we developed a next-generation sequencing approach to detect and quantify ctDNA in the blood of patients with the most common pediatric solid tumors. Methods: Detection of ctDNA requires assays sensitive to somatic events typically observed in the cancer type being studied. In pediatric solid tumors, structural variants are more common than recurrent point mutations. We adapted an ultralow passage whole-genome sequencing approach to capture copy number variants and a hybrid capture sequencing assay to detect translocations in liquid biopsy samples from pediatric patients. Results: Copy number changes seen by ultralow passage whole-genome sequencing enabled detection of ctDNA in patients with osteosarcoma, neuroblastoma, alveolar rhabdomyosarcoma, and Wilms tumor. In Ewing sarcoma, detection of the EWSR1 translocation was a more sensitive approach. For patients with samples collected at multiple time points, changes in ctDNA levels corresponded to treatment response. We also found that disease-specific genomic biomarkers of prognosis were detectable in ctDNA. Conclusion: This study demonstrates that liquid biopsy approaches that detect somatic structural variants are well suited to pediatric solid tumors. We show that children with the most common solid tumor malignancies have detectable levels of ctDNA, which may be used to track disease response and identify genomic subclassifiers of disease. Efforts to profile larger collections of clinically annotated specimens are under way to validate the clinical use of these assays.
Article
Purpose: The clinical utility of circulating tumor DNA (ctDNA) monitoring has been shown in tumors that harbor highly recurrent mutations. Leiomyosarcoma (LMS) represents a type of tumor with a wide spectrum of heterogeneous genomic abnormalities; thus, targeting hotspot mutations or a narrow genomic region for ctDNA detection may not be practical. Here we demonstrate a combinatorial approach that integrates different sequencing protocols for the orthogonal detection of single nucleotide variants (SNVs), small indels and copy number alterations (CNAs) in ctDNA. Experimental design: We employed Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for the analysis of SNVs and indels, together with a genome-wide interrogation of CNAs by Genome Representation Profiling (GRP). We profiled 28 longitudinal plasma samples and 25 tumor specimens from 7 patients with LMS. Results: We detected ctDNA in 6 of 7 of these patients with >98% specificity for mutant allele fractions down to a level of 0.01%. We show that results from CAPP-Seq and GRP are highly concordant, and the combination of these methods allows for more comprehensive monitoring of ctDNA by profiling a wide spectrum of tumor-specific markers. By analyzing multiple tumor specimens in individual patients obtained from different sites and at different times during treatment, we observed clonal evolution of these tumors that was reflected by ctDNA profiles. Conclusions: Our strategy allows for a comprehensive monitoring of a broad spectrum of tumor-specific markers in plasma. Our approach may be clinically useful not only in LMS but also in other tumor types that lack recurrent genomic alterations.
Article
Purpose Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with patient prognosis. Triple-negative breast cancer (TNBC) is characterized by few mutations but extensive somatic copy number alterations (SCNAs), yet little is known regarding SCNAs in metastatic TNBC. We sought to evaluate SCNAs in metastatic TNBC exclusively via cfDNA and determine if cfDNA tumor fraction is associated with overall survival in metastatic TNBC. Patients and Methods In this retrospective cohort study, we identified 164 patients with biopsy-proven metastatic TNBC at a single tertiary care institution who received prior chemotherapy in the (neo)adjuvant or metastatic setting. We performed low-coverage genome-wide sequencing of cfDNA from plasma. Results Without prior knowledge of tumor mutations, we determined tumor fraction of cfDNA for 96.3% of patients and SCNAs for 63.9% of patients. Copy number profiles and percent genome altered were remarkably similar between metastatic and primary TNBCs. Certain SCNAs were more frequent in metastatic TNBCs relative to paired primary tumors and primary TNBCs in publicly available data sets The Cancer Genome Atlas and METABRIC, including chromosomal gains in drivers NOTCH2, AKT2, and AKT3. Prespecified cfDNA tumor fraction threshold of ≥ 10% was associated with significantly worse metastatic survival (median, 6.4 v 15.9 months) and remained significant independent of clinicopathologic factors (hazard ratio, 2.14; 95% CI, 1.4 to 3.8; P < .001). Conclusion We present the largest genomic characterization of metastatic TNBC to our knowledge, exclusively from cfDNA. Evaluation of cfDNA tumor fraction was feasible for nearly all patients, and tumor fraction ≥ 10% is associated with significantly worse survival in this large metastatic TNBC cohort. Specific SCNAs are enriched and prognostic in metastatic TNBC, with implications for metastasis, resistance, and novel therapeutic approaches.
Article
Leiomyosarcoma (LMS) is one of the most common subtypes of soft tissue sarcoma in adults and can occur in almost any part of the body. Uterine leiomyosarcoma is the most common subtype of uterine sarcoma. Increased awareness of this unique histology has allowed for the development of drugs that are specific to LMS and has begun to shed light on the similarities and possible unique aspects of soft tissue and uterine LMS. In this review, we summarize the current understanding of the epidemiology, diagnosis, genomics, and treatment options for LMS.
Article
Uterine leiomyosarcomas (uLMS) are rare, aggressive malignancies for which limited treatment options are available. To gain novel molecular insights into uLMS and identify potential novel therapeutic targets, we characterized 84 uLMS samples for genome-wide somatic copy number alterations, mutations, gene fusions and gene expression, and performed a data integration analysis. We found that alterations affecting TP53, RB1, PTEN, MED12, YWHAE and VIPR2 were present in the majority of uLMS. Pathway analyses additionally revealed that the PI3K/AKT/mTOR, estrogen-mediated S-phase entry and DNA damage response signaling pathways, for which inhibitors have already been developed and approved, frequently harbored genetic changes. Furthermore, a significant proportion of uLMS was characterized by amplifications and overexpression of known oncogenes (CCNE1, TDO2), as well as deletions and reduced expression of tumor suppressor genes (PTEN, PRDM16). Overall, it emerged that the most frequently affected gene in our uLMS samples was VIPR2 (96%). Interestingly, VIPR2 deletion also correlated with unfavorable survival in uLMS patients (multivariate analysis; HR=4.5, CI=1.4-14.3, p=1.2E-02), while VIPR2 protein expression was reduced in uLMS versus normal myometrium. Moreover, stimulation of VIPR2 with its natural agonist VIP decreased SK-UT-1 uLMS cell proliferation in a dose-dependent manner. These data suggest that VIPR2, which is a negative regulator of smooth muscle cell proliferation, might be a novel tumor suppressor gene in uLMS. Our work further highlights the importance of integrative molecular analyses, through which we were able to uncover the genes and pathways most frequently affected by somatic alterations in uLMS. This article is protected by copyright. All rights reserved.