Kim et al. BMC Medical Genetics(2020) 21:200
https://doi.org/10.1186/s12881-020-01137-4

CASE REPORTOpen Access

Pathogenic NF1 truncating mutation andcopy number alterations in a
dedifferentiated liposarcoma with multiple
lung metastasis: a case report
Yoon-Seob Kim1,2,3, Sun Shin1,2,3, Seung-Hyun Jung2,3,4 and Yeun-Jun Chung1,2,3,5*Abstract
Background: Dedifferentiated liposarcoma (DDLPS), which accounts for an estimated 15–20% of liposarcomas, is ahigh-grade and aggressive malignant neoplasm, exhibiting a poor response to available therapeutic agents. However,genetic alteration profiles of DDLPS as well as the role of NF1 mutations have not been studied extensively.
Case presentation: The current study reports a patient presenting with rapidly growing DDLPS accompanied bymultiple lung and pleural metastases, in whom whole-exome sequencing revealed a NF1 truncating mutation of the
known pathogenic variant, c.C7486T, p.R2496X, as well as multiple copy number alterations (CNAs), including the wellknown 12q13–15 amplification, and multiple chromothripsis events encompassing potential cancer-related genes.
Conclusions: Our results suggest that, in addition to the 12q13–15 amplification, NF1 inactivation mutation and other
CNAs may contribute to DDLPS tumorigenesis accompanied by aggressive clinical features.
Keywords: Case report, Copy number alternation, Liposarcoma, Mutation, NF1, Copy number alteration

Background
Liposarcoma is the most common type of adult soft tissue
sarcoma. Dedifferentiated liposarcoma (DDLPS), which
accounts for an estimated 15–20% of liposarcomas, is ahigh-grade, aggressive disease that shows a poor response toavailable therapies [1]. Genetic analysis of DDLPS via The
Cancer Genome Atlas (TCGA) project revealed that, compared with other solid tumors, DDLPS carried frequent copy
number alterations (CNA) including recurrent amplification
at 12q13–15, and relatively lower somatic mutations with
only a few recurrently mutated genes, such as TP53 andATRX [2]. Regarding CNAs, DDLPS is characterized by* Correspondence: yejun@catholic.ac.kr
1
Department of Microbiology, College of Medicine, The Catholic University of
Korea, Seoul, Republic of Korea
2
Integrated Research Center for Genome Polymorphism, College of Medicine,
The Catholic University of Korea, Seoul, Republic of Korea
Full list of author information is available at the end of the article

highly recurrent amplifications in the 12q13–15 region that
contains the potential oncogenes, MDM2, CDK4, YEATS4,
and FRS2, and the adipocytic differentiation factors, DDIT,PTPRQ, and HMGA2 [2–6]. In addition to the 12q13–15
amplification, other CNAs such as the gain of 1p32 (JUN),and the loss of 17q11 (NF1) and Xq21 (ATRX) have been
identified in DDLPS [2–6]. However, genetic alteration profiles of DDLPS have not been studied extensively.
According to studies such as GENIE [7] and TCGA [2],NF1 mutations in DDLPS are rare, being limited to 2/179
cases in GENIE and 1/56 cases in TCGA. (Fig. S1A).
MDM4 amplification was observed in 0.6 and 5% of
DDLPS cases via the GENIE study [7] and the TCGA
study [2], respectively (Fig. S1A). In TCGA soft tissue
sarcoma project (N = 265) in cBioPortal database, patients
carrying NF1 truncating or missense mutations showedsignificantly lower NF1 mRNA expression levels than

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Kim et al. BMC Medical Genetics(2020) 21:200

those in non-mutated cases. (Mann-Whitney U test,
P = 0.012) (Fig. S1B). Patients carrying MDM4 gains
or amplifications showed significantly higher MDM4mRNA expression levels compared with those of
diploid or shallow deletion cases. (Mann-Whitney U
test, P < 0.001) (Fig. S1C).
Herein, we report a patient with rapidly growing
DDLPS with multiple lung and pleural metastasis, inwhom whole-exome sequencing (WES) revealed a NF1
truncating mutation of a known pathogenic variant andmultiple CNAs including MDM4 gain.

Case presentation
An eighty four-year old male visited the outpatient clinic
complaining of a painful mass that had been present on
his left thigh for 3 years. He was medically healthy and did
not have a previous medical or familial history of malignancy. There was no clinical sign of neurofibromatosis.
He had twice undergone excisional biopsies (18 and 3
years ago) of the mass at the same location under the

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presumed diagnosis of lipoma. Preoperative magnetic resonance imaging revealed an approximately 17.5 × 16.4 ×
30.2 cm sized, extensive, lobulated heterogeneous mass
with T2 high, T1 high signal intensity involving the left
thigh. A wide local excision was performed. Gross pathology of the tumor showed a well circumscribed tumor
mass with internal multi-lobulated areas (Fig. 1a). Multiple
lung and pleural metastases were diagnosed via chest
computed tomography (Fig. 1b). Histological findings
showed dedifferentiated areas with spindle cells without alipomatous portion and less dedifferentiated areas consisting of round cells with lipomatous portions, along with infiltrated polymorphonuclear cells (Fig. 1c). Based on theseclinical and pathological findings, the mass was diagnosed
as DDLPS. The patient died 2 months following surgery.
A DDLPS frozen tissue was obtained from the biobank ofSeoul St. Mary Hospital (Seoul, Republic of Korea). Genomic DNA was extracted by microdissection of tumor cellrich area (> 70% of tumor cell purity) and whole blood of
the patient using the DNeasy Blood & Tissue Kit (Qiagen,Fig. 1 Pathological and radiological features of the DDLPS case. a On gross pathology, tumor showed well circumscribed tumor mass withinternal multilobulated areas. b Multiple lung (blue arrow) and pleural metastases (red arrow) were detected by chest computed tomography. cHistological findings of representative tumor area showed dedifferentiated areas with spindle cells without lipogenic portion (left panel) and lessdedifferentiated areas with round cells with lipogenic portion (right panel) (hematoxylin and eosin, original magnification, × 100)

Kim et al. BMC Medical Genetics(2020) 21:200

Hilden, Germany). WES was performed using the Agilent
SureSelect Human All Exome 50 Mb Kit (Agilent Technologies, Santa Clara, CA) and Illumina HiSeq 2500 platform (Illumina, San Diego, CA). Data pre-processing wasdone using the best practices workflows of The Genome
Analysis toolkit (GATK, v4.1.1) (https://software.broadinsti
tute.org/gatk/) to align the sequence reads with the humanreference genome (UCSC hg19) and local realignmentswith base recalibration, and to identify somatic mutations.
The web ANNOVAR package was used to select somatic
mutations located in the exonic sequences and to predict

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their functional consequences [8]. In order to obtain reliable and robust mutation calling, the following variants
were eliminated: (i) read depth fewer than 20 in either thetumor or matched constitutional tissues; (ii) polymorphisms listed in the population databases of East Asians
with a minor allele frequency 0.1% or more; and (iii) variant
allele frequencies less than 5%. Catalogue of Somatic
Mutations in Cancer (COSMIC) mutation signatures wereobtained via a Mutalisk package [9] using known mutation
signatures of soft-tissue sarcoma [2] (signature 1, 2, 5, and13). To define CNAs, we used the ngCGH module andFig. 2 Identification of genomic alterations by WES from a DDLPS patient. a Identification of a somatic NF1 mutation. The red T letters indicate
the presence of a truncating mutation (c.7486C > T, p.Arg2496*, variant allele frequency 15.7%) in NF1 gene. b Genome-wide copy number
alternation profile of the DDLPS patient. X-axis represents individual chromosome, and Y-axis represents depth ratio (tumor/constitutional) in log2
scale. Red and blue lines indicate the threshold of copy gain and copy loss. Bold red line indicates the threshold of amplification. Red and blue
arrows represent the copy gain/amplification and loss, respectively. c Copy number alternation profiles of chromosome 1, 7, 8, 9, 12, and 20. Xaxis represents individual chromosome, and Y-axis represents depth ratio (tumor/constitutional) in log2 scale. Red arrows represent the copy gainor amplification regions where known DDLPS related genes are located. Chromothripsis events were observed in 7p, 9q, and 20q

Kim et al. BMC Medical Genetics(2020) 21:200

SNPRank Segmentation statistical algorithm in NEXUS
software 9.0 (Biodiscovery, El Segundo, CA). Segments
were classified as gains or losses when the log2 ratio was
greater than 0.25 or less than − 0.25, respectively. Amplification was defined as a log2 ratio greater than 1.0.
The average sequencing depths for tumor and constitutional DNA were 218X and 223X, respectively (Table S1).
A total of 36 non-silent mutations were identified in theexonic area (Table S2), which corresponded to a mutation
rate of 0.73 per Mb. The NF1 stop gain mutation(c.7486C > T, p.Arg2496*, variant allele frequency 15.7%)
was identified among the cancer-related genes listed in
the Cancer Gene Census of COSMIC database (Fig. 2a).
This variant was classified as ‘pathogenic’ in ClinVar DB(RCV000218957.1), and not reported in population levelvariant databases. There is no germline variant classified
as ‘pathogenic’ or ‘likely pathogenic’ in ClinVar DB.
Thirty-five regions affected by CNAs were found, whichcorrespond to 2% of the genome, including amplifications
on 12q13–15, which included known genes associatedwith DDLPS pathogenesis, such as DDIT3, CDK4,
MDM2, YEATS4, and FRS [2, 4–6] (Figs. 2b & c; Table
S3). In addition to 12q13–15 amplifications, copy gains oramplifications on 1q32, 5p13, 7p22, 8q11–12, 9q31,
12q15–21, 14q12, 19p12–13, and 20q12–13 as well as
copy losses on 2p11, 4p11-4q11, 7p21-p14, and 20q13.2
were observed. Several potential cancer-related genes,
such as MDM4, ELK4, SLC45A3, RAC1, KLF4, MAFB,
TOP1, PLCG1, and PTPRT, were located in the copy gain/
amplification regions (Fig. 2c; Table S3). Chromothripsis
events were also observed in 7p, 9q, and 20q (Fig. 2c). In amutational context, C > T mutations were predominantand signature decomposition analysis indicated predominant signatures 1 and 5 (clock-like mutational process)followed by a minor proportion of signatures 2 and 13(APOBEC-related), which was consistent with the resultsof a TCGA study on DDLPS [2] (Fig. S2).

Discussion and conclusions
Although genetic alteration of NF1 is commonly foundin liposarcomas (10–20%) [5, 6], inactivation of NF1 by amutation or a deletion may contribute to the aggressiveness of liposarcoma [5, 10]. Processes associated with theoccurrence of NF1 mutations in DDLPS remain unclear.
Using WES, we identified a pathogenic NF1 truncating
mutation with multiple CNAs in a DDLPS case exhibiting
aggressive clinical features. The NF1 truncating mutation
identified in this case was classified as a ‘pathogenic’ event(ClinVar) which could act as a driver. In spite of the tumor
being located in the extremities, a relatively favorable area[11], the patient presented with aggressive features of a
rapidly growing tumor mass accompanied by multiple
lung and pleural metastases.

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Decreased NF1 expression may lead to dysregulation of
the RAS/MAPK pathway, thus contributing to tumorigenesis of the sarcoma [12]. In addition to the known 12q13–
15 amplification, this case revealed other CNAs where
potential cancer related genes, such as MDM4, are located.
Amplification of MDM4 (1q32.1) is known to play a synergistic role by inducing the inactivation of TP53 and amplification of MDM2 [13]. Pissaloux et al. reported that a subsetof DDLPS exhibited MDM4 amplification as an oncogenic
alternative to MDM2 amplification [14]. Chromothripsis
events were observed in 7p, 9q, and 20q where potential
cancer related genes, such as RAC1, KLF4, MAFB, TOP1,
PLCG1, and PTPRT, are located. This result was compatible with that of a previous study which reported that 100%of liposarcomas (18/18) showed chromothripsis [15].
In conclusion, we report a DDLPS patient who presented
with aggressive clinical features. The patient harbored NF1
truncating mutations with multiple CNAs, including thewell-known 12q13–15 amplification, and multiple chromothripsis events. Further studies may be needed to elucidate
the role of NF1 inactivation mutations and multiple CNAs
in DDLPS tumorigenesis accompanied by aggressive clinical features.

Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12881-020-01137-4.
Additional file 1.
Abbreviations
CNA: Copy number alterations; DDLPS: Dedifferentiated liposarcoma;
TCGA: The Cancer Genome Atlas; WES: Whole exome sequencing
Acknowledgements
Not applicable.
Authors’ contributions
YSK and YJC wrote the manuscript. YJC conceived and designed the study.
YSK and SS participated in acquisition of data and sample preparation. YSK,SS, and SHJ carried out the molecular genetic studies and following analysis.
All authors read and approved the final manuscript.
Funding
This study was supported by grants from National Research Foundation of
Korea (2017R1E1A1A01074913, 2019R1A5A2027588, and2017M3C9A6047615). The funding body played no role in the design of thestudy and collection, analysis, and interpretation of data and in writing themanuscript.
Availability of data and materials
WES data were deposited in the SRA database (Project ID: PRJNA529696).
Human reference genome used in this study (UCSC hg19) can be
downloaded at the following url: https://hgdownload.soe.ucsc.edu/
downloads.html. Accession numbers of the putative pathogenic variant inthis study (NF1 c.7486C > T, p.Arg2496*) in ClinVar database is
RCV000218957.1. Functional annotation of genetic variants listed in
Supplementary Table S2 is generated by the web ANNOVAR package
available at the following url: http://wannovar.wglab.org/. Genes listed in The
Cancer Gene Census tier 1 curated by COSMIC project (v91 release) isavailable at the following url: https://cancer.sanger.ac.uk/census. All otherKim et al. BMC Medical Genetics(2020) 21:200

remaining data are available within the article and in the supplementary
data, or available from the authors upon reasonable request.
Ethics approval and consent to participate
This study was approved by the institutional review board of the Catholic
University of Korea (KC15TISI0966).
Consent for publication
The patient has provided written consent to publish this case report,
including medical data and images. A copy of the written consent forms isavailable for review by the Editor Office of this journal.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Microbiology, College of Medicine, The Catholic University of
Korea, Seoul, Republic of Korea. 2Integrated Research Center for Genome
Polymorphism, College of Medicine, The Catholic University of Korea, Seoul,
Republic of Korea. 3Precision Medicine Research Center, College of Medicine,
The Catholic University of Korea, Seoul, Republic of Korea. 4Department ofBiochemistry, The Catholic University of Korea, Seoul, Republic of Korea.
5
Biomedicine & Health Sciences, The Catholic University of Korea, Seoul,
Republic of Korea.
Received: 28 February 2020 Accepted: 1 October 2020

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