Pathogenic NF1 truncating mutation and copy number alterations in a dedifferentiated liposarcoma with multiple lung metastasis: a case report

Background Dedifferentiated liposarcoma (DDLPS), which accounts for an estimated 15–20% of liposarcomas, is a high-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 by multiple 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 well-known 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.

Herein, we report a patient with rapidly growing DDLPS with multiple lung and pleural metastasis, in whom whole-exome sequencing (WES) revealed a NF1 truncating mutation of a known pathogenic variant and multiple 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 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 a lipomatous portion and less dedifferentiated areas consisting of round cells with lipomatous portions, along with infiltrated polymorphonuclear cells (Fig. 1c). Based on these clinical 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 of Seoul St. Mary Hospital (Seoul, Republic of Korea). Genomic DNA was extracted by microdissection of tumor cell rich area (> 70% of tumor cell purity) and whole blood of the patient using the DNeasy Blood & Tissue Kit (Qiagen, 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 was done 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 human reference genome (UCSC hg19) and local realignments with 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 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 the tumor 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 were obtained via a Mutalisk package [9] using known mutation signatures of soft-tissue sarcoma [2] (signature 1, 2, 5, and 13). To define CNAs, we used the ngCGH module and 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.

Discussion and conclusions
Although genetic alteration of NF1 is commonly found in liposarcomas (10-20%) [5,6], inactivation of NF1 by a mutation or a deletion may contribute to the aggressiveness of liposarcoma [5,10]. Processes associated with the occurrence 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.
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 subset of 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 the well-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.