Targeted next-generation sequencing of circulating free DNA enables non-invasive tumor detection in myxoid liposarcomas
Molecular Cancer volume 21, Article number: 50 (2022)
Myxoid liposarcoma (MLS), a malignant soft-tissue tumor derived from lipocytes, is characterized by specific genetic translocations t(12;16) and t(12;22) on a background of few additional chromosomal changes . About 30% of patients with localized high-grade MLS will eventually develop distant metastases . Unlike other soft tissue sarcomas (STS), MLS exhibit a distinct pulmonary and extra-pulmonary metastatic pattern. Imaging for follow-up is thus extensive and requires whole-body Magnetic Resonance Imaging (MRI) or a combination of various imaging modalities .
Previously, we investigated the potential of circulating tumor DNA (ctDNA) to detect tumor recurrence and monitor treatment response . However, due to the unique nature of each translocation and few hotspot mutations, quantification of ctDNA was technically demanding and assays were not suitable for routine diagnostics.
To overcome these limitations, we developed targeted next generation sequencing (NGS) - based approaches, which allow ultrasensitive detection of MLS DNA in a routine diagnostic setting and prospective clinical trials. As standard NGS panels don’t cover common genetic alterations in MLS, we designed a lockdown panel which encompasses genes with a reported mutation frequency of at least 5%. The 36,541 base pair (bp) standard panel covers the introns of DDIT3, FUS and EWS where the t (12;16) and t (12;22) translocations occur, the TERT promoter region and mutation hotspots within exons from seven genes (Supplementary Fig. 1) [5,6,7,8]. Applying molecular barcodes for digital error correction  allowed the detection of mutations with a variant allele frequency (VAF) of 0.05% (Supplementary Fig. 2). 51 MLS tumors and two MLS cell lines (402-91 and 1765-92) were sequenced. Matched normal DNA was available for 23 tumors. Breakpoints could be identified in 49 tumors, and both cell lines. Translocations occurred in 87.7% between DDIT3 and FUS and in 8.3% between DDIT3 and EWSR1 (Fig. 1A). No translocations were observed in matched leukocyte DNA (sensitivity of 96% and specificity of 100%).
We could determine both breakpoints of the balanced translocations in 36 of the 51 tumors and both cell lines. This allowed us to determine if deletions or insertions occurred during the translocation event. We observed a mean loss of 7 bp (SD 64 bp) on chromosome 12 (DDIT3) and of 11 bp (SD 73 bp) on chromosome 16 (FUS) with a high intertumor variability (Fig. 1B).
Point mutations were detectable in 74.5% of tumors. Mutations in the TERT promoter region were prevalent in 73% of tumors. Thereof the C228T mutation occurred in 61% and the C250T mutation in 12% of analyzed tumors. PIK3CA mutations were found in 33% of MLS samples. Besides the well-known hotspot mutations in exon 9 (c.1624G > A, c.1633G > A, c.1633G > C, c1634A > G) and exon 20 (c.3140A > G) we identified less commonly annotated mutations in exon 5 (c.1035 T > A) and exon 8 (c. 1345C > A). Only two additional genes were mutated at low frequency in our cohort, TET2 (2%) and PTEN (4%) (Fig. 1C). Taken together, the lockdown panel could detect at least one mutation in 96% of all the tumor samples. Combining the breakpoints and point mutations, the panel detected an average 2.8 somatic mutations (1.7 breakpoints and 1.1 point mutations) per tumor, which can be targeted in circulating free DNA (cfDNA).
To determine the impact of tumor heterogeneity on ctDNA detection, spatially separate samples from two tumors (tumor 1 and 2 from Fig. 1A) were analyzed. Results were compared to matched white blood cells. Mutations with a VAF of at least 5 % were recorded. The two t (12;16) breakpoints were detected in all specimens. The presence of the major MLS driver translocation confirms its importance from tumor initiation to promotion. The consistency of patient individual breakpoints was previously reported for multifocal MLS . In contrast, there was marked intratumor heterogeneity for TERT promoter, PIK3CA and PTEN mutations (Fig. 1D). This supports the hypothesis that additional mutations seem to occur secondarily and evolve within different tumor subclones . Thus, tracking of breakpoint fragments in cfDNA promises detection of the primary tumor and all its potential metastases. In contrast, point mutations identified in the primary tumor might or might not be present in its metastases, depending of their clones of origin.
The assay was subsequently employed to quantify ctDNA in plasma samples of MLS patients. Nine plasma samples, collected during the 2-year treatment of patient 1 (see Fig. 1D) with a localized MLS who later developed metastatic disease were analyzed with the standard panel. The two PIK3CA mutations (c.1624G > A and c.3140A > G) were additionally quantified by droplet digital PCR. ctDNA levels decreased after tumor resection and increased when metastatic disease was detected. We observed a decline in ctDNA when radio/chemotherapy was initiated. However, with increasing tumor burden, concentrations rose again after several months (Fig. 2A and Supplementary Fig. 3 A). Serial ctDNA testing promises monitoring of treatment response in metastatic MLS. It might be especially beneficial for patients treated with immunotherapeutic agents, that challenge established imaging-based response assessment criteria [12, 13].
From the two PIK3CA mutations identified in the primary tumor, only c.3140A > G ctDNA correlated with t (12;16) ctDNA. This indicates that most metastases originated from a clone in the primary tumor, which harbored this mutation (Supplementary Fig. 4). As new treatment opportunities which target PIK3CA are only effective in PIK3CA mutated cells , liquid biopsy may enable us to identify patients with druggable metastases without the need of repeated biopsies.
Further mutations obtained by exome sequencing of individual tumors were added to the mutations already identified by the standard panel to lower the limit of detection (LoD) (Supplementary Fig. 5). These hybrid panels (exome panels) allowed us to monitor ctDNA of localized tumors as exemplified in the following scenario. Patient 2 received neoadjuvant radiotherapy and subsequently complete resection (tumor necrosis rate > 90%) of a localized MLS. Two plasma samples obtained at an interval of 9 days before initiation of radiotherapy showed similar concentrations of ctDNA (Fig. 2B). The third specimen obtained after radiotherapy demonstrated markedly decreased ctDNA. No ctDNA was detectable in the fourth sample collected after surgery. Analysis with the standard panel detected ctDNA in the initial plasma sample only. Calculating relative amounts, the fraction of ctDNA was between 0% and 0.05% (Supplementary Fig. 3 B). Detection of these minute amounts in limited plasma samples requires extremely sensitive assays. This was accomplished by exome panels, which target multiple mutations simultaneously, thus detecting more mutant copies in the same amounts of tumor DNA than ddPCR and the standard panel (Fig. 2C and Supplementary Fig. 5).
The impact of intertumor heterogeneity on ctDNA quantification was assessed in a patient who was initially presented with multifocal disease of his legs after previous resections at another hospital (Fig. 2D). In the course of his treatment, he suffered from four local recurrences before a small lung metastasis (0.3 cm3) was identified and resected. Exome sequencing was conducted from the initial leg tumor and the lung metastasis. The exome panels targeted 22 and 17 genomic regions respectively, and 6 mutations were identical in both panels. ctDNA in 15 plasma samples was quantified with both panels. During localized disease ctDNA fluctuated depending on the amount of viable tumor mass. The panel from the primary lesion performed superior during localized disease. The small lung metastasis however, led to markedly increased ctDNA concentrations which again decreased after resection. Both panels performed similarly during metastatic disease (Fig. 2E). Comparatively high ctDNA concentrations of lung metastases in MLS might enable detection of recurrence earlier by liquid biopsy than with imaging-based approaches [4, 15].
In this study, we present an approach for ctDNA monitoring in MLS patients in a routine diagnostic setting using a disease and patient-specific hybrid capture NGS technique. Quantification of ctDNA on the basis of cancer genomic profiling could help to predict tumor recurrence, and monitor tumor heterogeneity and treatment response in metastatic disease with minimal invasiveness and at affordable cost. The assay can easily be adapted to other translocation driven tumors, e.g. synovial sarcomas. Given our promising results, the methods we have described warrant investigations in prospective trials with larger cohorts, so they can timely be translated into clinical practice.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Soft tissue sarcoma
Circulating tumor DNA
Circulating free DNA
Next generation sequencing
Formalin-Fixed Paraffin-Embedded tissue
Homo sapiens (human) genome assembly GRCh38
Droplet digital PCR
Variant allele frequency
Magnetic Resonance Imaging
Limit of Detection
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The authors thank Dr. Pierre Åman, University of Gothenburg and Dr. Marcus Renner, University of Heidelberg for the kind donation of MLS cell lines (402-91 and 1765-92) and FFPE tissue samples. Dr. Dietmar Pfeifer and his team for the use of their NGS facility, technical assistance and advice. Marie Follo and the team of the Lighthouse Core Facility for their assistance with ddPCR. Prof. S. Laßmann and her team for use of the Fragment Analyzer and NGS facility. Prof. Börries and her team for bioinformatic support. Dr. A. Flörcken, principal investigator of the soft tissue sarcoma biobank (ZeBanC) and head of the interdisciplinary sarcoma board of the Comprehensive Cancer Center Charité (CCCC) for her support and contribution. This research has been conducted using biological samples and data obtained from the Central Biobank Charité (ZeBanC).
This study was supported by the German Research Foundation (DFG) (Grant number 396168587; BR 5712/1-1, CL 427/4-1, EI 866/7-1) and the Fördergesellschaft Forschung Tumorbiologie (Grant “Liquid-Biopsy-Initiative”). S.U.E. is a Heisenberg Professor of the DFG (EI 866/9-1). SUE is also supported by a personal DFG project grant (EI 866/10-1) that is not related to the study.
Ethics approval and consent to participate
The Ethics Committee of the Albert-Ludwigs-University of Freiburg, Germany, approved the study (study number 243/13 and 236/16). The design and performance of the study are in accordance with the Declaration of Helsinki. Signed informed consent was obtained from all participants before inclusion, allowing analysis of tumor tissue, blood samples and clinical data.
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Specifications of standard lockdown panel for MLS. Supplementary Figure 2. Evaluation of limit of detection (LoD). Supplementary Figure 3. Comparison of absolute and relative ctDNA quantification. Supplementary Figure 4. Impact of tumor heterogeneity on ctDNA detection. Supplementary Figure 5. Additional target mutations from exome sequencing increase sensitivity of tumor DNA detection. Supplementary Figure 6. Determination of sensitivity and specificity of standard panel.
Materials and Methods.
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Eisenhardt, A.E., Schmid, A., Esser, J. et al. Targeted next-generation sequencing of circulating free DNA enables non-invasive tumor detection in myxoid liposarcomas. Mol Cancer 21, 50 (2022). https://doi.org/10.1186/s12943-022-01523-x