lected primary tumor and plasma samples taken before and after surgery and at disease progression from a soft tissue sarcoma patient. Targeted resequencing was used to identify somatic mutations in the primary tumor and monitor the level of ctDNA from plasma samples during the course of the disease. Local recurrence or metastases after receiving potentially curative treatment is common, and early detection of these events is important for disease control. Recent technological advances make it possible to use blood plasma containing circulating cell-free tumor DNA (ctDNA) as a liquid biopsy. The following case report suggests how serial liquid biopsies can be used to monitor disease course and detect disease recurrence in a sarcoma patient. Case Presentation • A 55-year-old male presented with a rapidly growing, painful palpable mass in the left groin region, and a biopsy revealed a high-grade malignant spindle cell sarcoma. Magnetic resonance imaging performed 18 days before surgery revealed a 10.5 x 7.6 x 11.0 cm large intramuscular tumor. No metastases were detected on CT scans of the chest, abdomen and pelvic area performed 14 days before surgery. • Microscopic evaluation of a biopsy revealed a high-grade malignant spindle cell sarcoma. Due to extensive locoregional growth into the skeleton and intractable pain, a hemipelvectomy was performed. Small focus with metastatic disease was detected in two lymph nodes removed during the surgery. • Macroscopic examination showed a well demarcated nodular tumor with white and fleshy cutting surface with small necrotic areas and bleeding. Immunohistochemical analysis showed positive finding for CD99 and AE1/AE3, and negative staining for S-100, SMA, EMA and CD31. Cytogenetic analysis showed massive clonal chromosomal rearrangements, and PCR and FISH were negative for fusion genes normally seen in synovial sarcoma. • The differential diagnoses were synovial sarcoma and malignant peripheral nerve sheath tumor. Lymph node metastasis is more commonly seen in synovial sarcoma and the immunohistochemical finding is also in favor of a synovial sarcoma, but the genetic findings did not support that diagnosis. According to the WHO classi-fication, 22 the tumor was classified as an undifferentiated spindle cell sarcoma. Determining Somatic Mutations Targeted resequencing of the tumor and normal genomic DNA was performed. The sequencing revealed eight somatic mutations in the primary tumor. Among these, seven point mutations were identified in the genes COL2A1 (intronic), NF1 (p.K354R), PTGS2 (intronic), LRP2 (p.Q4132E), KRAS (p.G12V), PRRC2C (p.R1257G) and GATA6 (p.A29A), as well as a frameshift deletion in PRG4 (p.R791fs). Copy number analysis revealed a homozygote deletion of TP53. Targeted resequencing using a smaller ThunderBolts Cancer panel (Raindance Technologies, Billerica, Massachusetts, US) confirmed the identified KRAS mutation in the primary ���� The GIST and Sarcoma Journal tumor at an allele frequency of 66%, similar to the 60% frequency found using the 900 gene panel. Treatment With Surgery and Postoperative Findings The patient was scheduled for adjuvant chemotherapy, but repeated radiologic imaging six weeks postoperatively showed widespread macroscopic metastatic disease in the lungs and skeleton, as well as numerous soft tissue metastases in the pelvic region. Targeted resequencing of the plasma samples, using the NCGC 900 cancer gene panel, confirmed the presence of six of the eight above mutations in all three plasma samples with allele frequencies ranging from 2.1-75%. The level of total cfDNA was monitored during disease progression. High quantity of cfDNA was detected one day before surgery (110 ng/ml plasma), and a decrease was seen three days after surgery (76 ng/ml plasma). Six weeks after surgery, the quantity of cfDNA had increased to more than twice the initial level present before the surgery (316 ng/ml plasma). The ctDNA level was estimated from the somatic allele frequency of the recurrent mutations in the genes COL2A1, NF1, PTGS2, LRP2, KRAS and PRRC2C. The ctDNA level in plasma collected one day before the surgery (Plasma1) was high, and comparable to the level in primary tumour. Three days after surgery, the ctDNA level had dropped, but was still detectable in plasma (Plasma2). In the sample collected six weeks after surgery (Plasma3), there was again an increase in ctDNA level similar to the levels before surgery. When also taking into account the amount of cfDNA released, the number of mutated genomes per ml of plasma were three times higher at this time point than before surgery. This reflected the disease progression of the patient and correlated with the tumor burden, as multiple distant metastases were detected at this time. The patient’s general condition was considered too poor for administering chemotherapy, and he succumbed to the disease 13 weeks after surgery. Analyzing the Significance and Implications of ctDNA Findings In this study, we prospectively collected primary tumor and normal sample material at surgery and several plasma samples during the disease course of a high-grade soft tissue sarcoma patient. Targeted resequencing of the primary tumor and the normal sample identified eight somatic mutations of which six were also present in the plasma samples. Among the mutations, KRAS (p.G12V) and NF1 (p.K354R) were predicted by dbNSFP to have a deleterious effect on the protein function. It has been reported that simultaneous inactivation of TP53 and activation of KRAS induced quick formation of spindle-cell sarcoma in soft tissues in double transgenic mice. The homozygous deletion of TP53 found in the primary tumor strengthens the histology observed in the primary tumor. The patient in our study had an unusually aggressive spindle-cell sarcoma, supporting KRAS not only as biomarker, but as a driving gene of the disease progression.
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