Liquid Biopsies Monitor Disease Progression in a Sarcoma Patient

[Editor’s note: This report is adapted from Namløs HM, Zaikova O,Bjerkehagen B. Use of liquid biopsies to monitor disease progression in a sarcoma patient: a case report. BMC Cancer. 2017;17:29. DOI: 10.1186/s12885-016-2992-8. Additional material is included from PubMed sources.]

Although circulating tumor cells can be detected from the peripheral blood of cancer patients—and their prognostic value has been well established for meta-static colorectal, breast, and prostate cancer—information on their presence in patients affected by sarcomas is scarce. The discovery of EpCAM mRNA expression in different sarcoma cell lines and in a small cohort of metastatic sarcoma patients supports further investigations on these rare tumors to deepen the importance of circulating tumor cell (CTC) isolation.1 Although it is not clear whether EpCAM expression might be originally present on tumor sarcoma cells or acquired during the mesenchymal-epithelial transition, the discovery of EpCAM on circulating sarcoma cells opens a new scenario in CTC detection in patients affected by a rare mesenchymal tumor.

Sarcomas are relatively rare but particularly lethal tumors, with over 5,800 deaths per year in the United States,2 accounting for about 1% of all adult cancers and approximately 20% of pediatric solid malignancies.3 More than 50 subtypes of soft tissue malignant tumors of mesenchymal origin have been documented.4,5 The diagnosis is performed by hematoxylin and eosin staining of sections and immunohistochemistry. To date, radiographic imaging and PET are performed to detect recurrence and metastasis.

Advances in different technologies are transforming this picture. There are now new techniques available for molecular characterization of such tumor types Analyses such as Polymerase Chain Reaction (PCR), fluorescent in situ hybridization (FISH), Real-Time PCR, and next-generation sequencing (NGS) or the presence of reciprocal chromosomal translocations and fusion genes may be useful for diagnosis and treatment of sarcoma patients.6 Since most soft tissue sarcomas use the hematogenous dissemination to metastasize to lungs, liver, bones, and subcutaneous tissue whereas a small percentage may spread to lymph nodes,7,8 it is possible to isolate and characterize circulating tumor cells (CTCs) from whole blood based on their biological and/or physical properties.9 Furthermore, CTCs provide through their molecular evaluation an example of “liquid biopsy” useful for cancer patient care.10

The circulating cell-free tumor DNA (ctDNA) has been shown to contain the various tumor-specific alterations seen in the primary and metastatic tumors, and may more accurately represent the genetic profile of the whole tumor mass compared to DNA from a single biopsy of a heterogeneous lesion.11 By repeated sampling of liquid biopsies, somatic mutations identified in cfDNA can be used as unique non-invasive tumor-specific biomarkers for monitoring tumor burden throughout the disease course. Similar procedures are now in use for screening of fetal genetic aberrations using the mother’s blood, and in several cases aberrations from malignant tumors have been detected presymtomatic in pregnant women.12

Several reports have demonstrated that high-throughput sequencing of cfDNA may be used for prognosis and molecular stratification, early detection of recurrence and metastasis, monitoring response to treatment and identification of resistance mechanisms.13 Sequencing of cfDNA has been performed for cancers like colorectal, ovarian and breast, showing that the level of tumor-specific mutations reflects the course of the disease and the treatment response.14-16

Sarcomas make up a heterogeneous group of malignant tumors of mainly mesenchymal origin. The overall five-year survival of all soft tissue sarcoma patients is approximately 70%,17,18 and about 75% of soft tissue sarcomas are highly malignant. Soft tissue sarcomas often recur locally and/or metastasize, and the median time to local recurrence is around 1-1½ year and to metastasis about 1 year,19,20 both decreasing long-term survival. From a molecular genetics perspective, sarcomas are genetic diverse and may have numerous somatic mutations.21 The use of high-throughput sequencing of cfDNAs longitudinally collected during disease progression, making simultaneous screening for multiple mutations during the disease course possible, has not yet been reported for sarcomas.

As part of an ongoing prospective study,1 the study collected 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 detec-tion 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 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. NF1, a tumor suppressor that functions as a negative regulator of the Ras pathway, is among the most frequently mutated genes in several subtypes of sarcomas. Germline and somatic loss of NF1 in neurofibromatosis patients cause malignant peripheral nerve sheath tumors and GISTs. In addition, somatic NF1 mutations, including deletions, have been reported in a wide variety of pediatric and adult soft-tissue sarcomas with complex karyotypes . Although no therapeutics that target KRAS or NF1 are available, our study shows that repeated sampling using liquid biopsies opens new possibilities to identify and monitor biomarkers that can be used in targeted therapies.

The allele frequencies of the six mutated genes in the cfDNA represent the ctDNA level during disease progression. Three days after surgery, ctDNA was still detectable in the liquid biopsy. cfDNA has a rapid clearance, with reported half-life from 15 min to 13 h for fetal cfDNA in plasma. Although not detectable by CT before surgery, metastatic disease was detected in two lymph nodes removed during the hemipelvectomy, and a small amount of ctDNA detected was likely released from additional undiscovered local metastases that could not be detected by conventional diagnostic modalities. The patient had a very aggressive course of the disease, and metastases were detected both in soft tissue, skeleton and lungs only a few weeks after surgery. The plasma collected six weeks after surgery showed an increase in ctDNA relative to the levels before surgery, reflecting the presence of a tumor and rapid disease progression.

The cfDNA can originate from both normal and tumour cells. Based on the high mutated allele frequencies determined in plasma, the initial level of cfDNA is dominated by DNA from the tumor. Most of the cfDNA present three days after surgery is believed to originate from tissue injury and inflammation of normal cells as a consequence of the extensive surgery, which would explain the apparently higher normal contribution to the cfDNA at this time point. After six weeks, there was a large increase in cfDNA accompanied with an increase of the mutated allele frequencies. Thus, the quantities of cfDNA present in the plasma reflected the clinical status of the patient due to the fact that most of the cfDNA released during disease progression was tumour derived.

This study is the first report of using targeted resequencing of cfDNA from serial plasma samples to monitor disease progression in a soft tissue sarcoma patient. The findings show that the level of tumour-specific mutations in liquid biopsies is correlated to disease course in sarcomas, including clinical manifestation of metastatic disease. The longitudinally collected ctDNA allow for near real-time monitoring of the tumor genome during disease progression, and the ctDNA gives a good representation of the genomic profile of the tumor supporting the use of ctDNA from plasma as a liquid biopsy.

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