# CIRCUS: CIRCulating tumour cells in soft tissue Sarcoma - a short report

Robin J Young, Joanna E Chowdry, Denis Cochonneau, Dominique Heymann

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Robin J Young, Joanna E Chowdry, Denis Cochonneau, Dominique Heymann. CIRCUS: CIRCulating tumour cells in soft tissue Sarcoma - a short report. Cancer Drug Resistance, 2024, 10.20517/cdr.2024.149. hal-04866528

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# CIRCUS: CIRCulating tumour cells in soft tissue Sarcoma – a short report

Robin J. Young¹, Joanna E. Chowdry J², Denis Cochonneau³, Dominique Heymann²,³,⁴

## Affiliations

1. Sheffield Teaching Hospitals NHS Foundation Trust, UK

2. University of Sheffield, Medical School, S102RX, Sheffield, UK

3. Institut de Cancérologie de l'ouest, Tumour Heterogeneity and Precision Medicine, 44805, Saint-Herblain, France

4. Nantes Université, CNRS, UMR6286, US2B, 44322, Nantes, France

## Corresponding author

Dr Robin Young, Weston Park Cancer Centre, Sheffield Teaching Hospitals NHS Foundation Trust, S10 2SJ, UK; email: robin.young@nhs.net
Sarcomas are most likely to metastasise to the lung. To generate metastases, tumour cells must escape from the primary site, intravasate into a blood or lymphatic vessel, survive in the circulation, arrest in the capillaries at a new site, migrate into the interstitial space, and finally form a tumour at the new location. Circulating tumour cells (CTCs) can be detected in the peripheral blood. Due to the rarety of sarcoma, their diversity and the lack of specific biomarkers, CTCs have been predominantly investigated in carcinomas. Because CTCs exhibit a higher diameter than normal circulating blood cells, and lower deformfrequently larger and are less deformable. We performed a pilot study (IRAS ID: 235459) to test if these two parameters combined with the detection of mesenchymal and epithelial biomarkers could be used to isolate and quantify CTCs in peripheral blood samples collected from soft tissue sarcoma (STS) patients. 13 patients were recruited to this study; 7 patients with localised disease and 6 patients with metastatic disease. total circulating cell free DNA (cfDNA) level was alaos analysed. CTCs exhibited a high heterogeneity based on their mesenchymal and epithelial biomarkers. There was no significant difference in the number of CTCs between patients with localised versus metastatic disease. We observed no correlation between CTC numbers and cfDNA nor with the tumour size. However, the number of CTCs did correlate with the primary tumour size. The present study demonstrated the presence of CTCs in sarcoma patients with loclised and advanced disease. Further studies are needed to evaluate the prognostic significance of CTCs, and to study whether CTCs could be used as a biomarker to identify patients with localised disease who have a higher risk of developing metastases who may benefit most from adjuvant chemotherapy.
Introduction

Sarcomas are a rare heterogenous group of tumours of mesenchymal origin[1]. The biology of sarcoma subtypes vary widely; however, all sarcomas preferentially metastasise to the lungs, typically via haematological spread. The risk of developing metastases is dependent upon various tumour specific factors including tumour histology, size and grade, and occurs in approximately 50% of patients with localised high risk extremity soft tissue sarcoma (STS) [2]. Once patients develop metastatic disease, treatment is usually palliative and the prognosis is poor. A greater understanding of the metastatic process is needed to develop better treatments for STS.

Cancer cells that escape the primary tumour and intravasate into the bloodstream become circulating tumour cells (CTCs) that have the potential to seed into distant metastases [3]. CTCs can be detected in the peripheral blood, and the number of CTCs may correlate with clinical endpoints such as burden of metastatic disease and response to treatment [4]. As a window into the metastatic niche, the study of CTCs should improve our understanding of the metastatic process [5]. To date, most studies of CTCs have focused on patients with carcinomas, utilising tumour cell expression of cytokeratins such as epithelial cell adhesion molecule (EpCAM) to capture CTCs. Because CTCs are frequently larger and less deformable than normal circulating blood cells, cell size represents another potential characteristic for separating CTCs [6]. To date, there have been few studies of CTCs in sarcoma. This is partly due to the rarity of sarcomas, their heterogeneity, but also the absence of a sarcoma-specific CTC marker; though recently, vimentin has been suggested as one such marker [7].
We developed a workflow to isolate and analyse sarcoma CTCs based around tumour size, and performed a pilot study (IRAS ID: 235459) to isolate, quantify, and characterise CTCs in peripheral blood samples collected from patients presenting to the Sheffield Sarcoma Service with either localised or advanced extremity STS.
Materials and Methods

Patients presenting to the Sheffield Soft Tissue Sarcoma Service with a new diagnosis of localised or advanced extremity soft tissue sarcoma were eligible. Patient management was not protocol defined and treatment and follow up schedules were according to local and national guidelines [3]. Patient demographics, tumour histology, staging, and disease-free survival were extracted from medical records. Informed study consent and CTC blood sample collections were performed before initiation of any treatment (surgery, radiotherapy or chemotherapy). 20 mL peripheral blood samples were collected from patients in 2 x 10 mL Streck™ (Streck, USA) tubes and gently inverted ten times after collection to ensure adequate mixing with the preservative. The samples were enriched for CTCs within 96 hours of collection using a Parsortix™PR1 instrument (Angle ltd, UK); the blood sample was loaded on to a microfiltration cassette, which traps CTCs owing to their larger size in comparison to other blood components. The CTC isolate was then fixed in formaldehyde and stored at 4°C until analysis. CTCs were then separated out from other cells within the isolate and quantified utilising dielectrophoresis via the DEPArray™ (Menarini, Italy). Commercially available antibodies were employed to exclude contaminating white blood cell populations, using the white blood cell marker CD45 (1/20, PerCP/Cy5.5-mouse IgG1k anti human, clone HI30, Biolegend, USA). CTC expression of vimentin (Vim) was evaluated using anti-vimentin antibody (10 µg/mL, mouse anti-IgG2a anti human, clone VIM3B4, Dako, France) associated with an AF647-goat anti mouse IgG2a (1/1000, Invitrogen, Fisher Scientific, France), and pan cytokeratin (CK) expression evaluated using an anti-AE1/AE3 antibody (5 µg/mL, AF488-mouse IgG1k anti human, clone C-11, Biolegend).
Plasma extracted at the enrichment phase was aliquoted and stored at -20°C. Total cell free (cf)DNA was isolated by NucleoSnap™ cfDNA columns (Macherey-Nagel, France) and quantified by NanoDrop™ (Ozyme, France) according to manufacturer instructions; cfDNA levels averaged across paired replicate samples was reported.

## Statistical Analysis

Non-parametric tests were performed; the Mann-Whitney U test was used to compare populations and Spearman's Rho for correlations. p < 0.05 was considered significant.
## Results

13 patients [7 male, 6 female, median age 58yrs (39-82)] were recruited from 2019-2022; 7 patients with localised disease, 6 patients with advanced disease. Tumour histology was of 6 undifferentiated pleomorphic sarcoma (UPS), 2 myxofibrosarcoma, 2 myxoid liposarcoma, 1 de-differentiated liposarcoma, 1 leiomyosarcoma and 1 angiosarcoma; all 6 patients with metastatic disease had UPS (Tables 1, and 2).

Based on their size and their epithelial, mesenchymal biomarkers, the total number of CTCs and 3 subpopulations were enumerated: Vim⁺ CK⁺, Vim⁻ CK⁺, and Vim⁺ CK⁻ CTCs (Figure 1, Figure 2). CTCs were identified in all patients. There was a trend towards higher CTC numbers in patients with advanced disease, but this was not significant; the median number of total CTCs in patients with localised versus advanced disease was 3 (range 1-110) and 14.5 (range 2-133) respectively (p=0.32). The number of CTCs was not associated with tumour grade (median number of total CTCs for Trojani grade ≤2 versus 3 was 12 (range 1-133) and 10 (range 2-110) respectively (p=0.77). However, there was a significant positive relationship between the total number of CTCs and the primary tumour size ($r_s(8)=0.786; p=0.007$; Figure 3).

cfDNA levels ranged from 0.07ng/µl to 2.00ng/µl with a median of 0.19 ng/µl. Levels were similar in the two patient cohorts (median cfDNA levels in localised disease was 0.12ng/µl (range 0.07-2.00) and 0.28ng/µl (range 0.10-0.62) in advanced disease; Figure 4). No relationship was observed between either the cfDNA levels and the total number of CTCs
(r<sub>s</sub>(11)=-0.20; p=0.56), nor the cfDNA levels and the primary tumour size (r<sub>s</sub>(7)=-0.25; p=0.91).

The small sample size precluded any formal analysis, but there was no obvious
relationship between either the total number of CTCs or cfDNA and disease-free survival.
Discussion

This pilot study adds to the growing literature of CTCs in STS. Consistent with other recent
reports of CTCs in sarcoma, we found that CTCs were rare events with small numbers
isolated per sample [9-13].

As CTCs in sarcoma are rare, an efficient isolation process is needed for CTC capture. Our
protocol included an initial enrichment step to concentrate the blood samples for CTCs
utilising their physical properties. Parsortix™ microfiltration cartridges were used to trap
CTCs based on their increased size and lower deformability compared to normal cellular
blood components (white and red blood cells). A potential limitation of our workflow is
the loss of smaller CTCs at this step, although previous research indicates a high recovery
rate [6]. The enriched sample was then labelled using antibodies to target cell markers of
interest. Initial studies of CTCs in sarcoma focused on translocation-associated sarcomas
such as Ewing sarcoma [14] with isolation of CTCs based on tumour cell expression of the
characteristic fusion protein. Vimentin, a component of the cytoskeleton and universally
expressed in mesenchymal cells, has since been proposed as a pan-sarcoma CTC marker
[7]. However, sarcoma cells may also express cytokeratins, particularly within a
subpopulation of tumour cells undergoing mesenchymal-to-epithelial transition as part of
the metastatic process [8]. Thus, an isolation process identifying sarcoma CTCs exclusively
by vimentin expression may omit other CTC subpopulations of interest. We explored both
vimentin and CK marker expression, using the DEPArray™ to isolate and quantify these
cell populations from within the enriched blood samples. One of the strengths of our
study was to capture data on a broad population of CTCs, and a further advantage of our
process is that the DEPArray™ enables separation of different cell populations (e.g.
vimentin⁺/CK⁻ vs vimentin⁺/CK⁺) for further downstream analysis and characterisation. We
observed an important heterogeneity of CTCs with various expression of biomarkers that
may reflect a snapshot of the composition of the localised or/and metastatic tumour foci
at a given time [5]. Unfortunately, limited resources prohibited further molecular analyses
within this current study.

In keeping with some studies, our data supports CTC numbers correlate with tumour
burden [8]. However, other studies have indicated no relationship, and no association
with response to treatment [10,12]. The clinical value of CTCs in sarcoma thus remains
uncertain. A key clinical challenge in sarcoma is in identifying patients with high-risk
localised disease who may benefit most from intensive adjuvant chemotherapy. Existing
prognostic calculators already in clinical use such as the Sarculator [15] currently rely on
clinical characteristics such as tumour size, grade and histological subtype. Potentially,
CTCs could be useful as a prognostic marker to further refine patient selection for
adjuvant chemotherapy, but clearly further and larger studies are needed to explore this.
One of the largest reported studies to date of CTCs in sarcoma was a translational
endpoint within the TAPPAS study, a randomised phase III study of TRC105 +/− pazopanib
for advanced angiosarcoma [16]. CTC numbers did not change in response to treatment,
but they reported CTCs identified in the majority of evaluated patients [17]. Our study
included one patient with localised angiosarcoma. This patient had high numbers of CTCs
at baseline and subsequently quickly relapsed. Angiosarcoma is a very aggressive sarcoma,
and perhaps represents a promising subtype for the further study of CTCs and for
stratifying patients for adjuvant chemotherapy.

In summary, our pilot study demonstrated the feasibility of our process for isolating CTCs
from patients with STS and provides a platform for further studies in this area.

## Acknowledgements

We would like to thank the patients for participating in this study, and to Sarcoma UK for
funding this research (Grant number: SUK02.2017).

## References

[1] Grünewald TG, Alonso M, Avnet S et al. Sarcoma treatment in the era of molecular medicine. EMBO Mol Med. 2020. 12(11):e11131.

[2] Hayes AJ, Nixon IF, Strauss DC et al. UK guidelines for the management of soft tissue sarcomas. Br J Cancer. 2024. https://doi.org/10.1038/s41416-024-02674-y

[3] Tellez-Gabriel M, Brown HK, Young R et al. The Challenges of Detecting Circulating Tumor Cells in Sarcoma. Front Oncol. 2016. 6:202.

[4] Lin D, Shen L, Luo M et al. Circulating tumor cells: biology and clinical significance. Sig Transduct Target Ther. 2021. 6:404.
[5] Brown HK, Tellez-Gabriel M, Cartron PF, et al. Characterization of circulating tumor cells as a reflection of the tumor heterogeneity: myth of reality? Drug Discov Today 2019. 24(3):763-772.

[6] Hvichia GE, Parveen Z, Wagner C et al. A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells. Int J Cancer. 2016. 138(12):2894-904.

[7] Satelli A, Mitra A, Cutrera JJ et al. Universal marker and detection tool for human sarcoma circulating tumor cells. Cancer Res. 2014. 74(6):1645-50.

[8] Yang J, Du X, Wang G et al. Mesenchymal to epithelial transition in sarcomas. Eur J Cancer. 2014. 50(3):593-601.

[9] Le MC, Smith KA, Dopico PJ et al. Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas. Sci Rep. 2024. 14(1):12374.

[10] Blanchi J, Laroche-Clary A, Le Loarer F et al. Prognostic impact of circulating tumor cells in patients with soft-tissue sarcomas: a prospective study. Cancer Commun (Lond). 2023. 43(3):391-4.

[11] Vismara M, Reduzzi C, Silvestri M et al. Single-cell phenotypic and molecular characterization of circulating tumor cells isolated from cryopreserved peripheral blood mononuclear cells of patients with lung cancer and sarcoma. Clin Chem. 2022. 68(5):691-701.
[12] Hayashi M, Zhu P, McCarty G et al. Size-based detection of sarcoma circulating tumor cells and cell clusters. Oncotarget. 2017. 8(45):78965-77.

[13] Chinen LT, Mello CA, Abdallah EA et al. Isolation, detection, and immunomorphological characterization of circulating tumor cells (CTCs) from patients with different types of sarcoma using isolation by size of tumor cells: a window on sarcoma-cell invasion. Onco Targets Ther. 2014. 7:1609-17.

[14] Schleiermacher G, Peter M, Oberlin O et al. Increased risk of systemic relapses associated with bone marrow micrometastasis and circulating tumour cells in localized ewing tumor. J Clin Oncol. 2003. 21(1):85-91.

[15] Callegaro D, Miceli R, Bonvalot S et al. Development and external validation of a dynamic prognostic nomogram for primary extremity soft tissue sarcoma survivors. eClinicalMedicine. 2019. 22:17:100215.

[16] Jones RL, Ravi V, Brohl AS et al. Efficacy and safety of TRC105 plus pazopanib vs pazopanib alone for treatment of patients with advanced angiosarcoma. JAMA Oncol. 2022. 8(5):740-7.

[17] Ravi V, Brohl AS, Chawla SP et al. Detection of endoglin-expressing CTCs in patients enrolled in an adaptive enrichment phase 3 trial of TRC105 and pazopanib versus pazopanib alone in patients with advanced angiosarcoma (TAPPAS). J Clin Oncol. 2018. 36(15):suppl.e23570.
**Figure Legend**

**Figure 1:** Representative CTCs isolated in soft-tissue sarcoma patients. CTCs were isolated by using a double step based process including a pre enrichment by Parsortix™ followed by visualisation and isolation wthe the DEPARarry™ device after immunostaining. Three CTC populations were isolated: Vimentin (Vim)⁺ Cytokeratin (CK)⁺, Vim⁻ CK⁺ and Vim⁺ CK⁻.

**Figure 2:** Plots comparing CTC populations between patients with localised (Cohort A) and advanced disease (Cohort B). CTCs were preenriched 20 mL of blood by using Parsortix™ microfluidic system and enumerated by DEParray™ after immunostaining. CTCs were isolated from mesenchymal (Vimentin: Vim) and epithelial (Pan cytokeratin: CK) biomarkers. A) Vim⁺ CK⁺ CTCs, B) Vim⁻ CK⁺ CTCs, C) Vim⁺ CK⁻ CTCs, D) Total CTCs. All CTCs were CD45⁻.

**Figure 3:** Plot showing relationship between total number of CTCs and the primary tumour size (mm). After preenrichment by Parsortix™ device, Vimentin⁺ (Vim⁺) or/and pan cytokeratin⁺ (CK⁺), CD45⁻ CTCs were isolated by DEParray™. r<sub>s</sub>(8)=0.786; p=0.007.

**Figure 4:** Plot comparing cfDNA (ng/µl) in localised (Cohort A) and advanced disease (Cohort B).
<table><caption>Table 1: Clinical and biological parameters of the seven patients recruited with localised disease</caption><thead><tr><th rowspan="2">Age (yrs)</th><th rowspan="2">Sex</th><th rowspan="2">Tumour histology</th><th rowspan="2">Primary tumour site</th><th rowspan="2">Maximum tumour size (mm)</th><th rowspan="2">Trojani grade</th><th rowspan="2">Disease free at 3 years</th><th colspan="4">Number of CTCs</th><th rowspan="2">Total</th><th rowspan="2">cfDNA (ng/µl)</th></tr><tr><th>Vim<sup>+</sup> CK<sup>+</sup></th><th>Vim<sup>-</sup> CK<sup>+</sup></th><th>Vim<sup>+</sup> CK<sup>-</sup></th></tr></thead><tbody><tr><td>71</td><td>M</td><td>MFS</td><td>Rt arm</td><td>55</td><td>2</td><td>Y</td><td>1</td><td>9</td><td>2</td><td>12</td><td></td><td>0.12</td></tr><tr><td>54</td><td>M</td><td>MLPS</td><td>Rt thigh</td><td>170</td><td>1</td><td>Y</td><td>12</td><td>5</td><td>5</td><td>22</td><td></td><td>0.11</td></tr><tr><td>39</td><td>M</td><td>MLPS</td><td>Rt lower leg</td><td>118</td><td>2</td><td>N</td><td>1</td><td>0</td><td>2</td><td>3</td><td></td><td>0.19</td></tr><tr><td>58</td><td>F</td><td>AS</td><td>Rt breast</td><td>190</td><td>3</td><td>N</td><td>67</td><td>29</td><td>14</td><td>110</td><td></td><td>0.12</td></tr><tr><td>42</td><td>F</td><td>MFS</td><td>Lt knee</td><td>50</td><td>2</td><td>N</td><td>0</td><td>1</td><td>0</td><td>1</td><td></td><td>0.07</td></tr><tr><td>69</td><td>F</td><td>LMS</td><td>Lt thigh</td><td>29</td><td>3</td><td>Y</td><td>1</td><td>1</td><td>0</td><td>2</td><td></td><td>0.32</td></tr><tr><td>82</td><td>F</td><td>DDLPS</td><td>Lt thigh</td><td>40</td><td>2</td><td>N</td><td>1</td><td>0</td><td>0</td><td>1</td><td></td><td>2.00</td></tr></tbody></table>
AS: angiosarcoma, DDLPS: dedifferentiated liposarcoma LMS: leiomyosarcoma, MFS: myxofibrosarcoma,

MLPS: myxoid liposarcoma, Rt: right, Lt: left
<table><caption>Table 2: Clinical and biological parameters of for the six patients recruited with advanced disease</caption><thead><tr><th rowspan="2">Age (yrs)</th><th rowspan="2">Sex</th><th rowspan="2">Tumour histology</th><th rowspan="2">TNM stage</th><th rowspan="2">Sites of disease</th><th rowspan="2">Primary tumour size (mm)</th><th rowspan="2">Trojani grade</th><th colspan="3">Number of CTCs</th><th rowspan="2">Total</th><th rowspan="2">cfDNA (ng/µl)</th></tr><tr><th>Vim<sup>+</sup> CK<sup>+</sup></th><th>Vim<sup>-</sup> CK<sup>+</sup></th><th>Vim<sup>+</sup> CK<sup>-</sup></th></tr></thead><tbody><tr><td>73</td><td>M</td><td>UPS</td><td>TX N0 M1</td><td>Multiple lung + bilateral adrenal metastases</td><td>-</td><td>3</td><td>13</td><td>22</td><td>0</td><td>35</td><td>0.62</td></tr><tr><td>58</td><td>M</td><td>UPS</td><td>T4 N1 M0</td><td>Primary + Rt iliac LNs</td><td>220</td><td>2</td><td>42</td><td>91</td><td>0</td><td>133</td><td>NA</td></tr><tr><td>57</td><td>M</td><td>UPS</td><td>T4 N1 M1</td><td>Primary + Rt iliac LNs + solitary lung metastasis</td><td>170</td><td>2</td><td>0</td><td>2</td><td>10</td><td>12</td><td>0.23</td></tr><tr><td>62</td><td>F</td><td>UPS</td><td>T1 N0 M1</td><td>Local recurrence</td><td>43</td><td>3</td><td>5</td><td>9</td><td>3</td><td>17</td><td>0.10</td></tr></tbody></table>
<table><tr><td></td><td></td><td></td><td></td><td>+ multiple lung metastases</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>61</td><td>M</td><td>UPS</td><td>TX N0 M1</td><td>Soft tissue (regional recurrence)</td><td>-</td><td>3</td><td>2</td><td>0</td><td>0</td><td>2</td><td>0.32</td></tr><tr><td>42</td><td>F</td><td>UPS</td><td>TX N0 M1</td><td>Multiple lung metastases</td><td>-</td><td>3</td><td>3</td><td>0</td><td>0</td><td>3</td><td>NA</td></tr></table>

AS – angiosarcoma; DDLPS – de-differentiated liposarcoma; LMS – leiomyosarcoma; LNs – lymph nodes; MFS – myxofibrosarcoma; MLPS – myxoid liposarcoma; NA – sample not available; UPS – undifferentiated pleomorphic sarcoma.
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>