EGFR expression in circulating tumor cells from high-grade metastatic soft tissue sarcomas

Figures & data

Table 1. Sarcoma patients' clinic-pathological characteristics.

Variable	Nº.	%
Total number of patients	18	100
Age at entry study, y
Median (range)	49.33 (18 – 77)
Gender
Male	10	55.55
Female	8	44.44
Histological subtype
Synovial Sarcoma	7	38.88
Leiomyosarcoma	5	27.77
Pleomorphic Sarcoma	4	22.22
Liposarcoma	1	5.55
Rhabdomyosarcoma	1	5.55
Progression of disease after CTC collection
No	9	50
Yes	9	50
Median CTC/mL number (range)	2.0 (0 –11.0)
CTM baseline
No	13	72.22
Yes	5	27.77

Table 2. Patients' treatment, outcome and CTCs counting and characterization.

Patient ID	Histological subtype	Positivity ratio EGFR	Number of EGFR positive CTCs in 1ml of blood	Number of EGFR negative CTCs in 1ml of blood	CTC/1ml	CTM	Collection before the first-line treatment	Treatment received after CTC collection	Line of treatment
1	Pleomorphic Sarcoma	1	4	0	1.25	–	Yes	Gemcitabine + Docetaxel	1^st
2	Rhabdomyosarcoma	1	3	0	1	–	Yes	Ifosfamide + Doxorubicin	1^st
3	Liposarcoma	1	5	0	11.25	+	Yes	Doxorubicin Monotherapy	1^st
4	Synovial Sarcoma	0.7	5	1	4	+	Yes	Ifosfamide + Etoposide	1^st
5	Pleomorphic Sarcoma	1	2	0	4.5	+	Yes	Epirubicin + Ifosfamide	1^st
6^*	Leiomyosarcoma	1	1	0	0.75	–	No	Ifosfamide + Etoposide	2^nd
7^*	Pleomorphic Sarcoma	0.1	2	12	6.25	+	No	Dacarbazine + Gemcitabine	3^rd
8	Leiomyosarcoma	0.8	5	1	2	–	Yes	Ifosfamide + Doxorubicin	1^st
9^*	Leiomyosarcoma	0.3	1	2	0.75	–	No	Doxorubicin + Dacarbazine	2^nd
10	Synovial Sarcoma	0	0	7	2	–	No	Ifosfamide	2^nd
11	Synovial Sarcoma	1	2	0	1	–	Yes	Epirubicin + Ifosfamide	1^st
12	Leiomyosarcoma	1	1	0	0.93	–	Yes	Dacarbazine Monotherapy	1^st
13	Synovial Sarcoma	–	–	–	0	–	Yes	Ifosfamide + Doxorubicin	1^st
14	Synovial Sarcoma	0.5	1	1	1.87	–	No	Ifosfamide	2^nd
15	Leiomyosarcoma	0.5	2	2	9.6	–	No	Epirubicin + Ifosfamide	2^nd
16	Synovial Sarcoma	–	–	–	0	–	No	Ifosfamide	3^rd
17	Synovial Sarcoma	0.5	2	2	5	+	Yes	Epirubicin + Ifosfamide	1^st
18	Pleomorphic Sarcoma	0.5	2	2	5.66	–	No	Doxorubicin	2^nd

Positivity Ratio: The positivity ratio of EGFR was calculated as the number of EGFR-positive CTCs present on ISET membrane spot divided by the total CTCs present in the same spot.

CTC count per mL of blood: CTCs were counted in four spots of the membrane, which corresponds to 4 mL blood. After counting we calculated the mean of these four spots to obtain the amount of CTCs per1 mL of blood, according to Krebs et al. (2012).¹⁵

* patients that underwent resection of primary tumor and metastasis in first line treatment were excluded from the progression-free survival analysis.

Figure 1. A) Negative control, A-549 cell line “spiked” in healthy blood and negative for EGFR. B) Positive control, FaDu cell line “spiked” in healthy blood and stained for EGFR. C, D) Examples of an isolated CTC of sarcoma patient with cytomorphological features (negative staining for CD45, nucleus size ≥ 12 µm, hyperchromatic and irregular nucleus, visible presence of cytoplasm, and a high nucleus–cytoplasm ratio (Krebs, et al., 2012)¹⁵. E) Immunocytochemistry of CTC with anti-EGFR antibody and counterstaining with DAB. F) One CTM from STS patient observed in the blood filtered using the ISET.

Figure 2. Progression-Free Survival (PFS) in relation to EGFR staining on CTCs from sarcoma patients. A) PFS of all patients included. EGFR expression in STS patients (> 83% = positive EGFR staining on CTCs 2.2 months); ≤ 83% = negative EGFR staining on CTCs (NR) (p = 0.117). B) PFS including only patients treated in first line. EGFR + CTCs was 2.2 months versus NR, for EGFR- CTCs (p = 0.156). Notes: Dotted line: patients without expression of EGFR. Continuous line: patients with expression of EGFR. NR = not reached.

Krebs MG, Hou J-M, Sloane R, Lancashire L, Priest L, Nonaka D, Ward TH, Backen A, Clack G, Hughes A, et al. Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches. J Thorac Oncol Off Publ Int Assoc Study Lung Cancer. 2012;7(2):306–15.

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