Elevated levels of extracellular vesicles are associated with therapy failure and disease progression in breast cancer patients undergoing neoadjuvant chemotherapy

Figures & data

Figure 1. EV characterization by western blot and nanoparticle tracking analysis. (A) EV marker expression analysis for HSP70, CD63, TSG101, Syntenin, CD9, CD81, Cytochrome C (Cyt C) in EV fractions from two healthy persons (NP1, NP2), three BC patients pre-NACT (1-3) and post-NACT (4-6). In all analyses, HEK cell culture supernatant derived EVs served as positive control, for Cyt C detection MCF-7 cell lysate (*) was used as control. (B) The EV size [median (interquartile range) nm] did not differ pre- and post-NACT (Mann-Whitney test). (C) Healthy females presented lower EV levels [median (interquartile range) 10⁹/ml] compared to BC patients pre- and post-NACT. EV levels increased post-NACT in BC patients (Mann-Whitney test).

Table 1. Association of pre- and post-NACT EV levels with clinical parameters.

			EV concentration (10^9/ml)
		N	pre-NACT	post-NACT
Menopausal status^2	premenopausal	47	2064 (168 – 12980)	2340 (474 – 5360)
	perimenopausal	16	2579 (550 – 6160)	3210 (504 – 9600)
	postmenopausal	42	2004 (263 – 7648)	2483 (299 – 57280)
Histologic finding^2^,^3	ductal	77	2064 (263 – 12980)	2540 (299 – 23350)
	lobular	14	2004 (168 – 5285)	2401 (1620 – 57280)
	other	13	2264 (1070 – 5600)	2635 (950 – 3858)
Tumor grading^2^,^3	G1	8	1892 (263 – 2976)	2129 (880 – 3192)
	G2	46	1838 (168 – 12980)	2446 (474 – 57280)
	G3	49	2260 (395 – 7648)	2815 (299 – 23350)
Tumor size pre-NACT^2^,^3	cT1a – cT1c	24	1956 (263 – 7648)	2623 (639 – 23350)
	cT2	65	2012 (394 – 12980)	2352 (299 – 57280)
	>cT2	15	2472 (168 – 5285)	2517 (1182 – 3858)
Nodal status pre-NACT^2	cN0	56	1778 (168 – 7648)^*	2304 (504 – 23350)
	cN1	42	2247 (394 – 6160)^*	2613 (299 – 7664)
	cN2, cN3	7	2590 (1044 – 12980)^*	3275 (1182 – 57280)
Tumor size post-NACT^2^,^3	ypTis, ypT0	27	1695 (589 – 7648)	2610 (299 – 23350)
	ypT1a	10	1738 (1023 – 4400)	2470 (1224 – 5304)
	ypT1b, ypT1c	32	2225 (263 – 12980)	2529 (474 – 9600)
	ypT2	27	1995 (168 – 5285)	2370 (558 – 57280)
	> ypT2	8	3118 (551 – 4044)	2790 (1713 – 3858)
Nodal status post-NACT^2^,^3	yN0	68	1780 (168 – 7648)^*	2422 (504 – 23350)
	yN1	26	2431 (394 – 6160)^*	2668 (299 – 7664)
	yN2, yN3	10	2744 (551 – 12980)^*	3168 (474 – 57280)
Estrogen receptor^1	pos	74	2038 (168 – 12980)	2342 (299 – 23350)^*
	neg	31	2121 (395 – 7648)	3020 (524 – 57280)^*
Progesterone receptor^1	pos	66	1985 (168 – 12980)	2361 (474 – 23350)
	neg	39	2220 (395 – 7648)	2895 (299 – 57280)
HER2 status^1	pos	30	2169 (394 – 5600)	2419 (561 – 9440)
	neg	75	1995 (168 – 12980)	2520 (299 – 57280)
Tumor subtype (IHC)^2	ER-, PR-, HER2-	20	1797 (395 – 7648)	2958 (524 – 57280)
	ER-, PR-, HER2+	9	3110 (1593 – 5600)	3360 (1236 – 4552)
	ER+/PR+, HER2-	54	2004 (168 – 12980)	2361 (299 – 23350)
	ER+, PR+, HER2+	22	2022 (394 – 4032)	2256 (561 – 9440)
NACT regimen^2	CTX	70	2004 (168 – 12980)	2612 (474 – 57280)
	CTX + T	17	2478 (1020 – 4962)	2610 (1236 – 9440)
	CTX + T + L	7	2216 (1044 – 4400)	2304 (639 – 3360)
	CTX + A	4	2140 (1092 – 3510)	1813 (299 – 3425)
	HTX	7	1590 (263 – 4044)	960 (558 – 3858)
Response^1^,^3	response	95	1995 (168 – 7648)^*	2370 (299 – 57280)
	no response	8	3139 (1212 – 12980)^*	2973 (2520 – 4680)
Survival (OS)^1	alive	97	2012 (168 – 7648)	2370 (299 – 57280)^*
	dead	8	3207 (1160 – 12980)	3326 (2517 – 7664)^*
Recurrence (3y-PFS)^1^,^3	alive	83	1995 (168 – 12980)	2352 (299 – 57280)^**
	relapsed	8	1920 (1160 – 6160)	3850 (2517 – 23350)^**

EV levels pre-NACT [median (range) 10⁹/ml] were related to the lymph node infiltration pre- and post-NACT (Kruskal-Wallis test) and to the neoadjuvant therapy response (Mann-Whitney test). Post-NACT EV levels were associated with estrogen receptor expression (Mann-Whitney test), 3y-PFS (Mann-Whitney test) and OS (Mann-Whitney test). A – Avastin, CTX – chemotherapy, ER – estrogen receptor, HER2 – human epidermal growth factor receptor-2, HTX – hormonal therapy, L – Lapatinib, neg – negative, pos – positive, PR – progesterone receptor, T – Trastuzumab.

* p < 0.05.

** p < 0.01.

¹ Mann-Whitney test.

² Kruskal-Wallis test.

³ not all patients are included due to missing patient data.

Table 2. Post-NACT EV level cut-off discriminates between CTC subtypes. BC patients having post-NACT EV levels below the cut-off had a higher incidence of SL-CTCs, ALDH-1 positivity, ERCC1 positivity and ALDH-1/ERCC1 positivity. Data was evaluated by Fisher's exact test.

			EV concentration post-NACT
			cut-off value
			< 2484 ×10^9/ml		> 2484 ×10^9/ml
	n total		n	%	n	%	p-value^2
SL-CTC^1	56	positive	8	14	3	5	0.0463
		negative	15	27	30	54
ALDH-1^1	59	positive	6	10	1	2	0.0367
		negative	20	34	32	54
ERCC1^1	98	positive	15	15	9	9	0.1608
		negative	33	34	41	42
ALDH-1/ERCC1^1	61	positive	17	28	9	15	0.0085
		negative	10	16	25	41

¹ not all patients are included due to missing patient data.

² Fisher's exact test.

Figure 2. ROC analyses for cut-off determination for EV levels pre- and post-NACT and Kaplan-Meier survival analysis regarding 3y-PFS and OS. (A)/(C) By ROC analysis a consistent cut-off value of 2484 ×10⁹/ml for EV levels post-NACT was obtained for 3y-PFS and OS. (B/D) In Kaplan Meier analysis combined with the log rank test, BC patients having EV levels post-NACT above the determined cut-off value had a significantly reduced 3y-PFS and OS.

Figure 3. Association between CTC subtypes and EV levels post-NACT. EV concentrations post-NACT were reduced if the following CTC subpopulations were detectable: (A) SL-CTCs, (B) ALDH-1 positive, (C) ERCC1 positive and (D) ALDH-1/ERCC1 positive. Data is shown as EV levels [median (interquartile range) 10⁹/ml] and was evaluated by Mann-Whitney test. The dotted line indicates the cut-off value of 2484 ×10⁹ EVs/ml.

Table 3. Patient characteristics.

Total		105
Age		median: 51 years (18 – 83 years)
Menopausal status	premenopausal	47 (45%)
	perimenopausal	16 (15%)
	postmenopausal	42 (40%)
Histologic finding	ductal	77 (73%)
	lobular	14 (13%)
	other	13 (12%)
	unknown	1 (1%)
Tumor grading	G1	8 (8%)
	G2	46 (44%)
	G3	49 (47%)
	unknown	2 (2%)
Tumor size pre-NACT	cT1a-cT1c	24 (23%)
	cT2	65 (62%)
	> cT2	15 (14%)
	unknown	2 (2%)
Nodal status pre-NACT	cN0	56 (53%)
	cN1	42 (40%)
	cN2, cN3	7 (7%)
Tumor size post-NACT	ypTis, ypT0	27 (26%)
	ypT1a	10 (10%)
	ypT1b, ypT1c	32 (31%)
	ypT2	27 (26%)
	> ypT2	8 (8%)
	unknown	1 (1%)
Nodal status post-NACT	yN0	68 (65%)
	yN1	26 (25%)
	yN2, yN3	10 (10%)
	unknown	1 (1%)
Estrogen receptor	pos	74 (71%)
	neg	31 (30%)
Progesterone receptor	pos	66 (63%)
	neg	39 (37%)
HER2 status	pos	30 (29%)
	neg	75 (71%)
Tumor subtype (IHC)	ER-, PR-, HER2-	20 (19%)
	ER-, PR-, HER2+	9 (9%)
	ER+/PR+, HER2-	54 (51%)
	ER+, PR+, HER2+	22 (21%)
NACT regimen	CTX	70 (67%)
	CTX + T	17 (16%)
	CTX + T + L	7 (7%)
	CTX + A	4 (4%)
	HTX	7 (7%)
CTC pos	pre-NACT	23 (22%)
	post-NACT	9 (9%)
SL-CTC pos	pre-NACT	35 (33%)
	post-NACT	11 (11%)
Response	complete remission	22 (21%)
	partial remission	73 (70%)
	no remission	8 (8%)
	unknown	2 (2%)
Survival	OS	median: 52 months (4 – 82 months)
	alive	97 (92%)
	dead	8 (8%)
Recurrence	3-year PFS	median: 34 months (4 – 36 months)
	alive without relapse	83 (79%)
	relapse	8 (8%)
	unknown	14 (13%)

A – Avastin, CTC – Circulating tumor cells, CTX – chemotherapy, ER – estrogen receptor, HER2 – human epidermal growth factor receptor-2, HTX – hormonal therapy, L – Lapatinib, NACT – neoadjuvant chemotherapy, neg – negative, OS – overall survival, PFS – progression-free survival pos – positive, PR – progesterone receptor, SL-CTC – stem cell-like circulating tumor cell, T – Trastuzumab.