CD44+ Circulating Tumor Endothelial Cells Indicate Poor Prognosis in Pancreatic Ductal Adenocarcinoma After Radical Surgery: A Pilot Study

Figures & data

Figure 1 Detection of different subtypes of CTCs and CTECs expressing CD44 and vimentin in PDAC patients by SE-iFISH. (A) A CTC (DAPI+/CD45-/CD31-/vimentin-/CEP8+) has a positive expression of CD44; A CTC cluster (DAPI+/CD45-/CD31-/vimentin-/CEP8+) has a positive expression of CD44; A CTC (DAPI+/CD45-/CD31-/vimentin-/CEP8+) has a negative expression of CD44; A CTC cluster (DAPI+/CD45-/CD31-/vimentin+/CEP8+) has a negative expression of CD44. (B) A CTEC (DAPI+/CD45-/CD31+/vimentin-/CEP8+) has a positive expression of CD44; A CTEC cluster (DAPI+/CD45-/CD31+/vimentin+/CEP8+) has a positive expression of CD44; A CTEC (DAPI+/CD45-/CD31+/vimentin-/CEP8+) has a negative expression of CD44; A CTEC cluster (DAPI+/CD45-/CD31+/vimentin+/CEP8+) has a negative expression of CD44.

Table 1 Demographic Characteristics, Surgical Details and Pathological Details

Baseline Characteristics
Age (yr, ±SD)	59.41±9.62
Gender (Female)	33 (45.2%)
BMI (kg/m^2)	23.27±3.24
Smoke	27 (37.0%)
Alcohol	20 (27.4%)
Pancreatitis	4 (5.5%)
Diabetes	18 (24.7%)
Cardiovascular disease	4 (5.5%)
Obstructive jaundice	21 (28.8%)
Neoadjuvant chemotherapy	11 (15.1%)
Neoadjuvant radiotherapy	0
Surgical Details
Type of operation
Pancreaticoduodenectomy	48 (65.8%)
Distal pancreatectomy	23 (31.5%)
Total pancreatectomy	2 (2.7%)
Laparoscopic	26 (35.6%)
Operation time (min)	330.60±94.00
Blood loss (mL)	678.08±548.03
Transfusion (RBCs)	27 (37%)
Postoperative complications	29 (39.7%)
Adjuvant chemotherapy	67 (91.8%)
Adjuvant radiotherapy	7 (9.6%)
Pathological Details
Tumor size	3.58±1.76
Differentiation (poor)	31 (42.5%)
Margin status (not R0)	26 (35.6%)
Vascular infiltration	25 (34.2%)
Perineural infiltration	61 (83.6%)
Carcinoma embolus	34 (46.6%)
Total lymph nodes number	22.70±11.62
Positive lymph nodes number	1.11±1.45

Abbreviation: RBCs, red blood cells.

Figure 2 Flow diagram of patient enrollment.

Table 2 CTCs and CTECs Details

		Total	DFS<6 Months (n=16)	DFS≥6 Months (n=57)	P value
CA 19–9	Preoperative	238.45±327.35	270.87±273.99	229.35±342.47	0.657
	POD 7	59.45±81.38	75.48±64.03	56.10±84.82	0.521
	POM 1	117.40±427.72	415.77±846.19	28.52±84.82	0.002
CTCs	Preoperative	11.49±10.72	15.44±12.12	10.39±10.14	0.096
	POD 7	49.33±93.43	68.31±161.06	44.00±64.36	0.361
	POM 1	30.42±53.69	49.23±84.48	25.11±41.00	0.336
CD44+ CTCs	Preoperative	0.33±0.80	0.50±1.09	0.28±0.70	0.336
	POD 7	9.55±73.23	39.94±156.29	1.02±3.74	0.335
	POM 1	0.43±1.21	0.54±1.66	0.40±1.07	0.721
Vimentin+ CTCs	Preoperative	0.51±1.43	0.06±0.25	0.63±1.59	0.012
	POD 7	0.16±0.60	0.00±0.00	0.21±0.67	0.022
	POM 1	0.64±1.48	0.46±1.66	0.69±1.44	0.631
CTMs	Preoperative	0.52±1.40	0.56±1.54	0.51±1.37	0.894
	POD 7	0.74±1.23	0.94±1.28	0.68±1.22	0.473
	POM 1	0.46±0.91	0.62±1.04	0.41±0.88	0.487
CTECs	Preoperative	6.90±11.13	10.13±10.57	6.00±11.21	0.193
	POD 7	12.40±22.78	13.13±26.22	12.19±21.98	0.886
	POM 1	6.19±12.84	10.08±19.80	5.07±10.04	0.218
CD44+ CTECs	Preoperative	1.10±2.27	3.00±3.81	0.56±1.19	0.023
	POD 7	2.22±7.87	5.50±15.85	1.30±2.90	0.308
	POM 1	1.09±3.59	1.85±6.06	0.87±2.53	0.580
Vimentin+ CTECs	Preoperative	1.33±6.21	0.88±3.50	1.46±6.80	0.744
	POD 7	0.30±1.77	0.00±0.00	0.39±2.00	0.447
	POM 1	0.90±3.76	0.31±0.75	1.07±4.25	0.527
CTEMs	Preoperative	0.74±2.93	1.94±5.93	0.40±1.03	0.320
	POD 7	0.23±0.75	0.13±0.34	0.26±0.83	0.522
	POM 1	0.31±1.23	0.08±0.27	0.38±1.38	0.443

Note: Bold value: P < 0.05, considered statistically significant.

Abbreviations: CTCs, circulating tumor cells; CTMs, circulating tumor microemboli; CTECs, circulating tumor endothelial cells; CTEMs, circulating tumor endothelial microemboli.

Figure 3 The dynamics of (A) CA 19–9 levels, numbers of (B) CTCs, (C) CD44+ CTC, (D) vimentin+ CTC, (E) CTCM, and numbers of (F) CTECs (G) CD44+ CTEC, (H) vimentin+ CTEC, (I) CTEM in different phases (preoperative, POD7 and POM1). The patients were divided into early recurrence (ER) group (DFS<6 months) and late recurrence (LR) group (DFS≥6 months) according to the DFS.

Table 3 Univariate and Multivariate Cox Regression Analyses of Prognosis Factors (Clinicopathological Factors) for Disease Free Survival

	Univariate Analysis		Multivariate Analysis		Cutoff Value
Prognostic Factor	HR (95% CI)	P value	HR (95% CI)	P value
Age (years)	1.000 (0.968–1.033)	0.984
Gender (Female)	1.670 (0.921–3.027)	0.091
BMI (kg/m^2)	0.998 (0.915–1.089)	0.967
Obstructive jaundice	1.158 (0.613–2.186)	0.652
Smoke	2.070 (1.072–3.997)	0.030
Alcohol	1.054 (0.542–2.051)	0.876
Pancreatitis	0.825 (0.198–3.432)	0.791
Diabetes	2.422 (1.223–4.795)	0.011	2.656 (1.194–5.908)	0.017
Cardiovascular disease	1.693 (0.513–5.583)	0.387
Neoadjuvant chemotherapy	2.714 (1.173–6.279)	0.020
Type of operation		0.003
Pancreaticoduodenectomy	Ref.
Distal pancreatectomy	1.314 (0.681–2.538)	0.416
Total pancreatectomy	20.262 (3.614–113.587)	0.001
Laparoscopic	0.761 (0.411–1.410)	0.385
Operation time (min)	0.999 (0.995–1.002)	0.470
Blood loss (mL)	1.000 (1.000–1.001)	0.827
Transfusion (RBCs)	1.116 (0.602–2.068)	0.727
Postoperative complication	1.057 (0.562–1.989)	0.863
Adjuvant chemotherapy	1.800 (0.431–7.511)	0.420
Adjuvant radiotherapy	0.562 (0.201–1.578)	0.274
Tumor size	1.029 (0.910–1.165)	0.647
Differentiation (poor)	1.862 (1.023–3.389)	0.042
Margin status (not R0)	1.174 (0.632–2.182)	0.611
Vascular infiltration	1.018 (0.538–1.926)	0.957
Perineural infiltration	1.852 (0.661–5.189)	0.241
Carcinoma embolus	1.048 (0.575–1.911)	0.879
Total lymph nodes (numbers)	0.992 (0.961–1.024)	0.607
Positive lymph nodes (numbers)	1.749 (1.373–2.228)	<0.001	1.871 (1.388–2.522)	<0.001	2

Note: Bold value: P < 0.05, considered statistically significant.

Abbreviation: RBCs: red blood cells.

Table 4 Univariate and Multivariate Cox Regression Analyses of Prognosis Factors (CA19-9, CTCs and CTECs) for Disease Free Survival

	Univariate Analysis		Multivariate Analysis		Cutoff Value
Prognostic Factor	HR (95% CI)	P value	HR (95% CI)	P value
Preoperative CA19-9	1.000 (0.999–1.001)	0.686
Preoperative CTC	0.999 (0.974–1.025)	0.956
Preoperative CD44+ CTC	0.974 (0.665–1.426)	0.892
Preoperative Vimentin+ CTC	0.889 (0.630–1.254)	0.502
Preoperative CTM	1.027 (0.847–1.245)	0.786
Preoperative CTEC	0.999 (0.975–1.024)	0.940
Preoperative CD44+ CTEC	1.207 (1.077–1.353)	0.001	1.216 (1.064–1.390)	0.004	3
Preoperative Vimentin+ CTEC	1.005 (0.967–1.004)	0.810
Preoperative CTECM	1.062 (0.968–1.166)	0.203
POD7 CA19-9	1.001 (0.998–1.004)	0.633
POD7 CTC	1.000 (0.996–1.004)	0.939
POD7 CD44+ CTC	1.005 (1.001–1.008)	0.011
POD7 Vimentin+ CTC	0.738 (0.344–1.583)	0.435
POD7 CTM	0.927 (0.735–1.170)	0.523
POD7 CTEC	0.980 (0.958–1.002)	0.072
POD7 CD44+ CTEC	1.028 (0.986–1.072)	0.197
POD7 Vimentin+ CTEC	1.074 (0.922–1.236)	0.322
POD7 CTECM	0.571 (0.316–1.030)	0.063
POM1 CA19-9	1.001 (1.001–1.002)	<0.001	1.002 (1.001–1.002)	<0.001	89.6
POM1 CTC	1.003 (0.996–1.010)	0.399
POM1 CD44+ CTC	0.889 (0.643–1.228)	0.474
POM1 Vimentin+ CTC	0.940 (0.698–1.266)	0.683
POM1 CTM	1.285 (0.940–1.755)	0.116
POM1 CTEC	0.987 (0.957–1.018)	0.420
POM1 CD44+ CTEC	0.954 (0.840–1.083)	0.463
POM1 Vimentin+ CTEC	0.868 (0.680–1.108)	0.257
POM1 CTECM	0.730 (0.445–1.199)	0.214

Note: Bold value: P < 0.05, considered statistically significant.

Abbreviations: CTCs, circulating tumor cells; CTMs, circulating tumor microemboli; CTECs, circulating tumor endothelial cells; CTEMs, circulating tumor endothelial microemboli.

Table 5 Univariate and Multivariate Cox Regression Analyses of Prognosis Factors (Clinicopathological Factors) for Overall Survival

	Univariate Analysis		Multivariate Analysis		Cutoff Value
Prognostic Factor	HR (95% CI)	P value	HR (95% CI)	P value
Age (years)	0.967 (0.915–1.021)	0.226
Gender (Female)	2.325 (0.778–6.952)	0.131
BMI (kg/m2)	0.944 (0.800–1.115)	0.497
Obstructive jaundice	0.424 (0.094–1.916)	0.265
Smoke	46.586 (0.546–3976.136)	0.090
Alcohol	0.537 (0.120–2.404)	0.417
Pancreatitis	1.610 (0.206–12.596)	0.650
Diabetes	6.307 (1.917–20.758)	0.002	7.227 (1.916–27.265)	0.004
Cardiovascular disease	0.045 (0.000–857.323)	0.537
Neoadjuvant chemotherapy	5.083 (1.514–17.063)	0.009
Type of operation		0.005
Pancreaticoduodenectomy	Ref.
Distal pancreatectomy	1.368 (0.411–4.547)	0.609
Total pancreatectomy	18.353 (3.162–106.523)	0.001
Laparoscopic	0.212 (0.046–0.978)	0.047
Operation time (min)	0.999 (0.994–1.005)	0.823
Blood loss (mL)	1.000 (0.999–1.001)	0.605
Transfusion (RBCs)	1.601 (0.545–4.700)	0.392
Postoperative complication	0.774 (0.242–2.481)	0.667
Adjuvant chemotherapy	0.406 (0.089–1.847)	0.243
Adjuvant radiotherapy	0.451 (0.059–3.473)	0.445
Tumor size	1.131 (0.917–1.396)	0.249
Differentiation (poor)	2.460 (0.878–7.938)	0.084
Margin status (not R0)	0.997 (0.333–2.984)	0.996
Vascular infiltration	1.306 (0.435–3.914)	0.634
Perineural infiltration	2.090 (0.272–16.091)	0.479
Carcinoma embolus	0.834 (0.287–2.424)	0.739
Total lymph nodes (numbers)	1.021 (0.979–1.065)	0.331
Positive lymph nodes (numbers)	1.435 (0.993–2.073)	0.054

Note: Bold value: P < 0.05, considered statistically significant.

Abbreviation: RBCs, red blood cells.

Table 6 Univariate and Multivariate Cox Regression Analyses of Prognosis Factors (CA19-9, CTCs and CTECs) for Overall Survival

	Univariate Analysis		Multivariate Analysis		Cutoff Value
Prognostic Factor	HR (95% CI)	P value	HR (95% CI)	P value
Preoperative CA19-9	1.000 (0.999–1.002)	0.454
Preoperative CTC	1.011 (0.970–1.054)	0.603
Preoperative CD44+ CTC	1.122 (0.551–2.283)	0.751
Preoperative Vimentin+ CTC	0.506 (0.127–2.018)	0.335
Preoperative CTM	1.004 (0.725–1.391)	0.979
Preoperative CTEC	1.009 (0.971–1.049)	0.648
Preoperative CD44+ CTEC	1.204 (1.044–1.387)	0.010
Preoperative Vimentin+ CTEC	0.987 (0.904–1.079)	0.780
Preoperative CTECM	1.076 (0.985–1.175)	0.102
POD7 CA19-9	1.001 (0.996–1.007)	0.659
POD7 CTC	1.003 (0.999–1.007)	0.195
POD7 CD44+ CTC	1.005 (1.001–1.008)	0.011
POD7 Vimentin+ CTC	0.949 (0.303–2.973)	0.929
POD7 CTM	1.105 (0.768–1.590)	0.589
POD7 CTEC	1.001 (0.978–1.026)	0.915
POD7 CD44+ CTEC	1.040 (0.999–1.083)	0.058
POD7 Vimentin+ CTEC	0.977 (0.571–1.673)	0.933
POD7 CTECM	0.664 (0.230–1.918)	0.450
POM1 CA19-9	1.001 (1.000–1.002)	0.007	1.001 (1.000–1.002)	0.026	131.9
POM1 CTC	1.004 (0.991–1.017)	0.567
POM1 CD44+ CTC	1.030 (0.625–1.696)	0.907
POM1 Vimentin+ CTC	0.759 (0.382–1.510)	0.432
POM1 CTM	0.993 (0.511–1.930)	0.984
POM1 CTEC	1.014 (0.979–1.051)	0.435
POM1 CD44+ CTEC	1.060 (0.942–1.193)	0.335
POM1 Vimentin+ CTEC	0.825 (0.402–1.694)	0.600
POM1 CTECM	0.568 (0.074–4.334)	0.585

Note: Bold value: P < 0.05, considered statistically significant.

Abbreviations: CTCs, circulating tumor cells; CTMs, circulating tumor microemboli; CTECs, circulating tumor endothelial cells; CTEMs, circulating tumor endothelial microemboli.

Figure 4 The DFS were significantly different between two comparative groups divided by the independent prognostic factors: (A) history of diabetes, (B) positive lymph nodes > 2, (C) preoperative CD44+ CTECs > 3, (D) POM1 CA19-9 > 89.6 U/mL. The OS showed significantly different between two comparative groups divided by (E) history of diabetes, (F) POM1 CA19-9 > 131.9U/mL.