Pretreatment detection of circulating and tissue CD133⁺ CD44⁺ cancer stem cells as a prognostic factor affecting the outcomes in Egyptian patients with colorectal cancer

Figures & data

Table 1 Clinicopathologic characteristics of patients with nonmetastatic colorectal cancers

Characteristic	N	%
Age
Mean ± SD	45.24±2.41
Median	45
Min–max	17.0–80.0
Sex
Male:female	24:26	52:48
ECOG PS
1	21	42
2	19	38
3	10	20
Pathologic subtype
Adenocarcinoma	29	58
Mucoid carcinoma	2	4
Mucinous carcinoma	8	16
Signet ring carcinoma	11	22
Grade
1	5	10
2	27	54
3	6	12
4	12	24
Type of biopsy
Endoscopic	26	52
Incisional	7	14
Excisional	10	20
Punch biopsy	7	14
Perineural invasion
No	48	96
Yes	2	4
Lymphovascular invasion
No	43	86
Yes	7	14
CEA
Normal	12	24
High	38	76
T stage
1	3	6
2	6	12
3	22	44
4	16	32
Tx	3	6
N stage
0	8	16
1	19	38
2	14	28
Nx	9	18

Notes: Tx means that the primary lesion was completely excised at the time of colonoscopy and subsequently couldn’t be determined at time of surgery. Nx means inadequate number of LNs excised or complete absence of any LN from surgical specimens.

Abbreviations: CEA, carcinoembryonic antigen; ECOG PS, Eastern Cooperative Oncology Group Performance Status; N, lymph node; T, tumor.

Table 2 Distribution of tissue CD133⁺ CD44⁺ CSCs and tissue CD133⁻ CD44⁻ tumor cells among different cell cycle phases and their significance

Cell cycle phase	Tissue CD133^+ CD44^+ CSCs (mean ± SD, 43.61±3.606)	Tissue CD133^− CD44^− tumor cells (mean ± SD, 43.61±3.606)	P-value
G0/G1 phase	79.13±1.81 (53.0–99.0)	57.88±2.43 (19.0–73.0)	<0.02a
S phase	18.30±1.75 (0.0–45.0)	31.70±2.09 (19.73–100	<0.03a
G2/M phase	2.57±0.26 (0.0–6.5)	6.65±0.32 (0.0–12.35)	0.728

Note:

a Indicates significant.

Abbreviation: CSCs, cancer stem cells.

Figure 1 Flow cytometric detection of cancer colon circulating cancer stem cells.

Notes: (A) CD45 and side scatter histogram were used to select the CD45⁻cells. (B) The expression of CD133 and CD44 on CD45⁻ cells was then assessed. Circulating stem cells are CD45⁻ CD44⁺ CD133⁺.

Abbreviations: CSCs, cancer stem cells; PE, phycoerythrin.

Figure 2 Flow cytometric detection of cancer stem cells in primary tissue of cancer colon and cell cycles of sorted CD133⁺ CD44⁺ cells and CD133⁻ CD44⁻ cells.

Notes: (A, B) The expressions of CD133 and CD44 were assessed on tumor cells. Then, CD133⁺ CD44⁺ and CD133⁻ CD44⁻ cells were selected and sorted by using cell sorter of the FACSCalibur flow cytometer. (C, D) The cell cycle of isolated CD133⁺ CD44⁺ and CD133⁻ CD44⁻ cells.

Figure 3 Correlation between circulating CSCs and tissue CD133⁺ CD44⁺ CSCs (r=+0.677, P<0.001).

Abbreviations: CSCs, cancer stem cells; TSC, tissue cancer stem cells.

Figure 4 The median DFS of patients with nonmetastatic colorectal cancers.

Note: The median DFS of 50 patients with nonmetastatic colorectal cancers was 19±2.638 months (95% CI=13.840–24.160).

Abbreviation: DFS, disease-free survival.

Figure 5 The median OS of patients with colorectal cancers.

Note: The median OS of 50 patients with colorectal cancers was 23±1.755 months (95% CI=19.560–26.440).

Abbreviation: OS, overall survival.

Table 3 Univariate analysis of prognostic factors

Prognostic factors	DFS P-value	OS P-value
Age (years)	0.55	0.109
Sex	0.906	0.875
ECOG PS	<0.04a	<0.04a
Preoperative CEA	0.876	0.499
Pathologic subtype	0.235	0.471
Pathologic grade	0.320	0.377
T	<0.002b	<0.02a
N	<0.001c	<0.001c
Lymphovascular invasion	<0.04a	<0.01a
Perineural invasion	0.126	0.084

Notes:

a Indicates significant,

b indicates moderately significant, and

c indicates highly significant.

Abbreviations: CEA, carcinoembryonic antigen; DFS, disease-free survival; ECOG PS, Eastern Cooperative Oncology Group Performance Status; N, lymph node; OS, overall survival; T, tumor.

Table 4 The relations between circulating CSCs, tissue CD133⁺ CD44⁺ CSCs, and CD133⁻ CD44⁻ tumor cells with DFS and OS

Cell type	DFS	OS
Circulating CSCs	P<0.001,*** r=0.470	P<0.001,*** r=−0.487
CD133^+CD44^+ tissue CSCs	P<0.001,*** r=−0.548	P<0.001,*** r=−0.497
G0/G1 phase
Tissue CD133^+ CD44^+ CSCs	P<0.001***	P<0.003**
CD133^− CD44^− tumor cells	P=0.084	P=0.092
G2/M phase
Tissue CD133^+ CD44^+ CSCs Tissue CD133^− CD44^− tumor cells	P<0.004** P=0.09	P<0.01* P=0.07
S phase	P<0.001***	P<0.006**
Tissue CD133^+ CD44^+ CSCs	P<0.04*	P=0.334
CD133^− CD44^− tumor cells

Note:

* Significant,

** moderately significant,

*** highly significant.

Abbreviations: CSCs, cancer stem cells; DFS, disease-free survival; OS, overall survival.

Table 5 Multivariate analysis of different prognostic factors on DFS and OS

Factor	DFS		OS
	Standardized coefficient (beta)	P-value	Standardized coefficient (beta)	P-value
	ECOG PS	0.808	0.001***	0.738	0.001***
T	0.033	0.877	0.500	0.082
N	0.479	0.001***	0.557	0.002**
Lymphovascular invasion	–2.747	0.01*	–0.335	0.137
Circulating CSCs	–2.423	0.02*	–0.908	0.004**
Tissue CD133^+ CD44^+ CSCs	–2.951	0.006**	–0.862	0.031*

Note:

* Significant,

** moderately significant,

*** highly significant.

Abbreviations: CSCs, cancer stem cells; DFS, disease-free survival; ECOG PS, Eastern Cooperative Oncology Group Performance Status; N, lymph node; OS, overall survival; T, tumor.

Table 6 Relations between circulating CSCs and tissue CD133⁺ CD44⁺ CSCs with different clinical characteristics

Prognostic factors	Circulating CSCs P-value	Tissue CSCs P-value
Age (years)	0.329	0.478
Sex	0.532	<0.025*
ECOG PS	0.079	0.967
Preoperative CEA	0.693	0.619
Pathologic subtype	0.413	0.908
Pathologic grade	<0.004*	0.091
T	<0.004*	0.791
N	0.159	<0.045*
Lymphovascular invasion	0.287	0.414
Perineural invasion	0.426	0.236

Note:

* Significant.

Abbreviations: CEA, carcinoembryonic antigen; CSCs, cancer stem cells; ECOG PS, Eastern Cooperative Oncology Group Performance Status; N, lymph node; T, tumor.