Assessment of the Circulating Tumor Cells and Microsatellite Instability in Colorectal Cancer Patients: Prognostic and Diagnostic Value

Figures & data

Table 1 Clinico-Pathological Characteristics of the Assessed Colorectal Cancer Patients (n=100)

Characteristics	Patients, n (%)
Age (years)
Mean±SD	46.2±14.2
<46	48 (48)
≥46	52 (52)
Gender
Male	41(41)
Female	59 (59)
Diabetes mellitus
Non-diabetic	88 (88)
Diabetic	12 (12)
Hypertension
Non-hypertensive	87 (87)
Hypertensive	13 (13)
Family history
Absent	85 (85)
Present	15 (15)
T-stage
T1–2	24 (24)
T3–4	76 (76)
Stage
Early (I–II)	37 (37)
Late (III–IV)	63 (63)
Grade
I–II	75 (75)
III	25 (25)
Metastasis
M0	71 (71)
M1	29 (29)
Pathology
Adenocarcinoma	82 (82)
Mucinous	18 (18)
Laterality
Right colon	34 (34)
Left colon	31(31)
Transverse colon	4 (4)
Rectum	31 (31)
Treatment
Adjuvant	57 (57)
Neo-adjuvant	22 (22)
Palliative chemotherapy	29 (29)
Radiotherapy	10 (10)
Type of surgery (81)
Abdominoperineal resection	12 (14.7)
Low anterior resection	11 (13.5)
Left hemicolectomy	19 (23.5)
Palliative resection	5 (6.1)
Right hemicolectomy	25 (30.8)
Transverse colectomy	9 (11.1)

Figure 1 (A) The expression levels of MSI-high, MSI-low and MSS in colorectal cancer patients, (B) the mean count of CTCs in CRC patients before and after treatment, (C) the expression levels of CTCs (baseline, post-treatment, and serial CTCs change) in the patients.

Table 2 Association Between MSI and the Clinico-Pathological Features of the CRC Patients

Characteristics	MSI			P value^a
	Stable (n= 19)	MSI-Low (n=37)	MSI-High (n=44)
Age (years)
<46	9 (47.4)	15 (40.5)	24 (54.5)	0.45
≥46	10 (52.6)	22 (59.5)	20 (45.5)
Gender
Male	9 (47.4)	15 (40.5)	17 (38.6)	0.81
Female	10 (52.6)	22 (59.5)	27 (61.4)
DM
Non-diabetic	17 (89.5)	31 (83.6)	40 (90.9)	0.60
Diabetic	2 (10.5)	6 (16.2)	4 (9.1)
HTN
Non-hypertensive	17 (89.5)	31 (83.6)	39 (88.6)	0.76
Hypertensive	2 (10.5)	6 (16.2)	5 (11.4)
FH
Absent	16 (84.2)	31 (83.6)	38 (86.4)	0.94
Present	3 (15.8)	6 (16.2)	6 (13.6)
T-stage
T1–2	2 (10.5)	9 (24.3)	13 (29.5)	0.27
T3–4	17 (89.5)	28 (75.7)	31 (70.5)
Stage
Early (I–II)	6 (31.6)	7 (18.9)	24 (54.5)	0.004
III	4 (21.1)	13 (35.1)	16 (36.4)
IV	9 (47.3)	17 (45.9)	4 (9.1)
Grade
1–2	14 (73.7)	28 (75.7)	33 (75.0)	0.72
3	5 (26.3)	9 (24.3)	11 (25.0)
Metastasis
M0	10 (52.6)	21 (56.8)	40 (90.9)	<0.001
M1	9 (47.4)	16 (43.2)	4 (9.1)
Laterality
Right	7(36.8	13(35.1)	14(31.8)	0.82
Left	4(21.1)	10(27.0)	17(36.8)
Transverse	1(5.3)	1(2.7)	2(4.5)
Rectum	7(36.8)	13(35.1)	11(25.0)
Pathology
Adenocarcinoma	18(94.7)	29(78.4)	35(79.5)	0.27
Mucinous	1(5.3)	8(21.6)	9(20.5)

Notes: ^aChi-Square test, values in bold are statistically significant <0.05.

Abbreviation: MSI, microsatellite instability.

Table 3 Association Between the Baseline CTCs and Clinic-Pathological Features in CRC Patients

Characteristics	n	Baseline CTCs Count Median (Range)	P^a value	Baseline CTCs n (%)		P value
				<4 Cells	≥4 Cells
Age (years)
<46	48	6.0 (0–30)	0.33	19(48.7)	29(47.5)	0.91^c
≥46	52	5.0 (0–51)		20(51.3)	32(52.5)
Gender
Male	41	5.0 (0–30)	0.70	15(38.5)	26(42.6)	0.68^c
Female	59	6.0 (0–51)		24(61.5)	35(57.4)
DM
Non-diabetic	88	5.5 (0–50)	0.50	34(87.2)	54(88.4)	0.84^c
Diabetic	12	7.0 (0–51)		5(12.8)	7(11.6)
Hypertension
Non-hypertensive	87	5.0 (0–44)	0.06	36(92.3)	51(83.6)	0.21^c
Hypertensive	13	10.0 (1–51)		3(7.7)	10(16.4)
Family history
Absent	85	6.0 (0–51)	0.69	31(79.5)	54(88.4)	0.22^c
Present	15	3.0 (0–22)		8(20.5)	7(12.6)
T-stage
T1–2	24	4.0 (0–51)	0.59	11(28.2)	13(21.3)	0.43^c
T3–4	76	6.0 (0–50)		28(71.8)	48(78.7)
Stage
Early (I–II)	37	6.0 (0–44)	0.74	13(33.3)	24(39.3)	0.54^c
Late (III–IV)	63	5.0 (0–51)		26(66.7)	37(60.7)
Grade
1–2	75	5.0 (0–51)	0.24	30(76.9)	45(73.8)	0.72^c
3	25	7.0 (0–30)		9(23.1)	16(26.2)
Metastasis
M0	71	5.0 (0–51)	0.25	29(74.4)	42(68.9)	0.55^c
M1	29	7.0 (0–50)		10(25.6)	19(31.1)
Laterality
Right	34	6.0(0–51)	0.53^b	13(33.3)	21(34.4)	0.46^c
Left	31	8.0(0–44)		12(30.8)	19(31.1)
Transverse	4	5.0(4–18)		0(0.0)	4(6.6)
Rectum	31	4.0(0–50)		14(35.9)	17(27.9)
Pathology
Adenocarcinoma	82	5.0(0–51)	0.28^a	32(82.1)	50(82.0)	1.0^c
Mucinous	18	8.5(0–50)		7(17.9)	11(18.0)

Notes: ^aMann–Whitney test, ^bKruskal–Wallis test, ^cFischer’s exact test.

Abbreviation: CTCs, circulating tumor cells.

Table 4 Association Between Serial CTCs Level Changes and the Clinicopathological Features of the CRC Patients

Characteristics	n	Serial CTCs Level Changes n(%)		p value
		Decrease (n=74)	Increase (n=26)
Age (years)
<46	48	35(47.3)	13(50.0)	0.82^a
≥46	52	39(52.7)	13(50.0)
Gender
Male	41	28(37.8)	13(50.0)	0.36^a
Female	59	46(62.2)	13(50.0)
Diabetes Mellitus
Non-diabetic	88	65(87.8)	23(88.5)	1.0^a
Diabetic	12	9(12.2)	3(11.5)
Hypertension
Non-hypertensive	87	64(86.5)	23(88.5)	1.0^a
Hypertensive	13	10(13.5)	3(11.5)
Family History
Absent	85	65(87.8)	20(76.9)	0.21^a
Present	15	9(12.2)	6(23.1)
T-stage
T1-2	24	14(18.9)	10(38.5)	0.06^a
T3-4	76	60(81.1)	16(61.5)
Stage
Early (I–II)	37	31(41.9)	6(23.1)	0.10^a
Late (III–IV)	63	43(58.1)	20(76.9)
Grade
1-2	75	56(75.7)	19(73.1)	0.79^a
3	25	18(24.3)	7(26.9)
Metastasis
M0	71	60(81.1)	11(42.3)	0.001^a
M1	29	14(18.9)	15(57.7)
Laterality
Right	34	27(36.5)	7(26.9)	0.54^b
Left	31	20(27.0)	11(42.3)
Transverse	4	3(4.1)	1(3.8)
Rectum	31	24(32.4)	7(26.9)
Pathology
Adenocarcinoma	82	62(83.8)	20(76.9)	0.55^a
Mucinous	18	12(16.2)	6(23.1)

Notes: ^aFischer exact test, ^bChi-Square test, values in bold are statistically significant <0.05.

Abbreviation: CTCs, circulating tumor cells.

Figure 2 Association between PFS and (A) baseline CTCs count, (B) serial CTCs change, (C) post-treatment CTCs count. Association between OS and (D) baseline CTCs count, (E) serial CTCs change, (F) post-treatment CTCs count.

Figure 3 The impact of MSI levels in all CRC patients on (A) PFS and (B) OS rates. The impact of MSI levels in early-stage CRC patients on (C) PFS and (D) OS rates. The impact of MSI levels in late-stage CRC patients on (E) PFS and (F) OS rates.

Table 5 Univariate and Multivariate Survival Analysis for the Assessed CRC Patients

Parameters	Overall Survival			Progression-Free Survival
	HR	95% CI	p	HR	95% CI	p
Univariate
Age (years)  <46 vs ≥46	0.71	0.27–1.9	0.50	1.01	0.60–2.0	0.76
Gender  Male vs Female	2.6	0.94–7.1	0.067	1.37	0.75–2.5	0.31
DM  Diabetic vs Non-diabetic	1.04	0.24–4.6	0.96	2.7	1.2–5.8	0.012
Hypertension  HTN vs non-HTN	1.65	0.47–5.8	0.44	1.5	0.7–3.3	0.30
FH  Yes vs No	1.4	0.40–5.0	0.58	1.4	0.67–3.1	0.36
Stage  Late vs Early	9.6	1.3–72.5	0.029	3.47	1.6–7.5	0.002
Grade  3 vs 1–2	3.01	1.13–8.0	0.028	1.3	0.69–2.6	0.39
Metastasis  M1 vs M0	5.0	1.8–13.7	0.002	7.2	3.8–13.5	<0.001
T stage  T3-4 vs T1-2	1.29	0.37–4.5	0.69	0.58	0.30–1.2	0.11
LN  Present vs Absent	3.3	0.95–11.6	0.06	2.0	1.03–3.8	0.038
MSI  High vs Low and stable	2.9	1.19–7.2	0.019	1.6	1.04–2.05	0.033
Baseline CTCs Count  ≥4 vs <4/7 mL blood	0.46	0.17–1.2	0.12	1.3	0.7–2.5	0.39
Post-Treatment CTCs Level  ≥4 vs <4/7 mL blood	3.8	1.3–10.9	0.01	4.8	2.5–9.1	<0.001
Serial CTCs Change Levels  Increase vs Decrease	8.1	2.8–23.2	<0.001	6.8	3.5–12.9	<0.001
Multivariate
DM  Diabetic vs Non-diabetic				2.4	0.99–6.0	0.05
Hypertension  HTN vs Non-HTN				1.1	0.47–2.8	0.89
Stage  Late vs Early	5.7	0.67–49.6	0.11	3.3	0.99–10.8	0.051
Grade  3 vs 1–2	2.3	0.79–6.4	0.13
Metastasis  M1 vs M0	1.45	0.45–4.7	0.54	3.9	1.7–8.8	0.001
LN  Present vs Absent				2.6	1.01–7.04	0.047
MSI  High vs Low and MSS	1.87	0.71–4.9	0.21	0.84	0.51–1.4	0.50
Post-Treatment CTCs Level  ≥4 vs <4/7 mL blood	1.8	0.58–5.9	0.30	3.6	1.6–7.8	0.001
Serial CTCs Change Levels  Increase vs Decrease	4.1	1.26–13.5	0.012	5.1	2.3–11.3	<0.001

Note: Values in bold are statistically significant <0.05.

Abbreviations: CTCs, circulating tumor cells; MSI, microsatellite instability.

Figure 4 Correlation between MSI and CTCs count (Baseline and post-treatment). (A) Non-significant negative correlation between MSI-high and increase in number of base line CTCs (rho=−0.11, p=0.29). (B) shows significant positive correlation between MSI-high and increase in number of post-treatment CTCs (rho=0.399, p<0.001).

Abbreviation: rho, Spearman’s coefficient.