Circulating tumor cell levels are elevated in colorectal cancer patients with high tumor burden in the liver

Figures & data

Table 1 Patients' characteristics of stage IV CRC patients that were analyzed for presence of baseline CTCs

Total number of patients		24
Age	• Mean (SEM)	63.6 (2.5)
	• Median (range)	64 (42–88)
Gender	• Females	8 (33%)
	• Males	16 (67%)
Location of primary tumor	• Rectosigmoid	16 (67%)
	• Descending colon	2 (8%)
	• Transverse colon	1 (4%)
	• Right colon/cecum/others	5 (21%)
Tumor invasion depth of primary tumor (data available in 14/24 patients)	• pT1	0
	• pT2	1 (7%)
	• pT3	5 (36%)
	• pT4	8 (57%)
Nodal status of primary tumor (data available in 13/24 patients)	• pN0	1 (8%)
	• pN1	1 (8%)
	• pN2	11 (84)
Lymphatic invasion (data available in 12/24 patients)	• Absent	3 (25%)
	• Present	9 (75%)
Venous invasion (data available in 11/24 patients)	• Absent	4 (36%)
	• Present	7 (64%)
Perineural invasion (data available in 11/24 patients)	• Absent	6 (55%)
	• Present	5 (45%)
Histological differentiation (data available in 21/24 patients)	• Moderate	15 (72%)
	• Poor	3 (14%)
	• Undifferentiated	3 (14%)
Histology of adenocarcinomas (AC)	• AC	19 (79%)
	• AC with signet cell features	2 (8%)
	• AC with mucinous features	3 (13%)
Serum CEA level (ng/ml; normal <5)	• Mean (SEM)	370.9 (221.0)
	• Median (range)	8.0 (1.4–4967.7)
KRAS mutation (codon 12 or 13)		1/20 (5%)
BRAF mutation (codon 600)		3/19 (16%)
High EGFR expression		12/15 (80%)
High ERCC1 expression		1/14 (7%)
High TS expression		6/14 (43%)
Circulating tumor cells (CTCs)	• Detectable	17/24 (71%)
	• Mean (SEM)	6.3 (2.9)
	• Median (Range)	1 (0–56.3)
	• Low baseline:
	• 0	7/24 (29%)
	• 1–2	10 (42%)
	• High baseline:
	• ≥3	7 (29%)
Liver metastases present		15/24 (63%)
Tumor/liver volume ratio (data available in 11/15 patients)	• Low (<30%)	8/11 (73%)
	• High (≥30%)	3/11 (27%)

Table 2 List of the individual patients with CTC counts and tumor/liver volume ratio

Patient #	Sites of Metastases	CEA serum level (normal range: 0–5ng/ml)	Mean CTC (±SEM) per 7.5cc blood	CTCs low (0–2) or high (≥3)	Tumor/liver volume ratio: low (<30%) or high (≥30%)	Tumor/liver volume ratio (%)	Total liver volume (cc)	Tumor volume (cc)
1	Lymph nodes (retroperitoneal, abdominal, supraclavicular)	3.7	3 (±0.6)	high	—	—	—	—
2	Lymph nodes (abdominal)	1.6	0.67 (±0.3)	low	—	—	—	—
3	Liver, lymph nodes, omentum, bone	4.3	0.5 (±0.5)	low	low	0.3	1704	5
4	Liver, lymph nodes (retroperitoneal)	31.9	2.3 (±0.3)	low	low	0.2	1704	4
5	Bone	18.3	1 (±0.6)	low
6	Liver	4.7	0	low	low	0.8	1381	11
7	Liver, lung	4967.7	56.3 (±7.1)	high	high	54.3	3182	1729
8	Liver, lung, lymph nodes (mesenteric, mediastinal)	1.6	1 (±1)	low	low	5.1	2856	145
9	Liver	2246	42.7 (±3.0)	high	high	43.7	2831	1238
10	Liver	275.5	11.5 (±1.5)	high	low	2.8	1386	39
11	Liver, adrenal gland	7.9	0.5 (±0.5)	low	low	0.7	1682	12
12	Lung, lymph nodes (mediastinal, mesenteric, periaortic)	2.3	1 (0)	low	—	—	—	—
13	Lung	61.5	0	low	—	—	—	—
14	Lymph nodes (mediastinal)	3.4	0.3 (±0.3)	low	—	—	—	—
15	Lymph nodes (mediastinal)	93.6	0	low	—	—	—	—
16	Liver, lung, brain	7.2	4.5 (±0.5)	high	high	72.1	2808	2024
17	Liver	1.4	0	low	—	—	—	—
18	Liver, lung	2.7	1 (±1.0)	low	low	4.5	1346	61
19	Lung, lymph nodes	3.5	0	low	—	—	—	—
20	Liver, lymph nodes, adrenal glands	137.9	0	low	low	1.5	1123	17
21	Lymph nodes (retroperitoneal, abdominal, supraclavicular)	8.0	0	low	—	—	—	—
22	Liver, lymph nodes (abdominal)	86.2	2	low	—	—	—	—
23	Liver, lymph nodes, omentum, bone	492.4	7 (±2.0)	high	—	—	—	—
24	Liver, lymph nodes (retroperitoneal)	438.2	16	high	—	—	—	—

Figure 1. Baseline CTC levels correlate with tumor burden in the liver. To statistically analyze the association between CTC numbers and tumor burden in the liver, baseline CTCs were categorized into low (0–2) and high (≥3), and the tumor/liver volume ratio was log-transformed to assure the validity of statistical assumptions. A significant association between the log-transformed tumor/liver volume ratio and baseline low/high CTC levels by both parametric (¹two-sample T-test (P = 0.0036)) and non-parametric (Wilcoxon Rank-Sum test) (P = 0.0298) analysis was determined. As shown in Table 3, the tumor/liver volume ratio was dichotomized into low (<30%) and high (≥30%) tumor/liver volume ratio, which was also found to be significantly associated with baseline CTC levels (high/low).

Table 3 Correlation of categorized tumor/liver volume ratio and CEA serum level with baseline CTC numbers in stage IV CRC

	Total	Low CTC baseline (0–2)	High CTC baseline (≥3)	P value
Tumor/liver volume ratio
• Low (<30%)	8/11 (73%)	7 (100%)	1 (25%)	0.0242
• High (≥30%)	3/11 (27%)	0	3 (75%)
Serum CEA level (ng/ml)
• <2.5	4 (17%)	4 (24%)	0	0.0019
• 2.5–5	6 (25%)	5 (28%)	1 (14%)
• 5–50	5 (21%)	4 (24%)	1 (14%)
• 50–200	4 (17%)	4 (24%)	0
• >200	5 (21%)	0	5 (72%)

P values were calculated by Fisher's Exact test

Figure 2. Baseline CTC levels correlate with serum CEA levels. Baseline serum CEA levels were log-transformed to stabilize the variance and make the associated statistical assumption more valid. A significant association between log-transformed serum CEA levels and categorized baseline CTC counts (low: 0–2, high: ≥3) was determined by parametric (¹two-sample T-test (P = 0.0016)) and non-parametric analysis (Wilcoxon Rank-Sum test (P = 0.0092)). As shown in Table 3, baseline serum CEA levels were also categorized and the correlation analysis with low/high baseline CTC levels was also statistically significant.

Figure 3. Computer tomography (CT), CTC images and EpCAM immunostaining of CRC tumors. EpCAM is consistently expressed in CRC primary tumors, also in patients that have no CTCs detectable by EpCAM-based CellSearch® system. Shown are: mean CTC numbers (±SEM), the actual liver/tumor volume ratio, a CTC (CellSearch® system qualifies a cell as a CTC if it has an evident nucleus by DAPI and if it is EpCAM+, cytokeratin 8/18/19+, and CD45-), (A) CT scan of a patient with a high tumor/liver ratio (≥30%) and high baseline CTCs (≥3), (B) EpCAM immunostaining with a monoclonal antibody (BerEP4) and peroxidase method of the respective CRC primary tumor, (C) CT scan of a patient with a low high tumor/liver ratio (<30%) zero baseline CTCs, and (D) EpCAM immunostaining of the respective CRC primary tumor.