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Abstract

OBJECTIVE:

The present study investigates the differences in immune function, hemorheological alterations and prognostic evaluation in colorectal cancer (CRC) patients with different traditional Chinese medicine (TCM) syndromes.

METHODS:

A total of 128 patients, diagnosed as stage II and III of CRC, were recruited. They were assigned into three TCM syndromes: deficiency syndrome, excess syndrome, and syndrome of intermingled deficiency and excess, and another 53 healthy individuals were selected as the control. Flow cytometry was used to determine the peripheral blood lymphocyte subsets (the levels of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ , CD ${}_{8}^{+}$ , NK cells, and the ratios of CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ , Th1/Th2 and Tc1/Tc2). Whole blood viscosity (WBV), plasma viscosity (PV), hematocrit (Hct), erythrocyte sedimentation rate (ESR), plasma fibrinogen concentration (PFC) were measured using a fully-automatic blood rheological instrument. The univariate analysis and Cox regression analysis were conducted to evaluate the prognosis of CRC patients with different TCM syndromes.

RESULTS:

Compared with healthy individuals, CRC patients with three different syndromes had lower levels of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ , NK cells, and ratios of CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ , Th1/Th2 and Tc1/Tc2, but higher level of CD ${}_{8}^{+}$ , WBV, PV, Hct, ESR and PFC. Besides, patients with excess syndrome showed the highest levels of CD3 ${}^{+}$ , CD4 ${}^{+}$ and NK cells, and ratios of CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ , Th1/Th2 and Tc1/Tc2, but the lowest level of CD ${}_{8}^{+}$ among three syndromes, and those with deficiency syndrome showed an opposite trend. Compared with patients with excess syndrome, those with deficiency syndrome showed decreased WBV, PV, Hct, ESR and PFC. The pathological type, surgical approach, tumor node metastasis (TNM) stage, liver metastasis, TCM treatment time and different TCM syndromes were independent factors of prognostic survival in CRC patients except perioperative blood transfusion volume.

CONCLUSIONS:

Taken together, we conclude that patients with TCM deficiency syndrome has lower immune function and poorer prognosis while patients with TCM excess syndrome has higher immune function and better prognosis of CRC.

Keywords

Colorectal cancer traditional Chinese medicine syndrome immune function hemorheological alteration prognosis

1. Introduction

Colorectal cancer (CRC) is one of the leading malignancies influencing the digestive system, which is also the fourth reason for cancer-associated death with high incidence and high mortality in male and female of China [1, 2]. It is attributable to a great number of factors, such as genetics, lifestyle and environment, but in most cases, CRC is a non-hereditary disease that mainly results from somatic mutations in response to environmental factors [3]. Screening is likely to decrease the incidence rate of CRC with the help of promising tools for screening, such as virtual colonoscopy, colon capsule endoscopy and molecular stool and blood tests [4]. Moreover, the lymph node metastases, tumor differentiation, distant metastases, and tumor stage have been thought as prognostic and clinical factors of CRC [5]. CRC is the most common cancer of gastrointestinal cancer, and surgical resection seems to be the only curative choice for patients with gastrointestinal cancer, whereas the most of patients are diagnosed in advanced stages when the surgery is unavailable and may lead to a poor prognosis [6].

In recent years, traditional Chinese medicine (TCM) has been widely used as a therapeutic target for preventing and treating CRC [7]. TCM believes that all diseases lie in imbalance of people’s body and syndrome plays an important role in TCM due to all the therapies and formulas are determined on the basis of the patients’ syndrome condition [8]. In order to handle invading microorganisms, the innate immune system triggers inflammatory and antimicrobial reactions to protect body [9]. The immune function damage is in connection with the occurrence, metastasis, and prognosis of cancer [10]. Lymphocytes are central components of the immune system and lymphocyte subsets have produced a vital effect in the pathogenesis of CRC [11, 12]. Comparative hemorheology means that comparative variation is collected from blood composition and blood flow traits, moreover, comparative study may help understand variety of medical concepts and raise a new approach to medicine [13]. Over time there has been little emphasis given to study the combination of immune function, comparative hemorheology and TCM syndromes, and therefore, the objective of this study is to evaluate the role of different TCM syndromes in the diagnosis and prognosis of CRC.

2. Materials and methods

2.1 Ethics statement

The study was carried out with the approval of the Ethics Committee of the First Affiliated Hospital of Anhui Medical University. All participates were recruited by random selection and written informed consent was obtained prior to our study.

2.2 Study subjects

From June 2012 to January 2014, a total of 128 patients with CRC underwent colonoscopy and pathology in the Department of Gastrointestinal Surgery of the First Affiliated Hospital of Anhui Medical University were selected as the case group, including 65 cases of colon cancer and 63 cases of rectal cancer. The patients consisted of 79 males and 49 females with a mean age of 53.93 $\pm$ 11.57 years (ranged from 31 to 78 years), and their median age was 57 years. Another 53 healthy individuals tested in Physical Examination Center of the First Affiliated Hospital of Anhui Medical University were included into the control group over the same period, containing 33 males and 20 females with a mean age of 55.87 $\pm$ 11.62 years (ranged from 30 to 81 years), and the median age was 57 years. There was no significant difference in age and gender between the two groups (both $P>$ 0.05). Inclusion criteria were as follows: (1) patients with normal liver and kidney function, no concomitant metabolic, infectious or inflammatory bowel disease; (2) patients were not treated with steroids and immunosuppressants; (3) patients with no extra intestinal malignancy or history of chemotherapy or radiotherapy; (4) patients were in stage II and III of CRC. Exclusion criteria were as follows: (1) patients who did not meet the above inclusion criteria; (2) patients with severe dysfunction of vital organ (heart, lung, kidney and liver); (3) patients with psychiatric comorbidity or poor compliance; (4) patients who fail to be interviewed or contracted; (5) clinical data like gender, age, operation time or postoperative pathological findings was incomplete and irreparable, and thus we were unable to judge the stage, recurrence and metastasis time. TCM syndrome diagnostic standards contain the standard of excess and deficiency syndromes in “Diagnostics of traditional Chinese medicine” (a textbook for National Higher Institutions of Chinese Medicine) and classification of TCM syndrome of CRC at early and middle stages in “Diagnosis and treatment program for colorectal cancer (2012 edition)” by a key specialty cooperative group in the State Administration of traditional Chinese medicine. According to preoperative symptoms and diagnostic criteria of TCM, the subjects were divided into 3 different TCM syndromes: deficiency syndrome, excess syndrome, and syndrome of intermingled deficiency and excess.

2.3 Measurement of immune function

The immune function of three kinds of TCM syndromes in patients with CRC (deficiency syndrome, excess syndrome, syndrome of intermingled deficiency and excess, $n=$ 128) were detected. Meanwhile, 53 healthy individuals were taken as controls. Peripheral blood samples of patients with three different syndromes and normal subjects were taken, and flow cytometry (FACSaliur, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used to determine peripheral blood lymphocyte subsets, which included CD ${}_{3}^{+}$ (total T cells), CD ${}_{4}^{+}$ (helper T cells), CD ${}_{8}^{+}$ (cytotoxic T cells), natural killer (NK) cells, the ratios of CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ , Th1/Th2 and Tc1/Tc2. Firstly, fasting venous blood (2 ml) was obtained and anticoagulated by heparin. Then, the direct immunofluorescence on whole blood method was employed. Fluorescent labeled monoclonal antibodies (BD Pharmingen, San Diego, CA, USA) of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ , CD ${}_{8}^{+}$ , NK, Th1, Th2, Tc1 and Tc2 cells were added into tubes, every tube for 10 $\mu$ l. Subsequently, the tubes were added and mixed with 50 $\mu$ l blood individually, mixed, and followed by incubation in the dark at room temperature for 20 min. After that, the tubes were added and mixed with 2 ml hemolysin, followed by incubation in the dark at room temperature for 20 min again. The cells were centrifuged at 1000 r/min for 5 min and the supernatant was discarded. The cell were washed by phosphate buffer saline (PBS) (2 ml) and centrifuged at 1000 r/min for 5 min, then, the supernatant was removed. Paraformaldehyde (0.3 ml) with a concentration of 1% was added before measurement. The lymphocyte gate was set using forward scatter (FSC) as abscissa and side scatter (SSC) as ordinate, with 5000 cells were counted. FASCCFLLQHEST software was performed to detect and analyze immune function.

2.4 Determination of hemorheological alterations

Venous blood was extracted from normal subjects and CRC patients with three different syndromes, each for 3 ml. Whole blood viscosity (WBV) at high (200 s ${}^{-1}$ ), low (5 s ${}^{-1}$ ) shear rate, plasma viscosity (PV) ( $\eta$ p), hematocrit (Hct), erythrocyte sedimentation rate (ESR), plasma fibrinogen concentration (PFC) and other indicators were determined respectively using a fully-automatic blood rheological instrument (ZL – 6000, Beijing ZONCI Technology Development Co., Ltd, Beijing, China).

2.5 Data collection and follow-up

Clinical data were collected from outpatient electronic medical records. The general data like gender, age, surgical approach, perioperative blood transfusion volume, pathological type, tumor location, tumor node metastasis (TNM) stage, liver metastasis, and TCM treatment time were recorded in detail on the day of admission. Follow-up continued to the third year after surgery and the follow-up rate was 100%. Time for recurrence or metastasis was a period that started from the time of radical operation to the time of recurrence or metastasis. TCM treatment time was calculated during the first time to take TCM after radical operation to the time before metastasis or recurrence. Follow-up records of patients contains tumor marker, stool routine and occult blood, abdominal ultrasound, computed tomography (CT)/magnetic resonance image (MRI) examination records and the four diagnostic information when reexamination. The last follow-up was conducted by outpatient or telephone, and the deadline was January 2017.

2.6 Statistical analysis

The data were analyzed statistically using SPSS 19.0 software (IBM Corp Armonk, NY, USA), and measurement data were presented by mean $\pm$ standard deviation (SD). Data consistent with normal distribution between two groups was analyzed using $t$ -test and comparison among groups by one-way analysis of variance (ANOVA). Enumeration data were presented by the percentage and analyzed using Chi-square test. Kaplan-Meier analysis was used to draw the survival curves and log-rank method was used to compare survival curve between two groups or multiple groups. Cox regression analysis was applied to investigate the effects of multiple factors for prognosis of CRC. If the odds ratio (OR) was less than 1, the factor was considered as a protective factor; if the OR was more than 1, it was considered as a risk factor. $P<$ 0.05 was considered statistically significant.

3. Results

3.1 Baseline characteristics in healthy individuals and patients with deficiency syndrome, excess syndrome and intermingled deficiency and excess

As shown in Table 1, patients with three kinds of syndromes have no significant difference in gender or age when compared with healthy individuals (all $P>$ 0.05). Besides, gender, age, surgical approach, perioperative blood transfusion volume, pathological type, tumor location, TNM stage, liver metastasis and TCM treatment time showed no significant difference among patients with three different syndromes (all $P>$ 0.05).

Table 1
Comparison of baseline characteristics in healthy individuals and patients with deficiency syndrome, excess syndrome and intermingled deficiency and excess

Baseline characteristics	Deficiency syndrome	Excess syndrome	Syndrome of intermingled	Healthy individuals	$P$
	( $n=$ 42)	( $n=$ 39)	deficiency excess ( $n=$ 47)	( $n=$ 53)
Gender
Male $n$ (%)	28 (66.67%)	23 (58.97%)	28 (59.57%)	33 (62.26%)	0.883
Female $n$ (%)	14 (33.33%)	16 (41.03%)	19 (40.43%)	20 (37.74%)
Age (years old)
$\leqslant$ 55 $n$ (%)	19 (45.24%)	15 (38.46%)	17 (36.17%)	23 (43.40%)	0.803
$>$ 55 $n$ (%)	23 (54.76%)	24 (61.54%)	30 (63.83%)	30 (56.60%)
Tumor location
Colon $n$ (%)	25 (59.52%)	18 (46.15%)	22 (46.81%)	–	0.384
Rectum $n$ (%)	17 (40.48%)	21 (53.85%)	25 (53.19%)	–
Pathological type
Well differentiation $n$ (%)	21 (50.00%)	24 (61.54%)	26 (55.32%)	–	0.862
Moderate differentiation $n$ (%)	14 (33.33%)	11 (28.21%)	14 (29.79%)	–
Poor differentiation $n$ (%)	7 (16.67%)	4 (10.26%)	7 (14.89%)	–
TNM stage
II $n$ (%)	29 (69.05%)	30 (76.92%)	35 (74.47%)	–	0.711
III $n$ (%)	13 (30.95%)	9 (23.08%)	12 (25.53%)	–
Perioperative blood transfusion volume (ml)
$\leqslant$ 400 $n$ (%)	26 (61.90%)	26 (66.67%)	28 (59.57%)	–	0.792
$>$ 400 $n$ (%)	16 (38.10%)	15 (33.33%)	19 (40.43%)	–
Liver metastasis
H0 $n$ (%)	13 (30.95%)	12 (30.77%)	21 (44.68%)	–	0.464
H1 $n$ (%)	12 (28.57%)	11 (28.21%)	12 (25.53%)	–
H2 $n$ (%)	7 (16.67%)	7 (17.95%)	10 (21.28%)	–
H3 $n$ (%)	10 (23.81%)	9 (23.08%)	4 (8.51%)	–
Surgical approach
Laparotomy $n$ (%)	31 (73.81%)	34 (87.18%)	39 (82.98%)	–	0.284
Laparoscopic surgery $n$ (%)	11 (26.19%)	5 (12.82%)	8 (17.02%)	–
TCM treatment time (year)
$\leqslant$ 1 $n$ (%)	9 (21.43%)	8 (20.51%)	13 (27.66%)	–	0.688
$>$ 1 $n$ (%)	33 (78.57%)	31 (79.49%)	34 (72.34%)	–

3.2 Patients with excess syndrome have higher immune function

The results in Table 2 indicated that compared with healthy individuals, CRC patients with three different syndromes had low levels of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ , and NK, as well as ratios of Th1/Th2, CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ , and Tc1/Tc2 but a high level of CD ${}_{8}^{+}$ (all $P<$ 0.05). When patients with three different syndromes were compared, the levels of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ and NK cells in patients with excess syndrome was the highest, while with the deficiency syndrome was the lowest (all $P<$ 0.05). Moreover, the patients with deficiency syndrome had the highest level of CD ${}_{8}^{+}$ cell, while those with excess syndrome had the lowest level ( $P<$ 0.05). Besides, the syndrome of intermingled deficiency and excess always kept the middle position in the expression levels of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ , CD ${}_{8}^{+}$ and NK cells among three different groups. However, the ratios of Th1/Th2, CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ and Tc1/Tc2 were higher in patients with excess syndrome, while were smaller in those with deficiency syndrome ( $P<$ 0.05), and no significant difference was found in the syndrome of intermingled deficiency and excess ( $P>$ 0.05). From aforementioned, it was indicated that patients with deficiency syndrome have decreased immune function. Meanwhile, patients with excess syndrome may have higher immune function, and patients with syndrome of intermingled deficiency and excess have medium immune function.

3.3 Changes of hemorheological alterations in healthy individuals and patients with deficiency syndrome, excess syndrome and intermingled deficiency and excess

The findings in Table 3 demonstrated that in comparison to healthy individuals, patients with CRC had significant higher WBV, PV, Hct, ESR and PFC (all $P<$ 0.05), indicating that the blood of CRC patients was hypercoagulable. The WBV, PV, Hct, ESR and PFC were lower in patients with deficiency syndrome than those with excess syndrome (all $P<$ 0.05). Patient with syndrome of intermingled deficiency and excess had significant higher WBV and PFC than those

Table 2
Comparison of T lymphocyte subsets and NK cells in peripheral blood in healthy individuals and patients with deficiency syndrome, excess syndrome and intermingled deficiency and excess

Group	Case	CD ${}_{3}^{+}$		CD ${}_{4}^{+}$		CD ${}_{8}^{+}$		CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$		NK cell		Th1/Th2		Tc1/Tc2
Healthy individuals	53	69.58	$\pm$ 8.12	38.32	$\pm$ 5.07	20.04	$\pm$ 3.42	1.96	$\pm$ 0.41	20.48	$\pm$ 5.30	3.50	$\pm$ 1.57	15.35	$\pm$ 6.60
Deficiency syndrome	42	56.47	$\pm$ 3.99 ${}^{*\#}$	29.28	$\pm$ 3.74 ${}^{*\#}$	25.84	$\pm$ 3.22 ${}^{*\#}$	1.15	$\pm$ 0.21 ${}^{*\#}$	14.35	$\pm$ 3.49 ${}^{*\#}$	1.27	$\pm$ 0.37 ${}^{*\#}$	5.39	$\pm$ 2.13 ${}^{*\#}$
Excess syndrome	39	62.86	$\pm$ 8.57 ${}^{*}$	33.73	$\pm$ 4.00 ${}^{*}$	22.69	$\pm$ 3.22 ${}^{*}$	1.52	$\pm$ 0.31 ${}^{*}$	17.67	$\pm$ 4.03 ${}^{*}$	2.33	$\pm$ 0.61 ${}^{*}$	10.56	$\pm$ 5.57 ${}^{*}$
Syndrome of intermingled	47	59.46	$\pm$ 4.13 ${}^{*}$	31.45	$\pm$ 3.93 ${}^{*}$	24.14	$\pm$ 2.61 ${}^{*}$	1.32	$\pm$ 0.25 ${}^{*}$	15.91	$\pm$ 3.15 ${}^{*}$	1.89	$\pm$ 0.62 ${}^{*}$	8.06	$\pm$ 4.91 ${}^{*}$
deficiency and excess

Notes: ${}^{*}$ , $P<$ 0.05 compared with healthy individuals; ${}^{\#}$ , $P<$ 0.05 compared with patients with excess syndrome.

Table 3

Comparison of hemorheological alterations in healthy individuals and patients with deficiency syndrome, excess syndrome and intermingled deficiency and excess

Group	Case	Whole blood viscosity (mPa’s)				Plasma viscosity		Hematocrit		Erythrocyte sedimentation		Plasma fibrinogen
		High shear rate		Low shear rate		(mPa’s)		(%)		rate (mm/h)		concentration (g/l)
Healthy individuals	53	4.89	$\pm$ 0.13	6.35	$\pm$ 0.13	1.65	$\pm$ 0.11	35.36	$\pm$ 3.76	12.06	$\pm$ 4.78	284.05	$\pm$ 12.32
Deficiency syndrome	42	5.22	$\pm$ 0.07 ${}^{*\#}$	7.88	$\pm$ 0.16 ${}^{*\#}$	1.80	$\pm$ 0.05 ${}^{*\#}$	40.12	$\pm$ 3.79 ${}^{*\#}$	16.03	$\pm$ 5.98 ${}^{*\#}$	330.09	$\pm$ 14.55 ${}^{*\#}$
Excess syndrome	39	5.44	$\pm$ 0.16 ${}^{*}$	8.12	$\pm$ 0.28 ${}^{*}$	1.88	$\pm$ 0.16 ${}^{*}$	43.11	$\pm$ 4.20 ${}^{*}$	20.25	$\pm$ 8.22 ${}^{*}$	359.36	$\pm$ 28.12 ${}^{*}$
Syndrome of intermingled	47	5.27	$\pm$ 0.11 ${}^{*\#}$	8.00	$\pm$ 0.36 ${}^{*}$	1.83	$\pm$ 0.13 ${}^{*}$	41.62	$\pm$ 4.79 ${}^{*}$	17.15	$\pm$ 8.32 ${}^{*}$	342.10	$\pm$ 20.49 ${}^{*\#}$
deficiency and excess

Notes: ${}^{*}$ , $P<$ 0.05 compared with healthy individuals; ${}^{\#}$ , $P<$ 0.05 compared with patients with excess syndrome.

patients with deficiency syndrome, which were lower than those patients with excess syndrome (all $P<$ 0.05). There was no significant difference in WBV, PV, Hct, and ESR in patients with syndrome of intermingled deficiency and excess, deficiency syndrome and excess syndrome (all $P>$ 0.05).

3.4 Functional changes had greater influence on the quality of life of CRC patients with the deficiency syndrome by Karnofsky performance scores (KPS)

According to the KPS results in Table 4, there were 29 patients with CRC had a score of 90 accounted for 22.66% of the total cases, 68 patients had a score of 80 accounted for 53.13% of the total cases, and 31 patients had a score less than or equal to 70 accounted for 24.22% of the total cases. Among patients whose KPS $=$ 90, the patients with excess syndrome had the largest proportion while those with deficiency syndrome had the least proportion. Among patients whose KPS $=$ 70, the patients with excess syndrome had the least proportion while those with deficiency syndrome had the largest proportion. In addition, the patients with syndrome of intermingled deficiency and excess always occupied the middle ground. It was suggested that the functional changes had greater influence on the quality of life of CRC patients with deficiency syndrome.

Table 4
KPS for CRC patients with deficiency syndrome, excess syndrome and syndromes of intermingled deficiency and excess

Group	Case	90 scores	80 scores	$\leqslant$ 70 scores
Deficiency syndrome	42	5 (11.90%)	21 (50.00%)	16 (38.10%)
Excess syndrome	39	14 (35.90%)	22 (56.41%)	3 (7.69%)
Syndrome of intermingled deficiency and excess	47	10 (21.28%)	25 (53.19%)	12 (25.53%)
Total case	128	29 (22.66%)	68 (53.13%)	31 (24.22%)

Notes: KPS, Karnofsky Performance Scores; CRC, colorectal cancer.

3.5 Patients with excess syndrome have better prognosis

The clinicopathological features of CRC patients with different TCM syndromes were analyzed by univariate analysis (Table 5). The prognostic indicators included gender, age, surgical approach, perioperative blood transfusion volume, pathological type, tumor location, TNM stage, liver metastasis, and TCM treatment time. Among these, there was no significant difference in the prognosis of CRC between female and male patients, patients over 55 years and under 55 years, or patients with colon cancer and patients with rectal cancer (all $P>$ 0.05). With regard to pathological type, patients with higher degree of differentiation had a better prognosis. Moreover, patients in stage III had a poor prognosis compared with patients in stage II, suggesting that higher malignancy led to poor prognosis. Patients receiving laparoscopic surgery had a poor prognosis compared with patients receiving laparotomy. The larger the perioperative blood transfusion volume or the more severe the liver metastasis was, the poorer the prognosis was. In terms of TCM treatment time, the longer the time was, the better the prognosis was. Furthermore, patients with excess syndrome had a higher prognostic survival rate than patients with deficiency syndrome ( $P<$ 0.05) (Fig. 1).

Table 5
The univariate analysis of prognosis in CRC patients with different TCM syndromes

Clinicopathological features	Case	Three-year survival rate (%)	X ${}^{2}$	$P$
Gender
Male	79	48.1	1.631	0.202
Female	49	57.14
Age (years old)
$\leqslant$ 55	51	52.94	0.74	0.390
$>$ 55	77	50.65
Tumor location
Colon	65	49.23	0.186	0.667
Rectum	63	53.97
Pathological type
Well differentiation	71	69.01	14.987	0.001
Moderate differentiation	36	35.9
Poor differentiation	21	16.67
TNM stage
II	94	58.51	14.73	$<$ 0.001
III	34	32.35
Perioperative transfusion volume (ml)
$\leqslant$ 400	80	63.75	4.914	0.027
$>$ 400	48	31.25
Liver metastasis
H0	46	82.61	16.456	0.001
H1	35	40
H2	24	29.17
H3	23	30.43
Surgical approach
Laparotomy	104	58.65
Laparoscopic surgery	24	20.83	5.417	0.02
0TCM treatment time (year)
$\leqslant$ 1	30	26.67
$>$ 1	98	59.18	6.653	0.010
TCM syndromes
Deficiency syndrome	42	28.57
Excess syndrome	39	94.87	6.298	0.043
Syndrome of intermingled deficiency and excess	47	36.17

Notes: TCM, traditional Chinese medicine; CRC, colorectal cancer; TNM, tumor node metastasis.

Figure 1.

Survival curves of univariate analysis in patients with different TCM syndromes. Notes: A, survival curves of CRC patients with different pathological types; B, survival curves of CRC patients in different TNM stages; C, survival curves of CRC patients with different perioperative blood transfusion volume; D, survival curves of CRC patients with or without liver metastasis; E, survival curves of CRC patients with different surgical approach; F, survival curves of CRC patients having different TCM treatment time; G, survival curves of CRC patients with different TCM syndromes; H, the total survival curves of 128 CRC patients; TCM, traditional Chinese medicine; TNM, tumor node metastasis; CRC, colorectal cancer.

3.6 Cox regression analysis in prognosis of CRC patients with different TCM syndromes

Cox regression analysis was carried out using the prognostic factors of CRC patients with different TCM syndromes analyzed by univariate analysis. The factors affecting the prognosis of CRC were used as the independent variables and the prognostic survival time as dependent variable. As shown in Table 6, the pathological type, liver metastasis, TNM stage, TCM treatment time, surgical approach and different TCM syndromes were independent factors affecting the prognosis of CRC (all $P<$ 0.05). Moreover, TCM treatment time and surgical approach were protective factors, but pathological type, liver metastasis, TNM stage and different TCM syndromes were risk factors for CRC.

Table 6
Cox regression analysis of prognosis in CRC patients with different TCM syndromes

Factor	$\beta$	SE ( $\beta$ )	Wald	$P$	Exp ( $\beta$ )	95% CI. for EXP ( $\beta$ )
						Lower	Upper
Pathological type	1.267	0.404	9.825	0.002	3.550	1.608	7.840
Perioperative blood transfusion volume	$-$ 0.838	0.635	1.745	0.187	0.433	0.125	1.500
Liver metastasis	0.845	0.399	4.480	0.034	2.328	1.065	5.093
TNM stage	1.244	0.496	6.292	0.012	3.470	1.313	9.175
TCM treatment time	$-$ 0.963	0.461	4.364	0.037	0.382	0.155	0.942
Surgical approach	$-$ 2.052	0.365	31.628	$<$ 0.001	0.128	0.063	0.263
TCM syndrome	1.166	0.409	8.140	0.004	3.210	1.441	7.152

Notes: TCM, traditional Chinese medicine; CRC, colorectal cancer; CI, confidence interval; TNM, tumor node metastasis.

4. Discussion

CRC is a common malignant disease with morbidity and mortality that accounts for 600,000 deaths around the world every year [4]. At present, the survival rate of CRC patients diagnosed at early-stage has been increased greatly, which is owing to the application of several CRC screening tests, but the prognosis of CPC patients at late-stage remains poor [14]. We conducted this study to determine the relationship of different TCM syndromes with the immune function, hemorheological alterations and prognosis of patients with CRC, and found that patients with deficiency syndrome had lower immune function and poorer prognosis than patients with excess syndrome. Patients with the syndromes of intermingled deficiency and excess had proper prognosis. Moreover, our findings suggest that increasing TCM treatment time can improve the prognosis and survival rate of CRC patients.

Initially, a significant result of the present study was that patients with different syndromes had lower levels of lymphocyte subsets than healthy individuals, and the patients with excess syndrome had the highest levels of CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ , NK cells, as well as ratios of CD ${}_{4}^{+}$ /CD ${}_{8}^{+}$ , Th1/Th2 and Tc1/Tc2, and the lowest level of CD ${}_{8}^{+}$ , which suggested that patients with excess syndrome have better immune function. In line with our study, the immunoregulatory indexes CD ${}_{3}^{+}$ and CD ${}_{4}^{+}$ was decreased in patients with pancreatitis in comparison to the normal subjects [15]. Recently, a report has been published that the change of T lymphocyte subsets, such as CD ${}_{3}^{+}$ , CD ${}_{4}^{+}$ and CD ${}_{8}^{+}$ in immune system could take an important role in the immune function and prognosis of patients with lung cancer [10]. Another study has revealed a variety of tumor-infiltrating T-cell subsets are related with improved immune response in a way of molecular changes [16]. Besides, changes and expression of T cell immunoglobulin and mucin domain 3 (Tim-3) on peripheral NK cells play a key regulatory role in CRC patients [17]. One possible interpretation of this difference between patients with excess syndrome and patients with deficiency syndrome might be the change of Tim-3 level, which led to the different prognosis of CRC.

Another important finding of this study was that blood of CRC patients was in a hypercoagulable state, as patients with CRC had significant higher WBV, PV, Hct, ESR and PFC. Consistently, it has been reported that elevated haemorheological parameters (WBV, PV, Hct, ESR and PFC) are found in sickle cell anaemia patients in vaso-occulussive crisis [18]. The observation results are in accordance with the previous studies that activated coagulation is related with cancer formation and metastasis of different types; particularly, intravascular coagulation can be regarded as clinical biomarker for the diagnosis and prognosis of CRC patients [19, 20]. A review concluded that the hypercoagulable state was associated with CRC from three aspects, and the hypercoagulable conditions played an important role, because venous thrombocytosis, hyperfibrinogenemia or D-dimer enhancement are closely related to clinical progression and poor prognosis of cancer patients [21]. In addition, a former study explained that malignancy and the hemostatic system are interrelated, and there is an interaction between hemostasis and cancer biology [22]. From aforementioned, we can hypothesize that hypercoagulable state and hemorheological alterations can be used as viable biomarkers in the development of CRC.

Our study also suggested that the pathological type, liver metastasis, TNM stage, TCM treatment time, surgical approach and different TCM syndromes were independent factors influencing CRC prognosis, and that prolonging TCM treatment time improved the prognosis and survival rate of CRC patients. The data implied that TCM treatment is feasible to patients with CRC. As it is known to all, TCM has been applied for health care in China for centuries, which has been used to prevent, treat and cure disorders or diseases [23]. It has been previously shown that TCM has been widely applied in additional therapy for CRC; meanwhile, it has significant efficacy with regard to improvement of quality of life and survival rate [24]. Likewise, Chinese medicine is able to prolong survival rate in CRC patients with stage II and III [25]. Furthermore, a previous study suggested that TCM syndrome differentiation and treatment have significant influence on enhancing the prognosis of elderly CRC patients in stage II or III [7]. Based on these findings, a combination of TCM treatment method could provide a novel therapeutic target of CRC patients [26].

Taken together, we concluded that patients with TCM excess syndrome had higher immune function and better prognosis than patients with deficiency syndrome or with the syndrome of intermingled deficiency and excess, and TCM treatment time was an independent factor of CRC and enhancement of it was correlated to the prognosis of CRC. However, it should be noted that this study has examined only by small scale, therefore, further studies are needed to confirm our results.

Footnotes

Acknowledgments

This work was supported by the National Nature Science Foundation of China (Grant No. 81573942). We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.

Conflict of interest

The authors have declared that no competing interests exist.

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