J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website https://www.jocmr.org

Original Article

Volume 000, Number 000, July 2024, pages 000-000

A Complex Interplay of Tumor Microenvironment Could Enhance Cholangiocarcinoma Progression Even After Surgery: A Prospective Study

Figure 1. Flow cytometric detection of the checkpoint expression on T cells. (a) Forward and side scatter histogram was used to define monocytes and lymphocytes population. (b) CD68 and CD11b were assessed on the monocyte population to detect total macrophage. (c) Then CD4+ cells and CD8⁺ cells were assessed on lymphocytes and then gated. (d, e) The expression of CD279 (PD-1) was assessed on CD4⁺ and CD8⁺ cells. PD-1: programmed cell death protein 1.

Figure 2. Differential expression of PD-1 and macrophages between patients and healthy controls. Data are analyzed by Mann Whitney U-test. PD-1: programmed cell death protein 1.

Figure 4. Differential expression of immune cells between single and multiple sites of metastasis (Mann-Whitney test). PD-1: programmed cell death protein 1; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio.

Figure 5. Differential expression of CD11bCD68⁺ macrophages according to the macroscopic pattern (Kruskal Wallis test, P = 0.037).

Figure 6. Differential accumulation of immune cells according to grading. PD-1: programmed cell death protein 1; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio.

Figure 7. Differential expression of immune cells according to previous surgery. PD-1: programmed cell death protein 1; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio.

**Table 1.** Demographic Data of Patients
Demographic data	Descriptive
Data are expressed as mean, median, SD, number, and percentages, as well as ECOG. ECOG-PS: Eastern Cooperative Oncology Group-performance status; SD: standard deviation; Min: minimum; Max: maximum; BMI: body mass index; DM: diabetes mellitus; HTN: hypertension.
Age (median)	59 years
Mean ± SD	56.6 ± 10.8 years
Min -Max	34 - 77 years
Sex (male/female)	13/12 (1.1/1)
ECOG-PS
PS = 0	9 (36%)
PS = 1 - 2	16 (64%)
BMI
Underweight	9 (36%)
Normal weight	6 (24%)
Overweight	10 (40%)
Smoking
None	12 (48%)
Smoker	4 (16%)
Ex-smoker	9 (36%)
Comorbidities
None	2 (8%)
Diabetic	6 (24%)
Hypertensive	11 (44%)
Both DM and HTN	6 (24%)

Table 1. Demographic Data of Patients

Demographic data

Descriptive

Data are expressed as mean, median, SD, number, and percentages, as well as ECOG. ECOG-PS: Eastern Cooperative Oncology Group-performance status; SD: standard deviation; Min: minimum; Max: maximum; BMI: body mass index; DM: diabetes mellitus; HTN: hypertension.

Age (median)

59 years

Mean ± SD

56.6 ± 10.8 years

Min -Max

34 - 77 years

Sex (male/female)

13/12 (1.1/1)

ECOG-PS

PS = 0

9 (36%)

PS = 1 - 2

16 (64%)

BMI

Underweight

9 (36%)

Normal weight

6 (24%)

Overweight

10 (40%)

Smoking

None

12 (48%)

Smoker

4 (16%)

Ex-smoker

9 (36%)

Comorbidities

None

2 (8%)

Diabetic

6 (24%)

Hypertensive

11 (44%)

Both DM and HTN

6 (24%)

**Table 2.** Clinicopathologic Characteristics
Characteristics	Descriptive
Data are expressed as numbers and percentages. CCA: cholangiocarcinoma; iCCA: intrahepatic CCA; pCCA: perihilar CCA; dCCA: distal CCA.
Primary site
dCCA	10 (40%)
pCCA	10 (40%)
iCCA	5 (20%)
Sites of metastasis
Single site	17 (68%)
Multiple sites	8 (32%)
Macroscopic pattern
Mass-forming	5 (20%)
Periductal-infiltrating	7 (28%)
Intraductal-growing	13 (52%)
Grading of CCA
G1	4 (16%)
G2	10 (40%)
G3	11 (44%)
Previous surgery
No surgery	12 (48%)
Radical surgery	6 (24%)
Palliative surgery	7 (28%)
Response
Complete response	9 (36%)
Partial response	5 (20%)
Stable disease	7 (28%)
Progressive disease	4 (16%)
Outcome
Dead	10 (40%)
Alive	15 (60%)

Table 2. Clinicopathologic Characteristics

Characteristics

Descriptive

Data are expressed as numbers and percentages. CCA: cholangiocarcinoma; iCCA: intrahepatic CCA; pCCA: perihilar CCA; dCCA: distal CCA.

Primary site

dCCA

10 (40%)

pCCA

10 (40%)

iCCA

5 (20%)

Sites of metastasis

Single site

17 (68%)

Multiple sites

8 (32%)

Macroscopic pattern

Mass-forming

5 (20%)

Periductal-infiltrating

7 (28%)

Intraductal-growing

13 (52%)

Grading of CCA

4 (16%)

10 (40%)

11 (44%)

Previous surgery

No surgery

12 (48%)

Radical surgery

6 (24%)

Palliative surgery

7 (28%)

Response

Complete response

9 (36%)

Partial response

5 (20%)

Stable disease

7 (28%)

Progressive disease

4 (16%)

Outcome

Dead

10 (40%)

Alive

15 (60%)

**Table 3.** Comparison Between Patients and Controls Regarding Hematologic Indices
Index	Groups	Mean ± SD	P value
Data are expressed as mean ± SD and analyzed by Mann-Whitney test. SD: standard deviation; RBCs: red blood cells; Hb: hemoglobin; WBCs: white blood cells; NLR: neutrophil to lymphocyte ratio; MPV: mean platelet volume; MPR: mean platelet volume/platelet count ratio.
RBC	Patients	4.2 ± 0.43	0.012
	Healthy controls	4.8 ± 1.0
Hb	Patients	11.7 ± 1.2	0.038
	Healthy controls	13.01 ± 2.3
WBCs	Patients	6.85 ± 3.7	0.012
	Healthy controls	10.15 ± 4.7
Neutrophils, %	Patients	75.37 ± 8.9	0.6
	Healthy controls	77.14 ± 15.4
Lymphocytes, %	Patients	17.97 ± 5.6	0.4
	Healthy controls	15.27 ± 11.8
Monocytes, %	Patients	4.99 ± 3.4	0.05
	Healthy controls	7.84 ± 6.0
Platelet count	Patients	181.44 ± 44.8	< 0.001
	Healthy controls	278.1 ± 114.5
MPV	Patients	9.64 ± 1.2	0.001
	Healthy controls	10.89 ± 1.1
NLR	Patients	4.7 ± 1.9	0.012
	Healthy controls	8.5 ± 5.7
MPR	Patients	0.057 ± 0.02	0.8
	Healthy controls	0.059 ± 0.07

Table 3. Comparison Between Patients and Controls Regarding Hematologic Indices

Index

Groups

Mean ± SD

P value

Data are expressed as mean ± SD and analyzed by Mann-Whitney test. SD: standard deviation; RBCs: red blood cells; Hb: hemoglobin; WBCs: white blood cells; NLR: neutrophil to lymphocyte ratio; MPV: mean platelet volume; MPR: mean platelet volume/platelet count ratio.

RBC

Patients

4.2 ± 0.43

0.012

Healthy controls

4.8 ± 1.0

Patients

11.7 ± 1.2

0.038

Healthy controls

13.01 ± 2.3

WBCs

Patients

6.85 ± 3.7

0.012

Healthy controls

10.15 ± 4.7

Neutrophils, %

Patients

75.37 ± 8.9

0.6

Healthy controls

77.14 ± 15.4

Lymphocytes, %

Patients

17.97 ± 5.6

0.4

Healthy controls

15.27 ± 11.8

Monocytes, %

Patients

4.99 ± 3.4

0.05

Healthy controls

7.84 ± 6.0

Platelet count

Patients

181.44 ± 44.8

< 0.001

Healthy controls

278.1 ± 114.5

MPV

Patients

9.64 ± 1.2

0.001

Healthy controls

10.89 ± 1.1

NLR

Patients

4.7 ± 1.9

0.012

Healthy controls

8.5 ± 5.7

MPR

Patients

0.057 ± 0.02

0.8

Healthy controls

0.059 ± 0.07

**Table 4.** Correlations Between Survival and PD-1 Expression and Macrophages
Variable	PFS	CD4⁺	CD4⁺PD-1⁺	CD8⁺	CD8⁺PD-1⁺	CD11b⁺CD68⁺	NLR	MPR
^aSignificant. Spearman rho correlation, r: correlation coefficient. PFS: progression-free survival; PD-1: programmed cell death protein 1; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; M: macrophages.
PFS	r	1	0.472^a	-0.262	0.088	-0.428^a	-0.213	-0.48^a	0.133
	P		0.017	0.205	0.677	0.033	0.308	0.015	0.526
CD4⁺	r	0.472^a	1	-0.593	-0.084	-0.280	-0.569	-0.2	-0.11
	P	0.017		0.002	0.689	0.175	0.003	0.453	0.587
CD4⁺PD-1⁺	r	-0.262	-0.593	1	0.152	0.544	0.300	0.030	-0.1
	P	0.205	0.002		0.468	0.005	0.146	0.888	0.646
CD8⁺ T cells	r	0.088	-0.084	0.152	1	0.241	0.246	-0.15	-0.32
	P	0.677	0.689	0.468		0.246	0.236	0.462	0.116
CD8⁺PD-1⁺	r	-0.43^a	-0.280	0.544	0.241	1	0.29	-0.15	-0.26
	P	0.033	0.175	0.005	0.246		0.163	0.475	0.206
CD11b⁺CD68⁺ M	r	-0.213	-0.569	0.300	0.246	0.288	1	-0.3	0.258
	P	0.308	0.003	0.146	0.236	0.163		0.199	0.212
NLR	r	-0.5^a	-0.157	0.030	-0.154	-0.150	-0.27	1	-0.3
	P	0.015	0.453	0.888	0.462	0.475	0.199		0.191
MPR	r	0.133	-0.114	-0.097	-0.322	-0.262	0.258	-0.27	1
	P	0.526	0.587	0.646	0.116	0.206	0.212	0.191

Table 4. Correlations Between Survival and PD-1 Expression and Macrophages

Variable

PFS

CD4⁺

CD4⁺PD-1⁺

CD8⁺

CD8⁺PD-1⁺

CD11b⁺CD68⁺

NLR

MPR

^aSignificant. Spearman rho correlation, r: correlation coefficient. PFS: progression-free survival; PD-1: programmed cell death protein 1; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; M: macrophages.

PFS

0.472^a

-0.262

0.088

-0.428^a

-0.213

-0.48^a

0.133

0.017

0.205

0.677

0.033

0.308

0.015

0.526

CD4⁺

0.472^a

-0.593

-0.084

-0.280

-0.569

-0.2

-0.11

0.017

0.002

0.689

0.175

0.003

0.453

0.587

CD4⁺PD-1⁺

-0.262

-0.593

0.152

0.544

0.300

0.030

-0.1

0.205

0.002

0.468

0.005

0.146

0.888

0.646

CD8⁺ T cells

0.088

-0.084

0.152

0.241

0.246

-0.15

-0.32

0.677

0.689

0.468

0.246

0.236

0.462

0.116

CD8⁺PD-1⁺

-0.43^a

-0.280

0.544

0.241

0.29

-0.15

-0.26

0.033

0.175

0.005

0.246

0.163

0.475

0.206

CD11b⁺CD68⁺ M

-0.213

-0.569

0.300

0.246

0.288

-0.3

0.258

0.308

0.003

0.146

0.236

0.163

0.199

0.212

NLR

-0.5^a

-0.157

0.030

-0.154

-0.150

-0.27

-0.3

0.015

0.453

0.888

0.462

0.475

0.199

0.191

MPR

0.133

-0.114

-0.097

-0.322

-0.262

0.258

-0.27

0.526

0.587

0.646

0.116

0.206

0.212

0.191

**Table 5.** Disturbance of Immune Cells According to Responses to Treatment
Immune cells	Complete response	Partial response	Stable disease	Progressive disease	P value
Data are expressed as mean ± SD and analyzed by Robust test. NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; M: macrophages; SD: standard deviation; PD-1: programmed cell death protein 1.
CD4⁺ T cells	Mean ± SD	38.1 ± 5.8	25.3 ± 4.5	21.7 ± 4.9	19.33 ± 10.6	0.001
CD4⁺PD-1⁺	Mean ± SD	11.77 ±13.4	39.5 ± 32.2	32.47 ± 16.8	55.1 ± 27.9	0.04
CD8⁺ T cells	Mean ± SD	15.77 ± 7.1	18.8 ± 4.2	19.5 ± 5.2	21.5 ± 14.3	0.7
CD8⁺PD-1⁺	Mean ± SD	14.8 ± 8.5	18.9 ± 3.3	17.37 ± 7.4	66.53 ± 19.8	0.008
CD11b⁺CD68⁺ M	Mean ± SD	12.9 ± 2.3	16.5 ± 13.2	45.69 ± 20.8	39.0 ± 4.6	< 0.001
NLR	Mean ± SD	4.2 ± 0.9	5.3 ± 1.8	4.93 ± 3.0	5.0 ± 1.9	0.6
MPR	Mean ± SD	0.06 ± 0.01	0.05 ± 0.02	0.06 ± 0.02	0.05 ± 0.03	0.7

Table 5. Disturbance of Immune Cells According to Responses to Treatment

Immune cells

Complete response

Partial response

Stable disease

Progressive disease

P value

Data are expressed as mean ± SD and analyzed by Robust test. NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; M: macrophages; SD: standard deviation; PD-1: programmed cell death protein 1.

CD4⁺ T cells

Mean ± SD

38.1 ± 5.8

25.3 ± 4.5

21.7 ± 4.9

19.33 ± 10.6

0.001

CD4⁺PD-1⁺

Mean ± SD

11.77 ±13.4

39.5 ± 32.2

32.47 ± 16.8

55.1 ± 27.9

0.04

CD8⁺ T cells

Mean ± SD

15.77 ± 7.1

18.8 ± 4.2

19.5 ± 5.2

21.5 ± 14.3

0.7

CD8⁺PD-1⁺

Mean ± SD

14.8 ± 8.5

18.9 ± 3.3

17.37 ± 7.4

66.53 ± 19.8

0.008

CD11b⁺CD68⁺ M

Mean ± SD

12.9 ± 2.3

16.5 ± 13.2

45.69 ± 20.8

39.0 ± 4.6

< 0.001

NLR

Mean ± SD

4.2 ± 0.9

5.3 ± 1.8

4.93 ± 3.0

5.0 ± 1.9

0.6

MPR

Mean ± SD

0.06 ± 0.01

0.05 ± 0.02

0.06 ± 0.02

0.05 ± 0.03

0.7

**Table 6.** Differential Expression of Immune Cells According to Grading
Variable	G1	G2	G3	P value
Data are expressed as mean ± SD and analyzed by Robust test. M: macrophages; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; SD: standard deviation; PD-1: programmed cell death protein 1.
CD4⁺ T cells	Mean ± SD	36.3 ± 3.1	26.3 ± 8.9	26.4 ± 11.3	0.009
CD4⁺PD-1⁺T cells	Mean ± SD	9.6 ± 12.8	30.7 ± 24.3	36.91 ± 27.6	0.049
CD8⁺ T cells	Mean ± SD	21.4 ± 1.4	16.5 ± 4.8	18.9 ± 10.3	0.036
CD8⁺PD-1⁺ T cells	Mean ± SD	10.1 ± 6.03	19.8 ± 11.9	34.3 ± 26.6	0.027
CD11b⁺CD68⁺ M	Mean ± SD	12.7 ± 2.3	21.3 ± 9.7	37.4 ± 23.6	0.004
NLR	Mean ± SD	4.2 ± 1.5	6.0 ± 1.9	3.8 ± 1.5	0.06
MPR	Mean ± SD	0.06 ± 0.01	0.053 ± 0.01	0.059 ± 0.02	0.4

Table 6. Differential Expression of Immune Cells According to Grading

Variable

P value

Data are expressed as mean ± SD and analyzed by Robust test. M: macrophages; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; SD: standard deviation; PD-1: programmed cell death protein 1.

CD4⁺ T cells

Mean ± SD

36.3 ± 3.1

26.3 ± 8.9

26.4 ± 11.3

0.009

CD4⁺PD-1⁺T cells

Mean ± SD

9.6 ± 12.8

30.7 ± 24.3

36.91 ± 27.6

0.049

CD8⁺ T cells

Mean ± SD

21.4 ± 1.4

16.5 ± 4.8

18.9 ± 10.3

0.036

CD8⁺PD-1⁺ T cells

Mean ± SD

10.1 ± 6.03

19.8 ± 11.9

34.3 ± 26.6

0.027

CD11b⁺CD68⁺ M

Mean ± SD

12.7 ± 2.3

21.3 ± 9.7

37.4 ± 23.6

0.004

NLR

Mean ± SD

4.2 ± 1.5

6.0 ± 1.9

3.8 ± 1.5

0.06

MPR

Mean ± SD

0.06 ± 0.01

0.053 ± 0.01

0.059 ± 0.02

0.4

**Table 7.** Disturbances of Immune Cells According to Previous Surgery
Variable (mean ± SD)	No surgery	Radical surgery	Palliative surgery	P value
Data are expressed as mean ± SD and analyzed by Kruskal Wallis test^a, and Robust test. PD-1: programmed cell death protein 1; M: macrophages; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; SD: standard deviation.
CD4⁺ T cells	23.2 ± 9.8	27.2 ± 4.8	36.6 ± 8.0	0.01
CD4⁺PD-1⁺ T cells	37.7 ± 25.1	33.7 ± 31.9	13.9 ± 12.9	0.1
CD8⁺ T cells	19.8 ± 8.6	20.1 ±2.2	14.2 ± 7.8	0.2
CD8⁺PD-1⁺ T cells^a	34.4 ± 26.6	12.92 ± 5.6	17.9 ± 7.3	0.045
CD11b⁺CD68⁺ M	37.4 ± 21.2	19.9 ± 10.4	15.2 ± 10.8	0.022
NLR	3.97 ± 1.6	6.6 ± 2.3	4.5 ± 1.04	0.016
MPR	0.059 ± 0.02	0.05 ± 0.01	0.06 ± 0.01	0.3

Table 7. Disturbances of Immune Cells According to Previous Surgery

Variable (mean ± SD)

No surgery

Radical surgery

Palliative surgery

P value

Data are expressed as mean ± SD and analyzed by Kruskal Wallis test^a, and Robust test. PD-1: programmed cell death protein 1; M: macrophages; NLR: neutrophil to lymphocyte ratio; MPR: mean platelet volume/platelet count ratio; SD: standard deviation.

CD4⁺ T cells

23.2 ± 9.8

27.2 ± 4.8

36.6 ± 8.0

0.01

CD4⁺PD-1⁺ T cells

37.7 ± 25.1

33.7 ± 31.9

13.9 ± 12.9

0.1

CD8⁺ T cells

19.8 ± 8.6

20.1 ±2.2

14.2 ± 7.8

0.2

CD8⁺PD-1⁺ T cells^a

34.4 ± 26.6

12.92 ± 5.6

17.9 ± 7.3

0.045

CD11b⁺CD68⁺ M

37.4 ± 21.2

19.9 ± 10.4

15.2 ± 10.8

0.022

NLR

3.97 ± 1.6

6.6 ± 2.3

4.5 ± 1.04

0.016

MPR

0.059 ± 0.02

0.05 ± 0.01

0.06 ± 0.01

0.3

**Table 8.** Multivariate Analysis of Prognostic Factors of PFS
Variable	B	Wald	P value	HR	95.0% CI for HR
Data were analyzed using Cox regression using forward stepwise methods (likelihood ratio), HR = e^B. HR: hazard ratio; CI: confidence interval; NLR: neutrophil to lymphocyte ratio; PFS: progression-free survival.
Step 1	NLR	0.440	7.699	0.006	1.553	1.138	2.119
Step 2	Response		11.773	0.008
	Complete response	-3.144	10.762	0.001	0.043	0.007	0.282
	Partial response	-2.540	6.710	0.010	0.079	0.012	0.539
	Stable disease	-1.806	3.917	0.048	0.164	0.027	0.983
	Progressive disease	Reference
	NLR	0.556	7.945	0.005	1.743	1.184	2.565

Table 8. Multivariate Analysis of Prognostic Factors of PFS

Variable

Wald

P value

95.0% CI for HR

Lower

Upper

Data were analyzed using Cox regression using forward stepwise methods (likelihood ratio), HR = e^B. HR: hazard ratio; CI: confidence interval; NLR: neutrophil to lymphocyte ratio; PFS: progression-free survival.

Step 1

NLR

0.440

7.699

0.006

1.553

1.138

2.119

Step 2

Response

11.773

0.008

Complete response

-3.144

10.762

0.001

0.043

0.007

0.282

Partial response

-2.540

6.710

0.010

0.079

0.012

0.539

Stable disease

-1.806

3.917

0.048

0.164

0.027

0.983

Progressive disease

Reference

NLR

0.556

7.945

0.005

1.743

1.184

2.565