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://jocmr.elmerjournals.com

Original Article

Volume 16, Number 7-8, August 2024, pages 363-374


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

Figures

Figure 1.
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.
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 3.
Figure 3. PFS of 25 metastatic cholangiocarcinoma. PFS: progression-free survival
Figure 4.
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.
Figure 5. Differential expression of CD11bCD68+ macrophages according to the macroscopic pattern (Kruskal Wallis test, P = 0.037).
Figure 6.
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.
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.

Tables

Table 1. Demographic Data of Patients
 
Demographic dataDescriptive
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 ± SD56.6 ± 10.8 years
  Min -Max34 - 77 years
Sex (male/female)13/12 (1.1/1)
ECOG-PS
  PS = 09 (36%)
  PS = 1 - 216 (64%)
BMI
  Underweight9 (36%)
  Normal weight6 (24%)
  Overweight10 (40%)
Smoking
  None12 (48%)
  Smoker4 (16%)
  Ex-smoker9 (36%)
Comorbidities
  None2 (8%)
  Diabetic6 (24%)
  Hypertensive11 (44%)
  Both DM and HTN6 (24%)

 

Table 2. Clinicopathologic Characteristics
 
CharacteristicsDescriptive
Data are expressed as numbers and percentages. CCA: cholangiocarcinoma; iCCA: intrahepatic CCA; pCCA: perihilar CCA; dCCA: distal CCA.
Primary site
  dCCA10 (40%)
  pCCA10 (40%)
  iCCA5 (20%)
Sites of metastasis
  Single site17 (68%)
  Multiple sites8 (32%)
Macroscopic pattern
  Mass-forming5 (20%)
  Periductal-infiltrating7 (28%)
  Intraductal-growing13 (52%)
Grading of CCA
  G14 (16%)
  G210 (40%)
  G311 (44%)
Previous surgery
  No surgery12 (48%)
  Radical surgery6 (24%)
  Palliative surgery7 (28%)
Response
  Complete response9 (36%)
  Partial response5 (20%)
  Stable disease7 (28%)
  Progressive disease4 (16%)
Outcome
  Dead10 (40%)
  Alive15 (60%)

 

Table 3. Comparison Between Patients and Controls Regarding Hematologic Indices
 
IndexGroupsMean ± SDP 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.
RBCPatients4.2 ± 0.430.012
Healthy controls4.8 ± 1.0
HbPatients11.7 ± 1.20.038
Healthy controls13.01 ± 2.3
WBCsPatients6.85 ± 3.70.012
Healthy controls10.15 ± 4.7
Neutrophils, %Patients75.37 ± 8.90.6
Healthy controls77.14 ± 15.4
Lymphocytes, %Patients17.97 ± 5.60.4
Healthy controls15.27 ± 11.8
Monocytes, %Patients4.99 ± 3.40.05
Healthy controls7.84 ± 6.0
Platelet countPatients181.44 ± 44.8< 0.001
Healthy controls278.1 ± 114.5
MPVPatients9.64 ± 1.20.001
Healthy controls10.89 ± 1.1
NLRPatients4.7 ± 1.90.012
Healthy controls8.5 ± 5.7
MPRPatients0.057 ± 0.020.8
Healthy controls0.059 ± 0.07

 

Table 4. Correlations Between Survival and PD-1 Expression and Macrophages
 
VariablePFSCD4+CD4+PD-1+CD8+CD8+PD-1+CD11b+CD68+NLRMPR
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.
PFSr10.472a-0.2620.088-0.428a-0.213-0.48a0.133
P0.0170.2050.6770.0330.3080.0150.526
CD4+r0.472a1-0.593-0.084-0.280-0.569-0.2-0.11
P0.0170.0020.6890.1750.0030.4530.587
CD4+PD-1+r-0.262-0.59310.1520.5440.3000.030-0.1
P0.2050.0020.4680.0050.1460.8880.646
CD8+ T cellsr0.088-0.0840.15210.2410.246-0.15-0.32
P0.6770.6890.4680.2460.2360.4620.116
CD8+PD-1+r-0.43a-0.2800.5440.24110.29-0.15-0.26
P0.0330.1750.0050.2460.1630.4750.206
CD11b+CD68+ Mr-0.213-0.5690.3000.2460.2881-0.30.258
P0.3080.0030.1460.2360.1630.1990.212
NLRr-0.5a-0.1570.030-0.154-0.150-0.271-0.3
P0.0150.4530.8880.4620.4750.1990.191
MPRr0.133-0.114-0.097-0.322-0.2620.258-0.271
P0.5260.5870.6460.1160.2060.2120.191

 

Table 5. Disturbance of Immune Cells According to Responses to Treatment
 
Immune cellsComplete responsePartial responseStable diseaseProgressive diseaseP 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 cellsMean ± SD38.1 ± 5.825.3 ± 4.521.7 ± 4.919.33 ± 10.60.001
CD4+PD-1+Mean ± SD11.77 ±13.439.5 ± 32.232.47 ± 16.855.1 ± 27.90.04
CD8+ T cellsMean ± SD15.77 ± 7.118.8 ± 4.219.5 ± 5.221.5 ± 14.30.7
CD8+PD-1+Mean ± SD14.8 ± 8.518.9 ± 3.317.37 ± 7.466.53 ± 19.80.008
CD11b+CD68+ MMean ± SD12.9 ± 2.316.5 ± 13.245.69 ± 20.839.0 ± 4.6< 0.001
NLRMean ± SD4.2 ± 0.95.3 ± 1.84.93 ± 3.05.0 ± 1.90.6
MPRMean ± SD0.06 ± 0.010.05 ± 0.020.06 ± 0.020.05 ± 0.030.7

 

Table 6. Differential Expression of Immune Cells According to Grading
 
VariableG1G2G3P 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 cellsMean ± SD36.3 ± 3.126.3 ± 8.926.4 ± 11.30.009
CD4+PD-1+T cellsMean ± SD9.6 ± 12.830.7 ± 24.336.91 ± 27.60.049
CD8+ T cellsMean ± SD21.4 ± 1.416.5 ± 4.818.9 ± 10.30.036
CD8+PD-1+ T cellsMean ± SD10.1 ± 6.0319.8 ± 11.934.3 ± 26.60.027
CD11b+CD68+ MMean ± SD12.7 ± 2.321.3 ± 9.737.4 ± 23.60.004
NLRMean ± SD4.2 ± 1.56.0 ± 1.93.8 ± 1.50.06
MPRMean ± SD0.06 ± 0.010.053 ± 0.010.059 ± 0.020.4

 

Table 7. Disturbances of Immune Cells According to Previous Surgery
 
Variable (mean ± SD)No surgeryRadical surgeryPalliative surgeryP value
Data are expressed as mean ± SD and analyzed by Kruskal Wallis testa, 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 cells23.2 ± 9.827.2 ± 4.836.6 ± 8.00.01
CD4+PD-1+ T cells37.7 ± 25.133.7 ± 31.913.9 ± 12.90.1
CD8+ T cells19.8 ± 8.620.1 ±2.214.2 ± 7.80.2
CD8+PD-1+ T cellsa34.4 ± 26.612.92 ± 5.617.9 ± 7.30.045
CD11b+CD68+ M37.4 ± 21.219.9 ± 10.415.2 ± 10.80.022
NLR3.97 ± 1.66.6 ± 2.34.5 ± 1.040.016
MPR0.059 ± 0.020.05 ± 0.010.06 ± 0.010.3

 

Table 8. Multivariate Analysis of Prognostic Factors of PFS
 
VariableBWaldP valueHR95.0% CI for HR
LowerUpper
Data were analyzed using Cox regression using forward stepwise methods (likelihood ratio), HR = eB. HR: hazard ratio; CI: confidence interval; NLR: neutrophil to lymphocyte ratio; PFS: progression-free survival.
Step 1NLR0.4407.6990.0061.5531.1382.119
Step 2Response11.7730.008
Complete response-3.14410.7620.0010.0430.0070.282
Partial response-2.5406.7100.0100.0790.0120.539
Stable disease-1.8063.9170.0480.1640.0270.983
Progressive diseaseReference
NLR0.5567.9450.0051.7431.1842.565