Baseline immunity predicts prognosis of pancreatic cancer patients treated with WT1 and/or MUC1 peptide-loaded dendritic cell vaccination and a standard chemotherapy

Figures & data

Table 1. Patient characteristics

		n	%
Age	Median	64
	Range	41–83
Gender	Male	21	44
	Female	27	56
Performance status	0	29	60
	1	17	35
	2	2	4
Pancreatic tumor location	Head	22	46
	Body/Tail	26	54
Metastatic site	Local invasion	8	17
	Liver	16	33
	Lung	9	19
	Peritoneal	5	10
	Multiple	10	21
CA19-9 (U/ml)	Median	499.8
	Range	0.5–20379.8
CEA (ng/ml)	Median	7
	Range	2.0–281.4
Previous therapy	Chemotherapy	21	44
	Surgery	11	23
	Radiation	3	6
	None	13	27
Concurrent chemotherapy	GnP	33	69
	FOLFIRINOX	15	31
Postvaccination chemotherapy	S-1	10	16
	GEM	6	10
	GEM+S-1	3	4
	GnP	25	41
	FOLFIRINOX	12	20
	BSC	5	8

Abbreviations: CA19-9, carbohydrate antigen 19–9; CEA, carcinoembryonic antigen; S-1, tegafur gimeracil oteracil; GEM, gemcitabine; GnP, GEM+nab-paclitaxel; FOLFIRINOX, oxaliplatin, irinotecan, fluorouracil, and leucovorin; BSC, best supportive care.

Figure 1. Kaplan-Meier estimates of PFS and OS of patients treated with a combined DC vaccination with a chemotherapy. (a and b) PFS and OS of all patients. (c and d) PFS and OS of patients who showed positive immune responses in either WT1 or MUC1 ELIspot assay (solid line) and patients who showed negative immune responses in both assays (dotted line).

Table 2. Adverse events^†

	Any grade			Grade 3, 4
	n	%		n	%
Hematological AEs
Leukocytopenia	40	83		21	44
Neutropenia	35	73		22	46
Anemia	32	67		6	13
Thrombocytopenia	21	44		4	8
Nonhematological AEs
Anorexia	20	42		2	4
Diarrhea	14	29		0	0
Constipation	16	33		0	0
Nausea	22	46		0	0
Fatigue	33	48		1	2
Alopecia	21	44		0	0
Fever	19	40		0	0
Injection site reaction	48	100		0	0
Peripheral sensory neuropathy	20	42		0	0
Pain	18	38		0	0
ALT elevation	17	35		1	2
AST elevation	16	33		1	2
Bilirubin elevation	11	23		0	0
ALP elevation	10	21		0	0
Hypoalbuminemia	15	31		0	0

^†Adverse Events (AEs) listed are those that occurred in more than 20% of patients. The US National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 was used to classify the grades. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

Figure 2. IFNɤ ELIspot assay.

Table 3. Univariate analysis of the association of clinical factors with PFS and OS

Factor	Group	n	PFS			OS
			Median survival	p value		Median survival	p-value
Age	≧64	25	8.4 (4.4–12.6)	0.319		15.1 (9.7–23.8)	0.391
	≦64	23	4.9 (3.5–11.6)			13.1 (7.6–18.5)
Gender	Female	27	8.4 (4.2–13.4)	0.297		17.4 (7.9–23.9)	0.684
	Male	21	7.1 (3.7–10.0)			14.7 (7.6–23.1)
Performance status	1,2	19	8.4 (3.7–12.6)	0.705		15.1 (7.6–18.9)	0.308
	0	29	7.1 (4.2–12.6)			15.1 (7.8-NA)
Tumor location	Body/tail	26	7.1 (4.3–9.3)	0.439		15.1 (7.6–23.8)	0.688
	Head	22	8.8 (3.7–13.4)			15.1 (7.9–18.9)
Local invasion	No	41	8.8 (4.4–12.6)	0.614		15.1 (8.0–18.5)	0.75
	Yes	7	7.9 (3.0–10.0)			18.9 (4.8–23.8)
Liver metastasis	No	30	8.1 (3.7–10.0)	0.743		14.7 (7.8–18.9)	0.284
	Yes	18	7.1 (3.6-NA)			15.1 (7.7-NA)
Lung metastasis	No	40	7.9 (4.3–9.3)	0.469		15.1 (8.0–18.9)	0.448
	Yes	8	12.6 (2.8-NA)			23.9 (5.1-NA)
Peritoneum metastasis	No	43	8.4 (4.7–10)	0.921		15.1 (9.7–23.1)	0.215
	Yes	5	4.3 (3.2-NA)			7.9 (5.5-NA)
Multiple metastasis	No	38	7.9 (4.3–12.6)	0.532		17.4 (9.3–23.8)	0.0936
	Yes	10	8.1 (1.9–11.6)			13.4 (3.4–15.1)
Chemotherapy	No	27	8.8 (4.4–12.6)	0.546		15.1 (8.0–23.1)	0.987
	Yes	21	6.8 (3.5–13.4)			15.1 (6.6–23.8)
Surgery	No	37	9.2 (4.4–12.6)	0.122		14.7 (7.9–23.1)	0.848
	Yes	11	6.8 (2.6–8.1)			15.1 (5.2–23.8)
Radiation	No	45	8.1 (4.4–11.6)	0.739		15.1 (9.3–23.1)	0.767
	Yes	3	9.3 (3.2-NA)			18.5 (6.6-NA)
GEM+nab-PTX	No	15	7.1 (3.4–11.6)	0.753		14.7 (5.5-NA)	0.959
	Yes	33	8.4 (4.4–12.6)			15.1 (9.7–23.1)
FOLFIRINOX	No	33	8.4 (4.4–12.6)	0.753		15.1 (9.7–23.1)	0.959
	Yes	15	7.1 (3.4–11.6)			14.7 (5.5-NA)
Peptide	MUC1	13	12.6 (3.0-NA)	0.415		15.1 (4.8-NA)	0.511
	WT1+ MUC1	35	7.9 (4.4–9.3)			14.7 (7.9–23.1)

Parenthesis represents a 95% confidence interval. PFS, progression free survival; OS, overall survival; GEM+nab-PTX, gemcitabine+nab-paclitaxel; FOLFIRINOX, oxaliplatin, irinotecan, fluorouracil and leucovorin. MUC1, Mucin1; WT1, Wilms’ tumor 1.

Table 4. Univariate analysis of the association of laboratory factors with PFS and OS

Factor	Group	n	PFS			OS
			Median survival	p value		Median survival	p-value
WBC	≧4380/mm^3	24	8.4 (4.2–11.6)	0.479		14.7 (7.6–23.1)	0.621
	<4380/ mm^3	24	8.1 (3.7–13.4)			15.1 (8–23.8)
Hb	≧11.4 g/dL	24	8.4 (4.2–11.6)	0.99		15.1 (8–18.9)	0.808
	<11.4 g/dL	24	6.8 (3.7–12.6)			13.1 (7.1–23.9)
Plt	≧199/ mm^3	25	7.9 (4.2–9.3)	0.42		15.1 (7.6–23.8)	0.734
	<199/ mm^3	23	8.8 (3.7–12.6)			17.4 (9.3–23.1)
Alb	≧3.95 g/dL	24	8.1 (4.3–12.6)	0.596		17.4 (9.3–23.1)	0.447
	<3.95 g/dL	24	8.4 (3.5–12.6)			14.7 (7.1–23.9)
CRP	≧0.195 mg/dL	24	8.4 (4.4–12.6)	0.899		18.5 (7.1–23.)	0.857
	<0.195 mg/dL	24	7.9 (3.5–13.7)			15.1 (8–23.1)
CA19.9	≧499.8 U/ml	24	8.4 (4.2–12.6)	0.858		15.1 (7.7–18.9)	0.291
	<499.8 U/m	24	7.9 (3.7–12.6)			15.1 (7.9–23.8)
CEA	≧7.0 mg/ml	19	4.7 (3.0–9.3)	0.0444*		8.0 (5.1–18.5)	0.203
	<7.0 mg/ml	19	8.4 (4.9-NA)			18.9 (9.3–23.8)
NLR	≧5	11	4.2 (3.3-NA)	0.0206*		8.0 (6.6–15.1)	0.0164*
	<5	37	9.2 (6.8–12.6)			17.4 (13.1–23.8)
PNI	≧40	40	9.2 (6.8–12.6)	0.0174*		17.4 (13.4–23.1)	0.0109*
	<40	8	4.6 (1.9-NA)			7.4 (3.4–13.1)
mGPS	0	36	9.2 (6.8–12.6)	0.0169*		17.4 (13.4–23.1)	0.0251*
	1.2	12	4.4 (3.0–8.8)			7.4 (3.9–13.1)
PLR	≧150	32	6.8 (3.6–9.2)	0.0228*		13.1 (7.7–18.5)	0.0604
	<150	16	11.6 (4.4-NA)			18.9 (14.7-NA)
WT1	Weak	33	8.8 (4.3–11.6)	0.62		14.7 (7.9–23.1)	0.968
	Moderate	15	8.4 (3.5-NA)			17.4 (4.8–23.8)
MUC1	Weak	19	8.1 (3.7–12.6)	0.746		18.5 (7.1–23.1)	0.799
	Moderate	29	7.1 (4.2–13.4)			14.7 (7.9-NA)

Two groups were divided by the median value. The cutoff values for the NLR, PNI mGPS and PLR are determined on the basis of previous studies.^17,32–34 The classification of the intensity of staining in immunohistochemistry is determined on the basis of the report by Kanai et al.³⁵ Weak represents faint and barely perceptible staining in PDAC cells under high magnification (x200). Moderate represents moderate complete staining under low magnification (x40). Parenthesis represents a 95% confidence interval. PFS, progression free survival; OS, overall survival; WBC, white blood cell; Hb, hemoglobin; Plt, platelet; Alb, albumin; CRP, C-reactive protein; CA19-9, carbohydrate antigen 19–9; CEA, carcinoembryonic antigen; NLR, neutrophil to lymphocyte ratio; PNI, prognostic nutritional index; mGPS, modified Glasgow prognostic score; PLR, platelet to lymphocyte ratio; WT1, Wilms’ tumor 1 staining; MUC1, Mucin 1 staining. * represents statistically significant.

Table 5. Univariate analysis of the association of immune-related factors with PFS and OS

Factor	Group	n	PFS			OS
			Median survival	p value		Median survival	p-value
CD3	≧1253/mm^3	24	8.4 (4.3–13.4)	0.549		17.4 (7.8–23.1)	0.837
	<1253/mm^3	24	8.1 (3.5–12.6)			13.4 (7.6–23.1)
CD4	≧539/mm^3	24	8.4 (4.3–13.4)	0.533		17.4 (9.7–23.8)	0.158
	<539/mm^3	24	8.1 (3.6–11.6)			13.1 (6.9–18.5)
CD8	≧369/mm^3	24	7.9 (4.2–11.6)	0.436		15.1 (7.1–18.9)	0.553
	<369/mm^3	24	9.0 (3.7–12.6)			13.4 (8.0–23.9)
Th1	≧109.3/mm^3	24	8.8 (4.2–11.6)	0.944		17.4 (13.4–23.1)	0.235
	<109.3/mm^3	24	6.8 (3.5–12.6)			9.7 (6.9–23.1)
Th2	≧162.4/mm^3	24	7.9 (4.2–11.6)	0.708		15.1 (8.0–23.1)	0.909
	<162.4/mm^3	24	8.8 (3.7–12.6)			15.1 (6.6–23.9)
Treg	≧26.2/mm^3	24	4.7 (3.3–9.3)	0.00306*		9.3 (7.1–17.4)	0.0159*
	<26.2/mm^3	24	11.6 (7.1-NA)			18.9 (13.1-NA)
ɤδT	≧18.2/mm^3	24	7.9 (4.2–12.6)	0.529		17.4 (7.7–23.8)	0.492
	<18.2/mm^3	24	8.8 (3.7–11.6)			13.4 (7.8–18.5)
NK	≧129.3/mm^3	24	8.4 (4.2–10.0)	0.503		15.1 (7.9–18.9)	0.278
	<129.3/mm^3	24	7.1 (3.7–13.4)			17.4 (6.9-NA)
NKT	≧16.9/mm^3	24	8.1 (4.3–9.3)	0.493		15.1 (7.9–23.1)	0.686
	<16.9/mm^3	24	8.8 (3.5–13.4)			13.1 (6.9–23.9)
MDSC	≧6.2/mm^3	24	7.9 (4.3–11.6)	0.342		14.7 (7.8–18.9)	0.363
	<6.2/mm^3	24	9.3 (3.7–13.4)			18.5 (7.7–23.1)
MUC1 ELspot	≧12.8/10^5PBMC	24	12.6 (9.3-NA)	<0.0001*		23.1 (17.4-NA)	<0.0001*
	<12.8/10^5PBMC	23	3.7 (3.2–4.9)			7.6 (5.5–9.7)
WT1 ELIspot	≧9.3/10^4PBMC	16	12.6 (6.8-NA)	0.000914*		23.9 (15.1-NA)	<0.0001*
	<9.3/10^4PBMC	16	4.3 (3.3–8.8)			7.6 (5.5–13.1)
Skin reaction	≧30 mm	25	9.3 (7.1-NA)	0.0107*		18.9 (15.1–23.9)	0.00174*
	<30 mm	23	4.7 (3.6–9.2)			7.9 (6.6–15.1)

Two groups were divided by the median value. Parenthesis represents a 95% confidence interval. The cutoff values for the skin reaction is determined on the basis of previous studies.¹⁷ CD3, CD3⁺T cell; CD4, CD4⁺T cell; CD8, CD8⁺T cell; Th1, type 1 helper T cell; Th2, type 2 helper T cell; Treg, regulatory T cell; ɤδT, T cell receptor ɤ and δ chain⁺ T cell; NK. Natural killer cell; NKT, natural killer T cell; MDSC, myeloid-derived suppressor cell; MUC1, Mucin1; WT1, Wilms’ tumor 1; ELIspot, enzyme-linked immunospot. *represents statistically significant.

Table 6. Multivariate analysis

Factor	PFS			OS
	Hazard ratio	p value		Hazard ratio	p value
CEA (<7.0 mg/ml vs ≧7.0 mg/ml)	0.29 (0.07–1.29)	0.11
Treg (≧26.2/mm^3 vs <26.2/mm^3)	1.22 (0.20–7.49)	0.83		1.15 (0.31–4.19)	0.84
mGPS (1.2 vs 0)	0.27 (0.03–2.46)	0.25		3.04 (0.47–19.59)	0.24
NLR (<5 vs ≧5)	0.24 (0.05–1.27)	0.093		0.60 (0.14–2.45)	0.47
PNI (<40 vs ≧40)	3.24 (0.20–51.25)	0.4		0.36 (0.04–2.98)	0.34
MUC1 ELIspot (<12.8 vs≧12,8)	23.68 (1.99–282.00)	0.012*		19.26 (1.81–204.90)	0.014*
WT1 ELIspot (<9.3 vs ≧9.3)	0.51 (0.04–6.10)	0.6		6.11 (1.11–33.79)	0.038*
Skin reaction (<30 mm vs ≧30 mm)	4.91 (0.23–102.80)	0.3		4.43 (0.89–22.13)	0.07

Two groups in the CEA, Treg, MUC1 ELIspot and WT1 ELIspot were divided by the median value. The cutoff values for the mGPS, NLR, PNI and skin reaction are determined on the basis of previous studies.^{17,28,29,32,33} Parenthesis represents a 95% confidence interval. PFS, progression free survival; OS, overall survival; CEA, carcinoembryonic antigen; Treg, regulatory T cell; mGPS, modified Glasgow prognostic score; NLR, neutrophil to lymphocyte ratio; PNI, prognostic nutritional index; MUC1, Mucin1; WT1, Wilms’ tumor 1; ELIspot, enzyme-linked immunospot. *represents statistically significant.

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