Genomic, proteomic, and immunologic associations with a durable complete remission of measurable metastatic melanoma induced by a patient-specific dendritic cell vaccine

Figures & data

Figure 1. Delayed, durable, complete response of metastatic melanoma.

Table 1. Results of HLA-typing and for patient’s normal and malignant cells.^a

	PBMC	Tumor cells	PBMC	Tumor cells
	Allele 1	Allele 1	Allele 2	Allele 2
HLA-A	01:01:31	01:22N, 01:107	26:01:03	26:01:03
HLA-B	27:05:02	27:05:02	57:01:01	57:01:01
HLA-C	02:02:02	02:02:02	06:02:01	06:02:01
HLA-DQA1	01:05	01:05	02:01	02:01
HLA-DQB1	03:03:02	03:03:02	05:01:01	05:01:01
HLA-DRB1	09:02:02	07:01:01	10:01:01	10:01:01

^aBlood samples were obtained at baseline 1 week prior to first injection of DCV.

significance of bold values is: “Noteworthy variants”.

Table 2. Summary of genomic analysis for the short-term cell lines.

Tumor sample	2197	2243
Site from which tissue obtained	Axillary node	Abdominal soft tissue
When obtained after diagnosis	4 months	13 months
Total variants	3968	4376
Novel	3873 (97.6%)	4269 (97.6%)
Existing	95 (2.4%)	107 (2.4%)
Coding consequences
Missense variant	58%	60%
Synonymous variant	37%	33%
Stop gained	3%	3%
Stop lost	1%	0%
Frameshift variant	1%	0%
Consequences
Intron	52%	52%
Downstream	12%	12%
Non-coding	11%	11%
Upstream	11%	10%
NMG transcript	3%	3%
Intergenic	3%	2%
Missense	2%	2%
Regulatory	2%	2%
3 prime UTR	1%	1%
Others	3%	3%
Non-coding exon	–	3%
KEGG pathways potentially affected
Modified	172 (92%)	176 (95%)
Unmodified	14 (8%)	10 (5%)

NMG = National Molecular Microbiology Diagnostics User Group; UTR = untranslated region; KEGG = Kyoto Encyclopedia of Genes and Genomes.

Table 3. Mutated sites by numbers of mutations present in cells from autologous tumor cell line.

Sites of mutations	Significance
CTNNB1	β-catenin: coordinates cell-cell adhesion & gene transcription
GATA2	Regulates gene expression critical for embryogenesis & self-renewal
KDR	a vascular endothelial growth factor receptor, VEGFR-2
PRKCB	Protein Kinase C, tumor suppressor of aberrant signal transduction
NFKBIA	Inhibits NFKB which controls transcription, cytokine production, & apoptosis
PIK3CD	Enzymes that enhance cell growth, proliferation, motility & apoptosis
PAK1	P21 activated kinase, regulate cell proliferation, differentiation, motility, apoptosis, and development of dendrites & filopods
KITLG	KIT ligand stem cell factor, binds cKIT (CD117)
ITPR1	Tumor suppressor and induces apoptosis
FLT4	Encodes vascular endothelial growth factor receptors C and D
WNT2	WNT pathway signaling important in embryogenesis & oncogenesis
PGF	Placental growth factor, a member of the VEGF family
DCC	Tumor suppressor (deleted in colon cancer)
PHLPP1	Tumor suppressor (regulates PKC)
ITPR1	Inhibits cell proliferation and induces apoptosis
COL1A1	Type 1 collagen synthesis
COL1A2	Type 1 collagen synthesis

Tumor samples were obtained 4 and 13 months after initial diagnosis. In terms of prevalence of mutations, results were the same for cells from both cell cultures. Mutations are in rank order based on expression of the mutation.

Table 4. Serum marker levels in relation to normal control values at week-0 baseline week-0 (1 week before first vaccine injection) and week-4 (1 week after third weekly vaccine injection) and percent change.

Marker	Week-0	Week-4	Percent Change	Immune Effect
TNF-α	3.61	2.20	−39%	Produced by NK & CD4+, anti-tumor
IFN-γ	0.31	0.31	0	Produced by NK, NKT & activated T cells
IL-12p40	26.6	2.1	−92%	Produced by DC, induces NK,Th1, & CTL
IL-12p70	0.43	0.29	−30%	Produced by DC, drives Th1 response
IL-1a	1.07	0.61	−43%	Lymphocyte activator
IL-1b	1.84	2.14	+16%	Lymphocyte activator
IL-2	2.03	1.50	−26%	From T cells & DC, stimulates T cells
IL-3	2.46	2.76	+12%	Stimulates myeloid progenitor cells
IL-4	0.50	0.42	−16%	Induces Th2 cells
IL-5	3.41	3.54	+4%	Induces eosinophils in Th2 response
IL-6	1.67	1.28	−23%	From macrophages, proinflammatory
IL-7	0.72	0.69	−4%	Stimulates lymphoid cells: B, T, & NK
IL-8	1.53	1.14	−25%	Chemokine for monocytes & neurtrophils
IL-10	7.43	5.48	−26%	Anti-inflammatory
IL-13	2.89	2.55	−12%	Th2 response
IL-15	1.22	0.92	−25%	Similar to IL-2, NK enhancer
IL-16	5.01	4.05	−19%	Chemotactin for CD4+ & monocytes
IL-17	0.31	0.24	−23%	Secreted by Th17 in response to IL-23
IL-18	0.29	0.33	+14%	Induces Th1 & IFN-γ
IL-21	14.63	14.87	+2%	Enhances NK & CTL
IL-22	14.21	23.54	+66%	Major source is Th17 cells
IL-23	0.09	0.08	−11%	Induces Th4 to Th17
IL-27	0.93	0.91	−2%	Promotes Th1, suppresses Th4 & Th17
CD40L	2.80	3.48	+24%	Produced by activated T cells & other cells
TGF-β	1.59	0.88	−45%	Immune suppression
SAA	277	500	+81%	Inflammatory marker
GROa	5.31	6.86	+29%	Inflammation
TARC	0.71	3.04	+328%	GM-CSF-induced T-cell chemotactin
Gp130	0.86	1.54	+79%	IL-6 receptor family
TIMP-1	4.55	4.03	−11%	Tissue inhibitor of metalloproteinase
ICAM-1	4.26	2.59	−39%	TNF-induced adhesion molecule
CD163	0.59	0.76	+29%	Macrophage infiltration
β2mcg	1.64	0.86	−48%	Component of MHC1
IP10	1.45	6.97	+380%	IFNγ-induced chemo-attractant
PD-1	0.13	0.17	+31%	IFNγ-induced immune checkpoint
Galectin-3	0.20	0.26	+30%	Macrophage activation & angiogenesis
CRP	1.09	1.42	+30%	Acute phase reactant

Table 5. Rankings of serum markers by baseline levels relative to control values, and greatest percent changes after three vaccine injections.

Marker	Level greater than twice control	Marker	Level less than 33% of control	Marker	Level increased more than 20%	Marker	Level decreased more than 20%
SAA	277	IL23	0.09	IP-10	3.81	IL-12p40	−0.921
IL-12p40	26.6	PD-1	0.13	TARC	3.28	B2M	−0.476
IL-21	14.63	Galectin-3	0.20	SAA	0.805	Eotaxin	−0.454
IL-22	14.21	IL-18	0.29	gp130	0.791	TGF-β	–
MCP-3	8.82	IL17	0.31	IL-22	0.657	IL-1a	−0.430
IL-10	7.43	IFN-γ	0.31	PD-1	0.307	ICAM-1	−0.392
GROa	5.31			CRP	0.303	TNF-α	−0.391
IL-16	5.01			GROa	0.292	IL-12p70	−0.310
TIMP-1	4.55			CD163	0.288	IL-10	−0.262
ICAM-1	4.26			CD40L	0.243	IL-2	−0.261
TNF-α	3.61					IL-8	−0.255
IL-5	3.41					IL-15	−0.246
IL-13	2.89					IL-6	−0.234
CD40L	2.8
IL-3	2.46
IL-2	2.03

Figure 2. Summary of ELISPOT data.

Figure 3. Expression of immune markers.