1. No category

Pancreatic Cancer: Future Biologic
and Immunologic Approaches
John F. Gibbs, M.D.
Chief, Dept. of GI Surgery & Endoscopy
Roswell Park Cancer Institute
Objectives:
1. Risk factors for development of pancreatic cancer
2. Potential mechanisms of Gemcitabine resistance
3. Patient tumor-SCID model for evaluating therapeutic
approaches
4. The role of the uPA system cancer biology and
potential therapeutic implications
5. DC based immune modulation of CD8+ T Cells for
the generation of anti-tumor responses
• Pancreatic cancer is the Pancreatic Cancer Statistics (1
2nd most common GI
malignancy and 4th
50000
most common cause of
cancer death in the US.
37500
• Annual incidence of
pancreatic cancer is
25000
approximately 42,470
Incid
Deat
12500
cases.
• Mortality of pancreatic
0
cancer is approximately
12345678910
111
2131
4151
67
35,240 cases per year .
Year
(Jemal et al. Cancer J Clin 2009 )
B/
T
H
U
Pancreatic Cancer: The Reality!
Why is pancreas cancer so lethal?
Delay in Diagnosis
Incidence Rates
=
Mortality Rates
⇒
+
Aggressive Nature
+
Ineffective Systemic
Therapy
Pancreatic precursor lesions and genetic events involved in pancreatic adenocarcinoma
progression
Hezel A. F. et.al. Genes Dev. 2006;20:1218-1249
©2006 by Cold Spring Harbor Laboratory Press
Whipple Procedure
www.mayoclinic.org
Survival Following Resection for
Adenocarcinoma of the Pancreas
Author
Gudjonsson (1987)
No.
1-yr
3-yr
5-yr
Median
Patients Survival Survival Survival Survival
(%)
(%)
(%)
(mos)
2398
Crist & Cameron
(1987)
50
Kalser et al (1985)
43
3.8
40
20
18
12
20
Yeo et al. (1997)
282
67
30
20
18
Regine et al. (2008)
388
72
31
28
20.5
Patient tumor-SCID mouse xenograft model
1st passage
(TP#- 1p)
2nd passage
(TP#- 2p)
SCID
Normal Pancreas
Adenocarcinoma
Neuroendocrine
tumor
Targeting Cell Surface Death Receptors To Initiate
Apo2L/TRAIL
Apoptotic Signaling
Nature Reviews Drug Discovery 7,
1001-1012 (December 2008)
Avi Ashkenazi

Apo2L/TRAIL ( TNFrelated apoptosis
inducing ligand) is a
ligand for both DR4
and DR5

Ligand and antibodies
to DR4 and DR5 are
currently in pre-clinical
development and
Phase I clinical trials

Mouse models are
used for preclinical
evaluation.
Phase Ib study of recombinant human Apo2L/TRAIL plus
irinotecan and cetuximab or FOLFIRI in metastatic
colorectal cancer (mCRC) patients (pts): Preliminary
results J Clin Oncol 27:15s, 2009 (suppl; abstr 4129)
A SCID Mouse Patient Tumor Orthotopic Xenograft
Model of Pancreatic Cancer
(a) The mouse skin is incised on the
left flank.
(b) The pancreas is externalized to
expose its entire length.
(a)
(b)
(c) Pancreatic cancer cells are
injected.
(d) The abdominal wall muscles and
skin are closed.
(c)
(d)
Mets
(Sharma SSO 2010)
Experimental Design
Orthotopic Injection
Wait 94 Days
Randomize
Control (n=7)
Baseline MRI
MRI Weekly
MRI
Measurements
Treatment (n=7)
Saline 200µl daily IP
Metastasis Site
Apo2L/TRAIL
Metastasis Number
200µg/200µl IP daily
Implantation
Treatment
94
101
108
Day
115
Primary Tumor Volume
122
Metastasis Volume
MRI documents Change in Primary and Metastases
Tumor Volumes
Day 94
Day 101
Primary Tumor
Metastases
Control
Apo2L/TRAIL
Summary (1)
1) The patient derived xenograft maintain their
sensitivity to Apo2L/TRAIL at the orthotopic
primary location.
2) The metastases were also sensitive to Apo2L/TRAIL.
3) Early initiation of Apo2L/TRAIL treatment improves
survival.
Proteolytic enzymes as predictors of
aggressiveness in pancreatic cancer patients
The role of uPA in cell signaling and immunity
uPANCAM
uPA
Hp Hp
uPA
uPA
uPA
uPA
uPA
uPA
EGFR
uPAR
(MAPK)
uPA
p-Akt
(MST
24 mos)
p-Erk
(MST
10 mos)
uPA
SMRT(???)
uPA
uPA
DC
DC
Viral Immunization Induced CD8+
T Cell Response and Tumor
DC mediated viral immunization
Triad of Costimulatory Molecules
(TRICOM): B7-1, ICAM-1, LFA-3
PAI-1 WT/KO dendritic cells
?
Unknowns:
T cell expansion
Overall survival
Lymph nodes
Naïve T cells
Objective
• Determine whether PAI-1 expression of DC mediated
viral vaccination impacts T cell expansion and overall
survival in tumor bearing mice.
Experimental Approach
TRICOM 1.5 x 107 PFU
PAI-1 WT or KO
WT B6
-1 days
-8 days
0 day
Vaccinate
1 x 106 DCs
+/- TRICOM
Harvest
BMDCs
1) Flow
cytometry:
CD8+
Thy 1.1
2) Cell count
+5 days
Results
WT DCs - TRICOM
0.14%
Abs #10,500
CD8+
PAI KO DCs + TRICOM
0.02%
Abs #2,240
Thy 1.1
0.06%
Abs #2,520
WT DCs + TRICOM
Experimental Approach
PAI-1 WT or KO
TRICOM 1.5 E7 PFU
Days 73 and 92
MOSEC-OVA
Inject 1E7
MOSEC-OVA I.P.
Results
Overall survival
Conclusions
Summary (2)
 Virally induced DCs require PAI-1 to confer
CD8+ T cell clonal expansion.
 PAI-1 expression prolongs overall survival in
those tumor bearing mice vaccinated with
TRICOM.
Immune Modulation of CD8+ T Cells
for the Generation of Anti-Tumor
Responses
Instruc(ons program differen(a(on of naive CD8+ T cells
APC
B7
MHC/pep(de Prolifera(on/Differen(a(on
IL-­‐12
Death/Apoptosis
Naïve
CD8+ T cell
Ac(vated
Effector
IL-­‐7, IL-­‐15
Effector Memory
Func(onal state: Naïve Effector Memory
No-­‐ IFN-­‐γ, CTL Hi-­‐ IFN-­‐γ, CTL Hi/
Lo-­‐ IFN-­‐γ, CTL -­‐ Effector
CD62Lhi, CD44 lo CD62Llo, CD44hi CD62Lhi, CD44hi
0 hr 72-­‐144 hrs > 40 days •
Shaping and reshaping CD8+ T cell memory (2008) John T. Harty & Vladimir P. Badovinac . Nat. Rev. Immunol. 8:107 mTOR: A central integrator of instruc(ons and regulator of cell fate
mTOR: Mammalian Target of Rapamycin
Nutrients
Cellular stress
Growth factors
Energy levels
mTOR
Transcrip(on/
Transla(on
Survival
Cell Growth
and division
Cellular
differen(a(on
• TOR, a central regulator of cell growth. Schmelze, T. et. al. (2000) Cell 103, 253-­‐262 Dendritic Cell Immunotherapy
Provenge
•
First autologous immunotherapy to provide survival advantage:
•Design
• Multicenter phase 3 trial
• Inclusion: men with metastatic castration-resistant prostate cancer
• 512 pts randomly assigned in 2;1 ration to either receive sipuleucel-T or placebo
every 2 weeks for three infusions
•Results
•
•
•
•
22% relative reduction in death [95% CI 0.61 to 0.98; P=0.03] RECIST criteria
4.1-month benefit in median survival
Low toxicty: fever/chills/Headache
92.2% compliance with infusion protocol
Philip W. Kantoff, M.D. et al., Sipuleucel-T Immunotherapy for Castration-Resistant Prostate
Cancer. New England Journal of Medicine, 2010.
Objective: To determine the ability of
rapamycin mediated mTOR inhibition of
dendritic cells to augment memory CD8+ T
cell differentiation
Experimental Approach
Rapa/Ag (SIINFEKL)
LPS/TRICOM
BM progenitors
8 Days
BL/6
5 hrs
3 hrs
GM-CSF
BMDC
OT-1
FACS Analysis
•Activation markers
•Effector function
•CFSE Dilution
•Cell Counts
Rapamycin Treated DC’s Promote CD62LHI/CD44HI
phenotype
DC alone
10
10
10
10
10
LPS/Ag
4
10
3
10
20.5%
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
10
4
3
21.9%
2
1
0
10
0
CD44
10
10
10
10
4
10
3
10
26.9%
2
10
1
10
0
10
0
10
1
10
2
10
3
CD62L
1
10
2
10
3
10
4
LPS/Ag/Rapa 20ng
LPS/Ag/Rapa 2ng
10
10
10
4
10
4
3
32.5%
2
1
0
10
0
10
1
10
2
10
3
10
4
DC alone
LPS/Ag
104
104
103
103
102
102
101
101
100
100
100
100
50
45
10
4
10
10
10
10
10
1
2
10
3
10
4
10
100
0
10
CFSE
10
10
5
0
g
10
15
20n
101
20
DC Treatment
apa
102
25
g/R
102
30
LP
S/A
103
35
ng
103
3
10
104
a2
4
10
2
10
103
ap
104
1
10
102
LPS/Ag/Rapa 20ng
LPS/Ag/Rapa 2ng
100
100
101
g/R
104
LP
S/A
103
g
102
40
LP
S/A
101
INF-γ Positivity (MFI)
INF-γ
Rapamycin Treatment Augments Antigen Specific CD8+ T Cell
Expression and Absolute Cell Numbers for IFN-γ in a Dose
Dependent Manner
Rapamycin Treatment Augments Granzyme B Expression in
CD8+ T cells in Dose Dependent Manner
DC alone
LPS/Ag
1
10
2
10
3
10
4
10
10 0
10
10
10
10
4
10
3
10
2
10
1
10
10
3
10
4
4
3
2
1
0
0
10 0
10
2
10
LPS/Ag/Rapa 20ng
LPS/Ag/Rapa 2ng
10
1
10
1
10
2
10
10
3
10
4
10 0
10
CFSE
70
60
50
40
30
20
10
0
10
1
2
10
3
10
4
10
ng
10 0
10
0
20
0
80
1
LP
S/
Ag
Granzyme B
10
pa
1
10
90
/R
a
10
LP
S/
Ag
10
100
2
g
2
3
2n
10
pa
10
/R
a
10
3
LP
S/
Ag
10
4
Granzyme B Expression (MFI)
4
Inhibition of mTOR Induces MHC Class Switch in Rapamycin
Treated Dendritic Cells
Counts
Rapamycin inhibition of mTOR is dose dependent
in treated dendritic cells as measured by S6K
expression
Counts
MHC Class I/II, CD86 expression 24
hours post rapamycin treatment
Counts
H2Kb
S6K
DC alone
LPS/Ag
LPS/Ag/Rapa 2ng
LPS/Ag/Rapa 20ng
Counts
IAb
CD86
Summary (3)
• Rapamycin treated DC’s can augment CD8+
T cell responses In-vivo
• Rapamycin dosage as well as duration and
timing of administration can differentially
effect DC maturation and CD8+ T cell
differentiation
• DC immunization combined with Rapa may
augment CD8+ T cell anti-tumor responses
Conclusion
1. Clinicians & scientist interactions are instrumental in
providing context to hypothesis driven collaborative
research
2. A metastatic model of human pancreatic cancer is more
clinically relevant to evaluate therapeutic agents.
3. Overcoming pancreatic cancer biologic resistance and
immunosuppressive mechanisms will spawn novel
future therapeutic approaches
Acknowledgements
• TRAIL
–
–
–
–
–
Elizabeth Repasky
Bonnie Hylander
Shazhad Zafar
Rose Pitoniak
Rohit Sharma
• uPA
–
–
–
–
–
–
Thelma Hurd
Shashikumar Harvey
Gabor Markus
Massie Martinick
Parmvir Singh
James Corasanti
• Immunology
–
–
–
–
–
–
Protul Shrikant
Nicholas Yu
Joshua Kesterson
Joseph Vazzana
Rajesh Rao
Qingsheng Li, MD, PhD
• EGFR
– Milind Javle
– Jennifer Black
– Krisdeep Chadha