Developing PDTbased combination regimens for the treatment of pancreatic cancer

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Developing PDT-based combination regimens for the
treatment of pancreatic cancer
Tayyaba Hasan PhD
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2006 Estimated US Cancer Cases
Men720,280
Women679,510
31 Breast 12 Lung bronchus 11 Colon
rectum 6 Uterine corpus 4 Non-Hodgkin
lymphoma 4 Melanoma of skin 3
Thyroid 3 Ovary 2 Urinary bladder 2 Pancrea
s 22 All Other Sites
Prostate 33 Lung bronchus 13 Colon
rectum 10 Urinary bladder 6 Melanoma of
skin 5 Non-Hodgkin 4
lymphoma Kidney 3 Oral cavity 3 Leukemia 3 Pa
ncreas 2 All Other Sites 18
Excludes basal and squamous cell skin cancers
and in situ carcinomas except urinary
bladder. Source American Cancer Society, 2006.
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2006 Estimated US Cancer Deaths
Men291,270
Women273,560
Lung bronchus 31 Colon rectum 10 Prostate 9
Pancreas 6 Leukemia 4 Liver
intrahepatic 4bile duct Esophagus 4 Non-Hodgki
n 3 lymphoma
Urinary bladder 3 Kidney 3 All other sites
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26 Lung bronchus 15 Breast 10 Colon
rectum 6 Pancreas 6 Ovary 4 Leukemia
3 Non-Hodgkin lymphoma 3 Uterine
corpus 2 Multiple myeloma 2 Brain/ONS 23
All other sites
ONSOther nervous system. Source American Cancer
Society, 2006.
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Five-year Relative Survival () during Three
Time Periods By Cancer Site
1983-1985
1995-2001
Site
1974-1976
     

• All sites 50 53 65
• Breast (female) 75 78 88
• Colon 50 58 64
• Leukemia 34 41 48
• Lung and bronchus 12 14 15
• Melanoma 80 85 92
• Non-Hodgkin lymphoma 47 54 60
• Ovary 37 41 45
• Pancreas 3 3 5
• Prostate 67 75 100
• Rectum 49 55 65
• Urinary bladder 73 78 82

5-year relative survival rates based on follow
up of patients through 2002. Recent changes in
classification of ovarian cancer have affected
1995-2001 survival rates. Source Surveillance,
Epidemiology, and End Results Program, 1975-2002,
Division of Cancer Control and Population
Sciences, National Cancer Institute, 2005.
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What does the pancreas do?
Endocrine (10-15)
Exocrine (85-90)
Grays Anatomy of the Human Body
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Exocrine Pancreas (85-90)
Exocrine glands have ducts that carry their
secretions to specific locations.
Digestive gland that secretes digestive enzymes
into the duodenum through the pancreatic duct.
Grays Anatomy of the Human Body Robbins Basic
Pathology http//faculty.clintoncc.suny.edu/facult
y/Michael.Gregory/default.htm
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Endocrine Pancreas (10-15)
Endocrine glands do not have ducts to carry
secretions
Islets of Langerhans Secrete insulin and
glucagon Ductless Circulatory system carries
their hormones to target cells
http//www.bigeye.com/medical.htm
Insulin promotes the removal of glucose from the
blood for storage glycogen (muscle,
liver) fats (fat cells) protein Promotes the
buildup of fats and proteins and inhibits their
use as an energy source. Glucagon The effects
are opposite of insulin Raises the level of
glucose in the blood Normally secreted between
meals to maintain the concentration of blood.
glucose
NATURE REVIEWS CANCER VOLUME 2 DECEMBER 2002
http//faculty.clintoncc.suny.edu/faculty/Michael.
Gregory/default.htm
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Tumors of the Pancreas Usually Exocrine
No histological difference between sites
Carcinoma of the head Obstructs bile
duct Ulcerates duodenal mucosa Carcinoma of the
tail Remains silent longer Large widely
disseminated
60-70
5-10
10-15
Head
Neck/Body
Tail
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Diffuse tumors involving entire gland
Robbins Basic Pathology
NATURE REVIEWS CANCER VOLUME 2 DECEMBER 2002
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Adenocarcinoma of the Pancreas

• Over 95 of pancreatic tumors
• Average 5-year survival 5
• Extremely resistant to current therapies
• Chemotherapy
• Radiotherapy
• 15-20 eligible for surgery
• Whipple (pancreaticoduodenectomy)
• 5-year survival 20
• Management of most patients Palliation

Rustgi, AK. Bioch et Biophy Acta 97-101 (2005)
Robbins Basic Pathology
Cancer Statistics 2006
NATURE REVIEWS CANCER VOL 2 DECEMBER 2002
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• Mutant Kras
• gt90 of Panc Ca
• Rite of passage
• Autocrine EGF signaling
• Increased MAPK PI3K
• Increased EGFR ErbB2
• Increased prolif, survival, invasion

• Mutant CDKN2A (p16)
• 80-95 of sporadic Panc Ca
• Tumor-suppressor gene
• Mutation, deletion, hypermethylation
• Loss of INK4A and ARF
• Disrupt Rb and p53 tumor suppression pathways

KRAS CDKN2A

• Not seen in other tumors
• Molecular fingerprint

NATURE REVIEWS CANCER VOLUME 2 DECEMBER 2002
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Metastatic Pattern

• Liver
• Lungs
• Bones
• Peripancreatic nodes
• Gastric nodes
• Mesenteric nodes
• Omental nodes
• Portohepatic nodes
• Spleen
• Adrenal glands
• Vertebral column
• Transverse colon
• Stomach
• Extend through retroperitoneal spaces
• Infiltrate adjacent nerves

Robbins Basic Pathology
Grays Anatomy of the Human Body
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Pancreas
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Aim 2 Establish biodistribution pattern of the
photosensitizer in mouse models of pancreatic
cancer.
Aims
Aim 1 Evaluate responsiveness of pancreatic cell
lines to PDT and investigate PDT-based
combination regimens in biologically relevant
cultures.
Aim 3 Establish toxicology of PDT-dose in mouse
models for pancreatic cancer.
Aim 4 Determine PDT dose-response of pancreatic
tumors in mouse models.
Aim 5 Establish the minimum combination PDT and
chemotherapy doses required to achieve optimal
therapeutic outcome in mouse models
Aim 6 Test combination regimen determined from
Aim 5 on fresh patient tissue samples ex vivo
Aim 7 Strategies for targeted-PDT??
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Aim 1 Evaluate responsiveness of pancreatic cell
lines to PDT and investigate PDT-based
combination regimens in biologically relevant
cultures.
Hypothesis PDT leads to a more cytotoxic effect
than current standard chemotherapies such as
Gemcitabine and 5-Fluorouracil in pancreatic
cancer cells in monolayer. Three-dimensional cell
cultures will provide a more biologically
relevant system than monolayers to investigate
the efficacy of PDT-based combination regimens.
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Aim 1
10 pancreatic cell lines in monolayer

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Aim 1

• PDT combined with other therapies
• PDT C225
• PDT Gefitinib
• PDT Erlotinib
• PDT Gemcitabine

• PDT dose response
• BPD vs. ALA(?)
• Gemcit efficacy vs. PDT response
• 5-FU efficacy vs. PDT response

10 pancreatic cell lines in monolayer
3 cell lines in 3D cultures
Should be included as prelim data instead of as
Aim I?

• PS imaging
• LCM
• In vitro invasion metastasis model
• VEGF (?) imaging

Aim 2 PDT efficacy in vivo
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Aim 2 Establish biodistribution pattern of the
photosensitizer in mouse models of pancreatic
cancer.
Hypothesis Discrete incubation times will lead
to differences in photosensitizer localization
and concentrations within the tumor compartments,
and between tumor and surrounding normal tissue.
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Aim 2

• Biodistribution studies
• Extraction studies (?)
• Aurora (?)
• Intravital fluorescence microscopy (develop an
  endoscopic systemCORE)
• Confocal microscopy (?)

Establish pancreatic cancer animal models
Determine photosensitizer biodistribution and
tumor to normal ratios
DEPTH-SELECTIVE FIBER OPTIC PROBE FOR AURORA
Aim 3 Toxicology Studies

• Establish correlation between aurora extraction
  data (CORE will generate the correlation)
• Determine time-dependent compartmental
  distribution of PS
• Determine the effect of varying initial PS dose
  on concentrations in the tumor and surrounding
  tissue

Non-invasive PS monitoring ultrasound,
laproscope, OCT?
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Aim 3 Establish toxicology of PDT-dose in mouse
models for pancreatic cancer
Hypothesis Maximum tolerated PDT dose between
vascular- and extravascular- targeted PDT will be
different.
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Aim 3

• Toxicology studies
• Maximum Tolerated PDT Dose
• Aurora-based modification of light dose to
  maintain constant PDT dose
• (1 PS dose ?)
• Toxicology studies based on compartmentalization
  of PS
• Light delivery methods
• (interstitial vs. topical ?) (FOR SIMPLICITY,
  DELIVERY LIGHT TOPICALLY)

• Toxicology
• PDT total dose (light x PS)
• Vascular-targeted PDT
• Extravascular PDTC

• Toxicology
• PDT total dose (light x PS)
• Vascular-targeted PDT
• Extravascular PDTC

• Establish maximum tolerated PDT dose in
  vascular-targeted PDT
• Establish maximum tolerated PDT dose in
  extravascular PDT

Aim 4 Dose-dependent PDT efficacy
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Aim 4 Determine PDT dose-response of pancreatic
tumors in mouse models
Hypothesis Dose-dependent photodynamic
destruction of pancreatic tumors will affect
local tumor control and survival in mice, which
can be monitored by PET.
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Aim 4

• PDT Dose Response
• Correlation of PET with tumor burden
• PDT dose light x PS (Aurora)
• Monitor dose-dependent response of tumors to PDT
• Acute (PET) (find out why PET might not be
  feasible for pancreas)
• Long-term
• survival
• Metastases
• Tumor burden
• Develop a molecular imaging modality that can
  reliably predict treatment response.(molecular
  marker?)

PDT tumoricidal dose response
Investigate whether PDT alone upregulates any
molecular pathways that are relevant with the
candidates for chemotherapy agents.

• Correlate PDT dose with tumoricidal efficacy,
  survival and metastases
• Verify that PET can reliably measure acute
  treatment response
• Establish a molecular imaging modality to monitor
  PDT response in real-time and in vivo

Aim 5 Efficacy of PDT-based combination regimens
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Aim 5 Establish the minimum combination PDT and
chemotherapy doses required to achieve optimal
therapeutic outcome in mouse models
Hypothesis PDT-based combination regimens will
provide more effective and well tolerated tumor
destruction than individual monotherapies.
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Aim 5

• Efficacy of combination regimen
• PDT Gemcitabine (consider whether to keep
  gemcitabine as combination agent considering
  projects should look beyond current clinical
  practices) PDT ?
• Dose-dependence of combination therapy on acute
  tumor destruction? (PET/molecular imaging?)
• Compare treatment effect of combination regimens
  and individual monotherapies
• Acute (PET)
• Molecular imaging
• Long-term
• survival
• Metastases
• Tolerance to treatment (weight loss?)

Tumoricidal response of PDT-based combination
regimens
1.Sequence of PDT and chemo? 2.which additional
chemo agent (in addition to Gemcitabine)? 3.need
to look at cellular molecules that are regulated
post-PDT and find appropriate intervention
mechanisms.

• Determine the most effective combination regimen
  in vivo
• Establish the minimum dose required to achieve
  therapeutic efficacy?

PDT
Aim 6 Efficacy of combination regimen in patient
tissue
Chemo
Chemo Dose
Tumor Kill
Dose
PDT Dose
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Aim 6 Test combination regimen determined from
Aim 5 on fresh patient tissue samples ex vivo
Hypothesis Pancreatic tumor samples resected
from patients are responsive to the combination
regimen determined in aim 5
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Aim 6

• Efficacy of combination regimen
• PDT combination agent
• Evaluate treatment response in 4 groups (if size
  permits, a single tissue sample will be cut into
  4)
• No treatment
• PDT alone
• Second monotherapy
• Combination treatment
• MTT/MTS as viability assay
• Molecular markers
• IHC
• LCM
• PCR/gene array
• ELISA

Tumoricidal response of PDT-based combination
regimens
What is size of tissue sample? Will they all be
treated with PDT prior to resection? Do we want
to treat only the samples that were
non-responsive to PDT?
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??Aim 7 Strategies for targeted-PDT??
Targeted PDT treatments involve
photosensitizer-loaded C225 antibodies,
nanoparticles or polymers bearing molecules that
selectively bind to pancreatic cancer cell
surface components such as EGFR
C225 antibody carrier
Nanoparticle carrier
Polymer carrier
EGFR binding
photosensitizer
photosensitizer
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Enzyme-mediated PS activation on tumor surface
Conjugation quenching
H

H2N
N
Protease activation
peptide
Proximity quenching
peptide
Protease activation
peptide
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New specific aims

• Aim 1 molecular signatures affecting response to
  PDT in in vitro 3-D cultures to form a basis of
  developing combinations regimens.
• Aim 2 development of PDT-based combination
  regimens in in vitro 3-D cultures based on
  molecular studies (Aim 1) chemotherapy agent,
  sequence
• Aim 3 Establishment of PDT parameters for mouse
  models of pancreatic cancer (ie. Biodistribution,
  pharmacology)
• Aim 4 test PDT-based combination regimens in
  mouse models of pancreatic cancer and evaluate
  molecular response and molecular signature
  (therapeutic arm) dose response
• Aim 5 novel interventions(aptamer,c225,other),
  targeted PDT and bioinformatics

Ex vivo fresh samples from patients -gt deal in
core (in conjunction with Eds project and
possibly bioinformatics)
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Resistance to Gefitinib
Cancer Chemother Pharmacol. 2006 Mar 11 Activated
Src and Ras induce gefitantib resistance by
activation of Activated Src and Ras induce
gefitinib resistance by activation of signaling
pathways downstream of epidermal growth factor
receptor in human gallbladder adenocarcinoma
cells. Baoli Qin, et al
Results suggest that activated Ras and Src could
induce gefitinib resistance by activating either
or both of Akt and Erk signaling pathways, thus
providing signaling downstream of EGFR
Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, et
al. (2005) KRAS Mutations and Primary Resistance
of Lung Adenocarcinomas to Gefitinib or
Erlotinib. PLoS Med 2(1) e17
Gefitinib resistance in KRAS mutation however
EGFR mutants only some were resistant.