Biophysical Determinants of Photodynamic Therapy and Approaches to

Improve Outcome

Theresa M. Busch, Ph.D.Department of Radiation Oncology

University of Pennsylvania, Philadelphia, PA

What is Photodynamic Therapy?

PDT is a directed, light-based method of damaging malignant or otherwise abnormal tissues.

Image from Wikipedia

How Does it Work?

Energy transfer

3O2

Type 2 Reaction

1O2

Photosensitizer

hv

Photosensitizer3

Oxidation ofOrganic Substrates

How Does it Work?

Mechanisms of PDT action• Direct Cell Effects

• Direct 1O2-mediated toxicity to tumor cells• Indirect Effects

• Vascular damage• During light treatment• Delayed development within several hours after light

treatment• Stimulation of host immune responses.

Cell death may occur by apoptosis, necrosis, and/or autophagy

PDT Variables Photosensitizer

• Drug type• Dose• Drug-light interval

Light Delivery• Wavelength• Fluence• Fluence rate

What is it used for?FDA-Approved Indications (Oncology)

Obstructive esophageal cancer* Obstructive endobronchial lung

cancer* Microinvasive endobronchial lung

cancer Actinic keratosis Barrett’s esophagus/ high grade

dysplasia

*for palliative intent

Clinical Trials

Pleural spread of nonsmall cell lung cancer

Mesothelioma Intraperitoneal malignant tumors Head and Neck- pre-malignant through

advanced disease Brain tumors Skin cancer Prostate cancer

Heterogeneity in PDT

• Photosensitizer distribution• Tissue optical properties (light

distribution)• Microenvironment

• Tumor oxygenation• Vascular network

Heterogeneity in Photosensitizer Uptake:

A Lesson From the Intraperitoneal PDT Clinical Trial

Tissue Site Photofrin® Concentration (ng/mg) Normal

Median (Range, N) Tumor

Median (Range, N) Appendix 3.95(NA, N=1) NA

Fat 1.21(0.5-1.8, N=11) 4.25(4.0-4.5, N=2)

Gall Bladder 3.51(3.2-3.8, N=2) NA

Mucosa (Large Intestine) 5.75(4.8-6.7, N=2) NA

Large Intestine with mucosa 2.62(1.2-4.4, N=12) NA

Large Intestine without

mucosa

1.77(0.9-2.7, N=4) NA

Left Upper Quadrant NA 3.07(NA, N=1)

Liver 36.9(34.9-38.9, N=2) 1.71(NA, N=1)

Mesentery 2.68(NA, N=1) 2.87(2.0-4.9, N=5)

Muscle 0.82(NA, N=1) NA

Omental Bursa 0.96(0.3-3.0, N=15) 3.91(1.5-4.2, N=4)

Other 1.70(1.4-2.0, N=6) 2.06(0.4-6.0, N=37)

Pancreas 1.70(1.4-2.0, N=2) NA

Pelvis NA 3.54(NA, N=1)

Peritoneum 3.60(NA, N=1) 2.93(0.6-5.3, N=2)

Mucosa (Small Intestine) 3.61(2.6-4.3, N=4) NA

Small Intestine with mucosa 2.99(0.3-11.9, N=23) NA

Small Intestine without

mucosa

1.94(0.8-2.8, N=5) 0.66(NA, N=1)

Skin 0.91(0.3-2.3, N=4) NA

Spleen 21.5(15.7-23.2, N=3) 4.71(3.8-5.6, N=2)

Stomach 2.4(1.0-2.5, N=3) 2.95(1.9-4.0, N=2)

Hahn SM, et al. Clin Cancer Res 12:5464-70, 2006

How about light distribution?

Light absorption and scattering affects the fluence rate seen by the tissue.

Nor

mal

ized

flue

nce

rate

Distance (mm)

Tumor surface

3 mm depth

75 mW/cm2

630 nm

The tumor microenvironment is highly heterogeneous….

Busch TM, et al. Clin. Cancer Res. 10: 4630–4638, 2004

…. and PDT exacerbates heterogeneity in hypoxia

distribution

Control RIF Tumor During PDT5 mg/kg Photofrin

135 J/cm2, 75 mW/cm2

Busch TM, et al. Cancer Res. 62:, 7273-7279, 2002

Heterogeneity AboundsSo what to do?

ModifyModify

MonitorMonitor

Approach 1: Modify Light Delivery

Rationale: Lowering PDT fluence rate reduces the rate of

photochemical oxygen consumption. Better maintenance of tumor oxygenation during

illumination. Improves long-term tumor responses

Enhanced direct cell kill Enhanced vascular shutdown in the treatment field

Hypoxia Assay• EF3 and EF5 are nitroimidazole-

based drugs that binds to hypoxic cells as an inverse function of oxygen tension.

• Detection is by a fluorochrome-conjugated monoclonal antibody.

• Fluorescent micrographs are digitally analyzed for binding.

Section,Stain for EF3/5

Fluorescence microscopy

Labeling of Hypoxia during PDT• RIF murine tumor • EF3 at 52 mg/kg• Treated animals receive

Photofrin-PDT at 75 or 38 mW/cm2, 135 J/cm2

• Hoechst 33342 at 1.5 min before tumor excision

• Cryosectioning, immunohistochemistry, fluorescence microscopy

EF3

Hoechst

Hoechst (perfusion)Anti-EF3Anti-CD31Hoechst (tissue label)

PDT

Fluence rate effects on PDT-created hypoxia

0

50

100

150

Controls PDT

Surf Deep Surf Deep

38 mW/cm2

0

50

100

150

Controls PDT

Surf Deep Surf Deep

75 mW/cm2

EF

3 B

ind

ing

EF

3 B

ind

ing

Low fluence rate reduces intratumor heterogeneity in PDT-created hypoxia

0

0.5

1

1.5

2

2.5

3

Regional heterogeneity in EF3 bindingTumor Light PDT-75 PDT-38

*

*p<0.05 for a depth-dependent increase in hypoxia (one sided signed rank test)

Causes of depth-dependent hypoxia during PDT

Light distribution?

Nor

mal

ized

flue

nce

rate

Distance (mm)

Tumor surface

3 mm depth

Causes of depth-dependent hypoxia during PDT

Photosensitizer distribution?

0

2.5

5

7.5

10

12.5

Pho

tofr

in U

ptak

e (n

g/m

g)S D

Causes of depth-dependent hypoxia during PDT

Does not appear to be a result of photochemical oxygen consumption.

How about PDT-induced vascular effects?

Getting at heterogeneity in vascular response during PDT

• Diffuse Correlation Spectroscopy • Measures the temporal correlation of

fluctuations in the intensity of transmitted light (785 nm) to provide information on the motion of tissue scatters, e.g. red blood cells

• Data used to calculate relative blood flow, i.e. flow normalized to a pre-treatment baseline

• Monitoring throughout PDT is facilitated by a non-contact camera probe equipped with optical filters to block the 630 nm treatment light

• Separation distance between unique source-detector pairs determines the depth of tissue probed.

-0.4 -0.2 0 0.2 0.4-0.4

-0.2

0

0.2

0.4

cm

cm

1 2

3 4

5

6 7

8

9

10 11

12

13 I III

IV

II

Distance (mm)

sourcesdetectors

Substantial intratumor heterogeneity exists in PDT-created

vascular effects

0

0.5

1

1.5

2

2.5

-1000 0 1000 2000 3000

Normalized blood flow

Time (s)

PDT • PDT induces an initial increase in blood flow.

• PDT leads to significant depth-dependent intratumor heterogeneity in blood flow response during illumination.

Intratumor heterogeneity in vascular effects (controls)

0

0.5

1

1.5

2

2.5

-1000 0 1000 2000 3000Time (s)

Light

0

0.5

1

1.5

2

2.5

-1000 0 1000 2000 3000Time (s)

Light

Lower fluence rate reduces intratumor heterogeneity in relative blood flow during

PDT

Max rbf Max time (s) Min rbf Min time (s) CV (%) % of values

0.75-1.00

75 mW/cm2 1.72 ± 0.13 325 ± 57 0.47 ± 0.7 1195 ± 172 15 ± 3 13 ± 2

38 mW/cm2 1.76 ± 0.19 752 ± 175* 0.31 ± 0.03* 1647 ± 249 9 ± 1* 26 ± 5*

0

0.5

1

1.5

2

2.5

0 2000 4000Time (s)

PDT

0

0.5

1

1.5

2

2.5

-1000 0 1000 2000 3000

Normalized blood flow

Time (s)

PDT

Low fluence rate reduces intratumor heterogeneity in cytotoxic response.

0

10

20

30

40

50

60

****

Tumor Photofrin Light PDT- 0 h PDT- 2.5 h

38 mW/cm2

01 02 03 04 05 06 0***L ig h t P D T - 0 h P D T - 2 .5 h P D T - 8 h7 5 m W /c m2

0

10

20

30

40

50

60

* **

Light PDT- 0 h PDT- 2.5 h PDT- 8 h

75 mW/cm2

Low fluence rate improves long-term tumor response

% o

f an

imal

s w

ith t

umor

s <

400

mm

3

Lowering PDT fluence rate improves therapeutic outcome (summary)

Delivering a light dose more slowly provides Less intra-tumor heterogeneity in PDT-created

hypoxia during illumination Less intra-tumor heterogeneity in vascular

responses during illumination Greater direct cell kill of tumor cells Better long-term treatment response

Heterogeneity AboundsSo what to do?

ModifyModify

MonitorMonitor

Monitoring: Rationale

• PDT can create significant hypoxia in even vascular-adjacent tumor cells.

• Vascular monitoring, including oxygenation and/or blood flow, may be indicative of tumor response.

Monitoring: MethodsDiffuse optical spectroscopy

• Broadband reflectance spectroscopy with a noninvasive probe• Measures tissue optical properties in the range of 600-800 nm• Data used to calculate concentrations of oxyhemoglobin (HbO2) and deoxyhemoglobin

(Hb)• Tissue hemoglobin oxygen saturation (SO2 or StO2) = [HbO2]/[HbO2 + Hb]• In mouse tissues SO2 of 50% at pO2 of 40 mmHg

• Diffuse correlation spectroscopy with a non-contact probe• Measures temporal fluctuations in transmitted light (785 nm) to provide information on the

motion of tissue scatters, e.g. red blood cells • Data used to calculate relative blood flow, i.e. flow normalized to a pre-treatment baseline• Monitoring throughout PDT is facilitated by a non-contact camera probe equipped with

optical filters to block the 630 nm treatment light

PDT induces variable changes in tumor hemoglobin oxygen saturation

0

10

20

30

40

50

SO

2 (%

)

Before PDT After PDT

0 h 3 h

Pre- or post-PDT SO2 is not associated with tumor response

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

SO2 before PDT(%)

Tim

e-to

-400

mm

3 (d

ays)

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

SO2 after PDT (%)

Tim

e-to

-400

mm

3 (d

ays)

The PDT-induced change in SO2 in individual tumors is highly predictive of

response

0

5

10

15

20

25

0 0.5 1 1.5 2 2.5

y = 4.6034 + 9.4039x R 2= 0.72999

Relative-SO2

Tim

e-to

-400

mm

3 (d

ays)

Relative SO2=

SO2 after PDT

SO2 before PDT

Wang H-W, et al. Cancer Res. 64(20):7553-7561, 2004

The PDT-induced change in blood flow is highly predictive of response

0

0.5

1

1.5

2

2.5

-1000 0 1000 2000 3000

relative blood flow

Time (s)

PDT

Slope of decrease in blood flow

Tim

e to

a t

umor

vol

ume

of

400

mm

3 (d

ays)

Yu G, et al. Clin Cancer Res. 11:3543-52, 2005

Monitoring (Summary)

• Pre-existing tumor SO2 of similarly-sized tumors of the same line can be highly heterogeneous.

• PDT-induced changes in SO2 and blood flow can vary from tumor-to-tumor, even for the same PDT treatment conditions.

• Individualized measurement of PDT effect on blood flow or blood oxygenation in a given tumor is predictive of long term response in that animal.• Changes associated with better maintenance of tumor oxygen (smaller PDT-

induced decreases in SO2 or blood flow) lead to better tumor response.

• Diffuse optical spectroscopy, can be readily applied in the clinic and thereby may provide a means for the rapid, individualized assessment of PDT outcome.

Conclusions• Both and clinical and preclinical studies indicate that

tumors can be characterized by substantial heterogeneity in the essential components of PDT.

• MODIFICATION (e.g. light delivery or tumor microenvironment) can be used reduce physiologic, hemodynamic, and cytotoxic heterogeneity.

• MONITORING offers potential to optimize treatment through individualized, real-time dosimetry based on hemodynamic responses.

PDT at PennLaser Specialist/ManagerCarmen Rodriguez

BiostatisticsRosie MickMary Putt

Radiation OncologyEli GlatsteinStephen HahnRobert LustigJames MetzHarry QuonNeha VapiwalaKeith Cengel

Veterinary MedicineLilly DudaJolaine Wilson

SurgeryDouglas FrakerJoseph FriedbergScott CowanBert O’MalleyS. Bruce MalkowiczAra Chalian

Nursing CoordinatorsDebbie SmithSusan PrendergastMelissa Culligan

MedicineDan StermanColin GilespieAndrew HaasGregory Ginsberg

PhysicistsTimothy ZhuJarod FinlayAndreea DiMofte

Pre-clinical ResearchersTheresa BuschSydney EvansCameron KochStephen TuttleKeith CengelArjun YodhXioaman Xing

DermatologySteve Fakharzadeh

AcknowledgementsRadiation OncologySteve Hahn Eli GlatsteinKeith CengelCameron KochSydney Evans

Statistics/Image AnalysisE. Paul WileytoMary PuttKevin Jenkins

Physics and AstronomyArjun Yodh Xiaoman XingGuoqiang YuHsing-Wen Wang

Medical PhysicsTimothy ZhuJarod FinlayKen WangCarmen RodriguezAndreea Dimofte

Busch labElizabeth RickterShirron CarterMin YuanAmanda Maas

Grant Support (NIH)R01 CA 85831P01 CA 87971