Pre-clinical Imaging

Roger Lecomte, Ph.D.

Lecture 1: Thursday July 26, 2018, 9:15

Department of Nuclear R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 Medicine & Radiobiology 1 Duality of Interest Statement

IR&T Inc. – Co-founder & CSO Research contracts: Charles River, Bayer, Biogen

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 2 Outline

• Preclinical Imaging − Morphological/anatomical − Molecular imaging • Modalities − Overview (US, MRI, optical) − µCT − µSPECT − µPET • Applications − Multimodality imaging

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 3 Why Small Animal Imaging ?

• The rat and mouse host a large number of human diseases ∗ Opportunity to study disease progression / therapeutic response
under controlled conditions
non-invasively
in same animal
repetitively • Powerful tool for research into molecular pathways and biomarker identification ∗ Molecular targets, receptors & drug binding sites ∗ Relationship between genes ↔ phenotype ∗ Gene expression & gene therapy assessment • Bridge between in vitro / in vivo animal studies and Phase I trials ∗ PK/PD, ADME(T) ∗ Study of site, specificity, mechanism of action of new pharmaceuticals * Preclinical study of dose regimen, toxicity,… → faster screening of lead compounds (< time) → earlier decisions about compound’s suitability (< cost ) → smaller number of animals

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 4 Preclinical Imaging

In vivo
Non-invasive
Allows longitudinal follow up studies in same animal

Primarily anatomical Primarily molecular

 Ultrasound (US)  Optical (fluorescence, bioluminescence)  Magnetic resonance (MRI)  Single Photon Emission Computed  Computed tomography (CT) Tomography (SPECT)  Positron Emission Tomography (PET)

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 5 The Challenge of Preclinical Imaging Na 18 F Bone Scans

×××1/300 ×××1/10

75 kg human 200 g rat 20 g mouse Clinical PET scanner Preclinical PET scanner Mouse PET scanner (5-6 mm or ~1 cc) (1.3 mm or ~2 µl) (0.8 mm or ~0.5 µl)

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 6 A Anatomical Preclinical Imaging Modalities F Functional M Ultrasound Optical Molecular (Bioluminescence, fluorescence) Structure A 0.1 mm A Topography F Doppler µm to mm CT M Tissue Density, Z 3 A ~10 cells 20-50 µm ≠≠≠ quantitative F Fluoroscopy PET/SPECT F M MRI Radiotracer A F M ~1-2 mm H Concentration 0.1 mm <10 -12 mole BOLD, DCE = quantitative βββ-galactocidase 0.1 µmole H / µmole 31 P R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 7 In Vivo Pre-Clinical Imaging Modalities

MODALITY PARAMETERS RESOLUTION SENSITIVITY APPLICATIONS

OPTICAL Metabolism, Receptors, Tumor µm (in situ ) ~10 2-3 cells Fluorescence Visible/IR light progression, Gene expression, mm (external) 10 -9-10 -15 M Bioluminescence Therapy assessment Raman, IVM Acoustic impedance Anatomy, Blood flow 10 -15 US Speed / Attenuation <100 µm Tissue structural (mic robubbles) Photoacoustic Frequency shift characteristics

Anatomy Density ~20-50 µm ~µg Mineral content X-Ray CT Z Contrast media

1H concentration 0.1 µmole 1H Anatomy 31 MRI / MRS T1 & T 2 relaxation 25-100 µm µmole P Blood Flow Chemical shifts (>10 16 atoms) Conc./ Chemical State

Blood flow/volume, Perfusion SPECT Radiotracer µSPECT 0.1-2 mm 10 -12 mole Metabolism, PK/ADME 11 PET concentration µPET 1-2 mm (>10 atoms) Receptor concentration, Gene expression, Therapy assessment IVM: IntraVital Microscopy; US: UltraSound; CT: Computed Tomography; MRI/MRS: Magnetic Resonance Imaging/Spectroscopy; SPECT: Single Photon Emission Computed Tomography; PET: Positron Emission Tomography.

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 8 The Molecular Imaging Matrix*

CT

MRS

Resolution Limit Range

*Adapted from: SR Meikle et al , Small animal SPECT and its place in the matrix of molecular imaging technologies, Phys Med Biol 50:R45-R61;2005 JC Gore JC et al , Molecular imaging without radiopharmaceuticals? J Nucl Med 41(3):332-8;2009

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 9 Ultrasound Imaging

Vevo 2100 High-frequency US Photoacoustic high-frequency micro-US system from Visualsonics

Strengths:
Portable
Inexpensive
High spatial & time resolution

Non-targeted Targeted
Truly real-time (blood flow)
Good depth penetration
µbubbles contrast agent
Clinically translatable Weaknesses:  Limited to soft tissues (no bone or air structures) James & Gambhir. A molecular imaging primer: modalities,  Probe coupling to subject imaging agents, and applications. Physiol Rev 92:897-965;2012  Operator dependent

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 10 Magnetic Resonance Imaging (MRI)

Pre-clinical MRI: 4-11T (Earth magnetic field: 0.00003T)

Larmor frequency (H): !ͤ Ɣ ̼ͤ 1.5T: !ͤ Ɣ 64 ͇͂ͮ 7T: !ͤ Ɣ 300 ͇͂ͮ

With imaging agent

Pre-clinical MR scanner (SPIO)

Strengths:
High spatial resolution
High soft-tissue contrast
No ionizing radiation Weaknesses:  High cost (>1 M$)  Poor sensitivity  Long acquisition time

James & Gambhir. A molecular imaging primer: modalities, imaging agents, and applications. Physiol Rev 92:897-965;2012

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 11 Small Animal 7 Tesla MRI

7T MRI facility at the Sherbrooke Molecular Imaging Center

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 12 7T MRI Cerebral Imaging

Rat brain vascularisation Resovist (blood pool agent)

T2* Weighting Resolution: 117 µm Acquisition time: 30 min

In vivo Ex vivo Courtesy M. Lepage Université de Sherbrooke

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 13 Pharmacokinetic modeling with Gd-DTPA/MRI vs FDG/PET

Volume transfer constant

Extravascular volume fraction

Blood volume fraction Arterial input function measured with FDG and Gd-DTPA

Poulin et al , Magn Reson Med 2012 10.1002/mrm.24318

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 14 Optical Imaging

Bioluminescence Fluorescence Emerging variants • Cerenkov imaging • Raman imaging • Photoacoustic imaging • Intravital microscopy • Etc.

Strengths:
Inexpensive
High sensitivity
Multiplexing capabilities Weaknesses:  Poor spatial resolution Pre-treatment  Limited depth (mm to ~cm)  Non-quantitative (Surface weighted images

Post-treatment James & Gambhir. A molecular imaging primer: modalities, imaging agents, and applications. Physiol Rev 92:897-965;2012

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 15 Principle of µCT

• Digital X-ray camera DETECTOR • Phosphor (GOS) or CsI on CCD or CMOS photodiode or a-Si sensor matrix • µfocus 10-100µm • W or Mo anode • MOS Flat panel CONE • A-Se on TFT or CMOS BEAM • 20-80 KV readout array • 0.1-5 mA • Be filter • Semiconductor pixel detector array* • Resolution: 10-100 µm

* B. Mikulec, “Development of segmented semiconductor arrays for quantum imaging”, Nucl. Instrum. Meth. Phys. Res. A510, pp. 1-23, 2003.

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 16 CT Measurement Process

Transmission through tissues

 n  I === I 0 exp −−−x ∑∑∑µµµi   i===1 

n I0 ln === x ∑∑∑µµµi I i===1

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 17 CT Measurement

Image Display: Black +1000 ⇒ CT numbers* or Hounsfield Units (HU)

µi − µwater Ni = 1000 × µwater

⇒ Diagnostic quality ~5-10 HU (~1% SD)

* From G. Michael, “X-ray computed tomography”, Physics Education 36 (11), pp. 442-451, November 2001.

White R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 18 µCT Spatial Resolution

2 2 FWHM total ≈ FWHM d + FWHM x

x  1  ∆ FWHM M X FWHM d ≈ 2.35   x ≈ × f  M  2

FWHM d : detector resolution

FWHM x : projection blurring due to X-ray focal spot size Dx Xf ∆x : pixel size

Xf : focal spot size (FWHM) M : Magnification

M = (dxs + dsd ) / dxs

* M.J. Paulus et al., “High resolution X-ray computed tomography: an emerging tool for small animal cancer research”, Neoplasia 2(1-2), pp. 62-70, 2000

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 19 X-ray Energy in µCT

Attenuation Coefficient vs Energy Contrast Resolution vs Energy*

1000 )

-1 Bone Soft Tissue

100 Fat Water

10

1 Attenuation Coefficient (cm

0.1 0 20 40 60 80 100 X-ray Energy (keV)

• At low energy (µ ↑↑↑↑↑↑ ) contrast resolution limited by attenuation in subject • At higher energies (µ ↓↓↓↓↓↓ ) contrast resolution limited by low absorption in subject • Best contrast resolution at energy for which µ ~ 2/D

• For rats (D ~ 5-6 cm), Eopt ~ 30 keV; for mice (D ~ 2.5-3 cm), Eopt ~ 20 keV • Most studies performed at E ~ 40-60 keV

* M.J. Paulus et al., “High resolution X-ray computed tomography: an emerging tool for small animal cancer research”, Neoplasia 2 (1-2), pp. 62-70, Jan-April 2000. R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 20 µCT Systems

SkyScan-1078

ImTek MicroCAT II Stratec XCT Research SA

GE eXplore RS R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 21 Applications of µCT µCT Skeleton Image • Anatomy - Skeletal tissue - Adipose tissue - Thoracic imaging • Contrast enhanced soft tissue imaging - Vascular morphology - Abdominal tumor imaging - Renal morphology and function • Tissue density • Tissue composition

http://www.imtekinc.com/html/skeletal.html

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 22 Volume Rendering of Mouse Skull

À trouver

D0 D9

C. Lackas, https://commons.wikimedia.org/w/index.php?curid=5791359

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 23 Lung Tumor Follow-up in Mouse by CT

D0 D9

Kirsch et al, Int J Radiat Oncol Biol Phys 76:973–977, 2010

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 24 Contrast Agent in CT Imaging

Longitudinal micro-CT imaging of liposomal iodine biodistribution. Liposomes slowly accumulate in the subcutaneous tumor, liver, and spleen overD0 the course of 72 h. The white arrow points to the location of the D9 tumor in each image.

Ashton et al, In vivo small animal micro-CT using nanoparticle contrast agents, Front Pharmacol 00256, 2015

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 25 Contrast Agent in CT Imaging

EGFR- expressing tumors

3D micro-CT reconstructions of mice with EGFR-expressing tumors. Mice were injected with (A) saline, D9(B) non-targeted gold nanoparticles (GNPs), or (C) EGFR-antibody targeted gold nanoparticles. Increased CT enhancement was seen for both types of nanoparticles, but targeted nanoparticles showed significantly higher enhancement than non-targeted controls. Ashton et al, In vivo small animal micro-CT using nanoparticle contrast agents, Front Pharmacol 00256, 2015

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 26 Bone Repair Follow-up in Rat by µCT

 Empty/soft tissue (−1000 to +800)  Woven bone (+1200 to +1900)  Compact bone (>2700)

D0 D9 Di et al, A longitudinal low dose CT analysis of bone healing in mice: A pilot study, Adv Ortho ID791539, 2014

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 27 X-ray Computed Tomography (CT)

Advantages: – Relatively inexpensive, fast, efficient – High-resolution anatomical images – High contrast for bone – Iodinated or nanoparticles-based contrast agents can improve soft tissue contrast – Dual-energy CT for tissue composition (e.g., bone mineral density)
Excellent anatomical reference frame for other molecular imaging modalities Drawbacks: – High radiation exposure – Low soft-tissue contrast – Possible renal toxicity of contrast agents – Poor sensitivity (large mass needed to attenuate X-rays)

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 28 Pinhole SPECT

Detector Spatial Resolution in Pinhole SPECT

2 Object  ()a + b d   b 2 R ≅  e  +  R  Pinhole  a   i a 

a : pinhole-detector distance b : pinhole object distance

de : effective pinhole diameter R : detector intrinsic resolution b a i M : magnification = a/b

No physical limit of resolution !
Magnification enhances resolution  Sensitivity decreases as resolution improves  Long measuring time  Limited FOV  Images prone to distortions and artifacts

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 29 Dedicated µSPECT

PS-PMT + Array of NaI(Tl) 2×××2×××6 mm 3 Imaging FOV: 125 ×××125 mm 2 Square pinholes from 1 to 3 mm Spatial resolution: 1-2 mm FWHM 1-8 µl Sensitivity: 0.001-0.01%

Pharmaceutical: [99m Tc]-MDP Dose: ~1 mCi Pinhole: 1 mm square Distance (pinhole-center): 3.5 cm Views/Rotation: 128/360 degrees Acquisition time: 30 seconds/view or ~ 1 h

McElroy et al, IEEE Trans Nucl Sci 49:2139–47, 2002 http://www.gammamedica.com/products/a_spect/spect.html

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 30 Multi-Pinhole µSPECT

U-SPECT-I based on a triple-head 75 gold pinholes, 0.6 mm diam (expensive! ) clinical SPECT system Fixed object & camera, rotating multi-pinhole Spatial resolution: 0.35 mm FWHM 0.04 µl √ Extended effective FOV √ Improved sensitivity: 0.22% √ Reduced measuring time: ~1-3 min !!

Papillary Muscle

Cylinder with 75 gold pinhole focused at the center of cylinder

Cardiac perfusion study in mouse showing left (LV) and right (RV) ventricles according to 3 main axes (6 mCi 99mTc- tetrofosmin, 30 min acquisition at 30 min post-injection). (SNM Animal co-Image of the Year 2004)

Beekman et al, 2004 IEEE NSS/MIC ,, Rome, October 2004 Beekman et al, 2005 IEEE NSS/MIC, Puerto Rico, October 2005

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 31 µSPECT Cardiac Imaging

Quantification of LV perfusion, volumes, and motion in mice Ten time phases of a midventricular perfusion axial slice of a mouse throughout systole and diastole.

HR (bpm) 331 ± 35 EF (%) 60 ±9 EDV (µL) 50 ± 8 ESV (µL) 20 ± 6 SV (µL) 29.5 ± 6 CO (mL/min) 9.6 ± 1.6

QGS Cardiac Analysis Software Constantinesco et al, Assessment of left ventricular perfusion, volumes, and motion in mice using pinhole gated SPECT, J Nucl Med 46:1005-1011, 2005

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 32 µSPECT Imaging

http://www.milabs.com/

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 33 µSPECT Imaging

+ U-SPECT /CT 60 MBq 99m Tc, 90 min scan Dedicated triple-head Spiral bed trajectories NaI(Tl) SPECT system Attenuation correction

75 tungsten pinholes 0.25 /0.35 / 0.6 mm

Spatial resolution: 0.25-0.35 mm FWHM Efficiency: 0.038 -1.25% Ivashchenko et al, Mol Imaging 13:1-8, 2014

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 34 Whole-Body µSPECT Imaging

U-SPECT +/CT Dedicated triple-head NaI(Tl) SPECT system

54 lead pinholes 2 mm diam

Spatial resolution: 330 MBq 99m Tc-MDP, 90 min scan 0.85-1.1 mm FWHM Spiral bed trajectories Efficiency: 1.3% Attenuation correction

Ivashchenko et al, Mol Imaging 13:1-8, 2014

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 35 Ultra-high Resolution µSPECT Imaging

Image of “heart” phantom 450 µm

Co-registered SPECT/CT image of a mouse thyroid lit up due to the uptake of I-125 NaCl solution

• Ultra-high resolution gamma camera based on EMCCD sensor and DM tube • 100 µm pinhole • 60 µm intrinsic resolution

LJ Meng et al, Preliminary imaging performance of an ultra-high resolution pinhole SPECT system using an intensified EMCCD camera, 2006 IEEE NSS/MIC ; IEEE TNS 53:2376-84, 2006

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 36 µSPECT: Magnification vs Minification

High magnification Minification
Improved resolution
Improved sensitivity  Limited nb of pinholes − Object closer to detector  Low sensitivity − > nb pinholes without overlap → Requires higher spatial resolution detector

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 37

Energy Resolution of CZT compared with Pixelated NaI(Tl)

1.1 1 99m Tc NaI_2.2 0.9 NaI_1.5 0.8 CZT

0.7

0.6

0.5 0.4 NormalizedCounts 0.3 0.2

0.1 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Energy (keV)

1.1 1.1 Cd109 Cd109 1 Am241 1 Am241 Co57 0.9 0.9 Co57 Tc99m Tc99m 0.8 In111 0.8 In111 0.7 0.7 0.6 0.6

0.5 0.5

0.4 0.4 NormalizedCounts NormalizedCounts 0.3 0.3 0.2 0.2

0.1 0.1 0 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Energy (keV) Energy (keV) The better energy resolution of semiconductor (e.g. CZT) detectors enables characteristic energy peaks from different radioisotopes to be discriminated ( lower right panel ). This is hardly feasible with scintillator-based detectors (NaI) ( lower left panel ). Multi-Isotope SPECT with CT

99m Tc Bone (140 keV) 123 I Thyroid (159 keV) 201 Tl Heart (70 keV) X-ray CT

Excellent energy resolution of CZT enables separation of different radioisotope γ emissions µSPECT Configuration Pentagonal vs Hexagonal

• Better sampling of central FOV • Some loss of sensitivity at the periphery

Van Holen et al, Design and performance of a compact and stationary microSPECT system, Med Phys 40(11), 2013

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 41 µSPECT Imaging

Advantages: – Readily available, cheap, long half-lives radioisotopes (e.g., 99m Tc) – Easy translation to clinical applications – Good sensitivity (ng or picomoles of molecular probes) – Simultaneous multi-tracers imaging possible – Virtually no physical limit to spatial resolution Drawbacks: – Semi-quantitative – Very low efficiency at high resolution (~10 2 less than PET) – High radiation dose may be a confounding variable → Extra control groups without radiation exposure recommended

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 42 Positron Emission & Annihilation

Radioisotopes & Half-life 11 C (20 min) Detector 13 N (10 min) “Organic” Annihilation 15 O (2 min) + Photon βββ (511 keV) 18 F (110 min) 64 Cu (12.8 h) 61 Cu (3.4 h) 60 Cu (23 min) β+ - 89 Zr (78.4 h) ββ e 94 Positron Tc (52 m) Range 124 I (4.2 d) Non-colinearity 68 Ge/ 68 Ga (288 d/68 min) 180°±0.25° Generator Annihilation 82 Sr/ 82 Rb (25 d/76 sec) Photon . . (511 keV) . Detector

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 43 Positron Emission Tomography (PET)

Patient in PET scanner Brain PET Image

Time-Activity curves

Edge of tumour

Tumour Like conventional nuclear medicine, PET imaging enables the investigation of basic biochemistry and physiology in Brain vivo through observation of dynamic, functional

processes noninvasively. The tracer concentration as a concentration Radiotracer function of time can be readily measured. Time

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 44 Positron Emission Tomography (PET)

Advantages: – Coincidence detection eliminates the need for collimators – Inherently tomographic – “Organic” radioisotopes: 11 C, 13 N, 15 O, ( 18 F) – Quantitative and very sensitive (picomolar) →→→ Biological parameters can be measured at molecular level (Molecular Imaging ) – Measure of biochemistry & pharmacology in vivo without

perturbation Tracer kinetics ⇒⇒⇒ Cradiotracers << Cendogeneous or pharmacologic

Drawbacks: – Complex and expensive infrastructure – Short half-life of βββ+ radioisotopes (< 2 h) →→→ Cyclotron – Complex radiochemistry →→→ Fast, automated – Coincidence detection →→→ PET scanners →→→ Physical limit to resolution

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 45 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Geometric Coding Non- Positron Tomographic colinearity range reconstruction 1.1

FWHM : Full Width at Half Maximum a : Factor accounting for the resolution degradation due to tomographic reconstruction d : Detector size b : Detector positioning accuracy D : Distance between coincident detectors (~ ring diameter) r : Positron range in tissues

* Derenzo & Moses, “Critical instrumentation issues for resolution <2mm, high sensitivity brain PET”, in Quantification of Brain Function, Tracer Kinetics & Image Analysis in Brain PET , ed. Uemura et al, Elsevier, 1993, pp. 25-40.

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 46 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Positron 1 range

18 F ( E βββmax =0.64 MeV)

0.8 15 O ( E βββmax =1.73 MeV)

0.6

FWHM 0.4 RelativeAmplitude 0.2

FWTM 0 -4 -3 -2 -1 0 1 2 3 4 Distance (mm)

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 47 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Positron range 1

18 F ( E βββmax =0.64 MeV) LMH 15 O ( E βββmax =1.73 MeV)

0.1 LDH Amplitude Relative

0.01 -4 -3 -2 -1 0 1 2 3 4 Distance (mm)

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 48 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Positron range

Isotope T1/2 Eβββ+ Resolution (MeV) (mm FWHM) (mm FWTM) (mm rms ) # 11 C 20 min 0.96 0.188 1.86 0.39 13 N 10 min 1.19 0.282 2.53 0.57 15 O 122 sec 1.72 0.501 4.14 1.0 18 F 110 min 0.64 0.102 1.03 0.23 64 Cu 12.8 h 0.65 0.104 1.05 0.23 68 Ga 68 min 1.9 0.58 4.83 1.2 82 Rb 76 sec 3.4 1.27 10.5 2.6

# Worst case FWHM would be 2.35 ××× rms of distribution

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 49 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Positron range

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 50 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Non- colinearity

Diameter D Resolution # Scanner (cm) (mm) Whole-body 80 2.1 Brain 50 1.3 Animal 30 0.7 "Small animal" 15 0.33 ( # FWHM ≈ 0.5 D × tan 0.25 ° )

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 51 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Geometric d

d/2

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 52 Spatial Resolution in PET

FWHM = a ()d 2 2 + b2 + ().0 0022D 2 + r2 Coding

Light sharing: b ~ 2 mm b Charge sharing: b ~ 1 mm Individual coupling: b ~ 0 mm

Positioning accuracy ≠≠≠ Intrinsic (geometric) resolution!!

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 53 Spatial Resolution in PET

FWHM = a ()()d 2 2 + b2 + 0.0022 D 2 + r 2

Tomographic Geometric Coding Non- Positron reconstruction Detector Individual: ~0 mm colinearity range 1.1

Detector Physical limit ≤ 1.0 mm ⇒⇒⇒ ≈0.7 mm ≈0.4 - 0.7 mm

b ~ 0 b ~ 0.4 ( scaled ) d ≈≈≈ 1.2 mm d ≤≤≤ 0.8 mm

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 54 Coding effect in PET Scanners

Source size Subtracted Non-colinearity Hammersmith RatPET 3 Exact HR (UCLA) BaF /TMAE (CTI) 2 Tomitani SHR-2000 (VUB) (Hamamatsu) SHR-7700 A-PET(Philips) (Hamamatsu) ATLAS (NIH) MADPET 2 microPET (UCLA) HRRT Donner 600 (Munich) (Berkeley) ClearPET APD-BGO YAPPET X-PET (Sherbrooke) Ge eXplore X-PET microPET II Focus MADPET II Light Sharing (b ~ 2 mm) 1 HIDAC LabPET Electronic Coding (b ~ 1 mm) LabPET II

FWHMResolution (mm) Individual Coupling (b ~ 0 mm) Crystal Resolution (d/2) 0 0 1 2 3 4 5 Crystal Size (mm)

R. Lecomte, “Technology challenges in small animal PET imaging”, Nucl Instrum Meth Phys Res A, 527:157-165, 2004

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 55 Light/Charge Sharing vs Individual Pixels microPET II prototype LabPET (Tai et al, PMB 2003) (Bergeron et al, IEEE TNS 2008)

0.975 mm LSO 2 mm pixels 64-ch PMT + F.O. APD readout X-Y Analog decoding Parallel digital signal processing

FWHM = 1.22 mm FWHM = 1.21 mm

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 56 Sherbrooke Animal PET Scanner (1995-2009)

Cardiac PET Imaging Normal rat (270 g) 3.8 mCi 18 FDG 60 min ECG-gated acquisition

Non- Treated Treated

PET Imaging in Oncology Whole-Body PET Scan EMT-6 mammary tumors treated 18 F− + 18 FDG, 250 g rat by PhotoDynamic Therapy (PDT) BALB/c mouse (20 g) 400 µCi 18 FDG 60 sec R. Lecomte – 25 th Annual McGill Urology Research Day, September 16 th 2015 57 LabPET™ Scanner (2005)

1.2 mm / 1.8 µl resolution 3D Reconstruction + Physical Modeling

1st commercial APD-based scanner Rat 185 g, Na 18 F 31 MBq (bone tracer) 60 min acquisition @ 68 min post-injection Axial scan at 5 positions R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 58 18 FDG Whole-Body Scans

Rat Mouse 1 cm LabPET4 1 cm LabPET4 • 180 g rat • 19 g mouse • 58 MBq 18 FDG • 33 MBq 18 FDG • 60 min DAQ @ • 30 min DAQ @ 30 min post-iv 30 min post-iv • 10 overlapping • 5 overlapping beds (~6 min/ beds (~6 min/ bed) bed) • 30 MLEM • 30 MLEM iterations iterations

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 59 LabPET II Technology

FPGA-embedded signal processing unit

LYSO crystal arrays APD array 4.7 mm

LabPET II Mouse Scanner • 79 x 50 mm 2 Detector • 4 x 8 crystal & APD arrays module • 1.12 x 1.12 x 10.6 mm 3 LYSO • 1:1 coupling • Time-over-Threshold processing • Sensitivity: 3.3% CMOS 180 nm Mouse 21 g – Na 18 F 8.9 MBq • Energy resolution: 22% 64-channel ASICs 3D MLEM 40 iterations • Time resolution: 3 ns • Spatial resolution: 0.75 mm @ 5 mm

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 60 18 F-FDG Mouse whole-body scan LabPET II

Mouse F - 17 g Scan: 30 min after 30 min awake period Activity: 6.7 MBq @ 150 min p.i. 3D MLEM 30 iter.

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 61 Abnormal FDG Whole-Body Scan

LabPET 4 1 cm • 286 g rat on fatty diet • 60 MBq 18 FDG • 15 min DAQ @ 60 min post-iv • 13 overlapping beds (~1 min/ bed) • 30 MLEM iterations

It is essential to carefully control the physiological status of subjects !

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 62 11 C Imaging

LabPET 4 11 C-acetate 11 C-acetoacetate

• 170 g rat 1 cm • 183 g rat • 92 MBq 11 C- • 98 MBq 11 C- acetate acetoacetate • 40 min DAQ @ 2 • 40 min DAQ @ 2 min post-iv min post-iv • 11 overlapping • 11 overlapping beds @ (2-7) min beds @ (2-7) min from nose to tail from nose to tail • 10 MLEM • 10 MLEM iterations iterations

Tremblay et al, WMIC 2008 Poster #1687 Bergeron et al, WMIC 2008 Poster #421

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 63 11 C Imaging 11 C-acetate

Normal Rat

0.15

0.10 LV Anterior Segment iv Blood Pool • T½ = 20 min Bolus 0.05 • Fast uptake

Activity (counts/pixel/sec) • Fast washout 0.00 0 100 200 300 400 500 Time (sec)

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 64 Cardiovascular PET Imaging • Heart Function: • Blood transit time 13 11 • Blood flow ( NH 3, C-acetate) • Gated heart imaging 13 • Rest stress studies ( NH 3) • Heart Metabolism: • Oxidative metabolism (11 C-acetate) • Glucose metabolism (FDG) • Fatty acid metabolism (FTHA) • Pathological Conditions: • Infarct & Ischemia • Septic shock • Diabetes • Hypertension R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 65 ECG-Gated Myocardium PET Study in Rat

Normal Rat Infarcted Rat QGS Cardiac Analysis Software

Ejection Ejection Fraction Fraction 79 % 52 %

Ventricular Volume Ventricular Volume End Diastolic 506 µl End Diastolic 851 µl End Systolic 104 µl End Systolic 403 µl Stroke 402 µl Stroke 449 µl

Sherbrooke PET Scanner R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 66 Cardiac PET Imaging in Rat

Corridor4DM Cardiac Analysis Software 11 C-Acetate

18 F-FDG

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 67 (FDG) (FDG) Mouse Mouse

Triumph Cardiac PET Imaging inin Imaging Imaging PETPET CardiacCardiac

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 68 Cardiac Mouse Imaging with LabPET II

Left myocardium

C57BL/6 mouse • 3D-MLEM reconstruction 12.6 g • Analytical system matrix 8 MBq 18 FDG in 150 µl • 30 iterations 920 s static acquisition • 0.3 ××× 0.3 ××× 0.3 mm 3 pixels 45 min post-injection • No normalization

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 69 Multi-Tracer Cardiac PET Imaging Protocol

• 200 - 550 g Sprague & Dawley rats • Myocardial infarction by ligature of LAD coronary artery • Free access to food and water, Isoflurane anesthesia, iv caudal bolus • Dynamic scans at a fixed bed position through mid-LV plane • Static/gated scans at 3 bed positions + axial/transaxial sampling • Blood sampling at end of dynamic scans to calibrate blood pool • Corrections: decay, random, efficiency, residual (no scatter, no attenuation) • ML-EM reconstruction, 10-25 iterations, 0.475 mm pixel

13 N-ammonia 11 18 18 C-acetate FDG or FTHA Blood ~100 MBq / 100 g ~100 MBq / 100 g ~37 MBq / 100 g sampling

M u l t i – T r a c e r C a r d i a c P E T I m a g I n g P r o t o c o l ≈ Dynamic Static Dynamic Dynamic Static / Gating 15 min 10 min 15 min 30 min ~45 min

Oxygen Energy Cardiac Blood flow metabolism metabolism function

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 70 13 NH 3-PET Myocardial Blood Flow Studies

Normal 3 sec 6 sec 15 sec 60 sec Rat 300 g iv Femoral Injection

2 min 3 min 10 min 20 min

Infarcted Rat -15 sec 15 sec 45 sec 90 sec 436 g 8 weeks post-ligature iv Caudal Injection

3 min R.5 Lecomte min − Ohrid 2018 7Workshop min − July 25-28, 92018 min 71 11 C-Acetate Kinetic Modeling

Uptake ⇒⇒⇒ MBF = K 1 0.15 Normal Rat LV Anterior Segment ⇒ Fit Washout ⇒⇒ MVO 2 ~ k2 Blood Free Tracer 0.10 Metabolized Tracer

0.05 Activity (counts/pixel/sec) 0.00 0 100 200 300 400 500 Time (sec)

Arterial blood Ca K k4 1 k2

k Metabolic 3 Extravascular space space

CG CE

Tissue CT

* Buck, R.Wolpers, Lecomte Hutchins − Ohrid et 2018al., J. Workshop Nucl. Med. − July32: 1950-725-28, 2018(1991) 72 Myocardial Perfusion and Oxygen Metabolism in Rats with 11 C-Acetate

MBF MVO 2

Heart K1 (ml/g/min) k2 (1/min) Region Normal Infarct Normal Infarct (n = 7) (n = 11) (n = 7) (n = 11)

LV Anterior 4.7 ±1.0 3.1 ±1.0 1.39 ± 0.37 0.98 ± 0.51 LV Inferior 4.1 ±0.5 3.9 ±0.7 ~ 1.57 ± 0.34 0.94 ± 0.23 LV Lateral 7.1 ±2.1 3.2 ±1.2 1.75 ± 0.36 0.74 ± 0.25 LV Septal 6.8 ±0.9 7.9 ±2.0 ~ 1.21 ± 0.21 0.95 ± 0.30 Mean 5.7 ±1.5 4.5 ±2.3 1.48 ±0.23 0.90 ±0.11

* Bentourkia et al., IEEE TNS 49 (2002) 2322-2327 ≈≈≈ -40%

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 73 PET in ONCOLOGY

• Development of animal models ER+/ER KD breast tumors (F. Bénard) Glioma & BBB disruption (D. Fortin) 18 F-FDG Pain in bone tumor (P. Sarret, L. Gendron)

• Tumor imaging 18 F-estrogen analogs (J.E. van Lier) 64 Cu-labeled phthalocyanine (J.E. van Lier) Apoptosis (D. Hunting)

• Monitoring of therapeutic interventions Chemotherapy (D. Fortin, É. Turcotte) Hormonotherapy (É. Turcotte, F. Bénard) Photodynamic therapy (JE van Lier, A. Byrne) Cardiotoxicity in chemo- & anti-angiogenic therapy (A. Byrne, É. Turcotte) 18 F-Estrogen Analog R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 74 Tumor FDG Imaging

Mouse 18 FDG Scan

MC7-L1 ERα-KD

MC7-L1

MC4-L2 MC4-L2 ERα-KD Balb-c Mouse 18-23 g Tumors 3.5-4.5 mm diameter

⇒⇒⇒ Allows direct comparison under Michel Paquette, controlled conditions in same animal Université de Sherbrooke

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 75 Tumor Therapy Assessment

Day 0 Day 7 Day 14

11 C-Met

Letrozole (Aromatase inhibitor) FDG

MC7-L1 (ER+) MC7-L1 Day 0 Day 7 Day 14 ER α-Knockdown

11 C-Met Fulvestrant (Pure anti-estrogen)

FDG

11 C-Methionine : Protein synthesis (or indirect proliferation) 18 F-FDG : Glucose metabolism

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 76 Quantification 18 F-FDG & 11 C-Met FDG Control Fulvestrant

Letrozole Tamoxifen 11 C-Met

Control Fulvestrant

*; p<0,05 Letrozole Tamoxifen **; p<0,01 ***; p<0,005

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 77 PhotoDynamic Therapy (PDT) of Cancer

Photosensitizers Photofrin (Axcan Pharma) • Mixture of hematoporphyrin derivatives • Approved for clinical PDT in USA, Canada, Japan & European countries • Induces tumor necrosis mainly via vascular stasis (indirect effect )

Phthalocyanine (AlPcS 2) • Second generation photosensitizer • Induces tumor necrosis primarily via direct cell kill (direct effect )

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 78 Treatment Follow-up

% FDG Uptake relative to Control Tumor Size relative to Control

100 Photofrin (mg/kg) 90 250 Photofrin (mg/kg) 80 1.25 1.25 2.5 200 2.5 70 5 Tumor 5 60 10 Re-growth 10 150 50

40 100 30 Relative Size (%) Size Relative % Relative FDG Uptake FDG Relative % 20 50 10 0 0 1 h 24 h 1 week 3 week 1 h 24 h 1 week 3 week Time Time • Early scan → Poor predictor of outcome due to edema, latent cells surviving treatment... • At 24 hr: Low FDG uptake → tumor regression High FDG uptake → tumor re-growth • 1 week: Confirmatory R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 79 Tumor Response after PhotoDynamic Therapy

Photofrin AlPcS 2

30 min 2hrs 30 min 2hrs post PDT post PDT post PDT post PDT

BALB/c mice (20 g) 400 µCi 18 F-FDG Treated EMT-6 mammary tumors Scan: 60 sec, 30 min post-FDG Untreated (Control)

Lapointe et al, High-resolution PET imaging for in vivo monitoring of tumor response after photodynamic therapy in mice, J Nucl Med 40:876-882; 1999 R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 80 Tumor Response after PDT

Problems with this approach:

⇒ Effects of PDT in progress during FDG uptake

⇒ Does not allow PDT action mechanisms to be monitored in real-time

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 81 Real-Time Monitoring of PDT-Treated Tumors

• 300 g female rat implanted with two MAC mammary tumors • PDT on one tumor, 2 nd tumor as control • 18 FDG continuous infusion for 2 h, starting with 50 µCi/min • Dynamic PET imaging for 2 h (120 frames × 60 sec)

Administration iv PDT Tumor Photofrin 5 mg/kg implantation PET Imaging (2 h) AlPcS 2 1 mg/kg Base (30 min) 30 min P E T I m a g I n g P r o t o c o l 2-4 24 weeks hrs Continuous FDG Infusion (2 h)

Bérard et al, J Nucl Med 47(7):1119-1126, 2006

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 82 Real-Time Monitoring of PDT-Treated Tumors

450

400 Photofrin Control 350 Treated 300

250 Vascular stasis 200 Defense Reaction 150

100 Permanent TumorFDG units) Uptake(rel. 50 PDT vascular damage

0 0 20 40 60 80 100 120 Time (min) Control Photofrin Photofrin Induces tumor necrosis mainly via vascular stasis 30 min 60 min 90 min 120 min (indirect effect ) Constant FDG infusion during treatment R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 83 Real-Time Monitoring of PDT-Treated Tumors

140 AlPcS 2

120 Control Treated 100

80 Loss of 60 hexokinase activity

40

Tumor FDG Uptake (rel. units) Partial recovery reflects 20 PDT level of cell survival

0 0 20 40 60 80 100 120 Time (min)

Control AlPcS 2

Phthalocyanine (AlPcS 2) 2nd generation photosensitizer Tumor necrosis via direct cell kill 30 min 60 min 90 min 120 min (primarily direct effect ) Constant FDG infusion during treatment R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 84 PET Imaging of Apoptosis

Normal membrane polarity is maintained Externalisation of phosphatidylserine by translocase and floppase activity due to scramblase activation  Direct binding of labelled Extracellular Annexin-V yields poor space contrast in images
Pre-targeting strategy: Membrane Bleb Intracellular space Apoptosis: Annexin-V binds to phosphatidylserine 1. Floppase & Translocase inactivation present on the surface of membrane Cu-64 DOTA Biotin 2. Scramblase activation

Blankenberg et al., Nuclear Medicine Communications 21: 241-250 (2000) Streptavidin 1 3 PDT Annexin-V Biotin Apoptosis

Avidin Avidin Biotin

Cauchon et al, SNM 2004 , Young Investigator Competition 2 Cauchon et al, Eur J Nucl Med 34(2):247-258; 2007

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 85 PET Imaging of Apoptosis

No pre- No 3 steps targeting chase

Tumor Tumor No tumor uptake uptake uptake but poor good contrast contrast

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 86 Detection of Apoptosis after PDT

Apoptosis induced earlier after PDT

with ZnPcS 2 (direct effect) and at a later time with

AlPcS 2 (indirect or vascular effect).

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 87 PET/MRI of Rat Brain Tumor Model

7T MRI T1 PET /MRI FDG PET

Courtesy D. Fortin & M. Lepage Université de Sherbrooke

< 10 m

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 88 PET/MRI in PDT

PhotoDynamic Therapy (PDT): 180 g rat implanted with 2 tumors Porfimer sodium 5 µmol/kg 48 h post-injection: 200 mW/cm 2 @ 630 nm

PET: Control Treated MRI: LabPET4 tumor Myocardium tumor Varian 7T 24 h post-PDT 24 h post-PDT 37 MBq 18 FDG T1 10 min + @ Magnevist 50 min post-injection

Inflammation

Courtesy L. Tremblay & M. Lepage, Université de Sherbrooke → Sequential Imaging on MRI, then PET

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 89 PET/MRI in Pain

Tumor implanted in femoral PET PET MRI 7T: T1 area to induce pain Na 18 F + + 10 MBq MRI Magnevist Early stage (D15) o High Na 18 F in tumor area o Bone metabolism due to tumor blastic activity

Early stage (D18) o Decreased Na 18 F uptake in femoral tumor area o Increased uptake around tumor, indicates mixed (lytic and sclerotic) tumor activity

Early stage (D18) 11 C-Met o 11 C-methionine uptake 74 MBq indicative of high protein synthesis in outer layer of tumor

Louis Doré-Savard, P. Sarret, UdeS

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 90 PET/CT/MRI Multimodal Imaging PET CT MRI PET/CT PET/MRI

260 µCi 18 FES 80 kVp 7T 30 min @ 60 min 130 µA T2 Weighting M. Paquette, J. Rousseau & O. Sarrhini post-injection CRCHUS/Université de Sherbrooke R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 91 Conclusion

µCT, µSPECT and µPET imaging in small animals nearing or exceeding equivalent spatial definition of clinical imaging in humans ( ~10³ gain in spatial resolution )

Convergence of imaging modalities (PET/CT, PET/MRI…) appears inevitable for obtaining all potential benefits from PET

Still substantial progress to be made in detectors, electronics and system integration, but also in imaging protocols to fully exploit molecular imaging

R. Lecomte − Ohrid 2018 Workshop − July 25-28, 2018 92