and Instrumentation Group – Univ. Pisa Advances in PET technology Varese, 28 Febbraio 2008 Functional imaging and Instrumentation Group – Univ. Pisa Advances in PET technology for molecular imaging Alberto Del Guerra Professor of Medical Physics, Faculty of Medicine Head, and Director Specialty School in Medical Physics Head Functional Imaging and Instrumentation Group Department of Physics "E.Fermi' University of Pisa, Pisa, Italy e-mail: alberto.delguerra@df.unipi.it http://www.df.unipi.it/~fiig/ Department of Physics “E.Fermi” University of Pisa Center of Excellence AmbiSEN - Univ. Pisa INFN - Pisa

CONTENTS Molecular Imaging The Physics of PET The small animal scanner YAP-(S)PET Applications of the YAP-(S)PET scanner in molecular imaging Conclusions Acknowledgments

Imaging molecolare “Rappresentazione visuale, caratterizzazione e quantificazione dei processi biologici che avvengono in un essere vivente a livello cellulare e sub-cellulare”

Risorse ed obiettivi dell’imaging molecolare Sviluppo delle tecniche di biologia cellulare e molecolare Disponibilità di nuovi farmaci e probes ad alta specificità Sviluppo di strumentazione per imaging di piccoli animali Obiettivi: Sviluppo di metodi di imaging non invasivi che riflettano i processi cellulari e molecolari, (es. espressione genica o interazioni proteina-proteina) Visualizzazione di trafficking e targeting cellulare Ottimizzazione di terapie farmacologiche e geniche Follow-up delle malattie da un punto di vista molecolare ... e soprattutto=> Ottenere tali obiettivi in modo Rapido, Quantitativo e Riproducibile

Imaging molecolare: Interdisciplinare! Convergenza di varie metodologie di imaging, di biologia cellulare e molecolare, chimica, medicina e farmacologia, matematica e informatica e di varie tecnologie di fisica Anatomia Fisiologia Molecolare Ottico MN RMN US TAC

Concetto di probe 1924: Principio del radiotracciante La sostituzione di un atomo in una molecola con il suo analogo radioattivo (radioisotopo) non cambia significativamente il suo comportamento biologico Conseguenza: “il movimento, la distribuzione e la concentrazione di una molecola può essere misurata con rivelatori per radiazione” Estensione del concetto in imaging molecolare Si utilizzano opportuni “probes” molecolari come sorgente di contrasto per l’immagine. Questi sono solitamente ottenuti a partire da un composto affine che interagisce con il target di interesse con l’aggiunta di una componente che produce un segnale. György Hevesy (1885-1966) Premio Nobel per la Chimica (1943) “per il suo lavoro nell’utilizzo di isotopi come traccianti nello studio dei processi chimici”

Varie tecniche di imaging molecolare per piccoli animali Imaging PET di un ratto utilizzando 18F-FDG che mostra il metabolismo del glucosio Imaging TAC dell’addome di un topo dopo l’iniezione di un mezzo di contrasto iodato. Imaging SPECT dell’addome di un topo tramite 99mTc-methylene diphosphonate che mostra l’accumulo nelle ossa. Imaging ottico di un topo (D) che mostra la fluorescenza GFP dal fegato, addome, colonna vertebrale e cervello dovuta alla presenza di cellule tumorali immagine RMN pesata T2 del cervello di topo. Imaging ottico in bioluminescenza di un topo sovrapposta ad una fotografia dell’animale.

Principio della Tomografia a Emissione di Positroni (PET)

Formazione delle immagini a emissione di positroni Principio della tecnica PET I due rivelatori (fotomoltiplicatori e scintillatori) rivelano i due g misurando l’energia rilasciata ed il punto di impatto nel rivelatore Il circuito di coincidenza (AND in una certa finestra temporale) stabilisce se i due g provengono dall’annichilazione del positrone (coincidenza) Le posizioni di rivelazione nei rivelatori stabiliscono la linea lungo la quale è avvenuta l’annichilazione (linea di risposta o LOR).

Tipici radiotraccianti in PET Radioisotopi 11C(t1/2 = 20.4 min)  sostituzione isotopica 13N(t1/2 = 10.4 min)  sostituzione isotopica 15O(t1/2 = 2.5 min)  sostituzione isotopica 18F(t1/2 = 109.6 min)  sostituzione di un atomo di H Tracciante a-specifico: segue un processo biochimico 18F-FDG  tracciante di metabolismo ( Misura dell’attività metabolica: ricerca di processi anormali ) - 15O-H2O  tracciante di flusso sanguigno cerebrale Tracciante specifico: interagisce direttamente con un sito ricettore Segue uno specifico processo fisiologico o biochimico Es.: 11C-flumazenil  ricettori della benzodiazepina: Analisi di disturbi neurologici Misura dell’efficacia degli psicofarmaci In imaging molecolare si utilizzano principalmente traccianti specifici.

Limiti della tecnica PET Errori intrinseci Range del positrone Deviazione angolare Dipende dal radioisotopo Dipende dal raggio dell’anello (1.8 mm per 40 cm di raggio) <Ec > (MeV) <Range> in acqua FWHM (mm) 18F 0.242 1.4 mm 0.22 11C 0.385 1.7 mm 0.28 68Ga 0.740 3.0 mm 1.35 180° ± 0.25°

Spatial resolution requirements

PET Spatial resolution limitations Crystal Coding Non- colinearity Positron range Parallax error Intrinsic 1.25 : degradation due to tomographic reconstruction d : crystal size b : systematic inaccuracy of positioning scheme (range: 0-2 mm) D : coincident detector separation (~gantry diameter) r : effective source size, including positron range 0.55mm w/ 18F) p : Parallax error (radial elongation) How to achieve high spatial resolution? Individual detectors or “perfect coding” High granularity detectors (e.g. small crystal pixels) Parallax error reduction * 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.

Sensitivity requirements Imaging of low activity sources low uptake processes such as in gene research Possibility to study fast metabolic processes with characteristic time comparable with the scanning time Limitations Brain receptor saturation usually a maximum of 100 mCi can be injected to a mouse Limitation on the volume a maximum of 300 ml can be injected to a mouse Solutions Utilization of radionuclides with a very high specific activity such as PET short half-life radioisotopes: 15O (122 s), 13N (10 min), 11C (20 min), 18F (110min) High geometry efficiency (large solid angle covered by detectors) High detection efficiency (e.g. for crystals: high/medium Z, high density)

Strumentazione per “small animal PET” Tipicamente basata su rivelatori a scintillazione(LSO) e fotomoltiplicatori. La tecnologia più recente è orientata alla massimizzazione della sensibilità pur mantenendo una buona risoluzione. L’alta sensibilità si ottiene con cristalli scintillatori ad alta densità (alta probabilità di interazione) e alto Z (alta probabilità di interazione fotoelettrica). Sono necessarie tecnologie per limitare l’errore dovuto alla profondità di interazione nel cristallo (effetto di parallasse).

YAP-(S)PET II small animal scanner Scanner configuration Configuration: Four rotating heads Scintillator: YAlO3:Ce (YAP:Ce) Crystal size: 27 x 27 (1.5 x 1.5 x 20 mm3 each) Photodetector: Position Sensitive PMT Readout method: Resistive chain (4 channels) FoV size: 40.5 mm axial  40.5 mm Ø Collimators: (SPECT) Lead (parallel holes) Head-to-head distance: 10-15 cm Scanner installed at the “Institute of Clinical Physiology (IFC-CNR)” within the framework of the Center of Excellence AmbiSEN of the University of Pisa, Italy

Performance: system sensitivity The PET system sensitivity is measured with a linear source placed inside a metal tubes. The measure is repeated five times with increasing wall thickness. The system sensitivity at 125 mm head-to-head distance, averaged over the whole axial FOV, extrapolated from the accumulated sleeve measurements, is 1.25% per pair  2.50% per the four head scanner

Performance: absolute sensitivity Measured sensitivity PET: Measured with 18F-FDG High sensitivity energy window: ~25 cps/kBq @ CFOV (50-850 keV) (2.5%) High resolution energy window: ~12 cps/kBq @ CFOV (50-420 keV) (1.2%) SPECT: Measured with 99mTc: 37 cps/MBq (140-250 keV) Absolute sensitivity curve along the scanner axis in PET mode. The sensitivity is measured after energy cuts. The results are plotted against the actual position of the source along the axis. Two different curves are produced for different energy windows: 50-850 keV (high sensitivity) and 50-420 keV (high resolution).

Performance: PET spatial resolution Comparison of the radial, tangential, and axial FWHM of the reconstructed images, obtained with the FBP-2D (top left) using Single Slice (SSRB) and Fourier (FORE) rebinning (50-850 keV energy window). The spatial resolution is plotted against the radial offset. FBP We have used a 22Na point source of about 100 kBq. Volume resolution obtained for two axial positions (central plane and 10 mm axial offset using FORE+FBP).

Performance: transaxial resolution Derenzo Phantom (PET) 1.2 mm 1.2 mm 3.0 mm 1.5 mm 2.0 mm 2.5 mm 1.2 mm 3.0 mm 1.5 mm 2.0 mm 2.5 mm 1.5 mm 3.0 mm 2.0 mm 2.5 mm FORE+FBP 50-850 keV 3D-OSEM 50-850 keV The rods of the Derenzo phantom were filled with 18F solution. Both FBP+FORE (ramp filter) and 3D-OSEM reconstructions were used on a 0.3750.3750.750 mm3 voxel space. A high sensitivity energy window (50-850 keV) was used. 0.750 mm thick slices

Performance: Axial resolution Defrise Phantom (PET) Slice thickness 4 mm The Defrise phantom were filled with 18F solution. 3D-OSEM reconstructions was used on a 0.3750.3750.750 mm3 voxel space. A high sensitivity energy window (50-850 keV) was used. Volume view

Performance: Transaxial resolution Derenzo Phantom (SPECT) 3.0 mm 1.2 mm 1.5 mm The rods of the Derenzo phantom were filled with a 99mTc solution. FBP (ramp filter) reconstruction was used on a 0.3750.3751.5 mm3 voxel space. Sinograms were build using 140-250 keV energy window. 1.5 mm thick slices

Performance: Image quality NEMA I.Q. Phantom 8 mm  3 mm  2 mm  30 mm  1 mm  4 mm  5 mm  Drawing and picture of the NEMA Image Quality phantom for small animal PET scanners. The interior is has been filled with: PET mode: 300mCi of a 18F solution and scanned for 20 min. SPECT mode: 5 mCi of a 99mTc solution and scanned for 60 min.

Performance: Image quality NEMA I.Q. Phantom images (PET) 3D ML-EM reconstruction Voxel size 0.375 mm 0.375 mm (transaxial) 0.750 mm (axial) (E.W. 50-850 keV)

Uniformity and quantitation (PET) 20:1 10:1 1:1 Activity concentration Uniformity (std dev / mean) = 6%

Performance: Image quality NEMA I.Q. Phantom images (PET) Recovery coefficients obtained from hot bars in the IQ phantom Recovery coefficient = avg(maxROI)/meanUNIFORM ROI size = twice the rod diameter slice thickness (10 consecutive ROI’s were considered in the calculation)

Performance: Image quality NEMA I.Q. Phantom images (SPECT) EM coll. (50 it.) FBP (E.W. 140-250 keV) FBP

Small animal Imaging with the YAP-(S)PET scanner Brain Metabolism in Rats Heart Metabolism in Rats and Mice Heart Perfusion In Rats and Mice Bone metabolism in Rats and Mice Tumor Imaging in Rats and Mice Tumor Models in Mice (Breast Cancer) Neurology in Rats Myocardial Models in Rats

Brain metabolism in rat with 18F-FDG (PET) Transaxial sections (0.25 mm x 0.25 mm x 2.0 mm) Eye ball Olfactory bulbs Neostriatum Harderian glands Cerebral cortex Thalamus Inferior colliculus Cerebellum Salivary glands

Brain metabolism in rat Ipotyroidism study with 18F-FDG (PET) Normal Rat Rat with induced Ipotyroidism Normal rats (Wistar) were compared with rats with induced Ipotyroidism in terms of brain glucose consumption (FDG). The effect of the threatment with T3 has been also studied. The rats with induced Ipotyroidism shows a strongly reduced uptake in the harderian glands

Rat and mouse heart metabolism with 18F-FDG (PET) RAT (Pisa) The rat (Sprague-Dawley, 236 g) has been injected with 37 MBq (1 mCi) of 18F-FDG and scanned after 2h for 40min. Heart section details (contrast enhancement) MOUSE (Dijon) The mouse, 24 g has been injected with 30 MBq (0.8 mCi) of 18F-FDG and scanned after 25min for 33min. Heart section details (contrast enhancement)

Rat heart perfusion with 99mTc-Myoview (SPECT) Weight: 204 g Injected activity 8 mCi of 99mTc Myoview Acquisition start: 180 min post injection Scan time: 80 min Voxel 0.5 x 0.5 x 0.5 mm3 Voxel 0.5 x 0.5 x 0.5 mm3

Mouse heart perfusion with 99mTc-Myoview (SPECT) Weight: 33 g Injected activity 4 mCi of 99mTc Myoview Acquisition start: 90 min post injection Scan time 80 min. Voxel 0.5 x 0.5 x 2.0 mm3 Voxel 0.5 x 0.5 x 0.5 mm3 Voxel 0.5 x 0.5 x 2.0 mm3

Bone metabolism in rats with PET and SPECT (Mainz) SPECT (Ferrara) The rat (Sprague-Dawley, 200 g) has been injected with 480 MBq (13 mCi) of 99mTc-MDP and scanned after 2 h for 82 min (3 bed positions) The rat (200 g) has been injected with 48 MBq (1.3 mCi) of 18F- and scanned after 30 min for 30 min (2 bed positions)

Bone metabolism in mice with PET and SPECT PET (Mainz) 18F- Voxel size (0.25 x 0.25 x 1 mm) PET (Dijon) Transaxial slices (2 mm thick) Longitudinal slices SPECT (Ferrara) NaF 99mTc - MDP Voxel size (0.25 x 0.25 x 2 mm)

Tumour imaging in mice with 18F-FDG and 18F-Choline PET Tumor model: MAT-Ly-Lu – Prostatic tumor (subcutaneous) Body weight: 250g – Position: prone/left side down, head forward FDG F-Choline

Liver and kidney imaging in mice with 18F-Choline (PET) Horizontal sections (0.5 x 1 x 0.5 mm voxel) Transaxial sections (0.5 x 0.5 x 2 mm voxel) 3D rendering (maximum projections)

Tumor imaging: Human glioma in rat with 18F-FDG (PET) Normal Rat Rat with brain glioma Controls animals (Wistar) were compared with implanted rats using 18F-FDG. F98 Glioma model has been selected as tumor with infiltrative pattern. The methodology was able to image the tumor and giving the requested information on the position and dimension of the lesion.

Brain histological slice: Tumor Imaging: Human glioma in rat with 18F-FDG (PET) Tumor bearing rat (F98 line) injected with 37MBq of 18F-FDG. Uptake time 45 minutes, acquisition time 60 minutes. Brain histological slice: tumors and surrounding normal brain tissues were removed and treated following conventional preparative histological protocols to fixation and subsequent criosectioning. Coronal sections (0.5 x 0.5 x 2 mm voxel)

ed Istituto di Biostrutture e Bioimmagini CNR Tumor model: Nude mice model of carcinoma breast cancer with 99mTc-Sestamibi (SPECT) Nude mice with subcutaneous carcinoma breast cancer. The studies were performed before (Basal) and after (Post-therapy) the administration of citotoxic drugs. The SPECT acquisition were performed 1 hour after the injection of 99mTc-Sestamibi. Bladder Post-therapy Basal Tumor S. Del Vecchio et al., 2007. Universita’ degli Studi di Napoli “Federico II” ed Istituto di Biostrutture e Bioimmagini CNR

Tumor model: Mice model of breast cancer with 99mTc-Annexin V(SPECT) The RIII female mouse represents a model of genetically modified breast cancer induced by a virus (RIII virus, murine mammary tumor virus, MuMTV) which is transmitted from mother to daughter through breast feeding. The effect of Taxol ® was evaluated at different time points after the drug administration (1, 3, 6 and 24 hours), trying to understand when the highest uptake of 99mTc-Annexin V occurs, as indicator of Taxol induced apoptosis. The animals were i.v. injected in one of the caudal veins with a single dose of Taxol (0.02 mg/g, about 6mg/animal). After 1,3,6 and 24 hours from Taxol administration  37-55 MBq (1- 1.5 mCi) of 99mTc-Annexin V. One hour after radiotracer injection the animals were anaesthetized with intra-peritoneal injection of a mix of ketamine (60 mg/kg and 4.4 mg/kg) and fenobarbital (50 mg/kg). transaxial sagittal Nuclear Medicine Department, University of Pisa coronal

Neurology in rats: Striatal D2 receptors study with 18F-Fallypride (PET) Rat threated with receptor blocking Normal Rat Transaxial section Horizontal section Transaxial section Horizontal section Normal rats were compared with rats with receptor blocking (pre-treated with intraperitoneal injection of 50 mg/(kg body weight) of Haloperidol). All the animals were anesthetized with chloralhydrate 7% and injected via a lateral tail vein with 37 MBq of a high-affinity dopamine D2 receptor ligand 18-F-Fallypride: the acqusition started immediately and the activity in the striatum was monitored (performed at Mainz University). EM reconstruction: 40 iterations. A. Bartoli et al “Preliminary assessment of the imaging capability of the YAP–(S)PET small animal scanner in neuroscience”, NIM A 569, (2006) 488–491

scanner and a β-microprobe”, JCBFM, 2008, in press. Neurology in rats: 18F-MPPF 5HT1a receptors study at the University Hospital of Geneva Sprague-Dawley male rats underwent 18F-MPPF multiple injections: at o time: 1.5 mCi (55 MBq) of 18F-MPPF after 60 minutes: 1.5 mCi (55 MBq) of 18F-MPPF and 10 mg/kg of unlabeled MPPF after 115 minutes: 1.5 mCi (55 MBq) of 18F-MPPF and 110 mg/kg of unlabedeled MPPF. P. Millet et al “In vivo quantification of 5-HT-1A-[18]F]MPPF interactions in rats using the YAP-(S)PET scanner and a β-microprobe”, JCBFM, 2008, in press.

Neurology in rats: 18F-MPPF 5HT1a receptors study at the University Hospital of Geneva Results for this region: P. Millet et al “In vivo quantification of 5-HT-1A-[18]F]MPPF interactions in rats using the YAP-(S)PET scanner and a β-microprobe”, JCBFM, 2008, in press

Neurology in rats: Receptor study with 11C-Raclopride at San Raffaele Hospital, Milano, Italy Rat model of Huntington’s desease: monolateral lesion QA induced Day 0 - control 169 mCi (~6.2 MBq) injected, uptake time: 16 min, acquisition time: 45 minutes Day 8 after QA injection 108 mCi (~4.0 MBq) injected, uptake time: 26 min, acquisition time: 30 minutes Day 30 after QA injection 173 mCi (~6.4 MBq) injected, uptake time: 29 min, Male Wistar rats weighting 300 g were injected icv in the left striatum with 210 nmol of QA solution and in the right striatum with PBS 0.1 mol/l. Stereotaxic coordinates: AP=+ 1.5, L=+ 2.6, V=-7.0 mm from the Bregma, according to the atlas of Paxinos and Watson. Coronal Axial S. Belloli et al “Evaluation of three quinoline-carboxamide derivatives as potential radioligands for the in vivo pet imaging of neurodegeneration”, Neurochemistry International 44 (2004) 433–440

Myocardial studies of a rat model of ischemia and reperfusion Myocardial perfusion evaluation: 99mTc-Myoview 13 N-Ammonia Glucose metabolism: 18F-FDG Apoptosis: 99mTc-Annexin V Acute necrosis: 99mTc-Glucarate “Assessment of the imaging capability of the YAP-(S)PET small animal scanner in a rat model of ischemia and reperfusion”, Bartoli A., Lionetti V., Erba P.A., Fabbri S., Belcari N., Del Guerra A., Recchia F., Mariani G., Salvadori P. ESMI Naples (I), June 14-15, 2007

Rat myocardium perfusion studies with 99mTc-Myoview (SPECT) Rat injected with ~ 5 mCi of 99mTc-Myoview, 60 minutes uptake time, acquisition time 60 minutes, EM reconstruction

Blood Flow with 13N-Ammonia Rat injected with ~ 1 mCi of 13N-NH3, no uptake time, acquisition time 30 minutes, 3D-OSEM reconstruction

Glucose consumption with 18F-FDG Rat injected with ~ 1 mCi of 18F-FDG, 5 ml of glucosate at 5% 10-15 min before injection time, uptake time 45 minutes, acquisition time 45 minutes, EM reconstruction 10 iterations transaxial sagittal coronal

Tracer comparison study Myoview vs. Annexin on rat heart 99mTc-Annexin (low uptake in the heart) 99mTc-Myoview (high uptake in the heart) Fusion (feasible)

Model of rat heart with ischemia and subsequent re-perfusion w/ Dept Nuclear Medicine, Pisa Short axis Vertical long axis Horizontal long axis Injection: 300 MBq (8 mCi) of 99mTc-Myoview uptake time180 min, acquisition 48 min, reconstruction EM algorithm Injection: 300 MBq (8 mCi) of 99mTc-Annexin uptake time 90 min, acquisition 1 hour and half, reconstruction EM algorithm Fusion

Acute necrosis with 99mTc-Glucarate Rat injected with ~ 5-6 mCi of 99mTc-glucarate, uptake time 1 hour and half, acquisition time 1 hour and half, EM reconstruction 50 iterations with collimator model transaxial sagittal 3D rendering (maximum intensity projection) coronal

Small animal CT: technology Circular orbit (A) CT or Spiral CT (B) Rotating sample or rotating detectors Linear or flat panel detectors

“Small animal CT” Department of Physics, University of Pisa X-ray detector 1024 x 2048 pixels (48 mm each) 5 cm x 10 cm active area Maximum frame rate 2.7 fps Measured focus size: 7 mm FWHM 10lp/mm resolution X-ray source Fixed tungsten anode Maximum voltage: 60 kV Maximum power: 10 W Measured focus size: 7 mm FWHM Beam aperture: 32°

Applicazioni tipiche “small animal TAC” Organo malattia - ossa - Denti - Vasi sanguigni - tumori Campione/animale Biopsie Tessuti Piccoli animali (ratti / topi) in vitro e in vivo 3 mm 0.75 mm 40 mm vertebra 40 kVp, 1 mm Al, High-Speed continuous rotation protocol (5’ 00”) 500 views, full-scan, magnificazione 4x. Binning 2x2 Immagini ottenute con il prototipo dell’Università di Pisa

Conclusions Our experience with the YAP-(S)PET II indicates that its spatial resolution and sensitivity are adequate for molecular imaging investigation in both PET and SPECT modalities. The good image uniformity and linearity permit quantitative studies once the partial volume effect has been taken into account. The availability of both emission techniques on the same gantry allows multimodality study in a very easy and effective way. The future installation of an integrated CT will be a critical improvement for a better visualization of anatomical repere, attenuation correction and morphological characterization.

Acknowledgements #1 - FIIG Functional Imaging and Instrumentation Group Department of Physics”E.Fermi” University of Pisa, Pisa, Italy Francesca Attanasi (PhD student) Antonietta Bartoli (PhD student) Nicola Belcari (Res Assistant) Valter Bencivelli (Ass Professor) Laura Biagi (Post-doc) Maria G. Bisogni (Res Assistant) Manuela Camarda(PhD Student) Serena Fabbri (PhD Student) Alberto Del Guerra (Full Professor) Sebnem Erturk (PhD Student) Judy Fogli (PhD student) Gabriela Llosá (Marie Curie Fellow) Sara Marcatili (PhD Student) Sascha Moehrs (Post-Doc) Daniele Panetta (PhD Student) Michela Tosetti (Researcher) Valeria Rosso (Associate Professor) Sara Vecchio (PhD Student)

Acknowledgments #2 In alphabetical order: AdAcAp / Oncodesign (Dijon) Centro di Eccellenza AmbiSEN, University of PISA Istituto di Fisiologia Clinica del CNR, Pisa (Prof. Luigi Donato) ISE – Ingegneria dei Sistemi Elettronici, Pisa Ospedale S. Raffaele, Milano (Prof. F. Fazio) University of Ferrara University of Mainz (Prof. Frank Roesch) University of Pisa (dept of Endocrinology, dept of Nuclear Medicine) University of Napoli “Federico II” (Prof. Marco Salvatore) University Hospital Geneva (Prof. P. Millet) EMIL (European Molecular Imaging Laboratory) [FP6 NoE]

Thank you!