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UNIVERSITY OF HELSINKI REPORT SERIES IN PHYSICS

HU-P-D171

Proton magnetic resonance spectroscopy

of a boron neutron capture therapy 10B-carrier,

L-p-boronophenylalanine-fructose complex

Marjut Timonen

Department of PhysicsFaculty of Science

University of HelsinkiHelsinki, Finland

HUS Helsinki Medical Imaging CenterUniversity of Helsinki

Helsinki, Finland

ACADEMIC DISSERTATION

To be presented, with the permission ofthe Faculty of Science of the University of Helsinki,

for public examination in Auditorium D101,Physicum, Gustav Hällströmin katu 2 A

on January 22th, 2010, at 12 o’clock noon

Helsinki 2010

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ISSN 0356-0961ISBN 978-952-10-5648-2ISBN 978-952-10-5649-9 (pdf version)

Helsinki University PrintHelsinki 2010

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M. Timonen Proton magnetic resonance spectroscopy of a boron neutron capture therapy10B-carrier, L-p-boronophenylalanine-fructose complex, University of Helsinki, 2010,49p. + appendices, University of Helsinki, Report Series in Physics, HU-P-D171, ISSN0356-0961, ISBN 978-952-10-5648-2, ISBN 978-952-10-5649-9 (pdf version)

Classification (INSPEC): A8760I, A8760M, A8770E, A8770HKeywords: medical physics, 1H MRS, MRSI, BNCT, BPA-F

Abstract

Boron neutron capture therapy (BNCT) is a radiotherapy that has mainly been used to treatmalignant brain tumours, melanomas, and head and neck cancer. In BNCT, the patientreceives an intravenous infusion of a 10B carrier, which accumulates in the tumour area.The tumour is irradiated with epithermal or thermal neutrons, which result in a boronneutron capture reaction 10B(n,α)7Li. This generates heavy particles that damage tumourcells. In Finland, boronophenylalanine fructose (BPA-F) is used as the 10B-carrier. Theboron neutron capture reaction is responsible for the main therapeutic dose in BNCT, andtherefore all information about boron accumulation in the tumour area is valuable.Currently, the drifting of boron from blood to tumour as well as the spatial and temporalaccumulation of boron in the brain, are not precisely known. The BNCT treatmentplanning in Finland is based on estimations of boron concentrations in tumour and normalbrain. These concentratins, in turn, are based on whole blood boron concentration, andassumption of constant boron concentration ratios for blood-to-tumour (1:3.5) and blood-to-normal brain (1:1). During the irradiation, the whole blood boron concentration isestimated with kinetic models from the whole blood boron concentrations of previouslycollected periodic blood samples, where boron concentration is determined withinductively coupled plasma-atomic emission spectrometry (ICP-AES). However, usingthese constant concentration ratios of blood-to-tumour or blood-to-normal brain producessignificant uncertainties in estimating of the boron concentration in the normal brain andtumour. Therefore, new methods would be needed to determine the boron concentration inthe brain non-invasively. Proton magnetic resonance spectroscopy (1H MRS) could beused for selective BPA-F detection and quantification as aromatic protons of BPAresonate in the spectral region, which is clear of brain metabolite signals.

This study, which included both phantom and in vivo studies, examined the validity ofMRS as a tool for BPA detection. In the phantom study, BPA quantification was studied at1.5 and 3.0 T with single voxel 1H MRS, and at 1.5 T with magnetic resonance imaging(MRSI). The J-modulation of a coupled spin system of BPA aromatic protons complicatesthe determination of transverse relaxation time as well as the intensity measurement of theBPA signal when uncoupled resonance is used as the concentration reference. Therefore,the response curve of aromatic protons to the PRESS sequence was calculated and theresults were used for T2 determination and intensity corrections. The detection limit ofBPA was determined in phantom conditions at 1.5 T and 3.0 T with two different voxel

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sizes using single voxel 1H MRS, and at 1.5 T using MRSI. The gross tumour volume(GTV) may contain a resection cavity with clotted blood. The adverse effects of clottedblood on MRS were demonstrated with whole blood and blood plasma phantoms dopedwith BPA-F. In phantom conditions, BPA quantification accuracies of ± 5% and ± 15%were achieved with single voxel MRS using external or internal (internal water signal)concentration references, respectively. For MRSI, a quantification accuracy of <5% wasobtained using an internal concentration reference (creatine). The detection limits of BPAin phantom conditions for the PRESS sequence were 0.7 (3.0 T) and 1.4 mM (1.5 T) with203 mm3 single voxel MRS, 1.4 (3.0 T) and 2.2 mM (1.5 T) with 1.53 mm3 single voxelMRS, and 1.0 mM with acquisition-weighted MRSI (nominal voxel volume 10(RL) ×10(AP) × 7.5(SI) mm3), respectively. The blood-originated interference was prominent inthe whole blood/BPA-F spectrum, indicating that there are many challenges to BPAdetection in in vivo conditions, such as susceptibility effects caused by clotted blood in theresection cavities of operated tumours.

In the in vivo study, an MRSI or single voxel MRS or both were performed for tenpatients (patients 1-10) on the day of BNCT. Three patients had glioblastoma multiforme(GBM), and five patients had a recurrent or progressing GBM or anaplastic astrocytomagradus III, and two patients had head and neck cancer. For nine patients (patients 1-9),MRS/MRSI was performed 70-140 min after the second irradiation field, and for onepatient (patient 10), the MRSI study began 11 min before the end of the BPA-F infusionand ended 6 min after the end of the infusion. In comparison, single voxel MRS wasperformed before BNCT, for two patients (patients 3 and 9), and for one patient (patient9), MRSI was performed one month after treatment. For one patient (patient 10), MRSIwas performed four days before infusion. Signals in the tumour spectrum aromatic regionwere detected on the day of BNCT in three patients, indicating that in favourable cases, itis possible to detect BPA in vivo in the patient’s brain after BNCT treatment or at the endof BPA-F infusion. However, because the shape and position of the detected signals didnot exactly match the BPA spectrum detected in the in vitro conditions, unambiguousassignment of BPA is difficult. The opportunity to perform MRS immediately after theend of BPA-F infusion for more patients is necessary to evaluate the suitability of 1H MRSfor BPA detection or quantification for treatment planning purposes. However, it could bepossible to use MRSI as criteria in selecting patients for BNCT.

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Contents

Abstract 3

Contents 5

List of original publications and statement of involvement 7

Abbreviations 8

1. INTRODUCTION 10

1.1. Boron neutron capture therapy 10

1.2. Magnetic resonance spectroscopy 12

2. AIMS OF THE STUDY 16

3. MAGNETIC RESONANCE SPECTROSCOPY OFBORONOPHENYLALANINE FRUCTOSE 17

3.1 Localisation schemes of MRS 17

3.2 Quantification of metabolites in MRS 19

3.3 1H MRS of boronophenylalanine fructose 20

4. MATERIALS AND METHODS 22

4.1 Phantom studies 22

4.1.1. Phantoms 22

4.2.1. MR hardware and pulse sequences 22

4.2.2. Simulation studies 22

4.2.3. Relaxation times 23

4.2.4. Quantification 23

4.2.5. Detection limits 23

4.2.6. Effects of blood on 1H MRS quality 24

4.2 In vivo studies 24

4.2.1 Patients 24

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4.2.2. MRS/MRSI EXPERIMENTAL SETUP 24

5. RESULTS 28

5.1 Phantom studies: relaxation times, detection limits and quantification of BPA 28

5.2 Patient study: blood boron concentrations and in vivo BPA detection in thebrain 30

6. DISCUSSION 38

7. CONCLUSIONS 41

ACKNOWLEDGEMENTS 42

REFERENCES 44

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List of original publications and statement ofinvolvement

I Heikkinen S, Kangasmäki A, Timonen M, Kankaanranta L, Häkkinen AM,Lundbom N, Vähätalo J and Savolainen S. 1H MRS of a boron neutron capturetherapy 10B-carrier, L-p-boronophenylalanine-fructose complex, BPA-F: phantomstudies at 1.5 and 3.0 T. Phys Med Biol. 48 1027-1039 2003

II Timonen M, Kangasmäki A, Savolainen S and Heikkinen S. 1H MRS phantomstudies of BNCT 10B-carrier, BPA-F using STEAM and PRESS MRS sequences:Detection limit and quantification. Spectrosc-Int 18 133-142 2004

III Timonen M, Kankaanranta L, Lundbom N, Collan J, Kangasmäki A, KortesniemiM, Häkkinen AM, Lönngren A, Karjalainen S, Rasilainen M, Leinonen J, Huitti T,Jääskeläinen J, Kouri M, Savolainen S and Heikkinen S. 1H MRS studies in theFinnish boron neutron capture therapy project: detection of 10B-carrier, L-p-boronophenylalanine-fructose. Eur J Radiol. 56 154-159 2005

IV Timonen M, Savolainen S, and Heikkinen S. BPA quantification and detection inphantoms using three dimensional 1H magnetic resonance spectroscopic imaging.IFMBE Proc. 9 211-212 2005

V Timonen M, Kankaanranta L, Lundbom N, Kortesniemi M, Seppälä T, Kouri M,Savolainen S, and Heikkinen S. Acquisition-weighted MRSI for Detection andQuantification of BNCT 10B-carrier L-p-boronophenylalanine-fructose Complex, aPhantom Study. J Radiat Res 50 435-440 2009

All publications included in this thesis are the results of a group effort. In Study I, theauthor participated in the magnetic resonance spectroscopy measurements and analysis,critically revised the article and approved the final version to be published. In Studies II-V, the author participated in the study design, and performed the MRS/MRSImeasurements and data analysis, and wrote the articles.

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Abbreviations

BNCT Boron neutron capture therapyBPA-F Boronophenylalanine-fructoseBSH Sodium borocaptateCHESS Chemical shift selective imagingCho CholineDEFT Driven equilibrium Fourier transformFOV Field of viewFWHM Full width at half maximumGlc GlucoseGln GlutamineGlu GlutamateGlx Glutamate plus glutamineGTV Gross tumour volumeICP-AES Inductively coupled plasma-atomic emission spectrometerICRU International Commission of Radiation Units and MeasurementsIR Inversion recoveryISIS Image Selected In Vivo SpectroscopyLac LactateLASER Localisation by Adiabatic Selective RefocusingmI Myo-inositolMM MacromoleculeMRI Magnetic resonance imagingMRS Magnetic resonance spectroscopyMRSI Magnetic resonance spectroscopy imagingNAA N-acetylaspartateNMR Nuclear magnetic resonanceNRG Nuclear research & consultancy groupPEPSI Proton echo planar spectroscopic imagingPET Positron emission tomographyppm Parts per millionPRESS Point Resolved SpectroscopyPSF Point spread functionPGS Prompt gamma spectroscopySE Spin echosI Scyllo-InositolSLIM Spectral localisation by imagingSLOOP Spectral localisation with optimal pointspread functionSNR Signal-to-noise ratioSTEAM Stimulated echo acquisition modetCr Total creatine (creatine + phoshpocreatine)TE Time of echo

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TM Mixing timeTR Time of repetitionTSI Turbo spectroscopic imagingVOI Volume of interestWEFT Water eliminated Fourier transform

In this work, the abbreviation ppm (parts per million) has been used in two differentcontexts. The chemical shift is expressed in units of ppm, as it is independent of themagnetic field strength used. In addition, the whole blood boron concentration isexpressed in ppm (mg/kg) instead of mg/L, or mmol/L, as is the widespread practiceamong the BNCT community.

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1. INTRODUCTION

1.1. Boron neutron capture therapy

Boron neutron capture therapy (BNCT) is a bimodal radiotherapy that has traditionallybeen used to treat malignant brain tumours and melanomas (Diaz et al. 2000). In recentyears, BNCT has also been used to treat recurring head and neck carcinoma with goodresponse (Kankaanranta et al. 2007, Kato et al. 2004, Kouri et al. 2004). In BNCT, thepatient receives an intravenous infusion of a 10B carrier compound. As a result, boronaccumulates in the tumour area. After an appropriate time interval, when the boronconcentration in the tumour area has increased sufficiently relative to the surroundinghealthy tissue, the tumour is irradiated with epithermal or thermal neutrons. The boronneutron capture reaction 10B(n, α)7Li generates heavy particles that damage the tumourcells. The idea of clinical BNCT was illustrated for the first time in 1936 (Locher). Thefirst pioneering trials of BNCT were performed in the USA during 1951-1961 withunsatisfactory treatment results and adverse effects (Busse et al. 2001). The major reasonfor the disapointing results of the very first BNCT trials was the use of inadequate boron-carrier compounds and undeveloped thermal neutron generating facilities (Busse et al.2001). Subsequent trials in Japan were more promising (Nakagawa 2001). Clinical trialsbegan again in Europe and the US in late 1980s with boronophenylalaline (BPA) andsodium borocaptate (BSH) as boron carriers. In Finland, clinical trials began in 1999(Joensuu et al. 2003) with boronphenylalanine fructose (BPA-F).

The boron neutron capture reaction is responsible for the main therapeutic dose inBNCT, but the total biological effect is the sum of all dose components. The thermalneutrons also react with hydrogen 1H(n, )2H and nitrogen 14N(n,p)14C* in the tissue,producing a significant partion of the total patient dose. In addition, the reactor producesfast neutrons and gamma radiation with an energy range of 0-15 MeV. To estimate thebiological response of the tissue to the combination of all these doses, the concept ofweighted dose is used. The total biologically weighted dose DW is a sum of physical dosecomponents (Di) multiplied by weighting factors (wi) of each dose component in a tissue.The biologically weighted dose can be written (IAEA 2001):

(1) DW= wgDg + wBDB + wNDN + wnDn,

where Dg is the gamma dose, DB the boron capture dose, DN the nitrogen capture doseand Dn the dose from epithermal and fast neutrons. The unit for the biologically weightedtotal dose is Gy (W) where the W in parenthesis is used to emphasise the weightingwhereas the physical dose components are expressed in Gy unit. Detailed physicalquantities for BNCT dosimetry have previously been presented in doctoral theses by A.Kosunen (1999), M. Kortesniemi (2002), P. Ryynänen (2002), T. Seppälä (2002), P.

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Kotiluoto (2007) and J. Uusi-Simola (2009). The weighted boron dose was approximately90% of the total weighted dose for a tumour and 45% for a normal brain at the dosemaximum, according to a sample BNCT dose plan presented in the thesis by Seppälä(2002). The average planning target volume doses varied from 25 to 61 Gy (W) in theclinical trials for glioblastoma multiforme patients at the Finnish BNCT facility reportedby Joensuu et al. (2003). In BNCT of locally recurred head and neck cancer reported byKankaanranta et al. (2007), the median average tumor dose delivered to the gross tumorvolume during the first scheduled BNCT was 21 Gy (W) (range, 14–29 Gy [W]) andduring the second BNCT 20 Gy (W) (range, 15–24 Gy [W]). An average of 91% of thesetumor doses resulted from the boron neutron capture reactions.

According to ICRU (International Commission of Radiation Units and Measurements)guidelines, the patient dose uncertainty in external radiotherapy should not exceed 5%(ICRU 26). In BNCT, the uncertainty of the computed absorbed dose has been estimatedto be 20% (1 SD, standard deviation) in the normal brain (Seppälä 2002). The uncertaintyevaluated in estimating the boron concentration in normal tissue is 30% (Kortesniemi2002), which is the most important source of inaccuracies in BNCT dose computation.With no boron dose uncertainty, the uncertainty of the computed absorbed total dosewould be 8% (1 SD) (Seppälä 2002). It should be also taken into account that the 30%uncertainty (Kortesniemi 2002) in the boron concentration in normal tissue is estimatedusing the average dispersion of the boron concentration determined in brain tissue samplesby a limited number of patients (Coderre et al. 1998, Elowitz et al. 1998). The trueuncertainty of the boron concentration in the brain is unknown, and the uncertainty of theboron dose in the normal brain could therefore be even higher than the 30% reported. Themajority of European research groups involved in the development of BNCT haveprepared the report “Recommendations for the Dosimetry of Boron Neutron CaptureTherapy (BNCT)” published by the Nuclear Research & Consultancy Group (NRG). Thereport covers the guidelines for the basic characterisation and dosimetry of the epithermal(and thermal) neutron beams used in BNCT (Voorbraak and Järvinen 2003). However,this report includes no guidelines determining boron concentrations or calculations due tothe boron neutron capture reaction.

At the moment, the drifting of boron from blood to tumour and the spatial andtemporal accumulation of boron in the brain are not exactly known. In Finnish clinicalBNCT practice, the whole blood boron concentration is routinely determined during andafter BPA-F infusion from periodic blood samples using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Coderre and Morris 1999, Laakso et al. 2001,Kortesniemi 2002, Kortesniemi et al. 2004, Linko et al. 2008). The whole blood boronconcentration during irradiation is calculated from previously determined blood boronconcentrations using kinetic models (Kortesniemi 2002, Ryynänen 2002). The boronconcentration in the tumour and brain tissue during the irradiation are estimated usingsimple blood-to-brain and blood-to-tumour ratio values: 1:1 for blood-to-brain, and 1:3.5for blood-to-tumour (Coderre et al. 1998). In the Finnish BNCT of brain tumours, neutronirradiation was started approximately 45 min after the completion of infusion (Joensuu etal. 2003). The blood-to-brain and blood-to-tumour ratios used in Finland and in commonBNCT clinical practice are based on a study by Coderre et al. (1998). Coderre et al.

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(1998) and Elowitz et al. (1998) measured boron concentration invasively in surgicalsamples after BPA-F infusion. In patients with suspected or confirmed glioblastomamultiforme, the results of the histological findings indicated 1.4 (Elowitz et al. 1998) to 4(Coderre et al. 1998) mean blood-to-tumour boron concentration ratios as well as highvariation in the blood-to-tumour ratio inside particular tumours (Elowitz et al. 1998). Newnon-invasive methods are therefore needed to determine or monitor boron concentrationsin the brain.

Several studies have examined, the suitability of positron emission tomography (PET)for treatment planning purposes (Aihara et al. 2006, Imahori et al. 1998a, 1998b, 1998c,Nichols et al. 2002). In PET, the 18F-labelled BPA is detected in the brain. In Finland, 18FBPA PET studies have been used to study the suitability of patients, i.e. the accumulationof boron carrier for BNCT (Kankaanranta et al. 2007). Prompt gamma spectroscopy(PGS) has also been investigated for boron determination (Verbakel 2001, Verbakel et al.2003, Munck af Rosenschöld et al. 2001, Wittig et al. 2008). In addition, Bergenheim etal. (2005) have used microdialysis to study BPA distribution in glioblastoma patientsduring BNCT treatment. Several magnetic resonance techniques have been proposed forboron carrier detection. 11B or 10B magnetic resonance imaging (MRI) and MRspectroscopic methods have been reported for boron detection (Bendel et al. 1994, Bendeland Sauerwein 2001, Campanella et al. 2002, Kabalka et al. 1997). Recently, 19F magneticresonance spectroscopy (MRS) has also been used to detect 19F-labelled BPA (Porcari etal. 2008, 2009). Zuo et al. (1999) have proposed the possibility to apply clinically widelyused proton MR spectroscopy (1H MRS) for selective BPA-F detection and they detectedaromatic signals of BPA in vivo at 1.5 T. Later, Bendel et al. (2005a, 2005b) studied theMRS imaging (MRSI) of BPA-F with animals at high magnetic field.

1.2. Magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy (abbreviated as MRS in clinicalsciences) is a non-invasive tool to study tissue metabolism in vivo in a clinicalenvironment. Nowadays, MRS is used to investigate several diseases. The most commonapplication of 1H MRS in medicine is the investigation of neurological diseases(Danielsen and Ross 1999, Lin et al. 2005) such as brain tumours, epilepsy, stroke andneonatal hypoxia. 1H MRS can also applied in other body areas such as liver (Fischbachand Bruhn 2008), muscle (Boesch et al. 2006) and heart (Horn 2006). Phosphorus MRS(31P MRS) can study energy metabolism and has been used clinically in severalapplications as on the heart (Horn 2006), liver (Solga et al. 2005) and muscle (Mattei et al.2004). In addition, other nuclei - primarily carbon (13C), fluorine (19F) and sodium (23Na) -have been used in MRS, but mainly for research purposes (Babsky et al. 2008, Klomp etal. 2007, Kurhanewicz et al. 2008, Procissi et al. 2007).

The brain metabolites N-acetylaspartate (NAA), total creatine (creatine +phoshpocreatine; tCr), total choline (resonance composed mainly of choline-containingphospholipids; Cho) and lactate (Lac) are commonly monitored in in vivo 1H MRS duringthe investigation of neurological diseases. NAA is a neuronal amino acid that typically

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decreases in tumours and degenerative and inflammatory brain diseases (Danielsen andRoss 1999, Ross and Bluml 2001). Choline-containing phospholipids in membranes arereleased during active myelin breakdown or rapid cell proliferation (Danielsen and Ross1999). Therefore, increased Cho signal is detected in certain tumours during activeprocesses. Total creatine (tCr) is the sum of the creatine (Cr) and phosphocreatine (PCr)signals and is related to brain energy metabolism (Danielsen and Ross 1999, Ross andBluml 2001). The intensity of the tCr signal remains relatively constant and resistant tochanges, but major changes appear in destructive pathology, such as malignant tumours(Danielsen and Ross 1999). Lactate, a marker of anaerobic metabolism, is an end-productof oxidative metabolism (Danielsen and Ross 1999). Elevated lactate signals can bedetected after hypoxia (as in stroke), but also in many neurological diseases such astumours, Canavan disease, and intracerebral hemorrhage (Danielsen and Ross 1999).Several other metabolites, such as myo-inositol (mI), scyllo-Inositol (sI), Glucose (Glc),Glutamate (Glu), and Glutamine (Gln), can also be detected in the brain 1H MR spectrum(Ross and Bluml 2001).

The transverse relaxations times (T2) of different brain metabolite protons vary (Frahmet al. 1989, Mlynárik et al. 2001). In a typical clinical MRS study, the echo time (TE) ischosen according to the metabolites of interest. TE values of 20 to 40, 144 or 288 ms aretypical in the clinical setting. In the so-called “short TE spectra”, a TE value of 20 to 40ms is used, and several metabolites can be detected in the spectrum (Figure 1). In the“long TE spectra”, fewer signals can be detected (NAA, Cho, Cr, lac), and the spectrumbaseline is flat, as macromolecule and lipid signals have essentially decayed to noise level.

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Figure 1 A 3.0 T single voxel 1H MR spectrum of brain white matter (TE=37 ms and TR=2000ms) of a healthy adult. N-acetylaspartate (NAA), total creatine (tCr), total choline (Cho), myo-inositol (mI), glutamate plus glutamine (Glx), lactate (Lac), lipid (Lip) and macromolecule (MM)signals appear in the spectrum.

The protons of NMR-visible brain metabolites resonate mainly at a chemical shiftrange of 1 to 4 ppm (parts per million). This is the spectral region normally used forclinical studies. The protons of water molecules cause a major resonance at 4.7 ppm. Thewater signal is usually suppressed during the MRS sequence to avoid any overlap withlow-intensity metabolite signals and a distorted baseline, as the water concentration in thebrain is approximately 10 000–fold higher than the concentration of brain metabolites. Inadult white matter, the concentrations are approximately 6-9 mM for NAA, 1-2 mM forCho, and 5.2-5.7 mM for Cr (Frahm et al. 1989, Pan et al. 1998, Pouwels and Frahm1998, Tan et al. 1998, Wang and Li 1998, Hetherington et al. 1994, Graaf et al. 2007).

Aromatic protons resonate at 7 to 9 ppm. Only a few brain metabolite signals appear in“the aromatic region” of the 1H MRS spectrum (Aru`s et al. 1985, Gadian et al. 1986,Kreis et al. 1995, Mori et al. 1998, Rothman et al. 1997, Shungu and Glickson 1993).

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Exchangeable NAA amide protons resonate at ~7.9 ppm, the exchangeable amino protonsof Glu and protein at 6.8 ppm, and Cr at 6.71 ppm. In addition, ATP and nucleosidesresonate at 8.45 ppm and 8.22 ppm, homocarnosine at 8.00 and 7.04 ppm, and tryptophanat 7.29 ppm. However, the intensities of these signals are usually too low to be detectedwith typical measurement settings (voxel size and measurement duration). Therefore, thisspectral range is normally “clean of 1H signals” and is not used in clinical practice. Zuo etal. (1999) reported that aromatic signals of BPA at 7.0 to 7.5 ppm can be detected in vivousing 1H MRS. In theory, this offers much needed possibilities to study the spatial andtemporal distribution and concentration of BPA in the brain non-invasively. The aromaticproton signals of BPA would be ideal for BPA detection because normal brain metabolitesignals do not overlap with them. Bendel et al. (2005b) have optimised MRS and MRSIsequences for BPA detection, but have performed no studies on humans. Bendel et al.(2001) have studied 1H MRS detection of another clinically used 10B-carrier, BSH. Theproblem with 1H MRS detection is, however, that the broad signal of BSH protonscoincides almost precisely with those of lipid molecules.

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2. AIMS OF THE STUDY

The goal of this thesis was to

1. study BPA quantification with phantoms at 1.5 and 3.0 T using single voxel 1H MRS,and at 1.5 T using acquisition-weighted 1H MRSI (Papers I, IV and V).

2. determine the detection limit of BPA in phantom conditions at 1.5 T and 3.0 T usingsingle voxel 1H MRS and two different voxel sizes, and at 1.5 T using acquisition-weighted 1H MRSI (Papers II and V).

3. evaluate the validity of 1H MRS as a clinical tool for BNCT treatment planning (PaperIII and the thesis).

4. evaluate the clinical feasibility of 1H MRS-based BPA detection in BNCT.

This thesis presents the results of in vivo BPA-F 1H MRS measurements, includingpreviously unpublished data.

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3. MAGNETIC RESONANCE SPECTROSCOPY OFBORONOPHENYLALANINE FRUCTOSE

3.1 Localisation schemes of MRS

In MRS, the localisation of voxel is based on B0 magnetic field gradients and frequencyselective RF pulses. Point Resolved Spectroscopy (PRESS) (Bottomley 1987) andStimulated Echo Acquisition Mode (STEAM) (Frahm et al. 1987) are commonly usedlocalisation methods in clinical 1H MRS. PRESS uses one 90° and two 180° slice selectiveRF pulses (Figure 2), whereas STEAM is based on three 90° slice selective RF pulses. Useof the basic Spin Echo (SE) localisation method containing a 90° followed by one 180° RFpulse is also possible. However, in SE method, volume selection is possible only in twodimensions as a slice or a bar. Image Selected in vivo Spectroscopy (ISIS) is typically usedin 31P MRS studies (Ordidge et al. 1986). Localisation by Adiabatic Selective Refocusing(LASER) is a localisation scheme that utilizes adiabatic excitation and refocusing RFpulses (Garwood and DelaBarre 2001, Kinchesh and Ordidge 2005). The advantages ofLASER include improved tolerance to chemical shift artefacts and the effects of J-coupling in a coupled spin system. In neurological 1H MRS studies, frequency selectiveexcitation methods such as CHemical Shift Selective (CHESS) imaging (Haase et al.1985) are typically used for water suppression. Other water suppression techniques,include Water Eliminated Fourier Transform (WEFT) (Gupta 1976) or Driven EquilibriumFourier Transform method, which utilise differences in relaxation parameters (DEFT)(Becker et al. 1969).

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Figure 2 Schematic representation of the PRESS sequence.The timing of the 90° and 180°slice selective RF pulses (RF) and the three orthogonal gradients (Gx, Gy, Gz) are shown. Twopairs of crusher gradients surrounding 180° slice selective refocusing pulses ensure the selectionof the desired coherences, while simultaneously destroying all others.

Analoguously to MRI, in MRSI, phase encoding is used to collect spectral datacovering two- or tree-dimensional volume (i.e. one large volume is "divided" into severalsmaller voxels, each containing an MRS spectrum) (Brown et al. 1982, Maudsley et al.1983). MRSI is technically more challenging than single voxel MRS; the volumedefinition is less optimal, susceptibility to lipid contamination is higher, shimming thevolume of interest (VOI) can be difficult and the measurement duration can be relativelylong. In brain studies, an outer volume saturation and careful localisation of the VOI aretypically used for lipid suppression. In the body area, where the VOI can contain lipids,lipid magnetization can be destroyed using chemical shift selective pulses (as in CHESSwater suppression) or inversion-recovery (IR) methods.

The nominal voxel size of MRSI is defined by the field of view (FOV) divided by thenumber of phase encoding steps. In MRSI, however, due to few phase encoding samplingpoints, the point spread function (PSF) considerably influences the actual spatialresolution (Vikhoff-Baaz et al. 2001). To decrease the truncation artefact (Gibb’s ringingartefact) in spatial dimensions, the apodization function is usually applied before Fouriertransformation in the spatial dimensions. This, however, will widen the main peak andincrease the actual voxel size. Because the duration of a conventional MRSI acquisition isrelatively long, several fast MRSI methods have been developed. Conventional methods,such as circular or elliptical k-space sampling (Maudsley et al. 1994, Hugg et al. 1996),are usually available in clinical MR imagers. Some manufacturers also offer “weighted”acquisition methods (Pohmann and von Kienlin 2001) where k-space apodization isapplied already during the acquisition, thus requiring more than one average. In Proton

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Echo Planar Spectroscopic Imaging (PEPSI) (Mansfield 1984, Guilfoyle and Mansfield1985, Guilfoyle et al. 1989, Posse et al. 1994), various echoes are detected after a 180° RFpulse. As with turbo spin echo MRI, turbo spectroscopic imaging (TSI) uses extra 180°refocusing pulses to detect multiple echoes (Duyn and Moonen 1993). Nowadays, parallelimaging methods (Sodickson and Manning 1997, Pruessmann et al. 1999, Griswold et al.2002) are also available for clinical MRSI (Dydak et al. 2001, Dydak et al. 2003).Techniques such as Spectral Localisation by IMaging (SLIM) (Hu et al. 1988) or SpectralLocalisation with Optimal pOint Spread function (SLOOP) (Von Kielin and Mejia 1991)apply prior knowledge of the anatomy of the object to be measured in order to reduce themeasurement duration. It should be noted that in several fast MRSI methods, theshortening of measurement duration is achieved, at least, to some degree in cost of spatialresolution, signal-to-noise and/or spectral resolution.

3.2 Quantification of metabolites in MRS

In principle, MRS allows, determination of the absolute concentration of the metabolitesignal measured. However, various difficulties are involved in using MRS to reliablydetermine the absolute concentration (Graaf 2007). In quantification, a calibrationcompound with a known concentration must serve as a reference. Quantification can becarried out using either an external or an internal concentration reference. An internalwater signal commonly serves as the internal concentration reference. However, the watercontent of the tissue varies in pathology and between different tissue types (Graaf 2007).The external concentration references are based on separate phantom measurements, suchas those in the LCModel programme (Provencher 1993). The downside of the externalreference method is its sensitivity to various technical variables. In quantification,corrections are necessary due to differences in the T1 and T2 relaxation times of thereference and metabolite compounds (when TR is too short compared to the longest T1

and/or when long TE-values are used), diffusion, susceptibility, and also coil loading if aphantom replacement method is used. Relative concentrations (i.e. the intensity ratiobetween the NAA signals and the Cho signal) are commonly used in clinical practice (Linet al. 2005). The corrected intensity of metabolite signal Sm can be determined by (Graaf2007)

(2) Sm=SmmCT1,mCT2,mCnOe,mCADC,mCJ,mCloc,mCrf,m

where Smm is the intensity of the metabolite signal measured, and CT1,m CT2,m CnOe,m

CADC,m CJ,m Cloc,m and Crf,m are the correction factors of signals measured for partialsaturation due to incomplete T1 relaxation, T2 relaxation, nuclear Overhauser effects,diffusion, amplitude and phase modulation due to J-coupling, deviations from the ideallocalisation profile, amplitude and phase distortions due to specific RF pulse combinationssuch as binominal RF pulse, respectively. The corrected intensity of reference signal Sref

can be expressed by (Graaf 2007)

20

(3) Sref=SrmCT1,rCT2,rCnOe,rCADCrCJ,rCloc,rCrf,r

where Srm is the intensity of the reference signal measured, and CT1,r CT2,r CnOe,r CADCr

CJ,r Cloc,r and Crf,r are correction factors for the reference signal, as described in Eq. (2).

The absolute concentration [m] can be determined using equation (Graaf, 2007)

(4) [ ] gainavnref

m CCCrefSSm ][⋅

=

where Sm is the intensity of the corrected metabolite signal, Sfer is the intensity of thecorrected reference signal, [ref] is the concentration of the reference compound, Cn is thecorrection for the number of equivalent nuclei for each resonance, Cav is the correction forthe number of averages and Cgain is the correction for the receiver gain settings.

In an uncoupled spin system, if the PRESS sequence is used, the MRS signal intensityI can be expressed as

(5)

−=

−−12

0 1 TTR

TTE

eeII

where I0 corresponds to the MRS signal intensity of the theoretical situation whereTE=0.

For the STEAM sequence, the MRS signal intensity I can be expressed as

(6) 112/

20 1 T

TMT

TETMTRTTE

eeeII−−−

−−

−=

where TM is the mixing time. The relaxation times T1 and T2 can be solved using Eq.(5) from the data recorded with various TE and TR values.

3.3 1H MRS of boronophenylalanine fructose

Boronophenylalanine is an amino acid, a modified phenylalanine with a boronic acidgroup, -B(OH)2, in para-position to the phenyl ring. Due to the limited water solubility ofpure BPA at physiological pH, BPA infusion is administrated as a fructose complex (Moriet al. 1989, Yoshino et al. 1989). The likely structure of a BPA-fructose complex (BPA-F,molecular weigth 351.3 g/mol) appears in Figure 3 (Shull et al. 2000). As an amino acidanalogue, BPA can accumulate actively into tumour cells (Nakagawa et al. 2007). BPA is

21

considired to distribute uniformly in both the nucleus and the cytoplasm (Coderre et al.1998). In addition, partial disruption of the blood-brain barrier (BBB) within the tumourlikely increases boron accumulation in tumour (Yang et al. 1997). The four aromaticprotons of BPA form a strongly coupled spin system. At 1.5 T, the aromatic proton spinsystem consists of two pairs of chemically equivalent protons, AA’BB’. At 3.0 T, the spinsystem can be notated as AA’XX’ because the difference between resonance frequenciesis significantly higher than the coupling constant.

The coupled spin system causes difficulties in MRS when typical clinical MRSsequences are used (i.e. with non-zero TE). The phases and amplitudes of the signal aremodulated with changing TE. Therefore, the T2 determination is not so straightforward,and the signal intensity follows no simple exponential decay as a function of TE accordingEqs. (5) or (6).

O

O

NH3

B

OO

O

O

OHHO

Figure 3 The likely structure of a BPA-fructose complex in water at physiological pH (Shull etal. 2000).

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4. MATERIALS AND METHODS

4.1 Phantom studies

4.1.1. Phantoms

The phantoms contained aqueous BPA-F solutions with various BPA concentrations(details in Papers I, II, III, IV and V) and tissue modelling firm 2% agarose gel (IndubioseA37, BioSepra) with a BPA concentration of 7.2 mM (Paper I). To study the effects ofblood on 1H MRS quality, BPA-F phantoms containing whole blood and blood plasma,both with BPA concentration of 4.6 mM, were prepared (Paper III). In addition, wholeblood phantom with 100 mg of absorbable hemostat (Surgicel Fibrillar, oxidisedregenerated cellulose, Ethicon, Inc.) and blood plasma phantom were used. In order toincrease the coil loading to the level typically encountered in human MRS studies, a flaskcontaining BPA-F solution (or agarose gel with BPA-F) was placed in a spherical 4500 mlplastic phantom filled with 0.1 mM MnCl2-solution doped with NaCl (0.4%).

4.2.1. MR hardware and pulse sequences

The MRS and MRSI were performed with clinical 1.5 T and 3.0 T MR imagers (GE SignaHorizon LX EchoSpeed, GE Medical Systems, Fremont, GA, USA) (Papers I and II) anda 1.5 T MR imager (Siemens Magnetom Sonata, Siemens Medical Systems, Erlangen,Germany) (Papers III, IV and V). PRESS (Papers I, II, III, IV and V) and STEAM (PaperII) localisation sequences were used with the CHESS water suppression scheme.

4.2.2. Simulation studies

A Virtual NMR Spectrometer simulation program (Nicholas et al. 2000) was used tocalculate the response curves of the BPA aromatic proton spin system to the PRESS(Paper I) and STEAM (Paper II) sequences at 1.5 and 3.0 T. The response curvescalculated with various TE values (details in Papers I and II) served in T2 determinationusing Eq.

(7) 20)(

TTE

TEevolJTE eMMM−

−=

where MJ-evol(TE) is the calculated intensity at a given TE. This term can also serve tocorrect for J-evolution effects when an uncoupled resonance serves as a concentration

23

reference. In this case, the intensity correction factor due to J-evolution dTE can becalculated using Eq.

(8))(

)0(

TEevoJ

evolJTE M

Md

−

−=

where MJ-evol(0) is the intensity calculated at TE of 0 ms and MJ-evol(TE) is the intensitycalculated for the TE used in the measurement.

4.2.3. Relaxation times

The relaxation times T1 and T2 were determined for BPA-F in aqueous solution andagarose gel at 1.5 and 3.0 T (Papers I and II). In T1 determination, the MRS measurementswere carried out with various TR values while keeping TE constant and Eq. (5) (and Eq.(6) for the STEAM sequence in Paper II) was fitted to the signal intensities measured. T2

was determined by fitting Eq. (7) to the intensities measured with various TE. In addition,the T1 and T2 relaxation times of water in aqueous BPA-F phantom were determined usingEqs. (5) and (6), respectively.

4.2.4. Quantification

BPA quantification was studied with BPA-F agarose gel and aqueous BPA-F phantomsusing single voxel MRS at 1.5 T and 3.0 T (Papers I and II). The quantification wascarried out using Eq. (4) with external and internal concentration references. Intensitycorrections for T1 and T2 relaxation and J-evolution were applied. For comparison,quantification was also done without intensity corrections.

In addition, BPA quantification was studied using MRSI at 1.5 T (Papers IV and V). Inthe MRSI study, Cr was added to aqueous BPA-F phantom as an internal concentrationreference. For comparison, quantification was also done using single voxel MRS and thesame measurement duration used in the MRSI study (Papers IV and V). Intensitycorrections for T1 effects were performed, but not for T2 due to the relatively short TE.

4.2.5. Detection limits

The detection limit of BPA was evaluated in phantom conditions with a single voxel MRS(Paper II) and acquisition-weighted MRSI (Paper V). Single voxel MRS studies werecarried out at 1.5 T and 3.0 T using two different voxel volumes (15 mm3 and 20 mm3)and a clinically acceptable measurement duration (approximately 10 min, includingautomatic shimming and water suppression adjustments). The BPA detection limit, usingacquisition-weighted MRSI, was determined at 1.5 T using a ~17 min MRSI protocol with

24

a nominal spatial resolution of 10 (RL) x 10 (AP) x 7.5 (SI) mm3 (one-fold spatial zerofilling in the SI direction prior to Fourier transformation) (Paper V). An acquisition-weighted/elliptical scanning method with post-acquisition k-space filtering was used toreduce the measurement time significantly without losses in SNR.

4.2.6. Effects of blood on 1H MRS quality

Glioma patients usually undergo brain surgery prior to BNCT. Consequently, the grosstumour volume (GTV) often includes a resection cavity containing heamostatic agents andcoagulated blood that have detrimental effects on the MR spectrum quality. The blood-originated interference in the 1H MRS was studied with phantoms containing whole bloodand blood plasma with BPA-F, whole blood with absorbable haemostat, and blood plasma(Paper III).

4.2 In vivo studies

4.2.1 Patients

From December 2003 to February 2008, 1H MRS/MRSI was performed on the day ofBNCT for ten patients. Table 1 shows the patient age, sex and BNCT protocol. In BNCTprotocol P01, the patients exhibited newly diagnosed glioblastoma multiforme (GBM). Inprotocol P01, BNCT was given as the first postoperative radiation treatment. No otherconcomitant or adjuvant treatments were offered. In the BNCT protocol P03, patientsshowed recurrent or progressing GBM or anaplastic astrocytoma gradus III, and hadundergone prior cranial conventional external beam radiotherapy. Patients 2 and 3, whowere treated under the BNCT protocol HN, had inoperable, recurrent, histologicallyconfirmed head and neck cancer. Detailed criteria for protocols P01 and P03 appear in apaper by Joensuu et al. (2003), and for protocol HN in a paper by Kankaanranta et al.(2007).

All patients provided their written informed consent prior to the MRS. The study wasapproved by an institutional Ethics Committee (HUS40/E6/02) and the National Agencyfor Medicines, Finland.

4.2.2. MRS/MRSI EXPERIMENTAL SETUP

1H MRS and 3D 1H MRSI measurements were performed for nine patients with 1.5 T(Siemens Magnetom Sonata or Siemens Avanto, Erlangen, Germany), and 1H MRS forone patient with 3.0 T (Philips Intera Achieva, Best, Netherlands) MRI scanners. Astandard receiver birdcage head coil was used with a Siemens Magnetom Sonata, and a

25

transmit/receive head coil with a Philips Intera imager. Both transmit/receive (patient 9)and standard receive (patient 10) head coils were used with Siemens Avanto imager.PRESS localisation with a CHESS water suppression scheme was used. Spectral post-processing and lineshape fitting for patients 9 and 10 were carried out using the standardsoftware package of the MRI scanner (Siemens Syngo MMWP, VE23A).

3D MRSI studies were performed for patients 5 to 10 (Table 1) with 1.5 T MRIimagers using a TE of 30 ms and TR of 1500 ms. The number of averages was two, andthe number of phase encoding steps was 16 x 16 x 8. Weighted/elliptical sampling with a50% Hamming k-space post-acquisition filter was used. The VOI size was adjusted tocover the tumour and normal brain as much as possible, but also to avoid intracraniallipids and air cavities in of skull base. The total measurement duration of 3D MRSI was17 min. FOV and nominal voxel sizes appear in Table 1.

A single voxel MRS study was performed for patients 1 to 9. TE was 30 ms at 1.5 Tand 35 ms at 3.0 T measurements. TR was 1600 ms, except for patients 1 and 2, for whoma TR of 1500 ms was used. The nominal voxel size and number of averages appear in theTable 1.

MRS/MRSI studies were performed patients 1 to 9 after BNCT. In contrast, patients 3and 9 underwent single voxel MRS before BNCT alongside with treatment planning MRI.Patient 9 underwent a 3D MRSI one month after treatment with a one-month follow-upMRI. Patient 10 underwent a 3D MRSI during the infusion, begining 11 min before theend of the infusion and ending 6 min after the completion of the infusion. In contrast, 3DMRSI data were also collected alongside with treatment planning MRI four days beforethe infusion. The mean whole blood boron concentrations during the 1H MRSI/MRS(Table 5) were determined from the bi-exponential kinetic models based on the pre- andpost-MRSI whole blood samples analysed (Kortesniemi et al. 2004). The blood boronconcentration curves of patients 9 and 10, calculated using whole blood boronconcentrations measured by the ICP-AES (Laakso et al. 2001), appear in Figures 4 and 5.The timing of the irradiations and MRSI are indicated with dotted vertical lines.

Figure 4 The (ICP-AES) whole blood boron concentration (circles) measured from patient 9with estimated values (curve). The timing of irradiation is indicated with solid vertical lines andthe timing of MRSI with dotted vertical lines.

26

Figure 5 The (ICP-AES) whole blood boron concentration (circles) measured from patient 10with estimated values (curve). The timing of irradiation is indicated with solid vertical lines andthe timing of MRSI with dotted vertical lines.

27

Table 1. BNCT protocol and details of the MRS measurements of patients 1-10. In the BNCT protocol P01, the patients exhibited newly diagnosedglioblastoma multiforme (GBM), and BNCT was given as the first postoperative radiation treatment. In the BNCT protocol P03, patients showedrecurrent or progressing GBM or anaplastic strocytoma gradus III, and had undergone prior cranial conventional external beam radiotherapy. In theBNCT protocol HN, patients had inoperable, recurrent, histologically confirmed head and neck cancer.

Single Voxel MRS MRSIPt. Age Sex BNCT

prot.Field strength andMR imager

Vox. size cm3

(AP)x(RL)x(SI)NSA Nominal vox. size cm3

(AP)x(RL)x(SI)FOV cm3

(AP)x(RL)x(SI)1 65 F P01 1.5 T Sonata 1.8x1.8x1.8 304 - -2 44 M HN 1.5 T Sonata 2.0x2.0x2.0 256 - -3 61 M HN 1.5 T Sonata 1.8x1.8x1.8 304 - -4 42 F P03 3.0 T Achieva 2.0x2.0x2.0 176 - -5 35 M P03 1.5 T Sonata 1.7x1.5x1.5 304 1.0x1.0x0.75* 16x16x126 54 M P01 1.5 T Sonata 2.0x2.0x1.5 304 1.5x1.5x1.5 24x24x127 52 M P03 1.5 T Sonata 2.5x2.5x2.5 256 1.0x1.0x0.75* 16x16x128 65 M P01 1.5 T Sonata 1.8x2.4x1.7 304 1.0x1.0x0.75* 16x16x129 22 M P03 1.5T Avanto 2.0x1.8x2.0 128 1.1x1.1x0.75* 18x18x1210 48 M P03 1.5T Avanto - - 1.1x1.1x0.75* 18x18x12*one-fold spatial zero-filling in the SI direction

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5. RESULTS

5.1 Phantom studies: relaxation times, detection limits andquantification of BPA

The aromatic proton relaxation times of BPA in aqueous solution and 2% agarose gelphantom with a BPA concentration of 7.2 mM measured with PRESS sequence are shownin Table 2. The corresponding values for water appear in Papers I and II. The T2

determination of BPA in aqueous solution and water was carried out in both studiespresented in Papers I and II. The values shown in Table 2 are based on the study presentedin Paper I. The detection limits obtained for BPA at 1.5 and 3.0 T (Papers II and V) areshown in Table 3.

Table 2. The relaxation times of aromatic protons of BPA at 1.5 and 3.0 T.

BPA aqueous solution BPA agarose gel phantomT1(ms) T2(ms) T1(ms) T2(ms)

1.5 T 935 335 975 1803.0 T 785 225 870 175

Table 3. The detection limits for BPA at 1.5 and 3.0 T.

Detection limit (mM)Study Nominal voxel size

(mm3)1.5T 3.0T

Single voxel MRS 15x15x15 2.2 1.4Single voxel MRS 20x20x20 1.4 0.73D MSRI 10x10x7.5* 1.0 -*one-fold zero-filling in the SI direction

The BPA quantification results for aqueous BPA-F solutions and BPA-F agarose gelphantom (Papers I, II, IV and V) appear in Table 4, which shows the quantification resultsbased on an internal concentration reference. The results based on an externalconcentration reference appear in Paper I. The results of quantification based on theSTEAM sequence and corresponding quantification results without intensity corrections(relaxation and J-evolution effects) appear in Paper II.

29

Table 4. BPA quantification results for BPA aqueous solutions and BPA agarose gelphantom using the PRESS sequence and an internal concentration reference.

TruecBPA

(mM)

Measured cBPA

(mM)

MRS/MRSI Nominalvoxel size(mm3)

Phantom Concentrationreference

1.5 T 3.0 T

MRSI 10x10x7.5* BPA-F/Crsolution

Cr 3.0 2.89 -

SingleVoxel MRS

15×15×15 BPA-F/Crsolution

Cr 3.0 3.01 -

SingleVoxel MRS

18×18×18 BPA-F/Crsolution

Cr 3.0 3.00 -

SingleVoxel MRS

22×22×22 BPA-F/Crsolution

Cr 3.0 2.81 -

SingleVoxel MRS

20x20x20 aq BPA-Fsolution

internal water 5.8 6.3 5.9

SingleVoxel MRS

15x15x15 2% agarose gelwith BPA-F

internal water 7.2 8.5 7.7

SingleVoxel MRS

15x15x15 aq BPA-Fsolution

internal water 11.5 13.5 13.4

SingleVoxel MRS

20x20x20 aq BPA-Fsolution

internal water 23.1 - 23.2

*one-fold zero-filling in SI direction

Spectra measured from whole blood with 100 mg of absorbable hemostat, bloodplasma, whole blood and blood plasma doped with BPA-F (BPA concentration 4.6 mM)are presented in Figure 6. The blood-originated interference in the 1H MRS spectrum ofBPA is evident in spectrum D, which indicates that the presence of blood can obscure arelatively high BPA concentration. The small additional peaks located at 7.5–7.9 ppm inthe BPA aqueous solution spectra have increased intensities in the spectrum recorded fromthe blood plasma/BPA-F phantom (spectrum B), thus indicating possible changes in thestability of the BPA-F complex in the plasma environment.

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Figure 6 1.5 T 1H MR phantom spectra (Paper III). The spectrum from BPA-F aqueousphantom (A) with a BPA concentration of 23.1mM is approximately scaled to correspond to thesame BPA concentration (4.6 mM) as in blood plasma/BPA-F (spectrum B) and whole blood/BPA-F (spectrum D) phantoms. Spectra from blood plasma appear in (C) and whole blood in (E).

5.2 Patient study: blood boron concentrations and in vivo BPAdetection in the brain

The whole blood boron concentrations during the MRS measurements for patients 1 to 10appear in Table 5. The “Results of MRS” column indicates whether (+ or -) the signals(SNR>1) of the spectrum aromatic area were detected. The confirmation of aromaticsignals detection was done by visual inspection of the spectra (two independent viewersMT, SH). The criterion for a positive detection was the identification of the peak or peakpattern in the aromatic region (SNR>1). The results of the MRS/MRSI studies of patients1 and 2 have been published in Paper III.

31

Table 5. The estimated whole blood boron concentrations of patients 1 to 10 during MRS. The “BNCT protocol” column indicates the BNCTtreatment protocol of the patient: P01( newly diagnosed GBM, and BNCT was given as the first postoperative radiation treatment), P03 (recurrent orprogressing GBM or anaplastic strocytoma gradus III, and had undergone prior cranial conventional external beam radiotherapy) or HN ( inoperable,recurrent, histologically confirmed head and neck cancer). The “Results of MRS”column indicates whether (+ or -) the signals (SNR>1) in aromaticregion were detected in tumour on the day of BNCT. In the contralateral brain tissue, there where no detectable signals in the aromatic spectral region.

Pt. Age Sex BNCTprotocol

BPA-F infusion(mg BPA/kg)

Whole blood boronconcentration (mg/kg)during MRS

MRS timing (min)after the completionof BPA-F infusion

MRS timing(min) after thecompletion ofirradiation II

Results ofMRS

1 65 F P01 450 12 265 140 +2 44 M HN 400 14 170 125 -3 61 M HN 400 16 219 90 -4 42 F P03 450 16 203 69 -5 35 M P03 290 12 192 70 -6 54 M P01 500 19 204 89 -7 52 M P03 290 9 215 80 -8 65 M P01 500 25 216 80 -9 22 M P03 450 14 250 89 +10 48 M P03 450 38 ** ** +** MRSI study began 11 min before the completion of the infusion and ended 6 min after the end of the infusion

32

The tumour and contralateral brain tissue 3D MR spectra of patient 9 on the day ofBNCT (89 min after completion of the irradiation) (A and B) and one month after BNCT(E and F) appear in Figure 7. The spectra measured on the day of BNCT indicates that twosignals can be detected in the tumour spectrum. The spectra measured on the day of BNCTappear also without baseline correction (Figure 7 C and D). Figure 8 shows the tumourand contralateral brain tissue MRSI spectra of patient 10 before and during/after BPA-Finfusion (the MRSI of patient 10 began 11 min before completion of the infusion andended 6 min after completion of the infusion). For comparison, the spectrum of 3.0 mMBPA aqueous solution (with 10.1 mM creatine) appears in Figures 7 and 8. Figures 9 and10 show the metabolite maps based on the lineshape-fitting of the data from the aromaticregion area after BNCT (patient 9) or during/after BPA-F infusion (patient 10). Themetabolite maps indicate that aromatic signals were detected in several voxels in thetumour volumes of patients 9 and 10, whereas no signals were detected in the aromaticregion of the spectra recorded from normal brain tissue. Figure 11 presents “the metaboliteregion” (0.5 - 4.2 ppm) of spectra A and B presented in Figures 7 (above) and 8 (below),demonstrating the quality of the spectrum. The Full Width at Half Maximum (FWHM)Cho signal in the tumour spectrum was 3.9 Hz for patient 9 (Figure 11, spectrum at right,above) and 6.2 Hz for patient 10 (Figure 11, spectrum at right, below), respectively. InFigure 12, the spectrum from aqueous BPA-F/Cr solution (A) and the tumour spectra ofpatients 9 (B) and 10 (C) are shown again. The chemical shift scale is referenced withrespect to the creatine signal (3.03 ppm).

It would be interesting to comparing the results of our in vivo MRS study with PET,contrast enhanced MRI, radiation dose or the clinical outcome of the patients. However,the outcome of the BNCT treatment of these patients is still under analysis, except forpatient 2. The promising treatment results of patient 2 have been presented previously(Kouri et al 2004). However, we were not able to detect any characteristic BPA signals inthe brain spectra of patient 2.

33

Figure 7 The aromatic region of the 3D 1H MR spectra measured from tumour (A) andcontralateral brain tissue (B) after BNCT in patient 9. In spectra A and B, baseline correction hasbeen applied. The same data without baseline correction is shown in spectra C and D forcomparison. Two signals can be detected in the tumour spectrum (A). The tumour (E) andcontralateral brain tissue (F) spectra measured one month after BNCT and spectra 3.0 mM BPAaqueous solution (with 10.1mM creatine) (G) are also shown.

34

Figure 8 The aromatic region of the 3D 1H MR spectra measured from tumour (A) andcontralateral brain tissue (B) during/after BPA-F infusion (MRSI data collection began 11 minbefore and ended 6 min after the end of the infusion) in patient 10. The tumour (C) andcontralateral brain tissue spectra (D) measured four days before BNCT and spectra 3.0 mM BPAaqueous solution (with 10.1mM creatine) (E) are also shown.

35

Figure 9 The metabolite maps of patient 9. The metabolite maps are based on the Gaussianlineshape fitting of two peaks detected in the aromatic region after BNCT.

36

Figure 10 The metabolite maps of patient 10 based on the Gaussian lineshape fitting of thesignal detected in the aromatic region during/immediately after BPA-F infusion.

37

Figure 11 The metabolite region (0.5 - 4.2 ppm) of the 3D 1H MR spectra measured from thetumour (above, left) and from the contralateral brain tissue (above, right) from patient 9 afterBNCT. Spectra from the tumour (below, left) and contralateral brain tissue (below, right) ofpatient 10 measured during/immediately after BPA-F infusion are also shown.

Figure 12 3D 1H MRS spectra measured from 3.0 mM BPA aqueous solution (with 10.1mMcreatine) (A), the brain tumours of patients 9 (B) and 10 (C). Spectrum B was measured afterBNCT, and C during/directly after BPA-F infusion. Spectrum A appears in Figures 7 G and 8 E,spectrum B in Figure 7 A, and spectrum C in Figure 8 A.

38

6. DISCUSSION

Our phantom studies show that proton magnetic resonance spectroscopy is a promisingmethod for detecting BPA-F in in vivo conditions. Of the clinically common 1H MRSlocalisation schemes PRESS and STEAM, the PRESS scheme with short echo time(approximately 30 ms), was found to be optimal for detecting the aromatic protons of BPAand achieves maximal signal-to-noise of the weak BPA aromatic signals in low BPAconcentrations. If quantification is needed, corrections for T2 relaxation are unnecessary ifa short TE is used. Rather, T1 corrections are needed, since TR is typically set to anoptimised value (approximately 1.3 x T1, too short to omit T1 effects) for maximal SNR.

The results of the in vitro phantom studies were promising. The outcome of the in vivoMRS studies, however, was less satisfactory. Signals (SNR > 1) in the aromatic region ofthe spectrum were detected in three of ten patients (patients 1, 9 and 10). For patient 9,two distinct resonance lines were detected in the aromatic area (Figure 7 A), whereas forpatient 10, the corresponding resonance lines were broadened probably as a result ofpoorer magnetic field homogeneity adjustments (i.e. shimming), and only a broad singlepeak could be detected (Figure 8 A). The shape and position of the signals detected did notexactly match the BPA spectrum detected under in vitro conditions, thus renderingunambiguous assignment difficult (Figure 12). Possible explanations include changes inthe aromatic peak pattern in in vivo conditions due to some kind of dissociation of BPA-Fin the tissue environment and/or general chemical shift changes. Moreover, the low SNRof the detected aromatic signals can lead to a situation where noise contribution canchange the expected multiplet pattern. Despite the unclear multiplet pattern, the aromaticsignals detected in the in vivo spectra obviously originate from BPA, since no detectablesignals in the aromatic area of the spectra were recorded during the treatment planningMRI session (for patients 9 and 10) or one month after BNCT (patient 9).

The SNR of the detected in vivo signals was too low for quantification. BPAconcentrations in the brain were very close or below the in vivo detection limit of BPA.Using the blood boron concentration determined during the MRS/MRSI measurementsand blood-to-tumour BPA concentration ratio 3.5, the estimated tumour BPAconcentration would exceed 3 mM for every patient. The BPA detection limit determinedin phantom conditions varied from 0.7 mM to 2.2 mM (Papers II and IV). Naturally, thedetection limit is notably higher under in vivo conditions due to more challengingshimming environment. Bendel et al. (2005a) have extrapolated from high-field animalstudies that it would be possible to map concentrations of ~20 ppm boron from patientsin a clinical imager with about 1 cm3 spatial resolution. In water, 1 mM BPA equals 10ppm 10B, but in blood or tissue, this number is only an approximation because the preciserelationship between the two concentration units depends on the tissue/blood density. Theblood boron concentration during MRS (Table 2) varied from 9 to 38 ppm (mg/kgwhole

blood). This indicates that the tumour boron concentration would be 31.5-133.0 ppm if theblood-to-tumour concentration ratio would be the clinically proposed 1:3.5 during MRS.Naturally, the concentration ratio could differ during the time of the MRS study from thatduring the irradiation field (where a blood-to-tumour ratio of 1:3.5 is supposed).According to an animal study by Wang et al. (2004), the tumour-to-blood and tumour-to-

39

healthy brain 10B concentration ratios for F98 glioma decrease as a function of time fromthe maximum observed at 1 h post-injection. According to their study, two to four hoursafter injection, the ratio would be about two. Using tumour-to-blood boron concentrationratio of two, the boron concentration in the tumour would range from 18 to 50 ppm forpatients 1-9 during MRS. According to the estimated in vivo detection limit, even thoseconcentrations should be detectable in vivo. However, the detected signals with low SNRfor three patients and the lack of detectable aromatic signals for the rest of the patientssuggest that the BPA concentration is even lower or the in vivo detection limit of BPA issignificantly higher than the previously estimated values. It is difficult to explain thereason for the significantly higher detection limit of in vivo conditions as the spectrumregion from 1 to 4 ppm indicates normal quality (i.e. successful shimming, narrow lines)(Figure 11). In addition, some have suggested that the in vivo MRS detection limit of thelactate signal is ~1.5 mM (Dalsgaard et al. 2004). Obviously, it is difficult to directlycompare detection limits of the coupled lactate and coupled BPA aromatic protons signals.In lactate, three protons produce the doublet detected at 1.33 ppm. In BPA, four protonsproduce a “peak pattern” consisting of four main peaks (intensity ratio approx. 1:2:2:1).However, this roughly translates to a ~1.7 mM detection limit of BPA, which is in linewith values estimated from phantom results.

The MRS results suggest that the previously estimated blood-to-tumour concentrationratio of 1:3.5 is not necessarily valid during MRS. Alternatively, processes in the tumourlike possible binding of BPA to macromolecules or dissociation could, at least partially,reduce it’s MRS visibly and lower its signal intensity. Bendel et al. (2005a, b) haveshowed that dissociation from fructose complex to free BPA adds the additional peakpattern to the spectrum. This may also broaden the BPA signal in the in vivo spectrum,thus lowering the SNR and rising the detection limit of BPA. However, it was not possibleto determine which of these mechanisms was behind the low SNR of detected signals inour study.

Several different methods and techniques have been used to determine the distributionof BPA in the brain in vivo. PET with 18F BPA, prompt gamma spectroscopy (PGS),microdialysis and spectroscopic methods including 1H, 11B/10B and 19F MRS offerdifferent approaches to this problem. However, all of these methods bear their ownlimitations. The drawnback of PET is that 18F-labeled BPA is administrated as a bolus.There is no clinical evidence whether the bolus data are sufficiently valid to evaluate thetumour-to-brain concentration ratios (or absolute concentrations) obtained during theactual two-hour BPA-F infusion. In PGS, various technical challenges remain unsolved.Ideally, the detectors should be located in the irradiation room where they are prone toneutron irradiation. The main problem of 11B/10B MRS is the lack of 11B/10B detectioncapabilities in clinical imagers. Neither 19F NMR is a standard feature in clinicalinstruments. In addition, a separate infusion with a 11B-enriched carrier compound or theuse of 19F-labelled BPA is needed for 11B/19F spectroscopy/imaging. The 1H MRS ofBPA-F has many advantages: sensitivity of 1H detection, lack of spectral overlap andavailability in clinical scanners. In a study by Bergenheim et al. (2005), a microdialysismethod was applied to four patients who had received a 6-h BPA infusion. According tothat study, the maximal concentration ratio of the extracellular concentration of BPA in

40

tumour tissue vs. blood was 1.07. Obviously, the intracellular concentration of borondominates BNCT. MRS cannot discriminate between intracellular and extracellularspaces, but it can detect the concentration within the whole volume. The distribution ofbrain tissue volume is ~85% intracellular and ~15% extracellular (Ross and Bluml 2001).Consequently, comparing microdialysis results to the tumour boron concentrationmeasured by MRS is difficult.

The limitations of this study include its relatively low number of patients. In vivoMRS/MRSI measurements of ten patients were performed during four years and twomonths. Finding suitable patients for this study was difficult, since the tumour locationhad to be suitable and its volume sufficient for MRSI. Furthermore, performing MRS untilan average of 3.5 hours after completion of the BPA infusion for patients (except for one)proved impossible. The arrangement of MRS measurements during the BPA-F infusionprior to BNCT was demanding. Patients had to be transferred from the MRI imager to theBNCT facility for patient positioning and actual treatment immediately after MRS. Fromthe patient’s point of view, the day of BNCT was extremely exhausting even without MRSmeasurements. This also raised some limitations with respect to the selection of patientsfor MRS/MRSI. Also, the complicated shimming conditions due to resection cavities(especially those containing blood) in operated tumours naturally makes boron detectionmore challenging. The use of 3.0 T or even higher magnetic field MRS may improve thefeasibility of this method in clinical practice. In this study, 3.0 T 1H MRS was performedfor only one patient (patient 4), but detection of relevant signals in the aromatic area wasunsuccessful. Although increasing the magnetic field strength lowers the detection limit ofBPA in phantom conditions, and thus should improve the likelihood of detecting BPA invivo, challenges nevertless remain due to susceptibility effects when MRSI is performed inthe skull base area at high magnetic field.

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7. CONCLUSIONS

1. Under phantom conditions, BPA quantification accuracy of 5% can be achieved. BPAcan be quantified using either single voxel MRS or MRSI. Acquisition-weighted MRSIallows the detection of low BPA concentrations in a reasonable measurement time.

2. The BPA detection limit in phantoms suggests that estimated typical in vivo tumourBPA concentrations should lead to detectable signals.

3. Detecting BPA in vivo in a patient’s brain after a BNCT treatment or after thecompletion of BPA infusion is possible. However, in vivo conditions pose manychallenges for BPA detection. In cases when clotted blood is present in the resectioncavity or when to tumour is located near air cavities (e.g. the paranasal sinuses)susceptibility effects can disturb the MRS measurement. Unoperated tumours of relativelylarge volume are optimal for MRS-based BPA-F detection. It is not possible to evaluatethe value of MRS for BNCT treatment planning from our data. The opportunity to performMRS immediately after the completion of BPA-F infusion for more patients will beneeded to evaluate the suitability of 1H MRS for BPA detection or quantification in BNCTtrials.

4. MRSI may provide criteria for selecting patients for BNCT. If no indicative signal isdetected from the volume of interest located in a region feasible for good quality MRS,boron may not be accumulating sufficiently in the tumour to reach an appropriate radiationdose level in BNCT.

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ACKNOWLEDGEMENTS

This work was carried out as a part of the Finnish BNCT project. I owe my gratitude toProfessor Juhani Keinonen, Ph.D., the Head of the Department of Physics of theUniversity of Helsinki, and Docent Juhani Ahovuo, M.D., D.M.Sc., and Jyrki Putkonen,M.Sc.Tech., former and current Heads of the HUS Helsinki Medical Imaging Center ofthe Helsinki University Central Hospital for providing excellent research facilities.

I owe my warmest gratitude to my supervisors, Professor Sauli Savolainen, Ph.D. andDocent Sami Heikkinen, Ph.D., for their guidance and excellent support. Sauli’senthusiastic and encouraging attitude and his instinct “for focusing on the essentials” hashelped me many times in both hospital and research work over the years. And Sami’sexpertise in the field of nuclear magnetic resonance spectroscopy and his endless interestin scientific work has been essential to the accomplishment of this thesis.

I am grateful to the official referees of this thesis, Professors Jukka Jokisaari, Ph.D., andHannu Aronen M.D., D.M.Sc. Their invaluable comments and suggestions significantlyimproved this thesis.

I wish to express my deepest gratitude to the coauthors of the adjoining publications,especially to Leena Kankaanranta, M.D., Docent Nina Lundbom, M.D., D.M.Sc., DocentAki Kangasmäki, Ph.D., Docent Mika Kortesniemi, Ph.D. and Tiina Seppälä, Ph.D. I amalso grateful to Nina for her experienced advice on scientific writing and valuablecomments in proof-reading the summary.

The personnel of Boneca Ltd. are acknowledged for co-operation during the project.

I thank MRI technologists in the HUS Helsinki Medical Imaging Center. I also thank allmy friends and colleagues in HUS, especially Toni Ihalainen, Ph.Lic, Jouni Uusi-Simola,Ph.D., Docent Outi Sipilä, D.Sc.Tech., Eero Salli, D.Sc.Tech., Linda Kuusela M.Sc.,Yvonne Käser, M.Sc.Tech., and Juha Peltonen, M.Sc.Tech.

I also wish to thank all the patients who participated in this study.

I am deeply grateful to my friends and relatives for their support and interest in myresearch. I especially wish to thank Anne, Katja and Pauliina for their friendship andsupport during these years, as well as Piia for making my year on maternity leave evenmore enjoyable, which gave me new inspiration for my research work.

I owe my warmest gratitude to my parents, Tuulikki and Antero Timonen, for theirencouragement and support throughout these years. I also thank my brothers and theirfamilies for their support of my thesis, and I especially wish to thank to Annukka forkeeping the faith with my thesis and for being the “big sister” I never had.

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Finally, I owe my deepest gratitude to Jukka for his love, patience and unconditionalsupport throughout these years, and to my lovely daughter Maija, who turned my life“upside down” and gave new meaning of my life.

The financial support of the Academy of Finland, the State Subsidy for UniversityHospitals, and University of Helsinki are gratefully acknowledged.

December 2009 Marjut Timonen

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REFERENCES

Aihara T, Hiratsuka J, Morita N, Uno M, Sakurai Y, Maruhashi A, Ono K and Harada T2006 First clinical case of boron neutron capture therapy for head and neckmalignancies using 18F-BPA PET. Head Neck 28 850-855

Aru’s C, Chang Y-C and Ba´ra´ny M 1985 Proton nuclear magnetic resonance spectra ofexcised rat brain. Assignment of resonances. Physiol Chem Phys Med NMR 17 23–33

Babsky AM, Topper S, Zhang H, Gao Y, James JR, Hekmatyar SK and Bansal N 2008Evaluation of extra- and intracellular apparent diffusion coefficient of sodium in ratskeletal muscle: effects of prolonged ischemia. Magn Reson Med 59 485-91

Becker ED, Ferretti JA and Farrar DC 1969 Driven equiriblium Fourier transformspectroscopy. A new method for nuclear magnetic resonance signal enhancement. JAm Chem Soc 91 7784-7785

Bendel P, Margalit R, Coudinova N and Salomon Y 2005a Noninvasive quantitative invivo mapping and metabolism of boronophenylalanine (BPA) by nuclear magneticresonance (NMR) spectroscopy and imaging. Radiat Res 164 680-687

Bendel P, Margalit R and Salomon Y 2005b Optimized 1H MRS and MRSI methods forthe in vivo detection of boronophenylalanine. Magn Reson Med 53 1166-1171

Bendel P and Sauerwein W 2001 Optimal detection of the neutron capture therapy agentborocaptate sodium (BSH): a comparison between 1H and 10B NMR. Med Phys 28178-183

Bendel P, Zilberstein J and Salomon Y 1994 In vivo detection of a boron-neutron-captureagent in melanoma by proton observed 1H-10B double resonance. Magn Reson Med 32170-174

Bergenheim A T, Capala J, Roslin M and Henriksson R 2005 Distribution of BPA andmetabolic assessment in glioblastoma patients during BNCT treatment: a microdialysisstudy. J Neurooncol 71 287-93

Boesch C, Machann J, Vermathen P and Schick F 2006 Role of proton MR for the studyof muscle lipid metabolism. NMR Biomed 19 968-88

Bottomley PA 1987 Spatial localization in NMR spectroscopy in vivo. Ann NY Acad Sci508 333-348

Brown TR, Kincaid BM and Uqurbil K 1982 NMR chemical shift imaging in threedimensions. Proc Natl Acad Sci USA 79 3523-3526

Busse P, Zamenhof R, Harling O, Kaplan I, Kaplan J, Chuang C, Goorley J, Kiger III W,Riley K, Tang L, Solares G and Palmer M 2001 The Harward-MIT BNCT program, inFrontiers in Neutron Capture Therapy, M.F. Hawthorne, K. Shelly, and R.J. Wiersema,Editors. Kluwer Academic / Plenum Publishing Corporation: New York. p. 37-60

Campanella R, Capuani S, Fasano F, Maraviglia B 2002 Double resonance NMR imagingof BSH, In: Sauerwein W, Moss R, Wittig A, editors. Research and Development inNeutron Capture Therapy; 2002 Sep 8-13; Essen, Germany. Bologna, Italy: ModuzziEditore 963-965

Coderre JA, Chanana AD, Joel DD, Elowitz EH, Micca PL, Nawrocky MM, Chadha M,Gebbers J-O, Shady M, Peress NS and Slatkin DN 1998 Biodistribution ofboronophenylalanine in patients with glioblastoma multiforme: boron concentrationcorrelates with tumor cellularity. Radiat Res 149 163-170

Coderre JA and Morris GM 1999 The radiation biology of boron neutron capture therapy.Radiat Res 151 1-18

Dalsgaard M, Quistorff B, Danielsen E, Selmer C, Vogelsang T and Secher N 2004 Areduced cerebral metabolic ratio in exercise reflects metabolism and not accumulationof lactate within the human brain J Physiol. 554 571–578

45

Danielsen E and Ross BD 1999 Magnetic resonance spectroscopy diagnosis ofneurological diseases. New York: Marcel-Dekker 327 pages

Diaz AZ, Coderre JA, Chanana AD and Ma R 2000 Boron neutron capture therapy formalignant gliomas. Ann Med 32 81-85

Duyn JH and Moonen CTW 1993 Fast proton spectroscopic imaging of human brain usingmultiple spin echoes. Magn Reson Med 30 409–414

Dydak U, Weiger M, Pruessman KP, Meier D and Boesiger P 2001 Sensitivity-encodedspectroscopic imaging. Magn Reson Med 46 713-722

Dydak U, Pruessman KP, Weiger M, Tsao J, Meier D and Boesiger P 2003 Parallelspectroscopic imaging with spin-echo trains. Magn Reson Med 50 196-200

Elowitz EH, Bergland RM, Coderre JA, Joel DD, Chadha MM and Chanana AD 1998Biodistribution of p-boronophenylalanine in patients with glioblastoma multiforme foruse in boron neutron capture therapy. Neurosurgery 42 463-469

Fischbach F and Bruhn H 2008 Assessment of in vivo 1H magnetic resonancespectroscopy in the liver: a review. Liver Int 28 297-307

Frahm J Merboldt KD, and Hänicke W 1987 Localized proton spectroscopy usingstimulated echoes. J Magn Reson 72 502-508

Frahm J, Bruhn H, Gyngell ML, Merboldt KD, Hänicke W and Sauter R 1989 Localizedproton NMR spectroscopy in different regions of the human brain in vivo. Relaxationtimes and concentrations of cerebral metabolites. Magn Reson Med 11 47–63

Gadian DG, Proctor E, Williams SR, Cady EB and Gardiner RM 1986 Neurometaboliceffects of an inborn error of amino acid metabolism demonstrated in vivo by 1H NMR.Magn Reson Med 3 150–156

Garwood M and DelaBarre L 2001 The return of the frequency sweep: designing adiabaticpulses for contemporary NMR. J Magn Reson 153 155-177

Graaf RA 2007 In vivo NMR spectroscopy -2nd Edition Principles and techniques.London: John Wiley & Sons, Ltd

Griswold MA, Jakob PM, Heidemann RM, Nittka M, Jellus V, Wang J, Kiefer B andHaase A 2002 Generalized autocalibrating partially parallel acquisitions (GRAPPA).Magn Reson Med 47 1202-1210

Guilfoyle DN, Blamire A, Chapman B, Ordidge RJ and Mansfield P 1989 PEEP-a rapidmagnetic imaging method. Magn Reson Med 10 282-287

Guilfoyle DN and Mansfield P 1985 Chemical-shift imaging. Magn Reson Med 2 479-489Gupta RK 1976 Dynamic range problem in Fourier transform NMR. Modified WEFT

pulse sequence. J Magn Reson 24 461-465Haase A, Frahm J, Hänicke W and Matthaei D 1985 1H proton NMR chemical shift

selective (CHESS) imaging. Phys Med Biol 30 341–344Hetherington HP, Mason GF, Pan JW, Ponder SL, Vaughan JT, Twieg DB and Pohost

GM 1994 Evaluation of cerebral gray and white matter metabolite differences byspectroscopic imaging at 4.1T. Magn Reson Med 32 565-571

Horn M 2006 Cardiac magnetic resonance spectroscopy: a window for studyingphysiology. Methods Mol Med 124 225-48

Hu X, Levin DN, Lauterbur PC and Spraggins T 1988 SLIM: spectral localization byimaging. Magn Reson Med 8 314-322

Hugg JW, Maudsley AA, Weiner MW and Matson GB 1996 Comparison of k-spacesampling schemes for multidimensional MR spectroscopic imaging. Magn Reson Med36 469-473

Imahori Y, Ueda S, Ohmori Y, Kusuki T, Ono K, Fujii R and Ido T 1998a Fluorine-18-labeled fluoroboronophenylalanine PET in patients with glioma. JNM 39 325-333

46

Imahori Y, Ueda S, Ohmori Y, Sakae K, Kusuki T, Kobayashi T, Takagi M, Ono K, Ido Tand Fujii R 1998b Positron emission tomography-based neutron capture therapy usingboronophenylalanine for high-grade gliomas: part I. Clinical Cancer Research 4 1825-1832

Imahori Y, Ueda S, Ohmori Y, Sakae K, Kusuki T, Kobayashi T, Takagi M, Ono K, Ido Tand Fujii R 1998c Positron emission tomography-based neutron capture therapy usingboronophenylalanine for high-grade gliomas: part II. Clinical Cancer Research 4 1833-1841

International Atomic Energy Agency (IAEA) 2001 Current status of neutron capturetherapy, in IAEA-Tecdoc-1223., IAEA: Wien.

International Comission on Radiation Units and Measurement (ICRU) Determination ofabsorbed dose in a patient irradiated by beams of X or gamma rays in radiotherapyprocedures, Report 26 (Bethesda, MD: ICRU Publications, 1976).

Joensuu H, Kankaanranta L, Seppälä T, Auterinen I, Kallio M, Kulvik M, Laakso J,Vähätalo J, Kortesniemi M, Kotiluoto P, Seren T, Karila J, Brander A, Järviluoma E,Ryynänen P, Paetau A, Ruokonen I, Minn H, Tenhunen M, Jääskeläinen J, Färkkilä M,and Savolainen S 2003 Boron neutron capture therapy of brain tumors: clinical trials atthe Finnish facility using boronophenylalanine. J Neuro-Oncol 62 123-134

Kabalka GW, Tang C and Bendel P 1997 The role of boron MRI in boron neutron capturetherapy. J Neurooncol 33 153-161

Kankaanranta L, Seppälä T, Koivunoro H, Saarilahti K, Atula T, Collan J, Salli E,Kortesniemi M, Uusi-Simola J, Mäkitie A, Seppänen M, Minn H, Kotiluoto P,Auterinen I, Savolainen S, Kouri M and Joensuu H 2007 Boron Neutron CaptureTherapy in the Treatment of Locally Recurred Head and Neck Cancer. Int J RadiationOncology Biol Phys 69 475–482

Kato I, Ono K, Sakurai Y, Ohmae M, Maruhashi A, Imahori Y, Kirihata M, Nakazawa M,and Yura Y 2004 Effectiveness of BNCT for recurrent head and neck malignancies.Appl Radiat Isot 61 1069-1073

Kinchesh P and Ordidge RJ 2005 Spin-Echo MRS in humans at high field: LASERlocalisation using FOCI pulses. J Magn Reson 175 30-43

Klomp D, van Laarhoven H, Scheenen T, Kamm Y and Heerschap A 2007 Quantitative19F MR spectroscopy at 3 T to detect heterogeneous capecitabine metabolism in humanliver. NMR Biomed 20 485-492

Kortesniemi M Solutions for clinical implementation of boron neutron capture therapy inFinland, Helsinki, Finland: University of Helsinki, 2002 Ph.D. thesis, Report Series inPhysics HU-P-D95 https://oa.doria.fi/handle/10024/2586

Kortesniemi M, Seppälä T, Auterinen I and Savolainen S 2004 Enhanced blood boronconcentration estimation for BPA-F mediated BNCT. Appl Radiat Isot 61 823-827

Kosunen A. Metrology and quality of radiation therapy dosimetry of electron, photon andepithermal neutron beams. Helsinki, Finland: University of Helsinki, 1999. Ph.D.thesis, STUK-A164

Kotiluoto P Adaptive tree multigrids and simplified spherical harmonics approximation indeterministic neutral and charged particle transport, Helsinki, Finland: University ofHelsinki, 2007. Ph.D. thesis, VTT Publications 639 https://oa.doria.fi/handle/10024/5777

Kouri M, Kankaanranta L, Seppala T, Tervo L, Rasilainen M, Minn H, Eskola O,Vahatalo J, Paetau A, Savolainen S, Auterinen I, Jaaskelainen J and Joensuu H 2004Undifferentiated sinonasal carcinoma may respond to single-fraction boron neutroncapture therapy. Radiother Oncol 72 83-85

Kreis R, Pietz J, Penzien J, Herschkowitz N and Boesch C 1995 Identification andquantitation of phenylalanine in the brain of patients with phenylketonuria by means oflocalized in vivo 1H magnetic-resonance spectroscopy. J Magn Reson Ser B 107 242–251

47

Kurhanewicz J, Bok R, Nelson SJ and Vigneron DB 2008 Current and potentialapplications of clinical 13C MR spectroscopy. J Nucl Med 49 341-344

Laakso J, Kulvik M, Ruokonen I, Vähätalo J, Zilliacus R, Färkkilä M, and Kallio M 2001Atomic emission method for total boron in blood during neutron-capture therapy. ClinChem 47 1796-7803

Lin A, Ross BD, Harris K and Wong W 2005 Efficacy of proton magnetic resonancespectroscopy in neurological diagnosis and neurotherapeutic decision making.NeuroRx 2 197–214

Linko S, Revitzer H, Zilliacus R, Kortesniemi M, Kouri M and Savolainen S 2008 Borondetection from blood samples by ICP-AES and ICP-MS during boron neutron capturetherapy. Scand J Clin Lab Invest 68 696-702

Locher, GL 1936 Biological effects and therapeutic possibilities of neutrons. Am JRoentgenol/ Radium Ther 36 1-13

Mansfield P 1984 Spatial mapping of the chemical shift in NMR. Magn Reson Med1 370-386

Mattei JP, Bendahan D and Cozzone P 2004 P-31 magnetic resonance spectroscopy. Atool for diagnostic purposes and pathophysiological insights in muscle diseases.Reumatismo 56 9-14

Maudsley AA, Hilal SK, Perman WH and Simon HE 1983 Spatially resolved highresolution spectroscopy by “four dimensional” NMR. J Magn Reson 51 147-152

Maudsley AA, Matson GB, Hugg JW and Weiner MW 1994 Reduced phase encoding inspectroscopic imaging. Magn Reson Med 31 645-651

Mlynárik V, Gruber S and Moser E 2001 Proton T1 and T2 relaxation times of humanbrain metabolites at 3 Tesla. NMR Biomed 14 325-331

Mori S, Eleff SM, Pilatus U, Mori N and van Zijl PC 1998 Proton NMR spectroscopy ofsolvent-saturable resonances: a new approach to study pH effects in situ. Magn ResonMed 40 36–42

Mori Y, Suzuki A, Yoshino K and Kakihana H 1989 Complex formation ofpboronophenylalanine with some monosaccharides. Pigment Cell Research 2 273-277

Munck af Rosenschöld PM, Verbakel WF, Ceberg CP, Stecher-Rasmussen F and PerssonBR 2001 Toward clinical application of prompt gamma spectroscopy for in vivomonitoring of boron uptake in boron neutron capture therapy. Med Phys 28 787-795

Nakagawa N, Akai F, Fukawa N, Fujita Y, Suzuki M, Ono K, Taneda M 2007 Earlyeffects of boron neutron capture therapy on rat glioma models. Brain Tumor Pathol 247-13

Nakagawa, Y 2001 Boron neutron capture therapy. Recent aspect and new protocol inJapan, in Frontiers in Neutron Capture Therapy, M.F. Hawthorne, K. Shelly, and R.J.Wiersema, Editors., Kluwer Academic / Plenum Publishing Corporation: New York. p.73-79

Nicholas P, Fushman D, Ruchinsky V and Cowburn D 2000 The virtual NMRspectrometer: a computer program for efficient simulation of NMR experimentsinvolving pulsed field gradients. J Magn Reson 145 262-275

Nichols TL, Kabalka GW, Miller LF, Khan MK, and Smith GT 2002 Improved treatmentplanning for boron neutron capture therapy for glioblastoma multiforme usingfluorine-18 labeled boronophenylalanine and positron emission tomography. MedPhys 29 2351-2358

Ordidge RJ, Conelly A and Lohman JA 1986 Image selected in vivo spectroscopy (ISIS).A new technique for spatially selective NMR spectroscopy. J Magn Reson 66 283-294

Pan JW and Twieg DB 1998 Hetherington HP Quantitative spectroscopic imaging of thehuman brain. Magn Reson Med 40 363-369

48

Pohmann R and von Kienlin M 2001 Accurate phosphorus metabolite images of thehuman heart by 3D acquisition-weighted CSI Magn. Reson Med 45 817–826

Porcari P, Capuani S, D'Amore E, Lecce M, La Bella A, Fasano F, Campanella R,Migneco LM, Pastore FS and Maraviglia B 2008 In vivo 19F MRI and 19F MRS of 19F-labelled boronophenylalanine-fructose complex on a C6 rat glioma model to optimizeboron neutron capture therapy (BNCT). Phys Med Biol 53 6979–6989

Porcari P, Capuani S, D'Amore E, Lecce M, La Bella A, Fasano F, Migneco LM,Campanella R, Maraviglia B and Pastore FS 2009 In vivo 19F MR imaging andspectroscopy for the BNCT optimization. Appl Radiat Isot 67 S365-358

Posse S, DeCarli C and Le Bihan D 1994 Three-dimensional echo-planar MRspectroscopic imaging at short echo times in human brain. Radiology 192 733-738

Pouwels PJ and Frahm J 1998 Regional metabolite concentrations in human brain asdetermined by quantitative localized proton MRS. Magn Reson Med 39 53-60

Procissi D, Claus F, Burgman P, Koziorowski J, Chapman JD, Thakur SB, Matei C, LingCC and Koutcher JA 2007 In vivo 19F magnetic resonance spectroscopy and chemicalshift imaging of tri-fluoro-nitroimidazole as a potential hypoxia reporter in solidtumors. Clin Cancer Res 13 3738-3747

Provencher SW 1993 Estimation of metabolite concentrations from localized in vivoproton NMR spectra. Magn Reson Med 30 672-679

Pruessman KP, Weiger M, Scheidegger MB and Boesiger P 1999 SENSE: Sensitivityencoding for fast MRI. Magn Reson Med 42 952-962

Ross B and Bluml S 2001 Magnetic Resonance Spectroscopy of the Human Brain. AnatRec. 265 54-84

Rothman DL, Behar KL, Prichard JW and Petroff OA 1997 Homocarnosine and themeasurement of neuronal pH in patients with epilepsy. Magn Reson Med 38 924–929

Ryynänen P Kinetic Mathematical Models for the 111In-labelled Bleomycin Complex and10B in Boron Neutron Capture Therapy, Helsinki, Finland: University of Helsinki,2002. Ph.D. thesis, Report Series in Physics HU-P-D102https://oa.doria.fi/handle/10024/2655

Seppälä, T., Fir 1 Epithermal Neutron Beam Model and Dose Calculation for TreatmentPlanning in Neutron Capture Therapy, Helsinki, Finland: University of Helsinki, 2002.Ph.D. thesis, Report Series in Physics HU-P-D103 https://oa.doria.fi/handle/10024/2640

Shull BK, Spielvogel DE, Head G, Gopalaswamy R, Sankar S and Devito K 2000 Studieson the structure of the complex of the boron neutron capture therapy drug, L-p-boronophenylalanine, with fructose and related carbohydrates: Chemical and 13C NMRevidence for the -D-fructofuranose 2,3,6-(p-phenylalanyl-orthoborate) structure. JPharm Sci 89 215-222

Shungu DC and Glickson JD 1993 Sensitivity and localization enhancement inmultinuclear in vivo NMR spectroscopy by outer volume presaturation. Magn ResonMed 30 661–671

Sodickson DK and Manning WJ 1997 Simultaneus acquisition of spatial harmonics(SMASH): fast imaging with radiofrequency coil arrays. Magn Reson Med 38 591-607

Solga SF, Horska A, Clark JM and Diehl AM 2005 Hepatic 31P magnetic resonancespectroscopy: a hepatologist's user guide. Liver Int 25 490-500

Tan J, Bluml S, Hoang T, Dubowitz D, Mevenkamp G and Ross B 1998 Lack of effect oforal choline supplement on the concentrations of choline metabolites in human brain.Magn Reson Med 39 1005-1010

Uusi-Simola J. Finnish Dosimetric Practice for Epithermal Neutron Beam Dosimetry inBoron Neutron Capture Therapy, Helsinki, Finland: University of Helsinki 2009

49

Ph.D. thesis, Report Series in Physics HU-P-D160https://oa.doria.fi/handle/10024/44583

Verbakel WF 2001 Validation of the scanning -gamma-ray telescope for in vivo dosimetryand boron measurements during BNCT. Phys Med Biol 46 3269-3285

Verbakel W, Sauerwein W, Hideghety K and Stecher-Rasmussen F 2003 Boronconcentrations in brain during boron neutron capture therapy: in vivo measurementsfrom the phase I trial EORTC 11961 using a gammaray telescope. Int J Radiat OncolBiol Phys 55 743-755

Vikhoff-Baaz B, Starck G, Ljungberg M, Lagerstrand K, Forssell-Aronsson E and EkholmS 2001 Effects of k-space filtering and image interpolation on image fidelity in 1HMRSI. Magn Reson Imaging 19 1227-1234

Von Kielin M and Mejia R 1991 Spectral localization with optimal pointspread function. JMagn Reson 94 268-287

Voorbraak WP and Järvinen H (edit.) 2003 Recommendations for the Dosimetry of BoronNeutron Capture Therapy NRG, Petten 170 pages

Wang HE, Liao AH, Deng WP, Chang PF, Chen JC, Chen FD, Liu RS, Lee JS, andHwang JJ 2004 Evaluation of 4-borono-2-18F-fluoro-L-phenylalanine-fructose as aprobe for boron neutron capture therapy in glioma-bearing rat model. J Nucl Med 45302–308

Wang Y and Li SJ 1998 Differentiation of metabolic concentrations between gray matterand white matter of human brain by in vivo 1H magnetic resonance spectroscopy.MagnReson Med 39 28-33

Wittig A, Malago M, Collette L, Huiskamp R, Bührmann S, Nievaart V, Kaiser GM,Jöckel KH, Schmid KW, Ortmann U and Sauerwein WA 2008 Uptake of two 10B-compounds in liver metastases of colorectal adenocarcinoma for extracorporealirradiation with boron neutron capture therapy (EORTC Trial 11001). Int J Cancer 1221164-1171

Yang W, Barth RF, Rotaru JH, Moeschberger ML, Joel DD, Nawrocky MM, GoodmanJH 1997 Enhanced survival of glioma bearing rats following boron neutron capturetherapy with blood-brain barrier disruption and intracarotid injection ofboronophenylalanine. J Neuro-Oncol 33 59–70

Yoshino K, Suzuki A, Mori Y, Kakihana H, Honda C, Mishima Y, Kobayashi T andKanda K 1989 Improvement of solubility of p-Boronophenylalanine by complexformation with monosaccharides. Strahlentherapie und Onkologie 165 127-129

Zuo CS, Prasad PV, Busse P, Tang L and Zamenhof RG 1999 Proton nuclear magneticresonance measurement of p-boronophenylalanine (BPA): A therapeutic agent forboron neutron capture. Med Phys 26 1230-1236