ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1650

PET in Prostate Cancer –Detection, Tumour Biology andPrognosis

NARESH KUMAR REGULA

ISSN 1651-6206ISBN 978-91-513-0904-0urn:nbn:se:uu:diva-407053

Dissertation presented at Uppsala University to be publicly examined in H:sonHolmdahlsalen, Ingång 100, Dag Hammarskjöldsväg 8, Uppsala, Friday, 8 May 2020 at09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination willbe conducted in English. Faculty examiner: Professor Katrine Riklund (Umeå University,Department of Radiation Sciences).

AbstractRegula, N. K. 2020. PET in Prostate Cancer – Detection, Tumour Biology and Prognosis.Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine1650. 48 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0904-0.

Prostate cancer (PCa) is the most common non-cutaneous malignancy in men and the leadingcause of cancer-related deaths in Sweden. Despite the major advances, the current diagnosticmodalities fall short of standards, specifically, precise localization required for effectivemanagement of the PCa. positron emission tomography (PET) combined with computedtomography (CT) has evolved as a promising diagnostic imaging technique for PCa. Theprogression of the PCa is often associated with metabolic alterations and overexpression ofseveral proteins. Increased de novo fatty acid synthesis and prostate-specific membrane antigen(PSMA) overexpression are some of the distinctive features linked with PCa growth and thepotential targets for the development of PET radiotracers.

This thesis is based on four original articles and focuses on the utilization of some of severaldifferent PET tracers available to visualize PCa spread. The work can be divided into twodistinctive parts: (1) evaluate the prognostic value of 11C-acetate PET/CT towards survival inthe setting of biochemical relapse after surgery, investigate tumour biology using single-tissuecompartment model derived parametric images of 11C-acetate dynamic PET/CT both at patientand cell level and (2) the comparison of 68Ga-PSMA-11 PET/CT with 11C-acetate PET/CT and18F-NaF PET/CT in patients with PCa relapse depicting different aspects of PCa biology.

We demonstrated that quantification of 11C-acetate accumulation in PCa lesions was a strongpredictor of survival in patients with biochemical relapse. Furthermore, parametric imagesof 11C-acetate dynamic PET/CT enabled visualization of tumour biology exhibiting elevatedextraction of 11C-acetate associated with cancer aggressiveness also confirmed in in-vitrostudies. 68Ga-PSMA-11 PET/CT located more widespread disease and performed significantlybetter in locating lymph node and bone metastases compared to 11C-acetate PET/CT. Similarly,68Ga-PSMA-11 PET/CT was able to detect most of the bone lesions detected with 18F-NaF PET/CT along with additional soft tissue lesions.

In conclusion, we showed the role of 11C-acetate PET/CT in PCa prognosis with additionalunderstanding of tumour biology. Further, we successfully showed better performance of 68Ga-PSMA-11 PET/CT in locating PCa relapse and established it as a promising option for PCa re-staging.

Keywords: Prostate cancer, 11C-acetate, 68Ga-PSMA-11, 18F-NaF, PET/CT

Naresh Kumar Regula, Department of Surgical Sciences, Radiology, Akademiska sjukhuset,Uppsala University, SE-75185 Uppsala, Sweden.

© Naresh Kumar Regula 2020

ISSN 1651-6206ISBN 978-91-513-0904-0urn:nbn:se:uu:diva-407053 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-407053)

to my family, friends and teachers

తృ భవ తృ భవ ఆ ర భవ

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Regula, N., Häggman, M., Johansson, S., Sörensen, J. (2016)

Malignant lipogenesis defined by 11C-acetate PET/CT predicts prostate cancer-specific survival in patients with biochemical relapse after prostatectomy. Eur J Nucl Med Mol Imaging, 43(12):2131–2138

II Regula, N., Honarvar, H., Lubberink, M., Jorulf, H., Ladjevardi, S., Häggman, M., Antoni, G., Buijs, J., Velikyan, I., Sörensen J.(2020) Carbon flux as a measure of prostate cancer aggressiveness: [11C]-acetate PET/CT. Int J Med Sci, 17(2):214-223

III Regula, N., Kostaras, V., Johansson, S., Trampal, C., Lindström, E., Lubberink, M., Velikyan, I., Sörensen, J. (2020) Comparison of 68Ga-PSMA-11 PET/CT with 11C-acetate PET/CT in re-staging of prostate cancer relapse. Sci Rep, 10(1):4993

IV Regula, N., Kostaras, V., Johansson, S., Trampal, C., Lindström, E., Lubberink, M., Iyer, V., Velikyan, I., Sörensen, J. Comparison of 68Ga-PSMA-11 PET/CT with 18F-fluroide PET/CT for detection of bone metastatic disease in prostate cancer. Manuscript

Reprints were made with permission from the respective publishers.

Other papers not included in the thesis

I. Lindström, E., Velikyan, I., Regula, N., Alhusseinalkhudhur, A., Sundin, A., Sörensen, J., Lubberink, M. (2019) Regularized reconstruction of digital time-of-flight 68Ga-PSMA-11 PET/CT for the detection of recurrent disease in prostate cancer patients. Theranostics, 9(12):3476-3484

II. Carlsson, A., Regula, N., Sörensen, J., Thor, Andreas. [18F]-fluoride PET/CT for investigating bone healing around biomaterials and augmentations of bone in the facial skeleton. A pilot study on dental implant osseo integration, sinus membrane elevation, particulate bone graft and osseous free-falp healing. manuscript

Contents

Introduction ................................................................................................... 11 Background .............................................................................................. 11 Investigation of PCa recurrence ............................................................... 12 Current imaging techniques...................................................................... 13

TRUS ................................................................................................... 13 CT ........................................................................................................ 13 Magnetic Resonance Imaging (MRI) .................................................. 13

Bone scan ................................................................................................. 13 Positron emission tomography (PET) ...................................................... 14

Basic principles .................................................................................... 14 Quantification ...................................................................................... 15 Compartmental Model ......................................................................... 16

PET tracers for PCa in clinical use ........................................................... 16 18F-FDG ............................................................................................... 16 18F-Sodium Fluoride (18F-NaF) ............................................................ 17 11C- or 18F- choline ............................................................................... 17 18F-fluciclovine .................................................................................... 17 11C-acetate ............................................................................................ 17 68Ga-PSMA-11 .................................................................................... 19

Aims of the thesis.......................................................................................... 21

Methods ........................................................................................................ 22 Participants ............................................................................................... 22 PCa cell lines ............................................................................................ 22 Data acquisition ........................................................................................ 22

11C-acetate ............................................................................................ 23 68Ga-PSMA .......................................................................................... 23 18F-fluoride .......................................................................................... 23 In-vitro cell experiments ...................................................................... 23

Image analysis .......................................................................................... 23 Image analysis of static PET ................................................................ 23 Image analysis of dynamic PET .......................................................... 24 Analysis of 11C-acetate kinetics in-vitro .............................................. 24

Results ........................................................................................................... 25 Paper I ...................................................................................................... 25

Paper II ..................................................................................................... 27 Paper III .................................................................................................... 30 Paper IV ................................................................................................... 32

Discussion ..................................................................................................... 36

Conclusions and future perspectives ............................................................. 40

Acknowledgments......................................................................................... 42

References ..................................................................................................... 44

Abbreviations

1 TCM One tissue compartment model 2 TCM Two tissue compartment model BS Bone scan BSI Bone scan index CPS Counts per second CT Computed tomography DRE Digital rectal examination FASN Fatty acid synthetase LN Lymph node MRI Magnetic resonance imaging MIP Maximum intensity projection PET Positron emission tomography p.i Post injection PCa Prostate cancer PSA Prostate specific antigen PSMA Prostate specific membrane antigen SPECT Single photon emisstion tomography SUV Standardized uptake value TAC Time-activity curve TLA Total lesion (lipogenic) activity TTV Total tumour volume TRUS Transrectal ultrasonography TBF Tumour blood flow TV Tumour volume VT Volume of distribution VOI Volume of interest

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Introduction

Background Prostate cancer (PCa) is a major public health problem in Sweden, being the most common malignancy detected in men [1]. Every year around 10,000 men are newly diagnosed with PCa and over 2,300 die from PCa in Sweden [1]. PCa is primarily a disease affecting men over 50 years of age, most of them remain asymptomatic and have slow progress. Yet, PCa remains the fifth leading cause of cancer-related deaths in men worldwide [2].

The prostate is part of the male reproductive system located below the urinary bladder and in front of the rectum. The upper part is called the base, rests against the lower part of the urinary bladder, while the lower part is called the apex. It is divided into right and left lobes made up of different anatomical zones and surrounded by a thin connective tissue called the capsule. The peripheral zone is the largest area of the prostate and easily felt during a digital rectal examination (DRE). The majority of PCa (70%) develop from the peripheral zone (Figure 1).

Figure 1. A schematic overview of location and anatomy of the prostate gland.

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Prostate cancer screening One of the strategies to decrease the risk of PCa death is screeing with prostate-specific antigen (PSA). PSA, also called kallikrein-3, is a glycoprotein secreted by the epithelial cells of the prostate gland, normally present in small quantities in the serum of healthy men but upregulated in the presence of PCa. Since the discovery in the 1970s and clinical introduction in 1990s, the broad use of PSA, in combination with biopsy in individuals and mass screening, has resulted in a significant increase in early diagnosis and reduction in mortality [3, 4]. However, the extent of benefit with PSA screening is under continuous debate due to its poor sensitivity and limited diagnostic specificity, resulting in a substantial risk of overdiagnosis and overtreatment [5].

Prostate cancer diagnosis When a patient is suspected with PCa, the standard workup for the risk assessment and initial diagnosis include physical examination (DRE), measuring PSA level from blood sample followed by a core needle biopsy. During the biopsy, a needle is inserted either through the wall of the rectum (transrectal) or through the skin between the scrotum and anus (transperineal). Usually, 10-12 core samples from different parts of the prostate are collected, histochemically stained and microscopically analysed using Gleason score.

Once the diagnosis of PCa is established, PCa is approached with various treatment options. For smaller “low-risk” PCa, there is always the option of watchful waiting and active surveillance. For localized PCa, the treatment usually involves radical prostatectomy, and radiation therapy, e.g., internal brachytherapy or external beam therapy.

Investigation of PCa recurrence Following primary curative treatment, be it by surgery, 15 to 40% of patients will experience recurrent disease within 5 years [6, 7]. The first manifestation of PCa recurrence is a rise in the PSA level. Early detection of PCa recurrence is of utmost clinical relevance in terms of prognosis and still a major challenge in patient management.

Imaging plays an important role not only for primary staging but also for localizing and re-staging of PCa recurrence. Considering the amplified improvement in the imaging techniques, the goal of PCa imaging in the future will be more towards individualized patient management. The choice of treatment in the setting of PCa relapse is highly influenced by the distinction of local recurrence, distant metastatic disease, and a combination of both.

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Current imaging techniques conventional imaging techniques applied in the routine clinical workflow for PCa imaging include transrectal ultrasound (TRUS), computed tomography (CT) and magnetic resonance imaging (MRI).

TRUS TRUS has an essential role in measuring the prostate volume, guiding PCa biopsy and radioactive seed placement (brachytherapy) [8], but the use of TRUS for the detection of PCa recurrence is limited.

CT CT commonly used for initial staging of intermediate- to high-risk disease by identifying pelvic and retroperitoneal nodal disease [9]. However, its role in detecting low-volume recurrent disease is limited due to low sensitivity, especially at lower PSA level [10].

Magnetic Resonance Imaging (MRI) The role of MRI in the evaluation of patients with biochemical recurrence is increasing, especially the use of multiparametric MRI (mpMRI). MRI has many potential applications in PCa, including initial staging, biopsy guidance and re-staging. European Association of Urology (EAU) guidelines recommend the use of mpMRI in men with indications for biopsy to reduce over-diagnosis [11, 12]. The role of mpMRI in the detection of local recurrence depends on the size of the lesion and PSA level with sensitivity and specificity ranging from 71% to 100% and 74% to 100%, respectively [13, 14]. However, introduction of molecular imaging techniques like positron emission tomography (PET) that would allow visualization of biological processes enables the quantification of PCa progression on molecular level.

Bone scan The skeletal system is the most common site for distant metastasis and nearly 80% of patients with fatal PCa show bone metastasis [15]. Therefore, in clinics, bone scan (BS) also called skeletal scintigraphy is the most frequently used and least expensive modality for the diagnosis of bone metastasis. BS is used in PCa re-staging and is not appropriate for initial staging at PSA levels <10 ng/mL. On BS, a radioactive substance in trace amount [99mTc-methylene diphosphonate (MDP)] is injected into the patient’s vein and taken up in varying amounts by different sites of the skeletal system in proportion to the

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osteoblastic activity. The areas of increased abnormal bone metabolism appear as “hot spots” on BS. To obtain more information from BS, bone scan index (BSI), a quantitative metric developed at Memorial Sloan Kettering cancer centre, New York [16] and later automatized [17]. BSI served as a prognostic imaging biomarker of overall survival in patients with advanced castration-resistant PCa [18]. However, BS is only considered in conditions with high PSA levels (>10 ng/mL) or with symptoms of bone disease due to its poor performance at low PSA levels [19, 20]. To overcome problems with low specificity of BS, functional imaging techniques such as single photon emission computed tomography (SPECT) and PET are introduced and widely used.

Positron emission tomography (PET)

Basic principles PET is a non-invasive molecular imaging technique which allows quantitative in-vivo measurement of the distribution of so-called radio-ligands or tracers after intravenous administration. PET tracers are labelled with short-lived positron emitting isotopes and upon decay isotope emits a positron. The emitted positron travels at most few millimetres and collides with an electron, simultaneously releasing two photons with an energy of 511 keV in the opposite direction [21]. These photons are detected by an opposing pair of detectors if the signal coincides within a few nanoseconds. A simplified illustration of this process is shown in Figure 2.

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Figure 2. Schematic illustration of positron emission, positron and electron annihilation and the subsequent coincidence detection. Positron upon annihilates with electron in tissue, releasing two photons with an energy of 511 keV, detected by gamma ray detectors.

Quantification PET imaging produces a quantitative radioactive measurement of the physiological parameter of interest. This can be achieved either as a single static image collected at a specific time post-injection (p.i) or as a dynamic scan with full time-course of radioactivity measured over different time frames. To read a static PET image in clinical routine, standardized uptake value (SUV), a semiquantitative parameter is introduced. SUV is defined as the radioactive concentration in the region of interest divided by the injected activity divided by body weight. SUV represents how much tracer uptake exceeds in a specific tissue under uniform distribution. Though SUV is the simplest and the most frequently used measurement, true quantitative measurement of a given tracer in the system of interest is not possible.

Dynamic PET scan will show the rate of change of a tracer concentration in tissue throughout the course of PET study [22]. Time-activity curves (TAC) obtained from dynamic scan represents the time course of the radioactivity of a tracer in a specific volume of interest (VOI). This measured radioactivity concentration is a combination of different states of the tracer reflecting dynamic changes occurring in the tissue and these states are commonly referred to as “compartments”. Subsequent analysis using an appropriate “kinetic model” to represent different compartments results in more

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quantitative estimation of different parameters at voxel level called parametric imaging.

Compartmental model The most commonly used compartment models are one tissue compartment (1TCM) and two tissue compartment model (2TCM) as shown in Figure 3A and B, respectively.

Figure 3. Schematic illustration of a) the one tissue compartment model and b) two tissue compartment model. Cp, Ct, Cf and Cb represent radioactivity concentration in different compartments and K1, k2, k3 and k4 represent the rate constants.

As shown in Figure 3 each box is defined as compartment and several boxes are assumed to describe the tracer kinetics as per different compartmental model. Cp is usually referred as radioactivity concentration in blood or plasma measured by arterial blood sampling after tracer administration during the PET scan. Ct is the radioactivity concentration measured in tissue. Cf and Cb represent the free and bound tracer in the tissue, respectively. K1, k2, k3 and k4 represent the exchange rate of tracer between plasma or blood to tissue, and between different compartments. The pharmacokinetics of the given tracer can be explained by selecting the appropriate compartment model.

PET tracers for PCa in clinical use Several PET tracers targeting different aspects of PCa metabolism are currently available for clinical use.

18F-FDG 18F-FDG, a glucose analogue, takes the advantage of enhanced glycolysis, wherein rapidly growing cancer cells consume excess amounts of glucose to keep complex biological mechanisms and support cell survival. However, the

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slow-growing nature of PCa and urinary excretion of 18F-FDG limits its role in the primary staging and diagnosis and re-staging of PCa [23]. Nonetheless, FDG-PET is useful in specific scenarios such as diagnosis and staging of aggressive PCa, neuroendocrine differentiated and advanced castration-resistant PCa [24]. Due to the limited use of 18F-FDG PET in PCa, the focus is shifted towards the use of metabolic tracers.

18F-Sodium Fluoride (18F-NaF) 18F-NaF, a PET tracer that reflects osteoblastic activity, is used for identification of new bone formation [25]. Higher bone uptake and rapid clearance of 18F-NaF resulted in higher bone-to-background ratio, compared to a bone scan. This improved sensitivity and specificity of 18F-NaF PET in detecting bone lesions compared to bone scintigraphy. Furthermore, in clinics, 18F-NaF PET is used in combination with less-expensive and easily available diagnostic CT to rule out bone metastases and improve accuarcy. However, its bone-only detection capability limits the role of 18F-NaF PET in the era of evolving PCa specific tracers.

11C- or 18F- choline Choline is the precursor for the phospholipids, which constitutes major components of the cellular membrane. Overexpression of choline kinase to meet the increased demands of malignant cells is the biological basis for increased radiolabelled choline uptake. Three radiolabelled forms of choline such as 11C-, 18F- fluoromethyl and 18F-fluoroethyl choline are available for PET imaging in PCa with minor differences in diagnostic accuracy. Of these, 11C-Choline PET has extensively been investigated and showed a pooled detection rate of 62% in detecting site of PCa relapse [26]. The association between PSA level and detection rate of choline PET was studied and showed variable detection rate ranging from 36% to 73% according to PSA level [27].

18F-fluciclovine 18F-FACBC (fluciclovine) is an amino acid-based PET tracer exploits upregulated amino acid transporter activity. Several studies compared 18F-FACBC PET with choline PET and showed 18F-FACBC PET as an alternative and superior PET tracer in localizing recurrent lesions in PCa patients [28-30].

11C-acetate Of various available metabolic tracers, 11C-acetate is widely studied at our institution and around 3000 11C-acetate PET scans were performed. Acetate is a simple intermediate metabolite and the main carbon source for fatty acid and

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lipid synthesis. After entering the cell, acetate is activated to acetyl CoA. The known two major metabolic pathways of acetate are the Krebs cycle and de-novo fatty acid synthesis (Figure 4).

Figure 4. The fate of acetate in a cell. Acetate through different sources enters a cell and activated to acetyl CoA. A fraction of acetyl CoA undergoes oxidative metabolism in mitochondria and leaves as CO2. The other pathway incorporates acetate into phospholipids.

In Krebs cycle acetate undergoes oxidative metabolism and this pathway has been exploited with 11C-acetate PET to quantify myocardial oxidative metabolism [31-33]. However, cancer cells direct the use of acetate mainly towards de-novo lipogenesis.

Among different enzymes regulating fatty acid synthesis, fatty acid synthase (FASN) is the rate-limiting enzyme and extensively studied in the context of carcinogenesis. FASN is identified as an oncogene overexpressed in PCa and PCa aggressiveness is associated with its increased expression [34-37]. Several pre-clinical and clinical studies demonstrated a correlation of FASN expression with 11C-acetate and FASN upregulation is the basis for increased 11C-acetate uptake [36, 38, 39].

Historically, performance of acetate-PET was comparable to choline-PET in identifying recurrent PCa lesions. However, the use of various metabolic

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tracers including acetate is limited due to poor detection rate of PCa relapse at lower PSA level. This shift the focus towards the novel tracer 68Ga-PSMA-11.

68Ga-PSMA-11 Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, overexpressed 100-to 1000-fold in PCa cells compared to normal tissue. The expression of PSMA is shown even with negative or weak PSA staining and increases progressively in higher-grade tumours and metastatic or hormone-refractive disease. As a result, PSMA emerged as a promising target for PET imaging in PCa. Due to limitations in targeting PSMA with antibodies (ProstaScint®) for imaging, small molecule inhibitors targeting active substrate recognition site on the extracellular domain (Figure 5) became attractive for tracer development.

Figure 5. The structure of PSMA and its binding site for PSMA ligands. The commonly used radiolabelled small molecule PSMA ligands target the active substrate recognition site (yellow circle).

68Ga-PSMA-HBED-CC, also known as 68Ga-PSMA-11 is one of the small molecule inhibitors first described by Eder et al and the most commonly used PSMA inhibitor in PET imaging [40]. Since the introduction, 68Ga-PSMA-11 became widely popular and the results showed high detection rate of PCa

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recurrence even at low PSA levels [41-43]. Further, few studies evaluated 68Ga-PSMA-11 as a tool to predict recurrence in patients undergoing prostatectomy [44]. Based on accumulated data on superiority, many centres are driven towards 68Ga-PSMA-11 PET/CT use. Before implementing 68Ga-PSMA-11 PET/CT in the clinical routine, we need to establish documented evidence to prove its higher detection rate compared to the established 11C-acetate PET/CT.

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Aims of the thesis

The overall aims of this thesis were: 1) to investigate tumour biology in primary PCa patients using 11C-acetate PET/CT and evaluate its predictive role in PCa patients with biochemical recurrence after prostatectomy and 2) to evaluate performance of different tracers such as 11C-acetate, 68Ga-PSMA-11, 18F-NaF in identifying PCa relapse in patients after curative therapy. The aims of each study more specifically were:

i) To assess the predictive role of 11C-acetate PET/CT in PCa recurrent patients after prostatectomy towards survival.

ii) To investigate PCa tumour biology using parametric images derived

from dynamic 11C-acetate PET in primary PCa patients and correlate with findings from in-vitro cell experiments in real-time.

iii) To compare the performance of 11C-acetate and 68Ga-PSMA-11

PET/CT in identifying the biochemical recurrence of PCa. iv) To compare the performance of 18F-NaF and 68Ga-PSMA-11 PET/CT

in localizing bone metastases in patients with PCa relapse.

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Methods

Participants This thesis is based on data from a total of 200 subjects including both newly diagnosed and recurrent PCa cases. Briefly, the participants were recruited as follows: Paper I retrospectively included 121 patients (mean age: 72y) with biochemical recurrence after prostatectomy following inclusion criteria which include: biopsy-proven adenocarcinoma of prostate, prostatectomy as first-line curative treatment, biochemical relapse with rising PSA levels after surgery and with no secondary intervention for at least three months before scan. These patients were grouped into cohort I (patients with PCa specific deaths; n=22) and cohort II (rest; 89 alive patients along with 10 patients with PCa non-specific deaths). Paper II comprised 21 newly diagnosed histologically confirmed low-to-intermediate risk PCa patients (mean age: 65y). Paper III comprised 30 PCa patients (mean age: 69y) with biochemical relapse after primary curative therapy. In paper IV, 28 subjects (mean age: 70y) with confirmed metastatic disease to bone were recruited.

PCa cell lines The human PCa cell lines PC3 and DU145 (ATCC, Germany) were used in the in-vtro cell experiments (paper II).

Data acquisition Static PET scan from mid-thigh to skull base was acquired 10-minutes after injection of 11C-acetate (paper I, III). For patients in paper II simultaneous thirty-two-minute dynamic 11C-acetate PET was acquired. Static PET images were acquired around 60 minutes p.i of 68Ga-PSMA-11 (paper III) and 18F-NaF (paper IV). PET investigations were performed on a GE Discovery ST16 scanner (GE Healthcare, Waukesha, WI) with a spatial resolution of 5 mm at the centre of the field and with 3-minutes per bed position (Paper I-II) and on a Discovery MI PET/CT scanner (GE Healthcare, Waukesha, WI) with a spatial resolution of 4 mm at the centre of filed and with 2-minutes per bed position (Paper III-IV). Ordered subsets expectation maximization (OSEM)

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method along with all relevant corrections was used for image reconstruction of 11C-acetate and 18F-NaF PET. Whereas, for 68Ga-PSMA-11 PET image reconstruction block sequential regularized expectation maximization (BSREM) method with β-value 900 was used.

11C-acetate Static PET scan was acquired 10-minute after intravenous administration of 5 MBq/kg 11C-acetate (paper I) and 3.1 MBq/kg 11C-acetate (paper III). Simultaneously with the start of PET scan, 5 MBq/kg 11C-acetate was administered to the subjects in paper II and a 32-minute dynamic PET scan (32 frames; 12×5s, 6×10s, 4×30s, 4×60s, 2×120s, and 4×300s) was acquired.

68Ga-PSMA-11 In paper III, 2 MBq/kg 68Ga-PSMA-11 was administered intravenously and scans were acquired 1 hour after p.i. For subjects in paper IV PSMA-PET scans were acquired 1 hour after p.i of 1.6 MBq/kg 68Ga-PSMA-11.

18F-NaF In paper IV, 18F-NaF PET was performed 1 hour after administering 3.1 MBq/kg 18F-NaF intravenously.

In-vitro cell experiments 500-2000 kBq 11C-acetate was added to DU145 and PC3 cells, 11C-acetate uptake was measured for around 15 minutes and retention for around 30-45 minutes. A titration assay to check 11C-acetate uptake saturation was performed with an increasing amount of 11C-acetate such as 200, 600, 1800 and 3000 kBq. For competition assays, 1 MBq 11C-acetate was added to PC3 cells. To compare 11C-acetate uptake per cell between PC3 and DU145 cells, 3 MBq 11C-acetate was added.

Image analysis

Image analysis of static PET All static PET/CT images were analyzed using Hermes Hybrid Viewer (Hermes Medical Solutions AB, Stockholm, Sweden). All highly suspicious lesions with pathologic accumulation on 11C-acetate and 68Ga-PSMA-11 PET were identified and defined as local recurrence, pelvic or distant lymph node

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and bone metastasis. All pathological lesions suspicious for bone metastasis were identified on 18F-NaF PET. SUV was calculated for each lesion. Tumour volume (TV) was measured over a volume of interest (VOI) defined with a fixed threshold of 30% of SUVmax (paper I). In paper III, VOIs were defined by including all spatially connected voxels within a fixed threshold of 40% of SUVmax and a cut-off of SUV 3 (paper III). In paper IV, SUVmax was measured for 68Ga-PSMA-11 and 18F-NaF PET. Total tumour volume (TTV), defined as the sum of all individual TV, was measured on 11C-acetate and 68Ga-PSMA-11 PET (paper I, III). Total lesion activity, also named as total lipogenic activity (TLA) for 11C-acetate PET, was defined as the sum of SUV and TV of all lesions. In paper I, TLA was defined as sum of SUVmax and TV of all lesions. PET parameters used in this thesis work are highest SUVmax, TTV and TLA in paper I, early and late SUV in paper II, SUVmean, SUVmax, TTV, TLAmean and TLAmax in paper III and SUVmax in paper IV.

Image analysis of dynamic PET VOIager (GE Healthcare, Uppsala, Sweden) was used for dynamic 11C-acetate PET image analysis (paper II). Input function for the kinetic model was extracted from TAC derived from VOIs placed over iliac vessels. Parametric images of K1, k2 and VT, by implementing a simplified single-tissue compartment model, were generated to reflect transport rate (mL/cm3/min), oxidative metabolism (min-1) and anabolic metabolism (mL/cm3).

Analysis of 11C-acetate kinetics in-vitro LigandTracer® White (Ridgeview Instruments AB, Uppsala, Sweden) was used (paper II) to monitor the uptake and retention of 11C-acetate in PC3 and DU145 PCa cells in real-time. LigandTracer®, containing an inclined rotational platform to place a cell dish and the radioactivity detection unit facing the cell dish, measures the radioactive signal from incubated cells and the reference area per each rotation [45, 46]. Well-type thallium (Tl) quenched sodium iodide (NaI) scintillation counter was used (paper II) to measure 11C-acetate uptake per cell in PC3 and DU145 cells.

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Results

Paper I In this retrospective study on 121 patients with PCa recurrence after surgery, 11C-acetate PET/CT was able to detect at least one positive finding in 64 subjects (53%). Detection rate of 11C-acetate PET/CT in patients with PCa-specific death (cohort I; n=22) was 91% (n=20). All 11C-acetate PET/CT volumetric parameters were significantly higher in cohort I (Table 1).

Table 1. Volumetric parameters of 11C-acetate PET/CT in patients with at least one positive PET finding.

PET Parameter (mean±SD) Cohort I Cohort II p-value Highest SUVmax 7.5±2.9 5.7±3.5 0.04 TTV 7.9±12.0 1.2±2.0 0.02 TLA 80±137 11±25 0.04 Five-year-PCa-specific survival was significantly higher in patients with negative 11C-acetate PET/CT compared to PET positive patients (100% vs 80%; p<0.001) (Figure 6). A similar finding was also seen for all-cause survival (94% vs 76%; p=0.002).

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Figure 6. Kaplan-Meier survival curves showing 5-year PCa-specific survival in patients with negative (100%) and positive (80%) 11C-acetate PET. The interval between the date of 11C-acetate PET and the date of PCa-specific death was considered as PCa-specific survival.

A negative linear correlation between highest SUVmax and survival (r=-0.55, p=0.01), non-linear correlation for TLA (r=-0.75 p<0.001) and TTV (r=-78, p<0.001) towards survival was found in patients with PCa-specific death. Multivariate analysis showed TLA tertile was the strongest predictor of survival followed by post-operative Gleason Score sum and number of bone metastasis (Table 2).

Table 2. Cox regression analysis of factors predicting survival in patients with PCa relapse after prostatectomy and at least one finding with pathological uptake of 11C-acetate on PET/CT. HR: hazard ratios per unit change in regressor, CI: confidence interval, LN: lymph node.

Parameter Univariate analysis Multivariate analysis HR (95% CI) p-value HR (95% CI) p-value

Post-operative Gleason sum 1.77 (1.15-2.74) 0.01 1.84 (1.01-3.58) 0.05 Tertile of highest SUVmax 2.35 (1.29-4.55) <0.01 0.45 (0.14-1.53) 0.21 Tertile of TTV 2.74 (1.50-5.35) <0.01 0.68 (0.23-2.14) 0.50 Tertile of TLA 3.33 (1.77-6.83) <0.01 5.63 (1.22-27.68) 0.03 Number of distal LN metastases 1.29 (1.12-1.46) <0.01 1.11 (0.94-1.29) 0.18 Number of bone metastasis 2.16 (1.55-3.00) <0.01 1.74 (1.13-2.64) 0.01

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Stratification of PET volumetric parameters into tertiles followed by survival analysis showed a significant difference between tertile with the highest TLA (5-year survival: 51%) and lower and middle tertiles (Figure 7). Similarly, survival in patients of the tertile with the highest SUVmax and TTV was significantly lower, compared to tertiles with negative and lower tertiles.

Figure 7. Kaplan-Meier survival curves associated with total lipogenic activity (TLA) tertiles showing 5-year-PCa-specific survival. Patients were grouped as Negative (no signs of malignancy on PET, n=57) and TLA tertiles (tertile I: n=21, tertile II: n=22 and tertile III: n=21). Significant differences in survival among the tertiles were noted (tertile I vs II, p=0.02 and tertile II vs III, p=0.009).

Paper II In the dynamic 11C-acetate PET/CT study (paper II) parametric images of K1, k2 and VT were successfully produced using a 1TCM (Figure 8).

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Figure 8. parametric images of K1, k2 and VT dervied from dynamic 11C-acetate PET using single-tissue compartment model in axial, coral and sagittal plane from left to right, respectfully. Cancerous region in prostate showed significantly increased K1 (0.34 vs 0.23 mL/cm3/min; p<0.01) and VT (8.6 vs 5.4 mL/cm3; p<0.01) compared to normal prostate. Further, SUV is also significantly high in cancerous tissue (early SUV: 2.99 vs 1.99; p<0.01, late SUV: 3.04 vs 2.08; p<0.01) compared to normal prostate. No significant difference in oxidative metabolism between low-to-intermediate PCa and normal prostate (k2: 0.04 vs 0.05 min-1; p=0.26) was observed.

In-vitro studies showed that 11C-acetate uptake in PC3 and DU145 cells was proportional to 11C-acetate concentration and significantly higher in PC3 cells (2.94 vs 0.72 h-1, p=0.002) (Figure 9).

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Figure 9. 11C-acetate uptake measured with ligandTracer® with increasing 11C-acetate concentration (200, 600, 1800 and 3000 kBq; blue arrows) in PC3 (red curve) and DU145 (black curve). 11C-acetate retention was measured after 125 minutes.

11C-acetate uptake (counts per second (cps) per cell showed significantly higher uptake in rapidly growing PC3 cells compared to DU145 (0.008 vs 0.003 cps/cell; p<0.0001) (Figure 10).

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Figure 10. 11C-acetate uptake (counts per second (cps) normalized to the number of cells in PC3 (black) and DU145 (red). PC3 cells showing significantly high uptake.

Paper III In this prospective comparative study, 68Ga-PSMA-11 PET/CT identified 172 highly suspicious lesions in 25 patients compared to 126 lesions seen on 11C-acetate PET/CT in 25 patients (McNemar test, p=0.45). The detection rate of positive lesions using both 68Ga-PSMA-11 and 11C-acetate PET/CT was 77%. In particular, 68Ga-PSMA-11 PET/CT performed better in detecting PCa recurrence in lymph nodes (n= 107 vs 80; p=0.02) and bone metastasis (n= 54 vs 35; p=0.0001) compared to 11C-acetate PET/CT (Figure 11, 12).

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Figure 11. localizing lymph node (left column, white arrow) and bone metastasis (right column, white arrow) in the trans-axial plane of 11C-acetate (b, e) and 68Ga-PSMA-11 PET/CT (c, f) with respective CT images (a, d) in two different patients. A76-year-old 3+3 PCa patient with biochemical relapse (PSA 72 ng/mL) after curative therapy showed a small pelvic lymph node on CT (a), with negative uptake on 11C-acetate PET/CT (b) but detected by 68Ga-PSMA-11 PET/CT (c). In similar, a sclerotic bone lesion in the pelvis on CT (d) with no uptake on 11C-acetate PET/CT (e ) was visualized by 68Ga-PSMA-11 PET/CT (f) in a 74-year-old 3+4 PCa patients with PSA 28 ng/mL after intended curative therapy.

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Figure 12. Percentage of patients with at least one positive lymph node (a) and bone lesion (b) on both PET scans.

Pathological lesion uptake, TTV and TLA measured with 68Ga-PSMA-11 PET/CT were significantly higher compared to 11C-acetate PET/CT (Table 3). Stratifying the lesions based on the anatomical region also showed similar findings.

Table 3. Parameters derived from VOIs on both acetate and PSMA PET. Parameters (mean±SD)

11C-acetate PET 68Ga-PSMA-11

PET p-value

Highest SUVmean 3.7±1.9 7.5±6.4 0.004 Highest SUVmax 6.4±4.0 22.6±23.9 0.001 TTV 21.1±31.9 48.1±67.5 0.053 TLAmean 100.4±164.1 467.6±896.4 0.035 TLAmax 198.5±348.9 1587.2±3439.2 0.036 On univariate analysis, PSA velocity (PSAVel) showed a significant correlation with all PET parameters from 11C-acetate and 68Ga-PSMA-11 PET scans. Similarly, all PET volumetrics except SUVmax from both scans showed moderate but significant correlations with PSA at time of the scan. Both PET findings were presented during multidisciplinary conferences, wherein, 68Ga-PSMA-11 PET/CT influenced change in treatment strategy in 6 patients (20%), whereas 11C-acetate PET/CT influenced a change in 1 patient.

Paper IV In this study, 68Ga-PSMA-11 PET/CT was compared to 18F-NaF PET/CT to localize bone metastasis in 28 patients with high suspicion of cancerous spread to the bone. On both PET scans, seven patients (25%) were negative for bone lesions. However, in these patients, 68Ga-PSMA-11 PET/CT identified at least

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one local and or lymph node lesion and influenced treatment strategy (Figure 13).

Figure 13. A 75-year-old PCa patient having PSA relapse (PSA=42 ng/mL)after primary and secondary curative therapy. On referral, both fluoride (a) and PSMA (b) PET scans were negative for bone metastasis but lymph nodes in para-aortic region (c) showed positive uptake on PSMA-PET. Corresponding lymph node visualized on low-dose CT scan (d).

The overall spread of bone metastasis classified into axial and appendicular skeletal lesions based on location was identical on both PET scans, except in one patient, in whom 68Ga-PSMA-11 PET/CT showed bone lesions only in the axial skeleton. In total 18F-NaF PET/CT detected a higher number of bone lesions compared to 68Ga-PSMA-11 PET/CT (699 vs 579, p<0.001) (Figure 14). However, univariate analysis showed significant correlation of PSA at time of scan with tracer uptake of 68Ga-PSMA-11 PET/CT (SUVmax: r=0.58, p=0.01) but not with 18F-NaF PET/CT.

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Figure 14. Maximum intensity projection (MIP) images of 18F-NaF PET/CT (a) and 68Ga-PSMA-11 PET/CT (b) showing bone metastasis in a 65-year-old 4+3 hormone refractory PCa patient who underwent radiation, hormonal and chemotherapy. PET/CT in the trans-axial plane showed two sclerotic lesions (red arrows) on 18F-NaF PET/CT (C), whereas 68Ga-PSMA-11 PET/CT detected only one sclerotic lesion with reduced intensity (d, left red arrow). Corresponding CT showing respective sclerotic lesions in the trans-axial plane (e).

On comparison with the diagnostic CT, 68Ga-PSMA-11 PET/CT performed better in localizing soft tissue spread in the prostatic fossa and lymph nodes (Figure 15).

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Figure 15. Percentage and numbers of local recurrence in the prostatic fossa detected by CT and PSMA-PET(a). Percentage and number of patients with suspicious lymph nodes detected by CT and PSMA-PET(b. 1) followed by percentage and number of suspicious lymph nodes identified by CT and PSMA-PET(b.2).

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Discussion

The first step in this thesis was to investigate the prognostic role of 11C-acetate PET/CT to predict survival in biochemical relapse patients after prostatectomy. Though 11C-acetate PET/CT has previously been used for primary staging and re-staging of PCa, its role as a predictive tool of survival was not known. The results from Paper I clearly showed the association of 11C-acetate PET/CT on survival in PCa relapsed patients after surgery. This study highlighted that 11C-acetate PET/CT through visualizing disease spread identified patients at risk of early death. Similarly, a negative PET or minimal disease ruled out early death and showed a highly favourable outcome. From both clinician and patient perspectives, this finding could be useful to relieve anxiety in patients with negative PET findings but having a rise in PSA levels.

Considering the slow-progressive nature of PCa and other comorbidities, detection of distant metastasis is highly influential at relapse state. The current study with a doubled risk of relative death in the presence of bone metastasis provided evidence that this advanced and costly PET imaging technique was able to predict the outcome. Further, TLA which encompassed tumour volume and SUV to represent spatial and functional tumour burden turned out to be an independent prognostic factor of survival. Several previous studies established an association between upregulation of FASN and increased 11C-acetate uptake [38, 39, 47, 48]. Thus, inhibition of FASN seems to be a promising target for drug development [49-51]. In further support, the current study strengthened the link between malignant lipogenesis and outcome and advocates use of 11C-acetate PET/CT as a tool for new drug development targeting lipogenic tumours.

The results from paper II showed that a basis function implementation of single-tissue compartment model successfully yielded parametric images describing 11C-acetate kinetics in PCa patients. Similarly, LigandTracer®, a novel instrument was able to investigate in-vitro 11C-acetate kinetics in real-time. Both in-vitro and dynamic PET data from patients were able to show underlying biological changes such as 11C-acetate uptake and retention. Previous studies using other PET tracers quantified tumour blood flow (TBF) in PCa and showed that increase in TBF was associated with aggressiveness [52, 53]. In line with those studies, increased uptake (K1) of 11C-acetate in proportion to aggressiveness both at cell and patient-level supported the view of 11C-acetate uptake as an indicator of PCa growth. With evidence on inhibition of growth by driving cancer cells back into oxidative metabolism

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[54], determining the rate of oxidative metabolism in PCa is interesting. However, oxidative metabolism (k2), extracted from dynamic 11C-acetate PET/CT data did not significantly differ from cancerous to normal prostate. Both in-vitro and in-vivo evidence of increased retention, quantified by VT on PET scan further supported the notion on the incorporation of 11C-acetate into anabolic metabolism. The evidence of a similar pattern of uptake and retention from cell experiments and clinical study indicated use of 11C-acetate PET/CT to measure the extent of lipid synthesis and thus PCa progression.

Findings from these two studies on the use and quantification of 11C-acetate established 11C-acetate PET/CT as a predictive tool of prognosis in PCa relapse and as a biomarker for pharmacokinetic studies. However, in the advent of amplified development of new PCa specific tracers with improved detection rate and better sensitivity, the scope of use of 11C-acetate PET/CT is limited. Of various tracers developed to target PSMA, 68Ga-PSMA-11 is the most commonly used tracer and the results suggested that 68Ga-PSMA-PET/CT has higher detection rate compared to all other available metabolic tracers used in PCa diagnosis.

Evidence from Paper I about the impact of 11C-acetate PET/CT volumetrics on survival encouraged us to apply similar PET volumetrics for 68Ga-PSMA-PET/CT. On comparison, both 68Ga-PSMA-PET/CT and 11C-acetate PET/CT volumetrics showed significant and stronger correlation not only with PSA at time of PET scan but also with PSAvel. However, in this comparative study 68Ga-PSMA-PET/CT performed better, particularly, in localizing lymph node and bone metastases. The underlying mechanism for intensity uptake is different for both tracers. While 11C-acetate PET/CT denotes the on-going de-novo lipogenesis, 68Ga-PSMA-PET/CT reflects receptor expression. Hence, the presence of metabolically dormant but PSMA over-expressive PCa lesions might be one of the reasons for the higher detection rate of 68Ga-PSMA-PET/CT.

However, approximately 10% of PCa lesions appear negative on 68Ga-PSMA-PET/CT imaging [55] and little is known about this phenomenon. A recent study documented both intra and inter individual heterogeneity of PSMA expression and the corresponding altered gene expression [56]. In the light of heterogeneity, combining 68Ga-PSMA-PET/CT with 11C-acetate PET/CT or other metabolic PET would be beneficial in a subset of patients. The results from paper III provided further evidence on the use of 68Ga-PSMA-PET/CT as the most optimal first-line diagnostic PET tool currently available for PCa staging.

As PCa progresses, spread to the bone is common and approximately 80% of patients with advanced PCa are identified with bone metastasis [15]. Presence of bone metastasis has a significant impact on patient prognosis with reduced cancer-free-survival [57]. Hence, accurate detection of bone lesions is essential and among available bone-seeking nuclear medicine tracers, 18F-NaF is considered as “gold standard”. So far, limited studies compared the

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performance of 18F-NaF PET/CT with 68Ga-PSMA-PET/CT to locate bone metastasis [58-61]. The results from these studies were ranging from high sensitivity of 18F-NaF PET/CT to comparable accuracy. To establish further evidence on the use of 68Ga-PSMA-11 PET/CT, in paper IV we compared 18F-NaF and 68Ga-PSMA-11 PET/CT. The results from study IV showed that the majority of bone lesions identified on 18F-NaF PET/CT were also detected by 68Ga-PSMA-11 PET/CT (83%). Though the spread of bone metastasis was identical on both scans, 68Ga-PSMA-11 PET/CT influenced the choice of treatment strategy particularly in patients with negative and oligo-metastatic bone disease. The reason for a PET referral at PSA relapse is not only to locate bone lesions but also for non-osseous spread. As clearly seen in study IV, where both PET scans were negative for bone lesions but 68Ga-PSMA-11 PET/CT was able to detect lymph node and local spread. In this regards, 68Ga-PSMA-11 PET/CT has the advantage to assess both osseous and non-osseous spread in a single examination. To improve soft-tissue contrast, a diagnostic CT was performed immediately after 18F-NaF PET/CT but had low detectability in comparison to 68Ga-PSMA-11 PET/CT.

Biochemical relapse in the course of PCa progression is seen in up to 40% of patients after primary curative therapy. The classical definition for biochemical recurrence is when the PSA level increases over 0.2 ng/mL after prostatectomy and 2 ng/mL after radiation therapy as primary treatment [62, 63]. Use of PET imaging facilitates the visualization of metastatic spread in advance even before its clinical appearance. It was previously known that the spread of PCa relapse can be correlated with PSA levels. In support of this, through the study of this thesis work, we noticed an association between PET findings and PSA levels. In paper I, a highly favourable outcome was seen in subjects with minimal 11C-uptake even when PSA was well below 1 ng/mL and the detection rate was 31% at this PSA level. A significant correlation of PSA with early SUV and 11C-acetate uptake (K1) in low-to-intermediate PCa patients from paper II provided further evidence on the association of PET measurements and PSA levels.

In contrast, patient stratification at low PSA levels in 68Ga-PSMA-11 PET/CT comparative studies (paper III and paper IV) was not possible due to the limited number of subjects. However, a significant correlation between PSA and SUV measurements of all PET scans in both studies established the role of PSA on PET findings. Besides, a significant difference in PSA levels between patients with and without PET findings was also noticed in these studies. To further support, previous studies already showed the influence of PSA levels on the detection rate of 68Ga-PSMA-PET/CT [41, 43, 64, 65]. All these findings directed towards the critical role of PSA in patient management at PCa relapse.

Overall, this thesis work suffered a few limitations. One of the main limitations while conducting these studies was histopathological confirmation of positive lesions identified on PET imaging. Retrospective design, non-

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inclusion of high-risk aggressive PCa and lack of follow-up scan and or biopsy as standard in PSMA-PET comparative studies with a relatively low number of patients are the specific limitations for each study, respectively. Obtaining a biopsy sample was neither ethically nor practically feasible for all lesions and the collection of biopsy material was sought out only in patients in whom PET/CT showed high uptake suggestive of other diseases. For example, a patient in study III showed high intense uptake in the thyroid on both 11C-acetate and 68Ga-PSMA-11 PET/CT but histopathology later confirmed that lesion as oncocytoma of the thyroid.

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Conclusions and future perspectives

The first two studies presented in this thesis were focused on the utility of 11C-acetate PET/CT as a predictive tool and study of PCa biology using dynamic 11C-acetate PET. The performance and role of 68Ga-PSMA-11 PET/CT in PCa restaging and its establishment as a clinical routine was further investigated with comparative studies. The conclusions made from this work are

• PCa-specific survival in PCa relapsed patients after prostatectomy was predicted using 11C-acetate PET/CT (paper I). TLA, measured on 11C-acetate PET/CT to denote lipogenic activity, was the strongest independent predictor of survival. Similar volumetrics can be applied to measure tumour burden with other PET tracers used in PCa diagnosis (paper I).

• A simplified single-tissue compartment model was able to describe 11C-acetate kinetics (paper II). Similar pattern of uptake and retention of 11C-acetate in patients and cell experiments demonstrated the potential of 11C-acetate PET to measure PCa growth and aggressiveness (paper II).

• In comparison to 11C-acetate PET/CT, 68Ga-PSMA-11 PET/CT performed better in localizing PCa relapse, in particular, lymph nodes and bone metastasis (paper III).

• The majority of bone metastasis seen on 18F-NaF PET/CT were also seen with 68Ga-PSMA-11 PET/CT (paper IV).

Since the introduction in 2004, 11C-acetate PET/CT was widely used at our institution to make decisions in PCa patients. However, high sensitivity with improved detection rate favours the use of 68Ga-PSMA-11 PET/CT. In this thesis work, we documented evidence on better performance of 68Ga-PSMA-11 PET/CT and recommended to use 68Ga-PSMA-11 PET/CT in clinical routine for PCa diagnosis and re-staging in the setting of relapse. One of the future perspective regards to PSMA-PET/CT is its evolving role in treatment management. PSMA-PET can be established as a promising tool to evaluate PSMA targeted therapy (177Lu labelled PSMA) in advanced PCa patients. Emerging evidence is supporting PSMA-PET as a promising tool for therapy evaluation [66].

Another promising future perspective is development in PSMA targeted PET tracers. To overcome known limitations of 68Ga labelled PSMA such as

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high activity in the urinary bladder and relatively short half-time, 18F labelled PSMA tracers e.g., 18F-PSMA-1007, 18F-DCFPyL are developed and received increased attention. However, to establish the role of newly available PSMA PET tracer for therapy evaluation, prospective trials are needed which will span over several years to compile the evidence.

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Acknowledgments

This work was funded by grants obtained by Prof. Jens Sörensen from Cancerfonden. I feel extremely happy to thank everyone who is part of my journey towards finalizing this thesis, which would not have been possible without their support and help. My sincere apologies if I have missed mentioning someone.

I would especially like to extend my gratitude to my main supervisor Prof. Jens Sörensen, for his continuous guidance, supervision and support throughout my research career in Sweden. I thank you for choosing me as a doctoral student under your arms and paving the way into the research world. No words could explain the influence and motivation I gained from your kindness and gestures over these years, I believe no better supervisor I can dream of. I learnt a lot from you not only on how to be a good researcher and a good person in general. The time I spent and the support I received from you even before I began my journey as a researcher helped me to overcome issues to stay in Sweden and feel it as my second home. I am forever grateful for believing in me and providing with great opportunities.

My profound gratitude for the co-supervisors; Mark Lubberink, for your valuable suggestions and discussions in my research and for sharing your great expertise and knowledge and Irina Velikyan, for your guidance, all the time you spent on improving my manuscripts and for the interesting and great discussions not only on research but also on all range of subjects.

I thank all my co-authors who contributed in my publications, especially Silvia Johansson, for all your inputs and excellent discussions, Andreas Thor, Ante and Jos Buijs for the interesting projects and thank you for the collaboration.

Division of Biomedical radiation Sciences (BMS), for the master’s programme, Medical Nuclide Techniques, and the teaching Staff especially Prof. Vladimir Tolmachev, Prof. Anna Orlova, Olof Eriksson, Ola Åberg, Sergio Estrada, Marika Nester, Mohamad Altai and Prof. Bo Stenerlöw.

My classmates during my master’s programme with special mention to Alan Saaty, for his great friendship and sharing his experiences in Sweden.

Hadis, a friend and a person with a strong character. Thank you for sharing your knowledge and for showing me how to do lab experiments. My amazing friends and colleagues at “våning 5”, now “14B”, without you guys this would never have been possible.

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Marcos, my good friend and thank you for being there in my ups and downs and supporting me. You made the working place more fun with your jokes though sometimes sarcastic, spoiled me with your flying hobbies but shared your deep research knowledge. Without your teasing and continuous insist I would never join a gym. I wish you and Isabel as a couple have a great future forever.

Ezgi, a good friend and patient listener with good humour. Thank you for sharing your knowledge and helping me to learn new things.

Elin, a good friend always ready to help when I need. Thank you for helping me with the manuscript and motivating me to improve my innebandy game.

Ali, a great friend with a similar background. Thank you for sharing our common troubles with bureaucracy particularly with the migration agency and for those great coffee corridor meetings along with Marcos. My sincere thanks to Marcos and you for helping me to fix figures in the thesis.

My, a friend with extra talent in pole dancing. Without you, I never know “Mallakhamba” and thank you for helping with the information on thesis defence.

I want to thank all my past and present colleagues at the PET Centre, especially Adrian, Anders wall, Anders Sundin, Lieuwe, Mattias, Torsten, Victor, Gunnar, Achyut Ram, Anna, Mimmi, Kaisa, Hampus, Tomas and Mats for helping me.

I thank PPP for your great support to conduct our experiments, special mentioning to Ram, for our long discussions. It was always a great pleasure to meet you and talk in our common language.

Over the years, I gained many valuable friends through playing innebandy, badminton and cricket. It is impossible to name them all but thank you, everyone, for being part of my journey, for helping me to learn new skills and for the great memories and experiences.

I thank all the teachers I encountered throughout my life for educating me and for sharing your wisdom.

I thank my mom and dad for all the support you gave me since my childhood and for allowing to pursue my interest, though it was hard for me to leave you and come to Sweden. I thank my brother Anil and Kranthi vadina for their tremendous support throughout this journey. Thank you, Saanvi, and Ruthvik for giving me joy and brought my childhood back by allowing me to play with you.

Words are not enough to express my love and gratitude to my wife, Priya, for your love, patience, and constant support throughout my journey. Thank you for everything and being part of my life. A million thanks to Saatvik, you gave a completely new meaning to our lives, and I enjoy every moment of this new journey.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1650

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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