Linköping University Medical Dissertations No. 1196

Quantitative Evaluation of Contrast Agent Dynamics in Liver MRI

Nils Dahlström

Center for Medical Image Science and Visualization

Division of Radiological Sciences Department of Medicine and Health Sciences Faculty of Health Sciences Linköping University, SE-581 85 Linköping, Sweden

Linköping 2010

This work has been conducted within the Center for Medical Image Science and Visualization (CMIV) at Linköping University, Sweden. CMIV is acknowledged for provision of financial support and access to cutting-edge research infrastructure.

Quantitative Evaluation of Contrast Agent Dynamics in Liver MRI

Linköping University Medical Dissertations No. 1196 Copyright  Nils Dahlström, 2010, unless otherwise noted

Center for Medical Image Science and Visualization

Division of Radiological Sciences Department of Medicine and Health Sciences Faculty of Health Sciences Linköping University, Sweden

Published articles have been reprinted with the permission of the copyright holder.

ISBN 978-91-7393-338-4 ISSN 0345-0082

Printed by LiU-Tryck, Linköping, Sweden, 2010.

Cover: MRI image of the liver 20 minutes after Gd-EOB-DTPA contrast injection.

Abstract

ABSTRACT

The studies presented here evaluate the biliary, parenchymal and vascular enhancement effects of two T1-shortening liver-specific contrast agents, Gd-BOPTA and Gd-EOB-DTPA, in Magnetic Resonance Imaging (MRI) of healthy subjects and of patients.

Ten healthy volunteers were examined with both contrast agents in a 1.5 T MRI system using three-dimensional gradient echo sequences for dynamic imaging until five hours after injection. The enhancement of the common hepatic duct in contrast to the liver parenchyma was analyzed in the first study. This was followed by a study of the image contrasts of the hepatic artery, portal vein and middle hepatic vein versus the liver parenchyma.

While Gd-EOB-DTPA gave an earlier and more prolonged enhancement and image contrast of the bile duct, Gd-BOPTA achieved higher maximal enhancement and higher image contrast for all vessels studied during the arterial and portal venous phases. There was no significant difference in the maximal enhancement obtained in the liver parenchyma.

In a third study, another 10 healthy volunteers were examined with the same protocol in another 1.5 T MRI system. Using signal normalization and a more quantitative, pharmaco- kinetic analysis, the hepatocyte-specific uptake of Gd-EOB-DTPA and Gd-BOPTA was calcu- lated. A significant between-subjects correlation of the uptake estimates was found and the ratio of these uptake rates was of the same magnitude as has been reported in pre-clinical studies. The procedure also enabled quantitative analysis of vascular enhancement proper- ties of these agents. Gd-BOPTA was found to give higher vessel-to-liver contrast than Gd- EOB-DTPA when recommended doses were given.

In the final study, retrospectively gathered datasets from patients with hepatobiliary disease were analyzed using the quantitative estimation of hepatic uptake of Gd-EOB-DTPA described in the third study. The uptake rate estimate provided significant predictive ability in separating normal from disturbed hepatobiliary function, which is promising for future evaluations of regional and global liver disease.

In conclusion, the differing dynamic enhancement profiles of the liver-specific contrast agents presented here can be beneficial in one context and challenging in another. Diseases of the liver and biliary system may affect the vasculature, parenchyma or biliary excretion, or a combination of these. The clinical context in terms of the relative importance of vascular, hepatic parenchymal and biliary processes should therefore determine the contrast agent for each patient and examination. A quantitative approach to analysis of contrast-enhanced liver MRI examinations is feasible and may prove valuable for their interpretation.

Keywords: liver, spleen, hepatobiliary system, liver function, MRI, DCE-MRI, Gd- BOPTA, Gd-EOB-DTPA, pharmacokinetic, hepatocyte, relaxivity.

Till min familj

“Work expands so as to fill the time available for its completion.” C. Northcote Parkinson

“For five years I couldn't sleep if I lay on my left side. It felt like my guts weren't in the right place. It didn't hurt, it just felt weird knowing my internal organs might not be where they belonged. When I lay on my other side everything was fine. I don't know what changed, but now both sides work and my guts feel okay. I credit my spleen for fixing the problem because I don't know what else it's supposed to do and it rarely gets credit.” Scott Adams

List of Papers

LIST OF PAPERS

This thesis is based on the following four papers, which are referred to by their Roman numerals (I–IV).

I. Dahlström N, Persson A, Albiin N, Smedby Ö, Brismar TB. Contrast-Enhanced Magnetic Resonance Cholangiography with Gd-BOPTA and Gd-EOB-DTPA in Healthy Subjects. Acta Radiol. 2007 May; 48(4):362–8.

II. Brismar TB, Dahlström N, Edsborg N, Persson A, Smedby Ö, Albiin N. Liver Vessel Enhancement by Gd-BOPTA and Gd-EOB- DTPA – A Comparison in Healthy Volunteers. Acta Radiol. 2009 Sept; 50(7):709–15.

III. Dahlqvist Leinhard O, Dahlström N, Kihlberg J, Brismar TB, Smedby Ö, Lundberg P. Liver Specific Gd-EOB-DTPA vs. Gd- BOPTA Uptake in Healthy Subjects – A Novel and Quantitative MRI Analysis of Hepatic Uptake and Vascular Enhancement. Submitted to European Radiology on 21 Aug, 2010.

IV. Dahlström N, Dahlqvist Leinhard O, Sandström P, Brismar T, Lundberg P, Smedby Ö. Hepatic Uptake of Gd-EOB-DTPA in Patients with Varying Degree of Hepatobiliary Disease. Submitted to European Radiology on 21 Aug, 2010.

The contents of this thesis have been partially presented in Papers I and II, which are also part of the Licentiate Thesis: Dahlström N, Magnetic Resonance Imaging of the Hepatobiliary System Using Hepatocyte-Specific Contrast Media. Linköping University Electronic Press; 2009. Linköping Studies in Health Sciences. Thesis No 95.

vii Author Contributions

AUTHOR CONTRIBUTIONS

Paper I I participated in the planning of the volunteer examinations, image quality assurance, and the planning of the image review process, which involved three radiologists, including myself. I was responsible for final data assembly and analysis, with the assistance of Örjan Smedby in the planning of the statistical analysis. I wrote the first and final draft of the manuscript and managed the correspondence with the publishing journal.

Paper II I participated in the planning of the volunteer examinations, and the planning of the image review process. I participated in the data analysis and result assembly, and also in the editing of the manuscript.

Paper III I participated in the planning and management of the volunteer examinations, image dataset storage and handling, image quality assurance, and the planning of the image review process, in which I was responsible for correct measurement placement. I assisted Olof Dahlqvist Leinhard in developing parts of the analysis process, and contributed to quality assurance testing. I participated in the editing and submission of the manuscript.

Paper IV I planned and performed the retrospective retrieval of patient MRI examinations and the management of datasets prior to analysis, together with Olof Dahlqvist Leinhard. I performed the image review and participated in the processing, statistical analysis and interpretation of measurement data. I wrote the first and final draft of the manuscript and managed the manuscript submission.

viii Table of Contents

TABLE OF CONTENTS

1. INTRODUCTION 1 2. BACKGROUND 3

2.1. Hepatic and Biliary Disease 3 2.2. Imaging Modalities 5

2.2.1. Ultrasound (US) 5 2.2.2. Computed Tomography (CT) 6 2.2.3. Nuclear Medicine (NM) 7 2.2.4. Magnetic Resonance Imaging (MRI) 8

2.3. Contrast Media in Liver MRI 14

2.3.1. Safety of Gadolinium-Based Contrast Media 15 2.3.2. Gadolinium Contrast Media and Relaxivity 15 2.3.3. Contrast Administration and Bolus Timing 17 2.3.4. Gd-BOPTA (MultiHance®) 20 2.3.5. Gd-EOB-DTPA (Primovist®) 20 2.3.6. Contrast Agent Uptake Mechanisms 21

2.4. Dynamic Contrast-Enhanced MRI 24

2.4.1. Background 24 2.4.2. Enhancement 24 2.4.3. Semi-Quantitative Analysis 25 2.4.4. Quantitative Pharmacokinetic Analysis 26

2.5. Liver Function Tests, Prognostic Scores 29

3. AIMS 31 4. MATERIAL AND METHODS 33

4.1. Subjects 33

4.1.1. Paper I, II and III – Healthy Volunteers 33 4.1.2. Paper IV – Patients 33

ix Table of Contents

4.2. Contrast Media 35 4.3. MRI Technique 36 4.4. Image and Signal Intensity Analysis, Statistical Analysis 37

4.4.1. Paper I 37 4.4.2. Paper II 39 4.4.3. Paper III and IV 40

5. RESULTS 43

5.1. Paper I 43 5.2. Paper II 45 5.3. Paper III 47 5.4. Paper IV 49 5.5. Comparative Analyses – Papers I–III 53

6. DISCUSSION 55

6.1. Biliary Imaging 55 6.2. Vascular Imaging 57 6.3. Hepatic Parenchymal Enhancement 60

6.3.1. Semi-Quantitative analyses (Papers I, II) 60 6.3.2. Quantitative Analysis of Contrast Agent Uptake 61 6.3.3. Comparative Analyses – Papers I–III 67

6.4. Study Design and Limitations 68

6.4.1. Volunteer Sampling 68 6.4.2. Contrast Media Administration 68 6.4.3. MRI Protocol 70 6.4.4. Image Review, Documentation and Analysis 71

6.5. Clinical Applications, Future Studies 71

7. CONCLUSIONS 77 8. ACKNOWLEDGEMENTS 79 9. REFERENCES 81

x Abbreviations

ABBREVIATIONS

ANOVA Analysis of Variance ATP Adenosine Triphosphate AUC Area Under the Curve B0 The static magnetic field B1 The varying radiofrequency magnetic field produced by the RF-coil BBB Blood-Brain Barrier CAT Contrast Arrival Time CCC Cholangiocarcinoma CE-MRC Contrast-Enhanced Magnetic Resonance Cholangiography CHD Common Hepatic Duct CMIV Center for Medical Image Science and Visualization CNS Central Nervous System DCE-MRI Dynamic Contrast-Enhanced Magnetic Resonance Imaging ECF Extracellular fluid EES Extracellular Extravascular Space EPR Electronic Patient Records FA Flip Angle FDA United States Food and Drug Administration FOV Field Of View γ Gyromagnetic ratio [T-1s-1] GBCA Gadolinium-Based Contrast Agent Gd Gadolinium Gd-BOPTA Gadobenate dimeglumine Gd-DTPA Gadopentetic Acid Gd-EOB-DTPA Gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid GRE Gradient Echo HBV Hepatitis B Virus HCC Hepatocellular Carcinoma HCV Hepatitis C Virus HIDA Hepatobiliary Iminodiacetic Acid ICG Indocyanine Green In vitro In phantoms or tubes In vivo In the living body LSC Liver-to-Spleen Contrast ratio (synonymous with Q-LSC) MDCT Multi-Detector Computed Tomography MR Magnetic Resonance MRC(P) Magnetic Resonance Cholangio(-Pancreato)graphy MRI Magnetic Resonance Imaging MRP Multidrug Resistance Protein Mxy Transverse magnetization Mz Longitudinal magnetisation

xi Abbreviations

NAFLD Non-Alcoholic Fatty Liver Disease NASH Non-Alcoholic Steatohepatitis NMR Nuclear Magnetic Resonance NSF Nephrogenic Systemic Fibrosis NTCP Na+/Taurocholate Cotransporting Polypeptide OATP Organic Anion Transporting Polypeptide PD Proton density PET Positron Emission Tomography PSC Primary Sclerosing Cholangitis Q-LSC Quantitative Liver-to-Spleen Ratio (synonymous with LSC) r1 Longitudinal relaxivity [s-1mM-1] r2 Transverse relaxivity [s-1mM-1] R1 Longitudinal relaxation rate [s-1] R2 Transverse relaxation rate [s-1] RE Relative Enhancement RF Radio Frequency [MHz] ROI Region of Interest SBC Splenic Blood Clearance SD Standard Deviation SE Spin Echo SI Signal Intensity SPGRE Spoiled Gradient Echo SPIO Small Superparamagnetic Iron Oxide particles T Tesla – unit of magnetic field strength T1 Longitudinal relaxation time [s] T2 Transversal relaxation time [s] T2* Transversal relaxation time including B0 inhomogeneities effects [s] TE Echo Time [s] THRIVE T1-weighted high-resolution isotropic volume examination TR Repetition time [s] TTP Time To Peak UNOS United Network for Organ Sharing USPIO Ultrasmall Superparamagnetic Iron Oxide particles VIBE Volumetric Interpolated Breath-hold Examination VIF Vascular Input Function

xii Introduction

1. INTRODUCTION

The non-invasive methods for discovering and characterizing disease processes in the liver and the biliary system have improved greatly over the last decades due to the continuing development of computed tomography (CT), sonography or ultrasound (US), nuclear medicine (NM) and magnetic resonance imaging (MRI). Contrary to CT and NM, magnetic resonance imaging does not expose the patient to any ionizing radiation, and there are other advantages over US, such as greater volume coverage and less operator dependence. When body MRI was introduced in the early 1980s, its superior soft-tissue contrast was believed to provide sufficient diagnostic information for most needs, without the use of contrast media. Gadolinium-based and other contrast media were, however, soon shown to be of importance in disease detection and characterization. Liver-specific substances have also been developed and are now commonly used in various clinical situations. There is a continuous need for comparative studies to validate the choice of contrast medium and imaging strategy in different settings. With the introduction of Gd-EOB-DTPA (Primovist® 0.25 mmol/ml, Bayer Schering Pharma, Berlin, Germany) in 2004, there were two similar Gadolinium-based liver-specific compounds on the market, the other being Gd-BOPTA (MultiHance® 0.5 mmol/ml, Bracco Imaging, Milan, Italy). This thesis is aimed at evaluating the biliary, hepatic parenchymal and vascular enhancement effects of Gd-BOPTA and Gd-EOB-DTPA in MRI of healthy subjects and patients, employing semi-quantitative assessment and quantitative pharmacokinetic analysis.

1

Background – Hepatic and Biliary Disease

2. BACKGROUND

2.1. Hepatic and Biliary Disease

The liver is a large and complex organ with diverse functions, many of them critical for survival. When the liver is affected by disease, its considerable regenerative capacity allows many pathological processes to stay undetected for a long time. Diseases involving the liver and/or biliary system are generally referred to as hepatobiliary diseases. Disturbances initially arising in the biliary system can cause secondary or associated liver disease, and vice versa. Moreover, many types of cancer, e.g., colorectal and breast cancer, spread metastases to the liver. Metastatic disease can also stay subclinical for a long time, since a considerable portion of the liver has to be affected before liver function begins to fail (23). Hepatobiliary diseases can be classified in several ways, e.g., diffuse versus focal. Diffuse liver disease encompasses many different processes such as infection, autoimmune inflammation, fatty infiltration, metabolic disorders and certain genetic diseases. The dominant diffuse liver disease in the populations of industrialized regions is fatty infiltration, which can be caused by alcohol consumption but can also be present without association with alcohol, as non-alcoholic fatty liver disease, NAFLD (113). The latter is considered the hepatic aspect of insulin resistance and represents the spectrum from slight, diffuse fat accumulation (steatosis) via Non-Alcoholic Steatohepatitis (NASH), where there is also inflammation (60), to the formation of liver cirrhosis (94). In many parts of the world, infection with hepatitis B (HBV) and C (HCV) is very common and is the leading cause of cirrhosis (112). The inflammatory activity leads to liver fibrosis, which formerly was considered irreversible, but recent findings indicate that it may be reversible (100, 122). When detected early, before cirrhosis has developed, many of these conditions can be successfully treated and sometimes cured. Cirrhosis predisposes, however, to hepatocellular cancer (HCC), especially in chronic hepatitis B and C (143). Despite the absence of

3 Background – Hepatic and Biliary Disease cirrhosis in liver steatosis and NASH, these are also discussed as possible predisposing conditions to HCC (16, 44). In Europe and the United States, focal or multifocal malignant liver disease is most commonly caused by metastases from various cancer types, whereas in many regions of Asia and Africa, the primary liver cancers, primarily HCC, are more common than secondary metastases (1, 112). Diseases affecting the gallbladder, especially cholelithiasis and cholecystitis, are more common than bile duct stones and cholangitis (128). Cancer of the gallbladder or the intra- or extrahepatic bile ducts occurs less frequently than malignant disease in the liver (69). However, bile duct malignancy poses a significant clinical problem, since it may spread inconspicuously along the duct walls before producing a mass large enough to produce symptoms of biliary obstruction. Primary sclerosing cholangitis (PSC), a chronic diffuse inflammatory biliary disease of unknown origin but suspected of having an autoimmune component, causes long-standing inflammation of the duct walls and their surroundings, fibrosis with bile duct strictures and a greatly increased risk of bile duct cancer, cholangiocarcinoma (CCC) (15). At present, no curative treatment of PSC exists, aside from liver transplantation. An important goal of clinical observation is to detect CCC, which, if detected early, is an indication for liver transplantation. Following the progression of PSC is also important to adjust symptomatic treatment with medications and, in some cases, with dilatation of bile duct stenoses. Non-invasive imaging methods are of great importance for the detection and characterization of liver disease and also for evaluating hepatic vascular and segmental anatomy to provide a basis for surgical planning (84). The clinical surveillance programs of chronic hepatobiliary disease, e.g., HBV/HCV cirrhosis and PSC, rely on regular imaging in combination with biochemical and serologic tests. In many cases, biopsy is needed to characterize focal and diffuse liver disease, but since this is an invasive procedure, it is not feasible as a repeated test. There is an agreed- upon description of HCC as a successive change from benign parenchymal regenerative nodules to clearly malignant lesions, where arterial neovascularization is the most crucial sign of malignant change (157). The capacity of an imaging modality to reliably detect early pathological arterial

4 Background – Imaging Modalities vascularization is also important for the diagnosis of other hypervascular tumors, such as metastases from carcinoid, endocrine tumors, and renal cell carcinoma. Acute biliary conditions, such as biliary colic due to gallstones and/or cholecystitis, are nowadays primarily evaluated with ultrasound, complemented with other modalities if necessary, e.g., when percutaneous or endoscopic interventional treatment is required. In biliary imaging, the focus is on outlining the shape of bile ducts and gallbladder to detect stones, strictures or masses. At the same time, it is necessary to relate these findings to the surrounding structures, primarily the liver. Techniques to describe functional aspects of the biliary system will be briefly outlined in the following paragraphs. Overall, an optimal imaging modality needs to provide large volume coverage to include the entire liver and biliary system, a dynamic capacity to image changes in blood flow, contrast media enhancement and movement over time and image contrast and resolution suitable for the clinical situation.

2.2. Imaging Modalities

2.2.1. Ultrasound (US)

Diagnostic ultrasound, also called Sonography, uses high-frequency sound waves and pulses to form images of the body. The speed of sound depends on the tissue type and interfaces between tissues, which act as partial or total reflectors. The travel time of the ultrasound waves is interpreted as distance to the different interfaces; by using a range of frequencies, directions and pulse amplitudes in combination with advanced signal processing, two-, three- and even four-dimensional image data can be reconstructed. No known health risks are associated with exposure to diagnostic ultrasound (20). Thanks to its capacity for real-time imaging with high spatial and temporal resolution, it is useful in most areas of the body. Image quality is limited by total reflection by air- or gas-containing organs, skeletal or other calcified structures and by the depth of the object

5 Background – Imaging Modalities examined. Diagnostic ultrasound is considered more operator-dependent than other modalities. In upper abdominal imaging, ultrasound is an efficient means of examining the liver, gallbladder, bile ducts and pancreas. The introduction of ultrasound contrast media in the last decade has increased its accuracy and applications, e.g., in liver lesion detection and characterization, e.g., where small lesions are not fully characterized by multidetector computed tomography (MDCT) or MRI (37, 96, 166). Contrast-enhanced ultrasound (CEUS) adds functional information on normal and pathological vascular structures and on tissue perfusion in real time, which CT and MRI cannot provide at equal spatial or temporal resolution (22). Although the benefits and shortcomings of CEUS are well described and under discussion in many areas, there have been few studies so far that have compared modern CT and MRI techniques with CEUS, partly because ultrasound contrast is not yet approved by the U.S. Food and Drug Administration (FDA) and thus not available on the U.S. market (9, 71, 163). In a recent study by Larsen et al., CEUS did not perform better than MDCT in the detection of liver metastases from colorectal cancer (80). Several variants of US elastography have been developed. It provides an inexpensive non-invasive assessment of the elastic properties of the liver, but has low accuracy in the mild and moderate stages of fibrosis.

2.2.2. Computed Tomography (CT)

Introduced by Hounsfield in 1971 (50), computed tomography (CT) has developed from taking several minutes to produce a single two- dimensional image slice to almost instantly delivering time-resolved three- dimensional image datasets amounting to thousands of images. CT equipment consists of a rotating ring-like frame carrying the x-ray tube, which delivers fan-shaped radiation to the opposite side, where an array of small detector elements is located. The frame rotates continuously in its housing (gantry), while the patient table moves through the gantry opening. The detector array samples the radiation that has passed through the patient and this data is then processed to form a three-dimensional representation of the body. The image elements contain an attenuation value corresponding to the density or opacity to x-ray photons, measured on the

6 Background – Imaging Modalities

Hounsfield unit (HU) scale, which defines the attenuation of air as −1000 HU and that of water as 0 HU. Thus, all tissues have typical attenuation values, and direct comparisons between examinations can be made. While many structures are well delineated by CT, the contrast between soft tissues is low. Therefore, contrast media based on iodine are frequently used to improve the conspicuity of vessels and parenchymal organs. Computed tomography is a generally available modality in many parts of the world and is typically the first choice in examinations of the abdomen with a focus on malignant disease. Modern multidetector CT (MDCT) equipment (62) combined with power injectors for contrast agent delivery provides reliable imaging at high spatial resolution in several phases of vessel enhancement. The MDCT image data sets consist of volume elements (voxels) which are isotropic, i.e., have equal size in all three dimensions, enabling high-quality image reconstructions in any plane. The speed of modern MDCT equipment makes it possible to image the liver in one or several arterial-dominant phases as well as in the portal venous phase and later phases, if needed (40). However, examinations which include several phases can lead to a considerable dose of ionizing radiation, especially for those patients who are referred for regular follow- up, e.g., during or after chemotherapy. Specifically for biliary imaging purposes, ordinary contrast-enhanced MDCT image data from a portal venous or later phase can be processed using the minimum intensity projection (MinIP) technique. This will highlight the lowest-intensity parts of the image data, which normally correspond to the bile ducts and gallbladder (31, 70). Though not available in all countries, an intravenous cholangiographic contrast medium that is excreted by the biliary system, meglumine iotroxate (Biliscopin®, Bayer Schering Pharma, Berlin, Germany), can be used for contrast enhancement of the bile ducts in MDCT (35, 48, 111).

2.2.3. Nuclear Medicine (NM)

Nuclear medicine uses detectors sensitive to ionizing radiation from radioactive isotopes that have been introduced into the body. The isotopes, or rather the molecules – tracers – of which they are part, have different affinities for different organs or tissues. Organs and functions can be

7 Background – Imaging Modalities targeted by specific tracers to produce images and quantitative measures of tracer uptake and elimination. In hepatobiliary imaging, the hepatobiliary iminodiacetic acid (HIDA) scan is used to evaluate the elimination of HIDA, which mainly takes place in the liver (61). Positron Emission Tomography (PET) combined with CT in the same machine – CT/PET or PET-CT – has become increasingly available during recent years. Tracer compounds containing positron-emitting isotopes are introduced into the patient and detected by the PET equipment, which gives higher-resolution images than other NM techniques. These are then easily fused with morphological images obtained from the CT unit of the machine. So far, the role of CT/PET in hepatobiliary imaging is limited. It may assist in finding distant metastases of primary liver tumors that have tracer uptake (30). Galactose elimination analysis based on blood sampling (152) or a breath test (5) is used for assessing global liver function, since galactose metabolism occurs solely in the liver. Deriving from these techniques, experiments using PET for quantification of Fluorine-18 labeled galactose uptake and metabolism have been reported (138); if such an imaging technique is proven useful in patients, it may yield important information on regional functional metabolism.

2.2.4. Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging (MRI) is based on the electromagnetic interactions of the hydrogen nuclei – protons – in the body. All protons exhibit magnetic properties due to their electrical charge and spin. The MRI scanner consists of a superconducting electromagnetic coil producing a very strong stationary magnetic field, gradient coils providing additional and weaker but highly variable magnetic fields and antennas for sending and receiving radiofrequency waves to/from the body being examined. In very simplified terms, images are formed from weak radio waves emitted from the protons in the body, their frequency and phase representing different locations. To receive the proper signals, a great number of physical parameters need to be defined correctly in the MRI scanner, so that the relevant tissues are imaged with the intended characterization. The MRI process can be tuned to show different tissues with varying contrast, highlighting and suppressing structures in numerous ways. There

8 Background – Imaging Modalities is, however, no reference tissue or any well-defined range of signals from different tissues. In each experiment or scan, a grayscale image is finally formed, but the underlying values in the image pixels (the smallest image elements) lie on a unique scale for that scan. It is possible to perform MRI with a quantitative approach, but the methods have been time consuming and difficult to use. With newly developed MRI sequences this is becoming more practical, and quantitative comparison of tissue signals – instead of only visual judgment of image contrast – can be applied (32, 103, 159, 160).

Nuclear magnetic resonance (NMR) In quantum mechanics, proton spin can be viewed as the sum of the spins of a proton’s constituent elementary particles (quarks), but in the following text a simpler model will be used, regarding the proton as a charged rotating particle possessing a magnetic moment, similar to a bar magnet. The proton can also be called a magnetic dipole. When subjected to a strong magnetic field, protons will align along (parallel to) or against (antiparallel to) this field, in almost equal proportions. There will be a minute net surplus of protons in parallel orientation, which is the lower energy state, resulting in a net magnetization parallel to the external field. The stronger the magnetic field, the larger the net magnetization. The protons also exhibit a gyrating movement, precession, around the magnetic field axis, similar to how a gyroscope moves. The precession frequency is proportional to the external magnetic field strength according to the Larmor equation: γ = Bf (Eq. 1) 2π where f is the Larmor frequency, γ is the gyromagnetic ratio (constant for each nucleus), 42.6 MHz/Tesla for the proton and B is the external magnetic field strength, measured in Tesla (T). When the protons receive electromagnetic energy in the form of a radiofrequency (RF) wave or pulse, tuned to the Larmor frequency, the net magnetization vector will tip away from the external magnetic field. This is an unstable state, so as soon as the RF wave has ended, the net magnetization vector will return to equilibrium and at the same time, energy will be emitted from the protons in the form of RF. Hence, the

9 Background – Imaging Modalities system of aligned protons can temporarily absorb RF energy and later emit a detectable RF signal. This phenomenon is the foundation of Nuclear Magnetic Resonance, NMR, described in 1946 by Bloch and Purcell (11, 121).

MRI technique In magnetic resonance imaging (MRI), certain developments of the NMR technique make it possible to form images of the human body or objects. Lauterbur and Mansfield both described aspects of MRI that still constitute the basis of the technique today (38, 81, 92). The modern MRI scanner uses a constant strong magnetic field, typically with strength of 1.5 T, produced by a superconducting electromagnet. There are also three additional electromagnets, gradient coils, each designed to produce a transient magnetic field in one of three orthogonal directions, to be used separately or combined. This field can change quickly in amplitude and in direction. The MRI scanner also has radiofrequency coils – i.e., antennas – designed to deliver and receive RF energy. Usually, a large RF coil is used for delivery, and other, more flexible or specialized receiving RF coils of various shapes are placed near the patient. The MRI examination consists of a vast number of parameters defining complex programs – MRI sequences – for how gradient and RF energy will be used, how and when RF signals will be collected and, lastly, how the acquired data will be processed or reconstructed to form an image.

Basic layout of an MRI sequence If a magnetic gradient field is turned on in the same direction as the external magnetic field, it produces a gradual change in field strength along the head-to-feet orientation of the patient, here called the z-axis. The protons in the patient will thereby precess – resonate – at slightly different RF frequencies depending on their position along the z-axis. An RF pulse of a specific, limited frequency band can then excite all protons in a specific plane of limited width, on the z-axis or in any other orientation, which amounts to selecting a “slice” of protons in the patient.

10 Background – Imaging Modalities

After this slice-selecting gradient and RF pulse, the protons will start realigning to their equilibrium state. Using gradients perpendicular to the first one, in the x and y direction if the slice selecting gradient was applied in the z-direction, the precessions of the protons in the selected slice are manipulated so that the emitted RF signals differ in frequency and phase in a regular pattern. This means that the combination of a certain frequency and phase represents a certain position on the x-y-plane, i.e., in the selected slice. In this way, signals from different parts of the body can be localized in three dimensions (x, y, z) and used to form an image in which the bright- ness of the pixels represent the signal intensity (SI) of the corresponding tissue. Exchanging and combining the roles of the x, y and z gradients, images with arbitrary orientation may be produced (10).

RF signal conversion to image signal intensities The MRI scanner converts the weak RF signals to digital format by sampling, using an analogue to digital converter (ADC), and then performs several calculations to create images. The original RF signals are in this way translated to SI values defined for every image element (pixel or voxel). The range of RF energy received and thus the range of sampled SI values vary from one scan to another. To enable efficient data storage and optimal usage of the ADC, the initial values are converted to a different scale. Furthermore, image data may be stored and communicated in more than one data format. Therefore, the SI values of an image viewed on the console of the MRI scanner may be different from the values that are seen when the same image is viewed on another workstation. Some, but not all variants of signal rescaling are reversible. For quantitative analyses where SI measurements form the raw data, it is therefore important to use the original image datasets with signal intensities proportional to the RF signals. Alternatively, it is advisable to verify that the images one is using for the measurements are unperturbed by rescaling operations.

T1 and T2 relaxation

Two time constants, T1 and T2, describe the rate at which the net magneti- zation parallel/longitudinal (T1) and perpendicular/transversal (T2) to the external field returns to equilibrium, also referred to as relaxation (49).

11 Background – Imaging Modalities

T1 relaxation is caused by interaction between the excited protons and the local electromagnetic fields in the neighboring structures. One important interaction type is dipole-dipole, in which the proton is affected by another magnetic dipole tumbling in a frequency close to the Larmor frequency. As described in paragraph 2.3, this interaction is the basis of the effect of gadolinium-based contrast agents on T1 relaxation. The T1 constant represents the time it takes for the longitudinal magnetization, Mz, to reach 63% of its maximal value (Figure 1A).

T2 relaxation depends on the continuing dephasing of the precessing protons, the T2 constant being the time for the transversal magnetisation, Mxy, to fall to 37% of its original level (Figure 1B). Dephasing is caused by local magnetic field inhomogeneities and occurs at a faster rate than T1 relaxation. T2 is thus less than or equal to T1. The relaxation rate, R, is defined as the inverse of the time constant:

1 1 = = R1 and R2 (Eq. 2) T1 T2

Figure 1. A: Longitudinal relaxation. B: Transversal relaxation.

MRI sequences A vast number of MRI sequences have been developed. They can be classified as spin echo (SE) or gradient echo (GRE) sequences or as hybrids of SE and GRE. Briefly, an SE sequence can be described as using an RF pulse to refocus spins, while a GRE sequence applies varying gradient fields for the same purpose. The data is gathered in many repeated steps,

12 Background – Imaging Modalities i.e., in a sequential manner, where the repeat interval is called the repetition time (TR). During each TR, gradient changes and RF pulses are synchronized to optimize the echo, acquired at the Echo Time (TE), from longitudinal or transverse relaxation (or a combination of these) of the tissues of interest. The echo is related to RF energy emitted by the patient, in such a way that the transversal part of the magnetization vector induces a current in the receiving RF coil of the MRI scanner. Originally, MRI sequences typically used RF pulses long enough to flip the longitudinal magnetization a full 90 degrees. To increase speed and flexibility of modern sequences, this angle, called flip angle (FA), is often much smaller. All acquired echoes represent small portions of the data necessary for the formation of an image. This information is registered according to its frequency and phase in an abstract data representation called k-space. Low frequency information lies in the center of k-space and represents high contrast between large areas in the final image, while the periphery of k- space contains the data describing high spatial resolution but low contrast. Optimal high-contrast information is desirable, e.g., when a quickly passing bolus of contrast medium is imaged. Hence, MRI sequences can be designed to acquire the echoes carrying high-contrast information in various ways, which makes it possible to time not only the contrast medium delivery but also the acquisition of the most pertinent contrast enhancement.

The amplitude of the echo and its dependence on tissue T1 or T2, as well as the proton density (the number of protons in each volume element, voxel), is important for image quality. In abdominal imaging the time it takes to acquire a sequence is a crucial factor, since the abdominal organs move with the patient’s breathing. Imaging of the abdominal organs specifically requires that data acquisition be either synchronized with breathing movements or fast enough to be completed during one breath-hold. An important improvement is the development of fast T1-weighted GRE sequences that yield image data that is isotropic and of high resolution, i.e., three-dimensional image volumes instead of the two-dimensional slices several millimeters thick from traditional SE and GRE sequences. This sequence type includes an extra RF pulse designed to spoil the transverse magnetization before the excitation pulse, i.e., in order to avoid the T2- weighting that otherwise negatively influences the T1 contrast (95). The

13 Background – Contrast Media in Liver MRI present studies used two similar types of such a three-dimensional T1- weighted RF-spoiled GRE sequence (SPGRE): the volumetric interpolated breath-hold examination (VIBE) developed for Siemens MRI scanners and the T1-weighted high-resolution isotropic volume examination (THRIVE) used by Philips. These sequences are designed to provide an isotropic resolution of 1–2 mm and T1-weighting optimized for abdominal organs (126). The characteristics of the sequences depend on TR, TE and the flip angle (FA). Short TR values provide strong T1-weighting, so that the T1- shortening effects of contrast media can be better appreciated. TE is also short, to minimize signal loss and artifacts from dephasing. Normally, low flip angles around 10–15° are used, which allow a short TR and thus a short acquisition time. Depending on the application, different FAs are used. A lower FA than 10° may produce a lower signal with lower T1-weighting, while higher FA give more T1 weighting and a stronger signal, but take a longer time. Another sequence relevant to this work is Magnetic Resonance Cholangiopancreatography (MRCP), which is based on heavy T2-weighting (156). MRCP images are formed by the very large native contrast between the water (bile) in bile ducts and the much lower water signal of the surrounding structures, such as the liver parenchyma.

2.3. Contrast Media in Liver MRI

In 1988, Gd-DTPA (Magnevist®, Bayer Schering Pharma, Berlin, Germany, (161)) was the first Gadolinium-based contrast agent (GBCA) to be introduced in clinical MRI. It was followed by several compounds with similar properties and uses. These are distributed in the extracellular space and eliminated by the kidneys. The more recently developed contrast agents Gd-BOPTA (gadolinium benzyloxy-propionic tetraacetate or gadobenate dimeglumine, MultiHance® 0.5 mmol/ml, Bracco Imaging, Milan, Italy, (164)), available in Europe from 1998, and Gd-EOB-DTPA (gadolinium ethoxybenzyl diethylenetriamine- pentaacetic acid, Primovist® 0.25 mmol/ml, Bayer Schering Pharma, Berlin, Germany, (162)), introduced in Europe in 2004 and in the U.S. in 2008, can be used in the same way as the standard extracellular contrast media. In

14 Background – Contrast Media in Liver MRI addition, they are taken up by hepatocytes and excreted via the biliary system, i.e., they exhibit a hepatobiliary affinity and distribution. Another contrast agent excreted in the bile is Mangafodipir trisodium (Mn-DPDP, Teslascan®, GE Healthcare AS, Oslo, Norway, (168)), introduced in 1997. However, since it is administered as an infusion over 10–20 minutes, it cannot be used in early dynamic imaging. Furthermore, Endorem® (Guerbet, Roissy, France) and Resovist® (recently deregistered; Bayer Schering Pharma, Berlin, Germany), based on small (SPIO) or ultra-small (USPIO) iron oxide particles have been introduced as liver-specific agents (124) for T2-weighted imaging. They show high hepatic uptake, although this occurs in the Kupffer cells of the reticulo-endothelial system and not in the hepatocytes. In the following paragraphs, some background will be given on the Gadolinium-based contrast media, especially Gd-BOPTA and Gd-EOB- DTPA, and their use in liver MRI.

2.3.1. Safety of Gadolinium-Based Contrast Media

Gadolinium-based contrast media, including Gd-BOPTA and Gd-EOB- DTPA, are generally safe and well tolerated (7). The risk of inducing nephrogenic systemic fibrosis (NSF) in patients with low renal function is considered to be associated with any type of GBCA (24, 93, 145, 153), but neither Gd-BOPTA nor Gd-EOB-DTPA belongs to the group of contrast media that are contraindicated in patients with severely impaired renal function.

2.3.2. Gadolinium Contrast Media and Relaxivity

Gadolinium-based contrast media are normally used to shorten T1, a process which is accomplished by dipole-dipole interaction through the great magnetic moment of the Gd atom. Different Gd-based compounds have different tumbling rates and therefore different capacities to affect the T1 relaxation rate. This capacity is referred to as the relaxivity of the contrast agent and is measured in [s−1mM−1]. It is important to note that the effect on T1 relaxation here depends on the proximity of the tumbling rate of the contrast agent complex to the Larmor frequency, which, in its turn,

15 Background – Contrast Media in Liver MRI depends on the external magnetic field. This means that measurements of T1 relaxation performed at one magnetic field strength are not directly translatable to another. Contrast agents that are bound to plasma proteins have a lower tumbling rate, closer to the Larmor frequency, which permits more interaction between the Gd atom and the nearby water protons, and thus a greater change in relaxation (88). The traditional extracellular Gd-based contrast agents such as Gd- DTPA all have longitudinal relaxivity values – r1 – in plasma of around 4 mM−1s−1. Gd-BOPTA has frequently been referenced as having twice that relaxivity. The underlying measurements were, however, performed at 0.47 T (164), which is a common setup for in vitro relaxivity experiments. As reported by Cavagna et al. (18), in vivo measurements used in a computer model estimated the T1 relaxivity of Gd-BOPTA in rat liver to be 30 mM−1s−1, although data from two different groups of animals were used and the precision of the estimate was not stated. In measurements performed at the clinically more relevant field strength of 1.5 T, the additional relaxivity of Gd-BOPTA was in the order of +50%, rather than the alleged +100% (Table 1). The measured relaxivity values are also influenced by the different properties of the solvent or tissue holding the contrast medium. A comprehensive comparison in which 14 MRI contrast media were investigated at up to four different magnetic field strengths (0.47 T, 1.5 T, 3 T and 4.7 T) and in different solutions has been reported by Rohrer et al., who recommend that comparative measurements be performed in plasma (not water) and at clinically relevant field strengths: 1.5 T and 3 T (127). According to these measurements, Gd-EOB-DTPA and Gd-BOPTA have very similar relaxation rates.

16 Background – Contrast Media in Liver MRI

Table 1. Reported relaxivity values of Gd-BOPTA, Gd-EOB-DTPA and Gd-DTPA according to results available in the literature. The experiments reported by Rohrer in which 14 MRI contrast media were investigated at up to four different magnetic field strengths (0.47 T, 1.5 T, 3 T and 4.7 T) and in different solutions confirmed some relaxivity measures at 0.47 T from previously reported studies and provided more relevant measures performed at 1.5 T. Note the clearly higher relaxivity for both Gd-BOPTA and Gd-EOB-DTPA compared to Gd-DTPA. −1 −1 Relaxivity r1 [s mM ] at 1.5 T (63 MHz) Solution: Water Plasma Blood Liver

Contrast Gd- Gd-EOB- Gd- Gd-EOB- Gd- Gd-EOB- Gd- Gd-EOB- Gd-DTPA Gd-DTPA Gd-DTPA Medium: BOPTA DTPA BOPTA DTPA BOPTA DTPA BOPTA DTPA Rohrer 4.0 4.7 3.3 6.3 6.9 4.1 6.7 7.3 4.3

2005 (127) (3.8–4.2) (4.5–4.9) (3.1–3.5) (6.0–6.6) (6.5–7.3) (3.9–4.3) (6.3–7.1) (6.9–7.7) (4.0–4.6) −1 −1 Relaxivity r1 [s mM ] at 0.47 T (20 MHz) Rohrer 4.2 5.3 3.4 9.2 8.7 3.8 2005 (127) (3.9–4.4) (5.0–5.6) (3.2–3.6) (8.7–9.7) (8.3–9.1) (3.2–3.6) Vittadini 4.63 4.08 6.88 4.73 14.56 1988 (164) ±0.01 ±0.01 ±0.02 ±0.02 ±0.09 Schuhmann- 5.3 8.64 16.6 Giampieri ±0.33 ±0.47 ±1.10 1992 (134) De Haën 9.7 1999 (29)

Shuter 10.7* 1996 (137) ±0.5 Cavagna 30* 1990 (18) Bracco 4.4 10.8 2003 (55)

*Shuter and Cavagna reported values from experiments in rat liver.

2.3.3. Contrast Administration and Bolus Timing

Dynamic contrast-enhanced imaging aims at obtaining image sets from several successive time-points, requiring some sort of synchronization between imaging and contrast medium delivery. The first post-injection image acquisition is normally made to obtain an optimal enhancement of the arteries when the contrast bolus passes for the first time, so-called first- pass imaging. The time-points for the following acquisitions are then planned according to the clinical case, but will in most cases include a

17 Background – Contrast Media in Liver MRI portal venous phase at 45–50 seconds and an equilibrium or interstitial phase at 120–180 seconds post-injection. The simplest way to coordinate contrast media administration with imaging is to set a fixed delay, such as 15–20 seconds for the arterial phase, between contrast injection and the start of image acquisition. Due to the inter- and intraindividual variations in circulation, the imaging will sometimes start too early or too late for optimal arterial enhancement (79). The quality of the portal venous phase may also be affected, while later phases are generally not affected by a timing error of 5–10 seconds. The contrast medium may be injected manually or by a power injector or infusion pump. The timing can be individualized by using a test bolus of contrast, given at the same injection rate as the main bolus, followed by real-time imaging from which the operator can measure the time elapsed from the start of injection until peak enhancement. The same time delay is then used for timing the arterial phase acquisition in the dynamic imaging after the main bolus. The amount of contrast in the test bolus is normally considered too small to affect the image quality in the arterial phase, since the test bolus has been greatly diluted in the extracellular fluid volume. The special case of test bolus use with hepatocyte-specific contrast media will however be discussed later in this chapter. To achieve better synchronization between contrast injection and the MRI acquisition and a consistent injection rate, remotely controlled power injectors are helpful and nowadays considered necessary for routine clinical MRI. A technique that is being used more frequently, as more powerful MRI equipment capable of fast gradient switching is introduced is the automated bolus-detection technique. A real-time imaging acquisition runs during injection of the contrast medium by a power injector, while a software application on the MRI scanner continuously measures the signal intensity in the relevant anatomic region, typically the aorta. When the bolus arrives at that location, the signal increases, and the MRI scanner automatically triggers the first main image acquisition. Alternatively, the MRI operator can start the scan manually while monitoring the bolus progression in the real-time images. This technique was initially introduced

18 Background – Contrast Media in Liver MRI in MR angiography (MRA) of the abdominal aorta (118) but is now in use in various MRA applications and also in MRI of the liver. With the test bolus technique, there may be differences in contrast bolus arrival between the test bolus imaging and the acquisition of the arterial phase due to the imaging being done in different respiratory phases. The automated bolus- detection technique achieves better timing in this regard (53, 79). Techniques for obtaining more than one arterial phase have been described (65). In contrast-enhanced CT of the liver, the introduction of two or more early phases has been described since the introduction of fast MDCT equipment. The late arterial phase was considered more important than the early arterial phase when evaluating vascular, parenchymal and tumor enhancement (36, 65). Even the use of power injectors has undergone significant development. The single-head injectors used initially have largely been replaced by dual- head injectors, since most MRI protocols use saline to flush the contrast from the tubes and peripheral veins into the central circulation immediately after the contrast has been injected. Various strategies of injecting contrast have been described, but in general, for abdominal MRI, the contrast bolus is given at a flow rate of 1–3 mL/s, followed by the saline bolus at the same rate. The choice of injection rate depends primarily on the focus of the MRI exam and the speed of image acquisition. The first passage of contrast via the systemic veins, the heart and lungs and then to the systemic arteries spreads the bolus, lowering the peak concentration that can be achieved in a particular vessel. A high injection rate may then seem the natural choice, since it delivers the contrast agent in a more concentrated, “shorter” bolus. However, while MDCT can acquire several image volumes in only a few seconds, many MRI acquisitions take longer and are sensitive to large variations in signal, typically when the bolus is shorter than the time window of central k-space acquisition, leading to a so-called truncation artifact (89, 97). In this situation, the bolus should be optimized to deliver a high enough peak concentration while maintaining signal increase over the whole k-space center time. In MR- angiography, by comparison, the acquisitions are much faster which enables high injection rates of 3 mL/s or more while still avoiding this type of artifact. Another situation when a high injection rate is potentially

19 Background – Contrast Media in Liver MRI suboptimal is with the use of contrast agents that exhibit higher relaxivity due to their protein binding capacity. In the first pass, the local contrast concentration may be too high to provide an optimal equilibrium between bound and free fractions of the agent (117, 169).

2.3.4. Gd-BOPTA (MultiHance®)

Gd-BOPTA was introduced on the European market in 1998 and was approved by the FDA in 2004. Its indications include MRI of the central nervous system (CNS) and of the liver and contrast-enhanced MRA. Gd- BOPTA is distributed in the body like ordinary extracellular contrast media such as Gd-DTPA, but in the liver it is taken up by hepatocytes and excreted into the biliary canaliculi in an adenosine triphosphate (ATP)- dependent process involving a multispecific transporter protein (74). This allows extracellular space enhancement in early acquisitions and prolonged hepatocyte enhancement in delayed acquisitions. In humans, the dose percentage that is excreted via the hepatobiliary route is merely 2–4% (139), which means that the majority is eliminated by the kidneys. Despite this, enhancement of the biliary system is achieved 60–120 minutes after IV injection (165). In phase I studies, the mean elimination half-life ranged from 1.2 to 2.0 hours (139). Gd-BOPTA has higher relaxivity than standard extracellular Gd-based contrast media, due to its capacity to bind to plasma proteins (164). For Gd-BOPTA, the recommended dose for liver imaging is 0.05 mmol/kg (90) but in clinical routine the double dose is used in many centers (19, 43, 72, 73).

2.3.5. Gd-EOB-DTPA (Primovist®)

Gd-EOB-DTPA is a more recently approved liver-specific contrast agent, first released in 2004 in Europe and approved (labeled Eovist®) in the U.S. in 2008. Like Gd-BOPTA, it combines hepatocellular specificity with T1- relaxivity and extracellular behavior (134, 162). After intravenous injection, Gd-EOB-DTPA is first distributed into the extracellular space and then taken up by the hepatocytes. It is excreted unmetabolized in equal proportions by the kidneys and by ATP-dependent active transport in the hepatocytes to the biliary system, such that the hepatobiliary excretory proportion is approximately 10 times greater than for Gd-BOPTA. The

20 Background – Contrast Media in Liver MRI measured proportions of hepatobiliary and renal excretion were 42–51% and 43–53%, respectively (46). Renal excretion can be substituted with hepatobiliary elimination and vice versa. After a bolus injection of 0.025 mmol/kg (the recommended dose), the peak liver-specific enhancement is reached in about 20 minutes, followed by a plateau phase. The agent is cleared from the serum to reach a concentration below the limit of quantification 24 hours after injection, and its plasma half-life is one hour (46). In the safety phase I study by Hamm et al. (46), four doses of Gd-EOB- DTPA were tested: 0.010, 0.025, 0.05 and 0.1 mmol/kg. While the pharmacokinetic data suggested linear kinetics with no saturation of hepatic uptake at any dose, the highest dose (0.1 mmol/kg) produced susceptibility effects in liver imaging and was therefore considered excessive. Bollow et al. (13) also reported susceptibility effects at the highest two doses (0.05 and 0.1 mmol/kg) in a study on contrast-enhanced cholangiography. Recent reports by Zech (169) and Motosugi (97) have shown advantages to a lower injection rate and dilution, respectively, of the Gd-EOB-DTPA bolus. The low dose relative to standard extracellular contrast agents suggests that these adjustments should be made to avoid artifacts due to varying SI during central k-space acquisition. At our institution, the lower injection rate of 1 mL/s was chosen from the beginning as the standard for Gd-EOB-DTPA-enhanced MRI. In clinical imaging, the main application of Gd-EOB-DTPA is to aid in detection and characterization of focal lesions, utilizing the large image contrast between normal enhancing hepatocytes and lesions that do not contain functioning hepatocytes (51, 123).

2.3.6. Contrast Agent Uptake Mechanisms

Hepatocytes are polarized cells with two functionally distinct sides, the basolateral plasma membrane adjacent to the sinusoids, i.e., the blood and extracellular fluids, and the canalicular plasma membrane, which faces the smallest bile ducts, canaliculi. Bile salts, bilirubin and several other endo- and exogenous substances are transported from the sinusoids to the bile via a number of transport pathways (Figure 2).

21 Background – Contrast Media in Liver MRI

The uptake mechanisms of Gd-EOB-DTPA and Gd-BOPTA are not yet fully known but several details of organic anion transporting polypeptides (OATPs) located in the hepatocytic basolateral membrane have been reported in the last few years (26, 64, 78, 85, 91, 102, 116). Their transport mechanism is dependent on neither ATP hydrolysis nor any co-transport with sodium (Na+). Recently, it was found that Gd-EOB-DTPA is trans- ported into the hepatocyte by OATP1B1, OATP1B3 and Na+/taurocholate cotransporting polypeptide (NTCP), with the highest affinity for OATP1B3 (85). Another study established that the transport carried out by OATP1B3 is bidirectional (91). This has also been reported for the similar protein in the rat, oatp1, which was described by Müller et al. (101) as an “overflow system for amphipathic compounds, especially under pathological conditions such as cholestasis” (page G1289). Furthermore, the transport of Gd-EOB-DTPA from the cytosol to the bile ductule (canaliculus) across the canalicular membrane is mediated by the multidrug resistance protein MRP2, which is ATP-dependent and considered as a rate-limiting step in the excretion of several substances, including bilirubin. In the case of bile duct obstruction, changes have been observed in the expression of transport proteins. One such alteration is that another MRP, MRP3, located on the basolateral membrane is up-regulated, which is believed to provide a transport route for conjugated bilirubin (and other molecules) back to the bloodstream (6). Also, simple transmembrane diffusion occurs with bilirubin (171) and has been suggested as a possible uptake mechanism for Gd-EOB-DTPA. According to later reports, the main uptake of the latter is mediated by facilitated transport as described above, but diffusion cannot be completely ruled out as a low-capacity uptake pathway (91). Thus, based on observed affinities for different transport mechanisms in the hepatocyte, there exist several ways for Gd-EOB-DTPA to enter and exit the cell. Competitive inhibition at the transport protein, caused by similar uptake mechanisms for unconjugated bilirubin and substances like Indo- cyanine Green (ICG; see paragraph 2.5), which can inhibit bilirubin uptake, has been partially explored (110). This may explain the inhibited uptake of Gd-EOB-DTPA in the presence of increased serum bilirubin levels. ICG has been shown to inhibit the OATP2-mediated uptake of bilirubin but not OATP1B3-mediated transport (26). Since Gd-EOB-DTPA is transported by

22 Background – Contrast Media in Liver MRI more than one type of OATP, a competitive inhibition between ICG and Gd-EOB-DTPA at one or several transporters cannot be excluded.

Figure 2. Hepatobiliary transport of the contrast agent Gd-EOB-DTPA (blue), bilirubin (yellow; UC = unconjugated), bile salts (grey) and indocyanine green, ICG (green). The organic anion transporter polypeptides OATP1B1, OATP1B3 and OATP2 are together with the Na+/taurocholate cotransporting polypeptide NTCP and the multidrug resistant protein MRP3 located on the basolateral membrane. Simple transmembrane diffusion (TD), also occurs, for bilirubin and possibly also for Gd-EOB-DTPA. MRP2 is situated in the canalicular membrane and performs the transport into bile via an ATP-dependent process. Arrows illustrate a bidirectional transport capacity for OATP1B3. It is not clear if this also applies to OATP1B1. OATPs provide common uptake pathways for bilirubin, ICG and Gd-EOB-DTPA. Competitive inhibition may therefore occur (indicated by a minus sign) but the exact proportions for the different substances or pathways are not known. The MRP3, which is up- regulated in biliary obstruction, transports anions back to the extracellular space. The contrast agent Gd-BOPTA is also transported via OATPs, but the details are not known. NTCP take up bile salts, which are excreted to the bile via the bile salt export pump (BSEP, not shown). Interestingly, the increasing knowledge on hepatic transport proteins has led to new insights into cancer cellular biology. Hepatocellular cancer lesions with low, intermediate or high signal in the hepatobiliary contrast phase using Gd-EOB-DTPA were found by Kitao et al. to have an expression of OATP1B3 and MRP3 that correlated with the hepatocytic contrast uptake (75). In a study by Lee (82), OATP1B3 was seen to be overexpressed in the cytoplasm of colorectal tumor cells, while it was not expressed in normal colonic mucosa. There were signs that the OATP1B3

23 Background – Dynamic Contrast-Enhanced MRI protein interfered with the endogenous tumor suppressor protein p53 and that this effect was related to the transport function of the protein.

2.4. Dynamic Contrast-Enhanced MRI

2.4.1. Background

After a contrast agent has been intravenously administered to a patient, several different MRI techniques can be used to image the effects. The same applies to CT and US contrast agents and to various tracers in nuclear medicine. One rather straightforward application is in examinations of the CNS, where the blood-brain barrier (BBB) normally prevents the contrast agent from passing through the walls of the blood vessels. In most CNS tumors the BBB is damaged, leading to diffusion of the contrast agent into the extravascular space of the pathologic tissue. Therefore, after a few minutes this tissue is enhanced in signal relative to normal tissue. Dynamic contrast-enhanced MRI (DCE-MRI) is aimed at investigating such contrast agent effects over time (147). There is a growing interest in DCE-MRI analysis in oncology, for example, for breast and prostate cancer examination and treatment evaluation. In the following paragraphs, the T1- weighted type of DCE-MRI will be outlined.

2.4.2. Enhancement

Enhancement is a somewhat ambiguous term, and is used in different contexts of qualitative and quantitative MRI image analysis. When the measured quantity, the signal intensity, of a contrast-enhanced image is compared to the native SI of the original image (before the contrast agent is introduced) it is the relative enhancement (RE) that is described: ()()− preSIpostSI RE = (Eq. 3) ()preSI

When the SI at time t is divided by the SI before contrast, this is referred to as normalizing to the native SI. It is very similar to RE:

24 Background – Dynamic Contrast-Enhanced MRI

()postSI Normalized SI = RE += 1 (Eq. 4) ()preSI

Image contrast can be viewed as representing the visual appearance of structures in an image. The difference between the signal of two structures, e.g., a blood vessel and liver parenchyma, is calculated and then divided by the liver signal, for example (other formulas exist): ()()− liverSIvesselSI Image Contrast = (Eq. 5) ()liverSI

Just like RE and normalized SI, image contrast can be studied over time and characterized in terms of enhancement. As outlined in 2.2.4, it is important to consider where and how to make the SI measurements, so that no scaling errors are introduced.

2.4.3. Semi-Quantitative Analysis The purpose of a detailed analysis is to derive physiologically interpretable measures that describe the physiology and pathology as correctly as possible, i.e., with high accuracy, and with a high degree of reproducibility. This means that quantitative measures are needed instead of qualitative grading from the appearance of enhancement or non-enhancement of different structures. While MRI does not involve any ionizing radiation and has other advantages such as good volume coverage, the inherently large variability in the image formation is a challenge. The image data to be analyzed usually originates from regions of interest (ROIs) that are drawn and positioned manually by the observer. Signal intensity values of the image elements in the ROI can then be treated in different ways. A simple approach is to regard the body as a single compartment with inflow and outflow (elimination) of the contrast agent. The SI is then plotted over time in a time-intensity curve, from which the time to contrast arrival, the peak and mean signal amplitude and the time of peak enhancement can be read (Figure 3). The interpretation of such measures is ambiguous because there are many processes that can affect the signal in the image, one being the non-linear relationship between SI and contrast concentration. In addition, SI is influenced not only by the first

25 Background – Dynamic Contrast-Enhanced MRI passage of the bolus but also by the recirculation and diffusion of the contrast agent. The different physiologic processes that contribute to the shape of the time-intensity curve are therefore not distinguishable.

Figure 3. Examples of semi-quantitative measures. TTP: time to peak concentration; CAT: contrast arrival time; AUC: area under the curve. The area under the curve (AUC) can be calculated by integrating many short measurements of SI over a long time period. The area approximates the total amount of contrast that has entered the ROI and although it has better reproducibility than, for example, time-to-peak, it still has no clear physiological interpretation (58).

2.4.4. Quantitative Pharmacokinetic Analysis

In order to better quantify and separate some of the physiologic processes that occur, analysis of compartmental models has been introduced. Regarding the body as divided into a few defined compartments – the vascular plasma, the extracellular extravascular and the intracellular spaces – a compartmental model describes the various transport pathways for a contrast agent or tracer between these compartments (56). A two- compartment model is illustrated in Figure 4. Pharmacokinetic analysis is applied to calculate the transport rates and other parameters in order to describe the whole system (146). Ideally, if the initial amount and velocity of the tracer are known and the rates of tracer transport between all compartments are well defined, quantitative measures can be derived to

26 Background – Dynamic Contrast-Enhanced MRI represent, for instance, vessel wall permeability or the fraction of the tracer that is extracted from one compartment to another (148).

Figure 4. This example of a two-compartment model illustrates a tissue in which capillaries are permeable to the contrast agent, which is distributed in the vascular plasma space and the EES, whereas there is no uptake into the intracellular space. The contrast agent diffuses from the plasma to the EES and in the opposite direction (arrows). Physiological parameters such as blood flow and the permeability of the vessel wall decide the rate of transfer between the compartments (108, 146). The SI data can originate from ROIs or individual voxels. The SI values then have to be translated into changes in relaxation rate. This is a non- linear relationship that is dependent upon the type of MRI sequence that is used. The changes in relaxation rate most commonly translate into contrast concentration change according to a linear relationship (Eq. 6, (146)), but this is an approximation. Also, it is necessary to know the T1 value of the tissue before contrast administration, T10 or T1pre.

11 ⋅+= 1 Cr t (Eq. 6) TT 101 where T1 is the relaxation time (post-contrast), T10 is the pre-contrast T1, r1 is the relaxivity of the contrast agent and Ct is the contrast agent concentration in the tissue.

27 Background – Dynamic Contrast-Enhanced MRI

Although average values from former studies can be assumed to be good approximations, an individual quantitative mapping of T10 in the whole volume imaged would be preferred (159). In order to analyze the concentration changes in the tissue during the first minutes after contrast injection, knowledge is needed regarding the changes of contrast agent concentration in the blood plasma entering the tissue. This information is approximated using a vascular input function (VIF), which describes the fluctuations of concentration in the feeding vessel over time. From the variations in tissue SI (measured in ROIs or voxels), a deconvolution operation using the VIF as an approximation of an ideal (instantaneous) contrast injection can derive the contrast concentration change in the tissue, without the influence of the contrast agent circulating in the vessels (68). This approach is employed in, e.g., studies by Nilsson et al. (104), Ryeom et al. (130) and Jackson et al. (59). The VIF is usually sampled in a nearby artery leading to the tissue, but sometimes a more central vessel is chosen because of its larger size. In the liver, there is no ideal single vessel in which to sample the VIF, since there is a dual vascular supply, of which the portal vein provides about 75% and the hepatic artery 25%. To perform both portal venous and arterial sampling requires a very long breath-hold, which is excessive for clinical purposes. As an approximation, the VIF may be obtained from the portal vein (104) or the hepatic artery, as described by Jackson et al. (59). However, in the latter study, the specific purpose was to quantify the contrast leakage in hepatic malignant lesions with arterial supply. Furthermore, data of very high temporal resolution from a separate study population may be used to define a generic VIF used in the patient examinations (107). However, this approach risks masking important individual characteristics related to disease processes, such as tumors which vary in size and display varying vascularity and presence of necrosis. Accurate pharmacokinetic analysis in the initial phase benefits from a precise sampling of the VIF, preferably with a very high temporal resolution (57). The requirement of a high spatial resolution to enable measurements in small structures in clinical liver imaging is already a compromise since large volume coverage is needed. Thus, to achieve a high temporal resolution, a much smaller volume must be scanned, with lower

28 Background – Liver Function Tests, Prognostic Scores spatial resolution, potentially preventing the correct placement of the sampling ROI. A few minutes after the first pass, the tissue contrast concentration changes are slower, allowing acquisition times of 20–30 seconds sequences (one breath-hold), which is enough for high-resolution isotropic three- dimensional sequences covering the whole upper abdomen. Using a compartment model and a different pharmacodynamic approach, the vascular and extravascular extracellular distribution of a hepatocyte- specific contrast agent in the liver can be regarded as similar to that of the spleen. This is utilized in Papers III and IV.

2.5. Liver Function Tests, Prognostic Scores

Several methods have been developed to aid in clinical staging of and prognostication in hepatobiliary disease. In addition to serum levels of, e.g., liver enzymes and bilirubin, the clearance of specific substances that are eliminated by the liver can be measured. Of these, the indocyanine green (ICG) clearance test (131, 136) is well established in the planning of hepatic resection and transplantation. Indocyanine green (ICG) is a fluorescent dye that is exclusively eliminated by the hepatobiliary route. The ICG level measured 15 minutes after injection (ICG-R15) is largely dependent upon the hepatic perfusion, rendering high levels of retained ICG in cirrhosis. An elevated ICG-R15 value can reflect low liver function and a high risk of post-operative liver failure (135). In the complicated task of prioritizing liver transplant recipients, clinical scoring systems have an important role. The Child-Pugh (120) test and the model for liver disease (MELD) test (63) are today the most important, although the MELD test has replaced the Child-Pugh test in the united network for organ sharing (UNOS) allocation protocol used in the U.S. (155). These tests and scores all assess global liver function, and may therefore miss disease that is regionally advanced or in an early stage of its course.

29

Aims

3. AIMS

The primary aims A1–A8 of the papers I–IV were:

Paper I

A1. To evaluate and compare the time course of biliary enhancement in T1-weighted imaging with Gd-BOPTA and Gd-EOB-DTPA in normal healthy subjects.

Paper II

A2. To compare the time course of hepatic parenchymal and vascular enhancement in T1-weighted imaging with Gd-BOPTA and Gd- EOB-DTPA during the arterial, portal venous and hepatobiliary phases in normal healthy subjects.

Paper III

A3. To develop a procedure for quantifying the hepatocyte-specific contrast uptake during late time phases and to evaluate the procedure in examinations of healthy volunteers using both Gd- EOB-DTPA and Gd-BOPTA.

A4. To investigate the correlation between the uptake measures of these agents.

A5. To characterize the similarities and differences between Gd-EOB- DTPA and Gd-BOPTA pertaining to hepatic vascular enhance- ment.

Paper IV

A6. To evaluate the hepatic Gd-EOB-DTPA uptake in patients, investi- gating the relationship between the uptake measure and the hepatobiliary function, and to compare these results with similar analyses of variables based on unadjusted signal intensities (SI) and serum bilirubin.

31 Aims

A7. To evaluate the influence of SI normalization on liver-to-spleen SI ratios.

A8. To estimate blood plasma clearance through measurements in the spleen compared with data from healthy volunteers.

Complementary Aims The following complementary aims A9 and A10 were studied within the dissertation work:

A9. To compare relative enhancement values in Papers I, II and III where appropriate.

A10. To evaluate the bile duct enhancement in the patients studied for Paper IV in relation to the results of pharmacokinetic analysis.

32 Material and methods – Subjects

4. MATERIAL AND METHODS

4.1. Subjects

4.1.1. Paper I, II and III – Healthy Volunteers

After obtaining approval from the local ethics committee and written informed consent, 10 healthy volunteers were examined on two different occasions, during April and May 2005. Of these, four were men with an age range of 19–46 years (mean age, 30 years) and six were women with an age range of 20–45 years (mean age, 30 years). In order to rule out unknown liver and renal dysfunction, total serum bilirubin and creatinine were evaluated prior to MRI. All subjects completed both studies. For Paper III, another group of 10 healthy volunteers was examined in the same way but with a different MRI system. The group consisted of five women with an age range of 22–33 years (mean age, 25 years), and five men with an age range of 23–27 years (mean age, 25 years).

4.1.2. Paper IV – Patients

Twenty-two patients were evaluated retrospectively with approval from the local ethics committee. These patients had all been examined with Gd- EOB-DTPA-enhanced liver MRI for suspected hepatobiliary disease from December 2006 to August 2008, on the same MRI scanner as the subjects in Paper III. One examination was found to be incomplete due to technical failure and was excluded from the analysis. Therefore, datasets from 21 patients age 21–81 years (mean age, 55 years) – 13 men and 8 women – were included in the analysis (Table 2). The hepatobiliary function of the patients was interpreted to a clinical classification called clinical grade (levels 1, 2 and 3; refer to Table 1 of Paper IV) by an experienced liver surgeon. Relevant clinical data were read from the electronic patient records (EPR) to provide information on the final

33 Material and methods – Subjects diagnoses of the patients and to grade the hepatobiliary function that they possessed at the time of the MRI examination. Data for calculation of the Child-Pugh (120) and MELD (63) scores were retrieved from electronic records of blood laboratory tests and from information concerning the subjective parameters – ascites and hepatic encephalopathy – of the Child-Pugh score. An ordinal scale called the MELD range was defined, based on the application of MELD in the UNOS guidelines (155).

Table 2. Patient data, listed by clinical grade (ClGr) 1–3. Abbreviations INR = international normalized ratio (from prothrombin time); Bili = serum bilirubin; Crea = creatinine; ChP and ChPC = Child-Pugh score and class; MELD = model for end-stage liver disease; HCC = hepatocellular carcinoma; CCC = cholangiocarcinoma; PSC = primary sclerosing cholangitis.

Age, Cl MELD INR Bili Crea ChP ChPC MELD sex Gr Clinical Summary Range

21M 1 Ulcerative Colitis. Normal liver. 1.1 10 117 6 A 10 0

34F 1 Benign solitary liver cyst with 1.1 16 86 5 A 7 0 septations.

35F 1 Gall bladder carcinoma stage 1.0 8 96 5 A 7 0 1B.

37M 1 Moderate liver steatosis. 1.0 10 73 5 A 6 0 Normal bile ducts.

39M 1 Mild PSC. 1.0 10 77 5 A 6 0

61F 1 Crohn's disease. Normal liver. 0.9 4 69 5 A 6 0

64M 1 Severe liver steatosis. 0.9 6 95 5 A 6 0

Colon cancer metastases, 64M 1 adenocarcinoma. Three small 1.0 8 85 5 A 6 0 metastases resected.

81M 1 Solitary HCC, resected 2 years 0.9 5 64 5 A 6 0 earlier. No recurrence.

32M 2 PSC. CCC affecting bile ducts. 1.1 157 70 6 A 16 1

44M 2 PSC. CCC, gall bladder cancer. 1.0 23 101 5 A 9 0

34 Material and methods – Contrast Media

Age, Cl MELD INR Bili Crea ChP ChPC MELD sex Gr Clinical Summary Range

Liver transplant 10 years 47M 2 earlier due to PSC. PSC 2.2 21 118 6 A 19 2 recurrence in liver transplant.

68F 2 PSC, extra- and intrahepatic. 1.1 28 118 5 A 12 1 Ulcerative colitis. Portal vein thrombosis under 75M 2 treatment; partly resolved at 1.2 39 76 7 B 12 1 time of MR exam. Fully resolved five months later. Ulcerative Colitis. Normal liver. 38M 3 Small stone in CBD according 1.0 141 76 7 B 14 1 to MRCP. No stones after ERCP with EST. Cirrhosis. Perioperative bile duct damage 25 years earlier. 67F 3 Hepaticojejunostomy with 1.2 69 67 7 B 14 1 metal stents. Recurring non- patency problems. Old postoperative bile duct injury with right lobe atrophy. 69M 3 Bile duct stenoses. Two stones 1.0 76 133 9 B 16 1 in the CBD. Later underwent right lobe resection: CCC, Klatskin tumor.

71F 3 Pancreatic head cancer, 0.9 80 72 8 B 12 1 inoperable.

71F 3 Gall bladder tumor; 0.9 177 62 8 B 15 1 adenocarcinoma.

72F 3 CCC, Klatskin tumor, Bismuth 1.0 333 27 8 B 18 1 type 3A. Stone in common bile duct, Mirizzi's syndrome, plastic 73M 3 stent. Stone surgically 1.1 192 465* 10 C 33 3 removed. Renal failure; hemodialysis. *Commented in paragraph 6.3.2, on page 63.

4.2. Contrast Media

The amount of Gd-EOB-DTPA (Primovist® 0.25 mmol/ml, Schering, Berlin, Germany) used in all examinations was 0.025 mmol/kg. In studies I and II, Gd-BOPTA (MultiHance® 0.5 mmol/ml; Bracco Imaging, Milan, Italy) was

35 Material and methods – MRI Technique administered in the dose of 0.1 mmol/kg, while the dose was 0.05 mmol/kg in study III. In studies I and II, the test bolus technique was used for contrast bolus timing. One ml of contrast was delivered as a test bolus, followed by saline and the time delay for the arterial phase was determined by the time from the start of injection until the occurrence of contrast enhancement in the proximal abdominal aorta. The contrast bolus was injected by power injector at 2 mL/s, immediately followed by an equal amount of physiologic saline. Total acquisition time was 20 seconds, and the center of k-space was reached 8 seconds from the start. In study III, the contrast medium was administered intravenously by manual injection at a rate of approximately 1 mL/s and immediately followed by an equal amount of physiologic saline, in accordance with the standard clinical liver MRI protocol. The arterial phase acquisition was started after a fixed delay of approximately 20 seconds post-injection. In study IV, Gd-EOB-DTPA was administered by power injector at 1 mL/s, followed by saline. Arterial phase acquisition was started manually with the help of real-time bolus monitoring at the level of the upper abdominal aorta.

4.3. MRI Technique

In the first two studies, examinations were performed on a 1.5 T magnetic resonance system (Magnetom Vision, Siemens, Erlangen, Germany) combining the spine coil and the flexible phased-array body coil. Examinations were carried out in the early morning after more than seven hours of fasting. After one hour of scanning, a light meal containing less than 2 g of fat was allowed. Each examination lasted about six hours, and the two examinations for each subject were performed at least six days apart. The MRI protocol was designed by extending a standard protocol for contrast-enhanced examination of the liver by adding several late phases, resulting in a total of nine post-contrast axial breath-hold gradient-echo 3D T1-weighted VIBE (126) sequence acquisitions (TR 4.5 ms, TE 1.9 ms, flip angle 12°, FOV 40 cm, 120 slices of 1.7 mm thickness) at arterial and portal

36 Material and methods – Image and Signal Intensity Analysis, Statistical Analysis venous phases (48 seconds after the arterial phase) and 10, 20, 30, 40, 130, 240 and 300 minutes after injection. In Paper III, the same MRI protocol was used on another 1.5 T MRI system (Achieva, Philips Medical Systems, Best, the Netherlands). The standard axial breath-hold spoiled gradient-echo fat-saturated 3D T1- weighted sequence (THRIVE, TR = 5.2 ms, echo time (TE) = 2.6 ms, flip angle = 10°, scan time 30–40 seconds, FOV 350–400 × 250–280 mm2, matrix 256×115, reconstructed voxel size 1.6×1.6×1.6 mm3) was used. The MRI protocol for the patient examinations in Paper IV was similar to this protocol (Table 3).

Table 3. Parameters for the VIBE (Paper I and II) and THRIVE (Paper III ,IV) sequences.

Paper I, II Paper III Paper IV Parameter Volunteers Volunteers Patients TR [ms] 4.5 5.2 4.3 TE [ms] 1.9 2.5 2.1 Flip Angle [°] 12 10 10 Slice Thickness (Acquired) [mm] 3.3 3.3 4 Reconstructed voxel size [mm] 1.7 1.6 2 Acquisition Matrix 256/160 256/164 192/192 Percent Phase FOV 62 70 79 Coil Flexible phased-array Sense-Body Sense Body + Spine coil TorsoXL Reconstruction Diameter 400 400 395

4.4. Image and Signal Intensity Analysis, Statistical Analysis

4.4.1. Paper I

Three radiologists performed all measurements independently, using diagnostic workstations (PACS IDS5 v10.1, Sectra Imtec AB, Linköping, Sweden). Signal intensity measurements of the bile duct were made by placing a single measuring point in a central voxel in the common hepatic duct (CHD). The reviewers measured the mean SI of liver parenchyma by drawing circular regions of interest (ROI) of 0.7 cm2 in the same slice, at the

37 Material and methods – Image and Signal Intensity Analysis, Statistical Analysis same anteroposterior level as the measurement in the CHD, avoiding large vessels (Figure 5).

The bile duct-to-liver image contrast, CDuct-Liver, at the time-points from 10 minutes until 300 minutes post-injection was calculated as − = Duct SISI Liver C −LiverDuct (Eq. 7) SILiver

For all measurements, each reviewer documented his choice of image slice (in the data table) and measurement points (in the image); this information was not visible to the other reviewers. All data were anonymized, but there was no blinding of the subject alias during the image analysis, in order to ensure that SI values were registered under the correct alias in the data sheet. It was not possible to blind the reviewers to the time-point of the image, since contrast enhancement features, in general, are obvious to radiologists. Registration of all SI values, image slice number, time-point of image acquisition, etc. was done manually by reading digits from the image display and entering data in a structured spreadsheet template, one copy for each reviewer. The three spreadsheets were then merged. Simultaneous registration of redundant data from the images, e.g., both the image time stamp and the time-point of acquisition, was used to detect mistakes in data entry. To decrease the systematic variation in the placement of measurement points or ROIs, the reviewers were allowed to cross-reference image slices when choosing the representative slice for each image sequence. This assured the closest possible anatomical matching of the slices. Statistical analysis was performed in JMP 6.0 (SAS Institute Inc., Cary, NC, USA) using a mixed analysis of variance (ANOVA) model with the subject and the reviewer defined as random effects, testing for each time- point the null hypothesis of no difference in CDuct-Liver between Gd-BOPTA and Gd-EOB-DTPA within subjects. To evaluate the possible difference in late enhancement behavior, a similar linear model was applied to the CDuct-Liver values between 40 and 240 minutes post-injection, using time, subject (random effect) and reviewer (random effect) as model effects.

38 Material and methods – Image and Signal Intensity Analysis, Statistical Analysis

4.4.2. Paper II

Three radiologists performed all measurements independently, using diagnostic workstations (PACS IDS5 v10.1, Sectra Imtec AB, Linköping, Sweden) and working according to the same principles as in Paper I. Signal intensity measurements were made by placing ROIs in the common hepatic artery, the middle hepatic vein, a segmental branch of the right portal vein and the liver parenchyma, all in a single representative slice (Figure 6). The arterial and portal venous phases and the time-points from 10 minutes until 130 minutes post-injection were analyzed. The image contrast between the vessel and the liver parenchyma was calculated analogously to the bile duct image contrast (Eq. 7). Statistical analysis was performed for each time-point with a mixed ANOVA model with the subject and the reviewer defined as random effects. The signal intensity of liver parenchyma for Gd-BOPTA at 130 minutes versus Gd-EOB-DTPA at 10 minutes, representing the optimum for each contrast medium, was also compared using a similar model. These calculations were carried out in JMP 7.0 (SAS Institute, Cary, NC, USA).

Figure 5. Biliary enhancement measurements – Paper I. Axial T1-weighted 3D VIBE sequence through the liver, TR 4.5 ms and TE 1.9 ms. The arrow shows the contrast-enhanced common hepatic duct (CHD) and parenchymal enhancement at 130 minutes post-injection of Gd- BOPTA. A circular ROI is placed in liver parenchyma and a point of measurement is placed centrally in the CHD.

39 Material and methods – Image and Signal Intensity Analysis, Statistical Analysis

Figure 6. Vessel-to-liver contrast measurements – Paper II. Axial VIBE slice using Gd-BOPTA during the portal venous phase. MV = middle hepatic vein; PV = segmental branch of the portal vein; A = common hepatic artery; * = liver parenchyma, two regions.

4.4.3. Paper III and IV

Signal intensity curves were averaged from seven regions of interest (ROIs) in the liver and three in the spleen. The liver ROIs were placed in both the left and right lobes avoiding any large vessels or focal lesions. One ROI each was located in the splenic vein and the portal vein. Care was taken to preserve images in their original format (Philips PARREC), in order to enable analysis and comparison of signal measurements from different exams and patients. All ROI placements and SI measurements were made on a dedicated workstation running an in-house-developed application for Matlab r2007b (The MathWorks, Natick, Massachusetts, USA) to enable simultaneous anatomic matching of images from all dynamic sequences. The signal intensity of liver and spleen parenchyma, the common hepatic artery, the portal vein and the splenic vein were obtained before contrast agent administration, during the arterial and portal venous phases, and 10, 20, 30, and 40 minutes after intravenous contrast agent injection in volunteers, and up to 20 minutes post contrast in patients (study IV). In the following steps, the time series of SI from each ROI were recalculated to relaxation rate values, which were then applied in a pharmacokinetic model (Figure 7) to determine the relative accumulation FHep, of contrast agent in the hepatobiliary compartment.

40 Material and methods – Image and Signal Intensity Analysis, Statistical Analysis

• Pre-contrast normalization of SI

• Conversion of SI values to relaxation rates (R1) • Calculation of contrast concentration by pharmacokinetic analysis

From the time series of FHep, a rate estimate, KHep, of the hepatobiliary contrast uptake was determined by least square regression. A detailed, illustrated description is provided in Paper III.

Figure 7. Simple pharmacokinetic model. The grey boxes represent the liver and the spleen, respectively, where the measurement ROIs in the images give the signal intensities. The white boxes represent the compartments of the model, where plasma and EES are part of both the liver and the spleen. Arrows show the direction of contrast medium transport between compartments. The splenic intracellular compartment – “splenic tissue” – has no uptake. The eliminated contrast medium is collected in bile and urine, symbolized by white circles. Using a similar approach, the increases of splenic longitudinal relaxation rate relative to pre-contrast values, ∆R1Spleen, were calculated for each time point. The time series thus formed, excepting the arterial and venous phases, was fitted to a monoexponential function, to be interpreted as a model of blood clearance. The slope of the fit was defined as the splenic (blood) clearance rate, SBC.

41 Material and methods – Image and Signal Intensity Analysis, Statistical Analysis

In addition to the measurements reported in Paper IV, ROIs were also placed in the common hepatic duct. A bile duct measurement normalized to the pre-contrast SI (Eq. 4) was then defined for the time point 20 minutes after contrast administration: BileSI_N20. In the volunteer study (III), the SI values of liver and spleen ROIs 20 minutes after contrast agent injection were used to calculate the quantitative Liver-to-Spleen Contrast ratio, Q-LSC, according to the description by Motosugi et al. (99). The KHep value and Q-LSC ratio in each subject for both Gd-EOB-DTPA and Gd-BOPTA were compared using pairwise Pearson’s correlation and linear regression (MATLAB R2009a, The MathWorks, Inc. Massachusetts, USA). In the patient study (IV), the Q-LSC liver-to-spleen contrast ratios, here abbreviated as LSC, for the time-points of 10 (LSC10) and 20 (LSC20) were determined. The relationship of each of the parameters, KHep, LSC and bilirubin, to clinical grade was studied with ordinal logistic regression, with each parameter as a predictor variable in a separate model. Their associations with Child-Pugh class and MELD range were investigated in the same manner. One-way ANOVA was used to compare the group means of KHep, bilirubin, LSC and LSC_N representing clinical grade 1–3. Post-hoc testing was performed according to Games-Howell using BrightStat (140), due to unequal variances across groups. Spearman (ρ) and Pearson (r) correlation was calculated for the SBC, LSC20 and LSC_N20 parameters versus KHep. Except for the Games-Howell test, statistical calculations were performed in JMP 6.0 (SAS Institute Inc., Cary, NC, USA).

42 Results – Paper I

5. RESULTS

5.1. Paper I

The 20 MRI examinations were completed in a time span of 49 days without adverse reactions or subjective symptoms. Due to technical problems, one of the examinations did not include the acquisition at 20 minutes post- injection. The time delay between the administration of the test bolus and the start of the arterial phase acquisition was derived from the data documentation, to provide a means of estimating a possible effect of the test bolus on liver signal intensity before the main bolus. The mean delay ± standard deviation (SD) amounted to 13 ± 2.6 minutes for the Gd-BOPTA experiments and 15 ± 3.7 minutes for Gd-EOB-DTPA. Delays ranged from 0.8 to 5.4 minutes for 9 out of 10 subjects, and was 11 minutes for one subject. No significant differences in liver SI based on the type of contrast medium used for test bolus were detected. In the pre-contrast image series, there was no significant difference in CDuct-Liver between Gd-BOPTA and Gd-EOB-DTPA. At 10 min. post-injection, biliary enhancement was evident for Gd-EOB-DTPA (p<0.001), whereas none was seen for Gd-BOPTA. At the 20-min. time-point, slight biliary enhancement was noted for Gd-BOPTA (Paper I; Fig. 3), but CDuct-Liver was significantly higher for Gd-EOB-DTPA (p<0.05; Table 4). During the 30- min. and 40-min. time-points, CDuct-Liver increased for both contrast media, with no significant difference between them. From 130 to 300 min. after injection, CDuct-Liver was significantly higher for Gd-EOB-DTPA (p<0.01). After 40 min. post-injection, CDuct-Liver decreased (p<0.001) for Gd-BOPTA, whereas it increased for Gd-EOB-DTPA until 240 min. post-contrast injection (p<0.001; Figure 8).

43 Results – Paper I

Table 4. Mean and standard deviation (SD) of CDuct-Liver for Gd-BOPTA and Gd-EOB-DTPA (all subjects, all reviewers). In the rightmost column, p-values from the ANOVA are given: ***p<0.001, **p<0.01 and *p<0.05. Where p is non-significant, its exact value is reported.

Gd-BOPTA Gd-EOB-DTPA Delay Mean SD Mean SD

[min] CDuct-Liver CDuct-Liver CDuct-Liver CDuct-Liver n P 0 −0.57 0.14 −0.56 0.12 30 0.84 0.25 −0.46 0.24 −0.48 0.22 30 0.76 0.8 −0.56 0.15 −0.53 0.19 30 0.46 10 −0.30 0.18 0.29 0.26 30 *** 20 0.19 0.27 0.36 0.37 27 * 30 0.42 0.26 0.35 0.47 30 0.34 40 0.50 0.34 0.41 0.43 30 0.076 130 0.25 0.35 0.49 0.48 30 *** 240 0.22 0.39 0.68 0.57 30 *** 300 0.24 0.39 0.52 0.44 30 ***

Figure 8. (Paper I) Graph illustrating results shown in Table 4. Mean contrast (CDuct-Liver) between the common hepatic duct and liver parenchyma at 0, 10, 20, 30, 40, 130, 240 and 300 min. after injection of contrast medium (***: p<0.001; *: p<0.05; NS: not significant). From 40 to 240 min. post-injection, there is an increase of CDuct-Liver when using Gd-EOB-DTPA versus a decrease when using Gd-BOPTA (p<0.001, both)

44 Results – Paper II

5.2. Paper II

The signal intensity of the studied vessels was greater when using Gd- BOPTA than when using Gd-EOB-DTPA (Paper II: Table 1). When using Gd-EOB-DTPA, the vascular signal returned to a level close to the native state after 10 minutes, but when using Gd-BOPTA it remained almost three times higher than the native level for 40 minutes after administration. The image contrast of the hepatic artery measured in the arterial phase was significantly greater for Gd-BOPTA (Figure 9) than for Gd-EOB-DTPA (p<0.001, Figure 10 and Paper II; Table 2). Similarly, the portal vein was hyperintense in the portal venous phase with Gd-BOPTA, while it was isointense with Gd-EOB-DTPA. In later phases, the portal vein was almost isointense after Gd-BOPTA, but distinctly hypointense after Gd-EOB-DTPA (Figure 9, Figure 10). Also, the middle hepatic vein was hyperintense with Gd-BOPTA and isointense with Gd-EOB-DTPA, measured in the portal venous phase (p<0.001). Later, the image contrast of the middle hepatic vein was very low with Gd-BOPTA, while it was distinctly negative with Gd- EOB-DTPA. The liver parenchyma reached its highest signal intensity during the portal venous phase with Gd-BOPTA and at 10 minutes with Gd-EOB- DTPA. Until 130 minutes after Gd-BOPTA administration, there was no significant variation in signal intensity. For Gd-EOB-DTPA, the signal plateaued until 40 minutes post-injection, followed by a decrease to the 130- minute time-point. During this plateau, there was no significant difference in liver SI between the two contrast agents. The SI of liver parenchyma at 10 minutes after Gd-EOB-DTPA administration did not differ significantly from the liver SI at 130 minutes using Gd-BOPTA.

45 Results – Paper II

Figure 9. (Paper II) Image contrast of the portal vein, common hepatic artery and middle hepatic vein versus liver parenchyma after injection of Gd-BOPTA.

Figure 10. (Paper II) Image contrast of the portal vein, common hepatic artery and middle hepatic vein versus liver parenchyma after injection of Gd-EOB-DTPA.

46 Results – Paper III

5.3. Paper III

The SI measurements of all subjects were recalculated to R1 values which provided – via the pharmacokinetic model – the estimation of KHep for both Gd-EOB-DTPA and Gd-BOPTA for each subject. This stepwise calculation is illustrated in Paper III (Fig. 2). Mean values and SD of KHep were 0.032 ± 0.02 min−1 and 0.37 ± 0.19 min−1 for Gd-BOPTA and Gd-EOB-DTPA, respectively. While there was a correlation between KHep_Gd-EOB-DTPA and KHep_Gd-BOPTA with ρ = 0.64 (p<0.05), there was no correlation between the LSC ratios of Gd-EOB-DTPA and those of Gd-BOPTA (Figure 11). With the fitted regression intercept constrained to zero, the slope relating the uptake speed of Gd-EOB-DTPA to that of Gd-BOPTA was 11 ± 1.6. No strong correlation was observed between the LSC ratios and KHep values for Gd- BOPTA. A significant correlation, ρ = 0.64 (p<0.05), was observed between LSC and KHep for Gd-EOB-DTPA.

Figure 11. A. Correlation plot of the estimated KHep for all 10 subjects using Gd-EOB-DTPA and Gd-BOPTA, respectively, ρ = 0.64 (p<0.05). Fitted regression slope was 7.42 ± 3.66 (estimate ±standard error) and fitted intercept was 0.14 ± 0.12. With the intercept constrained to zero, the estimated slope was 11.0 ± 1.65. The results from the pre-clinical studies on Gd-EOB-DTPA and Gd-BOPTA indicate an expected slope of 10. B. Correlation plot of the quantitative liver-to-spleen contrast ratio, LSC, for all 10 subjects. No correlation between the contrast agents was observed. Note that the LSC values cannot be interpreted into physical properties of the contrast agent uptake.

47 Results – Paper III

In the vessels, the increase in relaxation rate, ∆R1, in the arterial and venous phases after Gd-EOB-DTPA administration, was 20.4% ± 14.3% (mean ± SD) higher than after the injection of Gd-BOPTA (p<0.02; Student’s t-test) after dose normalization (see Paper III; Fig. 5). However, the ratio of the mean vessel ∆R1 over the splenic ∆R1 was 1.85 ± 0.28 for Gd-EOB-DTPA and 2.37 ± 0.18 for Gd-BOPTA (Paper III; Fig. 4D), respectively, which was a significant difference (p<0.01; paired Student’s t-test).

Gd-BOPTA provided better vessel-to-liver contrast in the early time phases. In the late time phases, vessel-to-liver contrast was very low, i.e., a ratio very close to one, for Gd-BOPTA and inverted for Gd-EOB-DTPA, i.e., a lower signal in the vessels than in the liver. The blood clearance rates, SBC, showed no significant difference (p = 0.20; paired Student’s t-test) between the contrast agents in the late phases. Mean SBC ± SD was 0.028 ± 0.01 min−1 for Gd-EOB-DTPA and 0.022 ± 0.004 min−1 for Gd-BOPTA. When the SBC was calculated instead using monoexponential fitting from the arterial phase, the rate was significantly higher for Gd-EOB-DTPA (p<0.002).

The comparison of the splenic ∆R1 time series of this study with serum Gd-EOB-DTPA levels reported in a previous study by Schumann- Giampieri et al. 1997 (133) showed similar rates of clearance in the late phases from 10 minutes after contrast administration. With the simulation of a doubling of the Gd-EOB-DTPA dose in the pharmacokinetic model, the arterial and venous phase vessel-to-liver contrast was increased (Paper III; Fig. 7B) compared to that obtained with the standard dose used in the subject examinations.

48 Results – Paper IV

5.4. Paper IV

The rate estimate of hepatic Gd-EOB-DTPA uptake, KHep, was significantly different between patient groups, as indicated by clinical grades 1–3. The Games-Howell post hoc test showed a significant difference between grades 1 and 2 and between 1 and 3 (Table 5, Figure 12). The difference between the group means of LSC ratios at 10 and 20 minutes post-contrast (LSC10 and LSC20) and of bilirubin were also significant and the Games- Howell test showed a significant difference between clinical grade 1 and 3. There was, however, no significant difference between grades 1 and 2. For LSC10 and LSC20, though not for bilirubin, there was a difference between grades 2 and 3.

According to the ordinal logistic regression analysis, KHep was a significant predictor of clinical grade, with the highest chi-squared (goodness of fit) among the parameters tested and with the highest R2. As a predictor of Child-Pugh Class, KHep had higher χ2 and R2 than the parameters LSC10, LSC20 and bilirubin (Table 6). The normalized LSC_N10 and LSC_N20 ratios, however, gave higher χ2 and R2, surpassing even the results obtained with KHep in the prediction of Child-Pugh class. The results of the Games-Howell test on the normalized ratios showed a pattern similar to the result of the test on KHep. The pattern was similar in the analyses of these parameters as predictors of MELD range, with overall lower results. In all three analyses concerning clinical grade, Child-Pugh class and MELD range, bilirubin was the parameter with the lowest results. The results of the one-way ANOVA listed in Table 5 are also reported graphically by box plots together with the Games-Howell post-hoc test represented by brackets, in Figure 12.

A close correlation with KHep (Paper IV, Fig. 4) was found for the normalized ratio LSC_N20, Pearson’s r = 0.94 (p<0.001, confidence interval, CI: 0.85–0.98), Spearman’s ρ = 0.95 (p<0.001, CI: 0.87–0.98). The correlation with KHep for the LSC20 ratio was lower: r = 0.77 (p<0.001, CI: 0.49–0.91), ρ = 0.82 (p<0.001, CI: 0.59–0.93). The SBC only showed a significant difference between clinical grades 1 and 3. There was a moderate

49 Results – Paper IV correlation between SBC and KHep, r = 0.60 (p<0.01, CI: 0.21–0.82), ρ = 0.63 (p<0.01, CI: 0.26–0.84). In addition to the results reported in Paper IV, analyses based on biliary ROI measurements are here reported in the form of the BileSI_N20 parameter in Table 5 and Table 6. A plot of the hepatic uptake rate (KHep) by bile duct relative enhancement (normalized SI, BileSI_N20) is presented in Figure 14. In addition, KHep was plotted by bilirubin levels to illustrate the variation in these variables (Figure 13).

Table 5. A description of the patients’ classification according to clinical grade (as defined in Paper IV; Table 1) is provided in the first three columns. Values are displayed as means ± standard deviations, except for the MELD and Child-Pugh scores which are described by median and range. The F-ratio and R2 of one-way ANOVA are displayed. Post hoc comparisons of clinical grades 1, 2 and 3 according to the Games-Howell test are shown with significance levels indicated by ***p<0.001, **p<0.01 and *p<0.05. Where p is non- significant, its exact value is reported.

Clinical Grade ANOVA Games-Howell

1 2 3 F R2 1 vs 2 1 vs 3 2 vs 3

N 9 5 7

Age 48 ±20 53 ±18 66 ±12

MELD Score 6 [5–10] 12 [9–19] 15 [12–33]

Child-Pugh Score 6 [5–8] 6 [5–8] 6 [5 – 10]

P-Bilirubin [μmol/l] 9 ±4 54 ±58 153 ±94 11** 0.55 p=0.30 * p=0.11

−1 KHep Uptake rate [min ] 0.24 ±0.1 0.09±0.05 0.02 ±0.02 21*** 0.70 ** *** p=0.11

LSC10 Ratio 1.64 ±0.35 1.58 ±0.09 1.11 ±0.18 9** 0.50 p=0.90 ** ***

LSC20 Ratio 1.87 ±0.31 1.71 ±0.06 0.75 ±0.23 16*** 0.65 p=0.42 *** **

LSC_N10 Ratio 1.33 ±0.13 1.05 ±0.15 0.87 ±0.1 27*** 0.75 * *** p=0.12

LSC_N20 Ratio 1.45 ±0.16 1.14 ±0.15 0.92 ±0.11 26*** 0.75 * *** p=0.07

−1 SBC [min ] 0.08 ±0.05 0.04 ±0.02 0.04 ±0.02 5* 0.33 p=0.10 * p=0.87

BileSI_N20 7.8 ±3.9 5.0 ±1.1 1.1 ±0.5 13*** 0.62 p=0.22 ** **

50 Results – Paper IV

Figure 12. Box plots corresponding to one-way ANOVA results of the variables listed in Table 5, with patients grouped according to their clinical grade (ClinGr), indicated on the lowermost x-axes. Horizontal lines indicate overall means of the variables.

51 Results – Paper IV

Table 6. The value of MRI-based measures and bilirubin as predictors of clinical parameters as evaluated with ordinal logistic regression. Parameters in rows, dependent variables clinical grade, Child-Pugh class and MELD range in columns. Goodness of fit is indicated by the chi-square, with level of significance (***p<0.001, **p<0.01), and the ability of the model to explain the dependent variable is represented by the (pseudo)-R-squared (McFadden). MELD ranges M2 and M3 are represented by only one patient each. Clinical Grade Child-Pugh Class MELD Range 2 2 2 2 2 2 χ (p) R χ (p) R χ (p) R

KHep 27*** 0.61 30*** 0.88 15*** 0.35 LSC10 12*** 0.26 15*** 0.44 7** 0.17 LSC20 18*** 0.42 19*** 0.57 10** 0.24 LSC_N10 25*** 0.55 34*** 1.00 16*** 0.38 LSC_N20 24*** 0.56 27*** 0.83 15*** 0.37 BileSI_N20 24*** 0.57 22*** 0.69 10*** 0.27 Bilirubin 19*** 0.43 10** 0.3 8** 0.20

Figure 13. Scatter plot of KHep vs. bilirubin (Bili). Clinical grade indicated by: “+“ = 1; “◊” = 2; “□” = 3. While the patients with normal hepatobiliary function show variation in KHep, their separation vs. clinical grades 2 and 3 is still greater using KHep. With bilirubin, only groups 1 and 3 are significantly separated, cf. Table 5.

52 Results – Comparative Analyses – Papers I–III

Figure 14. Scatter plot of the hepatic uptake rate (KHep) by the bile duct relative enhancement at 20 minutes post-injection (BileSI_N20), with the clinical grade indicated by “+” = 1; “◊” = 2; “□”= 3. Biliary stasis patients show very low or no detected hepatobiliary Gd-EOB-DTPA accumulation according to KHep, nor any biliary contrast enhancement.

5.5. Comparative Analyses – Papers I–III

The datasets from the two groups (Papers I+II vs. Paper III) of healthy subjects were compared approximately according to the normalized SI of the liver parenchyma, the portal vein, the hepatic artery and the common hepatic duct. The comparison is presented graphically in Figure 15 as curves of enhancement over time (0–40 min) and shows good agreement of the Gd-EOB-DTPA curves except for the CHD enhancement, where the subjects in Paper III presented lower and somewhat later biliary enhancement. For Gd-BOPTA, there is a clear difference in amplitude in all of the compared measurements, amounting to approximately a factor of two. In the arterial and portal venous phases with Gd-EOB-DTPA, the normalized SI of the hepatic artery and portal vein are generally higher in the subjects of Paper II.

53 Results – Comparative Analyses – Papers I–III

Figure 15. Normalized SI measured in the liver parenchyma (Liver), the portal vein (VP), the hepatic artery (HA) and the common hepatic duct (CHD) from Papers I and II and Paper III are graphically compared from 0 to 40 minutes after contrast agent injection. Data series are labeled according to: A12 = study I+II; A3 = study III; solid lines: Gd-BOPTA; dashed lines: Gd- EOB-DTPA.

54 Discussion – Biliary Imaging

6. DISCUSSION

Imaging modalities are under continuous development and especially in liver imaging, the many aspects of the MRI technique itself and the diverse biological properties of the hepatobiliary system allow for a multitude of improvement strategies. One aim of these development efforts is to achieve the “one-stop shop” examination, providing all morphological and functional information that the clinician needs in a single and not-too- complicated study. In the studies performed here, two liver-specific contrast media developed at least partly with this focus were compared. Their enhancement dynamics were evaluated and related to different aspects of liver imaging. Through a quantitative approach, the analysis of the hepatic uptake can be improved and better suited for patient examinations.

6.1. Biliary Imaging

Functional information on biliary excretion can be obtained with both Gd- BOPTA and Gd-EOB-DTPA, since both are partly eliminated via the biliary system, where they can shorten the T1 of bile, providing enhancement of the bile ducts, gall bladder and intestine in T1-weighted imaging. The traditional method of imaging the biliary system is based on heavily T2-weighted magnetic resonance cholangiopancreatography (MRCP) sequences that give purely morphological information. When per- formed on modern MRI equipment with strong gradients and a capacity for respiratory triggered acquisition, high-quality three-dimensional MRCP acquisitions with high spatial resolution are achieved. Morphological imaging of the biliary system can also be performed with T1-weighted sequences, specifically three-dimensional T1-weighted SPGRE, after the administration of hepatobiliary contrast agents. This contrast-enhanced MR cholangiography (CE-MRC) has been shown to perform better than T2- weighted MRCP in the evaluation of bile duct anomalies in liver transplant donors (83).

55 Discussion – Biliary Imaging

Although it is in this way possible to depict contrast-enhanced bile ducts with high spatial resolution, Bollow (13), Carlos (17) and Lim (86) have reported that the peripheral branches of the bile ducts are not visua- lized as distinctly as in T2-weighted MRCP. This is due to the fact that, at the doses studied, the substances give a prolonged enhancement of functioning hepatocytes, i.e., hepatocytes in the immediate vicinity of the smaller ducts and ductules, lowering the image contrast of bile vs. parenchyma. Somewhat contradictory results were presented in another study by Lim (87), in which overall image quality scores were higher for Gd-BOPTA-enhanced 3D T1-weighted images than for 2D and 3D T2- weighted MRCP images. However, analogous to the previous studies mentioned, bile duct visualization with T2-weighted MRCP was superior to Gd-enhanced 3D T1-weighted imaging, except for the common bile duct. With 3 T MRI systems becoming more available, one can remark that with a higher magnetic field strength, both MRCP and CE-MRC can poten- tially be performed with higher quality. Koelblinger et al. reported significantly better delineation of intrahepatic bile ducts with 3 T compared to 1.5 T in Gd-EOB-DTPA-enhanced MR cholangiography (77). According to recently presented results by D’Souza et al. (abstract no. 2592, International Society for Magnetic Resonance in Medicine [ISMRM] – European Society for Magnetic Resonance in Medicine and Biology [ESMRMB] joint meeting 2010, Stockholm, Sweden), the visualization of the bile ducts may be further improved by applying a higher flip angle, 35–45 degrees instead of 10–15 degrees. Also at 1.5 T a higher flip angle may be beneficial for biliary imaging (4). In Paper I, biliary enhancement relative to the liver parenchyma was measured in the common hepatic duct. In normal subjects, the bile ducts are free from obstructions, so the biliary contrast concentration over time is likely to be similar in peripheral and central parts of the biliary system. Our so far limited clinical experience with CE-MRC indicates that a patient with obstruction of a duct branch will typically have ordinary excretion into the unaffected branches. When several or all branches are affected, the excretion will be delayed or nonexistent. To better recognize and quantify such delays, the semi-quantitative results from Paper I can provide some insight into the two patterns of normal biliary enhancement when Gd- BOPTA or Gd-EOB-DTPA is used. Also, the results indicate that because it

56 Discussion – Vascular Imaging offers an early time window for evaluation of suspected delays in biliary contrast excretion, Gd-EOB-DTPA is the more practical contrast medium. When using Gd-EOB-DTPA the excretion to bile is faster and provides a stronger enhancement of the biliary tree than Gd-BOPTA. However, with flexible scheduling at the MRI unit, normal and pathological biliary excretion can also be studied with Gd-BOPTA if the patient is scanned a second time 1–2 hours after contrast media administration (132). The high contrast between the common bile duct and liver parenchyma had an earlier onset and longer duration for Gd-EOB-DTPA. There was also a significant difference between the contrast increase (Gd-EOB-DTPA) and decrease (Gd-BOPTA) from 40 minutes to 240 minutes post-injection. Clini- cal hepatobiliary imaging that includes evaluation of the biliary elimination phase can therefore be performed in a shorter time when using Gd-EOB- DTPA. The vascular and early parenchymal phases of contrast-enhanced liver MRI are, however, important to obtain in liver imaging. Therefore, Paper II was planned to evaluate vascular enhancement dynamics of the same contrast media, primarily in the arterial and portovenous phases.

6.2. Vascular Imaging

In Paper II, both the vascular enhancement (Paper II; Table 1, Figs. 4A and 4B) of the hepatic artery, the portal vein and the middle hepatic vein and its contrast to the surrounding liver parenchyma (Figure 9, Figure 10) were significantly higher at the obtained imaging time-points when using Gd- BOPTA than when using Gd-EOB-DTPA. Ideally, the timing of the image acquisition should be optimized for each vessel studied. Here, the most critical timing problem occurs in the arterial phase. According to the standard protocol for liver MRI at this institution, the test bolus method was used. The timing of the portal venous phase is straightforward once the arterial phase is obtained. The acquisition is started around 60 seconds post-injection, as is commonly reported in the literature (19, 65, 66, 142). The hepatic vein is reached by the contrast agent almost as early as the portal vein, so that the optimal enhancement probably occurs just 10–15 seconds later. With an acquisition time of 20 seconds, this means that the

57 Discussion – Vascular Imaging hepatic vein is not imaged at its optimal time. A distinction is sometimes made between an early portal venous “inflow” phase and a later portal venous phase, where the former is aimed at providing portal venous enhancement before any significant enhancement is seen in the hepatic veins, while the latter is a more conventional phase in which both portal and hepatic veins are enhanced (36, 41). The setup used in these studies reflects the typical clinical situation, in which the arterial phase is the most important one to optimize, followed by a late portal venous phase. Optimal bolus timing for the arterial phase acquisition is more difficult to accomplish with Gd-EOB-DTPA than with standard extracellular agents or with Gd-BOPTA because of the lower dose. The bolus volume of Gd- EOB-DTPA is smaller, amounting to 7 ml instead of 14 ml in a typical case of a subject weighing 70 kg (170). A smaller bolus will give briefer enhancement of lower amplitude during the first passage, and will therefore be more difficult to image at the ideal moment when the center of k-space is acquired. Extending the bolus by dilution or a lowered injection rate may facilitate this task as suggested by Motosugi et al. and Zech et al. (97, 169). Another reason to extend the bolus is that there is a considerable risk of truncation artifacts (97) when a short bolus relative to the sampling time of the center of k-space causes the signal to vary greatly during sampling. Interestingly, the standard protocol of Motosugi’s group used a high injection rate, 3 mL/s. The bolus was diluted to a volume of 20 mL, which for a patient weighing 50 kg corresponds to lowering the concentration by a factor of four, roughly translatable to an injection of a standard bolus at a rate of 0.75 mL/s. This is in the same range as the lower rate tested by Zech et al., 1 mL/s, which performed better than a rate of 2 mL/s. Whether the recommended Gd-EOB-DTPA dose of 0.025 mmol/kg can be increased in order to facilitate early dynamic imaging has been discussed in recent studies by Halavaara (45) and by Tamada (142). If the Gd concentration were equal to that of Gd-BOPTA, there would be a risk of artifacts (46) due to increasing T2 effects (leading to signal loss from spin dephasing) at high Gd concentration where the contrast accumulates, i.e., in hepatocytes and in the biliary system. Recently presented data from Wehrli (e-poster no. 4582, ISMRM–ESMRMB 2010) suggest that a double dose of Gd-EOB-DTPA increases vessel conspicuity in both arterial and

58 Discussion – Vascular Imaging portovenous phases so that there is no significant difference compared to Gd-DTPA. Another, more unconventional strategy was reported by Wei (e-poster no. 4593, ISMRM-ESMRMB 2010), namely injecting both Gd-EOB-DTPA and Gd-DTPA at the same time, achieving better vessel en- hancement while also obtaining marked hepatocyte-specific enhancement. There are also published reports describing routine use of a higher dose in clinical MRI, e.g. by simply using one or two full 10 ml-vials, depending on patient weight (25). Another reason for considering a higher dose is that the initial dose-finding studies of Gd-EOB-DTPA were aimed at determining an optimal dose for liver lesion detection. This was recently pointed out by Huppertz (52), as an argument for allowing higher doses in vascular imaging. In studies I and II, Gd-BOPTA was used at the dose recommended for vascular and neurological studies (0.1 mmol/kg), which was the standard dose of the liver MRI protocol used at that institution. This dose is twice the recommended dose for liver MRI but is commonly used and the most frequently reported in recent studies (19, 43, 72, 73). In the studies presented here, no comparison with standard extracellular contrast agents, which are normally also given in the 0.1 mmol/kg dose, was performed. Other studies have reported superior vessel enhancement properties of Gd- BOPTA (39, 76, 119) due to its plasma protein binding capacity that gives about 50% higher relaxivity (Table 1). Gd-EOB-DTPA also binds to plasma protein and has at least as high a level of relaxivity as Gd-BOPTA, and would theoretically achieve the same improved vessel enhancement if given in the same dose. This hypothesis was partially explored in Paper III, in which the pharmacokinetic model was used to simulate the effects of a doubling of the dose of Gd-EOB-DTPA, i.e., to 0.05 mmol/kg. The results indicated that the vessel-to-liver contrast would be higher in the early imaging phases and comparable to the contrast obtained using Gd-BOPTA at the same dose. Although no significant differences in the pre-contrast SI of liver parenchyma were found between the contrast media in studes I and II, the risk of a test bolus of Gd-EOB-DTPA causing more pronounced T1- shortening of the liver parenchyma before the main bolus is given (which

59 Discussion – Hepatic Parenchymal Enhancement would decrease the image contrast vs. the liver) cannot be excluded (25, 47, 125).

6.3. Hepatic Parenchymal Enhancement

6.3.1. Semi-Quantitative analyses (Papers I, II)

Apart from enhancement being a somewhat ambiguous term in itself, as described in paragraph 2.4, enhancement of the liver parenchyma involves many different factors. Contrast agent effects take place in the vasculature, in the hepatocytes, in the EES, and in the small biliary ductules and larger bile ducts. The dual vascular supply of the liver and the only partly known transport pathways of the hepatocyte contribute to the complex dynamics of contrast agent distribution. In Papers I and II, enhancement refers to the development of image contrast over time, which was the main focus of the semi-quantitative analyses there. In Papers III and IV, enhancement refers to signal or relaxation rate changes from the pre-contrast situation (baseline), i.e., the relative enhancement. Although this aspect of enhancement can be studied from the SI-time curves in Papers I and II, no further analysis of relative enhancement was done in these. To enable more quantitative analyses, studies III and IV were designed to use contrast concentration estimates from recalculated, normalized signal values. In terms of hepatic parenchymal SI values as compared to the pre- contrast SI measured in studies I and II, the peak relative enhancement for Gd-BOPTA occurred at the portal venous phase, and no significant decrease occurred during the measurement time of 130 minutes after contrast media administration. For Gd-EOB-DTPA the highest enhancement was from 10 to 40 minutes after injection (Paper II; Fig. 4). This is in accordance with measurements taken in a phase I study from healthy volunteers by Hamm et al. (46), in which a “plateaulike enhancement” was described, from 20 minutes until 90 minutes post- injection. Initially, however, the high parenchymal SI is a combination of extracellular (including the vessels) and intracellular (hepatocytic) contrast agent effects. As reviewed in Paper IV, this phase is short for Gd-EOB-

60 Discussion – Hepatic Parenchymal Enhancement

DTPA, while Gd-BOPTA provides considerably more prolonged vessel enhancement. The more specific hepatobiliary enhancement occurs later (21, 42, 114, 132), especially for Gd-BOPTA, and enables a high contrast between the liver and lesions lacking normally functioning hepatocytes. In one of the few comparisons between Gd-EOB-DTPA and Gd-BOPTA that have been published so far, the contrast agents had similar performance in detecting HCC in patients with chronic liver disease (106). The hepatobiliary phase was acquired 20 minutes and three hours after contrast agent injection, respectively.

6.3.2. Quantitative Analysis of Contrast Agent Uptake

Papers III and IV A method for quantification of hepatocyte-specific contrast agent dynamics in the liver was developed for standard 3D T1-weighted SPGRE images acquired with high spatial resolution and full liver coverage. A measure of the contrast agent concentration in the tissue was obtained by the estimation of ∆R1. By relating the hepatic contrast agent concentration to the plasma concentration, as observed in the spleen, quantitative measurement of the late hepatic uptake was achieved.

The hepatic uptake rate KHep provided significant separation of patients without hepatobiliary disease from those with hepatobiliary disease, without (clinical grade 2) or with manifest biliary stasis (clinical grade 3). There was no significant difference between the two latter groups. In contrast, the serum bilirubin level and the simple liver-to-spleen ratio LSC showed a significant difference between the two patient groups with hepatobiliary disease, but no significant separation between these and patients with normal function (clinical grade 1). The apparently greater ability of KHep and the normalized LSC_N ratios to discriminate between clinical grades 1 and 2 may be an indication that the KHep estimate has a test response suitable for early detection of disturbed hepatobiliary function. The ratio of hepatobiliary uptake of Gd-EOB-DTPA to that of Gd- BOPTA was estimated through a fitted regression slope, which amounted to an uptake rate approximately ten times higher for Gd-EOB-DTPA, in agreement with pre-clinical studies (46, 139). The clearance of the contrast

61 Discussion – Hepatic Parenchymal Enhancement agent from the blood was determined from splenic ROI measurements and the higher clearance rate of Gd-EOB-DTPA is in line with its shorter plasma half-life compared to Gd-BOPTA. Moreover, there were similarities between the healthy volunteer group and the patients with normal hepatobiliary function as per the combination of low hepatobiliary uptake and prolonged vascular and splenic enhancement. In the patient study, the patient groups were better separated by the normalized liver-to-spleen ratio, LSC_N20, than by the non-normalized. The findings suggest that the pharmacokinetic analysis applied here yielded measures that have a connection to physiological properties, at least to a greater extent than simple ratios of liver and spleen SI values.

The effect of T2* relaxation on the accuracy of the ΔR1 was rather low according to our calculations, corresponding to a signal loss in the range of 2%, as described in paper IV.

The assumption of fixed intrinsic (pre-contrast) values, T1pre or T10, of the tissues is a simplification. The T1pre of liver used here was 586 ms, reported by de Bazelaire et al. (28), with a standard deviation of 39. Thomsen et al. (144) described a larger variation and also reported that the T1 is prolonged in cirrhosis patients. Values (mean ±SD) reported by van Lom et al., T1pre = 568 ±59 also show more variation (158). The addition of a quantitative T1- mapping sequence would provide the means to address interindividual variations as well as intraindividual changes, e.g., due to tissue alterations during treatment, in order to improve the pharmacokinetic analysis. The majority of quantitative DCE-MRI studies focus on the earliest phase of contrast transport and distribution. A different approach was described by Boss et al., who studied the SI of the liver, the spleen and the renal cortex in a late time interval 35–70 minutes post-contrast when the SI is steadily and linearly decreasing at a rate that was correlated to the renal glomerular filtration rate. This is similar to the splenic contrast clearance analysis in studies III and IV. In a study on liver perfusion using CT, Blomley et al. (12) demonstrated a method of subtracting the time- attenuation curve of the spleen from that of the liver, to obtain a curve of hepatic portal venous perfusion. The splenic perfusion was thus regarded “as a template for hepatic arterial blood flow” (page 425). Resembling this design, the pharmacokinetic analysis used in Paper III and IV regarded the

62 Discussion – Hepatic Parenchymal Enhancement blood and EES compartment of the spleen as similar to that of the liver. An obvious limitation of this approach is that splenic pathologies may affect the results. The practical significance of this limitation should be defined in prospective studies. The lack of accurate non-invasive measures of liver dysfunction was addressed by applying a clinician’s grading of the patient hepatobiliary function. A retrospective study by Tajima et al. (141) on differences between patients with chronic liver dysfunction and those with normal liver function has some similarities to our patient study in that the patient records formed the basis of patient grouping. In prospective study designs, additional tests and examinations, e.g., liver biopsies and ICG clearance tests, can improve the classification of hepatobiliary dysfunction. The subjective components (ascites, encephalopathy) of the Child-Pugh score, commonly referred to as a disadvantage of the test, were negative with only one exception. This patient had ascites, classified as clinical grade 3. This patient was exceptional also in that he was on hemodialysis due to renal failure. At the time of the examination (April 2007) cautionary advice concerning Gd-based contrast agents and the risk of Nephrogenic Systemic Sclerosis (NSF) had recently been distributed from the European Society of Urogenital Radiology (ESUR) (145).

Other investigations of hepatobiliary contrast uptake In experiments on rats, Tsuda et al. used a sequence with a temporal resolution of just over 6 seconds to describe the time-intensity curve of after Gd-EOB-DTPA and Gd-DTPA administration. A semi-quantitative analysis showed that the time to maximal RE, Tmax, was prolonged with non- alcoholic steatohepatitis (NASH) compared to non-alcoholic fatty liver disease (NAFLD). The elimination half-life, measured as a 50% decrease of RE in the later phases, was also longer in NASH (150). The analysis was based on SI values and it is possible that this experimental model could be further explored after recalculation to R1 changes and by using a pharmacokinetic model. In another experimental hepatic injury model in rabbits, Ryeom et al. studied the hepatic Gd-EOB-DTPA uptake with very high temporal resolution single-slice acquisitions and calculating the hepatic uptake

63 Discussion – Hepatic Parenchymal Enhancement fraction (HEF) by deriving a VIF from the aorta. An association between decreasing HEF and higher ICG levels in plasma (ICG retention) was seen in animals with hepatic injury. Nevertheless, relaxation rate changes are preferable to SI values in these calculations. Since Gd-EOB-DTPA and ICG have differing elimination (ICG is excreted purely by the biliary route) and VIF sampling of the portal circulation was not included, interpretation to physiological parameters is difficult. In addition, the time point of the ICG test was not specified, leading to some uncertainty whether interference with Gd-EOB-DTPA may have occurred. In a study by Planchamp et al., a relationship between pharmacokinetic parameters describing the Gd-BOPTA uptake in rat liver was shown to inversely correlate with the degree of liver fibrosis (115). Nilsson et al. (104) studied healthy volunteers using a dedicated MRI protocol with moderately high temporal resolution (12-second acquisitions). The liver parenchymal relative enhancement over time was deconvoluted, using a VIF sampled in the portal vein, to derive a parametric map of the liver displaying a HEF measure for each liver voxel. A substantial interindividual variation in the HEF values was noted. In a retrospective study on 198 patients who had undergone Gd-EOB- DTPA-enhanced MRI for evaluation of liver disease, Motosugi et al. described a correlation of the LSC, 20 minutes post- injection, with the ICG- R15 and with the Child-Pugh score (98). In view of the finding in Paper III of a significant correlation between LSC and KHep for Gd-EOB-DTPA, there may also be an association between KHep and ICG-R15.

Statistical analyses, Paper IV Ordinal logistic regression can be applied when the dependent variable (variable to be predicted) is ordinal and the independent variables or parameters (predictors) are continuous. In study IV, the clinical classification was on an ordinal scale since the patients with signs of hepatobiliary disease without biliary stasis can be regarded as an intermediate group between patients with normal function and patients with biliary stasis, but the distance between the groups is not exactly defined. In multiple regression analysis, several variables can be treated as potential effects in the regression model and included or omitted in a stepwise fashion. However, in logistic regression it is recommended to have

64 Discussion – Hepatic Parenchymal Enhancement at least ten observations – of individuals – per variable. In this study with 21 individual datasets, it was more reasonable to perform an analysis on only one variable at a time. The resulting (McFadden) pseudo-R2 (154) values represent the ability of the model to explain the dependent variable, with R2 = 0 signifying that the model predicts the data no better than a constant value of the dependent variable while R2 = 1 means that the model fits the data with no error. When comparing two models, McFadden's pseudo-R2 would be higher for the model with the greater ability to explain the dependent variable. In a larger sample, multiple logistic regression would be one possible method of exploring models containing more than one variable, but the reasoning behind the choice of variables must still be physiologically sound in order to obtain a useful model. Another limitation was that the independent variable MELD range had a skewed distribution since the two highest MELD ranges, M2 and M3, were represented by only one patient each. The corresponding ordinal regression analysis should therefore be interpreted with caution.

Findings related to contrast uptake mechanisms

In biliary stasis patients, FHep stayed at a level close to zero throughout the examination, equivalent to a KHep value near zero. At the cellular level, one can argue that if there were a very slow accumulation of contrast medium, FHep would have increased, while the splenic R1 would have decreased due to blood contrast agent clearance provided by renal excretion. Instead, our impression is that the liver and the spleen acquire similar appearances over time. Blood clearance is also slower than in a normal patient, causing the vessels to have a high signal and thus the image contrast between the vessels and the liver is low for a longer time. Theoretically, it is possible that there is still a small Gd-EOB-DTPA uptake even in biliary stasis. If so, it is apparently too low to be detected, i.e., considerably lower than that of Gd-BOPTA in healthy individuals. Even if Gd-EOB-DTPA enters the cell in the case of biliary stasis, the biochemical gradient driving the facilitated uptake is probably altered since the intracellular environment then contains increased levels of other anions and bilirubin. By this reasoning, the fraction of Gd-EOB-DTPA that can be bound to intracellular protein is markedly reduced, leading to equilibrium with little or no concentration gradient of the contrast agent. Both the

65 Discussion – Hepatic Parenchymal Enhancement bidirectional transporting capacity of OATPs (91, 101) and the (up- regulated) MRP3-mediated transport can enable this process. With very low intrahepatic concentrations of Gd-EOB-DTPA, the appearance on DCE- MRI would be that of absent hepatocytic uptake. The uptake mechanisms of Gd-BOPTA have been less studied than those of Gd-EOB-DTPA during the last decade. New techniques in molecular biology can possibly give new insights into this subject. In clinical MRI, Gd-BOPTA is currently used mainly as an extracellular contrast agent of high relaxivity for lesion characterization in general, and in all areas of vascular MRI, whereas it is more seldom used specifically for hepatobiliary enhancement. In study IV the patients with obstruction of the biliary system had a rather long-standing biliary stasis. In this category of patients the bilirubin level is always elevated, and quite often is high enough to cause obvious jaundice, while symptoms may be few or absent. In contrast, acute biliary obstruction mostly causes pain and nausea, which leads the patient to consult the clinician. These patients rarely have jaundice. In that case, when the bilirubin and bile salt excretion have been disturbed for only a short time, it is possible that the contrast medium uptake is affected in a different way. Tschirch (149) evaluated the quality of bile duct visualization with Gd- EOB-DTPA in cirrhosis patients. The quality was sufficient for morphologic description of the bile ducts in all control subjects, while less than half of the cirrhosis patients showed sufficient image quality. With increasing experience with Gd-EOB-DTPA, reports have emphasized that the presence of liver cirrhosis can affect the uptake and excretion of the agent (25). That differences in liver function affect the appearance of contrast enhancement is illustrated in Paper IV (Fig. 5) and is a reason for the radiologist to acquire specific knowledge about Gd-EOB-DTPA-enhanced MRI examinations. However, another aspect is that the patterns of varying contrast agent uptake and excretion may be explored further to provide diagnostic information on liver diseases that are otherwise difficult to grade. A prerequisite for such a development is most likely that a quantitative method of analysis is used.

66 Discussion – Hepatic Parenchymal Enhancement

Bile duct measurements of SI were not included in the pharmacokinetic analysis of Paper IV. However, to explore the relationship between estimations of contrast uptake and biliary excretion, a measurement in the common hepatic duct, normalized to the pre-contrast SI, was defined for the time point 20 minutes after contrast administration: BileSI_N20. As expected, the BileSI_N20 values were very low in all biliary stasis patients (Figure 14) whose mean value was significantly different from that of patients of clinical grade 1 and 2 (Table 5). The separation between the patients of clinical grades 1 and 2 was not significant. Larger studies are needed to explore if the combination of uptake measures such as KHep and the level of biliary enhancement can enable better grading of hepatobiliary disease without biliary stasis. In a more advanced pharmacokinetic model, measures of biliary enhancement as well as renal enhancement and clearance may be included.

6.3.3. Comparative Analyses – Papers I–III

The double dose of Gd-BOPTA used in Papers I and II compared to that used in Paper III is an obvious explanation of the much higher amplitude of the normalized SI in the liver, the hepatic artery, the portal vein and common hepatic duct measurements from Papers I and II (Figure 15). Despite the fact that the doses of Gd-EOB-DTPA were the same between the study populations, there was greater enhancement of the hepatic artery and the portal vein in the arterial and portal venous phases in study II. The main explanation for this difference is probably that a power injector with an injection rate of 2 mL/s was used for contrast agent administration in studies I and II, whereas study III used manual injection at 1 mL/s. Since the manual injection rate can be expected to have more variation than the rate delivered by a power injector, a more detailed comparison of injection rates is not feasible. On a more speculative note, the difference in common hepatic duct enhancement between the studies, seen for both contrast media, may in part be caused by the test bolus given in study I and II, which possibly contributed to a faster signal increase in the first 10 minutes of the examination.

67 Discussion – Study Design and Limitations 6.4. Study Design and Limitations

In the following paragraphs, certain areas of study design and limitations not covered in the previous paragraphs will be discussed.

6.4.1. Volunteer Sampling

Volunteers presumed to be healthy may have subclinical liver disease not reflected in pre-study laboratory tests. Also, significant changes in diet, alcohol intake and exercise affect the liver, which can result in varying degrees of fat accumulation and temporary abnormal laboratory liver test results (67). These factors may have an impact on the liver physiology and the imaging studies, but they are also represented in the general population. One way to obtain subjects more directly representative of normal physiology is to apply stricter exclusion criteria in the recruitment procedure, e.g., to avoid subjects who are in the middle of or have just performed a major change in their diet or exercise level.

6.4.2. Contrast Media Administration

One obvious complication of the study design relates to the inherent differences in the two clinical MRI protocols that formed the basis for the protocols used in studies I-III. The main advantage was that familiarity with the local procedures results in more consistent quality of performance. When using the test bolus technique with Gd-BOPTA and Gd-EOB- DTPA, approximately 5% and 50%, respectively, of the test bolus will be taken up by hepatocytes and excreted with the bile. This means that the liver parenchyma will already have increased signal intensity – albeit to a small extent – when the main bolus is administered. The greater volume of the main bolus combined with well-timed acquisition still results in prominent first-pass enhancement of the arteries and of the portal veins, since the parenchymal enhancement is small. In experimental situations the effect of the test bolus should, however, be discussed more thoroughly since it represents a methodological complication when judging the contrast enhancement in the liver parenchyma. Even a small dose will have accumulated in the liver when the main acquisitions start.

68 Discussion – Study Design and Limitations

In Study I no enhancement of the bile ducts due to the test bolus was seen in the earliest phases. No significant effect of the test bolus on the “pre-contrast” liver signal intensity was detected, although this problem was evaluated ad hoc and would have been better addressed by performing additional measurements before the main bolus injection. Another motivation to add more baseline measurements is that the pre-contrast scan was performed only once, which is a limitation since there is random variation in each scan within and between subjects. Performing 1–2 extra pre-contrast scans would have given the opportunity to control the intraindividual variation. As described in paragraph 2.3.3, the use of a fixed delay for the arterial phase acquisition in Paper III is a disadvantage compared to the test bolus method used in Paper I and II. The contrast agents were given in different doses. Gd-EOB-DTPA is not approved for use at any higher dose than the given dose, whereas both 0.05 and 0.1 mmol/kg are approved doses for Gd-BOPTA, the higher dose currently being used as the standard dose in many institutions. Even if the relationship between tissue SI and contrast agent concentration had been linear, the experiments in Paper I and II do not control for the influence of differing dosage. In other words, the contrast medium dose is a confounder. Studies I and II thus compare contrast media effects with the clinically used doses. The confounding effect causes uncertainty as to whether the choice of contrast agent is the sole cause of a significant difference in enhancement at a particular time-point; the effect seen is the combination of contrast agent and dose. The general shape of the contrast enhancement curve, i.e., the pattern of change over time, however, should be characteristic of the contrast agent and not affected by the dose. Although Gd-BOPTA was given in the “standard” dose of 0.05 mmol/kg in study III, the contrast enhancement dynamics showed properties similar to those described in study I and II, as reviewed in paragraph 6.3.3. In a clinical context, Gd-BOPTA is better suited for the assessment of vascularity since the doses that are available permit higher enhancement in blood vessels compared to standard Gd-based contrast media and to Gd- EOB-DTPA.

69 Discussion – Study Design and Limitations

In view of the general caution against the use of Gd-based contrast agents in patients with renal function deficit (145, 153), it is advisable to use as low a dose as possible. In patients with no renal impairment, the dose of Gd-BOPTA used here and in several other studies and also employed in MRA and in MRI of the CNS poses no known additional medical risk compared to other Gd-based compounds. For a more detailed evaluation of the benefits of the higher vs. the recommended dose in different patient groups, very large studies are likely to be needed, since good diagnostic performance has been reported for both doses.

6.4.3. MRI Protocol

The focus of the study MRI protocol was to use standard clinically used sequences instead of designing an experimental protocol. Although it was possible to add more phases to the protocol, the time-resolution of the acquisitions would have been too low for an adequate analysis of the first pass. This would require a specialized dynamic sequence with very short acquisitions, which means sacrificing volume coverage and spatial resolution. In the later phases, there were several minutes of waiting time between the sequences. In hindsight, we could have used these intermissions after the venous phase more efficiently, by sampling several additional phases, which would have given valuable information on the precision of the acquisitions. Newer MRI scanners with a field strength of 3 T are now becoming more common and although there are still challenges in upper abdominal imaging with artifacts and high RF energy deposition, the higher field strength can be used to shorten the acquisition time, increase the spatial resolution or increase the signal-to-noise ratio of the images. In addition to increasing the visibility of contrast-enhanced intrahepatic bile ducts (77), as mentioned in paragraph 6.1, using a higher flip angle than usual can increase the conspicuity of non-enhancing liver lesions, as reported by Bashir et al. (4). In the present studies, only standard flip angles were used in the VIBE and THRIVE sequences. In future studies as well as in the clinical context, it will certainly be of interest to evaluate higher flip angles.

70 Discussion – Clinical Applications, Future Studies

6.4.4. Image Review, Documentation and Analysis

We expected the measuring points to be affected by subjective judgment despite clearly defined instructions to the reviewers. To average the individual variations, more than one reviewer is needed. When choosing an image slice showing the relevant anatomy for ROI placement, different reviewers will sometimes choose different slices. Also, the positioning of a ROI can be accomplished in several ways. The measurement protocol allowed each reviewer to work in a consistent manner, with optimal control over anatomical matching of the image slices. In order to focus the statistical analysis on the difference between the contrast agents at a per time point basis, the analysis took into account the fact that the experiments were paired (each subject received both substances) and that observations pertaining to the same subject or reviewer were related. Some variation in the native signal intensity of bile has been described, in both T1- and T2-weighted imaging. Håkansson et al. (54) reported that 5% of a series of 319 patients had a high gallbladder signal relative to the liver on T1-weighted GRE, i.e., the opposite of the expected hypointense appearance. In studies I–II, all subjects had hypointense gallbladders on T1- weighted imaging before main contrast bolus administration. In study III there was one subject with a hyperintense gall bladder and six with some hyperintensity in the dependent portion due to sedimentation, while in study IV there were two patients with mixed gallbladder intensity. No significant difference in KHep values between subjects with high- vs. low- intensity gallbladders was seen, but it cannot be excluded that a significant effect would be noticed in a larger study population.

6.5. Clinical Applications, Future Studies

Great effort is being put into developing examination protocols for pre- operative staging and surgical planning, both for liver transplantation and for liver resection. An accurate three-dimensional mapping of all pathology along with relevant anatomical structures is necessary for precise visuali- zation of the various surgical problems that have to be solved. In large liver resections, especially in patients with advanced cirrhosis, liver function also influences surgical decisions. If the liver function is substantially depressed,

71 Discussion – Clinical Applications, Future Studies the risk of post-operative liver failure is higher since the remaining liver volume and total function (the hepatic reserve) are too small. In a comment on hepatic reserve assessment, Bennet et al. referred to a review of a large series of 1,800 consecutive patients over a period of 10 years who had had hepatic resection (8). They remarked that despite that the hepatic reserve was assessed by classification systems (Okuda (105) and Child-Pugh), as opposed to using more sophisticated metabolic or tracer uptake tests, the mortality was very low, 0.3%, and the mortality in general and specifically in cirrhotic patients had been reduced during the investigated period. This may be an indication that factors besides those revealed by pre-operative liver function testing, e.g., infections and cardiovascular complications, are more important for postoperative mortality. Although improvements in surgical technique and intensive care have been made and may account for a large portion of the reduced mortality, a thorough pre-operative assessment of both liver function and morphology may still prove valuable on peri- and postoperative care. Dynamic contrast-enhanced MRI is one of the many approaches that have been tried as a non-invasive diagnostic tool to stage diffuse liver disease, mainly liver fibrosis. The results presented by Tsuda et al. show some potential of Gd-EOB-DTPA in fibrosis staging in rats (151), but the results have so far not been replicated in humans. Methods using a combi- nation of Gd-based and USPIO/SPIO contrast agents have been described, but are currently only experimental (2). In MR elastography, sequences in which tissue displacement is encoded as phase shifts are synchronized with a pressure wave mechanically generated by a device positioned on the patient’s upper abdomen. The elastic properties of the parenchyma can then be calculated, since the speed of the pressure wave as it propagates through the liver is dependent upon the stiffness of the tissue. A high negative predictive value has been reported, which makes this one of the most promising MRI techniques for the detection and grading of fibrosis (14, 167) and probably more useful than current DCE-MRI methods. With the combination of high resolution MRCP with CE-MRC where both intra- and extrahepatic bile ducts are well visualized, the biliary excretory function can be evaluated on a segmental level. This can be of

72 Discussion – Clinical Applications, Future Studies interest especially for the monitoring of PSC patients. Quantitative com- parisons of hepatic contrast uptake measures, such as KHep, may also be of value both as a global and a regional assessment of liver function. However, further research on the normal intra- and interindividual variation is needed. Initially, examinations in PSC patients will probably have to be compared with a previous (baseline) study in qualitative terms, until there is sufficient experience from quantitative measures. Today, arterial enhancement is the most important finding in HCC detection and characterization, since normally – albeit dependent upon lesion size – it leads to the classification of the lesion as malignant, i.e., as an HCC rather than a regenerative nodule or other benign lesion. Nonetheless, as Bartolozzi et al. have pointed out (3), liver-specific contrast media may prove to be valuable in finding patients with focal lesions that have a high risk of malignancy but no arterial phase enhancement. It is likely that patients at many centers will undergo both an ICG retention test and Gd-EOB-DTPA-enhanced MRI. The finding that Gd-EOB- DTPA (and probably Gd-BOPTA) and ICG have common, or at least very similar, uptake pathways mediated by OATPs suggests a risk of interfe- rence. To counter this risk, a sufficient washout time should be included for either substance. For the same reason, there is some uncertainty as to how Gd-EOB-DTPA examinations should be interpreted in patients with ele- vated bilirubin levels. However, the similar uptake mechanisms and possible interference between ICG, bilirubin and Gd-EOB-DTPA can also represent an interesting mechanism to be further investigated. In particular, the similarities with ICG can be explored to define a quantitative Gd-EOB- DTPA enchanced MRI method where the global measure is comparable to the ICG retention test, whereas the regional measure enables further characterization of pathologic hepatobiliary function. Three out of the four studies reported in this thesis dealt with contrast enhancement only in healthy volunteers. Prospective studies in patients are underway, using dynamic examination of the liver and biliary system with Gd-EOB-DTPA combined with quantitative image analysis to explore the spectrum of hepatic uptake rates in different stages of hepatobiliary disease. To provide the best available image data for quantitative analysis of enhancement, there is room for improvement in the MRI sequence

73 Discussion – Clinical Applications, Future Studies design. A three-dimensional SPGRE sequence that acquires two echoes and is reconstructed with a two-point Dixon technique developed in our insti- tution (27, 33, 129) has shown promising results. It provides in- and out-of- phase images and also “water only” and “fat only” images with isotropic resolution (Figure 16, Figure 17).

Figure 16. Images reconstructed from a 3D T1-W GRE dual echo sequence using a two-point Dixon technique developed in our institution (129). The upper row shows the in-phase and out-of-phase base images (acquired 20 minutes after Gd-EOB-DTPA injection). There is visibly higher signal intensity of the central bile ducts compared to the liver parenchyma, due to biliary excretion of Gd-EOB-DTPA. The lower row shows reconstructed Water Only- and Fat Only images, before contrast injection. Sequence parameters: TR: 6.5 ms, TE: 2.3 ms/4.6 ms, FA: 13°, acquisition matrix 168 x 168, FOV 342 x 261, voxel size 2 x 2 x 2 mm.

The water-only reconstruction is well suited for DCE-MRI. A standard sequence such as THRIVE acquires signal during only a part of the sequence, due to the application of fat saturation pulses. In contrast, the new sequence uses the full acquisition time to collect signal and the water- only reconstruction effectively omits all fat-containing voxels. Hence, more signal can be acquired in the same time (Figure 17). Furthermore, to enable a more detailed analysis of early contrast phases, shorter scans with lower resolution but good volume coverage are being evaluated.

74 Discussion – Clinical Applications, Future Studies

Figure 17. Two 3D T1-weighted sequence types, from the same examination as in Figure 16. Upper row: standard THRIVE sequence before and 20 minutes after Gd-EOB-DTPA injection. Lower row: Water-only images reconstructed from an acquisition of a two-point Dixon sequence (129) pre- and post-contrast. Compared to standard THRIVE, there is better fat suppression and higher liver-to-spleen contrast after Gd-EOB-DTPA.

As discussed in Paper II, so-called blood pool contrast agents are of interest in further studies of the hepatic vasculature, since they are retained in the vasculature for a long time and, at the same time, have high T1 relaxivity in blood (34, 109). Unfortunately, the only approved agent Gadofosveset trisodium (Vasovist®, Bayer Schering Pharma AG, Berlin, Germany) is currently no longer available on the European market. From ongoing prospective investigations in our institution, there is a potential to achieve a more detailed evaluation of the quantitative MRI estimates, and to describe the test response in distinct hepatobiliary disease conditions. More and faster acquisitions have been added to the protocol to provide more insight into the intraindividual variation. Since the acquisition of quantitative voxel-based measures is likely improved, the characterization of regional function can be further explored. A valuable, if not necessary, ingredient for this development is a quantitative analytic approach to Gd-EOB-DTPA-enhanced MRI examina-

75 Discussion – Clinical Applications, Future Studies tions. At the same time, the versatility of Gd-BOPTA makes it the most robust choice in hepatic vascular and parenchymal imaging in order to detect and characterize transient but very critical signs of pathologic vascularization, e.g., in HCC diagnosis. Even if Gd-EOB-DTPA should be approved at a higher recommended dose in the future, Gd-BOPTA would still have the advantage since it can be used in the 0.1 mmol/kg dose, which is unlikely to become an alternative for Gd-EOB-DTPA. Compared to standard Gd-based contrast agents, both Gd-BOPTA and Gd-EOB-DTPA are at an advantage since their higher relaxivity enable adequate image enhancement at a lower dose, which is an important issue since the discovery of NSF. A combination of the clinical information available to the radiologist and the locally established methods of the MRI facility should determine how the examination is designed. As applications of hepatocyte-specific imaging with Gd-EOB-DTPA continue to develop, they may be introduced in the diagnosis and monitoring of non-malignant chronic liver diseases. Furthermore, the management of malignant liver disease is also likely to benefit from improved non-invasive evaluation of global and regional hepatobiliary function.

76 Conclusions

7. CONCLUSIONS

C1. The high contrast between the common bile duct and liver parenchyma had an earlier onset and longer duration for Gd- EOB-DTPA. Hepatobiliary imaging that includes evaluation of the biliary elimination phase can therefore be performed in a shorter time when using Gd-EOB-DTPA. C2. At the time-points studied and at the dosage used, Gd-BOPTA yields higher maximum enhancement of the hepatic artery, portal vein and middle hepatic vein than Gd-EOB-DTPA. Vascular imaging and characterization of lesion vascularity thus benefit from the use of Gd-BOPTA. C3. A signal rescaling procedure was developed to correct for the

non-linear SI response of T1-weighted SPGRE images and successfully implemented in the analysis of datasets from healthy volunteer subjects examined with a standard clinical abdominal DCE-MRI protocol. C4. The hepatobiliary uptake of Gd-EOB-DTPA was correlated to Gd- BOPTA. The approximately tenfold ratio of the hepatobiliary uptake using Gd-EOB-DTPA and Gd-BOPTA was in agreement with preclinical studies. Due to its higher rate of hepatobiliary uptake, Gd-EOB-DTPA is better suited for pharmacokinetic analysis related to liver function. C5. Gd-BOPTA provided better vessel-to-liver contrast in the early time phases than Gd-EOB-DTPA at the clinically approved doses. Pharmacokinetic analysis suggested that the low vessel-to-liver contrast during the early phases when using Gd-EOB-DTPA may be improved by doubling the dose. C6. The hepatic Gd-EOB-DTPA uptake rate, calculated from a pharmacokinetic model using quantitative measurements from

77 Conclusions

the liver and spleen, showed a significant association with the grade of hepatobiliary disease and the Child-Pugh score. It also provided a significant separation of patients with affected hepatobiliary function from those with normal function. The test can be applied to a standard MRI protocol and is a potential candidate for non-invasive quantitative investigation of hepatobiliary function. C7. Simple liver-to-spleen SI ratios and serum bilirubin displayed less association with the clinical grading and the Child-Pugh score. Signal intensity normalization of the liver-to-spleen ratios improved the results. C8. Blood Gd-EOB-DTPA clearance in patients graded as having normal hepatobiliary function was in agreement with data from healthy volunteers. Blood Gd-EOB-DTPA clearance was significantly lower in patients with biliary stasis. C9. Parameters measured in both groups of healthy volunteers were consistent with known differences in Gd-BOPTA dose given and in contrast agent administration. C10. The bile duct relative enhancement 20 minutes after contrast injection was very low in all biliary stasis patients, which were significantly separated from patients without biliary stasis. To closer relate the enhancement dynamics of the biliary system to other tissues, a more detailed pharmacokinetic modeling is required.

78 Acknowledgements

8. ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all of my colleagues and collaborators for their guidance, support and assistance. It has been a rewarding experience to take part in this research and to complete this thesis. Special thanks are due to:

My supervisor Örjan Smedby and co-supervisors Torkel Brismar and Anders Ynnerman for being inspiring, supportive and encouraging during all phases of these studies.

Anders Persson for helping to initiate this project, for introducing me to the field of Radiology and for always being an inspirational colleague and mentor.

Olof Dahlqvist Leinhard for giving invaluable input to this research, and for great teamwork.

My collaborators and co-authors Nils Albiin and Nick Edsborg, for scientific feedback and for carefully reviewing extensive amounts of images.

MR technologists at CMIV, Linköping University, and at the Dept. of Radiology, Karolinska University Hospital Huddinge, for performing studies of consistently high quality.

Johan Kihlberg, Peter Lundberg, Marcel Warntjes, Annika Hall, Anna Lindh, Petter Quick, Maria Engström, Maria Kvist, Anders Tisell, Claes Lundström, Mikael Forsgren, Bengt Norén and Ingela Allert for providing great support and a pleasant learning environment at CMIV.

My fellow PhD students in the CMIV Doctoral Programme, Master of Science students and all others working at CMIV, for giving cross-disciplinary insights on a daily basis.

Pia Säfström, Head of Department, colleagues and co-workers at the Dept. of Radiology, Linköping University Hospital, for creating a stimulating atmosphere for clinical work and scientific inspiration.

My residency supervisors Alf Johansson and Björn Relefors at the Department of Radiology, Hudiksvall, for being insightful, positive and empathic mentors and role models. Former Heads of Department, Karin Rydahl and Ingrid Bryntse, for providing scheduling, training and support necessary for the combination of clinical radiology and doctoral studies. Colleagues and co-workers at the departments of Radiology in Hälsingland for offering superb radiology training and a friendly working environment. Gävleborg County Research Council, for providing financial support.

Hans Reinerdahl, for his aesthetic, linguistic and common sense advice and for being a good friend.

My parents and my brother, for all love and support.

Finally, I would like to thank Linda, my love, for believing in me, and Gustav and Fredrik, for making every day an adventure.

79

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