Structural Changes & Relative Perfusion

Measurements in Crohn’s Disease

PhD dissertation

Rune T. Wilkens

Health Aarhus University

2016

Structural Changes & Relative Perfusion

Measurements in Crohn’s Disease PhD dissertation

Rune T. Wilkens

Health

Aarhus University

Department of Hepatology and Gastroenterology

Aarhus University Hospital

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Supervisors Klaus Krogh, Professor, MD, DMSc, PhD Department of Hepatology and Gastroenterology Aarhus University Hospital, Aarhus, Denmark Henning Glerup, MD, PhD Diagnostic Centre, University Research Clinic for Innovative patient pathways Silkeborg Regional Hospital, Silkeborg, Denmark Agnete Hedemann Nielsen, MD Diagnostic Centre, University Research Clinic for Innovative patient pathways Silkeborg Regional Hospital, Silkeborg, Denmark Anders Tøttrup, Associate Professor, MD, DMSc, PhD Department of Surgery Aarhus University Hospital, Aarhus, Denmark Assessment committee Odd Helge Gilja, Professor, MD, DMSc, PhD Department of Medicine, Section of Gastroenterology Haukelund University Hospital, Bergen, Norway Jens Kjeldsen, Professor, MD, PhD Department of Gastroenterology Odense University Hospital, Odense, Denmark Frank V Mortensen, Professor, MD, DMSc (chairman) Department of Surgery Aarhus University Hospital, Aarhus, Denmark Correspondence Rune T Wilkens Diagnostic Centre, University Research Clinic for Innovative patient pathways Silkeborg Regional Hospital Falkevej 1-3, Silkeborg, Denmark runetw@gmail.com

mailto:runetw@gmail.com

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Preface The present dissertation comprises the work of my PhD studies, performed at Silkeborg Regional Hospital,

where all the scans have been conducted and in close collaboration with Aarhus University Hospital, where all

the surgical procedures and the impedance planimetry procedures are performed.

It all began at a casual meeting in my apartment back in 2009 where Birgit Larsen and Henning Glerup inspired

me to take on the challenges with intestinal ultrasonography – which, according to their prediction, some day

might become the future in Danish gastroenterology. Henning Glerup later became my daily go to person and

supervisor. We have had endless discussions, visions and a common belief on innovation in gastroenterology. I

thank you both for introducing me into this world, and especially HG for being an inspiration as a supervisor,

doctor and as a person helping me through difficult times.

Klaus Krogh, whom I met at medical school and initially introduced me to research, was my obvious choice as

main supervisor. I’m grateful for all your hard work and timely manuscript editing in the critical phase, for your

advices (which I did not always follow) and your insight into this research world.

Agnete H Nielsen, I thank you for your constant believe in me, your motivation, inspiration and support.

Thanks to Anders Tøttrup, the IBD surgeon with a mind as a gastroenterologist but skills and passion like no

other surgeon.

Thank you to all of my great colleagues in Silkeborg, that helped me with recruitment and all the scans,

especially Valeriya P Hovgaard for MRE interpretation, John Hansen, Birgitte H Kristensen, Bonnie M Kirkegaard,

Susanne S Petersen and the rest of the nurses, secretaries and radiographers at Diagnostic Centre.

Thank you David A Peters, for building the software program for the MR analysis and for teaching me some of

this difficult technology. A special thanks also to Rikke H Hageman-Madsen for sticking to the project in crisis

and after seeking new challenges in Vejle, I’ve enjoyed our collaboration and all of our discussions.

The surgical department for helping me with recruitment, I thank Charlotte B Nørager, Daniel Kjær and Bodil

Sørensen, the fabulous nurses Margit Majgaard, Gitte K Sørensen, Chistina O Rasmussen.

The gastroenterologists from Silkeborg and Aarhus, especially Jens F Dahlerup, Lisbet A Christensen, Jørgen S

Agnholt, Christian L Hvas, Annett Canon, Ole K Bonderup, Sabine Becker, the study nurses Lisbet Gerdes and

Toto N Markussen and secretary Marianne Vonsild.

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My skilled and smart Chinese/Danish collaborations Donghua Liao and Hans Gregersen, I thank you for picking

your brains and all of your contributions.

Patients for participating in the study, thank you all.

A great thanks to ALL of my phd/research colleagues and friends for supporting me through this long process,

Lotte Fynne, Anne-Mette Haase, Sara Hebøll, Anne Grosen, Stine Hald, Tine N Gregersen, David Haldrup and

Cecillie B Siggaard, René D Østgaard (and all the rest) – also for distracting me at our basement office and

reminding me that research should be fun.

I’m delighted for all the personal and legal help, that was provided by Susanne Kudsk prior to and throughout

my studies. For the kindness and helpfulness by all my ultrasound co-workers, Lars Bolvig, Jeannette Slot and

Charlotte Strandberg.

I’m totally indebted by the kindness, hospitality, true friendship, inspiration and endless discussions I’ve had

with Stephanie R Wilson & Kerri L. Novak from Calgary, Canada. The outmost obligations to you and your

families for giving me adventures of my life. Also BIG regards to all my friends and collaborators from (University

of) Calgary, Luneburg (Christian Maaser and Frauke Petersen), Düren (Horst Kinkel) and Bergen (Kim Nylund).

Last but not least, I’m thankful for the love, understanding and support from my family and friends, that I’ve

been neglecting for so long. A special thanks to my beloved fiancé Trine for all your support, acceptance and

fruitful discussions.

Thank you

Rune Wilkens

May 2016, Aarhus

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Financial support This PhD study was financially supported by an unrestricted grant from AbbVie Denmark, Central Region Denmark Research Fund of Health Science, Regional Hospital Central Jutland, Research Fund, Crohn’s & Colitis Foundation Denmark, & Becket Fonden. AbbVie had no influence on study design, data analysis or interpretation, writing or submission of manuscripts. Conflict of interest RW has received travel grants from AbbVie Denmark and Takeda Denmark RW has received lecture grants, paid to his institution, from AbbVie Denmark and MSD Denmark The PhD thesis is based on the three papers listed below, referred to in the thesis by Roman numerals

I. Dynamic Contrast Enhanced Magnetic Resonance Enterography and Dynamic Contrast Enhanced Ultrasonography in Crohn’s Disease: An observational comparison study Wilkens R, Peters DA, Nielsen AH, Hovgaard VP, Glerup H, Krogh K (Manuscript submitted)

II. Validity of Contrast Enhanced Ultrasonography and Dynamic Contrast Enhanced MR

Enterography in the assessment of Crohn’s Disease Wilkens R, Hagemann-Madsen RH, Peters DA, Nielsen AH, Nørager CB, Glerup H, Krogh K (Ready for submission)

III. Which Cross-Sectional Imaging Parameters Predict Stiffness of Strictures in Crohn’s Disease

Wilkens R, Liao DH, Gregersen H, Nielsen AH, Peters DA, Nørager CB, Glerup H, Krogh K (Manuscript in preparation)

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Abbreviations AIF arterial input function AIU arbitrary intensity units AUC area under curve CD Crohn’s disease CDAI Crohn’s disease activity index CDEIS Crohn’s disease endoscopic index of severity CDI colour Doppler imaging CEUS contrast enhanced ultrasonography CRP C-reactive protein CSA cross sectional area dB decibel DCE-MRE dynamic contrast enhanced magnetic resonance enterography DCEUS dynamic contrast enhanced ultrasonography E Young’s modulus ESR erythrocyte sedimentation rate f-calpro faecal calprotectin fps frames per second HBI Harvey-Bradshaw index ICC intra-class correlation coefficient) LoA limits of agreement MaRIA Magnetic Resonance index of activity MEGS magnetic resonance enterography global score MR Magnetic Resonance MRCA magnetic resonance contrast agent MRE Magnetic Resonance Enterography MTT mean transit time MTTl mean transit time local NS not significant PROM patient reported outcome measurements PE peak enhancement QIBA Quantitative Imaging Biomarkers Alliance QoF quality of fit ROI region of interest RT rise time SES-CD Simple endoscopic score in Crohn’s disease TIC time intensity curve TOA time of arrival TTP time to peak WiAUC wash-in area under curve WiR wash-in rate WiPI wash-in perfusion index WiT wash-in time WoR wash-out rate WoT wash-out time WoAUC wash-out area under curve

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Table of Contents

English summary ...................................................................................................................................................... 8

Danish summary (Dansk resumé) ............................................................................................................................ 9

Background ............................................................................................................................................................ 10

Crohn’s Disease ................................................................................................................................................. 10

Chronic inflammatory bowel disease ............................................................................................................ 10

Disease manifestations .................................................................................................................................. 10

Disease course ............................................................................................................................................... 11

Defining disease severity ............................................................................................................................... 11

Inflammatory burden: Disease activity vs. fibrosis / chronicity .................................................................... 11

Diagnostic modalities and indices. .................................................................................................................... 12

Clinical assessment ........................................................................................................................................ 12

Biochemical evaluation.................................................................................................................................. 12

Endoscopic assessment ................................................................................................................................. 12

Imaging .......................................................................................................................................................... 13

Capsule Endoscopy ........................................................................................................................................ 15

Histology ........................................................................................................................................................ 15

Aims ....................................................................................................................................................................... 17

Hypotheses ............................................................................................................................................................ 17

Choice of methods ................................................................................................................................................. 18

Bowel Ultrasonography ..................................................................................................................................... 18

Dynamic Contrast Enhanced Ultrasonography ................................................................................................. 18

Magnetic Resonance Enterography .................................................................................................................. 24

Dynamic Contrast Enhanced Magnetic Resonance Enterography .................................................................... 25

Clinical assessment. ........................................................................................................................................... 26

Biochemical assessment .................................................................................................................................... 26

Faecal Calprotectin ............................................................................................................................................ 27

Histopathology .................................................................................................................................................. 27

Impedance planimetry ...................................................................................................................................... 28

Methods ................................................................................................................................................................ 29

Ultrasonography ................................................................................................................................................ 29

Contrast Enhanced Ultrasonography ................................................................................................................ 29

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Magnetic Resonance Enterography .................................................................................................................. 31

Dynamic Contrast Enhanced Magnetic Resonance Enterography .................................................................... 31

Clinical assessment ............................................................................................................................................ 32

Blood and stool Samples ................................................................................................................................... 32

Histopathology .................................................................................................................................................. 32

Impedance Planimetry ...................................................................................................................................... 34

In Vitro Ultrasonography ................................................................................................................................... 34

Data handling and Statistics .............................................................................................................................. 35

Ethical considerations ........................................................................................................................................ 36

Summary of results ................................................................................................................................................ 37

Paper I ................................................................................................................................................................ 37

Paper II ............................................................................................................................................................... 39

Paper III .............................................................................................................................................................. 43

Discussion .............................................................................................................................................................. 45

Technical considerations in relative perfusion measurements ......................................................................... 45

Contrast Enhanced Ultrasonography ............................................................................................................ 45

Dynamic Contrast Enhanced MR Enterography ............................................................................................ 46

Evaluation of Reference standards.................................................................................................................... 47

Clinical Disease Activity Indices ..................................................................................................................... 47

Biochemical ................................................................................................................................................... 48

Histopathological references ......................................................................................................................... 48

Impedance Planimetry................................................................................................................................... 49

General study limitations .................................................................................................................................. 50

Statistical ....................................................................................................................................................... 50

Overall study outcomes ..................................................................................................................................... 51

Paper I: ........................................................................................................................................................... 51

Paper II ........................................................................................................................................................... 51

Paper III .......................................................................................................................................................... 52

Conclusion ............................................................................................................................................................. 53

Further perspectives .............................................................................................................................................. 54

References ............................................................................................................................................................. 55

Appendices ............................................................................................................................................................ 68

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English summary Background: Measuring disease activity and severity in Crohn’s disease is a complex task, that relies on

objective measurements to guide both diagnosis and choice of treatment. Cross sectional imaging is becoming a

mandatory adjunction to gold standard ileocolonoscopy for mapping of disease. Increasing evidence is arising

for the applicability for dynamic contrast enhanced imaging techniques using both MR enterography and

particularly ultrasonography. The underlying hypothesis is the correlation between neoangiogenesis, perfusion

and acute inflammation. Most prior studies have investigated relative perfusion measurements utilizing either

mediocre methodology description or weak defined outcomes. We hypothesized, that ultrasonography and MR

enterography were equally good at predicting pathology and relative perfusion, that correlated with histology

and distensibility changes in affected bowel segments.

Aims: We aimed at investigating validity of structural changes and relative perfusion from ultrasonography and

MR enterography measurements in transmural Crohn’s disease.

Material and methods: We recruited patients with known Crohn’s disease with moderate to severe clinical

activity (study I) and patients referred to elective surgery for small bowel inflammation or complications (study II

& III). All patients were investigated with dynamic contrast enhanced ultrasonography and dynamic contrast

enhanced MR enterography. In study II we graded the histopathological changes of bowel specimens, and

specimens were investigated for distensibility stiffness in study III. These results were analysed for association to

structural imaging findings and time intensity curve parameters of the relative perfusion.

Results: In general, we found good relationship between the structural imaging findings; bowel wall thickness,

ulcers, and prestenotic dilatation with both histopathology and stiffness. Only moderate association was found

between the two dynamic contrast enhanced modalities, and only for some parameters. None of the relative

perfusion measurements were associated with histology and only initial slope of enhancement on MR

enterography associated with bowel wall stiffness.

Conclusion: Crohn’s disease patients can be reliably examined by both ultrasonography and MR enterography

for structural pathological changes, that reflect histological outcomes and stiffness. However, relative perfusion

measurements cannot be used interchangeably and are not predicting histological findings or bowel stiffness.

More methodological research is needed before dynamic contrast enhanced perfusion measurements can be

implemented as a separate part of standard of care.

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Danish summary (Dansk resumé)

Baggrund At måle sygdomsaktivitet og sværhedsgrad af Crohn’s sygdom er en vanskelige opgave, der afhænger

af objektive mål til at guide både diagnostik og behandlingsmetode. Som supplement til guld-standard ileo-

koloskopien er billeddiagnostiske undersøgelser efterhånden ved at blive obligatoriske til vurdering af

sygdomsudbredningen. Der er efterhånden stigende evidens for anvendeligheden af dynamiske

kontrastforstærkede billeddiagnostiske tekniker så som MR enterography og ultralyd. Den underliggende

hypotese er sammenhængen mellem karnydannelse, blodgennemstrømning og akut inflammation. De fleste

tidligere studier har undersøgt måling af relativ blodgennemstrømning, men anvendt enten middelmådig

metode beskrivelse eller svage definitioner af de anvendte endepunkter. Vores hypotese var, at ultralyd og MR

enterografi var ligeværdige til at forudsige patologi og relative blodgennemstrømning, som modsvarer

histologiske fund samt distensibilitet af afficerede tarmsegmenter.

Formål: Vores mål var at undersøge validiteten af strukturelle forandringer og relativ blodgennemstrømning

målt med ultralyd og MR enterography i tarmvæggen hos patienter med Crohn’s sygdom.

Materiale og metoder: Vi inkluderede patienter med kendt Crohn’s sygdom med moderat til svær klinisk

sygdomsaktivitet (studie I) og patienter henvist til elektiv kirurgi på grund af betændelse i eller komplikationer

relateret til tyndtarmen (studie II & III). Alle patienter blev undersøgt med kontrast ultralyd og dynamisk

kontrastforstærket MR enterography. I studie II graduerede vi de histopatologiske forandringer i tarmresektater

og i studie III undersøgte vi resektaterne for distensibilitets graden af stivhed. Disse resultater analyserede vi for

association til både de strukturelle billeddiagnostiske forandringer samt tids intensitets kurve parametre for den

relative blodgennemstrømning.

Resultater: Generelt fandt vi god sammenhæng mellem de strukturelle forandringer på de billeddiagnostiske

undersøgelser, som tarmvægs tykkelse, sår og præstenotisk dilatation, med både histologiske fund og graden af

stivheden. Mellem de 2 dynamiske kontrastforstærkede undersøgelses modaliteter fandt vi kun en moderat

sammenhæng, og det kun for enkelte parametre. Ingen af de relative blodgennemstrømningsparametre var

associeret til histologien og kun den initiale hældning fra MR skanningen associerede med tarmens vægstivhed.

Konklusion: Crohn patienter kan undersøges pålideligt med ultralyd og MR enterografi for strukturelle pato-

logiske forandringer. Relativ blodgennemstrømning kan ej sammenlignes mellem modaliteterne og kan ikke for-

udsige de histologiske forandringer eller tarmens grad af stivhed. Betydelig forskning kræves fortsat før måling

af tarmens blodgennemstrømningen kan implementeres som selvstændig parameter i sygdomsvurderingen.

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Background

Crohn’s Disease

Chronic inflammatory bowel disease

The two major chronic inflammatory bowel diseases are; Crohn’s Disease (CD) and Ulcerative Colitis. This thesis

will mainly focus on diagnosis, classification and assessment of CD. Where ulcerative colitis mainly affects the

rectum and the colon, CD may affect the entire intestinal tract from the mouth to the anus1. Additionally,

extraintestinal manifestations are common1. The pathogenesis of CD is not fully understood, but may involve

complex interactions between pathogenic bacteria, the intestinal epithelium, diminished intestinal mucosal

barrier and increased intestinal permeability1. The disease may be caused by environmental factors2 in

genetically susceptible individuals. A recent genetic association study even raises the probability, that

inflammatory bowel diseases should be further subdivided. Based on the individual persons DNA, CD could

potentially be better classified as CD with colonic involvement and CD with ileal involvement3.

Disease manifestations

Crohn’s Disease is a transmural disease characterized by intermittent periods of active disease and periods of

remission4. Traditionally, a rough disease classification from the Montreal working group5,6 divides patients into

age at onset (< 16 years (A1), 16-40 years (A2), and > 40 years (A3)), disease location (small bowel (L1), colon

(L2), small bowel and colon (L3), isolated upper disease (+L4)). Disease behaviour is further grouped as

inflammatory (B1), stricturing (B2) and penetrating (B3) disease with the possibility of a perianal disease

modifier (+p). Mucosal involvement is often the first sign of activity. Aphthous ulcers can progress into small,

large and confluent ulcerations involving large proportions of the bowel. However, CD is characterized by a

discontinuous involvement of the bowel, often containing skip lesions with intermittent normal bowel wall.

Diarrhoea, possibly with bloody stools, is often seen with active mucosal inflammation especially in the

colorectum. Stricturing disease with narrowing of the lumen and prestenotic dilatation can be seen in both

acute inflammation and, more commonly, in chronic disease1,7. Symptoms commonly associated with stricturing

disease are post prandial abdominal cramping, excessive bowel sounds (borborygmia), nausea and vomiting.

Patients often avoid certain types of food, that trigger discomfort and symptoms often resolve if food intake is

avoided8. Penetrating disease is characterized by luminal contents penetrating through the bowel wall, causing

phlegmons and abscesses in the mesentery or fistulas into other parts of the bowel or other epithelial lined

organs7. Apart from the localized involvement, there is a systemic autoimmune inflammatory response giving

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rise to general symptoms like fatigue, weight loss, work disability9,10 and extra intestinal manifestations mainly

affecting eyes, joints, and skin11.

Disease course

Although symptoms might diminish over time4, CD is generally considered a progressive disease12 going from

inflammation towards stricturing disease and penetrating disease. Thus, up to 70 % of patients will need surgery

within a 10 year period after diagnosis13. It is hypothesized that early diagnosis and aggressive medical

treatment may modify disease outcome14,15 as seen in rheumatology16. Furthermore, smoking doubles the risk

of surgery17 and smoking cessation is associated with a more benign disease course18.

Defining disease severity

Crohn’s Disease is a complex autoimmune disease with several diverse phenotypes and multiple aspects of

impact on the individual patients’ quality of life. Even though, evaluation of treatment efficacy has tended to be

a “one size fits all” approach. Clinical studies focus on corticosteroid-free remission14, mucosal healing, deep

remission19 and, recently, even transmural healing20. However, grading of CD severity is undergoing constant

debate and the “treat-to-target” approach, adopted from rheumatology, is increasingly promoted21,22. Driven by

the U.S. Food and Drug Administration recommendations for future trial endpoints, consensus is moving

towards the use of patient reported outcome measurements (PROMs) combined with objective measurements

of inflammation21. Recently, a group of experts proposed an even broader perspective on this complex

disease23, dividing severity into

Impact of the disease on the patient

Inflammatory burden

Disease course

In the following we will focus on methods for assessment of inflammatory burden.

Inflammatory burden: Disease activity vs. fibrosis / chronicity

Inflammatory burden may be summarized as the extent of disease combined with the actual amount of

inflammation or wall thickness. Some authors have proposed various scores to measure elements of this

entity24,25. Others are claiming to define activity26–28, without acknowledging the high concomitant presence of

both histologically active and chronic / fibrotic disease29. Existing treatment for CD is not effective for treating

fibrotic strictures30. Thus, a modality is needed to measure burden of disease, and additionally to differentiate

between disease mainly consisting of treatable active inflammation, and chronic fibrotic disease where surgery

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is needed. Since CD is exhibiting continues progression, grading indices should preferably give the amount of

activity or chronicity as a continuum rather than a dichotomous statement of active versus inactive disease.

Diagnostic modalities and indices.

Clinical assessment

Patient symptoms or clinical assessment are broadly used in research and everyday clinic to guide decision

making. The first and most widely used clinical index is the Crohn’s Disease activity index (CDAI)31. This index

consists of a seven days’ evaluation of number of stools per day, daily symptom severity, and general wellbeing

combined with assessment of extraintestinal manifestations, haemoglobin level, weight changes, use of

antispasmodic drugs and palpable abdominal mass. CDAI < 150 is regarded as clinical remission, 150-220 as

mild, 220-450 as moderate and > 450 as severe disease7. A simplified version was adopted in 1980 and called

the Harvey-Bradshaw Index (HBI)32. The advantage (and disadvantage) of this index is the momentary

assessment of the score on a daily basis. The indices have been the main outcome parameter in evaluation of

drug efficacy for decades33. However, treating beyond symptoms may improve long term patient outcomes15,19.

The STRIDE initiative suggested “resolution of pain” as the PROM of choice. However, the same group voted

that this cannot stand alone without more objective parameters like endoscopy21.

Biochemical evaluation

Systemic markers of inflammatory activity in blood samples have traditionally been haemoglobin, erythrocyte

sedimentation rate (ESR) and c-reactive protein (CRP)34. Stools can be analysed for calprotectin, a major protein

in neutrophilic granulocytes35 and a surrogate marker of intestinal inflammation in the small and large

intestine36,37. Faecal calprotectin (f-calpro) is a marker for predicting relapse after surgery38 and correlates better

with endoscopic indices than CRP and clinical assessment37,39. However, f-calpro does not provide information

on disease localization in the gut and it can be positive in the absence of other signs of inflammation and even

negative in patients with severe activity40.

Endoscopic assessment

Ileo-colonoscopy is regarded the gold standard for assessment of luminal CD. The best evaluated and validated

scores for grading activity are CD endoscopic index of severity (CDEIS)41 and Simple endoscopic score in CD (SES-

CD)28. CDEIS describes the rectum, sigmoid and left colon, transverse colon, right colon including cecum, and

terminal ileum with assessment of deep ulcers, superficial ulcers, percent of luminal surface involved by disease,

percent of luminal surface involved by ulcers, and ulcerated stricture or no- ulcerated stricture41. The simple

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score assesses the same segments semi quantitatively by scoring presence of ulcers from (0-3), extent of

ulcerated surface (0-3), extent of affected surface (0-3) and presence and type of narrowings (0-3)28. The scores

have been developed to evaluate treatment response in clinical trials42. A third, but unfortunately non-

validated, scoring system for assessment of the neo-terminal ileum, the Rutgeerts score43, is also commonly

used in clinical trials. The European Crohn’s and Colitis Organisation42 suggested the definition of mucosal

healing as CDEIS or SES-CD = 0. Unfortunately, no consensus exists and different cut-off values are being

proposed in the literature44–46. Although endoscopy is regarded gold standard, endoscopic scores have some

limitations. They are complex, cumbersome and typically requires post procedural time to evaluate42. The scores

have a bias towards classifying colonic disease as more severe than ileal disease, since only 26 out of 75 patients

had their ileum scored during the development phase41. Reproducibility is good for both scores only as long as

scoring is performed by experienced gastroenterologists47,48 and further standardization of their interpretation

is needed49.

Imaging

Traditionally, plain film small bowel follow through was the modality of choice for investigating the small

intestine for ulcerated mucosa, strictures and fistulas with excellent spatial resolution50. Today, this modality is

regarded obsolete due to poor visualization of transmural disease and because of radiation burden51. CT

enterography or enteroclysis have relatively high spatial resolution and high accuracy in detecting inflammation

and complications in CD52. This ionizing cross-sectional modality is widely accessible but the use should be

minimized due to the high radiation burden that exceeds the small bowel follow through by a factor five51. For

repeated examinations, especially in young adults or teenagers with CD, repeated CT can lead to a high

accumulated radiation dose53,54.

Magnetic Resonance Enterography

Magnetic resonance enterography (MRE) is an accurate technique for the diagnosis and determination of extent

and activity of CD55. T1-weighted gadolinium-enhanced imaging is usually added to conventional anatomical

imaging as part of standard MRI small bowel protocols and shows considerable promise as a marker of disease

activity56. Three MRE based activity scoring systems have been developed based on endoscopy or histology as

gold standard57. The Magnetic Resonance Index of Activity (MaRIA)58 is an index validated against endoscopy

and with capability to follow treatment efficacy59. The index encompasses bowel wall thickness, relative contrast

enhancement, bowel wall oedema and intestinal ulcers58 with a 90 % sensitivity and 94 % specificity for ulcer

healing and mucosal healing, respectively. The Magnetic Resonance Enterography Global Score (MEGS) was

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designed and initially validated against biopsy histopathology60 and later f-calpro61 and a combined clinical

reference standard62. The MEGS can distinguish responders from non-responders with a follow-up scan62. The

score is based on a multifactor semiquantitative score and a simplified score, described in more detail in

Methods of choice and Methods. The Nancy score includes diffusion-weighted imaging63, which is out of the

scope of this thesis. The Lemann score is also proposed for assessing digestive disease damage and is applicable

by both CT and MR24.

Dynamic Contrast Enhanced Magnetic Resonance Enterography

The chemical structure of the paramagnetic MR contrast agent (MRCA) includes a centrally placed gadolinium

ion, an octadentate ligand, and a coordinated water molecule. It changes the T1 relaxation time of extracellular

water molecules. MRCA is freely redistributed from the vascular to the interstitial space and cannot truly reflect

blood flow64–66. Dynamic contrast enhanced MRE (DCE-MRE) is commonly used to determine perfusion of the

brain as the vascular-brain-barrier keeps MRCA within the blood vessels. This is unfortunately not the case in

extra-cerebral MR contrast studies, where contrast extravasation makes assessment of blood volume difficult67.

Pixel-by-pixel or manually drawn regions of interest (ROIs) are used to build signal amplitude time intensity

curves (TICs), reflecting dynamic changes in T1 relaxation time. Calculating TIC parameters is easily done.

However, tissue extravasation of MRCA and difficulties in consistent imaging acquisition68 limit interpretation as

they do not reflect biophysiology69. Nevertheless, changes in signal intensity may be useful as relative

quantitative surrogate markers of tissue perfusion70,71.

Ultrasonography

B-mode ultrasound of the bowel with colour Doppler imaging (CDI), either with or without use of oral contrast

solution, has similar sensitivity and specificity as MRI and CT for assessment of inflammatory activity, extent and

location52. Furthermore, CDI correlates well with endoscopy72. Stenotic and penetrating disease can also be

detected by US55,73. A score has been proposed by Calabrese et al. for the evaluation of disease burden74.

However, the score is cumbersome and time consuming and does not differentiate between activity and

chronicity. Blood flow on US has traditionally been assessed with conventional CDI75,76. Although CDI is highly

sensitive to the detection of blood flow, it is better able to detect relatively fast-moving blood in larger blood

vessels than the lower perfusion level of the gut. Thus, CDI is inadequate for the quantitative assessment of

perfusion77 and can be falsely negative78.

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Dynamic Contrast Enhanced Ultrasonography

As with MRI and CT, intravenous contrast can be used to evaluate the bowel vasculature on US. Unlike the other

modalities, however, US has excellent temporal resolution and all contrast-enhanced ultrasonography (CEUS)

examinations are dynamic. Therefore, CEUS can be used to perform relative and absolute quantifications in

addition to real-time qualitative assessments of enhancement pattern and intensity79. Although many have tried

to establish a scoring system with various quality of reference standards78,80–83, none have so far been validated.

One of the main problems with the modality is the post-acquisition analysis. The procedure is far from

standardized and there is a lack of a solid methodology to analyse data from TICs with a high quality of fit and

reliability. This modality will be discussed in further details in Choice of methods.

Capsule Endoscopy

Three different scores exist for capsule endoscopy of the small bowel in CD42. The presence of more than three

ulcers in the small intestine has been regarded as diagnostic for CD84. The Capsule Endoscopy CDAI is a validated

score assessing small bowel inflammation, extend and narrowings, with strong correlation between observers

for the total score85. Finally, the Lewis score86 assess the small bowel in tertiles for villus oedema, number of

ulcers, extend and distribution of lesions. The great advantage is the low ulcer miss rate of 1 % yielding a high

sensitivity86. However, the consequence is a low positive predictive value at about 50 %45. Further, the modality

can by compromised by poor cleansing quality of the small bowel and there is a risk of capsule retention in

stenotic CD.87

Histology

Inflammation can be defined as local reaction to injury. In the inflammatory cascade, the contributions made by

hyperaemia, exudation of fluid, infiltrates carrying inflammatory cells, and cell proliferation vary with the type of

injury88. In classic histopathology active inflammation is regarded as epithelial damage in association with

neutrophils7,89,90. If the cause of injury does not disappear, homeostasis is disturbed and drives the active

inflammation in the direction of chronicity with activated myofibroblasts and intestinal fibroses91 rather than

successful intestinal healing where myofibroblasts undergo degradation92. For the diagnosis of CD7, colonoscopy

with multiple colonic and ileal biopsies (minimum of two per site) is established as first line procedure. Biopsies

are preferably taken from both involved and non-involved mucosa. European guidelines7 state that if

granulomas are detected, only one additional feature is needed for establishing a firm diagnosis (preferably

focal inflammation like architectural abnormalities). Tanaka et al. evaluated 70 histological features in colon

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biopsies from Japanese subjects and validated their index on Canadian subjects. They have suggested elements

for scoring IBD vs. non-IBD and for differentiating CD against ulcerative colitis93.

Gross examination of a resected specimen, typically reveals firm “creeping fat”, shown as sub-serosal fat

wrapping. The bowel wall is usually thickened and often narrowed over short or long distances. The mucosal

surface can be seen with aphthous or deep longitudinal ulcers, sometimes leaving a cobblestone apperance94.

Inflammatory changes of the serosa, including Crohn’s rosaries, can be seen in CD, which distinguishes it from

UC95. Abundant inflammation can be seen creating deep fissures, sinuses and fistulas94 which is more common

in ileum or ileocolonic disease90. Graham et al.96 showed that a strictured bowel segment contains more

collagen than a normal bowel segment within the same patient. The same group96 also found a relative increase

in type III collagen, produced by fibroblasts97, and type V collagen, produced mainly by adjacent smooth muscle

cells96,98.

17

Aims

1) To compare the following characteristics of CD obtained with US and MRE (study I):

a. Structural changes (e.g. thickness and length of disease)

b. Relative TIC perfusion (obtained with DCE-MRE and CEUS)

2) To compare the following findings on US and MRE with histopathology from CD stenoses (study II):

a. Structural changes (e.g. thickness and ulcers)

b. Relative TIC perfusion parameters (obtained by DCE-MRE and CEUS)

3) To compare the following findings on US and MRE with wall stiffness in CD stenoses (study III):

a. Structural changes (e.g. prestenotic dilatation and bowel wall pattern)

b. Relative TIC perfusion parameters (obtained with DCE-MRE and CEUS).

4) To investigate the repeatability of ROIs for CEUS analysis of small intestinal perfusion in CD (study I).

Hypotheses

1) Comparison of characteristics of CD obtained with US and MRE: a. There is no difference in structural changes found with US and MRE (e.g. bowel wall thickness

and length of disease) b. Relative TIC perfusion parameters are associated between CEUS and DCE-MRE for the initial

part of the TIC 2) US and MRE and histopathology from CD stenoses:

a. US and MRE are equally valid for identification of structural bowel wall changes like thickness

and ulcers b. Relative TIC perfusion parameters obtained by CEUS and DCE-MRE are equally valid and

correlate with active inflammation and inversely with chronic fibrotic changes 3) Correlations between US and MRE and wall stiffness in CD stenoses:

a. US and MRE can predict bowel wall stiffness from structural findings (presence of stricture and

prestenotic dilatation) b. Relative TIC perfusion parameters obtained by CEUS and DCE-MRE are inversely correlated with

wall stiffness. 4) The selection of ROI affects the repeatability of CEUS analysis of small intestinal perfusion in CD.

18

Choice of methods

As described above, the best modalities for non-invasive, radiation-free assessment of transmural localized ileal

CD are US and MRE54. As we wanted to explore the use of methods for assessment of relative perfusion, we

decided on CEUS and DCE-MRE. Considerations about experimental protocols and data analysis are given in the

following sections.

Bowel Ultrasonography

In experienced hands, ultrasound of the bowel may be possible even in the presence of intestinal gas. Usually,

an abdominal probe is used initially to get an overview while a medium to high frequency probe is used for

detailed examination of the site of interest99. The choice of probe depends on the investigator and the patient

composition. If the area of interest is deeply located, a low frequency probe will usually be needed. In our study,

most of the scans were performed with a linear 4-9 MHz matrix probe, which provided good details and

penetration, see Methods. With a high frequency probe it is usually possible to detect 5 to 9 sonographic layers

of the bowel wall that correlate relatively well with histology100,101. Nylund et al.101 has also shown that Crohn’s

rosaries appears as tiny echo poor bumps on the outer intestinal border on an ex-vivo ultrasound scan. Bowel

wall thickness is regarded as one of the most important factors for discriminating between normal and diseased

bowel with cut-offs between 2-4(5) mm for terminal ileum and colon52,102. However, a comprehensive study on

healthy volunteers revealed that in 95 % the bowel wall thickness from jejunum to sigmoid colon was between

0.5 – 1.8 mm103. In a meta-analysis, sensitivity for diagnosing IBD was found to be 91.0 % with a cut off at 3 mm

and 94 % for 4 mm. Specificity was also highest for the 4 mm cut-off. However, the meta-analysis was not

designed with the purpose to define the best cut-off and a lower cut-off value would intuitively give a higher

sensitivity but lower specificity. We decided to use 3 mm rather than 4 mm as a cut-off for recruitment of

patients into our studies, since we regarded patients with ≤ 3 mm as normal.

Dynamic Contrast Enhanced Ultrasonography

The US Probe The trade-off between resolution and penetration, which plagues all ultrasound exams, is even more

pronounced when performing CEUS. Although high frequency probes provide better image resolution for bowel

segments close to the abdominal wall, there is less backscatter signal intensity, more bubble destruction and

more signal attenuation104,105. Therefore, scanning with high frequency probes is ineffective in patients with high

BMI or deep bowel loops. Applying different probes and systems for comparison still needs to be evaluated for

repeatable performance. As for now selection of different probes and probe frequencies will not give

19

comparable results between patients or even between exams106. Therefore, we chose the 9L4 probe for all CEUS

examinations throughout the studies as it is a medium frequency probe allowing for sufficient amount of details

without highly sacrificing penetration.

Image Acquisition The imaging was performed by continuous acquisition and recording of data in a fixed location. This allows for

objective measurement of relative bowel perfusion as the intensity of backscatter signal correlates well with the

bubble concentration in a specific ROI107. Beam attenuation occurs with increasing volume of bubbles

necessitating appropriate dosing for low concentration of the contrast agent108. On the other hand, the

recommended contrast dose for bowel is higher than that for liver109. Since we generally use 1.2 ml for liver and

the equipment has a sensitive bubble detection, we chose 2.4 ml SonoVue® (Bracco Imaging, Milan, Italy) per

injection. In analysis of non-raw data, a high dynamic range of over 50 is required for good software de-log-

compression and thus accurate results108,110–113. Reduction in dynamic range/compression will produce higher

signal intensity, increase the risk of oversaturation and, thereby, limit reproducibility106,110,112. A constant low

mechanical index is required to preserve the bubble population105. Frame rates can easily be kept low with very

little effect on TIC114. We therefor decided on the maximum of dynamic range of 80, mechanical index < 0.1 and

framerate at 10 frames per second.

Choice of contrast administration At the time of study planning and approval, two main quantification techniques were described. Bolus injection

and infusion-burst-replenishment79. The advantages of the former are the simple setup, less use of contrast and

that it is unnecessary to have a pump and waiting for a steady state level of contrast to arrive. The advantages

of the latter are the ability to perform multiple repeated measurements and direct computation of relative

blood volume, mean transit time and thus a perfusion index. Due to simplification and practicality, we chose the

bolus injection, which was the most common use in prior publications assessing bowel wall perfusion115–117.

Deconvolution is a process that takes the arterial input function (AIF) into account, thus allowing to scale the

relative perfusion measurements accordingly118. In a phantom model, the reproducibility was comparable

between deconvolution and non-deconvolution, whereas the reproducibility was superior for deconvolution in a

mouse model. However, the even greater benefit is the possibility to obtain absolute measurements of blood

perfusion119. The central volume theorem states that

𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑓𝑓𝐵𝐵𝐵𝐵𝑓𝑓 = 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑣𝑣𝐵𝐵𝐵𝐵𝑣𝑣𝑣𝑣𝑣𝑣𝑀𝑀𝑣𝑣𝑀𝑀𝑀𝑀 𝑡𝑡𝑡𝑡𝑀𝑀𝑀𝑀𝑡𝑡𝑡𝑡𝑡𝑡 𝑡𝑡𝑡𝑡𝑣𝑣𝑣𝑣

(1)

Where blood flow is the main parameter of interest. In brief, in the deconvolution theory the equation is

20

𝐶𝐶(𝑡𝑡) = 𝜌𝜌𝑘𝑘𝐻𝐻∙ (𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑓𝑓𝐵𝐵𝐵𝐵𝑓𝑓) ∙ (𝐶𝐶𝑀𝑀(𝑡𝑡) ⊗𝑅𝑅(𝑡𝑡)) (2)

Where C and Ca are the concentration of contrast at the function of time t in the tissue and in the artery

respectfully. The letter ρ denotes the tissue density and kH the difference in haematocrit between arteries and

capillaries and R(t) is the residue function. Finally ⨂ is the tensor product. This is under several assumptions,

including ρ = 1 and kH = 1 and an instantaneous bolus injection. Through an algebraic approach using matrix

calculations, the AIF can be calculated and the blood flow measured. The details of this119,120 is outside the scope

of this thesis. Since the modality was not tested for CEUS in humans and the assumptions and calculations were

overwhelming without dedicated commercialized product at hand, we decided not to take the AIF into account.

Performing the Exam: There are many techniques which improve the acquisition and thereby the post-acquisition analysis. In order to

support in-plane motion compensation, alignment of the long axis of the probe in the plane of movement of the

bowel, caused by respiration, can be crucial. Therefore, in the majority of cases, a sagittal plane orientation is

preferred to compensate for expected diaphragmatic excursion with breathing. Also, a longitudinal orientation

of the transducer face to the bowel wall was preferred, as it provides more desirable ROIs. However, we tried

both directions on the bowel wall, since the transverse approach may be favourable in some occasions. In

general, longer acquisition time is preferable79,121, as more parameters can be measured due to a more

comprehensive TIC, especially the area under curve (AUC). We were not able to record longer than 90 seconds

from end of flush injection.

Post-acquisition: Dedicated software for detailed analysis of quantification data is required and offered by most ultrasound

manufactures. The majority of systems have the limitation of being vendor specific, allowing for analysis of their

own data only. Therefore, data from different systems cannot be compared. Platform neutral software allows

for analysis of data from all systems utilizing data in a proper format. The ability of such a system to neutralize

differences in quantification parameters across platforms is desirable but not yet validated in clinical trials. CEUS

equipment generally acquires raw native data in a linear format at the time of acquisition. However, images are

displayed in a log-compressed format. The transfer and export of data is generally in DICOM format, log-

compressed, including a picture or movie file, plus a header, containing the acquisition details79,113. Data analysis

should be based on linearized data only, which can be accomplished with optional special built-in software

programs. Alternately, the DICOM header information can be used to re-linearize acquisition data for analysis by

a specific de-log-compression data conversion with dedicated analytical software110–113. Other forms of data

21

such as AVI, MPEG and other video formats without acquisition information are less useful for analysis and

should be avoided. These video formats allow only a pixel-by-pixel analysis of log-compressed data and not an

intensity measurement from linear format. Since the build in option for Siemens was less useful for contrast

quantification, our main option to re-linearize data from DICOM format112 was the commercialized software,

VueBox® (Bracco Suisse SA, Geneva, Switzerland).

A TIC is constructed by calculating the change in mean ROI intensity over time, figure 1. Therefore, ROI size and

placement do play a crucial role in representing the overall mean intensity and tissue perfusion, figure 1122. A

small ROI size, positioned on the most enhanced segment of the bowel, does not show the true state of disease

activity in the bowel segment and a small ROI size is also more susceptible to motion related fluctuations in

intensity, figure 1. In addition, small ROI size may not have a high reproducibility rate due to variation in

intensity when ROI location is changed. A large ROI size may show a better approximation of overall disease

activity in the inflamed region by providing a better mean intensity, quality fitted curve, and measurement of

tissue perfusion. However, large ROI size may cover less perfused layers or segments of the bowel and

consequently result in a falsely lower peak enhancement. A useful feature for ROI placement optimization is an

in-plane motion compensation feature, which enables automatic detection of movement in the B-mode picture

and adjusts the ROI placement accordingly. In-plane motion compensation helps reducing the severe fluctuation

in TIC112, but it can be inaccurate when only small movements occur, and should thus not be used123. Single

frames can then be deleted due to severe displacement of ROI secondary to respiration or peristalsis. The

resultant curve will still be suitable even with very few frames114. Also, colour mapping of selected parameters

such as PE, RT and AUC is beneficial in ROI placement, figure 2. In study I, we therefore tested different ROI sizes

and numbers to determine the best repeatable ROI strategy. Some analytical software programs offer two types

of data presentation, a linear graph measured in arbitrary intensity units (AIU) and measurements or a graph in

decibels (dB). The linear graph shows the most realistic presentation of raw data, and consequently provides the

most reliable method for performing measurements. However, viewing the actual TIC in log-format

approximates more closely the visual interpretation of the enhancement79. Additionally, the dB values followed

a Gaussian distribution, which the AIU did not, see statistical analyses. TIC is analysed based on time

parameters, intensity parameters and a combination of them. These parameters should be calculated and

reported from the fitted curve on linear data. Due to the nature of the log-formula, curve fitting on log-data is

heavily susceptible to minor changes in the baseline and late wash out period. Therefore, log-fitted curves

should not be used79,121,124,125. Also, various mathematical equations are used for curve fitting in each graph

setting, especially for the linear data121.

22

Figure 2 Heat map

Colour mapping image on the left showing Peak Enhancement

with in the large circular Region of Interest. On the right side

the corresponding b-mode image is shown. In this case Peak

Enhancement is constant throughout the bowel wall layers and

thus a homogenous enhancement pattern.

Figure 1 TIC effect of size of region of interest (ROI).

Four different ROIs drawn are illustrated in the upper

right contrast enhanced image and the

corresponding time intensity curves are shown on

the graph. The largest ROI in blue shows the lowest

peak enhancement (PE) and the least signal

fluctuations. Smaller ROIs will have higher PEs but

show considerable signal fluctuation. Therefore, care

has to be taken when using very small ROIs

Figure 3 Measurable time intensity curve

parameters on contrast enhanced ultrasound.

See text for further details. Time parameters,

measured in seconds (s), are displayed in grey.

Intensity Parameters, measured in arbitrary

intensity units (AIU) on Linear scale and are

displayed in blue Combined parameters,

measured in AIU/s and Sum of AIUs are displayed

in green and red respectively.

23

The details and specifics of each model are beyond the scope of this thesis. We used the non-disclosed formula

from VueBox. The TIC parameters, that can be obtained from a bolus injection curve are shown in Figure 3 and

described in the following126.

Time Parameters: Time of arrival (TOA) or Time zero offset (t0) is the time from injection to arrival of the microbubble contrast

agent bolus in the ROI. It is a parameter heavily dependent on many factors like injection site/distance from the

heart, cardiac output and speed of contrast and flush injection127. Therefore, it should not be used as a relevant

clinical indicator. However, it is useful in calculating other time parameters. It is worth noting that some

programs start the upslope of the fitted curve from the first recorded frame and not TOA which should be taken

into consideration. Thus, manual elimination of the frames prior to TOA is necessary. To have consistent

measurement of TOA, timer and recording should be started at the same time. TOA can be useful in case of

reinjection and when breath hold timing is required.

Wash-in Time (WiT) and Rise Time (RT) are similar parameters, defined individually, based on quantification

software and thus, a clear definition of measured values is required in clinical studies. WiT is the interval from

TOA (start of the upslope) to the PE. RT is the time for which intensity varies from 5 % to 95 % or from 10 % to

90 % to PE (depending on software).

Time to Peak (TTP) is the time from injection to PE. The parameter is defined by TTP = TOA + WiT and should

therefore only be used for calculating the WiT. WiT and RT are often used interchangeably with TTP, which can

be misleading in data interpretation and analysis.

Wash-out Time (WoT) or Fall Time is the time from PE until total washout, which not always occurs within the

recording time.

Mean Transit Time (MTT) or Mean Transit Time local (MTTl) corresponds to the centre of gravity of the fitted

curve calculated using the programs specific perfusion model function, corrected for TOA. MTT can be

estimated by the time point where the AUC is divided into two halves, which for optimal estimation requires

total washout. Some programs, therefore, use the Full Width Half Max for the same estimation79.

Full Width Half Max: Is measured from the time between 50 % PE on the wash in slope to 50 % PE on wash out

slope. This parameter is often confused with or used for calculation of MTT/MTTl79,121.

Intensity Parameters Peak Enhancement (PE) refers to the highest enhancement level on the TIC and can be measured in AIU and dB.

Like other parameters, PE also should be accurately measured and calculated from linear data.

Intensity and combined parameters depend on the US system and many confounding factors discussed earlier.

24

Combined Parameters Area Under the Curve (AUC) is calculated from the linear curve only and shows the sum of all instant intensity

values on the fitted curve. It is believed that AUC reflects a reasonable approximation of disease activity.128,129

Since AUC is the sum of all instant intensity values, the same limitations which apply to measurement of PE will

apply to AUC when it comes to standardizing the calculation and its correlation with disease activity among

multiple CEUS centres. AUC can even be diverted into Wash-in AUC (WiAUC) and Wash out AUC (WoAUC).

AUC = WiAUC + WoAUC

Wash-in Rate (WiR) is the tangent of the wash in slope. Either perfectly calculated at the steepest part or

approximated by calculating difference in intensity values between two points on the curve divided by the time

between these two points, e.g. intensity 75%-25%

𝑊𝑊𝑊𝑊𝑅𝑅 =Y75%Wi− Y25%Wi

X75%Wi− X25%Wi

Wash-out Rate (WoR) is the tangent of the wash out slope. Either perfectly calculated at the steepest part or

approximated by calculating difference in intensity values between 2 points on the wash out curve (see WiR).

Wash-in Perfusion Index (WiPI) can be described as

𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 = 𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝐶𝐶𝑅𝑅𝑅𝑅

Since there is no consensus in the literature about what parameters to use, we tested most of the TIC

parameters against our outcome variables. TOA and TTP was consistently avoided and Fall Time deemed the

least important factor and it was thus avoided to minimize mass significance. During the analysis phase and

repeatability phase we found the MTTl very heterogeneous on the heat maps and less reproducible between

calculations. It was therefore not utilized for further correlation.

Magnetic Resonance Enterography

The use of oral contrast agents in MRE is essential to evaluate finer details in the bowel wall and minimize false

positive diagnostics. The choice of either enterography and enteroclysis is continuously debated. Enterography

is favoured by patients, since it is more tolerable and without the use of fluoroscopy to ensure correct

placement of the nasojejunal catheter130. Although enteroclysis is superior because of the distension of the

small intestine, especially the jejunum, the diagnostic accuracy and reproducibility are equal for the two

methods131. We have chosen enterography for better patient acceptance in our institution, which we also

applied in our studies. Like on ultrasonography, structural findings can be reported from MRE. MEGS and MaRIA

were the basis of our structural elements to record from the scans.

25

𝑀𝑀𝑀𝑀𝑀𝑀𝑊𝑊𝑀𝑀 (𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡) = 1.5 ∙ 𝑓𝑓𝑀𝑀𝐵𝐵𝐵𝐵 𝑡𝑡ℎ𝑊𝑊𝑖𝑖𝑖𝑖𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (𝑠𝑠𝑠𝑠) + 0.02 ∙ 𝑅𝑅𝐶𝐶𝑅𝑅 + 5 ∙ 𝐵𝐵𝑠𝑠𝐵𝐵𝑠𝑠𝑠𝑠𝑀𝑀 + 10 ∙ 𝑢𝑢𝐵𝐵𝑖𝑖𝑠𝑠𝑀𝑀𝑀𝑀𝑡𝑡𝑊𝑊𝐵𝐵𝑠𝑠

Where RCE is Relative contrast enhancement at 70 seconds.

Dynamic Contrast Enhanced Magnetic Resonance Enterography A variety of contrast-enhanced acquisition techniques and analyses are available for MRE. They range in

complexity from a single post-contrast dataset for radiologist-subjective assessment to rapid serial acquisitions

with full pharmacokinetic modelling68. However, all methods have limitations or may not be feasible. If we want

to analyse underlying pathophysiologic processes with DCE-MRE in an absolute quantitative manner,

parameters like the volume transfer constant Ktrans (that represents perfusion), extravascular volume fraction Ve

and Kep (the gadolinium washout rate) can be calculated using the two-compartment Tofts (extended) model or

variations of this model132.

𝐾𝐾𝑣𝑣𝑒𝑒 = 𝐾𝐾𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

𝑉𝑉𝑒𝑒 (3)

Reliable arterial input function (AIF) and temporal tissue sampling of ≤ 5 s / image are needed to fulfil the

requirements of this calculation68. The intrinsic pre-contrast T1 values of the area of interest and blood are also

needed. This is commonly performed using the variable flip angle technique133. The technique is unfortunately

far from perfect, since there is a variability of up to ±40 % of the flip angle in vivo. As a 15 % flip angle deviation

accounts for about 28 % error margin, this can lead to large uncertainties. In high temporal and resolution

images, signal-to-noise ratio further leads to measurement errors when comparing individual images to

Table 1 Magnetic Resonance Enterography Global Score (MEGS) Score 0 1 2 3

Mural thickness < 3 mm > 3 - 5 mm > 5 - 7 mm > 7 mm Mural T2 signal Equivalent to normal

bowel wall Minor increase in signal: bowel appears dark grey on fat-saturated images

Moderate increase in signal: bowel wall appears light grey on fat-saturated images

Marked increase in signal: bowel wall contains areas of white high signal approaching that of luminal content

Peri-mural T2 signal (mesenteric oedema)

Equivalent to normal mesentery

Increase in mesenteric signal but no fluid

Small fluid rim (≤ 2mm) Larger fluid rim (> 2 mm)

T1 Enhancement Equivalent to normal bowel wall

Minor enhancement: Bowel wall signal greater than normal small bowel but significantly less than nearby vascular structures

Moderate enhancement: bowel wall signal increased but somewhat less than nearby vascular structures

Marked enhancement: bowel wall signal approaches that of nearby vascular structures

Mural enhancement pattern N/A or homogenous Mucosal Layered Haustral loss (colon only) None 2/3 segment Multiplication factor per segment 0-5 cm x 1 5-15 cm x 1.5 >15 cm x 2 Additional score for extramural features 0 5 Lymph nodes (≥ 1 cm measured in shortest diameter)

Absent Present

Comb sign (linear densities on the mesenteric side of affected bowel segments)

Absent Present

Abscess Absent Present Fistula Absent Present

26

baseline. A recent study concludes that a simple signal difference strategy is more robust to the varying flip

angles observed in vivo than the underlying assumptions needed for Tofts model allowing for the technique to

be used in follow-up studies133. Sourbron et al. even state that Tofts model is useful in weakly vascularized or

highly perfused tissues only134. Absolute quantification of blood flow analyses in the bowel using DCE-MRE has

only been described in few studies71,135 and more simple methods are more often used.

We applied the variable flip angle technique (using the following degrees of the flip angle: 5, 10, 15, 20, 25) and

tested the reliability in the psoas muscle, expected to have a constant T1. However, we found the method

unreliable and decided to use the absolute signal difference instead. We chose the flip angle of the dynamic

sequence to be 24, based on prior recommendations136. We also considered applying the AIF for DCE-MRE.

However, there was a large variability even between aorta, iliac artery and superior mesenteric artery. Even

worse, the proportion of changes within arteries were vast between individuals, hampering the possibility to

choose the optimal artery for comparison.

To extract DCE-MRE values, a custom made program (RoiTool for Mac) was built in MATLAB® (MathWorks®,

Natick, MA). This program allows the examination of MRE DICOM files for the dynamic sequence and the

drawing of ROIs within all slices and frames. Since only the initial curves were regarded in our study, we did not

fit a specific curve type137.

Clinical assessment. We decided to assess all patients with the two most accepted clinical scores; CDAI and HBI. In study I, we chose

the defined cut-off for moderate disease (CDAI ≥ 220 or HBI ≥ 8) as inclusion criteria23. This was based on the

assumption that moderate to severe active disease would have a higher perfusion and because we aimed at

including patients with a more chronic or fibrotic state in study II, thereby covering a wider spectrum of CD.

Furthermore, by including mild disease, the bowel would potentially have more peristalsis and the bowel wall

could be thinner, giving rise to a lower success rate.

Biochemical assessment At our department routine biochemical assessment of CD includes ESR, haematocrit, haemoglobin, CRP,

leucocytes, vitamin D, albumin, and f-calpro (see below). Haematocrit is part of the CDAI score while CRP and

leucocytes are markers of systemic inflammation. Low vitamin D is also regarded as an indirect marker of

inflammation138, whereas albumin is a negative acute phase reactant and also related to malnutrition and post-

surgical complications139,140.

27

Faecal Calprotectin

F-calpro is a routine marker at our institution, and has been shown to help predicting recurrence after surgery38

and correlates with endoscopy and MRE141,142.

Histopathology Bowel wall thickness,

luminal diameter and

histological components of

inflammation or fibrosis

vary significantly along the

longitudinal axis of the intestine and maybe even within the

circumference143. We therefore decided to fixate the bowel

without cutting it open, allowing us to examine the full

circumference and the full transmural disease in one slice. The

disadvantage about this approach is the slightly increased

difficulties in cleansing of the bowel and proper fixation in

formaldehyde. We could overcome the limitations by dividing

exceptional long resected segments (e.g. > 50 cm) into two or

three segments, see Methods, and by using intraluminal wicks for

better central formaldehyde uptake and fixation. There are

several validated histological indices for ulcerative colitis144,145, but

still no optimal method for the grading of transmural inflammation and/or fibrosis in CD. Borley et al.143

described a semiquantitative score for both chronic and acute histological changes in the bowel wall, allowing

investigation of the heterogeneity of histology throughout the involved segment. The score is neither validated

nor produced with the purpose of assessing pure activity in CD. Other scores exist but are too complex146. We

further aimed at an area based score of tissue components like collagen, muscle, neutrophils etc. However, the

computer system intended for this purpose did not allow us to make these calculations within our time frame,

and we ended up with the semiquantitative approach for activity by Borley et al.143 and chronicity/fibrosis by

Chiorean et al.147, and Baumgart et al.148, Table 2, 3 & 4. Haematoxylin-eosin is the standard histopathology

staining, whereas Masson’s trichrome enhances the collagen (blue) and muscle tissue (red/purple).

Immunohistochemical staining on Ventana OptiView with anti-Desmin (Dako® clone D33, Glostrup, Denmark)

was performed to characterize smooth muscle cells and myofibroblasts149. Anti-CD34 (Confirm® clone

Table 2 Acute inflammatory features. Score Mucosal Ulceration score Oedema score Depth of neutrophil penetration 0 None None None 1 Aphthous ulceration only, < 7 mm diameter Mild Mucosa 2 Linear or rake ulceration Moderate Submucosa 3 Confluent or large ulceration Severe Muscularis propria 4 Serosa extramural fat

Table 3 Fibrostenotic features Score Mucosal Ulceration score 0 No or minimal fibrosis limited to submucosa

(< 25 % thickness) 1 Mild stricture (> 15 mm) with nondilated

lumen. Submucosal fibrosis and muscular hyperplasia > 25 % with preserved layers

2 Massive transmural fibrosis; effacement of normal layers; severe stricture.

Table 4 Collagen deposition Score Depth of excessive collagen deposition 0 No increased collagen 1 Increased in mucosa 2 Increased in muscularis mucosa 3 Increased in submucosa 4 Increased in muscularis propria 5 Increased in all layers incl. serosa

28

QBEnd/10, Roche®, Ventana Medical Systems, Inc., Tucson, AZ) stains vessels and was chosen for quantification

of micro vessel density within the bowel wall150.

Impedance planimetry

Stenoses in CD are usually located in the small intestine making in vivo mechanical testing difficult. Although

balloon dilatation is commonly utilized in strictures located at the terminal ileum or the neo-terminal ileum151

there is currently only a limited knowledge about the mechanical properties of CD, incl. strictures148,152,153.

Impedance planimetry is a valid method for computation of luminal cross sectional area (CSA) and pressure in

hollow organs like the gastrointestinal tract. During the last 25 years a large number of papers based on

impedance planimetry have described biomechanical properties of most regions of the GI tract in humans and

animals. Previously, the use of impedance planimetry was hampered by the cumbersome calibration procedure.

However, a recent device, EndoFLIP® probe (Crospon™, Galway, Ireland), has been introduced for impedance

planimetric description of mechanical properties of GI sphincters154–157. Based on the experience from our

institution, we chose impedance planimetric assessment with the EndoFLIP for description of mechanical

properties of resected small intestinal stenoses from patients with CD.

The length of stenoses in CD varies significantly. Therefore, we wanted pressure and not volume as the

independent parameter for distension. For that purpose, we had to bypass the EndoFLIP’s pumping mechanism,

as described in the methodology section. A limitation with the EndoFLIP probe is that it only detects the luminal

CSA. For advanced biomechanical evaluation the wall thickness has to be involved. Therefore, we combined

impedance planimetry with ultrasound allowing us to determine both the inner and outer CSA. Since change in

pH can influence muscle activity, we installed the specimen in a Krebs solution also allowing us for better US

visualisation. The point of the narrowest part of a stenosis was marked by acupuncture needles, since these are

very thin and only slightly affects the tissue with the additional benefit of being highly visible on

ultrasonography in a water bath. Thereby, we ensured that still images were taken at the same locations

regardless of pressure level.

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Methods Ultrasonography Participants were investigated after four hours fast. An

Acuson S3000 ultrasound machine with a 4-9 MHz linear

matrix transducer and a 1-6 MHz curvilinear transducer

was used (Siemens Medical Solutions, Malvern, PA). CDI

was set with a transmit frequency of 6.75 MHz, gain 1 dB,

pulsed repetition frequency 1099, low wall filter of 2, and

a scale of 6 cm/s.

Bowel wall thickness was measured in continues numbers

at the thickest site of the bowel wall without including

valvula conniventes. Length of disease was measured

within the lumen, drawn in free hand as illustrated in

Figure 4. As a standard bowel US examination, we further

examined bowel wall pattern (layered; partly disturbed;

disturbed), CDI after the Limberg classification76,

inflammatory fat (none; barely visible; < 2 cm; > 2 cm),

presence of ulcers (none; possible/small (< 1 mm);

large/transmural), lymph nodes (present or not), and

diameter (less than 1 cm or larger), presence of abscesses,

fistulas and strictures with prestenotic dilatation, Figure 5

& 6.

Contrast Enhanced Ultrasonography The consistent pulse centre frequency was set to 4 MHz,

the dynamic range at 80, the mechanical index was 0.06-

0.08 fixed throughout the examination, the frame rate was

10 frames per second (fps), the focal zone was placed

beneath the bowel loop123; contrast bolus injection was 2.4

ml SonoVue followed by a 5 ml saline flush over 2 seconds

in an 18 Gauge iv. catheter in the cubital vein. Scans were

recorded for 90 seconds. Scan plane was kept constant and

Figure 4 B-mode ultrasonography image showing terminal ileum and a free-hand length measurement.

Figure 5 B-mode ultrasonography image showing bowel wall thickening and a severe prestenotic dilatation.

Figure 6 B-mode ultrasonography image showing deep ulcer and Limberg 4 color Doppler signals.

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patients were instructed in gentle breathing. More than 5 minutes after first injection, the scan was repeated at

the same spot, but in a different scan plane to cover the segment in both the transverse and longitudinal axes.

The chosen bowel segments were either terminal ileum, neo-terminal ileum or proximal ileum. The location for

CEUS was determined as the most inflamed area of the segment according to a prior classification78,158. Cineloop files were exported in DICOM format, re-linearized, and quantified on VueBox 5.1 (Bracco Suisse SA, Geneva,

Switzerland) as earlier described82,112. If possible, four ROIs were drawn using the following criteria: the largest

possible ROI (ROI1) which reliably included the full bowel wall without measuring the lumen or surrounding

fatty tissue. Subsequently three smaller ROIs were chosen and had to be within the area of the large ROI while

still > 0.1 cm2. Within these (ROI2, ROI3 or ROI4) we identified the “highest peak enhancement ROI” and a mean

value of the three smaller ROIs ((ROI2 + ROI3 + ROI4)/3). Shapes and placement of ROIs were optimized to

obtain a quality of fit (QoF) >90 % or as high as possible. Built-in motion compensation was applied whenever

beneficial, Figure 7. Analyses were then compared for repeatability between the largest ROI, the maximum peak

ROI and mean of the three latter ROIs, exhibiting a QoF >85%, entitled mean ROI. The average (log-converted)

values of the best reproducible method were subsequently chosen for comparison with DCE-MRE results. Data

post processing was badge-analysed by the same investigator more than six months after the US and clinical

scoring of the last patient to ensure effective blinding of data.

Figure 7 VueBox post-processing. Dual contrast enhanced ultrasonography image with contrast mode on the left side and b-mode on the right site. Turquoise delineation marks the border for analysis and serves as the area for motion compensation. Four ROIs are drawn, ROI 1 (green) as the largest possible ROI. First injection (top left) and corresponding time intensity curves (bottom left). Second injection dual image and ROIs (top right) and corresponding time intensity curves (bottom right). NB Y-axis scale differs. Table on bottom right shows quality of fit.

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Magnetic Resonance Enterography Patients were instructed to fast for four

hours and drink one litre of oral contrast

one hour before the scan. Oral contrast

comprised a suspension of 125 ml

mannitol 15 % (Fresenius Kabi, Bad

Homburg, Germany) in 875 ml of tap

water, 30 ml psyllium HUSK® fibre, and ice

cubes. Peristalsis was suppressed by

intravenous injection of 20 mg Hyoscine

butylbromid (Buscopan®; Boehringer

Ingelheim, Ingelheim, Germany) prior to

non-dynamic sequences and repeated

before contrast injection. Images were

acquired using a 1.5 T MR unit (Avanto;

Siemens, Erlangen, Germany) with

patients in the prone position. The

intravenous contrast agent used was gadoterate meglumine (Dotarem®; Guerbet, Villepinte, France), 0.2 mg /

kg bodyweight at 5 ml/s followed by a 24 ml saline flush. Patients were instructed to hyperventilate prior to a

long breath hold followed by gentle breathing. MR scanning protocols can be seen in Appendix III.

In the first study, we did an interobserver correlation analysis for continues measures of maximum wall

thickness; continues measurements for length of disease involvement; type of enhancement pattern

(homogenous, layered, mucosal or absent); degree of oedema (based on bright mural T2 fat sat signal);

perimural T2 signal; presence or absence of fistulas, abscesses, lymph nodes, comb sign and ascites159. These

data were used to calculate the MEGS score, Figure 8. In article 2, only one reader examined the patient for the

same parameters as described above. Additionally, ulcers were classified and contrast signal intensity was

obtained to calculate the MaRIA score.

Dynamic Contrast Enhanced Magnetic Resonance Enterography The DCE-MRE analysis was performed within the same bowel segment examined by CEUS, using a RoiTool. ROI

selection was of moderate size encompassing the highest enhancement area within the bowel wall. No motion

compensation was applicable. Since the surrounding fatty tissue generally has a high signal intensity, all frames

Figure 8 MR Enterography T1 weighted sequence post contrast enhancement. Neoterminal ileum is clearly thickened and with homogenous enhancement. Additional overlapping images are from different slices

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were assessed to make sure that the ROI stayed within the bowel wall. ROIs were deleted in frames where

significant out-of-plane motion occurred. Based on these ROIs, a MRI physicist fitted a cubic spline curve,

defined the baseline length and the peak, and calculated the TIC parameters from intensity measurements. All

data were then exported in comma separated files for further import into Stata.

Clinical assessment Patients were asked about their smoking habits and symptoms within the last period of flair. Symptoms

assessed were; pain, nausea, vomiting, diarrhoea, bloody stools, weight loss, and fatigue. Symptoms were

graded semi quantitatively into none, mild, moderate and severe. The duration of the present period of flair was

reported. CDAI and HBI (appendix I & II) were recorded at the time of inclusion.

Blood and stool Samples At the day of CEUS, patients had routine blood samples taken. These included ESR, haematocrit, haemoglobin,

CRP, leucocytes, vitamin D, and albumin. At first visit, patients were instructed in taking a faecal sample and

send it to the central laboratory of Randers Regional Hospital, certified for analysis of f-calpro.

Histopathology The bowel segment was rinsed immediately after surgical excision. To preserve the luminal diameter, we did not

open the bowel segment longitudinally, but occasionally long segments were divided into two or three

segments, see Figure 9 & 10. Fixation was made in neutral buffered formaldehyde for 24-48 hours using

intraluminal wicks to ensure sufficient preservation. After fixation, all segments were transversally cut into slices

of 3-6 mm thickness, Figure 11 & 12. Representative slices (e.g. with greatest wall thickness and smallest lumen)

as whole mounted slices where then paraffin-embedded for sectioning and Haematoxylin-eosin staining. Based

on histological assessment of all slices, the slice with greatest wall thickness, smallest lumen, highest

inflammatory sub score, and highest fibrotic content was selected for digitalization. If the above mentioned

parameters did not all occur in one slice, two slices were selected. From these two sites, four new slices were

made and batch stained with 1) Haematoxylin-eosin, 2) Masson’s trichrome, 3) anti-Desmin, respectively 4) anti-

CD34. Slices were digitalized with NanoZoomer 2.0 HT and NanoZoomer 2.0-RS (Hamamatsu, Hamamatsu City,

Japan). Maximum wall thickness was measured digitally in the dedicated software program NDP.View2

(Hamamatsu, Hamamatsu City, Japan)160. In each patient, both slices were scored using the acute inflammatory

index described by Borley et al.143 and fibrosis by Chiorean et al.147 and collagen by Baumgart et al.148, see table

2, 3 & 4.

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Figure 9 Drawing of the intestine showing where the intestine is divided and the punctum maximum of disease.

Figure 10 Gross specimen

Figure 11 Full specimen cut in 3-6 mm slices

Figure 12 Close