Calcification as a prognostic factor for rupture risk of the abdominal aortic aneurysm

Author: Ruben V.C. BuijsStudent number: 1732722

Supervisor: Prof. dr. C. J. Zeebregts

University Medical Center Groningen, Department of Surgery (Division of Vascular Surgery)

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ABSTRACT (ENGLISH)Introduction: Abdominal aortic aneurysm (AAA) is a major cause of death in developed countries. Patients often lack clinical symptoms and many acute AAA patients do not survive rupture. Currently, the maximal AAA diameter in the horizontal plane is still taken as a prognostic measure for rupture and therapeutic interventions are initiated accordingly. However, recent studies claim this maximal diameter to be insufficiently accurate to be used for risk analysis. New diagnostic criteria need to be identified. In this study we aim to verify whether calcification of the AAA is associated to AAA rupture risk. Methods: A database with demographic and clinical characteristics of 239 AAA patients was constructed. Three groups were formed: elective (eAAA; n = 129), ruptured (rAAA; n = 73) and symptomatic non-ruptured (sAAA; n = 28) AAA patients. The Abdominal Aortic Calcification 8 score (AAC-8) was used to measure the severity of vascular calcification. Results: The AAA diameter (61 ± 12 vs. 74 ± 21; P < 0.001) and AAC-8 score (3.4 ± 2 vs. 4.7 ± 2.3; P < 0.001) were significantly different after univariate analysis of the eAAA and the combined rAAA and sAAA groups, respectively. Binary logistic regression analysis showed that larger AAA diameter (OR: 1.054/mm increase; P < 0.001), high AAC-8 score (OR: 1.38/point increase; P < 0.001) and also female gender (OR: 4.12; P = 0.02) were significantly correlated to a larger risk of developing a sAAA or rAAA.Conclusion: This study suggests that calcification degree is associated with AAA rupture risk. Future studies should clarify whether there is a causal relation between calcification and AAA rupture.

ABSTRACT (NEDERLANDS)Introductie: Abdominale aorta aneurysmata (AAA) zijn verantwoordelijk voor veel acute doden in ontwikkelde landen. Patiënten hebben weinig tot geen symptomen. AAA ruptuur kent een hoge mortaliteit. Huidige risico inschatting van een AAA geschiedt op basis van de maximale diameter van het aneurysma in het horizontale vlak. De kennis is afkomstig van populatie gebaseerde studies, maar maakt een individuele risico inschatting nog altijd vrij onnauwkeurig. Nieuwe diagnostische criteria zouden mogelijk nauwkeuriger werken. In dit onderzoek streven we de associatie tussen calcificatie en AAA ruptuur risico te bepalen.Methoden: Demografische en klinische factoren van 239 patiënten zijn retrospectief verzameld. Drie groepen zijn hieruit geïdentificeerd: electieve (eAAA; n = 129) AAA patiënten, geruptureerde (rAAA; n = 73) en symptomatische niet-geruptureerde (sAAA; n = 28) AAA patiënten. De Abdominal Aortic Calcification 8 score (AAC-8) is gebruikt om de mate van calcificatie in de aortawand te bepalen.Resultaten: AAA diameter (61 ± 12 vs. 74 ± 21; P < 0.001) en de AAC-8 score (3.4 ± 2 vs. 4.7 ± 2.3 P < 0.001) waren significant verschillend na univariate analyse van de eAAA en de gecombineerde rAAA en sAAA groepen, respectievelijk. Binaire logistische regressie analyse gaf aan dat een grotere AAA diameter (OR: 1.054/mm toename; P < 0.001), een hoge AAC-8 score (OR: 1.38/punt toename; P < 0.001) en vrouwelijk geslacht (OR: 4.12;

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P = 0.02) significant geassocieerd waren met een groter risico op het ontwikkelen van een sAAA of rAAA.Conclusie: De resultaten van deze studie suggereren dat calcificatie-graad is geassocieerd met AAA ruptuur risico. Verder onderzoek moet verduidelijken of er causaal verband is tussen calcificatie en AAA ruptuur.

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TABLE OF CONTENTS

1. INTRODUCTION..............................................................................................................4

1.1 Anatomy of the abdominal aorta.............................................................................4

1.2 Epidemiology and risk factors..................................................................................4

1.3 Pathophysiology and pathogenesis..........................................................................5

1.4 Current and experimental diagnostics.....................................................................6

1.5 Calcification of the abdominal aortic aneurysm.......................................................7

1.6 Pathophysiology of vascular calcification.................................................................7

1.7 Summary..................................................................................................................8

1.8 Aim and hypothesis..................................................................................................8

2. MATERIALS AND METHODS...........................................................................................9

2.1 Patient population and selection.............................................................................9

2.2 Computed tomography............................................................................................9

2.3 Calcification Score..................................................................................................10

2.4 Statistical analysis..................................................................................................10

2.5 Intra- and inter-observer variability.......................................................................10

3. RESULTS....................................................................................................................... 11

3.1 Patient characteristics............................................................................................11

3.2 Abdominal aortic aneurysm diameter....................................................................12

3.3 Abdominal aortic aneurysm calcification...............................................................13

3.4 “Gold standard” versus AAC-8...............................................................................14

4. DISCUSSION..................................................................................................................16

4.1 The Abdominal Aortic Calcification 8 Score............................................................16

4.2 Common riskfactors...............................................................................................17

4.3 AAA diameter versus the AAC-8 score...................................................................18

4.4 Calcification: protection or disruption?..................................................................18

4.5 Study limitations....................................................................................................19

4.6 Conclusion..............................................................................................................20

REFERENCES.....................................................................................................................21

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1.INTRODUCTION

The diagnosis “abdominal aortic aneurysm” (AAA) is currently primarily made by using either ultrasonography or computed tomography. The maximal aneurysm diameter is defined and once the threshold of 5.5 cm is reached, it usually will be considered for surgical repair (1). The theory behind this is that with an increasing aneurysm diameter, the rupture risk increases. It currently seems this theory is losing ground as a legitimate individual risk predictor (2-4). Research is ongoing to identify other markers that might correlate well with risk of rupture. Calcification is a well-documented entity in AAAs, but little is known about its implications for further risk of rupture. This chapter will elucidate the structural and (patho)physiological processes that determine the AAA and its relationship to calcification.

1.1 Anatomy of the abdominal aortaThe abdominal aorta originates from the thoracic aorta and starts its trajectory at the aortic hiatus in the diaphragm. The abdominal aorta extends to the aortic bifurcation at the sixth lumbar vertebra. It is positioned left of the median line and runs in parallel to the inferior vena cava. The major branches that extend out of the abdominal aorta are the renal arteries, the inferior and superior mesenteric artery, the celiac trunk and the iliac arteries. With these branches, the abdominal aorta is responsible for the blood circulation of the abdominal organs. The abdominal aorta differs in size depending on the gender, age and physique of the individual. The mean diameter is 2.0 centimetre and ranges from 1.4 to 3.0 cm.

1.2 Epidemiology and risk factorsAAA is a major cause of death in Western countries. It is the thirteenth largest cause of sudden death in the United States with over 15,000 deaths annually. In the United Kingdom, 8,000 yearly deaths are due to AAA rupture and in The Netherlands 775 yearly deaths are related to AAAs (5-7). AAA prevalence ranges from 1.3% to 8.9% for men versus 1% to 2.2% for women (8). Mortality is high, as an acute rupture leads to instant death in 50% of the cases and only 50% of patients who receive surgery will survive. Dutch epidemiological studies show how post-surgical survival rates decrease even further as the age of the patient increases. For example, the 28-day mortality is 47-59% for 70-74 year old patients versus a 91-92% mortality for patients over 85 years (9).

In search for an accurate risk assessment tool, several epidemiological studies found predictive factors for the occurrence and severity of AAAs. Tobacco use seems to be of great influence on AAA development. A direct dose- and exposure time related dependency is found for cigarette smoking and aneurysmal expansion (10). As shown above, men are up to five times more susceptible to AAA development than women, yet female patients show a relatively higher mortality (8). Racial factors also determine AAA risk, as Caucasians are twice as much at risk for an AAA as black people (11). AAA

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development is more often found in association with hypertension (odds ratio (OR): 1.15-1.25) and atherosclerosis (OR: 1.42-1.62).

Moreover, occurrence of AAA in first degree relatives increases AAA risk by 4.3 times (12). This non-environmental risk factor has been the subject of a large European genetics study in which over 4,500 individuals with AAA and almost 38,000 controls were included. This genome-wide study showed how the A allele of rs7025486 on chromosome 9q33 was associated with AAA (OR: 1.21, P = 0.000000000046) (13). Other studies too found an AAA frequency in first-degree family members of 15-19% versus 1-3% in unrelated individuals. Besides the genetic component, this could also be caused by familial traits that influence the exposure to environmental influences (14).

1.3 Pathophysiology and pathogenesisThe word “aneurysm” originates from the Greek word “aneurusma”, which means “dilatation”. The abdominal aortic aneurysm (AAA) is a result of local aortic wall dilatation distal and/or surrounding the renal arteries. Once the aortic diameter has grown over 1.5 times its original size or over 30 mm, it is considered an aneurysm (15). When only a specific region of the wall bulges outward, it is named a saccular aneurysm. A fusiform aneurysm is a dilatation over the entire circumference of the abdominal aorta. Dilatation of the aortic wall is a consequence of deterioration of the wall integrity. This integrity is the sum of the interactions of smooth muscle cells, cellular immunity and matrix proteins like collagen and elastin, among many different other cellular components.

Collagen and elastin are the main factors in maintaining the structural organization of the vessel wall. Mesenchymal cells in the aortic vessel wall increase and decrease the synthesis of these matrix proteins. The elastin fibres and the complex network it forms in coalescence with smooth muscle cells provide the aortic wall with tough, elastic properties. Elastin mass seems to increase with age, yet no definitive proof has been found of elastin neosynthesis after birth. Elastin fibres do have a long half-life due to its resilience to proteolysis. In aneurysmal aortas, elastin does not seem to increase appropriately to the increase of the aortic circumference. As the aneurysm size grows, elastin grows too but is organized differently and more dispersed than it is in healthy tissue. Collagen type 1 and 3 are both as important in providing a stretching but coherent structure. Contrary to elastin, collagen levels do increase relative to aneurysm size. Whether the organization of elastin and collagen are the cause or effect of aneurysmal development of the abdominal aorta remains ambiguous (16).

Proteolysis of these matrix proteins is theorized to weaken the vessel wall. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes that have been identified in AAAs. MMP-2 has shown to have collagenolytic properties whilst MMP-9 has both collagenolytic and elastinolytic activity (17). Research on MMP-9 and MMP-2 knockout mice showed that the knockout protected against CaCl-induced AAA formation in contrast to normal AAA development in wild type litter mates. Tissue

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inhibitor metalloproteinases (TIMPs) counteract the MMP activity. The TIMP and MMP equilibrium seems to be important for the aortic wall integrity, as knockout and overexpression studies have shown in the past (18-20).

Several specific diseases, though rare, can be the cause of specific AAA formation. Inborn disorders such as Marfan and Ehlers Danlos syndrome portray a highly impaired connective tissue physiology. Inflammatory diseases like Takayasu disease and chronic bacterial infections or trauma act via a different pathway (21-23). Non-specific causes of AAA too, are theorized to work mainly due to an inflammatory response of the vessel wall smooth muscle cells (SMCs) and immune cells from the bloodstream. In response to trauma or inflammation, these cells instigate proteolysis and attract MMPs, causing the equilibrium to shift towards proteolysis. Further worsening of the structural integrity is caused by development of atherosclerosis and thrombosis. Atherosclerosis attracts macrophages and other immune cells towards its foam cell core causing slow disintegration of the vessel intima. Thrombosis on the other hand causes thickening of the wall, leading to hypoxia in the deeper medial layers (24). In its turn, hypoxia attracts even more inflammatory cells. In summary, a strong positive feedback cycle seems to be at work. Yet it remains unclear what is causing some AAAs to worsen and others to stay intact despite these pathological processes.

1.4 Current and experimental diagnosticsAAA is diagnosed via three different routes. A suspect patient receives a physical examination in case of lower back pain, tenderness of the abdomen and a pulsating abdominal mass that is painful on palpation. Palpation of the abdomen is responsible for 30% of asymptomatic AAA diagnoses. Yet, as the sensitivity of this method is highly dependent on abdominal circumference and AAA size, the diagnosis is rarely based solely on this outcome. Usually ultrasonography and contrast CT are employed to confirm the diagnosis as both are almost 100% sensitive in finding AAAs (25). The second route is by coincidental imaging of AAA whilst searching for a different ailment in the abdominal and thoracic region. As such, magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasonography and (non-)contrast CT may demonstrate enlargement of the abdominal aorta. The last route is due to acute exsanguination caused by AAA rupture. This diagnosis is made in the emergency room, frequently after a contrast CT has been performed. While conscious, the patient has abdominal and lower back pain and a pulsating abdominal mass. Aneurysm rupture eventually leads to massive intra-abdominal bleeding, hypovolemic shock and possibly death.

After a non-ruptured AAA has been imaged, the diameter is the main determinant in the assessment whether a patient should be operated on or not. Clinically, an AAA diameter smaller than 5.0cm for women and smaller than 5.5cm for men is considered low-risk AAA. Larger aneurysms will generally be operated on, either by open or endovascular repair (EVAR). Treatment of small aneurysms is far more ambiguous. In large population studies, small aneurysms (< 5.0 cm) do not tend to rupture as soon as the larger ones

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(1). However, in individual cases, ruptures have been found in very small aneurysms whilst very large aneurysms remained undetected until after death due to other causes. Therefore, much research is focused to add more variables to the clinical checklist for AAA risk analysis.

Not only the potential of overlooking high risk AAAs, but also the risk of unnecessarily operating on an already frail and elderly population, is what drives AAA research to look beyond a diameter focused risk assessment. Over the years, rapid growth (>0.5 cm/6 months) and familial cases of AAA have been added to this list. Among the experimental variables are wall stress parameters like Von Mises stress and Peak Wall Stress using CT (26), 18-F fluorodeoxyglucose (FDG) uptake by inflammatory cells in AAAs using PET (27-29), Ultrasmall Paramagnetic Iron Oxide Particle (USPIO) uptake by inflammatory AAA cells using MRI (30, 31) and bio-optical imaging modalities using biomarkers specifically designed to bind AAA deteriorating factors (32, 33).

1.5 Calcification of the abdominal aortic aneurysmOne potential risk variable might not be as far from clinical implementation as the aforementioned experimental methods. Calcification of the arteries is a relatively common finding. Up to a third of all American males are afflicted with some degree of vascular calcification (34, 35). Aortic arch, thoracic aortic and abdominal aortic calcifications have all been linked to cardiovascular risk. Abdominal aortic aneurysms specifically have a strong independent association with cardiovascular disease and coronary heart disease. Though it seems to work independently of atherosclerosis and aneurysmal development, it is theorized there might also be interlinked influences between the three. It seems logical to assume calcification decreases elasticity, thereby lowering the aortic reactivity to passing blood and raising its risk of structural damage (36). On the other hand, studies have shown high levels of abdominal aortic calcification (more than 50%) to lower the expansion rate of the aneurysm, compared to less than 50% aortic calcification (37). This suggests that calcification might protect against eventual rupture.

1.6 Pathophysiology of vascular calcificationThe different layers of the aortic vessel wall have their own individual reactions to trauma or stress that could lead to calcification. The most externally located layer, the tunica adventitia, contains pericytes, mesenchymal stem cells and myofibroblasts which can all be influenced to differentiate towards osteogenic cells or have al calcifying effects on medial cells. An upregulation of osteoblastic transcription factors, an increase of bone morphogenic proteins and a decrease in calcification inhibitors could be found in these cells after stimulation. The tunica media is rich in smooth muscle cells (SMCs). SMCs are able to differentiate towards an osteochrondrogenic lineage if stimulated by the proper agents. Calcifying vascular cells are a subgroup of these vascular SMCs which may develop into osteoblast-like cells and concomitantly lose all smooth muscle-specific traits. This differentiation is often described as a result of inflammation (38-40).

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Inflammation acts on calcification in other ways too. Elastin degradation by certain MMPs produces degradation by-products, which in their turn attract inflammatory cells. Macrophages and leukocytes consequently affect the osteogenic differentiation of the vessel wall cells. These will then exude more MMPs, decreasing the vascular integrity and attracting even more inflammatory cells. Atherosclerosis is highly inflammation-dependent. Atherosclerosis-related angiogenesis is proposed to impact vascular calcification. The formation of new vessels within an atherosclerotic region could allow the aforementioned myofibroblasts and pericytes to be transported towards the intima and precipitate as osteogenic cells (41, 42).

1.7 SummaryTo summarize, AAAs are diagnosed using an insufficiently accurate instrument namely, the diameter of the lumen. Adding more variables to the assessment of rupture risk could decrease unnecessary treatments but also decrease the number of missed ruptures in small AAAs. Inflammation plays a pivotal role in AAA development. Calcification too is frequently found in vessels, but varies greatly in size and form for each patient. As calcification is tightly associated to the degree of inflammation, it could be proposed as a new variable for a more specific rupture risk assessment. Still, the current literature shows both a protective as a degenerative function of calcification.

1.8 Aim and hypothesisTherefore, the aim of this study is to assess whether aneurysm calcification is useful in identifying the risk of rupture. We hypothesize that the risk of rupture is associated to the degree of calcification of the AAA.

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2. MATERIALS AND METHODS2.1 Patient population and selectionThe patient population for this study was constructed out of a patient database at the Department of Surgery (Division of Vascular Surgery) of the University Medical Centre Groningen. This database contains all elective and acute AAA patients who were admitted between the first of January 2006 and the 18th of November 2011. For the purpose of this study, three types of patients were distinguished. Elective patients (eAAA) are patients who are diagnosed with having an AAA beyond the high risk diameter size of 5.0 cm. Symptomatic patients (sAAA) are patients who have been diagnosed with having lower back pain, tenderness of the abdomen and a pulsating abdominal mass that is painful on palpation but without signs of rupture. Patients with ruptured AAAs (rAAA) have been brought in acutely with signs of rupture in varying degrees of severity. rAAA and sAAA patient groups combined are called non-eAAA. Of these three patient groups, eAAAs were most frequently found and sAAAs the least. The ratio of these three groups was 243:98:32 for eAAA, rAAA and sAAA, respectively. After reassessment of the data from the raw database, 129 eAAAs, 73 rAAAs and 28 sAAAs were acquired. The fallout of these data was either due to missing pre-operative CT images (145 patients, the radiology database AquariusNet (21 patients) or due to a lack of patient records in the Poliplus database (1 patient).

To correct for interdependency of common clinical and demographic variables with rupture risk, the following factors were included: gender, age, length (cm), weight (kg), systolic and diastolic blood pressure (mmHg) and serum creatinine levels (umol/L). We also screened for comorbidities, aiming for: diabetes mellitus type 1 or 2 (DM), chronic obstructive pulmonary disease (COPD), known cardiovascular disease (CVD), nicotine abuse, family history of AAA and history of hypertension. Blood pressure, serum creatinine levels and weight were collected as close as possible to the time of AAA repair. To some extent, all these factors are known to affect cardiovascular pathology and were therefore included to correct for confounding (43-45).

2.2 Computed tomographyCT images were collected from the UMCG database and from peripheral hospitals in Groningen, Drenthe and Friesland. None of the images were acquired for the purpose of this study, but were taken as part of routine care. Protocols for single-detector CT scanning varied between different hospitals. A contrast-agent was used to visualize the abdominal aorta and accompanying vessels. The diagnosis AAA and exact diameter was concluded by independent radiologists. The AquariusNet Viewer Client V4.4.4.23 (AQNet) was used for the interpretation of the CT images. Before the interpretation, each separate image needed to be identified and copied to another database. To reliably image all calcification in each image, a Maximum Intensity Projection (MIP) was performed. This is a form of multiplanar reconstruction, stacking axial slices within a

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manually selected three-dimensional plane. With this method a volume is created in which calcification can be observed throughout the whole AAA.

2.3 Calcification ScoreCalcification measurement in the AAA wall was performed using the abdominal aortic calcium score (AAC-8). Though vascular calcium is an easily observable entity embedded in the vessel wall, constructing exact methods for quantification remains difficult. Fully quantitative measurement using the Agatston score was deemed impossible within the time span of the study. This will be elucidated in the discussion. The AAC-8 score was the second best option. The AAC-8 score is a modified version of the AAC-24 score. This latter method was developed by Kauppila et al in 1997 (46). The authors proposed this instrument as a simple, reliable and reproducible method to assess the severity of abdominal aortic calcification. The AAC-24 score is built up out of three separate scores. First, it measures calcification per lumbar vertebra, spanning from L1 to L4 (0-4). This is expanded by the presence of calcification in either the anterior or posterior wall of each segment (0-8). The severity of calcification in each anterior and posterior segment could eventually be graded to up to three points (0-24). No calcification constituted for zero points. The AAC-8 score bypasses this last step and therefore does not measure the individual segment calcification severity. It has been validated to perform on par with the AAC-24 score (47, 48). The main advantage of the AAC-8 score is the decreased amount of time that needs to be spent in the scoring process.

2.4 Statistical analysisStatistical analysis was performed using SPSS for Windows version 19 and STATA version 12. Demographic statistics were expressed as mean ± standard deviation (SD) for continuous variables for eAAA versus non-eAAA, respectively. Percentages were given for nominal variables and medians and interquartile range for skewed distributed variables (serum creatinine and AAA diameter). The demographic variables of non-eAAA patients and eAAA patients were compared. A Chi-square test was used to compare nominal variables. Student T-test was used to compare means of continuous data. Simple descriptive statistics provided normal distribution and univariate correlations. Logistic regression was performed for the association between the patient groups (eAAA vs. non-eAAA) as the dependent variable and explanatory variables such as AAC-8 score and demographic variables. Significance was set at P < 0.05. Using conditional backwards regression, the predictive value of several explanatory variables could be interpreted.

2.5 Intra- and inter-observer variabilityAs the interpretation of calcification is a semi-quantitative measurement, it is useful to perform an inter-observer variability test. 50 individuals were randomly selected from the database and their treatment and endpoints (election, symptoms or rupture) were blinded. A trained observer scored for calcification according to the standard protocol. The variability of the results was calculated using paired samples T-test. Slight variability was observed, but with the P-value varying from 0.193-1.00, no significance was found. To correct for intra-observer measurement variability, the AAC-8 score was measured

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again in a later stage by the main observer of this study. This too resulted in a slight, non-significant variability in results (P = 0.44-0.77).

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3. RESULTS3.1 Patient characteristics:Physiological and demographic characteristics of eAAA patients and sAAA combined with rAAA (non-eAAA) patients are summarized in table 1. 129 patients with elective AAA surgery, 79 patients with ruptured AAAs and 28 patients with symptomatic AAAs were analyzed. 7% of eAAA patients were female, compared to 15% in non-eAAA patients (P = 0.08). The mean age was insignificantly different (72 ± 9 vs. 73 ± 9 years; P = 0.68), as was the BMI (26 ± 5 for both; P = 0.92) and serum creatinine (132 ± 168 vs. 119 ± 84 mg/dL; P = 0.49). Body habitus (L3 diameter) differed marginally between the two groups (47 ± 3mm vs. 46 ± 4mm; P = 0.08). Systolic blood pressure had highly overlapping outcomes (136 ± 22 vs. 140 ± 26 mmHg; P = 0.22). Diastolic blood pressure too did not differ between groups (78 ± 12 vs. 78 ± 14 mmHg; P = 0.90) and the mean arterial pressure did not either (98 ± 14 vs. 99 ± 17 mmHg; P = 0.59). COPD (31% vs. 23%; P = 0.23), DM (19% vs. 22%; P = 0.74), hypertension (64% for both; P = 1), CVD (72% vs. 62%; P = 0.12) and familial AAA (2% vs. 5%; P = 0.75) were all insignificantly divided over the eAAA and non-eAAA groups. Current smokers (31% vs. 33%; P = 0.48), previous smokers (30% vs. 18%; P = 0.10) and patient who never smoked (39% vs. 49%; P = 0.11) were also similarly represented. Treatment of eAAA patients was 100% endovascular, whereas only 31% of non-eAAA patients received endovascular treatment. 8% of non-eAAA patients were excluded from treatment. The remaining 61% underwent open surgery.

Separate chi-square and T-tests were performed for sAAA patients and rAAA patients and the differences are stated in table 2. Pronounced differences were found for the representation of gender across the sAAA vs. rAAA patients. Of all rAAA patients, 8% was female, whereas of the sAAA patients 32% was female (P = 0.05). Body habitus differed slightly, but with high significance (47 ± 4 vs. 44 ± 5; P < 0.001). The diastolic blood pressure too, was significantly raised in sAAA patients (83 ± 20 mmHg) compared to rAAA patients (76 ± 21 mmHg; P = 0.04). Concomitantly, the mean arterial pressure was significantly raised slightly in sAAA patients (104 ± 15 mmHg) compared to rAAA patients (97 ± 17 mmHg; P = 0.05). Notable insignificant differences were found for familial occurrence of AAA (11% vs. 3%; P = 0.13) in sAAA and rAAA respectively. All other variables either did not differ or differed insignificantly.

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3.2

Abdominal aortic aneurysm diameterAll abdominal aortic aneurysms in this study were classified by their diameter. As this is the current gold standard, aneurysm size correlated with ruptured and symptomatic aneurysm as was expected. Though the standard deviation was wide, the mean aneurysm diameter was significantly larger in ruptured and symptomatic aneurysms than in elective aneurysms (61 ± 12mm vs. 74 ± 21mm; P < 0.001). The AAA diameter was also compared between sAAA and rAAA patients (Table 1 and 2). Patients with symptomatic aneurysms had a smaller mean diameter than rAAA patients, yet significance could not be attained (68 ± 20mm vs. 76 ± 21mm; P = 0.09).

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Table 1: Patients characteristics, measured data and common risk factors. Elective AAA patients were compared to combined ruptured and symptomatic AAA patientsAAC-8: Abdominal Aortic Calcification 8 score* P < 0.10*** P < 0.001

The abdominal aortic diameter is dependent of the body habitus of the patient. The L3 vertebral transverse diameter is a validated measure for the size of the patient’s body. A new variable was created that corrects for the patient habitus. The AAA diameter was divided by the transverse diameter of the L3 vertebra. A T-test for the effects of this variable provided the following results. When comparing eAAA and non-eAAA patients, the habitus-corrected AAA diameter size was still significantly lower in eAAA patients (1.29 ± 0.25 vs. 1.62 ± 0.48; P < 0.001).

3.3 Abdominal aortic aneurysm calcificationCalcification of the AAA was measured on a scale from 0 to 8. When comparing eAAA patients to non-eAAA patients, a highly significant difference was found using Student’s T-test. Patients with eAAAs had a mean AAC-8 score of 3.4 ± 2 points whilst rAAA patients had a score of 4.7 ± 2.3 points (P < 0.001). Between sAAA and rAAA patients, the difference was smaller and no longer significant (5.5 ± 2.3 points vs. 4.7 ± 2.3 points; P = 0.09).

Table 2: Patients characteristics, measured data and common risk factors. Ruptured AAA patients were compared to symptomatic AAA patients.AAC-8: Abdominal Aortic Calcification 8 score** P < 0.05*** P < 0.001

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3.4 “Gold standard” versus AAC-8:For the eAAA, sAAA and rAAA groups the AAA diameter measurements and AAC-8 score were plotted in an error bar (Graph 1). The mean AAA diameter was highest in the rAAA group (76 mm ± 5 mm), followed by the sAAA group (68 mm ± 7.5 mm). The AAA diameter was lowest in eAAA patients (60.5 mm ± 2.5 mm). AAC-8 scores were highest for sAAA patients (5.4 ± 0.85 95%-CI). Patients with rAAA had the next highest AAC-8 scores (4.7 ± 0.5). Patients with eAAA had the lowest mean AAC-8 score (3.3 ± 0.4) (Graph 2).

3.5 Correlation analysisCorrelation coefficients were first calculated in the eAAA and non-eAAA patient groups. Variables that remained significantly influential after stepwise backward Wald regression were AAA diameter, AAC-8 score and patient gender (Table 3). Female gender had a 4.12 (P = 0.02) increase of the odds. AAA diameter increased the odds of ending up in the rAAA or sAAA group by 1.054 (P < 0.001) for each millimeter the diameter increases. The AAC-8 score raised the odds of ending up in the rAAA or sAAA group by 1.38 (P < 0.001) for each AAC-8 point increase.

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Graph 1: Error bar graph of AAA diameter size for elective, ruptured and symptomatic AAAs.

Graph 2: Error bar graph of the AAC-8 score for elective, ruptured and symptomatic AAAs.

Table 3: Logistic regression Elective AAA vs. Ruptured and Symptomatic AAA*Female gender equals higher risk** For 1-mm difference*** For 1 point difference

A second stepwise backward Wald regression was performed for the rAAA vs. sAAA patient groups. Diameter (P = 0.072), gender (P = 0.37) and the AAC-8 score (P = 0.26) no longer showed significant correlation with either the sAAA or the rAAA group. However, body habitus did show significant correlation with AAA rupture (OR 1.042; P = 0.012) (Table 4).

Table 4: Logistic regression Ruptured AAA vs. Symptomatic AAA*Female gender equals higher risk** For 1-mm difference*** For 1 point difference

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4. DISCUSSION

In this study we aimed to assess the correlation of aneurysm calcification with aneurysm rupture risk. Univariate and logistic regressions results show that the mean AAC-8 score for calcification was significantly higher in ruptured and symptomatic patients compared to electively operated patients. Also, possible confounding factors appeared to be evenly distributed over the different groups. In this discussion these results will be evaluated and explained.

4.1 The Abdominal Aortic Calcification 8 ScoreThis study utilises the AAC-8 score, a method that has been frequently used in X-ray studies (46, 49). The AAC-8 score was developed because at the time there was no standardized protocol to assess the calcification of an AAA in radiographic images. The method could identify and quantify the degree of calcification in lumbar radiographs. Before Kauppila et al described and tested this method, Agatston et al had already constructed a method with a similar goal for ultrafast computed tomography in coronary arteries (50). Firstly, Hounsfield Units, a measure of radiodensity produced by CT, are scored in five groups: 0-129 Hounsfield Units equals 0, 130-199 Hounsfield Units equals 1, 200-299 Hounsfield Units equals 2, 300-399 equals 3 and 400 Hounsfield Units or more equals 4. The Hounsfield Units are measured over a certain amount of pixels, so these multiply the Hounsfield score. Each anatomic entity on CT spans a certain amount of CT slices of 3 mm thick. The score of each slice is summed and a total score is calculated. Callister et al produced an equal method, also based on Hounsfield Units, to calculate the total calcium volume (51). The Agatston and Callister scores were originally planned to be applied in this study since these methods provide highly specific quantitative calcium scores. What was not taken into consideration was the effect of an intra-luminal contrast agent that is used to visualize the vasculature in CT scans. The amount of Hounsfield Units showed by this contrast agent completely eclipses all the Hounsfield Units originating from aneurismal calcification. During the study, one PhD-student was tasked to construct an algorithm that would be able to automatically detract the contrast agent Hounsfield Units. This project is on-going, but cannot be used within this study yet. The AAC-8 score was therefore chosen as a semi-quantitative method for calcium measurement.

The strength of the AAC-8 score over the Agatston or Callister methods is its practicality in the clinical setting. Both methods need to be applied to non-contrast CT images. Pre-operative CTA imaging is essential for the preparation of AAA repair surgery so two CT images (with and without contrast) would need to be taken for each patient. This doubles the amount of radiation exposure, thus decreasing the applicability. Either the effect of the contrast needs to be excluded algorithmically or Dual Energy CT (DE CT) with contrast is required. The use of algorithms to adjust for contrast needs thorough research to assess whether the algorithm is representative of true non-contrast signal.

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This method, if validated, would provide a very useful new feature in the assessment of vascular calcification. Dual Energy CT scanning functions through the use of two X-ray emitting sources which send two different wavelengths of radiation at a 90 degree angle. This set-up enables the technician to separately view the signals of calcium and that of contrast agent and thus allowing for two sets of images during only one period of radiation exposure. Regrettably, this technique is relatively new and unavailable to most institutions and therefore decreases its practicality. The AAC-8 score bypasses these limitations and enables any clinician to quickly assess the amount of AAA calcification in both X-ray images as well as standard CT and CTA.

4.2 Common risk factorsWell published cardiovascular risk factors like smoking, hypertension, diabetes, male gender and high BMI did not prove to be significantly influential in the initial demographic and physiological results (Table 1). Smoking has often been associated with AAA development, thus it seems odd that almost no difference was found between current smoking habits in both eAAA and non-eAAA patients. The only significant difference found between elective AAA patients and ruptured or symptomatic AAA patients, was that of the treatment. Regrettably, the effect of different treatments is heavily biased by the selection of the groups. As elective patients prove to be a relatively low-risk population, endovascular repair is the method of choice. In other words, the complication risk of the AAA determines the surgical method instead of vice versa. Body habitus and gender varied somewhat between the two groups, but could not be considered significant with a P = 0.08. The results are nevertheless interesting. Body habitus, the L3 vertebra diameter, is dependent of gender as men tend to have a larger mean body habitus. It is therefore no surprise that the body habitus would follow the effect of gender. However, gender has been identified as a risk factor for AAA development so a larger difference was expected. This result does, on the other hand, negate the need of statistical correction for unobserved inter-variable effects. Table 1 concludes that the populations of both groups are highly similar in spite of their selection. Assuming this is true, then gender, smoking, cardiovascular morbidity and body habitus do not have an unobserved effect on diameter size in both groups.

When comparing sAAA patients to rAAA patients, significant differences do arise. Body habitus and gender present significantly different results in the two groups. The results for body habitus are perhaps due to the aforementioned effects of gender. In Table 2, it is clear that a significantly higher amount of women have had a sAAA and consequently show a decreased body habitus in the sAAA group compared to the rAAA group. Still, the fact that the percentage of female patients is significantly higher in the sAAA group in comparison to rAAA patients is an interesting observation. Previous research has found that men tend to develop AAA more often, while women have a higher AAA-related mortality (8). These results claim the opposite. Not only is there no statistical difference between the eAAA and non-eAAA groups, there are also less women with ruptured AAAs than with symptomatic AAAs. It can be theorized that this is the result of selection bias. Mortality was not measured for patients who passed away before

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reaching the hospital. Perhaps the low female AAA survival rates are mainly due to these cases, explaining how these deaths did not get included in this study.

4.3 AAA diameter versus the AAC-8 scoreInterestingly, the AAA diameter and the AAC-8 score had significantly different results when looking at non-elective and elective AAAs. Table 1 shows how the average AAA diameter is 13 mm larger for rAAA or sAAA patients in comparison to eAAA patients. The AAC-8 score also averaged 1.1 AAC point higher in sAAA and rAAA patients. Two error bar graphs were made to visualize the distribution of AAA diameter measurements and AAC-8 scores over the three sets of patients (Graph 1 and 2). These results suggest that a higher AAC-8 score is correlated with a higher chance of ending up in the ruptured or symptomatic AAA group. In other words, there appears to be a correlation between AAA rupture risk and the amount of calcification. Concomitantly, these graphs clearly show how the AAC-8 score mean decreases from sAAA to rAAA to eAAA. A clear overlap is visible between the sAAA and rAAA group, but both of these differ highly significantly from eAAA patients. For the AAA diameter measurements, the difference between rAAA and eAAA patients is also clearly significant. sAAA patients however, have a lower mean AAA diameter size and have a far broader 95%-confidence interval. Both the intervals of rAAA and eAAA patients overlap with those of sAAA patients.

These results solidify the current knowledge of the predictive value of AAA diameter size for AAA rupture. Yet it also visualizes an interesting difference between using diameter and the AAC-8 score. When comparing to rAAA and eAAA patients, why is the AAA diameter mean decreased in sAAA patients when the AAC-8 score average is higher in sAAA patients? Theoretically, it is logical to assume that the severity of wall deterioration and other developmental pathophysiology of the abdominal aorta, are higher for ruptured AAAs than for non-ruptured symptomatic AAAs. A second assumption would be that a larger diameter and a higher grade of calcification are equal to more severe pathophysiology. One can now deduce that sAAA patients should have a smaller AAA diameter size and a lower grade of calcification compared to rAAA patients. In this study however, this is only true for diameter size. We theorize that the AAC-8 calcification grade suffers from the inability to distinguish between solid calcium “plaques” and dispersed scattered calcification “spots”.

4.4 Calcification: protection or disruption?As discussed in the introduction, it is unclear whether calcification protects or weakens an AAA. One school of thought is that a calcium plaque acts as a shield, covering the inner AAA lesion from the influences of flowing blood. Others theorize that the calcification decreases the elasticity and compliance of the abdominal aortic wall, leading to the development of lesions. Studies focussing (52-55) on microcalcifications have shown how smaller calcium spots are highly dependent on local inflammation. In contrast with the more stable solid plaque or end-stage progression of calcification. The specific location of spotty calcifications could very well lead to acute disintegration of the AAA plaque (56-59).The current literature therefore suggests that the different

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forms of calcification have their separate effects on plaque progression. This would mean both of the aforementioned theorems might be equally true. To the observer, all calcification is visible in a two-dimensional plane while in reality it is the summation of calcification in a three-dimensional plane. This makes it difficult or even impossible to optically discriminate between solid plaques and scattered spots. Future research should focus on distinguishing between these two types of calcification. Perhaps this would give more or less weight to the AAC-8 score, leading to a more accurate methodology.

4.5 Study limitationsSeveral limitations of this research have been discussed above. Still some need mentioning and will be explained here. This study was performed on all UMCG-treated AAA patients from 2006 until this moment. This is both its strength and its limitation, as it enables a very strong selection bias. This is inherent to this type of retrospective research, but this specific illness enhances this bias even more. Because aneurysmal formation can stay quiescent right until the moment of rupture, it is logical to assume that there is a very large group of people currently walking around with an AAA. By choosing the elective AAA patients as a control group, we include patient who mostly by accident, got diagnosed with aneurysmal enlargement of the abdominal aorta. Still, for every one patient included there must be one, two or three AAA afflicted persons more who are not included. What happens in this very large population is unknown and might make a difference.

The selection bias acts in other ways too. The symptomatic and ruptured AAA groups exist largely due to the fact that these were not accidentally found earlier, so that they could be included in the elective group. The elective patients did not “get the chance” to develop a symptomatic or ruptured AAA. The eAAA group was treated before this could happen, the other groups were not. One could therefore state that age should have an effect on the groups, as an older patient would have been able to “wait” longer for an AAA to develop symptoms and younger persons would not. In addition to this, the entire eAAA patient group underwent endovascular repair (Table 1). In other words, for none of the patients the risk of rupture was deemed high enough to be considered for open surgery. This selection bias is noteworthy, yet since none of the patient demographics and clinical variables are significantly different for the three groups, excluding gender and body habitus, we do not expect this to be of great influence.

Lastly and perhaps most importantly, no causal assumptions can be made following the results of this study, regardless of any overt significant differences. As this study is a retrospective cohort study, the effect of endogeneity bias is unknown. A causal connection between calcification and ending up in the rAAA group could be theorized. But it is unknown whether rupture proneness itself has an effect on calcification. Better yet, we can assume the factors determining whether a plaque will rupture, also affect the severity of AAA calcification and vice versa. Unknown variables bias and endogeneity bias are present in this study, but no more than in similar studies.

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4.6 ConclusionThe aim of this study was to assess whether aneurysm calcification is useful in identifying the risk of rupture. Of all the variables that were included, only AAA diameter and the AAC-8 score were significantly different between the eAAA and non-eAAA groups. Gender and body habitus differed mostly between the sAAA and rAAA patient groups. It was also clearly visible how the symptomatic AAA patients were more accurately distinguished from elective AAA patients using the AAC-8 score compared to the AAA diameter. Regression analysis was able to show a highly significant correlation coefficient between lower AAC-8 scores and lower rupture risk (i.e. being in the eAAA group) and higher AAC-8 scores and higher rupture risk (i.e. being in the non-eAAA group). Our initial hypothesis was that the risk of rupture is associated to the degree of calcification of the AAA. Our results imply that there is indeed an association between calcification degree and AAA rupture risk and that therefore our hypothesis is true. Whether this is a causal connection is unclear. More basic research is needed to clarify this before arterial calcification can be validated as a true candidate for AAA risk assessment. In the future, more research is needed on both the AAC-8 score and on other calcification measurement methods like the Agatston or Callister method.

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