COMPARISON OF RADIOLOGICAL, MORPHOLOGICAL AND FUNCTIONAL ASSESSMENT OF METABOLIC CHANGES AMONG PATIENTS WITH PANCREATIC DISEASES

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

Edita Bieliūnienė

COMPARISON OF RADIOLOGICAL,

MORPHOLOGICAL AND FUNCTIONAL

ASSESSMENT OF METABOLIC CHANGES

AMONG PATIENTS WITH PANCREATIC

DISEASES

Doctoral Dissertation Medical and Health Sciences,

Medicine (M 001)

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Dissertation has been prepared at the Department of Radiology of Medical Academy of Lithuanian University of Health Sciences during the period of 2010–2019.

Scientific Supervisors:

2016–2019 Prof. Dr. Žilvinas Dambrauskas (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

2010–2016 Prof. Habil. Dr. Juozas Pundzius (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001).

Dissertation is defended at the Medical Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Rasa Verkauskienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001).

Members:

Prof. Dr. Rymantė Gleiznienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Asta Baranauskaitė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Nomeda Rima Valevičienė (Vilnius University, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Mindaugas Andrulis (Ulm University (Germany), Medical and Health Sciences, Medicine – M 001).

Dissertation will be defended at the open session of the Medical Research Council of the Lithuanian University of Health Sciences on the 11th_of December 2019, at 11 a.m. in the Auditorium of the Institute of Cardiology of the Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

Edita Bieliūnienė

RADIOLOGINIŲ, PATOMORFOLOGINIŲ

BEI FUNKCINIŲ TYRIMŲ PALYGINIMAS

VERTINANT METABOLINIUS AUDINIŲ

PAKITIMUS PACIENTAMS,

SERGANTIEMS KASOS LIGOMIS

Daktaro disertacija

Medicinos ir sveikatos mokslai, medicina (M 001)

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Disertacija rengta 2010–2019 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijos Radiologijos klinikoje.

Moksliniai vadovai:

2016–2019 m. prof. dr. Žilvinas Dambrauskas (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

2010–2016 m. prof. habil. dr. Juozas Pundzius (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001).

Disertacija ginama Lietuvos sveikatos mokslų universiteto medicinos mokslo krypties taryboje:

Pirmininkė

prof. dr. Rasa Verkauskienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001).

Nariai:

prof. dr. Rymantė Gleiznienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Asta Baranauskaitė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Nomeda Rima Valevičienė (Vilniaus universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Mindaugas Andrulis (Ulmo universitetas (Vokietija), medicinos ir sveikatos mokslai, medicina– M 001).

Disertacija bus ginama viešame medicinos mokslo krypties tarybos posėdyje. 2019 m. gruodžio 11 d. 11 val. Lietuvos sveikatos mokslų universiteto Kar-diologijos instituto posėdžių salėje.

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6 5. DISCUSSION ... 51 6. CONCLUSIONS ... 57 7. PRACTICAL RECOMMENDATIONS ... 58 SANTRAUKA ... 59 LIST OF LITERATURE ... 70 LIST OF PUBLICATIONS ... 76 APPENDICIES ... 99 Appendix 1 ... 99 Appendix 2 ... 100 Appendix 3 ... 101 Appendix 4 ... 103 Appendix 5 ... 104 Appendix 6 ... 108 CURRICULUM VITAE ... 110 ACKNOWLEDGEMENTS ... 111

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ABBREVIATIONS

ADC – Apparent diffusion coefficient

AP – Appetite loss

BMD – Bone mineral density

BMI – Body mass index

CP – Chronic pancreatitis

CT – Computed tomography

DI – Diarrhoea

DM – Diabetes mellitus

DWI – Diffusion-weighted imaging

DXA – Dual-energy X-ray absorptiometry

ECM – Extracellular matrix

EPI – Echo planar imaging

FA – Fatigue

FOV – Field of view

FS – Fat saturation

GRE – Gradient recalled echo

HU – Hounsfield units

MMPs – Matrix metalloproteinases

MRI – Magnetic resonance imaging

NSA – Number of signal averages

PA – Pain

PD – Proton density

PDAC – Pancreatic ductal adenocarcinoma and ampullary carcinoma PEI – Pancreatic exocrine insufficiency

PF – Pancreatic fibrosis

PF2 – Physical functioning

PSCs – Pancreatic stellate cell

QL2 – Global health status

QoL – Quality of life

ROC – Receiver operating characteristic

SI – Signal intensity

SMA – The area of skeletal muscle

T – Tesla

TE – Echo time

TF – Turbo factor

TI – Inversion time

TIMPs – Tissue inhibitors of metalloproteinases TIRM – Sequence using fat suppresion

TPV – Positive predictive value

TR – Repetition time

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1. INTRODUCTION

Chronic pancreatitis (CP) is marked by chronic progressive pancreatic inflammation, scarring and fibrosis, leading to loss and damage of ductal, endocrine, and exocrine cells. CP etiology has typically been categorized as obstructive, alcohol, hyperlipidemia, hereditary and idiopathic [74].

It was originally thought that CP was widely linked to alcohol, and was responsible for between 70–80% of the cases. Extreme intake results in an augmentation of the plasma levels of cytokines in CP, as opposed to the controls. They push pancreatic fibrogenesis and are therefore link to the progress of the same from acute pancreatitis to fibrosis and CP injury [70]. In the West, alcohol continues to a significant contributor to CP. In the last 100 years, alcohol’s frequency as an etiological element has risen from 19– 80%. Indian patients with the issue have been observed to be consumers of low-protein and low-fat diets, and do not consume alcohol at high rates, despite which they present with a high level of calcific CP which presents itself at an early age [61].

Smoking is associated independently with issues of CP like exocrine insufficiency, calcifications development, and ductal changes. The amount of smoking is more significant than the dose. Less than 5% of patients with pan-creatitis who undergo an examination are found to have autoimmune pancreas-titis [61].

CP is a supposed risk factor for pancreatic cancer. CP increases the risk of pancreatic cancer, but the association diminishes with follow-up in the long-term. Five years post diagnosis, these patients can have a risk of pancreatic cancer that is eight times as high [45]. “Pancreatic cancer develops in the setting of CP from all known etiologies but appears to require 30–40 years of inflammation before an appreciable percentage of patients develop pancreatic cancer [79].”

“Pancreatic cancer is a large contributor to cancer-linked deaths, coming in at the 7th_{spot for such issues globally. The toll it has is higher when we} looked at countries from the first world. Patients will often not show any symptoms till the disease is in an advanced phase. It is one of the most lethal malignant neoplasms, which led to 432,242 new deaths in 2018 (GLOBOCAN 2018 estimates). Around the world, 458,918 new cases of pancreatic cancer have been reported in 2018, and 355,317 new cases will show up by 2040 according to some estimates [79].”

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PSCs activation by pancreatic cancer cells leads to increased fibrosis pro-duction, which results in decreased micro vessel density and abnormal vascu-lature – in the case of cancer, the problem is that pancreatic stellate cell mediated hypoxia fibrosis cycle in the chemoresistance of pancreatic cancer [75].

The success of the pancreas surgery is also determined by the degree of fibrosis. One of the more common complications following pancreaticoduo-denectomy is a pancreatic fistula. A recent analysis also compared post-operative complications to the size and degree of fibrosis of the remnant gland. It is typically known that fibrotic pancreatic remnants that are normally found in CP facilitate anastomosis, while for pancreastic cancer, the soft and friable parenchyma of the remnant pancreas makes the pancreatic intestinal anastomosis hard to execute [14].

Pancreatic fibrosis (PF) is a histological feature of CP. The diagnosis of CP comes from morphological and clinical features as defined by the MANHEIM criteria [83].

Histologic evidence of fibrosis and parenchymal atrophy is the most specific diagnostic finding; however, it is rarely available. In addition, PF without inflammation or parenchymal destruction may be seen in asympto-matic individuals. This ‘bland fibrosis,’ which is found, associated with alcoholism, smoking, and ageing, should be distinguished from CP. While this bland fibrosis is clinically silent, radiologic appearances may be distinguishable from asymptomatic individuals [61].

It is important to note, however, that a correlation is not always observed between clinical manifestation and the extent of the fibrosis present. The examination of exocrine pancreatic function is conventionally conducted through use of unpleasant, invasive and costly processes. Measuring fecal elastaste-1 can be seen as a great alternative to such processes and has the merit of being implemented through random fecal specimens. Gauging this makes it possible for pancreatic exocrine deficiency to be excluded or diagnosed. Despite this, it presents itself in later stages of the illness [69].

The one method that should be implemented for accurate determination of PF levels is histological examination of pancreatic tissue. However, histological evaluation is not feasible in the routine diagnosis of patients suspected to have CP and it is challenging to properly determine the level of PF by non-invasive imaging tests. Noninvasive diagnostic methods, such as diffusion-weighted (DWI) magnetic resonance imaging (MRI) scans, could help to examine the levels of fibrosis in pancreatic tissue and confirm clinically suspected morphological pancreas related changes [30, 64].”

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The parenchymal CP changes revolve around pancreatic atrophy with replacement of the glandular elements by fat and fibrous tissue. Magnetic resonance imaging of CP has been zoomed in on finding particular focal pancreatic changes, such as inflammatory masses, pseudocysts, calcification, or pancreatic duct dilatation. Meanwhile focal changes are significant disease indicators, occur sporadically and are not present in all CP cases. On the other hand, pancreatic volume changes could be a reliable indicator of CP. Based on the known pathological changes, it is expected that a smaller sized pancreas would be characteristic of CP [38, 69].”

Advanced CP is never a problem in terms of diagnosis because of marked morphological alterations, including atrophy, ductal changes, calcification. This can prove to be a problem. However, this is more so true with slight morphological alterations, such as ductal changes. For CP that’s at a mild or early stage, the issue of the pancreas can look fine on an MRI. However, clinical suspicion can still exist. Here, new imaging biomarkers can prove helpful when looking at parenchymal changes that could help find augmented fibrosis, fat replacement, and any other indicators of early CP changes [44].

A primary reason for difficult paths towards finding proper treatment for the issue revolves around its markedly heterogeneous nature. The treatment certainly may not work with all that present with the issue [62].

Given this, finding fibrotic alterations before more serious issues present in the pancreas could help in finding early or mild forms of the illness, and help treatment ensure no further progression is made possible [30, 64, 97].

Furthermore, evaluating the issue in question is important if any issues or complications after the operation are to be predicted and prevented. Imaging techniques that can properly outline the degree of fibrosis and exocrine function can help surgeons adopt the right procedures as per the risk that’s at play. This will help build a realistic risk assessment about all the patients that are looking to undergo resection procedures. Therefore, acoustic radiation force impulse imaging or ultrasound elastography become quantitative, noninvasive processes that can help examine PF [39, 40].

MRI methods that are more advanced, such as Dixon or DWI, have been put forward as options that can help find the right information on the parenchymal organs composition [11]. A handful of studies look at the MRI imaging value when it comes to examining fibrosis in the liver. Sandrasegaran et al. found that ADC or apparent diffusion coefficient levels in cirrhotic liver are substantially smaller when looked at alongside values from an intact liver [63]. Bakan el al produced a study that showed that ADC value measurement helped develop information regarding liver fibrosis staging which was of substantial use [11]. Another study by Bonekamp et al found that liver ADC values shared an inverse correlation in terms of the fibrosis stage [17]. As far

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as this study is concerned, we have found only one piece of work that looked at Dixon and DWI use in pancreas, showing that non-invasive MRI can help produce information about steatosis and PF, providing the needed quantitative data needed [98].

“Both CP and pancreatic ductal adenocarcinoma (PDAC) may lead to cachexia, sarcopenia and osteopenia due to different mechanisms. These changes might impair the quality of life and/or limit treatment options and their effectiveness. Loss of adipose tissue (reflecting a negative energy balance) and reduction of skeletal muscle (reflecting a negative nitrogen balance) may or may not develop synchronously. Cachexia is associated with a lower tolerance for chemotherapy, which limits the total dose that can be delivered, number of symptomatic responses and any survival advantage that might be accrued [27, 86, 92].”

“Since both, computed tomography (CT) and MRI-based approaches are used in the diagnosis of the pancreatic disease, both modalities have also been extensively tested for the radiological assessment of body composition and metabolic changes in non-malignant and, more recently, malignant disease [27, 49].”

“The hypothesis of this study was that CT and MRI-based assessment of body composition and PF potentially could be a useful tool for routine detection of significant metabolic changes (loss of overall weight and fat, sarcopenia and osteopenia/osteoporosis) that affect the patients’ quality of life (QoL), treatment outcomes, thus mandate specific management.”

1.1. Aim of the study

The aim of the study was to test innovative imaging methods for early detection of clinically relevant pancreas fibrosis. This aim is achieved by completing 3 major tasks of the study.

1.2. Tasks of the study

1. To test the CT and MRI based methods for PF detection in the cohort of patients with histologically quantified grade of connective tissue

deposition in the pancreas.

2. To investigate the clinical relevance of PF in patients with pancreas diseases.

3. To correlate the pancreas fibrotic changes, sarcopenia and bone density changes with the QoL in the same cohort of patients.

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1.3. Novelty and originality of the study.

At this time there are just a few publications (PubMed search engine), which are analyzing prospectively collected patient’s data, in order to determine the most reliable radiological features, which help to diagnose the evaluation of PF and sarcopenia in patients with chronic pancreatic diseases. Our main purpose is to compare the sensitivity and specificity of several diagnostic methods and to add relevant information in diagnosing and prog-nosing the degree of PF, sarcopenia and osteoporosis development, which affects the patient’s QoL and accelerates disease progression. This is the first study, were prospectively collected clinical, pathomorphological and radiological diagnostic methods are analyzed and compared with each other.

1.4. Practical significance of the study

According to literature, progress within the analysis field of PF best-known intriguing cellular components, communication pathways and upstream regulators. Inhibition of fibrogenic processes discovered therapeu-tic effectivity in animal models, however their clinical application has not yet achieved. For this goal, identification of patients with early stage CP with novel diagnostic strategy are necessary [50, 87]. DWI MRI imaging is an emerging technology used to assess early parenchymal changes related to CP. ADC measurements will facilitate to differentiate between a disease-free pancreas and PF. Therefore, MRI could also be important to provide an early diagnosis of PF, thus patients is treated early or is given treatment choices which will prevent progression.”

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2. LITERATURE REVIEW

2.1. Pancreas and its functions

The pancreas is an organ located in the abdomen. It plays an essential role in converting the food we eat into fuel for the body’s cells. The pancreas has two main functions: an exocrine function that helps indigestion and an endocrine function that regulates blood sugar [89, 90]. The exocrine pancreas is necessary for most of the digestion of a meal and without it there is a substantial loss of digestion that results in malnutrition. The pancreas is connected by the nervous system containing both sensory neurons and effector neurons. The sensory neurons function for pain perception so that diseases of the pancreas cause significant pain while the effector neurons are involved in regulating the secretions of both the exocrine and endocrine pancreas. The two major cell types of the exocrine pancreas are the acinar cell and the ductal cell. The acinar cells are formed into a unit called the acinus which is connected to the ductal system composed of ductal cells. The stellate cell is a very slender star-shaped (hence the name stellate) cell that drapes itself around the acini, ducts and the Islets of Langerhans. In normal function pancreatic stellate cell (PSCs) are involved in directing proper formation of the epithelial structures. The PSCs is important because of its role in pathologic states [5, 9, 94].

In pathologic states such as CP and pancreatic cancer, the PSCs is trans-formed into a proliferating myofibroblastic cell type that synthesizes and secretes extracellular matrix proteins, pro-inflammatory cytokines and growth factors. In this transformed state PSCs promote the inflammation and fibrosis of both CP and pancreatic cancer that are key characteristics of these diseases [6].

2.2. Pancreatic fibrosis

PF begins with PSCs undergo morphological change after activation. Here, the rate of ECM deposition rises above the rate of degradation within the gland in question. Pancreatic cancer cells activate resulting in augmenta-tion in fibrosis [75]. PF’s definiaugmenta-tion is that it is a pancreatic atrophy that has had glandular elements replaced with fibrous and fat tissue subsequent to an injury. This typically occurs during CP.

“In a normal pancreas, PSCs may play a role in normal tissue architecture by regulating extracellular matrix turnover, given their known synthesis of ECM proteins and matrix degrading enzymes (matrix metalloproteinases

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(MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [76]. In response to pancreatic injury, PSCs transform into an active myofibroblast-like pheno-type, under the influence of activating factors such as alcohol and oxidant stress [3, 4, 54] or from products of injured cells including pro-inflammatory cytokines and growth factors [58, 95].”

They can serve as key participants in the pathobiology of the major disorders of the exocrine pancreas, including CP and pancreatic cancer. In these disorders, PSCs participate in disease pathogenesis after transforming from a quiescent state into an “activated” state (also known as a “myofibro-blast” state) [67]. PSCs have a key role in the extensive tissue fibrosis that accompanies CP and leads to destruction of the pancreas and loss of exocrine function [6, 46].

“Isolation and characterization of stromal cells from human PDAC and alcohol-induced C samples demonstrated that cells from both sources had the same characteristic morphology, cytofilament expression, and capacity to synthesize ECM proteins [9]. These results demonstrate that these 2 disorders contain common stromal elements and suggest similar mechanisms under-lying the development of fibrosis in CP and the desmoplasia in PDAC [67].” “PF plays fundamental roles in the pathogenesis of CP, which could be an attractive therapeutic target for the improvement of clinical outcome and maintenance of pancreatic functions.

Since activation of PSCs and their interaction with other cell types contribute to the pathogenesis of CP, therapeutic interventions against these interactions have been evaluated. However, the therapeutic interventions have limited benefit for patients with advanced CP, whose pancreatic parenchyma has already lost functional acinar and islet cells [75].

A large body of evidence has demonstration that when there is a bidi-rectional link between pancreatic cancer cells and PSCs, tumor progression becomes likely. Through the creation of fibrogenic and mitogenic mediators, the given cancer cells promote and attract the motility, proliferation and activation of PSCs. This results in augmented fibrosis production, which leads to a fall in abnormal vasculature and micro vessel density. This changed vasculature results in intratumoral and intrastromal hypoxia. In reaction to which, PSCs proliferate and ensure ECM deposition, leading to a cycle of hypoxia and fibrosis. Hypoxia can also augment PSCs activation and epithelial-mesenchymal transition (EMT). The two are contributors of overall chemoresistance. Moreover, this dense fibrosis and dysfunctional vasculature restricts chemotherapy agent delivery to the tumor [75].”

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2.2.1. Pancreatic fibrosis diagnostic

The assessment of exocrine pancreatic function has traditionally been carried out using invasive, unpleasant and expensive procedures. The measu-rement of fecal elastase-1 has been shown to be an excellent practical alterna-tive to these procedures and has the advantage of being carried out on random fecal specimens. Measurement of fecal elastase-1 allows pancreatic exocrine insufficiency to be diagnosed or excluded; however, it manifests in the advan-ced stages of the disease [69]. The only method that can be used to accurately determine the level of PF is histological examination of pancreatic tissue. However, histological examination is not feasible in the routine diagnosis of patients suspected to have CP and it remains challenging to reliably determine the level of PF by non-invasive imaging tests. Non-invasive diagnostic methods, such as DWI MRI scans, could help to evaluate the levels of fibrosis in pancreatic tissue and confirm clinically suspected morphological changes of the pancreas [30, 64].

DWI explores the random motion of water molecules in the body. The molecules are held outside the body in a container and are in persistent Brownian motion at random. This is known as free diffusion. On the other hand, water molecules and how they move in biological tissue is different because the interactions are modified and limited due to macromolecules and cell membranes. DWI is mostly used with a minimum of two b values (e.g., b = 0 s/mm² and other b values from 0 to 1,000 s/mm²) to enable meaningful interpretation. Typically, the degree of signal attenuation is linked to how large the b value gets. The bigger it is the bigger the signal attenuation is. By looking at the attenuation of signal intensity on images found through b values, it becomes possible to find tissue characterization based on varying water diffusion (Fig. 2.2.1.1) [47].”

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Fig. 2.2.1.1. ADC image of normal pancreas head (arrow)

A few studies have evaluated the value of MRI for the assessment of liver fibrosis. The study by Sandrasegaran et al. revealed that ADC values in cirrhotic liver are significantly lower compared with those in intact liver [81]. Bakan et al., found that the measurement of ADC values provides valuable information for liver fibrosis staging [10]. Bonekamp et al, concluded that liver ADC values inversely correlate with fibrosis stage [17]. To the best of our knowledge, only one study has evaluated the use of DWI and Dixon in the pancreas, indicating that non-invasive MRI parameters can yield quantita-tive information regarding pancreatic fibrosis and steatosis [98]. ADC values for patients with CP are lower than those found for patients with a normal pancreas. This finding is attributed to the replacement of normal pancreatic parenchyma with fibrous tissue and/or reduced exocrine function that may lower the amount of diffusible tissue water and result in decreased ADC [63].

Miller FH et al. revealed that chronic inflammation and fibrosis diminish the proteinaceous fluid content of the pancreas, resulting in a loss of the usual high signal intensity on T1-weighted fat-suppressed images (Fig. 2.2.1.2 and 2.2.1.3) [59].

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Fig. 2.2.1.2. Axial T1-weighted fat-suppressed spoiled gradient-echo image

shows abnormal low SI of pancreatic tail (arrow) while remainder of pancreas has normal bright signal intensity (Adapted from Miler FH et al.)

Fig. 2.2.1.3. Axial T1 fat-suppressed spoiled gradient-echo image shows

low SI pancreas due to CP (arrow) (Adapted from Miler FH et al.) 2.2.2. Fibrosis degree determination of pancreas

There are just a few clinical studies that analyzes the possibilities of MRI in PF and its degree determination.

Noda et al. (2016) performed a retrospective study of 29 patients, who underwent surgical resection of the pancreas, to evaluate the diagnostic value of noncontrast-enhanced MRI to grade PF and to assess hemoglobin A1c values. Patients were divided in 4 groups according to their HbA1c values

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and pancreatic fibrosis degree. The pancreas-to-muscle signal intensity ratio (SIR) on in- and opposed-phase T1-, T2-, and DWI, as well as the ADC were calculated. The PF grade and HbA1c value were negatively correlated with the SIR on opposed-phase T1-weighted images, however the significant differrence between ADC and SIR values in T1 and T2 wasn’t obtained. In this study authors didn’t search the correlation between ADC value and PF degree [64].

Watanabe et al. (2013) performed a retrospective study of 29 patients, who underwent surgical resection of the pancreas, to assess the potential value of MR imaging in evaluating PF and predicting the development of postoperative pancreatic fistula. Authors divided PF in 4 histological degrees and proved, that ADC value correlates with the histological degrees of PF. They found that ADC value were correlated with the PF grade (r = –0.69 and –0.60, respectively), there were 1.850 ± 0.23 10–3_{mm²/s, for F0, 1.730 ± 0.36} 10–3_{mm²/s for F1, 1.440 ± 0.27 10}–3_{mm²/s for F3 fibrosis grade [97].}

There were just few authors, who had measured pancreas ADC value in patients with CP. Obtained results were very diverse. Frokjaer et al. (2013) performed a retrospective study of 23 patients with CP, as well as 17 patients of control group, to evaluate the diagnostic value of DW MRI ADC value in the assessment of PF. Clinical, CP etiology and laboratory parameters were correlated alongside pain scores and image findings. The outcome demonstrated that people with CP, who presented with clinical and laboratory CP parameters that were typical, had a lower DW MRI ADC value. The study concluded that mean ADC 177.9 × 10–5_{mm²/s. Of the CP pancreatic tissue.} They found mean ADC 177.9 × 10–5_{mm²/s. of the CP pancreatic tissue. The} obtained results were statistically significant, so DW MRI is an emerging technology to assess early parenchymal changes associated with CP. ADC measurement can help differentiate between disease-free pancreas and CP [30].

Yoon JH et al. (2016) performed a retrospective study with total of 165 patients, who underwent preoperative 3-T MR imaging and subsequent pancreatectomy. There were four groups of PF, depended on DWI MRI ADC value. The research showed, that the estimated pancreatic fat fraction at MRI showed a moderate correlation with histologic results. With increasing degrees of fibrosis, pancreatic parenchyma showed a higher fat fraction (r = 0.35) [98].

In M. Fatih Akisik et al. research founded ADCs in the pancreas are reduced in patients with CP (mild, 168.6 × 10–5_{mm²/s; severe, 157.0 × 10}–5_mm²/s) compared with patients without pancreatitis (194.6 × 10–5_{mm²/s; P < 0.01) [1].} According to literature and our research, compared with CT, MRI is a more sensitive imaging tool for the diagnosis of CP. CT or ultrasound may be

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falsely negative in the detection of early CP because of their lack of detecting subtle ductal abnormalities. These tests fail to depict these parenchymal changes that are only detected with MRI.

2.3. Pancreatic fibrosis and metabolic changes 2.3.1. Pancreatic fibrosis and sarcopenia

Muscle function as a concept is changing. Previously, the skeletal muscle was thought to be linked with mobility, presenting a mechanical aspect in terms of its functionality. We know today that there are many functions for the muscles on a human body, including neurological, metabolically, endocrinal, and psychological [48].

Sarcopenia, derived from the Greek words for flesh (sarx) and loss (penia), is a condition of decreased skeletal muscle mass that can lead to a decline in physical ability [82].

The European Working Group on sarcopenia in older people recom-mends using the presence of low muscle mass and low muscle function (strength or performance) for the diagnosis of sarcopenia.

Sarcopenia is defined as depletion of skeletal muscle mass with a risk of adverse outcomes, such as physical disability and poor QoL. The process starts around the age of 40 and progresses at a rate of 8% loss of muscle tissue per decade until the age of 70, when muscle loss accelerates to 15% per decade [33].

Sarcopenia is associated with many clinical conditions, such as cancer (including pancreatic cancer), diabetes, acquired immune deficiency syndro-me, burns, chronic obstructive pulmonary disease, chronic heart failure, chronic renal failure, CP, rheumatoid arthritis and sepsis [7, 19, 85].

Peng P at al. measured total psoas area on preoperative cross-sectional imaging in 557 patients undergoing resection of PDAC. They found that sarcopenia was associated with increased risk of 3-year mortality and made a conclusion, that sarcopenia was a predictor of survival following pancreatic surgery, with sarcopenic patients having a 63% increased risk of death at 3 years. Sarcopenia was an objective measure of patient frailty that was strongly associated with long-term outcome independent of tumor-specific factors [71].

Body mass index (BMI) has several issues because it does not present a complete picture. A comprehensive examination of the body shows that lean tissues are wasting, and many patients do not register at the appropriate muscularity benchmark, coming in either higher or lower than the values outlined. This is important because it contradicts the levels needed to identify functional and mortality disability [49]. Approximate values for lean body

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mass are categorized as sarcopenic as per the range understand for many emaciated or wasted patient populations without or with the illness in question [55, 93].

A BMI <18.5 kg/m² is considered by many authorities to represent an individual at serious risk of undernutrition [84].

In the current literature it is becoming increasingly evident that concur-rent sarcopenia and high fat mass is a worst-case scenario [16, 26, 41, 42, 55, 78, 86], and this was clearly apparent in Tan, Benjamin H. L. et al. study group (albeit small), in which sarcopenic overweight/obese patients had the worst prognosis overall, even compared with patients who were sarcopenic and had a lower body weight. In this study, only 10% of individuals has BMI <18.5 kg/m². Given the prevalence of overweight/obesity (40%) it would seem unlikely that even in the presence of ongoing weight loss, the majority would reach this boundary at or near the time of death [86].

Ryuta Sh. Et al. study has demonstrated a clear relationship between sarcopenia and pancreatic exocrine insufficiency (PEI) in patients with pancreatic disease. They collected 221 patients with pancreatic disease, who underwent pancreatic surgery. Pancreatic exocrine function was assessed using a13C-labeled mixed triglyceride breath test. There was no significant relationship between clinical factors and PEI. On univariate analyses, the presence of sarcopenia (lowest vs highest quartile of L3 muscle mass index) was associated with PEI in both men (P < 0.001) and women (P = 0.012).

Sarcopenia is a common complication of CP, but in most patients, it is not recognised by conventional anthropometric parameters [66]. A wide variety of tests and tools are now available for characterization of sarcopenia in practice and in research. CT is the methods of choice for diagnostics of pancreatic cancer and inflammatory diseases of the pancreas. It is also a useful tool as one of the many methods to evaluate sarcopenia in these patients by using L3 skeletal muscle index, which can be measured using diagnostic CT-scans acquired during the routine care.

2.3.2. Pancreatic fibrosis and bone density changes

Osteoporosis is defined as a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, both conditions leading to an increase in bone fragility and fracture susceptibility [60].

Dual-energy X-ray absorptiometry (DXA) is typically deployed to evaluate at the molecular level, the body’s composition. The radiation exposure is also looked at and is thought to be safe and small for repeated intervention. During such a scan, the body is subjected to low-radiation

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X-rays of 2 varying photon energy levels. These are found through a detector that looks at photons and quantifies the size of absorption of energy by bone and soft tissue at every pixel in question [77].

Bone mineral density (BMD) results obtained by DXA are usually expressed as T-score or Z-score. T-score represents the number of standard deviations by which BMD varies from the mean value expected in a young healthy subject (20–30 years of age), whereas Z-score is the number of standard deviations by which BMD varies from the mean value typical for a person with the same age and sex. According to the World Health Organi-zation definition of osteoporosis, the disease can be diagnosed when T-score ≤−2.5 of DXA at the lumbar spine, femoral neck or distal third of the radius [48].

Appropriate amounts of vitamin D are needed for bone metabolism and bone mineral density levels to stay normal. How well this vitamin is absorbed into the into the body is linked with uptake of lipids, which can only happen if pancreatic enzymes are available in the right quantity, and at the appropriate place and time within the intestines [43]. In the Haas S. At al. study, a significant decrease was seen in serum vitamin D (25-OH cholecalciferol), along with a significant increase in deoxypyridinoline in urine. Bone mineral density was reduced in the majority of patients with CP. There was a correlation between pancreatic exocrine function and bone mineral density [37].

An article on nutritional therapy states, that CP is associated with osteo-porosis, sarcopenia, poor QoL and increased mortality [54].

Another study of Duggan et al. revealed, thatosteoporosis/osteopenia afflicts two-thirds of patients because of poor dietary intake of calcium and vitamin D, low physical activity, as well as chronic low-grade inflammation. Moreover, bone metabolism studies show increased bone resorption in CP [25].

Haaber et al. in their study did not find significant differences in BMD between pancreatitis patients with and without PEI, however, BMD as measured with DXA was significantly lower in the PEI group [35]. The percentage of patients with decreased bone mineral density (osteopathy) ranged from 34% to 100% in previous studies (Table 2.3.2.1) and did correlate with the severity of CP in at least one study [36]. Duggan SN at al. performed a systematic review and meta-analysis to determine the prevalence of osteoporosis and osteopenia in patients with CP and made conclusions, that almost 1 of 4 patients with CP have osteoporosis, and almost two-thirds of patients have either osteoporosis or osteopenia. Osteoporosis and osteopenia

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are underappreciated sources of morbidity in patients with CP. Bone health management guidelines are urgently required in patients with CP [24].

Table 2.3.2.1. Rates of osteopathy (osteoporosis, osteopenia, fractures) in

patients with CP (from Haas S. et al. study [37])

Year N o. o f p at ie nt s Me th od O ste op ath y to ta l (% ) O st eop or os is (%) O st eo pe ni a (% ) O st eom al ac ia (% ) U ns pe ci fie d (% ) Comments Ref.

1997 14 DXA 71 21 50 – – Men only Moran et al. [46]

2000 58 DXA 85 64 22 – – – Haaber et al. [45]

2003 42 DXA 100 – – – 100 Men only Mann et al. [47]

2008 73 DXA 39 5 26 8 – – Djusikova et al. [38]

2011 31 DXA 100 – – – – Sudeep et al. [48]

2012 62 DXA 34 – – – – Duggan et al. [44]

2013 N/a DXA – – – – – – Sikkens et al. [49]

2014 42

322 DXA, XR 64 – 16 – 76 – – – – – Men only Haas et al. [42] 2014 513 DXA,

XR 65 23 40 – – Systematic review Duggan et al. [50] N/a: not available.

2.3.3. Pancreatic fibrosis and exocrine or/and endocrine insufficiency

Patients with PEI suffer from malnutrition because of malabsorption and maldigestion. The assessment of PEI function has traditionally been carried out using invasive, unpleasant and expensive procedures. The measurement of fecal elastase-1 has been shown to be an excellent practical alternative to these procedures and has the advantage of being carried out on random fecal specimens.

Determination of fecal elastase-1 is highly sensitive in the diagnosis of PF [91]. Measurement of fecal elastase-1 allows PEI to be diagnosed or exclu-ded; however, it manifests in the advanced stages of the disease.

Partelli at al. examined the levels of fecal elastase-1 in 194 patients with pancreatic cancer. Half of all patients (97) had reduced fecal elastase-1 values and more than half of those (48) had extremely reduced fecal elastase-1 values

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(≤20 μg/g). This study showed that levels of fecal elastase-1 strongly corre-late with the reduced exocrine pancreatic function seen in advanced pan-creatic cancer [69].

PEI is defined as partial or complete loss of digestive enzyme and bicarbonate secretion. In CP this is caused by a progressive destruction of functioning pancreatic tissue. Progressive inflammatory destruction of pancreatic tissue in CP leads to reduced synthesis and secretion of pancreatic enzymes in response to food intake [74]. Pancreatic cancer is a typical condition in which normal PEI is impaired due to chronic obstructive damage to the secreting component of the organ [96].

Diabetes mellitus (DM) secondary to pancreatic issues is not considered during clinical practice. However, a large portion of endocrine pancreatic deficiency has been linked for a good number of the population, specifically those with diabetes [28].The subgroup of DM that occurs in conjunction with diseases of the endocrine pancreas is termed type 3c diabetes (or pancreato-genic diabetes) [2]. Due to its association with pancreatic disease, patients with type 3c diabetes tend to be undernourished and have nutrient deficien-cies. Managing this issue made difficult because of excessive intake of alcohol, malabsorption, and poor diets because of other issues such as anorexia, smoking, abdominal discomfort and more [21].

In contrast to the management of type 1 or type 2 diabetes, the endo-crinopathy in type 3c of DM is very complex and complicated by additional present comorbidities such as maldigestion and concomitant qualitative malnutrition [28].

2.4. Quality of life in patients with pancreatic diseases causing fibrosis

There are many different studies of patient’s QoL. Some of them are more focused on patients with CP QoL, independently from duration of the disease and interventional procedures, others are including these factors in their studies.

Health-related QoL is becoming a major issue in the evaluation of any therapeutic intervention in patients with chronic or hard to cure diseases. Pezzilli R at al. performed a study of all different types of pancreatic disease and revealed, that patients with CP have a substantially impaired QoL and, most importantly, the impairment of the QoL in younger patients is higher than in older ones with obvious economic consequences for society. The Medical Outcome Study 12-Item Short-Form Health Survey (SF-12) and European Organization for Research and Treatment of Cancer Quality of Life

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Questionnaire-C30 (EORTC QLQ-C30) questionnaires were used for the purpose of this study. One hundred and ninety-seven sex- and age-matched normative populations were also included in the study as a reference group. Of the 197 patients studied 164 (83.2%) had malignant disease and 33 had benign disease (16.8%). At initial evaluation, global health was significantly lower (P = 0.001) in the study population as compared to the normative population. At the end of the study, the QoL was not significantly different from the normative population, although the QoL of the 30 patients with benign disease was significantly better than that of the 72 patients with malignant disease [72].

In large prospective study Machicado JD et al. found that pain – espe-cially constant, pain-related disability/unemployment, current smoking, and concurrent co-morbidities to have a significant negative impact on the QoL of CP patients. They used data of 1,024 patients and the short Form-12 (SF-12) version 2 questionnaire. Physician-defined alcohol etiology, disease duration, pancreatic morphology, and prior endoscopic or surgical treatments did not independently affect QoL [52].

Bauer MR et al. have made a systematic review compared with healthy adults or population norms, adults with pancreatic cancer had worse QoL across all domains. Compared with patients with other cancer types, patients with pancreatic cancer evidenced worse psychological QoL. Physical and social QoL were either similar or more compromised than in patients with other cancers [13].

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3

.

STUDY POPULATION AND METHODS

3.1. Study population and its general characteristics

The prospective study was approved by the Kaunas Regional Biomedical Research Ethics Committee (Protocol No. BE-10-11). Informed consent was obtained in all cases, after oral and written information about the study had been provided. The permission from personal data protection inspection was obtained (No. IR-6945).

We prospectively collected data of 104 patients with CP (n = 63) and PDAC (n = 37). Four patients were excluded from the final analysis, because it was not possible to measure and calculate the degree of sarcopenia due to the poor quality of CT images and patient refusal to repeat the scanning. Fifty-two of them with cancer and complicated CP underwent surgery. All patients underwent preoperative abdominal CT and MRI scans. In 52 patients final diagnoses were consequently confirmed postoperatively by pathomorpho-logical examination of the surgical specimens. The stool samples for fecal elastase-1 estimation were collected at least 2 days before the surgery (n = 52)

or on the hospitalization day, if the patient wasn't operated (n = 52).

Also, all patients, who had surgery were categorized in 4 groups, depen-ding on the degree of PF. After that, obtained results were adapted for all other 52 patients who weren’t operated (with diagnosed CP) for metabolic changes analysis.

A control group of 52 randomly selected from databases patients with intact pancreatic tissue and no previous history of pancreatic disease such as acute or CP, pancreatic tumors, or other pathology. They underwent CT examination for other abdominal pathology. Gender distribution and age did not significantly differ between the control and investigation groups.

Sixty six males and 34 females with an average age of 55 years (range 22–89 years) and average BMI of 24 kg/m2_{(range 14-44) were enrolled in} the study.

DXA, MRI and CT imaging were performed in all cases. During the last inpatient stay all patients answered the same questionnaire for QoL assessment.

Inclusion criteria:

a) Determined CP or pancreatic cancer.

b) Patient is available for cooperation during the study.

c) Person is older than 18 years old and gives his written consent to be enrolled in biomedical study.

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Exclusion criteria:

a) No written consent of the patient to be enrolled in biomedical study was obtained.

b) Radiological images quality is insufficient for diagnostic.

c) Contraindications for MRI and/or CT scan (iodine sensitivity, impai-red renal or liver function).

d) Risk of allergy for contrast agent – bronchial asthma in anamnesis. e) Pregnancy.

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3.3. Methodology 3.3.1. CT examination

All CT examinations were performed with a 64-slice CT tomography unit GE Light Speed Pro (GE Healthcare, Milwaukee, WI, USA), with and without intravenous injection of 100 mL water-soluble iodine contrast medium (270– 320 mg/mL) in porto-venous phase. The slice thickness was 2.5 mm. All images were analysed at a window level of 40 Hounsfield units (HU) and window width of 300 HU.

Pancreatic tissue was referred as “atrophic” with typical enhancement. Atro-phic, with lesser enhancing pancreatic tissue (lesser than 80 HU in portovenous phase) was referred as “potentially fibrotic”, and pancreatic tissue with no enhan-cemet and with multiple calcifications (>10) – rated as “pancreatic fibrosis”.

The pancreas was identified based on the typical landmarks (splenic vein and superior mesenteric artery). Using the hand-outlined pancreas, we measured the total pancreas volume by use of the surface tool in the Vitrea workstation (Vitrea, Vital Images, Canon Group Company, USA).

Pancreatic volume was measured using the summation-of-areas method, in which the outer margin of the pancreas was manually drawn as a cutting line to remove all surrounding structures (tumor volume, cyst volume, dilated ducts etc. were excluded). This was repeated for every single slice containing pancreatic tissue. Total pancreatic volume (mL) was computed by summing up the volume from each slice (area from each slice × 2.5 m = volume) that included a piece of pancreatic tissue (Fig. 3.3.1.1). The measurement of pancreatic volume was carried out by a single investigator.

Fig. 3.3.1.1. Volume of the pancreas measured using

a summation-of-areas method

A – atrophic pancreatic tissue with dilated pancreatic duct and tumor; B – intact pancreatic tissue

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3.3.2. Assessment of skeletal muscle mass

A single axial CT image at the level of the third lumbar vertebrae was assessed to measure cross-sectional areas of skeletal muscle (m. psoas major, m. erector spinae, m. quadratus lumborum, m. obliquus externus abdominis, m. obliquus internus abdominis, m. travsversus abdominis, m. rectus abdo-minis) (Fig. 3.3.2.1 and 3.3.2.2). The tissue cross-sectional area at this level correlates with total body skeletal muscle distribution [33]. Images were analysed using NIH ImageJ (1.50i) software (Wayne Rasband, National Insti-tutes of Health, USA), which allows quantification of the tissue composition by using HU. The threshold for skeletal muscle was 150 to –29 HU. The area of skeletal muscle (SMA) was normalized for the height of the patient and the lumbar skeletal muscle index (L3 SMI) was calculated (cm²/m²) as SMA (cm2_{)/height (m}2_{) [90].}

Fig. 3.3.2.1. Axial CT image at the level of the L3 vertebrae

Structures that are segmented for the assessment of sarcopenia: 1 – m. rectus abdominis, 2 – subcutaneous adipose tissue (SAT), 3 – m. obliquus externus abdominis, 4 – m. obliquus externus abdominis, 5 –

m. obliquus internus abdominis, 6 – m. quadratus lumborum, 7 – m. psoas major, 8 – m. erector spinae, 9 – intramuscular adipose tissue (IAT), 10 – visceral adipose tissue (VAT), 11 – body of L3 vertebrae.

Fig. 3.3.2.2. Axial CT image at the level of the L3 vertebrae delineated

with Image J “freehands selection” tool outer (A) and inner (B) abdominal musculature and L3 vertebra (C)

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Based on skeletal muscle cut-off values defined from a large study in a healthy population, sarcopenia in patients was determined when the SMI was lower than 34.4 cm²/m² for females and, lower than 45.4 cm²/m² for males (Table 3.3.2.1) [18].

Table 3.3.2.1. Skeletal muscle cutoff values for sarcopenia diagnosis using

T10 to L5 measurements in a healthy US population [18]

VB Female Male P value

n Mean ± s. d. cutoff n Mean ± s. d. cutoff

SMA (cm2_{) T10 156 86.6 ± 16.0 54.6 122 135.3 ± 22.0 91.4 <0.001} T11 336 83.5 ± 15.5 52.4 241 131.7 ± 21.8 88.1 <0.001 T12 401 91.3 ± 17.6 56.1 299 141.2 ± 24.4 92.3 <0.001 L1 409 103.4 ± 16.7 70.1 315 159.2 ± 24.4 110.4 <0.001 L2 411 117.7 ± 17.9 81.9 315 183.9 ± 27.8 128.2 <0.001 L3 410 128.0 ± 17.9 92.2 317 195.2 ± 25.4 144.3 <0.001 L4 399 125.5 ± 16.6 92.4 305 177.1 ± 23.2 130.7 <0.001 L5 295 114.9 ± 16.7 81.5 211 176.0 ± 27.0 122.0 <0.001 SMI (cm2_/m2₎ T10 156 32.3 ± 5.9 20.4 122 42.2 ± 6.7 28.8 <0.001 T11 336 31.0 ± 5.9 19.2 241 41.1 ± 6.8 27.6 <0.001 T12 401 34.0 ± 6.6 20.8 299 44.1 ± 7.7 28.8 <0.001 L1 409 38.4 ± 6.2 25.9 315 49.7 ± 7.6 34.6 <0.001 L2 411 43.7 ± 6.7 30.4 315 57.4 ± 8.7 40.1 <0.001 L3 410 47.5 ± 6.6 34.4 317 60.9 ± 7.8 45.4 <0.001 L4 399 46.7 ± 6.2 34.2 305 55.3 ± 7.0 41.3 <0.001 L5 295 42.8 ± 6.1 30.6 211 54.7 ± 7.9 39.0 <0.001 SMRA (HU) T10 156 _{T11 336} 40.4 ± 6.9 _{43.4 ± 6.5} 26.5 122 _{30.4 241} 43.8 ± 5.7 _{46.5 ± 5.3} 32.4 <0.001 _{35.8 <0.001} T12 401 44.0 ± 6.4 31.3 299 48.2 ± 5.3 37.5 <0.001 L1 409 42.6 ± 6.4 29.8 315 47.5 ± 5.7 36.2 <0.001 L2 411 43.4 ± 5.5 32.5 315 48.1 ± 5.2 37.7 <0.001 L3 410 44.9 ± 5.3 34.3 317 49.0 ± 5.3 38.5 <0.001 L4 399 43.1 ± 5.2 32.7 305 47.6 ± 5.2 37.3 <0.001 L5 295 43.9 ± 5.0 33.9 211 49.9 ± 5.0 39.8 <0.001

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3.3.3. MRI Examination

All MRI examinations were performed by means of a 1.5 T Siemens Magnetom Area (Siemens Healthcare GmbH, Erlangen, Germany) using a torso multi-coil array. The included image sequences were as follows: (1) axial and coronal T2-weighted images (slice thickness, 5 mm; repetition time [TR]: 13.800 milliseconds; echo time [TE]: 89 milliseconds; flip angle, 90 degrees; matrix size 320×224; and field of view [FOV], 324 mm); (2) DWI with multiple b-values 50, 400, and 800 s/mm2_{(axial echo planar imaging,} slice thickness, 5 mm; TR, 6,000 milliseconds; TE, 52.2 milliseconds; matrix size 128×128; FOV, 400 mm), and ADC maps which were calculated auto-matically for each section by the imager software; and (3) axial T1-weighted images (slice thickness, 3 mm; TR, 7.14 milliseconds; TE, 2.38 milliseconds; flip angle, 10 degrees; and FOV, 380 mm).

Pancreatic tissue was identified on the T1, T2 and DWI sequences. Patients with CP were examined during their inpatient stay, 1–3 months before surgery, and patients with pancreatic cancer were examined on the day before surgery. The largest possible region of interest (ROI) was used for each patient and was repeated three times with different ROIs (Fig. 3.3.3.1). The ROI was kept at a maximum of 200 mm² and a minimum of 50 mm². The positions of the ROIs were guided by the axial T2-weighted images, and ducts, cysts, and artefacts were avoided.

In patients who underwent pancreas resection (n = 52), ADC and T1 signal intensity (SI) was measured at the anticipated resection margin selected under the surgeon’s supervision. CT scan images were evaluated and compared with MRI scan images for the identification of tumour margins. Measurements were performed approximately 1–3 cm from the margins of the tumour, in complex with the pancreas resection margin. The boundaries of each tumour were identified by means of contrast-enhanced CT scan and MRI T1 and DWI sequences. The average of the three measurements was accepted as the final ADC value of the segment. All ADC values were measured directly from the ADC map data on an independent workstation. Since fibrotic pancreatic tissue is known to have a more hypo intense SI in fat-suppressed T1-weighted imaging, quantitative T1 SI values were measu-red on the T1 Dixon water-only images in the same location as the ADC [15].

For the remaining patients, who have not undergone histological verifi-cation, ADC values were measured in the pancreas head/body projection (since histologically tested patients measurements were performed in a similar location).

All of the radiological examinations were evaluated by a radiologist with at least 10 years of experience who was blinded to the histology results.

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Fig. 3.3.3.1. Magnetic resonance imaging of pancreas

ADC map images (A, B, C) with ROI positioned in the different parts of pancreas for assessment of parenchymal fibrosis. Axial T1-weighted image (D),

with ROI positioned in the same location as ADC.

3.3.4. Assessment of bone density

Bone density was determined by using dual-energy X-ray absorption (DXA; QDR-1000, Hologic Instruments, Waltham, MA, USA) at the standard measurement sites in the lumbar spine and femur.

Results are presented as T scores and Z scores: normal bone density (T score >–1), osteopenia (T score from –1 to –2.5), and osteoporosis (T score <–2.5).

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3.3.5. Pancreatic exocrine function testing

The pancreatic exocrine function was tested on the day before surgery using the ScheBo® Pancreatic Elastase Stool test (ScheBo, Mannheim, Germany). Faecal elastase-1 test results exceeding 200–500 µg/g were consi-dered normal, whereas results less than 200 µg/g were consiconsi-dered moderate and less than 100 µg/g indicated strong PEI.

3.3.6. Morphometric analysis of the pancreatic tissue

The tissues from the pancreatic stump of the resection margin were obtained during surgery. Freshly remover samples were fixed in 10% formalin solution, dehydrated and embedded in paraffin. Paraffin embedded blocks were sectioned 4 μm thick sections. The sections of pancreatic tissue were processed with haematoxylin and eosin and Mason’s trichrome staining for the accurate estimation of connective tissue. Haematoxylin and eosin were used for the evaluation of general histological pancreatic tissue alterations, while Mason’s trichrome staining was used for the evaluation of PF degree and its relation to the intact pancreatic tissue (Fig. 3.3.6.1). Digital images of each slide were analysed by an experienced pathologist, using a semi quantitative measurement method with enlargement for light microscopy, using an Olympus BX63 microscope (BP72 camera, 1×1 cm area, ×40). Connective tissue stain positive areas were selected using “Select colour range” command in the Tools menu of the program Olympus cellSens Dimension 1.16 (2011, Germany). After images were transformed to red (normal pancreas tissue) and green (connective tissue) scale (Fig. 3.3.6.2) and analysed calculating the ratio of stained area against total area. Abnormalities were classified into four histological sub-groups (>10%, >25%, >50%, and >75% of fibrosis).

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Fig. 3.3.6.2. Image analysis in quantification of fibrotic changes

of pancreas

Red colour – normal pancreatic tissues, green colour – connective tissue.

3.3.7. Quality of life evaluation

We applied the EORTC QLQ-C30 (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30), which has been successfully used in other CP and pancreatic cancer studies [73].

The EORTC quality of life questionnaire is an integrated system for assessing the health-related QoL of cancer patients participating in interna-tional clinical trials. The core questionnaire, the QLQ-C30, is the product of more than a decade of collaborative research. EORTC QLQ-C30 version 3.0 is currently the standard version of the QLQ-C30 and should be used for all new studies unless investigators wish to maintain compatibility with previous studies, which used an earlier version of the QLQ-C30. The QLQ-C30 is composed of both multi-item scales and single-item measures [29].

These include five functional scales, three symptom scales, a global health status / QoL scale, and six single items. Each of the multi-item scales includes a different set of items – no item occurs in more than one scale. All of the scales and single-item measures range in score from 0 to 100. A high scale score represents a higher response level. Thus, a high score for a functional scale represents a high/healthy level of functioning, a high score for the global health status / QoL represents a high QoL, but a high score for a symptom scale/item represents a high level of symptomatology / problems. The principle for scoring these scales is the same in all cases: 1. Estimate the average of the items that contribute to the scale; this is the raw score. 2. Use a linear transformation to standardize the raw score, so that scores range from

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0 to 100; a higher score represents a higher (“better”) level of functioning or a higher (“worse”) level of symptoms [29]. For the analysis, we used the following scales: global health status (QL2), physical functioning (PF2), fatigue (FA), pain (PA), appetite loss (AP) and diarrhea (DI).

3.3.8. Statistical Analysis

Statistical analysis was performed using SPSS 22.0 (SPSS, Inc., Chicago, IL, USA) software and standard Windows environment Excel programs.

Descriptive statistics of qualitative variables are demonstrated in tables of frequencies, quantitive variables are demonstrated using medians and stan-dard deviations. For an assessment of the normality of quantitative variables data have been used Kolmogorov-Smirnov test. Two groups of quantitive variables with normal distribution were compared by using independent Stjudent t-test. Two groups of quantitive variables without normal distribu-tion were compared by using the non-parametric Mann-Whitney U test. In case of comparison of more than two groups of quantitive variables, the non-parametric Kruskal-Wallis test was used. In the same case of qualitative variables, Chi-squared test or Fisher's exact test were used. Wilcoxon signed-rank test was used in two dependent samples comparison. Analyzing the dependency in two quantitive variables we used Spearman correlation coeffi-cient. Nonparametric ROC curves were composed to assess cut-off values, sensitivity, and specificity.

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4. STUDY RESULTS

4.1. MRI and CT diagnostic value of pancreatic fibrosis

4.1.1. Association between MR imaging parameters and histological examination

MRI examination before surgery was performed for fifty-two patients, who were divided into four histological subgroups (>10%, >25%, >50%, and >75%). The highest degree of fibrosis was determined in the CP and pancreatic ductal adenocarcinoma patient groups and the fewest fibrotic changes were evident in patients with ampullary carcinoma.

A few representative clinical cases with 2%, 30% and 82% fibrotic tissue on digital images and MRI with measured ADC value demonstrated in Fig. 4.1.1.1.

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Fig. 4.1.1.1. Representative clinical cases with fibrotic tissue on digital

images and MRI DW images with measured ADC value

Patient – 22 years’ female, tumor of pancreas head (insulinoma). Pancreatic parenchyma contains 2% of fibrotic tissue (equal to normal tissue) (A). Image of DWI MRI, ADC value

at the resection margin 1.763 × 10–3_mm2_{/s (B). Patient – 64 years’ female, carcinoma of} the head of the pancreas. 30% of fibrotic tissue (C). DWI MR, ADC value at the resection

margin 1.381 × 10–3_mm2_{/s (D). Patient – 34 years’ male, chronic pancreatitis. Pancreas} containing 82% of fibrotic tissue (E). DWI MRI, ADC value was 1.173 × 10–3_mm2_{/s (F).}

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In our research, we measured the ADC value in DW sequences and T1SI values on T1 Dixon water-only images and compared the obtained results with a histological examination to determine the MRI value for determination of PF degree. Preoperative DW MRI ADC values of the first group of 52 patients, and the second group of 52 randomly selected patients with intact pancreatic tissue and no previous history of pancreatic disease such as acute or CP, pancreatic tumors, or other pathology with normal pancreatic tissue, were significantly different (P = 0.001).

We found that there was a significantly negative correlation between ADC value and histologically-determined PF (r= –0.752, P < 0.001), as well as a significant negative correlation between T1 SI value and histologically-determined PF (r = –0.631, P < 0.001) (Fig. 4.1.1.2, A–B). Interestingly, among the patients with PF <50% there was almost no single measurement of T1 SI less than 150, and none of the ADC values were below 1.20 × 10–_{³ mm²/s when fibrosis was <75%. The correlation between ADC and T1SI} values was significant; however, it was not exactly linear, revealing that ADC and T1SI values should be evaluated separately as predictors of real fibrotic changes (r = 0.599, P = 0.001) (Fig. 4.1.1.2, C).

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Fig. 4.1.1.2. The correlation between PF, ADC and T1SI values The negative correlation between ADC mean (×10–3_mm2_{/s) and histologically}

determined PF (%) (A) as well as negative correlation between T1 SI and histologically determined PF (B). Moreover, there was the significant

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The cut-off values were estimated for diagnosing histologically-detected fibrosis in order to assess the lowest possible levels of PF upon radiological examination.

We determined the cut-off values, ADC mean value and T1 SI mean value for diagnosing ≥10%, ≥25%, ≥50% and ≥75% fibrosis (Fig. 4.1.1.3 and 4.1.1.4). The ADC cut-off value in ≥10% PF was significantly higher (≤1.47 × 10–_³ mm²/s), and in ≥25%, ≥50% and ≥75%, the cut-off values were similar (≤1.331 × 10–_{³ mm²/s, ≤1.316 × 10}–_{³ mm²/s and ≤1.310 × 10}–_{³ mm²/s). The T1} SI cut-off values were significantly different in all PF groups: ≥10% (cut-off value was ≤201.15), ≥25% (≤172.1), ≥50% (≤159.9) and ≥75% (≤143.3).

Fig 4.1.1.3. ADC cut off values

Diagnosing ≥10% PF (≤1.47 × 10⁻³ mm²/s (A), ≥25% PF (≤1.331 × 10⁻³ mm²/s (B), ≥50% PF (≤1.316 × 10⁻³ mm²/s (C) and ≥75% PF (≤1.310 × 10⁻³ mm²/s) (D). AUC values were 0.969,

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Fig. 4.1.1.4. T1 SI cut off values

Diagnosing ≥10% PF (≤201.15) (A), ≥25% PF (≤172.1) (B), ≥50% PF (≤159.9) (C) and ≥75% PF (≤143.3) (D) PF. AUC values were 0.797, 0.793, 0.855 and 0.811 respectively.

We calculated the sensitivity and specificity of two combined MRI scanning techniques (ADC and T1 SI values) for the determination of PF. First, we measured ADC and T1 SI values separately and found the highest PF diagnostic sensitivity for ≥75% PF and the highest specificity for ≥10% PF. Cumulatively, both measurements provided significant sensitivity for diagnosing ≥75% PF as well as ≥10% PF (100% for diagnosing ≥75% PF and 97% for diagnosing ≥10% PF). The specificity here was higher for diagnosing ≥50% PF (88%) (Table 4.1.1.1). These results show that the combination of both measurements allows a more precise diagnosis to be determined.

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Table 4.1.1.1. The Sensitivity and Specificity of two Combined MRI Scanning

Techniques (ADC and T1 SI) in PF Diagnostic

Variable Sensitivity, % Specificity, %

≥10% fibrosis ADC 92 100 T1 83 75 ADC and T1 97 60 ≥25% fibrosis ADC 77 88 T1 77 70 ADC and T1 88 65 ≥50% fibrosis ADC 85 88 T1 78 84 ADC and T1 85 88 ≥75% fibrosis ADC 100 73 T1 67 86 ADC and T1 100 67

4.1.2. MRI for prediction of fibrosis

We used a linear regression equation (y = β₀ + β₁x₁ + β₂x₂) to predict the percent of PF from the two independent variables MRI ADC and T1SI values and found a significant correlation between regression-predicted values and histologically-determined PF (r = 0.806, P < 0.001). The coefficients obtained from the regression equation allow the percentage of suspected fibrosis to be predicted (Fig. 4.1.2.1, Table 4.1.2.1).

For example, when the ADC and T1 SI values are used in the equation: 242.010 + (–115.869 × 1.245) + (–0.222 × 154.71) = 63%, i.e. predicted PF. The histological examination revealed a PF of 70% for the same patient.

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Fig. 4.1.2.1. The significant correlation between Unstandardized predicted

values (%) of fibrosis percent using the estimated regression equation and histologically determined PF (r = 0.806, P = 0.001)

Table 4.1.2.1. The Coefficients Obtained from the Regression Equation Model Unstandartized coefficients Standartized coefficients t Significance

B Standard Error Beta

Constant 242.010 23.602 10.254 0.000

ADC –115.869 21.839 –0.587 –5.306 0.000

T1 SI –0.222 0.089 –0.275 –2.487 0.016

ADC and T1 SI values are considered as significant independent covariates in predicting PF by means of the regression equation: PF = 242.010 + (–115.869 × ADC) + (–0.222 × T1 SI).

4.1.3. Association between CT imaging parameters and histological examination

In our research, we measured the HU value in CT images in porto-venous phase and compared the obtained results with a histological examination in order to determine the CT value for determination of PF degree. We didn’t found a significantly correlation between CT HU value and histologically-determined PF (R² = 0.021, P = 0.302) (Fig. 4.1.3.1).

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Fig. 4.1.3.1. Correlation between CT HU in portovenous phase

and histologically determined PF

There was a significant correlation between the volume of the pancreas and PF histological quantification (P = 0.002). The lower volume of the pancreas was related to a higher percentage of fibrotic tissue in pancreatic tissue (Fig. 4.1.3.2).

Fig. 4.1.3.2. CT volumetry of the pancreas imaging

55 y/o female with CP diagnosed 7 years ago, in CT volumetry we can see atrophic pancreatic tissue with dilated pancreatic duct and a ductal concrement. The volume of the

pancreas here is 8.9 mL (A). A male of 69 y/o with CP diagnosed 3 years ago, in CT volumetry there is an intact pancreatic tissue, the volume of the pancreas 93.4 mL (B).

COMPARISON OF RADIOLOGICAL, MORPHOLOGICAL AND FUNCTIONAL ASSESSMENT OF METABOLIC CHANGES AMONG PATIENTS WITH PANCREATIC DISEASES

Edita Bieliūnienė