Effect of Aging and Diffuse Chronic Pancreatitis on Pancreas Elasticity Evaluated using Semiquantitative EUS Elastography

Abstract

Purpose: Endosonographic elastography has been introduced as a method of estimating the stiffness of pancreatic tumors. This prospective single-center study was conducted to evaluate changes in the stiffness of the pancreas related to age and diffuse chronic pancreatitis.

Patients and Methods: 46 individuals each up to age 60 (group 1) and over age 60 (group 2) with healthy pancreata and 26 patients with diffuse chronic pancreatitis (group 3) were included. Three elastograms were obtained in each case by endosonography in a defined section through the pancreatic body. Elastograms were further evaluated by histogram analysis. Mean strain values, based on a range from 0 (hardest) to 255 (softest), and their standard deviation were calculated from the histogram. The three groups were compared statistically with regard to pancreatic stiffness. A cut-off level for the diagnosis of chronic pancreatitis was defined by testing receiver operating characteristics curves.

Results: The strain values (mean, SD) measured in the pancreatic body in groups 1 – 3 were 110.2 (23.9), 80.0 (16.4), and 32.4 (11.9), respectively. Pairwise comparison of the groups revealed highly significant differences (p < 0.001). At a cut-off value of 50, the area under the curve was 0.993 for distinguishing between chronic pancreatitis and healthy pancreata in people aged over 60.

Conclusion: Semiquantitative elastography shows that pancreata become significantly harder during aging, but remain softer than in chronic pancreatitis. A cut-off value of 50 is suggested as a possible diagnostic criterion for diffuse chronic pancreatitis.

Zusammenfassung

Ziel: Studien zur endosonografischen Elastografie haben sich bislang überwiegend mit der Charakterisierung von Tumoren beschäftigt. In dieser monozentrischen prospektiven Studie haben wir untersucht, ob die Elastografie Änderungen der pankreatischen Gewebshärte infolge des Alterns und diffuser chronischer Pankreatitis nachweisen kann.

Patienten und Methoden: 46 Pankreasgesunde bis einschließlich 60 Jahre (Gruppe 1) oder über 60 Jahre (Gruppe 2) und 26 Patienten mit diffuser chronischer Pankreatitis (Gruppe 3) wurden untersucht. Bei jedem Probanden wurden drei Elastogramme eines endosonografischen Längsschnittes durch das Pankreaskorpus angefertigt. Mittels Histogrammanalyse wurde der mittlere Dehnungswert mit Standardabweichung bestimmt, wobei 0 die niedrigste und 255 die höchste Dehnbarkeit repräsentiert. Die drei Gruppen wurden auf Unterschiede der Dehnungswerte getestet. Mittels ROC (receiver operating characteristics)-Kurven wurde der beste cut-off-Wert für die Diagnose einer chronischen Pankreatitis ermittelt.

Ergebnisse: Die mittleren Dehnbarkeitswerte (Standardabweichung) waren in Gruppe 1 110,2 (23,9), in Gruppe 2 80,0 (16,4) und in Gruppe 3 32,4 (11,9). Alle Gruppen waren im paarweisen Vergleich signifikant verschieden (p < 0,001). Im Vergleich der Ergebnisse bei chronischer Pankreatitis mit den härteren Pancreata der älteren Gesunden zeigte die ROC-Kurve bei einem Wert unter 50 das größte Areal unter der Kurve (0,993).

Zusammenfassung: Semiquantitative Elastografie mittels Histogrammanalyse kann nachweisen, dass gesunde Pankreata mit zunehmendem Alter härter werden, aber weicher bleiben als bei chronischer Pankreatitis. Ein Dehnungswert unter 50 könnte ein diagnostisches Kriterium der chronischen Pankreatitis werden.

Key words

elastography - chronic pancreatitis - aging - endosonography

Introduction

The pancreas is a prime target for the use of endosonography. With a gastric and duodenal approach, the complete pancreas can be examined at high ultrasound frequencies, resulting in the best spatial resolution available to date for imaging of the organ. Endoscopic ultrasonography (EUS) is therefore the method of choice for visualizing small pancreatic tumors [1] [2], detecting early signs of chronic pancreatitis [3], or obtaining biopsy material under EUS guidance [4] [5] [6]. EUS now offers not only B-mode imaging, but even more sophisticated applications such as sensitive color Doppler EUS, contrast-enhanced EUS [7] [8], and endosonographic elastography [9] [10] [11].

EUS elastography makes it possible to differentiate between hard and soft tissue. Since malignant lesions often become stiffer due to their desmoplastic behavior, elastography has been used to diagnose malignant lymph nodes [12] [13] [14] and focal pancreatic lesions [9] [10] [11] [15] [16] [17], with promising results. It is known that chronic inflammation of the pancreas also induces fibrogenesis, resulting in fibrotic transformation and hence in changes in the mechanical properties of the tissue [18] [19]. Similar but less distinctive histologic changes have been reported for the pancreas in the course of aging [20].

Most studies on elastography published to date have concentrated on diagnosing malignant disease, and little is known about changes in the mechanical properties of tissue that result from aging and chronic inflammation.

This study was initiated for basic research of the properties of semiquantitative EUS elastography. The aims were to test firstly whether physiologic age-related differences in the stiffness of the healthy pancreas can be detected using this technique, and secondly whether the stiffness of pancreata with endosonographically probable diffuse chronic pancreatitis is significantly different from that of healthy pancreata. The answer to these questions could provide basic information for the potential use of elastography in the diagnosis and follow-up of chronic pancreatic disease.

Methods

Patients

All patients undergoing EUS in our department between April 2011 and September 2012 were screened for suitability to join the present study. When exclusion criteria were absent, they were asked to participate in the study. The participants signed informed consent forms for both the EUS examination and the study. The study was approved by the local ethics committee at the University of Witten/Herdecke.

Inclusion and exclusion criteria

Group 1 included individuals aged 18 – 60 with healthy pancreata. Any history of pancreatitis or focal pancreatic disease or any alcohol intake above 40 g/day in men or above 20 g/day in women, except on rare occasions, was regarded as an exclusion criterion.

Group 2 was defined in the same way as group 1, but only including individuals over the age of 60.

Group 3 consisted of patients with diffuse chronic pancreatitis that was known from the medical history. The patients had to have at least 4 of 11 positive criteria on EUS according to Catalano et al. [21] to support a diagnosis of chronic pancreatitis. Focal hypoechoic or anechoic lesions identified at the EUS examination were regarded as an exclusion criterion. The estimated lifetime amount of alcohol intake was recorded.

Any conditions that might impair elastographic recording as a result of shock-absorbent substances interposed between the scanner and the aorta, such as ascites, represented general exclusion criteria in all of the groups.

EUS examination

EUS elastography was performed with a longitudinal echoendoscope (Pentax FG 3870 UTK; Pentax Europe Ltd., Hamburg, Germany) along with the Hitachi Preirus ultrasound unit (Hitachi Medical Systems Europe, Ltd., Zug, Switzerland). All EUS examinations were performed as described by the same examiner (JJ).

Each examination was performed with the patient under conscious sedation and with adequate monitoring and was started in accordance with the given indication. The study-related examinations were carried out either during or at the end of the procedure, to ensure the shortest examination time possible.

The common software installed in the ultrasound unit was used for histogram analysis of the elastograms. In a pilot series, it was found that the best elastographic recordings could be obtained by scans from the posterior gastric wall in a section through the pancreatic body, the superior mesenteric artery, and the aorta. The frame rejection was set to the maximum value of 7 to exclude measurements impaired by artifacts. If no recordings were obtained with this preset, the frame rejection was reduced to a value that would allow elastographic measurements.

Elastography does not provide absolute values for tissue stiffness, due to the lack of calibration based on reference tissue with known stiffness. The elastographic sector was therefore maximized around the pancreatic body to include as much surrounding connective or fatty tissue as possible in order to establish the best possible standardization ([Fig. 1]). This procedure corresponds to the one recently proposed in the EFSUMB guidelines [22] [23].

Fig. 1 a–c Examples of histogram analysis of elastograms obtained from healthy pancreata in younger a and older b people and from patients with chronic pancreatitis c.

Abb. 1 a–c Repräsentative Histogrammanalysen von Elastogrammen bei jungen Pankreasgesunden a, älteren Pankreasgesunden b und Patienten mit diffuser chronischer Pankreatitis c.

After an elastogram had been obtained, the next step was to define a maximal rectangular area within the pancreatic body for histogram analysis ([Fig. 1]). The strain values for each pixel in this rectangle are assigned to 256 grades of stiffness on the x-axis of the histogram, with 0 representing the lowest and 255 representing the highest strain value. The y-axis displays the number of pixels according to each grade of stiffness. The mean and standard deviation (SD) for the distribution of the grade of stiffness are calculated by the software automatically. To compensate for possible variability, three elastograms were obtained in each case.

Statistics and sample size calculation

In a pilot study, the following mean values for stiffness were obtained in the histogram: 100 for healthy individuals up to 60 years of age, 86 for healthy individuals older than 60, and 60 for patients with diffuse chronic pancreatitis. On the basis of these data, the number of individuals needed to test for a difference between the three groups with a power of 90 % was n = 46 each for groups 1 and 2 and n = 26 for group 3.

Each histogram describes the mean stiffness within the square area expressed by the mean and its standard deviation. To determine the mean for the three elastograms obtained in one examination, a weighted mean had to be calculated for each case based on the mean and standard deviation of each of the three histograms. For group comparison, the weighted means were compared using the one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test for pairwise comparison. The significance level was set as p < 0.05. Pearson’s correlation test was applied to display the correlation between age and tissue stiffness for all healthy individuals from group 1 and group 2. The receiver operating characteristic (ROC) curve was used to evaluate the best cut-off value for distinguishing between patients with chronic pancreatitis and those with healthy pancreata. Statistical analysis was performed with the software STATISTICA Version 10.0 (StatSoft Inc, Tulsa, USA).

Results

Patients were recruited for the study consecutively as described above. After the EUS examination, 11 individuals had to be excluded from participation due to the following findings: cystic lesions (n = 4), hypoechoic focal disease (n = 2), and less than four EUS criteria for chronic pancreatitis in group 3 (n = 5). Recruitment was continued until all groups were completed. Indications for EUS of study participants were the following: Group 1: Examination of the biliopancreatic system (n = 33), evaluation of mural gastrointestinal masses (n = 5), fine-needle biopsy of lymph nodes (n = 6), other (n = 2). Group 2: Examination of the biliopancreatic system (n = 31), evaluation of mural gastrointestinal masses (n = 8), fine-needle biopsy of lymph nodes (n = 4), other (n = 3). Group 3: Evaluation of chronic pancreatitis (n = 26). The patient characteristics are shown in [Table 1].

Table 1 Characteristics of the three study groups.

	group 1 healthy, ≤ 60 y	group 2 healthy, > 60 y	group 3 chronic pancreatitis
patients (n)	46	46	26
mean age (SD)	44.2 (12.1) y	73.8 (7.9) y	55.4 (14.8) y
gender (male/female)	10/36	18/28	18/8
sum of positive CP criteria	0	0	7.0 ± 1.5
parenchymal criteria	0	0	4.1 ± 0.7
ductal criteria	0	0	3.0 ± 1.5
alcohol intake men > 40 g/day women > 20 g/day	n = 0 n = 0	n = 0 n = 0	n = 18 n = 4

CP, chronic pancreatitis

The patients with chronic pancreatitis in group 3 had 7.0 (1.5) positive EUS criteria (mean, SD), consisting of 4.1 (0.7) parenchymal and 3.0 (1.5) ductal criteria. Twenty-two patients were suffering from alcoholic chronic pancreatitis, with an estimated alcohol consumption of more than 40 g in men or 20 g in women per day for more than 5 years; four female patients had hereditary or idiopathic chronic pancreatitis. None of the patients had total pancreatic atrophy or large stones that would have made EUS elastography of the pancreatic body impossible.

All of the recordings were made at the maximal frame rejection of 7, except for one in a patient with chronic pancreatitis (frame rejection 5). The frame of the elastogram always included a wide area around the pancreatic body, providing reference tissue. In each case, three recordings were obtained for histogram analysis as described above.

The gender distribution among the three groups was not homogeneous ([Table 1]). Groups 1 and 2 consisted predominantly of women, but group 3 mainly included men. When the results of men and women within each group were compared, no significant differences in the mean strain value were found ([Table 2]). It therefore seems legitimate to assume that gender does not have a significant influence on tissue stiffness.

Table 2 Strain values in the three study groups, shown as means with standard deviation (SD). Total groups are statistically different in pairwise comparison (p< 0.001). Gender does not show any effect on the strain values.

strain values	group 1 healthy,≤ 60 y	group 2 healthy, > 60 y	group 3 chronic pancreatitis
total group	110.2 (23.9)	80.0 (16.4)	32.4 (11.9)
males	112.4 (27.0)	82.8 (16.0)	31.6 (11.1)
females	109.6 (22.9)	77.8 (17.2)	34.1 (13.4)

The mean stiffness values (mean, SD) for the pancreatic body in individuals up to age 60, over 60, and in patients with chronic pancreatitis were 110.2 (23.9), 80.0 (16.4), and 32.4 (11.9), respectively ([Table 2]). Among the 26 patients with chronic pancreatitis, nine patients (35 %) had a value below 25. [Fig. 2] illustrates the results in the form of a box plot.

Fig. 2 Box plot of the mean strain values comparing the three study groups.

Abb. 2 Boxplot-Diagramm der mittleren Dehnungswerte der drei Studiengruppen im Vergleich.

Pairwise comparison of the three groups using the one-way ANOVA followed by the LSD test showed that patients with chronic pancreatitis had significantly harder pancreata than people with healthy organs (p < 0.001). Among individuals with healthy pancreata, the stiffness was significantly higher in people over the age of 60 (p < 0.001).

Pearson’s correlation test for all healthy individuals from group 1 and group 2 shows that there seems to be a correlation between age and the stiffness of the healthy pancreas (r = -0.59). The scatterplot, however, reveals that there is substantial individual scattering ([Fig. 3]).

Fig. 3 Pearson’s correlation test of all healthy individuals from group 1 and group 2. Although there seems to be a certain correlation between age and the mean strain value (r = -0.59), the results show substantial individual scattering.

Abb. 3  Pearson’s Korrelations-Test aller Pankreasgesunden aus Gruppe 1 und Gruppe 2. Obwohl eine gewisse Korrelation zwischen Alter und Gewebshärte zu bestehen scheint (r = –0,59), ist die individuelle Streuung der Ergebnisse doch erheblich.

The ROC curve showed an area under the curve of 0.993 at a cut-off value of 50 for the mean strain value to distinguish between chronic pancreatitis and healthy pancreata in people over the age of 60. Individuals up to age 60 were excluded from this analysis, because they showed no overlap with patients suffering from chronic pancreatitis.

Discussion

This study shows that the effects of aging on the pancreas, previously reported in a histological study [20], are also reflected in semiqantitative pancreatic elastography. Detlefsen et al. [20] found that the amount of fibrotic tissue in the pancreas increases during the course of aging and is significantly higher in healthy organs of people over the age of 60 in comparison with individuals aged up to 60 years. Using the same age classes, the present study shows that healthy pancreata in older people are stiffer than those in individuals up to the age of 60. Nevertheless, the effect of aging is not uniform in the study population, since the scatterplot of the correlation test displays substantial individual differences ([Fig. 3]).

Former studies comparing histologic fibrosis of the pancreas and the number of B-mode EUS criteria for chronic pancreatitis found a low but statistically significant [24] or excellent [25] correlation. A recent study by Schrader et al. [26] reported that there was only poor correlation between the amount of pancreatic fibrosis and the elastographic findings in malignant pancreatic tumors. This finding might also be true for benign changes. In this context, it is important to consider that it is not only the amount of fibrosis that may be decisive for the stiffness of the tissue, but its structural arrangement as well. Unfortunately, direct comparison between the degree of fibrosis and the mean strain value was not possible in this study due to a lack of histological samples.

In relation to routine EUS, the most interesting finding in the present study may be the fact that the strain values for pancreata with diffuse chronic pancreatitis are significantly lower than those in healthy organs, even in older persons. The best cut-off level derived from the ROC curve for diagnosing chronic pancreatitis was 50. The study was not designed to investigate the correlation between the number of positive B-mode criteria and the strain value. This issue may be of clinical interest, also regarding patients with less than 4 positive criteria. For analysis more patients would have to be included.

In comparison with the pilot series, the mean strain value in patients with chronic pancreatitis was lower than expected. Nine patients (35 %) had values below 25, which in our experience are also found in patients with ductal adenocarcinoma of the pancreas. The only data published to date for histogram analysis of pancreatic elastograms were reported by Săftoiu et al. [10], who investigated focal pancreatic lesions. They used an inverse scale on the x-axis, with 0 representing the relatively softest tissue and 255 the hardest tissue. At a cut-off value of 175, above which the presence of a malignant tumor was assumed, they achieved a sensitivity of 93.8 % and a specificity of 63.6 % for the diagnosis of pancreatic malignancy. Shifting the cut-off level up to 190 caused the sensitivity to drop to 56.3 % and the specificity to increase to 90.9 % [10]. Transferring these cut-off levels to the scale used in the present study — i. e., values under 80 and 65, respectively — would mean that the results for all the patients with chronic pancreatitis in this study were within the range of malignancy. This raises questions regarding the usefulness of elastography for decision-making on potential malignancy.

It was decided to measure the tissue stiffness of the pancreas in a well-defined section through the pancreatic body to optimize and standardize the conditions for elastography as described above. Of course, this practice can only be correct if the parenchymal changes are diffusely distributed over the whole organ and the section chosen is therefore representative. In this scanning position, the aorta as the impulse generator is located directly opposite the scanner, with the pancreatic body in between. Using the maximal frame rejection is therefore certain to avoid recordings impaired by artifacts as much as possible. Earlier studies on elastography have used the lowest frame rejection of 1 to allow the recording of continuous elastography loops [9] [11] [16]. This practice often causes an impression of clouds coloring the image by chance, instead of producing reproducible elastograms, and has been criticized previously [27]. Despite our efforts to provide standard conditions, elastography without absolute calibration by tissue of known stiffness will always be impaired by subjective aspects like the choice of the elastogram sector and by the individual stiffness of the tissue around the pancreas. To overcome at least the intraindividual variabilities, we performed three measurements in each patient.

For further research, it would be of interest to study the diagnosis and outcome in patients with a mean strain value of 40 – 60, but with less than four B-mode criteria for chronic pancreatitis, in order to evaluate the usefulness of elastography for early diagnosis of chronic pancreatitis. The latter group was not admitted to this study, but no individual in groups 1 and 2 had to be excluded because of positive B-mode criteria. This indicates that positive B-mode criteria are rare events in healthy individuals. A second topic for further research is the use of semiquantitative elastography for intraindividual follow-up in patients with diffuse disease of the pancreas — e. g., idiopathic chronic pancreatitis or autoimmune pancreatitis.

The main limitation of the present study is its single-center design. The results, especially the diagnostic evaluation of chronic pancreatitis, require further testing in a prospective multicenter study. As mentioned above, the inhomogeneous gender distribution among the three groups is of no importance.

In conclusion, semiquantitative elastography can reveal changes in the mechanical properties of the pancreas caused by aging and chronic pancreatitis. The cut-off strain value of 50 may be useful in the diagnosis of chronic pancreatitis if focal disease has been excluded. The results could serve as the basis for further research.

Acknowledgements

The authors are grateful to Dr. Erich Reese for his support in statistical analysis.

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