Key words
ultrasound - adenocarcinoma - Crohn’s disease - strain ratio - ex-vivo
Introduction
Differentiation between benign and malignant tissue is a major challenge in medical
imaging. Tissue samples are usually necessary for a reliable diagnosis, but they may
be difficult to obtain. Pathological changes may alter tissue structure and hence
its elastic properties. New imaging modalities have been introduced as potential tools
for the discrimination between malignant lesions and benign tissue based on changes
in elasticity or strain distribution patterns [1]. Real-time elastography (RTE) is an ultrasound-based technique in which the distribution
of strain is mapped when soft tissue is exposed to repetitive stress. The method was
described by Ophir et al. and referred to as quasi-static elastography [2]. Several methods for registering strain or elasticity using different forces to
induce tissue deformation leading to recordable displacements or shear waves have
subsequently emerged [3]
[4]. These techniques have been used to characterize tissue in focal lesions, contracting
organs, such as the heart [5]
[6], stomach [7], and pancreas [8], or in liver fibrosis [9]
[10]. Hard tissue is characterized by low strain, while softer areas show higher strain.
RTE has shown promising results for differentiating between malignant and benign lesions
in breast lumps [11], prostate tumors [12]
[13], thyroid nodules [14]
[15]
[16], lymph nodes [17]
[18] and the pancreas [8]
[19]. Both malignant tumors and chronic inflammation may lead to the formation of soft
tissue fibrosis [20]
[21]. Fibrosis formation in malignant tumors is called desmoplasia and may contribute
to increased tissue hardness through several mechanisms involving tumor cells, stromal
cells and the extracellular matrix [22]. Malignant tumors are also usually characterized by increased interstitial pressure,
a feature that decreases quickly in devascularized tissue [23]
[24]
[25].
In this study, RTE was performed on surgically resected bowel specimens containing
benign or malignant lesions using one categorical classification system and two semi-quantitative
methods for relative strain assessment. Our aim was to evaluate whether RTE could
differentiate lesions caused by inflammation from malignant neoplastic lesions using
qualitative and semi-quantitative methods for strain assessment. Furthermore, we examined
whether strain ratio (SR) measurements were affected by changes in the elasticity
dynamic range (E-dyn). Finally, we also wanted to correlate elastography results to
a histological semi-quantification of fibrosis, inflammation parameters and tumor
description.
Material and methods
Patients referred for elective bowel resection were recruited for this feasibility
study from May 2008 to June 2009. All patients signed an informed consent to participate.
The study was approved by the Regional Committee for Medical and Health Research Ethics
in Western Norway and was conducted according to the Helsinki Declaration.
38 lesions from 27 patients were examined, 16 sections from Crohn’s stenoses in 9
different patients, 18 sections from adenocarcinomas in 16 patients and 4 sections
from adenomas in 3 different patients. One cancer patient had a synchronous adenoma
and another had two synchronous adenocarcinomas. Two adenomas were separate lesions
from the same patient. In one patient with a multilobular tumor, two different tumor
sections were included as separate lesions. In the Crohn’s disease specimens, separate
skip lesions as well as the proximal and distal end of some longer stenoses were included
as individual lesions.
The observer was not blinded to the indication for surgery, but the final diagnosis
was confirmed by histopathology after the examination and image evaluation.
Newly resected specimens were collected from the operating room, rinsed in water and
subsequently examined by ultrasound with RTE in a specially designated chamber. A
2-cm layer of hard paraffin wax covered the bottom of the chamber in order to limit
US reflections and to provide hold for the needles used for fastening the specimens.
A 1.5-cm layer of flexible agar (Agar No.1, 1 %, Oxoid Ltd, Basingstoke Hants, UK)
was placed on top of this to make a flexible and echo-free background for ultrasound
scanning. The scanning plane of a captured elastogram was marked on the specimen with
3 identically colored needles ([Fig. 1]). The specimen was then fixed in a 4 % formalin solution for 3 – 7 days before a
pathological examination was performed to obtain a histological confirmation of diagnosis.
Fig. 1 The surgical preparations were examined in a transparent plexiglass chamber with
the bottom covered with solid paraffin wax and a separate layer of 1.5 cm of 1 % agar.
The tissue was fixed to the agar layer with needles (yellow color) and the sections
examined with B-mode US and elastography were marked with three needles with the same
head-color (red, black, green) in order to define the position of the lesions and
the scanning direction.
Abb. 1 Die Untersuchung der chirurgischen Präparate erfolgte in einer transparenten Plexiglaskammer,
deren Boden mit einer festen Paraffinschicht und einer 1,5 cm dicken separaten Schicht
bestehend aus 1 % Agar bedeckt war. Das Gewebe wurde mit Nadeln (gelbe Farbe) auf
der Agarschicht fixiert und die mittels Graustufenultraschall und Elastografie untersuchten
Bereiche wurden mit drei gleichfarbigen Nadeln (Kopf in rot, schwarz, grün) markiert,
um die Position der Läsionen und die Messrichtung festzulegen.
Ultrasound equipment
A Hitachi Hi Vision 900 ultrasound scanner with software version V16 – 04 STEP 2 and
a L54 M linear probe with frequencies 9 – 13 MHz (Hitachi Medical Corporation, Tokyo,
Japan) were used in the study. The elastography modality was developed for free-hand
application, and small repetitive compression/decompression movements of the probe
were used to provide stress and induce tissue strain. With this method, relative strain
values in different areas are calculated based on the assumption that stress is evenly
distributed over the region of interest (ROI). A color map showing the strain in individual
image elements as compared to the mean strain of the whole ROI is produced. The color
distribution depends on the selected level of the E-dyn. A graphic indicator on the
display shows when there is sufficient strain to produce an elastogram. The applied
strain imaging algorithm records changes in echo positions between consecutive radio
frequency frames using the extended combined autocorrelation method (ECAM), which
is an extension of the original quasi-static elastography [26]
[27].
We used the following parameter settings for RTE: Frame reject: 6, noise reject: 4,
persistence: 2 – 5, smoothing: 2 – 3. The VAS and categorical scores were determined
using E-dyn level 4 (default level). SR examinations were also recorded at E-dyn levels
2 and 6. The frame rate varied between 9 and 14 depending on the selected ROI size.
Methods for strain assessment
We applied a categorical classification based on the color distribution within the
lesion as originally described for pancreatic lesions by Janssen et al. [28]. In this classification, three basic patterns of color distribution are recognized:
Type 1 is relatively homogeneous, type 2 has 2 or 3 different colors and type 3 has
a honeycomb pattern. The colors present are represented by the subsequent letters
usually indicating a declining proportion in the lesion: A = blue, B = green/yellow
and C = red. 32 lesions (92 %) were classified according to this system and fell into
five categories: 1B: homogeneously green, 2BA: green > blue, 2AB: blue > green, 1A:
homogeneously blue and 2 A-B-C: presence of blue, green and red without one of the
colors being classified as more prominent.
A visual analog scale (VAS) consisted of a 100-mm line where the observer marked the
assumed level of the lesion’s hardness as assessed by elastography at E-dyn level
4. If the lesion was regarded as equal to the surrounding tissue, the mark was set
at 50 mm. Lesions appearing harder were marked between 50 and 100 mm and softer lesions
were marked between 0 and 50 mm. Assessments using the VAS score and visual categorical
score were repeated by a trained observer blinded to the clinical data using video
clips of the elastography seen in Windows Media Player (Microsoft Inc., Ca, USA).
The results were compared to those obtained in the original scoring by the non-blinded
observer.
The third strain evaluation method was a semi-quantitative measurement of SR comparing
the reference tissue (B) and the lesion (A) using the following formula[]:
SR is a built-in scanner software function and was first introduced in breast scanning
and was called the “fat-lesion ratio”. It compares the mean strain in a user-selected
portion of the reference tissue with a similarly selected part of the lesion, assuming
that both areas have been subjected to the same amount of stress. We selected circular
areas of reference tissue at the same depth and with approximately the same size as
the lesion for all measurements ([Fig. 2]). In most cases, the central area of the lesion was selected for calculation (area
A). However, if the lesions had reproducible differences in tissue hardness, the hardest
area was selected as area A. Pericolic or mesenteric fat and connective tissue as
well as parts of the normal bowel wall could be included in the reference area. SR
measurements were performed on representative image frames with sufficient signal
quality. Measurements were usually repeated 3 times on E-dyn levels 2, 4 and 6, respectively,
thus yielding 9 SR measurements per lesion. However, only 3 SR measurements were recorded
in three lesions. (E-dyn scale: 1 – 8).
Fig. 2 Split-screen images showing B-mode ultrasound image on the right side and B-mode
image with elastogram on the left. The displayed colors represent the degree of strain
in the tissue covered by the region of interest. The strain ratio is calculated by
the mean strain in the reference tissue (area B) divided by the mean strain in the
lesion area (area A). The B-mode image is used for placing the areas A and B for strain
ratio calculation. Images represent: a: Adenoma (SR 1.58), b: Adenocarcinoma (SR 1.80) and c: Crohn's lesion (SR 3.73).
Abb. 2 Splitscreen-Darstellungen zeigen das B-Bild auf der rechten Seite und das B-Bild
mit Elastogramm zur Linken. Die dargestellten Farben stellen den Strain-Grad des Gewebes
in der „Region of Interest“ an. Die Strain-Ratio wird berechnet aus dem mittleren
Strain des Referenzgewebes (Bereich B) geteilt durch den mittleren Strain der Läsion
(Bereich A). Das B-Bild wird benötigt, um die Bereiche A und B zu platzieren und die
Strain-Ratio zu berechnen. Die Bilder stellen dar: a: Adenom (SR 1,58), b: Adenokarzinom (SR 1,80) und c: Crohn-Läsion (SR 3,73).
Pathological evaluation
One dedicated pathologist conducted all of the pathological examinations. The pathologist
was assisted by the person who performed the ultrasound to ensure the correct orientation
of tissue planes according to the previous US elastography examination during the
gross examination of the formalin-fixed specimens. Whole mount slices with a thickness
of 2 mm were taken according to the needle marks, inked for orientation and processed
by standard procedures. Haematoxylin-eosin-stained sections were investigated for
the following parameters of inflammation in cases of Crohn’s disease: ulceration,
granulation tissue, fissure ulcers, fistula, abscess, cryptitis, and lymphoplasmacytic
inflammation. A fibrosis score was determined in all cases. Lymphoplasmacytic inflammation
and fibrosis were graded semi-quantitatively as absent (0), slight (1), moderate (2),
or severe (3).
The fibrosis score was based on a semi-quantitative visual evaluation performed by
one pathologist. In order to validate this score, the same pathologist determined
a second score for all lesions one year after the first scoring. A Kappa score of
0.37 was obtained, indicating fair reproducibility of the fibrosis score.
Statistical analysis
The VAS scores were found to be within the limits of normal distribution for adenomas,
Crohn’s lesions and adenocarcinomas as tested with the Shapiro-Wilk test, and comparisons
between the groups were based on mean values with standard deviations in each group. When
evaluating SR differences, the median of repeated SR measurements in each lesion was
selected for further quantitative analysis. When comparing the three diagnostic groups
of lesions, the mean of these SR medians was calculated within each group. The median
SR results from Crohn’s lesions and adenocarcinomas were not distributed normally.
A log transformation was attempted, but did not yield a normal distribution. We had
included one to three separate lesions from the surgical specimens due to skip lesions
in Crohn’s lesions and some synchronous neoplastic lesions. Consequently, the untransformed
data were analyzed using a generalized estimating equations (GEE) model allowing analysis
of both related and unrelated observations. In this model, SR and VAS results for
Crohn’s lesions and adenomas were compared to the corresponding results for adenocarcinomas,
which served as a reference. For comparison of SR measurements at E-dyn levels 2,
4 and 6, we used a Spearman’s rho correlation and Wilcoxon-Mann-Whitney test for paired
differences between the medians of three recorded SR measurements at each E-dyn level.
The significance level was adjusted to apply for 3 measurements: p = 0.05/3 = 0.017. This
test also provided information about the repeatability of SR measurements in the individual
lesions.
The interobserver agreement for categorical scores was assessed using Cohen’s Kappa,
which expresses the agreement controlled for agreement by chance and is considered
a conservative measurement of interobserver agreement. The Kappa values were interpreted
in the following manner: 0 = no agreement, 0 – 0.20 = slight agreement, 0.21 – 0.40 = fair
agreement, 0.41 – 0.60 = moderate agreement, 0.61 – 0.80 = substantial agreement,
and 0.81–-1 = almost perfect agreement [29]. Continuous scores (VAS) were compared using correlation (Pearson’s) and limits
of agreement [30]. Statistical analyses were performed using SPSS 17 and IBM-SPSS 19 (Chicago, Ill,
USA).
Results
Categorical evaluation of strain images
The qualitative classification of color distribution in the elastogram showed reduced
strain in Crohn’s lesions as well as adenocarcinomas indicating increased tissue hardness
compared to surrounding tissue. Elastograms of an adenoma, an adenocarcinoma and a
Crohn’s lesion are shown in [Fig. 2a-c]. [Fig. 3] shows the distribution of qualitative strain image categories within each group
of lesions evaluated by observer 1. 29 of 33 scored cases were described as category
2AB or 2 BA.
Fig. 3 A categorical visual score based on the color distribution in the lesion of interest.
The bars indicate the number of lesions in each diagnostic group categorized by observer
1.
Abb. 3 Eine kategorische visuelle Bewertung aufgrund der Farbverteilung in der Läsion, die
von Interesse ist. Die Säulen geben die Anzahl der Herdläsionen in jeder diagnostischen
Gruppe an, kategorisiert durch Beobachter 1.
Semi-quantitative evaluation of strain images
The VAS evaluation showed no significant difference between Crohn’s lesions and adenocarcinomas,
while adenomas had a significantly lower VAS score indicating softer tissue. SR quantification
also could not demonstrate any difference between Crohn’s lesions and adenocarcinomas.
However, the four adenomas had a significantly lower SR than the adenocarcinomas.
The mean SR as well as median SR, intra-assay standard deviation, and VAS score for
each diagnostic group are summarized in [Table 1] and the GEE model details are listed for VAS and SR in appendices 1 and 2, respectively.
Appendix 1.
|
tissue strain by visual analog scale (VAS), n = 35
(GEE, linear model)
|
test of model effects
|
|
estimated marginal means
|
95 % confidence interval
|
coefficient
|
95 % confidence interval
|
p-value
|
|
diagnostic group
|
|
|
|
|
< 0.001
|
|
adenoma (n = 4)
|
59.8
|
(54.8, 64.7)
|
–15.472
|
(–23.910, –7.035)
|
|
|
Crohn’s lesion (n = 13)
|
81.6
|
(78.5, 84.8)
|
6.393
|
(–1.037, 13.823)
|
|
adenocarcinoma (n = 18)
|
75.2
|
(68.5, 82.0)
|
0
|
(reference)
|
Interobserver validation of categorical and semi-quantitative elastography
For the categorical evaluation, cine loops of 28 of the original 33 lesions were re-evaluated
by an observer blinded to the clinical data. Five cases were excluded from re-evaluation
because only still images were stored and not the original video files. A comparison
between observer 1 (original) and observer 2 (blinded) yielded a Kappa value of 0.38
for the categorical scale, indicating fair interobserver agreement. The second observer
also repeated the VAS scoring in 30 of the 35 cases that had previously been scored
by observer 1. There was a significant correlation between the two observers (Pearson’s
r = 0.55, p = 0.002). The correlation plot and limits of agreement between observer
1 and 2 are shown in [Fig. 4]. Moderately good correlations were found with better interobserver agreement and
correlation for higher values of VAS than for mid-range values.
Fig. 4 Correlation plot and limits of agreement plot of VAS score agreement between observer
1 and observer 2. Observer 2 was blinded to the clinical data. The agreement improved
with higher VAS scores.
Abb. 4 Korrelationsplot und „Limits of agreement“-Plot der Übereinstimmung von Beobachter
1 und 2 bei der VAS-Auswertung. Beobachter 2 kannte die klinischen Daten nicht (Verblindung).
Die Übereinstimmung verbesserte sich mit höherer VAS-Punktzahl.
Intraobserver validation of strain ratio measurements
The comparison between triplets of SR recorded in separate loops at different levels
of E-dyn is presented in [Table 2]. We found moderate to good correlations (Spearman’s rho = 0.47 – 0.82) between the
separate recordings with different E-dyn ranges. A comparison of the median SR showed
no significant differences between recorded SR measurements between E-dyn levels 2
and 6 or between E-dyn levels 4 and 6. However, there was a significant difference
in SR at E-dyn levels 2 and 4. The intra-assay standard deviations for all SR measurements
in individual lesions are listed according to diagnosis in [Table 1]. These values showed higher measurement variation for Crohn’s lesions than for adenocarcinomas.
Appendix 2.
|
tissue strain measured by strain ratio (SR), n = 38
(GEE, linear model)
|
test of model effects
|
|
estimated marginal means
|
95 % confidence interval
|
coefficient
|
95 % confidence interval
|
p-value
|
|
diagnostic group
|
|
|
|
|
< 0.001
|
|
adenoma (n = 4)
|
1.31
|
(1.20, 1.42)
|
–1.202
|
(–1.755, –0.649)
|
|
|
Crohn’s lesion (n = 16)
|
3.33
|
(0.79, 5.88)
|
0.821
|
(–0.783, 3.425)
|
|
adenocarcinoma (n = 18)
|
2.51
|
(1.95, 3.08)
|
0
|
(reference)
|
Assessment of fibrosis
The number of included patients, scanned sections, the macroscopic length and the
categorical fibrosis score for each of the three diagnostic groups are presented in
[Table 3]. There was no significant difference in the macroscopic length of Crohn’s stenoses
and the maximum length of the adenocarcinomas. The adenomas were smaller than the
adenocarcinomas and Crohn’s lesions. In two separate evaluations, one pathologist
visually categorized the fibrosis in 37 sections of the same lesions as 2 = “moderate
fibrosis” in 25 and 17 cases, respectively. Category 1 = “slight fibrosis” was used
7 and 13 times, respectively. The two separate evaluations with categorical fibrosis
scores 0 – 3 had a Kappa value of 0.37. In the Crohn’s lesions, the fibrosis score
indicated variations in fibrosis in different wall layers. The mean fibrosis scores
were as follows: submucosa –2.44, subserosa –1.75 and proper muscle –1.50.
Table 1
Mean and median strain ratio (SR) and visual analog scale for the three diagnostic
entities: adenoma, Crohn’s lesion and adenocarcinoma. The SR intra-assay standard
deviation is greater for Crohn’s lesions than for adenocarcinomas indicating a larger
measurement variation.
|
diagnosis
|
mean strain ratio[1] (SD)
|
median strain ratio (min, max)
|
SR intra-assay standard deviation
|
Mean VAS[2]
0 – 100 mm (SD)
|
|
adenoma (n = 4)
|
1.31 (0.58)
|
1.25 (0.68, 2.06)
|
0.35
|
59.8 (10.4)
|
|
Crohn’s lesion (n = 16)
|
3.33 (5.21)
|
2.09 (1.31, 22.75)
|
1.23
|
81.7 (6.3)
|
|
adenocarcinoma (n = 18)
|
2.51 (1.17)
|
2.18 (0.81, 5.34)
|
0.46
|
75.2 (13.3)
|
1 Mean SR: Crohn’s lesion vs. adenocinoma: p = 0.537. Mean SR: Adenoma vs. adenocarcinoma:
p < 0.001.
2 VAS: Crohn’s lesion vs. adenocarcinoma: p = 0.092. VAS: Adenoma vs. adenocarcinoma:
p < 0.001.
Assessment of inflammation in Crohn’s specimens
The patients diagnosed with Crohn’s disease all had pain as the main symptom with
stenotic findings in the small or large bowel confirmed by radiological imaging or
endoscopy. Eight of nine patients were non-responders to high-dose steroids or TNF-antagonists.
Two of the patients who underwent surgery in 2008 were reluctant to use TNF-antagonists
or prednisolone. Two patients who underwent surgery only 3 and 8 months after diagnosis
had suffered substantial weight loss of 10 and 15 kg, respectively. One Crohn’s patient
was operated on under the suspicion of cancer in the ascending colon, although histopathology
had not confirmed this prior to surgery. This patient had only received a 10-day course
of antibiotics prior to surgery. All Crohn’s lesions exhibited transmural inflammatory
changes. Lymphoplasmacytic inflammation was assessed as slight in 4/16 cases, moderate
in 8/16 cases and severe in 4/16 cases. Other inflammatory parameters were mucosal
ulcerations in 13/16 (81 %) cases, granulation tissue in 12/16 (75 %) cases, cryptitis
in 7/16 (44 %) cases, fissure ulcers in 4/16 (25 %) cases, fistulas in 3/16 (19 %)
cases and abscesses in 0/16 cases. Lesions with high overall inflammatory activity
did not differ from lesions with low overall inflammatory activity by fibrosis score.
Furthermore, there was no difference in SR or VAS between lesions with overall high
or low inflammatory activity. Inflammatory changes in the adenocarcinomas and adenomas
were not specifically evaluated. However, none of these specimens exhibited transmural
chronic inflammatory changes. Only one adenocarcinoma specimen also contained an area
of acute inflammation and abscess formation according to the original pathology report.
Strain evaluation by tumor stage and grade
The SR and VAS results were compared by tumor stage according to the TNM classification
for the 18 examined sections of malignant lesions. The results are summarized in [Table 4]. There was a tendency for a higher fibrosis score for tumors staged as T3 or T4. We
found no significant differences in SR and VAS between lesions staged as T3 or T4
compared to T1 and T2, but the included numbers were low. The tumor grade was described
as highly differentiated (> 95 % glandular structures) in 2 cases, moderately differentiated
(50 – 95 % glandular structures) in 9 cases and poorly differentiated (< 5 % glandular
structures) in 7 cases. When highly and moderately differentiated tumors were considered
as one group (n = 11) and compared to poorly differentiated tumors (n = 7), we found
no significant differences in the SR or VAS scores. The number of lesions given fibrosis
score 2 was 8/11 (73 %) for the highly and moderately differentiated adenocarcinomas
and 5/7 (71 %) for the poorly differentiated adenocarcinomas.
Table 2
Comparison of median strain ratio measurements of the same lesions with elasticity
dynamic range (E-dyn) levels 2, 4 and 6. The differences were not normally distributed.
The exact two-tailed Wilcoxon-Mann-Whitney test was used for the comparison of paired
differences. The significance level for differences was set to p ≤ 0.05/3 = 0.017
to adjust for multiple comparisons.
|
E-dyn levels
compared
|
Pairs
n
|
median SR (min-max)
|
Spearman’s correlation:
rho (p-value)
|
P-value of paired differences
|
|
E-dyn 4
E-dyn 2
|
34
|
2.25 (0.68 – 8.28)
1.74 (0.81 – 30.79)
|
0.47 (0.005)
|
0.008
|
|
E-dyn 4
E-dyn 6
|
35
|
2.25 (0.68 – 8.28)
2.06 (0.33–-22.75)
|
0.55 (0.001)
|
0.849 (n.s)
|
|
E-dyn 2
E-dyn 6
|
34
|
1.74 (0.81–-30.79)
2.06 (0.33 – 22.75)
|
0.82 (< 0.001)
|
0.022 (n.s)
|
Discussion
Differentiation between neoplastic and inflammatory lesions by RTE
RTE demonstrated low strain indicating harder tissue in resected lesions from both
colorectal carcinomas and Crohn’s disease. The hypothesis that strain imaging could
differentiate malignant neoplastic lesions from benign lesions caused by inflammation
was based upon observations that malignant tumors in other organs tended to become
harder than benign lesions [14]
[19]
[31]
[32]
[33]. Pathologists frequently observe increased fibrosis within and in the vicinity of
malignant lesions. A similar process also occurs in chronic inflammation in which
case fibroblasts and possibly other cells produce collagen-rich interstitium resulting
in scar tissue [34]. Such fibrogenic activity seems to be a dominant feature in stenotic lesions of
Crohn’s disease. A second contributor to the hard appearance of malignant tumors may
be high interstitial pressure [23]
[25]
[35]
[36], which decreases quickly after devascularization. Hence, its probable contribution
to differences in tissue elasticity was not measureable in our study on ex-vivo tissue
specimens. Crohn’s disease is usually subjected to surgery because of intractable
stenosis not responding to medical therapy. All Crohn’s lesions in our study had extensive
fibrosis, as demonstrated by histology. Hence, increased tissue hardness was visualized
in both Crohn’s lesions and adenocarcinomas. However, it was not possible to detect
any strain differences between benign transmural lesions caused by chronic inflammation
and malignant tumors.
Strain evaluation and fibrosis in adenocarcinomas
The number of adenocarcinomas in the study was limited. Hence, we could not find a
statistically significant difference in SR or VAS score, but there was a tendency
to increase the fibrosis score and SR with increasing tumor stage. With respect to
tumor grading, we would expect poorly differentiated tumors to have a higher fibrosis
score. However, no significant strain differences were found, even if both the SR
and VAS scores showed an insignificant tendency towards higher strain for poorly differentiated
tumors. The low number of lesions included may be the cause for this (type 2 error),
and larger studies are warranted.
Strain assessment
A majority of published papers on RTE have used visual categorical scoring based on
color distribution in the lesion as the main strain evaluation tool. In this study,
we applied a categorical classification based on a distribution pattern and predominance
of separate colors within the lesions. This classification was introduced by Janssen
et al. [28] for the evaluation of pancreatic lesions using endoscopic ultrasonography, and up
to five of the original categories were used in our study. The qualitative scoring
system could not reliably separate the diagnostic entities in this study. Observer
1 classified 50 % of the adenomas, 50 % of the Crohn’s lesions and 35 % of the adenocarcinomas
as 2BA, indicating more green than blue. Category 1B, with a homogeneously green presentation,
was only used for benign lesions, but only 3 of 16 benign lesions were categorized
as 1B. The interobserver reproducibility of categorical scoring was fair (Kappa: 0.38)
when the cine loops were re-evaluated by a second observer.
Elastograms were also evaluated with a continuous VAS score, anticipated to be more
fine-grained than the categorical score, and comprising an aspect of strain comparison
between the lesion and the surrounding reference tissue. Because none of the lesions
were perceived as softer than the reference tissue, the lower half of the scale was
not utilized. VAS evaluation had a moderate correlation (r = 0.55) between the original
and an observer blinded to clinical data. The difference between the observers was
larger for low scores (moderately hard and mixed lesions) than for high scores (harder
lesions) ([Fig. 4]). This coincides with an observation that two observers frequently scored the same
lesions as 2AB (inhomogeneous, blue > green) and 2BA (inhomogeneous, green > blue),
correspondingly, on the categorical scale.
The SR is based on a computer-based comparison of mean strain in two selected areas,
and may thus seem to be more objective than the VAS and the categorical scale. However,
the selection of calculation areas is still user-dependent, and the recorded strain
may be subject to significant variations especially in heterogeneous reference areas.
Uneven application of stress within the elastogram ROI may also influence the SR results.
Hence, SR evaluation is usually quoted as a semi-quantitative method. The SR data
([Table 1]) demonstrates a large spread in strain measurements within the groups of Crohn’s
lesions and adenocarcinomas. The variation between cases in the same group may express
measurement variation and also reflect a biological diversity in tissue strain for
that entity.
Reproducibility of SR measurements
The E-dyn level of RTE influences the color presentation of strain distribution profoundly
[37]. Consequently, it is crucial to keep this setting on a standardized level for the
visual comparison of strain images in different cases. Median SR recordings at E-dyn
4 vs. 6 and E-dyn 2 vs. 6 were not significantly different. A significant difference
was found for the SR recorded at E-dyn 2 vs. 4. This was an unexpected and probably
unreliable result since such a difference was not found with a larger E-dyn span between
2 and 6. We have previously shown that SR measurements are independent of E-dyn level
in a standardized liver-tissue-mimicking phantom [38]. Therefore, we interpreted the observed SR measurement variability as the result
of the general variability of this method in separate recordings of the same lesions
using a free-hand application, and not as a consequence of the selection of different
levels of E-dyn. More dedicated studies may be required to confirm the relationship
between E-dyn level and SR measurements in biological tissues.
Relevance
The differentiation between colorectal tumors and Crohn’s lesions predominantly in
the small intestines may seem to have limited clinical value. However, these lesions
were chosen as a model for lesions caused by benign inflammation and malignant neoplasms
in the bowel wall. We selected Crohn’s lesions because they are transmural and expand
the bowel wall in a similar manner to neoplasias. Rustemovic et al. have studied patients
with ulcerative colitis and compared them to patients with Crohn’s disease and healthy
controls in vivo [39]. They found a significantly higher SR in Crohn’s patients than in ulcerative colitis
patients with active rectal disease. In the case of Crohn’s disease, the patients
with active disease also had a significantly higher SR than those in remission, indicating
a substantial contribution from increased interstitial pressure. In the case of ulcerative
colitis, the inflammation is limited to the mucosa and submucosa and does not create
the transmural inflammation and fibrosis characteristic for Crohn’s disease. From
our point of view, the contribution of endoscopic RTE in the differentiation between
Crohn’s disease and ulcerative colitis may not provide more than visualization of
the transmural inflammation resulting in bowel wall thickening clearly visible by
B-mode EUS. However, further studies may investigate whether a possible clinical application
of EUS-elastography may be the evaluation of polyps and pseudo-polyps in ulcerative
colitis, and whether a thickened bowel wall with increased tissue hardness may indicate
the development of malignancy in a patient with ulcerative colitis under surveillance.
Very few adenomas were included in this study, as the main object was to evaluate
Crohn’s lesions and adenocarcinomas. However, we found that adenomas appeared to have
higher strain and, correspondingly, lower strain ratios than both adenocarcinomas
and Crohn’s lesions. The difference in strain properties between adenomas and adenocarcinomas
in the rectum is clinically relevant, and endorectal ultrasonography (ERUS) with elastography
can potentially be a valuable preoperative diagnostic tool [33]. ERUS is currently acknowledged as the best method for selecting patients for minimally
invasive rectal surgery such as endoscopic mucosal resection or transanal endoscopic
microsurgery [40]
[41]. The preoperative classification of early rectal adenocarcinomas and adenomas remains
challenging and there is still a need for improved methods in the staging of these
tumors.
Limitations of the study
This study was performed on surgically resected specimens, in which the contribution
of interstitial pressure as a source of tissue hardness is eliminated. Hence, the
ex-vivo setup may underestimate tissue hardness in vascularized tissue. However, all
surgical specimens were examined under the same conditions, and the contribution from
interstitial pressure should be reduced in a similar way for all samples. A second
limitation is that the primary observer was not blinded to the clinical diagnosis.
A second observer, blinded to clinical data, re-evaluated the elastography and B-mode
video loops. The second evaluation was performed during a short time span (days) while
the original scoring was performed after each examination spanning a period of 12
months. This may have caused some drifting in visual scoring over time, and may partly
explain the moderate agreement for VAS and the fair agreement for the categorical
score. The low number of adenomas included in this study (4) limits the validity of
the statistical difference found between adenomas and adenocarcinomas.
Conclusion
Free-hand RTE of newly resected surgical specimens could not differentiate stenotic
lesions caused by chronic inflammation in Crohn’s disease from adenocarcinomas on
the basis of differences in semi-quantitative strain measurements. The SR and VAS
score showed significant strain differences between adenocarcinomas and a limited
number of adenomas. Pathological evaluation showed increased tissue fibrosis in both
adenocarcinomas and Crohn’s lesions. The interobserver reproducibility of the visual
evaluation using a VAS score and categorical scale was fair to moderate. The intra-assay
reproducibility of SR was more reliable, but the potential influence of E-dyn settings
on SR measurements could not be unequivocally affirmed. No significant differences
in strain assessment were found on the basis of inflammation grade in Crohn’s lesions
and by tumor stage or grade in adenocarcinomas. Ultrasound elastography is a promising
clinical method, but increased strain may be observed in malignant and benign lesions
and further studies on strain data of different lesions in vivo are warranted.
Abbreviations
RTE:
real-time elastography
SR:
strain ratio
E-dyn:
elasticity dynamic range
VAS:
visual analog scale
Appendix
Table 3
Descriptive data for the diagnostic entities adenocarcinoma, Crohn’s lesion and adenoma
including the number of patients, number of lesions, macroscopic length and number
of lesions categorized using the 0 – 3 fibrosis score by the pathologist. One patient
had both an adenocarcinoma and an adenoma and was counted twice. A total of 27 patients
were included.
|
diagnosis
|
patients
|
lesions
|
macroscopic length (min-max) (cm)
|
fibrosis score (0 – 3)
|
|
Score
|
n
|
|
adenocarcinoma
|
16*
|
18
|
3.75 (1.3 – 7.5)
|
Score 0
|
0
|
|
Score 1
|
5
|
|
Score 2
|
13
|
|
Score 3
|
0
|
|
Crohn’s lesion
|
9
|
16
|
3.50 (2.0 – 15.0)
|
Score 0
|
0
|
|
Score 1
|
2
|
|
Score 2
|
10
|
|
Score 3
|
4
|
|
adenoma
|
3
|
4
|
2.35 (1.0 – 3.5)
|
Score 0
|
2
|
|
Score 1
|
0
|
|
Score 2
|
2
|
|
Score 3
|
0
|
Table 4
Fibrosis score, strain ratio and VAS score for adenocarcinomas according to tumor
stage.
|
adenocarcinomas,
T-stage
|
n
|
fibrosis score = 1
|
fibrosis score = 2
|
median SR
(min-max)
|
VAS score
(mean, SD)
|
|
T1
|
2
|
1
|
1
|
1.73 (1.62 – 1.84)
|
62.5 (4.95)
|
|
T2
|
2
|
1
|
1
|
1.56 (0.81 – 2.31)
|
72.0 (16.97)
|
|
T3
|
8
|
3
|
5
|
3.00 (1.19 – 5.34)
|
81.1 (11.05)
|
|
T4
|
6
|
0
|
6
|
2.41 (1.59 – 3.18)
|
72.2 (15.29)
|
