Study design, patient population and demographics
The study protocol of our prospective study was approved by our institutional review board and informed consent was obtained from the patients before the examination. Between August 2014 and July 2015, 23 patients (13 females and 10 males; mean age, 63.2 ± 7.9 years [range: 50–79 years]) with detection of a mass suspicious of PDAC in a previous CT and/or MRI and scheduled surgery with potentially curative intent were consecutively enrolled in our study. In all patients, indication for surgery had been made prior to and independently from the present study. The patients were scanned and evaluated prospectively, first with IVIM DW MRI, and second with CT perfusion, on the day before surgery.
Exclusion criteria were: general contraindications for the application of iodinated contrast agents, MR-unsafe foreign bodies, previous treatment for pancreatic carcinoma, inability to reproduce the breathing technique (see below), and/or denial of consent.
There was no histologically confirmed diagnosis at the time of inclusion in our study.
Out of the 23 total patients, the final histopathological diagnosis was PDAC in 20 patients, PNEN in one patient, anaplastic carcinoma in one patient, and mass-forming chronic pancreatitis in one patient. The three patients with histopathological diagnosis other than PDAC were excluded from our study. In one patient with PDAC the tumor area was missed in CT perfusion, therefore this patient was also excluded. CT perfusion and MRI diffusion could be evaluated in all remaining 19 patients. In 16 of these 19 patients, the tumor was located in the pancreatic head while 3 patients had a tumor in the pancreatic body and/ or tail. Among these 19 patients, resection was not possible and/or indicated due to infiltration of the superior mesenteric artery (SMA) in 1 patient, due to peritoneal metastases in 2 patients, and due to hepatic metastases in 1 patient. In these 4 patients in whom tumor resection was not performed, histological diagnosis was established from intraoperative biopsies from the part where the tumor infiltrated the SMA, or from the peritoneal/ hepatic metastases, respectively. In all 15 other patients, histological diagnosis was established from the resected primary tumor.
MR imaging and Post-processing
MR imaging was performed using a 1.5 T scanner (Magnetom Avanto, Siemens Medical Solutions) with a 6-element body-phased array coil and a 24-channel spine array coil. The pancreatic MR imaging protocol consisted of anatomic imaging sequences and diffusion weighted imaging (DWI) with 9 b-values (0, 50, 100, 150, 200, 300, 400, 600, and 800 s/mm
2, summarized in Table
1). This combination of b-values was chosen since it had proven feasible for assessment of tissue perfusion in correlation to MVD in PDAC [
19]. b-Values larger than 800 s/mm
2 were not chosen to minimize the kurtosis effect that becomes increasingly important at larger b-values [
21].
Table 1
Parameters of MR imaging
1) Anatomic MR imaging (performed in every patient) |
T1-weighted in/opposed phase | Inspiratory breath-hold | Upper abdomen | Transverse | 115 | 2.27 and 4.78 | 320 × 272 | 5 / 1 | 445 | |
HASTE-IR T2-weighted | Inspiratory breath-hold | Upper abdomen | Coronal | 1000 | 80 | 256 × 230 | 6 / 0.6 | 545 | |
HASTE T2-weighted | Expiratory breath-hold | Upper abdomen | Transverse | 680 | 95 | 320 × 320 | 4 / 0.4 | 505 | |
2) Diffusion weighted MR imaging (performed in every patient) |
ss-EPI | Expiratory breath-hold | Pancreas | Transverse | 2200 | 58 | 130 × 92 | 5 / 0.25 | 2260 | Pixel spacing: 2.7 mm/ 2.7 mm; Number of acquired slices per b-value: 14; b-values [s/mm2]: 0, 50, 100, 150, 200, 300, 400, 600, and 800; Number of excitations: 1 for b = 0 s/mm2, 2 for every other b-value; Number of diffusion-encoding gradient directions: 3; K-space based parallel imaging technique (GRAPPA); acceleration factor: 2; Fat saturation technique: spectral fat saturation. |
The acquisition was separated into blocks (b0, b50), (b0, b100) … (b0, b800). Each block was acquired in a single breath-hold in expiration (TA = 22 s) to avoid motion artifacts. No registration for correction of patient breathing-motion was applied. |
MITK Diffusion software version 2017.07 (Medical Imaging Interaction Toolkit, DKFZ Heidelberg,
www.MITK.org) was used for post-processing of DWI data [
22]. Among several possible approaches described by Klaasen et al. [
23], the following approach according to the IVIM model was chosen to calculate the perfusion fraction
f, pseudodiffusion coefficient D*, and diffusion coefficient D, as previously described [
19] (corresponding to Klaasen’s model no. 3). The signal was averaged within a region of interest for each b-value. Then the equation
$$ \frac{S_b}{S_0}=\left(1-f\right)\ast \exp \left(-b\ast D\right)+f\ast \exp \left(-b\ast \left(D+D\ast \right)\right) $$
was fitted to the data. Here, S
b stands for the signal with diffusion weighting and S
0 for the signal without diffusion weighting. Measurements at b-values greater than 170 s/mm
2 were used in a first step to estimate f and D. D* was then calculated in a second step by using exhaustive search.
Quantitative analysis of DWI was performed independently by two radiologists with at least 5 years of experience in abdominal imaging each (P.M. and F.F.), blinded to the other radiologist’s analysis and other patient information. Free-hand volumes of interest (VOIs) were drawn encompassing the tumor on DW images. The exact anatomical outline of the tumor was determined with the help of conventional CT images and/ or conventional biliary-pancreatic MR images. Calcifications (as detected by CT) and cystic/ necrotic tumor areas without enhancement (as detected by contrast enhanced CT) were excluded from the VOIs. When possible, upstream and downstream pancreatic parenchyma also was segmented by Reader 1 (P.M.). The reported values of f, D, and D* were derived from the generated VOIs in all cases.
CT imaging and Post-processing
Immediately after MR imaging, the patients were examined with CT imaging.
All examinations were carried out with a 2 × 64-slice CT scanner (Somatom Definition Flash; Siemens Medical Solutions), using the hydro-CT-technique [
24]. Patients were placed on the CT table in an oblique, 30°, right-sided down position. The acquisition protocol is summarized in Table
2.
Table 2
Parameters of CT imaging
1) Standard 3-phasic CT scan (only performed if patient didn’t have an in-house CT scan of the abdomen within previous 4 weeks) Application of 80 ml of nonionic iodinated contrast agent; chaser bolus: 40 ml saline solution; bolus tracking in suprarenal aorta (threshold: 100 HU). |
Native | Inspiratory breath-hold | Upper abdomen | 120 | 210 | 4 | 0.5 | Helical | 2 × 64 × 0.6 | 3 | Variable | I30f |
Arterial | Inspiratory breath-hold | Upper abdomen | 120 | 210 | 10 | 0.5 | Helical | 2 × 64 × 0.6 | 3 | Variable | I30f |
Portal-venous | Inspiratory breath-hold | Whole abdomen | 120 | 210 | 50 | 0.5 | Helical | 2 × 64 × 0.6 | 3 | Variable | I30f |
15 min break for contrast medium clearance (as applied in previous studies [ 35, 42]). Patient remains on CT table. Patient is instructed to a shallow breathing technique for reducing motion artefacts in the perfusion acquisition. |
2) Native scan for verification of the correct position of the examination volume for CT perfusion imaging (performed in every patient) |
Native | Shallow breathing | Pancreas | 120 | 210 | n.a. | 0.5 | Helical | 2 × 64 × 0.6 | 3 | Variable | I30f |
3) CT perfusion imaging (performed in every patient) Application of 80 ml of nonionic iodinated contrast agent; chaser bolus: 40 ml saline solution; flow rate: 5 ml/s. |
CT perfusion imaging * | Shallow breathing | Tumor area | 80 | 270 | 13 | 0.5 (full rotation); 1.5 (cycle time) | 34 | 32 × 0.6 | 3 × 5.0 | 0.6 / 0.6 | B30f |
Perfusion data were analyzed with a body-perfusion CT-tool (Body-PCT, Siemens Medical Solutions) at a multimodality workplace with the syngo.via imaging software version VB 30 (Siemens Medical Solutions).
The baseline definition for motion correction at any time step and for segmentation at time step zero was followed by the segmentation of an organ VOI and the definition of a circular region of interest (ROI) in the aorta for vascular identification. The mean tissue time-attenuation curve was derived automatically and based on these definitions and data the color-coded parameter maps were established and confirmed.
Two radiologists with at least 5 years of experience in interpreting abdominal images each (P.M. and F.F.), independently placed polygonal VOIs encompassing the tumor, blinded to the other radiologist’s analysis and other patient information. For exact CT VOI placement, the radiologists had access to the same set of conventional CT/MR images as provided for MRI VOI placement. When possible, upstream and downstream pancreatic parenchyma was also segmented by Reader 1 (P.M.). Calcifications and cystic/ necrotic tumor areas without enhancement were excluded from the VOIs. To avoid a potential bias, the time interval between IVIM DWI analysis and CT perfusion analysis was at least 3 months for each radiologist.
Using a deconvolution model the software calculated the following parameters:
$$ BF\ \left( blood\ flow\right)\ \left[\frac{ml}{100\ ml\ast \mathit{\min}}\right] $$
$$ PEM\ (permeability)\ \left[\frac{ml}{100\ ml\ast \mathit{\min}.}\right] $$
$$ BV\ \left( blood\ volume\right)\left[\frac{ml}{100\ ml}\right] $$
The dose-length-products (DLPs) were calculated from the volume CT dose index (CTDI
vol) - values and scan lengths:
$$ DLP={CTDI}_{vol}\ast scan\ length $$
For calculation of the effective dose (D
eff), the DLPs were multiplied with the corresponding conversion factor for abdominal CT-examinations [
25]:
$$ {D}_{eff}=\frac{0.015\ mSv}{mGy\ast cm}\ast DLP $$
Histology and immunohistochemistry for the assessment of the MVD
In resection specimens, the diagnosis of PDAC was established according to the criteria recommended by the World Health Organization (WHO) and pathological staining was provided using the Union internationale contre le cancer (UICC) criteria. Histopathological grading was based on combined assessment of growth pattern, mucin content, and mitoses. When heterogeneity (i.e. variation in the degree of differentiation) was seen, the highest grade was assigned.
TNM (tumor, node, metastasis) stages according to the 8th Edition of the UICC Manual and Grading (G) of the 15 resected tumors were as follows: T1 in 1 patient, T2 in 10 patients, T3 in 4 patients, N0 in 3 patients, N1 in 4 patients, N2 in 8 patients, M0/x in 14 patients, M1 in 1 patient. The histopathological grading was G2 in 8 patients, and G3 in 7 patients. Pathological tumor size in these 15 patients ranged from 1.8 cm to 6.1 cm (mean value: 3.35 cm ± 1.19 cm).
Because of the known tumor heterogeneity [
3], tissue selection for tissue-based analysis was performed as previously described. In 10 out of 15 patients who underwent resection for PDAC, representative whole tumor slides from formalin-fixed paraffin-embedded tissue were immunostained with a CD34-specific antibody (1:25, M7165, Dako), as previously described [
19]. In 4 patients, tissue slides of non-neoplastic pancreatic tissue were also immunostained with the CD34-specific antibody.
To generate digital slide images, tissue slides were scanned at 20x magnification using an Aperio slide scanner (Leica Biosystems Aperio). In 10 patients, a mean coherent tumor area of 45 mm
2 per tumor, and in 4 patients, representative non-neoplastic pancreatic tissue were then analyzed using the Aperio Microvessel Analysis software (Leica Biosystems Aperio) [
26], as previously described [
19]. Plausibility was confirmed by pathologists (blinded to clinical information). MVA was calculated as the proportion of the sum of all vessel areas to the total analyzed area, MVD was calculated as mean vessel count per mm
2.
Statistical analysis
Statistical data analysis was performed using MedCalc version 19.2.1 (MedCalc Software Ltd., Ostend, Belgium). Spearman rank correlation coefficients between IVIM-derived parameters, CT perfusion- derived parameters, and MVD/ MVA in tumors were calculated. As proposed by Campbell and Swinscow [
27], Spearman correlation was interpreted as very weak (0.00–0.19), weak (0.20–0.39), moderate (0.40–0.59), strong (0.60–0.79), or very strong (0.80–1.00). For comparing correlation coefficients, we used the test recommended by Meng et al., Steiger’s Z [
28]. For a two-tailed test, Z-scores greater than 1.96 or smaller than − 1.96 are considered statistically significant. Regression analysis was applied between
f and BF, between
f and MVD, as well as between BF and MVD, using linear regression models. Mann-Whitney U test was used for comparison of independent continuous variables while Wilcoxon test was used for comparison of dependent continuous variables. Inter-reader reliability was assessed by using the Intra-class Correlation Coefficient (ICC) with 95% confidence intervals (CI) and applying a 2-way ICC with random raters’ assumption reproducibility. As proposed by Song et al. [
29], ICC values were interpreted as poor (0.00–0.20), fair (0.21–0.40), moderate (0.41–0.60), good (0.61–0.80), or excellent (0.81–1.00). Receiver operating characteristic (ROC) curves were employed to analyze the diagnostic performance of DWI IVIM and CT perfusion parameters in distinguishing tumors from upstream parenchyma. Due to the small sample size of patients with downstream parenchyma (
n = 5), ROC curve analysis was not performed for distinguishing tumors from downstream parenchyma. The AUCs with 95% confidence intervals (CIs) were computed. Sensitivities and specificities of the ROC curves were calculated, and the optimal cut-off values were determined. The DeLong method [
30] was used for comparison of areas under the curves (AUCs). As proposed by Mandrekar [
31], AUC values were interpreted as acceptable (0.70–0.79), excellent (0.80–0.89), and outstanding (0.90–1.00), while an AUC of 0.5 suggests no discriminatory ability. Significance level was set at 0.05.