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The added value of intravoxel incoherent motion diffusion weighted imaging parameters in differentiating high-grade pancreatic neuroendocrine neoplasms from pancreatic ductal adenocarcinoma

The aim of the present study was to investigate the potential significance of intravoxel incoherent motion (IVIM)-diffusion weighted imaging (DWI) in differentiating high-grade pancreatic neuroendocrine neoplasms (pNENs) from pancreatic ductal adenocarcinoma (PDAC). A total of 50 patients, including 37 patients with PDAC and 13 patients with high-grade pNENs, underwent pancreatic multiple b-values DWI with 15 b-values including 0, 10, 20, 40, 60, 80, 100, 150, 200, 400, 800, 1,000, 1,200, 1,500 and 2,000 sec/mm2. Standard apparent diffusion coefficient (ADCstandard) and IVIM parameter [slow apparent diffusion coefficient (Dslow), fast apparent diffusion coefficient (Dfast), fraction of fast apparent diffusion coefficient (ƒ)] values of PDAC and pNENs were compared. P<0.05 was considered to indicate a statistically significant difference. Receiver operating characteristics analysis was performed in order to evaluate the diagnostic potential of IVIM parameters for differentiating high-grade pNENs from PDAC. Dslow of pNENs was significantly lower compared with that of PDAC (0.460 vs. 0.579×10−3 mm2/sec; P=0.001). Dfast of pNENs was significantly higher compared with that of PDAC (13.361 vs. 4.985×10−3 mm2/sec; P<0.001). Area under the curve of Dslow, Dfast and combined Dslow and Dfast was 0.793, 0.863 and 0.885 respectively. The specificity and sensitivity of Dslow≤0.472×10−3 mm2/sec were 97.3 and 53.9%, respectively, for differentiating high-grade pNENs from PDAC. The specificity and sensitivity of Dfast >9.58×10−3 mm2/sec were 91.9 and 69.2%, respectively, for differentiating high-grade pNENs from PDAC. When Dslow and Dfast were combined, the specificity and sensitivity for differentiating high-grade pNENs from PDAC were 76.9 and 100%, respectively. Taken together, these results indicated that the diffusion-associated parameter Dslow and the perfusion-associated parameter Dfast of IVIM-DWI may differentiate high-grade pNENs from PDAC with high diagnostic accuracy, and that IVIM-DWI may be a valuable biomarker in differentiating pancreatic neoplasms.


Introduction
Pancreatic neuroendocrine neoplasms (pNENs) are the second most commonly-occurring solid neoplasms in the pancreas, accounting for <3% of all pancreatic neoplasms (1). The reported annual incidence of pNENs is approximately 2.5-5 per million persons (2). As all pNENs have the potential to become malignant, surgery is the only curative treatment option for pNENs; thus, it is recommended to all patients (3)(4)(5). Compared with pancreatic ductal adenocarcinoma (PDAC), pNENs usually demonstrate more indolent biological behaviors; pNENs exhibit a more preferable response to chemotherapy, higher resectability and longer overall survival (6). Even advanced pNENs exhibit improved longer-term survival compared with PDAC (7). As PDAC is one of the leading causes of cancer-associated mortality worldwide, with mortality rates The added value of intravoxel incoherent motion diffusion weighted imaging parameters in differentiating high-grade pancreatic neuroendocrine neoplasms from pancreatic ductal adenocarcinoma of ~80.3% (8), preoperative adjuvant chemoradiation may improve the 1-and 2-year overall survival and disease free survival rates of patients (9). However, radiotherapy and chemotherapy induce numerous side effects, such as nausea, vomiting and bone marrow suppression. Therefore, it is imperative to accurately differentiate pNENs from PDAC prior to surgery. Contrast-enhancement (CE) CT and MRI are widely accepted techniques for differentiating pNENs from PDAC. PNENs usually present with rapid and notable enhancement in the arterial phase of the CT scan due to their rich vascularity (7). However, pNENs gradually lose angiogenic potential and their microvascular density (MVD) decreases as the disease progresses (10). High-grade pNENs demonstrate lower vascularity and MVD compared with grade-1 pNENs (11,12). Therefore, high-grade pNENs often exhibit hypo-enhancement or heterogeneous enhancement in early contrast-enhanced images (13). At present, the use of CE CT or MRI exhibit difficulties when differentiating high-grade pNENs from PDAC. Hypo-enhanced pNENs in arterial phase imaging are associated with poor differentiation, increased aggressiveness and decreased 5-year survival rate (14,15). High-grade pNENs including grade 2 and 3 tumors often exhibit local invasion and/or metastases (16,17).
Since Le Bihan et al (18) proposed the theory of intravoxel incoherent motion (IVIM), the phenomenon that tissue perfusion effects can be separated from true tissue diffusion in IVIM diffusion weighted imaging (IVIM-DWI) was determined (19,20). Owing to the increasing concern for nephrogenic systemic fibrosis (21), IVIM-DWI may be an effective alternative to determine perfusion in tissues without contrast agents (22). An increasing number of studies have demonstrated the value of IVIM-DWI for evaluating pancreatic neoplasms (23,24). In addition, studies have applied quantitative parameters derived from IVIM-DWI to predict histological characteristics of pNENs (25,26). However, to the best of our knowledge, no published studies that compare IVIM-DWI parameters between pNENs and PDAC are currently available. Therefore, the aim of the present study was to assess the diagnostic performance of IVIM-DWI parameters for distinguishing between high-grade pNENs and PDAC.

Materials and methods
Subjects. Ethical approval was acquired from the Institutional Ethics Committee Board of Xijing hospital (Xi'an, China) and written informed consent was obtained from all participants. A total of 90 patients with suspected pancreatic solid mass were considered for inclusion in the present study between May 2014 and April 2017. A total of 40 patients were excluded due to the following exclusion criteria: i) Patients diagnosed with diseases other than PDAC and high-grade pNENs (n=36) including grade 1 pNENs (n=25), solid pseudopapillary tumor (n=7), accessory spleen located in pancreas (n=1), cholangiocarcinoma located in pancreatic segment of common bile duct (n=2) and mass-forming chronic pancreatitis (n=1); ii) patients lost to the follow-up (n=4). The recruitment process is illustrated in Fig. 1.
Image acquisition. All patients were examined on a Discovery MR750 3.0 T whole-body MR scanner (GE Healthcare Life Sciences) using a 32-channel phased-array coil. Pancreatic MR sequences comprised axial fast spin echo transverse relaxation time-weighted images (T 2 WI) with fat-suppression, axial breath-hold 3D liver acquisition with volume acceleration Flex (LAVA Flex) and LAVA Flex with contrast (Omniscan; GE Healthcare Life Sciences). Pancreatic multiple b-value diffusion-weighted echo-planar imaging was performed with 15 b-values, including 0, 10, 20, 40, 60, 80, 100, 150, 200, 400, 800, 1,000, 1,200, 1,500 and 2,000 sec/mm 2 . The detailed parameters of each sequence are presented in Table I.
Image and data analysis. An abdominal radiologist with 11 years of MRI experience blinded to the histopathological results analyzed the acquired IVIM-DWI data. Following examination, all data were transmitted to a built-in AW 4.6 workstation (GE Healthcare Life Sciences) for post-processing. Functional maps of IVIM parameters and a standard apparent diffusion coefficient (ADC standard ) map were processed by mean ADC (MADC) programs on the built-in AW 4.6 workstation.
ADC standard was calculated using a mono-exponential model including the total b-values. The equation used was as follows: S(b)/S 0 =exp (-b x ADC standard ) (equation 1). IVIM parameters including slow apparent diffusion coefficient (D slow ), fast apparent diffusion coefficient (D fast ) and fraction of fast apparent diffusion coefficient (ƒ) were calculated using bi-exponential fitting by the segmented fitting method. The equation used was as follows, as proposed by Le Bihan (20): , where S(b) represents the mean signal intensity (SI) of a DW image according to a specific b-value, S 0 represents the mean SI of a DW image when b=0, D slow represents true tissue diffusivity while tissue microcapillary perfusion is mainly excluded, D fast represents the mean velocity of the flowing blood within capillaries and the microvascular architecture and ƒ represents the ratio of molecular diffusion within capillaries compared with the overall water molecular diffusion in a voxel. As D fast is larger compared with D slow by approximately one order of magnitude (22), -b x D fast would be >-3 when the b>200 sec/mm 2 , and the ƒ x exp (-b x D fast ) value would be >0.05x ƒ. Therefore, ƒ x exp (-b x D fast ) can be neglected and equation 2 can be expressed as follows: S(b)/S 0 =(1-ƒ) x exp (-b x D slow ) (equation 3). As b-values were >200 sec/mm 2 , S(b) was fitted using equation 3 according to a linear model, and D slow was calculated. The ƒ value was previously calculated according to equation 3; however, the accuracy was unacceptable. Therefore, the ƒ-value was recalculated according to equation 2. Subsequently, S(b) was fitted for all b-values according to equation 2 with the D slow value fixed by the nonlinear Levenberg-Marquardt method (19). When equation 2 was fitted, the initial estimated D fast value was set as 10x10 -3 mm 2 /sec, and the ƒ-value was set as the previous ƒ-value calculated from equation 3. The D fast and ƒ values were then acquired.
On the DW images that distinctly displayed the tumors, irregular regions of interest (ROIs) for each PDAC and pNENs were manually delineated along the edge of the tumor on three consecutive largest lesion slices. During measurement, particular efforts were made to exclude any areas of necrosis, the pancreatic duct and vessels within the tumor. Well-matched ROI copies were generated synchronously and appeared automatically on identical locations in every functional map of IVIM-DWI parameters and ADC standard by built-in MADC software on an AW 4.6 workstation (GE Healthcare Life Sciences). The processes of ROI setting on functional parameter maps are presented in Fig. 2. The mean value of the results of three measurements was used as the final result. The ROI area range of pNENs was between 22 and 1,379 mm 2 with a mean area of 372.22 mm 2 . The ROI area range of PDAC was between 136 and 1,025 mm 2 with a mean area of 324.58 mm 2 .
Statistical analysis. All analyses were performed using SPSS software version 17.0 for Windows (SPSS Inc.) and MedCalc software version 12.3 (MedCalc Software). The ADC standard and IVIM-DWI parameter values are presented as the mean ± standard deviation. ADC standard and IVIM-DWI parameter values were compared using an independent sample Student's t-test. Receiver operating characteristics (ROC) analysis was performed to evaluate the diagnostic performance of IVIM-DWI parameters and to determine the cut-off values using the maximum Youden index (the sum of specificity and sensitivity). P<0.05 was considered to indicate a statistically significant difference.

Results
Clinical and pathological characteristics of patients and lesions. The clinical and pathological characteristics of patients and lesions are summarized in Table II. A total of 50 patients including 13 cases of high-grade pNENs (grade 2, n=11; grade 3, n=2) and 37 cases of PDAC (well/moderately differentiated, n=21; poorly differentiated, n=11; confirmed by biopsy without pathological grades, n=5) confirmed by surgical pathology were included (27). The pNENs cohort (mean age, 55.1 years; age range, 42-75 years) included 8 male and 5 female patients. The clinical symptoms of the pNENs group included hypoglycemia, epigastric pain and discomfort, thrombocytopenia and splenomegaly. Among the pNENs group, one patient presented with painless jaundice. The PDAC cohort (mean age, 57.5 years; age range, 20-76 years) included 23 male and 14 female patients. The clinical symptoms of the PDAC group included marasmus, dyspepsia and abdominal and back pain. Among the PDAC group, 11 patients presented with jaundice.
Conventional MRI results. The conventional MRI results of PDAC and pNENs are presented in Table III. The mean long diameter of pNENs was 3.31 cm (range, 0.89-5.58 cm). Among the pNENs, 7 lesions were located in the head, 2 were in the body and 4 were in the tail of the pancreas. In the T 2 W images, 7 pNENs exhibited moderate hyperintensity, 4 exhibited slight hyperintensity and 2 exhibited moderate hypointensity. In the LAVA Flex water phase images, 12 pNENs exhibited moderate hypointensity and 1 presented with slight hypointensity. In the LAVA Flex with contrast images, all pNENs exhibited heterogeneous mild-to-moderate hypo-enhancement on arterial phase imaging. Representative conventional images of pNENs are presented in Fig. 3A-C. In the DW images, 12 pNENs exhibited hyperintensity and 1 displayed hypointensity. Among the 12 hyperintensive pNENs, 5 (41.7%) exhibited heterogeneous SI and 7 (58.3%) exhibited homogeneous SI in DWI. In addition, 8 of 12 (66.7%) hyperintensive pNENs exhibited high SI in all b-value DW images, including low b-value (b≤200 sec/mm 2 ), moderate b-value (200 sec/mm 2 <b≤1,500 sec/mm 2 ) and high b-value (b>1,500 sec/mm 2 ). In addition, 4 of 12 (33.3%) hyperintensive pNENs demonstrated high SI in low and moderate b-value DW images; however, they exhibited low SI in high b-value DW images. Representative DW images of pNENs are presented in Fig. 3D-F.

Comparison of IVIM-DWI parameters. IVIM-DWI parameters of PDAC and pNENs are presented in
ROC curves for differentiating pNENs from PDAC using D slow and D fast are presented in Fig. 6. The optimal cut-off values and AUC in differentiating pNENs from PDAC are listed in Table V. The AUC for D fast was slightly larger compared with that for D slow (0.863 vs. 0.793; P=0.499), whereas the AUC for combined D slow and D fast was slightly larger compared with that for D slow or D fast alone (0.885 vs. 0.793; P=0.257; 0.885 vs. 0.863; P=0.757, respectively). When D slow value was ≤0.472x10 -3 mm 2 /sec, the specificity and sensitivity for differentiating pNENs from PDAC were 97.3 and 53.9%, respectively. When D fast value was >9.58x10 -3 mm 2 /sec, the specificity and sensitivity for differentiating pNENs from PDAC were 91.9 and 69.2%, respectively. When D slow and D fast were combined, the specificity and sensitivity for differentiating high-grade pNENs from PDAC were 76.9 and 100%, respectively.

Discussion
The present study identified statistically significant differences in D fast and D slow between PDAC and pNENs. The differences in ADC standard and ƒ exhibited no significance between PDAC and pNENs. The ADC value is the most commonly used parameter for evaluating tissue diffusion that serves as a marker of cellularity (28,29), which is additionally decreased
The results of the present study demonstrated that D fast of PDAC was notably lower compared with that of pNENs. The ƒ of PDAC was slightly lower compared with that of pNENs. D fast is associated with blood flow and velocity within microcirculation (19,22). Parameter ƒ is associated with the proportion of protons in microcirculation within a voxel (29).
Poor blood supply leads to a lower blood volume and flow in PDAC compared with a normal pancreas (38). Due to abundant fibrosis and relatively sparse vascularity of PDAC, the enhancement degree of PDAC is lower compared with that of normal pancreatic tissue, especially in early post-contrast images (35,38). However, pNENs typically demonstrate fast and strong enhancement in early contrast-enhanced images due to abundant vascularity (7,39). A study by Kang et al (24) demonstrated that tissue perfusion may be a more important factor compared with diffusion in differentiating pNENs from PDAC. The results of the present study indicated that D fast and ƒ of pNENs were higher compared with those of PDAC due to different vascular perfusion, which was in agreement with the results obtained by Kang et al (24), although no significant differences in ƒ were identified between pNENs and PDAC. High-grade pNENs including grade 2 and 3 exhibit a lower vascularity and MVD (11)(12), which leads to a decreased ƒ value of pNENs, similar to that of PDAC. Unlike the study by Kang et al (24), which stated that D slow was not useful in differentiating PDAC and pNENs, the results obtained in the present study demonstrated that the D slow of PDAC was    significantly higher compared with that of pNENs. This may be due to Kang et al using only nine b-values in IVIM-DWI; however, 15 b-values were used in the present study. Therefore, in order to obtain high-quality IVIM-DWI, at least 10 b-values should be used (40).
The present study demonstrated that the optimal AUC was achieved by combining D slow and D fast (AUC, 0.885), which was followed by D fast and D slow alone (AUC, 0.863 and 0.793 respectively). The highest specificity was identified for D slow (97.3%), which was closely followed by D fast (91.9%). When Figure 5. ADC standard , D slow , D fast and ƒ values in pNENs and PDAC. The line represents the median. The central box represents the measurements from the lower (the 25th percentile) to the upper (the 75th percentile) quartile. Whiskers indicate the range from the minimum to the maximum parameter measurements. (A) * in the box-and-whisker plot revealed that the median D slow value of PDAC was significantly higher compared with that of pNENs. (B) ** in the box-and-whisker plot demonstrated that the median D fast value of PDAC is significantly lower compared with that of pNENs. Box-and-whisker plots revealed that no significant differences were observed between PDAC and pNENs for (C) ADC standard and (D) ƒ. pNENs, pancreatic neuroendocrine neoplasms; PDAC, pancreatic ductal adenocarcinoma; ADC, apparent diffusion coefficient; D slow , slow apparent diffusion coefficient; D fast , fast apparent diffusion coefficient; ƒ, fraction of fast apparent diffusion coefficient. D slow and D fast were combined, the sensitivity for differentiating high-grade pNENs from PDAC was 100.0%. Therefore, D slow and D fast may be the ideal screening indicators in differentiating high-grade pNENs from PDAC. There were certain limitations to the present study. For example, the sample size of the present study was relatively small, particularly regarding the limited number of high-grade pNENs due to its low incidence. However, all patients underwent surgery and were confirmed by histopathology. Secondly, the present study did not analyze the association between histopathology and IVIM-DWI parameters. This was due to the small sample size of different pathological grades pNENs and PDAC. Additional subjects need to be recruited in order to investigate the association between histopathology and IVIM-DWI parameters of pNENs and PDAC in further studies. In addition, dynamic contrast enhancement (DCE)-MRI was not performed in the present study due to the patients' preferences. The association between DCE-MRI and IVIM-DWI parameters will be investigated in our future studies. An additional limitation of the present study was that the b-values selected may have been suboptimal. Therefore, optimal b-values should be selected in any future studies to balance the least sampling time and the reliability of parameter estimation. Finally, a control group with normal pancreas was not included in the present study. This will be evaluated in further studies with larger populations.
In conclusion, the results of the present study demonstrated that the diffusion parameter D slow and the perfusion parameter D fast derived from IVIM-DWI may be reliable in differentiating high-grade pNENs from PDAC with high diagnostic accuracy. In addition, IVIM-DWI may be a potential technique to function as a surrogate biomarker in distinguishing pancreatic neoplasms. Figure 6. ROC curves of D slow and D fast for differentiating pNENs from PDAC. The diagonal line is the reference, which indicates the results for a test with 50% sensitivity and 50% specificity. The highest AUC was obtained for combined D slow and D fast , which was closely followed by D fast and D slow alone. ROC, receiver operating characteristics; D slow , slow apparent diffusion coefficient; D fast , fast apparent diffusion coefficient; AUC, area under the curve; pNENs, pancreatic neuroendocrine neoplasms; PDAC, pancreatic ductal adenocarcinoma.