Application of a novel targeting nanoparticle contrast agent combined with magnetic resonance imaging in the diagnosis of intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasm (IPMN) is a severe disease with macroscopic visible mucin secretion that primarily occurs in biliary tracts or pancreatic ducts. In comparison with standard diagnostic imaging, probing the molecular abnormalities associated with the initial stages of diseases rather than imaging the end effects markedly improves the accuracy of diagnosis. In the present study, magnetic resonance imaging (MRI) in combination with the contrast agent PEGylated magnetoliposome consisting of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) and target molecules of IPMN were investigated in the diagnosis of patients with suspected IPMN. The present investigation indicated that the novel targeting nanoparticle contrast agent targeted platelet-derived growth factor receptor-β and RET, and maintained a high affinity with tumor markers located on the IPMN surface. The novel targeting nanoparticle contrast agent combined with MRI exhibited increased sensitivity in diagnosing early-stage patients with IPMN. Furthermore, image quality was improved following the use of the novel targeting nanoparticle contrast agent combined with MRI compared with standard MRI. The targeting nanoparticle contrast agent retained sufficient affinity and was present for an adequate amount of time to observe the tumor mass in papillae using MRI. Notably, the targeting nanoparticle contrast agent was metabolized at 12 h post-injection. In conclusion, these outcomes indicate that the novel targeting nanoparticle contrast agent combined with MRI improved image quality and sensitivity compared with standard MRI, which suggests that this approach may be promising for clinical detection in patients with suspected IPMN.

Introduction

Intraductal papillary mucinous neoplasms (IPMNs) are rare pancreatic cystic tumors, and the morbidity and mortality rates associated with IPMNs have increased in recent years worldwide (1,2). Clinical investigations have revealed that there is a high prevalence of IPMN in extrapancreatic malignancies, and that IPMN is associated with autoimmune pancreatitis and others systemic diseases in patients (3). A previous study has explored the potential utility of a novel scoring formula for predicting malignant IPMN of the pancreas in order to simplify the treatment decision-making process in clinical settings (4). Various types of comprehensive therapies are available and target therapies have been regarded as the most important component of treatments for patients with IPMN; however, the 5-year survival rate of patients with IPMN is still poor (5,6). Notably, although a previous report has indicated that the imaging comparison of tubular and colloid pancreatic adenocarcinoma arising from IPMN on multidetector may be detected by computed tomography (CT) (7), diagnostic accuracy and sensitivity has not been improved for patients with early-stage IPMN. Furthermore, IPMN is associated with a spectrum of histologic and imaging findings, which have demonstrated the malignant transformation from adenoma to invasive carcinoma (8). These findings suggest that the diagnosis of patients with IPMN in the early stage is critical for providing efficient tumor treatment schemes and improving the survival rate.

A previous study has indicated that endoscopic diagnosis is the most commonly used diagnostic approach for IPMN of the bile duct (9). In recent years, contrast-enhanced ultrasound, CT, fluorodeoxyglucose-positron emission tomography (FDG-PET) and magnetic resonance imaging (MRI) have been widely used for diagnosing various types of human cancer (10). Among these diagnostic methods, MRI is considered the most accurate for the diagnosis of human tumors in situ (11). Studies have demonstrated multiple advantages of MRI for suspected cancer in patients (12–14). However, the resolution ratio and sensitivity of MRI is not sufficient for the diagnosis of patients with early-stage IPMN (15). Notably, nanoscale microbubble contrast agents have been indicated to improve the resolution and accuracy of MRI, which may be used to diagnose various tumors by using different contrast agents (16,17).

No reliable cyst fluid biomarkers have been identified in patients with IPMN (18). Magnetic resonance cholangiopancreatography using a specific nanoscale microbubble contrast agent has ameliorated the diagnosis of IPMN, which has allowed a clearer identification of the cystic lesion and improved diagnosis and treatment (19). Therefore, it was speculated that specific targeted nanoscale microbubbles may contribute to the efficacy and resolution of MRI in the diagnosis of patients with suspected IPMN.

Platelet-derived growth factor receptor (PDGFR)-β is overexpressed in pancreatic cancer, and targeting PDGFR may impair tumor growth, angiogenesis and metastasis (20). Various studies have demonstrated that RET is abundant in pancreatic cancer and that this may be associated in the progression IPMN (21–23).

In the present study, a novel nanoscale microbubble contrast agent targeting PDGFR-β and RET (CATPDR) was introduced for diagnosing patients with suspected IPMN. The accuracy and sensitivity of the combined use of CATPDR with MRI (CATPDR-MRI) compared with standard MRI alone in diagnosing clinical patients with suspected IPMN was determined. The present outcomes suggest that nanoscale microbubble contrast agent combined with MRI may be used for the early diagnosis and final confirmation of suspected cases.

Materials and methods

Ethics statement

The clinical design of the present study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Clinical Study of Beijing University of Chinese Medicine Third Affiliated Hospital (Beijing, China) (BUCMT20070612M25) and approved by the Ethics Committee of Cangzhou Central Hospital (Cangzhou, China). All patients provided their written, informed consent.

Patients

A total of 148 patients with suspected IPMN (aged 30.5–66.4 years old; 57 males: 91 females) and 150 healthy volunteers (aged 32.2–64.7 years old; 68 males: 82 females) were recruited for the present prospective analysis between May 2014 and June 2016 in Cangzhou Central Hospital (Cangzhou, China). The biochemical parameters (blood glucose and blood pressure) of patients with suspected IPMN indicated these patients were eligible for inclusion within the study. All patients were subjected to scanning for the detection of early-stage IPMN using CATPDR-MRI and standard MRI. Patients with a history of cancer were excluded from the study and patients with carcinoembryonic antigen (CEA) >5 µg/l and <10 µg/l were included in the study.

Nanoparticle contrast agent

PEGylated magnetoliposomes consisting of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) were produced using a two-step preparation method as described previously (24). A novel Gd-EOB-DTPA-encapsulated nanoparticle contrast agent with bispecific antibody targeting of PDGFR-β and RET (DS-72081; Shanghai AnYan Trading Co., Ltd., Shanghai, China) was used for the diagnosis of patients with suspected early-stage IPMN. PDGFR-β and RET were bound with the nanoparticles of Gd-EOB-DTPA using covalent bonds, as described previously (25). The nanoparticle contrast agent and Optison™ human serum albumin (GE healthcare, Chicago, IL, USA) was introduced by oral administration 30 min prior to the control MRI. The optimal dose of CATPDR (2–36 mg/kg, tested at 2 mg/kg intervals) was determined by the signal intensity as previously described (26).

MRI scanning

The MRI diagnosis system was used to diagnose IPMN in patients by using a preprogrammed setting. The preprogrammed setting was optimized to reach the optimal image formation. Pancreata of all patients were subjected to MRI using an MRI scanning system (Philips Medical Systems, Inc., Cleveland, OH, USA). The details of principles and settings of MRI were described in a previous study (27). CATPDR-MRI and MRI imaging was performed in all patients with suspected IPMN.

ELISA

A total of 10 ml blood was drawn from each patient and centrifuged at 4,000 × g for 15 min at 4°C. The plasma concentration of PDGFR-β (cat. no. DYC3096-2; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and RET (cat. no. E15070h; Wuhan Huamei Biological Engineering Co., Ltd., Wuhan, China) in patients was analyzed using commercialized ELISA kits according to the manufacturer's protocol. The results were recorded using an ELISA reader system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Image data analysis

Data of CATPDR-MRI and standard MRI image sets were analyzed using an MRI system. Existence of IPMN was diagnosed according to the MRI image. Patients with suspected early-stage IPMN were analyzed using a preparatory program (DynaCAD Breast Version 2.1; In vivo Corporation, Gainesville, FL, USA). Small IPMN tumor nodules were automatically calculated by drawing regions of tumor lesions.

Treatment of patients with suspected IPMN diagnosed by CATPDR-MRI

Patients with early-stage IPMN that were diagnosed using CATPDR-MRI received various treatments, including radiotherapy, chemotherapy, Chinese medicine, biological therapy and comprehensive therapy for 30 days. Certain patients received >1 type of treatment. The median overall survival and median progression-free survival were subsequently analyzed as described previously (28).

Western blotting

Tumor cells were homogenized in lysate buffer containing protease-inhibitor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and were centrifuged at 8,000 × g at 4°C for 10 min. The supernatant was used for analysis of the total protein using a bicinchoninic protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (20 µg) were separated on 15% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) as previously described (21). Protein was blocked with 5% bovine serum albumin reagent (BSA; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h at 37°C. For western blotting, primary rabbit anti-human antibodies against PDGFR-β (cat. no. ab32570), RET (cat. no. ab134100) and β-actin (cat. no. ab8226) (all 1:500 dilution; Abcam, Cambridge, UK), were incubated overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit immunoglobulin (Ig) G antibody (1:10,000; cat. no. HAF008; R&D Systems, Inc., Minneapolis, MN, USA) for 2 h at room temperature. A Ventana Benchmark automated staining system was used for analyzing protein expression (Olympus BX51; Olympus; Tokyo, Japan).

Immunofluorescence and histological staining

Once IPMN was confirmed in patients using CATPDR-MRI or MRI, the IPMN tumor cells from patients were isolated as previously described (29) and cultured in vitro using Minimum Essential Media (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) for 12 h at 37°C. Immunofluorescence procedures were previously reported in detail (30). Briefly, IPMN tumor cells (5×106) were subsequently incubated with targeting nanoparticle contrast agent (2 mg/ml) or PBS (2 mg/ml; control) for 30 min at 37°C. Following blocking with 5% BSA (Thermo Fisher Scientific, Inc.) for 1 h at 37°C the cells were incubated with rabbit anti-human antibodies against PDGFR-β, RET and β-actin (all 1:500 dilution) overnight at 4°C. This was followed by incubation with HRP-conjugated polyclonal anti-rabbit IgG antibodies (1:10,000) for 12 h at 4°C. Cells were observed using a fluorescence microscope (Olympus BX61).

Immunohistochemistry

Tumor tissues were fixed using 10% formaldehyde for 30 min at 37°C, washed with PBS and embedded in paraffin wax. Tissues were deparaffinized in xylene and rehydrated in graded series of alcohol. Antigen retrieval was performed at 95°C for 15 min. Immunohistochemical staining was performed using an avidin-biotin-peroxidase technique. Paraffin-embedded tissue sections (4-µm-thick) were prepared and epitope retrieval was performed for further analysis. The paraffin sections were subjected to hydrogen peroxide (3%) for 10–15 min at room temperature and blocked using a 2% bovine serum albumin (Thermo Fisher Scientific, Inc.) for 10–15 min at 37°C. Subsequently, the sections were incubated with goat anti-human PDGFR-β (1:1,000; ab220745) or RET (1:1,000; ab134110) primary antibodies (Abcam, Cambridge, MA, USA), respectively at 4°C for 12 h. Sections were then incubated with horseradish peroxidase-labeled secondary goat anti-rabbit secondary antibodies (1:2,000; ab150077; Abcam) for 2 h at 37°C and visualized using an LSM 510 confocal microscope (Zeiss AG, Oberkochen, Germany) at magnification, ×40.

Tumor tissues were fixed in situ overnight in 10% buffered formalin for 30 min at 37°C. The fixed tissues were embedded in paraffin and cut mid-sagittal (4-µm-thickness). For histological staining the 4-µm-thick tumor sections were stained with hematoxylin and eosin staining for 2 h at 37°C as previously reported (31). All sections were observed in three random fields in the confocal microscope at magnification, ×40. (Nikon E400, Nikon Corporation, Tokyo, Japan).

Statistical analysis

All data were presented as the mean ± or ± standard deviation of triplicate results as indicated. Data were analyses were performed using Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Unpaired data were analyzed using a Student's t-test and comparisons of data between multiple groups were analyzed by one-way analysis of variance followed by Tukey's honest significant difference post hoc test. Kaplan-Meier was used to estimate the survival rate during the 60-month long-term observation. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression levels of PDGFR-β and RET in plasma, IPMN cells and IPMN tissues in patients with suspected IPMN

Expression levels of PDGFR-β and RET in patients with IPMN were assessed. Results demonstrated that the plasma concentration of PDGFR-β and RET was significantly upregulated in patients with IPMN compared with the level in healthy volunteers (P<0.05; Fig. 1A and B). Western blot analysis indicated that protein expression levels of PDGFR-β and RET were significantly increased in IPMN cells compared with normal pancreatic ductal epithelial HPDE6-C7 cells (P<0.01; Fig. 1C and D). Postoperative pathological analysis indicated that PDGFR-β and RET protein expression levels were upregulated in IPMN tissue compared with normal pancreatic ductal tissue (Fig. 1E and F). These results suggest that PDGFR-β and RET may be regarded as potential targets for the diagnosis and treatment for patients with IPMN.

Figure 1.

Expression levels of PDGFR-β and RET in plasma, IPMN tissue and IPMN cells in patients with IPMN. Plasma concentration of (A) PDGFR-β and (B) RET in patients with suspected IPMN. Protein expression levels of (C) PDGFR-β and (D) RET in IPMN cells compared with normal pancreatic ductal epithelial HPDE6-C7 cells. Postoperative pathological analysis of (E) PDGFR-β and (F) RET expression levels in IPMN tissue. Magnification, ×40. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 as indicated. PDGFR-β, platelet-derived growth factor receptor-β; IPMN, intraductal papillary mucinous neoplasm.

Efficacy of CATPDR in IPMN cells

Combinative capabilities of CATPDR were analyzed in the present study. Contrast agent targeting of PDGFR-β and RET is a bispecific antibody, which may bind with both PDGFR-β and Ret compared with commercial anti-PDGFR-β or anti-RET (Fig. 2A and B). Results indicated that the fluorescence signals of PDGFR-β and Ret were weakened in IPMN cells not treated with CATPDR (Fig. 2C and D). Furthermore, the protein expression levels of PDGFR-β and RET were also significantly decreased following CATPDR treatment in IPMN cells compared with the level in the control (P<0.01; Fig. 2E and F). Collectively, these results suggest that CATPDR may significantly inhibit PDGFR-β and RET expression in IPMN cells.

Figure 2.

Characteristics of CATPDR in IPMN cells. Affinity of CATPDR with (A) PDGFR-β and (B) RET was determined using ELISA in vitro. Data are presented as the mean ± standard deviation. Effect of CATPDR on the expression levels of (C) PDGFR-β and (D) RET in IPMN cells was determined using immunofluorescence. Magnification, ×40. CATPDR significantly decreased the protein expression levels of (E) PDGFR-β and (F) RET in IPMN cells. Data are presented as the mean ± standard deviation. **P<0.01 as indicated. CATPDR, contrast agent targeting PDGFR-β and RET; IPMN, intraductal papillary mucinous neoplasm; PDGFR-β, platelet-derived growth factor receptor-β.

Efficacy of CATPDR-MRI in the early diagnosis of patients with suspected IPMN

In order to analyze the efficacy of CATPDR, a total of 148 patients with suspected IPMN were recruited to analyze the efficacy of CATPDR-MRI in the clinic. The characteristics of patients with suspected IPMN are summarized in Table I. The dose of CATPDR that achieved the optimum signal intensity for MIR detection was identified as 24 mg/kg, which was the minimum dose that generated the maximal signal intensity (Table II). Patients with suspected IPMN were required to orally take CATPDR prior to MRI. Results demonstrated that PDGFR-β and RET plasma concentration was significantly decreased in patients with suspected IPMN 30 min after CATPDR treatment (P<0.01; Fig. 3A and B). In addition, clinical analysis also indicated that blue fluorescent protein (BFP)-labeled-CATPDR adhered to IPMN cells, which contributed to the signal strength and resolution (Fig. 3C). Furthermore, clinical analysis of CATPDR-MRI results demonstrated that 80 patients (54.05%) were identified to have IPMN cancer and 68 (45.95%) patients were diagnosed as tumor free, as determined by CATPDR-MRI (Fig. 3D). Conversely, standard MRI only diagnosed 46 patients with IPMN cancer. These results suggest that CATPDR-MRI may increase the accuracy and sensitivity of MRI for diagnosis of early-stage IPMN in patients.

Table I.

Characteristics of patients with suspected intraductal papillary mucinous neoplasm.

	Sex of patients
Variable	Male	Female
Number of patients	57	91
Age range, years	32.4–63.4	30.5–66.4
Medical history of cancer	0	2
Blood pressure (mm Hg)	104.2±13.6	109.4±14.8
Blood glucose (mmol/l)	8.6±2.1	8.7±2.8
Diagnosis
MRI	57	91
CATPDR-MRI	57	91

CATPDR, contrast agent targeting platelet-derived growth factor receptor-β and RET; MRI, magnetic resonance imaging.

Table II.

Confirmation of targeting nanoparticle contrast agent dosage for patients with suspected intraductal papillary mucinous neoplasm.

	Dosage, mg/kg
Variable	2–12 (n=10)	14–24 (n=14)	26–36 (n=18)
Signal intensity	38.5±10.2	76.6±12.8	75.8±13.6
Sensitivity	35.2±13.4	72.5±13.8	76.4±12.4

The dosage was tested at 2 mg/kg intervals within each dose range.

Figure 3.

Use of CATPDR-MRI in the early diagnosis of patients with suspected IPMN. Changes of (A) PDGFR-β and (B) RET plasma concentration in patients with suspected IPMN pre- and post-CATPDR treatment. (C) Efficacy of CATPDR on signal strength and resolution of IPMN in IPMN cells. Magnification, ×40. (D) Diagnosis of patients suspected with early-stage IPMN was determined using CATPDR-MRI or MRI. Data are presented as the mean + standard deviation. **P<0.01 as indicated. CATPDR, contrast agent targeting PDGFR-β and RET; MRI, magnetic resonance imaging; IPMN, intraductal papillary mucinous neoplasm; PDGFR-β, platelet-derived growth factor receptor-β.

Pharmacodynamics of CATPDR in the plasma of patients with early-stage IPMN

The pharmacodynamics of CATPDR was investigated in the plasma of patients with suspected IPMN. Results demonstrated that the plasma concentration of CATPDR was decreased and metabolized within 12 h (<20 ng/ml; Fig. 4A). Patients with IPMN who had undergone CATPDR-MRI exhibited a notably decreased plasma concentration of PDGFR-β and RET following the administration of CATPDR, and these levels were recovered to normal levels within 20 h (Fig. 4B and C). These clinical data indicate that CATPDR-MRI may be a promising method for the diagnosis of early-stage IPMN.

Figure 4.

Pharmacodynamics of (A) CATPDR in the plasma of patients with IPMN. Plasma concentration of (B) PDGFR-β and (C) RET in patients with IPMN following CATPDR treatment for MRI detection. Data are presented as the mean ± standard deviation. CATPDR, contrast agent targeting PDGFR-β and RET; IPMN, intraductal papillary mucinous neoplasm; PDGFR-β, platelet-derived growth factor receptor-β; MRI, magnetic resonance imaging.

Histopathological analysis of CATPDR-MRI diagnosis in patients with suspected IPMN

Following the diagnosis of IPMN in patients using CATPDR-MRI, histopathological analysis was used to further confirm the diagnosis of CATPDR-MRI. The representative IPMN and incidence was studied in patients who were confirmed with IPMN as determined by CATPDR-MRI. As indicated in Fig. 5A, histopathological analysis was used to confirm the diagnosis of patients with IPMN diagnosed by CATPDR-MRI. Receiver operating characteristic curves were analyzed to compare the sensitivity between the two approaches and revealed that CATPDR-MRI had a markedly higher sensitivity than MRI (Fig. 5B). These outcomes suggest that the method of CATPDR-MRI for diagnosis of patients with IPMN is accurate and sensitive.

Figure 5.

Histopathological analysis of the accuracy of CATPDR-MRI diagnosis in patients with suspected IPMN. (A) Histopathological analysis identified patients with IPMN and was consistent with the diagnosis by CATPDR-MRI. Magnification, ×20. (B) Receiver operating characteristic curves were analyzed to determine the sensitivity and accuracy between CATPDR-MRI and MRI. CATPDR, contrast agent targeting platelet-derived growth factor receptor-β and RET; MRI, magnetic resonance imaging; IPMN, intraductal papillary mucinous neoplasm.

Survival rates of patients with IPMN diagnosed by CATPDR-MRI

Following the confirmation of IPMN diagnosis, patients with early-phase IPMN received various clinical treatments to inhibit tumor cell growth and aggressiveness. The survival rate of patients with IPMN (80 cases) diagnosed by CATPDR-MRI were determined. The clinical treatment methods for patients with IPMN are summarized in Table III. At the 60-month follow-up, it was observed that 68 patients (85.0%) were alive and tumor-free, and 8 patients (10.0%) still exhibited tumors. Only a total of 4 patients (5.0%) had succumbed to the disease in the present clinical investigation (Table IV). Notably, the median overall survival was 40 months (Fig. 6A) and median progression-free survival was 43.4 months (Fig. 6B). These outcomes suggest that the early-phase IPMN diagnosed by CATPDR-MR prolonged the survival and progression-free survival period following various anti-cancer treatments when compared with the mean survival.

Table III.

Treatment of patients with intraductal papillary mucinous neoplasm diagnosed by contrast agent targeting platelet-derived growth factor receptor-β and RET-magnetic resonance imaging.

Variable	Male, n	Female, n
Total number	57	91
Treatments
Radiotherapy	15	26
Chemotherapy	17	21
Chinese medicine	19	42
Biological therapy	24	28
Comprehensive therapy	36	56

Patients received >1 treatment.

Table IV.

Patient survival following diagnosis with contrast agent targeting platelet-derived growth factor receptor-β and RET-magnetic resonance imaging in a 60-month follow-up report.

	Outcome, n
Treatments	Tumor-free	Survival with tumor	Fatalities
Radiotherapy	13	1	1
Chemotherapy	7	0	2
Chinese medicine	12	2	1
Biological therapy	16	3	0
Comprehensive therapy	20	2	0

Figure 6.

Survival rates of patients with IPMN diagnosed by CATPDR-MRI. Evaluation of the (A) median overall survival and (B) median progression-free survival of patients diagnosed with early-stage IPMN by CATPDR-MRI. Kaplan-Meier was used to estimate the survival rate during the 60-month long-term observation. *P<0.05 and **P<0.01. IPMN, intraductal papillary mucinous neoplasm; CATPDR, contrast agent targeting platelet-derived growth factor receptor-β and RET; MRI, magnetic resonance imaging.

Discussion

Early diagnosis of cancer is vital for achieving the optimal treatment outcome for human cancer in the clinic (32,33). IPMN is a rare pancreatic cystic neoplasm and is typically difficult to diagnose due to shortage of knowledge and recognition of its initiation, development and treatments (34). Clinical investigations have demonstrated that monitoring tumor growth is beneficial for the inhibition of further IPMN recurrence for the majority of patients with late-stage cancer (9,35). Therefore, early diagnosis for patients with IPMN is beneficial for improving cancer treatments and survival time. In the present study, a novel CATPDR was used to locate and produce an enhanced signal intensity of IPMN tumors. The present study demonstrated that the expression levels of PDGFR-β and RET were significantly upregulated in patients with IPMN. Furthermore, CATPDR significantly inhibited PDGFR-β and RET expression levels and conglutinated on the surface of IPMN tumors, which enhanced the image resolution and accuracy generated by MRI.

Diagnostic techniques of endoscopy, contrast-enhanced ultrasound, FDG-PET, contrast-enhanced CT, chip technology and nuclear MRI have been widely used in the diagnosis of various types of human cancer (36–38). MRI has been demonstrated to have several advantages in diagnosing human cancer due to the optimization of accuracy rate and sensitivity in detection (39–41). However, MRI diagnosis is insufficient in the diagnostic progression of early-stage cancer in patients (42). The present study indicated that standard MRI detection provided limited accuracy and sensitivity for patients with suspected IPMN, as demonstrated by the lack of signal strength accumulated in the tumor lesions. However, the combination of CATPDR with MRI increased the accuracy rate, and improved the resolution and sensitivity in the diagnosis of patients with suspected IPMN.

PDGFR-β is a member of the PDGFRs that are located on tumor cell surface membranes and have regulatory effects on cancer cell proliferation, differentiation, growth and development (43,44). Gehmert et al (45) demonstrated that the PDGF-BB/PDGFR-β signaling pathway is associated with breast cancer cell migration and tumor metastasis. In the present study, it was identified that PDGFR-β was overexpressed in IPMN cells and may be used as a molecular target for IPMN diagnosis in the clinic. The findings suggest that microbubble contrast agent-bound bispecific antibody targeting of PDGFR-β enhances the signal strength in IPMN lesions in vivo.

RET proto-oncogene is reported to be overexpressed in the majority of cancer cells and serves an important role in tumor cell cycle arrest and aggressiveness (46,47). Previous results have indicated that targeting the receptor tyrosine kinase RET may inhibit ER-positive breast cancer xenografts (48). Previous results have suggested that RET is a possible key regulator in oral squamous cell carcinoma (OSCC) progression through persephin (PSPN)-RET-mitogen-activated protein kinase activation, and that PSPN overexpression may have diagnostic potential for OSCC (49). In the present study, results indicated that the receptor tyrosine kinase RET is overexpressed in IPMN cells and tumor tissues, and that it may be regarded as a molecular target in the diagnosis and treatment of patients with IPMN. Outcomes suggest that microbubble contrast agent-integrated bispecific antibody targeting of RET improves accuracy and image resolution in IPMN lesions in vivo.

In conclusion, the present study investigated the efficacy of CATPDR-MRI in the diagnosis of suspected early-stage IPMN in patients. Although it is important to distinguish pancreatic carcinoma from adenoma, the present study did not focus on distinguishing between pancreatic carcinoma and adenoma diagnosed by CATPDR-MRI. Notably, the results suggest that Gd-EOB-DTPA-integrated bispecific antibody targeting of PDGFR-β and Ret improves image resolution and signal intensity generated by MRI (50). These clinical outcomes indicate that CATPDR-MRI may be an efficient clinical approach in diagnosing patients with suspected early-stage IPMN via an accurate and sensitive assessment. The efficacy of CATPDR-MRI in diagnosing tumor size and type of IPMN in patients with suspected IPMN will be assessed in further work.