Validation of a Novel EUS-FNB-Derived Organoid Co-Culture System for Drug Screening in Patients with Pancreatic Cancer.

Cancer-associated fibroblasts (CAFs) have been shown to impact the chemosensitivity of patient-derived tumor organoids (PDTOs). However, the published literature comparing PDTO response to clinical outcome does not include CAFs in the models. Here, a co-culture model was created using PDTOs and CAFs derived from endoscopic ultrasound-guided fine-needle biopsies (EUS-FNBs) for potential use in drug screening applications. Co-cultures were established, and growth was compared to monocultures using image metrics and a commercially available assay. We were able to establish and expand validated malignant PDTOs from 19.2% of adenocarcinomas from EUS-FNBs. CAFs could be established from 25% of the samples. The viability of PDTOs in the mixed cell co-culture could be isolated using image metrics. The addition of CAFs promoted PDTO growth in half of the established co-cultures. These results show that co-cultures can be established from tiny amounts of tissue provided by EUS-FNB. An increased growth of PDTOs was shown in co-cultures, suggesting that the present setup successfully models CAF-PDTO interaction. Furthermore, we demonstrated that standard validation techniques may be insufficient to detect contamination with normal cells in PDTO cultures established from primary tumor core biopsies.

Targeted next-generation sequencing of EUS-guided through-the-needle-biopsy sampling from pancreatic cystic lesions.

BACKGROUND AND AIMS: Recent advances have introduced molecular subtyping of pancreatic cystic lesions (PCLs) as a possible amendment to the diagnostic algorithm. The study evaluated the feasibility and diagnostic accuracy of molecular analysis and subtyping of PCLs using the recently introduced EUS-guided through-the-needle-biopsy (TTNB) sampling. METHODS: We prospectively included 101 patients in the study who presented with PCLs >15 mm in the largest cross-section. EUS-guided TTNB samples were obtained by a micro-biopsy forceps introduced through a 19-gauge needle. The TTNB samples were analyzed by next-generation sequencing (NGS) for point mutations in tumor suppressors and oncogenes using a 51-gene customized hotspot panel. Sensitivity and specificity were calculated with the histologic diagnosis as reference. RESULTS: After initial microscopic evaluation of the samples, 91 patients had residual TTNB samples available for NGS. Of these, 49 harbored mutations, most frequently in KRAS and GNAS, reflecting an excess frequency of intraductal papillary mucinous neoplasms (IPMNs) in the study population. A sensitivity and specificity of 83.7% (95% confidence interval [CI], 70.3-92.7) and 81.8% (95% CI, 48.2-97.7), respectively, were demonstrated for the diagnosis of a mucinous cyst and 87.2% (95% CI, 74.2-95.2) and 84.6% (95% CI, 54.5-98.1) for the diagnosis of an IPMN. CONCLUSIONS: Thus, molecular analysis of TTNB samples by NGS has high sensitivity and specificity for diagnosing mucinous cysts and IPMNs. Although the procedure comes with a risk of adverse events of 9.9%, TTNB samples are a robust alternative to cyst fluid for a combined histologic and molecular diagnosis of PCLs. (Clinical trial registration number: NCT03578445.).

Clinical impact of endoscopic ultrasound-guided through-the-needle microbiopsy in patients with pancreatic cysts.

BACKGROUND: The limited data on the utility of endoscopic ultrasound (EUS)-guided through-the-needle biopsies (TTNBs) in patients with pancreatic cystic lesions (PCLs) originate mainly from retrospective studies. Our aim was to determine the clinical impact of TTNBs, their added diagnostic value, and the adverse event rate in a prospective setting. METHODS: This was a prospective, single-center, open-label controlled study. Between February 2018 and August 2019, consecutive patients presenting with a PCL of 15 mm or more and referred for EUS were included. Primary outcome was a change in clinical management of PCLs following TTNB compared with cross-sectional imaging and cytology. Adverse events were defined according to the ASGE lexicon. RESULTS: 101 patients were included. TTNBs led to a change in clinical management in 11.9 % of cases (n = 12). Of these, 10 had serous cysts and surveillance was discontinued, while one of the remaining two cases underwent surgery following diagnosis of a mucinous cystic neoplasm. The diagnostic yield of TTNBs for a specific cyst diagnosis was higher compared with FNA cytology (69.3 % vs. 20.8 %, respectively; P < 0.001). The adverse event rate was 9.9 % (n = 10; 95 % confidence interval 5.4 % - 17.3 %), with the most common event being acute pancreatitis (n = 9). Four of the observed adverse events were severe, including one fatal outcome. CONCLUSIONS: TTNBs resulted in a change of clinical management in about one in every 10 patients; however, the associated adverse event risk was substantial. Further studies are warranted to elucidate in which subgroups of patients the clinical benefit outweighs the risks.

Contrast-enhanced ultrasound for diagnosing, staging and assessment of operability of pancreatic cancer.

We have evaluated the usefulness of contrast-enhanced ultrasound (CEUS) for diagnosing, staging and assessment of operability of pancreatic head tumors. For some years CEUS has been used with great success for diagnosis of focal liver lesions but when we started our trial, it was still relatively untested in the pancreas. This PhD thesis is based on a methodological study, two clinical studies and an intra-/interobserver study. The methodological study consists of material collected from investigations made on 14 experimental pigs. First, we examined the pig pancreas with CEUS. Hereafter we repeated the CEUS examination after venous injection of the gastrointestinal hormones secretin and cholecystokinin. We investigated if the contrast-enhancement would intensify after hormone stimulation. The clinical studies consist of material collected from examinations of 49 patients referred to our hospital with the diagnosis, suspicion of pancreatic cancer. All patients had a conventional ultrasound examination and a CEUS examination. In addition, some of the patients also had a CEUS examination after stimulation with secretin and cholecystokinin. All patients had a 64-slice-CT examination and a biopsy was taken for histopathological verification. We studied whether CEUS was useful for assessment of tumor classification, tumor staging and tumor resectability. We also tested if hormone stimulation of the pancreas during CEUS could intensify contrast-enhancement of healthy pancreatic tissue and thus contribute to a better demarcation of a tumor. Finally, we tested the intra-/interobserver agreement of our visual interpretation of the contrast-enhanced ultrasound images and the concordance between the visual interpretation and histopathological test results. From the results of the methodological study it seemed possible to intensify contrast-enhancement using the gastrointestinal hormones by 3%. During the clinical studies it emerged that hormone stimulation did not improve the visual impression of the CEUS examinations in any cases. We found that CEUS may be a useful diagnostic tool in the diagnosis of pancreatic head tumors, with a sensitivity of 86% (CI: 79-96) in the diagnosis of pancreatic adenocarcinomas with histopathology as gold standard. Our intra-/interobserver agreements of the visual interpretation of tumor enhancement using CEUS showed substantial or almost perfect agreement with kappa values between 0.75 and 0.89. CEUS was useful for assessment of liver metastases but not as useful for assessment of the local tumor area and thereby not very useful for assessment of tumor resectability. By performing the US+CEUS and 64-CT we additionally found 35% and 45% respectively nonresectable patients of a group of patients, who were considered resectable on the primary radiological image material. In conclusion this thesis points to CEUS as a useful but not sufficient tool in the diagnosis, staging and assessment of operability of patients with pancreatic cancer.

Intra-/Interobserver Agreement of Enhancement Pattern for Pancreatic Head Lesions Using Contrast-Enhanced Ultrasound: According to EFSUMB Guidelines.

OBJECTIVE: To evaluate the intra-/interobserver agreement of the visual interpretation of contrast-enhanced ultrasound (CEUS) of pancreatic head lesions and its concordance with the histological test results. MATERIAL AND METHODS: Two observers (A + B) evaluated by simple visual interpretation 40 consecutive CEUS examinations of pancreatic head lesions and one of the observers evaluated the examinations twice (A1 + A2). The examinations were evaluated according to the criteria outlined in EFSUMB guidelines. The two experienced observers were blinded to histological evidence and clinical information of tumor type and to each other's results. RESULTS: The kappa value for the intraobserver evaluation between observer A1 and A2 was 0.89, equating to almost perfect agreement. The kappa value for the interobserver evaluation between observer A1 and B was 0.76 and between observer A2 and B it was 0.75, both equating to substantial agreement. Evaluation of the visual interpretation compared to the histological test result showed a positive predictive value for A1, A2 and B versus biopsy of 97%, 94% and 90% respectively and an accuracy of 83%, 88% and 73% respectively. CONCLUSIONS: Visual interpretation for assessment of contrast enhancement of pancreatic head lesions seemed to be an accurate method with reproducible results and good concordance with the histological test results.

Usefulness of contrast-enhanced transabdominal ultrasound for tumor classification and tumor staging in the pancreatic head.

OBJECTIVE: To evaluate contrast-enhanced ultrasound (CEUS) and compare it to ultrasound (US) and 64-slice-CT (64-CT) for diagnosing, staging and evaluation of resectability of pancreatic cancer. MATERIAL AND METHODS: US, CEUS and 64-CT were performed in 49 consecutive patients with pancreatic head tumors and with suspected cancer. After evaluation 44 patients had pancreatic head adenocarcinoma and 5 had chronic pancreatitis, all confirmed by histology. RESULTS: The sensitivity of US, CEUS and 64-CT for diagnosing malignant pancreatic head tumors was 89%, 86% and 93%, respectively, and the overall accuracy was 82%, 86% and 88% respectively. There was no significant difference in the malignant tumor size measurement between US and CEUS (p = 0.3619) or between US and 64-CT (p = 0.2129), but a significant difference was seen in the size measured by CEUS and 64-CT (p = 0.0197). The CEUS measurements on the tumor size were smaller. The overall accuracy for M staging of the patients who had surgery for adenocarcinoma was 86% and 90% for US + CEUS and 64-CT, respectively. By performing the CEUS and 64-CT we additionally found, respectively, 35% and 45% non-resectable patients of a group of patients, who were considered resectable on the primary radiological image material. CONCLUSIONS: CEUS may be a useful diagnostic tool in the diagnosis and staging of pancreatic head tumors. For the assessment of resectability CEUS did not prove useful. However, CEUS seemed very useful as an additional instrument in the detection of non-resectable patients already considered resectable on primary radiological image material.

Use of contrast-enhanced ultrasound imaging to detect the first draining lymph node (FDLN) in a swine model: correlation of imaging findings with the distance from the injection site to the FDLN.

OBJECTIVE: The purpose of this study was to investigate the use of contrast-enhanced ultrasound imaging (CEUS) to detect the first draining lymph node (FDLN) in a swine model and to determine whether the distance from the contrast agent injection site to the FDLN has any affect on the ability to detect contrast-enhanced FDLNs. METHODS: Thirteen swine (25-32 kg) were anesthetized during examinations and euthanized afterward. In every swine, 1 mL of a sulfur hexafluoride microbubble ultrasound contrast agent was bilaterally injected subcutaneously below a mammilla. The examined distances varied from 6 to 36 cm. The contrast-enhanced lymphatic channels were visualized with low-mechanical index CEUS and mapped from the injection site to the FDLN. After CEUS was performed, blue dye was injected in the same locations as the contrast agent, and dye-guided surgery was used to localize the FDLNs. To ensure the lymph node detected with the blue dye technique was the same found with CEUS, it was scanned again to confirm contrast enhancement. RESULTS: After 26 injections, 22 inguinal FDLNs were detected with CEUS and the blue dye technique. After 4 injections in 2 swine, contrast-filled lymphatic channels were identified with a course running toward the neck. In all cases but 1, the FDLNs received the contrast agent within 5 minutes. CONCLUSIONS: In this swine model, it was possible to use CEUS to locate the FDLNs. In these preliminary experiments, the distances from the contrast agent injection site to the FDLN did not affect the ability to detect the contrast-enhanced FDLNs.

Biopsy guided by real-time sonography fused with MRI: a phantom study.

OBJECTIVE: The purpose of our study was to test the accuracy of sonographically guided biopsies in a phantom of structures not visible on sonography but shown on MRI by using commercially available sonography systems with image fusion software. MATERIALS AND METHODS: A previously recorded MRI examination from a custom-made phantom was loaded into the sonography system. The phantom contained spheres that were invisible to sonography and contained red dye. The red dye was visible in the biopsy if it was successful. The images were coregistered using structures visible on both sonography and MRI, and biopsies were taken. The biopsy procedure was continued until a biopsy was successful, and the number of needle passes and time spent were registered. RESULTS: A total of 130 targets were hit. Ten minutes was used for loading the MRI data set and the coregistration; 94 of the 130 biopsies (72.3%) were successful at the first needle pass. The median number of needle passes until a successful biopsy was obtained was one (range, 1-7). CONCLUSION: The described method was successful in obtaining an adequate sample in a phantom.