Study design and ethics
This study is the retrospective study. Patients were not required to give informed consent because this study used anonymous clinical data obtained after each patient had agreed to medical activities by written consent. For full disclosure, the details of this study are published on the home page of Fukushima Medical University.
Patients
A total of 195 SPL patients who underwent EUS-FNA or EUS-FNB (EUS-FNAB) between January 2017 and March 2020 were enrolled in this study (Fig. 1). Among these patients, 89 SPL patients underwent EUS-FNAB using a 22-G needle (FNB: 28 patients, FNA: 61 patients). The SPLs of 58 patients were UR (FNB: 22 patients, FNA 36 patients).
Thirty-nine pancreatic cancer patients underwent EUS-FNAB after the MSI evaluation was covered by insurance in Japan. Among these patients, 16 patients had undergone the process of histological specimen acquisition but had received treatment at another hospital. Among the other 23 patients, EUS-FNB was performed in 9 patients, and EUS-FNA was performed in 14 patients.
EUS-FNAB procedures
The echoendoscope was gently inserted into the patient while in the left lateral decubitus position after sufficient sedation by the intravenous administration of midazolam. After the SPL was visualized on the monitor, the lack of blood flow on the aspiration line was confirmed using Doppler mode. Then, the needle was advanced to the SPL, and the stylet was removed. The needle was passed back and forth twenty times in the SPL while applying suction using a 20- or 10-ml syringe. The fanning method was utilized as much as possible for puncture [16]. Aspiration was repeated until sufficient material was confirmed by rapid onsite cytology (ROSE) [17, 18].
The following ultrasonography equipment and echoendoscopes were used in this study: EU-ME-1, EU-ME-2, GF-UC240AL-5, or GF-UCT260 (Olympus Medical Systems, Tokyo, Japan). The selection of the FNAB needle was as follows. For patients with an R SPL or a small SPL, a conventional FNA needle tended to be used because of its good penetration ability. Otherwise, the chosen needle was randomly selected by each endoscopist. The Franseen needle used for EUS-FNB in this study was an Acquire 22-G needle (Boston Scientific, MA, USA) (Fig. 2). The cutting surface of the Franseen needle (on the bottom of Fig. 2) is larger than that of the conventional aspiration needle (on the top of Fig. 2). The needles used for EUS-FNA in this study were an Expect 22-G needle (Boston Scientific, MA, USA), an EZ Shot 3 plus 22-G needle (Olympus Medical Systems), and an EchoTip 22-G needle (Cook Medical, Inc., NC, USA).
All procedures were performed by pancreaticobiliary specialists who had performed more than 200 EUS-FNAB procedures or beginners under the guidance of pancreaticobiliary specialists. The puncture needles were randomly chosen by each endoscopist.
Examination items
The patient characteristics (age, sex), SPL data (tumor size, tumor location, resectability), and procedural characteristics (puncture route, puncture number, histology, adverse events) were compared between patients who underwent EUS-FNB and those who underwent EUS-FNA. In the UR SPL patients, similar comparisons were performed. The tumor size was the maximum diameter measured by EUS or computed tomography (CT). UR SPLs were determined by the following characteristics: 1. contacts/invades the superior mesenteric vein or portal vein by more than 180 degrees with a range of contact/invasion exceeding the inferior duodenal angle; 2. contacts/invades the superior mesenteric artery or celiac artery by more than 180 degrees; 3. contacts/invades the common hepatic artery with a range of contact/invasion extending to the proper hepatic artery or celiac artery; 4. contacts/invades the aorta; and 5. distant metastases [19, 20]. R SPLs were finally diagnosed by surgery. On the other hand, UR SPLs were diagnosed by cytology or histology on EUS-FNAB. Malignancy was defined by class IV or V cytology.
In some UR SPL patients who underwent EUS-FNAB after the MSI evaluation was covered by insurance, the possibility of performing the MSI evaluation using the histological specimens was compared between EUS-FNB and EUS-FNA.
Requirements for histology and MSI evaluation
A histological diagnosis was attempted to be made using all the specimens submitted to department of pathology. To diagnose malignancy, the specimen must include tumor tissue that is not crushed or degenerated. If tumor tissues are fragmented or crushed, the diagnosis of malignancy become difficult. Furthermore, if a specimen contains inflammatory components, it become difficult to distinguish whether atypical cells are tumoral or reactive to inflammation.
To extract DNA for MSI evaluation, more than 2000 tumor cells are necessary. Additionally, more than 50% tumor cell content is needed. More than five unstained slide specimens of SPLs had to be prepared for the investigation of MSI. An MSI detection kit (FALCO HOLDINGS Co., Ltd., Kyoto, Japan) was used to measure MSI according to five mononucleotide markers (BAT-25, BAT-26, NR-21, NR-24, and MONO-27). When more than two markers were MSI positive, the tumor was defined as MSI-high.
Statistical analyses
Continuous variables that did not conform to a normal distribution and ordinal variables were compared by the Mann-Whitney U test. Nominal variables were compared by Fisher’s exact test. A P value < 0.05 was considered significantly different. All statistical analyses were performed with EzR (Saitama Medical Centre, Jichi Medical University, Saitama, Japan) [21].