Simultaneous Gemcitabine and Percutaneous CT-Guided Irreversible Electroporation for Locally Advanced Pancreatic Cancer

Background Irreversible electroporation (IRE) is a new local ablation technique for pancreatic cancer. The aim of this study is to analyse the safety and effectiveness of simultaneous gemcitabine and percutaneous CT-guided IRE for locally advanced pancreatic cancer (LAPC). Materials and Methods From October 2016 to January 2018, 61 patients with LAPC who received simultaneous gemcitabine and IRE therapy (GEM-IRE group, n = 31) or IRE alone therapy (IRE group, n = 30). Routine intravenous gemcitabine chemotherapy was performed 2 weeks after IRE in both groups. Results Technical success rates were 90.0% (27/30) and 93.3% (28/30) in the GEM-IRE and IRE groups. Compared with the IRE group, the GEM-IRE group exhibited longer overall survival (OS), local tumor progression free survival (LTPFS), and distant disease free survival (DDFS) from IRE (OS, 17.1 vs. 14.2 months, p=0.031; LTPFS, 14.6 vs. 10.2 months, p=0.045; DDFS, 15.4 vs. 11.7 months, p=0.071). Multivariate Cox regression analysis results suggested that tumor volume ≤37 cm3 and simultaneous gemcitabine with IRE were significant independent prognostic factors of OS, LTPFS, and DDFS. Four major adverse reactions occurred; all of them were resolved after symptomatic treatment. Conclusions Simultaneous gemcitabine and percutaneous CT-guided IRE therapy model was effective and well-tolerated therapeutic strategy in LAPC patients.


Introduction
It is projected that pancreatic cancer will become the second leading cause of death in the United States and Germany within the next decade [1,2]. Approximately 80% of patients are diagnosed with locally advanced pancreatic cancer (LAPC), and in fewer than 20% of these patients, surgical resection is possible [3,4], and a 5-year survival rate is less than 8% [5]. First-line chemotherapy regimens for LAPC include FOLFIRINOX, a combination of gemcitabine and albumin-bound paclitaxel, or just gemcitabine. However, the LAPC prognosis remains dismal, even with chemotherapy.
At present, minimally invasive treatments such as radiofrequency, microwave, and cryoablation have been demonstrated to be effective for LAPC patients [6][7][8].
ese types of technology, however, have the potential to damage bile ducts, pancreatic ducts, peripancreatic vessels, and the duodenum, which may result in limited benefit [9,10]. Irreversible electroporation (IRE) is a novel, minimally invasive ablation technique for soft tissue cancer, which uses high-voltage electrical pulses to develop irreversible small holes on the surface of the cell membrane, causing a disruption of homeostasis and cell death [11][12][13][14]. Currently, IRE has demonstrated distinct superiority and potency in the management of LAPC [15][16][17][18].
In recent years, there has been an explosion in the use of electrochemical therapy (ECT) in the treatment of solid tumors [19][20][21]. Usually, ECT combines poorly or not penetrating but strongly available cytotoxic agents with electroporation. Because it strengthens the transmission of molecules to the structure, ECT has a high potential for treating advanced solid tumors [22,23]. In the reversible electroporation (RE) zone, cell membrane permeability due to electroporation can promote drug diffusion into the cells [24][25][26][27], which may improve antitumor efficacy. On the other hand, a preclinical study has shown that IRE enhanced gemcitabine delivery into the RE zone and thereby reduced local recurrence [28].
Based on the above studies, a retrospective study about simultaneous gemcitabine and percutaneous CT-guided IRE for LAPC was conducted. In this study, we evaluated overall survival (OS), local tumor progression free survival (LTPFS), distant disease free survival (DDFS), and objective response rate (ORR) and adverse events after combination therapy, towards a more effective treatment strategy for LAPC.

Patients.
e patients received either simultaneous gemcitabine and percutaneous CT-guided IRE (GEM-IRE group) or IRE alone (IRE group). We executed this retrospective study with the approval of the Ethics Committee of Fuda Cancer Hospital and strict administration of the Declaration of Helsinki and the Declaration of Good Clinical Practice. We obtained written informed consent from each patient.

Procedure.
In all patients, preoperative bowel preparation was performed and anesthesia was inducted with etomidate (0.3 mg/kg), remifentanil (3-5 μg/kg, 1.2-1.6 mg/ h), and cisatracurium besilate (0.1 mg/kg). Anesthesia was administered by injection of intravenous cisatracurium besilate, remifentanil, and propofol with sevoflurane inhalation. To avoid cardiac arrhythmias, an electrocardiogram (ECG)-gating device was synchronized with pulse delivery during the cardiac refractory period. To prevent generalized muscle contractions, complete muscle relaxants were administered immediately before IRE delivery.
In the GEM-IRE group, before IRE treatment, gemcitabine (1000 mg/m 2 ) was given intravenously for 30 minutes. Once-weekly infusions of gemcitabine were administered for the first two weeks, followed by a one-week rest period. Subsequent cycles consisted of weekly injections for three continuous weeks, with a cycle of four weeks each. e therapy was maintained until disease progression, by mRECIST or intolerable toxicity [29]. Upon disease progression, palliative care was given.
During IRE, CT scanning and ultrasound were used to guide percutaneous insertion of the electrode. Pretreatment planning determines electrode-insertion mode, electrode number, and intraoperative parameters. e IRE parameters were set up as follows: 90-microsecond high-voltage (1500-3000 V), delivered between 9 pairs of paired monopolar electrodes with 2 cm exposed tips, totaling 90 pulses. Further treatment includes anti-infection treatments, stomach-preserving and liver protection therapies, and nutritional treatment. . Carbohydrate antigen 19-9 (CA19-9) was analyzed 30 days postintervention to evaluate the initial effect.

Technical Success.
Technical success was considered to be the ability to successfully deliver pulses (at least 90) to ensure a change in current of at least 5 A relative to the initial 10 pulses.

Follow-Up and Response
Assessment. Imaging follow-up was done using contrast-enhanced CT or magnetic resonance imaging (MRI) at 1, 3, and 6 months postintervention and then every 3 months. Imaging follow-up at 1 month evaluated the success of the technique. Imaging efficacy evaluation was based on mRECIST [29]. Complete response (CR) was considered as the absence of arterial enhancing lesions. A partial response (PR) was indicated by a 30% decrease in the size of the target lesion. Progressive disease (PD) was considered by a decrease of 20% in the size of the lesion. Stable disease (SD) was considered by lack of target lesions reduction to PR or increase to PD. We estimated the objective response rate (ORR) as ORR � (CR + PR)/total number × 100%.

OS.
e OS was the length of time from diagnosis and IRE treatment to death from any cause. e LTPFS was considered as the time from IRE treatment to local tumor progression. DDFS was considered as the time from IRE treatment to distant metastasis.

Safety.
Adverse events were assessed after 24 h using enhanced CT and ultrasound. Any adverse events that occurred within 30 days of treatment were graded depending on the CTCAE V4.0. CTCAE grades III-IV were classified as "severe." e change of AST, ALT, and ALP and routine blood tests were taken before the procedure and were repeated 1, 3, 7, and 14 days after the procedure to evaluate intervention safety.

Statistical Analysis.
Statistical analyses were performed with GraphPad Prism and SPSS 25.0 (IBM). We used the Fisher's exact test, the Mann-Whitney test, and the log-rank test to compare continuous data, categorical data, and survival curves. Wilcoxon matching was used to compare consecutive data tests in the same group. All statistical tests were two-sided. A P value of 0.05 indicates statistical significance.

Patient Parameters.
From October 2016 to January 2018, 84 patients were included in our study ( Figure 1). Among them, 23 patients were excluded because of chemoradiation within 1 month prior to the procedure (n � 12), iodine-125 seed treatment (n � 6), with metal duct stent or heart pacemaker (n � 3), and severe arrhythmia (n � 2). Overall, the GEM-IRE group (31 patients) received gemcitabine and IRE simultaneously, and the IRE group (30 patients) received IRE alone. During the follow-up, 1 patient lost to follow in the GEM-IRE group. Finally, 60 patients were analyzed. e clinical features of the 60 included patients are given in Table 1.

OS.
e median follow-up time was 20.4 months (3.0-28.4 months). e median OS from diagnosis in the GEM-IRE group was significantly longer than that of those in the IRE group (21.5 vs. 16.7 months, respectively; hazard ratio (HR), 0.520; p � 0.019; Figure 4(a)). e median OS from IRE was also significantly longer in the GEM-IRE group than that of those in the IRE group (17.1 vs. 14.2 months, respectively; HR, 0.54; p � 0.031; Figure 4( (Tables 5  and 6). e major adverse reactions included two cases of pancreatitis (grade III), one case of severe neutropenia (grade IV), and one case of gastroduodenal artery hemorrhage (grade IV) in the GEM-IRE group. e case of severe neutropenia was diagnosed on day 5 and that of gastroduodenal artery hemorrhage on day 11 after IRE. After a subcutaneous injection of 300 μg of recombinant human granulocyte colony-stimulating factor (G-CSF), the neutrophil level increased to 5.6 × 10 9 /L within 24 hours. e gastroduodenal artery hemorrhage cases were controlled by vascular interventional embolization with 560-710 μm of gelatin sponge particles and 2 mm and 3 mm coils. e differences in the frequencies of adverse events were not statistically significant in both groups (p > 0.05 for all; Table 6). e 90-day mortality rate was 0% overall.
For routine blood tests, the postoperative hemoglobin level continued to significantly decrease in both groups on days 3 and 7 (p < 0.000, Figure 5(a)). e neutrophil counts showed a significant increase on day 1 (p < 0.000, Figure 5(b)); however, the levels recovered to preoperative levels by day 3, 7. ere were significant differences in the platelet counts (p � 0.0035, Figure 5(c)) and white blood cell counts (p � 0.0179, Figure 5(d)) after treatment between the two groups at days 3 and 7 (p � 0.0035) ( Figure 5).
For liver function, on postoperation day 1, ALT ( Figure 6(a)) and AST (Figure 6(b)) values for both groups indicated a significant rising (p < 0.000 for both), but the levels decreased rapidly on day 3 and recovered to preoperative levels by day 7. Interestingly, the two groups did not differ statistically significantly (p > 0.05 for all). No obvious     Journal of Oncology changes in alkaline phosphatase (ALP) (Figure 6(c)) were observed after treatment.

Discussion
Recent chemotherapy regimens have improved OS for LAPC patients only by single-digit months [30]. Due to the insensitivity to conventional chemotherapy, gemcitabine administration is suppressed by the peritumor stroma and pancreatic cancer patients with low pancreatic hENT1 levels exhibit a significantly lower response to gemcitabine. To overcome this obstacle, ECT was developed. ECT utilizes high-intensity electrical pulses to enhance cell membrane permeability to injected chemotherapies. Although ECT has been applied to solid tumors for the past 2 decades and has been proven effective [31,32], this was the first study to investigate simultaneous gemcitabine combination with IRE. Recent studies suggest that IRE combination with chemotherapy improves survival, but they were all induction chemotherapy regimens before IRE or postoperative adjuvant chemotherapy [33][34][35].
Our study was the first to demonstrate the antitumor effects of simultaneously combining gemcitabine with IRE. Our data showed that the GEM-IRE group had a longer OS (21.5 months). According to statistics, the median OS ranged from 17.9-27.0 months in some previous retrospective studies [34,[36][37][38]. In the aforementioned retrospective studies, patients who underwent induction chemotherapy achieved stable disease. us, simultaneous gemcitabine and IRE treatment had some survival benefit regardless of patient condition. Using percutaneous IRE as first-line treatment for LAPC without prior systemic treatment, Mansson et al. [39] found that the median OS after diagnosis was 13.3 months compared to 21.5 months in our study, indicating that  simultaneous gemcitabine and IRE achieved better survival than percutaneous IRE as first-line LAPC treatment.
Interestingly, although the treatment success rate was similar within both groups, the short-term tumor response in the GEM-IRE group was higher than in the IRE group. erefore, this study showed that the simultaneous treatment of gemcitabine and IRE is a better option for patients with LAPC. e main reason for this phenomenon might be that the peak plasma values of gemcitabine and its deaminated metabolite occurr within 30 minutes of injection. Continued treatment with IRE at this point prolonged its peak value and enhanced drug toxicity. is is consistent with findings by Shamseddine et al. that intra-arterial plasma gemcitabine peaks at 30 minutes [40]. Membrane permeation by electroporation enhances drug entry into the cell, enhancing cytotoxicity [26,27,41,42]. ese factors highlight the potential value of ECT in LAPC treatment.
IRE enhances drug transmission to cells via breaking up the dense mesenchymal tissues of tumors [28,43]. Considering LAPC's high heterogeneity, local control of the tumor by RE ablation followed by neoadjuvant chemotherapy may improve survival rates. us, neoadjuvant chemotherapy followed by simultaneous gemcitabine and IRE may benefit LAPC patients more.
In terms of safety, most of the postoperative adverse reactions seen in this study were classified as grade I or II based on CTCAE V4.0 standards, while 4 cases were considered severe (grade III or grade IV). Between the 2 groups, there were no significant differences of adverse reactions (p > 0.05). Although IRE is known to preserve the structure of blood vessels, we observed a case of hemorrhage in the gastroduodenal artery during follow-up. Similar complications have been reported in other studies of about 4-7% [33][34][35]44].
is may be because the    duodenum was infiltrated by a pancreatic tumor and IRE ablation caused a duodenal ulcer, allowing gastric acid exudation and, ultimately, blood vessel rupture and bleeding.
us, the combined use of laparotomy and gastrointestinal anastomosis may be an effective preventive method. Additionally, after the first postintervention day, ALT and AST levels increased significantly, suggesting that neither treatment has long-term effects on liver function and that the combination therapy was safe for LAPC treatment.
e findings of our study have a few limitations. First is small sample size. In the future, multicenter, prospective, randomized controlled, large-sample clinical studies are needed to support our results. Second, our study did not directly compare our treatments with conventional chemotherapy, including FOLFIRINOX, gemcitabine-based chemotherapy. us, larger, multicenter studies are being developed to support our results.

Data Availability
All data are available upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.   Journal of Oncology