Efficacy and Safety of the Combination of Nano-Liposomal Irinotecan and 5-Fluorouracil/L-Leucovorin in Unresectable Advanced Pancreatic Cancer: A Real-World Study

Introduction: This retrospective study investigated the efficacy and safety of nano-liposomal irinotecan (nal-IRI) plus 5-fluorouracil/L-leucovorin (5-FU/l-LV) treatment in the second-line or later setting for advanced pancreatic cancer under real-world conditions. Methods: Between June 2020 and September 2021, a total of 44 patients with unresectable advanced pancreatic cancer treated with nal-IRI + 5-FU/l-LV in our affiliated hospitals were included. The prognosis, predictive factors (including systemic inflammation-based prognostic indicators), and adverse events were investigated. Results: The median age was 68 (interquartile range 62–73) years old, and 22 patients (50.0%) were male. Concerning tumor factors, 9 patients (20.5%) had local advanced disease and 35 patients (79.5%) had metastases. Twenty-five of the 44 patients were receiving second-line treatment, and 19 were receiving third-line or later treatment. The median overall survival (OS) and progression-free survival were 9.0 (range, 0.7–15.4) months and 4.4 (range, 0.6–15.4) months, respectively. The overall response rate was 5.3%. The disease control rate was 44.7%. Patients with a neutrophil-to-lymphocyte ratio of ≥2.7 had a significant risk of a poor OS (HR = 0.275, p = 0.017). Adverse events were manageable, although gastrointestinal symptoms and neutropenia were observed. The most common grade ≥3 adverse event was neutropenia, which was reported in 20% of patients. Conclusions: Nal-IRI + 5-FU/l-LV therapy was considered to be a useful regimen as second-line or later treatment for unresectable advanced pancreatic cancer, even in clinical practice.


Introduction
Despite recent advances in diagnostic technology and anticancer drugs, pancreatic cancer continues to have a poor prognosis worldwide [1]. It is the fourth leading cause of cancer-related death in the USA and Japan [1,2]. Approximately 70-90% of patients with pancreatic cancer are diagnosed at an advanced stage, and the 5-year overall survival (OS) rate is only 8-11% [3][4][5].
Nano-liposomal irinotecan (nal-IRI) consists of pegylated liposomes containing irinotecan sucrosofate salt, a topoisomerase I inhibitor [9,10]. Liposomal encapsulation reduces premature liver metabolism and conversion of irinotecan to the highly active SN-38 metabolite [9,10]. Nal-IRI exhibits a lower maximum concentration of free irinotecan in plasma, a longer half-life and a greater area under the curve in plasma for SN-38 [9,10] than non-liposomal irinotecan [9,10]. This prolongs tumor exposure to SN-38 above its antitumor activity threshold and increases the SN-38 levels in tumor tissue compared with plasma [9,10].
The phase III NAPOLI-1 trial demonstrated a better median OS with nal-IRI + 5-FU/L-leucovorin (5-FU/l-LV) than with 5-FU/l-LV (6.1 months vs. 4.2 months) [11,12]. A phase II trial in Japan also demonstrated a similar median OS of 6.3 months for nal-IRI + 5-FU/l-LV with a tolerable safety profile [13]. In Japan, this combination therapy has been available since June 2020 in the real-world setting. Therefore, there is limited post-approval real-world data regarding its efficacy, safety, and optimal sequencing in Japan. The NAPOLI-1 trial only enrolled patients who failed prior gemcitabine-based therapy.
Given the above, the present study investigated the efficacy and safety of nal-IRI + 5-FU/l-LV treatment under real-world conditions in Japanese patients. The predictive factors, including systemic inflammation-based prognostic indicators, were investigated.

Patients
Between June 2020 and September 2021, a total of 44 Japanese patients with advanced pancreatic cancer receiving nal-IRI + 5-FU/l-LV treatment at the Takasaki General Medical Center and its affiliated hospitals were included, and none were excluded from the current retrospective study. Patients were diagnosed with pancreatic cancer based on typical radiological findings or pathological findings. The authors retrospectively examined the medical records, collected patient characteristics and analyzed the outcomes, including the tumor response, OS, PFS, and adverse events (AEs).
Nal-IRI + 5-FU/l-LV treatment and the assessment of the tumor response and AEs. Nal-IRI (80 mg/m 2 ) was administered by intravenous infusion over 90 ± 10 min, followed by 200 mg/m 2 l-LV via intravenous infusion over 2 h and then 2,400 mg/m 2 5-FU via intravenous infusion over 46 ± 3 h, every 2 weeks. During screening, patients were tested for the presence of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1)*28 and UGT1A1*6 alleles to determine the starting dose for nal-IRI. A patient found to be homozygous with UGT1A1*28 or UGT1A1*6 or double heterozygous received a reduced starting dose of nal-IRI (60 mg/m 2 ). Treatment continued until the appearance of disease progression or unacceptable AEs.
Contrast-enhanced computed tomography or magnetic resonance imaging was carried out every 4-8 weeks. The tumor response was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The overall response rate (ORR) was defined as the sum of the complete response (CR), and partial response (PR). The disease control rate (DCR) was defined as the sum of the CR, PR, and stable disease rates. The OS was defined as the period from the day of initial nal-IRI + 5-FU/l-LV treatment to the day of death or last visit. The PFS was defined as the period from the day of initial nal-IRI + 5-FU/l-LV treatment to the day of the presence of disease progression or death.

Statistical Analyses
Categorical variables are presented as numbers and percentages, and continuous variables are presented as the median (interquartile range [IQR]). Differences between groups were analyzed by Fisher's exact probability test and the Mann-Whitney U test when a significant difference was obtained by the Kruskal-Wallis test. The prognosis was assessed using a Cox hazard analysis, the Kaplan-Meier method and a log-rank test. All statistical analyses were performed using the IBM SPSS (Statistics Package for Social Sciences) Statistics 25 software program (Chicago, IL, USA). p values of <0.05 were considered to indicate statistical significance.
DOI: 10.1159/000525742 significantly different from that in those treated with nal-IRI + 5-FU/l-LV as 3rd-line or later treatment (median 4.4 months; 95% CI 0.8-8.0 months; p = 0.583). Figure 2 shows the Kaplan-Meier curve of the OS according to the systemic inflammation-based prognostic indicators. The patients with an NLR < 2.7 (median, not reached) had a significantly better survival than those with an NLR ≥ 2.7 (median 6.0 months; 95% CI 4.98-6.95 months; p = 0.002; Fig. 2b). The patients with a CRP/Alb ratio <0.3 (median 9.8 months; 95% CI 7.82-11.71) also had a significantly better survival than those with a CRP/ Alb ratio ≥0.3 (median 5.1 months; 95% CI 0.0-10.31 months; p = 0.017; Fig. 2d). There were no significant differences in the OS regarding the mGPS score, PLR, or PNI. The OS in the patients treated with nal-IRI + 5-FU/ l-LV as 2nd-line treatment (median 9.0 months; 95% CI 6.2-11.8 months) was not significantly different from that in those treated with nal-IRI + 5-FU/l-LV as 3rd-line or later treatment (median 8.2 months; 95% CI 2.5-14.0 months; p = 0.802). Because the NLR calculated before the administration of nal-IRI + 5-FU/l-LV was a significant predictive factor for the prognosis, we also calculated the NLR at the time of the pancreatic cancer diagnosis (online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000525742). Five cases were missing data. Although the patients with an NLR of <2.7 (median 13.3 months; 95% CI 3.8-22.8 months) at the time of the pancreatic cancer diagnosis tended to have    better survival than those with an NLR ≥ 2.7 (median 6.8 months; 95% CI 2.7-10.9 months), the difference did not reach the statistical significance (p = 0.083). Online supplementary Figure 2 shows the PFS of earlier line treatment before nal-IRI + 5-FU/l-LV treatment. In the 24 patients treated with nal-IRI + 5-FU/l-LV as 2nd-line treatment, the median PFS of patients who received 1st-line treatment with GEM + Nab-PTX was 5.3 months (95% CI 4.4-6.1 months). The median OS of patients who received nal-IRI + 5-FU/l-LV as 2nd-line treatment was 9.0 months (95% CI 6.2-11.8 months). In the 13 patients treated with nal-IRI + 5-FU/l-LV as 3rdline treatment, the median PFS of patients who received 1st-line and 2nd-line treatment were 6.7 months (95% CI 3.9-9.6 months) and 6.7 months (95% CI 0.8-12.7), respectively. The median OS of patients who received nal-IRI + 5-FU/l-LV as 3rd-line treatment was 6.5 months (95% CI 1.2-11.8 months). First-line treatment included 8 patients with GEM + Nab-PTX, 4 patients with S-1, and 1 patient treated with mFOLFIRINOX. Second-line treatment included 5 patients treated with GEM + Nab-PTX, 4 patients treated with mFOLFIRINOX, 3 patients treated with S-1, and 1 patient treated with gemcitabine. The 6 patients treated with nal-IRI + 5-FU/l-LV as 4th-line or later treatment were excluded from this analysis. Although the PFS during 1st-line treatment in the patients with nal-IRI + 5-FU/l-LV as 3rd-line treatment tended to be better than in comparison to the patients who received nal-IRI + 5-FU/l-LV as 2nd-line treatment, the difference in PFS with 1st-line treatment was not statistically significant (online suppl. Fig. 2). Figure 3 shows the receiver operator characteristic analyses for the OS at the 6-month follow-up. The NLR (Fig. 3b, p = 0.019) and CRP/Alb ratio (Fig. 3d, p =  Online supplementary Figure 3 shows the Kaplan-Meier curve of the OS and PFS according to the UGT1A1 polymorphism. The OS in the patients with UGT1A1 non-poor metabolizer (median 9.0 months; 95% CI 7.2-10.9 months) was not significantly different from that in those with UGT1A1 poor metabolizer (median 4.1 months; 95% CI 0.0-10.9 months; p = 0.201; online suppl. Fig. 3a). The PFS in the patients with UGT1A1 non-poor metabolizer (median 4.4 months; 95% CI 1.8-7.1 months) was not significantly different from that in those with UGT1A1 poor metabolizer (median 2.0 months; 95% CI 0.0-6.0 months; p = 0.571; online suppl. Fig. 3b). Although it did not reach the statistical significances, the OS and PFS in the patients with UGT1A1 poor metabolizer tended to be worser than those in the patients with UG-T1A1 non-poor metabolizer. However, we cannot get the conclusive results because of small number of UGT1A1 poor metabolizer.

The ORR and DCR
The results associated with the tumor response are shown in Table 2. According to the RECIST, 2 patients (4.5%) had PR, 15 (34.1%) had stable disease (SD), 21 (47.8%) had PD, and 6 patients (13.6%) were not evaluable, while no patients had CR. Thus, the ORR and DCR in all patients were calculated to be 5.3% (2/38) and 44.7% (17/38), respectively. Table 3 shows predictive factors associated with the OS in patients treated with nal-IRI + 5-FU/l-LV by univariate  and multivariate analyses. According to univariate analyses, an NLR ≥ 2.7 (p = 0.004) and CRP/Alb ratio ≥ 0.3 (p = 0.023) were significant predictive factors of the OS. According to multivariate analyses, only an NLR ≥ 2.7 was selected as a significant predictive factor of the OS (p = 0.017).

AEs
The AEs during nal-IRI + 5-FU/l-LV treatment are summarized in Table 4. AEs were manageable, although gastrointestinal symptoms, such as nausea, loss of appetite, diarrhea, and constipation, were observed. The most frequent grade ≥3 AE was neutropenia, reported in 20% patients, followed by leukopenia, anemia, febrile neutropenia, and loss of appetite.

Discussion
The main finding of the present study was that nal-IRI + 5-FU/l-LV therapy was considered to be a useful regimen as second-line or later treatment for unresectable advanced pancreatic cancer, even in clinical practice. An NLR of ≥ 2.7 was a significant predictive factor for the OS. mGPS, modified Glasgow Prognostic Score; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; PNI, prognostic nutritional index. * p value <0.01. ** p value <0.05. In a systematic review of 71 studies in patients with unresectable advanced pancreatic cancer who received various second-line treatments, the median OS among all treatments ranged from 4.0 to 5.4 months [19]. Oxaliplatin has been investigated as a second-line treatment for patients with metastatic pancreatic cancer [20,21]. In the CONKO-3 trial, the median OS was 5.9 months for oxaliplatin + 5-FU/folinic acid as second-line treatment after first-line gemcitabine monotherapy [20]. In the PAN-CREOX phase 3 study, the median OS was 6.1 months for biweekly modified FOLFOX6 as second-line treatment after first-line gemcitabine monotherapy [21]. In the present study, the median OS was 9.0 months, which was considered to be satisfactory in comparison with the previous regimen of second-line treatment.
The median OS and PFS were reported to be 6.2 (95% CI 4.8-8.4) months and 3.1 (95% CI 2.7-4.2) months, respectively, in the NAPOLI-1 study [11,12]. A phase II trial in Japan also demonstrated a similar median OS and PFS of 6.3 and 2.7 months, respectively, with a tolerable safety profile [13]. The median OS and PFS in the current study seemed to be slightly better than those in NAPO-LI-1 study and the phase II trial in Japan. The ORR and DCR were previously reported to be 17% and 52%, respectively, in the NAPOLI-1 study [11,12], and a phase II trial in Japan demonstrated a similar ORR (17.5%) and DCR (52.5%) calculated based on the best response [13]. In the present study, the ORR and DCR were 5.3% and 44.7%, respectively, which were slightly worse than those values in previous reports. The American Society of Clinical Oncology Clinical Practice Guidelines for the treatment of metastatic pancreatic cancer recommend nal-IRI + 5-FU/l-LV as second-line therapy in patients previously treated with gemcitabine plus nab-paclitaxel [22], and the current National Comprehensive Cancer Network guidelines for the treatment of pancreatic cancer recommend nal-IRI + 5-FU/l-LV as category 1 second-line therapy for metastatic disease [23].
The NLR showed a significant difference as a predictive factor for the OS and response in the present study. Inflammation has recently been considered to play an essential role in cancer progression. A number of inflammation-based prognostic factors have been developed, including the GPS, mGPS, PLR, NLR, CRP/Alb ratio, and PNI [16][17][18][24][25][26]. Iwai et al. [27]. reported that a high NLR might be an independent indicator of a poor prognosis in patients with unresectable pancreatic cancer. In their study, the NLR was the best predictive factor among the GPS, mGPS, PLR, CRP/Alb ratio, and PNI [27]. Although all of these inflammation-based prognos-tic factors reached statistical significance in their study [27], only the NLR and CRP/Alb ratio reached statistical significance in the present study. In their receiver operator characteristic analyses for the OS at 6-month followup, the AUC area was the greatest for the NLR, followed in descending order by the CRP/Alb ratio, GPS, PNI, mGPS, and PLR [27]. The sequence of the AUC area in the present study was similar to the previous report: NLR, CRP/Alb ratio, mGPS, PNI, and PLR. If the number of patients were increased in our study, not only the NLR and CRP/Alb ratio but also other factors, namely the mGPS, PNI, and PLR, might have also shown statistical significance.
The mechanism underlying the relationship between the NLR and prognosis in patients with unresectable pancreatic cancer remains to be clarified. Neutrophils inhibit the immune response by lymphocytes, natural killer cells, or activated T cells [28,29], while lymphocytes reflect the immune response of the host to either infection or cancer. Tumor-infiltrating lymphocytes are reported to be associated with a good prognosis in patients with pancreatic ductal adenocarcinoma [30]. Baseline characteristics associated with long-term survivors who survived for more than 1 year in the NAPOLI-1 study [11,12] were a younger age, better performance status, lower NLR, lower CA19-9 level, and absence of liver metastases. Six of the 44 patients survived for more than 1 year from start of nal-IRI + 5-FU/LV treatment in the present study. Although the number of long-term survivors in our study was small, a lower NLR and lower CA19-9 level seemed to be associated with a long-term survival.
Liposomal encapsulation of nal-IRI reduces premature liver metabolism and conversion of irinotecan to the highly active SN-38 metabolite, a topoisomerase I inhibitor [9,10]. Nal-IRI can prolong the tumor exposure to SN-38 above its antitumor activity threshold in comparison to non-liposomal irinotecan [9,10]. Nanoparticles of SN-38 were recently reported to activate stimulator of interferon genes (STING) signaling pathway [31]. Interferon and the toll-like receptor 3 signaling pathway are potential pathways involved in the progression of pancreatic intraepithelial neoplasia to cancer and potential targets for pancreatic cancer treatment [32].
AEs were manageable, although gastrointestinal symptoms and blood cell AEs were observed. The most common grade ≥3 AE in this study was neutropenia (20.0%), followed by leukopenia (9.1%) and anemia (6.1%). In the NAPOLI-1 trial, grade ≥3 AEs included neutropenia (15.4%), a decreased white blood cell count (12.0%), and diarrhea (9.4%) [11,12]. DOI: 10.1159/000525742 The current study had several limitations. First, this was a retrospective study, and the number of patients was relatively small. Because the number of patients with prior irinotecan-based therapy was small, the effect of a history of irinotecan-based therapy could not be analyzed. Future studies should explore this point.
In conclusion, nal-IRI + 5-FU/l-LV therapy was considered to be a useful regimen as second-line or later treatment for unresectable advanced pancreatic cancer, even in clinical practice. An NLR of ≥ 2.7 was a significant predictive factor. Nal-IRI + 5-FU/l-LV therapy showed a good response with manageable AEs.

Statement of Ethics
This study protocol was approved by the Institutional Ethics Committee of National Hospital Organization Takasaki General Medical Center (2021-61) and each institution. It was conducted in compliance with the 1975 Declaration of Helsinki. Written informed consent was obtained from all patients before treatment, and this study received ethical approval for use of an opt-out methodology.