Safety and efficacy of peptide receptor radionuclide therapy with 177Lu-DOTA-EB-TATE in patients with metastatic neuroendocrine tumors

Rationale: This study aimed to assess the safety, efficacy, and survival of 177Lu-DOTA-EB-TATE in patients with metastatic neuroendocrine tumors (NETs). Methods: Thirty patients with metastatic NETs were prospectively enrolled and treated with 177Lu-DOTA-EB-TATE (3 intended cycles at 8 to 12-week intervals, 3.7 GBq/cycle). Treatment-related adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. The treatment response was graded according to RECIST 1.1 and PERCIST 1.0 criteria. Kaplan-Meier analysis was performed to calculate progression-free survival (PFS) and overall survival (OS). Results: Patients tolerated therapy well without acute adverse effects. During peptide receptor radionuclide therapy (PRRT), no grade 4 toxicity was observed in any of the patients; grade 3 hematotoxicity was recorded in 4 patients, including grade 3 thrombocytopenia in 4 patients (13.3%) and grade-3 anemia in 1 patient (3.3%); grade 3 hepatotoxicity was recorded in 1 (3.3%) patient, and no grade 2/3/4 nephrotoxicity was observed. On long-term follow-up, none of the patients developed grade 4 hematotoxicity or nephrotoxicity of any grade, reversible grade 3 hematotoxicity (thrombocytopenia) occurred in 1 patient. There was no incidence of leukemia or myelodysplastic syndrome for the duration of follow-up. Of 27 patients with RECIST-measurable disease, partial response and stable disease were seen in 9 and 14 patients, respectively, resulting in a response rate of 33.3% and disease control rate of 85.2%. Of 29 patients evaluable for response on 68Ga-DOTATATE PET/CT, 14 had partial response and 11 had stable disease, with a response rate of 48.3% and disease control rate of 86.2%. The follow-up period ranged from 5 to 57 months after the first 177Lu-DOTA-EB-TATE PRRT with a median follow-up of 46 months. The median PFS was 36 months, and the median OS was not reached. Ki-67 index of greater than 10% was associated with poorer PFS (P = 0.012). Conclusions: Our results suggest that PRRT with approximately 3.7 GBq 177Lu-DOTA-EB-TATE has acceptable toxicity profile and is effective in treating metastatic NET with high disease control rate. In addition, 177Lu-DOTA-EB-TATE achieved a favorable survival outcome with encouraging PFS.


Introduction
Neuroendocrine tumors (NETs) represent a heterogenous group of neoplasms that arise from neuroendocrine cells throughout the body. The incidence rate of these tumors has increased substantially in recent years [1,2]. The choice of appropriate treatment in patients with unresectable metastatic NET is limited, and over 50% of NET patients are at an advanced stage at the time of diagnosis [3].
The majority of NETs overexpress somatostatin receptors (SSTRs), predominantly subtype 2 (SSTR2), which provides the basis for the use of SSTR2-targeted peptide receptor radionuclide therapy (PRRT). PRRT is a form of radiolabeled somatostatin analogue therapy, delivering radionuclides directly to NET tumor cells with high expression of SSTRs. At present, 177 Lu-DOTATATE is the most frequently used radiopharmaceutical in patients with advanced NETs. Increasing evidence has confirmed the safety and efficacy of PRRT with 177 Lu-DOTATATE for patients with advanced, progressive, and SSTRpositive NETs [4][5][6]. In the prospective Phase 3 NETTER-1 trial, 177 Lu-DOTATATE plus 30 mg long-acting octreotide demonstrated remarkedly longer progression-free survival and overall survival in midgut NET patients compared to 60 mg long-acting octreotide, which led to the approval of 177 Lu-DOTATATE (Lutathera) by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) [7].
A potential disadvantage of 177 Lu-DOTATATE is its rapid elimination from the blood stream, leading to insufficient tumor uptake and retention [8]. To improve the efficacy of PRRT, Evans blue modified octreotate was synthesized and conjugated to DOTA chelator (DOTA-EB-TATE). Evans blue reversibly binds to serum albumin [9,10], thus extending the effective plasma half-life of 177 Lu-DOTA-TATE. In preclinical and first-in-human dosimetry studies, 177 Lu-DOTA-EB-TATE demonstrated much higher tumor dose compared with 177 Lu-DOTATATE [11,12]. Our prior dose escalation study showed that 177 Lu-DOTA-EB-TATE with doses of 3.7 GBq/cycle achieved acceptable safety and satisfactory therapeutic efficacy after one-cycle treatment, and the more recent study of 177 Lu-DOTA-EB-TATE with escalating doses in multiple cycles further supported this result [13,14]. In the present study, we aimed to further investigate the safety, efficacy, and long-term outcome of 177 Lu-DOTA-EB-TATE with planned dose of 3.7 GBq/cycle in patients with metastatic NETs.   with metastatic NETs were prospectively enrolled in this trial to evaluate the safety and efficacy of repeated cycles of 177 Lu-DOTA-EB-TATE. The inclusion criteria for PRRT were as follows: (1) histologically confirmed NETs with high SSTR2 expression determined by 68 Ga-DOTATATE PET/CT imaging (modified Krenning score 3 = lesion uptake > liver; or 4 = lesion uptake ≥ spleen [15]), performed within 2 weeks before therapy; (2) white blood cell (WBC) ≥ 3 × 10 9 /L; (3) platelets (PLT) ≥ 60 × 109/L; (4) hemoglobin (Hb) ≥ 90 g/L; (5) serum creatinine level < 150µmol/L; (6) patient's readiness to provide clinical information and follow-up. Exclusion criteria were as follows: (1) patients with no or low SSTR-expression on 68 Ga-DOTATATE PET/CT (Krenning score less than 3); (2) pregnancy; (3) breast-feeding patients; (4) patients with known previous malignancies.

Treatment regimen
DOTA-EB-TATE was labeled with 177 Lu using a previously published method [16]. The radioisotope 177 Lu, in the form of 177 LuCl 3 was purchased from LuMark®, IDB, Holland. Briefly, DOTA-EB-TATE (100 µg dissolved in 20 µL of absolute ethanol) was added to 200 µL 0.5 M NaOAc (pH 5.6), and then, the required amount of radioactivity of 177 LuCl 3 was added. The mixture was heated at 100 °C for 30 min and then purified by a preconditioned C18 light SEP-PAK cartridge and passed through a 0.22 µm aseptic filtration membrane directly into a sterile vial. The quality control was performed with analytical thin-layer chromatography (Bioscan, USA). CH3OH: NH 4 OAc (v/v 1:1) was used as the developing solution. The radiolabeling yield was greater than 90% and the radiochemical purity of 177 Lu-DOTA-EB-TATE was more than 95%. The administration of radiopharmaceutical was performed in the ward. Pre-set dose of radiopharmaceutical was slowly administered intravenously over 30 min. In order to protect the kidneys, the mixture of arginine and 5% glucose solution (25 g/L, 1000 mL) was administered at least 30 min before radiopharmaceutical administration and lasted for 4 h. The planned administered dose per cycle was 3.7 GBq. The treatment was planned for up to 3 cycles, and cycles were repeated at intervals of 8 to 12 weeks.

Safety evaluation
All patients were clinically monitored during the administration of radiopharmaceutical and for 3 days thereafter as inpatients. Laboratory examinations including hematological parameters, renal function tests, and liver function tests were performed before, 2 weeks, and 4 weeks after each cycle during the course of PRRT, as well as during follow-up (at 4-6 months intervals after completion of PRRT). Short-and long-term adverse events were graded in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0 (CTCAE 5.0). The rates of adverse events at baseline, 2 weeks, and 4 weeks after each cycle of therapy, and during follow-up were recorded.

Response evaluation
The treatment response assessment was performed 2-3 months after each cycle of PRRT. RECIST 1.1 criteria was applied to evaluate morphological response following PRRT with contrast-enhanced CT or MR. The molecular response was evaluated according to PERCIST 1.0 criteria [17] with 68 Ga-DOTATATE PET/CT. Partial response was defined as ≥ 30% reduction in tracer uptake, progressive disease was defined as ≥ 30% increase in tracer uptake or appearance of new lesions; and stable disease as neither partial response nor progressive disease on 68 Ga-DOTATATE PET/CT.

Progression-free survival and overall survival
Progression-free survival (PFS) was defined as the start of the first 177 Lu-DOTA-EB-TATE PRRT cycle to date of radiographically confirmed progression by RECIST 1.1 or PERCIST 1.0 criteria. Overall survival (OS) was defined as the start of the first PRRT to death or last follow-up.

Statistical analysis
All statistical analyses were carried out using SPSS Statistics for Windows version 26.0 (IBM Corp, Armonk, NY, USA). Continuous variables were expressed as mean ± standard deviations; non-continuous variables were expressed as counts and proportions. Differences between paired samples before and after treatment were compared using nonparametric testing or Student's test. Univariate Cox proportional hazards regression model was used to perform the survival analysis. Kaplan-Meier curves were used to depict the survival. All statistical tests were 2-tailed, and a P value less than 0.05 was considered statistically significant.

Patients
The details of patients' baseline clinical characteristics are listed in Table 1 (Supplemental  Table 1). In total, 30 patients who met the eligibility criteria were enrolled (17 men, 13 women). Of 30 patients, 18 patients (60%) had pancreatic NET, and the remaining 12 patients included rectal NET (n = 5), small intestinal NET (n = 4), paraganglioma (n = 2), and unknown primary site with metastases (n = 1). World Health Organization grades of tumor differentiation included grade 1 in 4 patients, grade 2 in 24 patients, and grade 3 in 2 patients. Approximately 3.7 GBq per cycle (average: 3.84 ± 0.70) of 177 Lu-DOTA-EB-TATE was administered. Of 30 patients, 17 did not complete 3 cycles of 177 Lu-DOTA-EB-TATE. Among those patients, 3 patients discontinued treatment because of hematotoxicity (grade 3 thrombocytopenia). Two patients withdrew voluntarily. Eight patients were delayed due to the COVID-19 lockdown. One patient discontinued treatment because of late enrollment time. Two patients discontinued treatment due to disease progression. One patient died due to pulmonary infection. The median cumulative administered activity was 8.97 GBq (range, 3.19-14.32 GBq) over a median of 2 cycles (range, 1-3). Treatment cycles and cumulative administered activity are summarized in Table 2.

Efficacy of 177 Lu-DOTA-EB-TATE
Of the 30 patients enrolled, tumor response assessment based on RECIST criteria was available for 27 patients after 177 Lu-DOTA-EB-TATE PRRT. Nine patients had partial response and 14 patients had stable disease, resulting in a response rate of 33.3% and a disease control rate of 85.2%. Four of those 27 patients had progressive disease. Tumor response assessment based on 68 Ga-DOTATATE PET/CT was available for 29 patients after 177 Lu-DOTA-EB-TATE PRRT. The partial response was seen in 14 patients, stable disease in 11 patients, and progressive disease in 4 patients, with a response rate of 48.3% and disease control rate of 86.2%. According to the primary site, pancreatic NET and non-pancreatic NET showed no statistically significant difference in treatment response referring to RECIST (response rate: 33.3% vs. 33.3%, P = 1.000; disease control rate: 88.9% vs. 77.8%, P = 0.582) and PERCIST criteria (response rate: 58.8% vs. 33.3%, P = 0.264; disease control rate: 94.1% vs. 75%, P = 0.279). Representative cases of 177 Lu-DOTA-EB-TATE PRRT efficacy on 68 Ga-DOTATATE PET/CT are shown in Figure 1 and Supplemental Figure 1.
Four patients received PRRT before (PRRT re-treatment). Three patients were previously treated with 177 Lu-DOTATATE PRRT (7.4 GBq/cycle), to which one showed partial response and two showed stable disease. One patient was previously treated with 177 Lu-DOTA-EB-TATE (1.11 GBq/cycle) and demonstrated progressive disease. After repeated PRRT with 3.7 GBq/cycle of 177 Lu-DOTA-EB-TATE, 3 patients (3/4) showed partial response and the remaining one patient (1/4) showed stable disease on 68 Ga-DOTATATE PET/CT. An example is shown in Figure 2.

Progression-free survival and overall survival
Follow-up data was available and ranges from 5 to 57 months after the start of the first PRRT in this study. The median follow-up period was 46 months (interquartile range, 13-50 months). The median PFS was 36 months, while the median OS was not reached. The observed PFS rates at 12 months, 24 months, and 36 months were 79.5%, 60.7%, and 47.2%, respectively; the observed OS rates at 12 months, 24 months, and 36 months were 92.9%, 79.7%, and 59.8%, respectively. In the univariate analysis, Ki-67 index was the only variable found to be significantly associated with PFS (Table 5). Therefore, a multivariate analysis was not performed. Of 14 patients with stable disease by RECIST criteria, those with partial response by PERCIST had longer PFS (median: not reached vs. 33 months; P = 0.817) and OS (median: not reached vs. 35 months; P = 0.109) than those with no response, whereas the difference was not statistically significant, probably due to the limited number of patients. Kaplan-Meier curves for PFS and OS after PRRT in this study are shown in Supplemental  Figures 2 and 3.

Discussion
Hematological and renal toxicity are considered as the main side effects and dose-limiting factors for PRRT. In this study, the rate of severe hematotoxicity rate was rather low with 13.3% of the patients experiencing grade 3 thrombocytopenia, and 3.3% experiencing grade 3 anemia, who presented with grade 3 anemia already at baseline. This rate was similar to the previously reported 177 Lu-DOTATATE PRRT studies that described grade 3/4 hematotoxicity rate of 3.1%-15% [7,[18][19][20][21][22][23][24]. Recently, Garske-Román et al. reported data on 200 NET patients receiving 177 Lu-DOTATATE, and grade 3/4 hematotoxicity was found in 30 patients (15%) [25]. In the study of Bergsma et al., grade 3/4 hematotoxicity was observed in 11% of patients treated with 177 Lu-DOTATATE PRRT [19]. For the 4 patients presented with grade 3 thrombocytopenia in this study, all were previously treated with multiple courses of chemotherapy or sulfatinib/everolimus, which are risk factors associated with hematotoxicity [26]. In addition, the platelet counts at baseline in 2 of them were only slightly higher than the lower limits (109 and 118 × 10 9 /L; normal 100-300 × 10 9 /L). These data confirmed the importance of baseline myeloid function and careful evaluation of risk factors for relevant toxicity. Regarding nephrotoxicity, no grade-2/3/4 toxicity was observed in our cohort, and renal function demonstrated by creatinine remained stable during the therapy.
Long-term hematotoxicity and nephrotoxicity are of particular concern in this trial. On follow-up, we observed low incidence of grade 3 hematotoxicity (3.4%) and no nephrotoxicity of any grade. Acute leukemia and MDS are rare delayed side effects and severe complications related to PRRT, which have been reported previously to occur in approximately 2% of patients [22]. In our study, none of the patients developed MDS or leukemia during the follow-up period. Our data confirm that PRRT with 177 Lu-DOTA-EB-TATE is a relatively safe therapeutic procedure in view of the low occurrences of shortand long-term toxicity.
In terms of efficacy, we observed a response rate of 33.3% and a disease control rate of 85.2% on RECIST criteria, and a response rate of 48.3% and a disease control rate of 86.2% on PERCIST criteria. These results are not inferior to that reported for 177 177 Lu-DOTATATE (dose: 5.55 to 7.4 GBq/cycle). They found a response rate of 30% and 31%, and a disease control rate of 90% and 88% on RECIST and PERCIST criteria, respectively [27]. Another study performed by Parghane et al. analyzed 57 GEP-NET patients receiving PRRT with 177 Lu-DOTATATE (dose: 7.4 GBq/cycle). They reported a response rate of 40% and 31%, and a disease control rate of 92.9% and 94.7% referring to RECIST and PERCIST criteria, respectively [28]. In a recent meta-analysis on the efficacy of 177 Lu-DOTATATE in NET patients, the pooled response rate and disease control rate were 27.58% and 79.14%, respectively, based on the RECIST criteria [29]. These findings suggested that 3.7 GBq/cycle of 177 Lu-DOTA-EB-TATE seems to be as effective as 5.55-7.4 GBq of 177 Lu-DOTATATE for tumor control.
Treatment options are limited for patients who experience recurrence after PRRT, which led to the investigation of potential practice of PRRT re-treatment. Several studies have reported a safety profile for PRRT re-treatment that is similar to initial PRRT [30][31][32]. However, the performance of PRRT re-treatment was reported to be poorer than initial PRRT [32,33]. The relatively poor performance of PRRT re-treatment was probably because patients have acquired a level of radio-resistance after previous PRRT. In the present study, 4 patients in our cohort were previously treated with PRRT, and repeated PRRT with 177 Lu-DOTA-EB-TATE performed well for tumor control. Our preliminary findings indicate the potential benefit of 177 Lu-DOTA-EB-TATE in PRRT re-treatment setting. In the future, it would make sense to apply 177 Lu-DOTA-EB-TATE to PRRT re-treatment patients to investigate its potential value in this special setting.
In the present study, we observed a median PFS of 36 months after a median follow-up of 46 months, while the median OS was not reached. Our result compares favorably with those of previous 177 Lu-DOTATATE studies, which reported a median PFS ranging from 26 to 37 months [4,18,23,34]. Ezzidin et al. in a retrospective study of 74 patients with metastatic NETs treated with 177 Lu-DOTATATE, reported a median PFS of 26 months [35]. In a large group of 443 NET patients treated with 177 177 Lu-DOTATATE and reported a median PFS of 37 months [36]. In the absence of a randomized trial comparing 177 Lu-DOTA-EB-TATE with 177 Lu-DOTATATE, these data seem to indicate that the survival benefit obtained with 177 Lu-DOTA-EB-TATE at lower dose is not inferior to 177 Lu-DOTATATE, at least in terms of PFS. We also found that Ki-67 index of greater than 10% was significantly associated with poorer PFS. A previous study has also reported that higher Ki-67 index was associated with poorer prognosis in NET patients treated with 177 Lu-DOTATATE PRRT [35], and this finding warrants further investigation. This study has several limitations. First, the main limitation of this study is the small population size, which might lead to some bias. The number of PRRT re-treatment patients was also limited. Still, our preliminary data were the first to report the potential benefit of 177 Lu-DOTA-EB-TATE in this setting, encouraging study with larger sample size to provide stronger evidence for the effect of 177 Lu-DOTA-EB-TATE in PRRT re-treatment patients. The second limitation is the lack of control group of standard PRRT with 177 Lu-DOTATATE. Thus, the performance of 177 Lu-DOTA-EB-TATE in this study was compared with the published literature. In the future, we will design a randomized controlled trial to compare 177 Lu-DOTA-EB-TATE and standard 177 Lu-DOTATATE to further determine the value of 177 Lu-DOTA-EB-TATE. The third limitation is the limited length of follow-up period. Despite this shortcoming, we were able to report the long-term toxicity and survival outcome of 177 Lu-DOTA-EB-TATE for the first time. Furthermore, the fact that most patients did not complete the planned 3 cycles due to various reasons may have affected the reported toxicity profile of 177 Lu-DOTA-EB-TATE in this study.

Conclusion
Our results suggest that PRRT with approximately 3.7 GBq 177 Lu-DOTA-EB-TATE has acceptable toxicity profile and is effective in treating metastatic NET with high disease control rate. In addition, 177 Lu-DOTA-EB-TATE achieved a favorable survival outcome with encouraging PFS. Future prospective randomized studies in larger number of NET patients comparing 177 Lu-DOTA-EB-TATE with standard 177 Lu-DOTATATE are warranted.