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Metronomic chemotherapy in patients with advanced neuroendocrine tumors: A single‐center retrospective analysis

Abstract Neuroendocrine tumors (NETs) are more commonly slow‐growing, therefore patients often receive chronic systemic therapies for tumor growth control and preservation of quality of life. Metronomic chemotherapy (mCT) is in line with this goal as it leads to stabilization of tumor growth over time without severe systemic toxicity. This is a retrospective analysis of patients with metastatic NETs receiving metronomic capecitabine (mCAP) or temozolomide (mTEM), at a NET‐referral center. The aims of the study were to explore activity and safety of mCT and relationships between some characteristics of the patient population and clinical outcomes. Among a total of 67 patients with metastatic well or moderately differentiated (W/M‐D) NETs, mostly gastroenteropancreatic (GEP) and nonfunctioning, 1.2 years (95% CI: 0.8–1.8) median progression‐free survival (mPFS), and 3.0 years (95% CI: 2.3–4.9) median overall survival (mOS) were observed. Disease control rate was 85%. Grade 3 adverse events occurred in 15% of patients in mCAP and 13% in mTEM, and were mostly hematological and gastrointestinal. At univariate and multivariate analysis none of the variables analyzed (treatment regimen, sex, age at diagnosis, site of primary tumor and metastases, number of previous mCT lines, baseline tumor status before mCT, Ki67 value) were significantly correlated to OS and PFS. Our retrospective study suggested that mCAP and mTEM can be active and well tolerated in patients with metastatic W/M‐D NETs, irrespective of the primary site, site of metastases, line of treatment and baseline tumor status.


| INTRODUCTION
Neuroendocrine tumors (NETs) represent a group of rare and well differentiated malignancies 1 with a very heterogeneous clinical behavior.
In the majority of cases, advanced NETs are relatively slow-growing, and patients often receive a sequence of systemic therapies in order to achieve tumor growth control and preserve quality of life.
Therefore, therapeutic schedules may play a crucial role in achieving these complex goals.
The therapeutic concept of administering agents continuously at lower doses is known as metronomic therapy. 2 The ability of tumor cells to mutate, developing resistance to different drugs, makes the metronomic chemotherapy (mCT) a way to potentially overcome possible changes in tumor behavior and evolution. 3 This could be mainly related to the reduction of the drug-free period, which represents a trigger for tumor cells to restart their progression. 4 The regular administration of mCT makes this therapy biologically active at any time point. Since mCT does not provide a cytotoxic effect, it maintains a significant proportion of sensitive alive cells within the tumor, meanwhile limiting the proliferation of the drug-resistant cell population. 5 The main role of mCT is also derived from its antiangiogenic mechanism of action. [6][7][8][9][10][11][12][13][14] In this context, vascular endothelial growth factor (VEGF) is one of the most important factors involved in the process of development of new tumor vessels. 13 Considering the high levels of VEGF secreted by neuroendocrine cells 15 which result in hypervascularization, NETs could potentially benefit from mCT. 16,17 A rich vascularization has been observed mainly in low-grade pancreatic NETs (panNETs) in comparison with high grade and other digestive epithelial neoplasms. [18][19][20] In addition, the metronomic schedule has proved to be effective on the immune system. Pre-and clinical studies showed that the antitumor effect has been linked to the depletion of immunosuppressive cells (T regulatory cells and myeloid-derived suppressor cells) 21 and mainly to the modulation of dendritic cells. Indeed, mCT induces dendritic cell maturation, which are crucial for the activation of an adaptive immune response, 22 and then enhances their function and the expression of tumor antigens and antigen-presenting molecules. Moreover, conversely to a conventional chemotherapy schedule, a regular low-dose of drug is deemed to interfere with a tumor's clonal evolution by targeting both tumor initiating cells and cancer stem cells. 8 The biological characteristics of advanced NETs represents the ideal basis for mCT application, with the aim to integrate the concepts of angiogenesis and angiogenetic machinery, tumoral microenvironment, cancer stem cells, and tumoral immunology. However, to date, most of the published body of evidence in this setting, include case reports, retrospective analyses 23,24 and very few phase II studies. [25][26][27][28] Furthermore, in these studies, mCT is often associated with other concomitant drugs and the patient population is heterogeneous in terms of primary site, biological features and previous treatments. For these reasons, despite the large use of chemotherapy in NETs, nowadays the use of metronomic schedules is not yet well defined in the main guidelines. 29 For these reasons, we conducted a retrospective analysis of patients with metastatic NETs from various primary sites treated with metronomic schedules of capecitabine (CAP) or temozolomide (TEM).
The aims were to evaluate the activity and safety of mCAP and mTEM, and to explore the relationships between some characteristics of the patient population and clinical outcomes.

| MATERIALS AND METHODS
This was a retrospective analysis of patients with a histological diagnosis of metastatic NET, receiving mCT at the European Institute of Oncology (IEO), ENETS-certified Center of Excellence (CoE) for gastroenteropancreatic (GEP) NETs. Patients treated with mCAP or mTEM from January 2007 to January 2020 were identified. They should have received mCT over a minimum period of one cycle, namely 12 weeks. The mCAP schedule could be 1000 mg/day continuously or 1500 mg/day continuously or 2000 mg/day continuously or 1000-2000 mg/day 2 weeks on/1 week off, orally taken two or three times per day arbitrarily set by physicians. The mTEM schedule could be 100 mg/day continuously or 100 mg/day 1 week on/1 week off or 2 weeks on / 1 week off, orally taken once per day, as determined by physician choice. The use of mCT was related to patient attitude or aspects such as performance status or comorbidities and represented an alternative option to "conventional" chemotherapy according to

| Statistical analysis
Continuous variables are reported as median and range, while categorical variables are reported as count and percentage. Progressionfree survival (PFS) is defined as the time from treatment initiation until disease progression or last contact without PD; overall survival (OS) is defined as the time between start of metronomic therapy and death for any cause or last contact. Survival curves for OS and PFS were estimated using the Kaplan-Meier method. The log-rank test was used to assess differences between groups. Median OS and PFS were reported with 95% confidence intervals (CIs). Univariate and multivariate Cox proportional hazard regression models were used to assess the association between treatment regimen and clinicopathological parameters with OS and PFS. All analyses were performed using SAS software version 9.4 (SAS Institute).

| Clinic-pathological characteristics
A total of 67 patients with metastatic NETs, with a median age of 54 years (range 12-80 years), predominantly males, were included.
All patients presented diagnosis of well or moderately differentiated   Table 1.
All patients received mCT, 76% of them mCAP and 24% mTEM; the various schedules are reported in Table S2. Most patients received mCT as more than a second-line therapy (Table S3). Concomitant SSA was administered in 52 patients (78%), while four patients underwent a concomitant nonsurgical locoregional treatment for liver metastases.
About 24% of patients had a baseline stable disease (SD), therefore, receiving mCT as a sort of "maintenance" treatment, whereas the others had a baseline radiological progressive disease (PD) according to RECIST criteria.
The DCR in the whole population was 85%, 87% in the mCAP group and 81% in the mTEM group. The analysis of DCR by site of the primary tumor showed rate of 88, 90 and 100% in the pancreas, midgut and GI extra-midgut, respectively, while a DCR of 69% was reported in lung population. Grouped by histological differentiation, a DCR of 82 and 92% was observed in well and moderately differentiated NETs, respectively (Table 3). Although the population of our study was heterogeneous in the site origin, we performed an efficacy analysis by arbitrarily distinguishing the whole population with a cutoff of Ki67 < 2, Ki67 2-10 by configuring "G2 low" NETs and Ki67 > 10 by configuring "G2 high" NETs, which showed an excellent DCR in all histopathological subgroups (Table S5).
Regarding the tolerability, about half of patients did not experience any grade of toxicity, whereas in the remaining patients most adverse events (AEs) were reported as mild to moderate. Six patients (15%) in mCAP group, and two (13%) in mTEM showed grade 3 (G3) AEs, such as diarrhea, neutropenia, stomatitis, renal failure, cutaneous and cardiac disorders ( Table 2). Furthermore, the analysis showed no correlation between SSA before mCT in any line treatment and survival outcomes (Tables S6 and S7).

| DISCUSSION
This retrospective study suggests that mCAP and mTEM can be active and well tolerated in patients with metastatic W/M-D NETs.
Interestingly, we observed an 85% DCR and more than 12 months of median treatment duration for more than half of the  Indeed, a potential advantage in ORR and DCR was noticed also in NET G3 patients. Lastly, the DCR did not differ significantly according to the primary site tumor, although we observed a lower DCR in lung disease than in gastrointestinal neoplasms. The overall DCR was promising, since more than half of the patients were still under treatment after 1 year of mCT.
Although the high rate of SD could be due to the SSA administration before mCT, we know from the literature that mCT also has no great capacity of inducing apoptosis, but due to their low drug levels, senescence can be one mechanism which occurs during mCT. 2 We did not observe significant correlations between survival and collected variablesincluding primary tumor site, treatment regimen,

| CONCLUSIONS
Our retrospective study revealed that mCAP and mTEM can be active