Immunotherapy for gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs): a 2021 update

Abstract

Neuroendocrine neoplasms (NENs) are a group of heterogeneous malignancies, arising from the neuroendocrine system. These neoplasms are divided into two distinct groups, the low-proliferating, well-differentiated neuroendocrine tumors (NETs), and the highly-proliferating, poorly-differentiated neuroendocrine carcinomas (NECs). Recent data demonstrate that the incidence of gastroenteropancreatic (GEP) neuroendocrine neoplasms, GEP-NETs and GEP-NECs, has increased exponentially over the last three decades. Although surgical resection is considered the best treatment modality, patients with GEP-NETs often present with advanced disease at diagnosis associated with a 5-year survival rate of 57% for well-differentiated tumors, and only 5.2% for small-cell tumors. Immunotherapy is a novel treatment approach, which has demonstrated effective and promising therapeutic results against several types of cancers. In the present study, we review the current ongoing clinical trials and to evaluate the efficacy of immunotherapy in GEP-NENs. Furthermore, we analyze the importance of tumor genetic profiling and its clinical implications in immunotherapy response.

Introduction

Neuroendocrine neoplasms (NENs) are rare tumors, accounting for approximately 7 per 100,000 of all cancers nowadays, whereas gastrointestinal NETs are responsible for about 70% of all NEN cases [1]. Based on clinical findings, molecular differences and proliferation rates, NENs are divided into two distinct groups, the low-proliferating, well-differentiated neuroendocrine tumors (NETs) and the poorly-differentiated neuroendocrine carcinomas (NECs) [2] while according to Ki-67 proliferation index, they are graded as Grade 1, 2 and 3 with Ki-67 < 3%, Ki-67 3–20% and Ki-67 > 20%, respectively [3].

GEP-NECs have been characterized as very aggressive malignancies, with a median survival of 5–10 months and a 5-year survival rate of less than 5%, with the therapeutic management of these patients being limited to chemotherapeutic regimens based on platinum-etoposide derivatives [4]. On the other hand, prognosis of GEP-NETs depends largely on the primary site with gastric neuroendocrine tumors (gastric-NETs), exhibiting a higher 5-year survival rate compared to both, colonic neuroendocrine tumors (colonic-NETs) and pancreatic neuroendocrine tumors (pancreatic-NETs); 64.1%, 54.6%, and 37.6%, respectively [5]. GEP-NETs may also display marked heterogeneity as far as the most suitable therapy is concerned, due to the fact that a significant proportion of these lesions may be functional, secreting specific hormones, manifesting as specific clinical syndromes, that should be addressed accordingly. [6].

Although surgical resection is often the first option for localized, non-metastatic disease followed by pathway-based systemic chemotherapy, patients with GEP-NENs often present with advanced disease at initial diagnosis with 5-year survival rates of 57% for well-differentiated tumors, and only 5.2% for small-cell tumors [7]. Traditional cytotoxic chemotherapeutic regimens, that are commonly used in other types of malignancies show limited effectiveness in most GEP-NETs, which are well-differentiated with only low to moderate proliferation rates [8]. Recent approvals of immunotherapy regimens by Food and Drug Administration (FDA) have expanded our management tools against many types of cancers, paving a novel pathway in surgical oncology [9]. It is well known, due to multiple studies that cancer cells escape immunosurveillance by increasing the expression of immune checkpoint receptors; Programmed cell Death protein-1 (PD-1) and its ligand; Programmed cell Death protein Ligand-1 (PD-L1) as well as cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), thereby preventing T- cells from eliminating tumor cells [10]. Development of antibodies that bind and inhibit such molecules, immune checkpoint inhibitors, have lately shown promising therapeutic outcomes (Fig. 1). Immunotherapy management may include the utilization of oncolytic viruses, in order to infect and destroy cancer cells [11]. Cancer-targeted newly developed vaccines, which activate immune response due to tumor-associated antigens (TAAs) presentation by major histocompatibility complex (MHC) class I molecules in antigen-presenting cells (APCs), may be an alternative approach. TAAs are usually originated either from whole-cell tumor lysates, full-length tumor proteins, recombinant tumor peptides or even DNA vaccines, in order to effectively activate immunity against cancer [12, 13]. Another approach, described as adoptive cell transfer (ACT), is also widely implemented. This approach uses cancer patients’ autologous TILs, identified ex vivo, stimulated and modified to be redirected toward relevant tumor antigens via engineered T-cell receptors (TCRs) or chimeric antigen receptors (CARs) that are being re-infused back to patients suffering from cancer [14]. Several clinical trials using immunotherapy in NENs are ongoing. In this review, we summarize key clinical trials conducted so far in NENs of the gastrointestinal tract.

Fig. 1.

Immune checkpoint inhibitors. a Binding of B7-1/B7-2 on antigen-presenting cells (APC) to CTLA-4 keeps the T-cells inactive to kill tumor cells. Blocking of CTLA-4 with an immune checkpoint inhibitor (anti-CTLA-4 antibody) allows T-cells activation and tumor destruction. b Binding of checkpoint proteins, PD-L1 on tumor cells to PD-1 keeps the T-cells inactive to kill tumor cells. Blocking of PD-L1 or PD-1 with an immune checkpoint inhibitor (anti-PD-L1 or anti-PD-1) allows T-cells activation and tumor destruction

Immune checkpoint inhibitors

PD-L1 expression is associated with high-grade classification (G3) and aggressive tumors, 0% in grade1, 78% in grade2 and 100% in grade3 [15, 16] while, PD-L2 was observed in a high proportion of neuroendocrine tumor cells [17]. According to these studies, checkpoint blockade may have a potential role in treating patients with this disease, and therefore, several clinical trials have been conducted and also reported on this topic (Table 1).

Table 1.

Immunotherapy clinical trials, single intervention

Intervention	Cancer stage	Clinical phase/identifier	Median progression­free survival (months, 95% CI)	Median overall survival (months, 95% CI)	Response rates (%, 95% CI)	Ref.
Pembrolizumab	PD-L1-positive Advanced/ metastatic carcinoid or well/moderately differentiated panNETs	Phase Ib/ NCT02054806	5.6 (95% CI, 3.5–10.7) 4.5 (95% CI, 3.6–8.3)	21.1 (95% CI, 9.1–22.4) 21.0 (95% CI, 20.2-NR)	12.0% (95% CI, 2.5–31.2%) 6.3% (95% CI, 0.2–30.2%)	[19]
	Advanced multiple cancer types including NETs	Phase II/NCT02628067	4.1 (95% CI, 3.5–5.4)	NR	3.7% (95% CI, 1.0–9.3%)	[20]
	Advanced multiple cancer types including NETs MSI-H/ dMMR cancer	Phase II/NCT02628067	4.1 (95% CI, 2.4–4.9)	23.5 (95% CI, 13.5-NR)	34.3% (95% CI, 28.3–40.8%)	[49]
	Metastatic high-grade G3-NEN (Ki-67 > 20%)	Phase II/NCT02939651	2.28 (95% CI, 1.67–3.28)	3.85 (95% CI, 3.25-NR)	NA	[21]
Spartalizumab	Well-differentiated gastrointestinal panNET poorly differentiated GEP-NEC	Phase II/NCT02955069	NA	NA	0%, 3.0%, 4.8%,	[23]
Toripalimab	Advanced NENs (NETs, NECs, mixed adenoneuroendocrine carcinoma)	Phase Ib/NCT03167853	2.8 (95% CI, 1.6–4.0)	NA	28.6%, all patients 83.3%, PD-L1 (≥ 10%)	[25]
	Unresectable, advanced NENs (Ki-67 ≥ 10%)	Phase Ib/NCT03167853	3.8, PD-L1 (≥ 10%) 2.2, PD-L1 < 10% (HR 0.50; 95% CI: 0.24–1.06)	9.1, PD-L1 (≥ 10%) 7.2, PD-L1 < 10% (HR 0.55; 95% CI: 0.24–1.23)	50.0% in PD-L1 (≥ 10%) 10.7% in PD-L1 < 10%	[26]
Avelumab	Advanced (NEC-G3) and moderately differentiated (NET-G3)	Phase II/NCT03352934	NA	4.2 (95% CI, 1–12)	NA	[27]

panNETs pancreatic neuroendocrine tumors, MSI-H/dMMR high microsatellite instability/mismatch repair, NEN neuroendocrine neoplasms, NEC neuroendocrine carcinomas, epanNETs extrapancreatic neuroendocrine tumors, NR Not reached, NA Non Applicable

Pembrolizumab; is an anti-PD-1 antibody [18]. Safety and efficacy has been evaluated so far with contradictory results. KEYNOTE-028 study (NCT02054806) including PD-L1-positive locally advanced; metastatic carcinoid; well-differentiated or moderately-differentiated panNETs patients, reported an antitumor activity with an objective response rate (ORR) at 12% (95% Confidence Interval (CI), 2.5–31.2%), and 6.3% (95% CI, 0.2–30.2%) for carcinoids and panNETs, respectively. Median progression-free survival (PFS) was reported at 5.6 months (95% CI, 3.5–10.7) and 4.5 months (95% CI, 3.6–8.3 months), and median overall survival (OS) was 21.1 months (95% CI, 9.1–22.4) and 21.0 months (95% CI, 20.2 months-nr) for carcinoids and panNETs, respectively (Table 1) [19]. On the other hand, KEYNOTE-158 (NCT02628067), including patients with NETs of the lung, appendix, small intestine, colon, rectum, and pancreas [20] as well as the phase II study (NCT02939651), on patients with metastatic high-grade neuroendocrine neoplasms (G3-NEN, Ki-67 > 20%) [21], showed limited antitumor activity. KEYNOTE-158 study reported an ORR that reached 3.7% (95% CI, 1.0–9.3%), including 4 out of 107 patients, having partial response. Three patients had pancreatic and one patient had a gastrointestinal NET of unknown primary; all of them were PD-L1-negative. PFS was 4.1 months (95% CI, 3.5– 5.4 months) and median OS was not reached at the time of data cut-off [20]. NCT02939651 study reported a median PFS of 2.28 months (95% CI, 1.67–3.28) and a median OS of 3.85 months (95% CI, 3.25–NR) [21]. Nevertheless, the effect of pembrolizumab is currently studied as monotherapy in a phase II study (NCT03901378), or in combination with lanreotide acetate in patients with progressive metastatic, well or moderately-differentiated GEP-NETs (NCT03043664). Unfortunately, the rationale that lanreotide would synergize with pembrolizumab in low/intermediate grade GEP-NETs, was not confirmed in PLANET study [22]. The primary end point was ORR, with 39% of patients showing stable disease and 52% of them progressive disease. Median PFS was 5.4 months (95% CI, 1.7–8.3) and median OS was not reached at a median follow-up of 15 months [22].

Spartalizumab; a humanized monoclonal antibody against PD-1 receptor was studied in a phase II, (NCT02955069), multi-center study that included 32, 20, and 21 patients with well-differentiated GI-NET, panNET and poorly differentiated GEP-NEC, respectively [23]. Authors reported an ORR of 0%, 3%, and 4.8%, respectively [23]. GEP-NEC had higher proportion of PD-L1 expression in immune cells (43%), compared to those with panNET (23%) and GI-NET (10%) [23].

Toripalimab (JS001); a recombinant mAb that prevents binding PD-L1 and PD-L2, was studied in a Phase Ib (NCT03167853) study [24] including patients with advanced or metastatic non-functional NETs (Ki-67 ≥ 10%), well-differentiated NETs and poorly-differentiated NECs. Zhang et al. reported an ORR of 28.6% for all patients; 40% in patients with NET, and 25% in patients with NEC and a median PFS of 2.8 months (95% CI 1.6–4.0 months). The PD-L1-positive rate was 28.6% (6 out of 21 patients enrolled in the study). For the 6 PD-L1-positive patients (2 with NET and 4 with NEC), the ORR was 83.3% by i-RECIST and 66.7% by RECIST (Table 1). Moreover, a reduction in tumor size (sum of the longest tumor diameters) was documented for 42.8% of all patients included in the study [25]. In 2020 Lu et al. reported that from 40 patients included in their study, eight patients had partial response and six patients had stable disease, with an ORR of 20%. Patients with PD-L1 expression ≥ 10% or high tumor mutational burden (TMB), had significantly better ORR (50%), compared to PD-L1 < 10% (10.7%) and TMB-low patients (75% vs. 16.1%, P = 0.03). Median OS was higher in the PD-L1-positive group (9.1 months), compared to 7.2 months in the PD-L1 < 10% group (HR 0.55; 95% CI: 0.24–1.23), as well as the median PFS; 3.8 months compared to 2.2 months, respectively (HR 0.50; 95% CI: 0.24–1.06). [26].

Avelumab, efficacy and safety of anti-PD-L1 antibody avelumab was evaluated in 29 patients with advanced NEC-G3 and moderately-differentiated NET-G3, including pancreas; genito-urinary tract; stomach and esophagus; colon and rectum; lung; head and neck; and papilla of Vater, phase II trial (NCT03352934) [27]. Fottner et al. reported a median OS of 4.2 months (95% CI, 1–12) and mainly mild to moderate Treatment-related adverse events (TRAEs) [27]. Assessment of the efficacy and safety of avelumab in combination with regorafenib, is currently in recruiting status (NCT03475953), while a phase II (NCT03147404) study in patients with metastatic grade 3 GEP-NEC as second-line treatment after failure of platinum plus etoposide has been completed; however, no results are publicly available yet.

Alternative approaches; Oncolytic viruses have been so far used in other cancer types [28]. Encouraging preclinical results of AdVince, an oncolytic adenovirus adapted for treatment of liver metastases from neuroendocrine cancers [29], resulted in a phase I/IIa clinical trial (NCT02749331) for patients with liver-dominant NETs, currently under the recruiting phase. The adenovirus is designed to use the promoter from human chromogranin A, to selectively replicate in neuroendocrine cells and eliminate NET cells [29]. On the other hand, Mandriani et al. presented unpublished data on ENETS Conference 2020, concerning adoptive cell transfer, the development of CAR T-cells directed against somatostatin receptor (SSTRs)- expressing NET cells [30]. A clinical trial currently under recruiting status (NCT03411915), use the anti-SSTR2/CD3 mAb (XmAb18087), a humanized, Fc domain-containing, bi-specific mAb targeting CD3 and somatostatin receptor 2. This mAb, XmAb18087, binds both T-cells and SSTR2-expressing cancer cells and, therefore, may result in a potent Cytotoxic T Lymphocytes (CTL) response against the SSTR2-expressing cancer cells. Finally, a phase I trial (NCT03879694), which is currently under recruiting status, evaluates the safety and the mechanisms of action of Survivin, long peptide vaccine (SVN53-67/M57-KLH Peptide Vaccine), along with sargramostim and octreotide acetate, a somatostatin analogue. None of these trails has presented any results so far.

Combined therapeutic strategies

Combination of different therapeutic strategies has been shown to be beneficial for other types of tumours, therefore, this strategy might play a role as potentially beneficial for GEP-NENs patients as well. Temozolomide and platinum doublets represent the cornerstone of therapy in patients with advanced G2/G3 NETs and NECs, respectively [31, 32]. The role of nivolumab, in combination with temozolomide (NCT03728361), or with carboplatin and etoposide (NCT03980925), is currently being studied. Combination of nivolumab and temozolomide demonstrated promising preliminary efficacy in NET in an interim analysis of NCT03728361 clinical trial. 3 out of 12 patients (ORR 25%) had partial response, 8 out of 12 patients (67%) had stable disease, while 1 patient (8%) showed progressive disease [33]. A Phase II trial (NCT03074513) evaluated the efficacy of atezolizumab, a humanized mAb against PD-L1, in combination with bevacizumab (Table 2). This study demonstrated moderate clinical activity and was well-tolerated in patients with advanced NETs. ORR was reported at 20% (95% CI 6–44%) in the panNET cohort and 15% (95% CI 3–38%) in the extra-panNET cohort, while the median PFS was 19.6 months (95% CI 10.6-NR) and 14.9 months (95% CI 6.1-NR), respectively (Table 2) [34]. On the other hand, combination of nivolumab and ipilimumab (CTLA-4 inhibitor) (NCT02834013, NCT02923934) demonstrated a significant clinical activity in subgroups of patients with advanced NETs (Table 2) [35, 36]. The DART phase II trial (NCT02834013) reported that the overall objective responses in high-grade tumors was 44%, compared to 0% in patients with low-to-intermediate grade tumors. The 6-month PFS was 31%, and median OS was 11 months (Table 2) [35] while, the NCT02923934 study, which was performed in advanced NETs, an ORR of 24% was reported, with a median PFS of 4.8 months and a median OS of 14.8 months [36]. Nivolumab and ipilimumab or ipilimumab alone, is also evaluated as a phase 2 study in NCT03591731 [37]. Tremelimumab, another anti- CTLA-4 antibody; in combination with durvalumab (anti-PD-L1 antibody) is currently studied in a phase II (NCT03095274) study [38]. Capdevila et al. reported that (DUNE trial; GETNE 1601) when durvalumab was combined with tremelimumab, showed modest activity with the i-RECIST overall response rates for G1/2 gastrointestinal, G1/2 pancreatic and G3 NENs of gastroenteropancreatic origin of 0%, 6.3%, and 9.1%, respectively, whereas no new safety concerns have been observed in this large population of advanced NENs (Table 2) [39].

Table 2.

Immunotherapy clinical trials, combined therapeutic strategies

Intervention	Cancer stage	Clinical phase/identifier	Median progression­free survival (months, 95% CI)	Median overall survival (months, 95% CI)	Response rates (%, 95% CI)	Ref.
Atezolizumab/Bevacizumab	Advanced (grade 1–2) panNETs epanNETs	Phase II/NCT03074513	19.6 (95% CI 10.6-NR), panNET 14.9 (95% CI 6.1-NR), epanNET	NA	20% (95% CI 6–44%), panNET 15% (95% CI 3–38%), epanNET	[34]
Ipilimumab, Nivolumab	Any grade, epNENs	Phase II/ NCT02834013	6-month PFS was 31%	11	NA	[35]
	Advanced NETs	Phase II/ NCT02923934	4.8 (95% CI, 2.7–10.5)	14.8 (95% CI, 4.1–21.3)	24%	[36]
Durvalumab, Tremelimumab	Advanced gastrointestinal G1/2, pancreatic G1/2 and gastroenteropancreatic G3-NENs	Phase II/ NCT03095274	NA	9-m rate 36.1%, gastroenteropancreatic G3	0%, gastrointestinal G1/2 6.3%, pancreatic G1/2 9.1%, gastroenteropancreatic G3	[39]

panNETs pancreatic neuroendocrine tumors, NEN neuroendocrine neoplasms, NEC neuroendocrine carcinomas, epanNETs extrapancreatic neuroendocrine tumors, NA Non Applicable

Predictive and prognostic biomarkers in GEP-NENs

The exact identification of implicated biomarkers in GEP-NENs represents the ‘holy grail’ for cancer research, in order to follow a more personalized therapy for patient subgroups that would benefit from immunotherapy and other targeted therapies. Currently, several ongoing clinical trials (NCT02586844, NCT02092714 and NCT03130205) provide preliminary data for diagnosis and patients’ management.

PD-L1 and/or PD-1 expression in tumor cells and/or TILs, is confronted in 75% of the cases of gastroenteropancreatic neuroendocrine neoplasms [40]. PD-1-, CD8-, and FOXP3-positive TILs were more frequently associated with PD-L1-positive tumors, than with PD-L1 negative tumors [40]. Apart from PD-1/PD-L1 expression, a common predictor of tumor response is microsatellite instability (MSI) status; random mutations occurring in small repetitive elements due to a defect in the mismatch repair deficiency (MMR) system [41]. MSI-High (MSI-H) status indicates accumulation of random mutations in genes, increasing neo-antigen formation, and therefore, promoting the expression of inflammatory cytokines and T-cell activation, thus rendering tumors susceptible to immunotherapy [42]. MSI is observed in gastro-esophageal cancer [43], pancreatic cancer [44], 10–15% of sporadic colorectal cancer [45], while House et al., reported that hypermethylation of the hMLH1 promoter was present in 23% of pan-NENs [46]. The aberrant promoter methylation of the MMR gene hMLH1, as well other MMR genes (MSH2, MSH6 and PMS2), are associated with MSI in cancer cells [41]. Microsatellite analysis of extracted DNA in GEP-NETs performed by Ghimenti et al. reported that among 16 patients in the study only one patient with panNET showed genomic instability, while loss of heterozygosity (LOH) was demonstrated in 50% of patients [47]. Almost similar results were presented by a more recent work in GEP-NET patients, where addition to LOH, MSI and the methylation status of various tumor associated genes were examined [48]. None of the tumors was MSI-H; 13.2% were low-grade unstable MSI-Low and 86.8% of tumors showed no mutations in any of the five microsatellite markers and, thus, were classified as MSI-Stable (MSS), while LOH was found in 22.2% of the analyzed patients [48]. However, Hasegawa et al. [40] reported that the expression of MMR proteins (MSH2, MSH6, PMS2, and MLH1) in tissue samples from GEP-NENs, indicate MSI. According to results reported recently by Marabelle et al., as part of the KEYNOTE-158 study, pembrolizumab treated patients with previously treated, advanced non-colorectal MSI-H/dMMR cancer, demonstrated clinical benefit among these patients [49]. The authors reported a median PFS and a median OS of 4.1 months (95% CI, 2.4–4.9 months) and 23.5 months (95% CI, 13.5-NR), respectively. The ORR was 34.3% (95% CI, 28.3%-40.8%), while grade 3–5 TRAEs were reported only in 14.6% of patients. Taken together, these results indicate that high expression of PD-L1 and/or PD-1, as well as MSI in the tumor microenvironment, may lead to an immunologically appropriate strategy for these tumors.

Conclusion

Although the incidence of GEP NENs remains low in general population, this number is expected to show incremental growth, due to improvements in diagnostic imaging and physician awareness. Nevertheless, diagnosis for these patients is still delayed. At the time of initial diagnosis, patients often present with advanced disease, therefore surgical resection, even though it is considered to be the mainstay of treatment, is usually performed with palliative intent with no chance to be curative. Another major disadvantage in the current therapeutic strategies is that the majority of GEP-NET tumors respond poorly to chemotherapy, thus necessitating identification of novel therapeutic strategies and molecular biomarkers to increase the effective treatment in these patients. Although immunotherapy is well reported as a powerful therapeutic weapon in other cancer types, the success in GEP-NENs is vague, with only few clinical trials demonstrating limited activity and improvement in therapeutic response. Moreover, strategies such as cancer vaccines and adoptive cell therapy are poorly developed. Lack of specific mutations and predictive biomarkers in GEP-NENs, in order to be used as TAAs, are considered limiting factors for researchers’ effort to develop efficient cancer vaccines. Microsatellite instability, which is prone to accumulation of random mutations, increasing neo-antigen formation, and therefore, promoting T-cell activation, shows limited activity in GEP-NENs. Interestingly enough, the presence of PD-L1 (PD-L1 ≥ 10%) was found in 30% of the tumor cells and these patients had significantly higher ORRs and OS rates after treatment with anti-PD-1 immune checkpoint inhibitors, pembrolizumab and toripalimab, indicating that PD-L1 is a plausible biomarker for prognosis, and predicting improved survival of these GEP-NENs. Finally, combination regiments of immune checkpoint inhibitors have shown promising results and are expected to make progress in the field of NEN, as patients with high grade NETs or NEC were shown to gain clinical benefit, but further proof of concept phase III studies is required.