Since the first result from a retrospective study by Aldinger et al. in 1978 [26], and despite the absence of prospective trials, EBRT is recommended because it has been shown to improve median OS in retrospective studies, including reports from large nationwide databases such as SEER [5,19,68–76]. Moreover, this improved prognosis with EBRT is obtained through a multimodal treatment as shown, for example, in the SEER database analysis by Song et al. which reported EBRT, surgery and chemotherapy as prognostic factors on OS in multivariate analysis in 433 stage IVc patients (hazard ratio (HR): 0.562, *p* ≤ 0.001) [76].

**Figure 3.** Initial treatment of stages IVa, IVb and IVc ATC, adapted from [2].

In order to provide the best survival benefit to the patients, EBRT dose matters. Indeed, the total dose delivered seems to be predictive of survival and local control in most of the studies, with 45–60 Gy providing an optimal control, whatever the type of fractionation, across different studies [5,59,60,77,78]. In a report of 31 patients with no distant disease at presentation who were treated by chemoradiotherapy (2 Gy daily fraction) +/− surgery, a total dose >50 Gy was associated with a significantly better median OS (9.3 vs. 1.6 months, *p* = 0.019) [79]. In the National Cancer Database (NCDB) analysis on 1288 patients with non-resected ATC, Pezzi et al. reported an improved one-year OS rate for Stage IVb and IVc patients who received 60 to 75 Gy as compared with patients treated with less than 60 Gy (31% versus 16%, *p* = 0.019) [73]. To better define the optimal dose, Nachalon et al. analyzed the total EBRT dose to the gross tumor in three different groups of 26 patients, using full dose (70 Gy), high palliative dose (50 Gy) and palliative dose (maximum 30 Gy), and found an association between dose and improved survival (*p* < 0.001) [80]. Following R0 or R1 resection, guidelines recommend that good-performance status patients with no evidence of metastatic disease who opt for aggressive management should be offered standard fractionation IMRT with concurrent systemic therapy [2].

Besides dose, fractionation modalities have been investigated for optimal tumor control in ATC patients. Bi-fractionation radiotherapy schedules can be either hyperfractionated (multiple daily doses not exceeding 1.5 Gy) or accelerated (twice-a-day dose of 1.8–2 Gy). These schedules have been used in an attempt to overcome fast progression and radioresistance of ATC [81]. However, there have been no randomized studies to compare standard with altered fractionation; such studies may not be feasible given the rarity of this disease. In 2002, Tennvall et al. published prospective results for patients treated by association of chemotherapy and hyper-fractionated EBRT (30 Gy before surgery and 16 Gy after, twice daily either as 2 × 1 Gy or 2 × 1.3 Gy per day or 46 Gy after, twice daily as 2 × 1.6 Gy) and debulking surgery. They found a trend for hyper-fractionated radiotherapy with a better OS outcome (9% of patients (5/55) always alive after two years) and 60% (33/55) of local control [82]. In 2006, Wang Y et al. reported results from 23 ATC patients treated with radical EBRT (dose > 40 Gy), with once-daily (*n* = 14) or twice-daily fractionation (*n* = 9, 1.5 Gy per fraction). They found that hyper-fractionated treatment was well tolerated with a longer median OS (13.6 months for twice-daily radiotherapy versus 1.3 months for the other group; however, this was without statistical significance (*p* = 0.3). There were also no difference in terms of local control in the two groups [83]. A prospective study using accelerated radiotherapy with two daily fractions by De Crevoisier et al. provided encouraging results with 7 patients out of 30 alive after a median follow-up of 45 months and 47% local control rate [63]. Although bi-fractionation may provide better local tumor control, there is no definitive evidence and no data to suggest an improved OS with these techniques. Data on ATC are scarce but we can extrapolate from head and neck cancer data where altered fractionation increases acute but not late toxicity and improves local control [84].

Hypofractionation with dose per fraction > 5 Gy has been reported to prevent death from local recurrence (*p* = 0.025) but with grade > 3 toxicities in a series of 33 patients and no improved OS [85]. However, because hyperfractionation or accelerated regimens require more intensive resources and the data supporting their efficacy are scarce, recent studies have used regimens close to those used in EBRT of other head and neck cancers in 2 Gy per fraction [60,78,86–89]. In recent guidelines from the Italian Society of Radiation Oncology (AIRO) and Spanish Thyroid Group, bifractionation and standard fractionations are listed as two possible EBRT options, whereas hyperfractionation is discouraged in ESMO guidelines [3,90,91]. ATA guidelines recommend standard fractionation but do not discourage hyperfractionation [2].

Although different fractionation techniques do not seem to have a major impact in management, high-accuracy radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) should also be used for decreasing toxicities [92,93]. Indeed, IMRT can achieve a better target coverage and reduced dose to the spinal cord [92]. In a monocentric retrospective analysis of 28 ATC patients treated with IMRT and 13 treated with three-dimensional conformal radiotherapy (3D-CRT), a better progression-free survival (PFS) (median 5.1 vs. 2.6 months, *p* = 0.049) and OS (*p* = 0.005 for both) were found in multivariate analysis, possibly related to an improved total dose (*p* = 0.005) without increased toxicity [94]. This lower toxicity rate was not found in all reports of IMRT [79]. This IMRT technique is important to deliver the optimal dose to the different treatment volumes which most often include the operative bed, the thyroid, and lymph node areas I to VI with the upper mediastinum up to the carina [63,78,79,89,95]. Few relapses at the edge of the radiotherapy field (less than 10% marginal relapses) have been reported [96,97]. The rate of loco-regional failure rate may relate to the extent of the EBRT field, as reported by Kim et al., with a five-year local control rate of 40% in ATC patients treated with a large extended field (*n* = 12) (such as described before) vs. 9% in case of limited field EBRT (*n* = 11) to involved site (tumor and node) (*p* = 0.04). [98]. We proposed EBRT by IMRT with a total dose of 66–70 Gy into the tumor volume with a 5 mm margin and 50 Gy to the bilateral level II–VI cervical nodes, and upper mediastinal nodes to the carina +/− area I, with an element of compromise therefore required in efforts to achieve acceptable toxicity.

The optimal timing of EBRT in the patient's treatment schedule with respect to surgery and chemotherapy/systemic treatment has not been defined. In patients with resectable stage IVa disease, adjuvant EBRT is recommended [2]. However, it is possible that preoperative radiotherapy can enable surgery (example in Figure 2). In a monocentric report of 79 patients treated between 1972 and 1998, Besic et al. found that the 12 patients treated by neoadjuvant chemo-radiotherapy, surgery and additional adjuvant EBRT (total dose of 45 to 64 Gy) had a better median OS (14.5 months as compared with the 26 patients treated with primary surgery followed by EBRT (7 months)) [99]. Although interesting, these results may reflect a selection bias with neoadjuvant treatment allowing for the selection of patients without primary refractoriness and thus, better prognosis. Indeed, Arora et al. found a longer cause-specific survival in cases of postoperative EBRT versus preoperative EBRT (*p* < 0.0001) in an analysis of PDTC and ATC patients from the SEER database [100]. Data are still needed to confirm the best timing for EBRT in patients with resectable disease.

Beside surgery, the other major question is the optimal systemic treatment to combine with EBRT and especially chemotherapy. Since the 1970s, several authors have described the results of combinations, and there are a large number of different protocols. One of the most recent analyses of the SEER database, which included an inverse probability weighting (IPW) to balance variables between groups, reported that radio-chemotherapy was associated with improved OS (adjusted HR = 0.69, 95% CI: 0.56–0.85, *p* < 0.001) versus EBRT alone; this difference remained significant within each subgroup stratified by surgical resection and distant metastasis [101]. The most recent and only randomized study in this setting is the RTOG 0912 trial which randomized 99 patients (56% Stage IVb) undergoing 2–3 weeks of weekly paclitaxel 80 mg/m2 with placebo or pazopanib 400 mg/day followed by concomitant 66 Gy EBRT and weekly 50 mg/m2 paclitaxel with placebo or pazopanib 300 mg/day [102]. The one-year OS rate was 29% in the placebo group and 37.1% in the pazopanib group (*p* = 0.283) in the 80% of patients who were randomized and eligible for this treatment. This randomized trial provides the prospective results of chemo-radiation therapy in ATC patients which are in line with those of the different retrospective studies which reported one-year OS rates between 30 and 50% with either doxorubicin or paclitaxel/docetaxel-based chemotherapy, with or without platin [63,65,74,103] (Table 1). No chemotherapy regimen has been shown to have a better impact on OS in the different retrospective studies published to date (Table 1). In the late 1990s and early 2000s, bi-fractionated radiotherapy (46 Gy, 1.6 Gy per fraction) associated with doxorubicin (10–20 mg/m2 weekly) was widely considered as the standard of care but at least 10 different chemotherapy regimens were found in different series or even in single-institution cohorts [57,67,79] (Table 1). De Crevoisier et al. have studied the association of doxorubicin (60 mg/m2) and cisplatin (120 mg/m2) with radiotherapy (40 Gy in 1.23 Gy per fraction twice a day) within a prospective cohort. They

reported a three-year OS of 27% (95% CI: 10–44%) and a median OS of 10 months [63]. In a German multicenter study, Wendler et al. found that any kind of systemic treatment (doxorubicin weekly, paclitaxel, paclitaxel and pemetrexed, paclitaxel and carboplatin, doxorubicin and cisplatin and tyrosin kinase inhibitor) was associated with a longer OS for IVc patients (HR: 0.23, 95% CI: 0.08–0.64, *p* = 0.005), and combined paclitaxel and pemetrexed was associated with a statistically significant likelihood of longer OS compared with other regimens (*p* < 0.0001) [5]. To summarize, most guidelines recommend, in eligible patients without targetable molecular alteration, the administration of chemo-radiation with either doxorubicin or paclitaxel/docetaxel +/− platin. This chemo-radiotherapy constitutes the standard induction treatment for fit stage IVb-IVc patients and has been referred to as "bridging therapy" in the recent ATA guidelines: a therapy that helps to stop the fast progression of the disease while all molecular explorations are undertaken and accessibility to targeted therapy/immunotherapy is evaluated [2]. Finally, for patients with *BRAF* mutations who are eligible for the dabrafenib/trametinib combination, the question of when to perform radiation therapy remains open as it is usually indicated to halt treatment during EBRT. However, a phase I/II trial in melanoma has shown that concurrent radiation could be feasible [104] and a phase I trial in ATC patients to evaluate the feasibility of concurrent EBRT and dabrafenib/trametinib combination (NCT03975231) is ongoing (Table 2).

## **Table1.**OutcomeofmultimodaltreatmentinATC.

tant sites.

**Table 2.** Ongoing clinical trials in ATC patients.

ATC: Anaplastic thyroid carcinoma, IMRT: Intensity-modulated radiation therapy, NA: non-applicable. Advanced ATCs, understanding advanced ATCs locally and/or at distant sites.

#### *4.3. Systemic Therapies: Failure of Chemotherapies, Success of Targeted Therapies and the Promises of Immunotherapy*

Chemotherapy and EBRT are independent prognostic factors that are associated with improved survival [4–6,14,16,117,118]. However, clinical trials comparing different chemotherapy regimens in ATC patients are scarce because of the disease's low incidence and aggressiveness limiting enrollment in clinical trials, leading to poor statistical power and a limited treatment time-frame. In the absence of molecular abnormalities, the most recent ATA Guidelines recommend starting with systemic therapy with genotoxic drugs such as paclitaxel and carboplatin combinations, cisplatin and doxorubicin combinations, docetaxel and doxorubicin combinations, paclitaxel alone, or doxorubicin alone [2,3,119,120]. Table 1 summarizes the studies on the different chemotherapy protocols in ATCs. Given the estimated doubling time of ATC is only 3–12 days, the interval between the administrations of the chemotherapeutic agent has to be short [63]. In this objective, some authors recommend using chemotherapeutic regimens at relatively short intervals (such as weekly administration compared with 3–4-week intervals). The poor prognosis of ATCs is often associated with primary chemoresistance which results in an average PFS of less than three months (Table 2). Indeed, though paclitaxel seems the most effective chemotherapeutic drug, chemo-resistance is common, which may be related to TAMs infiltration. TAMs occupy 50% of the tumor volume and provides paracrine signals via the CSF-1/CSF-1R axis, which promotes tumor progression and therapy resistance. Thus, targeting the CSF-1/CSF-1R pathway in TAMs was shown to restore the sensitivity of thyroid cancer cells to paclitaxel [121].

Due to this primary chemoresistance, other therapeutic strategies have been developed. High-throughput sequencing investigations have unveiled the molecular alterations of ATC opening the way to targeted therapies [7,14,28,29,61]. The recent approval of a combination therapy with the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib for patients with unresectable or metastatic *BRAFV600E*-positive ATC has generated enthusiasm in the field. Indeed, a phase II basket trial describing the efficacy and safety of dabrafenib plus trametinib has enrolled 36 ATC patients, the median age of whom was 71 years; 30/36 (83%) patients had undergone prior tumor radiation. The ORR was 56% (95% CI: 38.1–72.1%), including three complete responses and a median PFS and OS of 6.7 and 14.5 months, respectively, without new safety signals identified with this additional follow-up confirming the results of previous studies [8,9,122]. These results have led to approval by the FDA but not by the EMA. Unfortunately, this combination can only be offered to the 20–50% of ATC patients with *BRAFV600E* mutation and acquired resistance to BRAF inhibitors may develop via secondary mutations in the MAPK pathway or via the PI3K/AKT/mTOR pathway, or Hgf/Met activation, underlining the need for additional rationally designed approaches [123–125]. Much rarer than *BRAFV600E* mutations are *NTRK, ALK* and *RET* fusions which can be found in 2–3% of ATC patients. However, finding one of these alterations might greatly impact the prognosis of the few mutated patients given the very high response rates observed with their corresponding highly specific inhibitors in different basket trials. A pooled subgroup analysis of the larotrectinib trials (NCT02122913 and NCT02576431) reported a 29% response rate in two out of seven ATC patients; responses lasted for 3.7 and 10.2 months and the median OS was 14.1 months (95% CI: 2.6–NE) [126]. A long-lasting, dramatic response to the RET-specific inhibitor selpercatinib has been reported in a 73 year old patient with *CCD6-RET* fusion ATC [127]. Lasting responses to ALK inhibitor in patients with ALK fusions have also been reported [128]. Although these targeted therapies have demonstrated impressive activity, availability is a major issue in most countries except in the US, where approval has been obtained from the FDA. On 31 July 2020, a conditional marketing authorization valid throughout the European Union (EU) was issued for entrectinib for the treatment of adults with NTRK fusion-positive solid tumors that are locally advanced, metastatic or where surgical resection is likely to result in severe morbidity. To the best of our knowledge, no approvals have been obtained in ATC for RET inhibitors. All protocol regimens are presented in Table 3.

Inhibitors targeting the PI3K/AKT/mTOR pathway, such as everolimus, have been tested but the results were disappointing, with none of the seven patients included in a phase II study benefiting from treatment [129]. A multicenter, phase II trial of everolimus in locally advanced or metastatic thyroid cancer of all histologic subtypes included six ATC cases with only one response, and the others had progressive disease with a median PFS of 10 weeks [130]. Another phase II study evaluated the combination of sorafenib and temsirolimus in two patients with ATC, of which only one had an objective response [131]. To our knowledge, no trials have looked at mTOR inhibitors in the context of ATC with mutation in the PI3K/AKT/mTOR pathway. Antiangiogenic treatments such as lenvatinib [132], a multikinase inhibitor approved in differentiated thyroid cancers, have shown initially encouraging results but limited activity in subsequent studies, with a risk of bleeding and fistula as this disease often invades the trachea, esophagus and vessels, and are thus not recommended [2,77,133]. A recent prospective phase II trial was halted for futility as the minimum ORR threshold of 15% was not met upon interim analysis with a 2.9% response rate, a median PFS of 2.6 months and a median OS of 3.2 months [134].

Over the past few years, immuno-oncologic treatments, especially ICI (e.g., anti-PD-1, anti-PD-L1 and anti-CTLA-4) have revolutionized the field of anti-cancer therapies in many entities. PD-L1 has been suggested as a predictive biomarker of response to ICI in several cancers although its robustness has been questioned. As described above, PD-L1 is often expressed on ATC tumor cells, suggesting new treatment opportunities for ATC with immunotherapy [10,51]. Indeed, spartalizumab, an anti-PD-1 antibody, has been studied in ATC [10]. The response rate was 19% (five PR and three CR observed). The median OS

in the entire cohort was 5.9 months, with 40% of patients alive at one year. The median PFS was 1.7 months. Interestingly, those patients with PD-L1 expression of <1% had a median OS of 1.6 months and there were no responses in this group; however, those with PD-L1 expression of 1–49% and ≥50% had a median OS that had not been reached and an overall response rate of 18% (2/11) and 35% (6/17), respectively. The highest rate of response was observed in the subset of patients with PD-L1 > 50% (6/17; 35%). It should be noted that spartalizumab is not FDA- or EMA-approved and is not commercially available. The ACSé basket trial evaluated pembrolizumab in rare cancers in France and included a cohort of 16 ATC patients. The response rate was 25% with a median duration of response of 7.3 ymonths in responder patients [13]. These results differ from those of a phase II study of pembrolizumab combined with chemoradiotherapy as initial treatment which enrolled only three patients, because all three patients died within six months [53]. One might hypothesize that ICI would be more effective without concurrent radiation therapy but further data will be needed to evaluate the best timing for ICI initiation and its clinical efficacy, and clinical trials are ongoing (Table 2). Although effective, ICI will only benefit a small group of patients. Therefore, combination strategies have been developed. In a phase II umbrella study, anti PD-L1 atezolizumab has been studied in combination with either vemurafenib or cobimetinib for *BRAF*-mutated patients (cohort 1), cobimetinib alone for *RAS-* and *NF1*-mutated patients (cohort 2) and bevacizumab for patients with no mutation (cohort 3) in a prospective multi-arm trial [135]. Median OSs were not reached in cohort 1; these were 18.23 months in cohort 2 and 6.21 months in cohort 3. The response rate was 71% in cohort 1 and 7% in cohort 2. Therefore, combination of ICI with targeted therapies in ATC patients with molecular alterations is very promising. In a retrospective study, Diercks et al. analyzed six patients with metastatic ATC treated with multikinase inhibitors (lenvatinib) and ICI (pembrolizumab) and showed 66% with complete remissions (4/6), 16% with stable disease (1/6), and 16% with progressive disease (1/6). The median PFS was 16.8 months and the median OS was 17.3 months [11].

These optimistic data may lead to a systematic screening of PD-L1 and/or MMR status. However, it will first be necessary to define a specific expression score for PD-L1 expression in ATCs and to correlate it with the clinical benefit. Because the ATC immune microenvironment is an immunosuppressive medium, the development of immunotherapy combinations to improve these results will also be required. Clinical trials using immunotherapy in combination with other systemic agents are underway (Table 2).

**Table 3.** Treatment protocols in Anaplastic Thyroid Carcinoma.

**Figure 4.** Molecular and treatment landscape of ATC (based on [2]). ATC: Anaplastic Thyroid Carcinoma, L1 and L2: Line of treatment one or two, IMRT: Intensity-Modulated Radiation Therapy, PD-L1: Programmed Death-Ligand 1.

#### *4.4. Reappraisal of Surgery in the Era of Targeted Therapies*

There has been substantial debate about the appropriate role for surgery in the management of locally advanced and metastatic ATC. Operative treatment of local disease offers the best opportunity for prolonged survival if the neoplasm is intrathyroidal. When the neoplasm is extrathyroidal, the operative approach is controversial, as some have found that neither the extent of the operation nor the completeness of the tumor resection affects survival [63,77,136,137]. Complete resection is recommended whenever possible for patients with confined ATC (stage IVa/IVb) in whom R0/R1 resection is anticipated, if excessive morbidity can be avoided [2,3,138]. Lateral compartment lymphadenectomy should be performed only in the setting of complete macroscopic resection. Resection of the larynx, pharynx and esophagus are discouraged [56,67,139].

ATC patients present with extensively invasive primary tumors in between 85 and 95% of cases [68,140] justifying the use of neoadjuvant therapy by chemotherapy, chemoradiotherapy (Figure 3) or by targeted therapy [82,99,115,141]. In BRAFV600E-mutated ATC patients, the successful use of neoadjuvant dabrafenib plus trametinib with or without immunotherapy has been described in a case series of six patients that eventually underwent surgery [142,143]. In these patients, adjuvant therapy was prescribed with the *BRAF-*directed therapy or with chemoradiation after surgery. OS at 6 and 12 months was 100% and 83%, respectively, but the locoregional control rate was 100%. These data have revigorated interest toward primary resection upfront or following initial response to treatment in ATC patients.

#### **5. Conclusions**

ATCs are aggressive tumors occurring most often in the elderly. The survival outcomes of anaplastic thyroid carcinoma remain poor. Their diagnostic and therapeutic management must be initiated quickly and has to be coordinated within an expert center network. The objectives of the treatment are to fight against the risk of suffocation, to control the tumor mass and to ensure optimal treatment of symptoms within a multidisciplinary team involving endocrinologists, medical oncologists and radiotherapists, palliative care, geriatric oncologists, surgeons, and radiologists in order to offer appropriate care at each stage of the disease. Multimodal treatment combining surgery, radiotherapy, chemotherapy or targeted therapy can allow the control of the tumor. The results of immunotherapy are encouraging but its place is yet to be defined. With increasing knowledge of the tumor biology, the identification of the underlying molecular pathways, modifications of the transcriptome, proteome and associated immunomodulatory mechanisms of ATC (TAMs) on the one hand, and the emerging role of novel, molecular-based single/multi-targeted therapies on the other hand, a growing number of clinical trials can be noted. Prioritizing clinical trial enrollment will be a key factor in advancing care for patients with ATC.

**Author Contributions:** Conceptualization, A.J. and J.H.; methodology, A.J., A.E., J.H. and C.D.C.; data curation, A.E., A.A.G. and B.C.; writing—original draft preparation, A.J., A.E., J.H. and C.D.C.; writing—review and editing, A.J., J.H., C.D.C., A.A.G., B.C., F.D., P.B., D.H., L.L. (Livia Lamartina), E.B., L.L. (Ludovic Lacroix) and C.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We would like to thank Clio Baillet for providing the images in Figure 2.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Carcinoid Crisis: A Misunderstood and Unrecognized Oncological Emergency**

**Camilla Bardasi 1, Stefania Benatti 1, Gabriele Luppi 1, Ingrid Garajovà 2, Federico Piacentini 1, Massimo Dominici 1 and Fabio Gelsomino 1,\***

**Simple Summary:** In this review, the Authors are going to discuss the main highlights of the Carcinoid Crisis, an uncommon manifestation related to neuroendocrine tumors, focusing on the potential etiopathogenetic mechanisms, clinical implications, potential treatments and prophylaxis.

**Abstract:** Carcinoid Crisis represents a rare and extremely dangerous manifestation that can occur in patients with Neuroendocrine Tumors (NETs). It is characterized by a sudden onset of hemodynamic instability, sometimes associated with the classical symptoms of carcinoid syndrome, such as bronchospasm and flushing. Carcinoid Crisis seems to be caused by a massive release of vasoactive substances, typically produced by neuroendocrine cells, and can emerge after abdominal procedures, but also spontaneously in rare instances. To date, there are no empirically derived guidelines for the management of this cancer-related medical emergency, and the available evidence essentially comes from single-case reports or dated small retrospective series. A transfer to the Intensive Care Unit may be necessary during the acute setting, when the severe hypotension becomes unresponsive to standard practices, such as volemic filling and the infusion of vasopressor therapy. The only effective strategy is represented by prevention. The administration of octreotide, anxiolytic and antihistaminic agents represents the current treatment approach to avoid hormone release and prevent major complications. However, no standard protocols are available, resulting in great variability in terms of schedules, doses, ways of administration and timing of prophylactic treatments.

**Keywords:** crcinoid crisis; neuroendocrine tumors; hemodynamic instability; octreotide

#### **1. Introduction**

Neuroendocrine Tumors (NETs) are a heterogeneous family of neoplasms that can arise from any district of the body and can occur with an extremely wide range of symptoms and clinical manifestations, due to hormonal secretion. Carcinoid Syndrome (CS) is the most typical and common clinical presentation in functional NETs and is characterized by diarrhea, gastrointestinal discomforts, such as cramps and nausea, facial flushing with apparent peripheral cyanosis, eventual right-sided valvular heart disease preceded by palpitation and dyspnea with bronchospasm. Very rarely, patients with NETs can also exhibit a life-threatening occurrence known as Carcinoid Crisis (CC), generally described as a sudden onset of hemodynamic instability (prolonged hypertension or severe hypotension, unresponsive to standard practices), sometimes accompanied by characteristics of carcinoid syndrome, such as prolonged flushing, wheezing and hyperthermia. The underlying mechanism of CC is still not well known, but some Authors first hypothesized that CC could represent an extreme complication of the Carcinoid Syndrome caused by a sudden and massive release of tumor hormones that may be triggered by tumor manipulation or anesthesia [1,2].

**Citation:** Bardasi, C.; Benatti, S.; Luppi, G.; Garajovà, I.; Piacentini, F.; Dominici, M.; Gelsomino, F. Carcinoid Crisis: A Misunderstood and Unrecognized Oncological Emergency. *Cancers* **2022**, *14*, 662. https://doi.org/10.3390/ cancers14030662

Academic Editor: Karel Pacak

Received: 30 November 2021 Accepted: 26 January 2022 Published: 28 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

CC is mostly associated with foregut (respiratory tract, thymus, stomach, duodenum and pancreas) and midgut (small intestine, appendix and right colon) NETs [3]. Although theoretically every kind of tumor stress can cause CC, it typically occurs during invasive procedures, such as surgery and liver embolization [4], but it can also arise during clinical examination, biopsy, mammography [5], transesophageal echocardiography [6] or induction of anesthesia. Some cases of spontaneous onset have also been described [2,7,8]. The reported incidence of CC is about 7% in patients with NETs [9] undergoing abdominal surgery [10,11], but more recent works observed a higher number of cases, with a maximum incidence of 24–30% [12].

#### **2. Aim**

In this review, the Authors are going to discuss this uncommon manifestation, focusing on the potential etiopathogenetic mechanisms, clinical implications and potential treatments and prophylaxis.

#### **3. Materials and Methods**

The database PubMed was searched for the characteristics of CC using the term "carcinoid" combined with "crises" and "crisis" for publications with English abstracts up to September 2021. "Malignant carcinoid syndrome" was not considered. Studies included: (1) original articles, case series or case reports; (2) reporting at least one of the characteristics of Carcinoid Crisis (hemodynamic instability, continuous flushing, tachycardia predisposing to arrhythmias, bronchial wheezing, hyperthermia, peripheral cyanosis, severe diarrhea, central nervous system dysfunction with coma). In the section "Clinical Definition and Presentation", the Authors identified 12 case reports concerning CC.

#### **4. Clinical Definition and Presentation**

Carcinoid Crisis is an extremely rare life-threatening event, with little data published on this topic. The first report of CC dates back to 1964 [2], when Kahil et al. described a case of a 41-year-old woman who underwent surgical resection of a NET of the ileum and a few months later started to manifest episodes of flushing, cramps and pruritus, interpreted as malignant CS. She was treated with a peripheral serotonin antagonist, cyproheptadine and was trained to adopt a low-tryptophan diet, obtaining good control of symptoms. Thirteen days after the discontinuation of therapies, the woman manifested a sudden onset of apprehension, oppressive chest pain, abdominal cramps, frequent diarrhea, facial flushing, pruritus, paresthesia and hyperaesthesia, peripheral vascular collapse with pale cold cyanotic extremities and progressive extreme hypotension. Neither metaraminol, a potent vasoconstrictor, nor norepinephrine, was effective in blood pressure control. One hour after the onset of symptoms, a single intravenous injection of cyproheptadine was administered with a dramatic cessation of the thoracic and abdominal pain. The Authors named this an extremely dangerous condition of Carcinoid Crisis, thus suggesting for the first time that it could represent a serious complication of CS, caused by a sudden release of active substances by neuroendocrine cells, provoked by stress on tumor masses.

Table 1 summarizes published case reports of CC.

As assumed by Kahil et al., in the absence of external stress, neuroendocrine tumor cells produce a share of vasoactive peptides that provoke CS. Instead, when a greater stimulus takes place, a hormonal storm can trigger CC. As shown in Table 1, the most commonly reported primary tumor locations that can cause CC are lung and small bowel (ileum), which also represent the most common sites associated with CS, because of the overall major release of vasoactive peptides compared to other districts [3].

As previously shown in Table 1, not only direct manipulations of tumor mass, such as bronchoscopy [16,20], liver biopsy [13,16] or locoregional treatments [18,21], but also other kinds of tumor solicitations, such as the induction of anesthesia [14] or the infusion of radiotracers [17], can contribute to the rapid onset of CC. In addition, following the results of the NETTER-1 trial, the increasing use of peptide radionuclide therapy (PRRT) has led to a rise in CC due to tumor lysis [22], most frequently after the first cycle of treatment, and the main risk factors include large tumor burden, liver metastases, previous CS, carcinoid heart disease, advanced patient age, high chromogranin A levels and high 5-HIAA levels. Furthermore, in some cases, PRRT-induced CC can also occur long after therapy [24].

**Table 1.** Case reports of Carcinoid Crisis up to September 2021.

Furthermore, clinical presentation is remarkably variable. For instance, Kromas et al., described a case of a 31-year-old woman who presented chest pain, newly onset asthma and a sudden onset of hypotension and wheezing during rigid bronchoscopy [20]. Koopmans et al. reported the case of a 61-year-old woman who developed vomiting, accompanied by flushing, edema and severe hypotension during an 18F-DOPA PET scan [17]. The explanation proposed by the Canadian group of Seymour et al. is that each tumor secretes a specific cocktail of hormones, which leads to discrepancy regarding recognition and symptoms and contributes to the great clinical variety of CC [25].To date, there has been no international consensus on the most appropriate definition of CC. Clinicians continue to identify CC as a rapid onset of hemodynamic instability, unresponsive to conventional management associated with characteristics of CS, such as continuous flushing, tachycardia and arrhythmias, bronchial wheezing, hyperthermia, peripheral cyanosis, severe diarrhea and central nervous system dysfunction. Kinney et al. first clinically termed CC as severe hypotension with the systolic blood pressure (SBP) < 80 mmHg for more than 10 min, accompanied by the presence of flushing, urticaria, ventricular dysrhythmia, bronchospasm or acidosis, and they registered 15 cases of CC among 119 patients analyzed [9]. Subsequently, Condron et al. preferred to broaden the definition to a significant hemodynamic instability (SBP < 80 or >180 mmHg, heart rate > 120 bpm) or potential end-organ dysfunction not attributable to other causes and described a much higher incidence of CC, approaching 30% in their case series [11]. Seymour et al. also included bronchoconstriction and flushing in their definition of CC [25], while Massimino et al. combined the definitions of Kinney and Condron, considering all patients with systolic blood pressure (SBP) < 80 mmHg for more than 10 min) or presentation of hemodynamic instability (including hypotension, sustained hypertension or tachycardia) [10]. As a consequence, this variability in the definition of CC leads to the impossibility of determining the real incidence and the appropriate severity of these episodes, with no conclusive studies available.

The overhang of pressure values represents a minor common denominator among all definitions. The recent study of Condron et al. tried to better characterize the underlying physiological mechanism [26]. Assuming there are three putative hormonally driven pathways of the hypotension (the reduction in cardiac output due to pulmonary artery vasoconstriction, the coronary vessels vasoconstriction resulting in cardiac failure and peripheral vasodilatation with consequent hypovolemic shock), they concluded that the pathophysiology of CC appears consistent with distributive shock. Using intraoperative, transesophageal echocardiography, pulmonary artery catheterization and intraoperative blood collection, they observed a statistically significant reduction in systemic vascular resistance among all crises. The contextual rating of hormone levels during CC exhibited markedly diverse profiles, so the Authors concluded that another carcinoid-related manifestation, such as flushing or wheezing, is not mandatory to declare a CC.

Based on the results of this study, the scientific community is now working to clarify which is the precise etiopathogenetic mechanism behind the onset of CC and which endogen molecule is the major molecule responsible for hemodynamic instability to better classify and recognize this life-threatening event and to define the best clinical approach.

#### **5. Etiopathogenesis of Carcinoid Crisis**

As previously cited, Kahil et al. were the first Authors who approached the topic of CC. At the end of their experience, they concluded that an excessive amount of serotonin in the bloodstream, the principal amine responsible for CS, could cause CC as a consequence of increased tryptophan intake in diet or, less likely, of a release after spontaneous necrosis of the tumor. The treatment consisted of anti-serotonin/histamine agents instead of epinephrine and levarterenol, to avoid catecholamines use that can worsen or elicit CC [2]. Tryptophan is an essential precursor of serotonin, which is hydroxylated by the rate-limiting enzyme tryptophan hydroxylase and subsequently decarboxylated by an aromatic acid decarboxylase to serotonin [27] (Figure 1). It has been demonstrated that increased tryptophan intake in diet promotes a rise in peripheral serotonin.

The vast majority of peripheral serotonin is produced by enterochromaffin cells, the same cells constituting NET masses. Therefore, the rise of tryptophan in blood circulation determines an upregulation in NET cells, with a massive production of serotonin. This hormone has a wide range of peripheral effects (Figure 2).

Concerning the cardiovascular system, a direct effect on the smooth muscle of blood vessels can lead to either vasoconstriction or vasodilatation, depending on the particular vessels influenced: renal, placental and umbilical vessels respond with vasoconstriction, whereas coronary vessels and vessels of the skeletal muscle respond with vasodilatation. The consequent effect on the blood pressure consists of three phases: first, there is a brief early depressor phase; then comes the pressor phase, as serotonin increases the total peripheral resistance; and finally, when serotonin dilates the vessels of the skeletal muscles, a late depressor phase is observed. Serotonin also influences respiration by stimulating the carotid and aortic chemoreceptors. The result is a short-lasting increase in respiratory minute volume by direct stimulation of the smooth muscles of the bronchi, leading to bronchoconstriction. Furthermore, serotonin stimulates the gastrointestinal tract to greater motility, with increased tone or intense spastic contractions, colics and evacuation of the bowels [28].

**Figure 1.** Tryptophan metabolic pathway.

**Figure 2.** Peripheral effects of serotonin.

However, serotonin cannot be the unique molecule implicated in CC. Some reports denied the role of serotonin in flushing [29,30], one of the most typical symptoms associated with hemodynamic instability in CC. In addition, NETs originating from the respiratory tract and foregut do not express the aromatic acid decarboxylase, which converts 5-hydroxytryptophan to serotonin. Indeed, in this subtype of neoplasms, an atypical carcinoid syndrome can be observed characterized by patchy, sharply demarcated, serpiginous and cherry-red flushes (gastric NET) [31] or by prolonged flushing, lasting for hours or days, associated with disorientation, anxiety and tremor, lacrimation, salivation, hypotension, tachycardia, diarrhea, dyspnea, asthma and edema (Pulmonary NETs) [32]. Other potential mediators of CC are bradykinins, prostaglandins, tachykinins, substance P and histamine. Table 2 summarizes the main humoral molecules involved in the pathogenesis of CS/CC.

**Table 2.** Molecules implicated in CS/CC.

In CC, the most implicated compounds seem to be vasoactive peptides, such as serotonin, histamine, bradykinin, tachykinins and kallikrein. In particular, patients with carcinoid flushing exhibit elevated levels of bradykinin and kallikrein in the bloodstream [33], which are considered the most probably responsible for flushing, rather than serotonin.

The only available data concerning the incidence and possible biological onset of CC is derived from few, small studies on intraoperative CC, such as surgical resection or locoregional treatments. When tumor manipulation occurs, the stress response triggers the release of catecholamines from the adrenals or sympathetic neurons, which in turn contribute to the release of tumor products [10]. Although CC is more typical of functional NETs, it may also occur in nonfunctional tumors. The presence of liver metastases, older age, and, for intraoperative crises, anticipated long anesthesia time, represent the most significant risk factors associated with the onset of CC, as demonstrated by Massimino et al. [10] and subsequently prospectively confirmed by Condron et al. [11]. Recently, the Research Unit of the Oregon Health and Science University has evaluated hemodynamic parameters and serum hormone levels during elective major surgery in patients with NETs [26]. Forty-six patients with a high risk of CC (older age, liver involvement, long anesthesia) were enrolled. The patients presented pulmonary artery catheters inserted to track pulmonary artery pressure, cardiac output and systemic vascular resistance, in addition to transesophageal echocardiography probes inserted to supervise cardiac function, and had serial measurements of typical hormones considered to be implicated in CC (serotonin, histamine, bradykinin and kallikrein). Seventeen patients experienced CC with prolonged hypotension. The most significant finding was that the pre-incision serotonin level was significantly higher in patients who manifested the crisis, while there were no significant changes in the mean value of any of the four hormone levels during CC. Patients manifesting CC presented an increased risk of postoperative complications, particularly if the events continued for more than 10 min. Authors concluded that elevated pre-incision serum serotonin levels represent a novel marker for increased risk of CC, as well as the severity of the crisis, and they observed no evidence of any massive release of the evaluated hormones during CC, suggesting CC may be an entirely separate pathophysiologic entity from that of CS. However, since, to date, no other study has been carried out on this topic, this hypothesis needs further confirmation.

#### **6. Carcinoid Crisis Management**

#### *6.1. Octreotide*

Assuming that CC is caused by the massive release of hormones by tumor masses, octreotide has historically represented the mainstay of its treatment. This drug is a longacting synthetic octapeptide [34] that acts like somatostatin, a potent inhibitory peptide (Figure 3). The blockade of hormone releases, such as insulin, glucagon, gastrin and other gastrointestinal molecules, and the reduction of splanchnic and hepatic blood flow represent the mechanisms underlying the management of CC. However, the real role of octreotide in the management of CC is not well established yet, and the available data are partly contradictory.

The first report of using octreotide to treat CC dates back to 1985 [36], when Kvols et al. administered two intravenous boluses of 50 μg of this drug to a patient affected by a small bowel NET presenting life-threatening hypotension and prolonged flushing during abdominal surgery, unresponsive to intravenous fluids, intravenous calcium or intravenous conventional vasopressors. The Authors described a rapid resolution of the critical status and concluded that octreotide must be available in the operating room during surgery to rapidly manage CC. Furthermore, the same Authors conducted an explorative study on 25 patients with metastatic NET and documented CS [37] with the aim to evaluate the effect of this long-acting somatostatin analog, suggesting that this drug could be routinely safely used in the management of CS with excellent results.

**Figure 3.** Octreotide clinical effects (Figure adapted by Lamberts SW, van der Lely AJ, de Herder WW, Hofland LJ. Octreotide [35]).

However, further studies have questioned the real role of octreotide in the specific treatment of CC. Considering the pharmacodynamic profile of octreotide, this drug acts as a hormone release blocker and not as a hormone receptor blocker, therefore it should not neutralize the effect of circulating vasoactive peptides [38,39]. It should possibly prevent a worsening in the CC or prevent its occurrence at all. According to the most recent analysis put forward by Wann et al. [40], the rapid resolution of the acute episode described by Kvols et al. could also be explained by a delayed effect of epinephrine or other previously administered medications.

In 2001, Kinney et al. [9] evaluated the complication rate and outcomes of a larger series of patients with metastatic NET. Among the 119 subjects undergoing abdominal surgery included, 6 received only a preoperative octreotide dose, while 45 patients received octreotide intraoperatively, and 73 patients did not receive octreotide. A total of 15 out of 119 patients experienced perioperative complications, including 3 deaths, but none of the 45 subjects who received the intraoperative drug dose had an intraoperative complication. The researchers reported a statistically significant difference in terms of intraoperative complications between the 45 patients who received intraoperative octreotide and the 73 who did not (*p* = 0.023). They concluded that patients with metastatic carcinoid tumors can undergo abdominal surgery safely with an intraoperative octreotide dose, reporting a significantly global decrease in intraoperative complications such as CC.

Based on the possible preventive role of octreotide on CC, Massimino et al. retrospectively explored the use of octreotide prophylaxis in a group of 97 patients undergoing surgery during the years 2007–2011. A total of 87 patients (90%) received prophylactic octreotide (dose range 100–1100 mg, median 500 mg), and 56% received at least one additional intraoperative dose. Despite the use of octreotide, intraoperative complications occurred in 23 (24%) patients. Therefore, the obtained data greatly diverged from the results published by Kinney et al., In their series, 18 patients (19%) experienced prolonged hypotension, while 5 (5%) were reported to have had marked hemodynamic instability consistent with a CC. Intraoperative complications occurred with the same frequency among patients with functioning (21%) and non-functioning (28%) NET, and the presence of liver metastases was found to be a predictor of intraoperative complications. These findings suggest neither outpatient octreotide LAR nor single-dose preoperative bolus octreotide prevent all intraoperative complications [10].

In 2016, Woltering et al. [41] suggested a possible solution to improve octreotide effectiveness. Their retrospective report demonstrated a reduction in CC incidence by using a continuous infusion of high-dose octreotide during surgery. As anesthetic or surgical stimuli can potentially precipitate an unpredictable release of amines, in their protocols, the researchers administered a prophylactic preoperative 500 μg bolus of octreotide acetate along with a continuous intravenous intraoperative infusion for all NET patients undergoing surgical cytoreduction, regardless of the location of their primary tumor or their functional status. The rationale behind this choice is connected to octreotide pharmacokinetics: preoperative bolus of octreotide, with a half-life of 90–120 min, might not last long enough for protection against CC during long surgery. Without a continuous infusion, blood level would fall to 50% of the original octreotide concentration after 2 h and would be only 25% of the original concentration after 4 h. As result, Woltering et al. reported an incidence of CC of only 3.6%, concluding that continuous intraoperative octreotide infusion could significantly reduce the risk of CC onset.

To demonstrate the benefits of continuous octreotide infusion on CC prevention, Condron et al. [11] prospectively enrolled 127 patients (71% with liver metastases, 74% with CS) who underwent 150 operations with continuous octreotide infusions. Contrary to what was expected, 30% of the patients manifested CC, and the crises were significantly associated with the presence of liver metastases (*p* = 0.02) or a history of CS (*p* = 0.006). The rapid use of vasopressor was effective in reducing crisis duration, with a reduction in postoperative complications. These unexpected results could be explained by analyzing CC definitions. While Woltering et al. considered only episodes of hypotension lasting ≥10 min [41], Condron et al. registered all cases of hemodynamic instability (systolic blood pressure < 80 or >180 mmHg, heart rate > 120 beats per minute or display of any physiology that, if sustained, would be expected to lead to end-organ dysfunction, such as ventricular arrhythmias or bronchospasm) unattributable to any other causes [11]. Considering only episodes of hypotension lasting ≥10 min, as Woltering et al. did, Condron et al. would have reported only 8% of CC.

Other reports on the outcome of prophylactic octreotide were published in 2018 [42] and 2019 [12]. Kinney et al. retrospectively evaluated 169 patients undergoing partial hepatic resection for metastatic NET between 1997 and 2015, and 77% (130/169) of patients preoperatively received 500 μg of subcutaneous octreotide. In their report, there were no documented cases of CC; one patient developed clinical findings of an emerging CC but was successfully treated with doses of octreotide, and findings resolved in <10 min. Of note, in this case, CC was defined as a sudden or blunt onset of at least two of the following: flushing or urticaria that are not explained by an allergic reaction; bronchospasm or bronchodilator administration; hypotension (SBP < 80 mmHg for >10 min and treated with vasopressor) not explained by volume status or hemorrhage; tachycardia ≥120 beats per minute [42]. Analyzing only sustained hypotension, the incidence was 5.6%, but because none of those patients exhibited any other criteria, none were considered CC.

Finally, Kwon et al. reported a retrospective series of 75 patients with metastatic well-differentiated NETs who underwent liver resection, ablation or embolotherapy from 2012 to 2016. The CC was defined subjectively by clinical documentation of occurrence by any treating physician, including the anesthesiologist, surgeon or interventional radiologist, and it had to be associated with hemodynamic instability, defined as the presence of at least one of the following events sustained for more than 10 min during the procedure: hypotension (systolic blood pressure, 80 mm Hg) or tachycardia (heart rate, >120 beats per minute). CC was identified in 32% (24) of patients. Of note, the route and dose of

preprocedural octreotide administration varied widely. Neither long-acting octreotide, perioperative octreotide, intraoperative octreotide nor any combination was associated with a lower incidence of crisis. The Authors concluded that somatostatin analogs do not reliably prevent CC. One hypothesis is that CC may be a phenomenon physiologically distinct from CS, involving the release of a different distribution of vasoactive substances, against which different therapeutic agents can be used [12]. As previously mentioned, this proposal has been explored by Condron et al. [26].

Data are summarized in Table 3.

**Table 3.** Articles assessing the impact of octreotide in CC.

As reported in the most recent guidelines referring to clinical studies discussed so far, there are no standard octreotide regimens in the management of CC; subcutaneous administration of octreotide 100–200 mcg × 2–3 daily during surgery has been suggested for a minor procedure or lower-risk patients. However, intravenous octreotide infusions should also be readily available in the operating room to be used when deemed necessary. For major surgery, perioperative prophylactic treatment with intravenous octreotide, at the starting dose of 50–100 mcg/h (mean dose 100–200 mcg/h), is the standard regimen used by most clinical centers. Although this has not been substantiated by any prospective study, most experts start treatment with intravenous octreotide 12 h before the operation and escalate the dose as necessary until symptom control is achieved. This infusion continues for at least 48 h after the operation, with dose titration as clinically required [43].

Considering the increasing use of PRRT, several studies have been conducted to estimate the impact of octreotide in PRRT-induced CC. The incidence of CC during PRRT ranges between 1 and 10%, and, as previously mentioned in this review, specific risk factors have been defined, which, if present, expose the patient to a major rate of intratreatment complications [24]. In the clinical experience reported by de Kaizer et al., among 479 patients enrolled in the study, 7 cases of CC after the first cycle of PRRT were reported [44]. The treatment included high-dose octreotide, i.v. fluid replacement and corticosteroid. Despite additional precautions taken after their first therapy cycle (continuation of somatostatin analog, corticosteroids and reduction of administered dosage of 177Lu-octreotate), 3 patients developed a second CC after the subsequent cycle of PRRT. Analyzing the possible mechanism behind hormonal secretion during Lu-octreotate therapy (tumor lysis vs. discontinuation of short-acting somatostatin analog vs. emotional stress response to hospitalization vs. administration of arginine and lysine), the Authors concluded that hormonal crises should be managed with an infusion of somatostatin analogs i.v., fluids, corticosteroids and correction of electrolyte disturbances. More recently, Stenzel et al. came to the same conclusions [45]. Moreover, the Australian group of Tapia Rico et al. first proposed a protocol to prevent and manage severe CC and specifically defined which patients would benefit most ("high-risk patients") from premedication to PRRT therapy with corticosteroids and bolus dose of octreotide sc [46]. This work has been further enriched by De Olmo et al., who recently published the current procedure adopted for approaching patients undergoing PRRT [24]. The work of clinicians should start from the identification of risk factors for CC through:

In particular, in the case of bulky tumors, it is mandatory to consider a possible surgery or locoregional treatment before PRRT. In addition, it is essential to obtain good control of CS before the first cycle of 177Lu-octreotate.

As premedication, the Authors advise the administration of corticosteroids (dexamentasone 4–8 mg), antiemetic (ondansetron 4 mg), somatostatin analog (octreotide 100 μg s.c. or 50 μg i.v. bolus), antistaminic h1 (dexchlorpheniramine 5 mg i.v. in slow infusion) and antistaminic H2 (ranitidine 50 mg i.v. in slow infusion). In the event of an outbreak of CC, the infusion of Lu-DOTATATE should immediately stop, and a bolus of octreotide (100–500 μg) should be immediately administered, followed by a maintaining dose of 50–100 μg/h infusion. In case of severe hypotension, the Authors do not exclude the use of phenylephrine or vasopressor drugs.

#### *6.2. Vasopressors*

Based on clinical experiences [2,13,14,47], sympathomimetic drugs have always been avoided in the management of CC-related hypotension, since they may worsen it by triggering further release of peptides by tumor masses. Mason et al., in 1966, first observed vascular response to systemic epinephrine injection in the forearm in 7 NET patients. The Authors noted decreased systemic blood pressure, decreased vascular resistance and an elevated bradykinin level for 5 min after injection, although the response was quite variable among the 7 patients [47]. However, data supporting a widespread concern about the abuse of beta-adrenergic agonists remain very limited [48].

More recent experiences have tried to explore the real role of this class of drugs in the management of intraoperative CC [10,26,48]. Notably, Limbach et al. retrospectively examined the use of vasoactive medications during CC to determine whether an association between induction of a secondary CC and beta-adrenergic agonist administration could be detected. They observed no close correlation between the use of sympathomimetic drugs and secondary CC and, in addition, the duration of CC did not increase with administration of beta-adrenergic agonists [48].

Based on these reports and considering the recent findings, CC-related hypotension is due to a distributive shock, as previously discussed, and the administration of vasopressors, which determine a wider systemic effect (vs. octreotide alone, which has a vasoconstrictive effect only on splanchnic vessels), should be suggested when standard measures fail [40].

#### **7. Conclusions**

The CC remains a severe, sometimes fatal, acute manifestation of NETs. The rarity and unpredictability of this event has hampered the development of randomized controlled trials to define the best clinical and therapeutical approach. Data available in the literature are mainly derived from small retrospective studies or case reports, in which the clinical definition of CC is not consistent and universally accepted. Recently, the pathophysiological mechanism has also been questioned. According to some Authors, CC should be considered a completely separate event from CS, as it can be triggered by a different cocktail of hormones.

Current guidelines, such as the European ENETS and the American NANETS, continue to recommend the administration of octreotide to prevent and manage CC onset [42,49], although without a standard scheme, duration and dose specification. Furthermore, the use of vasopressors has been recently revised, as their administration during crises could accelerate the increase in blood pressure.

Based on recent evidence, some institutes stopped using octreotide during operations altogether instead of relying on vasopressors, including beta-adrenergic agonists [50]. Their experience on 195 patients demonstrates a rate of CC not significantly higher than that reported in previous studies. The Authors conclude that perioperative octreotide use may be safely stopped, owing to inefficacy, and the treatment of crisis should be replaced with intravenous fluids and vasopressors, which address the actual pathophysiology of the crisis, without concern about increasing crisis duration or rates of major postoperative complications.

Further efforts should be directed toward the understanding of the correct pathophysiology of CC to propose more specific, effective and established treatments.

**Author Contributions:** Conceptualization, C.B. and F.G.; methodology, C.B.; software, C.B.; validation, C.B., S.B., G.L., I.G., F.P., M.D. and F.G.; formal analysis, C.B.; investigation, C.B.; resources, C.B.; data curation, C.B.; writing—original draft preparation, C.B.; writing—review and editing, C.B., S.B. and F.G.; visualization, C.B.; supervision, F.G.; project administration, C.B. and F.G.; funding acquisition, F.G, F.P. and M.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** F.G. received honoraria for speaker/advisory roles from Servier, Eli Lilly, Iqvia, Merck Serono, Amgen and Bristol-Myers Squibb outside the present work; the other Authors declare no conflicts of interest.

#### **References**

## *Review* **Endocrine and Neuroendocrine Tumors Special Issue—Checkpoint Inhibitors for Adrenocortical Carcinoma and Metastatic Pheochromocytoma and Paraganglioma: Do They Work?**

**Camilo Jimenez 1,\*, Gustavo Armaiz-Pena 2, Patricia L. M. Dahia 3,4, Yang Lu 5, Rodrigo A. Toledo 6, Jeena Varghese 1 and Mouhammed Amir Habra 1**

**Simple Summary:** In the past decade, the landscape of cancer treatment has radically changed after the introduction of immunotherapy. Adrenocortical carcinoma and metastatic pheochromocytoma/paraganglioma are rare cancers with limited responses to traditional cancer treatments. The use of immunotherapy against these cancers has yielded a few responses when used alone or in combination with other drugs. We reviewed the current literature to summarize the role of immunotherapy in these rare cancers.

**Abstract:** Adrenocortical cancers and metastatic pheochromocytomas are the most common malignancies originating in the adrenal glands. Metastatic paragangliomas are extra-adrenal tumors that share similar genetic and molecular profiles with metastatic pheochromocytomas and, subsequently, these tumors are studied together. Adrenocortical cancers and metastatic pheochromocytomas and paragangliomas are orphan diseases with limited therapeutic options worldwide. As in any other cancers, adrenocortical cancers and metastatic pheochromocytomas and paragangliomas avoid the immune system. Hypoxia-pseudohypoxia, activation of the PD-1/PD-L1 pathway, and/or microsatellite instability suggest that immunotherapy with checkpoint inhibitors could be a therapeutic option for patients with these tumors. The results of clinical trials with checkpoint inhibitors for adrenocortical carcinoma or metastatic pheochromocytoma or paraganglioma demonstrate limited benefits; nevertheless, these results also suggest interesting mechanisms that might enhance clinical responses to checkpoint inhibitors. These mechanisms include the normalization of tumor vasculature, modification of the hormonal environment, and vaccination with specific tumor antigens. Combinations of checkpoint inhibitors with classical therapies, such as chemotherapy, tyrosine kinase inhibitors, radiopharmaceuticals, and/or novel therapies, such as vaccines, should be evaluated in clinical trials.

**Keywords:** adrenocortical cancer; metastatic pheochromocytoma; metastatic paraganglioma; checkpoint inhibitors; avelumab; ipilimumab; nivolumab; pembrolizumab

**Citation:** Jimenez, C.; Armaiz-Pena, G.; Dahia, P.L.M.; Lu, Y.; Toledo, R.A.; Varghese, J.; Habra, M.A. Endocrine and Neuroendocrine Tumors Special Issue—Checkpoint Inhibitors for Adrenocortical Carcinoma and Metastatic Pheochromocytoma and Paraganglioma: Do They Work? *Cancers* **2022**, *14*, 467. https://doi.org/ 10.3390/cancers14030467

Academic Editors: Alfredo Berruti, Vito Amoroso and Nicola Fazio

Received: 23 December 2021 Accepted: 15 January 2022 Published: 18 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

The adrenal glands are very important endocrine organs that are responsible for the regulation of many different physiological mechanisms that preserve human homeostasis and guarantee the individual's survival. These features include the modulation of the cellular responses to stress; the healing of damaged tissues; the protective responses of fighting and escape; the regulation of the corporal concentrations of acid and electrolytes; the modulation of the metabolism of glucose, fat, and proteins; and the maintenance of adequate blood pressure to satisfy metabolic needs. The adrenal glands regulate homeostasis through the synthesis and secretion of androgens, glucocorticoids, and mineralocorticoids, which are derived from the adrenal cortex, and catecholamines, which come from the adrenal medulla [1–3]. Embryologically, the adrenal cortex is derived from the intermediate mesoderm [4], and the adrenal medulla is derived from the neural crest cells close to the dorsal aorta [5]. The adrenal medulla is a modified autonomic sympathetic nervous system ganglion that, unlike other sympathetic ganglia, produces adrenaline and noradrenaline and releases these hormones directly into the bloodstream.

Primary malignant tumors may develop in the adrenal cortex or in the adrenal medulla. The most common cancers to develop in these regions are adrenocortical carcinoma (ACC), which is a tumor derived from the adrenal cortex, and metastatic pheochromocytoma, which is a tumor derived from the adrenal medulla. Similar tumors, called metastatic paragangliomas, develop in the extra-adrenal paraganglia and have genetic and molecular profiles similar to those of many metastatic pheochromocytomas [6], and current clinical trials study metastatic pheochromocytoma and metastatic paragangliomas together. For the purpose of this manuscript, we will consider metastatic pheochromocytomas and paragangliomas (MPPGL) as one tumor group.

ACCs are tumors associated with high proliferative rates and a common clinical phenotype of large, rapidly growing primary tumors that are associated with metastases in up to 80% of cases [7–9]. Conversely, pheochromocytomas and paragangliomas are usually characterized by lower proliferative rates than those observed in ACCs, and their metastatic spread is observed in up to 25% of cases [10,11]. Nevertheless, MPPGL tumors are usually large as primary tumors, and the metastases are frequently massive because the diagnosis of these tumors is frequently delayed [9,12]. Most ACCs and MPPGLs secrete excessive amounts of hormones, predisposing patients with these tumors to severe comorbidities. ACCs may secrete large amounts of glucocorticoids, mineralocorticoids, and/or androgens, which may lead to severe Cushing syndrome, hyperaldosteronism, and virilization [13,14], and MPPGLs may secrete excessive amounts of catecholamines, which may lead to severe cardiovascular and gastrointestinal diseases [15,16]. The combination of a large tumor burden, considerable tumor growth over time, and excessive hormonal secretion predisposes patients with ACC and MPPGL to a decreased quality of life and decreased overall survival rates [12–14]. In fact, only 15–44% of patients with ACC and 60% of patients with MPPGL are alive 5 years after initial diagnosis [7,17].

ACC and MPPGL are rare tumors. In the United States, approximately 200–300 new cases of ACC and 100–200 new cases of MPPGL are discovered every year [17,18]; by definition, ACC and MPPGL are orphan diseases, and, subsequently, the therapeutic options for advanced disease are limited [10,13]. ACC is mainly treated with a combination of systemic chemotherapy of cisplatin, doxorubicin, etoposide, and mitotane, and clinical responses are noted in approximately 30% of patients; ACC responses to chemotherapy usually have short duration, and treatment toxicity can be substantial [13]. Chemotherapy with cyclophosphamide, vincristine, and dacarbazine for MPPGL is widely available. However, response rates are also low, with approximately 37% of patients achieving partial response (PR), with at least a reduction of 30% of the tumor size when compared with baseline measurements, or disease stabilization; cures are exceptional, and treatment toxicity is also substantial [10,19,20]. Approximately 60–70% of MPPGLs express the noradrenaline transporter; therefore, these tumors are meta-iodine-benzyl-guanidine (MIBG)–avid [12,21]. The United States Food and Drug Administration (FDA) recently approved high-specificactivity MIBG (HSA-I-131-MIBG) for patients with MPPGL. HSA-I-131-MIBG demonstrated a clinical benefit rate (CBR), the proportion of patients who achieve a complete response or tumor disappearance, partial response, and disease stabilization as per RECIST 1.1., higher than 90%, with approximately 25% of patients exhibiting a PR and more than 60% of patients having stable disease with some degree of regression 1 year after treatment. Additionally, most patients who underwent HSA-I-131-MIBG treatment had improved blood pressure compared to baseline, and the toxicity of HSA-I-131-MIBG was acceptable [22]. This medication is only available in the United States and is not indicated for the treatment of patients with MPPGL that does not express the noradrenaline transporter [20].

Given this limited spectrum of therapeutic options, we need to identify other effective treatments for patients with ACC and MPPGL. Over the last decade, immunotherapy with checkpoint inhibitors has become one of the therapeutic pillars against cancer [23]. The results of phase 1 and 2 clinical trials with immunotherapy for ACC and MPPGL have revealed that immunotherapy is a potentially important treatment for patients with these tumors as well. In this review, we will discuss the rationale for the use of immunotherapy against ACC and MPPGL, the results of clinical trials with several checkpoint inhibitors against ACC and MPPGL, and potential mechanisms to induce or enhance an immune system response effective against ACC and MPPGL.

#### **2. Avoidance of the Immune System as a Hallmark of Cancer**

The hallmarks of cancer are the distinctive and complementary capabilities that enable tumor growth and metastatic dissemination and are the foundation for understanding the biology of any cancer. These hallmarks include the sustaining of the proliferative signaling of tumor cells, mechanisms that favor replicative immortality, genome instability and the presence of mutations, deregulation of cellular energetics and cell necrosis, tumorpromoting inflammation, the induction of abnormal vascular formation or angiogenesis, the activation of mechanisms of invasion and metastases, resistance to tumor cell death, the avoidance of growth suppressors, and the avoidance of the recognition of the cancer cell as such by the immune system [24]. Several features related to cancer cell biology theoretically lead to a destructive anti-cancer immune response. Cancer cells are characterized by the accumulation of a variable number of genetic alterations and the subsequent loss of normal cellular regulatory processes; cancer cells also accumulate neoantigens, antigens of differentiation, and cancer testis antigens, and a fraction of these antigens are bound to major histocompatibility class I molecules, allowing the immune system CD8+ T cells to recognize cancer cells [25,26].

Once the cancer cell is recognized as such, the cancer immunity cycle starts [27]. The cancer immunity cycle is a sequence of steps that must be initiated, allowed to proceed, and expanded to generate an anti-cancer immune response (Figure 1) [27]. The cycle starts with the release of antigens that are later presented to antigen-presenting cells, such as the dendritic cells, followed by the priming and activation of T cells in places, such as the lymph nodes.  The cancer immunity cycle has three important qualities: (1) adaptability, which is the capacity of the immune system to recognize the cancer cell; (2) specificity, which allows the immune system attack to be mainly focused on cancer cells, limiting toxic effects on the normal cells; and (3) memory, which guarantees that the immune system can more effectively recognize and destroy cancer cells that may develop again [27]. Tumors are, however, more than just cancer cells. Tumors are also composed of non-malignant cells that are recruited by the malignant cells to serve them. Together the malignant and non-malignant cells, the extracellular matrix and the tumor vasculature, and their complex communications create the tumor microenvironment.

**Figure 1.** The cancer immunity cycle and the mechanisms of action of checkpoint inhibitors and potential therapies that enhance immune system response. Abbreviations are as follows: PD1: programmed death cell protein 1; PD-L1: programmed death-ligand 1; CTLA-4: cytotoxic T-lymphocyte antigen-4; DC: dendritic cells; APC: antigen presenting cells; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor; TME: tumor microenvironment.

The cancer immunity cycle is a complex process [27] regulated by many inhibitory or stimulatory factors and pathways in the tumor microenvironment that determine the successful identification of cancer cells by the immune system. While many of these factors have been recognized, many others are unknown. Examples of inhibitory mechanisms of the cancer immunity cycle are the CTLA4 pathway, which prevents antigen-presenting cells from priming and activating T cells; the PD-L1/PD-1 pathway, which prevents the killing of cancer cells; vascular endothelial factors and the endothelin B receptor, which prevent T cells from infiltrating tumors and reduce the expression of the proteins of the major histocompatibility complex by the cancer cells (Figure 1) [27]. Subsequently, several medications that antagonize the activity of inhibitory factors and/or stimulate factors that enhance the cancer immunity cycle have been developed (Figure 1) [23]. Antibodies that block the actions of CTLA4, PD-1, and PD-L1, known as checkpoint inhibitors, such as ipilimumab (CTLA4 inhibitor), pembrolizumab (anti-PD-1 antibody), or nivolumab (anti–PD-L1 antibody), are currently indicated for the treatment of many different malignancies [23]. In some cases, checkpoint inhibitors alone have led to permanent remissions [23]. Antiangiogenic medications, such as cabozantinib, a tyrosine kinase inhibitor, may induce tumor vessel normalization, enhancing tumor infiltration and the recognition of cancer cell by immune T cells [28]. When combined with checkpoint inhibitors, antiangiogenic medications may lead to impressive clinical responses in clear cell renal cell carcinomas [29,30]. Interferon alpha or vaccines made with tumor antigens may facilitate antigen presentation [27]. Clinical trials combining checkpoint inhibitors and vaccines for different types of cancer are ongoing [31]. Chemotherapy, targeted therapies, radiation therapy, and radiopharmaceuticals may release tumor antigens that could be recognized by the immune system. Combinations of these classical systemic therapies with checkpoint inhibitors have been associated with clinical benefits in several malignancies [23,32,33].

In clinical trials, pembrolizumab demonstrated objective responses in patients with advanced clear cell renal cell carcinomas, lung adenocarcinomas, and melanomas that express PD-L1 [34]. Nevertheless, not all tumors that express PD-L1 exhibited objective responses [34]. Many tumors for which pembrolizumab is currently indicated are characterized by an inflamed tumor microenvironment and a high tumor mutation burden (TMB) [35]. ACC may exhibit an inflammatory environment and several somatic mutations [36]; nevertheless, these features are not as notable as the ones noted in the aforementioned cancers [35]. Pheochromocytomas and paragangliomas are associated with minimal or no tumor inflammation and, as such, are classified as "cold" tumors [35]. In addition, MPPGLs are mainly associated with monogenic germline and somatic mutations; in fact, up to 50% of MPPGLs are exclusively associated with germline mutations of the *SDHB* gene, and many MPPGLs have no recognized or a few additional somatic mutations [35]. These observations raise the question of whether immunotherapy with checkpoint inhibitors could be effective for ACC or MPPGL.

#### **3. Scientific Rationale for the Potential Use of Checkpoint Inhibitors for ACC or MPPGL**

Recent studies have found that some ACCs and MPPGLs express the programmed cell death ligands in tumor cell membranes or stromal cells in the tumor microenvironment [37–39]. Small immunohistochemical studies found PD-L1 expression in up to 70% of ACC samples [38] and 18% of MPPGL samples [37], suggesting that several patients with ACC and some patients with MPPGL may benefit from checkpoint inhibitors, such as avelumab, nivolumab, or pembrolizumab. In addition, it has been recognized that 3–18% of ACC cases are associated with somatic or germline mutations of DNA mismatch repair genes, such as *ML1*, *MSH2*, *MSH6*, and *PMS2* mutations (e.g., ACC-associated Lynch syndrome), and these mutations lead to microsatellite instability [40–42]. The use of checkpoint inhibitors seems to be an appealing option for this subset of ACC cases. In fact, the FDA approved the use of pembrolizumab for the treatment of any solid cancer associated with DNA mismatch repair gene mutations and microsatellite instability [43]. This approval was based on an impressive overall response rate (ORR) of pembrolizumab of approximately 40% (including some complete responses), with a duration of benefits longer than 6 months in 78% of patients treated with pembrolizumab across five clinical trials for several malignancies [43]. Nevertheless, these trials did not include ACC patients, so checkpoint inhibitors for ACC with DNA mismatch repair gene mutations and microsatellite instability must be evaluated through clinical trials. Importantly, microsatellite instability is associated with an increased TMB in approximately 4% of ACC [44].

In general, the cancer microenvironment is characterized by a rapid proliferation of the tumor cells that is unmatched by the available blood supply. To compensate, abnormal vessels develop; however, the supply of oxygen is still limited leading to hypoxia with subsequent stabilization of the inducible (alpha) subunit of hypoxia inducible factors (HIFs) [45]. HIFs activate the PD-L1 gene, which, in turn, induces tumor immune escape by suppressing the activity of cytolytic T cells [45]. Most MPPGL are characterized by an environment of pseudohypoxia [46]. Up to 50% of patients with MPPGL carry germline mutations of the subunit B of the succinate dehydrogenase gene (*SDHB*), and metastatic tumors also happen in carriers of other mutations involved in the regulation of the oxygen metabolism (e.g., *SDHA*, *SDHC*, *SDHD*, *FH*, and *VHL* genes) [46]. Furthermore, many apparently sporadic MPPGs are characterized by a microenvironment of pseudohypoxia, including those that carry activating mutations of *EPAS1*, the gene encoding for HIF2 alpha [46,47]. Therefore, it is worth exploring checkpoint inhibitors for patients with MPPGL. Of interest, there are rare cases of succinyl dehydrogenase gene mutation–associated

ACC [48]. However, because these cases are very rare, it is unlikely that pseudohypoxia is a major player determining a potential response to checkpoint inhibitors in ACC.

For a variety of reasons, checkpoint inhibitors are a potentially attractive therapy to orphan tumors [49]. Unlike other systemic therapies, immunotherapy with checkpoint inhibitors has been demonstrated as an effective treatment for many different cancers, irrespective of their embryological origin and histological characteristics [23]. In addition, clinical responses do not seem to always correlate with PD-L1 expression in the tumor cells and/or tumor microenvironment [50]. Adverse events associated with checkpoint inhibitors are, for the most part, acceptable and correctable with supportive measures [23,49,51]. Furthermore, there are no reliable preclinical models to predict the actions of immunotherapy in specific malignancies. The following sections describe the results of phase 1 and 2 clinical trials against ACC and MPPGL.

#### **4. Clinical Trials with Immune Checkpoint Inhibitors**

*4.1. Clinical Trials with Immune Checkpoint Inhibitors for ACC*

4.1.1. Avelumab

Avelumab, a PD-L1 inhibitor, was the first checkpoint inhibitor evaluated for ACC in clinical trials. Avelumab's pharmacokinetics, efficacy, and safety were evaluated in JAVELIN, a phase 1b international, multicenter clinical trial that included 50 patients with progressive metastatic ACC [52]. Objective responses (PR) were seen in 3 patients (6%), but the CBR was 48%. Almost half of the study participants received concomitant mitotane therapy, including two of the three patients with PR. A large majority of ACCs progressed over a short time; thus, the median progression-free survival (PFS) was only 2.6 months, and overall survival (OS) was 10.6 months [52]. Toxicity was acceptable, although 16% of patients had grade ≥3 adverse events. The study showed that 60% of tumors did not express PD-L1, and these patients exhibited shorter PFS and OS when compared with patients with PD-L1–positive tumors. Nevertheless, this difference was not statistically significant. The few PRs did not correlate with PD-L1 expression [52].

#### 4.1.2. Nivolumab

Nivolumab is a PD-1 inhibitor that was evaluated in a phase 2 investigator-initiated, single-center clinical trial for patients with ACC [53]. The primary endpoint of this trial was ORR. This small trial included 10 patients with progressive ACC who were either previously treated with platinum-based chemotherapy and/or mitotane or who were therapy naïve. The results of the study indicated that nivolumab did not elicit a response. ACC progressed in seven patients. Two patients had stable disease, one for a very short period and the other for 48 weeks. One patient had an unconfirmed PR; however, this patient withdrew from the trial because of a severe side effect. As expected, toxicity was acceptable overall and similar to what has been observed in clinical trials for nivolumab treatment of other malignancies. Finally, the PFS was only 1.8 months [53].

#### 4.1.3. Pembrolizumab

Like nivolumab, pembrolizumab is a PD-1 inhibitor. Pembrolizumab has been evaluated in two phase 2 clinical trials for patients with ACC. The first published phase 2 clinical trial with pembrolizumab included 16 patients with advanced ACC [54,55]. These patients previously underwent failed standard systemic therapy for ACC. The primary endpoint was the non-progression rate at 27 weeks. Two patients were not evaluable for the primary endpoint. Five patients (36%) did not have disease progression 27 weeks after treatment was initiated. The ORR was 14%, and the CBR was 57%. Tumor responses did not correlate with PD-L1 tumor expression, tumor-infiltrating lymphocytes, microsatellite instability, or tumor hormonal activity [55]. In fact, none of the patients had PD-L1 expression. Nevertheless, clinical observations suggest that clinical responses were more likely in patients with tumors that did not secrete hormones when compared with those with tumors associated with Cushing syndrome [55]. Nevertheless, tumors associated with Cushing syndrome

achieved either PR or stable disease, suggesting that combining pembrolizumab with medications that lower cortisol production may lead to better responses [55]. Pembrolizumab had severe side effects, including colitis and pneumonitis.

The second trial with pembrolizumab evaluated ORR as the primary endpoint and included 39 patients [56]. The ORR was 39%, and the CBR was 52%. However, the median PFS was only 2.1 months, and the median OS was 24.9 months. Serious adverse events were noted in 13% of patients. Positive tumor responses did not correlate with PD-L1 expression or microsatellite instability. This study did not provide information on tumor hormonal activity [56].

#### 4.1.4. Ipilimumab Plus Nivolumab

In a phase 2 clinical trial of ipilimumab plus nivolumab for patients with rare genitourinary tumors, a subset of 16 patients with advanced ACC was included. The ORR was only 6%; however, the CBR was almost 50%. The toxicity of ipilimumab plus nivolumab was acceptable [57]. The results of this study suggest that the combination of these two checkpoint inhibitors do not provide better responses than what is noticed in patients with ACC treated with single-agent pembrolizumab.

#### 4.1.5. Does Immunotherapy with Checkpoint Inhibitors Work for ACC?

In general, the ORRs for single-agent immune checkpoint inhibitors are low; however, the CBRs for these inhibitors are more impressive. Almost half of the patients treated with these therapies have shown at least disease stabilization, which is frequently associated with some degree of tumor regression for some time; in addition, the risk for significant toxicity of these inhibitors is much lower when compared with that of platinum-based chemotherapy. Furthermore, the few patients who respond to checkpoint inhibitors often enjoy a long duration of response [55,56], and a few cases of complete responses have been seen outside the clinical trials [58]. Nevertheless, it is currently very difficult to predict which patients may benefit from immunotherapy. Clinical trials have demonstrated that the expression of PD-L1 in ACC does not necessarily predict a positive clinical outcome; in fact, there are some patients with very impressive radiographic responses with ACC samples lacking PD-L1 expression [52,55]. Conversely, there are patients with PD-L1 expression in whom antitumor responses are not observed [55]. Although the absence of PD-L1 expression could represent a mechanism of tumor resistance to checkpoint inhibitors, observations from clinical trials indicate that this process is much more complicated.

However, other mechanisms of avoidance of the immune system have been proposed [59]. These mechanisms may include, but are likely not limited to, the frequently observed inactivation of the *P53* gene pathway in ACC due to somatic and occasional germline mutations of *P53* (as are common with Li-Fraumeni syndrome) [36,60]. These mutations may lead to the decreased recruitment of natural killer and other immune cells [61–63] or the sometimes-noted upregulation of the WNT/β-catenin pathway [36,60], which may impair adequate antigen presentation, chemotaxis, and tumor infiltration by T cells [59,64]. Nevertheless, clinical trials have not explored correlations with these molecular phenotypes, and these phenotypes may not necessarily predict an immunotherapy response. Moreover, ACC linked to excessive glucocorticoid secretion is associated with worse prognosis when compared with non-hormonally active ACC [7]. The glucocorticoidrelated toxicity suggests an increased risk for complications, such as osteoporosis and fractures, muscle weakness, hypertension, and especially immune suppression, which may predispose patients to systemic infections. Furthermore, ACC associated with Cushing syndrome exhibits elevated mitotic rates and, subsequently, more aggressive oncological behavior; these characteristics, together with the inherent comorbidity of Cushing syndrome, lead to lower overall survival rates compared with non-hormonally active tumors [36].

Considering the modest mutation burden of ACC, it is speculated that the effective immune targeting of ACC will require combination therapy or an engineered cellular therapy [65]. Emerging data show that checkpoint inhibitors combined with other treatments

may overcome the mechanisms of avoidance or resistance to the immune system. Based on the concern that excess cortisol creates an unfavorable atmosphere for immunotherapy [63], a phase 1b study is ongoing to evaluate the effect of combining pembrolizumab with relacorilant, a glucocorticoid receptor blocker (ClinicalTrials.gov Identifier: NCT04373265). In addition, checkpoint inhibitors may be combined with inhibitors of adrenal glucocorticoid synthesis, such as metyrapone.

The combination of mitotane, as the standard of care, with immune checkpoint inhibitors has been reported with avelumab, and this combination is likely safe, considering the relatively low risk of severe adverse events in the study [52]. A recent retrospective case series of six patients treated with pembrolizumab and mitotane found durable responses in the majority of patients, including two patients with a durable response rate [66]. This combination is of great interest because it can utilize the adrenolytic and steroid reduction properties of mitotane to make the tumors more susceptible to checkpoint inhibitor therapy.

In the past few years, the use of antiangiogenic agents has transformed the management of multiple malignancies. Targeting vascular endothelial growth factor receptor signaling, in combination with checkpoint inhibitors, e.g., by combining pembrolizumab and lenvatinib in renal cell carcinoma and endometrial carcinoma, has proven beneficial in multiple malignancies and resulted in exceptionally high response rates [67,68]. It is hypothesized that combining immunotherapy with antiangiogenic drugs has synergistic effects that enhance response rates [69]. A recent cases series evaluated the combination of lenvatinib with pembrolizumab in ACC. Despite undergoing many failed lines of therapy, some patients treated with this combination had durable responses to therapy, whereas checkpoint inhibitors and antiangiogenic therapy as single-agent therapies were unsuccessful [70].

#### *4.2. Clinical Trials with Immune Checkpoint Inhibitors for MPPGL*

#### 4.2.1. Pembrolizumab

A phase 2 clinical trial with pembrolizumab at 200 mg intravenously every 3 weeks explored the actions of PD-1 inhibition against MPPGL [71]. The primary endpoint of this trial was a non-progression rate at 27 weeks (9 cycles) greater than 20%, based on the Response Evaluation Criteria in Solid Tumors 1.1. Secondary endpoints included ORR, CBR, PFS, OS, safety, and correlations with PD-L1 expression and infiltrating mononuclear inflammatory cells in the primary tumor with disease response and genotype. The clinical trial included 11 patients with progressive MPPGL. Sixty-four percent of patients had apparently sporadic MPPGL, 18% had paraganglioma syndrome type 4 (germline *SDHB* mutations), 1 patient had paraganglioma syndrome type 1 (germline *SDHD* mutation), and 1 patient had a germline *PMS2* mutation. Sixty-four percent of patients had tumors that secreted noradrenaline. Fifty-five percent of the primary tumors were in the sympathetic extra-adrenal paraganglia, 36% of patients had pheochromocytomas, and 1 patient had a primary head and neck paraganglioma. Patients had an acceptable performance status, with an Eastern Cooperative Oncology Group performance score of ≤1. Only 28% of patients were naïve to therapy; most patients had previously undergone cyclophosphamide, vincristine, and dacarbazine chemotherapy, and treatment with HSA-I-131-MIBG, and/or tyrosine kinase inhibitors. Forty percent of patients had no evidence of disease progression at 27 weeks; however, the ORR was only 9%. The toxicity of pembrolizumab was acceptable, and there were no grade 4 or 5 side effects or cases of catecholamine crisis. The median PFS was 5.7 months, and the median OS was 19 months, with 55% of patients deceased at the time of publication of clinical trial results because of tumor progression [71]. There was no clear association between PD-L1 expression or tumor-infiltrating lymphocytes in the primary tumor with clinical response, genetic background, or hormonal activity.

A clinical assessment indicated that only two patients (18%) had an obvious benefit. One patient had a non-hormonally active tumor that achieved a confirmed immune-related partial response that persisted for longer than 2 years. The patient with the most impressive clinical response had paraganglioma syndrome type 4 metastatic paraganglioma associated with excessive noradrenaline secretion, overwhelming symptoms of catecholamine excess, and massive lymph node, lung, liver, and skeletal metastases. The patient had previously undergone cabozantinib treatment, which was complicated by severe hand and foot syndrome and a superinfection with pseudomonas aeruginosa. Cabozantinib was then discontinued, and the patient exhibited a rapid progression. Several metastases were palpable and visible on physical examination. The blood pressure was difficult to control, and the patient complained of palpitation, sweats, and headaches. The patient underwent pembrolizumab treatment. Four days after infusion, the metastases were no longer palpable or visible, the blood pressure normalized, and several antihypertensives were discontinued, as the patient complained of near syncopal episodes. Symptoms of catecholamine excess were no longer reported. Radiographic studies found a 56% tumor size reduction (Figure 2). The patient had elevated levels of liver enzymes, which delayed treatment with pembrolizumab. At the time of radiographic follow-up three months later, a new liver lesion was noted, and the patient discontinued his participation in the trial [71].

**Figure 2.** A 42-year-old man with metastatic paraganglioma underwent multiple surgery resection, post-surgical therapy with CVD × 6 months, cabozantinib × 8 months, then treatment with pembrolizumab. The pre-immunotherapy contrast enhanced CT (CECT) showed innumerable lymph node metastases in the chest, abdomen, and pelvis. At 2 months post initiation of immunotherapy, the lymph node metastases had significantly decreased by number and size (long and short arrows in images (**a**–**d**)). Representative axial CECT images ((**a**): pretherapy axial CECT of chest, (**b**): pretherapy axial CECT of pelvis, (**c**): post-therapy axial CECT of chest, (**d**): post-therapy axial CECT of pelvis) showed that the mediastinal (long and short arrows in (**a**,**c**)) and left common iliac (long arrow in (**b**,**d**)) lymph node metastases had significantly improved. The right paratracheal lymph node (long arrows in (**a**,**c**)) decreased from 4.3 × 3.5 cm to 1.5 × 1.2 cm, and the left common iliac lymph node (long arrows in (**b**,**d**)) decreased from 4.5 × 3.2 cm to 1.1 × 0.9 cm.

#### 4.2.2. Ipilimumab Plus Nivolumab

This phase 2 clinical trial included two patients with progressive MPPGL [57]. One patient did not experience a response to the therapy and exhibited disease progression. The other patient had stable disease for longer than 2 years, with excellent performance status, occasional fatigue, and no symptoms of tumor burden. However, the tumor size

did not decrease [57]. Whether the second patient exhibited disease stabilization because of immunotherapy or because of the nature of MPPGL tumors, which may become stable with no intervention despite initial growth, is still to be defined.

#### 4.2.3. Does Immunotherapy with Checkpoint Inhibitors Work for MPPGL?

The authors of the manuscript and others believe that checkpoint inhibitors and other types of immunotherapy can work for patients with MPPGL [72]. However, we need to better understand the mechanisms that may successfully activate the immune system. At this time, the clinical scientific experience with checkpoint inhibitors is limited to the definitive results of the small phase 2 clinical trial with single-agent pembrolizumab and the limited results of a phase 2 clinical trial that combined nivolumab and ipilimumab [71,72]. The results of these trials indicate that checkpoint inhibitors are associated with modest responses and that pembrolizumab alone or ipilimumab combined with nivolumab should not be considered as first-line therapies for patients with MPPGL [71,72]. Combining checkpoint inhibitors with other therapeutic modalities that enhance antigen recognition and/or facilitate vascular normalization could activate the immune system more successfully. Preliminary results of a phase 2 clinical trial with cabozantinib (the most potent antiangiogenic medication available in clinical practice) seem impressive and suggest that a substantial number of patients with MPPGL may benefit from this medication [28]. Similarly, a recent report described a patient who had a positive oncological response characterized by tumor size reduction and stabilization after receiving 40 mg of cabozantinib daily for 7.5 months. The patient was then treated with pembrolizumab with no response, followed by chemotherapy, which caused substantial toxicity. The patient later underwent a combined treatment of cabozantinib with nivolumab. Combining cabozantinib with nivolumab was associated with a PR, disease stabilization, the disappearance of the symptoms of catecholamine excess, and an acceptable toxicity, and the clinical benefits of this combined therapy lasted for 22 months. This case report suggests that cabozantinib could have induced some degree of tumor vascular normalization that facilitated the activation of the immune system [73].

In the phase 2 clinical trial with pembrolizumab, one patient had an impressive clinical response, raising the question of whether previous exposure to cabozantinib and/or the introduction of foreign bacterial antigens can activate the immune system against MPPGL. Pseudomonas aeruginosa is a Gram-negative bacterium, characterized by the presence of lipopolysaccharides in the outer layer and many other components that trigger or enhance an immune response. These bacteria may become trapped in the tumor microenvironment within the abnormal vessels, inducing an immune attack against tumor cells, thus supporting bacteria-based MPPGL immunotherapy [74,75].

A recent retrospective study found that patients with MPPGL may have a higher incidence of other malignancies, such as lung, prostate, melanoma, and colorectal cancers, when compared to the general population [76]; this finding suggests that some of the MPPGL tumorigenesis pathways and mechanisms of immune resistance could be similar to the ones observed in more common tumors for which immunotherapy has been demonstrated to be effective; thus, learning from the experience with the aforementioned malignancies may provide clues on how to treat MPPGL with immunotherapies.

#### **5. The Gut Microbiome and Peptide-Based Vaccination against ACC and MPPGL**

Peptide-based vaccination delivers immunogenic peptides, corresponding to tumorassociated or tumor-specific antigens, to elicit a T-cell immune response. It is challenging to generate a strong immune response against tumor-associated antigens (TAAs), mainly because the non-mutated tumor-associated antigens are part of the repertoire of selfantigens. To circumvent this problem, the immune response should target mutated, nonself-antigens.

Sequencing of the human fecal microbiota revealed that all TAAs had a closely structurally related "mimic" in the microbiome, with higher affinities for the MHC than the

corresponding TAA [77]. As these "mimics" are produced by bacteria, they have the potential to "pre-expose" any person and generate memory T-cells; the re-activation and subsequent expansion of these T cells can generate a robust response against the TAAs. The links between the microbiome, clinical response, and inhibition of cancer progression in cancer patients treated with targeted immunotherapies or with specific chemotherapeutic agents have already been emphasized [78]. The presence of commensal bacteria-specific memory T cells in the gut and in the periphery has been described as well [79]. These microbiome-derived peptides stimulate strong immune responses against TAAs and trigger in vivo tumor regression after vaccination.

The NCT04187404 trial (SPENCER Trial) is evaluating the vaccine EO2401 against ACC and MPPGL. This vaccine includes three microbiome-derived CD8+ epitopes mimicking parts of TAAs, such as the interleukin receptor alpha 2 (IL13Rα2), survivin (BIRC5), and the mammalian forkhead box M1 (FOXM1); these antigens are overexpressed and linked to clinical outcomes in ACC and MPPGL [80–87]. These antigens may induce an immune response against tumors of adrenal origin and have minimal to no expression in normal organs. The SPENCER trial is a multicenter, phase 1/2, first-in-human study to assess the safety, tolerability, immunogenicity, and preliminary efficacy of EO2401 in combination with nivolumab for patients with untreated or previously treated ACC or MPPGL. The initial data from the trial is awaited in 2022.

#### **6. Conclusions**

Immunotherapy for adrenal tumors, such as ACC and MPPGL, is at an early stage of development. Single-agent immunotherapy has led to some impressive and durable responses in patients with ACC; however, ORRs are generally low. In patients with MPPGL, responses have been uncommon, and the mechanisms of response are unclear. However, the failure of checkpoint inhibitors to elicit a response is not an indication of an absolute lack of success. Conversely, the failure of single-agent checkpoint inhibitor therapy represents an opportunity to identify the mechanisms that could lead to more successful treatment strategies. Combining checkpoint inhibitors with chemotherapy, mitotane, tyrosine kinase inhibitors, and/or vaccines for ACC or chemotherapy, tyrosine kinase inhibitors, and/or radiopharmaceuticals for MPPGL needs to be proactively explored.

**Author Contributions:** Conceptualization: C.J., G.A.-P., P.L.M.D., J.V. and M.A.H.; investigation: C.J. and M.A.H.; writing—original draft preparation: C.J.; writing—review and editing: C.J., G.A.-P., P.L.M.D., J.V., Y.L., R.A.T. and M.A.H.; supervision: C.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research did not receive external funding.

**Acknowledgments:** We thank Ashli Nguyen-Villarreal, Associate Scientific Editor, and Sarah Bronson, Scientific Editor, in the Research Medical Library at The University of Texas MD Anderson Cancer Center for editing this article.

**Conflicts of Interest:** C.J. is scientific advisor for HRA Pharma, Lantheus Pharmaceuticals, Merck Sharp and Dohme, and Pfizer Pharmaceuticals. C.J. has received research support from Enterome, Exelixis, Lantheus Pharmaceuticals, Merck Sharp and Dohme, Pfizer Pharmaceuticals, and Progenics; G.A.-P. does not have conflicts of interest; P.L.M.D. is a recipient of funds from the NIH (GM114102 and CA264248) and the Neuroendocrine Tumor Research Foundation and is the holder of the Robert Tucker Hayes Distinguished Chair in Oncology; J.V. does not have conflicts of interest; Y.L. does not have conflicts of interest; R.A.T. holds a Miguel Servet-I research contract by Institute of Health Carlos III (ISCIII) of the Ministry of Economy [grant number CP17/00199] and Competitiveness from the Spanish government and is supported by an Olga Torres Foundation emerging researcher grant, by the Swiss Bridge Award, and received a research grant from Novartis, Astrazeneca, Beigene (all pharma grants are not related to this project); M.A.H. received research support from Corcept Therapeutics Exelixis Inc.

#### **References**

## **Cardiovascular Toxicities Secondary to Biotherapy and Molecular Targeted Therapies in Neuroendocrine Neoplasms: A Systematic Review and Meta-Analysis of Randomized Placebo-Controlled Trials**

**Charalampos Aktypis 1,†, Maria-Eleni Spei 2,†, Maria Yavropoulou 2, Göran Wallin 3, Anna Koumarianou 4, Gregory Kaltsas 2, Eva Kassi 2,5 and Kosmas Daskalakis 2,3,\***

**Simple Summary:** Recent innovations in molecular pathogenesis of neuroendocrine neoplasms (NEN) and improvements in their multidisciplinary management, including the introduction of novel targeted therapies have contributed to favorable patient outcomes. Compared with traditional chemotherapy, targeted therapies have fewer toxicities and a more distinct safety profile. However, treatment-induced cardiovascular toxicities are occasionally critical issues in NEN management. Herein, we present a comprehensive summary of high quality randomized evidence with the methodology of a systematic review and quantitative meta-analysis on the safety profile of biotherapy and molecular targeted therapies in advanced and/or metastatic NEN with a special focus on cardiovascular toxicities in order to promote a patient-tailored approach and assist clinicians involved in the management of NEN patients.

**Abstract:** A broad spectrum of novel targeted therapies with prime antitumor activity and/or ample control of hormonal symptoms together with an overall acceptable safety profile have emerged for patients with metastatic neuroendocrine neoplasms (NENs). In this systematic review and quantitative meta-analysis, the PubMed, EMBASE, Cochrane Central Register of Controlled Trials and clinicaltrials.gov databases were searched to assess and compare the safety profile of NEN treatments with special focus on the cardiovascular adverse effects of biotherapy and molecular targeted therapies (MTTs). Quality/risk of bias were assessed using GRADE criteria. Placebocontrolled randomized clinical trials (RCTs) in patients with metastatic NENs, including medullary thyroid cancer (MTC) were included. A total of 3695 articles and 122 clinical trials registered in clinicaltrials.gov were screened. We included sixteen relevant RCTs comprising 3408 unique patients assigned to different treatments compared with placebo. All the included studies had a low risk of bias. We identified four drug therapies for NENs with eligible placebo-controlled RCTs: somatostatin analogs (SSAs), tryptophan hydroxylase (TPH) inhibitors, mTOR inhibitors and tyrosine kinase inhibitors (TKI). Grade 3 and 4 adverse effects (AE) were more often encountered in patients treated with mTOR inhibitors and TKI (odds ratio [OR]: 2.42, 95% CI: 1.87–3.12 and OR: 3.41, 95% CI: 1.46–7.96, respectively) as compared to SSAs (OR:0.77, 95% CI: 0.47–1.27) and TPH inhibitors (OR:0.77, 95% CI: 0.35–1.69). MTOR inhibitors had the highest risk for serious cardiac AE (OR:3.28, 95% CI: 1.66–6.48) followed by TKIs (OR:1.51, 95% CI: 0.59–3.83). Serious vascular

**Citation:** Aktypis, C.; Spei, M.-E.; Yavropoulou, M.; Wallin, G.; Koumarianou, A.; Kaltsas, G.; Kassi, E.; Daskalakis, K. Cardiovascular Toxicities Secondary to Biotherapy and Molecular Targeted Therapies in Neuroendocrine Neoplasms: A Systematic Review and Meta-Analysis of Randomized Placebo-Controlled Trials. *Cancers* **2021**, *13*, 2159. https://doi.org/ 10.3390/cancers13092159

Academic Editors: Alfredo Berruti, Vito Amoroso and Nicola Fazio

Received: 22 March 2021 Accepted: 28 April 2021 Published: 30 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

AE were more often encountered in NEN patients treated with mTOR inhibitors (OR: 1.72, 95% CI: 0.64–4.64) and TKIs (OR:1.64, 95% CI: 0.35–7.78). Finally, patients on TKIs were at higher risk for new-onset or exacerbation of pre-existing hypertension (OR:3.31, 95% CI: 1.87–5.86). In conclusion, SSAs and TPH inhibitors appear to be safer as compared to mTOR inhibitors and TKIs with regards to their overall toxicity profile, and cardiovascular toxicities in particular. Special consideration should be given to a patient-tailored approach with anticipated toxicities of targeted NEN treatments together with assessment of cardiovascular comorbidities, assisting clinicians in treatment selection and early recognition/management of cardiovascular toxicities. This approach could improve patient compliance and preserve cardiovascular health and overall quality of life.

**Keywords:** neuroendocrine neoplasms; molecular targeted therapies; mTOR inhibitors; somatostatin analogs; TPH inhibitors; meta-analysis

#### **1. Introduction**

Neuroendocrine neoplasms (NENs) comprise a group of diverse histopathological entities across different organs and systems, including the gastrointestinal system, the lungs, the adrenals and the thyroid. Although the majority of NENs are well differentiated (WD) and may exhibit a prolonged indolent course, some patients categories, e.g., the ones with medullary thyroid cancer (MTC), atypical lung carcinoids (LCs) and pancreatic NENs of higher proliferation may exhibit a more aggressive course [1,2]. Many NEN patients are diagnosed with established distant metastases or exhibit progress to stage IV under disease surveillance [3]. Systemic cytotoxic chemotherapy has a generally low response rate in patients with metastatic WD NENs of lower proliferation and MTC and is nevertheless associated with serious toxicities. On the other hand, prime anti-tumor activity and/or control of hormonal excess syndromes has been demonstrated for targeted agents in NENs, resulting in the approval of somatostatin analogs (SSAs) and tryptophan hydroxylase (TPH) inhibitors for gastroenteropancreatic NENs, also referred to as biotherapy, as well as novel molecular targeted therapies (MTTs), such as the mTOR inhibitor everolimus and a number of tyrosine kinase inhibitors (TKIs) with the latter being approved across a diverse spectrum of NEN primaries, including pancreatic NENs and MTC [4,5].

In the last decades, an increment in the prevalence of NENs along with a prolongation in life expectancy of these patients has been observed despite a rising prevalence of cardiovascular diseases in the elderly [1]. On the other hand, carcinoid heart disease, a rare cardiac manifestation involving the right-sided heart valves, constitutes a well-recognized sequela in patients with small intestinal neuroendocrine neoplasms (SI-NENs) often complicating the disease clinical course and eventually leading to right heart failure [6]. Finally, NEN metastases to the heart are rare, with associated clinical features ranging from asymptomatic patients to heart failure [7]. All these factors taken together with the cardiovascular side effects of different agents in the therapeutic NEN armamentarium may have a negative impact on patient outcomes, including quality of life and possibly survival outcomes. Nevertheless, the continual and occasionally prolonged nature of the administration of targeted agents leads to new challenges in their application with respect to the management of anticipated cardiovascular toxicities [5,8].

The most frequent side effects of SSAs consist mainly of gastrointestinal toxicities, with potentially a beneficial effect on cardiac parameters in the setting of acromegalic heart disease, a constellation of cardiac complications associated with acromegaly that involves nearly all aspects of the cardiovascular system [9]. In addition, SSAs and TPH inhibitors inhibit serotonin secretion from the tumor and subsequently lower 5-HIAA levels, relieving carcinoid syndrome, a rare secretory syndrome mainly associated with small intestinal and bronchial NENs that becomes manifest when serotonin and other vasoactive substances from the tumor enter the systemic circulation escaping hepatic degradation [4]. However, SSAs do not unequivocally reverse the progression of the carcinoid cardiac involvement

nor improve survival in the setting of carcinoid heart disease [10]. The role of the recently introduced telotristat ethyl for prevention or control of carcinoid heart disease remains largely unknown, but could be elucidated in the near future as we obtain further evidence from clinical trials.

With regards to pathophysiology, MTTs may induce cardiovascular adverse effects (AEs) as a result of "on-target" and "off-target" mechanisms [11,12]. The on-target toxicity mechanism implicates mainly the mTOR complex 1 pathway with the target of MTT playing a crucial role in oncogenesis and angiogenesis, but also in hypertrophic response and survival of cardiomyocytes [12]. Off-target toxicity on the other hand, implicates an unintentional inhibition of a kinase that is also important for cardiac cell survival or function. For example certain TKIs induce a cardiomyocyte damage-related lactate dehydrogenase (LDH) release, that is in turn associated with the binding specificity of TKIs to their molecular target [13].

The placebo-controlled RCTs on biotherapy and MTTs for NENs and MTC report treatment-related toxicities according to the National Cancer Institute Common Terminology Criteria for Adverse Events, and therefore constitute a complete resource of treatmentrelated toxicities [14–16]. The present overview provides a comprehensive summary of high quality randomized evidence with the methodology of a systematic review and quantitative meta-analysis on the distinct safety profile of biotherapy and MTTs in advanced and/or metastatic NEN with a special focus on cardiovascular toxicities in order to assist clinicians involved in the management of NEN patients.

#### **2. Results**

#### *2.1. Study Selection*

We screened 3695 titles and abstracts from PubMed, Excerpta Medica database (EM-BASE), Cochrane Central Register of Controlled Trials and additional 122 clinical trials in clinicaltrials.gov and identified 202 potentially eligible RCT reports (Figure 1). Some of the RCTs were reported in multiple publications or were posthoc analysis of the initial RCT; thus, they were excluded. After full article assessment, we included a total of 16 placebocontrolled RCTs reporting cardiovascular toxicities in the quantitative meta-analysis. Only patients with metastatic NET or MTC were included. The results on the safety profile of most RCTs were available from the published article or abstract and clinicaltrials.gov. A total of 3408 unique patients were recruited; four different categories of targeted agents were evaluated: SSAs, the TPH inhibitor telotrist etiprate, the mTOR inhibir everolimus and different TKIs. In particular, six RCTs addressed biotherapy (three RCTs on SSAs and three RCTs on telotrist etiprate) and ten RCTs addressed MTTs (the RADIANT-2, 3 and 4 trials on everolimus and seven RCTs on TKIs). All RCTs in the quantitative meta-analysis were industry sponsored. Study and patient characteristics are provided in Table 1 and Table S1, respectively.

**Table 1.** Characteristics of placebo-controlled randomized controlled trials included in the meta-analysis.

**Table 1.** *Cont*.

Abbreviations. ATA: American Thyroid Association; ENETS: European Neuroendocrine Tumor Society; ETA: European Thyroid Association; mTOR: Mechanistic target of rapamycin; n.a.: Non available; SSAs: Somatostatin analogs; TKI: Tyrosine kinase inhibitor; TPH: Tryptophan hydroxylase.

**Figure 1.** PRISMA flowchart of search results.

*2.2. Representation in International Guidelines*

Among the sixteen RCTs included in the present meta-analysis, six out of 14 RCTs on WD-NENs are included in the latest 2017 European Neuroendocrine Tumor Society (ENETS) [4], whereas one study on MTC, the ZETA trial, is included in both the 2015 Society American Thyroid Association (ATA) consensus guidelines [15] and the 2012 European Thyroid Association (ETA) guidelines for MTC (Table 1) [16].

#### *2.3. Risk of Bias Assessment*

In none of the included studies did we encounter high risk for bias in random sequence generation (selection bias), allocation concealment (selection bias), blinding participants and personnel (performance bias), blinding the outcome assessment (detection bias), incomplete outcome data (attrition bias), and selective reporting (reporting bias) (Table 2).

**Table 2.** Risk of bias summary: Authors' judgments about each risk of bias item for each included study following the Grading of Recommendations Assessment, Development and Evaluation (GRADE) Approach.

**Table 2.** *Cont*.

Each domain was judged as 'low risk of bias' (-), 'high risk of bias' (+), or 'unclear risk of bias' (?) in each study according to the Cochrane Handbook for Systematic Reviews of Interventions 1.

#### *2.4. Serious Toxicities' Profile*

Sixteen placebo-controlled RCTs compared grade 3 and 4 AE for four different categories of targeted agents in WD-NENs and MTC (Figure 2). TKIs exhibited a pooled odds ratio (OR) for serious AE of 3.41 (95% CI: 1.46–7.96). For surafatinib AE\_OR in pancreatic NENs was as high as 23.46 (95% CI: 9.99–55.09), whereas for sunitinib in the same subset of patients AE\_OR was 0.52 (95% CI: 0.27–0.99), as compared to placebo. In addition, the recently tested in phase III NEN trials axitinib and pazopanib demontrated a relatively high AE\_OR (axitinib AE\_OR: 7.08; 95% CI: 3.83–13.14 and pazopanib AE\_OR: 4.67; 95% CI: 1.31–16.71, respectively). For MTC, the effect estimates were AE\_OR: 2.97 (95% CI, 1.56–5.67) for vandetanib and 2.40 (95% CI: 1.43–4.04) for cabozatinib, as compared to placebo (Figure 2).The mTOR inhibitor everolimus exhibited a pooled OR for serious AE of 2.42 (95% CI: 1.87–3.12). With regards to biotherapy, SSAs demonstrated a pooled OR for serious AE as low as 0.77 (95% CI: 0.47–1.27), which was comparable to that of the TPH inhibitor telotristat etiprate (AE\_OR: 0.77; 95% CI: 0.35–1.69; Figure 2). Significant heterogeneity was observed across the subset of studies on TKI (I2 = 89.8%, *p*-value < 0.0001; Figure 2). A funnel plot was also produced demonstrating asymmetry (Figure S1A), that was mainly attributed to the recent studies on novel TKIs by Xu et al. on surufatinib in pancreatic NENs and by Carbonero et al. on axitinib in extra-pancreatic NENs (Galbraith's plot; Figure S1B) [29,30]. Egger's test showed no indication of publication bias across the included studies (*p*-value > 0.05). In the subset of studies on SSAs, TPH inhibitors and everolimus, we did not observe inter-study heterogeneity or publication bias (Figure 2 and Figure S1A–C).

**Figure 2.** Targeted Agents' Serious Toxicities profile per drug category in patients with Metastatic Well-Differentiated Neuroendocrine Neoplasms or Medullary Thyroid Cancer (Odds Ratios [OR] with 95% Confidence Intervals). Tyrosine kinase inhibitors exhibited the highest pooled OR: 3.41 (95% CI: 1.46–7.96) followed by everolimus (pooled OR: 2.42 [95% CI: 1.87–3.12]). Somatostatin analogues pooled OR was relatively low( 0.77 [95% CI: 0.47–1.27], same as that of telotristat etiprate (OR: 0.77 [95% CI: 0.35–1.69]).

#### *2.5. All Grade Toxicities' Profile*

We conducted a meta-analysis of AE of all grades reported in the included RCTs (Figure 3). Our findings showed comparable figures compared to serious toxicities analysis with TKIs and everolimus demontrating the highest risk of all grade toxicity (pooled TKI AE\_OR: 3.78; 95% CI: 1.35–10.56 across both WD-NEN and MTC diagnoses; and pooled everolimus AE\_OR: 3.91, 95% CI: 1.88–8.11). SSAs appeared to have the safest all grade toxicity profile (AE\_OR: 1.08, 95% CI: 0.52–2.23; Figure 3).

Significant heterogeneity was evident across the subset of studies on TKI (I2 = 77.7%, *p*value < 0.0001; Figure 3). A funnel plot was demonstrated signs of asymmetry (Figure S2A), that was mainly attributed to the study by Wells et al. on vandetanib in MTC and Xu et al. on surufatinib in pancreatic NENs (Galbraith's plot; Figure S2B). Egger's test showed no indication of publication bias across the included studies (*p*-value > 0.05). With regards to SSA, TPH inhibitors and everolimus, we did not observe inter-study heterogeneity or publication bias (Figure 3 and Figure S2A–C).

**Figure 3.** Targeted Agents' All Grade Toxicities profile per drug category in patients with Metastatic Well-Differentiated Neuroendocrine Neoplasms or Medullary Thyroid Cancer (Odds Ratios with 95% Confidence Intervals). Tyrosine kinase inhibitors (TKI) and everolimus demontrated the highest risk of all grade toxicity (pooled OR: 3.78 [95% CI: 1.35–10.56] and OR: 3.91 [95% CI: 1.88–8.11], respectively).

#### *2.6. Serious Cardiac Toxicities' Profile*

Eleven placebo-controlled RCTs compared grade 3 and 4 cardiac AE for all four different categories of targeted agents in WD-NENs and MTC, investigated in our metaanalysis (Figure 4). Everolimus exhibited a pooled OR for serious cardiac AE of 3.28 (95% CI: 1.66–6.48) followed by TKIs with a pooled AE\_OR of 1.51 (95% CI: 0.59–3.83). Within TKI analysis, sunitinib demonstrated the highest cardiac AE\_OR in pancreatic NEN patients with OR figures as high as 7.17 (95% CI: 0.36–140.90), as compared to placebo. Surufatinib in pancreatic NENs, as well as vandetanib and cabozatinib in MTC do not appear to confer a risk for serious cardiac AE in the included RCTs (Figure 4).

With regards to biotherapy, SSAs demonstrated a potential prophylactic effect with respect to serious cardiac AE with an OR as low as 0.07 (95% CI: 0.0–1.33), which was comparable to that of telotristat etiprate (AE\_OR: 0.21; 95% CI: 0.03–1.48; Figure 4).

Grade 3 and 4 cardiac AE in the 628 WD-NEN patients recieving everolimus in the intervention arm of the RADIANT trials included acute coronary syndrome (two patients), angina pectoris (two patients), cardiac arrest (two patients), cardiac failure (seven patients), congestive cardiac failure (six patients), cardio-respiratory arrest (two patients), left ventricular dysfunction/failure (two patients), right ventricular dysfunction (one patient), myocardial dysfunction (one patient), myocarditis (one patient), palpitations (one patient), tachycardia (one patient), pericardial efusion (two patients), tricuspid valve incompetence (one patient), mitral valve incompetence (one patient), pulmonary valve stenosis (one patient).

In patients receiving TKIs, Grade 3 and 4 cardiac AE included atrial flutter (one patient), atrial fibrillation (two patients), cardiac failure (three patients), cardiopulmonary failure (one patient), left ventricular dysfunction (one patient), supraventricular tachycardia (one patient), right ventricular dysfunction (one patient), bradycardia (one patient), arrhythmia (one patient), and pericarditis (one patient). Finally, we did not observe any inter-study heterogeneity or publication bias within the different pooled analyses across different targeted NEN treatments (Figure 4 and Figure S3A–C).

**Figure 4.** Targeted Agents' Serious Cardiac Toxicities profile per drug category in patients with Metastatic Well-Differentiated Neuroendocrine Neoplasms or Medullary Thyroid Cancer (Odds Ratios [OR] with 95% Confidence Intervals). Everolimus exhibited the highest pooled OR: 3.28 (95% CI: 1.66–6.48) followed by tyrosine kinase inhibitors with a pooled OR: 1.51 (95% CI: 0.59–3.83). Somatostatin analogs and telotristat etiprate demonstrated a potential prophylactic effect with with an OR: 0.07 (95% CI: 0.0–1.33) and OR: 0.21 (95% CI: 0.03–1.48), respectively.

#### *2.7. Serious Vascular Toxicities' Profile*

Nine placebo-controlled RCTs reported grade 3 and 4 vascular AE for WD-NENs treated with SSAs, everolimus or TKI and MTC treated with TKIs (Figure 5). Everolimus exhibited a pooled OR for serious vascular AE of 1.72 (95% CI: 0.64–4.64) followed by TKIs with a comparable pooled AE\_OR of 1.64 (95% CI: 0.35–7.78). Within TKI analysis, vandetanib and cabozatinib, the two TKIs approved for MTC demonstrated the highest vascular AE\_OR as high as 9.53 (95% CI: 0.55–164.60) for vandetanib and 5.82 (95% CI: 0.74–45.65) for cabozatinib, respectively when compared to placebo (Figure 5). With regards to biotherapy, SSAs in the CLARINET trial were not linked to a higher risk for serious vascular AE with an OR of 1.02 (95% CI: 0.14–7.39). The included placebo-controlled RCTs on telotristat etiprate did not report any serious vascular AEs.

The grade 3 and 4 vascular toxicities that were reported in the RADIANT trials included hypertension (two patients), hypotension (four patients), deep vein thrombosis (three patients), phlebitis, i.e., inflammation of the walls of a vein (one patient) and hematoma, i.e., a collection of blood outside of a blood vessel (one patient). Finally, the TKI trials reported the following serious vascular toxicities: hypertension (83 patients), hypertensive crisis (four patients), deep vein thrombosis (three patients) and hypotesion (one patient).

As in cardiac AEs' pooled analyses, we did not observe any inter-study heterogeneity or publication bias (Figure 5 and Figure S4A–C).

**Figure 5.** Targeted Agents' Serious Vascular Toxicities profile per drug category in patients with Metastatic Well-Differentiated Neuroendocrine Neoplasms or Medullary Thyroid Cancer (Odds Ratio [OR] with 95% Confidence Intervals). Molecular targeted therapies were linked with higher risk for Serious Vascular Toxicities (Everolimus pooled OR: 1.72 [95% CI: 0.64–4.64]; Tyrosine kinase inhibitorsTKIs pooled OR of 1.64 [95% CI: 0.35–7.78]).

#### *2.8. Hypertension Secondary to Molecular Targeted Therapies*

Nine placebo-controlled RCTs reported treatment-related hypertension, in particular new-onset or excarbation of pre-existing hyperension, in WD-NEN patients treated with everolimus or TKI and also MTC patients treated with TKIs (Figure 6). Everolimus was not linked to a significantly higher risk for treatment-related hypertension, as a pooled OR of 1.16 (95% CI: 0.14–9.45) was evident in RADIANT-2 and RADIANT-4 trials with only two cases of hypertension among 421 patients in the intervention arm. TKIs on the other hand exhibited a pooled OR for hypertension of 3.31 (95% CI: 1.87–5.86) with 193 patients with treatment-related hypertension among the 1021 patients in the intervention arm of these trials (Figure 6). Within TKI analysis, surufatinib in patients with pancreatic and extra-pancreatic NENs and cabozatinib in patients with MTC, demonstrated the highest ORs for treatment-related hypertension (SANET-p OR: 8.45; 95% CI: 2.86–24.96; SANET-ep OR: 3.82; 95% CI: 1.74–8.39; EXAM OR: 5.22; 95% CI: 0.31–104.34, respectively; Figure 6). The included placebo-controlled RCTs on biotherapy (SSAs and telotristat etiprate) did not report any treatment-related hypertension. Pooled analyses for hypertension were only available for everolimus and TKI, and did not reveal any inter-study heterogeneity or publication bias (Figure 6 and Figure S5A–C).

**Figure 6.** Targeted Agents' Risk Assessment for new-onset or exacerbation of pre-existing Hypertension per drug category in patients with Metastatic Well-Differentiated Neuroendocrine Neoplasms or Medullary Thyroid Cancer (Odds Ratios with 95% Confidence Intervals). Tyrosine kinsae inhibitors exhibited a pooled OR for hypertension of 3.31 (95% CI: 1.87–5.86).

#### **3. Discussion**