study design, which was not set up to provide a direct comparison between the two arms, and the use of a non-metronomic schedule for CAPE, which affected the toxicity results obtained. These are only some of the issues that require to be addressed to ensure that mCHT can be applied as a therapeutic strategy.

The need for an empirical basis for determining the optimal OBD and in monitoring the therapeutic activity of the drugs used in a metronomic schedule is a crucial point; for those drugs, such as CTX which is associated with a precise and known effect (angiogenesis), the OBD can be easily determined by assessing the maximum reduction in viable peripheral blood circulating VEGFR-2 or EPCs [66]. Monitoring of VEGFR-2 blood levels during treatment can also be easily achieved, as a useful dynamic biomarker to evaluate the response. Unfortunately, not all the drugs used in the different metronomic schedules are clearly associated with a precise mechanism of action, and not all the mechanisms are evaluable by a specific biomarker. For example, the well-known effect on T-reg down regulation observed with some metronomic schedules is only a partial and incomplete expression of the immune system stimulation.

In our opinion, the main limitation remains the absence of randomised studies for the vast majority of combinations and settings: fortunately, some trials [60] have filled this gap and some others are due, such as TEMPO BREAST (EudraCT number 2014-003860-19), which compared iv vs. a metronomic oral formulation of VNR as the first-line of treatment in 164 HR + /HER2-ABC patients randomly assigned to one of two treatment arms, and ME-TEORA (NCT02954055), a multi-center, randomised phase II trial that randomised women with ER-positive, HER2-negative (human epidermal growth factor receptor 2-negative) metastatic or locally relapsed breast cancer in a ratio of 1:1, to receive a metronomic regimen of VNR plus CTX and CAPE, or the conventional paclitaxel monotherapy, the results for which are awaited soon.

Another important shadow is the almost total absence of QoL evaluation, even in trials that enrolled large populations of ABC patients; this has been performed sporadically but, unfortunately, was not planned for in most of the prospective studies.

#### **4. Discussion**

mCHT for the treatment of breast cancer treatment started slowly at the beginning of the 2000s; at that time, most physicians believed that it was nothing more than a palliative therapy, confining its use to very late stages in care.

With increasing evidence coming from scientists all around the world who tested mCHT in single arm, proof-of-concept studies in cancers different from breast (e.g., lung, prostate, paediatric, head and neck and ovarian cancers, glioblastoma), it became evident that mCHT could play a different role.

Even after many years of clinical use, especially as part of treatment of ABCs, the use of mCHT remains a matter of debate in the scientific community. Physicians are divided between those who have adopted it in their clinical practice, based mainly on its safety profile, and those who remain fierce opponents, citing many different reasons. Research is required to look deeper into developing strategies to improve the efficacy of drugs and significantly reduce toxicity.

In this paper, we have reviewed the available data regarding mCHT in ABC patients, seeking to highlight the lights and shadows of this strategy. Among the "lights" of mCHT, we considered the safety profile of the different drugs as options for treatment of ABC patients, alone or in combination with targeted agents, and the data regarding QoL. As well covered in the literature analysis by Lien et al. [65], despite differences in reporting, mCHT has been found to lead to low toxicity rates, in particular no toxicity affected more than 6% of all pooled patients. This represents, in our opinion, the brightest light of mCHT.

In the preclinical setting, we evaluated as "lights," the well-described mechanisms that prevent or block angiogenesis, mainly by inhibiting VEGF and, to some extent, the effect of mCHT immunomodulation.

Among the "shadows", we discussed the absence of prospective randomised trials, the design and the end-points adopted in some studies, and the heterogeneity of existing schedules, even for the same drug. Recently, Mishra-Kalyani et al. considered the role of control arms in oncology [67]; they suggest that, even though randomised control trials allow for a comparison of treatment arms with minimal concern for confounding by known and unknown factors, in some situations, and for some strategies, a randomised study is not feasible. These authors suggest that, when such designs are not possible, the incorporation of external control data into the study design could get around the obstacle. In the absence of the possibility of conducting prospective randomised phase 3 studies in different settings of BC and for different populations (TNBC, Luminal, HER2 + ve), we hope that this suggested shortcut will allow a definitive comparison between standard and metronomic strategies.

From the preclinical point of view, the "shadows" are represented by all the aspects still to be clarified, including the different factors that might facilitate the use of combinatorial regimens of mCHT. These include understanding of the mechanisms of action of metronomic schedules and the recognition and validation of predictive biomarkers, which, if identified, would provide valuable support for clinical application.

Our aim is to encourage readers to extend the amount of available data collected on mCHT for the treatment of ABC patients and, perhaps, to push them to consider this strategy in their clinical practice for some patients, in some emergency situations, or in low- or middle-income countries, where a low-cost strategy, such as mCHT, might partially rebalance disparities.

#### **5. Conclusions**

The efficacy of other agents in mCHT treatment combined with antiangiogenic treatment should be investigated. An example of a promising approach is the use of Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib, which was developed to treat several blood cancers, but has recently been shown to act effectively in solid tumours [68,69]. These drugs hit multiple targets and are associated with numerous expected outcomes. It has already been shown that ibrutinib has an antimetastatic effect on MCF-7 cells by activating the MAPK/NF-kB/AP-1 pathway and inhibiting MMP-9 expression [70]; reducing the viability of Erb2-positive (Erb2+) breast cancer cell lines by inhibiting the phosphorylation of receptor tyrosine kinases ErbB1, ErbB2, ErbB3 [71]; inhibits xenograft tumour growth by decreasing HER2, BTK, Akt, Erk, and increasing cleaved caspase-3 [72]; and affects anti-tumour immunity by reprogramming myeloid-derived suppressor cells (MDSCs) to mature DCs thus preventing tumour growth and metastasis, as demonstrated in a murine model of breast cancer [73].

Preclinical models of breast cancer could be developed to gain a more in-depth understanding of the basis of resistance, relapse, or progression of mCHT therapy, as well as to perform precise therapeutic tests of mCHT treatment by using mice with metastatic disease or mice carrying patient-derived breast cancer tissue, to characterize and improve the efficacy of antitumor combination therapies in vivo, as suggested by Kerbel [74].

From this extensive analysis focused on lights and shadows, we can conclude that mCHT *per se* is not associated with severe toxicities, especially haematological or gastrointestinal effects, as detailed in the studies reported in this review, and as described in other similar papers [75,76]. However, mCHT loses this advantage, which is peculiar to this method, when different agents, with their related toxicities, or other chemotherapy drugs administered at standard doses, are co-administered [43,59].

Considering that improvement in the QoL of ABC patients, by reducing treatment toxicity, is one of the main goals of the Global Alliance Against Cancer, we strongly believe that current strategies, such as immunotherapy, should be studied in association with mCHT.

Global efforts should be combined so as not to throw away such a precious amount of data. More modern study designs should be used to clearly demonstrate if mCHT should be incorporated into our current strategies for treatment.

**Author Contributions:** Conceptualization, M.E.C. and M.G.C.; methodology, M.E.C. and M.G.C.; writing—original draft preparation, M.E.C., N.C., S.C., V.C., F.F.P., F.R. and M.G.C.; writing—review and editing, M.E.C. and M.G.C.; supervision, M.E.C. 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 acknowledge Maddalena Mongera for administrative and technical support and the Centro Studi Raffaella Trabattoni for donations in kind (APC).

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

#### **References**

## *Review* **Metronomic Chemotherapy in Prostate Cancer**

**Piotr J. Wysocki 1,2,\*, Maciej T. Lubas 2 and Malgorzata L. Wysocka 3,\***

**Abstract:** Despite the significant expansion of the therapeutic armamentarium associated with the introduction of novel endocrine therapies, cytotoxic agents, radiopharmaceuticals, and PARP inhibitors, progression of metastatic castration-resistant prostate cancer (mCRPC) beyond treatment options remains the leading cause of death in advanced prostate cancer patients. Metronomic chemotherapy (MC) is an old concept of wise utilization of cytotoxic agents administered continuously and at low doses. The metronomic is unique due to its multidimensional mechanisms of action involving: (i) inhibition of cancer cell proliferation, (ii) inhibition of angiogenesis, (iii) mitigation of tumor-related immunosuppression, (iv) impairment of cancer stem cell functions, and (v) modulation of tumor and host microbiome. MC has been extensively studied in advanced prostate cancer before the advent of novel therapies, and its actual activity in contemporary, heavily pretreated mCRPC patients is unknown. We have conducted a prospective analysis of consecutive cases of mCRPC patients who failed all available standard therapies to find the optimal MC regimen for phase II studies. The metronomic combination of weekly paclitaxel 60 mg/m2 i.v. with capecitabine 1500 mg/d p.o. and cyclophosphamide 50 mg/d p.o. was selected as the preferred regimen for a planned phase II study in heavily pretreated mCRPC patients.

**Keywords:** metronomic chemotherapy; prostate cancer; CRPC; castration; cyclophosphamide; capecitabine; paclitaxel; immunomodulation; angiogenesis; microbiome

**1. Standard Systemic Treatment of Advanced Prostate Cancer**

Endocrine therapy, basically androgen deprivation (ADT), is the standard treatment of prostate cancer (PC). This neoplasm arises from prostate gland epithelial cells located in lobules and ducts, whose growth and differentiation are tightly regulated by androgens, mainly testosterone. Therefore, well-differentiated prostate cancers are usually susceptible to androgen deprivation which allows for long-term inhibition of disease progression. However, many prostate cancers are poorly differentiated (Gleason ≥ 8 or grading ≥ 4), and therefore the sensitivity to endocrine treatment is low and short. Historically, surgical castration, and later, pharmacological castration, represented the only option of systemic treatment with proven activity in prostate cancer, and chemotherapy was generally assumed as not active in this disease [1]. The development of resistance to androgen deprivation (castration) is usually associated with increased expression and/or hypersensitivity of androgen receptors in PC cells and autocrine and paracrine production of androgens beyond the control hypothalamus–pituitary–gonadal axis [2,3]. Therefore, since, at the initial stage of castration resistance, PC cells demonstrate increased instead of decreased endocrine sensitivity, novel generations of hormonal agents like abiraterone acetate or irreversible androgen receptor (AR) blockers (enzalutamide, daralutamide, and apalutamide) could show high anticancer activity leading to prolonged disease control and improved patients' outcomes [4–8]. However, prostate cancer cells will ultimately become resistant to endocrine treatment by activating various hormone-unrelated molecular mechanisms,

**Citation:** Wysocki, P.J.; Lubas, M.T.; Wysocka, M.L. Metronomic Chemotherapy in Prostate Cancer. *J. Clin. Med.* **2022**, *11*, 2853. https:// doi.org/10.3390/jcm11102853

Academic Editors: Guido Bocci and Giulio Francia

Received: 26 April 2022 Accepted: 13 May 2022 Published: 18 May 2022

**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/).

leading to increased aggressiveness and proliferation. However, these features of castrationresistant prostate cancer reflect increased sensitivity of PC cells to antiproliferative cytotoxic agents such as microtubule inhibitors. Docetaxel was the first chemotherapy drug shown, in 2004, to improve the overall survival of metastatic castration-resistant prostate cancer (mCRPC) patients [9]. Additionally, due to its inhibitory impact on AR transcriptional activity, docetaxel's antitumor potential was also confirmed in metastatic castration-sensitive PC (mCSPC), where it was combined in first-line of treatment with androgen deprivation [10,11]. The increased chemotherapy sensitivity of CRPC cells, which results from AR-independency and increased proliferation rate, is not restricted to a single class of cytotoxic drugs (microtubule depolymerization inhibitors—taxanes). In hormone-refractory PC, various cytotoxic agents have demonstrated clinical activity, but only large phase III clinical studies on taxanes had sufficient power to prove a significant improvement of outcomes with the use of chemotherapy.

With the widespread of novel, highly active endocrine agents, the incidence of a previously rare phenomenon of neuroendocrine differentiation increased significantly [12]. Cells with neuroendocrine features occur in approx. 1% of primary prostate tumors, but neuroendocrine differentiation is present in 25% of end-stage metastatic castration-resistant disease cases [13]. The loss of adenocarcinoma phenotype and transformation into small cells with neuroendocrine features is one of the ways the androgen-deprived PC utilizes to get rid of androgen dependency. Characteristic features of small-cell neuroendocrine tumors are high aggressiveness, rapid proliferation, and sensitivity to chemotherapy platinum-based regimens usually used to treat neuroendocrine cancers such as small-cell lung cancer [14]. However, the sensitivity of prostate cancer to platinum compounds, which is atypical for this tumor besides neuroendocrine differentiation, can also result from prostate cancer genetic background. Inherited mutations of genes responsible for homologous recombination (homologous recombination deficiency—HRD) are relatively rare, with germinal mutations of the two most important cancer predisposition genes—*BRCA1* or *BRCA2* found in 0.4% and 1–2% of PC patients, respectively [15,16]. On the other hand, somatic or germinal mutations in genes responsible for double-strand DNA repair can be detected in 11.8% of tumor tissues from mCRPC patients [17]. Moreover, in a screening phase of a phase III study evaluating olaparib in mCRPC patients, at least one mutation/deletion (germline or somatic) within 15 genes responsible for homologous recombination was detected in 28% of patients (778 of 2792 tumors with HRD) [18]. Deleterious mutations in *BRCA1* and *BRCA2* genes in prostate cancer cells are predictive of the olaparib activity, which has been shown to significantly improve the overall survival of advanced mCRPC patients. However, recent data suggest the presence of HRD is also predictive of the activity of DNA-damaging cytotoxic agents. Therefore, in heavily pretreated mCRPC patients alkylating agents such as platinum compounds or cyclophosphamide can be more effective than previously, based on data from clinical studies conducted 2–3 decades ago, considered.

#### **2. Challenges Associated with the Use of Standard Chemotherapy in Prostate Cancer**

Prostate cancer is relatively resistant to chemotherapy. Before the introduction of docetaxel, the only available cytotoxic drug with any activity against prostate cancer was mitoxantrone [19]. However, when combined with prednisone, the benefit of this topoisomerase II inhibitor was superior to prednisone alone only in terms of pain control and quality of life, but not in terms of overall survival. In 2004, docetaxel was approved for the treatment of mCRPC patients based on two landmark studies—TAX327 and SWOG 9916, which demonstrated significant improvement in OS with docetaxel-based regimens compared to mitoxantrone [9,20]. Particularly, in TAX327 study, which compared 3-weekly docetaxel (75 mg/m2 q3w) with weekly docetaxel (30 mg/m2 q1w) and with mitoxantrone (all drugs combined with prednisone 10 mg/daily), the 3-weekly regimen significantly improved median OS compared to mitoxantrone (HR = HR = 0.79, 95%CI 0.67–0.93) [9]. The median OS was 19.2, 17.8, and 16.3 months, in docetaxel q3w, docetaxel q2w, and mitoxantrone arms, respectively. Additionally, both docetaxel-based regimens significantly

improved patients' quality of life (QOL) compared to mitoxantrone [21]. However, the significant improvement of outcomes with docetaxel administered at 3-week intervals came at the cost of increased toxicity. Grade ≥ 3 adverse events such as neutropenia, fatigue, diarrhea, neuropathy, stomatitis, dyspnea, and peripheral edema were significantly more frequent in docetaxel q3w than mitoxantrone arm.

The toxicity of standard docetaxel dose can be reduced by administering the drug in shorter intervals at a reduced dose. The TAX327 study has not confirmed the clinical utility of weekly docetaxel at 30 mg/m2, but a phase III study of a Scandinavian PROSTY group demonstrated at least similar activity of biweekly docetaxel 50 mg/m2 compared to the standard dosing. The biweekly administration of docetaxel was associated with significant improvement of the primary endpoint, which was time-to-treatment failure (TTF), compared to the 3-weekly regimen [22]. The median TTF was 5.6 months vs 4.9 months, for 2-weekly and 3-weekly regimens, respectively. Typical adverse events occurred less frequently with the lower, biweekly dose of docetaxel. Unexpectedly, secondary endpoints such as OS and PFS were significantly improved with biweekly docetaxel. There were statistically significant differences in overall QOL values and pain values favoring the biweekly treatment arm regarding patient-reported outcomes [23].

Over time, despite numerous attempts to improve the activity of docetaxel-based chemotherapy regimens by combining them with targeted agents, 3- or 2-weekly monotherapy with docetaxel remained the chemotherapy of choice for mCRPC patients [24–28].

Another taxane, cabazitaxel, is the second and the last cytotoxic agent with proven, significant activity in patients with advanced, castration-resistant prostate adenocarcinoma. A phase III TROPIC study comparing cabazitaxel 25 mg/m2 q3w (+prednisone) with mitoxantrone (+prednisone) in mCRPC patients following failure of docetaxel-based chemotherapy demonstrated significant superiority of cabazitaxel [29]. The median OS and PFS were 15.1 months vs. 12.7 months and 2.8 months vs 1.4 months in cabazitaxel and mitoxantrone arms, respectively. The hazard ratio for death of men treated with cabazitaxel compared with those taking mitoxantrone was 0.70 (95% CI 0.59–0.83). However, again the improvement in outcomes came at the cost of increased toxicity; 5% of patients treated with cabazitaxel and 2% treated with mitoxantrone died within 30 days of the last infusion. Still, none of the deaths in the cabazitaxel arm was associated with disease progression. The most frequent adverse events were associated with hematological toxicity (neutropenia, leukopenia, and anemia), and neutropenic fever occurred in 8% of patients. Subsequently, a non-inferiority phase III PROSELICA study aimed to compare a lower dose of cabazitaxel (20 mg/m2 q3w) with the standard dose [30]. The non-inferiority endpoint of at least 50% of OS benefit with low-dose cabazitaxel compared to the standard dose was achieved. Grade ≥ 3 adverse events were 39.7% for the low-dose cabazitaxel and 54.5% for the standard dose. Health-related quality of life did not differ between cohorts. However, significant differences were observed in favor of standard-dose cabazitaxel for PSA response and time to PSA progression (HR = 1.195; 95% CI 1.025 to 1.393).

There is no doubt that improvement in outcomes of prostate cancer patients achieved with taxane-based chemotherapy regimens came at the cost of toxicity, which, especially in elderly and fragile patients, may represent a significant drawback. However, attempts to optimize chemotherapy with dose reduction or decreased intensity may lead to inferior outcomes in at least a fraction of mCRPC patients in a real-world setting. Moreover, most mCRPC patients treated in clinical practice are old and present with a deteriorating performance status due to advanced disease and long-term systemic treatment. Therefore, after docetaxel treatment failure, many of them are no more candidates for further, intensive anticancer treatment, including but not restricted to cabazitaxel-based therapy. Since there is no further treatment option for many of such patients but hospice care, novel, low-toxic therapeutic approaches that would give a chance to improve survival and maintain quality of life, such as metronomic chemotherapy, are urgently needed.

#### **3. Metronomic Chemotherapy—Mechanisms of Action**

Metronomic chemotherapy (MC) is a concept of continuous administration of cytotoxic drugs at low doses. Unlike standard chemotherapy regimens, which usually use maximaltolerated doses (MTD) of chemotherapeutics and require long (usually >2 weeks) recovery periods, the metronomic chemotherapy safety profile allows for continuous, very frequent drug administration [31,32]. Metronomic chemotherapy is mainly based on oral drugs, which patients can take even several times a day. Standard chemotherapy regimens based on MTD are very useful for treating aggressive cancers with a high proliferation rate [33,34]. Highly proliferating tumors, in which 90–100% of cells are actively dividing, can wholly and rapidly respond to aggressive chemotherapy. However, the usual proliferation rate of most solid tumors in adults is lower, and Ki67 scores are far below 50% (often below 15% [35,36]), which means that far beyond 50% of tumor cells (non-proliferating cells) can survive administration of chemotherapy at MTD at wide intervals. In order to control such slowly proliferating tumors, tumor cells must be exposed continuously to antiproliferative agents which will impede cellular division whenever it occurs. It is thus clear that such a long-term exposure of tumor cells to anticancer agents can only be achieved by continuous administration of cytotoxic drugs. However, the MC not only means continuous inhibition of tumor-cell proliferation but also activation of other, clinically essential mechanisms of action, which represent crucial hallmarks of metronomic chemotherapy.

#### *3.1. Inhibition of Angiogenesis*

Angiogenesis is a crucial step in tumor development since its overgrowth beyond 2–3 mm in diameter must be associated with the development of tumor-associated vasculature [37]. The angiogenic switch is related to releasing proangiogenic factors by hypoxic cancer cells, which stimulate endothelial cells in nearby blood vessels to proliferate and migrate towards the tumor center. Some initial observations suggested that many cytotoxic drugs could inhibit the activation of endothelial cells, but the effect subsided during the withdrawal of medications in treatment-free intervals [38]. Therefore, the antiangiogenic potential of standard MTD-based chemotherapies is, at best, weak and intermittent. In contrast, metronomic chemotherapy administered continuously exerts continuous antiproliferative activity on endothelial cells, thus inhibiting their potential to create pathological vasculature. Numerous studies showed that metronomic chemotherapy inhibited the proliferation and circulation of endothelial cells and endothelial progenitor cells (CEPs) and reduced the differentiation of immature endothelial cells [39–41]. Moreover, MC can inhibit the secretion of proangiogenic factors by directly inhibiting hypoxia-inducible factor 1α (HIF1α) activity in tumor cells while simultaneously increasing the production of antiangiogenic molecules. It has been recently demonstrated that MTD-based chemotherapy can promote metastatic dissemination via activation of a transcriptional program dependent on HIF-1α and HIF-2α, and that this effect could be mitigated by switching from MTD to metronomic chemotherapy [42]. Bocci et al. demonstrated that metronomically administered cyclophosphamide inhibits the synthesis of thrombospondin-1 (TSP-1) and directly mediates growth arrest and apoptosis of endothelial cells.

Additionally, they showed the inhibition of angiogenesis in human tumor-bearing immunodeficient mice treated with low daily doses of CTX [43]. Later, it was demonstrated by several groups that the antiangiogenic effect is not restricted to cyclophosphamide but can be observed with many chemotherapy drugs administered in a metronomic manner [44,45]. Over the years, it turned out that metronomic chemotherapy stimulates the production of numerous antiangiogenic factors such as: endostatin, angiostatin, soluble VEGF receptors (sVEGFRs), and pigment epithelium-derived factor (PEDF) [46].

#### *3.2. Immunomodulation*

The immunological effects of chemotherapeutic drugs are extremely diverse and complex [47,48]. The classical assumption was that standard MTD-based chemotherapy is highly immunosuppressive, owning to its myelosuppressive activity. However, many

studies suggested that chemotherapy-induced myelosuppression correlates with improved outcomes [49]. In a retrospective analysis of the TROPIC trial, severe neutropenia in mCRPC patients with initially high NLR (neutrophil-to-lymphocyte ratio) treated with cabazitaxel was associated with significantly improved OS and PFS [50]. It is, however, unknown whether these improved outcomes resulted from depletion of immunosuppressive mononuclear cells or whether the neutropenia only reflected the high cytotoxic activity of cabazitaxel in particular patients. On the other hand, a significant amount of data supports the beneficial impact of metronomic chemotherapy on anticancer immune responses. The immunostimulatory effects of metronomic chemotherapy include (i) induction of immunogenic cancer cell death [51], (ii) preferential depletion of regulatory T (Treg) cells [52–55], (iii) enhancement of antigen-presentation through stimulation of dendritic cells activity (DC) [56] and increased immunogenicity of cancer cells [57], (iv) inhibition of myeloidderived suppressor cells (MDSC) [58], and (v) activation of immune effector cells, such as tumor-specific T cells [59,60] and γδT cells [61].

#### *3.3. Targeting Cancer Stem Cells*

Cancer stem cells (CSC) represent a population of cancer-initiating cells characterized by a slow proliferation rate, self-renewal capability, and resistance to chemotherapy and irradiation. In various in vitro models, metronomic chemotherapy impeded the survival and proliferation of CSCs. In a pancreatic tumor xenograft model, metronomic administration of cyclophosphamide reduced the number of CD133+ precursor cells and CD133+/CD44+/CD24+ cancer stem cells [62]. Similarly, metronomic, but not MTD-based, cyclophosphamide inhibited the function of C6 rat glioma CSCs [63]. In a preclinical model of luminal breast cancer, metronomic paclitaxel reduced the population of CD44+/CD24 stem cells [64]. Expansion of stem-like tumor-initiating cells correlates with the development of chemoresistance and metastatic potential of cancer cells. In animal models, MTD chemotherapy, regardless of cytotoxic agents used, induced persistent STAT-1 and NF-κB activity in carcinoma-associated fibroblasts leading to secretion of CXCR2-activating chemokines [65]. Stimulation of CXCR2 on cancer cells converts them into tumor-initiating CSC responsible for invasive behaviors and paradoxically enhanced tumor aggression after therapy. In contrast, the same overall dose of cytotoxic agents administered as an MC regimen prevented therapy-induced CXCR2 paracrine signaling, thus enhancing treatment response and extending the survival of tumor-bearing mice [65].

#### *3.4. Modulation of Gut and Tumor Microbiome*

Over the last few years, increasing attention has been paid to the host gut microbiome defined as the collection of genomes from all microorganisms in the gastrointestinal tract, especially within the intestines. However, it has been demonstrated that the microbiome in general (not only the gut, but also local or even intratumoral microbiome) is associated with the pathogenesis of various cancers such as colorectal, breast, ovarian, lung, and prostate cancers [66–70]. For example, compared to a healthy population, the fecal gut microbiome in CRC patients is enriched with *Prevotella*, *Collinsella*, and *Peptostreptococcus* and contained significantly lower concentrations of Escherichia-Shigella [70]. The gut microbiota of patients diagnosed with PC on transrectal biopsy was significantly enriched with *Bacteroides* spp. compared to patients with negative biopsy results [71]. In prostate cancer, the microbiota of tumor and peri-tumoral regions had higher relative abundance of *Staphylococcus* spp. than normal areas. Still, the normal areas had a higher abundance of *Streptococcus* spp. than the tumor and the peri-tumoral regions [72].

With the advent of novel immunotherapies, the role of the microbiome has become even more important in the context of its significant role in shaping responses to checkpoint inhibitors. The gut microbiota plays an important role in regulating systemic immune responses [73–75], and the role of intestinal microbiota in mediating immune activation in response to chemotherapeutic agents has been clearly demonstrated [76,77]. In an animal melanoma model, the presence of *Bifidobacterium* correlated with the activation of antitumor immune mechanisms by anti-PD1 checkpoint inhibitors, and oral administration of *Bifidobacterium* alone improved tumor control to the same degree as PD-L blockade [78]. Moreover, a combination of anti-PD-L1 antibody with oral administration of *Bifidobacterium* completely blocked the growth of melanoma tumors in mice. Many studies showed that administration of wide-spectrum antibiotics before initiation of treatment with checkpoint inhibitors could completely abolish the efficacy of immunotherapy in patients with various tumors [79–82]. Similar observations on the lack of activity of checkpoint inhibitors come from studies in germ-free animals that resemble the effect of gut microbiome eradication with the use of antibiotics. Transplantation of fecal microbiota from immune-responding melanoma patients into germ-free mice leads to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy [83]. Similarly, fecal transplantation from responding melanoma patients into germ-free or antibiotic-pretreated mice could restore antitumor immune responses and induce rejection of implanted tumors, which was not observed when such animals were transplanted with fecal microbiota from nonresponding patients [84]. The observation in animal models has been recently replicated in humans. In a phase II clinical trial, 15 melanoma patients refractory to anti PD-1 therapy received responder-derived fecal microbiota transplantation combined with pembrolizumab. Of the 15 treated patients, 6 achieved clinical benefit (one partial response and five disease stabilization) and durable changes in microbiome composition. Responding patients demonstrated an increased abundance of taxa known to be associated with response to anti-PD-1, increased CD8+ T cell activation, and decreased frequency of interleukin-8-expressing myeloid cells [85].

Escherichia coli, a common species found in benign prostate hyperplasia (BPH) tissue, induces inflammation and tissue damage leading to neoplastic transformation in prostate epithelial cells. Ma et al. demonstrated that specific microbes, such *as Listeria monocytogenes, Lactobacillus crispatus,* and *Thermus thermophilus*, prevented tumor formation and growth, while other microbes, such as *Nevskia ramosa* and *S. aureus*, displayed cancer-promoting properties by inducing inflammation, immunosuppression, and promoting prostate cancer stem cells (PCSC) responsible for the development of metastases [86]. Ma et al. have demonstrated that particular bacterial species—Listeria monocytogenes, Methylobacterium radiotolerans *JCM 2831*, *Xanthomonas albilineans GPE PC73*, and *Bradyrhizobium japonicum* correlated respectively with well-differentiated phenotype, earlier disease stage, lower PSA level, and lower AR expression. A particular species of *Bacteroides*, which is a genus of Gram-negative, anaerobic bacteria, may contribute to the development of high-risk prostate cancer.

The impact of metronomic chemotherapy and its difference from MTD-based microchemotherapy on the gut and intratumoral microbiome is not well recognized yet. An observational study by Zhu et al. suggested that gut microbiota diversity is higher in breast cancer patients than in healthy controls [87], but additional studies demonstrated that the diversity could be decreased with the use of metronomic capecitabine, but not MTD-dosed chemotherapy [88]. Since the impact of MC on tumors and host microbiomes can represent a novel mechanism action of MC, we have initiated a prospective analysis of the impact of various metronomic chemotherapy regimens in breast, ovarian, and prostate cancer patients, and the preliminary results shall be posted soon.

#### **4. Metronomic Chemotherapy in Prostate Cancer**

Various metronomic chemotherapy-based regimens have been evaluated in advanced PC patients over the last three decades. However, most studies were conducted before the introduction of novel hormonal or cytotoxic agents such as abiraterone acetate, enzalutamide, apalutamide, darolutamide, or cabazitaxel. All the above agents have been approved for the treatment of CRPC patients based on large, well-conducted phase III clinical trials. In comparison, the benefit of MC has been usually evaluated in small, non-randomized trials or retrospective analyses. Accordingly, metronomic chemotherapy should not be considered as a standard option at the early stages of mCRPC, if highly

active, well-tolerated novel hormonal agents are freely available. Therefore, MC represents a therapeutic option in mCRPC who failed all available therapies, are unfit for standard chemotherapies, or have no access to standard mCRPC treatments, e.g., in low- or middleincome countries [89]. Nevertheless, many studies demonstrated the activity of MC in advanced mCRPC patients before the advent of novel, active therapies dedicated for this population. Selected studies are summarized in Table 1. In addition to showing the antitumor activity of MC, which was reflected by biochemical responses (PSA decline) in 30–50% of patients, all of the summarized studies demonstrated a very favorable safety profile of MC. This observation underscores the clinical utility of MC, especially in elderly and frail patients, who represent the majority of mCRPC patients. In a recent study, Calvani et al. demonstrated that metronomic cyclophosphamide (50 mg/d p.o.) combined with corticosteroids (dexamethasone 1 mg/d p.o. or prednisone 10 mg/d p.o.) induced biochemical responses (at least 50% PSA reduction) in half of the studied population. However, only two analyzed patients could be considered standard contemporary candidates (pretreatment with docetaxel and novel hormonal agents) for metronomic chemotherapy. Therefore, it is still challenging to draw any conclusions regarding the actual utility of MC in heavily pretreated mCRPC patients since many of the old studies included mCRPC patients for whom, nowadays, at least 1–2 standard treatment options are routinely available. Additionally, most of the listed studies combined MC with steroids (especially dexamethasone), which further complicates the validation of the plain MC activity in mCRPC patients. Several studies have recently demonstrated that a simple switch from prednisone to dexamethasone in mCRPC patients treated with an abiraterone + prednisone combination can lead to profound biochemical responses [90–92]. This observation underscores the risk of overestimating the benefit of MC, especially when patients are simultaneously receiving dexamethasone as a part of an antitumor treatment strategy for the first time. There is only a single study evaluating the activity of metronomic chemotherapy in pretreated (docetaxel and ≥1 novel endocrine agent) mCRPC patients. In such mCRPC patients, single-agent cyclophosphamide led to a relatively low rate of PSA responses (16%), with PSF and OS of 4 and 8.1 months, respectively. This study demonstrates that the promising activity of MC observed before the era of novel therapies may not be easily reproducible in contemporary, standardly pretreated mCRPC patients.

**Table 1.** Studies evaluating metronomic chemotherapy in prostate cancer.

CTX—cyclophosphamide, VRB—vinorelbine, MTX—methotrexate, UFT—Tegafur/uracil, DXL—docetaxel, CAP—capecitabine, DEX—dexamethasone, PFS—progression-free survival, OS—overall survival, NR—not reported, 1—any PSA decrease, p.o.—oral administration, i.v.—intravenous administration, bid-twice daily, 2—typical, contemporary mCRPC patients (pretreated with DXL and novel endocrine agents).

#### **5. Our Hunt for an Optimal MC Regimen in mCRPC Patients**

We have conducted a prospective analysis of consecutive cases of patients treated with various MC regimens to find the most optimal MC combination for a subsequent phase II study. In order to define the real benefit of MC, none of the mCRPC patients was allowed to receive corticosteroids but all continued on pharmacological castration with LHRH analogs. The analysis included nine heavily pretreated patients (median two lines of systemic treatment for mCRPC) and the majority of patients were initially diagnosed with aggressive PC (median Gleason score of 9). Patients received the following combinations— (i) cyclophosphamide 50 mg/d p.o. + vinorelbine 30 mg q2d p.o. (two patients), (ii) cisplatin 25 mg/m2 i.v. qw + cyclophosphamide 50 mg/d p.o. (two patients), (iii) paclitaxel 60 mg/m2 qw + capecitabine 1500–2000 mg/d (depending on patient weight) p.o. (two patients), (iv) paclitaxel 60 mg/m2 qw + capecitabine 1500 mg/d p.o + cyclophosphamide 50 mg/d (three patients). The biochemical responses (PSA decline by ≥50%) were observed in 0%, 50%, 100%, and 100% of patients, respectively. The patients tolerated all analyzed regimens very well, with G1–2 myelotoxicity being the most common adverse event. Based on the prospective analysis of consecutive cases of mCRPC patients treated with MC, the paclitaxel + capecitabine + cyclophosphamide combination has been chosen for further evaluation in a planned phase II clinical trial.

The choice of paclitaxel as a compound for our MC regimen requires explanation. Although cabazitaxel (the new-generation taxoid) is an active and approved agent for the treatment of docetaxel-pretreated mCRPC [50,107,108] patients and studies on standarddose of paclitaxel failed to demonstrate its activity in prostate cancer [109]; some recent intriguing data justify the choice of paclitaxel as an element of our MC regimen. It is well known that paclitaxel, administered weekly, exerts robust antiangiogenic activity [110–112]. Our choice of paclitaxel in mCRPC is also justified by a recent study comparing weekly paclitaxel with cabazitaxel in advanced breast cancer. The study aimed to demonstrate the superiority of cabazitaxel 25 mg/m2 q3w over weekly paclitaxel as a first-line treatment in 158 patients with metastatic breast cancer, many of whom have previously received docetaxel in the neoadjuvant or adjuvant setting. Surprisingly, both drugs demonstrated similar OS, PFS, and ORR [113], thus suggesting the need for reevaluation of weekly paclitaxel in docetaxel-pretreated mCRPC patients.

#### **6. Conclusions**

Metronomic chemotherapy represents a unique treatment option for many advanced cancer patients, including those diagnosed with mCRPC. However, it should be not considered as a first-line option in chemotherapy-fit mCRPC patients due to the lack of robust, prospective data suggesting its equality to standard treatment options, including novel antiandrogen agents (abiraterone, enzalutamide, darolutamide, and apalutamide) [4,6–8,114], chemotherapeutic agents (docetaxel and cabazitaxel) [21,22,115], or novel targeted therapies (olaparib in HRD mCRPC or Lutetium-177–PSMA-617) [18,116]. The use of MC based on the current state of knowledge should be restricted, in a typical population of mCRPC patients, to later lines of treatment, where no standard treatment options nor dedicated clinical trials are available. Moreover, recent disappointing data on the lack of activity of novel immunotherapies in mCRPC when administered as single agents [117] represent an intriguing hint to combining checkpoint inhibitors with immunomodulating MC regimens. However, the MC remains an option for earlier lines of therapy in fragile, elderly patients in whom standard treatment options (especially bi- or three-weekly chemotherapy regimens) may be intolerable. There is also no doubt that MC, with its multidirectional mechanism of action, represents a treatment option for mCRPC patients in low- and middle-income patients who do not have access to still relatively novel and expensive anticancer agents. Therefore, studies on metronomic chemotherapy in various clinical settings are critical from a global perspective.

**Author Contributions:** Conceptualization, P.J.W. and M.L.W.; methodology, P.J.W. and M.T.L.; formal analysis, P.J.W.; investigation, P.J.W. and M.T.L.; data curation, M.T.L.; writing—original draft preparation, P.J.W. and M.L.W.; writing—review and editing, P.J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by Jagiielonian University—Medical College grant No N41/DBS/ 000706.

**Informed Consent Statement:** Informed consent was obtained from all subjects receiving metronomic treatment analyzed in the study.

**Conflicts of Interest:** The authors declare no conflict of interest. ONCOAID is a diagnostic company privately owned by Wysocka, which provides microbiome and laboratory diagnostics for cancer patients.

#### **References**

## *Review* **Cyclic Metronomic Chemotherapy for Pediatric Tumors: Six Case Reports and a Review of the Literature**

**Benjamin Carcamo 1,2,\* and Giulio Francia 3,\***

**Abstract:** We report a retrospective case series of six Hispanic children with tumors treated with metronomic chemotherapy. The six cases comprised one rhabdoid tumor of the kidney, one ependymoma, two medulloblastomas, one neuroblastoma, and a type II neurocytoma of the spine. Treatment included oral cyclophosphamide daily for 21 days alternating with oral etoposide daily for 21 days in a backbone of daily valproic acid and celecoxib. In one case, celecoxib was substituted with sulindac. Of the six patients, three showed complete responses, and all patients showed some response to metronomic therapy with only minor hematologic toxicity. One patient had hemorrhagic gastritis likely associated with NSAIDs while off prophylactic antacids. These data add to a growing body of evidence suggesting that continuous doses of valproic acid and celecoxib coupled with alternating metronomic chemotherapy of agents such as etoposide and cyclophosphamide can produce responses in pediatric tumors relapsing to conventional dose chemotherapy.

**Keywords:** metronomic chemotherapy; pediatric tumors; cyclophosphamide; etoposide; valproic acid

#### **1. Introduction**

Metronomic chemotherapy involves the continuous low-dose administration of chemotherapeutic drugs, and it is currently undergoing clinical evaluation for the treatment of various pediatric and adult malignancies [1–6]. For example, for adult tumors, a phase III clinical trial of metastatic colorectal cancer with maintenance metronomic capecitabine plus bevacizumab was reported, as were results of a phase III trial in early-stage breast cancer treated with bevacizumab plus metronomic cyclophosphamide and capecitabine [7,8]. For pediatric tumors, a number of phase II clinical trials have reported promising antitumor activity of metronomic chemotherapy, and these studies include those of Kieran and colleagues [9,10], of Andre and colleagues [11–17], and of the Baruchel group [18]. For both adult patients and pediatric tumors, the low toxicity that is frequently observed with this treatment strategy coupled with its (often) relatively low costs [4] make it an attractive treatment option. Preclinical studies [2,19,20] have shown that metronomic chemotherapy acts via inhibition of tumor angiogenesis, although additional mechanisms such as activation of the immune system have been proposed for regimens such as low-dose cyclophosphamide [21].

Metronomic chemotherapy may involve monotherapies, e.g., daily oral cyclophosphamide (CTX), but it can also be used to describe metronomic drug cocktails [22,23], including protocols that administer up to five different drugs [10] or more [24,25]. There are also evaluations involving the cyclical use of different drugs, an example of which is the work by Kieran and colleagues [9,10] on pediatric tumors. Here, we describe six children with pediatric cancer treated with cyclic alternating metronomic chemotherapy between 2002 and 2017.

**Citation:** Carcamo, B.; Francia, G. Cyclic Metronomic Chemotherapy for Pediatric Tumors: Six Case Reports and a Review of the Literature. *J. Clin. Med.* **2022**, *11*, 2849. https://doi.org/10.3390/jcm11102849

Academic Editors: Johannes Van Den Anker and Nikolas Herold

Received: 14 March 2022 Accepted: 13 May 2022 Published: 18 May 2022

**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/).

These children had failed standard first line and salvage therapy and had refused or had no access to phase I or II studies. They were the only patients treated with this regimen during this period of time. The intention of the treatment was to provide palliation to children with refractory cancer that had exhausted all the therapeutic options available at the time and when parents requested additional treatment to prolong their children's life. It was not done to prove the validity of a metronomic regimen. The parents where consented to use a palliative regimen without curative intent so that they would understand the expected side effects of the individual drugs. They understood that it was not known if this treatment would prolong their life or not, and that the palliation was not done in the context of a study.

Metronomic chemotherapy included alternating 21-day cycles of etoposide (VP16) and CTX, along with the continuous administration of celecoxib and valproic acid. Sometimes due to individual reasons only celecoxib or valproic acid was used and sometimes celecoxib was substituted with sulindac. Oral continuous CTX at 50 mg/m2/day had previously been used in metronomic regimens with minimal toxicity [26]. Oral VP16 at 50 mg/m2/day for 21 days was chosen on the basis of evidence that continuous low-dose etoposide could enhance tumor cytotoxicity while lowering the risk of acute myeloid leukemia [27], and that 21 days of this dose with a 1 week rest had achieved partial response with moderate toxicity in recurrent disseminated medulloblastoma [28]. Alternating cycles of these drugs were chosen to decrease hematologic toxicity from etoposide and the emergence of drug resistance to either drug alone. Dose reduction was allowed for hematologic toxicity. Celecoxib is a selective COX2 inhibitor that has been well tolerated without cardiac toxicity at a dose of 2–16 mg/kg/day for prevention of adenomatous polyps in children [29]. The COX2 pathway is known to be involved in angiogenesis [30], and inhibition of angiogenesis has been reported to produce enhance the antitumor effects of metronomic chemotherapy [31], for which celecoxib was chosen at a dose of 250 mg/m2/dose twice a day (equivalent to 8 mg/kg/day). Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor with a long safety record in children with seizures that has shown antitumor effects in preclinical models of medulloblastoma, for which it was chosen at the standard pediatric dose of 15 mg/kg/day in two divided doses [32,33]. Furthermore, a major reason for the choice of these drugs was also their low toxicity profile, low cost, and availability. Medications were administered as capsules (the exception was for patient 3, for whom the liquid preparation of etoposide was mixed with cranberry juice). Our results suggest that this approach showed promising antitumor activity in six pediatric cases, and we propose that a cyclical regimen of 21 day VP16 followed by 21 day oral CTX on a backbone of continuous celecoxib and VA be further evaluated in these settings.

#### *1.1. Patient 1*

Patient 1 was a Hispanic boy who presented with metastatic rhabdoid tumor of the kidney at 34 months of life (Figure 1). He had a 7 cm × 11 cm mass in the left kidney, retroperitoneum invasion, and extension into the renal vein and inferior vena cava. He had metastasis to retroperitoneal lymph nodes and lungs, as well as rare micro-aggregates of cohesive abnormal large mononuclear cells suggestive of metastatic malignant tumor in the bone marrow by morphology alone (INH1 was not done). Bone marrow karyotype was 46,XY with no abnormal clones. He received vincristine–irinotecan, followed by cyclophosphamide–doxorubicin, followed by high-dose methotrexate as per regimen UH1 of AREN0321. This was followed by radical left nephrectomy at week 15. Subsequent evaluation revealed a residual 2 mm lung nodule but otherwise complete response. He subsequently received radiation to lungs and abdomen, followed by cycles of cyclophosphamide– carboplatin–etoposide and vincristine–doxorubicin–cyclophosphamide. Evaluation at the end of this therapy revealed recurrent disease to the lungs (two nodules measuring 3 mm and 1.5 mm in diameter) and an abnormal bone marrow clone, t(5;17)(q13;p13) in 15% of the cells. This cytogenetic abnormality has been previously reported in treatmentrelated myeloid neoplasia [34], and its presence was worrisome for early development of

this condition from conventional chemotherapy and radiation, but there was no morphologic evidence of this entity at that time. No additional analysis was carried out, such as evaluation of INI1 loss. He was subsequently started on a metronomic regimen of VP16– CTX–celecoxib, and this maintenance regimen was effective at controlling the disease with no recurrent rhabdoid tumor or changes in the pulmonary nodules. Metronomic doses were as follows: VP16 37.5 mg/m2 PO daily days 1–21, followed CTX 50 mg/m2 PO daily days 22–42. Celecoxib was administered at 250 mg/m2 PO twice a day and VA 15 mg/kg/day divided into two doses (dose adjusted to achieve levels of 100–150 ug/mL). He subsequently developed expansion of the original bone marrow clone (5;17)(q11.2p11.2) to 80% of metaphase cells, for which metronomic therapy was changed to bevacizumab (5 mg/kg every 3 weeks), temozolomide (60 mg/m2/day on days 1–21), CTX (25 mg/m2/day on days 22–42), VA (15 mg/kg/day divided into two doses) and celecoxib (250 mg/m2 PO twice a day) after 17 months of treatment. He subsequently developed myelodysplastic changes consistent with treatment-related myeloid neoplasia, for which temozolomide and CTX were stopped 12 months later. These bone marrow myelodysplastic changes eventually progressed, in the absence of evidence of rhabdoid tumor reappearance, for which celecoxib–VA–bevacizumab were stopped, and he had a related donor allogenic bone marrow transplant 4 months later. He subsequently died of transplant-related infectious complications. He did not have evidence of recurrent rhabdoid tumor according to imaging studies at the time of his death. Autopsy was offered and declined. The rhabdoid tumor of this patient effectively responded to the metronomic chemotherapy for a period of 3 years.

**Figure 1.** Patient 1: CT with contrast shows a large 11 × 7 cm left renal tumor with retroperitoneal infiltration, regional metastatic retroperitoneal adenopathy, and extension to renal vein and inferior vena cava. The patient also had innumerable solid circumscribed masses throughout the lung parenchyma bilaterally (not shown).

#### *1.2. Patient 2*

Patient 2 was diagnosed with anaplastic ependymoma when he presented with right hemianopsia, severe vision loss, and a 12 cm × 8 cm × 8 cm left temporo-occipital tumor at 6 years of life. He had two consecutive surgeries to remove the tumor, but a 1 cm unresectable lesion was left behind. He then received 5940 cGy field radiation to the tumor bed. Three months after radiation, he developed seizures, transient right arm paralysis, and recurrent disease to the left cerebral hemisphere, for which he received vincristine, carboplatin, and cyclophosphamide following the guidelines of ACNS0121 [35]. Three months later, he developed progressive disease with a large infiltrative enhancing mass (~5 cm) in the left temporal, occipital, and parietal lobes with involvement of the left thalamus, internal capsule, left external capsule, and insula, as well as leptomeningeal metastasis to the thoracic spine. He had no access to Phase I or II studies and was unable to travel for lack of insurance and migratory status, for which treatment was changed to palliation chemotherapy with oral VP16 (50 mg/m2/day for 21 days) alternating with oral CTX (50 mg/m2/day for 21 days) and continuous celecoxib (120 mg/m2 PO bid) and VA. Celecoxib was subsequently changed to sulindac (8 mg/kg/day) due to drug access issues. He had an excellent response to palliation with resolution of all enhancing lesions and no evidence of residual disease after 2 years of therapy. (Figure 2). About this time VA was added to his seizure disorder management and continued until his last visit. He received 18 additional months of metronomic chemotherapy after complete response. He had no evidence of recurrent disease when he was lost to follow up 8 years from initial diagnosis and 4 years off metronomic chemotherapy. This patient showed complete response to the metronomic regimen.

**Figure 2.** Patient 2: MRI shows confluent encephalomalacia gliosis in the left temporal and occipital lobes. There was no evidence of mass or pathologic enhancement 8 years from diagnosis and 4 years off therapy.

#### *1.3. Patient 3*

Patient 3 was a Hispanic girl diagnosed with medulloblastoma, when she presented with ataxia, dysmetria, increased head circumference and hydrocephalus at 10 months of age. She had gross total resection of a 5 × 5 × 4.5 cm posterior fossa tumor. Postoperative MRI revealed gross total resection and partial decompression of the fourth, third, and lateral ventricles. Following surgery, imaging studies showed no evidence residual disease or leptomeningeal involvement. However, ventricular fluid had evidence of microscopic disease with a five-cell cluster of malignant cells. Cerebrospinal fluid was negative. She received induction chemotherapy with cisplatin, cyclophosphamide, vincristine, and oral etoposide, followed 4 months later by radiotherapy to the posterior fossa, followed by another 8 months of chemotherapy as per POG 9631 [36]. Six months after therapy was completed, microscopic relapse was documented in cerebrospinal fluid with three clusters of malignant cells. Imaging studies failed to document gross leptomeningeal involvement. She started metronomic chemotherapy for relapsed medulloblastoma with 21 days of oral VP16 (35 mg/m2/day) every 28 days. After 7 months of oral VP16, persistent microscopic disease was still present in the cerebrospinal fluid, and VA was added to the treatment. She achieved remission 5 months later, and serial cerebrospinal fluids remained consistently negative since then.  She was free of disease 5 years after metronomic chemotherapy was stopped.

#### *1.4. Patient 4*

Patient 4 was a Hispanic girl diagnosed with localized medulloblastoma without desmoplastic or anaplastic features when she presented with headaches, emesis, and blurred vision and a 4.9 × 4 × 4.5 cm heterogeneously enhancing tumor filling the fourth ventricle with associated moderate obstructive hydrocephalus at 5 years of age. Following gross total resection of the tumor, residual linear enhancement in the tumor bed was documented. She subsequently received 2340 cGy radiation to the craniospinal axis with a 3060 cGy boost to the posterior fossa (5400 cGy total) with concomitant vincristine after surgery. This was followed by 8 months of maintenance with vincristine, cisplatin, lomustine alternating with vincristine and cyclophosphamide as per ACNS0331 [37].

Three months off therapy, she developed a first relapse (Figure 3A) with recurrent tumors in the anterior horn of the left lateral ventricle (two), posterior horn of the left lateral ventricle (one), and anterior horn of the right lateral ventricle (one). CSF showed one abnormal cell. She was treated with 7 weeks of temozolomide followed by 1400 cGy of radiation to the ventricles and Gamma Knife surgery to a residual nodule, followed by autologous peripheral blood stem-cell transplantation (PBSCT), followed by 6 months of isotretinoin.

Three months later, she experienced a second relapse (Figure 3B) with a 1.7 × 1.4 × 0.5 cm enhancing tumor in the fourth ventricle, for which she was started on metronomic chemotherapy with alternating temozolomide (60 mg/m2/day; days 1–21) and CTX (30.9 mg/m2/day; days 22–42), daily celecoxib (250 mg/m2) and VA (15 mg/kg), and bevacizumab (5 mg/kg) every 3 weeks. After 3 months on metronomic chemotherapy (Figure 3C), a partial response was documented with improved enhancing lesions in the dorsal brainstem and inferior vermis. At 4 months (Figure 3D), she developed multiple cranial nerve neuropathies and heterogeneous enhancement with mild increased volume at the left midbrain at the level of the left cerebellar peduncle, for which temozolomide was changed to VP16 (24.6 mg/m2). This was complicated by hematologic toxicity requiring several treatment interruptions and VP16 dose reductions. After 8 months on treatment, MRI showed resolution of the enhancing tumor, an interval decrease in T2 changes, and diffusion restriction in the left cerebellar peduncle (Figure 3E). After 10 months on treatment (Figure 3F), she developed an extensive infiltrative tumor involving the dorsal mesencephalon, pons, and lower brainstem, and she died of her disease 2 months later. This heavily treated patient refractory to aggressive standard salvage therapy experienced 8 months of response and survived 12 months on metronomic chemotherapy.

ǻǼȱ

**Figure 3.** *Cont*.

ǻǼȱ

**Figure 3.** *Cont*.

**Figure 3.** Patient 4: (**A**) image shows and enhancing tumor in the anterior horn of the left ventricle when she presented with first relapse of medulloblastoma. She achieved complete remission with salvage therapy; (**B**) image shows an enhancing tumor involving the anterior aspect and floor of the fourth ventricle (arrows) when she presented with second relapse; (**C**) image shows response to 3 months of metronomic therapy with decreased size and intensity of enhancing lesions; (**D**) image shows interval progression of the tumor seen in T2 FLAIR resulting in a change from temozolomide to etoposide at 4 months of treatment; (**E**) image shows resolution of the tumor mass and T2 FLAIR changes at 8 months of metronomic therapy; (**F**) image shows progressive disease at 10 months of treatment.

#### *1.5. Patient 5*

This patient was a Hispanic boy diagnosed with metastatic neuroblastoma when he presented with an 8 × 12 cm right suprarenal mass, extensive retroperitoneal and mesenteric lymphadenopathy, spinal cord compression, and metastatic bone disease to the right scapula and body of L1 at 9 years of age. He was treated with high-dose cisplatin and etoposide, cyclophosphamide, Adriamycin and vincristine, ifosfamide and etoposide, and carboplatin and etoposide, followed by subtotal resection of the retroperitoneal tumor and right kidney as per ANBL00P1 [38]. PBSCT was not available due to a lack of funds, for which he received two cycles of topotecan–cyclophosphamide, followed by 2160 cGy radiation to the tumor bed and spine, followed by 6 months of isotretinoin. He had no evidence of residual disease at the end of treatment. Seven months later, he developed recurrent neuroblastoma involving the right external iliac lymph nodes, for which he started palliative metronomic chemotherapy with oral VP16 (50 mg/m2/day; days 1–21) alternating with oral CTX (75 mg/m2/day; days 22–41) and continuous sulindac (4 mg/kg/day). He had hematologic toxicity requiring interruptions and dose reductions of both VP16 and CTX. Evaluation showed decreased tumor size at 5 and 8 months of therapy. He subsequently developed progressive disease after 11 months on therapy, for which sulindac was changed to celecoxib (500 mg/m2/day). Two months later while off prophylactic antacids, the patient developed hemorrhagic gastritis, hematemesis, aspiration pneumonia, acute renal failure, and seizures, and died 13 months after starting metronomic chemotherapy.

#### *1.6. Patient 6*

This patient is a Hispanic girl found to have a large cervicomedullary tumor with a cervico-thoracic central syrinx with tumor seeding when she presented with right shoulder drop and right upper-extremity weakness at 3 years of age (Figure 4A). She had resection of the cervicomedullary portion of the tumor. Evaluation revealed a type 2 neurocytoma of the spine.

She subsequently received intensity-modulated radiotherapy to the tumor bed and residual cervico-thoracic tumor. Following radiation, a residual 11 mm expansile cervicomedullary nodule, centered at the C3–C4 level, was documented. Five months later, she developed progressive disease with increased size of the cervicomedullary nodule to 15 mm and development of a new second focus of enhancement on the ventral aspect of the spinal cord at C2. She was treated with topotecan, ifosfamide and carboplatin (TIP). She had significant hematologic toxicity and infectious complications during this treatment and no change in tumor size after nine cycles (Figure 4B), for which she was changed to metronomic chemotherapy with 21 days of temozolomide (60 mg/m2 day) aleternating with 21 days of CTX (50 mg/m2), VA (15 mg/kg), celecoxib (250 mg/m2/day) and bevacizumab (5 mg/kg IV every 3 weeks). Tumor response was noted 3 months after treatment with decreased tumor size to 9–10 mm (Figure 4C). Tumor size remained stable on subsequent studies, and temozolomide and CTX were stopped at 18 months, celecoxib and VA were stopped at 36 months, and bevacizumab was stopped at 48 months (Figure 4D). Metronomic chemotherapy was restarted after tumor progression was documented 10 months later (Table 1). She developed stable disease and treatment was stopped 15 months later.  Patient 6: (**A**) MRI shows a 4.5 cm homogeneously enhancing expansile intramedullary tumor involving the medulla and upper cervical cord down to the level of C3–C4 (arrow) with an elongated syrinx extending inferiorly to the T3–T4 level; (**B**) the tumor was removed and treated with radiation but came back, for which it was treated with nine cycles of topotecan–ifosfamide–carboplatin with no significant change in tumor size (arrow) but significant toxicity, for which treatment was changed to metronomic chemotherapy; (**C**) after 3 months on metronomic chemotherapy, the patient recovered from toxicity and the tumor was slightly decreased; (**D**) the tumor was stable at the end of 4 years of metronomic chemotherapy.

**Table 1.** Summary of the 6 pediatric cases, treatment regimens, and clinical course.

VP16 = etoposide, CTX = cyclophosphamide, VA = valproic acid, BV = bevacizumab, TMZ = temozolomide.

#### **2. Discussion**

Here, we report six cases of pediatric malignancies treated with metronomic chemotherapy that included alternating 21 days cycles of oral etoposide and cyclophosphamide in a backbone of celecoxib and/or valproic acid (Table 1). Because metronomic chemotherapy was used for palliation and not in the context of a study, some variation occurred due to medical and social issues such as individual choice, lack of insurance, and migration status. As noted, in some cases, bevacizumab and/or temozolomide were also administered. This treatment strategy produced significant tumor responses, including three complete responses. Three patients had hematologic toxicity with metronomic chemotherapy requiring interruptions and dose reductions. Two of these patients eventually progressed and died of their disease, and it is unclear whether the interruptions interfered with the metronomic effect. Patient 5 had hemorrhagic gastritis presumably from NSAIDs, for which this regimen should be used with prophylactic antacids as per current COG guidelines.

Three patients with brain tumors were treated. Unfortunately, they preceded easy access to molecular studies and, thus, are not reported according to the current WHO classification. The first patient (case 2) that used this regimen was treated in 2002. He had an anaplastic ependymoma that had failed surgery, radiation, and chemotherapy and had progressed with a large hemispheric tumor and spinal cord metastasis. He started treatment with alternating etoposide–cyclophosphamide on a backbone of celecoxib. Valproic acid was later added to the treatment for seizure control.  Two patients with medulloblastoma were treated. The first patient relapsed with clusters of malignant cells in CSF with normal imaging studies 6 months off therapy. The malignant cells persisted after 7 months of metronomic etoposide but resolved 5 months after adding valproic acid to the regimen. She remains free of disease 15 years off therapy. Since continuous celecoxib–valproic acid in the context of cyclic chemotherapy was well tolerated and the second patient may have responded to valproic acid, this medication was added to celecoxib in subsequent patients. The second medulloblastoma relapsed with brainstem involvement shortly after completing first- and second-line therapy. She was initially treated with alternating temozolomide–etoposide, bevacizumab, and continuous celecoxib–valproic acid but developed progressive disease 4 months later. She subsequently changed temozolomide to etoposide, achieved partial response 6 months later, and survived 12 months on metronomic chemotherapy. These three cases highlight the potential role of metronomic chemotherapy in refractory brain tumors. Recently, 29 children with refractory medulloblastoma were treated with a metronomic regimen that included alternating etoposide–cyclophosphamide, continuous thalidomide– celecoxib–fenofibrate, bevacizumab, and intraventricular liposomal cytarabine–etoposide (MEMMAT) [39]. EFS was 33% and 28% at 5 and 10 years, while OS was 44% and 39% at 5 and 10 years, respectively [39]. These results make MEMMAT the treatment of choice in refractory medulloblastoma. This regimen has some elements included in our regimen. For example, it contains alternating etoposide–cyclophosphamide and continuous celecoxib but lacks valproic acid. Since it is difficult to tell what elements in a complex metronomic regimen add to the activity, it is reasonable to explore our less complex regimen in patients that refuse invasive interventions or have poor access to care.

One patient with refractory metastatic neuroblastoma was treated after failing first-line therapy. He had no funds and was not able to travel to pursue second-line treatments including stem-cell transplant. He was treated with alternating etoposide–cyclophosphamide and sulindac. He had a partial response at 5 months but progressed at 11 months. Since then a regimen including cycles of 4 days of rapamycin–dasatinib followed by 5 days of irinotecan–temozolomide (RIST) showed impressive responses in refractory neuroblastoma with 90% response in 21 children including 12 CR and three PR, with 43% OS at 143 weeks [40]. While these results are impressive, rapamycin and dasatinib may not be readily available to patients with poor access to care. Most recently, 167 patients with high-risk stage 4 neuroblastoma without access to autologous stem-cell transplantation or anti-GD2 antibody therapy were treated with or without a metronomic regimen including continuous etoposide, cyclophosphamide, vinorelbine, topotecan, and celecoxib, including 106 patients in the metronomic arm and 61 in the non-metronomic arm. The 3 years of event-free survival (EFS) was 42.5% versus 29.4%, and overall survival (OS) was 71.1% versus 59.4%, respectively [41]. While the Corbacioglu et al. [40] report strongly supports RIST for refractory neuroblastoma, the report from Sun et al. [41] showing a benefit of oral metronomic chemotherapy comparable to autologous stem-cell transplant and anti-GD2 in high-risk Neuroblastoma suggests a possible role for metronomic chemotherapy in refractory neuroblastoma. Metronomic chemotherapy either with continuous drug delivery as reported by Sun et al. [41] or with alternating drugs as in this report needs to be further evaluated in these patients.

This report and previous reports by Kieran et al. [9,10], Andre et al. [11–13], Sharp et al. [18], Slavc et al. [39] and Sun [41], highlight possible metronomic regimen strategies for the treatment of refractory pediatric cancer. These metronomic strategies are summarized in Table 2.

**Table 2.** Summary of proposed metronomic regimens.

We have previously used molecular genetics to identify a driving metabolic pathway within refractory Inflammatory Myofibroblastic Tumor that was specifically targeted with the TKI Lorlatinib with good response [42]. We also used proteomics in a refractory childhood embryonal tumor with multilayered rosettes of the brain to identify a driving metabolic pathway that was targeted with the TKI desatinib that and resulted in gross total resection of the tumor and prolong survival [43]. Unfortunately we have also found that these heavily pretreated tumors frequently become resistant to TKI after a few months of response [42]. One way to overcome this difficulty could be to add a backbone of metronomic chemotherapy that would target the micro environment, malignant angiogenesis and drug resistance, while the TKI would target aberrant metabolic pathways in the tumor, increase chemotherapy susceptibility and induce apoptosis.

While it has been noted that a VEGFR targeting TKI may blunt the ability of metronomic chemotherapy [44] to activate the immune system (suggesting antagonistic activity), we have previously shown that a metronomic chemotherapy plus a neutralizing anti-VEGFR antibody preclinically delays the growth of B16 melanoma lung metastases [45]. In addition, effective combination of the TKI sorafenib with metronomic chemotherapy has also been reported preclinically [46].

Collectively, these observations have led us to propose a study that incorporates tyrosine kinase inhibitors (TKI) to metronomic chemotherapy. Our backbone of choice to compare with TKI is celecoxib-valproic acid based on our experience and the growing interest in anti-histone drugs that have undergone trials with responses in difficult pediatric tumors [47]. All patients will receive alternating cyclophosphamide and etoposide as described in this report. This study depicted in Figure 5 will involve screening refractory patient's tumors for metabolic targets, and treating the patients with alternating etoposidecyclophosphamide and either continuous celecoxib-valproic acid or continuous TKI if a targetable aberrant metabolic pathway is identified.

**Figure 5.** Schematic of future planned studies for pediatric cases eligible for metronomic chemotherapy. Proteomic and metabolic pathway analysis, where feasible, will be used to determine eligibility of patients to receive specific tyrosine kinase inhibitor (TKI) therapy coupled with metronomic chemotherapy, which involves alternating cycles of metronomic etoposide with metronomic cyclophosphamide. Patients not selected or not eligible for TKI-based therapies will receive valproic acid (VA) and celecoxib with metronomic chemotherapy as outlined in this manuscript.

The mechanisms via which metronomic chemotherapy produces antitumor effects are numerous [1] and are yet to be fully elucidated. Thus, in 2003, in collaboration with Bocci and colleagues [19], we reported that continuous low-dose CTX (as well as other drugs such as paclitaxel) can induce endothelial cells to overexpress the angiogenesis inhibitor protein thrombospondin-1. These data provide further evidence [48] for the antiangiogenic activity of metronomic chemotherapy. Other mechanisms include an alteration of intratumoral blood flow as observed following the administration of metronomic oral gemcitabine (LY2334737) as we previously observed in a preclinical model [20]. They also include the suppression of the homing of bone marrow-derived endothelial progenitor cells to the periphery of a tumor mass, as shown by Shaked and colleagues [49]. Additional mechanisms of action of metronomic chemotherapy, at least for drugs such as CTX, include immune activation, which likely occurs via suppression of regulatory T cells [21]. Furthermore, in collaboration with Emmenegger and colleagues [50], we reported that preclinical selection of tumors resistant to high-dose chemotherapy does not impair their response to subsequent metronomic regimens of the same drug [50]. These data suggest that mechanisms of tumor resistance to metronomic chemotherapy show little or no overlap with those for the same chemotherapeutics agent (e.g., CTX) given at pulsatile maximum tolerated doses. These results also suggest that a tumor relapsing to pulsatile high dose of a drug such as CTX may still show response to the same drug given on a metronomic schedule.

Metronomic chemotherapy has undergone a number of refinements since it was coined in 2000, to describe preclinical studies with drugs used in a continuous low-dose (as an antiangiogenic strategy or as a maintenance strategy), namely, CTX or vinblastine [48,51]. Since then, the concept of metronomic chemotherapy has evolved for the treatment of adult tumors, as well as in its application in pediatric oncology. Thus, for adult tumors, recent phase III clinical data in metastatic colorectal cancer showed improved progression-free survival in patients receiving maintenance low-dose capecitabine plus bevacizumab [7]. In addition, safety of administration was reported from a phase III clinical trial evaluating bevacizumab plus metronomic cyclophosphamide and capecitabine as first-line therapy in patients with HER2-negative advanced-stage breast cancer [8,52]. On the other hand, for pediatric tumors, reported protocols (as evident from a Pubmed search with the terms "metronomic" and "pediatric") include the long-term oral administration of daily low-dose

mercaptopurine and weekly low-dose methotrexate for children with ALL [11]. They also include a metronomic vincristine/CTX/methotrexate/VA regimen given to children with refractory cancer of various tumor types [53]. Recently, there have been additional reports of metronomic chemotherapy improving survival in high-risk neuroblastoma patients [41], as well as other pediatric malignancies including Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma [15], and studies have confirmed the low toxicity associated with this approach [54]. Alternating VP16 and cyclophosphamide metronomic regimens, with daily celecoxib, were combined with sirolimus in a phase I study of 18 pediatric relapsed or refractory solid and brain tumors, and the combination was found to be well tolerated [55]. It should be noted that among the first studies published on using chemotherapy metronomically for pediatric tumors were those of Kieran and colleagues [10] and of Andre and colleagues [13]. Thus, Kieran's group originally proposed continuous oral thalidomide and celecoxib with alternating oral etoposide and cyclophosphamide every 21 days. Recently, the same group reported a more complex strategy involving five drugs including metronomic CTX and etoposide, as shown in Table 2. Andre et al. proposed 2 weeks of metronomic etoposide followed by 2 weeks of metronomic CTX, plus celecoxib [13]. Because of the number of drugs involved in some regimens such as the one proposed by Kieran and colleagues (see Table 2), additional studies may be necessary to compare different permutations of the use of such drugs and the different orders in which such drugs are administered metronomically. Such studies may uncover whether specific combinations are important (e.g., CTX plus VA) or if the exact order of administration is critical (e.g., etoposide before CTX, or vice versa). In that respect, the six cases we report add data to a regimen of etoposide, CTX, and VA–celecoxib. Interestingly, with regard to VA–celecoxib as a backbone, we previously reported [20] that metronomic oral gemcitabine (LY2334737) has antitumor effects in preclinical mouse xenograft models. The LY2334737 oral gemcitabine prodrug is, once injected, metabolized into gemcitabine plus VA, suggesting that a metronomic oral gemcitabine schedule may unintentionally result in continuous administration of two drugs (gemcitabine and VA), although this possibility remains to be tested. Overall, these clinical and preclinical observations suggest that metronomic alternating regimens combined with continuous administration of celecoxib and VA have promising activity in tumors relapsing to pulsatile high-dose chemotherapy.

**Author Contributions:** Conceptualization, B.C.; writing—original draft preparation, B.C. and G.F.; writing—review and editing, B.C. and G.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** G.F. was supported by NIH-NCI SC2CA211029 and SC1 GM136630-01 grants.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Lidia Baiocchi, Guido Bocci, and Urban Emmenegger for their critical review of this manuscript. We also thank Karla Parra, Valerie Gallegos, and Paloma Valenzuela for assistance in assembling the documentation for this manuscript.

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

#### **Abbreviations**

cyclophosphamide (CTX), etoposide (VP16), valproic acid (VA).

 Docetaxel chemotherapy was the first life-prolonging treatment for mCRPC; however, the standard maximally tolerated dose (MTD) docetaxel regimen is often not considered for patients with mCRPC who are older and/or frail due to its toxicity. Low-dose metronomic chemotherapy (LDMC) is the frequent administration of typically oral and off-patent chemotherapeutics at low doses, which is associated with a superior safety profile and higher tolerability than MTD chemotherapy. We conducted a systematic literature review using the PUBMED, EMBASE, and MEDLINE electronic databases to identify clinical studies that examined the impact of LDMC on patients with advanced prostate cancer. The search identified 30 reports that retrospectively or prospectively investigated LDMC, 29 of which focused on mCRPC. Cyclophosphamide was the most commonly used agent integrated into 27/30 (90%) of LDMC regimens. LDMC resulted in a clinical benefit rate of 56.8 ± 24.5% across all studies. Overall, there were only a few non-hematological grade 3 or 4 adverse events reported. As such, LDMC is a well-tolerated treatment option for patients with mCRPC, including those who are older and frail. Furthermore, LDMC is considered more affordable than conventional mCRPC therapies. However, prospective phase III trials are needed to further characterize the efficacy and safety of LDMC in mCRPC before its use in practice.

**Keywords:** metronomic chemotherapy; metastatic castration-resistant prostate cancer; cyclophosphamide; side effects

#### **1. Introduction**

Cancer is amongst the most significant contributors to disease burden worldwide, with cancer incidence expected to double by 2035 [1]. The global cancer burden is greatest in low- and middle-income countries (LMIC), in which cancer incidence is rising most rapidly, where 75% of cancer deaths occur, yet where only 5% of the global spending on cancer is directed [1]. Prostate cancer follows this general trend as the second most common malignancy in men globally [2].

Localized prostate cancer is highly curable, but metastatic prostate cancer remains a fatal condition to date [3]. While prostate cancer is diagnosed at a median age of 66, prostate cancer-related deaths occur at a median age of 80 [4].

Because prostate cancer is an androgen-driven disease, androgen deprivation therapy (ADT) is the usual first-line therapy for metastatic prostate cancer, nowadays often combined with either docetaxel chemotherapy or androgen receptor signaling inhibitors (ARSi)

**Citation:** Parshad, S.; Sidhu, A.K.; Khan, N.; Naoum, A.; Emmenegger, U. Metronomic Chemotherapy for Advanced Prostate Cancer: A Literature Review. *J. Clin. Med.* **2022**, *11*, 2783. https://doi.org/10.3390/ jcm11102783

Academic Editors: Guido Bocci, Giulio Francia and Petros Grivas

Received: 22 March 2022 Accepted: 10 May 2022 Published: 15 May 2022

**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/).

such as abiraterone, apalutamide, or enzalutamide [5]. However, such patients will develop ADT-resistant prostate cancer eventually, referred to as metastatic castration-resistant prostate cancer (mCRPC), the lethal form of prostate cancer.

Docetaxel was established as the first life-prolonging and quality-of-life-improving therapy for mCRPC in 2004, providing a survival benefit of around three months [6,7]. This intravenous chemotherapeutic of the taxane family is usually administered as a three-weekly regimen at the maximum tolerated dose (MTD), which is associated with numerous acute and chronic side effects (e.g., myelosuppression, mucositis, and peripheral neuropathy). Thus, docetaxel is often not considered for older men who may have a lower tolerance than younger, healthier patients [8–11].

In recent years, abiraterone and enzalutamide have been approved as mCRPC treatment options, typically used in first line [3]. Both agents are relatively well tolerated, but acquired therapeutic resistance is the ultimate outcome of ARSi therapy in most instances [12,13]. Radium-223 and cabazitaxel chemotherapy represent treatment options for ARSi- and docetaxel-resistant mCRPC [14,15]. However, access to all these life-prolonging yet expensive prostate cancer therapies is limited, notably in LMIC [16,17]. Hence, there is a continued need for affordable mCRPC therapies, especially those suitable for the typically elderly and often patients with advanced prostate cancer who are frail. Low-dose metronomic chemotherapy (LDMC) possess many characteristics to fill this unmet need.

LDMC refers to the continuous administration of low doses of conventional chemotherapy drugs over a long period, usually via daily oral intake without scheduled treatment breaks, resulting in antiangiogenic and immunomodulatory anti-tumor effects amongst others [18]. Due to the low drug doses used, LDMC has a superior toxicity profile compared with MTD chemotherapy, including in people who are older and frail [19,20]. Furthermore, LDMC is relatively affordable owing to the use of off-patent drugs such as cyclophosphamide (CPA) and modest needs for monitoring treatment-associated side effects [21,22]. Herein, we summarize the findings of 30 studies on the role of LDMC in advanced prostate cancer.

#### **2. Materials and Methods**

#### *2.1. Search Strategy and Study Selection*

A systematic search of the PubMed, EMBASE, and MEDLINE electronic databases was conducted from inception up to 31 December 2021 to identify all relevant studies investigating the clinical impact of LDMC in patients with prostate cancer. The search strategy involved combining a methodological filter to specifically identify 'full text articles' using the search terms 'metronomic' and 'prostate cancer.' English-written literature was highly valued in the conduct of this review due to the ease of data extraction. However, non-English language was not a reason for exclusion. Additional studies were identified by screening the reference lists of review articles on LDMC. Titles and abstracts were screened for eligibility. The exclusion criteria are highlighted in Figure 1. Treatment regimens comprising at least one component administered without prespecified breaks were considered metronomic.

#### *2.2. Data Extraction and Statistical Analysis*

Two independent reviewers extracted information on study type and design, country of study, number of patients, patient demographics, treatment details, response criteria used and response rates, survival data, adverse events, and fatalities. Statistical analyses were computed using RStudio (RStudio for Macintosh, version 1.1.463). Graphs were created with Microsoft® Excel for Mac 16.45 (www.microsoft.com), Draw.io (Version 16.5.1; https: //app.diagrams.net), or GraphPad Prism (Version 9.3.1; https://www.graphpad.com).

**Figure 1.** Flow diagram of search strategy.

#### **3. Results**

#### *3.1. Study Selection*

Among the 234 reports identified during the initial search, there were 132 duplicates, leaving 102 abstracts for screening. Following the removal of 49 studies comprising reviews, and pre-clinical and mechanistic studies, we analyzed the full text of 53 articles. A further 23 studies were excluded, including letters, surveys, case reports, and studies that were not truly metronomic or prostate cancer specific. Overall, we identified 30 studies on the clinical use of LDMC for prostate cancer, as depicted in Figure 1. Key study details are outlined in Table 1.

#### *3.2. Study and Patient Characteristics*

LDMC studies in the field of prostate cancer cover the time period from 1993 until 2019, with the majority (23/30; 77%) published from 2010 onwards (Figure 2a). More than half of the studies analyzed were conducted in Europe (17/30; 57%), notably with ten of those (33% of all studies) in Italy. Moreover, eight studies (27%) were conducted in Asia, whereas only five reports (17%) were from North America and none were from Asia or South America. Eleven studies (37%) were retrospective analyses. Among the nineteen prospective studies, two (11%) were phase I [27,36], one (5%) was phase I/II [47], and sixteen (84%) were phase II studies [22,25,26,29,30,33,34,37,38,40–43,45,48,49]. Across both prospective and retrospective phase II type studies, the majority (23/26, 88%) were single arm trials [19,21–23,25,26,28–33,35,37,38,40–42,44,48,49].

The 30 studies comprise information on 973 patients overall, with 28 (range 8 to 74) being the median number of patients per study (Figure 2b). The median patient age per trial ranged from 60 to 83 years, of which 72.8 years was the median of the reported medians (Figure 2c). Of the 30 studies included, only one reported on men with biochemically recurrent (i.e., non-metastatic and castration-sensitive) prostate cancer (Figure 3) [22]. All other studies focused on patients with mCRPC and variable treatment history. Seven (23%) studies included chemotherapy-naïve participants [25,32,33,38,45,46,48], three reported on study subjects with or without prior chemotherapy (typically docetaxel) exposure [28,31,42], and nineteen did not provide details on prior therapies other than the use of ADT [19,21,23,24,26,27,29,30,34–37,39–41,43,44,47,49].

**Table 1.** Study characteristics.

\* = mean; N = sample size, N/A = not available.

**Figure 2.** *Cont.*

**Figure 2.** (**a**) Number of LDMC publications per year. (**b**) Number of patients per study. (**c**) Median patient age in years per study.

#### *3.3. Metronomic Treatment Regimens*

CPA was integrated into 27 of the 30 (90%) regimens (Figure 4) [19,21–25,27–35,37–44,46–49]. While six studies (20%) described the effects of CPA monotherapy [21,22,38,41,43,46], in the majority of reports, CPA was partnered with corticosteroids (19/30; 63.3%) [19,23–25,27,29–35,37,39,40,42,44,48,49]. Among the CPA/corticosteroid combination therapy studies, eight (27%) did not add further agents [23,24,27,31,35,37,40,49], the COX2 inhibitor celecoxib was added in three (10%) trials [19,34,44], and eight (27%) studies included other drugs (i.e., methotrexate, tegafur-uracil, etoposide, estramustine phosphate, and capecitabine) [25,29,30,32,33,39,42,48]. In two studies, CPA was combined with either thalidomide or lenalidomide (7%) [28,47].

**Figure 3.** Prostate cancer stage of patients of LDMC studies. mCRPC: metastatic castration-resistant prostate cancer.

Of the studies without a CPA backbone, a variety of agents were used. Di Desidero et al. studied vinorelbine with dexamethasone [26], Kubota et al. studied cisplatin and tegafururacil [36], and Tralongo et al. studied docetaxel or vinorelbine [45]. Of note, 24 of 30 (80%) treatment regimens comprised oral medications only [19,21–24,26–28,31–35,37–44,46,47,49].

#### *3.4. Outcomes*

To compare the effectiveness of the various LDMC regimens used, we extracted prostatespecific antigen (PSA) response rates and clinical benefit rates. Twenty-six trials provided information regarding patients' PSA levels (Figure 5a) [19,21,22,24–34,36–42,44–47,49]. The mean ± SD PSA response rate (i.e., at least a 50% treatment-related PSA decrease compared with baseline) was 33 ± 19.1%, while another 32.2 ± 16.5% of patients achieved stable PSA readings. One third of study patients (33.5 ± 19.4%) did not experience any biochemical benefit from LDMC.

**Figure 5.** (**a**) Biochemical response assessment based on prostate specific antigen (PSA) across 26 informative studies. PSA response was defined as a ≥50% treatment-related PSA decrease compared with baseline. (**b**) Clinical benefit rate (%) across 26 informative studies.

The mean clinical benefit rate reported across 26 studies was 56.8 ± 24.5% (range from 8.3% to 95.0%) (Figure 5b). Of note, the publications used variable definitions of "clinical benefit", with the most common being "sustained (≥6 months) absence of biochemical, clinical, and/or radiological progression".

Twenty studies reported the median overall survival of patients on LDMC regimens [19,21,23,25,26,29–31,34–37,39–41,43,44,46,47,49]. Fontana et al. observed the shortest median survival of 3.3 (95%CI: 2.2–4.2) months [29], while Derosa et al. described the longest median survival of 33.3 (95%CI: 23–35.6) months [25]. The median of medians of reported overall survival was 16.2 months.

#### *3.5. Toxicities and Adverse Events*

Twenty of the thirty studies used varying versions of the National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE) to grade toxicities observed among study participants undergoing LDMC [19,21–23,25–27,29,30,32,34,36–40,42–44,46]. One of the studies used criteria set by the World Health Organization (WHO) [45], while four studies reported adverse events without indicating the type of criteria used [28,47–49]. Five studies did not include information regarding toxicities and adverse events in relation to LDMC [24,31,33,35,41].

Figure 6 shows the percentage of study patients that experienced specific grade 3 or 4 (i.e., severe) toxicities reported in 15 informative clinical trials. Overall, hematological toxicities were more common than non-hematological adverse events. Instances of severe anemia were reported in nine trials, with a median of 8% of patients affected [21,27,28,34,37,38,42,43,45]. Grade 3/4 neutropenia was reported in eight studies, with a median of 5.5% of patients affected [25,27,32,37,38,43,45,47]. In the four studies listing severe lymphopenia, on average, around 20% of patients were affected. Asthenia was the most reported severe non-hematological adverse event, listed by five trials, with a median of 5.4% of patients affected [21,26,34,45,47].

**Figure 6.** Heatmap of grade 3 and 4 toxicities observed in LDMC studies.

Vorob'ev et al.'s retrospective study compared the side effects of MTD docetaxel (75 mg/m2 administered intravenously every three weeks; *n* = 30 patients) versus LDMC CPA (50 mg by mouth daily; *n* = 25 patients) [46]. There were far fewer and less severe side effects reported in the CPA cohort (using NCI-CTCAE version 3) in comparison with patients treated with docetaxel (Figure 7). While a high percentage of patients treated with docetaxel were affected by diarrhea, alopecia, grade 1–3 anemia, and grade 1–4 neutropenia, patients in the CPA cohort were primarily affected by grade 1–2 anemia and

148

grade 1–2 neutropenia, without severe (i.e., grade 3 or 4) cytopenia. Moreover, 16.7% of the docetaxel cohort stopped treatment due to adverse events, but no patient treated with CPA discontinued treatment because of side effects. Despite the distinct toxicity profiles, the mean overall survival was similar (>15 months) for both cohorts. However, MTD docetaxel resulted in a higher PSA response rate (46.7%) than LDMC CPA (12%). MTD docetaxel treatment was also slightly favored over LDMC CPA in terms of quality of life, measured with the Functional Assessment of Cancer Therapy-Prostate (FACT-P) questionnaire, and rate of pain response, based on a visual analogue scale, although these results were not statistically significant.

**Figure 7.** Depiction of the incidence of adverse events reported in patients undergoing conventional docetaxel versus metronomic cyclophosphamide therapy as reported by Vorob'ev et al., 2011 [46].

#### **4. Discussion**

The present literature review of 30 studies of LDMC for prostate cancer encompassing 973 patients illustrates several key findings. First, more than half of patients experience PSA responses or PSA stability and draw a clinical benefit from LDMC. Second, CPA is the most commonly used cytotoxic agent for metronomic purposes in prostate cancer (27/30 trials), as is the case in other cancer types [50]. The LDMC CPA studies characterize this classical alkylating agent as a convenient (oral mode of administration), well tolerated, and affordable (off-patent) treatment option that can be administered alone or in combination with other agents. Third, prostate cancer LDMC studies report low rates of severe (i.e., grade 3 or 4) adverse events. Hematological toxicity, notably lymphopenia, was more frequently observed than non-hematological adverse events. However, neither myelosuppression nor lymphopenia appear to be associated with an increased rate of infections. Furthermore, the low rate of typically mild LDMC-associated side effects compares favorably with the higher rate of adverse events seen with conventional MTD chemotherapy, including high-grade adverse events, as documented by Vorob'ev et al. [46].

Our study also reveals shortcomings and unmet needs regarding the clinical development of LDMC in prostate cancer. Foremost, there are no definite phase III clinical trials documenting the benefit of LDMC in prostate cancer, unlike in other malignancies

such as breast, head and neck, and colorectal cancer [51–58]. Moreover, the majority of the LDMC prostate cancer studies were single-arm trials describing relatively small and often heterogeneous patient cohorts. Second, LDMC was almost exclusively studied in later stages of prostate cancer and often after multiple lines of prior therapies, whereas phase III trials in other tumor types suggest that LDMC might be particularly suitable for maintenance therapy in earlier tumor stages [52,55,57]. Third, there are no validated predictive markers of response to LDMC [59]. However, anecdotal evidence of responses to metronomic CPA in patients with DNA repair deficient mCRPC warrant further study [60].

In current times, with limited resources and growing expenses for the treatment of early as well as advanced prostate cancer, drug costs are becoming an increasingly important consideration when choosing treatments options [61–63]. With the incidence of cancer surging and the rising economic burden of cancer treatment worldwide, there is a need for affordable treatment options [2,64]. This is especially important for resourcelimited countries, where mortality rates due to prostate cancer are rising, while decreasing in the more developed countries [65]. LDMC is an attractive treatment option in this respect. In Bocci et al.'s outcome analysis and cost comparison for conventional versus LDMC for metastatic breast cancer, LDMC was found to be more cost-effective due to several factors: LDMC (i) can be taken orally at home instead of administered during hospital visits, (ii) has lower incidences of adverse events, thereby decreasing related hospital and other healthcare visits, and (iii) is associated with lower administrative and health care provision costs due to a reduced need for medical attention [66].

Our systematic literature search did not identify studies comparing LDMC with ARSi therapy. Based on available evidence reported in the present analysis, it appears improbable that LDMC alone may provide a similar benefit to ARSi in advanced prostate cancer. Furthermore, ARSi are convenient oral albeit expensive medications that rarely result in severe side effects, even in patients who are older or frail [12,13,16,17]. However, LDMC might make ARSi therapy more affordable when integrated into intermittent ARSi regimens (e.g., LDMC maintenance therapy following ARSi induction). Aside from cost savings such LDMC use might also improve patient outcome by targeting ARSi-resistant prostate cancer cells [67,68]. Similarly, preliminary breast and ovarian cancer evidence suggests a possible role for combining LDMC using alkylating agents with poly (ADP-ribose) polymerase (PARP) inhibitors in patients with DNA-repair-deficient prostate cancer [69,70].

When extracting information from the 30 studies of LDMC for prostate cancer numerous limitations became apparent. Aside from the aforementioned lack of randomized controlled phase III trials, one third of studies were retrospective, and the majority of reports comprised relatively small single-arm studies. The definition of outcome measures such as clinical benefit rate varied across studies, rendering comparisons difficult. Not all reports contained information on adverse events. Finally, a wide variety of metronomic regimens were tested, although the use of CPA was a common denominator.

#### **5. Conclusions**

In conclusion, LDMC is a well-tolerated and cost-effective form of cancer therapy with documented anti-mCRPC effects. Because of mild toxicities and simple oral administration, LDMC can be regarded as an alternative treatment option especially for patients who are older or unfit and who are unable to tolerate conventional mCRPC therapies such as taxane chemotherapy. LDMC might also be considered in situations where ARSi are not available or affordable. Phase III trial evidence is needed to position LDMC with respect to other mCRPC therapies.

**Author Contributions:** Conceptualization, S.P. and U.E.; methodology, S.P. and U.E.; validation, S.P., A.K.S., N.K. and U.E; formal analysis, S.P., N.K., A.N. and U.E.; investigation, S.P., A.K.S., N.K. and U.E.; writing—original draft preparation, S.P. and A.K.S.; writing—review and editing, S.P., A.K.S., N.K., A.N. and U.E.; visualization, S.P., A.K.S. and N.K.; supervision, U.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** The preparation of this manuscript was financially supported by the Joseph and Silvana Melara Cancer Fund, Toronto/ON, Canada.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**

## *Review* **Metronomic Therapy in Oral Squamous Cell Carcinoma**

**Nai-Wen Su 1,2 and Yu-Jen Chen 2,3,4,\***

**Abstract:** Metronomic therapy is characterized by drug administration in a low-dose, repeated, and regular manner without prolonged drug-free interval. The two main anticancer mechanisms of metronomic therapy are antiangiogenesis and immunomodulation, which have been demonstrated in several delicate in vitro and in vivo experiments. In contrast to the traditional maximum tolerated dose (MTD) dosing of chemotherapy, metronomic therapy possesses comparative efficacy but greatlydecreases the incidence and severity of treatment side-effects. Clinical trials of metronomic anticancer treatment have revealed promising results in a variety cancer types and specific patient populations such as the elderly and pediatric malignancies. Oral cavity squamous cell carcinoma (OCSCC) is an important health issue in many areas around the world. Long-term survival is about 50% in locally advanced disease despite having high-intensity treatment combined surgery, radiotherapy, and chemotherapy. In this article, we review and summarize the essence of metronomic therapy and focus on its applications in OCSCC treatment.

**Keywords:** metronomic; oral cancer; oral squamous cell carcinoma

#### **1. Introduction**

Head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide, comprises heterogenous groups of tumors arising from the oral cavity, oropharynx, hypopharynx, and larynx [1]. In Taiwan, South–Central Asia, and parts of Europe, oral cavity squamous cell carcinoma (OCSCC) has a higher incidence (9–22 per 100,000) than tumors arising from other sites [2]. Smoking, alcohol consumption, and betel nut use are the major contributors in OCSCC carcinogenesis [3]. More importantly, OCSCC patients exposed to these traditional carcinogens tend to have poorer treatment outcomes than human papillomavirus (HPV)-related HNSCC patients [4]. In addition, the long-term (5 year) survival rate is unsatisfactory (approximately 50%). According to the GLOBO-CAN 2018 report, 117,384 of the 354,846 newly diagnosed OCSCC patients died from the disease [5]. Therefore, OCSCC poses a considerable burden to the healthcare systems in different regions [6–11]. The majority of OCSCC patients present with a locally advanced disease, and the main therapeutic strategy is tumor wide excision plus radical neck dissection, followed by adjuvant radiotherapy with or without chemotherapy [12,13]. This high-intensity treatment program results in significant acute or chronic adverse events [14]. Locoregional relapse and secondary primary HNSCC [15,16] are the most common recurrent patterns and curable if amenable to local treatment. However, the prognosis is grave once the disease is metastatic or attains platinum-refractory status. Although the therapeutic armamentarium is expanding, for example, anti-epidermal growth factor receptor (EGFR) monoclonal antibody and immune checkpoint inhibitors have been developed, the median overall survival is approximately 10–14 months [17–21].

**Citation:** Su, N.-W.; Chen, Y.-J. Metronomic Therapy in Oral Squamous Cell Carcinoma. *J. Clin. Med.* **2021**, *10*, 2818. https:// doi.org/10.3390/jcm10132818

Academic Editors: Giulio Francia and Guido Bocci

Received: 28 May 2021 Accepted: 24 June 2021 Published: 26 June 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/).

The most important chemotherapeutic agent in OCSCC treatment is platinum. In platinum-based concurrent chemoradiotherapy or palliative chemotherapy, administration of the maximum tolerated dose (MTD) of cisplatin(100 mg/m2 every 3 weeks) is generally considered a gold standard regimen [12,13,17]. The development of platinum-resistant OCSCC is an important factor that leads to treatment failure. Existence of cancer stem cells [22,23], increased angiogenesis [24,25], and unfavorable immune profile in the microenvironment [26,27] are the proposed mechanisms that contribute to platinum-resistant OCSCC and predict poor prognosis. Moreover, the MTD of cisplatin (100 mg/m2) is deemed as a highly toxic regimen because it has significant side-effects such as nephrotoxicity, ototoxicity, and neurotoxicity [28,29]. Therefore, novel therapeutic strategies are necessary to improve OCSCC treatment outcomes a step further [30]. Metronomic chemotherapy, one of the potentially promising treatments, is being studied and utilized in clinical practice for a variety of cancer types, including OCSCC [31–37]. The unique dosing schedule stems from preclinical studies, which have revealed the advantages of overcoming drug resistance and antiangiogenesis effects [38,39]. Clinically, metronomic chemotherapy induces drug sensitivity and, most importantly, it is accompanied by minimal adverse effects [40]. In this article, we briefly review the mechanism of action and clinical development of metronomic therapies in OCSCC.

#### **2. History of Metronomic Therapy**

Since the appearance of cytotoxic chemotherapeutic agents in the early 1960s, they have been playing a critical role in cancer treatment. The use of MTD to obtain the highest possible cancer cell killing is the mainstream method to calculate the chemotherapy dose [41]. The MTD chemotherapy originated from the great success of a pediatric leukemia treatment model [42]. However, MTD chemotherapy imposes great damage to the rapidly proliferating normal tissues such as hematopoietic or gastrointestinal epithelial cells. To limit the toxic effects and permit patient recovery, MTD chemotherapy should be administered with 3–4 weeks of drug-free interval only. Regrowth of the residual cancer cells is inevitable [43]. Moreover, complete eradication of cancer cells is rarely achieved with MTD chemotherapy owing to the development of drug resistance [44]. Decades of cancer research has revealed that cancer cells possess multiple genetic alterations and could have genomic evolution that contributes to resistance [45,46]. The cancer treatment is far more complex because components of the tumor microenvironment, such as immune cells, endothelial cells, and fibroblasts, communicate and relay pro-survival signals to malignant cells [47–49]. This adds more obstacles to our goal of cure or long-term disease control with MTD chemotherapy.

To evaluate the biological effects of administered chemotherapy, other than MTD, one unique dosing schedule—metronomic chemotherapy—was studied in preclinical cell and animal models. Metronomic dosing was defined as administration of low doses (1/3–1/10 of MTD) cytotoxic agents at more frequent intervals (no prolonged drug-free breaks) [50]. Initially, the metronomic chemotherapy was reported to inhibit cancer-associated angiogenesis and consequently promote tumor regression [51,52]. Simultaneously, Broder et al. demonstrated that this low-dose and frequent administration aids in overcoming drug resistance [53]. This regimen is more attractive because it has a lower toxicity profile than MTD [54]. In the following decade, more studies focused on the biological effects of metronomic chemotherapy.

#### **3. Metronomic Therapy: Mechanisms of Action**

#### *3.1. Antiangiogenic Effects*

Neoangiogenesis takes a central role during the growing phase of the primary tumor and supports the establishment of distant metastatic deposits. It has long been proposed as a potential target in cancer treatment [55,56]. Chemotherapeutic agents were found to have antiangiogenesis effects in a preclinical study, although the dosing schedule matters in this phenomenon [57]. Tumor-associated vascular endothelial cells are genetically

more stable than cancer cells. This is one of the possible explanations why metronomic chemotherapy more specifically causes endothelial cell apoptosis and less resistance development. Other proposed molecular mechanisms are the suppression of endothelial progenitor cell mobilization from the bone marrow [58] and an increase in the expression of thrombospondin-1 (TSP-1), which is an endogenous antiangiogenic factor [59]. Recently, metronomic chemotherapy was found to normalize the defective tumor vasculature [60]. Cyclophosphamide is the prototype of a chemotherapeutic agent possessing antiangiogenic property when administered in the metronomic schedule [61]. The same biological activity was proven for other agents such as taxanes, camptothecin, and vinca alkaloids in a variety of cancer types [62,63]. On the contrary, some studies have suggested that chemotherapeutic agents might induce acute mobilization of the bone marrow-derived circulating endothelial progenitor cells (EPCs), homing viable tumors and promoting their growth. Studies have provided evidence that coadministration of antiangiogenic agents might work in a synergistic fashion with chemotherapy for the suppression of tumor and emergence of resistance [64–67].

#### *3.2. Immunomodulatory Effects*

During the process of carcinogenesis and tumor progression, cancer cells may evade the immunosurveillance through the release of cytokines (such as transforming growth factor, vascular endothelial growth factor (VEGF), and interleukin-6) and recruitment of precancerous immune cells (such as regulatory T cell (Treg), myeloid derived suppressor cells (MDSC), and tumor associated macrophage-2) [68]. Studies have shown that the MTD of some traditional chemotherapeutic agents have immunomodulatory effects such as the induction of immunogenic cell death [69]. Through low-dose metronomic chemotherapy, studies found that different agents can deplete Treg and suppress MDSC [70–73]. In addition, it can promote dendritic cell maturation, an important step to educate the naïve cytotoxic T cells [74]. Some metronomic agents cause cell death and favorably promote the release of "eat me" signals (such as damage-associated molecular patterns, calreticulin, and high-mobility group box 1) [75]. All of these phenomena can possibly activate our immune cells to attack the tumor. However, our immune system is dynamic and, therefore, some study results might be controversial and require further clinical validation [76,77].

#### *3.3. Inhibition of Cancer Stem Cells*

Cancer cells, like their normal counterparts, contain a minority portion of cells that possess the ability of self-renewal and differentiation. These specific cancer cells are called cancer stem cells (CSCs), which are detected mainly with the expression of specific cell surface markers (CD24+, CD44+, or CD133+; alone or combined expression) [78]. CSCs have been strongly linked to chemoresistance or radio resistance and, consequently, anticancer treatment failure [79,80]. A few studies have revealed that metronomic chemotherapy can decrease the CSC population [81–83]. Vives et al. demonstrated that MTD chemotherapy followed by metronomic maintenance therapy with gemcitabine and cyclophosphamide successfully eliminated the CSCs in pancreatic cancer and ovarian cancer orthotopic models, respectively [84]. Incorporation of metronomic chemotherapy into or combined with current cancer treatment may aid in a further reduction in CSC-related resistance.

#### **4. Metronomic Therapy in OCSCC**

#### *4.1. Preclinical Evidence*

Although metronomic therapy has been applied in clinical trials or daily oncological practice, only one chemotherapeutic agent, S-1, has been tested completely in both in vitro and in vivo models. S-1 is composed of three compounds. One compound is a 5-fluorouracil (5-FU) prodrug, tegafur. The other two are gimeracil (inhibits the 5-FU degradation) and oteracil (lowers the gastrointestinal toxicity of 5-FU) [85]. In an in vitro study, Ferdous et al. first demonstrated that more frequent 5-FU dosing (16h treatment, 8h rest vs. 96h treatment, 48h rest or 48h treatment, 24h rest) did not result in more cytotoxic

cell death according to3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2*H* tetrazolium bromide assay. However, functional evaluations revealed that oral cancer treatment with more frequent 5-FU dosing (16h treatment, 8h rest) showed the highest expression of TSP-1 (antiangiogenesis molecule) and lowest expression of VEGF and CD44 (CSC marker). In the in vivo xenograft model, the usual S-1 schedules were 4weeks on followed by 2 weeks off and 2weeks on followed by 1weekoff, which were the same as the standard regimens in human. The metronomic dosing was 1dayon followed by 1dayoff. The results revealed that all three regimens resulted in similar tumor volume reductions. The nude mice in the two standard regimen groups had a lower weight than those in the metronomic treatment group. The xenograft tumors in the metronomic treatment group showed increased TSP-1 and decreased VEGF expression, which was consistent with the findings of the in vitro study. In addition, a reduction in CD44 expression was observed. The number of endothelial cells (based on CD31 expression) and the micro vessel density (MVD) both decreased in the metronomic treatment group tumors. The results of S-1 treatment in an oral tongue cancer model revealed that metronomic dosing resulted in comparable tumor inhibition. Considering the proof-of-concept viewpoint, metronomic dosing of S-1 demonstrated antiangiogenesis and cancer stem cell elimination effects. According to our knowledge, a variety of compounds extracted from traditional Chinese medicine possess anticancer effects [86]. Some of the studies have tested compounds in xenograft models with daily administration, which mimicked the metronomic dosing schedule. Most studies had a long treatment duration ranging from 3–8 weeks and no significant side-effects [87–93]. In our previous studies, we treated the oral cancer-bearing mice with cordycepin, a major biological compound from *Cordyceps sinensis*, alone or in combination with radiotherapy for 8 weeks. No weight loss was observed, and the hematological, renal, and hepatic functions were preserved [92,93].

#### *4.2. Clinical Evidence*

In the past two decades, the concept of metronomic therapy was translated into real practice, and clinical trials have finally proven its effectiveness and better tolerability in a variety of cancers [94–97] and specific populations [98,99]. Oral chemotherapeutic agents are the central pillars in most studies with different combinations, including targeted therapy [100], vascular disrupting agent [101], and even noncytotoxic agent [102]. We found two main clinical settings for the use of metronomic therapy in OCSCC; one was adjuvant/maintenance therapy after curative intent surgery or chemoradiotherapy, and the other was a palliative therapy in recurrent/metastatic status.

Three studies used metronomic fluorouracil drugs in adjuvant/maintenance therapy. Lin et al. conducted a prospective cohort study and reported that 80 OCSCC patients with stage III–IVa (T3–T4a and/or N0–N1) diseases received curative intent surgery. Forty patients received metronoic UFUR (each capsule contains tegafur 100 mg and uracil 224 mg) with a fixed dose of tegafur 150 mg/m2/day for 1 year, and another 40 patients were observational controls. The 4year disease-free survival (DFS) of the metronomic group versus the control group was 85.6% and 75.9% (*p* = 0.02). Simultaneously, they performed laboratory correlation by detecting the change in CEP (circulating endothelial precursor) cells, a marker of angiogenesis activity. The results revealed that patients treated with metronomic UFUR had a significant reduction in the detectable viable CEP cells, which might implicate an antiangiogenesis mechanism behind the survival benefits. The toxicities were mild and manageable, with skin rash (5%) being the most common side-effect, followed by hematological side-effects (2.5%) and nausea/vomiting (2.5%) [103]. Hsieh et al. [104] conducted a retrospective study of 356 stage III and IV OCSCC patients. All the patients received curative surgery followed by chemoradiotherapy, which suggested a more advanced disease condition. A total of 114 patients received tegafur/uracil (tegafur: 100–400 mg/day) as adjuvant therapy for 1–2 years if tolerable. The survival endpoints revealed that 5-year overall survival (OS) and DFS were 65% versus 48% (HR = 0.54, *p* = 0.0008) and 57% versus 41% (HR = 0.62, *p* = 0.0034) in the metronomic and control groups, respectively. In addition, longer UFUR treatment duration (≥12 months) and higher dose (300–400 mg/day) contributed to better survival benefits. The metronomic UFUR treatment seemed to decrease the occurrence of distant metastasis (OR = 4.3, *p* = 0.0015) compared with no UFUR treatment. Moreover, the toxicities were mild and well-tolerated, with skin rash (3.5%) being the most common side-effect. Another study used low-dose S-1 as adjuvant chemotherapy in locally advanced HNSCC patients [105]. The study retrospectively reviewed 52 stage III/IVa/IVb HNSCC patients who completed curative treatment and confirmed no residual tumor. Sixteen out of 52 patients had oral cancer. The S-1 dose was half of the usual standard dose (40 mg/day for body surface area (BSA) < 1.25 m2; 50 mg/day for 1.25 m2 ≤ BSA < 1.5 m2; 60 mg/day for BSA ≥ 1.5 m2) and was administered for 2 years. Forty-three (82.7%) patients received S-1 continuously for 2 years without dose reduction. Hematological toxicity was found in 98.1% of the patients; however, only two (3.8%) patients were grade 3. Nonhematological toxicity was found in 15 (28.8%) patients, and all cases were grade 1. The 3year DFS and OS rates were 82.6% and 94%, respectively.

Three studies examined the effectiveness and toxicity profiles using metronomic therapy in recurrent/metastatic OCSCC patients. Patil et al. conducted a prospective randomized phase II clinical trial, comparing metronomic chemotherapy with single agent (three-weekly cisplatin (75 mg/m2)) in recurrent/metastatic HNSCC patients. The metronomic chemotherapy consisted of weekly oral methotrexate (MTX; 15 mg/m2) and daily celecoxib (200 mg twice daily) [106]. MTX is one of the commonly used chemotherapeutic agents in HNSCC [107]. Celecoxib, a nonsteroidal anti-inflammatory agent, was found to have an antiangiogenesis effect in preclinical experiments [108,109]. A total of 110 patients were enrolled in the study, with 57 randomized to the metronomic arm and 53 to the cisplatin arm. The primary oral cancer patients accounted for 75–77% of the cohort in both the groups. The metronomic arm had a significantly longer progression-free survival (PFS; median PFS: 101 vs. 66 days, *p* = 0.014) and OS (median OS: 249 vs. 152 days, *p* = 0.02) compared with the cisplatin arm. Moreover, the response rate (RR) and clinical beneficial rate were better in the metronomic arm compared with the cisplatin arm: 12.3% versus 1.9% and 54.4% versus 32.1%, respectively. Fewer grade 3–4 adverse events were noted in the metronomic arm compared with the cisplatin arm (18.9% vs. 31.4%, *p* = 0.14), without statistical significance [110]. Due to the promising results of the phase II study, the same group did an open-label, parallel-group, noninferiority, randomized phase 3 trial. The treatments of the experimental arm and control arm were still MTX plus celecoxib versus three-weekly cisplatin (75 mg/m2). Overall, 418 patients (211 in the oral metronomic chemotherapy group and 207 in the intravenous cisplatin group) were included in the per-protocol analysis. At a median follow-up of 15.73 months, the primary endpoint, median overall survival, was 7.5 months in the oral metronomic chemotherapy group compared with 6.1 months in the intravenous cisplatin group (HR ratio for death 0.775; 95% confidence interval 0.616–0.974, *p* =0.029). Grade 3 or higher adverse events were observed in 37(19%) of 196 patients in the oral metronomic chemotherapy group versus 61(30%) of 202 patients in the cisplatin group [111]. This was the first phase III study to demonstrate that metronomic chemotherapy resulted in better survival and a preferable toxicity profile in recurrent/metastatic head and neck cancer. Actually, the oral cavity cancers consisted of about 80% of the overall population. The study design and its results undoubtedly added a more solid evidence to the application of metronomic treatment in OCSCC. In another study, a single arm phase I/II trial, Patil et al. determined the dose and evaluated the efficacy of triple metronomic chemotherapy in platinum-refractory oral cancer patients [110]. The regimen included fixed dose erlotinib (an anti-EGFR tyrosine kinase inhibitor; 150 mg/day) and celecoxib (200 mg twice daily). In phase I, they first determined the optimal biological dose (OBD) of MTX by de-escalating from weekly 15 mg/m2 and found 9 mg/m2 to be the OBD. Overall, 91 patients were enrolled in the study. The median PFS and OS were 4.6 and 7.17 months, respectively. The overall best RR was 42.9%, which was superior to the second line treatment (RR = 2.2–13.6%) in platinum-refractory HN- SCC [18,19,107]. The most common grade 3–4 toxicity was hyponatremia (14.8%), followed by elevation of aspartate transaminase (AST) and alanine transaminase (ALT) levels (4.5% and 5.7%, respectively).

#### **5. Advantages and Limitations of Metronomic Therapy in OCSCC**

Our data review revealed that metronomic therapy does achieve comparable disease control rate in OCSCC and has a favorable toxicity profile compared with standard systemic chemotherapy, such as platinum. However, more solid and high-level evidence (e.g., randomized phase III trials) is required to consolidate the role of metronomic therapy. In addition, we found that the optimal metronomic dosage for OCSCC is not well defined. Pharmacodynamic and pharmacokinetic analysis of the metronomic dosing might provide valuable information for determining the optimal dose [112–114]. Considering the viewpoint of healthcare economics, metronomic therapy truly curtails the cost [115]. The reasons for cost reduction are as follows: (1) metronomic agents are not newly developed, (2) enteral administration is performed, and (3) less high-grade toxicities occur that might avoid additional supportive agents or admission. These advantages of metronomic therapy might relieve the soaring healthcare cost and facilitate patients in areas with insufficient medical resources [116]. Although the enteral route for metronomic agents brings convenience and at-home treatment, it should be noted that some OCSCC patients have difficulty in swallowing. This issue matters and affects patient compliance if metronomic chemotherapy is used as adjuvant/maintenance therapy in OCSCC, which might last for 1–2 years [103,104].

#### **6. Future Perspectives of Metronomic Therapy in OCSCC**

Antiangiogenesis and immunomodulation are the two main mechanisms of action of metronomic therapy [117]. As far as we understand, these two mechanisms might have synergistic effects with contemporary immunotherapy [118,119]. Some preliminary experiments using combined metronomic chemotherapy and vaccine or oncolytic virus in advanced cancers have been reported [120,121]. Although not in OCSCC, some ongoing clinical trials are studying the use of current immune checkpoint inhibitor and metronomic chemotherapy combination in lung cancer (NCT03801304), breast cancer (NCT03007992), pediatric tumors (NCT03585465), and sarcomas (NCT02406781). The results are eagerly awaited, and this strategy can hopefully be tested in OCSCC patients.

#### **7. Conclusions**

Oral squamous cell carcinoma is a heterogenous disease with a high unmet medical requirement. The cure rate and accompanied complications have not improved despite high-intensity treatment. Metronomic therapy provides an alternative treatment choice in contrast to traditional chemotherapy. Its mechanism of action is as follows: angiogenesis inhibition, immunomodulation, and overcoming drug resistance. A comparative disease control rate and superior toxicity profile are its advantages. It is widely applicable in clinical patient care. However, randomized phase III trials comparing it with the standard care are required to demonstrate its effectiveness. We can anticipate that more studies focusing on metronomic therapy with novel agent combination, such as immunotherapy, will be conducted in the future and translated into clinical treatment benefits.

**Author Contributions:** Conceptualization, N.-W.S. and Y.-J.C.; writing—original draft preparation, N.-W.S.; writing—review and editing, Y.-J.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Ministry of Science and Technology (grant number: MOST 109-2314-B-195-003-MY3) and MacKay Memorial Hospital (grant numbers: MMH-E-109-13, MMH-E-110-13, TTMMH-109-05, and TTMMH-110-01), Taiwan.

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

### **References**

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