Trial Watch: Immunogenic cell death inducers for anticancer chemotherapy

Abstract

The term “immunogenic cell death” (ICD) is now employed to indicate a functionally peculiar form of apoptosis that is sufficient for immunocompetent hosts to mount an adaptive immune response against dead cell-associated antigens. Several drugs have been ascribed with the ability to provoke ICD when employed as standalone therapeutic interventions. These include various chemotherapeutics routinely employed in the clinic (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, cyclophosphamide and oxaliplatin) as well as some anticancer agents that are still under preclinical or clinical development (e.g., some microtubular inhibitors of the epothilone family). In addition, a few drugs are able to convert otherwise non-immunogenic instances of cell death into bona fide ICD, and may therefore be employed as chemotherapeutic adjuvants within combinatorial regimens. This is the case of cardiac glycosides, like digoxin and digitoxin, and zoledronic acid. Here, we discuss recent developments on anticancer chemotherapy based on ICD inducers.

Introduction

Ten years ago, we were the first to introduce the term “immunogenic cell death” (ICD) to indicate a functionally peculiar type of apoptosis that – in immunocompetent hosts – can elicit an immune response against dead cell-associated antigens in the absence of any adjuvant.^1,2 Indeed, the subcutaneous inoculation of cancer cells succumbing to doxorubicin (an anthracycline approved by regulatory agencies for the treatment of several tumors, see below) in vitro was sufficient to protect syngeneic mice against a re-challenge with malignant cells of the same type, but not with cancer cells of distinct origin.² Subsequent studies by us and others identified various mechanisms that underlie not only the ability of a specific stimulus to trigger bona fide ICD as opposed to a non-immunogenic instance of apoptosis, but also the capacity of the host to detect ICD and hence mount a therapeutically relevant immune response against dying cells.^1,3-6

Schematically, ICD itself relies on the coordinated emission of a series of damage-associated molecular patterns (DAMPs),^7-12 including the exposure of endoplasmic reticulum (ER) chaperones on the cell surface, the secretion of ATP and the release of the non-histone chromatin-binding protein high mobility group box 1 (HMGB1),^13-20 and immunostimulatory cytokines, such as type I interferons.²¹ When emitted in the correct spatiotemporal pattern,^22-24 such DAMPs recruit antigen-presenting cells, including dendritic cells, to the site of ICD and activate them to engulf dead cell-associated antigens, process and present them to CD4⁺ and CD8⁺ T lymphocytes in the context of co-stimulatory signals, resulting in the priming of a robust, antigen-specific immune response.^25-31 In line with this notion, the ability of cancer cells undergoing ICD to elicit a protective immune response upon inoculation to syngeneic mice is abrogated: (1) when the molecular pathways underlying the emission of the abovementioned DAMPs are pharmacologically or genetically inhibited in malignant cells;^13,32,33 as well as (2) in mice affected by relatively generalized forms or immunodeficiency or lacking specific components of the DAMP-sensing machinery, such as Toll-like receptor 4 (Tlr4) or type I interferon (α and β) receptor 1 (Ifnar1).^15,21,34 A more detailed description of these signal transduction pathways and cellular circuitries goes beyond the scope of this Trial Watch and can be found in several recent reviews.^1,3,4

Importantly, some – but not all – cell death inducers are capable of eliciting ICD,³⁵ and this property cannot be anticipated by structural or functional considerations.^3,36-38 Indeed, while cisplatin and oxaliplatin both exert cytostatic/cytotoxic effects as they induce inter- and intra-strand DNA adducts,^39-42 only the latter triggers bona fide ICD as it provokes a pre-mortem ER stress response.^43,44 Thus, although assays for the detection of surrogate ICD markers are available,⁴⁵ the gold standard approach for determining whether a cytotoxic intervention provokes bona fide ICD still relies on vaccination experiments involving murine cancer cells and syngeneic, immunocompetent mice.³ In addition, the ability of a specific stimulus to induce ICD can be inferred by testing its antineoplastic effects on tumors established in immunocompetent versus immunodeficient hosts.³ However, this approach cannot replace vaccination experiments as several therapeutic agents mediate optimal antineoplastic effects in immunocompetent hosts only as they have an off-target immunostimulatory activity but do not induce ICD.^46-48

So far, only a few stimuli have been ascribed with the ability to trigger ICD, encompassing both chemical and physical agents.^3,36 Interestingly, such bona fide ICD inducers include various anticancer chemotherapeutics that have been successfully employed in the clinic for several years (Table 1), like (1) doxorubicin, an anthracycline approved by the US Food and Drug Administration (FDA) for the treatment of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), breast carcinoma, gastric cancer, lymphoma, multiple myeloma (MM), neuroblastoma, ovarian carcinoma, small cell lung carcinoma, soft tissue and bone sarcomas, thyroid carcinoma, transitional cell bladder carcinoma and Wilms' tumor;^2,49 (2) epirubicin, an anthracycline licensed for use in breast carcinoma patients;^2,49 (3) idarubicin, an anthracycline currently employed for the treatment of AML;^19,49 (4) mitoxantrone, an anthracenedione licensed for use in individuals with AML, breast carcinoma, non-Hodgkin's lymphoma (NHL) and prostate carcinoma;^2,49 (5) bleomycin, a glycopeptide antibiotic commonly employed as a palliative treatment for Hodgkin's lymphoma, NHL, penile cancer, testicular cancer, and squamous carcinomas of the head and neck, cervix and vulva;⁵⁰ (6) bortezomib, a proteasomal inhibitor approved for use in subjects with MM and mantle cell lymphoma;^17,51,52 (7) cyclophosphamide, an alkylating agent nowadays employed for the treatment of ALL, AML, chronic lymphocytic leukemia, breast carcinoma, chronic myeloid leukemia (CML), lymphoma, MM, mycosis fungoides, neuroblastoma, ovarian carcinoma and retinoblastoma;⁵³ and (8) oxaliplatin, a platinum derivative approved for use in combination with 5-fluorouracil and folinic acid for the therapy of advanced colorectal carcinoma.^40,44,54 Moreover, at least in some cell types, ICD can be provoked by patupilone, an experimental microtubular inhibitor of the epothilone family,^55-57 and by 7A7, a monoclonal antibody (mAb) targeting the murine epidermal growth factor receptor (EGFR).^58,59 However, for the reasons mentioned above, FDA-approved epothilones (i.e., ixabepilone, which is licensed for the treatment of breast carcinoma)⁶⁰ and EGFR-targeting mAbs (i.e., cetuximab and panitumumab, which are currently employed for the treatment of head and neck cancer and colorectal carcinoma)^61-63 may not share this ability with patupilone and 7A7, respectively. Finally, it should be noted that some FDA-approved agents such as digoxin and digitoxin (which are licensed for the treatment of various cardiac disorders),⁶⁴ as well as zoledronic acid (which is commonly employed for the treatment of MM or hypercalcemia and bone lesions of oncological origin),⁶⁵ are very efficient at boosting the immunogenicity of otherwise non-immunogenic instances of cell death, although they are unable to elicit ICD per se.^66-69 These agents may be particularly relevant for the development of combinatorial chemotherapeutic regimens that actively engage the host immune system against malignant cells.

Table 1.

Immunogenic cell death inducers currently approved for cancer chemotherapy*

Drug	First approved	Indication(s)		Ref.
Bleomycin	<1995	HNSCC Lymphoma Penile cancer	SCC of the cervix SCC of the vulva Testicular cancer	50
Bortezomib	2003	Mantle cell lymphoma	Multiple myeloma	17,51,52
Cyclophosphamide	<1995	Breast carcinoma Leukemia Lymphoma Multiple myeloma	Mycosis fungoides Neuroblastoma Ovarian carcinoma Retinoblastoma	53
Doxorubicin	<1995	ALL AML Bladder carcinoma Bone sarcoma Breast carcinoma Gastric carcinoma Lymphoma	Multiple myeloma Neuroblastoma Ovarian carcinoma SCLC Soft tissue sarcoma Thyroid carcinoma Wilms' tumor	2,49
Epirubicin	1999	Breast carcinoma		2,49
Idarubicin	<1995	AML		19,49
Mitoxantrone	<1995	AML Breast carcinoma	NHL Prostate carcinoma	2,49
Oxaliplatin	1996	Colorectal carcinoma		44,54

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; HNSCC, head and neck squamous cell carcinoma; NHL, non-Hodgkin's lymphoma; SCC, squamous cell carcinoma; SCLC, small cell lung carcinoma; *by the US Food and Drug Administration or equivalent agency worldwide.

In the context of our monthly series,^70-72 this Trial Watch discusses recent developments on anticancer chemotherapy with ICD inducers. In line with this notion, irradiation and photodynamic therapy, 2 additional interventions that trigger bona fide ICD and are commonly employed for the treatment of several neoplasms,^73-82 will not be considered further here.

Update on the Development of ICD-Inducing Chemotherapeutics

Completed clinical trials. On 2015, Jan 6^th querying PubMed with the string “cancer AND (patients OR trial) AND (doxorubicin OR epirubicin OR idarubicin OR mitoxantrone OR bortezomib OR bleomycin OR cyclophosphamide OR oxaliplatin)” returned 48,701 entries, some 2,000 of which were published since the submission of our latest Trial Watch dealing with ICD-inducing chemotherapeutics (January 2014).⁸³ This figure obviously covers a number of preclinical research papers, review articles and editorials that is difficult to quantify with precision. Moreover, in a significant fraction of the clinical articles included in this figure, doxorubicin, epirubicin, idarubicin, mitoxantrone, bortezomib, bleomycin, cyclophosphamide and oxaliplatin are employed as part of standard chemotherapeutic regimens, in on-label indications (source (http://www.ncbi.nlm.nih.gov/pubmed). Among the clinical studies testing the safety and efficacy of ICD-inducing chemotherapeutics employed as off-label indications, we would like to highlight the works of: (1) Butts and collaborators (Cross Cancer Institute; Edmonton, Canada), who reported that the therapeutic activity of a tumor-targeting vaccine administered to non-small cell lung carcinoma (NSCLC) patients previously receiving cyclophosphamide-based chemotherapy may be influenced by the administration schedule (i.e., sequential vs. concurrent) of the latter;⁸⁴ (2) Roulstone and colleagues (The Institute of Cancer Research; London, UK), who demonstrated that the pre-administration of high-dose cyclophosphamide to individuals with solid tumors is unable to prevent the development of humoral, neutralizing immunity against oncolytic reoviruses;⁸⁵ (3) Bazzola and co-authors (Azienda Istituti Ospitalieri di Cremona; Cremona, Italy), who tested metronic cyclophosphamide combined with letrezole (a non-steroidal aromatase inhibitor) and sorafenib (a multi-targeted kinase inhibitor)^86,87 in primary breast cancer patients, with promising results;⁸⁸ (4) Lutz et al. (The Sidney Kimmel Cancer Center; Baltimore, MD, US), who demonstrated that low-dose cyclophosphamide converts the tolerogenic microenvironment of pancreatic adenocarcinoma into an immunogenic one, boosting the clinical activity of an irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic vaccine;^89-92 (5) Zheng and collaborators (Johns Hopkins University School of Medicine; Baltimore, MD, US), who – along similar lines – proved the capacity of low-dose cyclophosphamide to support the therapeutic activity of a vaccine composed of irradiated, allogeneic human colorectal cancer cells and GM-CSF-producing bystander cells;⁹³ (6) Hong and colleagues (University of Ulsan College of Medicine; Seoul, South Korea), who reported that, as compared to adjuvant folinic acid and 5-fluorouracil, adjuvant FOLFOX (folinic acid plus 5-fluorouracil plus oxaliplatin) is associated with an improved disease-free survival among patients with locally advanced rectal cancer after preoperative chemoradiotherapy and total mesorectal excision;⁹⁴ (7) Noh and co-workers (Yonsei University College of Medicine; Seoul, South Korea) and Yamada et al. (National Cancer Center Hospital; Tokyo, Japan), who demonstrated the clinical efficacy of oxaliplatin in combination with capecitabine or S-1, respectively, in patients with gastric carcinoma;^95,96 (8) Oettle and collaborators (Charitě Universitätsmedizin; Berlin, Germany) and O'Reilly and colleagues (Memorial Sloan-Kettering Cancer Center, New York, NY, US), who provided evidence in support of the therapeutic activity of oxaliplatin as part of neoadjuvant chemotherapeutic regimens for patients with refractory or chemotherapy-naïve pancreatic carcinoma;^97,98 (9) Straus and co-authors (Memorial Sloan-Kettering Cancer Center, New York, NY, US), who reported that the administration of liposomal doxorubicin to subjects with cutaneous T-cell lymphoma is associated with an objective responses rate that is among the highest ever reported for similar patient cohorts, while the subsequent application of bexarotene (a retinoid)^99-102 has negligible effects on response rate and duration.¹⁰³

Preclinical and translational advances. Within the abundant preclinical literature that has been published during the last 13 months on ICD-inducing chemotherapeutics, we found of particular interest the works of: (1) Pallasch and colleagues (Massachusetts Institute of Technology; Cambridge, MA, US), who demonstrated that cyclophosphamide induces an acute secretory phenotype in malignant cells, stimulating the release of various immunostimulatory cytokines that promotes a macrophage-driven, tumor-targeting innate immune response;^104-106 (2) Tavora and collaborators (Barts Cancer Institute; London, UK), who showed that the inhibition of protein tyrosine kinase 2 (PTK2, also known as FAK)^107-109 in the endothelial tumor compartment sensitizes cancer cells to doxorubicin-based chemotherapy;¹¹⁰ (3) Cottini and co-authors (Harvard Medical School; Boston, MA, US), who mechanistically involved the Hippo co-activator Yes-associated protein 1 (YAP1)^111-114 in the response of hematological tumors to DNA-damaging agents, including doxorubicin;^112,115 (4) Ichikawa et al. (Northwestern University School of Medicine; Chicago, Illinois, US) and Liu and colleagues (Harvard Medical School; Boston, MA, US), who demonstrated that the accumulation of iron within mitochondria^8,116-118 and consequent alterations in the activity of enzymes of the Krebs' cycle contribute to the cardiotoxicity of doxorubicin;^119,120 (5) Viaud and co-authors (Gustave Roussy Cancer Campus; Villejuif, France) and Iida and collaborators (National Cancer Institute; Frederick, MD, US), who proved that specific changes in the gut microbiota^121-124 induced by cyclophosphamide and oxaliplatin are responsible for their full-blown therapeutic activity as they favor the elicitation of anticancer immune responses;^125-127 (6) Morton et al. (Massachusetts Institute of Technology; Cambridge, MA, US), who developed a nanoparticle-based chemotherapy delivery system¹²⁸ that allows for the finely controlled release of up to 2 drugs, a vehicle that may be particular relevant for promoting ICD;¹²⁹ (7) Sistigu and co-authors (Gustave Roussy Cancer Campus; Villejuif, France), who demonstrated that the release of type I interferons from dying cancer cells is required for the host immune system to perceive such event as immunogenic and mount an adaptive immune response against dead cell-associated antigens;²¹ and Triulzi and collaborators (Fondazione IRCCS Istituto Nazionale dei Tumori; Milan, Italy), who identified a role for serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5, best known as maspin)^130-133 in the establishment of a collagen-enriched tumor microenvironment contributing to the resistance of (at least a subset of) breast carcinomas to doxorubicin.¹³⁴

Recently initiated clinical trials. Since the submission of our latest Trial Watch dealing with this topic (January 2014),⁸³ no less than 374 clinical studies involving ICD-inducing chemotherapeutics have been initiated (doxorubicin = 75 studies; epirubicin = 23 studies; idarubicin = 18 studies; mitoxantrone = 14 studies; bleomycin = 8 studies; bortezomib = 25 studies; cyclophosphamide = 128 studies; oxaliplatin = 83 studies). In the vast majority of these trials (250 studies), however, ICD inducers are employed as on-label therapeutic interventions, most often as (part of) the gold standard chemotherapeutic regimen given to the control arm of the study. These studies will not be discussed further here. In addition, no less than 125 clinical trials have recently been initiated to test the therapeutic profile of doxorubicin (14 studies), epirubicin (8 studies), idarubicin (3 studies), mitoxantrone (4 studies), bleomycin (2 studies), bortezomib (7 studies), cyclophosphamide (34 studies), and oxaliplatin (53 studies) employed as off-label chemotherapeutic interventions (source http://clinicaltrials.gov/).

In particular, doxorubicin is being tested in subjects with (1) breast carcinoma, who receive the drug in pegylated liposomal formulation combined with carboplatin, a cisplatin derivative approved for the treatment of NSCLC and ovarian carcinoma,^135,136 and paclitaxel, a microtubular inhibitor often employed in women with breast carcinoma^137,138 (NCT02315196); (2) hepatocellular carcinoma (HCC), most often in the context of transcatheter arterial chemoembolization (TACE)¹³⁹ (NCT02038296; NCT02070419; NCT02112656; NCT02125396; NCT02141906; NCT02147301; NCT02149771; NCT02182687; NCT02240771); (3) hepatic metastases from other solid tumors, again in liposomal formulation (NCT02181075); (4) melanoma, who receive doxorubicin as a standalone therapeutic intervention (NCT02094872); (5) MM, in the context of a multimodal chemoimmunotherapeutic regimen involving the immunomodulatory drug thalidomide^140,141 (NCT02128230); and (6) peritoneal carcinomatosis, who are treated with doxorubicin plus cisplatin as pressurized intraperitoneal aerosol chemotherapy (NCT02320448). The therapeutic profile of epirubicin is being evaluated in patients with (1) bladder carcinoma, who receive epirubicin as a standalone intravesical chemotherapeutic (NCT02214602); (2) gastric or gastroesophageal carcinoma, invariably as part of the so-called EOX regimen (epirubicin plus oxaliplatin plus capecitabine)¹⁴² (NCT02128243; NCT02177552; NCT02158988); (3) HCC, in the context of TACE (NCT02220088); (4) MM, as part of induction or tumor-reduction chemotherapy followed by stem cell mobilization and consolidation chemotherapy (NCT02288741); and (5) soft tissue sarcoma, who receive epirubicin in combination with conventional chemotherapeutics of trabectedin, a macrophage-repolarizing agent^143,144 (NCT02050919; NCT02066675). The clinical activity of idarubicin is being assessed in individuals with (1) CML, who receive idarubicin plus cladribine and cytarabine (2 inhibitors of nucleotide metabolism currently approved for the treatment of various forms of leukemia)¹⁴⁵ (NCT02115295); (2) myelodysplastic syndrome, in the context of cytarabine-based chemotherapy and donor lymphocyte infusion^146,147 (NCT02046122); and (3) HCC, who receive idarubicin in the form of drug-loaded microbeads (NCT02185768). Mitoxantrone is being tested in patients with: (1) ALL, in the context of combinatorial chemotherapeutic regimen (NCT02101853; NCT02303821); (2) lymphoma, who are treated with mitoxantrone as a single agent (NCT02131688); and (3) various solid tumors, who also receive mitoxantrone as standalone therapeutic intervention (NCT02043756). The clinical activity of bleomycin is being investigated in subjects with: (1) HCC, in the context of electrochemotherapy (NCT02291133); and (2) non-seminomatous malignant germ cell tumors, who receive bleomycin in combination with cisplatin-based chemotherapy (NCT02104986). The efficacy of bortezomib is being assessed in individuals with (1) various hematological malignancies, who often receive bortezomib as part of multimodal chemo- or immunotherapeutic regimens (NCT02037256; NCT02112916; NCT02208037; NCT02312102); (2) neuroblastoma, who are treated with bortezomib plus difluoromethylornithine (a hitherto experimental inhibitor of polyamine biosynthesis),^148,149 (NCT02139397); and (3) various solid tumors, who receive bortezomib as standalone therapeutic agent or combined with standard chemotherapy (NCT02211755; NCT02220049) (Table 2).

Table 2.

Clinical trials recently started to evaluate the therapeutic profile of doxorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin or bortezomib as off-label chemotherapeutic interventions*

Drug	Indication(s)	Phase	Status	Notes	Ref.
Doxorubicin	Breast carcinoma	II	Not yet recruiting	As PLD, combined with carboplatin and paclitaxel	NCT02315196
HCC	n.a.	Recruiting	In the context of TACE	NCT02141906
II	Active, not recruiting	In the context of TACE	NCT02182687
Not yet recruiting	In the context of TACE	NCT02147301
Withdrawn	In the context of TACE	NCT02070419
II/III	Recruiting	In the context of TACE	NCT02240771
III	Completed	In the context of TACE	NCT02038296
Not yet recruiting	In the context of TACE	NCT02125396
Recruiting	As thermosensitive liposomal doxorubicin	NCT02112656
In the context of TACE	NCT02149771
Hepatic metastases	I	Recruiting	As thermosensitive liposomal doxorubicin	NCT02181075
Melanoma	II	Recruiting	As single agent	NCT02094872
Multiple myeloma	II	Active, not recruiting	Combined with multimodalchemoimmunotherapy	NCT02128230
Peritoneal carcinomatosis	II	Not yet recruiting	Combined with cisplatin as PIPAC	NCT02320448
Epirubicin	Bladder carcinoma	IV	Recruiting	As intravesical standalone chemotherapeutic	NCT02214602
Gastric or gastroesophageal carcinoma	II	Recruiting	EOX regimen	NCT02177552
EOX plus cisplatin and S-1	NCT02128243
III	Recruiting	EOX regimen ± mitomycin C and cisplatin	NCT02158988
HCC	II/III	Recruiting	In the context of TACE	NCT02220088
Multiple myeloma	III	Completed	As a part of induction or tumor reduction chemotherapy followed by stem cell mobilization	NCT02288741
Soft tissue sarcoma	II	Recruiting	Combined with ifosfamide, sorafenib and RT	NCT02050919
Combined with trabectedin	NCT02066675
Idarubicin	AMLCML	II	Recruiting	Combined with cladribine and cytarabine	NCT02115295
AMLMDS	I	Recruiting	Combined with cytarabine and DLI	NCT02046122
HCC	II	Active, not recruiting	As idarubicin-loaded microbeads	NCT02185768
Mitoxantrone	ALL	I/II	Not yet recruiting	As part of combinatorial chemotherapy	NCT02303821
	III	Recruiting	As part of combinatorial chemotherapy	NCT02101853
Lymphoma	I	Recruiting	As single agent	NCT02131688
Solid tumors	I	Completed	As single agent	NCT02043756
Bleomycin	Liver cancer	I/II	Recruiting	Combined with electrochemotherapy	NCT02291133
NSMGCT	II	Not yet recruiting	Combined with cisplatin-based chemotherapy	NCT02104986
Bortezomib	Hematological malignancies	n.a.	Active, not recruiting	Combined with G-CSF in promoting stem cell mobilization	NCT02037256
I	Not yet recruiting	Combined with lenalidomide	NCT02312102
II	Recruiting	Combined with methotrexate and tacrolimus	NCT02208037
III	Recruiting	As part of combinatorial chemotherapy	NCT02112916
Neuroblastoma	I/II	Recruiting	Combined with difluoromethylornithine	NCT02139397
Solid tumors	I	Recruiting	Combined with clofarabine	NCT02211755
Completed	As single agent	NCT02220049

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; DLI, donor lymphocyte infusion; EOX, epirubicin plus oxaliplatin plus capecitabine; G-CSF, granulocyte colony-stimulating factor; HCC, hepatocellular carcinoma; MDS, myelodysplastic syndrome; n.a., not available; NSMGCT, non-seminomatous malignant germ cell tumor; PIPAC, pressurized intraperitoneal aerosol chemotherapy; PLD, pegylated liposomal doxorubicin; RT, radiation therapy; TACE, transcatheter arterial chemoembolization. *initiated after 2014, January 1^st.

The efficacy of cyclophosphamide as an off-label therapeutic intervention is being evaluated in subjects with: (1) colorectal carcinoma, often in the context of capecitabine-based chemotherapy^150,151 (NCT02271464; NCT02280694; NCT02298946); (2) medulloblastoma, who often are treated with cyclophosphamide plus a chemotherapeutic regimen based on cisplatin (NCT02066220; NCT02212574); (3) melanoma, to whom cyclophosphamide is administered as part of a lymphodepleting treatment followed by adoptive cell transfer^152-154 (NCT02062359; NCT02111863; NCT02278887); NSCLC, as part of various chemo- or immunotherapeutic regimens (NCT02049151; NCT02117024; NCT02133196; NCT02187367); (4) soft tissue sarcoma, as part of multimodal chemoimmunotherapy (NCT02059850; NCT02234050; NCT02306161); as well as breast carcinoma (NCT02276300), gastric carcinoma (NCT02276300; NCT02317471), ependydoma (NCT02265770), osteosarcoma (NCT02273583), pancreatic carcinoma (NCT02243371), prostate carcinoma (NCT02234921), testicular cancer (NCT02161692), germ cell tumors (NCT02161692), rhabdoid malignancies (NCT02114229), and various other solid tumors (NCT02054104; NCT02070406; NCT02096614; NCT02107963; NCT02111850; NCT02153905; NCT02159716; NCT02210104; NCT02223312; NCT02224599; NCT02280811), in the context of a heterogeneous panel of chemo-, radio- or immunotherapeutic regimens (Table 3).

Table 3.

Clinical trials recently started to evaluate the therapeutic profile of cyclophosphamide as an off-label chemotherapeutic intervention*

Indication(s)	Phase	Status	Notes	Ref.
Breast carcinoma Gastric carcinoma	I	Recruiting	Combined with GM-CSF, a peptide-based anticancer vaccine and imiquimod	NCT02276300
Colorectal carcinoma	I	Recruiting	Combined with a checkpoint blocker and RT	NCT02298946
II	Recruiting	As metronomic regimen combined with bevacizumab, capecitabine and FOLFOXIRI	NCT02271464
Not yet recruiting	Combined with capecitabine, celecoxib and methotrexate	NCT02280694
Ependymoma	II/III	Not yet recruiting	Combined with conventional chemotherapy and RT	NCT02265770
Gastric carcinoma	I/II	Recruiting	Combined with a HSP-based vaccine, oxaliplatin and S-1	NCT02317471
Germ cell tumorsTesticular cancer	II	Completed	Combined with cisplatin, etoposide and bleomycin ± carboplatin	NCT02161692
Medulloblastoma	II/III	Recruiting	Combined with conventional chemotherapy and RT	NCT02066220
n.a.	Not yet recruiting	Combined with conventional chemotherapy	NCT02212574
Melanoma	II	Recruiting	As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02062359
NCT02111863
III	Recruiting	As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02278887
NSCLC	II	Recruiting	As metronomic regimen combined witha cancer cell-based vaccine	NCT02117024
As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02133196
III	Active, not recruiting	Combined with tecemotide	NCT02049151
Not yet recruiting	Combined with an EGF-targeting vaccine	NCT02187367
Osteosarcoma	II	Recruiting	Combined with methotrexate	NCT02273583
Pancreatic carcinoma	II	Recruiting	Combined with multimodal immunotherapy	NCT02243371
Prostate cancer	I	Recruiting	Combined with imiquimod and a peptide-based anticancer vaccine	NCT02234921
Rhabdoid tumors	II	Recruiting	As a part of induction chemotherapyfollowed by alisertib and RT	NCT02114229
Soft tissue sarcoma	I	Recruiting	As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02059850
II	Not yet recruiting	Combined with multimodal immunochemotherapy	NCT02234050
Recruiting	Combined with multimodal immunochemotherapy	NCT02306161
Solid tumors	I	Not yet recruiting	As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02210104
Recruiting	As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02070406
NCT02096614
Combined with GD2-specificCAR-expressing T cells	NCT02107963
Combined with mesothelin-specificCAR-expressing T cells	NCT02159716
I/II	Not yet recruiting	Combined with GM-CSF and TAA-pulsed DCs	NCT02223312
	NCT02224599
Recruiting	As metronomic chemotherapy combined with celecoxib and followed by lysate-based vaccine	NCT02054104
As part of a conditioning regimen followed by adoptive cell transfer-based immunotherapy	NCT02111850
NCT02153905
NCT02280811

Abbreviations: CAR, chimeric antigen receptor; DC, dendritic cell; EGF, epidermal growth factor; FOLFOXIRI, folinic acid plus 5-fluorouracil plus oxaliplatin plus irinotecan; GM-CSF, granulocyte macrophage colony stimulating factor; HSP, heat shock protein; n.a., not available; NSCLC, non-small cell lung carcinoma; RT, radiation therapy; TAA, tumor-associated antigen. *initiated after 2014, January 1^st.

The clinical profile of off-label oxaliplatin is being assessed in patients with: (1) gastric, gastroesophageal or gastrointestinal carcinomas, most often in the context of either the so-called XELOX (capecitabine plus oxaliplatin)^155,156 or FOLFOX (folinic acid plus 5-fluorouracil plus oxaliplatin)^157,158 regimen (NCT02038621; NCT02048540; NCT02071043; NCT02114359; NCT02158988; NCT02191566; NCT02215837; NCT02226380; NCT02240524; NCT02250209; NCT02269904; NCT02289378; NCT02301481; NCT02317471; NCT02322593; NCT02037048; NCT02128243; NCT02177552; NCT02193594; NCT02213289; NCT02216149; NCT02241499; NCT02273713; NCT02287129; NCT02296671; NCT02102022; NCT02233205; NCT02268825; NCT02319018); (2) HCC, who often receive oxaliplatin combined with other conventional chemotherapeutics or with targeted anticancer agents (NCT02069041; NCT02089633; NCT02129322; NCT02149771); (3) lymphoma, invariably in the context of the so-called GEMOX (gemcitabine plus oxaliplatin) regimen^159,160 (NCT02080234; NCT02085655; NCT02181218); (4) pancreatic carcinoma, most frequently as part of the so-called FOLFIRINOX (folinic acid plus 5-fluorouracil plus irinotecan plus oxaliplatin) regimen^161,162 (NCT02035072; NCT02047474; NCT02080221; NCT02109341; NCT02125136; NCT02128100; NCT02143219; NCT02148549; NCT02172976; NCT02178709; NCT02195180; NCT02241551; NCT02243358; NCT02244489; NCT02311439); (5) breast carcinoma, who receive oxaliplatin as a standalone therapeutic agent (NCT02077998); and (6) biliary tract or gallbladder carcinoma, who are treated with the GEMOX regimen plus a MEK inhibitor¹⁶³ (NCT02105350) (Table 4).

Table 4.

Clinical trials recently started to evaluate the therapeutic profile of oxaliplatin as an off-label chemotherapeutic intervention*

Indication(s)	Phase	Status	Notes	Ref.
Biliary tract carcinoma Gallbladder carcinoma	I	Not yet recruiting	GEMOX regimen plus MEK inhibitor	NCT02105350
Breast carcinoma	0	Recruiting	As single agent	NCT02077998
Gastric cancer	I/II	Completed	FLOT regimen combined withgastrectomy ± bevacizumab	NCT02048540
Recruiting	Combined with a HSP-based vaccine, cyclophosphamide and S-1	NCT02317471
II	Completed	XELOX regimen	NCT02071043
Not yet recruiting	XELOX regimen plus paclitaxel	NCT02038621
SOX regimen	NCT02191566
Recruiting	FOLFOX regimen combined with autologous tumor lysate-pulsed DCs and CIK cells	NCT02215837
FOLFOX regimen	NCT02226380
XELOX regimen plus trastuzumab	NCT02250209
XELOX regimen	NCT02269904
FLOT regimen	NCT02289378
SOX regimen ± radiotherapy	NCT02301481
III	Not yet recruiting	Combined with TAS-118	NCT02322593
Recruiting	FOLFOX or XELOX regimen	NCT02114359
EOX regimen ± cisplatin and mitomycin C	NCT02158988
XELOX regimen plus D2 lymphadenectomy	NCT02240524
Gastroesophageal cancer	I/II	Not yet recruiting	FOLFOX or FLOT or FOLFIRI regimen combined with tumor-targeting antibodies	NCT02213289
Recruiting	XELOX regimen plus paclitaxel	NCT02273713
II	Not yet recruiting	SOX or XELOX regimen	NCT02216149
FOLFOX or PEMOX regimen	NCT02296671
Recruiting	FOLFOX regimen combined with RT± carboplatin and paclitaxel	NCT02037048
EOX or FOLFOX regimen pluscisplatin and S-1	NCT02128243
EOX regimen	NCT02177552
Combined with 5-FU and RT	NCT02241499
Combined with capecitabine, carboplatin, epirubicin, 5-FU, paclitaxel and RT	NCT02287129
II/III	Recruiting	SOX regimen ± radiotherapy	NCT02193594
Gastrointestinal cancer	I	Not yet recruiting	FOLFOX regimen plus alisertib	NCT02319018
Recruiting	FOLFOX regimen plus arginine deiminase	NCT02102022
I/II	Not yet recruiting	FOLFOX regimen plus pembrolizumab	NCT02268825
Recruiting	XELOX regimen plus gemcitabine	NCT02233205
Hepatocellular carcinoma	I	Recruiting	FOLFOX regimen plus ramucirumab	NCT02069041
II	Not yet recruiting	SOX regimen plus sorafenib	NCT02129322
Recruiting	PACOX regimen	NCT02089633
III	Recruiting	Combined with doxorubicin for TACE	NCT02149771
Lymphoma	I	Not yet recruiting	GEMOX regimen plus dexamethasone and G-CSF	NCT02181218
II	Recruiting	GEMOX regimen plus asparaginase plus RT	NCT02080234
III	Recruiting	GEMOX regimen plus asparaginase	NCT02085655
Pancreatic carcinoma	I	Recruiting	FIRINOX regimen	NCT02148549
XELOX regimen plus momelotinib	NCT02244489
I/II	Recruiting	FOLFOX regimen plus paclitaxel	NCT02109341
II	Not yet recruiting	FOLFIRINOX regimen plus RT	NCT02128100
FOLFIRINOX regimen	NCT02143219
FOLFOX regimen plus encapsulated asparaginase	NCT02195180
Recruiting	GEMOX regimen plus RT	NCT02035072
FOLFIRINOX regimen	NCT02047474
FOLFOX regimen plus abraxane	NCT02080221
FOLFIRINOX regimen plus gemcitabine and paclitaxel	NCT02125136
	NCT02241551
FOLFIRINOX regimen	NCT02178709
FOLFOX regimen plus gemcitabine and RT	NCT02243358
II/III	Not yet recruiting	FOLFIRINOX regimen plus natriumfolinate	NCT02172976
Recruiting	FOLFIRINOX regimen plus capecitabine and RT	NCT02311439

Abbreviations: 5-FU, 5-fluorouracil; CIK, cytokine-inducer killer; DC, dendritic cell; EOX, epirubicin plus oxaliplatin plus capecitabine; FIRINOX, 5-FU plus irinotecan plus oxaliplatin; FLOT, 5-FU plus oxaliplatin plus docetaxel; FOLFIRI, folinic acid plus 5-FU plus irinotecan; FOLFIRINOX, folinic acid plus 5-FU plus irinotecan plus oxaliplatin; FOLFOX, folinic acid plus 5-FU plus oxaliplatin; G-CSF, granulocyte colony-stimulating factor; GEMOX, gemcitabine plus oxaliplatin; HSP, heat shock protein; PACOX, pegylated human arginase plus XELOX; PEMOX, pemetrexed plus oxaliplatin; RT, radiation therapy; SOX, S-1 plus oxaliplatin; TACE, transcatheter arterial chemoembolization; XELOX, capecitabine plus oxaliplatin. *initiated after 2014, January 1^st.

Of note, the vast majority of these studies is ongoing, with a few notable exceptions. Thus, NCT02070419, a Phase II study investigating the therapeutic profile of doxorubicin-based TACE alone or combined with radiation therapy in HCC patients, has been withdrawn as the principal investigator left the institution. Moreover, NCT02038296, a Phase III trial testing different protocols for doxorubicin-based TACE in HCC patients, NCT02043756, a Phase I study investigating the pharmacokinetic, safety and preliminary efficacy of a peculiar pegylated variant of mitoxantrone given as a standalone therapeutic intervention to subjects with solid tumors, NCT02048540, a Phase I/II study testing oxaliplatin in combination with gastrectomy plus 5-fluorouracil, docetaxel and optional bevacizumab in subjects with gastric carcinoma, NCT02071043, a Phase II trial investigating the therapeutic profile of oxaliplatin plus capecitabine in individuals with gastric carcinoma, NCT02161692, a Phase II study testing cyclophosphamide combined with cisplatin, etoposide, bleomycin and optional carboplatin in patients with germ cell tumors or testicular cancer, NCT02220049, a Phase I trial investigating the safety and efficacy of bortezomib-based chemotherapy in subjects with solid tumors, and NCT02288741, a Phase III study testing epirubicin as part of induction or tumor reduction chemotherapy followed by stem cell mobilization in MM patients, have all been already completed (source http://clinicaltrials.gov/). The results of NCT02043756, which suggest that a pegylated variant of mitoxantrone is well tolerated by patients with solid tumors up to a dose of 18 mg/m² and may exert clinical efficacy,¹⁶⁴ and NCT02161692, which failed to meet the primary end point,¹⁶⁵ have already been published. Conversely, to the best of our knowledge, the results of NCT02038296, NCT02048540, NCT02071043, NCT02220049, and NCT02288741 have not been released yet.

Concluding Remarks

As discussed above, a bunch of clinically employed chemotherapeutics are able to trigger an immunogenic variant of apoptosis that – in immunocompetent hosts – triggers an adaptive immune response against dead cell-associated antigens.^3,36 Since these ICD inducers are not only approved by international regulatory agencies for use in subjects with various hematological and solid neoplasms, but also are part of consolidated therapeutic protocols, safety concerns are generally limited. Thus, these molecules are frequently included in clinical trials as (part of) the therapeutic regimen(s) administered to the control arm of the study. Moreover, FDA-approved ICD inducers are often investigated in off-label oncological indications, either as standalone therapeutic interventions or combined with other chemo-, radio- or immunotherapies. Consequently, a huge number of clinical studies involving chemical ICD inducers are initiated yearly.

Now, great efforts are being devoted to the development of combinatorial regimens relying on the co-administration of conventional or targeted anticancer agents plus one form of immunotherapy.^48,166 In this setting, it is tempting to hypothesize that the clinical profile of anticancer chemotherapy based on ICD inducers may be considerably ameliorated by the concomitant administration of various immunostimulatory interventions,¹⁶⁷ in particular checkpoint blockers such as the cytotoxic T lymphocyte-associated protein 4 (CTLA4)-targeting mAb ipilimumab and the programmed cell death 1 (PDCD1)-targeting mAbs pembrolizumab and nivolumab.^168-171 The results of several trials that have already been launched will clarify the actual clinical value of such combinatorial immunochemotherapeutic paradigms.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

Authors are supported by the Ligue contre le Cancer (équipe labelisée); Agence National de la Recherche (ANR); Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; AXA Chair for Longevity Research; Institut National du Cancer (INCa); Fondation Bettencourt-Schueller; Fondation de France; Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI).