Trial watch: Immunogenic cell death induction by anticancer chemotherapeutics

ABSTRACT

The expression “immunogenic cell death” (ICD) refers to a functionally unique form of cell death that facilitates (instead of suppressing) a T cell-dependent immune response specific for dead cell-derived antigens. ICD critically relies on the activation of adaptive responses in dying cells, culminating with the exposure or secretion of immunostimulatory molecules commonly referred to as “damage-associated molecular patterns”. Only a few agents can elicit bona fide ICD, including some clinically established chemotherapeutics such as doxorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, cyclophosphamide and oxaliplatin. In this Trial Watch, we discuss recent progress on the development of ICD-inducing chemotherapeutic regimens, focusing on studies that evaluate clinical efficacy in conjunction with immunological biomarkers.

KEYWORDS:

• antigen-presenting cell
• autophagy
• cytotoxic T lymphocyte
• endoplasmic reticulum stress
• damage-associated molecular pattern
• dendritic cell
• immune checkpoint blocker
• type I interferon

Introduction

The possibility that cancer cells might undergo an immunogenic form of regulated cell death (RCD) in response to selected stimuli has been proposed for the first time more than 10 years ago.¹ Since then, considerable efforts have been dedicated to the elucidation of the molecular and cellular mechanisms underlying immunogenic cell death (ICD), defined as a functionally distinct form of regulated cell death that facilitates (instead of suppressing) an adaptive immune response specific for dead cell-derived antigens.^1–7 Based on the available data, it is clear that ICD can facilitate T cell responses against a wide-spectrum of differentiation, over-expressed, and mutated tumor-associated antigens (TAAs).^1–4,8–13 However, the predominance of a fraction of TAA-specific T cells in driving ICD-based immunity might be regulated by: (1) the spatiotemporal expression patterns of specific TAAs within a tumor,^14–20 (2) the overall coverage of various TAAs by central or peripheral tolerance,^21–23 (3) the overall avidity of the T cell receptor (TCR) for specific TAA,^24–32 and (4) the general cellular and metabolic health of effector or memory T cell fractions.^33–38 Operationally, two experimental procedures have been established over the years to identify bona fide ICD inducers in vivo. First, bona fide ICD inducers display elevated efficacy against malignant cells growing in immunocompetent hosts, but are largely ineffective when the same tumors are established in immunocompromised animals.^39–43 Second, cancer cells succumbing to bona fide ICD in vitro are able to vaccinate syngeneic immunocompetent hosts against a subsequent challenge with living cancer cells of the same type.^1,3,39,44 Although the former approach (therapeutic setting) is rather convenient, it is intrinsically unable to discriminate between bona fide ICD inducers and molecules that exert other on-target immunostimulatory effects or drive off-target immunostimulation.^45–47 Thus, the only gold-standard approach to identify immunogenic instances of cell death relies on the latter approach (vaccination setting).³⁹ Since vaccination tests can only be performed with murine cancer cells and immunocompetent syngeneic hosts, however, surrogate approaches have been developed.^3,39,48 On the one side, surrogate biomarkers of ICD can be measured in (human and murine) cancer cells responding to putative ICD inducers.^{2–4,48–51} On the other side, malignant cells succumbing to a putative ICD inducer can be fed to dendritic cells (DCs),^2,44,52–55 followed by (1) phagocytosis assays^56–62; (2) assessment of activation markers on the DC surface (e.g., CD80, CD86, MHC Class II) and functional markers in conditioned media (e.g., interleukin-6 or IL6, IL1β, IL12p70)^54,63–72; or (3) functional cross-priming assays with syngeneic lymphocytes.^73–81 Although none of these assays (alone or in combination) can reliably predict the ability of a specific intervention to cause ICD, the use of surrogate approaches is highly convenient for screening purposes or when studies are focused on the human model.^2,3,44,48,82

A number of mechanisms regulate the capacity of a particular agent to drive bona fide ICD and the ability of the host to perceive such an instance of cell death as immunogenic, and hence respond with potentially curative TAA-specific adaptive immunity.³ At the level of cancer cells, ICD depends upon the timely emission of a constellation of immunomodulatory damage-associated molecular patterns (DAMPs).^40,83 In the case of chemotherapy-induced ICD, these include (but may not be limited to): (1) surface-exposed endoplasmic reticulum (ER) chaperones including calreticulin (CALR)^84–86; (2) extracellular ATP;^87–91 (3) extracellular high mobility group box 1 (HMGB1)^13,92; (4) extracellular annexin A1 (ANXA1)⁵⁵; (5) secreted type I interferon;^93–96 and (6) extracellular nucleic acids.⁹⁷ That said, ICD triggered by stimuli other than chemotherapy (e.g., radiation therapy, photodynamic therapy) is not necessarily associated with the same DAMPs.^3,40,98,99 Moreover, new DAMPs underlying the immunogenicity of specific instances of RCD are continuously being uncovered (see below).

When emitted in a proper spatiotemporal pattern, in combination with TAAs, and in the context of a tumor milieu amenable to immune intervention,^83,100–104 these DAMPs can efficiently recruit antigen-presenting cells including DCs to the tumor bed, facilitate the engulfment of dying cells or their corpses (along with their TAA-laden cargo) in the context of immunostimulatory signals (which promote DC maturation).^{13,41,44,93,99} Mature DCs acquire therefore the ability to cross-present processed TAAs to CD4⁺ and CD8⁺ T cells along with suitable co-stimulation, thereby resulting in engagement of TAA-specific immunity.^105–110 Accordingly, RCD can no longer be perceived as immunogenic when: (1) the intracellular stress responses regulating the emission of ICD-associated DAMPs are pharmacologically or genetically ablated in cancer cells; or (2) when the molecular machinery dedicated to DAMP detection is inhibited or ablated.^{13,44,84,91,93,97} Moreover, ICD-driven immunity can no longer operate in the presence of general immunological defects,¹¹¹ such as (1) an intrinsically low antigenicity of cancer cells, owing to low levels of TAAs or downregulation of MHC Class I molecules^112–119; (2) an increased immunological tolerance of the host, secondary to increased amounts of immunosuppressive cytokines,^120–128 or inhibitors of chemotaxis,^129–134 increased tumor infiltration by immunosuppressive cell populations,^135–145 or robust immune checkpoint activation;^{126,130,136–138,146} (3) a reduced persistence of TAA-specific CD8⁺ memory T cells due to peripheral tolerance;^{21,23,144,147–149} and (4) an intrinsically elevated resistance of cancer cells to lysis by immune effectors.^{136,147,150,151} Additional details about ICD-associated signaling pathways and resistance mechanisms can be found in various publications from us and others.^{2–4,7,40,152–155}

Of note, only a limited number of cell death inducers can elicit bona fide ICD, and this capacity cannot be predicted on the basis of structural or functional similarities. Thus, while cisplatin and oxaliplatin both induce RCD at least in part by forming inter- and intra-strand DNA adducts,¹⁵⁶ only the latter induces ICD.¹⁵⁷ Similarly, even though both melphalan and cyclophosphamide efficiently kill cancer cells by operating as DNA alkylating agents, only the latter drives ICD.¹⁵⁸ In both examples, the ability of a specific agent (e.g., oxaliplatin, cyclophosphamide) but not one of its alike (e.g., cisplatin, melphalan) to drive ICD can be explained by the differential activation of ER stress (and hence differential exposure of CALR in the course of RCD).^{100,157–159} Well established ICD inducers include commonly employed anticancer chemotherapeutics such as: (1) doxorubicin, an anthracycline approved by the US Food and Drug Administration (FDA) for treating acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small cell lung carcinoma, breast carcinoma, neuroblastoma, lymphoma, thyroid carcinoma, soft tissue and bone sarcomas, multiple myeloma (MM), gastric cancer, ovarian carcinoma, transitional cell bladder carcinoma and Wilms tumor;^{1,84,160–166} (2) epirubicin, an anthracycline licensed for use in breast carcinoma patients;^1,84,167,168 (3) idarubicin, an anthracycline generally employed against AML;^84,167–169 (4) mitoxantrone, an anthracenedione approved for the treatment of prostate carcinoma, AML, non-Hodgkin's lymphoma (NHL) and breast carcinoma;^1,84,167,168 (5) bleomycin, a glycopeptide antibiotic approved for the treatment of patients with NHL, testicular cancer, Hodgkin's lymphoma, penile cancer and squamous carcinomas of the cervix, head and neck or vulva;^167,168,170 (6) bortezomib, a proteasomal inhibitor approved for the therapy MM and mantle cell lymphoma (MCL);^171–181 (7) cyclophosphamide, a DNA-alkylating agent approved for use in patients with chronic myeloid leukemia (CML), AML, ALL, chronic lymphocytic leukemia, MM, ovarian carcinoma, breast carcinoma, mycosis fungoides, lymphoma, neuroblastoma, and retinoblastoma;^{177,182–191} and (8) oxaliplatin, a platinum-derivative licensed for the therapy of advanced colorectal carcinoma in combination with 5-fluorouracil and folinic acid.^{156,157,192–198} Moreover, there is some evidence that microtubule-targeting agents including taxanes and vinca alkaloids (which are commonly used for the treatment of multiple carcinomas) can stimulate ICD.^41,199

Along the lines of our Trial Watch series, here we discuss recent preclinical and clinical advances in the development of ICD-inducing chemotherapeutic regimens.²⁰⁰ Several other interventions that trigger bona fide ICD, such as radiation therapy administered according to specific regimens,^94,201–203 high hydrostatic pressure,^3,4 oncolytic virotherapy^204–208 and photodynamic therapy,^44,86,98,99 are not discussed here in further detail.

Recent preclinical developments

A high amount of preclinical and/or translational studies on chemotherapy-induced ICD has been published since the latest Trial Watch dealing with this topic (April 2015).⁵⁰ Of such an abundant scientific production from us and others, we found of particular significance the following works.

We documented that anthracycline-induced ICD critically relies on the release of ANXA1 by cancer cells, driving the late-stage phases of DC chemotaxis in a formyl peptide receptor 1 (FPR1)-dependent manner.⁵⁵ Accordingly, a loss-of-function single-nucleotide polymorphism in FPR1 was found to be associated with poor prognosis in cohorts of breast and colorectal carcinoma patients undergoing anthracycline or oxaliplatin-based chemotherapy.⁵⁵ Moreover, we found that combining chemotherapy-induced ICD with short-term fasting or caloric restriction mimetics (CRMs)⁹⁰ like hydroxycitrate or spermidine, boosts the efficacy of mitoxantrone and oxaliplatin in mice, a therapeutic improvement that is accompanied by decreased tumor infiltration by immunosuppressive CD4⁺CD25⁺FOXP3⁺ regulatory T (T_REG) cells.²⁰⁹ Along similar lines, Di Biase et al. (from University of Southern California, Los Angeles, CA, USA) observed that the combination of doxorubicin with a fasting-mimicking diet strongly delays breast cancer and melanoma progression as it increases the amounts of tumor-infiltrating cytotoxic T lymphocytes (CTLs) while concomitantly decreasing the expression levels of the immunosuppressive enzyme heme oxygenase-1 (HMOX1; also known as HO1).²¹⁰ We also discovered that cancer cells undergoing mitoxantrone-induced ICD trigger a pathogen response-like chemokine (PARC) signature characterized by the co-release of C-X-C motif chemokine ligand 1 (CXCL1), C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) (or homologues thereof), in thus far mimicking bacteria or virus infected cells.⁹⁷ Such a chemokine mixture is particularly efficient at recruiting neutrophils towards the dying cells (a process that appears to be evolutionarily conserved), paving the way to the CALR-dependent phagocytosis of dying cancer cells or corpses thereof, and the cytotoxic targeting of residual malignant cells.⁹⁷ We characterized a naturally-occurring preclinical model of cancer that exhibits intrinsic resistance against mitoxantrone-induced ICD in vivo secondary to a defect in CALR exposure,¹⁹⁹ and we documented that anthracyclines and oxaliplatin can trigger a necroptotic variant of ICD^211–215 in cancer cells expressing receptor interacting serine/threonine kinase 3 (RIP3K) and the pseudokinase mixed lineage kinase domain-like (MLKL).²¹⁵ Finally, we found that an engineered oncolytic vaccinia virus²¹⁶ can induce ICD-dependent antitumor immunity, which can be further potentiated by the co-administration of ICD-inducing chemotherapy or immune checkpoint blockers (ICBs),²¹⁶ and that pharmacological inhibition of signal transducer and activator of transcription 3 (STAT3)²¹⁷ signaling boosts the therapeutic efficacy of anthracyclines upon increased type I interferon (IFN) secretion.²¹⁷

Pfirschke and co-authors (from Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA, USA) found that autochthonous tumors lacking tumor-infiltrating lymphocytes (TILs) can be sensitized to immunological rejection via a Toll-like receptor 4 (TLR4)-dependent mechanism when suitable ICD inducers like oxaliplatin or cyclophosphamide are combined with ICBs targeting programmed cell death 1 (PDCD1; best known as PD-1)²¹⁸ and/or cytotoxic T-lymphocyte associated protein 4 (CTLA4).²¹⁸ Similarly, Stewart and colleagues (from MedImmune Ltd, Cambridge, United Kingdom) characterized a novel antibody directed against CD274 (best known as PD-L1) (MEDI4736), specifically engineered to prevent antibody-dependent cell-mediated cytotoxicity (ADCC),²¹⁹ which exhibits potent antitumor activity when combined with oxaliplatin (only in immunocompetent mice).²¹⁹ In a multi-modal combinatorial study, Rios-Doria and collaborators (from MedImmune, Gaithersburg, MD, USA) documented that liposomal doxorubicin induces potent ICD-associated innate and adaptive immune responses, in vivo, and effectively controls tumor growth, especially in combination with ICBs against CTLA4, PD-1, or its main ligand PD-L1 or with immunostimulatory fusion proteins targeting TNF receptor superfamily, member 4 (TNFRSF4; best known as OX40) or TNF receptor superfamily member 18 (TNFRSF18; best known as GITR).²²⁰ In a slightly different approach, Evans et al. (from Vaccinex, Inc., Rochester, NY, USA) reported that semaphorin 4D (SEMA4D) expression at the tumor invasive margin facilitates pro-tumorigenic inflammation, and that combining anti-SEMA4D monoclonal antibodies (mAbs) with ICBs or cyclophosphamide promotes immunological rejection in murine colorectal carcinoma models.²²¹ Finally, Blake et al. (from QIMR Berghofer Medical Research Institute, Herston, Australia) observed that inhibiting CD96 – a negative regulator of natural killer (NK) cell activity²²² – with ICBs or doxorubicin exerts superior antimetastatic effects in the lung.²²³

Musahl et al. (from Max Planck Institute for Molecular Genetics, Berlin, Germany) observed that the long non-coding RNA, ncRNA-RB1, positively regulates overall CALR expression levels so that, following anthracyclines-induced ICD, ncRNA-RB1 supports surface-exposed CALR driven phagocytosis.²²⁴ In a similar fashion, Colangelo and co-authors (from University of Sannio, Benevento, Italy) found that the microRNA miR-27a specifically suppresses the exposure of CALR by mitoxantrone and oxaliplatin in colorectal cancer cells by negatively affecting the intracellular CALR trafficking pathway.²²⁵ In a study exploring novel combinatorial regimens, Lu and colleagues (from University of Alabama, Birmingham, AL, USA) documented that combining an inhibitor of thrombospondin 1 (THBS1; also known as TSP1) and transforming growth factor beta 1 (TFGB1) signaling, i.e., SRI31277, with bortezomib mediates superior antineoplastic effects as compared to either agents alone.²²⁶ In a different combinatorial approach, Hsu and colleagues (from National Yang-Ming University, Taipei, Taiwan) revealed that pre-conditioning with low-dose doxorubicin or paclitaxel before adoptive cell transfer (ACT) significantly improves antitumor immunity upon inhibition of NF-kB-regulated immunosuppressive factors.²²⁷ Similarly, Koo and collaborators (from Catholic University of Korea, Bucheon, Republic of Korea) found that X-shaped double-stranded oligodeoxynucleotide molecules (so called “X-DNA”) that bind TLR9²²⁸ greatly enhance the antitumor efficacy of doxorubicin against colitis-associated colorectal carcinoma, via a mechanism that depends on DCs and T cells.²²⁹ Finally, Monk et al. (from University of Arizona College of Medicine-Phoenix, Phoenix, AZ, USA) documented that combining pegylated liposomal doxorubicin with a TLR8 agonist, i.e., motolimod, retards the growth of patient-derived ovarian carcinoma cells implanted in humanized mice.²³⁰

In a study exploring resistance mechanisms, Buondonno et al. (from University of Torino, Torino, Italy) documented that treating osteosarcoma cells that overexpress ATP binding cassette subfamily B member 1 (ABCB1) – a plasma membrane transporter that actively extrudes several chemotherapeutics including doxorubicin – with mitochondria-targeted doxorubicin overcomes chemoresistance to restore ICD and associated immune responses in vivo.²³¹ Finally, Shalapour and co-authors (from University of California San Diego, San Diego, CA, USA) reported that B cells producing class A immunoglobulins (IgAs), IL10 and PD-L1 in a TGFB1-dependent fashion act as immunosuppressive plasmocytes and prevent oxaliplatin-driven tumor rejection in different murine models of prostate cancer.²³² Thounaojam and colleagues (from Meharry Medical College, Nashville, TN, USA) found that bortezomib suppresses the growth of various solid tumors by upregulating the expression of various components of the Notch signaling pathway in lymphoid tissues including CD8⁺ CTLs, resulting in increased cytotoxic functions.^219,233 Guillerey and collaborators (from QIMR Berghofer Medical Research Institute, Herston, Australia) observed that anti-myeloma immune response elicited by bortezomib or cyclophosphamide critically relies on CD226, which is crucial for the effector and cytotoxic functions of NK and CD8⁺ T cells.²³⁴ Finally, Wong and collaborators (from National University of Singapore, Singapore) screened a library of chemotherapeutically active platinum derivatives and characterized a Pt(II) N-heterocyclic carbene complex as a putative inducer of ICD (pending in vivo validation), based on its capacity to trigger oxidative ER stress, CALR exposure, ATP secretion, and HMGB1 release.²³⁵

Taken together, these findings exemplify the attention currently focused around the molecular and cellular mechanisms through which the death of cancer cells responding to some (but not all) chemotherapeutics initiates a therapeutically relevant tumor-specific immune response.

Completed clinical trials

Since the publication of the latest Trial Watch dealing with this topic (April 2015), multiple peer-reviewed articles documented the outcome of clinical trials evaluating the efficacy of bona fide ICD-inducing chemotherapeutics (i.e., doxorubicin, epirubicin, idarubicin, mitoxantrone, bortezomib, bleomycin, cyclophosphamide or oxaliplatin) along with ICD-associated immunological biomarkers.^236,237 These publications were acquired from PubMed (http://www.ncbi.nlm.nih.gov/pubmed), and the initial list was manually curated to ensure relevance for this Trial Watch.

Several clinical studies demonstrated the beneficial immunostimulatory effects of ICD induction by chemotherapy. Loi et al. (from Peter MacCallum Cancer Centre, Melbourne, Australia) documented that increased tumor infiltration by T cells is associated with improved prognosis in patients affected by triple-negative breast cancer (TNBC) with residual disease following neoadjuvant chemotherapy (consisting of doxorubicin, cyclophosphamide, and in some cases paclitaxel).²³⁸ Cornelissen and collaborators (from Erasmus MC Cancer Institute, Rotterdam, Netherlands) reported that combining DC-based vaccines with metronomic cyclophosphamide (which efficiently reduces circulating T_REG cells) resulted in radiographic tumor control (and increased overall survival) in 8 out of 10 patients with malignant pleural mesothelioma patients.²³⁹ Schijns and colleagues (from Wageningen University, Wageningen, Netherlands) found that individuals with recurrent glioblastoma multiforme (GBM) receiving a vaccine composed of autologous antigens in combination with cyclophosphamide, experienced improved overall survival.²⁴⁰ Klein and co-authors (from Ludwig Institute for Cancer Research, Heidelberg, Australia) reported that cyclophosphamide promoted TAA-specific CD4⁺ T cell responses driven by a peptide-based vaccine targeting cancer/testis antigen 1B (CTAG1B; best known as NY-ESO-1)²⁴¹ in patients with advanced melanoma, in the absence of T_REG cell depletion.²³⁸ Murahashi et al. (from Kyushu University, Fukuoka, Japan) evaluated a multipeptide-based vaccine combined with escalating doses of cyclophosphamide in patients with locally advanced, metastatic and/or recurrent gastrointestinal, lung or cervical cancer, achieving increased overall survival accompanied by increased TAA-specific T cells and peripheral T_REG cell depletion.²⁴² Tanis and collaborators (from The Netherlands Cancer Institute, Amsterdam, Netherlands) found that patients with liver metastases from colorectal carcinoma obtain a survival benefit from the FOLFOX therapeutic regimen (consisting of folinic acid, fluorouracil and oxaliplatin), which is accompanied by increased amount of CTLs at invasive tumor margin, as well as with mast cell infiltration.²⁴³ Collectively, these reports demonstrate that ICD-inducing chemotherapeutic regimens can prolong the survival of some cancer patients, and this often correlates with biomarkers of ongoing anticancer immunity.

Alongside, a few articles documented negative immunological consequences of chemotherapeutic regimens (at least potentially) triggering ICD, such as the R-CHOP regimen (consisting of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). Specifically, Ito et al. (from Fujita Health University Hospital, Aichi, Japan) found that diffuse large B-cell lymphoma (DLBCL) patients receiving R-CHOP experienced immunosuppression for two or more years following treatment, mainly manifesting with marked decrease in circulating CD20⁺ B cells and CD4⁺ T cells.²⁴⁴ Brown and collaborators (from University of Oxford, John Radcliffe Hospital, Oxford, UK) reported that patients with DLBCL receiving R-CHOP exhibited decreased levels of major histocompatibility complex, class II, DR alpha (HLA-DRA) on DLBCL cells, correlating with inferior overall survival.²⁴⁵ On a relatively conciliatory note though, Wu and co-authors (from The Second Hospital of Lanzhou University, Lanzhou, China) found that the low circulating levels of monocytes and monocytic myeloid-derived suppressor cells (MDSCs) have profound positive prognostic impact on the overall survival of DLBCL patients treated with R-CHOP.²⁴⁶ It is tempting to speculate, yet remains to be formally investigated, that the immunosuppressive effects of R-CHOP originate from prednisone.²⁴⁷ It would therefore be interesting to see whether an “R-CHO” regimen deprived of prednisone might exert superior immunostimulatory and therapeutic effects.

Ongoing clinical trials

Official sources list no less than 58 clinical trials evaluating the clinical efficacy of bona fide ICD-inducing chemotherapeutic regimens in conjunction with relevant immune biomarkers initiated after April 2015 (since the publication of the latest Trial Watch dealing with this topic) (Fig. 1, Tables 1 and 2). These studies were retrieved from the ClinicalTrials.gov database (http://www.clinicaltrials.gov/), and the initial list was manually curated to ensure relevance for this Trial Watch.

Figure 1. Current clinical trials testing immunogenic cell death (ICD)-inducing chemotherapies in oncological indications. A. Distribution by immunological biomarker (biomarkers directly relevant for ICD are in bold). B. Distribution by main chemotherapeutic agent. C. Distribution by oncological indication. D. Number of clinical trials currently testing ICD-inducing chemotherapeutic regimens in combination with immunotherapy. CIK, cytokine-induced killer; CSF1, colony stimulating factor 1; DC, dendritic cell; mAb, monoclonal antibody; MDSC, myeloid-derived suppressor cell; NK, natural killer; TAA, tumor-associated antigen; TLR, Toll-like receptor; T_REG, regulatory T.

Table 1. Current clinical trials evaluating the therapeutic and immunological profile of ICD-inducing chemotherapeutic regimens.^*

Drug	Indication(s)	Phase	Status	Notes	Ref.
Bleomycin	Rectal cancer	II	Recruiting	As single agent in the context of electro-chemotherapy	NCT03040180
Bortezomib	Lung cancer	I	Not yet recruiting	Combined with a DRibble vaccine and DC/CIK therapy	NCT03057340
Doxorubicin	Breast carcinoma	I/II	Recruiting	Combined with epirubicin, cyclophosphamide, fluorouracil or methotrexate	NCT02897700
		II	Recruiting	Combined with decitabine, cyclophosphamide and paclitaxel, carboplatin and/or pembrolizumab	NCT02957968
			Recruiting	Combined with eribulin and cyclophosphamide	NCT02623972
			Recruiting	Combined with cyclophosphamide, ruxolitinib and paclitaxel	NCT02876302
	Pediatric solid tumors	I	Recruiting	As lyso-thermosensitive liposomal doxorubicin	NCT02536183
Epirubicin	Breast carcinoma	I/II	Recruiting	Combined with doxorubicin, cyclophosphamide, fluorouracil or methotrexate	NCT02897700
		II	Recruiting	Combined with paclitaxel, cyclophosphamide and MEDI4736	NCT02685059
				Combined with docetaxel, cyclophosphamide/ capecitabine or fluorouracil/cyclophosphamide	NCT02897050
				Combined with docetaxel, cyclophosphamide, fluorouracil, trastuzumab or pertuzumab	NCT03144947
				Combined with cyclophosphamide and fluorouracil	NCT02742051
				Combined with docetaxel, cyclophosphamide, paclitaxel, tocotrienol, trastuzumab or pertuzumab	NCT02909751
Idarubicin + mitoxantrone	Acute promyelocytic leukemia	III	Recruiting	Combined with arsenic trioxide, tretinoin, cytarabine, mercaptopurine and methotrexate	NCT02688140
Oxaliplatin	Colorectal carcinoma	n.s.	Recruiting	Combined with 5-fluorouracil and bevacizumab	NCT02817178
		I	Recruiting	Combined with trifluridine, bevacizumab and nivolumab	NCT02848443
		II	Suspended	Combined with 5-fluorouracil, leucovorin, bevacizumab, capecitabine, Ad-CEA vaccine and avelumab	NCT03050814
	Gastrointestinal cancer	IV	Not recruiting	Combined with cinobufotalin	NCT02860429
	Gastric cancer	II	Recruiting	Combined with capecitabine, epirubicin and pembrolizumab	NCT02918162
	Pancreatic cancer	I	Recruiting	Combined with folinic acid, irinotecan, 5-fluorouracil, paclitaxel, gemcitabine and a DC-based vaccine	NCT02548169

Abbreviations: Ad-CEA, adenovirus vector encoding carcinoembryonic antigen; CIK, cytokine-inducer killer; DC, dendritic cell; n.s., not specified

* Initiated between 1^st April 2015 and 30^th June 2017.

Table 2. Current clinical trials evaluating the therapeutic and immunological profile of cyclophosphamide-based chemotherapy.^*

Indication(s)	Phase	Status	Notes	Ref.
Acute lymphoblastic leukemia	II	Recruiting	Combined with standard-of-care chemotherapy	NCT02828358
Acute myeloid leukemia	III	Completed	Combined with standard-of-care chemotherapy and anti-lymphocyte globulin	NCT02461121
B-cell lymphoma	I/II	Recruiting	Combined with fludarabine and CD19-specific CAR-T cells	NCT03146533
Breast carcinoma	I	Unknown	Combined with radiotherapy	NCT02441270
		Recruiting	Combined with indomethacin, omeprazole, dietary supplements (e.g., multivitamins) and IRX-2	NCT02950259
	II	Recruiting	Combined with doxorubicin, paclitaxel, carboplatin and pembrolizumab	NCT02957968
			Combined with docetaxel, capecitabine, fluorouracil and epirubicin	NCT02897050
			Combined with everolimus, letrozole, fluorouracil and epirubicin	NCT02742051
		Not recruiting	Combined with peptide vaccination	NCT03012100
Chronic lymphocytic leukemia	II	Not recruiting	Combined with rituximab, fludarabine, bendamustine, lenalidomide and ACT	NCT02530515
DLBCL	I	Recruiting	Combined with fludarabine and CD19-specific CAR-T cells	NCT02431988
EBV-associated malignancies^**	I/II	Recruiting	Combined with fludarabine and PDCD1-deficient EBV-specific CTLs	NCT03044743
Esophageal cancer	II	Recruiting	Combined with PDCD1-deficient CTLs and IL2	NCT03081715
Gastrointestinal cancer	I	Not recruiting	Combined with ACT, IL2 and pembrolizumab	NCT02757391
Gynecological tumors	I	Recruiting	Combined with epacadostat and DPX-Survivac	NCT02785250
	II	Recruiting	Combined with pembrolizumab and bevacizumab	NCT02853318
	II	Not recruiting	Combined with PD0360324	NCT02948101
Hematological malignancies	I/II	Not recruiting	Combined with a DC-based vaccine and GM-CSF	NCT02709993
	III	Recruiting	Combined with tacrolimus and methotrexate	NCT02345850
Hepatocellular carcinoma	I/II	Not recruiting	Combined with IMA970 A plus CV8102	NCT03203005
Lung cancer	I	Withdrawn	Combined with DC-based vaccine	NCT02419170
		Not recruiting	Combined with radiotherapy	NCT02579005
		Recruiting	Combined with PDCD1-deficient CTLs and IL2	NCT02793856
Lung cancer and mesothelioma	I/II	Recruiting	Combined with ACT and IL2	NCT02408016
Melanoma	I/II	Recruiting	Combined with polyICLC and MHP vaccine	NCT02425306
	I	Recruiting	Combined with pembrolizumab and ONCOS-102	NCT03003676
Mesothelioma	I/II	Recruiting	Combined with premetrexed/cisplatin and ONCOS-102	NCT02879669
Multiple myeloma	I	Completed	Combined with lenalidomide and elotuzumab	NCT02655458
Neuroendocrine tumors	II	Recruiting	Combined with IFNα	NCT02838342
Osteosarcoma	I	Recruiting	Combined with sirolimus, methotrexate and zoledronic acid	NCT02517918
Prostate cancer	I	Recruiting	Combined with DNA-based vaccine	NCT02390063
			Combined with cytoxan and testosterone	NCT02995330
		Not recruiting	Combined with PDCD1-deficient CTLs and IL2	NCT02867345
Renal cell carcinoma	I	Not recruiting	Combined with PDCD1-deficient CTLs and IL2	NCT02867332
Sarcoma	II	Recruiting	Combined with pembrolizumab	NCT02406781
Solid tumors	I	Not recruiting	Combined with a TLR8 agonist and pegfilgrastim	NCT02650635
	I/II	Not recruiting	Combined with a DC-based vaccine and GM-CSF	NCT02705703
		Recruiting	Combined with fludarabine, neoantigen-specific T cells, PDCD1 inhibition and IL2	NCT03171220

Abbreviations: ACT, adoptive cell transfer; CAR, chimeric antigen receptor; CTL, cytotoxic T lymphocyte; DC, dendritic cell; DLBCL, diffuse large B-cell lymphoma; DPX, DepoVax™; EBV, Epstein-Barr virus; GM-CSF, granulocyte macrophage colony stimulating factor; IFN, interferon; IL, interleukin; MHP, melanoma helper peptides; polyICLC, polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose; TAA, tumor-associated antigen; TLR, Toll-like receptor

* Initiated between 1^st April 2015 and 30^th June 2017

** Including gastric carcinoma, nasopharyngeal carcinoma, T-cell lymphoma, Hodgkin lymphoma and DLBCL.

Our survey revealed that multiple ICD-relevant biomarkers are being investigated as immunological outcomes across recently initiated clinical trials based on ICD-inducing chemotherapeutic regimens (Fig. 1A). These immunological biomarkers include: (1) parameters of broad T cell immunophenotyping, such as the so-called “Immunoscore”,^248–250 and the abundance of tumor-infiltrating or circulating CD3⁺, CD4⁺ or CD8⁺ T cells, amongst others;^251–253 (2) the circulating levels of multiple cytokines, such as interferon, gamma (IFNG; best known as IFN-γ),²⁵⁴ IL6,²⁵⁵ and tumor necrosis factor (TNF);^256–258 (3) the amounts of TAA-specific T cells (via tetramer assays);^259,260 (4) the tumor mutational load and/or the abundance of predicted neo-antigens (via genome or whole-exome sequencing);^{115,138,261–265} and (5) the levels of circulating biomarkers of ICD (via proteomic or metabolomic assays).^49,266 Alongside, several immunological biomarkers with indirect relevance for ICD and its therapeutic outcomes are also being tested, including the abundance of circulating or tumor-infiltrating NK cells, MDSCs and T_REG cells, as well as the expression levels and activation status of immune checkpoints.^{48,261,267–273} In this setting, the profiling of T_REG cells is being performed in most cases to estimate the ability of cyclophosphamide to specifically target these immunosuppressive cells.^274–276 It will be interesting to see whether the peripheral profile (in terms of abundance and activation status) of CD4⁺ and/or CD8⁺ T cells can serve as reliable biomarker for the immunological consequences of chemotherapy.^4,277 Preclinical and (mostly retrospective) clinical data advocate that tumor-infiltrating T cells provide superior prognostic and/or predictive information,^251,267,278 but the identification of a reliable circulating biomarker would be a major asset for immunomonitoring.

A majority of the clinical trials surveyed here aim at testing cyclophosphamide, epirubicin, doxorubicin or oxaliplatin, typically as on-label therapeutic interventions and often as part of gold standard chemotherapeutic regimen (Fig. 1B, Tables 1 and 2). In some cases, the ICD-inducing potential of the chemotherapeutic regimen of choice was cited as part of the rational to the study. However, we were unable to identify any clinical trial specifically comparing the ICD-inducing potential of these agents with a relevant non-immunogenic chemotherapeutic. In a majority of cases, cyclophosphamide is administered with the primary aim of inhibiting or depleting (systemic) T_REG cells. However, considering the wide array of immunological biomarkers monitored in these clinical trials (Fig. 1A), the ICD-inducing potential of cyclophosphamide¹⁹¹ may also become apparent.

As for oncological indication, the studies we retrieved are currently enrolling patients with breast carcinoma (16 trials), lung cancer (in particular non-small cell lung carcinoma, NSCLC) (5 trials), colorectal carcinoma (3 trials), prostate cancer (3 trials), and other cancer types (31 trials) (Fig. 1C, Table 1 and 2). In general, solid tumors (51 trials) are preferred to hematological malignancies (8 trials). Possibly, this reflects, (1) the high sensitivity of several hematological tumors to standard-of-care treatments, and/or (2) the fact that hematological malignancies often stem from components of the immune system, resulting in compromised immune functions and limited susceptibility to multiple forms of immunotherapy and immunogenic chemotherapy.^279,280 In multiple clinical studies, bona fide ICD-inducing chemotherapeutic regimens are combined with agents that elicit ICD per se, such as radiation therapy (2 trials), or considerably boost the immunogenicity of cancer cells, such as taxanes or zoledronic acid (10 trials). Finally, in a limited amount of trials, ICD inducers are combined with targeted anticancer agents, including the inhibitor of JAK kinases ruxolitinib (1 trial) and the inhibitor of mechanistic target of rapamycin kinase (MTOR) rapamycin^281,282 (also known as sirolimus, or its derivative everolimus) (2 trials) (Tables 1 and 2).

Fitting well within the current oncology landscape, a majority of clinical trials surveyed for this Trial Watch combine ICD-inducing chemotherapeutic regimens with bona fide immunotherapies (Fig. 1D, Tables 1 and 2). These include: (1) immunostimulatory cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), IL2, IFN-α and IRX-2, a cell-free mixture of cytokines encompassing IL1, IL2, IL6, IL8, IL10, IL12, TNF, IFN-γ and colony stimulating factor 1 (CSF1, also called macrophage colony stimulating factor 1 or M-CSF); (2) ICBs such as the PD-1-targeting agents pembrolizumab²⁸³ and SHR-1210;²⁸⁴ and the PD-L1-targeting agents avelumab and MEDI4736;²⁸⁴ (3) the inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1);^138,285 (4) adoptively transferred T cells, often CD19-specific chimeric antigen receptor (CAR)-expressing T cells or PD-1-deficient T cells;²⁸⁶ (5) tumor-targeting mAbs, such as the CD20-specific agent rituximab,²⁸⁷ the vascular endothelial growth factor (VEGF)-targeting mAb bevacizumab,²⁸⁸ and various molecules targeting HER2;²⁸⁹ (6) anticancer vaccines;^{52,53,290,291} and (7) oncolytic viruses^{266,292–295} (Fig. 1D, Table 1 and 2).

Finally, most of the clinical trials we surveyed are ongoing (i.e., either actively recruiting or initiated/registered but not yet recruiting), with a few notable exceptions. NCT03050814 (a Phase 2 study testing oxaliplatin-based chemo(immuno)therapy in combination with PD-L1 blockade or bevacizumab in colorectal carcinoma patients) has been suspended pending further discussion with the US FDA, because an unspecified number of individuals died within 30 days of treatment. On a different note, NCT02419170 (a Phase I study testing DC-based vaccination plus cyclophosphamide in NSCLC patients) has been withdrawn prior to enrollment because the investigator manufacturing the vaccines left the concerned institution. Finally, NCT02461121 (a Phase III trial comparing cyclophosphamide-containing versus cyclophosphamide-free preconditioning in patients with acute myeloid leukemia allocated to transplantation) and NCT02655458 (a Phase I study involving cyclophosphamide-containing preconditioning in multiple myeloma patients) have been completed. However, to the best of our knowledge, the results of these studies have not been released yet.

Concluding remarks

A number of ICD-inducing chemotherapeutic regimens are currently approved by the US FDA or equivalent regulatory agencies worldwide for use in cancer patients. However, the widespread use of these treatments has been implemented mostly on empirical (rather than immunological) grounds.^{200,296–300} Indeed, the possibility that chemotherapy and other forms of treatment (including radiotherapy and photodynamic therapy) might promote an immunogenic form of cancer cell death has been overlooked for several decades.^301–303 Thus, current anticancer drugs have been developed in immunodeficient preclinical models and in clinical trials devoid of any form of immunomonitoring, mainly most often aimed at identifying maximum tolerated doses (MTDs).^298,304 Nonetheless, a majority of currently available anticancer agents mediate on-target or off-target immunostimulatory effects, which strongly argues against an irrelevant role for the immune system in the therapeutic effects of these treatments.^305–308 Moreover, in various cases, chemotherapeutics applied through multiple treatment cycles may negatively affect the immune system, by causing lymphopenia or leucopenia, thereby further compromising antitumor immune responses.^44,53,54 One of the major challenges for the future will be to identify doses and administration schedules that mediate maximal immunostimulatory effects.^297,309,310 Accumulating evidence suggest indeed that metronomic chemotherapy and hypofractionated radiation (rather than chemotherapy at the MTD and high single-dose radiation) exerts superior immunostimulatory (and hence therapeutic, at least in some settings) effects.^311–314 Alongside, it will be important to devise highly efficient combinatorial regimens that harness not only the ability of some treatments to drive ICD, but also the off-target immunostimulatory effects of a variety of agents. We are convinced that conventional therapeutic regimens – if properly employed – are a very powerful and relatively economical tool to drive clinically relevant anticancer immune responses.

Abbreviations

ACT	=	adoptive cell transfer
DAMP	=	damage-associated molecular pattern
DC	=	dendritic cell
ER	=	endoplasmic reticulum
FDA	=	Food and Drug Administration
GM-CSF	=	granulocyte-macrophage colony-stimulating factor
ICB	=	immune checkpoint blocker
ICD	=	immunogenic cell death
IFN	=	interferon
IL	=	interleukin
mAb	=	monoclonal antibody
MTD	=	maximum tolerated dose
NSCLC	=	non-small cell lung carcinoma
RCD	=	regulated cell death
TAA	=	tumor-associated antigen
TIL	=	tumor-infiltrating lymphocyte
TLR	=	Toll-like receptor

Acknowledgments

ADG is a recipient of FWO Postdoctoral (Renewal) Fellowship from FWO-Vlaanderen, Belgium and the POR award funds from KU Leuven. NR, SM, MLS are funded by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant (Agreement no. 642295 for NR and 675448 for SM/MLS). PA is supported by grants from FWO (G060713 N, G076617 N) and KU Leuven (C16/15/073). GK is supported by 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). LG is supported by WCMC (intramural funds) and Sotio a.c. (Prague, Czech Republic).