Trial Watch

Accumulating evidence suggests that the clinical efficacy of selected anticancer drugs, including conventional chemotherapeutics as well as targeted anticancer agents, originates (at least in part) from their ability to elicit a novel or reinstate a pre-existing tumor-specific immune response. One of the mechanisms whereby chemotherapy can stimulate the immune system to recognize and destroy malignant cells is commonly known as immunogenic cell death (ICD). Cancer cells succumbing to ICD are de facto converted into an anticancer vaccine and as such elicit an adaptive immune response. Several common chemotherapeutics share the ability of triggering ICD, as demonstrated in vaccination experiments relying on immunocompetent mice and syngeneic cancer cells. A large number of ongoing clinical trials involve such ICD inducers, often (but not always) as they are part of the gold standard therapeutic approach against specific neoplasms. In this Trial Watch, we summarize the latest advances on the use of cyclophosphamide, doxorubicin, epirubicin, oxaliplatin, and mitoxantrone in cancer patients, discussing high-impact studies that have been published during the last 13 months as well as clinical trials that have been initiated in the same period to assess the antineoplastic profile of these immunogenic drugs as off-label therapeutic interventions.


Introduction
Cancer is no longer considered as a purely cell-intrinsic disease, for several reasons. First, it has become clear that the development of both solid and hematological neoplasms critically relies upon an intimate crosstalk with non-malignant components of the tumor microenvironment, including endothelial, stromal, as well as immune cells. [1][2][3] Second, the notion that cancer would constitute a self entity and hence go completely unnoticed by the immune system has now been abandoned. [4][5][6] Research over the past two decades has indeed demonstrated that the immune system not only interacts with (and attempts to control) developing neoplasms, 4 but also (1) removes damaged and stressed cells, which are generally more prone to become malignant than their healthy counterparts (a process known as anticancer immunosurveillance); 7,8 and (2) plays a critical role in the response of various malignancies to therapy. [9][10][11] Both these facets of the complex interaction between (pre)neoplastic and non-malignant compartments of the tumor microenvironment have rapidly attracted interest as potential targets for the development of novel anticancer therapies, and some of these strategies have already entered the clinical practice. 2,[12][13][14] For instance, the monoclonal antibody bevacizumab, which is currently employed in patients with colorectal, lung, and renal carcinoma, 15,16 is specific for the vascular endothelial growth factor (VEGF), hence operating as an inhibitor of angiogenesis. 17,18 Along similar lines, ipilimumab, which has been approved by the US Food and Drug Administration (FDA) for the treatment of unresectable or metastatic melanoma in 2011, [19][20][21] inhibits the immunosuppressive receptor cytotoxic T lymphocyte-associated protein 4 (CTLA4), thereby exerting robust immunostimulatory effects. 22,23 Accumulating evidence suggests that the clinical efficacy of selected anticancer drugs, including conventional chemotherapeutics as well as targeted anticancer agents, originates (at least in part) from their ability to elicit a novel or reinstate a pre-existing tumor-specific immune response. One of the mechanisms whereby chemotherapy can provoke the immune system to recognize and destroy malignant cells is commonly known as immunogenic cell death (iCD). Cancer cells succumbing to iCD are de facto converted into an anticancer vaccine and as such elicit an adaptive immune response. Several common chemotherapeutics share the ability of triggering iCD, as demonstrated in vaccination experiments relying on immunocompetent mice and syngeneic cancer cells. A large number of ongoing clinical trials involve such iCD inducers, often (but not always) as they are part of the gold standard therapeutic approach against specific neoplasms. in this Trial watch, we summarize the latest advances on the use of cyclophosphamide, doxorubicin, epirubicin, oxaliplatin, and mitoxantrone in cancer patients, discussing high-impact studies that have been published during the last 13 months as well as clinical trials that have been initiated in the same period to assess the antineoplastic profile of these immunogenic drugs as off-label therapeutic interventions.
Importantly, besides mediating the antineoplastic effects of a variety of active and passive immunotherapeutic interventions, 24,25 the immune system appears to play a critical role in the response of several tumors to conventional therapeutic regimens as well as to targeted anticancer agents. 10,11 In support of this notion, high levels of tumor-infiltrating CD8 + T cells, alone or combined with limited amounts of intratumoral CD4 + CD25 + FOXP3 + regulatory T cells (Tregs), have been associated with improved disease outcome upon therapy in patients affected by a variety of solid neoplasms, including (but not limited to) breast carcinoma. 8,26,27 In addition, an ever increasing amount of preclinical data indicates that the efficacy of multiple anticancer agents and several forms of radiotherapy depends, at least in part, on an intact immune system. [28][29][30][31][32] Schematically, anticancer chemotherapeutics (as well as radiotherapy) can activate tumor-targeting immune responses that potentially eradicate the residual (chemo-or radioresistant) disease, hence leading to long-term clinical remissions, via 4, non-mutually exclusive mechanisms: (1) by directly stimulating the effector functions of innate or adaptive immune cells; (2) by inhibiting the immunosuppressive circuitries set in place by malignant cells to allow for tumor progression; (3) by enhancing the antigenicity of living cancer cells, their immunogenicity, or their susceptibility to immune effector mechanisms; or (4) by stimulating a peculiar type of apoptosis that results in the elicitation of adaptive immune responses against dead cellassociated antigens. 10,11,[33][34][35][36] The cellular and molecular circuitries involved in the capacity of selected chemotherapeutics to directly modulate the activity of immune cells and/or to increase the antigenicity of neoplastic cells, their immunogenicity or propensity to succumb to immune effectors have been reviewed elsewhere. 10,11,34 Immunogenic cell death (ICD) obligatorily relies on the emission of a spatiotemporally defined combination of signals by dying cells. [35][36][37][38][39] Such damage-associated molecular patterns (DAMPs) include (but presumably are not limited to): (1) the pre-apoptotic exposure of the endoplasmic reticulum chaperone calreticulin (CRT) to the outer leaflet of the plasma membrane; (2) the active secretion of ATP, which mainly occurs in the blebbing phase of the apoptotic program; and (3) the post-mortem release of the non-histone chromatin-binding protein high mobility group box 1 (HMGB1). 35,36,40 Altogether and in the correct order, these signals promote the uptake of apoptotic corpses by antigenpresenting cells including dendritic cells (DCs), the processing and presentation of dead cell-associated antigens, and the elicitation of an adaptive, interleukin (IL)-1β-, IL-17-, and interferon γ (IFNγ)-dependent immune response against such antigens. 35,36,40 A precise description of the molecular and cellular mechanisms that underlie the immunogenicity of cell death largely exceeds the scope of this Trial Watch and can be found elsewhere. 35,36,40 Importantly, although measuring CRT exposure, ATP secretion, and HMGB1 release can be useful to identify ICDinducing agents, 41,42 vaccination experiments constitute the gold standard approach to characterize immunogenic instances of cell death. 33,36 In this setting, immunocompetent mice are vaccinated with syngeneic cancer cells that are dying in response to a putative ICD inducer, and-one week later-challenged with living cancer cells of the same type. If a majority of these mice does not develop tumors, the malignant cells injected as a vaccine were indeed succumbing to ICD. 36 So far, only a few clinically relevant agents have been shown to trigger bona fide ICD. 43 These agents include (1) cyclophosphamide, an alkylating agent nowadays employed for the treatment of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), breast carcinoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), lymphoma, multiple myeloma (MM), mycosis fungoides, neuroblastoma, ovarian carcinoma, and retinoblastoma; 44 (2) doxorubicin, an anthracycline currently approved by the FDA for use in patients with ALL, AML, breast carcinoma, gastric cancer, lymphoma, MM, neuroblastoma, ovarian carcinoma, small cell lung carcinoma (SCLC), soft tissue and bone sarcomas, thyroid carcinoma, transitional cell bladder carcinoma, and Wilms' tumor; 45,46 (3) epirubicin, an anthracycline commonly employed for the treatment of breast carcinoma; 45,46 (4) idarubicin, an anthracycline licensed by the FDA for use in AML; 46,47 (5) mitoxantrone, an anthracenedione nowadays used in the clinic for the therapy of multiple sclerosis as well as acute leukemia, breast carcinoma, non-Hodgkin lymphoma (NHL), and prostate cancer; 45,46 (6) oxaliplatin, a platinum-containing drug licensed for use in combination with 5-fluorouracil and folinic acid in patients with advanced colorectal carcinoma; [48][49][50] and (7) patupilone (also known as epothilone B or EPO906) a microtubular inhibitor that has not yet been approved for use in humans. 8,51 Indeed, although clinically employed agents including various microtubular inhibitors other than epothilones (e.g., paclitaxel) and cardiac glycosides (e.g., digoxin, digitoxin) are very efficient at converting non-immunogenic instances of cell death into immunogenic ones, they are unable to trigger ICD as standalone interventions. 8,41,42 Along the lines of our Trial Watch series, 52,53 here we discuss recent discoveries related to the use of ICD inducers in cancer patients, presenting high-impact studies that have been published during the last 13 mo. In addition, we summarize the clinical trials that have been initiated in the same period to assess the antineoplastic profile of these immunogenic drugs as off-label therapeutic interventions.

Update on Clinical Reports
As cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, oxaliplatin, and patupilone are all approved by the FDA or other international regulatory agencies for the treatment of specific malignancies, the safety concerns related to the use of these molecules in cancer patients are limited. 54 This translates into a huge number of clinical trials investigating the possibility that ICD inducers might exert robust antineoplastic effects as off-label therapeutic interventions. During the last 13 mo, the results of no less than 44 studies assessing the efficacy of ICD inducers in (partially or completely) off-label clinical settings have been published in peer-reviewed scientific journals (source http://www.ncbi.nlm.nih.gov/pubmed) ( Table 1). In particular, cyclophosphamide has been tested as a possible alternative to standard therapeutic approaches in patients affected by (1) T-cell promyelocytic leukemia, as part of an induction chemotherapeutic cocktail including fludarabine and mitoxantrone; 55 (2) melanoma, according to a metronomic schedule in combination with low-dose IL-2; 56 (3) non-small cell lung carcinoma (NSCLC), as part of a therapeutic regimen encompassing a mucin 1-targeting vaccine; 57 or (4) various solid tumors, including colorectal carcinoma, in combination with low-dose IL-2 and/or tyrosine kinase receptor inhibitors such as sorafenib and imatinib. [58][59][60][61][62] Altogether, the results of these studies suggest that metronomic cyclophosphamide can be safely combined with conventional chemotherapeutic as well as with targeted anticancer agents and often results in immunological responses that may be therapeutically significant, at least in some patients.
The possibility that doxorubicin might promote therapeutic responses in off-label clinical settings has recently been investigated in individuals with (1) hepatocellular carcinoma (HCC), as a standalone palliative regimen compared with 5-fluorouracil, folinic acid, and oxaliplatin; 63 (2) non-metastatic osteosarcoma, in combination with high-dose ifosfamide (an alkylating mustard with a broad antineoplastic activity), cisplatin, and high-dose methotrexate; 64 (3) advanced prostate cancer, in the context of androgen deprivation therapy; 65 (4) tumors of the urothelial tract, either in its pegylated form as a single agent or combined with cisplatin, ifosfamide, and gemcitabine (a nucleoside analog licensed for the treatment of pancreatic cancer, NSCLC, breast carcinoma, and ovarian cancer); 66,67 as well as (5) in pediatric patients affected by relapsed or refractory extracranial nonhematopoietic solid tumors, in the context of oxaliplatin-based chemotherapy. 68 Conversely, epirubicin has been investigated as a potential alternative to standard therapeutic protocols in subjects bearing (1) advanced gastresophageal tumors, in combination with conventional cytotoxic agents and/or panitumumab, an FDA-approved monoclonal antibody specific for the epidermal growth factor receptor (EGFR); 69,70 or (2) transitional bladder carcinoma, as a second-line intervention for individuals who failed cisplatin-and gemcitabine-based first-line chemotherapy. 71 These anthracycline-based chemotherapeutic cocktails were generally well tolerated, but often failed to ameliorate disease outcome as compared with gold-standard therapeutic interventions.
During the last 13 mo, the clinical profile of oxaliplatin as an off label therapeutic intervention has been assessed in patients with (1) aggressive relapsed or refractory CLL, as part of a chemotherapeutic regimen involving fludarabine, cytarabine (a nucleoside analog approved for the treatment of various hematological malignancies), and rituximab (a CD20-targeting monoclonal antibody currently employed against CLL and NHL); 72 (2) refractory germ cell tumors, in combination with bevacizumab; 73 (3) breast carcinoma, combined with capecitabine (the precursor of 5-fluorouracil) or docetaxel (a microtubular inhibitor of the taxane family currently employed against various carcinomas); 74,75 (4) NSCLC, as part of a docetaxel-based chemotherapy; 76,77 (5) advanced nasopharyngeal carcinoma, coupled to radiation therapy; 78 (6) gastric or gastresophageal carcinoma, most frequently in the context of a chemotherapeutic cocktail involving docetaxel, capecitabine, or S-1 (an oral fluoropyrimidine currently approved for the treatment of gastric cancer); [79][80][81][82][83][84][85][86][87][88][89][90] (7) pancreatic, gallbladder, or biliary tract tumors, often in combination with gemcitabine-based chemotherapy; 91-94 (8) ovarian or bladder carcinoma, combined with conventional (often gemcitabine-based) therapeutic interventions; [95][96][97] or (9) various solid tumors, again in combination with cytotoxic chemotherapy. 98,99 Taken together, the results of these clinical trials, most of which were Phase I or II studies, indicate that oxaliplatin exerts promising antineoplastic effects in patients affected by several tumors other than colorectal carcinoma. Large, randomized Phase III trials will have to evaluate the true clinical profile of oxaliplatin in these settings.
During the last 13 mo, several publications have provided novel insights into the signaling pathways that underlie ICD and its translational relevance. For instance, we have dissected the molecular cascades whereby autophagy is responsible for the secretion of ATP in the course of ICD, 100,101 and demonstrated that both ATP and chemokine (C-C motif) ligand 2 (CCL2), [102][103][104] but not tumor necrosis factor α (TNFα), 105 are responsible for the therapeutically relevant accumulation of inflammatory DC-like cells within neoplastic lesions treated with ICD inducers. In addition, we and others have demonstrated a critical role for the gut microbiota in the therapeutic activity of cyclophosphamide and other immunostimulatory regimens. 106,107 The group headed by Patrizia Agostinis showed that in some circumstances autophagy may inhibit, rather than promote, ICD, 108 and that the signaling cascades elicited by hypericinbased photodynamic therapy (another bona fide ICD inducer) are amplified by selected members of the Bcl-2 protein family, 108 including phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, best known as NOXA) but not BCL2-like 11 (BCL2L11, best known as BIM). 109 The reasons why in some cases autophagy is critically required for ICD while in others appears to inhibit it remain unclear, but may relate to differences in the experimental models employed. Indeed, also the molecular cascades that underlie the pre-apoptotic exposure of calreticulin in response to anthracycline and hypericin-based photodynamic therapy overlap to a large extent, but not entirely. 110 Of note, recent data ascribe to the circulating levels of various ICDassociated molecules, including HMGB1 and one of its receptors, advanced glycosylation end product-specific receptor (AGER, best known as RAGE), prognostic or predictive relevance in patients with breast, pancreatic, or colorectal carcinoma. [111][112][113] Additional studies are required to fully understand the prognostic or predictive potential of circulating ICD markers.

Update on Clinical Trials Testing Immunogenic Cell Death Inducers
When this Trial Watch was being redacted (December 2013), official sources listed no less than 255 clinical trials launched after 2012, December 1st to evaluate the therapeutic profile of bona fide ICD inducers in cancer patients (source As part of non-myeloablative conditioning followed by haploidentical BMT NCT01804634 As part of non-myeloablative conditioning followed by ACT NCT01967823 iii Not yet recruiting Combined with celecoxib, etoposide and thalidomide NCT01858571 Recruiting Combined with standard chemotherapy ± G-CSF NCT01987596 waldenström's macroglobulinemia iii Not yet recruiting Combined with rituximab, dexamethasone ± bortezomib NCT01788020 Abbreviations: ACT, adoptive cell transfer; ASCT, allogeneic stem cell transplantation; BMT, bone marrow transplantation; CD137L, CD137 ligand; DSRCT, desmoplastic small round cell tumor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; iL-2, interleukin-2; SBRT; stereotactic body radiation therapy. *between 2012, December 1st and the day of submission. http://www.clinicaltrials.gov). One hundred 53 of these studies (83 involving cyclophosphamide, 63 doxorubicin, 14 epirubicin, 6 idarubicin, 7 mitoxantrone, and 32 oxaliplatin) were performed in on-label clinical settings, and hence will not be further discussed here. In addition, during the last 13 mo, 107 clinical trials have been launched to assess the clinical activity of ICD inducers in off-label settings. Of these studies, 27 involve cyclophosphamide, 17 doxorubicin, 6 epirubicin, 7 idarubicin, and 53 oxaliplatin. Cyclophosphamide is being tested as an off-label intervention (1) in melanoma patients, either as part of a fludarabine-and IL-2containing, non-myeloablative conditioning regimen combined to the adoptive transfer of tumor-infiltrating lymphocytes 24 NCT01955460; NCT01993719; NCT01995344), or given on a metronomic schedule in support of low-dose intravenous IL-2 58,59 (NCT01833767) or an allogeneic melanoma cell line engineered to express the immunostimulatory protein tumor necrosis factor (ligand) superfamily, member 9 (TNFSF9, best known as CD137L) 115 (NCT01898039); (2) in patients with various forms of sarcoma, combined with a vincristine-containing (radio)therapeutic regimen (NCT01864109; NCT01871766; NCT01946529); (3) in children affected by hematopoietic neoplasms, as part of a non-myeloablative conditioning regimen preceding allogeneic stem cell transplantation (ASCT) (NCT01824693), or solid tumors, in combination with other chemotherapeutics (NCT01858571; NCT01987596); (4) in medulloblastoma patients, as part of combinatorial induction or maintenance regimens (NCT01878617; NCT02017964); (5) in individuals with lung carcinoma, either at metronomic doses in support of conventional chemotherapy (NCT01947062) or in combination with a vaccine based on cancer cell-derived autophagosomes 116 (NCT01909752); and (6) in patients affected by a variety of other hematological and solid tumors, including Waldenström's macroglobulinemia (NCT01788020), glioblastoma (NCT01903330), esophageal carcinoma (NCT01795976), renal cell carcinoma (NCT01943188), endometrial carcinoma (NCT01918124), colorectal carcinoma (NCT01966289), and others (NCT01804634; NCT01967823) ( Table 2).
The safety and efficacy of oxaliplatin employed as an off-label therapeutic intervention are being assessed (1) Table 4).Of note, during the  last 13 mo no clinical trial has been initiated to evaluate the therapeutic profile of mitoxantrone in off-label oncological settings, and the status of only one of the studies discussed in our previous Trial Watches dealing with ICD inducers has changed since their publication. 124,125 Thus, official sources now list NCT01701375, testing mitoxantrone in combination with cytarabine and a cyclin-dependent kinase inhibitor (PD 0332991) in adults with relapsed and refractory acute leukemia or high-risk myelodysplastic syndrome, as terminated owing to sponsor withdrawal. Only 2 patients participated into this Phase I study, one of whom experienced relatively serious adverse effects including bone marrow aplasia and hyperbilirubinemia. Both these patients also suffered from less severe toxicities, including grade I-II mucositis (source http://www.clinicaltrials.gov).

Concluding Remarks
It has now become clear that several clinically successful anticancer agents share the unsuspected ability to activate, rather than inhibit, the immune system. 10,11 The molecular and cellular circuitries that underlie such an immunostimulatory activity include ICD, a particular case of apoptosis that results in the activation of an adaptive immune response specific for dead cell-associated antigens. 35,36,43 Cyclophosphamide, doxorubicin, epirubicin, idarubicin, mitoxantrone, and oxaliplatin are all currently approved by the US FDA and other international regulatory agencies for the treatment of some malignancies, and are all able to trigger ICD, as demonstrated by gold-standard vaccination experiments based on syngeneic tumor models. 35,36,43 Also patupilone belongs to the short list of bona fide ICD inducers, 8,9 but has not yet been approved for use in humans. Another epothilone, namely ixabepilone, is currently employed as a standalone therapeutic intervention in anthracycline-, taxaneand capecitabine-resistant breast carcinoma patients, or in combination with capecitabine for the treatment of anthracyclineand taxane-resistant locally advanced or metastatic breast carcinoma, 126,127 yet its ability to promote ICD remains unclear. As a matter of fact, the capacity of a given chemical to trigger the immunogenic demise of cancer cells cannot be anticipated by structural or functional considerations, as compounds that are as similar to each other as cisplatin and oxaliplatin have been shown to differ in this respect. 48,49 In line with this notion, although 7A7, a monoclonal antibody specific for murine EGFR, has been shown to trigger bona fide ICD, 128 whether clinically employed EGFR-targeting agents including panitumumab, cetuximab (2 Abbreviations: 3D-CRT, 3-dimensional conformal radiation therapy; 5-FU, 5 fluorouracil; DOS, docetaxel + oxaliplatin + S1; DOX, docetaxel + oxaliplatin + capecitabine; eOX, epirubicin + oxaliplatin + capecitabine; FLOT, 5-FU + oxaliplatin + docetaxel; FOLFiRiNOX, folinic acid + 5-FU + irinotecan + oxaliplatin; FOLFOX, folinic acid + 5-FU + oxaliplatin; G-FLiP, gemcitabine + 5FU + folinic acid + irinotecan + oxaliplatin; G-FLiP-DM, G-FLiP + docetaxel + mitomycin C; GemAOD, gemcitabine + oxaliplatin + pegaspargase + dexamethasone; GeMBRAX, albumin-bound paclitaxel + gemcitabine; GeMOX, gemcitabine + oxaliplatin; GOFL, gemcitabine + oxaliplatin, folinic acid + 5-FU; HAi, hepatic arterial infusion; HiPeC, hyperthermic intraperitoneal chemoperfusion; iMRT, intensity-modulated radiation therapy; iORT, intraoperative radiation therapy; n.a., not available; NK, natural killer; PBRT, proton beam radiation therapy; PeGPH20, pegylated recombinant human hyaluronidase; SBRT, stereotactic body radiotherapy; SOL, S-1 + oxaliplatin + leucovorin; SOX, S-1 + oxaliplatin; XeLOX, capecitabine + oxaliplatin. *between 2012, December 1st and the day of submission. monoclonal antibodies) 129,130 and erlotinib (a small compound that inhibits EGFR at the enzymatic level) 131,132 promote the immunogenic demise of cancer cells remains to be determined. It is interesting to note that the abovementioned ICD inducers as well as many other currently employed anticancer agents that have an immunostimulatory activity have been identified and developed empirically, based on their ability to mediate relatively selective antineoplastic effects in vitro and in vivo, in immunodeficient mice implanted with human cancer cells. 10 Thus, the immunostimulatory potential of all these compounds has gone unnoticed for decades, in part owing to the experimental models employed until now (and still very diffuse), which were/ are inapt to evaluate such a clinically relevant aspect of the pharmacology of a given agent. This also implies that many potentially efficient anticancer agents have never been fished out of large chemical libraries by standard screening procedures, or have gone lost at subsequent validation steps. We are deeply convinced that preclinical models that involve the immune system (for instance, syngeneic tumors developing in immunocompetent mice), or components thereof (for instance, cancer cells cultured in the presence of dendritic cells, macrophages, T cells, and/or NK cells), 10 and the systematic analysis of the immunological parameters that may affect the clinical response of patients to therapy (immunomonitoring) 133,134 are crucial for the discovery of next-generation chemotherapeutics, i.e., molecules that simultaneously hit cancer cells while exerting potent immunostimulatory effects. We have already bumped into some of these compounds in the past century, it is now time to go actively get the missing ones.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.