Combining low-dose or metronomic chemotherapy with anticancer vaccines: A therapeutic opportunity for lymphomas.

Therapeutic vaccination is regarded as a promising strategy against multiple hematological malignancies including lymphoma. However, this approach alone possesses limited potential for the treatment of established tumors. As several anticancer regimens relies on the combination of multiple drugs, it is reasonable to predict that also cancer vaccination will be most effective in the context of multimodal approaches. In particular, low-dose or metronomic chemotherapy could be coupled to anticancer vaccines to improve the efficacy of this immunotherapeutic interventions. This review summarizes recent findings in support of the use of anticancer vaccines combined with low-dose or metronomic chemotherapy for the treatment and management of lymphoid malignancies.

Introduction

Lymphomas are cancers of hematological origin that can be broadly classified into a Hodgkin’s and non-Hodgkin’s variant. Although most forms of Hodgkin’s lymphoma (HL) are curable with currently available treatment modality, aggressive forms of non-Hodgkin’s lymphoma (NHL) such as diffuse large B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, and mantle cell lymphoma are considered as incurable in advanced stages of the disease, and patients affected by these neoplasms undergo repeated remissions and relapses., Regardless of the subtype and stage of the disease, the treatment currently employed for most lymphomas relies on radiation therapy or combinatorial chemotherapeutic regimens that include prednisone plus the cytotoxic agents cyclophosphamide, doxorubicin, and vincristine as well as a tumor-targeting monoclonal antibody, such as the anti-CD20 agent rituximab. Such an immunotherapeutic regimen is commonly referred to as R-CHOP. Despite the enhanced overall survival exhibited by patients treated with R-CHOP, this regimen exerts substantial undesirable effects and may promote chemoresistance., Hence, less toxic and more effective modalities for the treatment of lymphoma patients are clearly needed.

Conventional chemotherapeutic agents were developed based on their capacity to directly kill malignant cells at a maximal tolerated dose (MTD), the highest amount of the drug associated with tolerable toxicity and manageable side effects. In this context, patients must go through long periods off therapy, allowing for their full recovery from the adverse effects of chemotherapy. These rest periods, however, also allow cancer cells to become resistant to chemotherapy and hence to drive a disease relapse. A new therapeutic paradigm has recently been established based on the administration of low-dose chemotherapeutics at short intervals (so-called, metronomic chemotherapy), in the absence of prolonged drug-free periods., Metronomic chemotherapy not only causes relatively mild and short-lived toxic effects, but also exerts an antiangiogenic activity had have been associated with robust antineoplastic effects in preclinical models of chemoresistant tumors. Accumulating evidence also indicates that some chemotherapeutic drugs given at a low dose are able to promote disease eradication by stimulating anticancer immune and selectively eliminating immunosuppressive cells (Table 1).

Table 1. Use of low-dose chemotherapy to overcome immunosuppression and immune evasion by lymphomas

Escape mechanism	Effects of low-dose chemotherapeutic agents
Reduced immunogenicity10,11	Cyclophosphamide, gemcitabine, doxorubicin- Promote the expression of MHC class I molecules   on cancer cells12- Stimulate the exposure of calreticulin on the cell   surface,13 as well as the release of HMGB113 and ATP14
Apoptosis resistance upon the downregulationof FAS and TRAILRs16,17	Etoposide, doxorubicin, cyclophosphamide- Promote the upregulation of FAS18 and TRAILRs19,21,22
Impaired cytotoxicity of NK cells resulting from the downregulation of NKG2D ligands24,25	Dacarbazine, doxorubicin, cisplatin- Induce the expression of NKG2D29 and CD226 ligands30
Increased frequency of immunosuppressive Tregs41,42 and MDSCs55,56	Cyclophosphamide, doxorubicin, paclitaxel,gemcitabine, 5-fluorouracil- Selectively kill Tregs43-45 and MDSCs57-59
Stimulation of angiogenesis61	Cyclophosphamide, doxorubicin, cisplatin- Promote the apoptotic demise of endothelial cells8- Inhibit the emigration of circulating   endothelial precursor cells from bone marrow65- Reduce the expression of VEGF66,67

Abbreviations: HMGB1, high mobility group box 1; MDSC, myeloid-derived suppressor cell; NK, natural killer; Treg, regulatory T cell; VEGF, vascular endothelial growth factor; TRAILR, tumor necrosis factor α-related apoptosis-inducing ligand receptor.

These studies provided a rationale for using low-dose or metronomic chemotherapy as a means to counteract the numerous mechanisms of immunological tolerance that are set in place by malignant lesions, and hence improve the antitumor efficacy of therapeutic anticancer vaccination. Here, we review preclinical and clinical studies involving a panel of cytotoxic agents used in low doses or according to a metronomic schedule, either as standalone interventions or in combination with therapeutic anticancer vaccines. While focusing our attention on lymphoma-related studies, we describe how low-dose or metronomic chemotherapy may improve the potential of therapeutic anticancer vaccination (Fig. 1).

Figure 1. Immunomodulatory properties of low-dose or metronomic chemotherapy and its potential for generating potent anticancer immune responses in combination with therapeutic anticancer vaccines. Multiple chemotherapeutic agents given at low doses can promote the initiation and/or persistence of tumor-targeting immune responses by upregulating the expression of MHC class I molecules on the surface of cancer cells (A); promoting the exposure or release of endogenous immunostimulatory calreticulin (CRT), ATP and high mobility group box 1 (HMGB1) (B); favoring the apoptotic demise of neoplastic cells by upregulating mannose-6-phosphate (M6P) receptors and death receptors on their surface (C,D); increasing the sensitivity of malignant cells to natural killer (NK) cells by triggering the expression of NKG2D ligands (E); enhancing the secretion of immunostimulatory cytokines that stimulate NKG2D expression on NK cells (F); selectively eradicate immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (G,H); and by exerting anti-angiogenic effects (I,J). Combining low-dose or metronomic chemotherapy with therapeutic anticancer vaccines can thus result in the elicitation of potent innate and adaptive immune responses against malignant cells. APC, antigen-presenting cells; EC, endothelial cell; GZMB, granzyme B; IL, interleukin; IFN, interferon; THBS1, thrombospondin 1; VEGF, vascular endothelial growth factor.

Preclinical Studies on the Immunomodulatory Effects of Low-Dose Chemotherapy

The therapeutic outcome of immunotherapy depends on the interplay between malignant cells and components of the immune system, and it is well recognized that tumors can evade immune responses by a multitude of mechanisms. Low-dose chemotherapy is likely to overcome these obstacles and hence promote the (re)establishment of antitumor immunity (Table 1). Generally, lymphomas are weakly immunogenic due to a decreased expression of MHC class I molecules on the surface of malignant cells, which prevent the recognition of tumor-associated antigens (TAAs) by cytotoxic T lymphocytes (CTLs)., Low-dose cyclophosphamide and gemcitabine have been shown to promote the expression of MHC class I molecules on the surface of cancer cells and thus to facilitate their antigen-dependent CTL-triggered demise. In the same study, the culture supernatants of cancer cells treated with low-dose gemcitabine were shown to promote the maturation and activation of dendritic cells (DCs) in vitro, in turn stimulating the proliferation of CD4+ and CD8+ T cells along with an increased production of interferon (IFN)γ. These findings suggest that low-dose chemotherapy can trigger the immunogenic demise of cancer cells by stimulating them to release immunostimulatory factors as they die. In this regard, Schiavoni and coworkers found that a low concentration of the active cyclophosphamide analog mafosfamide stimulates dying tumor cells to expose calreticulin (CRT) on the cell surface and to release the nuclear protein high mobility group box 1 (HMGB1). These events were deemed as signals for the induction of innate immune responses along with the maturation and expansion of conventional CD8α+ DCs. In a follow-up in vivo study, DCs from cyclophosphamide-treated mice were observed to infiltrate neoplastic lesions and to engulf apoptotic cancer cells before migrating to tumor-draining lymph nodes and eliciting a tumor-specific CTL responses through cross-priming. Doxorubicin has also been shown to trigger immunogenic cell death by facilitating the release of ATP from dying tumor cells, resulting in the activation of DCs and in the priming of anticancer immune responses.

It has recently been discovered that low-dose chemotherapy can increase the susceptibility of cancer to the cytotoxic activity of CTLs by allowing granzymes to enter target cells independently of perforin. In fact, low-dose paclitaxel, doxorubicin, and cisplatin were found to increase the permeability of cancer cells to granzyme B (GZMB) released by CTLs in vitro. This effect was solely attributed to the ability of low-dose chemotherapy to upregulate mannose-6-phosphate (M6P) receptors on malignant cells, hence facilitating the delivery of cytotoxic GZMB molecules. CTLs and natural killer (NK) cells also trigger the apoptotic demise of cancer cells upon the engagement of death receptors including FAS (CD95) and various members of the tumor necrosis factor α (TNFα)-related apoptosis-inducing ligand receptor (TRAILR) superfamily, with the respective ligands (e.g., FASL and TRAIL). However, several reports have revealed a marked downregulation or loss of FAS and TRAILRs in certain lymphomas, allowing malignant cells to evade the immunosurveillance mediated by CTLs and NK cells., Several chemotherapeutic drugs employed at low doses have been shown to restore the expression of these death receptors. For example, a low dose of etoposide reportedly upregulates the expression of FAS on the surface of human monocytic leukemia U937 cells, resulting in an increased FAS-dependent apoptotic responses. Low concentrations of doxorubicin or etoposide significantly increased TRAILR2 (also known as DR5) expression on human solid cancer cells, sensitizing them to TRAIL-mediated killing., Along similar lines, low-dose doxorubicin restored the sensitivity to TRAIL of an originally TRAIL-resistant variant of human prostatic cancer LNCaP cells upon the upregulation of TRAILR1 (also known as DR4). Furthermore, a single, low dose of cyclophosphamide sensitized malignant mesotheliomas to TRAIL-dependent CD8+ T cell- and NK cell-mediated cytotoxicity in vivo, resulting in the suppression of tumor growth.

NK cells play an important role in the recognition and elimination of (pre)malignant cells. Activating killer cell lectin-like receptor subfamily K, member 1 (KLRK1, best known as NKG2D) receptors can unleash the cytotoxic functions of NK cells, yet only when these signals overcome inhibitory signals delivered by MHC class I molecules or killer inhibitory receptor (KIR) ligands. NKG2D ligands are generally absent from normal cells, but are expressed on the surface of stress, infected or malignant cells. Lymphoma patients often exhibit decreased NK-cell activity, resulting in impaired cytotoxicity against malignant cells., It has been reported that malignant lymphomas are characterized by a significant downregulation of the NKG2D ligand MHC class I polypeptide-related sequence A (MICA) as this sheds from the plasma membrane, and patients with elevated circulating levels of MICA and similar molecules exhibit impaired NK-cell function., Once shed, these molecules are known to circumvent NK-dependent immunosurveillance mechanisms by blocking NKG2D receptors and promoting their downregulation., To elicit productive immune responses, lymphomas must be visible to NK cells and susceptible to their killing. Recently, dacarbazine was shown to trigger the expression of NKG2D ligands on the surface of cancer cells, leading to the activation of NK-cell cytotoxic functions and to IFNγ secretion in mice with melanoma. IFNγ boosts the immunogenicity of cancer cells by increasing the levels of MHC class I molecules on their surface and hence promoting their killing by CTLs. Interestingly, upon exposure to low-dose doxorubicin and cisplatin, multiple myeloma cells exhibit an increase susceptibility to NK-cell cytotoxicity, at least in part due to the upregulation of ligands for NKG2D and CD226, which is also involved in NK cell-mediated tumor cell killing.

Several cytokines including IFNα, interleukin (IL)-2, IL-12, and IL-15 have been shown to upregulate NKG2D receptors, a process that is associated with an amplification of NKG2D-mediated antitumor response.- Interestingly, low-dose cyclophosphamide has been reported to stimulate the expression of type 1 IFNs, i.e., IFNα and IFNβ, which are important stimuli for the proliferation and persistence of CD8+ T cells. Mice pretreated with low-dose cyclophosphamide were also found to produce immunostimulatory cytokines including IL-1β, IL-2, IL-7, IL-15, IL-21, IFNγ, and granulocyte macrophage colony-stimulating factor (GM-CSF), while secreting decreased amounts of immunosuppressive cytokines, such as IL-4 and IL-10. These immunological effects of low-dose cyclophosphamide exacerbated the antineoplastic potential of adoptively transferred immune cells against melanoma. Taken together, these observations indicate that some cytotoxic drugs such as cyclophosphamide at low doses can enhance the secretion of immunostimulatory cytokines, which in turn promote the expression of NKG2D receptors on the surface of NK cells to ultimately boost antitumor immunity.

Selective Eradication of Immunosuppressive Cell Populations by Low-Dose Chemotherapy

Regulatory T cells

Regulatory T cells (Tregs), which constitute about 5% of peripheral CD4+ T cells in both mice and humans, play a key role in the maintenance of peripheral tolerance and prevent the emergence of pathological autoimmune reactions. In tumor-bearing hosts, Tregs facilitate disease progression by curtailing NK and T-cell immunity through various mechanisms, including the expression of the inhibitory checkpoint regulator cytotoxic T lymphocyte-associated protein 4 (CTLA4) on their surface and the production of immunosuppressive mediators such as IL-10 and transforming growth factor β1 (TGFβ1).- Notably, lymphoma patients exhibit elevated levels of Tregs, and this contributes to the resistance of malignant cells to immunosurveillance., Interventions aimed at enhancing antitumor immune responses based on the depletion or neutralization of Tregs by chemotherapy have been intensively studied. Thus, low-dose cyclophosphamide was found to selectively kill Tregs in vivo, in a murine model of lymphoma. In this setting, effector T cells were activated along with Treg depletion and increased expression of IL-2. When mice with colon cancer were treated with low-dose cyclophosphamide plus doxorubicin, a significant suppression of tumor growth was associated with Tregs depletion and decreased secretion of TGFβ1 within the tumor microenvironment. Another study has demonstrated that low-dose paclitaxel decreases the viability of Tregs and their capacity to produce immunosuppressive cytokine in vivo, in a murine model of lung cancer, while leaving conventional T cells untouched.

Myeloid-derived suppressor cells

Under steady-state conditions, highly plastic immature myeloid cells arise from multipotent hematopoietic stem cells (HSCs) and develop into mature macrophages, dendritic cells, and granulocytes with diverse functions, through sequential steps of differentiation. Conversely, factors produced in the tumor microenvironment interfere with normal myelopoiesis, resulting in the accumulation of immature myeloid cells that exert immunosuppressive functions, which are cumulatively known as myeloid-derived suppressor cells (MDSCs), in the circulation, lymphoid tissues as well as within neoplastic lesions. In tumor bearing mice, co-expression of surface markers Gr-1+ and CD11b+ are commonly found on MDSCs. In humans, 2 major MDSC subsets have been characterized: CD11b+D14+HLA-DRloCD33+CD124+ monocytic MDSCs (M-MDSCs) and, CD11b+CD14−CD66b+CD15+CD124+ granulocytic/polymorphonuclear MDSCs (G-MDSCs/PMN-MDSCs)., The accumulation of these MDSCs in the peripheral blood and within neoplastic lesions has been reported in a large number of cancer patients.- MDSCs suppress innate and adaptive antitumor immune response by favoring the recruitment and expansion of Tregs, as well as by producing high levels of L-arginase, reactive oxygen species (ROS), inducible nitrogen oxide synthase (iNOS), and various immunosuppressive cytokines. Overall, these enzymes and soluble mediators promote neoangiogenesis and allow for the escape of immune cells from immunosurveillance, hence promoting disease progression.,- Of note, in lymphoma patients, an increased frequency of circulating MDSCs has been reported to correlate with aggressive disease and the inhibition of antitumor immunity.,

Clearly, both these immunosuppressive cell populations must be inhibited or eliminated in order to restore therapeutically-relevant antitumor immune responses. Some low-dose or metronomic chemotherapeutic regimens have been shown to selectively kill immunosuppressive cell populations. For example, low-dose gemcitabine appears to selectively inhibit tumor-associated MDSCs in mice bearing 4T1 mammary carcinomas, favoring the expansion of tumor-targeting T cells. Along similar lines, low-dose 5-flurouracil has been shown to provoke a major decrease in splenic and tumor-infiltrating MDSCs in mice bearing EL4 thymomas while leaving T cells, NK cells, dendritic cells, and B cells unaffected. Interestingly, the elimination of MDSCs by 5-flurouracil was associated with a boost in antitumor immunity, manifesting with an increment in IFNγ production by tumor-specific CD8+ T cells. Of note, metronomic chemotherapy with low-dose cyclophosphamide and gemcitabine has been demonstrated to deplete tumor-associated Tregs and MDSCs in mice bearing CT26 colon carcinomas, leading to antitumor T-cell response and tumor eradication, at least in some animals. As previously mentioned, doxorubicin is known for its ability to induce immunogenic cell death by allowing for the release of ATP from dying cancer cells and hence favoring the elicitation of antitumor immune responses. The release of ATP from malignant cells succumbing to doxorubicin was found to induce the differentiation of tumor-infiltrating myeloid cells toward CD11c+CD11b+Ly6Chi cells sharing some features with DCs, including the ability to efficiently engulf TAAs in situ and to elicit cellular immune responses against cancer cells. Taken together, these observations suggest that low-dose chemotherapy is a promising approach to restore antitumor immunity by eliminating or inhibiting immunosuppressive Tregs and MDSCs, thus promoting the differentiation of antigen-presenting cells and the elicitation of tumor-targeting CTL responses.

Anti-Angiogenic Effects of Low-Dose or Metronomic Chemotherapy

The expansion of the tumor vasculature is vital for the growth of hematological malignancies and increased angiogenesis is often correlated with poor prognosis in lymphoma patients., The major mediator of the angiogenic switch is vascular endothelial growth factor (VEGF), which is produced by lymphoma cells and binds VEGF receptors expressed on endothelial cells from pre-existing capillaries as well as on circulating endothelial precursor cells of bone marrow derivation. Metronomic chemotherapy has been shown to mediate beneficial anti-angiogenic effects. For example, Browder et al. administered cyclophosphamide on a metronomic schedule to mice affected by Lewis lung carcinoma or L1210 leukemia, and observed a substantial apoptotic response among endothelial cells of the growing tumor vasculature, resulting in the inhibition of angiogenesis. This effect was lost when the drug was given at the MTD. In addition, metronomic cyclophosphamide was 3 times more effective than the same drug given as a standard regimen in treating neoplastic lesions developing from cancer cells that had been made cyclophosphamide-resistant before inoculation.

Adopting the same metronomic schedule, Hamano et al. found that the anti-angiogenic effect of low-dose cyclophosphamide is mediated by the selective upregulation of thrombospondin 1 (THBS1) in malignant cells and tumor-associated endothelial cells. THBS1 is an endogenous inhibitor of angiogenesis that arrests the proliferation and triggers the apoptotic demise of endothelial cells. This finding was supported by results from Bocci and collegues, who observed that the plasma of SCID mice bearing human tumors contains higher levels of THBS1 upon the administration of metronomic low-dose cyclophosphamide. Most importantly, the anti-angiogenic and antitumor effects of metronomic cyclophosphamide were diminished in Thbs1−− mice, further corroborating the notion that THBS1 is a pivotal mediator of the therapeutic activity of low-dose cyclophosphamide. In addition, Bertolini et al. reported that the administration of metronomic cyclophosphamide to mice harboring human lymphomas decreases the abundance and viability of endothelial precursor cells, resulting in a significant reduction of tumor growth as compared with cyclophosphamide given at its MTD. Furthermore, metronomic doxorubicin as well as metronomic cisplatin have been shown to reduce the expression of VEGF, leading to a significant inhibition of tumor growth in murine models of adenocarcinoma and hepatocarcinoma.

Preclinical and Clinical Studies of Low-Dose Chemotherapy Alone and in Combination with Anticancer Vaccines

The efficacy of low-dose chemotherapy against lymphoma has been demonstrated in several preclinical studies. Mice bearing A20 lymphomas exhibted a prolonged survival and reduced rates of tumor growth upon treatment with low-dose cyclophosphamide (20 mg/kg). Bertolini et al. also observed a significant inhibition of tumor growth in mice bearing human mantle lymphomas upon the administration of cyclophosphamide in drinking water as a continuous low-dose of 20 mg/kg/day. In rat models of lymphoma, Rozados et al. showed that the metronomic administration of low-dose cyclophosphamide (10 mg/kg) i.p. results in tumor eradication in the absence of overt toxicity. This finding indicates that metronomic chemotherapy is an effective and safe alternative to conventional anticancer regimens.

Based on the results of these and other preclinical studies, numerous clinical trials have assessed the antineoplastic profile of metronomic chemotherapy in lymphoma patients. For instance, a regimen consisting of daily low-dose lomustine and chlorambucil, daily subcutaneous bleomycin and vincristine, plus methotrexate and dexamethasone given on an eight-week cycle (LBCMVD-56) has been evaluated in patients with refractory/relapsed lymphoma. The overall response rate (ORR) of these patients was 67%, encompassing 21% of complete responses (CRs), with a median overall survival of 13 mo. In a similar approach, Coleman et al. reported the clinical efficacy of continuous, low-dose oral prednisone (20 mg), etoposide (50 mg), procarbazine (50 mg), and cyclophosphamide (50 mg) (PEP-C) administered either daily, on alternate days, or on a fractionated basis, in heavily pretreated patients with refractory/relapsed lymphoma. In this study the ORR was 75%, 38% of patients achieved a CR, and minimal toxicity was reported. These clinical findings demonstrate that continuous low-dose chemotherapy is effective and well-tolerated by lymphoma patients. Objective responses and prolonged progression-free survival have also been reported among Hodgkin’s lymphoma patients treated with a combination of low-dose daily oral cyclophosphamide, weekly vinblastine and rofecoxib. As demonstrated in both preclinical and clinical settings, the efficacy of metronomic chemotherapy mainly relies on anti-angiogenic effects,,- coupled to the selective eradication of Tregs, enabling the restoration of antitumor T-cell and NK-cell activity.

Therapeutic vaccines, given their specificity, low toxicity and capability to harness the patient’s immune system to fight cancer, are being recognized as a potential complementary approach against solid and hematopoietic malignancies. Therapeutic vaccination is an active for of immunotherapy that can promote the elicitation of tumor-specific cellular immune responses, the production of tumor-targeting polyclonal antibodies as well as the establishment of memory immune responses against multiple TAAs, hence limiting the emergence of tumor immune escape variants. In a pilot study, Di Nicola et al. vaccinated indolent NHL patients undergoing disease relapse with autologous dendritic cells loaded with tumor cells. At a median follow up of 50.5 mo, 6 out of 18 patients manifested objective clinical responses including 3 CRs and 3 partial responses (PRs). Impressively, clinical responses were associated with a reduction in the levels of Tregs coupled to the induction of NK, T, and B-cell responses. BiovaxID, a patient-specific therapeutic anti-idiotype (ID) lymphoma vaccine conjugated to keyhole limpet hemocyanin (KLH), has recently been shown to provide clinical benefits to patients in a phase III study. At a median follow-up of 56.6 mo, the administration of ID-KLH together with GM-CSF to follicular lymphoma patients achieving a CR upon chemotherapy was associated with an impressive disease-free survival of 44.2 mo, compared with 30.6 mo for the control arm. Nevertheless, it is becoming apparent that anticancer vaccines administered as standalone therapeutic interventions have limited potential for the treatment of established tumors, owing to the intrinsic and/or acquired ability of neoplastic lesions to suppress the antitumor immune responses that are elicited by vaccination. Hence, low-dose or metronomic chemotherapy might be used in conjunction with anticancer vaccines in order to promote beneficial immunomodulatory effects and abrogate tumor-induced immunosuppression, thereby boosting vaccine-induced immune responses and achieving therapeutically-relevant antineoplastic effects.

The proof-of-principle in support of the use of this combinatorial immunochemotherapeutic strategy in lymphoma patients has been provided in a study in which a dendritic cell-based vaccine coupled to low-dose cyclophosphamide for 2 consecutive days was given to mice bearing A20 lymphomas, resulting in a significant improvement of survival and complete, long-term tumor regressions. Moreover, most of the mice in which the primary tumor was eradicated by combinatorial immunochemotherapy were resistant to a secondary tumor challenge, pointing to the development of long-lasting memory T-cell responses in response to vaccination. This approach was also shown to be effective at eradicating tumors that recurred after a period of regression following the initial vaccination. Along similar lines, a combination of low-dose cyclophosphamide and oligodeoxynucleotides containing unmethylated CG sequences administered intratumorally cured widely disseminated B-cell lymphomas in mice. The authors of this study found that CpG oligodeoxynucleotides enhance the presentation of TAAs by local DCs and hence induce tumor-specific CD8+ T-cell responses that are able to eradicate large and systemic lymphomas.

Our group has investigated the efficacy of combining metronomic cyclophosphamide with an NKT cell adjuvant-based vaccine against Eµ-myc-driven B-cell lymphomas in mice. In this setting, a single therapeutic vaccination with irradiated, α-galactosylceramide-loaded autologous tumor cells induced a large systemic spike of IFNγ coupled to a transient expansion of peripheral NKT and NK cells, globally leading to the suppression of tumor growth and to a prolonged protection against Eµ-myc-driven lymphoma. However, the therapeutic response to the vaccine was not durable and most mice succumbed to delayed disease relapse. In our latest study, cyclophosphamide was given to mice bearing Eµ-myc-driven lymphoma according to metronomic low-dose regimen (5 mg/kg, on alternative days for 2 weeks) prior to vaccination, in order to investigate the benefits of such a combinatorial immunochemotherapeutic approach. In preliminary experiments, mice that received cyclophosphamide followed by the NKT cell adjuvant-based vaccine were cured from disease, with no evidence of relapse. Conversely, mice treated with either the vaccine or metronomic chemotherapy alone succumbed to the lymphoma (Heng Sheng Sow, personal communication). These findings suggest that metronomic chemotherapy can enhance the efficacy of NKT adjuvants against B-cell lymphoma. The mechanisms underlying this effects, however, remain to be determined.

Published preclinical observations and our preliminary findings provide a rationale for combining low-dose or metronomic chemotherapy with anticancer vaccination for treating lymphoma. However, to the best of our knowledge, no clinical studies published so far have evaluated this strategy in lymphoma patients. Conversely, the therapeutic efficacy of this combinatorial immunochemotherapeutic approach has been explored in patients bearing solid tumors. Enhanced vaccine-induced immune responses have been observed in breast carcinoma patients treated with an allogeneic GM-CSF-secreting vaccine along with low doses of cyclophosphamide, given one day prior to vaccination, and doxorubicin, adminitered 1 week later. Moreover, the combination of metronomic cyclophosphamide with methotrexate and a 1E10 anti-idiotype vaccine has been reported to increase the clinical responses achieved by patients with metastatic breast cancer. Kandalaft et al. reported that the combination of a dendritic cell-based autologous vaccine with bevacizumab and metronomic cyclophosphamide provides clinical benefits to advanced recurrent ovarian cancer patients. In light of the feasibility and potential clinical activity demonstrated by this approach in patients with solid tumors, the possible therapeutic benefits of active immunotherapy and low-dose or metronomic chemotherapy for lymphoma patients are worthy further investigation.

Concluding Remarks

As demonstrated in multiple preclinical and clinical studies, including those discussed in this review, the multipronged immunostimulatory effects and good safety profile of low-dose or metronomic chemotherapy have gradually led to its use in cancer patients as an alternative to chemotherapy based on conventional doses and administration schedules. We speculate that low-dose or metronomic chemotherapy used in combination with therapeutic anticancer vaccines or other forms of immunotherapy will ameliorate disease outcomes as compared with standard, high-dose chemotherapy or immunotherapy administered as a standalone intervention. Additional studies are required to determine the repertoire of immunostimulatory effects mediated by various cytotoxic agents given at low doses or according to a metronomic schedule as well as the optimal dosage, delivery route, and schedule at which such effects can be obtained for combining these drugs with anticancer vaccines. The combination of anticancer vaccines with low-dose chemotherapy nowadays stands out as a promising therapeutic approach against a range of neoplasms, including lymphomas.