Introduction/background
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Granulocyte-macrophage-colony-stimulating factor (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF) belong to the family of hematopoietic cytokines. Their activities include stimulating the proliferation of granulocyte and/or macrophage progenitor cells, influencing differentiation, inducing maturation, and stimulating the functional activity of mature hematopoietic cells ([@b53-btt-2-13]; [@b76-btt-2-13]; [@b75-btt-2-13]). Colony stimulating factors (CSF) are not only important as mediators of the cellular response to immunologic or infectious insults but are also essential for maintaining basal hematopoiesis ([@b27-btt-2-13]; [@b32-btt-2-13]). G-CSF-deficient mice manifest a chronic neutropenia and an impaired response to infectious challenge leading to premature death ([@b68-btt-2-13]). GM-CSF-deficient mice, on the other hand, have normal levels of steady-state blood cell production, but exhibit defective phagocytosis and decreased oxygen radical production by granulocytes and macrophages; responses which are essential for the anti-bacterial defense ([@b116-btt-2-13]; [@b132-btt-2-13]; [@b66-btt-2-13]). These mice also exhibit decreased tumor necrosis factor-alpha and leukotriene secretion, abnormal para-bronchial accumulations of B and T lymphocytes, and decreased catabolism of alveolar surfactant lipids and proteins leading to a syndrome reminiscent of pulmonary alveolar proteinosis ([@b116-btt-2-13]; [@b84-btt-2-13]). Double G-CSF and GM-CSF knockout mice exhibited a greater degree of neutropenia, and had an increased mortality rate in the early post-natal period compared to mice deficient in G-CSF alone ([@b105-btt-2-13]). [@b30-btt-2-13] found that mice deficient in GM-CSF and gamma interferon (IFN-γ) acquired lymphoproliferative disorders and solid tumors in a background of chronic inflammation, supporting the relationship between inflammation and carcinogenesis.

Recombinant GM-CSF has made significant contributions in the supportive care of cancer patients, owing to enhanced myeloid recovery after cytotoxic chemotherapy. Recently GM-CSF has been successfully included in mobilization regimens for hematopoietic progenitor cell transplantation ([@b13-btt-2-13]; [@b70-btt-2-13]). Recent data on the effects of GM-CSF on dendritic cells has led to growing interest in its use as primary immunotherapy. The ability of GM-CSF to generate of type 1 dendritic cells (DC1), which can skew T-cells toward a Th-1 phenotype has been demonstrated and is an attractive approach toward generating anti-tumor effects ([@b34-btt-2-13]). Peripheral blood mononuclear cells (PBMC), T-cells and antigen presenting cells (APC) cultured with GM-CSF exhibited increased production of type 1 cytokines (interleukin-12, interferon-alpha, tumor necrosis factor-alpha) and decreased production of type 2 cytokines (interleukin-4 and interleukin-10) compared to cells treated with control media or G-CSF ([@b29-btt-2-13]). In addition, APC treated with GM-CSF induced higher proliferation of allogeneic T-cells compared to APC treated with G-CSF or control media ([@b29-btt-2-13]). The capacity of GM-CSF to skew the immune system toward Th1 effects in vitro suggests a role for GM-CSF in cell-mediated immune therapy and is currently being tested in-vivo. Dendritic cells (DC) have come to be recognized as the sensors of tissue injury, infection, or malignant transformation and as the agents responsible for the initial activation of the immune response ([@b74-btt-2-13]). DC are the antigen presenting cells of the immune system, have the capacity to express both HLA class I and HLA class II restricted peptides, and express the co-stimulatory molecules needed for T-cell activation ([@b6-btt-2-13]). A number of reports have shown that administration of GM-CSF can induce anti-tumor immune responses and tumor regressions. These immune activities are attributed to the action of GM-CSF on DC ([@b28-btt-2-13]; [@b130-btt-2-13]; [@b22-btt-2-13]). The limitations of GM-CSF as an immune adjuvant, and its modest clinical activity, have been attributed to discordance between the observed immune response measured in the laboratory and the clinical correlates of anti-tumor activity. The need to generate the co-stimulatory signals required to break immune tolerance, the proper dosing and timing of cytokines, the state of disease at time of treatment, and the role of concomitant chemotherapy are the topics of current investigation. In this review, we will discuss the role of GM-CSF as primary treatment and as an immune adjunct to the therapy of cancer.

Relationship between cancer and inflammation
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The connection between inflammation and malignant transformation has been recognized for over a century. In 1863, Dr. Rudolf Virchow recognized a possible relationship between chronic inflammation and the development of cancer, based on observations of inflammatory infiltrates and spontaneous regression of malignant tumors ([@b4-btt-2-13]; [@b104-btt-2-13]). In the late 1890s, William Coley, a surgeon in New York, observed tumor regressions in patients with cancer who recovered from acute skin infections. He then developed a vaccine composed of extracts of inactivated bacteria, which he administered to cancer patients with variable results ([@b80-btt-2-13]). He observed that tumor regressions were more common among patients who developed both a local and a systemic inflammatory response ([@b49-btt-2-13]). The premise was that non-specific activation of the immune system could lead to (specific) cytokine-mediated anti-tumor effects. While cytokines are credited with potent anti-tumor effects, a counteracting effect of tumor-secreted cytokines, and tumor-associated tolerizing T-cells, or immature DC has also been documented ([@b86-btt-2-13]; [@b126-btt-2-13]). Some solid tumors, for example, are capable of inducing immune tolerance via down-regulation of antigen-specific T-cell responses by tolerigenic APC ([@b20-btt-2-13]). Immature tumor-infiltrating DC are capable of compromising the tumor-specific immune response in draining lymph nodes ([@b86-btt-2-13]). The premise that the immune system can be manipulated in vivo supports a role for the manipulation of the cytokine/co-stimulatory signal milieu in the treatment of cancer using recombinant cytokines, such as GM-CSF.

Approaches will need to account for the level of maturation of tumor-associated DC, and the number of tumor-associated regulatory T-cells
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GM-CSF may enhance tumor-specific antigen presentation leading to better recognition of tumors by the immune system via effects on DC. However, its benefit has been limited to patients with minimal residual disease, and dose-escalation has been limited by significant systemic toxicities. The optimal use of cytokines may be directly in the tumor micro-environment. Furthermore, better quantitative measures of antigen-specific immune responses are needed ([@b57-btt-2-13]).

Role of GM-CSF in supportive therapy
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Numerous clinical trials have established the role of CSF in the prevention and treatment of febrile neutropenia ([@b37-btt-2-13]; [@b77-btt-2-13]; [@b90-btt-2-13]). The majority of clinical trials investigated the role of G-CSF in the supportive care of cancer patients; therefore, the current recommendations do not address the specific use of GM-CSF in this setting. The American Society of Clinical Oncology (ASCO) has provided guidelines for the use of CSF (either G-CSF or GM-CSF) in patients receiving chemotherapy for solid and hematological malignancies ([@b113-btt-2-13]). No recommendation was made regarding the equivalency of G-CSF and GM-CSF. Rowe et al reported a significant reduction of infectious complications in patients with acute myeloid leukemia (AML) who received GM-CSF after induction chemotherapy, compared to placebo ([@b94-btt-2-13]). However, Zittoun et al (2006) reported a decreased complete response (CR) rate in patients with AML who received GM-CSF with induction chemotherapy, indicating that the routine use of cytokines for acceleration of hematopoietic recovery may not always be indicated; and the use of CSF for priming of leukemic cells is not recommended.

Dendritic cells as regulators of immune responses
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DC play a central role in the initiation of innate and adaptive immune responses. Pattern recognition receptors, known as toll-like receptors (TLR), on the surface of DC are bound by proteins, lipids, and nucleic acids resulting in DC activation ([@b56-btt-2-13]; [@b25-btt-2-13]). Antigen-specific T-cell immune responses are initiated by DC when these bound antigens are internalized, degraded, and presented as processed peptides on the surface of HLA molecules ([@b46-btt-2-13]; [@b5-btt-2-13]; [@b6-btt-2-13]). Two main categories of peripheral blood and bone marrow derived DC have been described in humans, type 1, myeloid dendritic cells (DC1), and type 2 plasmacytoid dendritic cells (DC2) ([@b45-btt-2-13]). DC1 and DC2 differ in the type of cell differentiation markers and TLR expressed on their surface, their cytokine milieu, and their effect on polarizing T-cell immune responses ([@b92-btt-2-13]; [@b1-btt-2-13]; [@b25-btt-2-13]). The maturation status of DC is an important determinant of the type of immune response generated upon DC activation. For example, antigen presentation by immature DC leads to generation of anergic CD4+ T-cells ([@b63-btt-2-13]), and immuno-suppressive CD8+ T-cells with antigen-specificity ([@b42-btt-2-13]). Immature DC2 progenitors play a crucial role in the response to viral infection by releasing large amounts of alpha and beta interferon ([@b106-btt-2-13]; [@b35-btt-2-13]; [@b64-btt-2-13]; [@b16-btt-2-13]). The targeting of DC by synthetic TLR ligands is a topic of current clinical and pre-clinical research.

Generation of dendritic cells by GM-CSF
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Colony-stimulating factors can differentiate hematopoietic progenitor cells into specific DC lineages ([@b97-btt-2-13]; [@b45-btt-2-13]; [@b83-btt-2-13]; [@b92-btt-2-13]; [@b106-btt-2-13]; [@b10-btt-2-13]; [@b34-btt-2-13]). Hematopoietic stem cells cultured in GM-CSF and Flt3 can differentiate along a myelomonocytic lineage into DC1 ([@b34-btt-2-13]). CD14+ progenitor cells cultured in GM-CSF and IL-4 can also differentiate into immature DC1 ([@b34-btt-2-13]; [@b7-btt-2-13]). In contrast, treatment of hematopoietic progenitors with G-CSF and IL-3 can mobilize large numbers of (plasmacytoid) DC2 ([@b3-btt-2-13]). Thus, while GM-CSF and G-CSF have similar effects on the mobilization of neutrophils, they have significantly different effects on the mobilization and differentiation of DC1 and DC2, with culture in GM-CSF leading to differentiation of progenitors into DC1, and culture in G-CSF leading to differentiation of progenitors into DC2 ([@b3-btt-2-13]). Due to their effects on DC1 and DC2, these 2 cytokines are optimal agents for cellular immune therapy.

Cytokines and peripheral blood hematopoietic progenitor cell transplantation
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Peripheral blood as a source of stem cells for clinical stem cell transplantation was introduced by Korbling et al and Kessinger et al in the 1980s ([@b61-btt-2-13]; [@b58-btt-2-13]). CSF are now widely used for the mobilization of hematopoietic progenitor cells into the peripheral circulation, allowing collection of CD34+ cells for autologous and allogeneic hematopoietic progenitor cell transplantation. Essential differences have been noted between bone marrow (BMT) and peripheral blood grafts, owing to differences in the ratio of early pluripotent, self-renewing stem cells to lineage-committed, late progenitor cells, and the accessory cells in the grafts ([@b60-btt-2-13]). These differences may account for the observed differences in clinical outcomes after transplantation. A study by the European working group for blood and marrow transplant reported similar rates of overall survival, leukemia-free survival, and similar incidence of graft versus host disease (GVHD) when comparing bone marrow with cytokine-mobilized peripheral blood grafts. They did, however, observe improved platelet recovery with cytokine-mobilized peripheral graft compared to BMT ([@b102-btt-2-13]). A randomized, multi-center trial of cytokine-mobilized peripheral blood progenitor cell grafts versus BMT reported by [@b103-btt-2-13], found an increased risk of acute and chronic GVHD among recipients of cytokine-mobilized peripheral blood grafts, but no difference in survival compared to recipients of bone marrow transplants. In contrast, in another randomized trial comparing peripheral blood hematopoietic progenitor cell and bone marrow transplantation, Bensinger et al found more rapid neutrophil and platelet recovery and similar rates of acute and chronic GVHD. In that trial, the duration of chronic GVDH was longer among recipients of blood progenitor cell grafts. Furthermore, recipients of blood progenitor cell grafts had a higher estimated probability of overall survival and 2-year disease-free survival ([@b9-btt-2-13]). Interestingly, Urbini and colleagues found a higher number of CD34+ cells in peripheral blood grafts mobilized with G-CSF compared to bone marrow, and the dose of CD34+ cells infused correlated with the number of DC1 in peripheral blood grafts and DC2 in bone marrow allografts. In addition, among recipients of cytokine mobilized grafts, a significantly shorter overall survival and a trend toward lower disease free survival was noted among recipients of larger numbers of CD34+ cells ([@b118-btt-2-13]).

The fact that the incidence of acute GVHD in recipients of peripheral blood progenitor cell grafts was similar to, or only slightly higher than in BMT recipients, despite the higher content of T cells in the peripheral blood grafts, suggests that there may be quantitative differences in the other immune cellular components of peripheral blood grafts; such as, the presence of increased numbers of immunosuppressive DC2 in G-CSF mobilized peripheral blood grafts compared to bone marrow grafts ([@b3-btt-2-13]).

Clinical outcomes after hematopoietic progenitor cell transplantation: contribution of dendritic cells
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Recent growing interest in the role of accessory cells in hematopoietic progenitor cell grafts has led to further studies of the immune-modulatory effects of CSF on the constituents of the peripheral blood progenitor cell graft. Flt3 and GM-CSF administration led to mobilization of increased numbers of DC1 in the cellular apheresis product ([@b38-btt-2-13]). In contrast, administration of G-CSF lead to mobilization of increased numbers of DC2 cells in the grafts ([@b3-btt-2-13]). The clinical consequences of mobilizing more DC1 with GM-CSF and more DC2 with G-CSF remain unknown, and are the subject of current investigation.

Waller et al tested the hypothesis that the cellular constituents of the graft could affect clinical outcomes after bone marrow transplantation ([@b125-btt-2-13]). They performed a retrospective study of 113 patients with hematological malignancies who received non-T cell-depleted bone marrow grafts from HLA-matched siblings. After evaluating patient and disease-specific characteristics, characteristics of the graft constituents, and clinical outcomes, they reported that patients who received larger numbers of donor DC2 had significantly worse clinical outcomes, with lower event-free survival, less chronic GVHD, and an increased incidence of relapse than their counterparts who received fewer numbers of DC2 cells ([@b125-btt-2-13]).

The significance of absolute numbers of DC in post-transplant outcomes was also evaluated in a clinical study by Reddy and colleagues ([@b89-btt-2-13]). Fifty patients undergoing allogeneic transplantation for hematological disorders were evaluated. After evaluating the constituents of the grafts, as well as the type and number of DC in the peripheral blood early after engraftment, they noted that patients with lower absolute numbers of DC (\<4.97 cells/μL) at engraftment had worse clinical outcomes compared to patients with higher numbers of DC at engraftment, with lower overall survival (p = 0.002), increased incidence of relapse (p = 0.002), and a higher incidence of acute GVHD (p = 0.0005). Multivariable analysis confirmed that low DC count was independently associated with death (hazard ratio \[HR\], 3.8; p = 0.02), time to relapse (HR, 11.6; p = 0.001), and acute GVHD (HR, 3.3; p = 0.04). The effect was similar when DC1 were analyzed separately. However, when DC2 were analyzed separately, the effect was only significant for increased incidence of acute GVHD among patients with lower numbers of DC2 at engraftment. The independent effect of DC1 and DC2 was not confirmed in the multivariate analysis ([@b89-btt-2-13]).

Subsequently, Lonial and colleagues hypothesized that the combination of G-CSF and GM-CSF administered for the mobilization of stem cells after chemotherapy would reduce the content of DC2 in the autologous blood hematopoietic progenitor cell grafts compared with administration of G-CSF alone after chemotherapy ([@b70-btt-2-13]). They randomized 35 patients with lymphoma and multiple myeloma to receive either G-CSF or the combination of G-CSF plus GM-CSF after chemotherapy. Blood hematopoietic progenitor cell grafts were collected by large volume apheresis. They found a similar incidence of cytokine-related adverse events, and similar numbers of stem cells mobilized between the 2 treatment groups. There were minor differences with respect to the content of T cells in the apheresis products. However, grafts mobilized with the combination of GM-CSF plus G-CSF had significantly fewer DC2 and similar numbers of DC1 compared with grafts mobilized with G-CSF alone. A third cohort of patients received G-CSF and the sequential administration of GM-CSF 6 days later. Grafts from these patients had a markedly decreased DC2 content compared with grafts mobilized from patients treated with G-CSF alone or with the simultaneous administration of both cytokines. This preliminary trial formed the foundation for a randomized clinical trial where normal volunteer donors were randomized to receive either G-CSF or G-CSF plus GM-CSF in order to evaluate the impact of these cytokines on DC content, T-cell polarization, and immune function after allogeneic transplantation. Fifty patients were enrolled in the trial with 25 in the GM+G-CSF and 25 in the G-CSF alone arm. All patients were successfully mobilized. Among donors mobilized with G-CSF alone, the mean number of collections was 1.48 compared with 1.08 in the group receiving the combination of GM+G-CSF (p = 0.01). There was a trend toward a higher total cell dose in the G-CSF arm (p = 0.09). Two of the 25 donors in the G/GM group required more than 1 apheresis, and 8 of 25 donors in the G-CSF alone group required more than 1 collection to achieve an adequate number of CD34+ cells. Analysis of the T-cell and T-cell subset data revealed that in the group receiving G-CSF alone, there was a significantly higher percent and total T-cell, CD4+ and CD8+ cell content of the grafts when compared with the group receiving the GM+G-CSF combination. Among dendritic cell subsets in the grafts, there was a significantly lower percentage and fewer absolute numbers of DC2, as well as a lower delivered DC2 dose/kg for the group randomized to receive GM-CSF plus G-CSF compared with the group randomized to receive G-CSF alone (p \< 0.001). There was no significant difference in the DC1 content or the content of CD34+ cells between the 2 treatment arms. Proliferation of the graft in response to T and B-cell mitogens was measured on the graft itself. Cells were exposed to mitogens or control for 72 hours, and then thymidine incorporation was measured. So far, there are available data on mitogen stimulation for 32 patients, showing a trend toward more IL-12 secretion for G+GM-CSF mobilized grafts, and more IL-2 secretion for G-CSF mobilized grafts. There have been no differences in the incidence of GvHD, relapse or survival between the 2 cytokine arms to date ([@b70-btt-2-13]; [@b71-btt-2-13]).

These data, and data indicating that cross-presentation of antigen by DC2 may induce antigen-specific tolerance by T cells, suggest that the addition of GM-CSF to regimens during mobilization of peripheral blood progenitor cell grafts may be a clinically applicable strategy to enhance innate and acquired immunity after peripheral blood progenitor cell transplantation ([@b63-btt-2-13]; [@b42-btt-2-13]). Larger clinical trials are needed to determine the exact consequences of altering the DC1 and DC2 content of peripheral blood hematopoietic progenitor cell grafts. Those effects may include: incidence of acute and chronic GVHD, engraftment, graft rejection, graft versus leukemia effect and response to infection.

Role of GM-CSF in post transplant immune reconstitution
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Recent data on the differential effects of GM-CSF and G-CSF on the DC subsets in the graft, has inspired clinical studies to investigate whether the administration of these cytokines following autologous hematopoietic stem cell transplantation may influence the post-transplant reconstitution of cellular immunity. Fattorossi and colleagues conducted a randomized, prospective clinical trial to test for differences in immune recovery among 39 patients with ovarian and breast cancer who received either G-CSF or GM-CSF after high dose myeloablative chemotherapy and autologous transplantation. At day 12, GM-CSF was more efficient at up-regulating membrane molecules on phagocytic cells important for antibody-dependent cytotoxicity and for the uptake of immune complexes compared to treatment with G-CSF; and at day 80, a significantly higher proportion of mitogen-stimulated T cells from GM-CSF-treated patients expressed interleukin-2 receptor, and a higher proportion of these T cells were actively proliferating ([@b33-btt-2-13]). Recently, Gazitt and colleagues showed that among 29 non-Hodgkin's lymphoma (NHL) patients receiving cyclophosphamide plus GM-CSF, G-CSF or GM-CSF followed by G-CSF for stem cell collection, patients mobilized with the GM-CSF containing regimens mobilized higher numbers of DC, and had a higher probability of survival compared to patients receiving G-CSF alone (median of 55 months versus 15 months; p = 0.02). Of note, there was no difference in the ratio of DC1:DC2 between CSF regimens. This finding supports the hypothesis that higher numbers of DCs in the graft might be associated with prolonged survival of NHL patients after autologous transplantation ([@b40-btt-2-13]). Further studies in larger populations of patients are merited. More is known about the effects of G-CSF than the effects of GM-CSF on post-transplant immune reconstitution. Volpi and colleagues reported on the effects of G-CSF administration in the post-transplant setting among 43 patients with acute leukemia who received T-cell depleted peripheral blood progenitor cell transplants from HLA haploidentical related donors, compared to a cohort of 36 patients with acute leukemia who did not receive G-CSF after transplantation. They found significantly delayed recovery of Th1-type CD4+ T-cells (low IL-4 and IL-10, and high IL-12 receptor expression), a higher proportion of CD4+ T-cells with a Th2 phenotype (high levels of IL-4 and IL-10, and low levels of IL-12 receptor expression), and impaired production of IL-12 by dendritic cells, compared to patients who did not receive post-transplant G-CSF. T-cells from recipients of post-transplant G-CSF had significantly decreased in-vitro reactivity to fungal pathogens compared to T-cells from patients who did not receive post-transplant G-CSF. This finding suggests an increased susceptibility to opportunistic infections in the G-CSF treated cohort, given that Th1-responses are necessary for anti-fungal protection ([@b124-btt-2-13]). The effect of G-CSF on post-transplant immune reconstitution appeared to be dependent on G-CSF's influence on DC maturation and differentiation; given that administration of G-CSF following transplantation favored the appearance of IL-12-deficient DC which polarize T-cells toward Th2 responses. Fagnoni reported a similar effect of post-transplant G-CSF in children ([@b31-btt-2-13]). Ringden et al performed a retrospective analysis to determine the role of post-transplant treatment with G-CSF in patients with AML and acute lymphocytic leukemia (ALL) who received allogeneic BMT or peripheral blood grafts. They found that prophylactic, post-transplant treatment with G-CSF led to a higher risk of acute and chronic GVHD, higher transplant related mortality, and decreased overall survival and leukemia-free survival rates in patients who received BMT only. Post-transplant G-CSF led to faster engraftment of absolute neutrophil count but slower engraftment of platelets in transplant recipients irrespective of the type of graft ([@b91-btt-2-13]). These findings suggest that post-transplant administration of G-CSF may cause an imbalance in dendritic cell content or function, resulting in impaired cellular immunity in the early post-transplant period. This may lead to an increase in the incidence of GVHD ([@b31-btt-2-13]). Current guidelines support the use of colony stimulating factors for mobilization of autologous and allogeneic grafts and after peripheral blood progenitor cell transplantation in the autologous setting only ([@b113-btt-2-13]). Further studies are needed to support the addition of GM-CSF after allogeneic transplantation.

GM-CSF may improve antibody-dependent cell-mediated cytotoxicity
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The anti-CD20 antibody, rituximab, used alone or in combination with chemotherapy, is an established treatment for non-Hodgkin's lymphoma (NHL) ([@b21-btt-2-13]). Augmenting the expression of CD20 antigen on the tumor cells may increase the cell kill and therefore increase the effectiveness of the antibody ([@b120-btt-2-13]). Preliminary data suggest that GM-CSF can up-regulate the CD20 expression on lymphoid B cells in vitro, but these results have not been reproducible in vitro nor in vivo ([@b120-btt-2-13]; [@b15-btt-2-13]; [@b131-btt-2-13]).

Venugopal and colleagues performed experiments on cells from patients with chronic lymphocytic leukemia (CLL) where CLL cells were cultured with cytokines and the expression of CD 20 on the surface of the CLL cells was measured before and after cytokine exposure. They found a statistically significant up-regulation of CD20 antigen expression on CLL cells after culture with GM-CSF, IL-4, or TNF-alpha. Flow cytometry evaluation revealed an increase in fluorescence intensity as well in the percentage of cells expressing the antigen ([@b120-btt-2-13]). This led to further studies which have revealed promising, but inconsistent results. Olivieri and his group showed the feasibility of rescuing patients with NHL relapsing after autologous transplantation with a regimen containing rituximab, CHOP chemotherapy and GM-CSF. They reported a 75% overall response rate (60% complete remission, and 15% partial response) among 20 patients with aggressive NHL who relapsed after autologous transplantation ([@b82-btt-2-13]). Rapoport and colleagues reported promising results utilizing post-transplant rituximab and GM-CSF after autologous transplantation among a group of patients with advanced NHL and Hodgkin's disease ([@b87-btt-2-13]). However neither up-regulation of CD20 antigen, nor a change of the proportion of CD20 positive cells was observed after culture with GM-CSF in a study by [@b131-btt-2-13] on cells from 18 patients with CLL.

GM-CSF may augment the graft versus leukemia effect of allogeneic transplantation
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Relapse of acute leukemia after allogeneic transplantation remains a significant therapeutic challenge, affecting approximately one third of all patients with acute leukemia who receive allogeneic transplantation as a curative therapy. Salvage post-transplant maneuvers have focused on utilization of second transplants, but these are limited to a minority (10% in most series) of fit patients. Clinical and pre-clinical data has suggested a role for cytokine therapy in the induction of graft versus leukemia effects in the setting of post-transplant relapse ([@b110-btt-2-13]; [@b19-btt-2-13]; [@b12-btt-2-13]; [@b78-btt-2-13]; [@b59-btt-2-13]; [@b67-btt-2-13]). Improving the antigen-presenting capacity of leukemic blasts may lead to clinically-significant anti-leukemic effects. The feasibility of generating DC-like leukemic antigen presenting cells upon treatment with cytokines, including GM-CSF has been demonstrated ( [@b98-btt-2-13]; [@b78-btt-2-13]). The level of co-stimulatory molecule expression on leukemic blasts has been hypothesized to play a role in the capacity of leukemic blasts to present antigen to effector cells ([@b121-btt-2-13]; [@b129-btt-2-13]). A retrospective study at our institution reviewing the treatment of acute leukemia relapsed after allogeneic transplantation revealed promising results among a minority of patients treated with GM-CSF and interferon-alpha-2b ([@b2-btt-2-13]). A prospective clinical trial at our center is currently investigating the feasibility and activity of a regimen utilizing the combination of GM-CSF and interferon-alpha-2b after cytoreduction to treat acute leukemia relapsed after allogeneic transplantation. Correlative studies will test the hypothesis that GM-CSF and interferon-alpha-2b act by up-regulating co-stimulatory molecules on leukemic blasts, and down-regulating regulatory T-cells leading to improved antigen presentation and durable graft versus leukemia effects ([Figure 1](#f1-btt-2-13){ref-type="fig"}).

GM-CSF for the treatment of solid tumors
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Role of GM-CSF in the management of renal cell carcinoma
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Renal cell carcinoma (RCC) is known to be an immunogenic tumor. Interferon-alpha (IFN-alpha) has been established as the standard treatment for metastatic RCC with response rates ranging between 10 and 20%. High dose interleukin-2 has yielded similar results, but its use has been limited by significant toxicities ([@b17-btt-2-13]). Previous observations have indicated that GM-CSF can potentiate the effect of IL-2 on T-cell activation ([@b73-btt-2-13]; [@b44-btt-2-13]). Subsequently multiple trials have tested the activity of GM-CSF combinations for RCC with modest results. [Table 1](#t1-btt-2-13){ref-type="table"} summarizes activity and toxicities of GM-CSF in the treatment of renal cell carcinoma. Verra and colleagues studied the effect of simultaneous administration of low dose IL-2, IFN-alpha and GM-CSF in metastatic RCC in a phase I study and, subsequently in a multicenter phase II study, showing tolerability and promising efficacy with 19% overall responses (9% CR, 10% PR) ([@b24-btt-2-13]; [@b123-btt-2-13]). Recently, the same group conducted a phase I study of peri-operative low-dose IL-2, IFN-alpha, and GM-CSF in resectable RCC. In addition to determining the maximum tolerated dose of the cytokine combination in the peri-operative setting, the investigators studied the effects of the cytokines in the peripheral blood and at the tumor site. They found higher numbers of tumor-infiltrating T-cells and mature DC1 in tumors resected from patients who received peri-operative cytokines, compared to a control group of tumors resected from non-cytokine treated patients ([@b122-btt-2-13]). The advent of targeted therapy for RCC has marked a new paradigm in the treatment of this tumor ([@b79-btt-2-13]), and further studies may find a role for combining cytokines with the newer targeted agents.

Role of GM-CSF in the management of malignant melanoma
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Unresectable melanoma carries a poor prognosis with limited options for treatment ([@b85-btt-2-13]). GM-CSF has been shown to induce cytotoxic T-cells and activated DC at tumor sites and draining lymph nodes ([@b85-btt-2-13]). The use of GM-CSF in combination with IL-2 or IFN-alpha has yielded promising results, but is associated with significant systemic toxicity. [Table 2](#t2-btt-2-13){ref-type="table"} summarizes activity and toxicities of GM-CSF-containing regimens in the treatment of malignant melanoma. Delivery of therapy into local sites of disease may circumvent systemic toxicity and is the subject of current investigation.

GM-CSF and cancer vaccine development
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Addressing questions pertaining to the choice of vector, the specificity of the antigen, and the choice of co-stimulatory molecules is crucial to the optimal development of cancer vaccines. Tumor-associated antigens from tumor cells (both autologous and allogeneic), proteins, peptides, and nucleic acid have been used as immunogens. Genetically modified allogeneic tumor cells as well as recombinant viruses or bacterial genes have been utilized as vectors. Pre-existing immunity to the vector itself has limited the use of vaccines based on viral vectors ([@b93-btt-2-13]). Vaccination in the absence of the co-stimulatory signals necessary to break tolerance can lead to anergy. Therefore, some vectors have been designed to express not only tumor-associated antigens, but also, co-stimulatory molecules and cytokines. [@b28-btt-2-13] introduced GM-CSF as an important adjuvant in cancer vaccine trials, based on his observations that irradiated tumor cells expressing murine GM-CSF could stimulate potent, long-lasting, and tumor-specific immunity. In order to circumvent systemic toxicity and to increase immune responses, injection into the local tumor environment has been proposed. Hersch and colleagues used intra-tumor injection of HLA-B7/beta2-microglobulin genes as plasmid DNA in lipid into patients with malignant melanoma. In a phase I trial setting, they reported a 36% response at the locally injected tumor and a 19% systemic anti-tumor response ([@b48-btt-2-13]).Vaccine trials utilizing GM-CSF or engineering tumor cells to secrete GM-CSF showed encouraging results in the treatment of solid tumors including: malignant melanoma, breast carcinoma, pancreatic cancer, renal cell carcinoma, non-small cell carcinoma of the lung and prostate cancer ([@b101-btt-2-13]; [@b107-btt-2-13]; [@b52-btt-2-13]; [@b115-btt-2-13]; [@b26-btt-2-13]; [@b39-btt-2-13]; [@b65-btt-2-13]). Cassaday and colleagues performed a phase I study of immunization using particle-mediated epidermal delivery (PMED) of genes for gp100 and GM-CSF into uninvolved skin of melanoma patients. Two groups of 6 patients each were treated; group 1 received PMED with cDNA for gp100, and group 2 received PMED with cDNA for GM-CSF followed by PMED for gp100 at the same site. Biopsies were obtained and evaluated to assess transgene expression, gold-bead penetration, and dendritic cell infiltration. Exploratory studies included flow cytometric analyses of peripheral blood lymphocytes and evaluation of delayed-type hypersensitivity to gp100 peptide in HLA-A2 + patients. Local toxicity in both groups was mild and resolved within 2 weeks. No vaccine-related systemic toxicity was reported, including no induction of pathologic auto-antibodies. GM-CSF transgene expression in vaccinated skin sites was detected. GM-CSF and gp100 PMED yielded a greater infiltration of DC into vaccine sites than did gp100 PMED alone. Immunologic monitoring suggested modest activation of an anti-melanoma response ([@b14-btt-2-13]). This study demonstrated tolerability and induction of anti-melanoma immune responses with a local approach. Additional investigation utilizing this technique is warranted. Bendandi and colleagues tested the hypothesis that immune therapy is more effective in the setting of minimal residual disease (MRD). They documented clearance of the t(14,18) translocation by PCR from the peripheral circulation in 8 of 11 patients with lymphoma and MRD, after administration of a GM-CSF containing vaccine ([@b8-btt-2-13]). Currently our center participates in a multi-institutional trial of vaccine therapy for AML after remission induction. GM-CSF may enhance antigen presentation by recruiting DC to the (vaccine) site where antigen is taken up, processed, and presented to T-cells in draining lymph nodes, generating systemic tumor-specific responses ([@b11-btt-2-13]).

Cell-mediated vaccines: role of dendritic cells
===============================================

DC are ideal candidates for use in vaccination, owing to their role in antigen presentation. DC can be isolated from the peripheral circulation by FACS sorting or magnetic bead isolation, or can be generated in large quantities ex-vivo from peripheral blood progenitors in media supplemented with cytokines, including GM-CSF ([@b10-btt-2-13]). These DC may then be matured with cytokine culture prior to loading with antigen. They have yielded promising results and continue to be tested in the treatment of solid and hematological malignancies. Results from phase 1 and 2 clinical trials indicate that tumor-peptide loaded DC can induce clinically significant immune responses in patients with lymphoma and melanoma ([@b51-btt-2-13]; [@b47-btt-2-13]). Antigen-loaded DC as cancer vaccines have been limited by uncertainty regarding the best DC subtype to use, the optimal maturation status of the DC, the best site of administration (sub-cutaneous, intravenous, or intra-nodal) and the optimal schedule of administration. More research is needed in order to answer these questions and define the optimal use of GM-CSF as an adjuvant in cell-mediated vaccines.

Future directions
=================

Pre-clinical and clinical data support the role of GM-CSF as an immune adjuvant in the treatment of malignant solid and liquid tumors, but the challenge remains to devise combinations of cytotoxic and cytokine therapy which are synergistic in breaking immune tolerance, enhancing antigen presentation and up-regulating anti-tumor T-cell responses. Local production of GM-CSF by tumors in the setting of tumor-specific vaccination has shown promise in the induction of anti-tumor immune responses. However, the laboratory correlates of response; such as, the lymphocytic and inflammatory infiltrates that develop at the site of vaccination and cytokine injection, do not reproducibly correlate with improved clinical outcomes, and well designed translational studies are needed to better define the anti-tumor activity of GM-CSF and other cytokines. Vaccines and cytokine therapies are attractive for use in patients who cannot tolerate further cytotoxic chemotherapy, owing to their relatively low toxicity, and in patients whose tumors are in a minimal residual disease state.

The success of cytokine therapy will likely depend upon defining the most favorable combination of cytokines, the optimal site and route of administration, reaching a MRD status prior to cytokine therapy, and development of surrogate endpoints of anti-tumor activity that can be used to design subsequent clinical trials.

**Disclosures**

MA receives research support from Bayer Healthcare. SL has served as a consultant for, and received research support from, Berlex (now known as Bayer Healthcare).

![GM-CSF and IFN may upregulate co-stimulatory molecule expression on leukemic blasts leading to stimulation of cytotoxic T-cells and a GVL effect.\
**Abbreviations:** APC, antigen presenting cells; GVL, graft versus leukemia; GM-CSF, granulocyte-macrophage-colony-stimulating factor; IFN, interferon; CTL, cytotoxic T lymphocyte.](btt-2-13f1){#f1-btt-2-13}

###### 

Recent trials using GM-CSF in patients with renal cell carcinoma

  -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Author             Population                                 Immune therapy                                                         Dose of GM-CSF                           Outcomes                                                                                           Toxicities (most common)
  ------------------ ------------------------------------------ ---------------------------------------------------------------------- ---------------------------------------- -------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------
  [@b18-btt-2-13]    19 Metastatic RCC                          IL-2 0.5 MIU BID\                                                      150 μg/day SQ D 1--5                     PR: 4/19\                                                                                          Bone pain, asthenia, and fever
                                                                SQ D 6--15                                                                                                      SD: 11/19\                                                                                         
                                                                                                                                                                                mTTP = 9 months\                                                                                   
                                                                                                                                                                                2 year OS 42%\                                                                                     
                                                                                                                                                                                3 year OS 26%                                                                                      

  [@b123-btt-2-13]   59 Progressive RCC                         IL-2: 4 MIU/m^2^ SQ\                                                   2.5 μg/kg SQ\                            CR: 5/59\                                                                                          Flu-like symptoms, transient LFT elevations
                                                                INF-α :5MIU SQ × 12 days every 3 wks                                   D 1--12                                  PR: 6/59\                                                                                          
                                                                                                                                                                                mOS: 9.5 mos.                                                                                      

  [@b69-btt-2-13]    25 Metastatic RCC                          IL-2: 6 MIU/day SQ\                                                    13 patients also received GM-CSF\        IL-2 alone\                                                                                        More asthenia occurred in the IL-2 + GM group
                                                                6 D/wk × 4 wks\                                                        0.3 μg/kg D 1--3 each week of Il-2       PR: 3/13\                                                                                          
                                                                2 cycles (21-day intervals)\                                                                                    SD: 8/13\                                                                                          
                                                                Maintenance: 6 days/ month until progression                                                                    IL-2 + GM\                                                                                         
                                                                                                                                                                                PR: 3/12\                                                                                          
                                                                                                                                                                                SD: 6/12                                                                                           

  [@b112-btt-2-13]   21 (13 with metastatic RCC)                IL-2: 72,000 IU/kg\                                                    125 or 250 μg/m^2^/day\                  No PR or CR\                                                                                       Grade 3 confusion in 4 pts (3 on IL-2 alone)
                                                                TID × on D 2--6 and D16--20 ± GM-CSF                                   SQ D 1--7 and D 15--21                   SD: 4/13 with RCC                                                                                  

  [@b100-btt-2-13]   55 Metastatic RCC                          IL-2: 4.5 MU day 1--4 wks. 3 and 6\                                    400 μg SQ D 1--5 weeks 2 and 5           CR: 1/53,.\                                                                                        No toxicities greater than grade 2
                                                                INF-γ: 100 μg TIW wks 1 and 4                                                                                   PR: 4/53\                                                                                          
                                                                                                                                                                                SD: 14/53\                                                                                         
                                                                                                                                                                                mOS: 12 mos                                                                                        

  [@b128-btt-2-13]   10 Stage IV RCC Pilot study                IL-2: 4 MU/m^2^ SQ and INF-α: 5 MIU/m^2^\                              5 μg/kg SQ\                              PR 2/10\                                                                                           One grade 3 fever\
                                                                SQ multiple dosing schedules                                           D 1 + 4 week 1\                          1/10 mixed response                                                                                No other grade 3--4 toxicities
                                                                                                                                       D 1, 3, 5 week 2\                                                                                                                           
                                                                                                                                       D 1, 3, 5 weeks 3--5\                                                                                                                       
                                                                                                                                       D 1, 3, 5 weeks 7--9                                                                                                                        

  [@b117-btt-2-13]   13 Metastatic RCC, Phase I                 IL-6 1, 5, or 10 μg/kg/day D 1--14                                     3 μg/kg/day\                             No responses                                                                                       DLT: thrombocytosis and hyperbilirubinemia
                                                                                                                                       D1--14                                                                                                                                      

  [@b24-btt-2-13]    18\                                        IL-2: 1, 4, or 8 MIU/m^2^, and INF-α: 5 MIU SQ × 12 days every 3 wks   2.5 or 5 μg/kg/day SQ                    CR: 3/11\                                                                                          DLT: fever with chills, hypotension, fluid retention
                     11 RCC\                                                                                                                                                    SD: 5/11                                                                                           
                     7 melanoma\                                                                                                                                                                                                                                                   
                     Phase I                                                                                                                                                                                                                                                       

  [@b95-btt-2-13]    20 Metastatic RCC                          IL-2: 11 MIU SQ D1--4 weekly\                                          1.25 μg/day SQ\                          PR: 1\                                                                                             Grade 3 fever, fatigue, anorexia mucositis, and dermatitis\
                                                                INF-α: 10 MIU SQ 2 days/week\                                          D 1--14                                  SD: 3\                                                                                             One on-study death
                                                                c-RA 1 mg/kg daily orally for 4 weeks                                                                           1-year survival rate: 48%                                                                          

  [@b50-btt-2-13]    16 pts with RCC and pulmonary metastases   IL-2: 1.5, 2.25, or 4.5\                                               1.25, 2.25, or 2.5 g/kg/day SQ D 8--19   14 evaluable 0/14 had ≥50% shrinkage of total tumor burden nor reduction in pulmonary metastases   Grade 3--4 toxicities: lymphopenia, thrombocytopenia, elevated PT, thrombosis, hypotension, hypocalcemia, hyperglycemia, pain, constipation\
                                                                MIU/m^2^/day 96 h CIV\                                                                                                                                                                                             Grade 5: neurologic
                                                                D 1--4, 8--11, and 15--18                                                                                                                                                                                          
  -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

**Abbreviations:** c-RA, cis-retinoic acid; CR, complete response; D, days; INF-α, interferon alpha; INF-γ, interferon gamma; OR, overall response; PR, partial response; pt, patient; SQ, subcutaneous; IL-2, interleukin 2; PR, partial response; mOS, median overall survival; LFT, liver function test; wks, weeks; mTTP, median time to progression; CIV, continuous intra-venous infusion; PT, prothrombin time.

###### 

GM-CSF-containing regimens in patients with malignant melanoma

  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Author                             Population   Concurrent therapy                                               Dose of GM-CSF                                                      Outcomes                                                        Toxicities (most common)
  ---------------------------------- ------------ ---------------------------------------------------------------- ------------------------------------------------------------------- --------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------
  [@b72-btt-2-13]                    11           Thalidomide: intra-patient dose escalation 50--400 μg PO daily   125 μg/m^2^ SQ daily × 14 days                                      3 alive without recurrent disease                               Most common: fatigue, dizziness, somnolence, constipation

  [@b127-btt-2-13]                   31           Temozolamide: days 1--5\                                         125 μg/m^2^ SQ days 6--17                                           CR: 4\                                                          SAEs in 7 patients Most common toxicity: flu-like symptoms
                                                  INFα-2b: days 6--17\                                                                                                                 PR: 4\                                                          
                                                  IL-2: days 6--17                                                                                                                     SD: 7\                                                          
                                                                                                                                                                                       mOS: 15.9 mos                                                   

  [@b23-btt-2-13]                    74           Temozolamide: days 1--5\                                         2.5 μg/kg SQ days 6--17                                             CR: 4 s\                                                        DLT: thrombocytopenia\
                                                  INFα-2b: days 6--17\                                                                                                                 PR: 19\                                                         Grade 3--4 lymphopenia was observed\
                                                  IL-2: days 6--17\                                                                                                                    SD: 13\                                                         All patients developed flu-like syndrome
                                                  Repeated every 22 days in stable/ responding pts                                                                                     mOS: 252 day                                                    

  [@b44-btt-2-13]; [@b43-btt-2-13]   32           DTIC: day 1\                                                     2.5 μg/kg SQ days 2--12                                             CR: 4\                                                          Treatment was well tolerated
                                                  IL-2: days 6--17\                                                                                                                    PR: 6\                                                          
                                                  INFα-2b: days 6--17                                                                                                                  mOS: 8 mos                                                      

  [@b55-btt-2-13]                    28           Topotecan: 1.5 mg/m^2^ daily x 5 days\                           250 μg/m^2^ SQ QD post CHT\                                         CR: 0\                                                          Treatment was well tolerated
                                                  Cycle repeated every 21 days                                     250 μg/m^2^ SQ BID × 5 D prior to CHT; or none                      PR: 1                                                           

  [@b88-btt-2-13]                    32           A: None\                                                         A: 5 μg/kg BID SQ × 14 D\                                           Best response was SD:\                                          Dose alteration due to toxicity in 20% A and 4.7% B\
                                                  B: DTIC 800 mg/m^2^ D\                                           B: 5 μg/kg BID SQ D2-19, 21-day cycles                              A: 0\                                                           Grade 3 or 4 toxicity occurred in 40% A and 76.5% B
                                                  1 21-day cycles\                                                                                                                     B: 3\                                                           
                                                  Cross-over from A to B allowed for non- response/progression                                                                         OS\                                                             
                                                                                                                                                                                       A: 6.3 months\                                                  
                                                                                                                                                                                       B: 7.3 months                                                   

  [@b36-btt-2-13]                    7            Cisplatin, DTIC: D1\                                             5 μg/kg SQ D 2--7                                                   CR: 1\                                                          Treatment was well tolerated
                                                  IL-2: D 8--14\                                                                                                                       PR: 1\                                                          
                                                  INF-α: D 8,10,12,14\                                                                                                                 MR: 2                                                           
                                                  28 day cycles                                                                                                                                                                                        

  [@b41-btt-2-13]                    72           Temozolamide: D1--5\                                             250 μg SQ days 6--25                                                CR: 1\                                                          Significant toxicity with grade 2 and 4 thrombocytopenia and renal impairment
                                                  Cisplatin: D 1--3\                                                                                                                   PR: 11\                                                         
                                                  IL-2: D 1--4\                                                                                                                        mOS: 11 mos                                                     
                                                  INF-α: D 1--5\                                                                                                                                                                                       
                                                  28-day cycles                                                                                                                                                                                        

  [@b119-btt-2-13]                   19           Cisplatin: D 1--3\                                               450 μg/m^2^ SQ D\                                                   OR: 6\                                                          Grade 3--4 toxicities: bone marrow suppression, hypotension, pulmonary edema, confusion, and increased serum creatinine
                                                  DTIC: D 1--3\                                                    4,5,15,16 or 450 μg/m^2^ SQ\                                        CR: 2\                                                          
                                                  Tamoxifen: daily\                                                D 4,5,15,16 and 225 μg/m^2^ SQ D 6--10, 17--21 or 450 μg/m^2^ SQ\   mOS: 6.2 mos.                                                   
                                                  IL-2: D 6--10, 17--21\                                           D 4--10, 15--21                                                                                                                     
                                                  INFα-2b: days 6--10 and 17--21\                                                                                                                                                                      
                                                  28-day cycles                                                                                                                                                                                        

  [@b99-btt-2-13]                    40           INF-α: days 1,3,5\                                               20 μg/m^2^/day\                                                     CR: 9\                                                          Treatment was well tolerated
                                                  Carmustine: day 8\                                               SQ × 7 days following chemotherapy                                  PR: 11\                                                         
                                                  Cisplatin: days 8--10\                                                                                                               Survival benefit in responders: 22 mos. vs 8 mos.(p = 0.0001)   
                                                  DTIC: days 8--10\                                                                                                                                                                                    
                                                  Tamoxifen: daily 21-day cycles                                                                                                                                                                       
  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

**Abbreviations:** bid, twice daily; TIW, 3 times weekly; DTIC, dacarbazine; INF-α, interferon alpha; IL-2, interleukin 2; PD, progressive disease; PO, orally; PR, partial response; pt, patient; OR, overall response; mOS, median overall survival; SQ, subcutaneously; LFT, liver function test; wks, weeks; CHT, chemotherapy; D, days; DLT, dose-limiting toxicity; SAEs, serious adverse events.

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Clinical trials using GM-CSF transduced tumor cells as vaccines

  -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Authors                              Tumor type                                                                                                 Clinical results
  ------------------------------------ ---------------------------------------------------------------------------------------------------------- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  [@b111-btt-2-13]                     Melanoma\                                                                                                  -- Overall immune responses, including T-cell responses were superior in the GM-CSF arm, compared to the DC arm-- Helper T-cell responses were detected and correlated with T-cell reactivity to the melanoma peptides-- 2 PR in the GM-CSF arm, 1 in the DC arm. -2 SD in the GM-CSF arm and 1 in the DC arm-- mOS:14.8 months for patients in the GM-CSF arm and 6.2 months for the DC arm.
                                       Randomized comparison of multipeptide vaccine either in emulsion with GM-CSF or pulsed on monocytoid DCs   

  [@b81-btt-2-13]                      Renal cell carcinoma                                                                                       -- Increase in DTH response against autologous tumor cells-- 1 PR

  [@b108-btt-2-13];[@b109-btt-2-13]    Prostate cancer                                                                                            -- Increase in DTH response-- Induction of tumor specific T-cell and B-cell responses

  [@b62-btt-2-13]                      Melanoma                                                                                                   -- Increase in DTH response-- Increase in melanoma specific CTLs in most patients

  [@b115-btt-2-13]; [@b114-btt-2-13]   Melanoma                                                                                                   -- Increase in DTH response-- Eosinophilia-- TILs highly cytotoxic-- 1 CR, 1PR, 1MR, 3 minor responses

  [@b96-btt-2-13]                      Non-small cell lung Cancer                                                                                 -- Increase in DTH response-- T-cell and plasma cell infiltration of metastatic sites-- 5 SD, 1 mixed response

  [@b54-btt-2-13]                      Pancreatic cancer                                                                                          -- Increase in DTH response-- Increased systemic GM-CSF levels-- 2/5 patients receiving highest cell dose \>25 months in CR
  -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

**Abbreviations:** DTH, delayed type hypersensitivity; PR, partial response; CTLs, cytotoxic T lymphocytes; TILs, tumor infiltrating lymphocytes; MR, minor response; SD, stable disease; mOS, median overall survival; CR, complete response.
