Neutrophil to lymphocyte ratio (NLR) improves the risk assessment of ISS staging in newly diagnosed MM patients treated upfront with novel agents.

Recent reports identify the ratio between absolute neutrophil count (ANC) and absolute lymphocyte count (ALC), called neutrophil to lymphocyte ratio (NLR), as a predictor of progression-free survival (PFS) and overall survival (OS) in various malignancies. We retrospectively examined the NLR in a cohort of 309 newly diagnosed multiple myeloma (MM) patients treated upfront with novel agents. NLR was calculated using data obtained from the complete blood count (CBC) at diagnosis and subsequently correlated with PFS and OS. The median NLR was 1.9 (range 0.4-15.9). Higher NLR was independent of international staging system (ISS) stage, plasma cell infiltration or cytogenetics. The 5-year PFS and OS estimates were, respectively, 18.2 and 36.4 % for patients with NLR ≥ 2 versus 25.5 and 66.6 % in patients with NLR < 2. Among younger patients (age <65 years, N = 179), NLR ≥ 2 had a negative prognostic impact on both PFS and OS, in all ISS stages. By combining ISS stage and NLR in a model limited to young patients, we found that 19 % of the patients were classified as very low risk, 70 % standard risk and 11 % very high risk. The 5-year estimates were 39.3, 19.4 and 10.9 % for PFS and 95.8, 50.9 and 23.6 % for OS for very low, standard-risk and very high-risk groups. We found NLR to be a predictor of PFS and OS in MM patients treated upfront with novel agents. NLR can be combined with ISS staging system to identify patients with dismal outcome. However, larger cohorts and prospective studies are needed to use NLR as additional parameter to personalise MM therapy in the era of novel agents.

Cross-presentation of tumor antigens by bone marrow-derived antigen-presenting cells is the dominant mechanism in the induction of T-cell tolerance during B-cell lymphoma progression.

Tumor antigen-specific T-cell tolerance may limit the efficacy of therapeutic cancer vaccines. Direct presentation of antigens by tumor cells incapable of providing adequate costimulation to tumor-specific T cells has been suggested as the basis for this unresponsiveness. Using parent-into-F1 bone marrow (BM) chimeras, this study unambiguously demonstrates that the induction of this tolerant state requires T-cell recognition of tumor antigen presented by BM-derived antigen-presenting cells (APCs), not tumor cells themselves. In the absence of host APC presentation, tumor-specific T cells remained functional, even in the setting of antigen expressed by B-cell lymphomas residing in secondary lymphoid tissues. The intrinsic APC capacity of tumor cells has therefore little influence over T-cell priming versus tolerance, a decision that is regulated at the level of host APCs. (Blood. 2001;98:1070-1077)

New approaches to treating malignances with stem cell transplantation.

Stem cell transplantation has been successfully used to treat a wide variety of hematologic malignancies. New and exciting strategies being developed for use in conjunction with transplant will be useful in overcoming tumor resistance. It is now clear that a significant part of the antitumor effect of allogeneic stem cell transplantation is derived from the graft itself and is independent of the preparative regimen. Immune therapy derived from the donor's graft is uniquely suited for killing chemoresistant tumor cells and may prove to be an invaluable tool for decreasing the risk of relapse in patients with advanced disease. Among patients who have relapsed after allogeneic bone marrow transplantation (BMT), an immunologically based antitumor effect may be obtained simply by transfusing T cells obtained by leukopheresis of the original bone marrow donor. Referred to as donor leukocyte infusion (DLI), this technique has been used to obtain complete remissions in relapsed acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML). Another approach that uses the donor's graft to obtain a potent antitumor effect is the combination of nonmyeloablative BMT followed by immunotherapy with DLI. Numerous investigators are exploring ways of combining autologous BMT with immune therapy. Animal studies using tumor vaccines in conjunction with autologous transplantation offer a promising method for eliminating tumor. Patients undergoing autologous transplantation may have marrow that has been contaminated with tumor, which places them at a higher risk of relapse. Attempts have been made to eliminate contaminating tumor from the marrow by purging.

Sustaining the graft-versus-tumor effect through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing tumor vaccines.

For many cancers, autologous bone marrow transplantation (BMT) achieves a minimal residual disease state, yet relapse rates remain high. Using a syngeneic murine bone marrow transplant model, we demonstrate that vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing autologous tumor cells is effective in the post-BMT period and actually results in a greater tumor-free survival than vaccination in the nontransplant setting. Employing T cells specific for a model tumor-antigen, we find that transplantation of the tumor-bearing host results in a massive expansion and activation of tumor-specific T cells in the early posttransplant period, but this response rapidly declines in association with tumor progression. Immunization with irradiated GM-CSF tumor cells during the period of immune reconstitution results in the sustained amplification and activation of this response that closely correlates with freedom from relapse. These results demonstrate the feasibility of integrating GM-CSF vaccines in the postautologous BMT setting and suggest mechanisms that may contribute to the observed efficacy of immunization during the critical period of immune reconstitution.

In vivo blockade of CTLA-4 enhances the priming of responsive T cells but fails to prevent the induction of tumor antigen-specific tolerance.

The efficacy of therapeutic vaccination for the treatment of cancer is limited by peripheral tolerance to tumor antigens. In vivo blockade of CTLA-4, a negative regulator of T cell function, can induce the regression of established tumors and can augment the tumor rejection achieved through therapeutic vaccination. These outcomes may reflect enhanced tumor-specific T cell priming and/or interference with the development of tolerance to tumor antigens. We examined the effect of CTLA-4 blockade on the fate and function of T cells specific for a model tumor antigen in the tumor-bearing host. We found that while CTLA-4 blockade enhanced the priming of responsive T cells, it did not prevent the induction of tolerance to tumor antigens. These results demonstrate that there is a critical window in which the combination of CTLA-4 blockade and vaccination achieves an optimal response, and they point to mechanisms other than CTLA-4 engagement in mediating peripheral T cell tolerance to tumor antigens.

A universal granulocyte-macrophage colony-stimulating factor-producing bystander cell line for use in the formulation of autologous tumor cell-based vaccines.

Irradiated tumor cells transduced with the gene encoding the cytokine GM-CSF have been extensively studied as a vaccine formulation capable of priming systemic antitumor immune responses in the tumor-bearing host. In spite of the therapeutic promise of this vaccine strategy demonstrated in both animal models and early-phase clinical trials, clinical development has been limited by difficulties pertaining to the need to establish in culture the tumor of each patient and to perform individualized gene transfer. To circumvent these issues, we generated an HLA-negative human cell line producing large quantities of human GM-CSF for use as a universal bystander cell to be mixed with unmodified autologous tumor cells in the formulation of a vaccine. This line is easily propagated as a suspension culture in defined, serum-free medium. In a mouse model, we find that vaccination with a mixture of autologous tumor cells and an MHC-negative allogeneic GM-CSF-producing bystander cell primes antitumor immune responses that are equivalent or better than those achieved using autologous tumor cells directly transduced to secrete GM-CSF. This strategy greatly simplifies further clinical development of autologous tumor cell-based vaccines.

Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.

Tumor antigen-specific T-cell tolerance limits the efficacy of therapeutic cancer vaccines. Antigen-presenting cells mediate the induction of T-cell tolerance to self-antigens. We therefore assessed the fate of tumor-specific CD4+ T cells in tumor-bearing recipients after in vivo activation of antigen-presenting cells with antibodies against CD40. Such treatment not only preserved the responsiveness of this population, but resulted in their endogenous activation. Established tumors regressed in vaccinated mice treated with antibody against CD40 at a time when no response was achieved with vaccination alone. These results indicate that modulation of antigen-presenting cells may be a useful strategy for enhancing responsiveness to immunization.

Induction of antigen-specific T cell anergy: An early event in the course of tumor progression.

The priming of tumor-antigen-specific T cells is critical for the initiation of successful anti-tumor immune responses, yet the fate of such cells during tumor progression is unknown. Naive CD4(+) T cells specific for an antigen expressed by tumor cells were transferred into tumor-bearing mice. Transient clonal expansion occurred early after transfer, accompanied by phenotypic changes associated with antigen recognition. Nevertheless, these cells had a diminished response to peptide antigen in vitro and were unable to be primed in vivo. The development of antigen-specific T cell anergy is an early event in the tumor-bearing host, and it suggests that tolerance to tumor antigens may impose a significant barrier to therapeutic vaccination.

Tolerance and cancer: a critical issue in tumor immunology.

Self-antigens are the most relevant and abundant antigens to which the host's immune system must be tolerant. Induction and maintenance of tolerance to self-antigens is mediated by several mechanisms that prevent inappropriate damage to normal tissues. However, these same mechanisms may impose potential barriers for the full development of effective immune responses against antigens expressed by tumors. A critical issue in tumor immunology is whether antigen presented by a progressively expanding tumor cell population results in T-cell tolerance. Utilizing a T cell receptor transgenic mice specific for a model tumor antigen expressed on a B-cell lymphoma, recently we have obtained direct evidence supporting the existence of tumor-induced antigen-specific tolerance. A better identification and understanding of the factor(s) involved in tumor-induced tolerance has clear implications for the development of novel cancer immunotherapies aimed at safely breaking tolerance, for example, releasing the brakes on antitumor immune responses while still limiting the induction of undesirable autoimmune responses.

Ligand-independent tyrosine phosphorylation of EGF receptor and the erbB-2/neu proto-oncogene product is induced by hyperosmotic shock.

Treatment of intact cells with media containing high concentrations of ionic and non-ionic solutes induced increased tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and the protein product of the erbB-2/neu proto-oncogene. This self phosphorylation occurred in the absence of EGF or other growth factors. High concentrations of solutes did not activate phosphorylation of either isolated EGF receptor or EGF receptor solubilized by non-ionic detergents. No evidence for receptor dimerization was found in response to hyperosmotic shock. Since receptor dimers have been implicated in the EGF-induced activation of EGF receptor, hyperosmotic shock may activate EGF receptor by a different mechanism.

Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.

The epidermal growth factor receptor (EGF-R) and the erbB-2 proto-oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK-BR-3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB-2 protein. Phosphorylation of erbB-2 does not occur in cells lacking the EGF-R (MDA-MB-453). Phosphorylation of erbB-2 in SK-BR-3 cells is blocked if EGF is prevented from interacting with its receptor by specific monoclonal antibodies. While EGF induces the down-regulation of its receptor in SK-BR-3 cells, EGF has no effect on the stability of the erbB-2 protein. This result suggests that the erbB-2 protein is a substrate of the EGF-R and indicates the possibility of communication between these two proteins early in the signal transduction process.

Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent.

We have used the soluble covalent cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to examine the capacity of epidermal growth factor (EGF) to stimulate the dimerization of purified EGF receptor, of EGF receptor in membrane preparations and in intact A431 cells. The addition of EGF either to membranes from A431 cells or to EGF receptor which was purified from A431 cells by immunoaffinity chromatography caused the appearance of a cross-linked product of Mr 340,000 which was identified using EGF receptor-specific antibodies as an EGF receptor dimer. Three independent approaches including biosynthetic labeling, surface iodination, and immunoblotting experiments were utilized to follow EGF receptor dimerization in living A431 cells. These approaches provided consistent results indicating that EGF induced rapid dimerization of EGF receptor in living cells, suggesting that this process may play a role in transmembrane signalling mediated by EGF.

The calcium signal for BALB/MK keratinocyte terminal differentiation counteracts epidermal growth factor (EGF) very early in the EGF-induced proliferative pathway.

BALB/MK mouse epidermal keratinocytes require epidermal growth factor (EGF) for proliferation and terminally differentiate in response to high calcium concentrations. We show that EGF is an extremely potent mitogen, causing BALB/MK cultures to enter the cell cycle in a synchronous manner associated with a greater than 100-fold increase in DNA synthesis. Analysis of the expression of proto-oncogenes which have been reported to be activated during the cascade of events following growth factor stimulation of fibroblasts or lymphoid cells revealed a very rapid but transient 100-fold increase in c-fos RNA but little or no effect on the other proto-oncogenes analyzed. Exposure of EGF-synchronized BALB/MK cells to high levels of calcium was associated with a striking decrease in the early burst of c-fos RNA as well as the subsequent peak of cell DNA synthesis. Since the inhibitory effect of high calcium on c-fos RNA expression was measurable within 30 min, our studies imply that the EGF proliferative and calcium differentiation signals must interact very early in the pathway of EGF-induced proliferation. Our results also establish that c-fos RNA modulation is an important early marker of cell proliferation in epithelial as well as mesenchymal cells.