Interactions of tumor cells with dendritic cells: balancing immunity and tolerance.

Dendritic cells (DCs) are antigen-presenting cells specialized to initiate and maintain immunity and tolerance. DCs initiate immune responses in a manner that depends on signals they receive from pathogens, surrounding cells and their products. Most tumors are infiltrated by DCs. Thus, interactions between DCs and dying tumor cells may determine the balance between immunity and tolerance to tumor cells. In addition, DCs also display non-immunologic effects on tumors and the tumor microenvironment. Therefore, improved understanding of the cross talk between tumor cells and DCs may suggest new approaches to improve cancer therapy.

Systemic AL amyloidosis due to non-Hodgkin's lymphoma: an unusual clinicopathologic association.

Systemic AL amyloidosis (AL) is a disorder in which light chains form fibrillar deposits, leading to organ dysfunction and death. Rarely, AL has been associated with non-Hodgkin's lymphoma (NHL), although this association has not been well characterized. We report a series of six patients with AL associated with NHL, primarily lymphoplasmacytic lymphoma. Organ involvement was variable, with frequent bulky lymphadenopathy and visceral cavity deposits, but no cardiac involvement. Positron emission tomography scans were negative. Bone marrow and lymph node biopsies showed a mixed population of CD20+ lymphoid and CD138+ plasma cells. Serum free light chains were elevated, and correlated with response to therapy. Immunoglobulin light chain variable region (Ig VL) germline gene use was typical for AL, reflecting previously observed correlations between germline gene use and organ tropism. Five patients received rituximab-based therapies with two responses. Two patients underwent autologous stem cell transplantation with one complete haematological response. Four patients survive at 10-132 months from diagnosis. AL with NHL has distinctive clinical features but employs the same Ig VL gene repertoire as AL with clonal plasma cell dyscrasias. Serial serum free light chain levels are useful for tracking response to therapy. Treatments aimed at both lymphoid and plasma cell components appear warranted.

Syndecan-1 (CD138) immunoreactivity in bone marrow biopsies of multiple myeloma: shed syndecan-1 accumulates in fibrotic regions.

Syndecan-1 (CD138) mediates myeloma cell adhesion, and loss of syndecan-1 from the cell surface may contribute to myeloma proliferation and dissemination. Flow cytometry analysis of myeloma cells in bone marrow specimens shows heterogeneity in cell surface syndecan-1 expression. It is not known whether weaker expression correlates with more aggressive disease. However, recent reports suggest that variations in syndecan-1 staining intensity on myeloma cells may be an artifact of specimen handling. In this study, we evaluate syndecan-1 expression in bone marrow biopsy sections from 28 multiple myeloma patients, to elucidate the heterogeneity of syndecan-1 expression in situ. Immunoreactivity for syndecan-1, using the antibody B-B4 (CD138), was found in more than 95% of multiple myeloma cells in 27 of 28 biopsies. However, one biopsy had more than 50% CD138-negative cells and cells with weak CD138 expression were identified in the majority of cases. Loss of syndecan-1 did not appear to relate to myeloma cell differentiation. In addition, syndecan-1 was detected on intravascular and intrasinusoidal myeloma cells suggesting that loss of syndecan-1 may not be required for extramedullary dissemination. Bone marrow biopsies from nine additional patients, with variable CD138 staining intensity on myeloma cells as determined by flow cytometry, were studied by immunohistochemistry. The heterogeneous CD138 expression was confirmed in situ, with weakly positive cells concentrated in areas of reticulin fibrosis. These cells had a disrupted pattern of membrane staining in contrast to the strong linear membrane staining seen in the other multiple myeloma cells. In addition, the fibrotic stroma stained intensely for syndecan-1. Accumulation of syndecan-1 within the extracellular matrix of the marrow likely is derived by shedding of the molecule from the surface of myeloma cells. Because syndecan-1 can act to regulate the activity of heparan-binding growth factors, these reservoirs of syndecan-1 may play a critical role in promoting myeloma pathogenesis, or in regeneration of the tumor after chemotherapy.

Prognostic factors and response to fludarabine therapy in patients with Waldenström macroglobulinemia: results of United States intergroup trial (Southwest Oncology Group S9003).

Current information on Waldenström macroglobulinemia (WM) is based on retrospective or single-institution studies of patients requiring therapy. Between 1992 and 1998, 231 patients with WM were enrolled in a prospective observational multicenter clinical trial. Of these, 182 patients with symptomatic or progressive disease were treated with 4 to 8 cycles of therapy with a purine nucleoside analogue, fludarabine (FAMP; 30 mg/m(2) of body-surface area daily for 5 days every 28 days). A serum beta2-microglobulin (beta2M) level below 3 mg/L and a hemoglobin level of at least 120 g/L (12 g/dL) at presentation predicted a lower likelihood of requiring therapy. The overall rate of response to FAMP therapy was 36% (95% confidence interval, 29%-44%), with 2% complete remissions. Patients who were 70 years old or older had a substantially lower likelihood of response (odds ratio, 0.34; P =.004) than younger patients. On multivariate analysis, a serum beta2M level of 3 mg/L or higher, hemoglobin level below 120 g/L, and serum IgM level below 40 g/L [4 g/dL] were significant adverse prognostic factors for survival. We developed a simple staging system for WM by using these variables and identified 4 distinct subsets of patients with estimated 5-year overall survival rates of 87%, 64%, 53%, and 22%, and 5-year progression-free survival rates of 83%, 55%, 33%, and 12%. Prognosis in WM is highly variable and serum beta2M was the dominant predictor of a need for therapy and of survival. FAMP has activity against WM. Our staging system may provide guidance for a risk-based approach to the treatment of WM.

Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.

Immunostimulatory properties of dendritic cells (DCs) are linked to their maturation state. Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans. Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects. In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells. When pre- and postimmunization T cells were boosted in culture, there were greater numbers of MP-specific major histocompatibility complex tetramer-binding cells after immunization, but these had reduced interferon production and lacked killer activity. These data demonstrate the feasibility of antigen-specific inhibition of effector T cell function in vivo in humans and urge caution with the use of immature DCs when trying to enhance tumor or microbial immunity.

Active immunization of humans with dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells specialized to initiate T-cell immunity. The development of methods to generate large numbers of DCs has facilitated their application for immunotherapy. Recent studies have demonstrated the safety and immunogenicity of DCs in humans and have begun to outline the durability, kinetics, and nature of the elicited T-cell responses. However, DC-based immunotherapy remains a challenge and several parameters need to be examined to optimize immune responses, in order to maximize clinical efficacy against cancer and infectious diseases.

Mature dendritic cells boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes.

We have recently shown that a single injection of mature, antigen-pulsed, human dendritic cells (DCs) rapidly elicits CD4(+) and CD8(+) T-cell immunity in vivo. The DCs were pulsed with 2 foreign proteins, keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT), as well as an HLA A2.1-restricted influenza matrix peptide (MP). Responses to all 3 antigens peaked at 30-90 days after immunization and declined thereafter. To determine if the foreign helper proteins (TT and KLH) were essential for CD8(+) T-cell responses to the viral peptide, we reinjected 3 of the HLA-2.1 subjects with mature DCs pulsed with MP alone. All 3 volunteers showed a rapid boost in MP-specific immunity, and freshly sampled blood from 1 contained cytolytic T cells. In all 3 subjects, CD8(+) T-cell responses to booster DCs were faster and of greater magnitude than the responses to the first DC injection. Importantly, the T cells that proliferated after booster DC treatment secreted interferon-gamma upon challenge with much lower doses of viral peptide than those elicited after the first injection, indicating a higher functional avidity for the ligand. These data begin to outline the kinetics of T-cell immunity in response to DCs and demonstrate that booster injections of mature DCs enhance both qualitative and quantitative aspects of CD8(+) T-cell function in humans.

Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients.

The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs.

Waldenström's macroglobulinemia.

Waldenström's macroglobulinemia (WM) is a clinical syndrome with diverse prognoses, and not all patients require therapy at diagnosis. Serum beta2 microglobulin is a major prognostic determinant, and asymptomatic patients with low beta2 microglobulin levels and preserved hemoglobin can be observed over long periods without therapy. Low-dose alkylating agents and purine analogs are commonly employed as initial therapy but rarely yield complete remissions. Patients who are refractory to or have relapse after alkylator or purine analogue therapy can be salvaged with purine analogs. Improvement in outcome demands a comprehensive approach aimed at increasing and sustaining complete remissions. Such an approach should probably employ Rituxan (IDEX Pharmaceuticals, La Jolla, CA) in conjunction with induction therapy, peripheral stem cell procurement before purine analog therapy, and high-dose therapy followed by maintenance therapy with interferon.

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Recent advances in the treatment of multiple myeloma.

Multiple myeloma, a clonal B-cell malignancy, accounts for 10% of all hematologic cancers. In patients with multiple myeloma, median survival is approximately 30 to 36 months with conventional therapy, but high-dose chemotherapy resulted in better outcomes in a mature randomized trial. Pamidronate and other bisphosponates, used as supportive therapy, effectively reduce the incidence of skeletal events and decrease pain. They also have a role in disease control. Ongoing trials are now addressing such issues as further intensification with tandem transplantations, timing of transplantation, and decrease of malignant cell reinfusion by positive selection. Important laboratory observations have better defined the malignant clone and its interaction with the microenvironment. These observations will be important for the treatment of myeloma in the future.

Syndecan-1 (CD 138) in myeloma and lymphoid malignancies: a multifunctional regulator of cell behavior within the tumor microenvironment.

Syndecan-1 is a transmembrane proteoglycan expressed on the surface of tumor cells of various origins including myeloma, Hodgkin's disease, and certain human immunodeficiency virus (HIV) associated lymphomas. Functional studies in myeloma reveal that syndecan-1 may act as a multifunctional regulator of cell behavior in the tumor microenvironment; it mediates cell-cell adhesion, binding of myeloma cells to type I collagen, and inhibits tumor cell invasion into collagen gels. In addition, syndecan-1 is released from the surface of myeloma cells and this shed form of the molecule inhibits growth and induces apoptosis of myeloma cells and may modulate myeloma bone disease by inhibiting osteoclast formation and promoting osteoblast formation. In view of its effects on tumor cell growth, survival, adhesion and invasion and on bone cell differentiation, syndecan-1 may be an important potentially beneficial regulator of myeloma pathobiology. Further studies are needed to define the clinical significance of syndecan-1 in myeloma and to examine its functional significance in other lymphoid malignancies.

Anti-myeloma activity of pamidronate in vivo.

Two patients with progressive myeloma were treated with pamidronate disodium every 2-4 weeks. Pamidronate therapy resulted in a significant reduction of marrow plasmacytosis and plasma cell labelling index (PCLI), together with durable (> or = 20 months) stabilization of immunoglobulin (Ig) levels and an increase in bone mineral density in the first patient and > 50%, reduction in Ig levels and bone marrow plasmacytosis in the second. This, to our knowledge, is the first report of an anti-myeloma effect of bisphosphonates in humans and provides evidence that a therapeutic intervention largely directed at the myeloma microenvironment may alter the natural history of the disease.

Syndecan-1 is a multifunctional regulator of myeloma pathobiology: control of tumor cell survival, growth, and bone cell differentiation.

Multiple myeloma is characterized by an accumulation of malignant plasma cells in the bone marrow coupled with an altered balance of osteoclasts and osteoblasts, leading to lytic bone disease. Although some of the cytokines driving this process have been characterized, little is known about the negative regulators. We show that syndecan-1 (CD 138), a heparan sulfate proteoglycan, expressed on and actively shed from the surface of most myeloma cells, induces apoptosis and inhibits the growth of myeloma tumor cells and also mediates decreased osteoclast and increased osteoblast differentiation. The addition of intact purified syndecan-1 ectodomain (1 to 6 nmol/L) to myeloma cell lines in culture leads to induction of apoptosis and dose-dependent growth inhibition, with concurrent downregulation of cyclin D1. The addition of purified syndecan-1 in picomolar concentrations to bone marrow cells in culture leads to a dose-dependent decrease in osteoclastogenesis and a smaller increase in osteoblastogenesis. In contrast to the effect on myeloma cells, the effect of syndecan-1 on osteoclastogenesis only requires the syndecan-1 heparan sulfate chains and not the intact ectodomain, suggesting that syndecan's effect on myeloma and bone cells occurs through different mechanisms. When injected in severe combined immune deficient (scid) mice, control-transfected myeloma cells (ARH-77 cells) expressing little syndecan-1 readily form tumors, leading to hind limb paralysis and lytic bone disease. However, after the injection of syndecan-1-transfected ARH-77 cells, the development of disease-related morbidity and lytic bone disease is significantly inhibited. Taken together, our data demonstrate, both in vitro and in vivo, that syndecan-1 has a significant beneficial effect on the behavior of both myeloma and bone cells and therefore may represent one of the central molecules in the regulation of myeloma pathobiology.

Biologic and therapeutic determinants of bone mineral density in multiple myeloma.

The net impact of malignancy and anti-tumor therapy on bone resorption in myeloma is poorly understood because conventional skeletal radiographs are relatively insensitive for the diagnosis and monitoring of bone disease. We performed determinations of bone mineral density (BMD) at the lumbar spine, femoral neck and radial diaphysis by dual energy X ray absorptiometry (DEXA) in 168 consecutive patients with myeloma seen at our institution. Follow up studies were performed in 41 of these patients. A detailed analysis of patient and disease characteristics was performed to identify the determinants of BMD. Compared to normal age and sex matched controls, mean (+/- SE) BMD was significantly decreased at the lumbar spine (Z score -0.4 +/- 0.10) and femoral neck (Z score -1.0 +/- 0.10), but was surprisingly above normal at the radial diaphysis (Z score +0.35 +/- 0.10), a cortical bone site devoid of hematopoietic marrow, suggesting a differential bone preserving effect at this site. Lack of correlation between the BMD findings and the presence or extent of radiographically evident osteolytic lesions suggested the presence of a systemic bone disease. On multivariate analysis, duration of disease >12 months (p = 0.003) and female sex (p = 0.01) were independently associated with a lower BMD at the femoral neck/lumbar spine. On follow up DEXA (n = 41), BMD increased at > or = 1 site in 9 of 20 patients receiving bisphosphonates and in only 2 of 21 patients not receiving such therapy (p = 0.02). Similarly a decline in BMD at > or = 1 site was seen in 9 of 21 patients not receiving bisphosphonates, irrespective of the disease response status. Interval pamidronate therapy (p = 0.0007) and a low serum beta-2-microglobulin (< 2.5 mg/l) (p = 0.04) were the two most significant variables associated with an increase in BMD on multivariate analysis. These data suggest that myeloma is associated with a systemic bone disease with progressive generalized cancellous bone loss and a bone preserving effect on the radial cortical bone. The early use of bisphosphonates may improve myeloma related bone disease.

Incidence and impact of light chain associated (AL) amyloidosis on the prognosis of patients with multiple myeloma treated with autologous transplantation.

Little is known about the incidence of clinically occult AL amyloid in patients with multiple myeloma and its impact on prognosis of these patients. To address these issues, subcutaneous fat pad aspirates (SAFA) and bone marrow biopsies were evaluated for the presence of amyloid in a cohort of newly diagnosed patients with multiple myeloma prior to enrollment on a phase II study including tandem transplants. Organ directed biopsies were performed when clinically indicated. Presence of amyloid at > or = 1 site was noted in 32 patients (38%). SAFA was positive in 25 (31%), bone marrow in 8 patients (10%) and other organ sites in 7 patients. Patients with and without amyloid did not differ in disease characteristics, in particular no lambda predominance was observed in patients with amyloid. Event free survival (59+ vs 52 months; p = .9) and overall survival (59+ vs 66+ months; p = .9) were similar in both groups. Even the seven patients with symptomatic organ involvement with AL amyloid had a median overall survival of 38+ months. In conclusion, AL amyloidosis occurs more often than previously reported, but its presence does not influence the outcome of these patients after transplantation.

Treatment of AL-amyloidosis with dexamethasone plus alpha interferon.

Current therapy for primary systemic (AL) amyloidosis has only modest efficacy (response rate 25%) and because it includes alkylating agents, it has a significant leukemogenic potential (actuarial risk 21% at 3.5 years). We treated 9 consecutive patients with biopsy proven AL amyloidosis seen at our institution with pulse dexamethasone induction (40 mg on days 1-4, 9-12, 17-20 repeated q 35 days) for 3-6 cycles followed by maintenance alpha interferon 3-6 million units thrice weekly. Three patients also received maintenance dexamethasone (40 mg/day x 4 days q 4-8 weeks) for the first year. Improvement in > or = 1 AL organ involvement was seen in 8 of 9 patients. Of 7 patients with nephrotic range proteinuria, 6 had > or = 50% reduction in nonspecific proteinuria with a median time to response of 4 months (range 3-9 months). Marked improvement in organ function was also seen in 4 patients with gastrointestinal, hepatic and neuropathic involvement. However, none of the 2 patients with congestive heart failure improved. This dexamethasone plus alpha interferon regimen, devoid of leukemogenic potential, may lead to rapid and durable improvement in organ function in a significant proportion of patients with AL amyloidosis and deserves further evaluation as front line therapy.

Elevated levels of shed syndecan-1 correlate with tumour mass and decreased matrix metalloproteinase-9 activity in the serum of patients with multiple myeloma.

Sera from 20 myeloma patients and 12 normal controls were analysed for the presence of syndecan-1 and matrix metalloproteinase-9 (MMP-9). The level of syndecan-1 in the serum was elevated in 7/20 (3 5%) myeloma patients whilst 6/19 patients (31%) had decreased serum MMP-9 activity. The presence of increased syndecan-1 was associated with decreased serum MMP-9. Both elevated syndecan-1 and decreased MMP-9 were associated with higher marrow plasmacytosis, serum beta-2 microglobulin and paraprotein levels. These data provide evidence that the syndecan-1 ectodomain is shed in vivo. Quantitation of serum syndecan-1 may be a useful measure of tumour mass and may have important implications for myeloma biology.

Biology and therapy of immunoglobulin deposition diseases.

All forms of MIDD represent pathologic deposition of immunoglobulin as amorphous casts, crystals, congophilic fibrils (in AL amyloid), or punctate noncongophilic deposits (in LCDD/HCDD/LHCDD). Diagnosis is based on identification and immunohistochemical characterization of deposits and Congo red staining. Current information including development of novel in vitro and in vivo models suggests a contributory role of both protein and host factors in the pathogenesis of these disorders. In particular, primary structural features of the VL portions of the light chain molecule may affect not only the extent but also the morphologic type of protein deposits. Thus, certain types of light chains may be particularly pathogenic, although the nature or extent of proteolysis/processing involved in the pathogenesis of these deposits is yet unclear. Recent data also point to the importance of accessory molecules, cytokines, and host factors in this process. Newer therapeutic approaches using high-dose therapy with cytotoxic agents or dexamethasone appear promising, although these data need to be confirmed in a larger number of patients. The serendipitous discovery of I-DOX as an agent capable of promoting amyloid resorption provides another novel approach in patients with AL amyloidosis. Continued research on the mechanisms of deposition and resorption of these immunoglobulin deposits should provide important information that can be used to design strategies for more effective therapy and, ultimately, prevention of MIDD.