Madhav V. Dhodapkar

Antitumor Activity of Thalidomide in Refractory Multiple Myeloma

BACKGROUND Patients with myeloma who relapse after high-dose chemotherapy have few therapeutic options. Since increased bone marrow vascularity imparts a poor prognosis in myeloma, we evaluated the efficacy of thalidomide, which has antiangiogenic properties, in patients with refractory disease. METHODS Eighty-four previously treated patients with refractory myeloma (76 with a relapse after high-dose chemotherapy) received oral thalidomide as a single agent for a median of 80 days (range, 2 to 465). The starting dose was 200 mg daily, and the dose was increased by 200 mg every two weeks until it reached 800 mg per day. Response was assessed on the basis of a reduction of the myeloma protein in serum or Bence Jones protein in urine that lasted for at least six weeks. RESULTS The serum or urine levels of paraprotein were reduced by at least 90 percent in eight patients (two had a complete remission), at least 75 percent in six patients, at least 50 percent in seven patients, and at least 25 percent in six patients, for a total rate of response of 32 percent. Reductions in the paraprotein levels were apparent within two months in 78 percent of the patients with a response and were associated with decreased numbers of plasma cells in bone marrow and increased hemoglobin levels. The microvascular density of bone marrow did not change significantly in patients with a response. At least one third of the patients had mild or moderate constipation, weakness or fatigue, or somnolence. More severe adverse effects were infrequent (occurring in less than 10 percent of patients), and hematologic effects were rare. As of the most recent follow-up, 36 patients had died (30 with no response and 6 with a response). After 12 months of follow-up, Kaplan-Meier estimates of the mean (+/-SE) rates of event-free survival and overall survival for all patients were 22+/-5 percent and 58+/-5 percent, respectively. CONCLUSIONS Thalidomide is active against advanced myeloma. It can induce marked and durable responses in some patients with multiple myeloma, including those who relapse after high-dose chemotherapy.

Increased Expression of Interleukin 23 p19 and p40 in Lesional Skin of Patients with Psoriasis Vulgaris

Psoriasis is a type I–deviated disease characterized by the presence of interferon (IFN)-γ and multiple IFN-related inflammatory genes in lesions. Because interleukin (IL)-23 is now recognized to play a role in the recruitment of inflammatory cells in a T helper cell (Th)1-mediated disease, we examined psoriasis skin lesions for production of this newly described cytokine. IL-23 is composed of two subunits: a unique p19 subunit and a p40 subunit shared with IL-12. We found a reliable increase in p19 mRNA by quantitative reverse transcription polymerase chain reaction in lesional skin compared with nonlesional skin (22.3-fold increase; P = 0.001). The p40 subunit, shared by IL-12 and IL-23, increased by 11.6-fold compared with nonlesional skin (P = 0.003), but the IL-12 p35 subunit was not increased in lesional skin. IL-23 was expressed mainly by dermal cells and increased p40 immunoreactivity was visualized in large dermal cells in the lesions. Cell isolation experiments from psoriatic tissue showed strong expression of p19 mRNA in cells expressing monocyte (CD14+ CD11c+ CD83−) and mature dendritic cell (DC) markers (CD14− CD11c+ CD83+), whereas in culture, the mRNAs for p40 and p19 were strongly up-regulated in stimulated monocytes and monocyte-derived DCs, persisting in the latter for much longer periods than IL-12. Our data suggest that IL-23 is playing a more dominant role than IL-12 in psoriasis, a Th1 type of human inflammatory disease.

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.

Dendritic Cell Function in Vivo during the Steady State: A Role in Peripheral Tolerance

Abstract: The avoidance of autoimmunity requires mechanisms to actively silence or tolerize self reactive T cells in the periphery. During infection, dendritic cells are not only capturing microbial antigens, but also are processing self antigens from dying cells as well as innocuous environmental proteins. Since the dendritic cells are maturing in response to microbial and other stimuli, peptides will be presented from both noxious and innocuous antigens. Therefore it would be valuable to have mechanisms whereby dendritic cells, prior to infection, establish tolerance to those self and environmental antigens that can be processed upon pathogen encounter. In the steady state, prior to acute infection and inflammation, dendritic cells are in an immature state and not fully differentiated to carry out their known roles as inducers of immunity. These immature cells are not inactive, however. They continuously circulate through tissues and into lymphoid organs, capturing self antigens as well as innocuous environmental proteins. Recent experiments have provided direct evidence that antigen‐loaded immature dendritic in vivo silence T cells either by deleting them or by expanding regulatory T cells. In this way, it is proposed that the immune system overcomes at least some of the risk of developing autoimmunity and chronic inflammation. It is proposed that dendritic cells play a major role in defining immunologic self, not only centrally in the thymus but also in the periphery.

Sustained expansion of NKT cells and antigen-specific T cells after injection of α-galactosyl-ceramide loaded mature dendritic cells in cancer patients

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, α-galactosyl-ceramide (α-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of α-GalCer–pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-γ inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to α-GalCer–loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.

Antitumor Monoclonal Antibodies Enhance Cross-Presentation of Cellular Antigens and the Generation of Myeloma-specific Killer T Cells by Dendritic Cells

The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti–syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti–syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell–loaded mature DCs induced a strong CD8+ T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8+ T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti–syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1–specific response. Cross-presentation was inhibited by pretreatment of DCs with Fcγ receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

A Reversible Defect in Natural Killer T Cell Function Characterizes the Progression of Premalignant to Malignant Multiple Myeloma

We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. Vα24+Vβ11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-γ production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, α-galactosylceramide (α-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

Active immunization against cancer with dendritic cells: the near future.

DCs1 are antigen-presentin g cells that regulat e several components of the immune system. The mature or terminal stage of DC developmen t induces specific T-cell immunity and resistanc e to experimenta l tumors in vivo. However, DC maturation is induced by inflammator y and microbial stimuli, so it isunlikely that mature DCsnormally present antigens from tumor cells in cancer patients. Accordingly, clinical studies have begun in which DCs are generated ex vivo, charged with tumor antigens, exposed to maturation stimuli and reinfused to immunize patients. This approach has the potential to control response s to cancer antigens in a specific and nontoxic manner , in both vaccination and therapeuti c settings. DCs can mobilize several immune resistanc e mechanisms. These include CD8 CTLs, CD4 helper T cells, NK and NKT cells. Each of these lymphocytes recognizes target s through a distinct mechanis m and has the capacit y to kil l tumor cells and release valuabl e protective cytokines like IFN-g. CD4 T cells also provide essentia l help for the expansion and maintenanc e of CD8 cytolytic cells, while NK and NKT cells can eliminate target s that dampen presentatio n on MHC class I to escape CTL recognition. The stimulation and concerted action of these classes of lymphocytes can now be studied directly in patients, using ex vivo–derived DCs. Several findings have emerged from studies of healthy volunteers who have been immunized with DCs charged with model antigens, KLH protein and influenza virus matrix peptide. Mature DCs elicit a polarized Th1 type of CD4 T-cell response , only a single injection being required. Vaccination with antigen-bearing DCs also markedl y improves the functional affinity of CD8 T cells. These findings are important in the context of immunotherapy becaus e Th1 cells are more efficient helper cells in experimental model s of viral infection and tumors, while high-affinity CTLs shoul d improve recognition of tumor-derived peptides. An important caution also has surfaced when DCs are not adequately differentiated. Immature DCs can silence adaptive T-cell responses , e.g., by inducing IL-10–producing, T-reg cells. DC-based active immunization does not resul t in major shortterm toxicity in healthy subject sor cancer patients. Tumor-specific T-cell response s have been induced and detected in fresh blood specimens without the need for restimulation in vitro. However, the immune response s observed in the first protocol s are still smaller than those seen naturall y in acute viral infections. Occasional clinical regression s have been noted in these initial feasibility studies, particularl y in melanomas , pediatri c tumors, lymphomas, prostat e cancer s and renal cell cancers . This information, coupled with progres in DC biology, suggest s many ways to improve the efficacy of this new therapy. Some relevant topics include antigen loading and DC maturation procedures , frequency and route of DC injection, efficiency of DC homing to lymphoid tissues and their longevity once there and the role of distinct DC subsets . A valuabl e positive control in active immunization protocols is to include an aliquot of DCs pulsed with a viral peptide to verify that the DCs and the patient’s immune system are measurabl y competent. Most of the first vaccination protocol s have used DCs charged with synthetic, HLA-binding tumor peptides. Many methodsare in place to allow DCs to proces abroader array of peptides appropriate for the patient’s MHC haplotype. These include transfection (RNA, DNA and viral vectors) , select pathways of adsorptive endocytosis , exosomes and tumor cell fusion. An intriguing new approach involves the processing of whole tumor cells. Immature DCs are able to internalize tumor cells, including melanoma, EBV lymphoma and prostat e carcinoma cell lines. Following maturation, theprocessing of tumor cells leads to presentatio n of multiple epitopes on both MHC class I and II product s of DCs. Basic issues in human cancer immunology can be investigated with ex vivo–based DC immunization. Some topics for the near future are the extent to which tumor-specific tolerance is an obstacl e to immunetherapy and theneed to mobilizeseveral adaptive and innate mechanisms in tandem, including Th1 type CD4 helper T, NK and NKT cells. Progres is also being made in manipulating DCsdirectly in vivo. Thiswil l help to design vaccine adjuvant s that improve presentatio n of tumor cells in patients. Although tumorschallengetheimmunesystem in formidableways relative to infectious diseases , it is now feasibl e to use active immunotherap y and DC biology to manipulat e and study the human respons e to cancer.

Nivolumab in Patients With Relapsed or Refractory Hematologic Malignancy: Preliminary Results of a Phase Ib Study

PURPOSE Cancer cells can exploit the programmed death-1 (PD-1) immune checkpoint pathway to avoid immune surveillance by modulating T-lymphocyte activity. In part, this may occur through overexpression of PD-1 and PD-1 pathway ligands (PD-L1 and PD-L2) in the tumor microenvironment. PD-1 blockade has produced significant antitumor activity in solid tumors, and similar evidence has emerged in hematologic malignancies. METHODS In this phase I, open-label, dose-escalation, cohort-expansion study, patients with relapsed or refractory B-cell lymphoma, T-cell lymphoma, and multiple myeloma received the anti-PD-1 monoclonal antibody nivolumab at doses of 1 or 3 mg/kg every 2 weeks. This study aimed to evaluate the safety and efficacy of nivolumab and to assess PD-L1/PD-L2 locus integrity and protein expression. RESULTS Eighty-one patients were treated (follicular lymphoma, n = 10; diffuse large B-cell lymphoma, n = 11; other B-cell lymphomas, n = 10; mycosis fungoides, n = 13; peripheral T-cell lymphoma, n = 5; other T-cell lymphomas, n = 5; multiple myeloma, n = 27). Patients had received a median of three (range, one to 12) prior systemic treatments. Drug-related adverse events occurred in 51 (63%) patients, and most were grade 1 or 2. Objective response rates were 40%, 36%, 15%, and 40% among patients with follicular lymphoma, diffuse large B-cell lymphoma, mycosis fungoides, and peripheral T-cell lymphoma, respectively. Median time of follow-up observation was 66.6 weeks (range, 1.6 to 132.0+ weeks). Durations of response in individual patients ranged from 6.0 to 81.6+ weeks. CONCLUSION Nivolumab was well tolerated and exhibited antitumor activity in extensively pretreated patients with relapsed or refractory B- and T-cell lymphomas. Additional studies of nivolumab in these diseases are ongoing.

International prognostic scoring system for Waldenstrom macroglobulinemia

Recently, many new drugs have been developed for the treatment of Waldenström macroglobulinemia (WM). To optimize the treatment according to the prognosis and to facilitate the comparison of trials, we developed an International Prognostic Scoring System for WM in a series of 587 patients with clearly defined criteria for diagnosis and for initiation of treatment. The median survival after treatment initiation was 87 months. Five adverse covariates were identified: advanced age (>65 years), hemoglobin less than or equal to 11.5 g/dL, platelet count less than or equal to 100 x 10(9)/L, beta2-microglobulin more than 3 mg/L, and serum monoclonal protein concentration more than 7.0 g/dL. Low-risk patients (27%) presented with no or 1 of the adverse characteristics and advanced age, intermediate-risk patients (38%) with 2 adverse characteristics or only advanced age, and high-risk patients (35%) with more than 2 adverse characteristics. Five-year survival rates were 87%, 68%, and 36%, respectively (P < .001). The ISSWM retained its prognostic significance in subgroups defined by age, treatment with alkylating agent, and purine analog. Thus, the ISSWM may provide a means to design risk-adapted studies. However, independent validation and new biologic markers may enhance its significance.