P. Joy Ho

Response of myeloma to the proteasome inhibitor bortezomib is correlated with the unfolded protein response regulator XBP-1

Background Multiple myeloma, a malignancy of the antibody-secreting plasma cells, remains incurable by current therapy. However, the proteasome inhibitor bortezomib and other new drugs are revolutionizing its treatment. It remains unclear why myelomas are peculiarly sensitive to bortezomib, or what causes primary or acquired resistance. The ‘unfolded protein response’ is necessary for folding and assembly of immunoglobulin chains in both normal and malignant plasma cells, as well as for the disposal of incorrectly folded or unpaired chains via the ubiquitin-proteasome pathway. We tested the hypothesis that levels of transcription factor XBP-1, a major regulator of the unfolded protein response, predict response to bortezomib. Design and Methods Expression of XBP-1 and other regulators of the unfolded protein response were measured in myeloma and other cancer cell lines and two cohorts of patients with refractory myeloma and correlated with sensitivity/response to bortezomib. Bortezomib-resistant myeloma cell lines were derived and the effects on expression of unfolded protein response regulators, immunoglobulin secretion, proteasome activity and cross-resistance to cytotoxic drugs and tunicamycin determined. The consequences of manipulation of XBP-1 levels for sensitivity to bortezomib were tested. Results Low XBP-1 levels predicted poor response to bortezomib, both in vitro and in myeloma patients. Moreover, myeloma cell lines selected for resistance to bortezomib had down-regulated XBP-1 and immunoglobulin secretion. Expression of ATF6, another regulator of the unfolded protein response, also correlated with bortezomib sensitivity. Direct manipulation of XBP-1 levels had only modest effects on sensitivity to bortezomib, suggesting it is a surrogate marker of response to bortezomib rather than a target itself. Conclusions The unfolded protein response may be a relevant target pathway for proteasome inhibitors in the treatment of myeloma and its regulator XBP-1 is a potential response marker. (The BIR study was registered with Australian Clinical Trial Registry Number 12605000770662)

Either interleukin-12 or interferon-γ can correct the dendritic cell defect induced by transforming growth factor β1 in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44+) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up‐regulate the expression of the B7 co‐stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor β1 (TGFβ1) and interleukin (IL)‐10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti‐TGFβ1. As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up‐regulation was reduced in both stage I and stage III. It was demonstrated that both IL‐12 and interferon‐γ neutralized the failure to stimulate CD80 up‐regulation by huCD40LT in vitro. IL‐12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123−) suggesting that there would be a predominantly T‐helper cell type response. The addition of IL‐12 or interferon‐γ to future immunotherapy trials involving these patients should be considered.

Myeloid derived suppressor cells are numerically, functionally and phenotypically different in patients with multiple myeloma

Abstract Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells that have been implicated as inhibitors of lymphopoiesis in patients with malignancies. They have a consensus phenotype of CD33+/CD11b+/HLA-DRlo/− and can be further divided into CD15 + granulocytic (G-MDSC) and CD14 + monocytic (M-MDSC) subsets. We characterized MDSCs in patients with multiple myeloma (MM) and found a significant increase in G-MDSCs in the blood of patients with progressive MM. Flow-sorted MDSCs from patients with MM induced the generation of regulatory T cells (Treg). MDSCs from both patients with MM and aged-matched controls demonstrated a dose-dependent inhibition of lymphocyte proliferation in carboxyfluorescein succinimidyl ester (CFSE)-tracking experiments. Granulocyte colony stimulating factor (G-CSF) administered to induce stem cell mobilization caused an increase in the number of MDSCs in the peripheral blood of patients with MM and a concentration of these immune-suppressive cells in peripheral blood stem cell collections. MDSCs are likely to cause immune dysfunction in patients with MM.

International survey of T2* cardiovascular magnetic resonance in β-thalassemia major

Accumulation of myocardial iron is the cause of heart failure and early death in most transfused thalassemia major patients. T2* cardiovascular magnetic resonance provides calibrated, reproducible measurements of myocardial iron. However, there are few data regarding myocardial iron loading and its relation to outcome across the world. A survey is reported of 3,095 patients in 27 worldwide centers using T2* cardiovascular magnetic resonance. Data on baseline T2* and numbers of patients with symptoms of heart failure at first scan (defined as symptoms and signs of heart failure with objective evidence of left ventricular dysfunction) were requested together with more detailed information about patients who subsequently developed heart failure or died. At first scan, 20.6% had severe myocardial iron (T2*≤10ms), 22.8% had moderate myocardial iron (T2* 10–20ms) and 56.6% of patients had no iron loading (T2*>20ms). There was significant geographical variation in myocardial iron loading (24.8–52.6%; P<0.001). At first scan, 85 (2.9%) of 2,915 patients were reported to have heart failure (81.2% had T2* <10ms; 98.8% had T2* <20ms). During follow up, 108 (3.8%) of 2,830 patients developed new heart failure. Of these, T2* at first scan had been less than 10ms in 96.3% and less than 20ms in 100%. There were 35 (1.1%) cardiac deaths. Of these patients, myocardial T2* at first scan had been less than 10ms in 85.7% and less than 20ms in 97.1%. Therefore, in this worldwide cohort of thalassemia major patients, over 43% had moderate/severe myocardial iron loading with significant geographical differences, and myocardial T2* values less than 10ms were strongly associated with heart failure and death.

Myeloma skews regulatory T and pro-inflammatory T helper 17 cell balance in favor of a suppressive state

Abstract Discrepancies in the literature between regulatory T cell (Treg) and pro-inflammatory T helper 17 (Th17) numbers in multiple myeloma (MM) can be largely explained by technical differences in methodology and patient selection. In this study, Treg cells were defined as CD3+CD4+CD25++CD127lo cells. Patients with MM (n = 20) had a significant imbalance in Treg/Th17 ratio when compared with either aged-matched controls (n = 28) or other monoclonal gammopathies, and this was associated with a significantly worse survival. The percent Treg in bone marrow of patients with MM was higher than that in matched peripheral blood samples (p = 0.02), although FOXP3 expression within bone marrow T cells was lower (p = 0.02). We observed increased Treg function, both in vivo and in vitro, due at least partially to an increase in CTLA-4 expression by concurrent treatment with dexamethasone and immune modulatory compounds (iMiDs). We suggest that immunoregulatory balance is important during active chemotherapy and that conclusions related to the immunostimulatory effect of iMiDs based on in vitro testing must be considered with caution.

Regulatory T cells and multiple myeloma.

Many clinical observations point to active immunologic phenomena in patients with myeloma. These consist of active suppression of the hosts immune system and partially successful attempts by the hosts immune system to suppress the malignant B-cell population. Clinical conditions such as asymptomatic myeloma, which represents clinical presentation in the plateau phase of the disease, plateau establishment after conventional induction therapy without the ongoing need for therapy, and the positive prognostic importance of the presence of clones of cytotoxic T cells in the peripheral blood of some patients, suggest that host-tumor interaction is an active dynamic state. Regulatory T (Treg) cells comprise 5%-10% of peripheral CD4 T cells and are responsible for the control of autoimmune phenomena. Deficiency of the FoxP3 transcription factor, which normally characterizes Treg cells, leads to multiorgan autoimmune disorders in humans and mice. The role of Treg cells in the protection from malignancy is unclear, but their depletion can lead to the induction of tumor rejection in murine models, and their demonstration as tumorinfiltrating lymphocytes in malignancy point to a significant immunomodulator role. In myeloma, host-tumor immune interactions are complex. However, patients can clearly exhibit control of their B-cell malignancy for many years with stability of paraprotein levels, demonstrating a homeostasis between tumor and host. Whether Treg cells are playing a role in this homeostasis is unclear. At present, there is considerable debate in the literature regarding observations such as whether Treg cells are increased or decreased, functional or dysfunctional. In this review, we will discuss the potential importance of Treg cells and their role in myeloma, a disease characterized by a unique set of host-tumor interactions.

Gene Therapy in Patients with Transfusion-Dependent β-Thalassemia

Background Donor availability and transplantation‐related risks limit the broad use of allogeneic hematopoietic‐cell transplantation in patients with transfusion‐dependent β‐thalassemia. After previously establishing that lentiviral transfer of a marked β‐globin (βA‐T87Q) gene could substitute for long‐term red‐cell transfusions in a patient with β‐thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion‐dependent β‐thalassemia. Methods In two phase 1–2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion‐dependent β‐thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication‐competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. Results At a median of 26 months (range, 15 to 42) after infusion of the gene‐modified cells, all but 1 of the 13 patients who had a non–β0/β0 genotype had stopped receiving red‐cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a β0/β0 genotype or two copies of the IVS1‐110 mutation, the median annualized transfusion volume was decreased by 73%, and red‐cell transfusions were discontinued in 3 patients. Treatment‐related adverse events were typical of those associated with autologous stem‐cell transplantation. No clonal dominance related to vector integration was observed. Conclusions Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long‐term red‐cell transfusions in 22 patients with severe β‐thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB‐204 and HGB‐205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526.)

The myelodysplastic syndromes: diagnostic criteria and laboratory evaluation

INTRODUCTION The myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of the multipotent hemopoietic stem cell, characterized by clonal proliferation with ineffective and dysplastic hemopoiesis. The usual result is one or more peripheral blood cytopenias with morphological and functional abnormalities. Diagnosis is based on morphological examination of peripheral blood and marrow smears, with the exclusion of other causes of dysplasia such as Vitamin B12 or folate deficiencies, alcohol or drugs. Bone marrow trephine histology may also be helpful whilst more specialized investigations such as cytogenetics, immunophenotyping and hemopoietic cell culture have yielded insights not only into the etiology and pathogenesis of these disorders but are finding application in increasing diagnostic precision and identification of clinically significant subgroups. This paper presents a brief discussion of the pathogenesis of the MDS followed by an overview of the diagnostic criteria, the potential usefulness of specialized investigations and the implications for disease course and prognosis.

A CD2 high-expressing stress-resistant human plasmacytoid dendritic-cell subset

Human plasmacytoid dendritic cells (pDCs) were considered to be a phenotypically and functionally homogeneous cell population; however, recent analyses indicate potential heterogeneity. This is of major interest, given their importance in the induction of anti‐viral responses and their role in creating immunologically permissive environments for human malignancies. For this reason, we investigated the possible presence of human pDC subsets in blood and bone marrow, using unbiased cell phenotype clustering and functional studies. This defined two major functionally distinct human pDC subsets, distinguished by differential expression of CD2. The CD2hi and CD2lo pDCs represent discontinuous subsets, each with hallmark pDC functionality, including interferon‐alpha production. The rarer CD2hi pDC subset demonstrated a significant survival advantage over CD2lo pDC during stress and upon exposure to glucocorticoids (GCs), which was associated with higher expression of the anti‐apoptotic molecule BCL2. The differential sensitivity of these two human pDC subsets to GCs is demonstrated in vivo by a relative increase in CD2hi pDC in multiple myeloma patients treated with GCs. Hence, the selective apoptosis of CD2lo pDC during stress represents a novel mechanism for the control of innate responses.

Excellent outcomes for adolescents and adults with acute lymphoblastic leukemia and lymphoma without allogeneic stem cell transplant: the FRALLE-93 pediatric protocol

Abstract Adolescents and adults with acute lymphoblastic leukemia/lymphoma (ALL) have better outcomes when treated using pediatric protocols compared with treatment using adult protocols. We reviewed the progress and outcomes of 40 adolescents and adults up to 45 years of age, from three Australian centers, treated on the intensive French group for childhood ALL (FRALLE)-93 pediatric protocol. All except one patient achieved a morphologic complete remission following induction chemotherapy. Three-year overall survival for all-risk and standard-risk disease was 70% and 75%, respectively. The treatment protocol was generally well tolerated with no treatment related mortality. The FRALLE-93 pediatric protocol showed excellent overall survival for patients with standard-risk disease, without the need for allogeneic hematopoietic stem cell transplant in first remission.