Serum Free Light Chain Kinetics Is Predictive of Renal Response in Myeloma Patients With Renal Impairment-An ALLG Trial of Carfilzomib-Dexamethasone Therapy in Frontline and Relapse.

BACKGROUND AND PURPOSE: Renal impairment (RI) confers adverse prognosis in myeloma; its reversal and avoidance of dialysis are crucial. We investigated whether serum free light chain (SFLC) measurements can predict renal outcome, to enable change in therapy to optimize prognosis and avoid dialysis. PATIENTS AND METHODS: We investigated 36 myeloma patients (17 newly diagnosed [ND]; 19 relapsed refractory [RR]; with median of 5 prior lines) with eGFR 15-40 ml/min treated with carfilzomib (Cfz)-dexamethasone to determine whether SFLC kinetics can predict renal outcomes, and assess efficacy and tolerability. RESULTS: The change in involved SFLC at Cycle 2 Day 1 was significantly correlated with renal function; for every one log10 reduction in involved SFLC, eGFR increased by 9.0-15.0 mL/min at cycles 2-4, with SFLC reduction of 54%-78%. At a median follow-up of 30.6 months, renal outcomes were favorable-CRrenal 25%, MRrenal 36%. Disease responses (ND 100%, RR 75%), progression-free survival (ND 32.2 months, RR 11.1 months) and overall survival (ND not reached, RR 42.0 months) were comparable to patients without RI. There was significant toxicity, including Cfz-related cardiac impairment of 20% within a cohort with high co-morbidity, and a high incidence of infections. CONCLUSION: We propose that one log10 reduction in involved SFLC at Cycle 2 Day 1 is an appropriate target for reducing the risk of dialysis in myeloma patients with RI; below this threshold patients may benefit from a change in therapy. While Cfz-dexamethasone achieved favorable renal and disease outcomes, toxicity can be significant in this vulnerable cohort.

Single-cell analysis of the CD8+ T-cell compartment in multiple myeloma reveals disease specific changes are chiefly restricted to a CD69- subset suggesting potent cytotoxic effectors exist within the tumor bed.

Multiple myeloma (MM) is an incurable disease of the bone marrow (BM) characterized by the uncontrolled proliferation of neoplastic plasma cells. While CD8+ T cells have an established role in disease control, few studies have focused on these cells within the MM tumor microenvironment (TME). We analyzed CD8+ T cells in the BM and peripheral blood (PB) of untreated patients with MM and non-myeloma controls using flow cytometry, mass cytometry and single-cell RNA sequencing, using several novel bioinformatics workflows. Inter-tissue differences were most evident in the differential expression of Granzymes B and K, which were strongly associated with two distinct subsets of CD8+ T cells delineated by the expression of CD69, accounting for roughly 50% of BM-CD8+ T cells of all assessed cohorts. While few differences were observable between health and disease in the BM-restricted CD8CD69+ T-cell subset, the CD8+CD69- T-cell subset in the BM of untreated MM patients demonstrated increased representation of highly differentiated effector cells and evident compositional parallels between the PB, absent in age-matched controls, where a marked reduction of effector cells was observed. We demonstrate the transcriptional signature of BM-CD8+ T cells from patients with MM more closely resembles TCR-activated CD8+ T cells from age-matched controls than their resting counterparts.

Consensus guidelines and recommendations for infection prevention in multiple myeloma: a report from the International Myeloma Working Group.

Infection remains the leading cause of morbidity and mortality in patients with multiple myeloma because of the cumulative effect of disease, treatment, and host-related factors. Given that infectious risk is cumulative through the course of the disease, preventing infections is paramount. Optimal preventive strategies include vaccination against common pathogens, antimicrobial prophylaxis, infection control measures, and immunoglobulin replacement in a small subset of patients; however, there are no universally accepted guidelines for infection prevention. This Review provides a consensus statement from a panel of 36 experts with global representation, which was convened by The International Myeloma Society to review existing literature and current guidelines, address issues associated with the risk of infection and prevention of infectious complications in multiple myeloma in the context of emerging therapies, and offer recommendations for preventing these complications.

Treg and Oligoclonal Expansion of Terminal Effector CD8+ T Cell as Key Players in Multiple Myeloma.

The classical paradigm of host-tumor interaction, i.e. elimination, equilibrium, and escape (EEE), is reflected in the clinical behavior of myeloma which progresses from the premalignant condition, Monoclonal Gammopathy of Unknown Significance (MGUS). Despite the role of other immune cells, CD4+ regulatory T cells (Treg) and cytotoxic CD8+ T cells have emerged as the dominant effectors of host control of the myeloma clone. Progression from MGUS to myeloma is associated with alterations in Tregs and terminal effector CD8+ T cells (TTE). These changes involve CD39 and CD69 expression, affecting the adenosine pathway and residency in the bone marrow (BM) microenvironment, together with oligoclonal expansion within CD8+ TTE cells. In this mini-review article, in the context of earlier data, we summarize our recent understanding of Treg involvement in the adenosine pathway, the significance of oligoclonal expansion within CD8+ TTE cells and BM-residency of CD8+ TTE cells in MGUS and newly diagnosed multiple myeloma patients.

Biology and therapy of multiple myeloma.

Genetic sequencing of the myeloma genome has not revealed a specific disease-determining genetic alteration. Multiple disease subclones exist at diagnosis and vary in clinical importance with time and drug sensitivity. New diagnostic criteria have identified indications for early introduction of therapy. Autologous stem cell transplantation remains an essential component of therapy in young and fit patients. The use of continual suppressive (maintenance) therapy has been established as an important component in therapy. Immune therapies and the harnessing of the innate immune system offer great promise for future treatments. Since 2005, quality of life, supportive therapies, and survival have dramatically improved over a decade of remarkable progress. The common manifestations of multiple myeloma, such as bone pain, fatigue and weight loss, may be non-specific and are often initially ignored or missed by patients and medical practitioners.

A blood dendritic cell vaccine for acute myeloid leukemia expands anti-tumor T cell responses at remission.

Only modest advances in AML therapy have occurred in the past decade and relapse due to residual disease remains the major challenge. The potential of the immune system to address this is evident in the success of allogeneic transplantation, however this leads to considerable morbidity. Dendritic cell (DC) vaccination can generate leukemia-specific autologous immunity with little toxicity. Promising results have been achieved with vaccines developed in vitro from purified monocytes (Mo-DC). We now demonstrate that blood DC (BDC) have superior function to Mo-DC. Whilst BDC are reduced at diagnosis in AML, they recover following chemotherapy and allogeneic transplantation, can be purified using CMRF-56 antibody technology, and can stimulate functional T cell responses. While most AML patients in remission had a relatively normal T cell landscape, those who had received fludarabine as salvage therapy have persistent T cell abnormalities including reduced number, altered subset distribution, failure to expand, and increased activation-induced cell death. Furthermore, PD-1 and TIM-3 are increased on CD4T cells in AML patients in remission and their blockade enhances the expansion of leukemia-specific T cells. This confirms the feasibility of a BDC vaccine to consolidate remission in AML and suggests it should be tested in conjunction with checkpoint blockade.

CMRF-56(+) blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses.

There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56(+) BDC, we showed that transfection of hCMRF-56(+) BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56(+) BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56(+) BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8(+) T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56(+) BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56(+) BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

Response assessment in Waldenström macroglobulinaemia: update from the VIth International Workshop.

This report represents a further update of the consensus panel criteria for the assessment of clinical response in patients with Waldenström macroglobulinaemia (WM). These criteria have been updated in light of further data demonstrating an improvement in categorical responses with new drug regimens as well as acknowledgement of the fact that such responses are predictive of overall outcome. A number of key changes are proposed but challenges do however remain and these include the variability in kinetics of immunoglobulin M (IgM) reduction with different treatment modalities and the apparent discrepancy between IgM and bone marrow/tissue response noted with some regimens. Planned sequential bone marrow assessments are encouraged in clinical trials.

Thalidomide consolidation improves progression-free survival in myeloma with normal but not up-regulated expression of fibroblast growth factor receptor 3: analysis from the Australasian Leukaemia and Lymphoma Group MM6 clinical trial.

The translocation t(4;14) is associated with a poor prognosis in myeloma, but its effect in the setting of new drugs such as thalidomide, bortezomib and lenalidomide continues to be investigated, and the role of candidate genes such as FGFR3 (fibroblast growth factor receptor 3) is not yet clarified. In the Australasian Leukaemia and Lymphoma Group (ALLG) MM6 randomized study comparing consolidation thalidomide and prednisolone with prednisolone alone following autologous stem cell transplant, patients on consolidation thalidomide and prednisolone had superior progression-free (PFS) and overall survival (OS). We now show that thalidomide consolidation benefited both t(4;14)-positive (PFS 29 vs. 17 months, p =0.03) and -negative (52 vs. 24 months, p =0.04) disease. PFS for patients with normal FGFR3 expression was significantly better than for those with up-regulated FGFR3 (31 vs. 21 months, p =0.02). Consolidation thalidomide conferred an improved PFS in patients with normal FGFR3 expression (41 vs. 19 months, p =0.02), but there was no improvement in patients with up-regulated FGFR3 (31 vs. 29 months, p =0.76). We conclude that consolidation thalidomide may mitigate the poor prognostic effect of t(4;14), and improves PFS in normal but not up-regulated FGFR3 expression. Thus the level of FGFR3 expression provides additional prognostic information to t(4;14) in myeloma induction and consolidation therapy.

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).

The impact of mandatory fortification of flour with folic acid on the blood folate levels of an Australian population.

OBJECTIVE: To determine the impact that mandatory fortification with folic acid of wheat flour used in breadmaking has had on the blood folate levels of an Australian population since it was introduced in September 2009. DESIGN, SETTING AND PATIENTS: A retrospective analysis of serum and red blood cell (RBC) folate levels of 20,592 blood samples collected between April 2007 and April 2010 from a wide variety of inpatients and outpatients and analysed in a large public hospital diagnostic pathology laboratory. MAIN OUTCOME MEASURES: Prevalences of low levels of serum and RBC folate and monthly mean levels before and after introduction of mandatory fortification. RESULTS: Between April 2009 and April 2010, there was a 77% reduction in the prevalence of low serum folate levels (from 9.3% to 2.1%) in all samples tested and an 85% reduction in the prevalence of low RBC folate levels (from 3.4% to 0.5%). In April 2010, the prevalence of low RBC folate levels for females of childbearing age was 0.16% for all samples. There was a 31% increase in mean serum folate level (from 17.7 nmol/L to 23.1 nmol/L; t = 9.3, P < 0.01), and a 22% increase in mean RBC folate level (from 881 nmol/L to 1071 nmol/L). The greatest increment in mean serum folate levels occurred in September 2009, the month that mandatory fortification was introduced, although there was evidence of a gradual change during the preceding months. CONCLUSION: The introduction of mandatory fortification with folic acid has significantly reduced the prevalence of folate deficiency in Australia, including in women of childbearing age.

Clonal expansions of cytotoxic T cells exist in the blood of patients with Waldenstrom macroglobulinemia but exhibit anergic properties and are eliminated by nucleoside analogue therapy.

T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8(+)CD57(+) T-cell receptor Vbeta-positive (TCRVbeta(+))-restricted cytotoxic T-cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report that CTL clones with varying TCRVbeta repertoire are present in 70% of patients with Waldenström macroglobulinemia (WM; n = 20). Previous nucleoside analog (NA) therapy, associated with increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVbeta expansions (chi(2) = 11.6; P < .001), as 83% of patients without (n = 6) and only 7% with (n = 14) TCRVbeta expansions had received NA. Clonality of CD3(+)CD8(+)CD57(+)TCRVbeta(+)-restricted CTLs was confirmed by TCRVbeta CDR3 size analysis and direct sequencing. The differential expression of CD3(+)CD8(+)CD57(+)TCRVbeta(+) cells was profiled using DNA microarrays and validated at mRNA and protein level. By gene set enrichment analysis, CTL clones expressed not only genes from cytotoxic pathways (GZMB, PRF1, FGFBP2) but also genes that suppress apoptosis, inhibit proliferation, arrest cell-cycle G1/S transition, and activate T cells (RAS, CSK, and TOB pathways). Proliferation tracking after stimulation confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity, and nature of clonal CTLs in WM and highlight mechanisms that cause anergy in these cells.

Prognostically significant cytotoxic T cell clones are stimulated after thalidomide therapy in patients with multiple myeloma.

The expanded T cell clones are associated with a prolonged survival in patients with multiple myeloma. We sought to confirm this prognostic significance in a multicenter patient cohort and investigate the effect of thalidomide on clones and T regulatory cells (T(regs)). Blood was collected from 120 patients enrolled in a Phase III trial of maintenance therapy +/- thalidomide after autologous stem cell transplantation. TCR Vbeta repertoire analysis identified T cell expansions in 48% of patients pre-transplant and 68% after 8-month maintenance. T cell expansions, previously shown to be clonal, were predominantly CD8+ (93%) and all 24 TCR Vbeta families tested were represented. Thalidomide therapy was associated with a significant increase in the incidence of patients with multiple expansions (49% vs. 23%; chi2 = 6.8; p = 0.01). The presence of expansions regardless of therapy was associated with a significantly longer median progression free survival (PFS) (32.1 vs. 17.6 months; chi2 = 5.6; p = 0.02) and overall survival (OS) (chi2 = 3.9; p < 0.05). Median PFS in the thalidomide arm was 50.9 months for patients with expansions and 28.3 months for patients without expansions (chi2 = 19.4; p = 0.0002). Thalidomide did not appear to modulate T(reg) numbers. Expanded T cell clones are prognostically significant and have an impact on progression after thalidomide therapy in a proportion of patients.

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 host's immune system and partially successful attempts by the host's 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.

Transformation and progression of Waldenström's macroglobulinemia following cladribine therapy in two cases: natural evolution or iatrogenic causation?

We report two cases of Waldenström's macroglobulinemia with an unusual aggressive transformation following treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA), a purine analogue. The first patient developed transformation to a diffuse large-cell non-Hodgkin lymphoma, while the second developed extensive extramedullary involvement. Both patients displayed rapid progression following transformation and were refractory to chemotherapy. Both patients were pretreated with multiple courses of prednisone and chlorambucil, and transformation occurred shortly after therapy with cladribine. We propose that immune suppression from alkylating agents and purine analogues may have contributed to the unusual progression, resulting in a dismal outcome.

Multiple myeloma: the present and the future.

Major advances continue apace in therapy and in understanding its molecular pathogenesis.

Clonal cytotoxic T cells in myeloma.

Multiple myeloma (MM) is a malignant disease characterized by accumulation of morphologically recognizable plasma cells producing immunoglobulin (Ig) in the bone marrow. The occurrence of clonal T cells in MM, as defined by the presence of rearrangements in the T-cell receptor (TCR)-beta chains detected on Southern blotting, is associated with an improved prognosis. This review aims to describe the various ways in which we have demonstrated the presence of such T cell clones, and to describe the phenotype of these cells. Finally, the specificities of these clinically important CD8+ T cell populations will be discussed in the context of immunotherapy.

Prediction of high affinity class I-restricted multiple myeloma idiotype peptide epitopes.

Idiotypic determinants are potential patient-specific tumor antigens in multiple myeloma (MM). In this study, we have determined the DNA sequence of the variable region of the tumor immunoglobulin (Ig) in 6 patients with MM. We then selected high affinity class I-restricted T-cell peptide epitopes in tumor Ig using two different internet-based epitope prediction programs. High affinity binding peptides were identified by at least one program in 4 out of 6 patients. Of these 35 peptides, only 3 scored high by both analyses. Given that all 6 patients had expanded T-cell clones with a cytotoxic (CD57+CD8+CD28-perforin+) phenotype, known to be associated with a longer survival and postulated to recognise tumor epitopes, this analysis indicates that such clones are unlikely to be exclusively directed towards tumor immuoglobulin, and suggests the need to expand the scope of the search for tumor epitopes with the ability to stimulate cytotoxic T cells in vivo.