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Many researchers have speculated that the clinical progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is driven by defects in dendritic cell (DC) function. However, evidence supporting this assumption is controversial, and no mechanism for the putative DC dysfunction has been demonstrated thus far. We studied DC subsets from the bone marrow of MM patients compared with those of MGUS patients and control subjects. We found that myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) accumulate in the bone marrow during the MGUS-to-MM progression. After engulfment of apoptotic tumor plasma cells via CD91, bone marrow mDCs and pDCs mature and are able to activate tumor-specific CD8(+) T cells. However, by interacting directly with CD28 on live (nonapoptotic) tumor plasma cells, bone marrow mDCs downregulate the expression of proteasome subunits in these cells, thus enabling their evasion from human leukocyte antigen (HLA) class I-restricted CD8(+) T-cell killing. These results suggest that DCs play a dual, but opposing, role in MM: for one, DCs activate CD8(+) T cells against tumor plasma cells and, for the other, DCs protect tumor plasma cells from CD8(+) T-cell killing. This information should be taken into account in designing immunotherapy approaches to enhance immune surveillance in MGUS and to break down immune tolerance in MM., (© 2015 by The American Society of Hematology.)

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

Because tumor progression is angiogenesis-dependent, angiogenesis density was investigated by immunohistochemistry and computed image analysis in bone marrow (BM) biopsies of 45 newly diagnosed patients with Binet stage A B-cell chronic lymphocytic leukemia (BCLL) and correlated to upstaging and progression-free survival during a 40-month follow-up period. Their microvessel areas and counts were significantly higher than those of patients with anemia due to iron or vitamin B(12) deficiencies. A cutoff value of 0.90 mm(2) x 10(-2) or greater of the microvessel area identified patients with earlier upstaging and shorter progression-free survival. When the cutoff was applied to the Rai subclassification, both Rai 0 and Rai I-II patients who upstaged and shortened the progression-free survival were classified correctly. Information of this type was not given by the microvessel counts. The cutoff did not correlate with other predictors representative of tumor mass or disease progression. The microvessel area correlated with the expression of angiogenic vascular endothelial growth factor (VEGF) by tumor tissue, and serum levels of VEGF were found to be of prognostic value. A causal relationship between risk of progression and BM angiogenesis in BCLL is suggested. A risk stratification inside Rai is proposed. The prognostic usefulness of BM angiogenesis in patients with BCLL is envisaged.

Anemia of variable severity occurs in more than two-thirds of patients with multiple myeloma (MM). Besides the altered cytokine network, chronic erythropoietn deficiency, blood loss and hemolysis, we have shown that deregulated myeloma cell apoptosis contributes to progressive destruction of the erythroid matrix by inducing erythroblast cytotoxicity. To exert this effect, highly malignant plasma cells overexpress both Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which efficiently trigger the death of immature erythroblasts. However, this Fas-L/TRAIL-based anemia occurs in particular in patients with severely progressive MM, thus suggesting that these apoptogen receptors may characterize a peculiar cytotoxic-apoptogenic phenotype of malignancy. Immunophenotyping of myeloma cells could help to identify patients with a higher risk of erythropoiesis exhaustion.

We identified STAT1 gain of function (GOF) in a 32-year-old female with pallor, weakness, cough, and dyspnea admitted to our Division of Medicine. She had severe oral ulcers (OU), type 1 diabetes (T1DM), and pancytopenia. Bone marrow (BM) biopsy showed the absence of erythroid precursors. Peripheral blood parameters such as neutrophils < 500/mL, reticulocytes < 2%, and BM hypo-cellularity allowed to diagnose severe aplastic anemia. A heterozygous variant (p.520T>C, p.Cys174Arg) of STAT1 was uncovered. Thus, p.Cys174Arg mutation was investigated as potentially responsible for the patient's inborn immunity error and aplastic anemia. Although STAT1 GOF is rare, aplastic anemia is a more common condition; therefore, we explored STAT1 functional role in the pathobiology of BM failure. Interestingly, in a cohort of six patients with idiopathic aplastic anemia, enhanced phospho-STAT1 levels were observed on BM immunostaining. Next, the most remarkable features associated with STAT1 signaling dysregulation were examined: in both pure red cell aplasia and aplastic anemia, CD8+ T cell genetic variants and mutations display enhanced signaling activities related to the JAK-STAT pathway. Inborn errors of immunity may represent a paradigmatic condition to unravel crucial pathobiological mechanisms shared by common pathological conditions. Findings from our case-based approach and the phenotype correspondence to idiopathic aplastic anemia cases prompt further statistically powered prospective studies aiming to elucidate the exact role and theragnostic window for JAK/STAT targeting in this clinical context. Nonetheless, we demonstrate how a comprehensive study of patients with primary immunodeficiencies can lead to pathophysiologic insights and potential therapeutic approaches within a broader spectrum of aplastic anemia cases. [ABSTRACT FROM AUTHOR]

It was believed that vasculogenesis occurred only during embryo life and that postnatal formation of vessels arose from angiogenesis. Recent findings demonstrate the existence of Endothelial Precursor Cells (EPCs), which take partin postnatal vasculogenesis. EPCs are recruited from the bone marrow under the stimulation of growth factors and cytokines and reach the sites of neovascularization in both physiological and pathological conditions such as malignancies where they contribute to the “angiogenic switch” and tumor progression. An implementation of circulating EPCs in the bloodstream of patients with haematological malignancies has been demonstrated. This increase is strictly related to the bone marrow microvessel density and correlated with a poor prognosis. The EPCs characterization is a very complex process and still under investigation. This literature review aims to provide an overview of the functional and biological role of EPCs in haematological malignancies and to investigate their potential as a new cancer therapeutic target. [ABSTRACT FROM AUTHOR]

The crosstalk of myeloma cells with accessory cells drives the expansion of malignant plasma cell clones and the hyperactivation of osteoclastogenesis that occurs in multiple myeloma (MM). These reciprocal interactions promote defective dendritic cell (DC) function in terms of antigen processing, clearance of tumor cells, and efficacy of the immune response. Thus, myeloma cells exert immune suppression that explains, at least in part, the failure of therapeutic approaches, including DC vaccination. Impairment of DCs depends on high bone marrow levels of cytokines and adhesion molecules that affect both maturation and expression of costimulatory molecules by DCs. Moreover, DCs share with osteoclasts (OCs) a common ontogenetic derivation from the monocyte lineage, and thus may undergo OC-like transdifferentiation both in vitro and in vivo. Immature DCs (iDCs) induce clonogenic growth of malignant plasma cells while displaying OC-like features, including the ability to resorb bone tissue once cultured with myeloma cells. This OC-like transdifferentiation of iDCs is dependent on the activation of both the receptor activator of nuclear factor κB (RANK)-RANK ligand RANK-L) and CD47-thrombospondin (TSP)-I axes, although interleukin 17-producing T helper-17 clones within the bone microenvironment may also take part in this function. Therefore, iDCs allied with malignant plasma cells contribute to MM osteoclastogenesis, although other molecules released by tumor cells may independently contribute to the bone-resorbing machinery. [ABSTRACT FROM AUTHOR]

Umbilical cord (UC) mesenchymal stem cells (MSCs) are being currently investigated as an alternative to bone marrow (BM) MSCs for bone repair and regeneration. Here, we describe the gene regulation of their differentiation to osteogenic, adipogenic, and chondrogenic precursors and demonstrate their tendency to differentiate toward the osteoblast lineage. Fibroblast-like cells from the Warthon’s Jelly were cultured with dedicated media to obtain osteogenic-, adipogenic-, and chondrogenic-differentiated cells. After induction, a typical fibroblast-like shape with condensed fibers of F-actin was early noted in osteogenic-induced UC-MSCs, whereas those differentiating to adipocytes were flat with minor cytoskeleton relevance. Real-time PCR measured the transcription of master genes of the three lineages, thus revealing a remarkable up-regulation of Runx2 in osteogenic-induced cells with respect to both PPARg and SOX9 for adipogenic- and chondrogenic-differentiating UC-MSCs. However, TAZ, a coactivator of the nuclear transcription of Runx2 previously detected in BM-MSCs, was expressed in osteogenic- and, at lower magnitude, in adipogenic-induced cells, in keeping with its role in the reciprocal control of the differentiation between osteogenic- and adipogenic-induced cells. Its differential role in these cells was confirmed by its accumulation as protein product in the nuclei to activate Runx2 in osteogenic-differentiating UC-MSCs. These data emphasize the predominant expression by UC-MSCs of genes engaged in the osteogenic differentiation and their tendency to differentiate into osteoblasts, being similar in this respect to BM-MSCs. They may, thus, constitute a promising option for bone remodeling in regenerative medicine. [ABSTRACT FROM AUTHOR]

Hyperactive osteoclastogenesis is a hallmark of multiple myeloma, a B cell neoplasia homing to bone marrow and resulting in multiple osteolytic lesions and skeleton devastation. We provide evidence that myeloma cells can themselves act as osteoclasts in vitro. By extending standard cultures of U-266 and MCC-2 myeloma cell lines, we found that subsets of adherent cells also expressed the osteoclast phenotype, including multinuclear morphology, cytoplasmic tartrate-resistant acid phosphatase, the calcitonin receptor and a specific osteoclast antigen. These subsets resorbed bone substrates by producing osteoclast enzymes as well as the characteristic redistribution of F-actin in their cytoskeleton, thus forming the sealing zone that is adopted by adherent osteoclasts to generate the acidified environment essential for bone resorption. Neither the phenotype nor the functional properties of osteoclasts were detected in parental non-adherent cells. In adherent cultures osteoclastogenesis was associated with deregulated expression of both receptor activator of nuclear transcription factor (NF)- κB (RANK) and its ligand RANK-L, which triggers cell maturation in osteoclast precursors. Resorption of bone substrates was prevented by a neutralising anti-RANK-L antibody. Our data indicate that osteoclast-like transformation of both U-266 and MCC-2 cellular models of human myeloma is dependent on RANK-L stimulation. [ABSTRACT FROM AUTHOR]

The erythrocyte water channel aquaporin 1 (AQP1) is expressed in multiple absorptive and secretory epithelia including the capillary endothelia. Immunoblot analysis showed that bone marrow biopsies of patients with active multiple myeloma (MM) display significantly higher levels of AQP1 than those from patients with non-active MM, whose values are higher, but to a lesser extent, than those of patients with monoclonal gammopathies of undetermined significance (MGUS). Values of MGUS overlapped those of patients with anaemia as a result of iron or vitamin B12 deficiencies (called ‘benign anaemias’). Immunohistochemistry and computerized image analysis of AQP1 highlighted bone marrow microvessels whose area per microscopic field was significantly greater in patients with active MM, and always larger than and closely correlated with the microvessel area when assessed with factor VIII-related antigen/von Willebrand's factor (FVIII–VWF). The intensity of AQP1 expression by microvessels evaluated using image analysis was significantly greater in active than non-active MM and in the latter over MGUS or benign anaemias. It is suggested that, among plasma cell tumours, AQP1 expression is preferentially associated with microvessels of MM and that the highest degree of expression occurs in active MM in step with enhanced angiogenesis, in which AQP1 recognizes more immature neovessels than FVIII–VWF. It may, perhaps, favour angiogenesis in a positive loop and, hence, MM progression, and thus be applied for therapeutic vascular targeting. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) line the bone marrow microvasculature and are in close contact with CD8+ T cells that come and go across the permeable capillaries. Because of these intimate interactions, we investigated the capacity of EC to act as antigen-presenting cells (APC) and modulate CD8+ T cell activation and proliferation in bone marrow of patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance. We found that EC from MM patients show a phenotype of semi-professional APC given that they express low levels of the co-stimulatory molecules CD40, CD80 and CD86, and of the inducible co-stimulator ligand (ICOSL). In addition, they do not undergo the strong switch from immunoproteasome to standard proteasome subunit expression which is typical of mature professional APC such as dendritic cells. EC can trap and present antigen to CD8+ T cells, stimulating a central memory CD8+ T cell population that expresses Foxp3 and produces high amounts of IL-10 and TGF-β. Another CD8+ T cell population is stimulated by professional APC, produces IFN-γ, and exerts antitumor activity. Thus, two distinct CD8+ T cell populations coexist in the bone marrow of MM patients: the first population is sustained by EC, expresses Foxp3, produces IL-10 and TGF-β, and exerts pro-tumor activity by negatively regulating the second population. This study adds new insight into the role that EC play in MM biology and describes an additional immune regulatory mechanism that inhibits the development of antitumor immunity and may impair the success of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Waldenström''s macroglobulinemia (WM) is defined as a B-cell lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone marrow associated with a monoclonal IgM component in the serum. Its clinical presentation is marked by diffuse clonal cell expansion, as well as by the physical and chemical properties of the monoclonal component, its autoantibody activity and possible tissue deposition. Initiation of treatment is not determined by the monoclonal IgM level, nor the extent of bone marrow infiltration, but confined to symptomatic patients. Their median overall survival ranges from 5 to 10 years. Poor outcome predictors include advanced age, low hemoglobin levels, low platelet count, high β2-microglobulin and high concentration of the serum monoclonal component. First-line therapeutic approaches include alkylating agents (chlorambucil, melphalan, cyclophosphamide), nucleoside analogs (fludarabine, cladribrine), and rituximab, whether singly or combined. Thalidomide-based regimens and bortezomib have also been assessed, and new agents such as bendamustine and everolimus are being investigated. We review these general features and describe our series of 121 patients with clearly established WM. [Copyright &y& Elsevier]

Many researchers have speculated that the clinical progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is driven by defects in dendritic cell (DC) function. However, evidence supporting this assumption is controversial, and no mechanism for the putative DC dysfunction has been demonstrated thus far. We studied DC subsets from the bone marrow of MM patients compared with those of MGUS patients and control subjects. We found that myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) accumulate in the bone marrow during the MGUS-to-MM progression. After engulfment of apoptotic tumor plasma cells viaCD91,bonemarrowmDCs andpDCsmature and are able to activate tumor-specific CD8+ T cells. However, by interacting directly with CD28 onlive (nonapoptotic) tumor plasma cells, bone marrow mDCs downregulate the expression of proteasome subunits in these cells, thus enabling their evasion from human leukocyte antigen (HLA) class I-restricted CD8+ T-cell killing. These results suggest that DCs play a dual, but opposing, role in MM: for one, DCs activate CD8+ T cells against tumor plasmacells and, for the other, DCs protect tumor plasmacells from CD8+ T-cell killing. This information should be taken in to account in designing immunotherapy approaches to enhance immune surveillance in MGUS and to break down immune tolerance in MM. [ABSTRACT FROM AUTHOR]

Abstract: Bone disease in multiple myeloma (MM) leads to progressive devastation of the skeleton and is the most severe cause of morbidity. Its pathogenetic mechanisms are not fully defined, though the current evidence points to hyperactivation of osteoclasts (OC) in presence of a major defect of bone repairing in erosion sites due to osteoblast (OB) impairment. Bone resorption, however, is promoted by early OB, namely stromal cells that respond to chronic stimulation by myeloma cells by enhancing marrow levels of RANKL and other osteoclastogenic factors and thus accelerating the maturation of OC progenitors. In myeloma bone disease (MBD), OBs are systematically defeated by a number of inhibiting effects induced by the malignant clone within the marrow microenvironment. Thus, MBD primarily affects the OB lineage, particularly in overt MM, where serum markers of osteoblastogenesis, such as osteocalcin and osteoprotegerin, are extremely low in contrast with their slight increase in inactive MM. These markers, in association with others of bone turnover (RANKL, MIP-1α, type I collagen telopeptides such as NTX and CTX) may be used in the clinical assessment of MBD as well as to monitor the efficacy of bisphosphonate in delaying the progressive skeletal destruction. [Copyright &y& Elsevier]