Bone Marrow Stroma and Vascular Contributions to Myeloma Bone Homing.

PURPOSE OF THE REVIEW: Herein we dissect mechanisms behind the dissemination of cancer cells from primary tumor site to the bone marrow, which are necessary for metastasis development, with a specific focus on multiple myeloma. RECENT FINDINGS: The ability of tumor cells to invade vessels and reach the systemic circulation is a fundamental process for metastasis development; however, the interaction between clonal cells and the surrounding microenvironment is equally important for supporting colonization, survival, and growth in the secondary sites of dissemination. The intrinsic propensity of tumor cells to recognize a favorable milieu where to establish secondary growth is the basis of the "seed and soil" theory. This theory assumes that certain tumor cells (the "seeds") have a specific affinity for the milieu of certain organs (the "soil"). Recent literature has highlighted the important contributions of the vascular niche to the hospitable "soil" within the bone marrow. In this review, we discuss the crucial role of stromal cells and endothelial cells in supporting primary growth, homing, and metastasis to the bone marrow, in the context of multiple myeloma, a plasma cell malignancy with the unique propensity to primarily grow and metastasize to the bone marrow.

Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs.

BACKGROUND: A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes. METHODS: DNA was prepared from blood of 203 healthy adults (range: 18-91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests. RESULTS: The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR. CONCLUSIONS: Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies.

Sequential monitoring of lymphocyte subsets and of T-and-B cell neogenesis indexes to identify time-varying immunologic profiles in relation to graft-versus-host disease and relapse after allogeneic stem cell transplantation.

T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet. Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B <2.4%, CD4+TCM cells <45%; HR 0.5, p = 0.001); PC2 (at aGVHD onset: CD4+>44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA<1, total Treg<4, TregEM <2 cells/µl; HR 0.5, p = 0.002). Chronic GVHD was associated with one PC (TregEM <2 cells/µl at day+28, CD8+TEMRA<43% at day+90, immature B cells<6 cells/µl and KRECs<11710/ml at day+180; HR 0.4, P = 0.001). Two PC correlated with relapse: PC1 (pre-SCT: CD4+ <269, CD4+TCM <120, total Treg <18, TregCM <8 cells/µl; HR 4.0, p = 0.02); PC2 (pre-SCT mature CD19+ >69%, switched memory CD19+ = 0 cells and KRECs<6614/ml at +90; HR 0.1, p = 0.008). All these immunologic parameters were independent indicators of chronic GVHD and relapse, also considering the possible effect of previous steroid-therapy for acute GVHD. Specific time-varying immunologic profiles were associated to GVHD and relapse. Pre-SCT host immune-microenvironment and changes of B cell homeostasis could influence GVH- and Graft-versus-Tumor reactions. The paradoxical increase of EM Treg in PB of patients with GVHD could be explained by their compartmentalization outside lymphoid tissues, which are of critical relevance for regulation of GVH reactions.

MxA RNA quantification in febrile patients who underwent hematopoietic cell transplantation for primary immunodeficiency.

The presence of myxovirus resistance protein A (MxA) RNA was studied in 55 febrile children with primary immunodeficiency, 27 of whom underwent hematopoietic cell transplantation, and in 28 age-matched controls. The level of MxA RNA was above the cutoff, established as the 95th percentile found in controls, with primary immunodeficiency either undergoing transplantation or not in febrile patients, and with a documented diagnosis of infection by adenovirus, cytomegalovirus, Epstein-Barr virus, respiratory syncytial virus, and rotavirus. The presence of rare viral infections, unrecognized among those that more frequently occur in patients with primary immunodeficiency and in patients undergoing transplantation, may explain the high MxA RNA levels observed in some patients with fever but undetectable genomes or antibodies for the more common viruses. The level of MxA in febrile patients with acute graft versus host disease was below the cutoff, with a median level comparable with that observed in patients with primary immunodeficiency, who did not undergo transplantation and were without fever and infections, but significantly lower compared with controls. The level of MxA was well correlated with viral infections in follow-up samples. These data indicate that the measurement of MxA RNA is simple and useful to detect viral infections and in distinguishing them from acute graft versus host disease after allogeneic cell transplantation.

The different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine deaminase deficiency.

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle(+) lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA-treated patients, it was accompanied by a significant and progressive decrease in circulating CD19(+) lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle(+) cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.

Quantification of newly produced B and T lymphocytes in untreated chronic lymphocytic leukemia patients.

BACKGROUND: The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes. METHODS: The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test. RESULTS: KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs+ cells was preserved. Furthermore, the observed increase of CD4+ lymphocytes could be ascribed to the accumulation of CD4+ cells with effector memory phenotype. CONCLUSIONS: The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.

Dacarbazine treatment before peptide vaccination enlarges T-cell repertoire diversity of melan-a-specific, tumor-reactive CTL in melanoma patients.

Combination of chemotherapy and immunotherapy to increase the effectiveness of an antitumor immune response is currently regarded as an attractive antitumor strategy. In a pilot clinical trial, we have recently documented an increase of melanoma antigen A (Melan-A)-specific, tumor-reactive, long-lasting effector-memory CD8(+) T cells after the administration of dacarbazine (DTIC) 1 day before peptide vaccination in melanoma patients. Global transcriptional analysis revealed a DTIC-induced activation of genes involved in the immune response and leukocyte activation. To identify the possible mechanisms underlying this improved immune response, we have compared the endogenous and the treatment-induced anti-Melan-A response at the clonal level in patients treated with the vaccine alone or with DTIC plus vaccine. We report a progressive widening of T-cell receptor (TCR) repertoire diversity, accompanied by high avidity and tumor reactivity, only in Melan-A-specific T-cell clones of patients treated with chemoimmunotherapy, with a trend toward longer survival. Differently, patients treated with vaccine alone showed a tendency to narrowing the TCR repertoire diversity, accompanied by a decrease of tumor lytic activity in one patient. Collectively, our findings indicate that DTIC plus vaccination shapes the TCR repertoire in terms of diversity and antitumor response, suggesting that this combined therapy could be effective in preventing melanoma relapse.

Transfer of myxovirus-protein-A mRNA assay for interferon-ß bioactivity measurement in multiple sclerosis patients to routine laboratory practice. A 4-year experience.

BACKGROUND: As new biomarkers are validated and their significance in the natural history of specific diseases is established, these technologies can be rapidly transferred to clinical application. Since it has been shown that a single post-interferon-ß (IFNß) injection measurement of myxovirus-protein-A (MxA) mRNA correlates with IFNß bioactivity in IFNß treated patients with multiple sclerosis (MS), we had previously validated an assay for its quantification. METHODS: We introduced a real-time PCR relative quantification assay into routine clinical practice and measured MxA mRNA expression in 564 samples from 500 unselected IFNß treated MS patients over a 4-year period. RESULTS: We confirmed that the assay is reproducible over time, and found that the percentage of patients lacking MxA mRNA induction is comparable to that described in studies performed worldwide after patient selection by pre-screening for the presence of anti-IFNß antibodies. CONCLUSIONS: In view of its simplicity and reproducibility, this MxA assay is an alternative to anti-IFNß antibody determinations for use in routine clinical practice.

Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation.

A major problem in the field of stem cell transplantation is the difficulty to monitor the efficacy of immune reconstitution. By modifying the widely used method of measuring T-cell receptor excision circles (TRECs) and the recently proposed kappa-deleting recombination excision circles (KRECs) assay, we set up a duplex Real-Time PCR that allowed the simultaneous quantification of newly produced T and B cells in children with primary immunodeficiency undergone to transplantation. We found that lymphocyte recovery involves the mobilization of both new T and B cells from production and maturation sites, and that the increase of TRECs and KRECs can be or strictly associated or independent one from the other. Some patients showed a "lymphocyte rebound" which is followed by a progressive decrease of newly produced T and B lymphocytes starting about 2years after transplantation. In other patients, TRECs and KRECs number remained very low for the entire period of study.

Upregulation of myxovirus-resistance protein A: a possible marker of type I interferon induction in systemic sclerosis.

OBJECTIVE: .To examine whether myxovirus-resistance protein A (MxA) mRNA expression, commonly considered a reliable marker of Type I interferon (IFN) bioactivity, is modified in patients with systemic sclerosis (SSc); if it is associated to specific clinical features; and if its modulation is accompanied by modulation of mRNA for the Type I IFN receptor (IFNAR). METHODS: Quantification of mRNA for MxA and the subunit IFNAR1 and isoforms of IFNAR2 was performed by real-time polymerase chain reaction in 50 patients with SSc. Results were compared with those obtained from healthy controls and patients with another autoimmune disease such as multiple sclerosis. RESULTS: . Levels of MxA mRNA above the 99th percentile of values found in healthy controls were observed in 9 out of 50 patients with SSc (p < 0.001). Induced MxA expression was significantly associated with some features of more severe disease, such as lower forced vital capacity and the presence of ischemic digital ulcers. No differences in the levels of IFNAR were found within MxA-induced and MxA-non-induced patients, but there was a direct correlation between levels of MxA and the soluble isoform of IFNAR2. CONCLUSION: . Our results show induction of MxA expression in some patients with SSc, which correlates with the presence of ischemic ulcers and other signs of worse disease, suggesting a potential role of Type I IFN in the pathogenesis of this disease and/or its complications.

Type I interferon-dependent gene MxA in perinatal HIV-infected patients under antiretroviral therapy as marker for therapy failure and blood plasmacytoid dendritic cells depletion.

BACKGROUND: To determine the role of interferon-alpha in controlling HIV infection we phenotypically and functionally analyzed circulating plasmacytoid dendritic cells (pDC), which are known to be the highest interferon-alpha producing cells, in 33 perinatally infected HIV+ patients undergoing standard antiretroviral therapy. METHODS: Circulating pDC were identified by flow cytometry using anti-BDCA-2 monoclonal antibody and by measuring BDCA-2 mRNA by real-time PCR, while tissue-resident pDC were identified by immunohistochemistry. mRNA for interferon-alpha and MxA, a gene that is specifically induced by interferon-alpha, was quantified in peripheral blood cells by real-time PCR, while serum interferon-alpha protein was measured by ELISA. RESULTS: While median values of pDC, both in terms of percentage and absolute number, were not statistically different from age-matched controls, interferon-alpha mRNA was increased in HIV-infected patients. However, in a group of patients with long disease duration, having a low number of both pDC and CD4+ lymphocytes and a significant increase of serum interferon-alpha, MxA mRNA was produced at high level and its expression directly correlated with HIV RNA copy numbers. Furthermore in patients displaying a low CD4+ blood cell count, a severe depletion of pDC in the tonsils could be documented. CONCLUSION: HIV replication unresponsive to antiretroviral treatment in perinatal-infected patients with advanced disease and pDC depletion may lead to interferon-alpha expression and subsequent induction of MxA mRNA. Thus, the latter measurement may represent a valuable marker to monitor the clinical response to therapy in HIV patients.

Modulation of IFNAR1 mRNA expression in multiple sclerosis patients.

Interferon-beta receptor (IFNAR) is composed of 2 subunits, IFNAR1 and IFNAR2, the latter of which is expressed as functional (IFNAR2.2), non-functional (IFNAR2.1) and soluble (IFNAR2.3) isoform. Real-Time PCR analysis of mRNA for all IFNAR components in multiple sclerosis patients naïve for therapy and undergoing long-term treatment with interferon-beta shows that IFNAR1 mRNA level is lower than in healthy controls. If long-term treated patients are divided according to the production of mRNA for Myxovirus protein-A, a marker of interferon-beta bioactivity, IFNAR1 mRNA reaches the values observed in controls only in Myxovirus protein-A-induced patients. Since chronic cell stimulation by interferon-beta induces IFNAR protein down-regulation, we suggest that the increase of IFNAR1 mRNA might serve as a mechanism for counterbalancing the loss of protein receptor, enhancing, at least in this sub-group of patients, cell responsiveness to interferon-beta.

Decreased type I interferon receptor-soluble isoform in antiretroviral-treated HIV-positive children.

We developed a real-time PCR assay to simultaneously measure the mRNA level of type I interferon (IFN) receptor (IFNAR) components in peripheral blood cells of children with chronic immune stimulation due to HIV infection. All patients were undergoing antiretroviral therapy and were divided into two groups on the basis of the induction of MxA mRNA, a marker of type I IFN bioactivity. We found that IFNAR-2 subunit mRNA was higher than that of the IFNAR-1 subunit, that the mRNA for the IFNAR-2.2 functional isoform was more expressed than that for the truncated IFNAR-2.1 isoform, and both were much more represented than that of the IFNAR-2.3 soluble isoform. We also demonstrated that soluble isoform mRNA was significantly diminished in the subgroup of patients with MxA mRNA below the cutoff value (determined as the 99th percentile of MxA measured in healthy controls). These results suggest that downregulation of the soluble receptor isoform, which would not compete with the functional isoform for binding to the target cytokine, would give type I IFN, eventually induced in these patients in the case of viral reactivation, the opportunity to promptly exert its antiviral activity.

T-lymphocyte production, function, and death in children who recovered from opsoclonus-myoclonus syndrome.

Reduced CD4+ lymphocytes have been recently found in peripheral blood of children with active opsoclonus-myoclonus syndrome. The authors identified 2 children who recovered from this syndrome, one of whom showed reduced CD4+ lymphocytes 2 years after the disease onset. Except for a decrease of "naive" CD45RA+ CD4+ population and a mild restriction of T-cell heterogeneity in this patient, probably related to the immune response to viral infections, no alterations of T-cell homeostasis and function were found in either child. Therefore, the decrease of CD4+ cells may persist after clinical recovery, but the causes of this abnormality cannot be ascribed to intrinsic T-cell defects.

T-cell immune reconstitution after hematopoietic stem cell transplantation for HIV-associated lymphoma.

BACKGROUND: One of the major concern for high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) for HIV-associated lymphoma is that posttransplant immunosuppression might worsen immune defects of HIV individuals. Since the introduction of highly active antiretroviral therapy has made HSCT possible also in these patients, we analyzed whether the immune system already compromised by HIV infection might support an efficient T-cell recovery after HSCT. METHODS: The kinetics and the extent of T-cell reconstitution were investigated before and after HSCT in four patients with HIV-related lymphoma (one with Hodgkin's Disease and three with non-Hodgkin's lymphoma) by measuring the thymic output, the level of IL-7 and the heterogeneity of T-cell repertoire. T-cell competence was gauged at two functional levels: by determining the number of T-cell divisions and by measuring IFN-gamma production. RESULTS: The thymus of transplanted patients can be capable of generating new T cells, but there is no relationship between increasing number of newly produced lymphocytes and modification of IL-7 level. Various T-cell subsets, expressing different T-cell receptor variable beta genes, were preferentially expanded in CD8 population and most of them showed a restricted diversity. Furthermore, CD3 lymphocytes showed heterogeneous behaviors in terms of proliferative capability and IFN-gamma production. CONCLUSIONS: High-dose therapy and HSCT in HIV patients under highly active antiretroviral therapy does not worsen the immune defects. On the contrary, in the presence of some conditions (including the type of hematologic malignancy, the therapy compliance, and the immune status before transplantation), high-dose therapy and HSCT might support the improvement of immune conditions.