Analysis of Lymphocyfic Infiltration in Uveal Melanoma

Among 27 uveal melanomas, five were found to contain tumor infiltrating lymphocytes (TILs). Four had high levels of lymphocytes, and the fifth had comparatively low levels but adequate numbers for comprehensive analysis. The TILs were analyzed by flow cytometry to determine the relative proportions of lymphocyte subsets and markers of lymphocyte activation. The results show the predominance of T-suppressor/cytotoxic lymphocytes and insignificant levels of B-cells present in the infiltrate. The T-suppressor/cytotoxic cells were generally activated to a higher degree than the T-helper cells when assayed for levels of the histocompatibility antigen, HLA-DR. T-helper cells expressed more interleukin (IL-2) receptor (Tac) than T-suppressor/cytotoxic cells. Invest Ophthalmol Vis Sci 31: [2106][2107][2108][2109][2110]1990 Uveal melanomas, like their cutaneous counterpart, are considered to be relatively susceptible to immunologic influences because of reports of spontaneous regression, 1 -2 of the development of vitiligo and halo nevi, 5 Such intraocular transplantation can be prevented by prior adoptive transfer of humoral or cellular immunity to the recipient. " 14 Some investigators report the predominance of the T-cytotoxic/suppressor cell subpopulation; 1213 others found a majority of T-helper/inducer cells. 10 " 14 There is, however, no convincing evidence that a more favorable prognosis is associated with lymphocytic infiltration in uveal melanomas, 715 and it remains uncertain whether tumor infiltrating lymphocytes (TILs) play a significant role in tumor immunity. The availability of precisely defined monoclonal-antibody markers and flow-cytometric techniques makes it From the *Department of Biochemistry and the fTennent Institute of Ophthalmology, University of Glasgow, Glasgow, Scotland. Reprint requests: Fiona H. Durie, Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, UK. possible to do a detailed objective analysis of a large number of lymphocytes in any tumour. We characterized the phenotype of the TILs and studied the expression of the histocompatibility antigen, HLA-DR, and interleukin (IL-2) receptor (Tac), which are markers of activation, on the surface of T-helper and T-suppressor/cytotoxic lymphocytes in ocular melanoma. Materials and Methods Preparation of Cells Tissue was obtained from five choroidal melanomas which were removed either by local resection or by enucleation. Slices were cut from the apical part of the fresh specimen after preliminary examination, and these were transferred to RPMI-1640 medium (Gibco, Grand Island, NY). Spilled cells were teased out using a sterile needle. The cells were then harvested, washed by centrifugation at 1000 rpm for 15 min, counted, and adjusted to 1X10 6 cells/ml. The remaining cell suspensions of TILs and tumor cells were cryopreserved in medium containing 90% fetal calf serum and 10% dimethylsulfoxide and stored in liquid nitrogen until use. Four of the five patients consented to venipuncture, and peripheral blood lymphocytes were separated on a discontinuous ficoll density gradient. 1617 Preparation of Samples Cell suspensions of TILs and tumor cells were washed and resuspended in 1 ml of phosphate-buffered saline. Cells (50 ii\) were incubated on ice with appropriate fluorescein isothiocyanate (FITC)-conjugated leu series monoclonal antibody and phycoerythrin (PE) conjugated antibody (Becton-Dickinson, 2106 Downloaded from iovs.arvojournals.org on 06/28/201

Recommendations for acquisition, interpretation and reporting of whole body low dose CT in patients with multiple myeloma and other plasma cell disorders: a report of the IMWG Bone Working Group

Whole Body Low Dose CT (WBLDCT) has important advantages as a first-line imaging modality for bone disease assessment in patients with plasma cell disorders and has been included in the 2014 International Myeloma Working Group (IMWG) criteria for multiple myeloma (MM) definition. Nevertheless, standardization guidelines for the optimal use of WBLDCT in MM patients are still lacking, preventing its more widespread use, both in daily practice and clinical trials. The aim of this report by the Bone Group of the IMWG is to provide practical recommendations for the acquisition, interpretation and reporting of WBLDCT in patients with multiple myeloma and other plasma cell disorders

Engaging service users and carers in health and social care education: : challenges and opportunities in the Chinese Community

This is an accepted manuscript of an article published by Taylor & Francis Group in Social Work Education on 25 June 2010, available online at: http://dx.doi.org/10.1080/02615479.2010.491542.Service users' and carers' involvement in health and social care education has become a mainstream activity in Britain. However, members from black and minority ethnic communities (BME) remain under-represented in this area of participation. In this article, we will take the readers across the globe to explore the difficulties and opportunities of engaging such an under-represented group, the Chinese community. The journey will begin in Britain where barriers to engagement of service users and carers from the Chinese community will be discussed. We will then travel to Hong Kong, a cosmopolitan city, where successful engagement in work with Chinese service users and carers will be explored. Throughout the journey, we will highlight the importance of the consideration of cultural factors, particularly Confucian beliefs such as social harmony and collectivism, when working with Chinese people. We will also fully explore the issue of ‘trust’ as a culturally laden concept in Chinese societies and its significance for successful engagement in work with Chinese service users and carers in different parts of the world.Peer reviewedFinal Accepted Versio

Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p < 0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p < 0.0001) and a shorter survival in MM patients with active disease requiring treatment (p <= 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM

Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3

The restricted bone marrow (BM) localisation of multiple myeloma (MM) cells most likely results from a specific homing influenced by chemotactic factors, combined with the proper signals for growth and survival provided by the BM microenvironment. In analogy to the migration and homing of normal lymphocytes, one can hypothesise that the BM homing of MM cells is mediated by a multistep process, involving the concerted action of adhesion molecules and chemokines. In this study, we report that primary MM cells and myeloma derived cell lines (Karpas, LP-1 and MM5.1) express the chemokine receptor CCR2. In addition, we found that the monocyte chemotactic proteins (MCPs) MCP-1, -2 and -3, three chemokines acting as prominent ligands for CCR2, are produced by stromal cells, cultured from normal and MM BM samples. Conditioned medium (CM) from BM stromal cells, as well as MCP-1, -2 and -3, act as chemoattractants for human MM cells. Moreover, a blocking antibody against CCR2, as well as a combination of neutralizing antibodies against MCP-1, -2 and -3, significantly reduced the migration of human MM cells to BM stromal cell CM. The results obtained in this study indicate the involvement of CCR2 and the MCPs in the BM homing of human MM cells. (C) 2003 Cancer Research UK

Endothelial progenitor cells display clonal restriction in multiple myeloma

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V(H)). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V(H )primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM

International Myeloma Working Group risk stratification model for smoldering multiple myeloma (SMM)

Smoldering multiple myeloma (SMM) is an asymptomatic precursor state of multiple myeloma (MM). Recently, MM was redefined to include biomarkers predicting a high risk of progression from SMM, thus necessitating a redefinition of SMM and its risk stratification. We assembled a large cohort of SMM patients meeting the revised IMWG criteria to develop a new risk stratification system. We included 1996 patients, and using stepwise selection and multivariable analysis, we identified three independent factors predicting progression risk at 2 years: serum M-protein >2 g/dL (HR: 2.1), involved to uninvolved free light-chain ratio >20 (HR: 2.7), and marrow plasma cell infiltration >20% (HR: 2.4). This translates into 3 categories with increasing 2-year progression risk: 6% for low risk (38%; no risk factors, HR: 1); 18% for intermediate risk (33%; 1 factor; HR: 3.0), and 44% for high risk (29%; 2–3 factors). Addition of cytogenetic abnormalities (t(4;14), t(14;16), +1q, and/or del13q) allowed separation into 4 groups (low risk with 0, low intermediate risk with 1, intermediate risk with 2, and high risk with ≥3 risk factors) with 6, 23, 46, and 63% risk of progression in 2 years, respectively. The 2/20/20 risk stratification model can be easily implemented to identify high-risk SMM for clinical research and routine practice and will be widely applicable

Calculating Stage Duration Statistics in Multistage Diseases

Many human diseases are characterized by multiple stages of progression. While the typical sequence of disease progression can be identified, there may be large individual variations among patients. Identifying mean stage durations and their variations is critical for statistical hypothesis testing needed to determine if treatment is having a significant effect on the progression, or if a new therapy is showing a delay of progression through a multistage disease. In this paper we focus on two methods for extracting stage duration statistics from longitudinal datasets: an extension of the linear regression technique, and a counting algorithm. Both are non-iterative, non-parametric and computationally cheap methods, which makes them invaluable tools for studying the epidemiology of diseases, with a goal of identifying different patterns of progression by using bioinformatics methodologies. Here we show that the regression method performs well for calculating the mean stage durations under a wide variety of assumptions, however, its generalization to variance calculations fails under realistic assumptions about the data collection procedure. On the other hand, the counting method yields reliable estimations for both means and variances of stage durations. Applications to Alzheimer disease progression are discussed