A paleodemographic assessment of mortality and fertility rates during the second demographic transition in rural central Indiana.

OBJECTIVES: Since its inception, skeletally based paleodemographic research has emphasized the utility of biocultural models for interpreting the dynamic relationship between the sociocultural and ecological forces accompanying demographic transitions and shaping populations' health and well-being. While the demographic transition associated with the Neolithic Revolution has been a common focus in bioarcheology, the present study analyzes human skeletal remains from a large 19th century cemetery in central Indiana to examine population dynamics during the second demographic transition, a period generally characterized by decreasing fertility rates and improvements in life expectancy. This study demonstrates the potential to methodologically identify regional variations in the timing and interactions between broad-scale socioeconomic changes and technological advancements that characterized the time period through observed changes in survivorship and fertility based on age-at-death distributions. MATERIALS AND METHODS: This study uses three temporally distinct samples (AD 1827-1869; 1870-1889; 1890-1935) from the Bethel Cemetery (n = 503). Kaplan-Meier survival analyses with a log- rank tests are utilized to evaluate survivorship and mortality over time. Next, Cox proportional hazard analyses are employed to examine the interaction between sex and time as covariates. Finally, the D0-14/D ratio is applied to estimate fertility for each of the three temporally bounded cohorts. RESULTS: The Kaplan-Meier survival analyses and Cox proportional hazard modeling revealed statistically significant differences in survivorship between the three time periods. Age-specific mortality rates are reduced among adult female and male age classes in this rural community over the course of the 19th and early 20th centuries, resulting in the increasing life expectancies associated with the second demographic transition. While mortality in early adulthood was common during the first time period and decreases thereafter, sex was not identified as a meaningful covariate. The proportion of juveniles in the three temporal samples indicate that fertility rates were higher than national averages for the better part of the 19th century and subsequently declined around the turn of 20th century for this community. CONCLUSIONS: The results indicate temporal differences between the three periods, demonstrating increased survivorship and decreased mortality and fertility over time. These findings corroborate two key features of the second demographic transition characterized by the move from high rates of both fertility and mortality to reduced rates and a general easing of demographic pressures. The observed trends likely reflect improvements in health, coinciding the industrial advance and economic development within and around Indianapolis. While the socioeconomic factors characterizing the Industrial Revolution drove demographic shifts that parallel an equally important epidemiological transition, potential regional differences are discussed to highlight variability in the timing of demographic transitions. The paleodemographic methods utilized in this study demonstrate improved accuracy and efficacy, which ultimately advances researchers' potential to disentangle population-specific socioeconomic factors that may contribute to asymmetrical experiences of health and mortality.

Dental microwear texture analysis of Homo sapiens sapiens: Foragers, farmers, and pastoralists.

OBJECTIVES: The current study seeks to determine if a sample of foragers, farmers, and pastoralists are distinguishable based on their dental microwear texture signatures. MATERIALS AND METHODS: The study included a sample of 719 individuals from 51 archeological sites (450 farmers, 192 foragers, 77 pastoralists). All were over age 12 and sexes were pooled. Using a Sensofar® white-light confocal profiler we collected dental microwear texture analysis (DMTA) data from a single first or second molar from each individual. We leveled and cleaned data clouds following standard procedures and analyzed the data with Sfrax® and Toothfrax® software. The DMTA variables were complexity and anisotropy. Statistics included ANOVA with partial eta squared and Hedges's g. We also performed a follow-up K-means cluster analysis. RESULTS: We found significant differences between foragers and farmers and pastoralists for complexity and anisotropy, with foragers having greater complexity than either the farmers or the pastoralists. The farmers and pastoralists had greater anisotropy than the foragers. The Old World foragers had significantly higher anisotropy values than New World foragers. Old and New World farmers did not differ. Among the Old World farmers, those dating from the Neolithic through the Late Bronze Age had higher complexity values than those from the Iron Age through the medieval period. The cluster analysis discerned foragers and farmers but also indicated similarity between hard food foragers and hard food farmers. DISCUSSION: Our findings reaffirm that DMTA is capable of distinguishing human diets. We found that foragers and farmers, in particular, differ in their microwear signatures across the globe. There are some exceptions, but nothing that would be unexpected given the range of human diets and food preparation techniques. This study indicates that in general DMTA is an efficacious means of paleodietary reconstruction in humans.

Identification of TFG (TRK-fused gene) as a putative metastatic melanoma tumor suppressor gene.

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation "hotspot" at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

On the relationship of dental microwear to dental macrowear.

Dental microwear analysts have demonstrated that hard diets leave numerous microscopic pits on occlusal surfaces. The relationship between occlusal pitting and gross macrowear, however, is not well known. The current study seeks to elucidate the relationship between dental microwear and macrowear by determining if microscopically pitted teeth are associated with greater expressions of macrowear. This study examined microwear and macrowear on mandibular second molars from 60 prehistoric adult Native Americans representing three dietary regimes (foraging, mixed economy, and agriculture). Initially, two dental microwear feature variables were studied: percentage of pits and mean scratch width. Standard macrowear scores ranged from 4 to 40. ANOVAs suggested that neither of the microwear variables was affected by age or sex, but age affected macrowear scores. Because of this, the sample had a balanced number of young and old adults (i.e., those below and above skeletal age 35). A Pearson's correlation showed no covariation between scratch width and the percentage of pits. Regression analysis indicated that macrowear was not a function of the percentage of pits. However, a significant positive relationship was found between dental macrowear and scratch width. A post priori test found a significant negative relationship between macrowear and the total number of scratches. It is concluded, then, that wide scratches remove more enamel and dentin than do numerous pits, although both cause dental wear. It is suggested here that the term "abrasive" be used to describe those microwear profiles that lead to heavy macrowear and have relatively wide scratches.

Pseudopathological vertebral changes in a young individual from Herculaneum (79 C.E.).

Post mortem abnormal modification of bone are known as pseudopathologies. The geochemical characteristic of the burial soil and/or the presence of biological agents may produce marked changes in bone preservation. This could be the case for a young individual, E74, from Herculaneum, which was a Roman town near Naples completely destroyed by the volcanic eruption of Mt. Vesuvius in 79 CE. E74 is an incomplete skeleton of a male individual of 7-8 years of age. Its second and third cervical vertebrae, the eighth thoracic vertebra and the first lumbar vertebra show a septum dividing the vertebral foramen. This condition could be diagnosed as diastematomyelia that consists of the splitting of the spinal cord or cauda equina. In particular Type I malformations consist of two hemicords separated into two dural tubes by a bone septum. The gross anatomy and histological aspects of the vertebrae and their septa were investigated through macroscopic, microscopic, radiographic and chemical analyses. The results demonstrate that the vertebral septum is constituted by three layers of inorganic substances deposited at different times on a thin, probably organic, substrate (original meninges?). The central layer contain framboidal pyrite, that is a sedimentary mineral rarely found in ancient human skeletons. The septum splitting the vertebral canal of individual E74 is consequent to a taphonomic event and is not due to a pathological condition. Distinguishing between ante and post mortem alterations can be a challenging exercise even for the experienced paleopathologists and, as this case indicates, paleopathological diagnoses should be supported by detailed examinations.

Dendritic cell immunotherapy for stage IV melanoma.

Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell 'vaccine' in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enrolled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings.

Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4.

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

Generation of CD8(+) T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy.

Adoptive T-cell therapy of cancer often fails due to the tumor cells' immune escape mechanisms, like antigen loss or down-regulation. To anticipate immune escape by loss of a single antigen, it would be advantageous to equip T cells with multiple specificities. To study the possible interference of 2 T-cell receptors (TCRs) in one cell, and to examine how to counteract competing effects, we generated TETARs, CD8(+) T cells expressing two additional T-cell receptors by simultaneous transient transfection with 2 TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognizing a patient-specific, individual mutation of CCT6A (chaperonin containing TCP1, subunit 6A) termed "CCT6A(m) TCR." These CD8(+) T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibition was observed. Murinisation of the CCT6A(m) TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. Taken together, we generated functional, dual-specific CD8(+) T cells directed against a common melanoma-antigen and an individually mutated antigen for the use in personalised adoptive T-cell therapy of melanoma. The intended therapy would involve repetitive injections of the RNA-transfected cells to overcome the transiency of TCR expression. In case of autoimmunity-related side effects, a cessation of treatment would result in a disappearance of the introduced receptors, which increases the safety of this approach.

Exome Sequencing to Predict Neoantigens in Melanoma.

The ability to use circulating peripheral blood cells and matched tumor sequencing data as a basis for neoantigen prediction has exciting possibilities for application in the personalized treatment of cancer patients. We have used a high-throughput screening approach, combining whole-exome sequence data, mRNA microarrays, and publicly available epitope prediction algorithm output to identify mutated proteins processed and displayed by patient tumors and recognized by circulating immune cells. Matched autologous melanoma cell lines and peripheral blood mononuclear cells were used to create mixed lymphocyte tumor cell cultures, resulting in an expansion of tumor-reactive T cells to use for mutated peptide screening. Five patients were investigated, three of whom had a durable complete response (CR; 15+ years) in an autologous melanoma-pulsed dendritic cell clinical trial. We identified seven mutated antigens in total that stimulated T-effector memory cells in two of the five patients. While the procedure did not result in clinically applicable neoantigens for all patients, those identified were likely important in tumor clearance, leading to durable CR. The nature of the screening process allows results to be obtained rapidly and is easily applicable to a wide variety of different tumor types.

miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma.

To identify 'melanoma-specific' microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA 'pull-down' experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

High efficiency ex vivo cloning of antigen-specific human effector T cells.

While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

Melanomas of unknown primary have a mutation profile consistent with cutaneous sun-exposed melanoma.

Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients present with metastatic disease without an evident primary site. To determine their likely site of origin, we combined exome sequencing from 33 MUPs to assess the total rate of somatic mutations and degree of UV mutagenesis. An independent cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed that the majority of MUPs exhibited high somatic mutation rates, high ratios of C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of 101, 32%) mutations, collectively indicating a mutation profile consistent with cutaneous sun-exposed melanomas. These data suggest that a significant proportion of MUPs arise from regressed or unrecognized primary cutaneous melanomas or arise de novo in lymph nodes from nevus cells that have migrated from the skin.

Light microscopy of microfractures in burned bone.

Bone color changes depending upon the taphonomic agents that affect it. Burning turns bone black, brown, blue, gray, and white as the bone's temperature increases and collagen is lost. It also creates diagnostic fractures that are visible at the gross level. Usually heat-altered bone is readily identified as such, but there are times when dark organic stains can mimic bone that is charred black. This paper provides a means to observe and quantify microfractures in burned bone for those instances when macroscopic observations fail to clarify if a bone fragment is actually burned; specifically, it distinguishes charred from organically stained bone. It is based upon a study of 50 calcined (burned to a bright white), charred, and organically stained bone fragments (n = 150) that were viewed with a standard stereomicroscope at ×30. Microfractures are readily discerned on charred and calcined bones, being more common on the latter; they are not present on organically stained bone. Charred bones may have their cortical surfaces obscured by a microlayer of adhering soft tissues as well as by microflaking of the external circumferential lamella. Overall, the methods described here allow the detection of microfractures on bone using a very simple approach and readily available technology.

A preliminary assessment of using a white light confocal imaging profiler for cut mark analysis.

White light confocal microscopy creates detailed 3D representations of microsurfaces that can be qualitatively and quantitatively analyzed. The study describes its application to the analysis of cut marks on bone, particularly when discerning cuts made by steel tools from those made by stone. The process described comes from a study where cuts were manually made on a cow rib with seven cutting tools, four stone (an unmodified chert flake, a chert biface, a bifacially ground slate fragment, and an unsharpened piece of slate), and three steel (a Swiss Army Knife, a serrate steak knife, and a serrate saw). Kerfs were magnified ×20 and 3D data clouds were generated using a Sensofar(®) White Light Confocal Profiler (WLCP). Kerf profiles and surface areas, volumes, mean depths, and maximum depths were calculated with proprietary software (SensoScan(®) and SolarMap(®)). For the most part, the stone tools make shallower and wider cuts. Kerf floors can be studied at higher magnifications; they were viewed at ×100. When comparing the kerf floors of the unsharpened slate and the serrate steak knife it was found that the slate floor was more uneven, but the serrate steak knife generated more overall relief. Although preliminary, the approach described here successfully distinguishes stone and steel tools; the authors conclude that the WLCP is a promising technology for cut mark analysis because of the very detailed 3D representations it creates and the numerous avenues of analysis it provides.

A high-throughput panel for identifying clinically relevant mutation profiles in melanoma.

Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient's tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n = 154, 57%), NRAS (n = 55, 20%), CDK4 (n = 8, 3%), PTK2B (n = 7, 2.5%), and ERBB4 (n = 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs.

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing.

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

Antigens for cancer immunotherapy.

Progress in tumor immunology has not been translated to effective immunotherapies for cancer. Most of the current effort in basic and clinical research concentrates on generating effective immune responses against model or well characterized antigens, yet vaccines targeting defined antigens have been less clinically successful than those based on whole tumor cells or their extracts. This review considers characteristics of proteins that determine how effectively they might serve as targets of immune control, and how different sources of antigens have fared in clinical trials.

Immunological characteristics correlating with clinical response to immunotherapy in patients with advanced metastatic melanoma.

Current treatment options for advanced metastatic melanoma are limited to experimental regimen that provide poor survival outcomes. Immunotherapy is a promising alternative and we recently reported a clinical trial in which 6 out of 19 patients enrolled had objective clinical responses to a fully autologous melanoma/dendritic cell vaccine. The mechanism of the vaccine is not well understood, but we hypothesized that general immunocompetence may be a determinant of clinical response. We therefore examined the immune status of an expanded series of 21 patients who displayed varying clinical responses to the melanoma/dendritic cell vaccine. Immunocompetence was assessed using in vitro assays of lymphocyte function: survival, proliferation and cytokine responses to mitogen stimulation as well as T-cell receptor zeta expression and lymphocyte subset analysis. Although lymphocytes from patients mostly performed comparably to age-matched and sex-matched controls, in some assays we identified significant differences between complete clinical responders and other patients, both before and following vaccination. Surprisingly, before vaccination, only lymphocytes from clinical responder patients showed impaired in vitro survival. Following vaccination, T lymphocyte survival improved and cells recovered their ability to produce the Th1-associated cytokines TNF and IFN-gamma in response to anti-CD3 stimulation in vitro. No increase in Th1 cytokine production was observed in lymphocytes from patients who experienced partial clinical responses or progressive disease. We conclude that, before vaccination, patients who go on to have complete responses have immune characteristics suggestive of high cell turnover and low Th1-associated cytokine production, and that these can be reversed with vaccination. These results have potential implications for future immunotherapeutic strategies.

Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine.

Advanced metastatic melanoma is incurable by standard treatments, but occasionally responds to immunotherapy. Recent trials using dendritic cells (DC) as a cellular adjuvant have concentrated on defined peptides as the source of antigens, and rely on foreign proteins as a source of help to generate a cell-mediated immune response. This approach limits patient accrual, because currently defined, non-mutated epitopes are restricted by a small number of human leucocyte antigens. It also fails to take advantage of mutated epitopes peculiar to the patient's own tumour, and of CD4+ T lymphocytes as potential effectors of anti-tumour immunity. We therefore sought to determine whether a fully autologous DC vaccine is feasible, and of therapeutic benefit. Patients with American Joint Cancer Committee stage IV melanoma were treated with a fully autologous immunotherapy consisting of monocyte-derived DC, matured after culture with irradiated tumour cells. Of 19 patients enrolled into the trial, sufficient tumour was available to make treatments for 17. Of these, 12 received a complete priming phase of six cycles of either 0.9x10(6) or 5x10(6) DC/intradermal injection, at 2-weekly intervals. Where possible, treatment continued with the lower dose at 6-weekly intervals. The remaining five patients could not complete priming, due to progressive disease. Three of the 12 patients who completed priming have durable complete responses (average duration 35 months+), three had partial responses, and the remaining six had progressive disease (WHO criteria). Disease regression was not correlated with dose or with the development of delayed type hypersensitivity responses to intradermal challenge with irradiated, autologous tumour. However, plasma S-100B levels prior to the commencement of treatment correlated with objective clinical response ( P=0.05) and survival (log rank P<0.001). The treatment had minimal side-effects and was well tolerated by all patients. Mature, monocyte-derived DC preparations exposed to appropriate tumour antigen sources can be reliably produced for patients with advanced metastatic melanoma, and in a subset of those patients with lower volume disease their repeated administration results in durable complete responses.