Moreover, tumour infiltration by suppressive myeloid cells was strongly diminished. These insights into the regulation of DC functionality reveal MK2 as a targetable pathway for DC-centred immunomodulatory cancer therapies.Maintaining dendritic cells (DC) in a state of dysfunction represents a key mechanism by which tumour cells evade recognition and elimination by the immune system. Limited knowledge about the intracellular mediators of DC dysfunction restricts success of therapies aimed at reactivating a DC-driven anti-tumour immune response. Using a cell type-specific murine knock-out model, we have identified MAPK-activated protein kinase 2 (MK2) as a major guardian of a suppressive DC phenotype in the melanoma tumour microenvironment. MK2 deletion in CD11c While the myeloid lineage represents a crucial line of immune defence in the absence of a tumour, tumour-driven distortion of myelopoiesis, resulting in severely altered myeloid phenotypes, is a key mechanism of tumour immune evasion5. One hallmark of altered myelopoiesis is the skewing of dendritic cell (DC) differentiation to an expansion of myeloid-derived suppressor cells (MDSCs)6 and multiple tumour-derived factors have been identified to drive such myeloid deviation7. The accumulation of this heterogeneous population of immature myeloid cells and progenitors is strongly associated with tumour progression and unfavourable prognosis across multiple cancer types9.The role of myeloid cells in promoting tumour progression by contributing to an immunosuppressive microenvironment has been well-established10. Although several pathways have been implicated in DC susceptibility to tumour-derived factors14, many questions remain open as to which intracellular molecules drive the manifestation of a dysfunctional DC phenotype. This poses considerable limitations to therapeutic strategies aimed at restoring DC functionality in tumours, for example through the delivery of maturation stimuli such as toll-like receptor (TLR) agonists16.DCs are potent antigen-presenting cells (APCs) that are crucial for the orchestration of T cell-mediated tumour elimination. In the tumour microenvironment (TME), however, DCs frequently exhibit a defective phenotype, characterized by markers of immaturity and immunosuppressive activity+ DC sub-population has recently been identified to represent a minor, yet the most pivotal APC population mediating anti-tumour immunity in murine models of melanoma18. High interleukin (IL)-12 secretion19, enhanced CD8+ cross-priming activity20, and the capacity to transport intact tumour antigens to lymph nodes (LN)22, highlight the importance of CD103+ DCs in the priming of an effective cytotoxic anti-tumour T cell response. In melanoma patients, elevated numbers of BDCA3/CD141hi DCs, the equivalent counterpart to murine CD103+ DCs in humans, correlate with better prognosis24. Since the function of certain DC subsets and phenotypes in different cancer types are still not fully resolved, gaining a more thorough understanding of tumour-driven DC plasticity is of urgent interest.An intratumoural CD10325. p38-MK2 signalling constitutes a major inflammatory axis with MK2 being responsible for the production of multiple cytokines and chemokines. The pivotal pro-inflammatory function of MK2 in macrophages has been described in various models of systemic inflammation27. With regard to tumour development, systemic MK2 deletion has been shown to result in reduced skin carcinogenesis28 and resistance to inflammation-induced colon carcinoma29. Moreover, MK2 acts as cell cycle regulator, coming into play upon p53 mutation31. In this context, MK2 and several of its downstream effectors have been identified to mediate resistance of tumours to therapy-induced apoptosis32. Altogether, emerging evidence supports the idea of pharmacological MK2 inhibition as a viable treatment option for both inflammatory and malignant diseases. However, in distinct tissue contexts MK2 has been proposed to mediate negative feedback signalling and dampen on-going inflammatory responses35. Consistently, we have previously reported a Th1-attenuating function of MK2 in DCs in response to TLR ligation36. DC-specific loss of MK2 promotes severe autoimmunity, suggesting a cell type-specific protective role of MK2 in preventing host damage caused by excessive inflammation. However, taking into consideration its diverse functions, whether DC-expressed MK2 modulates anti-tumour immune responses has not been answered to date.MAPK-activated protein kinase 2 (MK2) is the main downstream target of p38 MAPK+ lineage-specific MK2 deletion (CD11c-Cre Mapkapk2flox/flox) and found that MK2 is implicated in immunosuppressive DC activity within the melanoma microenvironment in response to TLR stimulation. Ablation of MK2 in the CD11c+ lineage reduced intratumoural DC and MDSC accumulation and promoted the expansion of phenotypically mature CD103+ DCs, which favoured the priming of CD8+ over CD4+ T cells. Along with enhanced infiltration of these T cells into tumours, CD11c-specific loss of MK2 resulted in the generation of a clonally expanded, less exhausted T cell population, altogether reinstating immune control over tumour progression.In the present study we therefore set out to address this unresolved question. We used a murine system of CD11cΔCD11c and wild-type (WT) littermate control mice by subcutaneous injection of the murine melanoma cell line B16-F10, and monitored tumour growth until tumours reached 15 mm in diameter and animals were euthanized. Since we did not detect any difference in either tumour growth  as a potent TLR4 agonist subcutaneously in close proximity to the inguinal (draining) lymph nodes (dLN) on days 5 and 9 post-tumour cell injection. Compared to PBS control groups, tumour growth was delayed in these animals but again no significant differences were observed between genotypes . However, tumours of MK2ΔCD11c mice showed an overall reduced presence of myeloid cell populations as compared to WT littermate controls when immunized with LPS + tumour cell lysate , which remained unaffected by DC-specific loss of MK2. The monocytic subset , to which immunosuppressive function, association with poor prognosis and accumulation in advanced melanoma have been ascribed9, was reduced to 3.0 (±2.6) % in MK2ΔCD11c mice as compared to 6.5 (±2.9) % in WT controls  remained constant  accumulated at significantly lower numbers in tumours of MK2ΔCD11c mice , MDSCs (CD11b+ Gr-1+) and the remaining CD11b+ CD11c− Gr-1− myeloid population from tumours and spleens of melanoma-bearing WT mice at day 12 post-tumour cell injection  to 2.3 (±0.4) pg/mL in MK2ΔCD11c mice  compartment more closely with regard to cell surface expression of CD11b and CD103 revealed a robust decline in CD11b+ DCs in MK2ΔCD11c mice upon LPS + lysate immunization  and –inhibitory  surface molecules as compared to CD103− DCs  upon MK2 knock-out in CD11c+ DCs  % in WT to 37.8 (±11.2) % in MK2ΔCD11c mice  repertoire of MK2ion Fig. . This obice Fig. , which sion Fig. . Additioion Fig. , represeion Fig. , indicat+ T cells at the tumour site, tumour-infiltrating effector T cells exhibit a less exhausted phenotype with reduced expression of immune checkpoint receptors PD-1 and TIM-3. Finally, high clonality of the intratumoural TCR pool indicates effective expansion of tumour-reactive T cell clones, together enabling sustained tumour growth control.In conclusion, loss of MK2 in the DC lineage results in the induction of a potent tumour-targeted cytotoxic T cell response upon local simultaneous delivery of a TLR agonist and tumour antigen. Besides increased priming and accumulation of CD810. We identified the p38 downstream effector kinase MK2 as an intracellular mediator of tumour-driven DC malfunction in the presence of TLR-activating signals and tumour antigen. This has important implications with regard to therapeutic strategies aiming to maintain or restore DC maturity. The administration of adjuvant DC-activating danger signals, including TLR agonists, is currently being harnessed to enhance responses to different treatment regimens16. However, limited success has been achieved so far. One explanation is provided by the observation that following transient activation, a majority of DCs revert to a state of malfunction, presumably due to complex interactions within intracellular signalling circuits. Even though many factors in the TME have been identified to induce DC dysfunction and modulate their response to TLR ligation41, the intracellular mediators translating these signals into a regulatory phenotype are unknown. Our finding herein of significantly diminished tumour growth in MK2ΔCD11c mice in response to TLR activation and delivery of tumour antigen highlights MK2 as one potential candidate.Tumour cells develop intricate mechanisms to evade recognition by the immune system. The highly versatile nature of DCs makes them prone to manipulation by tumour factors. As a result, tumour-associated DCs often display a dysfunctional phenotype and contribute to tumour immune escape40. Our previous studies revealed MK2 as a molecular switch mediating a shift from inflammatory to immunosuppressive DC activity upon prolonged TLR stimulation, which proved to be crucial for the prevention of autoimmune reactions emerging from an excessive inflammatory response36. Consistently, others have reported similar patterns of MK2 involvement in negative feedback signalling in other cell types35. These observations are contrasted by studies revealing that systemic loss of MK2 causes enhanced susceptibility to infections27 and reduces inflammation-induced tumour growth29. Collectively, these aspects underline the importance of MK2 as a central mediator of inflammation, with a crucial feedback function in DCs that is required for the tight control and timely resolution of inflammatory processes. This function, however, is essentially linked to simultaneous antigen stimulation. The significance of such self-limiting features of negative regulation have recently been highlighted with reference to other key inflammatory pathways42. Consequently, our findings suggest that MK2 represents an intracellular immune checkpoint in DCs. While MK2 is required for DC-mediated immune homeostasis, tumour cells seem to hijack this function to arrest DCs in a dysfunctional state. Several tumour-derived molecules act as major drivers of DC malfunction43 and engage the p38-MK2 pathway for signal transduction45, exemplifying how MK2 could be exploited to relay environmental cues to modulate DC behaviour. Based on these observations, we propose that MK2 signalling conveys tumour-driven DC suppression and restricts DC activation by TLR agonists in the tumour stroma.Tumours are capable of exploiting immune regulatory mechanisms that are required to maintain peripheral tolerance in a healthy organism, as exemplified by prominent immune checkpoint molecules11, one specific DC subset has recently emerged as the pivotal APC population mediating rejection of melanoma cells. Designated as DC2 by Broz et al., these cells are CD103+ and exhibit a signature of enhanced stimulatory activity22. We show here that CD103-expressing DCs are enriched among the overall MK2-deficient DC population. In accordance with their high T cell-engaging capacity, we detected a robust increase in MHC-II concomitant with alterations in co-stimulatory and inhibitory molecules on the surface of these APCs as compared to their CD103− counterparts, which was amplified upon MK2 ablation. Most strikingly, PD-L1 expression was significantly diminished, and the ratio of MHC-II+ PD-L1+ double positive cells over PD-L1− cells was tilted in favour of PD-L1− APCs upon loss of MK2. Considering the importance of antigen presentation for successful priming of tumour-reactive CTLs, this shift indicates improved antigen-specific T cell activation in the absence of MK2. The implications of these observations are particularly interesting as PD-L1 has been suggested as the dominant pathway of immunosuppression by tumour-associated DCs15. Moreover, phenotypically mature CD103+ DCs have been reported to be indispensable for response to immune checkpoint therapy targeting PD-L1 in melanoma21. Our findings here delineate the importance of MK2 in the regulation of stimulatory and inhibitory surface receptors on such DCs and suggest MK2 being responsible for a limited expansion of stimulatory DCs within the intratumoural DC population.While abundance and phenotype of DC populations vary greatly across cancer types21. We found that MK2 deletion resulted in differential regulation of a number of chemokine receptors on DCs, with CCR7 and CXCR4 representing the most highly upregulated examples. While CCR7 represents a general key regulator for DC LN migration, CXCR4 is required for the movement of cutaneous DCs and epidermal Langerhans cells47. In our melanoma model, elevated expression of CXCR4 upon MK2 deletion correlated with an accumulation of DCs in dLNs. Furthermore, the proportion of stimulatory CD103-expressing DCs was increased in lymphoid tissues. Taken together, this suggests improved trafficking of tumour-associated DCs bearing a mature, stimulatory signature to regional LN in the absence of MK2.Corresponding to their enhanced stimulatory signature, CD103-expressing DCs are crucial for the transport of intact tumour antigens to regional dLNs+ DCs is an elevated CD8+ cross-priming activity, highlighting their significance in directing a cytotoxic anti-tumour T cell response20. Accompanying elevated numbers of CD103+ DCs upon MK2 deletion, we observed a shift in both intratumoural and LN-resident T cell populations towards a higher presence of CD8+ T cells upon immunization with LPS + tumour antigen. Regarding T cell functionality, we previously reported enhanced proliferation and cytotoxicity of CD8+ T cells following priming by MK2-deficient DCs in the context of inflammation36. The observation here that ablation of MK2 in the CD11c+ lineage yielded enhanced accumulation of CD8+ T cells at the tumour, which exhibited a clonally enriched TCR repertoire, suggests successful expansion of tumour-directed T cell clones. Even more importantly, MK2-deficient DCs induced lower levels of inhibitory receptors PD-1 and TIM-3 on both CD4+ and CD8+ T cells. Together, the sum of these changes in the T cell response appears sufficient to enable tumour-reactive T cells to regain control over tumour progression.Another hallmark of melanoma-associated CD103+ Gr-1+ immature myeloid cells or MDSCs represents one of the most prominent immunosuppressive mediators that accelerates tumour immune evasion. Loss of MK2 in the CD11c+ lineage reduced overall myeloid tumour infiltration and led to diminished numbers of intratumoural MDSCs. Low serum levels of myelo-attracting chemokines CCL3 and CCL4 furthermore suggest an involvement of MK2 in DC-mediated recruitment of suppressive myeloid cells into the TME. Moreover, release of CCL4 has been linked to recruitment of regulatory T cells48 and tumour-promoting Th17 cells49. Nevertheless, we cannot exclude the possibility that MK2 impacts directly on tumour-driven distortion of myeloid differentiation, making DCs susceptible to assume an MDSC-like phenotype. Supporting this hypothesis, we found MK2 expression to be elevated in MDSCs as compared to other myeloid cell types. This increase correlated with markers of immunosuppression TGF-β, Arg-1 and IL-1037. Consistently, our prior studies revealed IL-10 secretion from DCs to be stringently regulated by MK2 upon TLR-induced inflammation36. However, CD11c-driven MK2 ablation did not affect the phenotype of MDSCs itself, hence suggesting a dominant effect on the mobilization and recruitment of suppressive myeloid cells to the TME.Besides an impaired T cell response, a heterogeneous population of CD11b+ T cell response, and (iii) mediating expansion of suppressive myeloid populations at the tumour site. In tumour cells, the identification of MK2 as an alternative cell cycle checkpoint, mediating resistance to apoptosis upon p53 mutation, has led to the emergence of MK2 as a promising target for combinatorial cancer therapies26. When envisioning administration of pharmacological MK2 inhibitors, it will therefore be crucial to dissect concurrent effects on immune cells both within tumour stroma and periphery. Our data suggest a synergistic effect of MK2 inhibition on DC-orchestrated anti-tumour immune responses. The combination of MK2 inhibitors with conventional anti-cancer therapies on the one hand, and immune modulatory strategies aiming at DC re-activation on the other, holds considerable promise. Future preclinical studies exploring different therapy combinations will thus be of critical importance to evaluate the potential of targeting MK2 for the treatment of cancer.In summary, we show that MK2 expression in tumour-associated DCs contributes to tumour immune evasion on three levels Fig. : (i) hinMapkapk2flox/flox (MK2ΔCD11c) mice were generated and genotyped as previously described36. C57BL/6N wild-type mice were purchased from the Division for Laboratory Animal Science and Genetics of the Medical University of Vienna . For all experiments, female and male mice at 8–14 weeks of age were used. All animal experiments were performed in accordance with the institutional guidelines and approved by the Animal Care and Use Committee of the Medical University of Vienna (GZ: 856861/2013/16).CD11c-Cre 5 B16-F10 cells injected subcutaneously into left and right lateral flanks of mice. Five days post-tumour cell injection mice were immunized with either PBS, LPS  alone or in combination with either B16-F10 or GL-261  whole cell lysate (0.4 mg/kg) subcutaneously proximal to the inguinal lymph nodes. Immunization was repeated four days later. Cell lysates were prepared by four intervening freeze/thaw cycles. Tumour volume was measured using a digital calliper and calculated as V = (width2 × length)/2, with length representing the largest and width representing the perpendicular diameter.The B16-F10 murine melanoma cell line was purchased from the ATCC (CRL-6475) and maintained in DMEM + GlutaMAX™ (Gibco) supplemented with 10% FCS (Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). 3–4 passages post-thawing, cells were tested for mycoplasma  and subsequently 10+ 7-AAD− CD11c+ MHC-IIhi), MDSCs (CD45.2+ 7-AAD− CD11c− CD11b+ Gr-1hi) and other myeloid cells (CD45.2+ 7-AAD− CD11c− CD11b+ Gr-1−) were sorted from whole spleen and tumour single cell suspensions on a BD FACSAria™ II flow cytometer.On day 12 post tumour cell injection, animals were euthanized and organs isolated. Single cell suspensions of whole spleen and tumour tissue were obtained by enzymatic digestion using 10 mg/mL collagenase D and 20 U/mL DNase I (both Roche), followed by red blood cell lysis. Tumour-draining  LN were meshed and filtered at 70 μm. Cells were stained with the following antibodies: CD45.2-APC-eFluor780 (104), CD11b-APC (M1/70), CD11c-eFluor450 (N418), CD3ε-FITC (145-2C11), CD4-PerCP-eFluor710 (RM4-5), CD4-PerCP-Cy5.5 (RM4-5), CD4-eVolve605 (RM4-5), CD8a-PE (53–6.7), CD45R (B220)-Alexa Fluor 700 (RA3-6B2), NK1.1-PE-Cy7 (PK136), Gr-1-FITC (RB6-8C5), CD274 (PD-L1)-PE-Cy7 (MIH5), CD273 (PD-L2)-PerCP-eFluor710 (122), MHC-II I-Ab-FITC (AF6-120.1), MHC-II I-Ab-PerCP-eFluor710 (AF6-120.1), CD279 (PD-1)-PE-Cy7 (J43), CD279 (PD-1)-APC-eFluor780 (J43), TIM-3-PE (8B.2C12), CD197 (CCR7)-Alexa Fluor 700 (4B12), CD184 (CXCR4)-PerCP-eFluor710 (2B11) ; CD8a-Brilliant Violet 510 (53-6.7), F4/80-PerCP (BM8), CD86-Brilliant Violet 650 (GL-1), CD103-Brilliant Violet 510 (2E7) ; CD3ε-PE-CF594 (145-2C11), Ly6C-Brilliant Violet 605 (AL21), Ly6G-PE (1A8), CD80-PE-CF594 (16-10A1) . Dead cells were stained using 7-AAD (eBioscience). Flow cytometry was performed on a BD LSRFortessa™ flow cytometer and data analysed using FlowJo 10.0.5 (Treestar). Live DCs (CD45.2Mapkapk2 (Mm01288465_m1), Tgfb1 (Mm01178820_m1), Il10 (Mm01288386_m1), Cxcr4 (Mm01996749_s1), Ccr7 (Mm99999130_s1), Ccl3 (Mm00441259_g1), Ccl4 (Mm00443111_m1), Tbx21 (Mm00450960_m1) and Ifng (Mm01168134_m1) normalized to Ubc (Mm02525934_g1) as a housekeeping control.RNA was isolated from fresh sorted myeloid cell populations or frozen splenocyte single cell suspensions using the RNeasy Mini Kit (Qiagen) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Quality control of RNA isolated from frozen samples was done using the Experion™ RNA StdSens Assay (Bio-Rad). TaqMan probes (Life Technologies) were used to quantify expression of 50 and pulsed for four hours with 10 μg/mL B16-F10 freeze/thaw lysate in the presence of 100 ng/mL LPS . BMDCs were then cultured with CD3+ T cells isolated from spleens of C57BL/6N wild-type mice  for four days at a 1:5 ratio to generate tumour-specific T cells. After four days, T cells were harvested. DCs were sorted from tumour (tDCs) single cell suspensions on day 12 post-tumour cell injection following dead cell removal (Miltenyi Biotec) on a BD FACSAria™ II flow cytometer. tDCs were then co-incubated with tumour-specific T cells at a 1:10 ratio in 96-well U-bottom plates and cultures analysed by flow cytometry five days later. T cells stimulated with 1 μL αCD3/CD28 Dynabeads (Life Technologies)/100,000 cells served as activation control.For assessment of T cell activation, bone marrow-derived dendritic cells (BMDCs) were generated as described previouslyMurine sera collected on day 12 post-tumour cell injection were analysed using the ProcartaPlex™ Multiplex Immunoassay technology (Affymetrix) combining simplex kits to detect IFN-γ, IL-6, IL-12p70, IL-17A, IL-23, MIP-1α (CCL3) and MIP-1β (CCL4) according to the manufacturer’s instructions.+ cells were purified from whole spleen single cell suspensions of MK2ΔCD11c and WT littermate control mice by magnetic bead isolation (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were stimulated with 100 ng/mL LPS  for 4–24 hours. RNA was isolated and submitted for quality control , labelling and measurement on a Mouse Gene 2.0 ST Array (Affymetrix) to the Core Facility Genomics at the Medical University of Vienna (kindly performed by Markus Jeitler). Data were normalized by RMA Sketch  and analysed using one-way between-subject ANOVA (unpaired) with a p value < 0.05 designating statistical significance.CD11cDNA was isolated from formalin-fixed paraffin-embedded B16-F10 tumours using the AllPrep DNA/RNA FFPE Kit (Qiagen) and submitted for amplification and survey level sequencing of TCRB CDR3 via the immunoSEQ platform (Adaptive Biotechnologies). Data were normalized and analysed using the immunoSEQ analyzer software toolset.t-test. RT-qPCR data were analysed by Mann-Whitney U test. Tumour growth curves were analysed by repeated-measures two-way ANOVA and Bonferroni correction for multiple comparisons. Significance levels are indicated as ns (not significant), *P < 0.05, **P < 0.01; ***P < 0.001 and ****P < 0.0001. All statistical analyses were done using GraphPad Prism 6.0.All animal experiments included 5–8 mice per group and were repeated independently at least twice. Tumour size was measured in a blinded manner with regard to experimental groups. Randomization of mice to groups was done based on tumour size on day 5 post-tumour cell inoculation. Data are presented throughout as mean ± SEM with each symbol representing one individual animal and were analysed by two-tailed unpaired or paired Student’s The microarray datasets are available in the GEO repository (accession number: GEO:GSE96628). All other datasets generated during the current study are available from the corresponding authors upon reasonable request.Supplementary information


The effect of endostatin combined with a small dose of 32P-colloidal on tumour growth in vivo was evaluated and the potential mechanism underlying the combined therapy was explored. We found that 32P-colloid combined with endostatin exhibited higher inhibitory effect upon tumour growth compared with application of 32P-colloid or endostatin alone, although three therapies all significantly inhibited tumour growth compared with saline control group. The higher inhibitory effect of 32P-colloid combined with endostatin upon tumour growth might be attributed to a synergistic effect of inhibiting angiogenesis by endostatin and inducing apoptosis by 32P-colloid, as demonstrated by microvessel density (MVD) and apoptotic index (AI) measurement. Combined therapy of 32P-colloid and endostatin probably serves as a novel and efficacious therapy of tumour growth.Eighty healthy male Wistar rats, aged 5 weeks, weighing 100–120 g, were utilized for establishing tumour-bearing models by immediate Walker-256 cancerous ascites injection and randomly divided to four groups ( However, the range of clinical application is still highly restricted due to severe side effects or alternative unfavourable factors. How to alleviate the damages to normal tissues during cancer treatment is to be urgently resolved. Recently, internal radiation therapy with radiopharmaceuticals has gradually captivated widespread attention due to multiple advantages, such as focused and high efficient therapy, small radiation dose and minor damage to adjacent tissues . Folkman32P radioactive colloid with particle diameter 20–50 nm and radiochemical purity of 99% was purchased from Beijing Atomic Hi-Tech Co.The secreted endostatin plasmid (pCMV-ssEndostatin) was a gift from the Key Laboratory of Radiobiology, Ministry of Health, School of Public Health, Jilin University, China. Lipofectamine 2000 was purchased from Invitrogen. The rabbit antibody against mouse VIII factor related antigen was purchased from Fuzhou Maixin Biotech. Co. The TUNEL kit was purchased from Wuhan Boster Company. Materia Medica, Chinese Academy of Medical Sciences, China.The rat breast cancer cell line Walker-256 was provided by Institute of 5–106 cells) was injected subcutaneously into the outer side of the right hind root of rat. At 7 days after inoculation, the longest diameter (L) and maximum vertical diameter (W) of tumours were measured with callipers and the tumour volume was calculated by V (mm3)=L × W2/2. The rats with similar tumour volume were randomly divided to four groups (n=20) treated with 0.2 ml solution containing saline, 32P-colloid (0.3 mCi), endostatin gene (20 μg) and endostatin gene combined with colloid 32P respectively. The tumour volume was measured at the indicated time after treatment and the tumour growth rate (f) was calculated as the ratio of tumour volume after treatment (Vt) and tumour volume before treatment (V0): f=Vt/V0.Healthy male Wistar rats, 5-week old, weighing 100–120 g, were provided by the Experimental Animal Center at the First Affiliated Hospital of Harbin Medical University. Under sterile conditions, 0.3–0.5 ml of Walker-256 cancerous ascites  and tumour morphology was observed under light microscopy.Under immunochemical observation, the tumour cells with positive VEGF were stained as yellow and brown cytoplasm. Five fields with high expression of endostatin were selected. Subsequently, the ratio of positive-VEGF cells/total cells was calculated. The average value of the five visual fields was obtained as the final positive rate of VEGF. Immunochemical staining of the tumour cells with positive VEGF was subsequently performed to confirm the accuracy of the calculated outcomes.One representative section of tumour from each rat was stained for factor VIII-related antigen using a standard immunohistochemistry technique. The vascular distribution in the whole slice was first observed at 40 times magnification and five regions with the most intensive microvascular distribution, namely ‘hot spots’, were chosen and the number of vessels in each region was counted at 200 times magnification. The average counting of the five regions was indicated as microvessel density (MVD). Endothelial cells or endothelial cell clusters which were brown stained, vessel lumen and erythrocytes were not judged as microvessel.TUNNEL assay was used to measure the Apoptotic Index (AI). Briefly, the paraffin-embedded tissue sections were deparaffinized and dehydrated. A portion of 50 μl of TUNEL reaction mixture was added to tissue sections. Then, on each slide, 50 μl of conversion agent (POD) and 50–100 μl of substrate solution were added successively and slides were incubated for 10 min at room temperature. Slides were then dehydrated, cleared and mounted for co-focal microscopy observation. Positive signal was defined as the presence of a distinct fluorescence staining within the nuclei. AI was determined by counting a total of at least 1000 nuclei subdivided in 20 fields chosen randomly at 400 times magnification. Apoptotic cells were identified by TUNEL in conjunction with characteristic morphological changes, such as cell shrinkage, membrane blebbing and chromatin condensation, to distinguish apoptotic cells and apoptotic bodies from necrotic cells, which were not considered as apoptotic cells. AI was calculated by dividing the number of apoptotic cells by the total number of counted cells in each slide.x±S (mean ±standard deviation). SNK-q test was used for statistical analysis of tumour growth rate, tumour MVD and AI. SPSS13.0 statistical software was used for all data analysis (SPSS).All quantification data were expressed as 32P-colloid or endostatin alone group, the animals were generally in good condition without abnormalities in hair and body colour, and presented with slow tumour growth rate and high mobility. Interestingly, the tumours in the 32P-colloid combined with endostatin group were unpalpable and flaky and significantly reduced in volume, and some tumours formed scabs and fell off at late stage.In the saline control group, the tumours were obviously enlarged with a magenta colour. At late stage, the rats exhibited extremely poor status with sparse hair and thin epidermis. Tumours on these rats were not movable and ulcerations were noted on tumour surface showing white fish flesh-like tissues. Most rats died within 14 days after treatment. In the The expression level of endostatin protein was detected by Western blot. The results revealed that the expression level of endostatin in the p-CMV-endostatin group (lane 3) was significantly up-regulated compared with those in the PBS (lane 1) and empty plasmid groups (lane 2), as illustrated in The expression levels of endostatin mRNA among different groups were quantitatively measured by RT-PCR. The results revealed that the expression levels of endostatin mRNA in the endostatin gene (lane 3) and 32P+endostatin groups (lane 4) were significantly up-regulated compared with those in the control (lane 1) and 32P groups (lane 2), as shown in 32P group was (70.08±15.53)%, which was the highest among all groups, followed by (57.68±13.58)% in the control group, (35.46±12.45)% in the endostatin gene group and (18.60±8.62)% in the 32P+endostatin group, which was the lowest among four groups. The results of VEGF positive rate were consistent with immunohistochemical staining findings (32P group was the largest among four groups.The results demonstrated that the positive rate of VEGF in the findings . The tumP<0.001). There was no statistical significance in tumour growth rate between the 32P-colloid and endostatin gene groups (P>0.05). However, the tumours in the 32P-colloid combined with endostatin gene group grew at a slower rate compared with either 32P-colloid or endostatin gene group (P<0.01).The tumour growth rates at D7 and D14 after treatment were shown in 32P-colloid and endostatin gene groups, whereas extensive necrosis and loosely arranged tumour cells were observed in the 32P-colloid combined with endostatin gene group . The MVD in the 32P-colloid combined with endostatin group was the smallest, significantly differing from those in the 32P-colloid or endostatin gene alone group (P<0.05).A large amount of vascular endothelial cells were stained in the peripheral region of the tumour in the control saline group, whereas the number of stained endothelial cells in three treatment groups was significantly decreased . After tP<0.001). No significant difference was observed between the 32P-colloid and endostatin gene groups (P>0.05), whereas the AI in the 32P-colloid combined with endostatin gene group was significantly higher than those in 32P-colloid and endostatin gene groups (both P<0.01) (The representative imaging for AI measurement in each group was shown in  P<0.01) E.in vivo anti-tumour effect of 32P-colloids combined with endostatin was demonstrated. 32P is a pure beta-emitter, with an average length of 3-4 mm in soft tissues, 99% of the energy accumulated in the tumour tissue 4 mm adjacent to the injection site with a half-life of 14.28 days. Due to these characteristics, 32P has been widely used in internal radiation therapy for tumour treatment [In the present study, the reatment .32P colloid upon tumour growth [32P can directly kill tumour cells. After injected into the tumours, 32P colloid is accumulated mainly in the tumour tissues due to the limited lymphatic drainage, resulting in much higher radioactivity within the tumour tissues than that in adjacent tissues. Subsequently, the tumour cells will be delivered with an absorbed lethal dose, whereas the adjacent tissues will be spared from irreversible radiation damage. In addition, because of relatively long half-life and retention in tumour tissues, 32P colloid can continuously irradiate tumour cells and lead to retention effect to overcome the radiation resistance acquired by DNA-damage repair. Secondly, 32P can induce tumour cell apoptosis, possibly through the DNA damage initiated by hydroxyl radicals. More importantly, internal radiation therapy rarely induces inflammation because of relative low dosage compared with the single high-dose external irradiation. This opinion could be supported by our results in the present study. Most rats in three treatment groups were in good condition and the tumour grew slowly without significant radiation damage or systemic symptoms. Thirdly, 32P is probably capable of inhibiting tumour angiogenesis. Compared with pure ionic colloidal solution, 32P has a larger particle diameter, which makes it easier to stay in the local neovascularization and cause lethal damage to peripheral vascular endothelial cells by continuous irradiation. In this way, the tumour regrowth and recurrence will be reduced because of disrupting the development of collateral circulation, which was also supported by the facts that the tumour growth rate and MVD in the 32P treatment were significantly lower compared with those in the control group.Several mechanisms have been proposed for the inhibitory effect of r growth . Firstly32P-colloid or endostatin alone could inhibit tumour growth, both therapies failed to completely prevent tumour growth, indicating the limitations of 32P-colloid or endostatin treatment alone. It has been reported that 32P-colloid inhibited tumour growth in a dose-dependent manner [32P-colloid can kill more tumour cells, but also increases the risk of radiation injury. Endostatin targets the nascent endothelial cells rather than the tumour cells. Therefore, it can only indirectly inhibit tumour growth. In our study, endostatin was injected into the well-established tumour mass, in which most vascular endothelial cells were in a relative static state, had decreased proliferation ability and lacked sensitivity to endostatin. Bergers et al. [Although t manner . A highes et al.  have dems et al. ,13, sugg32P-colloid combined with endostatin group was the lowest among all groups, even significantly lower than that in either 32P-colloid or endostatin alone group, suggesting that endostatin could enhance the effect of 32P-colloid on killing tumour cells, which was further supported by the massive tumour cell necrosis, sparsely arranged nascent tumour cells and significantly declined MVD in the 32P-colloid combined with endostatin group as compared with other groups. The results in this study were consistent with previous report [The tumour growth rate in the s report ,15.32P-colloid combined with endostatin on killing tumour cells may be explained by the follow causes. First, it is more easily to transfect endostatin plasmid into the 32P-colloid-treated tumour cells. Second, the endothelial cells which are in static state and insensitive to endostatin could initiate the process of DNA damage repair after 32P-colloid irradiation and become sensitive to endostatin during this process. Third, in order to repair cell damage caused by 32P-colloid, a larger amount of angiogenesis is required but it could be inhibited by continuous expression of endostatin in our experiment.The synergic effect of 32P-colloid significantly enhances the inhibitory effects of 32P-colloid at a relatively low dosage on tumour growth, and thus reduces the radiation injury. In summary, 32P-colloid combined with endostatin achieved high in vivo inhibitory effects upon tumour growth, which may provide a novel strategy for tumour treatment.Due to the side effects of anti-cancer medications constantly result in the failure of anti-tumour therapy, reducing the medication dosage under the premise to ensure the clinical efficacy is critical for successful treatment. Our study demonstrated that the combination of endostatin and


Progenitor cell analysis coreMolecular Phenotyping coreStem Cell COBRE Grant P30GM106391Vascular Biology COBRE Grant P30GM103392Upon publication of the original article , it was This has since been acknowledged and corrected in this erratum.


TARDOX is a Phase I single center study of ultrasound triggered targeted drug delivery in adult oncology patients with incurable liver tumours. This proof of concept study is designed to demonstrate the safety and feasibility of targeted drug release and enhanced delivery of doxorubicin from thermally sensitive liposomes (ThermoDox®) triggered by mild hyperthermia induced by focused ultrasound in liver tumours. A key feature of the study is the direct quantification of the doxorubicin concentration before and after ultrasound exposure from tumour biopsies, using high performance liquid chromatography (HPLC).The study is conducted in two parts: Part 1 includes minimally-invasive thermometry via a thermistor or thermocouple implanted through the biopsy co-axial needle core, to confirm ultrasound-mediated hyperthermia, whilst Part 2 is carried out without invasive thermometry, to more closely mimic the ultimately intended clinical implementation of the technique. Whilst under a general anaesthetic, adult patients with incurable confirmed hepatic primary or secondary (metastatic) tumours receive a single cycle of ThermoDox®, immediately followed by ultrasound-mediated hyperthermia in a single target liver tumour. For each patient in Part 1, the HPLC-derived total doxorubicin concentration in the ultrasound-treated tumour is directly compared to the concentration before ultrasound exposure in that same tumour. For each patient in Part 2, as the tumour biopsy taken before ultrasound exposure is not available, the mean of those Part 1 tumour concentrations is used as the comparator. Success of the study requires at least a two-fold increase in the total intratumoural doxorubicin concentration, or final concentrations over 10 μg/g, in at least 50% of all patients receiving the drug, where tissue samples are evaluable by HPLC. Secondary outcome measures evaluate safety and feasibility of the intervention. Radiological response in the target tumour and control liver tumours are analysed as a tertiary outcome measure, in addition to plasma pharmacokinetics, fluorescence microscopy and immunohistochemistry of the biopsy samples.If this early phase study can demonstrate that ultrasound-mediated hyperthermia can effectively enhance the delivery and penetration of chemotherapy agents intratumorally, it could enable application of the technique to enhance therapeutic outcomes across a broad range of drug classes to treat solid tumours.NCT02181075, Edura-CT Identifier: 2014-000514-61.ClinicalTrials.gov Identifier: Ethics Number: 14/NE/0124. A major challenge of systemic chemotherapy is the delivery of a therapeutic dose to the target tumour without exceeding the maximum tolerated dose in other tissues. Device-targeted drug delivery has seen decades of pre-clinical development but could now be at the dawn of clinical adoption, as a generic tool for overcoming the challenges of delivering existing and emerging therapeutics to solid tumours –3. A sigp value of 0.05), post-hoc subgroup analysis demonstrated a 53% risk improvement in overall survival in the subset of patients having received RFA treatment for 45 min or more with ThermoDox®. The ongoing OPTIMA study is a lead Randomized, Double Blind, Dummy-Controlled Phase III clinical study of ThermoDox® used in combination with standardized RFA for 45 min or more for solitary Hepatocellular Carcinoma (HCC). The OPTIMA study was initiated in June 2014 and is estimated to enrol about 550 patients. The primary endpoint is overall survival with progression free survival as a secondary endpoint. ThermoDox® is also currently under investigation in recurrent chest wall cancer, where a superficial hyperthermia device is placed in contact with the skin (DIGNITY study) and in paediatric solid tumours in combination with HIFU.Small animal tumour models have demonstrated that MR guided HIFU systems can non-invasively generate tissue hyperthermia, releasing circulating low thermosensitive liposomes (LTSLs), as confirmed by co-encapsulation with paramagnetic MRI contrast agents (“dose painting”) –14. TherUnlike RFA, focused ultrasound (FUS) is a non-invasive clinical treatment modality. FUS has been shown to be safe , 16, higThe TARDOX study explores the safety and feasibility of using an extracorporeal ultrasound-guided FUS device at sub-ablative powers to induce highly localized hyperthermia in liver tumours to trigger the release and enhance the delivery of systemically circulating ThermoDox®.This proof of concept study aims to determine whether triggered release and enhanced delivery of doxorubicin from ThermoDox® (‘drug’) using mild hyperthermia generated non-invasively by FUS is clinically feasible, safe and efficacious, for a given systemic dose.This is a Phase I prospective non-randomised safety cohort study with all patients recruited from a single UK site (Oxford). The study has an open label design with all participants receiving systemic ThermoDox® and ultrasound-guided FUS targeted at a single target liver tumour using the Model JC200 Focused Ultrasound Tumour Therapeutic System , which is clinically approved (CE-marked) for tumour therapy in Europe and China.2. After a minimum of five patients have received the Part 1 intervention with real-time thermometry, data will be reviewed by the Trial Management Group (TMG) to confirm readiness to proceed without real-time thermometry in Part 2 of the study. Subject to TMG approval, Part 2, which does not require implantation of a thermometry device, and instead uses predictions from Part 1 data to set the FUS parameters, will be opened to run in parallel to Part 1. Targeted drug delivery in Part 2 thus proceeds completely non-invasively, and this part of the study is designed to more closely reflect how the therapy might be implemented in routine clinical practice.The study is split into two parts. Part 1 is designed to identify optimal FUS exposure parameters for a range of tumour locations within the liver, using real-time thermometry data from an implanted thermometry device (a thermistor or thermocouple). Patients in Part 1 receive a single cycle of ThermoDox® intravenously, at a dose of of 50 mg/mAll participants from both Parts of the study will be included in the endpoint analysis. The primary endpoint relates to evidencing enhanced delivery of doxorubicin from ThermoDox® at the target tumour site, by comparing intratumoural concentrations of the drug before and after FUS exposure. The doxorubicin concentration will be directly determined from tissue biopsies of the target tumour, using a Good Laboratory Practice-validated high performance liquid chromatography (HPLC) assay, based on previously published methods , 19.Adult patients with incurable primary or secondary liver tumour(s) are eligible for the study.Pathologically confirmed advanced solid tumour with liver metastasis or primary liver tumour (hepatocellular carcinoma or cholangiocarcinoma). At least one liver tumour must be over 1 cm in diameter, should be visible using conventional ultrasound imaging onboard the Model JC200 Focused Ultrasound Tumour Therapeutic System and amenable to ultrasound-guided biopsy.Progressed or remained stable on conventional chemotherapy.Male or female, of age ≥ 18 years.Life expectancy of ≥3 months.Left Ventricular Ejection Fraction (LVEF) ≥ 50% on echocardiogram.Have not received radiotherapy to the target area within the preceding 12 months.World Health Organisation (WHO) performance status of ≤1.Able and willing to give written informed consent, indicating that they are aware of the investigational nature of this study and potential risks, and able to comply with the protocol for the duration of the study, including scheduled follow-up visits and examinations.Have surgery or other procedure requiring general anaesthesia planned to be undertaken during the period of the study.Serious illness including, but not limited to, congestive heart failure ; life threatening cardiac arrhythmia; or myocardial infarction or cerebral vascular accident within the last 6 months.On-going significant infection .Uncontrolled diabetes.2 or a lifetime dose of epirubicin over 900 mg/m2, or any dose of both.Have received a lifetime dose of doxorubicin over 450 mg/mPregnant or breast-feeding women. In women of childbearing potential, a negative pregnancy test (serum) is required within 30 days prior to study intervention.Female participants of child bearing potential and male participants whose partner is of child bearing potential who are not willing to practice an acceptable form of contraception  during the study and for 6 months thereafter. Women whose partner has or men who have undergone a vasectomy must use a second form of birth control.Known allergic reactions to any of the drugs or liposomal components or intravenous imaging agents to be used in this study.Portal or hepatic vein tumour invasion/thrombosis.Any inadequate haematological or biochemical indices, as shown in Table Have contraindications to receiving doxorubicin including prior sensitivity  attributed to anthracyclines or other liposomal drugs.Use of chemotherapy or of an investigational drug within 30 days or 5 half-lives, whichever is longer, preceding the intervention.Child-Pugh Class C liver disease, or Class A-B with encephalopathy and/or refractory ascites.HIV positive.Haemochromatosis.Contrast-induced nephropathy.Suspected liver haemangioma or other vascular tumour, tense ascites.Other medical or psychiatric conditions or laboratory abnormalities that the investigator considers would make the patient a poor trial candidate.Evaluation of previous cross-sectional imaging is required to ensure availability of potential tumour targets before proceeding to screening. Screening consists of an ultrasound examination, routine blood tests, an echocardiogram and a pre-operative assessment to ensure suitability for general anaesthetic Fig. . Tumours18F-FDG) positron emission tomography / CT (PET-CT) and dynamic contrast-enhanced MRI (DCE-MRI).Baseline imaging is performed in the week prior to intervention and consists of perfusion CT, fluorodeoxyglucose  or dot (shot-by-shot) mode, using the on-board plane-by-plane treatment planning tool to encompass all or part of the target tumour volume that can be spanned in a 30-min period. Hyperthermia is delivered with a clinically approved 0.95 MHz transducer, with a focal length of 145 mm and a diameter of 200 mm, resulting in a focus with a transverse 3-dB beam-width of 1.2 mm and an axial 3-dB length of 9.5 mm, using up to the full range of acoustic powers (32-400 W) and duty cycles (10–100%) available on the device to achieve at least 30 min of cumulative hyperthermia in the range of 40–42 °C in the target region. The specific treatment parameters will naturally vary depending on tumour anatomy.The ability to achieve targeted release of doxorubicin from ThermoDox® at the tumour site is determined by direct analysis of tumour biopsies taken during intervention, using a GLP HPLC assay. Plasma samples obtained during the intervention are used to evaluate doxorubicin pharmacokinetics.For Part 1 of the study, to achieve real-time thermometry of the target tumour and obtain biopsies at the same tumour site, a co-axial needle is first inserted in the target tumour under the guidance of a separate, hand-held diagnostic ultrasound system . This co-axial needle can be instrumented with either a thermometry probe or a biopsy needle, thus eliminating the need for repeated skin punctures and minimising patient risk.With the patient under general anaesthesia, the target liver tumour is first localised using the ultrasound guidance system that is integral to the FUS device, through an intercostal space or subcostally. In order to minimise respiratory movement of the target tumour during the intervention, appropriate anaesthetic techniques, such as high-frequency jet ventilation , are emp2 in 250 mL of normal saline or 5% dextrose over a 30-min infusion. In Part 1, biopsies of the target tumour are taken before and immediately after the drug infusion, and again following FUS-mediated delivery  in the liver, which receive ThermoDox® alone, are selected as controls against which radiological response in the FUS-targeted tumour (target tumour) are compared.The intervention, recovery and follow-up steps for both parts of the study are summarised in Fig. The primary outcome measure for the study is the concentration of intratumoural doxorubicin at the targeted tumour site, following FUS-mediated hyperthermia. In Part 1 the comparator biopsy is taken post-drug infusion but before FUS exposure. In Part 2 the average concentration of all HPLC-evaluable Part 1 comparator biopsies is used to assess the fold-increase. To be evaluable for the primary endpoint, the tumour must have received attempted hyperthermia following drug administration. In addition, for intratumoural biopsy samples to be suitable for HPLC analysis, the tissue must be of sufficient mass, and the HPLC analytical technique must have produced validated quantifiable data.Secondary endpoints relate to feasibility of inducing controlled FUS-mediated targeted hyperthermia in the target tumour non-invasively in Part 1 and, in addition, adverse event monitoring for 30 days in both parts. Radiological response is an exploratory (tertiary) endpoint, reflecting that only a single cycle of chemotherapy is being administered and that enhanced delivery, rather than response, is the primary aim of this early study.The TARDOX study is intended to recruit up to 28 evaluable participants. No formal sample size calculation has been performed. Sample sizes have been designed to reflect the fact that this is a Phase I proof-of-concept study, and thus only small patient numbers are required. The sample size is designed to be large enough to demonstrate statistically meaningful enhanced drug delivery but small enough to ensure that participants are not unnecessarily recruited. The sample size is also in line with the available resources for this study (funding).In Part 1, feasibility of targeted hyperthermia in the target liver tumour is assessed using an implanted thermometry device for real-time thermometry assessment. For Part 1 treatments, the cumulative equivalent in minutes (CEM43) thermal dose threshold for cell death  is calcuIn addition, to explore if  any potential delayed radiological response can be attributed to a chemo-ablative process rather than direct tumour ablation secondary to high intensity FUS, a post-intervention DCE-MRI is performed with 36 h of treatment. The primary objective is analysed using a quantitative outcome measure, as per the primary endpoint; the concentration of intratumoural doxorubicin obtained from tumour biopsy samples.All participants from both Parts of the study who received the intervention will be included in the primary endpoint analysis, thus evaluated on an intention to treat basis. To satisfy the primary endpoint, demonstration of a two-fold increase over the comparator, or an absolute value exceeding 10 μg/g, of the concentration of intratumoural doxorubicin at the treated tumour site following attempted FUS-induced hyperthermia, is required in at least 50% of all evaluable participants. To be included in this analysis, intra-tumoural drug concentrations from biopsy samples must have been successfully analyzed by HPLC.Preclinical studies in a variety of human xenograft tumour types for long-circulating liposomes with a half-life of more than 10 h have demonstrated that, in the absence of ultrasound, the passive intratumoral accumulation of systemically administered liposomal doxorubicin increases by no more than 100% in the first 2 h following intravenous administration, even in the leakiest tumour type investigated . In the Safety assessments are performed for 30 days post-intervention and consist of clinical, haematological and biochemical review. Adverse events are assessed for expectedness and causality to the drug and also to FUS, and classified according to the Common Toxicity Criteria Adverse Events .If this early phase study can demonstrate that ultrasound-mediated hyperthermia can safely and effectively enhance the delivery and penetration of chemotherapy agents, it could enable application of the technique to enhance therapeutic outcomes across a broad range of drug classes to treat primary and metastatic solid tumours.


Adult-type fibrosarcoma is a rare and highly aggressive subtype of soft tissue sarcomas. Due to the existence of other spindle-cell shaped sarcomas, its diagnosis is always one of exclusion. The likelihood of misdiagnoses between similar tumour entities is high, and often leads to inappropriate tumour treatment. We summarize here the main features of fibrosarcoma. When fibrosarcoma is appropriately diagnosed, the patient`s overall prognosis is generally quite poor. Fibrosarcoma is characterized by its low sensitivity towards radio- and chemotherapy as well as by its high rate of tumour recurrences. Thus it is important to identify new methods to improve treatment of this tumour entity. We discuss some promising new directions in fibrosarcoma research, specifically focusing on more effective targeting of the tumour microenvironment. Communication between tumour cells and their surrounding stromal tissue play a crucial role in cancer progression, invasion, metastasis and chemosensitivity. The therapeutic potential of targeting the tumour microenvironment is addressed. Fibrosarcoma is a rare, highly malignant tumour of mesenchymal cell origin. It derives from pathologically transformed spindle shaped fibroblasts with an excessively high division rate. , 2 AccorThe diagnosis of fibrosarcoma is by one of exclusion . Using iTo help ensure an accurate diagnosis and thus appropriate treatment plan, we summarize below the diagnostic steps and the typical anamnestic, clinical, imaging, histologic and immunohistochemical features of fibrosarcoma. The highly aggressive character of this tumor, its low response rate to chemotherapy, and the high rate of tumour recurrences contribute to its poor prognosis. The investigation of new treatment strategies is needed. We specifically discuss the potential importance of targeting the tumour microenvironment as a means of controlling tumor growth and chemosensitivity.A definitive cause of fibrosarcoma has not been identified. Nevertheless, genetic mutations and somePatients with unclear and potentially malignant soft tissue masses should be thoroughly examined for information about its location, size, shape, consistency and relationship to the surrounding tissue. Determination of the range of motion of nearby joints, a complete neurovascular examination as well as palpation of regional lymph nodes in view of tumour metastasis should be part of the physical examination , 20.Fibrosarcoma mainly arises in regions consisting of collagen-rich connective tissue. Often, adult-type fibrosarcoma occurs in the lower extremities, especially in the area around the thighs and knees, the arms and the trunk, , 21. In If the anamnesis and physical examination suggest a potential soft tissue tumour, radiological imaging Table  is the nAlternatively, computed tomography (CT) or X-ray can help detect bone involvement. CT of the chest and abdomen/pelvis or an MRT of the whole body or positron emission tomography (PET)-CT can assist in the detection of distant metastases. CT is also recommended for retroperitoneal located tumours., 22, MorIn radiological imaging, fibrosarcomas appear as unspecific, often intramuscular localized, ovoid lesions. Its margins are slightly irregular. A fibrosThere are different biopsy procedures for soft tissue sarcomas. It can be done by incisional or excisional biopsies conducted via open surgery. A minimal invasive procedure such as fine needle aspiration (FNA) biopsy or the core needle biopsy can be used –25. The Most soft tissue sarcomas can be assigned to one of the following five histomorphologic groups: pleomorphic pattern, epitheloid cell pattern, myxoid pattern, small round cell pattern and spindle cell pattern , 29. SpiHistopathology alone is not sufficient for a clear distinction between fibrosarcoma and other spindle-cell neoplasms. Immunohistochemistry (IHC) is applied in the diagnostics of fibrosarcoma where specific antibody reagents allow the detection of differential diagnostically important tumor markers , 34–36, The positive staining of vimentin demonstrates the mesenchymal origin of fibrosarcoma. Desmin, alpha smooth muscle actin (α-SMA) and muscle specific actin (MSA) belong to the most common myogenic markers. The S-100 protein is a neuronal marker that serves to exclude malignant peripheral nerve sheath tumors (MPNST). Positive CD 31, CD34 and factor VIII (von Willebrand factor) immunostains point to vascular malignancies e.g. spindle-cell angiosarcomas. Epithelial markers such as the epithelial membrane antigen (EMA), and cytokeratins can be helpful in the differential diagnosis of sarcomatoid carcinomas , 10. In In fibrosarcoma, vimentin, is often the only positively stained marker , 35, 3. Ki-67, a cell cycle–associated nuclear antigen, is also used as a diagnostic marker for fibrosarcoma , 43, 44.The lack of specifity of IHC markers has driven the search for additional biomarkers. In this context, microRNAs (miRNAs) have recently gained importance. Micro RNAs (miRNAs) are a class of small noncoding RNAs that bind to the 3′ untranslated regions (UTRs) of messenger RNA (mRNA), where they negatively regulate translation. It has been suggested that more than 60% of all mRNAs are controlled by miRNAs suggesting that they are major players in the posttranscriptional gene regulation. MalignanFibrosarcomas and soft tissue sarcomas are prognostically evaluated by taking into account the age of the patient, the tumour size, depth and malignancy, the involvement of nerves, vessels, bone, the collagen density, as well as the metastatic potential and the formation of tumour recurrences , 26, 21.Surgery represents the standard therapy of localized soft tissue sarcomas . The surIn addition to surgery, radiotherapy and/or hyperthermia therapy, chemotherapy is a major category of treatment. By targeting and killing rapidly dividing and proliferating cells, such as malignant tumour cells, chemotherapeutical agents are broadly used to stabilize disease and for tumour remission. Adjuvant chemotherapeutic therapy in soft tissue sarcomas is very controversial, , 21 and Chemotherapy in patients with advanced stage fibrosarcomas is based on anthracyclines as the first-line treatment. In this context, doxorubicin is the most widely applied drug. Besides doxorubicin, response rates above 15% can be reached by actinomycin D and ifosfamide , 52. A nIn contrast to adjuvant chemotherapy, neoadjuvant treatment has been shown to be more effective. Patients with high-grade fibrosarcomas can benefit from a presurgical MAID  treatment .As we have seen, the aggressive character, lack of treatment response to chemotherapy and the high rate of tumour recurrences contribute to the poor prognosis of patients with fibrosarcoma. Therefore, major efforts have been made to identify new ways to slow its proliferation and migration and to increase tumor sensitivity towards apoptosis inducing drugs such as the doxorubicin.To this end, the tumour surrounding tissue, or stroma, has been the focus of new therapy approaches. The tumour microenvironment consists of two main components, the cellular component comprising tumor associated fibroblasts, smooth muscle cells, adipocytes, endothelial cells and immune cells. The second component is the extracellular matrix (ECM) which fills the intercellular space. The ECM consists mainly of proteoglycans, fibrous proteins, adhesion molecules and proteases, and is characterized by being high dynamic . The intMatrix metalloproteinases (MMPs) , 56 are So there is a general positive correlation between the amount of MMPs present and tumour progression. The higher the MMP concentration, the more advanced the cancer - the poorer the prognosis and overall survival. Controlling MMP activity within the tumour microenvironment is one way to locally control fibrosarcoma growth and metastasis.As a means to control ECM degradation and tumour progression, a series of broad spectrum MMPs inhibitors such as batimastat (BB-94), marimastat (BB-2516), GM 6001, CT1746, KB-R7787, prinomastat (AG3340), BMS275291, BAY 12-9566, CGS 27023A were developed. However, due to the high levels of side effects such as the musculoskeletal syndrome, most clinical phase III studies had to prematurely be curtailed , 66. As N-Isopropoxy-Aminoacetohydroxamic (ARP 101) selectively inhibits MMP2 activity which is strongly increased in fibrosarcoma. In vitro, ARP 101 resulted in a decrease of tumour invasion [The MMPs expression profile is generally tumour-specific. Human fibrosarcoma cells express extracellular MMPs 1, 2, 3, 7 and 9 as well as the membrane type MMPs 14, 15, 16 , 63, MMPinvasion . Selectiin vitro treatment of human fibrosarcoma cells with TIMP-1-GPI showed an inhibition of cell proliferation and migration as well as in an increased cell apoptosis and enhanced sensitivity to chemotherapy agents [Because these enzymes play such central roles in tissue homeostasis, pronounced side effects are seen with systemically applied MMPs-inhibitors (MMPI) . To helpy agents .Fibrosarcoma shows pronounced resistance towards apoptosis inducing chemotherapeutic agents . An incrde novo development of drug resistance [The reduced fibrosarcoma response towards therapeutic agents is linked in part to interactions between the tumour cells and their microenvironment . This phsistance . It is isistance . The EMDsistance . The autsistance .Collagen type I plays an important role in CAM-DR in fibrosarcomas. Two type I collagens have been found within the tumour tissue . The hetRecently the ‘cancer stem cell hypothesis’ has gained importance in cancer research , 88, It The tumour microenvironment raises hopes for new sarcoma treatments . This isThe phase III placebo-controlled clinical trial of Anlotinib, a multi-target tyrosine kinase inhibitor, is another example of a clinical trial in progress to find a more effective treatment of advanced soft-tissue sarcoma by targeting the tumour microenvironment –104.The diagnosis of fibrosarcoma is one of exclusion, and an accurate diagnosis is a prerequisite for the design of an adequate treatment plan. Consequently, understanding the typical characteristics and features of this tumour type is important for accurate diagnosis. New markers such as miRNA expression profiles may represent an additional supportive diagnostic step in the identification of fibrosarcoma.Due to its aggressive nature, the overall survival rate for a patient with adult-type fibrosarcoma is poor. The best prognosis is seen when the following points can be addressed: (i) complete surgical tumour resection with histological tumour-free margins (R0), (ii) the use of agents that lead to a reduction in tumour proliferation and migration and/or inducing remission via neoadjuvant or adjuvant therapy, (iii) preventing tumour invasion and metastasis.A tumour`s proliferation and migration significantly correlates with the composition of the surrounding ECM components. The high concentration of MMPs within the tumour tissue results in a highly increased degradation of the ECM which consequently facilitates tumour growth as well as its spread to distant sites. In this context, intratumoral injections especially of the recently developed TIMP-1-GPI fusion protein  may reprSimilar to other treatment strategies the ones discussed above have their pros and cons Figure . Hence tIt should also be kept in mind that all the approaches mentioned in this article are pieces of the puzzle. The complexity of cancer is multidimensional. Therefore, the tumour microenvironment is only one dimension in the treatment of fibrosarcoma.Targeting the tumour initiating cancer stem cells may be an important step in the treatment of fibrosarcoma. At present fibrosarcoma stem cell markers, even in combination, still fail to identify all CSC present in a sample. Further work in this field is urgently needed.


Patients who increase stoke volume or cardiac index more than 10 or 15% after a fluid challenge are usually considered fluid responders. Assessment of fluid responsiveness prior to volume expansion is critical to avoid fluid overload, which has been associated with poor outcomes. Maneuvers to assess fluid responsiveness are well established in mechanically ventilated patients; however, few studies evaluated maneuvers to predict fluid responsiveness in spontaneously breathing patients. Our objective was to perform a systematic review of literature addressing the available methods to assess fluid responsiveness in spontaneously breathing patients. Studies were identified through electronic literature search of PubMed from 01/08/2009 to 01/08/2016 by two independent authors. No restrictions on language were adopted. Quality of included studies was evaluated with Quality Assessment of Diagnostic Accuracy Studies tool. Our search strategy identified 537 studies, and 9 studies were added through manual search. Of those, 15 studies  were included in this analysis. In total, 649 spontaneously breathing patients were assessed for fluid responsiveness. Of those, 340 (52%) were deemed fluid responsive. Pulse pressure variation during the Valsalva maneuver (∆PPV) of 52% (AUC ± SD: 0.98 ± 0.03) and passive leg raising-induced change in stroke volume (∆SV-PLR) > 13% (AUC ± SD: 0.96 ± 0.03) showed the highest accuracy to predict fluid responsiveness in spontaneously breathing patients. Our systematic review indicates that regardless of the limitations of each maneuver, fluid responsiveness can be assessed in spontaneously breathing patients. Further well-designed studies, with adequate simple size and power, are necessary to confirm the real accuracy of the different methods used to assess fluid responsiveness in this population of patients.The online version of this article (10.1186/s13613-018-0365-y) contains supplementary material, which is available to authorized users. Intravascular volume expansion is a common intervention in critically ill patients . Patient2O  OR “hemodynamics”[All Fields]) AND (“respiration”[MeSH Terms] OR “respiration”[All Fields] OR “cell respiration”[MeSH Terms] OR (“cell”[All Fields] AND “respiration”[All Fields]) OR “cell respiration”[All Fields]) AND (“cardiac output”[MeSH Terms] OR (“cardiac”[All Fields] AND “output”[All Fields]) OR “cardiac output”[All Fields]))). Literature search was limited to a period of time (01/08/2009 to 01/08/2016) and to “human.” No restrictions on language were adopted. Additionally, we hand-searched the reference lists of the included studies to identify other relevant studies.Prospective studies that reported sensitivity, specificity, cutoff value of each maneuver to assess fluid responsiveness, number of patients included and frequency of fluid responsiveness and non-fluid responsiveness patients were included in this systematic review. Review articles, editorials, studies assessing fluid responsiveness during mechanical ventilation and studies that did not report outcomes of interest were excluded.Two authors independently screened all retrieved citations by reviewing their titles and abstracts (RCFC and FTM). Then, the reviewers independently evaluated the full-text manuscripts for eligibility using a standardized form. Reviewers independently extracted the relevant data from the full-text manuscripts and assessed the risk of bias using a standardized form. Any disagreement between the authors was resolved by a third author (ASN).The quality of each study was evaluated by the Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS) . DetailsThe primary objective was to report the available methods to assess fluid responsiveness in spontaneously breathing patients.Secondary objectives were to assess diagnostic performance and build a receiver operating characteristics curve (ROC curve) of methods available to assess fluid responsiveness in spontaneously breathing patients.FB);  , 19, 20.The number of patients included, study design, setting, inclusion and exclusion criteria, time and type of fluid infused, the best cutoff value of each maneuver and definition of fluid responders were extracted from published studies. The accuracy of each diagnostic test was assessed with sensitivity (Sens), specificity (Spec), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR +), negative likelihood ratio (LR −), AUC along with its standard deviation (SD) or 95% confidence interval (95% CI). Whenever not reported, accuracy, PPV, NPV, LR + and LR − were calculated using the Review Manager (RevMan) [computer program]—version 5.3—Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014.A receiver operator characteristics curve (ROC curve) was constructed using the sensitivity and specificity of each maneuver extracted from included study using Meta-DiSc version 1.4  . MethodsThe initial search strategy identified 537 studies Fig. . After sCharacteristics of included studies are presented in Tables Out of 649 spontaneously breathing patients assessed for fluid responsiveness, 340 patients 52%) were responders. In 12 studies [12/15 (80%)], only spontaneous breathing patients without any type of ventilatory support were included (572 patients) , 24–29. % were reFluid challenge was performed in seven (46.6%) studies through an I.V. infusion of 500 ml of saline; , 27, 28 Adopted definitions of fluid responsiveness were an increase in SV > 10%  or > 15%®) are summarized in Table Thirty-four maneuvers for predicting fluid responsiveness in spontaneously breathing patients were reported Tables  and 2. SOut of 34 reported maneuvers for predicting fluid responsiveness in spontaneously breathing patients, 13 (38%) maneuvers had excellent accuracy (AUC from 0.9 to 1), 9 26%) had adequate accuracy (AUC from 0.8 to 0.89), 6 (18%) had fair accuracy (AUC from 0.7 to 0.79), 5 (15%) had poor accuracy (AUC from 0.6 to 0.69) and 1 maneuver (3%) was classified as failure (AUC from 0.5 to 0.59) ,  ∆SV-PLR ∆VSP of 52% [AUC ± SD: 0.98 ± 0.03]  had the TE > 13% [AUC ± SD: 0.96 ± 0.03] had the highest accuracy, while ∆SV ≥ 10% [AUC (95% CI) 0.57(0.34–0.78)] had the worst accuracy to predict fluid responsiveness in mechanically ventilated patients in a spontaneous mode   of reported maneuvers were deemed adequate or excellent to predict fluid responsiveness in spontaneous breathing patients without ventilatory support and 60% (3/5) were deemed excellent in mechanically ventilated patients in a spontaneous mode. Moreover, approximately half of the patients included in this study were not fluid responsive. This finding reinforces the importance of assessing fluid responsiveness in critically ill patients prior to intravascular volume expansion, thus avoiding unnecessary exposure to additional fluids.In patients with an invasive arterial line in place, dynamic parameters such as ∆PP in association with a maneuver that magnifies cyclic changes in intrathoracic pressures, i.e., deep inspiration or forced inspiratory breathing, represent important tools to assess fluid responsiveness continuously and with minimal inter-rater variability. , 20 EchoReversible and noninvasive maneuvers that magnify cyclic changes in intrathoracic pressures and on transpulmonary pressure, such as Valsalva or deep inspiration maneuver, in association with ∆PP or echocardiographic measurements, improve the accuracy of the maneuvers without adverse effects, allowing clinicians at the bedside to assess preload dependency , 19. NevFurthermore, transforming a continuous diagnostic index, such as ∆PP and ∆SV, into binary variables  represents an important limitation of all methods to assess fluid responsiveness . The decOur study has limitations. First, it is important to emphasize that the results of this systematic review should be interpreted in the context of the included studies. Furthermore, studies with small sample size, carried out in different clinical scenarios and with a heterogeneous methodology, were included in this systematic review. Finally, systematic reviews are subject to publication bias, which may exaggerate the conclusion of the study if publication is related to the strengths of the results.In conclusion, our systematic review suggests that regardless of the limitations of each maneuver, fluid responsiveness could be assessed in spontaneously breathing patients. Further research with adequate sample size and power are necessary to confirm the real accuracy of the different methods available to assess fluid responsiveness in this population of critically ill patients.Additional file 1. A pdf file containing quality of each study was evaluated by the Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS), a receiver operating characteristic curve of methods to assess fluid responsiveness in spontaneous breathing patients without any ventilatory support and in mechanically ventilated patients during a spontaneous mode.


Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients.A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients.P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/μl identified two distinct prognostic groups . Patients with cfDNA ≥89 pg/μl had shorter OS  and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden . In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(μl*cm3), P = 0.024).Baseline cfDNA concentration correlated with pre-treatment tumour burden  in plasma was performed in metastatic melanoma patients. Paired blood collections and scan assessments were studied. CfDNA was measured by means of droplet digital Polymerase Chain Reaction and tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients.•Baseline plasma cfDNA level correlates to tumour volume.•Plasma cfDNA level and is a surrogate biomarker for tumour burden and tumour burden changes after treatment.•Plasma cfDNA level is a prognostic marker for survival in metastatic melanoma patients. Key Message: Plasma cfDNA level correlates to tumour volume and is a surrogate biomarker for tumour burden and a prognostic marker for survival in metastatic melanoma patients.1Circulating cell-free DNA (cfDNA) is short fragment  double stranded DNA that is present in blood and other body fluids One explanation for the current lack of evidence to directly correlate tumour burden and cfDNA levels is that precise evaluations of tumour burden are not routinely performed. This is because assessing tumour burden in individual patients is demanding and requires time-consuming procedures to measure all metastatic lesions. Metastatic load in melanoma is considered an important prognostic and predictive factor in melanoma and surrogate biomarkers are currently used for clinical purposes, including the number of metastatic sites and Response Evaluation Criteria in Solid Tumours (RECIST) marker lesion measurement One of the most powerful uses of cfDNA is related to the identification of tumour-specific mutations that are derived from the cancer cells. The analysis of this circulating tumour DNA (ctDNA) allows application of liquid biopsies for personalised strategies In the present study, we examined the relationship between cfDNA, ctDNA and tumour burden in patients with metastatic melanoma. We used computed tomography (CT) and/or magnetic resonance imaging (MRI) scans to determine the total tumour burden in the patients and then compared this to cfDNA levels. Intriguingly, we did not find a correlation between ctDNA and cfDNA, but did find a correlation between cfDNA and tumour burden. Our study shows the potential of cfDNA as biomarker of tumour burden in metastatic melanoma patients, and we show that cfDNA is a biomarker for prognosis and response to treatment.22.1A prospective longitudinal biomarker cohort study was performed in collaboration between Cancer Research UK Manchester Institute and The Christie NHS Foundation Trust. Ethical approval was granted by the Manchester Cancer Research Centre (MCRC) Biobank Access Committee (Protocol number 13RIMA01). All patients gave written informed consent. Inclusion criteria were the diagnosis of metastatic melanoma, patients naïve for systemic oncological treatments or with an interval from therapy (in the adjuvant or metastatic setting) of at least 2 years, to be longitudinally followed up during treatment. Patients were studied with paired blood collections and scan assessments performed before treatment initiation and at treatment response evaluation (at 12–16 weeks).2.2Tumour burden estimation was performed with computed tomography (CT), magnetic resonance imaging (MRI) or positron-emission tomography coupled with CT (PET-CT) (slide thickness 3 mm). Scans were evaluated by a radiologist as per RECIST 1.1 and then independently reviewed for metastases volume analysis; Picture Archiving and Communication System Centricity Enterprise Web Viewer ) was used. Tumour burden was assessed as the sum of all three-dimensional metastatic lesions volumes , plasma was separated and frozen within 4 h of phlebotomy. DNA was extracted from plasma using QIAamp Circulating Nucleic Acid Kits (Qiagen). CfDNA was quantified by means of measurement of a reference gene, RNAseP, by quantitative polymerase chain reaction (PCR) using the TaqMan RNAseP Assay (Life Technologies); in 33 patients, ctDNA was additionally quantified by means of droplet digital PCR Bio-Rad QX200 targeted to a specific mutation in melanoma cells and expressed as variant allele frequency (VAF), the fraction of mutant droplets among the total number of mutant and wild-type droplets, as previously described 2.4P = 0.425, where a P value < 0.05 would reject the hypothesis of normality; 2 = 0.35).After checking the assumptions of normality distribution  was used to identify the cut off of baseline cfDNA concentration for overall survival less than one year.Kaplan–Meier method was used for survival analysis; groups were compared with log-rank test. Analysis of HR was performed with multivariate Cox regression and ANOVA to determine the significance level; proportional hazard hypothesis was verified by Schoenfeld residuals test resulting N = 38).Enrolment continued to fulfillment of sample size for all study parts. All patients were included in the prognostic study. 43 patients were included in the baseline biomarkers study; patients with paired assessments at baseline and first re-assessment were included in the biomarker study for tumour burden and treatment response if the treatment was not stopped or changed in the meantime, for example because of clinical progression or toxicity, .3BRAF, NRAS, KIT) in this patient population, and the treatment modalities administered reflect the standard of care for patients at that time. The median progression free survival was 4.8 months (range 1.1–15.3) and the median overall survival was 17.3 months (range 2.9–28.3)   with a m0.85 cm3 , Table 2P < 0.001) between baseline plasma total cfDNA and pre-treatment tumour burden (P = 0.339) (P = 0.386) , but not glomerular filtration rate or white blood cell count (P = 0.135 and P = 0.080 respectively)  B or WBC = 0.386) C. MultipP = 0.002). Note, the correlation was consistent across all treatment types  and observed a significant correlation , aggregated central quartiles  and upper quartile (≥262.9 pg/μL N = 11), including all 43 patients in this analysis. We found that overall survival differed if cfDNA was low (22.7 months), intermediate (10.4 months) or high    . To faci= 0.032) . Above t= 0.014) B.Fig. 3PP = 0.009) but LDH was not  = 1.001 (1–1.002); 0.277). We then performed a multivariate Cox regression analysis using cfDNA and LDH as categorical variables  and cfDNA was confirmed to be a better prognostic factor  (P = 0.213).Next, we performed multivariate Cox regression analysis to determine if cfDNA or LDH is the better prognostic factor in our patients. Firstly we analyzed cfDNA and LDH as continuous covariates without bias produced by grouping and found that cfDNA was significantly associated with the hazard of death  = 1.002 [1.001–1.004]; = 0.004) , than LD3) as the optimal cut-off of the shedding coefficient for survival shorter than one year (3]) had worse prognosis than those with a low cfDNA shedding coefficient , with a median overall survival of 11.6 months and 27.9 months respectively (P = 0.024)  impacted on prognosis. To assess if patients with similar tumour burden had a worse outcome if their baseline plasma cfDNA level was relatively higher. We identified 8 pg/(μL*cmone year  and obse= 0.024) C.BRAF, NRAS or KIT mutations, and also in tumours that were wild type for all of the three genes, but did not find any correlation between plasma total cfDNA concentration and mutation group for these therapeutically important driver oncogenes    A. In add= 0.162) B.Fig. 4P4In this study we demonstrated that there is a correlation between plasma total cfDNA and tumour burden in melanoma patients. By longitudinal sampling, we further showed that the correlation between cfDNA and tumour burden reflected the changes in tumour volumes that occurred with therapeutic intervention. Critically, we showed that baseline levels of total cfDNA were prognostic for OS, and that patients whose tumours released higher concentrations of cfDNA per unit volume had a worse prognosis. Thus, we concluded that plasma total cfDNA is a reliable surrogate biomarker for tumour burden and for changes that occur in tumour burden during treatment. We also showed that in a patient who presented only brain lesions, total cfDNA reflected response to treatment, demonstrating that metastatic sites in the central nervous system also contribute to total ctDNA in the plasma.Intriguingly, we did not observe a correlation between total cfDNA and individual mutant driver oncogene DNA levels (VAF) in the plasma. Notably, tumour mass visualized by radiological investigations contains not only cancer cells carrying specific driver mutations, but also a vast array of stromal and inflammatory cells that do not harbour those specific mutations. Both cell populations are in constant turnover and are likely to contribute disproportionally to the cfDNA in the blood. Moreover, the proportion of cancer cells to stromal cells within individual tumours will be different, further affecting the amount of specific mutant DNA within the total ctDNA for an individual patient. Furthermore, there is considerable clonal variation between the cancer cells within an individual tumour, and the evolutionary selective pressure imposed by treatment and immuno-surveillance can increase or decrease the cancer cell clonality within disease localization, again affecting the proportion of specific mutant to total ctDNA in the blood.One of the limits of the present study is the relatively small sample size, allowing the analysis of only two covariates in the prognostic study. A larger number of patients would be needed to include more prognostic factors for melanoma. Furthermore, treatment options for melanoma continue to improve over time, and whether our findings remain valid with emerging treatment options is unknown.LDH is a well-established prognostic factor in advanced melanoma, though why this is, is not clearly understood. LDH level is routinely considered when advising on treatment options and likely outcome, and has maintained its relevance in the revised AJCC Staging System for melanoma 5In conclusion we show that cfDNA should be considered as a prognostic factor and a biomarker that could be used as a surrogate for tumour burden and prognosis with treatment in melanoma patients.Wellcome Trust [100282/Z/12/Z] and the Cancer Research UK Manchester Institute [C5759/A12328]. SV was supported by the ESMO Clinical Research Fellowship with the aid of a grant from Novartis. Any views, opinions, findings, conclusions, or recommendations expressed in this material are those solely of the author(s) and do not necessarily reflect those of the Wellcome Trust, the Cancer Research UK Manchester Institute, ESMO or Novartis.This work was supported by the The authors do not have any conflicts of interest.