Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive.In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most.We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques  on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms.We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims. Introduction of single cell analysis led to paradigm shifts in almost all fields of biology and medical sciences as it allows for an accurate representation of the cell-to-cell heterogeneity instead an average measure of an entire cell population . In cancCTCs are tumor cells that have separated from primary tumor or current metastases and have infiltrated the systemic blood circulation . QuantifIndividual CTCs can be investigated using a combination of whole genome amplification (WGA) and next generation sequencing (NGS) to determine copy number aberrations (CNAs) and gene mutations. However, single cell genomics is associated with certain technical challenges, such as introduction of WGA- and NGS-associated errors. Different technologies for WGA and NGS are currently available but their effectiveness in combination is currently unknown, as well as influence of material preservation on downstream analysis. Suitability of a certain WGA-NGS combination for a particular downstream analysis should be extensively investigated in order to establish a powerful and reliable tool for single cell genomics.WGA is required for molecular profiling of CTCs since a single cell does not contain enough DNA for direct biomolecular investigation. WGA can be performed by different techniques, such as PCR-based, multiple-displacement amplification (MDA)-based, and a combination of MDA pre-amplification and PCR-amplification. Unlike exponential gain in the first two WGA methods, combined MDA-PCR provides quasi-linear amplification –7. The aAn important factor influencing WGA is material preservation, in particular blood preservation. EDTA-preserved blood requires processing as soon as possible . CirculaDownstream analysis of amplified DNA can be performed by massive parallel sequencing using NGS in order to identify SNPs (single nucleotide polymorphisms), indels (insertions-deletions), loss of heterozygosity, structural variations, and CNAs.Single cell analysis of genomic aberrations by array-CGH is hampered. The necessity of the pre-selected targets’ analysis on template, obtained by random and incomplete genome amplification during WGA –14 resulExisting NGS platforms differ by library preparation and signal detection methods. Illumina's HiSeq machines exploit sequencing-by-synthesis approach , 18. CurIn this study, we evaluated different protocols, including different methods of preservation, WGA and sequencing to identify an optimal process for single cell sequencing. We compared our findings against unamplified DNA from bulk cell pellets to quantitatively define the impact of different protocols on single cell sequencing. In order to determine the impact of WGA method, we evaluated three different commercially available WGA kits and measured DNA quality and yield. In order to investigate the performance and compatibility of NGS platforms with whole exome sequencing of WGA single cell DNA, we compared the detection of genomic variants  from single SK-BR-3 cells spiked and re-captured from EDTA-preserved blood. In order to investigate the influence of material fixatives, we evaluated detection of genomic variants from single SK-BR-3 cells spiked and re-captured from EDTA-preserved vs. CellSave preserved blood. Next, we evaluated the limit of detection and consistency of genomic variant detection with increasing amounts of starting material (i.e. increasing numbers of pooled cells). Finally, we demonstrate proof of principle by evaluating genomic variants detected from CTCs collected from breast cancer patients. Our findings indicate the technical and biological variability in genomic variant detection from single cell sequencing and suggest optimized protocols dependent on starting material and objective .GAPDH gene, of the tested WGA kits are presented in Table Three WGA kits  were used to amplify single cell samples of 4 groups: A) 10 individual SK-BR-3 cells spiked and picked from EDTA-preserved blood; B) 10 individual SK-BR-3 cells spiked and picked from CellSave-preserved blood; C) 10 single SK-BR-3 cells picked from FFPE SK-BR-3 cells; and D) 10 individual CTCs picked from EDTA-collected blood of breast cancer patients. In total, 120 single cells were individually processed by WGA. DNA yield and success rate, as measured by multiplex PCR of GAPDH multiplex-PCR demonstrated a 100% success rate for the experiment with EDTA tubes, CellSave tubes, and FFPE experiments, whereas the amplification of the patients’ CTCs demonstrated a success of 70% for CTCs (Table PCR-based WGA (Ampli1 kit) demonstrated an average DNA yield of 7.07 μg, 5.86 μg, 6.74 μg and 4.69 μg for the 4 different 10-sample sets respectively with the average DNA yield 6.09 μg Table . The GAPCs Table . The aveGenomic variants detected from single cells recovered from EDTA-preserved blood were analyzed to compare sequencing platforms and WGA methods. Variants detected in single cell analyses were compared to variants detected in bulk cell pellets without WGA as a gold standard. We report sequencing quality statistics (e.g. read depth), the total number of single nucleotide variants (SNVs) and indels detected, including both previously reported SNPs and indels and novel variants, the allelic dropout rate and the sensitivity and positive predictive value (PPV) of detection compared against unamplified DNA as metrics to compare different protocols. Sequencing with HiSeq2000 platform produced more reads and provided higher depth and breadth of target base coverage, higher mapping rates, and lower duplicate rates compared to IonProton. Comparing the applied WGA procedures, the highest numbers of clean reads, mapping and duplicate rates were observed for MDA-based WGA kit. The complete characteristics of NGS data are presented in r < 0.7), strong (r > 0.8) and weak (r < 0.3) correlation with genomic DNA, respectively . The highest number of novel SNPs was observed for the cell amplified with combined MDA-PCR technique and HiSeq2000-sequenced (4711 SNPs). Among the samples sequenced with the same NGS platform, more known indels were identified in samples amplified with PCR-based WGA . The fraction of known indels was the highest for PCR-based WGA-HiSeq2000 analysis (15.3%) Table . CorrelaAllelic dropout (ADO) rates demonstrated dependence on both WGA and sequencing platform Table . ADO ratTo investigate the detection limit with increasing amount of starting material for WGA, as well as the influence of CellSave preservative on WGA and NGS performance we analyzed duplicates of 1, 3, 5, and 10 pooled SK-BR-3 cells amplified with PCR-based and MDA-PCR combined WGA techniques and sequenced on Illumina's Hiseq2000. The whole obtained data is presented in Comparison between single cells obtained from EDTA- and CellSave-collected blood revealed lower numbers of the total and known identified SNPs and indels, and higher number of novel SNPs and indels in cells from CellSave-preserved blood. Sensitivity of SNP and indel calling was lower for single cells from CellSave tubes in comparison to single cells obtained from EDTA-preserved blood Table . The oveAnalyses of pooled cells demonstrated that the numbers of the identified total SNPs/mutations increased with increasing number of pooled cells for experiments with PCR-based WGA and decreased for experiments with MDA-PCR WGA technique, statistically significant for MDA-PCR experiments only. In contrast, the numbers of the identified known SNPs/mutations increased with increasing number of pooled cells for both WGA kits, however statistically significant between different groups of pooled cells for MDA-PCR only. Moreover, the rate of change of detection of total and known SNPs with increasing number of cells was found to be different with PCR-based and MDA-PCR combined WGA techniques. PCR-based WGA technique appears to have more variability in performance as indicated by the variance in the number of total SNPs detected Figure . VariancSensitivity of SNP and indel analyses increased with increasing number of pooled cells. The effect was statistically significant for MDA-PCR, but not PCR-based WGA as indicated by significant correlation. However, the differences in kits’ performance were not significantly different by these metrics Figure .Samples analyzed with either kit showed similar ADO rates  . With eaCorrelation between CNA profiles of genomic DNA and analyzed samples increased along with the number of pooled cells for both WGA kits, however, this effect was not statistically significant. There was no significant difference in the rate of change of performance between the two kits Figure .The obtained results suggest that the PCR-based WGA technique is superior to the MDA-PCR technique for SNP and indel analysis in single cells Table . NotablyCHEK2, PRAME, and KIT were present in both CTCs, mutation in gene FGFR2 was detected in CTC-1 only and in gene TP53 – in CTC-2 only , has certain limitations. Site-specific digestion of template DNA prior to PCR by the MseI enzyme  results Commercially available exome enrichment kits have not been optimized for WGA products. The usage of fragmented WGA DNA as template might drastically reduce capturing efficacy. Moreover, a significant fraction of template DNA can be nonspecifically enriched outside target regions, varying from kit to kit –27, causAlthough samples amplified with MDA-based WGA technique (REPLI-g kit) demonstrated the highest DNA yield from a single cell, the quality of the obtained DNA was remarkably low and insufficient for appropriate SNP/mutation, indel, and CNA analyses. Based on our experience and observations of de Bourcy et al.  and BergSingle cells from EDTA-collected blood demonstrated higher sensitivity for SNP/mutation and indel analyses, than single cells from CellSave-preserved blood. Since EDTA-collected blood requires timely processing after collection , CellSavThe concordance of the identified SNPs/mutations in 1, 3, 5 and 10 cells from CellSave-preserved blood with the reference was invariant to the WGA technique used and improved with increasing number of pooled cells. For MDA-PCR amplified DNA, the sensitivity r ≥ 0.8) with unamplified DNA (Table r = 0.1 and r = 0.7) (Table r < 0.7)  Table . The exp.8 Table .A recent study from our lab has demonstrated genetic heterogeneity within a cancer cell line upon sequencing single cells . TherefoFGFR2 and TP53 genes might indicate clonal evolution of the tumor. Further single cell genomic research and improved WGA methods may enable us to investigate cancer evolution during tumor development and under therapy pressure leading to treatment resistance using CTC sequencing.Sequencing CTCs from cancer patients has been suggested as a “liquid biopsy” that could be used to study tumor heterogeneity and find therapy associated markers . In our First, we investigated performance of 3 WGA kits, representing 3 WGA methods, in 4 groups of source material, differing by origin and preservation method. The 4 sources of material included: A) individual SK-BR-3 cells obtained from EDTA-preserved blood; B) individual SK-BR-3 cells obtained from CellSave-preserved blood; C) single SK-BR-3 cells picked from FFPE SK-BR-3 cells; and D) individual CTCs obtained from EDTA-preserved blood from a breast cancer patient.WGA was performed using PCR-based Ampli1 , combined MDA-PCR PicoPlex , and MDA-based REPLI-g  WGA kits according to the manufacturers’ recommendations. After DNA yield and quality per WGA kit were estimated, DNA of single cells from each WGA group was used for whole exome NGS on 2 platforms. Briefly, 3 SK-BR-3 cells, obtained from EDTA-preserved blood and amplified with Ampli1, PicoPlex, and REPLI-g kit, were analyzed with both HiSeq200 and IonProton platforms.Based on results obtained from initial pilot experiments, the IonProton platform and Repli-G WGA kit were excluded from further experiments. The second round of experiments included WGA of single and pooled cells in duplicates and NGS of obtained DNA in order to investigate the performance and the limit of detection with increasing amounts of material. Duplicates of 1, 3, 5, and 10 pooled SK-BR-3 cells obtained from CellSave-preserved blood and amplified with Ampli1 and PicoPlex kits were sequenced on HiSeq2000.Subsequently, a proof of principle experiment was performed on 2 individual CTCs obtained from EDTA-collected blood of a breast cancer patient. The cells were individually amplified with PicoPlex WGA kit and sequenced on HiSeq2000 . In totaThe breast cancer cell line SK-BR-3 was acquired from ATCC and cultivated under prescribed conditions. The cells were harvested using trypsin/EDTA , washed and resuspended in PBS  for further experiments. Genomic DNA was extracted using the Blood&Cell Culture DNA Mini Kit . The same cell line was previously formalin-fixed, paraffin-embedded, and stored for over 3 years to simulate archival material.Blood from healthy individuals and metastatic breast cancer patients was obtained from the Department of Transfusion Medicine and Department of Gynecology at the University Medical Center Hamburg-Eppendorf, respectively. All study participants gave written informed consent. The examination of blood from breast cancer patients was approved by the local ethics review board Aerztekammer Hamburg (OB/V/03). Breast cancer patients’ blood was sampled in EDTA collection tubes . Blood from healthy donors was collected either in EDTA or CellSave tubes  and spiked with SK-BR-3 cells to simulate CTCs.The patient who received her CTCs analyzed by NGS was diagnosed with primary metastatic breast cancer in 2012 at the age of 69 years. The primary tumor was strongly positive for the ER (> 80% of the cells with nuclear positivity) and HER2-negative. Metastatic lesions were detected in the lung and in the spine. Chemotherapy with Paclitaxel weekly was started, however after the first week the therapy was switched to the endocrine therapy with Letrozole based on the patient's wish.Blood samples collected in EDTA tubes were processed within 2 hours. Blood samples collected in CellSave tubes were stored for 24–30 hours at room temperature before being processed. Mononuclear cells from both cancer patients’ and healthy donors’ blood spiked with SK-BR-3 cells were enriched by Ficoll density gradient centrifugation as previously described , fixed wSingle cells were picked by micromanipulation  with the use of glass capillaries, allowing for the isolation of individual cells. Each individual cell was transferred in 1μl of PBS into the cap of a 0.2 ml PCR tube and stored at −80°C overnight. In order to obtain samples with pooled 3, 5 and 10 cells, every single cell was picked individually and transferred into a 0.2 ml PCR tube without touching the liquid already present in the tube, until the desired number of cells was riched.FFPE SK-BR-3 material was cut in 5 μm thin sections and preprocessed as described before , 37. CroWGA was performed according to the manufacturers’ recommendations using 3 different kits: PCR-based Ampli1 , combined MDA-PCR PicoPlex , and MDA-based REPLI-g  WGA kits. The WGA products after Ampli1 and PicoPlex underwent cleanup with NucleoSpin Gel and PCR Clean-up kit . REPLI-g WGA products were cleaned according to the QIAGEN recommendations with ethanol for the FFPE samples and spin columns  for the blood samples.GAPDH gene as described elsewhere [DNA concentration was measured with a Nanodrop1000 . Nanodrop was calibrated according to the manufacturer's recommendations using the CF-1 Calibration Fluid . The quality control of the WGA products was assessed by a multiplex PCR of the lsewhere  with minhttp://www.ebi.ac.uk/ena/data/view/PRJEB11307Amplified DNA was investigated with whole exome sequencing on HiSeq2000 and IonProton platforms, unamplified DNA of the SK-BR-3 cells was sequenced with HiSeq2000. Sequence data are available atRaw data from the Ampli1- and PicoPlex-amplified samples underwent adapter clipping. PCR adapters of the Ampli1 kit are ligated to DNA sticky ends after MseI restriction of T^TAA sites , therefoFurther data analysis was done according to the GATK Best Practices recommendations , 40, detSensitivity, specificity, positive and negative predictive values of the variant calling analysis were evaluated based on the schema presented on Venn diagrams were created with the used of BioVenn web application .Allelic dropout (ADO) rate was calculated as follows: heterozygous SNPs in sample, present in reference, divided by their sum with homozygous SNPs in sample, present in reference as heterozygous SNPs.CNAs were evaluated using Control-FREEC  with a wWe comprehensively tested the effectiveness of WGA of single cells for exome sequencing by NGS. As an aspect of testing, we evaluated 3 WGA techniques, 2 NGS platforms, and the influence of material fixation for long term preservation. Although MDA-based WGA technique (REPLI-g kit) yielded the highest DNA amount, DNA quality was not adequate for SNP/mutation, indel, and CNA analysis.PCR-based WGA technique (Ampli1 kit) combined with Illumina's HiSeq2000 platform demonstrated the best concordance with unamplified DNA for SNP/mutation and indel calling, both for EDTA- and CellSave-preserved cells with ADO rates 9–79%, mostly dependent on the amount of starting material. However, performance of the MDA-PCR combining WGA technique (PicoPlex kit) significantly improves with the number of pooled cells (increasing amount of input DNA), whereas performance of the PCR-based WGA technique did not significantly improve with increase of input material in any case.The CNA profiles produced with MDA-PCR combining WGA technique on both HiSeq2000 and IonProton, independent of blood preservative, resembled unamplified DNA the most. Performance of CNA analysis of MDA-PCR combining WGA technique is not affected by input amount.Our study shows the feasibility of genomic analysis of single cells isolated from differently preserved material, enabling advanced diagnostics such as on CTCs during cancer treatment for companion diagnostics.


In this minireview, we discuss how cGAS–STING and RIG-I–MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways are currently being targeted for clinical application in oncology.The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8 The innate immune system provides the first line of defense against pathogen infection. It relies on a small set of germ line-encoded sensors named pattern recognition receptors (PRRs), which are deputized to detection of pathogen-associated molecular patterns (PAMPs) and danger damage-associated molecular patterns (DAMPs) signals.Nucleic acid (NA)-sensing is an essential mechanism of the innate immunity that utilizes cytosolic receptors to detect extranuclear DNA or extracellular RNA as DAMPs signals .In mammalian cells, two paradigmatic cytosolic NA-sensing pathways are the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) and the RIG-I-like receptors (RLRs)-MAVS pathways, which are responsible for cytosolic DNA and RNA sensing, respectively , 3. The + T lymphocytes  cross-presentation in DCs. Type-I IFNs production by DCs actually represents the link between NAs sensing and effective Ags cross-presentation to CD8immunity . Type-I immunity –17. Sincimmunity , 18, autimmunity –21.Endosomal tumor-derived NAs escape into the DC cytosol through a yet not completely understood mechanism. Specific internalizing receptors such as CLEC9A and CD205 and high-mobility group box 1 protein can mediate uptake of genetic material from dying tumor cells and affect subsequent endosomal trafficking –24. LikePresence of NA sensor proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. Actually the expression of RIG-I was significantly downregulated in human hepatic carcinoma (HCC) tissues , 26.Clinical studies revealed that the expression of STING was significantly reduced in HCC tissues compared to the controls, and lower expression of STING was associated with a more advanced tumor stages and a worse survival  as well Furthermore, PRRs mediate the response to NAs in several vaccination approaches, including DNA immunization. As route of dsDNA intracellular entry, DNA immunization leads to expression of key components of the cytosolic NA-sensing pathways.+ T cells, and NK cells into the TME.Recent data showed that natural and synthetic agonists of NA-sensing pathways could trigger cell death in malignant cells and recruit immune cells, such as DCs, CD8In this minireview, we will highlight the newest insights from preclinical studies demonstrating the relevance of manipulating the cGAS-STING and RLRs-MAVS signaling pathways for cancer treatment and how these pathways are currently being targeted pharmacologically Figure .Clinical evaluation of these innate immune modulators, with agonists alone and in combination with other immunomodulatory agents demonstrates the high translational potential for cGAS-STING and RLRs-MAVS signaling pathway engagement. In Table Results from completed early phase clinical trials with human STING and RIG-I agonists showed biologic and therapeutic effects in patients, leading to combination clinical trials with checkpoint inhibitors , 43.Regulation and function of the cGAS-STING pathway has been reviewed elsewhere , 46–50, ex vivo, have been found to produce type-I IFNs  injection of cGAMP in CT26 colon adenocarcinoma-bearing mice. The antitumor activity of cGAMP relied on DC activation and CD8-priming . More re-priming  after i.Canonical cyclic-dinucleotides (CDNs), as direct agonists for STING, show a poor ability to activate human STING. Therefore, an increasing number of synthetic CDNs that potently activate all human STING variants have been designed in recent years , 58.New synthetic CDNs agonist has shown potent antitumor efficiency in various tumor models such as B16F10 melanoma, 4T1 mammary adenocarcinoma, and CT26 colon carcinoma, with regression of established tumor, metastasis rejection, and establishment of long-term immune memory .Recently, many preclinical studies draw a blueprint for the application of STING agonists in tumor therapy in the context of combination therapies.in vivo therapeutic efficacy in several models of established cancer. Antitumor activity was STING dependent and corresponded to activation of DCs and tumor antigen-specific CD8+ T cells. STINGVAX combined with PD-1 blockade induced regression of poorly immunogenic tumors that did not respond to PD-1 blockade alone (Strategies that combine STING immunotherapy with other immunomodulatory agents are being explored in mouse models. The antitumor efficacy of cGAMP administered by i.t. injection into B16.F10 tumors was enhanced when combined with anti-programmed death-1 (PD-1) and anti-cytotoxic T-lymphocyte associated-4 (CTLA-4) antibodies . In othede alone . STING ade alone , 63.The focus of STING pathway agonists for clinical use has thus far centered on their role as vaccine adjuvants and as cancer immunotherapeutic agents for treatment of solid tumors. However, induction of type-I IFNs and other inflammatory cytokines through STING pathway activation results in potent leukemia-specific immunity, culminating in impressive improvements in survival of preclinical acute myeloid leukemia models. Thus, Curran et al. provided solid rationale for clinical translation of STING agonists as immune therapy for leukemia and other hematologic cancers .in vivo activation of STING in T cells provokes type-I IFNs production and ISGs expression mimicking the response of innate cells , resulting in the adjuvant effect for the encoded antigen  resulted in enhanced tumor rejection and protection against B16 melanoma challenge .The concerted stimulation of innate immune PRRs by DNA vaccines can achieve a more potent and broader activation of the immune responses and a long-lasting protective adaptive immunity.Plasmodium falciparum, in the same plasmid backbone enhanced the antigen-specific humoral immune responses, but failed to improve cellular immune responses (Co-expressing TBK1 and the serine repeat antigen, a candidate vaccine antigen expressed in the blood-stages of esponses .Favorable safety profile and potential clinical benefit were achieved after the phase I clinical trial on the activity of CYL-02, a non-viral gene therapy product that sensitizes pancreatic cancer cells to gemcitabine, a chemotherapic acting as a STING pathway agonist, and a non-invasive biomarkers for patient selection was identified .RIG-I and MDA5-activating DNA vaccines can elicit apoptotic and immunostimulatory effects and, thus, could induce growth inhibition or apoptosis of multiple types of cancer cells. Plasmid vector backbones expressing composite immunostimulatory RNAs that act as synergic RIG-I agonists lead to type-I IFNs production , 94.The spatiotemporal orchestration of innate stimulation with antigen cross-presentation in APCs represents a crucial challenge in reaching a strong tumor specific T-cell response, which in turn is crucial for cancer immunotherapy.Several studies suggest that cytosolic NA-sensing plays a central role in inducing and bridging innate immunity and adaptive immune responses against tumors and that triggering innate immune system contribute to counteract tumor-induced immunosuppression. Employment of RIG-I/MDA5 and cGAS-STING agonists could represent a novel strategy for cancer immunotherapy.The role of cGAS-STING and RLRs-MAVS pathways in tumor immunity remains complex and numerous questions still remain unanswered.Noteworthy, some studies suggest that STING activation may induce a suppressive TME, contributing to tumor growth and metastasis and that STING agonists may not be effective against all tumors, particularly those with tolerogenic responses to DNA and low tumor antigenicity , 96. SenIn head and neck squamous cell carcinoma, dose-dependent activation of RIG-I resulted in divergent effects on cancer cell proliferation. Actually, low dose of dsRNA promoted NF-kB- and Akt-dependent cell proliferation and metastasis .The functional consequences of the cGAS-STING and RIG-I-MAVS pathways regulation/activation in different cells within the TME require deeper characterization. Targeting multiple pathways may be required for efficacious therapeutic responses in some patients and the crosstalk between RIG-I and STING pathways through direct interactions between downstream signaling components may amplify the innate response.The complex role of STING and RIG-I signaling in cancer underlines how innate immune pathways in the TME alter tumorigenesis in distinct tumors, with effects in designing efficacious immunotherapy trials.SI: searched for literature articles, conceived and wrote the manuscript, conceived the figure, and revised and approved the final version of the manuscript. DF: performed search for clinical trials, collected and analyzed clinical trials data, prepared the tables, conceived and prepared the figure, contributed to the manuscript preparation and editing, and revised and approved the final version of the manuscript. MR: searched for literature articles, conceived and wrote the manuscript, edited the manuscript, and revised and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Experiencing multiple types of victimization (poly-victimization) during adolescence is associated with onset of psychotic experiences. However, many poly-victimized adolescents will not develop such subclinical phenomena and the factors that protect them are unknown. This study investigated whether individual, family, or community-level characteristics were associated with an absence of psychotic experiences amongst poly-victimized adolescents.Participants were from the Environmental Risk (E-Risk) Longitudinal Twin Study, a nationally-representative cohort of 2232 UK-born twins. Exposure to seven different types of victimization between ages 12–18 was ascertained using a modified Juvenile Victimization Questionnaire at age 18. Adolescents were also interviewed about psychotic experiences at age 18. Protective factors were measured at ages 12 and 18.Exposure to poly-victimization during adolescence was associated with age-18 psychotic experiences , but more than a third of the poly-victimized adolescents reported having no psychotic experiences (40.1%). Greater social support was found to be protective against adolescent psychotic experiences amongst those exposed to poly-victimization. Notably, social support was also generally associated with a reduced likelihood of age-18 psychotic experiences in the whole sample .Increasing social support from friends and family appears to be an important area for preventive interventions targeting adolescent psychotic experiences. Such prevention efforts would be most effectively targeted at poly-victimized adolescents who are at high-risk of developing psychotic phenomena.


Virtual-assisted lung mapping (VAL-MAP) is a novel marking technique to assist sub-lobar resection of small hardly palpable lung tumors. Here, we present the first case of synchronous bilateral multiple lung adenocarcinomas that were successfully resected with VAL-MAP navigation.A 73-year-old female with multiple nodules  was referred. Complete resection was achieved with left lower lobectomy in combination with sub-lobar resections  in which resection lines with securing adequate margins were determined with VAL-MAP navigation.VAL-MAP is useful in sub-lobar resections including complex segmentectomy for multiple lung adenocarcinomas. Recent prevalence of lung cancer screening with computed tomography (CT) has contributed to early detection of multiple lung cancer , and 122Virtual-assisted lung mapping (VAL-MAP) is a novel bronchoscopic marking technique to assist sub-lobar resections of small hardly palpable lung tumors , 4, and The VAL-MAP navigation was approved by the Ethics Committee of the University of Occupational and Environmental Health, Japan (H26–003), and was performed after written informed consent from the patient.A 72-year-old female never-smoker was referred for resection of bilateral multiple lung nodules revealed on chest CT Fig. . As eachWe achieved complete resection of all nodules with two-staged thoracoscopic operations. First, we performed right-sided operation consisting of S3 segmentectomy and complex segmentectomy of S6 plus S8 with VAL-MAP navigation. Marking spots were designed to navigate ideal resection lines in segmentectomies beyond anatomical inter-segmental planes with adequate resection margins. For example, two VAL-MAP markings, one indicating the place of S6 tumor and another indicating the “turning point” during lung stapling lines, were made for S6+S8 segmentectomy Fig. . During We presented the first case of synchronous bilateral adenocarcinomas that were completely resected with lobectomy in combination with sub-lobar resections with VAL-MAP navigation. VAL-MAP is useful not only in wedge resection to identify small hardly palpable lung tumors with adequate resection margin but also in segmentectomy to determine resection lines with securing adequate margins. As compared with previous methods to determine resection margins in segmentectomy such as the usage of infiltration-deflation lines and injection of indocyanine green into selected pulmonary artery, VAL-MAP has an advantage of easy performing under thoracoscopic surgery without any special instrument. In addition, there is no need for lung inflation that may be easily performed by anesthetists and surgeons who are not familiar with infiltration-deflation techniques. More importantly, VAL-MAP has another advantage of capability to flexibly determine ideal resection lines beyond anatomical inter-segmental planes to provide adequate resection margins, as shown in the present case for complex segmentectomy of S6 plus S8 as well as for simple segmentectomy of S3. Despite these advantages, VAL-MAP navigation have several limitations such as difficulty in securing deep resection margins . More soVAL-MAP is useful in sub-lobar resections for multiple lung adenocarcinomas.


MicroRNA-874 (miR-874) is downregulated in several human cancers and has been suggested to be a tumor suppressor gene. However, the molecular mechanism of miR-874 downregulation in breast cancer has not been well elucidated. Here we aimed to study the aberrant hyper-methylation of CpG sites with the utility of miR-874 downreregulation in breast cancer and evaluate the clinical function of miR-874 as a prognostic marker. The miR-874 expressions in cells and tissues of two breast cancer lines were measured by real-time PCR. The DNA methylation status of the miR-874 promoter region in 19 pairs of breast cancer and adjacent normal samples was analyzed with Sequenom EpiTYPER MassArray. To evaluate whether miR-874 is a potential prognostic marker in breast cancer, we also explored the clinical long-time follow-up records from The Cancer Genome Atlas (TCGA). We found miR-874 expression was downregulated in 47 pairs of breast cancer tissues. Moreover, univariate and multivariate analysis revealed miR-874 expression may be a prognostic biomarker of overall survival in breast cancer patients. Preconditioning with 5-Aza-CdR in two cell lines elevated miR-874 expressions. The data from Sequenom EpiTYPER MassArray showed that DNA methylation of the promoter region of miR-874 was upregulated and accompanied by decreased miR-874 expression, which was further confirmed by TCGA. After comprehensive considerations, we think miR-874, which might be served as a prognostic biomarker, is mediated by DNA methylation. Breast cancer is one of the most commonly diagnosed cancers and a major cause of cancer-related death in women worldwide . The exiMicroRNAs (miRNAs) are short highly-conserved small non-coding RNA molecules of 19 to 25 nucleotides that regulate post-transcriptional gene expressions. By targeting complementary binding sites within the 3′-untranslated region (3′-UTR) of target messenger RNAs, they impair or inhibit translation and promote degradation –6. MiRNARecently, several reports show that miRNA-874 (miR-874) expression is downregulated and functions as a tumor suppressor in several cancers, including non-small cell lung cancer (NSCLC) , breast Accumulating evidences demonstrate that the dysregulation of miRNA subsets is attributed to genetic and epigenetic alterations , 21. AmoIt is demonstrated that miR-874 expression is correlated with tumor size, tumor/node/metastasis (TNM) staging and lymph node metastasis . Given iP < 0.01). Accordingly, we used the median miR-874 expression as a cutoff point and divided the 47 breast cancer patients into a high-expression group (n = 24) and a low-expression group (n = 23). Furthermore, we find miR-874 expression is associated with pathological differentiation, TNM staging and lymph node metastasis in breast cancer tissues. As depicted in Figure t-test . Considering the small sample size and some samples without long-time follow-up records, we also applied the clinical parameters with long-time follow-up records to investigate the association between miR-874 expression and OS from an external breast cancer cohort in TCGA (n = 1089). As depicted in Table P < 0.05), which proves the database is convictive and appropriate compared to the above results. Moreover, patients with high miR-874 expression exhibit better OS   shows that miR-874 expressions are downregulated in 47 pairs of breast cancer samples and matched paranormal tissues Figure . The miRroup n = 4 and a l1) Table . Collecthttps://genome-cancer.ucsc.edu/) show that the DNA methylation levels in the miR-874 promoter region are remarkably elevated in breast cancer tissues compared to paratumor tissues  show a significant negative correlation between miR-874 expression and methylation levels in its 300-upstream region . Comprehensively, these data suggest that DNA hyper-methylation in the upstream region of miR-874 might play a significant role in miR-874 expression downregulation in breast cancer.To further explore the upstream molecular mechanism of down-regulated miR-874 expression in breast cancer, we assessed the DNA methylation status of the miR-874 promoter region in both breast cancer cells and tissues. As depicted in Figure in vivo [Increasing evidence indicates that miRNAs could act as oncogenes or tumor suppressors and are involved in both biological and pathological processes in several cancers, including proliferation, angiogenesis and metastasisI , 30. Morin vivo . TherefoAs reported, miR-874 expression is remarkably down-regulated and miR-874 may function as an oncogenic or tumor suppressor in various malignancies. Several studies partially substantiated the downstream mechanisms of miR-874 altering cell processes. However, the upstream mechanism underlying how miR-874 is down- regulated in breast cancer still has not been well elucidated. Recent studies suggest many tumor-suppressor miRNAs are downregulated due to promoter hyper- methylation of the CpG islands in several cancers , 35. To After comprehensive considerations, we think miR-874, which might serve as a prognostic biomarker or potential therapeutic strategy, is mediated by DNA methylation in breast cancer patients.Breast cancer specimens and their matched normal breast tissues were collected from patients treated at Jiangsu Cancer Hospital, Affiliated Hospital of Nanjing Medical University between 2014 and 2016. The specimens were snap- frozen in liquid nitrogen. The study protocol was approved by the Ethics Committee of Jiangsu Cancer Hospital, and informed consent was obtained from all patients.2. After 24 hours of serum starvation, the two cell lines were separately seeded in six-well plates, allowed to attach for 3 or 5 days and treated with 0, 5 or 10 μM 5-Aza-CdR.Human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from American Type Culture Collection . Both cell lines were cultured in the Roswell Park Memorial Institute 1640 medium  supplemented with 10% fetal bovine serum  at 37°C in a humidified atmosphere with 5% COTotal RNA was extracted using an RNAsimple Total RNA kit  according to the manufacturer's instructions. The concentration and quality of the RNA were measured on a Nanodrop 2000 spectrophotometer  at the ultraviolet wavelengths of 260 and 280 nm (260/280 nm). Mature miR-874 levels were quantified using TaqMan microRNA Assay (Applied Biosystems). Expression of miRNA was analyzed using a MiR-X miRNA qRT-PCR SYBR Kit  on Roche LightCycler 480 II, with the manufacturer-provided mRQ 3′ Primer and miRNA-specific forward primer. The miRNA expressions in cells and tissues were normalized using U6 snRNA as an endogenous control. The cycle threshold (Ct) of each gene was normalized to the internal control, and the relative fold change was calculated using the ΔΔCt method in triplicate.http://MethHC.mbc.nctu.edu.tw). The discriminating methylation levels of the three CpG sites , the miR-874 expression and clinic-pathologic data in breast cancer and non-tumor tissues were analyzed on basis of the gene chip data (TCGA_BRCA_ hMethyl450) and (TCGA_BRCA_miRNA) from Cancer Browser (https://genome-cancer.ucsc.edu/).Data were downloaded from the TCGA portal and processed partly with MethHC from Institute of Bioinformatics and Systems Biology at National Chiao Tung University, Hsinchu, Taiwan .Genomic DNAs were extracted from 26 paired breast cancer specimens and matched normal tissues using a genomic DNA rapid extraction kit . The quality and quantity of DNA were evaluated by gel electrophoresis and a NanoDrop spectrophotometer , respectively, but 7 of the 26 paired tissues were not qualified. Genomic DNA from each specimen was isolated using a QIAamp DNA Mini Kit (Qiagen) and modified by bisulfite using an Epitect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. Methylation of the miR-874 promoter was quantitatively analyzed on Sequenom MassARRAY , which employs matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and RNA base-specific cleavage. Pairs of primers on basis of the reverse complemented strand of miR-874  were used to amplify base pairs -193–(+29) around miR-874. Each forward primer was added with a 10-mer tag (5′-AGG AAGAGAG-3′) and each reverse primer with a T7-promoter tag (5-CAGTAATAC GACTCACTATAG GGAGAAGGCT-3′) for transcription t-test and paired t-test were applied to evaluate methylation level of miR-874. A linear regression was performed to infer the correlation between the methylation level and expression of miR-874. The connection between miR-874 expressions and clinical parameters were analyzed by the chi2 test. Survival curves were exhibited using the Kaplan–Meier method, the log-rank test and Cox's proportional. Statistical significance was set at P < 0.05.All statistical analyses were performed using SPSS version 19.0 software . Student's


Restoration of tissue homeostasis by controlling stem cell aging is a promising therapeutic approach for geriatric disorders. The molecular mechanisms underlying age-related dysfunctions of specific types of adult tissue stem cells (TSCs) have been studied, and various microRNAs were recently reported to be involved. However, the central roles of microRNAs in stem cell aging remain unclear. Interest in this area was sparked by murine heterochronic parabiosis experiments, which demonstrated that systemic factors can restore the functions of TSCs. Age-related changes in secretion profiles, termed the senescence-associated secretory phenotype, have attracted attention, and several pro- and anti-aging factors have been identified. On the other hand, many microRNAs are linked with the age-dependent dysregulations of various physiological processes, including “stem cell aging.” This review summarizes microRNAs that appear to play common roles in stem cell aging. Overcoming age-related diseases and elongating the healthy lifespan are emerging issues for aging societies. Dysfunctions of aged tissue stem cells (TSCs) contribute to loss of tissue homeostasis, including reductions in lymphopoiesis and the long-term repopulating abilities of hematopoietic stem/progenitor cells (HSCs) , 2, the We previously identified growth differentiation factor 6  as a regenerative factor secreted by young MSCs , 22. UprmiR-17 family members play essential and pleiotropic roles in development, metabolism, diseases, tumorigenesis, and aging , 24. We lin-4, a Caenorhabditis elegans homolog of miR-125b, is a heterochronic gene and generates the temporal pattern of many cell lineages during development [lin-4 elongates lifespan, whereas loss-of mutation accelerates tissue aging and shortens it.A myeloid skewing phenotype and a decline in engraftment capability have long been recognized as age-related dysfunctions of old HSCs . miR-125elopment , and is elopment . OverexpChronic inflammation accelerates systemic aging . miR-181Recent studies have revealed that a part of microRNAs appear to play common roles in stem cell aging Table . In factSome microRNAs have similar functions in different types of TSCs. Downregulation of these specific-microRNAs induces similar age-related dysfunctions of TSCs. These microRNAs may define the “young competence” by specifying the signal pathways with suppression of their regulon, including signal mediators and transcription factors. Further investigation of the roles of the other microRNAs in stem cell aging will help to elucidate the central molecular machinery of the aging and develop the next-generation therapeutic methods for geriatric diseases.


Targeted therapeutic agents, such as inhibitors of epithelial growth factor receptor (EGFR), have transformed the management of non-small cell lung cancer (NSCLC) patients. MET-amplified NSCLC cells display resistance to EGFR-targeting agents, but are addicted to MET signaling for survival and proliferation and are sensitive to MET inhibition. However, responsive cancer cells invariably develop resistance to MET-targeted treatment.The tumor microenvironment plays a major role in resistance to anticancer therapy. We demonstrated that fibroblasts block the response of MET-amplified NSCLC cells to the MET kinase inhibitor, JNJ38877605 in an HGF-dependent manner. Thus, MET-amplified NSCLC cells become addicted to HGF upon pharmacological inhibition of MET. HGF restored phosphorylation of MET, EGFR and RON, and maintained pro-survival AKT and ERK signaling in MET-inhibited cells.We developed a small molecule inhibitor of pro-HGF activation, SRI31215, which acts as a triplex inhibitor of the pro-HGF activating proteases matriptase, hepsin and HGF activator (HGFA). SRI31215 blocked crosstalk between tumor cells and fibroblasts and overcame fibroblast-mediated resistance to MET inhibition by preventing fibroblast-mediated reactivation of AKT and ERK signaling. Structurally unrelated triplex inhibitors of matriptase, hepsin and HGFA that we developed in parallel showed similar biological activity.Our data suggest that simultaneous inhibition of HGF and MET is required to overcome resistance to MET inhibitors in MET-amplified NSCLC cells. This provides a rationale for the development of novel combination therapeutic strategies for the treatment of NSCLC patients with MET amplification. Lung cancer remains a leading cause of cancer-related deaths worldwide. Oncogenic EGFR activation, commonly observed in non-small cell lung cancer (NSCLC), drives cell proliferation and survival. Indeed, EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib have become an important therapeutic modality for the treatment of NSCLC patients with EGFR mutations –3. HowevMET amplifications have been found in ∼20% of NSCLC patients with acquired resistance to gefitinib or erlotinib , 11. METIn addition to cell-autonomous mechanisms of resistance, factors in the tumor microenvironment have been shown to block the response to therapy , 20. StrNeutralizing anti-HGF antibodies that are being tested, alone or in combination with other therapies, appear to be well tolerated in clinical trials , 28. Thein vivo [HGF is secreted as a single chain inactive precursor called pro-HGF. Proteolytic conversion of pro-HGF to the heterodimeric active HGF is required to trigger ligand-dependent MET signaling. This is achieved by one of the trypsin-like serine proteases, matriptase, hepsin or HGF activator (HGFA), which are expressed by tumor cells . A protein vivo –35. We din vivo , 36.In this study, we established that recombinant HGF or HGF-producing fibroblasts protect MET-amplified NSCLC cells from MET-targeting therapy by maintaining AKT and ERK signaling in MET-inhibited cells. We demonstrated that novel inhibitors of HGF activation overcome fibroblast-mediated resistance to MET inhibition in MET-amplified lung cancer cells. This highlights the need to include agents blocking the biological activity of HGF, such as inhibitors of pro-HGF activation, into therapeutic regimens for MET-amplified NSCLC patients.Whereas MET-amplified lung cancer cells do not respond to EGFR inhibition, their growth is strongly inhibited by MET tyrosine kinase inhibitors, confirming that these cells are addicted to MET signaling , 14. AccHGF has been shown to mediate resistance to several targeted therapeutic agents such as inhibitors of EGFR, BRaf and HER2 , 37. HerThese data indicated that MET-amplified NSCLC cells rely on HGF for survival upon pharmacological inhibition of MET. We have shown earlier that SRI31215, a triplex inhibitor of matriptase, hepsin and HGFA, Figure . ConsistThe levels of matriptase and hepsin, two of the pro-HGF converting enzymes, are significantly elevated in lung cancer compared to normal lung tissue . MoreoveHGF is a key growth factor in the tumor micro-environment that inhibits the response of cancer cells to targeted therapy . Cancer-Consistent with our finding that HGF and pro-HGF block the activity of JNJ38877605 Figure , conditiLike HGF neutralizing antibodies, SRI31215 blocked the ability of fibroblasts to rescue EBC-1 and H1993 cells from JNJ38877605-induced cell death Figure . AccordiTo understand the mechanism whereby HGF elicits resistance to JNJ38877605, EBC-1 cells were treated with JNJ38877605 alone or in the presence of recombinant HGF or fibroblasts and SRI31215. As expected, JNJ38877605 inhibited the expression of p-MET, p-EGFR, p-Gab1, p-ERK 1/2 and p-AKT Figure . We demoCollectively, our data show that fibroblasts elicit resistance to MET-targeted therapy through HGF in MET-amplified lung cancer cells. We showed that HGF is sufficient to sustain MET and EGFR signaling and downstream activation of ERK and AKT upon MET inhibition. Inhibitors of pro-HGF that we developed blocked the pro-survival activity of fibroblasts, indicating that dual inhibition of MET and HGF is required to overcome resistance to MET-targeted therapy in MET-amplified NSCLC.MET activation promotes growth, survival and stimulates epithelial-mesenchymal transition which leads to enhanced migration of cancer cells . Indeed,We have shown previously that SRI31215 impedes crosstalk between colon cancer cells and fibroblasts and thus inhibits the tumor-promoting activities of fibroblasts in colon cancer . Here weThus, while HGF (or fibroblasts) do not further stimulate MET signaling  or promoTo understand how HGF signals in MET-amplified lung cancer cells when MET kinase activity is inhibited, we used the Proteome Profiler Human Phospho-RTK array (R&D Systems) to investigate the phosphorylation profiles of 49 different receptor tyrosine kinases. Additionally, we used Phospho-Kinase Array (R&D Systems) containing 46 kinases and their substrates.Inhibition of MET kinase by JNJ38877605 blocked MET phosphorylation, however we showed that JNJ38877605 also significantly reduced phosphorylation of EGFR, RON Figure , AXL andHGF restored phosphorylation of MET, EGFR and RON in MET-inhibited EBC-1 cells Figure , but didHow does HGF signal in MET-inhibited cells? It is possible that due to MET overexpression in MET-amplified cells there is always some residual MET in MET- inhibited cells, which responds to HGF stimulation. Indeed, we showed that HGF reactivates MET in JNJ38877605-treated cells Figure .The tumor-microenvironment plays a major role in resistance to targeted therapy. In this study we demonstrated that HGF, commonly produced by cancer-associated fibroblasts, confers resistance to MET kinase inhibitors in MET-amplified NSCLC cells. We showed that a novel inhibitor of pro-HGF activation, SRI31215, blocks the crosstalk between cancer cells and fibroblasts and overcomes resistance to anti-MET therapy in MET-amplified NSCLC cells.The availability of targeted therapeutic options, such as EGFR inhibitors, has improved the management of patients with EGFR mutant NSCLC. However, only a fraction of patients shows a good clinical response to EGFR inhibitors and virtually all patients develop resistance to therapy –43. AcquThe tumor microenvironment has been shown to elicit resistance to therapy. HGF, which is commonly overexpressed in the tumor microenvironment, is a frequent source of resistance to targeted therapy –21. HGF Here we demonstrated that HGF also drives resistance to anti-MET therapy in MET-amplified lung cancer cells Figure . Thus, MNeutralizing anti-HGF antibodies that are being tested, alone or in combination with other therapies, appear to be well tolerated. They have shown encouraging results, yielding partial response in cancer patients , 48–51. We demonstrated previously that SRI31215 effectively inhibits HGF-dependent MET activation, proliferation, epithelial-mesenchymal transition and migration of cancer cells and overcomes fibroblast-mediated resistance to EGFR inhibitors in colon cancer cells . Here wein vitro and in vivo [Several MET kinase inhibitors have been developed and have entered clinical trials. In preclinical studies cancer cells rapidly develop resistance to MET kinase inhibitors, which will probably translate to resistance in patients. It has been demonstrated that discontinuation of MET tyrosine kinase inhibitors results in increased and prolonged MET activation, coupled to enhanced tumor growth  in vivo , suggest in vivo .D1228V mutation [The mechanisms whereby cancer cells develop resistance to MET kinase inhibitors are not well understood. Recently, a lung adenocarcinoma patient with MET amplification developed resistance to dual anti-EGFR/MET therapy due to an acquired METmutation . We demoIn summary, our data underscore a key role of the tumor microenvironment in acquired resistance to MET kinase inhibitors in MET-amplified NSCLC cells. We demonstrated that MET-amplified NSCLC cells become addicted to HGF under pharmacological MET inhibition. Because HGF is frequently overexpressed in the tumor microenvironment, our work highlights the need to include inhibitors of biological activity of HGF into therapeutic regimen in MET-amplified NSCLC patients.Primary antibodies used were anti-MET, anti-pMET, anti-EGFR, anti-pEGFR, anti-GAB1, anti-pGAB1, anti-AKT, anti-pAKT, anti-pERK1/2,  and anti-β-actin (Sigma). The antibody arrays used were Proteome Profiler Human Phospho-Receptor Tyrosine Kinase (RTK) (ARY001B) and Phospho-Kinase Array (ARY003B) kits from R&D Systems. Recombinant HGF and pro-HGF were purchased from R&D Systems. The MET kinase inhibitor, JNJ38877605 was purchased from Selleck Chemicals (Selleckchem). HGF-specific and nonspecific (NSP) siRNAs were purchased from Dharmacon.2. Conditioned medium from fibroblasts was prepared from 18Co fibroblasts as reported before [A549, WI38 and H1993 cells were obtained from ATCC and EBC-1 cells were purchased from XenoTech, KS. EBC-1, A549 and WI38 cells were maintained in minimum essential medium (MEM) and H1993 cells were cultured in RPMI medium. Growth media were supplemented with 10% fetal bovine serum, L-glutamine and antibiotics and cells were maintained under standard cell culture conditions at 37°C and 5% COd before . Brieflyd before . Conflue4 cells/well in serum-free media. Transwells were placed in wells (24-well format) containing 10% serum-containing media and incubated for 24h. Cells that migrated were stained for 10 minutes at room temperature, transferred to wells containing 200 μl of extraction buffer and incubated for 10 minutes on a shaker. The optical density (OD) of stained cells was measured at 560 nm.Cytoselect cell migration assay kit was purchased from Cell Biolabs and the assay was performed according to the manufacturer's instructions. EBC-1 cells were seeded in transwells (8 μM pore size inserts) at a density of 7.5×10® luminescent assay kit from Promega was used to assess cell viability. Cells were seeded in 96-well plates at a density of 1×104 cells per well and treated as indicated. Cell viability was assessed 24, 48 and 72 hours after treatment, following the manufacturer's protocol.CellTiter GloImmunoblotting was performed using standard procedures. Proteins were separated by SDS-PAGE. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature (RT), and immunoblotted with primary antibodies in 5% BSA-TBST overnight at 4°C. Following washing with TBST buffer, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour and then washed with TBST. Membranes were developed using the ECL chemiluminescent detection system (Amersham).7 cells/mL. Array membranes that had been spotted with capture antibodies were blocked with blocking buffer and then incubated with 300 μg of cell lysates overnight. Arrays were washed to remove unbound proteins followed by incubation with detection antibodies conjugated to horseradish peroxidase. Phosphorylated proteins were detected using chemiluminescent reagents and quantified using the Image Quant LAS 4000 array analysis software .The phosphorylation profiles of kinases and their protein substrates were determined using Proteome Profiler Human Phospho-Receptor Tyrosine Kinase (RTK) and Human Phospho-Kinase Array kits (R&D Systems), following the manufacturers protocol. Serum-starved EBC-1 cells were treated with JNJ38877605 for 6h in the absence or presence of recombinant HGF. Cells were rinsed with PBS and then solubilized with lysis buffer at a concentration of 1×10EBC-1 cells were seeded in a 6-well plate DMEM growth medium. Upon reaching 80-90% confluence, growth medium was changed to Opti-MEM and cells were transfected with HGF-specific siRNA (Dharmacon) using Lipofectamine 2000 (Invitrogen). Non-specific (NSP), nontargeting siRNA (Dharmacon) was used as a negative control. Both HGF and NSP siRNAs were delivered at a final concentration of 25 nM. Growth medium was changed to DMEM without antibiotics after overnight transfection. HGF silencing was confirmed by immunoblotting.p < 0.05 was considered statistically significant.All experiments were repeated at least three times. Results are expressed as the mean ± SEM as indicated. Statistical analyses using two-tailed student's t-test were performed with the GraphPad Prism 5.0 software.


Two illustrative cases of acute central retinal artery occlusion (CRAO) are presented with multimodal imaging, including fluorescein angiography (FA) and commercially available optical coherence tomography angiography (OCT-A). In both patients, retinal ischemia was imaged well using both FA and OCT-A, and the two imaging studies provided comparable pictures. OCT-A provides useful information for the diagnosis and management of patients with acute CRAO, without the need for dye injection.  Central retinal artery occlusion (CRAO) results from obstruction of blood flow due to embolic, thrombotic, inflammatory, or traumatic causes. In some eyes with CRAO, visual loss is relatively less severe due to sparing of the cilioretinal artery .In most patients, the diagnosis of CRAO may be made with ophthalmoscopy alone, although ancillary testing is frequently used to confirm the diagnosis and to document the findings at presentation. Multimodal imaging includes fluorescein angiography (FA), spectral domain optical coherence tomography (SD-OCT), and optical coherence tomography angiography (OCT-A). Although FA has been used traditionally to evaluate the retinal circulation, OCT-A is an emerging technology that provides clinically useful information.The present manuscript uses OCT-A to identify the pathologic features in two illustrative cases of CRAO. In both patients, OCT-A was performed using the commercially available Cirrus 5000 with AngioPlex  with no subsequent image processing. A 6 × 6 mm slab was used for all images.A 77-year-old male with a history of atrial fibrillation and nonneovascular age-related macular degeneration (AMD) presented about 4 hours following acute visual loss in the left eye. Best corrected visual acuity (BCVA) was count fingers. Fundus examination revealed macular drusen as well as mild macular whitening and an early cherry red spot . SD-OCT An 81-year-old male with a history of hypertension presented about 13 hours following acute visual loss in the left eye. BCVA was 20/50. Fundus examination revealed macular whitening in a pattern consistent with CRAO with cilioretinal artery sparing . SD-OCT OCT-A characteristics of CRAO have been reported previously –5. TheseOCT-A offers several advantages compared with traditional FA. OCT-A is noninvasive and has no risks of allergy . In mostIn patients with acute CRAO in whom an adequate OCT-A can be obtained, FA may not be necessary. Since many of these patients have serious systemic vascular diseases, OCT-A is an easily performed, quick, noninvasive alternative to FA.


In a continuous and mutual exchange of information, cancer cells are invariably exposed to microenvironment transformation. This continuous alteration of the genetic, molecular and cellular peritumoral stroma background has become as critical as the management of primary tumor progression events in cancer cells. The communication between stroma and tumor cells within the extracellular matrix is one of the triggers in colon and liver carcinogenesis. All non- codingRNAs including long non-coding RNAs, microRNAs and ultraconserved genes play a critical role in almost all cancers and are responsible for the modulation of the tumor microenvironment in several malignant processes such as initiation, progression and dissemination. This review details the involvement of non codingRNAs in the evolution of human colorectal carcinoma and hepatocellular carcinoma in relationship with the microenvironment. Recent research has shown that a considerable number of dysregulated non- codingRNAs could be valuable diagnostic and prognostic biomarkers in cancer. Therefore, more in-depth knowledge of the role non- codingRNAs play in stroma-tumor communication and of the complex regulatory mechanisms between ultraconserved genes and microRNAs supports the validation of future effective therapeutic targets in patients suffering from hepatocellular and colorectal carcinoma, two distinctive entities which share quite a lot common non-coding RNAs. Non-coding RNAs (ncRNAs) are a heterogeneous class of transcribed RNA moleculesfrom non-(protein)-coding regions, which lack an open reading frame and consequently have no apparent protein-coding ability. Based on the size of the functional RNA molecule, regulatory ncRNAs are classified analytically as long ncRNAs , which are relatively less well-described, and small ncRNAs  [In recent years, the study and understanding of the tumor microenvironment has become as important as the modulation of cancer cell progression itself . ColorecAlthough advancements have been made with regard to available therapy benefits, there are only modest improvements in the survival rates of CRC and HCC patients due to the lack of early detection and excellent prognostic indicators .Molecules secreted by HCC, the most frequent liver tumor type, lead to the activation of hepatic stellate cells (HSCs) that remodel peritumoral stroma and the composition of the extracellular matrix (ECM) . The mutIn addition, the communication between stroma and tumor cells within the ECM opens the way to carcinogenesis in the liver and colon. Besides the modulation of HSCs in ECM turnover, activated stellate cells are an important determinant of hepatic pro-inflammatory cytokines such as transforming growth factor- beta (TGF-β), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). They also recruit immunoinflammatory cells, mono- and polimorphonuclear leukocytes which, in their turn, produce certain chemokines including C-C chemokine receptor type (CCR5), Regulated on Activation Normal T Cell Expressed and Secreted (RANTES), monocyte chemotactic protein 1 (MCP-1) and CC chemokine ligand 21 (CCL21) .Moreover, targeting the microenvironment containing CAFs, endothelial cells, adipocytes, immune cells and cancer stem cells (CSC), particularly the crosstalk between tumor cells and stromal cells, has emerged as an encouraging cancer therapeutic approach . This strategy would be relevant for both HCC and CRC, which frequently develop in a setting of chronic inflammatory process and microenvironment redesign, especially associated with hepatic fibrosis to which HSC and CAFs greatly contribute , 10, 11 Thereby, the HSC “metamorphosis” represents a key cell-reprogramming event that favors tumor progression. Additionally, the amount of activated HSCs from the tumorigenic HCC environment after surgical resection could predict early recurrence and poor clinical outcome in HCC patients. The angiogenin secreted by HCC cells binds directly to the promoter region of DNA, thus inducing the biogenesis of angiogenic factors such as VEGF and FGF-2, which are important in cell growth and tumor proliferation. Consequently, targeting the angiogenin signaling pathway with small-molecule inhibitors such as non- coding RNAs could be required to cancel the activating capacity of HSCs and thus control HCC progression , 12.Lately it has become obvious that the dialogue between stroma and tumor cells is not simply composed of cell-matrix adhesion and signaling secreted proteins. Lipid membrane bound exosomes are recognized vesicular transporters secreted from both stromal and cancer cells that control the gene expression of neighboring cells. Generally, exosomes deliver their protein and RNA load and adjust gene expression in the recipient cells. Among their cargo miRNAs emerge as important players because they are almost stable compared to proteins and mRNA. Furthermore, they accumulate to a level that can generate a durable biological effect .Thus, noCRC and HCC sharecommon non-coding RNAs despite major differences between them in terms of clinical aspects and treatment. These non-coding RNAs are emerging as key potential biomarkers in both types of cancer thus providing us with a new perspective to our understanding of their molecular mechanisms, at the same time also providing new therapeutic opportunities. This review presents a genomic standpoint on the alterations of non-coding RNAs in liver and CRC tumor microenvironment. It also highlights the significance of the interaction between tumor and stromal cells through the activation of HSCs / CAFs, which have a key role in tumor initiation and development.The lncRNAs family contains multiple classes of RNAs, which are nuclear RNAs greater than 200 nucleotides involved in the regulation of cellular processes such as apoptosis, proliferation and metastases  Figures.Conversely, microRNAs are an abundant class of endogenous small RNA molecules, 20-25 nucleotides in length, which act as oncogenes and tumor suppressors and thus have crucial roles in carcinogenesis , 15. SimRecent research revealed two key roles of miRNAs in the dynamics of tumor microenvironment: in tumor cells, they modify the peritumoral stroma and the composition of the ECM through their own mechanisms while in neighboring cells, they imprint cancer hallmark Figure . MoleculPrevious studies indicated that miR-34a inhibits tumor growth, miR-21 promotes apoptosis resistance of tumor cells proliferation while the miR-200 family is strongly associated with the epithelial- mesenchymal transition (EMT) , 19. In miRNA- 26a is a new HCC and CRC angiogenesis suppressor and a possible therapeutic target influencing the hepatocyte growth factor (HGF) - cMet pathway. It also inhibits the expression of the vascular endothelial growth factor A (VEGFA) in cancer cells. Moreover, the miR-26 down-regulation increases the angiogenic potential of these types of cancers. HGF was identified as a target of miR-26a and its activation antagonizes the effects induced by the up-regulation of miR-26a .Therefore, miR-26a partially exerted its anti-angiogenesis effect by blocking the HGF-receptor (cMet) and its signaling pathway, thus consequently suppressing VEGFA production in HCC cells and modifying vascular endothelial growth factor receptor 2 (VEGFR2)-signaling in endothelial cells. In conclusion, HCC patients with low hepatocyte growth factor (HGF), low VEGFA, high miR-26a levels or low microvessel density in tumor cells have a better prognosis with longer overall survival and time to recurrence. In multivariate analysis, it was demonstrated that miR-26a, alone or in combination with HGF, is an independent prognostic indicator for time to recurrence and overall survival in HCC patients  Figure .miR- 26a also decreases the glucose metabolism of CRC cells by direct targeting of the pyruvate dehydrogenase protein X component (PDHX), which blocks the conversion of pyruvate to acetyl coenzyme A in the Krebs cycle. The overexpression of miR-26a in tumor cells strongly improved the accumulation of pyruvate and reduced the production of acetyl coenzyme A. At the same time, the inhibition of miR-26a expression developed opposite biological effects .in vitro models showed that miR-195 not only suppresses the ability of HCC cells to develop the migration and capillary formation of endothelial cells but also directly decrease the ability of HCC cells to migrate and invade the ECM gel [Another promising HCC biomarker with a considerable therapeutic potential is inflamma-miR-195, which suppresses HCC angiogenesis and metastasis if overexpressed in tumor tissues. Both loss-of-function and gain-of-function research of  ECM gel .down-regulation of miR-195 elevated CARMA3 protein expression, whereas miR-195 up-regulation abolished the Caspase recruitment domain  protein expression in CRC cells through NF-kB activity. Research based on mouse models indicated that induced expression of miR-195 dramatically reduced tumor microvessel densities and inhibited both pulmonary and intrahepatic metastasis. Thereafter, miR-195 directly decreases the expression of the proangiogenic VEGF and the prometastatic factors VAV2 and CDC42 from the Rho-family GTP-ases, which activates cellular actin dynamics in manycellular functions , 25  and modulating the cell cycle [In contrast, the up-regulation of miR-21 expression in HCC tissues promotes tumorigenesis as well as resistance to antitumor 5FU and interferon α combination therapy . Moreovell cycle .Metastasis is responsible for the rapid progress and death by HCC. miR-100 down-regulation in HCC and CRC tissues was strongly associated with venous invasion, poorer cell differentiation and shorter recurrence-free survival /signaling pathway, which was correlated with expanded tumorigenesis, although the anti- tumorigenic response is unclear as the limited pre-clinical data available remains controversial [There are some examples of microRNAs with an essential impact on angiogenesis and ECM remodeling. Equally, miR-155 has a pivotal role in modulating liver-cancer-associated mesenchymal stem cell (LC-MSCs), the invasion promoting effect being attenuated by a miR-155 antagonist . MiR-155oversial .via vascular endothelial growth factor-A (VEGF-A). miR- 9 could function as a microtumor promoter gene in HCC because its up-regulation was significantly correlated with aggressive clinicopathological parameters [Another EMT- regulatory miRNA is miR-9, a novel prognostic biomarker for HCC which modulates angiogenesis rameters , 41, 42.rameters .miR- 135b up-regulation in HCC tissues facilitates the generation of an IL-17- producing immunophenotype by interfering with the normal lymphocyte differentiation program. Additionally, the reversion-inducing-cysteine-rich protein with kazal motifs (RECK) and ecotropic viral integration site 5 (EVI5) were identified as the functional and direct targets of miR-135b in HCC. heat shock transcription factor 1 (HSF1) directly activates the miR-135b expression, consequently enhancing HCC invasiveness .The miR-135b blockage in CRC experimental mouse models diminishes tumor evolution by suppressing the genes involved in proliferation, apoptosis and invasion. Recent research identified miR-135b as a key effector of oncogenic pathways and a promising target for CRC therapy . The devIn order to determine the role of miRs in a cell program, it was shown that miR-29b sensitizes HCC cells to apoptosis by directly targeting two anti-apoptotic molecules such as Bcl-2 and Mcl-1. Similarly, miR-29b deregulation is involved in CRC. The down-regulation of miR-29b angiogenesis by affecting the endothelial cells. This effect is due to an increase in MMP-2 expression, which promotes VEGFR signaling in the endothelial cells , 48. miRUnderstanding the two-way communication between stroma and tumor is essential because extracellular miRNAs could inhibit tumor development and prognosis in the HCC microenvironment . miR-122For instance, miR-210 and miR-155, which are overexpressed in HCC and CRC, promote the metastatic potential of HCC cells by targeting the vacuole membrane protein 1 (VMP1), in this way being an example of tumor- to- stroma communication  by increasing the expression of matrix metalloproteinases (MMP) or by promoting angiogenesis , 55 Fig.Examples of ncRNAs included in exosomes are: miRNAs and long non-coding RNA (lncRNA), which are powerful regulators of cell signaling pathways, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long intergenic non-coding RNA (lincRNA), ribosomal RNA (rRNA), piwi-interacting RNA (piRNA), circular RNA (circ RNA) and transfer RNA (tRNA) , 57, 58.in vivo and in vitro studies. On the one hand, the intravenous administration of B-cell derived exosomes loaded with miR-155 correlated with significantly increased miR-155 expression in the liver of miR-knockout mice compared with controls [Research demonstrated that like hormones, exosomal miRNAs secreted by a donor cell were taken up by a recipient cell and remained functional both controls , 59. On controls . A stromcontrols , 61.1/S-phase transition through the activation of the protein kinase B/ Forkhead box O3 transcription factor (Akt/FOXO3a) signaled by targeting antagonists of Phosphatidylinositol-3-Kinase (PI3K)/Akt such as the proteinphosphatases PHLPP1 and PHLPP2. miR-224 also up-regulates cyclin-D1 and down-regulates cyclin-dependent kinase inhibitorsp27Kip1 and p21Cip1. Therefore, miR-224 promotes CRC tumor growth and metastasisviatargeting SMAD4. Alone or in combination with SMAD4, miR-224 could be an independent prognostic marker for the survival of CRC patients [miR-142-3p and miR-224 could be considered important predictive and prognostic markers in CRC and HCC as they are secreted in the circulatory system through exosomal compartments. miR-224 accelerates the Gpatients  - targeted therapy in CRC patients  . H19 alters the activation of miR-200 in cooperation with a specific protein complex . WorkingGrowth arrest specific transcript 5 (GAS5) was significantly associated with HCC and CRC prognosis, it is used as a potential and independent biomarker for predicting clinical outcome , 79. OthHCC- derived exosomes have selective enrichment of lncRNA, especially in lnc-RNA TUC339 and lnc-RNA regulator of reprogramming (ROR), which are involved in tumor cell reprogramming. These lncRNAs were demonstrated to modulate cellular responses to chemotherapy and for their potential as disease biomarkers , 84-85. Another oncogenic lncRNA, HEIH is significantly associated with HCC evolution and works as an independent biomarker for predicting disease progression in HCC patients , 88. Lncproliferating cell nuclear antigen antisense RNA1 (PCNA- AS1) promotes tumor cell growth and is highly up-regulated in this type of cancer while LncRNA-down-regulated expression by hepatitis B virus X-HBx- protein (DREH) is down-regulated especially in HBV-related HCC tissues and is also dramatically decreased in HCC patients with poor prognosis -91.The expression of maternally expressed gene 3 (MEG3), which is down-regulated in HCC, may be regulated by microRNA-29 low expression in tumor (LET) is also down-regulated in HCC and contributes to hypoxia-mediated invasionATB- lncRNA activated by TGF-β is highly expressed in HCC and strongly associated with poor prognosis in HCC patientsTherefore, the discovery of the impact of non-coding RNAs in HCC and CRC has led to useful applications in the clinical management of HCC, future treatment being represented by a cocktail of T-cell modulators and vaccines enriched with the molecular targets of blockers in cancer-signaling pathways ; 92-97In contrast to miRNAs, which were largely studied for their roles in carcinogenesis, lncRNAs are less described. However, this review reveals the most specific non-coding RNAs in CRC and underlines their potential role in prevention and early detection  Table 22.c- Myc (a well-known transcription factor) activates the gene expression of lncRNA CRC-associated transcript 1 and 2 (CCAT1 and CCAT2), thus contributing to CRC tumorigenesis and metastasis. It is also a potential target for lncRNA direct therapy, being able to modify the clinical course of the disease [99Pseudogenes of transcripts such as myosin light chain kinase pseudogene 1 (MYLKP1), octamer binding transcription factor 4 pseudogene 1 (POU5F1P1) and phosphatase and tensin homolog 1 pseudogene 1 (PTENP1) which target the tumor stroma in HCC are often deregulated in cancer tissues compared to normal cells. They are normally involved in CRC progression and significantly associated with survival time in the third stage of cancer .SnoRNAs are a subclass of small non-coding RNAs (sncRNAs) (60 - 300 nt) involved in the adjustment of ribosomal RNAs (rRNAs) and transcribed by RNA polymerase II. Certain snoRNAs have different expressions in many human cancers including HCC and could serve as reference genes able to validate the expression of miRNAs -102.This review focuses on studies verifying the association of snoRNAs with tumorigenesis in CRC. A positive correlation was established between p53 expression and snoRNAs concentrations derived from GAS5. At the same time, a significant correlation was found between miR-34a expression levels and both snoRNA U47 and snoRNA U44 .Ultraconserved genes (UCGs) are another class of non-coding RNAs involved in CRC and HCC initiation and progression. They represent a special subset of mainly non-coding transcripts which are absolutely conserved during evolution and which can also regulate miRNAs by direct interaction. Fingerprints of differential UCG expression classify CRC and could be involved in metastasis  Table 22.Examples of the most important UCGs highly expressed in CRC include uc 29, uc 73, uc111, uc 112, uc 134, uc 206, uc 230, uc 292, uc 339, uc 34, uc 388, uc 399 and uc 420. Furthermore, the intensity of uc 73 effects on survival and tumor cell proliferation in CRC corresponded to the degree of down-regulation by specific siRNAs. This UCG acts as an oncogene which raises the number of malignant cells as a result of reduced apoptosis rate in CRC cells .HCC and CRC cell- derived exosomes contain ucRNAs and could be taken up by other HCC and CRC cells resulting in ucRNA intercellular transfer with consequent adjustment of cellular behavior . MoreoveLncRNA-LncRNA-identified as two ucRNAs with an altered gene expression in HCC in order to activate modified cell growth in hepatocytes. However, TUC 339 is more enhanced within extracellular MVs released by hepatic cells than TUC 338, which has different functions. biologically active signaling carriers. Moreover, the exosomes-mediated transfer of ucRNAs such as , 82.In addition, uc001ncr and AX800134 were selected as potential biomarkers in hepatitis B virus (HBV)-positive HCC due to their significantly high-expression in both tissues and serum samples compared with controls, especially in early-stage disease or in patients with alpha-fetoprotein (AFP) ≤ 400 ng/ml .We have revealed common small non-coding RNA molecules  in HCC and CRC, which suppress the expression of multiple genes involved in tumor- stromal interactions, immune invasion and tumor angiogenesis. Furthermore, we included their reported role and target genes in both types of cancer in a generic table  or the hepatocyte growth factor. CAFs also secret fibroblast growth factor-2 (FGF-2)that activates angiogenesis and induces the metastatic capability and invasiveness of the cancer cells. Epithelial-mesenchymal transition (EMT) is a cellular program leading to cancer cell metastasis that is promoted by CAFs in both CRC and HCC. Exosomes containing microRNAs are secreted from both cancers and stromal cells and they suppress the gene expression of neighboring cells.The tumor microenvironment (TME) is composed of fibroblasts, blood vessels, support cells, signaling molecules, immune cells and the extracellular matrix (ECM). Cancer cells (CC) grow in TME that favors tumor growth and contributes to therapy resistance. Major cell types in the tumor microenvironment are cancer-associated fibroblasts (CAFs), endothelial cells and immune cells, all of which communicate with cancer cells. One way that these cell types promote angiogenesis is by altering the expression of miRNAs either in associated normal cells or in cancer cells. Up-and down-regulation in miRNA expression can occur even through the direct communication between cells through exosomal miRNAs.Chronic inflammation driven by the NF-kB signaling pathway is a critical component of cancer progression. The inflammatory microenvironment of tumor cells is a key participant in metastasis. In addition, tumor exosomes containing long non-coding RNAs, which are overexpressed in HCC and CRC, are reliable players in the formation of the microenvironment by ◦ initiating the inflammatory process ◦ triggering angiogenesis ◦ enhancing the metastatic evolution of the primary tumor ◦ preparing the “premetastatic niche,” which dictates the pattern of metastatic spread in the new anatomical location.


Patients with anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) mount a humoral and cellular immune response against ALK. More than 90% of children and adolescents with ALK-positive ALCL have detectable anti-ALK antibodies in serum or plasma, and the antibody titer inversely correlates with the risk of relapse. ALK-specific CD8 and CD4 T cell responses have been described in patients with ALK-positive ALCL. Vaccination with ALK DNA led to protection against lymphoma growth in a murine model. Collectively, these data suggest that the ALK-specific immune response is involved in the control of the disease. The characteristics of the humoral and cellular immune response against ALK as well as tumor immune escape mechanisms have been increasingly investigated. However, tumor and host factors contributing to the individual immune response against ALK are still largely unknown. Depending on the individual strength of the immune response and its determinants, individualized immunological approaches might be appropriate for the consolidation of ALCL patients. Strategies such as ALK vaccination could be effective for those with a pre-existing anti-tumor immunity, while an allogeneic blood stem cell transplantation or check-point inhibition could be effective for others. Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a biologically defined disease of children and young adults . It accoClinically, ALK-positive ALCL is characterized by a high incidence of B-symptoms (60%) and extra-nodal involvement (60%), particularly in the skin, lung, bone, and soft tissue ,15,16,17Wildtype ALK is a tyrosine kinase receptor belonging to the insulin receptor super family. During embryogenesis, ALK is highly expressed mainly in the nervous system but its presence in adult normal tissue is almost absent ,28. The Clinical, histopathological, and clinical laboratory observations provided the first hints that ALCL might provoke an immune response in patients.More than 60% of children and adolescents with ALK-positive ALCL present with B-symptoms, suggesting an unspecific stimulation of the immune system by the lymphoma ,16. MoreThe very low relapse rate of 10–20% in patients with progressive or relapsed ALCL after allogeneic SCT suggests that a graft versus ALCL effect may exist ,31,32,33Tumor cells in ALK-positive ALCL cells are often surrounded by abundant reactive bystander cells which suggest an accompanying immune reaction ,35. The Pro-inflammatory cytokines are detected in the serum of ALCL patients at the time of diagnosis ,38,39. AHumoral immune responses against tumor-associated antigens have been described in many cancers . Autoantp < 0.001; The analysis of pretreatment anti-ALK antibodies in 95 ALK-positive pediatric ALCL patients, enrolled in clinical studies with comparable short-pulse chemotherapies, confirmed that more than 90% of the patients had measurable anti-ALK antibody titers at diagnosis compared to only one of 99 controls . When caA systematic analysis of the course of anti-ALK antibody titers was performed during standard short-pulse chemotherapy in 122 pediatric NPM-ALK positive ALCL patients . The perThe presence of anti-ALK antibodies was also observed in ALCL patients with variant ALK fusion partners, ALK-positive diffuse large B cell lymphoma, and in ALK-positive non-small cell lung carcinoma (NSCLC) ,51. EpitIt is not clear so far whether ALK-specific autoantibodies may act against tumor cells or are merely a surrogate marker for the strength of the patients’ overall cellular immune response against ALCL. A direct anti-tumor effect of autoantibodies has been shown for antibodies against cell surface antigens. Examples include HER2 autoantibodies that suppress the activity of the HER2 receptor in some patients with HER2-positive breast or ovarian cancer after vaccination with HER2 specific peptides . HoweverALK is spontaneously recognized as tumor antigen in ALK-positive ALCL patients. Several studies demonstrated the presence and persistence not only of an antibody response to ALK but also of ALK-specific CD8 and CD4 T cells in patients with ALK-positive ALCL.The immunogenic potential of ALK in initiating a cytotoxic T cell (CTL) response was first demonstrated in a study by Passoni et al. . In a reA CTL response to ALK in ALK-positive ALCL patients was subsequently reported by Ait-Tahar et al. . In thisThe natural frequency and functional phenotype of circulating anti-ALK CD8 T cell precursors in the peripheral blood of healthy donors and ALK-positive ALCL patients was assessed by tetrameric MHC/peptide analysis, IFN-γ ELISPOT assay, and in vitro lysis of ALK-positive target cells . High frMemory CD8 T cells are required for protective anti-tumor immunity ,57,58. R5 cells). However, the combination of chemotherapy (a single dose of doxorubicin) with following ALK vaccination significantly enhanced the survival of mice challenged intravenously with 1 × 106 ALK-positive lymphoma cells before therapy [Chiarle et al. examined the in vivo potential and clinical relevance of vaccination with ALK cDNA against ALK-positive ALCL in mice . BALB/c  therapy . This st therapy . The immAs a prerequisite for the clinical development of immunotherapeutic strategies, we recently examined NPM-ALK-specific CD8 T cell responses in NPM-ALK-positive ALCL patients in remission after chemotherapy . To circTaken together, these in vitro and in vivo data reveal the existence of ALK-specific CD8 T cell responses in ALK-positive ALCL. The possible role of these CTLs in lymphoma protection, however, needs to be investigated in more detail.The presence of IgG antibodies and CD8 memory T cells against ALK in patients suggests the involvement of CD4 T cells in the anti-ALK immune response ,62. A stThe paucity of data regarding ALK-specific CD4 T cell response in ALK-positive ALCL patients demands further characterization of the CD4 T cell response in these patients.In cancer patients, a protective anti-tumor response is impaired. Cells of the immune system may not detect tumor antigens or recognize them as self rather than foreign. Also, immunosuppressive factors from the tumor microenvironment or the tumor itself might dampen tumor-reactive effector cells ,65. WhilHowever, even if neoplastic cells are visible to the immune system, during carcinogenesis, cancer cells acquire complex immune escape mechanisms protecting themselves from immune surveillance. Some ALK-dependent immune escape mechanisms have been described for ALK-positive ALCL. A direct molecular link between ALK activation and suppression of the anti-tumor immune response has been shown by the Wasik group. In this study, it was demonstrated that NPM-ALK induces the expression of immunosuppressive programmed death-ligand 1 (PD-L1) in ALCL cells through STAT3 . In anotALK-positive ALCL cell lines have been shown to secrete TGF-beta, IL-10 and express FoxP3 . ElevateThe increasing evidence for the existence and clinical relevance of an autologous immune response against ALK implies that immunotherapeutic approaches might be an effective therapeutic intervention for the treatment of ALK-positive ALCL patients.Certain chemotherapeutic drugs exhibit immune stimulatory effects. By applying this knowledge, classical therapies could be further developed to a backbone with less acute and long-term toxicity. Even the current intensive polychemotherapy may boost the anti-tumor immune response in some patients, as suggested by the persisting ALK-antibody titers and observations of measurable anti-ALK T cell responses after—but not before—chemotherapy in a few patients ,72. CyclThe classical immunotherapy in leukemia and lymphoma is allogeneic SCT. Its efficacy as a consolidation therapy for children with ALCL relapses has been well established ,31,32,33Since ALK-positive ALCLs express PD-L1 ,67, immuMore specific immunotherapies targeting ALCL surface markers or ALK include anti-CD30 chimeric antigen receptor (CAR) T cells, vaccination strategies against ALK, and possible ALK-specific T cell therapies. CD30-specific CAR T cells have been tested in mouse models and a phase I clinical trial so far . One patChiarle et al. showed the in vivo potential of a vaccination therapy with truncated ALK DNA, as well as this approach in combination with chemotherapy, in a mouse model . The preTools for the selection and cultivation of antigen-specific T cells for adoptive immunotherapy have been established for several cancers . HoweverThe available data suggest that immunotherapies targeting ALK have a high potential to revolutionize treatment strategies for ALK-positive ALCL in the future. One of the major challenges is the definition of the most suitable immunotherapy for an individual patient. It is currently unclear which group of patients might benefit from a vaccination or unspecific immune stimulation for the induction of life-long tumor control and which patients require an allogeneic SCT for cure. The deciphering of the host and tumor factors influencing the strength and efficacy of an autologous immune response against ALK-positive ALCL might help individual decision-making regarding consolidation therapy in the future.Vaccination against ALK presumably has a very low safety risk and likely needs a pre-existent boostable immune response to be effective in the time before relapses occur. Vaccination studies could, therefore, be of benefit and should be studied for adults and children with low-risk ALCL. The focus for studies against very high risk or relapsed ALCL might include the optimization of allogeneic SCT and check-point inhibitors.Both the cellular and humoral immune response in ALCL has become increasingly elucidated in the past years. However, more work is needed to further unravel the immunology of ALK-related malignancies. For the successful development of a vaccine, the antigenic ALK epitopes recognized by cells of the adaptive immune system need to be characterized.