Circulating in China and 158 other countries and areas, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public health. However, efforts to identify effectively supportive therapeutic drugs and treatments has been hampered by our limited understanding of host immune response for this fatal disease. To characterize the transcriptional signatures of host inflammatory response to SARS-CoV-2 (HCoV-19) infection, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients. Our results reveal distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release such as CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B. Furthermore, SARS-CoV-2 induced activation of apoptosis and P53 signalling pathway in lymphocytes may be the cause of patients’ lymphopenia. The transcriptome dataset of COVID-19 patients would be a valuable resource for clinical guidance on anti-inflammatory medication and understanding the molecular mechansims of host response. The recent ongoing outbreak of viral pneumonia (COVID-19) has sparked worldwide concern. First reported in December 2019 in Wuhan, COVID-19 spread rapidly across other areas and caused a major epidemic with 80,894 confirmed cases including 3237 deaths in China by 17 March 17 2020. A novel highly infectious coronavirus, officially called the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was subsequently identified as the causative pathogen of this outbreak by deep sequencing and etiological investigations . As globDespite increasing global threats of COVID-19, the host immune response against SARS-CoV-2 infection remains poorly understood. Viral RNAs are recognized by the innate immune system through three major classes of cytoplasmic pattern recognition receptors: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), which trigger the expression of interferon (IFN) and activation of anti-viral effectors such as Natural Killer cells, T CD8+ cells and macrophages . CoronavTranscriptomic analyses of cells upon virus infection are extremely useful to identify the host immune response dynamics and gene regulatory networks ,19. In tThis study was approved by the Ethics Committee of the Zhongnan Hospital of Wuhan University. The RNA-seq analyses of BALF samples and PBMC were performed on existing samples collected during standard diagnostic tests, posing no extra burden to patients.Whole blood and PBMC (Ficoll preparation) were obtained from 3 SARS-CoV-2 patients and 3 healthy donors at Zhongnan Hospital of Wuhan University. Peripheral blood sample (4 ml) from each patient and healthy control was drawn into vacutainer tubes. The Ficoll density gradient centrifugation method was used to separate the PBMC. We diluted the blood with 1× phosphate-buffered saline (PBS) 1:1 and then transferred it to the ficoll tube. After centrifugation , the buffy coat of PBMC cells was pooled and transferred into a 15-ml falcon. PBMC was then washed twice with 10 ml PBS and centrifuged at 250× g for 10 minutes. The precipitate was collected and the total RNA was extracted.2% lidocaine was injected into the segment of the lung for local anesthesia. 100 ml fractions of room temperature sterile saline were instilled into the right middle lobe or the left lingular segment of the lung. BALF was retrieved by gentle syringe suction and put into sterile containers.Total RNA was isolated from cells and fluid with TRIzol and TRIzol LS reagents respectively under the instruction of the manufacturer.One microgram of total RNAs were used as input. Messenger RNAs were purified using oligo-dTs covalently coupled magnetic beads. Then, the RNAs were fragmented into small pieces by heating. The expected size was 180 nt to 250 nt. First-strand of cDNA was synthesized in the presence of specific chemicals to ensure that only RNAs were used as template. Double strand cDNAs were purified with Agencourt AMPure XP beads after the reaction. DNA library was constructed through end-repair, adaptor-ligation and PCR amplification. The intermediate products were size-selected after the adaptor-ligation using two rounds of Agencourt AMPure XP beads. Qualified double-strand DNA library was transformed into single-stranded circular DNA library through DNA-denaturation and circularization. DNA nanoballs (DNBs) were generated from single-stranded circular DNA using rolling circle amplification (RCA). The DNBs were qualified using Qubit 2.0. Qualified DNBs were loaded on the flow cell and sequenced with MGISEQ-2000 platform  for 4 PBMC samples (P1-3 and N1). Another two PBMC healthy samples (N2 and N3) were sequenced with Illumina NovaSeq platform. The RNA-seq data for BALF healthy control samples  were downloaded from the NCBI SRA database with accession numbers: SRR10571724, SRR10571730, and SRR10571732 [https://github.com/ENCODE-DCC/long-rna-seq-pipeline). The human un-mappable reads were then mapped to the SARS-CoV-2 genome (GenBank MN988668) as previously reported [RNA-seq reads were firstly mapped to rRNA sequences to remove potential rRNA reads using STAR (v2.7.2b)  with thereported . PCR repreported . RNA-seqreported , and visreported  with cusp-value < 0.05 and fold-change > 2. The expressed genes , were selected and normalized to counts per millions (CPM) for further analysis.Gene expression was calculated by featureCounts in SubReads package (v1.5.3)  with thep-values were corrected for multiple hypothesis tests using the Benjamini and Hochberg method to control the false discovery rate (FDR). To inspect genes in specific GO-term and pathway, the GO and KEGG pathway annotation were downloaded from Gene Ontology Resource (Last updated: 22 February 2020) [Functional enrichment analysis was performed on the list of differentially expressed genes by using the clusterProfiler package (v 3.14.3)  to deterry 2020)  and KEGGry 2020) , respecthttps://github.com/zhouyulab/ncov/.The source codes for the analysis are available at the https://bigd.big.ac.cn/) [The raw sequencing data from this study have been deposited in the Genome Sequence Archive  in BIG D.ac.cn/) , BeijingTo investigate the impact and mechanism of SARS-CoV-2 infections in patients, we exploited RNA-seq to detect transcriptome changes in both PMBC and BALF samples from healthy donors and COVID-19 patients  in the Supplementary Tables 1 and 2 for BALF and PBMC samples, respectively. Consistent with our previous report focusing on viral genome assembly , BALF paFor BALF samples, we detected 9609 expressed genes, in which 679 genes are up-regulated and the 325 genes are decreased in patients compared with healthy individuals . The differentially expressed genes are represented in scaled heatmap comparing patients to healthy controls for both BALF  indicated cell count reduction of various types of immune cells including lymphocytes in patients’ blood. Previous clinical and autopsy reports indicated that patients’ lymphocytes have been greatly reduced . We founhttp://www.altanalyze.org). In order to illustrate the differences between the two cell types, we classified genes with different trends between the BALF samples and PBMC , which was also supported by the expression data base of bone marrow-derived blood cells (p < 0.05) when BALF and PBMC samples of COVID-19 patients were compared to control, respectively. As expected, these genes fall into categories including humoral immune response, lymphocyte mediated immunity and complement activation, which all play important roles in restricting viral infection.The infection of SARS-CoV-2 can cause severe pulmonary disease and complications with significant morbidities and mortalities. Currently, there is no optimal treatment or effective drug for this fatal lung disease. Our understanding of the host immune response to SARS-CoV-2 infection is limited, making it difficult to design novel therapeutics. Viral infection usually causes massive alterations in the host transcriptome, leading to aberrant host cell metabolism and modulated immune response which is ideal for viral replication ,33. UsinInflammatory response in viral pneumonia can be a double-edged sword. Although beneficial inflammation is necessary for the local tissues to fight infection, exacerbated inflammatory responses in pneumonia patients result in excessive release of pro-inflammatory cytokines known as “cytokine storm”, leading to detrimental outcomes such as diffuse alveolar damage and fibrosis, progressive respiratory failure and multiple organ dysfunction,34,35. Ttocilizumab has been proposed as a treatment in clinical practice by ameliorating inflammation of COVID-19 patients. Of note, we noticed that relatively lower expression levels of IL6R was observed in BALF of COVID-19 patients compared with healthy individual (Supplementary File 1), and there are no significant differences in PBMC (Induction of inflammatory cytokines is tightly controlled to prevent tissue damage and maintain a proper immune homeostasis. It has been shown that pro-inflammatory cytokines IL-6, which is required for the regulation of inflammatory response, B-cell differentiation and antibody production, was increased in SARS patients ,40. LaboMoreover, we found that anti-inflammatory cytokines IL-10 and TGF-b were also induced upon SARS-CoV-2 virus infection, resembling the high TGF-b activity found in SARS patients ,41. TGF-Lymphopenia have been found in a large portion of SARS and COVID-19 patients, although the underlying mechanism remains unclear ,6,44–47.In summary, the present work demonstrated that SARS-CoV-2 virus infection stimulates a unique transcriptome profile in COVID-19 patients BALF and PBMC. Additionally, the cytokine expression profile suggests excessive pro-inflammatory cytokine release might be a hallmark of COVID-19 patients. Furthermore, the COVID-19 patients BALF and PBMC RNA-seq dataset provides a useful resource to the community for further thorough analyses.YC, KL and YZ conceptualized the study design; YX and HWJ recruited the patients, collected specimens, collected demographic and clinical data; AJ, HW, YQL and MZ constructed and sequenced the RNA-seq libraries; YL and MG did the laboratory tests; DW, JY, ZT, YZ, LC and MS analysed the sequencing data; LC, YL and YC interpreted the results; YL and LC wrote the initial drafts of the manuscript; YL, YC, YZ and KL revised the manuscript. All authors read and approved the final manuscript.


Six patients wereinitially evaluated for possible cardiac symptoms such as chest pain orpalpitations; all 16 had evidence of cardiac effects, including electrocardiogramabnormalities such as arrhythmias, elevated troponin levels, or echocardiographicevidence of left or right ventricular dysfunction. Thirteen patients hadgastrointestinal symptoms on admission; five had dermatologic manifestations onadmission, including three with mucositis. Despite minimal respiratory symptoms, 10patients had pulmonary ground glass opacities, and six had pleural effusionsidentified on chest imaging. Inflammatory markers. All patients had markedly elevated laboratorymarkers of inflammation, including CRP  and ferritin , as well as markers ofcoagulopathy including D-dimer . Tenpatients had absolute lymphocyte counts lower than normal range .SARS-CoV-2 test results. Ten patients received positive SARS-CoV-2 PCRtest results at the time of initial assessment for MIS-A, seven of whom also hadserologic evidence of infection (positive antibody test results) at that time. Sixpatients received negative SARS-CoV-2 PCR test results; of those, four had positiveanti-SARS-CoV-2 antibody test results when first evaluated. Two patients hadpositive SARS-CoV-2 PCR test results 14 and 37 days before admission, negative PCRresults at the time of admission, and no known antibody testing. Three additionalpatients had positive SARS-CoV-2 PCR test results 25–41 days before admissionand continued positive PCR test results at the time of admission.Treatment. Seven patients were treated with intravenous immunoglobulin,10 with corticosteroids, and two with the interleukin-6 inhibitor, tocilizumab. Tenpatients required intensive care; seven required inotropes or vasopressors, and onerequired mechanical circulatory support . Three patientsrequired endotracheal intubation and mechanical ventilation, and two patientsdied.Three published case series were identified describing adult patients withmanifestations consistent with MIS-A  associated withpositive SARS-CoV-2 test results .Findings indicate that adult patients of all ages with current or previous SARS-CoV-2infection can develop a hyperinflammatory syndrome resembling MIS-C. Althoughhyperinflammation and extrapulmonary organ dysfunction have been described inhospitalized adults with severe COVID-19, these conditions are generally accompaniedby respiratory failure (The pathophysiology of MIS in both children and adults is currently unknown. Eight of27 (30%) adults described in this report and 45% of 440 children with MIS-C reportedto CDC through July 29, 2020, . Because patients described in this reviewrepresent a convenience sample from a small number of jurisdictions, conclusionscannot be made regarding the true burden or determinants of MIS-A in differentgroups; further research is needed.All but one of the patients with MIS-A described in this report belonged to racial orethnic minority groups. Long-standing health and social inequities have resulted inincreased risk for infection and severe outcomes from COVID-19 in communities ofcolor.The majority (24 of 27) of patients with MIS-A survived, similar to those with MIS-C,associated with receiving care in acute, often intensive, health care settings.Because of the potential therapies that might benefit these patients as described inthese case reports, clinicians should consider MIS-A within a broader differentialdiagnosis when caring for adult patients with clinical and laboratory findingsconsistent with the working MIS-A case definition.The findings in this report are subject to at least three limitations. First, casesdescribed here were voluntarily reported or published and therefore are notrepresentative of the true clinical spectrum or racial/ethnic distribution of thisemerging syndrome. Additional cases might not have been reported or published;others might have remained unrecognized because of absence of COVID-like symptoms,lack of antibody testing, or negative test results. Second, the working casedefinition excludes patients with severe respiratory dysfunction to distinguishMIS-A from severe COVID-19; however, the two conditions might overlap in some cases.Finally, the working case definition for this syndrome is potentially nonspecific,and some patients with other disease processes might have been misclassified ashaving MIS-A.Clinicians and health departments should consider MIS-A in adults with signs andsymptoms compatible with the current working MIS-A case definition. Antibody testingfor SARS-CoV-2 might be needed to confirm previous COVID-19 infection in patientswho do not have positive SARS-CoV-2 PCR or antigen test results. Findings in thisconvenience sample emphasize the importance of collecting race/ethnicity data oncase reports at the jurisdictional level. As with children, it is important thatmultidisciplinary care be considered to ensure optimal treatment. In the process oflearning more from MIS-A cases, the working case definition might need to be revisedin order to systematically conduct a call for cases. Further research is needed tounderstand the pathogenesis and long-term effects of this newly described condition.Ultimately, the recognition of MIS-A reinforces the need for prevention efforts tolimit spread of SARS-CoV-2. Multisystem inflammatory syndrome in children (MIS-C) is a rare but severecomplication of SARS-CoV-2 infection in children and adolescents. Since June2020, several case reports and series have been published reporting asimilar multisystem inflammatory syndrome in adults (MIS-A).Cases reported to CDC and published case reports and series identify MIS-A inadults, who usually require intensive care and can have fatal outcomes.Antibody testing was required to identify SARS-CoV-2 infection inapproximately one third of 27 cases.Clinical suspicion and indicated SARS-CoV-2 testing, including antibodytesting, might be needed to recognize and treat adults with MIS-A. Furtherresearch is needed to understand the pathogenesis and long-term effects ofthis condition. Ultimately, the recognition of MIS-A reinforces the need forprevention efforts to limit spread of SARS-CoV-2.


Severe coronavirus disease 2019 (COVID-19) causes an uncontrolled activation of the innate immune response, resulting in acute respiratory distress syndrome and systemic inflammation. The effects of COVID-19–induced inflammation on cancer cells and their microenvironment are yet to be elucidated. Here, we formulate the hypothesis that COVID-19–associated inflammation may generate a microenvironment favorable to tumor cell proliferation and particularly to the reawakening of dormant cancer cells (DCCs). DCCs often survive treatment of primary tumors and populate premetastatic niches in the lungs and other organs, retaining the potential for metastatic outgrowth. DCCs reawakening may be promoted by several events associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, including activation of neutrophils and monocytes/macrophages, lymphopenia and an uncontrolled production of pro-inflammatory cytokines. Among pro-inflammatory factors produced during COVID-19, neutrophil extracellular traps (NETs) released by activated neutrophils have been specifically shown to activate premetastatic cancer cells disseminated in the lungs, suggesting they may be involved in DCCs reawakening in COVID-19 patients. If confirmed by further studies, the links between COVID-19, DCCs reactivation and tumor relapse may support the use of specific anti-inflammatory and anti-metastatic therapies in patients with COVID-19 and an active or previous cancer. Since the beginning of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, several studies have investigated the susceptibility and mortality of COVID-19 in cancer patients. A recent meta-analysis reported a high mortality in patients with COVID-19 and cancer . While t+ T cells that may contribute to release pre-metastatic cells from immune-mediated control  protein priming . Followigression –22. Secogression . An altegression . RAS imbgression . Importagression . Moreovegression , contribgression Figure 1gression . NF-κB igression .COVID-19 patients admitted to intensive care units usually develop acute respiratory distress and cytokine release syndrome (CRS), a life-threatening toxicity that may lead to sustained fever, edema, neurologic symptoms, organ failure and shock . Cytokin+ and CD8+ tumor-inﬁltrating lymphocytes, as well as stimulating tumor growth, angiogenesis and metastasis  and a functional impairment of NK cells have been consistently detected in severe COVID-19 cases  have been proposed as a feasible strategy to prevent DCCs reactivation . In parallel to preclinical studies, in our opinion it will be crucial to investigate all the clinical effects of COVID-19 in cancer patients. While the first large studies in this field have focused on the susceptibility and mortality of COVID-19 in cancer patients  Investigator Grant to AZ (AIRC IG 2017 Ref: 20744).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.


Next-generation sequencing (NGS) is emerging as a new method for the detection of clinically significant copy number variants (CNVs). In this study, we developed and validated rapid CNV-sequencing (rCNV-seq) for clinical application in prenatal diagnosis. Low-pass whole-genome sequencing was performed on PCR libraries prepared from amniocyte genomic DNA. From 10–40 ng of input DNA, PCR-free libraries consistently produced sequencing data with high unique read mapping ratios, low read redundancy, low coefficient of variation for all chromosomes and high genomic coverage. In validation studies, reliable and accurate CNV detection using PCR-free-based rCNV-seq was demonstrated for a range of common trisomies and sex chromosome aneuploidies as well as microdeletion and duplication syndromes. In reproducibility studies, CNV copy number and genomic intervals closely matched those defined by chromosome microarray analysis. Clinical testing of genomic DNA samples from 217 women referred for prenatal diagnosis identified eight samples (3.7%) with known chromosome disorders. We conclude that PCR-free-based rCNV-seq is a sensitive, specific, reproducible and efficient method that can be used in any NGS-based diagnostic laboratory for detection of clinically significant CNVs. There are over 100 recurrent chromosome disorders that affect the human population . While tThe most common whole chromosome aneuploidies of the newborn are trisomies T21 (Down’s syndrome), T18 (Edward’s syndrome) and T13 (Patau’s syndrome) and the sex chromosome aneuploidies 45,XO (Turner’s syndrome), 47,XXX (Triple X syndrome), 47,XXY (Klinefelter’s syndromes) and 47,XYY (Jacob’s syndrome) . On the In prenatal diagnosis practice, women with a high fetal risk for a chromosome abnormality are usually referred for invasive testing by amniocentesis at 15–16 weeks gestation or by chorionic villous sampling at 10–11 weeks gestation. High risk is generally inferred by either an abnormal maternal serum screening score, advanced maternal age or presence of a soft ultrasound marker or an ultrasound structural abnormality . InvasivIn the field of molecular diagnostics, CMA is still the preferred methodology for identification of clinically significant CNVs in prenatal samples. To advance the clinical application of NGS, improvements in the sensitivity, specificity, reproducibility and versatility of NGS-based CNV detection methods are still needed before this technology can be generally considered for routine chromosome testing. One essential part of the NGS workflow is the library preparation method, with most CNV-seq tests currently using a low DNA input (50–200 ng) and a PCR step to amplify genomic fragments. As an alternative approach, we developed and validated a PCR-free-based rapid CNV-seq (rCNV-seq) method suitable for analysis of low nanograms amounts of genomic DNA from uncultured amniocytes. In a prospective study of 217 patients referred for chromosome testing, we show that rCNV-seq of the amniotic cell genomic DNA can reliably and accurately detect clinically significant CNVs.All blood and amniocentesis samples were collected at the Prenatal Care Unit of Peking Union Medical College Hospital (PUMCH). The research study was approved by the Ethics Committee for Drug Clinical Trials in PUMCH , and written informed consent was obtained from each patient. For validation studies, peripheral blood samples (2 mL) were taken from patients with known chromosome disorders and genomic DNA (gDNA) extracted using AxyPrep Mag Tissue-Blood gDNA kit . For patients with a suspected chromosome abnormality due to indications such as advanced maternal age, an abnormal maternal serum screening result or a structural abnormality revealed by ultrasound, invasive testing was performed on amniocentesis samples (10 mL of amniotic fluid) obtained at 15–16 weeks gestation. The fluid was centrifuged, and gDNA from the amniocyte cell pellet was purified using the AxyPrep Mag Tissue-Blood gDNA kit. Prior to CNV testing, amniocyte DNA was checked for maternal cell contamination (MCC) using an STR-based semi quantitative PCR assay ; MCC levAn overview of the three library construction methods used in this study is shown in Single end sequencing was performed on the NextSeq CN500 platform  with a run time of 6.5 h to generate approximately 5 million raw 45 bp reads per sample. Raw reads were then edited to remove artificial adaptor sequences, and the true 36 bp genome sequences were then mapped to the hg19 reference genome using the Burrows and Wheeler algorithm . On averhttp://projects.tcag.ca/variation), OMIM (Online Mendelian Inheritance in Man) (http://www.omim.org), PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and UCSC   were used as a reference source of CNVs. The pathogenicity of detected CNVs was assessed following ACMG guidelines [Genomic variant databases including DGV (Database of Genomic Variants), idelines . Cultured amniocytes were karyotyped by standard procedures . CytogenCMA was performed using the CytoScan™ HD Array Kit  according to the recommended protocol. The array contains more than 2.6 million SNPs and can detect copy number changes across the genome at a resolution of 25–50 kb. Genomic DNA samples were labeled and hybridized to the array according to the manufacturer’s recommended protocol. Fluorescence signals were scanned using the GeneChip scanner  and chromosome copy number changes called by Applied Biosystems™ GeneChip Command Console Software (version 3.2.2).p < 0.0001) and PCR-soni (p < 0.0001) libraries. Both PCR-free-frag and PCR-free-soni libraries had a significantly lower read redundancy rate compared to the PCR-soni libraries (p < 0.0001). The coefficient of variation (CV) achieved with PCR-free-frag libraries was also lower across all chromosomes and for each individual chromosome, compared to PCR-free-soni and PCR-soni libraries. The median read sequencing depth, indicative of increased bin read numbers, was also higher for PCR-free-frag compared to PCR-free-soni and PCR-soni libraries. Further, the Guanidine/Cytosine (GC) content of the sequencing reads was higher for PCR-free-frag than PCR-free-soni. Lastly, the Q30 values were significantly higher for PCR-free-frag compared to PCR-free-soni (p < 0.0001) and PCR-soni (p < 0.0001). Based on evaluation of these key Quality Control (QC) sequencing indicators, rCNV-seq using PCR-free-frag libraries showed the highest performance values.The performance of PCR-free-frag-based libraries (rCNV-seq) and two control PCR-free-soni and PCR-soni libraries (CNV-seq)  was bioiWe further assessed the impact of input gDNA amount on library yield, unique mapping ratio and redundancy for PCR-free rCNV-seq . Mean DNOur newly designed rCNV-seq method based on PCR-free-frag libraries was further evaluated for the ability to detect known chromosome disorders, previously detected by CMA. These included single samples identified with trisomies T21, T18 and T13 and sex chromosome aneuploidies (SCAs) 45,XO, 47,XXX, 47,XXY and 47,XYY. In addition, we also selected MMS samples including three cases each of Cri du Chat and William–Beuren syndrome, two cases each of Wolf–Hirschhorn and Di George syndrome and one case each of Prader-Willi/Angelman, Smith–Magenis and Miller–Dieker syndrome. Further, we also included four additional samples with variants of uncertain significance (VOUS) involving CNVS < 1 Mb in size. In these experiments, we used a low DNA input of 40 ng as the starting template for PCR-free-frag library construction. Following rCNV-seq analysis of all samples, the specific CNV that was originally identified by CMA was also detected by rCNV-Seq . In addiIn detailed reproducibility experiments using 10 replicate samples of 10ng for library construction, we further evaluated the reliability of rCNV-seq to correctly detect the precise CNV interval, benchmarking it against the CNV interval previously defined by CMA . For 45,To assess the diagnostic performance of rCNV-seq, 40 ng of amniocyte gDNA was analyzed from 217 pregnant women referred for chromosome testing for a variety of different clinical indications . MCC cheIn this study, we developed rCNV-seq based on a PCR library free sequencing methodology for detection of clinically significant CNVs in prenatal samples. The optimized rCNV-seq method accommodates input DNA sheared enzymatically without additional size selection steps and then combines the end repair and dA (adenine)-tailing into one step, removing the need for a PCR process and making library preparation much faster and easier . EquallyOne of the main drivers for reliable and accurate calling of CNVs is the quality and depth of the sequencing data that are binned across each chromosome. In this regard, rCNV-seq based on PCR-free libraries demonstrated improved performance over current CNV-seq methods, showing an average Q30 for the sequencing data as high as 94%. Compared to PCR, PCR-free-frag libraries achieved a higher proportion of uniquely mapped reads (fewer duplicate reads), a lower read redundancy and a smaller CV for all chromosomes, which are important parameters to improve the signal-to-noise ratio and allow more accurate detection of CNVs and their correct copy number. In addition, rCNV-seq exhibited some additional advantages for implementation of the method into molecular diagnostic laboratories with NGS capacity. Firstly, PCR-free libraries can be generated in around 2 h compared to 4–5 h for current PCR-free and PCR-dependent commercial methods. Thus, from sample receipt in the morning and with a sequencing run time of 6.5 h on the Next-Seq platform, it is possible to generate same day results and improves the overall efficiency of laboratory workflow and staff time. Second, the method can generate representative libraries from a wide range of input DNA amounts, improving versatility for the analysis of different sample types and compromised samples where the gDNA is limiting. Benchmarking against CMA, rCNV-seq demonstrated a high degree of accuracy and reproducibility. For the majority of CNVs tested, there was a high concordance between the two methods for correctly calling the CNV interval and copy number. There were some exceptions where the CNV interval varied up to 33% for the genome location called. These differences are probably related to the inherent limitations of both techniques, whereby CMA has reduced probe coverage for some genome positions, whereas rCNV-seq has reduced coverage in highly repetitive regions. While high sensitivity and specificity is an important parameter for calling CNVs, reproducibility is also a key factor for defining the resolution of CNV detection, since each rCNV-seq analysis is based on a set of randomly mapped sequencing reads mapped across the 24 chromosomes. Previous studies have shown that while CNVs as small as 0.1 Mb can be detected by CNV-seq , there wBased on our findings, rCNV-seq is also suitable as an alternative to CMA for routine prenatal testing where a gDNA sample is generally available from cultured amniocytes. However, there are also clinical situations whereby rCNV-seq has some clear advantages over CMA. Firstly, some women are referred for an invasive chromosome test close to the legal limit for termination of pregnancy, which is usually 20 weeks in most countries . SecondlPCR-free-based rCNV-seq is a rapid, sensitive, specific and efficient method that can be used in any NGS-based diagnostic laboratory to achieve a reliable and accurate CNV diagnosis. Further studies on a large number of amniocentesis samples are needed to define the true clinical utility and versatility of rCNV-seq for prenatal chromosome testing.


Work over the past 30 years has shown that lipid-activated nuclear receptors form a bridge between metabolism and immunity integrating metabolic and inflammatory signaling in innate immune cells. Ligand-induced direct transcriptional activation and protein-protein interaction-based transrepression were identified as the most common mechanisms of liganded-nuclear receptor-mediated transcriptional regulation. However, the integration of different next-generation sequencing-based methodologies including chromatin immunoprecipitation followed by sequencing and global run-on sequencing allowed to investigate the DNA binding and ligand responsiveness of nuclear receptors at the whole-genome level. Surprisingly, these studies have raised the notion that a major portion of lipid-sensing nuclear receptor cistromes are not necessarily responsive to ligand activation. Although the biological role of the ligand insensitive portion of nuclear receptor cistromes is largely unknown, recent findings indicate that they may play roles in the organization of chromatin structure, in the regulation of transcriptional memory, and the epigenomic modification of responsiveness to other microenvironmental signals in macrophages. In this review, we will provide an overview and discuss recent advances of our understanding of lipid-activated nuclear receptor-mediated non-classical or unorthodox actions in macrophages. A generally accepted paradigm in endocrinology has been over the last 30 years that if not the sole, but the main function of lipid-sensing nuclear receptors is to translate microenvironmental chemical cues into distinct biological effects through tight regulation of gene transcription. In the 1980s, the cloning of the first nuclear receptors including the glucocorticoid and estrogen receptors was followed by the identification of dozens of evolutionarily related proteins and initiated the development of this concept. Although at the time the newly identified proteins showed similar domain structure containing DNA binding, ligand binding, and transactivation domains, initially the nature of their endogenous chemical ligands was unknown. Therefore, these proteins were called ″orphan″ receptors. The approach termed ″reverse endocrinology″ (having the receptor first and looking for the ligand later) allowed the discovery of dietary lipid-derived, often lower-affinity endogenous ligands, for many of them including liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) . Since tin vitro and in vivo which can be switched depending on changing microenvironmental milieu  , 18. Genin vitro and in vivo pieces of evidence indicate that different microenvironmental signals can interact with each other at the epigenomic level affecting genome-wide chromatin accessibility, cofactor binding, and enhancer activity in human and murine macrophages. These complex interactions decisively influence transcriptomic profiles of macrophages resulting in complex macrophage phenotypes under different physiological and pathological conditions . Many microenvironmental signals including pathogen-derived molecules, cytokines, and lipids can activate SDTFs turning on signal-specific gene expression programs –32. Tollnditions –40. It iThe nuclear receptor superfamily contains various transcription factors that act as SDTF and play a crucial role in the signal translation from constantly changing lipid microenvironment to gene expression alterations. This functional complexity is based on the evolutionarily conserved protein structure. All nuclear receptors consist of N-terminal ligand-independent activation function (AF-1), DNA binding (DBD), hinge or linker, ligand binding (LBD), and ligand-dependent terminal activation (AF-2) domains. Highly conserved two zinc-finger motifs containing DBDs are responsible for the recognition and binding of specific DNA sequences known as hormone response elements. More diverse LBDs recognize receptor-specific lipid ligands and form dimerization surfaces, while AF-2 domains within LBDs create a binding surface for coactivator and corepressor complexes .The nuclear receptor superfamily includes both classical endocrine receptors such as receptors for steroid hormones, thyroid hormones, and fat-soluble vitamin A or D and various orphan receptors whose ligands were initially unknown. Since their discovery, many orphan receptors become ″adopted″ by identification of their specific endogenous ligands for instance oxysterols for LXRs or short-chain fatty acids for PPARs. However, several receptors remained orphans without known endogenous ligands . DespiteSeveral lipid-sensing nuclear receptors including LXRs, PPARγ, PPARδ, and their heterodimerization partners RXRα and β are expressed in macrophages and their expression levels and ligand-dependent activities are tightly regulated by various microenvironmental signals –48. The in vivo relevance. However, several pieces of evidence show that 9-cis-13,14-dihydro retinoic acid meets better the criterion of a physiological RXR ligand , 21 in the fruit fly, 33 in the amphioxus, 47 in the rat, 49 in the mouse, and 48 in the human genome  is routinely used to study the genome-wide binding of transcription factors and cofactors as well as post-translational histone modification patterns in eukaryotic cells. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) is suitable for the identification of open chromatin regions, while Global Run-On Sequencing (GRO-seq) can detect and quantify the nascent RNA expression –70. TherElovl5, Abca1, and Abcg1 was diminished in the absence of PU.1. Conversely, PU.1 binding, and the active enhancer mark H3K4m1 pattern did not show any differences in LXRα/β double deficient BMDMs  are associated with significantly regulated nascent RNA expression following RXR agonist LG268 treatment in non-polarized murine BMDMs. In the case of the remaining part of the RXR cistrome, RXR binding is observed at transcriptionally silent (GRO-seq negative) or transcriptionally active (GRO-seq positive) but LG268 insensitive genomic regions. These findings suggest that a significant part of RXR cistrome is ligand insensitive or just responds to ligands of heterodimerization partners . AlthougIt has been previously described that IL-4 can enhance the ligand-dependent activity of PPARγ in human and murine alternatively polarized macrophages through three different mechanisms including EGR2 transcription factor-dependent activation of its expression, induction of endogenous ligand producing mechanisms, and direct protein-protein interaction with IL-4-activated STAT6 –90. Nevevia the binding of endogenous metabolites to non-canonical ligand-binding surfaces cannot be completely excluded.Even though the above-mentioned studies indicate that a part of lipid-sensing nuclear receptor cistromes is ligand insensitive, the factors affecting their enhancer-specific ligand sensitivity are partially enigmatic. Our knowledge about the three-dimensional structure of lipid sensing nuclear receptors is essential to solving this enigma. In the last two decades, the structural biologists extensively studied the three-dimensional structures of lipid-sensing nuclear receptor complexes such as PPARγ:RXR and LXR : RXR heterodimers contributing to the better understanding of their interactions between various ligands, co-factors, or DNA –98. InitIt has been also demonstrated that the nucleotide sequence of the binding motif can modulate the three-dimensional nuclear receptor structures and their interactions with DNA influencing the ligand sensitivity. Studying the PPARγ cistrome in alternatively polarized murine BMDMs, we found that the extended DR1 motif was significantly enriched at the synthetic agonist rosiglitazone-activated enhancers. In contrast, ligand insensitive regulatory regions lack this extension and harbor a shorter, more canonical RXR binding site . It has via inhibited recruitment of Thyroid hormone receptor-associated protein 3 (THRAP3)  , 106. It(THRAP3) , 108. Thin vivo chemical affinity capture and massively parallel DNA sequencing (Chem-seq) is suitable method to identify the genomic sites bound by small chemical molecules including nuclear receptor activity modifier molecules . ZC was supported by the Premium Postdoctoral Fellowship Program of the Hungarian Academy of Sciences.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.


Among the major mechanisms involved in tumorigenesis, DNA methylation is an important epigenetic modification impacting both genomic stability and gene expression. Methylation of promoter-proximal CpG islands (CGIs) and transcriptional silencing of tumor suppressors represent the best characterized epigenetic changes in neoplastic cells. The global cancer-associated effects of DNA hypomethylation influence chromatin architecture and reactivation of repetitive elements. Moreover, recent analyses of cancer cell methylomes highlight the role of the DNA hypomethylation of super-enhancer regions critically controlling the expression of key oncogenic players. We will first summarize some basic aspects of DNA methylation in tumorigenesis, along with the role of dysregulated DNA methyltransferases and TET (Ten-Eleven Translocation)-family methylcytosine dioxygenases. We will then examine the potential contribution of epimutations to causality and heritability of cancer. By reviewing some representative genes subjected to hypermethylation-mediated silencing, we will survey their oncosuppressor functions and roles as biomarkers in various types of cancer. Epithelial-to-mesenchymal transition (EMT) and the gain of stem-like properties are critically involved in cancer cell dissemination, metastasis, and therapeutic resistance. However, the driver vs passenger roles of epigenetic changes, such as DNA methylation in EMT, are still poorly understood. Therefore, we will focus our attention on several aspects of DNA methylation in control of EMT and metastasis suppressors, including both protein-coding and noncoding genes. During multistep tumor progression, both mutational and nonmutational genomic changes result in the selection and expansion of cancer cell subclones exhibiting selective advantage in the tumor microenvironment (TME), in both primary and secondary sites of cancer growth . In addiPioneering studies on cancer-associated epigenetic reprogramming were mainly focused on DNA methylation, while the interactions between DNA methylation, repressive histone modifications, recruitment of epigenetic readers and nucleosome remodeling represent relatively recent areas of investigation ,3.Herein, we will first give an overview of alterations of DNA methylation in cancer cells and then focus on some recent findings on the role of DNA methylation in EMT and metastasis mechanisms.Aberrant DNA methylation is the prevalent epigenetic dysregulation in cancer and consists of both losses (DNA hypomethylation) and gains (DNA hypermethylation) of 5-methyl-cytosine within the CpG dinucleotides. Methylome analyses have shown that, compared to the surrounding healthy tissues, nearly every cancer type at advanced stages holds large hypomethylated regions , along wApcmin model of intestinal tumorigenesis, indicate that DMRs  contribute to the distinction between the early epigenetic alterations occurring in adenomas, compared to the late events promoting tumor progression. These analyses reveal a DNA methylation signature partly conserved in human colorectal cancer [Apc functional loss [This general conclusion mainly results from comparisons between heterogenous tissue specimens (tumor vs surrounding mucosa), rather than specific cell types. However, multiple lines of evidence show that DNA hypomethylation further correlates with the cancer-cell-type specificity and tumor progression stages. For example, recent works based on the murine l cancer . Remarkanal loss .As summarized in N-methyltransferase EZH2 (enhancer of zest homologue 2) binding and repressive marks. Gene-poor domains encompassing constitutively repressed regions are recruited at the nuclear periphery trough lamina association. The repatterning of DNA methylation and heterochromatin structures (loss of histone methylation within LADs and LOCKs) is integrated with an extensive reorganization of the nuclear architecture, with an impact on cancer-associated gene expression [The hypomethylated CpGs, which affect both transcription and genome stability, are concentrated in very large hypomethylated domains spreading across vast regions of the cancer cell genome. Hypomethylated blocks largely overlap with LOCKs (large organized chromatin K-modifications marked by H3K27me3 and H3K9me2/me3) and LADs (lamina-associated domains) , which rpression .DNA hypomethylation contributes to genomic instability through mechanisms reflecting a whole reorganization of the global genome architecture. In cancer cells, DNA hypomethylation corresponds to hotspots of chromosomal breaks and largely overlaps with repetitive sequences localized within centromeric, pericentromeric, and subtelomeric chromosomal regions, thus resulting in the reactivation of repeat elements. Reactivation of centromeric satellite repeats (microsatellites) and dormant repeat elements, such as the long interspersed nuclear elements (LINEs) is a major determinant of chromosomal instability. Moreover, DNA hypomethylation of LINEs and other transposable elements increases the odds of transposition to other genetic loci, thus contributing to genomic rearrangements by retrotransposition .In addition to the well-established effects of large-scale DNA hypomethylation on genome architecture and chromosomal instability, recent analyses point to the specific function of DNA hypomethylation of distal regulatory sites as a determinant of cancer gene dysregulation . Genome-Recent genome-wide analyses  have been focused on super-enhancer regions, defined as clusters of transcriptional enhancers exhibiting maximal levels of histone H3K27 acetylation and BRD4 recruitment , exhibitCEBPB , representing a major driver of global transcriptional reprogramming in Hepatocellular carcinoma (HCC) tumor progression [The causal links between cancer-associated enhancer hypomethylation are supported by analysis of enhancer-associated DMRs in various tumors. In hepatic carcinogenesis, among 369 differentially methylated enhancers, a key role is played by the recurrently hypomethylated enhancer of gression . Hypermethylation of promoter-associated CGIs likely represents the best studied epigenetic change in tumorigenesis. CGIs are genomic regions at least 200 bp long, with 50% GC dinucleotides and an observed-to-expected CpG ratio > 0.6. CGIs are flanked on both sides by (approx. 2kb-long) CGI shores, regions of DNA with a low density of CpG dinucleotides, where most of the cancer-associated methylation differences are detectable .Hypermethylation of promoter-associated CGIs has been predominantly associated with the transcriptional silencing of TSGs or mismatch repair genes involved in most cancer-relevant pathways . DNA metCancers can be classified according to their degree of methylation, and the tumors with high degrees of methylation represent a clinically and etiologically distinct group characterized by epigenetic instability. Specifically, in some tumors the concurrent multiple promoter hypermethylation of tumor-related genes across the genome is referred to as CIMP (CGI methylator phenotype) . RemarkaThe cancer-associated epigenomic reprogramming, involving global DNA hypomethylation and reorganization of heterochromatic regions and hypermethylation of promoter-associated CGIs and CIMP, results from somatic genetic mutations and/or expression changes of various epigenetic regulators . Among D5-hydroxymethylcytosine (5-hmC) is a recently identified cytosine modification, resulting from the oxidation of 5-mC, catalyzed by the TET family of methylcytosine dioxygenases . TET enzymes are responsible for the successive oxidation of 5-mC to 5hmC, 5fC (5-formylcytosine), and 5caC (5-carboxylcytosine). These residues are the intermediates in the demethylation process in which the TDG (thymine DNA glycosylase), by hydrolyzing the bond between the base and deoxyribose ring, produces an AP (A-Pyrimidinic) site that is replaced by an unmethylated cytosine by BER (base-excision repair) ,27.Mutations of DNMTs and the resulting dysregulation of genomic methylation are involved in neoplastic transformation and tumor progression. DNMTs abnormalities are associated with a large variety of tumors in which they cause aberrant patterns of DNA methylation . Common In agreement with the DNMT3A role in hematopoietic stem cell differentiation , DNMT3A In a variety of tumors  overexpression rather than mutation of DNMTs  results in hypermethylation and oncogenic activation. Heterogeneous degrees of DNMT1 overexpression are associated with primary colon cancer cells, while DNMT3B overexpression has been related to CIMP-high colon cancer, although the overexpression of the protein not always parallels the RNA levels .IDH1/2 genes, encoding the isocitric dehydrogenases metabolic enzymes, result in the accumulation of 2-hydroxyglutarate, which inhibits the TET protein enzyme activity. The evidence that in some tumors, such as AML (acute myeloid leukemia) TET2 and IDH mutations are mutually exclusive, support their involvement in the same pathway. Accordingly, the TET2- and IDH-mutated clinical samples exhibit similar DNA methylation profiles [More recently, the role of TET proteins in neoplastic transformation has been established by the identification of inactivating mutations, mainly affecting TET2, in various types of cancer with a higher prevalence of hematological malignancies with respect to solid tumors ,27. Moreprofiles ,34.Dnmt1 causes DNA hypomethylation and genomic instability leading to aggressive T-cell-induced tumorigenesis, associated with increased mutation rates and aneuploidies [Dnmt1, whose reduction resulted in increased tumor incidence. In addition, the lack of de novo methyltransferase activity accelerates oncogene-driven carcinogenesis, indicating that also Dnmt3a and Dnmt3b may act as oncosuppressor genes [Despite the number of mechanistic aspects of tumor-associated DNA demethylation that deserve further elucidation, data from model organisms supports the oncogenic function of the loss of DNA methylation. A seminal evidence has been provided by mouse models in which a strong hypomorphic allele or deletion of ploidies . Functioor genes .Dnmt1 can exert oncosuppressor or tumor promoter activity depending on the cancer cell context. In murine models, the MYC transgene induces T-cell lymphomagenesis, while the MLL-AF9 (mixed lineage leukemia-ALL1-fused gene from chromosome 9) chimeric oncoprotein drives AML. Loss of Dnmt1 delays lymphomagenesis and leukemogenesis by suppressing normal hematopoiesis and impairing tumor cell proliferation and leukemic stem cell self-renewal. Therefore, in this context, DNMT1 may be important for tumor maintenance [DNMTs genes have been described in many tumors and related to aggressiveness and therapeutic resistance, the frequency of DNMT1 somatic mutations is relatively low  [Nevertheless, the role of maintenance methylation in tumor development is controversial, since ntenance ,37. The  of AML) ,39,40.Epigenetic aberrations represent an emerging mechanism that plays a pivotal role in carcinogenesis. The term epimutation  describecis-acting DNA alterations [cis-acting genetic mutations. Epimutations due to germline variants are widely distributed in all normal somatic tissues. Constitutional epimutations are epigenetic abnormalities arising in early stages of embryonic development and result in a mosaic of both mitotically and meiotically heritable changes. Somatic (de novo) epimutations accumulate in somatic cells in response to environmental stresses and/or ageing. In cancer-affected individuals, epigenetic abnormalities can be selectively found in tumor cells or detected in preneoplastic lesions as well in macroscopically normal tissues adjacent to or within the same organ as the tumor site. The detection of cancer-related somatic epitypes in normal tissues strongly suggests that some epigenetic alterations are acquired prior to carcinogenesis. Somatic epitypes have also been observed in cancer-free individuals and associated with ageing [Epimutations include primary epimutations (such as promoter methylation) where no DNA alterations are detected, and secondary epimutations, occurring in concert with (and caused by) a local erations . Inherith ageing . Environh ageing .Somatic epimutations can participate to cancer initiation or contribute to cancer progression by activating oncogenes and prometastatic genes and/or by inactivating growth-inhibitory genes or key TSGs. Silencing of TSGs by de novo aberrant promoter DNA hypermethylation is an early oncogenic event that contributes to clonal expansion of the preneoplastic cell populations and precedes the onset of malignant growth. Epigenetic abnormalities can also represent secondary consequence of neoplastic transformation due to somatic genetic mutations hitting some epigenetic regulator(s). Germline and constitutional epimutations can precede and predispose to cancer development and/or represent a tumor-initiating event. Therefore, constitutional and germline epimutations provide an alternative mechanism to genetic mutation for cancer predisposition and inheritability, thus contributing to the individual cancer susceptibility. However, evidence linking epimutations to cancer risk has been reported for a limited number of genes .H19-IGF2 genomic imprinting in Wilms’ tumor. Abnormal methylation of the imprinting control region (ICR) on the maternal allele leads to LOI of the H19-ICR locus. The consequently reduced expression of the maternally expressed noncoding transcript H19 allows the activation of IGF2. Therefore, H19 works as a tumor suppressor, by preventing the biallelic expression of IGF2 [Observational studies in cancer-affected families point to the possible role of intergenerational inheritability of constitutional epimutation in human tumorigenesis. Loss of imprinting (LOI) in Wilms’ tumors represents a seminal example of constitutional epimutation. LOI refers to the altered methylation states leading to either biallelic expression or complete silencing of the genes that are normally expressed monoallelically in a parent-of-origin-specific manner. A paradigmatic example is the dysregulated  of IGF2 .The tumor suppressor genes silenced by aberrant promoter hypermethylation encode a wide range of protein products, including cell cycle inhibitors (INK4A/p16 and Rb), DNA repair factors, detoxifying enzymes , angiogenesis inhibitors , cell–cell adhesion receptors , metalloprotease inhibitors , and many others ,46. MoreCDKN2A (cyclin-dependent kinase inhibitor 2A) gene, encoding the INK4A/p16 cell cycle inhibitor, blocking the CDK4/6 activity. CDKN2A promoter hypermethylation is responsible for the inactivation of the wild-type allele in CRC cells in which the other allele has been lost by deletion. INK4A methylation and loss of p16 expression is an early event in breast and lung cancer . Accordingly, in breast cancer specimens, INK4a promoter hypermethylation is detectable even in histologically normal human mammary epithelia [RB1 in human retinoblastoma represents another key example of promoter hypermethylation as the second hit. Similar mechanisms affect various TSGs in tumors associated with familial cancer syndromes caused by heterozygous germline mutations [In agreement with the Knudson two-hit hypothesis , hypermepithelia . Inactivutations .MLH1 (mutL homolog-1), MSH2 (mutS homolog 2), MGMT (O6-methylguanine-DNA methyltransferase), and BRCA1/2 (breast cancer gene 1 and 2) [The major TSGs implicated in DNA repair mechanisms include 1 and 2) .MLH1 promoter hypermethylation and epigenetic silencing is implicated in different types of cancers characterized by mismatch repair (MMR) deficiency, which causes insertions or deletions in repeated sequences. Microsatellite instability (MSI) is detected in 90% of hereditary form and 10–15% of sporadic CRCs . HNPCC  or Lynch syndrome is mainly due to germline pathogenic variant in MLH1 or MSH2 mismatch-repair genes [MLH1 [MLH1 methylation occurs quite rarely in hereditary forms (Lynch syndrome) and cancer risk associated with the epigenetic mosaicism needs to be ascertained. In contrast, MLH1 hypermethylation is highly frequent in sporadic CRC with MSI. Therefore, methylated MLH1 can represent a specific marker for sporadic MSI tumors, which can be used to select patients for genetic testing for Lynch syndrome [ir genes , except es [MLH1 . Neverthsyndrome .MSH2 mismatch-repair gene is very frequent and associated with relapse in multiple cancers, constitutional MSH2 epimutation in families with Lynch syndrome is a rare event [MSH2 methylation in colon mucosa and stemming CRC cells has been found associated with a deletion in an upstream gene  [While methylation of the promoter region of the re event . Remarkaolecule) .MGMT gene, encoding the O-6-methylguanine-DNA methyltransferase, essential for reversing the addition of alkyl groups to guanine residues. Therefore, its involvement in CRC predisposition remains unexplored and warrants further research [MGMT promoter methylation is found associated with a SNP localized in the first exon [Similarly, in hereditary CRC no germline or constitutional epimutations have been detected so far in the research ,54. In srst exon .BRCA1 promoter methylation has been reported in 30–35% of all triple-negative breast cancers with germline BRCA1/2 wild-type status, mainly the basal-like subtype. BRCA1 methylation in endoderm- and mesoderm-derived normal tissues both in patients and in cancer-free individuals supports the incidence of constitutional epimutations . In som variant . MLH1 and CDKN2A(p16) methylation combined with the CIMP status exhibit clinical and prognostic value in colorectal cancer [Genome-wide DNA sequencing and methylome analyses made available by international consortia (such as the TCGA project), confirm the much higher frequency of tumor-associated epigenetic changes compared to genetic mutations. The number of genes silenced by cancer-associated promoter hypermethylation has dramatically increased during the years, reaching almost 10% of the CGI-containing promoters. Therefore, genome-wide CGIs methylation profiling, in addition to providing novel biomarkers for diagnosis and prognostic predictions, is a valuable tool for the identification of novel oncosuppressor genes. For example, analyses of l cancer .MLH1 promoter methylation is a well-established biomarker for multiple solid tumors, including esophageal, NSCL, gastric, bladder, and papillary thyroid cancer. In CRC, methylated MLH1 is one of the major epigenetic biomarkers along with other genes involved in the tumor progression and metastasis, such as CDKN2A/p16(INK4A), CDKN2A/p14(ARF), MGMT, TIMP, THBS1 3, and THSD1 (thrombospondin type 1 domain containing 1), identified in genome-wide screening for methylation-silenced genes, along with meta-analysis of clinical data. CDKN2A hypermethylation correlates with tumor progression, metastasis, and overall survival, thus representing a promising diagnostic and prognostic biomarker in HNSCC (head and neck squamous cell carcinoma) [In addition to CRC, rcinoma) .(GALR1 and GALR2) [TACR1) [SST and SSTR1) [SST hypermethylation is also detectable in esophageal, gastric, colon, and renal cancer in which the prognostic power of GALR1 and GALR2 epigenetic modifications is further confirmed [MGMT (90%), DAPK (death-associated protein kinase) (91%) and CDH1 (E-cadherin) (81%) (but not p16) is a frequent event, although not predictive of mortality or second primary cancer [In addition to the well-known tumor suppressors, novel genes have emerged as candidate TSGs and novel biomarkers. For example, aberrant methylation and silencing of neuropeptides and GPCRs, including galanin and galanin receptors d GALR2) , tachyki [TACR1) , and somd SSTR1)  are commonfirmed . Among ty cancer .MLH1 hypermethylation confers resistance to multiple chemotherapeutic drugs in ovarian and colorectal cancer. In human gliomas, MGMT downregulation correlates with poor prognosis but also with drug-responsiveness. Accordingly MGMT promoter methylation is associated with sensitivity to alkylating agents (temozolomide) [BRCA genes predicts sensitivity to PARP inhibitors, which are highly effective in the treatment of BRCA-positive breast and ovarian cancers (reviewed in [The prognostic relevance of the above-mentioned epigenetic biomarkers is further highlighted by their predictive role in the responses to chemotherapeutic treatments. olomide) . Similariewed in ).Recent strategies rely on the genome-wide methylation profiling aimed at the identification of novel prognostic signatures. This is crucial for discriminating the prospectively localized from the tumors at high risk for metastatic progression.Representative examples include breast and prostate cancer. Genome-wide DNA methylation profiles have been generated from almost 300 tumor tissue specimens and validated in independent datasets and in large numbers of samples from TCGA. These studies have provided a prognostic signature based on methylation changes of 15 CGIs, which correlates with survival of patients diagnosed with invasive breast tumors or DCIS  . SimilarAmong the innovative protocols aimed at minimally invasive cancer diagnosis, the immunoprecipitation-based cfMeDIP-seq (cell-free methylated DNA immuno-precipitation sequencing) allows to perform cost-effective methylome analyses on small amounts of circulating cfDNA (cell-free DNA). Thus, the large-scale DNA methylation changes allow the detection and classification of several tumor types in patients with early-stage disease .Metastatic colonization is a complex process, resulting from multiple sequential steps: cells detachment from primary tumor, tissue invasion, intravasation, survival in blood or lymphatic stream, extravasation, and formation of micro-metastases that eventually progress to clinically apparent metastases .According to the current model of parallel progression of primary tumors and metastasis , based oEMT, along with the reverse mechanism , represents the trans-differentiation process, which allows cancer cells to switch reversibly between epithelial and mesenchymal phenotypes . EMT depCDH1), which is essential for the intercellular contacts supporting epithelial integrity, represents one of the key hallmarks of EMT [Loss of the major component of adherens junctions, E-cadherin  raises the question on the relationship between EMT transcriptional control and epigenetic modifications at the CDH1 locus. The time-dependent response of immortalized human mammary cells to sustained EMT-inducing treatments shows that E-cadherin transcriptional repression precedes the methylation of the CDH1 CGI [CDH1 silencing strongly correlates with the promoter methylation of other genes, such as ESR1 , silenced in a subset of invasive breast cancer . Accordingly, both CDH1 and ESR1 are represented in a signature of genes silenced by promoter methylation, which characterizes the TNBC, basal-like, and claudin-low breast cancers [DNMT3B overexpression. The hyper-methylator phenotype, characterizing a distinct cluster of basal-like breast cancers, co-segregates with the worst prognosis due to the high rate of metastases [ESR1 and TWIST1) are targeted by DNA methylation in response to microenvironmental TGF-beta favors the role of deterministic vs stochastic mechanisms, as responsible for the epigenetic reprogramming in the phenotypically plastic cell populations undergoing EMT [The well characterized CDH1 CGI . The in  cancers . These rtastases . Moreoveoing EMT . HRAS-transformed (MCF10A-derived) cell lines. In this breast cancer progression system, increased activity of the TGF-beta-Smad2 signaling pathway is associated with the DNA methylation-mediated silencing of a gene subset including CDH1. Along with the mesenchymal cancer cell phenotype, inhibition of TGF-beta signaling is able to revert the methylation status and expression of select genes by inhibiting the DNMT1 and DNMT3B binding to the CDH1 and other promoter regions [Mesenchymal transformation of human breast cancer cells can take place in vivo, without TGF-beta treatment, in a mouse xenograft model of  regions .CDH1 and COL1A1 (Collagen Alpha-1 Chain), are paralleled by the TGF-beta-induced expression and activity of DNMTs . Accordingly, the treatment with a DNMT inhibitor prevents the TGF-beta-induced EMT [The global changes of DNA methylome in the TGF-beta induced EMT in ovarian cancer cells show that the promoter methylation and decreased expression of EMT hallmarks, such as uced EMT .INSIG1, insulin-induced gene 1) by the TET1 dioxygenase, induced by hypoxia in multiple cancer cell lines. Interestingly, in addition to its role in demethylating 5-mC, TET1 also works as a coactivator of HIF1, participating to the transcriptional induction of the hypoxia-responsive genes [Among the well-characterized EMT-inducing factors, hypoxia, as TGF-beta, results in a global epigenetic reprogramming and changes of the DNA methylome. The hypoxia-induced EMT depends on the demethylation of a subset of genes (with a major role played by ve genes .HMGA2), the HMGI-C chromatin remodeling activity contributes to the Cdh1 hypermethylation by recruiting Dnmt3a on the Cdh1 promoter, in addition to upregulating the Dnmt3a expression [In a mouse cell line, in which EMT is driven by the constitutive expression of a nonhistone chromatin component , several relevant metastasis suppressor genes are differentially methylated and transcriptionally silenced in metastatic lesions with respect to primary tumors  is another metastasis suppressor impinging on the proinvasive MAPK pathway, by inhibiting the Raf-1 (but not B-Raf) kinase activity . The pror growth .SHISA3 gene product inhibits the TRIM21 (tripartite motif containing 21 E3 ubiquitin-proteinigase)-dependent degradation of SGSM1 , which negatively controls the MEK/ERK signaling. When SHISA3 is silenced, the increased SGSM1 polyubiquitylation and degradation abrogates one of the brakes impinging on the MAPK pathway. Accordingly, SHISA3 is downregulated in various tumor types, including nasopharyngeal carcinoma (NPC) in which the SHISA3 promoter hypermethylation correlates with the SHISA3 ability to suppress the NPC in vitro invasion and in vivo lymph node metastasis [Among the more recently characterized metastasis suppressors affecting the MAPK pathway, the tastasis . RECK encodes for a membrane glycoprotein , which negatively regulates the ECM-degrading metalloproteases. RECK is silenced by DNMT3b-mediated promoter methylation in lung cancer cells in which RECK suppresses invasiveness [The metastasis suppressor siveness  and invesiveness , PDAC —which inhibits breast  cancer, in addition to cancer metastasis without affecting in vitro and in vivo tumor growth—participates to the mSin3 histone deacetylase transcriptional repressor complex. Interestingly, in addition to correlating with the pathological staging in breast cancer and NSCLC [BRMS1 (breast cancer metastasis suppressor 1) promoter methylation represents a relevant example of prognostic biomarker detectable by liquid biopsies in both cfDNA [Other metastasis-suppressor gene products act as transcriptional regulators. In particular, nd NSCLC ,104, theth cfDNA .The prognostic value of DNA methylation analysis in cfDNA has been recently applied to the prediction of therapeutic resistance in multiple solid tumors. In addition to the ability to disseminate and metastasize, the EMT also confers resistance to both cytotoxic and targeted therapies. Since the DNA methylation-driven mesenchymal transition correlates with therapeutic resistance, monitoring of DNA methylation changes in EMT genes allows to predict the tumor responsiveness and acquired resistance to sorafenib in patients with advanced hepatocellular carcinoma . The rolIn addition to protein-coding genes, the search for metastasis suppressors has been extended to noncoding RNAs. By correlating the methylation of CGIs and expression profiles of miRNA-containing genes in metastatic cancer cell lines treated with demethylating drugs, several miRNAs reactivated by promoter demethylation were identified and characterized for their in vitro and in vivo anti-invasive and antimetastatic activity  Table 2Table 2.The interplay between the EMT-TFs and miRNAs is a major control mechanism of EMT, invasion and metastasis. The EMT-TFs (E-cadherin repressors) are connected to miRNAs by double-negative feedback loops in which the miRNA targets the EMT-TF, which in turn, transcriptionally inhibits the cognate miRNA. Well-studied examples include the miR-200-ZEB1/2, miR-34a/b/c-SNAIL, miR-15a/16-1-AP4, etc. These regulatory feedback loops are further controlled by the p53 tumor suppressor, which, as transcriptional inducer of the three miRNA families , shifts the balance towards the miRNAs, thus contributing to the maintenance of the epithelial state ,108.During tumor progression, promoter hypermethylation is a major mechanism of silencing of the miR-34 family of oncosuppressor miRNAs transcribed from two genetic loci, encoding for miR-34a and miR-34bc ,111,112.ZEB1, and/or ZEB2, thus interrupting the miR-200-ZEB1/2 negative feedback loop. Interestingly, in an in vitro EMT cell system, prolonged TGF-beta treatment induces the DNA methylation of the five miR-200 promoter regions [CDH1 transcription by both direct and indirect mechanisms, mediated by hypermethylation of the CDH1 promoter and/or DNA hypermethylation-mediated transcriptional downregulation of the miRNAs targeting the E-cadherin repressors (EMT-TFs).The miR-200 family members, encoded by two miRNA clusters localized on chromosome 1  and chromosome 12 (miR-200c and miR-141), are highly expressed in cancer cells with epithelial features and downregulated in phenotypically mesenchymal cells, in which they are repressed by the EMT-TFs ZEB1 and ZEB2. Extracellular EMT-inducing cytokines, such as TGF-beta, transcriptionally stimulate  regions  in agree regions ,120,121. regions ,123. Mor regions . In summCRB3 and LGL2, involved in the control of epithelial cell polarity. The CGI hypermethylation, along with the associated histone modifications of the two loci encoding the five miR-200 family members, is a dynamic process, which can be reversibly triggered by extracellular cues, such as TGF-beta. In addition, the methylation-mediated silencing of miR-200 takes place in experimental models of liver metastasis. Accordingly, in laser micro-dissected human colorectal cancer samples miR-200 CpG hypermethylation and silencing are detectable in mesenchymal cells  [Although E-cadherin represents the best characterized target of ZEB1/2 in EMT, the miR-200-ZEB1/2 axis also controls other key genes, such as  stroma) .Along with miR-200 and miR-34 family members, silencing of other CpG-hypermethylated miRNAs favors the metastatic process through several proinvasive pathways. In pancreatic cancer, hypermethylation of the miR-124 promoter region  correlates with tumor progression and metastasis, through mechanisms at least partially mediated by miR-124. miR-124 targets the RAS-superfamily GTPase Rac1, a well-characterized driver of cell motility, which in turn, drives cell migration and invasion through the MKK4-JNK-c-Jun pathway in pancreatic cancer cells .DNA methylation is the major control mechanism of genomic imprinting. The 14q32 locus encompasses three paternally expressed protein-coding genes and a large variety of maternally expressed noncoding genes , also including a cluster of 54 miRNAs. In CRC cells, both genetic and pharmacological strategies reactivate the 14q32 miRNA cluster. Drug-induced reactivation is mediated by the MEG3 -DMR regulatory element, which controls the transcription of the 14q32 miRNAs, through mechanisms dependent on the recruitment of CTCF (CCCTC-binding factor) to the demethylated MEG3-DMR. Ectopic re-expression of four members of the 14q32 miRNA cluster is sufficient to inhibit liver metastasis by colorectal cancer cells in agreement with clinical data showing the association of increased 14q32 miRNAs expression with limited metastatic spread and better prognosis .It is well established that the EMT generates cells with properties of stem cells . AccordiBesides being a promising new technique for early cancer diagnosis and treatment, noninvasive liquid biopsy has strongly impacted the studies on the role of DNA methylation, EMT, and metastasis. In particular, various methods have been developed to detect and isolate the CTCs from easy-access small blood samples for downstream analysis. Protocols based on physical features (size- and density-based methods), antibody-mediated capturing (immunoaffinity-based), or functional assays (or combinations of these methods) allow real-time information on tumor staging (metastatic vs. nonmetastatic) and the molecular profiling of CTCs.As expected on the basis of the role played by EMT in intravasation and anoikis-resistance, CTCs from patients affected by the major solid tumors exhibit prevalent expression of mesenchymal vs epithelial markers .CTCs isolated from breast cancer patients include cell populations predominantly expressing mesenchymal markers but also epithelial and E/M subpopulations, coexpressing both epithelial and mesenchymal markers) . The detThe evidence that the mesenchymal CTCs derived from breast tumors are present as both single cells and multicellular clusters  agrees wRecently, to understand the role of CTC clusters in metastasis mechanisms, the genome-wide methylation landscape of single CTCs and CTC clusters has been investigated in samples from both breast cancer patients and mouse models. One major difference is represented by the methylation of the genomic binding sites for stemness-associated transcription factors , which are specifically hypomethylated (while Polycomb target genes are hypermethylated) in CTC clusters compared to single CTCs. Moreover, the clustering of CTCs is a determinant of methylome remodeling and hypomethylation of the stemness TFBSs . These results were obtained by the treatments with drugs that dissociate the CTCs clusters. Accordingly, the same drugs inhibit the formation of metastasis in mouse models and the DNA hypomethylation profile of CTCs clusters correlates with a subset of breast cancers exhibiting shorter PFS  .The contrast between these findings and the key role of EMT in metastasis is only apparent if considering the lines of evidence showing that the hybrid E/M, rather than the fully mesenchymal phenotype, is essential for maximal tumorigenicity of TNBC cells . RemarkaSOX2 and NANOG, is higher within eDMRs than within differentially methylated CGIs or promoters in agreement with the findings obtained in CTCs [The extensive analysis of eDMRs  in multiple datasets representing both primary  tumors shows that most methylation variation occurs at enhancers. eDMRs analysis allows the classification of primary tumors according to the organ system and correlates with the likelihood of metastasis. The eDMRs plasticity is associated with the prognosis of melanoma patients. Interestingly, mimicking the melanoma bone metastasis microenvironment (by coculture with osteoblasts) recapitulates the expression changes of the eDMRs-associated genes altered in bone metastasis . In addi in CTCs .The EMT and the upregulation of pluripotency transcription factors result in the gain of stem-like features, which characterize the CSC population, responsible for tumor initiation, metastatic dissemination and therapeutic resistance.NANOG shows ubiquitous expression correlating with the hypomethylation of two of the CGIs associated with the NANOG promoter in response to the miR-135a/DNMT1/SMYD4 axis. The switch from non-CSCs to CSCs is associated with miR-135a upregulation. miR-135a, in turn, directly targets DNMT1, thus allowing SMYD4 to bind the unmethylated NANOG promoter and activate its expression. Moreover, the proinflammatory cytokine TNF-alpha triggers the FOXM1 (Forkhead Box M1)-mediated induction of miR-135a, enabling the transformation of non-CSCs (Nanog−) into CSCs (Nanog+). Therefore, the TNF-alpha/FOXM1/miR-135a/DNMT1/SMYD4 pathway links the control of NANOG expression to the inflammatory TME [Recent findings point to the key role of DNA methylation in the switch between CSCs and non-CSCs. In CSCs, tory TME .NANOG promoter, the silencing of the H1.0 (encoding the H1.0 linker histone) is required for maintenance of the self-renewing CSCs fraction. According to single-cell gene expression and methylation analysis of the H1F0 locus in clinical samples, compared to various TCGA datasets, in multiple tumor types H1.0 is heterogeneously expressed in non-CSC tumor cells, while the self-renewing CSC population exhibits the lowest levels of the protein. These changes result from differential methylation of an enhancer region (characterized by high H3K27ac) within a CGI shore, upstream to the H1F0 promoter. The lack of H1.0 causes the destabilization of nucleosome-DNA interactions and coordinated de-repression of neighboring genes, resulting in the activation of transcriptional networks involved in cancer cell self-renewal [In contrast with the cancer cell stemness-associated demethylation of the -renewal .We have summarized several aspects concerning the gene expression changes associated with abnormal DNA methylation in neoplastic transformation with a focus on DNA methylation in EMT and metastasis mechanisms.DNA methylation represents one of the key epigenetic players, along with the recently emerged noncoding RNAs, including the relatively well-characterized miRNAs. As above described, the EMT-associated gene expression programs are finely tuned by multiple feedback loops involving the major metastasis-suppressor miRNAs and several EMT-TFs.DNMTs and methyl-CpG binding proteins, which in turn, control the transcription of miRNA-coding genes [Similarly, a novel regulatory layer is represented by the mutual regulation of miRNAs and DNA methylation, involving the miRNA-mediated regulation of both ng genes . These mBRCA1 tumor suppressor [As summarized in this review, DNA methylation critically controls the expression of a wide range of oncosuppressor and metastasis suppressors. Functional validation generally relies on the effect of global gene reactivation by treatment with demethylating agents and/or ectopic overexpression of individual genes. However, several recently developed methods, derived from the CRISPR-Cas9 editing tool and based on the sequence-specific recruitment of the catalytic domain of the TET1 dioxygenase, allow the targeted demethylation at specific sites . This apppressor . These sThe application of diagnostic and prognostic tools based on DNA methylation analyses depends on the availability of low-cost methods for mapping DNA methylation at single-nucleotide resolution along with genomic datasets, such as the TCGA database, which represents most tumor types in very large cohorts of patients. The integration of genome-wide DNA methylation analyses with expression profiling and clinical data will require increasingly powerful computational algorithms. For example, the recently developed RESET  has been exploited for obtaining a pan-cancer landscape of aberrant DNA methylation, by integrating the DNA-methylation data and cis-transcriptional changes across 6000 human tumors from 24 cancer types . By idenMoreover, promising future perspectives include the studies on DNA-methylation in therapeutic resistance. Given the role of EMT in controlling the fraction of therapeutically resistant CSCs, it will be important to define the DNA-methylation changes implicated in the mechanisms linking EMT, CSCs, and therapeutic resistance . AccordiThe analyses of tumor-associated DNA methylomes in very low amounts of cfDNA is a key advantage of liquid biopsies. This approach, however, differing from the analysis of DNA from CTCs, does not allow us to correlate the DNA methylation changes with the cancer cell phenotypes. On the other hand, the possibility to investigate the EMT and stem-cell markers in CTCs offers the opportunity to study the in vivo role and prognostic correlations of the DNA methylation changes in the cell fraction implicated in metastatic dissemination . Therefo


One patient showed sustained, highly increased NFL levels  in the initial assessment when radiological signs of axonal injury were still absent. Anti-SSA(Ro)-antibodies were found in the serum of three patients, while two patients additionally presented intrathecal anti-SSA(Ro)-antibody production. Elevated CSF-NFL levels and intrathecal synthesis of anti-SSA(Ro)-antibodies were associated with a relapsing and treatment-resistant disease course. Conclusion: Inflammatory spinal cord lesions associated with pSS are a rare but serious disease leading to severe disability. NFL and anti-SSA(Ro)-antibodies in CSF might serve as prognostic biomarkers and should be routinely assessed in patients with pSS.Objective: To identify radiological and laboratory hallmarks in patients with primary Sjögren’s syndrome (pSS) presenting with spinal cord involvement. Methods: Clinical and laboratory routine parameters were analyzed in a retrospective multicenter case series of four patients who developed myelitis associated with pSS. Serological and cerebrospinal fluid (CSF) measurements of pSS associated anti-SSA(Ro)-antibodies were initiated, and CSF neurofilament light chain (NFL) levels were assessed. NFL values were compared with results from 15 sex- and age-matched healthy controls. Radiological assessment was performed using multi-sequence spinal cord magnetic resonance imaging. Results: Three of the four patients initially developed neurological signs suggestive of myelitis and were subsequently diagnosed with pSS. All patients presented a longitudinal spinal T2-hyperintense lesion in the cervical spinal cord, whereas only two patients showed pleocytosis and oligoclonal bands in the CSF. Median (range) CSF-NFL levels were significantly elevated in patients compared to controls  vs. 585 pg/mL (357–729),  Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by lymphocytic infiltrations in secretory glands and extraglandular neurological manifestations ,2. The aAnti-SSA(Ro)-antibodies can be found in approximately 50% of patients suffering from pSS. These antibodies have a high diagnostic value and seem to be associated with extraglandular disease manifestations, although seronegative patients with CNS involvement have been described ,7,8,9. IThe levels of neurofilament light chain (NFL) in serum or cerebrospinal fluid (CSF) are thought to reflect neuroaxonal damage and are proposed to have a predictive value concerning outcomes in patients with inflammatory and autoimmune diseases associated with myelitis ,12.Thus, we aimed to identify clinical, radiological, and laboratory hallmarks, including new potential biomarkers, in a detailed case series of patients with pSS presenting with spinal cord involvement.Patients were recruited at the Departments of Neurology of the University Hospitals Magdeburg, Münster, and Hannover, all located in Germany. CSF and serum samples from 15 sex- and age-matched healthy controls recruited in Magdeburg were included for comparison. All controls reported non-specific complaints and underwent lumbar puncture (LP) during a routine diagnostic examination conducted to rule out any neurological condition. None of the controls was diagnosed with a specific neurological disorder or showed any specific abnormalities during the neurological examination. In addition to the clinical classification, all controls fulfilled the following laboratory criteria defining a normal CSF: < 5 cells/μL, < 2.0 mmol/L lactate, no disruption of the blood-CSF-barrier function , no oligoclonal bands (OCB), and no intrathecal synthesis of the immunoglobulins (Ig) IgG, IgA, and IgM . In addiFor all patients, a LP was performed at each respective study site according to standard protocols. The LP for all controls was conducted in Magdeburg. At each study site, CSF cells were counted manually using a Fuchs-Rosenthal chamber. Concentrations of Albumin, IgG, IgM, and IgA in serum and CSF were measured by kinetic nephelometry. Intrathecal synthesis of IgG, IgA, and IgM was calculated according to Reiber’s revised hyperbolic function. Isoelectric focusing was applied to detect CSF specific oligoclonal bands. All samples for CSF-NFL and antibody measurements were stored at −80 °C and shipped on dry ice. CSF-NFL levels were determined in Magdeburg using commercially available ELISA kits . Each measurement was performed together with a blank and a commercial positive and negative control provided by the manufacturer. Anti-SSA(Ro)-antibodies both in sera and CSF were measured in Hannover using EliA . Samples were measured in serial procedures and not in batches.Multi-sequence MRI (1.5 Tesla) of the spinal cord was performed in all patients and included a sagittal T2-weighted turbo-spin echo, a short tau inversion recovery sequence, and a T1-weighted postcontrast sequence covering the cervical and upper thoracic cord. Additionally, axial T2-weighted turbo-spin echo sequences were used. Furthermore, multi-sequence MRI of the head was performed in patients 1, 3, and 4, and included an axial T1- (pre- and postcontrast), a T2-weighted turbo-spin echo sequence and an axial diffusion-weighted imaging sequence. Moreover, a 3D sagittal T2-FLAIR-weighted sequence was applied in patients 1 and 4 and an axial T2-FLAIR-weighted sequence in patient 3.This study was performed according to the Declaration of Helsinki and approved by the local ethics committees . All patients gave written informed consent.Demographic, clinical, and laboratory findings are provided in A 39-year-old Caucasian female with known hypothyroidism (without evidence of a Hashimoto thyroiditis), alopecia of undeterminable cause, and sicca symptoms developed progressive sensory ataxia of the lower limbs and a mild paraparesis in 10/2017. MRI examinations of the brain and spinal cord were inconspicuous in 10/2017 and 12/2017. Lower limb somatosensory evoked potentials (SEP) showed axonal affection, whereas motor and sensory neurographies provided no evidence of neuropathy. Repeated CSF investigations revealed a pleocytosis (12–45 cells/µL) and OCB in CSF A. Immuno2 body surface area). During therapy, an additional left-sided T2-hyperintense lesion, without T1-gadolinium enhancement, was detected in the posterior column of the cervical and thoracic spinal cord -antibodies under treatment with hydroxychloroquine and intermittent use of oral glucocorticosteroids (GCS), developed a mild dysesthesia of the upper and lower limbs and slight sensory ataxia in 2016. Lower limb SEP, peripheral nerve conduction studies of upper and lower limbs, and CSF analysis were inconspicuous. The cervical MRI identified a T2-hyperintense longitudinally extensive spinal cord lesion without T1-gadolinium enhancement in the posterior column as the cause for neurological symptoms C,D. DiseA 55-year-old Caucasian female with known primary biliary cholangitis developed a progressive dysesthesia of the upper and lower limbs and a mild tetraparesis in 2018. The spinal cord MRI showed a long segment T2 lesion without T1-gadolinium-enhancement in the lateral pyramidal tracts and lateral spinothalamic tracts of the cervical spine E,F. The 2 body surface), oral GCS, and finally with rituximab , under which disease progression could be stopped.Pathological changes in lower limb SEPs were recorded, whereas nerve conduction examinations of upper and lower limbs showed no impairment of motor and sensory nerves. CSF measurements did not reveal inflammatory signs. In line with the evidence of elevated anti-alpha-fodrin antibodies, objective xerophthalmia, and a pathological minor salivary gland histology, an inflammatory spinal cord lesion associated with pSS was diagnosed. Therapy was initiated with IV cyclophosphamide -antibodies in sera and CSF. An elevated antibody specificity index (ASI) was detectable in patients 1 (2.2) and 4 (5.1), while patient 2 showed no elevated ASI (1.0). We could not detect anti-SSA(Ro)-antibodies in the serum or CSF of patient 3.Although CSF-NFL levels in patient 2 were normal , the medDue to the aggressive disease course involving the CNS, all patients were extensively assessed, including radiological, serological, and CSF analysis.Previous reports have described a rare occurrence of spinal cord manifestations associated with pSS ,19,20. TTo date, the relationship between anti-SSA(Ro)-antibodies and neurological manifestations in pSS is not sufficiently understood, in particular since both antibody-negative courses and isolated antibody-positive associated myelitis have been described ,19,24,25CSF-NFL measurements have recently been discovered to possess biomarker qualities for different neurological disorders and can be applied to assess early axonal damage and reflect early disease activity , even inIn conclusion, physicians need to keep in mind that in rare cases, imaging findings can be absent, particularly in the early disease course, or difficult to detect since fine longitudinal tracts can be involved in an isolated manner. However, absent radiological findings should not prevent from further diagnostic procedures necessary to identify a possible pSS. Moreover, our case series implies that CSF-NFL and intrathecal synthesis of anti-SSA(Ro)-antibodies are reliable and early markers for severe spinal cord involvement associated with pSS. We therefore strongly suggest that both values are routinely assessed to predict pSS-related CNS involvement even in the absence of radiological findings or systemic disease activity. Nevertheless, this study is limited because of its low number of analyzed patients, which is a common problem in rare diseases. Further investigations in larger cohorts are needed to verify the pattern of spinal cord affection and to clarify the association between the clinical course, NFL levels and the intrathecal production of anti-SSA(Ro)-antibodies.