Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB. Retroviruses (Retroviridae family) are characterized by a replication cycle in which the viral RNA genome is reverse-transcribed and integrated into a host cell’s nuclear genome to form the provirus. The integration of retroviruses into germline DNA can lead to the formation of vertically transmittable proviral sequences known as endogenous retroviruses (ERVs). Such proviruses maintain the potential to generate multiple germ line copies, either by infectious cycles or retrotransposition , 2. Vertgag (group-specific antigen), pol (transcriptase/polymerase) and env (envelope), which are flanked by long terminal repeat sequences (LTRs). Most of this viral DNA is inactivated by deletions or by mutations that have led to the disruption of open reading frames (ORF). Nevertheless, there are a few HERVs with complete and intact open reading frames for the generation of viral proteins  and widely used in many laboratories  based gene expression data from the LL-100 panel of leukemia and lymphoma cell lines were made available, which covers 22 different entities including T cell, B cell and myeloid malignancies and thus the entire spectrum of these diseases. This dataset includes cell lines from the following entities: acute myeloid leukemia (AML) with myelocytic, monocytic, erythroid, and megakaryocytic differentiation; chronic myeloid leukemia (CML) with myeloid or lymphoid blast crises; multiple myeloma/plasma cell leukemia (PCL); T cell and pre B cell acute lymphoblastic leukemia (ALL); Hodgkin lymphoma; Burkitt lymophoma/B cell ALL; chronic lymphocytic leukemia/prolymphocytic leukemia (CLL/PLL); activated B cell (ABC) and germinal center (GC) subtypes of diffuse large B cell lymphoma (DLBCL); hairy cell leukemia (HCL); mantle cell lymphoma (MCL); primary effusion lymphoma (PEL); primary mediastinal B cell lymphoma (PMBL); anaplastic large cell lymphoma (ALCL). The panel is completed by cell lines from myeloproliferative neoplasm (MPN), NK cell and mature T cell malignancies. The LL-100 panel consists of well-characterized and authenticated cell lines that are publicly available  elements were collected from the nucleotide database from the National Center for Biotechnology Information (NCBI). The corresponding reference publications and accession numbers are listed in the https://bioedit.software.informer.com), the created sequence was saved as a fasta file, which was then used as the HERV reference genome for mapping. We have added this fasta file named Viruses21.fasta in the supplement.To quantify HERV expression, a synthetic virus metagenome was assembled to be used for mapping analysis. Because endogenous viral sequences can influence the development of cancer and immune diseases by transcription of The cell lines of the LL-100 panel are authenticated and free of contamination by mycoplasma or non-inherent viruses. Furthermore, the method of RNA isolation and sequencing are identical in all cell lines. Therefore, this dataset allows comparative studies without methodical impact. The RNA-seq data were downloaded from the NCBI Short Read Archive (SRA) under BioProject PRJEB30312 .HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin B (UBB) were used. Analogous to the HERV family specific FPKM, the housekeeping gene specific FPKM was calculated and HERV family specific FPKM were normalized to this value (see Supplement).To analyze the sequencing data they were uploaded to the Galaxy platform . With FaThree paired-end RNA-seq datasets  from human pluripotent embryonic stem cells (ESCs) from the BioProject PRJNA296379 were analyzed . The rea2 atmosphere in a humidified 37°C incubator and cultured in RPMI , supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin . The cells were passaged in 75cm2 flask twice a week.The five Hodgkin lymphoma cell lines L-1236, L-428, L-540, KM-H2 and HDLM-2 were obtained from the German Collection of Microorganisms and Cell Cultures GmbH . Cell lines were grown in 5% COTotal RNA was extracted using the NucleoSpin RNA Mini kit for RNA purification  according to manufacturer’s instructions. Quality and concentration of RNA were determined by visualization of rRNA bands in agarose gel electrophoresis and with a NanoDrop 2000 spectrophotometer .To quantify HERVFc-1 transcripts, 2 μg of total RNA and 100 μM Oligo-dT primer were used for cDNA synthesis  following the manufacturer’s instructions. A sample without reverse transcription was also prepared to check for genomic DNA contamination (noRT control).For the qRT-PCR 5 μL 5xGreen GoTaq buffer, 0.2 μl GoTaq polymerase , 16.8 μl nuclease-free water, 0.5 μl of 10 mM dNTPs , 0.25 μl forward primer (25 μM), 0.25 μl reverse primer (25 μM) and 2 μl cDNA were prepared. The amplification was performed under the following conditions: 94°C for 30 s, 60°C for 30 s, 72°C for 45 s (40 cycles). The experiment was repeated four times for the gene expression analysis.-ΔΔCt method (HPRT1) as reference gene for normalization. noRT controls were used for exclusion of genomic DNA amplifications. The following primers from Eurofins Genomics GmbH (Ebersberg/Germany) were used: HERVFc-1_forward: 5’-CTC CCC ATC TCT CTG GTG C-3’ and HERVFc-1_reverse: 5’-TGA GGA GGC TGG TTT CTC TAA G-3’; HPRT1 _forward: 5’-ACC AGT CAA CAG GGG ACA TAA-3’ and HPRT1_reverse: 5’-CTT CGT GGG GTC CTT TTC ACC-3’.Relative gene expression was calculated with the 2t method  using hyData visualization and One-way ANOVA followed by Dunnett’s multiple comparisons test for statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA. Statistical significances are symbolized as asterisks: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).We analyzed the RNA-seq data of 100 cell lines from the LL-100 panel covering 22 entities of human leukemia and lymphoma . Eight REBNA-1 in the EBV-positive cell lines DAUDI, RAJI, VAL, YT, HG-3, JVM-3, JVM-13, MEC-1, PGA-1, BONNA-12, HAIR-M, HC-1, GRANTA-519 and JVM-2 , Human herpes virus 8 (HHV8) or xenotropic MuLV-related virus (XMRV). To demonstrate the functionality of our method we first analyzed all cell lines for the presence of these exogenous viruses. As expected, we could detect reads mapping against the EBV gene pression . In the oduction . PEL casoduction . XMRV wagag, pol and env genes to our virus metagenome. We found only one known full-length HERVE sequence known as ERVE-1 in the human NCBI nucleotide database. Within the human genome there is a large number of HERVE related sequences that are 90-93% identical to the known full-length ERVE-1 sequence  virus , of whicThe endogenous retrovirus group 48 member 1 (ERVH48-1/ERV-Fb) is part of the HERVF family, whose family members are preferentially expressed in the human placenta . Our datThe activation of endogenous retroviral LTRs plays a fundamental role in the maintenance of pluripotency and induction of an antiviral state in stem cells . TherefoIn both stem cell types, nine HERV families and elements are each significantly upregulated compared to PBMCs. The HERVK family, HERV-like family, HERVV family, HERVT family were all highly significantly upregulated  in both stem cell types 3. The hThe HERVFc subfamily is part of the larger HERVF family . The humTo confirm the results obtained in the mapping analyses and to further verify the functionality of our mapping approach, the expression of HERVFc-1 in the Hodgkin lymphoma cell lines L-428, L-540, L-1236, KM-H2 and HDLM-2 was analyzed by RT-PCR. According to the mapping results for HERVFc-1 , suggesting that the HERVE family is expressed according to the transcriptional program of human tissues and human cancer cells , respectively, is regulated by upstream LTRs belonging to the ERV9 group of HERVs, which have an anti-oncogenic effect  and ZS/2018/12/96169 (HC) from European Regional Development Fund within the local program “Sachsen-Anhalt WISSENSCHAFT Schwerpunkte”. We acknowledge the financial support of the Open Access Publication Fund of the Martin Luther University Halle-Wittenberg.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.


Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC. Disease carcinogenesis and prevention substantially distinctive between esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), with obvious differences in histological types2. ESCC accounts for up to 90% of esophageal cancer in the Asian population, which is characterized by high invasion or distant metastasis3. Although advances in ESCC treatments approaches such as surgery, chemotherapy, and radiotherapy, the overall 5-year survival rate is still unsatisfied4. Therefore, it is urgent to investigate the new biomarkers and treatments for ESCC.Esophageal cancer (EC) is one of the most aggressive cancer worldwide and contributes the sixth highest cancer-related mortality rate5. Accumulating studies have reported lncRNAs are aberrantly expressed in human cancers and participate critically roles in diverse biological processes7. Mechanistically, lncRNAs function as important regulators in gene expression networks in multiple ways, such as chromatin modification, transcription, and posttranscriptional regulation, which mainly depend on their subcellular localization8. For example, HNF1A-AS1 in gastric cancer9 and TTN-AS1 in non-small cell lung cancer (NSCLC) progression10 functioning as the ceRNA molecular is widely reported in cancer development. Besides, lncRNAs regulate their target gene expressions via binding with RNA-binding proteins (RBPs)12. DiGeorge syndrome critical region gene 5 (DGCR5), also known as linc00037, is a lncRNA located on chromosome22 (22q11.21), which is first reported in Huntington’s disease. Emerging studies indicate that higher expression of DGCR5 is particularly important in the progression of laryngeal carcinoma13 and gallbladder cancer14, suggesting DGCR5 may be an oncogenic lncRNA in cancers. By contrast, DGCR5 acts as a tumor suppressor in cervical cancer15, gastric cancer16, and bladder cancer17. All these seemingly contradictory findings prove the heterogeneity roles of DGCR5 in different biological scenarios. As for ESCC, research works have confirmed that some aberrantly expressed lncRNAs related to ESCC development19. However, the functions and mechanisms of DGCR5 in ESCC remain unknown.Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides, most of which lack valid coding capacity20. Importantly, aberrant AS events are frequently observed in a diverse spectrum of tumor types, which is independent of the already characterized genetic alterations21. Serine- and arginine-rich splicing factor 1 (SRSF1) belongs to the splicing factor of the SR family, and participates in posttranscriptional gene regulation through mRNA alternative splicing22. Our previous research has demonstrated that SRSF1 could mediate the aberrant AS of bridging integrator-1 (BIN1) to neutralize its tumor-suppressing functions in NSCLC23. Nevertheless, the concrete function and upstream regulatory mechanisms of SRSF1 in ESCC still remain elusive. Recently, the interactions between lncRNAs and splicing factors are regarded to be important for initiating and maintaining the alternative splicing process24. Nevertheless, the potential function of DGCR5 on ESCC and whether it relates to the AS events via SRSF1-regulated have never been discovered.Alternative splicing (AS) is a posttranscriptional process enabling to regulate the generation of various mRNA and protein products, playing a vital role in the process of development and differentiationThe aim of this study is to characterize the biological functions of DGCR5 in ESCC and investigate the underlying mechanisms associated with alternative splicing. In this study, we first identified an upregulated expression of DGCR5 in ESCC. Gain-of-function and loss-of-function experiments investigated the carcinogenesis of DGCR5 on ESCC cells in vitro and in vivo. Then, the interaction between DGCR5 and SRSF1 was analyzed to explore the impact of DGCR5 on the alternative splicing process of myeloid cell leukemia-1 (Mcl-1). Taken above, our results shed new light on the role of DGCR5 in the tumorigenesis and progression of ESCC.https://cancergenome.nih.gov/) and found that DGCR5 was significantly upregulated in ESCC tissues compared with normal tissues , more lymph node metastasis (P = 0.0008) and higher TNM stage (P = 0.0008), while not related with age and gender (Table P = 0.045), lymph node metastasis (P = 0.047), and DGCR5 expression (P = 0.003) were significantly associated with the prognosis of ESCC patients  online database to identify proteins that interacted with DGCR5. As showed SRSF1 mediating the aberrant AS of BIN1 in NSCLC in our previous study, SRSF1 protein caught our attention for its role in splicing regulation in cancers (Supplementary Table P < 0.01)  and subcellular fractionation. The results showed DGCR5 primarily localized in the nucleus of TE1 and Kyse170 cells Fig. . Since lIn order to explore whether oncogenic splicing factor SRSF1 is regulated by DGCR5, we detect SRSF1 expression on ESCC cells when DGCR5 overexpression or knockdown. The consequence showed that there was no correlation between DGCR5 and SRSF1 mRNA level Fig. . However27. In addition, considering Mcl-1 has been related to SRSF128, we focused on Mcl-1 as a potential target. Then we confirmed Mcl-1 mRNA expression was significantly upregulated in ESCC samples . Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis identified that DGCR5 had a great correlation with the apoptosis signaling pathway Fig. . Moreoveles Fig. . Furtherls Figs.  and 2F, ls Figs. . Then wels Figs. . Therefo31. To analyze whether the alternative splicing of Mcl-1 regulated by SRSF1 protein in ESCC cells, we successfully constructed SRSF1 overexpressed or knockdown in the TE1 and Kyse170 cells  and a full-length isoform Mcl-1L (antiapoptotic) Fig. . We then32, we then detect the function of the DGCR5/SRSF1/Mcl-1 axis on caspase-3 proteins. The results showed no obvious change was observed in the cleaved-caspase-3  model by subcutaneously injecting Kyse170 cells with vector or interfered DGCR5 into 5-week-old nude mice. Tumor volumes were observed 23 days after injection. We found that tumor volumes and weights in silencing the DGCR5 group were significantly suppressed compared with the control Fig. . DGCR5 e33 In this study, we confirmed DGCR5 was highly expressed in ESCC tissues, which was an unfavorable prognosis. Then, we examined that elevated expression of DGCR5 was positively correlated with deeper invasion range, more lymph node metastasis, and higher TNM stage. Knockdown of DGCR5 significantly decreased the proliferation, migration, and invasion ability, while promoted apoptosis of ESCC cells in vitro and in vivo. In addition, we defined the novel mechanism that DGCR5 could connect with the oncogenic splicing factor SRSF1 protein and increase its stability, thus involving the alternative splicing of Mcl-1 mRNA in ESCC cells  and Mcl-1L (antiapoptotic), in which the latter variant is predominant in cancers. In particular, Mcl-1 is highly expressed in ESCC and correlates with poor prognosis49. As previous studies provided, Mcl-1 could be alternatively spliced to exert antiapoptotic functions by SRSF1 protein in breast cancer50. In this study, we analyzed the intensive correlation between SRSF1 and Mcl-1 expression in ESCC. Our data revealed that SRSF1 regulated the apoptosis signaling pathway by participating in the alternative splicing of Mcl-1 and triggered the elevation of Mcl-1L subtype in ESCC, providing the novel insight into the role of SRSF1 in Mcl-1 alternative splicing of ESCC cells.Aberrant AS is demonstrated as a key mechanism that generates multiple RNA variants to regulate protein diversity and enlarge the complexity regulation in many diseases, including cancers51. However, the number of current studies on the correlation of lncRNAs with the regulation of AS events in ESCC is limited. Through bioinformatics analysis, we found that DGCR5 was related to the apoptosis-related signaling pathway. In addition, our research indicated that DGCR5 regulated ESCC cell apoptosis through the Mcl-1 pathway. Then, we explored the influence of SRSF1 on Mcl-1 alternative splicing through DGCR5-mediated in ESCC cells. Our results found that SRSF1 protein was responsible for the regulation of the malignant biological behavior of DGCR5, thereby facilitating the splicing of Mcl-1. These findings demonstrated that the role of the regulatory network between DGCR5/SRSF1/Mcl-1 in ESCC. Moreover, caspase-3 is a downstream target gene of the apoptosis pathway that playing a crucial role in the caspase family52. Then we detected the expression of the cleaved-caspase-3 protein in ESCC cells to determine whether it was essential for the DGCR5/SRSF1/Mcl-1axis. Nevertheless, we found that DGCR5 promoted apoptosis of ESCC cells through a caspase-3-independent pathway. Collectively, our findings showed that the interaction between DGCR5 and SRSF1 increased the expression of Mcl-1L via mediating alternative splicing and promoted ESCC cells apoptosis via a caspase-3-independent pathway.LncRNAs have been confirmed to directly participate in posttranslational regulation of splicing factors in cancersIn summary, we determined upregulated expression of DGCR5 in ESCC and verified its great significance for ESCC patients. DGCR5 led to the progression of ESCC cell tumorigenesis both in vitro and in vivo. Moreover, our findings revealed the molecular mechanism that DGCR5 involved in tumorigenesis of ESCC via SRSF1-mediated alternative splicing of Mcl-1. Overall, our study certificated DGCR5/SRSF1/Mcl-1axis could act as a novel regulatory factor in the carcinogenesis of ESCC.Seventy pairs of ESCC tissues and matched normal tissues were obtained from radical surgery of ESCC patients in the Fourth Hospital of Hebei Medical University  between March 2017 and April 2018. None of the ESCC patients received radiotherapy, chemotherapy, or immunotherapy before surgery. The samples were diagnosed independently by two pathologists. We collected and updated patients’ clinical information every 3 months by follow-up. This study was approved by the ethics committee of the Fourth Hospital of Hebei Medical University.2 incubator. All experiments were performed with mycoplasma-free cells.All human ESCC cell lines  were obtained from the Research center of the Fourth hospital of Hebei Medical University . ESCC cell lines were cultured in RPMI-1640  supplemented with 10% fetal bovine serum  in the humidified atmosphere of the 5% COTotal RNA was extracted using the Trizol reagent . PARIS Kit  was used to isolate the nuclear and cytoplasmic RNA. In total, 2 μg of the total RNA performed to reverse transcription with reverse transcriptase . The cDNA was subjected to PCR amplification  using primers designed for human DGCR5 and Mcl-1, then were separated using 2% agarose gels and visualized with ethidium bromide. SYBR Green Master Mix  was used for qRT-PCR in ABI Quant Studio TM 6 Flex. The relative expression of lncRNA and mRNA levels were normalized to GAPDH or small nuclear U6, respectively, and then analyzed by the 2 − ΔCt or 2 − ΔΔCt method. All primers are listed in Supplementary Tables 10. The siRNA sequences are as follows Supplementary Table DGCR5, SRSF1, or Mcl-1 were, respectively, cloned into pCDNA3.1 plasmid for DGCR5, SRSF1, or Mcl-1 overexpression, which were purchased from GenePharma . DGCR5 and SRSF1 siRNAs were also synthesized by GenePharma. Transfection was conducted with Lipofectamine 2000 (Invitrogen). Cell transfection was performed as we previously described3 cells/well for CCK-8 assays, then was performed as we previously described10. For the colony-formation assays, 1 × 103 transfected ESCC cells were inoculated into six-well plates and cultured at 37 °C for 14 days. The colonies were fixed with 4% paraformaldehyde, then stained with 1% crystal violet and counted.Transfected cells were seeded into 96-well plates at a density of 1 × 105 transfected cells suspended in 200 μl of serum-free RPMI-1640 medium were seeded in the upper chamber , and 600 μl RPMI-1640 medium supplemented with 10% FBS (BI) was placed in the lower chamber. After incubation for 36 h, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, then stained with 0.1% crystal violet for 20 min. Cells on the lower side of the filters were defined as migration or invasion cells and counted at ×200 magnification in three random fields of each filter. We performed invasion and migration assays as previously described10.For invasion and migration assays, the upper chamber of each insert coated with 20 μl Matrigel to form a matrix barrier or not, 2 × 1010.Apoptosis assays were assayed with flow cytometry (FCM) using the FITC Annexin V Apoptosis Detection Kit I , which was performed as previously describedTE1 and Kyse170 cells were seeded until cell confluence reached 60–70%, fixed in 4% paraformaldehyde for 20 min, and then added proteinase K (20 μg/ml) to cover cells and incubate for 30 min at 37 °C. Rabbit serum was added for blocking after hybridization with RNA probe . Then anti-DIG-HRP was incubated at 37 °C for 40 min. TSA chromogenic reagent added and reacted in the dark for 5 min at room temperature. DAPI was incubated for 8 min in the dark, and then a fluorescence microscope was mounted.10.The EZ-Magna RIP kit  was used for RNA immunoprecipitation (RIP) according to the manufacturer’s instructions. TE1 cells were lysed in RIP buffer consisting of 150 mM KCL, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, 5 mM DTT, 0.5% Triton X-100, supplemented with RNase inhibitor Ribolock and proteinase inhibitor cocktail. The lysate was mixed with SRSF1 antibody  or normal IgG coupled beads and left under rotation at 4 °C. Beads were subsequently washed in lysis buffer and the input RNA was purified, and thus analyzed by RT-PCR and qRT-PCR using the primer of DGCR5. RIP assay was performed as we previously describedTotal proteins were extracted and lysed with RIPA . Nuclear and cytoplasmic proteins were extracted with the nuclear and cytoplasmic protein extraction kit . Proteins (30 μg) were separated by 15% SDS-PAGE and transferred onto PVDF membranes , then blocked with 5% skimmed milk and incubated overnight at 4 °C with the following antibodies: anti-SRSF1 , Mcl-1 , Mcl-1 long-form for the molecular weight of 40 kDa and Mcl-1 short-form was 32 kDa. Bcl-2 , BAX , Caspase-3 , Histone-H3 , or β-actin . Then the species-matched secondary antibodies were incubated for 1 h at 37 °C and the proteins were detected using BeyoECLPlus .53. The rabbit polyclonal antibody against human SRSF1  and the mouse polyclonal antibody against human Mcl-1  were used for detection. The staining was visualized according to the 0–4 semi-quantitative system, which based on the percentage of positive cells and the intensity of staining. The total scores were determined by multiplying the percentage score and intensity score and considered as a low expression for the score of 0–4 and high expression for the score of 5–12. The immunostaining score was assessed by two pathologists, independently.IHC analysis was performed as we previously describedn = 5/group). In total, 4 × 106 Kyse170 cells with different transfected were suspended in 200 μl serum-free RPMI-1640, then subcutaneously injected into the right flank of each mouse. The tumor was measured every 3 days. The mice were sacrificed after 23 days, then tumors were weighed and processed for further analysis. Tumor volume was calculated as follows: V (volume) = (length × width2)/2. All animal experiments were performed under approval by the Fourth Hospital of Hebei Medical University Animal Care Commission.Four-week-old male BLAB/c nude mice were randomly divided into two groups . The measurement data are presented as mean ± standard error. The Student’s Supplementary figure legendsUnivariate Cox regression analysis of the relationship between clinicopathological features and survival rate of ESCC patientsMultivariate Cox regression analysis of the relationship between clinicopathological features and survival rate of ESCC patientsList of DGCR5 target proteins by starBase V2.0Expression of SRSF1 staining between ESCC tissues and matched adjacent tissues n (%)Real Time PrimersRT-PCR primerssiRNA sequencesDGCR5 overexpression promoted ESCC progressionDGCR5 promotes ESCC cells migration in vitro, and the subcellular location of DGCR5 in ESCC cells.DGCR5 had no effect on the expression of SRSF1 protein in cytoplasm.The expression of SRSF1 at mRNA and protein levels were examined by transfection of si-SRSF1 or SRSF1 on TE1 and Kyse170 cells.DGCR5 promotes the expression of Mcl-1 by activation of SRSF1on TE1 and Kyse170 cells.


ARF−/− cell line to be resistant to one of four classical chemotherapy agents: doxorubicin, vincristine, paclitaxel, and cisplatin. We then surveyed collateral responses to acquisition of resistance to these agents. Although numerous collateral sensitivities have been documented for antibiotics and targeted cancer therapies, we observed only one collateral sensitivity: half of cell lines that acquired resistance to paclitaxel also acquired a collateral sensitivity to verapamil. However, we found that the mechanism of this collateral sensitivity was unrelated to the mechanism of paclitaxel resistance. Interestingly, we observed heterogeneity in the phenotypic response to acquisition of resistance to most of the drugs we tested, most notably for paclitaxel, suggesting the existence of multiple different states of resistance. Surprisingly, this phenotypic heterogeneity in paclitaxel resistant cell lines was unrelated to transcriptomic heterogeneity among those cell lines. These features of phenotypic and transcriptomic heterogeneity must be taken into account in future studies of treated tumor subclones and in design of chemotherapy combinations.Chemotherapy resistance is a major obstacle to curing cancer patients. Combination drug regimens have shown promise as a method to overcome resistance; however, to date only some cancers have been cured with this method. Collateral sensitivity—the phenomenon whereby resistance to one drug is co-occurrent with sensitivity to a second drug—has been gaining traction as a promising new concept to guide rational design of combination regimens. Here we evolved over 100 subclones of the Eµ-Myc; p19 The antibiotic resistance field is facing a similar problem—rates of antibiotic resistance doubled between 2002 and 2014, and there are 23,000 deaths in the US each year due to antibiotic-resistant infections, according to the Centers for Disease Control and Prevention2. In response, researchers in the antibiotic resistance field have studied cases where resistance to one antibiotic causes, or is co-occurrent with, sensitivity to a second antibiotic—termed collateral sensitivity3. In theory, using collaterally sensitive drug pairs in combination or sequentially could select against the emergence of resistant microbes and thus reduce or prevent the emergence of antibiotic resistance4.Since the 1950s, clinicians have combated intrinsic chemotherapy resistance by treating patients with combinations of chemotherapies that have non-overlapping toxicities thereby allowing for higher doses. However, this method has resulted in an overall 5-year survival rate across all cancers of only 68%5. The following year, Andrew Dhawan and colleagues characterized collateral sensitivities to several tyrosine kinase inhibitors (TKIs) in ALK-positive non-small cell lung cancer (NSCLC). They found that cell lines resistant to first-line TKIs are often sensitized to non-TKIs including the classical chemotherapies etoposide and pemetrexed6. In 2020 a study from the same group showed that resistance to combinations of classical chemotherapies in Ewing’s sarcoma causes resistance to the drugs in the combinations, as well as to actinomycin D, and potentially sensitivity to SP-2509, a lysine-specific demethylase 1 inhibitor7. Recently, Mueller et al. found that resistance to PRMT5 inhibition causes collateral sensitivity to paclitaxel due to upregulation of Stmn28.Recently, the concept of collateral sensitivity has begun to be explored in the context of cancer chemotherapy as well. For example, in 2016, Zhao et al. found that in Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL), resistance to dasatinib, a BCR-ABL1 inhibitor, can cause collateral sensitivity to crizotinib, foretinib, vandetanib, and cabozantinib—cMET and/or VEGFR inhibitors9. Additionally, there is experimental and clinical data suggesting that, at least in some cases, cisplatin resistance can cause sensitivity to paclitaxel, and vice versa12. The mechanism behind this effect remains unclear, however, this combination has been found to be effective to treat patients with ovarian, breast, lung, skin, and head and neck tumors13.While the growing knowledge of collateral sensitivities to targeted therapies is encouraging, most patients today are treated with at least one classical chemotherapy. A close review of the literature reveals several examples of likely collateral sensitivities between classical therapies. Vinblastine and paclitaxel have opposing mechanisms of action—the former destabilizes microtubules, and the latter stabilizes microtubules. They also have opposing mechanisms of resistance, vinblastine resistance can result from stabilizing mutations in α- and β-tubulin, while paclitaxel resistance can result from destabilizing mutations in α- and β-tubulin. When these resistance mechanisms are present, vinblastine-resistant cell lines are sensitive to paclitaxel, and vice versaARF−/− cell line which is a murine model for human Burkitt’s lymphoma16. We chose to investigate classical therapies which are commonly used in the clinic, several of which are used to treat Burkitt’s lymphoma, and several of which have collateral sensitivities described in the literature17. The drugs we studied are doxorubicin, a topoisomerase II poison, vincristine, a microtubule destabilizing agent, paclitaxel, a microtubule stabilizing agent, and cisplatin, a DNA crosslinker20.While there are individual examples of collateral sensitivities to classical chemotherapies, there has not been a comprehensive survey of collateral sensitivities to single-agents in this class of drug, as has been done for antibiotics and targeted therapies. Furthermore, the dearth of known mutations that predict resistance to classical chemotherapies motivated us to define a proxy of resistant state by using the collateral effect of resistance to classical chemotherapies on sensitivity to other agents. Here, we survey collateral responses to acquired resistance to several classical therapies in the Eµ-Myc; p19Using a 96-well plate-based drug pulse and stepwise dose increase protocol, we were able to create > 15 evolutionary replicates of stable cell lines resistant to each of these four drugs. We created a protocol to measure the resulting lines’ sensitivities to a panel of 20 chemotherapies with high precision. We found only one collateral sensitivity in a subset of the cell lines we evolved. This work also revealed marked heterogeneity in collateral changes in drug sensitivity between cell lines evolved to be resistant to the same drug. Surprisingly, transcriptomic profiling of cell lines resistant to paclitaxel was unable to explain the phenotypic heterogeneity. This heterogeneity in resistance acquisition needs to be taken into account when designing classical and salvage combination regimens in order to optimize patient outcome. Similarly, potential discordance between phenotype and transcriptome must be considered when studying transcriptome as a proxy for functional biology.ARF−/− cell line as a tractable model because it is chemo-sensitive, has a short doubling time of ~ 12 hours, and grows in suspension so it can be easily manipulated in small culture wells15.To study collateral effects of resistance to classical chemotherapies, we developed a protocol allowing us to evolve many cell lines to resistance to one therapy in parallel. In these experiments we used the murine Eµ-Myc; p19To evolve resistance to classical chemotherapies, 10,000 cells were treated with a dose of drug expected to kill 85% of cells over the course of three days in 96-well plates. After three days, killing was assessed using PI staining via flow cytometry and drug was removed from cells. Cells were allowed to recover for four days in untreated media and wells were split as they became confluent. On day four, the plate was split to leave approximately 10,000 cells in each well which were treated again with a dose expected to kill 85% of cells based on the killing achieved the previous week. Remaining cells from the split were frozen and saved at −80 °C  , regulation of apoptosis via Bcl2 (with venetoclax), and glutathione levels (with buthionine sulfoximine). We first performed dose response curves with these compounds alone to identify the highest non-toxic dose to use in the combination treatment. Unexpectedly, we found that Paclitaxel Resistance Group 2 cell lines are significantly more sensitive to verapamil as a single agent than Paclitaxel Resistance Group 1 cell lines. In other words, Paclitaxel Resistance Group 2 cell lines are collaterally sensitive to verapamil Fig. B,C.FigurIn addition to blocking Abcb1-mediated drug efflux, verapamil is also a calcium channel blocker. Therefore, we tested the hypothesis that calcium flux or signaling may differ between the two groups of paclitaxel resistant cell lines. We compared expression of calcium channels and calcium-dependent kinases between the paclitaxel resistant groups. We found that the calcium channel Cacna1e is more highly expressed in Paclitaxel Resistance Group 2 Fig.  and S4.Finally, to assess the role of Abcb1 drug efflux, Bcl2 signaling, and glutathione levels on paclitaxel resistance, we next performed paclitaxel dose response curves in the presence or absence of the highest non-toxic dose of the three compounds that inhibit these mechanisms of resistance. Treatment with 0.5 μM venetoclax or 10 μM buthionine sulfoximine did not affect paclitaxel sensitivity in any of our cell lines  did not exhibit the broad cross-resistance to other chemotherapeutics expected in cell lines with higher expression of Abcb1. Specifically, cell lines in Paclitaxel Resistance Group 1 had either no or only moderate decreases in sensitivity to Abcb1 substrates including etoposide, doxorubicin, vincristine, 5-FU, and cisplatin, and increased sensitivity to the Abcb1 substrate actinomycin D. Interestingly, verapamil did not fully ameliorate paclitaxel resistance in the 2nd group of paclitaxel resistant cell lines. This group of cell lines (Paclitaxel Resistance Group 2) has increased expression of class III β-tubulin, an alteration that has been widely implicated in paclitaxel resistance both in the lab and in the clinic35. Although these cell lines do not have different levels of MRP-1  is collaterally sensitive to verapamil. We suspect that this collateral sensitivity is not due to drug efflux per se, because both paclitaxel resistance groups have similar levels of Abcb1 expression and similar effects of verapamil co-treatment with paclitaxel. While determination of the mechanism of this collateral sensitivity is beyond the scope of this study, we have several hypotheses that could explain these observations. The first is that the cell lines in Paclitaxel Resistance Group 2 may be creating an endogenous toxic compound that is normally effluxed by Abcb1. The second is that verapamil has been shown to induce apoptosis by increasing glutathione efflux via MRP-1Examination of the changes in drug sensitivity across our DMSO control cell lines revealed several curious effects of our culturing system. First, we observed heterogeneity in sensitivity to several drugs including statins, YM155, sorafenib, 5-FU, chlorambucil, and topotecan. We also observed heterogeneity in gene expression among the DMSO control cell lines, although differences between control cell lines were not concordant in transcriptomic and phenotypic space, echoing what we observed when comparing the paclitaxel resistant cell lines. Specifically, the DMSO control lines only shared nearest neighbors in drug sensitivity and transcriptome PCA space in two out of 12 cases Fig. .40.We observed that some aspect of the growth conditions during the evolution of resistance, such as the extended culturing time, or culturing in the presence of DMSO caused increased sensitivity to two statins and to YM155 in many of the control cell lines. The mechanism behind the control cell lines’ ‘collateral’ sensitivities to the statins and YM155 is unknown. Recently there has been evidence in the literature that statins may be effective as cancer therapies; however, these results need to be carefully considered if culturing techniques can affect cell lines’ statin sensitivity42. Current combination regimens are effective largely because the drugs within them act independently of each other, and drug interactions like collateral sensitivity are rare.After removing artifact drugs for which the DMSO control cell lines exhibited changes in sensitivity, we observed many collateral resistances in our resistant cell lines, but only one collateral sensitivity in eight out of 112 cell lines. Intriguingly, this result supports a recently revived theory termed independent action which posits that drug combinations are effective not because of synergy or other interaction between drugs, but rather because one drug will be effective for each individual cell or tumor. Under this paradigm, drug combinations are effective simply because multiple drugs represent multiple ‘shots on goal’ for an individual in a population with heterogeneous and difficult-to-predict drug sensitivitiesThe fact that collateral sensitivity to classical chemotherapies is rare even in the single agent setting tested here underlines the reality that our understanding of the short- and long-term effects of these drugs is lacking. In contrast, resistance to targeted therapies is much better understood, and in fact targeted therapies induce collateral sensitivities. However, in the clinic, poor response to 1st-line treatment with classical chemotherapy is predictive of failure of 2nd-line therapies. This observation predicts the result we observed but is complicated by the fact that most patients are treated with more than one agent. Perhaps the early use of classical chemotherapies in treatment is problematic and should instead be a last resort. Instead, to extend patient survival, frontline therapy should consist of combinations of targeted therapies which have more predictable resistance mechanisms and can result in treatable collateral sensitivities.45, as well as to targeted chemotherapy resistance46. While antibiotics and targeted therapies are thought to interact relatively specifically with their targets, classical chemotherapies are thought to be more promiscuous and thus have a wider distribution of binding affinities for entities within the cell49. Our results lead us to hypothesize that collateral sensitivities result from strong evolutionary pressure on one or a small number of specific targets within the cell. In these cases, resistance can result from on-target mutations or target bypass, both of which can produce novel and exploitable sensitivities52. In contrast, the diffuse evolutionary pressure exerted by a compound with many binding partners would not be addressed by any single mutation or bypass mechanism as many cellular processes are affected. In this case, resistance may instead result from a more general state change, for example, to an anti-apoptotic state which would not be vulnerable to inhibition of any specific node within the cell. This paradigm leads to our observed result—rare collateral sensitivities to classical chemotherapies, while others have observed collateral sensitivities to antibiotics and targeted therapies.The sparsity of collateral sensitivities to classical chemotherapies is surprising, as collateral sensitivity has been widely documented in response to antibiotic resistanceOur results, showing the sparsity of collateral sensitivities to classical chemotherapy resistance, motivates further work to better understand mechanisms of action and resistance to these agents. Furthermore, efforts are needed to understand which combinations of classical chemotherapies work well together in order to rationally design novel combination regimens. This work is complicated by our observation of heterogeneity in phenotypic response to acquisition of classical chemotherapy resistance which is not explained by differences in gene expression. The discussion of heterogeneity in cancer has previously focused on inter-patient heterogeneity, heterogeneity between metastases within a patient, and intra-tumor heterogeneity. We have uncovered a new dimension of heterogeneity which needs to be further explored—heterogeneity in response and resistance to chemotherapy Fig. .Figure 7ARF−/−-B-cell lymphomas16  were cultured in B-cell medium . All cell lines were routinely tested for mycoplasma contamination using MycoAlert (Lonza). All drugs were obtained from LC Laboratories, Sigma-Aldrich, Calbiochem, or Tocris Biosciences. All drugs were dissolved in DMSO except cisplatin, which was dissolved in 0.9% saline.Murine Eµ-Myc; p1910,000 cells were seeded into each well of 96-well plates , then treated with a dose of a chemotherapeutic to kill 85% of cells after three days. After treatment, viability was assessed via PI staining and FACS. Cells were spun out of drug and allowed to recover in drug-free media for 4 days. During recovery, cells were split as individual wells’ media color started to change. On day 4, all wells were split to approximately 10,000 cells per well and the removed cells were frozen at −80 °C in PCR plates. The cycle was repeated for 12–18 weeks.Cells were seeded in cell-culture treated 384-well plates , then cells were treated as indicated. Cell viability was measured after 72 h using resazurin sodium salt (Sigma). Resazurin was used at 0.008 mg/mL and plates were analyzed 6 h after addition. Fluorescence was measured using the Tecan M200 Pro at an excitation of 550 nm and emission of 600 nm.inf is the viability at high doses of the drug, and A0 is the viability with low or no drug. Sensitivity metrics were generated for each replicate individually, which were then used for further statistical analyses.The following analysis was performed in Matlab: median fluorescence of wells without cells was subtracted from all wells on the same plate. Viability was then normalized to cell growth by dividing all corrected fluorescence values by the median corrected fluorescence of wells containing untreated cells. Viability data was then fit to the following four-parameter hill curve:Total RNA was prepared from the paclitaxel resistant cell lines, the DMSO control cell lines, and the parental cell line using the RNeasy Mini Kit (Qiagen), per the manufacturer’s protocol, and included the on-column DNAse step (Qiagen).6]N10T30VN-3' where 5Biosg = 5’ biotin, [BC6] = 6 bp barcode specific to each sample/well, N10 = Unique Molecular Identifiers, Integrated DNA technologies), to generate two subpools of 15 samples each.RNA samples were quantified and quality assessed using an Advanced Analytical Fragment Analyzer. The initial steps were performed on a Tecan EVO150.10 ng of total RNA was used for library preparation. 3’DGE-custom primers 3V6NEXT-bmc#1-15 were added to a final concentration of 1 µM.  followed by incubation at 42 °C for 90 min and inactivation at 80 °C for 10 min.Following the template switching reaction, cDNA from 15 wells containing unique well identifiers were pooled together and cleaned using RNA Ampure beads at 1.0×. cDNA was eluted with 17 µl of water followed by digestion with Exonuclease I at 37 °C for 30 min, and inactivated at 80 °C for 20 min.54.Second strand synthesis and PCR amplification was done by adding the Advantage 2 Polymerase Mix (Clontech) and the SINGV6 primer  directly to the exonuclease reaction. 8 cycles of PCR were performed followed by clean up using regular SPRI beads at 0.6×, and eluted with 20 µl of EB. Successful amplification of cDNA was confirmed using the Fragment AnalyzerIllumina libraries were then produced using standard Nextera tagmentation substituting P5NEXTPT5-bmc primer  in place of the normal N500 primer.Final libraries were cleaned using SPRI beads at 0.7× and quantified using the Fragment Analyzer and qPCR before being loaded for paired-end sequencing using the Illumina NextSeq500 in paired-end mode (26/50 nt reads).55. Single-end reads were aligned to the GRCm38 mm10 assembly with gencode M25 annotation using STAR version 2.5.1b56. Gene counts from STAR were used to perform differential gene expression analysis with R version 3.6 using edgeR version 3.32.058. Gene expression differences at the total gene level were considered significant at an FDR of < 0.01.Quality control was done with FastQC version 0.11.459 using the hallmark gene sets in MSigDB60 with an FDR correction and a p-value cutoff of 0.05. enrichplot was used to make the GSEA plots61.GSEA was performed on the described differentially expressed genes using clusterProfilerRNA was prepared from the indicated cell lines using the Quick-RNA Miniprep Kit (Zymo Research) and reverse transcribed with SuperScript IV Reverse Transcriptase (Thermo Fischer). Real-time PCR reactions were performed using PrimeTime Gene Expression Master Mix (IDT) on a LightCycler 480 II (Roche). Data were analyzed using the ΔΔCT method, and relative mRNA levels were normalized to Pgk1 levels. Primers used were IDT Predesigned Taqman Gene Expression Arrays for for Bcl-xL (Mm00437783_m1), Mcp-1 (Mm00441242_m1), Abcb1a (Mm00440761_m1), Abcb1b (Mm00440736_m1), and Pgk1 (Mm.PT.39a.22214854).The indicated cell lines were seeded in 6-well tissue culture plates for 24H. The media from those plates was centrifuged to remove cells, supernatant collected, and stored at −80 °C until measuring of IL-6 levels with a kit from Thermo Fisher (88-7064-88).Statistics were performed using GraphPad Prism 5 (GraphPad Software Inc) and R version 3.6. All error bars represent standard error of the mean.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Information.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.


Epstein–Barr virus (EBV)-associated lymphoproliferative disorders have been garnering attention in recent years. EBV infects people primarily in their first years of life. The viral genome is maintained in a latent phase within the host cells, usually B-lymphocytes. The immunosuppression of different origins alters homeostasis between the virus and the host, enabling EBV-linked lymphoproliferative disorders to appear. Entities with different biological behaviors often share some morphological and phenotypic features, making the diagnosis complicated. Nodal and extra-nodal sites, including the gastrointestinal tract may be involved. This review, divided into three parts, aims to summarize the available clinical, pathological, molecular and therapeutic data on EBV-associated lymphoproliferative disorders involving the gastrointestinal tract. In this part of the review, we discuss plasmablastic lymphoma, extra-cavitary primary effusion lymphoma and Burkitt lymphoma.Epstein–Barr virus (EBV) is a common pathogen infecting people primarily early in life. The virus has the ability to persist throughout a person’s life, usually in B lymphocytes. Conditions of immunodeficiency as well as the introduction of immunosuppressive therapies and the advent of transplant technologies has brought immunodeficiency-associated lymphoproliferative disorders into view, which are often driven by EBV. The group of EBV-associated lymphoproliferative disorders includes different entities, with distinct biological features, ranging from indolent disorders, which may even spontaneously regress, to aggressive lymphomas requiring prompt and adequate treatment. These disorders are often diagnostically challenging due to their overlapping morphology and immunophenotype. Both nodal and extra-nodal sites, including the gastrointestinal tract, may be involved. This review, divided in three parts, summarizes the clinical, pathological, molecular features and treatment strategies of EBV-related lymphoproliferative disorders occurring in the gastrointestinal tract and critically analyzes the major issues in the differential diagnosis. In this part of the review, we discuss plasmablastic lymphoma, extra-cavitary primary effusion lymphoma and Burkitt lymphoma. EBV represents the most common persistent virus in humans, as after primary infection in the first years of life, the virus has the ability to latently persist in B lymphoid cells ,10,11,12While EBV is the common denominator of entities with different pathological features and clinical behaviors, other factors, including the state of the host immune system, iatrogenic immunosuppression (IS), age-related immune-senescence and genetic alterations, play important roles in the pathogenesis of EBV-associated LPDs ,10,11,12The incidence of different entities varies according to the geographical areas.Some EBV-positive LPDs, such as EBV-positive diffuse large B-cell lymphoma, not otherwise specified  and Burkitt lymphoma (BL), have a higher incidence in areas with high rates of EBV infection . DependiFor therapeutic reasons, the correct identification of different lymphoid entities linked to EBV is essential, and it may be diagnostically challenging as different entities share morphological and immunophenotypic features.EBV is associated with many tumors, including epithelial, mesenchymal and hematopoietic neoplastic proliferations.The EBV-associated lymphoid disorders are summarized in The gastrointestinal tract (GIT) is a common site of EBV-driven LPDs.In this review, we focus on LPDs that develop in the GIT, in which EBV detection is considered a diagnostic parameter. Some of these entities may even develop in inflammatory bowel disease (IBD). The mechanism underlying the association of IBD with EBV-associated hematopoietic disorders still remains not completely clarified ,24,25,26Our review consists of three parts. In this part, we describe the clinicopathological features of plasmablastic lymphoma (PBL), extra-cavitary primary effusion lymphoma (EC-PEL) and Burkitt lymphoma (BL).In part 1 of the review, we discussed EBVMCU, EBV-positive DLBCL, NOS and cHL .In part 3, we will analyze, post-transplant lymphoproliferative disorders (PTLDs); chronic active EBV (CAEBV) infection of T- and NK-cell types; and extra-nodal NK/T-cell lymphoma (ENKTL), nasal type .The exact role of EBV in the pathogenesis and development of many LPDs is often not completely elucidated.Among the entities discussed in the present paper, BL represents the first example of a virus-associated lymphoma. Due to the different associations of EBV in the three variants of BL and the restrictive latency pattern with only the expression of EBNA-1, some authors suggest that the virus may not be the initiating event for BL, with the oncogenic capacity of EBNA1 being controversial ,30. RecePEL is strictly linked to Kaposi Sarcoma Herpes Virus/Human Herpes Virus 8 (KSHV/HHV8), which is considered the main driving force in its pathogenesis; EBV is not present in all cases of PEL and it is supposed to play a cofactor role .MYC gene rearrangement [Similar to PEL, in PBL, EBV is identified in a proportion of cases and it is likely to have a pathogenetic role in conjunction with other factors such as angement . FurtherPBL, initially considered a morphological variant of diffuse large B-cell lymphoma (DLBCL), was identified as a distinct entity starting in the 2008 WHO classification .PBL is a high-grade lymphoma initially reported in the jaw and oral cavity of young, HIV-positive males ,34,35.It may develop even in the context of other causes of IS, including autoimmune disorders, post-transplant, iatrogenic IS and age-related immune-senescence ,37. ImmuThe disease shows a male prevalence of 4–5:1, occurring at any age, although HIV-positive individuals may develop PBL at a younger age (median 42 years) compared with PBL associated with other conditions of IS (median 60) ,38.PBL is predominantly an extra-nodal disease, with a predilection for the oral cavity in HIV-positive individuals, with the GIT being the second most common site. Other extra-nodal sites such as the skin, bone, genitourinary tract, sino-nasal tract, central nervous system (CNS), liver, lung and orbits are more rarely reported ,58,59,60Nodal involvement is rare, particularly in HIV-positive cases, whereas it is more frequently observed in post-transplant cases .EBV is likely to have a pathogenetic role in PBL occurrence. Particularly in the context of HIV infection, a large number of PBL cases harbor EBV as documented by EBV-encoded ribonucleic acid (EBER) in situ hybridization (ISH) . HoweverThe GIT represents the most common extraoral site, accounting for approximately 30% of extraoral PBL .In GI PBL, male patients are affected almost twice as often as female  and the The rectum and anal canal have been reported as the most commonly involved sites, particularly in HIV-positive patients ,43. In aThe association between IBD and non-Hodgkin lymphomas, including PBL, is well-known. The risk of lymphoma in IBD patients seems to be related to the treatment more than to the disease itself, in particular with thiopurines and to a lesser extent with anti- tumor necrosis factor α (anti-TNFα) ,44,45.In GIT, PBL presents, commonly, as a mass and/or a polypoid lesion  and, morPlasmablasts (PBs) are large cells with eccentrically located large nuclei, evident nucleoli, basophilic or amphophilic cytoplasm, often with a perinuclear clear halo.However, PBL shows a wide morphologic spectrum ranging from cohesive and monomorphic proliferation of large cells with immunoblastic, plasmablastic or anaplastic features to proliferation of cells with a clear-cut plasmacytic differentiation .Geographic necrosis can be present, and a starry sky pattern with apoptotic bodies and tingible body macrophages is common.The neoplastic cells express a plasma cell phenotype  .CD45 and B-cell markers (CD20 and PAX5) are generally negative  or weaklMYC-rearranged cases, suggesting alternative mechanisms of MYC activation and protein expression. EBER is positive in 60–75% of cases, more frequently in HIV-positive and post-transplant patients. In EBER-positive cases, LMP1 is rarely expressed, indicating type I latency pattern. The latency pattern type II is observed in PBL arising in HIV-positive individuals or in transplantation [MYC rearrangement and a better outcome than EBER-negative PBL [MYC protein is usually overexpressed and not limited to the antation . EBER-potive PBL ,59,60.c-MYC plays important roles in cellular proliferation, protein synthesis and apoptosis. About 50% of PBL cases show MYC rearrangement, generally with an Ig gene partner, and are associated with MYC protein overexpression by immunohistochemistry [MYC amplification and deletion are also detected in 11% and 4% of PBL cases, respectively [MYC rearrangement usually occurs in association with a positive EBER status, CD10 expression and PRDM1 gene mutation and in the absence of mutations in the MYC promoter region [PRDM1 gene, encoding for the BLIMP1 protein often expressed by PBL, has been found in some cases, and it acts as an MYC regulator [MYC in PBL plays an important pathogenetic role in association with EBV infection [Clonal immunoglobulin heavy chain (IGH) rearrangement is usually identified, even when immunoglobulin (Ig) expression is not demonstrable by immunohistochemistry. IGH variable region genes (IGHV) may have somatic hypermutation or may be unmutated with a germline configuration . c-MYC phemistry . MYC ampectively . MYC rear region ,49,50. Megulator . The oncIn the current WHO classification, there are distinct lymphomas with plasmablastic morphology and immunophenotype to be considered in the differential diagnosis with PBL, including EC-PEL, EBV-positive DLBCL, NOS, BL, anaplastic lymphoma kinase positive large B-cell lymphoma (ALK-positive LBCL) and plasmablastic myeloma (PM) ,61,62,63PEL, in its solid/extra-cavitary variant, usually involves lymph nodes, although it is reported even at extra-nodal sites, including the GIT. Similar to PBL, EC-PEL occurs often in immunocompromised patients and typically expresses plasma cell-related markers, CD30 and EMA, whereas B-cell antigens are often negative, although more frequently expressed in the extra-cavitary variant. Unlike in PBL usually expressing immunoglobulin (often IgG), cytoplasmic immunoglobulin are generally absent in PEL, although reported with higher expression in EC-PEL. In PEL, EBV infection is present mainly in HIV-positive individuals. KSHV/HHV8 expression in PEL allows for differential diagnosis with PBL, which is consistently HHV8-negative .Unlike PBL, EBV-positive DLBCL, NOS shows a pan B-cell phenotype, strongly expressing CD20 and PAX5, in the absence of plasma cell-associated markers ,53.MYC rearrangement. However, the phenotype is clearly distinct as BL expresses CD20 and CD10.BL shows some similarities with PBL such as frequent occurrence in HIV-positive hosts, the presence of plasmacytic differentiation, EBER positivity and ALK-positive LBCL is a high-grade lymphoma usually involving lymph nodes and mediastinum, although extra-nodal sites may be involved. Patients are usually diagnosed in the advanced stage. The neoplastic cells show an immunoblastic or plasmablastic morphology and a rather striking intrasinusoidal growth pattern. Similar to PBL, the neoplastic cells strongly express plasma cell-related markers and EMA and are negative or focally positive for B-cell associated markers and CD45. Unlike PBL, which is often CD30 and EBV positive, ALK-positive DLBCL is usually negative or weakly positive for CD30 and is always EBV-negative. The lymphoma cells are strongly positive for the ALK protein with a restricted granular cytoplasmic staining pattern, which is indicative of t or CTLC-ALK fusion protein. Few cases show cytoplasmic, nuclear and nucleolar ALK staining associated with the NPM-ALK fusion protein .PM and PBL share morphological and immunophenotypic features with the expression of plasma cell-related markers. The presence of IS and a positive EBV status favor PBL. On the other hand, the identification of multiple lytic bone lesions and renal dysfunction plus laboratory data such as monoclonal paraproteinemia, hypercalcemia and light chain proteins in urine support a diagnosis of PM. CD56 and cyclin-D1 expression by neoplastic cells is also in favor of PM. However, in some cases, especially with a solitary lesion, a definitive distinction cannot be made, and according to the current WHO, the use of a descriptive diagnosis such as “plasmablastic neoplasm consistent with PBL or PM” is acceptable .In some PBL cases, the aberrant expression of T-cell markers represents a potential pitfall which that lead to an erroneous diagnosis of T-cell lymphoma.The main distinguishing features of PBL, PEL and BL are summarized in MYC abnormalities; therefore, targeted therapies of oncogenic MYC are considered a potential treatment worthy of evaluation [PBL shows a dismal outcome with a median survival of 6–11 months. Currently, there is no standard therapy for PBL patients. Due to the frequent CD20 negativity, rituximab has no role in PBL, and the CHOP  scheme, used in DLBCL, is considered inadequate. More intensive regimens such as hyper-CVAD-MA , CODOX-M/IVAC , COMB  and EPOCH  are used . Furtheraluation . Both EBaluation . Anti-PDPEL is an aggressive large B-cell lymphoma classically presenting exclusively as serous effusions in the pleural, pericardial or peritoneal cavities in the absence of an identifiable tumor mass . AlthougIn a minority of cases (approximately 5%), lymphomas indistinguishable for PEL present as solid tumor masses outside body cavities and have been recognized as EC-PEL in the current WHO classification ,73,74,75KSHV/HHV8 is the pathogen involved in all cases of PEL in both the serous-based classic PEL and EC-PEL; HHV8 detection is a required diagnostic criterion for the disease.Similar to classic PEL, the extra-cavitary variant most often occurs in HIV-infected individuals or in severe immunodeficiency of some kind. Besides the context of HIV infection, IS may be a consequence of either drugs, for instance following transplantation, or aging ,76. The In the context of HIV infection, the neoplastic cells are often co-infected by HHV8 and EBV, whereas co-infection is documented more infrequently in HIV-negative cases ,73,74,75Similar to EBV, KSHV/HHV8 is a gamma human herpes virus that is lymphotropic and oncogenic.Unlike EBV, which is ubiquitous, KSHV/HHV8 is endemic in certain geographic areas such as sub-Saharan Africa, Latin America, the Caribbean, the Mediterranean and Middle Eastern countries.The virus is transmitted through saliva, but it may also be transmitted sexually and even vertically through breast milk. Other possible routes of transmission are blood transfusion, intravenous drug use and transplant ,76.Epidemiologic evidence supports a fecal–oral route of spread and viral DNA has been identified in rectal mucosa from HIV-positive individuals .Similar to EBV, HHV8 may infect cells such as endothelial cells and lymphocytes, persisting throughout a person’s life in a latent form. When the host immune control is altered, HHV8 may reactivate the lytic replicative cycle producing viremia .The virus was initially identified as having a pathogenetic role in Kaposi sarcoma (KS) , and subEC-PEL may occur in any part of the GIT, including the anorectal site, although the stomach and large bowel are more often involved ,73,74,75The tumor may present as a mass; a polypoid lesion; or a thickening of the gastrointestinal (GI) wall, with or without nodal involvement ,73,74,75In GIT, EC-PEL have also been reported before the development of effusions  and follThe histology of EC-PEL is indistinguishable from classic PEL.The neoplastic cells are large-sized resembling immunoblasts (IBs) or PBs or displaying an anaplastic morphology .PEL in its classic form usually fails to express pan-B-cell markers such as CD19, CD20, CD79α and PAX5 as well as surface and cytoplasmic immunoglobulin. The classic form usually expresses CD45/LCA.The extra-cavitary variant may express B-cell markers (25% vs. <5%), although not in all cases , as wellThe neoplastic cells generally express plasma cell-related markers, including CD138, MUM18  and MUM1The lymphoma cells are positive for HHV8 in all cases. HHV8 is usually demonstrated by immunohistochemistry, detecting the expression of HHV8-encoded latency-associated nuclear antigen 1 (LANA-1) protein .HHV8 can also be documented by in situ hybridization or polymerase chain reaction (PCR) analyses.EBV co-infection is identified by EBER in 50–80% of PEL. In non-HIV infected individuals, PEL may be EBV-negative. Despite positivity for EBV by EBER, EBV-latent membrane protein 1 (LMP1) is usually absent, showing a type I latency pattern, similar to PBL.MYC, BCL2 and BCL6 loci are usually absent. Structural alterations in the MYC gene are absent; however, MYC protein overexpression may be seen, probably due to the activity of HHV8-encoded latent proteins [Clonal immunoglobulin (IG) gene rearrangement is present, indicating a B-cell origin. In some cases, T-cell receptor (TCR) gene rearrangement may be found in addition to IG genes. HHV8 viral genomes are present in all cases. Rearrangements at the proteins   and activation of the NF-kB, JAK/STAT and PI3K/AKT/mTOR pathways involved in the survival of PEL [HHV8-encoded latent proteins, including latency-associated nuclear antigen-1 (LANA-1), LANA-2/vIRF-3, viral cyclin, viral FLICE inhibitory protein (v-FLIP) and kaposin, are considered to play significant roles in PEL. These proteins are related to inhibition of apoptosis ; EBV-positive DLBCL, NOS; PBL; HHV8-positive DLBCL, NOS; ALK-positive LBCL; and PM.The anaplastic morphology and the intrasinusoidal growth observed in some cases of PEL  in combiPerforming a panel of immunostainings including plasma cell-related markers and, in particular, HHV8 helps to avoid missing the diagnosis of EC-PEL; molecular analysis may further support the diagnosis.Unlike the classic form of PEL, which is usually negative for B-cell markers, EC-PEL often expresses some B-cell markers and should be differentiated from EBV-positive DLBCL, NOS. HHV8 positivity represents the diagnostic criterion for PEL.The distinction of EC-PEL from PBL has been discussed in the section on PBL.PEL, in both the classic and extra-cavitary variants, belongs to the spectrum of HHV8-related lymphoid disorders including HHV8-positive MCD ; GLPD 8,82,83; aAmong these HHV8-linked entities, HHV8-positive DLBCL, NOS is differentially diagnosed with EC-PEL.HHV8-positive DLBCL, NOS is an aggressive lymphoma usually developing in HIV-positive hosts, mainly in the setting of HHV8-positive MCD and involves lymph nodes, the spleen and the liver, although extra-nodal sites and BM may be affected [The neoplastic cells of HHV8-positive DLBCL, NOS have the same MCD phenotype with characteristic expressions of IgM lambda, MUM1/IRF4 and CD38, and is absent or weak positivity for B-cell markers, CD30 and CD138.Unlike HHV8-positive DLBCL, NOS, PEL usually expresses CD138, EMA and CD30 and lacks cytoplasmic immunoglobulin.In contrast with PEL, ALK-positive LBCL is always negative for HHV8 and EBER, and CD30 is negative or only weakly and focally positive; ALK staining confirms the diagnosis of ALK-positive LBCL.The distinction between PEL and PM frequently relies on clinical correlation. The presence of lytic bone lesions and a serum monoclonal protein favors the diagnosis of PM.The outcome of PEL is dismal with a median survival of less than 6 months despite intensive chemotherapy.EC-PEL in HIV-positive hosts seems to have a slightly better outcome than patients with classic PEL, with a median survival of 11 months vs. 3 months . The stuBL is a highly aggressive B-cell lymphoma, named after the surgeon who first identified it in the 1950s in Uganda as a rapidly growing neoplasm in children with jaw and abdominal masses ,89,90.In the current WHO classification, there are three epidemiological subtypes of BL: endemic, sporadic and immunodeficiency-related .MYC oncogene [BL is the first historical example of a virus-associated human neoplasm and shows activation of the oncogene . The strEndemic BL occurs in certain geographical area  with a strict epidemiological correlation with malaria and the EBV genome is identified in >95% of cases of endemic BL .Plasmodium falciparum and even with other viruses, such as herpesviruses HHV5 and HHV8 [Local environmental factors play a contributory role in BL development ,92. In eand HHV8 ,92.Endemic BL is the most common pediatric malignancy in equatorial Africa and Papua New Guinea, with a male predilection (M:F = 2:1) and a median age of 7 years at presentation . The earSporadic BL is observed worldwide, generally in children and young adults, although three peaks of age at 10, 40 and 70 years are observed; there is a male prevalence (M:F = 2–3:1) ,95. In EImmunodeficiency-related BL occurs in middle-aged HIV-positive patients, often early in the disease course when the count of CD4+ T cells is still high and the use of HAART has not decreased BL incidence . ImmunodBL involves mainly extra-nodal sites with some differences among the three forms.In endemic BL, the most frequently involved site at presentation is the head and neck area , although the ileocecal region is also frequently involved.In sporadic BL, the disease more frequently presents as an abdominal mass  and more uncommonly in the facial region. Other sites involved in both sporadic and endemic forms are the gonads, kidneys and breasts. Lymph node presentation is rare but observed more frequently in adults.The immunodeficiency-associated BL shows nodal and BM involvement more often. The presentation of the disease as acute leukemia is rare in the endemic form. All of the variants of BL may involve the CNS.BL often affects the GIT, although the frequency of GIT involvement varies among the three forms.Primary GI involvement is observed more frequently in the sporadic form.The most frequently involved GI site is the small bowel at the ileo-cecal region. The stomach and appendix are more rarely involved.Due to the significantly elevated proliferation rate of BL, patients generally present with a bulky disease.BL of the bowel appears as a mass or segmental wall thickening. The intestinal tract involvement usually presents with intussusception, leading to signs of obstruction; bleeding and sometimes malnourishment may be present ,98. In tBL typically consists of a diffuse cohesive proliferation of monomorphic medium-sized cells with round nuclei, finely clumped chromatin and multiple small nucleoli. The cytoplasm is deeply basophilic and contains lipid vacuoles more easily seen in imprint preparations .Frequent mitotic figures and apoptosis are present; the starry sky pattern is indicative of the high proliferative index and is due to the presence of numerous tingible-body macrophages.Some cases have a more variable morphology with greater nuclear irregularity, whereas a subset of cases show plasmacytic differentiation, especially in HIV-positive patients.BL cells have a full B-cell phenotype with strong positivity for CD20, CD79alpha, PAX5, CD19 and IgM expression with light chain restriction. The cells express germinal center markers (CD10 and BCL6) and are usually negative for BCL2. In some cases, there is an atypical phenotype with the absence of CD10, CD5 positivity and weak BCL2 expression. The proliferation rate is very high (ki67 > 95%) and there is strong MYC protein overexpression. EBER is positive  in the mMYC proto-oncogene and one of the IG genes is identified in BL, irrespective of the epidemiological form and of whether or not BL carries EBV.The reciprocal chromosomal translocation between the MYC translocations may occur even in other types of lymphoma and are not specific of BL.However, MYC gene, is involved in the three most common translocations. In the majority of BL (about 80%), there is the translocation ) that transposes the telomeric region of chromosome 8 to the IGH on chromosome 14.A breakpoint in the long arm of chromosome 8 at the 8q24 locus, next to or within the Other translocations such as t  and t  are present in a subset of cases.MYC, whereas in the endemic form, the breakpoints are dispersed several hundred kilobases further upstream of the gene [MYC rearrangement [MYC-negative cases, stringent clinical, morphological and immunophenotypic criteria need to be used to rule out lymphomas that may simulate BL. Some of these cases lacking MYC rearrangement represent the provisional entity Burkitt-like lymphoma with 11 q aberration [In sporadic and immunodeficiency-related BL, the breakpoints are usually within or close to the gene ,100. Aboangement . Accordierration .MYC, some direct consequences are cell growth and proliferation, which may cause lymphoma genesis [From the activation of oncogenes such as  genesis .MYC may also induce cell death by apoptosis and an irreversible block to proliferation named senescence [However, activated nescence . LymphomMYC translocation, leading to the inhibition of the proapoptotic signaling [EBV promotes epigenetic changes of the proapoptotic protein named Bim, inhibiting Bim function and, hence, favoring cell survival ,100. In ignaling ,100.MYC and BCL2 and/or BCL6 rearrangements, so called double-hit and triple-hit lymphomas.BL need to be differentiated from high-grade B-cell lymphoma with A subset of these lymphomas (about 50%) shows morphological features closely mimicking BL or has features in between DLBCL and BL.The diffuse proliferation of monomorphic, medium-sized cells simulates BL although the cytoplasm is probably less basophilic than those in BL and the cytoplasmic vacuoles are absent. BL can be ruled on the basis of the immunophenotype (often strong BCL2 expression in contrast to the absent or weak BCL2 expression in BL) and molecular genetic findings.A subset of BL shows plasmacytic features and needs to be distinguished from other lymphomas with plasma cell differentiation such as PBL and PEL. Unlike these neoplasms often lacking B-cell markers, BL shows a pan-B-cell phenotype.MYC rearrangements. Compared with BL, this subset of lymphomas often involves lymph nodes and shows a certain degree of nuclear pleomorphism.Of note, the provisional entity of Burkitt-like lymphoma with 11 q aberration has to be excluded in cases resembling BL but lacking Current treatments employ intensive, multi-agent regimens that have significantly improved the overall survival of BL patients to almost 90% of cases .The chemotherapeutic schemes involve CODOX-M/IVAC  and DA-EPOCH-R . Modified Berlin–Frankfurt–Münster (BMF) chemotherapeutic regimens plus rituximab have been adopted in adult patients affected by BL, with high rates of complete response (CR) and durable responses . Due to Clinicians and pathologists should be aware of the complex and expanding spectrum of EBV-linked LPDs that may arise even in the GIT. These entities are often difficult to correctly diagnose not only due to their rarity but also due to their overlapping morphologic and immunophenotypic features shared by different entities.Their diagnoses should be based on the evaluation of multiple parameters such as clinical data, morphology, immunophenotyping and genetic analysis.


R = 0.953–0.999) and in women (R = 0.813–0.982), except for VT1–V1 compared with FT1–F1, which demonstrated a moderate correlation in women . Only VT1–V1 and FT1–F1 in women displayed a significant difference (p = 0.001). Therefore, this is the first study to simultaneous record VFSS and noninvasive signals by FSR sensor. The correlation of temporal parameters between these two tests was strong. This finding is valuable for future applications of this noninvasive swallowing study tool.Small flexible force-sensing resistor (FSR) sensors can detect laryngeal excursion during swallowing, but the detected laryngeal excursion has not been correlated with videofluoroscopic swallowing study (VFSS) results. Here, we tested the correlation of temporal parameters between the laryngeal excursion recording by FSR sensor and the hyoid motion recording by VFSS under simultaneously swallowing test recordings. Swallowing measurements were recorded in a radiological suite by simultaneously using VFSS and FSR sensors to detect hyoid motion and laryngeal excursion, respectively. Volunteers sat with their head vertical to the Frankfort plane. Two FSR sensors, each for detecting thyroid cartilage excursion and thumb pressing, were placed. VFSS images and FSR sensor signals during single 5-mL barium liquid (30% wt/volume %) bolus swallowing were collected and analyzed for four swallows per participant. In total, 15 men (28.0 ± 4.1 years old); 14 women (28.4 ± 4.2 years old) were recruited. Temporal parameters between VFSS and noninvasive system demonstrated a strong correlation by Pearson’s correlation analysis: in men ( Noninvasive swallowing studies, a new trend, have several advantages. Because it does not involve radiation exposure, it is suitable for studying early, subtle, or mild dysphagia , 2, and Recently, noninvasive oropharyngeal swallowing study tools are being used, and various noninvasive sensors are used to detect and record thyroid cartilage excursion from the skin surface. These sensors include piezoelectric sensors, accelerators, bend sensors, force-sensing resistor (FSR) sensors , 4–9, anOur team used piezoelectric , 9 and FDetecting thyroid cartilage excursion is a key feature of noninvasive swallowing study. A study demonstrated that excursion of thyroid cartilage is synchronized with hyoid motion during VFSS . When swThe results from noninvasive swallowing studies using FSR sensors to detect the thyroid cartilage excursion, however, were not correlated with those from VFSS. Therefore, our team synchronized these two systems to verify if the temporal parameters between the systems were correlated and postulated that laryngeal excursion detected using FSR sensors is synchronized with the hyoid motion during VFSS. This study aimed to identify the relationship between the temporal swallowing parameters of laryngeal excursion signals detected using FSR sensors and hyoid motion images detected during VFSS, which were recorded simultaneously in young volunteers without dysphagia.The Ethics Committee of Chang-Gung Memorial Hospital approved the study protocol (No. 201602016B0C101). Each participant was introduced to and thoroughly explained the study’s aims and procedures and asked to sign the informed consent form.All participants were young and healthy. Exclusion criteria included a history of dysphagia, cardiopulmonary disease, neurological disease, chronic indigestion disorder, diseases of the head and neck, and smoking in the previous 5 years. All volunteers were administered the Functional Oral Intake Scale (FOIS)  and EatiThe ambulatory noninvasive swallowing respiration assessment system based on the MP150 system  was sent to radiological suites and set up for simultaneous recording by using a VFSS equipment .Two FSR sensors were used—one to detect thumb pressing and the other to detect thyroid cartilage excursion. Noninvasive swallowing and respiration coordination signal monitoring based on the Biopac system was set up in our laboratory when FSR sensors were used to detect thyroid cartilage excursion in previous studies , 3, 7–9.For the VFSS, the recording speed was 30 frames/s in the lateral view and the time duration was 1/30-s interval per image.To overcome the problem of synchronizing the two systems Biopac system and VFSS, another FSR sensor, on the upper end of a handheld stick designed to detect a thumb pressing, was used. These thumb pressing reference points enabled the analysis of timepoints and durations between the two systems. The noninvasive oropharyngeal swallowing signals, including those detected using FSR sensors, were simultaneously recorded during VFSS and analyzed offline later using LabView .Acqknowledge  for the MP150 system was used for recording swallowing signals, and LabView was used for the subsequent offline analysis.To validate FSR sensor detection of temporal swallowing parameters, an ambulatory noninvasive swallowing study system (based on the MP150 system) was used and a VFSS was performed simultaneously in radiological suites. In this study, a specially designed FSR sensor on one side of a small stick was held in the palm to detect thumb pressings as the reference time events for analysis. Volunteers were equipped with two ECG adhesive electrodes for submental sEMG, a nasal cannula for respiration, an FSR sensor for the laryngeal excursion, and an FSR sensor for thumb pressing—all of which were connected onto the MP150 system. These validation study procedures included an FSR sensor held in the volunteer’s hand, which was pressed with the thumb twice—before swallowing and once after swallowing—and another FSR sensor was fixed onto the anterior neck at thyroid cartilage level to detect thyroid cartilage excursion during 5-mL barium swallowing in VFSS. During VFSS, the volunteers sat on a chair with their head vertical to the Frankfort plane. Few small water boluses at room temperature were dispensed to volunteers for the setting up a noninvasive measurement system and to familiarize the volunteers with the test procedure. Liquid barium  bolus was administered using a 20-mL disposable syringe into the mouth of the volunteers. All participants were instructed to hold the FSR sensor fixed on one side of small stick in their hands and hold the fed 5-mL barium bolus in their mouths; they were then asked to press the FSR sensor and swallow the barium bolus on verbal instruction by the radiologist in the following order: “press” (thumb press the FSR sensor on the stick), “press,” “swallow,” and “press”. In total, four separate single boluses of 5-mL barium were recorded each individually by using the two systems simultaneously. In the lateral view in the VFSS, the dynamic images of oropharyngeal swallowing were recorded with 30 images/s. All VFSS videos were recorded and saved in an picture archiving and communication system (PACS). After the study was completed, the copies of VFSS videos from PACS were exported for later offline analysis. The recorded swallowing and respiration signals were exported for subsequent offline analysis  during VFSS  along wi, and V3 FSR sensors were used to detect thyroid cartilage excursion during swallowing. A W-shaped waveform with two “V”s was recorded. F1 is the onset time of W, F2 is the turning point of W, and F3 is the offset time. A semiautomatic analysis software based on LabView was used, which detected and analyzed all three swallowing-related signals  locked in the same frame of three different traces  and combThe point VT1 in VFSS and FT1 by FSR sensor were defined as timepoint zero Fig. . Consecup-value of < 0.01 for a two-tailed T test.Statistical analyses were performed using SPSS statistical package . To determine the correlation between the hyoid motion during VFSS and thyroid cartilage excursion by using FSR in a noninvasive system, Pearson’s correlation coefficients (ϒs) were calculated. Statistical significance between the parameters of the two study systems was taken as a R = 0.838–0.990, all p < 0.001; data not shown). No significant differences were noted in the temporal parameters between the two tests for each corresponding time duration , except for the onset of hyoid motion (VT1–V1) and of thyroid cartilage excursion (FT1–F1), which demonstrated a significant difference .The temporal parameters hyoid motion detected during VFSS and thyroid cartilage excursion detected using FSR sensors demonstrated highly significant correlation . In women, the correlation was strong for all , except VT1–V1 and FT1–F1, which displayed moderate correlation . There were no significant differences in temporal parameters between the two systems, except for that in one temporal parameter (VT1–V1 to FT1–F1) in women that displayed a statistically significant difference but a high correlation. Unlike in the study on the correlation of bend sensor and VFSS , we simuFindings on the effects of gender on swallowing thus far have been inconclusive: some have indicated differences in swallowing parameters between genders, whereas have not –33. ThesWhen simultaneously recording the swallowing signals of a noninvasive FSR sensor swallowing study system and VFSS, some technical issues need to be solved using hardware and software. To synchronize the two systems, a handheld stick with an FSR sensor, connected to one of the multiple MP150 system channels, was used in this study. Our handheld stick design could be used in future swallowing studies to create timepoint references to aid in synchronizing FSR with VFSS or other recording methods.This noninvasive swallowing study method using FSR sensors to detect thyroid cartilage excursion of swallowing is reliable and efficient at indirectly detecting the temporal parameters of oropharyngeal swallowing, which has uses in clinical application and future studies. This system has multiple channels to simultaneously record, and time-lock, swallowing-related physiological signals including submental sEMG, nasal airflow, and uncalibrated measurement of global laryngeal excursion, which is adequate to detect a swallowing event. Swallowing events were analyzed but the detailed parameters were not. The reliability of the detection of swallowing events has been demonstrated, and thus, this noninvasive system can be used to analyze piecemeal deglutition and dysphagia limits . These fSwallowing study objective instruments all have various advantages and limitations. The numerous facets of swallowing function studies include temporal parameters , 29, dynThe noninvasive swallowing study without radiation exposure is suitable for posttherapy monitoring of the immediate effects. Furthermore, the advantages of noninvasive swallowing study systems include the possibility of a larger quantity of swallowing events data that can be collected for a cross-section study, a larger number of repeated measurements over a short period in follow-up studies, and studies for swallowing data collection over a long follow-up duration. It is also useful in long-term objective follow-up studies. Another valuable potential application of a noninvasive swallowing system is in feedback training , 38. OurNoninvasive systems are, however, not without limitations. The noninvasive system cannot record through the oropharyngeal anatomic structures and hyoid displacement, UES relaxation, bolus passage, penetration, aspiration, and residuals after swallowing were recorded during VFSS . These fDeveloping automatic analysis software systems to analyze the dynamic images and temporal parameters of swallowing would be valuable in both VFSS and noninvasive swallowing study systems. Using artificial intelligence to solve this semiautomatic analysis problem may lead noninvasive swallowing studies to another frontier.This is the first study to concurrently record noninvasive swallowing signals and perform VFSS. We provided a new method for researchers to conduct correlation studies between noninvasive swallowing studies and VFSS.Our results demonstrated a strong correlation in the temporal parameters of swallowing between noninvasive surface sensors and VFSS. This is crucial for further usage of this noninvasive swallowing study tool in studies and clinically.