Serological responses of patients with nasopharyngeal carcinoma to an N-terminal Epstein-Barr virus DNA polymerase protein expressed in prokaryotic cells

A 1.7-kb cDNA clone, pGEM-cDP, was isolated from a cDNA library of IUdR-induced p3HR1 cells. It contains the upstream nucleotide sequence of the Epstein-Barr virus (EBV) DNA polymerase gene from 156,859 to 155,088, and was subcloned into expression vector pET3cp* by the polymerase chain reaction, giving the plasmid pDP1. Using a T7 RNA polymerase expression system, a 77-kD polypeptide was produced from pDP1 inEscherichia coli and specific hyperimmune serum was generated in mice. The truncated EBV DNA polymerase was shown to possess the authentic antigenicity by an indirect immunofluorescence assay and by immunoblotting using EBV-containing cells as antigens. Serum from nasopharyngeal carcinoma (NPC) patients and healthy donors was examined for antibodies against the 77-kD polypeptide by Western blot analyses and ELISAs. About 70% NPC patients were positive, while less than 15% of healthy persons showed weak reactivities in ELISAs.     

Improved long-term results of intensity-modulated radiotherapy for a non-endemic European nasopharyngeal carcinoma cohort: single-center retrospective study

PurposeReport our matured outcomes of European nasopharyngeal carcinoma (NPC) treatment from a non-endemic region in the IMRT era.MethodsWe reviewed 109 consecutive patients with biopsy proven NPC treated between 2009 and 2013. All received IMRT as per RTOG 0615. Toxicity was scored accordingly to CTCAE 4.03. Platinum-based chemotherapy was delivered following the Intergroup 0099.ResultsMedian age of 53 years; 97% Caucasian; 74% male; 72% WHO grade III; 43% T1; 14% T2; 18% T3, 25% T4; 17% N0; 17% N1; 39% N2; 27% N3. Compliance to adjuvant chemotherapy was 88%. With a median follow up of 56 months, the 4-year local control was 90.2% (88.6% for T1; 100% for T2; 85% for T3; and 91.7% for T4), the 4-year distant metastases-free survival was 86% and an overall survival rate was 77%. Local control and survival were better in G3 (p < 0.001 and p = 0.032, respectively). Xerostomia was the most frequent late toxicity in 55% (n = 60). Hypothyroidism requiring hormonal reposition occurred in 15.5% (n = 17). From the 36 deaths, 20 were due to distant metastases, 3 grade 5 toxicity, 2 from local progression, 5 non-cancer deaths and unknown cause in the remaining 6. On multivariable analysis, age (p = 0.017), local recurrence and distant metastases were associated with death (p < 0.001, both).ConclusionOur matured data from the IMRT era showed a major improvement from our 3D cohort series reaching excellent local and regional control, even in T4. Local recurrences, despite few, and distant metastases were correlated with the risk of death.     

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.     

The prognostic value of CD3+ tumor-infiltrating lymphocytes for stage II colon cancer according to use of adjuvant chemotherapy: A large single-institution cohort study

BackgroundHigh tumor infiltrating lymphocytes (TILs) density was previously shown to be associated with favorable prognosis for patients with colon cancer (CC). However, the impact of TILs on overall survival (OS) of stage II CC patients who received adjuvant chemotherapy (ADJ) or not (no-ADJ) is unknown. We assessed the prognostic value of CD3+ TILs in stage II CC patients according to whether they had ADJ or not.MethodsPatients treated with curative surgery for stage II CC (2002–2013) were selected from the Santa Maria alle Scotte Hospital registry. TILs at the invasive front, center of tumor, and stroma were determined by immunohistochemistry and manually quantified as the rate of TILs/total tissue areas. High TILs (H-TILs) was defined as >20%. Patients were categorized as high or low TILs (L-TILs) and ADJ or no-ADJ.ResultsOf the 678 patients included, 137 (20%) received ADJ and 541 (80%) did not. The distribution of the 4 groups were: 16% (L-TIL/ADJ), 64% (L-TIL/no-ADJ), 5% (H-TIL/ADJ), 15% (H-TIL/no-ADJ). Compared to H-TILs/no-ADJ, ADJ patients showed a significantly increased OS (P<.01) regardless of the TILs rate whereas L-TILs/no-ADJ had significantly decreased OS and higher risk of death (HR=1.41; 95% CI, 1.06–1.88; P<.0001). On multivariable analysis, the unfavorable prognostic value of L-TILs (vs. H-TILs) for no-ADJ patients was confirmed (HR=1.36; 95% CI 1.02, 1.82; P=.0373).ConclusionLow CD3+ TILs rate was associated with shorter OS in those with stage II colon cancer who did not receive adjuvant therapy. Low CD3+ TILs could be considered an additional risk factor for still ADJ-untreated stage II CC patients, which could facilitate clinical decision making.     

Use of plasma Epstein-Barr virus DNA monitoring as a tumor marker in follow-up of patients with nasopharyngeal carcinoma: preliminary results and report of two cases

Recent studies suggest that plasma Epstein-Barr virus (EBV) DNA may reflect tumor burden in patients with nasopharyngeal cancer. A prospective study was initiated to investigate this correlation in 125 patients (34 pretreatment [Group A], 78 in remission [Group B] and 13 relapsed [Group C]) and 19 healthy controls. In group A, EBV DNA was detected in plasma samples of 24 (70%) patients. In Group B, EBV DNA was detected in 7 patients (range 77-13,731 copies/mL) and further imaging in all but one of these patients revealed active disease confirmed by ultrasound-guided fine-needle biopsy. There was only one false-positive case; this patient is currently under follow-up. Here we describe 2 of the 7 patients with detectable plasma EBV DNA in whom recurrence was documented by PET scan during follow-up. Our results showed that in group B the positive predictive value of quantitative analysis of plasma EBV DNA was 85%. Quantitative analysis of EBV DNA in plasma seems to become an integral part of screening, staging, monitoring, and prediction of relapse in patients with nasopharyngeal carcinoma. However, previous studies cannot be considered definitive and more reports on the use of this technique are urgently needed from both endemic and non-endemic regions.     

Molecular events associated with epithelial to mesenchymal transition of nasopharyngeal carcinoma cells in the absence of Epstein-Barr virus genome

Epithelial-mesenchymal transition (EMT) is an important process in tumor metastasis. The EMT-related events associated with metastasis of NPC in the absence of EBV have not been elucidated. We established an EBV-negative NPC cell line from a bone marrow biopsy of an NPC patient. Using a Matrigel system we isolated an invasive and non-invasive sublines, designated NPC-BM29 and NPC-BM00. NPC-BM29 acquired an invasive-like phenotype characterized by EMT, marked by down-regulation of E-cadherin and β-catenin with concomitant increased expression of Ets1. NPC-BM29 cells expressed ≥ 10-fold higher of MMP-9 than NPC-BM00 cells. NPC-BM29 cells grew better in 2% serum than NPC-BM00 cells, with a population doubling-time of 26.8 h and 30.7 h, respectively. A marked reduction in colony-formation ability of NPC-BM00 cells compared to NPC-BM29 was observed. Wound-healing assay revealed that NPC-BM29 cells displayed higher motility than NPC-BM00 and the motility was further enhanced by cell treatment with TPA, a PKC activator. Cell surface markers and tumor-associated molecules, AE3, MAK6 and sialyl-Tn, were up-regulated in NPC-BM29 cells, whereas the expression of HLA-DR and CD54 was significantly increased in NPC-BM00 cells. NPC-BM29 consistently released higher levels of IL-8 and IL-10 than NPC-BM00, with low levels of IL-1α expression in both cell lines. Higher level of VEGF production was detected in NPC-BM00 than NPC-BM29 cells. These data show that EBV is not required for exhibiting multiple metastatic phenotypes associated with EMT. More studies that target right molecules/signalings associated with the EMT may offer new therapeutic intervention options for NPC invasion and metastasis.     

Proteome profiling of Epstein-Barr virus infected nasopharyngeal carcinoma cell line: identification of potential biomarkers by comparative iTRAQ-coupled 2D LC/MS-MS analysis

Epstein-Barr virus (EBV) has been implicated in the development of nasopharyngeal carcinoma (NPC), a squamous cell carcinoma with high-occurrence in Southeast Asia and southern China. However, the underlying relationship of EBV and NPC squamous cell remains obscure. In this study, we employ a comparative iTRAQ-coupled 2D LC-MS/MS system to analyze the protein profile of NPC cell line upon EBV infection. Based on the proteome data and Western blot validation, 12 proteins were found to be significantly up-regulated and associated with signal transduction, cytoskeleton formation, metabolic pathways and DNA bindings. The interactions among NPC and EBV proteins were further analyzed and protein networks were established. Based on the functions of differentially expressed proteins, a metabolic pathway was proposed to elucidate their relationship in cytoskeleton formation, cell proliferation and apoptosis. Our results suggested a new proteome platform to analyze EBV's role in NPC squamous cell line. And these differentially expressed proteins may hold the promise as potential biomarkers for NPC diagnostics and treatment.     

Initiation of lytic DNA replication in Epstein-Barr virus: search for a common family mechanism

Herpesviruses are a complex family of dsDNA viruses that are a major cause of human disease. All family members share highly related viral replication proteins, such as DNA polymerase, ssDNA-binding proteins and processivity factors. Consequently, it is generally thought that lytic replication occurs through a common and conserved mechanism. However, considerable evidence indicates that proteins controlling initiation of DNA replication vary greatly among the herepesvirus subfamilies. In this article, we focus on some of the known mechanisms that regulate Epstein-Barr virus lytic-cycle replication, and compare this to other herpesvirus family members. Our reading of the literature leads us to conclude that diverse viral mechanisms generate a common nucleoprotein prereplication structure that can be recognized by a highly conserved family of viral replication enzymes.     

Prolonged low dose IL-2 and thalidomide in progressive metastatic renal cell carcinoma with concurrent radiotherapy to bone and/or soft tissue metastasis: a phase II study

Metastatic renal cell cancer is one of the immuno-sensitive tumors. Apart from the immuno-modulating agents IFN and IL-2, thalidomide has been reported to be effective in this type of cancer. However, bone metastases and bulky metastases, show limited response to immunotherapy, are often site of recurrent disease and are therefore often treated later with radiotherapy. In this phase II study, we evaluated toxicity and efficacy of the combination of continuous low dose (1 mIU/m²) s.c. IL-2 and thalidomide (200 mg once daily) in 22 patients with progressive metastatic renal cell cancer. In addition, 13 soft tissue lesions and two bone metastases in 13 patients were concurrently treated with fractionated radiotherapy. T cell number and activation in blood was measured by immunoflowcytometry. Nearly all patients developed grade 1–2 toxicity consisting of fatigue, sensory neuropathy, constipation and dizziness. Five patients had a grade 3–4 toxic event: four patients with deep venous thrombosis requiring anticoagulant therapy, and one patient who developed radiation myelopathy. On systemic response evaluation ten patients showed ongoing SD with a mean progression free survival of 9 months. One patient showed a PR (at an irradiated site). Regarding local response to irradiation, seven lesions showed a PR for a mean time period of 8.7 months, whereas seven were stable for 6 months. The radiation response of one lesion was not evaluable. Immunoflowcytometry showed an increase in number and activation of lymphocytes (mainly Natural Killer—NK-cells), which was absent or even decreased in irradiated patients. The combination of sc. low dose IL-2, thalidomide and radiotherapy is feasible, but relatively toxic and does not lead to higher responses at non-irradiated sites. The combination of immunotherapy and concurrent radiotherapy is effective at 60% of the relatively large evaluable sites. Progressive myelopathy developed in one patient, possibly due to radiotherapy in combination with thalidomide.     

Fluctuations of epstein-barr virus serological antibodies and risk for nasopharyngeal carcinoma: a prospective screening study with a 20-year follow-up

Background

The impact of variation of Epstein-Barr virus (EBV) antibody titers before the development of nasopharyngeal carcinoma (NPC) is still unclear. We analyzed the fluctuations of antibodies against EBV before histopathological diagnosis to assess the risk of NPC and aimed to provide a reliable basis for screening in high risk populations.

Methods

This study was based on a population-based screening program in Sihui County in Guangdong Province of China. A total of 18,986 subjects were recruited in 1987 and 1992, respectively. Baseline and repeated serological tests were performed for IgA antibodies against EBV capsid antigen (VCA/IgA) and early antigen (EA/IgA). Follow-up until the end of 2007 was accomplished through linkage with population and health registers. Cox proportional hazards regression model was used to estimate the relative risk of NPC in association with EBV antibodies. Time-dependent receiver operating characteristic curve (ROC) analysis was used to further evaluate the predictive ability.

Results

A total of 125 NPCs occurred during an average of 16.9 years of follow-up. Using baseline information alone or together with repeated measurements, serological levels of VCA/IgA and EA/IgA were significantly associated with increased risks for NPC, with a striking dose-response relationship and most prominent during the first 5 years of follow-up. Considering the fluctuant types of serological titers observed during the first three tests, relative risk was highest among participants with ascending titers of EBV VCA/IgA antibodies with an adjusted hazard ratio (HR) of 21.3 (95% confidence interval [CI] 7.1 to 64.1), and lowest for those with decreasing titers (HR = 1.5, 95% CI 0.2 to 11.4), during the first 5 years of follow-up. Time-dependent ROC analysis showed that VCA/IgA had better predictive performance for NPC incidence than EA/IgA.

Conclusion

Our study documents that elevated EBV antibodies, particularly with ascending titers, are strongly associated with an increased risk for NPC.     

The effect of adding a lipid tail to tyrosine-azobenzenearsonate: In vivo and in vitro consequences for cell-mediated immunity

Coupling lipid tails of increasing length to the tyrosine-azobenzene-arsonate molecule resulted in a decrease in in vivo immunogenicity. When used to induce in vitro stimulation of sensitized lymphocytes a decrease in dose was required to produce an optimal response as the lipid sidechain increased in size. This was attributable to the cytotoxic effects produced by the detergent-like activity of the large lipid side-chain derivatives. When used for pulsing of macrophages the stickiness of the larger lipid derivatives made them more effective at low doses and times for initiation of lymphocyte stimulation.     

Treatment of stage IIIB squamous-cell lung cancer with pembrolizumab combined with chemotherapy to achieve pCR: a case report

Lung cancer is currently the leading cause of global cancer-related deaths and its incidence increases every year. Most squamous-cell lung cancers are in the advanced stage at diagnosis. This study reported a case of a 66-year-old man diagnosed with unresectable stage IIIB squamous-cell lung cancer (cT2aN3M0), who was treated with pembrolizumab combined with paclitaxel (albumin-bound) + nedaplatin. After four cycles, a follow-up chest computed tomography (CT) scan showed the disappearance of the original right lower lobe lung mass with cavitation-like changes, and a follow-up chest CT scan 6w later revealed a solid nodule measuring approximately 1.5 cm in diameter within the cavity. Subsequent surgical excision of the residual primary lesion was performed to achieve complete pathological remission. The patient was treated with pembrolizumab combined with paclitaxel (albumin-bound) + nedaplatin and achieved complete pathological remission with surgical excision.     

A comprehensive DNA barcode database for Central European beetles with a focus on Germany: adding more than 3500 identified species to BOLD

Beetles are the most diverse group of animals and are crucial for ecosystem functioning. In many countries, they are well established for environmental impact assessment, but even in the well‐studied Central European fauna, species identification can be very difficult. A comprehensive and taxonomically well‐curated DNA barcode library could remedy this deficit and could also link hundreds of years of traditional knowledge with next generation sequencing technology. However, such a beetle library is missing to date. This study provides the globally largest DNA barcode reference library for Coleoptera for 15 948 individuals belonging to 3514 well‐identified species (53% of the German fauna) with representatives from 97 of 103 families (94%). This study is the first comprehensive regional test of the efficiency of DNA barcoding for beetles with a focus on Germany. Sequences ≥500 bp were recovered from 63% of the specimens analysed (15 948 of 25 294) with short sequences from another 997 specimens. Whereas most specimens (92.2%) could be unambiguously assigned to a single known species by sequence diversity at CO1, 1089 specimens (6.8%) were assigned to more than one Barcode Index Number (BIN), creating 395 BINs which need further study to ascertain if they represent cryptic species, mitochondrial introgression, or simply regional variation in widespread species. We found 409 specimens (2.6%) that shared a BIN assignment with another species, most involving a pair of closely allied species as 43 BINs were involved. Most of these taxa were separated by barcodes although sequence divergences were low. Only 155 specimens (0.97%) show identical or overlapping clusters.     

The cooperative binding of large ligands to a one-dimensional lattice: the steric hindrance effect

The cooperative binding of monomeric ligands to a long lattice of a linear polymer with complete or partial steric hindrance is treated using a matrix method. Results and typical calculations of the model are represented. Non-saturated cooperative binding as well as two-step (biphasic) binding isotherms can be interpreted by the steric hindrance model. This is applicable to the analysis of the binding of surfactants to polymer. The usefulness and the limitation also are discussed.     

Marker rescue of a transformation-negative Epstein-Barr virus recombinant from an infected Burkitt lymphoma cell line: a method useful for analysis of genes essential for transformation.

A Burkitt lymphoma cell line infected in vitro with a transformation-defective mutant recombinant Epstein-Barr virus (EBV) was used to attempt marker rescue of transformation competence by transfection with cloned wild-type DNA. EBV replication was induced in the transfected cells, and wild-type EBV DNA recombined via flanking homologous sequences adjacent to the deletion, resulting in a virus which transformed primary B lymphocytes in vitro. This strategy should be useful for molecular genetic analysis of the role of part or all of any gene in cell growth transformation.