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Epstein-Barr virus (EBV) is an oncogenic human herpes virus that was discovered in 1964. Viral non-coding RNAs, such as Bam HI-A rightward fragment-derived microRNAs (BART miRNAs) or Bam HI-H rightward fragment 1-derived miRNAs (BHRF1 miRNA) in EBV-infected cells have been recently reported. Host miRNAs are also upregulated upon EBV infection. Viral and host miRNAs are important in maintaining viral infection and evasion of host immunity. Although miRNAs in EBV-infected cells often promote cell proliferation by targeting apoptosis or cell cycle, this review focuses on the regulation of the recognition of the host immune system. This review firstly describes the location and organization of two clusters of viral miRNAs, then describes evasion from host immune surveillance systems by modulating viral gene expression or inhibiting innate and acquired immunity by viral miRNAs as well as host miRNAs. Another topic is the enigmatic depletion of viral miRNAs in several types of EBV-infected tumor cells. Finally, this review introduces the strong correlation of nasopharyngeal cancer cases with a newly identified single nucleotide polymorphism that enhances BART miRNA promoter activity., (Copyright © 2020 Iizasa, Kim, Kartika, Kanehiro and Yoshiyama.)

Epstein-Barr virus-positive gastric cancer (EBVGC) comprises approximately 9% of all gastric cancers and is associated with a low prevalence of lymph node metastasis (LNM). Given that limited data concerning LNM in EBV-related early GC are available, EBV status is not considered an indicator for endoscopic submucosal dissection (ESD). In this review, we focused on pT1 EBVGC and on gastric carcinoma with lymphoid stroma (GCLS), and discuss expanded ESD indications and curative resection criteria. In pT1b EBVGC, the incidence of LNM was low (6/180 patients, 3.3%; 95% confidence interval [CI] 1.2-7.1), especially in lymphovascular invasion-negative EBVGC (1/109 patients, 0.9%). No patients with pT1a EBVGC had LNM (0/38 patients, 0%; 95% CI 0-7.6), even those who did not meet the current curative ESD criteria. Although the frequency of LNM in GCLS was low (5.0-10.6%), the incidence of LNM in non-EBV GCLS was relatively high (10.0-20.0%); therefore, EBV status can be considered a more important factor than GCLS. In summary, the clinicopathological characteristics of EBVGC differ from those of conventional GC, and EBV negativity is a risk factor for LNM in early GC. Therefore, patients in this group are likely to be promising candidates for ESD, and we recommend that EBV status evaluation be included in early GC treatment guidelines., (© 2019 Japan Gastroenterological Endoscopy Society.)

Objectives: A subset of vulvar carcinomas (VC) are associated with human papillomavirus (HPV) DNA. This trait can be used to identify tumor markers for patient's follow-up. A large diversity of HPV prevalence in VC has been reported, but no data are available concerning the insertional HPV status in this tumor type. Therefore, we have used an innovative next generation sequencing (NGS)-based CaptHPV method able to provide an extensive characterization of HPV DNA in tumors., Material and Methods: Tumor tissue specimens from 55 patients with VC were analyzed using p16 immunohistochemistry, in situ hybridization, polymerase chain reaction, and CaptHPV-NGS assays., Results: Our analyses showed that 8 (14.5%) of 55 cases were associated with HPV 16 DNA. No other HPV genotypes were identified. The HPV genome was in a free episomal state only in one case and both episomal and integrated into the tumor cell genome in 7. There was a single insertion in 5 cases and multiple sites, scattered at different chromosomal loci in two. ISH data suggest that some of these might reflect tumor heterogeneity. Viral integration targeted cellular genes among which were TP63, CCDC148, LOC100133091, PKP1, and POLA2. Viral integration at the PKP1 locus was associated with partial gene deletion, and no PKP1 protein was detected in tumor tissue., Conclusions: Using the NGS-based innovative capture-HPV approach, we established a cartography of HPV 16 DNA in 8 VC cases and identified novel genes targeted by integration that may be used as specific tumor markers. In addition, we established a rationale strategy for optimal characterization of HPV status in VC.

Human papillomaviruses (HPVs) are the causative agents of carcinoma of the uterine cervix. A number of HPV genotypes have been associated with cervical cancer and almost all tumors associated with HPV show strong p16 expression. However, there is little information on the possible impact of the HPV genotype and p16 immunostaining on the clinicopathological features or their prognostic value in cervical carcinoma. We evaluated a series of 194 patients with HPV-positive cervical cancers treated at our institution, focusing on the clinicopathological features and the relationship of the HPV genotypes and p16 immunostaining with the prognosis. A single HPV type was identified in 149 (77%) tumors, multiple HPV infection was detected in 30 cases (15%), and undetermined HPV type/s were identified in 15 (8%) carcinomas. HPV 16 and/or 18 were detected in 156 (80%) tumors. p16 was positive in 186 (96%) carcinomas, but eight tumors (4%) were negative for p16 (seven squamous cell carcinomas, one adenocarcinoma); 5/8 caused by HPV 16 and/or 18. Patients with HPV 16 and/or 18 were younger (49 ± 15 vs. 57 ± 17 years, p < 0.01) and more frequently had nonsquamous tumors than patients with other HPV types (24% [37/156] vs. 0% [0/38]; p = 0.01). Neither the HPV type nor multiple infection showed any prognostic impact. Patients with p16-negative tumors showed a significantly worse overall survival than women with p16-positive carcinomas (45 vs. 156 months, p = 0.03), although no significant differences in disease-free survival were observed. In the multivariate analysis, negative p16 immunostaining was associated with a worse overall survival together with advanced FIGO stage and lymph node metastases. In conclusion, the HPV genotype has limited clinical utility and does not seem to have prognostic value in cervical cancer. In contrast, a negative p16 result in patients with HPV-positive tumors is a prognostic marker associated with a poor overall survival.

Epstein-Barr virus (EBV) has been associated with about 9% of all gastric carcinomas, but its role in gastric carcinogenesis remains unclear since there is lack of evidence of EBV presence in pre-neoplastic lesions of gastric mucosa. This study intends to determine the prevalence of EBV in gastric dysplasia and superficial neoplasia to clarify whether EBV infection is an early or late event in gastric cancer development. This retrospective study included a total of 242 gastric lesions from 199 consecutive patients who were referred for endoscopic resection. The histological classification of lesions includes 137 low- and high-grade dysplasia and 105 superficial carcinomas. EBV infection was investigated by EBER-ISH. Results showed that EBV was not detected in any epithelial cells of any case with dysplasia or superficial carcinomas, although we observed the presence of a small number of EBV-infected lymphocytes in 2.1% of all lesions. These results showed that EBV is not present in gastric dysplasia neither in superficial carcinomas suggesting that EBV carcinogenesis is a late event in well/moderately differentiated gastric carcinogenesis.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC), originally known as HPV-related carcinoma with adenoid cystic carcinoma-like features, is a recently described neoplasm that presents only in the sinonasal tract, displays features of both a surface-derived carcinoma and a salivary gland carcinoma, and is associated with high-risk HPV, specifically HPV type 33. Majority of the cases display high-grade histologic features, but HMSC paradoxically behaves in a relatively indolent fashion. Distinguishing HMSC from other histologic mimickers is essential as the management and prognosis are significantly different. In this article, we present a unique case of HMSC and review the literature.

Human papillomavirus (HPV)-related carcinoma with adenoid cystic-like features is a rare, recently recognized entity restricted to the sinonasal tract. By definition, it is associated with high-risk HPV infection, particularly with HPV type 33. In most cases, tumors are composed of dual cell populations, including predominant basaloid myoepithelial cells and usually inconspicuous ductal cells. Solid components with focal cribriform or tubular patterns, abrupt keratinization within tumor nests, and squamous dysplasia of the surface epithelium are characteristics of HPV-related carcinoma with adenoid cystic-like features. The immunohistochemistry of p16 followed by high-risk HPV testing may help in the differential diagnosis. Recent studies have demonstrated that the morphologic features of this entity are more diverse than initially believed. Surgical resection is the prime alternative for treatment. According to the limited data, the prognosis of this disease may be better than that of other sinonasal carcinomas.

The inheritance of mutated suppressor genes, such as BRCA1 and BRCA2, is acknowledged as an etiological factor in hereditary breast carcinoma (HBC). Two different molecular mechanisms are possible; the Knudson's "two hits" or the gene haploinsufficiency. Etiology of sporadic breast carcinoma (SBC) is not known, although data support the possible role of the betaretrovirus Mouse Mammary Tumor Virus (MMTV). This study analyzes the presence of MMTV exogenous sequences in two representative groups of HBC and SBC, excluding any contamination by murine and retroviral material and endogenous betaretroviruses. The 30.3% of 56 SBC contained MMTV sequences, against the 4.2% of 47 HBC ( p < 0.001). Cases positive for viral sequences showed the presence of p14, signal peptide of the MMTV envelope precursor. This result was expected based on the fact that HBCs, having a specific genetic etiology, do not need the action of a carcinogenetic viral agent. Moreover, the striking results obtained by comparing two groups of vastly different tumors represent an additional element of quality control: the distinction between HBC and SBC is so well-defined that results cannot be ascribed to mere coincidence. This paper strengthens the hypothesis for a viral etiology for human sporadic breast carcinoma.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a recently described distinctive clinicopathologic entity defined by association to high risk HPV, localization to sinonasal tract and close histologic resemblance to salivary gland tumors. Lack of awareness of its pathologic features and biology among pathologists and oncologists make this entity susceptible to misdiagnosis and erroneous management. Herein, we illustrate a case of HMSC of the nasal cavity associated with heretofore unreported subtype HPV-52 and discuss the challenges associated with diagnosis and management of this rare tumor. A 48-year-old woman with intermittent epistaxis for 6 months presented with a nasal mass and underwent middle turbinectomy. Histology showed a tumor with features typical of adenoid cystic carcinoma (ACC) in the form of basaloid cells and cribriform architecture. However, careful inspection revealed findings uncommon in ACC; such as surface pagetoid tumor spread, areas of solid sheets of myoepithelial cells accompanied by increased mitotic figures which prompted immunohistochemistry. Multidirectional differentiation into ductal (CK7, AE1/AE3) and myoepithelial (p63, p40, S100, calponin) lineage together with strong and diffuse immunopositivity for p16 distinguished this tumor from ACC. HPV genotyping was positive for high risk HPV subtype HPV52, which confirmed the diagnosis of HMSC. HPV-related multiphenotypic sinonasal carcinoma is an under-recognized unique clinicopathologic entity that needs awareness to avoid mistaking it for commoner salivary gland tumors. Making accurate diagnosis of this newly-described tumor is imperative in order to understand its biology and to develop optimal therapeutic strategies.

Pulmonary lymphoepithelioma-like carcinoma (LELC) is a rare and distinct subtype of primary lung cancer characterized by Epstein-Barr virus (EBV) infection. Herein, we reported the mutational landscape of pulmonary LELC using whole-exome sequencing, targeted deep sequencing and single-nucleotide polymorphism arrays. We identify a low degree of somatic mutation but widespread existence of copy number variations. We reveal predominant signature 2 mutations and frequent loss of type I interferon genes that are involved in the host-virus counteraction. Integrated analysis shows enrichment of genetic lesions affecting several critical pathways, including NF-κB, JAK/STAT, and cell cycle. Notably, multi-dimensional comparison unveils that pulmonary LELC resemble NPC but are clearly different from other lung cancers, natural killer/T-cell lymphoma or EBV-related gastric cancer in terms of genetic features. In all, our study illustrates a distinct genomic landscape of pulmonary LELC and provides a road map to facilitate genome-guided personalized treatment.

Epstein-Barr virus (EBV) is implicated in the tumorigenesis of a variety of malignancies, including Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma (NPC). EBV+ lymphoepithelioma-like cholangiocarcinoma (LELCC) is a rare type of intrahepatic cholangiocarcinoma with a distinct pathology and poorly understood treatment options. Morphologically, this neoplasm resembles undifferentiated NPC, a commonly EBV+ tumour with a prominent lymphoid infiltrate. Almost all of the current literature regarding LELCC describes early stage tumours that are treated surgically and achieve good outcomes. In contrast, this report documents a late stage LELCC treated unsuccessfully with systemic chemotherapy., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Papillomaviruses (PVs) are ubiquitous host- and site-specific viruses. PV infections in cats are associated with oral papillomas, viral plaques, Bowenoid in situ carcinomas (BISCs), squamous cell carcinomas and sarcoids; this association is primarily based on PCR detection of PV DNA within said lesions. PV DNA is frequently detectable on normal feline skin; thus, it is possible that some of the implicated DNA is commensal rather than associated with lesion formation. Therefore, the aim of the present study was to use fluorescence in situ hybridization (FISH) to localize PV DNA within feline BISCs, to provide additional evidence that PV infection may influence the development of these neoplasms., Methods: FISH probes targeting Felis catus papillomavirus type 2 (FcaPV2) DNA were used to localize FcaPV2 DNA within 42 BISCs from which FcaPV2 DNA had previously been amplified via PCR., Results: Fifteen of 42 BISC lesions (35.7%) demonstrated intralesional FcaPV2 using FISH. Probe annealing was predominantly located within the nuclei of koilocytes found in the upper strata of the epidermis. Probes were typically scattered multifocally within the lesions; most commonly this was near the periphery of the BISCs., Conclusions and Relevance: These results confirm that a proportion of BISCs contain FcaPV2 DNA. These results further support a causative association between FcaPV2 and BISCs in cats.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a distinct, newly-described sinonasal tract neoplasm characterized by a salivary gland tumor-like appearance with myoepithelial and ductal cells, frequent surface squamous dysplasia, and relatively indolent behavior. When considering a diagnosis of HMSC, aggressive high-grade salivary gland carcinomas, particularly those with a basaloid morphology such as basal cell adenocarcinoma and adenoid cystic carcinoma, enter the differential diagnosis. The full morphologic and immunophenotypic profile of HMSC continues to be unraveled. In this series of ten cases, we demonstrate that this tumor has consistent, strong immunohistochemical expression of LEF-1 yet lacks nuclear expression of β-catenin, and also has consistent yet variable expression of MYB protein. While LEF-1 expression may be a useful diagnostic adjunct, it can also be a pitfall, as other salivary tumors such as basal cell adenocarcinoma have been previously shown to express LEF-1. Additionally, MYB protein expression is not a discriminatory marker when trying to separate HMSC from adenoid cystic carcinoma.

The involvement of human papillomavirus (HPV) in laryngeal and hypopharyngeal lymphoepithelial carcinoma was investigated in a series of ten cases (seven laryngeal and three hypopharyngeal), retrieved from the files of three tertiary hospitals in the 2000-2017 period, through polymerase chain reaction with SPF10 primers and INNO-LiPA HPV Genotyping Extra II (Innogenetics). Epstein-Barr virus (EBV) was tested in all cases with in situ hybridization INFORM EBER Probe (Ventana Medical Systems). p16 and p53 expression were immunohistochemically analyzed. Calculated annual incidence was 0.013/100,000, and prevalence was 0.2% of laryngeal and hypopharyngeal carcinomas. All cases were EBV negative. HPV was detected in five cases, three of which also overexpressed p16. HPV16 was detected in four cases, and HPV58 in one case. Five cases were HPV negative, only one of these five overexpressed p16. No recurrence was observed in nine cases during follow-up. The 5-year disease-specific-survival rate was 100%. Mean overall survival was 87 months. Lymphoepithelial carcinoma of the larynx and hypopharynx are not related to EBV. Simultaneous HPV+/p16+ is consistent with HPV causation in a fraction of laryngeal and hypopharyngeal lymphoepithelial carcinomas.

Breast cancer (BC) is a complex and heterogeneous disease whose evolution depends on the tumor-host interaction. This type of cancer occurs when the mammary cells begin to grow wildly and become able to invade nearby tissues and/or promote metastases. Mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice. The identification of MMTV-like sequences and antigens in human mammary carcinoma has supported the theory that a virus homologous to MMTV (namely, HMTV) may be involved in human BC, but the role of retroviral elements in this disease remains elusive, as results from different research groups were contradictory. In the present review we present works for and against the involvement of HMTV in BC and discuss possible causes of divergences among studies. In the final section we fit current data regarding this issue to stablished causality criteria. We conclude that there is convincing data supporting the association of HMTV with BC, however there is still a need for epidemiological and basic research studies focusing on carcinogenic mechanisms for this virus in humans to fully understand its role in BC. This knowledge may open the way for the development of new preventive and therapeutic approaches in human BC., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Epstein-Barr virus (EBV)-associated lymphoepithelioma-like gastric carcinoma (LELGC) is a rare primary stomach tumor, which has overlapping imaging features with mass forming gastric carcinoma (GC). The aim of our study was to present the computed tomography (CT) findings and clinical features of EBV-associated LELGC to increase awareness of this entity.The CT findings and clinical features of 4 patients with pathologically documented EBV-associated LELGC were retrospectively analyzed.Among the 4 patients, 3 were male, and 1 was female. The medium age was 51 years old. All cases were single lesions including 1 was in the gastric cardia, 1 was in the gastric body, and 2 were in the gastric antrum. A focal thickening of the gastric wall was conducted, with a large thickness-to-length ratio. The low-density stripe of the normal gastric wall abruptly terminated at the edge of the lesion. The CT images of 4 cases showed inhomogeneous density with the radiodensity values ranging from 25 to 48 HU. In addition, an ulcer was demonstrated with an irregular base and slightly raised borders in all cases. Enhancement after injection of contrast material was heterogeneous enhancement (n = 3) or homogeneous (n = 1). After enhancement, obvious enhancement was seen in 1 case, moderate enhancement was seen in 3 cases, with the peak value of the tumor in the portal phase. No evidence of lymph node involvement and distant invasion was observed.Although LELGC is quite rare, it should be considered in differential diagnosis of early GC, advanced GC, and lymphoma. The relatively typical CT appearance, combined the age and sex of patients, can suggest the diagnosis of LELGC.

Objective: To analyze the pathological features and their influence on the clinical outcome of non-nasopharyngeal EBV-associated carcinomas. Methods: One hundred and twenty cases of non-nasopharyngeal EBV-associated carcinoma confirmed by in situ hybridization were identified at Zhejiang Cancer Hospital from January 1, 2006 to May 1, 2018, and the clinicopathological data were collected and analyzed using Kaplan-Meier survival analysis, Cox univariate and multivariate analysis. Results: One hundred and twenty cases were involved in the study; the male to female ratio was 1∶1; patients' age range was 24 to 89 years (median 50 years). The primary sites were large parotid glands (62 cases), lung(26 cases), stomach(15 cases), and others (oral, oropharynx, larynx, cervix, liver; totally 17cases). Non-nasopharyngeal EBV-associated cancer could be divided into two histological types according to the amount of interstitial lymphocytes: type Ⅰ was "lymphoepithelial-like carcinoma" and rich in stromal lymphocytes; type Ⅱ lacked lymphocytic infiltration. Ninety-eight primary tumor samples could be classified morphologically: 43 cases were as type Ⅰ and 55 cases as typeⅡ; the distribution of type Ⅰ was 57.4% (27/47) in large parotid glands, 20.8% (5/24) in lung, 4/13 in stomach, and 7/14 in other sites. Complete treatment and survival data were obtained for 114 patients. According to the TNM staging criteria of WHO, 52 patients were at early stages (Ⅰ-Ⅱ) and 62 were at advanced stages (Ⅲ-Ⅳ); 102 patients underwent surgery. Seventy-four patients received adjuvant chemotherapy before or after surgery, and 52 patients received local radiotherapy. Kaplan-Meier survival analysis showed that patients with type Ⅱ EBV-associated carcinoma had a worse prognosis than patients with type Ⅰtumors ( P= 0.010 2). In addition, vascular invasion( P= 0.021 8),neural recidivism( P= 0.000 1),advanced stage( P= 0.017 1),lymph node metastasis ( P= 0.005 0) and chemotherapy ( P= 0.013 2) were poor prognostic factors; female patients had better survival than male ( P= 0.028 4). Cox multivariate regression analysis found that lymph node metastasis (95 %CI : 1.489-13.830, P= 0.007 6) and neural recidivism (95 %CI : 1.228-6.544, P= 0.014 7) were independent adverse prognostic factors. Cox multivariate regression analysis after stratification by site revealed that radiotherapy was a preferable prognostic factor for EBV-associated carcinoma of the large salivary glands (95% CI : 0.003-0.569, P= 0.016 8). Conclusion: EBV associated carcinoma can be divided into two types, for which type Ⅰ was with abundant interstitial lymphocytes and type Ⅱ was lack of interstitial lymphocytes. TypeⅡ EBV-associated carcinoma has a worse prognosis than type Ⅰ. Radiation therapy can prolong the survival time of patients with primary EBV-associated carcinoma of large salivary glands.

Introduction:: Recent reported evidence indicates that vocal cord carcinoma is evolving similarly to oropharyngeal cancer with an increasing number of patients without a smoking history having human papillomavirus (HPV) disease. Observations also suggest that an increasing number of patients who present with glottic carcinoma are younger than has been reported in the past. Therefore, an investigation was done to examine the incidence of glottic carcinoma in patients 30 years old (y/o) or younger., Methods:: A retrospective review was done with Institutional Review Board approval to evaluate the incidence of patients 30 y/o or younger presenting with glottic carcinoma in 2 symmetric-length time periods over 28 years. These data were comprised from glottic cancer patients evaluated by the senior author (S.M.Z.) at the Massachusetts Eye and Ear Infirmary (July 1990-June 2004) and subsequently at the Massachusetts General Hospital (July 2004-June 2018). HPV testing was done on those patients identified as having a disease process at 30 y/o or younger., Results:: Between July 1990 and June 2018, 353 patients were diagnosed with glottic carcinoma. From July 1990 to June 2004, there were 112 patients, with none being 30 y/o or younger. From July 2004 to June 2018, 241 patients were diagnosed with glottic carcinoma; 11 patients (7 females, 4 males) were 30 y/o or younger. Of the 11 patients, 3 (1 female, 2 males) were 10 to 19 y/o, 3 (2 females, 1 male) were 20 to 25 y/o, and 5 (4 females, 1 male) were 26 to 30 y/o. Moreover, 10 of the 11 cases were tested and were positive for high-risk HPV. None of the 11 glottic cancer patients had been previously treated for benign recurrent respiratory papillomatosis although it was initially suspected prior to biopsy due to the morphology of the lesions and the patients' young age. Three of 11 had a history of smoking; all 3 had less than 3 pack-years. One of the 11 glottic cancer patients was treated with serial Cidofovir injections that resulted in dramatic acceleration in the growth of the cancer., Conclusion:: Historically, glottic carcinoma is considered to be a tobacco-induced disease associated with a multidecade process of initiation, promotion, transformation, and progression. However, recent published evidence shows that glottic carcinoma can be an HPV-related disease with increasing incidence in nonsmokers. It isn't surprising that alternate malignant pathways may have a different timeline. In this investigation, an increased incidence of HPV-positive glottic cancer in patients 30 y/o or younger was documented in the past 14 years. This finding further supports the concept that glottic carcinoma is an evolving disease, and it demonstrates the increasing importance of discriminating potential glottic carcinomas in young patients from benign low-risk HPV recurrent respiratory papillomatosis.

Virus-associated carcinomas of the head and neck represent an unusual confluence of infections spread by viral transmission and cellular dysregulation resulting in carcinogenesis. While much remains to be elucidated about the exact progression from infection to cancer, a basic framework of viral biology can complement the pathologist's understanding of morphology. This context informs the pathologist's everyday practice, including selecting ancillary studies, communicating prognostically relevant findings, and participating in treatment planning. By comparing and contrasting the salient features of human papillomavirus-associated oropharyngeal carcinoma and Epstein-Barr virus-associated nasopharyngeal carcinoma, this review summarizes recent evidence to guide current practice., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lymphoepithelioma-like carcinoma (LELC) of the lung is a rare Epstein-Barr virus (EBV)-associated carcinoma. It is histologically characterized by a syncytial growth pattern with marked lymphocytic infiltration that is indistinguishable from the histology observed in undifferentiated nasopharyngeal carcinomas. However, it has been noted that LELC can display nonclassic morphology and lack significant lymphocytic infiltration. In this study, we conducted a comprehensive clinicopathologic analysis of 61 patients with pulmonary LELC and performed automatic quantification of the lymphocytic infiltrate using the IHC Profiler software. We demonstrated that pulmonary LELCs have a morphologically continuous spectrum, ranging from classic poorly differentiated tumors with intense lymphocytic infiltration to nonclassic morphology with little lymphocytic infiltration. These EBV-associated tumors represent a distinct entity and usually occur in female and nonsmoking patients. Tumors with low lymphocytic infiltration can closely resemble nonkeratinizing squamous cell carcinoma and tend to be larger in size, have higher maximum standardized uptake values on radiography, and exhibit shorter times to recurrence than those with high lymphocytic infiltration. Through detailed pathologic examination, we observed several distinct morphologic features in pulmonary LELCs, including granulomatous inflammation, focal keratinization, spread through alveolar spaces, and lepidic spreading pattern. We also found that patients with tumors exhibiting granulomatous inflammation have favorable outcomes; however, spread through alveolar spaces did not significantly correlate with prognosis. As many of these "LELCs" do not resemble undifferentiated nasopharyngeal carcinoma or lymphoepithelioma, we propose using an alternative term, EBV-associated pulmonary carcinoma, to encompass the entire morphologic spectrum of this distinct disease entity.

Objectives: Lymphoepithelioma-like carcinoma (LELC) of the uterine cervix is a rare tumor. The goal of this study was to evaluate a series of cases of cervical LELC and to investigate possible association with human papillomavirus (HPV) and/or Epstein-Barr virus (EBV)., Methods: Immunohistochemistry for p63, p16, human leukocyte antigen-D related (HLA-DR), and B-cell lymphoma 2 (BCL-2); in situ hybridization (ISH) for EBV and HPV; and polymerase chain reaction (PCR) genotyping were performed. Mismatch repair (MMR) studies and PD-L1 status were obtained., Results: We found eight cases of LELC. Tumors demonstrated sheets of cells containing vesicular nuclei, amphiphilic cytoplasm, and dense peri- and intratumoral lymphocytic infiltrates. All tumors stained for p63, p16, and HLA-DR; two also stained for BCL-2. When combining ISH and PCR results, seven tumors were HPV positive; they were all Epstein-Barr encoding region negative. All cases were MMR intact, and most overexpressed PD-L1., Conclusions: This study shows that cervical LELCs are associated with HPV and not EBV.

Background: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial., Objectives: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16 INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated., Materials and Methods: Tissue samples from 31 submandibular gland lesions were evaluated. p16 INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed., Results: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16 INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16 INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers., Conclusions: HPV-DNA positivity and p16 INK4A -immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.

Objective: The exfoliative cell analyzer, LC-1000 (Sysmex Corporation, Japan), is a medical device that presents the cell proliferation index and 23 research parameters as indicators of cellular proliferative potential. The objective was to evaluate the clinical usability of qualitative assessment by LC-1000 compared with cytology, the human papillomavirus (HPV) test, and histology as gold standard., Study Design: Women that visited 3 sites between July 2015 and March 2017 were registered. The primary endpoint in this study was the comparison between LC-1000 measurement and HPV test for sensitivity and specificity for cervical intraepithelial neoplasia 2+ (CIN2+). A tree model algorithm was newly constructed by a statistical method and its relationship with histological results was evaluated., Results: The sensitivity and specificity of LC-1000 were 78.3 and 74.1%, while those of the HPV test were 94.7 and 85.4%, respectively. A tree model comprising five categories was constructed. The proportion of advanced lesions was higher with the change in the rank classification results from 1 to 5. The positive predictive values of CIN2+ in the categories 4 and 5 were high. Despite the small number of subjects, cancer was undetected in categories 1 and 2. In addition, the comparison with follow-up results in 19 women assessed as CIN1 showed that the rate of progression in the categories 3-5 was 50% (7/14); progression in the categories 1 and 2 was 0% (0/5)., Conclusions: LC-1000 may be useful for cervical cancer screening as an index to qualitatively evaluate CIN and cancer based on the changes in characteristics of cells., (© 2019 S. Karger AG, Basel.)

Renal transplant recipients are at increased risk of developing urologic malignancies, some of which are associated with prolonged BK virus infection. We report two cases of BK virus-associated carcinoma with variant morphological patterns (clear cell adenocarcinoma of the bladder and micropapillary urothelial carcinoma of the pelvicaliceal system) arising in the urinary tract of renal transplant recipients. In both cases, the diagnosis was initially established on cytologic specimens: on urine cytology in one patient and on fine needle aspiration of an inguinal lymph node in the other patient. The unusual cytologic features of both cases (multiple morphologies in one patient and micropapillary pattern in the other), co-occurrence of decoy cells in the urine of one patient and the occurrence of these tumors in renal transplant recipients raised the possibility of BK polyomavirus-associated malignancy and led to confirmatory SV40 immunostains that were positive. These cases expand the morphologic variants of BK virus-associated urologic malignancies diagnosed in solid organ transplant patients. While differentiating BK virus-infected cells from malignant cells in urine cytology specimens is a diagnostic challenge, greater awareness of their possible co-existence is vital, as this could be the only chance for an early diagnosis., (© 2018 Wiley Periodicals, Inc.)

Background/aim: Epstein-Barr virus (EBV) is an oncovirus that is commonly associated with the development of lymphomas and epithelial carcinomas. In the era of immunotherapy, histological evaluation of EBV-related cancers is currently a multi-sample, multi-technique process requiring separate time-consuming detection of EBV-encoded small RNAs by in situ hybridisation (ISH), and parallel labelling of sections for cancer-associated protein markers., Methods: Using EBV-associated tumours as proof-of-concept for feasibility, here we developed an approach that allows simultaneous detection of EBV RNAs and multiple protein markers such as PD-L1, EBV-LMP, CD8, CD4, CD20, CD30 and CD15on a single tissue section based on our recently reported automated staining protocol., Results: We successfully combined multiplex immunofluorescence (mIF) to detect 3 abovementioned protein markers involved in cancer, with ISH, and applied the protocol to f tissue samples from patients diagnosed with EBV-associated pulmonary lymphoepithelioma-like carcinoma (LELC), gastric carcinoma and Hodgkin's Lymphoma. Empowered by the Vectra 3 Automated Quantitative Pathology Imaging System, we demonstrate the utility and potential of this integrated approach to concurrently detect and quantitate viral RNA and protein biomarkers of immune and tumour cells., Conclusion: This study represents an important step forward in the research and diagnosis of EBV-associated cancers, and could be readily modified to include other proteins and RNA markers to apply to other malignancies. More importantly, the novel automated ISH-mIF protocol that we detailly described here could also be readily reproduced by most of the diagnostic and research lab to future projects that aim to look at both RNA and protein markers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vulvar carcinoma is the fourth most common gynecological malignancy. Two separate carcinogenic pathways are suggested, where one is associated with the human papillomavirus (HPV) and HPV16 the most common genotype. The aim of this study was to evaluate HPV-markers in a set of primary tumors, metastases and recurrent lesions of vulvar squamous cell carcinomas (VSCC). Ten HPV16-positive VSCC with metastatic regional lymph nodes, distant lymphoid/hematogenous metastases or local recurrent lesions were investigated for HPV genotype, HPV16 variant, HPV16 viral load, HPV16 integration and HPV16 E2BS3 and 4 methylation. In all 10 analyzed case series, the same HPV genotype (HPV16), HPV16 variant and level of viral load were detected in all lesions within a patient case. Primary tumors with a high E2/E6 ratio were found to have fewer vulvar recurrences and/or metastases after diagnosis and treatment. Also, a significantly lower viral load was evident in regional lymph nodes compared to primary tumors. The data presented strengthens the evidence for a clonal HPV-induced pathway for vulvar carcinoma., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

About 10 % of gastric carcinoma worldwide is associated with EBV, which is defined as EBV-associated gastric carcinoma (EBVaGC). To date, EBV sequence data from EBVaGC in Guangdong, China, an endemic area of nasopharyngeal carcinoma (NPC), are not available. In the present study, two EBV genomes from EBVaGC specimens from Guangdong (designated as GDGC1 and GDGC2) were determined by next-generation sequencing, de novo assembly and joining of contigs by Sanger sequencing. In addition, we sequenced EBV from two Korean EBVaGC cell lines, YCCEL1 and SNU-719. Genomic diversity, including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), phylogenetic analysis and rates of protein evolution, was performed using bioinformatics software. The four gastric carcinoma-derived EBV (GC-EBV) were all type I. Compared with the reference EBV genome, a total of 1815 SNPs (146 indels), 1519 SNPs (106 indels), 1812 SNPs (126 indels) and 1484 SNPs (106 indels) were found in GDGC1, GDGC2, YCCEL1 and SNU-719, respectively. These variations were distributed across the entire genome, especially in latent genes. In contrast, the sequences of promoters and non-coding RNAs were strictly conserved. Phylogenetic analyses suggested the presence of at least two parental lineages of EBV among the GC-EBV genomes. Rates of protein evolution analyses showed that lytic genes were under purifying selection; in contrast, latency genes were under positive selection. In conclusion, this study determined the EBV genomes in EBVaGC from Guangdong and performed a detailed genome-wide analysis of GC-EBV, which would be helpful for further understanding of the relationship between EBV genomic variation and EBVaGC carcinogenesis.

Objective: Uterine Cervical Cancer (UCC) screening has changed with the introduction of the High Risk Human Papilloma Virus test (HRHPV) and its evaluation is necessary. The objective of this study is to analyze the effectiveness of UCC screening with activities aimed at early detection and treatment to modify the natural history of the process and improve its prognosis., Methods: Cytology and HR-HPV (co-testing) were performed according to the SEGO protocol of 2010 between 2011 and 2015 with follow-up until 2017. The HR-HPV DNA test was HC2 Hybrid Capture (Digene®) at the beginning (16.1% of the cases) and Cobas 4800 (Roche®) afterwards. Target population: Barbastro´s health area. The initial treatment was conization with loop (LLETZ). Sensitivity and Positive Predictive Value of tests were studied, as well as the association between demographic and pathological variables., Results: 238 high-grade dysplasias (HSIL) or more (CIN2+) were detected with a mean age of 37.9±10.3 years and 60.0% were genotype 16 and/or 18 positive. 220 patients (92.4%) underwent conization completed thereafter with reconization or hysterectomy in 25 cases (11.4%). HSIL was diagnosed in 220 cases (92.4%) and invasive carcinoma in 18 (7.6%), 7 microinvasive (2.9%). 14.4% of cones had no HSIL (negative cone) and 83.2% got free margins. 52.0% had involvement in a single quadrant and the mean horizontal extension was 3.5±3.1mm. Only in 14 (6.7%) patients the disease (HR-HPV positive) persisted after treatment. A statistically significant association was found in our cases between affected borders and age over 45 years (p=0.005)., Conclusions: The co-test has detected small preinvasive lesions, localized in a single quadrant and microinvasive cancers . Loop conization was effective, achieving the cure of 93.3% of the patients., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Viral infections cause at least 10%-15% of all human carcinomas. Over the last century, the elucidation of viral oncogenic roles in many cancer types has provided fundamental knowledge on carcinogenetic mechanisms and established a basis for the early intervention of virus-related cancers. Meanwhile, rapidly evolving genome-editing techniques targeting viral DNA/RNA have emerged as novel therapeutic strategies for treating virus-related carcinogenesis and have begun showing promising results. This review discusses the recent advances of genome-editing tools for treating tumorigenic viruses and their corresponding cancers, the challenges that must be overcome before clinically applying such genome-editing technologies, and more importantly, the potential solutions to these challenges.

In recent years, there has been increased interest in carcinomas of the urologic tract, that demonstrate association with the polyoma virus BK arising in immunosuppressed individuals, though the nature of this association is uncertain. To begin to understand this phenomenon, we reviewed the clinical, morphological, and immunohistochemical features of 11 carcinomas of the urologic tract, mainly urothelial (N = 9) and collecting duct carcinomas (N = 2), occurring during immunosuppression, and expressing polyoma virus T-antigen by immunohistochemistry. These were compared to a control group of carcinomas (N = 8), also arising during immunosuppression, but without T-antigen expression. A subset of both groups were also studied by hybrid capture-based DNA sequencing, probing not only for 479 cancer-related human genes, but also for polyoma and other viral sequences. Polyoma T-antigen-expressing tumors arose in 7 males and 4 females, at a median age of 66, and were aggressive, high-grade tumors with more than 1 variant morphologic pattern identified in 81% of cases, and a majority (73%) presenting at high stage category (>pT3). Diffuse polyoma T-antigen staining was seen in 91% of cases, with co-localization of aberrant p53 staining in 89%. Sequencing detected a lower number of deleterious mutations among T-antigen-expressing cases (average 1.62; 1/8 with TP53 mutation) compared to control cases (average 3.5, 2/4 with TP53 mutation). Only BK virus was detected with clonal integration and breakpoints randomly distributed across the human and viral genomes in 5/5 of the polyoma T-antigen-expressing carcinomas, and in none of the controls (0/4). In summary, these findings identify aggressive clinicopathologic features of polyoma T-antigen-expressing carcinomas, document BK as the strain involved, and associate BK viral integration with T-antigen expression and p53 aberrancy. While the apparent randomness of viral insertion sites is functionally unclear, the differing rates of mutations between T-antigen-expressing and control cases is intriguing.

Nasopharyngeal carcinoma (NPC) is a metastatic Epstein-Barr virus (EBV)-associated cancer that expresses the viral oncogenic protein, latent membrane protein 1 (LMP1). During epithelial metastasis, detached cells must overcome anoikis-induced cell death and gain the ability to reattach and restore growth potential. Anoikis assays have revealed cell survival mechanisms during suspension, but few studies have tracked the fate of cells surviving anoikis-inducing conditions. In this study, a modified anoikis assay was used to examine if the expression of LMP1 confers the recovery of epithelial cells from anoikis. Cells expressing LMP1 mutants and strains were evaluated for distinguishing properties in survival during suspension, reattachment, and outgrowth potential. Expression of LMP1 promoted the outgrowth of the NPC cell line HK1 following anoikis induction that was not attributed to enhanced cell survival in suspension or reattachment. The mechanism of LMP1-induced outgrowth required Akt signaling and the conserved PXQXT motif on LMP1, which activates Akt. Deletion of any of the three LMP1 C-terminal activation regions (CTAR) abrogated anoikis recovery, suggesting that additional LMP1-regulated signaling pathways are likely involved. Of the seven LMP1 strains, only B958, China1, and Med + promoted HK1 outgrowth from anoikis. This distinguishing biological property segregates LMP1 strains into two categories (anoikis recovering and nonrecovering) and suggests that LMP1 strain-specific sequences may be important in determining metastatic outgrowth potential in NPC tumors. IMPORTANCE LMP1 is one of the most divergent sequences in the EBV genome and phylogenetically segregates into seven LMP1 strains. The LMP1 strains differ in geographical distribution and NPC tumor prevalence, but the molecular basis for this potential selection is not clear. While there are signaling motifs conserved in all LMP1 sequences from circulating EBV isolates, phylogenetic studies of NPC also suggest that there may be sequence selection for tumor-associated LMP1 strains and polymorphisms. The present study describes a modified anoikis assay that can distinguish LMP1 strains into two groups by biological properties. The pleiotropic LMP1 signaling properties and sequence diversity may offer a unique opportunity to illuminate the complex mechanisms of metastasis. Although the host genomic landscape is variable between NPC tumors, the present functional-mapping studies on LMP1 support the notion that viral proteins could serve as molecular tool kits and potentially reveal sequence-associated risk factors in NPC metastatic progression., (Copyright © 2018 American Society for Microbiology.)

Objectives/hypothesis: Human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma (OPSCC) are distinct disease entities. Prognostic factors specific to each entity have not been adequately explored. Goals for this study were: 1) to determine whether HPV-positive and HPV-negative OPSCCs have distinct prognostic factors, and 2) to explore the prognostic significance of sex and race in OPSCC after HPV stratification STUDY DESIGN: Retrospective case series., Methods: A retrospective review of 239 incident OPSCC patients from 1995 to 2012, treated at Johns Hopkins and University of California-San Francisco was conducted. Women and nonwhite races were oversampled. All analyses were stratified by tumor HPV in situ hybridization status. The effects of sex and race on survival were considered in Kaplan-Meier and unadjusted and adjusted Cox regression models., Results: One hundred thirty-four (56.1%) OPSCC patients were HPV positive. On univariate analysis, women had better overall survival than men among HPV-positive (hazard ratio [HR]: 0.47, 95% confidence interval [CI]: 0.20-1.07; P = .06) but not HPV-negative (HR: 0.73, 95% CI: 0.43-1.24; P = .24) OPSCCs. On multivariate analysis, women with HPV-positive OPSCCs remained at lower risk of death (adjusted hazard ratio [aHR]: 0.34, 95% CI: 0.12-0.96; P = .04). Survival did not vary significantly by race among HPV-positive patients. Among HPV-negative patients, Hispanic patients had significantly better survival in unadjusted (HR: 0.27, 95% CI: 0.08-0.91; P = .04) but not adjusted (aHR: 0.93, 95% CI: 0.11-7.36; P = .94) analysis., Conclusions: Women with HPV-positive OPSCC may have improved overall survival compared to men. Sex does not play a prognostic role in HPV-negative OPSCC. There are no differences in prognosis by race among HPV-positive or HPV-negative patients., Level of Evidence: 4 Laryngoscope, E287-E295, 2018., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Purpose: The measuring Epstein-Barr virus (EBV) DNA is an important predictor of nasopharyngeal carcinoma (NPC). This study evaluated the predictive value of pretreatment serum amyloid A (SAA) and C-reactive protein (CRP) comparing with EBV DNA in patients with NPC., Materials and Methods: In an observational study of 419 non-metastatic NPC patients, we prospectively evaluated the prognostic effects of pretreatment SAA, CRP, and EBV DNA on survival. The primary endpoint was progress-free survival (PFS)., Results: The median level of SAA and CRP was 4.28 mg/L and 1.88 mg/L, respectively. For the highSAA group (&gt; 4.28 mg/L) versus the low-SAA (≤ 4.28 mg/L) group and the high-CRP group (&gt; 1.88 mg/L) versus the low-CRP (≤ 1.88 mg/L) group, the 5-year PFS was 64.5% versus 73.1% (p=0.013) and 65.2% versus 73.3% (p=0.064), respectively. EBV DNA detection showed a superior predictive result, the 5-year PFS in the EBV DNA ≥ 1,500 copies/mL group was obviously different than the EBV DNA &lt; 1,500 copies/mL group (62.2% versus 77.8%, p &lt; 0.001). Multifactorial Cox regression analysis confirmed that in the PFS, the independent prognostic factors were including EBV DNA (hazard ratio [HR], 1.788; p=0.009), tumour stage (HR, 1.903; p=0.021), and node stage (HR, 1.498; p=0.049), but the SAA and CRP were not included in the independent prognostic factors., Conclusion: The results of SAA and CRP had a certain relationship with the prognosis of NPC, and the prognosis of patients with high level of SAA and CRP were poor. However, the predictive ability of SAA and CRP was lower than that of EBV DNA.

Purpose: Little is known about combination of the circulating Epstein-Barr viral (EBV) DNA and tumor volume in prognosis of stage II nasopharyngeal carcinoma (NPC) patients in the intensity modulated radiotherapy (IMRT) era. We conducted this cohort study to evaluate the prognostic values of combining these two factors., Materials and Methods: By Kaplan-Meier, we compare the differences of survival curves between 385 patients with different EBV DNA or tumor volume levels, or with the combination of two biomarkers mentioned above., Results: Gross tumor volume of cervical lymph nodes (GTVnd, p &lt; 0.001) and total tumor volume (GTVtotal, p &lt; 0.001) were both closely related to pretreatment EBV DNA, while gross tumor volume of nasopharynx (GTVnx, p=0.047) was weakly related to EBV DNA. EBV DNA was significantly correlated with progress-free survival (PFS, p=0.005), locoregional-free survival (LRFS, p=0.039), and distant metastasis-free survival (DMFS, p=0.017), while GTVtotal, regardless of GTVnx and GTVnd, had a significant correlation with PFS and LRFS. The p-values of GTVtotal for PFS and LRFS were 0.008 and 0.001, respectively. According to GTVtotal and pretreatment EBV DNA level, patients were divided into a low-risk group (EBV DNA 0 copy/mL, GTVtotal &lt; 30 cm3; EBV DNA 0 copy/mL, GTVtotal ≥ 30 cm3; or EBV DNA &gt; 0 copy/mL, GTVtotal &lt; 30 cm3) and a high-risk group (EBV DNA &gt; 0 copy/mL, GTVtotal ≥ 30 cm3). When patients in the low-risk group were compared with those in the high-risk group, 3-year PFS (p=0.003), LRFS (p=0.010), and DMFS (p=0.031) rates were statistically significant., Conclusion: Pretreatment plasma EBV DNA and tumor volume were both closely correlated with prognosis of stage II NPC patients in the IMRT era. Combination of EBV DNA and tumor volume can refine prognosis and indicate for clinical therapy.

The Epstein-Barr virus latent membrane protein 1 (EBV‑LMP1) is an oncoviral protein that plays an important role in oncogenic transformation in EBV‑associated nasopharyngeal carcinoma (NPC). Our previous studies demonstrated that LMP1 increased VEGFA expression and upregulated angiogenesis in NPC. Vasculogenic mimicry (VM) is a mechanism by which tumor cells can obtain nutrients to survive, and VM has been observed in numerous types of tumors. However, the occurrence and significance of VM in NPC and the relationship between LMP1 and VM have not yet been evaluated. In the present study, we observed that it was almost impossible for LMP1-negative NPC cells to form tubular structures, whereas LMP1-positive NPC cells were able to form tubular structures. Moreover, VEGFA was found to be involved in VM formation in LMP1-positive NPC cells. Knockdown of LMP1 or VEGFR1 distinctly disrupted tubular structures, whereas inhibition of VEGFR2 did not affect the process, indicating that VEGFR1 not VEGFR2 signaling was involved in LMP1-mediated VM formation. Furthermore, the data of immunohistochemistry (IHC) and CD34/PAS double staining in a tumor tissue array showed that LMP1 was positively correlated with VEGFR1 and VM. Meanwhile, after analyzing the clinicopathological features, we found that VM formation was associated with a poor prognosis in NPC patients. These results suggest that VM formation is increased by EBV‑LMP1 via VEGF/VEGFR1 signaling and provide additional information to clarify the role of EBV‑LMP1 in NPC pathophysiology.

Epstein-Barr virus (EBV) can establish a life-long latent infection in the host and is associated with various human malignancies, including nasopharyngeal carcinoma (NPC), the most common cancer originated from nasopharynx. EBV nuclear antigen 1 (EBNA1) is the only viral protein absolutely demanded for segregation, replication, transcription and maintenance of EBV viral genome in host cells. Baicalein, a bioactive flavonoid compound purified from the root of Scutellariae baicaleinsis, displays anti-inflammatory, immunosuppressive, and anti-tumor properties. In this study, the therapeutic effects and functional mechanism of baicalein on EBV-positive human NPC were determined. Cell Counting Kit-8 assays and cell formation colony were performed to investigate that baicalein can suppress proliferation of EBV-infected human NPC cells. Flow cytometric and hoechst 33258 staining results indicated that baicalein induced cell cycle arrest and apoptosis. Western blotting results demonstrated that baicalein down-regulates EBNA1 expression but not reduces the stability and half-life of EBNA1 in EBV-infected NPC cells. Additionally, the mRNA level of EBNA1 was examined by real time-PCR, the activity of EBNA1 Q promoter (Qp) was determined by dual luciferase reporter assay. Considering that transcription factor specificity protein 1 (Sp1) can maintain EBNA1 Qp active. Further analyses also elucidated that baicalein inhibits the expression of Sp1 while knock-down Sp1 by specific shRNAs decreases the expression and transcription levels of EBNA1. Therefore, the results suggested that baicalein may decrease EBNA1 expression level in EBV-positive NPC cells via inhibiting the activity of EBNA1 Q-promoter while over-expression of EBNA1 attenuate the inhibitory effect of baicalein. Finally, it was found that baicalein may strongly reduce growth of tumor in the mouse xenograft model of EBV-positive NPC. These results indicated that baicalein inhibits growth of EBV-positive NPC by repressing the activity of EBNA1 Q-promoter. Baicalein may be used as a therapeutic agent to treat EBV-positive NPC., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Small cell carcinoma of the uterine cervix is a rare and highly malignant tumor, and its etiopathogenesis is strongly related to high-risk HPV infections., Methods: The clinicopathological data of 30 cases of cervical primary small cell carcinoma were retrospectively analyzed. In situ hybridization, polymerase chain reaction and reverse dot-blot hybridization were employed to detect HPV DNA in both small cell carcinoma and other coexisting epithelial tumors. Immunohistochemistry was used to detect the protein expression of p16 and p53., Results: Amongst 30 patients with cervical primary small cell carcinoma, 15 patients simultaneously exhibited other types of epithelial tumors, including squamous cell carcinoma, adenocarcinoma, squamous cell carcinoma in situ, and adenocarcinoma in situ. Most tumor cells infected with HPV presented integrated patterns in the nuclei by in situ hybridization. HPV DNA was detected in every small cell carcinoma case (100%) by polymerase chain reaction and reverse dot blot hybridization. 27 cases (90%) harbored type 18, and 15 (50%) displayed multiple HPV18 and 16 infections. The prevalence of HPV 18 infection in small cell carcinoma was higher than in cervical squamous and glandular epithelial neoplasms (P = 0.002). However, similar infection rates of HPV 16 were detected in both tumors (P = 0.383). Both small cell carcinoma and other types of epithelial tumors exhibited strong nuclear and cytoplasmic staining for p16 in all cases. Three cases of small cell carcinoma revealed completely negative p53 immunohistochemical expression in 15 cases of composite tumors, which suggested TP53 nonsense mutation pattern. The pure small cell carcinoma of uterine cervix had similar mutation or wild type pattern for TP53 compared with composite tumor (P = 0.224)., Conclusions: Cervical small cell carcinomas are often associated with squamous or glandular epithelial tumors, which might result from multiple HPV infections, especially HPV 16 infection. Multiple HPV infections were not correlated with tumor stage, size, lymphovascular invasion, lymph node metastasis, or prognosis. Furthermore, careful observation of specimens is very important in finding little proportion of small cell carcinoma in the composite lesions, specifically in cervical biopsy specimens, in order to avoid the missed diagnosis of small cell carcinoma.

Multiple factors are associated with human papillomavirus (HPV) infection related cervical anomalies and its progression to cervical carcinoma (CaCx), but data vary with respect to the underlying HPV genotype and with population being studied. No data are available regarding the role of immunological imbalance in HPV infected CaCx pathogenesis from Northeast India, which has an ethnically distinct population, and was aimed to be addressed through this study. The study included 76 CaCx cases, 25 cervical intraepithelial neoplasia (CIN) cases, and 50 healthy female controls. HPV screening and genotyping were performed by PCR. Differential expression of tumor necrosis factor alpha (TNF-α) was studied at serum level by enzyme-linked immunosorbent assay and tissue level by immunohistochemistry and messenger RNA (mRNA) level by real-time PCR. The data were correlated with interferon gamma (IFN-γ) and NF-κβp65 levels at protein level, as well as HPV16 E6 and E7 expression at transcript level statistically. HPV infection and HPV16 genotype were predominant in the studied cohort. TNF-α was found to be downregulated at both mRNA and protein levels in CaCx cases compared to controls; and the gradient downregulation correlated with progression of the disease from normal→CIN→CaCx. TNF-α expression correlated with insufficient modulation of both IFN-γ and NF-κβp65. The HPV16 E6 and E7 transcripts were found to be sharply upregulated in CaCx cases strongly inversely correlated with the TNF-α expression. Significant role of TNF-α downregulation associated with insufficient IFN-γ and total NF-κβp65 modulation and the resulting significant upregulation of viral transcripts E6 and E7 are key to the HPV16 infection mediated CaCx pathogenesis in northeast Indian patients.

Background/aim: Malignant tumors of the salivary glands are rare and heterogeneous, with more than 20 subtypes, and classified mainly by histopathology. Their diagnosis is often challenging and their etiology unknown. Here, the possible association between human polyomaviruses (PyVs) and one or more salivary gland tumor subtypes was examined., Materials and Methods: Ninety-one primary tumors, including 12 subtypes and eight corresponding metastases, were analyzed for the presence of DNA of 10 different human PyV species by a bead-based multiplex assay using polymerase chain reaction and Luminex analyses., Results: Three samples, one adenocarcinoma (not otherwise specified), one adenoid cystic carcinoma, and one mucoepidermoid carcinoma were found to be positive. However, the amount of MCPyV DNA in these tumors was estimated to be less than one genome per tumor cell., Conclusion: The analysis of DNA from 10 human PyVs in a large number of malignant salivary gland cancers did not implicate any of these human PyVs as an important causative agent in any of the 12 subtypes studied., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Pancreatic cancer (i.e., pancreatic ductal adenocarcinoma, PDAC) is an important healthcare issue and a highly lethal disease. Thus, almost 80% of patients with PDAC will die within one year after diagnosis. Several factors including smoking, obesity, advanced age, diabetes mellitus and chronic pancreatitis have been associated with increased risk of PDAC. Hepatitis B virus (HBV) infection is also considered as a risk factor for PDAC development in some studies. However, the role of HBV infection in PDAC is poorly explored. The present paper reviews the current relevant literature exploring the impact of HBV infection in PDAC. Assessment of HBV infection impact in PDAC is challenging because its effects could be easily underestimated. Indeed, the role played by occult B infection (OBI) and intrinsic difficulties to detect HBV antigens or DNA in pancreatic tissue remains major limitations to further progress. To date a significant proportion of available literature suggests the potential oncogenic role of HBV in PDAC but experimental evidences remain scarce. Remarkably, it appears that HBV infection might influence some clinical and pathological features of patients with PDAC. Future researches to better define the role of HBV infection in developing PDAC are urgently needed., (Celsius.)

Aims: To assess differences in methylation profiles, and thus pathogenesis, between Epstein-Barr virus (EBV)-positive and negative nasopharyngeal carcinomas (NPCs). Also, promoter hypermethylation is a common phenomenon in early carcinogenesis to inactivate tumour suppressor genes. Since epigenetic changes are reversible, the therapeutic application of methylation inhibitors could provide treatment options., Methods: We evaluated promoter hypermethylation profiles of 22 common tumour suppressor genes in 108 NPCs using methylation-specific multiplex ligation-dependent probe amplification. Correlation between methylation, clinicopathological features (including EBV) and survival was examined. Cluster analysis was also performed., Results: Hypermethylation of RASSF1A and ESR1 was significantly more frequent in EBV-positive NPC, while hypermethylation of DAPK1 was more frequent in EBV-negative NPC. In logistic regression, age, with EBV-positive NPC occurring at earlier age, and RASSF1, with RASSF1 hypermethylation being more frequent in EBV-positive NPC, remained significant. In EBV-positive NPC, hypermethylation of RASSF1A predicted worse overall survival (OS) (HR 3.058,95% CI 1.027 to 9.107). In EBV-negative NPC, hypermethylated adenomatous polyposis coli (APC) was a predictor of poor disease-free survival (DFS) (HR 6.868, 95% CI 2.142 to 22.022)., Conclusion: There are important epigenetic differences between EBV-negative and EBV-positive NPCs, with EBV-negative NPC having a more similar hypermethylation profile to other head and neck squamous cell carcinomas than EBV-positive NPC. Hypermethylation of RASSF1A might contribute to worse OS in EBV-positive NPC, and may be an important event in the pathogenesis of EBV-infected NPC. Hypermethylation of APC might contribute to worse DFS in EBV-negative NPC., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objective: The purpose of this study was to identify human Cytomegalovirus (HCMV) in tissue blocks obtained from patients with nasopharyngeal carcinoma (NPC)., Materials and Methods: Formalin-fixed paraffin wax processed NPC tissue were obtained from 150 tissue blocks and retrospectively investigated for the presence of HCMV using polymerase chain reaction., Results: Of the 150 NPC tissue specimens, HCMV was identified in 53/150 (35.3%) of the samples. Out of the 53 samples infected with HCMV, 33/97 (34%) were among males and 20/53 (37.7%) were among females. Of the 53 positive samples, 36/53 (68%) were found to harbor Epstein-Barr virus (EBV)., Conclusion: The present study has shown a relatively considerable association between HCMV and NPC. The great majority of samples sheltering HCMV were also found to hide EBV, which proposes the potentiality of EBV over HCMV., Competing Interests: There are no conflicts of interest

Our previous study found that smoking was associated with an elevated level of the antibody against VCA in the Epstein-Barr virus (EBV) lytic phase, which was an important predictive marker of the risk of nasopharyngeal carcinoma (NPC). It remained unknown whether environmental factors were associated with the levels of other EBV antibodies, such as Zta-IgA, EA-IgA, EBNA1-IgA, and LMP1-IgA, in the lytic and latent infection periods. We aimed to investigate the possible environmental inducers that could affect EBV antibody levels in two independent healthy male populations from endemic NPC areas in South China (N=1498) and non-endemic NPC areas in North China (N=1961). We performed ELISA and immunoenzymatic assays to test the levels of antibodies specific to the EBV antigens. The seropositive rates of antibodies against the antigens expressed in both the EBV latent and lytic infection periods, namely, LMP1-IgA, EBNA1-IgA, and Zta-IgA, in endemic areas (28.65%, 5.43% and 14.49%, respectively) were significantly higher than those in non-endemic areas (14.43%, 1.07% and 6.32%, respectively). Smoking was associated with higher seropositivity for EBNA1-IgA (OR=1.47, 95% CI=1.12-1.93) and Zta-IgA (OR=1.28, 95% CI=0.99-1.66), with dose-response effects, while not associated with the levels of LMP1-IgA. In conclusion, smoking was an important environmental factor, which associated with increased levels of EBNA1-IgA, and Zta-IgA., (Copyright © 2018 German Center for Neurodegenerative Diseases (DZNE). Published by Elsevier B.V. All rights reserved.)

In this case of suspected sphenoid wing meningioma, Ga-DOTATATE PET/CT showed a somatostatin receptor (SSR)-expressing tumor with extension to the nasopharynx and SSR-expressing cervical lymph nodes. Subsequent biopsy from the nasopharynx revealed an Epstein-Barr virus (EBV)-associated, undifferentiated World Health Organization type 3 nasopharyngeal carcinoma (NPC), a potential clinical pitfall due to the reported high SSR expression of this tumor subtype. In consideration of the high target-to-background contrast, SSR ligands might be superior to F-FDG for EBV-associated NPC PET imaging, particularly at the skull base. Somatostatin receptor ligands might furthermore offer interesting theranostic possibilities for patients with advanced/extensive EBV-associated NPC.

Background/aims: To investigate the etiology of esophageal cancer (EC) related with human papillomavirus (HPV) infection., Materials and Methods: Fresh surgically resected tissue samples and clinical information were obtained from 189 patients. Genomic DNA was extracted, and HPV was detected using polymerase chain reaction (PCR) with HPV L1 gene primers of MY09/11; HPV16 was detected using HPV16 E6 type-specific primer sets. Copies of HPV16 E2, E6, and the human housekeeping gene β-actin were tested using quantitative PCR to analyze the relationship between HPV16 integration and esophageal squamous cell carcinoma and the relationship between the HPV16 integration status and clinical information of patients., Results: Of the 189 samples, 168 HPV-positive samples were detected, of which 76 were HPV16 positive. Among the HPV16 positive samples, 2 cases (E2/E6 ratio>1) were 2.6% (2/76) purely episomal, 65 (E2/E6 ratio between 0 and 1) were 85.6% (65/76) mixture of integrated and episomal, and 9 (E2/E6 ratio=0) were 11.8% (9/76) purely integrated. The results indicate that integration of HPV16 was more common in the host genome than in the episome genome. The prevalence rate of HPV16 integration is increasing with the pathological stage progression of esophageal carcinoma (EC)., Conclusion: A high prevalence of HPV16 suggested that HPV16 has an etiological effect on the progress of EC. Integration of HPV16 is more common than episome genome in the host cells, indicating that continuous HPV infection is the key to esophageal epithelial cell malignant conversion and canceration.

Aims: Tumour-associated macrophages (TAMs) and regulatory T cells (Tregs) form a special niche supporting tumour progression, and both correlate with worse survival in head and neck cancers. However, the prognostic role of TAM and Tregs in nasopharyngeal carcinoma (NPC) is still unknown. Therefore, we determined differences in TAMs and Tregs in different NPC subtypes, and their prognostic significance., Methods: Tissue of 91 NPCs was assessed for TAMs and Tregs by determination of CD68, CD163, CD206 and FOXP3 expression in the tumour microenvironment. Clinicopathological correlations were assessed using Pearson X 2 test, Fisher's exact test, analysis of variance and Mann-Whitney U test. Survival was analysed using Kaplan-Meier curves and Cox regression., Results: CD68 and FOXP3 counts were higher in Epstein-Barr virus (EBV)-positive NPC, while CD68-/FOXP3-, CD163+/FOXP3- and CD206+/FOXP3- infiltrates were more common in EBV-negative NPC. In the whole NPC group, CD68-/FOXP3- correlated with worse overall survival (OS), and after multivariate analysis high FOXP3 count showed better OS (HR 0.352, 95% CI 0.128 to 0.968). No difference in M2 counts existed between EBV-positive and negative NPC., Conclusions: FOXP3, a Treg marker, seems to be an independent prognostic factor for better OS in the whole NPC group. Therefore, immune-based therapies targeting Tregs should be carefully evaluated. M2 spectrum macrophages are probably more prominent in EBV-negative NPC with also functional differences compared with EBV-positive NPC., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) which is prevalent in South China, and its association with systemic lupus erythematosus (SLE) or other autoimmune diseases has not been studied in the mainland of China. The EBV serological tests have been performed on patients with various diseases or manifestations for years at our institution and their values need to be evaluated., Methods: For routine medical purposes, anti-EB viral capsid antigen (VCA) IgG, IgA and IgM antibodies, anti-EBV diffuse early antigen (EA-D) IgA antibodies, and anti-EBV nuclear antigen-1(EBNA-1) IgG antibodies were tested with commercial enzyme-linked immunosorbent assay (ELISA) in patients visiting Peking Union Medical College Hospital between 2013 and 2017. The test results were analyzed in this retrospective study., Results: There were a total of 11122 serum samples available to be tested in the study. As indicators of past EBV infection, the prevalence of VCA-IgG/EBNA1-IgG were 66.6%/58.5%, 84.3%/78.8%, 92.9%/87.0% and 98.5%/95.4% in patients aged under 5 years, 6-10 years, 11-20 years and 21-30 years old, respectively, and these values maintained at this highest rate as age increased further. The prevalence of VCA-IgM, as a parameter of acute EBV infection, was 14.6%, 10.2%, 10.4%, 6.3% and 3.1% in patients aged under 5 years, 6-10 years,11-20 years, 21-30 years, 31-40 years old, respectively, and decreased to 2%~3% in older patients. Patients with elevated serum liver enzymes were more likely to have a higher prevalence of EA/D IgA antibody (P < 0.01) and young patients (≤30 years) with lymphadenopathy were more likely to have higher prevalence of VCA-IgM antibody (P < 0.01). The prevalence of VCA-IgA and EAD-IgA were 87.0% and 59.2% in NPC patients, respectively, and both were significantly higher (P < 0.001) than that in non-NPC patients. The prevalence of VCA-IgA was 45.4% and 25.6% in SLE patients and patients with other autoimmune diseases, respectively, which were significantly (P < 0.001) and mildly (P = 0.039) higher than their controls. In pediatric SLE patients between 6 and10 years old, the prevalence of VCA-IgG, VCA-IgA and EBNA1-IgG was 100%, 59.5% and 100%, respectively, all being significantly higher than the age (6-10y) related controls (P< 0.01). In the 705 cerebral spinal fluid (CSF) specimens, VCA-IgG, VCA-IgM, VCA-IgA and EAD-IgA were found to be positive in 12.1%, 0.15%, 0.25% and 0.25%, respectively. There were 157 paired specimens (CSF and serum were collected simultaneously) and VCA-IgG was identified as positive in 12.7% of the CSF and 100% of the serum specimens., Conclusions: Around 98% of Chinese patients were infected with EBV before 30 years of age and the highest rate of acute EBV infection were observed in patients under 5 years old. EBV infection was found to be associated with elevated serum liver enzymes, NPC and SLE. Acute anti-EBV antibody was valued for young patients with lymphadenopathy but limited value for CNS neuropathy.