EBER Research Articles

Classic Hodgkin Lymphoma with Marked Granulomatous Reaction: A Clinicopathologic Study of 20 Cases.

Granulomatous reactions can be associated with various types of lymphoma, most commonly classic Hodgkin lymphoma (CHL). In some cases, the granulomatous reaction is extensive, obscuring the presence of neoplastic cells and potentially leading to delayed diagnosis and treatment. It is unknown if this subgroup of CHL has any clinicopathologic features. Here, we assessed the clinical and pathological features of 20 cases of CHL with a marked granulomatous reaction, defined in this study as granulomas representing > 50% of the total cellularity/space of the specimen. This cohort of patients showed a male predominance (M:F ratio = 1.9:1) and 75% of patients were older than 40 years. Nineteen (95%) patients presented with lymphadenopathy with the neck/supraclavicular areas being most commonly involved (11/19; 58%). Advanced stage (III-IV) disease and B symptoms were present in 69% and 64% of patients, respectively. The morphologic features of these neoplasms fit best with mixed cellularity type. The Hodgkin and Reed-Sternberg (HRS) cells were positive for CD30, PAX5 (weak), pSTAT3 (80%), CD15 (70%), PD-L1 (67%), EBV-encoded small RNA (EBER)/LMP1 (50%) and CD20 (42%), and were negative for CD3, CD5, CD45, ALK and pERK. The histiocytes of the granulomas were positive for PD-L1 (67%), pSTAT3 (50%), and were negative for pERK and cyclin D1. Next generation sequencing using a 162-gene panel was negative for mutations in 4 cases. With a median follow-up of 58.9 months (range, 3.4-199.2 months), the median overall survival was 111.0 months and the 5-year overall survival was 78%. In summary, patients with CHL and a marked granulomatous reaction can present a diagnostic challenge and the pathologist must be alert to the possible presence of CHL to avert potential misdiagnosis. The histiocytes in the granulomas frequently express PD-L1, likely through the activation of JAK2/STAT3 pathway, suggesting a potential role for PD-1 blockade therapy in these patients.

Infection pattern and immunological characteristics of Epstein-Barr virus latent infection in cervical squamous cell carcinoma.

Previous studies reported the association between Epstein-Barr virus (EBV) and cervical squamous cell carcinoma (CSCC), but its infection pattern and clinical significance unclear. This study aimed to comprehensively investigate the infection pattern, clinicopathology, outcomes, and immunology of this entity in central China. We evaluated a total of 104 untreated CSCC tumor tissue specimens using in situ hybridization for EBV-encoded small RNAs (EBERs), and by employing flowcytometry fluorescence hybridization for human papillomavirus (HPV) genotyping. The expression of EBV latency proteins and immune biomarkers was evaluated and quantified by immunohistochemistry. EBERs transcripts were detected in 21 (20.2%) cases overall (in malignant epithelial cells of 13 cases and in lymphocytes of 8 cases). EBV belonged to latency type I infection in CSCC. The high-risk (HR)-HPV was detected in all of EBV-positive CSCC, and the difference of detection rate of HR-HPV was significant when compared with EBV-negative CSCC (p = 0.001). The specific clinicopathology with increased frequency of advanced clinical stages, tumor-positive lymph nodes, neural invasion, and increased infiltration depth (all p value < 0.05) were observed in cases with EBV. However, EBV infection was found to have no impact on prognosis of patients with CSCC. Increased densities of forkhead box P3 (FoxP3)+-tumor infiltrating lymphocytes (TILs) (p = 0.005) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)+-TILs (p = 0.017) and higher expression of programmed cell death-1 (PD-1) (p = 0.002) and programmed cell death-1 ligand 1 (PD-L1) (p = 0.040) were associated with EBV latent infection in CSCC, and these immunological changes were more likely to be associated with the infection in lymphocytes rather than tumor cells. Moreover, in patients with HPV-positive CSCC, similar significant differences were still found. In conclusions, EBV-positive CSCC may have specific infection pattern and clinicopathology and can exhibit an immunosuppressive microenvironment dominated by Treg cells aggregation and immune checkpoint activation.

Loss of viral genome with altered immune microenvironment during tumour progression of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A162 RARE PRESENTATION OF EXTRANODAL NK/T-CELL LYMPHOMA INVOLVING STOMACH AND EYE: CASE REPORT AND LITERATURE REVIEW

Abstract Background Extranodal NK/T-cell lymphoma (ENKTL) is a rare and aggressive form of non-Hodgkin lymphoma. ENKTL are predominantly localized to nasal and upper aerodigestive sites, but extranasal involvement including gastrointestinal tract are rarely seen. Small and large intestines are primary sites of gastrointestinal ENKTL. Gastric involvements are exceedingly rare accounting for less than 5% of all gastrointestinal ENKTL. Purpose We present a literature review on gastrointestinal ENKTL and a case report of gastrointestinal bleed secondary to ENKTL involving stomach and left orbit. Method Case report and literature review. Result(s) 33-year-old female was admitted to a tertiary hospital with 3-week history of epigastric pain and left periorbital swelling. Abdominal CT showed edema and thickening of gastric folds. Head CT showed grossly enlarged left lateral rectus muscle and periorbital soft tissue swelling suggestive of left orbital pseudotumor. Esophagogastroduodenoscopy revealed multifocal Forrest classification II-C ulcerations throughout her stomach and duodenum. Biopsies from stomach showed gastric mucosa with extensive infiltration by an atypical lymphoid cell population. Immunohistochemistry demonstrated high grade lymphoid cells with uniform expression of CD2, CD3, CD30, CD56, TIA1, perforin and granzyme B. EBV-encoded small RNA in-situ hybridization (EBER ISH) was strongly positive. Findings were consistent with gastric ENKTL. Left orbital biopsy revealed similar morphology and phenotypic features consistent with concurrent ENKTL involvement of her orbit. Patient was initiated on intravenous corticosteroids, but unfortunately developed hemorrhagic shock secondary to gastrointestinal bleeding from gastric ulcer and passed away. Gastrointestinal ENKTL is a rare presentation of a rare disease. Gastric involvement is especially rare and described only in few case reports. Gastrointestinal ENKTL are often initially asymptomatic but can progress to abdominal pain, bleeding and even bowel perforation. Due its nonspecific clinical features and rarity, diagnosis can be difficult and requires careful examination by an experienced pathologist. This aggressive lymphoma is characterized by positive CD2, CD3, CD30, CD56, TIA, granzyme B, perforin and EBER ISH. Optimal treatment approach remains unclear due to lack of prospective clinical studies. Currently, treatment modalities used for other lymphomas including radiotherapy and non-anthracycline-based chemotherapy are used. Despite treatment, prognosis is grim with median overall survival period of 7-8 months. Image Conclusion(s) We present a case of gastric and orbital ENKTL with gastric ulcer bleeding. Gastric ENKTL disease is a rare presentation of a rare disease. Due to non-specific clinical features, diagnosis is often difficult and relies on careful pathology examination by experienced pathologist. Prognosis is poor without optimal treatment approach due to rarity of disease and lack of validated data. Please acknowledge all funding agencies by checking the applicable boxes below None Disclosure of Interest None Declared INFLAMMATORY BOWEL DISEASES MECHANISMS AND TREATMENTS

Quantitative analysis of Epstein-Barr virus DNA in plasma and stomach biopsies of patients with gastric cancer.

Epstein-Barr virus (EBV) associated gastric carcinoma (EBVaGC) is a subtype of gastric cancer with distinct histological and molecular features. The study aimed to assess the EBV DNA copy number and the prevalence of EBVaGC in gastric cancer samples taken from Iranian patients. The next aim was to assess whether the DNA and microRNAs EBV are present in plasma. EBV load was analyzed in 68 gastric cancer biopsies and compared with the results of EBV-encoded small RNA in situ hybridization (EBER-ISH) test in these patients. After the detection of 6 EBV miRNAs in gastric tissue by stem-loop RT-PCR, plasma samples were evaluated for the viral load and EBV miRNAs. Four gastric cancer cases were EBER -ISH positive (5.8%), with a significantly higher viral load than the remaining cases, 47,781 vs. 1909 copies/μg of tissue DNA. Here, was also found a significant difference in plasma EBV load between EBER-positive and EBER-negative cases. Although EBV miRNAs were detectable in all the EBER-positive tumors, the test did not detect any of these miRNAs among the plasma samples tested. Our data indicate that the prevalence of EBVaGC among Iranian patients with gastric cancer is lower than the global prevalence and although none of the EBV miRNAs were detected in plasma, evaluation of EBV microRNAs in tumor tissue, especially miR-BART7-3p, may constitute useful biomarkers for diagnosis of EBVaGC.

Primary spinal extranodal NK/T-cell lymphoma diagnosed by fine-needle aspiration and core biopsy.

We report an extremely rare case of primary spinal extranodal NK/T-cell lymphoma (PSENKTL). A 54-year-old man presented with fever of unknown etiology for 2 months, with clinical evidence of hemophagocytic lymphohistiocytosis. Imaging studies revealed multiple spinal lesions without evidence of disease in other body sites. Fine-needle aspiration and core biopsy (FNACB) of a paraspinal mass showed a monotonous population of intermediate lymphocytes with abundant cytoplasm with fine granules, round to slightly irregular nuclei, and inconspicuous nucleoli. Core biopsy revealed diffuse infiltration by cells with a NK cell phenotype, positive Epstein-Barr virus-encoded small RNA, and negative T-cell receptor gene rearrangement. Bone marrow biopsy showed the presence of hemophagocytosis without evidence of lymphoma. The disease disseminated to the small bowel late in the clinical course and the patient died shortly after admission. This unusual case was diagnosed by FNACB and raised awareness of ENKTL as a differential diagnosis in spinal lesions.

Descriptive Study of Tumor Immune Microenvironment and Epstein-Barr Virus Infection in Gastric Adenocarcinoma

Introduction. The Cauca region, in Colombia, has a high incidence of gastric cancer and there are few studies describing tumor microenvironment in gastric cancer samples obtained from this geographic region. Aim. The aim of this study was to describe the infiltration of CD3+, CD8+ T cells, the expression of programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1), and Epstein-Barr virus infection in biopsies from Cauca patients with advanced intestinal-type gastric adenocarcinoma. Methodology. This is a descriptive cross-sectional study of 24 gastrectomy samples out of 48 samples analyzed. Expression of CD3, CD8, PD-1, and PD-L1 was analyzed using immunohistochemistry, and infection by Epstein-Barr virus was analyzed by Epstein-Barr virus-encoded small RNA in-situ hybridization (EBER-ISH) in gastric tissue samples on formalin-fixed, paraffin-embedded. Finally, the immunoscore and the Combined Positive Score were calculated. Results. ITGA samples were 21.8% ± 13.6% and 14.8% ± 14.8% CD3 + and CD8 + positive respectively, 100% had low PD-1 expression, CPS score for PDL-1, 91.7% had &lt;1 and 8.3% had &gt;1. In EBER-ISH, 20.8% were positive, and in immunoscore, 16.7% had a score &gt; 25%. Conclusion. This paper reports advanced ITGA cases with an atypical tumor microenvironment, compared to previously reported data. It is important to consider that the tumor microenvironment in gastric cancer is heterogeneous, which makes the analysis of microsatellite instability in this type of sample relevant.

Establishment and characterization of two Epstein-Barr virus-positive gastric cancer cell lines with epitheliotropic M81 strain undergoing distinct viral and altered cellular expression profiles.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer (GC) distinguished by the presence of the EBV genome and limited viral gene expression within malignant epithelial cells. EBV infection is generally thought to be a relatively late event following atrophic gastritis in carcinogenesis, which implies the heterogeneity of EBVaGC. To facilitate the study of the role of EBV in EBVaGC, we established two EBV-positive GC cell lines (AGS-EBV and HGC27-EBV) with an epitheliotropic EBV strain M81 and characterized viral and cellular gene expression profiles in comparison to SNU719, a naturally derived EBV-positive GC cell line. Like SNU719, AGS-EBV and HGC27-EBV stably maintained their EBV genomes and expressed EBV-encoded small RNAs and nuclear antigen EBNA1. Comprehensive analysis of the expression of EBV-encoded miRNAs within the BamHI-A region rightward transcript region, and the transcripts of EBV latent and lytic genes in cell lines, as well as xenografts, reveals that AGS-EBV and HGC27-EBV cells undergo distinct viral expression profiles. A very small fraction of AGS-EBV and SNU719 cells can spontaneously produce infectious progeny virions, while HGC27-EBV does not. AGS-EBV (both M81 and Akata) cells largely mimic SNU719 cells in viral gene expression profiles, and altered cellular functions and pathways perturbed by EBV infection. Phylogenetic analysis of the EBV genome shows both M81 and Akata EBV strains are closely related to clinical EBVaGC isolates. Taken together, these two newly established EBV-positive GC cell lines can serve as models to further investigate the role of EBV in different contexts of gastric carcinogenesis and identify novel therapeutics against EBVaGC.

Analysis of clinical features, treatment, and prognosis of primary Xlymphoepithelioma-like carcinoma of the lung.

Primary pulmonary lymphoepithelioma-like carcinoma (PPLELC) is a rare lung malignancy occurring most frequently in young non-smokers from Southeast Asia. Given its low incidence, PPLELC clinical features, treatment methods, and the factors affecting its prognosis remain elusive. To date, PPLELC data are mainly derived from clinical case reports, and no cohort studies are available. Therefore, we retrospectively analyzed a group of PPLELC cases and summarized the clinical features of patients, treatment responses, and the factors affecting patient prognosis. A total of 91 patients having primary pulmonary lymphoepithelioma-like carcinoma were recruited in this study. These included sex, age, place of birth, smoking history, pre-treatment symptoms, tumor location, tumor markers, maximum tumor diameter, treatment regimen, lymph node presence metastasis after an operation, pathological picture, immunohistochemistry, genetic findings, and tumor stage grading. We determined the overall survival (OS), progression-free survival (PFS), basic clinical characteristics, treatment option, treatment response, and recurrence pattern among the patients. In addition, we understood the influence of sex, age, tumor, nodes, and metastases (TNM) stage, tumor size, and surgery over patient prognosis. Primary pulmonary lymphoepithelioma-like carcinoma is more common among young non-smokers, with a slightly higher incidence in women than in men. The expression of Epstein-Barr virus-encoded small RNA (EBER), pancytokeratin (PCK), Cytokeratin 5/6 (CK5/6), and tumor protein 63 (P63) was positive in immunohistochemistry. Serum cytokeratin 19 fragment antigen (CYFRA21-1) and Epstein-Barr DNA (EB-DNA) could be used as markers to diagnose primary pulmonary lymphoepithelioma-like carcinoma. TNM stage and surgery were independent prognostic factors. Primary pulmonary lymphoepithelioma-like carcinoma is rare, showing a good prognosis.

Epstein-Barr virus detection in endoscopic submucosal dissection-proven early gastric cancer with mixed-type histology.

Early gastric cancer (EGC) with mixed-type histology is a significant risk factor for additional surgery after endoscopic submucosal dissection (ESD). On the other hand, Epstein-Barr virus-associated gastric cancer (EBVaGC) with mixed-type histology is a favorable risk factor with regard to lymph node metastasis. We performed EBV detection in 13 ESD-proven lesions in 13 cases of early gastric cancer with mixed-type histology using EBV-encoded small RNA1 in situ hybridization (EBER1 ISH). EBVaGC was diagnosed in only one (7.7%) of the tested lesions. That EBVaGC patient underwent surgery and there was no residual lesion and no lymph metastasis. EBVaGC is not frequent in EGC with mixed-type histology. EBV testing of gastric biopsy specimens seems not to be useful to predict the mixed-type histology results of ESD. However, EBV testing for ESD specimens of EGC with mixed-type histology is expected to be useful for avoiding excessive additional surgery.

Copy Number Alterations of Chromosome 9p24.1 in Elderly Patients with Advanced-Stage Classic Hodgkin Lymphoma Who Received ABVD: An Ancillary Analysis of Multi-Center Retrospective Study in Japan (HORIZON study)

Introduction Copy number alterations (CNAs) of chromosome 9p24.1 are frequently observed in patients (pts) with classic Hodgkin lymphoma (cHL), which result in increased expression of programmed death ligand-1 on Reed-Sternberg (RS) cells. The amplification of 9p24.1 was found in approximately 50% of pts with advanced-stage cHL, and was an independent prognostic factor for worse progression-free survival (PFS) (Roemer MG, et al. JCO. 2016). In addition, 9p24.1 CNAs is thought to be one of the potential predictive markers for response of immune checkpoint blockade for cHL (Gerhard-Hartmann E, et al. BJH. 2022). However, the incidence or prognostic impact of 9p24.1 CNAs in elderly pts with newly diagnosed advanced-stage cHL remains unclear. In this ancillary analysis of a multi-center retrospective study (HORIZON study, UMIN000033264), we evaluated clinical impact of 9p24.1 CNAs in elderly pts with newly diagnosed advanced-stage cHL who received a doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD). Methods The key eligibility criteria of the current analysis were as follows: 1) pts with histologically diagnosed cHL between 2007 and 2016 in each institution; 2) advanced-stage cHL (stage III, IV or IIB with bulky or extranodal lesion); 3) age at diagnosis > 60 years; 4) had received at least one cycle of ABVD as initial treatment including a modified ABVD regimen (dacarbazine dose reduced to 250 mg/m2); and 5) had available specimen for tissue fluorescence in situ hybridization (FISH) analysis. All specimens were centrally reviewed by 3 experienced hematopathologists and confirmed the diagnosis of cHL. In the tissue FISH analysis, CNAs of the 9q24.1 loci along with the expression of CD30 were simultaneously detected using a combination of immunofluorescence with Fluorescence Immunophenotyping and interphase Cytogenetics as a Tool for Investigation of Neoplasia (FICTION), as described previously (Martínez-Ramírez A, et al. Leukemia. 2004). A median number of 50 RS cells (range: 30 to 74 RS cells) per case were analyzed. According to the previous report (Roemer MG, et al. JCO. 2016), nuclei with a target:control probe ratio of ≥ 3:1 were defined as amplification, and those with a probe ratio of > 1:1 but , < 3:1 were classified as relative copy gain. Nuclei with a probe ratio of 1:1 but more than two copies of each probe were defined as polysomic for chromosome 9p. In each case, the percent and magnitude of 9p24.1 amplification, copy gain, polysomy, and normal copy numbers (disomy) were noted. Cases were classified by the highest observed level of 9p24.1 CNAs. As survival analysis, overall survival (OS), PFS, and event-free survival (EFS; defined as the time from a diagnosis of cHL to disease progression or relapse, subsequent systemic chemotherapy for cHL, or death due to any cause), were evaluated. Results Among 99 eligible pts, the median age was 71 years (interquartile range [IQR] 65-77). The numbers of pts with an ECOG performance status (PS) 2 or more and B symptoms were 17 (17%) and 50 (51%), respectively. Sixty pts (61%) had mixed cellularity cHL, and 28 pts (28%) had nodular sclerosis cHL. Forty-five pts (45%) were positive for EBV-encoded small RNA (EBER) in situ hybridization. Hypoalbuminemia [serum albumin <4 g/dL] and anemia [hemoglobin <10.5 g/dL], were observed in 84 (85%), and 37 pts (37%), respectively. All the 99 pts had 9p24.1 CNAs; 67 pts (68%) had amplification, and the remaining 32 pts (32%) had copy gain (Figure 1). Hypoalbuminemia and anemia were significantly more frequent among pts with 9p24.1 amplification. The incidence of 9p24.1 amplification in EBER-positive cases tend to be higher than that of EBER-negative cases (78% in pts with EBER- positive vs. 59% in pts with EBER-negative, P=0.056). With a median follow-up duration of 52 months, the estimated OS, PFS, and EFS at 5 years in pts with 9p24.1 amplification were 80.1%, 64.2%, 59.9%, respectively. The estimated 5-year OS, PFS, and EFS of pts with 9p24.1 copy gain were 71.2%, 58.4%, 55.7%, respectively. No statistical differences were observed regarding these survival parameters between those pts with amplification and copy gain. Conclusion This ancillary analysis showed that hypoalbuminemia and anemia were significantly more frequent among pts with 9p24.1 amplification. Nevertheless, 9p24.1 amplification was not a prognostic factor for OS, PFS, and EFS in elderly pts with advanced-stage cHL treated with ABVD in the present study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Significance of Epstein-Barr Virus in Peripheral T-Cell Lymphoma, Not Otherwise Specified

Introduction: The prognostic implication of Epstein-Barr virus (EBV) in patients with peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is unclear. This study aimed to identify its clinical impact on survival outcomes in newly diagnosed patients with PTCL-NOS. Methods: Between January 2007 and March 2020, 145 patients were newly diagnosed with PTCL-NOS at Asan Medical Center, Seoul, Korea. A total of 131 patients who underwent baseline blood real-time polymerase chain reaction (PCR) for EBV-DNA were included in this analysis. Real-time blood PCR for EBV-DNA was performed using whole blood (WB) or plasma samples prior to treatments. All patients were tested for WB EBV-DNA, of which 52 were also tested for plasma EBV-DNA. In addition, the presence of EBV was assessed by EBV-encoded small RNA (EBER) in situ hybridization (ISH) in tissue samples from 121 patients. Progression-free survival (PFS) was defined as time from the date of diagnosis to disease progression or death of any cause, and overall survival (OS) was defined as time from the date of diagnosis to death of any cause. Survival curves were estimated by Kaplan-Meier method and compared using log-rank test. Results: The median age at diagnosis was 60 years (range, 19-86 years), 84 patients (64.1%) were male, and 108 patients (82.4%) had advanced stage diseases (stage III or IV). A total of 55 patients (42.0%) were positive for WB EBV-DNA in the entire cohort (n=131), 9 patients (17.3%, 9/52) were positive for plasma EBV-DNA, and 59 patients (48.8%, 59/121) were positive for EBER ISH. Among 52 patients who were tested for both WB and plasma PCR, 16 patients (30.8%) showed discordant results, all of them with positive results for WB PCR but negative results for plasma PCR. There was no significant difference in baseline characteristics between EBV positive and negative groups according to WB EBV-DNA (n=131) and EBER ISH (n=121), while higher proportion of patients with positive plasma EBV-DNA had bone marrow involvement (77.8% vs. 23.3%, p = 0.003) and thrombocytopenia (37.5% vs. 7.5%, p = 0.050) compared to patients with negative plasma EBV-DNA. With a median follow-up duration of 80.5 months (95% confidence interval [CI], 63.8-130), the median PFS and OS of the entire cohort were 8.1 months (95% CI, 5.8-10.2) and 18.6 months (95% CI, 13.1-28.0), respectively. Patients with positive WB EBV-DNA showed worse PFS compared to those with negative WB EBV-DNA (median, 5.0 vs. 9.6 months, p = 0.046), although there was no significant difference in OS (positive vs. negative: median, 13.0 vs. 20.8 months, p = 0.337). There was no significant difference in survival outcomes according to positivity of EBER ISH (positive vs. negative: median PFS, 6.7 vs. 9.7 months, p = 0.389; median OS, 15.7 vs. 21.2 months, p = 0.565) or plasma EBV-DNA (positive vs. negative: median PFS, 5.4 vs. 9.3 months, p = 0.268; median OS, 13.3 vs. 30.5 months, p = 0.325). In the multivariate analysis, positive WB EBV-DNA showed a trend towards worse PFS with hazard ratio of 1.54 (95% CI, 0.99-2.38, p = 0.055), but there was no association with OS (p = 0.713). Conclusion: Our study demonstrated that EBV positivity as measured by EBER-ISH, WB, or plasma PCR in PTCL-NOS was not significantly associated with survival. Nevertheless, there was a trend towards worse PFS in patients who were positive for WB EBV-DNA. Thus, further investigations are warranted in larger cohorts to determine its clinical implications.

Clinical Features, Phenotypic Markers and Outcomes of Diffuse Large B-Cell Lymphoma between HIV-Infected and HIV-Uninfected Chinese Patients.

Background: The effect of HIV infection on the clinicopathological characteristics of diffuse large B-cell lymphoma (DLBCL) remains debatable. Methods: Fifty-three HIV-infected and ninety-three HIV-uninfected DLBCL patients were enrolled in the retrospective study by propensity score matching for sex, age, body mass index and international prognostic index (IPI) at a ratio of 1:2. The clinicopathological characteristics were compared between the two groups. Results: HIV-infected DLBCL patients had lower white blood cell counts [×109/L; 4.4 (3.4-5.6) vs. 6.1 (4.2-8.2), p &lt; 0.001], platelet counts (×109/L; 184.7 ± 89.3 vs. 230.0 ± 113.9, p = 0.014) and serum albumin (g/L; 37.3 ± 6.9 vs. 41.3 ± 6.2, p &lt; 0.001) but higher incidences of central nervous system (CNS) involvement (9.4% vs. 1.1%, p = 0.014), bone marrow involvement (24.5% vs. 11.5%, p = 0.044) and Epstein-Barr viremia (61.1% vs. 26.7%, p = 0.002) than HIV-uninfected patients. In terms of histopathology, HIV-infected patients had higher positivity of Epstein-Barr virus-encoded small RNA (EBER) (41.7% vs. 6.7%, p = 0.002), but lower CD20 (90.2% vs. 98.7%, p= 0.029) and CD79a (23.1% vs. 53.7%, p &lt; 0.001) expression. The overall response rate (ORR) at the end of chemotherapy (70.2% vs. 87.8%, p= 0.012) and 1-year overall survival (OS) (61.7% vs. 84.2%, log-rank p = 0.006) in HIV-infected patients were significantly lower than those in HIV-uninfected patients. Multivariate analysis suggested IPI ≤2.0 [adjusted odds ratio (AOR) (95% confidence interval): 5.0 (1.2-21.2), p = 0.030] was associated with ORR, hypoalbuminemia [AOR: 3.3 (1.3-9.1), p = 0.018] and CNS involvement [AOR: 3.3 (1.0-10.5), p = 0.044] were associated with reduced 1-year OS in HIV-infected patients. Conclusion: HIV-infected DLBCL patients have unique blood profiles and phenotypic markers. Low ORR and 1-year OS were observed in HIV-infected DLBCL patients in our study, even in the HAART era.

Epstein-Barr Virus Detection and LMP-1 Expression in Thai Patients with Oral Squamous Cell Carcinoma.

To date, high-risk human papillomavirus (HPV) is primarily linked to oropharyngeal carcinoma, but only a small subset of oral squamous cell carcinoma (OSCC) is truly driven by high-risk HPV. In addition, Epstein-Barr virus (EBV) is another potential oncogenic virus for OSCC development. This study aims to investigate the role of EBV infection in Thai patients with OSCC. Forty-seven formalin-fixed paraffin-embedded specimens of OSCC were obtained. EBV DNA was detected by polymerase chain reaction analysis using primers for LMP-1 region of EBV. EBV-positive OSCC cases were subjected to LMP-1 immunohistochemical analysis and EBV-encoded small RNA (EBER) in situ hybridization to determine EBV cellular localization in OSCC. LMP-1 immunohistochemical analysis was also performed in all EBV-negative OSCC cases. Of the 47 OSCC specimens, ten (21%) exhibited EBV DNA by PCR analysis. Seven of ten (70%) EBV-positive specimens showed high-grade LMP-1 expression by immunohistochemistry. However, no EBER expression was detected in all EBV-positive OSCC specimens. In EBV-negative specimens, LMP-1 was also negative except in 3 specimens which showed low grade expression of LMP-1. The prevalence of EBV infection in OSCC in this group of Thai patients was 21%. Most of EBV-positive OSCC cases showed LMP-1 expression but a lack of EBER expression. From our findings, we presume that EBV may take some roles in OSCC development in this group of participants.

The value of a nomogram based on clinical data and contrast enhanced CT radiomics in the preoperative prediction of Epstein-Barr virus-associated gastric carcinoma

Objective: To explore the value of a nomogram based on clinical data and enhanced CT radiomics in the prediction of Epstein-Barr virus-associated gastric carcinoma(EBVaGC). Methods: The data of 136 patients, including 100 males and 36 females, aged [M (Q1, Q3)] 65 (53, 71) years, with gastric cancer confirmed by surgery and pathology were retrospectively analyzed. According to Epstein-Barr virus-encoded small RNA (EBER) in situ hybridization, those patients were divided into Epstein-Barr virus (EBV) positive group (n=32) and EBV negative group (n=104). All patients underwent multi-phase enhanced CT scanning before surgery and randomly assigned to the training group (n=95) and validation group (n=41) in a ratio of 7︰3. MaZda software was used to extract radiomics features of enhanced CT images. The intra-group correlation coefficient (ICC), variance analysis and minimum absolute shrinkage and selection algorithm (LASSO) regression were used to reduce the dimensionality of the radiomics features, and then the radiomics score (Radscore) was calculated. The nomogram model was based on combined clinical data, morphological features and Radscore. The predictive power of the nomogram was evaluated according to the area under the receiver operating characteristic curve (AUC), and the net clinical benefit of the nomogram was evaluated by the decision curve and calibration curves were drawn according to the data of the training group and the validation group to analyze the consistency of the nomogram model. Results: After selection, six optimal radiomics features were obtained, including Mean, Skewness, S(1, 0) Sum entropy, S(1, 1) Contrast, 99% percentile and S(2, 2)Angular second moment. Radscore of EBV positive group were higher than that of the EBV negative group (training group: 3.78±0.83 vs 2.80±0.98; validation group: 3.81±0.47 vs 2.94±0.95) (both P<0.05) both in the training group and validation group. The AUC of the radiomics model in training group and validation group were 0.773(95%CI:0.612-0.962)and 0.792(95%CI:0.597-0.927)respectively,and the sensitivity and specificity were 63.6% and 93.1%, 70.0% and 87.1%, respectively. The AUC of the nomogram model based on clinical data and radiomics in the training group and the validation group were 0.883(95%CI:0.644-0.984) and 0.851(95%CI:0.715-0.996), respectively. The nomogram model showed superior predictive performance (both P<0.05). Conclusion: The nomogram model based on clinical data and radiomics has better efficacy in the prediction of Epstein-Barr virus associated gastric cancer.

Prediction of Epstein-Barr Virus Status in Gastric Cancer Biopsy Specimens Using a Deep Learning Algorithm

Epstein-Barr virus (EBV)-associated gastric cancer (EBV-GC) is 1 of 4 molecular subtypes of GC and is confirmed by an expensive molecular test, EBV-encoded small RNA in situ hybridization. EBV-GC has 2 histologic characteristics, lymphoid stroma and lace-like tumor pattern, but projecting EBV-GC at biopsy is difficult even for experienced pathologists. To develop and validate a deep learning algorithm to predict EBV status from pathology images of GC biopsy. This diagnostic study developed a deep learning classifier to predict EBV-GC using image patches of tissue microarray (TMA) and whole slide images (WSIs) of GC and applied it to GC biopsy specimens from GCs diagnosed at Kangbuk Samsung Hospital between 2011 and 2020. For a quantitative evaluation and EBV-GC prediction on biopsy specimens, the area of each class and the fraction in total tissue or tumor area were calculated. Data were analyzed from March 5, 2021, to February 10, 2022. Evaluation metrics of predictive model performance were assessed on accuracy, recall, precision, F1 score, area under the receiver operating characteristic curve (AUC), and κ coefficient. This study included 137 184 image patches from 16 TMAs (708 tissue cores), 24 WSIs, and 286 biopsy images of GC. The classifier was able to classify EBV-GC image patches from TMAs and WSIs with 94.70% accuracy, 0.936 recall, 0.938 precision, 0.937 F1 score, and 0.909 κ coefficient. The classifier was used for predicting and measuring the area and fraction of EBV-GC on biopsy tissue specimens. A 10% cutoff value for the predicted fraction of EBV-GC to tissue (EBV-GC/tissue area) produced the best prediction results in EBV-GC biopsy specimens and showed the highest AUC value (0.8723; 95% CI, 0.7560-0.9501). That cutoff also obtained high sensitivity (0.895) and moderate specificity (0.745) compared with experienced pathologist sensitivity (0.842) and specificity (0.854) when using the presence of lymphoid stroma and a lace-like pattern as diagnostic criteria. On prediction maps, EBV-GCs with lace-like pattern and lymphoid stroma showed the same prediction results as EBV-GC, but cases lacking these histologic features revealed heterogeneous prediction results of EBV-GC and non-EBV-GC areas. This study showed the feasibility of EBV-GC prediction using a deep learning algorithm, even in biopsy samples. Use of such an image-based classifier before a confirmatory molecular test will reduce costs and tissue waste.

Case report: Reactive Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis and thrombosis with thrombocytopenia syndrome following SARS-CoV-2 vaccination and treated with intravenous immunoglobulin

Viral reactivation was previously reported after severe acute respiratory syndrome coronavirus‐2 (SARS-CoV-2) infection but was seldom documented after SARS-CoV-2 vaccination, except varicella-zoster virus and cytomegalovirus. Here, we present a case of reactive Epstein–Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) and thrombosis with thrombocytopenia syndrome after receiving SARS-CoV-2 mRNA vaccination. Antiplatelet factor 4 antibody was detected, and the bone marrow study showed hemophagocytosis and was positive in the immunohistochemistry staining for EBV-encoded small nuclear RNAs and negative staining for CD3 and CD56 markers of small lymphocytes. The high percentage of CD38 high/HLA-DR+ cells among CD8+ T cells further confirmed HLH. After intravenous administration of immunoglobulin, the clinical symptoms, D-dimer level, fibrinogen, platelet count, EBV-DNA titer, and anti-PF4 level were all improved. Further investigation into the pathogenesis of vaccine-associated EBV reactivation, such as TNF-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6), is warranted.

EXTRANODAL NK/T-CELL LYMPHOMA: A CASE REPORT

To present a case of a rare and challenging disease with an atypical presentation. Methods: Data were collected on eletronical and physical medical records. A 44-year-old-man was referred to the hematology clinic with a 6-month history of a painlesscutaneous plaque, that progressed to an extensive erythematous tumour lesion with central ulceration and necrosis. During this period, the patient experienced unexplained fever, night sweats and weight loss. His baseline labortatory parameters were normal, including complete blood counts, renal function, calcium and lactate dehydrogenase. A skin biopsy was performed and revealed diffuse infiltration of the skin by medium and large-sized cells with irregularly contoured nuclei, homogenous chromatin, minute nucleolus and scarce cytoplasm, extending from the papillary dermis to the subcutaneous tissue. Immunohistochemical staining showed that the cells expressed CD3, CD56 and CD5 and were negative for CD20, CD4, CD8, CD7 and CD30. In situ hybridization for EBV-encoded small RNA (EBER) showed diffuse positivity, which supported the diagnosis of Extranodal NK/T-cell lymphoma. Positron emission tomography scan (PET/CT) showed an expansive tumour lesion above the right elbow, with SUV of 21. There were no other sites of glycolytic hypermetabolism, including lymph nodes and nasal region. Bone marrow biopsy was not performed. He was treated with 5 cycles of SMILE chemotherapy (methylprednisolone, methotrexate, ifosfamide, L-asparaginase and etoposide) and achieved complete remission. He was then submitted to related allogeneic stem cell transplantation, followed by consolidation radiotherapy at the tumor site and remains alive and in remission of the lymphoma 25 months after the procedure, with mild cutaneous and hepatic chronic graft versus host disease. Extranodal NK/T-cell lymphoma is characterized by vascular damage, necrosis, cytotoxicphenotype and EBV association, with median patient age of 44-54 years. The most common involved site is the upper aerodigestive tract, specially the nasal area. Extranasal dissemination can occur with the skin involvement being one of the most frequent sites and associated with a worse prognosis. Other unfavourable prognostic factors include advanced-stage disease, high KI-67 proliferation index (40-65%) and high circulating EBV DNA levels. Treatment is based on staging. Radiotherapy is the backbone of treatment of localized disease, often combined with chemotherapy. In advanced disease, multiagent chemotherapy, with asparaginase containing regimes are used, followed by consolidation with allogeneic stem cell transplantation when feasible. We presented a NK/ T-cell lymphoma case with a rare and exuberant cutaneous presentation that was successfully treated with aggressive chemotherapy followed by stem cell transplantation.

ACUTE FIBRINOUS AND ORGANIZING PNEUMONIA (AFOP) PRESENTING AS DIFFUSE CAVITARY LESIONS IN THE SETTING OF EPSTEIN BARR VIRUS (EBV)-INDUCED HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS (HLH)

ACUTE FIBRINOUS AND ORGANIZING PNEUMONIA (AFOP) PRESENTING AS DIFFUSE CAVITARY LESIONS IN THE SETTING OF EPSTEIN BARR VIRUS (EBV)-INDUCED HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS (HLH)

High plasma EBV-DNA load and positive EBER status associated with viral recurrence and persistent infection in early treatment of lymphoma.

The relationship between plasma EBV-DNA load (PEDL) and Epstein-Barr virus (EBV)-encoded small RNA (EBER) during the early treatment of lymphoma remains unclear. We explored discrepancies in PEDL and variables associated with EBER and evaluated the consistency between EBER and qualitative analysis of PEDL (qPEDL). Serial measurements of PEDL were performed to determine the dynamic changes of PEDL in early treatment of lymphoma. As a result, the median PEDL of non-Hodgkin's lymphoma NKT cell subtype (NHL-NKT) was higher than that of non-Hodgkin's lymphoma B cell subtype (NHL-B), the median PEDL of extranodal NK/T cell lymphoma (ENKTCL) was higher than that of diffuse large B cell lymphoma (DLBCL), and the median PEDL of EBER positive was higher than that of EBER negative. Age, Ki-67 ≧ 80%, Bcl-2 ≧ 80%, p53, and qPEDL were related to EBER. The PEDL could distinguish NHL-B, DLBCL, NHL-NKT, and ENKTCL from other lymphoma subtypes. EBER-positive patients spent more time with viral "turn negative (TN)" and "continuous positive (CP)" and less time with viral "continuous negative (CN)." The median PEDL of CP was higher than that of TN. In conclusion, although EBER affects the levels of PEDL in general, it has poor concordance with qPEDL. Our results show, for the first time, that high PEDL and positive EBER present a strong association with viral recurrence and persistent infection in the early treatment of lymphoma.