Epstein-Barr Virus Encoded RNA Positivity Research Articles

Utility of Epstein-Barr Viral Load in Blood for Diagnosing and Predicting Prognosis of Lymphoma: A Comparison with Epstein-Barr Virus–Encoded RNA in Situ Hybridization

Epstein-Barr virus (EBV) is a ubiquitous pathogen that persists in a small portion of B cells after primary infection and is etiologically associated with multiple lymphoma subtypes. We evaluated the clinical utility of EBV real-time quantitative PCR in comparison with the widely used Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (ISH) method in 912 patients with four lymphoma subtypes: diffuse large B-cell lymphoma (DLBCL), extranodal natural killer/T-cell lymphoma (ENKTCL), peripheral T-cell lymphoma (PTCL), and Hodgkin lymphoma. We also assessed the impact of EBV positivity determined from each method or a combination of both methods on mortality using Kaplan-Meier survival analysis and Cox proportional hazard regression. EBV real-time quantitative PCR identified more positive cases than EBER-ISH for all subtypes, except ENKTCL. EBV DNA-positive patients with ENKTCL and PTCL displayed poorer overall survival (OS) than EBV DNA-negative patients (P=0.0016 and P=0.0013, respectively). In addition, among those with EBER-positive DLBCL and ENKTL and those with EBER-negative PTCL, OS was significantly worse for EBV DNA-positive patients (P=0.027, P=0.0016, and P=0.0018, respectively). EBER positivity was associated with worse OS for DLBCL (P=0.037), in reanalyses including only the 862 patients with unambiguous EBER-ISH results. Overall, EBV DNA positivity is a more effective prognostic marker than EBER-ISH status for patients with certain lymphoma subtypes.

EBV-Positive Plasmacytomas Involving a Nasopharyngeal Angiofibroma in an Adolescent

We report comprehensive characterization of an unusual collision tumor comprising extramedullary plasmacytomas and nasopharyngeal angiofibroma in a pediatric patient, which has yet to be reported. Histologically, the nasopharyngeal angiofibroma showed typical morphology with a diffuse, dense plasmacytic infiltrate in the stroma. The neoplastic plasma cells showed a spectrum of well-differentiated, plasmablastic, and anaplastic morphology, Epstein-Barr virus encoded RNA (EBER) positivity, and aberrant immunophenotype. Fluorescence in situ hybridization using a plasma cell myeloma targeted panel detected gains of 1q21.3 (CKS1B, x3), 3q27 (BCL6, x4), and 11q22.3 (ATM, x3), but no rearrangement of ALK and MYC. A 50-gene next generation sequencing lymphoma panel failed to detect any pathogenic mutation. Plasmacytoma with EBER positivity and plasmablastic morphology must be distinguished from plasmablastic lymphoma due to different clinical management and prognosis. This case highlights the importance of a thorough pathological evaluation of a mass lesion with synchronous neoplastic processes.

Lymphoid enhancer binding factor 1 (LEF1) expression is significantly higher in Hodgkin lymphoma associated with Richter syndrome relative to de novo classic Hodgkin lymphoma

Lymphoid enhancer binding factor 1 (LEF1) is consistently upregulated in chronic lymphocytic leukemia (CLL) and in a subset of large B cell lymphoma. Knowledge of LEF1 expression in Hodgkin lymphoma is limited. In this study, we used immunohistochemistry to survey LEF1 expression in various subsets of Hodgkin lymphoma, de novo classic Hodgkin lymphoma (CHL) (n = 43), Hodgkin lymphoma associated with Richter syndrome (HL-RS) (n = 20), and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) (n = 9). LEF1 expression was significantly higher in HL-RS compared with de novo CHL (12/20, 60% vs. 12/43, 28%; p = 0.0248). Only a single case (1/9; 11%) of NLPHL showed LEF1 expression. Epstein-Barr virus encoded RNA (EBER) was detected in 17 (40%) cases of de novo CHL and 14 (70%) HL-RS. Notably, we identified a correlation between LEF1 expression and EBER positivity (p = 0.0488). We concluded that LEF1 is commonly positive in CHL but not in NLPHL, and such a distinction may be helpful in this differential diagnosis. The higher frequency of LEF1 upregulation in HL-RS relative to de novo CHL suggests that these neoplasms might have different underlying pathogenic mechanisms and warrants further investigation.

Detection of Epstein-Barr Virus in 130 Cases of Eyelid Sebaceous Gland Carcinoma Using In Situ Hybridization.

Purpose In this study, we aimed to investigate the presence of Epstein–Barr virus (EBV) in the eyelid sebaceous gland carcinoma (SGC) and its association with the clinicopathologic features. Methods One hundred and thirty paraffin-embedded SGC specimens were retrieved from the Clinical Pathology Department of Zhongshan Ophthalmic Center. Epstein–Barr virus-encoded RNA (EBER) was detected with in situ hybridization (ISH) using the Leica BOND system autostainer. The age and gender distributions of all patients were analyzed and compared with earlier reports. Pearson's χ2 and Fisher's exact tests were used to determine the association between clinicopathological features such as age, gender, laterality eye, tumor basal dimension, degree of tumor differentiation, and EBER positivity. Likewise, the relationship between the grade and tumor basal dimension in EBER-positive SGC of the eyelid was analyzed. Results Thirty-four out of one hundred and thirty (26.2%) eyelid SGC specimens were positively stained for EBER. The age range of highest incidence was 46–75 years, and the female to male ratio was 1 : 0.9. No significant correlation was found between EBER-positivity and age (p = 0.5370), gender (p = 0.4758), and degree of tumor differentiation (p = 0.7787). However, EBV positivity was strongly correlated with the right eye (p = 0.0287), the tumor basal dimension (p = 0.0001). EBV positivity grade presented statistically associated with tumor size (p = 0.0329). Conclusion We conclude that ISH is a sensitive method to identify EBV in SGC of the eyelid. A possible causal association of EBV in SGC patients is suggested by high frequency of EBER-ISH positivity and its association with the clinicopathologic features.

Methotrexate-associated Lymphoproliferative Disorders in Patients With Rheumatoid Arthritis: Clinicopathologic Features and Prognostic Factors.

Methotrexate (MTX) carries a risk of lymphoproliferative disorders (LPDs), but MTX-associated LPDs (MTX-LPDs) can resolve spontaneously after MTX withdrawal. However, the precise clinicopathologic features of MTX-LPD remain unclear. We aimed to investigate the clinicopathologic characteristics, outcomes, and prognostic factors for histologic types of MTX-LPD. Paraffin-embedded tissue samples of 219 patients with MTX-LPD were analyzed. In total, 30,33,106, and 26 had reactive lymphoid hyperplasia (RH), polymorphic-LPD (Poly-LPD), diffuse large B-cell lymphomas (DLBCLs), and classic Hodgkin lymphoma (CHL), respectively. The clinicopathologic features of RH, Poly-LPD, DLBCLs, and CHL were as follows: extranodal involvement: 13.8% (4/29), 36.4% (12/33), 69.5% (73/105), and 15.4% (4/26); Epstein-Barr virus encoded RNA positivity: 55.2% (16/29), 71.9% (23/32), 45.3% (48/106), and 76.9% (20/26); necrosis: 0% (0/29), 51.5% (17/33), 34.3% (36/105), and 12.0% (3/25); and Hodgkin Reed-Sternberg-like cells: 17.2% (5/29), 50% (14/28), and 19.8% (21/106). The median duration from MTX withdrawal to the time of disease regression was 10.4, 3.0, 4.2, and 2.7 months for RH, Poly-LPD, DLBCLs, and CHL. After MTX withdrawal, progression-free survival was the greatest for RH, followed by for Poly-LPD, DLBCL, and CHL (all P<0.05). Overall survival did not differ significantly between the groups. On univariate analysis, the predictive factors for progression-free survival included plasma cell infiltrate for CHL, eosinophil infiltrate, age above 70 years, and extensive necrosis for Poly-LPD, while they were Epstein-Barr virus encoded RNA positivity and International Prognostic Index risk for DLBCL on multivariate analysis. In conclusion, histologic categorization and histology-specific factors could be useful for predicting MTX-LPD progression after MTX withdrawal.

Clinical Significance of BCL2, C-MYC, and BCL6 Genetic Abnormalities, Epstein-Barr Virus Infection, CD5 Protein Expression, Germinal Center B Cell/Non-Germinal Center B-Cell Subtypes, Co-expression of MYC/BCL2 Proteins and Co-expression of MYC/BCL2/BCL6 Proteins in Diffuse Large B-Cell Lymphoma: A Clinical and Pathological Correlation Study of 120 Patients.

Background: Clinical significance of germinal center B-cell (GCB) and non-GCB sub-categorization, expression of MYC, BCL2, BCL6, CD5 proteins and Epstein Barr virus encoded RNA (EBER) positivity in diffuse large B-cell lymphoma (DLBCL) remain controversial. Could these biomarkers accurately identify high risk DLBCL patients? Are MYC, BCL2 and BCL6 proteins expression feasible as baseline testing to predict c-Myc, BCL2 or BCL6 gene rearrangements?Aims: To investigate prognostic values of GCB/non-GCB sub-categorization, Double Protein Expression Lymphoma (DPL), Triple Protein Expression Lymphoma (TPL), positivity of CD5 protein and EBER in patients with DLBCL disease. To evaluate correlation between BCL2 , c-Myc and BCL6 gene rearrangements with BCL2, MYC and BCL6 proteins expression.Methods: Diagnostic tissue samples of 120 DLBCL patients between January 2012 to December 2013 from four major hospitals in Malaysia were selected. Samples were subjected to immunohistochemical staining, fluorescent in-situ hybridization (FISH) testing, and central pathological review. Pathological data were correlated with clinical characteristics and treatment outcome.Results: A total of 120 cases were analysed. Mean age of diagnosis was 54.1 years ± 14.6, 64 were males, 56 were females, mean follow up period was 25 months (ranged from 1 to 36 months). Of the 120 cases, 74.2% were non-GCB whereas 25.8% were GCB, 6.7% were EBER positive, 6.7% expressed CD5 protein, 13.3% were DPL and 40% were TPL. The prevalence of c-Myc, BCL2, BCL6 gene rearrangements were 5.8%, 5.8%, and 14.2%, respectively; and 1.6% were Double Hit Lymphoma (DHL). EBER positivity, DPL, TPL, c-Myc gene rearrangement, BCL2 gene rearrangement, extra copies of BCL2 gene and BCL6 gene rearrangement were associated with shorter median overall survival (P<0.05). IPI score was the significant determinants of median overall survival in DPL and TPL (P<0.05). CD5 protein expression and GCB/non-GCB sub-categorization did not affect treatment outcome (P>0.05). Overall, c-Myc, BCL2 and BCL6 gene rearrangements showed weak correlation with expression of MYC, BCL2 and BCL6 proteins (P>0.05). Fluorescent in situ hybridization is the preferred technique for prediction of treatment outcome in DLBCL patients.Conclusion: c-Myc, BCL2, and BCL6 gene rearrangements, EBER expression, DHL, TPL and IPI score are reliable risk stratification tools. MYC, BCL2 and BCL6 proteins expression are not applicable as baseline biomarkers to predict c-Myc, BCL2, and BCL6 gene rearrangements.

CD5 positivity is an independent adverse prognostic factor in elderly patients with diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. Age over 60 years is one of the five parameters of the International Prognostic Index (IPI), which is the most important clinical prognostic predictor in DLBCL. A previous study on German DLBCL patients over 60 years of age showed that immunoblastic morphology, but not germinal center B cell-like (GCB)/non-GCB subtype, correlated with short survival. We collected 174 DLBCL cases over 60 years of age in Taiwan and performed immunophenotyping and detection of Epstein-Barr virus (EBV)-encoded RNA (EBER) by in situ hybridization. Of the cases, 5.2 % were positive for CD5 and 5.7 % positive for EBER. Neither immunoblastic morphology nor GCB/non-GCB subtype correlated with survival. In univariate analysis, adverse prognostic factors included IPI ≥ 3 (P < 0.000001), B symptoms (P = 0.000075), bone marrow/peripheral blood involvement (P = 0.017), EBER positivity (P = 0.0013), and CD5 positivity (P = 0.016). In multivariate analysis, CD5 positivity was the only independent adverse prognostic factor (HR = 3.16; 95 % CI = 1.34-7.47; P = 0.0087) in addition to IPI ≥ 3 (HR = 3.07; 95 % CI = 1.84-5.11; P = 0.000018). Surprisingly, despite an overall 5.2 % incidence of central nervous system (CNS) relapse in our patients, none of the CD5+ cases experienced CNS relapse (P = 1.00). This is in stark contrast to the more frequent CNS relapse in Japanese CD5+ DLBCL patients. EBER positivity was associated with IPI ≥ 3 (P = 0.010), stage III-IV (P = 0.0082), and B symptoms (P = 0.011). In multivariate analysis, EBER positivity was not an independent adverse prognostic factor (P = 0.81), its effect being due likely to accompanying adverse clinical parameters.

Clinical characteristics and treatment outcome in a Taiwanese population of patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

Epstein-Barr virus-positive diffuse large B-cell lymphoma is a provisional entity in the 2008 World Health Organization classification of tumors of hematopoietic and lymphoid tissues. Reports on the characteristics and clinical outcome of this disease in different geographic regions showed great disparities. To define the clinical characteristics as well as the prognostic impact of Epstein-Barr virus infection on diffuse large B-cell lymphoma in Taiwan, we retrospectively investigated the Epstein-Barr virus status of 89 patients with newly diagnosed diffuse large B-cell lymphoma in our institute. Using a cutoff point of positive nuclear staining of Epstein-Barr virus-encoded RNA-1-in situ hybridization in ≥20% of the examined cells, we identified 15 cases (16.9%) of the entire study cohort as Epstein-Barr virus-positive diffuse large B-cell lymphoma. The clinical and laboratory features were not different between Epstein-Barr virus-positive and -negative diffuse large B-cell lymphoma patients. Univariate analysis showed patients with diffuse large B-cell lymphoma that were either Epstein-Barr virus-positive or had activated B-cell-like features had an inferior overall survival. Older age, advanced stage and lymphoma with activated B-cell-like features or Epstein-Barr virus-encoded RNA positivity were independent prognostic factors affecting overall survival on multivariate analysis. Patients with two or three of these adverse-risk factors were considered high risk and fared far worse than patients with no or only one adverse factor. Taken together, we demonstrated that a higher frequency of Epstein-Barr virus association was detected in a Taiwanese cohort of diffuse large B-cell lymphoma patients, and Epstein-Barr virus-encoded RNA positivity was shown to add important prognostic value in these patients.

Primary Leiomyosarcoma of the Pancreas

Primary pancreatic leiomyosarcomas are rare lesions and not well described, yet they are the most common primary pancreatic sarcoma. English-language medical literature reports 29 cases as single cases or small series. A systematized nomenclature of medicine (SNOMED) search of Mayo Clinic surgical pathology files from 1994 to 2006 identified 22 primary pancreatic leiomyosarcomas. Nine patients with pancreatic leiomyosarcoma were diagnosed and treated at our institution (5 males and 4 females; mean age at diagnosis, 63 y; range, 39 to 87 y) are described, with a literature review. In situ hybridization for Epstein-Barr virus (EBV)-encoded RNA (EBER) was conducted in all cases to exclude EBV-associated smooth muscle tumor (EBV-SMT). Seven of the 9 patients presented with abdominal pain, weight loss, and jaundice. Seven tumors (mean, 10.7 cm; range, 1.0 to 30 cm) were located in the pancreatic head and 2 in the tail. Histologic findings of primary pancreatic leiomyosarcomas (7 spindle and 2 epithelioid) were similar to leiomyosarcomas of other sites. All tumors stained positive for smooth muscle actin and desmin and negative for KIT. No case showed EBER positivity. Pancreaticoduodenectomy was done in 4 patients; 3 patients had palliative procedures, and 2 had biopsy only. No lymph node metastasis was identified in 4 resected tumors, but liver metastases were present in 4 patients. All patients died; 5 deaths were known to be disease related (overall mean survival, 31 months; range, 5 to 98 mo). Historical cases showed similar clinicopathologic findings. These pancreatic leiomyosarcoma lesions have the same morphologic features as their counterparts of other sites. EBER testing should be conducted--especially for pediatric patients--to rule out EBV-SMT. The tumor is likely to metastasize to liver but not regional lymph nodes. Extensive surgical resection should be advocated, even when morphologic results show a low-grade lesion.

Distinguishing angioimmunoblastic T‐cell lymphoma from peripheral T‐cell lymphoma, unspecified, using morphology, immunophenotype and molecular genetics

To identify distinguishing histological, immunophenotypic and molecular genetic features between angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma (PTL). Nodal T-cell lymphomas examined (n =137), included AITL (n = 89), PTL (n = 22), anaplastic large cell lymphoma (n = 16) and 'AITL/PTL indeterminate' (n = 10) with overlapping features between AITL and PTL, showing morphology typical of AITL but lacking follicular dendritic cell expansion. Immunohistochemistry for CD3, CD20, CD21 and CD10, in situ hybridization for Epstein-Barr virus encoded RNA (EBER) and polymerase chain reaction for T-cell and B-cell clonality analysis were performed. Of the AITLs, 74/89 showed typical morphology, whereas 15/89 showed hyperplastic follicles. AITL and 'AITL/PTL indeterminate' showed a polymorphous infiltrate and prominent vascularity in all cases. In both groups, CD10 was present in the majority and clear cells and EBER positivity were specific (but not universal) features lacking in PTL. Detection of T-cell clonality was significantly higher in AITL (90%) compared with PTLu (59%). Clear cells and EBV infection (when present) are useful distinguishing features and CD10 a sensitive and specific marker of AITL. Hyperplastic follicles are present in a significant minority of AITL. AITL/PTL indeterminate probably falls within the spectrum of AITL rather than PTL.

In situ localization of epstein-barr virus encoded RNA in non-nasal/nasopharyngeal CD56-positive and CD56-negative T-cell lymphomas

We recently reported a group of non-nasal/nasopharyngeal hematolymphoid malignancies expressing the natural killer cell marker CD56, characterized by frequent extranodal localization, angiocentricity, and aggressive clinical course ( Hum Pathol 23:798–804, 1992). Because we have shown a very strong association of Epstein-Barr virus (EBV) with CD56-positive T-cell lymphomas of the nose nasopharynx, we asked whether a similar association also occurs with the non-nasal CD56-positive T-cell lymphomas. In situ localization of EBV encoded RNA (EBER) was performed on paraffin sections of 15 such cases, including the nine previously reported cases and six new cases (three showing prominent hepatosplenic involvement and three showing involvement of one or more extranodal sites, such as the parotid gland, tonsils, gastrointestinal tract, skeletal muscle, and testis). A case was considered positive when the majority of the tumor cells showed nuclear signal. Ten cases showed EBER positivity, and all but one of them were negative for CD3 and other T-cell markers, except CD2. Only one of four cases showing a CD3-positive phenotype was EBER positive. Of the remaining two CD3-negative EBER-negative cases, one showed a histiocytic phenotype and the other was positive for multiple T-cell markers. Among 15 cases of CD56-negative non-nasal peripheral T-cell lymphoma studied for comparison, six were CD3-negative, among which three showed EBER positivity. All nine CD3-positive cases were EBER negative. Five cases (three CD3 positive and two CD3 negative) showed rare isolated (< 1%) EBER-positive tumor cells. We conclude that among non-nasal T-cell lymphomas, EBV is strongly correlated with CD56 positivity (66.7% v 20%), and the positive cases almost always show an immunophenotype identical to that commonly observed in nasal lymphomas (CD2 positive, CD3 negative, and CD56 positive). Thus, EBV may play an etiologic role in these CD56-positive lymphomas. There is also a correlation between EBER positivity and CD3 negativity, irrespective of the CD56 status. The presence of isolated EBER-positive cells in CD56-negative T-cell lymphomas, occurring at a frequency similar to that reported in the European population, probably represents secondary infection of tumor cells.