Prognostic significance of Epstein–Barr virus DNA detection in pretreatment serum in diffuse large B‐cell lymphoma

It is still a matter of debate whether detection of Epstein–Barr virus (EBV) DNA in pretreatment serum has clinical implications for diffuse large B‐cell lymphoma. For this study, we measured EBV DNA load in pretreatment serum from 127 diffuse large B‐cell lymphoma patients without any underlying immunodeficiency to evaluate its effects on clinical manifestations and prognosis. Anthracycline‐based chemotherapy in combination with rituximab was given as initial therapy for 119 patients (94%). Epstein–Barr virus DNA was detected in 15 patients (12%), who were older (P = 0.005) and tended to be at a more advanced disease stage (P = 0.053). They showed significantly worse progression‐free survival (PFS) and overall survival (OS) than other patients (P < 0.001 each). This effect remained significant (P = 0.004 and P = 0.027, respectively) after adjustment for age, lactate dehydrogenase, performance status, stage, and extranodal sites. The status of EBV‐encoded small RNA in situ hybridization was known for 123 patients; 6 of 8 positive patients (75%) and 9 of 115 negative patients (8%) had detectable EBV DNA in pretreatment serum. While patients positive for EBV‐encoded small RNA had significantly worse PFS and OS than negative patients (P = 0.001 and P = 0.029, respectively), EBV DNA detection in pretreatment serum was associated with poorer PFS and OS even for the 115 patients negative for EBV‐encoded small RNA (P < 0.001 each). These findings suggest that EBV DNA detection in pretreatment serum may have an adverse prognostic impact for patients with diffuse large B‐cell lymphoma.     

Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma association is not only restricted to elderly patients

Diffuse large B‐cell lymphoma (DLBCL), the most common group of malignant lymphomas, account for 30% of adult non‐Hodgkin lymphomas. The 2008 World Health Organization (WHO) classification included a new entity, Epstein‐Barr virus (EBV)+ DLBCL of the elderly, affecting patients aged 50 years or older. However, some reports of younger EBV+ DLBCL cases, without evidence of underlying immunosuppression, can be found. The role of EBV in tumor microenvironment composition in DLBCL is still not well understood. Our aim was to assess EBV presence and latency pattern as well as tumor T‐cell population in an adult DLBCL series of Argentina. The study was conducted on biopsies from 75 DLBCL patients. EBERs expression was performed by in situ hybridization, while EBV gene expression was analyzed using real‐time polymerase chain reaction. LMP1, LMP2A, EBNA2, EBNA3A, CD4, CD8 and Foxp3 expression was assessed by immunohistochemistry. Nine percent of cases showed EBV expression, with similar frequency among patients younger than 50 years and 50 years or older (13% and 8%, respectively). T‐cell subsets were not altered by EBV presence. Latency type II was the most frequently observed, together with lytic gene expression in EBV+ DLBCL, with ≥20% of EBERs+ cells. These findings suggest that EBV+ DLBCL in our series was similar to the previously described in Asia and Latin‐America, displaying latency II or III expression profile and no age‐specific characteristics. Finally, EBV+ DLBCL may be an entity that is not only restricted to patients who are older than 50 years of age, in consequence the age cutoff revision may be a current goal.     

Epstein–Barr virus presence in pediatric diffuse large B‐cell lymphoma reveals a particular association and latency patterns: Analysis of viral role in tumor microenvironment

Non‐Hodgkin's lymphoma represents 6–10% of pediatric malignancies, and diffuse large B‐cell lymphoma (DLBCL) is one of the three major subtypes. The 2008 WHO classification included a new entity, Epstein–Barr virus (EBV)‐positive DLBCL of the elderly, affecting patients >50 years. It has been demonstrated that EBV may play a role in tumor microenvironment composition, disturbing antitumor immune response and disease progression. As most studies were performed in adults, our aim was to assess EBV presence and latency pattern, as well as T‐cell microenvironment in a pediatric DLBCL series of Argentina. The study was conducted on formalin‐fixed paraffin‐embedded biopsies from 25 DLBCL patients. EBV‐encoded small nuclear early regions (EBERs) expression was performed by in situ hybridization, whereas EBV gene expression was analyzed using real‐time PCR. Epstein–Barr virus latent membrane proteins (LMP)1, LMP2A, CD3, CD4, CD8 and Foxp3 expression were assessed by immunohistochemistry (IHC). Forty percent of cases showed EBV expression, with a significantly higher incidence among patients <10 years (p = 0.018), and with immunosuppressed (p = 0.023). T‐cell subsets were not altered by EBV presence. Full EBV latency antigen expression (latency type III) was the most frequently pattern observed, together with BZLF1 lytic gene expression. One patient showed II‐like pattern (LMP1 without LMP2A expression). Based exclusively on IHC, some patients showed latency II/III (EBERs and LMP1 expression) or I (EBERs only). These findings suggest that EBV association in our series was higher than the previously demonstrated for elderly DLBCL and that EBV latency pattern could be more complex from those previously observed. Therefore, EBV could be an important cofactor in pediatric DLBCL lymphomagenesis.     

The prognostic significance of EBV DNA load and EBER status in diagnostic specimens from diffuse large B‐cell lymphoma patients

Epstein–Barr virus (EBV)‐encoded small RNA in situ hybridization (EBER‐ISH) is a widely accepted method to evaluate EBV involvement in diffuse large B‐cell lymphoma (DLBCL), although little is known regarding associations between EBV DNA load and the EBER status and whether EBV DNA load data provide additional clinical information. In this study, we quantified EBV DNA load in diagnostic specimens from DLBCL patients diagnosed at our hospital to evaluate clinical implications of EBV DNA load in diagnostic specimens as contrasted with EBER‐ISH. Among 140 DLBCL patients without underlying immunodeficiency, 51 were evaluable for both EBER and EBV DNA load, 83 for EBER only and one for EBV DNA load only. The median EBV DNA load was 708 copies/µg. Although EBV DNA load was significantly higher for EBER‐positive patients than for EBER‐negative patients (p < 0.001), EBV DNA was detected in up to 72% of EBER‐negative patients. Progression‐free survival and overall survival were significantly worse for patients with EBV DNA load above 700 copies/µg than for those with EBV DNA load below 700 copies/µg (p = 0.009 and p = 0.003); they were also significantly worse for EBER‐positive patients than for EBER‐negative patients (p < 0.001 and p = 0.001). Even among EBER‐negative patients, higher EBV DNA load conferred worse progression‐free survival and overall survival (p = 0.041 and p = 0.013). These findings indicate that EBV DNA load in diagnostic specimens is not a simple surrogate for the EBER status and may be a potential biomarker associated with EBV involvement and prognosis in DLBCL. Copyright © 2015 John Wiley & Sons, Ltd.     

利妥昔单抗时代弥漫大B细胞淋巴瘤的预后影响因素

目的 弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)是非霍奇金淋巴瘤中最常见的一种类型,临床表现、免疫表型、分子遗传学特征和预后均具有显著的异质性,近年来靶向药物利妥昔单抗联合常规化疗的运用,改善了DLBCL患者的预后.本研究总结在利妥昔单抗使用背景下DLBCL预后影响因素的研究进展.方法 应用PubMed及万方数据库检索系统,以“利妥昔单抗、弥漫大B细胞淋巴瘤和预后因素”作为关键词,检索2007-2016年相关文献,检索到英文文献322篇,中文文献38篇.纳入标准:(1) DLBCL的研究内容;(2)利妥昔单抗时代DLBCL预后影响因素.剔除标准:(1)无原始数据的综述;(2)个例报道;(3)重复内容的文章.根据纳入标准和剔除标准,符合分析文献37篇.结果 DLBCL是非霍奇金淋巴瘤中最常见的一大类型,临床表现、免疫表型、分子遗传学特征及预后均具有显著的异质性,利妥昔单抗前时代,常规化疗能使约40％的患者获得长期缓解,近年来靶向CD20药物利妥昔单抗的运用使DLBCL患者的缓解率明显提高,预后显著改善,同时也改变了一些指标在预后评估系统中的地位.结论 重新评估DLBCL预后影响因素,以及建立更为完善的预后评估体系,将有助于更准确的对DLBCL患者行危险度分层,并给与相应治疗,以进一步改善患者的疗效和生存.     

Epstein–Barr virus‐induced epigenetic alterations following transient infection

Epstein–Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBV's contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV‐negative, transiently infected clones to uninfected controls identified expression changes in more than 1,000 genes. Among downregulated genes, several genes known to be deoxyribonucleic acid (DNA) methylated in cancer were identified including E‐cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV‐positive cells were retained after viral loss, indicating an epigenetic effect. Repression of PYCARD expression was a result of increased promoter CpG methylation, whereas loss of E‐cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E‐cadherin promoter. Rather, repression of E‐cadherin was consistent with the formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5′ end of the E‐cadherin gene was observed in an EBV‐negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, whereas its own contribution to the oncogenic process is masked.     

Epstein-barr virus-positive diffuse large B-cell lymphoma of the elderly: what we know so far

Epstein-Barr virus-positive (EBV-positive) diffuse large B-cell lymphoma (DLBCL) of the elderly is a newly described lymphoproliferative disorder recently included as a "provisional" entity in the most current WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. The objective of this review is to provide a thorough and current summary of the existing knowledge of this subtype of DLBCL. We will review and discuss the incidence of EBV expression in DLBCL, the pathogenesis behind EBV-driven malignant transformation of B cells, the different EBV latency patterns associated with DLBCL, the distinct pathologic characteristics of EBV-positive DLBCL, the potential predictive and prognostic value of EBV tumoral status in patients with DLBCL, and potential strategies for the treatment of this rare entity, which is characterized by a suboptimal response to therapy and poor survival rate.     

Diffuse large B‐cell lymphoma

BACKGROUND:

The objective of this study was to compare the clinical features and prognosis of patients with diffuse large B‐cell lymphoma (DLBCL) of Waldeyer ring (WR‐DLBCL) and patients with lymph node DLBCL (N‐DLBCL).

METHODS:

One hundred eighty‐one patients with WR‐DLBCL and N‐DLBCL were reviewed. There were 57 patients with stage I disease, 83 patients with stage II disease, 26 patients with stage III disease, and 15 patients with stage IV disease. Among them, 101 patients had primary N‐DLBCL, and 80 patients had primary WR‐DLBCL.

RESULTS:

Patients with WR‐DLBCL and N‐DLBCL usually presented at an older age and had localized disease, a low frequency of B symptoms, a good performance status, and a low‐risk International Prognostic Index (IPI) score. Compared with patients who had N‐DLBCL, patients who had WR‐DLBCL presented with more stage II disease and lower tumor burdens. The overall response rate after treatment was similar in both groups. The 5‐year overall survival (OS) and progression‐free survival (PFS) rates were 76% and 61% in patients with WR‐DLBCL, respectively, and 56% and 50% in patients with N‐DLBCL, respectively (P = .119 for OS; P = .052 for PFS). IPI scores and elevated β2‐microglobulin and LDH levels were associated with a poor prognosis for patients who had WR‐DLBCL; whereas bulky tumor, elevated β2‐microglobulin levels, and IPI scores were associated with poor OS for patients who had N‐DLBCL.

CONCLUSIONS:

The current results supported the continued inclusion of WR‐DLBCL as a lymph node group in the staging of DLBCL. Patients with WR‐DLBCL had clinical features and prognosis similar to those of patients with N‐DLBCL. Cancer 2009. © 2009 American Cancer Society.     

De novo diffuse large B‐cell lymphoma with a CD20 immunohistochemistry‐positive and flow cytometry‐negative phenotype: Molecular mechanisms and correlation with rituximab sensitivity

CD20 is expressed in most B-cell lymphomas and is a critical molecular target of rituximab. Some B-cell lymphomas show aberrant CD20 expression, and rituximab use in these patients is controversial. Here we show both the molecular mechanisms and the clinical significance of de novo diffuse large B-cell lymphomas (DLBCL) that show a CD20 immunohistochemistry (IHC)-positive and flow cytometry (FCM)- negative (IHC[+]/FCM[−]) phenotype. Both IHC and FCM using anti-CD20 antibodies L26 and B1, respectively, were analyzed in 37 of the 106 cases of de novo DLBCL; 8 (22%) of these cases were CD79a(+)/CD20(+) with IHC and CD19(+)/CD20(−) with FCM. CD20 (MS4A1) mRNA expression was significantly lower in IHC(+)/FCM(−) cells than in IHC(+)/FCM(+) cells (P = 0.0005). No genetic mutations were detected in MS4A1 promoter and coding regions. Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(−) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed. FCM using rituximab detected CD20 more efficiently than B1. No significant difference was observed between IHC(+)/FCM(−) and IHC(+)/FCM(+) patients in overall survival (P = 0.664). Thus, lower expression of CD20 mRNA is critical for the CD20 IHC(+)/FCM(−) phenotype. Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC. FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(−) cases.     

Primary diffuse large B‐cell lymphoma of the tonsil

BACKGROUND: The purpose was to evaluate the prognostic factors and treatment outcome of Indian patients with primary diffuse large B-cell lymphoma (DLBCL) of the tonsil treated at a single institution. METHODS: In all, 121 patients with DLBCL of the tonsil, treated at the Tata Memorial Hospital, Mumbai, India, from January 1990 to December 2002, were included. The median age was 45 years and the majority of patients (68%) were males. Systemic symptoms were present in 12% of patients; 28% presented with stage I and 67% had stage II disease. Treatment consisted of a combination of chemotherapy (CTh) and radiotherapy (RT) for the majority of patients (69.4%). Among those receiving RT, 64% received an RT dose of > or =45 Gy. RESULTS: After a median follow-up of 62 months, disease-free survival (DFS) and overall survival (OS) were 66.4% and 81.6%, respectively. Significant prognostic factors included: WHO performance score > or =2 (OS: 72.1% vs 95.6%, P = .016), bulky tumors (OS: 68.5% vs 86.9%, P = .001), presence of B-symptoms (OS: 36.7% vs 79.6%, P < .001), and Ann Arbor stage. On multivariate analysis; WHO performance score > or =2 (hazard ratio [HR], 4.27; 95% confidence interval [CI], 1.20-15.12), and B symptoms (HR, 6.27; 95% CI, 2.38-16.48), retained statistical significance. CTh + RT resulted in a significantly better outcome than those treated with CTh alone (OS: 85.7% vs 70.7%, P = .008). The complete response (P = .053), DFS (P = .039), and OS (P = .014) rates were significantly better for patients receiving an RT dose > or =45 Gy. CONCLUSIONS: Tumor bulk, WHO performance score, the presence of B symptoms, and Ann Arbor stage significantly influence outcome. A combined modality treatment, consisting of CTh and RT (with an RT dose of > or =45 Gy), results in a satisfactory outcome in patients with this uncommon neoplasm.     

Burkitt lymphoma versus diffuse large B‐cell lymphoma: a practical approach

Burkitt Lymphoma (BL) is listed in the World Health Organization (WHO) classification of lymphoid tumours as an “aggressive B‐cell non‐Hodgkin's lymphoma”, characterized by a high degree of proliferation of the malignant cells and deregulation of the c‐MYC gene. The main diagnostic challenge in BL is to distinguish it from diffuse large B‐cell lymphoma (DLBCL). While in children BL and DLBCL types probably do not differ clinically, and the differential diagnosis between BL and DLBCL may theoretically appear clear‐cut, in adults daily practice shows the existence of cases that have morphological features, immunophenotypic and cytogenetics intermediate between DLBCL and BL, and cannot be classified with certainty in these categories. Distinguishing between BL and DLBCL is critical, as the two diseases require different management. This review summarizes the current practical approach, including the use of a large panel of antibodies, and cytogenetic and molecular diagnostic techniques, to distinguish between BL, DLBCL and the provisional category of “B‐cell lymphoma, unclassificable, with features intermediate between diffuse large B‐cell lymphoma and Burkitt lymphoma”, now listed in the updated WHO classification. Copyright © 2009 John Wiley & Sons, Ltd.     

The therapeutic effect of rituximab on CD5‐positive and CD5‐negative diffuse large B‐cell lymphoma

The prognosis of diffuse large B‐cell lymphoma (DLBCL) has improved markedly in recent years of rituximab era. The prognosis of de novo CD5‐positive DLBCL is reported to be poor, but the effect of rituximab on this type of lymphoma remains unclear. To investigate the effect of rituximab on CD5‐positive DLBCL, we collected DLBCL patients and analysed prognostic factors. A total of 157 patients with DLBCL who were immunophenotyped with flow‐cytometry (FCM) and treated with chemotherapy were subjected to analysis. Those treated with radiotherapy alone or with supportive therapy only were not included. Patients diagnosed in 2003 or later were treated with rituximab combined chemotherapy. There were 95 males and 62 females. Their age ranged from 20 to 91 years old, and the median was 65 years. Nineteen patients were diagnosed as having de novo CD5‐positive DLBCL. Rituximab was given alongside chemotherapy in 85 patients. Of these, 11 were positive for CD5 and 74 were negative. The addition of rituximab improved the overall survival (OS) of DLBCL patients (2‐year OS: 82% vs. 70%, p = 0.01). For CD5‐negative DLBCL, patients treated with rituximab showed 2‐year OS of 84%, which was significantly better than those treated without rituximab (70%, p = 0.008). However, for CD5‐positive DLBCL, the prognosis was not statistically different between the patients treated with and without rituximab (59% vs. 50%, p = 0.72). Although rituximab improved the prognosis of DLBCL, such improvement was restricted to the CD5‐negative group. Further investigation is required to improve the prognosis of patients with CD5‐positive DLBCL. Copyright © 2009 John Wiley & Sons, Ltd.     

Burkitt lymphoma versus diffuse large B‐cell lymphoma: a practical approach

Burkitt Lymphoma (BL) is listed in the World Health Organization (WHO) classification of lymphoid tumours as an ‘aggressive B‐cell non‐Hodgkin's lymphoma’, characterized by a high degree of proliferation of the malignant cells and deregulation of the c‐MYC gene. The main diagnostic challenge in BL is to distinguish it from diffuse large B‐cell lymphoma (DLBCL). While in children BL and DLBCL types probably do not differ clinically, and the differential diagnosis between BL and DLBCL may theoretically appear clear‐cut, in adults daily practice shows the existence of cases that have morphological features, immunophenotypic and cytogenetics intermediate between DLBCL and BL, and cannot be classified with certainty in these categories. Distinguishing between BL and DLBCL is critical, as the two diseases require different management. This review summarizes the current practical approach, including the use of a large panel of antibodies, and cytogenetic and molecular diagnostic techniques, to distinguish between BL, DLBCL and the provisional category of ‘B‐cell lymphoma, unclassifiable, with features intermediate between diffuse large B‐cell lymphoma and Burkitt lymphoma’, now listed in the updated WHO classification. Copyright © 2009 John Wiley & Sons, Ltd.     

Multicentre phase II study of CyclOBEAP plus rituximab in patients with diffuse large B‐cell lymphoma

The R‐CHOP regimen has been found to improve the outcome of diffuse large B‐cell lymphoma (DLBCL). However, it does not provide a satisfactory treatment outcome in the high‐risk group. We previously administered the CyclOBEAP regimen to patients with DLBCL, and reported its safety and efficacy. The R‐CyclOBEAP regimen was administered over a total period of 12 weeks, and rituximab 375 mg/m² was given every 2 weeks. There were 101 eligible patients. CR was achieved in 96 patients (95%). The 5‐year overall survival (OS) rate was 85% and progression‐free survival (PFS) rate was 76%. When the patients were divided according to the IPI, the 5‐year OS and PFS rates did not significantly differ among the risk groups. The 5‐year PFS of the germinal centre B‐cell group was 80% and that of the non‐GCB group was 74% (NS). Univariate analysis showed that the presence of B symptoms, extranodal lesions ≧2, and sIL‐2R were significant poor prognostic factors. Grade 4 neutropenia was observed in 91 patients and thrombocytopenia in 9 patients. The addition of rituximab to CyclOBEAP therapy may enhance the effect of CyclOBEAP therapy for DLBCL. Copyright © 2010 John Wiley & Sons, Ltd.     

Circulating Epstein‐Barr virus–encoded micro‐RNAs as potential biomarkers for nasal natural killer/T‐cell lymphoma

Nasal natural killer/T‐cell lymphoma (NNKTL) is an Epstein‐Barr virus (EBV)–associated malignancy and is characterized by local invasion and widespread dissemination, with a consequent poor prognosis. Micro‐RNAs (miRNAs) play roles in the pathogenesis of several malignancies by regulating gene expression and have been recently identified as stable entities in serum. Here, we investigated the value of circulating EBV‐miRNAs as biomarkers for NNKTL. Sera of patients with NNKTL were subjected to miRNA polymerase chain reaction (PCR)–array analysis, after which serum EBV‐miRNA levels were verified using quantitative PCR. The latter analysis revealed high miR‐BART2‐5p, miR‐BART7‐3p, miR‐BART13‐3p, and miR‐BART1‐5p expression levels in sera of patients with NNKTL and indicated accurate values for discriminating patients with NNKTL from healthy controls. Levels of these 4 EBV‐miRNAs, which were secreted from NNKTL cells, significantly decreased after treatment compared with those before treatment. Furthermore, a high circulating miR‐BART2‐5p level was associated with disease progression and poor prognosis in patients with NNKTL. Our findings demonstrate that circulating EBV‐miRNAs, particularly miR‐BART2‐5p, may serve as potential diagnostic and prognostic biomarkers in patients with NNKTL.     

Antiviral therapy improves overall survival in hepatitis C virus‐infected patients who develop diffuse large B‐cell lymphoma

Chronic Hepatitis C virus (HCV) infection is associated with increased incidence of non‐Hodgkin lymphoma. Several studies have demonstrated regression of indolent lymphoma with antiviral therapy (AVT) alone. However, the role of AVT in HCV‐infected patients with diffuse large B‐cell lymphoma (DLBCL) is unclear. We therefore analyzed AVT's impact on oncologic outcomes of HCV‐infected patients (cases) who developed DLBCL. Cases seen at our institution (June 2004–May 2014) were matched with uninfected counterparts (controls) and then divided according to prior AVT consisting of interferon‐based regimens. We studied 304 patients (76 cases and 228 controls). More cases than controls had extranodal (79% vs. 72%; p = 0.07) and upper gastrointestinal (GI; 42% vs. 24%; p = 0.004) involvement. Cases never given AVT had DLBCL more refractory to first‐line chemotherapy than that in the controls (33% vs. 17%; p = 0.05) and exhibited a trend toward more progressive lymphoma at last examination compared to controls (50% vs. 32%; p = 0.09) or cases given AVT (50% vs. 27%; p = 0.06). Cases never given AVT had worse 5‐year overall survival (OS) rates than did the controls (HR, 2.3 [95% CI, 1.01–5.3]; p = 0.04). Furthermore, AVT improved 5‐year OS rates among cases in both univariate (median [Interquartile range]: 39 [26–56] vs. 16 [6–41] months, p = 0.02) and multivariate analyses (HR = 0.21 [95% CI, 0.06–0.69]; p = 0.01). This study highlights the negative impact of chronic HCV on survival of DLBCL patients and shows that treatment of HCV infection is associated with a better cancer response to chemotherapy and improves 5‐year OS.