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.     

Response and survival benefit with chemoimmunotherapy in Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma

Epstein‐Barr virus (EBV)‐positive diffuse large B‐cell lymphoma (DLBCL) is a haematologic malignancy with poor prognosis when treated with chemotherapy. We evaluated response and survival benefits of chemoimmunotherapy in EBV‐positive DLBCL patients. A total of 117 DLBCL patients were included in our retrospective analysis; 33 were EBV‐positive (17 treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R‐CHOP] and 16 with CHOP), and 84 were EBV‐negative (all treated with R‐CHOP). The outcomes of interest were complete response (CR) and overall survival (OS) in EBV‐positive DLBCL patients (R‐CHOP versus CHOP) and in DLBCL patients treated with R‐CHOP (EBV‐positive vs EBV‐negative). There were no differences in the clinical characteristics between EBV‐positive and EBV‐negative DLBCL patients. Among EBV‐positive DLBCL patients, R‐CHOP was associated with higher odds of CR (OR 3.14, 95% CI 0.75‐13.2; P = .10) and better OS (hazard ratio 0.30, 95% confidence interval [CI] 0.09‐0.94; P = .04). There were no differences in CR rate (OR 0.52, 95% CI 0.18‐1.56; P = .25) or OS (hazard ratio 0.93, 95% CI 0.32‐2.67; P = .89) between EBV‐positive and EBV‐negative DLBCL patients treated with R‐CHOP. Based on our study, the addition of rituximab to CHOP is associated with improved response and survival in EBV‐positive DLBCL patients. Epstein‐Barr virus status does not seem to affect response or survival in DLBCL patients treated with R‐CHOP.     

Frequent downregulation of BTB and CNC homology 2 expression in Epstein–Barr virus‐positive diffuse large B‐cell lymphoma

Diffuse large B‐cell lymphoma (DLBCL) is the most common B‐cell lymphoma subtype, and the Epstein–Barr virus (EBV)‐positive subtype of DLBCL is known to show a more aggressive clinical behavior than the EBV‐negative one. BTB and CNC homology 2 (BACH2) has been highlighted as a tumor suppressor in hematopoietic malignancies; however, the role of BACH2 in EBV‐positive DLBCL is unclear. In the present study, BACH2 expression and its significance were studied in 23 EBV‐positive and 43 EBV‐negative patient samples. Immunohistochemistry revealed BACH2 downregulation in EBV‐positive cases (P < 0.0001), although biallelic deletion of BACH2 was not detected by FISH. Next, we analyzed the contribution of BACH2 negativity to aggressiveness in EBV‐positive B‐cell lymphomas using FL‐18 (EBV‐negative) and FL‐18‐EB cells (FL‐18 sister cell line, EBV‐positive). In BACH2‐transfected FL‐18‐EB cells, downregulation of phosphorylated transforming growth factor‐β‐activated kinase 1 (pTAK1) and suppression in p65 nuclear fractions were observed by Western blot analysis contrary to non‐transfected FL‐18‐EB cells. In patient samples, pTAK1 expression and significant nuclear p65, p50, and p52 localization were detected immunohistochemically in BACH2‐negative DLBCL (P < 0.0001, P = 0.006, and P = 0.001, respectively), suggesting that BACH2 downregulation contributes to constitutive activation of the nuclear factor‐κB pathway through TAK1 phosphorylation in BACH2‐negative DLBCL (most EBV‐positive cases). Although further molecular and pathological studies are warranted to clarify the detailed mechanisms, downregulation of BACH2 may contribute to constitutive activation of the nuclear factor‐κB pathway through TAK1 activation.     

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.     

A sporadic case of CTLA4 haploinsufficiency manifesting as Epstein–Barr virus-positive diffuse large B-cell lymphoma

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is a coinhibitory receptor that plays an essential role in maintaining immune system homeostasis by suppressing T-cell activation. We report a sporadic case of CTLA4 haploinsufficiency in a patient with Epstein–Barr virus-positive diffuse large B-cell lymphoma and subsequent benign lymphadenopathy. A missense mutation in exon 2 of the CTLA4 gene (c.251T>C, p.V84A) was found in the patient’s peripheral blood and buccal cell DNA, but not in her parents’ DNA. CTLA4 expression decreased in the peripheral regulatory T cells upon stimulation, whereas CTLA4 and PD-1-positive T cell subsets increased, possibly to compensate for the defective CTLA4 function. This case suggests that some adult lymphoma patients with no remarkable medical history have primary immune disorder. As immune-targeted therapies are now widely used for the treatment of malignancies, it is increasingly important to recognize the underlying primary immune disorders to properly manage the disease and avoid unexpected complications of immunotherapies.     

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.     

Mean platelet volume predicts prognosis in patients with diffuse large B‐cell lymphoma

To determine the prognostic value of baseline mean platelet volume (MPV) in diffuse large B‐cell lymphoma (DLBCL) patients. We retrospectively analyzed 161 DLBCL patients who received R‐CHOP chemotherapy. The associations between MPV and clinicopathological factors were assessed. A low MPV (MPV ≤ 9.1 fl, cut‐off was calculated by receiver operating characteristics) was not associated with any other clinicopathological factors. * Patients with MPV ≤ 9.1 fl experienced a shorter progression‐free survival (PFS) (2‐year PFS rate, 60.6% vs 84.0%, P = 0.003) and overall survival (OS) (2‐year OS rate, 70.4% vs 87.9%, P = 0.030), compared with those with MPV > 9.1 fl. The multivariate analysis demonstrated that MPV ≤ 9.1 fl was an independent prognostic factor of OS (Hazard Ratio [HR] = 0.588, P = 0.045) and PFS (HR = 0.456, P = 0.010). Therefore, we demonstrated that low baseline MPV is an independent prognostic marker of poor outcome in patients with DLBCL.     

Spatiotemporal homogeneity and distinctness of the T‐cell receptor β‐chain repertoires in Epstein–Barr virus‐associated primary and metastatic nasopharyngeal carcinomas

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)‐associated lymphoepithelioma. The aim of this study was to characterize the homogeneity and distinctness of the T‐cell repertoires within and between primary and metastatic NPCs. We used ultra‐deep sequencing of the hypervariably rearranged antigen‐binding CDR3 regions of T‐cell receptor beta (TCR_beta) to comprehensively profile the T‐cell repertoires in NPC patients receiving definitive chemoradiotherapy with long‐term follow‐up. We observed not only various spatially heterogeneous patient‐specific TCR_beta clone compositions that changed with time but also several commonly enriched TCR_beta subclones that were constantly shared between primary NPCs in the head and neck regions, locally recurrent tumors after treatment and later‐developed distant metastatic tumors in the liver, lung and bone. Comparison of the overlap frequency of the T‐cell clonality between TCR_beta repertoires enabled us to calculate the pairwise genetic distance between primary NPCs of different patients and different sites of metastatic or recurrent NPCs. The constructed NPC phylogeny clearly differentiated the low‐risk patients without relapse from the high‐risk patients with distant metastasis after chemoradiotherapy. In contrast to the rather low frequency of nonsilent somatic mutations in NPC cells, the degrees of similarity and divergence of NPC‐infiltrating lymphocyte TCR_beta repertoires among different patients showed prognostication. Moreover, the persistent presence of commonly NPC‐shared in‐frame TCR_beta CDR3 gene sequences spatiotemporally identified in the NPC‐infiltrating lymphocytes within varied EBV‐positive NPCs and their metastases suggest the existence of frequently shared epitopes of neoantigens virally or nonvirally displayed on cancer cells, thereby providing opportunities for the development of precisely tumor‐targeted immunotherapy for distant metastasis.     

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.