Epstein-Barr virus infection and altered control of apoptotic pathways in posttransplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLD) represent a spectrum of lymphoid diseases complicating the clinical course of transplant recipients. Most PTLD are Epstein-Barr virus (EBV) associated with viral latency type III. Several in vitro studies have revealed an interaction between EBV latency proteins and molecules of the apoptosis pathway. Data on human PTLD regarding an association between Bcl-2 family proteins and EBV are scarce. We analyzed 60 primary PTLD for expression of 8 anti- (Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic proteins (Bak and Bax), the so-called BH3-only proteins (Bad, Bid, Bim, and Puma), as well as the apoptosis effector cleaved PARP by immunohistochemistry. Bim and cleaved PARP were both significantly (p = 0.001 and p = 5.251e-6) downregulated in EBV-positive compared to EBV-negative PTLD [Bim: 6/40 (15%), cleaved PARP: 10/43 (23%), vs. Bim: 13/16 (81%), cleaved PARP: 12/17 (71%)]. Additionally, we observed a tendency toward increased Bcl-2 protein expression (p = 0.24) in EBV-positive PTLD. Hence, we provide evidence of a distinct regulation of Bcl-2 family proteins in EBV-positive versus negative PTLD. The low-expression pattern of the proapoptotic proteins Bim and cleaved PARP together with the high-expression pattern of the antiapoptotic protein Bcl-2 by trend in EBV-positive tumor cells suggests disruption of the apoptotic pathway by EBV in PTLD, promoting survival signals in the host cells.

p52 Activation in monomorphic B-cell posttransplant lymphoproliferative disorder/diffuse large B-cell lymphoma without BAFF-R expression.

Posttransplantation lymphoproliferative disorders (PTLD) are associated with Epstein-Barr virus (EBV) and activate the NF-κB pathway. B-cell activating factor (BAFF) modulates cell growth and survival in non-Hodgkin's lymphomas. However, there are few studies of EBV, BAFF/BAFF-R signaling, and NF-κB1 and NF-κB2 pathway activation in PTLD. Diffuse large B-cell lymphomas (DLBCL) in two different clinical contexts, immunocompetent patients (DLBCL/IC; n = 30) or posttransplantation solid-organ recipients (DLBCL/PTLD; n = 21), were characterized histogenically as germinal center (GC) or non-germinal center (NGC). Expression of BAFF, BAFF-R, and NF-κB proteins p50 and p52 and the presence or absence of EBV were compared in these clinical contexts. Regardless of the GC or NGC pattern of DLBCL, BAFF-R was expressed in 37% of DLBCL/IC but in only 4.8% of DLBCL/PTLD. p52 was expressed in DLBCL/PTLD/NGC (12 of 19 cases) as compared with DLBCL/IC/NGC (0 of 18 cases). This pattern might be related to the presence of EBV and latent membrane protein 1 because p52 expression was observed primarily in EBV-positive DLBCL/PTLD cases expressing latent membrane protein 1. Thus, the activation profile or NGC pattern of DLBCL/PTLD was not associated with BAFF/BAFF-R expression, whereas nuclear p52 related to NF-κB2 pathway activation might be linked to EBV.

Diagnosis of Burkitt lymphoma using an algorithmic approach--applicable in both resource-poor and resource-rich countries.

Distinguishing Burkitt lymphoma (BL) from B cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma (DLBCL) and BL (DLBCL/BL), and DLBCL is challenging. We propose an immunohistochemistry and fluorescent in situ hybridization (FISH) based scoring system that is employed in three phases - Phase 1 (morphology with CD10 and BCL2 immunostains), Phase 2 (CD38, CD44 and Ki-67 immunostains) and Phase 3 (FISH on paraffin sections for MYC, BCL2, BCL6 and immunoglobulin family genes). The system was evaluated on 252 aggressive B-cell lymphomas from Europe and from sub-Saharan Africa. Using the algorithm, we determined a specific diagnosis of BL or not-BL in 82%, 92% and 95% cases at Phases 1, 2 and 3, respectively. In 3·4% cases, the algorithm was not completely applicable due to technical reasons. Overall, this approach led to a specific diagnosis of BL in 122 cases and to a specific diagnosis of either DLBCL or DLBCL/BL in 94% of cases that were not diagnosed as BL. We also evaluated the scoring system on 27 cases of BL confirmed on gene expression/microRNA expression profiling. Phase 1 of our scoring system led to a diagnosis of BL in 100% of these cases.

Lymphomas in sub-Saharan Africa--what can we learn and how can we help in improving diagnosis, managing patients and fostering translational research?

Approximately 30 000 cases of non-Hodgkin lymphoma (NHL) occur in the equatorial belt of Africa each year. Apart from the fact that Burkitt lymphoma (BL) is very common among children and adolescents in Africa and that an epidemic of human immunodeficiency virus (HIV) infection is currently ongoing in this part of the world, very little is known about lymphomas in Africa. This review provides information regarding the current infrastructure for diagnostics in sub-Saharan Africa. The results on the diagnostic accuracy and on the distribution of different lymphoma subsets in sub-Saharan Africa were based on a review undertaken by a team of lymphoma experts on 159 fine needle aspirate samples and 467 histological samples during their visit to selected sub-Saharan African centres is presented. Among children (<18 years of age), BL accounted for 82% of all NHL, and among adults, diffuse large B-cell lymphoma accounted for 55% of all NHLs. Among adults, various lymphomas other than BL, including T-cell lymphomas, were encountered. The review also discusses the current strategies of the International Network of Cancer Treatment and Research on improving the diagnostic standards and management of lymphoma patients and in acquiring reliable clinical and pathology data in sub-Saharan Africa for fostering high-quality translational research.

Genetic variants in the noncoding region of RPS19 gene in Diamond-Blackfan anemia: potential implications for phenotypic heterogeneity.

Mutations in the RPS19 gene have been identified in 25% of individuals affected by Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia characterized by an aregenerative anemia and a variety of malformations. More than 60 mutations in the five coding exons of RPS19 have been described to date. We previously reported a mutation (c.-1 + 26G>T) and an insertion at -631 upstream of ATG (c.-147_-146insGCCA) in the noncoding region. Because DBA phenotype is extremely heterogeneous from silent to severe and because haploinsufficiency seems to play a role in this process, it is likely that genetic variations in the noncoding regions affecting translation of RPS19 can modulate the phenotypic expression of DBA. However, to date, very few studies have addressed this question comprehensively. In this study, we performed detailed sequence analysis of the RPS19 gene in 239 patients with DBA and 110 of their relatives. We found that 6.2% of the patients with DBA carried allelic variations upstream of ATG: 3.3% with c.-1 + 26G>T; 2.5% with c.-147_-146insGCCA; and 0.4% with c.-174G>A. Interestingly, the c.-147_-146insGCCA, which has been found in a black American and French Caribbean control population, was not found in 500 Caucasian control chromosomes we studied. However, it was found in association with the same haplotype distribution of four intronic polymorphisms in our patients with DBA. Although a polymorphism, the frequency of this variant in the patients with DBA and its association with the same haplotype raises the possibility that this polymorphism and the other genetic variations in the noncoding region could play a role in DBA pathogenesis.

The mechanisms underlying MMR deficiency in immunodeficiency-related non-Hodgkin lymphomas are different from those in other sporadic microsatellite instable neoplasms.

The spectrum of tumors showing microsatellite instability (MSI) has recently been enlarged to sporadic neoplasms whose incidence is favored in the context of chronic immunosuppression. We investigated the biological, therapeutic and clinical features associated with MSI in immunodeficiency-related non-Hodgkin lymphomas (ID-RL). MSI screening was performed in 275 ID-RL. MSI ID-RL were further analyzed for MMR gene expression and for BRAF/KRAS mutations since these genes are frequently altered in MSI cancers. We also assessed the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme whose inactivation has been reported in lymphomas and may help in the selection of MMR deficient clones. Unlike other sporadic MSI neoplasms, MSI ID-RL (N = 17) presented with heterogeneous MMR defects and no MLH1 promoter methylation. About one third of these tumors presented with normal expression of MLH1, MSH2, MSH6 and PMS2. They accumulated BRAF activating mutations (33%). Unlike other ID-RL, MSI ID-RL were primarily EBV-negative NHL of T-cell origin, and arose after long-term immunosuppression in patients who received azathioprine as part of their immunosuppressive regimen (p = 0.05) and/or who exhibited methylation-induced loss of expression of MGMT in tumor cells (p= 0.02). Overall, these results highlight that, in the context of deficient immune status, some MSI neoplasms arise through alternative mechanism when compared to other sporadic MSI neoplasms. They give the exact way how to make the diagnosis of MSI in these tumors and may help to define biological and clinicalrisk factors associated with their emergence in such a clinicalcontext.

Hodgkin lymphoma-associated hemophagocytic syndrome: a disorder strongly correlated with Epstein-Barr virus.

The retrospective study of 34 patients with Hodgkin lymphoma-associated hemophagocytic syndrome led us to define this association as a specific disorder. Its characteristics are male predominance (male-to-female sex ratio, 3.3:1), immunodeficiency-like histological features (lymphocyte depletion, 45% of cases; mixed cellularity Hodgkin lymphoma subtype, 40%), and strong association with Epstein-Barr virus (94%).

Ten novel Diamond-Blackfan anemia mutations and three polymorphisms within the rps19 gene.

INTRODUCTION: A total of 25% of patients presenting with Diamond-Blackfan anemia (DBA) carry mutations in the rps19 gene, which encodes protein RPS19 of the small ribosomal subunit. The other DBA cases carry mutations in other, unknown gene(s). MATERIALS AND METHODS: We searched mutations in 48 DBA families or isolated patients based on PCR of exons of the rps19 gene and automatic sequencing. We also studied three novel intronic polymorphisms in 85 persons (most of the patients and their relatives, when the latter could be investigated). RESULTS: We identified 10 new mutations within the rps19 gene. We found no obvious correlation between the clinical expression and the nature of the mutation. Besides, we found three polymorphisms within the rps19 gene. Polymorphisms a, b and c were (i) a one-base insertion (+c at position +79) in intron 2, (ii) the g-->c substitution at position +89 also in intron 2 and (iii) the g-->a substitution at position +14 in intron 4. Inheritance studies showed that the polymorphisms were transmitted en bloc, thus defining the --- haplotypes (changes absent) and the +++ haplotypes (changes present). The percentages of each haplotype were about 50% in families and isolated persons with DBA, as well as in controls. CONCLUSION: For the 10 novel mutations found in the rps19 gene, there were no obvious genotype-phenotype correlations. The transmission of the polymorphisms was en bloc and the studies did not suggest any clinical correlates at this stage.

Characteristics of B-cell lymphomas in HIV/HCV-coinfected patients during the combined antiretroviral therapy era: an ANRS CO16 LYMPHOVIR cohort study.

Hepatitis C virus (HCV) infection is frequent among HIV-infected patients. We describe, the characteristics of 6 HIV/HCV-coinfected patients with B-cell non-Hodgkin lymphoma (NHL) included in a prospective cohort study of HIV-related lymphomas. Five of the 6 cases had features of marginal zone/lymphoplasmacytic NHL versus 1 of 33 HIV only-infected patients. Remarkably, anti-HCV treatment led to a hematological response in a patient with splenic marginal zone lymphoma. This supports the role of chronic antigenic stimulation by HCV on lymphomagenesis and further evaluation of HCV antiviral therapy in coinfected patients with NHL.

Postmortem diagnosis of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare etiology for congenital anemia, but this diagnosis should be considered when aregenerative hypoplastic anemia occurs in infancy. A term infant girl received a red blood cell transfusion at birth for neonatal anemia (hemoglobin 75 g/L) initially attributed to abruptio placentae. There were no additional investigations. Hemoglobin gradually decreased during the first 4 weeks of life, leading to severe anemia and death despite transfusions. A postmortem diagnosis of DBA was made by extraction of DNA collected on blood filter paper showing a deletion in RPS19 gene. Neonatal anemias should be carefully investigated and close follow-up should be performed during the first months of life, even if there is an obvious hemorrhagic etiology.

A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells.

The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.