Irina Bonzheim

Geographic variation in the prevalence of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly: a comparative analysis of a Mexican and a German population

Epstein–Barr virus (EBV)-positive diffuse large B-cell lymphoma of the elderly was included as a provisional entity in the 2008 WHO lymphoma classification. Most reports of this disease come from Asia and little is known about it in other regions of the world, including Latin America. Therefore, in this study, 305 diffuse large B-cell lymphomas in patients above 50 years were analyzed, 136 from Mexico and 169 from Germany. EBV was detected by Epstein–Barr early RNA (EBER) in situ hybridization. Only cases with EBER+ in the majority of tumor cells were regarded as EBV+ diffuse large B-cell lymphoma. The prevalence of EBV+ diffuse large B-cell lymphoma in Mexican patients was found to be 7% (9 of 136), whereas only 2% (4 of 169) of the German cases were positive. The median age at diagnosis was 66 years in the Mexican cohort, as opposed to 77 years in the German group. The site of presentation was in both groups predominantly nodal in nine cases (70%) and extranodal in four cases (30%). Of the 13 EBV+ cases, 10 (77%) were classified as polymorphic and 3 (23%) as monomorphic type. The polymorphic cases showed a non-germinal center B-cell immunophenotype (CD10− MUM1+). Twelve cases (92%) were LMP1 positive and two (15%) expressed EBNA2. An interesting finding was the high frequency of EBV type B with the LMP1 30 bp deletion found in the Mexican cases (50%). Eight of the 11 evaluable cases were B-cell monoclonal by polymerase chain reaction. In summary, we found a similar prevalence of EBV+ diffuse large B-cell lymphoma of the elderly in a Mexican population compared with what has been reported in Asian countries, and in contrast to the low frequency in Western populations (1–3%). However, compared with the Asian series, the Mexican patients were younger at diagnosis, presented predominantly with nodal disease and rarely expressed EBNA2 protein.

Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR

Mutations of components of the mitogen-activated protein kinase pathway, mainly BRAF, are common in serous ovarian borderline tumors, whereas high-grade serous ovarian carcinomas rarely show this feature. With the advent of specific kinase inhibitors active against BRAF-mutated cancers, rapid and sensitive detection of the BRAF V600E, by far the most common mutation of this gene, is of great practical relevance. Currently, BRAF mutations are detected by DNA-based techniques. Recently, a monoclonal antibody (VE1) specific for the BRAF V600E protein suitable for archival tissues has been described. In this study, we compared detection of the V600E mutation in serous ovarian tumors by VE1 immunostaining and by allele-specific polymerase chain reaction. All 141 cases of high-grade serous ovarian cancer showed negative or rarely weak, diffuse background VE1 immunostaining, and BRAF wild type was confirmed by molecular analysis in all tested cases. In contrast, 1 (14%) of 7 low-grade serous carcinomas and 22 (71%) of 31 serous borderline tumors revealed moderate to strong VE1 positivity. Immunostaining was clearly evaluable in all cases with sufficient tumor cells, and only rare cases with narrow cytoplasm were difficult to interpret. The V600E mutation was confirmed by allele-specific polymerase chain reaction and sequencing in all VE1-positive cases. Two VE1-positive cases with low epithelial cell content required repeat microdissection to confirm the presence of the mutation. Immunohistochemistry with the VE1 antibody is a specific and sensitive tool for detection of the BRAF V600E mutation in serous ovarian tumors and may provide a practical screening test, especially in tumor samples with low epithelial content.

Hydroa vacciniforme-like lymphoma: a chronic EBV+ lymphoproliferative disorder with risk to develop a systemic lymphoma

Hydroa vacciniforme-like lymphoma (HVLL) is an Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disorder of childhood that occurs mainly in Central and South America and Asia. We present the clinicopathological features of 20 Mexican children with HVLL with a median age of 8 years at diagnosis (range, 1-15). All patients presented with skin lesions involving sun-exposed areas, but not exclusively. Fever, lymphadenopathy, and hepatosplenomegaly were often observed. Most patients were treated with immunomodulators and/or immunosuppressive agents, resulting in temporary remission. For 13 patients follow-up was available for a median of 3 years (range, 1 month-13 years). Three patients with long follow-up (9-13 years) are alive with disease. Four patients died, 2 after developing systemic lymphoma. Histologically, the skin showed a predominantly angiocentric and periadnexal Epstein-Barr early RNA+ lymphoid infiltrate with variable atypia and subcutaneous involvement. Fifteen patients showed a T-cell phenotype (12, αβ; 2, γδ; 1, silent phenotype) and monoclonal T-cell receptor-γ rearrangements, whereas 6 exhibited a natural killer (NK)-cell phenotype. Four patients had hypersensitivity to mosquito bites. One patient showed both phenotypes. HVLL is an EBV-associated lymphoproliferative disorder of αβ-, γδ-, or NK-cell phenotype with a broad clinical spectrum, usually prolonged clinical course, and risk for progression to systemic disease.

Epstein-Barr virus-positive diffuse large B-cell lymphomas of the elderly.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly has been included in the 2008 WHO classification as a new provisional entity and is defined as blastic, clonal B-cell proliferation associated with EBV occurring in patients over 50 years of age, presumably due to senescence of the immune system. Secondary immunodeficiencies must be excluded. Morphologically, the predominant polymorphic subtype shows a mixed proliferation of large transformed cells, plasma cells, plasmablasts, lymphocytes, and commonly Reed-Sternberg (RS)-like cells, whereas the monomorphic subtype reveals sheets of large cells. An additional characteristic feature is large areas of “geographical” necrosis. EBV+ DLBCL of the elderly is positive for pan-B cell markers and often for CD30. EBV infection detected by Epstein Barr early RNA in situ hybridization should be present in the majority of tumor cells. Most cases express LMP1 and up to 32% EBNA2, the latter a sign of an impaired immune system. The most challenging differential diagnosis is EBV+ classical Hodgkin lymphoma in older patients. Strong, homogeneous expression of B-cell markers, including transcription factors OCT2 and BOB.1, and lack of CD15 support a diagnosis of EBV+ DLBCL. EBV+ DLBCL of the elderly accounts for 8% to 10% of DLBCL in Asian countries, but seems to be uncommon in Western populations, indicating an ethnic or geographic predisposition. Clinically, patients may present with nodal or extranodal involvement. B-symptoms and poor performance status are common, and prognosis is significantly inferior compared with EBV- cases. Whether EBV+DLBCL of the elderly represents a true entity or only a DLBCL variant remains to be determined.

C/EBPβ expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the STAT3 signaling pathway

Background Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein formed of nucleophosmin (NPM) and ALK. Recently, we reported the abnormal expression of the transcription factor CCAAT/enhancer binding protein-beta (C/EBPβ) in ALK-positive anaplastic large cell lymphomas, and demonstrated its dependence on NPM-ALK activity. Design and Methods In this study, the role of C/EBPβ in proliferation and survival of ALK-positive anaplastic large cell lymphomas was investigated, as well as the mechanism of its expression and activity. Highly effective short hairpin RNA sequences and/or pharmacological inhibitors were used to abrogate the expression or activity of C/EBPβ, signal transducer and activator of transcription 3 (STAT3), AKT, extracellular signal-related kinase 1/2 (ERK1/2) and mammalian target of rapamycin (mTOR). Results Interference with C/EBPβ expression resulted in a dramatic decrease in cell proliferation in ALK-positive anaplastic large cell lymphomas, with a mild induction of apoptosis after 6 days. Down-regulation of STAT3 resulted in a marked decrease in C/EBPβ mRNA and protein levels with impairment in cell proliferation and viability, underscoring the important role of these two proteins in ALK-mediated oncogenesis. Additionally, we demonstrated that reduction of ERK1/2 activity led to C/EBPβ Thr235 dephosphorylation and moderate growth retardation. The AKT/mTOR signaling pathway did not have any influence on C/EBPβ expression or C/EBPβ phosphorylation. Conclusions These findings reveal the convergence of STAT3 and ERK1/2 signaling pathways activated by NPM-ALK in mediating the regulation of C/EBPβ expression, a transcription factor central to NPM-ALK transformation.

High frequency of MYD88 mutations in vitreoretinal B-cell lymphoma: a valuable tool to improve diagnostic yield of vitreous aspirates

Vitreoretinal diffuse large B-cell lymphoma is a rare disorder, occurring as primary ocular disease or as secondary involvement by primary central nervous system lymphoma. It is usually diagnosed by cytologic, immunocytochemical, and molecular examination of vitreous aspirates. However, distinguishing vitreoretinal diffuse large B-cell lymphoma from uveitis remains difficult, and clonality analysis may be either unsuccessful or misleading. Diffuse large B-cell lymphoma arising in immune-privileged sites (eg, the central nervous system) shows a high frequency of MYD88 mutations. Therefore, we retrospectively assessed the frequency of MYD88 mutations in vitreoretinal lymphoma (VRL) and their diagnostic potential in 75 vitrectomy samples of 69 patients, and validated our results in a separate cohort (n = 21). MYD88 mutations were identified in 20 of 29 (69%) clinically, histologically, and molecularly confirmed VRL, including 6 cases of the test cohort initially diagnosed as reactive (3/6) or suspicious (3/6) for lymphoma. MYD88 mutations, especially L265P, are very frequent in VRL and their detection significantly improves the diagnostic yield of vitrectomy specimens.

Disturbed expression of the T-cell receptor/CD3 complex and associated signaling molecules in CD30+ T-cell lymphoproliferations

Background CD30+ T-cell lymphoproliferations comprise a spectrum of clinically heterogeneous entities, including systemic anaplastic large cell lymphomas (ALK− and ALK+) and primary cutaneous CD30+ T-cell lymphoproliferative disorders. While all these entities are characterized by proliferation of highly atypical, anaplastic CD30+ T cells, the expression of T-cell specific antigens in the tumor cells is not consistently detectable. Design and Methods We evaluated biopsies from 19 patients with primary cutaneous CD30+ lymphoproliferative disorders, 38 with ALK− and 33 with ALK+ systemic anaplastic large cell lymphoma. The biopsies were examined for the expression of T-cell receptorαβ/CD3 complex (CD3γ, δ, ɛ, ζ), transcription factors regulating T-cell receptor expression (ATF1, ATF2, TCF-1, TCF-1α/LEF-1, Ets1), and molecules of T-cell receptor-associated signaling cascades (Lck, ZAP-70, LAT, bcl-10, Carma1, NFATc1, c-Jun, c-Fos, Syk) using immunohistochemistry. Results In comparison to the pattern in 20 peripheral T-cell lymphomas, not otherwise specified, we detected a highly disturbed expression of the T-cell receptor/CD3 complex, TCF-1, TCF-1α/LEF-1, Lck, ZAP-70, LAT, NFATc1, c-Jun, c-Fos and Syk in most of the systemic anaplastic large cell lymphomas. In addition, primary cutaneous CD30+ lymphoproliferative disorders showed such a similar expression pattern to that of systemic anaplastic large cell lymphomas, that none of the markers we investigated can reliably distinguish between these CD30+ T-cell lymphoproliferations. Conclusions Severely altered expression of the T-cell receptor/CD3 complex, T-cell receptor-associated transcription factors and signal transduction molecules is a common characteristic of systemic and cutaneous CD30+ lymphoproliferations, although the clinical behavior of these entities is very different. Since peripheral T-cell lymphomas, not otherwise specified retain the full expression program required for functioning T-cell receptor signaling, the differential expression of a subset of these markers might be of diagnostic utility in distinguishing peripheral T-cell lymphomas, not otherwise specified from the entire group of CD30+ lymphoproliferations.

MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity.

Recurrent mutations in MYD88 have been identified in >90% of lymphoplasmacytic lymphoma (LPL). Recently, WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome‐like mutations in CXCR4 have been described in 28% of LPL cases, and seem to impact clinical presentation and response to therapy. We investigated the presence of the MYD88 L265P mutation in 90 decalcified, formalin‐fixed, paraffin‐embedded (FFPE) bone marrow (BM) biopsies, including 51 cases of LPL, 14 cases of B‐cell chronic lymphocytic leukaemia (CLL), 13 cases of marginal zone lymphoma (MZL) and 12 normal controls. In addition, the C‐terminal domain of CXCR4 was sequenced in LPL cases. MYD88 L265P was found in 49/51 (96%) LPL cases and in 1/13 (7·6%) MZL (splenic type), whereas all CLL samples remained negative. The two MYD88 wild type LPL cases were associated with cold agglutinin disease. Mutations in CXCR4 were detected in 17/47 (36·2%) LPL cases, which showed a higher extent of BM infiltration and lower leucocyte counts (P = 0·02), haemoglobin (P = 0·05) and platelet counts (P = 0·01). In conclusion the detection of MYD88 L265P mutation in FFPE samples is reliable and useful for subtyping small B‐cell lymphomas in BM biopsies. In addition, the presence of CXCR4 mutations identifies a subgroup of LPL patients with higher disease activity.

A unique case of follicular lymphoma provides insights to the clonal evolution from follicular lymphoma in situ to manifest follicular lymphoma

To the editor: Follicular lymphoma in situ (FLIS) is characterized by the presence of a B-cell population with immunophenotypic and genotypic features of follicular lymphoma (FL) but exclusively localized to germinal centers (GCs) in morphologically reactive lymph nodes.[1][1] FLIS lesions are

Increasing genomic and epigenomic complexity in the clonal evolution from in situ to manifest t(14;18)-positive follicular lymphoma

Follicular lymphoma (FL) is characterized besides the t(14;18)(q32;q21), by recurrent chromosomal alterations and somatic mutations. In this study, we analyzed cases of FL in situ (FLIS) without manifest FL (mFL), partial involvement by FL (PFL) and paired cases of FLIS and mFL to detect possible early chromosomal imbalances, mutations, as well as DNA-methylation patterns of genomic regions of selected genes. We demonstrate that all paired FLIS and mFL cases were clonally related, based on IGH rearrangement patterns and BCL2 breakpoint sequences. FLIS and PFL had no or few secondary chromosomal imbalances detectable by array comparative genomic hybridization (FLIS 0.8 copy number alterations (CNA)/case; PFL 2.0 CNA/case; mFL 6.3 CNA/case) and a lower level of DNA methylation of genes recurrently de novo methylated in lymphomas, as compared with mFL. EZH2 Tyr641 mutations were detected in a subset of both FLIS (2/9) and PFL (1/3) cases. In conclusion, these findings provide evidence that FLIS represents a FL precursor lesion of long-lived clonal B cells carrying the t(14;18) with no or few secondary genetic changes. Our data suggest that there may be more than one distinct lesion driving the progression from FLIS to manifest lymphoma.