Optimal Minimal Panels of Immunohistochemistry for Diagnosis of B-Cell Lymphoma for Application in Countries With Limited Resources and for Triaging Cases Before Referral to Specialist Centers.

OBJECTIVES: Establish and validate optimal minimal immunohistochemistry panels for usage in a staged algorithmic manner for precise diagnosis of B-cell lymphomas in countries with limited resources. Suggest short panels of immunostains to be used in referring units that refer suspected lymphomas to specialist diagnostic centers in resourceful countries. METHODS: Significant proportion of six B-cell lymphomas has characteristic morphology requiring a short panel of confirmatory immunostains. The rest would go through five different algorithms. RESULTS: 812 cases in which a B-cell lymphoma or an HIV-associated lymphoma was suspected on morphological grounds were evaluated. This led to arriving at a specific diagnosis of 799 B-cell lymphomas. A correct diagnosis was achievable in 69% cases with the application of three to five antibodies; others required additional work-up. CONCLUSIONS: The panels/algorithms assist pathologists in practicing lymphoma diagnostics in countries with limited resources and in making lymphoma referrals to specialist centers.

Bone marrow manifestations in multicentric Castleman disease.

This study aimed to document the morphological and immunophenotypic features, and describe the diagnostic features of bone marrow (BM) involvement in human herpes virus 8 Multicentric Castleman disease (HHV8-MCD). BM trephine biopsy (BMTB) specimens from 28 patients were revisited. Samples were evaluated for expression of CD3, CD20, CD138, CD68R, glycophorin C, CD42b, HHV8-latency-associated nuclear antigen (LANA1), Epstein-Barr virus-encoded small RNA and light chains. Presence of significant numbers of HHV8-LANA1(+) lymphoid/plasmacytic cells, noted in 10/28 cases, was indicative of BM involvement and was associated with low CD4 and CD8 counts in peripheral blood. The characteristic morphological appearance of MCD seen in lymph nodes is a rare finding in BMTB. 4/5 cases with lymphoid aggregates were involved by MCD, whereas 6/23 cases without lymphoid aggregates were involved by MCD (P = 0·023). 9/18 cases with hypercellular marrow were involved by MCD, whilst only 1/8 cases with normo/hypocellular marrow showed involvement by MCD (P = 0·070). While 9/21 cases with increased marrow reticulin were involved by MCD, none of the cases with no increase in reticulin were involved by MCD (P = 0·080). Reactive plasmacytosis is a frequent finding. We conclude that bone marrow is involved in a significant proportion of patients with MCD (36%), and involvement can be identified by HHV8-LANA1 immunohistochemistry.

Myofibroblastoma of the female breast with admixed but distinct foci of spindle cell lipoma: a case report.

Mammary myofibroblastoma (MFB) is a rare benign spindle neoplasm that affects both sexes with a male predominance. It can exhibit a wide range of histological patterns. We report a case of epithelioid/spindle MFB of the female breast with admixed, but distinct, foci of spindle cell lipoma. Whilst all the spindle cells within the tumour expressed CD34, AR, ER, BCL2, and CD10, only those within the myofibroblastoma expressed desmin and only those within the lipomatous areas expressed S100. This finding, to our knowledge, is a novel one that has not been reported before.

Posttransplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLDs) are a group of diseases that range from benign polyclonal to malignant monoclonal lymphoid proliferations. They arise secondary to treatment with immunosuppressive drugs given to prevent transplant rejection. Three main pathologic subsets/stages of evolution are recognised: early, polymorphic, and monomorphic lesions. The pathogenesis of PTLDs seems to be multifactorial. Among possible infective aetiologies, the role of EBV has been studied in depth, and the virus is thought to play a central role in driving the proliferation of EBV-infected B cells that leads to subsequent development of the lymphoproliferative disorder. It is apparent, however, that EBV is not solely responsible for the "neoplastic" state. Accumulated genetic alterations of oncogenes and tumour suppressor genes (deletions, mutations, rearrangements, and amplifications) and epigenetic changes (aberrant hypermethylation) that involve tumour suppressor genes are integral to the pathogenesis. Antigenic stimulation also plays an evident role in the pathogenesis of PTLDs. Plasmacytoid dendritic cells (PDCs) that are critical to fight viral infections have been thought to play a pathogenetically relevant role in PTLDs. Furthermore, regulatory T cells (Treg cells), which are modulators of immune reactions once incited, seem to have an important role in PTLDs where antigenic stimulation is key for the pathogenesis.

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors.

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc-/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

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.

BCL3 rearrangement, amplification and expression in diffuse large B-cell lymphoma.

AIMS: Aim of the study is to investigate diffuse large B-cell lymphoma (DLBCL) for the presence of BCL3 gene rearrangement and protein expression and to correlate these with immunophenotypic subsets of DLBCL. We aimed to investigate the pathogenetic implication of BCL3 in DLBCL. METHODS AND RESULTS: Tissue microarray sections from 78 DLBCLs were evaluated for BCL3 protein expression using immunohistochemistry and for BCL3 and IGH rearrangement using Fluorescent in situ hybridisation (FISH) with split-apart probes. BCL3 expression was positive in 36/78 cases, of which BCL3 rearrangement was seen seen in one case. Three additional cases showed evidence of trisomy of BCL3/chromosome 19, and two of these three cases showed BCL3 expression. The four cases with FISH-detectable abnormalities showed MUM1 expression and had a non-germinal center (GC) phenotype. The median [and inter-quartile range (IQR)] percentage of BCL3-positive cells in MUM1-positive and MUM1-negative subsets was 65% (5-85%) and 5% (0-20%), respectively (P < 0.001). The median (IQR) percentage of BCL3-positive cells among GC and non-GC subsets of DLBCLs was 12% (12-81%) and 60% (6-87%), respectively (P = 0.022). CONCLUSION: Rearrangement or amplification involving the BCL3 gene is a rare event in DLBCL but is likely to play a role in the pathogenesis of a minority of de novo DLBCL. BCL3 over-expression is more frequent and occurs in the absence of rearrangement or amplification and is a feature of the non-GC subset of DLBCL.

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.

Presence of monoclonal T-cell populations in B-cell post-transplant lymphoproliferative disorders.

As has been previously shown, the lack of immune surveillance plays a major role in the unchecked proliferation of Epstein-Barr virus (EBV)-infected B cells in the pathogenesis of B-cell post-transplant lymphoproliferative disorders. We hypothesised that the lack of immune surveillance should possibly also affect T cells, and this should lead to subsequent emergence of T-cell clones. The presence of both B- and T-cell clones in post-transplant lymphoproliferative disorders samples has rarely been demonstrated in the past. We systematically evaluated 26 B-cell post-transplant lymphoproliferative disorder, 23 human immune deficiency virus-associated B-cell lymphoma and 10 immune-competent diffuse large B-cell lymphoma samples for B- and T-cell clonality (polymerase chain reaction and heteroduplex analysis using BIOMED-2 protocol), T-cell subsets (immunohistochemistry) and EBV association (in situ hybridisation using EBER). One-half of B-cell post-transplant lymphoproliferative disorders showed evidence of monoclonal T-cell expansion, and among the T cells present in the tissue samples, CD8-positive cells predominated. Although 9/13 (69%) B-cell post-transplant lymphoproliferative disorders with the presence of monoclonal T-cell population had a CD4:CD8 ratio of ≤0.4, 0/13 of the cases without monoclonal T-cell expansion had a ratio ≤0.4 (P = 0.002). Only 2/26 (8%) demonstrated significant cytological atypia in the CD3/CD8-positive cells. There was no association between EBV and presence of T-cell clones. T-cell clones were not identified in lymphomas other than B-cell post-transplant lymphoproliferative disorders. Among 53.8% cases of EBV-positive B-cell post-transplant lymphoproliferative disorders with associated clonal expansion of T-cells tested, none had EBV-positive T cells. We conclude that half of B-cell post-transplant lymphoproliferative disorders are associated with clonal expansion of CD8-positive T cells, most of which do not amount to the coexistence of a T-cell post-transplant lymphoproliferative disorders.

Epstein-Barr virus (EBV) genotypes among human immunodeficiency virus (HIV)-related B-cell lymphomas and B-cell post-transplant lymphoproliferative disorders (B-PTLD)--late-onset lymphomas, especially in the HIV setting, are associated with type-B-EBV.

We investigated 26 B-cell post-transplant lymphoproliferative disorders (B-PTLD) and 15 human immunodeficiency virus-related aggressive B-cell lymphomas (HIV-BCL) from England that were associated with Epstein-Barr virus (EBV) for the polymorphic sequences of the EBV-encoded nuclear antigen 3C (EBNA3C) gene to distinguish the two different EBV strains. Type-A-EBV was identified in 92% of B-PTLDS and in 53% of HIV-BCL (P = 0.003). Among HIV-BCL, patients associated with type-B-EBV had been HIV positive for significantly longer when compared to those associated with type-A (P = 0.037) although there were no correlations with ethnicity, CD4 cell counts or plasma HIV viral load.