Another look at follicular lymphoma: immunophenotypic and molecular analyses identify distinct follicular lymphoma subgroups.

AIMS: The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl-2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B-cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype. METHODS AND RESULTS: Two hundred and five follicular lymphomas were investigated with immunohistochemistry and fluorescence in-situ hybridization (FISH). The use of three distinct anti-bcl-2 antibodies together with CD10 expression data and FISH analysis for bcl-2 and bcl-6 rearrangements allowed subclassification of follicular lymphoma into four distinct subgroups: (i) CD10-positive/bcl-2-positive, (ii) CD10-positive/bcl-2-negative, (iii) CD10-negative/bcl-2-positive, and (iv) CD10-negative/bcl-2-negative. All cases were bcl-6-positive. STMN1 (stathmin) was shown to be helpful in diagnosing bcl-2-negative and/or CD10-negative follicular lymphomas, and in their distinction from marginal zone B-cell lymphoma. CONCLUSIONS: Combined immunohistological and molecular analyses reveal that follicular lymphomas showing an atypical immunophenotypic and molecular profile exist, and we demonstrate that STMN1 represents a novel useful diagnostic marker for these.

An integrated genomic and expression analysis of 7q deletion in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoproliferative disorder characterised by 7q32 deletion, but the target genes of this deletion remain unknown. In order to elucidate the genetic target of this deletion, we performed an integrative analysis of the genetic, epigenetic, transcriptomic and miRNomic data. High resolution array comparative genomic hybridization of 56 cases of SMZL delineated a minimally deleted region (2.8 Mb) at 7q32, but showed no evidence of any cryptic homozygous deletion or recurrent breakpoint in this region. Integrated transcriptomic analysis confirmed significant under-expression of a number of genes in this region in cases of SMZL with deletion, several of which showed hypermethylation. In addition, a cluster of 8 miRNA in this region showed under-expression in cases with the deletion, and three (miR-182/96/183) were also significantly under-expressed (P<0.05) in SMZL relative to other lymphomas. Genomic sequencing of these miRNA and IRF5, a strong candidate gene, did not show any evidence of somatic mutation in SMZL. These observations provide valuable guidance for further characterisation of 7q deletion.

BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas.

The genetics and pathogenesis of splenic marginal zone lymphoma are poorly understood. The lymphoma lacks chromosome translocation, and approximately 30% of cases are featured by 7q deletion, but the gene targeted by the deletion is unknown. A recent study showed inactivation of A20, a "global" NF-κB negative regulator, in 1 of 12 splenic marginal zone lymphomas. To investigate further whether deregulation of the NF-κB pathway plays a role in the pathogenesis of splenic marginal zone lymphoma, we screened several NF-κB regulators for genetic changes by PCR and sequencing. Somatic mutations were found in A20 (6/46=13%), MYD88 (6/46=13%), CARD11 (3/34=8.8%), but not in CD79A, CD79B and ABIN1. Interestingly, these genetic changes are largely mutually exclusive from each other and MYD88 mutation was also mutually exclusive from 7q deletion. These results strongly suggest that deregulation of the TLR (toll like receptor) and BCR (B-cell receptor) signaling pathway may play an important role in the pathogenesis of splenic marginal zone lymphoma.

Importance of expert central review in the diagnosis of lymphoid malignancies in a regional cancer network.

PURPOSE: The accurate diagnosis of hematologic malignancies remains a challenging area for histopathologists. In 2003, the North Central London Lymphoma network was established to provide a centralized expert review service for general histopathologists based in peripheral nonspecialist hospitals. By studying samples sent for review, we sought to assess the diagnostic and clinical impact of centralized expert review in lymphoma diagnosis. METHODS: A total of 1,949 patient samples were subject to expert review between 2003 and 2008. Diagnostic discordance rates were identified after expert review, and the impact on patient management was assessed by a hematologic oncology specialist. RESULTS: An overall discordance rate of 27.3% was identified. Among the 10 most commonly referred lymphoid malignancies, the discordance rate varied between 3.6% and 34.1%. A small but significant number of reactive (n = 17) and malignant (n = 5) discordant diagnoses were also identified. Expert central review resulted in a major change to patient management in 2.1% of patients and prevented delays in treatment in 9.3% of patients. During the 6-year study, the discordance rate improved significantly, decreasing from 32% to 13%. Although centralized review incurred additional costs, these were relatively small compared with the costs associated with treatment. CONCLUSION: This retrospective study demonstrates the importance of expert central review in the accurate diagnosis and timely management of lymphoid malignancies. Furthermore, it shows that where full centralization of services is not viable, a network comprising nonspecialist hospitals linked to a center providing expert review is a practical alternative to full unification of services at a single location and can help prevent local deskilling.

Primary effusion lymphoma: genomic profiling revealed amplification of SELPLG and CORO1C encoding for proteins important for cell migration.

Primary effusion lymphoma (PEL) is associated with Kaposi sarcoma herpesvirus (KSHV) but its pathogenesis is poorly understood. Many KSHV-associated products can deregulate cellular pathways commonly targeted in cancer. However, KSHV infection alone is insufficient for malignant transformation. PEL also lacks the chromosomal translocations seen in other lymphoma subtypes. We investigated 28 PELs and ten PEL cell lines by 1 Mb resolution array comparative genomic hybridization (CGH) and found frequent gains of 1q21-41 (47%), 4q28.3-35 (29%), 7q (58%), 8q (63%), 11 (32%), 12 (61%), 17q (29%), 19p (34%), and 20q (34%), and losses of 4q (32%), 11q25 (29%), and 14q32 (63%). Recurrent focal amplification was seen at several regions on chromosomes 7, 8, and 12. High-resolution chromosome-specific tile-path array CGH confirmed these findings, and identified selectin-P ligand (SELPLG) and coronin-1C (CORO1C) as the targets of a cryptic amplification at 12q24.11. Interphase FISH and quantitative PCR showed SELPLG/CORO1C amplification (>4 extra copies) and low levels of copy number gain (1-4 extra copies) in 23% of PELs, respectively. Immunohistochemistry revealed strong expression of both SELPLG and coronin-1C in the majority of PELs, irrespective of their gene dosage. SELPLG is critical for cell migration and chemotaxis, while CORO1C regulates actin-dependent processes, thus important for cell motility. Their overexpression in PEL is expected to play an important role in its pathogenesis.

The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation.

BACKGROUND: T follicular helper (T(FH)) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T(FH) cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified. DESIGN AND METHODS: In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas. RESULTS: Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T(FH)-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T(FH)-associated molecules. CONCLUSIONS: ICOS is a useful molecule for identifying T(FH) cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T(FH)-like profile) suggests its inclusion in the antibody panel for diagnosing T(FH)-derived lymphomas. Our findings provide further evidence that the histological spectrum of T(FH)-derived lymphomas is broader than previously assumed.

Successful treatment of HIV-associated multicentric Castleman's disease and multiple organ failure with rituximab and supportive care: a case report.

INTRODUCTION: Multicentric Castleman's Disease (MCD), a lymphoproliferative disorder associated with Human Herpes Virus-8 (HHV-8) infection, is increasing in incidence amongst HIV patients. This condition is associated with lymphadenopathy, polyclonal gammopathy, hepato-splenomegaly and systemic symptoms. A number of small studies have demonstrated the efficacy of the anti-CD20 monoclonal antibody, rituximab, in treating this condition. CASE PRESENTATION: We report the case of a 46 year old Zambian woman who presented with pyrexia, diarrhoea and vomiting, confusion, lymphadenopathy, and renal failure. She rapidly developed multiple organ failure following the initiation of treatment of MCD with rituximab. Following admission to intensive care (ICU), she received prompt multi-organ support. After 21 days on the ICU she returned to the haematology medical ward, and was discharged in remission from her disease after 149 days in hospital. CONCLUSION: Rituximab, the efficacy of which has thus far been examined predominantly in patients outside the ICU, in conjunction with extensive organ support was effective treatment for MCD with associated multiple organ failure. There is, to our knowledge, only one other published report of its successful use in an ICU setting, where it was combined with cyclophosphamide, adriamycin and prednisolone. Reports such as ours support the notion that critically unwell patients with HIV and haematological disease can benefit from intensive care.

Splenic marginal zone lymphoma: characterization of 7q deletion and its value in diagnosis.

The diagnosis of splenic marginal zone lymphoma (SMZL) is frequently a challenge, due to its lack of specific histological features and immunophenotypic markers, and the existence of other poorly characterized splenic lymphomas defying classification. Moreover, the clinical outcome of SMZL is variable, with 30% of cases pursuing an aggressive clinical course, the prediction of which remains problematic. Thus, there is a real need for biomarkers in the diagnosis and prognostication of SMZL. To search for genetic markers, we comprehensively investigated the genomic profile, TP53 abnormalities, and immunoglobulin heavy gene (IGH) mutation in a large cohort of SMZLs. 1 Mb resolution array comparative genomic hybridization (aCGH) on 25 SMZLs identified 7q32 deletion (44%) as the most frequent copy number change, followed by gains of 3q (32%), 8q (20%), 9q34 (20%), 12q23-24 (8%), and chromosome 18 (12%), and losses of 6q (16%), 8p (12%), and 17p (8%). High-resolution chromosome 7 tile-path aCGH on 17 SMZLs with 7q32 deletion identified by 1 Mb aCGH or interphase FISH screening mapped the minimal common deletion to a 3 Mb region at 7q32.1-32.2. Although it is not yet possible to identify the genes targeted by the deletion, interphase FISH screening showed that the deletion was seen in SMZL (19/56 = 34%) and splenic B-cell lymphoma/leukaemia unclassifiable (3/9 = 33%), but not in 39 cases of other splenic lymphomas including chronic lymphocytic leukaemia (n = 14), hairy cell leukaemia (4), mantle cell lymphoma (12), follicular lymphoma (6), and others. In SMZL, 7q32 deletion was inversely correlated with trisomy 18, but not associated with other copy number changes, TP53 abnormalities, or IGH mutation status. None of the genetic parameters examined showed significant and independent association with overall or event-free survival. In conclusion, 7q32 deletion is a characteristic feature of SMZL, albeit seen in isolated cases of splenic B-cell lymphoma/leukaemia unclassifiable, and its detection may help the differential diagnosis of splenic B-cell lymphomas.

Chronic lymphocytic leukemia with paraimmunoblastic transformation - with comparative genomic hybridization and review of the literature.

Richter's transformation (RT) is the development of a high-grade lymphoma in patients with B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We report an extremely rare case with paraimmunoblastic transformation. A 78-year-old Taiwanese male had Rai stage 1 and Binet stage B CLL/SLL involving skin, peripheral blood (PB), and bone marrow (BM) with paraimmunoblastic transformation in the lymph node. Molecular/genomic studies showed the same clonal origin of tumor tissues at various locations with trisomy 12 and a deletion of chromosome 13q14. Interestingly, there seemed to be no additional chromosomal aberrations in the transformed nodal tissue, suggesting that the micro-environment rather than an additional genetic lesion contributed to the transformation. The patient received chemotherapy and was alive with disease after 33.5 months.

Follicular lymphoma of the thyroid gland.

The majority of lymphomas arising in the thyroid gland are mucosa-associated lymphoid tissue lymphomas and diffuse large B-cell lymphomas, which arise from a background of chronic lymphocytic thyroiditis. Follicular lymphoma may also present in the thyroid gland, but its clinicopathologic features at this site are not well characterized, leading to difficulties in diagnosis and clinical management. We have addressed this problem by studying the clinical, morphologic, immunophenotypic, and genetic features of 22 such cases. All cases showed morphology characteristic of follicular lymphoma, however, in many the interfollicular neoplastic infiltrate was particularly prominent and all lymphomas contained readily identifiable and often striking lymphoepithelial lesions, features heretofore considered indicative of mucosa-associated lymphoid tissue lymphoma at this site. Furthermore, 13 of 18 cases for which sufficient evidence was available had clinical and/or histologic evidence of chronic lymphocytic thyroiditis. Analysis of genetic and immunohistochemical features identified 2 distinct groups. In 1 group, similar to typical adult follicular lymphoma, cases carried a t(14;18)/IGH-BCL2 and/or expressed Bcl-2, and were mostly CD10-positive and of World Health Organization (WHO) grade 1 to 2. Follicular lymphomas in the other group lacked IGH-BCL2 and Bcl-2 expression, were often of WHO grade 3 and were often CD10-negative, similar to the minority of follicular lymphomas previously described that are Bcl-2-negative and are often encountered at other extranodal sites. The 2 groups differed in clinical stage at presentation, 11 patients in the former group but none in the latter group having disease beyond the thyroid gland. Appreciation of the spectrum of morphologic, immunophenotypic, and genetic characteristics of follicular lymphoma presenting in the thyroid gland should aid both diagnosis and clinical management.

Classification of lymphoid neoplasms: the microscope as a tool for disease discovery.

In the past 50 years, we have witnessed explosive growth in the understanding of normal and neoplastic lymphoid cells. B-cell, T-cell, and natural killer (NK)-cell neoplasms in many respects recapitulate normal stages of lymphoid cell differentiation and function, so that they can be to some extent classified according to the corresponding normal stage. Likewise, the molecular mechanisms involved the pathogenesis of lymphomas and lymphoid leukemias are often based on the physiology of the lymphoid cells, capitalizing on deregulated normal physiology by harnessing the promoters of genes essential for lymphocyte function. The clinical manifestations of lymphomas likewise reflect the normal function of lymphoid cells in vivo. The multiparameter approach to classification adopted by the World Health Organization (WHO) classification has been validated in international studies as being highly reproducible, and enhancing the interpretation of clinical and translational studies. In addition, accurate and precise classification of disease entities facilitates the discovery of the molecular basis of lymphoid neoplasms in the basic science laboratory.

Novel markers of normal and neoplastic human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are involved in innate immunity (eg, by secreting interferons) and also give rise to CD4+CD56+ hematodermic neoplasms. We report extensive characterization of human pDCs in routine tissue samples, documenting the expression of 19 immunohistologic markers, including signaling molecules (eg, BLNK), transcription factors (eg, ICSBP/IRF8 and PU.1), and Toll-like receptors (TLR7, TLR9). Many of these molecules are expressed in other cell types (principally B cells), but the adaptor protein CD2AP was essentially restricted to pDCs, and is therefore a novel immunohistologic marker for use in tissue biopsies. We found little evidence for activation-associated morphologic or phenotypic changes in conditions where pDCs are greatly increased (eg, Kikuchi disease). Most of the molecules were retained in the majority of pDC neoplasms, and 3 (BCL11A, CD2AP, and ICSBP/IRF8) were also commonly negative in leukemia cutis (acute myeloid leukemia in the skin), a tumor that may mimic pDC neoplasia. In summary, we have documented a range of molecules (notably those associated with B cells) expressed by pDCs in tissues and peripheral blood (where pDCs were detectable in cytospins at a frequency of <1% of mononuclear cells) and also defined potential new markers (in particular CD2AP) for the diagnosis of pDC tumors.

The pattern of genomic gains in salivary gland MALT lymphomas.

BACKGROUND AND OBJECTIVES: Salivary gland mucosa-associated lymphoid tissue (MALT) lymphomas typically lack chromosomal translocations and the molecular genetics underlying their development is unknown. The aim of this sudy was to investigate chromosomal changes in these lymphomas. DESIGN AND METHODS: We performed comparative genomic hybridisation using DNA samples extracted from microdissected tumour cells in 19 salivary gland MALT lymphomas. Recurrent chromosomal changes were further verified by interphase fluorescence in situ hybridization (FISH). RESULTS: Chromosomal gains were much more common than losses. Recurrent gains were found at 1p32-ter (42%), 9q33-34 (84%), 11q11-13 (42%), 17 (58%) and 18q21-22 (42%). Among these, the recurrent gains at 9q34, 11q11-13 and 18q21 were nearly the exclusive gain of the corresponding chromosome. Notably, chromosomal gains at 9q34, 11q13 and 18q21 were frequently concurrent, with 12/19 cases affecting at least two of the three loci. The genomic gains at these chromosomal regions were further confirmed by interphase FISH with probes targeting the TRAF2 and CARD9 (9q34), RELA and CCND1 (11q13), and MALT1 (18q21) loci. INTERPRETATION AND CONCLUSIONS: Salivary gland MALT lymphomas show a conserved pattern of chromosomal gains, which appear to target genes positively modulating cell survival and proliferation.

Distinct comparative genomic hybridisation profiles in gastric mucosa-associated lymphoid tissue lymphomas with and without t(11;18)(q21;q21).

t(11;18)(q21;q21) occurs specifically in mucosa-associated lymphoid tissue (MALT) lymphoma and the translocation generates a functional API2-MALT1 fusion product that activates nuclear factor (NF)kappaB. t(11;18) positive lymphomas usually lack the chromosomal aberrations and microsatellite alterations frequently seen in the translocation-negative MALT lymphomas. To further understand their genetic differences, we investigated gastric MALT lymphomas with and without t(11;18) by comparative genomic hybridisation. In general, both chromosomal gains and losses were far more frequent in t(11;18)-negative (median = 3.4 imbalances) than t(11;18)-positive cases (median = 1.6 imbalances), with gains being more frequent than losses. Recurrent chromosomal gains involving whole or major parts of a chromosome were seen for chromosomes 3, 12, 18 and 22 (23%, 19%, 19% and 27% respectively). Discrete recurrent chromosomal gains were found at 9q34 (11/26 = 42%). Bioinformatic analysis of genes mapping to 9q34 revealed potential targets. Among them, TRAF2 and CARD9 are known interaction partners of BCL10, playing a role in NFkappaB activation. Interphase fluorescent in situ hybridisation confirmed genomic gain of the TRAF2, CARD9 and MALT1 loci in 5/6 and 2/2 cases showing chromosomal gains at 9q34 and 18q21 respectively. The results further highlight the genetic difference between MALT lymphomas with and without t(11;18). Moreover, our findings suggest that genomic gain of genes that modulate NFkappaB activation, such as MALT1, TRAF2 and CARD9, may play a role in the pathogenesis of the translocation-negative MALT lymphoma.