Syk-induced phosphatidylinositol-3-kinase activation in Epstein-Barr virus posttransplant lymphoproliferative disorder.

Posttransplant lymphoproliferative disorder (PTLD)-associated Epstein-Barr virus (EBV)+ B cell lymphomas are serious complications of solid organ and bone marrow transplantation. The EBV protein LMP2a, a B cell receptor (BCR) mimic, provides survival signals to virally infected cells through Syk tyrosine kinase. Therefore, we explored whether Syk inhibition is a viable therapeutic strategy for EBV-associated PTLD. We have shown that R406, the active metabolite of the Syk inhibitor fostamatinib, induces apoptosis and cell cycle arrest while decreasing downstream phosphatidylinositol-3'-kinase (PI3K)/Akt signaling in EBV+ B cell lymphoma PTLD lines in vitro. However, Syk inhibition did not inhibit or delay the in vivo growth of solid tumors established from EBV-infected B cell lines. Instead, we observed tumor growth in adjacent inguinal lymph nodes exclusively in fostamatinib-treated animals. In contrast, direct inhibition of PI3K/Akt significantly reduced tumor burden in a xenogeneic mouse model of PTLD without evidence of tumor growth in adjacent inguinal lymph nodes. Taken together, our data indicate that Syk activates PI3K/Akt signaling which is required for survival of EBV+ B cell lymphomas. PI3K/Akt signaling may be a promising therapeutic target for PTLD, and other EBV-associated malignancies.

Lack of association of tumor-associated macrophages with clinical outcome in patients with classical Hodgkin's lymphoma.

BACKGROUND: A recent study demonstrated that an increased number of CD68+ macrophages were correlated with primary treatment failure, shortened progression-free survival (PFS) and disease-specific survival (DSS) in patients with classical Hodgkin's lymphoma (cHL). PATIENTS AND METHODS: The aim of the present study was to verify the relationship between the number of CD68+ and CD163+ macrophages with clinical outcomes in a cohort of 265 well-characterized patients with cHL treated uniformly with the standard doxorubicin, bleomycin, vinblastine and dacarbazine chemotherapy regimen. Two pairs of hematopathologists carried out independent pathological evaluations of tissue microarray slides. RESULTS: There were no associations between clinical characteristics and the expression of CD68 or CD163. However, higher levels of CD68 and CD163 expression were correlated with the presence of Epstein-Barr virus-positive Hodgkin tumor cells (P = 0.01 and 0.037, respectively). The expression of CD68 or CD163 was not associated with either the PFS or the DSS. CONCLUSION: CD68 and CD163 expression require further evaluation before their use can be recommended for prognostic stratification of patients with cHL.

International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% (specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01-0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

Nuclear signaling by interleukin-6.

The molecular analysis of the regulation of nuclear proteins induced by interleukin-6 has provided new insights into this largely unknown signal transduction pathway. Transcription factors of the CCAAT/enhancer-binding protein and AP-1 families, as well as the octamer-binding proteins and the tumor suppressor gene product pRB, are regulated by interleukin-6 in a cell type specific manner, suggesting that they may play a role in the nuclear signaling by interleukin-6.

Molecular impact of selective NFKB1 and NFKB2 signaling on DLBCL phenotype.

Diffuse large B-cell lymphoma (DLBCL) has been categorized into two molecular subtypes that have prognostic significance, namely germinal center B-cell like (GCB) and activated B-cell like (ABC). Although ABC-DLBCL has been associated with NF-κB activation, the relationships between activation of specific NF-κB signals and DLBCL phenotype remain unclear. Application of novel gene expression classifiers identified two new DLBCL categories characterized by selective p100 (NF-κB2) and p105 (NF-κB1) signaling. Interestingly, our molecular studies showed that p105 signaling is predominantly associated with GCB subtype and histone mutations. Conversely, most tumors with p100 signaling displayed ABC phenotype and harbored ABC-associated mutations in genes such as MYD88 and PIM1. In vitro, MYD88 L265P mutation promoted p100 signaling through TAK1/IKKα and GSK3/Fbxw7a pathways, suggesting a novel role for this protein as an upstream regulator of p100. p100 signaling was engaged during activation of normal B cells, suggesting p100's role in ABC phenotype development. Additionally, silencing p100 in ABC-DLBCL cells resulted in a GCB-like phenotype, with suppression of Blimp, IRF4 and XBP1 and upregulation of BCL6, whereas introduction of p52 or p100 into GC cells resulted in differentiation toward an ABC-like phenotype. Together, these findings identify specific roles for p100 and p105 signaling in defining DLBCL molecular subtypes and posit MYD88/p100 signaling as a regulator for B-cell activation.

Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma.

Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed-Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-gamma2 (absent from Reed-Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.

Natural killer/natural killer-like T-cell lymphoma, CD56+, presenting in the skin: an increasingly recognized entity with an aggressive course.

PURPOSE: To describe and identify the clinical and pathologic features of prognostic significance for natural killer (NK) and NK-like T-cell (NK/T-cell) lymphoma presenting in the skin. PATIENTS AND METHODS: This study was a retrospective review of 30 patients with CD56+ lymphomas initially presenting with cutaneous lesions, with analysis of clinical and histopathologic parameters. RESULTS: The median survival for all patients was 15 months. Those with extracutaneous manifestations at presentation (11 patients) had a shorter median survival of 7.6 months as compared with those without extracutaneous involvement (17 patients), who had a more favorable median survival of 44.9 months (P =.0001). Age, gender, extent of cutaneous involvement, and initial response to therapy had no statistically significant effect on survival. Seven patients (24%) had detectable Epstein-Barr virus (EBV) within neoplastic cells. The patients with tumor cells that coexpress CD30 (seven patients) have not yet reached a median survival after 35 months of follow-up as compared with those with CD30- tumor cells (20 patients), who had a median survival of 9.6 months (P <.02). Routine histopathologic characteristics had no prognostic significance nor did the presence of CD3epsilon, EBV, or multidrug resistance. CONCLUSION: NK/T-cell lymphoma is an aggressive neoplasm; however, a subset with a more favorable outcome is identified in this study. The presence of extracutaneous disease at presentation is the most important clinical variable and portends a poor prognosis. The extent of initial skin involvement does not reliably predict outcome. Patients from the United States with NK/T-cell lymphoma presenting in the skin have a low incidence of demonstrable EBV in their tumor cells. Patients with coexpression of CD30 in CD56 lymphomas tend to have a more favorable outcome.

The Epstein-Barr Virus (EBV) in T Cell and NK Cell Lymphomas: Time for a Reassessment.

While Epstein-Barr virus (EBV) was initially discovered and characterized as an oncogenic virus in B cell neoplasms, it also plays a complex and multifaceted role in T/NK cell lymphomas. In B cell lymphomas, EBV-encoded proteins have been shown to directly promote immortalization and proliferation through stimulation of the NF-κB pathway and increased expression of anti-apoptotic genes. In the context of mature T/NK lymphomas (MTNKL), with the possible exception on extranodal NK/T cell lymphoma (ENKTL), the virus likely plays a more diverse and nuanced role. EBV has been shown to shape the tumor microenvironment by promoting Th2-skewed T cell responses and by increasing the expression of the immune checkpoint ligand PD-L1. The type of cell infected, the amount of plasma EBV DNA, and the degree of viral lytic replication have all been proposed to have prognostic value in T/NK cell lymphomas. Latency patterns of EBV infection have been defined using EBV-infected B cell models and have not been definitively established in T/NK cell lymphomas. Identifying the expression profile of EBV lytic proteins could allow for individualized therapy with the use of antiviral medications. More work needs to be done to determine whether EBV-associated MTNKL have distinct biological and clinical features, which can be leveraged for risk stratification, disease monitoring, and therapeutic purposes.

Utility of paraffin section immunohistochemistry for C-KIT (CD117) in the differential diagnosis of systemic mast cell disease involving the bone marrow.

Systemic mast cell disease is characterized by an abnormal infiltration of mast cells involving several parenchymal organs and the bone marrow. Its spectrum of clinical and histologic presentation is highly variable and is not necessarily correlated with prognosis. Mast cell disorders presenting as atypical infiltrates in the bone marrow may simulate or be associated with other hematolymphoid malignancies, from which they must be distinguished. The paucity of reliable histochemical and immunohistochemical markers for the detection of mast cells in paraffin sections further confounds this diagnosis. The authors have employed immunohistochemistry for the C-KIT encoded tyrosine kinase receptor protein, CD117, for detection of mast cells on paraffin sections of 89 bone marrow specimens including systemic mast cell disease and other disorders. CD117 staining was found in all cases of mast cell disorders (seven of seven), and in one case of chronic myelogenous leukemia in blast crisis. None of the other myeloid disorders tested (0 of 16), or any of the cases of Hodgkin's disease (0 of 12), B-cell lymphomas (0 of 32), T-cell lymphomas (0 of 3), or histiocytic proliferations (0 of 3) showed staining for CD117. CD117 expression is effective in the separation of mast cell disease from disorders that may simulate it histologically.

Aggressive cutaneous NK and NK-like T-cell lymphomas: clinicopathologic, immunohistochemical, and molecular analyses of 12 cases.

Natural killer (NK) and NK-like T-cell lymphomas are rare hematolymphoid malignancies that predominate in the upper aerodigestive system. They also involve other extranodal sites, including the skin. Primary cutaneous manifestations of NK and NK-like T-cell lymphomas are uncommon, and the clinicopathologic features are poorly understood. We have studied 12 patients of varied ethnic backgrounds with CD56-positive lymphomas in the skin. Six patients subsequently progressed to disseminated disease. These lymphomas showed the following immunophenotype: CD56+, CD43+, TCRb-, CD3-/+, CD20-, CD30-/+, CD4-, and CD8-. Two cases exhibited T-cell receptor gene rearrangements supporting a T-cell origin for these lymphomas, whereas the remaining 10 cases were likely derived from NK cells. Our results show inconsistent association of these lymphomas with Epstein-Barr virus (EBV), the multidrug resistance phenotype, and expression of P53. In addition, we found a previously unreported correlation between lymphomas harboring EBV mRNA and the expression of the multidrug resistance phenotype. These lymphomas were aggressive and were associated with rapid clinical progression, treatment failure, multiple relapses, and an average survival of 15 months from the time of diagnosis. Our results indicate the importance of recognizing this disease as a distinct subset of aggressive cutaneous lymphomas that may be diagnosed on the basis of morphology, immunophenotype, and gene rearrangement studies.

Blastic/blastoid transformation of follicular lymphoma: immunohistologic and molecular analyses of five cases.

Progression of follicular lymphoma to a higher-grade malignancy frequently heralds a poor prognosis. Clinical transformation is variably accompanied by a spectrum of histologic changes characterized by alteration in growth and cytology. Although several cytogenetic events and potential oncogenes have been documented in this progression, the underlying molecular mechanisms are largely unknown. We present five patients with an unusual histologic transformation of follicular lymphoma manifested by blastic/blastoid morphology. This transformation is histologically distinct from other types of transformation of follicular lymphoma. All five cases exhibited the t(14;18) translocation and expressed the BCL-2 protein. In addition, two of the five patients showed increased levels of the p53 protein within neoplastic cells implicating a possible role for this oncogene in blastic/blastoid transformation. The lack of BCL-1 and myeloid antigens by immunohistochemistry and flow cytometry studies served to distinguish blastic/blastoid transformation of follicular lymphoma from its morphologic mimics. This distinction is clinically important because lymphoblastic and myeloid leukemias require significantly different therapeutic modalities and show better prognosis. Moreover, the lack of Epstein-Barr virus-specific mRNA suggests that this virus is unlikely to participate in blastic/blastoid transformation of follicular lymphoma.

Epstein-Barr virus infection is associated with endothelial Bcl-2 expression in transplant liver allografts.

INTRODUCTION: In liver transplant recipients with Epstein-Barr virus (EBV) disease, we reported a low rate of acute rejection after stopping or markedly lowering immunosuppression. This observation led to the hypothesis that EBV, as a means of viral persistence, induces expression of antiapoptotic factors and these factors, in turn, confer protection to the transplanted organ. Bcl-2, an antiapoptotic factor induced by EBV in various host cells, is not normally expressed in the liver. We questioned whether bcl-2 is expressed in the transplanted liver and whether its expression is modified by EBV. MATERIALS AND METHODS: Retrospective liver biopsy specimen from liver transplant patients diagnosed with EBV (n=12) were examined for the presence of bcl-2 by immunohistochemistry and compared with EBV (-) transplant (n=15), and nontransplant (n=13) livers. RESULTS: The most significant finding was the presence of endothelial bcl-2 expression in the majority of EBV (+) transplant samples examined (67%) and its relative absence in the other two groups (P<0.005). There was also bcl-2 expression in the hepatocytes and lymphocytes of the majority of transplant liver samples, irrespective of EBV status. DISCUSSION: We have identified a strong association between EBV infection and endothelial bcl-2 expression in transplant livers. We also found that transplantation, in itself, was associated with bcl-2 expression in the hepatocytes and lymphocytes of liver allografts.

Aggressive natural killer-like T-cell malignancy with leukemic presentation following solid organ transplantation.

NK-like T-cell malignancies are part of a spectrum of lymphoproliferative diseases that complicate immunosuppression associated with solid organ transplantation. We describe 2 patients with long-standing immunosuppression following solid organ transplantation. Both patients had systemic symptoms that included fever, myalgia, and weight loss. Organ involvement and lymphadenopathy were not initially observed. Unique to these 2 cases are the initial leukemic symptoms, which led to further characterization and identification of NK-like T-cell malignancies. Both patients exhibited an anomalous T/NK phenotype, CD56 positivity, and atypical blastic architecture of the large granular lymphocytes. Clonal rearrangement of T-cell receptor genes was detected in both patients. In 1 patient, a cytogenetic abnormality involving 8q24 was demonstrated. The disease course in both patients was aggressive, with involvement of multiple sites and rapid demise. This study emphasizes the importance of including NK-like T-cell malignancies in the differential diagnosis of lymphoproliferative disorders associated with immunosuppression and recognizing that an aggressive clinical course may follow leukemic presentation of disease.

Durable remission in recurrent T-cell-rich B-cell lymphoma with the anti-CD20 antibody rituximab.

A diagnostic continuum exists between lymphocyte-predominant Hodgkin's disease, T-cell-rich B-cell lymphoma (TCRBCL), and diffuse large B-cell lymphoma. While TCRBCLs are uncommon, their clinical and morphologic presentation can mimic other Hodgkin's and non-Hodgkin's lymphomas from which they must be distinguished for diagnosis and treatment. We present an unusual case of a 30-year-old man with recurrent TCRBCL arising from lymphocyte-predominant Hodgkin's disease with remarkable response to treatment with the anti-CD20 antibody, rituximab.

A unique AML1 (CBF2A) rearrangement, t(1;21)(p32;q22), observed in a patient with acute myelomonocytic leukemia.

The AML1 (CBFA2) gene is the most frequent target of chromosomal rearrangements observed in human acute leukemia. These rearrangements include the commonly reported t(8;21)(q22;q22) or AML1/ETO fusion in AML-M2, the t(3;21)(q26;q22) or AML1 fusion with one of three genes, MDS1, EAP or EVI1, in therapy-related AML and MDS, as well as in blast crisis in CML and the t(12;21)(p13;q22) or TEL/AML1 fusion in B-cell ALL. In addition to the t(3;21), other AML1 translocations have also been reported in therapy-related MDS and AML, particularly after treatment with topoisomerase II inhibitors. AML1 gene rearrangements have also been observed less frequently with numerous other chromosomal partners. Here, we describe a patient with AML-M4 and a previously unreported rearrangement involving the AML1 locus and an unknown locus on the short arm of chromosome 1 at 1p32.

Natural killer cell precursor acute lymphoma/leukemia presenting in an infant.

Lymphoma/leukemia derived from immature natural killer (NK) cells occur most commonly in adults and are characterized by blastic cytologic features and an aggressive outcome. Predilection for extranodal sites and absence of the Epstein-Barr virus associated with mature NK cell malignancies further distinguish this entity. We present a NK precursor acute lymphoma presenting with multiple masses in an infant without circulating blasts or marrow replacement by disease. The diagnostic difficulty arose from several factors, including young age, presentation with multiple masses, blastic cytologic features mistaken for a small, round, blue cell tumor, and the absence of lineage-specific markers. The CD56+, CD34+, CD33+, MPO-, cytoplasmic CD3+, CD45-, CD7-, HLA-DR-, and TdT- immunophenotype of this neoplasm overlaps with previously reported cases of myeloid/NK precursor acute leukemia and blastic NK cell lymphoma/leukemia. This case emphasizes the need for a strong index of suspicion to recognize this rare entity and to distinguish it from solid tumors and other hematolymphoid neoplasms that occur in infancy.

Angiocentric lymphomas (lymphomatous vasculitis).

Angiocentric lymphomas are a heterogeneous spectrum of hematolymphoid malignancies that share a particular histologic characteristic, namely, an angiocentric or perivascular growth pattern. They include a variety of T-, B-, and natural killer-cell derived lymphomas that digress in many clinicopathologic features, immunophenotype, and prognosis. The term angiocentric lymphomas was initially used to refer to natural killer and natural killer-like T-cell lymphomas that show a prominent angiocentric growth pattern. With better immunophenotypic and molecular characterization together with evolving knowledge regarding their biology and pathogenesis, these lymphomas have now been reclassified. Apart from morphology, many features pertinent to the diagnosis of natural killer and natural killer-like T-cell lymphomas are shared by other peripheral T-cell and B-cell lymphomas, and by a subset of leukemias. The salient clinicopathologic features of natural killer and natural killer-like T-cell lymphomas together with the inherent difficulty of their identification and an integrated approach to their diagnosis are outlined in this article.

Expression of the CD2AP adaptor molecule in normal, reactive and neoplastic human tissue.

AIMS: To study the expression of CD2-associated protein (CD2AP), an adaptor protein involved in T-cell signalling and renal function, in normal, reactive and neoplastic human lymphoid tissues. METHODS AND RESULTS: We used immunohistochemical techniques to evaluate monoclonal antibodies against CD2AP on over 400 formalin fixed paraffin embedded tissue blocks retrieved from the host institutions of three authors. The samples tested included normal, reactive and neoplastic lymphoid tissue. In lymphoid tissues, strong CD2AP staining was observed in plasmacytoid dendritic cells (pDCs), weak and variable in mantle zone B cells and moderate in rare germinal center cells. CD2AP labeled cortical and rare medullary thymocytes and isolated mononuclear cells in bone marrow trephines. Furthermore, epithelial and endothelial cells expressed CD2AP. Among neoplasms, the greatest number of CD2AP-positive cases were found in diffuse large B cell (21/94), NK T-cell lymphomas (7/67), "blastic plasmacytoid dendritic cell neoplasms" (9/10) and some types of solid tumor. CONCLUSIONS: Our finding that mature peripheral T cells are CD2AP-negative but immature cortical thymocytes are positive may prove useful for diagnostic purposes. Moreover, our results demonstrate that CD2AP represents a useful marker of normal and neoplastic pDC and may be used in a diagnostic panel in reactive or neoplastic lymphoid proliferations.

Selective loss of B-cell phenotype in lymphocyte predominant Hodgkin lymphoma.

The neoplastic Reed-Sternberg cells characteristic of classical Hodgkin's lymphoma (cHL) are of B-cell origin but they almost always show striking loss of a range of B-cell-associated molecules. In contrast, the neoplastic cells found in lymphocyte predominant Hodgkin's lymphoma (LPHL) (L&H cells) are traditionally thought of as possessing the full repertoire of features associated with germinal centre B cells (eg BCL-6 expression, 'ongoing' Ig gene mutation). In the present paper, we report an extensive phenotypic analysis of L&H cells which revealed down-regulation of a number of markers associated with the B-cell lineage (eg CD19, CD37) and with the germinal centre maturation stage (eg PAG, LCK). The promoter methylation status of three of these down-regulated genes (CD10, CD19, and LCK) was further studied in microdissected L&H cells, and this revealed that their promoters were unmethylated. In contrast, these genes showed promoter methylation in cell lines derived from CHL. Further investigation of the mechanisms responsible for the deregulation of these molecules in L&H cells may provide new insights into the genetic abnormalities underlying LPHL.

Loss of CD19 expression in B-cell neoplasms.

AIMS: To investigate whether an antibody against an intracellular epitope can detect CD19 in routine biopsy specimens and thus to document in detail its expression in human lymphomas. METHOD AND RESULTS: A polyclonal antibody to the C terminus of CD19 was used to immunostain paraffin-embedded samples of normal and neoplastic lymphoid tissues. CD19 was widely expressed in normal B cells and in extramedullary plasma cells. It was found in most B-cell neoplasms, but expression in follicular lymphoma was weak (33/69) or negative (four cases). Similarly, CD19 expression in diffuse large B-cell lymphomas was weak (28/56) or negative (eight cases). In T-cell-rich B-cell lymphomas, CD19 was also weak (4/10) or negative (three cases). CD19 was often absent in post-transplant B lymphoproliferative disease, classical Hodgkin's disease and plasma cell neoplasms. An unexpected finding was the frequent absence of CD19 in the neoplastic cells in lymphocyte predominant Hodgkin's disease. CONCLUSIONS: CD19 can now be detected in routine biopsy specimens. In contrast to the classical pan-B marker CD20, CD19 is not always strongly expressed in B-cell neoplasms. Furthermore, the lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin's disease (which express most B-cell-associated markers) commonly lack CD19.