CD28 expressed on malignant plasma cells induces a prosurvival and immunosuppressive microenvironment.

Interactions between the malignant plasma cells of multiple myeloma and stromal cells within the bone marrow microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal bone marrow-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce prosurvival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance, the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor-prognosis subgroups and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell directly transduces a prosurvival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal dendritic cell to produce the prosurvival cytokine IL-6 (involving novel cross-talk with the Notch pathway) and the immunosuppressive enzyme IDO. These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, point to similar interaction for normal plasma cells, and suggest novel therapeutic strategies to target malignant and pathogenic (e.g., in allergy and autoimmunity) plasma cells.

Platelet satellitism and dual surface immunoglobulin light-chain expression in circulating splenic marginal zone lymphoma cells.

Platelet satellitism is believed to be an in vitro phenomenon induced at room temperature in ethylenediamine tetraacetic acid-anticoagulated blood. Most reports involve neutrophils; involvement with circulating lymphoma cells are exceedingly rare. Normally, mature B cells exhibit allelic exclusion in which a single class of surface immunoglobulin light chains (either κ or λ) is expressed. The simultaneous expression of both κ and λ immunoglobulin light chains is rare. Herein, we report the unusual case of a patient with splenic marginal zone lymphoma in which circulating lymphoma cells express dual surface immunoglobulin light chains and exhibit platelet satellitism. In addition to clinical findings, a comprehensive analysis of the peripheral blood including correlated light and electron microscopy as well as flow cytometry are described.

Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R-CHOP.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by variable clinical outcomes. Outcome prediction at the time of diagnosis is of paramount importance. Previously, we constructed a 6-gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies. However, the standard therapy has evolved into rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Herein, we evaluated the predictive power of a paraffin-based 6-gene model in R-CHOP-treated DLBCL patients. RNA was successfully extracted from 132 formalin-fixed paraffin-embedded (FFPE) specimens. Expression of the 6 genes comprising the model was measured and the mortality predictor score was calculated for each patient. The mortality predictor score divided patients into low-risk (below median) and high-risk (above median) subgroups with significantly different overall survival (OS; P = .002) and progression-free survival (PFS; P = .038). The model also predicted OS and PFS when the mortality predictor score was considered as a continuous variable (P = .002 and .010, respectively) and was independent of the IPI for prediction of OS (P = .008). These findings demonstrate that the prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment and that the model can be applied to routine FFPE tissue from initial diagnostic biopsies.

EUS-guided biopsy for the diagnosis and classification of lymphoma.

BACKGROUND: EUS-guided FNA and Tru-cut biopsy (TCB) is highly accurate in the diagnosis of lymphoma. Subclassification, however, may be difficult in low-grade non-Hodgkin lymphoma and Hodgkin lymphoma. OBJECTIVE: To determine the yield of EUS-guided biopsy to classify lymphoma based on the World Health Organization classification of tumors of hematopoietic lymphoid tissues. DESIGN: Retrospective study. SETTING: Tertiary referral center. PATIENTS: A total of 24 patients referred for EUS-guided biopsy who had a final diagnosis of lymphoma or "highly suspicious for lymphoma." INTERVENTIONS: EUS-guided FNA and TCB combined with flow cytometry (FC) analysis. MAIN OUTCOMES MEASUREMENT: Lymphoma subclassification accuracy of EUS guided biopsy. RESULTS: Twenty-four patients were included in this study. Twenty-three patients underwent EUS-FNA, and 1 patient had only TCB. Twenty-two underwent EUS-TCB combined with FNA. EUS correctly diagnosed lymphoma in 19 out of 24 patients (79%), and subclassification was determined in 16 patients (66.6%). Flow cytometry correctly identified B-cell monoclonality in 95% (18 out of 19). In 1 patient diagnosed as having marginal-zone lymphoma by EUS-FNA/FC only, the diagnosis was changed to hairy cell leukemia after a bone marrow biopsy was obtained. EUS had a lower yield in nonlarge B-cell lymphoma (only 9 out of 15 cases [60%]) compared with large B-cell lymphoma (78%; P = .3 [Fisher exact test]). LIMITATIONS: Retrospective, small number of patients. CONCLUSION: EUS-guided biopsy has a lower yield to correctly classify Hodgkin lymphoma and low-grade lymphoma compared with high-grade diffuse large B-cell lymphoma.

Prognostic significance of VEGF, VEGF receptors, and microvessel density in diffuse large B cell lymphoma treated with anthracycline-based chemotherapy.

Vascular endothelial growth factor-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of lymphoma cell proliferation and/or survival induced by autocrine vascular endothelial growth factor receptor-mediated signaling. We have recently shown that diffuse large B cell lymphomas expressing high levels of vascular endothelial growth factor protein also express high levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether tumor vascularity, or expression of vascular endothelial growth factor protein and its receptors, contribute to patient outcomes. Our results show that increased tumor vascularity is associated with poor overall survival (P=0.047), and is independent of the international prognostic index. High expression of vascular endothelial growth factor receptor-1 by lymphoma cells by contrast is associated with improved overall survival (P=0.044). The combination of high vascular endothelial growth factor and vascular endothelial growth factor receptor-1 protein expression by lymphoma cells identifies a subgroup of patients with improved overall (P=0.003) and progression-free (P=0.026) survival; these findings are also independent of the international prognostic index. The prognostic significance of overexpression of this ligand-receptor pair suggests that autocrine signaling via vascular endothelial growth factor receptor-1 may represent a survival or proliferation pathway in diffuse large B cell lymphoma. Dependence on autocrine vascular endothelial growth factor receptor-1-mediated signaling may render a subset of diffuse large B-cell lymphomas susceptible to anthracycline-based therapy.

Ocular Adnexal Lymphoma: no evidence for bacterial DNA associated with lymphoma pathogenesis.

Ocular Adnexal Lymphomas (OALs) are the most common tumors of the eye, the majority being extranodal mucosa-associated lymphoid tissue (MALT) lymphomas. Association with Chlamydia psittaci was described in some geographic areas. OAL response to antibiotic therapy was reported in cases not harboring Chlamydia psittaci DNA, suggesting that other bacterial infection might be implicated in the pathogenesis. We examined 49 MALT OALs for bacterial DNA using two distinct polymerase chain reaction techniques based on universal bacterial primers. No bacterial DNA that could be implicated in OAL pathogenesis was detected, suggesting that bacterial infection is not associated with OAL in South Florida.

Optimization of RNA extraction from formalin-fixed, paraffin-embedded lymphoid tissues.

Comprehensive analysis of gene expression using RNA extracted from frozen lymphoma specimens is becoming increasingly important for understanding disease pathogenesis, disease subclassification, and prognostication. As paraffin tissues are widely available whereas frozen specimens are not, development of gene expression analysis based on RNA extracted from paraffin-embedded tissues would facilitate application of the accumulated knowledge to a sample type that is typical of clinical practice. In the present study, we have developed and optimized methods of RNA extraction from paraffin-embedded lymphoid tissues. In contrast to previously suggested methods of RNA extraction from paraffin, our method uses sodium dodecyl sulfate that better preserves the extracted RNA and is optimized for more complete proteinase K digestion to release RNA from its complexes with protein. These modifications yield long RNA fragments up to 2000 bp enabling amplification of long amplicons. This allows usage of paraffin specimens for molecular rescue of RNA transcripted from rearranged clonal immunoglobulin genes-an advance that may increase the eligibility of lymphoma patients for immunotherapeutic approaches. Furthermore, real-time polymerase chain reaction analysis of expression of genes implicated in determination of prognosis of diffuse large B-cell lymphoma patients demonstrated an extremely high correlation (R>0.90) in normalized gene expression between paired frozen and formalin-fixed, paraffin-embedded specimens. Similarly, good correlation was also observed in gene array studies. These results suggest that the methods of RNA extraction we propose are suitable for giving accurate real-time quantitative reverse transcriptase-polymerase chain reaction results, array gene expression profiling, and molecular rescue of RNA transcripted from rearranged immunoglobulin genes for diagnostic and immunotherapeutic approaches.

Anaphylactic reaction to platelet transfusion as the initial symptom of an undiagnosed systemic mastocytosis: a case report and review of the literature.

INTRODUCTION: The association between anaphylactic reactions and systemic mastocytosis is well documented. However, platelet transfusion has not previously been reported as a potential elicitor of anaphylaxis in the context of systemic mastocytosis. CASE PRESENTATION: We describe the clinicopathological findings of a 59-year-old Latin American man who presented to the emergency room with fatigue, leukocytosis, thrombocytopenia and mild hepatosplenomegaly. He developed two separate, temporally associated and severe anaphylactic reactions after receiving platelet transfusions. The result of a laboratory investigation for clerical errors and Coombs test was negative. Pre- and post-transfusion urine samples were negative for hemolysis. Bone marrow biopsy and aspirate smears performed demonstrated involvement by systemic mastocytosis, which had been previously undiagnosed. CONCLUSIONS: We posit the transfusion reaction to be an anaphylactic reaction to transfused products as a result of heightened allergic sensitivity due to the underlying systemic mastocytosis. To the best of our knowledge, this is the first reported case of a severe anaphylactic-type reaction to blood products occurring in the setting of a previously undiagnosed systemic mastocytosis. Furthermore, it seems there are no published studies closely examining the relationship between hematopoietic neoplasms and transfusion reactions in general.

Oral and extraoral plasmablastic lymphoma: similarities and differences in clinicopathologic characteristics.

Plasmablastic lymphoma (PBL), initially characterized as an aggressive lymphoma arising in the jaw and oral mucosa in HIV-infected patients, was recently reported to occur with extraoral manifestations, heterogeneous histologic findings, and variable association with immunodeficiency states. We reviewed clinical, morphologic, and immunophenotypic features of 13 cases of PBL to determine whether these different subtypes represent distinct morphologic and clinical entities. Two distinct subtypes of PBL were identified and classified as oral and extraoral PBL. The oral PBLs were strongly associated with HIV infection and commonly demonstrated plasmablastic morphologic features without plasmacytic differentiation. Extraoral PBLs tended to occur in patients with underlying non-HIV-related immunosuppression and universally demonstrated plasmacytic differentiation. The patients with oral PBL demonstrated better overall survival compared with patients with extraoral PBL (P = .02). Our findings suggest that PBL with oral and extraoral manifestation represent 2 distinct clinicopathologic entities.

High rate and prolonged duration of complete remissions induced by rituximab, methotrexate, doxorubicin, cyclophosphamide, vincristine, ifosfamide, etoposide, cytarabine, and thalidomide (R-MACLO-IVAM-T), a modification of the National Cancer Institute 89-C-41 regimen, in patients with newly diagnosed mantle cell lymphoma.

Novel therapeutic approaches are needed in mantle cell lymphoma (MCL). We conducted a phase II study in MCL testing an intensive regimen, R-MACLO-IVAM-T, a modification of the NCI 89-C-41 protocol. Newly diagnosed patients were treated with rituximab, methotrexate, doxorubicin, cyclophosphamide, and vincristine (cycle 1) followed by rituximab, ifosfamide (and mesna), etoposide, and cytarabine (cycle 2). These two cycles were repeated once, and patients achieving complete response (CR) received maintenance thalidomide. Among the 22 patients enrolled, 21 completed two or more cycles and achieved a CR. Three patients relapsed, while 17 are alive and relapse-free after a median follow-up of 37 months (range 19-65 months). Two patients died: one from sepsis during cycle 1 and another at 38 months while in remission from MCL. The progression-free survival at 3 years was 78% (95% CI: 51-91%). These results compare favorably with previously reported outcomes suggesting that durable remissions can be achieved without myeloablative therapy.

LMO2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with anthracycline-based chemotherapy with and without rituximab.

PURPOSE: The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab. PATIENTS AND METHODS: DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome. RESULTS: In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis. CONCLUSION: We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

CD28-mediated regulation of multiple myeloma cell proliferation and survival.

Although interactions with bone marrow stromal cells are essential for multiple myeloma (MM) cell survival, the specific molecular and cellular elements involved are largely unknown, due in large part to the complexity of the bone marrow microenvironment itself. The T-cell costimulatory receptor CD28 is also expressed on normal and malignant plasma cells, and CD28 expression in MM correlates significantly with poor prognosis and disease progression. In contrast to T cells, activation and function of CD28 in myeloma cells is largely undefined. We have found that direct activation of myeloma cell CD28 by anti-CD28 mAb alone induces activation of PI3K and NFkappaB, suppresses MM cell proliferation, and protects against serum starvation and dexamethasone (dex)-induced cell death. Coculture with dendritic cells (DCs) expressing the CD28 ligands CD80 and CD86 also elicits CD28-mediated effects on MM survival and proliferation, and DCs appear to preferentially localize within myeloma infiltrates in primary patient samples. Our findings suggest a previously undescribed myeloma/DC cell-cell interaction involving CD28 that may play an important role in myeloma cell survival within the bone marrow stroma. These data also point to CD28 as a potential therapeutic target in the treatment of MM.

IRF-4 and c-Rel expression in antiviral-resistant adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a generally fatal malignancy. Most ATLL patients fare poorly with conventional chemotherapy; however, antiviral therapy with zidovudine (AZT) and interferon alpha (IFN-alpha) has produced long-term clinical remissions. We studied primary ATLL tumors and identified molecular features linked to sensitivity and resistance to antiviral therapy. Enhanced expression of the proto-oncogene c-Rel was noted in 9 of 27 tumors. Resistant tumors exhibited c-Rel (6 of 10; 60%) more often than did sensitive variants (1 of 9; 11%). This finding was independent of the disease form. Elevated expression of the putative c-Rel target, interferon regulatory factor-4 (IRF-4), was observed in 10 (91%) of 11 nonresponders and in all tested patients with c-Rel+ tumors and occurred in the absence of the HTLV-1 oncoprotein Tax. In contrast, tumors in complete responders did not express c-Rel or IRF-4. Gene rearrangement studies demonstrated the persistence of circulating T-cell clones in long-term survivors maintained on antiviral therapy. The expression of nuclear c-Rel and IRF-4 occurs in the absence of Tax in primary ATLL and is associated with antiviral resistance. These molecular features may help guide treatment. AZT and IFN-alpha is a suppressive rather than a curative regimen, and patients in clinical remission should remain on maintenance therapy indefinitely.

Ocular adnexal lymphoma: a clinicopathologic study of a large cohort of patients with no evidence for an association with Chlamydia psittaci.

Non-Hodgkin lymphomas are among the most common primary tumors occurring in the ocular adnexa. Herein, we present a 14-year single-institution experience in 62 patients with primary ocular adnexal lymphomas (OALs). Association with Chlamydia psittaci infection is examined in 57 tumor specimens. Extranodal marginal zone lymphoma (EMZL) was the most frequent histologic subtype (89%). The majority of patients with EMZL (84%) presented with stage E-extranodal (IE), however only 16% had an advanced stage. All stage IE patients were treated with local radiotherapy, whereas patients with disseminated disease received systemic therapy with or without local irradiation. All but 1 patient with EMZL achieved complete remission (CR). During a median follow-up of 52 months (range, 3-153 months), the estimated 5-year overall survival (OS) and freedom from progression (FFP) were 96% and 79%, respectively. During the follow-up, 22% of patients relapsed, mainly in extranodal sites, and 4% transformed to diffuse large B-cell lymphoma. None of the patients exhibited local orbital failure in the radiation field. None of the OAL specimens harbored C psittaci DNA. Our study demonstrates that EMZLs, accounting for the majority of primary OALs, are characterized by an indolent natural history with frequent, continuous extranodal relapses. In South Florida, OALs are not associated with C psittaci infections.

Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation.

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

The utility of a bone marrow biopsy in diagnosing the source of fever of unknown origin in patients with AIDS.

A bone marrow (BM) aspiration and biopsy is often believed to be a needed diagnostic procedure in the work-up of patients with fever of unknown origin (FUO), especially in the setting of AIDS. Is it worthwhile to proceed with this invasive diagnostic method? Clinical information obtained on 104 patients in whom AIDS had been previously diagnosed and who had been admitted with a presumptive diagnosis of FUO was retrospectively analyzed. Seventy-two cases met the inclusion criteria. A BM aspiration and biopsy had a low sensitivity as a diagnostic tool even in patients who had abnormal hematologic parameters. BM biopsy was also not helpful in diagnosing non-Hodgkin lymphoma (NHL) cases in this study. Although the incidence of NHL has risen since the emergence of HIV, the predominant types of lymphoma seen in AIDS patients are intermediate/high-grade lymphomas rather than low grade, and consequently, the incidence of BM involvement is low, decreasing the sensitivity of a BM biopsy as a diagnostic tool. These observations were validated in this study. The majority of BM biopsies in this series revealed diagnostic features of infections. This observation can likely be related to the high prevalence of HIV/AIDS patients in this community and opportunistic infections associated with this disease.